JP2025503994A - Coronavirus vaccine - Google Patents

Abstract

The present disclosure relates to the field of RNA for preventing or treating coronavirus infections. In particular, the present disclosure relates to methods and agents for vaccination against coronavirus infections and for inducing an effective coronavirus antigen-specific immune response, such as an antibody and/or T cell response. Specifically, in one embodiment, the present disclosure relates to a method for inducing an immune response in a subject against a coronavirus S protein, in particular the S protein of SARS-CoV-2, comprising administering to said subject an RNA encoding a peptide or protein comprising an epitope of the SARS-CoV-2 spike protein (S protein), i.e., a vaccine RNA encoding a vaccine antigen.

Description

The present disclosure relates to the field of RNA for preventing or treating coronavirus infections. In particular, the present disclosure relates to methods and agents for vaccination against coronavirus infections and for inducing effective coronavirus antigen-specific immune responses, such as antibody and/or T cell responses. These methods and agents are particularly useful for preventing or treating coronavirus infections. Administration of the RNA disclosed herein to a subject can protect the subject from coronavirus infection. Specifically, in one embodiment, the present disclosure relates to a method comprising administering to a subject RNA encoding a peptide or protein comprising an epitope of the SARS-CoV-2 spike protein (S protein), i.e., a vaccine RNA encoding a vaccine antigen, to induce an immune response in the subject against a coronavirus S protein, particularly the S protein of SARS-CoV-2. Administering the RNA encoding the vaccine antigen to the subject can provide the vaccine antigen (after expression of the RNA by appropriate target cells) for inducing an immune response to the vaccine antigen (and disease-associated antigens) in the subject.

Coronaviruses are single-stranded positive-stranded RNA ((+)ssRNA) enveloped viruses that encode a total of four structural proteins: spike protein (S), envelope protein (E), membrane protein (M), and nucleocapsid protein (N). The spike protein (S protein) is responsible for receptor recognition, cell binding, infection via the endosomal pathway, and genome release driven by fusion of the viral membrane with the endosomal membrane. Although the sequences between different family members differ, conserved regions and motifs exist within the S protein, dividing the S protein into two subdomains: S1 and S2. The S1 domain recognizes virus-specific receptors and binds to target host cells, while the S2 domain, which has a transmembrane domain, is responsible for membrane fusion. In several coronavirus isolates, receptor binding domains (RBDs) have been identified and the general structure of the S protein has been defined (Figure 1).

In December 2019, an outbreak of pneumonia of unknown cause occurred in Wuhan, China, and a novel coronavirus (Severe Acute Respiratory Syndrome coronavirus 2, SARS-CoV-2) was identified as the underlying cause. The genetic sequence of SARS-CoV-2 was made available to the WHO and the public (MN908947.3), and the virus was classified into the Betacoronavirus subfamily. Sequence analysis revealed a phylogenetic tree that revealed a closer relationship to Severe Acute Respiratory Syndrome (SARS) virus isolates than to another coronavirus infecting humans, namely Middle East Respiratory Syndrome (MERS) virus.

SARS-CoV-2 infection and the resulting disease COVID-19 have spread globally, affecting an increasing number of countries. On March 11, 2020, the WHO characterized the COVID-19 outbreak as a pandemic. As of December 1, 2020, there have been more than 63 million confirmed cases of COVID-19 worldwide, with more than 1.4 million deaths, and 191 countries/territories affected. The ongoing pandemic remains a significant challenge to global public health and economic stability.

All individuals are at risk of infection as there is no pre-existing immunity to SARS-CoV-2. Following infection, some, but not all, individuals develop protective immunity in terms of neutralizing antibody responses and cell-mediated immunity. However, it is currently unknown to what extent and for how long this protection lasts. According to the WHO, 80% of infected individuals recover without needing hospital care, 15% develop more severe disease, and 5% require intensive care. Increasing age and underlying medical conditions are considered risk factors for developing severe disease.

Symptoms of COVID-19 are generally accompanied by cough and fever, and chest radiographs show ground-glass opacities or patchy shadows. However, many patients present without fever or radiographic changes and the infection may be asymptomatic, which is relevant for controlled transmission. In symptomatic subjects, disease progression may lead to acute respiratory distress syndrome requiring mechanical ventilation and subsequent multiple organ failure and death. Common symptoms in hospitalized patients (ordered from most to least frequent) include fever, dry cough, shortness of breath, fatigue, myalgia, nausea/vomiting or diarrhea, headache, weakness, and nosebleeds. In approximately 3% of individuals with COVID-19, anosmia (loss of smell) or anageusia (loss of taste) may be the only presenting symptom.

All ages can present with the disease, but the case fatality rate (CFR) is elevated, especially in those over 60 years of age. Comorbidities are also associated with increased CFR, including cardiovascular disease, diabetes, hypertension, and chronic respiratory disease. Healthcare workers are overrepresented among COVID-19 patients due to occupational exposure to infected patients.

In most cases, molecular tests are used to detect SARS-CoV-2 and confirm infection. Reverse transcription polymerase chain reaction (RT-PCR) testing methods targeting SARS-CoV-2 viral RNA are the gold standard in vitro method for diagnosing suspected cases of COVID-19. Samples to be tested are collected by swabbing from the nose and/or throat.

Among other things, the present disclosure provides insight into the immune responses elicited by exposure (e.g., by vaccination and/or infection) to different SARS-CoV-2 variants or immunogenic polypeptides (e.g., S proteins), or immunogenic fragments thereof. For example, in some embodiments, administration of RNA encoding the S proteins of the BA.2 and/or BA.4/5 omicron SARS-CoV-2 variants, or immunogenic fragments thereof, can result in improved immune responses, including, for example, improved neutralization of omicron BA.4 and/or omicron BA.5 SARS-CoV-2 variants and/or broader cross-neutralization of variants of concern (e.g., omicron variants) (e.g., increased neutralization titers against more variants of concern (e.g., omicron variants)). In some embodiments, the present disclosure provides insight that a bivalent coronavirus vaccine (e.g., a bivalent BA.4/5 vaccine comprising a first RNA encoding a SARS-CoV-2 S protein of the Wuhan strain, or an immunogenic fragment thereof, and a second RNA encoding a SARS-CoV-2 S protein, or an immunogenic fragment thereof, comprising one or more mutations characteristic of the BA.4/5 omicron variant) can provide broader cross-neutralization against the SARS-CoV-2 Wuhan strain and certain variants thereof (e.g., in some embodiments, variants that are prevalent and/or spreading rapidly in a relevant jurisdiction, e.g., certain omicron variants) in certain subjects compared to a monovalent coronavirus vaccine (e.g., a vaccine comprising an RNA encoding a SARS-CoV-2 S protein of a coronavirus strain or variant thereof). In some embodiments, such broader cross-neutralization may be observed in unvaccinated subjects. In some embodiments, such broader cross-neutralization may be observed in subjects without a coronavirus infection (e.g., SARS-CoV-2 infection). In some embodiments, such broader cross-neutralization may be observed in subjects who have previously received a SARS-CoV-2 vaccine (e.g., in some embodiments, an RNA vaccine encoding a SARS-CoV-2 S protein, e.g., in some embodiments of the Wuhan strain). In some embodiments, such broader cross-neutralization may be observed in young pediatric subjects (e.g., subjects aged 6 months to less than 2 years and/or 2 years to less than 5 years).

In some embodiments, the present disclosure provides insight that exposure to at least two certain SARS-CoV-2 variants or immunogenic polypeptides (e.g., S protein), or immunogenic fragments thereof, may result in synergistic improvements in immune responses (e.g., higher neutralizing titers, broader cross-neutralization, and/or immune responses that are less susceptible to immune escape) compared to exposure to other combinations of one SARS-CoV-2 strain and/or SARS-CoV-2 variants. In some embodiments, the present disclosure provides insight that exposure to the S protein or immunogenic fragment thereof from the Wuhan strain (e.g., by vaccination and/or infection) and exposure to the Omicron BA. In some embodiments, the present disclosure provides the insight that exposure to the S protein or immunogenic fragment thereof from the Wuhan strain (e.g., by vaccination and/or infection) and exposure to the S protein or immunogenic fragment thereof from the Omicron BA.4 or BA.5 variants (e.g., by vaccination and/or infection) can result in a synergistic improvement in the immune response (e.g., higher neutralization titers, broader cross-neutralization, and/or an immune response that is less susceptible to immune escape) compared to exposure to other combinations of one SARS-CoV-2 strains and/or SARS-CoV-2 variants. In some embodiments, the present disclosure provides the insight that exposure to the S protein or immunogenic fragment thereof from the Wuhan strain (e.g., by vaccination and/or infection) and exposure to the S protein or immunogenic fragment thereof from the Omicron BA.4 or BA.5 variants (e.g., by vaccination and/or infection) can result in a synergistic improvement in the immune response (e.g., higher neutralization titers, broader cross-neutralization, and/or an immune response that is less susceptible to immune escape) compared to exposure to other combinations of one SARS-CoV-2 strains and/or SARS-CoV-2 variants. In some embodiments, the present disclosure provides the insight that (i) exposure (e.g., by vaccination and/or infection) to an S protein from a strain/variant selected from the Wuhan strain, alpha variant, beta variant, delta variant, Omicron BA.1, and sublineages derived from any of the foregoing strains/variants, or immunogenic fragments thereof, in combination with (ii) exposure (e.g., by vaccination and/or infection) to an S protein from a strain/variant selected from the group consisting of Omicron BA.2, Omicron BA.4, Omicron BA.5, and sublineages derived from any of the foregoing strains/variants, can result in a synergistic improvement in the immune response (e.g., higher neutralization titers, broader cross-neutralization, and/or an immune response less susceptible to immune escape) compared to exposure to other combinations of one SARS-CoV-2 strain and/or SARS-COV-2 variant.

The present disclosure also provides important insights into how immune responses develop in subjects following exposure (e.g., vaccination and/or infection) to multiple different SARS-CoV-2 strains. Among other things, disclosed herein is the finding that different combinations of SARS-CoV-2 variants induce different immune responses. Specifically, the present disclosure provides the insight that exposure to certain combinations of SARS-CoV-2 variants can induce improved immune responses (e.g., higher neutralizing titers, broader cross-neutralization, and/or immune responses that are less susceptible to immune escape). In some embodiments, improved immune responses can be produced when a subject is delivered two or more antigens (e.g., as polypeptides or RNA encoding such polypeptides), each of which has few shared epitopes. In some embodiments, an improved immune response may be produced when a subject is delivered (e.g., as a polypeptide or RNA encoding such a polypeptide) a combination of SARS-CoV-2 S proteins that share 50% or less (e.g., 40% or less, 30% or less, 20% or more) epitopes (e.g., including amino acid mutations) that can be bound by neutralizing antibodies. In some embodiments, an improved immune response may be produced by delivering (a) a SARS-CoV-2 S protein or an immunogenic fragment thereof from the Wuhan strain of SARS-CoV-2, an alpha variant, a beta variant, or a delta variant, and (b) an S protein or an immunogenic fragment thereof from the SARS-CoV-2 omicron variant, as a polypeptide or RNA encoding such a polypeptide. In some embodiments, an improved immune response may be produced by delivering, as a polypeptide or RNA encoding such polypeptide, (a) a SARS-CoV-2 S protein or an immunogenic fragment thereof from the Wuhan strain, an alpha variant, a beta variant, or a delta variant of SARS-CoV-2, and (b) an S protein or an immunogenic fragment thereof of a SARS-CoV-2 omicron variant that is not a BA.1 omicron variant. In some embodiments, an improved immune response may be produced by delivering, as a polypeptide or RNA encoding such polypeptide, (a) an S protein or an immunogenic fragment thereof from the Wuhan strain, an alpha variant, a beta variant, a delta SARS-CoV-2 variant, or a BA.1 omicron variant, and (b) an S protein or an immunogenic fragment thereof of a SARS-CoV-2 omicron variant that is not a BA.1 omicron variant. In some embodiments, an improved immune response may be produced by delivering, as a polypeptide or RNA encoding such a polypeptide, (a) the SARS-CoV-2 S protein or an immunogenic fragment thereof from the Wuhan strain, alpha variant, beta variant, or delta variant, and (b) the S protein or an immunogenic fragment thereof of the BA.2 or BA.4 or BA.5 SARS-CoV-2 omicron variant.

In some embodiments, the present disclosure also provides insight that administration of multiple doses (e.g., at least two, at least three, at least four, or more doses) of a coronavirus vaccine described herein (e.g., a bivalent vaccine described herein, such as a bivalent BA.4/5 vaccine) may provide certain beneficial effect(s) on antibody affinity for one or more SARS-CoV-2 strains or variants thereof. In some embodiments, such beneficial effect(s) on antibody affinity may be observed for antibodies to certain omicron variants. By way of example only, in some embodiments, such beneficial effect(s) on antibody affinity may be observed for antibodies to certain omicron variants that share at least one or more common epitopes with, for example, the Wuhan strain.

Also disclosed herein are compositions that can provide improved immune responses (e.g., immune responses with broader cross-neutralizing activity, more potent neutralization, and/or immune responses that are less susceptible to immune escape). In some embodiments, the compositions described herein include two or more antigens or nucleic acids (e.g., RNA) encoding such antigens that have few shared epitopes. In some embodiments, the compositions described herein deliver, as a polypeptide or nucleic acid encoding such a polypeptide, a combination of SARS-CoV-2 S proteins or immunogenic fragments thereof that share 50% or less (e.g., 40% or less, 30% or less, 20% or less, or more) of epitopes that can be bound by neutralizing antibodies (e.g., including amino acid mutations). In some embodiments, the compositions described herein comprise (a) an RNA encoding a SARS-CoV-2 S protein or an immunogenic fragment thereof from the Wuhan strain, an alpha variant, a beta variant, or a delta variant, and (b) an RNA encoding an S protein or an immunogenic fragment thereof from an omicron variant of SARS-CoV-2 (e.g., in some embodiments, an S protein from the BA.1, BA.2, or BA.4/5 omicron variants) or an immunogenic fragment thereof. In some embodiments, the compositions described herein comprise (a) an RNA encoding a SARS-CoV-2 S protein or an immunogenic fragment thereof from the Wuhan strain, an alpha variant, a beta variant, or a delta variant, and (b) an RNA encoding an S protein or an immunogenic fragment thereof of an omicron variant of SARS-CoV-2 that is not the BA.1 omicron variant. In some embodiments, the compositions described herein comprise (a) an RNA encoding a SARS-CoV-2 S protein or an immunogenic fragment thereof from the Wuhan strain, an alpha variant, a beta variant, or a delta variant, or an S protein from the omicron variant of SARS-CoV-2 that is not the BA.1 omicron variant. The compositions described herein comprise (a) an RNA encoding a SARS-CoV-2 S protein of the Wuhan strain of SARS-CoV-2, an alpha variant, a beta variant, or a delta variant, and (b) an RNA encoding an S protein or an immunogenic fragment thereof of a BA.1 omicron variant that is not a BA.1 omicron variant. In some embodiments, the compositions described herein comprise (a) an RNA encoding a SARS-CoV-2 S protein of the Wuhan strain of SARS-CoV-2, an alpha variant, a beta variant, or a delta variant, and (b) an RNA encoding an S protein or an immunogenic fragment thereof of a BA.2 or BA.4 or BA.5 omicron variant of SARS-CoV-2. In some embodiments, the compositions described herein comprise RNA encoding an S protein or an immunogenic fragment thereof from the BA.2 omicron variant of SARS-CoV-2. In some embodiments, the compositions comprise RNA encoding an S protein or an immunogenic fragment thereof from the BA.4 or BA.5 omicron variant of SARS-CoV-2.

SARS-CoV-2 is an RNA virus with four structural proteins. One of them, the spike protein, is a surface protein that binds to angiotensin-converting enzyme 2 (ACE-2) present in host cells. Therefore, the spike protein is considered to be a relevant antigen for vaccine development.

BNT162b2 (SEQ ID NO: 20) is an mRNA vaccine for the prevention of COVID-19, which has demonstrated greater than 95% efficacy in preventing COVID-19. The vaccine is made with 5'-capped mRNA encoding the full-length SARS-CoV-2 spike glycoprotein (S) encapsulated in lipid nanoparticles (LNPs). The final product is presented as an injectable dispersion concentrate containing BNT162b2 as the active agent. Other ingredients are ALC-0315 (4-hydroxybutyl)azanediyl)bis(hexane-6,1-diyl)bis(2-hexyldecanoate), ALC-0159 (2-[(polyethylene glycol)-2000]-N,N-ditetradecylacetamide), 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), cholesterol, potassium chloride, potassium dihydrogen phosphate, sodium chloride, disodium phosphate hydrate, sucrose, and water for injection.

In some embodiments, a different buffer may be used in place of PBS. In some embodiments, the buffer is formulated in a Tris buffer. In some embodiments, the formulation includes ALC-0315 (4-hydroxybutyl)azanediyl)bis(hexane-6,1-diyl)bis(2-hexyldecanoate), ALC-0159 (2-[(polyethylene glycol)-2000]-N,N-ditetradecylacetamide), DSPC (1,2-distearoyl-sn-glycero-3-phosphocholine), cholesterol, sucrose, trometamol (Tris), trometamol hydrochloride, and water.

In some embodiments, the concentration of RNA in the pharmaceutical RNA preparation is about 0.1 mg/ml. In some embodiments, about 30 ug of RNA is administered by administering about 200 uL of the RNA preparation. In some embodiments, the RNA in the pharmaceutical RNA preparation is diluted prior to administration (e.g., diluted to a concentration of about 0.05 mg/ml). In some embodiments, the administration volume is about 200 μl to about 300 μl. In some embodiments, the RNA in the pharmaceutical RNA preparation is formulated in about 10 mM Tris buffer and about 10% sucrose.

In some embodiments, the concentration of RNA in the pharmaceutical RNA preparation is about 0.1 mg/ml and is formulated in about 10 mM Tris buffer, about 10% sucrose, and a dose of about 10 μg, or the RNA is administered by diluting the pharmaceutical RNA preparation about 1:1 and administering about 200 μl of the diluted pharmaceutical RNA preparation. In some embodiments, the concentration of RNA in the pharmaceutical RNA preparation is about 0.1 mg/ml and is formulated in about 10 mM Tris buffer, about 10% sucrose, and a dose of about 10 μg of RNA, and is administered by diluting the pharmaceutical RNA preparation about 1:5.75 and administering about 200 μl of the diluted pharmaceutical RNA preparation.

The sequence of the Wuhan strain SARS-CoV-2 S protein disclosed herein was selected based on the sequence of "SARS-CoV-2 isolate Wuhan-Hu-1" GenBank: MN908947.3 (complete genome) and GenBank: QHD43416.1 (spike surface glycoprotein).

In some embodiments, the active agent consists of a single strand 5' cap codon optimized mRNA that is translated into the spike antigen of SARS-CoV-2. In some embodiments, the encoded spike antigen protein sequence contains two proline mutations that stabilize an antigenically optimal pre-fusion confirmation (P2S). In some embodiments, the RNA does not contain any uridines, e.g., modified N1-methyl pseudouridine can be used in place of uridine in the RNA synthesis. The mRNA disclosed herein can be translated into the SARS-CoV-2 S protein in a host cell. The S protein is then expressed on the cell surface where it can induce an adaptive immune response. The S protein has been identified as a target for neutralizing antibodies against the virus and can be considered a relevant vaccine component.

BNT162b2 can be administered intramuscularly (IM) in two 30 μg doses of diluted vaccine solution given approximately 21 days apart (e.g., for adult unvaccinated subjects (i.e., subjects aged 12 years or older who have not previously received a SARS-CoV-2 vaccine)).

The recent emergence of novel circulating variants of SARS-CoV-2 raises serious concerns regarding the geographic and temporal effectiveness of vaccine interventions. One of the earliest variants to emerge and rapidly become dominant globally was D614G.

The alpha variant (also known as B.1.1.7, VOC202012/01, 501Y.V1 or GRY) was initially detected in the UK. The alpha variant has a number of mutations, including several mutations in the S gene. It has been shown to be more transmissible in nature, with an estimated growth rate of 40-70% higher than other SARS-CoV-2 lineages in several countries (Volz et al., 2021, Nature, https://doi.org/10.1038/s41586-021-03470-x; Washington et al., 2021, Cell https://doi.org/10.1016/j.cell.2021.03.052).

The beta variant (also known as B.1.351 or GH/501Y.V2) was first detected in South Africa. The beta variant has several mutations in the S gene. Three of these mutations are sites in the RBD that are associated with immune evasion: N501Y (shared with alpha), and E484K and K417N.

The gamma variant (also known as P.1 or GR/501Y.V3) was first detected in Brazil. The gamma variant has several mutations that affect the spike protein, including two mutations shared with the beta (N501Y and E484K) as well as a different mutation at position 417 (K417T).

The delta variant (also known as B.1.617.2 or G/478K.V1) was first documented in India. The delta variant has several point mutations affecting the spike protein, including P681R (a mutation position shared with alpha and adjacent to the furin cleavage site) and L452R, which is within the RBD and has been linked to increased binding to ACE2 and neutralizing antibody resistance. There is also a deletion in the spike protein at positions 156/157.

These four VOCs have become prevalent worldwide and have become the predominant variants in the geographic regions in which they were first identified.

On November 24, 2021, the Omicron (B.1.1.529) variant was first reported to WHO from South Africa.

Since its first appearance in November 2021, SARS-CoV-2 Omicron and its sublineages have had a major impact on the epidemiological situation of the COVID-19 pandemic (WHO Technical Advisory Group on SARS-CoV-2 Virus Evolution (TAG-VE): Classification of Omicron (B.1.1.259): SARS-CoV-2 Variant of Concern (2021), WHO Headquarters (HQ), WHO Health Emergency Programme, Enhancing Response to Omicron SARS-CoV-2 variants: Technical brief and priority actions for Member States (2022)). The first omicron variant BA., which results in the loss of many neutralizing antibody epitopes. Due to significant modifications of the spike (S) glycoprotein of BA.1 (M. Hoffmann et al., "The Omicron variant is highly resistant against antibody mediated neutralization: Implications for control of the COVID-19 pandemic", Cell 185, 447-456.e11 (2022)), 1 was able to partially escape immunity based on the previously established SARS-CoV-2 wild-type strain (Wuhan-Hu-1) (V. Servellita, et al., "Neutralizing 30 immunity in vaccine breakthrough infections from the SARS-CoV-2 Omicron and Delta variants", Cell 185, 1539-1548.e5 (2022); Y. Cao et al., "Omicron escapes the majority of existing SARS-CoV-2 neutralizing antibodies", Nature 602, 657-663 (2022)). Thus, breakthrough infection with Omicron is more common in vaccinated individuals than with previous variants of concern (VOCs). In many countries around the world, Omicron BA.1 has been replaced by the BA.2 variant, while BA.1.1 and BA. Other variants, such as 3, have gained momentum temporarily and/or locally but have not become globally dominant (S. Xia et al., "Origin, virological features, immune evasion and intervention of SARS-CoV-2 Omicron sublineages. Signal Transduct. Target. Ther. 7, 241 (2022); H. Gruell et al., "SARS-CoV-2 Omicron sublineages exhibit distinct antibody escape patterns, Cell Host Microbe 7, 241 (2022). ) Omicron BA.2.12.1 has since replaced BA.2 as the dominant species in the United States, while BA.4 and BA.5 have dominated in Europe, parts of Africa, and Asia/Pacific. 2 has become dominant (H. Gruell et al., "SARS-CoV-2 Omicron sublineages exhibit distinct antibody escape patterns," Cell Host Microbe 7, 241 (2022), European Centre for Disease Prevention and Control, Weekly COVID-19 country overview-Country overview report: Week 31 2022 (2022), J. Hadfield et al., "SARS-CoV-2 Omicron sublineages exhibit distinct antibody escape patterns," Cell Host Microbe 7, 241 (2022), European Centre for Disease Prevention and Control, Weekly COVID-19 country overview-Country overview report: Week 31 2022 (2022), J. Hadfield et al., "SARS-CoV-2 Omicron sublineages exhibit distinct antibody escape patterns," Cell Host Microbe 7, 241 (2022), European Centre for Disease Prevention and Control, Weekly COVID-19 country overview-Country overview report: Week 31 2022 (2022), J. Hadfield et al., "SARS-CoV-2 Omicron sublineages exhibit distinct antibody escape patterns," Cell Host Microbe 7, 241 (2022), al., "Nextstrain: Real-time tracking of pathogen evolution," Bioinformatics 34, 4121-4123 (2018)). Currently, Omicron XBB. BA. 1.5 is predominant worldwide, including in the United States (Centers for Disease Control and Prevention. COVID Data Tracker. Atlanta, GA: US Department of Health and Human Services, CDC; 2023, January 22. https://covid.cdc.gov/coviddata-tracker (2022)). Omicron has acquired many modifications (amino acid exchanges, insertions, or deletions) in the S glycoprotein, some of which are shared among all Omicron VOCs and some of which are specific to one or more Omicron sublineages. Antigenically, BA. 2.12.1 is closely related to BA. Although BA.4 and BA.5 show high similarity to BA.2 but not to BA.1, BA.4 and BA.5 are significantly different from their ancestor BA.2 along their lineage and even more different from BA.1 (A.Z. Mykytyn et al., "Antigenic cartography of SARS-CoV-2 reveals that Omicron BA.1 and BA.2 are antigenically distinct," Sci. Immunol. 7, eabq4450 (2022).). The main differences between BA.1 and BA.2 include Δ143-145, L212I, or ins214EPE in the S glycoprotein N-terminal domain, and G446S or G496S in the receptor binding domain (RBD). The amino acid changes T376A, D405N, and R408S in the RBD are, in turn, shared with BA.2 and its progeny, but not found in BA.1. In addition, some modifications are specific to individual BA.2 progeny VOCs, including L452Q in the case of BA.2.12.1, or L452R and F486V in the cases of BA.4 and BA.5 (BA.4 and BA.5 encode 30 identical S sequences). Most of these shared VOC-specific modifications have been shown to play important roles in immune escape from monoclonal antibodies and polyclonal sera against the wild-type S glycoprotein. In particular, the BA.4/BA.2/BA.5 VOCs are not shared with BA.2. 5-specific modifications are strongly involved in the immune evasion of these VOCs (P. Wang et al., "Antibody resistance of SARS-CoV-2 variants B.1.351 and B.1.1.7. Nature 593, 130-135 (2021); Q. Wang et al., "Antibody evasion by SARS-CoV-2 Omicron subvariants BA.2.12.1, BA.4, & BA.5. Nature 608, 5 603-608 (2022)).

The present disclosure generally encompasses immunotherapeutic treatment of a subject comprising administration of an RNA, i.e., a vaccine antigen, that encodes an amino acid sequence, i.e., a vaccine antigen, comprising a SARS-CoV-2 S protein, an immunogenic variant thereof, or an immunogenic fragment of a SARS-CoV-2 S protein or an immunogenic variant thereof, i.e., an antigenic peptide or protein. Thus, the vaccine antigen comprises an epitope of a SARS-CoV-2 S protein for inducing an immune response in a subject against a coronavirus S protein, particularly a SARS-CoV-2 S protein. The RNA encoding the vaccine antigen is administered (following expression of the polynucleotide by appropriate target cells) to provide an antigen for induction, i.e., stimulation, priming, and/or expansion, of an immune response, e.g., antibodies and/or immune effector cells, that are targeted to the target antigen (coronavirus S protein, particularly a SARS-CoV-2 S protein) or its progressive products. In one embodiment, the immune response induced in accordance with the present disclosure is a B cell-mediated immune response, i.e., an antibody-mediated immune response. Additionally or alternatively, in one embodiment, the immune response induced in accordance with the present disclosure is a T cell-mediated immune response. In one embodiment, the immune response is an anti-coronavirus, particularly an anti-SARS-CoV-2, immune response.

The vaccines described herein comprise as an active ingredient a single stranded RNA that can be translated into a protein upon entry into the recipient's cells. In addition to a wild type or codon optimized sequence encoding an antigen sequence, the RNA may contain one or more structural elements (e.g., a 5' cap, a 5' UTR, a 3' UTR, a poly(A) tail, or a combination thereof) that are optimized for maximum effectiveness of the RNA with respect to stability and translation efficiency. In one embodiment, the RNA contains all of these elements. In one embodiment, a cap1 structure may be utilized as a specific capping structure at the 5' end of the RNA drug substance. In one embodiment, beta-S-ARCA(D1)(m ₂ ^7,2'-O GppSpG) or m ₂ ^7,3'-O Gppp(m ₁ ^2'-O )ApG may be utilized as a specific capping structure at the 5' end of the RNA drug substance. As the 5'-UTR sequence, the 5'-UTR sequence of human alpha-globin mRNA (e.g., SEQ ID NO: 12), optionally with an optimized "Kozak sequence" to increase translation efficiency, may be used. As the 3'-UTR sequence, a combination of two sequence elements (FI elements) derived from the "amino-terminal enhancer of split" (AES) mRNA (called F) and the mitochondrially encoded 12S ribosomal RNA (called I) (e.g., SEQ ID NO: 13) placed between the coding sequence and the poly(A) tail to ensure higher maximum protein levels and long-term persistence of the mRNA may be used. These were identified by an ex vivo selection process for sequences that confer RNA stability and enhance total protein expression (see WO 2017/060314, incorporated herein by reference). Alternatively, the 3'-UTR may be two repeated 3'-UTRs of human beta-globin mRNA.

Additionally or alternatively, in some embodiments, the RNA comprises a poly(A) tail comprising a length of at least 90 adenosine nucleotides (e.g., at least about 100 adenosine nucleotides, at least about 110 adenosine nucleotides, at least about 120 adenosine nucleotides, at least about 130 adenosine nucleotides, or more). In some embodiments, the poly(A) tail may comprise a length of about 90 to about 150 adenosine nucleotides (e.g., about 100 to about 150 adenosine nucleotides). In some embodiments, the poly(A) tail may comprise an interrupted poly(A) tail. For example, in some such embodiments, a poly(A) tail measuring about 90 to about 120 nucleotides (e.g., about 110 nucleotides in length) may be used (e.g., a poly(A) tail comprising SEQ ID NO: 14), consisting of a stretch of about 30 adenosine residues (e.g., about 28, about 29, about 30, about 31, or about 32 adenosine residues), followed by a linker sequence of about 10 nucleotides (e.g., about 9, about 10, or about 11 random nucleotides) and another about 70 adenosine nucleotides (e.g., about 65, about 66, about 67, about 68, about 69, about 70, about 71, about 72, about 73, about 74, or about 75 adenosine nucleotides). This poly(A) tail sequence was designed to improve RNA stability and translation efficiency.

Furthermore, in some embodiments, a nucleotide sequence encoding a secretory signal peptide (sec) may be fused to the antigen coding region, preferably in such a way that the sec is translated as an N-terminal tag. In one embodiment, the sec corresponds to the secretory signal peptide of the SARS-CoV-2 S protein. In some embodiments, a sequence encoding a short linker peptide consisting mainly of the amino acids glycine (G) and serine (S), commonly used in fusion proteins, may be used as the GS/linker to join the secretory signal and the antigenic polypeptide.

The vaccine RNA described herein may be complexed with proteins and/or lipids, preferably lipids, to generate RNA particles for administration. When combinations of different RNAs are used, the RNAs may be complexed together or may be complexed separately with proteins and/or lipids to generate RNA particles for administration.

In one aspect, the disclosure features a composition or medical preparation that includes RNA encoding a SARS-CoV-2 S polypeptide or an immunogenic fragment thereof, the SARS-CoV-2 S polypeptide or fragment includes: (a) an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO:1 and including one of the following sets of amino acid substitutions relative to SEQ ID NO:1: (1) D985P, V987P, F817P, A892P, A899P, and A942P, (2) K986P, V987P, F817P, A892P, A899P, and A942P, (3) D98 5P, V987P, F817P, A892P, A899P, A942P, D614G, R682G, R683S, and R685S, (4) K986P, V987P, F817P, A892P, A899P, A942P, D614G, R682G, R683S, and R685S, (5) D985P, K986P, V987P, F817P, A892P, A899P, A942P, D614G, R682G, R683S, and R685S, (6) D985P, V987P, F817P, A892P, (7) K986P, V987P, F817P, A892P, A899P, A942P, R682G, R683S, and R685S; or (8) D985P, K986P, V987P, F817P, A892P, A899P, A942P, R682G, R683S, and R685S; (b) at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more similar to SEQ ID NO: 69. or an amino acid sequence having 100% identity and containing one of the following sets of amino acid substitutions relative to SEQ ID NO:69: (1) D982P, V984P, F814P, A889P, A896P, and A939P; (2) K983P, V984P, F814P, A889P, A896P, and A939P; (3) D982P, V984P, F814P, A889P, A896P, A939P, R679G, R680S, and R682S; (4) K983P, V984P, F814P, A889P, (c) an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO:70 and including one of the following sets of amino acid substitutions relative to SEQ ID NO:70: (1) D982P, K983P, V984P, F814P, A889P, A896P, A939P, R679G, R680S, and R682S; 82P, V984P, F814P, A889P, A896P, and A939P, (2) K983P, V984P, F814P, A889P, A896P, and A939P, (3) D982P, V984P, F814P, A889P, A896P, A939P, R679G, R680S, and R682S, (4) K983P, V984P, F814P, A889P, A896P, A939P, R679G, R680S, and R682S, or (5) D982P, K983P, V984P, F81 or (d) an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 104 or 105 and including one of the following sets of amino acid substitutions relative to SEQ ID NO: 104 or 105: (1) D980P, V982P, F812P, A887P, A894P, and A937P; (2) K980P, V982P, F812P, A887P, A894P, and A937P; 81P, V982P, F812P, A887P, A894P, and A937P, (3) D980P, V982P, F812P, A887P, A894P, A937P, R677G, R678S, and R680S, (4) K981P, V982P, F812P, A887P, A894P, A937P, R677G, R678S, and R680S, or (5) D980P, K981P, V982P, F812P, A887P, A894P, A937P, R677G, R678S, and R680S.

In some embodiments, the RNA comprises modified nucleosides in place of uridines. In some embodiments, the RNA comprises modified uridines in place of all uridines. In some embodiments, the RNA comprises N1-methyl-pseudouridine (m1ψ) in place of all uridines. In some embodiments, the RNA comprises a 5' cap. In some embodiments, the 5' cap is or comprises m ₂ ^7,3'-O Gppp(m ₁ ^2'-O )ApG.

In some embodiments, the RNA comprises a 5'-UTR that is or comprises a modified human alpha-globin 5'-UTR. In some embodiments, the RNA comprises a 5'-UTR that comprises the nucleotide sequence of SEQ ID NO:12, or a nucleotide sequence that is at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identical to the nucleotide sequence of SEQ ID NO:12.

In some embodiments, the RNA comprises a 3'-UTR that is or includes a first sequence from a split amino-terminal enhancer (AES) messenger RNA and a second sequence from a mitochondrially encoded 12S ribosomal RNA. In some embodiments, the RNA comprises a 3'-UTR that includes a nucleotide sequence of SEQ ID NO:13, or a nucleotide sequence that is at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identical to the nucleotide sequence of SEQ ID NO:13.

In some embodiments, the RNA comprises a polyA sequence. In some embodiments, the polyA sequence comprises at least 100 nucleotides. In some embodiments, the polyA sequence comprises 30 adenine nucleotides followed by 70 adenine nucleotides, the 30 adenine nucleotides and the 70 adenine nucleotides being separated by a linker sequence. In some embodiments, the polyA sequence comprises or consists of a nucleotide sequence of SEQ ID NO:14, or a nucleotide sequence that is at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identical to the nucleotide sequence of SEQ ID NO:14.

In some embodiments, the RNA is or is to be formulated for intramuscular administration. In some embodiments, the RNA is or is to be formulated as a particle. In some embodiments, the particle is a lipid nanoparticle (LNP) or lipoplex (LPX) particle. In some embodiments, the LNP comprises ((4-hydroxybutyl)azanediyl)bis(hexane-6,1-diyl)bis(2-hexyldecanoate), 2-[(polyethylene glycol)-2000]-N,N-ditetradecylacetamide, 1,2-distearoyl-sn-glycero-3-phosphocholine, and cholesterol. In some embodiments, the lipoplex particle can be obtained by mixing the RNA with liposomes.

In some embodiments, the RNA is mRNA or saRNA.

In some embodiments, the composition or medical preparation is a pharmaceutical composition. In some embodiments, the composition or medical preparation is a vaccine.

In some embodiments, the pharmaceutical composition further comprises one or more pharma- ceutically acceptable carriers, diluents, and/or excipients.

In another aspect, the disclosure provides a composition or medical preparation comprising a first RNA encoding a first SARS-CoV-2 S polypeptide or an immunogenic fragment thereof and a second RNA encoding a second SARS-CoV-2 S polypeptide or an immunogenic fragment thereof, wherein (a) the first SARS-CoV-2 S polypeptide or fragment comprises an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO:1, and (b) the second SARS-CoV-2 S polypeptide or fragment comprises an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO:1. The S polypeptide or fragment has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO:1 and comprises an amino acid sequence that includes one of the following sets of amino acid substitutions relative to SEQ ID NO:1: (1) D985P, V987P, F817P, A 892P, A899P, and A942P, (2) K986P, V987P, F817P, A892P, A899P, and A942P, (3) D985P, V987P, F817P, A892P, A899P, A942P, D614G, R682G, R683S, and R685S, (4) K986P, V987P, F817P, A89 2P, A899P, A942P, D614G, R682G, R683S, and R685S, (5) D985P, K986P, V987P, F817P, A892P, A899P, A942P, D614G, R682G, R683S, and R685S, (6) D985P, V987P, F817P, A892P, A899P, A942 P, R682G, R683S, and R685S, (7) K986P, V987P, F817P, A892P, A899P, A942P, R682G, R683S, and R685S, or (8) D985P, K986P, V987P, F817P, A892P, A899P, A942P, R682G, R683S, and R685S.

In one aspect, the disclosure features a composition or medical preparation that includes a first RNA encoding a first SARS-CoV-2 S polypeptide or an immunogenic fragment thereof, and a second RNA encoding a second SARS-CoV-2 S polypeptide or an immunogenic fragment thereof, wherein (a) the first SARS-CoV-2 S polypeptide or fragment comprises an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO:1, and (b) the second SARS-CoV-2 S polypeptide or fragment comprises an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO:1. The S polypeptide or fragment has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO:69 and comprises an amino acid sequence that includes one of the following sets of amino acid substitutions relative to SEQ ID NO:69: (1) D982P, V984P, F814P, A889P, A896P, and A939P; (2) K983P, V984P, F814P, A889P, A939P; 896P, and A939P, (3) D982P, V984P, F814P, A889P, A896P, A939P, R679G, R680S, and R682S, (4) K983P, V984P, F814P, A889P, A896P, A939P, R679G, R680S, and R682S, or (5) D982P, K983P, V984P, F814P, A889P, A896P, A939P, R679G, R680S, and R682S.

Another aspect of the disclosure features a composition or medical preparation that includes a first RNA encoding a first SARS-CoV-2 S polypeptide or an immunogenic fragment thereof, and a second RNA encoding a second SARS-CoV-2 S polypeptide or an immunogenic fragment thereof, wherein (a) the first SARS-CoV-2 S polypeptide or fragment comprises an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO:1, and (b) the second SARS-CoV-2 S polypeptide or fragment comprises an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO:1. The S polypeptide or fragment has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO:70 and comprises an amino acid sequence containing one of the following sets of amino acid substitutions relative to SEQ ID NO:70: (1) D982P, V984P, F814P, A889P, A896P, and A939P; (2) K983P, V984P, F814P, A889P, A939P; 896P, and A939P, (3) D982P, V984P, F814P, A889P, A896P, A939P, R679G, R680S, and R682S, (4) K983P, V984P, F814P, A889P, A896P, A939P, R679G, R680S, and R682S, or (5) D982P, K983P, V984P, F814P, A889P, A896P, A939P, R679G, R680S, and R682S.

Another aspect of the disclosure features a composition or medical preparation that includes a first RNA encoding a first SARS-CoV-2 S polypeptide or an immunogenic fragment thereof, and a second RNA encoding a second SARS-CoV-2 S polypeptide or an immunogenic fragment thereof, wherein (a) the first SARS-CoV-2 S polypeptide or fragment comprises an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO:1, and (b) the second SARS-CoV-2 S polypeptide or fragment comprises an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO:1. The S polypeptide or fragment has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 104 or 105 and comprises an amino acid sequence that includes one of the following sets of amino acid substitutions relative to SEQ ID NO: 104 or 105: (1) D980P, V982P, F812P, A887P, A894P, and A937P; (2) K981P, V982P, F812P, A887P, A894P, and A937P, (3) D980P, V982P, F812P, A887P, A894P, A937P, R677G, R678S, and R680S, (4) K981P, V982P, F812P, A887P, A894P, A937P, R677G, R678S, and R680S, or (5) D980P, K981P, V982P, F812P, A887P, A894P, A937P, R677G, R678S, and R680S.

Another aspect of the present disclosure provides a composition or medical preparation comprising a first RNA encoding a first SARS-CoV-2 S polypeptide or an immunogenic fragment thereof, and a second RNA encoding a second SARS-CoV-2 S polypeptide or an immunogenic fragment thereof, wherein (a) the first SARS-CoV-2 S polypeptide or fragment comprises an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO:69, and (b) the second SARS-CoV-2 S polypeptide or fragment comprises an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO:69. The S polypeptide or fragment has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO:1 and comprises an amino acid sequence that includes one of the following sets of amino acid substitutions relative to SEQ ID NO:1: (1) D985P, V987P, F817P, A 892P, A899P, and A942P, (2) K986P, V987P, F817P, A892P, A899P, and A942P, (3) D985P, V987P, F817P, A892P, A899P, A942P, D614G, R682G, R683S, and R685S, (4) K986P, V987P, F817P, A89 2P, A899P, A942P, D614G, R682G, R683S, and R685S, (5) D985P, K986P, V987P, F817P, A892P, A899P, A942P, D614G, R682G, R683S, and R685S, (6) D985P, V987P, F817P, A892P, A899P, A942 P, R682G, R683S, and R685S, (7) K986P, V987P, F817P, A892P, A899P, A942P, R682G, R683S, and R685S, or (8) D985P, K986P, V987P, F817P, A892P, A899P, A942P, R682G, R683S, and R685S.

Another aspect of the disclosure features a composition or medical preparation that includes a first RNA encoding a first SARS-CoV-2 S polypeptide or an immunogenic fragment thereof, and a second RNA encoding a second SARS-CoV-2 S polypeptide or an immunogenic fragment thereof, wherein (a) the first SARS-CoV-2 S polypeptide or fragment comprises an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO:69, and (b) the second SARS-CoV-2 S polypeptide or fragment comprises an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO:69. The S polypeptide or fragment has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO:69 and comprises an amino acid sequence that includes one of the following sets of amino acid substitutions relative to SEQ ID NO:69: (1) D982P, V984P, F814P, A889P, A896P, and A939P; (2) K983P, V984P, F814P, A889P, A939P; 896P, and A939P, (3) D982P, V984P, F814P, A889P, A896P, A939P, R679G, R680S, and R682S, (4) K983P, V984P, F814P, A889P, A896P, A939P, R679G, R680S, and R682S, or (5) D982P, K983P, V984P, F814P, A889P, A896P, A939P, R679G, R680S, and R682S.

Another aspect of the disclosure features a composition or medical preparation that includes a first RNA encoding a first SARS-CoV-2 S polypeptide or an immunogenic fragment thereof, and a second RNA encoding a second SARS-CoV-2 S polypeptide or an immunogenic fragment thereof, wherein (a) the first SARS-CoV-2 S polypeptide or fragment comprises an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO:69, and (b) the second SARS-CoV-2 S polypeptide or fragment comprises an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO:69. The S polypeptide or fragment has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO:70 and comprises an amino acid sequence containing one of the following sets of amino acid substitutions relative to SEQ ID NO:70: (1) D982P, V984P, F814P, A889P, A896P, and A939P; (2) K983P, V984P, F814P, A889P, A939P; 896P, and A939P, (3) D982P, V984P, F814P, A889P, A896P, A939P, R679G, R680S, and R682S, (4) K983P, V984P, F814P, A889P, A896P, A939P, R679G, R680S, and R682S, or (5) D982P, K983P, V984P, F814P, A889P, A896P, A939P, R679G, R680S, and R682S.

Another aspect of the disclosure features a composition or medical preparation that includes a first RNA encoding a first SARS-CoV-2 S polypeptide or an immunogenic fragment thereof, and a second RNA encoding a second SARS-CoV-2 S polypeptide or an immunogenic fragment thereof, wherein (a) the first SARS-CoV-2 S polypeptide or fragment comprises an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO:69, and (b) the second SARS-CoV-2 S polypeptide or fragment comprises an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO:69. The S polypeptide or fragment has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 104 or 105 and comprises an amino acid sequence that includes one of the following sets of amino acid substitutions relative to SEQ ID NO: 104 or 105: (1) D980P, V982P, F812P, A887P, A894P, and A937P; (2) K981P, V982P, F812P, A887P, A894P, and A937P, (3) D980P, V982P, F812P, A887P, A894P, A937P, R677G, R678S, and R680S, (4) K981P, V982P, F812P, A887P, A894P, A937P, R677G, R678S, and R680S, or (5) D980P, K981P, V982P, F812P, A887P, A894P, A937P, R677G, R678S, and R680S.

Another aspect of the disclosure features a composition or medical preparation that includes a first RNA encoding a first SARS-CoV-2 S polypeptide or an immunogenic fragment thereof, and a second RNA encoding a second SARS-CoV-2 S polypeptide or an immunogenic fragment thereof, wherein (a) the first SARS-CoV-2 S polypeptide or fragment comprises an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO:70, and (b) the second SARS-CoV-2 S polypeptide or fragment comprises an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO:70. The S polypeptide or fragment has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO:1 and comprises an amino acid sequence that includes one of the following sets of amino acid substitutions relative to SEQ ID NO:1: (1) D985P, V987P, F817P, A 892P, A899P, and A942P, (2) K986P, V987P, F817P, A892P, A899P, and A942P, (3) D985P, V987P, F817P, A892P, A899P, A942P, D614G, R682G, R683S, and R685S, (4) K986P, V987P, F817P, A89 2P, A899P, A942P, D614G, R682G, R683S, and R685S, (5) D985P, K986P, V987P, F817P, A892P, A899P, A942P, D614G, R682G, R683S, and R685S, (6) D985P, V987P, F817P, A892P, A899P, A942 P, R682G, R683S, and R685S, (7) K986P, V987P, F817P, A892P, A899P, A942P, R682G, R683S, and R685S, or (8) D985P, K986P, V987P, F817P, A892P, A899P, A942P, R682G, R683S, and R685S.

Another aspect of the disclosure features a composition or medical preparation that includes a first RNA encoding a first SARS-CoV-2 S polypeptide or an immunogenic fragment thereof, and a second RNA encoding a second SARS-CoV-2 S polypeptide or an immunogenic fragment thereof, wherein (a) the first SARS-CoV-2 S polypeptide or fragment comprises an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO:70, and (b) the second SARS-CoV-2 S polypeptide or fragment comprises an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO:70. The S polypeptide or fragment has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO:69 and comprises an amino acid sequence that includes one of the following sets of amino acid substitutions relative to SEQ ID NO:69: (1) D982P, V984P, F814P, A889P, A896P, and A939P; (2) K983P, V984P, F814P, A889P, A939P; 896P, and A939P, (3) D982P, V984P, F814P, A889P, A896P, A939P, R679G, R680S, and R682S, (4) K983P, V984P, F814P, A889P, A896P, A939P, R679G, R680S, and R682S, or (5) D982P, K983P, V984P, F814P, A889P, A896P, A939P, R679G, R680S, and R682S.

Another aspect of the disclosure features a composition or medical preparation that includes a first RNA encoding a first SARS-CoV-2 S polypeptide or an immunogenic fragment thereof, and a second RNA encoding a second SARS-CoV-2 S polypeptide or an immunogenic fragment thereof, wherein (a) the first SARS-CoV-2 S polypeptide or fragment comprises an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO:70, and (b) the second SARS-CoV-2 S polypeptide or fragment comprises an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO:70. The S polypeptide or fragment has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO:70 and comprises an amino acid sequence containing one of the following sets of amino acid substitutions relative to SEQ ID NO:70: (1) D982P, V984P, F814P, A889P, A896P, and A939P; (2) K983P, V984P, F814P, A889P, A939P; 896P, and A939P, (3) D982P, V984P, F814P, A889P, A896P, A939P, R679G, R680S, and R682S, (4) K983P, V984P, F814P, A889P, A896P, A939P, R679G, R680S, and R682S, or (5) D982P, K983P, V984P, F814P, A889P, A896P, A939P, R679G, R680S, and R682S.

Another aspect of the disclosure features a composition or medical preparation that includes a first RNA encoding a first SARS-CoV-2 S polypeptide or an immunogenic fragment thereof, and a second RNA encoding a second SARS-CoV-2 S polypeptide or an immunogenic fragment thereof, wherein (a) the first SARS-CoV-2 S polypeptide or fragment comprises an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO:70, and (b) the second SARS-CoV-2 S polypeptide or fragment comprises an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO:70. The S polypeptide or fragment has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 104 or 105 and comprises an amino acid sequence that includes one of the following sets of amino acid substitutions relative to SEQ ID NO: 104 or 105: (1) D980P, V982P, F812P, A887P, A894P, and A937P; (2) K981P, V982P, F812P, A887P, A894P, and A937P, (3) D980P, V982P, F812P, A887P, A894P, A937P, R677G, R678S, and R680S, (4) K981P, V982P, F812P, A887P, A894P, A937P, R677G, R678S, and R680S, or (5) D980P, K981P, V982P, F812P, A887P, A894P, A937P, R677G, R678S, and R680S.

In one aspect, the disclosure provides a composition or medical preparation comprising a first RNA encoding a first SARS-CoV-2 S polypeptide or an immunogenic fragment thereof, and a second RNA encoding a second SARS-CoV-2 S polypeptide or an immunogenic fragment thereof, wherein (a) the first SARS-CoV-2 S polypeptide or fragment comprises an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 104 or SEQ ID NO: 105, and (b) the second SARS-CoV-2 S polypeptide or fragment comprises an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 104 or SEQ ID NO: 105, and The S polypeptide or fragment has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO:1 and comprises an amino acid sequence that includes one of the following sets of amino acid substitutions relative to SEQ ID NO:1: (1) D985P, V987P, F817P, A 892P, A899P, and A942P, (2) K986P, V987P, F817P, A892P, A899P, and A942P, (3) D985P, V987P, F817P, A892P, A899P, A942P, D614G, R682G, R683S, and R685S, (4) K986P, V987P, F817P, A89 2P, A899P, A942P, D614G, R682G, R683S, and R685S, (5) D985P, K986P, V987P, F817P, A892P, A899P, A942P, D614G, R682G, R683S, and R685S, (6) D985P, V987P, F817P, A892P, A899P, A942 P, R682G, R683S, and R685S, (7) K986P, V987P, F817P, A892P, A899P, A942P, R682G, R683S, and R685S, or (8) D985P, K986P, V987P, F817P, A892P, A899P, A942P, R682G, R683S, and R685S.

Another aspect of the disclosure features a composition or medical preparation that includes a first RNA encoding a first SARS-CoV-2 S polypeptide or an immunogenic fragment thereof, and a second RNA encoding a second SARS-CoV-2 S polypeptide or an immunogenic fragment thereof, wherein (a) the first SARS-CoV-2 S polypeptide or fragment comprises an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO:104 or SEQ ID NO:105, and (b) the second SARS-CoV-2 S polypeptide or fragment comprises an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO:104 or SEQ ID NO:105, and The S polypeptide or fragment has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO:69 and comprises an amino acid sequence that includes one of the following sets of amino acid substitutions relative to SEQ ID NO:69: (1) D982P, V984P, F814P, A889P, A896P, and A939P; (2) K983P, V984P, F814P, A889P, A939P; 896P, and A939P, (3) D982P, V984P, F814P, A889P, A896P, A939P, R679G, R680S, and R682S, (4) K983P, V984P, F814P, A889P, A896P, A939P, R679G, R680S, and R682S, or (5) D982P, K983P, V984P, F814P, A889P, A896P, A939P, R679G, R680S, and R682S.

Another aspect of the disclosure features a composition or medical preparation that includes a first RNA encoding a first SARS-CoV-2 S polypeptide or an immunogenic fragment thereof, and a second RNA encoding a second SARS-CoV-2 S polypeptide or an immunogenic fragment thereof, wherein (a) the first SARS-CoV-2 S polypeptide or fragment comprises an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO:104 or SEQ ID NO:105, and (b) the second SARS-CoV-2 S polypeptide or fragment comprises an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO:104 or SEQ ID NO:105, and The S polypeptide or fragment has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO:70 and comprises an amino acid sequence containing one of the following sets of amino acid substitutions relative to SEQ ID NO:70: (1) D982P, V984P, F814P, A889P, A896P, and A939P; (2) K983P, V984P, F814P, A889P, A939P; 896P, and A939P, (3) D982P, V984P, F814P, A889P, A896P, A939P, R679G, R680S, and R682S, (4) K983P, V984P, F814P, A889P, A896P, A939P, R679G, R680S, and R682S, or (5) D982P, K983P, V984P, F814P, A889P, A896P, A939P, R679G, R680S, and R682S.

Another aspect of the disclosure features a composition or medical preparation that includes a first RNA encoding a first SARS-CoV-2 S polypeptide or an immunogenic fragment thereof, and a second RNA encoding a second SARS-CoV-2 S polypeptide or an immunogenic fragment thereof, wherein (a) the first SARS-CoV-2 S polypeptide or fragment comprises an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO:104 or SEQ ID NO:105, and (b) the second SARS-CoV-2 S polypeptide or fragment comprises an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO:104 or SEQ ID NO:105, and The S polypeptide or fragment has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 104 or 105 and comprises an amino acid sequence that includes one of the following sets of amino acid substitutions relative to SEQ ID NO: 104 or 105: (1) D980P, V982P, F812P, A887P, A894P, and A937P; (2) K981P, V982P, F812P, A887P, A894P, and A937P, (3) D980P, V982P, F812P, A887P, A894P, A937P, R677G, R678S, and R680S, (4) K981P, V982P, F812P, A887P, A894P, A937P, R677G, R678S, and R680S, or (5) D980P, K981P, V982P, F812P, A887P, A894P, A937P, R677G, R678S, and R680S.

Another aspect of the disclosure features a composition or medical preparation that includes a first RNA encoding a first SARS-CoV-2 S polypeptide or an immunogenic fragment thereof, and a second RNA encoding a second SARS-CoV-2 S polypeptide or an immunogenic fragment thereof, wherein (a) the first SARS-CoV-2 S polypeptide or fragment comprises an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO:1 and including the following substitutions relative to SEQ ID NO:1: D985P, V987P, F817P, A892P, A899P, and A942P; and (b) the second SARS-CoV-2 S polypeptide or fragment comprises an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO:1 and including the following substitutions relative to SEQ ID NO:1: D985P, V987P, F817P, A892P, A899P, and A942P. The S polypeptide or fragment has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO:69 and comprises an amino acid sequence that includes one of the following sets of amino acid substitutions relative to SEQ ID NO:69: (1) D982P, V984P, F814P, A889P, A896P, and A939P; (2) K983P, V984P, F814P, A889P, A939P; 896P, and A939P, (3) D982P, V984P, F814P, A889P, A896P, A939P, R679G, R680S, and R682S, (4) K983P, V984P, F814P, A889P, A896P, A939P, R679G, R680S, and R682S, or (5) D982P, K983P, V984P, F814P, A889P, A896P, A939P, R679G, R680S, and R682S.

Another aspect of the disclosure features a composition or medical preparation that includes a first RNA encoding a first SARS-CoV-2 S polypeptide or an immunogenic fragment thereof, and a second RNA encoding a second SARS-CoV-2 S polypeptide or an immunogenic fragment thereof, wherein (a) the first SARS-CoV-2 S polypeptide or fragment comprises an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO:1 and including the following substitutions relative to SEQ ID NO:1: K986P, V987P, F817P, A892P, A899P, and A942P; (b) the second SARS-CoV-2 S polypeptide or fragment comprises an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO:1 and including the following substitutions relative to SEQ ID NO:1: K986P, V987P, F817P, A892P, A899P, and A942P; The S polypeptide or fragment has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO:69 and comprises an amino acid sequence that includes one of the following sets of amino acid substitutions relative to SEQ ID NO:69: (1) D982P, V984P, F814P, A889P, A896P, and A939P; (2) K983P, V984P, F814P, A889P, A939P; 896P, and A939P, (3) D982P, V984P, F814P, A889P, A896P, A939P, R679G, R680S, and R682S, (4) K983P, V984P, F814P, A889P, A896P, A939P, R679G, R680S, and R682S, or (5) D982P, K983P, V984P, F814P, A889P, A896P, A939P, R679G, R680S, and R682S.

Another aspect of the disclosure features a composition or medical preparation that includes a first RNA encoding a first SARS-CoV-2 S polypeptide or an immunogenic fragment thereof, and a second RNA encoding a second SARS-CoV-2 S polypeptide or an immunogenic fragment thereof, wherein (a) the first SARS-CoV-2 S polypeptide or fragment comprises an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO:1 and including the following substitutions relative to SEQ ID NO:1: D985P, V987P, F817P, A892P, A899P, A942P, D614G, R682G, R683S, and R685S; (b) the second SARS-CoV-2 S polypeptide or fragment comprises an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO:1 and including the following substitutions relative to SEQ ID NO:1: D985P, V987P, F817P, A892P, A899P, A942P, D614G, R682G, R683S, and R685S; The S polypeptide or fragment has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO:69 and comprises an amino acid sequence that includes one of the following sets of amino acid substitutions relative to SEQ ID NO:69: (1) D982P, V984P, F814P, A889P, A896P, and A939P; (2) K983P, V984P, F814P, A889P, A939P; 896P, and A939P, (3) D982P, V984P, F814P, A889P, A896P, A939P, R679G, R680S, and R682S, (4) K983P, V984P, F814P, A889P, A896P, A939P, R679G, R680S, and R682S, or (5) D982P, K983P, V984P, F814P, A889P, A896P, A939P, R679G, R680S, and R682S.

Another aspect of the disclosure features a composition or medical preparation that includes a first RNA encoding a first SARS-CoV-2 S polypeptide or an immunogenic fragment thereof, and a second RNA encoding a second SARS-CoV-2 S polypeptide or an immunogenic fragment thereof, wherein (a) the first SARS-CoV-2 S polypeptide or fragment comprises an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO:1 and including the following substitutions relative to SEQ ID NO:1: K986P, V987P, F817P, A892P, A899P, A942P, D614G, R682G, R683S, and R685S; (b) the second SARS-CoV-2 S polypeptide or fragment comprises an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO:1 and including the following substitutions relative to SEQ ID NO:1: K986P, V987P, F817P, A892P, A899P, A942P, D614G, R682G, R683S, and R685S; The S polypeptide or fragment has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO:69 and comprises an amino acid sequence that includes one of the following sets of amino acid substitutions relative to SEQ ID NO:69: (1) D982P, V984P, F814P, A889P, A896P, and A939P; (2) K983P, V984P, F814P, A889P, A939P; 896P, and A939P, (3) D982P, V984P, F814P, A889P, A896P, A939P, R679G, R680S, and R682S, (4) K983P, V984P, F814P, A889P, A896P, A939P, R679G, R680S, and R682S, or (5) D982P, K983P, V984P, F814P, A889P, A896P, A939P, R679G, R680S, and R682S.

Another aspect of the disclosure features a composition or medical preparation that includes a first RNA encoding a first SARS-CoV-2 S polypeptide or an immunogenic fragment thereof, and a second RNA encoding a second SARS-CoV-2 S polypeptide or an immunogenic fragment thereof, the composition or medical preparation including: (a) a first RNA encoding a first SARS-CoV-2 S polypeptide or an immunogenic fragment thereof; The S polypeptide or fragment comprises an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO:1 and including the following substitutions relative to SEQ ID NO:1: D985P, K986P, V987P, F817P, A892P, A899P, A942P, D614G, R682G, R683S, and R685S; (b) a second SARS-CoV-2 The S polypeptide or fragment has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO:69 and comprises an amino acid sequence that includes one of the following sets of amino acid substitutions relative to SEQ ID NO:69: (1) D982P, V984P, F814P, A889P, A896P, and A939P; (2) K983P, V984P, F814P, A889P, A939P; 896P, and A939P, (3) D982P, V984P, F814P, A889P, A896P, A939P, R679G, R680S, and R682S, (4) K983P, V984P, F814P, A889P, A896P, A939P, R679G, R680S, and R682S, or (5) D982P, K983P, V984P, F814P, A889P, A896P, A939P, R679G, R680S, and R682S.

Another aspect of the disclosure features a composition or medical preparation that includes a first RNA encoding a first SARS-CoV-2 S polypeptide or an immunogenic fragment thereof, and a second RNA encoding a second SARS-CoV-2 S polypeptide or an immunogenic fragment thereof, wherein (a) the first SARS-CoV-2 S polypeptide or fragment comprises an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO:1 and including the following substitutions relative to SEQ ID NO:1: D985P, V987P, F817P, A892P, A899P, A942P, R682G, R683S, and R685S; (b) the second SARS-CoV-2 S polypeptide or fragment comprises an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO:1 and including the following substitutions relative to SEQ ID NO:1: D985P, V987P, F817P, A892P, A899P, A942P, R682G, R683S, and R685S; The S polypeptide or fragment has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO:69 and comprises an amino acid sequence that includes one of the following sets of amino acid substitutions relative to SEQ ID NO:69: (1) D982P, V984P, F814P, A889P, A896P, and A939P; (2) K983P, V984P, F814P, A889P, A939P; 896P, and A939P, (3) D982P, V984P, F814P, A889P, A896P, A939P, R679G, R680S, and R682S, (4) K983P, V984P, F814P, A889P, A896P, A939P, R679G, R680S, and R682S, or (5) D982P, K983P, V984P, F814P, A889P, A896P, A939P, R679G, R680S, and R682S.

Another aspect of the disclosure features a composition or medical preparation that includes a first RNA encoding a first SARS-CoV-2 S polypeptide or an immunogenic fragment thereof, and a second RNA encoding a second SARS-CoV-2 S polypeptide or an immunogenic fragment thereof, wherein (a) the first SARS-CoV-2 S polypeptide or fragment comprises an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO:1 and including the following substitutions relative to SEQ ID NO:1: K986P, V987P, F817P, A892P, A899P, A942P, R682G, R683S, and R685S; (b) the second SARS-CoV-2 S polypeptide or fragment comprises an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO:1 and including the following substitutions relative to SEQ ID NO:1: K986P, V987P, F817P, A892P, A899P, A942P, R682G, R683S, and R685S; The S polypeptide or fragment has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO:69 and comprises an amino acid sequence that includes one of the following sets of amino acid substitutions relative to SEQ ID NO:69: (1) D982P, V984P, F814P, A889P, A896P, and A939P; (2) K983P, V984P, F814P, A889P, A939P; 896P, and A939P, (3) D982P, V984P, F814P, A889P, A896P, A939P, R679G, R680S, and R682S, (4) K983P, V984P, F814P, A889P, A896P, A939P, R679G, R680S, and R682S, or (5) D982P, K983P, V984P, F814P, A889P, A896P, A939P, R679G, R680S, and R682S.

Another aspect of the disclosure features a composition or medical preparation that includes a first RNA encoding a first SARS-CoV-2 S polypeptide or an immunogenic fragment thereof, and a second RNA encoding a second SARS-CoV-2 S polypeptide or an immunogenic fragment thereof, wherein (a) the first SARS-CoV-2 S polypeptide or fragment comprises an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO:1 and including the following substitutions relative to SEQ ID NO:1: D985P, K986P, V987P, F817P, A892P, A899P, A942P, R682G, R683S, R685S, (b) the second SARS-CoV-2 The S polypeptide or fragment has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO:69 and comprises an amino acid sequence that includes one of the following sets of amino acid substitutions relative to SEQ ID NO:69: (1) D982P, V984P, F814P, A889P, A896P, and A939P; (2) K983P, V984P, F814P, A889P, A939P; 896P, and A939P, (3) D982P, V984P, F814P, A889P, A896P, A939P, R679G, R680S, and R682S, (4) K983P, V984P, F814P, A889P, A896P, A939P, R679G, R680S, and R682S, or (5) D982P, K983P, V984P, F814P, A889P, A896P, A939P, R679G, R680S, and R682S.

Another aspect of the disclosure features a composition or medical preparation that includes a first RNA encoding a first SARS-CoV-2 S polypeptide or an immunogenic fragment thereof, and a second RNA encoding a second SARS-CoV-2 S polypeptide or an immunogenic fragment thereof, wherein (a) the first SARS-CoV-2 S polypeptide or fragment comprises an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO:1 and including the following substitutions relative to SEQ ID NO:1: D985P, V987P, F817P, A892P, A899P, and A942P; and (b) the second SARS-CoV-2 S polypeptide or fragment comprises an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO:1 and including the following substitutions relative to SEQ ID NO:1: D985P, V987P, F817P, A892P, A899P, and A942P. The S polypeptide or fragment has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO:70 and comprises an amino acid sequence that includes one of the following sets of amino acid substitutions relative to SEQ ID NO:70: (1) D982P, V984P, F814P, A889P, A896P, and A939P; (2) K983P, V984P, F814P, A889P, A939P; 896P, and A939P, (3) D982P, V984P, F814P, A889P, A896P, A939P, R679G, R680S, and R682S, (4) K983P, V984P, F814P, A889P, A896P, A939P, R679G, R680S, and R682S, or (5) D982P, K983P, V984P, F814P, A889P, A896P, A939P, R679G, R680S, and R682S.

Another aspect of the disclosure features a composition or medical preparation that includes a first RNA encoding a first SARS-CoV-2 S polypeptide or an immunogenic fragment thereof, and a second RNA encoding a second SARS-CoV-2 S polypeptide or an immunogenic fragment thereof, wherein (a) the first SARS-CoV-2 S polypeptide or fragment comprises an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO:1 and including the following substitutions relative to SEQ ID NO:1: K986P, V987P, F817P, A892P, A899P, and A942P; (b) the second SARS-CoV-2 S polypeptide or fragment comprises an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO:1 and including the following substitutions relative to SEQ ID NO:1: K986P, V987P, F817P, A892P, A899P, and A942P; The S polypeptide or fragment has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO:70 and comprises an amino acid sequence that includes one of the following sets of amino acid substitutions relative to SEQ ID NO:70: (1) D982P, V984P, F814P, A889P, A896P, and A939P; (2) K983P, V984P, F814P, A889P, A939P; 896P, and A939P, (3) D982P, V984P, F814P, A889P, A896P, A939P, R679G, R680S, and R682S, (4) K983P, V984P, F814P, A889P, A896P, A939P, R679G, R680S, and R682S, or (5) D982P, K983P, V984P, F814P, A889P, A896P, A939P, R679G, R680S, and R682S.

Another aspect of the disclosure features a composition or medical preparation that includes a first RNA encoding a first SARS-CoV-2 S polypeptide or an immunogenic fragment thereof, and a second RNA encoding a second SARS-CoV-2 S polypeptide or an immunogenic fragment thereof, wherein (a) the first SARS-CoV-2 S polypeptide or fragment comprises an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO:1 and including the following substitutions relative to SEQ ID NO:1: D985P, V987P, F817P, A892P, A899P, A942P, D614G, R682G, R683S, and R685S; (b) the second SARS-CoV-2 S polypeptide or fragment comprises an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO:1 and including the following substitutions relative to SEQ ID NO:1: D985P, V987P, F817P, A892P, A899P, A942P, D614G, R682G, R683S, and R685S; The S polypeptide or fragment has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO:70 and comprises an amino acid sequence that includes one of the following sets of amino acid substitutions relative to SEQ ID NO:70: (1) D982P, V984P, F814P, A889P, A896P, and A939P; (2) K983P, V984P, F814P, A889P, A939P; 896P, and A939P, (3) D982P, V984P, F814P, A889P, A896P, A939P, R679G, R680S, and R682S, (4) K983P, V984P, F814P, A889P, A896P, A939P, R679G, R680S, and R682S, or (5) D982P, K983P, V984P, F814P, A889P, A896P, A939P, R679G, R680S, and R682S.

Another aspect of the disclosure features a composition or medical preparation that includes a first RNA encoding a first SARS-CoV-2 S polypeptide or an immunogenic fragment thereof, and a second RNA encoding a second SARS-CoV-2 S polypeptide or an immunogenic fragment thereof, wherein (a) the first SARS-CoV-2 S polypeptide or fragment comprises an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO:1 and including the following substitutions relative to SEQ ID NO:1: K986P, V987P, F817P, A892P, A899P, A942P, D614G, R682G, R683S, and R685S; (b) the second SARS-CoV-2 S polypeptide or fragment comprises an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO:1 and including the following substitutions relative to SEQ ID NO:1: K986P, V987P, F817P, A892P, A899P, A942P, D614G, R682G, R683S, and R685S; The S polypeptide or fragment has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO:70 and comprises an amino acid sequence that includes one of the following sets of amino acid substitutions relative to SEQ ID NO:70: (1) D982P, V984P, F814P, A889P, A896P, and A939P; (2) K983P, V984P, F814P, A889P, A939P; 896P, and A939P, (3) D982P, V984P, F814P, A889P, A896P, A939P, R679G, R680S, and R682S, (4) K983P, V984P, F814P, A889P, A896P, A939P, R679G, R680S, and R682S, or (5) D982P, K983P, V984P, F814P, A889P, A896P, A939P, R679G, R680S, and R682S.

Another aspect of the disclosure features a composition or medical preparation that includes a first RNA encoding a first SARS-CoV-2 S polypeptide or an immunogenic fragment thereof, and a second RNA encoding a second SARS-CoV-2 S polypeptide or an immunogenic fragment thereof, the composition or medical preparation including: (a) a first RNA encoding a first SARS-CoV-2 S polypeptide or an immunogenic fragment thereof; The S polypeptide or fragment comprises an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO:1 and including the following substitutions relative to SEQ ID NO:1: D985P, K986P, V987P, F817P, A892P, A899P, A942P, D614G, R682G, R683S, and R685S; (b) a second SARS-CoV-2 The S polypeptide or fragment has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO:70 and comprises an amino acid sequence that includes one of the following sets of amino acid substitutions relative to SEQ ID NO:70: (1) D982P, V984P, F814P, A889P, A896P, and A939P; (2) K983P, V984P, F814P, A889P, A939P; 896P, and A939P, (3) D982P, V984P, F814P, A889P, A896P, A939P, R679G, R680S, and R682S, (4) K983P, V984P, F814P, A889P, A896P, A939P, R679G, R680S, and R682S, or (5) D982P, K983P, V984P, F814P, A889P, A896P, A939P, R679G, R680S, and R682S.

Another aspect of the disclosure features a composition or medical preparation that includes a first RNA encoding a first SARS-CoV-2 S polypeptide or an immunogenic fragment thereof, and a second RNA encoding a second SARS-CoV-2 S polypeptide or an immunogenic fragment thereof, wherein (a) the first SARS-CoV-2 S polypeptide or fragment comprises an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO:1 and including the following substitutions relative to SEQ ID NO:1: D985P, V987P, F817P, A892P, A899P, A942P, R682G, R683S, and R685S; (b) the second SARS-CoV-2 S polypeptide or fragment comprises an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO:1 and including the following substitutions relative to SEQ ID NO:1: D985P, V987P, F817P, A892P, A899P, A942P, R682G, R683S, and R685S; The S polypeptide or fragment has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO:70 and comprises an amino acid sequence that includes one of the following sets of amino acid substitutions relative to SEQ ID NO:70: (1) D982P, V984P, F814P, A889P, A896P, and A939P; (2) K983P, V984P, F814P, A889P, A939P; 896P, and A939P, (3) D982P, V984P, F814P, A889P, A896P, A939P, R679G, R680S, and R682S, (4) K983P, V984P, F814P, A889P, A896P, A939P, R679G, R680S, and R682S, or (5) D982P, K983P, V984P, F814P, A889P, A896P, A939P, R679G, R680S, and R682S.

Another aspect of the disclosure features a composition or medical preparation that includes a first RNA encoding a first SARS-CoV-2 S polypeptide or an immunogenic fragment thereof, and a second RNA encoding a second SARS-CoV-2 S polypeptide or an immunogenic fragment thereof, wherein (a) the first SARS-CoV-2 S polypeptide or fragment comprises an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO:1 and including the following substitutions relative to SEQ ID NO:1: K986P, V987P, F817P, A892P, A899P, A942P, R682G, R683S, and R685S; (b) the second SARS-CoV-2 S polypeptide or fragment comprises an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO:1 and including the following substitutions relative to SEQ ID NO:1: K986P, V987P, F817P, A892P, A899P, A942P, R682G, R683S, and R685S; The S polypeptide or fragment has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO:70 and comprises an amino acid sequence that includes one of the following sets of amino acid substitutions relative to SEQ ID NO:70: (1) D982P, V984P, F814P, A889P, A896P, and A939P; (2) K983P, V984P, F814P, A889P, A939P; 896P, and A939P, (3) D982P, V984P, F814P, A889P, A896P, A939P, R679G, R680S, and R682S, (4) K983P, V984P, F814P, A889P, A896P, A939P, R679G, R680S, and R682S, or (5) D982P, K983P, V984P, F814P, A889P, A896P, A939P, R679G, R680S, and R682S.

Another aspect of the disclosure features a composition or medical preparation that includes a first RNA encoding a first SARS-CoV-2 S polypeptide or an immunogenic fragment thereof, and a second RNA encoding a second SARS-CoV-2 S polypeptide or an immunogenic fragment thereof, wherein (a) the first SARS-CoV-2 S polypeptide or fragment comprises an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO:1 and including the following substitutions relative to SEQ ID NO:1: D985P, K986P, V987P, F817P, A892P, A899P, A942P, R682G, R683S, and R685S; (b) the second SARS-CoV-2 S polypeptide or fragment comprises an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO:1 and including the following substitutions relative to SEQ ID NO:1: D985P, K986P, V987P, F817P, A892P, A899P, A942P, R682G, R683S, and R685S; The S polypeptide or fragment has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO:70 and comprises an amino acid sequence that includes one of the following sets of amino acid substitutions relative to SEQ ID NO:70: (1) D982P, V984P, F814P, A889P, A896P, and A939P; (2) K983P, V984P, F814P, A889P, A939P; 896P, and A939P, (3) D982P, V984P, F814P, A889P, A896P, A939P, R679G, R680S, and R682S, (4) K983P, V984P, F814P, A889P, A896P, A939P, R679G, R680S, and R682S, or (5) D982P, K983P, V984P, F814P, A889P, A896P, A939P, R679G, R680S, and R682S.

Another aspect of the disclosure features a composition or medical preparation that includes a first RNA encoding a first SARS-CoV-2 S polypeptide or an immunogenic fragment thereof, and a second RNA encoding a second SARS-CoV-2 S polypeptide or an immunogenic fragment thereof, wherein (a) the first SARS-CoV-2 S polypeptide or fragment comprises an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO:1 and including the following substitutions relative to SEQ ID NO:1: D985P, V987P, F817P, A892P, A899P, and A942P; (b) the second SARS-CoV-2 S polypeptide or fragment comprises an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO:1 and including the following substitutions relative to SEQ ID NO:1: D985P, V987P, F817P, A892P, A899P, and A942P; The S polypeptide or fragment has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 104 or 105 and comprises an amino acid sequence that includes one of the following sets of amino acid substitutions relative to SEQ ID NO: 104 or 105: (1) D980P, V982P, F812P, A887P, A894P, and A937P; (2) K981P, V982P, F812P; A887P, A894P, and A937P, (3) D980P, V982P, F812P, A887P, A894P, A937P, R677G, R678S, and R680S, (4) K981P, V982P, F812P, A887P, A894P, A937P, R677G, R678S, and R680S, or (5) D980P, K981P, V982P, F812P, A887P, A894P, A937P, R677G, R678S, and R680S.

Another aspect of the disclosure features a composition or medical preparation that includes a first RNA encoding a first SARS-CoV-2 S polypeptide or an immunogenic fragment thereof, and a second RNA encoding a second SARS-CoV-2 S polypeptide or an immunogenic fragment thereof, wherein (a) the first SARS-CoV-2 S polypeptide or fragment comprises an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO:1 and including the following substitutions relative to SEQ ID NO:1: K986P, V987P, F817P, A892P, A899P, and A942P; (b) the second SARS-CoV-2 S polypeptide or fragment comprises an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO:1 and including the following substitutions relative to SEQ ID NO:1: K986P, V987P, F817P, A892P, A899P, and A942P; The S polypeptide or fragment has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 104 or 105 and comprises an amino acid sequence that includes one of the following sets of amino acid substitutions relative to SEQ ID NO: 104 or 105: (1) D980P, V982P, F812P, A887P, A894P, and A937P; (2) K981P, V982P, F812P; A887P, A894P, and A937P, (3) D980P, V982P, F812P, A887P, A894P, A937P, R677G, R678S, and R680S, (4) K981P, V982P, F812P, A887P, A894P, A937P, R677G, R678S, and R680S, or (5) D980P, K981P, V982P, F812P, A887P, A894P, A937P, R677G, R678S, and R680S.

Another aspect of the disclosure features a composition or medical preparation that includes a first RNA encoding a first SARS-CoV-2 S polypeptide or an immunogenic fragment thereof, and a second RNA encoding a second SARS-CoV-2 S polypeptide or an immunogenic fragment thereof, wherein (a) the first SARS-CoV-2 S polypeptide or fragment comprises an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO:1 and including the following substitutions relative to SEQ ID NO:1: D985P, V987P, F817P, A892P, A899P, A942P, D614G, R682G, R683S, and R685S; (b) the second SARS-CoV-2 S polypeptide or fragment comprises an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO:1 and including the following substitutions relative to SEQ ID NO:1: D985P, V987P, F817P, A892P, A899P, A942P, D614G, R682G, R683S, and R685S; The S polypeptide or fragment has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 104 or 105 and comprises an amino acid sequence that includes one of the following sets of amino acid substitutions relative to SEQ ID NO: 104 or 105: (1) D980P, V982P, F812P, A887P, A894P, and A937P; (2) K981P, V982P, F812P; A887P, A894P, and A937P, (3) D980P, V982P, F812P, A887P, A894P, A937P, R677G, R678S, and R680S, (4) K981P, V982P, F812P, A887P, A894P, A937P, R677G, R678S, and R680S, or (5) D980P, K981P, V982P, F812P, A887P, A894P, A937P, R677G, R678S, and R680S.

Another aspect of the disclosure features a composition or medical preparation that includes a first RNA encoding a first SARS-CoV-2 S polypeptide or an immunogenic fragment thereof, and a second RNA encoding a second SARS-CoV-2 S polypeptide or an immunogenic fragment thereof, wherein (a) the first SARS-CoV-2 S polypeptide or fragment comprises an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO:1 and including the following substitutions relative to SEQ ID NO:1: K986P, V987P, F817P, A892P, A899P, A942P, D614G, R682G, R683S, and R685S; (b) the second SARS-CoV-2 S polypeptide or fragment comprises an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO:1 and including the following substitutions relative to SEQ ID NO:1: K986P, V987P, F817P, A892P, A899P, A942P, D614G, R682G, R683S, and R685S; The S polypeptide or fragment has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 104 or 105 and comprises an amino acid sequence that includes one of the following sets of amino acid substitutions relative to SEQ ID NO: 104 or 105: (1) D980P, V982P, F812P, A887P, A894P, and A937P; (2) K981P, V982P, F812P; A887P, A894P, and A937P, (3) D980P, V982P, F812P, A887P, A894P, A937P, R677G, R678S, and R680S, (4) K981P, V982P, F812P, A887P, A894P, A937P, R677G, R678S, and R680S, or (5) D980P, K981P, V982P, F812P, A887P, A894P, A937P, R677G, R678S, and R680S.

Another aspect of the disclosure features a composition or medical preparation that includes a first RNA encoding a first SARS-CoV-2 S polypeptide or an immunogenic fragment thereof, and a second RNA encoding a second SARS-CoV-2 S polypeptide or an immunogenic fragment thereof, the composition or medical preparation including: (a) a first RNA encoding a first SARS-CoV-2 S polypeptide or an immunogenic fragment thereof; (b) a second SARS-CoV-2 polypeptide or fragment comprising an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO:1 and including the following substitutions relative to SEQ ID NO:1: D985P, K986P, V987P, F817P, A892P, A899P, A942P, D614G, R682G, R683S, and R685S; The S polypeptide or fragment has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 104 or 105 and comprises an amino acid sequence that includes one of the following sets of amino acid substitutions relative to SEQ ID NO: 104 or 105: (1) D980P, V982P, F812P, A887P, A894P, and A937P; (2) K981P, V982P, F812P; A887P, A894P, and A937P, (3) D980P, V982P, F812P, A887P, A894P, A937P, R677G, R678S, and R680S, (4) K981P, V982P, F812P, A887P, A894P, A937P, R677G, R678S, and R680S, or (5) D980P, K981P, V982P, F812P, A887P, A894P, A937P, R677G, R678S, and R680S.

Another aspect of the disclosure features a composition or medical preparation that includes a first RNA encoding a first SARS-CoV-2 S polypeptide or an immunogenic fragment thereof, and a second RNA encoding a second SARS-CoV-2 S polypeptide or an immunogenic fragment thereof, wherein (a) the first SARS-CoV-2 S polypeptide or fragment comprises an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO:1 and including the following substitutions relative to SEQ ID NO:1: D985P, V987P, F817P, A892P, A899P, A942P, R682G, R683S, and R685S; (b) the second SARS-CoV-2 S polypeptide or fragment comprises an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO:1 and including the following substitutions relative to SEQ ID NO:1: D985P, V987P, F817P, A892P, A899P, A942P, R682G, R683S, and R685S; The S polypeptide or fragment has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 104 or 105 and comprises an amino acid sequence that includes one of the following sets of amino acid substitutions relative to SEQ ID NO: 104 or 105: (1) D980P, V982P, F812P, A887P, A894P, and A937P; (2) K981P, V982P, F812P, A887P, A894P, and A937P, (3) D980P, V982P, F812P, A887P, A894P, A937P, R677G, R678S, and R680S, (4) K981P, V982P, F812P, A887P, A894P, A937P, R677G, R678S, and R680S, or (5) D980P, K981P, V982P, F812P, A887P, A894P, A937P, R677G, R678S, and R680S.

Another aspect of the disclosure features a composition or medical preparation that includes a first RNA encoding a first SARS-CoV-2 S polypeptide or an immunogenic fragment thereof, and a second RNA encoding a second SARS-CoV-2 S polypeptide or an immunogenic fragment thereof, wherein (a) the first SARS-CoV-2 S polypeptide or fragment comprises an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO:1 and including the following substitutions relative to SEQ ID NO:1: K986P, V987P, F817P, A892P, A899P, A942P, R682G, R683S, and R685S; (b) the second SARS-CoV-2 S polypeptide or fragment comprises an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO:1 and including the following substitutions relative to SEQ ID NO:1: K986P, V987P, F817P, A892P, A899P, A942P, R682G, R683S, and R685S; The S polypeptide or fragment has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 104 or 105 and comprises an amino acid sequence that includes one of the following sets of amino acid substitutions relative to SEQ ID NO: 104 or 105: (1) D980P, V982P, F812P, A887P, A894P, and A937P; (2) K981P, V982P, F812P; A887P, A894P, and A937P, (3) D980P, V982P, F812P, A887P, A894P, A937P, R677G, R678S, and R680S, (4) K981P, V982P, F812P, A887P, A894P, A937P, R677G, R678S, and R680S, or (5) D980P, K981P, V982P, F812P, A887P, A894P, A937P, R677G, R678S, and R680S.

Another aspect of the disclosure features a composition or medical preparation that includes a first RNA encoding a first SARS-CoV-2 S polypeptide or an immunogenic fragment thereof, and a second RNA encoding a second SARS-CoV-2 S polypeptide or an immunogenic fragment thereof, wherein (a) the first SARS-CoV-2 S polypeptide or fragment comprises an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO:1 and including the following substitutions relative to SEQ ID NO:1: D985P, K986P, V987P, F817P, A892P, A899P, A942P, R682G, R683S, and R685S; (b) the second SARS-CoV-2 S polypeptide or fragment comprises an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO:1 and including the following substitutions relative to SEQ ID NO:1: D985P, K986P, V987P, F817P, A892P, A899P, A942P, R682G, R683S, and R685S; The S polypeptide or fragment has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 104 or 105 and comprises an amino acid sequence that includes one of the following sets of amino acid substitutions relative to SEQ ID NO: 104 or 105: (1) D980P, V982P, F812P, A887P, A894P, and A937P; (2) K981P, V982P, F812P; A887P, A894P, and A937P, (3) D980P, V982P, F812P, A887P, A894P, A937P, R677G, R678S, and R680S, (4) K981P, V982P, F812P, A887P, A894P, A937P, R677G, R678S, and R680S, or (5) D980P, K981P, V982P, F812P, A887P, A894P, A937P, R677G, R678S, and R680S.

Another aspect of the disclosure features a composition or medical preparation that includes a first RNA encoding a first SARS-CoV-2 S polypeptide or an immunogenic fragment thereof, and a second RNA encoding a second SARS-CoV-2 S polypeptide or an immunogenic fragment thereof, wherein (a) the first SARS-CoV-2 S polypeptide or fragment comprises an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO:69 and including the following substitutions relative to SEQ ID NO:69: D982P, V984P, F814P, A889P, A896P, and A939P; (b) the second SARS-CoV-2 S polypeptide or fragment comprises an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO:69 and including the following substitutions relative to SEQ ID NO:69: D982P, V984P, F814P, A889P, A896P, and A939P; The S polypeptide or fragment has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO:70 and comprises an amino acid sequence that includes one of the following sets of amino acid substitutions relative to SEQ ID NO:70: (1) D982P, V984P, F814P, A889P, A896P, and A939P; (2) K983P, V984P, F814P, A889P, A939P; 896P, and A939P, (3) D982P, V984P, F814P, A889P, A896P, A939P, R679G, R680S, and R682S, (4) K983P, V984P, F814P, A889P, A896P, A939P, R679G, R680S, and R682S, or (5) D982P, K983P, V984P, F814P, A889P, A896P, A939P, R679G, R680S, and R682S.

Another aspect of the disclosure features a composition or medical preparation that includes a first RNA encoding a first SARS-CoV-2 S polypeptide or an immunogenic fragment thereof, and a second RNA encoding a second SARS-CoV-2 S polypeptide or an immunogenic fragment thereof, wherein (a) the first SARS-CoV-2 S polypeptide or fragment comprises an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO:69 and including the following substitutions relative to SEQ ID NO:69: K983P, V984P, F814P, A889P, A896P, and A939P; and (b) the second SARS-CoV-2 S polypeptide or fragment comprises an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO:69 and including the following substitutions relative to SEQ ID NO:69: K983P, V984P, F814P, A889P, A896P, and A939P. The S polypeptide or fragment has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO:70 and comprises an amino acid sequence that includes one of the following sets of amino acid substitutions relative to SEQ ID NO:70: (1) D982P, V984P, F814P, A889P, A896P, and A939P; (2) K983P, V984P, F814P, A889P, A939P; 896P, and A939P, (3) D982P, V984P, F814P, A889P, A896P, A939P, R679G, R680S, and R682S, (4) K983P, V984P, F814P, A889P, A896P, A939P, R679G, R680S, and R682S, or (5) D982P, K983P, V984P, F814P, A889P, A896P, A939P, R679G, R680S, and R682S.

Another aspect of the disclosure features a composition or medical preparation that includes a first RNA encoding a first SARS-CoV-2 S polypeptide or an immunogenic fragment thereof, and a second RNA encoding a second SARS-CoV-2 S polypeptide or an immunogenic fragment thereof, wherein (a) the first SARS-CoV-2 S polypeptide or fragment comprises an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO:69 and including the following substitutions relative to SEQ ID NO:69: D982P, V984P, F814P, A889P, A896P, A939P, R679G, R680S, and R682S; (b) the second SARS-CoV-2 S polypeptide or fragment comprises an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO:69 and including the following substitutions relative to SEQ ID NO:69: D982P, V984P, F814P, A889P, A896P, A939P, R679G, R680S, and R682S; The S polypeptide or fragment has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO:70 and comprises an amino acid sequence that includes one of the following sets of amino acid substitutions relative to SEQ ID NO:70: (1) D982P, V984P, F814P, A889P, A896P, and A939P; (2) K983P, V984P, F814P, A889P, A939P; 896P, and A939P, (3) D982P, V984P, F814P, A889P, A896P, A939P, R679G, R680S, and R682S, (4) K983P, V984P, F814P, A889P, A896P, A939P, R679G, R680S, and R682S, or (5) D982P, K983P, V984P, F814P, A889P, A896P, A939P, R679G, R680S, and R682S.

Another aspect of the disclosure features a composition or medical preparation that includes a first RNA encoding a first SARS-CoV-2 S polypeptide or an immunogenic fragment thereof, and a second RNA encoding a second SARS-CoV-2 S polypeptide or an immunogenic fragment thereof, wherein (a) the first SARS-CoV-2 S polypeptide or fragment comprises an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO:69 and including the following substitutions relative to SEQ ID NO:69: K983P, V984P, F814P, A889P, A896P, A939P, R679G, R680S, and R682S; (b) the second SARS-CoV-2 S polypeptide or fragment comprises an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO:69 and including the following substitutions relative to SEQ ID NO:69: K983P, V984P, F814P, A889P, A896P, A939P, R679G, R680S, and R682S; The S polypeptide or fragment has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO:70 and comprises an amino acid sequence that includes one of the following sets of amino acid substitutions relative to SEQ ID NO:70: (1) D982P, V984P, F814P, A889P, A896P, and A939P; (2) K983P, V984P, F814P, A889P, A939P; 896P, and A939P, (3) D982P, V984P, F814P, A889P, A896P, A939P, R679G, R680S, and R682S, (4) K983P, V984P, F814P, A889P, A896P, A939P, R679G, R680S, and R682S, or (5) D982P, K983P, V984P, F814P, A889P, A896P, A939P, R679G, R680S, and R682S.

Another aspect of the disclosure features a composition or medical preparation that includes a first RNA encoding a first SARS-CoV-2 S polypeptide or an immunogenic fragment thereof, and a second RNA encoding a second SARS-CoV-2 S polypeptide or an immunogenic fragment thereof, wherein (a) the first SARS-CoV-2 S polypeptide or fragment comprises an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO:69 and including the following substitutions relative to SEQ ID NO:69: D982P, K983P, V984P, F814P, A889P, A896P, A939P, R679G, R680S, and R682S; (b) the second SARS-CoV-2 S polypeptide or fragment comprises an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO:69 and including the following substitutions relative to SEQ ID NO:69: D982P, K983P, V984P, F814P, A889P, A896P, A939P, R679G, R680S, and R682S; The S polypeptide or fragment has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO:70 and comprises an amino acid sequence that includes one of the following sets of amino acid substitutions relative to SEQ ID NO:70: (1) D982P, V984P, F814P, A889P, A896P, and A939P; (2) K983P, V984P, F814P, A889P, A939P; 896P, and A939P, (3) D982P, V984P, F814P, A889P, A896P, A939P, R679G, R680S, and R682S, (4) K983P, V984P, F814P, A889P, A896P, A939P, R679G, R680S, and R682S, or (5) D982P, K983P, V984P, F814P, A889P, A896P, A939P, R679G, R680S, and R682S.

Another aspect of the disclosure features a composition or medical preparation that includes a first RNA encoding a first SARS-CoV-2 S polypeptide or an immunogenic fragment thereof, and a second RNA encoding a second SARS-CoV-2 S polypeptide or an immunogenic fragment thereof, wherein (a) the first SARS-CoV-2 S polypeptide or fragment comprises an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO:69 and including the following substitutions relative to SEQ ID NO:69: D982P, V984P, F814P, A889P, A896P, and A939P; (b) the second SARS-CoV-2 S polypeptide or fragment comprises an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO:69 and including the following substitutions relative to SEQ ID NO:69: D982P, V984P, F814P, A889P, A896P, and A939P; The S polypeptide or fragment has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 104 or 105 and comprises an amino acid sequence that includes one of the following sets of amino acid substitutions relative to SEQ ID NO: 104 or 105: (1) D980P, V982P, F812P, A887P, A894P, and A937P; (2) K981P, V982P, F812P; A887P, A894P, and A937P, (3) D980P, V982P, F812P, A887P, A894P, A937P, R677G, R678S, and R680S, (4) K981P, V982P, F812P, A887P, A894P, A937P, R677G, R678S, and R680S, or (5) D980P, K981P, V982P, F812P, A887P, A894P, A937P, R677G, R678S, and R680S.

Another aspect of the disclosure features a composition or medical preparation that includes a first RNA encoding a first SARS-CoV-2 S polypeptide or an immunogenic fragment thereof, and a second RNA encoding a second SARS-CoV-2 S polypeptide or an immunogenic fragment thereof, wherein (a) the first SARS-CoV-2 S polypeptide or fragment comprises an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO:69 and including the following substitutions relative to SEQ ID NO:69: K983P, V984P, F814P, A889P, A896P, and A939P; and (b) the second SARS-CoV-2 S polypeptide or fragment comprises an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO:69 and including the following substitutions relative to SEQ ID NO:69: K983P, V984P, F814P, A889P, A896P, and A939P. The S polypeptide or fragment has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 104 or 105 and comprises an amino acid sequence that includes one of the following sets of amino acid substitutions relative to SEQ ID NO: 104 or 105: (1) D980P, V982P, F812P, A887P, A894P, and A937P; (2) K981P, V982P, F812P; A887P, A894P, and A937P, (3) D980P, V982P, F812P, A887P, A894P, A937P, R677G, R678S, and R680S, (4) K981P, V982P, F812P, A887P, A894P, A937P, R677G, R678S, and R680S, or (5) D980P, K981P, V982P, F812P, A887P, A894P, A937P, R677G, R678S, and R680S.

Another aspect of the disclosure features a composition or medical preparation that includes a first RNA encoding a first SARS-CoV-2 S polypeptide or an immunogenic fragment thereof, and a second RNA encoding a second SARS-CoV-2 S polypeptide or an immunogenic fragment thereof, wherein (a) the first SARS-CoV-2 S polypeptide or fragment comprises an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO:69 and including the following substitutions relative to SEQ ID NO:69: D982P, V984P, F814P, A889P, A896P, A939P, R679G, R680S, and R682S; (b) the second SARS-CoV-2 S polypeptide or fragment comprises an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO:69 and including the following substitutions relative to SEQ ID NO:69: D982P, V984P, F814P, A889P, A896P, A939P, R679G, R680S, and R682S; The S polypeptide or fragment has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 104 or 105 and comprises an amino acid sequence that includes one of the following sets of amino acid substitutions relative to SEQ ID NO: 104 or 105: (1) D980P, V982P, F812P, A887P, A894P, and A937P; (2) K981P, V982P, F812P; A887P, A894P, and A937P, (3) D980P, V982P, F812P, A887P, A894P, A937P, R677G, R678S, and R680S, (4) K981P, V982P, F812P, A887P, A894P, A937P, R677G, R678S, and R680S, or (5) D980P, K981P, V982P, F812P, A887P, A894P, A937P, R677G, R678S, and R680S.

Another aspect of the disclosure features a composition or medical preparation that includes a first RNA encoding a first SARS-CoV-2 S polypeptide or an immunogenic fragment thereof, and a second RNA encoding a second SARS-CoV-2 S polypeptide or an immunogenic fragment thereof, wherein (a) the first SARS-CoV-2 S polypeptide or fragment comprises an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO:69 and including the following substitutions relative to SEQ ID NO:69: K983P, V984P, F814P, A889P, A896P, A939P, R679G, R680S, and R682S; (b) the second SARS-CoV-2 S polypeptide or fragment comprises an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO:69 and including the following substitutions relative to SEQ ID NO:69: K983P, V984P, F814P, A889P, A896P, A939P, R679G, R680S, and R682S; The S polypeptide or fragment has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 104 or 105 and comprises an amino acid sequence that includes one of the following sets of amino acid substitutions relative to SEQ ID NO: 104 or 105: (1) D980P, V982P, F812P, A887P, A894P, and A937P; (2) K981P, V982P, F812P; A887P, A894P, and A937P, (3) D980P, V982P, F812P, A887P, A894P, A937P, R677G, R678S, and R680S, (4) K981P, V982P, F812P, A887P, A894P, A937P, R677G, R678S, and R680S, or (5) D980P, K981P, V982P, F812P, A887P, A894P, A937P, R677G, R678S, and R680S.

Another aspect of the disclosure features a composition or medical preparation that includes a first RNA encoding a first SARS-CoV-2 S polypeptide or an immunogenic fragment thereof, and a second RNA encoding a second SARS-CoV-2 S polypeptide or an immunogenic fragment thereof, wherein (a) the first SARS-CoV-2 S polypeptide or fragment comprises an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO:69 and including the following substitutions relative to SEQ ID NO:69: D982P, K983P, V984P, F814P, A889P, A896P, A939P, R679G, R680S, and R682S; (b) the second SARS-CoV-2 S polypeptide or fragment comprises an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO:69 and including the following substitutions relative to SEQ ID NO:69: D982P, K983P, V984P, F814P, A889P, A896P, A939P, R679G, R680S, and R682S; The S polypeptide or fragment has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 104 or 105 and comprises an amino acid sequence that includes one of the following sets of amino acid substitutions relative to SEQ ID NO: 104 or 105: (1) D980P, V982P, F812P, A887P, A894P, and A937P; (2) K981P, V982P, F812P; A887P, A894P, and A937P, (3) D980P, V982P, F812P, A887P, A894P, A937P, R677G, R678S, and R680S, (4) K981P, V982P, F812P, A887P, A894P, A937P, R677G, R678S, and R680S, or (5) D980P, K981P, V982P, F812P, A887P, A894P, A937P, R677G, R678S, and R680S.

Another aspect of the disclosure features a composition or medical preparation that includes a first RNA encoding a first SARS-CoV-2 S polypeptide or an immunogenic fragment thereof, and a second RNA encoding a second SARS-CoV-2 S polypeptide or an immunogenic fragment thereof, wherein (a) the first SARS-CoV-2 S polypeptide or fragment comprises an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO:70 and including the following substitutions relative to SEQ ID NO:70: D982P, V984P, F814P, A889P, A896P, and A939P; (b) the second SARS-CoV-2 S polypeptide or fragment comprises an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO:70 and including the following substitutions relative to SEQ ID NO:70: D982P, V984P, F814P, A889P, A896P, and A939P; The S polypeptide or fragment has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 104 or 105 and comprises an amino acid sequence that includes one of the following sets of amino acid substitutions relative to SEQ ID NO: 104 or 105: (1) D980P, V982P, F812P, A887P, A894P, and A937P; (2) K981P, V982P, F812P; A887P, A894P, and A937P, (3) D980P, V982P, F812P, A887P, A894P, A937P, R677G, R678S, and R680S, (4) K981P, V982P, F812P, A887P, A894P, A937P, R677G, R678S, and R680S, or (5) D980P, K981P, V982P, F812P, A887P, A894P, A937P, R677G, R678S, and R680S.

Another aspect of the disclosure features a composition or medical preparation that includes a first RNA encoding a first SARS-CoV-2 S polypeptide or an immunogenic fragment thereof, and a second RNA encoding a second SARS-CoV-2 S polypeptide or an immunogenic fragment thereof, wherein (a) the first SARS-CoV-2 S polypeptide or fragment comprises an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO:70 and including the following substitutions relative to SEQ ID NO:70: K983P, V984P, F814P, A889P, A896P, and A939P; (b) the second SARS-CoV-2 S polypeptide or fragment comprises an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO:70 and including the following substitutions relative to SEQ ID NO:70: K983P, V984P, F814P, A889P, A896P, and A939P; The S polypeptide or fragment has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 104 or 105 and comprises an amino acid sequence that includes one of the following sets of amino acid substitutions relative to SEQ ID NO: 104 or 105: (1) D980P, V982P, F812P, A887P, A894P, and A937P; (2) K981P, V982P, F812P; A887P, A894P, and A937P, (3) D980P, V982P, F812P, A887P, A894P, A937P, R677G, R678S, and R680S, (4) K981P, V982P, F812P, A887P, A894P, A937P, R677G, R678S, and R680S, or (5) D980P, K981P, V982P, F812P, A887P, A894P, A937P, R677G, R678S, and R680S.

Another aspect of the disclosure features a composition or medical preparation that includes a first RNA encoding a first SARS-CoV-2 S polypeptide or an immunogenic fragment thereof, and a second RNA encoding a second SARS-CoV-2 S polypeptide or an immunogenic fragment thereof, wherein (a) the first SARS-CoV-2 S polypeptide or fragment comprises an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO:70 and including the following substitutions relative to SEQ ID NO:70: D982P, V984P, F814P, A889P, A896P, A939P, R679G, R680S, and R682S; (b) the second SARS-CoV-2 S polypeptide or fragment comprises an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO:70 and including the following substitutions relative to SEQ ID NO:70: D982P, V984P, F814P, A889P, A896P, A939P, R679G, R680S, and R682S; The S polypeptide or fragment has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 104 or 105 and comprises an amino acid sequence that includes one of the following sets of amino acid substitutions relative to SEQ ID NO: 104 or 105: (1) D980P, V982P, F812P, A887P, A894P, and A937P; (2) K981P, V982P, F812P; A887P, A894P, and A937P, (3) D980P, V982P, F812P, A887P, A894P, A937P, R677G, R678S, and R680S, (4) K981P, V982P, F812P, A887P, A894P, A937P, R677G, R678S, and R680S, or (5) D980P, K981P, V982P, F812P, A887P, A894P, A937P, R677G, R678S, and R680S.

Another aspect of the disclosure features a composition or medical preparation that includes a first RNA encoding a first SARS-CoV-2 S polypeptide or an immunogenic fragment thereof, and a second RNA encoding a second SARS-CoV-2 S polypeptide or an immunogenic fragment thereof, wherein (a) the first SARS-CoV-2 S polypeptide or fragment comprises an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO:70 and including the following substitutions relative to SEQ ID NO:70: K983P, V984P, F814P, A889P, A896P, A939P, R679G, R680S, and R682S; (b) the second SARS-CoV-2 S polypeptide or fragment comprises an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO:70 and including the following substitutions relative to SEQ ID NO:70: K983P, V984P, F814P, A889P, A896P, A939P, R679G, R680S, and R682S; The S polypeptide or fragment has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 104 or 105 and comprises an amino acid sequence that includes one of the following sets of amino acid substitutions relative to SEQ ID NO: 104 or 105: (1) D980P, V982P, F812P, A887P, A894P, and A937P; (2) K981P, V982P, F812P; A887P, A894P, and A937P, (3) D980P, V982P, F812P, A887P, A894P, A937P, R677G, R678S, and R680S, (4) K981P, V982P, F812P, A887P, A894P, A937P, R677G, R678S, and R680S, or (5) D980P, K981P, V982P, F812P, A887P, A894P, A937P, R677G, R678S, and R680S.

Another aspect of the disclosure features a composition or medical preparation that includes a first RNA encoding a first SARS-CoV-2 S polypeptide or an immunogenic fragment thereof, and a second RNA encoding a second SARS-CoV-2 S polypeptide or an immunogenic fragment thereof, wherein (a) the first SARS-CoV-2 S polypeptide or fragment comprises an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO:70 and including the following substitutions relative to SEQ ID NO:70: D982P, K983P, V984P, F814P, A889P, A896P, A939P, R679G, R680S, and R682S; (b) the second SARS-CoV-2 S polypeptide or fragment comprises an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO:70 and including the following substitutions relative to SEQ ID NO:70: D982P, K983P, V984P, F814P, A889P, A896P, A939P, R679G, R680S, and R682S; The S polypeptide or fragment has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 104 or 105 and comprises an amino acid sequence that includes one of the following sets of amino acid substitutions relative to SEQ ID NO: 104 or 105: (1) D980P, V982P, F812P, A887P, A894P, and A937P; (2) K981P, V982P, F812P; A887P, A894P, and A937P, (3) D980P, V982P, F812P, A887P, A894P, A937P, R677G, R678S, and R680S, (4) K981P, V982P, F812P, A887P, A894P, A937P, R677G, R678S, and R680S, or (5) D980P, K981P, V982P, F812P, A887P, A894P, A937P, R677G, R678S, and R680S.

The present disclosure provides, among other things, the insight that incorporation of a D985P mutation rather than a K986P mutation can improve protein expression and/or immunogenicity (e.g., improve neutralization response). In some embodiments, incorporation of D985P rather than K986P can provide such improvement when combined with one or more other proline mutations (e.g., one or more proline mutations disclosed herein). In some embodiments, incorporation of D985P rather than K986P can provide such improvement when combined with V987P (e.g., one or more proline mutations disclosed herein). In some embodiments, incorporation of D985P rather than K986P can provide such improvement when combined with one or more (e.g., all) of F817P, A892P, A899P, A942P, and V987P.

In some embodiments, the present disclosure provides the insight that RNAs encoding SARS-CoV-2 S proteins containing one or more proline mutations (e.g., one or more of the proline mutations disclosed herein and/or a combination of proline mutations) and a mutated furin cleavage site can provide an improved immune response (e.g., an improved immune response compared to a similar or identical construct containing an intact furin cleavage site).

In some embodiments, the first RNA and the second RNA each include a modified nucleoside in place of a uridine. In some embodiments, the first RNA and the second RNA each include a modified uridine in place of every uridine. In some embodiments, the first RNA and the second RNA each include an N1-methyl-pseudouridine (m1ψ) in place of every uridine.

In some embodiments, the first RNA and the second RNA each comprise a 5' cap. In some embodiments, the 5' cap comprises m ₂ ^{7,3' -O} Gppp(m ₁ ^2'-O )ApG.

In some embodiments, the first RNA and the second RNA each comprise a 5'-UTR that is or comprises a modified human alpha-globin 5'-UTR. In some embodiments, the first RNA and the second RNA each comprise a 5'-UTR that comprises the nucleotide sequence of SEQ ID NO:12, or a nucleotide sequence that is at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identical to the nucleotide sequence of SEQ ID NO:12.

In some embodiments, the first RNA and the second RNA each comprise a 3'-UTR that is or comprises a first sequence from a split amino-terminal enhancer (AES) messenger RNA and a second sequence from a mitochondrially encoded 12S ribosomal RNA. In some embodiments, the first RNA and the second RNA each comprise a 3'UTR that comprises a nucleotide sequence of SEQ ID NO:13, or a nucleotide sequence that is at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identical to the nucleotide sequence of SEQ ID NO:13.

In some embodiments, the first RNA and the second RNA each comprise a polyA sequence. In some embodiments, the first RNA and the second RNA each comprise a polyA sequence comprising at least 100 nucleotides. In some embodiments, the first RNA and the second RNA each comprise 30 adenine nucleotides followed by 70 adenine nucleotides, the 30 adenine nucleotides and the 70 adenine nucleotides being separated by a linker sequence. In some embodiments, the first RNA and the second RNA each comprise a polyA sequence comprising or consisting of the nucleotide sequence of SEQ ID NO:14, or a nucleotide sequence that is at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identical to the nucleotide sequence of SEQ ID NO:14.

In some embodiments, the first RNA and the second RNA are each formulated or to be formulated for intramuscular administration.

In some embodiments, the first RNA and the second RNA are each formulated or to be formulated as particles. In some embodiments, the first RNA and the second RNA are each formulated as lipid nanoparticles (LNPs) or lipoplex (LPX) particles. In some embodiments, the LNPs include ((4-hydroxybutyl)azanediyl)bis(hexane-6,1-diyl)bis(2-hexyldecanoate), 2-[(polyethylene glycol)-2000]-N,N-ditetradecylacetamide, 1,2-distearoyl-sn-glycero-3-phosphocholine, and cholesterol.

In some embodiments, the first RNA and the second RNA are formulated in separate LNPs. In some embodiments, the first RNA and the second RNA are formulated in the same LNP. In some embodiments, lipoplex particles can be obtained by mixing RNA with liposomes.

In some embodiments, the first RNA and the second RNA are each mRNA. In some embodiments, the first RNA and the second RNA are each saRNA.

In some embodiments, the composition or medical preparation is a pharmaceutical composition. In some embodiments, the composition or medical preparation is a vaccine.

In some embodiments, the pharmaceutical composition further comprises one or more pharma- ceutically acceptable carriers, diluents, and/or excipients.

In another aspect, the present disclosure provides a method of inducing an immune response in a subject, comprising inducing an immune response in a subject by administering to the subject a composition or pharmaceutical preparation described herein.

In some embodiments, the SARS-CoV-2 S polypeptide comprises an amino acid sequence that does not include a D985P substitution relative to SEQ ID NO:1, an amino acid sequence that does not include a D982P substitution relative to SEQ ID NO:69 or SEQ ID NO:70, or an amino acid sequence that does not include a D980P substitution relative to SEQ ID NO:104 or SEQ ID NO:105.

In some embodiments, the method further comprises administering a second RNA encoding a second SARS-CoV-2 S polypeptide or immunogenic fragment thereof, wherein the second SARS-CoV-2 S polypeptide or immunogenic fragment is an omicron variant SARS-CoV-2 S polypeptide that is not the BA.1 omicron variant.

In some embodiments, the method further comprises administering a second, different RNA encoding a second SARS-CoV-2 S polypeptide or immunogenic fragment thereof, wherein the second SARS-CoV-2 S polypeptide or fragment is selected from the SARS-CoV-2 S polypeptides or fragments described herein.

Another aspect of the disclosure provides a method of inducing an immune response in a subject, comprising inducing an immune response in the subject by administering to the subject a composition or pharmaceutical formulation described herein. In some embodiments, the SARS-CoV-2 S polypeptide comprises an amino acid sequence that does not include a D985P substitution relative to SEQ ID NO:1, an amino acid sequence that does not include a D982P substitution relative to SEQ ID NO:69 or SEQ ID NO:70, or an amino acid sequence that does not include a D980P substitution relative to SEQ ID NO:104 or SEQ ID NO:105.

In some embodiments, the method further comprises administering a second composition or medical preparation, the second composition or medical preparation comprising RNA encoding a SARS-CoV-2 S polypeptide or immunogenic fragment of an omicron variant that is not the BA.1 omicron variant.

In some embodiments, the method further comprises administering a second composition or medical preparation, the second composition or medical preparation comprising a third RNA encoding a third SARS-CoV-2 S polypeptide or immunogenic fragment thereof, and a fourth RNA encoding a fourth SARS-CoV-2 S polypeptide or immunogenic fragment thereof.

In some embodiments, the third RNA encodes a SARS-CoV-2 S polypeptide or immunogenic fragment thereof that is a first or second SARS-CoV-2 S polypeptide or immunogenic fragment thereof recited in any one of claims 24-102, and the third RNA encodes a SARS-CoV-2 S polypeptide or fragment that is different from the first SARS-CoV-2 S polypeptide or fragment encoded by the first RNA and/or different from the second SARS-CoV-2 S polypeptide or fragment encoded by the second RNA.

In some embodiments, the fourth RNA encodes a SARS-CoV-2 S polypeptide or immunogenic fragment thereof that is a first or second SARS-CoV-2 S polypeptide or immunogenic fragment thereof recited in any one of claims 24-102, and the fourth RNA encodes a SARS-CoV-2 S polypeptide or fragment that is different from the first SARS-CoV-2 S polypeptide or fragment encoded by the first RNA and/or different from the second SARS-CoV-2 S polypeptide or fragment encoded by the second RNA.

In some embodiments, the third RNA encodes a SARS-CoV-2 S polypeptide or immunogenic fragment thereof that is a first or second SARS-CoV-2 S polypeptide or immunogenic fragment thereof recited in any one of claims 24-102, and the third RNA encodes a SARS-CoV-2 S polypeptide or fragment that is different from the first SARS-CoV-2 S polypeptide or fragment encoded by the first RNA and different from the second SARS-CoV-2 S polypeptide or fragment encoded by the second RNA.

In some embodiments, the fourth RNA encodes a SARS-CoV-2 S polypeptide or immunogenic fragment thereof that is a first or second SARS-CoV-2 S polypeptide or immunogenic fragment thereof recited in any one of claims 24-102, and the fourth RNA encodes a SARS-CoV-2 S polypeptide or fragment that is different from the first SARS-CoV-2 S polypeptide or fragment encoded by the first RNA and/or different from the second SARS-CoV-2 S polypeptide or fragment encoded by the second RNA.

The third RNA encodes a SARS-CoV-2 S polypeptide or an immunogenic fragment thereof that is the first or second SARS-CoV-2 S polypeptide or an immunogenic fragment thereof recited in any one of claims 24 to 102, the third RNA encodes a SARS-CoV-2 S polypeptide or a fragment thereof that is different from the first SARS-CoV-2 S polypeptide or fragment encoded by the first RNA and different from the second SARS-CoV-2 S polypeptide or fragment encoded by the second RNA, the fourth RNA encodes a SARS-CoV-2 S polypeptide or an immunogenic fragment thereof that is the first or second SARS-CoV-2 S polypeptide or an immunogenic fragment thereof recited in any one of claims 24 to 102, and the fourth RNA encodes a SARS-CoV-2 S polypeptide or an immunogenic fragment thereof that is the first SARS-CoV-2 S polypeptide or an immunogenic fragment thereof recited in any one of claims 24 to 102, and the fourth RNA encodes a SARS-CoV-2 S polypeptide or an immunogenic fragment thereof that is the first SARS-CoV-2 S polypeptide or an immunogenic fragment thereof encoded by the first RNA. It encodes a SARS-CoV-2 S polypeptide or fragment that is distinct from the S polypeptide or fragment and that is distinct from a second SARS-CoV-2 S polypeptide or fragment encoded by a second RNA.

In some embodiments, each of the first, second, third, and fourth RNAs encodes a different SARS-CoV-2 S polypeptide or immunogenic fragment thereof.

In another aspect, a monovalent vaccine described herein can be administered with a bivalent vaccine described herein. For example, in some embodiments, a method of inducing an immune response comprises administering to a subject (i) a composition or medical preparation described herein comprising an RNA encoding a SARS-CoV-2 S polypeptide or immunogenic fragment thereof described herein, and (ii) a composition or medical preparation comprising at least a first RNA encoding a first SARS-CoV-2 S polypeptide or immunogenic fragment thereof described herein and a second RNA encoding a second SARS-CoV-2 S polypeptide or immunogenic fragment thereof. [DDM1] In some embodiments, the monovalent vaccine and the bivalent vaccine can be administered at least 3 weeks apart, including, for example, at least 4 weeks, at least 5 weeks, at least 6 weeks, at least 7 weeks, at least 8 weeks, or more. In some embodiments, the monovalent and bivalent vaccines can be administered at least three months apart, including, for example, at least four months, at least five months, at least six months, at least seven months, at least eight months, or more. In some embodiments, the monovalent and bivalent vaccines can be administered in a single session in different arms. In some embodiments, the monovalent and bivalent vaccines can be administered in a single injection as a trivalent vaccine (e.g., mixing the monovalent and bivalent vaccines together prior to administration).

Overview of S protein organization of SARS-CoV-2 S protein. The sequence within the S1 subunit consists of the signal sequence (SS) and the receptor binding domain (RBD), which is the key subunit within the S protein involved in binding to the human cell receptor ACE2. The S2 subunit contains the S2 protease cleavage site (S2'), followed by the fusion peptide (FP) for membrane fusion, heptad repeats (HR1 and HR2) with a central helix (CH) domain, a transmembrane domain (TM) and a cytoplasmic tail (CT). Exemplary SARS-CoV-2 vaccine constructs. Based on the complete and wild-type S protein, we designed different constructs encoding (1) the complete protein with mutations in close proximity to the first heptad repeat (HRP1) containing stabilizing mutations that preserve the neutralization sensitive site, (2) the S1 domain, or (3) only the RB domain (RBD). To further stabilize the protein fragment, a fibritin domain (F) was fused to the C-terminus. All constructs start with a signal peptide (SP) to ensure Golgi transport to the cell membrane. General structure of a particular RNA vaccine. Schematic diagram of the general structure of a particular RNA vaccine with 5' cap, 5' and 3' untranslated regions, coding sequence with essential secretory signal peptide and GS linker, and poly(A) tail. Note that the individual elements are not drawn exactly to scale compared to their respective sequence lengths. UTR = untranslated region, sec = secretory signal peptide, RBD = receptor binding domain, GS = glycine-serine linker. General structure of a particular RNA vaccine. Schematic of the general structure of a particular RNA drug substance with 5' cap, 5' and 3' untranslated regions, coding sequence with essential secretory signal peptide and GS linker, and poly(A) tail. Note that the individual elements are not drawn exactly to scale compared to their respective sequence lengths. GS = glycine-serine linker, UTR = untranslated region, Sec = secretory signal peptide, RBD = receptor binding domain. General structure of certain RNA vaccines. Schematic diagram of the general structure of an RNA vaccine with 5' cap, 5' and 3' untranslated regions, coding sequence for Venezuelan Equine Encephalitis Virus (VEEV) RNA-dependent RNA polymerase replicase, and SARS-CoV-2 antigen with a unique secretory signal peptide and GS linker, and poly(A) tail. Note that the individual elements are not drawn exactly to scale compared to their respective sequence lengths. UTR = untranslated region, Sec = secretory signal peptide, RBD = receptor binding domain, GS = glycine-serine linker. Schematic diagram of the S protein organization of the SARS-CoV-2 S protein and constructs for the development of a SARS-CoV-2 vaccine. Based on the wild-type S protein, two different transmembrane-anchored RBD-based vaccine constructs were designed encoding an RBD fragment fused to the T4 fibritin trimerization domain (F) and a unique transmembrane domain (TM). Construct (1) starts with the SARS-CoV-2-S signal peptide (SP, AA 1-19 of the S protein), whereas construct (2) starts with the human Ig heavy chain signal peptide (huSec) to ensure Golgi transport to the plasma membrane. Anti-S protein IgG responses at days 6, 14, and 21 after immunization with LNP-C12 formulated modRNA encoding transmembrane anchored RBD-based vaccine constructs. BALB/c mice were immunized IM once with 4 μg of LNP-C12 formulated transmembrane anchored RBD-based vaccine constructs (surrogate to BNT162b3c/BNT162b3d). At days 6, 14, and 21 after immunization, animals were bled and serum samples were analyzed for total anti-S1 (left) and anti-RBD (right) antigen-specific immunoglobulin G (IgG) measured via ELISA. Different serum dilutions were included in the graphs for days 6 (1:50), 14 (1:300), and 21 (1:900). Each point on the graph represents one mouse, and all mouse samples were measured in duplicate (group size n=8, mean + SEM included in groups). Neutralization of SARS-CoV-2 pseudovirus 6, 14, and 21 days after immunization with LNP-C12 formulated modRNA encoding transmembrane anchored RBD-based vaccine constructs. BALB/c mice were immunized IM once with 4 μg of LNP-C12 formulated transmembrane anchored RBD-based vaccine constructs (surrogate to BNT162b3c/BNT162b3d). Animals were bled and sera were tested for SARS CoV-2 pseudovirus neutralization 6, 14, and 21 days after immunization. Graph shows pVN ₅₀ serum dilution (50% reduction in infectious events compared to positive control without serum). One point on the graph represents one mouse. All mouse samples were measured in duplicate. Group size n=8. Mean+SEM is indicated by horizontal bars with whiskers for each group. LLOQ, lower limit of quantification. ULOQ, upper limit of quantification. Pseudovirus neutralization titers (pVNT50) of sera collected 21 days after the second dose and 1 month after the third dose of BNT162b2 against VSV-SARS-CoV-2-S pseudoviruses bearing Wuhan Hu-1 reference or Omicron BA.1 lineage spike proteins. N=19-20 sera from immunized subjects collected either 21 days after the second BNT162b2 dose or 1 month after the third BNT162b2 dose were tested. For values below the limit of detection (LOD, ₁₀ ), the LOD/2 value was plotted. Group GMT (values above bars) with 95% confidence intervals are shown. CD8+ T cell epitopes in the BNT162b2 vaccine remain largely unaffected by omicron variant mutations. The number of previously identified MHC-I epitopes affected in various variants of concern (VOCs) is shown. Approximately 80% of previously identified CD8+ epitopes are unaffected by omicron BA.1 variant mutations, suggesting that two doses of BNT162b2 may still induce protection against severe disease. Neutralization of Omicron BA.1 after two doses of BNT162b2 and a variant-specific booster. Shown is neutralization of Omicron BA.1 variants from serum of patients who received two doses of BNT162b2 and (i) a third booster dose of BNT162b2, or (ii) a third booster dose of RNA encoding a spike protein with an alpha or delta variant mutation, or a third booster dose of both a spike protein containing an alpha mutation and a spike protein containing a delta mutation. These values are derived from separate neutralization GMTs from pseudovirus testing. Also shown is a schematic illustrating the process for developing new SARS-CoV-2 variant-specific vaccines. Longitudinal analysis of neutralizing antibody responses to VSV-SARS-CoV-2-S pseudoviruses bearing Wuhan or Omicron BA.1 variant spike proteins in a subset of study participants. Sera from n=9 participants extracted 21 days after dose 2, before dose 3, 1 month after dose 3, and 3 months after dose 3 were tested. Each serum was tested in duplicate and individual geometric mean 50% pseudovirus neutralization titers (GMT) were calculated. For values below the limit of detection (LOD), the LOD/2 value was assigned. Group GMTs (table values) and 95% confidence intervals per time point are shown. Analysis of HLA class I T cell epitope conservation between Wuhan and Omicron BA.1 variants. HLA class I restricted spike protein epitopes with T cell reactivity identified based on their recognition by CD8+ T cells and reported in the IEDB (n=244) are plotted by their position (top) along the spike protein (bottom). Epitope adaptation is located by the amino acid position of the epitope's center, epitopes conserved in both variants are marked in light grey (n=208), while epitopes spanning the Omicron BA.1 mutation site are marked in dark grey (n=36). NTD=N-terminal domain, RBD=receptor binding domain, FCS=furin cleavage site. S1 and S2 regions of the spike protein are indicated. Schematic diagram of an exemplary vaccination regimen. Cohorts, sample collection, and experimental settings for characterization of immune responses in Omicron BA.1 breakthrough cases. Blood samples were collected from four cohorts: Omicron-naive individuals vaccinated two or three times with BNT162b2, and individuals vaccinated two or three times with BNT162b2 who subsequently developed breakthrough infection with Omicron BA.1. PBMCs and serum were isolated within the Omicron-naive cohort at the indicated time points after most recent vaccination, and for the convalescent cohort, the time from most recent vaccination to Omicron BA.1 infection and infection to PBMCs and serum isolation are shown (all values are designated as median ranges). Serum neutralization capacity was assessed using pseudovirus and live virus neutralization tests. SARS-CoV-2 spike-specific B _MEM cells were assessed by a flow cytometry-based B cell phenotypic assay using bulk PBMCs. N/A, not applicable. Omicron BA.1 breakthrough infection in individuals vaccinated twice and three times with BNT162b2 induces broad neutralization of Omicron BA.1, BA.2, and other VOCs. Sera were collected from individuals vaccinated twice 22 days after the second dose (BNT162b2 ² ) (open circles), from individuals vaccinated three times 28 days after the third dose (BNT162b2 ³ ) (filled circles), from individuals vaccinated twice and with Omicron BA.1 breakthrough infection 46 days after infection (BNT162b2 ² + Omi) (open triangles), and from individuals vaccinated three times and with Omicron BA.1 breakthrough infection 44 days after infection (BNT162b2 ³ + Omi) (filled triangles). Sera were tested in duplicate and (A) _pVN50 geometric mean titers (GMT) for 50% pseudovirus neutralization (B) _VN50 GMT for 50% virus neutralization (C) geometric mean ratios for SARS-CoV-2 variants of concern (VOC) and Wuhan _VN50 GMT are shown. For values below the limit of detection (LOD), the LOD/2 value was plotted. Values above the violin plot represent group GMT. Wuhan neutralization group GMT was compared to titers against the indicated variants and SARS-CoV-1 using the nonparametric Friedman test with Dunn's multiple comparisons correction. Multiplicity adjusted p-values are shown. (A) _pVN50 GMT for Wuhan, VOC and SARS-CoV-1 pseudoviruses. (B) _VN50 GMT for live SARS-CoV-2 Wuhan, beta, delta, and omicron BA.1. (C) Group geometric mean ratios with 95% confidence intervals for all cohorts shown in (B). Ibid. B _MEM cells from individuals vaccinated twice and three times with BNT162b2 broadly recognize VOCs and are further boosted by Omicron BA.1 breakthrough infection. PBMC samples from a twice vaccinated individual (BNT162b2 2 ) 22 days after the second dose (open squares) and 5 months after the second dose (open circles), a sample from a three times vaccinated individual (BNT162b2 ³ ) 84 days after the third dose (filled circles), a sample from a twice vaccinated, Omicron BA.1 breakthrough infected individual (BNT162b2 ² +Omi) 46 days after infection (open triangles), and a sample from a three times vaccinated, Omicron BA.1 breakthrough infected individual (BNT162b2 ² +Omi) 44 days after infection (open triangles). Samples (filled triangles) from one breakthrough infected individual (BNT162b23+Omi) were analyzed by flow cytometry for the frequency of SARS-CoV-2 specific B _MEM cells (B _MEM -CD3-CD19+CD20+IgD-CD38 ^mid/low ) via B cell bait staining. (A) Schematic of one-dimensional staining of B MEM cells with fluorochrome-labeled SARS-CoV-2 S protein tetramer baits allowing differentiation of variant recognition. The frequency of Wuhan or VOC full-length S protein- (B) and RBD- (C) specific B _MEM cells in the four populations was analyzed. The frequency of variant-specific B _MEM cells was normalized to the frequency of Wuhan for S protein (D) and RBD- (E) binding. (F) The frequency ratio _{of RBD protein-specific B MEM cells to full-length S protein-specific B MEM cells is} shown. Omicron BA.1 breakthrough infection of individuals vaccinated twice and three times with BNT162b2 boosts BMEM primarily against conserved epitopes that are extensively shared between the Wuhan S protein and other VOCs, rather than strictly Omicron S-specific epitopes. _PBMC samples from a twice vaccinated individual (BNT162b2 2 ) 22 days after the second dose (open squares) and 5 months after the second dose (open circles), a sample from a three times vaccinated individual (BNT162b2 ³ ) 84 days after the third dose (filled circles), a sample from a twice vaccinated, Omicron BA.1 breakthrough infected individual (BNT162b2 ² +Omi) 46 days after infection (open triangles), and a sample from a three times vaccinated, Omicron BA.1 breakthrough infected individual (BNT162b2 ² +Omi) 44 days after infection (open triangles). Samples (filled triangles) from one breakthrough infected individual (BNT162b2 ⁺ Omi) were analyzed by flow cytometry for SARS-CoV-2 specific memory B cell (B _MEM -CD3-CD19+CD20+IgD-CD38 ^medium/low ) frequency via B cell bait staining (schematic diagram shown in (A)). (B) Representative flow diagrams of Omicron BA.1 and Wuhan S protein binding and RBD binding for each of the four populations examined are shown. Frequencies of B _MEM binding Omicron BA.1, Wuhan, or both (shared) shown for full-length S protein in (C) and RBD shown in (D) for Omicron BA.1 experienced and Omicron BA.1 naive BNT162b2 vaccinated 2 and 3 times. (E) Venn diagram visualizing the combinatorial (Boolean) gating strategy to identify cross-reactive B MEMs simultaneously recognizing all four variants (All 4+ve) and B _MEMs recognizing _{only the Omicron BA.1 S protein (Omicron-only) or only the Wuhan S protein (Wuhan-only). The frequency of these three B MEM subgroups} was compared for the full-length S protein (F) and RBD (G) in the four different populations studied. The RBD variant recognition patterns by B _MEMs were assessed through a Boolean flow cytometry gating strategy, and the frequency of recognition of Wuhan and/or variant RBD combinations is displayed in (H) for all Omicron-convalescent subjects (pooled 2- and 3-dose vaccinated, n=13). (I) Conserved sites within the RBD domain recognized by RBD-specific B _MEMs after Omicron BA.1 breakthrough infection. Mean values are shown in C, D, F, and G. n = number of individuals per group. Ibid. Ibid. Ibid. Omicron BA.1 breakthrough infection in individuals vaccinated with other approved COVID-19 vaccines or combination regimens results in immune sera that broadly neutralize SARS-CoV-1 in addition to Omicron BA.1, BA.2, and other VOCs. Sera were collected from 10 individuals vaccinated with other approved COVID-19 vaccines or combination regimens at a median of 43 days post-infection (grey diamonds). Sera were tested in duplicate and individual 50% pseudoviral neutralization ( _pVN50 ) geometric mean titers (GMT) against SARS-CoV-2 Wuhan, alpha, beta, delta, and Omicron BA.1 and BA.2 variants, as well as SARS-CoV-1, were plotted. For values below the limit of detection (LOD), the LOD/2 value was plotted. Values above the violin plot represent group GMT. Wuhan neutralization group GMTs were compared with titers against the indicated variants and SARS-CoV-1 using a non-parametric Friedman test with Dunn's multiple comparison correction. Multiplicity adjusted p-values are shown. Approved vaccines include AZD1222, BNT162b2 (in some embodiments, as part of a 4-dose series), Ad26.COV2.S, mRNA-1273 (administered as a 2- or 3-dose series), and combinations thereof. 50% neutralization titers of sera collected one month after a fourth dose of BNT162b2 or an omicron-specific booster. Subjects previously administered two doses of BNT162b2 and a third (booster) dose of BNT162b2 (30 ug) showed a 50% neutralization titer of SARS-CoV-2 from (i) the omicron variant at a dose (30 ug). Subjects received RNA encoding an S protein (e.g., as described herein (referred to herein as an "OmicronBA.1-specific RNA vaccine"), or (ii) BNT162b2 as a fourth (booster) dose. Subject serum was collected one month after administration of the fourth (booster) dose. Group GMTs (values above the bars) with 95% confidence intervals are shown. "b2" refers to serum from subjects who received a Wuhan-specific RNA vaccine as the fourth (booster) dose of BNT162b2. "OMI" refers to serum from subjects who received an OmicronBA.1-specific fourth (booster) dose. "FFRNT" refers to sera from subjects who received the 4th dose of the Omicron BA.1 specific RNA vaccine as the 4th dose. Also shown is the fold change in titer from before to after the 4th dose (pre-vaccination/post-vaccination fold increase), and the ratio of the geometric mean ratio (GMR) and geometric mean fold increase (GMFR) observed in subjects who received the 4th dose of the Omicron BA.1 specific RNA vaccine as the 4th dose compared to subjects who received BNT162b2 as the 4th dose of the Omicron BA.1 specific RNA vaccine as the 4th dose. "FFRNT" refers to fluorescent focus reduction neutralization test. SARS-CoV-2 from the variants indicated in the figure. Neutralization data was obtained using FFRNT assays with virus particles containing the S protein. (A) Comparison of neutralizing antibody titers against SARS-CoV-2-S pseudoviruses containing SARS-CoV-2 S proteins with mutations characteristic of the Omicron BA.1 variant, excluding sera from subjects previously or currently infected with SARS-CoV-2. (B) Comparison of neutralizing antibody titers against SARS-CoV-2 pseudoviruses containing SARS-CoV-2 S proteins with mutations characteristic of the Omicron BA.1 variant, as determined by antigen or PCR assays, respectively, in sera from populations including subjects previously or currently infected with SARS-CoV-2. (C) Comparison of neutralizing antibody titers against SARS-CoV-2 pseudoviruses containing SARS-CoV-2 S proteins with mutations characteristic of the Omicron BA.1 variant, as determined by antigen or PCR assays, respectively, in sera from populations including subjects previously or currently infected with SARS-CoV-2. (D) Comparison of neutralizing antibody titers against a SARS-CoV-2 pseudovirus comprising the SARS-CoV-2 S protein from the Wuhan strain in sera from a population that includes individuals previously or currently infected with SARS-CoV-2, as determined by antigen or PCR assays, respectively. (E) Comparison of neutralizing antibody titers against a SARS-CoV-2 pseudovirus comprising the SARS-CoV-2 S protein with a mutation characteristic of the delta variant, as determined by antigen or PCR assays, in sera from a population that includes individuals previously or currently infected with SARS-CoV-2. (F) Comparison of neutralizing antibody titers (determined by antigen assay or PCR assay, respectively) in sera from populations including subjects previously or currently infected with SARS-CoV-2 against a SARS-CoV-2 pseudovirus containing a SARS-CoV-2 protein with a mutation characteristic of the delta variant. Ibid. Ibid. Ibid. Ibid. Ibid. Neutralization of SARS-CoV-2 pseudovirus 7 days after immunization with modRNA encoding variant-specific S proteins. Mice were immunized twice with LNP-formulated vaccines comprising (i) BNT162b2 (encoding a SARS-CoV-2 S protein from the Wuhan strain), (ii) RNA encoding a SARS-CoV-2 S protein with mutations characteristic of the Omicron BA.1 variant (Omi), (iii) RNA encoding an S protein with mutations characteristic of the delta variant, (iv) a combination of BNT162b2 and RNA encoding a protein with mutations characteristic of the Omicron BA.1 variant (B2+Omi), or (v) RNA encoding a SARS-CoV-2 S protein with mutations characteristic of the delta variant and RNA encoding a SARS-CoV-2 S protein with mutations characteristic of the Omicron BA.1 variant (delta+Omi). Seven days after the second immunization, animals were bled and sera were tested for neutralization of SARS-CoV-2-S pseudoviruses containing SARS-CoV-2 S protein from the Wuhan strain or SARS-CoV-2 S protein with mutations characteristic of beta, delta, or omicron BA.1 variants. Graph shows pVN ₅₀ serum dilution (50% reduction in infectious events compared to positive control without serum). One point on the graph represents one mouse. All mouse samples were measured in duplicate. Mean + SEM is indicated by whiskered horizontal bars for each group. LLOD, lower limit of detection. ULOD, upper limit of detection. RNA encoding a SARS-CoV-2 S protein with mutations characteristic of the beta variant increases neutralizing antibody titers against SARS-CoV-2 when administered to patients who had previously received two doses of a vaccine encoding the Wuhan strain of SARS-CoV-2 S protein. Subjects who had previously received two doses of an RNA vaccine encoding the Wuhan strain of SARS-CoV-2 S protein were administered a third and fourth dose of an RNA vaccine encoding a SARS-CoV-2 S protein with mutations characteristic of the beta variant. Neutralizing antibody titers were measured prior to administration of the RNA vaccine encoding the SARS-CoV-2 S protein of the Wuhan strain (D1-PreVax), one month after administration of a second dose of the RNA vaccine encoding the SARS-CoV-2 S protein of the Wuhan strain (M1PD2), one month after administration of a third dose of the RNA vaccine encoding a SARS-CoV-2 S protein with a mutation characteristic of the SARS-CoV-2 beta variant, and one month after administration of a fourth dose of the RNA vaccine encoding a SARS-CoV-2 S protein with a mutation characteristic of the SARS-CoV-2 beta variant. The third and fourth doses were administered one month apart from each other. GMFR refers to the geometric mean fold increase and is a measure of the increase in neutralizing antibody titers since the previous vaccine dose (e.g., GMFR after dose 2 (PD2) is a measure of the increase in neutralizing antibody titers compared to before administration of any vaccine (pre-vax)). (A) Neutralizing antibody titers measured in a virus neutralization assay using virus particles containing the SARS-CoV-2 S protein of the Wuhan strain. (B) Neutralizing antibody titers measured in a virus neutralization assay using virus particles containing the SARS-CoV-2 S protein with a mutation characteristic of a beta variant. Ibid. 50% neutralization titers of sera collected 7 days after a fourth dose of BNT162b2, an Omicron BA.1-specific booster, or a bivalent vaccine. Subjects who previously received two doses of BNT162b2 (30 ug), and a third (booster) dose of BNT162b2 (30 ug) were randomized to receive (i) a 30 ug dose of BNT162b2 (encoding the SARS-CoV-2 S protein from the Wuhan strain), (ii) a 60 ug dose of BNT162b2, (iii) a 30 ug dose of Omicron BA.1-specific booster, or a bivalent vaccine. (iii) a 60 ug dose of an RNA encoding a SARS-CoV-2 S protein having a mutation characteristic of the Omicron BA.1 variant, (iv) a 30 ug dose of a bivalent vaccine comprising 15 ug of BNT162b2 and 15 ug of an RNA encoding a SARS-CoV-2 S protein including a mutation characteristic of the Omicron BA.1 variant, or (v) a 30 ug dose of a bivalent vaccine comprising 30 ug of BNT162b2 and 30 ug of an RNA encoding a SARS-CoV-2 S protein including a mutation characteristic of the Omicron BA.1 variant, and RNA encoding the S protein. Geometric mean ratios (GMR) of titers in serum from subjects collected 7 days after administration of the fourth dose. "b2" refers to serum from subjects who received the Wuhan-specific RNA vaccine as the fourth dose of BNT162b2. "OMI" refers to serum from subjects who received the Omicron BA.1-specific fourth dose. "Bivalent" refers to serum from subjects who received the composition comprising BNT162b2 and RNA encoding the SARS-CoV-2 S protein as the fourth dose, including a mutation characteristic of the Omicron BA.1 variant. Also shown is the fold increase in titer from before administration of the fourth dose to 7 days after administration of the fourth dose (*fold increase). "FFRNT" refers to fluorescent focus reduction neutralization test. SARS-CoV-2 with mutations characteristic of the variants shown in the figure Neutralization data was obtained using the FFRNT assay with virus particles containing the S protein. LLOQ refers to the lower limit of quantification and ULOQ refers to the upper limit of quantification. (A) Comparison of neutralizing antibody titers against SARS-CoV-2 pseudoviruses containing SARS-CoV-2 S proteins with mutations characteristic of the Omicron BA.1 variant. Sera from subjects previously or currently infected with SARS-CoV-2 are excluded. (B) Comparison of neutralizing antibody titers against SARS-CoV-2 pseudoviruses containing SARS-CoV-2 S proteins with mutations characteristic of the Omicron BA.1 variant in sera from populations including subjects previously or currently infected with SARS-CoV-2 (as determined by antibody testing or PCR assay, respectively). (C) Comparison of neutralizing antibody titers against SARS-CoV-2 pseudoviruses comprising the Wuhan strain of SARS-CoV-2 S protein, excluding sera from subjects previously or currently infected with SARS-CoV-2. (D) Comparison of neutralizing antibody titers against SARS-CoV-2 pseudoviruses comprising the Wuhan strain of SARS-CoV-2 S protein in sera from populations including individuals previously or currently infected with SARS-CoV-2. (E) Comparison of neutralizing antibody titers against SARS-CoV-2 pseudoviruses comprising the SARS-CoV-2 S protein with a mutation characteristic of the delta variant, excluding sera from subjects previously or currently infected with SARS-CoV-2. (F) Comparison of neutralizing antibody titers against a SARS-CoV-2 pseudovirus containing a SARS-CoV-2 S protein with a mutation characteristic of the delta variant in serum from a population including subjects previously or currently infected with SARS-CoV-2. (G) Comparison of neutralizing antibody titers against a SARS-CoV-2 pseudovirus containing a SARS-CoV-2 S protein with a mutation characteristic of the delta variant in subjects receiving 60 ug of BNT162b2, 30 ug of RNA encoding a SARS-CoV-2 S protein with a mutation characteristic of the Omicron BA.1 variant (OMI 30 ug), 60 ug of RNA encoding a SARS-CoV-2 S protein with a mutation characteristic of the Omicron BA.1 variant (OMI 60 ug), 15 ug of BNT162b2, and 15 ug of Omicron BA.1 as a fourth dose compared to subjects receiving 30 ug of BNT162b2 as a fourth dose. Geometric mean rise (GMR) of neutralizing antibodies observed in subjects administered 30 ug of a bivalent vaccine (Bivalent 30 ug) comprising 30 ug of BNT162b2 and 30 ug of Omicron BA.1 variant and an RNA encoding a SARS-CoV-2 S protein with a mutation characteristic of the SARS-CoV-2 S protein. Results are presented for both population pools excluding and including subjects previously or currently infected with SARS-CoV-2. Ibid. Ibid. Ibid. Ibid. Ibid. Ibid. Reactogenicity of certain exemplary RNAs (formulated in LNPs) at a given dose: Subjects receiving a 60ug dose of RNA encoding the SARS-CoV-2 S protein were more likely to show injection site pain and a similar systemic reaction than subjects receiving a 30ug dose of RNA. Subjects were administered 30ug or 60ug of RNA encoding the SARS-CoV-2 S protein from the Wuhan strain (BNT162b2, corresponding to groups G1 and G2, respectively), 30ug or 60ug of RNA encoding a SARS-CoV-2 S protein with a mutation characteristic of the Omicron BA.1 variant (BNT162b2 OMI, corresponding to groups G3 and G4, respectively), 15ug of RNA encoding the SARS-CoV-2 S protein from the Wuhan strain and 15ug of Omicron BA. Patients were administered 30 ug of a bivalent vaccine (BNT162B2 (15 ug) + BNT162b2 OMI (15 ug), corresponding to group G5) containing 30 ug of RNA encoding a SARS-CoV-2 S protein from the Wuhan strain and 30 ug of Omicron BA. 1 variant, or 60 ug of a bivalent vaccine (BNT162b2 (30 ug) + BNT162b2 OMI (30 ug), corresponding to group G6) containing 30 ug of RNA encoding a SARS-CoV-2 S protein from the Wuhan strain and 30 ug of Omicron BA. 1 variant. (A) Local reactions including redness, swelling, and pain at the injection site observed within 7 days of injection. The 60 ug Omicron BA. 1 variant was administered at 30 ug of the bivalent vaccine (BNT162b2 (30 ug) + BNT162b2 OMI (30 ug), corresponding to group G6) containing 30 ug of RNA encoding a SARS-CoV-2 S protein from the Wuhan strain and 30 ug of Omicron BA. 1 variant. (B) Local reactions including redness, swelling, and pain at the injection site observed within 7 days of injection. The 60 ug Omicron BA. 1 variant was administered at 30 ug of the bivalent vaccine (BNT162b2 (30 ug) + BNT162b2 OMI (30 ug), corresponding to group G6) compared to the other doses tested. Increased pain at the injection site was found in subjects who received RNA encoding SARS-CoV-2 S protein containing mutations characteristic of the mono-variant or bivalent vaccines. (B) Systemic reactions observed within 7 days of injection, including fever, fatigue, headache, chills, vomiting, diarrhea, myalgia, arthralgia, and medication use. Systemic reactions by 7 days were observed to be broadly similar across the different groups. Fatigue tended to be higher following administration of the 60ug dose compared to the 30ug dose. Ibid. Omicron BA.1 breakthrough infection of individuals vaccinated two and three times with BNT162b2 induces broad neutralization of Omicron BA.1, BA.2, and other VOCs, but to a lesser extent BA.4 and BA.5. This figure is an extension of FIG. 16, which includes data neutralization activity against Omicron BA.4 and BA.5. As described in FIG. 16, sera were tested in duplicate and the 50% pseudovirus neutralization ( _pVN50 ) geometric mean titers (GMT) (in A and B) and the geometric mean ratios of SARS-CoV-1 _pVN50 GMT normalized to SARS-CoV-2 variants of concern (VOCs) and Wuhan _pVN50 GMT were plotted. For values below the limit of detection (LOD), the LOD/2 value was plotted. Values above the violin plots represent group GMT. Wuhan neutralization group GMT was compared to titers against the indicated variants and SARS-CoV-1 using a nonparametric Friedman test with Dunn's multiple comparison correction. Multiplicity-adjusted p-values are shown. (A) pVN _{50 GMT against Wuhan, VOC, and SARS-CoV-1 pseudoviruses in patients who received two or three doses of BNT162b2. (B) pVN 50} GMT against Wuhan, VOC, and SARS-CoV-1 pseudoviruses in patients who received two or three doses of _BNT162b2 and who were previously infected with the Omicron BA.1 variant of SARS-CoV-2. (C) Group geometric mean ratios with 95% confidence intervals for all cohorts shown in (A) and (B). Ibid. Omicron BA.1 breakthrough infection in individuals vaccinated with other approved COVID-19 vaccines or combination regimens results in immune sera that broadly neutralize Omicron BA.1, BA.2, and other VOCs, but to a lesser extent BA.4 and BA.5. This figure is an extension of FIG. 19, which includes data neutralizing activity against Omicron BA.4 and BA.5. As described in FIG. 19, sera were tested in duplicate and individual 50% pseudoviral neutralization (pVN50) geometric mean titers ( _GMT ) against SARS-CoV-2 Wuhan, alpha, beta, delta, and Omicron BA.1, BA.2, and BA.4/5 variants, as well as SARS-CoV-1, were plotted. For values below the limit of detection (LOD), the LOD/2 value was plotted. Values above the violin plots represent group GMT. Wuhan neutralization group GMT was compared with titers against the indicated variants and SARS-CoV-1 using the nonparametric Friedman test with Dunn's multiple comparisons correction. Multiplicity-adjusted p-values are shown. Sequences of the RBD of the SARS-CoV-2 Wuhan strain and its variants. The top sequence corresponds to the Wuhan, the second sequence corresponds to the alpha variant, the third sequence corresponds to the delta variant, the fourth sequence corresponds to the Omicron BA.1 variant, and the fifth sequence corresponds to the Omicron BA.4/5 variant. Variant-specific amino acid modifications are shown in bold. Cohorts and sample collection for the study described in Example 14. Schematic diagram for testing immune responses in 3-vaccinated patients (i) omicron-naive, (ii) infected with BA.1 omicron variant, or (iii) infected with BA.2 omicron variant. Blood samples were taken from three cohorts: omicron-naive individuals vaccinated 3 times with BNT162b2 (BNT162b2 ³ ), and individuals vaccinated in a homologous or heterologous 3-dose regimen who subsequently had a breakthrough infection with omicron either during the BA.1 predominance in Germany (November 2021-January 2022, all Vax+BA.1) or during the BA.2 predominance (March-May 2022, all Vax+BA.2, blue). Serum (droplets) was isolated in the Omicron-naive cohort at the indicated time points after most recent vaccination, and for the convalescent cohort, the time from most recent vaccination to Omicron infection and the time from infection to serum isolation are shown. All values are designated as median ranges. Serum neutralization capacity was assessed using a pseudovirus neutralization test. 50% pseudovirus neutralization ( _pVN50 ) geometric mean titers (GMT) from BNT162b2 ³ and All Vax+Omi BA.1 breakthrough infected cohorts. Sera were collected from Omicron-naive BNT162b2 vaccinated individuals 28 days after the third dose (BNT162b2 ³ , circles) and from vaccinated individuals who subsequently developed Omicron BA.1 breakthrough infection (all Vax+Omi BA.1, triangles) at a median of 43 days post-infection. 50% pseudovirus neutralization ( _pVN50 ) geometric mean titers (GMT) for Omicron-naive individuals are plotted in (A) and for BA.1 breakthrough infected individuals in (B). This data was compared with the BA.1 breakthrough infected cohorts. Except for the 2.12.1 neutralization data, data were previously published in Quandt et al. (e.g., "Omicron BA.1 breakthrough infection drives cross-variant neutralization and memory B cell formation against conserved epitopes." Science immunology, eabq2427 (2022), doi:10.1126/sciimmunol.abq2427). Sera were tested in duplicate. For values below the limit of detection (LOD), the LOD/2 value was plotted. Values above the violin plot represent group GMT. Wuhan neutralization group GMTs were compared with titers against the indicated variants and SARS-CoV-1 using the nonparametric Friedman test with Dunn's multiple comparison correction. Multiplicity-adjusted p-values are shown. Omicron BA.2 breakthrough infection in previously vaccinated individuals refocuses neutralization against Omicron BA.2 and BA.2-derived subvariants BA.2.12.1 and BA.4/BA.5. Sera were collected from individuals vaccinated three times with BNT162b2 who subsequently had an Omicron BA.1 breakthrough infection at a median of 44 days post-infection (BNT162b2 ³ + Omi BA.1, triangles) and from individuals vaccinated three times with BNT162b2 who subsequently had an Omicron BA.2 breakthrough infection 38 days post-infection (BNT162b2 ³ + Omi BA.2, squares). Plotted are 50% pseudovirus neutralization ( _pVN50 ) geometric mean titers (GMT) (in A,B) and geometric mean ratios of SARS-CoV-1 _pVN50 GMT normalized to SARS-CoV-2 variants of concern (VOC) and Wuhan pVN50 GMT (in C). pVN50 GMT and geometric mean ratio data for Omicron-naive BNT162b2-vaccinated individuals (BNT162b2 ³ , circles) and Omicron BA.1 breakthrough-infected BNT162b2-vaccinated individuals (BNT162b2 ₃ , circles) are consistent with those reported by Quandt et al., with the exception of the BA.2.12.1 neutralization data. (Previously published in "Omicron BA.1 breakthrough infection drives cross-variant neutralization and memory B cell formation against conserved epitopes." Science immunology, eabq2427 (2022), doi:10.1126/sciimmunol.abq2427). Sera were tested in duplicate. For values below the limit of detection (LOD), the LOD/2 value was plotted. Values above the violin plot represent group GMT. Wuhan neutralization group GMTs were compared with titers against the indicated variants and SARS-CoV-1 using the nonparametric Friedman test with Dunn's multiple comparison correction. Multiplicity-adjusted p-values are shown. (A, B) _pVN50 GMTs against Wuhan, VOC and SARS-CoV-1 pseudoviruses. (C) Group geometric mean ratios with 95% confidence intervals. Ibid. Characterization of SARS-CoV-2 S glycoprotein used in VSV-SARS-CoV-2 pseudovirus-based neutralization assay. Wuhan-Hu-1 isolate SARS-CoV-2 S glycoprotein (GenBank: QHD43416.1) was used as reference. The amino acid position, amino acid description (single letter code), and type of mutation (substitution, deletion, insertion) are indicated. NTD, N-terminal domain; RBD, receptor binding domain; Δ, deletion; ins, insertion; *, cytoplasmic domain truncated for the C-terminal 19 amino acids. SARS-CoV-2 Omicron sublineage spike glycoprotein amino acid sequence changes. The amino acid position, amino acid description (one letter code), and type of mutation (substitution, deletion, insertion) are shown. White text in boxes indicates amino acid substitutions by sublineage; Δ, deletion; ins, insertion; NTD, N-terminal domain; RBD, receptor binding domain. Immunization protocol for studies with VOC boosters. BALB/c mice were immunized with two doses (1 ug each) of the original BNT162b2 vaccine, followed by at least one dose (1 ug total) of a monovalent, bivalent, or trivalent booster dose of either: (a) original BNT162b2 ("BNT162b2"), (b) BNT162b2 OMI BA.1 ("OMI BA.1"), (c) BNT162b2 OMI BA.4/5 ("OMI BA.4/5"), or combinations thereof, according to the indicated schedule. Baseline Grouped Neutralization GMT. Sera collected from mice immunized as shown in Figure 33 (day 104, pre-boost) were evaluated for geometric mean titers of neutralizing antibodies against the various strains. Data are presented grouped by cohort. Baseline Staggered Neutralization GMT. Sera collected from mice immunized as shown in Figure 33 (day 104, pre-boost) were assessed for geometric mean titers of neutralizing antibodies against the various strains. Data are presented in a staggered format (i.e., for each strain for which neutralization was assessed). Baseline cross-neutralization. Sera collected from mice immunized as shown in Figure 33 (day 104, pre-boost) were assessed for geometric mean titers of neutralizing antibodies against the various strains. Cross-neutralization results are presented as calculated variant/Wuhan reference GMT ratios. Geometric mean fold increase in GMT after boost. Sera collected from mice immunized as shown in Figure 33 (day 111, 7 days after boost) were evaluated for the geometric mean fold increase in GMT of neutralizing antibodies against various strains. Grouped Neutralization GMTs Post-Boost. Sera collected from mice immunized as shown in Figure 33 (day 111, 7 days post-boost) were evaluated for the geometric mean fold increase in GMTs of neutralizing antibodies against the various strains. Data are presented grouped by cohort. Cross-neutralization after boost. Sera collected from mice immunized as shown in Figure 33 (day 111, 7 days post-boost) were evaluated for the geometric mean fold increase in GMT of neutralizing antibodies against various strains. Cross-neutralization results are presented as calculated variant/Wuhan reference GMT ratios. Exemplary spike protein amino acid mutations. Modified amino acid residues are shown and used to generate RNA vaccines encoding variant coronavirus spike proteins. In some instances, such amino acid modifications can be combined with other amino acid residue modifications, as shown under "Mutation" and "Mutation Type" in FIG. 41. Amino acid positions are numbered relative to the S protein sequence from the Wuhan sequence (SEQ ID NO: 1). In some embodiments, various combinations of amino acid mutations as described herein can be applied to different coronavirus S proteins or immunogenic fragments thereof. Exemplary spike protein variants. Exemplary combinations of spike protein mutations are shown, including modified amino acid residues, types of mutations, and furin mutations (682/683/684/685 RRAR to GSAS). RNA constructs encoding exemplary combinations of spike protein mutations were evaluated for S protein expression, CR3022 epitope response, and ACE2 response. Amino acid positions are numbered relative to the S protein sequence from the Wuhan sequence (SEQ ID NO:1). In some embodiments, various combinations of amino acid mutations as described herein can be applied to different coronavirus S proteins or immunogenic fragments thereof. Effect of RNA encoding exemplary spike protein variants on neutralization against various coronavirus strains and/or variants. RNA encoding exemplary spike protein variants (e.g., containing a P6' backbone as shown in Figure 40, D614G, and furin site mutations (682/683/684/685 RRAR to GSAS)) stimulated higher neutralization titers across a range of VOCs. BNT162b5 format bivalent (Wuhan + BA.4/5) is more immunogenic than BNT162b2 format bivalent (Wuhan + BA.4/5). Mice were administered two doses of BNT162b2 21 days apart, followed by vaccination with either (i) BNT162b2, (ii) a bivalent vaccine comprising a first RNA encoding the Wuhan spike protein and a second RNA encoding a SARS-CoV-2 spike protein comprising a mutation characteristic of the BA.4/5 omicron variant, wherein the spike protein encoded by each of the first and second RNAs also comprises the mutations K986P and V987P ("BNT162b2 bivalent (BA.4/5)"); or (iii) a first RNA encoding the Wuhan spike protein and a second RNA encoding a SARS-CoV-2 spike protein comprising a mutation characteristic of the BA.4/5 omicron variant, wherein the spike protein encoded by each of the first and second RNAs also comprises the mutations K986P and V987P ("BNT162b2 bivalent (BA.4/5)"). and a second RNA encoding a SARS-CoV-2 spike protein containing mutations characteristic of the Wuhan and Omicron variants BA.1, BA.2, BA.2.12.1, and BA.4/5, where the S protein encoded by each of the first and second RNAs also contains the P6' mutations (D985P, V987P, F817P, A892P, A899P, and A942P), D614G, and a furin cleavage site mutation (682/683/684/685 RRAR vs. GSAS). Serum samples were taken one month after the third dose and were neutralized using a pseudovirus neutralization assay (50% pVNT titer) to detect the presence of SARS-CoV-2 variants BA.1, BA.2, BA.2.12.1, and BA.4/5. Neutralization titers were determined for 4/5. Bivalent BNT162b5 provides improved immune responses in vaccine-experienced human subjects. Human subjects who previously received three doses of BNT162b2 (encoding the Wuhan strain of SARS-CoV-2 S protein and including the K986P and V987P mutations) were administered a bivalent vaccine comprising: (i) a first RNA encoding the Wuhan spike protein and a second RNA encoding a SARS-CoV-2 spike protein including mutations characteristic of the BA.1 omicron variant, where the spike protein encoded by each of the first and second RNAs also includes the K986P and V987P mutations ("BNT162b2 bivalent Omi BA.1"); or (ii) a bivalent vaccine comprising a first RNA encoding the Wuhan spike protein and a second RNA encoding a SARS-CoV-2 spike protein including mutations characteristic of the BA.1 omicron variant, where the spike protein encoded by each of the first and second RNAs also includes the K986P and V987P mutations ("BNT162b2 bivalent Omi BA.1"). and a second RNA encoding a SARS-CoV-2 spike protein containing mutations characteristic of the omicron BA.2 variant, where the S protein encoded by each of the first and second RNAs also contains the P6' mutations (D985P, V987P, F817P, A892P, A899P, and A942P), D614G, and a mutation at the furin cleavage site (682/683/684/685 RRAR to GSAS) ("BNT162b5 Bivalent (BA.2)"). Serum was collected one month after administration of the RNA vaccine, and neutralization titers were collected for the Wuhan ("WT"), omicron BA.1 ("BA.1"), or omicron BA.2 ("BA.2") SARS-CoV-2 variants. Titers are shown for (A) all subjects, (B) subjects who showed evidence of prior SARS-CoV-2 infection at the time of administration of the SARS-CoV-2 vaccine, and (C) subjects who showed no evidence of prior SARS-CoV-2 infection at the time of administration of the SARS-CoV-2 vaccine. Titer values are shown above each bar. Titers were collected using a fluorescent focus reduction neutralization titer (FFRNT) assay. "1MPD4" refers to 1 month after dose 4. "WT" refers to the Wuhan strain. "LLOQ" stands for lower limit of quantification. Ibid. Ibid. Bivalent BNT162b5 and BNT162b6 provide improved immune responses when administered as a booster to vaccine-experienced mice. Mice administered two doses of BNT162b2 (encoding the Wuhan strain of SARS-CoV-2 S protein and including the K986P and V987P mutations) were administered (i) a bivalent vaccine comprising a first RNA encoding the Wuhan spike protein and a second RNA encoding a SARS-CoV-2 spike protein including mutations characteristic of the BA.4/5 omicron variant, where the spike protein encoded by each of the first and second RNAs also includes the K986P and V987P mutations ("BNT162b2 bivalent BA.4/5"); (ii) a bivalent vaccine comprising a first RNA encoding the Wuhan spike protein and a second RNA encoding a SARS-CoV-2 spike protein including mutations characteristic of the BA.4/5 omicron variant, where the spike protein encoded by each of the first and second RNAs also includes the K986P and V987P mutations ("BNT162b2 bivalent BA.4/5"); and a second RNA encoding a SARS-CoV-2 spike protein comprising mutations characteristic of the BA.4/5 omicron variant, wherein the S protein encoded by each of the first and second RNAs also comprises the P6' mutations (D985P, V987P, F817P, A892P, A899P, and A942P), D614G, and a mutation at the furin cleavage site (682/683/684/685 RRAR to GSAS) ("BNT162b5 Bivalent (BA.4/5)"); or (iii) a first RNA encoding a Wuhan spike protein and a second RNA encoding a SARS-CoV-2 spike protein comprising mutations characteristic of the BA.4/5 omicron variant, wherein the S protein encoded by each of the first and second RNAs also comprises the P6' mutations (D985P, V987P, F817P, A892P, A899P, and A942P), D614G, and a mutation at the furin cleavage site (682/683/684/685 RRAR to GSAS) ("BNT162b5 Bivalent (BA.4/5)"). and a second RNA encoding a SARS-CoV-2 spike protein containing mutations characteristic of the SARS-CoV-2 BA.4/5 omicron variant, where the S protein encoded by each of the first and second RNAs also contains the P6 mutations (K986P, V987P, F817P, A892P, A899P, and A942P), D614G, and mutations at the furin cleavage site (682/683/684/685 RRAR to GSAS), and mutations introducing a disulfide bond (T547C, N978C) ("BNT162b6 Bivalent (BA.4/5)"). Serum was collected one month after administration of the RNA vaccine and neutralization titers were determined for a collection of variants of concern (indicated in the legend). The 50% neutralization titers ("50% pVNT titers") are shown. "LOD" stands for limit of detection. FIGURE 46. In vitro characterization of exemplary SARS-CoV-2 variants. Shown are protein expression, ACE2 binding, and CR3022 binding (neutralizing antibodies) for certain exemplary SARS-CoV-2 variants that contain mutations described herein (mutations present in each SARS-CoV-2 variant listed in Table 34). FIGURE 47. In vitro characterization of exemplary SARS-CoV-2 variants. Shown are protein expression, ACE2 binding, and CR3022 binding (neutralizing antibodies) for certain exemplary SARS-CoV-2 variants that contain mutations described herein (mutations present in each SARS-CoV-2 variant listed in Table 34). Ibid. Ibid. Figure 45. Bivalent BNT162b6 and BNT162b7 provide improved immune responses when administered as a booster to unvaccinated mice. Mice were vaccinated with (i) a bivalent vaccine comprising a first RNA encoding the Wuhan spike protein and a second RNA encoding a SARS-CoV-2 spike protein comprising mutations characteristic of the BA.4/5 omicron variant, where the spike protein encoded by each of the first and second RNAs also comprises the K986P and V987P mutations ("BNT162b2 bivalent BA.4/5"); (ii) a bivalent vaccine comprising a first RNA encoding the Wuhan spike protein and a second RNA encoding a SARS-CoV-2 spike protein comprising mutations characteristic of the BA.4/5 omicron variant, where the spike protein encoded by each of the first and second RNAs also comprises the K986P and V987P mutations ("BNT162b2 bivalent BA.4/5"); (iii) a bivalent vaccine comprising a first RNA encoding a SARS-CoV-2 spike protein comprising mutations characteristic of the BA.4/5 omicron variant, wherein the S protein encoded by each of the first and second RNAs also comprises the P6' mutations (D985P, V987P, F817P, A892P, A899P, and A942P), D614G, and a mutation at the furin cleavage site (682/683/684/685 RRAR to GSAS) ("BNT162b5 Bivalent (BA.4/5)"); a bivalent vaccine comprising a first RNA encoding a SARS-CoV-2 spike protein comprising mutations characteristic of the SARS-CoV-2 .4/5 omicron variant, wherein the S protein encoded by each of the first and second RNAs also comprises the P6 mutations (K986P, V987P, F817P, A892P, A899P, and A942P), D614G, a furin cleavage site mutation (682/683/684/685 RRAR to GSAS), and mutations to introduce disulfide bonds (T547C, N978C) ("BNT162b6 Bivalent (BA.2)"); or (iv) a first RNA encoding a Wuhan spike protein and a BA. A bivalent vaccine ("BNT162b7 Bivalent (BA.4/5")) was administered comprising a second RNA encoding a SARS-CoV-2 spike protein containing mutations characteristic of the SARS-CoV-2 omicron variant, where the S protein encoded by each of the first and second RNAs also contained the P6 mutations (K986P, V987P, F817P, A892P, A899P, and A942P), D614G, a furin cleavage site mutation (682/683/684/685 RRAR to GSAS), and mutations to introduce a disulfide bond (T547C, S982C). Sera were collected one month after administration of the RNA vaccine and neutralization titers were determined for a collection of variants of concern (indicated in the legend). The 50% neutralization titers ("50% pVNT titers") are shown in (A) and (B), and neutralization titers relative to BNT162b2 titers are shown in (C). Ibid.

Although the present disclosure is described in detail below, it should be understood that the disclosure is not limited to the specific methodology, protocols, and reagents described herein, as these may vary. It should also be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present disclosure, which is limited only by the appended claims. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art.

Preferably, the terms used herein are defined as described in "A multilingual glossary of biotechnical terms: (IUPAC Recommendations)", H. G. W. Leuenberger, B. Nagel, and H. Kolbli, Eds., Helvetica Chimica Acta, CH-4010 Basel, Switzerland, (1995).

The practice of the present disclosure employs, unless otherwise indicated, conventional chemical, biochemical, cell biology, immunological, and recombinant DNA techniques described in the literature of the art (see, e.g., Molecular Cloning: A Laboratory Manual, 2nd Edition, J. Sambrook et al. eds., Cold Spring Harbor Laboratory Press, Cold Spring Harbor 1989).

The following describes elements of the present disclosure. Although these elements are listed with specific embodiments, it should be understood that they may be combined in any manner and in any number to create additional embodiments. The various described examples and embodiments should not be construed to limit the present disclosure to only the embodiments expressly described. This description should be understood to disclose and encompass embodiments combining the explicitly described embodiments with any number of the disclosed elements. Furthermore, any permutation and combination of all described elements should be considered to be disclosed by this description unless the context dictates otherwise.

Several documents are cited throughout the text of this specification. Each document cited herein (including all patents, patent applications, scientific publications, manufacturer's specifications, instructions, etc.), whether supra or infra, is hereby incorporated by reference in its entirety. Nothing herein should be construed as an admission that the present disclosure is not entitled to antedate such disclosure.

DEFINITIONS The following provide definitions that apply to all aspects of this disclosure. The following terms have the following meanings unless otherwise indicated. Any undefined terms have their art-recognized meaning.

The term "about" means approximately or approximately, and in one embodiment, in the context of a numerical value or range described herein, means ±20%, ±10%, ±5%, or ±3% of the recited or claimed numerical value or range.

The terms "a," "an," and "the," and similar referents as used in the context of describing this disclosure (particularly in the context of the claims) should be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context. The recitation of ranges of values herein is merely intended to serve as a shorthand method of individually referring to each individual value falling within the range. Unless otherwise indicated herein, each individual value is incorporated herein as if it were individually recited herein. All methods described herein may be performed in any suitable order unless otherwise indicated herein or clearly contradicted by context. The use of any and all examples or exemplary language (e.g., "etc.") provided herein is intended merely to better illustrate the disclosure and does not limit the scope of the claims. No language in this specification should be construed as indicating any non-claimed element essential to the practice of the disclosure.

Unless otherwise expressly specified, the term "comprises" is used in the context of this document to indicate that in addition to the members of the list introduced by "comprises", further members may optionally be present. However, it is contemplated as a specific embodiment of the present disclosure that the term "comprises" encompasses the possibility that further members are not present, i.e., for the purposes of this embodiment, "comprises" should be understood to have the meaning of "consisting of" or "consisting essentially of".

As used herein, terms such as "reduce," "decrease," "inhibit," or "impair" refer to an overall reduction or ability to cause an overall reduction in levels, preferably of at least 5%, at least 10%, at least 20%, at least 50%, at least 75%, or even more. These terms include complete or essentially complete inhibition, i.e., reduction to zero or reduction to essentially zero.

Terms such as "increase," "enhance," or "exceed" preferably relate to an increase or enhancement of at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 80%, at least 100%, at least 200%, at least 500%, or even more.

According to the present disclosure, the term "peptide" includes oligo- and polypeptides and refers to a substance that contains about 2 or more, about 3 or more, about 4 or more, about 6 or more, about 8 or more, about 10 or more, about 13 or more, about 16 or more, about 20 or more, and up to about 50, about 100, or about 150 consecutive amino acids linked together via peptide bonds. The term "protein" or "polypeptide" refers to large peptides, particularly peptides having at least about 150 amino acids, although the terms "peptide", "protein", and "polypeptide" are generally used synonymously herein.

A "therapeutic protein" when provided to a subject in a therapeutically effective amount has a positive or beneficial effect on a condition or disease state of the subject. In one embodiment, a therapeutic protein has curative or palliative properties and may be administered to improve, alleviate, reduce, reverse, delay onset, or reduce the severity of one or more symptoms of a disease or disorder. A therapeutic protein may have prophylactic properties and may be used to delay the onset of a disease or reduce the severity of such a disease or pathological condition. The term "therapeutic protein" includes whole proteins or peptides and may also refer to therapeutically active fragments thereof. It may also include therapeutically active variants of proteins. Examples of therapeutically active proteins include, but are not limited to, antigens for vaccination and immune stimulants such as cytokines.

"Fragment" refers to an amino acid sequence (peptide or protein) and relates to a portion of the amino acid sequence, i.e. a sequence that represents an amino acid sequence truncated at the N-terminus and/or C-terminus. A C-terminally truncated fragment (N-terminal fragment) is obtainable, for example, by translation of a truncated open reading frame lacking the 3' end of the open reading frame. A N-terminally truncated fragment (C-terminal fragment) is obtainable, for example, by translation of a truncated open reading frame lacking the 5' end of the open reading frame, as long as the truncated open reading frame contains an initiation codon that serves to initiate translation. A fragment of an amino acid sequence comprises, for example, at least 50%, at least 60%, at least 70%, at least 80%, at least 90% of the amino acid residues from the amino acid sequence. A fragment of an amino acid sequence preferably comprises at least 6, in particular at least 8, at least 12, at least 15, at least 20, at least 30, at least 50 or at least 100 consecutive amino acids from the amino acid sequence.

As used herein, "variant" refers to an amino acid sequence that differs from a parent amino acid sequence by at least one amino acid modification. The parent amino acid sequence can be a naturally occurring or wild-type (WT) amino acid sequence, or can be a modified version of the wild-type amino acid sequence. Preferably, the variant amino acid sequence has at least one amino acid modification compared to the parent amino acid sequence, e.g., 1 to about 20 amino acid modifications compared to the parent, preferably 1 to about 10 or 1 to about 5 amino acid modifications.

As used herein, "wild-type" or "WT" or "native" refers to an amino acid sequence found in nature, including allelic variants. A wild-type amino acid sequence, peptide, or protein has an amino acid sequence that has not been intentionally modified.

For the purposes of this disclosure, a "variant" of an amino acid sequence (peptide, protein or polypeptide) includes amino acid insertion variants, amino acid addition variants, amino acid deletion variants, and/or amino acid substitution variants. The term "variant" includes all mutants, splice variants, post-translational modification variants, conformations, isoforms, allelic variants, species variants, and species homologs, particularly those that occur naturally. The term "variant" specifically includes fragments of an amino acid sequence.

Amino acid insertion variants include the insertion of a single or two or more amino acids in a particular amino acid sequence. In the case of amino acid sequence variants with insertions, one or more amino acid residues are inserted at a specific site in the amino acid sequence, although random insertion with appropriate screening of the resulting product is also possible. Amino acid addition variants include amino- and/or carboxy-terminal fusions of one or more amino acids, such as 1, 2, 3, 5, 10, 20, 30, 50, or more amino acids. Amino acid deletion variants are characterized by the removal of one or more amino acids from a sequence, for example, the removal of 1, 2, 3, 5, 10, 20, 30, 50, or more amino acids. The deletion may be at any position in the protein. Amino acid deletion variants, including deletions at the N-terminus and/or C-terminus of a protein, are also referred to as N-terminus and/or C-terminus truncation variants. Amino acid substitution variants are characterized by at least one residue in the sequence being removed and another residue being inserted in its place. Modifications at positions in the amino acid sequence that are not conserved between homologous proteins or peptides and/or replacement of amino acids with other amino acids with similar properties are preferred. Preferably, the amino acid changes in the peptides and protein variants are conservative amino acid changes, i.e., substitutions of similarly charged or uncharged amino acids. Conservative amino acid changes involve the substitution of one of the amino acid families associated with their side chains. Naturally occurring amino acids are generally classified into four families: acidic (aspartate, glutamate), basic (lysine, arginine, histidine), non-polar (alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), and uncharged polar (glycine, asparagine, glutamine, cysteine, serine, threonine, tyrosine) amino acids. Phenylalanine, tryptophan, and tyrosine are sometimes jointly classified as aromatic amino acids. In one embodiment, conservative amino acid substitutions include substitutions within the following groups:
Glycine, Alanine;
Valine, isoleucine, leucine;
Aspartic acid, glutamic acid;
Asparagine, Glutamine;
Serine, Threonine;
Lysine, arginine; and phenylalanine, tyrosine.

Preferably, the degree of similarity, preferably identity, between a given amino acid sequence and an amino acid sequence that is a variant of the given amino acid sequence will be at least about 60%, 70%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%. The degree of similarity or identity is preferably given for an amino acid region that is at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or about 100% of the full length of the reference amino acid sequence. For example, if the reference amino acid sequence consists of 200 amino acids, the degree of similarity or identity is given, in some embodiments, for at least about 20, at least about 40, at least about 60, at least about 80, at least about 100, at least about 120, at least about 140, at least about 160, at least about 180, or about 200 consecutive amino acids. In some embodiments, the degree of similarity or identity is given for the entire length of the reference amino acid sequence. Alignment to determine sequence similarity, preferably sequence identity, can be performed using tools known in the art, preferably using best sequence alignment, for example, Align using standard settings, preferably EMBOSS::Needle, Matrix:Blosum62, Gap Open 10.0, Gap Extend 0.5.

"Sequence similarity" refers to the percentage of amino acids that are identical or represent conservative amino acid substitutions. "Sequence identity" between two amino acid sequences refers to the percentage of amino acids that are identical between the sequences. "Sequence identity" between two nucleic acid sequences refers to the percentage of nucleotides that are identical between the sequences.

"% identical", "% identity" or similar terms are intended to refer in particular to the percentage of nucleotides or amino acids that are identical in optimal alignment between the sequences being compared. The percentage is purely statistical, and the differences between the two sequences may be, but are not necessarily, randomly distributed over the entire length of the sequences being compared. Comparison of two sequences is usually performed by comparing the sequences after optimal alignment over a segment or "comparison window" to identify local regions of corresponding sequences. Optimal alignment for comparison is performed manually or with the aid of the local homology algorithm by Smith and Waterman, 1981, Ads App. Math. 2, 482, by Neddleman and Wunsch, 1970, J. Mol. Biol. 48, 443, by Pearson and Lipman, 1988, Proc. Natl Acad. Sci. USA 88, 2444, or with the aid of computer programs using said algorithms (GAP, BESTFIT, FASTA, BLAST P, BLAST N, and TFASTA in the Wisconsin Genetics software package (Genetics Computer Group, 575 Science Drive, Madison, Wis.)). In some embodiments, the percent identity of two sequences is determined using the BLASTN or BLASTP algorithms available at the National Center for Biotechnology Information (NCBI) website (e.g., blast.ncbi.nlm.nih.gov/Blast.cgi?PAGE_TYPE=BlastSearch&BLAST_SPEC=blast2seq&LINK_LOC=align2seq). In some embodiments, the algorithm parameters used for the BLASTN algorithm on the NCBI website include: (i) expectation threshold set to 10, (ii) word size set to 28, (iii) maximum match of query range set to 0, (iv) match/mismatch score set to 1, -2, (v) gap cost set to linear, and (vi) a filter for low complexity regions is used. In some embodiments, the algorithm parameters used for the BLASTP algorithm on the NCBI website include: (i) expectation threshold set to 10, (ii) word size set to 3, (iii) maximum match of query range set to 0, (iv) matrix set to BLOSUM62, (v) gap costs set to Existence: 11 Extension: 1, and (vi) conditional composition score matrix adjustment.

Percentage identity is obtained by determining the number of identical positions that the compared sequences correspond to, dividing this number by the number of positions being compared (e.g., the number of positions in the reference sequence), and multiplying this result by 100.

In some embodiments, the degree of similarity or identity is given for a region that is at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or about 100% of the entire length of the reference sequence. For example, if the reference nucleic acid sequence consists of 200 nucleotides, the degree of identity is given for at least about 100, at least about 120, at least about 140, at least about 160, at least about 180, or about 200 contiguous nucleotides in some embodiments. In some embodiments, the degree of similarity or identity is given for the entire length of the reference sequence.

Homologous amino acid sequences according to the present disclosure exhibit identity of at least 40%, particularly at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, and preferably at least 95%, at least 98, or at least 99% of the amino acid residues.

The amino acid sequence variants described herein may be readily prepared by one of skill in the art, for example, by recombinant DNA manipulation. The manipulation of DNA sequences to prepare peptides or proteins having substitutions, additions, insertions, or deletions is described in detail, for example, in Sambrook et al. (1989). Furthermore, the peptides and amino acid variants described herein may be readily prepared with the aid of known peptide synthesis techniques, such as, for example, solid phase synthesis and similar methods.

In one embodiment, the fragment or variant of an amino acid sequence (peptide or protein) is preferably a "functional fragment" or "functional variant". The term "functional fragment" or "functional variant" of an amino acid sequence relates to any fragment or variant that exhibits one or more functional properties identical or similar to those of the amino acid sequence from which it is derived, i.e. functionally equivalent. With respect to an antigen or antigen sequence, one particular function is one or more immunogenic activities exhibited by the amino acid sequence from which the fragment or variant is derived. The term "functional fragment" or "functional variant" as used herein refers in particular to a variant molecule or sequence that includes an amino acid sequence that is modified by one or more amino acids compared to the amino acid sequence of the parent molecule or sequence and is still capable of performing one or more functions of the parent molecule or sequence, e.g., the function of eliciting an immune response. In one embodiment, the modifications in the amino acid sequence of the parent molecule or sequence do not significantly affect or alter the characteristics of the molecule or sequence. In different embodiments, the function of the functional fragment or variant may be reduced but still significantly present, for example, the immunogenicity of the functional variant may be at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% of the parent molecule or sequence. However, in other embodiments, the immunogenicity of the functional fragment or variant may be enhanced compared to the parent molecule or sequence.

An amino acid sequence (peptide, protein, or polypeptide) "derived from" a specified amino acid sequence (peptide, protein, or polypeptide) refers to the origin of the first amino acid sequence. Preferably, an amino acid sequence derived from a particular amino acid sequence has an amino acid sequence that is identical, essentially identical, or homologous to the particular sequence or a fragment thereof. An amino acid sequence derived from a particular amino acid sequence may be a variant of the particular sequence or a fragment thereof. For example, it will be understood by those skilled in the art that antigens suitable for use herein may be modified such that the sequence varies from the naturally occurring sequence or the native sequence from which they are derived while retaining the desired activity of the native sequence.

As used herein, "educational material" or "instructions" includes publications, recordings, drawings, or any other medium of expression that can be used to communicate the usefulness of the disclosed compositions and methods. The educational material of the kits of the present disclosure can be, for example, affixed to a container containing the disclosed compositions or shipped together with a container containing the composition. Alternatively, the educational material may be shipped separately from the container with the intention that the educational material and the composition will be used cooperatively by the recipient.

"Isolated" means altered or removed from the natural state. For example, a nucleic acid or peptide that is naturally present in a living animal is not "isolated," but the same nucleic acid or peptide that has been partially or completely separated from the coexisting materials of its natural state is "isolated." An isolated nucleic acid or protein can exist in a substantially purified form, or can exist in a non-native environment, such as, for example, a host cell.

The term "recombinant" in the context of this disclosure means "made by genetic engineering." Preferably, a "recombinant entity," such as a recombinant nucleic acid, in the context of this disclosure does not occur in nature.

As used herein, the term "naturally occurring" refers to the fact that an object can be found in nature. For example, a peptide or nucleic acid that is present in an organism (including viruses), can be isolated from a natural source, and has not been intentionally modified by man in a laboratory is naturally occurring.

"Physiological pH" as used herein refers to a pH of about 7.5.

The term "genetic modification" or simply "modification" includes the transfection of cells with a nucleic acid. The term "transfection" relates to the introduction of a nucleic acid, particularly RNA, into a cell. For the purposes of this disclosure, the term "transfection" also includes the introduction of a nucleic acid into a cell, or the uptake of a nucleic acid by such a cell, which may be present in a subject, e.g., a patient. Thus, according to the present disclosure, cells for transfection of a nucleic acid as described herein can be present in vitro or in vivo, e.g., the cell can form part of an organ, tissue, and/or organism of a patient. According to the present disclosure, transfection can be transient or stable. In some applications of transfection, it is sufficient that the transfected genetic material is expressed transiently. RNA can be transfected into a cell to transiently express its encoded protein. Since the nucleic acid introduced in the transfection process is not usually integrated into the nuclear genome, the foreign nucleic acid is diluted or degraded through mitosis. Cells that allow episomal amplification of the nucleic acid greatly reduce the dilution rate. If it is desired that the transfected nucleic acid actually remains in the genome of the cell and its daughter cells, stable transfection must be performed. Such stable transfection can be achieved by using a virus-based or transposon-based system for transfection. In general, the nucleic acid encoding the antigen is transiently transfected into the cell. RNA can be transfected into the cell to transiently express its encoded protein.

The term "seroconversion" includes a 4-fold or greater increase from pre-vaccination to 1 month after dose 2.

Coronaviruses are enveloped, positive-sense, single-stranded RNA ((+)ssRNA) viruses. They have the largest genomes (26-32 kb) of any known RNA virus and are phylogenetically divided into four genera (α, β, γ, and δ), with the betacoronaviruses further subdivided into four lineages (A, B, C, and D). Coronaviruses infect a wide range of avian and mammalian species, including humans. Some human coronaviruses generally cause mild respiratory illness, although more severe disease may occur in infants, the elderly, and immunocompromised patients. Middle East Respiratory Syndrome Coronavirus (MERS-CoV) and Severe Acute Respiratory Syndrome Coronavirus (SARS-CoV), which belong to the betacoronavirus lineages C and B, respectively, are highly pathogenic. Both viruses emerged from animal reservoirs into human populations within the past 15 years and caused outbreaks with high case fatality rates. The outbreak of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), which causes atypical pneumonia (coronavirus disease 2019, COVID-19), has been raging in China since mid-December 2019 and has developed into a public health emergency of international concern. SARS-CoV-2 (MN908947.3) belongs to the betacoronavirus lineage B. It shares at least 70% sequence similarity with SARS-CoV.

In general, coronaviruses have four structural proteins: envelope (E), membrane (M), nucleocapsid (N), and spike (S). E and M proteins have important functions in virus assembly, while N protein is required for viral RNA synthesis. An important glycoprotein, S, is involved in virus binding and entry into target cells. S protein is synthesized as a single-stranded inactive precursor that is cleaved into two non-covalently associated subunits, S1 and S2, by furin-like host proteases in the producing cells. The S1 subunit contains a receptor-binding domain (RBD) that recognizes the host cell receptor. The S2 subunit contains a fusion peptide, two heptad repeats, and a transmembrane domain, all of which are required to mediate fusion of the viral and host cell membranes by undergoing extensive conformational rearrangements. The S1 and S2 subunits trimerize to form a large pre-fusion spike.

The S precursor protein of SARS-CoV-2 can be proteolytically cleaved into S1 (685 aa) and S2 (588 aa) subunits. The S1 subunit contains the receptor binding domain (RBD) that mediates viral entry into susceptible cells via the host angiotensin-converting enzyme 2 (ACE2) receptor.

Antigens Many embodiments of the present disclosure include the use of RNA encoding an amino acid sequence comprising the SARS-CoV-2 S protein, an immunogenic variant thereof, or an immunogenic fragment of the SARS-CoV-2 S protein or an immunogenic variant thereof. Thus, the RNA encodes a peptide or protein comprising at least an epitope SARS-CoV-2 S protein or an immunogenic variant thereof to induce an immune response in a subject against the coronavirus S protein, particularly the SARS-CoV-2 S protein. Amino acid sequences comprising the SARS-CoV-2 S protein, an immunogenic variant thereof, or an immunogenic fragment of the SARS-CoV-2 S protein or an immunogenic variant thereof (i.e., antigenic peptides or proteins) are also designated herein as "vaccine antigens,""peptide and protein antigens,""antigenicmolecules," or simply "antigens." The SARS-CoV-2 S protein, an immunogenic variant thereof, or an immunogenic fragment of the SARS-CoV-2 S protein or an immunogenic variant thereof, is also designated herein as an "antigenic peptide or protein" or an "antigenic sequence."

The SARS-CoV-2 coronavirus full-length spike (S) protein from the first detected SARS-CoV-2 strain (referred to herein as the Wuhan strain) consists of 1273 amino acids and has the amino acid sequence according to SEQ ID NO:1:
MFVFLVLLPLVSSQCVNLTTRTQLPPAYTNSFTRGVYYPDKVFRSSVLHSTQDLFLPFFSNVTWFHAIHVSGTNGTKRFD NPVLPFNDGVYFASTEKSNIIRGWIFGTTLDSKTQSLLIVNNATNVVIKVCEFQFCNDPFLGVYYHKNNKSWMESEFRVY SSANNCTFEYVSQPFLMDLEGKQGNFKNLREFVFKNIDGYFKIYSKHTPINLVRDLPQGFSALEPLVDLPIGINITRFQT LLALHRSYLTPGDSSSSGWTAGAAAYYVGYLQPRTFLLKYNENGTITDAVDCALDPLSETKCTLKSFTVEKGIYQTSNFRV QPTESIVRFPNITNLCPFGEVFNATRFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGVSPTKLNDLCFTNVYADSF VIRGDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPC NGVEGFNCYFPLQSYGFQPTNGVGYQPYRVVVVLSFELLHAPATVCGPKKSTNLVKNKCVNFNFNGLTGTGVLTESNKKFL PFQQFGRDIADTTDAVRDPQTLEILDITPCSFGGVSVITPGTNTSNQVAVLYQDVNCTEVPVAIHADQLTPTWRVYSTGS NVFQTRAGCLIGAEHVNNSYECDIPIGAGICASYQTQTNSPRRARSVASQSIIAYTMSLGAENSVAYSNNSIAIPTNFTI SVTTEILPVSMTKTSVDCTMYICGDSTECSNLLLQYGSFCTQLNRALTGIAVEQDKNTQEVFAQVKQIYKTPPIKDFGGF NFSQILPDPSKPSKRSFIEDLLFNKVTLADAGFIKQYGDCLGDIAARDLICAQKFNGLTVLPPLLTDEMIAQYTSALLAG TITSGWTFGAGAALQIPFAMQMAYRFNGIGVTQNVLYENQKLIANQFNSAIGKIQDSLSSTASALGKLQDVVNQNAQALN TLVKQLSSNFGAISSSVLNDILSRLDKVEAEVQIDRLITGRLQSLQTYVTQQLIRAAEIRASANLAATKMSECVLGQSKRV DFCGKGYHLMSFPQSAPHGVVFLHVTYVPAQEKNFTTAPAICHDGKAHFPREGVFVSNGTHWFVTQRNFYEPQIITTDNT FVSGNCDVVIGIVNNTVYDPLQPELDSFKEELDKYFKNHTSPDVDLGDISGINASVVNIQKEIDRLNEVAKNLNESLIDLQELGKYEQYIKWPWYIWLGFIAGLIAIVMVTIMLCCMTSCCSCLKGCCSCGSCCKFDEDDSEPVLKGVKLHYT (SEQ ID NO: 1)

For purposes of this disclosure, the above sequences are considered to be wild-type or Wuhan SARS-CoV-2 S protein amino acid sequences. Unless otherwise indicated, position numbering in the SARS-CoV-2 S protein given herein refers to the amino acid sequence according to SEQ ID NO:1. One of skill in the art reading this disclosure will be able to determine the location of corresponding positions in SARS-CoV-2 S protein variants.

Table 1 below includes additional exemplary S proteins from various coronavirus variants, including alpha, beta, gamma, delta, and omicron variants (including omicron BA.1, BA.2, and BA.4/5). Unless otherwise specified, "omicron variant" as used herein refers to any omicron variant, including, for example, the omicron variants described herein and their progeny.

The amino acid sequences were obtained from the UniProt database, accessible via the World Wide Web at uniprot.org, or the GenBank database, accessible via the World Wide Web at ncbi.nlm.nih.gov, and the UniProt or GenBank database accession numbers for each spike protein sequence are included in Table 1. These amino acid sequences correspond to the amino acid sequences of naturally occurring coronavirus spike proteins. In some aspects, the amino acid sequence of a naturally occurring coronavirus spike protein encoded by an RNA construct described herein can be modified to produce an immunogenic polypeptide comprising a variant coronavirus spike protein that is a modification of a naturally occurring coronavirus spike protein or a fragment thereof, as described herein. For example, in some aspects, the amino acid sequence of a naturally occurring coronavirus spike protein encoded by an RNA construct described herein is substituted to produce an immunogenic polypeptide comprising a variant coronavirus spike protein that is a modification of a naturally occurring coronavirus spike protein or a fragment thereof, as described herein.

Similar to the amino acid sequences of native coronavirus spike proteins, the amino acid sequences of the spike proteins of these SARS-CoV-2 variants (including, for example, alpha, beta, gamma, delta, and omicron variants (including omicron BA.1, BA.2, BA.4/5)) encoded by the RNA constructs described herein can be modified at corresponding positions, as described herein, to produce immunogenic polypeptides that include variant coronavirus spike proteins that are modifications of native variant coronavirus spike proteins or fragments thereof. For example, in some aspects, the amino acid sequences of the spike proteins of these SARS-CoV-2 variants encoded by the RNA constructs described herein are substituted to produce immunogenic polypeptides that include variant coronavirus spike proteins that are modifications of variant coronavirus spike proteins or fragments thereof, as described herein. Additional variants not specifically described below are also contemplated. For example, any variant coronavirus spike protein having 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or greater sequence identity to a native coronavirus spike protein sequence encoded by an RNA construct described herein can be modified (e.g., substituted) at the corresponding positions described herein to produce an immunogenic polypeptide comprising a variant coronavirus spike protein that is a modification of the native coronavirus spike protein or a fragment thereof.

In some embodiments, the coronavirus spike protein sequence comprises SEQ ID NO:105, shown below, which is a SARS-CoV-2 (Omicron BA.4/5) sequence represented by SEQ ID NO:104 (see Table 1), but differs by one amino acid at position 403 and contains a R403S mutation.
MFVFLVLLPLVSSQCVNLITRTQSYTNSFTRGVYYPDKVFRSSVLHSTQDLFLPFFSNVTWFHAISGTNGTKRFDNPVL PFNDGVYFASTEKSNIIRGWIFGTTLDSKTQSLLIVNNATNVVIKVCEFQFCNDPFLDVYYHKNNKSWMESEFRVYSSAN NCTFEYVSQPFLMDLEGKQGNFKNLREFVFKNIDGYFKIYSKHTPINLGRDLPQGFSALEPLVDLPIGINITRFQTLLAL HRSYLTPGDSSSSGWTAGAAAYYVGYLQPRTFLLKYNENGTITDAVDCALDPLSETKCTLKSFTVEKGIYQTSNFRVQPTE SIVRFPNITNLCPFDEVFNATRFASVYAWNRKRISNCVADYSVLYNFAPFFAFKCYGVSPTKLNDLCFTNVYADSFVIR GNEVSQIAPGQTGNIADYNYKLPDDFTGCVIAWNSNKLDSKVGGNYNYRYRLFRKSNLKPFERDISTEIYQAGNKPCNGV AGVNCYFPLQSYGFRPTYGVGHQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKCVNFNFNGLTGTGVLTESNKKFLPFQ QFGRDIADTTDAVRDPQTLEILDITPCSFGGVSVITPGTNTSNQVAVLYQGVNCTEVPVAIHADQLTPTWRVYSTGSNVF QTRAGCLIGAEYVNNSYECDIPIGAGICASYQTQTKSHRRARSVASQSIIAYTMSLGAENSVAYSNNSIAIPTNFTISV TTEILPVSMTKTSVDCTMYICGDSTECSNLLLQYGSFCTQLKRALTGIAVEQDKNTQEVFAQVKQIYKTPPIKYFGGFNF SQILPDPSKPSKRSFIEDLLFNKVTLADAGFIKQYGDCLGDIAARDLICAQKFNGLTTVLPPLLTDEMIAQYTSALLAGTI TSGWTFGAGAALQIPFAMQMAYRFNGIGVTQNVLYENQKLIANQFNSAIGKIQDSLSSTASALGKLQDVVNHNAQALNTL VKQLSSKFGAISSVLNDILSRLDPPEAEVQIDRLITGRLQSLQTYVTQQLIRAAEIRASANLAATKMSECVLGQSKRVDF CGKGYHLMSFPQSAPHGVVFLHVTYVPAQEKNFTTAPAICHDGKAHFPREGVFVSNGTHWFVTQRNFYEPQIITTDNTFV SGNCDVVIGIVNNTVYDPLQPELDSFKEELDKYFKNHTSPDVDLGDISGINASVVNIQKEIDRLNEVAKNLNESLIDLQELGKYEQYIKWPWYIWLGFIAGLIAIVMVTIMLCCMTSCCSCLKGCCSCGSCCKFDEDDSEPVLKGVKLHYT (SEQ ID NO: 105)

Coronavirus Spike Protein Modifications In certain embodiments, a full-length spike (S) protein (e.g., the full-length S protein of SEQ ID NO:1) is modified to stabilize the native prefusion conformation. Certain mutations that stabilize prefusion conformation are known in the art, as disclosed, for example, in WO2022/266010A1, WO2021/243122A2, and Hsieh, Ching-Lin, et al. ("Structure-based design of prefusion-stabilized SARS-CoV-2 spikes," Science 369.6510(2020):1501-1505), the contents of each of which are incorporated herein by reference in their entireties. In some embodiments, the SARS-CoV-2 S protein may be stabilized by introducing one or more glycine mutations (e.g., one or more glycine mutations at the crown of the helical turn region of the S protein, the 12 amino acids between the heptad region 1 (HR1) and the central helix (CH) or heptad region 2 (HR2) regions of the S2 subunit, and/or at one or more of L984, D985, K986, and V987 (positions relative to SEQ ID NO:1). In some embodiments, the spike protein comprises a glycine mutation at each of L984, D985, K986, and V987 (i.e., at positions corresponding to these residues in SEQ ID NO:1). In some embodiments, the SARS-CoV-2 S protein may be stabilized by introducing one or more proline mutations. In some embodiments, the SARS-CoV-2 S protein comprises a proline substitution at residues 986 and/or 987 of SEQ ID NO:1. In some embodiments, the SARS-CoV-2 S protein comprises a proline substitution at one or more of residues 817, 892, 899, and 942 of SEQ ID NO: 1. In some embodiments, the SARS-CoV-2 S protein comprises a proline substitution at each of residues 817, 892, 899, and 942 of SEQ ID NO: 1. In some embodiments, the SARS-CoV-2 S protein comprises a proline substitution at each of residues 817, 892, 899, 942, 986, and 987 of SEQ ID NO: 1.

In some embodiments, the SARS-CoV-2 S protein comprises a proline substitution at residues 985 and/or 987 of SEQ ID NO:1. In some embodiments, the SARS-CoV-2 S protein comprises a proline substitution at each of residues 817, 892, 899, 942, 985, and 987 of SEQ ID NO:1.

In some embodiments, stabilization of the pre-fusion conformation can be obtained by introducing two consecutive proline substitutions at AS residues 986 and 987 in the full-length spike protein. Specifically, a spike (S) protein stabilized protein variant is obtained in such a way that the amino acid residue at position 986 is exchanged to proline and the amino acid residue at position 987 is also exchanged to proline. In one embodiment, the SARS-CoV-2 S protein variant in which the prototypical pre-fusion conformation is stabilized comprises the amino acid sequence shown in SEQ ID NO:7.
MFVFLVLLPLVSSQCVNLTTRTQLPPAYTNSFTRGVYYPDKVFRSSVLHSTQDLFLPFFSNVTWFHAIHVSGTNGTKRF DNPVLPFNDGVYFASTEKSNIIRGWIFGTTLDSKTQSLLIVNNATNVVIKVCEFQFCNDPFLGVYYHKNNKSWMESEFRV YSSANNCTFEYVSQPFLMDLEGKQGNFKNLREFVFKNIDGYFKIYSKHTPINLVRDLPQGFSALEPLVDLPIGINITRF QTLLALHRSYLTPGDSSSGWTAGAAAYYVGYLQPRTFLLKYNENGTITDAVDCALDPLSETKCTLKSFTVEKGIYQTSNF RVQPTESIVRFPNITNLCPFGEVFNATRFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGVSPTKLNDLCFTNVYA DSFVIRGDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGS TPCNGVEGFNCYFPLQSYGFQPTNGVGYQPYRVVVVLSFELLHAPATVCGPKKSTNLVKNKCVNFNFNGLTGTGVLTESN KKFLPFQQFGRDIADTTDAVRDPQTLEILDITPCSFGGVSVITPGTNTSNQVAVLYQDVNCTEVPVAIHADQLTPTWRVY STGSNVFQTRAGCLIGAEHVNNSYECDIPIGAGICASYQTQTNSPRRARSVASQSIIAYTMSLGAENSVAYSNNSIAIP TNFTISVTTEILPVSMTKTSVDCTMYICGDSTECSNLLLQYGSFCTQLNRALTGIAVEQDKNTQEVFAQVKQIYKTPPIK DFGGFNFSQILPDPSKPSKRSFIEDLLFNKVTLADAGFIKQYGDCLGDIAARDLICAQKFNGLTVLPPLLTDEMIAQYT SALLAGTITSGWTFGAGAALQIPFAMQMAYRFNGIGVTQNVLYENQKLIANQFNSAIGKIQDSLSSTASALGKLQDVVNQ NAQALNTLVKQLSSNFGAISSVLNDILSRLDPPEAEVQIDRLITGRLQSLQTYVTQQLIRAAEIRASANLAATKMSECV LGQSKRVDFCGKGYHLMSFPQSAPHGVVFLHVTYVPAQEKNFTTAPAICHDGKAHFPREGVFVSNGTHWFVTQRNFYEPQ IITTDNTFVSGNCDVVIGIVNNTVYDPLQPELDSFKEELDKYFKNHTSPDDVDLGDISGINASVVNIQKEIDRLNEVAKNL NESLIDLQELGKYEQYIKWPWYIWLGFIAGLIAIVMVTIMLCCMTSCCSCLKGCCSCGSCCKFDEDDSEPVLKGVKLHYT
(SEQ ID NO:7)

In some embodiments, the spike protein can be modified to block pre-fusion to post-fusion conformational changes (referred to herein as a "pre- and post-fusion block"). In some embodiments, a pre- and post-fusion block can be introduced by introducing two cysteine mutations at residues close to each other in the folded protein (e.g., at positions close to each other in the pre-fusion conformation of the spike protein). Examples of pre- and post-fusion block mutations include L984C-A989C and I980C-Q992C.

In some embodiments, the spike protein may be modified to reduce "shedding" (i.e., to reduce separation of the S1 and S2 subunits). In some embodiments, the spike protein may be modified to reduce shedding by introducing a mutation at the furin cleavage site (e.g., one or more mutations at residues 682-685 of SEQ ID NO:1) such that the furin protease can no longer bind to and/or cleave the S protein. In some embodiments, the S protein may be modified to reduce shedding by introducing a mutation at each of residues 682, 683, and 685 (e.g., introducing mutations at (i) R682G, R683S, and R685S, or (ii) R682Q, R683Q, and R685Q).

In some embodiments, the S protein can be modified to reduce shedding by introducing cysteine mutations that can form disulfide bonds (e.g., by introducing cysteine mutations at positions close to each other in the folded conformation of the S protein, e.g., at residues A570 and N960).

In some embodiments, one or more modifications may be introduced into the spike protein to stabilize the "up" conformation (referred to herein as an "RBD-up" mutation). Without wishing to be bound by theory, it is believed that the up conformation of the SARS-CoV-2 spike protein increases the exposure of neutralization-sensitive residues. Thus, mutations that stabilize the up conformation may produce a more immunogenic vaccine.

Table 2 below lists various combinations of amino acid modifications that can be introduced into the coronavirus spike protein sequences disclosed above, thus producing polynucleotides (e.g., RNA) that encode immunogenic polypeptides that include coronavirus spike proteins that are variants of native coronavirus spike proteins or fragments thereof. The "+" symbol indicates the inclusion of a particular modification in a particular S protein sequence from a coronavirus strain or variant (e.g., a SARS-CoV-2 strain and/or variant listed in Table 1). In some examples, a spike protein sequence may contain any combination of the modifications in Table 2 below. The amino acid positions shown in Table 2 are numbered relative to SEQ ID NO:1 (Wuhan), SEQ ID NO:69 (Omicron BA.1), SEQ ID NO:70 (Omicron BA.2), and SEQ ID NO:104 (Omicron BA.4/5). Corresponding amino acid positions in spike protein sequences from other coronavirus variants (e.g., alpha, beta, or delta variants) can be determined by alignment with SEQ ID NO:1 (see, e.g., Table 5).

Table 3 below lists various combinations of amino acid modifications that can be introduced into the coronavirus spike protein sequences disclosed above, thus producing polynucleotides (e.g., RNA) that encode immunogenic polypeptides that include coronavirus spike proteins that are variants of native coronavirus spike proteins or fragments thereof. Table 3 lists the positions of amino acid modifications (with respect to the Wuhan spike protein sequence according to SEQ ID NO: 1) similar to Table 2, and Table 3 also includes the specific amino acid residues that are substituted with native amino acid residues. The "+" symbol indicates the inclusion of a specific modification in a particular S protein sequence from a coronavirus strain or variant (e.g., a SARS-CoV-2 strain and/or variant listed in Table 1). In some examples, the coronavirus spike protein variants encoded by the RNA vaccines may include any combination of the modifications in Table 2 above, for example, any of the specific substitutions shown in Table 3. The amino acid positions shown in Table 2 are numbered relative to SEQ ID NO: 1 (Wuhan), SEQ ID NO: 69 (Omicron BA.1), SEQ ID NO: 70 (Omicron BA.2), and SEQ ID NO: 104 (Omicron BA.4/5). Corresponding amino acid positions in spike protein sequences from other coronavirus variants (e.g., alpha, beta, or delta variants) can be determined by alignment with SEQ ID NO:1 (see, e.g., Table 5).

Table 4 below lists various combinations of amino acid modifications that can be introduced into the coronavirus spike protein sequences disclosed herein (see, e.g., Table 1) to produce polynucleotides (e.g., RNA) that encode immunogenic polypeptides that include coronavirus spike proteins that are variants of native coronavirus spike proteins or fragments thereof. The "+" symbol indicates the inclusion of a particular modification in a particular S protein sequence from a coronavirus strain or variant (e.g., a SARS-CoV-2 strain and/or variant listed in Table 1). In some examples, a spike protein sequence may contain any combination of the modifications in Table 4 below. The amino acid positions shown in Table 4 correspond to SEQ ID NO:69 (SARS-CoV-2 Omicron UFO69279.1 (BA.1, formerly B.1.1.529) in Table 1), and corresponding amino acid positions in other coronavirus spike proteins can be determined by sequence alignment (see, e.g., the alignment of various coronavirus spike protein sequences in Table 5).

In some embodiments, the amino acid corresponding to amino acid 326 of SEQ ID NO:69 (SARS-CoV-2 Omicron UFO69279.1 (BA.1, formerly B.1.1.529) may be substituted to produce a variant coronavirus spike protein encoded by an RNA as described herein. In some embodiments, the amino acid corresponding to amino acid 326 of SEQ ID NO:69 may be substituted with a serine residue to produce a variant coronavirus spike protein encoded by an RNA as described herein. The substitution with a serine residue at 326 may be referred to herein as 326S.

In some embodiments, the amino acid corresponding to amino acid 364 of SEQ ID NO:69 (SARS-CoV-2 Omicron UFO69279.1 (BA.1, formerly B.1.1.529) may be substituted to produce a variant coronavirus spike protein encoded by an RNA as described herein. In some embodiments, the amino acid corresponding to amino acid 364 of SEQ ID NO:69 may be substituted with a phenylalanine residue to produce a variant coronavirus spike protein encoded by an RNA as described herein. The substitution with a phenylalanine residue at 364 may be referred to herein as 364F.

In some embodiments, the amino acid corresponding to amino acid position 567 of SEQ ID NO:69 may be substituted to produce a variant coronavirus spike protein encoded by the RNA described herein. In some embodiments, the amino acid corresponding to amino acid position 567 of SEQ ID NO:69 may be substituted with a cysteine residue to produce a variant coronavirus spike protein encoded by the RNA described herein. The substitution with a cysteine residue at 567 may be referred to herein as 567C.

In some embodiments, the amino acid corresponding to amino acid 611 of SEQ ID NO:69 may be substituted to produce a variant coronavirus spike protein encoded by the RNA described herein. In some embodiments, the amino acid corresponding to amino acid 611 of SEQ ID NO:69 may be substituted with a glycine residue to produce a variant coronavirus spike protein encoded by the RNA described herein. The substitution with a glycine residue at 611 may be referred to herein as 611G.

In some embodiments, the amino acid corresponding to amino acid position 814 of SEQ ID NO:69 may be substituted to produce a variant coronavirus spike protein encoded by the RNA described herein. In some embodiments, the amino acid corresponding to amino acid position 814 of SEQ ID NO:69 may be substituted with a phenylalanine residue to produce a variant coronavirus spike protein encoded by the RNA described herein. The substitution with a phenylalanine residue at 814 may be referred to herein as 814P.

In some embodiments, the amino acid corresponding to amino acid 840 of SEQ ID NO:69 may be substituted to produce a variant coronavirus spike protein encoded by the RNA described herein. In some embodiments, the amino acid corresponding to amino acid 840 of SEQ ID NO:69 may be substituted with an asparagine residue to produce a variant coronavirus spike protein encoded by the RNA described herein. The substitution of an asparagine residue at 840 may be referred to herein as 840N.

In some embodiments, the amino acid corresponding to amino acid 851 of SEQ ID NO:69 may be substituted to produce a variant coronavirus spike protein encoded by the RNA described herein. In some embodiments, the amino acid corresponding to amino acid 851 of SEQ ID NO:69 may be substituted with a phenylalanine residue to produce a variant coronavirus spike protein encoded by the RNA described herein. The substitution with a phenylalanine residue at 851 may be referred to herein as 851F.

In some embodiments, the amino acid corresponding to amino acid position 889 of SEQ ID NO:69 may be substituted to produce a variant coronavirus spike protein encoded by the RNA described herein. In some embodiments, the amino acid corresponding to amino acid position 889 of SEQ ID NO:69 may be substituted with a proline residue to produce a variant coronavirus spike protein encoded by the RNA described herein. The substitution of a proline residue at 889 may be referred to herein as 889P.

In some embodiments, the amino acid corresponding to amino acid 896 of SEQ ID NO:69 may be substituted to produce a variant coronavirus spike protein encoded by the RNA described herein. In some embodiments, the amino acid corresponding to amino acid 896 of SEQ ID NO:69 may be substituted with a proline residue to produce a variant coronavirus spike protein encoded by the RNA described herein. The substitution at 896 with a proline residue may be referred to herein as 896P.

In some embodiments, the amino acid corresponding to amino acid position 939 of SEQ ID NO:69 may be substituted to produce a variant coronavirus spike protein encoded by the RNA described herein. In some embodiments, the amino acid corresponding to amino acid position 939 of SEQ ID NO:69 may be substituted with a proline residue to produce a variant coronavirus spike protein encoded by the RNA described herein. The substitution of a proline residue at 939 may be referred to herein as 939P.

In some embodiments, the amino acid corresponding to amino acid 957 of SEQ ID NO:69 may be substituted to produce a variant coronavirus spike protein encoded by the RNA described herein. In some embodiments, the amino acid corresponding to amino acid 957 of SEQ ID NO:69 may be substituted with a cysteine residue to produce a variant coronavirus spike protein encoded by the RNA described herein. The substitution with a cysteine residue at 957 may be referred to herein as 957C.

In some embodiments, the amino acid corresponding to amino acid position 977 of SEQ ID NO:69 may be substituted to produce a variant coronavirus spike protein encoded by the RNA described herein. In some embodiments, the amino acid corresponding to amino acid position 977 of SEQ ID NO:69 may be substituted with a cysteine residue to produce a variant coronavirus spike protein encoded by the RNA described herein. The substitution with a cysteine residue at 977 may be referred to herein as 977C.

In some embodiments, the amino acid corresponding to amino acid 981 of SEQ ID NO:69 may be substituted to produce a variant coronavirus spike protein encoded by the RNA described herein. In some embodiments, the amino acid corresponding to amino acid 981 of SEQ ID NO:69 may be substituted with a cysteine residue to produce a variant coronavirus spike protein encoded by the RNA described herein. The substitution with a cysteine residue at 981 may be referred to herein as 981C.

In some embodiments, the amino acid corresponding to amino acid position 982 of SEQ ID NO:69 may be substituted to produce a variant coronavirus spike protein encoded by the RNA described herein. In some embodiments, the amino acid corresponding to amino acid position 982 of SEQ ID NO:69 may be substituted with a proline residue to produce a variant coronavirus spike protein encoded by the RNA described herein. The substitution with a proline residue at 982 may be referred to herein as 982P.

In some embodiments, the amino acid corresponding to amino acid position 983 of SEQ ID NO:69 may be substituted to produce a variant coronavirus spike protein encoded by the RNA described herein. In some embodiments, the amino acid corresponding to amino acid position 983 of SEQ ID NO:69 may be substituted with a proline residue to produce a variant coronavirus spike protein encoded by the RNA described herein. The substitution with a proline residue at 983 may be referred to herein as 983P.

In some embodiments, the amino acid corresponding to amino acid 984 of SEQ ID NO:69 may be substituted to produce a variant coronavirus spike protein encoded by the RNA described herein. In some embodiments, the amino acid corresponding to amino acid 984 of SEQ ID NO:69 may be substituted with a proline residue to produce a variant coronavirus spike protein encoded by the RNA described herein. The substitution at 983 with a proline residue may be referred to herein as 984P.

In some embodiments, the amino acid corresponding to amino acid position 986 of SEQ ID NO:69 may be substituted to produce a variant coronavirus spike protein encoded by the RNA described herein. In some embodiments, the amino acid corresponding to amino acid position 986 of SEQ ID NO:69 may be substituted with a cysteine residue to produce a variant coronavirus spike protein encoded by the RNA described herein. The substitution with a cysteine residue at 986 may be referred to herein as 986C.

In some embodiments, the amino acid corresponding to amino acid position 989 of SEQ ID NO:69 may be substituted to produce a variant coronavirus spike protein encoded by the RNA described herein. In some embodiments, the amino acid corresponding to amino acid position 989 of SEQ ID NO:69 may be substituted with a cysteine residue to produce a variant coronavirus spike protein encoded by the RNA described herein. The substitution of 989 with a cysteine residue may be referred to herein as 989C.

In some embodiments, the variant spike protein encoded by the RNA described herein is selected from the group consisting of 326, 364, 567, 611, 814, 840, 851, 889, 896, 900, 910, 920, 930, 940, 950, 960, 970, 980, 990, 1000, 1010, 1020, 1030, 1040, 1050, 1060, 1070, 1080, 1090, 1109, 1200, 1210, 1220, 1230, 1240, 1250, 1260, 1270, 1280, 1290, 1300, 1310, 1320, 1330, 1340, 1350, 1360, 1370, 1380, 1390, 1400, 1410, 1420, 1430, 1440, 1450, 1460, 1470, 1480, 1490, 1500, 1510, 1520, 1530, 1540, 1550, 1560, 1570, 1580, 1590, 1600, 1610, 1620, 1630, 1640, 1650, 1660, 1670, 1680, 1690, 1700, 1710, 1720, 1730, 1740 and at least or up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, and/or 18 of the following modifications at position 939, and in some embodiments the modifications at that position or the corresponding position 326 are serine, 364 is phenylalanine, 567 is cysteine, 611 is glycine, 814 is proline, 840 is asparagine, 851 is phenylalanine, and nin, 889 is proline, 896 is proline, 939 is proline, 957 is cysteine, 977 is cysteine, 981 is cysteine, 982 is proline, 983 is proline, 984 is proline, 986 is cysteine, and 989 is cysteine, and in a further embodiment the variant spike protein is SARS-CoV-2 omicron (BA.1, formerly B.1. 1.529) spike protein UniProt Accession No. UFO69279.1 has at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% sequence identity to the spike protein. In some examples, the modifications described herein, alone or in combination with any one or more additional modifications described herein, may produce an RNA (e.g., as described herein) that encodes an immunogenic polypeptide that includes a variant coronavirus spike protein that is a variant of a native coronavirus spike protein or a fragment thereof. In some embodiments, these modifications can (a) increase the adoption by the RBD of the variant coronavirus spike protein of the RBD-up conformation to expose more neutralization-sensitive epitopes on the spike protein, (b) decrease the adoption by the RBD of the variant coronavirus spike protein of the RBD-down conformation, (c) increase the expression of the variant coronavirus spike protein compared to the native coronavirus spike protein, (d) increase the adoption of the prefusion conformation, (e) decrease the shedding of the S1 subunit of the variant coronavirus spike protein, and/or (f) improve the localization of the variant coronavirus spike protein to the host cell membrane.

The mutations described herein and, for example, in Tables 2A, 2B, and 2C may be introduced into the S protein sequence of other coronavirus strains or variant sequences, or immunogenic fragments thereof, and the corresponding positions may be determined by sequence alignment with SEQ ID NO:69 (see, for example, Table 5).

In some embodiments, the variant spike protein encoded by the RNA described herein has at least or at most one of the following modifications at positions 326, 364, 567, 611, 814, 840, 851, 889, 896, 939, 957, 977, 981, 982, 983, 984, 986, 989 as shown in the corresponding amino acids in the SARS-CoV-2 Omicron (BA.1, formerly B.1.1.529) spike protein, UniProt Accession No. UFO69279.1, or another coronavirus spike protein: and/or 18, in some embodiments the modification at that position or the corresponding 326 position is to any amino acid except phenylalanine, 364 is any amino acid except valine, 567 is any amino acid except alanine, 611 is any amino acid except glycine, 814 is any amino acid except phenylalanine, 840 is any amino acid except aspartic acid, and 851 is any amino acid except lysine. , 889 is any amino acid except alanine, 896 is any amino acid except alanine, 939 is any amino acid except alanine, 957 is any amino acid except asparagine, 977 is any amino acid except isoleucine, 981 is any amino acid except leucine, 982 is any amino acid except aspartic acid, 983 is any amino acid except lysine, 984 is any amino acid except valine, 986 is any amino acid except alanine, and 989 is any amino acid except glutamine. and in further embodiments, the variant spike protein has at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% sequence identity to SARS-CoV-2 Omicron (BA.1, formerly B.1.1.529) spike protein, UniProt Accession No. UFO69279.1. In some examples, the modifications described herein, alone or in combination with any one or more additional modifications described herein, may produce an RNA encoding an isolated immunogenic polypeptide that includes a variant coronavirus spike protein that is a variant of a native coronavirus spike protein or a fragment thereof. In some embodiments, these modifications can (a) increase the recruitment by the RBD of the variant coronavirus spike protein of the RBD-up conformation to expose more neutralization-sensitive epitopes on the spike protein, (b) decrease the recruitment by the RBD of the variant coronavirus spike protein of the RBD-down conformation, (c) increase the expression of the variant coronavirus spike protein compared to the native coronavirus spike protein, (d) increase the adoption of the prefusion conformation, (e) decrease the shedding of the S1 subunit of the variant coronavirus spike protein, and/or (f) improve the localization of the variant coronavirus spike protein to the host cell membrane.

The amino acids in each human coronavirus spike protein sequence and their corresponding positions relative to SEQ ID NO:1 can be determined based on an alignment of the protein sequences. Table 5 below is an alignment of the human coronavirus spike protein sequences (e.g., the spike protein sequences in Table 1). The highlighted positions in the alignment below correspond to the positions of the modified amino acids identified in Table 2 above.

Coronavirus Variants Those of skill in the art will be aware of the various spike variants and/or resources documenting them. For example, the following strains, their SARS-CoV-2 S protein amino acid sequences, and in particular their modifications compared to the wild-type SARS-CoV-2 S protein amino acid sequence, e.g., compared to SEQ ID NO: 1, are useful herein:

B.1.1.7 ("Variants of Concern 202012/01" (VOC-202012/01)
B.1.1.7 is a variant of SARS-CoV-2 that was first detected in October 2020 during the COVID-19 pandemic in the UK in samples taken the previous month, and quickly began to spread by mid-December. This correlates with a significant increase in COVID-19 infection rates in the UK, which is thought to be due, at least in part, to an N501Y change in the receptor-binding domain of the spike glycoprotein, which is required for binding to ACE2 on human cells. The B.1.1.7 variant is defined by 23 mutations: 13 nonsynonymous mutations, 4 deletions, and 6 synonymous mutations (i.e., there are 17 mutations that change the protein and 6 that do not). B.1.1.7 Spike protein changes in 1.1.7 include deletion 69-70, deletion 144, N501Y, A570D, D614G, P681H, T716I, S982A, and D1118H.

B. 1.351 (501.V2)
The B. 1.351 lineage and colloquially known as the South African COVID-19 variant is a variant of SARS-CoV-2. Preliminary results indicate that this variant may have increased transmissibility. The B. 1.351 variant is defined by multiple spike protein changes, including L18F, D80A, D215G, deletion 242-244, R246I, K417N, E484K, N501Y, D614G, and A701V. There are three mutations in the spike region of the B. 1.351 genome that are of particular interest: K417N, E484K, and N501Y.

B. 1.1.298 (cluster 5)
B. 1.1.298 was discovered in North Jutland, Denmark, and is believed to have been transmitted from minks to humans via mink farms. Several different mutations in the spike protein of the virus have been identified. Specific mutations include the deletion 69-70, Y453F, D614G, I692V, M1229I, and optionally S1147L.

P. 1 (B.1.1.248)
Lineage B.1.1.248, known as the Brazilian variant, is one of the SARS-CoV-2 variants designated the P.1 lineage. P.1 has several S protein modifications [L18F, T20N, P26S, D138Y, R190S, K417T, E484K, N501Y, D614G, H655Y, T1027I, V1176F] and is similar to the variant B.1.351 from South Africa at certain critical RBD positions (K417, E484, N501).

B. 1.427/B. 1.429 (CAL.20C)
Lineage B.1.427/B.1.429, also known as CAL.20C, is defined by the following modifications in the S-protein: S13I, W152C, L452R, and D614G (of which the L452R modification is of particular concern). The CDC lists B.1.427/B.1.429 as a "variant of concern."

B. 1.525
B.1.525 has the same E484K modification found in the P.1 and B.1.351 variants, and the same ΔH69/ΔV70 deletion found in B.1.1.7 and B.1.1.298. It also has the modifications D614G, Q677H, and F888L.

B. 1.526
B. 1.526 was detected as an emerging lineage of virus isolates in the New York area that shares mutations with previously reported variants. The most common set of spike mutations in this lineage are L5F, T95I, D253G, E484K, D614G, and A701V.

The table below provides an overview of circulating SARS-CoV-2 strains that are VOI/VOC.

B.1.1.529
B.1.529 ("Omicron") was first detected in South Africa in November 2021. Omicron was found to grow approximately 70 times faster than the delta variant and quickly became the dominant strain of SARS-CoV-2 worldwide. Since its initial detection, several sublineages have arisen. The current Omicron variants of concern, along with certain characteristic mutations associated with each S protein (mutation positions shown relative to SEQ ID NO:1), are listed below in Table 3A. In some embodiments, the BA.4 and BA.5 variants have the same S protein amino acid sequence, in which case the term "BA.4/5" may be used to refer to the amino acid sequence of the S protein that can be found in either BA.4 or BA.5. Similarly, in some embodiments, the BA.4.6 and BF.7 variants have the same protein amino acid sequence, in which case the term "BA.4.6/BF.7" may refer to the amino acid sequence of the S protein that can be found in either BA.4.6 or BF. It may be used to refer to the amino acid sequence of the S protein present in any of the seven strains.

In addition to the omicron variants described above, additional variants of BA.5 (e.g., such variants include BF.14) have been observed that contain one or more of the following mutations in the S protein (positions shown relative to SEQ ID NO:1): E340X (e.g., E340K), R346X (e.g., R346T, R346I, or R346S), K444X (e.g., K444N or K444T), V445X, 5 N450D, and S:N460X (e.g., N460K).

In some embodiments, the RNA described herein comprises a nucleotide sequence encoding a SARS-CoV-2 S protein that includes one or more (e.g., including 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more) mutations characteristic of an omicron variant (e.g., one or more mutations of an omicron variant listed in Table 3A). In some embodiments, the RNA comprises a nucleotide sequence encoding a SARS-CoV-2 S protein that includes one or more (e.g., including 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more) mutations listed in Table 3A. In some such embodiments, the one or more mutations may be derived from two or more variants as listed in Table 3A. In some embodiments, the RNA comprises a nucleotide sequence encoding a SARS-CoV-2 S protein that includes each of the mutations identified in Table 3A as being characteristic of a particular Omicron variant (e.g., in some embodiments, the RNA comprises a nucleotide sequence encoding a SARS-CoV-2 S protein that includes each of the mutations listed in Table 3A as being characteristic of Omicron BA.1, BA.2, BA.2.12.1, BA.4/5, BA.2.75, BA.2.75.1, BA.4.6, BQ.1.1, XBB, XBB.1, XBB.2, or XBB.1.3).

In some embodiments, the RNA disclosed herein comprises a nucleotide sequence encoding an immunogenic fragment of a SARS-CoV-2 S protein (e.g., the RBD) and includes one or more mutations characteristic of a SARS-CoV-2 variant (e.g., the Omicron variant described herein). For example, in some embodiments, the RNA comprises a nucleotide sequence encoding the RBD of the S protein of a SARS-CoV-2 variant (e.g., a region of the S protein corresponding to amino acids 327-528 of SEQ ID NO:1, the region including one or more mutations within the region that are characteristic of a variant of concern).

In some embodiments, the RNA encodes a SARS-CoV-2 S protein that includes a subset of the mutations listed in Table 3A. In some embodiments, the RNA encodes a SARS-CoV-2 S protein that includes the mutations listed in Table 3A that are most common in a particular variant (e.g., mutations detected in at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% of sequences collected to date for a given variant sequenced). The prevalence of a mutation can be determined, for example, based on published sequences (e.g., sequences collected by GISAID and made publicly available).

In some embodiments, the RNA described herein encodes a SARS-CoV-2 S protein that includes one or more mutations characteristic of the BA.4/5 variant. The one or more mutations characteristic of the 4/5 variant include T19I, Δ24-26, A27S, ΔO24-26, A27S, Δ69/70, G142D, G142D, V213G, G339D, S371F, S373P, S375F, T376A, D405N, K417N, N440K, L452R, S477N, T478K, E484A, F486V, Q498R, N501Y, Y505H, D614G, H655Y, N679K, P681H, N764K, D796Y, Q954H, and N969K. The SARS-CoV-2 S protein contains one or more mutations characteristic of the BA.4/5 variant, excluding R408S. In some embodiments, the RNA described herein encodes a SARS-CoV-2 S protein containing one or more (e.g., including 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more) mutations characteristic of the BA.4/5 variant, excluding R408S.

In some embodiments, the RNA described herein encodes a SARS-CoV-2 S protein that includes one or more (e.g., including 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more) mutations characteristic of the BA.2.75 variant. The one or more mutations characteristic of the 2.75 variant include T19I, Δ24-26, A27S, G142D, K147E, W152R, F157L, I210V, V213G, G257S, G339H, S371F, S373P, S375F, T376A, D405N, R408S, K417N, N440K, G446S, N460K, S477N, T478K, E484A, F486V, Q498R, N501Y, Y505H D614G, H655Y, N679K, P681H, N764K, D796Y, Q954H, and N969K. In some embodiments, the RNA described herein encodes a SARS-CoV-2 S protein that includes one or more mutations characteristic of the BA.4/BA.5 variant, excluding R408S. In some embodiments, the RNA described herein encodes a SARS-CoV-2 S protein that includes one or more (e.g., including 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more) mutations characteristic of the BA.4/BA.5 variant, excluding R408S and N354D. In some embodiments, the RNA described herein encodes a SARS-CoV-2 S protein that includes one or more (e.g., including 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more) mutations characteristic of the BA.2.75 variant. In some embodiments, the one or more mutations characteristic of a BA.2.75 variant include T19I, Δ24-26, A27S, G142D, K147E, W152R, F157L, I210V, V213G, G257S, G339H, S371F, S373P, S375F, T376A, D405N, R408S, K417N, N440K, G446S, N460K, S477N, T478K, E484A, Q498R, N501Y, Y505H D614G, H655Y, N679K, P681H, N764K, Q954H, and N969K. In some embodiments, the RNA described herein encodes a SARS-CoV-2 S protein that includes one or more (e.g., including 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more) mutations characteristic of the BA.2.75 variant, excluding N354D. In some embodiments, the RNA described herein encodes a SARS-CoV-2 S protein that includes one or more (e.g., including 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more) mutations characteristic of the BA.2.75 variant, excluding D796Y. In some embodiments, the RNA described herein encodes a SARS-CoV-2 S protein that includes one or more (e.g., including 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more) mutations characteristic of the BA.2.75 variant, excluding N354D. 2.75 Encodes a SARS-CoV-2 S protein that contains one or more (e.g., including 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more) mutations characteristic of the variant, excluding D796Y and N354D.

In some embodiments, the RNA described herein encodes a SARS-CoV-2 S protein that includes one or more mutations characteristic of the BA.2.75.2 variant. In some embodiments, the one or more mutations characteristic of the BA.2.75.2 variant include T19I, Δ24-26, A27S, G142D, K147E, W152R, F157L, I210V, V213G, G257S, G339H, R346T, N354D, S371F, S373P, S375F, T376A, D405N , R408S, K417N, N440K, G446S, N460K, S477N, T478K, E484A, F486S, Q498R, N501Y, Y505H, D614G, H655Y, N679K, P681H, N764K, D796Y, Q954H, N969K, and D1199N. In some embodiments, the RNA described herein encodes a SARS-CoV-2 S protein that includes one or more (e.g., including 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more) mutations characteristic of the BA.2.75.2 variant, excluding R346T.

In some embodiments, the RNA described herein encodes a SARS-CoV-2 S protein that includes one or more mutations characteristic of the BA.4.6 or BF.7 variant. In some embodiments, the RNA described herein encodes a SARS-CoV-2 S protein that includes one or more mutations characteristic of the BA.4.6 or BF.7 variant. The one or more mutations characteristic of the 7 variants include T19I, Δ24-26, A27S, Δ69/70, G142D, V213G, G339D, R346T, S371F, S373P, S375F, T376A, D405N, K417N, N440K, L452R, S477N, T478K, E484A, F486V, Q498R, N501Y, Y505H, D614G, H655Y, N679K, P681H, N764K, D796Y, Q954H, and N969K. In some embodiments, the RNA described herein is selected from the group consisting of BA.4.6 or BF. In some embodiments, the RNA described herein encodes a SARS-CoV-2 S protein that includes one or more (including, for example, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more) mutations characteristic of the BA.4.6 or BF.7 variant, excluding R408S. In some embodiments, the RNA described herein encodes a SARS-CoV-2 S protein that includes one or more (including, for example, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more) mutations characteristic of the BA.4.6 or BF.7 variant, excluding N65 ... It encodes a SARS-CoV-2 S protein that contains one or more (e.g., including 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more) mutations characteristic of the seven variants, excluding N658S and R408S.

In some embodiments, the RNA described herein encodes a SARS-CoV-2 S protein that includes one or more mutations characteristic of an omicron XBB variant. In some embodiments, the one or more mutations characteristic of an omicron XBB variant are T19I, Δ24-26, A27S, V83A, G142D, Δ144, H146Q, Q183E, V213E, G339H, R346T, L368I, S371F, S373P, S375F, T376A, D405N , R408S, K417N, N440K, V445P, G446S, N460K, S477N, T478K, E484A, F486S, F490S, Q498R, N501Y, Y505H, D614G, H655Y, N679K, P681H, N764K, D796Y, Q954H, and N969K.

In some embodiments, the RNA described herein encodes a SARS-CoV-2 S protein that includes one or more mutations characteristic of an Omicron XBB.1 variant. In some embodiments, the one or more mutations characteristic of an Omicron XBB.1 variant include G252V. In some embodiments, the one or more mutations characteristic of an Omicron XBB.1 variant include T19I, Δ24-26, A27S, V83A, G142D, Δ144, H146Q, Q183E, V213E, G252V, G339H, R346T, L368I, S371F, S373P, S375F, T376A, D405N, R408S , K417N, N440K, V445P, G446S, N460K, S477N, T478K, E484A, F486S, F490S, Q498R, N501Y, Y505H, D614G, H655Y, N679K, P681H, N764K, D796Y, Q954H, and N969K. In some embodiments, the RNA described herein encodes a SARS-CoV-2 S protein that includes one or more (e.g., including 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more) mutations characteristic of the Omicron XBB.1 variant, excluding Q493R. In some embodiments, the RNA described herein encodes a SARS-CoV-2 S protein that contains one or more (e.g., including 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more) mutations characteristic of the Omicron XBB variant, excluding Q493R and G252V.

In some embodiments, the RNA described herein encodes a SARS-CoV-2 S protein that includes one or more mutations characteristic of an Omicron XBB.2 variant. In some embodiments, the one or more mutations characteristic of an Omicron XBB.2 variant include D253G. In some embodiments, the one or more mutations characteristic of an Omicron XBB.2 variant include T19I, Δ24-26, A27S, V83A, G142D, Δ144, H146Q, Q183E, V213E, D253G, G339H, R346T, L368I, S371F, S373P, S375F, T376A, D405N, R408S, K4 Includes 17N, N440K, V445P, G446S, N460K, S477N, T478K, E484A, F486S, F490S, Q493R, Q498R, N501Y, Y505H, D614G, H655Y, N679K, P681H, N764K, D796Y, Q954H, and N969K.

In some embodiments, the RNA described herein encodes a SARS-CoV-2 S protein that includes one or more mutations characteristic of an Omicron XBB.1.3 variant. In some embodiments, the one or more mutations characteristic of an Omicron XBB.1.3 variant include G252V and A484T. In some embodiments, the one or more mutations characteristic of an Omicron XBB.1.3 variant include T19I, Δ24-26, A27S, V83A, G142D, Δ144, H146Q, Q183E, V213E, G252V, G339H, R346T, L368I, S371F, S373P, S375F, T376A, D405N, R408S, K Includes 417N, N440K, V445P, G446S, N460K, S477N, T478K, A484T, F486S, F490S, Q493R, Q498R, N501Y, Y505H, D614G, H655Y, N679K, P681H, N764K, D796Y, Q954H, and N969K.

In some embodiments, the RNA described herein encodes a SARS-CoV-2 S protein that includes one or more mutations characteristic of the BQ.1.1 variant. The one or more mutations characteristic of the 1.1 variant include T19I, Δ24-26, A27S, Δ69/70, G142D, V213G, G339D, S371F, S373P, S375F, T376A, D405N, R408S, K417N, N440K, K444T, L452R, N463K, S477N, T478K, E484A, F486V, Q498R, N501Y, Y505H, D614G, H655Y, N679K, P681H, N764K, D796Y, Q954H, and N969K. In some embodiments, the RNA described herein is selected from the group consisting of BQ. 1.1 Encodes a SARS-CoV-2 S protein that contains one or more (e.g., including 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more) mutations characteristic of a variant.

In one embodiment, the vaccine antigen described herein comprises, consists essentially of, or consists of the spike protein (S) of SARS-CoV-2, a variant thereof, or a fragment thereof, and includes one or more of the mutations characteristic of SARS-CoV-2 variants (e.g., one or more of the mutations associated with the omicron variant listed in Table 3A).

In one embodiment, the vaccine antigen comprises (a) an amino acid sequence of amino acids 17-1273 of SEQ ID NO: 1 or 7, an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of amino acids 17-1273 of SEQ ID NO: 1 or 7, or an immunogenic fragment of the amino acid sequence of amino acids 17-1273 of SEQ ID NO: 1 or 7, or an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to an immunogenic fragment of the amino acid sequence of amino acids 17-1273 of SEQ ID NO: 1 or 7, and (b) one or more mutations associated with a SARS-CoV-2 variant of concern (e.g., one or more mutations listed in Table 3A). In one embodiment, the vaccine antigen comprises an amino acid sequence from amino acids 17 to 1273 of SEQ ID NO: 1 or 7, including one or more mutations associated with a SARS-CoV-2 variant of concern (e.g., one or more mutations listed in Table 3A).

In one embodiment, the vaccine antigen comprises (a) an amino acid sequence of amino acids 17-1273 of SEQ ID NO:80, an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of amino acids 17-1273 of SEQ ID NO:80, or an immunogenic fragment of the amino acid sequence of amino acids 17-1273 of SEQ ID NO:80, or an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to an immunogenic fragment of the amino acid sequence of amino acids 17-1273 of SEQ ID NO:80, and (b) one or more of the mutations listed in Table 3A. In one embodiment, the vaccine antigen comprises an amino acid sequence of amino acids 17-1273 of SEQ ID NO:80, and includes one or more mutations associated with a SARS-CoV-2 variant of concern (e.g., one or more mutations listed in Table 3A).

In one embodiment, the RNA encoding the vaccine antigen is selected from the group consisting of (i) the nucleotide sequence of nucleotides 49 to 3819 of SEQ ID NO: 2, 8, or 9, a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of nucleotides 49 to 3819 of SEQ ID NO: 2, 8, or 9, or a fragment of the nucleotide sequence of nucleotides 49 to 3819 of SEQ ID NO: 2, 8, or 9, or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to a fragment of the nucleotide sequence of nucleotides 49 to 3819 of SEQ ID NO: 2, 8, or 9, and/or (ii) the nucleotide sequence of SEQ ID NO: 1 or 7. or an immunogenic fragment of an amino acid sequence from amino acid 17 to 1273 of SEQ ID NO: 1 or 7, or an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence from amino acid 17 to 1273 of SEQ ID NO: 1 or 7; and (b) a nucleotide sequence encoding a SARS-CoV-2 S protein that includes one or more mutations associated with a SARS-CoV-2 variant of concern (e.g., one or more mutations listed in Table 3A). In one embodiment, the RNA encoding the vaccine antigen (a) encodes an amino acid sequence that includes (i) a nucleotide sequence from nucleotide 49 to 3819 of SEQ ID NO: 2, 8, or 9, and/or (ii) an amino acid sequence from amino acid 17 to 1273 of SEQ ID NO: 1 or 7, and (b) includes one or more mutations characteristic of SARS-CoV-2 of concern (e.g., one or more mutations listed in Table 3A).

In one embodiment, the RNA encoding the vaccine antigen comprises (i) a nucleotide sequence of nucleotides 49-3819 of SEQ ID NO:81, a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of nucleotides 49-3819 of SEQ ID NO:81, or a fragment of the nucleotide sequence of nucleotides 49-3819 of SEQ ID NO:81, or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to a fragment of the nucleotide sequence of nucleotides 49-3819 of SEQ ID NO:81, and/or (ii) amino acids 17-1 of SEQ ID NO:80. or an immunogenic fragment of the amino acid sequence of amino acids 17-1273 of SEQ ID NO:80, or an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of amino acids 17-1273 of SEQ ID NO:80; and (b) comprising one or more mutations associated with a SARS-CoV-2 variant of concern (e.g., one or more mutations listed in Table 3A). In one embodiment, the RNA encoding the vaccine antigen (a) comprises a nucleotide sequence encoding an amino acid sequence that includes (i) nucleotides 49-3819 of SEQ ID NO: 81 and/or (ii) amino acid sequence 17-1273 of SEQ ID NO: 80 or 7, and (b) includes one or more mutations characteristic of a SARS-CoV-2 variant of concern (e.g., one or more mutations listed in Table 3A).

In one embodiment, the vaccine antigen comprises, consists essentially of, or consists of the SARS-CoV-2 spike S1 fragment (S1) (the S1 subunit of the spike protein (S) of SARS-CoV-2), a variant thereof, or a fragment thereof, including one or more mutations of the SARS-CoV-2 variants described herein.

In one embodiment, the vaccine antigen comprises an amino acid sequence of amino acids 17-683 of SEQ ID NO:1, an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of amino acids 17-683 of SEQ ID NO:1, or an immunogenic fragment of the amino acid sequence of amino acids 17-683 of SEQ ID NO:1, or an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to an immunogenic fragment of the amino acid sequence of amino acids 17-683 of SEQ ID NO:1, and includes one or more mutations characteristic of SARS-CoV-2 variants (e.g., one or more mutations listed in Table 3A). In one embodiment, the vaccine antigen comprises an amino acid sequence of amino acids 17-683 of SEQ ID NO:1, and includes one or more mutations characteristic of SARS-CoV-2 variants (e.g., one or more mutations listed in Table 3A).

In one embodiment, the vaccine antigen comprises an amino acid sequence of amino acids 17-683 of SEQ ID NO:80, an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of amino acids 17-683 of SEQ ID NO:1, an immunogenic fragment of the amino acid sequence of amino acids 17-683 of SEQ ID NO:1, or an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to an immunogenic fragment of the amino acid sequence of amino acids 17-683 of SEQ ID NO:80, and one or more mutations characteristic of SARS-CoV-2 variants (e.g., one or more mutations listed in Table 3A). In one embodiment, the vaccine antigen comprises an amino acid sequence of amino acids 17-683 of SEQ ID NO:80, and one or more mutations characteristic of SARS-CoV-2 variants (e.g., one or more mutations listed in Table 3A).

Vaccine Antigens and Combinations Thereof In one embodiment, the vaccine antigens described herein comprise, consist essentially of, or consist of the spike protein (S) of SARS-CoV-2, a variant thereof, or a fragment thereof.

In one embodiment, the vaccine antigen comprises the amino acid sequence of amino acids 17-1273 of SEQ ID NO: 1 or 7, an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of amino acids 17-1273 of SEQ ID NO: 1 or 7, or an immunogenic fragment of the amino acid sequence of amino acids 17-1273 of SEQ ID NO: 1 or 7, or an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of amino acids 17-1273 of SEQ ID NO: 1 or 7. In one embodiment, the vaccine antigen comprises the amino acid sequence of amino acids 17-1273 of SEQ ID NO: 1 or 7.

In one embodiment, the RNA encoding the vaccine antigen comprises: (i) a nucleotide sequence of nucleotides 49-3819 of SEQ ID NO:2, 8, or 9, a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of nucleotides 49-3819 of SEQ ID NO:2, 8, or 9, or a fragment of the nucleotide sequence of nucleotides 49-3819 of SEQ ID NO:2, 8, or 9, or a fragment of the nucleotide sequence of nucleotides 49-3819 of SEQ ID NO:2, 8, or 9, or a fragment of the nucleotide sequence of nucleotides 49-3819 of SEQ ID NO:2, 8, or 9, and/or (ii) encodes an amino acid sequence comprising the amino acid sequence of amino acids 17-1273 of SEQ ID NO: 1 or 7, an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of amino acids 17-1273 of SEQ ID NO: 1 or 7, or an immunogenic fragment of the amino acid sequence of amino acids 17-1273 of SEQ ID NO: 1 or 7, or an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of amino acids 17-1273 of SEQ ID NO: 1 or 7. In one embodiment, the RNA encoding the vaccine antigen (i) comprises the nucleotide sequence of nucleotides 49-3819 of SEQ ID NO: 2, 8, or 9, and/or (ii) encodes an amino acid sequence comprising the amino acid sequence of amino acids 17-1273 of SEQ ID NO: 1 or 7.

In one embodiment, the vaccine antigen comprises, consists essentially of, or consists of the SARS-CoV-2 spike S1 fragment (S1) (the S1 subunit of the spike protein (S) of SARS-CoV-2), a variant thereof, or a fragment thereof.

In one embodiment, the vaccine antigen comprises the amino acid sequence of amino acids 17-683 of SEQ ID NO:1, an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of amino acids 17-683 of SEQ ID NO:1, or an immunogenic fragment of the amino acid sequence of amino acids 17-683 of SEQ ID NO:1, or an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of amino acids 17-683 of SEQ ID NO:1. In one embodiment, the vaccine antigen comprises the amino acid sequence of amino acids 17-683 of SEQ ID NO:1.

In one embodiment, the RNA encoding the vaccine antigen is (i) a nucleotide sequence of nucleotides 49-2049 of SEQ ID NO:2, 8, or 9, a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of nucleotides 49-2049 of SEQ ID NO:2, 8, or 9, or a fragment of the nucleotide sequence of nucleotides 49-2049 of SEQ ID NO:2, 8, or 9, or a fragment of the nucleotide sequence of nucleotides 49-2049 of SEQ ID NO:2, 8, or 9, or a fragment of the nucleotide sequence of nucleotides 49-2049 of SEQ ID NO:2, 8, or 9, %, or 80% identity to the amino acid sequence of amino acids 17-683 of SEQ ID NO:1, and/or (ii) encodes an amino acid sequence comprising the amino acid sequence of amino acids 17-683 of SEQ ID NO:1, an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of amino acids 17-683 of SEQ ID NO:1, or an immunogenic fragment of the amino acid sequence of amino acids 17-683 of SEQ ID NO:1, or an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of amino acids 17-683 of SEQ ID NO:1. In one embodiment, the RNA encoding the vaccine antigen (i) comprises the nucleotide sequence of nucleotides 49-2049 of SEQ ID NO:2, 8, or 9, and/or (ii) encodes an amino acid sequence comprising the amino acid sequence of amino acids 17-683 of SEQ ID NO:1.

In one embodiment, the vaccine antigen comprises the amino acid sequence of amino acids 17-685 of SEQ ID NO:1, an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of amino acids 17-685 of SEQ ID NO:1, or an immunogenic fragment of the amino acid sequence of amino acids 17-685 of SEQ ID NO:1, or an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of amino acids 17-685 of SEQ ID NO:1. In one embodiment, the vaccine antigen comprises the amino acid sequence of amino acids 17-685 of SEQ ID NO:1.

In one embodiment, the RNA encoding the vaccine antigen is (i) a nucleotide sequence of nucleotides 49-2055 of SEQ ID NO:2, 8, or 9, a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of nucleotides 49-2055 of SEQ ID NO:2, 8, or 9, or a fragment of the nucleotide sequence of nucleotides 49-2055 of SEQ ID NO:2, 8, or 9, or a fragment of the nucleotide sequence of nucleotides 49-2055 of SEQ ID NO:2, 8, or 9, or a fragment of the nucleotide sequence of nucleotides 49-2055 of SEQ ID NO:2, 8, or 9, %, or 80% identity to the amino acid sequence of amino acids 17-685 of SEQ ID NO:1, and/or (ii) encodes an amino acid sequence comprising the amino acid sequence of amino acids 17-685 of SEQ ID NO:1, an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of amino acids 17-685 of SEQ ID NO:1, or an immunogenic fragment of the amino acid sequence of amino acids 17-685 of SEQ ID NO:1, or an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of amino acids 17-685 of SEQ ID NO:1. In one embodiment, the RNA encoding the vaccine antigen (i) comprises the nucleotide sequence of nucleotides 49-2055 of SEQ ID NO:2, 8, or 9, and/or (ii) encodes an amino acid sequence comprising the amino acid sequence of amino acids 17-685 of SEQ ID NO:1.

In one embodiment, the vaccine antigen comprises, consists essentially of, or consists of the receptor binding domain (RBD) of the S1 subunit of the spike protein (S), variants thereof, or fragments thereof, of SARS-CoV-2. The amino acid sequence of amino acids 327-528 of SEQ ID NO:1, variants thereof, or fragments thereof, is also referred to herein as the "RBD" or "RBD domain."

In one embodiment, the vaccine antigen comprises the amino acid sequence of amino acids 327-528 of SEQ ID NO:1, an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of amino acids 327-528 of SEQ ID NO:1, or an immunogenic fragment of the amino acid sequence of amino acids 327-528 of SEQ ID NO:1, or an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of amino acids 327-528 of SEQ ID NO:1. In one embodiment, the vaccine antigen comprises the amino acid sequence of amino acids 327-528 of SEQ ID NO:1.

In one embodiment, the RNA encoding the vaccine antigen is (i) a nucleotide sequence of nucleotides 979-1584 of SEQ ID NO:2, 8, or 9, a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of nucleotides 979-1584 of SEQ ID NO:2, 8, or 9, or a fragment of the nucleotide sequence of nucleotides 979-1584 of SEQ ID NO:2, 8, or 9, or a fragment of the nucleotide sequence of nucleotides 979-1584 of SEQ ID NO:2, 8, or 9, or a fragment of the nucleotide sequence of nucleotides 979-1584 of SEQ ID NO:2, 8, or 9, %, or 80% identity to the amino acid sequence of amino acids 327-528 of SEQ ID NO:1, and/or (ii) encodes an amino acid sequence comprising the amino acid sequence of amino acids 327-528 of SEQ ID NO:1, an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of amino acids 327-528 of SEQ ID NO:1, or an immunogenic fragment of the amino acid sequence of amino acids 327-528 of SEQ ID NO:1, or an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of amino acids 327-528 of SEQ ID NO:1. In one embodiment, the RNA encoding the vaccine antigen (i) comprises the nucleotide sequence of nucleotides 979-1584 of SEQ ID NO:2, 8, or 9, and/or (ii) encodes an amino acid sequence comprising the amino acid sequence of amino acids 327-528 of SEQ ID NO:1.

According to certain embodiments, the signal peptide is fused directly or via a linker to the SARS-CoV-2 S protein, its variants, or its fragments, i.e., antigenic peptide or protein. Thus, in one embodiment, the signal peptide is fused to the above-mentioned amino acid sequence derived from the SARS-CoV-2 S protein or its immunogenic fragment (antigenic peptide or protein) constituted by the above-mentioned vaccine antigen.

Such signal peptides typically exhibit a length of about 15-30 amino acids and are preferably, but not limited to, sequences located at the N-terminus of an antigenic peptide or protein. A signal peptide as defined herein preferably allows the transport of an antigenic peptide or protein encoded by an RNA to a defined cellular compartment, preferably the cell surface, the endoplasmic reticulum (ER) or an endosomal-lysosomal compartment. In one embodiment, a signal peptide sequence as defined herein includes, but is not limited to, the signal peptide sequence of SARS-CoV-2 S protein, in particular a sequence comprising the amino acid sequence of amino acids 1-16 or 1-19 of SEQ ID NO: 1, or a functional variant thereof.

In one embodiment, the signal sequence comprises the amino acid sequence of amino acids 1-16 of SEQ ID NO:1, an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of amino acids 1-16 of SEQ ID NO:1, or a functional fragment of the amino acid sequence of amino acids 1-16 of SEQ ID NO:1, or an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of amino acids 1-16 of SEQ ID NO:1. In one embodiment, the signal sequence comprises the amino acid sequence of amino acids 1-16 of SEQ ID NO:1.

In one embodiment, (i) the RNA encoding the signal sequence is a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of nucleotides 1 to 48 of SEQ ID NO: 2, 8, or 9, or a fragment of ... or (ii) encodes an amino acid sequence comprising the amino acid sequence of amino acids 1-16 of SEQ ID NO:1, an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of amino acids 1-16 of SEQ ID NO:1, or a functional fragment of the amino acid sequence of amino acids 1-16 of SEQ ID NO:1, or an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of amino acids 1-16 of SEQ ID NO:1. In one embodiment, the RNA encoding the signal sequence (i) comprises the nucleotide sequence of nucleotides 1-48 of SEQ ID NO:2, 8, or 9, and/or (ii) encodes an amino acid sequence comprising the amino acid sequence of amino acids 1-16 of SEQ ID NO:1.

In one embodiment, the signal sequence comprises the amino acid sequence of amino acids 1 to 19 of SEQ ID NO:1, an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of amino acids 1 to 19 of SEQ ID NO:1, or a functional fragment of the amino acid sequence of amino acids 1 to 19 of SEQ ID NO:1, or an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of amino acids 1 to 19 of SEQ ID NO:1. In one embodiment, the signal sequence comprises the amino acid sequence of amino acids 1 to 19 of SEQ ID NO:1.

In one embodiment, (i) the RNA encoding the signal sequence is a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of nucleotides 1 to 57 of SEQ ID NO: 2, 8, or 9, or a fragment of ... or (ii) encodes an amino acid sequence comprising the amino acid sequence of amino acids 1-19 of SEQ ID NO:1, an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of amino acids 1-19 of SEQ ID NO:1, or a functional fragment of the amino acid sequence of amino acids 1-19 of SEQ ID NO:1, or an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of amino acids 1-19 of SEQ ID NO:1. In one embodiment, the RNA encoding the signal sequence (i) comprises the nucleotide sequence of nucleotides 1-57 of SEQ ID NO:2, 8, or 9, and/or (ii) encodes an amino acid sequence comprising the amino acid sequence of amino acids 1-19 of SEQ ID NO:1.

In some embodiments, the RNA comprises a sequence encoding a signal peptide. In some embodiments, a signal peptide sequence as defined herein includes, but is not limited to, an immunoglobulin signal peptide sequence, e.g., an immunoglobulin heavy chain variable region signal peptide sequence, and the immunoglobulin may be a human immunoglobulin. Specifically, in some embodiments, a signal peptide sequence as defined herein includes a sequence comprising the amino acid sequence of amino acids 1-22 of SEQ ID NO: 31, or a functional variant thereof.

In some embodiments, the signal peptide sequence is functional in mammalian cells. In some embodiments, the signal sequence utilized is "endogenous" in that it is naturally associated (e.g., linked) with the encoded polypeptide. In some embodiments, the signal sequence utilized is heterologous to the encoded polypeptide, e.g., is not a naturally occurring part of the polypeptide (e.g., protein) whose sequence is included in the encoded polypeptide. In some embodiments, the signal peptide is a sequence that is typically characterized by a length of about 15-30 amino acids. In many embodiments, the signal peptide is located at the N-terminus of the encoded polypeptide described herein, but is not limited thereto. In some embodiments, the signal peptide preferably enables the transport of the polypeptides encoded by the RNA of the present disclosure with which they are associated to a defined cellular compartment, preferably the cell surface, the endoplasmic reticulum (ER), or the endosomal-lysosomal compartment. In some embodiments, the signal sequence is selected from S1S2 signal peptide (aa1-16 or aa1-19), immunoglobulin secretory signal peptide (aa1-22), HSV-1 gD signal peptide (MGGAAARLGAVILFVVIVGLHGVRSKY), HSV-2 gD signal peptide (MGRLTSGVGTAALLVVAVGLRVVCA), human SPARC signal peptide, human insulin isoform 1 signal peptide, human albumin signal peptide, etc. Those of skill in the art will recognize other secretory signal peptides, for example, as disclosed in WO2017/0810822220 (e.g., SEQ ID NOs: 1-1115 and 1728, or fragment variants thereof), as well as WO2019/008001. In some embodiments, the RNA sequence encodes an epitope that may include or be otherwise linked to a signal sequence (e.g., a secretory sequence), such as those listed in Table A, or a sequence having at least 1, 2, 3, 4, or 5 amino acid differences thereto. In some embodiments, a signal sequence such as MFVFLVLLPLVSSQCVNLT, or a sequence having at least 1, 2, 3, 4, or up to 5 amino acid differences thereto, is utilized. In some embodiments, a sequence such as MFVFLVLLPLVSSQCVNLT, or a sequence having 1, 2, 3, 4, or up to 5 amino acid differences thereto, is utilized. In some embodiments, the signal sequence is selected from those included in Table A below and/or encoded by the sequences in Table B below.

In one embodiment, the signal sequence comprises the amino acid sequence of amino acids 1 to 22 of SEQ ID NO:31, an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of amino acids 1 to 22 of SEQ ID NO:31, or a functional fragment of the amino acid sequence of amino acids 1 to 22 of SEQ ID NO:31, or an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of amino acids 1 to 22 of SEQ ID NO:31. In one embodiment, the signal sequence comprises the amino acid sequence of amino acids 1 to 22 of SEQ ID NO:31.

In one embodiment, the RNA encoding the signal sequence comprises: (i) a nucleotide sequence of nucleotides 54-119 of SEQ ID NO:32, a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of nucleotides 54-119 of SEQ ID NO:32, or a fragment of the nucleotide sequence of nucleotides 54-119 of SEQ ID NO:32, or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of nucleotides 54-119 of SEQ ID NO:32. and/or (ii) an amino acid sequence comprising the amino acid sequence of amino acids 1-22 of SEQ ID NO:31, an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of amino acids 1-22 of SEQ ID NO:31, or a functional fragment of the amino acid sequence of amino acids 1-22 of SEQ ID NO:31, or an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of amino acids 1-22 of SEQ ID NO:31. In one embodiment, the RNA encoding the signal sequence (i) comprises the nucleotide sequence of nucleotides 54-119 of SEQ ID NO:32, and/or (ii) encodes an amino acid sequence comprising the amino acid sequence of amino acids 1-22 of SEQ ID NO:31.

Such a signal peptide is preferably used to facilitate secretion of the encoded antigenic peptide or protein. More preferably, a signal peptide as defined herein is fused to the encoded antigenic peptide or protein as defined herein.

Thus, in a particularly preferred embodiment, the RNA described herein comprises at least one coding region encoding an antigenic peptide or protein and a signal peptide, the signal peptide being preferably fused to the antigenic peptide or protein, more preferably to the N-terminus of the antigenic peptide or protein described herein.

In one embodiment, the vaccine antigen comprises the amino acid sequence of SEQ ID NO: 1 or 7, an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of SEQ ID NO: 1 or 7, or an immunogenic fragment of the amino acid sequence of SEQ ID NO: 1 or 7, or an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of SEQ ID NO: 1 or 7. In one embodiment, the vaccine antigen comprises the amino acid sequence of SEQ ID NO: 1 or 7.

In one embodiment, the RNA encoding the vaccine antigen is (i) a nucleotide sequence of SEQ ID NO: 2, 8, or 9, a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of SEQ ID NO: 2, 8, or 9, or a fragment of the nucleotide sequence of SEQ ID NO: 2, 8, or 9, or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of SEQ ID NO: 2, 8, or 9 and/or (ii) encodes an amino acid sequence comprising the amino acid sequence of SEQ ID NO: 1 or 7, an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of SEQ ID NO: 1 or 7, or an immunogenic fragment of the amino acid sequence of SEQ ID NO: 1 or 7, or an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of SEQ ID NO: 1 or 7. In one embodiment, the RNA encoding the vaccine antigen (i) comprises the nucleotide sequence of SEQ ID NO: 2, 8, or 9, and/or (ii) encodes an amino acid sequence comprising the amino acid sequence of SEQ ID NO: 1 or 7.

In one embodiment, the vaccine antigen comprises the amino acid sequence of SEQ ID NO:7, an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of SEQ ID NO:7, or an immunogenic fragment of the amino acid sequence of SEQ ID NO:7, or an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of SEQ ID NO:7. In one embodiment, the vaccine antigen comprises the amino acid sequence of SEQ ID NO:7.

In one embodiment, the RNA encoding the vaccine antigen comprises: (i) a nucleotide sequence of SEQ ID NO: 15, 16, 19, 20, 24, or 25, a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of SEQ ID NO: 15, 16, 19, 20, 24, or 25, or a fragment of ... 97%, 96%, 95%, 90%, 85%, or 80% identity, and/or (ii) encodes an amino acid sequence comprising the amino acid sequence of SEQ ID NO: 7, an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of SEQ ID NO: 7, or an immunogenic fragment of the amino acid sequence of SEQ ID NO: 7, or an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of SEQ ID NO: 7. In one embodiment, the RNA encoding the vaccine antigen (i) comprises the nucleotide sequence of SEQ ID NO: 15, 16, 19, 20, 24, or 25, and/or (ii) encodes an amino acid sequence comprising the amino acid sequence of SEQ ID NO: 7.

In one embodiment, the vaccine antigen comprises the amino acid sequence of amino acids 1-683 of SEQ ID NO:1, an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of amino acids 1-683 of SEQ ID NO:1, or an immunogenic fragment of the amino acid sequence of amino acids 1-683 of SEQ ID NO:1, or an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of amino acids 1-683 of SEQ ID NO:1. In one embodiment, the vaccine antigen comprises the amino acid sequence of amino acids 1-683 of SEQ ID NO:1.

In one embodiment, the RNA encoding the vaccine antigen is (i) a nucleotide sequence of nucleotides 1 to 2049 of SEQ ID NO:2, 8, or 9, a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of nucleotides 1 to 2049 of SEQ ID NO:2, 8, or 9, or a fragment of the nucleotide sequence of nucleotides 1 to 2049 of SEQ ID NO:2, 8, or 9, or a fragment of the nucleotide sequence of nucleotides 1 to 2049 of SEQ ID NO:2, 8, or 9, or a fragment of the nucleotide sequence of nucleotides 1 to 2049 of SEQ ID NO:2, 8, or 9, %, or 80% identity to the amino acid sequence of SEQ ID NO:1, and/or (ii) encodes an amino acid sequence comprising the amino acid sequence of amino acids 1 to 683 of SEQ ID NO:1, an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of amino acids 1 to 683 of SEQ ID NO:1, or an immunogenic fragment of the amino acid sequence of amino acids 1 to 683 of SEQ ID NO:1, or an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of amino acids 1 to 683 of SEQ ID NO:1. In one embodiment, the RNA encoding the vaccine antigen (i) comprises the nucleotide sequence of nucleotides 1 to 2049 of SEQ ID NO:2, 8, or 9, and/or (ii) encodes an amino acid sequence comprising the amino acid sequence of amino acids 1 to 683 of SEQ ID NO:1.

In one embodiment, the vaccine antigen comprises the amino acid sequence of amino acids 1-685 of SEQ ID NO:1, an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of amino acids 1-685 of SEQ ID NO:1, or an immunogenic fragment of the amino acid sequence of amino acids 1-685 of SEQ ID NO:1, or an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of amino acids 1-685 of SEQ ID NO:1. In one embodiment, the vaccine antigen comprises the amino acid sequence of amino acids 1-685 of SEQ ID NO:1.

In one embodiment, the RNA encoding the vaccine antigen is (i) a nucleotide sequence of nucleotides 1 to 2055 of SEQ ID NO: 2, 8, or 9, a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of nucleotides 1 to 2055 of SEQ ID NO: 2, 8, or 9, or a fragment of the nucleotide sequence of nucleotides 1 to 2055 of SEQ ID NO: 2, 8, or 9, or a fragment of the nucleotide sequence of nucleotides 1 to 2055 of SEQ ID NO: 2, 8, or 9, or a fragment of the nucleotide sequence of nucleotides 1 to 2055 of SEQ ID NO: 2, 8, or 9, %, or 80% identity to the amino acid sequence of SEQ ID NO:1, and/or (ii) encodes an amino acid sequence comprising the amino acid sequence of amino acids 1 to 685 of SEQ ID NO:1, an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of amino acids 1 to 685 of SEQ ID NO:1, or an immunogenic fragment of the amino acid sequence of amino acids 1 to 685 of SEQ ID NO:1, or an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of amino acids 1 to 685 of SEQ ID NO:1. In one embodiment, the RNA encoding the vaccine antigen (i) comprises the nucleotide sequence of nucleotides 1 to 2055 of SEQ ID NO:2, 8, or 9, and/or (ii) encodes an amino acid sequence comprising the amino acid sequence of amino acids 1 to 685 of SEQ ID NO:1.

In one embodiment, the vaccine antigen comprises the amino acid sequence of SEQ ID NO:3, an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of SEQ ID NO:3, or an immunogenic fragment of the amino acid sequence of SEQ ID NO:3, or an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of SEQ ID NO:3. In one embodiment, the vaccine antigen comprises the amino acid sequence of SEQ ID NO:3.

In one embodiment, the RNA encoding the vaccine antigen comprises (i) a nucleotide sequence of SEQ ID NO:4, a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of SEQ ID NO:4, or a fragment of the nucleotide sequence of SEQ ID NO:4, or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of SEQ ID NO:4, and/or (ii) an amino acid sequence comprising the amino acid sequence of SEQ ID NO:3, an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of SEQ ID NO:3, or an immunogenic fragment of the amino acid sequence of SEQ ID NO:3, or an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of SEQ ID NO:3. In one embodiment, the RNA encoding the vaccine antigen (i) comprises the nucleotide sequence of SEQ ID NO:4, and/or (ii) encodes an amino acid sequence comprising the amino acid sequence of SEQ ID NO:3.

In one embodiment, the vaccine antigen comprises the amino acid sequence of amino acids 1-221 of SEQ ID NO:29, an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of amino acids 1-221 of SEQ ID NO:29, or an immunogenic fragment of the amino acid sequence of amino acids 1-221 of SEQ ID NO:29, or an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of amino acids 1-221 of SEQ ID NO:29. In one embodiment, the vaccine antigen comprises the amino acid sequence of amino acids 1-221 of SEQ ID NO:29.

In one embodiment, the RNA encoding the vaccine antigen comprises: (i) a nucleotide sequence of nucleotides 54-716 of SEQ ID NO:30, a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of nucleotides 54-716 of SEQ ID NO:30, or a fragment of the nucleotide sequence of nucleotides 54-716 of SEQ ID NO:30, or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of nucleotides 54-716 of SEQ ID NO:30. and/or (ii) encodes an amino acid sequence comprising the amino acid sequence of amino acids 1-221 of SEQ ID NO:29, an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of amino acids 1-221 of SEQ ID NO:29, or an immunogenic fragment of the amino acid sequence of amino acids 1-221 of SEQ ID NO:29, or an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of amino acids 1-221 of SEQ ID NO:29. In one embodiment, the RNA encoding the vaccine antigen (i) comprises the nucleotide sequence of nucleotides 54-716 of SEQ ID NO:30, and/or (ii) encodes an amino acid sequence comprising the amino acid sequence of amino acids 1-221 of SEQ ID NO:29.

In one embodiment, the vaccine antigen comprises the amino acid sequence of amino acids 1-224 of SEQ ID NO:31, an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of amino acids 1-224 of SEQ ID NO:31, or an immunogenic fragment of the amino acid sequence of amino acids 1-224 of SEQ ID NO:31, or an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of amino acids 1-224 of SEQ ID NO:31. In one embodiment, the vaccine antigen comprises the amino acid sequence of amino acids 1-224 of SEQ ID NO:31.

In one embodiment, the RNA encoding the vaccine antigen comprises: (i) a nucleotide sequence of nucleotides 54-725 of SEQ ID NO:32, a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of nucleotides 54-725 of SEQ ID NO:32, or a fragment of the nucleotide sequence of nucleotides 54-725 of SEQ ID NO:32, or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of nucleotides 54-725 of SEQ ID NO:32. and/or (ii) encodes an amino acid sequence comprising the amino acid sequence of amino acids 1-224 of SEQ ID NO:31, an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of amino acids 1-224 of SEQ ID NO:31, or an immunogenic fragment of the amino acid sequence of amino acids 1-224 of SEQ ID NO:31, or an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of amino acids 1-224 of SEQ ID NO:31. In one embodiment, the RNA encoding the vaccine antigen (i) comprises the nucleotide sequence of nucleotides 54-725 of SEQ ID NO:32, and/or (ii) encodes an amino acid sequence comprising the amino acid sequence of amino acids 1-224 of SEQ ID NO:31.

Multimerization Domains In some embodiments, the RNA utilized as described herein comprises a sequence encoding a multimerization element (e.g., a heterologous multimerization element). In some embodiments, the heterologous multimerization element comprises a dimerization, trimerization, or tetramerization element. In some embodiments, the multimerization element is one described in WO2017/081082 (e.g., SEQ ID NOs: 1116-1167, or a fragment or variant thereof). Exemplary trimerization and tetramerization elements include, but are not limited to, engineered leucine zippers, fibritin foldon domains from enterobacteriaceae phage T4, GCN4pll, GCN4-pll, and p53. In some embodiments, the encoded polypeptide(s) provided are capable of forming a trimeric complex. For example, the encoded polypeptide(s) utilized may comprise a domain that allows for the formation of a multimeric complex, such as, for example, a trimeric complex of amino acid sequences that comprise the encoded polypeptide(s) described herein. In some embodiments, the domain that allows for the formation of a multimeric complex comprises a trimerization domain, e.g., a trimerization domain described herein. In some embodiments, the encoded polypeptide(s) may be modified, e.g., by adding the "foldon" trimerization domain from T4-fibritin, to increase its immunogenicity.

According to certain embodiments, the trimerization domain is fused to the SARS-CoV-2 S protein, its variants, or its fragments, i.e., antigenic peptides or proteins, either directly or via a linker, e.g., a glycine/serine linker. Thus, in one embodiment, the trimerization domain is fused to the above amino acid sequence derived from the SARS-CoV-2 S protein or its immunogenic fragments (antigenic peptides or proteins) constituted by the above vaccine antigens (which can optionally be fused to a signal peptide as described above).

Such trimerization domains are preferably, but not limited to, located at the C-terminus of an antigenic peptide or protein. The trimerization domains defined herein preferably allow trimerization of antigenic peptides or proteins encoded by RNA. Examples of trimerization domains defined herein include, but are not limited to, the natural trimerization domain of T4 fibritin, Foldon. The C-terminal domain of T4 fibritin (Foldon) is essential for the formation of the fibritin trimer structure and can be used as an artificial trimerization domain. In one embodiment, the trimerization domains defined herein include, but are not limited to, a sequence comprising the amino acid sequence of amino acids 3-29 of SEQ ID NO: 10, or a functional variant thereof. In one embodiment, the trimerization domains defined herein include, but are not limited to, a sequence comprising the amino acid sequence of SEQ ID NO: 10, or a functional variant thereof.

In one embodiment, the trimerization domain comprises the amino acid sequence of amino acids 3 to 29 of SEQ ID NO: 10, an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of amino acids 3 to 29 of SEQ ID NO: 10, or a functional fragment of the amino acid sequence of amino acids 3 to 29 of SEQ ID NO: 10, or an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of amino acids 3 to 29 of SEQ ID NO: 10. In one embodiment, the trimerization domain comprises the amino acid sequence of amino acids 3 to 29 of SEQ ID NO: 10.

In one embodiment, the RNA encoding the trimerization domain comprises (i) a nucleotide sequence of nucleotides 7-87 of SEQ ID NO:11, a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of nucleotides 7-87 of SEQ ID NO:11, or a fragment of the nucleotide sequence of nucleotides 7-87 of SEQ ID NO:11, or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of nucleotides 7-87 of SEQ ID NO:11. and/or (ii) encodes an amino acid sequence comprising the amino acid sequence of amino acids 3-29 of SEQ ID NO:10, an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of amino acids 3-29 of SEQ ID NO:10, or a functional fragment of the amino acid sequence of amino acids 3-29 of SEQ ID NO:10, or an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of amino acids 3-29 of SEQ ID NO:10. In one embodiment, the RNA encoding the trimerization domain (i) comprises the nucleotide sequence of nucleotides 7-87 of SEQ ID NO:11, and/or (ii) encodes an amino acid sequence comprising the amino acid sequence of amino acids 3-29 of SEQ ID NO:10.

In one embodiment, the trimerization domain comprises the amino acid sequence of SEQ ID NO: 10, an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of SEQ ID NO: 10, or a functional fragment of the amino acid sequence of SEQ ID NO: 10, or an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of SEQ ID NO: 10. In one embodiment, the trimerization domain comprises the amino acid sequence of SEQ ID NO: 10.

In one embodiment, the RNA encoding the trimerization domain comprises (i) a nucleotide sequence of SEQ ID NO:11, a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of SEQ ID NO:11, or a fragment of the nucleotide sequence of SEQ ID NO:11, or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of SEQ ID NO:11. and/or (ii) encodes an amino acid sequence comprising the amino acid sequence of SEQ ID NO: 10, an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of SEQ ID NO: 10, or a functional fragment of the amino acid sequence of SEQ ID NO: 10, or an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of SEQ ID NO: 10. In one embodiment, the RNA encoding the trimerization domain (i) comprises the nucleotide sequence of SEQ ID NO: 11, and/or (ii) encodes an amino acid sequence comprising the amino acid sequence of SEQ ID NO: 10.

Such trimerization domains are preferably used to promote trimerization of the encoded antigenic peptide or protein. More preferably, a trimerization domain as defined herein is fused to an antigenic peptide or protein as defined herein.

Thus, in a particularly preferred embodiment, the RNA described herein comprises at least one coding region encoding an antigenic peptide or protein and a trimerization domain as defined herein, which is preferably fused to the antigenic peptide or protein, more preferably to the C-terminus of the antigenic peptide or protein.

In one embodiment, the vaccine antigen comprises the amino acid sequence of SEQ ID NO:5, an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of SEQ ID NO:5, or an immunogenic fragment of the amino acid sequence of SEQ ID NO:5, or an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of SEQ ID NO:5. In one embodiment, the vaccine antigen comprises the amino acid sequence of SEQ ID NO:5.

In one embodiment, the RNA encoding the vaccine antigen comprises (i) a nucleotide sequence of SEQ ID NO:6, a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of SEQ ID NO:6, or a fragment of the nucleotide sequence of SEQ ID NO:6, or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of SEQ ID NO:6, and/or (ii) an amino acid sequence comprising the amino acid sequence of SEQ ID NO:5, an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of SEQ ID NO:5, or an immunogenic fragment of the amino acid sequence of SEQ ID NO:5, or an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of SEQ ID NO:5. In one embodiment, the RNA encoding the vaccine antigen (i) comprises the nucleotide sequence of SEQ ID NO:6, and/or (ii) encodes an amino acid sequence comprising the amino acid sequence of SEQ ID NO:5.

In one embodiment, the RNA encoding the vaccine antigen is (i) a nucleotide sequence of SEQ ID NO: 17, 21, or 26, a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of SEQ ID NO: 17, 21, or 26, or a fragment of the nucleotide sequence of SEQ ID NO: 17, 21, or 26, or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of SEQ ID NO: 17, 21, or 26 %, or 80% identity to the amino acid sequence of SEQ ID NO:5, and/or (ii) encodes an amino acid sequence comprising an amino acid sequence of SEQ ID NO:5, an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of SEQ ID NO:5, or an immunogenic fragment of the amino acid sequence of SEQ ID NO:5, or an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of SEQ ID NO:5. In one embodiment, the RNA encoding the vaccine antigen (i) comprises the nucleotide sequence of SEQ ID NO:17, 21, or 26, and/or (ii) encodes an amino acid sequence comprising the amino acid sequence of SEQ ID NO:5.

In one embodiment, the vaccine antigen comprises the amino acid sequence of SEQ ID NO: 18, an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of SEQ ID NO: 18, or an immunogenic fragment of the amino acid sequence of SEQ ID NO: 18, or an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of SEQ ID NO: 18. In one embodiment, the vaccine antigen comprises the amino acid sequence of SEQ ID NO: 18.

In one embodiment, the vaccine antigen comprises the amino acid sequence of amino acids 1-257 of SEQ ID NO:29, an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of amino acids 1-257 of SEQ ID NO:29, or an immunogenic fragment of the amino acid sequence of amino acids 1-257 of SEQ ID NO:29, or an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of amino acids 1-257 of SEQ ID NO:29. In one embodiment, the vaccine antigen comprises the amino acid sequence of amino acids 1-257 of SEQ ID NO:29.

In one embodiment, the RNA encoding the vaccine antigen comprises: (i) a nucleotide sequence of nucleotides 54-824 of SEQ ID NO:30, a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of nucleotides 54-824 of SEQ ID NO:30, or a fragment of the nucleotide sequence of nucleotides 54-824 of SEQ ID NO:30, or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of nucleotides 54-824 of SEQ ID NO:30. and/or (ii) encodes an amino acid sequence comprising the amino acid sequence of amino acids 1 to 257 of SEQ ID NO:29, an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of amino acids 1 to 257 of SEQ ID NO:29, or an immunogenic fragment of the amino acid sequence of amino acids 1 to 257 of SEQ ID NO:29, or an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of amino acids 1 to 257 of SEQ ID NO:29. In one embodiment, the RNA encoding the vaccine antigen (i) comprises the nucleotide sequence of nucleotides 54 to 824 of SEQ ID NO:30, and/or (ii) encodes an amino acid sequence comprising the amino acid sequence of amino acids 1 to 257 of SEQ ID NO:29.

In one embodiment, the vaccine antigen comprises the amino acid sequence of amino acids 1-260 of SEQ ID NO:31, an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of amino acids 1-260 of SEQ ID NO:31, or an immunogenic fragment of the amino acid sequence of amino acids 1-260 of SEQ ID NO:31, or an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of amino acids 1-260 of SEQ ID NO:31. In one embodiment, the vaccine antigen comprises the amino acid sequence of amino acids 1-260 of SEQ ID NO:31.

In one embodiment, the RNA encoding the vaccine antigen comprises: (i) a nucleotide sequence of nucleotides 54-833 of SEQ ID NO:32, a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of nucleotides 54-833 of SEQ ID NO:32, or a fragment of the nucleotide sequence of nucleotides 54-833 of SEQ ID NO:32, or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of nucleotides 54-833 of SEQ ID NO:32. and/or (ii) encodes an amino acid sequence comprising the amino acid sequence of amino acids 1-260 of SEQ ID NO:31, an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of amino acids 1-260 of SEQ ID NO:31, or an immunogenic fragment of the amino acid sequence of amino acids 1-260 of SEQ ID NO:31, or an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of amino acids 1-260 of SEQ ID NO:31. In one embodiment, the RNA encoding the vaccine antigen (i) comprises the nucleotide sequence of nucleotides 54-833 of SEQ ID NO:32, and/or (ii) encodes an amino acid sequence comprising the amino acid sequence of amino acids 1-260 of SEQ ID NO:31.

In one embodiment, the vaccine antigen comprises the amino acid sequence of amino acids 20-257 of SEQ ID NO:29, an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of amino acids 20-257 of SEQ ID NO:29, or an immunogenic fragment of the amino acid sequence of amino acids 20-257 of SEQ ID NO:29, or an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of amino acids 20-257 of SEQ ID NO:29. In one embodiment, the vaccine antigen comprises the amino acid sequence of amino acids 20-257 of SEQ ID NO:29.

In one embodiment, the RNA encoding the vaccine antigen comprises: (i) a nucleotide sequence of nucleotides 111-824 of SEQ ID NO:30, a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of nucleotides 111-824 of SEQ ID NO:30, or a fragment of the nucleotide sequence of nucleotides 111-824 of SEQ ID NO:30, or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of nucleotides 111-824 of SEQ ID NO:30. and/or (ii) encodes an amino acid sequence comprising the amino acid sequence of amino acids 20-257 of SEQ ID NO:29, an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of amino acids 20-257 of SEQ ID NO:29, or an immunogenic fragment of the amino acid sequence of amino acids 20-257 of SEQ ID NO:29, or an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of amino acids 20-257 of SEQ ID NO:29. In one embodiment, the RNA encoding the vaccine antigen (i) comprises the nucleotide sequence of nucleotides 111-824 of SEQ ID NO:30, and/or (ii) encodes an amino acid sequence comprising the amino acid sequence of amino acids 20-257 of SEQ ID NO:29.

In one embodiment, the vaccine antigen comprises the amino acid sequence of amino acids 23-260 of SEQ ID NO:31, an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of amino acids 23-260 of SEQ ID NO:31, or an immunogenic fragment of the amino acid sequence of amino acids 23-260 of SEQ ID NO:31, or an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of amino acids 23-260 of SEQ ID NO:31. In one embodiment, the vaccine antigen comprises the amino acid sequence of amino acids 23-260 of SEQ ID NO:31.

In one embodiment, the RNA encoding the vaccine antigen comprises: (i) a nucleotide sequence of nucleotides 120-833 of SEQ ID NO:32, a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of nucleotides 120-833 of SEQ ID NO:32, or a fragment of the nucleotide sequence of nucleotides 120-833 of SEQ ID NO:32, or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of nucleotides 120-833 of SEQ ID NO:32. and/or (ii) encodes an amino acid sequence comprising the amino acid sequence of amino acids 23-260 of SEQ ID NO:31, an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of amino acids 23-260 of SEQ ID NO:31, or an immunogenic fragment of the amino acid sequence of amino acids 23-260 of SEQ ID NO:31, or an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of amino acids 23-260 of SEQ ID NO:31. In one embodiment, the RNA encoding the vaccine antigen (i) comprises the nucleotide sequence of nucleotides 120-833 of SEQ ID NO:32, and/or (ii) encodes an amino acid sequence comprising the amino acid sequence of amino acids 23-260 of SEQ ID NO:31.

Transmembrane Domain In some embodiments, the RNA described herein comprises a sequence encoding a membrane associated element (e.g., a heterologous membrane associated element), such as a transmembrane domain. The transmembrane domain can be N-terminal, C-terminal, or internal to the encoded polypeptide. The coding sequence for the transmembrane element is typically positioned in frame (i.e., in the same reading frame), 5', 3', or internal to the coding sequence of the sequence to which it is linked (e.g., a sequence encoding a polypeptide(s)). In some embodiments, the transmembrane domain comprises or is the transmembrane domain of influenza virus hemagglutinin (HA), HIV-1 Env, equine infectious anemia virus (EIAV), murine leukemia virus (MLV), mouse mammary tumor virus, vesicular stomatitis virus (VSV) G protein, rabies virus, or a seven transmembrane domain receptor.

According to certain embodiments, the transmembrane domain is fused to the SARS-CoV-2 S protein, its variants, or fragments thereof, i.e., antigenic peptide or protein, either directly or via a linker, e.g., a glycine/serine linker. Thus, in one embodiment, the transmembrane domain is fused to a SARS-CoV-2 S polypeptide or an immunogenic fragment thereof (antigenic peptide or protein), which can optionally be fused to a signal peptide and/or trimerization domain as described above.

Secretion Signal Such a transmembrane domain is preferably, but not limited to, located C-terminal to the antigenic peptide or protein. Preferably, such a transmembrane domain, if present, is located C-terminal to the trimerization domain. In one embodiment, the trimerization domain is present between the SARS-CoV-2 S protein, variant, or fragment thereof, i.e., the antigenic peptide or protein, and the transmembrane domain.

The transmembrane domain defined herein preferably allows anchoring of the antigenic peptide or protein encoded by the RNA into the cell membrane.

In one embodiment, the transmembrane domain sequence defined herein includes, but is not limited to, the transmembrane domain sequence of the SARS-CoV-2 S protein, in particular a sequence comprising the amino acid sequence of amino acids 1207 to 1254 of SEQ ID NO:1, or a functional variant thereof.

In one embodiment, the transmembrane domain sequence comprises the amino acid sequence of amino acids 1207-1254 of SEQ ID NO:1, an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of amino acids 1207-1254 of SEQ ID NO:1, or a functional fragment of the amino acid sequence of amino acids 1207-1254 of SEQ ID NO:1, or an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of amino acids 1207-1254 of SEQ ID NO:1. In one embodiment, the transmembrane domain sequence comprises the amino acid sequence of amino acids 1207-1254 of SEQ ID NO:1.

In one embodiment, the RNA encoding the transmembrane domain sequence is (i) a nucleotide sequence of nucleotides 3619-3762 of SEQ ID NO:2, 8, or 9, a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of nucleotides 3619-3762 of SEQ ID NO:2, 8, or 9, or a fragment of the nucleotide sequence of nucleotides 3619-3762 of SEQ ID NO:2, 8, or 9, or a fragment of the nucleotide sequence of nucleotides 3619-3762 of SEQ ID NO:2, 8, or 9, or a fragment of the nucleotide sequence of nucleotides 3619-3762 of SEQ ID NO:2, 8, or 9, %, or 80% identity to the amino acid sequence of amino acids 1207-1254 of SEQ ID NO: 1, and/or (ii) encodes an amino acid sequence comprising the amino acid sequence of amino acids 1207-1254 of SEQ ID NO: 1, an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of amino acids 1207-1254 of SEQ ID NO: 1, or a functional fragment of the amino acid sequence of amino acids 1207-1254 of SEQ ID NO: 1, or an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of amino acids 1207-1254 of SEQ ID NO: 1. In one embodiment, the RNA encoding the transmembrane domain sequence (i) comprises the nucleotide sequence of nucleotides 3619-3762 of SEQ ID NO: 2, 8, or 9, and/or (ii) encodes an amino acid sequence comprising the amino acid sequence of amino acids 1207-1254 of SEQ ID NO: 1.

In one embodiment, the vaccine antigen comprises the amino acid sequence of amino acids 1 to 311 of SEQ ID NO:29, an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of amino acids 1 to 311 of SEQ ID NO:29, or an immunogenic fragment of the amino acid sequence of amino acids 1 to 311 of SEQ ID NO:29, or an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of amino acids 1 to 311 of SEQ ID NO:29. In one embodiment, the vaccine antigen comprises the amino acid sequence of amino acids 1 to 311 of SEQ ID NO:29.

In one embodiment, the RNA encoding the vaccine antigen comprises: (i) a nucleotide sequence of nucleotides 54-986 of SEQ ID NO:30, a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of nucleotides 54-986 of SEQ ID NO:30, or a fragment of the nucleotide sequence of nucleotides 54-986 of SEQ ID NO:30, or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of nucleotides 54-986 of SEQ ID NO:30. and/or (ii) encodes an amino acid sequence comprising the amino acid sequence of amino acids 1 to 311 of SEQ ID NO:29, an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of amino acids 1 to 311 of SEQ ID NO:29, or an immunogenic fragment of the amino acid sequence of amino acids 1 to 311 of SEQ ID NO:29, or an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of amino acids 1 to 311 of SEQ ID NO:29. In one embodiment, the RNA encoding the vaccine antigen (i) comprises the nucleotide sequence of nucleotides 54 to 986 of SEQ ID NO:30, and/or (ii) encodes an amino acid sequence comprising the amino acid sequence of amino acids 1 to 311 of SEQ ID NO:29.

In one embodiment, the vaccine antigen comprises the amino acid sequence of amino acids 1 to 314 of SEQ ID NO:31, an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of amino acids 1 to 314 of SEQ ID NO:31, or an immunogenic fragment of the amino acid sequence of amino acids 1 to 314 of SEQ ID NO:31, or an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of amino acids 1 to 314 of SEQ ID NO:31. In one embodiment, the vaccine antigen comprises the amino acid sequence of amino acids 1 to 314 of SEQ ID NO:31.

In one embodiment, the RNA encoding the vaccine antigen comprises: (i) a nucleotide sequence of nucleotides 54-995 of SEQ ID NO:32, a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of nucleotides 54-995 of SEQ ID NO:32, or a fragment of the nucleotide sequence of nucleotides 54-995 of SEQ ID NO:32, or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of nucleotides 54-995 of SEQ ID NO:32. and/or (ii) encodes an amino acid sequence comprising the amino acid sequence of amino acids 1 to 314 of SEQ ID NO:31, an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of amino acids 1 to 314 of SEQ ID NO:31, or an immunogenic fragment of the amino acid sequence of amino acids 1 to 314 of SEQ ID NO:31, or an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of amino acids 1 to 314 of SEQ ID NO:31. In one embodiment, the RNA encoding the vaccine antigen (i) comprises the nucleotide sequence of nucleotides 54 to 995 of SEQ ID NO:32, and/or (ii) encodes an amino acid sequence comprising the amino acid sequence of amino acids 1 to 314 of SEQ ID NO:31.

In one embodiment, the vaccine antigen comprises the amino acid sequence of amino acids 20-311 of SEQ ID NO:29, an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of amino acids 20-311 of SEQ ID NO:29, or an immunogenic fragment of the amino acid sequence of amino acids 20-311 of SEQ ID NO:29, or an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of amino acids 20-311 of SEQ ID NO:29. In one embodiment, the vaccine antigen comprises the amino acid sequence of amino acids 20-311 of SEQ ID NO:29.

In one embodiment, the RNA encoding the vaccine antigen comprises: (i) a nucleotide sequence of nucleotides 111-986 of SEQ ID NO:30, a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of nucleotides 111-986 of SEQ ID NO:30, or a fragment of the nucleotide sequence of nucleotides 111-986 of SEQ ID NO:30, or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of nucleotides 111-986 of SEQ ID NO:30. and/or (ii) encodes an amino acid sequence comprising the amino acid sequence of amino acids 20-311 of SEQ ID NO:29, an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of amino acids 20-311 of SEQ ID NO:29, or an immunogenic fragment of the amino acid sequence of amino acids 20-311 of SEQ ID NO:29, or an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of amino acids 20-311 of SEQ ID NO:29. In one embodiment, the RNA encoding the vaccine antigen (i) comprises the nucleotide sequence of nucleotides 111-986 of SEQ ID NO:30, and/or (ii) encodes an amino acid sequence comprising the amino acid sequence of amino acids 20-311 of SEQ ID NO:29.

In one embodiment, the vaccine antigen comprises the amino acid sequence of amino acids 23-314 of SEQ ID NO:31, an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of amino acids 23-314 of SEQ ID NO:31, or an immunogenic fragment of the amino acid sequence of amino acids 23-314 of SEQ ID NO:31, or an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of amino acids 23-314 of SEQ ID NO:31. In one embodiment, the vaccine antigen comprises the amino acid sequence of amino acids 23-314 of SEQ ID NO:31.

In one embodiment, the RNA encoding the vaccine antigen comprises: (i) a nucleotide sequence of nucleotides 120-995 of SEQ ID NO:32, a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of nucleotides 120-995 of SEQ ID NO:32, or a fragment of the nucleotide sequence of nucleotides 120-995 of SEQ ID NO:32, or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of nucleotides 120-995 of SEQ ID NO:32. and/or (ii) encodes an amino acid sequence comprising the amino acid sequence of amino acids 23-314 of SEQ ID NO:31, an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of amino acids 23-314 of SEQ ID NO:31, or an immunogenic fragment of the amino acid sequence of amino acids 23-314 of SEQ ID NO:31, or an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of amino acids 23-314 of SEQ ID NO:31. In one embodiment, the RNA encoding the vaccine antigen (i) comprises the nucleotide sequence of nucleotides 120-995 of SEQ ID NO:32, and/or (ii) encodes an amino acid sequence comprising the amino acid sequence of amino acids 23-314 of SEQ ID NO:31.

In one embodiment, the RNA encoding the vaccine antigen comprises (i) a nucleotide sequence of SEQ ID NO:30, a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of SEQ ID NO:30, or a fragment of the nucleotide sequence of SEQ ID NO:30, or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of SEQ ID NO:30. and/or (ii) encodes an amino acid sequence comprising the amino acid sequence of SEQ ID NO:29, an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of SEQ ID NO:29, or an immunogenic fragment of the amino acid sequence of SEQ ID NO:29, or an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of SEQ ID NO:29. In one embodiment, the RNA encoding the vaccine antigen (i) comprises the nucleotide sequence of SEQ ID NO:30, and/or (ii) encodes an amino acid sequence comprising the amino acid sequence of SEQ ID NO:29.

In one embodiment, the RNA encoding the vaccine antigen comprises: (i) a nucleotide sequence of SEQ ID NO:32, a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of SEQ ID NO:32, or a fragment of the nucleotide sequence of SEQ ID NO:32, or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of SEQ ID NO:32. and/or (ii) encodes an amino acid sequence comprising the amino acid sequence of SEQ ID NO:31, an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of SEQ ID NO:31, or an immunogenic fragment of the amino acid sequence of SEQ ID NO:31, or an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of SEQ ID NO:31. In one embodiment, the RNA encoding the vaccine antigen (i) comprises the nucleotide sequence of SEQ ID NO:32, and/or (ii) encodes an amino acid sequence comprising the amino acid sequence of SEQ ID NO:31.

In one embodiment, the vaccine antigen comprises the amino acid sequence of SEQ ID NO:28, an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of SEQ ID NO:28, or an immunogenic fragment of the amino acid sequence of SEQ ID NO:28, or an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of SEQ ID NO:28. In one embodiment, the vaccine antigen comprises the amino acid sequence of SEQ ID NO:28.

In one embodiment, the RNA encoding the vaccine antigen comprises (i) a nucleotide sequence of SEQ ID NO:27, a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of SEQ ID NO:27, or a fragment of the nucleotide sequence of SEQ ID NO:27, or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of SEQ ID NO:27. and/or (ii) encodes an amino acid sequence comprising the amino acid sequence of SEQ ID NO:28, an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of SEQ ID NO:28, or an immunogenic fragment of the amino acid sequence of SEQ ID NO:28, or an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of SEQ ID NO:28. In one embodiment, the RNA encoding the vaccine antigen (i) comprises the nucleotide sequence of SEQ ID NO:27, and/or (ii) encodes an amino acid sequence comprising the amino acid sequence of SEQ ID NO:28.

In one embodiment, the vaccine antigen comprises the amino acid sequence of SEQ ID NO:49, an amino acid sequence having at least 99.5%, 99%, 98.5%, 98%, 98.5%, or 97% identity to the amino acid sequence of SEQ ID NO:49, or an immunogenic fragment of the amino acid sequence of SEQ ID NO:49, or an amino acid sequence having at least 99.5%, 99%, 98.5%, 98%, 98.5%, or 97% identity to the amino acid sequence of SEQ ID NO:49. In one embodiment, the vaccine antigen comprises the amino acid sequence of SEQ ID NO:49. The amino acid sequence of SEQ ID NO:49 corresponds to the amino acid sequence of the full-length S protein of Omicron BA.1, which contains proline residues at positions 986 and 987 of SEQ ID NO:49.

In one embodiment, the RNA encoding the vaccine antigen comprises (i) a nucleotide sequence of SEQ ID NO:50, a nucleotide sequence having at least 99.5%, 99%, 98.5%, 98%, 98.5%, or 97% identity to the nucleotide sequence of SEQ ID NO:50, or a fragment of the nucleotide sequence of SEQ ID NO:50, or a nucleotide sequence having at least 99.5%, 99%, 98.5%, 98%, 98.5%, or 97% identity to the nucleotide sequence of SEQ ID NO:50, and/or (ii) an amino acid sequence comprising the amino acid sequence of SEQ ID NO:49, an amino acid sequence having at least 99.5%, 99%, 98.5%, 98%, 98.5%, or 97% identity to the amino acid sequence of SEQ ID NO:49, or an immunogenic fragment of the amino acid sequence of SEQ ID NO:49, or an amino acid sequence having at least 99.5%, 99%, 98.5%, 98%, 98.5%, or 97% identity to the amino acid sequence of SEQ ID NO:49. In one embodiment, the RNA encoding the vaccine antigen (i) comprises the nucleotide sequence of SEQ ID NO:50 and/or (ii) encodes an amino acid sequence comprising the amino acid sequence of SEQ ID NO:49. The nucleotide sequence of SEQ ID NO:50 is a nucleotide sequence designed to encode the amino acid sequence of the full-length S protein from Omicron BA.1, which has proline residues at positions 986 and 987 of SEQ ID NO:49.

In one embodiment, the RNA encoding the vaccine antigen comprises (i) a nucleotide sequence of SEQ ID NO:51, a nucleotide sequence having at least 99.5%, 99%, 98.5%, 98%, 98.5%, or 97% identity to the nucleotide sequence of SEQ ID NO:51, or a fragment of the nucleotide sequence of SEQ ID NO:51, or a nucleotide sequence having at least 99.5%, 99%, 98.5%, 98%, 98.5%, or 97% identity to the nucleotide sequence of SEQ ID NO:51, and/or (ii) an amino acid sequence comprising the amino acid sequence of SEQ ID NO:49, an amino acid sequence having at least 99.5%, 99%, 98.5%, 98%, 98.5%, or 97% identity to the amino acid sequence of SEQ ID NO:49, or an immunogenic fragment of the amino acid sequence of SEQ ID NO:49, or an amino acid sequence having at least 99.5%, 99%, 98.5%, 98%, 98.5%, or 97% identity to the amino acid sequence of SEQ ID NO:49. In one embodiment, the RNA encoding the vaccine antigen (i) comprises the nucleotide sequence of SEQ ID NO:51, and/or (ii) encodes an amino acid sequence comprising the amino acid sequence of SEQ ID NO:49. The nucleotide sequence of SEQ ID NO:51 corresponds to an RNA construct (e.g., including the 5'UTR, S protein coding sequence, 3'UTR, and polyA tail) that encodes the amino acid sequence of the full-length S protein from Omicron BA.1, having proline residues at positions 986 and 987 of SEQ ID NO:49.

In one embodiment, the vaccine antigen comprises the amino acid sequence of SEQ ID NO:55, an amino acid sequence having at least 99.5%, 99%, 98.5%, 98%, 98.5%, or 97% identity to the amino acid sequence of SEQ ID NO:55, or an immunogenic fragment of the amino acid sequence of SEQ ID NO:55, or an amino acid sequence having at least 99.5%, 99%, 98.5%, 98%, 98.5%, or 97% identity to the amino acid sequence of SEQ ID NO:55. In one embodiment, the vaccine antigen comprises the amino acid sequence of SEQ ID NO:55.

In one embodiment, the RNA encoding the vaccine antigen comprises (i) a nucleotide sequence of SEQ ID NO:56, a nucleotide sequence having at least 99.5%, 99%, 98.5%, 98%, 98.5%, or 97% identity to the nucleotide sequence of SEQ ID NO:56, or a fragment of the nucleotide sequence of SEQ ID NO:56, or a nucleotide sequence having at least 99.5%, 99%, 98.5%, 98%, 98.5%, or 97% identity to the nucleotide sequence of SEQ ID NO:56, and/or (ii) an amino acid sequence comprising the amino acid sequence of SEQ ID NO:55, an amino acid sequence having at least 99.5%, 99%, 98.5%, 98%, 98.5%, or 97% identity to the amino acid sequence of SEQ ID NO:55, or an immunogenic fragment of the amino acid sequence of SEQ ID NO:55, or an amino acid sequence having at least 99.5%, 99%, 98.5%, 98%, 98.5%, or 97% identity to the amino acid sequence of SEQ ID NO:55. In one embodiment, the RNA encoding the vaccine antigen (i) comprises the nucleotide sequence of SEQ ID NO:56 and/or (ii) encodes an amino acid sequence comprising the amino acid sequence of SEQ ID NO:55.

In one embodiment, the RNA encoding the vaccine antigen comprises (i) a nucleotide sequence of SEQ ID NO:57, a nucleotide sequence having at least 99.5%, 99%, 98.5%, 98%, 98.5%, or 97% identity to the nucleotide sequence of SEQ ID NO:57, or a fragment of the nucleotide sequence of SEQ ID NO:57, or a nucleotide sequence having at least 99.5%, 99%, 98.5%, 98%, 98.5%, or 97% identity to the nucleotide sequence of SEQ ID NO:57, and/or (ii) an amino acid sequence comprising the amino acid sequence of SEQ ID NO:55, an amino acid sequence having at least 99.5%, 99%, 98.5%, 98%, 98.5%, or 97% identity to the amino acid sequence of SEQ ID NO:55, or an immunogenic fragment of the amino acid sequence of SEQ ID NO:55, or an amino acid sequence having at least 99.5%, 99%, 98.5%, 98%, 98.5%, or 97% identity to the amino acid sequence of SEQ ID NO:55. In one embodiment, the RNA encoding the vaccine antigen (i) comprises the nucleotide sequence of SEQ ID NO:57, and/or (ii) encodes an amino acid sequence comprising the amino acid sequence of SEQ ID NO:55.

In one embodiment, the vaccine antigen comprises the amino acid sequence of SEQ ID NO:58, an amino acid sequence having at least 99.5%, 99%, 98.5%, 98%, 98.5%, or 97% identity to the amino acid sequence of SEQ ID NO:58, or an immunogenic fragment of the amino acid sequence of SEQ ID NO:58, or an amino acid sequence having at least 99.5%, 99%, 98.5%, 98%, 98.5%, or 97% identity to the amino acid sequence of SEQ ID NO:58. In one embodiment, the vaccine antigen comprises the amino acid sequence of SEQ ID NO:58.

In one embodiment, the RNA encoding the vaccine antigen comprises (i) a nucleotide sequence of SEQ ID NO:59, a nucleotide sequence having at least 99.5%, 99%, 98.5%, 98%, 98.5%, or 97% identity to the nucleotide sequence of SEQ ID NO:59, or a fragment of the nucleotide sequence of SEQ ID NO:59, or a nucleotide sequence having at least 99.5%, 99%, 98.5%, 98%, 98.5%, or 97% identity to the nucleotide sequence of SEQ ID NO:59, and/or (ii) an amino acid sequence comprising the amino acid sequence of SEQ ID NO:58, an amino acid sequence having at least 99.5%, 99%, 98.5%, 98%, 98.5%, or 97% identity to the amino acid sequence of SEQ ID NO:58, or an immunogenic fragment of the amino acid sequence of SEQ ID NO:58, or an amino acid sequence having at least 99.5%, 99%, 98.5%, 98%, 98.5%, or 97% identity to the amino acid sequence of SEQ ID NO:58. In one embodiment, the RNA encoding the vaccine antigen (i) comprises the nucleotide sequence of SEQ ID NO:59, and/or (ii) encodes an amino acid sequence comprising the amino acid sequence of SEQ ID NO:58.

In one embodiment, the RNA encoding the vaccine antigen comprises (i) a nucleotide sequence of SEQ ID NO:60, a nucleotide sequence having at least 99.5%, 99%, 98.5%, 98%, 98.5%, or 97% identity to the nucleotide sequence of SEQ ID NO:60, or a fragment of the nucleotide sequence of SEQ ID NO:60, or a nucleotide sequence having at least 99.5%, 99%, 98.5%, 98%, 98.5%, or 97% identity to the nucleotide sequence of SEQ ID NO:60, and/or (ii) an amino acid sequence comprising the amino acid sequence of SEQ ID NO:58, an amino acid sequence having at least 99.5%, 99%, 98.5%, 98%, 98.5%, or 97% identity to the amino acid sequence of SEQ ID NO:58, or an immunogenic fragment of the amino acid sequence of SEQ ID NO:58, or an amino acid sequence having at least 99.5%, 99%, 98.5%, 98%, 98.5%, or 97% identity to the amino acid sequence of SEQ ID NO:58. In one embodiment, the RNA encoding the vaccine antigen (i) comprises the nucleotide sequence of SEQ ID NO: 60, and/or (ii) encodes an amino acid sequence comprising the amino acid sequence of SEQ ID NO: 58.

In one embodiment, the vaccine antigen comprises the amino acid sequence of SEQ ID NO:61, an amino acid sequence having at least 99.5%, 99%, 98.5%, 98%, 98.5%, or 97% identity to the amino acid sequence of SEQ ID NO:61, or an immunogenic fragment of the amino acid sequence of SEQ ID NO:61, or an amino acid sequence having at least 99.5%, 99%, 98.5%, 98%, 98.5%, or 97% identity to the amino acid sequence of SEQ ID NO:61. In one embodiment, the vaccine antigen comprises the amino acid sequence of SEQ ID NO:61.

In one embodiment, the RNA encoding the vaccine antigen comprises (i) a nucleotide sequence of SEQ ID NO:62, a nucleotide sequence having at least 99.5%, 99%, 98.5%, 98%, 98.5%, or 97% identity to the nucleotide sequence of SEQ ID NO:62, or a fragment of the nucleotide sequence of SEQ ID NO:62, or a nucleotide sequence having at least 99.5%, 99%, 98.5%, 98%, 98.5%, or 97% identity to the nucleotide sequence of SEQ ID NO:62, and/or (ii) an amino acid sequence comprising the amino acid sequence of SEQ ID NO:61, an amino acid sequence having at least 99.5%, 99%, 98.5%, 98%, 98.5%, or 97% identity to the amino acid sequence of SEQ ID NO:61, or an immunogenic fragment of the amino acid sequence of SEQ ID NO:61, or an amino acid sequence having at least 99.5%, 99%, 98.5%, 98%, 98.5%, or 97% identity to the amino acid sequence of SEQ ID NO:61. In one embodiment, the RNA encoding the vaccine antigen (i) comprises the nucleotide sequence of SEQ ID NO: 62, and/or (ii) encodes an amino acid sequence comprising the amino acid sequence of SEQ ID NO: 61.

In one embodiment, the RNA encoding the vaccine antigen comprises (i) a nucleotide sequence of SEQ ID NO:63, a nucleotide sequence having at least 99.5%, 99%, 98.5%, 98%, 98.5%, or 97% identity to the nucleotide sequence of SEQ ID NO:63, or a fragment of the nucleotide sequence of SEQ ID NO:63, or a nucleotide sequence having at least 99.5%, 99%, 98.5%, 98%, 98.5%, or 97% identity to the nucleotide sequence of SEQ ID NO:63, and/or (ii) an amino acid sequence comprising the amino acid sequence of SEQ ID NO:61, an amino acid sequence having at least 99.5%, 99%, 98.5%, 98%, 98.5%, or 97% identity to the amino acid sequence of SEQ ID NO:61, or an immunogenic fragment of the amino acid sequence of SEQ ID NO:61, or an amino acid sequence having at least 99.5%, 99%, 98.5%, 98%, 98.5%, or 97% identity to the amino acid sequence of SEQ ID NO:61. In one embodiment, the RNA encoding the vaccine antigen (i) comprises the nucleotide sequence of SEQ ID NO: 63, and/or (ii) encodes an amino acid sequence comprising the amino acid sequence of SEQ ID NO: 61.

In one embodiment, the vaccine antigen comprises the amino acid sequence of SEQ ID NO:52, an amino acid sequence having at least 99.5%, 99%, 98.5%, 98%, 98.5%, or 97% identity to the amino acid sequence of SEQ ID NO:52, or an immunogenic fragment of the amino acid sequence of SEQ ID NO:52, or an amino acid sequence having at least 99.5%, 99%, 98.5%, 98%, 98.5%, or 97% identity to the amino acid sequence of SEQ ID NO:52. In one embodiment, the vaccine antigen comprises the amino acid sequence of SEQ ID NO:52.

In one embodiment, the RNA encoding the vaccine antigen comprises (i) a nucleotide sequence of SEQ ID NO:53, a nucleotide sequence having at least 99.5%, 99%, 98.5%, 98%, 98.5%, or 97% identity to the nucleotide sequence of SEQ ID NO:53, or a fragment of the nucleotide sequence of SEQ ID NO:53, or a nucleotide sequence having at least 99.5%, 99%, 98.5%, 98%, 98.5%, or 97% identity to the nucleotide sequence of SEQ ID NO:53, and/or (ii) an amino acid sequence comprising the amino acid sequence of SEQ ID NO:52, an amino acid sequence having at least 99.5%, 99%, 98.5%, 98%, 98.5%, or 97% identity to the amino acid sequence of SEQ ID NO:52, or an immunogenic fragment of the amino acid sequence of SEQ ID NO:52, or an amino acid sequence having at least 99.5%, 99%, 98.5%, 98%, 98.5%, or 97% identity to the amino acid sequence of SEQ ID NO:52. In one embodiment, the RNA encoding the vaccine antigen (i) comprises the nucleotide sequence of SEQ ID NO:53, and/or (ii) encodes an amino acid sequence comprising the amino acid sequence of SEQ ID NO:52.

In one embodiment, the RNA encoding the vaccine antigen comprises (i) a nucleotide sequence of SEQ ID NO:54, a nucleotide sequence having at least 99.5%, 99%, 98.5%, 98%, 98.5%, or 97% identity to the nucleotide sequence of SEQ ID NO:54, or a fragment of the nucleotide sequence of SEQ ID NO:54, or a nucleotide sequence having at least 99.5%, 99%, 98.5%, 98%, 98.5%, or 97% identity to the nucleotide sequence of SEQ ID NO:54, and/or (ii) an amino acid sequence comprising the amino acid sequence of SEQ ID NO:52, an amino acid sequence having at least 99.5%, 99%, 98.5%, 98%, 98.5%, or 97% identity to the amino acid sequence of SEQ ID NO:52, or an immunogenic fragment of the amino acid sequence of SEQ ID NO:52, or an amino acid sequence having at least 99.5%, 99%, 98.5%, 98%, 98.5%, or 97% identity to the amino acid sequence of SEQ ID NO:52. In one embodiment, the RNA encoding the vaccine antigen (i) comprises the nucleotide sequence of SEQ ID NO:54, and/or (ii) encodes an amino acid sequence comprising the amino acid sequence of SEQ ID NO:52.

In one embodiment, the RNA encoding the vaccine antigen comprises (i) a nucleotide sequence of SEQ ID NO: 83, a nucleotide sequence having at least 99.5%, 99%, 98.5%, 98%, 98.5%, or 97% identity to the nucleotide sequence of SEQ ID NO: 83, or a fragment of the nucleotide sequence of SEQ ID NO: 83, or a nucleotide sequence having at least 99.5%, 99%, 98.5%, 98%, 98.5%, or 97% identity to the nucleotide sequence of SEQ ID NO: 83, and/or (ii) an amino acid sequence comprising the amino acid sequence of SEQ ID NO: 80, an amino acid sequence having at least 99.5%, 99%, 98.5%, 98%, 98.5%, or 97% identity to the amino acid sequence of SEQ ID NO: 80, or an immunogenic fragment of the amino acid sequence of SEQ ID NO: 80, or an amino acid sequence having at least 99.5%, 99%, 98.5%, 98%, 98.5%, or 97% identity to the amino acid sequence of SEQ ID NO: 80. In one embodiment, the RNA encoding the vaccine antigen (i) comprises the nucleotide sequence of SEQ ID NO: 83, and/or (ii) encodes an amino acid sequence comprising the amino acid sequence of SEQ ID NO: 80.

In one embodiment, the RNA encoding the vaccine antigen comprises (i) a nucleotide sequence of SEQ ID NO: 103, a nucleotide sequence having at least 99.5%, 99%, 98.5%, 98%, 98.5%, or 97% identity to the nucleotide sequence of SEQ ID NO: 103, or a fragment of the nucleotide sequence of SEQ ID NO: 103, or a nucleotide sequence having at least 99.5%, 99%, 98.5%, 98%, 98.5%, or 97% identity to the nucleotide sequence of SEQ ID NO: 103. and/or (ii) encodes an amino acid sequence comprising the amino acid sequence of SEQ ID NO: 100, an amino acid sequence having at least 99.5%, 99%, 98.5%, 98%, 98.5%, or 97% identity to the amino acid sequence of SEQ ID NO: 100, or an immunogenic fragment of the amino acid sequence of SEQ ID NO: 100, or an amino acid sequence having at least 99.5%, 99%, 98.5%, 98%, 98.5%, or 97% identity to the amino acid sequence of SEQ ID NO: 100. In one embodiment, the RNA encoding the vaccine antigen (i) comprises the nucleotide sequence of SEQ ID NO: 103, and/or (ii) encodes an amino acid sequence comprising the amino acid sequence of SEQ ID NO: 100.

In one embodiment, the RNA encoding the vaccine antigen comprises (i) a nucleotide sequence of SEQ ID NO:98, a nucleotide sequence having at least 99.5%, 99%, 98.5%, 98%, 98.5%, or 97% identity to the nucleotide sequence of SEQ ID NO:98, or a fragment of the nucleotide sequence of SEQ ID NO:98, or a nucleotide sequence having at least 99.5%, 99%, 98.5%, 98%, 98.5%, or 97% identity to the nucleotide sequence of SEQ ID NO:98, and/or (ii) an amino acid sequence comprising the amino acid sequence of SEQ ID NO:95, an amino acid sequence having at least 99.5%, 99%, 98.5%, 98%, 98.5%, or 97% identity to the amino acid sequence of SEQ ID NO:95, or an immunogenic fragment of the amino acid sequence of SEQ ID NO:95, or an amino acid sequence having at least 99.5%, 99%, 98.5%, 98%, 98.5%, or 97% identity to the amino acid sequence of SEQ ID NO:95. In one embodiment, the RNA encoding the vaccine antigen (i) comprises the nucleotide sequence of SEQ ID NO: 98 and/or (ii) encodes an amino acid sequence comprising the amino acid sequence of SEQ ID NO: 95.

In one embodiment, the vaccine antigen comprises a continuous sequence of the SARS-CoV-2 coronavirus spike (S) protein that consists of or consists essentially of the above amino acid sequence derived from the SARS-CoV-2 S protein or an immunogenic fragment thereof (antigenic peptide or protein) constituted by the vaccine antigen. In one embodiment, the vaccine antigen comprises a continuous sequence of the SARS-CoV-2 coronavirus spike (S) protein of no more than 220 amino acids, 215 amino acids, 210 amino acids, or 205 amino acids.

In one embodiment, the RNA encoding the vaccine antigen is a nucleoside modified messenger RNA (modRNA) described herein as BNT162b1 (RBP020.3), BNT162b2 (RBP020.1 or RBP020.2), or BNT162b3 (e.g., BNT162b3c). In one embodiment, the RNA encoding the vaccine antigen is a nucleoside modified messenger RNA (modRNA) described herein as RBP020.2. In one embodiment, the RNA encoding the vaccine antigen is a nucleoside modified messenger RNA (modRNA) described herein as BNT162b3 (e.g., BNT162b3c).

In one embodiment, the RNA encoding the vaccine antigen is a nucleoside-modified messenger RNA (modRNA) and (i) comprises the nucleotide sequence of SEQ ID NO:21, a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of SEQ ID NO:21, and/or (ii) encodes an amino acid sequence comprising the amino acid sequence of SEQ ID NO:5, or an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of SEQ ID NO:5. In one embodiment, the RNA encoding the vaccine antigen is a nucleoside-modified messenger RNA (modRNA) and (i) comprises the nucleotide sequence of SEQ ID NO:21, and/or (ii) encodes an amino acid sequence comprising the amino acid sequence of SEQ ID NO:5.

In one embodiment, the RNA encoding the vaccine antigen is a nucleoside-modified messenger RNA (modRNA) and (i) comprises a nucleotide sequence of SEQ ID NO: 19 or 20, a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of SEQ ID NO: 19 or 20, and/or (ii) encodes an amino acid sequence comprising an amino acid sequence of SEQ ID NO: 7, or an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of SEQ ID NO: 7. In one embodiment, the RNA encoding the vaccine antigen is a nucleoside-modified messenger RNA (modRNA) and (i) comprises a nucleotide sequence of SEQ ID NO: 19 or 20, and/or (ii) encodes an amino acid sequence comprising an amino acid sequence of SEQ ID NO: 7.

In one embodiment, the RNA encoding the vaccine antigen is a nucleoside-modified messenger RNA (modRNA) and (i) comprises a nucleotide sequence of SEQ ID NO:20, a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of SEQ ID NO:20, and/or (ii) encodes an amino acid sequence comprising an amino acid sequence of SEQ ID NO:7, or an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of SEQ ID NO:7. In one embodiment, the RNA encoding the vaccine antigen is a nucleoside-modified messenger RNA (modRNA) and (i) comprises a nucleotide sequence of SEQ ID NO:20, and/or (ii) encodes an amino acid sequence comprising an amino acid sequence of SEQ ID NO:7.

In one embodiment, the RNA encoding the vaccine antigen is a nucleoside-modified messenger RNA (modRNA) and (i) comprises a nucleotide sequence of SEQ ID NO: 30, a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of SEQ ID NO: 30, and/or (ii) encodes an amino acid sequence comprising an amino acid sequence of SEQ ID NO: 29, or an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of SEQ ID NO: 29. In one embodiment, the RNA encoding the vaccine antigen is a nucleoside-modified messenger RNA (modRNA) and (i) comprises a nucleotide sequence of SEQ ID NO: 30, and/or (ii) encodes an amino acid sequence comprising an amino acid sequence of SEQ ID NO: 29.

In one embodiment, the RNA encoding the vaccine antigen is a nucleoside-modified messenger RNA (modRNA) and (i) comprises a nucleotide sequence of SEQ ID NO:50, a nucleotide sequence having at least 99.5%, 99%, 98.5%, 98%, 98.5%, or 97% identity to the nucleotide sequence of SEQ ID NO:50, and/or (ii) encodes an amino acid sequence comprising an amino acid sequence of SEQ ID NO:49, or an amino acid sequence having at least 99.5%, 99%, 98.5%, 98%, 98.5%, or 97% identity to the amino acid sequence of SEQ ID NO:49. In one embodiment, the RNA encoding the vaccine antigen is a nucleoside-modified messenger RNA (modRNA) and (i) comprises a nucleotide sequence of SEQ ID NO:50, and/or (ii) encodes an amino acid sequence comprising an amino acid sequence of SEQ ID NO:49.

In one embodiment, the RNA encoding the vaccine antigen is a nucleoside-modified messenger RNA (modRNA) and (i) comprises the nucleotide sequence of SEQ ID NO:51, a nucleotide sequence having at least 99.5%, 99%, 98.5%, 98%, 98.5%, or 97% identity to the nucleotide sequence of SEQ ID NO:51, and/or (ii) encodes an amino acid sequence comprising the amino acid sequence of SEQ ID NO:49, or an amino acid sequence having at least 99.5%, 99%, 98.5%, 98%, 98.5%, or 97% identity to the amino acid sequence of SEQ ID NO:49. In one embodiment, the RNA encoding the vaccine antigen is a nucleoside-modified messenger RNA (modRNA) and (i) comprises the nucleotide sequence of SEQ ID NO:51, and/or (ii) encodes an amino acid sequence comprising the amino acid sequence of SEQ ID NO:49.

In one embodiment, the RNA encoding the vaccine antigen is a nucleoside-modified messenger RNA (modRNA) and (i) comprises the nucleotide sequence of SEQ ID NO:57, a nucleotide sequence having at least 99.5%, 99%, 98.5%, 98%, 98.5%, or 97% identity to the nucleotide sequence of SEQ ID NO:57, and/or (ii) encodes an amino acid sequence comprising the amino acid sequence of SEQ ID NO:55, or an amino acid sequence having at least 99.5%, 99%, 98.5%, 98%, 98.5%, or 97% identity to the amino acid sequence of SEQ ID NO:55. In one embodiment, the RNA encoding the vaccine antigen is a nucleoside-modified messenger RNA (modRNA) and (i) comprises the nucleotide sequence of SEQ ID NO:57, and/or (ii) encodes an amino acid sequence comprising the amino acid sequence of SEQ ID NO:55.

In one embodiment, the RNA encoding the vaccine antigen is a nucleoside-modified messenger RNA (modRNA) and (i) comprises a nucleotide sequence of SEQ ID NO: 60, a nucleotide sequence having at least 99.5%, 99%, 98.5%, 98%, 98.5%, or 97% identity to the nucleotide sequence of SEQ ID NO: 60, and/or (ii) encodes an amino acid sequence comprising an amino acid sequence of SEQ ID NO: 58, or an amino acid sequence having at least 99.5%, 99%, 98.5%, 98%, 98.5%, or 97% identity to the amino acid sequence of SEQ ID NO: 58. In one embodiment, the RNA encoding the vaccine antigen is a nucleoside-modified messenger RNA (modRNA) and (i) comprises a nucleotide sequence of SEQ ID NO: 60, and/or (ii) encodes an amino acid sequence comprising an amino acid sequence of SEQ ID NO: 58.

In one embodiment, the RNA encoding the vaccine antigen is a nucleoside-modified messenger RNA (modRNA) and (i) comprises the nucleotide sequence of SEQ ID NO: 63, a nucleotide sequence having at least 99.5%, 99%, 98.5%, 98%, 98.5%, or 97% identity to the nucleotide sequence of SEQ ID NO: 63, and/or (ii) encodes an amino acid sequence comprising the amino acid sequence of SEQ ID NO: 61, or an amino acid sequence having at least 99.5%, 99%, 98.5%, 98%, 98.5%, or 97% identity to the amino acid sequence of SEQ ID NO: 61. In one embodiment, the RNA encoding the vaccine antigen is a nucleoside-modified messenger RNA (modRNA) and (i) comprises the nucleotide sequence of SEQ ID NO: 63, and/or (ii) encodes an amino acid sequence comprising the amino acid sequence of SEQ ID NO: 61.

In one embodiment, the RNA encoding the vaccine antigen is a nucleoside-modified messenger RNA (modRNA) and (i) comprises the nucleotide sequence of SEQ ID NO:53, a nucleotide sequence having at least 99.5%, 99%, 98.5%, 98%, 98.5%, or 97% identity to the nucleotide sequence of SEQ ID NO:53, and/or (ii) encodes an amino acid sequence comprising the amino acid sequence of SEQ ID NO:52, or an amino acid sequence having at least 99.5%, 99%, 98.5%, 98%, 98.5%, or 97% identity to the amino acid sequence of SEQ ID NO:52. In one embodiment, the RNA encoding the vaccine antigen is a nucleoside-modified messenger RNA (modRNA) and (i) comprises the nucleotide sequence of SEQ ID NO:53, and/or (ii) encodes an amino acid sequence comprising the amino acid sequence of SEQ ID NO:52.

In one embodiment, the RNA encoding the vaccine antigen is a nucleoside-modified messenger RNA (modRNA) and (i) comprises the nucleotide sequence of SEQ ID NO:54, a nucleotide sequence having at least 99.5%, 99%, 98.5%, 98%, 98.5%, or 97% identity to the nucleotide sequence of SEQ ID NO:54, and/or (ii) encodes an amino acid sequence comprising the amino acid sequence of SEQ ID NO:52, or an amino acid sequence having at least 99.5%, 99%, 98.5%, 98%, 98.5%, or 97% identity to the amino acid sequence of SEQ ID NO:52. In one embodiment, the RNA encoding the vaccine antigen is a nucleoside-modified messenger RNA (modRNA) and (i) comprises the nucleotide sequence of SEQ ID NO:54, and/or (ii) encodes an amino acid sequence comprising the amino acid sequence of SEQ ID NO:52.

As used herein, the term "vaccine" refers to a composition that induces an immune response upon inoculation into a subject. In some embodiments, the induced immune response provides protective immunity.

In one embodiment, the RNA encoding the antigen molecule is expressed in the cells of the subject to provide the antigen molecule. In one embodiment, the expression of the antigen molecule is on the cell surface or in the extracellular space. In one embodiment, the antigen molecule is presented in the context of MHC. In one embodiment, the RNA encoding the antigen molecule is transiently expressed in the cells of the subject. In one embodiment, after administration of the RNA encoding the antigen molecule, expression of the RNA encoding the antigen molecule in the muscle occurs, in particular after intramuscular administration of the RNA encoding the antigen molecule. In one embodiment, after administration of the RNA encoding the antigen molecule, expression of the RNA encoding the antigen molecule in the spleen occurs. In one embodiment, after administration of the RNA encoding the antigen molecule, expression of the RNA encoding the antigen molecule occurs in antigen presenting cells, preferably professional antigen presenting cells. In one embodiment, the antigen presenting cells are selected from the group consisting of dendritic cells, macrophages, and B cells. In one embodiment, after administration of the RNA encoding the antigen molecule, there is no or essentially no expression of the RNA encoding the antigen molecule in the lungs and/or liver. In one embodiment, after administration of the RNA encoding the antigen molecule, expression of the RNA encoding the antigen molecule in the spleen is at least 5 times the amount of expression in the lungs.

In some embodiments, following administration to a subject, particularly following intramuscular administration, the methods and agents, e.g., mRNA compositions, described herein result in delivery of RNA encoding a vaccine antigen to lymph nodes and/or the spleen. In some embodiments, the RNA encoding a vaccine antigen is detectable in the lymph nodes and/or spleen 6 hours or more, preferably up to 6 days or more, following administration.

In some embodiments, following administration to a subject, particularly following intramuscular administration, the methods and agents, e.g., mRNA compositions, described herein result in delivery of RNA encoding a vaccine antigen to B cell follicles, cortical sinuses, and/or T cell zones, particularly B cell follicles and/or cortical sinuses of lymph nodes.

In some embodiments, following administration to a subject, particularly following intramuscular administration, the methods and agents, e.g., mRNA compositions, described herein result in delivery of RNA encoding a vaccine antigen to B cells (CD19+), cortical sinus macrophages (CD169+) and/or dendritic cells (CD11c+) in the T cell zone and intermediate sinus of a lymph node, particularly B cells (CD19+) and/or cortical sinus macrophages (CD169+) of the lymph node.

In some embodiments, following administration to a subject, particularly following intramuscular administration, the methods and agents, e.g., mRNA compositions, described herein result in delivery of RNA encoding a vaccine antigen to the white pulp and/or spleen.

In some embodiments, following administration to a subject, particularly following intramuscular administration, the methods and agents, e.g., mRNA compositions, described herein result in delivery of RNA encoding a vaccine antigen to B cells, DCs (CD11c+), particularly those surrounding B cells, and/or macrophages in the spleen, particularly B cells and/or DCs (CD11c+).

In one embodiment, the vaccine antigen is expressed in lymph nodes and/or spleen, particularly in cells of said lymph nodes and/or spleen.

Peptide and protein antigens suitable for use according to the present disclosure typically include peptides or proteins that include an epitope of the SARS-CoV-2 S protein or a functional variant thereof to induce an immune response. The peptide or protein or epitope may be derived from a target antigen, i.e., an antigen against which an immune response is elicited. For example, the peptide or protein antigen, or an epitope contained in the peptide or protein antigen, may be the target antigen or a fragment or variant of the target antigen. The target antigen may be a coronavirus S protein, in particular the SARS-CoV-2 S protein.

An antigen molecule or a processed product, e.g., a fragment thereof, may bind to an antigen receptor, such as a BCR or TCR, carried by an immune effector cell, or to an antibody.

Peptide and protein antigens, i.e., peptide and protein antigens provided to a subject according to the present disclosure by administering RNA encoding the vaccine antigen, preferably result in the induction of an immune response, e.g., a humoral immune response and/or a cellular immune response, in the subject provided with the peptide or protein antigen. The immune response is preferably directed against a target antigen, in particular a coronavirus S protein, in particular a SARS-CoV-2 S protein. Thus, the vaccine antigen may comprise the target antigen, a variant thereof, or a fragment thereof. In one embodiment, such a fragment or variant is immunologically equivalent to the target antigen. In the context of the present disclosure, the term "fragment of an antigen" or "variant of an antigen" refers to an agent that results in the induction of an immune response that targets an antigen, i.e., the target antigen. Thus, the vaccine antigen may correspond to or comprise the target antigen, or may correspond to or comprise a fragment of the target antigen, or may correspond to or comprise an antigen that is homologous to the target antigen or a fragment thereof. Thus, according to the present disclosure, the vaccine antigen may comprise an immunogenic fragment of the target antigen or an amino acid sequence that is homologous to an immunogenic fragment of the target antigen. An "immunogenic fragment of an antigen" according to the present disclosure preferably relates to a fragment of an antigen that is capable of inducing an immune response against the target antigen. The vaccine antigen may be a recombinant antigen.

The term "immunologically equivalent" means that an immunologically equivalent molecule, such as an immunologically equivalent amino acid sequence, exhibits the same or essentially the same immunological properties and/or exerts the same or essentially the same immunological effect, e.g., with respect to the type of immunological effect. In the context of the present disclosure, the term "immunologically equivalent" is preferably used with respect to the immunological effect or properties of the antigen or antigen variant used for immunization. For example, an amino acid sequence is immunologically equivalent to a reference amino acid sequence if, when exposed to the immune system of a subject, the amino acid sequence elicits an immune response with specificity reactive with the reference amino acid sequence.

"Activated" or "stimulated" as used herein refers to the state of an immune effector cell, such as a T cell, that has been stimulated sufficiently to induce detectable cell proliferation. Activation may also be associated with the initiation of signaling pathways, induced cytokine production, and detectable effector function. The term "activated immune effector cell" refers, among other things, to an immune effector cell that is undergoing cell division.

The term "priming" refers to the process by which an immune effector cell, such as a T cell, first comes into contact with its specific antigen, triggering differentiation into an effector cell, such as an effector T cell.

The term "clonal expansion" or "expansion" refers to a process by which a specific entity is multiplied. In the context of the present disclosure, the term is preferably used in the context of an immune response in which immune effector cells are stimulated by an antigen, proliferate, and the specific immune effector cells that recognize that antigen are amplified. Preferably, clonal expansion results in differentiation of immune effector cells.

The term "antigen" relates to an agent comprising an epitope against which an immune response can be generated. The term "antigen" includes, inter alia, proteins and peptides. In one embodiment, the antigen is presented by a cell of the immune system, such as an antigen-presenting cell, such as a dendritic cell or a macrophage. The antigen or its processing product, such as a T-cell epitope, is in one embodiment bound by a T-cell or B-cell receptor, or by an immunoglobulin molecule, such as an antibody. Thus, the antigen or its processing product may react specifically with an antibody or a T-lymphocyte (T-cell). In one embodiment, the antigen is a viral antigen, such as a coronavirus S protein, e.g., SARS-CoV-2 S protein, and the epitope is derived from such an antigen.

The term "viral antigen" refers to any viral component that has antigenic properties, i.e., is capable of eliciting an immune response in an individual. A viral antigen can be a coronavirus S protein, e.g., SARS-CoV-2 S protein.

The term "expressed on the cell surface" or "associated with the cell surface" means that a molecule, such as an antigen, is associated with and located in the plasma membrane of a cell, with at least a portion of the molecule facing the extracellular space of the cell and accessible from the outside of the cell, for example, by an antibody located on the outside of the cell. In this context, the portion is preferably at least 4, preferably at least 8, preferably at least 12, more preferably at least 20 amino acids. The association can be direct or indirect. For example, the association can be by one or more transmembrane domains, one or more lipid anchors, or by interaction with any other protein, lipid, sugar, or other structure that can be found on the outer leaflet of the plasma membrane of the cell. For example, a molecule that associates with the surface of a cell can be a transmembrane protein with an extracellular portion, or a protein that associates with the surface of a cell by interacting with another protein that is a transmembrane protein.

"Cell surface" or "surface of a cell" is used according to its normal meaning in the art and thus includes the exterior of a cell that is accessible for binding by proteins and other molecules. An antigen is expressed on the surface of a cell if it is located on the surface of that cell and is accessible for binding by an antigen-specific antibody that is attached to the cell.

The term "extracellular portion" or "exodomain" in the context of the present disclosure refers to a portion of a molecule, such as a protein, that faces the extracellular space of a cell and is preferably accessible from the outside of said cell, e.g., by a binding molecule, such as an antibody, that is located on the outside of the cell. Preferably, the term refers to one or more extracellular loops or domains, or fragments thereof.

The term "epitope" refers to a portion or fragment of a molecule, such as an antigen, that is recognized by the immune system. For example, an epitope may be recognized by a T cell, a B cell, or an antibody. An epitope of an antigen may include a continuous or discontinuous portion of the antigen and may be about 5 to about 100, e.g., about 5 to about 50, more preferably about 8 to about 30, and most preferably about 8 to about 25 amino acids in length, e.g., an epitope may preferably be 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 amino acids in length. In one embodiment, an epitope is about 10 to about 25 amino acids in length. The term "epitope" includes T cell epitopes.

The term "T cell epitope" refers to a portion or fragment of a protein that is recognized by a T cell when presented in the context of an MHC molecule. The term "major histocompatibility complex" and the abbreviation "MHC" refer to a complex of genes that includes MHC class I and MHC class II molecules and is present in all vertebrates. MHC proteins or molecules are important in signaling between lymphocytes and antigen-presenting or diseased cells in an immune response; MHC proteins or molecules bind peptide epitopes and present them for recognition by the T cell receptor on T cells. Proteins encoded by MHC are expressed on the surface of cells and present both self antigens (peptide fragments from the cell itself) and non-self antigens (e.g., fragments of invading microorganisms) to T cells. In the case of class I MHC/peptide complexes, the binding peptide is typically about 8 to about 10 amino acids in length, although longer or shorter peptides may be effective. For class II MHC/peptide complexes, the binding peptides are typically about 10 to about 25 amino acids in length, particularly about 13 to about 18 amino acids in length, although longer and shorter peptides may be effective.

Peptide and protein antigens can be 2-100 amino acids, for example, 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50 amino acids in length. In some embodiments, peptides can be more than 50 amino acids. In some embodiments, peptides can be more than 100 amino acids.

The peptide or protein antigen can be any peptide or protein that can induce or increase the ability of the immune system to develop antibody and T cell responses against the peptide or protein.

In one embodiment, the vaccine antigen is recognized by an immune effector cell. Preferably, when recognized by an immune effector cell, the vaccine antigen, in the presence of an appropriate co-stimulatory signal, can induce stimulation, priming, and/or expansion of immune effector cells bearing an antigen receptor that recognizes the vaccine antigen. In the context of the embodiments of the present disclosure, the vaccine antigen is preferably presented or present on the surface of a cell, preferably an antigen-presenting cell. In one embodiment, the antigen is presented by a diseased cell, such as a virus-infected cell. In one embodiment, the antigen receptor is a TCR that binds to an epitope of the antigen presented in the context of MHC. In one embodiment, binding of the TCR, when expressed by and/or present on a T cell, to an antigen presented by a cell, such as an antigen-presenting cell, results in stimulation, priming, and/or expansion of the T cell. In one embodiment, binding of the TCR, when expressed by and/or present on a T cell, to an antigen presented on a diseased cell results in cytolysis and/or apoptosis of the diseased cell, and the T cell preferably releases cytotoxic factors, such as perforin and granzymes.

In one embodiment, the antigen receptor is an antibody or B cell receptor that binds to an epitope within the antigen. In one embodiment, the antibody or B cell receptor binds to a natural epitope of the antigen.

Bivalent Vaccine Combinations Multiple different spike proteins (S) of SARS-CoV-2 variants as described herein can be delivered in combination, for example, by a bivalent RNA vaccine that includes at least one RNA encoding two or more spike proteins (S) or any variants thereof (e.g., as described herein). Exemplary combinations of spike proteins are described herein and are shown, for example, in the table below. A bivalent vaccine can include any of these described combinations in any of the spike proteins encoded by the RNA vaccine. Additionally, the mutations described herein (e.g., in Tables 2A, 2B, and 2C) can be included in any of the various coronavirus strains described herein, and additionally, any additional known coronavirus strains (see, e.g., the World Health Organization (WHO) database for tracking SARS-CoV-2 variants at https://www.who.int/activities/tracking-SARS-CoV-2-variants).

Exemplary spike protein variants (where the mutations described herein apply to various strains of the coronavirus spike protein sequence) are shown in Table 7 below.

According to the present disclosure, in some embodiments, the RNA vaccine comprises at least one RNA encoding one or more coronavirus spike proteins (e.g., spike protein variants described in Table 7). In some embodiments, the RNA vaccine comprises at least two RNAs, each encoding a different coronavirus spike protein (e.g., spike protein variants described in Table 7). The coronavirus spike protein antigens may be administered as single-stranded 5'-capped mRNAs that are translated into the respective proteins upon entry into the cells of the subject receiving the RNA. Preferably, the RNA comprises structural elements optimized for maximum efficacy of the RNA with respect to stability and translation efficiency (5' cap, 5' UTR, 3' UTR, poly(A) sequence).

In one embodiment, beta-S-ARCA (D1) is utilized as a specific capping structure at the 5' end of the RNA. In one embodiment, m27,3' ^- OGppp (m12'-O)ApG is utilized as a specific capping structure at the 5' end of the RNA. In one embodiment, the 5'-UTR sequence is derived from human alpha-globin mRNA, optionally with an optimized "Kozak sequence" to increase translation efficiency. In one embodiment, a combination of two sequence elements (FI elements) derived from the "amino-terminal enhancer of split" (AES) mRNA (called F) and the mitochondrially encoded 12S ribosomal RNA (called I) are placed between the coding sequence and the poly(A) sequence to ensure higher maximum protein levels and long-term persistence of the mRNA. In one embodiment, two repeated 3'-UTRs derived from human beta-globin mRNA are placed between the coding sequence and the poly(A) sequence to ensure higher maximum protein levels and long-term persistence of the mRNA. In one embodiment, a poly(A) sequence measuring 110 nucleotides in length is used, consisting of a stretch of 30 adenosine residues followed by a 10 nucleotide linker sequence and another 70 adenosine residues, which was designed to improve RNA stability and translation efficiency.

RNA vaccines encoding any of the coronavirus spike protein variants described herein (and in, e.g., Table 7) may include any of the other nucleic acid modifications and RNA assembly components described herein.

In some embodiments, the RNA molecules may be formulated in lipid nanoparticles (LNPs) to form a bivalent vaccine (e.g., the two RNA populations are mixed prior to LNP formulation, or each RNA is formulated in a separate LNP composition and subsequently mixed together).

Exemplary combinations of spike protein variants described herein (e.g., as shown in Table 7) may be utilized in a bivalent RNA vaccine. Exemplary combinations of spike proteins that may be utilized in a bivalent RNA vaccine are shown in Table 8 below.

Nucleic Acids The term "polynucleotide" or "nucleic acid" as used herein is intended to include DNA and RNA, e.g., genomic DNA, cDNA, mRNA, recombinantly produced and chemically synthesized molecules. Nucleic acids can be single-stranded or double-stranded. RNA includes in vitro transcribed RNA (IVT RNA) or synthetic RNA. According to the present disclosure, polynucleotides are preferably isolated.

The nucleic acid may be contained in a vector. The term "vector" as used herein includes any vector known to those skilled in the art, including plasmid vectors, cosmid vectors, phage vectors such as lambda phage, viral vectors such as retrovirus, adenovirus, or baculovirus vectors, or artificial chromosome vectors such as bacterial artificial chromosomes (BAC), yeast artificial chromosomes (YAC), or P1 artificial chromosomes (PAC). Such vectors include expression and cloning vectors. Expression vectors include plasmids and viral vectors, and generally contain a desired coding sequence and appropriate DNA sequences required for expression of an operably linked coding sequence in a particular host organism (e.g., bacteria, yeast, plants, insects, or mammals) or in an in vitro expression system. Cloning vectors are generally used to manipulate and amplify a particular desired DNA fragment and may lack functional sequences required for expression of the desired DNA fragment.

In one embodiment of all aspects of the present disclosure, the RNA encoding the vaccine antigen is expressed in cells, such as antigen-presenting cells, of the subject being treated to provide the vaccine antigen.

The nucleic acids described herein may be recombinant and/or isolated molecules.

In this disclosure, the term "RNA" refers to a nucleic acid molecule that includes ribonucleotide residues. In a preferred embodiment, the RNA contains all or most of the ribonucleotide residues. As used herein, "ribonucleotide" refers to a nucleotide that has a hydroxyl group at the 2' position of a β-D-ribofuranosyl group. RNA includes, but is not limited to, double-stranded RNA, single-stranded RNA, isolated RNA such as partially purified RNA, essentially pure RNA, synthetic RNA, recombinantly produced RNA, and modified RNA that differs from naturally occurring RNA by the addition, deletion, substitution, and/or modification of one or more nucleotides. Such modifications may refer to the addition of non-nucleotide material to internal RNA nucleotides or to the end(s) of the RNA. It is also contemplated herein that the nucleotides in the RNA may be non-standard nucleotides such as chemically synthesized nucleotides or deoxynucleotides. In this disclosure, these modified RNAs are considered analogs of naturally occurring RNA.

In certain embodiments of the present disclosure, the RNA is messenger RNA (mRNA), which refers to an RNA transcript that codes for a peptide or protein. As established in the art, mRNA generally includes a 5' untranslated region (5'-UTR), a peptide coding region, and a 3' untranslated region (3'-UTR). In some embodiments, the RNA is produced by in vitro transcription or chemical synthesis. In one embodiment, the mRNA is produced by in vitro transcription using a DNA template, where DNA refers to a nucleic acid that contains deoxyribonucleotides.

In one embodiment, the RNA is in vitro transcribed RNA (IVT-RNA) and can be obtained by in vitro transcription of a suitable DNA template. The promoter for controlling the transcription can be any promoter of any RNA polymerase. The DNA template for in vitro transcription can be obtained by cloning a nucleic acid, in particular a cDNA, and introducing it into a suitable vector for in vitro transcription. The cDNA can be obtained by reverse transcription of the RNA.

In certain embodiments of the present disclosure, the RNA is a "replicon RNA" or simply a "replicon", in particular a "self-replicating RNA" or a "self-amplifying RNA". In a particularly preferred embodiment, the replicon or self-replicating RNA is derived from or includes elements derived from a ssRNA virus, in particular a positive-stranded ssRNA virus such as an alphavirus. Alphaviruses are typical representatives of positive-stranded RNA viruses. Alphaviruses replicate in the cytoplasm of infected cells (for a review of the alphavirus life cycle, see Jose et al., Future Microbiol., 2009, vol. 4, pp. 837-856). The total genome length of many alphaviruses typically ranges from 11,000 to 12,000 nucleotides, and the genomic RNA typically has a 5' cap and a 3' poly(A) tail. The genome of an alphavirus encodes nonstructural proteins (involved in transcription, modification, and replication of viral RNA and involved in protein modification) as well as structural proteins (forming the virus particle). There are typically two open reading frames (ORFs) in the genome. The four nonstructural proteins (nsP1-nsP4) are typically co-encoded by the first ORF beginning near the 5' end of the genome, while the alphavirus structural proteins are co-encoded by the second ORF found downstream of the first ORF and extending near the 3' end of the genome. Typically, the first ORF is larger than the second ORF, the ratio being approximately 2:1. In cells infected by alphaviruses, only the nucleic acid sequences encoding the nonstructural proteins are translated from the genomic RNA, while the genetic information encoding the structural proteins is translatable from subgenomic transcripts, which are RNA molecules similar to eukaryotic messenger RNA (mRNA, Gould et al., 2010, Antiviral Res., vol. 87 pp. 111-124). After infection, i.e., early in the viral life cycle, the (+) strand genomic RNA acts directly like a messenger RNA for the translation of an open reading frame encoding the nonstructural polyprotein (nsP1234). Alphavirus-derived vectors have been proposed for the delivery of foreign genetic information to target cells or organisms. In a simple approach, the open reading frame encoding the alphavirus structural proteins is replaced by an open reading frame encoding the protein of interest. Alphavirus-based trans-replication systems rely on alphavirus nucleotide sequence elements on two separate nucleic acid molecules, one encoding the viral replicase and the other capable of replicating by said replicase in trans (hence the name trans-replication system). Trans-replication requires the presence of both of these nucleic acid molecules in a given host cell. The nucleic acid molecule capable of replicating by the replicase in trans must contain certain alphavirus sequence elements to allow recognition by the alphavirus replicase and RNA synthesis.

In one embodiment, the RNA described herein can have modified nucleosides. In some embodiments, the RNA includes a modified nucleoside in place of at least one (e.g., all) uridine.

The term "uracil," as used herein, describes one of the nucleobases that can occur in RNA nucleic acids. The structure of uracil is:

The term "uridine," as used herein, describes one of the nucleosides that can occur in RNA. The structure of uridine is:

UTP (uridine 5'-triphosphate) has the following structure:

Pseudo-UTP (pseudo-uridine 5'-triphosphate) has the following structure:

"Pseudouridine" is an example of a modified nucleoside that is an isomer of uridine in which uracil is attached to the pentose ring via a carbon-carbon bond instead of a nitrogen-carbon glycosidic bond.

Another exemplary modified nucleoside is N1-methyl-pseudouridine (m1Ψ), which has the following structure:

N1-methyl-pseudo-UTP has the following structure:

Another exemplary modified nucleoside is 5-methyl-uridine (m5U), which has the following structure:

In some embodiments, one or more uridines in the RNA described herein are replaced by a modified nucleoside. In some embodiments, the modified nucleoside is a modified uridine.

In some embodiments, the RNA includes a modified nucleoside in place of at least one uridine. In some embodiments, the RNA includes a modified nucleoside in place of each uridine.

In some embodiments, the modified nucleosides are independently selected from pseudouridine (ψ), N1-methyl-pseudouridine (m1ψ), and 5-methyl-uridine (m5U). In some embodiments, the modified nucleoside comprises pseudouridine (ψ). In some embodiments, the modified nucleoside comprises N1-methyl-pseudouridine (m1ψ). In some embodiments, the modified nucleoside comprises 5-methyl-uridine (m5U). In some embodiments, the RNA may comprise two or more types of modified nucleosides, the modified nucleosides are independently selected from pseudouridine (ψ), N1-methyl-pseudouridine (m1ψ), and 5-methyl-uridine (m5U). In some embodiments, the modified nucleoside comprises pseudouridine (ψ) and N1-methyl-pseudouridine (m1ψ). In some embodiments, modified nucleosides include pseudouridine (Ψ) and 5-methyl-uridine (m5U). In some embodiments, modified nucleosides include N1-methyl-pseudouridine (m1Ψ) and 5-methyl-uridine (m5U). In some embodiments, modified nucleosides include pseudouridine (Ψ), N1-methyl-pseudouridine (m1Ψ), and 5-methyl-uridine (m5U).

In some embodiments, the modified nucleoside replacing one or more, e.g., all, uridines in the RNA is 3-methyl-uridine (m ³ U), 5-methoxy-uridine (mo ⁵ U), 5-aza-uridine, 6-aza-uridine, 2-thio-5-aza-uridine, 2-thio-uridine (s 2 U), 4-thio-uridine (s ⁴ U), 4-thio-pseudouridine, 2-thio-pseudouridine, 5-hydroxy-uridine (ho ⁵ U), 5-aminoallyl-uridine, 5-halo-uridine (e.g., 5-iodo-uridine or 5-bromo-uridine), uridine 5-oxyacetic acid (cmo ^{5 U), uridine 5-oxyacetic acid methyl ester (mcmo 5 U), 5-carboxymethyl-uridine (cm 5} U), 1-carboxymethyl-pseudouridine, 5-carboxyhydroxymethyl-uridine (chm 5 U), 5-carboxymethyl-uridine (cm ^{5 U), 1-carboxymethyl-pseudouridine, 5-carboxyhydroxymethyl-uridine (chm 5 U), 5-aminoallyl-uridine, 5-halo-uridine (e.g., 5-iodo-uridine or 5-bromo-uridine), uridine 5-oxyacetic acid (cmo 5} U), uridine 5-oxyacetic acid methyl ester (mcmo 5 U), 5-carboxymethyl-uridine (cm 5 U), 1-carboxymethyl-pseudouridine, 5-carboxyhydroxymethyl-uridine (chm ⁵ U), 5-aminoallyl-uridine, 5-halo-uridine (e.g., 5-iodo-uridine or 5-bromo-uridine), uridine 5-oxyacetic acid methyl ester (mcmo ⁵ U), 5-carboxymethyl-uridine (cm ⁵ U), U), 5-carboxyhydroxymethyl-uridine methyl ester (mchm ⁵ U), 5-methoxycarbonylmethyl-uridine (mcm ⁵ U), 5-methoxycarbonylmethyl-2-thio-uridine (mcm ⁵ s ² U), 5-aminomethyl-2-thio-uridine (nm ⁵ s ² U), 5-methylaminomethyl-uridine (mnm ⁵ U), 1-ethyl-pseudouridine, 5-methylaminomethyl-2-thio-uridine (mnm ⁵ s ² U), 5-methylaminomethyl-2-seleno-uridine (mnm ⁵ se ² U), 5-carboxymethyl-uridine (ncm ⁵ U), 5-carboxymethylaminomethyl-uridine (cmnm ⁵ U), 5-carboxymethylaminomethyl-2-thio-uridine (cmnm ⁵ s 2 ^U) , U), 5-propynyl-uridine, 1-propynyl-pseudouridine, 5-taurinomethyl-uridine (τm ⁵ U), 1-taurinomethyl-pseudouridine, 5-taurinomethyl-2-thio-uridine (τm5s2U), 1-taurinomethyl-4-thio-pseudouridine), 5-methyl-2-thio-uridine (m ⁵ s ² U), 1-methyl-4-thio-pseudouridine (m ¹ s ⁴ ψ), 4-thio-1-methyl-pseudouridine, 3-methyl-pseudouridine (m ³ ψ), 2-thio-1-methyl-pseudouridine, 1-methyl-1-deaza-pseudouridine, 2-thio-1-methyl-1-deaza-pseudouridine, dihydrouridine (D), dihydropseudouridine, 5,6-dihydrouridine, 5-methyl-dihydrouridine (m ⁵ D), 2-thio-dihydrouridine, 2-thio-dihydropseudouridine, 2-methoxy-uridine, 2-methoxy-4-thio-uridine, 4-methoxy-pseudouridine, 4-methoxy-2-thio-pseudouridine, N1-methyl-pseudouridine, 3-(3-amino-3-carboxypropyl)uridine (acp ³ U), 1-methyl-3-(3-amino-3-carboxypropyl)pseudouridine (acp ³ ψ), 5-(isopentenylaminomethyl)uridine (inm ⁵ U), 5-(isopentenylaminomethyl)-2-thio-uridine (inm ⁵ s ² U), α-thio-uridine, 2'-O-methyl-uridine (Um), 5,2'-O-dimethyl-uridine (m ⁵ Um), 2'-O-methyl-pseudouridine (ψm), 2-thio-2'-O-methyl-uridine (s ² Um), 5-methoxycarbonylmethyl-2'-O-methyl-uridine (mcm ⁵ Um), 5-carbamoylmethyl-2'-O-methyl-uridine (ncm ⁵ Um), 5-carboxymethylaminomethyl-2'-O-methyl-uridine (cmnm ⁵ Um), 3,2'-O-dimethyl-uridine (m ³ Um), 5-(isopentenylaminomethyl)-2'-O-methyl-uridine (inm ⁵ Um), 1-thio-uridine, deoxythymidine, 2'-F-ara-uridine, 2'-F-uridine, 2'-OH-ara-uridine, 5-(2-carbomethoxyvinyl)uridine, 5-[3-(1-E-propenylamino)uridine, or any other modified uridine known in the art.

In one embodiment, the RNA includes other modified nucleosides or includes additional modified nucleosides, such as modified cytidine. For example, in one embodiment, in the RNA, 5-methylcytidine is partially or completely replaced by cytidine, preferably completely. In one embodiment, the RNA includes 5-methylcytidine and one or more selected from pseudouridine (ψ), N1-methyl-pseudouridine (m1ψ), and 5-methyl-uridine (m5U). In one embodiment, the RNA includes 5-methylcytidine and N1-methyl-pseudouridine (m1ψ). In some embodiments, the RNA includes 5-methylcytidine in place of each cytidine and N1-methyl-pseudouridine (m1ψ) in place of each uridine.

In some embodiments, the RNA according to the present disclosure includes a 5'-cap. In one embodiment, the RNA of the present disclosure does not have an uncapped 5'-triphosphate. In one embodiment, the RNA may be modified with a 5'-cap analog. The term "5'-cap" refers to the structure found on the 5' end of an RNA (e.g., mRNA) molecule and generally consists of a guanosine nucleotide connected to the RNA (e.g., mRNA) via a 5' to 5'-triphosphate bond. In one embodiment, the guanosine is methylated at the 7 position. Providing the RNA with a 5'-cap or 5'-cap analog may be accomplished by in vitro transcription, where the 5'-cap is co-transcriptionally expressed within the RNA strand or may be attached to the RNA post-transcriptionally using a capping enzyme.

In some embodiments, an RNA (e.g., an mRNA) comprises cap0, cap1, or cap2, preferably cap1 or cap2, more preferably cap1. According to the present disclosure, the term "cap0" comprises the structure "m ⁷ GpppN", where N is any nucleoside having an OH moiety at the 2' position. According to the present disclosure, the term "cap1" comprises the structure "m ⁷ GpppNm", where Nm is any nucleoside having an OCH ₃ moiety at the 2' position. According to the present disclosure, the term "cap2" comprises the structure "m ⁷ GpppNmNm", where each Nm is independently any nucleoside having an OCH ₃ moiety at the 2' position.

In some embodiments, the building block cap for RNA is m ₂ ^7,3′-O Gppp(m ₁ ^2′-O )ApG (sometimes referred to as m ₂ ^7,3′O G(5′)ppp(5′)m ^2′-O ApG) and has the following structure:

The following is an exemplary Cap1 RNA, which contains RNA and m ₂ ^7,3'O G(5')ppp(5')m ^2'-O ApG:

The following is another exemplary Cap1 RNA (without cap analog):

In some embodiments, the RNA is modified with a "Cap0" structure, using a cap analog anti-reverse cap (ARCA Cap(m ₂ ^7,3'O G(5')ppp(5')G)), which in one embodiment has the following structure:

The following is an exemplary Cap0 RNA ^{that contains RNA and m27,3'OG} ₍ 5')ppp(5')G:

In some embodiments, the "Cap0" structure is generated using the cap analog beta-S-ARCA (m ₂ ^7,2'O G(5')ppSp(5')G) having the following structure:

The following is ^an exemplary Cap0 RNA containing beta-S-ARCA ( _m27,2'OG (5')ppSp(5')G) and RNA:

The "D1" diastereomer of beta-S-ARCA or "beta-S-ARCA(D1)" is the diastereomer of beta-S-ARCA that elutes first on an HPLC column compared to the D2 diastereomer of beta-S-ARCA (beta-S-ARCA(D2)) and therefore exhibits a shorter retention time (see WO 2011/015347, incorporated herein by reference).

Particularly preferred caps are beta-S-ARCA(D1)(m ₂ ^7,2'-O GppSpG) or m ₂ ^7,3'-O Gppp(m ₁ ^2'-O )ApG.

In some embodiments, an RNA according to the present disclosure comprises a 5'-UTR and/or a 3'-UTR. The term "untranslated region" or "UTR" refers to a region in a DNA molecule that is transcribed but not translated into an amino acid sequence, or the corresponding region in an RNA molecule, such as an mRNA molecule. An untranslated region (UTR) can be present 5' (upstream) of an open reading frame (5'-UTR) and/or 3' (downstream) of an open reading frame (3'-UTR). If present, the 5'-UTR is located at the 5' end upstream of the start codon of the protein coding region. The 5'-UTR is downstream of the 5'-cap (if present), e.g., directly adjacent to the 5'-cap. If present, the 3'-UTR is located at the 3' end downstream of the termination codon of the protein coding region, although the term "3'-UTR" preferably does not include a poly(A) sequence. Thus, the 3'-UTR is upstream of the poly(A) sequence (if present), e.g., directly adjacent to the poly(A) sequence.

In some embodiments, the RNA comprises a 5'-UTR comprising a nucleotide sequence of SEQ ID NO:12, or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of SEQ ID NO:12.

In some embodiments, the RNA comprises a 3'-UTR comprising a nucleotide sequence of SEQ ID NO:13, or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of SEQ ID NO:13.

A particularly preferred 5'-UTR comprises the nucleotide sequence of SEQ ID NO: 12. A particularly preferred 3'-UTR comprises the nucleotide sequence of SEQ ID NO: 13.

In some embodiments, the RNA according to the present disclosure includes a 3'-poly(A) sequence.

As used herein, the term "poly(A) sequence" or "poly(A tail)" refers to an uninterrupted or interrupted sequence of adenylate residues typically located at the 3' end of an RNA molecule. Poly(A) sequences are known to those of skill in the art and may follow the 3'-UTR in the RNAs described herein. Uninterrupted poly(A) sequences are characterized by consecutive adenylate residues. Uninterrupted poly(A) sequences are typical in nature. The RNAs disclosed herein can have poly(A) sequences attached to the free 3' end of the RNA after transcription by a template-independent RNA polymerase, or poly(A) sequences encoded by DNA and transcribed by a template-dependent RNA polymerase.

Poly(A) sequences of approximately 120 A nucleotides have been demonstrated to have beneficial effects on the levels of RNA in transfected eukaryotic cells, as well as on the levels of protein translated from open reading frames present upstream (5') of the poly(A) sequence (Holtkamp et al., 2006, Blood, vol. 108, pp. 4009-4017).

The poly(A) sequence may be of any length. In some embodiments, the poly(A) sequence comprises, consists essentially of, or consists of at least 20, at least 30, at least 40, at least 80, or at least 100, and up to 500, up to 400, up to 300, up to 200, or up to 150 A nucleotides, particularly about 120 A nucleotides. In this context, "consists essentially of" means that the majority of the nucleotides in the poly(A) sequence, typically at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, by number of nucleotides in the poly(A) sequence, are A nucleotides, but allows for the remaining nucleotides to be nucleotides other than A nucleotides, e.g., U nucleotides (uridylate), G nucleotides (guanylate), or C nucleotides (cytidylate). In this context, "consists of" means that all nucleotides in the poly(A) sequence are A nucleotides, i.e., 100% by number of nucleotides in the poly(A) sequence. The term "A nucleotide" or "A" refers to adenylate.

In some embodiments, the poly(A) sequence is attached during RNA transcription, e.g., during preparation of in vitro transcribed RNA, based on a DNA template that contains repeated dT nucleotides (deoxythymidylate) in the strand complementary to the coding strand. The DNA sequence that encodes the poly(A) sequence (coding strand) is referred to as a poly(A) cassette.

In some embodiments, the poly(A) cassette present in the coding strand of DNA consists essentially of dA nucleotides, but is interrupted by a random sequence of four nucleotides (dA, dC, dG, and dT). Such random sequences can be 5-50, 10-30, or 10-20 nucleotides long. Such cassettes are disclosed in WO 2016/005324 A1, which is incorporated herein by reference. Any of the poly(A) cassettes disclosed in WO 2016/005324 A1 may be used in certain embodiments of the present disclosure. A poly(A) cassette consisting essentially of dA nucleotides, but interrupted by a random sequence having an equal distribution of the four nucleotides (dA, dC, dG, dT) and having a length of, for example, 5-50 nucleotides, exhibits at the DNA level a constant growth of plasmid DNA in E. coli, and at the RNA level, still associates and contains beneficial properties with respect to supporting RNA stability and translation efficiency. Thus, in some embodiments, the poly(A) sequences contained in the RNA molecules described herein consist essentially of A nucleotides, but are interrupted by random sequences of four nucleotides (A, C, G, U). Such random sequences can be 5-50, 10-30, or 10-20 nucleotides in length.

In some embodiments, no nucleotides other than A nucleotides are adjacent to the poly(A) sequence at its 3' end, i.e., the poly(A) sequence is not masked or is not followed by nucleotides other than A at its 3' end.

In some embodiments, the poly(A) sequence may comprise at least 20, at least 30, at least 40, at least 80, or at least 100, and up to 500, up to 400, up to 300, up to 200, or up to 150 nucleotides. In some embodiments, the poly(A) sequence may consist essentially of at least 20, at least 30, at least 40, at least 80, or at least 100, and up to 500, up to 400, up to 300, up to 200, or up to 150 nucleotides. In some embodiments, the poly(A) sequence may consist of at least 20, at least 30, at least 40, at least 80, or at least 100, and up to 500, up to 400, up to 300, up to 200, or up to 150 nucleotides. In some embodiments, the poly(A) sequence comprises at least 100 nucleotides. In some embodiments, the poly(A) sequence comprises about 150 nucleotides. In some embodiments, the poly(A) sequence comprises about 120 nucleotides.

In some embodiments, the poly(A) sequence included in the RNA described herein is an interrupted poly(A) sequence, e.g., as described in WO 2016/005324, the entire contents of which are incorporated herein by reference for purposes described herein. In some embodiments, the poly(A) sequence includes a stretch of at least 20 adenosine residues (e.g., including at least 30, at least 40, at least 50, at least 60, at least 70, or more adenosine residues), followed by a linker sequence (e.g., in some embodiments including non-A nucleotides) and another stretch of at least 20 adenosine residues (e.g., including at least 30, at least 40, at least 50, at least 60, at least 70, or more adenosine residues). In some embodiments, such linker sequences can be 3-50 nucleotides in length, or 5-25 nucleotides in length, or 10-15 nucleotides in length. In some embodiments, the poly(A) sequence comprises a stretch of about 30 adenosine residues, followed by a linker sequence having a length of about 5-15 nucleotides (e.g., in some embodiments including non-A nucleotides), and another stretch of about 70 adenosine residues.

In some embodiments, the RNA comprises a poly(A) sequence comprising the nucleotide sequence of SEQ ID NO:14, or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of SEQ ID NO:14.

A particularly preferred poly(A) sequence comprises the nucleotide sequence of SEQ ID NO:14.

According to the present disclosure, the vaccine antigens are preferably administered as single-stranded 5'-capped mRNA that is translated into the respective protein upon entry into the cells of the subject receiving the RNA. Preferably, the RNA contains structural elements optimized for maximum efficacy of the RNA with respect to stability and translation efficiency (5'-cap, 5'-UTR, 3'-UTR, poly(A) sequence).

In one embodiment, beta-S-ARCA (D1) is utilized as a specific capping structure at the 5' end of the RNA. In one embodiment, m ₂ ^7,3'-O Gppp (m ₁ ^2'-O ) ApG is utilized as a specific capping structure at the 5' end of the RNA. In one embodiment, the 5'-UTR sequence is derived from human alpha-globin mRNA, optionally with an optimized "Kozak sequence" to increase translation efficiency. In one embodiment, a combination of two sequence elements (FI elements) derived from the "amino-terminal enhancer of split" (AES) mRNA (called F) and the mitochondrially encoded 12S ribosomal RNA (called I) are placed between the coding sequence and the poly(A) sequence to ensure higher maximum protein levels and long-term persistence of the mRNA. In one embodiment, two repeated 3'-UTRs derived from human beta-globin mRNA are placed between the coding sequence and the poly(A) sequence to ensure higher maximum protein levels and long-term persistence of the mRNA. In one embodiment, a poly(A) sequence measuring 110 nucleotides in length is used, consisting of a stretch of 30 adenosine residues followed by a 10 nucleotide linker sequence and another 70 adenosine residues, which was designed to improve RNA stability and translation efficiency.

In one embodiment of all aspects of the present disclosure, RNA encoding the vaccine antigen is expressed in cells of the subject being treated to provide the vaccine antigen. In one embodiment of all aspects of the present disclosure, the RNA is transiently expressed in cells of the subject. In one embodiment of all aspects of the present disclosure, the RNA is in vitro transcribed RNA. In one embodiment of all aspects of the present disclosure, expression of the vaccine antigen is at the cell surface. In one embodiment of all aspects of the present disclosure, the vaccine antigen is expressed and presented in the context of MHC. In one embodiment of all aspects of the present disclosure, expression of the vaccine antigen is in the extracellular space, i.e., the vaccine antigen is secreted.

In the context of this disclosure, the term "transcription" refers to the process by which the genetic code in a DNA sequence is transcribed into RNA. The RNA can then be translated into a peptide or protein.

According to the present disclosure, the term "transcription" includes "in vitro transcription", which relates to a process in which RNA, particularly mRNA, is synthesized in vitro in a cell-free system, preferably using a suitable cell extract. Preferably, a cloning vector is applied for the generation of the transcript. These cloning vectors are generally designated as transcription vectors and, according to the present disclosure, are encompassed by the term "vector". According to the present disclosure, the RNA used in certain embodiments of the present disclosure is preferably an in vitro transcribed RNA (IVT-RNA) and can be obtained by in vitro transcription of a suitable DNA template. The promoter for controlling the transcription can be any promoter of any RNA polymerase. Specific examples of RNA polymerases are T7, T3, and SP6 RNA polymerases. Preferably, the in vitro transcription according to the present disclosure is controlled by a T7 or SP6 promoter. The DNA template for in vitro transcription can be obtained by cloning a nucleic acid, particularly a cDNA, and introducing it into a suitable vector for in vitro transcription. The cDNA can be obtained by reverse transcription of the RNA.

With respect to RNA, the terms "expression" or "translation" refer to the process in a cell's ribosomes where a chain of mRNA directs the assembly of a sequence of amino acids to make a peptide or protein.

In one embodiment, following administration of the RNA described herein, e.g., RNA formulated as an RNA lipid particle, at least a portion of the RNA is delivered to the target cell. In one embodiment, at least a portion of the RNA is delivered to the cytoplasm of the target cell. In one embodiment, the RNA is translated by the target cell to produce the peptide or protein it encodes. In one embodiment, the target cell is a spleen cell. In one embodiment, the target cell is an antigen presenting cell, such as a professional antigen presenting cell in the spleen. In one embodiment, the target cell is a dendritic cell or a macrophage. RNA particles, such as the RNA lipid particles described herein, can be used to deliver RNA to such target cells. Thus, the present disclosure also relates to a method for delivering RNA to a target cell in a subject, comprising administering to the subject an RNA particle described herein. In one embodiment, the RNA is delivered to the cytoplasm of the target cell. In one embodiment, the RNA is translated by the target cell to produce the peptide or protein encoded by the RNA.

"Encode" refers to the inherent property of a particular sequence of nucleotides in a polynucleotide, such as a gene, cDNA, or mRNA, to serve as a template for the synthesis of other polymers and macromolecules in biological processes having either a defined sequence of nucleotides (i.e., rRNA, tRNA, and mRNA) or a defined sequence of amino acids and biological properties derived therefrom. Thus, a gene codes for a protein when transcription and translation of the mRNA corresponding to that gene produces the protein in a cell or other biological system. Both the coding strand, which is the nucleotide sequence identical to the mRNA sequence and usually provided in a sequence listing, and the non-coding strand, which is used as a template for transcription of a gene or cDNA, can be referred to as encoding the protein or other product of that gene or cDNA.

In one embodiment, the RNA encoding the vaccine antigen administered according to the present disclosure is non-immunogenic. RNA encoding an immune stimulant may be administered according to the present disclosure to provide an adjuvant effect. The RNA encoding the immune stimulant may be a standard RNA or a non-immunogenic RNA.

The term "non-immunogenic RNA" as used herein refers to, for example, RNA that, upon administration to a mammal, does not induce a response by the immune system or induces a weaker response than that induced by the same RNA, i.e., standard RNA (stdRNA), which differs only in the modifications that render the non-immunogenic RNA non-immunogenic and in that it is not targeted to a therapy. In a preferred embodiment, the non-immunogenic RNA, also referred to herein as modified RNA (modRNA), is rendered non-immunogenic by incorporating modified nucleosides into the RNA that inhibit RNA-mediated activation of innate immune receptors and by eliminating double-stranded RNA (dsRNA).

In order to make non-immunogenic RNA non-immunogenic by incorporating modified nucleosides, any modified nucleoside can be used as long as it reduces or suppresses the immunogenicity of RNA.Particularly preferred is the modified nucleoside that suppresses the RNA-mediated activation of innate immune receptors.In one embodiment, the modified nucleoside comprises the replacement of one or more uridines with the nucleoside that comprises modified nucleobase.In one embodiment, the modified nucleobase is modified uracil. In one embodiment, the nucleoside comprising a modified nucleobase is 3-methyl-uridine (m ³ U), 5-methoxy-uridine (mo ⁵ U), 5-aza-uridine, 6-aza-uridine, 2-thio-5-aza-uridine, 2-thio-uridine (s ² U), 4-thio-uridine (s ⁴ U), 4-thio-pseudouridine, 2-thio-pseudouridine, 5-hydroxy-uridine (ho ⁵ U), 5-aminoallyl-uridine, 5-halo-uridine (e.g., 5-iodo-uridine or 5-bromo-uridine), uridine 5-oxyacetic acid (cmo ⁵ U), uridine 5-oxyacetic acid methyl ester (mcmo ⁵ U), 5-carboxymethyl-uridine (cm ⁵ U), 1-carboxymethyl-pseudouridine, 5-carboxyhydroxymethyl-uridine (chm ⁵ U), U), 5-carboxyhydroxymethyl-uridine methyl ester (mchm ⁵ U), 5-methoxycarbonylmethyl-uridine (mcm ⁵ U), 5-methoxycarbonylmethyl-2-thio-uridine (mcm ⁵ s ² U), 5-aminomethyl-2-thio-uridine (nm ⁵ s ² U), 5-methylaminomethyl-uridine (mnm ⁵ U), 1-ethyl-pseudouridine, 5-methylaminomethyl-2-thio-uridine (mnm ⁵ s ² U), 5-methylaminomethyl-2-seleno-uridine (mnm ⁵ se ² U), 5-carboxymethyl-uridine (ncm ⁵ U), 5-carboxymethylaminomethyl-uridine (cmnm ⁵ U), 5-carboxymethylaminomethyl-2-thio-uridine (cmnm ⁵ s 2 ^U) , U), 5-propynyl-uridine, 1-propynyl-pseudouridine, 5-taurinomethyl-uridine (τm ⁵ U), 1-taurinomethyl-pseudouridine, 5-taurinomethyl-2-thio-uridine (τm5s2U), 1-taurinomethyl-4-thio-pseudouridine), 5-methyl-2-thio-uridine (m ⁵ s ² U), 1-methyl-4-thio-pseudouridine (m ¹ s ⁴ ψ), 4-thio-1-methyl-pseudouridine, 3-methyl-pseudouridine (m ³ ψ), 2-thio-1-methyl-pseudouridine, 1-methyl-1-deaza-pseudouridine, 2-thio-1-methyl-1-deaza-pseudouridine, dihydrouridine (D), dihydropseudouridine, 5,6-dihydrouridine, 5-methyl-dihydrouridine (m ⁵ D), 2-thio-dihydrouridine, 2-thio-dihydropseudouridine, 2-methoxy-uridine, 2-methoxy-4-thio-uridine, 4-methoxy-pseudouridine, 4-methoxy-2-thio-pseudouridine, N1-methyl-pseudouridine, 3-(3-amino-3-carboxypropyl)uridine (acp ³ U), 1-methyl-3-(3-amino-3-carboxypropyl)pseudouridine (acp ³ ψ), 5-(isopentenylaminomethyl)uridine (inm ⁵ U), 5-(isopentenylaminomethyl)-2-thio-uridine (inm ⁵ s ² U), α-thio-uridine, 2'-O-methyl-uridine (Um), 5,2'-O-dimethyl-uridine (m ⁵ Um), 2'-O-methyl-pseudouridine (ψm), 2-thio-2'-O-methyl-uridine (s ² Um), 5-methoxycarbonylmethyl-2'-O-methyl-uridine (mcm ⁵ Um), 5-carbamoylmethyl-2'-O-methyl-uridine (ncm ⁵ Um), 5-carboxymethylaminomethyl-2'-O-methyl-uridine (cmnm ⁵ Um), 3,2'-O-dimethyl-uridine (m ³ Um), 5-(isopentenylaminomethyl)-2'-O-methyl-uridine (inm ⁵ In a particularly preferred embodiment, the nucleoside comprising a modified nucleobase is selected from the group consisting of pseudouridine (ψ), N1-methyl-pseudouridine (m1ψ) or 5-methyl-uridine (m5U), in particular N1-methyl-pseudouridine.

In one embodiment, the replacement of one or more uridines with a nucleoside comprising a modified nucleobase includes replacement of at least 1%, at least 2%, at least 3%, at least 4%, at least 5%, at least 10%, at least 25%, at least 50%, at least 75%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% of the uridines.

During synthesis of RNA (e.g., mRNA) by in vitro transcription (IVT) using T7 RNA polymerase, atypical activity of the enzyme generates significant amounts of aberrant products, including double-stranded RNA (dsRNA). dsRNA activates effector enzymes that induce inflammatory cytokines and lead to protein synthesis inhibition. dsRNA can be removed from RNA, such as IVT RNA, by, for example, ion-pair reversed-phase HPLC using nonporous or porous C-18 polystyrene-divinylbenzene (PS-DVB) matrices. Alternatively, an enzyme-based method can be used that uses E. coli RNase III, which specifically hydrolyzes dsRNA, but not ssRNA, thereby eliminating dsRNA contaminants from IVT RNA preparations. Additionally, dsRNA can be separated from ssRNA by using cellulose materials. In one embodiment, the RNA preparation is contacted with a cellulosic material and the ssRNA is separated from the cellulosic material under conditions that allow binding of the dsRNA to the cellulosic material and that do not allow binding of the ssRNA to the cellulosic material.

As used herein, the term "remove" or "removal" refers to a feature in which a population of a first substance, such as non-immunogenic RNA, is separated from the vicinity of a population of a second substance, such as dsRNA, where the population of the first substance is not necessarily devoid of the second substance, and the population of the second substance is not necessarily devoid of the first substance. However, a population of a first substance that is characterized by the removal of the population of the second substance has a measurably lower content of the second substance compared to a non-separated mixture of the first and second substances.

In one embodiment, removal of dsRNA from the non-immunogenic RNA includes removal of dsRNA such that less than 10%, less than 5%, less than 4%, less than 3%, less than 2%, less than 1%, less than 0.5%, less than 0.3%, or less than 0.1% of the RNA in the non-immunogenic RNA composition is dsRNA. In one embodiment, the non-immunogenic RNA is free or essentially free of dsRNA. In some embodiments, the non-immunogenic RNA composition comprises a purified preparation of single-stranded nucleoside-modified RNA. For example, in some embodiments, the purified preparation of single-stranded nucleoside-modified RNA is substantially free of double-stranded RNA (dsRNA). In some embodiments, the purified preparation is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or at least 99.9% single-stranded nucleoside-modified RNA relative to all other nucleic acid molecules (DNA, dsRNA, etc.).

In one embodiment, the non-immunogenic RNA is translated in a cell more efficiently than a standard RNA having the same sequence. In one embodiment, translation is enhanced 2-fold compared to its unmodified counterpart. In one embodiment, translation is enhanced by a factor of 3-fold. In one embodiment, translation is enhanced by a factor of 4-fold. In one embodiment, translation is enhanced by a factor of 5-fold. In one embodiment, translation is enhanced by a factor of 6-fold. In one embodiment, translation is enhanced by a factor of 7-fold. In one embodiment, translation is enhanced by a factor of 8-fold. In one embodiment, translation is enhanced by a factor of 9-fold. In one embodiment, translation is enhanced by a factor of 10-fold. In one embodiment, translation is enhanced by a factor of 15-fold. In one embodiment, translation is enhanced by a factor of 20-fold. In one embodiment, translation is enhanced by a factor of 50-fold. In one embodiment, translation is enhanced by a factor of 100-fold. In one embodiment, translation is enhanced by a factor of 200-fold. In one embodiment, translation is enhanced by a factor of 500-fold. In one embodiment, translation is enhanced by a factor of 1000 fold. In one embodiment, translation is enhanced by a factor of 2000 fold. In one embodiment, the factor is 10-1000 fold. In one embodiment, the factor is 10-1000 fold. In one embodiment, the factor is 10-200 fold. In one embodiment, the factor is 10-300 fold. In one embodiment, the factor is 10-500 fold. In one embodiment, the factor is 20-1000 fold. In one embodiment, the factor is 30-1000 fold. In one embodiment, the factor is 50-1000 fold. In one embodiment, the factor is 100-1000 fold. In one embodiment, the factor is 200-1000 fold. In one embodiment, the factor is any other significant amount or range of amounts.

In one embodiment, the non-immunogenic RNA exhibits significantly less innate immunogenicity than a standard RNA having an identical sequence. In one embodiment, the non-immunogenic RNA exhibits 2-fold less innate immune response than its unmodified counterpart. In one embodiment, the innate immunogenicity is reduced by a factor of 3. In one embodiment, the innate immunogenicity is reduced by a factor of 4. In one embodiment, the innate immunogenicity is reduced by a factor of 5. In one embodiment, the innate immunogenicity is reduced by a factor of 6. In one embodiment, the innate immunogenicity is reduced by a factor of 7. In one embodiment, the innate immunogenicity is reduced by a factor of 8. In one embodiment, the innate immunogenicity is reduced by a factor of 9. In one embodiment, the innate immunogenicity is reduced by a factor of 10. In one embodiment, the innate immunogenicity is reduced by a factor of 15. In one embodiment, the innate immunogenicity is reduced by a factor of 20. In one embodiment, the innate immunogenicity is reduced by a factor of 50 fold. In one embodiment, the innate immunogenicity is reduced by a factor of 100 fold. In one embodiment, the innate immunogenicity is reduced by a factor of 200 fold. In one embodiment, the innate immunogenicity is reduced by a factor of 500 fold. In one embodiment, the innate immunogenicity is reduced by a factor of 1000 fold. In one embodiment, the innate immunogenicity is reduced by a factor of 2000 fold.

The term "exhibits significantly less innate immunogenicity" refers to a reduction in detectable innate immunogenicity. In one embodiment, the term refers to a reduction such that an effective amount of the non-immunogenic RNA may be administered without eliciting a detectable innate immune response. In one embodiment, the term refers to a reduction such that the non-immunogenic RNA may be administered repeatedly without eliciting an innate immune response sufficient to detectably reduce production of the protein encoded by the non-immunogenic RNA. In one embodiment, the reduction is such that the non-immunogenic RNA may be administered repeatedly without eliciting an innate immune response sufficient to eliminate detectable production of the protein encoded by the non-immunogenic RNA.

"Immunogenicity" is the ability of a foreign substance, such as RNA, to elicit an immune response in humans or other animals. The innate immune system is the component of the immune system that is relatively non-specific and immediate. It is one of the two main components of the vertebrate immune system, along with the adaptive immune system.

As used herein, "endogenous" refers to any material that originates from or is produced within an organism, cell, tissue, or system.

As used herein, the term "exogenous" refers to any material that is introduced or produced from outside an organism, cell, tissue, or system.

As used herein, the term "expression" is defined as the transcription and/or translation of a particular nucleotide sequence.

As used herein, the terms "linked," "fused," or "fusion" are used interchangeably. These terms refer to the joining of two or more elements or components or domains.

Codon Optimization/Increased G/C Content In some embodiments, an amino acid sequence comprising a SARS-CoV-2 S protein, an immunogenic variant thereof, or an immunogenic fragment of a SARS-CoV-2 S protein or an immunogenic variant thereof described herein is encoded by a coding sequence that is codon-optimized and/or has an increased G/C content compared to a wild-type coding sequence. This includes embodiments in which one or more sequence regions of the coding sequence are codon-optimized and/or have an increased G/C content compared to the corresponding sequence region of a wild-type coding sequence. In one embodiment, the codon optimization and/or increased G/C content preferably does not change the sequence of the encoded amino acid sequence.

The term "codon optimization" refers to the modification of codons in the coding region of a nucleic acid molecule to reflect the typical codon usage of a host organism, preferably without modifying the amino acid sequence encoded by the nucleic acid molecule. Within the context of the present disclosure, the coding region is preferably codon optimized for optimal expression in a subject treated using the RNA molecules described herein. Codon optimization is based on the discovery that translation efficiency is also determined by the different frequencies of occurrence of tRNAs in a cell. Thus, the sequence of the RNA can be modified such that, instead of "rare codons", codons for which frequently occurring tRNAs are available are inserted.

In some embodiments of the present disclosure, the guanosine/cytosine (G/C) content of the coding region of the RNA described herein is increased compared to the G/C content of the corresponding coding sequence of the wild-type RNA, and the amino acid sequence encoded by the RNA is preferably not modified compared to the amino acid sequence encoded by the wild-type RNA. This modification of the RNA sequence is based on the fact that the sequence of any RNA region to be translated is important for the efficient translation of that RNA (e.g., mRNA). Sequences with an increased G (guanosine)/C (cytosine) content are more stable than sequences with an increased A (adenosine)/U (uracil) content. With regard to the fact that several codons code for one and the same amino acid (the so-called degeneration of the genetic code), it is possible to determine the codons that are most favorable for stability (the so-called alternative codon usage). Depending on the amino acid encoded by the RNA, there are various possibilities for the modification of the RNA sequence compared to its wild-type sequence. In particular, codons containing A and/or U nucleotides can be modified by replacing these codons with other codons that code for the same amino acids but that do not contain A and/or U or that do not contain a lower content of A and/or U nucleotides.

In various embodiments, the G/C content of the coding region of the RNA described herein is increased by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 55%, or even more, compared to the G/C content of the coding region of the wild-type RNA. In some embodiments, the G/C content of the coding region is increased by about 10% to about 60% (e.g., about 20% to about 60%, about 30% to about 60%, about 40% to about 60%, about 50% to about 60%, or about 51%, about 52%, about 53%, about 54%, about 55%, about 56%, about 57%, about 58%, about 59%, or about 60%), compared to the G/C content of the coding region of the wild-type RNA.

In some embodiments, the RNA disclosed herein comprises a sequence disclosed herein (e.g., SEQ ID NO: 9) modified to encode one or more mutations characteristic of a SARS-CoV-2 variant (e.g., BA.2 or BA.4/5 omicron variant). In some embodiments, the RNA can be modified to encode one or more mutations characteristic of a SARS-CoV-2 variant by making as few nucleotide changes as possible. In some embodiments, the RNA can be modified to encode one or more mutations characteristic of a SARS-CoV-2 variant by introducing mutations that result in hyper-codon optimization and/or increased G/C content.

In some embodiments, one or more mutations characteristic of a SARS-CoV-2 variant are introduced into a full-length S protein (e.g., an S protein comprising SEQ ID NO:1). In some embodiments, one or more mutations characteristic of a SARS-CoV-2 variant are introduced into a full-length S protein having one or more proline mutations that increase the stability of the pre-fusion confirmation. For example, in some embodiments, proline substitutions are made at positions corresponding to positions 986 and 987 of SEQ ID NO:1. In some embodiments, proline substitutions are made at positions corresponding to positions 985 and 987 of SEQ ID NO:1. In some embodiments, at least four proline substitutions are made. In some embodiments, at least four of such proline mutations include mutations at positions corresponding to residues 817, 892, 899, and 942 of SEQ ID NO:1. In some embodiments, such SARS-CoV-2 proteins that include proline substitutions at positions corresponding to residues 817, 892, 899, and 942 of SEQ ID NO:1 may further include proline substitutions at positions corresponding to residues 986 and 987 of SEQ ID NO:1. In some embodiments, such SARS-CoV-2 proteins that include proline substitutions at positions corresponding to residues 817, 892, 899, and 942 of SEQ ID NO:1 may further include proline substitutions at positions corresponding to residues 985 and 987 of SEQ ID NO:1. In some embodiments, one or more mutations characteristic of a SARS-CoV-2 variant are introduced onto an immunogenic fragment of the S protein (e.g., the RBD).

Embodiments of Administered RNA In some embodiments, the disclosure provides an RNA (e.g., an mRNA) that comprises an open reading frame encoding a polypeptide comprising at least a portion of a SARS-CoV-2 S protein. The RNA is suitable for intracellular expression of the polypeptide. In some embodiments, such an encoded polypeptide comprises a sequence corresponding to the complete S protein. In some embodiments, such an encoded polypeptide does not comprise a sequence corresponding to the complete S protein. In some embodiments, the encoded polypeptide comprises a sequence corresponding to a receptor binding domain (RBD). In some embodiments, the encoded polypeptide comprises a sequence corresponding to an RBD and further comprises a trimerization domain (e.g., a trimerization domain as disclosed herein, such as a fibritin domain). In some embodiments, the RBD comprises a signaling domain (e.g., a signaling domain disclosed herein). In some embodiments, the RBD comprises a transmembrane domain (e.g., a transmembrane domain disclosed herein). In some embodiments, the RBD comprises a signaling domain and a trimerization domain. In some embodiments, the RBD comprises a signaling domain, a trimerization domain, and a transmembrane domain.

In some embodiments, the encoded polypeptide comprises sequences corresponding to two receptor binding domains. In some embodiments, the encoded polypeptide comprises sequences corresponding to two receptor binding domains in tandem in an amino acid chain, as disclosed, for example, in Dai, Lianpan, et al. "A universal design of betacoronavirus vaccines against COVID-19, MERS, and SARS," Cell 182.3 (2020): 722-733, the contents of which are incorporated herein by reference in their entirety.

In some embodiments, the SARS-CoV-2 S protein or immunogenic fragment thereof contains one or more mutations to modify, add, or remove glycosylation sites, e.g., as described in WO2022/221835A2, US2022/0323574A1, WO2022/266012A1, or WO2022/195351A1.

In some embodiments, the compositions or medical preparations described herein include RNA encoding an amino acid sequence that includes the SARS-CoV-2 S protein, an immunogenic variant thereof, or an immunogenic fragment of the SARS-CoV-2 S protein or an immunogenic variant thereof. Similarly, the methods described herein include administration of such RNA.

The active platform for use herein is based on antigen-encoding RNA vaccines to induce robust neutralizing antibodies and concomitant/concurrent T cell responses to achieve protective immunity, preferably with minimal vaccine doses. The administered RNA is preferably in vitro transcribed RNA.

Three different RNA platforms are particularly preferred: unmodified uridine containing mRNA (uRNA), nucleoside modified mRNA (modRNA), and self-amplifying RNA (saRNA). In a particularly preferred embodiment, the RNA is in vitro transcribed RNA. In some embodiments, the uRNA is mRNA. In some embodiments, the modRNA is mRNA.

Below, embodiments of these three different RNA platforms are described, and certain terms used in describing the elements thereof have the following meanings.
S1S2 protein/S1S2 RBD: sequences encoding the respective antigens of SARS-CoV-2.

nsP1, nsP2, nsP3, and nsP4: Wild-type sequences encoding the Venezuelan equine encephalitis virus (VEEV) RNA-dependent RNA polymerase replicase, and the subgenomic promoter plus conserved sequence elements supporting replication and translation.

virUTR: A viral untranslated region that encodes part of the subgenomic promoter and sequence elements that support replication and translation.

hAg-Kozak: 5'-UTR sequence of human alpha-globin mRNA with an optimized "Kozak sequence" to improve translation efficiency.

Sec: Sec corresponds to a secretory signal peptide (sec) that directs translocation of the nascent polypeptide chain to the endoplasmic reticulum. In some embodiments, such a secretory signal peptide comprises the unique S1S2 secretory signal peptide of the SARS-CoV-2 S protein. In some embodiments, such a secretory signal peptide is a secretory signal peptide from a non-S1S2 protein, such as an immunoglobulin secretory signal peptide (aa 1-22), an HSV-1 gD signal peptide (MGGAAARLGAVILFVVIVGLHGVRSKY), an HSV-2 gD signal peptide (MGRLTSGVGTAALLVVAVGLRVVCA), a human SPARC signal peptide, a human insulin isoform 1 signal peptide, a human albumin signal peptide, or any other signal peptide described herein.

Glycine-serine linker (GS): A sequence that codes for a short linker peptide consisting mainly of the amino acids glycine (G) and serine (S), commonly used in fusion proteins.

Fibritin: A partial sequence of T4 fibritin (foldon) used as an artificial trimerization domain.

TM: The TM sequence corresponds to the transmembrane portion of the protein. The transmembrane domain may be N-terminal, C-terminal, or internal to the encoded polypeptide. The coding sequence for the transmembrane element is typically located in frame (i.e., in the same reading frame), 5', 3', or internal to the coding sequence of the sequence to which it is linked (e.g., the sequence encoding the polypeptide(s)). In some embodiments, the transmembrane domain comprises or is a transmembrane domain of influenza virus hemagglutinin (HA), HIV-1 Env, equine infectious anemia virus (EIAV), murine leukemia virus (MLV), mouse mammary tumor virus, vesicular stomatitis virus (VSV) G protein, rabies virus, or a seven transmembrane domain receptor. In some embodiments, the transmembrane portion of the protein is from the S1S2 protein.

FI elements: The 3'-UTR is a combination of two sequence elements derived from the "amino-terminal enhancer of split" (AES) mRNA (termed F) and the mitochondrially encoded 12S ribosomal RNA (termed I). These were identified by an ex vivo selection process for sequences that confer RNA stability and enhance total protein expression.

A30L70: A poly(A) tail measuring 110 nucleotides in length, consisting of 30 adenosine residues followed by a 10 nucleotide linker sequence and a stretch of another 70 adenosine residues designed to enhance RNA stability and translation efficiency in dendritic cells.

In general, the vaccine RNA described herein may comprise, from 5' to 3', one of the following structures:
cap-5'-UTR-vaccine antigen coding sequence-3'-UTR-poly(A)
or Cap-hAg-Kozak-vaccine antigen-coding sequence-FI-A30L70.

In some embodiments, the vaccine antigens described herein may comprise a full-length S protein or an immunogenic fragment thereof (e.g., the RBD). In some embodiments in which the vaccine antigen comprises a full-length S protein, its secretory signal peptide and/or transmembrane domain may be replaced by a heterologous secretory signal peptide (e.g., as described herein) and/or a heterologous transmembrane domain (e.g., as described herein).

In some embodiments, the vaccine antigens described herein may comprise, from N-terminus to C-terminus, one of the following structures:
signal sequence-RBD-trimerization domain or signal sequence-RBD-trimerization domain-transmembrane domain.

The RBD and the trimerization domain may be separated by a linker, in particular a GS linker, such as a linker having the amino acid sequence GSPGSGSGS. The trimerization domain and the transmembrane domain may be separated by a linker, in particular a GS linker, such as a linker having the amino acid sequence GSGSGS.

The signal sequence can be a signal sequence as described herein. The RBD can be an RBD domain as described herein. The trimerization domain can be a trimerization domain as described herein. The transmembrane domain can be a transmembrane domain as described herein.

In one embodiment,
the signal sequence comprises the amino acid sequence of amino acids 1 to 16 or 1 to 19 of SEQ ID NO:1, or the amino acid sequence of amino acids 1 to 22 of SEQ ID NO:31, or an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity thereto;
the RBD comprises an amino acid sequence of amino acids 327-528 of SEQ ID NO:1, or an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity thereto;
the trimerization domain comprises an amino acid sequence of amino acids 3 to 29 of SEQ ID NO: 10, or an amino acid sequence of SEQ ID NO: 10, or an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity thereto;
The transmembrane domain comprises the amino acid sequence of amino acids 1207-1254 of SEQ ID NO:1, or an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity thereto.

In one embodiment,
the signal sequence comprises the amino acid sequence of amino acids 1 to 16 or 1 to 19 of SEQ ID NO:1, or the amino acid sequence of amino acids 1 to 22 of SEQ ID NO:31;
the RBD comprises the amino acid sequence of amino acids 327-528 of SEQ ID NO:1;
the trimerization domain comprises the amino acid sequence of amino acids 3 to 29 of SEQ ID NO: 10, or the amino acid sequence of SEQ ID NO: 10;
The transmembrane domain comprises the amino acid sequence of amino acids 1207-1254 of SEQ ID NO:1.

In some embodiments, an RNA polynucleotide that includes a sequence encoding a vaccine antigen (e.g., a SARS-CoV-2 S protein, an immunogenic variant thereof, or an immunogenic fragment of a SARS-CoV-2 S protein or an immunogenic variant thereof) or that includes an open reading frame encoding a vaccine antigen (e.g., a SARS-CoV-2 S protein, an immunogenic variant thereof, or an immunogenic fragment of a SARS-CoV-2 S protein or an immunogenic variant thereof), such as the nucleotide sequence of SEQ ID NO:50 or the nucleotide sequence of SEQ ID NO:53, a variant or fragment thereof, further includes a 5' cap (e.g., a 5' cap including a cap 1 structure), a 5' UTR sequence (e.g., a 5' UTR sequence including a nucleotide sequence of SEQ ID NO:12), a 3' UTR sequence (e.g., a 3' UTR sequence including a nucleotide sequence of SEQ ID NO:13), and a polyA sequence (e.g., a polyA sequence including a nucleotide sequence of SEQ ID NO:14). In some embodiments, the RNA polynucleotide is formulated in a composition comprising ((4-hydroxybutyl)azanediyl)bis(hexane-6,1-diyl)bis(2-hexyldecanoate), cholesterol, distearoylphosphatidylcholine, and (2-[(polyethylene glycol)-2000]-N,N-ditetradecylacetamide).

The RNA described herein or the RNA encoding the vaccine antigen described herein can be unmodified uridine containing mRNA (uRNA), nucleoside modified mRNA (modRNA), or self-amplifying RNA (saRNA). In one embodiment, the RNA described herein or the RNA encoding the vaccine antigen described herein is a nucleoside modified mRNA (modRNA).

Variant-Specific Vaccines In some embodiments, the RNA disclosed herein encodes an S protein that includes one or more mutations that are characteristic of a SARS-CoV-2 variant. In some embodiments, the RNA encodes a SARS-CoV-2 S protein that includes one or more mutations that are characteristic of an alpha variant. In some embodiments, the RNA encodes a SARS-CoV-2 S protein that includes one or more mutations that are characteristic of a beta variant. In some embodiments, the RNA encodes a SARS-CoV-2 S protein that includes one or more mutations that are characteristic of a delta variant. In some embodiments, the RNA encodes a SARS-CoV-2 S protein that includes one or more mutations that are characteristic of an omicron variant (e.g., an S protein that includes one or more mutations that are characteristic of a BA.1, BA.2, or BA.4/5 omicron variant). In some embodiments, the RNA encodes a SARS-CoV-2 S protein that includes one or more mutations that are characteristic of a BA.1 omicron variant. In some embodiments, the RNA encodes a SARS-CoV-2 S protein that includes one or more mutations that are characteristic of a BA. In some embodiments, the RNA encodes a SARS-CoV-2 S protein that includes one or more mutations that are characteristic of the BA.2 omicron variant. In some embodiments, the RNA encodes a SARS-CoV-2 S protein that includes one or more mutations that are characteristic of the BA.2.12.1 omicron variant. In some embodiments, the RNA encodes a SARS-CoV-2 S protein that includes one or more mutations that are characteristic of the BA.3 omicron variant. In some embodiments, the RNA encodes a SARS-CoV-2 S protein that includes one or more mutations that are characteristic of the BA.4 omicron variant. In some embodiments, the RNA encodes a SARS-CoV-2 S protein that includes one or more mutations that are characteristic of the BA.5 omicron variant.

Unmodified uridine RNA (uRNA)
The active ingredient of unmodified messenger RNA (uRNA) drug substances is a single-stranded mRNA that is translated upon entry into a cell. In addition to the sequence encoding the coronavirus vaccine antigen (i.e., open reading frame), each uRNA preferably contains common structural elements (5'-cap, 5'-UTR, 3'-UTR, poly(A) tail) that are optimized for maximum effectiveness of the RNA with respect to stability and translation efficiency. A preferred 5'-cap structure is beta-S-ARCA(D1) (m ₂ ^7,2'-O GppSpG). The preferred 5'-UTR and 3'-UTR comprise the nucleotide sequence of SEQ ID NO:12 and the nucleotide sequence of SEQ ID NO:13, respectively. A preferred poly(A) tail comprises the sequence of SEQ ID NO:14.

Different embodiments of this platform are:

Figure 3 illustrates the general structure of antigen-encoding RNA.

Nucleoside-modified messenger RNA (modRNA)
The active ingredient of nucleoside-modified messenger RNA (modRNA) drug substances is a single-stranded mRNA that is translated upon entry into cells. In addition to the sequence encoding the coronavirus vaccine antigen (i.e., open reading frame), each modRNA contains common structural elements optimized for maximum effectiveness of the RNA as a uRNA (5'-cap, 5'-UTR, 3'-UTR, poly(A)-tail). Compared to uRNA, modRNA contains 1-methyl-pseudouridine instead of uridine. The preferred 5' cap structure is m ₂ ^7,3'-O Gppp(m ₁ ^2'-O )ApG. The preferred 5'-UTR and 3'-UTR contain the nucleotide sequences of SEQ ID NO:12 and SEQ ID NO:13, respectively. The preferred poly(A) tail contains the sequence of SEQ ID NO:14. An additional purification step is applied to the modRNA to reduce dsRNA contaminants generated during the in vitro transcription reaction.

Different embodiments of this platform are:

FIG. 4 illustrates the general structure of an RNA encoding an antigen.

Nucleotide sequence of RBP020.11 (Beta-specific vaccine) The nucleotide sequence is shown with individual sequence elements as indicated in bold. In addition, the sequence of the translated protein is shown in italics below the coding nucleotide sequence (* = stop codon). Red text indicates point mutations in the nucleotide and amino acid sequences.

The sequence of RBP020.11 is also shown in Table 9. Additional sequences of exemplary RNA constructs encoding SARS-CoV-2 spike sequence variants are shown in Tables 8-18. For each variant, the spike protein sequence and encoding DNA and RNA sequences are provided. Additionally, exemplary full-length RNA vaccines and corresponding DNA sequences are provided. In the full-length sequences provided in these tables (7-18a), and other DNA or RNA sequences provided herein, "U" may represent naturally occurring uridine or modified uridine, e.g., pseudouridine. Additionally, note that in the full-length RNA vaccine sequences provided in Tables 7-18a and their corresponding DNA sequences, a polyA tail is included in the sequence. In accordance with the present disclosure herein, in some embodiments, the RNA and DNA sequences described herein may include polyA tails that are shorter or longer than those indicated by, for example, at least 1, at least 2, at least 3, at least 4 nucleotides, and up to at least 10 "A" nucleotides.

In some embodiments, an RNA construct encoding a spike protein from a coronavirus variant listed in Tables 7-18a has the structure shown below:
m ₂ ^7,3'-O Gppp(m ₁ ^2'-O )ApG)-hAg-Kozak-antigen-FI-A30L70, where the encoded "antigen" is the viral spike protein (S1S2 protein) of SARS-CoV-2 (S1S2 full length protein) as shown in Tables 7-18a.

Nucleotide sequence of RBP020.14 (Alpha-specific vaccine) The nucleotide sequence is shown with individual sequence elements as indicated in bold. In addition, the sequence of the translated protein is shown in italics below the coding nucleotide sequence (* = stop codon). Red text indicates point mutations in both the nucleotide and amino acid sequences.
The sequence of RBP020.14 is also shown in Table 10.

Nucleotide sequence of RBP020.16 (Delta-specific vaccine) The nucleotide sequence is shown with individual sequence elements as indicated in bold. In addition, the sequence of the translated protein is shown in italics below the coding nucleotide sequence (* = stop codon). Red text indicates point mutations in both the nucleotide and amino acid sequences.

The sequence of RBP020.14 is also shown in Table 11.

Self-amplifying RNA (saRNA)
The active ingredient of a self-amplifying mRNA (saRNA) drug substance is a single-stranded RNA that self-amplifies upon entry into a cell, after which the coronavirus vaccine antigen is translated. In contrast to uRNA and modRNA, which preferably code for a single protein, the coding region of saRNA contains two open reading frames (ORFs). The 5'-ORF codes for an RNA-dependent RNA polymerase, such as the Venezuelan Equine Encephalitis Virus (VEEV) RNA-dependent RNA polymerase (replicase). The replicase ORF is followed by a 3' subgenomic promoter and a second ORF that codes for the antigen. In addition, the saRNA UTR contains 5' and 3' conserved sequence elements (CSEs) required for self-amplification. saRNA contains common structural elements (5' cap, 5'-UTR, 3'-UTR, poly(A) tail) that are optimized for maximum effectiveness of the RNA as a uRNA. In some embodiments, the saRNA preferably comprises a uridine. In some embodiments, the saRNA comprises one or more nucleoside modifications described herein. A preferred 5' cap structure is beta-S-ARCA(D1)(m ₂ ^7,2'-O GppSpG).

In some embodiments, the saRNA described herein encodes a single antigen (e.g., one SARS-CoV-2 S polypeptide). In some embodiments, the saRNA utilized in accordance with the present disclosure encodes two or more antigens (e.g., two or more SARS-CoV-2 S polypeptides). In some embodiments, the saRNA encodes two S polypeptides, each from a different SARS-CoV-2 variant.

In some embodiments, the saRNA platform may offer certain advantages over other RNA platforms. For example, in some embodiments, saRNA may offer extended duration of antigen expression, lower dose levels, improved tolerability, and/or increased antigen dosage while maintaining robust antibody and T cell responses.

Cytoplasmic delivery of saRNA initiates an alphavirus-like life cycle. However, because saRNA does not encode alphavirus structural proteins required for genome packaging or cell entry, the generation of replication-competent viral particles is highly unlikely or impossible. Replication does not involve any intermediate step that generates DNA. Therefore, the use/uptake of saRNA does not pose a risk of genome integration or other permanent genetic recombination in target cells. Moreover, saRNA itself effectively activates the innate immune response via recognition of dsRNA intermediates, thereby preventing its persistent replication.

Different embodiments of this platform are:

Figure 5 illustrates the general structure of RNA encoding an antigen.

In some embodiments, the vaccine RNA described herein comprises a nucleotide sequence selected from the group consisting of SEQ ID NOs: 15, 16, 17, 19, 20, 21, 24, 25, 26, 27, 30, and 32. Particularly preferred vaccine RNAs described herein comprise a nucleotide sequence selected from the group consisting of SEQ ID NOs: 15, 17, 19, 21, 25, 26, 30, and 32, e.g., SEQ ID NOs: 17, 19, 21, 26, 30, and 32.

In some embodiments, the RNA described herein is formulated in a lipid nanoparticle, lipoplex, polyplex (PLX), lipidated polyplex (LPLX), liposome, or polysaccharide nanoparticle. In some embodiments, the RNA described herein is preferably formulated in a lipid nanoparticle (LNP). In one embodiment, the LNP comprises a cationic lipid, a neutral lipid, a steroid, a polymer-conjugated lipid, and RNA. In one embodiment, the cationic lipid is ALC-0315, the neutral lipid is DSPC, the steroid is cholesterol, and the polymer-conjugated lipid is ALC-0159. The preferred mode of administration is intramuscular administration, more preferably in an aqueous cryoprotectant buffer for intramuscular administration. The pharmaceutical product is preferably a preservative-free, sterile dispersion of RNA formulated in lipid nanoparticles (LNP) in an aqueous cryoprotectant buffer for intramuscular administration.

In different embodiments, the pharmaceutical comprises the ingredients indicated below, preferably in the proportions or concentrations indicated below.
[1] ALC-0315 = ((4-hydroxybutyl)azanediyl)bis(hexane-6,1-diyl)bis(2-hexyldecanoate)/6-[N-6-(2-hexyldecanoyloxy)hexyl-N-(4-hydroxybutyl)amino]hexyl 2-hexyldecanoate [2] ALC-0159 = 2-[(polyethylene glycol)-2000]-N,N-ditetradecylacetamide/2-[2-(ω-methoxy(polyethylene glycol 2000)ethoxy]-N,N-ditetradecylacetamide [3] DSPC = 1,2-distearoyl-sn-glycero-3-phosphocholine q.s. = appropriate amount (amount that may be sufficient)

ALC-0315:
ALC-0159:
DSP C:
cholesterol:

In some embodiments, the particles disclosed herein are formulated in a solution comprising 10 mM Tris and 10% sucrose, optionally having a pH of about 7.4. In some embodiments, the particles disclosed herein are formulated in a solution comprising about 103 mg/ml sucrose, about 0.20 mg/ml tromethamine (Tris base), and about 1.32 mg/ml Tris.

In some embodiments, the composition comprises:
(a) about 0.1 mg/mL of RNA comprising an open reading frame encoding a polypeptide comprising a SARS-CoV-2 protein or an immunogenic fragment or variant thereof;
(b) about 1.43 mg/ml ALC-0315;
(c) about 0.18 mg/ml ALC-0159;
(d) about 0.31 mg/ml DSPC;
(e) about 0.62 mg/ml cholesterol;
(f) about 103 mg/ml sucrose;
(g) tromethamine (Tris base) at about 0.20 mg/ml;
(h) about 1.32 mg/ml of Tris(hydroxymethyl)aminomethane hydrochloride (Tris HCl); and (i) a sufficient amount of water.

In one embodiment, the ratio of RNA (e.g., mRNA) to total lipids (N/P) is 6.0-6.5, e.g., about 6.0 or about 6.3.

Nucleic Acid-Containing Particles The nucleic acids described herein, for example, RNA encoding vaccine antigens, may be formulated and administered as particles.

In the context of the present disclosure, the term "particle" relates to a structured entity formed by a molecule or a molecular complex. In one embodiment, the term "particle" relates to a micro- or nano-sized structure, e.g., a micro- or nano-sized compact structure dispersed in a medium. In one embodiment, the particle is a nucleic acid-containing particle, such as a particle containing DNA, RNA, or a mixture thereof.

Electrostatic interactions between positively charged molecules, such as polymers and lipids, and negatively charged nucleic acids are involved in particle formation. This results in complexation and spontaneous formation of nucleic acid particles. In one embodiment, the nucleic acid particles are nanoparticles.

As used in this disclosure, "nanoparticles" refers to particles having an average diameter suitable for parenteral administration.

A "nucleic acid particle" can be used to deliver a nucleic acid to a desired target site (e.g., a cell, tissue, organ, etc.). A nucleic acid particle can be formed from at least one cationic lipid or cationically ionizable lipid or lipid-like material, at least one cationic polymer such as protamine, or a mixture thereof, and a nucleic acid. In some embodiments, exemplary nucleic acid particles include lipid nanoparticles, polyplexes (PLX), rapidate polyplexes (LPLX), (LNP)-based and lipoplex (LPX)-based formulations, liposomes, or polysaccharide nanoparticles. In some embodiments, RNA encoding an amino acid sequence comprising the SARS-CoV-2 S protein, an immunogenic variant thereof, or an immunogenic fragment of the SARS-CoV-2 S protein or an immunogenic variant thereof is formulated as an LNP. In some embodiments, the LNPs comprise one or more cationically ionizable lipids, one or more neutral lipids (e.g., in some embodiments, a sterol, such as, for example, cholesterol, and/or a phospholipid), and one or more polymer-conjugated lipids. In some embodiments, the formulation comprises ALC-0315 (4-hydroxybutyl)azanediyl)bis(hexane-6,1-diyl)bis(2-hexyldecanoate), ALC-0159 (2-[(polyethylene glycol)2000]-N,N-ditetradecylacetamide), DSPC (1,2-distearoyl-sn-glycero-3-phosphocholine), cholesterol, sucrose, trometamol (Tris), trometamol hydrochloride, and water. The RNA particles described herein include nanoparticles. In some embodiments, exemplary nanoparticles include lipid nanoparticles, lipoplexes, polyplexes (PLX), lipidated polyplexes (LPLX), liposomes, or polysaccharide nanoparticles. Polyplexes (PLX), polysaccharide nanoparticles, and liposomes are all delivery technologies well known to those of skill in the art. See, e.g., Lachelt, Ulrich, and Ernst Wagner. “Nucleic acid therapeutics using polyplexes: a journey of 50 years (and beyond)”Chemical reviews 115.19 (2015): 11043-11078, Plucinski, Alexander, Zan Lyu, and Bernhard VKJ Schmidt, “Polysaccharide nanoparticles: from ”Journal of Materials Chemistry B (2021), and Tenchov, Rumiana, et al. "Lipid Nanoparticles-From Liposomes to mRNA Vaccine Delivery, a Landscape of Research Diversity and Advancement," ACS nanooo15.11 (2021):16982-17015, respectively (the contents of each are incorporated by reference in their entirety). In some embodiments, the concentration of RNA in the pharmaceutical RNA preparation is about 0.1 mg/ml. In some embodiments, the concentration of RNA in the pharmaceutical RNA preparation is about 30 μg/ml to about 100 μg/ml. In some embodiments, the concentration of RNA in the pharmaceutical RNA preparation is about 50 μg/ml to about 100 μg/ml.

Without intending to be bound by any theory, it is believed that cationic lipids or cationic ionizable lipids or lipid-like materials, and/or cationic polymers complex with nucleic acids to form aggregates, which aggregate to yield colloidally stable particles.

In one embodiment, the particles described herein further comprise at least one lipid or lipid-like material other than a cationic lipid or a cationic ionizable lipid or lipid-like material, at least one polymer other than a cationic polymer, or a mixture thereof.

In some embodiments, the nucleic acid particles include two or more types of nucleic acid molecules, the molecular parameters of which may be similar or different from each other, for example, with respect to molar mass or basic structural elements such as molecular structure, capping, coding regions, or other features.

The nucleic acid particles described herein, in one embodiment, may have an average diameter ranging from about 30 nm to about 1000 nm, from about 50 nm to about 800 nm, from about 70 nm to about 600 nm, from about 90 nm to about 400 nm, or from about 100 nm to about 300 nm.

The nucleic acid particles described herein may exhibit a polydispersity index of less than about 0.5, less than about 0.4, less than about 0.3, or less than or equal to about 0.2. By way of example, the nucleic acid particles may exhibit a polydispersity index in the range of about 0.1 to about 0.3 or about 0.2 to about 0.3.

For RNA-lipid particles, the N/P ratio gives the ratio of the number of nitrogen groups in the lipid to the number of phosphate groups in the RNA. It correlates with the charge ratio, since usually the nitrogen atom (depending on the pH) is positively charged and the phosphate group is negatively charged. The N/P ratio is pH dependent if a charge balance exists. Lipid formulations are frequently formed with N/P ratios above 4 up to 12, since positively charged nanoparticles are considered to be favorable for transfection. The RNA is then considered to be fully bound to the nanoparticles.

The nucleic acid particles described herein can be prepared using a wide variety of methods that can include obtaining a colloid from at least one cationic lipid or cationically ionizable lipid or lipid-like material, and/or at least one cationic polymer, and mixing the colloid with nucleic acid to obtain a nucleic acid particle.

The term "colloid" as used herein refers to a type of homogeneous mixture in which the dispersed particles do not settle. The insoluble particles in the mixture are microscopic and have a particle size between 1 and 1000 nanometers. The mixture may be referred to as a colloid or a colloidal suspension. The term "colloid" refers only to the particles in the mixture, not to the suspension as a whole.

For the preparation of colloids comprising at least one cationic lipid or cationic ionizable lipid or lipid-like material, and/or at least one cationic polymer method, conventionally used and appropriately adapted methods for preparing liposomal vesicles are applicable herein. The most commonly used methods for preparing liposomal vesicles share the following basic steps: (i) dissolving lipid in an organic solvent, (ii) drying the resulting solution, and (iii) hydration of the dried lipid (using various aqueous media).

In the film hydration method, the lipids are first dissolved in a suitable organic solvent and dried to produce a thin film at the bottom of the flask. The resulting lipid film is hydrated using a suitable aqueous medium to produce a liposome dispersion. Additionally, an additional reduction step may be included.

Reverse phase evaporation is an alternative method to film hydration for preparing liposomal vesicles that involves the formation of a water-in-oil emulsion between the aqueous phase and the lipid-containing organic phase. Brief sonication of this mixture is necessary for homogenization of the system. Removal of the organic phase under reduced pressure results in a milky gel that then transforms into a liposomal suspension.

The term "ethanol injection technique" refers to a process in which an ethanol solution containing lipids is rapidly injected through a needle into an aqueous solution. This action disperses the lipids throughout the solution and promotes lipid structure formation, e.g., lipid vesicle formation, such as liposome formation. In general, the RNA lipoplex particles described herein can be obtained by adding RNA to a colloidal liposome dispersion. Using the ethanol injection technique, such a colloidal liposome dispersion is formed, in one embodiment, as follows: an ethanol solution containing lipids, such as cationic lipids and additional lipids, is injected into an aqueous solution under stirring. In one embodiment, the RNA lipoplex particles described herein are accessible without an extrusion step.

The term "extrusion" or "extrusion" refers to the creation of particles having a fixed cross-sectional shape. In particular, it refers to the reduction of particles whereby the particles are forced through a filter having defined pores.

Other methods that have organic solvent-free properties may be used to prepare colloids in accordance with the present disclosure.

LNPs typically contain four components: an ionizable cationic lipid, a neutral lipid such as a phospholipid, a steroid such as cholesterol, and a polymer-conjugated lipid such as a polyethylene glycol (PEG) lipid. Each component participates in payload protection and allows for effective intracellular delivery. LNPs can be prepared by mixing lipids, rapidly dissolved in ethanol, with nucleic acid in an aqueous buffer.

The term "mean diameter" refers to the average hydrodynamic diameter of the particles as measured by dynamic laser light scattering (DLS) with data analysis using a so-called cumulative algorithm, which results in the so-called Z- _average , which has the dimension of length, and the polydispersity index (PI), which is dimensionless (Koppel, D., J. Chem. Phys. 57, 1972, pp 4814-4820, ISO 13321). Herein, the "mean diameter", "diameter" or "size" of a particle is used synonymously with this value of the Z- _average .

The "polydispersity index" is preferably calculated on the basis of dynamic light scattering measurements by so-called cumulative analysis, as mentioned in the definition of "average diameter". Under certain prerequisites, it can be taken as a measure of the overall size distribution of the nanoparticles.

Different types of nucleic acid-containing particles have been previously described as suitable for delivery of nucleic acids in particulate form (e.g., Kaczmarek, J.C. et al., 2017, Genome Medicine 9, 60). For non-viral nucleic acid delivery vehicles, nanoparticle encapsulation of nucleic acids can physically protect the nucleic acid from degradation and, depending on the specific chemistry, aid in cellular uptake and endosomal escape.

The present disclosure describes particles comprising a nucleic acid, at least one cationic lipid or cationically ionizable lipid or lipid-like material, and/or at least one cationic polymer that associates with the nucleic acid to form a nucleic acid particle, as well as compositions comprising such particles. The nucleic acid particles may contain nucleic acid complexed in different forms by non-covalent interactions to the particle. The particles described herein are not viral particles, and in particular are infectious viral particles, i.e., they are not capable of viral infection of cells.

Suitable cationic lipids or cationically ionizable lipids or lipid-like materials and cationic polymers form nucleic acid particles and are included in the term "particle-forming components" or "particle-forming agents." The term "particle-forming components" or "particle-forming agents" refers to any component that associates with nucleic acid to form a nucleic acid particle. Such components include any component that can be part of a nucleic acid particle.

In some embodiments, a nucleic acid-containing particle (e.g., lipid nanoparticle (LNP)) comprises two or more RNA molecules, each comprising a different nucleic acid sequence. In some embodiments, a nucleic acid-containing particle comprises two or more RNA molecules, each encoding a different immunogenic polypeptide or immunogenic fragment thereof. In some embodiments, the two or more RNA molecules present in the nucleic acid-containing particle comprise a first RNA molecule encoding an immunogenic polypeptide or immunogenic fragment thereof from a coronavirus and a second RNA molecule encoding an immunogenic polypeptide or immunogenic fragment thereof from an infectious disease pathogen (e.g., a virus, a bacterium, a parasite, etc.). For example, in some embodiments, the two or more RNA molecules present in the nucleic acid-containing particle comprise a first RNA molecule encoding an immunogenic polypeptide or immunogenic fragment thereof from a coronavirus (e.g., in some embodiments, SARS-CoV-2 Wuhan strain or a variant thereof, e.g., SARS-CoV-2 having one or more mutations characteristic of an omicron variant), and a second RNA molecule encoding an immunogenic polypeptide or immunogenic fragment thereof from an influenza virus.

In some embodiments, the nucleic acid-containing particle comprises a first RNA molecule encoding an immunogenic polypeptide or immunogenic fragment thereof from a first coronavirus (e.g., as described herein) and a second RNA molecule encoding an immunogenic polypeptide or immunogenic fragment thereof from a second coronavirus (e.g., as described herein). In some embodiments, the first coronavirus is different from the second coronavirus. In some embodiments, the first and/or second coronavirus are independently from the SARS-CoV-2 Wuhan strain or a variant thereof, e.g., SARS-CoV-2 having one or more mutations characteristic of the Omicron variant. In some embodiments, the two or more RNA molecules present on the nucleic acid-containing particle each encode an immunogenic polypeptide or immunogenic fragment thereof from the same and/or different strains and/or variants of coronavirus (e.g., in some embodiments, SARS-CoV-2 strains or variants). For example, in some embodiments, two or more RNA molecules present on the nucleic acid-containing particle each encode a different immunogenic polypeptide or immunogenic fragment thereof from a coronavirus membrane protein, a coronavirus nucleocapsid protein, a coronavirus spike protein, a coronavirus nonstructural protein, and/or a coronavirus accessory protein. In some embodiments, such immunogenic polypeptides or immunogenic fragments thereof may be derived from the same or different coronaviruses (e.g., in some embodiments, the SARS-CoV-2 Wuhan strain or variants thereof, e.g., in some embodiments, variants having one or more mutations characteristic of common variants such as the Omicron variant). In some embodiments, the nucleic acid-containing particle comprises a first RNA molecule encoding a SARS-CoV-2 S protein or immunogenic fragment thereof from a first strain or variant, and a second RNA molecule encoding a SARS-CoV-2 S protein or immunogenic fragment thereof from a second strain or variant, the second strain or variant being different from the first strain or variant.

In some embodiments, a nucleic acid-containing particle (e.g., in some embodiments, an LNP described herein) comprises a first RNA molecule encoding a SARS-CoV-2 S protein from the Wuhan strain and a second RNA molecule encoding a SARS-CoV-2 S protein that includes one or more mutations characteristic of an omicron variant (e.g., a BA.1, BA.2, BA.3, BA.4, or BA.5 omicron variant). In some embodiments, a nucleic acid-containing particle comprises a first RNA molecule encoding a SARS-CoV-2 S protein from the Wuhan strain and a second RNA molecule encoding a SARS-CoV-2 S protein that includes one or more mutations characteristic of an omicron BA.1 variant. In some embodiments, a nucleic acid-containing particle comprises a first RNA molecule encoding a SARS-CoV-2 S protein from the Wuhan strain and a second RNA molecule encoding a SARS-CoV-2 S protein that includes one or more mutations characteristic of an omicron BA.1 variant. In some embodiments, the nucleic acid-containing particle comprises a first RNA molecule encoding a SARS-CoV-2 S protein from the Wuhan strain and a second RNA molecule encoding a SARS-CoV-2 S protein that includes one or more mutations characteristic of the Omicron BA.2 variant. In some embodiments, the nucleic acid-containing particle comprises a first RNA molecule encoding a SARS-CoV-2 S protein from the Wuhan strain and a second RNA molecule encoding a SARS-CoV-2 S protein that includes one or more mutations characteristic of the Omicron BA.3 variant. In some embodiments, the nucleic acid-containing particle comprises a first RNA molecule encoding a SARS-CoV-2 S protein from the Wuhan strain and a second RNA molecule encoding a SARS-CoV-2 S protein that includes one or more mutations characteristic of the Omicron BA.4 or BA.5 variant.

In some embodiments, the nucleic acid-containing particle comprises a first RNA molecule encoding a SARS-CoV-2 S protein from a first omicron variant and a second RNA molecule encoding a SARS-CoV-2 S protein that includes one or more mutations characteristic of a second omicron variant. In some embodiments, the nucleic acid-containing particle comprises a first RNA molecule encoding a SARS-CoV-2 S protein from a BA.1 omicron variant and a second RNA molecule encoding a SARS-CoV-2 S protein that includes one or more mutations characteristic of a BA.2 omicron variant. In some embodiments, the nucleic acid-containing particle comprises a first RNA molecule encoding a SARS-CoV-2 S protein from a BA.1 omicron variant and a second RNA molecule encoding a SARS-CoV-2 S protein that includes one or more mutations characteristic of a BA.3 omicron variant. In some embodiments, the nucleic acid-containing particle comprises a first RNA molecule encoding a SARS-CoV-2 S protein from a BA.1 omicron variant and a second RNA molecule encoding a SARS-CoV-2 S protein that includes one or more mutations characteristic of a BA.3 omicron variant. In some embodiments, the nucleic acid-containing particle comprises a first RNA molecule encoding a SARS-CoV-2 S protein from the BA.1 omicron variant and a second RNA molecule encoding a SARS-CoV-2 S protein that includes one or more mutations characteristic of the BA.4 or BA.5 omicron variant. In some embodiments, the nucleic acid-containing particle comprises a first RNA molecule encoding a SARS-CoV-2 S protein from the BA.2 omicron variant and a second RNA molecule encoding a SARS-CoV-2 S protein that includes one or more mutations characteristic of the BA.3 omicron variant. In some embodiments, the nucleic acid-containing particle comprises a first RNA molecule encoding a SARS-CoV-2 S protein from the BA.2 omicron variant and a second RNA molecule encoding a SARS-CoV-2 S protein that includes one or more mutations characteristic of the BA.4 or BA.5 omicron variant. In some embodiments, the nucleic acid-containing particle comprises a first RNA molecule encoding a SARS-CoV-2 S protein from the BA.2 omicron variant and a second RNA molecule encoding a SARS-CoV-2 S protein that includes one or more mutations characteristic of the BA.4 or BA.5 omicron variant. The SARS-CoV-2 S protein includes a first RNA molecule encoding a SARS-CoV-2 S protein from the BA.3 omicron variant, and a second RNA molecule encoding a SARS-CoV-2 S protein that includes one or more mutations characteristic of the BA.4 or BA.5 omicron variant.

In some embodiments, the first RNA molecule encoding a SARS-CoV-2 S protein from the Wuhan strain comprises a nucleotide sequence encoding the amino acid sequence of SEQ ID NO:7. In some embodiments, the first RNA molecule encoding a SARS-CoV-2 S protein from the Wuhan strain comprises a nucleotide sequence that is at least 80% identical (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical) to SEQ ID NO:9. In some embodiments, the first RNA molecule encoding a SARS-CoV-2 S protein from the Wuhan strain comprises a nucleotide sequence that is at least 80% identical (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical) to SEQ ID NO:20. In some embodiments, a first RNA molecule encoding a SARS-CoV-2 S protein from the Wuhan strain comprises a nucleotide sequence that encodes an amino acid sequence that is at least 80% identical (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical) to SEQ ID NO: 7. In some embodiments, a second RNA molecule encoding a SARS-CoV-2 S protein having one or more mutations characteristic of an omicron variant comprises a nucleotide sequence that encodes the amino acid sequence of SEQ ID NO: 49. In some embodiments, the second RNA molecule encoding a SARS-CoV-2 S protein comprising one or more mutations characteristic of an omicron variant comprises a nucleotide sequence that is at least 80% identical (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical) to SEQ ID NO: 50. In some embodiments, the second RNA molecule encoding a SARS-CoV-2 S protein comprising one or more mutations characteristic of an omicron variant comprises a nucleotide sequence that is at least 80% identical (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical) to SEQ ID NO: 51. In some embodiments, the second RNA molecule encoding a SARS-COV-2 S protein containing one or more mutations characteristic of an omicron variant comprises a nucleotide sequence encoding an amino acid sequence that is at least 80% identical (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical) to SEQ ID NO:49.

In some embodiments, the nucleic acid-containing particle (e.g., in some embodiments, an LNP as described herein) comprises a first RNA molecule comprising a nucleotide sequence encoding an amino acid sequence of SEQ ID NO:7 or an amino acid sequence that is at least 80% (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or more) identical to SEQ ID NO:7, and a second RNA molecule comprising a nucleotide sequence encoding an amino acid sequence of SEQ ID NO:49 or an amino acid sequence that is at least 80% (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or more) identical to SEQ ID NO:49.

In some embodiments, a nucleic acid-containing particle (e.g., in some embodiments, an LNP as described herein) comprises a first RNA molecule comprising a nucleotide sequence of SEQ ID NO:9 or a nucleotide sequence that is at least 80% (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or more) identical to SEQ ID NO:9, and a second RNA molecule comprising a nucleotide sequence of SEQ ID NO:50 or a nucleotide sequence that is at least 80% (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or more) identical to SEQ ID NO:50.

In some embodiments, a nucleic acid-containing particle (e.g., in some embodiments, an LNP as described herein) comprises a first RNA molecule comprising a nucleotide sequence of SEQ ID NO:20 or a nucleotide sequence that is at least 80% (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or more) identical to SEQ ID NO:20, and a second RNA molecule comprising a nucleotide sequence of SEQ ID NO:51 or a nucleotide sequence that is at least 80% (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or more) identical to SEQ ID NO:51.

In some embodiments, the nucleic acid-containing particle (e.g., in some embodiments, an LNP as described herein) comprises a first RNA molecule comprising a nucleotide sequence encoding an amino acid sequence of SEQ ID NO:7 or an amino acid sequence that is at least 80% (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or more) identical to SEQ ID NO:7, and a second RNA molecule comprising a nucleotide sequence encoding an amino acid sequence of SEQ ID NO:55, 58, or 61 or an amino acid sequence that is at least 80% (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or more) identical to SEQ ID NO:55, 58, or 61.

In some embodiments, the nucleic acid-containing particle (e.g., in some embodiments, an LNP as described herein) comprises a first RNA molecule comprising a nucleotide sequence of SEQ ID NO:9 or a nucleotide sequence that is at least 80% (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or more) identical to SEQ ID NO:9, and a second RNA molecule comprising a nucleotide sequence of SEQ ID NO:56, 59, or 62 or a nucleotide sequence that is at least 80% (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or more) identical to SEQ ID NO:56, 59, or 62.

In some embodiments, a nucleic acid-containing particle (e.g., in some embodiments, an LNP as described herein) comprises a first RNA molecule comprising a nucleotide sequence of SEQ ID NO:20 or a nucleotide sequence that is at least 80% (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or more) identical to SEQ ID NO:20, and a second RNA molecule comprising a nucleotide sequence of SEQ ID NO:57, 60, or 63 or a nucleotide sequence that is at least 80% (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or more) identical to SEQ ID NO:57, 60, or 63.

In some embodiments, the nucleic acid-containing particle (e.g., in some embodiments, an LNP as described herein) comprises a first RNA molecule comprising a nucleotide sequence encoding an amino acid sequence of SEQ ID NO:58 or an amino acid sequence that is at least 80% (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or more) identical to SEQ ID NO:58, and a second RNA molecule comprising a nucleotide sequence encoding an amino acid sequence of SEQ ID NO:49, 55, or 61 or an amino acid sequence that is at least 80% (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or more) identical to SEQ ID NO:49, 55, or 61.

In some embodiments, a nucleic acid-containing particle (e.g., in some embodiments, an LNP as described herein) comprises a first RNA molecule comprising a nucleotide sequence of SEQ ID NO:59 or a nucleotide sequence that is at least 80% (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or more) identical to SEQ ID NO:59, and a second RNA molecule comprising a nucleotide sequence of SEQ ID NO:50, 56, or 62 or a nucleotide sequence that is at least 80% (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or more) identical to SEQ ID NO:50, 56, or 62.

In some embodiments, a nucleic acid-containing particle (e.g., in some embodiments, an LNP as described herein) comprises a first RNA molecule comprising a nucleotide sequence of SEQ ID NO:60 or a nucleotide sequence that is at least 80% (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or more) identical to SEQ ID NO:60, and a second RNA molecule comprising a nucleotide sequence of SEQ ID NO:51, 57, or 63 or a nucleotide sequence that is at least 80% (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or more) identical to SEQ ID NO:51, 57, or 63.

In some embodiments, the nucleic acid-containing particle (e.g., in some embodiments, an LNP as described herein) comprises a first RNA molecule comprising a nucleotide sequence encoding an amino acid sequence of SEQ ID NO:49 or an amino acid sequence that is at least 80% (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or more) identical to SEQ ID NO:49, and a second RNA molecule comprising a nucleotide sequence encoding an amino acid sequence of SEQ ID NO:55 or 61 or an amino acid sequence that is at least 80% (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or more) identical to SEQ ID NO:55 or 61.

In some embodiments, the nucleic acid-containing particle (e.g., in some embodiments, an LNP as described herein) comprises a first RNA molecule comprising a nucleotide sequence of SEQ ID NO:50 or a nucleotide sequence that is at least 80% (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or more) identical to SEQ ID NO:50, and a second RNA molecule comprising a nucleotide sequence of SEQ ID NO:56 or 62 or a nucleotide sequence that is at least 80% (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or more) identical to SEQ ID NO:56 or 62.

In some embodiments, a nucleic acid-containing particle (e.g., in some embodiments, an LNP as described herein) comprises a first RNA molecule comprising a nucleotide sequence of SEQ ID NO:51 or a nucleotide sequence that is at least 80% (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or more) identical to SEQ ID NO:51, and a second RNA molecule comprising a nucleotide sequence of SEQ ID NO:57 or 63 or a nucleotide sequence that is at least 80% (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or more) identical to SEQ ID NO:57 or 63.

In some embodiments, the nucleic acid-containing particle (e.g., in some embodiments, an LNP as described herein) comprises a first RNA molecule comprising a nucleotide sequence encoding an amino acid sequence of SEQ ID NO:55 or an amino acid sequence that is at least 80% (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or more) identical to SEQ ID NO:55, and a second RNA molecule comprising a nucleotide sequence encoding an amino acid sequence of SEQ ID NO:61 or an amino acid sequence that is at least 80% (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or more) identical to SEQ ID NO:61.

In some embodiments, a nucleic acid-containing particle (e.g., in some embodiments, an LNP as described herein) comprises a first RNA molecule comprising a nucleotide sequence of SEQ ID NO:56 or a nucleotide sequence that is at least 80% (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or more) identical to SEQ ID NO:56, and a second RNA molecule comprising a nucleotide sequence of SEQ ID NO:62 or a nucleotide sequence that is at least 80% (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or more) identical to SEQ ID NO:62.

In some embodiments, a nucleic acid-containing particle (e.g., in some embodiments, an LNP as described herein) comprises a first RNA molecule comprising a nucleotide sequence of SEQ ID NO:57 or a nucleotide sequence that is at least 80% (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or more) identical to SEQ ID NO:57, and a second RNA molecule comprising a nucleotide sequence of SEQ ID NO:63 or a nucleotide sequence that is at least 80% (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or more) identical to SEQ ID NO:63.

In some embodiments, particles (e.g., in some embodiments, LNPs) containing nucleic acids (e.g., RNA) encoding different polypeptides can be formed by mixing multiple (e.g., at least two, at least three, or more) RNA molecules with particle-forming components (e.g., lipids). In some embodiments, the nucleic acids (e.g., RNA) encoding different polypeptides can be mixed (e.g., in some embodiments, in substantially equal proportions, e.g., in some embodiments, in a 1:1 ratio when two RNA molecules are present) prior to mixing with the particle-forming components (e.g., lipids).

In some embodiments, two or more RNA molecules, each encoding a different polypeptide (e.g., as described herein), can be mixed with a particle forming agent to form a nucleic acid-containing particle as described above. In alternative embodiments, two or more RNA molecules, each encoding a different polypeptide (e.g., as described herein), can be formulated into separate particle compositions that are then mixed together. For example, in some embodiments, individual populations of nucleic acid-containing particles, each population comprising RNA molecules encoding a different immunogenic polypeptide or immunogenic fragment thereof (e.g., as described herein), can be formed separately and then mixed together, e.g., prior to filling into vials during the manufacturing process or immediately prior to administration (e.g., by the administering medical practitioner). Thus, in some embodiments, described herein are compositions comprising two or more particle populations (e.g., in some embodiments, lipid nanoparticles), each population comprising at least one RNA molecule encoding a different immunogenic polypeptide or immunogenic fragment thereof (e.g., SARS-CoV-2 S protein or fragment thereof from different variants). In some embodiments, each population may be provided in the composition in a desired ratio (e.g., in some embodiments, each population may be provided in the composition in an amount that provides the same amount of RNA molecules).

Cationic Polymers Given their high degree of chemical flexibility, polymers are commonly used materials for nanoparticle-based delivery. Typically, cationic polymers are used to electrostatically condense negatively charged nucleic acids to nanoparticles. These positively charged groups are often composed of amines that change protonation state in the pH range of 5.5-7.5, which is believed to lead to an ionic imbalance that results in endosomal rupture. Polymers such as poly-L-lysine, polyamidoamine, protamine, and polyethyleneimine, as well as naturally occurring polymers such as chitosan, have all been applied to nucleic acid delivery and are suitable as cationic polymers herein. In addition, some researchers have synthesized polymers specifically for nucleic acid delivery. Poly(β-amino esters) have become widely used in nucleic acid delivery due to their ease of synthesis and biodegradability. Such synthetic polymers are also suitable as cationic polymers herein.

As used herein, "polymer" is given its ordinary meaning, i.e., a molecular structure containing one or more repeating units (monomers) connected by covalent bonds. The repeating units may all be identical, or in some cases, there may be more than one type of repeating unit present within the polymer. In some cases, the polymer is biologically derived, i.e., a biopolymer such as a protein. In some cases, additional moieties, e.g., targeting moieties such as those described herein, may also be present within the polymer.

When more than one type of repeat unit is present in a polymer, the polymer is said to be a "copolymer." It is understood that a polymer as used herein may be a copolymer. The repeat units forming the copolymer may be arranged in any manner. For example, the repeat units may be arranged in a random order, alternating, or as a "block" copolymer, i.e., one or more regions each containing a first repeat unit (e.g., a first block), one or more regions each containing a second repeat unit (e.g., a second block), etc. A block copolymer may have two (diblock copolymer), three (triblock copolymer), or more distinct blocks.

In certain embodiments, the polymer is biocompatible. A biocompatible polymer is typically a polymer that does not cause significant cell death at moderate concentrations. In certain embodiments, a biocompatible polymer is biodegradable, i.e., the polymer can be chemically and/or biologically degraded within a physiological environment, such as within the body.

In certain embodiments, the polymer may be protamine or a polyalkyleneimine, particularly protamine.

The term "protamine" refers to any of a variety of relatively low molecular weight, strongly basic proteins that are rich in arginine and specifically associate with DNA instead of somatic histones in the sperm cells of various animals (e.g., fish). In particular, the term "protamine" refers to a protein found in fish sperm that is strongly basic, soluble in water, does not coagulate by heat, and yields primarily arginine upon hydrolysis. In purified form, it is used in long-acting formulations of insulin and to neutralize the anticoagulant effect of heparin.

In accordance with the present disclosure, the term "protamine" as used herein is meant to include any protamine amino acid sequence obtained or derived from natural or biological sources, including fragments thereof and multimeric forms of said amino acid sequence or fragments thereof, as well as polypeptides that are artificial, specifically designed for a particular purpose, and cannot be isolated from natural or biological sources (synthesized).

In one embodiment, the polyalkyleneimine comprises polyethyleneimine and/or polypropyleneimine, preferably polyethyleneimine. A preferred polyalkyleneimine is polyethyleneimine (PEI). The average molecular weight of PEI is preferably 0.75·10 ² to 10 ⁷ Da, preferably 1000 to 10 ⁵ Da, more preferably 10000 to 40000 Da, more preferably 15000 to 30000 Da, even more preferably 20000 to 25000 Da.

According to the present disclosure, linear polyalkyleneimines such as linear polyethyleneimine (PEI) are preferred.

Cationic polymers (including polycationic polymers) contemplated for use herein include any cationic polymer that can electrostatically bind to nucleic acids. In one embodiment, cationic polymers contemplated for use herein include any cationic polymer with which nucleic acids can be associated, for example, by forming a complex with the nucleic acid or by forming a vesicle in which the nucleic acid is surrounded or encapsulated.

The particles described herein may also include polymers other than cationic polymers, i.e., non-cationic polymers and/or anionic polymers. Collectively, anionic and neutral polymers are referred to herein as non-cationic polymers.

Lipids and Lipid-Like Materials The terms "lipid" and "lipid-like materials" are broadly defined herein as molecules that contain one or more hydrophobic moieties or groups, and optionally also one or more hydrophilic moieties or groups. Molecules that contain hydrophobic and hydrophilic moieties are also frequently referred to as amphiphiles. Lipids are usually poorly soluble in water. In an aqueous environment, the amphiphilic nature allows the molecules to self-assemble into organized structures and different phases. One of these phases consists of lipid bilayers, as they exist in vesicles, multilamellar/unilamellar liposomes, or membranes in an aqueous environment. Hydrophobicity can be imparted by the inclusion of non-polar groups, including, but not limited to, long-chain saturated and unsaturated aliphatic hydrocarbon groups, and such groups substituted by one or more aromatic, alicyclic, or heterocyclic group(s). The hydrophilic groups can include polar and/or charged groups, including carbohydrates, phosphates, carboxylates, sulfates, amino, sulfhydryl, nitro, hydroxyl, and other similar groups.

As used herein, the term "amphiphilic" refers to a molecule that has both polar and non-polar portions. Often, an amphipathic compound has a polar head attached to a long hydrophobic tail. In some embodiments, the polar portion is soluble in water and the non-polar portion is insoluble in water. In addition, the polar portion may have either a formal positive charge or a formal negative charge. Alternatively, the polar portion may have both a formal positive charge and a formal negative charge and is a zwitterion or inner salt. For purposes of this disclosure, an amphipathic compound may be, but is not limited to, one or more natural or non-natural lipids and lipid-like compounds.

The term "lipid-like material", "lipid-like compound", or "lipid-like molecule" refers to substances that are structurally and/or functionally related to lipids, but may not be considered lipids in the strict sense. For example, the term includes compounds that can form amphiphilic layers due to their presence in vesicles, multilamellar/unilamellar liposomes, or membranes in an aqueous environment, and includes surfactants, or synthetic compounds that have both hydrophilic and hydrophobic moieties. In general, the term refers to molecules that contain hydrophilic and hydrophobic moieties with different structural structures that may or may not resemble lipids. As used herein, the term "lipid" should be construed to encompass both lipids and lipid-like materials, unless otherwise indicated herein or clearly contradicted by context.

Specific examples of amphiphilic compounds that may be included in the amphiphilic layer include, but are not limited to, phospholipids, aminolipids, and sphingolipids.

In certain embodiments, the amphiphilic compound is a lipid. The term "lipid" refers to a group of organic compounds characterized by being insoluble in water but soluble in many organic solvents. In general, lipids can be classified into eight categories: fatty acids, glycerolipids, glycerophospholipids, sphingolipids, glycolipids, polyketides (derived from the condensation of ketoacyl subunits), sterol lipids, and prenol lipids (derived from the condensation of isoprene subunits). The term "lipid" is sometimes used as a synonym for fat, which is a subgroup of lipids called triglycerides. Lipids also encompass molecules such as fatty acids and their derivatives (including trilipids, dilipids, monoglycerides, and phospholipids), as well as sterol-containing metabolites such as cholesterol.

Fatty acids, or fatty acid residues, are a diverse group of molecules made of a hydrocarbon chain terminating in a carboxylic acid group; this arrangement gives the molecule a polar hydrophilic end and a non-polar hydrophobic end that is insoluble in water. The carbon chain, which is typically 4-24 carbons long, can be saturated or unsaturated and can be attached to functional groups containing oxygen, halogens, nitrogen, and sulfur. When fatty acids contain double bonds, they can be either cis or trans geometric isomers, which significantly affects the configuration of the molecule. Cis double bonds cause the fatty acid chain to bend, and this effect is compounded with more double bonds in the chain. Other major lipid classes in the fatty acid category are fatty acid esters and fatty acid amides.

Glycerolipids are composed of mono-, di-, and tri-substituted glycerols, the best known being the fatty acid triesters of glycerol called triglycerides. The word "triacylglycerol" is sometimes used synonymously with "triglyceride." In these compounds, the three hydroxyl groups of glycerol are typically each esterified with a different fatty acid. A further subclass of glycerolipids is represented by glycosylglycerols, characterized by the presence of one or more sugar residues attached to glycerol via glycosidic bonds.

Glycerophospholipids are amphipathic molecules (containing both hydrophobic and hydrophilic regions) that contain a glycerol core linked by ester bonds to two fatty acid-derived "tails" and a phosphate ester bond to a "head" group. Examples of glycerophospholipids, commonly referred to as phospholipids (although sphingomyelin is also classified as a phospholipid), are phosphatidylcholine (PC, also known as GPCho or lecithin), phosphatidylethanolamine (PE or GPEtn), and phosphatidylserine (PS or GPSer).

Sphingolipids are a complex family of compounds that share a common structural feature, the sphingoid base backbone. The major sphingoid base in mammals is commonly referred to as sphingosine. Ceramides (N-acyl-sphingoid bases) are a major subclass of sphingoid base derivatives with amide-linked fatty acids. The fatty acids are typically saturated or monounsaturated with chain lengths of 16-26 carbon atoms. The major phosphosphingolipid in mammals is sphingomyelin (ceramide phosphocholine), insects contain primarily ceramide phosphoethanolamine, and fungi have phytoceramide phosphoinositol and mannose-containing head groups. Glycosphingolipids are a diverse family of molecules consisting of one or more sugar residues linked via glycosidic bonds to a sphingoid base. Examples of these are simple and complex glycosphingolipids such as cerebrosides and gangliosides.

Sterol lipids, such as cholesterol and its derivatives, or tocopherol and its derivatives, are important components of membrane lipids, along with glycerophospholipids and sphingomyelins.

Glycolipids describe compounds in which fatty acids are directly linked to a sugar backbone, forming structures compatible with membrane bilayers. In glycolipids, monosaccharides replace the glycerol backbone present in glycerolipids and glycerophospholipids. The best known glycolipids are the acylated glucosamine precursors of the lipid A component of lipopolysaccharides in gram-negative bacteria. Typical lipid A molecules are disaccharides of glucosamine, which are derivatized with up to seven fatty acyl chains. The minimal lipopolysaccharide required for growth of E. coli is Kdo2-lipid A, a hexacylated disaccharide of glucosamine glycosylated with two 3-deoxy-D-manno-octulosonic acid (Kdo) residues.

Polyketides are synthesized by polymerization of acetyl and propionyl subunits by classical enzymes, as well as iterative and multimodular enzymes that share mechanistic features with fatty acid synthases. They include numerous secondary metabolites and natural products from animal, plant, bacterial, fungal, and marine sources and have a great diversity of structures. Many polyketides are cyclic molecules whose backbones are often further modified by glycosylation, methylation, hydroxylation, oxidation, or other processes.

According to the present disclosure, lipids and lipid-like materials can be cationic, anionic, or neutral. Neutral lipids or lipid-like materials exist in an uncharged or neutral zwitterionic form at a selected pH.

Cationic lipid or cationically ionizable lipid or lipid-like material The nucleic acid particles described herein may comprise at least one cationic lipid or cationically ionizable lipid or lipid-like material as particle forming agent.The cationic lipid or cationically ionizable lipid or lipid-like material contemplated for use herein includes any cationic lipid or cationically ionizable lipid or lipid-like material that can electrostatically bind to nucleic acid.In one embodiment, the cationic lipid or cationically ionizable lipid or lipid-like material contemplated for use herein can associate with nucleic acid by forming a complex with nucleic acid or forming a vesicle in which nucleic acid is surrounded or enclosed.

As used herein, "cationic lipid" or "cationic lipid-like material" refers to a lipid or lipid-like material that has a net positive charge. Cationic lipids or lipid-like materials bind to negatively charged nucleic acids through electrostatic interactions. Generally, cationic lipids have a lipophilic moiety, e.g., a sterol, an acyl chain, a diacyl chain or more acyl chains, and the head group of the lipid typically carries the positive charge.

In certain embodiments, the cationic lipid or lipid-like material has a net positive charge only at certain pHs, particularly acidic pHs, while preferably having no net positive charge, preferably no charge, i.e., neutral at a different, preferably higher, pH, such as physiological pH. This ionizable behavior is believed to enhance efficacy by aiding in endosomal escape and reducing toxicity, compared to particles that remain cationic at physiological pH.

For purposes of this disclosure, such "cationically ionizable" lipids or lipid-like materials are encompassed by the term "cationic lipids or lipid-like materials," unless the context indicates differently.

In one embodiment, the cationic lipid or cationically ionizable lipid or lipid-like material comprises a head group that is positively charged or contains at least one nitrogen atom (N) that can be protonated.

Examples of cationic lipids include 1,2-dioleoyl-3-trimethylammonium propane (DOTAP); N,N-dimethyl-2,3-dioleyloxypropylamine (DODMA), 1,2-di-O-octadecenyl-3-trimethylammonium propane (DOTMA), 3-(N-(N',N'-dimethylaminoethane)-carbamoyl)cholesterol (DC-Chol), dimethyldioctadecylammonium (DDAB); 1,2-dioleoyl-3-dimethylammonium-propane (DODAP); 1,2-diacyloxy-3-dimethylammonium propane; 1,2-dialkyloxy-3-dimethylammonium propane; dioctadecyldimethylammonium chloride ( DODAC), 1,2-distearyloxy-N,N-dimethyl-3-aminopropane (DSDMA), 2,3-di(tetradecoxy)propyl-(2-hydroxyethyl)-dimethylazanium (DMRIE), 1,2-dimyristoyl-sn-glycero-3-ethylphosphocholine (DMEPC), 1,2-dimyristoyl-3-trimethylammonium propane (DMTAP), 1,2-dioleyloxypropyl-3-dimethyl-hydroxyethylammonium bromide (DORIE), and 2,3-dioleoyloxy-N-[2(sperminecarboxamido)ethyl]-N,N-dimethyl-1-propanammonium trifluoroacetate (DOSPA), 1,2-dilinoleyloxy-N,N- Dimethylaminopropane (DLinDMA), 1,2-dilinolenyloxy-N,N-dimethylaminopropane (DLenDMA), dioctadecylamidoglycylspermine (DOGS), 3-dimethylamino-2-(cholest-5-ene-3-beta-oxybutane-4-oxy)-1-(cis,cis-9,12-octadecadienoxy)propane (CLinDMA), 2-[5'-(cholest-5-ene-3-beta-oxy)-3'-oxapentoxy]-3-dimethyl-1-(cis,cis-9',12'-octadecadienoxy)propane (CpLinDMA), N,N-dimethyl-3,4-dioleyloxybenzylamine (DMOBA), 1,2-N,N'-dioleylcarbamyl-3- Dimethylaminopropane (DOcarbDAP), 2,3-dilinoleoyloxy-N,N-dimethylpropylamine (DLinDAP), 1,2-N,N'-dilinoleylcarbamyl-3-dimethylaminopropane (DLincarbDAP), 1,2-dilinoleoylcarbamyl-3-dimethylaminopropane (DLinCDAP), 2,2-dilinoleyl-4-dimethylaminomethyl-[1,3]-dioxolane (DLin-K-DMA), 2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane (DLin-K-XTC2-DMA), 2,2-dilinoleyl-4-(2-dimethylaminoethyl)-[1,3]-dioxolane (DLin-KC2-DMA), heptatriaco DLin-MC3-DMA), N-(2-hydroxyethyl)-N,N-dimethyl-2,3-bis(tetradecyloxy)-1-propanaminium bromide (DMRIE), (±)-N-(3-aminopropyl)-N,N-dimethyl-2,3-bis(cis-9-tetradecenyloxy)-1-propanaminium bromide (GAP-DMORIE), (±)-N-(3-aminopropyl)-N,N-dimethyl-2,3-bis(dodecyloxy)-1-propanaminium bromide (GAP-DLRIE), (±)-N-(3-aminopropyl)-N,N-dimethyl-2,3-bis(tetradecyloxy)- 1-propanaminium bromide (GAP-DMRIE), N-(2-aminoethyl)-N,N-dimethyl-2,3-bis(tetradecyloxy)-1-propanaminium bromide (βAE-DMRIE), N-(4-carboxybenzyl)-N,N-dimethyl-2,3-bis(oleoyloxy)propanaminium (DOBAQ), 2-({8-[(3β)-cholest-5-en-3-yloxy]octyl}oxy)-N,N-dimethyl-3-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]propanaminium-1-amine (octyl-CLinDMA), 1,2-dimyristoyl-3-dimethylammonium-propane (DMDAP), 1,2-dipalmitoyl-3-dimethylammonium-propane (DMDAP), dimethylammonium-propane (DPDAP), N1-[2-((1S)-1-[(3-aminopropyl)amino]-4-[di(3-aminopropyl)amino]butylcarboxamido)ethyl]-3,4-di[oleyloxy]-benzamide (MVL5), 1,2-dioleoyl-sn-glycero-3-ethylphosphocholine (DOEPC), 2,3-bis(dodecyloxy)-N-(2-hydroxyethyl)-N,N-dimethylpropane-1-ammonium bromide (DLRIE), N-(2-aminoethyl)-N,N-dimethyl-2,3-bis(tetradecyloxy)propane-1-aminium bromide (DMORIE), di(Z)-non-2-en-1-yl)8,8'-((((2(dimethylaminium (2-dodecyl)ethyl)thio)carbonyl)azanediyl)dioctanoate (ATX), N,N-dimethyl-2,3-bis(dodecyloxy)propan-1-amine (DLDMA), N,N-dimethyl-2,3-bis(tetradecyloxy)propan-1-amine (DMDMA), di((Z)-non-2-en-1-yl)-9-((4-(dimethylaminobutanoyl)oxy)heptadecanedioate (L319), N-dodecyl-3-((2-dodecylcarbamoyl-ethyl)-{2-[(2-dodecylcarbamoyl-ethyl)-2-{(2-dodecylcarbamoyl-ethyl)-[2-(2-dodecylcarbamoyl-ethylamino)-ethyl]-amino}-ethylamino]propionamide (Lipidoid 98N _12-5 ), 1-[2-[bis(2-hydroxydodecyl)amino]ethyl-[2-[4-[2-[bis(2hydroxydodecyl)amino]ethyl]piperazin-1-yl]ethyl]amino]dodecan-2-ol (Lipidoid C12-200).

In some embodiments, the cationic lipids may comprise about 10 mol% to about 100 mol%, about 20 mol% to about 100 mol%, about 30 mol% to about 100 mol%, about 40 mol% to about 100 mol%, or about 50 mol% to about 100 mol% of the total lipid present in the particle.

Additional lipid or lipid-like material The particles described herein may also include lipid or lipid-like material other than cationic lipid or cationic ionizable lipid or lipid-like material, i.e. non-cationic lipid or lipid-like material (including non-cationic non-ionizable lipid or lipid-like material). Collectively, anionic and neutral lipid or lipid-like material are referred to herein as non-cationic lipid or lipid-like material. In addition to ionizable/cationic lipid or lipid-like material, optimization of the formulation of nucleic acid particles by adding other hydrophobic moieties such as cholesterol and lipid can enhance particle stability and efficacy of nucleic acid delivery.

Additional lipids or lipid-like materials may be incorporated, which may or may not affect the overall charge of the nucleic acid particle. In certain embodiments, the additional lipids or lipid-like materials are non-cationic lipids or lipid-like materials. Non-cationic lipids may include, for example, one or more anionic lipids and/or neutral lipids. As used herein, "anionic lipid" refers to any lipid that is negatively charged at a selected pH. As used herein, "neutral lipid" refers to any of several lipid species that exist in either an uncharged or neutral zwitterionic form at a selected pH. In preferred embodiments, the additional lipid comprises one of the following neutral lipid components: (1) a phospholipid, (2) cholesterol or a derivative thereof, or (3) a mixture of a phospholipid and cholesterol or a derivative thereof. Examples of cholesterol derivatives include, but are not limited to, cholesterol, cholestanone, cholestenone, coprostanol, cholesteryl-2'-hydroxyethyl ether, cholesteryl-4'-hydroxybutyl ether, tocopherol and derivatives thereof, and mixtures thereof.

Specific phospholipids that can be used include, but are not limited to, phosphatidylcholine, phosphatidylethanolamine, phosphatidylglycerol, phosphatidic acid, phosphatidylserine, or sphingomyelin. Such phospholipids include, in particular, diacylphosphatidylcholines, such as distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dimyristoylphosphatidylcholine (DMPC), dipentadecanoylphosphatidylcholine, dilauroylphosphatidylcholine, dipalmitoylphosphatidylcholine (DPPC), diarachidoylphosphatidylcholine (DAPC), dibehenoylphosphatidylcholine (D BPC), ditricosanoyl phosphatidylcholine (DTPC), dilignoceroyl phosphatidylcholine (DLPC), palmitoyloleoyl-phosphatidylcholine (POPC), 1,2-di-O-octadecenyl-sn-glycero-3-phosphocholine (18:0 diether PC), 1-oleoyl-2-cholesterylhemisuccinoyl-sn-glycero-3-phosphocholine (OChemsPC), 1-hexadecyl-sn-glycero-3-phosphocholine (C16 Lyso PC), and phosphatidylethanolamines, especially diacylphosphatidylethanolamines, such as dioleoylphosphatidylethanolamine (DOPE), distearoyl-phosphatidylethanolamine (DSPE), dipalmitoyl-phosphatidylethanolamine (DPPE), dimyristoyl-phosphatidylethanolamine (DMPE), dilauroyl-phosphatidylethanolamine (DLPE), diphytanoyl-phosphatidylethanolamine (DPyPE), and even phosphatidylethanolamine lipids with different hydrophobic chains.

In certain preferred embodiments, the additional lipid is DSPC or DSPC and cholesterol.

In certain embodiments, the nucleic acid particles include both a cationic lipid and an additional lipid.

In one embodiment, the particles described herein include a polymer-conjugated lipid, such as a pegylated lipid. The term "pegylated lipid" refers to a molecule that includes both a lipid portion and a polyethylene glycol portion. Pegylated lipids are known in the art.

Without wishing to be bound by theory, the amount of at least one cationic lipid compared to the amount of at least one additional lipid can affect important nucleic acid particle characteristics such as nucleic acid charge, particle size, stability, tissue selectivity, and bioactivity. Thus, in some embodiments, the molar ratio of at least one cationic lipid to at least one additional lipid is about 10:0 to about 1:9, about 4:1 to about 1:2, or about 3:1 to about 1:1.

In some embodiments, non-cationic lipids, particularly neutral lipids (e.g., one or more phospholipids and/or cholesterol), may comprise from about 0 mol% to about 90 mol%, from about 0 mol% to about 80 mol%, from about 0 mol% to about 70 mol%, from about 0 mol% to about 60 mol%, or from about 0 mol% to about 50 mol% of the total lipid present in the particle.

Lipoplex Particles In certain embodiments of the present disclosure, the RNA described herein may be present in an RNA lipoplex particle.

In the context of the present disclosure, the term "RNA lipoplex particle" refers to a particle containing lipids, particularly cationic lipids, and RNA. Electrostatic interactions between positively charged liposomes and negatively charged RNA result in complexation and spontaneous formation of RNA lipoplex particles. Positively charged liposomes may generally be synthesized using a cationic lipid, such as DOTMA, and an additional lipid, such as DOPE. In one embodiment, the RNA lipoplex particle is a nanoparticle.

In certain embodiments, the RNA lipoplex particles include both a cationic lipid and an additional lipid. In an exemplary embodiment, the cationic lipid is DOTMA and the additional lipid is DOPE.

In some embodiments, the molar ratio of the at least one cationic lipid to the at least one additional lipid is about 10:0 to about 1:9, about 4:1 to about 1:2, or about 3:1 to about 1:1. In specific embodiments, the molar ratio can be about 3:1, about 2.75:1, about 2.5:1, about 2.25:1, about 2:1, about 1.75:1, about 1.5:1, about 1.25:1, or about 1:1. In an exemplary embodiment, the molar ratio of the at least one cationic lipid to the at least one additional lipid is about 2:1.

The RNA lipoplex particles described herein, in one embodiment, have an average diameter in the range of about 200 nm to about 1000 nm, about 200 nm to about 800 nm, about 250 to about 700 nm, about 400 to about 600 nm, about 300 nm to about 500 nm, or about 350 nm to about 400 nm. In certain embodiments, the RNA lipoplex particles have an average diameter of about 200 nm, about 225 nm, about 250 nm, about 275 nm, about 300 nm, about 325 nm, about 350 nm, about 375 nm, about 400 nm, about 425 nm, about 450 nm, about 475 nm, about 500 nm, about 525 nm, about 550 nm, about 575 nm, about 600 nm, about 625 nm, about 650 nm, about 700 nm, about 725 nm, about 750 nm, about 775 nm, about 800 nm, about 825 nm, about 850 nm, about 875 nm, about 900 nm, about 925 nm, about 950 nm, about 975 nm, or about 1000 nm. In one embodiment, the RNA lipoplex particles have an average diameter in the range of about 250 nm to about 700 nm. In another embodiment, the RNA lipoplex particles have an average diameter ranging from about 300 nm to about 500 nm. In an exemplary embodiment, the RNA lipoplex particles have an average diameter of about 400 nm.

The RNA lipoplex particles and compositions comprising the RNA lipoplex particles described herein are useful for delivery of RNA to target tissues after parenteral administration, particularly after intravenous administration. The RNA lipoplex particles may be prepared using liposomes, which may be obtained by injecting a solution of lipids in ethanol into water or a suitable aqueous phase. In one embodiment, the aqueous phase has an acidic pH. In one embodiment, the aqueous phase comprises acetic acid, for example in an amount of about 5 mM. Liposomes may be used to prepare the RNA lipoplex particles by mixing the liposomes with the RNA. In one embodiment, the liposomes and the RNA lipoplex particles comprise at least one cationic lipid and at least one additional lipid. In one embodiment, the at least one cationic lipid comprises 1,2-di-O-octadecenyl-3-trimethylammonium propane (DOTMA) and/or 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP). In one embodiment, the at least one additional lipid comprises 1,2-di-(9Z-octadecenoyl)-sn-glycero-3-phosphoethanolamine (DOPE), cholesterol (Chol) and/or 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC). In one embodiment, the at least one cationic lipid comprises 1,2-di-O-octadecenyl-3-trimethylammonium propane (DOTMA) and the at least one additional lipid comprises 1,2-di-(9Z-octadecenoyl)-sn-glycero-3-phosphoethanolamine (DOPE). In one embodiment, the liposomes and RNA lipoplex particles comprise 1,2-di-O-octadecenyl-3-trimethylammonium propane (DOTMA) and 1,2-di-(9Z-octadecenoyl)-sn-glycero-3-phosphoethanolamine (DOPE).

Spleen-targeted RNA lipoplex particles are described in WO 2013/143683 (herein incorporated by reference). It has been found that RNA lipoplex particles having a net negative charge can be used to preferentially target spleen tissue or spleen cells, such as antigen-presenting cells, particularly dendritic cells. Thus, following administration of the RNA lipoplex particles, RNA accumulation and/or RNA expression occurs in the spleen. Thus, the RNA lipoplex particles of the present disclosure can be used to express RNA in the spleen. In one embodiment, following administration of the RNA lipoplex particles, no or essentially no RNA accumulation and/or RNA expression occurs in the lung and/or liver. In one embodiment, following administration of the RNA lipoplex particles, RNA accumulation and/or RNA expression occurs in antigen-presenting cells, such as professional antigen-presenting cells, in the spleen. Thus, the RNA lipoplex particles of the present disclosure can be used to express RNA in such antigen-presenting cells. In one embodiment, the antigen-presenting cells are dendritic cells and/or macrophages.

Lipid Nanoparticles (LNPs)
In one embodiment, the nucleic acid, such as the RNA described herein, is administered in the form of a lipid nanoparticle (LNP). LNPs may include any lipid capable of forming a particle to which one or more nucleic acid molecules are attached or in which one or more nucleic acid molecules are encapsulated.

In one embodiment, the LNPs include one or more cationic lipids and one or more stabilizing lipids. Stabilizing lipids include neutral lipids and pegylated lipids.

In one embodiment, the LNPs comprise cationic lipids, neutral lipids, steroids, polymer-conjugated lipids, and RNA encapsulated within or associated with the lipid nanoparticles.

In one embodiment, the LNPs comprise 40-55 mol percent, 40-50 mol percent, 41-49 mol percent, 41-48 mol percent, 42-48 mol percent, 43-48 mol percent, 44-48 mol percent, 45-48 mol percent, 46-48 mol percent, 47-48 mol percent, or 47.2-47.8 mol percent cationic lipid. In one embodiment, the LNPs comprise about 47.0, 47.1, 47.2, 47.3, 47.4, 47.5, 47.6, 47.7, 47.8, 47.9, or 48.0 mol percent cationic lipid.

In one embodiment, the neutral lipid is present at a concentration ranging from 5 to 15 molar percent, 7 to 13 molar percent, or 9 to 11 molar percent. In one embodiment, the neutral lipid is present at a concentration of about 9.5, 10, or 10.5 molar percent.

In one embodiment, the steroid is present in a concentration ranging from 30 to 50 molar percent, 35 to 45 molar percent, or 38 to 43 molar percent. In one embodiment, the steroid is present in a concentration of about 40, 41, 42, 43, 44, 45, or 46 molar percent.

In one embodiment, the LNPs contain 1-10 mole percent, 1-5 mole percent, or 1-2.5 mole percent of polymer-conjugated lipid.

In one embodiment, the LNPs comprise 40-50 molar percent cationic lipid, 5-15 molar percent neutral lipid, 35-45 molar percent steroid, 1-10 molar percent polymer-conjugated lipid, and RNA encapsulated within or associated with the lipid nanoparticle.

In one embodiment, the mole percentage is determined based on the total moles of lipid present in the lipid nanoparticle.

In one embodiment, the neutral lipid is selected from the group consisting of DSPC, DPPC, DMPC, DOPC, POPC, DOPE, DOPG, DPPG, POPE, DPPE, DMPE, DSPE, and SM. In one embodiment, the neutral lipid is selected from the group consisting of DSPC, DPPC, DMPC, DOPC, POPC, DOPE, and SM. In one embodiment, the neutral lipid is DSPC.

In one embodiment, the steroid is cholesterol.

In one embodiment, the polymer-conjugated lipid is a pegylated lipid. In one embodiment, the pegylated lipid has the following structure:
or a pharma- ceutically acceptable salt, tautomer, or stereoisomer thereof, wherein:
R ¹² and R ¹³ are each independently a linear or branched, saturated or unsaturated alkyl chain containing from 10 to 30 carbon atoms, the alkyl chain optionally interrupted by one or more ester linkages, and w has an average value in the range of 30 to 60. In one embodiment, R ¹² and R ¹³ are each independently a linear, saturated alkyl chain containing from 12 to 16 carbon atoms. In one embodiment, w has an average value in the range of 40 to 55. In one embodiment, the average w is about 45. In one embodiment, R ¹² and R ¹³ are each independently a linear, saturated alkyl chain containing about 14 carbon atoms, and w has an average value of about 45.

In one embodiment, the pegylated lipid is, for example, DMG-PEG 2000, having the following structure:

In some embodiments, the cationic lipid component of the LNP has the structure of formula (III):
or a pharma- ceutically acceptable salt, tautomer, prodrug, or stereoisomer thereof, wherein:
One of L ¹ and L ² is -O(C=O)-, -(C=O)O-, -C(=O)-, -O-, -S(O) _x -, -S-S-, -C(=O)S-, SC(=O)-, -NR ^a C(=O)-, -C(=O)NR ^a -, NR ^a C(=O)NR ^a -, -OC(=O)NR ^a -, or -NR ^a C(=O)O-; and the other of L ¹ and L ² is -O(C=O)-, -(C=O)O-, -C(=O)-, -O-, -S(O) _x -, -S-S-, -C(=O)S-, SC(=O)-, -NR ^a C(=O)-, -C(=O)NR ^a -, NR ^a C(=O)NR ^a -, -OC(=O)NR ^a - or -NR ^a C(=O)O-, or a direct bond;
G ¹ and G ² are each independently an unsubstituted C ₁ -C ₁₂ alkylene or C ₁ -C ₁₂ alkenylene;
^G3 is C ₁ -C ₂₄ alkylene, C ₁ -C ₂₄ alkenylene, C ₃ -C ₈ cycloalkylene, C ₃ -C ₈ cycloalkenylene;
R ^a is H or C ₁ -C ₁₂ alkyl;
R ¹ and R ² are each independently a C ₆ -C ₂₄ alkyl or a C ₆ -C ₂₄ alkenyl;
R ³ is H, OR ⁵ , CN, -C(=O)OR ⁴ , -OC(=O)R ⁴ , or -NR ⁵ C(=O)R ⁴ ;
^R4 is _C1 - _C12 alkyl;
^R5 is H or _C1 - _C6 alkyl;
x is 0, 1, or 2.

In some of the foregoing embodiments of formula (III), the lipid has one of the following structures (IIIA) or (IIIB):
In the formula,
A is a 3- to 8-membered cycloalkyl or cycloalkylene ring;
R ⁶ , at each occurrence, is independently H, OH, or C ₁ -C ₂₄ alkyl;
n is an integer ranging from 1 to 15.

In some of the foregoing embodiments of formula (III), the lipid has structure (IIIA), and in other embodiments, the lipid has structure (IIIB).

In other embodiments of formula (III), the lipid has one of the following structures (IIIC) or (IIID):
In the formula, y and z are each independently an integer ranging from 1 to 12.

In any of the foregoing embodiments of formula (III), one of L ¹ or L ² is -O(C=O)-. For example, in some embodiments, each of L ¹ and L ² is -O(C=O)-. In several different embodiments of any of the foregoing, L ¹ and L ² are each independently -(C=O)O- or -O(C=O)-. For example, in some embodiments, each of L ¹ and L ² is -(C=O)O-.

In some different embodiments of formula (III), the lipid has one of the following structures (IIIE) or (IIIF):

In some of the foregoing embodiments of formula (III), the lipid has one of the following structures (IIIG), (IIIH), (IIII), or (IIIJ):

In some of the foregoing embodiments of formula (III), n is an integer ranging from 2 to 12, e.g., from 2 to 8 or from 2 to 4. For example, in some embodiments, n is 3, 4, 5, or 6. In some embodiments, n is 3. In some embodiments, n is 4. In some embodiments, n is 5. In some embodiments, n is 6.

In some other of the foregoing embodiments of formula (III), y and z are each independently an integer in the range of 2 to 10. For example, in some embodiments, y and z are each independently an integer in the range of 4 to 9 or 4 to 6.

In some of the foregoing embodiments of formula (III), R ⁶ is H. In other of the foregoing embodiments, R ⁶ is C ₁ -C ₂₄ alkyl. In other embodiments, R ⁶ is OH.

In some embodiments of formula (III), ^G3 is unsubstituted. In other embodiments, G3 is substituted. In various different embodiments, ^G3 is a straight chain C ₁ -C ₂₄ alkylene or a straight chain C ₁ -C ₂₄ alkenylene.

In some other foregoing embodiments of formula (III), R ¹ or R ² , or both, are C ₆ -C ₂₄ alkenyl. For example, in some embodiments, R ¹ and R ² each independently have the structure:
In the formula,
R ^7a and R ^7b , at each occurrence, are independently H or C ₁ -C ₁₂ alkyl;
a is an integer from 2 to 12;
R ^7a , R ^7b and a are each selected such that R ¹ and R ² each independently contain from 6 to 20 carbon atoms. For example, in some embodiments, a is an integer ranging from 5 to 9 or 8 to 12.

In some of the foregoing embodiments of formula (III), at least one occurrence of R ^7a is H. For example, in some embodiments, R ^7a is H at each occurrence. In other different embodiments of the foregoing, at least one occurrence of R ^7b is C ₁ -C ₈ alkyl. For example, in some embodiments, C ₁ -C ₈ alkyl is methyl, ethyl, n-propyl, iso-propyl, n-butyl, iso-butyl, tert-butyl, n-hexyl, or n-octyl.

In different embodiments of formula (III), R ¹ or R ² , or both, have one of the following structures:

In some of the foregoing embodiments of formula (III), R ³ is OH, CN, -C(=O)OR ⁴ , -OC(=O)R ⁴ , or -NHC(=O)R ^4. In some embodiments, R ⁴ is methyl or ethyl.

In various different embodiments, the cationic lipid of formula (III) has one of the structures set forth in the table below.

In some embodiments, the LNP comprises a lipid of formula (III), RNA, a neutral lipid, a steroid, and a pegylated lipid. In some embodiments, the lipid of formula (III) is compound III-3. In some embodiments, the neutral lipid is DSPC. In some embodiments, the steroid is cholesterol. In some embodiments, the pegylated lipid is ALC-0159.

In some embodiments, the cationic lipid is present in the LNP in an amount of about 40 to about 50 mole percent. In one embodiment, the neutral lipid is present in the LNP in an amount of about 5 to about 15 mole percent. In one embodiment, the steroid is present in the LNP in an amount of about 35 to about 45 mole percent. In one embodiment, the pegylated lipid is present in the LNP in an amount of about 1 to about 10 mole percent.

In some embodiments, the LNPs comprise compound III-3 in an amount of about 40 to about 50 mole percent, DSPC in an amount of about 5 to about 15 mole percent, cholesterol in an amount of about 35 to about 45 mole percent, and ALC-0159 in an amount of about 1 to about 10 mole percent.

In some embodiments, the LNPs comprise compound III-3 in an amount of about 47.5 mole percent, DSPC in an amount of about 10 mole percent, cholesterol in an amount of about 40.7 mole percent, and ALC-0159 in an amount of about 1.8 mole percent.

In various different embodiments, the cationic lipid has one of the structures shown in the table below.

In some embodiments, the LNPs comprise a cationic lipid as shown in the table above, e.g., a cationic lipid of formula (B) or formula (D), particularly a cationic lipid of formula (D), RNA, a neutral lipid, a steroid, and a pegylated lipid. In some embodiments, the neutral lipid is DSPC. In some embodiments, the steroid is cholesterol. In some embodiments, the pegylated lipid is DMG-PEG 2000.

In one embodiment, the LNPs comprise a cationic lipid that is an ionizable lipid-like material (lipidoid). In one embodiment, the cationic lipid has the following structure:
The N/P value is preferably at least about 4. In some embodiments, the N/P value ranges from 4 to 20, 4 to 12, 4 to 10, 4 to 8, or 5 to 7. In one embodiment, the N/P value is about 6.

The LNPs described herein, in one embodiment, may have an average diameter ranging from about 30 nm to about 200 nm, or from about 60 nm to about 120 nm.

RNA Targeting Some aspects of the present disclosure involve targeted delivery of the RNA disclosed herein (eg, RNA encoding vaccine antigens and/or immunostimulants).

In one embodiment, the present disclosure involves targeting the lungs. Targeting to the lungs is particularly preferred when the administered RNA is an RNA encoding a vaccine antigen. The RNA can be delivered to the lungs, for example, by administering by inhalation the RNA, which can be formulated as a particle, e.g., lipid particle, as described herein.

In one embodiment, the present disclosure involves targeting the lymphatic system, particularly the secondary lymphatic organs, and more particularly the spleen. Targeting the lymphatic system, particularly the secondary lymphatic organs, and more particularly the spleen, is particularly preferred when the administered RNA is RNA encoding a vaccine antigen.

In one embodiment, the target cell is a spleen cell. In one embodiment, the target cell is an antigen presenting cell, such as a professional antigen presenting cell in the spleen. In one embodiment, the target cell is a dendritic cell in the spleen.

The "lymphatic system" is a part of the circulatory system and an important part of the immune system that includes a network of lymphatic vessels that transport lymph. The lymphatic system consists of lymphoid organs, a conducting network of lymphatic vessels, and circulating lymph. Primary or central lymphoid organs generate lymphocytes from immature precursor cells. The thymus and bone marrow constitute the primary lymphoid organs. Secondary or peripheral lymphoid organs, including lymph nodes and the spleen, maintain mature naive lymphoid cells and initiate adaptive immune responses.

RNA may be delivered to the spleen by so-called lipoplex formulations, in which the RNA is bound to liposomes containing cationic lipids and optionally additional or helper lipids to form an injectable nanoparticle formulation. Liposomes can be obtained by injecting a solution of lipids in ethanol into water or a suitable aqueous phase. RNA lipoplex particles may be prepared by mixing liposomes with RNA. RNA lipoplex particles targeted to the spleen are described in WO 2013/143683 (incorporated herein by reference). It has been found that RNA lipoplex particles with a net negative charge can be used to preferentially target splenic tissue or splenic cells, such as antigen-presenting cells, particularly dendritic cells. Thus, after administration of the RNA lipoplex particles, RNA accumulation and/or RNA expression in the spleen occurs. Thus, the RNA lipoplex particles of the present disclosure can be used to express RNA in the spleen. In one embodiment, after administration of the RNA lipoplex particles, no or essentially no RNA accumulation and/or RNA expression in the lung and/or liver occurs. In one embodiment, administration of the RNA lipoplex particles results in RNA accumulation and/or RNA expression in antigen-presenting cells, such as professional antigen-presenting cells in the spleen. Thus, the RNA lipoplex particles of the present disclosure can be used to express RNA in such antigen-presenting cells. In one embodiment, the antigen-presenting cells are dendritic cells and/or macrophages.

The charge of the RNA lipoplex particles of the present disclosure is the sum of the charge present in the at least one cationic lipid and the charge present in the RNA. The charge ratio is the ratio of the positive charge present in the at least one cationic lipid to the negative charge present in the RNA. The charge ratio of the positive charge present in the at least one cationic lipid to the negative charge present in the RNA is calculated by the following equation: Charge ratio = [(cationic lipid concentration (mol)) * (total number of positive charges in the cationic lipid)] / [(RNA concentration (mol)) * (total number of negative charges in the RNA)].

The spleen-targeted RNA lipoplex particles described herein at physiological pH preferably have a net negative charge, e.g., a charge ratio of positive to negative charges of about 1.9:2 to about 1:2, or about 1.6:2 to about 1:2, or about 1.6:2 to about 1.1:2. In certain embodiments, the charge ratio of positive to negative charges in the RNA lipoplex particles at physiological pH is about 1.9:2.0, about 1.8:2.0, about 1.7:2.0, about 1.6:2.0, about 1.5:2.0, about 1.4:2.0, about 1.3:2.0, about 1.2:2.0, about 1.1:2.0, or about 1:2.0.

The immune stimulant may be provided to the subject by administering to the subject RNA encoding the immune stimulant in a formulation for preferential delivery of the RNA to the liver or liver tissue. Delivery of RNA to such target organs or tissues is preferred, particularly when it is desired to express large amounts of the immune stimulant and/or when a systemic presence of particularly significant amounts of the immune stimulant is desired or required.

RNA delivery systems have an inherent preference for the liver. This concerns lipid-based particles, cationic and neutral nanoparticles, especially lipid nanoparticles such as liposomes, nanomicelles and lipophilic ligands in bioconjugates. Liver accumulation is caused by discontinuities in the hepatic vasculature or lipid metabolism (liposomes and lipid or cholesterol conjugates).

For in vivo delivery of RNA to the liver, a drug delivery system may be used to transport the RNA to the liver by preventing degradation of the RNA. For example, polyplex nanomicelles consisting of a poly(ethylene glycol) (PEG) coated surface and an RNA (e.g., mRNA)-containing core are useful systems because the nanomicelles provide excellent in vivo stability of RNA under physiological conditions. Furthermore, the stealth properties provided by the polyplex nanomicelle surface consisting of a high density PEG pallisard effectively evade host immune defenses.

Examples of immune stimulants suitable for targeting to the liver are cytokines involved in the proliferation and/or maintenance of T cells. Examples of suitable cytokines include IL2 or IL7, fragments and variants thereof, and fusion proteins of these cytokines, fragments and variants, such as extended PK cytokines.

In another embodiment, the RNA encoding the immune stimulant may be administered in a formulation to preferentially deliver the RNA to the lymphatic system, particularly to secondary lymphoid organs, more particularly to the spleen. Delivery of the immune stimulant to such a target tissue is preferred, particularly when the presence of the immune stimulant in this organ or tissue is desired (e.g., when the immune stimulant is required to induce an immune response, particularly during T cell priming, such as a cytokine, or for activation of resident immune cells), but it is not desired that the immune stimulant be present systemically, particularly in significant amounts (e.g., because the immune stimulant has systemic toxicity).

Examples of suitable immune stimulants are cytokines involved in T cell priming. Examples of suitable cytokines include IL12, IL15, IFN-α, or IFN-β, fragments and variants thereof, and fusion proteins of these cytokines, fragments, and variants, such as extended PK cytokines.

Immune Stimulants In one embodiment, the RNA encoding the vaccine antigen may be non-immunogenic. In this and other embodiments, the RNA encoding the vaccine antigen may be co-administered with an immune stimulant or an RNA encoding an immune stimulant. The methods and medicaments described herein are particularly effective when the immune stimulant is attached to a pharmacokinetic modifier (hereinafter referred to as an "extended pharmacokinetic (PK)" immune stimulant). The methods and medicaments described herein are particularly effective when the immune stimulant is administered in the form of an RNA encoding the immune stimulant. In one embodiment, the RNA is targeted to the liver for systemic availability. Hepatocytes can be efficiently transfected and large amounts of protein can be produced.

An "immunostimulant" is any substance that stimulates the immune system by inducing activation or increasing the activity of any of the components of the immune system, especially immune effector cells. An immune stimulant may be pro-inflammatory.

According to one aspect, the immune stimulant is a cytokine or variant thereof. Examples of cytokines include interferons such as interferon-alpha (IFN-α) or interferon-gamma (IFN-γ), interleukins such as IL2, IL7, IL12, IL15, and IL23, colony stimulating factors such as M-CSF and GM-CSF, and tumor necrosis factor. According to another aspect, the immune stimulant comprises an adjuvant type immune stimulant such as an APC Toll-like receptor agonist or a costimulatory/cell adhesion membrane protein. Examples of Toll-like receptor agonists include costimulatory/adhesion proteins such as CD80, CD86, and ICAM-1.

Cytokines are a category of small proteins (approximately 5-20 kDa) that are important in cell signaling. Their release affects the behavior of surrounding cells. As immunomodulators, cytokines participate in autocrine, paracrine, and endocrine signaling. Cytokines include chemokines, interferons, interleukins, lymphokines, and tumor necrosis factors, but generally do not include hormones or growth factors (although there is some overlap in the term). Cytokines are produced by a wide range of cells, including immune cells such as macrophages, B lymphocytes, T lymphocytes, and mast cells, as well as endothelial cells, fibroblasts, and various stromal cells. A given cytokine may be produced by more than one type of cell. Cytokines act through receptors and are particularly important in the immune system, where they regulate the balance of humoral and cell-based immune responses, and they regulate the maturation, growth, and responsiveness of specific cell populations. Some cytokines enhance or inhibit the action of other cytokines in complex ways.

According to the present disclosure, the cytokine may be a naturally occurring cytokine, or a functional fragment or variant thereof. The cytokine may be a human cytokine and may be derived from any vertebrate, particularly any mammal. One particularly preferred cytokine is interferon-α.

Interferons (IFNs) are a group of signaling proteins made and released by host cells in response to the presence of several pathogens, such as viruses, bacteria, parasites, and tumor cells. In a typical scenario, a cell infected with a virus releases interferon to mount an antiviral defense in nearby cells.

Based on the type of receptor through which they signal, interferons are typically divided into three classes: type I interferons, type II interferons, and type III interferons.

All type I interferons bind to a specific cell surface receptor complex known as the IFN-α/β receptor (IFNAR), which consists of the IFNAR1 and IFNAR2 chains.

The type I interferons present in humans are IFNα, IFNβ, IFNε, IFNκ, and IFNω. Generally, type I interferons are produced when the body recognizes an invading virus. They are produced by fibroblasts and monocytes. Once released, type I interferons bind to specific receptors on target cells, which leads to the expression of proteins that prevent the virus from producing and replicating its RNA and DNA.

IFNα proteins are mainly produced by plasmacytoid dendritic cells (pDCs). They are mainly involved in innate immunity against viral infections. The genes responsible for their synthesis are derived from 13 subtypes called IFNA1, IFNA2, IFNA4, IFNA5, IFNA6, IFNA7, IFNA8, IFNA10, IFNA13, IFNA14, IFNA16, IFNA17, IFNA21. These genes are found together in a cluster on chromosome 9.

IFNβ proteins are produced in large quantities by fibroblasts. They have antiviral activity that is primarily involved in the innate immune response. Two types of IFNβ have been described, IFNβ1 and IFNβ3. Natural and recombinant forms of IFNβ1 have antiviral, antibacterial, and anticancer properties.

Type II interferons (IFNγ in humans), also known as immune interferons, are activated by IL12. In addition, type II interferons are released by cytotoxic T cells and T helper cells.

Type III interferons signal through a receptor complex consisting of IL10R2 (also called CRF2-4) and IFNLR1 (also called CRF2-12). Although more recently discovered than types I and II IFNs, recent information demonstrates the importance of type III IFNs in several types of viral or fungal infections.

In general, type I and type II interferons are responsible for regulating and activating the immune response.

According to the present disclosure, the type I interferon is preferably IFNα or IFNβ, more preferably IFNα.

According to the present disclosure, the interferon can be a naturally occurring interferon, or a functional fragment or variant thereof. The interferon can be a human interferon and can be derived from any vertebrate, particularly any mammal.

Interleukins (IL) are a group of cytokines (secreted proteins and signaling molecules) that can be divided into four major groups based on distinguishing structural features. However, their amino acid sequence similarity is rather weak (typically 15-25% identity). The human genome encodes over 50 interleukins and related proteins.

According to the present disclosure, the interleukin can be a naturally occurring interleukin, or a functional fragment or variant thereof. The interleukin can be a human interleukin and can be derived from any vertebrate, particularly any mammal.

Extended PK Groups The immunostimulatory polypeptides described herein can be prepared as fusion or chimeric polypeptides comprising an immunostimulatory moiety and a heterologous polypeptide (i.e., a polypeptide that is not an immunostimulatory agent). The immunostimulatory agent may be fused to an extended PK group that increases the circulating half-life. Non-limiting examples of extended PK groups are described below. It should be understood that other PK groups that increase the circulating half-life of an immunostimulatory agent, such as a cytokine or variant thereof, are also applicable to the present disclosure. In certain embodiments, the extended PK group is a serum albumin domain (e.g., mouse serum albumin, human serum albumin).

As used herein, the term "PK" is an acronym for "pharmacokinetic" and encompasses the properties of a compound including, by way of example, absorption, distribution, metabolism, and elimination by a subject. As used herein, an "extended PK group" refers to a protein, peptide, or moiety that, when fused to or administered together with a biologically active molecule, increases the circulating half-life of the biologically active molecule. Examples of extended PK groups include serum albumin (e.g., HSA), immunoglobulin Fc or Fc fragments and variants thereof, transferrin and variants thereof, and human serum albumin (HSA) binders (such as those disclosed in U.S. Publication Nos. 2005/0287153 and 2007/0003549). Other exemplary extended PK groups are disclosed in Kontermann, Expert Opin Biol Ther, 2016 Jul;16(7):903-15, which is incorporated herein by reference in its entirety. As used herein, an "extended PK" immunostimulant refers to an immunostimulant moiety combined with an extended PK group. In one embodiment, the extended PK immunostimulant is a fusion protein in which the immunostimulant moiety is linked or fused to the extended PK group.

In certain embodiments, the serum half-life of the extended PK immunostimulant is increased compared to the immunostimulant alone (i.e., the immunostimulant not fused to an extended PK group). In certain embodiments, the serum half-life of the extended PK immunostimulant is at least 20, 40, 60, 80, 100, 120, 150, 180, 200, 400, 600, 800, or 1000% longer compared to the serum half-life of the immunostimulant alone. In certain embodiments, the serum half-life of the extended PK immunostimulant is at least 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 6, 7, 8, 10, 12, 13, 15, 17, 20, 22, 25, 27, 30, 35, 40, or 50 times longer than the serum half-life of the immunostimulant alone. In certain embodiments, the serum half-life of the extended PK immunostimulant is at least 10 hours, 15 hours, 20 hours, 25 hours, 30 hours, 35 hours, 40 hours, 50 hours, 60 hours, 70 hours, 80 hours, 90 hours, 100 hours, 110 hours, 120 hours, 130 hours, 135 hours, 140 hours, 150 hours, 160 hours, or 200 hours.

As used herein, "half-life" refers to the time required for the serum or plasma concentration of a compound, such as a peptide or protein, to be reduced by 50% in vivo, for example due to degradation and/or clearance or sequestration by natural mechanisms. Suitable extended PK immunostimulants for use herein are stabilized in vivo and their half-life is increased, for example, by fusion to serum albumin (e.g., HSA or MSA), which resists degradation and/or clearance or sequestration. The half-life can be determined by any method known per se, such as pharmacokinetic analysis. Suitable techniques will be clear to the skilled artisan and may, for example, generally include the steps of administering a suitable dose of the amino acid sequence or compound to a subject, periodically collecting blood or other samples from the subject, determining the level or concentration of the amino acid sequence or compound in the blood sample, and calculating from the (plot of) the data so obtained the time until the level or concentration of the amino acid sequence or compound is reduced by 50% compared to the initial level at the time of administration. Further details are provided in standard handbooks such as, for example, Kenneth, A. et al., Chemical Stability of Pharmaceuticals: A Handbook for Pharmacists and in Peters et al., Pharmacokinetic Analysis: A Practical Approach (1996). See also Gibaldi, M. et al., Pharmacokinetics, 2nd Rev. Edition, Marcel Dekker (1982).

In certain embodiments, the extended PK group comprises serum albumin or a fragment thereof, or a variant of serum albumin or a fragment thereof (all of which are included in the term "albumin" for purposes of this disclosure). The polypeptides described herein may be fused to albumin (or a fragment or variant thereof) to form an albumin fusion protein. Such albumin fusion proteins are described in U.S. Publication No. 2007/0048282.

As used herein, an "albumin fusion protein" refers to a protein formed by the fusion of at least one molecule of albumin (or a fragment or variant thereof) to at least one molecule of a protein, such as a therapeutic protein, particularly an immune stimulant. Albumin fusion proteins can be produced by translation of a nucleic acid in which a polynucleotide encoding a therapeutic protein is joined in frame with a polynucleotide encoding albumin. The therapeutic protein and albumin were once part of the albumin fusion protein and each can be referred to as a "portion," "region," or "moiety" of the albumin fusion protein (e.g., a "therapeutic protein portion" or an "albumin protein portion"). In a highly preferred embodiment, the albumin fusion protein comprises at least one molecule of a therapeutic protein (including, but not limited to, a mature form of a therapeutic protein) and at least one molecule of albumin (including, but not limited to, a mature form of albumin). In one embodiment, the albumin fusion protein is processed by host cells, such as cells of a target organ for the administered RNA (e.g., liver cells), and secreted into the circulation. Processing of the nascent albumin fusion protein that occurs in the secretory pathway of the host cell used to express the RNA may include, but is not limited to, signal peptide cleavage, disulfide bond formation, proper folding, carbohydrate addition and processing (e.g., N- and O-linked glycosylation), specific proteolytic cleavage, and/or assembly into multimeric proteins. The albumin fusion protein is preferably encoded by the RNA in an unprocessed form, particularly with a signal peptide at the N-terminus, and subsequent secretion by the cell is preferably in a processed form, particularly with the signal peptide cleaved. In the most preferred embodiment, the "processed form of the albumin fusion protein" refers to an albumin fusion protein product that has undergone N-terminal signal peptide cleavage, also referred to herein as a "mature albumin fusion protein."

In a preferred embodiment, the albumin fusion protein comprising a therapeutic protein has a higher plasma stability compared to the plasma stability of the same therapeutic protein when not fused to albumin. Plasma stability typically refers to the period of time that a therapeutic protein is administered in vivo and transported into the bloodstream, and the period of time that the therapeutic protein is degraded and cleared from the bloodstream and ultimately enters organs such as the kidney or liver that clear the therapeutic protein from the body. Plasma stability is calculated in terms of the half-life of the therapeutic protein in the bloodstream. The half-life of a therapeutic protein in the bloodstream can be readily determined by common assays known in the art.

As used herein, "albumin" collectively refers to an albumin protein or amino acid sequence, or an albumin fragment or variant, having one or more functional activities (e.g., biological activities) of albumin. In particular, "albumin" refers to human albumin or a fragment or variant thereof, particularly the mature form of human albumin, or albumin or a fragment thereof from other vertebrates, or variants of these molecules. Albumin may be derived from any vertebrate, particularly any mammal, such as human, bovine, ovine, or porcine. Non-mammalian albumins include, but are not limited to, hen and salmon. The albumin portion of the albumin fusion protein may be derived from a different animal than the therapeutic protein portion.

In certain embodiments, the albumin is human serum albumin (HSA), or a fragment or variant thereof, such as those disclosed in US 5,876,969, WO 2011/124718, WO 2013/075066, and WO 2011/0514789.

The terms human serum albumin (HSA) and human albumin (HA) are used interchangeably herein. The terms "albumin" and "serum albumin" are broader and include human serum albumin (and fragments and variants thereof) as well as albumins from other species (and fragments and variants thereof).

As used herein, a fragment of albumin sufficient to extend the therapeutic activity or plasma stability of a therapeutic protein refers to a fragment of albumin of sufficient length or structure to stabilize or extend the therapeutic activity or plasma stability of the protein, such that the plasma stability of the therapeutic protein portion of the albumin fusion protein is extended or extended compared to the plasma stability of the unfused state.

The albumin portion of the albumin fusion protein may include the full length of the albumin sequence, or may include one or more fragments thereof that can stabilize or extend therapeutic activity or plasma stability. Such fragments may be 10 or more amino acids in length, or may include about 15, 20, 25, 30, 50, or more contiguous amino acids from the albumin sequence, or may include some or all of a particular domain of albumin. For example, one or more fragments of HSA spanning the first two immunoglobulin-like domains may be used. In a preferred embodiment, the HSA fragment is the mature form of HSA.

Generally speaking, an albumin fragment or variant is at least 100 amino acids long, preferably at least 150 amino acids long.

According to the present disclosure, the albumin can be naturally occurring albumin, or a fragment or variant thereof. The albumin can be human albumin and can be derived from any vertebrate, particularly any mammal.

Preferably, the albumin fusion protein comprises albumin as the N-terminal portion and a therapeutic protein as the C-terminal portion. Alternatively, an albumin fusion protein comprising albumin as the C-terminal portion and a therapeutic protein as the N-terminal portion may be used. In other embodiments, the albumin fusion protein has a therapeutic protein fused to both the N-terminus and the C-terminus of albumin. In a preferred embodiment, the therapeutic proteins fused at the N-terminus and the C-terminus are the same therapeutic protein. In another preferred embodiment, the therapeutic proteins fused at the N-terminus and the C-terminus are different therapeutic proteins. In one embodiment, the different therapeutic proteins are both cytokines.

In one embodiment, the therapeutic protein(s) are conjugated to albumin via (a) a peptide linker(s). The linker peptide between the fusion moieties may provide greater physical separation between the moieties, thus maximizing the accessibility of the therapeutic protein moiety to bind, for example, to its cognate receptor. The linker peptide may be composed of amino acids such that it is flexible or more rigid. The linker sequence may be cleavable by a protease or chemically.

As used herein, the term "Fc region" refers to the portion of a native immunoglobulin formed by the Fc domains (or Fc portions) of each of its two heavy chains. As used herein, the term "Fc domain" refers to a portion or fragment of a single immunoglobulin (Ig) heavy chain in which the Fc domain does not include an Fv domain. In certain embodiments, the Fc domain begins at the hinge region immediately upstream of the papain cleavage site and ends at the C-terminus of the antibody. Thus, a complete Fc domain includes at least a hinge domain, a CH2 domain, and a CH3 domain. In certain embodiments, the Fc domain includes at least one of a hinge (e.g., upper, middle, and/or lower hinge region) domain, a CH2 domain, a CH3 domain, a CH4 domain, or a variant, portion, or fragment thereof. In certain embodiments, the Fc domain includes a complete Fc domain (i.e., a hinge domain, a CH2 domain, and a CH3 domain). In certain embodiments, the Fc domain includes a hinge domain (or a portion thereof) fused to a CH3 domain (or a portion thereof). In certain embodiments, the Fc domain comprises a CH2 domain (or a portion thereof) fused to a CH3 domain (or a portion thereof). In certain embodiments, the Fc domain consists of a CH3 domain or a portion thereof. In certain embodiments, the Fc domain consists of a hinge domain (or a portion thereof) and a CH3 domain (or a portion thereof). In certain embodiments, the Fc domain consists of a CH2 domain (or a portion thereof) and a CH3 domain. In certain embodiments, the Fc domain consists of a hinge domain (or a portion thereof) and a CH2 domain (or a portion thereof). In certain embodiments, the Fc domain lacks at least a portion of the CH2 domain (e.g., all or a portion of the CH2 domain). An Fc domain herein generally refers to a polypeptide comprising all or a portion of the Fc domain of an immunoglobulin heavy chain. This includes, but is not limited to, polypeptides comprising the entire CH1, hinge, CH2, and/or CH3 domains, as well as fragments of such peptides comprising, for example, only the hinge, CH2, and CH3 domains. The Fc domain may be derived from any species and/or any subtype of immunoglobulin, including, but not limited to, human IgG1, IgG2, IgG3, IgG4, IgD, IgA, IgE, or IgM antibodies. Fc domains encompass natural Fc and Fc variant molecules. As described herein, it will be understood by those skilled in the art that any Fc domain may be modified to vary in amino acid sequence from the native Fc domain of a naturally occurring immunoglobulin molecule. In certain embodiments, the Fc domain has reduced effector function (e.g., FcγR binding).

The Fc domains of the polypeptides described herein may be derived from different immunoglobulin molecules. For example, the Fc domains of the polypeptides may comprise a CH2 and/or CH3 domain derived from an IgG1 molecule, and a hinge region derived from an IgG3 molecule. In another example, the Fc domains may comprise a chimeric hinge region derived, in part, from an IgG1 molecule and in part from an IgG3 molecule. In another example, the Fc domains may comprise a chimeric hinge region derived, in part, from an IgG1 molecule and in part from an IgG4 molecule.

In certain embodiments, the extended PK group comprises an Fc domain or a fragment thereof, or a variant of an Fc domain or a fragment thereof (all of which are included in the term "Fc domain" for purposes of this disclosure). The Fc domain does not contain a variable region that binds to an antigen. Fc domains suitable for use in this disclosure may be obtained from a number of different sources. In certain embodiments, the Fc domain is derived from a human immunoglobulin. In certain embodiments, the Fc domain is derived from a human IgG1 constant region. However, it is understood that the Fc domain may be derived from an immunoglobulin of another mammalian species, including, for example, rodent (e.g., mouse, rat, rabbit, guinea pig) or non-human primate (e.g., chimpanzee, macaque) species.

Furthermore, the Fc domain (or a fragment or variant thereof) can be derived from any immunoglobulin class, including IgM, IgG, IgD, IgA, and IgE, and any immunoglobulin isotype, including IgG1, IgG2, IgG3, and IgG4.

A variety of Fc domain gene sequences (e.g., mouse and human constant region gene sequences) are available in the form of publicly available deposits. Constant region domains, including Fc domain sequences, can be selected that lack specific effector functions and/or have specific modifications to reduce immunogenicity. Many sequences of antibodies and antibody-encoding genes have been published, and suitable Fc domain sequences (e.g., hinge, CH2, and/or CH3 sequences, or fragments or variants thereof) can be derived from these sequences using art-recognized techniques.

In certain embodiments, the extended PK group is a serum albumin binding protein, such as those described in US 2005/0287153, US 2007/0003549, US 2007/0178082, US 2007/0269422, US 2010/0113339, WO 2009/083804, and WO 2009/133208, which are incorporated by reference in their entireties. In certain embodiments, the extended PK group is transferrin, as disclosed in US 7,176,278 and US 8,158,579, which are incorporated by reference in their entireties. In certain embodiments, the extended PK group is a serum immunoglobulin binding protein, such as those disclosed in US 2007/0178082, US 2014/0220017, and US 2017/0145062, which are incorporated herein by reference in their entirety. In certain embodiments, the extended PK group is a fibronectin (Fn)-based scaffold domain protein that binds to serum albumin, such as those disclosed in US 2012/0094909, which is incorporated herein by reference in its entirety. Methods for making fibronectin-based scaffold domain proteins are also disclosed in US 2012/0094909. A non-limiting example of an Fn3-based extended PK group is Fn3 (HSA), an Fn3 protein that binds to human serum albumin.

In certain aspects, extended PK immunostimulants suitable for use according to the present disclosure may employ one or more peptide linkers. As used herein, the term "peptide linker" refers to a peptide or polypeptide sequence that connects two or more domains (e.g., an extended PK moiety and an immunostimulatory moiety) within the linear amino acid sequence of a polypeptide chain. For example, a peptide linker may be used to connect an immunostimulatory moiety to an HSA domain.

Linkers suitable for fusing an extended PK group, for example, to an immunostimulant, are well known in the art. Exemplary linkers include glycine-serine-polypeptide linkers, glycine-proline-polypeptide linkers, and proline-alanine polypeptide linkers. In certain embodiments, the linker is a glycine-serine-polypeptide linker, i.e., a peptide consisting of glycine and serine residues.

In addition to or instead of the heterologous polypeptides described above, the immunostimulatory polypeptides described herein can contain sequences encoding a "marker" or "reporter." Examples of marker or reporter genes include β-lactamase, chloramphenicol acetyltransferase (CAT), adenosine deaminase (ADA), aminoglycoside phosphotransferase, dihydrofolate reductase (DHFR), hygromycin-B-phosphotransferase (HPH), thymidine kinase (TK), β-galactosidase, and xanthine guanine phosphoribosyltransferase (XGPRT).

Pharmaceutical Compositions The agents described herein may be administered in a pharmaceutical composition or medicament, and may be administered in the form of any suitable pharmaceutical composition.

In one embodiment, the pharmaceutical composition described herein is an immunogenic composition for inducing an immune response against a coronavirus in a subject. For example, in one embodiment, the immunogenic composition is a vaccine.

In one embodiment of all aspects of the disclosure, the components described herein, such as RNA encoding a vaccine antigen, may be administered in a pharmaceutical composition that may include a pharma- ceutically acceptable carrier and, optionally, one or more adjuvants, stabilizers, etc. In one embodiment, the pharmaceutical composition is for therapeutic or prophylactic treatment, e.g., for use in the treatment or prevention of a coronavirus infection.

The term "pharmaceutical composition" refers to a formulation comprising a therapeutically active agent, preferably together with a pharma- ceutically acceptable carrier, diluent, and/or excipient. The pharmaceutical composition is useful for treating, preventing, or reducing the severity of a disease or disorder by administration of the pharmaceutical composition to a subject. Pharmaceutical compositions are also known in the art as medicinal formulations.

The pharmaceutical compositions of the present disclosure may include or be administered with one or more adjuvants. The term "adjuvant" refers to a compound that prolongs, enhances, or promotes an immune response. Adjuvants include a heterogeneous group of compounds such as oil emulsions (e.g., Freund's adjuvant), mineral compounds (such as alum), bacterial products (such as Bordetella pertussis toxin), or immune stimulating complexes. Examples of adjuvants include, but are not limited to, LPS, GP96, CpG oligodeoxynucleotides, growth factors, and cytokines (monokines, lymphokines, interleukins, chemokines, etc.). Cytokines may be IL1, IL2, IL3, IL4, IL5, IL6, IL7, IL8, IL9, IL10, IL12, IFNα, IFNγ, GM-CSF, LT-a. Further known adjuvants are aluminum hydroxide, Freund's adjuvant, or oils such as Montanide® ISA 51. Other suitable adjuvants for use in the present disclosure include lipopeptides such as Pam3Cys.

Pharmaceutical compositions according to the present disclosure are generally applied in "pharmaceutical effective amounts" and "pharmaceutical acceptable preparations."

The term "pharmaceutical acceptable" refers to the non-toxicity of a material that does not interact with the action of the active ingredients of a pharmaceutical composition.

The term "pharmacologically effective amount" or "therapeutically effective amount" refers to an amount that alone or together with further doses achieves the desired response or the desired effect. In the case of the treatment of a particular disease, the desired response preferably relates to the inhibition of the course of the disease. This includes slowing the progression of the disease, and in particular, halting or reversing the progression of the disease. The desired response in the treatment of a disease may also be the delay or prevention of the onset of the disease or condition. The effective amount of the compositions described herein will depend on the individual parameters of the patient, including the condition being treated, the severity of the disease, age, physiological condition, size and weight, duration of treatment, type of concomitant therapy (if any), the particular route of administration and similar factors. Thus, the dose of the compositions described herein administered may depend on various such parameters. If the response in the patient is insufficient with the initial dose, a higher dose (or an effectively higher dose achieved by a different, more localized route of administration) can be used.

The pharmaceutical compositions of the present disclosure may contain salts, buffers, preservatives, and optionally other therapeutic agents. In one embodiment, the pharmaceutical compositions of the present disclosure include one or more pharma- ceutically acceptable carriers, diluents, and/or excipients.

Suitable preservatives for use in the pharmaceutical compositions of the present disclosure include, but are not limited to, benzalkonium chloride, chlorobutanol, parabens, and thimerosal.

As used herein, the term "excipient" refers to a substance that may be present in a pharmaceutical composition of the present disclosure but is not an active ingredient. Examples of excipients include, but are not limited to, a carrier, binder, diluent, lubricant, thickener, surface active agent, preservative, stabilizer, emulsifier, buffer, flavoring agent, or coloring agent.

The term "diluent" refers to a diluent and/or thinning agent. Further, the term "diluent" includes any one or more of a fluid, liquid, or solid suspension, and/or mixed medium. Examples of suitable diluents include ethanol, glycerol, and water.

The term "carrier" refers to a component, which may be natural, synthetic, organic, or inorganic, with which an active ingredient is combined to facilitate, enhance, or enable administration of a pharmaceutical composition. As used herein, a carrier may be one or more compatible solid or liquid fillers, diluents, or encapsulating substances suitable for administration to a subject. Suitable carriers include, but are not limited to, sterile water, Ringer's, Ringer's lactate, sterile sodium chloride solution, isotonic saline, polyalkylene glycols, hydrogenated naphthalenes, and especially biocompatible lactide polymers, lactide/glycolide copolymers, or polyoxyethylene/polyoxypropylene copolymers. In one embodiment, the pharmaceutical composition of the present disclosure comprises isotonic saline.

Pharmaceutically acceptable carriers, excipients, or diluents for therapeutic use are well known in the pharmaceutical arts and are described, for example, in Remington's Pharmaceutical Sciences, Mack Publishing Co. (A.R Gennaro edit. 1985).

Pharmaceutical carriers, excipients, or diluents can be selected with regard to the intended route of administration and standard pharmaceutical practice.

In one embodiment, the pharmaceutical compositions described herein may be administered intravenously, intraarterially, subcutaneously, intradermally, or intramuscularly. In certain embodiments, the pharmaceutical compositions are formulated for local or systemic administration. Systemic administration may include enteral administration with absorption via the digestive tract, or parenteral administration. As used herein, "parenteral administration" refers to administration in any manner other than via the digestive tract, such as intravenous injection. In a preferred embodiment, the pharmaceutical composition is formulated for intramuscular administration. In another embodiment, the pharmaceutical composition is formulated for systemic administration, e.g., intravenous administration.

As used herein, the term "co-administration" refers to the process by which different compounds or compositions (e.g., RNA encoding an antigen and RNA encoding an immunostimulant) are administered to the same patient. The different compounds or compositions can be administered simultaneously, substantially simultaneously, or sequentially.

The pharmaceutical compositions and products described herein may be provided as a freeze concentrate for injectable solution, e.g., at a concentration of 0.50 mg/mL. In one embodiment, for preparation of the injectable solution, the pharmaceutical product is thawed and diluted with an isotonic sodium chloride solution (e.g., 0.9% NaCl, saline), e.g., by a one-step dilution process. In some embodiments, a bacteriostatic sodium chloride solution (e.g., 0.9% NaCl, saline) cannot be used as a diluent. In some embodiments, the diluted pharmaceutical product is an off-white suspension. The concentration of the final solution for injection varies depending on the respective dose level to be administered.

In one embodiment, administration is performed within 6 hours after the start of preparation due to the risk of microbial contamination and considering the multi-dose approach of the preparation process. In one embodiment, two conditions are allowed during this 6 hour period: room temperature for preparation, handling, and transportation, and 2-8°C for storage.

The compositions described herein may be shipped and/or stored under temperature controlled conditions, e.g., at or below about 4-5°C, at or below about -20°C, at or below -70°C ± 10°C (e.g., -80°C to -60°C), e.g., utilizing a cooling system (e.g., which may be or may include dry ice) to maintain the desired temperature. In one embodiment, the compositions described herein are shipped in temperature controlled thermal shippers. Such shippers may include GPS-enabled thermal sensors to track the location and temperature of each shipment. The compositions may be stored, e.g., by refilling with dry ice.

Treatment In one aspect, the disclosure provides methods and medicaments for inducing an adaptive immune response against coronavirus in a subject comprising administering an effective amount of a composition comprising RNA encoding a coronavirus vaccine antigen described herein.

In one embodiment, the methods and medicaments described herein provide immunity in a subject against a coronavirus, a coronavirus infection, or a disease or disorder associated with a coronavirus. In another aspect, the present disclosure thus provides methods and medicaments for treating or preventing an infection, disease, or disorder associated with a coronavirus.

In one embodiment, the methods and medicaments described herein are administered to a subject having an infection, disease, or disorder associated with coronavirus. In one embodiment, the methods and medicaments described herein are administered to a subject at risk of developing an infection, disease, or disorder associated with coronavirus. For example, the methods and medicaments described herein may be administered to a subject at risk of contacting coronavirus. In one embodiment, the methods and medicaments described herein are administered to a subject who lives in, has traveled, or is expected to travel to a geographic area where coronavirus is prevalent. In one embodiment, the methods and medicaments described herein are administered to a subject who is in contact with, or is expected to be in contact with, another person who lives in, has traveled, or is expected to travel to a geographic area where coronavirus is prevalent. In one embodiment, the methods and medicaments described herein are administered to a subject who has been knowingly exposed to coronavirus through occupational or other contact. In one embodiment, the coronavirus is SARS-CoV-2. In some embodiments, the methods and medicaments described herein are administered to a subject who has evidence of previous exposure and/or infection with or cross-reactivity with SARS-CoV-2 and/or antigens or epitopes thereof. For example, in some embodiments, the methods and medicaments described herein are administered to subjects in which antibodies, B cells, and/or T cells reactive with one or more epitopes of the SARS-CoV-2 spike protein are detectable and/or have been detected.

For a composition to be useful as a vaccine, it must induce an immune response to a coronavirus antigen in a cell, tissue, or subject (e.g., a human). In some embodiments, the composition induces an immune response to a coronavirus antigen in a cell, tissue, or subject (e.g., a human). In some cases, the vaccine induces a protective immune response in a mammal. The therapeutic compounds or compositions of the present disclosure can be administered prophylactically (i.e., to prevent a disease or disorder) or therapeutically (i.e., to treat a disease or disorder) to a subject suffering from or at risk of developing (or susceptible to) a disease or disorder. Such subjects can be identified using standard clinical methods. In the context of the present disclosure, prophylactic administration occurs prior to the onset of overt clinical symptoms of a disease, such that the disease or disorder is prevented or, alternatively, its progression is delayed. In the context of the medical field, the term "preventing" encompasses any activity that reduces the burden of mortality or morbidity due to a disease. Prevention can occur at the primary, secondary, and tertiary prevention levels. While primary prevention avoids the onset of disease, secondary and tertiary levels of prevention encompass activities aimed at preventing disease progression and the appearance of symptoms, as well as reducing the adverse effects of an already established disease by restoring function and reducing disease-related complications.

As used herein, the term "dose" generally refers to the "dosage amount" that relates to the amount of RNA administered per administration, i.e., per dosage.

In some embodiments, administration of the immunogenic compositions or vaccines of the present disclosure may be performed by a single dose or may be boosted by multiple doses.

In some embodiments, the regimen described herein comprises at least one dose. In some embodiments, the regimen comprises a first dose and at least one subsequent dose. In some embodiments, the first dose is in the same amount as at least one subsequent dose. In some embodiments, the first dose is in the same amount as all subsequent doses. In some embodiments, the first dose is in a different amount than at least one subsequent dose. In some embodiments, the first dose is in a different amount than all subsequent doses. In some embodiments, the regimen comprises two doses. In some embodiments, the regimen provided consists of two doses. In some embodiments, the regimen comprises three doses.

In one embodiment, the disclosure contemplates administration of a single dose. In one embodiment, the disclosure contemplates administration of a priming dose followed by one or more booster doses. The booster dose or first booster dose may be administered 7-28 days or 14-24 days after administration of the priming dose. In some embodiments, the first booster dose may be administered 1 week to 3 months (e.g., 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, 11 weeks, 12 weeks) after administration of the priming dose. In some embodiments, the subsequent booster dose may be administered at least 1 week or more, including, for example, at least 2 weeks, at least 3 weeks, at least 4 weeks, at least 5 weeks, at least 6 weeks, at least 7 weeks, at least 8 weeks, at least 9 weeks, at least 10 weeks, at least 11 weeks, at least 12 weeks, or more, after the preceding booster dose. In some embodiments, subsequent booster doses may be administered at intervals of about 5-9 weeks or 6-8 weeks. In some embodiments, at least one subsequent booster dose (e.g., after a first booster dose) may be administered at least 3 months or more, including, for example, at least 4 months, at least 5 months, at least 6 months, at least 7 months, at least 8 months, at least 9 months, at least 10 months, or more, after the preceding dose.

In some embodiments, a subsequent dose given to an individual (e.g., as part of a primary or booster regimen) may have the same amount of RNA as previously given to the individual. In some embodiments, a subsequent dose given to an individual (e.g., as part of a primary or booster regimen) may have a different amount of RNA compared to the amount previously given to the individual. For example, in some embodiments, the subsequent dose may be higher or lower than the previous dose based on consideration of various factors including, for example, immunogenicity and/or reactogenicity induced by the previous dose, disease prevalence, etc. In some embodiments, the subsequent dose may be at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or more higher than the previous dose. In some embodiments, the subsequent dose may be at least 1.5 times, at least 2 times, at least 2.5 times, at least 3 times, or more higher than the previous dose. In some embodiments, a subsequent dose may be at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90% or more higher than the previous dose, In some embodiments, a subsequent dose may be at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70% or more lower than the previous dose. In some embodiments, an amount of 0.1 μg to 300 μg, 0.5 μg to 200 μg, or 1 μg to 100 μg, e.g., about 1 μg, about 2 μg, about 3 μg, about 4 μg, about 5 μg, about 6 μg, about 7 μg, about 8 μg, about 9 μg, about 10 μg, about 15 μg, about 20 μg, about 25 μg, about 30 μg, about 35 μg, about 40 μg, about 45 μg, about 50 μg, about 55 μg, about 60 μg, about 70 μg, about 80 μg, about 90 μg, or about 100 μg of the RNA described herein may be administered per dose (e.g., at a given dose).

In some embodiments, an amount of 60 μg or less, 55 μg or less, 50 μg or less, 45 μg or less, 40 μg or less, 35 μg or less, 30 μg or less, 25 μg or less, 20 μg or less, 15 μg or less, 10 μg or less, 5 μg or less, 3 μg or less, 2.5 μg or less, or 1 μg or less of the RNA described herein may be administered per dose (e.g., at a given dose).

In some embodiments, an amount of at least 0.25 μg, at least 0.5 μg, at least 1 μg, at least 2 μg, at least 3 μg, at least 4 μg, at least 5 μg, at least 10 μg, at least 15 μg, at least 20 μg, at least 25 μg, at least 30 μg, at least 40 μg, at least 50 μg, or at least 60 μg of the RNA described herein may be administered per dose (e.g., in a given dose). In some embodiments, an amount of at least 3 ug of the RNA described herein may be administered in at least one of the given doses. In some embodiments, an amount of at least 10 ug of the RNA described herein may be administered in at least one of the given doses. In some embodiments, an amount of at least 15 ug of the RNA described herein may be administered in at least one of the given doses. In some embodiments, an amount of at least 20 ug of the RNA described herein may be administered in at least one of the given doses. In some embodiments, an amount of at least 25ug of the RNA described herein may be administered in at least one of the given doses. In some embodiments, an amount of at least 30ug of the RNA described herein may be administered in at least one of the given doses. In some embodiments, an amount of at least 50ug of the RNA described herein may be administered in at least one of the given doses. In some embodiments, an amount of at least 60ug of the RNA described herein may be administered in at least one of the given doses. In some embodiments, a combination of the aforementioned amounts may be administered in a regimen comprising two or more doses (e.g., the previous dose and the subsequent dose may be different amounts as described herein). In some embodiments, a combination of the aforementioned amounts may be administered in a primary regimen and a booster regimen (e.g., different doses may be given in the primary regimen and the booster regimen).

In some embodiments, an amount of 0.25 μg to 60 μg, 0.5 μg to 55 μg, 1 μg to 50 μg, 5 μg to 40 μg, or 10 μg to 30 μg of the RNA described herein may be administered per dose. In some embodiments, an amount of 3 μg to 30 μg of the RNA described herein may be administered in at least one of the given doses. In some embodiments, an amount of 3 μg to 20 μg of the RNA described herein may be administered in at least one of the given doses. In some embodiments, an amount of 3 μg to 15 μg of the RNA described herein may be administered in at least one of the given doses. In some embodiments, an amount of 3 μg to 10 μg of the RNA described herein may be administered in at least one of the given doses. In some embodiments, an amount of 10 μg to 30 μg of the RNA described herein may be administered in at least one of the given doses.

In some embodiments, the regimen administered to the subject may include multiple doses (e.g., at least two doses, at least three doses, or more). In some embodiments, the regimen administered to the subject may include a first dose and a second dose given at least two weeks apart, at least three weeks apart, at least four weeks apart, or more apart. In some embodiments, such doses may be spaced at least one month, at least two months, at least three months, at least four months, at least five months, at least six months, at least seven months, at least eight months, at least nine months, at least ten months, at least eleven months, at least twelve months, or more apart. In some embodiments, the doses may be administered several days apart, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60 or more days apart. In some embodiments, doses may be administered about 1 to about 3 weeks apart, or about 1 to about 4 weeks apart, or about 1 to about 5 weeks apart, or about 1 to about 6 weeks apart, or about 1 to more than 6 weeks apart. In some embodiments, doses may be separated by a period of about 7 to 60 days, such as about 14 to about 48 days. In some embodiments, the minimum number of days between doses may be about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, or more. In some embodiments, the maximum number of days between doses may be about 60, 59, 58, 57, 56, 55, 54, 53, 52, 51, 50, 49, 48, 47, 46, 45, 44, 43, 42, 41, 40, 39, 38, 37, 36, 35, 34, 33, 32, 31, 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, or less. In some embodiments, the doses may be spaced apart by about 21 to about 28 days. In some embodiments, the doses may be spaced apart by about 19 to about 42 days. In some embodiments, the doses may be spaced apart by about 7 to about 28 days. In some embodiments, the doses may be spaced apart by about 14 to about 24 days. In some embodiments, the doses may be spaced apart by about 21 to about 42 days.

In some embodiments, the vaccination regimen includes a first dose and a second dose. In some embodiments, the first dose and the second dose are administered at least 21 days apart. In some embodiments, the first dose and the second dose are administered at least 28 days apart.

In some embodiments, the vaccination regimen includes a first dose and a second dose, and the amount of RNA administered in the first dose is the same as the amount of RNA administered in the second dose. In some embodiments, the vaccination regimen includes a first dose and a second dose, and the amount of RNA administered in the first dose is different from the amount of RNA administered in the second dose.

In some embodiments, the vaccination regimen includes a first dose and a second dose, and the amount of RNA administered in the first dose is less than the amount of RNA administered in the second dose. In some embodiments, the amount of RNA administered in the first dose is 10%-90% of the amount of RNA administered in the second dose. In some embodiments, the amount of RNA administered in the first dose is 10%-50% of the amount of RNA administered in the second dose. In some embodiments, the amount of RNA administered in the first dose is 10%-20% of the amount of RNA administered in the second dose. In some embodiments, the first dose and the second dose are administered at least 2 weeks apart, at least 3 weeks apart, at least 4 weeks apart, at least 5 weeks apart, at least 6 weeks apart, or more apart. In some embodiments, the first dose and the second dose are administered at least 3 weeks apart.

In some embodiments, the first dose comprises less than about 30ug of RNA and the second dose comprises at least about 30ug of RNA. In some embodiments, the first dose comprises about 1 to less than about 30ug of RNA (e.g., about 0.1, about 1, about 3, about 5, about 10, about 15, about 20, about 25, or less than about 30ug of RNA) and the second dose comprises about 30 to about 100ug of RNA (e.g., about 30, about 40, about 50, or about 60ug of RNA). In some embodiments, the first dose comprises about 1 to about 20ug of RNA, about 1 to about 10ug of RNA, or about 1 to about 5ug of RNA and the second dose comprises about 30 to about 60ug of RNA.

In some embodiments, the first dose comprises about 1 to about 10 ug of RNA (e.g., about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, or about 10 ug of RNA) and the second dose comprises about 30 to about 60 ug of RNA (e.g., about 30, about 35, about 40, about 45, about 50, about 55, or about 60 ug of RNA).

In some embodiments, the first dose comprises about 1 ug of RNA and the second dose comprises about 30 ug of RNA. In some embodiments, the first dose comprises about 3 ug of RNA and the second dose comprises about 30 ug of RNA. In some embodiments, the first dose comprises about 5 ug of RNA and the second dose comprises about 30 ug of RNA. In some embodiments, the first dose comprises about 10 ug of RNA and the second dose comprises about 30 ug of RNA. In some embodiments, the first dose comprises about 15 ug of RNA and the second dose comprises about 30 ug of RNA.

In some embodiments, the first dose comprises about 1 ug of RNA and the second dose comprises about 60 ug of RNA. In some embodiments, the first dose comprises about 3 ug of RNA and the second dose comprises about 60 ug of RNA. In some embodiments, the first dose comprises about 5 ug of RNA and the second dose comprises about 60 ug of RNA. In some embodiments, the first dose comprises about 6 ug of RNA and the second dose comprises about 60 ug of RNA. In some embodiments, the first dose comprises about 10 ug of RNA and the second dose comprises about 60 ug of RNA. In some embodiments, the first dose comprises about 15 ug of RNA and the second dose comprises about 60 ug of RNA. In some embodiments, the first dose comprises about 20 ug of RNA and the second dose comprises about 60 ug of RNA. In some embodiments, the first dose comprises about 25ug of RNA and the second dose comprises about 60ug of RNA. In some embodiments, the first dose comprises about 30ug of RNA and the second dose comprises about 60ug of RNA.

In some embodiments, the first dose comprises less than about 10ug of RNA and the second dose comprises at least about 10ug of RNA. In some embodiments, the first dose comprises about 0.1 to less than about 10ug of RNA (e.g., about 0.1, about 0.5, about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, or less than about 10ug of RNA) and the second dose comprises about 10 to about 30ug of RNA (e.g., about 10, about 15, about 20, about 25, or about 30ug of RNA). In some embodiments, the first dose comprises about 0.1 to about 10ug of RNA, about 1 to about 5ug of RNA, or about 0.1 to about 3ug of RNA and the second dose comprises about 10 to about 30ug of RNA.

In some embodiments, the first dose comprises about 0.1 to about 5 ug of RNA (e.g., about 0.1, about 0.5, about 1, about 2, about 3, about 4, about 5 ug of RNA) and the second dose comprises about 10 to about 20 ug of RNA (e.g., about 10, about 12, about 14, about 16, about 18, about 20 ug of RNA).

In some embodiments, the first dose comprises about 0.1 ug of RNA and the second dose comprises about 10 ug of RNA. In some embodiments, the first dose comprises about 0.3 ug of RNA and the second dose comprises about 10 ug of RNA. In some embodiments, the first dose comprises about 1 ug of RNA and the second dose comprises about 10 ug of RNA. In some embodiments, the first dose comprises about 3 ug of RNA and the second dose comprises about 10 ug of RNA.

In some embodiments, the first dose comprises less than about 3ug of RNA and the second dose comprises at least about 3ug of RNA. In some embodiments, the first dose comprises about 0.1 to less than about 3ug of RNA (e.g., about 0.1, about 0.2, about 0.3, about 0.5, about 0.6, about 0.7, about 0.8, about 0.9, about 1.0, about 1.5, about 2.0, or about 2.5ug of RNA) and the second dose comprises about 3 to about 10ug of RNA (e.g., about 3, about 4, about 5, about 6, or about 7, about 8, about 9, or about 10ug of RNA). In some embodiments, the first dose comprises about 0.1 to about 3ug of RNA, about 0.1 to about 1ug of RNA, or about 0.1 to about 0.5ug of RNA and the second dose comprises about 3 to about 10ug of RNA.

In some embodiments, the first dose comprises about 0.1 to about 1.0 ug of RNA (e.g., about 0.1, about 0.2, about 0.3, about 0.4, about 0.5, about 0.6, about 0.7, about 0.8, about 0.9, or about 1.0 ug of RNA) and the second dose comprises about 1 to about 3 ug of RNA (e.g., about 1.0, about 1.5, about 2.0, about 2.5, or about 3.0 ug of RNA).

In some embodiments, the first dose comprises about 0.1 ug of RNA and the second dose comprises about 3 ug of RNA. In some embodiments, the first dose comprises about 0.3 ug of RNA and the second dose comprises about 3 ug of RNA. In some embodiments, the first dose comprises about 0.5 ug of RNA and the second dose comprises about 3 ug of RNA. In some embodiments, the first dose comprises about 1 ug of RNA and the second dose comprises about 3 ug of RNA.

In some embodiments, the vaccination regimen includes a first dose and a second dose, and the amount of RNA administered in the first dose is greater than the amount of RNA administered in the second dose. In some embodiments, the amount of RNA administered in the second dose is 10%-90% of the first dose. In some embodiments, the amount of RNA administered in the second dose is 10%-50% of the first dose. In some embodiments, the amount of RNA administered in the second dose is 10%-20% of the first dose. In some embodiments, the first dose and the second dose are administered at least 2 weeks apart, at least 3 weeks apart, at least 4 weeks apart, at least 5 weeks apart, at least 6 weeks apart, or more apart. In some embodiments, the first dose and the second dose are administered at least 3 weeks apart.

In some embodiments, the first dose comprises at least about 30ug of RNA and the second dose comprises less than about 30ug of RNA. In some embodiments, the first dose comprises about 30 to about 100ug of RNA (e.g., about 30, about 40, about 50, or about 60ug of RNA) and the second dose comprises about 1 to about 30ug of RNA (e.g., about 0.1, about 1, about 3, about 5, about 10, about 15, about 20, about 25, or about 30ug of RNA). In some embodiments, the second dose comprises about 1 to about 20ug of RNA, about 1 to about 10ug of RNA, or about 1 to 5ug of RNA. In some embodiments, the first dose comprises about 30 to about 60 ug of RNA and the second dose comprises about 1 to about 20 ug of RNA, about 1 to about 10 ug of RNA, or about 0.1 to about 3 ug of RNA.

In some embodiments, the first dose comprises about 30 to about 60 ug of RNA (e.g., about 30, about 35, about 40, about 45, about 50, about 55, or about 60 ug of RNA) and the second dose comprises about 1 to about 10 ug of RNA (e.g., about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, or about 10 ug of RNA).

In some embodiments, the first dose comprises about 30ug of RNA and the second dose comprises about 1ug of RNA. In some embodiments, the first dose comprises about 30ug of RNA and the second dose comprises about 3ug of RNA. In some embodiments, the first dose comprises about 30ug of RNA and the second dose comprises about 5ug of RNA. In some embodiments, the first dose comprises about 30ug of RNA and the second dose comprises about 10ug of RNA. In some embodiments, the first dose comprises about 30ug of RNA and the second dose comprises about 15ug of RNA.

In some embodiments, the first dose comprises about 60ug of RNA and the second dose comprises about 1ug of RNA. In some embodiments, the first dose comprises about 60ug of RNA and the second dose comprises about 3ug of RNA. In some embodiments, the first dose comprises about 60ug of RNA and the second dose comprises about 5ug of RNA. In some embodiments, the first dose comprises about 60ug of RNA and the second dose comprises about 6ug of RNA. In some embodiments, the first dose comprises about 60ug of RNA and the second dose comprises about 10ug of RNA. In some embodiments, the first dose comprises about 60ug of RNA and the second dose comprises about 15ug of RNA. In some embodiments, the first dose comprises about 60ug of RNA and the second dose comprises about 20ug of RNA. In some embodiments, the first dose comprises about 60ug of RNA and the second dose comprises about 25ug of RNA. In some embodiments, the first dose comprises about 60ug of RNA and the second dose comprises about 30ug of RNA.

In some embodiments, the first dose comprises at least about 10ug of RNA and the second dose comprises less than about 10ug of RNA. In some embodiments, the first dose comprises about 10 to about 30ug of RNA (e.g., about 10, about 15, about 20, about 25, or about 30ug of RNA) and the second dose comprises about 0.1 to less than about 10ug of RNA (e.g., about 0.1, about 0.5, about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, or less than about 10ug of RNA). In some embodiments, the first dose comprises about 10 to about 30ug of RNA, or about 0.1 to about 3ug of RNA, and the second dose comprises about 1 to about 10ug of RNA, or about 1 to about 5ug of RNA.

In some embodiments, the first dose comprises about 10 to about 20 ug of RNA (e.g., about 10, about 12, about 14, about 16, about 18, about 20 ug of RNA) and the second dose comprises about 0.1 to about 5 ug of RNA (e.g., about 0.1, about 0.5, about 1, about 2, about 3, about 4, or about 5 ug of RNA).

In some embodiments, the first dose comprises about 10ug of RNA and the second dose comprises about 0.1ug of RNA. In some embodiments, the first dose comprises about 10ug of RNA and the second dose comprises about 0.3ug of RNA. In some embodiments, the first dose comprises about 10ug of RNA and the second dose comprises about 1ug of RNA. In some embodiments, the first dose comprises about 10ug of RNA and the second dose comprises about 3ug of RNA.

In some embodiments, the first dose comprises at least about 3ug of RNA and the second dose comprises less than about 3ug of RNA. In some embodiments, the first dose comprises about 3 to about 10ug of RNA (e.g., about 3, about 4, about 5, about 6, or about 7, about 8, about 9, or about 10ug of RNA) and the second dose comprises about 0.1 to less than about 3ug of RNA (e.g., about 0.1, about 0.2, about 0.3, about 0.5, about 0.6, about 0.7, about 0.8, about 0.9, about 1.0, about 1.5, about 2.0, or about 2.5ug of RNA). In some embodiments, the first dose comprises about 3 to about 10ug of RNA and the second dose comprises about 0.1 to about 3ug of RNA, about 0.1 to about 1ug of RNA, or about 0.1 to about 0.5ug of RNA.

In some embodiments, the first dose comprises about 1 to about 3 ug of RNA (e.g., about 1, about 1.5, about 2.0, about 2.5, or about 3.0 ug of RNA) and the second dose comprises about 0.1 to about 0.3 ug of RNA (e.g., about 0.1, about 0.2, about 0.3, about 0.4, about 0.5, about 0.6, about 0.7, about 0.8, about 0.9, or about 1.0 ug of RNA).

In some embodiments, the first dose comprises about 3ug of RNA and the second dose comprises about 0.1ug of RNA. In some embodiments, the first dose comprises about 3ug of RNA and the second dose comprises about 0.3ug of RNA. In some embodiments, the first dose comprises about 3ug of RNA and the second dose comprises about 0.6ug of RNA. In some embodiments, the first dose comprises about 3ug of RNA and the second dose comprises about 1ug of RNA.

In some embodiments, the vaccination regimen includes at least two doses (e.g., at least three doses, including at least four doses or more). In some embodiments, the vaccination regimen includes three doses. In some embodiments, the time interval between the first dose and the second dose can be the same as the time interval between the second dose and the third dose. In some embodiments, the time interval between the first dose and the second dose can be longer than the time interval between the second dose and the third dose, including, for example, days or weeks (e.g., at least 3 days, at least 4 days, at least 5 days, at least 6 days, at least 1 week, at least 2 weeks, at least 3 weeks, at least 4 weeks, at least 5 weeks, at least 6 weeks, or more). In some embodiments, the time interval between the first and second doses may be shorter than the time interval between the second and third doses, e.g., by days or weeks (including, e.g., at least 3 days, at least 4 days, at least 5 days, at least 6 days, at least 1 week, at least 2 weeks, at least 3 weeks, at least 4 weeks, at least 5 weeks, at least 6 weeks, or more). In some embodiments, the time interval between the first and second doses may be shorter than the time interval between the second and third doses, e.g., by at least 1 month (including, e.g., at least 2 months, at least 3 months, at least 4 months, at least 5 months, at least 6 months, at least 7 months, at least 8 months, at least 9 months, at least 10 months, at least 11 months, at least 12 months, or more).

In some embodiments, the last dose of the primary regimen and the first dose of the booster regimen are given at least 2 months, at least 3 months, at least 4 months, at least 5 months, at least 6 months, at least 7 months, at least 8 months, at least 9 months, at least 10 months, at least 11 months, at least 12 months, or more apart. In some embodiments, the primary regimen may include two doses. In some embodiments, the primary regimen may include three doses.

In some embodiments, the first dose and the second dose (and/or other subsequent doses) may be administered by intramuscular injection. In some embodiments, the first dose and the second dose (and/or other subsequent doses) may be administered in the deltoid muscle. In some embodiments, the first dose and the second dose (and/or other subsequent doses) may be administered in the same arm.

In some embodiments, the RNA (e.g., mRNA) compositions described herein are administered (e.g., by intramuscular injection) as two successive doses (e.g., 0.3 mL each) spaced 21 days apart. In some embodiments, the RNA (e.g., mRNA) compositions described herein are administered (e.g., by intramuscular injection) as two successive doses (e.g., 0.2 mL each) spaced 21 days apart. In some embodiments, the RNA (e.g., mRNA) compositions described herein are administered (e.g., by intramuscular injection) as three successive doses (e.g., 0.3 mL or less, e.g., including 0.2 mL), the doses being given at least three weeks apart. In some embodiments, the first and second doses may be administered three weeks apart, while the second and third doses may be administered at a longer time interval than between the first and second doses, e.g., at least four weeks or more apart (including at least five weeks, at least six weeks, at least seven weeks, at least eight weeks, at least nine weeks, or more). In some embodiments, each dose is about 60ug. In some embodiments, each dose is about 50ug. In some embodiments, each dose is about 30ug. In some embodiments, each dose is about 25ug. In some embodiments, each dose is about 20ug. In some embodiments, each dose is about 15ug. In some embodiments, each dose is about 10ug. In some embodiments, each dose is about 3ug.

In some embodiments, at least one dose given in a vaccination regimen (e.g., a primary vaccination regimen and/or a booster vaccination regimen) is about 60ug. In some embodiments, at least one dose given in a vaccination regimen (e.g., a primary vaccination regimen and/or a booster vaccination regimen) is about 50ug. In some embodiments, at least one dose given in a vaccination regimen (e.g., a primary vaccination regimen and/or a booster vaccination regimen) is about 30ug. In some embodiments, at least one dose given in a vaccination regimen (e.g., a primary vaccination regimen and/or a booster vaccination regimen) is about 25ug. In some embodiments, at least one dose given in a vaccination regimen (e.g., a primary vaccination regimen and/or a booster vaccination regimen) is about 20ug. In some embodiments, at least one dose given in a vaccination regimen (e.g., a primary vaccination regimen and/or a booster vaccination regimen) is about 15ug. In some embodiments, at least one dose given in a vaccination regimen (e.g., a primary vaccination regimen and/or a booster vaccination regimen) is about 10 ug. In some embodiments, at least one dose given in a vaccination regimen (e.g., a primary vaccination regimen and/or a booster vaccination regimen) is about 3 ug.

In one embodiment, an amount of about 60 μg of the RNA described herein is administered per dose. In one embodiment, an amount of about 50 μg of the RNA described herein is administered per dose. In one embodiment, an amount of about 30 μg of the RNA described herein is administered per dose. In one embodiment, an amount of about 25 μg of the RNA described herein is administered per dose. In one embodiment, an amount of about 20 μg of the RNA described herein is administered per dose. In one embodiment, an amount of about 15 μg of the RNA described herein is administered per dose. In one embodiment, an amount of about 10 μg of the RNA described herein is administered per dose. In one embodiment, an amount of about 5 μg of the RNA described herein is administered per dose. In one embodiment, an amount of about 3 μg of the RNA described herein is administered per dose. In one embodiment, at least two of such doses are administered. For example, the second dose may be administered about 21 days after administration of the first dose.

In some embodiments, the efficacy of an RNA vaccine described herein (e.g., administered in two doses, where the second dose may be administered about 21 days after administration of the first dose, and may be administered in an amount of, e.g., about 30 μg per dose) is at least 70%, at least 80%, at least 90, or at least 95% beginning 7 days after administration of the second dose (e.g., beginning 28 days after administration of the first dose, where the second dose is administered 21 days after administration of the first dose). In some embodiments, such efficacy is observed in populations of at least 50 years of age, at least 55 years of age, at least 60 years of age, at least 65 years of age, at least 70 years of age, or older. In some embodiments, the efficacy of an RNA vaccine described herein (administered in two doses, the second dose may be administered about 21 days after administration of the first dose, e.g., in an amount of about 30 μg per dose) in a population of at least 65 years of age, such as 65-80 years, 65-75 years, or 65-70 years of age, beginning 7 days after administration of the second dose (e.g., beginning 28 days after administration of the first dose, if the second dose is administered 21 days after administration of the first dose) is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, or at least 95%. Such efficacy may be observed over a period of up to 1 month, 2 months, 3 months, 6 months, or even longer.

In one embodiment, vaccine efficacy is defined as the percent reduction in the number of subjects (vaccinated vs. non-vaccinated) with evidence of infection.

In one embodiment, efficacy is assessed through surveillance for potential cases of COVID-19. If at any time a patient develops acute respiratory illness, for purposes of this specification, the patient may be considered to have potential COVID-19 disease. Evaluation may include nasal (middle turbinate) swabs, which may be tested using a reverse transcription polymerase chain reaction (RT-PCR) test to detect SARS-CoV-2. In addition, clinical information and local standard of care trial results may be evaluated.

In some embodiments, efficacy assessment may utilize the definition of SARS-CoV-2 associated cases:
Confirmed COVID-19: Presence of at least one of the following symptoms and a positive SARS-CoV-2 NAAT (nucleic acid amplification-based test) during or within 4 days before or after the symptom period: fever, new or increased cough, new or increased shortness of breath, chills, new or increased muscle pain, new loss of taste or smell, sore throat, diarrhea, vomiting.

Alternatively or additionally, in some embodiments, efficacy assessment may utilize a SARS-CoV-2 associated case definition as defined by the CDC in which one or more of the following additional symptoms may be considered: fatigue, headache, stuffy or runny nose, nausea.

In some embodiments, efficacy assessment may utilize a definition of SARS-CoV-2 associated severe cases.
● Confirmed severe COVID-19: Confirmed COVID-19 and presence of at least one of the following: clinical signs at rest indicating severe systemic illness (e.g., RR ≥ 30 breaths/min, HR ≥ 125 breaths/min, SpO2 _≤ 93% on interface level room air, or _PaO2 / _FiO2 < 300 mmHg); respiratory failure (which may be defined as requiring high-flow oxygen, noninvasive ventilation, mechanical ventilation, or ECMO); evidence of shock (e.g., SBP < 90 mmHg, DBP < 60 mmHg, or requiring vascular compression agents); significant acute renal, hepatic, or neurological dysfunction; admission to the ICU; death.

Alternatively or additionally, in some embodiments, serological definitions can be used for patients without clinical presentation of COVID-19: for example, confirmed seroconversion to SARS-CoV-2 without confirmed COVID-19: for example, a positive N-binding antibody result in a patient with a previous negative N-binding antibody result.

In some embodiments, any or all of the following assays may be performed on the serum sample: SARS-CoV-2 neutralization assay, S1 binding IgG level assay, RBD binding IgG level assay, N binding antibody assay.

In one embodiment, the methods and medicaments described herein are administered to a pediatric population. In various embodiments, the pediatric population includes or consists of subjects under 18 years of age, e.g., 5-18 years of age, 12-18 years of age, 16-18 years of age, 12-16 years of age, or 5-12 years of age. In various embodiments, the pediatric population includes or consists of subjects under 5 years of age, e.g., 2-5 years of age, 12-24 months of age, 7-12 months of age, or 6 months of age. In some such embodiments, the RNA (e.g., mRNA) compositions described herein are administered to subjects under 2 years of age, e.g., 6 months to 2 years of age. In some such embodiments, the RNA (e.g., mRNA) compositions described herein are administered to subjects under 6 months of age, e.g., 1 month to 4 months of age. In some embodiments, the dosing regimen (e.g., dose and/or dosing schedule) for the pediatric population may vary for different age groups. For example, in some embodiments, subjects aged 6 months to 4 years may be administered according to a primary regimen comprising at least three doses, where the first two doses are administered at least three weeks apart (e.g., including at least four weeks, at least five weeks, at least six weeks, or more), followed by a third dose administered at least eight weeks after the second dose (e.g., including at least nine weeks, at least ten weeks, at least eleven weeks, at least twelve weeks, or more). In some such embodiments, at least one dose administered is 3 ug of RNA as described herein. In some embodiments, subjects aged 5 years or older may be administered according to a primary regimen comprising at least two doses, where the two doses are administered at least three weeks apart (e.g., including at least three weeks, at least four weeks, at least five weeks, at least six weeks, or more). In some such embodiments, at least one dose administered is 10 ug of RNA as described herein. In some embodiments, subjects 5 years of age or older who are immunocompromised (e.g., in some embodiments, subjects who have undergone a solid organ transplant or who have been diagnosed with a condition believed to correspond to an immunocompromised state) may be administered according to a primary regimen comprising at least three doses, with the first two doses being administered at least three weeks apart (e.g., at least three weeks, at least four weeks, at least five weeks, at least six weeks, or more), followed by a third dose administered at least four weeks after the second dose (e.g., at least five weeks, at least six weeks, at least seven weeks, at least eight weeks, at least nine weeks, at least ten weeks, at least eleven weeks, at least twelve weeks, or more).

In some embodiments, the RNA (e.g., mRNA) compositions described herein are administered to subjects aged 12 years or older, and each dose is about 30ug. In some embodiments, the RNA (e.g., mRNA) compositions described herein are administered to subjects aged 12 years or older (including, for example, 18 years or older), and each dose is greater than 30ug (including, for example, 35ug, 40ug, 45ug, 50ug, 55ug, 60ug, 65ug, 70ug, or more). In some such embodiments, the RNA (e.g., mRNA) compositions described herein are administered to subjects aged 12 years or older, and each dose is about 60ug. In some such embodiments, the RNA (e.g., mRNA) compositions described herein are administered to subjects aged 12 years or older, and each dose is about 50ug. In one embodiment, the pediatric population includes or consists of subjects aged 12 to 18 years (including subjects aged 16 to 18 years and/or subjects aged 12 to 16 years). In this embodiment, the treatment can include two vaccinations, spaced 21 days apart, and in one embodiment, the vaccine is administered in an amount of 30 μg of RNA per dose, for example by intramuscular administration. In some embodiments, higher doses are administered to older pediatric patients and adults, for example patients 12 years of age or older, compared to younger children or infants, for example 2-5 years of age, 6 months to 2 years of age, or under 6 months of age. In some embodiments, higher doses are administered to children who are 2-5 years of age, compared to toddlers and/or infants who are, for example, 6 months to 2 years of age, or under 6 months of age.

In one embodiment, the pediatric population includes or consists of subjects aged 5-18 years (including subjects aged 12-18 years and/or subjects aged 5-12 years). In this embodiment, the treatment can include two vaccinations 21 days apart, and in various embodiments, the vaccine is administered in an amount of 10 μg, 20 μg, or 30 μg of RNA per dose, e.g., by intramuscular administration. In some such embodiments, the RNA (e.g., mRNA) compositions described herein are administered to subjects aged 5-11 years, and each dose is about 10 ug. In some embodiments, each dose includes about 5 ug of RNA encoding a SARS-CoV-2 S protein of a first variant and about 5 ug of RNA encoding a SARS-CoV-2 S protein of a second variant. In some embodiments, each dose contains about 5ug of RNA encoding the SARS-CoV-2 S protein of the Wuhan strain and about 5ug of RNA encoding the SARS-CoV-2 S protein of the omicron variant. In some embodiments, each dose contains about 5ug of RNA encoding the SARS-CoV-2 S protein of the Wuhan strain (e.g., RNA comprising SEQ ID NO:20) and about 5ug of RNA encoding the SARS-CoV-2 S protein of the BA.1 omicron variant (e.g., RNA comprising SEQ ID NO:93). In some embodiments, each dose contains about 5ug of RNA encoding the SARS-CoV-2 S protein of the Wuhan strain (e.g., RNA comprising SEQ ID NO:20) and about 5ug of RNA encoding the SARS-CoV-2 S protein of the BA.4/5 omicron variant (e.g., RNA comprising SEQ ID NO:103).

In one embodiment, the pediatric population includes or consists of subjects under 5 years of age (including subjects between 2 and 5 years of age, subjects between 12 and 24 months of age, subjects between 7 and 12 months of age, subjects between 6 and 12 months of age, and/or subjects under 6 months of age).

In some embodiments, a pediatric patient can be administered a dose (e.g., a first, second, third, fourth, or fifth dose) comprising about 3 μg, about 6 μg, about 10 μg, about 20 μg, or about 30 μg of RNA (e.g., monovalent or multivalent RNA). In some embodiments, a pediatric patient is administered a multivalent vaccine comprising two or more RNAs, each RNA encoding a SARS-CoV-2 S protein associated with a different variant (e.g., a bivalent vaccine comprising about 3 μg, about 6 μg, about 10 μg, about 20 μg, or about 30 μg of total RNA). In some embodiments, a pediatric patient is administered a multivalent vaccine comprising two RNAs, each RNA encoding a SARS-CoV-2 S protein associated with a different variant (e.g., a bivalent vaccine comprising about 1.5 μg, about 3 μg, about 5 μg, about 10 μg, or about 15 μg of each RNA). In some embodiments, pediatric subjects are administered a subsequent dose (e.g., a second, third, fourth, or fifth dose) that contains a higher amount of RNA than the previous dose. For example, in some such embodiments, a pediatric subject is administered a subsequent dose (e.g., a third dose administered as a booster) that is 1-10 times the previous dose (e.g., 1-5 times, 2-5 times, 2-4 times, about 1.5 times, about 2 times, about 2.5 times, about 3 times, about 3.5 times, about 4 times, about 4.5 times, about 5 times, about 5.5 times, about 6 times, about 6.5 times, about 7.5 times, about 8 times, about 8.5 times, about 9 times, about 9.5 times, or about 10 times the previous dose).

In this embodiment, the treatment may include two vaccinations (e.g., 21-42 days apart, e.g., 21 days apart), and in various embodiments, the vaccine is administered, e.g., by intramuscular administration, in an amount of about 3 μg, about 6 μg, about 10 μg, about 20 μg, or about 30 μg of RNA per dose. In some such embodiments, the RNA (e.g., mRNA) compositions described herein are administered to subjects aged 2 to less than 5 years, and each dose is about 3 ug. In some such embodiments, the RNA (e.g., mRNA) compositions described herein are administered to subjects aged from about 6 months to less than 5 years, and each dose is about 3 ug. In some embodiments, each dose comprises about 1.5 ug of RNA encoding a SARS-CoV-2 S protein of a first variant and about 1.5 ug of RNA encoding a SARS-CoV-2 S protein of a second variant. In some embodiments, each dose comprises about 1.5ug of RNA encoding the SARS-CoV-2 S protein of the Wuhan strain and about 1.5ug of RNA encoding the SARS-CoV-2 S protein of the omicron variant. In some embodiments, each dose comprises about 1.5ug of RNA encoding the SARS-CoV-2 S protein of the Wuhan strain (e.g., RNA comprising SEQ ID NO:83) and about 1.5ug of RNA encoding the SARS-CoV-2 S protein of the BA.2 omicron variant (e.g., RNA comprising SEQ ID NO:98). In some embodiments, each dose comprises about 1.5ug of RNA encoding the SARS-CoV-2 S protein of the Wuhan strain (e.g., RNA comprising SEQ ID NO:83) and about 1.5ug of RNA encoding the SARS-CoV-2 S protein of the BA.4/5 omicron variant (e.g., RNA comprising SEQ ID NO:103).

In some such embodiments, an RNA (e.g., mRNA) composition described herein is administered to a subject about 6 months to less than about 5 years of age, and each dose is about 6ug. In some embodiments, each dose comprises about 3ug of RNA encoding a SARS-CoV-2 S protein of a first variant and about 3ug of RNA encoding a SARS-CoV-2 S protein of a second variant. In some embodiments, each dose comprises about 3ug of RNA encoding a SARS-CoV-2 S protein of the Wuhan strain and about 3ug of RNA encoding a SARS-CoV-2 S protein of the Omicron variant. In some embodiments, each dose comprises about 3ug of RNA encoding a SARS-CoV-2 S protein of the Wuhan strain (e.g., RNA comprising SEQ ID NO:83) and about 3ug of RNA encoding a SARS-CoV-2 S protein of the BA.2 Omicron variant (e.g., RNA comprising SEQ ID NO:98). In some embodiments, each dose contains about 3 ug of RNA encoding the SARS-CoV-2 S protein of the Wuhan strain (e.g., RNA comprising SEQ ID NO:83) and about 3 ug of RNA encoding the SARS-CoV-2 S protein of the BA.4/5 omicron variant (e.g., RNA comprising SEQ ID NO:103).

In some such embodiments, an RNA (e.g., mRNA) composition described herein is administered to a subject from about 6 months to less than about 5 years of age, and each dose is about 10 ug. In some embodiments, each dose comprises about 5 ug of RNA encoding a SARS-CoV-2 S protein of a first variant and about 5 ug of RNA encoding a SARS-CoV-2 S protein of a second variant. In some embodiments, each dose comprises about 5 ug of RNA encoding a SARS-CoV-2 S protein of the Wuhan strain and about 5 ug of RNA encoding a SARS-CoV-2 S protein of the Omicron variant. In some embodiments, each dose comprises about 5 ug of RNA encoding a SARS-CoV-2 S protein of the Wuhan strain (e.g., RNA comprising SEQ ID NO: 83) and about 5 ug of RNA encoding a SARS-CoV-2 S protein of the BA.2 Omicron variant (e.g., RNA comprising SEQ ID NO: 98). In some embodiments, each dose contains about 5 ug of RNA encoding the SARS-CoV-2 S protein of the Wuhan strain (e.g., RNA comprising SEQ ID NO:83) and about 5 ug of RNA encoding the SARS-CoV-2 S protein of the BA.4/5 omicron variant (e.g., RNA comprising SEQ ID NO:103).

In some embodiments, the RNA (e.g., mRNA) compositions described herein are administered to subjects aged 12 years or older, and at least one dose given in a vaccination regimen (e.g., a primary vaccination regimen and/or a booster vaccination regimen) is about 60ug. In some embodiments, the RNA (e.g., mRNA) compositions described herein are administered to subjects aged 12 years or older, and at least one dose given in a vaccination regimen (e.g., a primary vaccination regimen and/or a booster vaccination regimen) is about 30ug. In some embodiments, the RNA (e.g., mRNA) compositions described herein are administered to subjects aged 12 years or older, and at least one dose given in a vaccination regimen (e.g., a primary vaccination regimen and/or a booster vaccination regimen) is about 15ug. In some embodiments, the RNA (e.g., mRNA) compositions described herein are administered to subjects aged 5 to less than 12 years old, and at least one dose given in a vaccination regimen (e.g., a primary vaccination regimen and/or a booster vaccination regimen) is about 10ug. In some embodiments, the RNA (e.g., mRNA) compositions described herein are administered to subjects aged 2 to under 5 years and at least one dose given in the vaccination regimen (e.g., primary vaccination regimen and/or booster vaccination regimen) is about 3ug. In some embodiments, the RNA (e.g., mRNA) compositions described herein are administered to subjects aged 6 months to under 2 years and at least one dose given in the vaccination regimen (e.g., primary vaccination regimen and/or booster vaccination regimen) is about 3ug or less (e.g., including 2ug, 1ug, or less). In some embodiments, the RNA (e.g., mRNA) compositions described herein are administered to infants under 6 months and at least one dose given in the vaccination regimen (e.g., primary vaccination regimen and/or booster vaccination regimen) is about 3ug or less (e.g., including 2ug, 1ug, 0.5ug, or less).

In some embodiments, an RNA (e.g., mRNA) composition described herein is administered as a single dose (e.g., by intramuscular injection). In some embodiments, the single dose comprises a single RNA encoding a SARS-CoV-2 S protein or an immunogenic fragment thereof (e.g., the RBD domain). In some embodiments, the single dose comprises at least two RNAs described herein, e.g., each RNA encoding a SARS-CoV-2 S protein or an immunogenic fragment thereof (e.g., the RBD domain) from a different strain. In some embodiments, such at least two RNAs described herein may be administered as a single mixture. For example, in some such embodiments, two separate RNA compositions described herein may be mixed to produce a single mixture prior to injection. In some embodiments, such at least two RNAs described herein may be administered as two separate compositions, e.g., administered at different injection sites (e.g., different arms, or different sites on the same arm).

In some embodiments, the dose administered to a subject in need thereof may comprise administration of a single RNA (e.g., mRNA) composition described herein.

In some embodiments, the dose administered to a subject in need thereof may include administration of at least two or more (e.g., including at least three or more) different pharmaceuticals/formulations. For example, in some embodiments, the at least two or more different pharmaceuticals/formulations may include at least two different RNA (e.g., mRNA) compositions (e.g., in some embodiments, each comprising a different RNA construct) described herein.

In some embodiments, the RNA (e.g., mRNA) compositions disclosed herein may be administered in conjunction with a vaccine targeting a different infectious agent. In some embodiments, the different infectious agent is one that increases the likelihood that a subject will experience adverse symptoms when co-infected with SARS-CoV-2 and the infectious agent. In some embodiments, the infectious agent is one that increases the infectivity of SARS-CoV-2 when a subject is co-infected with SARS-CoV-2 and the infectious agent. In some embodiments, at least one RNA (e.g., mRNA) composition described herein may be administered in conjunction with a vaccine targeting influenza. In some embodiments, at least two or more different pharmaceuticals/formulations may include at least one RNA (e.g., mRNA) composition described herein and a vaccine targeting a different infectious agent (e.g., influenza vaccine). In some embodiments, the different pharmaceuticals/formulations are administered separately. In some embodiments, such different pharmaceuticals/formulations are administered separately at different sites on the subject (e.g., in different arms of the subject) at the same time (e.g., in the same vaccination session).

In one embodiment, at least two doses are administered. For example, the second dose may be administered about 21 days after administration of the first dose.

In some embodiments, at least one single dose is administered. In some embodiments, such a single dose is administered to a subject who may have previously received one or more doses or a complete regimen of, for example, a SARS-CoV-2 vaccine (e.g., a BNT162b2 vaccine [including, e.g., as described herein], an mRNA-1273 vaccine, an Ad26.CoV2.S vaccine, a ChAdxOx1 vaccine, an NVX-CoV2373 vaccine, a CvnCoV vaccine, a GAM-COVID0Vac vaccine, a CoronaVac vaccine, a BBIBP-CorV vaccine, an Ad5-nCoV vaccine, a zf2001 vaccine, an SCB-2019 vaccine, a JNJ78436735 vaccine, or other approved RNA (e.g., mRNA) or adeno-vector vaccine, etc.). Alternatively or additionally, in some embodiments, a single dose is administered to a subject exposed and/or infected with SARS-CoV-2. In some embodiments, at least one single dose is administered to a subject who has received one or more doses or a complete regimen of a SARS-CoV-2 vaccine and who has been exposed and/or infected with SARS-CoV-2.

At least one single dose is administered to a subject who has received one or more doses of a prior SARS-CoV-2 vaccine, in some particular embodiments, such prior SARS-CoV-2 vaccine is a different vaccine, or a different form (e.g., formulation) and/or dose of a vaccine having the same activity (e.g., BNT162b2), and in some such embodiments, such subject has not received a full regimen of such prior vaccine and/or has experienced one or more undesirable reactions or effects to one or more received doses of such prior vaccine. In some particular embodiments, such prior vaccine is or includes a higher dose(s) of the same activity (e.g., BNT162b2). Alternatively or additionally, in some such embodiments, such subject was exposed to and/or infected with SARS-CoV-2 prior to completion (but in some embodiments after initiation) of a full regimen of such prior vaccine.

In one embodiment, at least two doses are administered. For example, the second dose may be administered about 21 days after administration of the first dose.

In one embodiment, at least three doses are administered. In some embodiments, such a third dose is administered at a time period after the second dose that is comparable (e.g., the same) as the time period between the first and second doses. For example, in some embodiments, the third dose may be administered about 21 days after administration of the second dose. In some embodiments, the third dose is administered at a longer time period after the second dose than the second dose is after the first dose. In some embodiments, the three-dose regimen is administered to an immunocompromised patient, such as a cancer patient, an HIV patient, a patient who has undergone and/or is undergoing immunosuppressant therapy (e.g., an organ transplant patient). In some embodiments, the length of time between the second and third doses (e.g., the second and third doses administered to an immunocompromised patient) is at least about 21 days (e.g., at least about 28 days).

In some embodiments, the vaccination regimen includes administering the same amount of RNA in different doses (e.g., in the first and/or second and/or third and/or subsequent doses). In some embodiments, the vaccination regimen includes administering different amounts of RNA in different doses. In some embodiments, one or more subsequent doses are greater than one or more previous doses (e.g., in situations where a decrease in vaccine efficacy from one or more previous doses is observed and/or where immune escape due to a variant (e.g., a variant described herein) that is prevalent or rapidly spreading in the relevant jurisdiction at the time of administration is observed). In some embodiments, one or more subsequent doses may be at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or more greater than one or more previous doses, provided that the safety and/or tolerability of such doses are clinically acceptable. In some embodiments, one or more subsequent doses may be at least 1.1-fold, at least 1.5-fold, at least 2-fold, at least 3-fold, at least 4-fold, or more than one or more previous doses, provided that the safety and/or tolerability of such doses are clinically acceptable. In some embodiments, one or more subsequent doses may be less than one or more previous doses (e.g., if a patient experienced a negative reaction after one or more previous doses and/or was exposed to and/or infected with SARS-CoV-2 between a previous dose and a subsequent dose). In some embodiments, one or more subsequent doses may be at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, or more than one or more previous doses. In some embodiments where different doses are utilized, they are related to each other by identity and/or dilution with a common stock as described herein.

In some embodiments, when at least two or more doses are administered (e.g., at least two doses administered in a primary regimen, at least two doses administered in a booster regimen, or at least one dose administered in a primary regimen and at least one dose in a booster regimen), the same RNA composition described herein may be administered in such doses, and each of such doses may be the same or different (as described herein). In some embodiments, at least two or more doses are administered (e.g., at least two doses are administered in a primary regimen, at least two doses are administered in a booster regimen, or at least one dose is administered in a primary regimen and at least one dose is administered in a booster regimen), different RNA compositions described herein (e.g., different encoded viral polypeptides from different coronavirus clades or from different strains of the same coronavirus clade; different construct elements (e.g., 5' cap, 3' UTR, 5' UTR, etc.); different formulations (e.g., different excipients and/or buffers (e.g., PBS vs. Tris)); different LNP compositions; or combinations thereof) may be administered in such doses, each of which may be the same or different (e.g., as described herein).

In some embodiments, a subject is administered two or more RNAs (e.g., as part of either a primary or booster regimen), and the two or more RNAs are administered on the same day or at the same clinic visit. In some embodiments, the two or more RNAs are administered in separate compositions, for example, by administering each RNA to a separate part of the subject (e.g., by intramuscular administration to different arms of the subject or different sites on the same arm of the subject). In some embodiments, the two or more RNAs are mixed prior to administration (e.g., mixed immediately prior to administration, e.g., by the administering practitioner). In some embodiments, the two or more RNAs are formulated together (e.g., by (a) mixing separate populations of LNPs, each population containing a different RNA, or (b) mixing the two or more RNAs prior to LNP formulation, such that each LNP contains two or more RNAs). In some embodiments, the two or more RNAs include RNA encoding a coronavirus S protein or an immunogenic fragment thereof (e.g., RBD or other relevant domain) from one strain (e.g., the Wuhan strain) and a variant that is prevalent or spreading rapidly in the relevant jurisdiction at the time of administration (e.g., a variant described herein). In some embodiments, such a variant is an omicron variant (e.g., a BA.1, BA.2, or BA.3 variant). In some embodiments, the two or more RNAs include a first RNA and a second RNA that have been shown to elicit a broad immune response in a subject. In some embodiments, the two or more RNAs include RNA encoding a SARS-CoV-2 S protein from the Wuhan strain and RNA encoding a SARS-CoV-2 S protein from the BA.1 omicron variant. In some embodiments, the two or more RNAs include RNA encoding a SARS-CoV-2 S protein from the Wuhan strain and RNA encoding a SARS-CoV-2 S protein from the BA.2 omicron variant. In some embodiments, the two or more RNAs comprise an RNA encoding a SARS-CoV-2 S protein from the Wuhan strain and an RNA encoding a SARS-CoV-2 S protein from a BA.4 or BA.5 omicron variant. In some embodiments, the two or more RNAs comprise an RNA encoding a SARS-CoV-2 S protein from a BA.1 omicron variant and an RNA encoding a SARS-CoV-2 S protein from a BA.2 omicron variant. In some embodiments, the two or more RNAs comprise an RNA encoding a SARS-CoV-2 S protein from a BA.1 omicron variant and an RNA encoding a SARS-CoV-2 S protein from a BA.4 or BA.5 omicron variant. In some embodiments, the two or more RNAs comprise an RNA encoding a SARS-CoV-2 S protein from a BA.2 strain and an RNA encoding a SARS-CoV-2 S protein from a BA. In some embodiments, the two or more RNAs include an RNA encoding a SARS-CoV-2 S protein from the Wuhan strain, an alpha variant, a beta variant, or a delta variant, or a sublineage derived therefrom; and an RNA encoding a SARS-CoV-2 S protein from the BA.2, BA.4, or 5 omicron variant, or a sublineage derived therefrom.

In some embodiments, a subject may be administered a dose comprising any one of combinations 1-66 listed in the table below. In some embodiments, such combinations may be administered using an LNP formulation, where the first RNA and the second RNA are encapsulated within the same LNP or within separate LNPs. In some embodiments, such combinations may be administered as separate LNP formulations (e.g., by administration to the subject at separate sites).

In some embodiments, the vaccination regimen includes a first vaccination regimen (e.g., a primary regimen) comprising at least two doses of an RNA composition described herein (e.g., the second dose may be administered about 21 days after administration of the first dose), and a second vaccination (e.g., a booster regimen) comprising a single or multiple doses (e.g., two doses) of an RNA composition described herein. In some embodiments, the doses of the booster regimen are related to the doses of the primary regimen by identity with or dilution from a common stock described herein. In various embodiments, the booster regimen is administered (e.g., initiated) at least 1 week, at least 2 weeks, at least 3 weeks, at least 1 month, at least 2 months, at least 3 months, at least 4 months, at least 5 months, or at least 6 months, at least 7 months, at least 8 months, at least 9 months, at least 10 months, at least 11 months, at least 12 months, or more after administration of the primary regimen, e.g., after completion of a primary regimen comprising at least two doses. In various embodiments, the booster regimen is administered (e.g., initiated) 1-12 months, 2-12 months, 3-12 months, 4-12 months, 6-12 months, 1-6 months, 1-5 months, 1-4 months, 1-3 months, or 2-3 months after administration of the primary regimen, e.g., after completion of a primary regimen comprising at least two doses. In various embodiments, the booster regimen is administered (e.g., initiated) 1-60 months, 2-48 months, 2-24 months, 3-24 months, 6-18 months, 6-12 months, or 5-7 months after administration of the primary regimen, e.g., after completion of a two-dose primary regimen. In some embodiments, each dose of the primary regimen is about 60 μg per dose. In some embodiments, each dose of the primary regimen is about 50 μg per dose. In some embodiments, each dose of the primary regimen is about 30 μg per dose. In some embodiments, each dose of the primary regimen is about 25 μg per dose. In some embodiments, each dose of the primary regimen is about 20 μg per dose. In some embodiments, each dose of the primary regimen is about 15 μg per dose. In some embodiments, each dose of the primary regimen is about 10 μg per dose. In some embodiments, each dose of the primary regimen is about 3 μg per dose. In some embodiments, each dose of the booster regimen is the same as the dose of the primary regimen. In some embodiments, each dose of the booster regimen contains the same amount of RNA as the dose administered in the primary regimen. In some embodiments, at least one dose of the booster regimen is the same as the dose of the primary regimen. In some embodiments, at least one dose of the booster regimen contains the same amount of RNA as at least one dose of the primary regimen. In some embodiments, at least one dose of the booster regimen is lower than the dose of the primary regimen. In some embodiments, at least one dose of the booster regimen contains a lower amount of RNA than the dose of the primary regimen. In some embodiments, at least one dose of the booster regimen is higher than the dose of the primary regimen. In some embodiments, at least one dose of the booster regimen includes a higher amount of RNA than the dose of the primary regimen.

In some embodiments, a booster regimen (e.g., as described herein) is administered to a pediatric patient (e.g., a patient between 2 and 5 years of age, a patient between 5 and 11 years of age, or a patient between 12 and 15 years of age). In some embodiments, a booster regimen is administered to a pediatric patient who is between 6 months and less than 2 years of age. In some embodiments, a booster regimen is administered to a pediatric patient who is less than 6 months of age. In some embodiments, a booster regimen is administered to a pediatric patient who is between 6 months and less than 5 years of age. In some embodiments, a booster regimen is administered to a pediatric patient who is between 2 years and less than 5 years of age. In some embodiments, a booster regimen is administered to a pediatric patient who is between 5 years and less than 12 years of age. In some embodiments, a booster regimen is administered to a pediatric patient who is between 12 years and less than 16 years of age. In some embodiments, each dose of the pediatric booster regimen comprises about 3 μg of RNA. In some embodiments, each dose of the pediatric booster regimen comprises about 6 μg of RNA. In some embodiments, each dose of the pediatric booster regimen comprises about 10 μg of RNA. In some embodiments, each dose of the pediatric booster regimen comprises about 15 μg of RNA. In some embodiments, each dose of the pediatric booster regimen comprises about 20 μg of RNA. In some embodiments, each dose of the pediatric booster regimen comprises about 25 μg of RNA. In some embodiments, each dose of the pediatric booster regimen comprises about 30 μg of RNA. In some embodiments, the booster regimen is administered to a non-pediatric patient (e.g., a patient aged 16 years or older, a patient aged 18-64 years, and/or a patient aged 65 years or older). In some embodiments, each dose of the non-pediatric booster regimen comprises about 3 ug of RNA, about 10 ug of RNA, about 25 μg of RNA, about 30 μg of RNA, about 40 μg of RNA, about 45 μg of RNA, about 50 μg of RNA, about 55 μg of RNA, or about 60 μg of RNA. In some embodiments, the same booster regimen may be administered to both pediatric and non-pediatric patients (e.g., a patient aged 12 years or older). In some embodiments, a booster regimen administered to a non-pediatric patient is administered in a formulation and dose related by identity or by dilution to the formulation and dose of the primary regimen previously received by the patient. In some embodiments, a non-pediatric patient receiving a booster regimen at a lower dose than the primary regimen may experience an adverse reaction to one or more doses of such primary regimen and/or may be exposed to and/or infected with SARS-CoV-2 between such primary regimen and such booster regimen, or between a dose of such primary regimen and such booster regimen. In some embodiments, pediatric and non-pediatric patients may receive a booster regimen at a higher dose than the primary regimen when a decrease in vaccine efficacy at lower doses is observed and/or when immune escape of a variant that is prevalent and/or spreading rapidly in the relevant jurisdiction at the time of administration is observed.

In some embodiments, one or more doses of the booster regimen are different from the doses of the primary regimen. For example, in some embodiments, the doses administered may correspond to the age of the subject, and the patient may age from one treatment age group to the next treatment age group. Alternatively or additionally, in some embodiments, the doses administered may correspond to the condition of the patient (e.g., an immunocompromised state), and a different dose may be selected for one or more doses of the booster regimen than for the primary regimen (e.g., due to an intervening cancer treatment, infection with HIV, or receipt of immunosuppressive therapy associated with, for example, an organ transplant). In some embodiments, at least one dose of the booster regimen may contain a higher amount of RNA than at least one dose administered in the primary regimen (e.g., in situations where a decrease in vaccine efficacy from one or more earlier doses is observed and/or where immune escape due to a variant (e.g., a variant described herein) that is prevalent or spreading rapidly in the relevant jurisdiction at the time of administration is observed).

In some embodiments, the primary regimen may involve one or more 3ug doses, and the booster regimen may involve one or more 10ug doses, and/or one or more 20ug doses, or one or more 30ug doses. In some embodiments, the primary regimen may involve one or more 3ug doses, and the booster regimen may involve one or more 3ug doses. In some embodiments, the primary regimen may involve two or more 3ug doses (e.g., at least two doses each containing 3ug of RNA and administered about 21 days apart from each other), and the booster regimen may involve one or more 3ug doses. In some embodiments, the primary regimen may involve one or more 10ug doses, and the booster regimen may involve one or more 20ug doses, and/or one or more 30ug doses. In some embodiments, the primary regimen may involve one or more 10ug doses and the booster regimen may involve one or more 10ug doses. In some embodiments, the primary regimen may involve two or more 10ug doses (e.g., two doses each containing 10ug of RNA administered about 21 days apart) and the booster regimen may involve one or more 10ug doses. In some embodiments, the primary regimen may involve one or more 20ug doses and the booster regimen may involve one or more 30ug doses. In some embodiments, the primary regimen may involve one or more 20ug doses and the booster regimen may involve one or more 20ug doses. In some embodiments, the primary regimen may involve one or more 30ug doses and the booster regimen may involve one or more 30ug doses. In some embodiments, the primary regimen may involve two or more 30ug doses (e.g., two doses each containing 30ug of RNA administered about 21 days apart), and the booster regimen may also involve one or more 30ug doses. In some embodiments, the primary regimen may involve two or more 30ug doses (e.g., two doses each containing 30ug of RNA administered about 21 days apart), and the booster regimen may also involve one or more 50ug doses. In some embodiments, the primary regimen may involve two or more 30ug doses (e.g., two doses each containing 30ug of RNA administered about 21 days apart), and the booster regimen may involve one or more 60ug doses.

In some embodiments, the subject is administered a booster regimen comprising at least one 30ug dose of RNA. In some embodiments, the subject is administered a booster regimen comprising at least one 30ug dose of RNA encoding a SARS-CoV-2 S protein from the Wuhan strain of SARS-CoV-2 (e.g., BNT162b2). In some embodiments, the subject is administered a booster regimen comprising at least one 30ug dose of RNA encoding a SARS-CoV-2 S protein having one or more mutations characteristic of a SARS-CoV-2 variant (e.g., a variant described herein). In some embodiments, the subject is administered a booster regimen comprising at least one 30ug dose of RNA encoding a SARS-CoV-2 S protein having one or more mutations characteristic of an omicron variant (e.g., a BA.1, BA.2, BA.3, or BA.4, or BA.5 omicron variant). In some embodiments, the subject is administered a booster regimen comprising at least one dose of 30ug of RNA, the 30ug of RNA comprising RNA encoding a SARS-CoV-2 S protein from the Wuhan strain and RNA encoding a SARS-CoV-2 S protein comprising a mutation characteristic of a SARS-CoV-2 variant (e.g., in some embodiments, the subject is administered a booster regimen comprising at least one dose comprising 15ug of RNA encoding a SARS-CoV-2 S protein from the Wuhan strain and 15ug of RNA encoding a SARS-CoV-2 S protein with one or more mutations characteristic of an Omicron variant). In some embodiments, the subject is administered a booster regimen comprising at least one dose comprising 15ug of RNA encoding a SARS-CoV-2 S protein from the Wuhan strain and 15ug of BA. In some embodiments, the subject is administered a booster regimen comprising at least one dose comprising 15 ug of RNA encoding a SARS-CoV-2 S protein from the Wuhan strain and 15 ug of RNA encoding a SARS-CoV-2 S protein with one or more mutations characteristic of the BA.1 omicron variant. In some embodiments, the subject is administered a booster regimen comprising at least one dose comprising 15 ug of RNA encoding a SARS-CoV-2 S protein from the Wuhan strain and 15 ug of RNA encoding a SARS-CoV-2 S protein with one or more mutations characteristic of the BA.2 omicron variant. In some embodiments, the subject is administered a booster regimen comprising at least one dose comprising 15 ug of RNA encoding a SARS-CoV-2 S protein from the Wuhan strain and 15 ug of RNA encoding a SARS-CoV-2 S protein with one or more mutations characteristic of the BA.3 omicron variant. In some embodiments, the subject is administered a booster regimen comprising at least one dose comprising 15 ug of RNA encoding a SARS-CoV-2 S protein from the Wuhan strain and 15 ug of RNA encoding a SARS-CoV-2 S protein with one or more mutations characteristic of the BA. The patient is administered a booster regimen comprising at least one dose of RNA encoding a SARS-CoV-2 S protein having one or more mutations characteristic of the BA.4 or BA.5 omicron variant.

In some embodiments, the subject is administered a booster regimen comprising at least one dose comprising 15 ug of RNA encoding a SARS-CoV-2 S protein from the BA.1 omicron variant and 15 ug of RNA encoding a SARS-CoV-2 S protein with one or more mutations characteristic of the BA.2 omicron variant. In some embodiments, the subject is administered a booster regimen comprising at least one dose comprising 15 ug of RNA encoding a SARS-CoV-2 S protein from the BA.1 omicron variant and 15 ug of RNA encoding a SARS-CoV-2 S protein with one or more mutations characteristic of the BA.3 omicron variant. In some embodiments, the subject is administered a booster regimen comprising at least one dose comprising 15 ug of RNA encoding a SARS-CoV-2 S protein from the BA.1 omicron variant and 15 ug of RNA encoding a SARS-CoV-2 S protein with one or more mutations characteristic of the BA.4 or BA. The patient will be administered a booster regimen that includes at least one dose of RNA encoding a SARS-CoV-2 S protein that has one or more mutations characteristic of the 5-omicron variant.

In some embodiments, the subject is administered a booster regimen comprising at least one dose comprising 15 ug of RNA encoding a SARS-CoV-2 S protein from the BA.2 omicron variant and 15 ug of RNA encoding a SARS-CoV-2 S protein with one or more mutations characteristic of the BA.4 or BA.5 omicron variant. In some embodiments, the subject is administered a booster regimen comprising at least one dose comprising 15 ug of RNA encoding a SARS-CoV-2 S protein from the BA.3 omicron variant and 15 ug of RNA encoding a SARS-CoV-2 S protein with one or more mutations characteristic of the BA.4 or BA.5 omicron variant.

In some embodiments, subjects are administered a booster regimen comprising two or more 30ug doses of RNA administered at least two months apart from each other. For example, in some embodiments, subjects are administered a booster regimen comprising two 30ug doses of RNA encoding a SARS-CoV-2 S protein having one or more mutations characteristic of an omicron variant (e.g., a BA.1, BA.2, or BA.4, or BA.5 omicron variant).

In some embodiments, a subject is administered (i) a primary regimen comprising at least two 30 ug doses of RNA encoding a SARS-CoV-2 S protein from the Wuhan strain, the two doses being administered at least approximately 21 days apart, and (ii) a booster regimen comprising a 30 ug dose of RNA encoding a SARS-CoV-2 S protein having one or more mutations characteristic of an omicron variant of SARS-CoV-2 (e.g., a BA.1, BA.2, BA.3, BA.4, or BA.5 omicron variant), the booster regimen being administered at least two months (e.g., including at least three months, at least four months, at least five months, at least six months, or more) after completion of the primary regimen. In some embodiments, subjects receive (i) a primary regimen comprising at least two 30ug doses of RNA encoding the SARS-CoV-2 S protein from the Wuhan strain (the two doses are administered at least approximately 21 days apart), and (ii) a booster regimen comprising 30ug doses of RNA including 15ug of RNA encoding the SARS-CoV-2 S protein from the Wuhan strain and 15ug of RNA encoding a SARS-CoV-2 S protein having one or more mutations characteristic of the omicron variant.

In some embodiments, a subject is administered (i) a primary regimen comprising at least two 30 ug doses of RNA encoding a SARS-CoV-2 S protein from the Wuhan strain, the two doses being administered at least approximately 21 days apart, and (ii) a booster regimen comprising a 30 ug dose of RNA encoding a SARS-CoV-2 S protein having one or more mutations characteristic of the BA.1 omicron variant of SARS-CoV-2, the booster regimen being administered at least two months (including, e.g., at least three months, at least four months, at least five months, at least six months, or more) after completion of the primary regimen. In some embodiments, subjects receive (i) a primary regimen comprising at least two 30ug doses of RNA encoding the SARS-CoV-2 S protein from the Wuhan strain (the two doses are administered at least approximately 21 days apart), and (ii) a booster regimen comprising 30ug doses of RNA including 15ug of RNA encoding the SARS-CoV-2 S protein from the Wuhan strain and 15ug of RNA encoding a SARS-CoV-2 S protein having one or more mutations characteristic of the BA.1 omicron variant.

In some embodiments, a subject is administered (i) a primary regimen comprising at least two 30 ug doses of RNA encoding a SARS-CoV-2 S protein from the Wuhan strain, the two doses being administered at least approximately 21 days apart, and (ii) a booster regimen comprising a 30 ug dose of RNA encoding a SARS-CoV-2 S protein having one or more mutations characteristic of the BA.2 omicron variant of SARS-CoV-2, the booster regimen being administered at least two months (including, e.g., at least three months, at least four months, at least five months, at least six months, or more) after completion of the primary regimen. In some embodiments, subjects receive (i) a primary regimen comprising at least two 30ug doses of RNA encoding the SARS-CoV-2 S protein from the Wuhan strain, the two doses being administered at least approximately 21 days apart, and (ii) a booster regimen comprising 30ug doses of RNA including 15ug of RNA encoding the SARS-CoV-2 S protein from the Wuhan strain and 15ug of RNA encoding a SARS-CoV-2 S protein having one or more mutations characteristic of the BA.2 omicron variant.

In some embodiments, a subject is administered (i) a primary regimen comprising at least two 30 ug doses of RNA encoding a SARS-CoV-2 S protein from the Wuhan strain, the two doses being administered at least approximately 21 days apart, and (ii) a booster regimen comprising a 30 ug dose of RNA encoding a SARS-CoV-2 S protein having one or more mutations characteristic of the BA.4 or BA.5 omicron variant of SARS-CoV-2, the booster regimen being administered at least two months (including, e.g., at least three months, at least four months, at least five months, at least six months, or more) after completion of the primary regimen. In some embodiments, subjects receive (i) a primary regimen comprising at least two 30ug doses of RNA encoding the SARS-CoV-2 S protein from the Wuhan strain, the two doses being administered at least approximately 21 days apart, and (ii) a booster regimen comprising 30ug doses of RNA comprising 15ug of RNA encoding the SARS-CoV-2 S protein from the Wuhan strain and 15ug of RNA encoding a SARS-CoV-2 S protein having one or more mutations characteristic of the BA.4 or BA.5 omicron variant.

In some embodiments, subjects receive (i) a primary regimen comprising at least two 30ug doses of RNA encoding a SARS-CoV-2 S protein from the Wuhan strain, the two doses being administered at least approximately 21 days apart, and (ii) a booster regimen comprising 30ug doses of RNA including 15ug of RNA encoding a SARS-CoV-2 S protein from the BA.1 omicron variant and 15ug of RNA encoding a SARS-CoV-2 S protein having one or more mutations characteristic of the BA.2 omicron variant.

In some embodiments, subjects receive (i) a primary regimen comprising at least two 30ug doses of RNA encoding a SARS-CoV-2 S protein from the Wuhan strain, the two doses being administered at least approximately 21 days apart, and (ii) a booster regimen comprising 30ug doses of RNA including 15ug of RNA encoding a SARS-CoV-2 S protein from the BA.1 omicron variant and 15ug of RNA encoding a SARS-CoV-2 S protein having one or more mutations characteristic of the BA.4 or BA.5 omicron variant.

In some embodiments, subjects receive (i) a primary regimen comprising at least two 30ug doses of RNA encoding a SARS-CoV-2 S protein from the Wuhan strain, the two doses being administered at least approximately 21 days apart, and (ii) a booster regimen comprising 30ug doses of RNA including 15ug of RNA encoding a SARS-CoV-2 S protein from the BA.2 omicron variant and 15ug of RNA encoding a SARS-CoV-2 S protein having one or more mutations characteristic of the BA.4 or BA.5 omicron variant.

In some embodiments, subjects receive (i) a primary regimen comprising at least two 30 ug doses of RNA encoding a SARS-CoV-2 protein from the Wuhan strain (the two doses are administered at least approximately 21 days apart), and (ii) a booster regimen comprising at least one 30 ug dose of RNA encoding a SARS-CoV-2 S protein from a non-BA.1 omicron variant.

In some embodiments, a subject is administered (i) a primary regimen comprising at least one 30ug dose of RNA encoding a SARS-CoV-2 S protein from the Wuhan strain, the two doses being administered at least approximately 21 days apart, and (ii) a booster regimen comprising at least two 30ug doses of RNA encoding a SARS-CoV-2 S protein having one or more mutations characteristic of the omicron variant, the booster regimen being administered at least two months (including, for example, at least three months, at least four months, at least five months, at least six months, or more) after completion of the primary regimen, the two booster doses being administered at least two months apart from each other.

In some embodiments, the subject is administered a booster regimen comprising at least one 50ug dose of RNA. In some embodiments, the subject is administered a booster regimen comprising at least one 50ug dose of RNA encoding a SARS-CoV-2 S protein from the Wuhan strain (e.g., BNT162b2). In some embodiments, the subject is administered a booster regimen comprising at least one 50ug dose of RNA encoding a SARS-CoV-2 S protein having one or more mutations characteristic of a SARS-CoV-2 variant (e.g., a variant described herein). In some embodiments, the subject is administered a booster regimen comprising at least one 50ug dose of RNA encoding a SARS-CoV-2 S protein having one or more mutations characteristic of an Omicron variant. In some embodiments, the subject is administered at least one booster regimen comprising a 50ug dose of RNA, the 50ug of RNA comprising an RNA encoding a SARS-CoV-2 S protein from the Wuhan strain and an RNA encoding a SARS-CoV-2 S protein comprising a mutation characteristic of a SARS-CoV-2 variant (e.g., in some embodiments, the subject is administered a booster regimen comprising a 50ug dose of RNA comprising 25ug of an RNA encoding a SARS-CoV-2 S protein from the Wuhan strain and 25ug of an RNA encoding a SARS-CoV-2 S protein with one or more mutations characteristic of an Omicron variant).

In some embodiments, the subject is administered a booster regimen comprising at least one dose comprising 25 ug of RNA encoding a SARS-CoV-2 S protein from the Wuhan strain and 25 ug of RNA encoding a SARS-CoV-2 S protein with one or more mutations characteristic of the BA.1 omicron variant. In some embodiments, the subject is administered a booster regimen comprising at least one dose comprising 25 ug of RNA encoding a SARS-CoV-2 S protein from the Wuhan strain and 25 ug of RNA encoding a SARS-CoV-2 S protein with one or more mutations characteristic of the BA.2 omicron variant. In some embodiments, the subject is administered a booster regimen comprising at least one dose comprising 25 ug of RNA encoding a SARS-CoV-2 S protein from the Wuhan strain and 25 ug of RNA encoding a SARS-CoV-2 S protein with one or more mutations characteristic of the BA.2 omicron variant. The subject is administered a booster regimen comprising at least one dose comprising RNA encoding a SARS-CoV-2 S protein having one or more mutations characteristic of the BA.3 omicron variant. In some embodiments, the subject is administered a booster regimen comprising at least one dose comprising 25 ug of RNA encoding a SARS-CoV-2 S protein from the Wuhan strain and 25 ug of RNA encoding a SARS-CoV-2 S protein having one or more mutations characteristic of the BA.4 or BA.5 omicron variant.

In some embodiments, the subject is administered a booster regimen comprising at least one dose comprising 25 ug of RNA encoding a SARS-CoV-2 S protein from the BA.1 omicron variant and 25 ug of RNA encoding a SARS-CoV-2 S protein with one or more mutations characteristic of the BA.2 omicron variant. In some embodiments, the subject is administered a booster regimen comprising at least one dose comprising 25 ug of RNA encoding a SARS-CoV-2 S protein from the BA.1 omicron variant and 25 ug of RNA encoding a SARS-CoV-2 S protein with one or more mutations characteristic of the BA.3 omicron variant. In some embodiments, the subject is administered a booster regimen comprising at least one dose comprising 25 ug of RNA encoding a SARS-CoV-2 S protein from the BA.1 omicron variant and 25 ug of RNA encoding a SARS-CoV-2 S protein with one or more mutations characteristic of the BA.4 or BA. The patient will be administered a booster regimen that includes at least one dose of RNA encoding a SARS-CoV-2 S protein that has one or more mutations characteristic of the 5-omicron variant.

In some embodiments, the subject is administered a booster regimen comprising at least one dose comprising 25 ug of RNA encoding a SARS-CoV-2 S protein from the BA.2 omicron variant and 25 ug of RNA encoding a SARS-CoV-2 S protein with one or more mutations characteristic of the BA.4 or BA.5 omicron variant. In some embodiments, the subject is administered a booster regimen comprising at least one dose comprising 25 ug of RNA encoding a SARS-CoV-2 S protein from the BA.3 omicron variant and 25 ug of RNA encoding a SARS-CoV-2 S protein with one or more mutations characteristic of the BA.4 or BA.5 omicron variant.

In some embodiments, a subject is administered (i) a primary regimen comprising two 30 ug doses of RNA encoding a SARS-CoV-2 S protein from the Wuhan strain (the two doses are administered approximately 21 days apart), and (ii) at least one 50 ug dose of RNA encoding a SARS-CoV-2 S protein having one or more mutations characteristic of an omicron variant of SARS-CoV-2 (e.g., a BA.1, BA.2, BA.4, or BA.5 omicron variant), where the booster regimen is administered at least two months (e.g., including at least three months, at least four months, at least five months, at least six months, or more) after completion of the primary regimen. In some embodiments, a subject is administered (i) a primary regimen comprising two 30ug doses of RNA encoding a SARS-CoV-2 S protein from the Wuhan strain, the two doses being administered approximately 21 days apart, and (ii) a booster regimen comprising at least one 50ug dose of RNA, the 50ug RNA comprising 25ug of RNA encoding a SARS-CoV-2 S protein from the Wuhan strain and 25ug of RNA encoding a SARS-CoV-2 S protein having one or more mutations characteristic of an omicron variant (e.g., a BA.1, BA.2, BA.4, or BA.5 variant), the booster regimen being administered at least two months (e.g., including at least three months, at least four months, at least five months, at least six months, or more) after completion of the first booster regimen.

In some embodiments, a subject is administered (i) a primary regimen comprising at least two 30 ug doses of RNA encoding a SARS-CoV-2 S protein from the Wuhan strain, the two doses being administered at least approximately 21 days apart, and (ii) a booster regimen comprising a 50 ug dose of RNA encoding a SARS-CoV-2 S protein having one or more mutations characteristic of the BA.1 omicron variant of SARS-CoV-2, the booster regimen being administered at least two months (including, e.g., at least three months, at least four months, at least five months, at least six months, or more) after completion of the primary regimen. In some embodiments, subjects receive (i) a primary regimen comprising at least two 30ug doses of RNA encoding the SARS-CoV-2 S protein from the Wuhan strain (the two doses are administered at least approximately 21 days apart), and (ii) a booster regimen comprising 50ug doses of RNA including 25ug of RNA encoding the SARS-CoV-2 S protein from the Wuhan strain and 25ug of RNA encoding a SARS-CoV-2 S protein having one or more mutations characteristic of the BA.1 omicron variant.

In some embodiments, a subject is administered (i) a primary regimen comprising at least two 30 ug doses of RNA encoding a SARS-CoV-2 S protein from the Wuhan strain, the two doses being administered at least approximately 21 days apart, and (ii) a booster regimen comprising a 50 ug dose of RNA encoding a SARS-CoV-2 S protein having one or more mutations characteristic of the BA.2 omicron variant of SARS-CoV-2, the booster regimen being administered at least two months (including, e.g., at least three months, at least four months, at least five months, at least six months, or more) after completion of the primary regimen. In some embodiments, subjects receive (i) a primary regimen comprising at least two 30ug doses of RNA encoding the SARS-CoV-2 S protein from the Wuhan strain, the two doses being administered at least approximately 21 days apart, and (ii) a booster regimen comprising 50ug doses of RNA, including 25ug of RNA encoding the SARS-CoV-2 S protein from the Wuhan strain and 25ug of RNA encoding a SARS-CoV-2 S protein having one or more mutations characteristic of the BA.2 omicron variant.

In some embodiments, a subject is administered (i) a primary regimen comprising at least two 30 ug doses of RNA encoding a SARS-CoV-2 S protein from the Wuhan strain, the two doses being administered at least approximately 21 days apart, and (ii) a booster regimen comprising a 50 ug dose of RNA encoding a SARS-CoV-2 S protein having one or more mutations characteristic of the BA.4 or BA.5 omicron variant of SARS-CoV-2, the booster regimen being administered at least two months (including, e.g., at least three months, at least four months, at least five months, at least six months, or more) after completion of the primary regimen. In some embodiments, subjects receive (i) a primary regimen comprising at least two 30ug doses of RNA encoding the SARS-CoV-2 S protein from the Wuhan strain, the two doses being administered at least approximately 21 days apart, and (ii) a booster regimen comprising 50ug doses of RNA, including 25ug of RNA encoding the SARS-CoV-2 S protein from the Wuhan strain and 25ug of RNA encoding a SARS-CoV-2 S protein having one or more mutations characteristic of the BA.4 or BA.5 omicron variant.

In some embodiments, subjects receive (i) a primary regimen comprising at least two 30ug doses of RNA encoding a SARS-CoV-2 S protein from the Wuhan strain (the two doses are administered at least approximately 21 days apart), and (ii) a booster regimen comprising 50ug doses of RNA including 25ug of RNA encoding a SARS-CoV-2 S protein from the BA.1 omicron variant and 25ug of RNA encoding a SARS-CoV-2 S protein having one or more mutations characteristic of the BA.2 omicron variant.

In some embodiments, subjects receive (i) a primary regimen comprising at least two 30ug doses of RNA encoding a SARS-CoV-2 S protein from the Wuhan strain, the two doses being administered at least approximately 21 days apart, and (ii) a booster regimen comprising 50ug doses of RNA, including 25ug of RNA encoding a SARS-CoV-2 S protein from the BA.1 omicron variant and 25ug of RNA encoding a SARS-CoV-2 S protein having one or more mutations characteristic of the BA.4 or BA.5 omicron variant.

In some embodiments, subjects are administered (i) a primary regimen comprising at least two 30ug doses of RNA encoding a SARS-CoV-2 S protein from the Wuhan strain, the two doses being administered at least approximately 21 days apart, and (ii) a booster regimen comprising 50ug doses of RNA, including 25ug of RNA encoding a SARS-CoV-2 S protein from the BA.2 omicron variant and 25ug of RNA encoding a SARS-CoV-2 S protein having one or more mutations characteristic of the BA.4 or BA.5 omicron variant.

In some embodiments, the subject is administered a booster regimen comprising at least one 60ug dose of RNA. In some embodiments, the subject is administered a booster regimen comprising 60ug of RNA encoding a SARS-CoV-2 S protein from the Wuhan variant. In some embodiments, the subject is administered 60ug of RNA encoding a SARS-CoV-2 S protein having one or more mutations characteristic of a SARS-CoV-2 variant (e.g., a variant described herein). In some embodiments, the subject is administered a booster regimen comprising 60ug of RNA encoding a SARS-CoV-2 S protein having one or more mutations characteristic of an omicron variant (e.g., a BA.1, BA.2, BA.4, or BA.5 omicron variant). In some embodiments, the subject is administered a booster regimen comprising a 60ug dose of RNA, the RNA comprising a first RNA encoding a SARS-CoV-2 S protein from the Wuhan strain, and at least one additional RNA encoding a SARS-CoV-2 S protein comprising a mutation characteristic of a SARS-CoV-2 variant (e.g., in some embodiments, the subject is administered a booster regimen comprising 30ug of RNA encoding a SARS-CoV-2 S protein from the Wuhan strain, and 30ug of RNA encoding a SARS-CoV-2 S protein having one or more mutations characteristic of an Omicron variant (e.g., a BA.1, BA.2, BA.4, or BA.5 variant)).

In some embodiments, the subject is administered a booster regimen comprising at least one dose comprising 30 ug of RNA encoding a SARS-CoV-2 S protein from the Wuhan strain and 30 ug of RNA encoding a SARS-CoV-2 S protein with one or more mutations characteristic of the BA.1 omicron variant. In some embodiments, the subject is administered a booster regimen comprising at least one dose comprising 30 ug of RNA encoding a SARS-CoV-2 S protein from the Wuhan strain and 30 ug of RNA encoding a SARS-CoV-2 S protein with one or more mutations characteristic of the BA.2 omicron variant. In some embodiments, the subject is administered a booster regimen comprising at least one dose comprising 30 ug of RNA encoding a SARS-CoV-2 S protein from the Wuhan strain and 30 ug of RNA encoding a SARS-CoV-2 S protein with one or more mutations characteristic of the BA.2 omicron variant. The subject is administered a booster regimen comprising at least one dose comprising RNA encoding a SARS-CoV-2 S protein having one or more mutations characteristic of the BA.3 omicron variant. In some embodiments, the subject is administered a booster regimen comprising at least one dose comprising 30 ug of RNA encoding a SARS-CoV-2 S protein from the Wuhan strain and 30 ug of RNA encoding a SARS-CoV-2 S protein having one or more mutations characteristic of the BA.4 or BA.5 omicron variant.

In some embodiments, the subject is administered a booster regimen comprising at least one dose comprising 30 ug of RNA encoding a SARS-CoV-2 S protein from the BA.1 omicron variant and 30 ug of RNA encoding a SARS-CoV-2 S protein with one or more mutations characteristic of the BA.2 omicron variant. In some embodiments, the subject is administered a booster regimen comprising at least one dose comprising 30 ug of RNA encoding a SARS-CoV-2 S protein from the BA.1 omicron variant and 30 ug of RNA encoding a SARS-CoV-2 S protein with one or more mutations characteristic of the BA.3 omicron variant. In some embodiments, the subject is administered a booster regimen comprising at least one dose comprising 30 ug of RNA encoding a SARS-CoV-2 S protein from the BA.1 omicron variant and 30 ug of RNA encoding a SARS-CoV-2 S protein with one or more mutations characteristic of the BA.4 or BA. The patient will be administered a booster regimen that includes at least one dose of RNA encoding a SARS-CoV-2 S protein that has one or more mutations characteristic of the 5-omicron variant.

In some embodiments, the subject is administered a booster regimen comprising at least one dose comprising 30 ug of RNA encoding a SARS-CoV-2 S protein from the BA.2 omicron variant and 30 ug of RNA encoding a SARS-CoV-2 S protein with one or more mutations characteristic of the BA.4 or BA.5 omicron variant. In some embodiments, the subject is administered a booster regimen comprising at least one dose comprising 30 ug of RNA encoding a SARS-CoV-2 S protein from the BA.3 omicron variant and 30 ug of RNA encoding a SARS-CoV-2 S protein with one or more mutations characteristic of the BA.4 or BA.5 omicron variant.

In some embodiments, a subject is administered (i) a primary regimen comprising two 30 ug doses of RNA encoding the SARS-CoV-2 S protein from the Wuhan strain of SARS-CoV-2, the two doses being administered at least approximately 21 days apart, and (ii) a booster regimen comprising at least one 60 ug dose of RNA encoding the SARS-CoV-2 S protein from the Wuhan strain of SARS-CoV-2, the booster regimen being administered at least two months (including, e.g., at least three months, at least four months, at least five months, at least six months, or more) after completion of the primary regimen.

In some embodiments, a subject is administered (i) a primary regimen comprising two 30 ug doses of RNA encoding a SARS-CoV-2 S protein from the Wuhan strain (the two doses are administered approximately 21 days apart), and (ii) a booster regimen comprising at least one 60 ug dose of RNA encoding a SARS-CoV-2 S protein having one or more mutations characteristic of an omicron variant of SARS-CoV-2 (e.g., a BA.1, BA.2, BA.4, or BA.5 omicron variant), wherein the booster regimen is administered at least two months (e.g., including at least three months, at least four months, at least five months, at least six months, or more) after completion of the primary regimen. In some embodiments, the subject is administered (i) a primary regimen comprising two 30 ug doses of RNA encoding a SARS-CoV-2 S protein from the Wuhan strain, the two doses being administered approximately 21 days apart, and (iii) a booster regimen comprising at least one 60 ug dose of RNA comprising 30 ug of RNA encoding a SARS-CoV-2 S protein having one or more mutations characteristic of the omicron variant of SARS-CoV-2 and 30 ug of RNA encoding a SARS-CoV-2 S protein from the Wuhan strain, the second booster regimen being administered at least two months (e.g., including at least three months, at least four months, at least five months, at least six months, or more) after completion of the first booster regimen.

In some embodiments, a subject is administered (i) a primary regimen comprising at least two 30 ug doses of RNA encoding a SARS-CoV-2 S protein from the Wuhan strain, the two doses being administered at least approximately 21 days apart, and (ii) a booster regimen comprising a 60 ug dose of RNA encoding a SARS-CoV-2 S protein having one or more mutations characteristic of the BA.1 omicron variant of SARS-CoV-2, the booster regimen being administered at least two months (including, e.g., at least three months, at least four months, at least five months, at least six months, or more) after completion of the primary regimen. In some embodiments, subjects receive (i) a primary regimen comprising at least two 30ug doses of RNA encoding the SARS-CoV-2 S protein from the Wuhan strain (the two doses are administered at least approximately 21 days apart), and (ii) a booster regimen comprising a 60ug dose of RNA comprising 30ug of RNA encoding the SARS-CoV-2 S protein from the Wuhan strain and 30ug of RNA encoding a SARS-CoV-2 S protein having one or more mutations characteristic of the BA.1 omicron variant.

In some embodiments, a subject is administered (i) a primary regimen comprising at least two 30 ug doses of RNA encoding a SARS-CoV-2 S protein from the Wuhan strain, the two doses being administered at least approximately 21 days apart, and (ii) a booster regimen comprising a 60 ug dose of RNA encoding a SARS-CoV-2 S protein having one or more mutations characteristic of the BA.2 omicron variant of SARS-CoV-2, the booster regimen being administered at least two months (including, e.g., at least three months, at least four months, at least five months, at least six months, or more) after completion of the primary regimen. In some embodiments, subjects receive (i) a primary regimen comprising at least two 30ug doses of RNA encoding the SARS-CoV-2 S protein from the Wuhan strain (the two doses are administered at least approximately 21 days apart), and (ii) a booster regimen comprising a 60ug dose of RNA comprising 30ug of RNA encoding the SARS-CoV-2 S protein from the Wuhan strain and 30ug of RNA encoding a SARS-CoV-2 S protein having one or more mutations characteristic of the BA.2 omicron variant.

In some embodiments, a subject is administered (i) a primary regimen comprising at least two 30 ug doses of RNA encoding a SARS-CoV-2 S protein from the Wuhan strain, the two doses being administered at least approximately 21 days apart, and (ii) a booster regimen comprising a 60 ug dose of RNA encoding a SARS-CoV-2 S protein having one or more mutations characteristic of the BA.4 or BA.5 omicron variant of SARS-CoV-2, the booster regimen being administered at least two months (including, e.g., at least three months, at least four months, at least five months, at least six months, or more) after completion of the primary regimen. In some embodiments, subjects receive (i) a primary regimen comprising at least two 30ug doses of RNA encoding the SARS-CoV-2 S protein from the Wuhan strain (the two doses are administered at least approximately 21 days apart), and (ii) a booster regimen comprising a 60ug dose of RNA comprising 30ug of RNA encoding the SARS-CoV-2 S protein from the Wuhan strain and 30ug of RNA encoding a SARS-CoV-2 S protein having one or more mutations characteristic of the BA.4/5 omicron variant.

In some embodiments, subjects receive (i) a primary regimen comprising at least two 30ug doses of RNA encoding a SARS-CoV-2 S protein from the Wuhan strain (the two doses are administered at least approximately 21 days apart), and (ii) a booster regimen comprising 60ug doses of RNA, including 30ug of RNA encoding a SARS-CoV-2 S protein from the BA.1 omicron variant and 30ug of RNA encoding a SARS-CoV-2 S protein having one or more mutations characteristic of the BA.2 omicron variant.

In some embodiments, subjects are administered (i) a primary regimen comprising at least two 30ug doses of RNA encoding a SARS-CoV-2 S protein from the Wuhan strain, the two doses being administered at least approximately 21 days apart, and (ii) a booster regimen comprising 60ug doses of RNA, including 30ug of RNA encoding a SARS-CoV-2 S protein from the BA.1 omicron variant and 30ug of RNA encoding a SARS-CoV-2 S protein having one or more mutations characteristic of the BA.4 or BA.5 omicron variant.

In some embodiments, subjects are administered (i) a primary regimen comprising at least two 30ug doses of RNA encoding a SARS-CoV-2 S protein from the Wuhan strain, the two doses being administered at least approximately 21 days apart, and (ii) a booster regimen comprising 60ug doses of RNA, including 30ug of RNA encoding a SARS-CoV-2 S protein from the BA.2 omicron variant and 30ug of RNA encoding a SARS-CoV-2 S protein having one or more mutations characteristic of the BA.4 or BA.5 omicron variant.

In some embodiments, patients are administered a primary regimen comprising two 30 ug doses administered approximately 21 days apart, and a booster regimen comprising at least one 60 ug dose of RNA (e.g., 60 ug of RNA encoding a SARS-CoV-2 S protein from the Wuhan strain, 60 ug of RNA encoding a SARS-CoV-2 S protein having one or more mutations characteristic of an omicron variant (e.g., the BA.1, BA.2, BA.4, or BA.5 omicron variant), or 30 ug of RNA encoding a SARS-CoV-2 S protein from the Wuhan strain and 30 ug of RNA encoding a SARS-CoV-2 S protein having one or more mutations characteristic of an omicron variant). In some embodiments, patients are administered a primary regimen comprising two 30 ug doses administered approximately 21 days apart, and a booster regimen comprising at least one 50 ug dose of RNA (e.g., 50 ug of RNA encoding a SARS-CoV-2 S protein from the Wuhan strain, 50 ug of RNA encoding a SARS-CoV-2 S protein with one or more mutations characteristic of the Omicron variant, or 25 ug of RNA encoding a SARS-CoV-2 S protein from the Wuhan strain and 25 ug of RNA encoding a SARS-CoV-2 S protein with one or more mutations characteristic of the Omicron variant). In some embodiments, patients are administered a primary regimen comprising two 30ug doses administered approximately 21 days apart, and a booster regimen comprising at least one 30ug dose of RNA (e.g., 30ug of RNA encoding a SARS-CoV-2 S protein from the Wuhan strain, 30ug of RNA encoding a SARS-CoV-2 S protein with one or more mutations characteristic of the Omicron variant, or 15ug of RNA encoding a SARS-CoV-2 S protein from the Wuhan strain and 15ug of RNA encoding a SARS-CoV-2 S protein with one or more mutations characteristic of the Omicron variant).

In some embodiments, the primary regimen may involve one or more 30ug doses and the booster regimen may involve one or more 20ug doses, one or more 10ug doses, and/or one or more 3ug doses. In some embodiments, the primary regimen may involve one or more 20ug doses and the booster regimen may involve one or more 10ug doses, and/or one or more 3ug doses. In some embodiments, the primary regimen may involve one or more 10ug doses and the booster regimen may involve one or more 3ug doses. In some embodiments, the primary regimen may involve one or more 3ug doses and the booster regimen may involve one or more 3ug doses.

In some embodiments, the booster regimen includes a single dose, for example, for patients who experienced an adverse reaction while receiving the primary regimen.

In some embodiments, the same RNA is used in the booster regimen as is used in the primary regimen. In some embodiments, the RNA used in the primary and booster regimens is BNT162b2.

In some embodiments, different RNA is used in the booster regimen compared to that used in the primary regimen administered to the same subject. In some embodiments, BNT162b2 is used in the primary regimen but not in the booster regimen. In some embodiments, BNT162b2 is used in the booster regimen but not in the primary regimen. In some embodiments, similar BNT162b2 constructs can be used in the primary and booster regimens, except that the RNA constructs used in the primary and booster regimens encode a SARS-CoV-2 S protein (or an immunogenic portion thereof) of a different SARS-CoV-2 strain (e.g., as described herein).

In some embodiments, BNT162b2 is used in the primary or booster regimen, but not both, and if a different RNA is used for the other, such different RNA may be an RNA that encodes the same SARS-CoV-2 S protein but has a different codon optimization or other different RNA sequence. In some embodiments, such different RNA may encode a SARS-CoV-2 S protein (or an immunogenic portion thereof) of a different SARS-CoV-2 strain, e.g., a variant strain discussed herein. In some such embodiments, such variant is endemic or spreading rapidly in the relevant jurisdiction. In some embodiments, such a different RNA may be an RNA encoding a SARS-CoV-2 S protein or variant thereof (or any immunogenic portion thereof) that includes one or more mutations described herein for a SARS-CoV-2 S protein variant, particularly an S protein variant such as a naturally occurring S protein variant, and in some such embodiments, the SARS-CoV-2 variant may be selected from the group consisting of VOC-202012/01, 501. V2, Cluster 5, and B. 1.1.248. In some embodiments, the SARS-CoV-2 variant may be selected from the group consisting of VOC-202012/01, 501. V2, Cluster 5, and B. 1.1.248, B. 1.1.7, B. 1.617.2, and B. 1.1.529. In some embodiments, the booster regimen includes at least one dose of RNA encoding a SARS-CoV-2 S protein (or an immunogenic fragment thereof) of a variant that is rapidly spreading in the relevant jurisdiction at the time of administration. In some such embodiments, the variant encoded by the RNA administered in the booster regimen may be different from the variant encoded by the RNA administered in the primary regimen.

In some embodiments, the booster regimen includes administering (i) an RNA encoding the same SARS-CoV-2 S protein (or an immunogenic fragment thereof) as the RNA administered in a dose of the primary regimen (e.g., an RNA encoding a SARS-CoV-2 S protein (or an immunogenic fragment thereof) from the SARS-CoV-2 Wuhan strain), and (ii) an RNA encoding a SARS-CoV-2 S protein (or an immunogenic fragment thereof) of a variant that is rapidly spreading in the relevant jurisdiction at the time of administration of a dose (e.g., a SARS-CoV-2 S protein (or an immunogenic fragment thereof) from one of the SARS-CoV-2 variants described herein).

In some embodiments, the booster regimen includes multiple doses (e.g., at least two doses, at least three doses, or more). For example, in some embodiments, the first dose of the booster regimen may include RNA encoding the same SARS-CoV-2 S protein (or an immunogenic fragment thereof) administered in the primary regimen, and the second dose of the booster regimen may include RNA encoding the SARS-CoV-2 S protein of a variant that is rapidly spreading in the relevant jurisdiction at the time of administration. In some embodiments, the first dose of the booster regimen may include RNA encoding the SARS-CoV-2 S protein (or an immunogenic fragment thereof) of a variant that is rapidly spreading in the relevant jurisdiction at the time of administration, and the second dose of the booster regimen may include RNA encoding the same SARS-CoV-2 S protein (or an immunogenic fragment thereof) administered in the primary regimen. In some embodiments, the booster regimen includes multiple doses, where the RNA encoding the S protein of the variant that is spreading rapidly in the relevant jurisdiction is administered in a first dose, and the RNA encoding the S protein administered in the primary regimen is administered in a second dose.

In some embodiments, the doses in the booster regimen (e.g., the first and second doses, or any two consecutive doses) are administered at least two weeks apart, including, for example, at least three weeks, at least four weeks, at least five weeks, at least six weeks, at least seven weeks, at least eight weeks, at least nine weeks, at least ten weeks, at least eleven weeks, at least twelve weeks, at least thirteen weeks, at least fourteen weeks, at least fifteen weeks, at least sixteen weeks, or more. In some embodiments, the doses in the booster regimen (e.g., the first and second doses, or any two consecutive doses) are administered approximately two to one hundred and sixty-eight weeks apart. In some embodiments, the doses in the booster regimen (e.g., the first and second doses, or any two consecutive doses) are administered approximately three to twelve weeks apart. In some embodiments, the doses in the booster regimen (e.g., the first and second doses, or any two consecutive doses) are administered approximately four to ten weeks apart. In some embodiments, the doses in the booster regimen (e.g., the first and second doses, or any two consecutive doses) are administered approximately 6-8 weeks apart (e.g., about 21 days apart, or about 6-8 weeks apart). In some embodiments, the first and second doses are administered on the same day (e.g., by intramuscular injection at different sites in the subject).

In such embodiments, the booster regimen may optionally further include a third and fourth dose administered approximately 2-8 weeks after the first and second doses (e.g., about 21 days after the first and second doses, or about 6 weeks to about 8 weeks after the first and second doses), where the third and fourth doses are administered on the same day (e.g., by intramuscular injection at different sites in the subject) and include the same RNA administered in the first and second doses of the booster regimen.

In some embodiments, multiple booster regimens may be administered. In some embodiments, a booster regimen is administered to a patient who has previously received a booster regimen.

In some embodiments, the second booster regimen is administered to a patient who has previously received a first booster regimen, and the amount of RNA administered in at least one dose of the second booster regimen is greater than the amount of RNA administered in at least one dose of the first booster regimen.

In some embodiments, the second booster regimen includes administering at least one dose of 3ug of RNA. In some embodiments, the second booster regimen includes administering at least one dose of 5ug of RNA. In some embodiments, the second booster regimen includes administering at least one dose of 10ug of RNA. In some embodiments, the second booster regimen includes administering at least one dose of 15ug of RNA. In some embodiments, the second booster regimen includes administering at least one dose of 20ug of RNA. In some embodiments, the second booster regimen includes administering at least one dose of 25ug of RNA. In some embodiments, the second booster regimen includes administering at least one dose of 30ug of RNA. In some embodiments, the second booster regimen includes administering at least one dose of 50ug of RNA. In some embodiments, the second booster regimen includes administering at least one dose of 60 ug of RNA.

In some embodiments, the subject is administered a primary regimen comprising two doses of 30ug RNA administered approximately 21 days apart, and a booster regimen comprising at least one dose of approximately 30ug RNA. In some embodiments, the subject is administered a primary regimen comprising two doses of 30ug RNA administered approximately 21 days apart, and a booster regimen comprising at least one dose of approximately 50ug RNA. In some embodiments, the subject is administered a primary regimen comprising two doses of 30ug RNA administered approximately 21 days apart, and a booster regimen comprising at least one dose of approximately 60ug RNA.

In some embodiments, the subject is administered a primary regimen comprising two doses of 30ug RNA administered approximately 21 days apart, a first booster regimen comprising at least one dose of approximately 30ug RNA, and a second booster regimen comprising at least one dose of approximately 30ug RNA. In some embodiments, the subject is administered a primary regimen comprising two doses of 30ug RNA administered approximately 21 days apart, a first booster regimen comprising at least one dose of approximately 30ug RNA, and a second booster regimen comprising at least one dose of approximately 50ug RNA. In some embodiments, the subject is administered a primary regimen comprising two doses of 30ug RNA administered approximately 21 days apart, a first booster regimen comprising at least one dose of approximately 30ug RNA, and a second booster regimen comprising at least one dose of approximately 60ug RNA. In some embodiments, the first booster regimen includes two doses of RNA, each dose includes RNA encoding a spike protein from a different SARS-CoV-2 variant. In some embodiments, the first booster regimen includes two doses of RNA, each dose includes RNA encoding a spike protein from a different SARS-CoV-2 variant, and the two doses of RNA are administered on the same day. In some embodiments, the two doses of RNA are administered in a single composition (e.g., by mixing a first composition including RNA encoding a spike protein from a first SARS-CoV-2 variant with a second composition including RNA encoding a spike protein from a second SARS-CoV-2 variant).

In some embodiments, a subject is administered a booster regimen comprising a first dose comprising RNA encoding a spike protein from the Wuhan strain of SARS-CoV-2 and a second dose comprising RNA encoding a spike protein including a mutation from a variant that is prevalent and/or rapidly spreading in the relevant jurisdiction when the booster regimen is administered, where the first and second doses of RNA may be administered on the same day. In some embodiments, a subject is administered a booster regimen comprising a first dose comprising RNA encoding a spike protein from the Wuhan strain of SARS-CoV-2 and a second dose comprising RNA encoding a spike protein including a mutation from an alpha variant of SARS-CoV-2, where the first and second doses may be administered on the same day. In some embodiments, a subject is administered a booster regimen comprising a first dose comprising RNA encoding a spike protein from the Wuhan strain of SARS-CoV-2 and a second dose comprising RNA encoding a spike protein comprising a mutation from a beta variant of SARS-CoV-2, where the first and second doses may be administered on the same day. In some embodiments, a subject is administered a booster regimen comprising a first dose comprising RNA encoding a spike protein from the Wuhan strain of SARS-CoV-2 and a second dose comprising RNA encoding a spike protein comprising a mutation from a delta variant of SARS-CoV-2, where the first and second doses may be administered on the same day. In some embodiments, a subject is administered a booster regimen comprising a first dose comprising RNA encoding a spike protein from the Wuhan strain of SARS-CoV-2 and a second dose comprising RNA encoding a spike protein comprising a mutation from the Omicron variant of SARS-CoV-2, where the first and second doses may be administered on the same day. Such a booster regimen may be administered, for example, to a subject previously administered a primary dosing regimen and/or to a subject previously administered a primary dosing regimen and a booster regimen.

In some embodiments, the subject is administered a first booster regimen comprising a first dose of 15ug of RNA encoding the spike protein from the Wuhan variant and a second dose of 15ug of RNA encoding the spike protein from the Omicron variant of SARS-CoV-2, where the first and second doses are administered on the same day (e.g., the compositions comprising the RNA are mixed prior to administration and the mixture is then administered to the patient). In some embodiments, the subject is administered a first booster regimen comprising a first dose of 25ug of RNA encoding the spike protein from the Wuhan variant and a second dose of 25ug of RNA encoding the spike protein from the Omicron variant of SARS-CoV-2. In some embodiments, the first and second doses are optionally administered on the same day. In some embodiments, a subject is administered a first booster regimen comprising a first dose of 25ug of RNA encoding the spike protein from the Wuhan variant and a second dose of 25ug of RNA encoding the spike protein from the Omicron variant of SARS-CoV-2. In some embodiments, the first and second doses are administered on the same day. In some embodiments, a subject is administered a first booster regimen comprising a first dose of 30ug of RNA encoding the spike protein from the Wuhan variant and a second dose of 30ug of RNA encoding the spike protein from the Omicron variant of SARS-CoV-2, optionally, the first and second doses being administered on the same day (e.g., in separate administrations or as an administration of a multivalent vaccine). In some embodiments, such a first booster regimen is administered to a subject who has previously been administered a primary regimen comprising two doses of 30 ug of RNA administered about 21 days apart, and the first booster regimen is administered at least 3 months (e.g., at least 4 months, at least 5 months, or at least 6 months) after administration of the primary regimen.

In some embodiments, the subject is administered a second booster regimen comprising a first dose of 15ug of RNA encoding the spike protein from the Wuhan variant and a second dose of 15ug of RNA encoding the spike protein from the Omicron variant of SARS-CoV-2, where the first and second doses are administered on the same day (e.g., the compositions comprising the RNA are mixed prior to administration to form a multivalent vaccine, and the mixture is then administered to the patient). In some embodiments, the subject is administered a second booster regimen comprising a first dose of 25ug of RNA encoding the spike protein from the Wuhan variant and a second dose of 25ug of RNA encoding the spike protein from the Omicron variant of SARS-CoV-2, where the first and second doses are optionally administered on the same day (e.g., via administration of a multivalent vaccine or via administration of separate compositions). In some embodiments, a subject is administered a second booster regimen comprising a first dose of 25ug of RNA encoding the spike protein from the Wuhan variant and a second dose of 25ug of RNA encoding the spike protein from the Omicron variant of SARS-CoV-2. In some embodiments, a subject is administered a second booster regimen comprising a first dose of 30ug of RNA encoding the spike protein from the Wuhan variant and a second dose of 30ug of RNA encoding the spike protein from the Omicron variant of SARS-CoV-2, the first and second doses optionally being administered on the same day (e.g., via administration of a multivalent vaccine or via administration of separate compositions). In some embodiments, such a second booster regimen is administered to a subject who previously received a primary regimen comprising two doses of 30ug of RNA administered about 21 days apart. In some embodiments, such a second booster regimen is administered to a subject who has previously received a primary regimen comprising two doses of 30 ug RNA administered about 21 days apart, and a first booster regimen comprising one dose of 30 ug RNA, and the second booster regimen is administered at least 3 months (e.g., at least 4 months, at least 5 months, or at least 6 months) after administration of the first booster regimen.

In some embodiments, patients receiving a dose(s) of an RNA composition described herein are monitored for one or more specific conditions, e.g., after administration of one or more doses. In some embodiments, such condition(s) may be or include allergic reaction(s) (particularly in subject(s) with relevant allergies or a history of allergic reactions), myocarditis (inflammation of the heart muscle, particularly if the subject is a young male and/or may have previously experienced such inflammation), pericarditis (inflammation of the outer lining of the heart, particularly if the subject is a young male and/or may have previously experienced such inflammation), fever, bleeding (particularly if the subject is known to have a bleeding disorder or is undergoing therapy with a blood thinner). Alternatively or additionally, patients who may receive more detailed monitoring may be or include patients who affect the immune system, are pregnant or planning to become pregnant, are breastfeeding, have received another COVID-19 vaccine, and/or have experienced injection-related fainting, are immunocompromised, or are undergoing treatment with a drug that affects the immune system. In some embodiments, a patient is monitored for myocarditis after administration of one of the compositions disclosed herein. In some embodiments, a patient is monitored for pericarditis after administration of one of the compositions disclosed herein. The patient may be monitored and/or treated for the condition using the current standard of care.

In some embodiments, efficacy for the RNA (e.g., mRNA) compositions described (e.g., as described herein) in pediatric populations can be assessed by various metrics described herein, including, but not limited to, incidence of COVID-19 per 1000 person-years in subjects without serological or virological evidence of prior SARS-CoV-2 infection, e.g., geometric mean ratio (GMR) of SARS CoV-2 neutralizing titers measured 7 days after the second dose, etc.

In some embodiments, pediatric populations described herein (e.g., ages 12-16 years) may be monitored for the development of multisystem inflammatory syndrome (MIS) (e.g., inflammation in different body parts, such as the heart, lungs, kidneys, brain, skin, eyes, and/or gastrointestinal organs) following administration of an RNA composition (e.g., mRNA) described herein. Exemplary symptoms of MIS in children may include, but are not limited to, fever, abdominal pain, vomiting, diarrhea, neck pain, rash, bloodshot eyes, excessive fatigue, and combinations thereof.

In one embodiment, the RNA administered as described above is a nucleoside modified messenger RNA (modRNA) described herein as BNT162b1 (RBP020.3), BNT162b2 (RBP020.1 or RBP020.2), or BNT162b3 (e.g., BNT162b3c). In one embodiment, the RNA administered as described above is a nucleoside modified messenger RNA (modRNA) described herein as RBP020.2. In one embodiment, the RNA encoding the vaccine antigen is a nucleoside modified messenger RNA (modRNA) described herein as BNT162b3 (e.g., BNT162b3c).

In one embodiment, the RNA administered as described above is a nucleoside-modified messenger RNA (modRNA) and (i) comprises the nucleotide sequence of SEQ ID NO:21, a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of SEQ ID NO:21, and/or (ii) encodes an amino acid sequence comprising the amino acid sequence of SEQ ID NO:5, or an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of SEQ ID NO:5. In one embodiment, the RNA administered as described above is a nucleoside-modified messenger RNA (modRNA) and (i) comprises the nucleotide sequence of SEQ ID NO:21, and/or (ii) encodes an amino acid sequence comprising the amino acid sequence of SEQ ID NO:5.

In one embodiment, the RNA administered as described above is a nucleoside-modified messenger RNA (modRNA) and (i) comprises a nucleotide sequence of SEQ ID NO: 19 or 20, a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of SEQ ID NO: 19 or 20, and/or (ii) encodes an amino acid sequence comprising an amino acid sequence of SEQ ID NO: 7, or an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of SEQ ID NO: 7. In one embodiment, the RNA administered as described above is a nucleoside-modified messenger RNA (modRNA) and (i) comprises a nucleotide sequence of SEQ ID NO: 19 or 20, and/or (ii) encodes an amino acid sequence comprising an amino acid sequence of SEQ ID NO: 7.

In one embodiment, the RNA administered as described above is a nucleoside-modified messenger RNA (modRNA) and (i) comprises a nucleotide sequence of SEQ ID NO:20, a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of SEQ ID NO:20, and/or (ii) encodes an amino acid sequence comprising an amino acid sequence of SEQ ID NO:7, or an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of SEQ ID NO:7. In one embodiment, the RNA administered as described above is a nucleoside-modified messenger RNA (modRNA) and (i) comprises a nucleotide sequence of SEQ ID NO:20, and/or (ii) encodes an amino acid sequence comprising an amino acid sequence of SEQ ID NO:7.

In one embodiment, the RNA administered as described above is a nucleoside-modified messenger RNA (modRNA) and (i) comprises a nucleotide sequence of SEQ ID NO: 30, a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of SEQ ID NO: 30, and/or (ii) encodes an amino acid sequence comprising an amino acid sequence of SEQ ID NO: 29, or an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of SEQ ID NO: 29. In one embodiment, the RNA administered as described above is a nucleoside-modified messenger RNA (modRNA) and (i) comprises a nucleotide sequence of SEQ ID NO: 30, and/or (ii) encodes an amino acid sequence comprising an amino acid sequence of SEQ ID NO: 29.

In one embodiment, the administered RNA is a nucleoside-modified messenger RNA (modRNA) that (i) comprises the nucleotide sequence of SEQ ID NO:20 and/or (ii) encodes an amino acid sequence that comprises the amino acid sequence of SEQ ID NO:7, and is administered in an amount of about 30 μg per dose. In one embodiment, at least two of such doses are administered. For example, the second dose may be administered about 21 days after administration of the first dose.

In some embodiments, the population treated with the RNA described herein comprises, consists essentially of, or consists of subjects at least 50 years of age, at least 55 years of age, at least 60 years of age, or at least 65 years of age. In some embodiments, the population treated with the RNA described herein comprises, consists essentially of, or consists of subjects aged 55-90, 60-85, or 65-85.

In some embodiments, the period between administered doses is at least 7 days, at least 14 days, or at least 21 days. In some embodiments, the period between administered doses is 7 to 28 days, e.g., 14 to 23 days.

In some embodiments, no more than 5 doses, no more than 4 doses, or no more than 3 doses of the RNA described herein may be administered to a subject.

In some embodiments, the methods and medicaments described herein are administered (in a regimen, e.g., in doses, frequency of doses, and/or number of doses) such that the adverse event (AE), i.e., any undesired medical event in the patient, e.g., any undesirable and unintended signs, symptoms, or illnesses associated with the use of the pharmaceutical product, whether or not related to the pharmaceutical product, is mild or moderate in intensity. In some embodiments, the methods and medicaments described herein are administered such that the adverse event (AE) can be managed with an intervention, e.g., treatment with paracetamol or other drugs that provide analgesia, antipyretic (fever reduction), and/or anti-inflammatory effects, e.g., nonsteroidal anti-inflammatory drugs (NSAIDs), e.g., aspirin, ibuprofen, and naproxen. Paracetamol or "acetaminophen," which is not classified as an NSAID, exerts a weak anti-inflammatory effect and can be administered as an analgesic in accordance with the present disclosure.

In some embodiments, the methods and medicaments described herein provide a neutralizing effect in a subject against a coronavirus, a coronavirus infection, or a disease or disorder associated with a coronavirus.

In some embodiments, the methods and medicaments described herein after administration to a subject induce an immune response in the subject that blocks or neutralizes coronavirus. In some embodiments, the methods and medicaments described herein after administration to a subject induce the production of antibodies, such as IgG antibodies, in the subject that block or neutralize coronavirus. In some embodiments, the methods and medicaments described herein after administration to a subject induce an immune response in the subject that blocks or neutralizes coronavirus S protein that binds to ACE2. In some embodiments, the methods and medicaments described herein after administration to a subject induce the production of antibodies in the subject that block or neutralize coronavirus S protein that binds to ACE2.

In some embodiments, the methods and medicaments described herein following administration to a subject induce a geometric mean concentration (GMC) of an RBD domain-binding antibody, such as an IgG antibody, of at least 500 U/ml, 1000 U/ml, 2000 U/ml, 3000 U/ml, 4000 U/ml, 5000 U/ml, 10000 U/ml, 15000 U/ml, 20000 U/ml, 25000 U/ml, 30000 U/ml, or even more. In some embodiments, the elevated GMC of the RBD domain-binding antibody persists for at least 14 days, 21 days, 28 days, 1 month, 3 months, 6 months, 12 months, or even more.

In some embodiments, the methods and medicaments described herein after administration to a subject induce a geometric mean titer (GMT) of neutralizing antibodies, such as IgG antibodies, of at least 100 U/ml, 200 U/ml, 300 U/ml, 400 U/ml, 500 U/ml, 1000 U/ml, 1500 U/ml, or more. In some embodiments, the elevated GMT of neutralizing antibodies persists for at least 14 days, 21 days, 28 days, 1 month, 3 months, 6 months, 12 months, or even longer.

As used herein, the term "neutralization" refers to an event in which a binding agent, such as an antibody, binds to a biologically active site of a virus, such as a receptor binding protein, thereby inhibiting viral infection of a cell. As used herein, the term "neutralization" with respect to coronaviruses, and in particular the coronavirus S protein, refers to an event in which a binding agent, such as an antibody, binds to the RBD domain of the S protein, thereby inhibiting viral infection of a cell. In particular, the term "neutralization" refers to an event in which a binding agent eliminates or significantly reduces the toxicity (e.g., ability to infect a cell) of the virus of interest.

The type of immune response generated in response to an antigenic challenge can generally be distinguished by the subset of T helper (Th) cells involved in the response. Immune responses can be broadly divided into two types: Th1 and Th2. Th1 immune activation is optimized against intracellular infections such as viruses, whereas Th2 immune responses are optimized for humoral (antibody) responses. Th1 cells produce interleukin 2 (IL-2), tumor necrosis factor (TNFα), and interferon gamma (IFNγ). Th2 cells produce IL-4, IL-5, IL-6, IL-9, IL-10, and IL-13. Th1 immune activation is most desired in many clinical situations. Vaccine compositions specifically designed to induce Th2 or humoral immune responses are generally not effective against most viral diseases.

In some embodiments, the methods and medicaments described herein after administration to a subject induce or promote a Th1-mediated immune response in the subject. In some embodiments, the methods and medicaments described herein after administration to a subject induce or promote a cytokine profile in the subject that is typical of a Th1-mediated immune response. In some embodiments, the methods and medicaments described herein after administration to a subject induce or promote the production of interleukin 2 (IL-2), tumor necrosis factor (TNFα) and/or interferon gamma (IFNγ) in the subject. In some embodiments, the methods and medicaments described herein after administration to a subject induce or promote the production of interleukin 2 (IL-2) and interferon gamma (IFNγ) in the subject. In some embodiments, the methods and medicaments described herein after administration to a subject do not induce or promote a Th2-mediated immune response in the subject or induce or promote a Th2-mediated immune response in the subject to a significantly lesser extent compared to the induction or promotion of a Th1-mediated immune response. In some embodiments, the methods and medicaments described herein following administration to a subject do not induce or promote a cytokine profile that is typical of a Th2-mediated immune response in the subject, or induce or promote a cytokine profile that is typical of a Th2-mediated immune response in the subject to a significantly lesser extent as compared to the induction or promotion of a cytokine profile that is typical of a Th1-mediated immune response. In some embodiments, the methods and medicaments described herein following administration to a subject do not induce or promote the production of IL-4, IL-5, IL-6, IL-9, IL-10, and/or IL-13, or induce or promote the production of IL-4, IL-5, IL-6, IL-9, IL-10, and/or IL-13 in the subject to a significantly lesser extent as compared to the induction or promotion of interleukin 2 (IL-2), tumor necrosis factor (TNFα), and/or interferon gamma (IFNγ) in the subject. In some embodiments, the methods and agents described herein, following administration to a subject, do not induce or promote the production of IL-4 or induce or promote the production of IL-4 in the subject to a significantly lesser extent than the induction or promotion of interleukin 2 (IL-2) and interferon gamma (IFNγ) in the subject.

In some embodiments, the methods and medicaments described herein after administration to a subject induce an antibody response in the subject, particularly a neutralizing antibody response, that targets a panel of different S protein variants, such as SARS-CoV-2 S protein variants, particularly naturally occurring S protein variants. In some embodiments, the panel of different S protein variants includes at least 5, at least 10, at least 15, or even more S protein variants. In some embodiments, such S protein variants include variants with amino acid modifications within the RBD domain and/or variants with amino acid modifications outside the RBD domain. In one embodiment, such S protein variants include a SARS-CoV-2 S protein or a naturally occurring variant thereof, wherein the amino acid corresponding to position 321 (Q) of SEQ ID NO:1 is S. In one embodiment, such S protein variants include a SARS-CoV-2 S protein or a naturally occurring variant thereof, wherein the amino acid corresponding to position 321 (Q) of SEQ ID NO:1 is L. In one embodiment, such an S protein variant comprises a SARS-CoV-2 S protein or a naturally occurring variant thereof, wherein the amino acid corresponding to position 341 (V) of SEQ ID NO:1 is I. In one embodiment, such an S protein variant comprises a SARS-CoV-2 S protein or a naturally occurring variant thereof, wherein the amino acid corresponding to position 348 (A) of SEQ ID NO:1 is T. In one embodiment, such an S protein variant comprises a SARS-CoV-2 S protein or a naturally occurring variant thereof, wherein the amino acid corresponding to position 354 (N) of SEQ ID NO:1 is D. In one embodiment, such an S protein variant comprises a SARS-CoV-2 S protein or a naturally occurring variant thereof, wherein the amino acid corresponding to position 359 (S) of SEQ ID NO:1 is N. In one embodiment, such an S protein variant comprises a SARS-CoV-2 S protein or a naturally occurring variant thereof, wherein the amino acid corresponding to position 367 (V) of SEQ ID NO:1 is F. In one embodiment, such an S protein variant comprises a SARS-CoV-2 S protein or a naturally occurring variant thereof, wherein the amino acid corresponding to position 378 (K) of SEQ ID NO:1 is S. In one embodiment, such an S protein variant comprises a SARS-CoV-2 S protein or a naturally occurring variant thereof, wherein the amino acid corresponding to position 378 (K) of SEQ ID NO:1 is R. In one embodiment, such an S protein variant comprises a SARS-CoV-2 S protein or a naturally occurring variant thereof, wherein the amino acid corresponding to position 408 (R) of SEQ ID NO:1 is I. In one embodiment, such an S protein variant comprises a SARS-CoV-2 S protein or a naturally occurring variant thereof, wherein the amino acid corresponding to position 409 (Q) of SEQ ID NO:1 is E. In one embodiment, such an S protein variant comprises a SARS-CoV-2 S protein or a naturally occurring variant thereof, wherein the amino acid corresponding to position 435 (A) of SEQ ID NO:1 is S. In one embodiment, such an S protein variant comprises a SARS-CoV-2 S protein or a naturally occurring variant thereof, wherein the amino acid corresponding to position 439 (N) of SEQ ID NO:1 is K. In one embodiment, such an S protein variant comprises a SARS-CoV-2 S protein or a naturally occurring variant thereof, wherein the amino acid corresponding to position 458 (K) of SEQ ID NO:1 is R. In one embodiment, such an S protein variant comprises a SARS-CoV-2 S protein or a naturally occurring variant thereof, wherein the amino acid corresponding to position 472 (I) of SEQ ID NO:1 is V. In one embodiment, such an S protein variant comprises a SARS-CoV-2 S protein or a naturally occurring variant thereof, wherein the amino acid corresponding to position 476 (G) of SEQ ID NO:1 is S. In one embodiment, such an S protein variant comprises a SARS-CoV-2 S protein or a naturally occurring variant thereof, wherein the amino acid corresponding to position 477 (S) of SEQ ID NO:1 is N. In one embodiment, such an S protein variant comprises a SARS-CoV-2 S protein or a naturally occurring variant thereof, wherein the amino acid corresponding to position 483 (V) of SEQ ID NO:1 is A. In one embodiment, such an S protein variant comprises a SARS-CoV-2 S protein or a naturally occurring variant thereof, wherein the amino acid corresponding to position 508 (Y) of SEQ ID NO:1 is H. In one embodiment, such an S protein variant comprises a SARS-CoV-2 S protein or a naturally occurring variant thereof, wherein the amino acid corresponding to position 519 (H) of SEQ ID NO:1 is P. In one embodiment, such an S protein variant comprises a SARS-CoV-2 S protein or a naturally occurring variant thereof, wherein the amino acid corresponding to position 614 (D) of SEQ ID NO:1 is G.

In some embodiments, the methods and medicaments described herein after administration to a subject induce an antibody response in the subject, particularly a neutralizing antibody response, that targets SARS-CoV-2 S protein variants, particularly S protein variants such as naturally occurring S protein variants that contain a mutation at a position corresponding to position 501 (N) of SEQ ID NO:1. In one embodiment, the amino acid corresponding to position 501 (N) of SEQ ID NO:1 is Y.

The S protein variant comprising a mutation at a position corresponding to position 501 (N) of SEQ ID NO:1 may comprise one or more additional mutations. Such one or more additional mutations may be selected from mutations at positions corresponding to the following positions of SEQ ID NO:1: 69 (H), 70 (V), 144 (Y), 570 (A), 614 (D), 681 (P), 716 (T), 982 (S), 1118 (D), 80 (D), 215 (D), 484 (E), 701 (A), 18 (L), 246 (R), 417 (K), 242 (L), 243 (A), and 244 (L). In one embodiment, the amino acid corresponding to position 69 (H) of SEQ ID NO:1 is deleted. In one embodiment, the amino acid corresponding to position 70 (V) of SEQ ID NO:1 is deleted. In one embodiment, the amino acid corresponding to position 144 (Y) of SEQ ID NO:1 is deleted. In one embodiment, the amino acid corresponding to position 570 (A) of SEQ ID NO:1 is D. In one embodiment, the amino acid corresponding to position 614 (D) of SEQ ID NO:1 is G. In one embodiment, the amino acid corresponding to position 681 (P) of SEQ ID NO:1 is H. In one embodiment, the amino acid corresponding to position 716 (T) of SEQ ID NO:1 is I. In one embodiment, the amino acid corresponding to position 982 (S) of SEQ ID NO:1 is A. In one embodiment, the amino acid corresponding to position 1118 (D) of SEQ ID NO:1 is H. In one embodiment, the amino acid corresponding to position 80 (D) of SEQ ID NO:1 is A. In one embodiment, the amino acid corresponding to position 215 (D) of SEQ ID NO:1 is G. In one embodiment, the amino acid corresponding to position 484 (E) of SEQ ID NO:1 is K. In one embodiment, the amino acid corresponding to position 701 (A) of SEQ ID NO:1 is V. In one embodiment, the amino acid corresponding to position 18 (L) of SEQ ID NO:1 is F. In one embodiment, the amino acid corresponding to position 246 (R) of SEQ ID NO:1 is I. In one embodiment, the amino acid corresponding to position 417 (K) of SEQ ID NO:1 is N. In one embodiment, the amino acid corresponding to position 242 (L) of SEQ ID NO:1 is deleted. In one embodiment, the amino acid corresponding to position 243 (A) of SEQ ID NO:1 is deleted. In one embodiment, the amino acid corresponding to position 244 (L) of SEQ ID NO:1 is deleted.

In some embodiments, the methods and medicaments described herein, following administration to a subject, induce an antibody response in the subject, particularly a neutralizing antibody response, that targets VOC-202012/01.

In some embodiments, the methods and medicaments described herein after administration to a subject induce an antibody response in the subject, particularly a neutralizing antibody response, that targets an S protein variant that includes the following mutations at positions corresponding to the following positions in SEQ ID NO:1: deletion 69-70, deletion 144, N501Y, A570D, D614G, P681H, T716I, S982A, and D1118H.

In some embodiments, the methods and medicaments described herein, following administration to a subject, induce an antibody response in the subject, particularly a neutralizing antibody response, that targets 501.V2.

In some embodiments, the methods and medicaments described herein after administration to a subject induce an antibody response in the subject, particularly a neutralizing antibody response, that targets an S protein variant that includes the following mutations at positions corresponding to the following positions in SEQ ID NO:1: D80A, D215G, E484K, N501Y, and A701V, and optionally, L18F, R246I, K417N, and deletion 242-244. The S protein variant may also include a D->G mutation at a position corresponding to position 614 in SEQ ID NO:1.

In some embodiments, the methods and medicaments described herein, following administration to a subject, induce an antibody response in the subject, particularly a neutralizing antibody response, that targets SARS-CoV-2 S protein variants, particularly S protein variants, such as naturally occurring S protein variants that contain deletions at positions corresponding to positions 69 (H) and 70 (V) of SEQ ID NO:1.

In some embodiments, the S protein variants comprising deletions at positions corresponding to positions 69 (H) and 70 (V) of SEQ ID NO:1 may comprise one or more additional mutations. Such one or more additional mutations may be selected from mutations at positions corresponding to the following positions of SEQ ID NO:1: 144 (Y), 501 (N), 570 (A), 614 (D), 681 (P), 716 (T), 982 (S), 1118 (D), 80 (D), 215 (D), 484 (E), 701 (A), 18 (L), 246 (R), 417 (K), 242 (L), 243 (A), 244 (L), 453 (Y), 692 (I), 1147 (S), and 1229 (M). In one embodiment, the amino acid corresponding to position 144 (Y) of SEQ ID NO:1 is deleted. In one embodiment, the amino acid corresponding to position 501 (N) of SEQ ID NO:1 is Y. In one embodiment, the amino acid corresponding to position 570 (A) of SEQ ID NO:1 is D. In one embodiment, the amino acid corresponding to position 614 (D) of SEQ ID NO:1 is G. In one embodiment, the amino acid corresponding to position 681 (P) of SEQ ID NO:1 is H. In one embodiment, the amino acid corresponding to position 716 (T) of SEQ ID NO:1 is I. In one embodiment, the amino acid corresponding to position 982 (S) of SEQ ID NO:1 is A. In one embodiment, the amino acid corresponding to position 1118 (D) of SEQ ID NO:1 is H. In one embodiment, the amino acid corresponding to position 80 (D) of SEQ ID NO:1 is A. In one embodiment, the amino acid corresponding to position 215 (D) of SEQ ID NO:1 is G. In one embodiment, the amino acid corresponding to position 484 (E) of SEQ ID NO:1 is K. In one embodiment, the amino acid corresponding to position 701 (A) of SEQ ID NO:1 is V. In one embodiment, the amino acid corresponding to position 18 (L) of SEQ ID NO:1 is F. In one embodiment, the amino acid corresponding to position 246 (R) of SEQ ID NO:1 is I. In one embodiment, the amino acid corresponding to position 417 (K) of SEQ ID NO:1 is N. In one embodiment, the amino acid corresponding to position 242 (L) of SEQ ID NO:1 is deleted. In one embodiment, the amino acid corresponding to position 243 (A) of SEQ ID NO:1 is deleted. In one embodiment, the amino acid corresponding to position 244 (L) of SEQ ID NO:1 is deleted. In one embodiment, the amino acid corresponding to position 453 (Y) of SEQ ID NO:1 is F. In one embodiment, the amino acid corresponding to position 692 (I) of SEQ ID NO:1 is V. In one embodiment, the amino acid corresponding to position 1147 (S) of SEQ ID NO:1 is L. In one embodiment, the amino acid corresponding to position 1229 (M) of SEQ ID NO:1 is I.

In some embodiments, the methods and medicaments described herein, following administration to a subject, induce an antibody response in the subject, particularly a neutralizing antibody response, that targets VOC-202012/01.

In some embodiments, the methods and medicaments described herein after administration to a subject induce an antibody response in the subject, particularly a neutralizing antibody response, that targets an S protein variant that includes the following mutations at positions corresponding to the following positions in SEQ ID NO:1: deletion 69-70, deletion 144, N501Y, A570D, D614G, P681H, T716I, S982A, and D1118H.

In some embodiments, the methods and medicaments described herein, following administration to a subject, induce an antibody response in the subject, particularly a neutralizing antibody response, that targets "cluster 5."

In some embodiments, the methods and medicaments described herein, following administration to a subject, induce an antibody response in the subject, particularly a neutralizing antibody response, that targets an S protein variant that includes the following mutations corresponding to the following positions in SEQ ID NO:1: deletion 69-70, Y453F, I692V, M1229I, and optionally S1147L.

In some embodiments, the methods and medicaments described herein after administration to a subject induce an antibody response in the subject, particularly a neutralizing antibody response, that targets SARS-CoV-2 S protein variants, particularly S protein variants such as naturally occurring S protein variants that contain a mutation at a position corresponding to position 614 (D) of SEQ ID NO:1. In one embodiment, the amino acid corresponding to position 614 (D) of SEQ ID NO:1 is G.

In some embodiments, the S protein variant comprising a mutation at position corresponding to 614 (D) of SEQ ID NO:1 may comprise one or more additional mutations. Such one or more additional mutations may be selected from mutations at positions corresponding to the following positions of SEQ ID NO:1: 69 (H), 70 (V), 144 (Y), 501 (N), 570 (A), 681 (P), 716 (T), 982 (S), 1118 (D), 80 (D), 215 (D), 484 (E), 701 (A), 18 (L), 246 (R), 417 (K), 242 (L), 243 (A), 244 (L), 453 (Y), 692 (I), 1147 (S), and 1229 (M). In one embodiment, the amino acid corresponding to position 69 (H) of SEQ ID NO:1 is deleted. In one embodiment, the amino acid corresponding to position 70 (V) of SEQ ID NO:1 is deleted. In one embodiment, the amino acid corresponding to position 144 (Y) of SEQ ID NO:1 is deleted. In one embodiment, the amino acid corresponding to position 501 (N) of SEQ ID NO:1 is Y. In one embodiment, the amino acid corresponding to position 570 (A) of SEQ ID NO:1 is D. In one embodiment, the amino acid corresponding to position 681 (P) of SEQ ID NO:1 is H. In one embodiment, the amino acid corresponding to position 716 (T) of SEQ ID NO:1 is I. In one embodiment, the amino acid corresponding to position 982 (S) of SEQ ID NO:1 is A. In one embodiment, the amino acid corresponding to position 1118 (D) of SEQ ID NO:1 is H. In one embodiment, the amino acid corresponding to position 80 (D) of SEQ ID NO:1 is A. In one embodiment, the amino acid corresponding to position 215 (D) of SEQ ID NO:1 is G. In one embodiment, the amino acid corresponding to position 484 (E) of SEQ ID NO:1 is K. In one embodiment, the amino acid corresponding to position 701 (A) of SEQ ID NO:1 is V. In one embodiment, the amino acid corresponding to position 18 (L) of SEQ ID NO:1 is F. In one embodiment, the amino acid corresponding to position 246 (R) of SEQ ID NO:1 is I. In one embodiment, the amino acid corresponding to position 417 (K) of SEQ ID NO:1 is N. In one embodiment, the amino acid corresponding to position 242 (L) of SEQ ID NO:1 is deleted. In one embodiment, the amino acid corresponding to position 243 (A) of SEQ ID NO:1 is deleted. In one embodiment, the amino acid corresponding to position 244 (L) of SEQ ID NO:1 is deleted. In one embodiment, the amino acid corresponding to position 453 (Y) of SEQ ID NO:1 is F. In one embodiment, the amino acid corresponding to position 692 (I) of SEQ ID NO:1 is V. In one embodiment, the amino acid corresponding to position 1147 (S) of SEQ ID NO:1 is L. In one embodiment, the amino acid corresponding to position 1229 (M) of SEQ ID NO:1 is I.

In some embodiments, the methods and medicaments described herein, following administration to a subject, induce an antibody response in the subject, particularly a neutralizing antibody response, that targets VOC-202012/01.

In some embodiments, the methods and medicaments described herein after administration to a subject induce an antibody response in the subject, particularly a neutralizing antibody response, that targets an S protein variant that includes the following mutations at positions corresponding to the following positions in SEQ ID NO:1: deletion 69-70, deletion 144, N501Y, A570D, D614G, P681H, T716I, S982A, and D1118H.

In some embodiments, the methods and medicaments described herein after administration to a subject induce an antibody response in the subject, particularly a neutralizing antibody response, that targets an S protein variant that includes the following mutations at positions corresponding to the following positions in SEQ ID NO:1: D80A, D215G, E484K, N501Y, D614G, and A701V, and optionally, L18F, R246I, K417N, and deletion 242-244.

In some embodiments, the methods and medicaments described herein after administration to a subject induce an antibody response in the subject, particularly a neutralizing antibody response, that targets SARS-CoV-2 S protein variants, particularly S protein variants such as naturally occurring S protein variants that contain mutations at positions corresponding to positions 501 (N) and 614 (D) of SEQ ID NO:1. In one embodiment, the amino acid corresponding to position 501 (N) of SEQ ID NO:1 is Y and the amino acid corresponding to position 614 (D) of SEQ ID NO:1 is G.

In some embodiments, the S protein variant comprising mutations at positions corresponding to positions 501 (N) and 614 (D) of SEQ ID NO:1 may comprise one or more additional mutations. Such one or more additional mutations may be selected from mutations at positions corresponding to the following positions of SEQ ID NO:1: 69 (H), 70 (V), 144 (Y), 570 (A), 681 (P), 716 (T), 982 (S), 1118 (D), 80 (D), 215 (D), 484 (E), 701 (A), 18 (L), 246 (R), 417 (K), 242 (L), 243 (A), 244 (L), 453 (Y), 692 (I), 1147 (S), and 1229 (M). In one embodiment, the amino acid corresponding to position 69 (H) of SEQ ID NO:1 is deleted. In one embodiment, the amino acid corresponding to position 70 (V) of SEQ ID NO:1 is deleted. In one embodiment, the amino acid corresponding to position 144 (Y) of SEQ ID NO:1 is deleted. In one embodiment, the amino acid corresponding to position 570 (A) of SEQ ID NO:1 is D. In one embodiment, the amino acid corresponding to position 681 (P) of SEQ ID NO:1 is H. In one embodiment, the amino acid corresponding to position 716 (T) of SEQ ID NO:1 is I. In one embodiment, the amino acid corresponding to position 982 (S) of SEQ ID NO:1 is A. In one embodiment, the amino acid corresponding to position 1118 (D) of SEQ ID NO:1 is H. In one embodiment, the amino acid corresponding to position 80 (D) of SEQ ID NO:1 is A. In one embodiment, the amino acid corresponding to position 215 (D) of SEQ ID NO:1 is G. In one embodiment, the amino acid corresponding to position 484 (E) of SEQ ID NO:1 is K. In one embodiment, the amino acid corresponding to position 701 (A) of SEQ ID NO:1 is V. In one embodiment, the amino acid corresponding to position 18 (L) of SEQ ID NO:1 is F. In one embodiment, the amino acid corresponding to position 246 (R) of SEQ ID NO:1 is I. In one embodiment, the amino acid corresponding to position 417 (K) of SEQ ID NO:1 is N. In one embodiment, the amino acid corresponding to position 242 (L) of SEQ ID NO:1 is deleted. In one embodiment, the amino acid corresponding to position 243 (A) of SEQ ID NO:1 is deleted. In one embodiment, the amino acid corresponding to position 244 (L) of SEQ ID NO:1 is deleted. In one embodiment, the amino acid corresponding to position 453 (Y) of SEQ ID NO:1 is F. In one embodiment, the amino acid corresponding to position 692 (I) of SEQ ID NO:1 is V. In one embodiment, the amino acid corresponding to position 1147 (S) of SEQ ID NO:1 is L. In one embodiment, the amino acid corresponding to position 1229 (M) of SEQ ID NO:1 is I.

In some embodiments, the methods and medicaments described herein, following administration to a subject, induce an antibody response in the subject, particularly a neutralizing antibody response, that targets VOC-202012/01.

In some embodiments, the methods and medicaments described herein after administration to a subject induce an antibody response in the subject, particularly a neutralizing antibody response, that targets an S protein variant that includes the following mutations at positions corresponding to the following positions in SEQ ID NO:1: deletion 69-70, deletion 144, N501Y, A570D, D614G, P681H, T716I, S982A, and D1118H.

In some embodiments, the methods and medicaments described herein after administration to a subject induce an antibody response in the subject, particularly a neutralizing antibody response, that targets an S protein variant that includes the following mutations at positions corresponding to the following positions in SEQ ID NO:1: D80A, D215G, E484K, N501Y, D614G, and A701V, and optionally, L18F, R246I, K417N, and deletion 242-244.

In some embodiments, the methods and medicaments described herein, following administration to a subject, induce an antibody response in the subject, particularly a neutralizing antibody response, that targets a SARS-CoV-2 S protein variant, particularly an S protein variant, such as a naturally occurring S protein variant that includes a mutation at a position corresponding to position 484 (E) of SEQ ID NO:1. In one embodiment, the amino acid corresponding to position 484 (E) of SEQ ID NO:1 is K.

In some embodiments, an S protein variant comprising a mutation at a position corresponding to position 484 (E) of SEQ ID NO:1 may comprise one or more additional mutations. Such one or more additional mutations may be selected from mutations at positions corresponding to the following positions of SEQ ID NO:1: 69 (H), 70 (V), 144 (Y), 501 (N), 570 (A), 614 (D), 681 (P), 716 (T), 982 (S), 1118 (D), 80 (D), 215 (D), 701 (A), 18 (L), 246 (R), 417 (K), 242 (L), 243 (A), 244 (L), 453 (Y), 692 (I), 1147 (S), 1229 (M), 20 (T), 26 (P), 138 (D), 190 (R), 417 (K), 655 (H), 1027 (T), and 1176 (V). In one embodiment, the amino acid corresponding to position 69 (H) of SEQ ID NO:1 is deleted. In one embodiment, the amino acid corresponding to position 70 (V) of SEQ ID NO:1 is deleted. In one embodiment, the amino acid corresponding to position 144 (Y) of SEQ ID NO:1 is deleted. In one embodiment, the amino acid corresponding to position 501 (N) of SEQ ID NO:1 is Y. In one embodiment, the amino acid corresponding to position 570 (A) of SEQ ID NO:1 is D. In one embodiment, the amino acid corresponding to position 614 (D) of SEQ ID NO:1 is G. In one embodiment, the amino acid corresponding to position 681 (P) of SEQ ID NO:1 is H. In one embodiment, the amino acid corresponding to position 716 (T) of SEQ ID NO:1 is I. In one embodiment, the amino acid corresponding to position 982 (S) of SEQ ID NO:1 is A. In one embodiment, the amino acid corresponding to position 1118 (D) of SEQ ID NO:1 is H. In one embodiment, the amino acid corresponding to position 80 (D) of SEQ ID NO:1 is A. In one embodiment, the amino acid corresponding to position 215 (D) of SEQ ID NO:1 is G. In one embodiment, the amino acid corresponding to position 701 (A) of SEQ ID NO:1 is V. In one embodiment, the amino acid corresponding to position 18 (L) of SEQ ID NO:1 is F. In one embodiment, the amino acid corresponding to position 246 (R) of SEQ ID NO:1 is I. In one embodiment, the amino acid corresponding to position 417 (K) of SEQ ID NO:1 is N. In one embodiment, the amino acid corresponding to position 242 (L) of SEQ ID NO:1 is deleted. In one embodiment, the amino acid corresponding to position 243 (A) of SEQ ID NO:1 is deleted. In one embodiment, the amino acid corresponding to position 244 (L) of SEQ ID NO:1 is deleted. In one embodiment, the amino acid corresponding to position 453 (Y) of SEQ ID NO:1 is F. In one embodiment, the amino acid corresponding to position 692 (I) of SEQ ID NO:1 is V. In one embodiment, the amino acid corresponding to position 1147 (S) of SEQ ID NO:1 is L. In one embodiment, the amino acid corresponding to position 1229 (M) of SEQ ID NO:1 is I. In one embodiment, the amino acid corresponding to position 20 (T) of SEQ ID NO:1 is N. In one embodiment, the amino acid corresponding to position 26 (P) of SEQ ID NO:1 is S. In one embodiment, the amino acid corresponding to position 138 (D) of SEQ ID NO:1 is Y. In one embodiment, the amino acid corresponding to position 190 (R) of SEQ ID NO:1 is S. In one embodiment, the amino acid corresponding to position 417 (K) of SEQ ID NO:1 is T. In one embodiment, the amino acid corresponding to position 655 (H) of SEQ ID NO:1 is Y. In one embodiment, the amino acid corresponding to position 1027 (T) of SEQ ID NO:1 is I. In one embodiment, the amino acid corresponding to position 1176 (V) of SEQ ID NO:1 is F.

In some embodiments, the methods and medicaments described herein, following administration to a subject, induce an antibody response in the subject, particularly a neutralizing antibody response, that targets 501.V2.

In some embodiments, the methods and medicaments described herein after administration to a subject induce an antibody response in the subject, particularly a neutralizing antibody response, that targets an S protein variant that includes the following mutations at positions corresponding to the following positions in SEQ ID NO:1: D80A, D215G, E484K, N501Y, and A701V, and optionally, L18F, R246I, K417N, and deletion 242-244. The S protein variant may also include a D->G mutation at a position corresponding to position 614 in SEQ ID NO:1.

In some embodiments, the methods and medicaments described herein, following administration to a subject, induce an antibody response in the subject, particularly a neutralizing antibody response, that targets "B.1.1.28".

In some embodiments, the methods and medicaments described herein, following administration to a subject, induce an antibody response in the subject, particularly a neutralizing antibody response, that targets "B.1.1.248".

In some embodiments, the methods and medicaments described herein after administration to a subject induce an antibody response in the subject, particularly a neutralizing antibody response, that targets an S protein variant that includes the following mutations at positions corresponding to the following positions in SEQ ID NO:1: L18F, T20N, P26S, D138Y, R190S, K417T, E484K, N501Y, H655Y, T1027I, and V1176F.

In some embodiments, the methods and medicaments described herein after administration to a subject induce an antibody response in the subject, particularly a neutralizing antibody response, that targets SARS-CoV-2 S protein variants, particularly S protein variants such as naturally occurring S protein variants that contain mutations at positions corresponding to positions 501 (N) and 484 (E) of SEQ ID NO:1. In one embodiment, the amino acid corresponding to position 501 (N) of SEQ ID NO:1 is Y and the amino acid corresponding to position 484 (E) of SEQ ID NO:1 is K.

In some embodiments, an S protein variant comprising a mutation at a position corresponding to 501 (N) and 484 (E) of SEQ ID NO:1 may comprise one or more additional mutations. Such one or more additional mutations may be selected from mutations at positions corresponding to the following positions of SEQ ID NO:1: 69 (H), 70 (V), 144 (Y), 570 (A), 614 (D), 681 (P), 716 (T), 982 (S), 1118 (D), 80 (D), 215 (D), 701 (A), 18 (L), 246 (R), 417 (K), 242 (L), 243 (A), 244 (L), 453 (Y), 692 (I), 1147 (S), 1229 (M), 20 (T), 26 (P), 138 (D), 190 (R), 417 (K), 655 (H), 1027 (T), and 1176 (V). In one embodiment, the amino acid corresponding to position 69 (H) of SEQ ID NO:1 is deleted. In one embodiment, the amino acid corresponding to position 70 (V) of SEQ ID NO:1 is deleted. In one embodiment, the amino acid corresponding to position 144 (Y) of SEQ ID NO:1 is deleted. In one embodiment, the amino acid corresponding to position 570 (A) of SEQ ID NO:1 is D. In one embodiment, the amino acid corresponding to position 614 (D) of SEQ ID NO:1 is G. In one embodiment, the amino acid corresponding to position 681 (P) of SEQ ID NO:1 is H. In one embodiment, the amino acid corresponding to position 716 (T) of SEQ ID NO:1 is I. In one embodiment, the amino acid corresponding to position 982 (S) of SEQ ID NO:1 is A. In one embodiment, the amino acid corresponding to position 1118 (D) of SEQ ID NO:1 is H. In one embodiment, the amino acid corresponding to position 80 (D) of SEQ ID NO:1 is A. In one embodiment, the amino acid corresponding to position 215 (D) of SEQ ID NO:1 is G. In one embodiment, the amino acid corresponding to position 701 (A) of SEQ ID NO:1 is V. In one embodiment, the amino acid corresponding to position 18 (L) of SEQ ID NO:1 is F. In one embodiment, the amino acid corresponding to position 246 (R) of SEQ ID NO:1 is I. In one embodiment, the amino acid corresponding to position 417 (K) of SEQ ID NO:1 is N. In one embodiment, the amino acid corresponding to position 242 (L) of SEQ ID NO:1 is deleted. In one embodiment, the amino acid corresponding to position 243 (A) of SEQ ID NO:1 is deleted. In one embodiment, the amino acid corresponding to position 244 (L) of SEQ ID NO:1 is deleted. In one embodiment, the amino acid corresponding to position 453 (Y) of SEQ ID NO:1 is F. In one embodiment, the amino acid corresponding to position 692 (I) of SEQ ID NO:1 is V. In one embodiment, the amino acid corresponding to position 1147 (S) of SEQ ID NO:1 is L. In one embodiment, the amino acid corresponding to position 1229 (M) of SEQ ID NO:1 is I. In one embodiment, the amino acid corresponding to position 20 (T) of SEQ ID NO:1 is N. In one embodiment, the amino acid corresponding to position 26 (P) of SEQ ID NO:1 is S. In one embodiment, the amino acid corresponding to position 138 (D) of SEQ ID NO:1 is Y. In one embodiment, the amino acid corresponding to position 190 (R) of SEQ ID NO:1 is S. In one embodiment, the amino acid corresponding to position 417 (K) of SEQ ID NO:1 is T. In one embodiment, the amino acid corresponding to position 655 (H) of SEQ ID NO:1 is Y. In one embodiment, the amino acid corresponding to position 1027 (T) of SEQ ID NO:1 is I. In one embodiment, the amino acid corresponding to position 1176 (V) of SEQ ID NO:1 is F.

In some embodiments, the methods and medicaments described herein, following administration to a subject, induce an antibody response in the subject, particularly a neutralizing antibody response, that targets 501.V2.

In some embodiments, the methods and medicaments described herein after administration to a subject induce an antibody response in the subject, particularly a neutralizing antibody response, that targets an S protein variant that includes the following mutations at positions corresponding to the following positions in SEQ ID NO:1: D80A, D215G, E484K, N501Y, and A701V, and optionally, L18F, R246I, K417N, and deletion 242-244. The S protein variant may also include a D->G mutation at a position corresponding to position 614 in SEQ ID NO:1.

In some embodiments, the methods and medicaments described herein, following administration to a subject, induce an antibody response in the subject, particularly a neutralizing antibody response, that targets "B.1.1.248".

In some embodiments, the methods and medicaments described herein after administration to a subject induce an antibody response in the subject, particularly a neutralizing antibody response, that targets an S protein variant that includes the following mutations at positions corresponding to the following positions in SEQ ID NO:1: L18F, T20N, P26S, D138Y, R190S, K417T, E484K, N501Y, H655Y, T1027I, and V1176F.

In some embodiments, the methods and medicaments described herein after administration to a subject induce an antibody response in the subject, particularly a neutralizing antibody response, that targets SARS-CoV-2 S protein variants, particularly S protein variants such as naturally occurring S protein variants that contain mutations at positions corresponding to positions 501 (N), 484 (E), and 614 (D) of SEQ ID NO:1. In one embodiment, the amino acid corresponding to position 501 (N) of SEQ ID NO:1 is Y, the amino acid corresponding to position 484 (E) of SEQ ID NO:1 is K, and the amino acid corresponding to position 614 (D) of SEQ ID NO:1 is G.

In some embodiments, an S protein variant comprising mutations at positions corresponding to positions 501 (N), 484 (E), and 614 (D) of SEQ ID NO:1 may comprise one or more additional mutations. Such one or more additional mutations may be selected from mutations at positions corresponding to the following positions of SEQ ID NO:1: 69 (H), 70 (V), 144 (Y), 570 (A), 681 (P), 716 (T), 982 (S), 1118 (D), 80 (D), 215 (D), 701 (A), 18 (L), 246 (R), 417 (K), 242 (L), 243 (A), 244 (L), 453 (Y), 692 (I), 1147 (S), 1229 (M), 20 (T), 26 (P), 138 (D), 190 (R), 417 (K), 655 (H), 1027 (T), and 1176 (V). In one embodiment, the amino acid corresponding to position 69 (H) of SEQ ID NO:1 is deleted. In one embodiment, the amino acid corresponding to position 70 (V) of SEQ ID NO:1 is deleted. In one embodiment, the amino acid corresponding to position 144 (Y) of SEQ ID NO:1 is deleted. In one embodiment, the amino acid corresponding to position 570 (A) of SEQ ID NO:1 is D. In one embodiment, the amino acid corresponding to position 681 (P) of SEQ ID NO:1 is H. In one embodiment, the amino acid corresponding to position 716 (T) of SEQ ID NO:1 is I. In one embodiment, the amino acid corresponding to position 982 (S) of SEQ ID NO:1 is A. In one embodiment, the amino acid corresponding to position 1118 (D) of SEQ ID NO:1 is H. In one embodiment, the amino acid corresponding to position 80 (D) of SEQ ID NO:1 is A. In one embodiment, the amino acid corresponding to position 215 (D) of SEQ ID NO:1 is G. In one embodiment, the amino acid corresponding to position 701 (A) of SEQ ID NO:1 is V. In one embodiment, the amino acid corresponding to position 18 (L) of SEQ ID NO:1 is F. In one embodiment, the amino acid corresponding to position 246 (R) of SEQ ID NO:1 is I. In one embodiment, the amino acid corresponding to position 417 (K) of SEQ ID NO:1 is N. In one embodiment, the amino acid corresponding to position 242 (L) of SEQ ID NO:1 is deleted. In one embodiment, the amino acid corresponding to position 243 (A) of SEQ ID NO:1 is deleted. In one embodiment, the amino acid corresponding to position 244 (L) of SEQ ID NO:1 is deleted. In one embodiment, the amino acid corresponding to position 453 (Y) of SEQ ID NO:1 is F. In one embodiment, the amino acid corresponding to position 692 (I) of SEQ ID NO:1 is V. In one embodiment, the amino acid corresponding to position 1147 (S) of SEQ ID NO:1 is L. In one embodiment, the amino acid corresponding to position 1229 (M) of SEQ ID NO:1 is I. In one embodiment, the amino acid corresponding to position 20 (T) of SEQ ID NO:1 is N. In one embodiment, the amino acid corresponding to position 26 (P) of SEQ ID NO:1 is S. In one embodiment, the amino acid corresponding to position 138 (D) of SEQ ID NO:1 is Y. In one embodiment, the amino acid corresponding to position 190 (R) of SEQ ID NO:1 is S. In one embodiment, the amino acid corresponding to position 417 (K) of SEQ ID NO:1 is T. In one embodiment, the amino acid corresponding to position 655 (H) of SEQ ID NO:1 is Y. In one embodiment, the amino acid corresponding to position 1027 (T) of SEQ ID NO:1 is I. In one embodiment, the amino acid corresponding to position 1176 (V) of SEQ ID NO:1 is F.

In some embodiments, the methods and medicaments described herein after administration to a subject induce an antibody response in the subject, particularly a neutralizing antibody response, that targets an S protein variant that includes the following mutations at positions corresponding to the following positions in SEQ ID NO:1: D80A, D215G, E484K, N501Y, A701V, and D614G, and optionally, L18F, R246I, K417N, and deletion 242-244.

In some embodiments, the methods and medicaments described herein, following administration to a subject, induce an antibody response in the subject, particularly a neutralizing antibody response, that targets SARS-CoV-2 S protein variants, particularly S protein variants, such as naturally occurring S protein variants that contain deletions at positions corresponding to positions 242 (L), 243 (A), and 244 (L) of SEQ ID NO:1.

In some embodiments, an S protein variant comprising a deletion at positions corresponding to 242 (L), 243 (A), and 244 (L) of SEQ ID NO:1 may comprise one or more additional mutations. Such one or more additional mutations may be selected from mutations at positions corresponding to the following positions of SEQ ID NO:1: 69 (H), 70 (V), 144 (Y), 501 (N), 570 (A), 614 (D), 681 (P), 716 (T), 982 (S), 1118 (D), 80 (D), 215 (D), 484 (E), 701 (A), 18 (L), 246 (R), 417 (K), 453 (Y), 692 (I), 1147 (S), 1229 (M), 20 (T), 26 (P), 138 (D), 190 (R), 417 (K), 655 (H), 1027 (T), and 1176 (V). In one embodiment, the amino acid corresponding to position 69 (H) of SEQ ID NO:1 is deleted. In one embodiment, the amino acid corresponding to position 70 (V) of SEQ ID NO:1 is deleted. In one embodiment, the amino acid corresponding to position 144 (Y) of SEQ ID NO:1 is deleted. In one embodiment, the amino acid corresponding to position 501 (N) of SEQ ID NO:1 is Y. In one embodiment, the amino acid corresponding to position 570 (A) of SEQ ID NO:1 is D. In one embodiment, the amino acid corresponding to position 614 (D) of SEQ ID NO:1 is G. In one embodiment, the amino acid corresponding to position 681 (P) of SEQ ID NO:1 is H. In one embodiment, the amino acid corresponding to position 716 (T) of SEQ ID NO:1 is I. In one embodiment, the amino acid corresponding to position 982 (S) of SEQ ID NO:1 is A. In one embodiment, the amino acid corresponding to position 1118 (D) of SEQ ID NO:1 is H. In one embodiment, the amino acid corresponding to position 80 (D) of SEQ ID NO:1 is A. In one embodiment, the amino acid corresponding to position 215 (D) of SEQ ID NO:1 is G. In one embodiment, the amino acid corresponding to position 484 (E) of SEQ ID NO:1 is K. In one embodiment, the amino acid corresponding to position 701 (A) of SEQ ID NO:1 is V. In one embodiment, the amino acid corresponding to position 18 (L) of SEQ ID NO:1 is F. In one embodiment, the amino acid corresponding to position 246 (R) of SEQ ID NO:1 is I. In one embodiment, the amino acid corresponding to position 417 (K) of SEQ ID NO:1 is N. In one embodiment, the amino acid corresponding to position 453 (Y) of SEQ ID NO:1 is F. In one embodiment, the amino acid corresponding to position 692 (I) of SEQ ID NO:1 is V. In one embodiment, the amino acid corresponding to position 1147 (S) of SEQ ID NO:1 is L. In one embodiment, the amino acid corresponding to position 1229 (M) of SEQ ID NO:1 is I. In one embodiment, the amino acid corresponding to position 20 (T) of SEQ ID NO:1 is N. In one embodiment, the amino acid corresponding to position 26 (P) of SEQ ID NO:1 is S. In one embodiment, the amino acid corresponding to position 138 (D) of SEQ ID NO:1 is Y. In one embodiment, the amino acid corresponding to position 190 (R) of SEQ ID NO:1 is S. In one embodiment, the amino acid corresponding to position 417 (K) of SEQ ID NO:1 is T. In one embodiment, the amino acid corresponding to position 655 (H) of SEQ ID NO:1 is Y. In one embodiment, the amino acid corresponding to position 1027 (T) of SEQ ID NO:1 is I. In one embodiment, the amino acid corresponding to position 1176 (V) of SEQ ID NO:1 is F.

In some embodiments, the methods and medicaments described herein, following administration to a subject, induce an antibody response in the subject, particularly a neutralizing antibody response, that targets 501.V2.

In some embodiments, the methods and medicaments described herein after administration to a subject induce an antibody response in the subject, particularly a neutralizing antibody response, that targets an S protein variant that includes the following mutations at positions corresponding to the following positions in SEQ ID NO:1: D80A, D215G, E484K, N501Y, A701V, and deletions 242-244, and optionally, L18F, R246I, and K417N. The S protein variant may also include a D->G mutation at a position corresponding to position 614 in SEQ ID NO:1.

In some embodiments, the methods and medicaments described herein after administration to a subject induce an antibody response in the subject, particularly a neutralizing antibody response, that targets a SARS-CoV-2 S protein variant, particularly an S protein variant, such as a naturally occurring S protein variant that includes a mutation at a position corresponding to position 417 (K) of SEQ ID NO:1. In one embodiment, the amino acid corresponding to position 417 (K) of SEQ ID NO:1 is N. In one embodiment, the amino acid corresponding to position 417 (K) of SEQ ID NO:1 is T.

In some embodiments, an S protein variant comprising a mutation at a position corresponding to position 417 (K) of SEQ ID NO:1 may comprise one or more additional mutations. Such one or more additional mutations may be selected from mutations at positions corresponding to the following positions of SEQ ID NO:1: 69 (H), 70 (V), 144 (Y), 501 (N), 570 (A), 614 (D), 681 (P), 716 (T), 982 (S), 1118 (D), 80 (D), 215 (D), 484 (E), 701 (A), 18 (L), 246 (R), 242 (L), 243 (A), 244 (L), 453 (Y), 692 (I), 1147 (S), 1229 (M), 20 (T), 26 (P), 138 (D), 190 (R), 655 (H), 1027 (T), and 1176 (V). In one embodiment, the amino acid corresponding to position 69 (H) of SEQ ID NO:1 is deleted. In one embodiment, the amino acid corresponding to position 70 (V) of SEQ ID NO:1 is deleted. In one embodiment, the amino acid corresponding to position 144 (Y) of SEQ ID NO:1 is deleted. In one embodiment, the amino acid corresponding to position 501 (N) of SEQ ID NO:1 is Y. In one embodiment, the amino acid corresponding to position 570 (A) of SEQ ID NO:1 is D. In one embodiment, the amino acid corresponding to position 614 (D) of SEQ ID NO:1 is G. In one embodiment, the amino acid corresponding to position 681 (P) of SEQ ID NO:1 is H. In one embodiment, the amino acid corresponding to position 716 (T) of SEQ ID NO:1 is I. In one embodiment, the amino acid corresponding to position 982 (S) of SEQ ID NO:1 is A. In one embodiment, the amino acid corresponding to position 1118 (D) of SEQ ID NO:1 is H. In one embodiment, the amino acid corresponding to position 80 (D) of SEQ ID NO:1 is A. In one embodiment, the amino acid corresponding to position 215 (D) of SEQ ID NO:1 is G. In one embodiment, the amino acid corresponding to position 484 (E) of SEQ ID NO:1 is K. In one embodiment, the amino acid corresponding to position 701 (A) of SEQ ID NO:1 is V. In one embodiment, the amino acid corresponding to position 18 (L) of SEQ ID NO:1 is F. In one embodiment, the amino acid corresponding to position 246 (R) of SEQ ID NO:1 is I. In one embodiment, the amino acid corresponding to position 242 (L) of SEQ ID NO:1 is deleted. In one embodiment, the amino acid corresponding to position 243 (A) of SEQ ID NO:1 is deleted. In one embodiment, the amino acid corresponding to position 244 (L) of SEQ ID NO:1 is deleted. In one embodiment, the amino acid corresponding to position 453 (Y) of SEQ ID NO:1 is F. In one embodiment, the amino acid corresponding to position 692 (I) of SEQ ID NO:1 is V. In one embodiment, the amino acid corresponding to position 1147 (S) of SEQ ID NO:1 is L. In one embodiment, the amino acid corresponding to position 1229 (M) of SEQ ID NO:1 is I. In one embodiment, the amino acid corresponding to position 20 (T) of SEQ ID NO:1 is N. In one embodiment, the amino acid corresponding to position 26 (P) of SEQ ID NO:1 is S. In one embodiment, the amino acid corresponding to position 138 (D) of SEQ ID NO:1 is Y. In one embodiment, the amino acid corresponding to position 190 (R) of SEQ ID NO:1 is S. In one embodiment, the amino acid corresponding to position 655 (H) of SEQ ID NO:1 is Y. In one embodiment, the amino acid corresponding to position 1027 (T) of SEQ ID NO:1 is I. In one embodiment, the amino acid corresponding to position 1176 (V) of SEQ ID NO:1 is F.

In some embodiments, the methods and medicaments described herein, following administration to a subject, induce an antibody response in the subject, particularly a neutralizing antibody response, that targets 501.V2.

In some embodiments, the methods and medicaments described herein after administration to a subject induce an antibody response in the subject, particularly a neutralizing antibody response, that targets an S protein variant that includes the following mutations at positions corresponding to the following positions in SEQ ID NO:1: D80A, D215G, E484K, N501Y, A701V, and K417N, and optionally, L18F, R246I, and deletion 242-244. The S protein variant may also include a D->G mutation at a position corresponding to position 614 in SEQ ID NO:1.

In some embodiments, the methods and medicaments described herein, following administration to a subject, induce an antibody response in the subject, particularly a neutralizing antibody response, that targets "B.1.1.248".

In some embodiments, the methods and medicaments described herein after administration to a subject induce an antibody response in the subject, particularly a neutralizing antibody response, that targets an S protein variant that includes the following mutations at positions corresponding to the following positions in SEQ ID NO:1: L18F, T20N, P26S, D138Y, R190S, K417T, E484K, N501Y, H655Y, T1027I, and V1176F.

In some embodiments, the methods and medicaments described herein after administration to a subject induce an antibody response in the subject, particularly a neutralizing antibody response, that targets SARS-CoV-2 S protein variants, particularly S protein variants such as naturally occurring S protein variants that include mutations at positions corresponding to positions 417 (K) and 484 (E) and/or 501 (N) of SEQ ID NO:1. In one embodiment, the amino acid corresponding to position 417 (K) of SEQ ID NO:1 is N, the amino acid corresponding to position 484 (E) of SEQ ID NO:1 is K, and/or the amino acid corresponding to position 501 (N) of SEQ ID NO:1 is Y. In one embodiment, the amino acid corresponding to position 417 (K) of SEQ ID NO:1 is T, the amino acid corresponding to position 484 (E) of SEQ ID NO:1 is K, and/or the amino acid corresponding to position 501 (N) of SEQ ID NO:1 is Y.

In some embodiments, an S protein variant comprising a mutation at a position corresponding to positions 417 (K) and 484 (E), and/or 501 (N) of SEQ ID NO:1 may comprise one or more additional mutations. Such one or more additional mutations may be selected from mutations at positions corresponding to the following positions of SEQ ID NO:1: 69 (H), 70 (V), 144 (Y), 570 (A), 614 (D), 681 (P), 716 (T), 982 (S), 1118 (D), 80 (D), 215 (D), 701 (A), 18 (L), 246 (R), 242 (L), 243 (A), 244 (L), 453 (Y), 692 (I), 1147 (S), 1229 (M), 20 (T), 26 (P), 138 (D), 190 (R), 655 (H), 1027 (T), and 1176 (V). In one embodiment, the amino acid corresponding to position 69 (H) of SEQ ID NO:1 is deleted. In one embodiment, the amino acid corresponding to position 70 (V) of SEQ ID NO:1 is deleted. In one embodiment, the amino acid corresponding to position 144 (Y) of SEQ ID NO:1 is deleted. In one embodiment, the amino acid corresponding to position 570 (A) of SEQ ID NO:1 is D. In one embodiment, the amino acid corresponding to position 614 (D) of SEQ ID NO:1 is G. In one embodiment, the amino acid corresponding to position 681 (P) of SEQ ID NO:1 is H. In one embodiment, the amino acid corresponding to position 716 (T) of SEQ ID NO:1 is I. In one embodiment, the amino acid corresponding to position 982 (S) of SEQ ID NO:1 is A. In one embodiment, the amino acid corresponding to position 1118 (D) of SEQ ID NO:1 is H. In one embodiment, the amino acid corresponding to position 80 (D) of SEQ ID NO:1 is A. In one embodiment, the amino acid corresponding to position 215 (D) of SEQ ID NO:1 is G. In one embodiment, the amino acid corresponding to position 701 (A) of SEQ ID NO:1 is V. In one embodiment, the amino acid corresponding to position 18 (L) of SEQ ID NO:1 is F. In one embodiment, the amino acid corresponding to position 246 (R) of SEQ ID NO:1 is I. In one embodiment, the amino acid corresponding to position 242 (L) of SEQ ID NO:1 is deleted. In one embodiment, the amino acid corresponding to position 243 (A) of SEQ ID NO:1 is deleted. In one embodiment, the amino acid corresponding to position 244 (L) of SEQ ID NO:1 is deleted. In one embodiment, the amino acid corresponding to position 453 (Y) of SEQ ID NO:1 is F. In one embodiment, the amino acid corresponding to position 692 (I) of SEQ ID NO:1 is V. In one embodiment, the amino acid corresponding to position 1147 (S) of SEQ ID NO:1 is L. In one embodiment, the amino acid corresponding to position 1229 (M) of SEQ ID NO:1 is I. In one embodiment, the amino acid corresponding to position 20 (T) of SEQ ID NO:1 is N. In one embodiment, the amino acid corresponding to position 26 (P) of SEQ ID NO:1 is S. In one embodiment, the amino acid corresponding to position 138 (D) of SEQ ID NO:1 is Y. In one embodiment, the amino acid corresponding to position 190 (R) of SEQ ID NO:1 is S. In one embodiment, the amino acid corresponding to position 655 (H) of SEQ ID NO:1 is Y. In one embodiment, the amino acid corresponding to position 1027 (T) of SEQ ID NO:1 is I. In one embodiment, the amino acid corresponding to position 1176 (V) of SEQ ID NO:1 is F.

In some embodiments, the methods and medicaments described herein, following administration to a subject, induce an antibody response in the subject, particularly a neutralizing antibody response, that targets 501.V2.

In some embodiments, the methods and medicaments described herein after administration to a subject induce an antibody response in the subject, particularly a neutralizing antibody response, that targets an S protein variant that includes the following mutations at positions corresponding to the following positions in SEQ ID NO:1: D80A, D215G, E484K, N501Y, A701V, and K417N, and optionally, L18F, R246I, and deletion 242-244. The S protein variant may also include a D->G mutation at a position corresponding to position 614 in SEQ ID NO:1.

In some embodiments, the methods and medicaments described herein, following administration to a subject, induce an antibody response in the subject, particularly a neutralizing antibody response, that targets "B.1.1.248".

In some embodiments, the methods and medicaments described herein after administration to a subject induce an antibody response in the subject, particularly a neutralizing antibody response, that targets an S protein variant that includes the following mutations at positions corresponding to the following positions in SEQ ID NO:1: L18F, T20N, P26S, D138Y, R190S, K417T, E484K, N501Y, H655Y, T1027I, and V1176F.

In some embodiments, the methods and medicaments described herein, following administration to a subject, induce an antibody response in the subject, particularly a neutralizing antibody response, that targets the Omicron (B.1.1.529) variant.

In some embodiments, the methods and medicaments described herein after administration to a subject induce an antibody response, particularly a neutralizing antibody response, in the subject that targets S protein variants that include at least 10, at least 15, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 26, at least 27, at least 28, at least 29, at least 30, at least 31, at least 32, at least 33, at least 34, at least 35, at least 36, or at least 37 of the following mutations: T547K, H655 Y, D614G, N679K, P681H, N969K, S373P, S371L, N440K, G339D, G446S, N856K, N764 K, K417N, D796Y, Q954H, T95I, A67V, L981F, S477N, G496S, T478K, Q498R, Q493R, E484A, N501Y, S375F, Y505H, V143del, H69del, V70del, N211del, L212I, ins214EPE, G142D, Y144del, Y145del, L141del, Y144F, Y145D, G142del (compared to sequence number 1).

In some embodiments, the methods and medicaments described herein, following administration to a subject, induce an antibody response, particularly a neutralizing antibody response, in the subject that targets S protein variants that include at least 10, at least 15, at least 20, at least 21, at least 22, at least 23, at least 24, or all of the following mutations: T547K, H655Y, D614G, N679K, P681H, N969K, S373P, S371L, N440K, G339D, G446S, N856K, N764K, K417N, D796Y, Q954H, T95I, A67V, L981F, S477N, G496S, T478K, Q498R, Q493R, E484A (relative to SEQ ID NO:1). The S protein variant may include at least one, at least two, at least three, at least four, at least five, or all of the following mutations: N501Y, S375F, Y505H, V143del, H69del, V70del (relative to SEQ ID NO:1); and/or at least one, at least two, at least three, at least four, at least five, or all of the following mutations: N211del, L212I, ins214EPE, G142D, Y144del, Y145del (relative to SEQ ID NO:1). In some embodiments, the S protein variant may include at least one, at least two, at least three, or all of the following mutations: L141del, Y144F, Y145D, G142del (relative to SEQ ID NO:1).

In some embodiments, the methods and medicaments described herein following administration to a subject induce an antibody response, particularly a neutralizing antibody response, in the subject that targets S protein variants that comprise at least 10, at least 15, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 26, at least 27, at least 28, at least 29, at least 30, at least 31, at least 32, or at least 33 of the following mutations:
A67V, Δ69-70, T95I, G142D, Δ143-145, Δ211, L212I, ins214EPE, G339D, S371L, S373P, S375F, K417N, N440K, G446S, S477N, T478K, E484A, Q493R, G496S, Q498R, N501Y, Y505H, T547K, D614G, H655Y, N679K, P681H, N764K, D796Y, N856K, Q954H, N969K, and L981F (relative to SEQ ID NO:1).

In some embodiments, the methods and medicaments described herein after administration to a subject induce an antibody response in the subject, particularly a neutralizing antibody response, that targets the S protein variants comprising the following mutations:
A67V, Δ69-70, T95I, G142D, Δ143-145, Δ211, L212I, ins214EPE, G339D, S371L, S373P, S375F, K417N, N440K, G446S, S477N, T478K, E484A, Q493R, G496S, Q498R, N501Y, Y505H, T547K, D614G, H655Y, N679K, P681H, N764K, D796Y, N856K, Q954H, N969K, and L981F (relative to SEQ ID NO:1).

In some embodiments, the methods and medicaments described herein after administration to a subject induce an antibody response in the subject, particularly a neutralizing antibody response, that targets the S protein variants comprising the following mutations:
A67V, Δ69-70, T95I, G142D, Δ143-145, Δ211, L212I, ins214EPE, G339D, S371L, S373P, S375F, S477N, T478K, E484A, Q493R, G496S, Q498R, N501Y, Y505H, T547K, D614G, H655Y, N679K, P681H, N764K, D796Y, N856K, Q954H, N969K, and L981F (relative to SEQ ID NO:1).

In some embodiments, the methods and medicaments described herein, following administration to a subject, induce an immune response in the subject (a cellular and/or antibody response, particularly a neutralizing antibody response) that targets the Omicron (B.1.1.529) variant.

In some embodiments, the methods and medicaments described herein, following administration to a subject, induce an immune response (a cellular and/or antibody response, particularly a neutralizing antibody response) in the subject that targets S protein variants that include at least 10, at least 15, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 26, at least 27, at least 28, at least 29, at least 30, at least 31, at least 32, at least 33, at least 34, at least 35, at least 36, or at least 37 of the following mutations: T54 7K, H655Y, D614G, N679K, P681H, N969K, S373P, S371L, N440K, G339D, G446S, N856 K, N764K, K417N, D796Y, Q954H, T95I, A67V, L981F, S477N, G496S, T478K, Q498R, Q4 93R, E484A, N501Y, S375F, Y505H, V143del, H69del, V70del, N211del, L212I, ins214EPE, G142D, Y144del, Y145del, L141del, Y144F, Y145D, G142del (compared to sequence number 1).

In some embodiments, the methods and medicaments described herein, following administration to a subject, induce an immune response (cellular and/or antibody response, particularly a neutralizing antibody response) in the subject that targets S protein variants that include at least 10, at least 15, at least 20, at least 21, at least 22, at least 23, at least 24, or all of the following mutations: T547K, H655Y, D614G, N679K, P681H, N969K, S373P, S371L, N440K, G339D, G446S, N856K, N764K, K417N, D796Y, Q954H, T95I, A67V, L981F, S477N, G496S, T478K, Q498R, Q493R, E484A (relative to SEQ ID NO: 1). The S protein variant may include at least one, at least two, at least three, at least four, at least five, or all of the following mutations: N501Y, S375F, Y505H, V143del, H69del, V70del (relative to SEQ ID NO:1); and/or at least one, at least two, at least three, at least four, at least five, or all of the following mutations: N211del, L212I, ins214EPE, G142D, Y144del, Y145del (relative to SEQ ID NO:1). In some embodiments, the S protein variant may include at least one, at least two, at least three, or all of the following mutations: L141del, Y144F, Y145D, G142del (relative to SEQ ID NO:1).

In some embodiments, the methods and medicaments described herein after administration to a subject induce an immune response in the subject (a cellular and/or antibody response, particularly a neutralizing antibody response) that targets S protein variants that comprise at least 10, at least 15, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 26, at least 27, at least 28, at least 29, at least 30, at least 31, at least 32, or at least 33 of the following mutations:
A67V, Δ69-70, T95I, G142D, Δ143-145, Δ211, L212I, ins214EPE, G339D, S371L, S373P, S375F, K417N, N440K, G446S, S477N, T478K, E484A, Q493R, G496S, Q498R, N501Y, Y505H, T547K, D614G, H655Y, N679K, P681H, N764K, D796Y, N856K, Q954H, N969K, and L981F (relative to SEQ ID NO:1).

In some embodiments, the methods and medicaments described herein following administration to a subject induce an immune response in the subject (a cellular and/or antibody response, particularly a neutralizing antibody response) that targets S protein variants that include the following mutations:
A67V, Δ69-70, T95I, G142D, Δ143-145, Δ211, L212I, ins214EPE, G339D, S371L, S373P, S375F, K417N, N440K, G446S, S477N, T478K, E484A, Q493R, G496S, Q498R, N501Y, Y505H, T547K, D614G, H655Y, N679K, P681H, N764K, D796Y, N856K, Q954H, N969K, and L981F (relative to SEQ ID NO:1).

In some embodiments, the methods and medicaments described herein following administration to a subject induce an immune response in the subject (a cellular and/or antibody response, particularly a neutralizing antibody response) that targets S protein variants that comprise the following mutations:
A67V, Δ69-70, T95I, G142D, Δ143-145, Δ211, L212I, ins214EPE, G339D, S371L, S373P, S375F, S477N, T478K, E484A, Q493R, G496S, Q498R, N501Y, Y505H, T547K, D614G, H655Y, N679K, P681H, N764K, D796Y, N856K, Q954H, N969K, and L981F (relative to SEQ ID NO:1).

As used herein, the term "amino acid corresponding to position ..." refers to an amino acid position number in the SARS-CoV-2 S protein, particularly the amino acid position number corresponding to the amino acid sequence set forth in SEQ ID NO:1. The phrase "relative to SEQ ID NO:1" is equivalent to "at a position corresponding to the following position in SEQ ID NO:1". Corresponding amino acid positions in other coronavirus S protein variants, such as SARS-CoV-2 S protein variants, can be found by alignment with the SARS-CoV-2 S protein, particularly the amino acid sequence set forth in SEQ ID NO:1. It is believed to be well known in the art how to align sequences or segments in a sequence, thereby determining corresponding positions in a sequence for amino acid positions according to the present disclosure. Standard sequence alignment programs such as ALIGN, ClustalW, or similar programs can be used, typically with default settings.

In some embodiments, the panel of different S protein variants to which the antibody response is targeted comprises at least 5, at least 10, at least 15, or even more S protein variants selected from the group consisting of the Q321S, V341I, A348T, N354D, S359N, V367F, K378S, R408I, Q409E, A435S, K458R, I472V, G476S, V483A, Y508H, H519P, and D614G variants described above. In some embodiments, the panel of different S protein variants to which the antibody response is targeted includes all S protein variants from the above Q321S, V341I, A348T, N354D, S359N, V367F, K378S, R408I, Q409E, A435S, K458R, I472V, G476S, V483A, Y508H, H519P, and D614G variants.

In some embodiments, the panel of different S protein variants to which the antibody response is targeted comprises at least 5, at least 10, at least 15, or even more S protein variants selected from the group consisting of the Q321L, V341I, A348T, N354D, S359N, V367F, K378R, R408I, Q409E, A435S, N439K, K458R, I472V, G476S, S477N, V483A, Y508H, H519P, and D614G variants described above. In some embodiments, the panel of different S protein variants to which the antibody response is targeted includes all S protein variants from the above Q321L, V341I, A348T, N354D, S359N, V367F, K378R, R408I, Q409E, A435S, N439K, K458R, I472V, G476S, S477N, V483A, Y508H, H519P, and D614G variants.

In some embodiments, the SARS-CoV-2 S protein, an immunogenic variant thereof, or an immunogenic fragment of the SARS-CoV-2 S protein or an immunogenic variant thereof, e.g., as encoded by the RNA described herein, comprises one or more of the mutations described herein for a SARS-CoV-2 S protein variant, particularly an S protein variant, such as a naturally occurring S protein variant. In one embodiment, the SARS-CoV-2 S protein, an immunogenic variant thereof, or an immunogenic fragment of the SARS-CoV-2 S protein or an immunogenic variant thereof, e.g., as encoded by the RNA described herein, comprises a mutation at a position corresponding to position 501 (N) of SEQ ID NO:1. In one embodiment, the amino acid corresponding to position 501 (N) of SEQ ID NO:1 is Y. In some embodiments, e.g., the SARS-CoV-2 S protein, an immunogenic variant thereof, or an immunogenic fragment of a SARS-CoV-2 S protein or an immunogenic variant thereof, encoded by an RNA described herein, comprises one or more mutations, e.g., all mutations, of a SARS-CoV-2 S protein of a SARS-CoV-2 variant selected from the group consisting of VOC-202012/01, 501.V2, Cluster 5, and B.1.1.248. In some embodiments, for example, the SARS-CoV-2 S protein, an immunogenic variant thereof, or an immunogenic fragment of the SARS-CoV-2 S protein or an immunogenic variant thereof encoded by the RNA described herein comprises an amino acid sequence having an alanine substitution at position 80, a glycine substitution at position 215, a lysine substitution at position 484, a tyrosine substitution at position 501, a valine substitution at position 701, a phenylalanine substitution at position 18, an isoleucine substitution at position 246, an asparagine substitution at position 417, a glycine substitution at position 614, a deletion at positions 242-244, and proline substitutions at positions 986 and 987 of SEQ ID NO:1.

In some embodiments, for example, the SARS-CoV-2 S protein, immunogenic variants thereof, or immunogenic fragments of the SARS-CoV-2 S protein or immunogenic variants thereof encoded by the RNA described herein, comprises at least 10, at least 15, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 26, at least 27, at least 28, at least 29, at least 30, at least 31, at least 32, at least 33, at least 34, at least 35, at least 36, or at least 37 of the following mutations: T547K, H655Y, D614G, N679K, P681H, N969 K, S373P, S371L, N440K, G339D, G446S, N856K, N764K, K417N, D796Y, Q954H, T95I, A67V, L981F, S477N, G496S, T478K, Q498R, Q493R, E484A, N501Y, S375F, Y505H, V143del, H69del, V70del, N211del, L212I, ins214EPE, G142D, Y144del, Y145del, L141del, Y144F, Y145D, G142del (compared to SEQ ID NO: 1). In some embodiments, for example, the SARS-CoV-2 S protein, an immunogenic variant thereof, or an immunogenic fragment of the SARS-CoV-2 S protein or an immunogenic variant thereof encoded by an RNA described herein that includes the mutation comprises K986P and V987P compared to SEQ ID NO:1.

In some embodiments, the SARS-CoV-2 S protein, an immunogenic variant thereof, or a SARS-CoV-2 S protein encoded by, for example, an RNA described herein. Immunogenic fragments of the S protein or immunogenic variants thereof include at least 10, at least 15, at least 20, at least 21, at least 22, at least 23, at least 24, or all of the following mutations: T547K, H655Y, D614G, N679K, P681H, N969K, S373P, S371L, N440K, G339D, G446S, N856K, N764K, K417N, D796Y, Q954H, T95I, A67V, L981F, S477N, G496S, T478K, Q498R, Q493R, E484A (relative to SEQ ID NO:1). The SARS-CoV-2 S protein, variant, or fragment may include at least one, at least two, at least three, at least four, at least five, or all of the following mutations: N501Y, S375F, Y505H, V143del, H69del, V70del (relative to SEQ ID NO:1), and/or at least one, at least two, at least three, at least four, at least five, or all of the following mutations: N211del, L212I, ins214EPE, G142D, Y144del, Y145del (relative to SEQ ID NO:1). In some embodiments, the SARS-CoV-2 S protein, variant, or fragment may include at least one, at least two, at least three, or all of the following mutations: L141del, Y144F, Y145D, G142del (relative to SEQ ID NO:1). In some embodiments, for example, the SARS-CoV-2 S protein, an immunogenic variant thereof, or an immunogenic fragment of the SARS-CoV-2 S protein or an immunogenic variant thereof encoded by an RNA described herein that includes the mutation comprises K986P and V987P compared to SEQ ID NO:1.

In some embodiments, for example, the SARS-CoV-2 S protein, immunogenic variants thereof, or immunogenic fragments of the SARS-CoV-2 S protein or immunogenic variants thereof encoded by the RNA described herein comprises at least 10, at least 15, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 26, at least 27, at least 28, at least 29, at least 30, at least 31, at least 32, or at least 33 of the following mutations:
A67V, Δ69-70, T95I, G142D, Δ143-145, Δ211, L212I, ins214EPE, G339D, S371L, S373P, S375F, K417N, N440K, G446S, S477N, T478K, E484A, Q493R, G496S, Q498R, N501Y, Y505H, T547K, D614G, H655Y, N679K, P681H, N764K, D796Y, N856K, Q954H, N969K, and L981F (relative to SEQ ID NO:1). In some embodiments, for example, the SARS-CoV-2 S protein, an immunogenic variant thereof, or an immunogenic fragment of the SARS-CoV-2 S protein or an immunogenic variant thereof encoded by an RNA described herein that includes such mutations, includes K986P and V987P compared to SEQ ID NO:1.

In some embodiments, for example, the SARS-CoV-2 S protein, an immunogenic variant thereof, or an immunogenic fragment of the SARS-CoV-2 S protein or an immunogenic variant thereof, encoded by the RNA described herein, comprises the following mutations:
A67V, Δ69-70, T95I, G142D, Δ143-145, Δ211, L212I, ins214EPE, G339D, S371L, S373P, S375F, K417N, N440K, G446S, S477N, T478K, E484A, Q493R, G496S, Q498R, N501Y, Y505H, T547K, D614G, H655Y, N679K, P681H, N764K, D796Y, N856K, Q954H, N969K, and L981F (relative to SEQ ID NO:1).

In some embodiments, for example, the SARS-CoV-2 S protein, an immunogenic variant thereof, or an immunogenic fragment of the SARS-CoV-2 S protein or an immunogenic variant thereof encoded by an RNA described herein that includes such mutations includes K986P and V987P compared to SEQ ID NO:1.

In some embodiments, for example, the SARS-CoV-2 S protein, an immunogenic variant thereof, or an immunogenic fragment of the SARS-CoV-2 S protein or an immunogenic variant thereof, encoded by an RNA described herein, comprises the following mutations:
A67V, Δ69-70, T95I, G142D, Δ143-145, Δ211, L212I, ins214EPE, G339D, S371L, S373P, S375F, S477N, T478K, E484A, Q493R, G496S, Q498R, N501Y, Y505H, T547K, D614G, H655Y, N679K, P681H, N764K, D796Y, N856K, Q954H, N969K, and L981F (relative to SEQ ID NO:1).

In some embodiments, for example, the SARS-CoV-2 S protein, an immunogenic variant thereof, or an immunogenic fragment of the SARS-CoV-2 S protein or an immunogenic variant thereof encoded by an RNA described herein that includes such mutations includes K986P and V987P compared to SEQ ID NO:1.

In some embodiments, for example, the SARS-CoV-2 S protein, an immunogenic variant thereof, or an immunogenic fragment of the SARS-CoV-2 S protein or an immunogenic variant thereof, encoded by an RNA described herein, comprises the following mutations:
A67V, Δ69-70, T95I, G142D, Δ143-145, Δ211, L212I, ins214EPE, G339D, S371L, S373P, S375F, K417N, N440K, G446S, S477N, T478K, E484A, Q493R, G496S, Q498R, N501Y, Y505H, T547K, D614G, H655Y, N679K, P681H, N764K, D796Y, N856K, Q954H, N969K, L981F, K986P, and V987P (relative to SEQ ID NO:1).

In some embodiments, for example, the SARS-CoV-2 S protein, an immunogenic variant thereof, or an immunogenic fragment of the SARS-CoV-2 S protein or an immunogenic variant thereof, encoded by the RNA described herein, comprises the following mutations:
A67V, Δ69-70, T95I, G142D, Δ143-145, Δ211, L212I, ins214EPE, G339D, S371L, S373P, S375F, S477N, T478K, E484A, Q493R, G496S, Q498R, N501Y, Y505H, T547K, D614G, H655Y, N679K, P681H, N764K, D796Y, N856K, Q954H, N969K, L981F, K986P, and V987P (relative to SEQ ID NO:1).

In some embodiments, for example, the SARS-CoV-2 S protein, an immunogenic variant thereof, or an immunogenic fragment of the SARS-CoV-2 S protein or an immunogenic variant thereof, encoded by the RNA described herein, comprises the following mutations:
In some embodiments, spike changes in Omicron BA.2 variants include T19I, Δ24-26, A27S, G142D, V213G, G339D, S371F, S373P, S375F, T376A, D405N, R408S, K417N, N440K, S477N, T478K, E484A, Q493R, Q498R, N501Y, Y505H, D614G, H655Y, N679K, P681H, N764K, D796Y, Q954H, N969K, K986P, and V987P compared to SEQ ID NO:1.

In some embodiments, for example, the SARS-CoV-2 S protein, an immunogenic variant thereof, or an immunogenic fragment of the SARS-CoV-2 S protein or an immunogenic variant thereof, encoded by the RNA described herein, comprises the following mutations:
T19I, Δ24-26, A27S, Δ69/70, G142D, V213G, G339D, S371F, S373P, S375F, T376A, D405N, R408S, K417N, N440K, L452R, S477N, T478K, E484A, F486V, Q498R, N501Y, Y505H, D614G, H655Y, N679K, P681H, N764K, D796Y, Q954H, N969K, K986P, and V987P (relative to SEQ ID NO:1). In some embodiments, administration of a variant-specific vaccine (e.g., a variant-specific vaccine disclosed herein) may result in an improved immune response in a patient compared to administration of a vaccine that encodes or includes the SARS-CoV-2 S protein from the Wuhan strain, or an immunogenic fragment thereof. In some embodiments, administration of a variant-specific vaccine may result in induction of a broader immune response in a subject (e.g., inducing a stronger neutralizing response against a greater number of SARS-CoV-2 variants and/or a neutralizing response that recognizes epitopes in a greater number of SARS-CoV-2 variants) compared to a patient administered a vaccine that includes or encodes the SARS-CoV-2 S protein from the Wuhan strain (or an immunogenic fragment thereof). In certain embodiments, a broader immune response may be induced when a variant-specific vaccine is administered in combination with a vaccine that includes or encodes a SARS-CoV-2 S protein from a different variant or the Wuhan strain (e.g., in some embodiments, a broader immune response may be induced when a variant-specific vaccine is administered in combination with a vaccine that includes or encodes a SARS-CoV-2 S protein from the Wuhan strain, or a SARS-CoV-2 S protein that includes a mutation that is characteristic of a different SARS-CoV-2 variant). For example, a broader immune response may be induced when an RNA vaccine encoding a SARS-CoV-2 S protein from the Wuhan strain is administered in combination with an RNA vaccine encoding a SARS-CoV-2 S protein with a mutation that is characteristic of the Omicron variant. In another embodiment, a broader immune response may be induced when an RNA vaccine encoding a SARS-CoV-2 S protein containing one or more mutations characteristic of the delta variant is administered in combination with an RNA vaccine encoding a SARS-CoV-2 S protein containing one or more mutations characteristic of the omicron variant. In such embodiments, a "broader" immune response may be defined as compared to patients administered a vaccine that includes or encodes a SARS-CoV-2 S protein from a single variant (e.g., an RNA vaccine encoding a SARS-CoV-2 S protein from the Wuhan strain). Vaccines that include or encode S proteins from different SARS-CoV-2 variants, or immunogenic fragments thereof, may be administered in combination by administering them at different times (e.g., administering a vaccine that encodes a SARS-CoV-2 S protein from the Wuhan strain and a vaccine that encodes a SARS-CoV-2 S protein with one or more mutations characteristic of the variant strains at different times, e.g., both are administered as part of a primary regimen or as part of a booster regimen, or one is administered as part of a primary regimen and one is administered as part of a booster regimen). In some embodiments, vaccines that include or encode S proteins from different SARS-CoV-2 variants, or immunogenic fragments thereof, may be administered in combination by administering a multivalent vaccine (e.g., a composition that includes RNA encoding a SARS-CoV-2 S protein from the Wuhan strain and RNA encoding a SARS-CoV-2 S protein with a mutation characteristic of the Omicron variant). In some embodiments, a variant-specific vaccine may induce a superior immune response (e.g., induce higher concentrations of neutralizing antibodies) against the variant that the vaccine is specifically designed to immunize against, as well as an immune response against one or more other variants. In some such embodiments, the immune response against the other variant(s) may be similar to or greater than that observed with a vaccine that encodes or includes the SARS-CoV-2 S protein from the Wuhan strain.

In some embodiments, the geometric mean ratio (GMR) or geometric mean fold increase (GMFR) of neutralizing antibodies induced by a variant-specific vaccine is at least 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, or 2.0 (e.g., 1.1-4, 1.1-3.5, 1.1-3, 1.5-3, or 1.1-1.5) times higher than that induced by a non-variant-specific vaccine (e.g., measured 1 day to 3 months after immunization, 7 days to 2 months after administration, about 7 days after administration, or about 1 month after administration).

In some embodiments, the SARS-CoV-2 S protein, immunogenic variant thereof, or immunogenic fragment of the SARS-CoV-2 S protein or immunogenic variant thereof, comprising the mutation, e.g., encoded by an RNA described herein, comprises the amino acid sequence of SEQ ID NO:49, an amino acid sequence having at least 99.5%, 99%, 98.5%, 98%, 98.5%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of SEQ ID NO:49, or an immunogenic fragment of the amino acid sequence of SEQ ID NO:49, or an amino acid sequence having at least 99.5%, 99%, 98.5%, 98%, 98.5%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of SEQ ID NO:49. In some embodiments, the SARS-CoV-2 S protein, an immunogenic variant thereof, or an immunogenic fragment of the SARS-CoV-2 S protein or an immunogenic variant thereof, including the mutation, e.g., encoded by an RNA described herein, comprises the amino acid sequence of SEQ ID NO:49.

In some embodiments, the SARS-CoV-2 S protein, immunogenic variant thereof, or immunogenic fragment of the SARS-CoV-2 S protein or immunogenic variant thereof, comprising the mutation, e.g., encoded by an RNA described herein, comprises the amino acid sequence of SEQ ID NO:52, an amino acid sequence having at least 99.5%, 99%, 98.5%, 98%, 98.5%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of SEQ ID NO:52, or an immunogenic fragment of the amino acid sequence of SEQ ID NO:52, or an amino acid sequence having at least 99.5%, 99%, 98.5%, 98%, 98.5%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of SEQ ID NO:52. In some embodiments, the SARS-CoV-2 S protein, an immunogenic variant thereof, or an immunogenic fragment of the SARS-CoV-2 S protein or an immunogenic variant thereof, including the mutation, e.g., encoded by an RNA described herein, comprises the amino acid sequence of SEQ ID NO:52.

In some embodiments, the methods and agents, e.g., mRNA compositions, described herein, following administration to a subject, induce a cell-mediated immune response (e.g., a CD4+ and/or CD8+ T cell response). In some embodiments, T cells are induced that recognize one or more epitopes (e.g., MHC class I restricted epitopes) selected from the group consisting of LPFNDGVYF, GVYFASTEK, YLQPRTFLL, QPTESIVRF, CVADYSVLY, KCYGVSPTK, NYNYLYRLF, FQPTNGVGY, IPFAMQMAY, RLQSLQTYV, GTHWFVTQR, VYDPLQPEL, QYIKWPWYI, and KWPWYIWLGF. In one embodiment, T cells that recognize the epitope YLQPRTFLL are induced. In one embodiment, T cells that recognize the epitope NYNYLYRLF are induced. In one embodiment, T cells that recognize the epitope QYIKWPWYI are induced. In one embodiment, T cells that recognize the epitope KCYGVSPTK are induced. In one embodiment, T cells that recognize the epitope RLQSLQTYV are induced. In some embodiments, the methods and agents, e.g., mRNA compositions, described herein are administered according to a regimen that achieves the induction of such T cells.

In some embodiments, the methods and agents described herein, e.g., mRNA compositions, following administration to a subject induce a cell-mediated immune response (e.g., CD4+ and/or CD8+ T cell response) that is detectable after 15 weeks, 16 weeks, 17 weeks, 18 weeks, 19 weeks, 20 weeks, 21 weeks, 22 weeks, 23 weeks, 24 weeks, or 25 weeks after administration, e.g., using two doses of RNA described herein (the second dose may be administered about 21 days after administration of the first dose). In some embodiments, the methods and agents described herein, e.g., mRNA compositions, are administered according to a regimen that achieves such induction of a cell-mediated immune response.

In one embodiment, vaccination against coronavirus as described herein, e.g., using the RNA described herein, which may be administered in two doses of 30 μg/dose, e.g., administered 21 days apart, e.g., in the amounts and regimens described herein, may be repeated after a period of time, e.g., using the same or a different vaccine as used in the first vaccination, once a diminished protection against coronavirus infection is observed. Such a period of time may be at least 6 months, 1 year, 2 years, etc. In one embodiment, the same RNA used in the first vaccination is used in the second or further vaccination, but at a lower dose or less frequent administration. For example, the first vaccination may include vaccination using a dose of about 30 μg per dose, in one embodiment at least two of such doses are administered (e.g., the second dose may be administered about 21 days after administration of the first dose), and the second or further vaccination may include vaccination using a dose of less than about 30 μg per dose, in one embodiment only one of such doses is administered. In one embodiment, a different RNA used in a first vaccination is used in a second or further vaccination, for example, BNT162b2 is used in a first vaccination and BNT162B1 or BNT162b3 is used in a second or further vaccination.

In one embodiment, the vaccination regimen includes a first vaccination using at least two doses of the RNA described herein, e.g., two doses of the RNA described herein (the second dose can be administered about 21 days after administration of the first dose), and a second vaccination using a single or multiple doses, e.g., two doses of the RNA described herein. In various embodiments, the second vaccination is administered 3-24 months, 6-18 months, 6-12 months, or 5-7 months after administration of the first vaccination, e.g., after the initial two-dose regimen. The amount of RNA used in each dose of the second vaccination can be equal to or different from the amount of RNA used in each dose of the first vaccination. In one embodiment, the amount of RNA used in each dose of the second vaccination is equal to the amount of RNA used in each dose of the first vaccination. In one embodiment, the amount of RNA used in each dose of the second vaccination and the amount of RNA used in each dose of the first vaccination is about 30 μg per dose. In one embodiment, the same RNA used in the second vaccination is used as in the first vaccination.

In one embodiment, the RNA used for the first vaccination and the second vaccination is BNT162b2.

In some embodiments, when the RNA used in the first vaccination and the second vaccination is BNT162b2, the objective is to induce an immune response that targets SARS-CoV-2 variants, including but not limited to the Omicron (B.1.1.529) variant. Thus, in some embodiments, when the RNA used in the first vaccination and the second vaccination is BNT162b2, the objective is to protect the subject from infection with SARS-CoV-2 variants, including but not limited to the Omicron (B.1.1.529) variant.

In one embodiment, a different RNA as used in the first vaccination is used in the second vaccination. In one embodiment, the RNA used in the first vaccination is BNT162b2 and the RNA used in the second vaccination is an RNA encoding a SARS-CoV-2 S protein of a SARS-CoV-2 variant strain, such as a strain discussed herein. In one embodiment, the RNA used in the first vaccination is BNT162b2 and the RNA used in the second vaccination is an RNA encoding a SARS-CoV-2 S protein of a SARS-CoV-2 variant strain that is circulating or spreading rapidly at the time of the second vaccination. In one embodiment, the RNA used for the first vaccination is BNT162b2 and the RNA used for the second vaccination is RNA encoding the SARS-CoV-2 S protein, an immunogenic variant thereof, or an immunogenic fragment of the SARS-CoV-2 S protein or an immunogenic variant thereof that includes one or more of the mutations described herein for SARS-CoV-2 S protein variants, particularly S protein variants such as naturally occurring S protein variants. In one embodiment, the RNA used for the first vaccination is BNT162b2 and the RNA used for the second vaccination is RNA encoding the SARS-CoV-2 S protein, an immunogenic variant thereof, or an immunogenic fragment of the SARS-CoV-2 S protein or an immunogenic variant thereof that includes one or more of the mutations described herein for SARS-CoV-2 S protein variants, particularly S protein variants such as naturally occurring S protein variants. The RNA encodes an immunogenic fragment of the SARS-CoV-2 S protein or an immunogenic variant thereof, including one or more mutations, for example all mutations, of the SARS-CoV-2 S protein of a SARS-CoV-2 variant selected from the group consisting of SARS-CoV-2 S protein of SARS-CoV-2 variants ...

In one embodiment, the RNA used for the first vaccination encodes a polypeptide comprising an amino acid sequence having proline residue substitutions at positions 986 and 987 of SEQ ID NO:1, and the RNA used for the second vaccination encodes a polypeptide comprising an amino acid sequence having an alanine substitution at position 80, a glycine substitution at position 215, a lysine substitution at position 484, a tyrosine substitution at position 501, a valine substitution at position 701, a phenylalanine substitution at position 18, an isoleucine substitution at position 246, an asparagine substitution at position 417, a glycine substitution at position 614, a deletion at positions 242 to 244, and proline substitutions at positions 986 and 987 of SEQ ID NO:1.

In one embodiment, the RNA used for the first vaccination encodes a polypeptide comprising an amino acid sequence with proline residue substitutions at positions 986 and 987 of SEQ ID NO:1, and the RNA used for the second vaccination is an RNA encoding a polypeptide comprising an amino acid sequence with the following mutations in SEQ ID NO:1:
A67V, Δ69-70, T95I, G142D, Δ143-145, Δ211, L212I, ins214EPE, G339D, S371L, S373P, S375F, K417N, N440K, G446S, S477N, T478K, E484A, Q493R, G496S, Q498R, N501Y, Y505H, T547K, D614G, H655Y, N679K, P681H, N764K, D796Y, N856K, Q954H, N969K, L981F, K986P, and V987P (relative to SEQ ID NO:1).

In one embodiment, the RNA used for the first vaccination encodes a polypeptide comprising an amino acid sequence with the following mutation in SEQ ID NO:1: residue substitutions at positions 986 and 987 of SEQ ID NO:1, and the RNA used for the second vaccination is an RNA encoding a polypeptide comprising an amino acid sequence with the following mutation in SEQ ID NO:1:
T19I, Δ24-26, A27S, G142D, V213G, G339D, S371F, S373P, S375F, T376A, D405N, R408S, K417N, N440K, S477N, T478K, E484A, Q493R, Q498R, N501Y, Y505H, D614G, H655Y, N679K, P681H, N764K, D796Y, Q954H, N969K, K986P, and V987P (compared to SEQ ID NO:1).

In one embodiment, the RNA used for the first vaccination encodes a polypeptide comprising an amino acid sequence with proline residue substitutions at positions 986 and 987 of SEQ ID NO:1, and the RNA used for the second vaccination is an RNA encoding a polypeptide comprising an amino acid sequence with the following mutations in SEQ ID NO:1:
T19I, Δ24-26, A27S, Δ69/70, G142D, V213G, G339D, S371F, S373P, S375F, T376A, D405N, R408S, K417N, N440K, L452R, S477N, T478K, E484A, F486V, Q498R, N501Y, Y505H, D614G, H655Y, N679K, P681H, N764K, D796Y, Q954H, N969K, K986P, and V987P (relative to SEQ ID NO:1).

In one embodiment, the RNA used for the first vaccination encodes a polypeptide comprising an amino acid sequence having the following mutation in SEQ ID NO:1:
A67V, Δ69-70, T95I, G142D, Δ143-145, Δ211, L212I, ins214EPE, G339D, S371L, S373P, S375F, K417N, N440K, G446S, S477N, T478K, E484A, Q493R, G496S, Q498R, N501Y, Y505H, T547K, D614G, H655Y, N679K, P681H, N764K, D796Y, N856K, Q954H, N969K, L981F, K986P, and V987P, the RNA used for the second vaccination encodes a polypeptide comprising an amino acid sequence with the following mutations in SEQ ID NO:1:
T19I, Δ24-26, A27S, G142D, V213G, G339D, S371F, S373P, S375F, T376A, D405N, R408S, K417N, N440K, S477N, T478K, E484A, Q493R, Q498R, N501Y, Y505H, D614G, H655Y, N679K, P681H, N764K, D796Y, Q954H, N969K, K986P, and V987P (compared to SEQ ID NO:1).

In one embodiment, the RNA used for the first vaccination encodes a polypeptide comprising an amino acid sequence having the following mutation in SEQ ID NO:1:
A67V, Δ69-70, T95I, G142D, Δ143-145, Δ211, L212I, ins214EPE, G339D, S371L, S373P, S375F, K417N, N440K, G446S, S477N, T478K, E484A, Q493R, G496S, Q498R, N501Y, Y505H, T547K, D614G, H655Y, N679K, P681H, N764K, D796Y, N856K, Q954H, N969K, L981F, K986P, and V987P, the RNA used for the second vaccination encodes a polypeptide comprising an amino acid sequence with the following mutations in SEQ ID NO:1:
T19I, Δ24-26, A27S, Δ69/70, G142D, V213G, G339D, S371F, S373P, S375F, T376A, D405N, R408S, K417N, N440K, L452R, S477N, T478K, E484A, F486V, Q498R, N501Y, Y505H, D614G, H655Y, N679K, P681H, N764K, D796Y, Q954H, N969K, K986P, and V987P (relative to SEQ ID NO:1).

In one embodiment, the RNA used for the first vaccination encodes a polypeptide comprising an amino acid sequence having the following mutation in SEQ ID NO:1:
T19I, Δ24-26, A27S, G142D, V213G, G339D, S371F, S373P, S375F, T376A, D405N, R408S, K417N, N440K, S477N, T478K, E484A, Q493R, Q498R, N501Y, Y505H, D614G, H655Y, N679K, P681H, N764K, D796Y, Q954H, N969K, K986P, and V987P (relative to SEQ ID NO:1), the RNA used for the second vaccination encodes a polypeptide comprising an amino acid sequence having the following mutations in SEQ ID NO:1:
T19I, Δ24-26, A27S, Δ69/70, G142D, V213G, G339D, S371F, S373P, S375F, T376A, D405N, R408S, K417N, N440K, L452R, S477N, T478K, E484A, F486V, Q498R, N501Y, Y505H, D614G, H655Y, N679K, P681H, N764K, D796Y, Q954H, N969K, K986P, and V987P (relative to SEQ ID NO:1).

In one embodiment, the RNA used in the first vaccination encodes a polypeptide comprising the amino acid sequence of SEQ ID NO:7, and the RNA used in the second vaccination encodes a polypeptide comprising the amino acid sequence of SEQ ID NO:49.

In one embodiment, the RNA used for the first vaccination comprises the nucleotide sequence of SEQ ID NO: 20, and the RNA used for the second vaccination comprises the nucleotide sequence of SEQ ID NO: 51.

In one embodiment, the RNA used in the first vaccination encodes a polypeptide comprising the amino acid sequence of SEQ ID NO:7, and the RNA used in the second vaccination encodes a polypeptide comprising the amino acid sequence of SEQ ID NO:55, 58, or 61.

In one embodiment, the RNA used for the first vaccination comprises the nucleotide sequence of SEQ ID NO: 20, and the RNA used for the second vaccination comprises the nucleotide sequence of SEQ ID NO: 57, 60, or 63.

In one embodiment, the RNA used in the first vaccination encodes a polypeptide comprising the amino acid sequence of SEQ ID NO:58, and the RNA used in the second vaccination encodes a polypeptide comprising the amino acid sequence of SEQ ID NO:49, 55, or 61.

In one embodiment, the RNA used for the first vaccination comprises the nucleotide sequence of SEQ ID NO: 60, and the RNA used for the second vaccination comprises the nucleotide sequence of SEQ ID NO: 51, 57, or 63.

In one embodiment, the RNA used in the first vaccination encodes a polypeptide comprising the amino acid sequence of SEQ ID NO: 49, and the RNA used in the second vaccination encodes a polypeptide comprising the amino acid sequence of SEQ ID NO: 55 or 61.

In one embodiment, the RNA used for the first vaccination comprises the nucleotide sequence of SEQ ID NO: 51, and the RNA used for the second vaccination comprises the nucleotide sequence of SEQ ID NO: 57 or 63.

In one embodiment, the RNA used in the first vaccination encodes a polypeptide comprising the amino acid sequence of SEQ ID NO:55, and the RNA used in the second vaccination encodes a polypeptide comprising the amino acid sequence of SEQ ID NO:61.

In one embodiment, the RNA used for the first vaccination comprises the nucleotide sequence of SEQ ID NO:57, and the RNA used for the second vaccination comprises the nucleotide sequence of SEQ ID NO:63.

In one embodiment, the RNA used for the first vaccination encodes a polypeptide comprising an amino acid sequence with proline residue substitutions at positions 986 and 987 of SEQ ID NO:1, and the RNA used for the second vaccination is an RNA encoding a polypeptide comprising an amino acid sequence with the following mutations in SEQ ID NO:1:
A67V, Δ69-70, T95I, G142D, Δ143-145, Δ211, L212I, ins214EPE, G339D, S371L, S373P, S375F, S477N, T478K, E484A, Q493R, G496S, Q498R, N501Y, Y505H, T547K, D614G, H655Y, N679K, P681H, N764K, D796Y, N856K, Q954H, N969K, L981F, K986P, and V987P (relative to SEQ ID NO:1). In some embodiments, the polypeptide encoded by the RNA used in the second vaccination further comprises proline residue substitutions at positions corresponding to 986 and 987 of SEQ ID NO:1.

In one embodiment, the RNA used in the first vaccination encodes a polypeptide comprising the amino acid sequence of SEQ ID NO:7, and the RNA used in the second vaccination encodes a polypeptide comprising the amino acid sequence of SEQ ID NO:52.

In one embodiment, a subject is administered two 30ug doses of a primary regimen comprising an RNA comprising a sequence encoding a polypeptide comprising the amino acid sequence of SEQ ID NO:7, and at least one 30ug dose of a booster regimen comprising an RNA comprising a sequence encoding a polypeptide comprising the amino acid sequence of SEQ ID NO:7, the booster regimen being administered at least two months (e.g., at least three months, at least four months, at least five months, or at least six months) after administration of the primary regimen, and the subject has optionally previously administered a first booster regimen comprising a 30ug dose of an RNA comprising a sequence encoding a polypeptide comprising the amino acid sequence of SEQ ID NO:7.

In one embodiment, a subject is administered two 30ug doses of a primary regimen comprising an RNA comprising a sequence encoding a polypeptide comprising the amino acid sequence of SEQ ID NO:7, and at least one 50ug dose of a booster regimen comprising an RNA comprising a sequence encoding a polypeptide comprising the amino acid sequence of SEQ ID NO:7, the booster regimen being administered at least two months (e.g., at least three months, at least four months, at least five months, or at least six months) after administration of the primary regimen, and the subject has optionally previously administered a first booster regimen comprising a 30ug dose of an RNA comprising a sequence encoding a polypeptide comprising the amino acid sequence of SEQ ID NO:7.

In one embodiment, a subject is administered two 30ug doses of a primary regimen comprising an RNA comprising a sequence encoding a polypeptide comprising the amino acid sequence of SEQ ID NO:7, and at least one 60ug dose of a booster regimen comprising an RNA comprising a sequence encoding a polypeptide comprising the amino acid sequence of SEQ ID NO:7, the booster regimen being administered at least two months (e.g., at least three months, at least four months, at least five months, or at least six months) after administration of the primary regimen, and the subject has optionally previously administered a first booster regimen comprising a 30ug dose of an RNA comprising a sequence encoding a polypeptide comprising the amino acid sequence of SEQ ID NO:7.

In one embodiment, a subject is administered two 30ug doses of a primary regimen comprising an RNA comprising a sequence encoding a polypeptide comprising the amino acid sequence of SEQ ID NO:7, and at least one 30ug dose of a booster regimen comprising an RNA comprising a sequence encoding a polypeptide comprising the amino acid sequence of SEQ ID NO:49, the booster regimen being administered at least two months (e.g., at least three months, at least four months, at least five months, or at least six months) after administration of the primary regimen, and the subject has optionally previously administered a first booster regimen comprising a 30ug dose of an RNA comprising a sequence encoding a polypeptide comprising the amino acid sequence of SEQ ID NO:7.

In one embodiment, a subject is administered at least two 30ug doses of a primary regimen comprising an RNA comprising a sequence encoding a polypeptide comprising the amino acid sequence of SEQ ID NO:7, and at least two 30ug doses of a booster regimen comprising an RNA comprising a sequence encoding a polypeptide comprising the amino acid sequence of SEQ ID NO:49, in some embodiments, the two doses of the booster regimen are administered at least two months apart (e.g., at least three months, at least four months, at least five months, or at least six months apart). In some embodiments, such a subject may have previously been administered a 30ug dose of an RNA comprising a sequence encoding a polypeptide comprising the amino acid sequence of SEQ ID NO:7 as a booster dose.

In one embodiment, a subject is administered two 30ug doses of a primary regimen comprising RNA comprising a sequence encoding a polypeptide comprising the amino acid sequence of SEQ ID NO:7, and at least one 50ug dose of a booster regimen comprising RNA comprising a nucleotide sequence encoding the amino acid sequence of SEQ ID NO:49, the booster regimen being administered at least two months (e.g., at least three months, at least four months, at least five months, or at least six months) after administration of the primary regimen, and the subject has optionally previously administered a first booster regimen comprising a 30ug dose of RNA comprising a sequence encoding a polypeptide comprising the amino acid sequence of SEQ ID NO:7.

In one embodiment, a subject is administered two 30ug doses of a primary regimen comprising RNA comprising a sequence encoding a polypeptide comprising the amino acid sequence of SEQ ID NO:7, and at least one 60ug dose of a booster regimen comprising RNA comprising a nucleotide sequence encoding the amino acid sequence of SEQ ID NO:49, the booster regimen being administered at least two months (e.g., at least three months, at least four months, at least five months, or at least six months) after administration of the primary regimen, and the subject has optionally previously administered a first booster regimen comprising a 30ug dose of RNA comprising a sequence encoding a polypeptide comprising the amino acid sequence of SEQ ID NO:7.

In some embodiments, a subject is administered a primary regimen comprising two doses of 30ug of RNA (e.g., administered about 21 days after each other), each 30ug of RNA comprising 15ug of RNA comprising a nucleotide sequence encoding the amino acid sequence of SEQ ID NO:7 and 15ug of RNA comprising a nucleotide sequence encoding the amino acid sequence of SEQ ID NO:49. In some embodiments, such a primary regimen is administered to an unvaccinated subject.

In some embodiments, a subject is administered a primary regimen comprising two doses of 30 ug of RNA (e.g., administered about 21 days after each other), each 30 ug dose of RNA comprising a nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 49. In some embodiments, such a primary regimen is administered to an unvaccinated subject.

In one embodiment, the subject is administered a primary regimen comprising two 30ug doses of RNA comprising a sequence encoding a polypeptide comprising the amino acid sequence of SEQ ID NO:7, and at least one dose of a booster regimen comprising 30ug of RNA, the 30ug RNA comprising 15ug of RNA comprising a nucleotide sequence encoding the amino acid sequence of SEQ ID NO:7, and 15ug of RNA comprising a nucleotide sequence encoding the amino acid sequence of SEQ ID NO:49, the two RNAs optionally being administered in the same composition, the booster regimen being administered at least two months (e.g., at least three months, at least four months, at least five months, or at least six months) after administration of the primary regimen, and the subject has optionally previously administered a first booster regimen comprising a 30ug dose of RNA comprising a sequence encoding a polypeptide comprising the amino acid sequence of SEQ ID NO:7.

In one embodiment, the subject is administered a primary regimen comprising two 30ug doses of RNA comprising a sequence encoding a polypeptide comprising the amino acid sequence of SEQ ID NO:7, and at least one dose of a booster regimen comprising 50ug of RNA, the 50ug RNA comprising 25ug of RNA comprising a nucleotide sequence encoding the amino acid sequence of SEQ ID NO:7, and 25ug of RNA comprising a nucleotide sequence encoding the amino acid sequence of SEQ ID NO:49, the two RNAs optionally being administered in the same composition (e.g., a formulation comprising both RNAs), the booster regimen being administered at least two months (e.g., at least three months, at least four months, at least five months, or at least six months) after administration of the primary regimen, and the subject has optionally previously administered a first booster regimen comprising a 30ug dose of RNA comprising a sequence encoding a polypeptide comprising the amino acid sequence of SEQ ID NO:7.

In one embodiment, the subject is administered a primary regimen comprising two 30ug doses of RNA comprising a sequence encoding a polypeptide comprising the amino acid sequence of SEQ ID NO:7, and at least one dose of a booster regimen comprising 60ug of RNA, the 60ug RNA comprising 30ug of RNA comprising a nucleotide sequence encoding the amino acid sequence of SEQ ID NO:7, and 30ug of RNA comprising a nucleotide sequence encoding the amino acid sequence of SEQ ID NO:49, the two RNAs optionally being administered in the same composition, the booster regimen being administered at least two months (e.g., at least three months, at least four months, at least five months, or at least six months) after administration of the primary regimen, and the subject has optionally previously administered a first booster regimen comprising a 30ug dose of RNA comprising a sequence encoding a polypeptide comprising the amino acid sequence of SEQ ID NO:7.

In one embodiment, the RNA used for the first vaccination comprises the nucleotide sequence of SEQ ID NO: 20, and the RNA used for the second vaccination comprises the nucleotide sequence of SEQ ID NO: 54.

In one embodiment, a subject is administered two 30ug doses of a primary regimen comprising RNA comprising the nucleotide sequence of SEQ ID NO:20 and at least one 30ug dose of a booster regimen comprising RNA comprising the nucleotide sequence of SEQ ID NO:20, where the booster regimen is administered at least two months (e.g., at least three months, at least four months, at least five months, or at least six months) after administration of the primary regimen, and the subject has optionally previously administered a first booster regimen comprising a 30ug dose of RNA comprising the nucleotide sequence of SEQ ID NO:20.

In one embodiment, a subject is administered a primary regimen comprising two 30ug doses of RNA comprising the nucleotide sequence of SEQ ID NO:20 and at least one dose of 50ug of a booster regimen comprising RNA comprising the nucleotide sequence of SEQ ID NO:20, where the booster regimen is administered at least two months (e.g., at least three months, at least four months, at least five months, or at least six months) after administration of the primary regimen, and the subject has optionally previously administered a first booster regimen comprising 30ug of RNA comprising the nucleotide sequence of SEQ ID NO:20.

In one embodiment, a subject is administered two 30ug doses of a primary regimen comprising RNA comprising the nucleotide sequence of SEQ ID NO:20 and at least one 60ug dose of a booster regimen comprising RNA comprising the nucleotide sequence of SEQ ID NO:20, the booster regimen being administered at least two months (e.g., at least three months, at least four months, at least five months, or at least six months) after administration of the primary regimen, and the subject has optionally previously been administered a first booster regimen comprising 30ug of RNA comprising the nucleotide sequence of SEQ ID NO:20.

In one embodiment, a subject is administered a primary regimen comprising two 30ug doses of RNA comprising the nucleotide sequence of SEQ ID NO:20 and at least one 30ug dose of a booster regimen comprising RNA comprising the nucleotide sequence of SEQ ID NO:51, the booster regimen being administered at least two months (e.g., at least three months, at least four months, at least five months, or at least six months) after administration of the primary regimen, and the subject has optionally previously administered a first booster regimen comprising a 30ug dose of RNA comprising the nucleotide sequence of SEQ ID NO:20.

In one embodiment, a subject is administered a primary regimen comprising two 30ug doses of RNA comprising the nucleotide sequence of SEQ ID NO:20 and at least one 50ug dose of a booster regimen comprising RNA comprising the nucleotide sequence of SEQ ID NO:51, the booster regimen being administered at least two months (e.g., at least three months, at least four months, at least five months, or at least six months) after administration of the primary regimen, and the subject has optionally previously administered a first booster regimen comprising a 30ug dose of RNA comprising the nucleotide sequence of SEQ ID NO:20.

In one embodiment, a subject is administered a primary regimen comprising two 30ug doses of RNA comprising the nucleotide sequence of SEQ ID NO:20 and at least one 60ug dose of a booster regimen comprising RNA comprising the nucleotide sequence of SEQ ID NO:51, the booster regimen being administered at least two months (e.g., at least three months, at least four months, at least five months, or at least six months) after administration of the primary regimen, and the subject has optionally previously been administered a first booster regimen comprising a 30ug dose of RNA comprising the nucleotide sequence of SEQ ID NO:20.

In one embodiment, the subject is administered two 30ug doses of a primary regimen comprising RNA comprising the nucleotide sequence of SEQ ID NO:20 and at least one 30ug dose of a booster regimen comprising RNA, the 30ug RNA comprising 15ug of RNA comprising the nucleotide sequence of SEQ ID NO:20 and 15ug of RNA comprising the nucleotide sequence of SEQ ID NO:51, the two RNAs optionally being administered in the same composition, the booster regimen being administered at least two months (e.g., at least three months, at least four months, at least five months, or at least six months) after administration of the primary regimen, and the subject optionally having previously administered a first booster regimen comprising a 30ug dose of RNA comprising the nucleotide sequence of SEQ ID NO:20.

In one embodiment, the subject is administered a primary regimen comprising two 30ug doses of RNA comprising the nucleotide sequence of SEQ ID NO:20 and a booster regimen comprising at least one dose comprising 50ug of RNA, the 50ug RNA comprising 25ug of RNA comprising the nucleotide sequence of SEQ ID NO:20 and 25ug of RNA comprising the nucleotide sequence of SEQ ID NO:51, the two RNAs optionally being administered in the same composition, the booster regimen being administered at least two months (e.g., at least three months, at least four months, at least five months, or at least six months) after administration of the primary regimen, and the subject has optionally previously administered a first booster regimen comprising a 30ug dose of RNA comprising the nucleotide sequence of SEQ ID NO:20.

In one embodiment, the subject is administered a primary regimen comprising two 30ug doses of RNA comprising the nucleotide sequence of SEQ ID NO:20 and a booster regimen comprising at least one dose comprising 60ug of RNA, the 60ug of RNA comprising 30ug of RNA comprising the nucleotide sequence of SEQ ID NO:20 and 30ug of RNA comprising the nucleotide sequence of SEQ ID NO:51, the two RNAs optionally being administered in the same composition, the booster regimen being administered at least two months (e.g., at least three months, at least four months, at least five months, or at least six months) after administration of the primary regimen, and the subject has optionally previously administered a first booster regimen comprising a 30ug dose of RNA comprising the nucleotide sequence of SEQ ID NO:20.

In one embodiment, a subject is administered a primary regimen comprising two 30ug doses of RNA comprising the nucleotide sequence of SEQ ID NO:20 and at least one 30ug dose of a booster regimen comprising RNA comprising the nucleotide sequence of SEQ ID NO:57, the booster regimen being administered at least two months (e.g., at least three months, at least four months, at least five months, or at least six months) after administration of the primary regimen, and the subject has optionally previously administered a first booster regimen comprising a 30ug dose of RNA comprising the nucleotide sequence of SEQ ID NO:20.

In one embodiment, a subject is administered a primary regimen comprising two 30ug doses of RNA comprising the nucleotide sequence of SEQ ID NO:20 and at least one 50ug dose of a booster regimen comprising RNA comprising the nucleotide sequence of SEQ ID NO:57, the booster regimen being administered at least two months (e.g., at least three months, at least four months, at least five months, or at least six months) after administration of the primary regimen, and the subject has optionally previously administered a first booster regimen comprising a 30ug dose of RNA comprising the nucleotide sequence of SEQ ID NO:20.

In one embodiment, a subject is administered a primary regimen comprising two 30ug doses of RNA comprising the nucleotide sequence of SEQ ID NO:20 and at least one 60ug dose of a booster regimen comprising RNA comprising the nucleotide sequence of SEQ ID NO:57, the booster regimen being administered at least two months (e.g., at least three months, at least four months, at least five months, or at least six months) after administration of the primary regimen, and the subject has optionally previously administered a first booster regimen comprising a 30ug dose of RNA comprising the nucleotide sequence of SEQ ID NO:20.

In one embodiment, a subject is administered a primary regimen comprising two 30ug doses of RNA comprising the nucleotide sequence of SEQ ID NO:20 and at least one 30ug dose of a booster regimen comprising RNA comprising the nucleotide sequence of SEQ ID NO:60, where the booster regimen is administered at least two months (e.g., at least three months, at least four months, at least five months, or at least six months) after administration of the primary regimen, and the subject has optionally previously administered a first booster regimen comprising a 30ug dose of RNA comprising the nucleotide sequence of SEQ ID NO:20.

In one embodiment, a subject is administered a primary regimen comprising two 30ug doses of RNA comprising the nucleotide sequence of SEQ ID NO:20 and at least one 50ug dose of a booster regimen comprising RNA comprising the nucleotide sequence of SEQ ID NO:60, where the booster regimen is administered at least two months (e.g., at least three months, at least four months, at least five months, or at least six months) after administration of the primary regimen, and the subject has optionally previously administered a first booster regimen comprising a 30ug dose of RNA comprising the nucleotide sequence of SEQ ID NO:20.

In one embodiment, a subject is administered a primary regimen comprising two 30ug doses of RNA comprising the nucleotide sequence of SEQ ID NO:20 and at least one 60ug dose of a booster regimen comprising RNA comprising the nucleotide sequence of SEQ ID NO:60, where the booster regimen is administered at least two months (e.g., at least three months, at least four months, at least five months, or at least six months) after administration of the primary regimen, and the subject has optionally previously administered a first booster regimen comprising a 30ug dose of RNA comprising the nucleotide sequence of SEQ ID NO:20.

In one embodiment, a subject is administered a primary regimen comprising two 30ug doses of RNA comprising the nucleotide sequence of SEQ ID NO:20 and at least one 30ug dose of a booster regimen comprising RNA comprising the nucleotide sequence of SEQ ID NO:63, the booster regimen being administered at least two months (e.g., at least three months, at least four months, at least five months, or at least six months) after administration of the primary regimen, and the subject has optionally previously administered a first booster regimen comprising a 30ug dose of RNA comprising the nucleotide sequence of SEQ ID NO:20.

In one embodiment, a subject is administered a primary regimen comprising two 30ug doses of RNA comprising the nucleotide sequence of SEQ ID NO:20 and at least one 50ug dose of a booster regimen comprising RNA comprising the nucleotide sequence of SEQ ID NO:63, the booster regimen being administered at least two months (e.g., at least three months, at least four months, at least five months, or at least six months) after administration of the primary regimen, and the subject has optionally previously administered a first booster regimen comprising a 30ug dose of RNA comprising the nucleotide sequence of SEQ ID NO:20.

In one embodiment, a subject is administered a primary regimen comprising two 30ug doses of RNA comprising the nucleotide sequence of SEQ ID NO:63 and at least one 60ug dose of a booster regimen comprising RNA comprising the nucleotide sequence of SEQ ID NO:57, the booster regimen being administered at least two months (e.g., at least three months, at least four months, at least five months, or at least six months) after administration of the primary regimen, and the subject has optionally previously administered a first booster regimen comprising a 30ug dose of RNA comprising the nucleotide sequence of SEQ ID NO:20.

In one embodiment, the vaccination regimen includes a first vaccination with two doses of RNA encoding a polypeptide comprising an amino acid sequence with proline residue substitutions at positions 986 and 987 of SEQ ID NO:1 administered about 21 days apart, and a second vaccination with a single or multiple doses of RNA encoding a polypeptide comprising an amino acid sequence with proline residue substitutions at positions 986 and 987 of SEQ ID NO:1 administered about 4-12 months, 5-12 months, or 6-12 months after the first two-dose regimen. In one embodiment, each RNA dose includes 30 μg of RNA. In this embodiment, the objective in one embodiment is to induce an immune response that targets SARS-CoV-2 variants, including but not limited to the Omicron (B.1.1.529) variant. Thus, in this embodiment, the objective in one embodiment is to protect a subject from infection with SARS-CoV-2 variants, including but not limited to the Omicron (B.1.1.529) variant.

In one embodiment, the vaccination regimen includes a first vaccination with two doses of RNA encoding a polypeptide comprising an amino acid sequence having proline residue substitutions at positions 986 and 987 of SEQ ID NO:1 administered at an interval of about 21 days, and a second vaccination with a single or multiple doses of RNA encoding a polypeptide comprising an amino acid sequence having an alanine substitution at position 80, a glycine substitution at position 215, a lysine substitution at position 484, a tyrosine substitution at position 501, a valine substitution at position 701, a phenylalanine substitution at position 18, an isoleucine substitution at position 246, an asparagine substitution at position 417, a glycine substitution at position 614, a deletion at positions 242-244, and proline substitutions at positions 986 and 987 of SEQ ID NO:1 administered about 6-12 months after the administration of the first vaccination, i.e., the initial two-dose regimen. In one embodiment, each RNA dose includes 30 μg of RNA.

In one embodiment, the vaccination regimen comprises a first vaccination with two doses of RNA encoding a polypeptide comprising an amino acid sequence with proline residue substitutions at positions 986 and 987 of SEQ ID NO:1, administered about 21 days apart, and a second vaccination with a single or multiple doses of RNA encoding a polypeptide comprising an amino acid sequence with the following mutations of SEQ ID NO:1, administered, for example, about 6-12 months after the administration of the first vaccination, i.e., the initial two-dose regimen:
A67V, Δ69-70, T95I, G142D, Δ143-145, Δ211, L212I, ins214EPE, G339D, S371L, S373P, S375F, K417N, N440K, G446S, S477N, T478K, E484A, Q493R, G496S, Q498R, N501Y, Y505H, T547K, D614G, H655Y, N679K, P681H, N764K, D796Y, N856K, Q954H, N969K, L981F, K986P, and V987P (relative to SEQ ID NO: 1). In one embodiment, each RNA dose comprises 30 μg of RNA.

In one embodiment, the vaccination regimen comprises a first vaccination with two doses of RNA encoding a polypeptide comprising an amino acid sequence with proline residue substitutions at positions 986 and 987 of SEQ ID NO:1, administered about 21 days apart, and a second vaccination with a single or multiple doses of RNA encoding a polypeptide comprising an amino acid sequence with the following mutations of SEQ ID NO:1, administered, for example, about 6-12 months after the administration of the first vaccination, i.e., the initial two-dose regimen:
A67V, Δ69-70, T95I, G142D, Δ143-145, Δ211, L212I, ins214EPE, G339D, S371L, S373P, S375F, S477N, T478K, E484A, Q493R, G496S, Q498R, N501Y, Y505H, T547K, D614G, H655Y, N679K, P681H, N764K, D796Y, N856K, Q954H, N969K, L981F, K986P, and V987P (relative to SEQ ID NO: 1). In one embodiment, each RNA dose comprises 30 μg of RNA. In some embodiments, the encoded polypeptide further comprises proline residue substitutions at positions corresponding to 986 and 987 of SEQ ID NO:1.

In one embodiment, the vaccination regimen comprises a first vaccination with at least two doses of RNA encoding a polypeptide comprising an amino acid sequence with proline residue substitutions at positions 986 and 987 of SEQ ID NO:1, administered about 21 days apart, and a second vaccination with a single or multiple doses of RNA encoding a polypeptide comprising an amino acid sequence with the following mutations of SEQ ID NO:1, administered, for example, about 6-12 months after the administration of the first vaccination, i.e., the initial two-dose regimen:
T19I, Δ24-26, A27S, G142D, V213G, G339D, S371F, S373P, S375F, T376A, D405N, R408S, K417N, N440K, S477N, T478K, E484A, Q493R, Q498R, N501Y, Y505H, D614G, H655Y, N679K, P681H, N764K, D796Y, Q954H, N969K, K986P, and V987P (relative to SEQ ID NO: 1). In one embodiment, the or at least one RNA dose comprises 30 μg of RNA.

In one embodiment, the vaccination regimen comprises a first vaccination with at least two doses of RNA encoding a polypeptide comprising an amino acid sequence with proline residue substitutions at positions 986 and 987 of SEQ ID NO:1, administered about 21 days apart, and a second vaccination with a single or multiple doses of RNA encoding a polypeptide comprising an amino acid sequence with the following mutations of SEQ ID NO:1, administered, for example, about 6-12 months after the administration of the first vaccination, i.e., the initial two-dose regimen:
T19I, Δ24-26, A27S, Δ69/70, G142D, V213G, G339D, S371F, S373P, S375F, T376A, D405N, R408S, K417N, N440K, L452R, S477N, T478K, E484A, F486V, Q498R, N501Y, Y505H, D614G, H655Y, N679K, P681H, N764K, D796Y, Q954H, N969K, K986P, and V987P (relative to SEQ ID NO: 1). In one embodiment, the or at least one RNA dose comprises 30 μg of RNA.

In one embodiment, the vaccination regimen comprises a first vaccination with at least two doses of RNA encoding a polypeptide comprising an amino acid sequence having the following mutation in SEQ ID NO:1:
A67V, Δ69-70, T95I, G142D, Δ143-145, Δ211, L212I, ins214EPE, G339D, S371L, S373P, S375F, K417N, N440K, G446S, S 477N, T478K, E484A, Q493R, G496S, Q498R, N501Y, Y505H, T547K, D614G, H655Y, N679K, P681H, N764K, D796Y, N856K, Q9 54H, N969K, L981F, K986P, and V987P, the two doses of the first vaccination are administered about 21 days apart, and the vaccination regimen includes, for example, a second vaccination with a single or multiple doses of RNA encoding a polypeptide comprising an amino acid sequence having the following mutation in SEQ ID NO:1, administered about 6-12 months after the administration of the first vaccination, i.e., the initial two-dose regimen:
T19I, Δ24-26, A27S, G142D, V213G, G339D, S371F, S373P, S375F, T376A, D405N, R408S, K417N, N440K, S477N, T478K, E484A, Q493R, Q498R, N501Y, Y505H, D614G, H655Y, N679K, P681H, N764K, D796Y, Q954H, N969K, K986P, and V987P (relative to SEQ ID NO: 1). In one embodiment, the or at least one RNA dose comprises 30 μg of RNA.

In one embodiment, the vaccination regimen comprises a first vaccination with at least two doses of RNA encoding a polypeptide comprising an amino acid sequence having the following mutation in SEQ ID NO:1:
A67V, Δ69-70, T95I, G142D, Δ143-145, Δ211, L212I, ins214EPE, G339D, S371L, S373P, S375F, K417N, N440K, G446S, S 477N, T478K, E484A, Q493R, G496S, Q498R, N501Y, Y505H, T547K, D614G, H655Y, N679K, P681H, N764K, D796Y, N856K, Q9 54H, N969K, L981F, K986P, and V987P, the two doses of the first vaccination are administered about 21 days apart, and the vaccination regimen includes, for example, a second vaccination with a single or multiple doses of RNA encoding a polypeptide comprising an amino acid sequence having the following mutation in SEQ ID NO:1, administered about 6-12 months after the administration of the first vaccination, i.e., the initial two-dose regimen:
T19I, Δ24-26, A27S, Δ69/70, G142D, V213G, G339D, S371F, S373P, S375F, T376A, D405N, R408S, K417N, N440K, L452R, S477N, T478K, E484A, F486V, Q498R, N501Y, Y505H, D614G, H655Y, N679K, P681H, N764K, D796Y, Q954H, N969K, K986P, and V987P (relative to SEQ ID NO: 1). In one embodiment, the or at least one RNA dose comprises 30 μg of RNA.

In one embodiment, the vaccination regimen comprises a first vaccination with at least two doses of RNA encoding a polypeptide comprising an amino acid sequence having the following mutation in SEQ ID NO:1:
T19I, Δ24-26, A27S, G142D, V213G, G339D, S371F, S373P, S375F, T376A, D405N, R408S, K417N, N440K, S477N, T478K, E484A, Q493R, Q498R, N501Y, Y505H, D614G, H655Y, N679K, P681H, N764K, D796Y, Q954H, N969K, K986P, and V987P (relative to SEQ ID NO: 1), the two doses of the first vaccination are administered about 21 days apart, and the vaccination regimen includes, for example, a second vaccination with a single or multiple doses of RNA encoding a polypeptide comprising an amino acid sequence having the following mutation in SEQ ID NO: 1, administered about 6-12 months after the administration of the first vaccination, i.e., the initial two-dose regimen:
T19I, Δ24-26, A27S, Δ69/70, G142D, V213G, G339D, S371F, S373P, S375F, T376A, D405N, R408S, K417N, N440K, L452R, S477N, T478K, E484A, F486V, Q498R, N501Y, Y505H, D614G, H655Y, N679K, P681H, N764K, D796Y, Q954H, N969K, K986P, and V987P (relative to SEQ ID NO: 1). In one embodiment, the or at least one RNA dose comprises 30 μg of RNA.

In one embodiment, the second vaccination provides a boost in the immune response.

In one embodiment, the RNA described herein is co-administered with other vaccines. In some embodiments, the RNA described herein is co-administered with a composition comprising one or more T cell epitopes of SARS-CoV-2 or RNA encoding same. In some embodiments, the RNA described herein is co-administered with one or more T cell epitopes derived from the M protein, N protein, and/or ORF1ab protein of SARS-CoV-2, or RNA encoding same, e.g., a composition disclosed in WO2021/188969, the contents of which are incorporated herein by reference in their entirety. In some embodiments, an RNA described herein (e.g., administered with an RNA encoding a SARS-CoV-2 S protein containing a mutation characteristic of the BA.1, BA.2, or BA.4/5 omicron variants, optionally with an RNA encoding a SARS-CoV-2 S protein of the Wuhan variant) may be co-administered with a T-string construct described in WO2021/188969 (e.g., an RNA encoding SEQ ID NO: RS C7p2full in WO2021/188969). In some embodiments, an RNA described herein and a T-string construct described in WO2021/188969 are administered in a combination comprising up to about 100 ug of RNA combined. In some embodiments, the subject is administered at least two doses, each dose comprising one or more RNAs described herein (e.g., in some embodiments, 15ug each) and a T-string construct (e.g., an RNA encoding SEQ ID NO: RS C7p2full of WO2021/188969), e.g., two or more RNAs described herein, and an RNA encoding SEQ ID NO: RS C7p2full in a total amount of RNA of about 100ug or less. In some embodiments, each dose comprises 5ug of a T-string construct and 3ug of an RNA described herein (e.g., 3ug of a bivalent RNA vaccine described herein). In some embodiments, each dose comprises 5ug of a T-string construct and 6ug of an RNA described herein (e.g., 3ug of a bivalent RNA vaccine described herein). In some embodiments, each dose comprises 5ug of a T-string construct and 10ug of an RNA described herein (e.g., 10ug of a bivalent RNA vaccine described herein). In some embodiments, each dose contains 5 ug of the T-string construct and 30 ug of an RNA described herein (e.g., 30 ug of a bivalent RNA vaccine described herein).

In some embodiments, each dose comprises 10ug of the T-string construct and 3ug of the RNA described herein (e.g., 3ug of a bivalent RNA vaccine described herein). In some embodiments, each dose comprises 10ug of the T-string construct and 6ug of the RNA described herein (e.g., 3ug of a bivalent RNA vaccine described herein). In some embodiments, each dose comprises 10ug of the T-string construct and 10ug of the RNA described herein (e.g., 10ug of a bivalent RNA vaccine described herein). In some embodiments, each dose comprises 10ug of the T-string construct and 30ug of the RNA described herein (e.g., 30ug of a bivalent RNA vaccine described herein).

In some embodiments, each dose comprises 15ug of the T-string construct and 3ug of the RNA described herein (e.g., 3ug of a bivalent RNA vaccine described herein). In some embodiments, each dose comprises 15ug of the T-string construct and 6ug of the RNA described herein (e.g., 3ug of a bivalent RNA vaccine described herein). In some embodiments, each dose comprises 10ug of the T-string construct and 15ug of the RNA described herein (e.g., 10ug of a bivalent RNA vaccine described herein). In some embodiments, each dose comprises 15ug of the T-string construct and 30ug of the RNA described herein (e.g., 30ug of a bivalent RNA vaccine described herein).

In some embodiments, the two doses are administered, for example, at least 4 weeks or more apart (including, for example, at least 5 weeks, at least 6 weeks, at least 7 weeks, at least 8 weeks, at least 9 weeks, at least 10 weeks, at least 11 weeks, or at least 12 weeks, or more) from each other. In some embodiments, the subject is administered at least three doses, each dose comprising one or more RNAs described herein (e.g., 30 ug each) and a T-string construct (e.g., an RNA encoding SEQ ID NO: RS C7p2full of WO2021/188969). In some embodiments, each of the three doses comprises up to about 100 ug of RNA in total. In some embodiments, the first and second doses, and the second and third doses, each independently, are administered at least 4 weeks or more apart (including, for example, at least 5 weeks, at least 6 weeks, at least 7 weeks, at least 8 weeks, at least 9 weeks, at least 10 weeks, at least 11 weeks, or at least 12 weeks, or more) from each other.

In some embodiments, the RNA and T-string constructs described herein may be co-administered as separate formulations (e.g., by IM injection at separate injection sites). In some embodiments, the RNA and T-string constructs described herein may be co-administered as a co-formulation (e.g., a co-formulation in which each RNA is encapsulated in a separate LNP, a co-formulation in which the RNA described herein is encapsulated in a first LNP and the T-string construct is encapsulated in a second LNP, or a co-formulation in which all RNAs are encapsulated together in the same LNP (e.g., by mixing all RNAs prior to LNP formulation)).

In some embodiments, the RNA compositions described herein are co-administered with one or more vaccines against a non-SARS-CoV-2 disease. In some embodiments, the RNA compositions described herein are co-administered with one or more vaccines against a non-SARS-CoV-2 viral disease. In some embodiments, the RNA compositions described herein are co-administered with one or more vaccines against a non-SARS-CoV-2 respiratory disease. In some embodiments, the non-SARS-CoV-2 respiratory disease is a non-SARS-CoV-2 coronavirus, influenza virus, Pneumoviridae virus, or Paramyxoviridae virus. In some embodiments, the Pneumoviridae virus is a respiratory syncytial virus or a metapneumovirus. In some embodiments, the metapneumovirus is a human metapneumovirus (hMPV). In some embodiments, the Paramyxoviridae virus is a parainfluenza virus or a henipavirus. In some embodiments, the parainfluenza virus is PIV3. In some embodiments, the non-SARS-CoV-2 coronavirus is a betacoronavirus (e.g., SARS-CoV-1). In some embodiments, the non-SARS-CoV-2 coronavirus is a Merbecovirus (e.g., MERS-CoV virus).

In some embodiments, the RNA compositions described herein are co-administered with an RSV vaccine (e.g., an RSV A or RSV B vaccine). In some embodiments, the RSV vaccine delivers (e.g., comprises or encodes) an immunogenic fragment of an RSV fusion protein (F), an RSV attachment protein (G), an RSV small hydrophobic protein 20 (SH), an RSV matrix protein (M), an RSV nucleoprotein (N), an RSV M2-1 protein, an RSV large polymerase (L), and/or an RSV phosphoprotein (P), or an immunogenic variant thereof, or a nucleic acid (e.g., RNA) encoding any one of them. In some embodiments, the RSV vaccine delivers a pre-fusion stabilized F protein. In some embodiments, the RSV vaccine delivers a bivalent stabilized pre-fusion F protein (e.g., sequences based on RSV A and RSV B strains, e.g., RSVpreF as described in Falsey A., et al. J. Infect Dis 2022:225(12):2056-2066, Walsh E., et al. J. Infect Dis 2022:225(8):1357-1366, and/or Baber J., et al. J. Infect Dis 2022 May 11; jiac 189, the contents of each of which are incorporated herein by reference in their entirety).

In some embodiments, the RNA compositions described herein are co-administered with an influenza vaccine. In some embodiments, the influenza vaccine is an alpha influenza virus, a beta influenza virus, a gamma influenza virus, or a delta influenza virus vaccine. In some embodiments, the vaccine is an influenza A virus, an influenza B virus, an influenza C virus, or an influenza D virus vaccine. In some embodiments, the influenza A virus vaccine comprises a hemagglutinin selected from H1, H2, H3, H4, H5, H6, H7, H8, H9, H10, H11, H12, H13, H14, H15, H16, H17, and H18, or an immunogenic fragment or variant thereof, or a nucleic acid (e.g., RNA) encoding any one of them. In some embodiments, the influenza A vaccine comprises or encodes a neuraminidase (NA) selected from N1, N2, N3, N4, N5, N6, N7, N8, N9, N10, and N11, or an immunogenic fragment or variant thereof, or a nucleic acid (e.g., RNA) encoding any one of them. In some embodiments, the influenza vaccine comprises at least one influenza virus hemagglutinin (HA), neuraminidase (NA), nucleoprotein (NP), matrix protein 1 (M1), matrix protein 2 (M2), nonstructural protein 1 (NS1), nonstructural protein 2 (NS2), nuclear export protein (NEP), polymerase acidic protein (PA), polymerase basic protein PB1, PB1-F2, and/or polymerase basic protein 2 (PB2), or an immunogenic fragment or variant thereof, or a nucleic acid (e.g., RNA) encoding any one of them.

Exemplary influenza vaccines that can be administered in combination with the RNA described herein include commercially available influenza vaccines, inactivated influenza vaccines (e.g., Fluzone®, Fluzone high-dose quadrivalent®, Fluzone quadrivalent®, Fluzone intradermal quadrivalent®, Fluzone quadrivalent southern hemisphere®, Fluad®, Fluad quadrivalent®, Afluria Quardivalent®, Fluarix Quadrivalent®, FluLaval ... In some embodiments, the RNA disclosed herein may be administered in combination with one or more RNAs, each RNA encoding one or more antigenic polypeptides associated with an influenza virus (e.g., an HA or NA protein). Exemplary RNA vaccines are known in the art (see, e.g., Feldman, Robert A., et al. "mRNA vaccines against H10N8 and H7N9 influenza viruses of Pandemic potentially immunogenic and well tolerated in healthy adults in phase 1 randomized clinical trials." Vaccine 37.25 (2019): 3326-3334 (the contents of which are incorporated herein by reference in their entirety).

In some embodiments, the RNA compositions described herein are administered in combination with saRNA encoding an influenza antigen. In some embodiments, the RNA compositions described herein are administered in combination with saRNA encoding a single influenza antigen (e.g., one HA polypeptide, one NA polypeptide, or one SARS-CoV-2 S polypeptide). In some embodiments, the RNA compositions described herein are co-administered with saRNA encoding two or more antigens (e.g., two or more HA polypeptides, two or more NA polypeptides, one or more HA polypeptides and one or more NA polypeptides, one or more NA polypeptides and one or more SARS-CoV-2 S polypeptides). In some embodiments, the RNA compositions described herein are co-administered with saRNA encoding two HA polypeptides, each derived from a different influenza strain. In some embodiments, the RNA compositions described herein are co-administered with saRNA encoding two NA polypeptides, each derived from a different influenza strain. In some embodiments, the saRNA encodes an HA polypeptide and an NA polypeptide, each from the same influenza strain (see, e.g., "Pfizer Near-Term Launches+High-Value Pipeline Day", published December 2006). 12, 2022; chrome-extension://efaidnbmnnnnibpcajpcglclefindmkaj/https://s28.q4cdn.com/781576035/files/doc_presentation/2022/12/B/Pfizer-Near-Term-Launches-High-Value-Pipeline-Day-Presentation_6pm_v2.pdf).

In some embodiments, the nucleotide sequence encoding the antigen polypeptide is located downstream of the saRNA replicase gene. In some embodiments, the saRNA comprises a nucleotide sequence encoding an HA antigen and a nucleotide sequence encoding an NA antigen, and the nucleotide sequence encoding the HA antigen is located upstream of the nucleotide sequence encoding the NA antigen.

In some embodiments, the RNA compositions described herein are co-administered with an RSV vaccine, an influenza vaccine, or an RSV vaccine and an influenza vaccine.

In some embodiments, the RNA compositions provided herein and the other injectable vaccine(s) are administered at different times. In some embodiments, the RNA compositions provided herein are administered simultaneously with the other injectable vaccine(s). In some such embodiments, the RNA compositions provided herein and at least one other injectable vaccine(s) are administered at different injection sites. In some embodiments, the RNA compositions provided herein are not mixed with any other vaccines in the same syringe. In some embodiments, the RNA compositions provided herein are not combined with other coronavirus vaccines as part of a vaccination against a coronavirus, e.g., SARS-CoV-2.

The term "disease" refers to an abnormal condition that affects an individual's body. A disease is often interpreted as a medical condition associated with certain symptoms and signs. A disease may be caused by factors that originate from an external source, such as an infectious disease, or may be caused by an internal malfunction, such as an autoimmune disease. In humans, "disease" is often used more broadly to refer to any condition that causes pain, dysfunction, distress, social problems, or death to the affected individual, or similar problems for people who come into contact with the individual. In this broader sense, it sometimes includes injuries, disabilities, disorders, syndromes, infections, isolated symptoms, deviant behavior, and atypical variations in structure and function, which in other contexts and for other purposes may be considered as distinct categories. Diseases usually affect individuals not only physically, but also emotionally, as living with many diseases can change one's perspective on life and personality.

In the present context, the terms "treatment", "treating" or "therapeutic intervention" relate to the management and treatment of a subject with the aim of combating a condition such as a disease or disorder. The term is intended to include the full range of treatments for a given condition suffered by a subject, such as the administration of therapeutically active compounds to alleviate symptoms or complications, to delay the progression of a disease, disorder or condition, to relieve or alleviate symptoms and complications, and/or to cure or eliminate a disease, disorder or condition, as well as to prevent a condition, where prevention is understood as the management and care of an individual for the purpose of combating a disease, condition or disorder, and includes the administration of active compounds to prevent the onset of symptoms or complications.

The term "therapeutic treatment" refers to any treatment that improves the health status and/or extends (increases) the lifespan of an individual. The treatment may eliminate the disease in an individual, halt or delay the onset of the disease in an individual, inhibit or delay the onset of the disease in an individual, reduce the frequency or severity of symptoms in an individual, and/or reduce recurrence in an individual who currently has or has previously had the disease.

The term "prophylactic treatment" or "preventive treatment" refers to any treatment intended to prevent a disease from occurring in an individual. The terms "prophylactic treatment" or "preventive treatment" are used interchangeably herein.

The terms "individual" and "subject" are used interchangeably herein. They refer to a human or another mammal (e.g., a mouse, rat, rabbit, dog, cat, cow, pig, sheep, horse, or primate) that may or may not have a disease or disorder, but may be afflicted or susceptible to a disease or disorder. In many embodiments, the individual is a human. Unless otherwise specified, the terms "individual" and "subject" do not denote a particular age, and thus encompass adults, seniors, children, and newborns. In some embodiments, the term "subject" includes humans that are at least 50 years old, at least 55 years old, at least 60 years old, at least 65 years old, at least 70 years old, or older. In some embodiments, the term "subject" includes humans that are at least 65 years old, e.g., 65-80 years old, 65-75 years old, or 65-70 years old. In embodiments of the present disclosure, an "individual" or "subject" is a "patient."

The term "patient" refers to an individual or subject for treatment, particularly an affected individual or subject.

In one embodiment of the present disclosure, the objective is to provide an immune response to coronavirus and to prevent or treat coronavirus infection.

A pharmaceutical composition comprising an RNA encoding a peptide or protein comprising an epitope can be administered to a subject to induce an immune response in the subject against an antigen comprising the epitope, which may be therapeutic or partially or completely protective. Those skilled in the art will know that one of the principles of immunotherapy and vaccination is based on the fact that an immune protective response against a disease is generated by immunizing a subject with an antigen or epitope that is immunologically relevant for the disease to be treated. Thus, the pharmaceutical compositions described herein are applicable to the induction or enhancement of an immune response. Thus, the pharmaceutical compositions described herein are useful in the prophylactic and/or therapeutic treatment of diseases involving antigens or epitopes.

As used herein, "immune response" refers to the body's integrated response to an antigen or a cell expressing an antigen, and may refer to a cellular immune response and/or a humoral immune response. The immune system is divided into the more primitive innate immune system and the adaptive or adaptive immune system of vertebrates, each of which contains humoral and cellular components.

"Cell-mediated immunity", "cellular immunity", "cellular immune response", or similar terms are meant to include cellular responses to cells characterized by the expression of antigens, particularly those cells characterized by the presentation of antigens with class I or class II MHC. The cellular response involves immune effector cells, particularly cells called T cells or T lymphocytes that act as either "helpers" or "killers". Helper T cells (also called CD4 ⁺ T cells) play a central role by regulating the immune response, while killer cells (also called cytotoxic T cells, cytolytic T cells, CD8 ⁺ T cells or CTLs) kill diseased cells, such as virus-infected cells, and prevent the production of further diseased cells.

Immune effector cells include any cells that are responsive to vaccine antigens. Such responsiveness includes activation, differentiation, proliferation, survival, and/or adaptation of one or more immune effector functions. Cells include, in particular, cells with lytic capacity, in particular lymphocyte cells, preferably T cells, in particular cytotoxic lymphocytes, preferably selected from cytotoxic T cells, natural killer (NK) cells, and lymphokine-activated killer (LAK) cells. Upon activation, each of these cytotoxic lymphocytes causes the destruction of a target cell. For example, cytotoxic T cells cause the destruction of a target cell by either or both of the following means: First, upon activation, T cells release cytotoxins such as perforin, granzymes, and granulysin. Perforin and granulysin create pores in the target cell, and granzymes trigger a caspase cascade in the cytoplasm that enters the cell and induces apoptosis (programmed cell death) of the cell. Second, apoptosis can be induced via Fas-Fas ligand interactions between T cells and target cells.

The term "effector function" in the context of the present disclosure includes any function mediated by components of the immune system that results in, for example, neutralization of a pathogenic agent, such as a virus, and/or killing of a diseased cell, such as a virus-infected cell. In one embodiment, an effector function in the context of the present disclosure is a T cell-mediated effector function. Such functions include, in the case of helper T cells (CD4 ⁺ T cells), the release of cytokines and/or activation of CD8 ⁺ lymphocytes (CTLs) and/or B cells, and in the case of CTLs, specific cytolytic killing of cells, i.e., cells characterized by expression of an antigen, e.g., via apoptosis or perforin-mediated cytolysis, production of cytokines such as IFN-γ and TNF-α, and antigen-expressing target cells.

The term "immune effector cells" or "immunoreactive cells" in the context of the present disclosure relates to cells that exert effector functions during an immune response. An "immune effector cell" in one embodiment is capable of binding to an antigen, such as an antigen presented in the context of MHC on a cell or expressed on the surface of a cell, and mediating an immune response. For example, immune effector cells include T cells (cytotoxic T cells, helper T cells, tumor infiltrating T cells), B cells, natural killer cells, neutrophils, macrophages, and dendritic cells. Preferably, in the context of the present disclosure, an "immune effector cell" is a T cell, preferably a CD4 ⁺ and/or CD8 ⁺ T cell, most preferably a CD8 ⁺ T cell. According to the present disclosure, the term "immune effector cell" also includes cells that can mature into immune cells (e.g., T cells, in particular T helper cells, or cytolytic T cells) upon suitable stimulation. Immune effector cells include CD34 ⁺ hematopoietic stem cells, immature and mature T cells, and immature and mature B cells. Differentiation of T cell precursors into cytolytic T cells upon exposure to antigen is similar to clonal selection in the immune system.

"Lymphoid cells" are cells capable of producing an immune response, such as a cellular immune response, or precursors of such cells, and include lymphocytes, preferably T lymphocytes, lymphoblasts, and plasma cells. Lymphoid cells may be immune effector cells as described herein. A preferred lymphoid cell is a T cell.

The terms "T cell" and "T lymphocyte" are used interchangeably herein and include T helper cells (CD4+ T cells), including cytolytic T cells, and cytotoxic T cells (CTL, CD8+ T cells). The term "antigen-specific T cell" or similar terms refers to a T cell that recognizes an antigen against which the T cell is targeted and preferably exerts an effector function of the T cell.

T cells belong to a group of white blood cells known as lymphocytes and play a central role in cell-mediated immunity. They can be distinguished from other lymphocyte types, such as B cells and natural killer cells, by the presence of a special receptor on the cell surface called the T cell receptor (TCR). The thymus is the main organ responsible for the maturation of T cells. Several different subsets of T cells have been found, each with different functions.

T helper cells assist other white blood cells in immunological processes, including, among other functions, maturation of B cells into plasma cells and activation of cytotoxic T cells and macrophages. These cells are also known as CD4+ T cells because they express the CD4 glycoprotein on their surface. Helper T cells are activated when peptide antigens are presented to them by MHC class II molecules expressed on the surface of antigen-presenting cells (APCs). Once activated, they rapidly divide and secrete small proteins called cytokines that regulate or assist in active immune responses.

Cytotoxic T cells destroy virus-infected and tumor cells and are also involved in transplant rejection. These cells are also known as CD8+ T cells because they express the CD8 glycoprotein on their surface. These cells recognize targets by binding to antigens associated with MHC class I, which is present on the surface of almost every cell in the body.

The majority of T cells have a T cell receptor (TCR) that exists as a complex of several proteins. The TCR of T cells binds to major histocompatibility complex (MHC) molecules and can interact with immunogenic peptides (epitopes) presented on the surface of target cells. Specific binding of the TCR triggers a signal cascade within the T cell, leading to proliferation and differentiation into mature effector T cells. The actual T cell receptor is composed of two separate peptide chains produced from independent T cell receptor alpha and beta (TCRα and TCRβ) genes, called α- and β-TCR chains. γδ T cells (gamma delta T cells) represent a small subset of T cells that have separate T cell receptors (TCR) on their surface. However, in γδ T cells, the TCR is composed of one γ chain and one δ chain. This T cell population is much less common than αβ T cells (2% of all T cells).

"Humoral immunity" or "humoral immune response" is the aspect of immunity mediated by macromolecules found in extracellular fluids, such as secreted antibodies, complement proteins, and certain antimicrobial peptides. It is contrasted with cell-mediated immunity. The aspect involving antibodies is often called antibody-mediated immunity.

Humoral immunity refers to antibody production and its associated accessory processes, including Th2 activation and cytokine production, germinal center formation and isotype switching, affinity maturation and memory cell generation. It also refers to antibody effector functions, including pathogen neutralization, classical complement activation, and opsonization to facilitate phagocytosis and pathogen elimination.

In a humoral immune response, first, B cells mature in the bone marrow and acquire B cell receptors (BCRs) that are displayed in large numbers on the cell surface. These membrane-bound protein complexes carry antibodies that are specific for antigen detection. Each B cell has a unique antibody that binds to an antigen. Mature B cells migrate from the bone marrow to lymph nodes or other lymphoid organs, where they begin to encounter pathogens. When a B cell encounters an antigen, the antigen binds to the receptor and is taken up into the B cell by endocytosis. The antigen is again processed and presented on the surface of the B cell by MHC-II proteins. The B cell waits for a helper T cell (TH) to bind to the complex. This binding activates the TH cell, which then releases cytokines that induce the B cell to divide rapidly, creating thousands of identical clones of B cells. These daughter cells become either plasma cells or memory cells. The memory B cells remain inactive here, and if these memory B cells later encounter the same antigen through reinfection, they will divide and form plasma cells. On the other hand, plasma cells produce a large number of antibodies, which are freely released into the circulation. These antibodies encounter antigens and bind to them. This may prevent chemical interactions between host and foreign cells, or form bridges between their antigenic sites and prevent their proper function, or their presence may attract macrophages or killer cells to attack and phagocytose them.

The term "antibody" includes immunoglobulins comprising at least two heavy (H) chains and two light (L) chains interconnected by disulfide bonds. Each heavy chain is composed of a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region. Each light chain is composed of a light chain variable region (abbreviated herein as VL) and a light chain constant region. The VH and VL regions can be further subdivided into regions of hypervariability called complementarity determining regions (CDRs), interspersed with more conserved regions called framework regions (FRs). Each VH and VL is composed of three CDRs and four FRs, arranged from the amino terminus to the carboxy terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy and light chains contain the binding domains that interact with antigens. The constant region of the antibody can mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (Clq) of the classical complement system. The antibody preferably specifically binds to an antigen.

Antibodies expressed by B cells are sometimes referred to as BCRs (B cell receptors) or antigen receptors. The five members of this class of proteins are IgA, IgG, IgM, IgD, and IgE. IgA is the primary antibody present in body secretions such as saliva, tears, breast milk, gastrointestinal secretions, and mucous secretions of the respiratory and genitourinary tracts. IgG is the most common prevalent antibody. IgM is the major immunoglobulin produced in the primary immune response in most subjects. It is the most efficient immunoglobulin in agglutination, complement fixation, and other antibody responses, and is important in defense against bacteria and viruses. IgD is an immunoglobulin with no known antibody function, but which may serve as an antigen receptor. IgE is an immunoglobulin that mediates immediate hypersensitivity by triggering the release of mediators from mast cells and basophils upon exposure to allergens.

"Antibody heavy chain," as used herein, refers to the larger of the two polypeptide chains present in an antibody molecule.

"Antibody light chain" as used herein refers to the smaller of the two types of polypeptide chains present in antibody molecules in their naturally occurring conformations; kappa and lambda light chains refer to the two major antibody light chain isotypes.

The present disclosure contemplates an immune response that may be protective, preventive, prophylactic, and/or therapeutic. As used herein, "inducing [or inducing] an immune response" may indicate that there was no immune response to a particular antigen prior to induction, or that there was a basal level of an immune response to a particular antigen prior to induction that was enhanced following induction. Thus, "inducing [or inducing] an immune response" includes "enhancing [or enhancing] an immune response."

The term "immunotherapy" relates to the treatment of a disease or condition by inducing or enhancing an immune response. The term "immunotherapy" includes antigen immunization or antigen vaccination.

The terms "immunization" or "vaccination" describe the process of administering an antigen to an individual for the purpose of inducing an immune response, e.g., for therapeutic or prophylactic reasons.

The term "macrophage" refers to a subgroup of phagocytes produced by differentiation of monocytes. Activated by inflammation, immune cytokines, or microbial products, macrophages nonspecifically engulf and kill foreign pathogens within the macrophage by hydrolytic and oxidative attack, resulting in degradation of the pathogen. Peptides from degraded proteins are displayed on the macrophage cell surface where they can be recognized by T cells, and they can directly interact with antibodies on the B cell surface, resulting in T cell and B cell activation, leading to further stimulation of the immune response. Macrophages belong to a class of antigen-presenting cells. In one embodiment, the macrophages are splenic macrophages.

The term "dendritic cells" (DC) refers to another subtype of phagocytes that belong to the class of antigen-presenting cells. In one embodiment, dendritic cells are derived from hematopoietic bone marrow progenitors. These progenitors are first converted into immature dendritic cells. These immature cells are characterized by high phagocytosis and low T cell activation potential. Immature dendritic cells constantly sample the surrounding environment for pathogens such as viruses and bacteria. Upon contact with presentable antigens, they are activated into mature dendritic cells and begin to migrate to the spleen or lymph nodes. Immature dendritic cells phagocytose pathogens, break down their proteins into small pieces, and upon maturation, present the fragments on their cell surface using MHC molecules. At the same time, they upregulate cell surface receptors that function as co-receptors in T cell activation, such as CD80, CD86, and CD40, greatly enhancing their ability to activate T cells. They also upregulate CCR7, a chemotactic receptor that induces dendritic cells to migrate through the bloodstream to the spleen or through the lymphatic system to lymph nodes. Here, they function as antigen-presenting cells, activating helper and killer T cells as well as B cells by presenting antigens together with non-antigen-specific costimulatory signals. Thus, dendritic cells can actively induce T-cell or B-cell-related immune responses. In one embodiment, the dendritic cells are splenic dendritic cells.

The term "antigen-presenting cell" (APC) refers to any cell that is capable of displaying, acquiring, and/or presenting at least one antigen or antigen fragment on (or at) its cell surface. Antigen-presenting cells can be distinguished in professional and non-professional antigen-presenting cells.

The term "professional antigen-presenting cells" refers to antigen-presenting cells that constitutively express major histocompatibility complex class II (MHC class II) molecules necessary for interaction with naive T cells. When a T cell interacts with the MHC class II molecule complex on the membrane of the antigen-presenting cell, the antigen-presenting cell produces costimulatory molecules that induce T cell activation. Professional antigen-presenting cells include dendritic cells and macrophages.

The term "non-professional antigen-presenting cells" refers to antigen-presenting cells that do not constitutively express MHC class II molecules, but do so upon stimulation by certain cytokines, such as interferon-gamma. Exemplary non-professional antigen-presenting cells include fibroblasts, thymic epithelial cells, thyroid epithelial cells, glial cells, pancreatic beta cells, or vascular endothelial cells.

"Antigen processing" refers to the degradation of an antigen into progressive products that are fragments of the antigen (e.g., protein degradation into peptides) and the association (e.g., via binding) of one or more of these fragments with MHC molecules for presentation to specific T cells by cells such as antigen-presenting cells.

The term "disease associated with an antigen" refers to any disease associated with an antigen, e.g., a disease characterized by the presence of an antigen. The disease associated with an antigen may be an infectious disease. As noted above, the antigen may be a disease-associated antigen, such as a viral antigen. In one embodiment, the disease associated with an antigen is a disease involving cells that express the antigen, preferably on the cell surface.

The term "infectious disease" refers to any disease that can be transmitted from individual to individual or organism to organism and is caused by a microbial agent (e.g., the common cold). Infectious diseases are known in the art and include, for example, viral, bacterial, or parasitic diseases, which are diseases caused by viruses, bacteria, or parasites, respectively. In this regard, infectious diseases can be, for example, hepatitis, sexually transmitted diseases (e.g., chlamydia or gonorrhea), tuberculosis, HIV/Acquired Immune Deficiency Syndrome (AIDS), diphtheria, Hepatitis B, Hepatitis C, cholera, Severe Acute Respiratory Syndrome (SARS), avian influenza, and influenza.

Exemplary Dosing Regimens In some embodiments, the compositions and methods disclosed herein can be used according to the exemplary vaccination regimen illustrated in FIG.

Primary Dosing Regimen In some embodiments, the subject is administered a primary dosing regimen. The primary dosing regimen can include one or more doses. For example, in some embodiments, the primary dosing regimen includes a single dose (PD ₁ ). In some embodiments, the primary dosing regimen includes a first dose (PD ₁ ) and a second dose (PD _{2 ). In some embodiments, the primary dosing regimen includes a first dose, a second dose, and a third dose (PD 3 )} . In some embodiments, the primary dosing regimen includes a first dose, a second dose, a third dose, and one or more additional doses (PD _n ) of any one of the pharmaceutical compositions described herein.

In some embodiments, PD ₁ comprises administering 1-100 ug of RNA. In some embodiments, PD ₁ comprises administering 1-60 ug of RNA. In some embodiments, PD ₁ comprises administering 1-50 ug of RNA. In some embodiments, PD ₁ comprises administering 1-30 ug of RNA. In some embodiments, PD ₁ comprises administering about 3 ug of RNA. In some embodiments, PD ₁ comprises administering about 5 ug of RNA. In some embodiments, PD ₁ comprises administering about 10 ug of RNA. In some embodiments, PD ₁ comprises administering about 15 ug of RNA. In some embodiments, PD ₁ comprises administering about 20 ug of RNA. In some embodiments, PD ₁ comprises administering about 30 ug of RNA. In some embodiments, PD ₁ comprises administering about 50 ug of RNA. In some embodiments, PD ₁ comprises administering about 60 ug of RNA.

In some embodiments, PD ₂ comprises administering 1-100 ug of RNA. In some embodiments, PD ₂ comprises administering 1-60 ug of RNA. In some embodiments, PD ₂ comprises administering 1-50 ug of RNA. In some embodiments, PD ₂ comprises administering 1-30 ug of RNA. In some embodiments, PD ₂ comprises administering about 3 ug. In some embodiments, PD _{2 comprises administering about 5 ug of RNA. In some embodiments, PD 2} comprises administering about 10 ug of RNA. In some embodiments, PD ₂ comprises administering about 15 ug of RNA. In some embodiments, PD ₂ comprises administering about 20 ug of RNA. In some embodiments, PD ₂ comprises administering about 30 ug of RNA. In some embodiments, PD ₂ comprises administering about 50 ug of RNA. In some embodiments, PD ₂ comprises administering about 60 _ug of RNA.

In some embodiments, the PD ₃ comprises administering 1-100 ug of RNA. In some embodiments, the PD ₃ comprises administering 1-60 ug of RNA. In some embodiments, the PD ₃ comprises administering 1-50 ug of RNA. In some embodiments, the PD ₃ comprises administering 1-30 ug of RNA. In some embodiments, the PD ₃ comprises administering about 3 ug of RNA. In some embodiments, the PD ₃ comprises administering about 5 ug of RNA. In some embodiments, the PD ₃ comprises administering about 10 ug of RNA. In some embodiments, the PD ₃ comprises administering about 15 ug of RNA. In some embodiments, the PD ₃ comprises administering about 20 ug of RNA. In some embodiments, the PD ₃ comprises administering about 30 ug of RNA. In some embodiments, the PD ₃ comprises administering about 50 ug of RNA. In some embodiments, the PD ₃ comprises administering about 60 ug of RNA.

In some embodiments, PD _n comprises administering 1-100 ug of RNA. In some embodiments, PD _n comprises administering 1-60 ug of RNA. In some embodiments, PD _n comprises administering 1-50 ug of RNA. In some embodiments, PD _{n comprises administering 1-30 ug of RNA. In some embodiments, PD n} comprises administering about 3 ug of RNA. In some embodiments, PD _n comprises administering about 5 ug of RNA. In some embodiments, PD _n comprises administering about 10 ug of RNA. In some embodiments, PD _n comprises administering about 15 ug of RNA. In some embodiments, PD _n comprises administering about 20 ug of RNA. In some embodiments, _{PD n comprises administering about 30 ug of RNA. In some embodiments, PD n comprises administering about 50 ug of RNA. In some embodiments, PD n comprises administering about} 60 ug of RNA.

In some embodiments, PD ₁ comprises RNA encoding a SARS-CoV-2 spike protein or immunogenic fragment thereof from the Wuhan strain. In some embodiments, PD ₁ comprises RNA encoding a spike protein or immunogenic fragment thereof from a SARS-CoV-2 strain that is prevalent and/or spreading rapidly in a relevant jurisdiction. In some embodiments, PD ₁ comprises RNA encoding a SARS-CoV-2 spike protein or immunogenic fragment thereof that includes one or more mutations from an alpha variant. In some embodiments, PD ₁ comprises RNA encoding a SARS-CoV-2 spike protein or immunogenic fragment thereof that includes one or more mutations from a delta variant. In some embodiments, PD ₁ comprises RNA encoding a SARS-CoV-2 spike protein or immunogenic fragment thereof that includes one or more mutations from a beta variant. In some embodiments, PD ₁ comprises RNA encoding a SARS-CoV-2 spike protein or immunogenic fragment thereof that includes one or more mutations from an omicron variant. In some embodiments, PD ₁ comprises RNA encoding a SARS-CoV-2 spike protein or an immunogenic fragment thereof from the Wuhan strain, and one or more additional RNAs encoding a spike protein or an immunogenic fragment thereof from a SARS-CoV-2 strain that is prevalent and/or spreading rapidly in a relevant jurisdiction. In some embodiments, PD ₁ comprises RNA encoding a SARS-CoV-2 spike protein or an immunogenic fragment thereof from the Wuhan strain, and RNA encoding a SARS-CoV-2 spike protein or an immunogenic fragment thereof that includes one or more mutations from an alpha variant. In some embodiments, PD ₁ comprises RNA encoding a SARS-CoV-2 spike protein or an immunogenic fragment thereof from the Wuhan strain, and RNA encoding a SARS-CoV-2 spike protein or an immunogenic fragment thereof that includes one or more mutations from a beta variant. In some embodiments, the PD ₁ comprises RNA encoding a SARS-CoV-2 spike protein or an immunogenic fragment thereof from the Wuhan strain and RNA encoding a SARS-CoV-2 spike protein or an immunogenic fragment thereof comprising one or more mutations from the delta variant.In some embodiments, the PD ₁ comprises RNA encoding a SARS-CoV-2 spike protein or an immunogenic fragment thereof from the Wuhan strain and RNA encoding a SARS-CoV-2 spike protein or an immunogenic fragment thereof comprising one or more mutations from the omicron variant.

In some embodiments, PD ₂ comprises RNA encoding a SARS-CoV-2 spike protein or immunogenic fragment thereof from the Wuhan strain. In some embodiments, PD ₂ comprises RNA encoding a spike protein or immunogenic fragment thereof from a SARS-CoV-2 strain that is prevalent and/or spreading rapidly in a relevant jurisdiction. In some embodiments, PD _{2 comprises RNA encoding a SARS-CoV-2 spike protein or immunogenic fragment thereof that includes one or more mutations from an alpha variant. In some embodiments, PD 2 comprises} RNA encoding a SARS-CoV-2 spike protein or immunogenic fragment thereof that includes one or more mutations from a delta variant. In some embodiments, PD ₂ comprises RNA encoding a SARS-CoV-2 spike protein or immunogenic fragment thereof that includes one or more mutations from a beta variant. In some embodiments, PD ₂ comprises RNA encoding a SARS-CoV-2 spike protein or immunogenic fragment thereof that includes one or more mutations from an omicron variant. In some embodiments, PD ₂ comprises RNA encoding a SARS-CoV-2 spike protein or an immunogenic fragment thereof from the Wuhan strain, as well as one or more additional RNAs encoding a spike protein or an immunogenic fragment thereof from a SARS-CoV-2 strain that is prevalent and/or spreading rapidly in the relevant jurisdiction. In some embodiments, PD ₂ comprises RNA encoding a SARS-CoV-2 spike protein or an immunogenic fragment thereof from the Wuhan strain, and RNA encoding a SARS-CoV-2 spike protein or an immunogenic fragment thereof that includes one or more mutations from an alpha variant. In some embodiments, PD ₂ comprises RNA encoding a SARS-CoV-2 spike protein or an immunogenic fragment thereof from the Wuhan strain, and RNA encoding a SARS-CoV-2 spike protein or an immunogenic fragment thereof that includes one or more mutations from a beta variant. In some embodiments, PD ₂ comprises RNA encoding a SARS-CoV-2 spike protein or an immunogenic fragment thereof from the Wuhan strain and RNA encoding a SARS-CoV-2 spike protein or an immunogenic fragment thereof comprising one or more mutations from the delta variant.In some embodiments, PD ₂ comprises RNA encoding a SARS-CoV-2 spike protein or an immunogenic fragment thereof from the Wuhan strain and RNA encoding a SARS-CoV-2 spike protein or an immunogenic fragment thereof comprising one or more mutations from the omicron variant.

In some embodiments, the PD ₃ comprises RNA encoding a SARS-CoV-2 spike protein or immunogenic fragment thereof from the Wuhan strain. In some embodiments, the PD ₃ comprises RNA encoding a spike protein or immunogenic fragment thereof from a SARS-CoV-2 strain that is prevalent and/or spreading rapidly in a relevant jurisdiction. In some embodiments, the PD ₃ comprises RNA encoding a SARS-CoV-2 spike protein or immunogenic fragment thereof that includes one or more mutations from an alpha variant. In some embodiments, the PD ₃ comprises RNA encoding a SARS-CoV-2 spike protein or immunogenic fragment thereof that includes one or more mutations from a delta variant. In some embodiments, the PD ₃ comprises RNA encoding a SARS-CoV-2 spike protein or immunogenic fragment thereof that includes one or more mutations from a beta variant. In some embodiments, the PD ₃ comprises RNA encoding a SARS-CoV-2 spike protein or immunogenic fragment thereof that includes one or more mutations from an omicron variant. In some embodiments, the PD ₃ comprises an RNA encoding a SARS-CoV-2 spike protein or an immunogenic fragment thereof from the Wuhan strain, and one or more additional RNAs encoding a spike protein or an immunogenic fragment thereof from a SARS-CoV-2 strain that is prevalent and/or spreading rapidly in the relevant jurisdiction. In some embodiments, the PD ₃ comprises an RNA encoding a SARS-CoV-2 spike protein or an immunogenic fragment thereof from the Wuhan strain, and an RNA encoding a SARS-CoV-2 spike protein or an immunogenic fragment thereof that includes one or more mutations from an alpha variant. In some embodiments, the PD ₃ comprises an RNA encoding a SARS-CoV-2 spike protein or an immunogenic fragment thereof from the Wuhan strain, and an RNA encoding a SARS-CoV-2 spike protein or an immunogenic fragment thereof that includes one or more mutations from a beta variant. In some embodiments, the PD ₃ comprises RNA encoding a SARS-CoV-2 spike protein or an immunogenic fragment thereof from the Wuhan strain and RNA encoding a SARS-CoV-2 spike protein or an immunogenic fragment thereof comprising one or more mutations from the delta variant.In some embodiments, the PD ₃ comprises RNA encoding a SARS-CoV-2 spike protein or an immunogenic fragment thereof from the Wuhan strain and RNA encoding a SARS-CoV-2 spike protein or an immunogenic fragment thereof comprising one or more mutations from the omicron variant.

In some embodiments, the PD _n comprises RNA encoding a SARS-CoV-2 spike protein or immunogenic fragment thereof from the Wuhan strain. In some embodiments, the PD _n comprises RNA encoding a spike protein or immunogenic fragment thereof from a SARS-CoV-2 strain that is circulating and/or spreading rapidly in the relevant jurisdiction. In some embodiments, the PD _{n comprises RNA encoding a SARS-CoV-2 spike protein or immunogenic fragment thereof that includes one or more mutations from an alpha variant. In some embodiments, the PD n comprises} RNA encoding a SARS-CoV-2 spike protein or immunogenic fragment thereof that includes one or more mutations from a delta variant. In some embodiments, the PD _n comprises RNA encoding a SARS-CoV-2 spike protein or immunogenic fragment thereof that includes one or more mutations from a beta variant. In some embodiments, the PD _n comprises RNA encoding a SARS-CoV-2 spike protein or immunogenic fragment thereof that includes one or more mutations from an omicron variant. In some embodiments, the PD _n comprises an RNA encoding a SARS-CoV-2 spike protein or an immunogenic fragment thereof from the Wuhan strain, and one or more additional RNAs encoding a spike protein or an immunogenic fragment thereof from a SARS-CoV-2 strain that is prevalent and/or spreading rapidly in the relevant jurisdiction. In some embodiments, the PD _n comprises an RNA encoding a SARS-CoV-2 spike protein or an immunogenic fragment thereof from the Wuhan strain, and an RNA encoding a SARS-CoV-2 spike protein or an immunogenic fragment thereof that includes one or more mutations from an alpha variant. In some embodiments, the PD _n comprises an RNA encoding a SARS-CoV-2 spike protein or an immunogenic fragment thereof from the Wuhan strain, and an RNA encoding a SARS-CoV-2 spike protein or an immunogenic fragment thereof that includes one or more mutations from a beta variant. In some embodiments, PD _n comprises RNA encoding a SARS-CoV-2 spike protein or an immunogenic fragment thereof from the Wuhan strain and RNA encoding a SARS-CoV-2 spike protein or an immunogenic fragment thereof comprising one or more mutations from the delta variant.In some embodiments, PD _n comprises RNA encoding a SARS-CoV-2 spike protein or an immunogenic fragment thereof from the Wuhan strain and RNA encoding a SARS-CoV-2 spike protein or an immunogenic fragment thereof comprising one or more mutations from the omicron variant.

In some embodiments, PD ₁ , PD ₂ , PD ₃ , and PD _n can each independently comprise a plurality (e.g., at least two) of the RNA (e.g., mRNA) compositions described herein. In some embodiments, PD ₁ , PD ₂ , PD ₃ , and PD _n can each independently comprise a first and a second RNA (e.g., mRNA) composition. In some embodiments, at least one of the plurality of RNA (e.g., mRNA) compositions comprises BNT162b2 (e.g., as described herein). In some embodiments, at least one of the plurality of RNA (e.g., mRNA) compositions comprises RNA (e.g., mRNA) encoding a SARS-CoV-2 S protein, or an immunogenic fragment thereof, from a different SARS-CoV-2 variant. In some embodiments, at least one of the plurality of RNA (e.g., mRNA) compositions comprises RNA (e.g., mRNA) encoding a SARS-CoV-2 S protein, or an immunogenic fragment thereof, from the Wuhan strain of SARS-CoV-2. In some embodiments, at least one of the plurality of RNA (e.g., mRNA) compositions comprises an RNA encoding a SARS-CoV-2 S protein or immunogenic fragment thereof that includes one or more mutations from a variant that is prevalent and/or rapidly spreading in a relevant jurisdiction. In some embodiments, at least one of the plurality of RNA (e.g., mRNA) compositions comprises an RNA (e.g., mRNA) encoding a SARS-CoV-2 S protein or immunogenic fragment thereof that includes one or more mutations from an alpha variant. In some embodiments, at least one of the plurality of RNA (e.g., mRNA) compositions comprises an RNA (e.g., mRNA) encoding a SARS-CoV-2 S protein or immunogenic fragment thereof that includes one or more mutations from a delta variant. In some embodiments, at least one of the plurality of RNA (e.g., mRNA) compositions comprises an RNA (e.g., mRNA) encoding a SARS-CoV-2 S protein or immunogenic fragment thereof that includes one or more mutations from an omicron variant.

In some embodiments, the multiple RNA (e.g., mRNA) compositions provided at PD ₁ , PD ₂ , PD ₃ , and/or PD _n can each independently comprise at least two different RNA (e.g., mRNA) constructs (e.g., differing in protein coding sequence). For example, in some embodiments, the multiple RNA (e.g., mRNA) compositions provided at PD ₁ , PD ₂ , PD ₃ , and/or PD _n can each independently comprise an RNA (e.g., mRNA) encoding a SARS-CoV-2 S protein, or an immunogenic fragment thereof, from the Wuhan strain of SARS-CoV-2, as well as an RNA (e.g., mRNA) encoding a SARS-CoV-2 S protein, or an immunogenic fragment thereof, that includes one or more mutations from a variant that is prevalent and/or spreading rapidly in the relevant jurisdiction. In some embodiments, the multiple RNA (e.g., mRNA) compositions provided at PD ₁ , PD ₂ , PD ₃ , and/or PD _n can each independently comprise an RNA (e.g., mRNA) encoding a SARS-CoV-2 S protein or an immunogenic fragment thereof from the Wuhan strain of SARS-CoV-2, as well as an RNA (e.g., mRNA) encoding a SARS-CoV-2 S protein or an immunogenic fragment thereof that includes one or more mutations from a variant that is prevalent and/or rapidly spreading in the relevant jurisdiction. In some such embodiments, the variant can be an alpha variant. In some such embodiments, the variant can be a delta variant. In some such embodiments, the variant can be an omicron variant.

In some embodiments, each of the multiple RNA (e.g., mRNA) compositions provided at PD ₁ , PD ₂ , PD ₃ , and/or PD _n can independently comprise at least two RNAs (e.g., mRNAs), each encoding a SARS-CoV-2 S protein or immunogenic fragment thereof that includes one or more mutations from a distinct variant that is prevalent and/or spreading rapidly in the relevant jurisdiction. In some embodiments, each of the multiple RNA (e.g., mRNA) compositions provided at PD ₁ , PD ₂ , PD ₃ , and/or PD _n can independently comprise an mRNA encoding a SARS-CoV-2 S protein or immunogenic fragment thereof from an alpha variant, and an RNA (e.g., mRNA) encoding a SARS-CoV-2 S protein or immunogenic fragment thereof that includes one or more mutations from a delta variant. In some embodiments, each of the multiple RNA (e.g., mRNA) compositions provided at PD ₁ , PD ₂ , PD ₃ , and/or PD _n can independently comprise an RNA (e.g., mRNA) encoding a SARS-CoV-2 S protein or immunogenic fragment thereof from an alpha variant, and an RNA (e.g., mRNA) encoding a SARS-CoV-2 S protein or immunogenic fragment thereof that includes one or more mutations from a delta variant. In some embodiments, each of the multiple RNA (e.g., mRNA) compositions provided at PD ₁ , PD ₂ , PD ₃ , and/or PD _n can independently comprise an RNA (e.g., mRNA) encoding a SARS-CoV-2 S protein or immunogenic fragment thereof from an alpha variant, and an RNA (e.g., mRNA) encoding a SARS-CoV-2 S protein or immunogenic fragment thereof that includes one or more mutations from a delta variant.

In some embodiments, PD ₁ , PD ₂ , PD ₃ , and/or PD _n each comprise a plurality of RNA (e.g., mRNA) compositions, and each RNA (e.g., mRNA) composition is administered separately to the subject. For example, in some embodiments, each RNA (e.g., mRNA) composition is administered via intramuscular injection at a different injection site. For example, in some embodiments, a first and second RNA (e.g., mRNA) composition given in PD ₁ , PD ₂ , PD ₃ , and/or PD _n are administered separately via intramuscular injection into different arms of the subject.

In some embodiments, PD ₁ , PD ₂ , PD ₃ , and/or PD _n comprises administering a plurality of RNA molecules, each RNA molecule encoding a spike protein comprising a mutation from a different SARS-CoV-2 variant, and the plurality of RNA molecules are administered to the subject in a single formulation. In some embodiments, the single formulation comprises RNA encoding a spike protein or an immunogenic fragment thereof from the Wuhan strain, and RNA encoding a SARS-CoV-2 spike protein or an immunogenic fragment thereof comprising one or more mutations from an alpha variant. In some embodiments, the single formulation comprises RNA encoding a SARS-CoV-2 spike protein or an immunogenic fragment thereof from the Wuhan strain, and RNA encoding a SARS-CoV-2 spike protein or an immunogenic fragment thereof comprising one or more mutations from a beta variant. In some embodiments, a single formulation comprises RNA encoding a SARS-CoV-2 spike protein or an immunogenic fragment thereof from the Wuhan strain, and RNA encoding a SARS-CoV-2 spike protein or an immunogenic fragment thereof that includes one or more mutations from the delta variant. In some embodiments, a single formulation comprises RNA encoding a SARS-CoV-2 spike protein or an immunogenic fragment thereof from the Wuhan strain, and RNA encoding a SARS-CoV-2 spike protein or an immunogenic fragment thereof that includes one or more mutations from the omicron variant.

In some embodiments, the length of time between PD ₁ and PD ₂ (PI ₁ ) is at least about 1 week, at least about 2 weeks, at least about 3 weeks, or at least about 4 weeks. In some embodiments, PI ₁ is from about 1 week to about 12 weeks. In some embodiments, PI ₁ is from about 1 week to about 10 weeks. In some embodiments, PI ₁ is from about 2 weeks to about 10 weeks. In some embodiments, PI ₁ is from about 2 weeks to about 8 weeks. In some embodiments, PI ₁ is from about 3 weeks to about 8 weeks. In some embodiments, PI ₁ is from about 4 weeks to about 8 weeks. In some embodiments, PI ₁ is from about 6 weeks to about 8 weeks. In some embodiments, PI ₁ is from about 3 to about 4 weeks. In some embodiments, PI ₁ is about 1 week. In some embodiments, PI ₁ is about 2 weeks. In some embodiments, PI ₁ is about 3 weeks. In some embodiments, PI ₁ is about 4 weeks. In some embodiments, PI ₁ is about 5 weeks. In some embodiments, PI ₁ is about 6 weeks. In some embodiments, PI ₁ is about 7 weeks. In some embodiments, PI ₁ is about 8 weeks. In some embodiments, PI ₁ is about 9 weeks. In some embodiments, PI ₁ is about 10 weeks. In some embodiments, PI ₁ is about 11 weeks. In some embodiments, PI ₁ is about 12 weeks.

In some embodiments, the length of time between PD ₂ and PD ₃ (PI ₂ ) is at least about 1 week, at least about 2 weeks, or at least about 3 weeks. In some embodiments, PI ₂ is from about 1 week to about 12 weeks. In some embodiments, PI ₂ is from about 1 week to about 10 weeks. In some embodiments, PI ₂ is from about 2 weeks to about 10 weeks. In some embodiments, PI ₂ is from about 2 weeks to about 8 weeks. In some embodiments, PI ₂ is from about 3 weeks to about 8 weeks. In some embodiments, PI ₂ is from about 4 weeks to about 8 weeks. In some embodiments, PI ₂ is from about 6 weeks to about 8 weeks. In some embodiments, PI ₂ is from about 3 to about 4 weeks. In some embodiments, PI ₂ is about 1 week. In some embodiments, PI ₂ is about 2 weeks. In some embodiments, PI ₂ is about 3 weeks. In some embodiments, PI ₂ is about 4 weeks. In some embodiments, PI ₂ is about 5 weeks. In some embodiments, PI ₂ is about 6 weeks. In some embodiments, PI ₂ is about 7 weeks. In some embodiments, PI ₂ is about 8 weeks. In some embodiments, PI ₂ is about 9 weeks. In some embodiments, PI ₂ is about 10 weeks. In some embodiments, PI ₂ is about 11 weeks. In some embodiments, PI ₂ is about 12 weeks.

In some embodiments, the length of time (PI _n ) between PD ₃ and a subsequent dose that is part of the primary dosing regimen, or between doses for any doses beyond PD ₃ , is each separately and independently selected from about 1 week or more, about 2 weeks or more, or about 3 weeks or more. In some embodiments, PI _n is about 1 week to about 12 weeks. In some embodiments, PI _n is about 1 week to about 10 weeks. In some embodiments, PI _n is about 2 weeks to about 10 weeks. In some embodiments, PI _n is about 2 weeks to about 8 weeks. In some embodiments, PI _n is about 3 weeks to about 8 weeks. In some embodiments, PI _n is about 4 weeks to about 8 weeks. In some embodiments, PI _n is about 6 weeks to about 8 weeks. In some embodiments, PI _n is about 3 to about 4 weeks. In some embodiments, PI ₂ is about 1 week. In some embodiments, PI _n is about 2 weeks. In some embodiments, PI _n is about 3 weeks. In some embodiments, PI _n is about 4 weeks. In some embodiments, PI _n is about 5 weeks. In some embodiments, PI _n is about 6 weeks. In some embodiments, PI _n is about 7 weeks. In some embodiments, PI _n is about 8 weeks. In some embodiments, PI _n is about 9 weeks. In some embodiments, PI _n is about 10 weeks. In some embodiments, PI _n is about 11 weeks. In some embodiments, PI _n is about 12 weeks.

In some embodiments, one or more compositions administered on PD ₁ are formulated in a Tris buffer. In some embodiments, one or more compositions administered on PD ₂ are formulated in a Tris buffer. In some embodiments, one or more compositions administered on PD ₃ are formulated in a Tris buffer. In some embodiments, one or more compositions administered on PD _n are formulated in a Tris buffer.

In some embodiments, the primary dosing regimen comprises administering two or more RNA (e.g., mRNA) compositions described herein, where at least two of the RNA (e.g., mRNA) compositions have different formulations. In some embodiments, the primary dosing regimen comprises PD ₁ and PD ₂ , where PD ₁ comprises administering RNA (e.g., mRNA) formulated in Tris buffer, and PD ₂ comprises administering RNA (e.g., mRNA) formulated in PBS buffer. In some embodiments, the primary dosing regimen comprises PD ₁ and PD ₂ , where PD ₁ comprises administering RNA (e.g., mRNA) formulated in Tris buffer, and PD ₂ comprises administering RNA (e.g., mRNA) formulated in PBS buffer.

In some embodiments, one or more of the RNA (e.g., mRNA) compositions provided in PD ₁ , PD ₂ , PD ₃ , and/or PD _n may be administered in conjunction with another vaccine. In some embodiments, the other vaccine is for a disease that is not COVID-19. In some embodiments, the disease is one that increases the deleterious effects of SARS-CoV-2 when a subject is co-infected with the disease and SARS-CoV-2. In some embodiments, the disease is one that increases the transmission rate of SARS-CoV-2 when a subject is co-infected with the disease and SARS-CoV-2. In some embodiments, the other vaccine is a different commercially available vaccine. In some embodiments, the different commercially available vaccine is an RNA vaccine. In some embodiments, the different commercially available vaccine is a polypeptide-based vaccine. In some embodiments, another vaccine (e.g., as described herein) and one or more RNA (e.g., mRNA) compositions given at PD ₁ , PD ₂ , PD ₃ , and/or PD _n are administered separately, e.g., via intramuscular injection, in some embodiments at different injection sites. For example, in some embodiments, an influenza vaccine described herein and one or more SARS-CoV-2 RNA (e.g., mRNA) compositions given at PD ₁ , PD ₂ , PD ₃ , and/or PD _n are administered separately via intramuscular injection into different arms of a subject.

Booster Dosing Regimens In some embodiments, the methods of vaccination disclosed herein include one or more booster dosing regimens. A booster dosing regimen disclosed herein includes one or more doses. In some embodiments, the booster dosing regimen is administered to a patient who has been administered a primary dosing regimen (e.g., as described herein). In some embodiments, the booster dosing regimen is administered to a patient who has not received a pharmaceutical composition disclosed herein. In some embodiments, the booster dosing regimen is administered to a patient who has previously been vaccinated with a COVID-19 vaccine different from the vaccine administered in the primary dosing regimen.

In some embodiments, the length of time between the primary dosing regimen and the booster dosing regimen is at least 1 week, at least 2 weeks, at least 3 weeks, at least 4 weeks, at least 5 weeks, at least 6 weeks, at least 7 weeks, at least 8 weeks, at least 9 weeks, at least 10 weeks, at least 11 weeks, at least 12 weeks, at least 2 months, at least 3 months, at least 4 months, at least 5 months, at least 6 months, at least 7 months, at least 8 months, at least 9 months, at least 10 months, at least 11 months, or at least 12 months or more. In some embodiments, the length of time between the primary dosing regimen and the booster dosing regimen is about 1 month. In some embodiments, the length of time between the primary dosing regimen and the booster dosing regimen is at least about 2 months. In some embodiments, the length of time between the primary dosing regimen and the booster dosing regimen is at least about 3 months. In some embodiments, the length of time between the primary dosing regimen and the booster dosing regimen is at least about 4 months. In some embodiments, the length of time between the primary dosing regimen and the booster dosing regimen is at least about 5 months. In some embodiments, the length of time between the primary dosing regimen and the booster dosing regimen is at least about 6 months. In some embodiments, the length of time between the primary dosing regimen and the booster dosing regimen is from about 1 month to about 48 months. In some embodiments, the length of time between the primary dosing regimen and the booster dosing regimen is from about 1 month to about 36 months. In some embodiments, the length of time between the primary dosing regimen and the booster dosing regimen is from about 1 month to about 24 months. In some embodiments, the length of time between the primary dosing regimen and the booster dosing regimen is from about 2 months to about 24 months. In some embodiments, the length of time between the primary dosing regimen and the booster dosing regimen is from about 3 months to about 24 months. In some embodiments, the length of time between the primary dosing regimen and the booster dosing regimen is from about 3 months to about 18 months. In some embodiments, the length of time between the primary dosing regimen and the booster dosing regimen is about 3 months to about 12 months. In some embodiments, the length of time between the primary dosing regimen and the booster dosing regimen is about 6 months to about 12 months. In some embodiments, the length of time between the primary dosing regimen and the booster dosing regimen is about 3 months to about 9 months. In some embodiments, the length of time between the primary dosing regimen and the booster dosing regimen is about 5 months to about 7 months. In some embodiments, the length of time between the primary dosing regimen and the booster dosing regimen is about 6 months.

In some embodiments, the subject is administered a booster dosing regimen. The booster dosing regimen can include one or more doses. For example, in some embodiments, the booster dosing regimen includes a single dose (BD ₁ ). In some embodiments, the booster dosing regimen includes a first dose (BD ₁ ) and a second dose (BD ₂ ). In some embodiments, the booster dosing regimen includes a first dose, a second dose, and a third dose (BD ₃ ). In some embodiments, the booster dosing regimen includes a first dose, a second dose, a third dose, and one or more additional doses (BD _n ) of any one of the pharmaceutical compositions described herein.

In some embodiments, the BD ₁ comprises administering 1-100 ug of RNA. In some embodiments, the BD ₁ comprises administering 1-60 ug of RNA. In some embodiments, the BD ₁ comprises administering 1-50 ug of RNA. In some embodiments, the BD ₁ comprises administering 1-30 ug of RNA. In some embodiments, the BD ₁ comprises administering about 3 ug of RNA. In some embodiments, the BD ₁ comprises administering about 5 ug of RNA. In some embodiments, the BD ₁ comprises administering about 10 ug of RNA. In some embodiments, the BD ₁ comprises administering about 15 ug of RNA. In some embodiments, the BD ₁ comprises administering about 20 ug of RNA. In some embodiments, the BD ₁ comprises administering about 30 ug of RNA. In some embodiments, the BD ₁ comprises administering about 50 ug of RNA. In some embodiments, BD ₁ comprises administering about 60 ug of RNA.

In some embodiments, BD ₂ comprises administering 1-100 ug of RNA. In some embodiments, BD ₂ comprises administering 1-60 ug of RNA. In some embodiments, BD ₂ comprises administering 1-50 ug of RNA. In some embodiments, BD ₂ comprises administering 1-30 ug of RNA. In some embodiments, BD ₂ comprises administering about 3 ug. In some embodiments, BD 2 comprises administering about 5 ug of RNA. In some embodiments, BD ₂ comprises administering about 10 ug of RNA. In some embodiments, BD ₂ comprises administering about 15 ug of RNA. In some embodiments, BD ₂ comprises administering about 20 ug of RNA. In some embodiments, BD ₂ comprises administering about 30 ug of RNA. In some embodiments, BD ₂ comprises administering about 50 _ug of RNA. In some embodiments, BD ₂ comprises administering about 60 ug of RNA.

In some embodiments, the BD ₃ comprises administering 1-100ug of RNA. In some embodiments, the BD ₃ comprises administering 1-60ug of RNA. In some embodiments, the BD ₃ comprises administering 1-50ug of RNA. In some embodiments, the BD ₃ comprises administering 1-30ug of RNA. In some embodiments, the BD ₃ comprises administering about 3ug of RNA. In some embodiments, the BD ₃ comprises administering about 5ug of RNA. In some embodiments, the BD ₃ comprises administering about 10ug of RNA. In some embodiments, the BD ₃ comprises administering about 15ug of RNA. In some embodiments, the BD ₃ comprises administering about 20ug of RNA. In some embodiments, the BD ₃ comprises administering about 30ug of RNA. In some embodiments, the BD ₃ comprises administering about 50ug of RNA. In some embodiments, BD ₃ comprises administering about 60 ug of RNA.

In some embodiments, BD _n comprises administering 1-100 ug of RNA. In some embodiments, BD _n comprises administering 1-60 ug of RNA. In some embodiments, BD _n comprises administering 1-50 ug of RNA. In some embodiments, BD _{n comprises administering 1-30 ug of RNA. In some embodiments, BD n} comprises administering about 3 ug of RNA. In some embodiments, BD _n comprises administering about 5 ug of RNA. In some embodiments, BD _n comprises administering about 10 ug of RNA. In some embodiments, BD _n comprises administering about 15 ug of RNA. In some embodiments, _{BD n comprises administering about 20 ug of RNA. In some embodiments, BD n comprises administering about 30 ug of RNA. In some embodiments, BD n comprises administering about} 60 ug of RNA. In some embodiments, BD _n comprises administering about 50 ug of RNA.

In some embodiments, BD ₁ comprises RNA encoding a SARS-CoV-2 spike protein or an immunogenic fragment thereof from the Wuhan strain. In some embodiments, BD ₁ comprises RNA encoding a spike protein or an immunogenic fragment thereof from a SARS-CoV-2 strain that is prevalent and/or spreading rapidly in the relevant jurisdiction. In some embodiments, BD _{1 comprises RNA encoding a SARS-CoV-2 spike protein or an immunogenic fragment thereof that includes one or more mutations from an alpha variant. In some embodiments, BD 1 comprises} RNA encoding a SARS-CoV-2 spike protein or an immunogenic fragment thereof that includes one or more mutations from a delta variant. In some embodiments, BD ₁ comprises RNA encoding a SARS-CoV-2 spike protein or an immunogenic fragment thereof that includes one or more mutations from a beta variant. In some embodiments, BD ₁ comprises RNA encoding a SARS-CoV-2 spike protein or an immunogenic fragment thereof that includes one or more mutations from an omicron variant.

In some embodiments, BD ₁ comprises an RNA encoding a SARS-CoV-2 spike protein or an immunogenic fragment thereof from the Wuhan strain, and one or more additional RNAs encoding a spike protein or an immunogenic fragment thereof from a SARS-CoV-2 strain that is prevalent and/or spreading rapidly in the relevant jurisdiction. In some embodiments, BD ₁ comprises an RNA encoding a SARS-CoV-2 spike protein or an immunogenic fragment thereof from the Wuhan strain, and an RNA encoding a SARS-CoV-2 spike protein or an immunogenic fragment thereof that includes one or more mutations from the alpha variant. In some embodiments, BD ₁ comprises an RNA encoding a SARS-CoV-2 spike protein or an immunogenic fragment thereof from the Wuhan strain, and an RNA encoding a SARS-CoV-2 spike protein or an immunogenic fragment thereof that includes one or more mutations from the delta variant. In some embodiments, BD ₁ comprises RNA encoding a SARS-CoV-2 spike protein or an immunogenic fragment thereof from the Wuhan strain, and RNA encoding a SARS-CoV-2 spike protein or an immunogenic fragment thereof comprising one or more mutations from the beta variant.In some embodiments, BD ₁ comprises RNA encoding a SARS-CoV-2 spike protein or an immunogenic fragment thereof from the Wuhan strain, and RNA encoding a SARS-CoV-2 spike protein or an immunogenic fragment thereof comprising one or more mutations from the omicron variant.

In some embodiments, BD ₂ comprises RNA encoding a SARS-CoV-2 spike protein or immunogenic fragment thereof from the Wuhan strain. In some embodiments, BD ₂ comprises RNA encoding a spike protein or immunogenic fragment thereof from a SARS-CoV-2 strain that is prevalent and/or spreading rapidly in the relevant jurisdiction. In some embodiments, BD _{2 comprises RNA encoding a SARS-CoV-2 spike protein or immunogenic fragment thereof that includes one or more mutations from an alpha variant. In some embodiments, BD 2 comprises} RNA encoding a SARS-CoV-2 spike protein or immunogenic fragment thereof that includes one or more mutations from a delta variant. In some embodiments, BD ₂ comprises RNA encoding a SARS-CoV-2 spike protein or immunogenic fragment thereof that includes one or more mutations from a beta variant. In some embodiments, BD ₂ comprises RNA encoding a SARS-CoV-2 spike protein or immunogenic fragment thereof that includes one or more mutations from an omicron variant. In some embodiments, BD ₂ comprises an RNA encoding a SARS-CoV-2 spike protein or an immunogenic fragment thereof from the Wuhan strain, and one or more additional RNAs encoding a spike protein or an immunogenic fragment thereof from a SARS-CoV-2 strain that is prevalent and/or spreading rapidly in the relevant jurisdiction. In some embodiments, BD ₂ comprises an RNA encoding a SARS-CoV-2 spike protein or an immunogenic fragment thereof from the Wuhan strain, and an RNA encoding a SARS-CoV-2 spike protein or an immunogenic fragment thereof that includes one or more mutations from an alpha variant. In some embodiments, BD ₂ comprises an RNA encoding a SARS-CoV-2 spike protein or an immunogenic fragment thereof from the Wuhan strain, and an RNA encoding a SARS-CoV-2 spike protein or an immunogenic fragment thereof that includes one or more mutations from a delta variant. In some embodiments, BD ₂ comprises RNA encoding a SARS-CoV-2 spike protein or an immunogenic fragment thereof from the Wuhan strain and RNA encoding a SARS-CoV-2 spike protein or an immunogenic fragment thereof comprising one or more mutations from a beta variant.In some embodiments, BD ₂ comprises RNA encoding a SARS-CoV-2 spike protein or an immunogenic fragment thereof from the Wuhan strain and RNA encoding a SARS-CoV-2 spike protein or an immunogenic fragment thereof comprising one or more mutations from an omicron variant.

In some embodiments, BD ₃ comprises RNA encoding a SARS-CoV-2 spike protein or immunogenic fragment thereof from the Wuhan strain. In some embodiments, BD ₃ comprises RNA encoding a spike protein or immunogenic fragment thereof from a SARS-CoV-2 strain that is prevalent and/or spreading rapidly in the relevant jurisdiction. In some embodiments, BD _{3 comprises RNA encoding a SARS-CoV-2 spike protein or immunogenic fragment thereof that includes one or more mutations from an alpha variant. In some embodiments, BD 3} comprises RNA encoding a SARS-CoV-2 spike protein or immunogenic fragment thereof that includes one or more mutations from a delta variant. In some embodiments, BD ₃ comprises RNA encoding a SARS-CoV-2 spike protein or immunogenic fragment thereof that includes one or more mutations from a beta variant. In some embodiments, BD ₃ comprises RNA encoding a SARS-CoV-2 spike protein or immunogenic fragment thereof that includes one or _more mutations from an omicron variant.

In some embodiments, BD ₃ comprises an RNA encoding a SARS-CoV-2 spike protein or an immunogenic fragment thereof from the Wuhan strain, and one or more additional RNAs encoding a spike protein or an immunogenic fragment thereof from a SARS-CoV-2 strain that is prevalent and/or spreading rapidly in the relevant jurisdiction. In some embodiments, BD ₃ comprises an RNA encoding a SARS-CoV-2 spike protein or an immunogenic fragment thereof from the Wuhan strain, and an RNA encoding a SARS-CoV-2 spike protein or an immunogenic fragment thereof that includes one or more mutations from an alpha variant. In some embodiments, BD ₃ comprises an RNA encoding a SARS-CoV-2 spike protein or an immunogenic fragment thereof from the Wuhan strain, and an RNA encoding a SARS-CoV-2 spike protein or an immunogenic fragment thereof that includes one or more mutations from a delta variant. In some embodiments, BD ₃ comprises RNA encoding a SARS-CoV-2 spike protein or an immunogenic fragment thereof from the Wuhan strain and RNA encoding a SARS-CoV-2 spike protein or an immunogenic fragment thereof comprising one or more mutations from the beta variant.In some embodiments, BD ₃ comprises RNA encoding a SARS-CoV-2 spike protein or an immunogenic fragment thereof from the Wuhan strain and RNA encoding a SARS-CoV-2 spike protein or an immunogenic fragment thereof comprising one or more mutations from the Omicron variant.

In some embodiments, BD _n comprises RNA encoding a SARS-CoV-2 spike protein or immunogenic fragment thereof from the Wuhan strain. In some embodiments, BD _n comprises RNA encoding a spike protein or immunogenic fragment thereof from a SARS-CoV-2 strain that is prevalent and/or spreading rapidly in a relevant jurisdiction. In some embodiments, BD _n comprises RNA encoding a SARS-CoV-2 spike protein or immunogenic fragment thereof that includes one or more mutations from an alpha variant. In some embodiments, BD _n comprises RNA encoding a SARS-CoV-2 spike protein or immunogenic fragment thereof that includes one or more mutations from a delta variant. In some embodiments, BD _n comprises RNA encoding a SARS-CoV-2 spike protein or immunogenic fragment thereof that includes one or more mutations from a beta variant. In some embodiments, BD _n comprises RNA encoding a SARS-CoV-2 spike protein or immunogenic fragment thereof that includes one or more mutations from an omicron variant.

In some embodiments, the BD _n comprises an RNA encoding a SARS-CoV-2 spike protein or an immunogenic fragment thereof from the Wuhan strain, and one or more additional RNAs encoding a spike protein or an immunogenic fragment thereof from a SARS-CoV-2 strain that is prevalent and/or spreading rapidly in the relevant jurisdiction. In some embodiments, the BD _n comprises an RNA encoding a SARS-CoV-2 spike protein or an immunogenic fragment thereof from the Wuhan strain, and an RNA encoding a SARS-CoV-2 spike protein or an immunogenic fragment thereof that includes one or more mutations from the alpha variant. In some embodiments, the BD _n comprises an RNA encoding a SARS-CoV-2 spike protein or an immunogenic fragment thereof from the Wuhan strain, and an RNA encoding a SARS-CoV-2 spike protein or an immunogenic fragment thereof that includes one or more mutations from the delta variant. In some embodiments, BD _n comprises RNA encoding a SARS-CoV-2 spike protein or an immunogenic fragment thereof from the Wuhan strain and RNA encoding a SARS-CoV-2 spike protein or an immunogenic fragment thereof comprising one or more mutations from the beta variant.In some embodiments, BD _n comprises RNA encoding a SARS-CoV-2 spike protein or an immunogenic fragment thereof from the Wuhan strain and RNA encoding a SARS-CoV-2 spike protein or an immunogenic fragment thereof comprising one or more mutations from the omicron variant.

In some embodiments, BD ₁ , BD ₂ , BD ₃ , and BD _n can each independently comprise a plurality (e.g., at least two) of RNA (e.g., mRNA) compositions as described herein. In some embodiments, BD ₁ , BD ₂ , BD ₃ , and BD _n can each independently comprise a first and a second RNA (e.g., mRNA) composition. In some embodiments, BD ₁ , BD ₂ , BD ₃ , and BD _n can each independently comprise a plurality (e.g., at least two) of RNA (e.g., mRNA) compositions, where at least one of the plurality of RNA (e.g., mRNA) compositions comprises BNT162b2 (e.g., as described herein). In some embodiments, at least one of the plurality of RNA (e.g., mRNA) compositions comprises RNA (e.g., mRNA) encoding a SARS-CoV-2 S protein or immunogenic fragment thereof from a different SARS-CoV-2 variant (e.g., a variant that is prevalent or spreading rapidly in a relevant jurisdiction, e.g., a variant disclosed herein). In some embodiments, at least one of the plurality of RNA (e.g., mRNA) compositions comprises RNA (e.g., mRNA) encoding a SARS-CoV-2 S protein or immunogenic fragment thereof from the Wuhan strain of SARS-CoV-2. In some embodiments, at least one of the plurality of RNA (e.g., mRNA) compositions comprises RNA encoding a SARS-CoV-2 S protein or immunogenic fragment thereof that includes one or more mutations from a variant that is prevalent and/or spreading rapidly in a relevant jurisdiction. In some embodiments, at least one of the plurality of RNA (e.g., mRNA) compositions comprises an RNA (e.g., mRNA) encoding a SARS-CoV-2 S protein or immunogenic fragment thereof that includes one or more mutations from an alpha variant. In some embodiments, at least one of the plurality of RNA (e.g., mRNA) compositions comprises an RNA (e.g., mRNA) encoding a SARS-CoV-2 S protein or immunogenic fragment thereof that includes one or more mutations from a delta variant. In some embodiments, at least one of the plurality of RNA (e.g., mRNA) compositions comprises an RNA (e.g., mRNA) encoding a SARS-CoV-2 S protein or immunogenic fragment thereof that includes one or more mutations from an omicron variant.

In some embodiments, the multiple RNA (e.g., mRNA) compositions provided with BD ₁ , BD ₂ , BD ₃ , and/or BD _n can each independently comprise at least two different mRNA constructs (e.g., RNA constructs having different protein coding sequences). For example, in some embodiments, the multiple RNA (e.g., mRNA) compositions provided with BD ₁ , BD ₂ , BD ₃ , and/or BD _n can each independently comprise an RNA (e.g., mRNA) encoding a SARS-CoV-2 S protein, or an immunogenic fragment thereof, from the Wuhan strain of SARS-CoV-2, as well as an RNA (e.g., mRNA) encoding a SARS-CoV-2 S protein, or an immunogenic fragment thereof, that includes one or more mutations from a variant that is prevalent and/or spreading rapidly in a relevant jurisdiction. In some embodiments, the multiple RNA (e.g., mRNA) compositions provided by BD ₁ , BD ₂ , BD ₃ , and/or BD _n can each independently comprise an RNA (e.g., mRNA) encoding a SARS-CoV-2 S protein or an immunogenic fragment thereof from the Wuhan strain of SARS-CoV-2, as well as an RNA (e.g., mRNA) encoding a SARS-CoV-2 S protein or an immunogenic fragment thereof that includes one or more mutations from a variant that is prevalent and/or rapidly spreading in the relevant jurisdiction. In some such embodiments, the variant can be an alpha variant. In some such embodiments, the variant can be a delta variant. In some such embodiments, the variant can be an omicron variant.

In some embodiments, the multiple RNA (e.g., mRNA) compositions provided with BD ₁ , BD ₂ , BD ₃ , and/or BD _n can each independently comprise at least two RNAs (e.g., mRNAs), each encoding a SARS-CoV-2 S protein or immunogenic fragment thereof that includes one or more mutations from a distinct variant that is prevalent and/or spreading rapidly in a relevant jurisdiction. In some embodiments, the multiple RNA (e.g., mRNA) compositions provided with BD ₁ , BD ₂ , BD ₃ , and/or BD _n can each independently comprise an mRNA encoding a SARS-CoV-2 S protein or immunogenic fragment thereof from an alpha variant, and an RNA (e.g., mRNA) encoding a SARS-CoV-2 S protein or immunogenic fragment thereof that includes one or more mutations from a delta variant. In some embodiments, a plurality of RNA (e.g., mRNA) compositions provided with BD ₁ , BD ₂ , BD ₃ , and/or BD _n can each independently comprise an RNA (e.g., mRNA) encoding a SARS-CoV-2 S protein or an immunogenic fragment thereof from the alpha variant, and an RNA (e.g., mRNA) encoding a SARS-CoV-2 S protein or an immunogenic fragment thereof comprising one or more mutations from the omicron variant. In some embodiments, a plurality of RNA (e.g., mRNA) compositions provided with BD ₁ , BD ₂ , BD ₃ , and/or BD _n can each independently comprise an RNA (e.g., mRNA) encoding a SARS-CoV-2 S protein or an immunogenic fragment thereof from the delta variant, and an RNA (e.g., mRNA) encoding a SARS-CoV-2 S protein or an immunogenic fragment thereof comprising one or more mutations from the omicron variant.

In some embodiments, multiple RNA (e.g., mRNA) compositions provided with _BD1 , _BD2 , _BD3 , and/or _BDn are separately administered to a subject via intramuscular injection, e.g., in some embodiments, at different injection sites. For example, in some embodiments, a first and a second RNA (e.g., mRNA) composition provided with _BD1 , _BD2 , _BD3 , and/or _BDn are separately administered to a subject via intramuscular injection into different arms.

In some embodiments, the length of time between BD ₁ and BD ₂ (BI ₁ ) is at least about 1 week, at least about 2 weeks, at least about 3 weeks, or at least about 4 weeks. In some embodiments, BI ₁ is from about 1 week to about 12 weeks. In some embodiments, BI ₁ is from about 1 week to about 10 weeks. In some embodiments, BI ₁ is from about 2 weeks to about 10 weeks. In some embodiments, BI ₁ is from about 2 weeks to about 8 weeks. In some embodiments, BI ₁ is from about 3 weeks to about 8 weeks. In some embodiments, BI ₁ is from about 4 weeks to about 8 weeks. In some embodiments, BI ₁ is from about 6 weeks to about 8 weeks. In some embodiments, BI ₁ is from about 3 to about 4 weeks. In some embodiments, BI ₁ is about 1 week. In some embodiments, BI ₁ is about 2 weeks. In some embodiments, BI ₁ is about 3 weeks. In some embodiments, BI ₁ is about 4 weeks. In some embodiments, BI ₁ is about 5 weeks. In some embodiments, BI ₁ is about 6 weeks. In some embodiments, BI ₁ is about 7 weeks. In some embodiments, BI ₁ is about 8 weeks. In some embodiments, BI ₁ is about 9 weeks. In some embodiments, BI ₁ is about 10 weeks.

In some embodiments, the length of time between BD ₂ and BD ₃ (BI ₂ ) is at least about 1 week, at least about 2 weeks, or at least about 3 weeks. In some embodiments, BI ₂ is from about 1 week to about 12 weeks. In some embodiments, BI ₂ is from about 1 week to about 10 weeks. In some embodiments, BI ₂ is from about 2 weeks to about 10 weeks. In some embodiments, BI ₂ is from about 2 weeks to about 8 weeks. In some embodiments, BI ₂ is from about 3 weeks to about 8 weeks. In some embodiments, BI ₂ is from about 4 weeks to about 8 weeks. In some embodiments, BI ₂ is from about 6 weeks to about 8 weeks. In some embodiments, BI ₂ is from about 3 to about 4 weeks. In some embodiments, BI ₂ is about 1 week. In some embodiments, BI ₂ is about 2 weeks. In some embodiments, BI ₂ is about 3 weeks. In some embodiments, BI ₂ is about 4 weeks. In some embodiments, BI ₂ is about 5 weeks. In some embodiments, BI ₂ is about 6 weeks. In some embodiments, BI ₂ is about 7 weeks. In some embodiments, BI ₂ is about 8 weeks. In some embodiments, BI ₂ is about 9 weeks. In some embodiments, BI ₂ is about 10 weeks.

In some embodiments, the length of time (BI _n ) between BD ₃ and any subsequent doses that are part of a booster dosing regimen, or between doses for any doses beyond BD ₃ , is each separately and independently selected from about 1 week or more, about 2 weeks or more, or about 3 weeks or more. In some embodiments, BI _n is about 1 week to about 12 weeks. In some embodiments, BI _n is about 1 week to about 10 weeks. In some embodiments, BI _n is about 2 weeks to about 10 weeks. In some embodiments, BI _n is about 2 weeks to about 8 weeks. In some embodiments, BI _n is about 3 weeks to about 8 weeks. In some embodiments, BI _n is about 4 weeks to about 8 weeks. In some embodiments, BI _n is about 6 weeks to about 8 weeks. In some embodiments, BI _n is about 3 to about 4 weeks. In some embodiments, BI _n is about 1 week. In some embodiments, BI _n is about 2 weeks. In some embodiments, BI _n is about 3 weeks. In some embodiments, BI _n is about 4 weeks. In some embodiments, BI _n is about 5 weeks. In some embodiments, BI _n is about 6 weeks. In some embodiments, BI _n is about 7 weeks. In some embodiments, BI _n is about 8 weeks. In some embodiments, BI _n is about 9 weeks. In some embodiments, BI _n is about 10 weeks.

In some embodiments, one or more compositions administered on BD ₁ are formulated in a Tris buffer. In some embodiments, one or more compositions administered on BD ₂ are formulated in a Tris buffer. In some embodiments, one or more compositions administered on BD ₃ are formulated in a Tris buffer. In some embodiments, one or more compositions administered on BD ₃ are formulated in a Tris buffer.

In some embodiments, the booster dosing regimen comprises administering two or more RNA (e.g., mRNA) compositions described herein, where at least two of the RNA (e.g., mRNA) compositions have different formulations. In some embodiments, the booster dosing regimen comprises BD ₁ and BD ₂ , where BD ₁ comprises administering RNA (e.g., mRNA) formulated in Tris buffer, and BD ₂ comprises administering RNA (e.g., mRNA) formulated in PBS buffer. In some embodiments, the booster dosing regimen comprises BD ₁ and BD ₂ , where BD ₁ comprises administering RNA (e.g., mRNA) formulated in PBS buffer, and BD ₂ comprises administering RNA (e.g., mRNA) formulated in Tris buffer.

In some embodiments, one or more RNA (e.g., mRNA) compositions provided in BD ₁ , BD ₂ , BD ₃ , and/or BD _n may be administered in conjunction with another vaccine. In some embodiments, the other vaccine is for a disease that is not COVID-19. In some embodiments, the disease is one that increases the deleterious effects of SARS-CoV-2 when a subject is co-infected with the disease and SARS-CoV-2. In some embodiments, the disease is one that increases the transmission rate of SARS-CoV-2 when a subject is co-infected with the disease and SARS-CoV-2. In some embodiments, the other vaccine is a different commercially available vaccine. In some embodiments, the different commercially available vaccine is an RNA vaccine. In some embodiments, the different commercially available vaccine is a polypeptide-based vaccine. In some embodiments, another vaccine (e.g., as described herein) and one or more RNA (e.g., mRNA) compositions provided in BD ₁ , BD ₂ , BD ₃ , and/or BD _n are administered separately, e.g., via intramuscular injection, in some embodiments, at different injection sites. For example, in some embodiments, an influenza vaccine described herein and one or more SARS-CoV-2 RNA (e.g., mRNA) compositions provided in BD ₁ , BD ₂ , BD ₃ , and/or BD _n are administered separately via intramuscular injection into different arms of a subject.

Additional Booster Regimens In some embodiments, the methods of vaccination disclosed herein include administering two or more booster dosing regimens. In some embodiments, it may be necessary to administer two or more booster dosing regimens to increase the neutralizing antibody response. In some embodiments, two or more booster dosing regimens may be required to combat SARS-CoV-2 strains that have been shown to be more likely to evade the immune response elicited by a vaccine that the patient previously received. In some embodiments, additional booster dosing regimens are administered to patients that have been determined to produce low concentrations of neutralizing antibodies. In some embodiments, additional booster dosing regimens are administered to patients (e.g., immunocompromised patients, cancer patients, and/or organ transplant patients) that have been determined to be more likely to be susceptible to SARS-CoV-2 infection despite previous vaccination.

The above description of the first booster dosing regimen also describes one or more additional booster dosing regimens. The time interval between the first booster dosing regimen and the second booster dosing regimen, or between subsequent booster dosing regimens, can be any of the above-mentioned acceptable time intervals between the primary dosing regimen and the first booster dosing regimen.

In some embodiments, the dosing regimen includes a primary regimen and a booster regimen, and at least one dose given in the primary regimen and/or booster regimen includes a composition comprising an RNA encoding an S protein or an immunogenic fragment thereof from a variant (e.g., an Omicron variant as described herein) that is prevalent or spreading rapidly in the relevant jurisdiction. For example, in some embodiments, the primary regimen includes at least two doses of BNT162b2 (e.g., encoding the Wuhan strain), given, for example, at least three weeks apart, and the booster regimen includes at least one dose of a composition comprising an RNA encoding an S protein or an immunogenic fragment thereof from a variant (e.g., an Omicron variant as described herein) that is prevalent or spreading rapidly in the relevant jurisdiction. In some such embodiments, such doses of the booster regimen may further include RNA encoding an S protein or an immunogenic fragment thereof from the Wuhan strain, which may be administered together with RNA encoding an S protein or an immunogenic fragment thereof from a variant that is prevalent or rapidly spreading in the relevant jurisdiction (e.g., an Omicron variant as described herein) as a single mixture or as two separate compositions, e.g., in a 1:1 weight ratio. In some embodiments, the booster regimen may also include at least one dose of BNT162b2, which may be administered as a first booster dose or a subsequent booster dose.

In some embodiments, the RNA composition described herein is given as a booster at a higher dose than the dose given during the primary regimen (primary dose) and/or the dose given for the first booster (if present). For example, in some embodiments, such a dose may be 60ug, or in some embodiments, such a dose may be higher than 30ug and lower than 60ug (e.g., 55ug, 50ug, or less). In some embodiments, the RNA composition described herein is given as a booster at least 3-12 months, or 4-12 months, or 5-12 months, or 6-12 months after the last dose (e.g., the last dose of the primary regimen or the first dose of the booster regimen). In some embodiments, the primary dose and/or the first booster dose (if present) may comprise BNT162b2 at, for example, 30ug per dose.

In some embodiments, the RNA compositions described herein include RNA encoding a polypeptide set forth in SEQ ID NO:49 or an immunogenic fragment thereof, or a variant thereof (e.g., having at least 70% or more (e.g., including at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or more) identity to SEQ ID NO:49). In some embodiments, the RNA compositions include RNA comprising a sequence of SEQ ID NO:50 or a variant thereof (e.g., having at least 70% or more (e.g., including at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or more) identity to SEQ ID NO:50). In some embodiments, the RNA compositions include RNA comprising a sequence of SEQ ID NO:51 or a variant thereof (e.g., having at least 70% or more (e.g., including at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or more) identity to SEQ ID NO:51).

In some embodiments, the RNA compositions described herein include RNA encoding a polypeptide set forth in SEQ ID NO:55 or an immunogenic fragment thereof, or a variant thereof (e.g., having at least 70% or more (e.g., including at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or more) identity to SEQ ID NO:55). In some embodiments, the RNA compositions include RNA comprising a sequence of SEQ ID NO:56 or a variant thereof (e.g., having at least 70% or more (e.g., including at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or more) identity to SEQ ID NO:56). In some embodiments, the RNA compositions include RNA comprising a sequence of SEQ ID NO:57 or a variant thereof (e.g., having at least 70% or more (e.g., including at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or more) identity to SEQ ID NO:57).

In some embodiments, the RNA compositions described herein include RNA encoding a polypeptide set forth in SEQ ID NO:58 or an immunogenic fragment thereof, or a variant thereof (e.g., having at least 70% or more (e.g., including at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or more) identity to SEQ ID NO:58). In some embodiments, the RNA compositions include RNA comprising a sequence of SEQ ID NO:59 or a variant thereof (e.g., having at least 70% or more (e.g., including at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or more) identity to SEQ ID NO:59). In some embodiments, the RNA compositions include RNA comprising a sequence of SEQ ID NO:60 or a variant thereof (e.g., having at least 70% or more (e.g., including at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or more) identity to SEQ ID NO:60).

In some embodiments, the RNA compositions described herein include RNA encoding a polypeptide set forth in SEQ ID NO:61 or an immunogenic fragment thereof, or a variant thereof (e.g., having at least 70% or more (e.g., including at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or more) identity to SEQ ID NO:61). In some embodiments, the RNA composition includes RNA comprising a sequence of SEQ ID NO:62 or a variant thereof (e.g., having at least 70% or more (e.g., including at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or more) identity to SEQ ID NO:62). In some embodiments, the RNA composition includes RNA comprising a sequence of SEQ ID NO:63 or a variant thereof (e.g., having at least 70% or more (e.g., including at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or more) identity to SEQ ID NO:63).

In some embodiments, the formulations disclosed herein can be used to implement any of the dosing regimens set forth in Table 28 (below).

In some embodiments of certain exemplary dosing regimens described in Table 28 above, an RNA composition described herein (e.g., comprising an RNA encoding a variant described herein) is given in the first dose of the primary regimen. In some embodiments of certain exemplary dosing regimens described in Table 28 above, an RNA composition described herein (e.g., comprising an RNA encoding a variant described herein) is given in the second dose of the primary regimen. In some embodiments of certain exemplary dosing regimens described in Table 28 above, an RNA composition described herein (e.g., comprising an RNA encoding a variant described herein) is given in the first and second doses of the primary regimen. In some embodiments of certain exemplary dosing regimens described in Table 28 above, an RNA composition described herein (e.g., comprising an RNA encoding a variant described herein) is given in the first dose of the booster regimen. In some embodiments of certain exemplary dosing regimens described in Table 28 above, an RNA composition described herein (e.g., comprising an RNA encoding a variant described herein) is given in the second dose of the booster regimen. In some embodiments of certain exemplary dosing regimens described in Table 28 above, an RNA composition described herein (e.g., comprising an RNA encoding a variant described herein) is given in the first and second doses of a booster regimen. In some embodiments of certain exemplary dosing regimens described in Table 28 above, an RNA composition described herein (e.g., comprising an RNA encoding a variant described herein) is given in the first and second doses of a primary regimen and at least one dose of a booster regimen. In some embodiments of certain exemplary dosing regimens described in Table 28 above, an RNA composition described herein (e.g., comprising an RNA encoding a variant described herein) is given in at least one dose (e.g., comprising at least two doses) of a booster regimen, and BNT162b2 is given in the primary regimen. In some embodiments of certain exemplary dosing regimens described in Table 28 above, an RNA composition described herein (e.g., comprising an RNA encoding a variant described herein) is given in the second dose of the booster regimen, and BNT162b2 is given in the primary regimen and in the first dose of the booster regimen. In some embodiments, an RNA composition described herein (e.g., comprising an RNA encoding a variant described herein) comprises an RNA encoding a polypeptide set forth in SEQ ID NO:49 or an immunogenic fragment thereof, or a variant thereof (e.g., having at least 70% or more (e.g., including at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or more) identity to SEQ ID NO:49). In some embodiments, the RNA compositions described herein (e.g., including RNA encoding a variant described herein) include RNA comprising a sequence of SEQ ID NO: 50 or a variant thereof (e.g., having at least 70% or more (e.g., including at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or more) identity to SEQ ID NO: 50). In some embodiments, the RNA compositions described herein (e.g., including RNA encoding a variant described herein) include RNA comprising a sequence of SEQ ID NO: 51 or a variant thereof (e.g., having at least 70% or more (e.g., including at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or more) identity to SEQ ID NO: 51).

In some embodiments, the RNA compositions described herein include RNA encoding a polypeptide set forth in SEQ ID NO:55 or an immunogenic fragment thereof, or a variant thereof (e.g., having at least 70% or more (e.g., including at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or more) identity to SEQ ID NO:55). In some embodiments, the RNA compositions include RNA comprising a sequence of SEQ ID NO:56 or a variant thereof (e.g., having at least 70% or more (e.g., including at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or more) identity to SEQ ID NO:56). In some embodiments, the RNA compositions include RNA comprising a sequence of SEQ ID NO:57 or a variant thereof (e.g., having at least 70% or more (e.g., including at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or more) identity to SEQ ID NO:57).

In some embodiments, the RNA compositions described herein include RNA encoding a polypeptide set forth in SEQ ID NO:58 or an immunogenic fragment thereof, or a variant thereof (e.g., having at least 70% or more (e.g., including at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or more) identity to SEQ ID NO:58). In some embodiments, the RNA compositions include RNA comprising a sequence of SEQ ID NO:59 or a variant thereof (e.g., having at least 70% or more (e.g., including at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or more) identity to SEQ ID NO:59). In some embodiments, the RNA compositions include RNA comprising a sequence of SEQ ID NO:60 or a variant thereof (e.g., having at least 70% or more (e.g., including at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or more) identity to SEQ ID NO:60).

In some embodiments, the RNA compositions described herein include RNA encoding a polypeptide set forth in SEQ ID NO:61 or an immunogenic fragment thereof, or a variant thereof (e.g., having at least 70% or more (e.g., including at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or more) identity to SEQ ID NO:61). In some embodiments, the RNA composition includes RNA comprising a sequence of SEQ ID NO:62 or a variant thereof (e.g., having at least 70% or more (e.g., including at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or more) identity to SEQ ID NO:62). In some embodiments, the RNA composition includes RNA comprising a sequence of SEQ ID NO:63 or a variant thereof (e.g., having at least 70% or more (e.g., including at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or more) identity to SEQ ID NO:63).

In some embodiments, such RNA compositions described herein (e.g., including RNA encoding a variant described herein) can further include RNA encoding an S protein or immunogenic fragment thereof of a different strain (e.g., Wuhan strain). By way of example, in some embodiments, the second dose of a booster regimen of Regimen Nos. 9-11 described in Table 28 above can include an RNA composition described herein (e.g., in one embodiment described in this embodiment, including RNA encoding a variant described herein, such as Omicron) and a BNT162b2 construct, e.g., in a 1:1 weight ratio.

In some embodiments of Regimen No. 6 as described in Table 28 above, the first and second doses of the primary regimen and the first and second doses of the booster regimen each comprise an RNA composition as described herein (e.g., RNA encoding a variant described herein, such as omicron, e.g., in one embodiment, RNA as described in this Example). In some such embodiments, the second dose of the booster regimen may not be necessary.

In some embodiments of Regimen No. 6 as described in Table 28 above, the first and second doses of the primary regimen and the first and second doses of the booster regimen each comprise an RNA composition as described herein (e.g., RNA encoding a variant described herein, such as omicron, e.g., in one embodiment, RNA as described in this Example). In some such embodiments, the second dose of the booster regimen may not be necessary.

In some embodiments of Regimen No. 6 as described in Table 28 above, the first and second doses of the primary regimen each include a BNT162b2 construct, and the first and second doses of the booster regimen each include an RNA composition described herein (e.g., RNA encoding a variant described herein, such as omicron, e.g., in one embodiment, RNA as described in this Example). In some such embodiments, the second dose of the booster regimen may not be necessary.

In some embodiments of Regimen No. 6 as described in Table 28 above, the first and second doses of the primary regimen and the first dose of the booster regimen each comprise a BNT162b2 construct, and the second dose of the booster regimen comprises an RNA composition as described herein (e.g., RNA encoding a variant described herein, such as omicron, e.g., in one embodiment, RNA as described in this Example).

Certain exemplary embodiments:
1. A composition or medical preparation comprising an RNA encoding an amino acid sequence comprising the SARS-CoV-2 S protein, an immunogenic variant thereof, or an immunogenic fragment of said SARS-CoV-2 S protein or said immunogenic variant thereof.

2. The composition or medical preparation of embodiment 1, wherein the immunogenic fragment of the SARS-CoV-2 S protein comprises the S1 subunit of the SARS-CoV-2 S protein or the receptor binding domain (RBD) of the S1 subunit of the SARS-CoV-2 S protein.

3. The composition or medical preparation of embodiment 1 or 2, wherein the amino acid sequence comprising the SARS-CoV-2 S protein, an immunogenic variant thereof, or an immunogenic fragment of the SARS-CoV-2 S protein or the immunogenic variant thereof is encoded by a coding sequence that is codon-optimized and/or has an increased G/C content compared to a wild-type coding sequence, and wherein the codon-optimization and/or the increase in the G/C content preferably does not change the sequence of the encoded amino acid sequence.

4.
(i) SARS-CoV-2 S protein, an immunogenic variant thereof, or the SARS-CoV-2 and/or (ii) the RNA encoding an immunogenic fragment of an S protein or said immunogenic variant thereof comprises the nucleotide sequence of nucleotides 979 to 1584 of SEQ ID NO:2, 8, or 9, a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of nucleotides 979 to 1584 of SEQ ID NO:2, 8, or 9, or a fragment of the nucleotide sequence of nucleotides 979 to 1584 of SEQ ID NO:2, 8, or 9, or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of nucleotides 979 to 1584 of SEQ ID NO:2, 8, or 9, and/or The composition or medical preparation of any one of embodiments 1 to 3, wherein the immunogenic fragment of S protein or said immunogenic variant thereof comprises the amino acid sequence of amino acids 327-528 of SEQ ID NO:1, an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to said amino acid sequence of amino acids 327-528 of SEQ ID NO:1, or an immunogenic fragment of said amino acid sequence of amino acids 327-528 of SEQ ID NO:1, or an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to said amino acid sequence of amino acids 327-528 of SEQ ID NO:1.

5.
(i) the RNA encoding a SARS-CoV-2 S protein, an immunogenic variant thereof, or an immunogenic fragment of the SARS-CoV-2 S protein or of the immunogenic variant thereof comprises the nucleotide sequence of nucleotides 111-986 of SEQ ID NO:30, a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of nucleotides 111-986 of SEQ ID NO:30, or a fragment of the nucleotide sequence of nucleotides 111-986 of SEQ ID NO:30, or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of nucleotides 111-986 of SEQ ID NO:30; and/or (ii) a SARS-CoV-2 5. The composition or medical preparation of any one of embodiments 1 to 4, wherein the S protein, immunogenic variant thereof, or immunogenic fragment of said SARS-CoV-2 S protein or said immunogenic variant thereof comprises the amino acid sequence of amino acids 20 to 311 of SEQ ID NO:29, an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to said amino acid sequence of amino acids 20 to 311 of SEQ ID NO:29, or an immunogenic fragment of said amino acid sequence of amino acids 20 to 311 of SEQ ID NO:29, or an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to said amino acid sequence of amino acids 20 to 311 of SEQ ID NO:29.

6.
(i) the RNA encoding a SARS-CoV-2 S protein, an immunogenic variant thereof, or an immunogenic fragment of the SARS-CoV-2 S protein or of the immunogenic variant thereof comprises the nucleotide sequence of nucleotides 49 to 3819 of SEQ ID NO:2, 8, or 9, a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of nucleotides 49 to 3819 of SEQ ID NO:2, 8, or 9, or a fragment of the nucleotide sequence of nucleotides 49 to 3819 of SEQ ID NO:2, 8, or 9, or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of nucleotides 49 to 3819 of SEQ ID NO:2, 8, or 9, and/or (ii) a SARS-CoV-2 6. The composition or medical preparation of any one of embodiments 1 to 5, wherein the S protein, immunogenic variant thereof, or immunogenic fragment of said SARS-CoV-2 S protein or said immunogenic variant thereof comprises the amino acid sequence of amino acids 17 to 1273 of SEQ ID NO: 1 or 7, an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to said amino acid sequence of amino acids 17 to 1273 of SEQ ID NO: 1 or 7, or an immunogenic fragment of said amino acid sequence of amino acids 17 to 1273 of SEQ ID NO: 1 or 7, or an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to said amino acid sequence of amino acids 17 to 1273 of SEQ ID NO: 1 or 7.

7. The composition or medical preparation of any one of embodiments 1 to 6, wherein the amino acid sequence comprising the SARS-CoV-2 S protein, an immunogenic variant thereof, or an immunogenic fragment of the SARS-CoV-2 S protein or the immunogenic variant thereof comprises a secretory signal peptide.

8. The composition or medical preparation of embodiment 7, wherein the secretory signal peptide is fused, preferably N-terminally, to the SARS-CoV-2 S protein, an immunogenic variant thereof, or an immunogenic fragment of the SARS-CoV-2 S protein or the immunogenic variant thereof.

9.
(i) the RNA encoding the secretory signal peptide is a nucleotide sequence of nucleotides 1 to 48 of SEQ ID NO: 2, 8, or 9, a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of nucleotides 1 to 48 of SEQ ID NO: 2, 8, or 9, or a fragment of the nucleotide sequence of nucleotides 1 to 48 of SEQ ID NO: 2, 8, or 9, or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of nucleotides 1 to 48 of SEQ ID NO: 2, 8, or 9. 9. The composition or medical preparation of embodiment 7 or 8, comprising said nucleotide sequence; and/or (ii) said secretory signal peptide comprises the amino acid sequence of amino acids 1 to 16 of SEQ ID NO:1, an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of amino acids 1 to 16 of SEQ ID NO:1, or an immunogenic fragment of the amino acid sequence of amino acids 1 to 16 of SEQ ID NO:1, or an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of amino acids 1 to 16 of SEQ ID NO:1.

10.
(i) the RNA encoding the SARS-CoV-2 S protein, an immunogenic variant thereof, or an immunogenic fragment of the SARS-CoV-2 S protein or of the immunogenic variant thereof comprises the nucleotide sequence of SEQ ID NO:6, a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of SEQ ID NO:6, or a fragment of the nucleotide sequence of SEQ ID NO:6, or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of SEQ ID NO:6; and/or (ii) the RNA encoding the SARS-CoV-2 S protein, an immunogenic variant thereof, or an immunogenic fragment of the SARS-CoV-2 S protein or of the immunogenic variant thereof comprises the nucleotide sequence of SEQ ID NO:6, a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of SEQ ID NO:6. 10. The composition or medical preparation of any one of the preceding embodiments, wherein the immunogenic fragment of S protein or said immunogenic variant thereof comprises the amino acid sequence of SEQ ID NO:5, an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to said amino acid sequence of SEQ ID NO:5, or an immunogenic fragment of said amino acid sequence of SEQ ID NO:5, or an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to said amino acid sequence of SEQ ID NO:5.

11.
(i) the RNA encoding the SARS-CoV-2 S protein, an immunogenic variant thereof, or an immunogenic fragment of the SARS-CoV-2 S protein or of the immunogenic variant thereof comprises the nucleotide sequence of nucleotides 54-986 of SEQ ID NO:30, a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of nucleotides 54-986 of SEQ ID NO:30, or a fragment of the nucleotide sequence of nucleotides 54-986 of SEQ ID NO:30, or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of nucleotides 54-986 of SEQ ID NO:30; and/or (ii) the RNA encoding the SARS-CoV-2 S protein, an immunogenic variant thereof, or an immunogenic fragment of the SARS-CoV-2 S protein or of the immunogenic variant thereof comprises the nucleotide sequence of nucleotides 54-986 of SEQ ID NO:30, 11. The composition or medical preparation of any one of embodiments 1 to 10, wherein the immunogenic fragment of S protein or said immunogenic variant thereof comprises the amino acid sequence of amino acids 1 to 311 of SEQ ID NO:29, an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to said amino acid sequence of amino acids 1 to 311 of SEQ ID NO:29, or an immunogenic fragment of said amino acid sequence of amino acids 1 to 311 of SEQ ID NO:29, or an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to said amino acid sequence of amino acids 1 to 311 of SEQ ID NO:29.

12. The composition or medical preparation according to any one of the preceding embodiments, wherein the RNA comprises a modified nucleoside instead of uridine, in particular the modified nucleoside is selected from pseudouridine (ψ), N1-methyl-pseudouridine (m1ψ), and 5-methyl-uridine (m5U), in particular the modified nucleoside is N1-methyl-pseudouridine (m1ψ).

13. The composition or medical preparation of any one of embodiments 1 to 12, wherein the RNA comprises a 5' cap.

14. The composition or medical preparation of any one of embodiments 1 to 13, wherein the RNA encoding an amino acid sequence comprising a SARS-CoV-2 S protein, an immunogenic variant thereof, or an immunogenic fragment of the SARS-CoV-2 S protein or the immunogenic variant thereof comprises a 5'UTR comprising a nucleotide sequence of SEQ ID NO: 12, or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of SEQ ID NO: 12.

15. The composition or medical preparation of any one of embodiments 1 to 14, wherein the RNA encoding an amino acid sequence comprising a SARS-CoV-2 S protein, an immunogenic variant thereof, or an immunogenic fragment of the SARS-CoV-2 S protein or the immunogenic variant thereof comprises a 3'UTR comprising a nucleotide sequence of SEQ ID NO: 13, or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of SEQ ID NO: 13.

16. The composition or medical preparation of any one of embodiments 1 to 15, wherein the RNA encoding an amino acid sequence comprising a SARS-CoV-2 S protein, an immunogenic variant thereof, or an immunogenic fragment of the SARS-CoV-2 S protein or the immunogenic variant thereof comprises a polyA sequence.

17. The composition or medical preparation of embodiment 16, wherein the polyA sequence comprises at least 100 nucleotides.

18. The composition or medical preparation of embodiment 16 or 17, wherein the polyA sequence comprises or consists of the nucleotide sequence of SEQ ID NO: 14.

19. The composition or medical preparation of any one of embodiments 1 to 18, wherein the RNA is or is to be formulated as a liquid, solid, or a combination thereof.

20. The composition or medical preparation according to any one of the preceding embodiments, wherein said RNA is formulated or is to be formulated for injection.

21. The composition or medical preparation of any one of embodiments 1 to 20, wherein the RNA is or is to be formulated for intramuscular administration.

22. The composition or medical preparation of any one of embodiments 1 to 21, wherein the RNA is or is to be formulated as a particle.

23. The composition or medical preparation of embodiment 22, wherein the particle is a lipid nanoparticle (LNP) or lipoplex (LPX) particle.

24. The composition or medical preparation of embodiment 23, wherein the LNP particles comprise ((4-hydroxybutyl)azanediyl)bis(hexane-6,1-diyl)bis(2-hexyldecanoate), 2-[(polyethylene glycol)-2000]-N,N-ditetradecylacetamide, 1,2-distearoyl-sn-glycero-3-phosphocholine, and cholesterol.

25. The composition or medical preparation of embodiment 23, wherein the RNA lipoplex particles are obtainable by mixing the RNA with liposomes.

26. The composition or medical preparation of any one of embodiments 1 to 25, wherein the RNA is mRNA or saRNA.

27. A composition or medical preparation according to any one of embodiments 1 to 26, which is a pharmaceutical composition.

28. The composition or medical preparation of any one of embodiments 1 to 27, which is a vaccine.

29. The composition or medical preparation of embodiment 27 or 28, wherein the pharmaceutical composition further comprises one or more pharma- ceutically acceptable carriers, diluents, and/or excipients.

30. A composition or medical preparation according to any one of embodiments 1 to 26, which is a kit.

31. The composition or medical preparation of embodiment 30, wherein the RNA and optionally the particle-forming component are in separate vials.

32. The composition or medical preparation of embodiment 30 or 31, further comprising instructions for use of the composition or medical preparation to induce an immune response against coronavirus in a subject.

33. A composition or medical preparation according to any one of embodiments 1 to 32 for pharmaceutical use.

34. The composition or medical preparation of embodiment 33, wherein the pharmaceutical use comprises inducing an immune response against coronavirus in a subject.

35. The composition or medical preparation of embodiment 33 or 34, wherein the pharmaceutical use comprises therapeutic or prophylactic treatment of a coronavirus infection.

36. A composition or medical preparation according to any one of embodiments 1 to 35, for administration to a human.

37. The composition or medical preparation of any one of embodiments 32 to 36, wherein the coronavirus is a betacoronavirus.

38. The composition or medical preparation of any one of embodiments 32 to 37, wherein the coronavirus is a sarbecovirus.

39. The composition or medical preparation of any one of embodiments 32 to 38, wherein the coronavirus is SARS-CoV-2.

40. A method of inducing an immune response to a coronavirus in a subject, the method comprising administering to the subject a composition comprising RNA encoding an amino acid sequence comprising a SARS-CoV-2 S protein, an immunogenic variant thereof, or an immunogenic fragment of the SARS-CoV-2 S protein or the immunogenic variant thereof.

41. The method of embodiment 40, wherein the immunogenic fragment of the SARS-CoV-2 S protein comprises the S1 subunit of the SARS-CoV-2 S protein or the receptor binding domain (RBD) of the S1 subunit of the SARS-CoV-2 S protein.

42. The method of embodiment 40 or 41, wherein the amino acid sequence comprising the SARS-CoV-2 S protein, an immunogenic variant thereof, or an immunogenic fragment of the SARS-CoV-2 S protein or the immunogenic variant thereof is encoded by a coding sequence that is codon-optimized and/or has an increased G/C content compared to a wild-type coding sequence, and the codon optimization and/or the increase in G/C content preferably does not change the sequence of the encoded amino acid sequence.

43.
(i) SARS-CoV-2 S protein, an immunogenic variant thereof, or the SARS-CoV-2 and/or (ii) the RNA encoding an immunogenic fragment of an S protein or said immunogenic variant thereof comprises the nucleotide sequence of nucleotides 979 to 1584 of SEQ ID NO: 2, 8, or 9, a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of nucleotides 979 to 1584 of SEQ ID NO: 2, 8, or 9, or a fragment of the nucleotide sequence of nucleotides 979 to 1584 of SEQ ID NO: 2, 8, or 9, or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of nucleotides 979 to 1584 of SEQ ID NO: 2, 8, or 9. The method of any one of embodiments 40-42, wherein the immunogenic fragment of S protein or said immunogenic variant thereof comprises the amino acid sequence of amino acids 327-528 of SEQ ID NO:1, an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to said amino acid sequence of amino acids 327-528 of SEQ ID NO:1, or an immunogenic fragment of said amino acid sequence of amino acids 327-528 of SEQ ID NO:1, or an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to said amino acid sequence of amino acids 327-528 of SEQ ID NO:1.

44.
(i) the RNA encoding a SARS-CoV-2 S protein, an immunogenic variant thereof, or an immunogenic fragment of the SARS-CoV-2 S protein or of the immunogenic variant thereof comprises the nucleotide sequence of nucleotides 111-986 of SEQ ID NO:30, a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of nucleotides 111-986 of SEQ ID NO:30, or a fragment of the nucleotide sequence of nucleotides 111-986 of SEQ ID NO:30, or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of nucleotides 111-986 of SEQ ID NO:30, and/or (ii) a SARS-CoV-2 The method of any one of embodiments 40-43, wherein the S protein, immunogenic variant thereof, or immunogenic fragment of the SARS-CoV-2 S protein or immunogenic variant thereof comprises the amino acid sequence of amino acids 20-311 of SEQ ID NO:29, an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of amino acids 20-311 of SEQ ID NO:29, or an immunogenic fragment of the amino acid sequence of amino acids 20-311 of SEQ ID NO:29, or an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of amino acids 20-311 of SEQ ID NO:29.

45.
(i) the RNA encoding a SARS-CoV-2 S protein, an immunogenic variant thereof, or an immunogenic fragment of the SARS-CoV-2 S protein or of the immunogenic variant thereof comprises the nucleotide sequence of nucleotides 49 to 3819 of SEQ ID NO:2, 8, or 9, a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of nucleotides 49 to 3819 of SEQ ID NO:2, 8, or 9, or a fragment of the nucleotide sequence of nucleotides 49 to 3819 of SEQ ID NO:2, 8, or 9, or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of nucleotides 49 to 3819 of SEQ ID NO:2, 8, or 9, and/or (ii) a SARS-CoV-2 The method of any one of embodiments 40-44, wherein the S protein, immunogenic variant thereof, or immunogenic fragment of the SARS-CoV-2 S protein or immunogenic variant thereof comprises the amino acid sequence of amino acids 17-1273 of SEQ ID NO: 1 or 7, an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of amino acids 17-1273 of SEQ ID NO: 1 or 7, or an immunogenic fragment of the amino acid sequence of amino acids 17-1273 of SEQ ID NO: 1 or 7, or an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of amino acids 17-1273 of SEQ ID NO: 1 or 7.

46. The method of any one of embodiments 40 to 45, wherein the amino acid sequence comprising the SARS-CoV-2 S protein, an immunogenic variant thereof, or an immunogenic fragment of the SARS-CoV-2 S protein or the immunogenic variant thereof comprises a secretory signal peptide.

47. The method of embodiment 46, wherein the secretory signal peptide is fused, preferably N-terminally, to the SARS-CoV-2 S protein, an immunogenic variant thereof, or an immunogenic fragment of the SARS-CoV-2 S protein or the immunogenic variant thereof.

48.
(i) the RNA encoding the secretory signal peptide is a nucleotide sequence of nucleotides 1 to 48 of SEQ ID NO: 2, 8, or 9, a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of nucleotides 1 to 48 of SEQ ID NO: 2, 8, or 9, or a fragment of the nucleotide sequence of nucleotides 1 to 48 of SEQ ID NO: 2, 8, or 9, or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of nucleotides 1 to 48 of SEQ ID NO: 2, 8, or 9. and/or (ii) the secretory signal peptide comprises an amino acid sequence of amino acids 1 to 16 of SEQ ID NO:1, an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of amino acids 1 to 16 of SEQ ID NO:1, or an immunogenic fragment of the amino acid sequence of amino acids 1 to 16 of SEQ ID NO:1, or an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of amino acids 1 to 16 of SEQ ID NO:1.

49.
(i) the RNA encoding the SARS-CoV-2 S protein, an immunogenic variant thereof, or an immunogenic fragment of the SARS-CoV-2 S protein or of the immunogenic variant thereof comprises the nucleotide sequence of SEQ ID NO:6, a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of SEQ ID NO:6, or a fragment of the nucleotide sequence of SEQ ID NO:6, or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of SEQ ID NO:6; and/or (ii) the RNA encoding the SARS-CoV-2 S protein, an immunogenic variant thereof, or an immunogenic fragment of the SARS-CoV-2 S protein or of the immunogenic variant thereof comprises the nucleotide sequence of SEQ ID NO:6, a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of SEQ ID NO:6. The method of any one of embodiments 40-48, wherein the immunogenic fragment of S protein or said immunogenic variant thereof comprises the amino acid sequence of SEQ ID NO:5, an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of SEQ ID NO:5, or an immunogenic fragment of the amino acid sequence of SEQ ID NO:5, or an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of SEQ ID NO:5.

50.
(i) the RNA encoding the SARS-CoV-2 S protein, an immunogenic variant thereof, or an immunogenic fragment of the SARS-CoV-2 S protein or of the immunogenic variant thereof comprises the nucleotide sequence of nucleotides 54-986 of SEQ ID NO:30, a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of nucleotides 54-986 of SEQ ID NO:30, or a fragment of the nucleotide sequence of nucleotides 54-986 of SEQ ID NO:30, or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of nucleotides 54-986 of SEQ ID NO:30; and/or (ii) the RNA encoding the SARS-CoV-2 S protein, an immunogenic variant thereof, or an immunogenic fragment of the SARS-CoV-2 S protein or of the immunogenic variant thereof comprises the nucleotide sequence of nucleotides 54-986 of SEQ ID NO:30, 50. The method of any one of embodiments 40-49, wherein the immunogenic fragment of S protein or said immunogenic variant thereof comprises the amino acid sequence of amino acids 1 to 311 of SEQ ID NO:29, an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to said amino acid sequence of amino acids 1 to 311 of SEQ ID NO:29, or an immunogenic fragment of said amino acid sequence of amino acids 1 to 311 of SEQ ID NO:29, or said amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to said amino acid sequence of amino acids 1 to 311 of SEQ ID NO:29.

51. The method of any one of embodiments 40 to 49, wherein the RNA comprises a modified nucleoside instead of uridine, in particular the modified nucleoside is selected from pseudouridine (ψ), N1-methyl-pseudouridine (m1ψ), and 5-methyl-uridine (m5U), in particular the modified nucleoside is N1-methyl-pseudouridine (m1ψ).

52. The method of any one of embodiments 40 to 51, wherein the RNA comprises a cap.

53. The method of any one of embodiments 40 to 52, wherein the RNA encoding an amino acid sequence comprising a SARS-CoV-2 S protein, an immunogenic variant thereof, or an immunogenic fragment of the SARS-CoV-2 S protein or the immunogenic variant thereof comprises a 5'UTR comprising a nucleotide sequence of SEQ ID NO: 12 or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of SEQ ID NO: 12.

54. The method of any one of embodiments 40 to 53, wherein the RNA encoding an amino acid sequence comprising a SARS-CoV-2 S protein, an immunogenic variant thereof, or an immunogenic fragment of the SARS-CoV-2 S protein or the immunogenic variant thereof comprises a 3'UTR comprising a nucleotide sequence of SEQ ID NO: 13 or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of SEQ ID NO: 13.

55. The method of any one of embodiments 40 to 54, wherein the RNA encoding an amino acid sequence comprising a SARS-CoV-2 S protein, an immunogenic variant thereof, or an immunogenic fragment of the SARS-CoV-2 S protein or the immunogenic variant thereof comprises a polyA sequence.

56. The method of embodiment 55, wherein the polyA sequence comprises at least 100 nucleotides.

57. The method of embodiment 55 or 56, wherein the polyA sequence comprises or consists of the nucleotide sequence of SEQ ID NO: 14.

58. The method of any one of embodiments 40 to 57, wherein the RNA is formulated as a liquid, a solid, or a combination thereof.

59. The method of any one of embodiments 40 to 58, wherein the RNA is administered by injection.

60. The method of any one of embodiments 40 to 59, wherein the RNA is administered by intramuscular administration.

61. The method of any one of embodiments 40 to 60, wherein the RNA is formulated as particles.

62. The method of embodiment 61, wherein the particle is a lipid nanoparticle (LNP) or lipoplex (LPX) particle.

63. The method of embodiment 62, wherein the LNP particles comprise ((4-hydroxybutyl)azanediyl)bis(hexane-6,1-diyl)bis(2-hexyldecanoate), 2-[(polyethylene glycol)-2000]-N,N-ditetradecylacetamide, 1,2-distearoyl-sn-glycero-3-phosphocholine, and cholesterol.

64. The method of embodiment 62, wherein the RNA lipoplex particles are obtainable by mixing the RNA with liposomes.

65. The composition or medical preparation of any one of embodiments 40 to 64, wherein the RNA is mRNA or saRNA.

66. The method of any one of embodiments 40 to 65, which is a method for vaccination against coronavirus.

67. The method of any one of embodiments 40 to 66, which is a method for therapeutic or prophylactic treatment of a coronavirus infection.

68. The method of any one of embodiments 40 to 67, wherein the subject is a human.

69. The method of any one of embodiments 40 to 68, wherein the coronavirus is a betacoronavirus.

70. The method of any one of embodiments 40 to 69, wherein the coronavirus is a sarbecovirus.

71. The method of any one of embodiments 40 to 70, wherein the coronavirus is SARS-CoV-2.

72. The method of any one of embodiments 40 to 71, wherein the composition is a composition described in any one of embodiments 1 to 39.

73. A composition or medical preparation according to any one of embodiments 1 to 39 for use in a method according to any one of embodiments 40 to 72.

74. An immunogenic composition comprising a lipid nanoparticle (LNP) comprising RNA, wherein the RNA encodes a polypeptide of SEQ ID NO: 49 and comprises a nucleotide sequence of SEQ ID NO: 50 or a nucleotide sequence that is at least 80% identical (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 97%, at least 98%, or more than 99%) to SEQ ID NO: 50, wherein the RNA is
(a) a modified uridine,
(b) comprising a 5'cap;
The immunogenic composition, wherein the LNP comprises ((4-hydroxybutyl)azanediyl)bis(hexane-6,1-diyl)bis(2-hexyldecanoate), 2-[(polyethylene glycol)-2000]-N,N-ditetradecylacetamide, 1,2-distearoyl-sn-glycero-3-phosphocholine, and cholesterol.

75. The immunogenic composition of embodiment 74, wherein the nucleotide sequence includes modified uridines in place of all uridines.

76. The immunogenic composition of embodiment 74 or 75, wherein each of the modified uridines is N1-methyl-pseudouridine.

77. The RNA has the following characteristics:
5' untranslated region (UTR) comprising SEQ ID NO: 12;
77. The immunogenic composition of any one of embodiments 74-76, further comprising: a 3' untranslated region (UTR) comprising SEQ ID NO: 13; and at least one, at least two, or all of a polyA sequence of at least 100 A nucleotides.

78. The immunogenic composition of embodiment 77, wherein the polyA sequence comprises 30 adenine nucleotides followed by 70 adenine nucleotides, the 30 adenine nucleotides and the 70 adenine nucleotides being separated by a linker sequence.

79. The immunogenic composition of embodiment 77, wherein the polyA sequence comprises SEQ ID NO: 14.

80. The immunogenic composition of any one of embodiments 76 to 79, wherein the RNA comprises SEQ ID NO:51.

81. An immunogenic composition comprising a lipid nanoparticle (LNP) comprising RNA, wherein the RNA encodes a polypeptide of SEQ ID NO:55 and comprises a nucleotide sequence of SEQ ID NO:56 or a nucleotide sequence that is at least 80% (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 97%, at least 98%, or more than 99%) identical to SEQ ID NO:56, wherein the RNA is
(a) a modified uridine,
(b) comprises a 5'cap;
The immunogenic composition, wherein the LNP comprises ((4-hydroxybutyl)azanediyl)bis(hexane-6,1-diyl)bis(2-hexyldecanoate), 2-[(polyethylene glycol)-2000]-N,N-ditetradecylacetamide, 1,2-distearoyl-sn-glycero-3-phosphocholine, and cholesterol.

82. The immunogenic composition of embodiment 81, wherein the nucleotide sequence includes modified uridines in place of all uridines.

83. The immunogenic composition of embodiment 81 or 82, wherein each of the modified uridines is N1-methyl-pseudouridine.

84. The RNA has the following characteristics:
5' untranslated region (UTR) comprising SEQ ID NO: 12;
The immunogenic composition of any one of embodiments 81-83, further comprising a 3' untranslated region (UTR) comprising SEQ ID NO: 13, and at least one, at least two, or all of a polyA sequence of at least 100 A nucleotides.

85. The immunogenic composition of embodiment 84, wherein the polyA sequence comprises 30 adenine nucleotides followed by 70 adenine nucleotides, the 30 adenine nucleotides and the 70 adenine nucleotides being separated by a linker sequence.

86. The immunogenic composition of embodiment 85, wherein the polyA sequence comprises SEQ ID NO:14.

87. The immunogenic composition of any one of embodiments 81 to 86, wherein the RNA comprises SEQ ID NO:57.

88. An immunogenic composition comprising a lipid nanoparticle (LNP) comprising RNA, wherein the RNA encodes a polypeptide of SEQ ID NO:58 and comprises a nucleotide sequence of SEQ ID NO:59 or a nucleotide sequence that is at least 80% (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 97%, at least 98%, or more than 99%) identical to SEQ ID NO:59, wherein the RNA is
(a) a modified uridine,
(b) comprises a 5'cap;
The immunogenic composition, wherein the LNP comprises ((4-hydroxybutyl)azanediyl)bis(hexane-6,1-diyl)bis(2-hexyldecanoate), 2-[(polyethylene glycol)-2000]-N,N-ditetradecylacetamide, 1,2-distearoyl-sn-glycero-3-phosphocholine, and cholesterol.

89. The immunogenic composition of embodiment 88, wherein the nucleotide sequence includes modified uridines in place of all uridines.

90. The immunogenic composition of embodiment 88 or 89, wherein each of the modified uridines is N1-methyl-pseudouridine.

91. The RNA has the following characteristics:
5' untranslated region (UTR) comprising SEQ ID NO: 12;
91. The immunogenic composition of any one of embodiments 88-90, further comprising: a 3' untranslated region (UTR) comprising SEQ ID NO: 13; and at least one, at least two, or all of a polyA sequence of at least 100 A nucleotides.

92. The immunogenic composition of embodiment 91, wherein the polyA sequence comprises 30 adenine nucleotides followed by 70 adenine nucleotides, the 30 adenine nucleotides and the 70 adenine nucleotides being separated by a linker sequence.

93. The immunogenic composition of embodiment 92, wherein the polyA sequence comprises SEQ ID NO:14.

94. The immunogenic composition of any one of embodiments 88 to 93, wherein the RNA comprises SEQ ID NO: 60.

95. An immunogenic composition comprising a lipid nanoparticle (LNP) comprising RNA, wherein the RNA encodes a polypeptide of SEQ ID NO:61 and comprises a nucleotide sequence of SEQ ID NO:62 or a nucleotide sequence that is at least 80% (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 97%, at least 98%, or more than 99%) identical to SEQ ID NO:62, and wherein the RNA is
(a) a modified uridine,
(b) comprising a 5'cap;
The immunogenic composition, wherein the LNP comprises ((4-hydroxybutyl)azanediyl)bis(hexane-6,1-diyl)bis(2-hexyldecanoate), 2-[(polyethylene glycol)-2000]-N,N-ditetradecylacetamide, 1,2-distearoyl-sn-glycero-3-phosphocholine, and cholesterol.

96. The immunogenic composition of embodiment 95, wherein the nucleotide sequence includes modified uridines in place of all uridines.

97. The immunogenic composition of embodiment 95 or 96, wherein each of the modified uridines is N1-methyl-pseudouridine.

98. The RNA has the following characteristics:
5' untranslated region (UTR) comprising SEQ ID NO: 12;
98. The immunogenic composition of any one of embodiments 95-97, further comprising a 3' untranslated region (UTR) comprising SEQ ID NO:13, and at least one, at least two, or all of a polyA sequence of at least 100 A nucleotides.

99. The immunogenic composition of embodiment 98, wherein the polyA sequence comprises 30 adenine nucleotides followed by 70 adenine nucleotides, the 30 adenine nucleotides and the 70 adenine nucleotides being separated by a linker sequence.

100. The immunogenic composition of embodiment 99, wherein the polyA sequence comprises SEQ ID NO:14.

101. The immunogenic composition of any one of embodiments 95 to 100, wherein the RNA comprises SEQ ID NO: 63.

102. An immunogenic composition comprising a first RNA and a second RNA,
the first RNA encodes a polypeptide of SEQ ID NO:7 and comprises a nucleotide sequence of SEQ ID NO:9 or a nucleotide sequence that is at least 80% identical (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 97%, at least 98%, or 99% or more) to said SEQ ID NO:9;
the second RNA encodes a polypeptide of SEQ ID NO:49 and comprises a nucleotide sequence of SEQ ID NO:50, or a nucleotide sequence that is at least 80% identical (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 97%, at least 98%, or 99% or more) to SEQ ID NO:50;
Each of the first RNA and the second RNA is
(a) a modified uridine, and (b) a 5' cap,
The immunogenic composition, wherein the first RNA and the second RNA are formulated in a lipid nanoparticle (LNP), and the LNP comprises ((4-hydroxybutyl)azanediyl)bis(hexane-6,1-diyl)bis(2-hexyldecanoate), 2-[(polyethylene glycol)-2000]-N,N-ditetradecylacetamide, 1,2-distearoyl-sn-glycero-3-phosphocholine, and cholesterol.

103. The immunogenic composition of embodiment 102, wherein the first RNA and the second RNA are formulated in the same lipid nanoparticle.

104. The immunogenic composition of embodiment 102, wherein the first RNA and the second RNA are formulated in separate lipid nanoparticles.

105. The immunogenic composition of any one of embodiments 102 to 104, wherein each of the first RNA and the second RNA comprises modified uridines in place of all uridines.

106. The immunogenic composition of any one of embodiments 102 to 105, wherein each of the modified uridines is N1-methyl-pseudouridine.

107. The first RNA and the second RNA each independently have the following characteristics:
5' untranslated region (UTR) comprising SEQ ID NO: 12;
107. The immunogenic composition of any one of embodiments 102-106, further comprising a 3' untranslated region (UTR) comprising SEQ ID NO: 13, and at least one, at least two, or all of a polyA sequence of at least 100 A nucleotides.

108. The immunogenic composition of embodiment 107, wherein the polyA sequence comprises 30 adenine nucleotides followed by 70 adenine nucleotides, the 30 adenine nucleotides and the 70 adenine nucleotides being separated by a linker sequence.

109. The immunogenic composition of embodiment 107, wherein the polyA sequence comprises SEQ ID NO: 14.

110. The immunogenic composition of any one of embodiments 102 to 109, wherein the first RNA comprises SEQ ID NO: 20 and the second RNA comprises SEQ ID NO: 51.

111. An immunogenic composition comprising a first RNA and a second RNA,
the first RNA encodes a polypeptide of SEQ ID NO:7 and comprises a nucleotide sequence of SEQ ID NO:9 or a nucleotide sequence that is at least 80% identical (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 97%, at least 98%, or 99% or more) to said SEQ ID NO:9;
the second RNA encodes a polypeptide of SEQ ID NO: 55, 58, or 61 and comprises a nucleotide sequence of SEQ ID NO: 56, 59, or 62, or a nucleotide sequence that is at least 80% identical (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 97%, at least 98%, or 99% or more) to said SEQ ID NO: 56, 59, or 62;
Each of the first RNA and the second RNA is
(a) a modified uridine, and (b) a 5' cap,
The immunogenic composition, wherein the first RNA and the second RNA are formulated in a lipid nanoparticle (LNP), and the LNP comprises ((4-hydroxybutyl)azanediyl)bis(hexane-6,1-diyl)bis(2-hexyldecanoate), 2-[(polyethylene glycol)-2000]-N,N-ditetradecylacetamide, 1,2-distearoyl-sn-glycero-3-phosphocholine, and cholesterol.

112. The immunogenic composition of embodiment 111, wherein the first RNA and the second RNA are formulated in separate lipid nanoparticles.

113. The immunogenic composition of embodiment 111, wherein the first RNA and the second RNA are formulated in the same lipid nanoparticle.

114. The immunogenic composition of any one of embodiments 111 to 113, wherein the first RNA and the second RNA each contain modified uridines in place of all uridines.

115. The immunogenic composition of any one of embodiments 111 to 114, wherein each of the modified uridines is N1-methyl-pseudouridine.

116. The first RNA and the second RNA each independently have the following characteristics:
5' untranslated region (UTR) comprising SEQ ID NO: 12;
116. The immunogenic composition of any one of embodiments 111-115, further comprising: a 3' untranslated region (UTR) comprising SEQ ID NO: 13; and at least one, at least two, or all of a polyA sequence of at least 100 A nucleotides.

117. The immunogenic composition of any one of embodiments 111 to 116, wherein the polyA sequence comprises 30 adenine nucleotides followed by 70 adenine nucleotides, the 30 adenine nucleotides and the 70 adenine nucleotides being separated by a linker sequence.

118. The immunogenic composition of embodiment 117, wherein the polyA sequence comprises SEQ ID NO: 14.

119. The immunogenic composition of any one of embodiments 111 to 118, wherein the first RNA comprises SEQ ID NO: 9 and the second RNA comprises SEQ ID NO: 56.

120. The immunogenic composition of any one of embodiments 111 to 118, wherein the first RNA comprises SEQ ID NO: 9 and the second RNA comprises SEQ ID NO: 59.

121. The immunogenic composition of any one of embodiments 111 to 118, wherein the first RNA comprises SEQ ID NO: 9 and the second RNA comprises SEQ ID NO: 62.

122. The immunogenic composition of any one of embodiments 111 to 118, wherein the first RNA comprises SEQ ID NO: 20 and the second RNA comprises SEQ ID NO: 57.

123. The immunogenic composition of any one of embodiments 111 to 118, wherein the first RNA comprises SEQ ID NO: 20 and the second RNA comprises SEQ ID NO: 60.

124. The immunogenic composition of any one of embodiments 111 to 118, wherein the first RNA comprises SEQ ID NO: 20 and the second RNA comprises SEQ ID NO: 63.

125. An immunogenic composition comprising a first RNA and a second RNA,
the first RNA encodes a polypeptide of SEQ ID NO:58 and comprises a nucleotide sequence of SEQ ID NO:59 or a nucleotide sequence that is at least 80% identical (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 97%, at least 98%, or 99% or more) to said SEQ ID NO:59;
the second RNA encodes a polypeptide of SEQ ID NO: 49, 55, or 61 and comprises a nucleotide sequence of SEQ ID NO: 50, 56, or 62, or a nucleotide sequence that is at least 80% identical (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 97%, at least 98%, or 99% or more) to said SEQ ID NO: 50, 56, or 62;
Each of the first RNA and the second RNA is
(a) a modified uridine, and (b) a 5' cap,
The immunogenic composition, wherein the first RNA and the second RNA are formulated in a lipid nanoparticle (LNP), and the LNP comprises ((4-hydroxybutyl)azanediyl)bis(hexane-6,1-diyl)bis(2-hexyldecanoate), 2-[(polyethylene glycol)-2000]-N,N-ditetradecylacetamide, 1,2-distearoyl-sn-glycero-3-phosphocholine, and cholesterol.

126. The immunogenic composition of embodiment 125, wherein the first RNA and the second RNA are formulated in separate lipid nanoparticles.

127. The immunogenic composition of embodiment 125, wherein the first RNA and the second RNA are formulated in the same lipid nanoparticle.

128. The immunogenic composition of any one of embodiments 125 to 127, wherein the first RNA and the second RNA each contain modified uridines in place of all uridines.

129. The immunogenic composition of any one of embodiments 125 to 128, wherein each of the modified uridines is N1-methyl-pseudouridine.

130. The first RNA and the second RNA each independently have the following characteristics:
5' untranslated region (UTR) comprising SEQ ID NO: 12;
130. The immunogenic composition of any one of embodiments 125-129, further comprising a 3' untranslated region (UTR) comprising SEQ ID NO: 13, and at least one, at least two, or all of a polyA sequence of at least 100 A nucleotides.

131. The immunogenic composition of embodiment 130, wherein the polyA sequence comprises 30 adenine nucleotides followed by 70 adenine nucleotides, the 30 adenine nucleotides and the 70 adenine nucleotides being separated by a linker sequence.

132. The immunogenic composition of embodiment 130, wherein the polyA sequence comprises SEQ ID NO: 14.

133. The immunogenic composition of any one of embodiments 125 to 132, wherein the first RNA comprises SEQ ID NO:59 and the second RNA comprises SEQ ID NO:50.

134. The immunogenic composition of any one of embodiments 125 to 132, wherein the first RNA comprises SEQ ID NO:59 and the second RNA comprises SEQ ID NO:56.

135. The immunogenic composition of any one of embodiments 125 to 132, wherein the first RNA comprises SEQ ID NO:59 and the second RNA comprises SEQ ID NO:62.

136. The immunogenic composition of any one of embodiments 125 to 132, wherein the first RNA comprises SEQ ID NO: 60 and the second RNA comprises SEQ ID NO: 51.

137. The immunogenic composition of any one of embodiments 125 to 132, wherein the first RNA comprises SEQ ID NO: 60 and the second RNA comprises SEQ ID NO: 57.

138. The immunogenic composition of any one of embodiments 125 to 132, wherein the first RNA comprises SEQ ID NO: 60 and the second RNA comprises SEQ ID NO: 63.

139. An immunogenic composition comprising a first RNA and a second RNA,
the first RNA encodes a polypeptide of SEQ ID NO:49 and comprises a nucleotide sequence of SEQ ID NO:50, or a nucleotide sequence that is at least 80% identical (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 97%, at least 98%, or 99% or more) to SEQ ID NO:50;
the second RNA encodes a polypeptide of SEQ ID NO: 55 or 61 and comprises a nucleotide sequence of SEQ ID NO: 56 or 62, or a nucleotide sequence that is at least 80% identical (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 97%, at least 98%, or 99% or more) to said SEQ ID NO: 56 or 62;
Each of the first RNA and the second RNA is
(a) a modified uridine, and (b) a 5' cap,
The immunogenic composition, wherein the first RNA and the second RNA are formulated in a lipid nanoparticle (LNP), and the LNP comprises ((4-hydroxybutyl)azanediyl)bis(hexane-6,1-diyl)bis(2-hexyldecanoate), 2-[(polyethylene glycol)-2000]-N,N-ditetradecylacetamide, 1,2-distearoyl-sn-glycero-3-phosphocholine, and cholesterol.

140. The immunogenic composition of embodiment 139, wherein the first RNA and the second RNA are formulated in separate lipid nanoparticles.

141. The immunogenic composition of embodiment 139, wherein the first RNA and the second RNA are formulated in the same lipid nanoparticle.

142. The immunogenic composition of any one of embodiments 139 to 141, wherein the first RNA and the second RNA each contain modified uridines in place of all uridines.

143. The immunogenic composition of any one of embodiments 139 to 142, wherein each of the modified uridines is N1-methyl-pseudouridine.

144. The first RNA and the second RNA each independently have the following characteristics:
5' untranslated region (UTR) comprising SEQ ID NO: 12;
144. The immunogenic composition of any one of embodiments 139-143, further comprising: a 3' untranslated region (UTR) comprising SEQ ID NO: 13; and at least one, at least two, or all of a polyA sequence of at least 100 A nucleotides.

145. The immunogenic composition of embodiment 144, wherein the polyA sequence comprises 30 adenine nucleotides followed by 70 adenine nucleotides, the 30 adenine nucleotides and the 70 adenine nucleotides being separated by a linker sequence.

146. The immunogenic composition of embodiment 144, wherein the polyA sequence comprises SEQ ID NO: 14.

147. The immunogenic composition of any one of embodiments 139 to 146, wherein the first RNA comprises SEQ ID NO:50 and the second RNA comprises SEQ ID NO:56.

148. The immunogenic composition of any one of embodiments 139 to 146, wherein the first RNA comprises SEQ ID NO:50 and the second RNA comprises SEQ ID NO:62.

149. The immunogenic composition of any one of embodiments 139 to 146, wherein the first RNA comprises SEQ ID NO:51 and the second RNA comprises SEQ ID NO:57.

150. The immunogenic composition of any one of embodiments 139 to 146, wherein the first RNA comprises SEQ ID NO:51 and the second RNA comprises SEQ ID NO:63.

151. An immunogenic composition comprising a first RNA and a second RNA,
the first RNA encodes a polypeptide of SEQ ID NO:55 and comprises a nucleotide sequence of SEQ ID NO:56, or a nucleotide sequence that is at least 80% identical (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 97%, at least 98%, or 99% or more) to SEQ ID NO:56;
the second RNA encodes a polypeptide of SEQ ID NO:61 and comprises a nucleotide sequence of SEQ ID NO:62, or a nucleotide sequence that is at least 80% identical (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 97%, at least 98%, or 99% or more) to SEQ ID NO:62;
Each of the first RNA and the second RNA is
(a) a modified uridine, and (b) a 5' cap,
The immunogenic composition, wherein the first RNA and the second RNA are formulated in a lipid nanoparticle (LNP), and the LNP comprises ((4-hydroxybutyl)azanediyl)bis(hexane-6,1-diyl)bis(2-hexyldecanoate), 2-[(polyethylene glycol)-2000]-N,N-ditetradecylacetamide, 1,2-distearoyl-sn-glycero-3-phosphocholine, and cholesterol.

152. The immunogenic composition of embodiment 151, wherein the first RNA and the second RNA are formulated in separate lipid nanoparticles.

153. The immunogenic composition of embodiment 151, wherein the first RNA and the second RNA are formulated in the same lipid nanoparticle.

154. The immunogenic composition of any one of embodiments 151 to 153, wherein the first RNA and the second RNA each contain modified uridines in place of all uridines.

155. The immunogenic composition of any one of embodiments 151 to 154, wherein each of the modified uridines is N1-methyl-pseudouridine.

156. The first RNA and the second RNA each independently have the following characteristics:
5' untranslated region (UTR) comprising SEQ ID NO: 12;
156. The immunogenic composition of any one of embodiments 151-155, further comprising a 3' untranslated region (UTR) comprising SEQ ID NO: 13, and at least one, at least two, or all of a polyA sequence of at least 100 A nucleotides.

157. The immunogenic composition of embodiment 156, wherein the polyA sequence comprises 30 adenine nucleotides followed by 70 adenine nucleotides, the 30 adenine nucleotides and the 70 adenine nucleotides being separated by a linker sequence.

158. The immunogenic composition of embodiment 156, wherein the polyA sequence comprises SEQ ID NO: 14.

159. The immunogenic composition of any one of embodiments 151 to 158, wherein the first RNA comprises SEQ ID NO:57 and the second RNA comprises SEQ ID NO:63.

160. The immunogenic composition of any one of embodiments 74-159, wherein the 5'-cap is or comprises m ₂ ^7,3'-O Gppp(m ₁ ^2'-O )ApG.

161. The immunogenic composition of any one of embodiments 74 to 160, wherein the LNP comprises about 40 to about 50 mole percent ((4-hydroxybutyl)azanediyl)bis(hexane-6,1-diyl)bis(2-hexyldecanoate), about 35 to about 45 mole percent cholesterol, about 5 to about 15 mole percent 1,2-distearoyl-sn-glycero-3-phosphocholine, and about 1 to about 10 mole percent 2-[(polyethylene glycol)-2000]-N,N-ditetradecylacetamide.

162. The immunogenic composition of any one of embodiments 74 to 161, wherein the composition comprises a plurality of LNPs, and the average diameter of the plurality of LNPs is from about 30 nm to about 200 nm or from about 60 nm to about 120 nm (e.g., as determined by dynamic light scattering measurements).

163. A method for inducing an immune response against SARS-CoV-2, comprising administering an immunogenic composition according to any one of embodiments 74 to 162.

164. The method of embodiment 163, wherein the immune response is elicited against an omicron variant of SARS-CoV-2.

165. The method of embodiment 163, wherein the immune response is elicited against a beta variant of SARS-CoV-2.

166. The method of embodiment 163, wherein the immune response is elicited against an alpha variant of SARS-CoV-2.

167. The method of embodiment 163, wherein the immune response is elicited against a delta variant of SARS-CoV-2.

168. The method of embodiment 163, wherein the immune response is elicited against the Wuhan strain, omicron variant, beta variant, alpha variant, and delta variant of SARS-CoV-2.

Citation of documents and works referenced herein is not intended as an admission that any of the foregoing is related to prior art. All statements regarding the contents of these documents are based on information available to the applicants and do not constitute any admission as to the accuracy of the contents of these documents.

The following description is presented to enable those skilled in the art to make and use the various embodiments. Descriptions of specific devices, techniques, and applications are provided only as examples. Various modifications to the examples described herein will be readily apparent to those skilled in the art, and the general principles defined herein may be applied to other examples and applications without departing from the spirit and scope of the various embodiments. Thus, the various embodiments are not intended to be limited to the examples described and shown herein, but are to be accorded the scope consistent with the claims.

Example 1: Immunogenicity study of BNT162b3 variants BNT162b3c and BNT162b3d To get an idea about the potential efficacy of transmembrane anchored RBD based vaccine antigens (schematic diagram in FIG. 6, BNT162b3c (1) and BNT162b3d (2)), BALB/c mice were immunized intramuscularly once with 4 μg LNP-C12 formulated mRNA or buffer as control. Non-clinical LNP-C12 formulated mRNA was used as a surrogate for BNT162b3 variants BNT162b3c and BNT162b3d. The immunogenicity of the RNA vaccine was investigated focusing on the antibody immune response.

ELISA data 6, 14, and 21 days after the first immunization show early dose-dependent immune activation against the S1 protein and receptor binding domain (Figure 7). Sera obtained 6, 14, and 21 days after immunization show high SARS-CoV-2 pseudovirus neutralization, correlating with increased IgG antibody titers (Figure 8).

Example 2: Neutralization of SARS-CoV-2 Omicron strain (also known as B.1.1.529) by human sera induced by BNT162b2 vaccine Materials and Methods:
Recombinant replication-deficient VSV vectors encoding green fluorescent protein (GFP) and luciferase (Luc) instead of VSV-glycoprotein (VSV-G) were used to identify SARS-CoV-2 spike (S) from the Wuhan-Hu-1 isolate (GenBank: QHD43416.1) and mutations found in the S protein of the Omicron (B.1.1.529) lineage (A67V, Δ69-70, T95I, G142D, Δ143-145, Δ211, L212I, ins214E, Δ215I, Δ216I, Δ217I, Δ218I, Δ219I, Δ220I, Δ221I, Δ2 ... The variant spikes harboring the following mutations were pseudotyped according to published pseudotyping protocols: PE, G339D, S371L, S373P, S375F, K417N, N440K, G446S, S477N, T478K, E484A, Q493R, G496S, Q498R, N501Y, Y505H, T547K, D614G, H655Y, N679K, P681H, N764K, D796Y, N856K, Q954H, N969K, L981F). Briefly, HEK293T/17 monolayers transfected to express the respective SARS-CoV-2 S truncation of the C-terminal cytoplasmic 19 amino acids (SARS-CoV-2-S(CΔ19)) were inoculated with VSVΔG-GFP/Luc vector. After 2 h incubation at 37°C, the inoculum was removed and cells were washed with PBS before adding bait supplemented with anti-VSV-G antibody (clone 8G5F11, Kerafast) to neutralize residual input virus. VSV-SARS-CoV-2 pseudovirus-containing medium was collected 20 h post-inoculation, 0.2 μm filtered, and stored at -80°C.

For pseudovirus neutralization assays, 40,000 Vero 76 cells were seeded per 96-well. Sera were serially diluted 1:2 in culture medium starting at a 1:10 dilution (dilution range 1:10 to 1:10,240). At approximately 200 TU in the assay, VSV-SARS-CoV-2-S pseudoparticles were diluted in culture medium for fluorescence focus unit (ffu) counts. Serum dilutions were mixed 1:1 with pseudovirus for 30 min at room temperature before being added to Vero 76 cell monolayers in 96-well plates and incubated at 37°C for 16-24 h. Supernatants were removed and cells were lysed with luciferase reagent (Promega). Luminescence was recorded and neutralization titers were calculated as the interaction of the highest serum dilution that still resulted in a 50% reduction in luminescence. Results were reported as the GMT of duplicates. If no neutralization was observed, an arbitrary titer value of 5 (half the limit of detection [LOD]) was reported.

Sera (N=19-20) were collected from subjects 21 days after receiving a second 30 μg dose or one month after receiving a third 30 μg dose of BNT162b2. Each serum was tested for its neutralizing antibody titers against wild-type SARS-CoV-2 Wuhan Hu-1 and Omicron lineage (B.1.1.529) spike protein-pseudotyped VSV by a 50% neutralization assay (pVNT50). The Omicron strain spike protein used in the neutralization assay had the following amino acid changes compared to the Wuhan reference: A67V, Δ69-70, T95I, G142D, Δ143-145, Δ211, L212I, ins214EPE, G339D, S371L, S373P, S375F, K417N, N440K, G446S, S477N, T478K, E484A, Q493R, G496S, Q498R, N501Y, Y505H, T547K, D614G, H655Y, N679K, P681H, N764K, D796Y, N856K, Q954H, N969K, L981F.

BNT162b2-immune sera generated 21 days after the second dose showed effective neutralization of the SARS-CoV-2 Wuhan Hu-1 pseudotype reference. However, a greater than 25-fold reduction in neutralizing titers against the Omicron variant was observed compared to the Wuhan reference (geometric mean titers [GMT] of 6 vs. 155). Importantly, the third dose significantly increased neutralizing antibody titers against the Omicron strain pseudovirus by 25-fold. Thus, neutralizing titers against the Omicron variant pseudovirus after three doses of BNT162b2 were comparable to neutralizing titers against the wild-type strain observed in sera from individuals who received two doses of BNT162b2 (GMT of 154 vs. 155).

Example 3: Additional Data for Neutralization of SARS-CoV-2 Omicron Lineage (aka B.1.1.529) Pseudovirus by Human Sera Elicited by BNT162b2 Vaccine In addition to the studies and data as described in Example 2, a longitudinal analysis of neutralization titers was also performed in an independent smaller subset of subjects. Sera collected 21 days after dose 2 showed a 19.6-fold reduction in GMT against the Omicron variant compared to the Wuhan reference pseudovirus (Figure 12, GMT of 6 vs. 118). Sera obtained from study participants immediately prior to receiving a third dose of BNT162b2 (median 251 days after dose 2) had substantially reduced neutralization titers against the Wuhan pseudovirus (GMT of 14), although omicron-specific titers were below the limit of detection. The third dose of BNT162b2 resulted in a significant increase in neutralization titers against the Wuhan pseudovirus (GMT of 254), with an increase in neutralization titers against Omicron one month after dose 3 resulting in a greater than 26.6-fold increase compared to titers 21 days after dose 2 (GMT of 160 vs. 6). In all nine subjects, reduced but effective neutralization of Omicron was observed up to three months after three doses (3.2-fold reduction compared to one month after dose 3, GMT of 50 vs. 160), while Wuhan-specific neutralization GMTs remained stable.

In summary, a third dose of BNT162b2 boosts omicron neutralization capacity to a level similar to that observed after two doses against the Wuhan variant. Thus, this data indicates that providing a third dose of BNT162b2 can improve protection against infection with the omicron variant.

Example 4 Neutralization of Other SARS-CoV-2 Lineage Pseudoviruses by Human Sera Elicited by BNT162b2 Vaccine As described in Examples 2 and 3, each serum was also tested for its neutralizing antibody titers against beta and delta lineage spike protein pseudotyped VSV by 50% neutralization assay (pVNT50) (data not shown).

A recombinant replication-deficient VSV vector encoding green fluorescent protein (GFP) and luciferase (Luc) instead of VSV-glycoprotein (VSV-G) was used to identify the Wuhan-Hu-1 isolate SARS-CoV-2 spike (S) (GenBank: QHD43416.1) and mutations found in the S protein of the beta lineage (mutations: L18F, D80A, D21 5G, R246I, Δ242-244, K417N, E484K, N501Y, D614G, A701V), and the Delta lineage (mutations: T19R, G142D, Δ157/158, K417N, L452R, T478K, D614G, P681R, D950N, K986P, V987P) were pseudotyped according to published pseudotyping protocols.

For sera collected 21 days after the second dose of BNT162b2, the _PVNT50 was reduced approximately 6.7-fold (24 vs. 155 GMT) for the beta variant and approximately 2.2-fold (73 vs. 155 GMT) for the delta variant compared to the Wuhan variant, but was significantly higher than the neutralization response against the delta variant. The third dose of BNT162b2 also increased neutralizing activity against the beta and delta pseudoviruses, with GMTs of 279 and 413, respectively.

Example 5: T cell epitope preservation in omicron spike variants In addition to humoral immunity, T cell mediated immunity is another layer of defense to prevent COVID-19, especially severe cases. The previous observation that efficacy against disease was already established about 12 days after the first dose of BNT162b2, before the second dose was administered and before the onset of high neutralizing titers, further highlights the potential protective role of T cell responses. Previous reports have shown that CD8 T cell responses in individuals vaccinated with BNT162b2 are polyepitopic.

To assess the risk of immune evasion of CD8+ T cell responses by omicron, we investigated a set of HLA class I restricted T cell epitopes from the Wuhan spike protein sequence reported to be immunogenic in the Immune Epitope Database (IEDB, n=244) (the procedure used to identify these epitopes is described in the following paragraphs). Despite the large number of mutations in the omicron spike protein, 85.25% (n=208) of the described epitopes were not affected at the amino acid sequence level, indicating that most targets of the T cell response elicited by BNT162b2 may still be conserved in omicron variants (Figure 13). Early laboratory studies have confirmed that CD8+ T cell recognition of omicron epitopes is conserved in COVID-19-recovered individuals exposed early in the pandemic and that omicron VOCV has not evolved extensive T cell escape mutations at this time.

To estimate the rate of nonsynonymous mutations in T cell epitopes in the spike glycoprotein, the immune epitope database (https://www.iedb.org/) was used to obtain epitopes confirmed for T cell reactivity in experimental assays. The database was filtered using the following criteria: Organism: SARS-COV2, Antigen: Spike glycoprotein, Positive assay, No B cell assay, No MHC assay, MHC restriction type: Class I, Host: Homo sapiens (human). The resulting table was filtered by removing epitopes "derived from reactive overlapping peptide pools," as well as epitopes longer than 14 amino acids, to restrict the data set to only minimal confirmed epitopes. Of the 251 unique epitope sequences obtained with this approach, 244 were found in the Wuhan strain spike glycoprotein. Of these, 36 epitopes (14.75%) contained positions reported to be mutated in Omicron by sequence analysis as disclosed herein. The results are summarized in FIG. 10. Also shown are the number of predicted MHC-I epitopes mutated in each of the alpha, beta, gamma, and delta SARS-CoV-2 variants. FIG. 13 shows the location of T cell epitopes within the spike protein and indicates which epitopes are conserved or mutated in the spike protein from the Omicron variants.

Example 6: Exemplary Dosing Regimen In some embodiments, the compositions and methods disclosed herein can be used according to the exemplary vaccination regimen illustrated in FIG.

Primary Dosing Regimen In some embodiments, the subject is administered a primary dosing regimen. The primary dosing regimen can include one or more doses. For example, in some embodiments, the primary dosing regimen includes a single dose (PD ₁ ). In some embodiments, the primary dosing regimen includes a first dose (PD ₁ ) and a second dose (PD _{2 ). In some embodiments, the primary dosing regimen includes a first dose, a second dose, and a third dose (PD 3 )} . In some embodiments, the primary dosing regimen includes a first dose, a second dose, a third dose, and one or more additional doses (PD _n ) of any one of the pharmaceutical compositions described herein.

In some embodiments, PD ₁ comprises administering 1-100 ug of RNA. In some embodiments, PD ₁ comprises administering 1-60 ug of RNA. In some embodiments, PD ₁ comprises administering 1-50 ug of RNA. In some embodiments, PD ₁ comprises administering 1-30 ug of RNA. In some embodiments, PD ₁ comprises administering about 3 ug of RNA. In some embodiments, PD ₁ comprises administering about 5 ug of RNA. In some embodiments, PD ₁ comprises administering about 10 ug of RNA. In some embodiments, PD ₁ comprises administering about 15 ug of RNA. In some embodiments, PD ₁ comprises administering about 20 ug of RNA. In some embodiments, PD ₁ comprises administering about 30 ug of RNA. In some embodiments, PD ₁ comprises administering about 50 ug of RNA. In some embodiments, PD ₁ comprises administering about 60 ug of RNA.

In some embodiments, PD ₂ comprises administering 1-100 ug of RNA. In some embodiments, PD ₂ comprises administering 1-60 ug of RNA. In some embodiments, PD ₂ comprises administering 1-50 ug of RNA. In some embodiments, PD ₂ comprises administering 1-30 ug of RNA. In some embodiments, PD ₂ comprises administering about 3 ug. In some embodiments, PD _{2 comprises administering about 5 ug of RNA. In some embodiments, PD 2} comprises administering about 10 ug of RNA. In some embodiments, PD ₂ comprises administering about 15 ug of RNA. In some embodiments, PD ₂ comprises administering about 20 ug of RNA. In some embodiments, PD ₂ comprises administering about 30 ug of RNA. In some embodiments, PD ₂ comprises administering about 50 ug of RNA. In some embodiments, PD ₂ comprises administering about 60 _ug of RNA.

In some embodiments, the PD ₃ comprises administering 1-100 ug of RNA. In some embodiments, the PD ₃ comprises administering 1-60 ug of RNA. In some embodiments, the PD ₃ comprises administering 1-50 ug of RNA. In some embodiments, the PD ₃ comprises administering 1-30 ug of RNA. In some embodiments, the PD ₃ comprises administering about 3 ug of RNA. In some embodiments, the PD ₃ comprises administering about 5 ug of RNA. In some embodiments, the PD ₃ comprises administering about 10 ug of RNA. In some embodiments, the PD ₃ comprises administering about 15 ug of RNA. In some embodiments, the PD ₃ comprises administering about 20 ug of RNA. In some embodiments, the PD ₃ comprises administering about 30 ug of RNA. In some embodiments, the PD ₃ comprises administering about 50 ug of RNA. In some embodiments, the PD ₃ comprises administering about 60 ug of RNA.

In some embodiments, PD _n comprises administering 1-100 ug of RNA. In some embodiments, PD _n comprises administering 1-60 ug of RNA. In some embodiments, PD _n comprises administering 1-50 ug of RNA. In some embodiments, PD _{n comprises administering 1-30 ug of RNA. In some embodiments, PD n} comprises administering about 3 ug of RNA. In some embodiments, PD _n comprises administering about 5 ug of RNA. In some embodiments, PD _n comprises administering about 10 ug of RNA. In some embodiments, PD _n comprises administering about 15 ug of RNA. In some embodiments, PD _n comprises administering about 20 ug of RNA. In some embodiments, _{PD n comprises administering about 30 ug of RNA. In some embodiments, PD n comprises administering about 50 ug of RNA. In some embodiments, PD n comprises administering about} 60 ug of RNA.

In some embodiments, PD ₁ comprises RNA encoding a SARS-CoV-2 spike protein or immunogenic fragment thereof from the Wuhan strain. In some embodiments, PD ₁ comprises RNA encoding a spike protein or immunogenic fragment thereof from a SARS-CoV-2 strain that is prevalent and/or spreading rapidly in a relevant jurisdiction. In some embodiments, PD ₁ comprises RNA encoding a SARS-CoV-2 spike protein or immunogenic fragment thereof that includes one or more mutations from an alpha variant. In some embodiments, PD ₁ comprises RNA encoding a SARS-CoV-2 spike protein or immunogenic fragment thereof that includes one or more mutations from a delta variant. In some embodiments, PD ₁ comprises RNA encoding a SARS-CoV-2 spike protein or immunogenic fragment thereof that includes one or more mutations from a beta variant. In some embodiments, PD ₁ comprises RNA encoding a SARS-CoV-2 spike protein or immunogenic fragment thereof that includes one or more mutations from an omicron variant. In some embodiments, PD ₁ comprises RNA encoding a SARS-CoV-2 spike protein or an immunogenic fragment thereof from the Wuhan strain, and one or more additional RNAs encoding a spike protein or an immunogenic fragment thereof from a SARS-CoV-2 strain that is prevalent and/or spreading rapidly in a relevant jurisdiction. In some embodiments, PD ₁ comprises RNA encoding a SARS-CoV-2 spike protein or an immunogenic fragment thereof from the Wuhan strain, and RNA encoding a SARS-CoV-2 spike protein or an immunogenic fragment thereof that includes one or more mutations from an alpha variant. In some embodiments, PD ₁ comprises RNA encoding a SARS-CoV-2 spike protein or an immunogenic fragment thereof from the Wuhan strain, and RNA encoding a SARS-CoV-2 spike protein or an immunogenic fragment thereof that includes one or more mutations from a beta variant. In some embodiments, the PD ₁ comprises RNA encoding a SARS-CoV-2 spike protein or an immunogenic fragment thereof from the Wuhan strain and RNA encoding a SARS-CoV-2 spike protein or an immunogenic fragment thereof comprising one or more mutations from the delta variant.In some embodiments, the PD ₁ comprises RNA encoding a SARS-CoV-2 spike protein or an immunogenic fragment thereof from the Wuhan strain and RNA encoding a SARS-CoV-2 spike protein or an immunogenic fragment thereof comprising one or more mutations from the omicron variant.

In some embodiments, PD ₂ comprises RNA encoding a SARS-CoV-2 spike protein or immunogenic fragment thereof from the Wuhan strain. In some embodiments, PD ₂ comprises RNA encoding a spike protein or immunogenic fragment thereof from a SARS-CoV-2 strain that is prevalent and/or spreading rapidly in a relevant jurisdiction. In some embodiments, PD _{2 comprises RNA encoding a SARS-CoV-2 spike protein or immunogenic fragment thereof that includes one or more mutations from an alpha variant. In some embodiments, PD 2 comprises} RNA encoding a SARS-CoV-2 spike protein or immunogenic fragment thereof that includes one or more mutations from a delta variant. In some embodiments, PD ₂ comprises RNA encoding a SARS-CoV-2 spike protein or immunogenic fragment thereof that includes one or more mutations from a beta variant. In some embodiments, PD ₂ comprises RNA encoding a SARS-CoV-2 spike protein or immunogenic fragment thereof that includes one or more mutations from an omicron variant. In some embodiments, PD ₂ comprises RNA encoding a SARS-CoV-2 spike protein or an immunogenic fragment thereof from the Wuhan strain, as well as one or more additional RNAs encoding spike protein or an immunogenic fragment thereof from a SARS-CoV-2 strain that is prevalent and/or spreading rapidly in the relevant jurisdiction. In some embodiments, PD ₂ comprises RNA encoding a SARS-CoV-2 spike protein or an immunogenic fragment thereof from the Wuhan strain, and RNA encoding a SARS-CoV-2 spike protein or an immunogenic fragment thereof that includes one or more mutations from an alpha variant. In some embodiments, PD ₂ comprises RNA encoding a SARS-CoV-2 spike protein or an immunogenic fragment thereof from the Wuhan strain, and RNA encoding a SARS-CoV-2 spike protein or an immunogenic fragment thereof that includes one or more mutations from a beta variant. In some embodiments, PD ₂ comprises RNA encoding a SARS-CoV-2 spike protein or an immunogenic fragment thereof from the Wuhan strain and RNA encoding a SARS-CoV-2 spike protein or an immunogenic fragment thereof comprising one or more mutations from the delta variant.In some embodiments, PD ₂ comprises RNA encoding a SARS-CoV-2 spike protein or an immunogenic fragment thereof from the Wuhan strain and RNA encoding a SARS-CoV-2 spike protein or an immunogenic fragment thereof comprising one or more mutations from the omicron variant.

In some embodiments, the PD ₃ comprises RNA encoding a SARS-CoV-2 spike protein or immunogenic fragment thereof from the Wuhan strain. In some embodiments, the PD ₃ comprises RNA encoding a spike protein or immunogenic fragment thereof from a SARS-CoV-2 strain that is prevalent and/or spreading rapidly in a relevant jurisdiction. In some embodiments, the PD ₃ comprises RNA encoding a SARS-CoV-2 spike protein or immunogenic fragment thereof that includes one or more mutations from an alpha variant. In some embodiments, the PD ₃ comprises RNA encoding a SARS-CoV-2 spike protein or immunogenic fragment thereof that includes one or more mutations from a delta variant. In some embodiments, the PD ₃ comprises RNA encoding a SARS-CoV-2 spike protein or immunogenic fragment thereof that includes one or more mutations from a beta variant. In some embodiments, the PD ₃ comprises RNA encoding a SARS-CoV-2 spike protein or immunogenic fragment thereof that includes one or more mutations from an alpha variant. In some embodiments, the PD ₃ comprises an RNA encoding a SARS-CoV-2 spike protein or an immunogenic fragment thereof from the Wuhan strain, and one or more additional RNAs encoding a spike protein or an immunogenic fragment thereof from a SARS-CoV-2 strain that is prevalent and/or spreading rapidly in the relevant jurisdiction. In some embodiments, the PD ₃ comprises an RNA encoding a SARS-CoV-2 spike protein or an immunogenic fragment thereof from the Wuhan strain, and an RNA encoding a SARS-CoV-2 spike protein or an immunogenic fragment thereof that includes one or more mutations from an alpha variant. In some embodiments, the PD ₃ comprises an RNA encoding a SARS-CoV-2 spike protein or an immunogenic fragment thereof from the Wuhan strain, and an RNA encoding a SARS-CoV-2 spike protein or an immunogenic fragment thereof that includes one or more mutations from a beta variant. In some embodiments, the PD ₃ comprises RNA encoding a SARS-CoV-2 spike protein or an immunogenic fragment thereof from the Wuhan strain and RNA encoding a SARS-CoV-2 spike protein or an immunogenic fragment thereof comprising one or more mutations from the delta variant.In some embodiments, the PD ₃ comprises RNA encoding a SARS-CoV-2 spike protein or an immunogenic fragment thereof from the Wuhan strain and RNA encoding a SARS-CoV-2 spike protein or an immunogenic fragment thereof comprising one or more mutations from the omicron variant.

In some embodiments, the PD _n comprises RNA encoding a SARS-CoV-2 spike protein or immunogenic fragment thereof from the Wuhan strain. In some embodiments, the PD _n comprises RNA encoding a spike protein or immunogenic fragment thereof from a SARS-CoV-2 strain that is circulating and/or spreading rapidly in the relevant jurisdiction. In some embodiments, the PD _{n comprises RNA encoding a SARS-CoV-2 spike protein or immunogenic fragment thereof that includes one or more mutations from an alpha variant. In some embodiments, the PD n comprises} RNA encoding a SARS-CoV-2 spike protein or immunogenic fragment thereof that includes one or more mutations from a delta variant. In some embodiments, the PD _n comprises RNA encoding a SARS-CoV-2 spike protein or immunogenic fragment thereof that includes one or more mutations from a beta variant. In some embodiments, the PD _n comprises RNA encoding a SARS-CoV-2 spike protein or immunogenic fragment thereof that includes one or more mutations from an omicron variant. In some embodiments, the PD _n comprises an RNA encoding a SARS-CoV-2 spike protein or an immunogenic fragment thereof from the Wuhan strain, and one or more additional RNAs encoding a spike protein or an immunogenic fragment thereof from a SARS-CoV-2 strain that is prevalent and/or spreading rapidly in the relevant jurisdiction. In some embodiments, the PD _n comprises an RNA encoding a SARS-CoV-2 spike protein or an immunogenic fragment thereof from the Wuhan strain, and an RNA encoding a SARS-CoV-2 spike protein or an immunogenic fragment thereof that includes one or more mutations from an alpha variant. In some embodiments, the PD _n comprises an RNA encoding a SARS-CoV-2 spike protein or an immunogenic fragment thereof from the Wuhan strain, and an RNA encoding a SARS-CoV-2 spike protein or an immunogenic fragment thereof that includes one or more mutations from a beta variant. In some embodiments, PD _n comprises RNA encoding a SARS-CoV-2 spike protein or an immunogenic fragment thereof from the Wuhan strain and RNA encoding a SARS-CoV-2 spike protein or an immunogenic fragment thereof comprising one or more mutations from the delta variant.In some embodiments, PD _n comprises RNA encoding a SARS-CoV-2 spike protein or an immunogenic fragment thereof from the Wuhan strain and RNA encoding a SARS-CoV-2 spike protein or an immunogenic fragment thereof comprising one or more mutations from the omicron variant.

In some embodiments, PD ₁ , PD ₂ , PD ₃ , and PD _n can each independently comprise a plurality (e.g., at least two) of the RNA (e.g., mRNA) compositions described herein. In some embodiments, PD ₁ , PD ₂ , PD ₃ , and PD _n can each independently comprise a first and a second RNA (e.g., mRNA) composition. In some embodiments, at least one of the plurality of RNA (e.g., mRNA) compositions comprises BNT162b2 (e.g., as described herein). In some embodiments, at least one of the plurality of RNA (e.g., mRNA) compositions comprises RNA (e.g., mRNA) encoding a SARS-CoV-2 S protein, or an immunogenic fragment thereof, from a different SARS-CoV-2 variant. In some embodiments, at least one of the plurality of RNA (e.g., mRNA) compositions comprises RNA (e.g., mRNA) encoding a SARS-CoV-2 S protein, or an immunogenic fragment thereof, from the Wuhan strain of SARS-CoV-2. In some embodiments, at least one of the plurality of RNA (e.g., mRNA) compositions comprises an RNA encoding a SARS-CoV-2 S protein or immunogenic fragment thereof that includes one or more mutations from a variant that is prevalent and/or rapidly spreading in a relevant jurisdiction. In some embodiments, at least one of the plurality of RNA (e.g., mRNA) compositions comprises an RNA (e.g., mRNA) encoding a SARS-CoV-2 S protein or immunogenic fragment thereof that includes one or more mutations from an alpha variant. In some embodiments, at least one of the plurality of RNA (e.g., mRNA) compositions comprises an RNA (e.g., mRNA) encoding a SARS-CoV-2 S protein or immunogenic fragment thereof that includes one or more mutations from a delta variant. In some embodiments, at least one of the plurality of RNA (e.g., mRNA) compositions comprises an RNA (e.g., mRNA) encoding a SARS-CoV-2 S protein or immunogenic fragment thereof that includes one or more mutations from an omicron variant.

In some embodiments, the multiple RNA (e.g., mRNA) compositions provided at PD ₁ , PD ₂ , PD ₃ , and/or PD _n can each independently comprise at least two different RNA (e.g., mRNA) constructs (e.g., differing in protein coding sequence). For example, in some embodiments, the multiple RNA (e.g., mRNA) compositions provided at PD ₁ , PD ₂ , PD ₃ , and/or PD _n can each independently comprise an RNA (e.g., mRNA) encoding a SARS-CoV-2 S protein, or an immunogenic fragment thereof, from the Wuhan strain of SARS-CoV-2, as well as an RNA (e.g., mRNA) encoding a SARS-CoV-2 S protein, or an immunogenic fragment thereof, that includes one or more mutations from a variant that is prevalent and/or spreading rapidly in the relevant jurisdiction. In some embodiments, the multiple RNA (e.g., mRNA) compositions provided at PD ₁ , PD ₂ , PD ₃ , and/or PD _n can each independently comprise an RNA (e.g., mRNA) encoding a SARS-CoV-2 S protein or an immunogenic fragment thereof from the Wuhan strain of SARS-CoV-2, as well as an RNA (e.g., mRNA) encoding a SARS-CoV-2 S protein or an immunogenic fragment thereof that includes one or more mutations from a variant that is prevalent and/or rapidly spreading in the relevant jurisdiction. In some such embodiments, the variant can be an alpha variant. In some such embodiments, the variant can be a delta variant. In some such embodiments, the variant can be an omicron variant.

In some embodiments, each of the multiple RNA (e.g., mRNA) compositions provided at PD ₁ , PD ₂ , PD ₃ , and/or PD _n can independently comprise at least two RNAs (e.g., mRNAs), each encoding a SARS-CoV-2 S protein or immunogenic fragment thereof that includes one or more mutations from a distinct variant that is prevalent and/or spreading rapidly in the relevant jurisdiction. In some embodiments, each of the multiple RNA (e.g., mRNA) compositions provided at PD ₁ , PD ₂ , PD ₃ , and/or PD _n can independently comprise an mRNA encoding a SARS-CoV-2 S protein or immunogenic fragment thereof from an alpha variant, and an RNA (e.g., mRNA) encoding a SARS-CoV-2 S protein or immunogenic fragment thereof that includes one or more mutations from a delta variant. In some embodiments, each of the multiple RNA (e.g., mRNA) compositions provided at PD ₁ , PD ₂ , PD ₃ , and/or PD _n can independently comprise an RNA (e.g., mRNA) encoding a SARS-CoV-2 S protein or immunogenic fragment thereof from an alpha variant, and an RNA (e.g., mRNA) encoding a SARS-CoV-2 S protein or immunogenic fragment thereof that includes one or more mutations from a delta variant. In some embodiments, each of the multiple RNA (e.g., mRNA) compositions provided at PD ₁ , PD ₂ , PD ₃ , and/or PD _n can independently comprise an RNA (e.g., mRNA) encoding a SARS-CoV-2 S protein or immunogenic fragment thereof from an alpha variant, and an RNA (e.g., mRNA) encoding a SARS-CoV-2 S protein or immunogenic fragment thereof that includes one or more mutations from a delta variant.

In some embodiments, PD ₁ , PD ₂ , PD ₃ , and/or PD _n each comprise a plurality of RNA (e.g., mRNA) compositions, and each RNA (e.g., mRNA) composition is administered separately to the subject. For example, in some embodiments, each RNA (e.g., mRNA) composition is administered via intramuscular injection at a different injection site. For example, in some embodiments, a first and second RNA (e.g., mRNA) composition given in PD ₁ , PD ₂ , PD ₃ , and/or PD _n are administered separately via intramuscular injection into different arms of the subject.

In some embodiments, PD ₁ , PD ₂ , PD ₃ , and/or PD _n comprises administering a plurality of RNA molecules, each RNA molecule encoding a spike protein comprising a mutation from a different SARS-CoV-2 variant, and the plurality of RNA molecules are administered to the subject in a single formulation. In some embodiments, the single formulation comprises RNA encoding a spike protein or an immunogenic fragment thereof from the Wuhan strain, and RNA encoding a SARS-CoV-2 spike protein or an immunogenic fragment thereof comprising one or more mutations from an alpha variant. In some embodiments, the single formulation comprises RNA encoding a SARS-CoV-2 spike protein or an immunogenic fragment thereof from the Wuhan strain, and RNA encoding a SARS-CoV-2 spike protein or an immunogenic fragment thereof comprising one or more mutations from a beta variant. In some embodiments, a single formulation comprises RNA encoding a SARS-CoV-2 spike protein or an immunogenic fragment thereof from the Wuhan strain, and RNA encoding a SARS-CoV-2 spike protein or an immunogenic fragment thereof that includes one or more mutations from the delta variant. In some embodiments, a single formulation comprises RNA encoding a SARS-CoV-2 spike protein or an immunogenic fragment thereof from the Wuhan strain, and RNA encoding a SARS-CoV-2 spike protein or an immunogenic fragment thereof that includes one or more mutations from the omicron variant.

In some embodiments, the length of time between PD ₁ and PD ₂ (PI ₁ ) is at least about 1 week, at least about 2 weeks, at least about 3 weeks, or at least about 4 weeks. In some embodiments, PI ₁ is from about 1 week to about 12 weeks. In some embodiments, PI ₁ is from about 1 week to about 10 weeks. In some embodiments, PI ₁ is from about 2 weeks to about 10 weeks. In some embodiments, PI ₁ is from about 2 weeks to about 8 weeks. In some embodiments, PI ₁ is from about 3 weeks to about 8 weeks. In some embodiments, PI ₁ is from about 4 weeks to about 8 weeks. In some embodiments, PI ₁ is from about 6 weeks to about 8 weeks. In some embodiments, PI ₁ is from about 3 to about 4 weeks. In some embodiments, PI ₁ is about 1 week. In some embodiments, PI ₁ is about 2 weeks. In some embodiments, PI ₁ is about 3 weeks. In some embodiments, PI ₁ is about 4 weeks. In some embodiments, PI ₁ is about 5 weeks. In some embodiments, PI ₁ is about 6 weeks. In some embodiments, PI ₁ is about 7 weeks. In some embodiments, PI ₁ is about 8 weeks. In some embodiments, PI ₁ is about 9 weeks. In some embodiments, PI ₁ is about 10 weeks. In some embodiments, PI ₁ is about 11 weeks. In some embodiments, PI ₁ is about 12 weeks.

In some embodiments, the length of time between PD ₂ and PD ₃ (PI ₂ ) is at least about 1 week, at least about 2 weeks, or at least about 3 weeks. In some embodiments, PI ₂ is from about 1 week to about 12 weeks. In some embodiments, PI ₂ is from about 1 week to about 10 weeks. In some embodiments, PI ₂ is from about 2 weeks to about 10 weeks. In some embodiments, PI ₂ is from about 2 weeks to about 8 weeks. In some embodiments, PI ₂ is from about 3 weeks to about 8 weeks. In some embodiments, PI ₂ is from about 4 weeks to about 8 weeks. In some embodiments, PI ₂ is from about 6 weeks to about 8 weeks. In some embodiments, PI ₂ is from about 3 to about 4 weeks. In some embodiments, PI ₂ is about 1 week. In some embodiments, PI ₂ is about 2 weeks. In some embodiments, PI ₂ is about 3 weeks. In some embodiments, PI ₂ is about 4 weeks. In some embodiments, PI ₂ is about 5 weeks. In some embodiments, PI ₂ is about 6 weeks. In some embodiments, PI ₂ is about 7 weeks. In some embodiments, PI ₂ is about 8 weeks. In some embodiments, PI ₂ is about 9 weeks. In some embodiments, PI ₂ is about 10 weeks. In some embodiments, PI ₂ is about 11 weeks. In some embodiments, PI ₂ is about 12 weeks.

In some embodiments, the length of time (PI _n ) between PD ₃ and a subsequent dose that is part of the primary dosing regimen, or between doses for any doses beyond PD ₃ , is each separately and independently selected from about 1 week or more, about 2 weeks or more, or about 3 weeks or more. In some embodiments, PI _n is about 1 week to about 12 weeks. In some embodiments, PI _n is about 1 week to about 10 weeks. In some embodiments, PI _n is about 2 weeks to about 10 weeks. In some embodiments, PI _n is about 2 weeks to about 8 weeks. In some embodiments, PI _n is about 3 weeks to about 8 weeks. In some embodiments, PI _n is about 4 weeks to about 8 weeks. In some embodiments, PI _n is about 6 weeks to about 8 weeks. In some embodiments, PI _n is about 3 to about 4 weeks. In some embodiments, PI ₂ is about 1 week. In some embodiments, PI _n is about 2 weeks. In some embodiments, PI _n is about 3 weeks. In some embodiments, PI _n is about 4 weeks. In some embodiments, PI _n is about 5 weeks. In some embodiments, PI _n is about 6 weeks. In some embodiments, PI _n is about 7 weeks. In some embodiments, PI _n is about 8 weeks. In some embodiments, PI _n is about 9 weeks. In some embodiments, PI _n is about 10 weeks. In some embodiments, PI _n is about 11 weeks. In some embodiments, PI _n is about 12 weeks.

In some embodiments, one or more compositions administered on PD ₁ are formulated in a Tris buffer. In some embodiments, one or more compositions administered on PD ₂ are formulated in a Tris buffer. In some embodiments, one or more compositions administered on PD ₃ are formulated in a Tris buffer. In some embodiments, one or more compositions administered on PD _n are formulated in a Tris buffer.

In some embodiments, the primary dosing regimen comprises administering two or more RNA (e.g., mRNA) compositions described herein, where at least two of the RNA (e.g., mRNA) compositions have different formulations. In some embodiments, the primary dosing regimen comprises PD ₁ and PD ₂ , where PD ₁ comprises administering RNA (e.g., mRNA) formulated in Tris buffer, and PD ₂ comprises administering RNA (e.g., mRNA) formulated in PBS buffer. In some embodiments, the primary dosing regimen comprises PD ₁ and PD ₂ , where PD ₁ comprises administering RNA (e.g., mRNA) formulated in Tris buffer, and PD ₂ comprises administering RNA (e.g., mRNA) formulated in PBS buffer.

In some embodiments, one or more of the RNA (e.g., mRNA) compositions provided in PD ₁ , PD ₂ , PD ₃ , and/or PD _n may be administered in conjunction with another vaccine. In some embodiments, the other vaccine is for a disease that is not COVID-19. In some embodiments, the disease is one that increases the deleterious effects of SARS-CoV-2 when a subject is co-infected with the disease and SARS-CoV-2. In some embodiments, the disease is one that increases the transmission rate of SARS-CoV-2 when a subject is co-infected with the disease and SARS-CoV-2. In some embodiments, the other vaccine is a different commercially available vaccine. In some embodiments, the different commercially available vaccine is an RNA vaccine. In some embodiments, the different commercially available vaccine is a polypeptide-based vaccine. In some embodiments, another vaccine (e.g., as described herein) and one or more RNA (e.g., mRNA) compositions given at PD ₁ , PD ₂ , PD ₃ , and/or PD _n are administered separately, e.g., via intramuscular injection, in some embodiments at different injection sites. For example, in some embodiments, an influenza vaccine described herein and one or more SARS-CoV-2 RNA (e.g., mRNA) compositions given at PD ₁ , PD ₂ , PD ₃ , and/or PD _n are administered separately via intramuscular injection into different arms of a subject.

Booster Dosing Regimens In some embodiments, the methods of vaccination disclosed herein include one or more booster dosing regimens. A booster dosing regimen disclosed herein includes one or more doses. In some embodiments, the booster dosing regimen is administered to a patient who has been administered a primary dosing regimen (e.g., as described herein). In some embodiments, the booster dosing regimen is administered to a patient who has not received a pharmaceutical composition disclosed herein. In some embodiments, the booster dosing regimen is administered to a patient who has previously been vaccinated with a COVID-19 vaccine different from the vaccine administered in the primary dosing regimen.

In some embodiments, the length of time between the primary dosing regimen and the booster dosing regimen is at least 1 week, at least 2 weeks, at least 3 weeks, at least 4 weeks, at least 5 weeks, at least 6 weeks, at least 7 weeks, at least 8 weeks, at least 9 weeks, at least 10 weeks, at least 11 weeks, at least 12 weeks, at least 2 months, at least 3 months, at least 4 months, at least 5 months, at least 6 months, at least 7 months, at least 8 months, at least 9 months, at least 10 months, at least 11 months, or at least 12 months or more. In some embodiments, the length of time between the primary dosing regimen and the booster dosing regimen is about 1 month. In some embodiments, the length of time between the primary dosing regimen and the booster dosing regimen is at least about 2 months. In some embodiments, the length of time between the primary dosing regimen and the booster dosing regimen is at least about 3 months. In some embodiments, the length of time between the primary dosing regimen and the booster dosing regimen is at least about 4 months. In some embodiments, the length of time between the primary dosing regimen and the booster dosing regimen is at least about 5 months. In some embodiments, the length of time between the primary dosing regimen and the booster dosing regimen is at least about 6 months. In some embodiments, the length of time between the primary dosing regimen and the booster dosing regimen is from about 1 month to about 48 months. In some embodiments, the length of time between the primary dosing regimen and the booster dosing regimen is from about 1 month to about 36 months. In some embodiments, the length of time between the primary dosing regimen and the booster dosing regimen is from about 1 month to about 24 months. In some embodiments, the length of time between the primary dosing regimen and the booster dosing regimen is from about 2 months to about 24 months. In some embodiments, the length of time between the primary dosing regimen and the booster dosing regimen is from about 3 months to about 24 months. In some embodiments, the length of time between the primary dosing regimen and the booster dosing regimen is from about 3 months to about 18 months. In some embodiments, the length of time between the primary dosing regimen and the booster dosing regimen is about 3 months to about 12 months. In some embodiments, the length of time between the primary dosing regimen and the booster dosing regimen is about 6 months to about 12 months. In some embodiments, the length of time between the primary dosing regimen and the booster dosing regimen is about 3 months to about 9 months. In some embodiments, the length of time between the primary dosing regimen and the booster dosing regimen is about 5 months to about 7 months. In some embodiments, the length of time between the primary dosing regimen and the booster dosing regimen is about 6 months.

In some embodiments, the subject is administered a booster dosing regimen. The booster dosing regimen can include one or more doses. For example, in some embodiments, the booster dosing regimen includes a single dose (BD ₁ ). In some embodiments, the booster dosing regimen includes a first dose (BD ₁ ) and a second dose (BD ₂ ). In some embodiments, the booster dosing regimen includes a first dose, a second dose, and a third dose (BD ₃ ). In some embodiments, the booster dosing regimen includes a first dose, a second dose, a third dose, and one or more additional doses (BD _n ) of any one of the pharmaceutical compositions described herein.

In some embodiments, the BD ₁ comprises administering 1-100 ug of RNA. In some embodiments, the BD ₁ comprises administering 1-60 ug of RNA. In some embodiments, the BD ₁ comprises administering 1-50 ug of RNA. In some embodiments, the BD ₁ comprises administering 1-30 ug of RNA. In some embodiments, the BD ₁ comprises administering about 3 ug of RNA. In some embodiments, the BD ₁ comprises administering about 5 ug of RNA. In some embodiments, the BD ₁ comprises administering about 10 ug of RNA. In some embodiments, the BD ₁ comprises administering about 15 ug of RNA. In some embodiments, the BD ₁ comprises administering about 20 ug of RNA. In some embodiments, the BD ₁ comprises administering about 30 ug of RNA. In some embodiments, the BD ₁ comprises administering about 50 ug of RNA. In some embodiments, BD ₁ comprises administering about 60 ug of RNA.

In some embodiments, BD ₂ comprises administering 1-100 ug of RNA. In some embodiments, BD ₂ comprises administering 1-60 ug of RNA. In some embodiments, BD ₂ comprises administering 1-50 ug of RNA. In some embodiments, BD ₂ comprises administering 1-30 ug of RNA. In some embodiments, BD ₂ comprises administering about 3 ug. In some embodiments, BD 2 comprises administering about 5 ug of RNA. In some embodiments, BD ₂ comprises administering about 10 ug of RNA. In some embodiments, BD ₂ comprises administering about 15 ug of RNA. In some embodiments, BD ₂ comprises administering about 20 ug of RNA. In some embodiments, BD ₂ comprises administering about 30 ug of RNA. In some embodiments, BD ₂ comprises administering about 50 _ug of RNA. In some embodiments, BD ₂ comprises administering about 60 ug of RNA.

In some embodiments, the BD ₃ comprises administering 1-100ug of RNA. In some embodiments, the BD ₃ comprises administering 1-60ug of RNA. In some embodiments, the BD ₃ comprises administering 1-50ug of RNA. In some embodiments, the BD ₃ comprises administering 1-30ug of RNA. In some embodiments, the BD ₃ comprises administering about 3ug of RNA. In some embodiments, the BD ₃ comprises administering about 5ug of RNA. In some embodiments, the BD ₃ comprises administering about 10ug of RNA. In some embodiments, the BD ₃ comprises administering about 15ug of RNA. In some embodiments, the BD ₃ comprises administering about 20ug of RNA. In some embodiments, the BD ₃ comprises administering about 30ug of RNA. In some embodiments, the BD ₃ comprises administering about 50ug of RNA. In some embodiments, BD ₃ comprises administering about 60 ug of RNA.

In some embodiments, BD _n comprises administering 1-100 ug of RNA. In some embodiments, BD _n comprises administering 1-60 ug of RNA. In some embodiments, BD _n comprises administering 1-50 ug of RNA. In some embodiments, BD _{n comprises administering 1-30 ug of RNA. In some embodiments, BD n} comprises administering about 3 ug of RNA. In some embodiments, BD _n comprises administering about 5 ug of RNA. In some embodiments, BD _n comprises administering about 10 ug of RNA. In some embodiments, BD _n comprises administering about 15 ug of RNA. In some embodiments, _{BD n comprises administering about 20 ug of RNA. In some embodiments, BD n comprises administering about 30 ug of RNA. In some embodiments, BD n comprises administering about} 60 ug of RNA. In some embodiments, BD _n comprises administering about 50 ug of RNA.

In some embodiments, BD ₁ comprises RNA encoding a SARS-CoV-2 spike protein or an immunogenic fragment thereof from the Wuhan strain. In some embodiments, BD ₁ comprises RNA encoding a spike protein or an immunogenic fragment thereof from a SARS-CoV-2 strain that is prevalent and/or spreading rapidly in the relevant jurisdiction. In some embodiments, BD _{1 comprises RNA encoding a SARS-CoV-2 spike protein or an immunogenic fragment thereof that includes one or more mutations from an alpha variant. In some embodiments, BD 1 comprises} RNA encoding a SARS-CoV-2 spike protein or an immunogenic fragment thereof that includes one or more mutations from a delta variant. In some embodiments, BD ₁ comprises RNA encoding a SARS-CoV-2 spike protein or an immunogenic fragment thereof that includes one or more mutations from a beta variant. In some embodiments, BD ₁ comprises RNA encoding a SARS-CoV-2 spike protein or an immunogenic fragment thereof that includes one or more mutations from an omicron variant.

In some embodiments, BD ₁ comprises an RNA encoding a SARS-CoV-2 spike protein or an immunogenic fragment thereof from the Wuhan strain, and one or more additional RNAs encoding a spike protein or an immunogenic fragment thereof from a SARS-CoV-2 strain that is prevalent and/or spreading rapidly in the relevant jurisdiction. In some embodiments, BD ₁ comprises an RNA encoding a SARS-CoV-2 spike protein or an immunogenic fragment thereof from the Wuhan strain, and an RNA encoding a SARS-CoV-2 spike protein or an immunogenic fragment thereof that includes one or more mutations from the alpha variant. In some embodiments, BD ₁ comprises an RNA encoding a SARS-CoV-2 spike protein or an immunogenic fragment thereof from the Wuhan strain, and an RNA encoding a SARS-CoV-2 spike protein or an immunogenic fragment thereof that includes one or more mutations from the delta variant. In some embodiments, BD ₁ comprises RNA encoding a SARS-CoV-2 spike protein or an immunogenic fragment thereof from the Wuhan strain, and RNA encoding a SARS-CoV-2 spike protein or an immunogenic fragment thereof comprising one or more mutations from the beta variant.In some embodiments, BD ₁ comprises RNA encoding a SARS-CoV-2 spike protein or an immunogenic fragment thereof from the Wuhan strain, and RNA encoding a SARS-CoV-2 spike protein or an immunogenic fragment thereof comprising one or more mutations from the omicron variant.

In some embodiments, BD ₂ comprises RNA encoding a SARS-CoV-2 spike protein or immunogenic fragment thereof from the Wuhan strain. In some embodiments, BD ₂ comprises RNA encoding a spike protein or immunogenic fragment thereof from a SARS-CoV-2 strain that is prevalent and/or spreading rapidly in the relevant jurisdiction. In some embodiments, BD _{2 comprises RNA encoding a SARS-CoV-2 spike protein or immunogenic fragment thereof that includes one or more mutations from an alpha variant. In some embodiments, BD 2 comprises} RNA encoding a SARS-CoV-2 spike protein or immunogenic fragment thereof that includes one or more mutations from a delta variant. In some embodiments, BD ₂ comprises RNA encoding a SARS-CoV-2 spike protein or immunogenic fragment thereof that includes one or more mutations from a beta variant. In some embodiments, BD ₂ comprises RNA encoding a SARS-CoV-2 spike protein or immunogenic fragment thereof that includes one or more mutations from an omicron variant. In some embodiments, BD ₂ comprises an RNA encoding a SARS-CoV-2 spike protein or an immunogenic fragment thereof from the Wuhan strain, and one or more additional RNAs encoding a spike protein or an immunogenic fragment thereof from a SARS-CoV-2 strain that is prevalent and/or spreading rapidly in the relevant jurisdiction. In some embodiments, BD ₂ comprises an RNA encoding a SARS-CoV-2 spike protein or an immunogenic fragment thereof from the Wuhan strain, and an RNA encoding a SARS-CoV-2 spike protein or an immunogenic fragment thereof that includes one or more mutations from an alpha variant. In some embodiments, BD ₂ comprises an RNA encoding a SARS-CoV-2 spike protein or an immunogenic fragment thereof from the Wuhan strain, and an RNA encoding a SARS-CoV-2 spike protein or an immunogenic fragment thereof that includes one or more mutations from a delta variant. In some embodiments, BD ₂ comprises RNA encoding a SARS-CoV-2 spike protein or an immunogenic fragment thereof from the Wuhan strain and RNA encoding a SARS-CoV-2 spike protein or an immunogenic fragment thereof comprising one or more mutations from a beta variant.In some embodiments, BD ₂ comprises RNA encoding a SARS-CoV-2 spike protein or an immunogenic fragment thereof from the Wuhan strain and RNA encoding a SARS-CoV-2 spike protein or an immunogenic fragment thereof comprising one or more mutations from an omicron variant.

In some embodiments, BD ₃ comprises RNA encoding a SARS-CoV-2 spike protein or immunogenic fragment thereof from the Wuhan strain. In some embodiments, BD ₃ comprises RNA encoding a spike protein or immunogenic fragment thereof from a SARS-CoV-2 strain that is prevalent and/or spreading rapidly in the relevant jurisdiction. In some embodiments, BD ₃ comprises RNA encoding a SARS-CoV-2 spike protein or immunogenic fragment thereof that includes one or more mutations from an alpha variant. In some embodiments, BD ₃ comprises RNA encoding a SARS-CoV-2 spike protein or immunogenic fragment thereof that includes one or more mutations from a delta variant. In some embodiments, BD ₃ comprises RNA encoding a SARS-CoV-2 spike protein or immunogenic fragment thereof that includes one or more mutations from a beta variant. In some embodiments, BD ₃ comprises RNA encoding a SARS-CoV-2 spike protein or immunogenic fragment thereof that includes one or more mutations from an omicron variant.

In some embodiments, BD ₃ comprises an RNA encoding a SARS-CoV-2 spike protein or an immunogenic fragment thereof from the Wuhan strain, and one or more additional RNAs encoding a spike protein or an immunogenic fragment thereof from a SARS-CoV-2 strain that is prevalent and/or spreading rapidly in the relevant jurisdiction. In some embodiments, BD ₃ comprises an RNA encoding a SARS-CoV-2 spike protein or an immunogenic fragment thereof from the Wuhan strain, and an RNA encoding a SARS-CoV-2 spike protein or an immunogenic fragment thereof that includes one or more mutations from an alpha variant. In some embodiments, BD ₃ comprises an RNA encoding a SARS-CoV-2 spike protein or an immunogenic fragment thereof from the Wuhan strain, and an RNA encoding a SARS-CoV-2 spike protein or an immunogenic fragment thereof that includes one or more mutations from a delta variant. In some embodiments, BD ₃ comprises RNA encoding a SARS-CoV-2 spike protein or an immunogenic fragment thereof from the Wuhan strain and RNA encoding a SARS-CoV-2 spike protein or an immunogenic fragment thereof comprising one or more mutations from the beta variant.In some embodiments, BD ₃ comprises RNA encoding a SARS-CoV-2 spike protein or an immunogenic fragment thereof from the Wuhan strain and RNA encoding a SARS-CoV-2 spike protein or an immunogenic fragment thereof comprising one or more mutations from the Omicron variant.

In some embodiments, BD _n comprises RNA encoding a SARS-CoV-2 spike protein or immunogenic fragment thereof from the Wuhan strain. In some embodiments, BD _n comprises RNA encoding a spike protein or immunogenic fragment thereof from a SARS-CoV-2 strain that is prevalent and/or spreading rapidly in a relevant jurisdiction. In some embodiments, BD _n comprises RNA encoding a SARS-CoV-2 spike protein or immunogenic fragment thereof that includes one or more mutations from an alpha variant. In some embodiments, BD _n comprises RNA encoding a SARS-CoV-2 spike protein or immunogenic fragment thereof that includes one or more mutations from a delta variant. In some embodiments, BD _n comprises RNA encoding a SARS-CoV-2 spike protein or immunogenic fragment thereof that includes one or more mutations from a beta variant. In some embodiments, BD _n comprises RNA encoding a SARS-CoV-2 spike protein or immunogenic fragment thereof that includes one or more mutations from an omicron variant.

In some embodiments, the BD _n comprises an RNA encoding a SARS-CoV-2 spike protein or an immunogenic fragment thereof from the Wuhan strain, and one or more additional RNAs encoding a spike protein or an immunogenic fragment thereof from a SARS-CoV-2 strain that is prevalent and/or spreading rapidly in the relevant jurisdiction. In some embodiments, the BD _n comprises an RNA encoding a SARS-CoV-2 spike protein or an immunogenic fragment thereof from the Wuhan strain, and an RNA encoding a SARS-CoV-2 spike protein or an immunogenic fragment thereof that includes one or more mutations from the alpha variant. In some embodiments, the BD _n comprises an RNA encoding a SARS-CoV-2 spike protein or an immunogenic fragment thereof from the Wuhan strain, and an RNA encoding a SARS-CoV-2 spike protein or an immunogenic fragment thereof that includes one or more mutations from the delta variant. In some embodiments, BD _n comprises RNA encoding a SARS-CoV-2 spike protein or an immunogenic fragment thereof from the Wuhan strain and RNA encoding a SARS-CoV-2 spike protein or an immunogenic fragment thereof comprising one or more mutations from a beta variant, In some embodiments, BD _n comprises RNA encoding a SARS-CoV-2 spike protein or an immunogenic fragment thereof from the Wuhan strain and RNA encoding a SARS-CoV-2 spike protein or an immunogenic fragment thereof comprising one or more mutations from an omicron variant.

In some embodiments, BD ₁ , BD ₂ , BD ₃ , and BD _n can each independently comprise a plurality (e.g., at least two) of RNA (e.g., mRNA) compositions as described herein. In some embodiments, BD ₁ , BD ₂ , BD ₃ , and BD _n can each independently comprise a first and a second RNA (e.g., mRNA) composition. In some embodiments, BD ₁ , BD ₂ , BD ₃ , and BD _n can each independently comprise a plurality (e.g., at least two) of RNA (e.g., mRNA) compositions, where at least one of the plurality of RNA (e.g., mRNA) compositions comprises BNT162b2 (e.g., as described herein). In some embodiments, at least one of the plurality of RNA (e.g., mRNA) compositions comprises RNA (e.g., mRNA) encoding a SARS-CoV-2 S protein or immunogenic fragment thereof from a different SARS-CoV-2 variant (e.g., a variant that is prevalent or spreading rapidly in a relevant jurisdiction, e.g., a variant disclosed herein). In some embodiments, at least one of the plurality of RNA (e.g., mRNA) compositions comprises RNA (e.g., mRNA) encoding a SARS-CoV-2 S protein or immunogenic fragment thereof from the Wuhan strain of SARS-CoV-2. In some embodiments, at least one of the plurality of RNA (e.g., mRNA) compositions comprises RNA encoding a SARS-CoV-2 S protein or immunogenic fragment thereof that includes one or more mutations from a variant that is prevalent and/or spreading rapidly in a relevant jurisdiction. In some embodiments, at least one of the plurality of RNA (e.g., mRNA) compositions comprises an RNA (e.g., mRNA) encoding a SARS-CoV-2 S protein or immunogenic fragment thereof that includes one or more mutations from an alpha variant. In some embodiments, at least one of the plurality of RNA (e.g., mRNA) compositions comprises an RNA (e.g., mRNA) encoding a SARS-CoV-2 S protein or immunogenic fragment thereof that includes one or more mutations from a delta variant. In some embodiments, at least one of the plurality of RNA (e.g., mRNA) compositions comprises an RNA (e.g., mRNA) encoding a SARS-CoV-2 S protein or immunogenic fragment thereof that includes one or more mutations from an omicron variant.

In some embodiments, the multiple RNA (e.g., mRNA) compositions provided with BD ₁ , BD ₂ , BD ₃ , and/or BD _n can each independently comprise at least two different mRNA constructs (e.g., RNA constructs having different protein coding sequences). For example, in some embodiments, the multiple RNA (e.g., mRNA) compositions provided with BD ₁ , BD ₂ , BD ₃ , and/or BD _n can each independently comprise an RNA (e.g., mRNA) encoding a SARS-CoV-2 S protein, or an immunogenic fragment thereof, from the Wuhan strain of SARS-CoV-2, as well as an RNA (e.g., mRNA) encoding a SARS-CoV-2 S protein, or an immunogenic fragment thereof, that includes one or more mutations from a variant that is prevalent and/or spreading rapidly in a relevant jurisdiction. In some embodiments, the multiple RNA (e.g., mRNA) compositions provided by BD ₁ , BD ₂ , BD ₃ , and/or BD _n can each independently comprise an RNA (e.g., mRNA) encoding a SARS-CoV-2 S protein or an immunogenic fragment thereof from the Wuhan strain of SARS-CoV-2, as well as an RNA (e.g., mRNA) encoding a SARS-CoV-2 S protein or an immunogenic fragment thereof that includes one or more mutations from a variant that is prevalent and/or rapidly spreading in the relevant jurisdiction. In some such embodiments, the variant can be an alpha variant. In some such embodiments, the variant can be a delta variant. In some such embodiments, the variant can be an omicron variant.

In some embodiments, the multiple RNA (e.g., mRNA) compositions provided with BD ₁ , BD ₂ , BD ₃ , and/or BD _n can each independently comprise at least two RNAs (e.g., mRNAs), each encoding a SARS-CoV-2 S protein or immunogenic fragment thereof that includes one or more mutations from a distinct variant that is prevalent and/or spreading rapidly in a relevant jurisdiction. In some embodiments, the multiple RNA (e.g., mRNA) compositions provided with BD ₁ , BD ₂ , BD ₃ , and/or BD _n can each independently comprise an mRNA encoding a SARS-CoV-2 S protein or immunogenic fragment thereof from an alpha variant, and an RNA (e.g., mRNA) encoding a SARS-CoV-2 S protein or immunogenic fragment thereof that includes one or more mutations from a delta variant. In some embodiments, a plurality of RNA (e.g., mRNA) compositions provided with BD ₁ , BD ₂ , BD ₃ , and/or BD _n can each independently comprise an RNA (e.g., mRNA) encoding a SARS-CoV-2 S protein or an immunogenic fragment thereof from the alpha variant, and an RNA (e.g., mRNA) encoding a SARS-CoV-2 S protein or an immunogenic fragment thereof comprising one or more mutations from the omicron variant. In some embodiments, a plurality of RNA (e.g., mRNA) compositions provided with BD ₁ , BD ₂ , BD ₃ , and/or BD _n can each independently comprise an RNA (e.g., mRNA) encoding a SARS-CoV-2 S protein or an immunogenic fragment thereof from the delta variant, and an RNA (e.g., mRNA) encoding a SARS-CoV-2 S protein or an immunogenic fragment thereof comprising one or more mutations from the omicron variant.

In some embodiments, multiple RNA (e.g., mRNA) compositions provided with _BD1 , _BD2 , _BD3 , and/or _BDn are separately administered to a subject via intramuscular injection, e.g., in some embodiments, at different injection sites. For example, in some embodiments, a first and a second RNA (e.g., mRNA) composition provided with _BD1 , _BD2 , _BD3 , and/or _BDn are separately administered to a subject via intramuscular injection into different arms.

In some embodiments, the length of time between BD ₁ and BD ₂ (BI ₁ ) is at least about 1 week, at least about 2 weeks, at least about 3 weeks, or at least about 4 weeks. In some embodiments, BI ₁ is from about 1 week to about 12 weeks. In some embodiments, BI ₁ is from about 1 week to about 10 weeks. In some embodiments, BI ₁ is from about 2 weeks to about 10 weeks. In some embodiments, BI ₁ is from about 2 weeks to about 8 weeks. In some embodiments, BI ₁ is from about 3 weeks to about 8 weeks. In some embodiments, BI ₁ is from about 4 weeks to about 8 weeks. In some embodiments, BI ₁ is from about 6 weeks to about 8 weeks. In some embodiments, BI ₁ is from about 3 to about 4 weeks. In some embodiments, BI ₁ is about 1 week. In some embodiments, BI ₁ is about 2 weeks. In some embodiments, BI ₁ is about 3 weeks. In some embodiments, BI ₁ is about 4 weeks. In some embodiments, BI ₁ is about 5 weeks. In some embodiments, BI ₁ is about 6 weeks. In some embodiments, BI ₁ is about 7 weeks. In some embodiments, BI ₁ is about 8 weeks. In some embodiments, BI ₁ is about 9 weeks. In some embodiments, BI ₁ is about 10 weeks.

In some embodiments, the length of time between BD ₂ and BD ₃ (BI ₂ ) is at least about 1 week, at least about 2 weeks, or at least about 3 weeks. In some embodiments, BI ₂ is from about 1 week to about 12 weeks. In some embodiments, BI ₂ is from about 1 week to about 10 weeks. In some embodiments, BI ₂ is from about 2 weeks to about 10 weeks. In some embodiments, BI ₂ is from about 2 weeks to about 8 weeks. In some embodiments, BI ₂ is from about 3 weeks to about 8 weeks. In some embodiments, BI ₂ is from about 4 weeks to about 8 weeks. In some embodiments, BI ₂ is from about 6 weeks to about 8 weeks. In some embodiments, BI ₂ is from about 3 to about 4 weeks. In some embodiments, BI ₂ is about 1 week. In some embodiments, BI ₂ is about 2 weeks. In some embodiments, BI ₂ is about 3 weeks. In some embodiments, BI ₂ is about 4 weeks. In some embodiments, BI ₂ is about 5 weeks. In some embodiments, BI ₂ is about 6 weeks. In some embodiments, BI ₂ is about 7 weeks. In some embodiments, BI ₂ is about 8 weeks. In some embodiments, BI ₂ is about 9 weeks. In some embodiments, BI ₂ is about 10 weeks.

In some embodiments, the length of time (BI _n ) between BD ₃ and any subsequent doses that are part of a booster dosing regimen, or between doses for any doses beyond BD ₃ , is each separately and independently selected from about 1 week or more, about 2 weeks or more, or about 3 weeks or more. In some embodiments, BI _n is about 1 week to about 12 weeks. In some embodiments, BI _n is about 1 week to about 10 weeks. In some embodiments, BI _n is about 2 weeks to about 10 weeks. In some embodiments, BI _n is about 2 weeks to about 8 weeks. In some embodiments, BI _n is about 3 weeks to about 8 weeks. In some embodiments, BI _n is about 4 weeks to about 8 weeks. In some embodiments, BI _n is about 6 weeks to about 8 weeks. In some embodiments, BI _n is about 3 to about 4 weeks. In some embodiments, BI _n is about 1 week. In some embodiments, BI _n is about 2 weeks. In some embodiments, BI _n is about 3 weeks. In some embodiments, BI _n is about 4 weeks. In some embodiments, BI _n is about 5 weeks. In some embodiments, BI _n is about 6 weeks. In some embodiments, BI _n is about 7 weeks. In some embodiments, BI _n is about 8 weeks. In some embodiments, BI _n is about 9 weeks. In some embodiments, BI _n is about 10 weeks.

In some embodiments, one or more compositions administered on BD ₁ are formulated in a Tris buffer. In some embodiments, one or more compositions administered on BD ₂ are formulated in a Tris buffer. In some embodiments, one or more compositions administered on BD ₃ are formulated in a Tris buffer. In some embodiments, one or more compositions administered on BD ₃ are formulated in a Tris buffer.

In some embodiments, the booster dosing regimen comprises administering two or more RNA (e.g., mRNA) compositions described herein, where at least two of the RNA (e.g., mRNA) compositions have different formulations. In some embodiments, the booster dosing regimen comprises BD ₁ and BD ₂ , where BD ₁ comprises administering RNA (e.g., mRNA) formulated in Tris buffer, and BD ₂ comprises administering RNA (e.g., mRNA) formulated in PBS buffer. In some embodiments, the booster dosing regimen comprises BD ₁ and BD ₂ , where BD ₁ comprises administering RNA (e.g., mRNA) formulated in PBS buffer, and BD ₂ comprises administering RNA (e.g., mRNA) formulated in Tris buffer.

In some embodiments, one or more RNA (e.g., mRNA) compositions provided in BD ₁ , BD ₂ , BD ₃ , and/or BD _n may be administered in conjunction with another vaccine. In some embodiments, the other vaccine is for a disease that is not COVID-19. In some embodiments, the disease is one that increases the deleterious effects of SARS-CoV-2 when a subject is co-infected with the disease and SARS-CoV-2. In some embodiments, the disease is one that increases the transmission rate of SARS-CoV-2 when a subject is co-infected with the disease and SARS-CoV-2. In some embodiments, the other vaccine is a different commercially available vaccine. In some embodiments, the different commercially available vaccine is an RNA vaccine. In some embodiments, the different commercially available vaccine is a polypeptide-based vaccine. In some embodiments, another vaccine (e.g., as described herein) and one or more RNA (e.g., mRNA) compositions provided in BD ₁ , BD ₂ , BD ₃ , and/or BD _n are administered separately, e.g., via intramuscular injection, in some embodiments, at different injection sites. For example, in some embodiments, an influenza vaccine described herein and one or more SARS-CoV-2 RNA (e.g., mRNA) compositions provided in BD ₁ , BD ₂ , BD ₃ , and/or BD _n are administered separately via intramuscular injection into different arms of a subject.

Additional Booster Regimens In some embodiments, the methods of vaccination disclosed herein include administering two or more booster dosing regimens. In some embodiments, it may be necessary to administer two or more booster dosing regimens to increase the neutralizing antibody response. In some embodiments, two or more booster dosing regimens may be required to combat SARS-CoV-2 strains that have been shown to be more likely to evade the immune response elicited by a vaccine that the patient previously received. In some embodiments, additional booster dosing regimens are administered to patients that have been determined to produce low concentrations of neutralizing antibodies. In some embodiments, additional booster dosing regimens are administered to patients (e.g., immunocompromised patients, cancer patients, and/or organ transplant patients) that have been determined to be more likely to be susceptible to SARS-CoV-2 infection despite previous vaccination.

The above description of the first booster dosing regimen also describes one or more additional booster dosing regimens. The time interval between the first booster dosing regimen and the second booster dosing regimen, or between subsequent booster dosing regimens, can be any of the above-mentioned acceptable time intervals between the primary dosing regimen and the first booster dosing regimen.

In some embodiments, the dosing regimen includes a primary regimen and a booster regimen, and at least one dose given in the primary regimen and/or booster regimen includes a composition comprising an RNA encoding an S protein or an immunogenic fragment thereof from a variant (e.g., an Omicron variant as described herein) that is prevalent or spreading rapidly in the relevant jurisdiction. For example, in some embodiments, the primary regimen includes at least two doses of BNT162b2 (e.g., encoding the Wuhan strain), given, for example, at least three weeks apart, and the booster regimen includes at least one dose of a composition comprising an RNA encoding an S protein or an immunogenic fragment thereof from a variant (e.g., an Omicron variant as described herein) that is prevalent or spreading rapidly in the relevant jurisdiction. In some such embodiments, such doses of the booster regimen may further include RNA encoding an S protein or an immunogenic fragment thereof from the Wuhan strain, which may be administered together with RNA encoding an S protein or an immunogenic fragment thereof from a variant that is prevalent or rapidly spreading in the relevant jurisdiction (e.g., an Omicron variant as described herein) as a single mixture or as two separate compositions, e.g., in a 1:1 weight ratio. In some embodiments, the booster regimen may also include at least one dose of BNT162b2, which may be administered as a first booster dose or a subsequent booster dose.

In some embodiments, the RNA composition described herein is given as a booster at a higher dose than the dose given during the primary regimen (primary dose) and/or the dose given for the first booster (if present). For example, in some embodiments, such a dose may be 60ug, or in some embodiments, such a dose may be higher than 30ug and lower than 60ug (e.g., 55ug, 50ug, or less). In some embodiments, the RNA composition described herein is given as a booster at least 3-12 months, or 4-12 months, or 5-12 months, or 6-12 months after the last dose (e.g., the last dose of the primary regimen or the first dose of the booster regimen). In some embodiments, the primary dose and/or the first booster dose (if present) may comprise BNT162b2 at, for example, 30ug per dose.

In some embodiments, the RNA compositions described herein include RNA encoding a polypeptide set forth in SEQ ID NO:49 or an immunogenic fragment thereof, or a variant thereof (e.g., having at least 70% or more (e.g., including at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or more) identity to SEQ ID NO:49). In some embodiments, the RNA compositions include RNA comprising a sequence of SEQ ID NO:50 or a variant thereof (e.g., having at least 70% or more (e.g., including at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or more) identity to SEQ ID NO:50). In some embodiments, the RNA compositions include RNA comprising a sequence of SEQ ID NO:51 or a variant thereof (e.g., having at least 70% or more (e.g., including at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or more) identity to SEQ ID NO:51).

In some embodiments, the RNA compositions described herein include RNA encoding a polypeptide set forth in SEQ ID NO:55 or an immunogenic fragment thereof, or a variant thereof (e.g., having at least 70% or more (e.g., including at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or more) identity to SEQ ID NO:55). In some embodiments, the RNA compositions include RNA comprising a sequence of SEQ ID NO:56 or a variant thereof (e.g., having at least 70% or more (e.g., including at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or more) identity to SEQ ID NO:56). In some embodiments, the RNA compositions include RNA comprising a sequence of SEQ ID NO:57 or a variant thereof (e.g., having at least 70% or more (e.g., including at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or more) identity to SEQ ID NO:57).

In some embodiments, the RNA compositions described herein include RNA encoding a polypeptide set forth in SEQ ID NO:58 or an immunogenic fragment thereof, or a variant thereof (e.g., having at least 70% or more (e.g., including at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or more) identity to SEQ ID NO:58). In some embodiments, the RNA compositions include RNA comprising a sequence of SEQ ID NO:59 or a variant thereof (e.g., having at least 70% or more (e.g., including at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or more) identity to SEQ ID NO:59). In some embodiments, the RNA compositions include RNA comprising a sequence of SEQ ID NO:60 or a variant thereof (e.g., having at least 70% or more (e.g., including at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or more) identity to SEQ ID NO:60).

In some embodiments, the RNA compositions described herein include RNA encoding a polypeptide set forth in SEQ ID NO:61 or an immunogenic fragment thereof, or a variant thereof (e.g., having at least 70% or more (e.g., including at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or more) identity to SEQ ID NO:61). In some embodiments, the RNA compositions include RNA comprising a sequence of SEQ ID NO:62 or a variant thereof (e.g., having at least 70% or more (e.g., including at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or more) identity to SEQ ID NO:62). In some embodiments, the RNA compositions include RNA comprising a sequence of SEQ ID NO:63 or a variant thereof (e.g., having at least 70% or more (e.g., including at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or more) identity to SEQ ID NO:63).

In some embodiments, the formulations disclosed herein can be used to implement any of the dosing regimens set forth in Table 28 (below).

In some embodiments of certain exemplary dosing regimens described in Table 28 above, an RNA composition described herein (e.g., comprising an RNA encoding a variant described herein) is given in the first dose of the primary regimen. In some embodiments of certain exemplary dosing regimens described in Table 28 above, an RNA composition described herein (e.g., comprising an RNA encoding a variant described herein) is given in the second dose of the primary regimen. In some embodiments of certain exemplary dosing regimens described in Table 28 above, an RNA composition described herein (e.g., comprising an RNA encoding a variant described herein) is given in the first and second doses of the primary regimen. In some embodiments of certain exemplary dosing regimens described in Table 28 above, an RNA composition described herein (e.g., comprising an RNA encoding a variant described herein) is given in the first dose of the booster regimen. In some embodiments of certain exemplary dosing regimens described in Table 28 above, an RNA composition described herein (e.g., comprising an RNA encoding a variant described herein) is given in the second dose of the booster regimen. In some embodiments of certain exemplary dosing regimens described in Table 28 above, an RNA composition described herein (e.g., comprising an RNA encoding a variant described herein) is given in the first and second doses of a booster regimen. In some embodiments of certain exemplary dosing regimens described in Table 28 above, an RNA composition described herein (e.g., comprising an RNA encoding a variant described herein) is given in the first and second doses of a primary regimen and at least one dose of a booster regimen. In some embodiments of certain exemplary dosing regimens described in Table 28 above, an RNA composition described herein (e.g., comprising an RNA encoding a variant described herein) is given in at least one dose (e.g., comprising at least two doses) of a booster regimen, and BNT162b2 is given in the primary regimen. In some embodiments of certain exemplary dosing regimens described in Table 28 above, an RNA composition described herein (e.g., comprising an RNA encoding a variant described herein) is given in the second dose of the booster regimen, and BNT162b2 is given in the primary regimen and in the first dose of the booster regimen. In some embodiments, an RNA composition described herein (e.g., comprising an RNA encoding a variant described herein) comprises an RNA encoding a polypeptide set forth in SEQ ID NO:49 or an immunogenic fragment thereof, or a variant thereof (e.g., having at least 70% or more (e.g., including at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or more) identity to SEQ ID NO:49). In some embodiments, the RNA compositions described herein (e.g., including RNA encoding a variant described herein) include RNA comprising a sequence of SEQ ID NO: 50 or a variant thereof (e.g., having at least 70% or more (e.g., including at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or more) identity to SEQ ID NO: 50). In some embodiments, the RNA compositions described herein (e.g., including RNA encoding a variant described herein) include RNA comprising a sequence of SEQ ID NO: 51 or a variant thereof (e.g., having at least 70% or more (e.g., including at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or more) identity to SEQ ID NO: 51).

In some embodiments, the RNA compositions described herein include RNA encoding a polypeptide set forth in SEQ ID NO:55 or an immunogenic fragment thereof, or a variant thereof (e.g., having at least 70% or more (e.g., including at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or more) identity to SEQ ID NO:55). In some embodiments, the RNA compositions include RNA comprising a sequence of SEQ ID NO:56 or a variant thereof (e.g., having at least 70% or more (e.g., including at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or more) identity to SEQ ID NO:56). In some embodiments, the RNA compositions include RNA comprising a sequence of SEQ ID NO:57 or a variant thereof (e.g., having at least 70% or more (e.g., including at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or more) identity to SEQ ID NO:57).

In some embodiments, the RNA compositions described herein include RNA encoding a polypeptide set forth in SEQ ID NO:58 or an immunogenic fragment thereof, or a variant thereof (e.g., having at least 70% or more (e.g., including at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or more) identity to SEQ ID NO:58). In some embodiments, the RNA compositions include RNA comprising a sequence of SEQ ID NO:59 or a variant thereof (e.g., having at least 70% or more (e.g., including at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or more) identity to SEQ ID NO:59). In some embodiments, the RNA compositions include RNA comprising a sequence of SEQ ID NO:60 or a variant thereof (e.g., having at least 70% or more (e.g., including at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or more) identity to SEQ ID NO:60).

In some embodiments, the RNA compositions described herein include RNA encoding a polypeptide set forth in SEQ ID NO:61 or an immunogenic fragment thereof, or a variant thereof (e.g., having at least 70% or more (e.g., including at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or more) identity to SEQ ID NO:61). In some embodiments, the RNA compositions include RNA comprising a sequence of SEQ ID NO:62 or a variant thereof (e.g., having at least 70% or more (e.g., including at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or more) identity to SEQ ID NO:62). In some embodiments, the RNA compositions include RNA comprising a sequence of SEQ ID NO:63 or a variant thereof (e.g., having at least 70% or more (e.g., including at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or more) identity to SEQ ID NO:63).

In some embodiments, such RNA compositions described herein (e.g., including RNA encoding a variant described herein) can further include RNA encoding an S protein or immunogenic fragment thereof of a different strain (e.g., Wuhan strain). By way of example, in some embodiments, the second dose of a booster regimen of Regimen Nos. 9-11 described in Table 28 above can include an RNA composition described herein (e.g., in one embodiment described in this embodiment, including RNA encoding a variant described herein, such as Omicron) and a BNT162b2 construct, e.g., in a 1:1 weight ratio.

In some embodiments of Regimen No. 6 as described in Table 28 above, the first and second doses of the primary regimen and the first and second doses of the booster regimen each comprise an RNA composition as described herein (e.g., RNA encoding a variant described herein, such as omicron, e.g., in one embodiment, RNA as described in this Example). In some such embodiments, the second dose of the booster regimen may not be necessary.

In some embodiments of Regimen No. 6 as described in Table 28 above, the first and second doses of the primary regimen and the first and second doses of the booster regimen each comprise an RNA composition as described herein (e.g., RNA encoding a variant described herein, such as omicron, e.g., in one embodiment, RNA as described in this Example). In some such embodiments, the second dose of the booster regimen may not be necessary.

In some embodiments of Regimen No. 6 as described in Table 28 above, the first and second doses of the primary regimen each include a BNT162b2 construct, and the first and second doses of the booster regimen each include an RNA composition as described herein (e.g., RNA encoding a variant described herein, such as omicron, e.g., in one embodiment, RNA as described in this Example). In some such embodiments, the second dose of the booster regimen may not be necessary.

In some embodiments of Regimen No. 6 as described in Table 28 above, the first and second doses of the primary regimen and the first dose of the booster regimen each comprise a BNT162b2 construct, and the second dose of the booster regimen comprises an RNA composition as described herein (e.g., RNA encoding a variant described herein, such as omicron, e.g., in one embodiment, RNA as described in this Example).

Example 7: Omicron breakthrough infection drives cross-variant neutralization and memory B cell formation This example shows that omicron breakthrough infection in individuals vaccinated two and three times with BNT162b2 drives cross-variant neutralization and memory B cell formation, including the production of neutralizing antibodies and B cell responses to omicron variants. One of skill in the art reading this example will understand that such findings can be extended to administering an RNA (e.g., mRNA) vaccine comprising RNA encoding a SARS-CoV-2 S protein having a mutation (e.g., as described herein) characteristic of an omicron variant to a subject who has previously received two or three doses of a SARS-CoV-2 vaccine (e.g., developed in some embodiments based on the S protein from the Wuhan-Hu-1 strain).

Omicron is the most evolutionarily distinct SARS-CoV-2 variant of concern (VOC) to date. To address how Omicron breakthrough infection may reshape SARS-CoV-2 recognition in vaccinated individuals, we investigated the effect of Omicron breakthrough infection on serum neutralization and B _MEM cell antigen recognition in individuals vaccinated two and three times with BNT162b2. Omicron breakthrough infection induced broad neutralization of VOCs, including Omicron, with substantially more potent neutralization compared to Omicron-naive two and three vaccinees. Broad recognition of VOCs by B _MEM cells from individuals vaccinated two and three times with BNT162b2 was boosted by Omicron breakthrough infection and was primarily directed at conserved epitopes that are broadly shared between variants, rather than Omicron-specific epitopes. The data presented herein demonstrate that Omicron breakthrough infection efficiently expands neutralizing antibody and/or B cell responses to multiple variants and that a vaccine adapted to the Omicron S protein may be able to reshape the immune repertoire.

Introduction Containment of the current COVID-19 pandemic requires the generation of durable and sufficiently broad immunity to provide protection against circulating and future variants of SARS-CoV-2. Neutralizing antibody titers against SARS-CoV-2, as well as antibody binding to the spike (S) glycoprotein and its receptor binding domain (RBD), are thought to be correlates of protection against infection (D. S. Khoury et al., “Neutralizing antibody levels are highly predictive of immune protection from symptomatic SARS-CoV-2 infection,” Nature medicine. 27, 1205-1211 (2021), doi: 10.1038/s41591-021-01377-8, and P. B. Gilbert et al., “Neutralizing antibody levels are highly predictive of immune protection from symptomatic SARS-CoV-2 infection,” Nature medicine. 27, 1205-1211 (2021), doi: 10.1038/s41591-021-01377-8, and P. B. Gilbert et al., “Neutralizing antibody levels are highly predictive of immune protection from symptomatic SARS-CoV-2 infection,” Nature medicine. al. , “Immune correlates analysis of the mRNA-1273 COVID-19 vaccine efficacy clinical trial,” Science (New York, N.Y.). 375, 43-50 (2022), doi:10.1126/science. abm3425). Currently available vaccines are based on the ancestral Wuhan-Hu-1 strain and induce antibodies with a neutralizing capacity that exceeds the breadth induced by infection with the Wuhan strain or variants of concern (VOCs) (K. Roltgen et al., "Immune imparting, breeding of variant recognition, and germinal center response in human SARS-CoV-2 infection and vaccination," Cell (2022), doi:10.1016/j.cell.2022.01.018). However, the protective titers decreased over time (J.P. Evans et al., "Neutralizing antibody responses elicited by SARS-CoV-2 mRNA vaccination when over time and are boosted by breakthrough infection," Science translational medicine, eabn8057 (2022), doi:10.1126/scitranslmed.abn8057; S. Yamayoshi et al., "Antibody titers against SARS-CoV-2 mRNA vaccination when over time and are boosted by breakthrough infection," Science translational medicine, eabn8057 (2022), doi:10.1126/scitranslmed.abn8057). SARS-CoV-2 decline, but do not disappear for several months, “EclinicalMedicine, 32, 100734 (2021), doi:10.1016/j.eclinm.2021.100734, W.N. Chia et al., “Dynamics of SARS-CoV-2 neutralizing antibody responses and duration of immunity, “The Lancet Microbe, 2, e240-e249 (2021), doi: 10.1016/S2666-5247(21)00025-2, Y. Goldberg et al., "Waning Immunity after the BNT162b2 Vaccine in Israel," The New England journal of medicine. 385, e85 (2021), doi: 10.1056/NEJMoa2114228, Regular booster vaccination is thought to be necessary to induce recall immunity and maintain efficacy against new VOCs (A.R. Falsey et al., "SARS-CoV-2 Neutralization with BNT162b2 Vaccine Dose 3, “The New England journal of medicine. 385, 1627-1629 (2021), doi:10.1056/NEJMc2113468, A. Choi et al., “Safety and immunogenicity of SARS-CoV-2 variants. mRNA vaccine boosters in healthy adults, “Nature medicine. 27, 2025-2031 (2021), doi:10.1038/s41591-021-01527-y, and N. Andrews et al. , “Effectiveness of COVID-19 booster vaccines against COVID-19 related symptoms, hospitalization and death in England, “Nature medicine (2022), doi:10.1038/s41591-022-01699-1.

Long-lived memory B ( _BMEM ) cells are the basis of recall responses upon antigen re-encounter, either by infection or booster vaccination. The low affinity selection mechanism during the germinal center reaction and the continuous hypermutation of B _MEM cells play an important role in the maintenance and evolution of antiviral antibody responses to variants, expanding the breadth of viral variant recognition over time (W. E. Purtha, et al., "Memory B cells, but not long-lived plasma cells, possess antigen specificities for viral escape mutants," The Journal of experimental medicine, 208, 2599-2606 (2011) doi: 10.1084/jem.20110740, and Y. Adachi ... al. , “Distinct germinal center selection at local sites shapes memory B cell response to viral escape, “The Journal of experimental medicine. 212, 1709-1723 (2015), doi:10.1084/jem. 20142284).

How vaccine-mediated protective immunity evolves over time and is modified by repeated exposure to COVID-19 vaccines and infections with increasingly divergent viral variants is particularly relevant with the emergence of antigenically distinct VOCs. Omicron is the most evolutionarily distant reported VOC, harboring an unprecedented number of amino acid changes in the S glycoprotein, including at least 15 amino acid changes in the RBD and extensive changes in the N-terminal domain (NTD). These modifications are predicted to affect most neutralizing antibody epitopes. Furthermore, Omicron is highly transmissible and distinct from its sublineages BA.1 and BA.2. 2 is spreading rapidly around the world and is expected to overtake Delta as the predominant circulating VOC within weeks (W. Dejnirattisai et al., "SARS-CoV-2 Omicron-B.1.1.529 leads to widespread escape from neutralizing antibody responses," Cell. 185, 467-484.e15 (2022), doi: 10.1016/j.cell.2021.12.046, and M. Hoffmann et al., "The Omicron variant is highly resistant against antibody-mediated neutralization, “Cell.185, 447-456.e11 (2022), doi:10.1016/j.cell.2021.12.032).

To date, more than 1 billion people worldwide have been vaccinated with the mRNA-based COVID-19 vaccine BNT162b2, receiving either a primary two-dose series or a further booster. This vaccine has contributed substantially to a pattern of herd immunity in many areas where further immune editing and the effectiveness of currently circulating variants are building.

To characterize the effect of Omicron breakthrough infection on serum neutralizing activity and size and breadth of _BMEM cells, blood samples from individuals vaccinated two or three times with BNT162b2 were studied.

Because understanding antigen-specific B cell memory pools is a key determinant of an individual's ability to respond to emerging variants, this data can help guide vaccine development.

Results and Discussion Cohort and Sample Collection Blood samples were sourced from the BNT162b2 vaccine trial biological specimen collection and a biobank of samples collected prospectively from vaccinated individuals with subsequent SARS-CoV-2 omicron breakthrough infection. Samples were selected to investigate biomarkers in four independent groups: individuals vaccinated (i) twice or (ii) three times with BNT162b2 without previous or breakthrough infection at the time of sample collection (BNT162b2 ² , BNT162b2 ³ ); and individuals vaccinated (iii) twice or (iv) three times with BNT162b2 who experienced a breakthrough infection with the SARS-CoV-2 Omicron variant after a median of approximately 5 months or 4 weeks, respectively (BNT162b2 ² +Omi, BNT162b2 ³ +Omi) (see Materials and Methods below). Immune sera were used to characterize Omicron infection-associated changes in the magnitude and breadth of serum neutralizing activity. PBMCs were used to characterize the VOC specificity of peripheral B _MEM cells that recognize each full-length SARS-CoV-2 S protein or its RBD (Figure 15).

Omicron breakthrough infection in individuals vaccinated two and three times with BNT162b2 induces broad neutralization of Omicron BA.1, BA.2, and other VOCs.
To assess the neutralizing activity of immune sera, two orthogonal test systems were used: a well-characterized pseudovirus neutralization test (pVNT) to investigate the breadth of inhibition of virus entry in growth-deficient settings, and a live SARS-CoV-2 neutralization test (VNT) designed to assess neutralization during multicycle replication of the authentic virus with antibodies maintained throughout the test period. 2. We assessed breadth using pseudoviruses carrying S proteins containing mutations characteristic of other SARS-CoV-2 VOCs (Wuhan, alpha, beta, delta), while we used pseudoviruses carrying the S protein of SARS-CoV-1 (T. Li et al., "Phyloge- netic supertree reveals detailed evolution of SARS-CoV-2," Scientific reports, 10, 22366 (2020), doi:10.1038/s41598-020-79484-8) to detect potential pan-sarbecovirus neutralizing activity (C.-W. Tan et al., "Pan-Sarbecovirus Neutralizing Antibodies in BNT162b2-Immunized SARS-CoV-1 Survivors, “The New England journal of medicine, 385, 1401-1406 (2021), doi:10.1056/NEJMoa2108453).

As previously reported (A. R. Falsey et al., “SARS-CoV-2 Neutralization with BNT162b2 Vaccine Dose 3,” The New England journal of medicine, 385, 1627-1629 (2021), doi: 10.1056/NEJMc2113468 and C.-W. Tan et al., “Pan-Sarbecovirus Neutralizing Antibodies in BNT162b2-Immunized SARS-CoV-1 Survivors,” The New England journal of medicine. 385, 1401-1406 (2021), doi:10.1056/NEJMoa2108453), in omicron-naive, two-vaccinated individuals, the 50% pseudovirus neutralization ( _pVN50 ) geometric mean titers (GMTs) of beta and delta VOCs were reduced, with neutralization of both omicron sublineages virtually undetectable. In omicron-naive, three-vaccinated individuals, _pVN50 GMTs against all tested VOCs were substantially higher, with robust neutralization of alpha, beta, and delta variants. GMTs against omicron BA.1 were significantly lower compared to Wuhan (GMT 160 vs. 398), but titers against omicron BA.2 were also significantly reduced at 211. Thus, three vaccinations induced similar levels of neutralization against the two Omicron sublineages (Figure 16, A) (A. Muik et al., "Neutralization of SARS-CoV-2 Omicron by BNT162b2 mRNA vaccine-elicited human sera," Science (New York, N.Y.), 375, 678-680 (2022), doi:10.1126/science.abn7591; C.-W. Tan et al., "Pan-Sarbecovirus Neutralizing Antibodies in BNT162b2-Immunized SARS-CoV-1 Survivors, “The New England journal of medicine, 385, 1401-1406 (2021), doi:10.1056/NEJMoa2108453, J. Liu et al., “BNT162b2-elicited neutralization of B. 1.617 and other SARS-CoV-2 variants, “Nature, 596, 273-275 (2021), doi:10.1038/s41586-021-03693-y, A. Muik et al., “Neutralization of SARS-CoV-2 lineage B. 1.1.7 Pseudovirus by BNT162b2 vaccine-elicited human sera, “Science (New York, N.Y.), 371, 1152-1153 (2021), doi:10.1126/science. al. , “Neutralizing Activity of BNT162b2-Elicited Serum, “The New England journal of medicine, 384, 1466-1468 (2021), doi:10.1056/NEJMc2102017).

Omicron breakthrough infection had a significant effect on the magnitude and breadth of neutralizing antibody responses in both twice- and three-times-vaccinated individuals, with slightly higher _pVN50 GMTs observed in three-times-vaccinated individuals (FIG. 16, A). The pVN50 GMTs of twice-vaccinated individuals with breakthrough infection against Omicron BA.1 and BA.2 were 100-fold and 35-fold higher than those of Omicron-naive twice-vaccinated individuals. Immune sera from twice-vaccinated individuals with breakthrough infection had broad neutralizing activity, with higher pVN50 GMTs against beta and delta than observed in Omicron-naive three-times-vaccinated individuals (GMT 740 vs. 222 and 571 vs. 370).

The effect of Omicron breakthrough infection on neutralization of Omicron BA.1 and BA.2 pseudoviruses was less pronounced when looking at 3-dose vaccinated individuals (approximately 7-fold and 4-fold increase in neutralization compared to 3-dose Omicron-naive vaccinated individuals). _pVN50 GMTs for Omicron BA.1, BA.2, and Delta were 1029, 836, and 1103 in 3-dose vaccinated Omicron breakthrough infected individuals compared to 160, 211, and 370 in 3-dose Omicron-naive vaccinated individuals. GMTs for all SARS-CoV-2 VOCs, including Beta and Omicron, were close to the titers for the Wuhan criteria but were significantly reduced in 3-dose vaccinated Omicron-naive individuals.

Similarly, sera from vaccinated Omicron-naive individuals had no or poor _{pVN 50} titers against the phylogenetically more distant SARS-CoV-1, but convalescent sera from twice- and even more notably three-times-vaccinated Omicron-infected individuals robustly neutralized the SARS-CoV-1 pseudovirus (Figure 16, A and B). Nine of 18 breakthrough-infected individuals (four twice-vaccinated and five three times-vaccinated) had SARS-CoV-1 pVN50 GMTs equal to or greater than those against the Wuhan criteria in Omicron-naive twice-vaccinated individuals (GMT > 120).

Authentic live SARS-CoV-2 virus neutralization assays performed with Wuhan, beta, delta, and Omicron BA.1 pseudoviruses also showed similar findings (Figure 16, B). In individuals vaccinated two and three times with BNT162b2, Omicron infection was associated with strong increased neutralizing activity against Omicron BA.1 with 50% virus neutralization ( _VN50 ) GMTs in the same range as the Wuhan strain (Figure 16, B; GMT 493 vs. 381, and GMT 538 vs. 613). Similarly, individuals vaccinated two and three times with Omicron convalescent showed similar levels of neutralization against other variants (e.g., GMT 493 and 729 against beta), indicating a broad range of neutralizing activity.

Taken together, these data demonstrate that SARS-CoV-2 omicron breakthrough infection induces a deep breadth of neutralizing activity in vaccine-experienced individuals, a finding that is further supported by the calculated ratios of _VN50 GMTs for the Wuhan strain and SARS-CoV-2 VOCs (Figure 16, C). Omicron-naive individuals vaccinated twice and to a lesser extent three times with BNT162b2 showed marked differences in neutralizing potency against VOCs, whereas neutralizing activity in omicron-recovery subjects was comparable with similar breadth and high potency against all variant strains tested.

Similarly, Omicron breakthrough infection had a similarly broad neutralization-enhancing effect in individuals vaccinated with other approved COVID-19 vaccines or heterologous regimens (Figure 19, Table 29).

B _MEM cells from individuals vaccinated two and three times with BNT162b2 broadly recognize VOCs and are further boosted by Omicron breakthrough infection.
Next, the phenotype and quantity of SARS-CoV-2 S protein-specific B cells were investigated. A flow cytometry-based B cell phenotyping assay was used for differential detection of variant-specific S protein-binding B cells in bulk PBMCs. All S protein-specific and RBD-specific B cells in peripheral blood were found to be of B _MEM phenotype (B _MEM , CD 20 high CD ^{38 int/neg} ), since no antigen-specific plasma cells or naive B cells were detected (data not shown). Thus, the assay allowed differentiation for each of the SARS-CoV- ² variants between B _MEM cells recognizing the complete S protein or its RBD, which is a hotspot for amino acid modifications, and variant-specific antigenic epitopes ( FIG. 17 , A).

The overall frequency of antigen-specific B _MEM cells varied across the different groups. The frequency of B _MEM cells in omicron-naive, twice-vaccinated individuals was low early after vaccination and increased over time: at 5 months, compared with 3 weeks after the second BNT162b2 dose, S protein-specific B _MEM cells nearly quadrupled and RBD-specific tripled across all VOCs, reaching amounts similar to those observed in omicron-naive, 3-vaccinated individuals (FIGS. 17, B and C).

Two- or three-times BNT162b2-vaccinated individuals with SARS-CoV-2 omicron breakthrough infection showed strongly increased frequencies of B _MEM cells, which were higher than those of omicron-naive three-times-vaccinated individuals (Fig. 17, B and D).

In all groups, including omicron-uninfected and omicron-infected individuals, B _MEM cells against the omicron BA.1 S protein were detectable at frequencies comparable to those against the Wuhan and other VOCs tested (Fig. 17, B and D), whereas the frequency of B _MEM cells against the omicron BA.1 RBD was slightly lower compared to the other variants (Fig. 17, C and E).

The ratio of RBD protein to S protein binding in the different groups was then compared and found to be biased towards S protein recognition for Omicron BA.1 VOCs, especially in the Omicron-naive group (Figure 17, F). In the Omicron-experienced group, this ratio was higher, indicating that Omicron breakthrough infection improved Omicron BA.1 RBD recognition.

Omicron breakthrough infections in individuals vaccinated two and three times with BNT162b2 primarily boost B MEM against conserved _epitopes that are extensively shared between the Wuhan S protein and other VOCs, rather than strictly Omicron S-specific epitopes.

These findings indicate that Omicron infection in vaccinated individuals not only boosts neutralizing activity against Omicron BA.1 and B _MEM cells, but also broadly enhances immunity against a variety of VOCs. To investigate the specificity of the antibody response at the cellular level, multiparameter analysis of B _MEM cells stained with fluorescently labeled variant-specific S or RBD proteins was performed.

A combinatorial gating strategy was applied to distinguish _BMEM cell subsets that could identify only single variant-specific epitopes of Wuhan, alpha, delta, or omicron BA.1 from those that could specify any given combination of them (Figure 18, A).

In the first analysis, B _MEM cell recognition of Wuhan and Omicron BA.1 S and RBD proteins was evaluated (FIG. 18, B, C, and D). The SARS-CoV-2 Omicron variant has 37 amino acid alterations in the S protein compared to the Wuhan parental strain, which has 15 alterations in the RBD, the immunodominant target of neutralizing antibodies induced by COVID-19 vaccines or SARS-CoV-2 infection.

Staining with full-length S protein showed that the greatest proportion of B _MEM cells from Omicron-naive, two-vaccinated, and even more predominantly, three-vaccinated individuals were directed against epitopes shared by both the Wuhan and Omicron BA.1 SARS-CoV-2 variants. Consistent with the observation that vaccination with BNT162b2 can induce an immune response against wild-type epitopes that do not recognize the corresponding altered epitopes in the Omicron BA.1 S protein (FIG. 18, B and C), in most individuals, a smaller but clearly detectable proportion of B MEM cells that recognized only the Wuhan S protein or the RBD was found. Consistent with the lack of exposure, no _{B MEM cells} were detected that bound only to the Omicron BA.1 S or RBD proteins in these Omicron-naive individuals.

In Omicron-convalescent individuals, the frequency of _BMEM cells recognizing S protein epitopes shared between Wuhan and Omicron BA.1 was significantly higher than in Omicron-naive individuals (FIG. 18, B and C). In most of these subjects, only a small percentage of Wuhan S protein-specific _BMEM cells, as well as a slightly lower frequency of Omicron BA.1 variant S protein-specific cells, were found.

A similar but slightly different pattern was observed by B cell staining with tagged RBD protein (Fig. 18, B and D). Again, Omicron breakthrough infection in 2/3 vaccinated individuals was found to predominantly boost B _MEM cells reactive to conserved epitopes. A modest boost in Wuhan-specific reactivity was observed, but only a small population of Omicron-RBD-specific B _MEM cells was detected in individuals tested (Fig. 18, D).

Next, a combinatorial gating approach was used to identify subsets of S protein or RBD-binding B MEM cells that bind exclusively to Wuhan or Omicron BA.1 or that bind to common epitopes that are broadly conserved across all four variants of Wuhan, alpha, delta, and Omicron BA.1 (Figure 18, E). Across all four study groups, the frequency of B _MEM cells that recognize S protein epitopes was found to be conserved across all tested variants and accounted for the largest portion of the pool of S protein-binding B _MEM cells (Figure 18, F, all 4+ve). The S protein of the Wuhan strain does not have exclusive amino acid changes that distinguish it from the spike proteins of alpha, delta, or Omicron BA.1 VOCs. Thus, B _MEM cells that exclusively recognize the Wuhan S protein were rarely detected in any individual (Figure 18, F). In some individuals with Omicron breakthrough infection, Omicron BA.1 VOCs were found to be conserved across all tested variants and accounted for the largest portion of the pool of S protein-binding B _MEM cells (Figure 18, F). Although a small percentage of _BMEM cells was detected that bound exclusively to the OmicronBA.1 S protein (FIG. 18, F), very few individuals displayed a strictly OmicronBA.1 RBD-specific response (FIG. 18, G).

These findings indicate that SARS-CoV-2 omicron breakthrough infection in vaccinated individuals does not induce large numbers of omicron-specific B _MEM cells, but rather primarily expands the broad B _MEM cell repertoire against conserved S protein and RBD epitopes.

To further analyze this response, we characterized the B _MEM subsets directed against the RBD. A combinatorial Boolean gating approach was used to identify B _MEM cells with distinct binding patterns in the spectrum of strictly variant-specific and common epitopes shared by several variants. Multiple sequence alignments revealed that the Omicron BA.1 RBD deviates from the conserved RBD sequence region in Wuhan, alpha, and delta by 13 single amino acid modifications. All Omicron-recovered individuals were found to have a robust frequency of B _MEM cells that recognized the Wuhan, alpha, and delta VOC RBDs but not the Omicron BA.1 RBD, while B _MEM cells reacting exclusively with the Omicron BA.1 RBD were nearly absent in the majority of these individuals (Figure 18, H). No significant differences were observed between Omicron BA.1 and alpha RBDs, or Omicron BA.1 RBDs. 1 and delta RBD exclusively were not detected in _BMEM cells.

Furthermore, we identified two additional subsets of RBD-specific B _MEM cells in all individuals. One subset was characterized by binding to Wuhan, alpha, and Omicron BA.1, but not to delta, RBDs. The other population showed binding to Wuhan and alpha, but not to Omicron BA.1 or delta RBDs (Figure 18, H). Sequence alignment identified L452R as the only RBD mutation unique to delta that was not shared by the other three variant RBDs (Figure 18, I top). Similarly, we found that the only RBD site conserved in Wuhan and alpha, but altered in delta and Omicron BA.1, was T478K (Figure 18, I bottom). Both L452R and T478K alterations are known to be associated with evasion of vaccine-induced neutralizing antibody responses. Notably, B _{MEM cells were not detected in all combinatorial subgroups where multiple sequence alignment failed to identify unique epitopes in the RBD sequences that met the Boolean selection criteria (e.g., Wuhan only, or Wuhan and Omicron BA.1, but not alpha, delta). These findings indicate that the B MEM cell} response to the RBD is driven by specificity induced through prior vaccination with BNT162b2, and is not substantially redirected against new RBD epitopes mutated in the Omicron variants following infection.

SUMMARY SARS-CoV-2 omicron is a partial immune escape variant with an unprecedented number of amino acid alterations in the S protein at the site of neutralizing antibody binding, distinguishing it from previously reported variants. Recent neutralizing antibody mapping and molecular modeling studies strongly support the functional relevance of these alterations, and their importance is confirmed by the observation that twice-vaccinated individuals have no detectable neutralizing activity against SARS-CoV-2 omicron.

The findings presented herein show that omicron breakthrough infection of vaccinated individuals not only boosts neutralizing activity against omicron and _BMEM cells, but also broadly enhances immunity to a variety of VOCs and provides insight into how broad-spectrum immunity is achieved.

The data presented herein indicate that early exposure to the Wuhan strain S protein may have shaped the formation of B _MEM cells and imprinted the formation of novel B _MEM cell responses to the more distinctive epitopes of the omicron variant. Similar observations have been reported from vaccinated individuals who experienced breakthrough infection with the delta variant (K. Roltgen et al., "Immune imprinting, breeding of variant recognition, and germinal center response in human SARS-CoV-2 infection and vaccination," Cell (2022), doi:10.1016/j.cell.2022.01.018.). As demonstrated in this example, omicron breakthrough infection does not induce large numbers of strictly omicron-specific B _MEM cells, but rather primarily expands the broad B _MEM cell repertoire against conserved S protein and RBD epitopes.

Thus, omicron breakthrough infection in twice-vaccinated individuals results in an expansion of the pre-existing B _MEM cell pool, similar to a third dose booster vaccination. However, there are clear differences in the immune response patterns induced by the homologous vaccine booster compared to omicron breakthrough infection. Despite the focus of the B cell memory response on conserved epitopes, omicron breakthrough infection results in a more substantial increase in antibody neutralizing titers against omicron, as well as significant cross-neutralization of both ancestral and novel SARS CoV-2 variants. These effects are particularly pronounced in twice-vaccinated individuals.

Without wishing to be bound by theory, three findings may point to potentially complementary and synergistic mechanisms responsible for these results.
First, an overall increase in S protein-specific B _MEM cells. Omicron-recovered individuals vaccinated twice had a higher frequency of B _MEM cells and higher neutralizing antibody titers against all VOCs compared to vaccinated three times. That breakthrough infection elicits a stronger neutralizing antibody response than the third vaccine dose in twice-vaccinated individuals is not clear from previous studies describing breakthrough infections with other variants (Evans et al., Science Translational Medicine (2022) 14, eabn8057), which may be explained by poor neutralization of Omicron variants at the early stage of infection, which may cause increased or prolonged antigen exposure of the immune system to the modified S protein.

Second, a stronger bias towards RBD-specific B _MEM cell responses. Omicron breakthrough infection promotes proportionally more pronounced boosting of RBD-specific B _MEM cells than B _MEM cells that recognize S protein-specific epitopes outside the RBD. Thus, Omicron-infected individuals have a significantly higher ratio of RBD/S protein-specific B _MEM cells compared to vaccinated Omicron-uninfected individuals. The RBD is the major domain of the S protein that binds to the SARS-CoV-2 receptor ACE2 and has multiple neutralizing antibody binding sites within the region that is not affected by Omicron modifications, e.g., position L452. Increased focus of the immune response against this domain may promote B _MEM cells producing neutralizing antibodies against RBD epitopes that are not modified in Omicron.

Third, induction of broadly neutralizing antibodies. The majority of sera from omicron-convalescent individuals, but not from omicron-naive individuals, were found to robustly neutralize SARS-CoV-1. This may indicate that omicron infection in vaccinated individuals stimulates B _{MEM cells to form neutralizing antibodies against spike protein epitopes conserved in the SARS-CoV-1 and SARS-CoV-2 families. It was reported that broadly neutralizing antibodies were present in SARS-CoV-1-infected individuals vaccinated with BNT162b2. Such pan-cervecovirus} immune responses are likely driven by neutralizing antibodies against highly conserved S protein domains. The greater antigenic distance of the omicron spike protein from other SARS-Cov-2 strains may facilitate targeting of conserved subdominant neutralizing epitopes, such as those recently described to be located in the C-terminal portion of the spike protein.

Taken together, these results indicate that despite possible imprinting of an immune response by prior vaccination, preformed B cell memory pools can be refocused and quantitatively remodeled upon exposure to heterologous S protein to allow neutralization of variants that evade previously established neutralizing antibody responses.

In conclusion, although the data are based on samples from individuals exposed to Omicron S protein as a result of infection, the findings presented here support that a vaccine adapted to Omicron S protein could similarly reshape the B cell memory repertoire and therefore may be more beneficial than an extended booster series with existing Wuhan-Hu-1 spike-based vaccines.

Materials and Methods Participant Recruitment and Sample Collection Individuals from the SARS-CoV-2 omicron-naive BNT162b2 vaccinated two (BNT162b2 ² ) and three (BNT162b2 ³ ) cohorts provided informed consent as part of their participation in clinical trials (as part of the Phase 1/2 trial BNT162-01 [NCT04380701], the Phase 2 rollover trial BNT162-14 [NCT04949490], or the BNT162-17 [NCT05004181] trial).

Participants from the SARS-CoV-2 Omicron convalescent 2- and 3-dose vaccinated cohorts (BNT162b2 ² +Omi cohort and BNT162b2 ³ +Omi cohort, respectively) and individuals vaccinated with other approved COVID-19 vaccines or in combination regimens with subsequent Omicron breakthrough infection were recruited from the University Hospital of Goethe University Frankfurt to provide blood samples and clinical data for the study as part of a research program recruiting patients who experienced Omicron breakthrough infection after vaccination against COVID-19. Omicron strain infection was confirmed by variant-specific PCR or sequencing, and participants were asymptomatic at the time of blood collection.

The time points of sample collection are shown in Figure 15.

Serum was isolated by centrifugation at 2000 × g for 10 min and stored frozen until use. Li-heparin blood samples were isolated by density gradient centrifugation using Ficoll-Paque PLUS (Cytiva) and then stored frozen until use.

VSV-SARS-CoV-2 S variant pseudovirus generation. Recombinant replication-deficient vesicular stomatitis virus (VSV) vectors encoding green fluorescent protein (GFP) and luciferase instead of the VSV-glycoprotein (VSV-G) were cloned into SARS-CoV-1 spike (S) (UniProt Ref: P59594) and the Wuhan reference strain (NCBI Ref: P59595). Ref: 43740568), alpha variant (mutations: Δ69/70, Δ144, N501Y, A570D, D614G, P681H, T716I, S982A, D1118H), beta variant (mutations: L18F, D80A, D215G, Δ242-244, R246I, K417N, E484K, N501Y, D614G, A701V), delta variant (mutations: T19R, G142D, E156G, Δ157/158, K417N, L452R, T478K, D614G, P681R, D950N) Omicron BA. 1 variant (mutations: A67V, Δ69/70, T95I, G142D, Δ143-145, Δ211, L212I, ins214EPE, G339D, S371L, S373P, S375F, K417N, N440K, G446S, S477N, T478K, E484A, Q493R, G496S, Q498R, N501Y, Y505H, T547K, D614G, H655Y, N679K, P681H, N764K, D796Y, N856K, Q954H, N969K, L981F), or Omicron BA. SARS-CoV-2 derived from one of the two variants (mutations: T19I, Δ24-26, A27S, G142D, V213G, G339D, S371F, S373P, S375F, T376A, D405N, R408S, K417N, N440K, S477N, T478K, E484A, Q493R, Q498R, N501Y, Y505H, D614G, H655Y, N679K, P681H, N764K, D796Y, Q954H, N969K) were pseudotyped according to the published pseudotyping protocol (M. Berger Rentsch, G. Zimmer, A vesicular stomatitis Virus replica-based bioassay for the rapid and sensitive determination of multi-species type I interferon. PloS one. 6, e25858 (2011), doi:10.1371/journal. bone. 0025858).

Briefly, HEK293T/17 monolayers (ATCC® CRL-11268™) cultured in Dulbecco's modified Eagle's medium (DMEM) with GlutaMAX™ (Gibco) supplemented with 10% heat-inactivated bovine serum (FBS [Sigma-Aldrich]) (referred to as medium) were transfected with Sanger sequence-verified SARS-CoV-1 or variant-specific SARS-CoV-2S expression plasmids using Lipofectamine LTX (Life Technologies) according to the manufacturer's instructions. For 24 hours, VSV-G complemented VSVΔG vectors were transfected. After incubation at 37°C with 7.5% _CO2 for 2 h, cells were washed twice with phosphate-buffered saline (PBS) and then medium supplemented with anti-VSV-G antibody (clone 8G5F11, Kerafast Inc.) was added to neutralize residual VSV-G-complemented input virus. VSV-SARS-CoV-2-S pseudotype-containing medium was harvested 20 h after seeding, passed through a 0.2 μm filter (Nalgene), and stored at -80°C. Pseudovirus batches were titrated on Vero 76 cells (ATCC® CRL-1587™) grown in medium. The relative luciferase units induced by a defined amount of the Wuhan spike pseudovirus reference batch previously described in Muik et al. (Muik et al., "Neutralization of SARS-CoV-2 lineage B.1.1.7 pseudovirus by BNT162b2 vaccine-elicited human sera. Science (New York, N.Y.) 371, 1152-1153 (2021), doi:10.1126/science.abg6105"), corresponding to an infectious titer of 200 transducing units (TU) per mL, was used as a comparison. The input volume of the SARS-CoV-2 variant pseudovirus batches was calculated to normalize the infectious titers based on relative luciferase units to the reference.

Pseudovirus Neutralization Assay Vero 76 cells were seeded in 96-well white flat-bottom plates (Thermo Scientific) at 40,000 cells/well in medium 4 hours prior to the assay and incubated at 37°C _with 7.5% CO2. Each serum was serially diluted twice in medium, the first dilution being 1:5 (Omicron uninfected, vaccinated twice and three times with BNT162b2, dilution range 1:5 to 1:5,120) or 1:30 (Omicron breakthrough infection followed by BNT162b2 vaccinated twice and three times, dilution range 1:30 to 1:30,720). VSV-SARS-CoV-2-S/VSV-SARS-CoV-1-S particles were diluted in medium to obtain 200 TU in the assay. Serum dilutions were mixed 1:1 with pseudovirus (n=2 technical replicates per serum per pseudovirus) for 30 min at room temperature before being added to Vero 76 cell monolayers and incubated at 37° C. with 7.5% CO ₂ for 24 h. Supernatants were removed and cells were lysed with luciferase reagent (Promega). Luminescence was recorded on a CLARIOstar® Plus microplate reader (BMG Labtech) and neutralization titers were calculated as the interaction of the highest serum dilution that still resulted in a 50% reduction in luminescence. Results were expressed as the geometric mean titer (GMT) of duplicates. When no neutralization was observed, an arbitrary titer value of half the limit of detection [LOD] was reported.

Live SARS-CoV-2 Neutralization Assay SARS-CoV-2 virus neutralization titers were determined by a cytopathic effect (CPE)-based microneutralization assay at VisMederi S.r.l. (Siena, Italy). Briefly, heat-inactivated serum samples from participants were serially diluted 1:2 (starting at 1:10) and incubated at 37°C for 1 h with 100 TCID50 of live Wuhan-like SARS-CoV-2 virus strain 2019-nCOV/ITALY-INMI1 (GenBank: MT066156), betavirus strain human nCoV19 isolate/England ex-SA/HCM002/2021 (mutations: D80A, D215G, Δ242-244, K417N, E484K, N501Y, D614G, A701V), sequence-verified Delta strains isolated from nasopharyngeal swabs (mutations: T19R, G142D, E156G, Δ157/158, L452R, T478K, D614G, P681R, R682Q, D950N) or Omicron BA. 1 strain hCoV-19/Belgium/rega-20174/2021 (mutations: A67V, Δ69/70, T95I, G142D, Δ143-145, Δ211, L212I, ins214EPE, G339D, S371L, S373P, S375F, K417N, N440K, G446S, S477N, T47 SARS-CoV-2 S protein (SEQ ID NO: 1) was incubated with SARS-CoV-2 S (SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4) and the 2019-nCOV/ITALY-INMI1 strain S protein (SEQ ID NO: 1) was incubated with SARS-CoV-2 S protein (S ...1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4) and the 2019-nCOV/ITALY-INMI1 strain S protein (SEQ ID NO: 1) was incubated with SARS-CoV-2 S protein (SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4) and the 2019-nCOV/ITALY-INMI1 strain S protein (SEQ ID NO: 1) was incubated with SARS-CoV-2 S protein (SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4) and the 2019-nCOV/ITALY-IN Vero E6 (ATCC® CRL-1586™) cell monolayers were inoculated with serum/virus mixtures in 96-well plates and incubated for 3 days (2019-nCOV/ITALY-INMI1 strain) or 4 days (beta, delta, and omicron BA.1 variant strains) to allow infection with non-neutralizing viruses. Plates were observed under an inverted light microscope and wells were scored as positive for SARS-CoV-2 infection (i.e., exhibited CPE) or negative for SARS-CoV-2 infection (i.e., cells were viable without CPE). Neutralization titers were determined as the interaction of the highest serum dilution that protected >50% of cells from CPE and reported as the GMT of duplicates. If no neutralization was observed, an arbitrary titer value of 5 (half the limit of detection [LOD]) was reported.

Detection and characterization of SARS-CoV-2 specific B cells using flow cytometry. Spike/RBD specific B cells were detected using recombinant biotinylated SARS-CoV-2 spike (Acro Biosystems: Wuhan-SPN-C82E9, alpha-SPN-C82E5, delta-SPN-C82Ec, Omicron-SPN-C82Ee) and RBD (Acro Biosystems: Wuhan-SPD-B28E9, alpha-SPD-C82E6, delta-SPD-C82Ed, Omicron-SPD-C82E4) proteins. Recombinant spike and RBD proteins were tetramerized with fluorescently labeled streptavidin (BioLegend, BD Biosciences) at a 4:1 molar ratio for 1 h in the dark at 4° C. Samples were then spun down for 10 min at 4° C. to remove any final precipitate.

For flow cytometry analysis, PBMCs were thawed and ^5x106 cells were seeded per sample in 96 U-bottom plates. Cells were blocked for Fc receptor binding (Human BD Fc Block™, BD Biosciences) and assayed with free biotin (D-biotin, Invitrogen, 1 μM) in flow buffer (DPBS (Gibco) supplemented with 2% FBS (Sigma), 2 mM EDTA (Sigma-Aldrich)) for 20 min at 4°C. Cells were washed and labeled in the dark with BCR bait tetramers (2 μg/ml for Spike and 0.25 μg/ml for RBD protein) supplemented with free biotin in flow buffer (D-biotin, Invitrogen, 2 μg/ml) for 1 h at 4°C. Cells were washed with flow buffer and stained for viability (fixed viability dye eFluor™ 780, eBioscience) and surface markers (CD3-clone: UCHT1 (BD Biosciences), CD4-clone: SK3 (BD Biosciences), CD185 (CXCR5)-clone: RF8B2 (BioLegend), CD279 (PD-1)-clone: EH12.1 (BD Biosciences), CD278 (ICOS)-clone: C398.4A (BioLegend), CD19-clone: SJ25C1 (BD Biosciences), CD20-clone: 2H7 (BD Biosciences), CD21-clone: B-ly4 (BD Biosciences)). Biosciences), CD27-clone: L128 (BD Biosciences), CD38-clone: HIT2 (BD Biosciences), CD11c-clone: S-HCL-3 (BD Biosciences), CD138-clone: MI15 (BD Biosciences), IgG-clone: G18-145 (BD Biosciences), IgM-clone: G20-127 (BD Biosciences), IgD-clone: IA6-2 (BD Biosciences), CD14-clone: MφP9 (BD Biosciences, dump channel), CD16-clone: 3G8 (BD Samples were stained for 20 min at 4 °C in flow buffer supplemented with Brilliant Stain Buffer Plus (BD Biosciences, dump channel) for 1 h at 4 °C. Samples were washed and fixed with BDTM stabilizing fixative (BD Biosciences, according to manufacturer's instructions) before data acquisition on a BD Symphony A3 flow cytometer. FCS 3.0 files were exported from BD Diva software and analyzed using FlowJo software (version 10.7.1).

Debris and doublets were identified via FSC/SSC. Dead cells and monocytes (CD14, CD16- viability/dump channel) were then excluded. CD19 positive B cells were analyzed for IgD and CD27 expression, thereby identifying naïve B cells as IgD ⁺ cells with a Boolean "make no gate" function. Within non-naïve B cells, plasmablasts (CD38 ^high CD20 ^low ) and memory B cells (B _MEM CD38 ^medium/low CD20 ^high ) were differentiated. _{B MEM} cells were analyzed for B cell bait binding. SARS-CoV-2 spike reactivity was assessed by gating on each spike/RBD variant tested by plotting against the CD20 signal. Bait gates were overlaid on total B _MEM cells and displayed as NxN-Plots for the four bait channels.

Statistical Analysis The statistical method of aggregation used for analysis of antibody titers was the geometric mean, and for the ratio of SARS-CoV-2 VOC titers and Wuhan titers, the geometric mean and corresponding 95% confidence interval. The use of the geometric mean accounts for the non-normal distribution of antibody titers, which spans several orders of magnitude. A paired signed rank test of the geometric mean neutralizing antibody titers of groups with a common control group was performed using the Friedman test with Dunn's correction for multiple comparisons. Flow cytometry frequencies were analyzed and tables were exported from FlowJo software (version 10.7.1.). Statistical analysis of cumulative memory B cell frequencies was the mean and standard error of the mean (SEM). All statistical analyses were performed using GraphPad Prism software version 9.

Example 8: Induced Antibody Responses of Vaccines Encoding SARS-CoV-2 S Proteins from the Omicron Variant To test the efficacy of an RNA vaccine encoding a SARS-CoV-2 S protein that includes one or more mutations characteristic of the omicron variant, subjects who had previously received a primary regimen comprising two doses of 30 ug of RNA encoding a SARS-CoV-2 S protein from the Wuhan strain (e.g., BNT162b2) and a booster regimen comprising a dose of 30 ug of RNA encoding a SARS-CoV-2 S protein from the Wuhan strain (i.e., a Wuhan-specific booster, e.g., BNT162b2) were administered with either (i) 30 ug of RNA encoding a SARS-CoV-2 S protein from the Wuhan strain (e.g., BNT162b2), or (ii) 30 ug of a SARS-CoV-2 S protein encoding a SARS-CoV-2 S protein that includes one or more mutations characteristic of the omicron variant. An additional booster dose was administered (the dose administered as part of the second booster regimen is referred to as the "fourth dose" in the figure) comprising RNA encoding the S protein (i.e., an omicron-specific booster, e.g., RNA encoding a SARS-CoV-2 S protein comprising the amino acid sequence of SEQ ID NO:49 and/or the nucleotide sequence of SEQ ID NO:50 and/or 51). Serum was collected from the subjects at the time of administration of the second booster regimen and one month thereafter.

Neutralizing antibody titers were determined using a fluorescent focus reduction neutralization test ("FFRNT"). Suitable FFRNT assays are known in the art and include, for example, the assay described in Zou J, Xia H, Xie X, et al. "Neutralization against Omicron SARS-CoV-2 from previous non-Omicron infection," Nat Commun 2022;13:852, the entire contents of which are incorporated herein by reference. Additional exemplary neutralization assays include those described in the preceding Examples and in Bewley, Kevin R., et al. “Quantification of SARS-CoV-2 neutralizing antibody by wild-type plaque reduction neutralization, microneutralization and pseudotyped virus neutralization assays.” Nature Protocols 16.6 (2021): 3114-3140. As shown in FIG. 20, A, subjects who received a second booster regimen containing a dose of RNA encoding a SARS-CoV-2 S protein containing a mutation characteristic of the omicron variant were able to reduce or neutralize a dose of the Wuhan strain of SARS-CoV-2. Compared to subjects who received a second booster regimen containing RNA encoding the S protein, subjects who received the omicron-specific booster showed a significant increase in the concentration of neutralizing antibodies against the omicron variant. Specifically, subjects who received the omicron-specific booster showed a 1.79-fold higher GMR and a 2.31-fold higher GMFR than those observed in subjects who received a fourth dose of RNA encoding the SARS-CoV-2 S protein from the Wuhan strain. The superior immune response induced by the omicron-specific booster against the omicron variant was further increased in subjects who were previously infected with SARS-CoV-2 (as determined by antigen testing) or currently infected with SARS-CoV-2 (as determined by PCR). See FIG. 20, B, which shows that a subject population including previously and/or currently infected subjects exhibited a 2.94-fold higher GMR and a 1.97-fold higher GMFR ratio than that observed in a subject population including previously and/or currently infected subjects administered an RNA vaccine encoding the SARS-CoV-2 S protein from the Wuhan strain.

Using the same serum samples described above, pseudovirus neutralization assays were also performed using pseudoviruses containing the SARS-CoV-2 S protein from the Wuhan strain. Subjects administered RNA encoding the SARS-CoV-2 S protein from the Wuhan strain (e.g., BNT162b2) exhibited similar titers of neutralizing antibodies as those observed in subjects administered the Omicron-specific booster, demonstrating that the two vaccines are at least as effective in inducing an antibody response against the Wuhan strain. See FIG. 20C, which shows that the GMR and GMFR observed in subjects administered the Wuhan-specific booster (e.g., BNT162b2) are similar to those observed in subjects administered the Omicron-specific booster (OMI). In subjects previously infected with SARS-CoV-2 (e.g., as determined by antigen assay) or currently infected with SARS-CoV-2 (e.g., as determined by PCR assay), subjects who received an omicron-specific booster showed improved immune responses compared to subjects who received a Wuhan-specific booster. See FIG. 20D, which shows that the GMR for subjects who received an omicron-specific booster was about 1.4-fold higher than subjects who received a Wuhan-specific booster.

Subjects who received the omicron-specific booster also demonstrated superior immune responses to the delta variant in pseudovirus neutralization assays. See Figure 20E, which shows that the GMFR for subjects who received the omicron-specific booster was approximately 1.20-fold higher than that observed in subjects who received the Wuhan-specific booster. The superior immune response induced by the omicron-specific booster to the delta variant was further increased in sera from subjects who were previously and/or currently infected with SARS-CoV-2. See Figure 20F.

Example 9: Immunogenicity study of vaccines encoding S proteins of SARS-CoV-2 variants in unvaccinated subjects To test the immunogenicity of various variant-specific vaccines in unvaccinated subjects, unvaccinated mice were vaccinated with (a) saline (negative control), (b) an RNA vaccine encoding a SARS-CoV-2 S protein from the Wuhan strain, (c) an RNA vaccine encoding a SARS-CoV-2 S protein with mutations characteristic of the omicron variant (Omi), (d) an RNA vaccine encoding a SARS-CoV-2 S protein with mutations characteristic of the delta variant (Delta), (e) a bivalent vaccine comprising RNA encoding a SARS-CoV-2 S protein from the Wuhan strain and a SARS-CoV-2 S protein containing mutations characteristic of the omicron variant (b2+Omi), and (f) a SARS-CoV-2 S protein with mutations characteristic of the delta variant. Patients were immunized twice with a bivalent vaccine (Delta + Omi) containing RNA encoding the S protein and RNA encoding the SARS-CoV-2 S protein with a mutation characteristic of the Omicron variant. The immunogenicity of the RNA vaccine was investigated by focusing on antibody immune responses.

Serum was obtained 7 days after immunization and analyzed using a pseudovirus neutralization assay (e.g., the assay described in Example 2) using a pseudovirus containing a SARS-CoV-2 S protein from the Wuhan strain, a SARS-CoV-2 S protein containing mutations characteristic of a beta variant, a SARS-CoV-2 S protein containing mutations characteristic of a delta variant, or a SARS-CoV-2 S protein containing mutations characteristic of an omicron variant. As shown in Figure 21, the bivalent vaccine was found to elicit the broadest immune response in unvaccinated mice.

Example 10: Vaccine encoding a SARS-CoV-2 S protein containing one or more mutations characteristic of a beta variant induces antibody responses in subjects previously administered an RNA vaccine encoding a SARS-CoV-2 S protein from the Wuhan strain. To test the efficacy of an RNA vaccine encoding a SARS-CoV-2 S protein containing one or more mutations characteristic of a beta variant, subjects previously administered a primary regimen containing two doses (30ug each) of an RNA encoding a SARS-CoV-2 S protein from the Wuhan strain (in this example, BNT162b2 (SEQ ID NO:20)) were administered two booster doses, each containing 30ug of an RNA encoding a SARS-CoV-2 S protein containing one or more mutations characteristic of a beta variant (hereafter referred to as a beta-specific vaccine). In this example, construct RBP020.11 was administered as the beta-specific vaccine. In this example, the two booster doses were administered approximately one month apart, however, in some embodiments, the two booster doses can be administered at least 3 weeks apart, at least 4 weeks apart, at least 5 weeks apart, at least 6 weeks apart, at least 7 weeks apart, at least 8 weeks apart, or longer apart (e.g., according to the exemplary dosing regimens described herein).

Serum was collected from subjects prior to administration of BNT162b2, one month after administration of the two primary doses of BNT162b2, one month after administration of the first dose of the beta-specific vaccine, and one month after administration of the second dose of the beta-specific vaccine. Neutralizing antibody titers against pseudoviruses containing the Wuhan strain of SARS-CoV-2 S protein or SARS-CoV-2 S protein containing one or more mutations characteristic of a beta variant were measured using a pseudovirus neutralization assay (results shown in FIG. 22). Subjects showed increased neutralizing antibody titers against both the Wuhan strain and beta variant of SARS-CoV-2 after administration of the third and fourth doses of the beta-specific vaccine.

Example 11: Vaccine encoding a SARS-CoV-2 S protein containing one or more mutations characteristic of a beta variant induces antibody responses in unvaccinated subjects. To test the efficacy of an RNA vaccine encoding a SARS-CoV-2 S protein containing one or more mutations characteristic of a beta variant in unvaccinated subjects, subjects who had not previously received a SARS-CoV-2 vaccine and who showed no evidence of previous or current infection with SARS-CoV-2 (e.g., as assessed by antibody and/or PCR testing) were administered two doses (30 ug each) of RNA encoding a SARS-CoV-2 S protein containing one or more mutations characteristic of a beta variant (in this example, RBP020.11). Serum was collected one month after administration of the second dose, and neutralizing antibody titers were measured using a virus neutralization assay using virus particles containing either the SARS-CoV-2 S protein from the Wuhan strain or a SARS-CoV-2 S protein with one or more mutations characteristic of a beta variant. Tables 22 and 23 below show the results of the neutralization assay against the beta variant (neutralization assay results against the Wuhan strain are not shown). As shown in the tables, compared to unvaccinated subjects who received two doses of an RNA vaccine encoding a SARS-CoV-2 S protein from the Wuhan strain (in this example, BNT162b2), the RNA vaccine encoding a SARS-CoV-2 S protein with mutations characteristic of a beta variant was found to induce a significantly stronger antibody response against the beta variant.

Example 12: Induced Antibody Responses and Reactogenicity of BNT162b2 or Omicron-Specific Vaccine as Monovalent, Bivalent, and High Dose in Participants 55 Years of Age and Older To test the efficacy and safety of (i) higher doses of an RNA vaccine (e.g., as described herein), (ii) an RNA vaccine encoding a SARS-CoV-2 S protein with one or more mutations characteristic of the omicron variant (omicron-specific vaccine), and (iii) a bivalent vaccine comprising RNA encoding a SARS-CoV-2 S protein from the Wuhan variant and RNA encoding a SARS-CoV-2 S protein with one or more mutations characteristic of the omicron variant, subjects who had previously received at least one dose of an RNA vaccine encoding a SARS-CoV-2 S protein of the Wuhan strain were administered one of several booster doses (e.g., as described herein). Specifically, subjects who had previously received two doses of 30 ug of an RNA vaccine encoding the SARS-CoV-2 S protein from the Wuhan strain (in this example, BNT162b2), a third dose of 30 ug of an RNA encoding the SARS-CoV-2 S protein of the Wuhan strain (also in this example, BNT162b2),
(a) 30 ug of RNA encoding the SARS-CoV-2 S protein from the Wuhan strain;
(b) 60 ug of RNA encoding the SARS-CoV-2 S protein from the Wuhan strain;
(c) 30 ug of omicron-specific vaccine;
(d) 60 ug of omicron-specific vaccine;
e) 30 ug of a bivalent RNA vaccine comprising 15 ug of RNA encoding the SARS-CoV-2 S protein from the Wuhan strain and 15 ug of RNA encoding a SARS-CoV-2 S protein containing a mutation characteristic of the Omicron variant (Omicron-adapted bivalent vaccine); or (f) 60 ug of a bivalent RNA vaccine comprising 30 ug of RNA encoding the SARS-CoV-2 S protein from the Wuhan strain and 30 ug of RNA encoding a SARS-CoV-2 S protein containing a mutation characteristic of the Omicron variant (Omicron-adapted bivalent vaccine).

In this example, for the fourth dose, the RNA encoding the SARS-CoV-2 S protein from the Wuhan variant is BNT162b2, and the RNA encoding the SARS-CoV-2 S protein having a mutation characteristic of the Omicron variant comprises the nucleotide sequence of SEQ ID NO:51.

Serum samples were collected at the time of administration of the fourth dose and 7 days thereafter and were tested for neutralizing antibody titers against virus particles containing the SARS-CoV-2 S protein from the Wuhan strain or SARS-CoV-2 S protein containing mutations characteristic of the delta or omicron variants.

Neutralizing antibody titers were determined using a fluorescent focus reduction neutralization test ("FFRNT"). Suitable FFRNT assays are known in the art, as discussed in Example 8. Neutralizing responses are shown in Figure 23.

As shown in FIG. 23(A), subjects receiving a fourth dose of 30 ug of the omicron-specific vaccine showed increased neutralizing antibodies against the omicron variant compared to subjects receiving a fourth dose of 30 ug of BNT162b2. Administering 60 ug of RNA increased the neutralizing response for both BNT162b2 and the omicron-specific vaccine, with the 60 ug omicron-specific vaccine showing a stronger immune response against the omicron variant. As shown in FIG. 23(B), a similar effect was observed in a population that included subjects previously or currently infected with SARS-CoV-2 (e.g., as determined by antibody and PCR testing, respectively).

Figures 23 (C-D) provide data on neutralizing responses to the Wuhan strain of SARS-CoV-2 in a population of subjects excluding those previously or currently infected with SARS-CoV-2 (Figure 23(C)) and a population of subjects that includes these subjects (Figure 23(D)).

Figures 23 (E-F) provide data on neutralizing responses to delta variants in a subject population excluding subjects previously or currently infected with SARS-CoV-2 (Figure 23(E)) and a subject population that includes these subjects (Figure 23(F)).

Figure 23(G) shows the neutralization response compared to subjects administered a fourth dose of 30ug BNT162b2. As can be seen in the table, the Omicron-specific vaccine induced a strong response against the Omicron variant and a response that was at least equivalent to the response of BNT162b2 for the other variants. The bivalent vaccine (Omicron-adapted bivalent vaccine) generated a strong immune response at both the 30ug and 60ug doses against each SARS-CoV-2 variant tested.

The reactivity of the fourth dose tested was also monitored in patients for 7 days after administration of the fourth dose. Figure 24(A) shows the local immune responses observed in the different groups of subjects as indicated. As can be seen in the figure, the 60ug dose of omicron-specific vaccine and bivalent vaccine were found to be more likely to cause pain at the injection site compared to that observed with the other booster doses tested, although pain was rated as mild or moderate for both doses. Redness and swelling responses were low and comparable for each dose tested.

Figure 24(B) shows the systemic immune response observed in subjects from different groups, as indicated. Systemic responses (characterized by fever, fatigue, headache, chills, vomiting, diarrhea, muscle pain, or joint pain) were similar at each dose, but fatigue tended to be higher at the 60ug dose.

The immune responses and reactogenicity of Omicron-adapted vaccines (monovalent and bivalent vaccines as described in this example) as booster doses are also confirmed in a Phase 2/3 trial involving over 1,000 participants aged 56 years and older. Omicron-adapted vaccines (monovalent or bivalent, and 30ug or 60ug) given as booster doses induced substantially higher neutralizing antibody responses against OmicronBA.1 compared to those induced by BNT162b2 (encoding the SARS-CoV-2 S protein from the Wuhan strain). The predefined criterion for superiority was measured by a neutralization geometric mean titer ratio (GMR) of the lower limit of the 95% confidence interval >1. The geometric mean ratios (GMRs) of the monovalent omicron-specific vaccines (30 μg and 60 μg) compared to those induced by BNT162b2 (encoding the SARS-CoV-2 S protein from the Wuhan strain) were 2.23 (95% CI: 1.65, 3.00) and 3.15 (95% CI: 2.38, 4.16). The GMRs of the omicron-adapted bivalent 30 μg and 60 μg vaccines (as described in this example) compared to those induced by BNT162b2 (encoding the SARS-CoV-2 S protein from the Wuhan strain) were 1.56 (95% CI: 1.17, 2.08) and 1.97 (95% CI: 1.45, 2.68), respectively. Monovalent Omicron-specific vaccines at 30 μg and 60 μg achieved a lower 95% confidence interval of GMR >1.5, demonstrating superiority over Omicron and meeting the regulatory requirement for superiority.

One month after administration, booster doses of Omicron BA.1-adapted monovalent vaccine (30 μg and 60 μg) increased neutralization geometric mean titers (GMTs) against Omicron BA.1 by 13.5 and 19.6-fold over pre-booster dose levels, while booster doses of Omicron-adapted bivalent vaccine (30 μg and 60 μg) conferred 9.1 and 10.9-fold increases in neutralization GMTs against Omicron BA.1. Both Omicron-adapted vaccines (e.g., monovalent and bivalent vaccines) were well tolerated in participants who received one or the other Omicron-adapted vaccine and showed a similar good safety and tolerability profile as BNT162b2 (encoding the SARS-CoV-2 S protein from the Wuhan strain).

Additionally, in a SARS-CoV-2 live virus neutralization assay tested on sera from participants aged 56 years and older who received an Omicron-adapted vaccine (e.g., a monovalent or bivalent vaccine as described in this Example), the sera also neutralized Omicron BA.4/BA.5 at lower titers than Omicron BA.1.

Example 13: Omicron breakthrough infection drives cross-variant neutralizing and memory B cell formation, but to a lesser extent against Omicron BA.4 and BA.5.
New Omicron sublineages carrying further modifications of the SARS-CoV-2 S protein continue to arise and have been designated BA.4 and BA.5 by the European Centre for Disease Prevention and Control (ECDC) on May 12, 2022. 5 were considered VOCs (European Centre for Disease Prevention and Control, Epidemiological update: SARS-CoV-2 Omicron sub-lines BA.4 and BA.5 (2022) (available at https://www.ecdc.europa.eu/en/news-events/epidemiological-update-sarscov-2-omicron-sub-lines-ba4-and-ba5)).

This Example 13 is an extension of Example 7, in which serum samples collected from BA.1 breakthrough cases as described in Example 7 were further analyzed for their neutralizing activity against omicron BA.4 and BA.5 variants.

As described in Example 7, in Omicron-naive, twice-vaccinated individuals, the 50% pseudoviral neutralization ( _pVN50 ) geometric mean titers (GMT) of beta and delta VOCs were found to be reduced compared to the Wuhan strain, while neutralization of Omicron sublines BA.1 and BA.2 was virtually undetectable. In this example, Figure 25(a) shows that neutralization titers of BA.4/5 were also virtually undetectable in twice-vaccinated BA.1 breakthrough patients.

As described in Example 7, Omicron-naive, 3-vaccinated individuals showed substantially higher _pVN50 GMTs against all tested VOCs compared to 2-vaccinated individuals. Robust neutralization of alpha, beta, and delta variants was observed, while neutralization of Omicron BA.1 and BA.2 was reduced compared to Wuhan (GMT 160 and 211 vs. 398). As shown in FIG. 25(A) of this example, neutralization of Omicron BA.4/5 was further reduced in Omicron-naive, 3-vaccinated patients (GMT 74), corresponding to a 5-fold lower titer compared to the Wuhan strain.

As shown in Figure 25(b), Omicron BA.1 breakthrough infection was found to have only a slight boosting effect on neutralization of BA.4/5. In patients vaccinated twice, pVN50 GMTs against Omicron BA.4/5 were significantly lower than those against Wuhan (GMT 135 vs. 740). A similar pattern was observed with BA.1 convalescent and control sera from individuals vaccinated three times. As described in Example 7, BA.1 convalescent sera showed high _pVN50 GMTs against previous SARS-CoV-2 VOCs, including beta (1182), Omicron BA.1 (1029), and Omicron BA.2 (836), which were close to the titers against the Wuhan reference (1182). In contrast, as shown in Figure 25(b), BA.1 convalescent sera showed high _pVN50 GMTs against previous SARS-CoV-2 VOCs, including beta (1182), Omicron BA.1 (1029), and Omicron BA.2 (836), which were close to the titers against the Wuhan reference (1182). Neutralization of BA.4/5 in 3-vaccinated individuals with 1 breakthrough infection was significantly reduced compared to the Wuhan strain, with a _pVN50 GMT of 197, a 6-fold reduction versus the Wuhan strain.

Notably, in all cohorts, neutralizing titers against BA.4/5 were closer to the low levels observed against the phylogenetically more distant SARS-CoV-1 pseudovirus than those seen against Wuhan. Comparing the ratios of SARS-CoV-2 VOC and SARS-CoV-1 _pVN50 GMT normalized to Wuhan (Figure 25(c)), it is striking that breakthrough infection with Omicron BA.1 does not result in more efficient cross-neutralization of Omicron BA.4/5 in two- and three-vaccinated individuals. Taken together, these data demonstrate that Omicron BA.1 breakthrough infection in vaccine-experienced individuals mediates broadly neutralizing activity against BA.1, BA.2, and several previous SARS-CoV-2 variants, but not BA.4/5.

As shown in FIG. 26, similar results were found for patients who had previously received a non-BNT162b2 vaccine and had a BA.1 breakthrough infection.

As described in Example 7, Omicron BA.1 breakthrough infection in BNT162b2 vaccinated individuals was found to produce potent neutralizing activity against Omicron BA.1, BA.2 and previous SARS-CoV-2 VOCs, primarily by expanding B _MEM cells to epitopes that are broadly shared across different SARS-CoV-2 strains. These data demonstrate that the vaccination imprinted B _MEM cell pool is plastic enough to be remodeled upon exposure to heterologous SARS-CoV-2 S proteins. Selective expansion of B _MEM cells that recognize shared epitopes allows for effective neutralization of most variants that evade previously established immunity, although susceptibility to escape by variants that acquire modifications at previously conserved sites may be enhanced. ... harboring the additional modifications L452R and F486V in the RBD. The marked reduction in neutralizing activity against 4/5 pseudoviruses supports a mechanism of immune evasion due to loss of the few remaining conserved epitopes.

Discussion Surprisingly, and contrary to the results observed in Example 7, neutralization of Omicron sublineages BA.4 and BA.5 was not enhanced in BA.1 breakthrough patients, but instead had titers comparable to that against the more phylogenetically distant SARS-CoV-1. Although this example focused on individuals vaccinated with the BNT162b2 mRNA vaccine, a similar observation has been recently reported in individuals vaccinated with CoronaVac (a whole inactivated virus vaccine developed by Sinovac Biotech), where Omicron BA.4/5 neutralized BA. It has been suggested that this may circumvent the humoral immune boost that mediates infection (Y. Cao et al., BA.2.12.1, BA.4 and BA.5 escape antibodies elicited by Omicron infection, bioRxiv: the preprint server for biology (2022)).

This disclosure provides insight into how immunity to multiple variants in BA.1 breakthrough cases is achieved, why Omicron BA.4 and BA.5 sublineages can partially evade neutralization, and provides vaccination protocols and techniques to enhance protection across coronavirus strains and lineages, particularly including Omicron lineages (e.g., including BA.4 and/or BA.5). Without wishing to be bound by any particular theory, this disclosure proposes that early exposure to the Wuhan strain S protein can shape the formation of B _MEM cells and imprint on novel B _MEM cell responses that recognize epitopes characteristic of Omicron BA.1 variants.

Omicron BA.1 breakthrough infection in BNT162b2-vaccinated individuals primarily expands a broad B _{MEM cell} repertoire against conserved SARS-CoV-2 S protein and RBD epitopes rather than inducing strictly Omicron BA.1-specific B _MEM cells. Compared to immune responses induced by homologous vaccine boosters, Omicron BA.1 breakthrough infection results in a more substantial increase in antibody neutralizing titers against Omicron and robust cross-neutralization of many SARS CoV-2 variants.

As discussed in Example 7, one potential explanation for the broad neutralization induced by BA.1 breakthrough infection is the induction of broadly neutralizing antibodies. Sera from Omicron BA.1 convalescent vaccinated individuals were found to neutralize SARS-CoV-2 Omicron BA.4/5 and SARS-CoV-1 to a much lower extent than previous SARS-CoV-2 VOCs, including BA.1 and BA.2. This finding indicates that Omicron BA.1 infection in vaccinated individuals stimulates B MEM cells to produce neutralizing antibodies against S protein epitopes conserved in SARS-CoV- ₂ variants up to Omicron BA.2, but mostly lost in BA.4/5, and mostly not shared by SARS-CoV-1.

Omicron BA from a previous SARS-CoV-2 strain. 1 The greater antigenic distance of the S protein may be due, for example, to a cryptic site in a part of the RBD that is distinct from the receptor binding motif (Li, Tingting, et al. "Cross-neutralizing antibodies bind a SARS-CoV-2 cryptic site and resist circulating variants," Nature communications 12.1 (2021): 1-12, and Yuan, Meng, et al. "A highly conserved cryptic epitope in the receptor binding domains of SARS-CoV-2 and SARS-CoV" Science 368.6491 (2020): 630-633) or the membrane-proximal S glycoprotein subunit designated S2 (Pinto, Dora, et al. "Broad betacoronavirus neutralization by a stem helix-specific human antibody." Science 373.6559 (2021): 1109-1116 Li, Wenwei, et al. "Structural basis and mode of action for two broadly neutralizing antibodies against SARS-CoV-2 emerging variants of concern. "Cell reports 38.2 (2022): 110210, Hurlburt, Nicholas K., et al. "Structural definition of a pan-sarbecovirus neutralizing epitope on the spike S2 subunit. "Communications biology 5.1 (2022): 1-13), which may facilitate targeting of conserved subdominant neutralizing epitopes.

As noted in Example 7, individuals infected with Omicron BA.1 appear to have a significantly higher RBD/S protein-specific B _MEM cell ratio compared to vaccinated Omicron-uninfected individuals. Omicron BA.1 carries multiple S protein modifications in the major neutralizing antibody binding sites of the NTD (such as del69/70 and del143-145), dramatically reducing the targeting surface for memory B cell responses in this region. Although the Omicron BA.1 RBD has multiple modifications, several neutralizing antibody binding sites are unaffected (20). Expansion of B MEM _cells producing neutralizing antibodies against RBD epitopes that are not modified in Omicron BA.1, such as the one at position L452 shown in this example, can help rapidly restore neutralization of BA.1 and BA.2 variants. Importantly, the potent neutralization of Omicron BA.1 and BA.2 is consistent with the Omicron BA.1 RBD. This should not conceal the fact that neutralizing _BMEM immune responses in convalescent vaccinated individuals are driven by a small number of epitopes. The markedly reduced neutralizing activity against Omicron BA.4/5 pseudoviruses carrying the additional modifications L452R and F486V in the RBD demonstrates a mechanism of immune evasion due to loss of the few remaining conserved epitopes. On the other hand, further sublineages carrying the L452 modification (e.g., BA.2.12.1) have been reported to evade humoral immunity induced by BA.1 breakthrough infection (Y. Cao et al., cited above).

The present disclosure proposes that immunity in the early stages of Omicron BA.1 infection in vaccinated individuals can be based on the recognition of conserved epitopes and can be narrowly focused on a small number of neutralizing sites that are not modified in Omicron BA.1 and BA.2. Such a narrow immune response carries a high risk that these small number of epitopes may be lost by acquiring further modifications during the ongoing evolution of Omicron, which may result in immune escape, as experienced by sub-lineages BA.2.12.1, BA.4, and BA.5 (Y. Cao et al., cited above, and K. Khan et al., Omicron sub-lines BA.4/BA.5 escape BA.1 infection elicited neutralizing immunity (2022)). Importantly, Omicron BA.1 infection is not immune to the small number of neutralizing sites that are conserved in vaccinated individuals. Omicron BA.1 breakthrough infection does not appear to reduce the entire spectrum of (Wuhan) S glycoprotein-specific memory B cells, as memory B cells that do not recognize .1 S remain detectable in the blood at similar frequencies. Wuhan-specific (non-Omicron BA.1 reactive) B _MEM cells were consistently detected in Omicron BA.1 breakthrough infected individuals at similar levels as Omicron uninfected 2/3 vaccinated individuals. Without wishing to be bound by any particular theory, the present disclosure notes that these findings may reflect an increase in the total B _MEM cell repertoire due to selective expansion of B _MEM cells that recognize a shared epitope.

This example provides, among other things, insight that a subject infected with the Wuhan strain or administered at least one dose (e.g., at least two, including at least three doses) of a vaccine(s) adapted for the Wuhan strain (e.g., but not limited to, a protein-based vaccine or an RNA-based vaccine such as BNT162b2, Moderna mRNA-1273, etc.) may be more beneficial to receive at least one dose of a vaccine (e.g., a protein- or RNA-based vaccine) adapted for a strain other than Omicron BA.1. In some embodiments, the vaccine adapted for a strain other than Omicron BA.1 may be or may include a vaccine adapted for Omicron BA.4 and/or Omicron BA.5. This example provides, among other things, insight that a subject with no prior SARS-CoV-2 infection may be more beneficial to receive at least one dose of a vaccine adapted for the Wuhan strain (e.g., in some embodiments, an RNA vaccine such as BNT162b2) and at least one dose of a vaccine adapted for the Omicron BA. The present invention also provides insight that it may be desirable to administer a combination of vaccines, including a vaccine adapted to a strain other than the one described herein. In some embodiments, such combinations of vaccines may be administered as primary and/or booster doses administered separately at different times, e.g., in some embodiments, by a predetermined time period (e.g., according to a particular dosing regimen described herein). In some embodiments, such combinations of vaccines may be administered as a single multivalent vaccine.

Materials and Methods Serum samples, neutralization assays, and all other experiments described in this example were performed as in Example 7. A recombinant replication-defective vesicular stomatitis virus (VSV) vector encoding green fluorescent protein (GFP) and luciferase instead of the VSV-glycoprotein (VSV-G) was used to generate the SARS-CoV-2 S variant pseudovirus with the following mutations for the Wuhan strain: Pseudotyped with the S protein: T19I, Δ24-26, A27S, Δ69/70, G142D, V213G, G339D, S371F, S373P, S375F, T376A, D405N, R408S, K417N, N440K, L452R, S477N, T478K, E484A, F486V, Q498R, N501Y, Y505H, D614G, H655Y, N679K, P681H, N764K, D796Y, Q954H, N969K).

Example 14. Omicron BA.2 breakthrough infection in vaccinated individuals induces broad cross-neutralization against Omicron BA.1, BA.2, and other VOCs, including BA.4 and BA.5.
This example shows that BA.2 omicron breakthrough infection in individuals vaccinated three times with BNT162b2 surprisingly drives superior cross-variant neutralization, including improved production of neutralizing antibodies against BA.4/5 omicron variants, compared to BA.1 breakthrough infection in individuals vaccinated three times with BNT162b2. Thus, among other things, the present disclosure demonstrates the feasibility of defining immune synergistic categories of coronavirus strains and/or sequences (e.g., spike protein sequences).

In some embodiments of the improved coronavirus vaccination strategies provided by the present disclosure, a subject is exposed to each of at least two different such synergistic categories. In some embodiments, the subject is infected, has been infected, or is infected with a virus of a first category and receives at least one dose of a vaccine of a second category characterized by immunological synergy with the first category. Alternatively or additionally, in some embodiments, the subject receives or has received doses of a first and a second vaccine of such first and second categories. In some embodiments, the vaccines of the different categories may be administered separately (e.g., at different times and/or to different sites on the subject). In some embodiments, the vaccines of the different categories may be administered together (e.g., substantially simultaneously and/or at about or exactly the same site and/or in a single composition).

Compared to Omicron BA.1 breakthrough infection in vaccinated individuals, which induces lower neutralization to Omicron BA.4/BA.5 compared to neutralization to other SARS-CoV-2 variants (including, for example, the Wuhan-Hu-1 strain, alpha variants, beta variants, delta variants, Omicron BA.1, Omicron BA.2, and Omicron BA.2.11.2), this example shows that BA.2 Omicron breakthrough infection in individuals vaccinated with BNT162b2 surprisingly drives superior cross-variant neutralization, including improved production of neutralizing antibodies to BA.4/5 Omicron variants. Thus, in some embodiments, the present disclosure demonstrates that, among other things, SARS-CoV-2 strains and/or variants may be grouped into at least two distinct categories such that a subject exposed to a SARS-CoV-2 strain and/or variant from each of such two distinct categories may benefit from the immune synergistic protection conferred by such two distinct categories. In some embodiments, a first category of SARS-CoV-2 strains/variants includes the Wuhan-Hu-1 strain, alpha variants, beta variants, delta variants, Omicron BA.1, and sub-variants derived from the aforementioned strains and/or variants, while a second category includes Omicron BA.2, Omicron BA.2.12.1, Omicron BA.4/BA.5, and sub-variants derived from the aforementioned strains and/or variants. Thus, in some embodiments, the present disclosure provides the insight that, among other things, a combination of a first vaccine (e.g., an mRNA vaccine as described herein encoding a spike protein polypeptide) that includes or delivers at least one dose (e.g., including at least one, at least two, at least three, at least four, or more) of a SARS-CoV-2 spike protein polypeptide having a sequence characteristic of a first category as described above, with a second vaccine (e.g., an mRNA vaccine as described herein encoding a spike protein polypeptide) that includes or delivers at least one dose (e.g., including at least one, at least two, at least three, at least four, or more) of a SARS-CoV-2 spike protein polypeptide having a sequence characteristic of a second category as described above, can synergistically provide superior cross-variant neutralization, including enhanced production of neutralizing antibodies against the BA.4/5 omicron variant.

In some embodiments, the present disclosure specifically teaches the surprising efficacy of administering at least one dose of a vaccine (e.g., an mRNA vaccine encoding a spike protein polypeptide as described herein) that includes or delivers a SARS-CoV-2 spike protein polypeptide having a sequence characteristic of the BA.2 omicron variant to a subject who has received at least one (e.g., two, three, or more) doses of a vaccine (e.g., a vaccine encoding a spike protein polypeptide as described herein) that includes or delivers a SARS-CoV-2 spike protein polypeptide having a sequence characteristic of the Wuhan-Hu-1 strain.

The emergence of SARS-CoV-2 omicron variants (VOCs) of concern in November 2021 (Reference 1) can be considered a turning point in the COVID-19 pandemic due to their ability to substantially escape previously established immunity. Omicron BA.1, which replaced delta within weeks as the predominant circulating VOC, acquired significant modifications in the receptor binding domain (RBD) and N-terminal domain (NTD) (Reference 2). These changes led to the loss of many epitopes recognized by neutralizing antibodies (References 3-4), dramatically compromising humoral immunity induced by vaccines based on the ancestral Wuhan strain or exposure to the ancestral strain or previous variants (References 5-7). BA.1 was subsequently replaced by the BA.2 variant, which in turn gave rise to further sublineages. BA.4 and BA.5 derived from BA.2. BA.5 is now the predominant variant in many countries around the world, and several studies suggest significant changes in antigenic properties compared to BA.2, and especially compared to BA.1 (References 8-9). BA.4 and BA.5 are referred to herein as BA.4/5, as they share the same S glycoprotein sequence. Many of the amino acid changes in the RBD are shared between the omicron sublineages, but the modifications within the NTD of BA.2-derived sublineages, including BA.4/5, are mostly distinct from those found in BA.1 (Figure 32).

The majority of the world population has been immunized with vaccines adapted to the Wuhan strain, including, for example, mRNA vaccines such as BNT162b2 and mRNA-1273 (Reference 10), thus essentially forming SARS-CoV-2 herd immunity. However, the emergence of the immune escape variant Omicron BA. 1 has led to a sharp increase in the occurrence of breakthrough infections in vaccinated individuals. It has been reported that SARS-CoV-2 variant breakthrough infections can reshape humoral immunity and thereby modulate neutralizing antibody titers against other variants (References 8, 11, 12). However, as previously reported, BA. 1 breakthrough infections may not provide robust immunity against Omicron BA. 4/5.

Certain Findings To determine whether BA.2 breakthrough infection refocuses immunity against Omicron BA.2 and BA.2-derived sublineages such as BA.4/5, the magnitude and breadth of neutralizing antibody responses were examined in samples from individuals (All Vax+Omi BA.2) who had received a three-dose vaccination scheme with an mRNA vaccine (BNT162b2/mRNA-1273) and subsequently experienced a SARS-CoV-2 breakthrough infection between March and May 2022 (during which BA.2 lineages were predominant in Germany). Such findings have important implications for ongoing vaccine design efforts, as containment of the COVID-19 pandemic will require the generation of durable and sufficiently broad immunity to provide protection against current and future variants of SARS-CoV-2.

Two reference cohorts were generated from data previously published in Quandt et al. (Ref. 12), including (i) individuals vaccinated three times with BNT162b2 (BNT162b2 ³ ) with no prior or breakthrough SARS-CoV-2 infection at the time of sample collection, and (ii) individuals vaccinated three times with an mRNA vaccine with subsequent breakthrough infection during a period of Omicron BA.1 dominance (All Vax + Omi BA.1).

Breakthrough infection with SARS-CoV-2 Omicron BA.1 and BA.2 occurred at a median of approximately 4 months or 3 weeks, respectively, after three vaccinations with mRNA-based COVID-19 vaccines (BNT162b2, mRNA-1273, or heterologous regimens including both vaccines; AllVax+Omi BA.1, AllVax+Omi BA.2) (Figure 28). Immune sera used to characterize serum neutralizing activity were collected at a median of 28 days post-vaccination for the BNT162b2 ³ cohort, 43 days post-BA.1 breakthrough for the AllVax+Omi BA.1 cohort, and 39 days post-BA.2 breakthrough for the AllVax+Omi BA.2 cohort. Median ages of the cohorts were similar (32-38 years). 2.12.1 Neutralization data was generated from serum samples from cohorts for this study, BNT162b ³ and All Vax+Om BA.1.

To assess the neutralizing activity of immune sera, a pseudovirus neutralization test (pVNT) was used, e.g., as described in refs. 13, 14. Pseudoviruses bearing the S glycoproteins of SARS-CoV-2 Wuhan, alpha, beta, delta, omicron BA.1, BA.2, BA.2.12.1, and the recently emerged omicron sublineages BA.4 and BA.5 were applied to assess neutralization breadth. BA.4 and BA.5 are referred to herein as BA.4/5, since they share identical S glycoprotein sequences, including the major modifications L452R and F486V. In addition, SARS-CoV (referred to herein as SARS-CoV-1, ref. 15) was assayed to detect potential pan-sarbecovirus neutralizing activity.

As previously reported in reference 12, the 50% pseudovirus neutralization ( _pVN50 ) geometric mean titers (GMT) of immune sera from SARS-CoV-2-naive, 3-times vaccinated individuals against Omicron BA.1 and BA.2 were significantly reduced compared to the Wuhan strain (GMT 160 and 221 vs. 398). Neutralizing activity against BA.2.12.1 and BA.4/5 was further reduced (GMT 111 and 74), corresponding to a 5.4-fold lower titer for BA.4/5 compared to the Wuhan strain (Figure 29(A)).

Omicron BA.2 breakthrough infection significantly increased _pVN50 GMTs against BA.2 and BA.2.12.1 compared to SARS-CoV-2 uninfected 3-vaccinated immune sera, such that neutralization of BA.2 after breakthrough infection was comparable to the Wuhan strain (Figure 30(B-C)). Similarly, BA.1 breakthrough infection significantly increased neutralization of BA. BA.2 convalescent sera conferred robust neutralizing activity against the Wuhan strain (Figure 29(B), Figure 30(A). Importantly, _pVN50 GMT against BA.4/5 in BA.2 convalescent sera was lower against the Wuhan strain (GMT 391 vs. 922, i.e., a 2.4-fold reduction), but this reduction was still lower than that observed in the Omicron-uninfected BNT162b2 3 cohort, whose sera exhibited a 5.4-fold reduction in BA.4/5 neutralizing activity (Figure 30(C)). In contrast, _pVN50 GMT against BA.4/5 and Wuhan following BA.1 breakthrough infection was significantly higher than that observed in the Omicron-uninfected BNT162b2 ³ cohort, whose sera exhibited a 5.4-fold reduction in BA.4/5 neutralizing activity (Figure 30(C)). The GMTs were 266 and 1327, respectively (i.e., a 5-fold reduction, FIG. 29(B)). Thus, Omicron BA.1 breakthrough infection of 3-vaccinated individuals did not result in more efficient cross-neutralization of Omicron BA.4/5 compared to 3-vaccinated Omicron-naive individuals. In both cohorts, neutralization titers against BA.4/5 were closer to the lower levels observed against the phylogenetically more distant SARS-CoV-1 than those seen against Wuhan (FIG. 29). Of note, _pVN50 GMTs against the Wuhan strain following BA.1 breakthrough infection were slightly higher than those observed for BA.2 breakthrough infection (GMT 1327 vs. 922), which, without wishing to be bound by any particular theory, may be related to the longer interval between the third vaccination and infection (median 22 days for BA.1 and 127.5 days for BA.2) (FIG. 30).

Separate analyses were performed that included only individuals vaccinated three times with BNT162b2 (BA.2 or BA.1 breakthrough infection, or Omicron-naive). In these analyses, similar observations were made regarding BA.4/5 neutralizing activity: _pVN50 GMT against BA.4/5 in BA.2 convalescent sera was 2.4-fold higher than the Wuhan strain, but was reduced 6-fold after BA.1 breakthrough infection (Figure 30). Relative neutralization of BA.2 and BA.2.12.1 was comparable in BA.2 and BA.1 convalescent sera, although neutralizing activity against these variants remained slightly higher than that seen in Omicron-naive sera.

Immune sera from 3-vaccinated Omicron-naive individuals had broad neutralizing activity against ancestral SARS-CoV-2 VOCs. Neutralizing activity against beta was slightly higher in BA.1 convalescent sera, whereas alpha and delta neutralization was not affected by BA.1 or BA.2 breakthrough infection (Figure 30(C)).

Taken together, these data demonstrate that Omicron BA.2 breakthrough infection in vaccine-experienced individuals mediates broad neutralizing activity against BA.1, BA.2, BA.2.12.1, and several ancestral SARS-CoV-2 variants. Furthermore, neutralizing activity against BA.4/5 is provided to a greater extent than BA.1 convalescent sera, although at a lower level against the Wuhan criteria.

Recent studies have demonstrated that omicron BA.1 breakthrough infection in individuals vaccinated with mRNA vaccines (BNT162b2 or mRNA-1273) boosts serum neutralizing titers not only against the ancestral Wuhan strain but also against VOCs, including BA.2 (refs. 8, 11, 12). This effect was seen in individuals vaccinated three times, but was particularly pronounced in individuals vaccinated twice, whose sera contained little or no neutralizing activity against BA.2. However, BA.1 breakthrough infection did not elicit strong neutralizing activity against BA.4/5, the VOCs that have now established global dominance. Without wishing to be bound by a particular theory, this immune escape is due to amplification and/or recall of pre-existing neutralizing antibody responses that recognize epitopes not present in omicron sublines BA.2.12.1, BA.4, and BA.5.

This example provides, inter alia, insight that BA.2 breakthrough infection triggers recall responses that mediate enhanced neutralization of BA.2-derived sublineages, including BA.4/5, and shows that the higher S protein sequence similarity between BA.2, BA.2.12.1, and BA.4/5 drives more efficient cross-neutralization compared to breakthrough infections with more distant BA.1 variants. Despite the importance of vaccination with currently approved Wuhan-derived vaccines, such as BNT162b2, which provide effective protection from severe disease with current VOCs, including Omicron BA.1 and BA.2, the present findings of broad cross-neutralizing activity against current VOCs, including BA.4/5, following BA.2 breakthrough infection, inter alia, support the use of vaccines adapted to Wuhan strain sequences or variant sequences from the same immunologically related categories as described above (e.g., alpha strains, beta strains, delta strains, Omicron BA.1), and BA. This provides insight that vaccine combinations adapted to two variant sequences or variant sequences from the same immunologically related categories as those described above (e.g., Omicron BA.2.12.1, Omicron BA.4/BA.5) can provide enhanced cross-neutralizing activity against variants from two different categories. In some embodiments, this example provides evidence supporting the implementation of a licensing procedure modeled on that of seasonal influenza vaccines that uses up-to-date epidemiological data to select COVID-19 vaccine strains. In some embodiments, this example further provides evidence supporting the establishment of rapid strain selection for seasonal updates of COVID-19 vaccines, similar to the selection process implemented by the World Health Organization (WHO) Global Influenza Surveillance and Response System (GISRS), and/or agreement on an accelerated approval pathway based on surrogate immunogenicity endpoints.

Neutralizing titers from subjects who were vaccinated against SARS-CoV-2 and experienced a BA.1 or BA.2 breakthrough infection are shown in Figure 30(A) and (B), respectively, and the GMRs for both groups of subjects are shown in Figure 30(C). As shown in Figures 30(A) and (B), sera from subjects who were previously vaccinated against SARS-CoV-2 and experienced a breakthrough infection with either BA.1 or BA.2 were found to have significant neutralizing titers against pseudoviruses containing the SARS-CoV-2 S protein of the Wuhan strain, alpha variant, beta variant, delta variant, and omicron BA.1 variant. As previously mentioned, neutralizing titers against BA.2 were somewhat lower in sera from BA.1 breakthrough patients (GMT of 875 for BA.2 vs. 1327 for Wuhan strain) and were significantly higher for BA.2.12.1 and BA. The neutralization titers against omicron BA.1 are somewhat higher in BA.1 breakthrough patients than in BA.2 breakthrough patients (GMR of 0.76 compared to 0.60), whereas the neutralization titers against omicron BA.2 are higher in BA.2 breakthrough patients than in BA.1 breakthrough patients (GMR of 0.94 vs. 0.66). However, surprisingly, the neutralization responses against BA.4/5 are higher than those against BA.1 breakthrough patients (GMR of 0.94 vs. 0.66). The GMR was significantly higher in BA.2 breakthrough patients (GMR of 0.39 in BA.2 breakthrough patients compared to GMR of 0.2 in BA.1 breakthrough patients). Thus, the present disclosure documents that a broader immune response can be elicited by BA.2 breakthrough infection compared to BA.1 breakthrough infection in subjects vaccinated against SARS-CoV-2, and teaches that administering a booster vaccine containing RNA encoding an S protein containing mutations characteristic of the BA.2 omicron variant can achieve surprising and unexpected benefits.

Furthermore, the present disclosure provides the insight that, given the similarity between the S protein sequences of the BA.2 and BA.4/5 variants, particularly broad immunization (i.e., synergistic immunization as described herein) may also be achieved by combining a vaccination dose that includes or delivers a BA.4 and/or BA.5 variant spike sequence with a vaccination dose that includes or delivers a Wuhan spike sequence.

In some embodiments, these findings suggest that synergistic categories of coronavirus strains and/or variant sequences (e.g., SARS-CoV-2 strains and/or variant sequences) may be defined in some embodiments based on, for example, shared amino acid modifications in the S glycoprotein of the coronavirus strains and/or variant sequences. For example, many of the amino acid changes in the RBD of the S protein are shared among Omicron sublineages (e.g., BA.1, BA.2, BA.2.12.1, and BA.4/5), while modifications within the NTD of BA.2 and BA.2-derived sublineages, including BA.4/5, are largely distinct from those found in BA.1. Thus, in some embodiments, synergistic categories of coronavirus strains and/or variant sequences (e.g., SARS-CoV-2 strains and/or variant sequences) may be defined based on the extent of shared amino acid modifications present with the NTD of the S protein. For example, in some embodiments where two SARS-CoV-2 strain and/or variant sequences share at least 50% (including, e.g., at least 60%, at least 70%, at least 80%, at least 90%, or more) of the amino acid mutations present in the NTD of the S protein, both the SARS-CoV-2 strain and variant sequences can be grouped into the same category. In some embodiments where two SARS-CoV-2 strain and/or variant sequences share 50% or less (including, e.g., 45% or less, 40% or less, 30% or less, or less) of the amino acid mutations present in the NTD of the S protein, both the SARS-CoV-2 strain and variant sequences can be grouped into different categories. In particular, the present findings provide the insight that by exposing a subject (e.g., via infection and/or vaccination) to at least two antigens of different synergistic categories (e.g., as shown in the table below), a more robust immune response can be generated (e.g., expanding the spectrum of cross-neutralization against different variants and/or generating an immune response that is less prone to immune escape).

For example, in some embodiments, a vaccine combination may be administered to an unvaccinated subject with no history of infection, at least two of which are each adapted to a SARS-CoV-2 strain of a different synergistic category (e.g., as described herein). In some embodiments, such a vaccine combination may be administered as a first dose and a second dose that are administered at different times, e.g., in some embodiments, a predetermined period of time (e.g., according to certain dosing regimens described herein), spaced apart. In some embodiments, such a vaccine combination may be administered as a multivalent vaccine. In some embodiments, a subject infected or vaccinated with a SARS-CoV-2 strain of one category may be administered with a vaccine adapted to a SARS-CoV-2 strain of a different category (e.g., as described herein). In some embodiments, such a vaccine may be a polypeptide-based or RNA-based vaccine.

Although the findings are based on retrospective analyses of samples from different studies, with relatively small sample sizes and cohorts that were not fully matched with respect to individual immunization intervals and demographic characteristics such as age and sex, the findings provide useful insights for vaccine design and vaccination strategies to improve cross-neutralization against a broader spectrum of SARS-CoV-2 variants.

Materials and Methods Participant Recruitment and Sample Collection SARS-CoV-2-naive BNT162b2 triple vaccinated (BNT162b2 ³ ) cohort individuals provided informed consent as part of their participation in the Phase 2 clinical trial BNT162-17 (NCT05004181). Individuals with Omicron BA.1 or BA.2 breakthrough infection (All Vax + Omi BA.1 and All Vax + Omi BA.2 cohorts) were vaccinated three times with, for example, one or more doses of BNT162b2, Moderna mRNA-1273, AstraZeneca ChAdOx1-S recombinant vaccine, or combinations thereof, and were recruited to provide blood samples and clinical data for the study. Omicron infections were confirmed by variant-specific PCR either between November 2021 and mid-January 2022 (All Vax + Omi BA.1) or between March 2022 and May 2022, sometimes with sublineages BA.1 or BA.2 predominating, respectively (Reference 24). Infections in certain participants in this study (e.g., at least seven participants) were further characterized by genomic sequencing, which confirmed Omicron BA.1 or BA.2 infection.

Participants were asymptomatic at the time of blood collection. Table 32 summarizes the characteristics of vaccinated individuals analyzed for neutralizing antibody responses. All participants had no documented SARS-CoV-2 infection prior to vaccination.

Serum was isolated by centrifugation of collected blood at 2000 x g for 10 minutes and stored frozen until use.

VSV-SARS-CoV-2 S variant pseudovirus generation. A recombinant replication-deficient vesicular stomatitis virus (VSV) vector encoding green fluorescent protein (GFP) and luciferase instead of the VSV-glycoprotein (VSV-G) was cloned into the SARS-CoV-1 S glycoprotein (UniProt Ref: P59594) and the Wuhan reference strain (NCBI Ref: P59595). Ref: 43740568), alpha variant (modifications: Δ69/70, Δ144, N501Y, A570D, D614G, P681H, T716I, S982A, D1118H), beta variant (modifications: L18F, D80A, D215G, Δ242-244, R246I, K417N, E484K, N501Y, D614G, A701V), delta variant (modifications: T19R, G142D, E156G, Δ157/158, K417N, L452R, T478K, D614G, P681R, D950N) Omicron BA. 1 variant (modifications: A67V, Δ69/70, T95I, G142D, Δ143-145, Δ211, L212I, ins214EPE, G339D, S371L, S373P, S375F, K417N, N440K, G446S, S477N, T478K, E484A, Q493R, G496S, Q498R, N501Y, Y505H, T547K, D614G, H655Y, N679K, P681H, N764K, D796Y, N856K, Q954H, N969K, L981F), Omicron BA. 2 variants (modifications: T19I, Δ24-26, A27S, G142D, V213G, G339D, S371F, S373P, S375F, T376A, D405N, R408S, K417N, N440K, S477N, T478K, E484A, Q493R, Q498R, N501Y, Y505H, D614G, H655Y, N679K, P681H, N764K, D796Y, Q954H, N969K), Omicron BA. 2.12.1 variant (modifications: T19I, Δ24-26, A27S, G142D, V213G, G339D, S371F, S373P, S375F, T376A, D405N, R408S, K417N, N440K, L452Q, S477N, T478K, E484A, Q493R, Q498R, N501Y, Y505H, D614G, H655Y, N679K, P681H, S704L, N764K, D796Y, Q954H, N969K), or Omicron BA. SARS-CoV-2 S glycoproteins derived from any of the 4/5 variants (modifications: T19I, Δ24-26, A27S, Δ69/70, G142D, V213G, G339D, S371F, S373P, S375F, T376A, D405N, R408S, K417N, N440K, L452R, S477N, T478K, E484A, F486V, Q498R, N501Y, Y505H, D614G, H655Y, N679K, P681H, N764K, D796Y, Q954H, N969K) were used for pseudotyping according to published pseudotyping protocols (ref. 49).

A diagram of the SARS-CoV-2 S glycoprotein modifications is shown in Figure 31, and a separate alignment of S glycoprotein modifications in the Omicron sublineage is shown in Figure 32. Briefly, HEK293T/17 monolayers (ATCC® CRL-11268™) cultured in Dulbecco's Modified Eagle's Medium (DMEM) with GlutaMAX™ (Gibco) supplemented with 10% heat-inactivated bovine serum (FBS [Sigma-Aldrich]) (referred to as medium) were transfected with Sanger sequence-verified SARS-CoV-1 or variant-specific SARS-CoV-2S expression plasmids using Lipofectamine LTX (Life Technologies) according to the manufacturer's instructions. At 24 h, VSV-G complemented the VSVΔG vector. After incubation at 37° C. with 7.5% CO2 for 2 h, cells were washed twice with phosphate-buffered saline (PBS) and then medium supplemented with anti-VSV-G antibody (clone 8G5F11, Kerafast Inc.) was added to neutralize residual VSV-G-complemented input virus. VSV-SARS-CoV-2-S pseudotype-containing medium was harvested 20 h after seeding, passed through a 0.2 μm filter (Nalgene) and stored at −80° C. Pseudovirus batches were titrated on Vero 76 cells (ATCC® CRL-1587™) cultured in medium. As previously described by Muik et al. The relative luciferase units induced by a defined volume of the Wuhan S glycoprotein pseudovirus reference batch, corresponding to an infectious titer of 200 transducing units (TU) per mL, as described in , 2021, was used as a comparison. The input volume of the SARS-CoV-2 variant pseudovirus batch was calculated to normalize the infectious titer based on the relative luciferase units to the reference.

Pseudovirus Neutralization Assays Vero 76 cells were seeded in 96-well white flat-bottom plates (Thermo Scientific) at 40,000 cells/well in medium 4 hours prior to the assay and incubated at 37°C with 7.5% CO2. Each individual serum was serially diluted twice in medium, the first dilution being 1:5 (Omicron-naive, 3x BNT162b2 vaccinated; dilution range 1:5 to 1:5,120) or 1:30 (3x vaccinated after subsequent Omicron BA.1 or BA.2 breakthrough infection; dilution range 1:30 to 1:30,720). For the SARS-CoV-1 pseudovirus assay, all individual sera were initially diluted 1:5 (dilution range 1:5 to 1:5,120). VSV-SARS-CoV-2-S/VSV-SARS-CoV-1-S particles were diluted in medium to obtain 200 TU in the assay. Serum dilutions were mixed 1:1 with pseudovirus (n=2 technical replicates per serum per pseudovirus) for 30 min at room temperature before being added to Vero 76 cell monolayers and incubated at 37°C with 7.5% CO2 for 24 h. Supernatants were removed and cells were lysed with luciferase reagent (Promega). Luminescence was recorded on a CLARIOstar® Plus microplate reader (BMG Labtech) and neutralization titers were calculated as the interaction of the highest serum dilution that still resulted in a 50% reduction in luminescence. Results were expressed as the geometric mean titer (GMT) of duplicates. When no neutralization was observed, an arbitrary titer value of half the limit of detection [LOD] was reported.

Statistical Analysis The statistical method of agglutination used to analyze antibody titers is the geometric mean and for the ratios of SARS-CoV-2 VOC titers and Wuhan titers, the geometric mean and corresponding 95% confidence intervals. The use of the geometric mean accounts for the non-normal distribution of antibody titers, which spans several orders of magnitude. Paired signed rank tests of the geometric mean neutralizing antibody titers of groups with a common control group were performed using the Friedman test with Dunn's correction for multiple comparisons. All statistical analyses were performed using GraphPad Prism software version 9.

References cited in Example 14 1. WHO Technical Advisory Group on SARS-CoV-2 Virus Evolution (TAG-VE), Classification of Omicron (B.1.1.529): SARS-CoV-2 Variant of Concern (2021).
2. WHO Headquarters (HQ), WHO Health Emergencies Programme, Enhancing response to Omicron SARS-CoV-2 variant: Technical brief and priority actions for Member States (2022).
3. M. Hoffmann et al. , The Omicron variant is highly resistant against antibody-mediated neutralization. Cell. 185, 447-456. e11 (2022), doi:10.1016/j. cell. 2021.12.032.
4. W. Dejnirattisai et al. , SARS-CoV-2 Omicron-B. 1.1.529 leads to widespread escape from neutralizing antibody responses. Cell. 185, 467-484. e15 (2022), doi:10.1016/j. cell. 2021.12.046.
5. V. Servellita et al. , Neutralizing immunity in vaccine breakthrough infections from the SARS-CoV-2 Omicron and Delta variants. Cell. 185, 1539-1548. e5 (2022), doi:10.1016/j. cell. 2022.03.019.
6. C. Kurhade et al. , Neutralization of Omicron BA. 1. BA. 2, and BA. 3 SARS-CoV-2 by 3 doses of BNT162b2 vaccine. Nature communications. 13, 255 (2022), doi:10.1038/s41467-022-30681-1.
7. Y. Cao et al. , Omicron escapes the majority of existing SARS-CoV-2 neutralizing antibodies. Nature. 602, 657-663 (2022), doi:10.1038/s41586-021-04385-3.
8. Y. Cao et al. , B.A. 2.12.1, BA. 4 and BA. 5 escape antibodies elicited by Omicron infection. Nature (2022), doi:10.1038/s41586-022-04980-y.
9. N. P. Hachmann et al. , Neutralization Escape by SARS-CoV-2 Omicron Subvariants BA. 2.12.1, BA. 4, and BA. 5. The New England journal of medicine (2022), doi:10.1056/NEJMc2206576.
10. E. Mathieu et al. , A global database of COVID-19 vaccinations. Nature human behavior. 5, 947-953 (2021), doi:10.1038/s41562-021-01122-8.
11. C. I. Kaku et al. , Recall of pre-existing cross-reactive B cell memory following Omicron BA. 1 breakthrough infection. Science immunology, eabq3511 (2022), doi:10.1126/sciimmunol. abq3511.
12. J. Quandt et al. , Omicron BA. 1 breakthrough infection drives cross-variant neutralization and memory B cell formation against conserved epitopes. Science immunology, eabq2427 (2022), doi:10.1126/sciimmunol. abq2427.
13. A. Muik et al. , Neutralization of SARS-CoV-2 Omicron by BNT162b2 mRNA vaccine-elicited human sera. Science (New York, NY). 375, 678-680 (2022), doi:10.1126/science. abn7591.
14. A. Muik et al. , Neutralization of SARS-CoV-2 lineage B. 1.1.7 pseudovirus by BNT162b2 vaccine-elicited human sera. Science (New York, NY). 371, 1152-1153 (2021), doi:10.1126/science. abg6105.
15. C. -W. Tan et al. , Pan-Sarbecovirus Neutralizing Antibodies in BNT162b2-Immunized SARS-CoV-1 Survivors. The New England journal of medicine. 385, 1401-1406 (2021), doi:10.1056/NEJMoa2108453.

Example 15: Further Update on Immune Responses Elicited by Vaccines Encoding SARS-CoV-2 S Proteins from the Omicron Variant Following the experiments described in Example 8, additional subjects were enrolled in clinical trials investigating RNA vaccines encoding SARS-CoV-2 S proteins containing one or more mutations characteristic of the BA.1 Omicron variant. In this example, subjects (ages 18-55 with or without evidence of prior infection) were administered a second booster (fourth dose) of either 30 ug of RNA encoding the SARS-CoV-2 S protein of the Wuhan strain (in this example, BNT162b2) or 30 μg of RNA encoding a SARS-CoV-2 S protein having one or more mutations characteristic of the omicron variant (in this example, BNT162b2 OMI, which encodes a SARS-CoV-2 S protein having one or more mutations characteristic of the BA.1 omicron variant and includes SEQ ID NOs:50 and 51, encoding the amino acid sequence of SEQ ID NO:49).

In a primary immunogenicity analysis of participants without evidence of prior infection, BNT162b2 OMI (N=132) elicited superior neutralizing antibody responses against BA.1 omicron SARS-CoV-2 virus compared to BNT162b2 (N=141). The BNT162b2 OMI GMT against BA.1 omicron was 1929 (CI: 1632, 2281) compared to 1100 (CI: 932, 1297) for BNT162b2, a GMT ratio of 1.75 (95% CI: 1.39, 2.22).

Compared to BNT162b2, BNT162b2 OMI elicited a neutralizing antibody response similar to the Wuhan strain of SARS-CoV-2. BNT162b2 OMI GMT was 11997 (CI: 10554, 13638) compared to BNT162b2 GMT 12009 (CI: 10744, 13425).

The data suggest that the Omicron monovalent vaccine as a second booster vaccination (fourth dose) improves neutralizing antibody responses to BA.1 Omicron compared to an RNA vaccine encoding the S protein of the Wuhan strain and does not adversely affect neutralizing antibody responses to the Wuhan strain of SARS-CoV-2.

Example 16. Immunological Impact of VOC Vaccination This example describes the immunological impact of administering a BNT162b2 vaccine encoding a spike protein from a particular variant of concern ("VOC"). In particular, this example describes the immunological impact of administering a "booster dose" to subjects (in this example, mice) who have received two doses (i.e., according to established model immunization protocols) of the "original" BNT162b2 vaccine (i.e., encoding the Wuhan spike protein as described herein).

Figure 33 shows the immunization protocol utilized in this example. Specifically, BALB/c mice were immunized twice with BNT162b2 (1 ug dose each), followed at a later time point with BNT162b2/VOC (1 ug dose each). Immunizations were performed up to 3 or 4 times. Animals were bled periodically to analyze antibody immune responses by ELISA and pseudovirus neutralization assays. At the end of the trial, animals were euthanized and T cell responses in the spleen were analyzed.

Boosting was performed with: (a) original BNT162b2 ("BNT162b2"), (b) BNT162b2 OMI BA.1 ("OMI BA.1"), (c) BNT162b2 OMI BA.4/5 ("OMI BA.4/5"), (d) BNT162b2 + OMI BA.4/5 (0.5ug each), (e) OMI BA.1 + OMI BA.4/5 (0.5ug each), and (f) BNT162b2 + OMI BA.1 + OMI BA.4/5 (0.33ug each).

Omicron variants BA.4 and BA.5 were reported to have first circulated in January 2022 and were becoming the predominant variant by June 2022. Both of these lineages contain the amino acid substitutions F486V and R493Q. Preliminary studies suggest a significant change in the antigenic characteristics of BA.4 and BA.5 compared to BA.1 and BA.2, and especially compared to BA.1. Furthermore, as an increasing trend in the proportion of BA.5 variants is observed in certain locations (e.g., Portugal), the number of COVID-19 cases and test positivity rates are also increasing. The present disclosure proposes that BA.4/5 (considered together in this example, given their common spike protein mutations) may represent escape VOCs. The present disclosure provides a basis for the identification of related BA. This demonstrates the particular advantages of a dosing regimen (e.g., as described herein and specifically exemplified in the present examples) that includes one or more doses of a vaccine that includes or delivers (e.g., via expression of an administered RNA) a spike protein that includes a 4/5 sequence (e.g., amino acid substitution).

Figures 34 and 35 present baseline (pre-boost) geometric mean titers ("GMTs") compared to various SARS-CoV-2 strains as indicated. As can be seen, baseline immunization of the different mouse cohorts was comparable. Specifically, group GMTs per pseudovirus were consistently observed within the same expected range between cohorts, with no greater than about 2-fold differences observed. As noted above, consistent with observations made in human populations, neutralization GMTs against the Wuhan strains were considerably higher (up to GMTs of 3,044) compared to those against VOCs. Overall, the order of GMTs was:
It was.

Figure 36 shows baseline (determined on day 104, pre-boost) cross-neutralization analysis, demonstrating comparable baseline immunization of the cohorts with respect to cross-neutralization potential. Specifically, at baseline, the calculated variant/Wuhan reference GMT ratios showed that cross-neutralization potential was relatively comparable between cohorts (with only one outlier in the BNT162b2 monovalent group, BA.1 neutralization was observed). Also consistent with observations in human populations, BA.1 = BA.2 > BA.2.12.1 > BA.4/5.

Figures 37-39 present data obtained 7 days after boosting and document the remarkable efficacy of BA.4/5, especially monovalent BA.4/5, in achieving significant geometric mean fold increases in GMT (Figures 37 and 38) as well as effective cross-neutralization (Figure 39). As can be seen, BNT162b2 booster immunization resulted in comparable titer increases (3.9-7.1 fold) against all VOCs, while monovalent BA.1 and BA.4/5 boosters resulted in much stronger increases in homologous VOC titers (16.8-fold for BA.1 and 67.3-fold for BA.4/5).

The monovalent BA.4/5 booster was the most effective at driving titer increases across the pseudovirus panel tested. The bivalent boosters showed a similar but attenuated trend compared to the monovalent VOC booster, and among the bivalent boosters, the b2+BA.4/5 combination was the most effective at driving broad cross-neutralization. The trivalent booster (b2+BA.1+BA.4/5) was superior to the bivalent boosters and provided intermediate immunization between the bivalent b2+BA.4/5 and monovalent BA.4/5 boosters.

FIG. 39 presents, among other things, the calculated variant/Wuhan reference GMT ratios, which show:
(i) the BNT162b2 booster results in relatively poor cross-neutralization, especially of BA.2 and progeny (BA.2.12.1, BA.4/5); (ii) the BA.1 booster results in excellent cross-neutralization of BA.1, but still relatively poor neutralization of BA.2.12.1, BA.4/5; (iii) the BA.4/5 booster results in balanced pan-omicron neutralization, with very encouraging neutralization of BA.2, BA.2.12.1 and BA.4/5. Bivalent boosters showed a similar but attenuated trend compared to the monovalent VOC boosters, with the b2+BA.4/5 combination being the most effective at driving broad cross-neutralization of the bivalent boosters, and the trivalent boosters (b2+BA.1+BA.4/5) being the most effective at driving broad cross-neutralization of BA. The immunization induced cross-neutralization equivalent to that of the 1/BA.1/BA.4/5 booster.

The present specification demonstrates the remarkable efficacy of BA.4/5 immunization (specifically, for example, BNT162b2+BA.4/5 immunization with sequences provided herein). Additionally, the present specification demonstrates the efficacy of BA.4/5 immunization in monovalent, bivalent, and trivalent formats, and documents the surprising efficacy of monovalent BA.4/5.

The present disclosure specifically demonstrates the remarkable utility of one or more BA. 4/5 doses administered to subjects previously immunized (e.g., with the Wuhan vaccine, e.g., with at least (or exactly) two doses of the Wuhan vaccine). Without wishing to be bound by any particular theory, the present disclosure demonstrates that the immunological characteristics of the Omicron BA. 4/5 spike may be particularly useful or effective in immunizing subjects, particularly including subjects immunized (e.g., via prior administration of one or more vaccine doses and/or prior infection) with the Wuhan strain (and/or one or more strains immunologically related to the Wuhan strain), including vaccination with one or more (e.g., 1, 2, 3, 4, or more) doses of the original BNT162b2.

Example 17. Selection and characterization of exemplary spike protein variants.
This example describes the design and characterization of various spike variant sequences for use in an RNA vaccine (e.g., a vaccine comprising RNA, e.g., in some embodiments, mRNA, that encodes a spike protein sequence from a coronavirus, e.g., SARS-CoV-2).

As described herein, in some embodiments, certain mutations may be introduced into the SARS-CoV-2 spike protein coding sequence that contribute to increased immunogenicity of the spike protein antigen, for example, by improving the expression of the spike protein, improving the stability of the spike protein, improving the stability of certain conformations of the spike protein (e.g., improving the stabilization of the pre-fusion conformation), and/or increasing the number of neutralization-sensitive epitopes on the spike protein. Certain mutations are described, for example, in WO2021/243122A2 and Hsieh, Ching-Lin, et al. ("Structure-based design of prefusion-stabilized SARS-CoV-2 spikes," Science 369.6510 (2020): 1501-1505), the contents of each of which are incorporated herein by reference in their entirety.

In this example, various combinations of proline substitutions were introduced into the amino acid sequence of the SARS-CoV-2 S protein. Exemplary combinations of such combinations of proline mutations are shown in FIG. 40. The positions of the mutations as listed in FIG. 40 are shown with respect to the spike protein sequence according to SEQ ID NO:1 (SARS-CoV-2 Wuhan, i.e., wild-type strain). The spike protein sequence containing the proline mutations corresponding to K986P and V987P in the Wuhan strain is designated as "P2". The spike protein sequence containing the proline mutations corresponding to K986P, V987P, F817P, A892P, A899P, and A942P in the Wuhan strain is designated as "P6". The spike protein sequence containing the proline mutations corresponding to D985P and V987P in the Wuhan strain is designated as "P2". The spike protein sequence containing proline mutations corresponding to V987P, F817P, A892P, A899P, and A942P is designated as "P5". The spike protein sequence containing proline mutations corresponding to D985P, V987P, F817P, A892P, A899P, and A942P is designated as "P6". The spike protein sequence containing proline mutations corresponding to D985P, K986P, F817P, A892P, A899P, A942P is designated as "P6". The spike protein sequence containing proline mutations corresponding to D985P, K986P, V987P, F817P, A892P, A899P, A942P is designated as "P7".

Various combinations of proline mutations described herein (e.g., as described in FIG. 40) can be introduced into a coronavirus spike protein or immunogenic fragment thereof. In this example, various combinations of proline mutations described in FIG. 40 can be introduced into the S protein of SARS-CoV-2 strains and variants, or immunogenic fragments thereof, as shown in Table 1.

Alternatively or additionally, additional mutations may be introduced into a coronavirus spike protein or immunogenic fragment thereof, e.g., in some embodiments, the S protein or immunogenic fragment thereof of SARS-CoV-2 strains and variants. In some embodiments, such additional mutations are selected, for example, for (a) increasing the adoption of the RBD-up conformation to expose more neutralization-sensitive epitopes on the spike protein, (b) decreasing the adoption of the RBD-down conformation, (c) increasing expression of the variant coronavirus spike protein compared to a reference (e.g., native or wild-type) coronavirus spike protein, (d) increasing the adoption of the prefusion conformation, (e) decreasing shedding of the S1 subunit of the variant coronavirus spike protein, and/or (f) improving localization of the variant coronavirus spike protein to the host cell membrane. Exemplary such mutations are disclosed in FIG. 41 under the columns "Mutation" and "Variant Type." In some embodiments, the furin cleavage site in a coronavirus protein or immunogenic fragment thereof that includes the amino acid sequence of RRAR can optionally be mutated to the amino acid sequence (GSAS).

Figure 41 shows the mutations introduced into the coronavirus S protein and certain combinations of mutations in the mRNA constructs encoding such coronavirus S protein variants. S protein expression, ACE2 binding, and CR3022 ^* epitope binding (Yuan et al., "A highly conserved cryptic epitope in the receptor-binding domains of SARS-CoV-2 and SARS-CoV (2020) 368:630) were assessed for each mRNA encoding the S protein variants (see Figure 41). Surprisingly, as shown in Figure 41, incorporation of the D985P mutation, but not the K986P mutation, was found to improve protein expression, CR3022 response, and ACE2 response, respectively (compare data for variant E and variant I).

Example 18. Neutralization of various SARS-CoV-2 VOCs by RNA vaccines encoding exemplary spike protein variants.
This example describes the immunological effects of administering an RNA vaccine encoding an exemplary spike protein variant of a SARS-CoV-2 strain or VOC. In particular, this example describes the immunological effects of administering at least one dose (e.g., including at least two doses or at least three doses) to a subject (in this example, a mouse).

Specifically, mice were administered at least one dose of: (i) BNT162b2; (ii) RNA encoding the S protein of the Wuhan strain having at least the mutations K986P, V987P, and D614G at the furin cleavage site (R682G, R683S, R685S) (Figure 42 "P2 with D614G + furin mutant"); (iii) RNA encoding the S protein of the Wuhan strain having at least the mutations D985P, V987P, F817P, A892P, A899P, A942P, and D614G (designated as "P6' with D614G+intact furin" in FIG. 42), and (iv) RNA encoding the S protein of the Wuhan strain with at least the mutations D985P, V987P, F817P, A892P, A899P, A942P, and D614G, as well as mutations at the furin cleavage site (R682G, R683S, R685S) (designated as "P6' with D614G+furin mutants" in FIG. 42). Animals were bled approximately one month after receiving the second dose of vaccine, and serum was analyzed to determine antibody immune responses, e.g., using a pseudovirus neutralization assay described herein. Specifically, as shown in FIG. 42, neutralizing antibody titers against the Wuhan (wild type), beta variant, delta variant, and Omicron BA.1 variant were evaluated for each RNA vaccine tested.

42 shows that an exemplary RNA construct encoding a SARS-CoV-2 S protein having at least mutations at positions corresponding to D985P, V987P, F817P, A892P, A899P, A942P, and D614G of the Wuhan strain (e.g., SEQ ID NO:1) and at the furin cleavage site (R682G, R683S, R685S) stimulated higher neutralization titers across VOCs including Wuhan, beta variants, delta variants, and omicron BA.1 variants. As described in Tables 2A-2B, such mutations are numbered relative to the Wuhan spike protein sequence (SEQ ID NO:1), although the location of the specific mutations may vary as described herein depending on the strain of the SARS-CoV-2 spike protein sequence (see, e.g., the alignment in Table 5). Surprisingly, as shown in FIG. 42, RNA encoding the SARS-CoV-2 S protein containing the set of P6' mutations, the D614G mutation, and an intact furin cleavage site was found to generate an immune response similar to or worse than BNT162b2, while incorporating the furin site mutation in the same construct was found to significantly increase the immune response compared to BNT162b2. The immune response was particularly improved for SARS-CoV-2 variants, indicating that BNT162b5 is able to induce an immune response with broader cross-neutralization compared to BNT162b2. Similar experiments performed with the BA.4/5 adapted bivalent vaccine in unvaccinated mice did not reproduce these results, although improved immune responses were still observed for the BQ.1.1 and XBB omicron variants (data shown in FIG. 47).

Example 19: Immunogenicity Study of Vaccines Encoding SARS-CoV-2 S Protein Variants in Vaccine-Experienced Healthy Subjects This example describes a study that evaluated the safety, tolerability, and immunogenicity of BNT162b, an RNA-based SARS-CoV-2 vaccine candidate, given as a booster dose in adults to prevent COVID-19.

Evaluations were performed on subjects including:
● Healthy people aged 18 to 55 (those who may have had pre-existing conditions, as long as they are stable).
Have received one booster dose of a U.S.-authorized COVID-19 vaccine (e.g., BNT162b2, Moderna COVID-19 vaccine, etc.), with the last dose being ≥90 days prior to the first study visit.

In some embodiments, subjects were excluded from the study if they had at least one of the following characteristics:

• History of severe adverse reactions related to vaccines and/or severe allergic reactions (e.g., anaphylaxis) to any component of the study vaccine.
• Known or suspected immunodeficiency.
Bleeding diathesis or condition associated with prolonged bleeding that, in the opinion of the investigator, contraindicates intramuscular injection.
●Pregnant or breastfeeding women.
• Other medical or psychiatric conditions or laboratory abnormalities that may have increased the risk of study participation or that, in the investigator's judgment, made the participant unsuitable for the study.
- Chronic systemic treatment with known immunosuppressive drugs (including cytotoxic agents or systemic corticosteroids) or radiation therapy within 60 days prior to study vaccination and through the end of the study.
• Receipt of blood/plasma products, immunoglobulins, or monoclonal antibodies within 60 days prior to, or planned receipt throughout, the study.
Participation in another study involving the study intervention within 28 days of randomization.
- Anticipated participation in another study within 28 days of receipt of the research intervention in this study.

Exemplary Dosing Regimen:
Subjects in this study received one of two investigational vaccines: BNT162b5 bivalent or BNT162b2 bivalent.

As described herein, BNT162b2 bivalent (WT/OMI BA.1) refers to a composition comprising (i) an RNA encoding an S protein from the Wuhan strain, and (ii) an RNA encoding an S protein from the Omicron BA.1 variant, both S protein sequences containing two proline mutations at positions corresponding to K986P and V987P in the Wuhan sequence (SEQ ID NO:1), respectively. In some embodiments, such an RNA vaccine sequence encoding an S protein from the Wuhan sequence with two proline mutations is set forth in SEQ ID NO:20 (and the corresponding nucleotide sequence encoding the S protein with two proline mutations is set forth in SEQ ID NO:9, and the amino acid sequence of the S protein with two proline mutations is set forth in SEQ ID NO:7). ... Such an RNA encoding an S protein from the 1 variant is set forth in SEQ ID NO:51 (the corresponding nucleotide sequence encoding the S protein from OmicronBA.1 with two proline mutations is set forth in SEQ ID NO:50, and the amino acid sequence of the S protein from OmicronBA.1 with two proline mutations is set forth in SEQ ID NO:49). In some embodiments, both types of RNA molecules may be formulated into lipid nanoparticles (LNPs) to form a bivalent vaccine (e.g., the two RNA populations are mixed prior to LNP formulation, or each RNA is formulated in a separate LNP composition and then mixed together).

As described herein, BNT162b5 bivalent (WT/OMI BA.2) refers to a composition comprising (i) RNA encoding an S protein from the Wuhan strain, and (ii) RNA encoding an S protein from the Omicron BA.2 variant, both S protein sequences containing (A) six proline mutations at positions corresponding to F817P, A892P, A899P, A942P, D985P, and V987P, (B) furin cleavage site mutations at positions corresponding to R682G, R683S, and R685S, and (C) a D614G mutation, all of which mutation positions are numbered relative to the Wuhan sequence (SEQ ID NO:1). In some embodiments, such an RNA sequence encoding an S protein from the Wuhan sequence having the aforementioned mutations (A), (B), and (C) is set forth in SEQ ID NO:83 (and the corresponding nucleotide sequence encoding an S protein having the aforementioned mutations (A), (B), and (C) is set forth in SEQ ID NO:81, and the amino acid sequence of the S protein having the aforementioned mutations (A), (B), and (C) is set forth in SEQ ID NO:80). In some embodiments, such an RNA sequence encoding an S protein from the Omicron BA.2 sequence having the aforementioned mutations (A), (B), and (C) is set forth in SEQ ID NO:98 (and the corresponding nucleotide sequence encoding an S protein from an Omicron BA.2 variant having the aforementioned mutations (A), (B), and (C) is set forth in SEQ ID NO:96, and the amino acid sequence of the S protein from an Omicron BA.2 variant having the aforementioned mutations (A), (B), and (C) is set forth in SEQ ID NO:95). In some embodiments, both types of RNA molecules may be formulated in lipid nanoparticles (LNPs) to form a bivalent vaccine (e.g., the two RNA populations are mixed prior to LNP formulation, or each RNA is formulated in a separate LNP composition and then mixed together).

In this study, subjects received a single 30 microgram dose of the investigational vaccine as described above, and blood samples were collected at the indicated times post-dose to assess neutralizing antibody titers against various SARS-CoV-2 strains and variants (including, for example, Wuhan, Omicron BA.1, Omicron BA.2, etc.).

Exemplary primary outcome measures:
● Percentage of participants reporting local reactions [time frame: 7 days after study vaccination] injection site pain, redness, and swelling self-reported in electronic diaries ● Percentage of participants reporting systemic events [time frame: 7 days after study vaccination] fever, fatigue, headache, chills, vomiting, diarrhea, new or worsening muscle pain, and new or worsening joint pain self-reported in electronic diaries ● Percentage of participants reporting adverse events [time frame: 1 month after study vaccination].
Percentage of participants reporting serious adverse events [time frame: 6 months after study vaccination].
• Geometric mean titers (GMT) of SARS-CoV-2 omicron (BA.2), omicron (BA.1), and reference strain neutralizing antibody levels for BNT162b5 bivalent (WT/OMI BA.2) 30 μg and BNT162b2 bivalent (WT/OMI BA.1) 30 μg measured in a central laboratory [time frame: pre-study vaccination and 1 week, 1 month, 3 months, and 6 months post-study vaccination].
Geometric mean fold increase (GMFR) of SARS-CoV-2 Omicron (BA.2), Omicron (BA.1), and reference strain neutralizing antibody levels for BNT162b5 bivalent (WT/OMI BA.2) 30 μg and BNT162b2 bivalent (WT/OMI BA.1) 30 μg. [Time frame: pre-study vaccination to 1 week, 1 month, 3 months, and 6 months post-study vaccination]
Percentage of participants with seroresponse to BNT162b5 bivalent (WT/OMI BA.2) 30 μg and BNT162b2 bivalent (WT/OMI BA.1) 30 μg for GMT of SARS-CoV-2 Omicron (BA.2), Omicron (BA.1), and reference strain neutralizing antibody levels. [Time frames: 1 week, 1 month, 3 months, 6 months after study vaccination]

As shown in Figure 44(A)-(C), neutralizing titers against the Wuhan strain were higher in subjects administered BNT162b5 compared to BNT162b2 one month after administration of the SARS-CoV-2 vaccine (GMFR 4.1 vs. 3.0 for all participants, GMFR 8.2 vs. 5.6 for subjects with no evidence of infection prior to vaccine administration, and GMFR 2.8 vs. 2.1 for subjects with evidence of prior infection). Similarly, neutralizing titers in subjects administered BNT162b5 were increased against the Omicron BA.1 (GMFR 5.5 vs. 4.2) and BA.2 (GMFR 5.1 vs. 3.2) variants. This effect was observed for all patients, regardless of prior infection status, with the benefit provided by BNT162b5 being particularly pronounced in subjects with no evidence of prior infection (see Figure 44C). Thus, this clinical trial data confirms the effects observed in previously described mouse and in vitro studies, specifically, BNT162b5 is able to induce improved immune responses and/or broader cross-neutralization compared to BNT162b2.

Example 20: Safety/immunogenicity study of a vaccine encoding the S protein of SARS-CoV-2 variants in healthy subjects The recent evolution of SARS-CoV-2 has led to the emergence of new viral variants with multiple mutations in the S protein, which may be associated with lower efficacy of some of the current vaccines. Thus, this study provides a new approach to overcome the decline in immunity and/or the development of modified vaccines.

This example describes a study evaluating the safety, tolerability, and immune response of an exemplary COVID-19 RNA vaccine as a booster to a COVID-19 vaccine in experienced healthy adults (e.g., age 56 years or older).

The COVID-19 RNA vaccines tested include the B162b5 bivalent vaccine alone or in combination with a T-string RNA construct (e.g., an RNA construct that includes a sequence encoding at least two or more T-cell epitopes). Exemplary T-string constructs are described, for example, in WO 2021/188969, the contents of each of which are incorporated herein by reference in their entirety.

In some embodiments, the B162b5 bivalent vaccine tested in this study is a composition comprising (i) RNA encoding an S protein from the Wuhan sequence, and (ii) RNA encoding an S protein from an omicron variant (e.g., BA.1, BA.2, or BA.4/5), both S protein sequences containing (A) six proline mutations corresponding to F817P, A892P, A899P, A942P, D985P, and V987P, respectively, (B) furin mutations at R682G, R683S, R685S, and (C) a D614G mutation, all of which mutation positions are numbered relative to the Wuhan sequence (SEQ ID NO:1). In some embodiments, both types of RNA molecules (i) and (ii) may be formulated in lipid nanoparticles (LNPs) to form a bivalent vaccine (e.g., the two RNA populations are mixed prior to LNP formulation, or each RNA is formulated in a separate LNP composition and subsequently mixed together).

In some embodiments, the RNA encoding the S protein from the Wuhan sequence used in this study is described in Example 19. In some embodiments, the RNA encoding the S protein from the Omicron BA.2 sequence used in this study is described in Example 19. In some embodiments, the RNA vaccine sequence encoding the S protein from the Omicron BA.1 sequence having the aforementioned mutations (A), (B), and (C) is described in SEQ ID NO:93 (and the corresponding nucleotide sequence encoding the S protein from the Omicron BA.1 sequence having the aforementioned mutations (A), (B), and (C) is described in SEQ ID NO:91, and the amino acid sequence of the S protein from the Omicron BA.1 variant having the aforementioned mutations (A), (B), and (C) is described in SEQ ID NO:90). ... The RNA vaccine sequence encoding the S protein from the OmicronBA.4/5 sequence is set forth in SEQ ID NO:103 (and the corresponding nucleotide sequence encoding the S protein from the OmicronBA.4/5 sequence having the aforementioned mutations (A), (B), and (C) is set forth in SEQ ID NO:101, and the amino acid sequence of the S protein from the OmicronBA.4/5 sequence having the aforementioned mutations (A), (B), and (C) is set forth in SEQ ID NO:100).

In some embodiments, subjects vaccinated with one of the COVID-19 RNA vaccine candidates described herein include patients who have received three doses of an approved vaccine (e.g., BNT162b2, Moderna COVID-19 vaccine, or other COVID mRNA or protein-based vaccine). In some embodiments, subjects are administered at least one dose of BNT162b5 (e.g., in some embodiments, 30 ug each), alone or in combination with a dose of a T-string construct (e.g., as described herein, e.g., an RNA encoding SEQ ID NO: RS C7p2full of WO2021/188969), e.g., a combination of BNT162b5 and an RNA encoding SEQ ID NO: RS C7p2full, for a total of up to about 100 ug RNA. In some embodiments, the subject is administered at least two doses of BNT162b5 (e.g., in some embodiments, 30 ug each), alone or in combination with a T-string construct (e.g., an RNA encoding SEQ ID NO: RS C7p2full as described herein, e.g., of WO2021/188969), e.g., a combination of each dose of BNT162b5 and an RNA encoding SEQ ID NO: RS C7p2full up to about 100 ug RNA total, the two doses being administered, e.g., at least 4 weeks or more apart (including, e.g., at least 5 weeks, at least 6 weeks, at least 7 weeks, at least 8 weeks, at least 9 weeks, at least 10 weeks, at least 11 weeks, or at least 12 weeks or more) from each other. In some embodiments, the subject is administered at least three doses of BNT162b5 (e.g., in some embodiments, 30ug each), alone or in combination with a T-string construct (e.g., an RNA encoding SEQ ID NO: RS C7p2full, as described herein, e.g., in WO2021/188969), e.g., a combination of each dose of BNT162b5 and an RNA encoding SEQ ID NO: RS C7p2full, up to about 100ug total RNA, wherein the first and second doses, and the second and third doses, are each independently administered at least 4 weeks or more apart (e.g., at least 5 weeks, at least 6 weeks, at least 7 weeks, at least 8 weeks, at least 9 weeks, at least 10 weeks, at least 11 weeks, or at least 12 weeks, or more).

In some embodiments, the dose is administered by intramuscular injection, for example, in the deltoid muscle of the subject's arm. In some embodiments, the combination of BNT162b5 and the T-string construct may be administered simultaneously as a single injection.

Exemplary characterization assays for evaluation:
Subjects are evaluated at various times during and after treatment for safety and any adverse reactions. Safety is assessed by physical evaluation: A (complete) physical examination includes at least skin, lymph node, cardiovascular, respiratory, gastrointestinal, and neurological evaluations. A brief (symptom-oriented) physical examination. A brief physical examination includes an overall health assessment. If there are obvious signs of pathology or if the subject shows any signs or symptoms, a detailed physical examination is required. Vital signs (including systolic/diastolic blood pressure, pulse rate, respiratory rate, and oral temperature) are evaluated at various times.

Electrocardiogram: A standard 12-lead ECG is recorded.

Evaluation of the intensity of local reactions: Pain (perceived) at the injection site is rated as absent, mild, moderate, or severe. Erythema/redness and induration/swelling are measured and then classified as absent, mild, moderate, or severe during analysis.

Assessment of the intensity of systemic reactions: Symptoms of systemic reactions are assessed as absent, mild, moderate, or severe. Investigations of "confirmed COVID-19" cases are also being conducted.

Serological testing for SARS-CoV-2 N-binding antibodies: At screening, blood will be collected for serological testing for SARS-CoV-2 N-binding antibodies by serum SARS-CoV-2 nucleocapsid protein-specific antibody immunoassay, the results of which may be useful for stratification.

SARS-CoV-2 SequencingSARS-CoV-2 Genome SequencingArchival swabs will be collected for SARS-CoV-2 genome sequencing. Outcomes will include SARS-CoV-2 S protein sequence and/or full genome sequence, assigned to known viral variants (if possible).

Adverse events of special interest (AESI), serious or non-serious, are among the scientific and medical concerns specific to this study. Exemplary AESIs include:
Myocarditis (all levels of certainty including "probable case" (1-3) according to the Brighton Collaboration Case Definition, https://brightoncollaboration.us/myocardis-case-definition-update/)
Pericarditis (all levels of certainty including "probable case" (1-3) according to the Brighton Collaboration Case Definition, https://brightoncollaboration.us/myocardis-case-definition-update/)
Anaphylaxis Thromboembolic events (e.g., deep vein thrombosis, stroke, myocardial infarction)
Immune thrombocytopenia Immune-based neurological events (e.g., optic neuropathy, Guillain-Barré syndrome)
Genetics: Blood samples (blood and/or isolated peripheral blood mononuclear cells) are collected. In some embodiments, these biological samples are used for human leukocyte antigen typing of the subject to allow additional analyses, e.g., characterization of T cell receptor and B cell receptor sequences, to characterize the fine specificity of the immune response. These blood samples can also be used to analyze the transcriptional response to administration of COVID-19 RNA vaccine candidates using RNA sequencing methods. Analysis of the immune response can provide a better understanding of the mechanisms underlying reactogenicity, as well as the induction of antibody and T cell responses.

Immune response: The study will evaluate immune responses at various time points.

Exemplary humoral immune response assessments may include:
Characterization of antigen-specific serum antibody titers, binding and functionality kinetics using e.g. ELISA and virus neutralization tests (VNT).
Cell-mediated immune response assessment includes:
Using antigen-derived peptide pools encoded by the RNA component of the vaccine, assessment of antigen-specific CD4 and CD8 T cells, including transcriptional composition profiling and functional characteristics such as effector function as measured by expression of cytokines such as (but not limited to) IL-2, IFN-γ, TNF-α, and the activation marker CD40L.
• Enumeration of antigen-specific IFN-γ secreting cells assessed using assays including intracellular cytokine staining and enzyme-linked immunosorbent spot (ELISpot) assay.

Additional characterization/assessment: Additional characterization/assessment may include, but is not limited to, phenotypic or functional characterization of antigen-specific T or B cells (e.g., by flow cytometry-based phenotyping including multimer staining), transcriptomic activity and analysis of T and B cell receptor repertoires (e.g., by next generation sequencing, single cell RNA sequencing), and multiplex cytokine analysis. Such analysis may also include human leukocyte antigen typing. Phenotypic or functional characterization of other immune cell populations that may be relevant to understanding vaccine-induced immune responses may also be included in the study.

Profiling of antibody Fc-mediated effector functions may be performed using a systems serology approach. This analysis may include, but is not limited to, evaluation of antigen-specific antibody affinity, isotype and subclass, antibody Fc receptor binding, and antibody functionality such as antibody-dependent cellular cytotoxicity, antibody-dependent natural killer cell activation, and antibody-dependent cellular phagocytosis (ADCP).

The studies may also include characterization of the molecular and cellular networks that influence innate and adaptive immunity to the antigens encoded by the RNA components of the vaccine. This analysis may include characterization of the transcriptional signatures induced by the antigens encoded by the RNA components of the vaccine by RNA sequencing (transcriptome analysis), complemented by evaluation of potential changes in cellular composition and levels of cytokines, chemokines or inflammatory markers induced by administration of the COVID-19 RNA vaccine candidate.

Example 21. Neutralization of Various SARS-CoV-2 VOCs by RNA Vaccines Encoding Exemplary Spike Protein Variants in Vaccine-Experienced Subjects This example describes the immunological impact of administering an RNA vaccine encoding an exemplary spike protein variant of a SARS-CoV-2 strain or VOC. In particular, this example describes the immunological impact of administering at least one dose (e.g., including at least two doses or at least three doses) to subjects (in this example, mice) who have previously received one or more doses of another SARS-CoV-2 vaccine delivering SARS-CoV-2 spike proteins including K986P and V987P (in this example, BNT162b2).

Mice were first immunized with two doses of BNT162b2 and then administered (i) a bivalent vaccine comprising two RNAs, each encoding a SARS-CoV-2 S protein containing the K986P and V987P mutations, where one of the RNAs encodes the SARS-CoV-2 S protein of the Wuhan strain and the other RNA encodes a SARS-CoV-2 S protein containing mutations characteristic of the Omicron BA.4/5 variant ("BNT162b2 bivalent BA.4/5" in FIG. 43); or (iii) a first RNA encoding a SARS-CoV-2 S protein of the Wuhan strain and a second RNA encoding a SARS-CoV-2 S protein containing mutations characteristic of the Omicron BA.4/5 variant ("BNT162b2 bivalent BA.4/5" in FIG. 43). A bivalent RNA vaccine was administered comprising a second RNA encoding an S protein, wherein each of the first and second RNAs comprised the P6′ set of mutations (D985P, V987P, F817P, A892P, A899P, A942P), D614G, and a furin cleavage site mutation (R682G, R683S, and R685S) (designated as “BNT162b5 bivalent BA.4/5” in FIG. 43 ). Mutations shown relative to the quality (e.g., SEQ ID NO:1). Animals were bled after administration of the third dose of vaccine and serum was analyzed to determine antibody immune responses, e.g., using a pseudovirus neutralization assay described herein. Specifically, as shown in FIG. 43, neutralizing antibody titers against Wuhan (WT) and Omicron variants BA.1, BA.2, BA.2.12.1, and BA.4/5 were evaluated for each RNA vaccine tested.

Similar to the results shown in FIG. 42, the results in FIG. 43 show that the P6', D614G, and furin cleavage site mutations can induce a stronger immune response compared to K986P and V987P in the context of a variant-adapted vaccine (e.g., an Omicron-adapted bivalent vaccine) administered as a booster dose to vaccine-experienced subjects. Again, a stronger immune response was found to be induced by BNT162b5 compared to BNT162b2 for each of the variants tested. The immune response was particularly improved for the Omicron BA.4/5 variant, where the combination of the P6', D614G, and furin cleavage site mutations induced an immune response approximately 2.8-fold higher than that induced by BNT162b2. Surprisingly, the immune response was higher against the unmatched variants (e.g., the immune response induced against the Omicron BA.1 variant was higher than against any variant in the BNT162b2 bivalent vaccine), demonstrating that the P6', D614G, and furin site mutations are able to induce a broader cross-neutralizing response than the variant-adapted version of BNT162b2.

The above experiment was repeated with additional spike protein variants. Specifically, in an empirical repeat, mice were first immunized with two doses of BNT162b2 and then administered one of the following vaccines as a third dose:
(i) BNT162b2
(ii) a bivalent vaccine comprising two RNAs, each encoding a SARS-CoV-2 S protein containing the K986P and V987P mutations, where one of the RNAs encodes the SARS-CoV-2 S protein of the Wuhan strain and the other RNA encodes a SARS-CoV-2 S protein containing mutations characteristic of the Omicron BA.4/5 variant ("BNT162b2 bivalent BA.4/5" in FIG. 43);
(iii) a bivalent RNA vaccine comprising a first RNA encoding a SARS-CoV-2 S protein of the Wuhan strain and a second RNA encoding a SARS-CoV-2 S protein comprising mutations characteristic of the Omicron BA.4/5 variant, wherein each of the first and second RNAs comprises a set of P6′ mutations (D985P, V987P, F817P, A892P, A899P, A942P), D614G, and a mutation at the furin cleavage site (R682G, R683S, and R685S) (designated as “BNT162b5 bivalent BA.4/5 in FIG. 43 ); or (iv) a bivalent RNA vaccine comprising a first RNA encoding a SARS-CoV-2 S protein of the Wuhan strain and a SARS-CoV-2 S protein comprising mutations characteristic of the Omicron BA.4/5 variant. and a second RNA encoding an S protein, wherein each of the first and second RNAs comprises a set of P6 mutations (K986P, V987P, F817P, A892P, A899P, A942P), D614G, mutations in the furin cleavage site (R682G, R683S, and R685S), and mutations to introduce disulfide bonds (T547C and N978C) (designated as "BNT162b6 Bivalent BA.4/5" in Figure 45).

Mutations shown compared to the Wuhan S protein (e.g., SEQ ID NO:1).

Again, serum was collected one month after the third dose was administered, and neutralizing antibody titers against Wuhan (wild type), delta variants, and omicron variants BA.1, BA.2, BA.2.12.1, BA.4/5, BA.4.6, BA.2.75.2, BQ.1.1, and XBB were assessed for each RNA vaccine tested, as shown in Figures 45(A) and (B). Titers are also shown in Table 33 below.

Figures 45(A) and (B), and the titers in Table 33 above, again demonstrate that RNA encoding a SARS-CoV-2 S protein containing the set of P6' mutations (D985P, V987P, F817P, A892P, A899P, A942P), D614G, and furin site mutations (R682G, R683S, R685S) can induce an improved immune response (e.g., higher neutralization titers) when administered to vaccine-experienced subjects, compared to RNA encoding a SARS-CoV-2 S protein containing K986P and V987P.

Example 22: Additional exemplary spike protein variants Additional spike proteins were expressed, purified, and characterized in vitro using the assays described in the previous examples. Table 34 (below) lists additional S protein variants that were expressed and purified, along with certain characteristic mutations present in the same.

Constructs #1-87 were expressed, purified, and characterized in vitro using the assays described in the previous examples. The results of these assays are shown in Figure 46.

Variants were also designed in which two or more cysteine residues were introduced to allow disulfide bond formation. Specifically, mutants were prepared that had any one of the following five interprotomer disulfide bonds: (i) A570C-N960C, (ii) A570C-K964C, (iii) A570C-S967C, (iv) T547C-N978C, and (v) T547C-S982C. The mutants also included a D614G mutation, a proline mutation, and a furin site mutation, and are listed in Table 35 below.

The sequences of the constructs provided in Table 34 above are shown in Table 36 below. Sequences are shown with and without certain modifications introduced for expression and purification purposes (modifications include, for example, C-terminal truncation folds, Precession protease cleavage sites, etc.). Modifications for expression are included in the upper sequence of each line and omitted in the lower sequence. Modifications for expression are also indicated in the legend provided with each line. Modifications introduced to improve immunogenicity are also indicated with each line and are the same for both the upper and lower sequences.

Based on the in vitro results shown in Figure 46, constructs 70, 41, 84, 57, 75, 50, and 85 in Table 34 were selected for in vivo immunogenicity testing. Construct 50 in Table 34 demonstrated the greatest immunogenicity of the variants tested.

Example 23. Neutralization of various SARS-CoV-2 VOCs by RNA vaccines encoding exemplary spike protein variants.
This example provides data demonstrating the immunogenicity of additional SARS-CoV-2 variants described herein, particularly certain exemplary constructs listed in Table 34 above.

The study described in this Example was conducted to evaluate the immunogenicity of a new modified mRNA construct design of the protein variant described in Example 22 above. The modRNA tested in this Example contained N1-methylpseudouracil and was encapsulated in lipid nanoparticles (LNPs). Groups of 10 unvaccinated mice each were immunized with the LNP formulation in a two-dose series. Serum was collected on days 29 and 49 for neutralizing antibody responses. As shown in Tables 37 and 38 below, the interprotomer A570C-N960C was shown to provide improved immunogenicity.

Claims (120)

1. A composition or medical preparation comprising RNA encoding a SARS-CoV-2 S polypeptide or an immunogenic fragment thereof, said SARS-CoV-2 S polypeptide or fragment comprising:
(a) an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO:1 and containing one of the following sets of amino acid substitutions relative to SEQ ID NO:1:
(1) D985P, V987P, F817P, A892P, A899P, and A942P;
(2) K986P, V987P, F817P, A892P, A899P, and A942P;
(3) D985P, V987P, F817P, A892P, A899P, A942P, D614G, R682G, R683S, and R685S;
(4) K986P, V987P, F817P, A892P, A899P, A942P, D614G, R682G, R683S, and R685S;
(5) D985P, K986P, V987P, F817P, A892P, A899P, A942P, D614G, R682G, R683S, and R685S;
(6) D985P, V987P, F817P, A892P, A899P, A942P, R682G, R683S, and R685S;
(7) K986P, V987P, F817P, A892P, A899P, A942P, R682G, R683S, and R685S; or (8) D985P, K986P, V987P, F817P, A892P, A899P, A942P, R682G, R683S, and R685S;
(b) an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO:69 and containing one of the following sets of amino acid substitutions relative to SEQ ID NO:69:
(1) D982P, V984P, F814P, A889P, A896P, and A939P;
(2) K983P, V984P, F814P, A889P, A896P, and A939P;
(3) D982P, V984P, F814P, A889P, A896P, A939P, R679G, R680S, and R682S;
(4) K983P, V984P, F814P, A889P, A896P, A939P, R679G, R680S, and R682S; or (5) D982P, K983P, V984P, F814P, A889P, A896P, A939P, R679G, R680S, and R682S;
(c) an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 70 and containing one of the following sets of amino acid substitutions relative to SEQ ID NO: 70:
(1) D982P, V984P, F814P, A889P, A896P, and A939P;
(2) K983P, V984P, F814P, A889P, A896P, and A939P;
(3) D982P, V984P, F814P, A889P, A896P, A939P, R679G, R680S, and R682S;
(4) K983P, V984P, F814P, A889P, A896P, A939P, R679G, R680S, and R682S; or (5) D982P, K983P, V984P, F814P, A889P, A896P, A939P, R679G, R680S, and R682S; or (d) an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 104 or 105 and including one of the following sets of amino acid substitutions relative to SEQ ID NO: 104 or 105:
(1) D980P, V982P, F812P, A887P, A894P, and A937P;
(2) K981P, V982P, F812P, A887P, A894P, and A937P;
(3) D980P, V982P, F812P, A887P, A894P, A937P, R677G, R678S, and R680S;
(4) K981P, V982P, F812P, A887P, A894P, A937P, R677G, R678S, and R680S; or (5) D980P, K981P, V982P, F812P, A887P, A894P, A937P, R677G, R678S, and R680S. The composition or medical preparation of claim 1, wherein the RNA comprises modified nucleosides in place of uridine. The composition or medical preparation of claim 1 or 2, wherein the RNA contains modified uridines in place of all uridines. The composition or medical preparation of any one of claims 1 to 3, wherein the RNA contains N1-methyl-pseudouridine (m1ψ) in place of all uridines. The composition or medical preparation of any one of claims 1 to 4, wherein the RNA comprises a 5' cap. 6. The composition or medical preparation of claim 5, wherein the 5' cap is or comprises m ₂ ^7,3'-O Gppp(m ₁ ^2'-O )ApG. The composition or medical preparation of any one of claims 1 to 6, wherein the RNA comprises a 5'-UTR that is or comprises a modified human alpha-globin 5'-UTR. The composition or medical preparation of any one of claims 1 to 7, wherein the RNA comprises a 5'UTR comprising a nucleotide sequence of SEQ ID NO:12 or a nucleotide sequence that is at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identical to the nucleotide sequence of SEQ ID NO:12. The composition or medical preparation of any one of claims 1 to 8, wherein the RNA comprises a 3'-UTR that is or includes a first sequence from a split amino-terminal enhancer (AES) messenger RNA and a second sequence from a mitochondrially encoded 12S ribosomal RNA. The composition or medical preparation of any one of claims 1 to 9, wherein the RNA comprises a 3'UTR comprising a nucleotide sequence of SEQ ID NO:13 or a nucleotide sequence that is at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identical to the nucleotide sequence of SEQ ID NO:13. The composition or medical preparation according to any one of claims 1 to 10, wherein the RNA comprises a polyA sequence. The composition or medical preparation of claim 11, wherein the polyA sequence comprises at least 100 nucleotides. The composition or medical preparation of claim 11 or 12, wherein the polyA sequence comprises 30 adenine nucleotides followed by 70 adenine nucleotides, the 30 adenine nucleotides and the 70 adenine nucleotides being separated by a linker sequence. The composition or medical preparation of any one of claims 11 to 13, wherein the polyA sequence comprises or consists of the nucleotide sequence of SEQ ID NO:14 or a nucleotide sequence that is at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identical to the nucleotide sequence of SEQ ID NO:14. A composition or medical preparation according to any one of claims 1 to 14, wherein the RNA is or is to be formulated for intramuscular administration. A composition or medical preparation according to any one of claims 1 to 15, wherein the RNA is or is to be formulated as a particle. The composition or medical preparation of claim 16, wherein the particle is a lipid nanoparticle (LNP) or lipoplex (LPX) particle. The composition or medical preparation of claim 17, wherein the LNP comprises a cationic ionizable lipid, a neutral lipid, a sterol, and a polymer-lipid conjugate. The composition or medical preparation of claim 17, wherein the lipoplex particles are obtainable by mixing the RNA with liposomes. The composition or medical preparation according to any one of claims 1 to 19, wherein the RNA is mRNA or saRNA. A composition or medical preparation according to any one of claims 1 to 20, which is a pharmaceutical composition. A composition or medical preparation according to any one of claims 1 to 21, which is a vaccine. The composition or medical preparation of claim 21 or 22, wherein the pharmaceutical composition further comprises one or more pharma- ceutically acceptable carriers, diluents, and/or excipients. 1. A composition or medical preparation comprising a first RNA encoding a first SARS-CoV-2 S polypeptide or an immunogenic fragment thereof, and a second RNA encoding a second SARS-CoV-2 S polypeptide or an immunogenic fragment thereof,
(a) the first SARS-CoV-2 S polypeptide or fragment comprises an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO:1;
(b) the second SARS-CoV-2 S polypeptide or fragment has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO:1 and the following set of amino acid substitutions relative to SEQ ID NO:1:
(1) D985P, V987P, F817P, A892P, A899P, and A942P;
(2) K986P, V987P, F817P, A892P, A899P, and A942P;
(3) D985P, V987P, F817P, A892P, A899P, A942P, D614G, R682G, R683S, and R685S;
(4) K986P, V987P, F817P, A892P, A899P, A942P, D614G, R682G, R683S, and R685S;
(5) D985P, K986P, V987P, F817P, A892P, A899P, A942P, D614G, R682G, R683S, and R685S;
(6) D985P, V987P, F817P, A892P, A899P, A942P, R682G, R683S, and R685S;
(7) K986P, V987P, F817P, A892P, A899P, A942P, R682G, R683S, and R685S; or (8) D985P, K986P, V987P, F817P, A892P, A899P, A942P, R682G, R683S, and R685S. 1. A composition or medical preparation comprising a first RNA encoding a first SARS-CoV-2 S polypeptide or an immunogenic fragment thereof, and a second RNA encoding a second SARS-CoV-2 S polypeptide or an immunogenic fragment thereof,
(a) the first SARS-CoV-2 S polypeptide or fragment comprises an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO:1;
(b) the second SARS-CoV-2 S polypeptide or fragment has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO:69 and has the following set of amino acid substitutions relative to SEQ ID NO:69:
(1) D982P, V984P, F814P, A889P, A896P, and A939P;
(2) K983P, V984P, F814P, A889P, A896P, and A939P;
(3) D982P, V984P, F814P, A889P, A896P, A939P, R679G, R680S, and R682S;
(4) K983P, V984P, F814P, A889P, A896P, A939P, R679G, R680S, and R682S; or (5) D982P, K983P, V984P, F814P, A889P, A896P, A939P, R679G, R680S, and R682S. 1. A composition or medical preparation comprising a first RNA encoding a first SARS-CoV-2 S polypeptide or an immunogenic fragment thereof, and a second RNA encoding a second SARS-CoV-2 S polypeptide or an immunogenic fragment thereof,
(a) the first SARS-CoV-2 S polypeptide or fragment comprises an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO:1;
(b) the second SARS-CoV-2 S polypeptide or fragment has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO:70 and has the following set of amino acid substitutions relative to SEQ ID NO:70:
(1) D982P, V984P, F814P, A889P, A896P, and A939P;
(2) K983P, V984P, F814P, A889P, A896P, and A939P;
(3) D982P, V984P, F814P, A889P, A896P, A939P, R679G, R680S, and R682S;
(4) K983P, V984P, F814P, A889P, A896P, A939P, R679G, R680S, and R682S; or (5) D982P, K983P, V984P, F814P, A889P, A896P, A939P, R679G, R680S, and R682S. 1. A composition or medical preparation comprising a first RNA encoding a first SARS-CoV-2 S polypeptide or an immunogenic fragment thereof, and a second RNA encoding a second SARS-CoV-2 S polypeptide or an immunogenic fragment thereof,
(a) the first SARS-CoV-2 S polypeptide or fragment comprises an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO:1;
(b) the second SARS-CoV-2 S polypeptide or fragment has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 104 or 105 and has the following set of amino acid substitutions relative to SEQ ID NO: 104 or 105:
(1) D980P, V982P, F812P, A887P, A894P, and A937P;
(2) K981P, V982P, F812P, A887P, A894P, and A937P;
(3) D980P, V982P, F812P, A887P, A894P, A937P, R677G, R678S, and R680S;
(4) K981P, V982P, F812P, A887P, A894P, A937P, R677G, R678S, and R680S; or (5) D980P, K981P, V982P, F812P, A887P, A894P, A937P, R677G, R678S, and R680S. 1. A composition or medical preparation comprising a first RNA encoding a first SARS-CoV-2 S polypeptide or an immunogenic fragment thereof, and a second RNA encoding a second SARS-CoV-2 S polypeptide or an immunogenic fragment thereof,
(a) the first SARS-CoV-2 S polypeptide or fragment comprises an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO:69;
(b) the second SARS-CoV-2 S polypeptide or fragment has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO:1 and the following set of amino acid substitutions relative to SEQ ID NO:1:
(1) D985P, V987P, F817P, A892P, A899P, and A942P;
(2) K986P, V987P, F817P, A892P, A899P, and A942P;
(3) D985P, V987P, F817P, A892P, A899P, A942P, D614G, R682G, R683S, and R685S;
(4) K986P, V987P, F817P, A892P, A899P, A942P, D614G, R682G, R683S, and R685S;
(5) D985P, K986P, V987P, F817P, A892P, A899P, A942P, D614G, R682G, R683S, and R685S;
(6) D985P, V987P, F817P, A892P, A899P, A942P, R682G, R683S, and R685S;
(7) K986P, V987P, F817P, A892P, A899P, A942P, R682G, R683S, and R685S; or (8) D985P, K986P, V987P, F817P, A892P, A899P, A942P, R682G, R683S, and R685S. 1. A composition or medical preparation comprising a first RNA encoding a first SARS-CoV-2 S polypeptide or an immunogenic fragment thereof, and a second RNA encoding a second SARS-CoV-2 S polypeptide or an immunogenic fragment thereof,
(a) the first SARS-CoV-2 S polypeptide or fragment comprises an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO:69;
(b) the second SARS-CoV-2 S polypeptide or fragment has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO:69 and has the following set of amino acid substitutions relative to SEQ ID NO:69:
(1) D982P, V984P, F814P, A889P, A896P, and A939P;
(2) K983P, V984P, F814P, A889P, A896P, and A939P;
(3) D982P, V984P, F814P, A889P, A896P, A939P, R679G, R680S, and R682S;
(4) K983P, V984P, F814P, A889P, A896P, A939P, R679G, R680S, and R682S; or (5) D982P, K983P, V984P, F814P, A889P, A896P, A939P, R679G, R680S, and R682S. 1. A composition or medical preparation comprising a first RNA encoding a first SARS-CoV-2 S polypeptide or an immunogenic fragment thereof, and a second RNA encoding a second SARS-CoV-2 S polypeptide or an immunogenic fragment thereof,
(a) the first SARS-CoV-2 S polypeptide or fragment comprises an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO:69;
(b) the second SARS-CoV-2 S polypeptide or fragment has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO:70 and has the following set of amino acid substitutions relative to SEQ ID NO:70:
(1) D982P, V984P, F814P, A889P, A896P, and A939P;
(2) K983P, V984P, F814P, A889P, A896P, and A939P;
(3) D982P, V984P, F814P, A889P, A896P, A939P, R679G, R680S, and R682S;
(4) K983P, V984P, F814P, A889P, A896P, A939P, R679G, R680S, and R682S; or (5) D982P, K983P, V984P, F814P, A889P, A896P, A939P, R679G, R680S, and R682S. 1. A composition or medical preparation comprising a first RNA encoding a first SARS-CoV-2 S polypeptide or an immunogenic fragment thereof, and a second RNA encoding a second SARS-CoV-2 S polypeptide or an immunogenic fragment thereof,
(a) the first SARS-CoV-2 S polypeptide or fragment comprises an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO:69;
(b) the second SARS-CoV-2 S polypeptide or fragment has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 104 or 105 and has the following set of amino acid substitutions relative to SEQ ID NO: 104 or 105:
(1) D980P, V982P, F812P, A887P, A894P, and A937P;
(2) K981P, V982P, F812P, A887P, A894P, and A937P;
(3) D980P, V982P, F812P, A887P, A894P, A937P, R677G, R678S, and R680S;
(4) K981P, V982P, F812P, A887P, A894P, A937P, R677G, R678S, and R680S; or (5) D980P, K981P, V982P, F812P, A887P, A894P, A937P, R677G, R678S, and R680S. 1. A composition or medical preparation comprising a first RNA encoding a first SARS-CoV-2 S polypeptide or an immunogenic fragment thereof, and a second RNA encoding a second SARS-CoV-2 S polypeptide or an immunogenic fragment thereof,
(a) the first SARS-CoV-2 S polypeptide or fragment comprises an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO:70;
(b) the second SARS-CoV-2 S polypeptide or fragment has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO:1 and the following set of amino acid substitutions relative to SEQ ID NO:1:
(1) D985P, V987P, F817P, A892P, A899P, and A942P;
(2) K986P, V987P, F817P, A892P, A899P, and A942P;
(3) D985P, V987P, F817P, A892P, A899P, A942P, D614G, R682G, R683S, and R685S;
(4) K986P, V987P, F817P, A892P, A899P, A942P, D614G, R682G, R683S, and R685S;
(5) D985P, K986P, V987P, F817P, A892P, A899P, A942P, D614G, R682G, R683S, and R685S;
(6) D985P, V987P, F817P, A892P, A899P, A942P, R682G, R683S, and R685S;
(7) K986P, V987P, F817P, A892P, A899P, A942P, R682G, R683S, and R685S; or (8) D985P, K986P, V987P, F817P, A892P, A899P, A942P, R682G, R683S, and R685S. 1. A composition or medical preparation comprising a first RNA encoding a first SARS-CoV-2 S polypeptide or an immunogenic fragment thereof, and a second RNA encoding a second SARS-CoV-2 S polypeptide or an immunogenic fragment thereof,
(a) the first SARS-CoV-2 S polypeptide or fragment comprises an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO:70;
(b) the second SARS-CoV-2 S polypeptide or fragment has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO:69 and has the following set of amino acid substitutions relative to SEQ ID NO:69:
(1) D982P, V984P, F814P, A889P, A896P, and A939P;
(2) K983P, V984P, F814P, A889P, A896P, and A939P;
(3) D982P, V984P, F814P, A889P, A896P, A939P, R679G, R680S, and R682S;
(4) K983P, V984P, F814P, A889P, A896P, A939P, R679G, R680S, and R682S; or (5) D982P, K983P, V984P, F814P, A889P, A896P, A939P, R679G, R680S, and R682S. 1. A composition or medical preparation comprising a first RNA encoding a first SARS-CoV-2 S polypeptide or an immunogenic fragment thereof, and a second RNA encoding a second SARS-CoV-2 S polypeptide or an immunogenic fragment thereof,
(a) the first SARS-CoV-2 S polypeptide or fragment comprises an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO:70;
(b) the second SARS-CoV-2 S polypeptide or fragment has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO:70 and has the following set of amino acid substitutions relative to SEQ ID NO:70:
(1) D982P, V984P, F814P, A889P, A896P, and A939P;
(2) K983P, V984P, F814P, A889P, A896P, and A939P;
(3) D982P, V984P, F814P, A889P, A896P, A939P, R679G, R680S, and R682S;
(4) K983P, V984P, F814P, A889P, A896P, A939P, R679G, R680S, and R682S; or (5) D982P, K983P, V984P, F814P, A889P, A896P, A939P, R679G, R680S, and R682S. 1. A composition or medical preparation comprising a first RNA encoding a first SARS-CoV-2 S polypeptide or an immunogenic fragment thereof, and a second RNA encoding a second SARS-CoV-2 S polypeptide or an immunogenic fragment thereof,
(a) the first SARS-CoV-2 S polypeptide or fragment comprises an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO:70;
(b) the second SARS-CoV-2 S polypeptide or fragment has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 104 or 105 and has the following set of amino acid substitutions relative to SEQ ID NO: 104 or 105:
(1) D980P, V982P, F812P, A887P, A894P, and A937P;
(2) K981P, V982P, F812P, A887P, A894P, and A937P;
(3) D980P, V982P, F812P, A887P, A894P, A937P, R677G, R678S, and R680S;
(4) K981P, V982P, F812P, A887P, A894P, A937P, R677G, R678S, and R680S; or (5) D980P, K981P, V982P, F812P, A887P, A894P, A937P, R677G, R678S, and R680S. 1. A composition or medical preparation comprising a first RNA encoding a first SARS-CoV-2 S polypeptide or an immunogenic fragment thereof, and a second RNA encoding a second SARS-CoV-2 S polypeptide or an immunogenic fragment thereof,
(a) the first SARS-CoV-2 S polypeptide or fragment comprises an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO:104 or SEQ ID NO:105;
(b) the second SARS-CoV-2 S polypeptide or fragment has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO:1 and the following set of amino acid substitutions relative to SEQ ID NO:1:
(1) D985P, V987P, F817P, A892P, A899P, and A942P;
(2) K986P, V987P, F817P, A892P, A899P, and A942P;
(3) D985P, V987P, F817P, A892P, A899P, A942P, D614G, R682G, R683S, and R685S;
(4) K986P, V987P, F817P, A892P, A899P, A942P, D614G, R682G, R683S, and R685S;
(5) D985P, K986P, V987P, F817P, A892P, A899P, A942P, D614G, R682G, R683S, and R685S;
(6) D985P, V987P, F817P, A892P, A899P, A942P, R682G, R683S, and R685S;
(7) K986P, V987P, F817P, A892P, A899P, A942P, R682G, R683S, and R685S; or (8) D985P, K986P, V987P, F817P, A892P, A899P, A942P, R682G, R683S, and R685S. 1. A composition or medical preparation comprising a first RNA encoding a first SARS-CoV-2 S polypeptide or an immunogenic fragment thereof, and a second RNA encoding a second SARS-CoV-2 S polypeptide or an immunogenic fragment thereof,
(a) the first SARS-CoV-2 S polypeptide or fragment comprises an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO:104 or SEQ ID NO:105;
(b) the second SARS-CoV-2 S polypeptide or fragment has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO:69 and has the following set of amino acid substitutions relative to SEQ ID NO:69:
(1) D982P, V984P, F814P, A889P, A896P, and A939P;
(2) K983P, V984P, F814P, A889P, A896P, and A939P;
(3) D982P, V984P, F814P, A889P, A896P, A939P, R679G, R680S, and R682S;
(4) K983P, V984P, F814P, A889P, A896P, A939P, R679G, R680S, and R682S; or (5) D982P, K983P, V984P, F814P, A889P, A896P, A939P, R679G, R680S, and R682S. 1. A composition or medical preparation comprising a first RNA encoding a first SARS-CoV-2 S polypeptide or an immunogenic fragment thereof, and a second RNA encoding a second SARS-CoV-2 S polypeptide or an immunogenic fragment thereof,
(a) the first SARS-CoV-2 S polypeptide or fragment comprises an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO:104 or SEQ ID NO:105;
(b) the second SARS-CoV-2 S polypeptide or fragment has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO:70 and has the following set of amino acid substitutions relative to SEQ ID NO:70:
(1) D982P, V984P, F814P, A889P, A896P, and A939P;
(2) K983P, V984P, F814P, A889P, A896P, and A939P;
(3) D982P, V984P, F814P, A889P, A896P, A939P, R679G, R680S, and R682S;
(4) K983P, V984P, F814P, A889P, A896P, A939P, R679G, R680S, and R682S; or (5) D982P, K983P, V984P, F814P, A889P, A896P, A939P, R679G, R680S, and R682S. 1. A composition or medical preparation comprising a first RNA encoding a first SARS-CoV-2 S polypeptide or an immunogenic fragment thereof, and a second RNA encoding a second SARS-CoV-2 S polypeptide or an immunogenic fragment thereof,
(a) the first SARS-CoV-2 S polypeptide or fragment comprises an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO:104 or SEQ ID NO:105;
(b) the second SARS-CoV-2 S polypeptide or fragment has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 104 or 105 and has the following set of amino acid substitutions relative to SEQ ID NO: 104 or 105:
(1) D980P, V982P, F812P, A887P, A894P, and A937P;
(2) K981P, V982P, F812P, A887P, A894P, and A937P;
(3) D980P, V982P, F812P, A887P, A894P, A937P, R677G, R678S, and R680S;
(4) K981P, V982P, F812P, A887P, A894P, A937P, R677G, R678S, and R680S; or (5) D980P, K981P, V982P, F812P, A887P, A894P, A937P, R677G, R678S, and R680S. 1. A composition or medical preparation comprising a first RNA encoding a first SARS-CoV-2 S polypeptide or an immunogenic fragment thereof, and a second RNA encoding a second SARS-CoV-2 S polypeptide or an immunogenic fragment thereof,
(a) the first SARS-CoV-2 S polypeptide or fragment comprises an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO:1 and including the following substitutions relative to SEQ ID NO:1: D985P, V987P, F817P, A892P, A899P, and A942P;
(b) the second SARS-CoV-2 S polypeptide or fragment has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO:69 and has the following set of amino acid substitutions relative to SEQ ID NO:69:
(1) D982P, V984P, F814P, A889P, A896P, and A939P;
(2) K983P, V984P, F814P, A889P, A896P, and A939P;
(3) D982P, V984P, F814P, A889P, A896P, A939P, R679G, R680S, and R682S;
(4) K983P, V984P, F814P, A889P, A896P, A939P, R679G, R680S, and R682S; or (5) D982P, K983P, V984P, F814P, A889P, A896P, A939P, R679G, R680S, and R682S. 1. A composition or medical preparation comprising a first RNA encoding a first SARS-CoV-2 S polypeptide or an immunogenic fragment thereof, and a second RNA encoding a second SARS-CoV-2 S polypeptide or an immunogenic fragment thereof,
(a) the first SARS-CoV-2 S polypeptide or fragment comprises an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO:1 and including the following substitutions relative to SEQ ID NO:1: K986P, V987P, F817P, A892P, A899P, and A942P;
(b) the second SARS-CoV-2 S polypeptide or fragment has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO:69 and has the following set of amino acid substitutions relative to SEQ ID NO:69:
(1) D982P, V984P, F814P, A889P, A896P, and A939P;
(2) K983P, V984P, F814P, A889P, A896P, and A939P;
(3) D982P, V984P, F814P, A889P, A896P, A939P, R679G, R680S, and R682S;
(4) K983P, V984P, F814P, A889P, A896P, A939P, R679G, R680S, and R682S; or (5) D982P, K983P, V984P, F814P, A889P, A896P, A939P, R679G, R680S, and R682S. 1. A composition or medical preparation comprising a first RNA encoding a first SARS-CoV-2 S polypeptide or an immunogenic fragment thereof, and a second RNA encoding a second SARS-CoV-2 S polypeptide or an immunogenic fragment thereof,
(a) the first SARS-CoV-2 S polypeptide or fragment comprises an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO:1 and including the following substitutions relative to SEQ ID NO:1: D985P, V987P, F817P, A892P, A899P, A942P, D614G, R682G, R683S, and R685S;
(b) the second SARS-CoV-2 S polypeptide or fragment has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO:69 and has the following set of amino acid substitutions relative to SEQ ID NO:69:
(1) D982P, V984P, F814P, A889P, A896P, and A939P;
(2) K983P, V984P, F814P, A889P, A896P, and A939P;
(3) D982P, V984P, F814P, A889P, A896P, A939P, R679G, R680S, and R682S;
(4) K983P, V984P, F814P, A889P, A896P, A939P, R679G, R680S, and R682S; or (5) D982P, K983P, V984P, F814P, A889P, A896P, A939P, R679G, R680S, and R682S. 1. A composition or medical preparation comprising a first RNA encoding a first SARS-CoV-2 S polypeptide or an immunogenic fragment thereof, and a second RNA encoding a second SARS-CoV-2 S polypeptide or an immunogenic fragment thereof,
(a) the first SARS-CoV-2 S polypeptide or fragment comprises an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO:1 and including the following substitutions relative to SEQ ID NO:1: K986P, V987P, F817P, A892P, A899P, A942P, D614G, R682G, R683S, and R685S;
(b) the second SARS-CoV-2 S polypeptide or fragment has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO:69 and has the following set of amino acid substitutions relative to SEQ ID NO:69:
(1) D982P, V984P, F814P, A889P, A896P, and A939P;
(2) K983P, V984P, F814P, A889P, A896P, and A939P;
(3) D982P, V984P, F814P, A889P, A896P, A939P, R679G, R680S, and R682S;
(4) K983P, V984P, F814P, A889P, A896P, A939P, R679G, R680S, and R682S; or (5) D982P, K983P, V984P, F814P, A889P, A896P, A939P, R679G, R680S, and R682S. 1. A composition or medical preparation comprising a first RNA encoding a first SARS-CoV-2 S polypeptide or an immunogenic fragment thereof, and a second RNA encoding a second SARS-CoV-2 S polypeptide or an immunogenic fragment thereof,
(a) the first SARS-CoV-2 S polypeptide or fragment comprises an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO:1 and including the following substitutions relative to SEQ ID NO:1: D985P, K986P, V987P, F817P, A892P, A899P, A942P, D614G, R682G, R683S, and R685S;
(b) the second SARS-CoV-2 S polypeptide or fragment has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO:69 and has the following set of amino acid substitutions relative to SEQ ID NO:69:
(1) D982P, V984P, F814P, A889P, A896P, and A939P;
(2) K983P, V984P, F814P, A889P, A896P, and A939P;
(3) D982P, V984P, F814P, A889P, A896P, A939P, R679G, R680S, and R682S;
(4) K983P, V984P, F814P, A889P, A896P, A939P, R679G, R680S, and R682S; or (5) D982P, K983P, V984P, F814P, A889P, A896P, A939P, R679G, R680S, and R682S. 1. A composition or medical preparation comprising a first RNA encoding a first SARS-CoV-2 S polypeptide or an immunogenic fragment thereof, and a second RNA encoding a second SARS-CoV-2 S polypeptide or an immunogenic fragment thereof,
(a) the first SARS-CoV-2 S polypeptide or fragment comprises an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO:1 and including the following substitutions relative to SEQ ID NO:1: D985P, V987P, F817P, A892P, A899P, A942P, R682G, R683S, and R685S;
(b) the second SARS-CoV-2 S polypeptide or fragment has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO:69 and has the following set of amino acid substitutions relative to SEQ ID NO:69:
(1) D982P, V984P, F814P, A889P, A896P, and A939P;
(2) K983P, V984P, F814P, A889P, A896P, and A939P;
(3) D982P, V984P, F814P, A889P, A896P, A939P, R679G, R680S, and R682S;
(4) K983P, V984P, F814P, A889P, A896P, A939P, R679G, R680S, and R682S; or (5) D982P, K983P, V984P, F814P, A889P, A896P, A939P, R679G, R680S, and R682S. 1. A composition or medical preparation comprising a first RNA encoding a first SARS-CoV-2 S polypeptide or an immunogenic fragment thereof, and a second RNA encoding a second SARS-CoV-2 S polypeptide or an immunogenic fragment thereof,
(a) the first SARS-CoV-2 S polypeptide or fragment comprises an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO:1 and including the following substitutions relative to SEQ ID NO:1: K986P, V987P, F817P, A892P, A899P, A942P, R682G, R683S, and R685S;
(b) the second SARS-CoV-2 S polypeptide or fragment has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO:69 and has the following set of amino acid substitutions relative to SEQ ID NO:69:
(1) D982P, V984P, F814P, A889P, A896P, and A939P;
(2) K983P, V984P, F814P, A889P, A896P, and A939P;
(3) D982P, V984P, F814P, A889P, A896P, A939P, R679G, R680S, and R682S;
(4) K983P, V984P, F814P, A889P, A896P, A939P, R679G, R680S, and R682S; or (5) D982P, K983P, V984P, F814P, A889P, A896P, A939P, R679G, R680S, and R682S. 1. A composition or medical preparation comprising a first RNA encoding a first SARS-CoV-2 S polypeptide or an immunogenic fragment thereof, and a second RNA encoding a second SARS-CoV-2 S polypeptide or an immunogenic fragment thereof,
(a) the first SARS-CoV-2 S polypeptide or fragment comprises an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO:1 and including the following substitutions relative to SEQ ID NO:1: D985P, K986P, V987P, F817P, A892P, A899P, A942P, R682G, R683S, R685S;
(b) the second SARS-CoV-2 S polypeptide or fragment has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO:69 and has the following set of amino acid substitutions relative to SEQ ID NO:69:
(1) D982P, V984P, F814P, A889P, A896P, and A939P;
(2) K983P, V984P, F814P, A889P, A896P, and A939P;
(3) D982P, V984P, F814P, A889P, A896P, A939P, R679G, R680S, and R682S;
(4) K983P, V984P, F814P, A889P, A896P, A939P, R679G, R680S, and R682S; or (5) D982P, K983P, V984P, F814P, A889P, A896P, A939P, R679G, R680S, and R682S. 1. A composition or medical preparation comprising a first RNA encoding a first SARS-CoV-2 S polypeptide or an immunogenic fragment thereof, and a second RNA encoding a second SARS-CoV-2 S polypeptide or an immunogenic fragment thereof,
(a) the first SARS-CoV-2 S polypeptide or fragment comprises an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO:1 and including the following substitutions relative to SEQ ID NO:1: D985P, V987P, F817P, A892P, A899P, and A942P;
(b) the second SARS-CoV-2 S polypeptide or fragment has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO:70 and has the following set of amino acid substitutions relative to SEQ ID NO:70:
(1) D982P, V984P, F814P, A889P, A896P, and A939P;
(2) K983P, V984P, F814P, A889P, A896P, and A939P;
(3) D982P, V984P, F814P, A889P, A896P, A939P, R679G, R680S, and R682S;
(4) K983P, V984P, F814P, A889P, A896P, A939P, R679G, R680S, and R682S; or (5) D982P, K983P, V984P, F814P, A889P, A896P, A939P, R679G, R680S, and R682S. 1. A composition or medical preparation comprising a first RNA encoding a first SARS-CoV-2 S polypeptide or an immunogenic fragment thereof, and a second RNA encoding a second SARS-CoV-2 S polypeptide or an immunogenic fragment thereof,
(a) the first SARS-CoV-2 S polypeptide or fragment comprises an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO:1 and including the following substitutions relative to SEQ ID NO:1: K986P, V987P, F817P, A892P, A899P, and A942P;
(b) the second SARS-CoV-2 S polypeptide or fragment has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO:70 and has the following set of amino acid substitutions relative to SEQ ID NO:70:
(1) D982P, V984P, F814P, A889P, A896P, and A939P;
(2) K983P, V984P, F814P, A889P, A896P, and A939P;
(3) D982P, V984P, F814P, A889P, A896P, A939P, R679G, R680S, and R682S;
(4) K983P, V984P, F814P, A889P, A896P, A939P, R679G, R680S, and R682S; or (5) D982P, K983P, V984P, F814P, A889P, A896P, A939P, R679G, R680S, and R682S. 1. A composition or medical preparation comprising a first RNA encoding a first SARS-CoV-2 S polypeptide or an immunogenic fragment thereof, and a second RNA encoding a second SARS-CoV-2 S polypeptide or an immunogenic fragment thereof,
(a) the first SARS-CoV-2 S polypeptide or fragment comprises an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO:1 and including the following substitutions relative to SEQ ID NO:1: D985P, V987P, F817P, A892P, A899P, A942P, D614G, R682G, R683S, and R685S;
(b) the second SARS-CoV-2 S polypeptide or fragment has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO:70 and has the following set of amino acid substitutions relative to SEQ ID NO:70:
(1) D982P, V984P, F814P, A889P, A896P, and A939P;
(2) K983P, V984P, F814P, A889P, A896P, and A939P;
(3) D982P, V984P, F814P, A889P, A896P, A939P, R679G, R680S, and R682S;
(4) K983P, V984P, F814P, A889P, A896P, A939P, R679G, R680S, and R682S; or (5) D982P, K983P, V984P, F814P, A889P, A896P, A939P, R679G, R680S, and R682S. 1. A composition or medical preparation comprising a first RNA encoding a first SARS-CoV-2 S polypeptide or an immunogenic fragment thereof, and a second RNA encoding a second SARS-CoV-2 S polypeptide or an immunogenic fragment thereof,
(a) the first SARS-CoV-2 S polypeptide or fragment comprises an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO:1 and including the following substitutions relative to SEQ ID NO:1: K986P, V987P, F817P, A892P, A899P, A942P, D614G, R682G, R683S, and R685S;
(b) the second SARS-CoV-2 S polypeptide or fragment has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO:70 and has the following set of amino acid substitutions relative to SEQ ID NO:70:
(1) D982P, V984P, F814P, A889P, A896P, and A939P;
(2) K983P, V984P, F814P, A889P, A896P, and A939P;
(3) D982P, V984P, F814P, A889P, A896P, A939P, R679G, R680S, and R682S;
(4) K983P, V984P, F814P, A889P, A896P, A939P, R679G, R680S, and R682S; or (5) D982P, K983P, V984P, F814P, A889P, A896P, A939P, R679G, R680S, and R682S. 1. A composition or medical preparation comprising a first RNA encoding a first SARS-CoV-2 S polypeptide or an immunogenic fragment thereof, and a second RNA encoding a second SARS-CoV-2 S polypeptide or an immunogenic fragment thereof,
(a) the first SARS-CoV-2 S polypeptide or fragment comprises an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO:1 and including the following substitutions relative to SEQ ID NO:1: D985P, K986P, V987P, F817P, A892P, A899P, A942P, D614G, R682G, R683S, and R685S;
(b) the second SARS-CoV-2 S polypeptide or fragment has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO:70 and has the following set of amino acid substitutions relative to SEQ ID NO:70:
(1) D982P, V984P, F814P, A889P, A896P, and A939P;
(2) K983P, V984P, F814P, A889P, A896P, and A939P;
(3) D982P, V984P, F814P, A889P, A896P, A939P, R679G, R680S, and R682S;
(4) K983P, V984P, F814P, A889P, A896P, A939P, R679G, R680S, and R682S; or (5) D982P, K983P, V984P, F814P, A889P, A896P, A939P, R679G, R680S, and R682S. 1. A composition or medical preparation comprising a first RNA encoding a first SARS-CoV-2 S polypeptide or an immunogenic fragment thereof, and a second RNA encoding a second SARS-CoV-2 S polypeptide or an immunogenic fragment thereof,
(a) the first SARS-CoV-2 S polypeptide or fragment comprises an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO:1 and including the following substitutions relative to SEQ ID NO:1: D985P, V987P, F817P, A892P, A899P, A942P, R682G, R683S, and R685S;
(b) the second SARS-CoV-2 S polypeptide or fragment has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO:70 and has the following set of amino acid substitutions relative to SEQ ID NO:70:
(1) D982P, V984P, F814P, A889P, A896P, and A939P;
(2) K983P, V984P, F814P, A889P, A896P, and A939P;
(3) D982P, V984P, F814P, A889P, A896P, A939P, R679G, R680S, and R682S;
(4) K983P, V984P, F814P, A889P, A896P, A939P, R679G, R680S, and R682S; or (5) D982P, K983P, V984P, F814P, A889P, A896P, A939P, R679G, R680S, and R682S. 1. A composition or medical preparation comprising a first RNA encoding a first SARS-CoV-2 S polypeptide or an immunogenic fragment thereof, and a second RNA encoding a second SARS-CoV-2 S polypeptide or an immunogenic fragment thereof,
(a) the first SARS-CoV-2 S polypeptide or fragment comprises an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO:1 and including the following substitutions relative to SEQ ID NO:1: K986P, V987P, F817P, A892P, A899P, A942P, R682G, R683S, and R685S;
(b) the second SARS-CoV-2 S polypeptide or fragment has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO:70 and has the following set of amino acid substitutions relative to SEQ ID NO:70:
(1) D982P, V984P, F814P, A889P, A896P, and A939P;
(2) K983P, V984P, F814P, A889P, A896P, and A939P;
(3) D982P, V984P, F814P, A889P, A896P, A939P, R679G, R680S, and R682S;
(4) K983P, V984P, F814P, A889P, A896P, A939P, R679G, R680S, and R682S; or (5) D982P, K983P, V984P, F814P, A889P, A896P, A939P, R679G, R680S, and R682S. 1. A composition or medical preparation comprising a first RNA encoding a first SARS-CoV-2 S polypeptide or an immunogenic fragment thereof, and a second RNA encoding a second SARS-CoV-2 S polypeptide or an immunogenic fragment thereof,
(a) the first SARS-CoV-2 S polypeptide or fragment comprises an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO:1 and including the following substitutions relative to SEQ ID NO:1: D985P, K986P, V987P, F817P, A892P, A899P, A942P, R682G, R683S, and R685S;
(b) the second SARS-CoV-2 S polypeptide or fragment has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO:70 and has the following set of amino acid substitutions relative to SEQ ID NO:70:
(1) D982P, V984P, F814P, A889P, A896P, and A939P;
(2) K983P, V984P, F814P, A889P, A896P, and A939P;
(3) D982P, V984P, F814P, A889P, A896P, A939P, R679G, R680S, and R682S;
(4) K983P, V984P, F814P, A889P, A896P, A939P, R679G, R680S, and R682S; or (5) D982P, K983P, V984P, F814P, A889P, A896P, A939P, R679G, R680S, and R682S. 1. A composition or medical preparation comprising a first RNA encoding a first SARS-CoV-2 S polypeptide or an immunogenic fragment thereof, and a second RNA encoding a second SARS-CoV-2 S polypeptide or an immunogenic fragment thereof,
(a) the first SARS-CoV-2 S polypeptide or fragment comprises an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO:1 and including the following substitutions relative to SEQ ID NO:1: D985P, V987P, F817P, A892P, A899P, and A942P;
(b) the second SARS-CoV-2 S polypeptide or fragment has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 104 or 105 and has the following set of amino acid substitutions relative to SEQ ID NO: 104 or 105:
(1) D980P, V982P, F812P, A887P, A894P, and A937P;
(2) K981P, V982P, F812P, A887P, A894P, and A937P;
(3) D980P, V982P, F812P, A887P, A894P, A937P, R677G, R678S, and R680S;
(4) K981P, V982P, F812P, A887P, A894P, A937P, R677G, R678S, and R680S; or (5) D980P, K981P, V982P, F812P, A887P, A894P, A937P, R677G, R678S, and R680S. 1. A composition or medical preparation comprising a first RNA encoding a first SARS-CoV-2 S polypeptide or an immunogenic fragment thereof, and a second RNA encoding a second SARS-CoV-2 S polypeptide or an immunogenic fragment thereof,
(a) the first SARS-CoV-2 S polypeptide or fragment comprises an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO:1 and including the following substitutions relative to SEQ ID NO:1: K986P, V987P, F817P, A892P, A899P, and A942P;
(b) the second SARS-CoV-2 S polypeptide or fragment has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 104 or 105 and has the following set of amino acid substitutions relative to SEQ ID NO: 104 or 105:
(1) D980P, V982P, F812P, A887P, A894P, and A937P;
(2) K981P, V982P, F812P, A887P, A894P, and A937P;
(3) D980P, V982P, F812P, A887P, A894P, A937P, R677G, R678S, and R680S;
(4) K981P, V982P, F812P, A887P, A894P, A937P, R677G, R678S, and R680S; or (5) D980P, K981P, V982P, F812P, A887P, A894P, A937P, R677G, R678S, and R680S. 1. A composition or medical preparation comprising a first RNA encoding a first SARS-CoV-2 S polypeptide or an immunogenic fragment thereof, and a second RNA encoding a second SARS-CoV-2 S polypeptide or an immunogenic fragment thereof,
(a) the first SARS-CoV-2 S polypeptide or fragment comprises an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO:1 and including the following substitutions relative to SEQ ID NO:1: D985P, V987P, F817P, A892P, A899P, A942P, D614G, R682G, R683S, and R685S;
(b) the second SARS-CoV-2 S polypeptide or fragment has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 104 or 105 and has the following set of amino acid substitutions relative to SEQ ID NO: 104 or 105:
(1) D980P, V982P, F812P, A887P, A894P, and A937P;
(2) K981P, V982P, F812P, A887P, A894P, and A937P;
(3) D980P, V982P, F812P, A887P, A894P, A937P, R677G, R678S, and R680S;
(4) K981P, V982P, F812P, A887P, A894P, A937P, R677G, R678S, and R680S; or (5) D980P, K981P, V982P, F812P, A887P, A894P, A937P, R677G, R678S, and R680S. 1. A composition or medical preparation comprising a first RNA encoding a first SARS-CoV-2 S polypeptide or an immunogenic fragment thereof, and a second RNA encoding a second SARS-CoV-2 S polypeptide or an immunogenic fragment thereof,
(a) the first SARS-CoV-2 S polypeptide or fragment comprises an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO:1 and including the following substitutions relative to SEQ ID NO:1: K986P, V987P, F817P, A892P, A899P, A942P, D614G, R682G, R683S, and R685S;
(b) the second SARS-CoV-2 S polypeptide or fragment has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 104 or 105 and has the following set of amino acid substitutions relative to SEQ ID NO: 104 or 105:
(1) D980P, V982P, F812P, A887P, A894P, and A937P;
(2) K981P, V982P, F812P, A887P, A894P, and A937P;
(3) D980P, V982P, F812P, A887P, A894P, A937P, R677G, R678S, and R680S;
(4) K981P, V982P, F812P, A887P, A894P, A937P, R677G, R678S, and R680S; or (5) D980P, K981P, V982P, F812P, A887P, A894P, A937P, R677G, R678S, and R680S. 1. A composition or medical preparation comprising a first RNA encoding a first SARS-CoV-2 S polypeptide or an immunogenic fragment thereof, and a second RNA encoding a second SARS-CoV-2 S polypeptide or an immunogenic fragment thereof,
(a) the first SARS-CoV-2 S polypeptide or fragment comprises an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO:1 and including the following substitutions relative to SEQ ID NO:1: D985P, K986P, V987P, F817P, A892P, A899P, A942P, D614G, R682G, R683S, and R685S;
(b) the second SARS-CoV-2 S polypeptide or fragment has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 104 or 105 and has the following set of amino acid substitutions relative to SEQ ID NO: 104 or 105:
(1) D980P, V982P, F812P, A887P, A894P, and A937P;
(2) K981P, V982P, F812P, A887P, A894P, and A937P;
(3) D980P, V982P, F812P, A887P, A894P, A937P, R677G, R678S, and R680S;
(4) K981P, V982P, F812P, A887P, A894P, A937P, R677G, R678S, and R680S; or (5) D980P, K981P, V982P, F812P, A887P, A894P, A937P, R677G, R678S, and R680S. 1. A composition or medical preparation comprising a first RNA encoding a first SARS-CoV-2 S polypeptide or an immunogenic fragment thereof, and a second RNA encoding a second SARS-CoV-2 S polypeptide or an immunogenic fragment thereof,
(a) the first SARS-CoV-2 S polypeptide or fragment comprises an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO:1 and including the following substitutions relative to SEQ ID NO:1: D985P, V987P, F817P, A892P, A899P, A942P, R682G, R683S, and R685S;
(b) the second SARS-CoV-2 S polypeptide or fragment has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 104 or 105 and has the following set of amino acid substitutions relative to SEQ ID NO: 104 or 105:
(1) D980P, V982P, F812P, A887P, A894P, and A937P;
(2) K981P, V982P, F812P, A887P, A894P, and A937P;
(3) D980P, V982P, F812P, A887P, A894P, A937P, R677G, R678S, and R680S;
(4) K981P, V982P, F812P, A887P, A894P, A937P, R677G, R678S, and R680S; or (5) D980P, K981P, V982P, F812P, A887P, A894P, A937P, R677G, R678S, and R680S. 1. A composition or medical preparation comprising a first RNA encoding a first SARS-CoV-2 S polypeptide or an immunogenic fragment thereof, and a second RNA encoding a second SARS-CoV-2 S polypeptide or an immunogenic fragment thereof,
(a) the first SARS-CoV-2 S polypeptide or fragment comprises an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO:1 and including the following substitutions relative to SEQ ID NO:1: K986P, V987P, F817P, A892P, A899P, A942P, R682G, R683S, and R685S;
(b) the second SARS-CoV-2 S polypeptide or fragment has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 104 or 105 and has the following set of amino acid substitutions relative to SEQ ID NO: 104 or 105:
(1) D980P, V982P, F812P, A887P, A894P, and A937P;
(2) K981P, V982P, F812P, A887P, A894P, and A937P;
(3) D980P, V982P, F812P, A887P, A894P, A937P, R677G, R678S, and R680S;
(4) K981P, V982P, F812P, A887P, A894P, A937P, R677G, R678S, and R680S; or (5) D980P, K981P, V982P, F812P, A887P, A894P, A937P, R677G, R678S, and R680S. 1. A composition or medical preparation comprising a first RNA encoding a first SARS-CoV-2 S polypeptide or an immunogenic fragment thereof, and a second RNA encoding a second SARS-CoV-2 S polypeptide or an immunogenic fragment thereof,
(a) the first SARS-CoV-2 S polypeptide or fragment comprises an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO:1 and including the following substitutions relative to SEQ ID NO:1: D985P, K986P, V987P, F817P, A892P, A899P, A942P, R682G, R683S, and R685S;
(b) the second SARS-CoV-2 S polypeptide or fragment has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 104 or 105 and has the following set of amino acid substitutions relative to SEQ ID NO: 104 or 105:
(1) D980P, V982P, F812P, A887P, A894P, and A937P;
(2) K981P, V982P, F812P, A887P, A894P, and A937P;
(3) D980P, V982P, F812P, A887P, A894P, A937P, R677G, R678S, and R680S;
(4) K981P, V982P, F812P, A887P, A894P, A937P, R677G, R678S, and R680S; or (5) D980P, K981P, V982P, F812P, A887P, A894P, A937P, R677G, R678S, and R680S. 1. A composition or medical preparation comprising a first RNA encoding a first SARS-CoV-2 S polypeptide or an immunogenic fragment thereof, and a second RNA encoding a second SARS-CoV-2 S polypeptide or an immunogenic fragment thereof,
(a) the first SARS-CoV-2 S polypeptide or fragment comprises an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO:69 and including the following substitutions relative to SEQ ID NO:69: D982P, V984P, F814P, A889P, A896P, and A939P;
(b) the second SARS-CoV-2 S polypeptide or fragment has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO:70 and has the following set of amino acid substitutions relative to SEQ ID NO:70:
(1) D982P, V984P, F814P, A889P, A896P, and A939P;
(2) K983P, V984P, F814P, A889P, A896P, and A939P;
(3) D982P, V984P, F814P, A889P, A896P, A939P, R679G, R680S, and R682S;
(4) K983P, V984P, F814P, A889P, A896P, A939P, R679G, R680S, and R682S; or (5) D982P, K983P, V984P, F814P, A889P, A896P, A939P, R679G, R680S, and R682S. 1. A composition or medical preparation comprising a first RNA encoding a first SARS-CoV-2 S polypeptide or an immunogenic fragment thereof, and a second RNA encoding a second SARS-CoV-2 S polypeptide or an immunogenic fragment thereof,
(a) the first SARS-CoV-2 S polypeptide or fragment comprises an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO:69 and including the following substitutions relative to SEQ ID NO:69: K983P, V984P, F814P, A889P, A896P, and A939P;
(b) the second SARS-CoV-2 S polypeptide or fragment has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO:70 and has the following set of amino acid substitutions relative to SEQ ID NO:70:
(1) D982P, V984P, F814P, A889P, A896P, and A939P;
(2) K983P, V984P, F814P, A889P, A896P, and A939P;
(3) D982P, V984P, F814P, A889P, A896P, A939P, R679G, R680S, and R682S;
(4) K983P, V984P, F814P, A889P, A896P, A939P, R679G, R680S, and R682S; or (5) D982P, K983P, V984P, F814P, A889P, A896P, A939P, R679G, R680S, and R682S. 1. A composition or medical preparation comprising a first RNA encoding a first SARS-CoV-2 S polypeptide or an immunogenic fragment thereof, and a second RNA encoding a second SARS-CoV-2 S polypeptide or an immunogenic fragment thereof,
(a) the first SARS-CoV-2 S polypeptide or fragment comprises an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO:69 and including the following substitutions relative to SEQ ID NO:69: D982P, V984P, F814P, A889P, A896P, A939P, R679G, R680S, and R682S;
(b) the second SARS-CoV-2 S polypeptide or fragment has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO:70 and has the following set of amino acid substitutions relative to SEQ ID NO:70:
(1) D982P, V984P, F814P, A889P, A896P, and A939P;
(2) K983P, V984P, F814P, A889P, A896P, and A939P;
(3) D982P, V984P, F814P, A889P, A896P, A939P, R679G, R680S, and R682S;
(4) K983P, V984P, F814P, A889P, A896P, A939P, R679G, R680S, and R682S; or (5) D982P, K983P, V984P, F814P, A889P, A896P, A939P, R679G, R680S, and R682S. 1. A composition or medical preparation comprising a first RNA encoding a first SARS-CoV-2 S polypeptide or an immunogenic fragment thereof, and a second RNA encoding a second SARS-CoV-2 S polypeptide or an immunogenic fragment thereof,
(a) the first SARS-CoV-2 S polypeptide or fragment comprises an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO:69 and including the following substitutions relative to SEQ ID NO:69: K983P, V984P, F814P, A889P, A896P, A939P, R679G, R680S, and R682S;
(b) the second SARS-CoV-2 S polypeptide or fragment has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO:70 and has the following set of amino acid substitutions relative to SEQ ID NO:70:
(1) D982P, V984P, F814P, A889P, A896P, and A939P;
(2) K983P, V984P, F814P, A889P, A896P, and A939P;
(3) D982P, V984P, F814P, A889P, A896P, A939P, R679G, R680S, and R682S;
(4) K983P, V984P, F814P, A889P, A896P, A939P, R679G, R680S, and R682S; or (5) D982P, K983P, V984P, F814P, A889P, A896P, A939P, R679G, R680S, and R682S. 1. A composition or medical preparation comprising a first RNA encoding a first SARS-CoV-2 S polypeptide or an immunogenic fragment thereof, and a second RNA encoding a second SARS-CoV-2 S polypeptide or an immunogenic fragment thereof,
(a) the first SARS-CoV-2 S polypeptide or fragment comprises an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO:69 and including the following substitutions relative to SEQ ID NO:69: D982P, K983P, V984P, F814P, A889P, A896P, A939P, R679G, R680S, and R682S;
(b) the second SARS-CoV-2 S polypeptide or fragment has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO:70 and has the following set of amino acid substitutions relative to SEQ ID NO:70:
(1) D982P, V984P, F814P, A889P, A896P, and A939P;
(2) K983P, V984P, F814P, A889P, A896P, and A939P;
(3) D982P, V984P, F814P, A889P, A896P, A939P, R679G, R680S, and R682S;
(4) K983P, V984P, F814P, A889P, A896P, A939P, R679G, R680S, and R682S; or (5) D982P, K983P, V984P, F814P, A889P, A896P, A939P, R679G, R680S, and R682S. 1. A composition or medical preparation comprising a first RNA encoding a first SARS-CoV-2 S polypeptide or an immunogenic fragment thereof, and a second RNA encoding a second SARS-CoV-2 S polypeptide or an immunogenic fragment thereof,
(a) the first SARS-CoV-2 S polypeptide or fragment comprises an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO:69 and including the following substitutions relative to SEQ ID NO:69: D982P, V984P, F814P, A889P, A896P, and A939P;
(b) the second SARS-CoV-2 S polypeptide or fragment has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 104 or 105 and has the following set of amino acid substitutions relative to SEQ ID NO: 104 or 105:
(1) D980P, V982P, F812P, A887P, A894P, and A937P;
(2) K981P, V982P, F812P, A887P, A894P, and A937P;
(3) D980P, V982P, F812P, A887P, A894P, A937P, R677G, R678S, and R680S;
(4) K981P, V982P, F812P, A887P, A894P, A937P, R677G, R678S, and R680S; or (5) D980P, K981P, V982P, F812P, A887P, A894P, A937P, R677G, R678S, and R680S. 1. A composition or medical preparation comprising a first RNA encoding a first SARS-CoV-2 S polypeptide or an immunogenic fragment thereof, and a second RNA encoding a second SARS-CoV-2 S polypeptide or an immunogenic fragment thereof,
(a) the first SARS-CoV-2 S polypeptide or fragment comprises an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO:69 and including the following substitutions relative to SEQ ID NO:69: K983P, V984P, F814P, A889P, A896P, and A939P;
(b) the second SARS-CoV-2 S polypeptide or fragment has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 104 or 105 and has the following set of amino acid substitutions relative to SEQ ID NO: 104 or 105:
(1) D980P, V982P, F812P, A887P, A894P, and A937P;
(2) K981P, V982P, F812P, A887P, A894P, and A937P;
(3) D980P, V982P, F812P, A887P, A894P, A937P, R677G, R678S, and R680S;
(4) K981P, V982P, F812P, A887P, A894P, A937P, R677G, R678S, and R680S; or (5) D980P, K981P, V982P, F812P, A887P, A894P, A937P, R677G, R678S, and R680S. 1. A composition or medical preparation comprising a first RNA encoding a first SARS-CoV-2 S polypeptide or an immunogenic fragment thereof, and a second RNA encoding a second SARS-CoV-2 S polypeptide or an immunogenic fragment thereof,
(a) the first SARS-CoV-2 S polypeptide or fragment comprises an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO:69 and including the following substitutions relative to SEQ ID NO:69: D982P, V984P, F814P, A889P, A896P, A939P, R679G, R680S, and R682S;
(b) the second SARS-CoV-2 S polypeptide or fragment has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 104 or 105 and has the following set of amino acid substitutions relative to SEQ ID NO: 104 or 105:
(1) D980P, V982P, F812P, A887P, A894P, and A937P;
(2) K981P, V982P, F812P, A887P, A894P, and A937P;
(3) D980P, V982P, F812P, A887P, A894P, A937P, R677G, R678S, and R680S;
(4) K981P, V982P, F812P, A887P, A894P, A937P, R677G, R678S, and R680S; or (5) D980P, K981P, V982P, F812P, A887P, A894P, A937P, R677G, R678S, and R680S. 1. A composition or medical preparation comprising a first RNA encoding a first SARS-CoV-2 S polypeptide or an immunogenic fragment thereof, and a second RNA encoding a second SARS-CoV-2 S polypeptide or an immunogenic fragment thereof,
(a) the first SARS-CoV-2 S polypeptide or fragment comprises an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO:69 and including the following substitutions relative to SEQ ID NO:69: K983P, V984P, F814P, A889P, A896P, A939P, R679G, R680S, and R682S;
(b) the second SARS-CoV-2 S polypeptide or fragment has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 104 or 105 and has the following set of amino acid substitutions relative to SEQ ID NO: 104 or 105:
(1) D980P, V982P, F812P, A887P, A894P, and A937P;
(2) K981P, V982P, F812P, A887P, A894P, and A937P;
(3) D980P, V982P, F812P, A887P, A894P, A937P, R677G, R678S, and R680S;
(4) K981P, V982P, F812P, A887P, A894P, A937P, R677G, R678S, and R680S; or (5) D980P, K981P, V982P, F812P, A887P, A894P, A937P, R677G, R678S, and R680S. 1. A composition or medical preparation comprising a first RNA encoding a first SARS-CoV-2 S polypeptide or an immunogenic fragment thereof, and a second RNA encoding a second SARS-CoV-2 S polypeptide or an immunogenic fragment thereof,
(a) the first SARS-CoV-2 S polypeptide or fragment comprises an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO:69 and including the following substitutions relative to SEQ ID NO:69: D982P, K983P, V984P, F814P, A889P, A896P, A939P, R679G, R680S, and R682S;
(b) the second SARS-CoV-2 S polypeptide or fragment has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 104 or 105 and has the following set of amino acid substitutions relative to SEQ ID NO: 104 or 105:
(1) D980P, V982P, F812P, A887P, A894P, and A937P;
(2) K981P, V982P, F812P, A887P, A894P, and A937P;
(3) D980P, V982P, F812P, A887P, A894P, A937P, R677G, R678S, and R680S;
(4) K981P, V982P, F812P, A887P, A894P, A937P, R677G, R678S, and R680S; or (5) D980P, K981P, V982P, F812P, A887P, A894P, A937P, R677G, R678S, and R680S. 1. A composition or medical preparation comprising a first RNA encoding a first SARS-CoV-2 S polypeptide or an immunogenic fragment thereof, and a second RNA encoding a second SARS-CoV-2 S polypeptide or an immunogenic fragment thereof,
(a) the first SARS-CoV-2 S polypeptide or fragment comprises an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO:70 and including the following substitutions relative to SEQ ID NO:70: D982P, V984P, F814P, A889P, A896P, and A939P;
(b) the second SARS-CoV-2 S polypeptide or fragment has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 104 or 105 and has the following set of amino acid substitutions relative to SEQ ID NO: 104 or 105:
(1) D980P, V982P, F812P, A887P, A894P, and A937P;
(2) K981P, V982P, F812P, A887P, A894P, and A937P;
(3) D980P, V982P, F812P, A887P, A894P, A937P, R677G, R678S, and R680S;
(4) K981P, V982P, F812P, A887P, A894P, A937P, R677G, R678S, and R680S; or (5) D980P, K981P, V982P, F812P, A887P, A894P, A937P, R677G, R678S, and R680S. 1. A composition or medical preparation comprising a first RNA encoding a first SARS-CoV-2 S polypeptide or an immunogenic fragment thereof, and a second RNA encoding a second SARS-CoV-2 S polypeptide or an immunogenic fragment thereof,
(a) the first SARS-CoV-2 S polypeptide or fragment comprises an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO:70 and including the following substitutions relative to SEQ ID NO:70: K983P, V984P, F814P, A889P, A896P, and A939P;
(b) the second SARS-CoV-2 S polypeptide or fragment has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 104 or 105 and has the following set of amino acid substitutions relative to SEQ ID NO: 104 or 105:
(1) D980P, V982P, F812P, A887P, A894P, and A937P;
(2) K981P, V982P, F812P, A887P, A894P, and A937P;
(3) D980P, V982P, F812P, A887P, A894P, A937P, R677G, R678S, and R680S;
(4) K981P, V982P, F812P, A887P, A894P, A937P, R677G, R678S, and R680S; or (5) D980P, K981P, V982P, F812P, A887P, A894P, A937P, R677G, R678S, and R680S. 1. A composition or medical preparation comprising a first RNA encoding a first SARS-CoV-2 S polypeptide or an immunogenic fragment thereof, and a second RNA encoding a second SARS-CoV-2 S polypeptide or an immunogenic fragment thereof,
(a) the first SARS-CoV-2 S polypeptide or fragment comprises an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO:70 and including the following substitutions relative to SEQ ID NO:70: D982P, V984P, F814P, A889P, A896P, A939P, R679G, R680S, and R682S;
(b) the second SARS-CoV-2 S polypeptide or fragment has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 104 or 105 and has the following set of amino acid substitutions relative to SEQ ID NO: 104 or 105:
(1) D980P, V982P, F812P, A887P, A894P, and A937P;
(2) K981P, V982P, F812P, A887P, A894P, and A937P;
(3) D980P, V982P, F812P, A887P, A894P, A937P, R677G, R678S, and R680S;
(4) K981P, V982P, F812P, A887P, A894P, A937P, R677G, R678S, and R680S; or (5) D980P, K981P, V982P, F812P, A887P, A894P, A937P, R677G, R678S, and R680S. 1. A composition or medical preparation comprising a first RNA encoding a first SARS-CoV-2 S polypeptide or an immunogenic fragment thereof, and a second RNA encoding a second SARS-CoV-2 S polypeptide or an immunogenic fragment thereof,
(a) the first SARS-CoV-2 S polypeptide or fragment comprises an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO:70 and including the following substitutions relative to SEQ ID NO:70: K983P, V984P, F814P, A889P, A896P, A939P, R679G, R680S, and R682S;
(b) the second SARS-CoV-2 S polypeptide or fragment has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 104 or 105 and has the following set of amino acid substitutions relative to SEQ ID NO: 104 or 105:
(1) D980P, V982P, F812P, A887P, A894P, and A937P;
(2) K981P, V982P, F812P, A887P, A894P, and A937P;
(3) D980P, V982P, F812P, A887P, A894P, A937P, R677G, R678S, and R680S;
(4) K981P, V982P, F812P, A887P, A894P, A937P, R677G, R678S, and R680S; or (5) D980P, K981P, V982P, F812P, A887P, A894P, A937P, R677G, R678S, and R680S. 1. A composition or medical preparation comprising a first RNA encoding a first SARS-CoV-2 S polypeptide or an immunogenic fragment thereof, and a second RNA encoding a second SARS-CoV-2 S polypeptide or an immunogenic fragment thereof,
(a) the first SARS-CoV-2 S polypeptide or fragment comprises an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO:70 and including the following substitutions relative to SEQ ID NO:70: D982P, K983P, V984P, F814P, A889P, A896P, A939P, R679G, R680S, and R682S;
(b) the second SARS-CoV-2 S polypeptide or fragment has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 104 or 105 and has the following set of amino acid substitutions relative to SEQ ID NO: 104 or 105:
(1) D980P, V982P, F812P, A887P, A894P, and A937P;
(2) K981P, V982P, F812P, A887P, A894P, and A937P;
(3) D980P, V982P, F812P, A887P, A894P, A937P, R677G, R678S, and R680S;
(4) K981P, V982P, F812P, A887P, A894P, A937P, R677G, R678S, and R680S; or (5) D980P, K981P, V982P, F812P, A887P, A894P, A937P, R677G, R678S, and R680S. The composition or medical preparation of any one of claims 24 to 78, wherein the first RNA and the second RNA each contain a modified nucleoside in place of uridine. The composition or medical preparation of any one of claims 24 to 79, wherein the first RNA and the second RNA each contain modified uridines in place of all uridines. The composition or medical preparation of any one of claims 24 to 80, wherein the first RNA and the second RNA each contain N1-methyl-pseudouridine (m1ψ) in place of all uridines. The composition or medical preparation of any one of claims 24 to 81, wherein the first RNA and the second RNA each include a 5' cap. 83. The composition or medical preparation of claim 82, wherein the 5' cap comprises _m27,3' ^-O Gppp( _m12' ^-O )ApG. The composition or medical preparation of any one of claims 24 to 83, wherein the first RNA and the second RNA each comprise a 5'-UTR that is or comprises a modified human alpha-globin 5'-UTR. The composition or medical preparation of any one of claims 24 to 84, wherein the first RNA and the second RNA each comprise a 5'UTR comprising a nucleotide sequence of SEQ ID NO:12 or a nucleotide sequence that is at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identical to the nucleotide sequence of SEQ ID NO:12. The composition or medical preparation of any one of claims 24 to 85, wherein the first RNA and the second RNA each comprise a 3'-UTR that is or includes a first sequence from a split amino-terminal enhancer (AES) messenger RNA and a second sequence from a mitochondrially encoded 12S ribosomal RNA. The composition or medical preparation of any one of claims 24 to 86, wherein the first RNA and the second RNA each comprise a 3'UTR comprising a nucleotide sequence of SEQ ID NO:13 or a nucleotide sequence that is at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identical to the nucleotide sequence of SEQ ID NO:13. The composition or medical preparation according to any one of claims 24 to 87, wherein the first RNA and the second RNA each contain a polyA sequence. The composition or medical preparation of claim 88, wherein the first RNA and the second RNA each comprise a polyA sequence comprising at least 100 nucleotides. The composition or medical preparation of claim 88 or 89, wherein the first RNA and the second RNA each comprise 30 adenine nucleotides followed by 70 adenine nucleotides, the 30 adenine nucleotides and the 70 adenine nucleotides being separated by a linker sequence. The composition or medical preparation of any one of claims 88 to 90, wherein the first RNA and the second RNA each comprise a polyA sequence comprising or consisting of a nucleotide sequence of SEQ ID NO:14 or a nucleotide sequence that is at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identical to the nucleotide sequence of SEQ ID NO:14. The composition or medical preparation according to any one of claims 24 to 91, wherein the first RNA and the second RNA are each formulated for intramuscular administration or are to be formulated for intramuscular administration. The composition or medical preparation according to any one of claims 24 to 92, wherein the first RNA and the second RNA are each formulated as particles or are to be formulated as particles. 94. The composition or medical preparation of claim 93, wherein the first RNA and the second RNA are each formulated as a lipid nanoparticle (LNP) or lipoplex (LPX) particle. The composition or medical preparation of claim 94, wherein the LNP comprises a cationic ionizable lipid, a neutral lipid, a sterol, and a polymer-lipid conjugate. The immunogenic composition of claim 94 or 95, wherein the first RNA and the second RNA are formulated in separate LNPs. The immunogenic composition of claim 94 or 95, wherein the first RNA and the second RNA are formulated in the same LNP. The composition or medical preparation of claim 94, wherein the lipoplex particles are obtainable by mixing the RNA with liposomes. The composition or medical preparation according to any one of claims 24 to 98, wherein the first RNA and the second RNA are each mRNA, or the first RNA and the second RNA are each saRNA. A composition or medical preparation according to any one of claims 24 to 99, which is a pharmaceutical composition. A composition or medical preparation according to any one of claims 24 to 100, which is a vaccine. The composition or medical preparation of claim 100 or 101, wherein the pharmaceutical composition further comprises one or more pharma- ceutically acceptable carriers, diluents, and/or excipients. A method for inducing an immune response in a subject, comprising inducing an immune response in the subject by administering to the subject a composition or medical preparation according to any one of claims 1 to 23. The method of claim 103, wherein the SARS-CoV-2 S polypeptide comprises an amino acid sequence that does not contain a D985P substitution relative to SEQ ID NO:1, an amino acid sequence that does not contain a D982P substitution relative to SEQ ID NO:69 or SEQ ID NO:70, or an amino acid sequence that does not contain a D980P substitution relative to SEQ ID NO:104 or SEQ ID NO:105. The method of claim 103 or 104, further comprising administering a second RNA encoding a second SARS-CoV-2 S polypeptide or immunogenic fragment thereof, wherein the second SARS-CoV-2 S polypeptide or immunogenic fragment is an omicron variant SARS-CoV-2 S polypeptide that is not the BA.1 omicron variant. The method of claim 103 or 104, further comprising administering a second, different RNA encoding a second SARS-CoV-2 S polypeptide or an immunogenic fragment thereof, wherein the second SARS-CoV-2 S polypeptide or fragment is selected from the SARS-CoV-2 S polypeptides or fragments listed in claim 1. A method for inducing an immune response in a subject, comprising inducing an immune response in the subject by administering to the subject a composition or medical preparation according to any one of claims 24 to 102. The method of claim 107, wherein the SARS-CoV-2 S polypeptide comprises an amino acid sequence that does not contain a D985P substitution relative to SEQ ID NO:1, an amino acid sequence that does not contain a D982P substitution relative to SEQ ID NO:69 or SEQ ID NO:70, or an amino acid sequence that does not contain a D980P substitution relative to SEQ ID NO:104 or SEQ ID NO:105. The method of claim 107 or 108, further comprising administering a second composition or medical preparation, the second composition or medical preparation comprising RNA encoding a SARS-CoV-2 S polypeptide or immunogenic fragment of an omicron variant that is not the BA.1 omicron variant. The method of claim 107 or 108, further comprising administering a second composition or medical preparation, the second composition or medical preparation comprising a third RNA encoding a third SARS-CoV-2 S polypeptide or an immunogenic fragment thereof, and a fourth RNA encoding a fourth SARS-CoV-2 S polypeptide or an immunogenic fragment thereof. The method of claim 110, wherein the third RNA encodes a SARS-CoV-2 S polypeptide or an immunogenic fragment thereof that is a first or second SARS-CoV-2 S polypeptide or an immunogenic fragment thereof recited in any one of claims 24 to 102, and the third RNA encodes a SARS-CoV-2 S polypeptide or fragment that is different from the first SARS-CoV-2 S polypeptide or fragment encoded by the first RNA and/or different from the second SARS-CoV-2 S polypeptide or fragment encoded by the second RNA. The method of claim 110, wherein the fourth RNA encodes a SARS-CoV-2 S polypeptide or an immunogenic fragment thereof that is a first or second SARS-CoV-2 S polypeptide or an immunogenic fragment thereof recited in any one of claims 24 to 102, and the fourth RNA encodes a SARS-CoV-2 S polypeptide or fragment that is different from the first SARS-CoV-2 S polypeptide or fragment encoded by the first RNA and/or different from the second SARS-CoV-2 S polypeptide or fragment encoded by the second RNA. The method of claim 110, wherein the third RNA encodes a SARS-CoV-2 S polypeptide or an immunogenic fragment thereof that is a first or second SARS-CoV-2 S polypeptide or an immunogenic fragment thereof recited in any one of claims 24 to 102, and the third RNA encodes a SARS-CoV-2 S polypeptide or fragment that is different from the first SARS-CoV-2 S polypeptide or fragment encoded by the first RNA and different from the second SARS-CoV-2 S polypeptide or fragment encoded by the second RNA. The method of claim 110, wherein the fourth RNA encodes a SARS-CoV-2 S polypeptide or an immunogenic fragment thereof that is a first or second SARS-CoV-2 S polypeptide or an immunogenic fragment thereof recited in any one of claims 24 to 102, and the fourth RNA encodes a SARS-CoV-2 S polypeptide or fragment that is different from the first SARS-CoV-2 S polypeptide or fragment encoded by the first RNA and different from the second SARS-CoV-2 S polypeptide or fragment encoded by the second RNA. The third RNA encodes a SARS-CoV-2 S polypeptide or an immunogenic fragment thereof, which is a first or second SARS-CoV-2 S polypeptide or an immunogenic fragment thereof as listed in any one of claims 24 to 102, the third RNA encodes a SARS-CoV-2 S polypeptide or fragment that is different from the first SARS-CoV-2 S polypeptide or fragment encoded by the first RNA, and different from the second SARS-CoV-2 S polypeptide or fragment encoded by the second RNA, the fourth RNA encodes a SARS-CoV-2 S polypeptide or an immunogenic fragment thereof, which is a first or second SARS-CoV-2 S polypeptide or an immunogenic fragment thereof as listed in any one of claims 24 to 102, and the fourth RNA encodes a SARS-CoV-2 S polypeptide or an immunogenic fragment thereof, which is a first or second SARS-CoV-2 S polypeptide or an immunogenic fragment thereof as listed in any one of claims 24 to 102, and the fourth RNA encodes the first SARS-CoV-2 S polypeptide or an immunogenic fragment thereof as listed in any one of claims 24 to 102, The method of claim 110, which encodes a SARS-CoV-2 S polypeptide or fragment that is distinct from the S polypeptide or fragment and that is distinct from the second SARS-CoV-2 S polypeptide or fragment encoded by the second RNA. The method of claim 115, wherein each of the first, second, third, and fourth RNAs encodes a different SARS-CoV-2 S polypeptide or an immunogenic fragment thereof. A method of inducing an immune response in a subject, comprising inducing an immune response in the subject by administering to the subject (i) a composition or medical preparation according to any one of claims 1 to 23, and (ii) a composition or medical preparation according to any one of claims 24 to 102. 117. The method of claim 117, wherein the subject receiving the composition or medical preparation of (ii) has previously been administered the composition or medical preparation of (i). 117. The method of claim 117, wherein the subject receiving the composition or medical preparation of (i) has previously been administered the composition or medical preparation of (ii). 117. The method of claim 117, wherein the composition or medical preparation of (i) and the composition or medical preparation of (ii) are administered together as a multivalent vaccine.