WO2023148256A1

WO2023148256A1 - Inactivated sars-cov-2 virus vaccine

Info

Publication number: WO2023148256A1
Application number: PCT/EP2023/052534
Authority: WO
Original assignee: Valneva Austria Gmbh
Priority date: 2022-02-02
Filing date: 2023-02-02
Publication date: 2023-08-10

Abstract

Described herein are SARS-CoV-2 vaccines and compositions and methods of producing and administering said vaccines to subjects in need thereof.

Description

INACTIVATED SARS-CoV-2 VIRUS VACCINE

FIELD OF THE INVENTION

The disclosure relates to SARS-CoV-2 vaccines and compositions and methods for producing said vaccines and administering the vaccines to subjects for the generation of an anti-SARS-CoV-2 immune response.

BACKGROUND OF THE INVENTION

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2; hereinafter the “virus”) was detected for the first time in China around November 2019. Since then, the virus has caused a global pandemic. The natural reservoir are bats and the virus belongs to the Coronaviridae family, genus Betacoronavirus (betaCoV). The virus has a ssRNA genome, 29,903 bp (wild type, Wuhan-Hu-1: GenBank Reference sequence: NC_045512.2 and MN908947; A new coronavirus associated with human respiratory disease in China. 2020 Wu, et al. Nature 579:265-269) encoding for 9,860 amino acids, 25 non-structural proteins and 4 structural proteins: spike (S), envelope (E), membrane (M) and nucleocapsid (N) proteins. The virus has a variable size of between 60 to 140 nm in diameter. It is enveloped and sensitive to UV, heat, and lipid solvents. It has 89% nucleotide identity with bat SARS-like-CoVZXC21 and 81% nucleotide identity with human SARS-CoV. Evidence suggests that this virus spreads when an infected person coughs small droplets - packed with the virus - into the air. These can be breathed in, or cause an infection if one touches a surface they have landed on, then one’s eyes, nose or mouth. In addition, other vectors may exist, and the virus may be transmitted by blood transfusion, transplacentally, and through sexual transmission. Though the symptoms of SARS-CoV-2 virus infection (also herein referred to as COVID-19, COVID or COVID-19 disease) may be mild, and include typically fever and cough, it can also be asymptomatic or in the other extreme it can be severe or fatal, i.e. lethal. The key symptoms are usually high temperature, cough and breathing difficulties. Although there are meanwhile specific treatments and vaccines available for COVID, it still presents a substantial public health threat. Furthermore, various escape mutants have emerged (e.g. alpha, beta, delta and/or omicron and further future emerging strains may occur) which further worsen the situation and thus this unfortunate development needs to be addressed as well. Another problem is that there is a substantial vaccine hesitancy for COVID-19 vaccines. E.g. in the U.S. it is estimated that the vaccine hesitancy (including unsure subjects) is up to 27% of the population (see CDC data https://data.cdc.gOv/stories/s/Vaccine-Hesitancy-for-COVID-19/cnd2-a6zw/).

Thus, there is a need for a broadly protective vaccine that can overcome vaccine hesitancy as much as possible and can be produced at high volume. A universal vaccine based on a classical inactivation approach of the virus or virus mixture wherein the virus is optimized for high volume manufacturing could serve as such a vaccine, providing protection against various strains of SARS-CoV-2 as well as giving subjects a choice of using a well-established vaccine technology.

SUMMARY OF THE INVENTION

Accordingly, the present invention provides an improved inactivated SARS-CoV-2 vaccine capable of generating neutralizing antibodies against a native homologous and/or heterologous SARS-CoV-2 particles and/or is capable of raising an effective T-Cell response against a native homologous and/or heterologous SARS-CoV-2 particle in a human subject. Whilst extensive effort has already been invested by research groups throughout the world in developing a SARS-CoV-2 vaccine, most approaches have focused on subunit vaccines (e.g. encoding the SARS-CoV-2 S protein or fragments thereof), live attenuated vaccines or recombinant DNA or RNA vaccines encoding viral proteins. However, there has been little interest in whole virus, inactivated vaccine approaches, and a successful inactivated SARS-CoV-2 vaccine has not yet been fully developed. In so far as an inactivated vaccine approach has been contemplated, the use of typical inactivating agents (e.g. formaldehyde) and adjuvants (e.g. alum) under standard conditions may have drawbacks which hinder development of an effective vaccine candidate. Moreover, there is a risk that such a vaccine candidate could result in antibody-dependent enhancement (ADE) of SARS-CoV-2 disease, enhanced vaccine associated respiratory disease (VAERD); enhanced respiratory disease (ERD) and/or Th2 type immunopathology possibly resulting from the hypersensitivity responses to SARS-CoV-2 components. A further drawback of the existing vaccines is the emerging variants or variants of concern (“VOC”; see WHO definition) for which the existing vaccines do not provide a good or only a reduced protection. Furthermore, it seems that frequent boostering (e.g. every 4 months) is required by the existing vaccines, e.g. mRNA vaccines to provide for an ongoing protection. The present invention aims to address these problems and thus to produce a safe and effective whole virus, inactivated SARS-CoV-2 vaccine that overcomes the drawbacks of the prior art.

Thus in one aspect the present invention provides a SARS-CoV-2 vaccine comprising at least two or exactly two different beta-propiolactone-inactivated SARS-CoV-2 particles; wherein the vaccine is capable of generating neutralizing antibodies to a native homologous and/or heterologous SARS-CoV- 2 particle and/or is capable of raising an effective T-Cell response against a native homologous and/or heterologous SARS-CoV-2 particle in a human subject. Preferably a native surface conformation of the SARS-CoV-2 particle is preserved in the vaccine and/or the furin cleavage site activity is reduced or eliminated by passaging out the furin site and/or introducing mutations in the cleavage site. In another aspect the present invention provides a SARS-CoV-2 vaccine comprising at least two or exactly two different beta-propiolactone-inactivated SARS-CoV-2 particles; wherein a native surface conformation of the SARS-CoV-2 particle is preserved in the vaccine, such that the vaccine is capable of generating neutralizing antibodies against native SARS-CoV-2 particles and/or other immunological responses in a human subject that are able to protect partly or fully more than 50%, preferably more than 60%, more than 70%, more than 80%, more than 90% of said vaccinated human subjects.

In particular, the present invention aims to provide optimally inactivated SARS-CoV-2 particles, which are incapable of replication and infection of human cells, but which retain immunogenic epitopes of viral surface proteins and are thus suitable for generating protective immunity in vaccinated subjects. By optimizing the inactivation process and other steps in the production of the vaccine, including the selection of an appropriate adjuvant, a novel vaccine composition can be obtained that preserves a native surface conformation of SARS-CoV-2 particles and which reduces the risk of negative effects such as ADE, VAERD, ERD and immunopathology. Such vaccine compositions are described in more detail below.

In a further particular embodiment, the invention aims to provide an optimal combination of optimally inactivated different SARS-CoV-2 particles, which are incapable of replication and infection of human cells, but which retain immunogenic epitopes of viral surface proteins and are thus suitable for generating protective immunity in vaccinated subjects. By an optimal combination of different and optimally inactivated SARS-CoV-2 particles, an improved vaccine composition can be obtained that is capable of generating neutralizing antibodies against a native homologous and/or heterologous SARS- CoV-2 particle and/or is capable of raising an effective T-Cell response against a native homologous and/or heterologous SARS-CoV-2 particle in a human subject that are able to protect partly or fully more than 50%, preferably more than 60%, more than 70%, more than 80%, more than 90% of said vaccinated human subjects.

Each of the limitations of the invention can encompass various embodiments of the invention. It is therefore anticipated that each of the limitations of the invention involving any one element or combinations of elements can be included in each aspect of the invention. This invention is not limited in its application to the details of construction and the arrangement of components set forth in the following description or illustrated in the drawings. The invention is capable of other embodiments and of being practiced or of being carried out in various ways.

BRIEF DESCRIPTION OF THE DRAWINGS The accompanying drawings are not intended to be drawn to scale. The figures are illustrative only and are not required for enablement of the disclosure. For purposes of clarity, not every component may be labeled in every drawing. In the drawings:

Figure 1. The process for production of the inactivated SARS-CoV-2 vaccine of the current invention. Steps include cell buildup of Vero host cells, infection of Vero cells with SARS-CoV-2, virus harvest, DNA reduction, primary inactivation, purification, optional secondary inactivation and formulation with adjuvant.

Figure 2. During the course of the SARS-CoV-2 pandemic, SARS-CoV-2 genomic sequences from isolates from around the world have been reported including the recent new variants or lineages such as the UK B 1.1.7, Brazilian Pl, Californian B.1.427/B. 1.429 and South African B.1.351 lineages. The accession numbers and origins of complete SARS-CoV-2 genomic sequences are provided in tabular form, along with accession numbers for the corresponding orflab polyprotein and S protein, when available (- or no entry = not available).

Figure 3. A preferred set-up for the sucrose gradient centrifugation used as a polishing step for the SARS-CoV-2 vaccine of the invention.

Figure 4. Total IgG in response to SARS-CoV-2 vaccine. Coating antigens: SI (A), receptor binding domain of spike protein (B) and nucleoprotein (C). Endpoint titer: absorbance of 3 -fold the blank used as cut-off (dashed line).

Figure 5. IgGl and IgG2a titers in response to SARS-CoV-2 vaccine adjuvanted with alum. Antibody titers specific to SI protein were determined by ELISA. The concentrations were determined by comparison with a mAb subclass standard curve.

Figure 6. Production process delivers high density and intact spike proteins. Shown are electron micrographs of the SARS-CoV-2 inactivated drug substance produced according to Example 1. About 1-1.5 10⁷ viral particles per AU.

Figure 7. Comparison of Size-Exclusion-Chromatography and SDS-PAGE profiles of SARS-CoV-2 and JEV drug substance. High purity (>95%) according to SDS-PAGE (silver stain, reduced) and monomer virus (>95%) according to SE-HPLC. Difference in retention time due to different virus particle size (JEV (IXIARO) about 50nm, SARS-CoV-2 about lOOnm). Figure 8. A) DNA sequence corresponding to the RNA sequence of a wild type isolate, also referred to as Wuhan or reference sequence (SEQ ID NO: 1); B) DNA sequence corresponding to the RNA sequence of a wild type isolate from INMI (SEQ ID NO: 9).

Figure 9. DNA sequence corresponding to the RNA sequence of a Delta typed isolate B.1.617.2 (SEQ ID NO: 2).

Figure 10. DNA sequence corresponding to the RNA sequence of an Omicron typed isolate (SEQ ID NO: 3). (PAC-IHU-49242.3 IHU Marseille isolate hCoV-19/France/PAC-IHU-

49242/2021|EPI_ISL_7308635|2021-12-01 plus 36n+18bp UTR5* and 21bp+45n UTR3' from virus EPI_ISL_803385 [SARS-CoV-2] plus N assignment with consensus of VB-2022-156-O.)

Figure 11. DNA sequence corresponding to the RNA sequence of an Omicron typed isolate (SEQ ID NO: 4). (Rega-20174.2 rega-20174 Severe acute respiratory syndrome coronavirus 2, hCoV- 19/Belgium/rega-20174/2021|EPI_ISL_6794907.2|2021-l 1-24, partial genome [SARS-CoV-2] UTR 573' fdled with n.)

Figure 12. S-protein sequence of a wild typed isolate from INMI (SEQ ID NO: 5).

Figure 13. Generation of virus seed banks with reduced or eliminated furin cleavage activity.

Figure 14. Monovalent SARS-CoV-2 vaccine-immune human sera neutralize the Delta and Omicron VOCs. For methods, see Hoffmann et al, Cell 2021 Apr 29; 184(9):2384-2393. Solid bars indicate geometric mean titer. Dashed line indicates lowest dilution tested (1 :25). Statistical analysis by Kruskal- Wallis test with Dunn’s multiple comparisons. ** P<0.0I, ***P<0.00I.

Figure 15. Phase 3 clinical study design described in Example 9.

Figure 16. Phase 2/3 clinical study design described in Example 10. Primary endpoints refer to a) GMT fold-rise for neutralizing antibodies against SARS-CoV-2 at Day 15 following a single booster dose with monovalent SARS-CoV-2 vaccine, and b) Frequency and severity of solicited AEs (local and systemic reactions) within 7 days after the booster vaccination with monovalent SARS-CoV-2 vaccine. Figure 17. Study design for NHP challenge study. Three groups of 8 animals each; Two dose groups for SARS-CoV-2 vaccine (10 AU & 40 AU, formulated with 0.5 mg/dose Al³⁺ and 1 mg Thl responsestimulating adjuvant per dose added directly before administration) and a placebo group (DPBS). The SARS-CoV-2 challenge strain is BetaCoV/France/IDF/0372/2020 (Maisonmasse et al., Hydroxychloroquine use against SARS-CoV-2 infection in non-human primates, 2020, Nature 585:584-587). Methods and timing of testing: Hematology on d-28, dO, d7, dl4, d21, d28, d35, d47, d49, d50, d51, d54, d62. Ab response (EUISA, IF A) on d-28, dO, dl4, d21, d28, d35, d47, d54, d62. T cell response (ICS, EUISPOT) on d-28, dO, dl4, d35, d54, d62. Cytokine response (LUMINEX) on d47, d49, d50, d51, d54, d62. SWABS (viral load (qRT-PCR-genomic + subgenomic): nasal & tracheal swabs on d35, d49, d50, d51, d54, d57, d62; rectal swabs at baseline and on d2, d7, d 15. BAL viral load (qRT-PCR-genomic + subgenomic): d50. Euthanasia: lung harvest, viral load (qRT-PCR - genomic + subgenomic): d54, d62. CT scans: d35, d50, d57.

DETAILED DESCRIPTION OF THE INVENTION

Embodiments of the present invention are directed to a SARS-CoV-2 vaccine or immunogenic composition comprising at least two or exactly two different inactivated SARS-CoV-2 particles. Typically, the inactivated SARS-CoV-2 particles are whole virus, inactivated particles, i.e. the inactivated virus particles are derived from whole native SARS-CoV-2 particles that have been inactivated. As used herein “SARS-CoV-2” refers to the SARS-CoV-2 virus and “SARS-CoV-2 particles” typically refers to whole SARS-CoV-2 viral particles, i.e. virions and includes also variants of SARS-CoV-2.

In some embodiments of the present invention, the SARS-CoV-2 particles are inactivated without substantially modifying their surface structure. In other words, a native surface conformation of the SARS-CoV-2 particles is retained in the inactivated virus particles. It has been found that by optimizing an inactivation process, e.g. using beta-propiolactone, infectivity of native SARS-CoV-2 particles can be substantially abrogated without adversely affecting their antigenicity and/or immunogenicity. Thus, the present invention provides in one aspect an inactivated virus vaccine (e.g. a beta-propiolactone - inactivated virus vaccine) that is capable of generating neutralizing antibodies against a native homologous and/or heterologous SARS-CoV-2 particle and/or is capable of raising an effective T-Cell response against a native homologous and/or heterologous SARS-CoV-2 particle in a human subject.

In one embodiment, the SARS-CoV-2 particles are inactivated by a method that preferentially targets viral RNA. By this it is meant that e.g. the inactivation step modifies viral RNA more than viral proteins. Thus, the inactivated SARS-CoV-2 particles may comprise replication-deficient viral RNA, i.e. the viral RNA is modified in the inactivation step such that the inactivated particles are incapable of replicating. By utilizing an inactivation method that preferentially targets viral RNA, the present invention advantageously allows the preservation of immunogenic epitopes in viral surface proteins.

Preferably, the inactivation method spares viral (surface) proteins relative to viral RNA, e.g. the viral surface proteins (e.g. the spike (S) protein) may comprise fewer or more infrequent modifications resulting from the inactivation step compared to viral RNA. For instance, a lower proportion of amino acid residues in the viral surface proteins (e.g. S protein) may be modified by the inactivation step compared to the proportion of modified nucleotide residues in the viral RNA. In some embodiments, the proportion of modified amino acid residues in the viral surface proteins (e.g. S protein) may be at least 5%, 10%, 20%, 30%, 50%, 70% or 90% lower than the proportion of modified nucleotide residues in the viral RNA. By “modifications” or “modified residues” it is meant to refer to non-native residues that are not present in the native SARS-CoV-2 particles, e.g. chemical (covalent) modifications of such residues resulting from the inactivation step.

In one embodiment, the viral RNA is inactivated by alkylation and/or acylation, i.e. the modifications in the SARS-CoV-2 inactivated particles comprise alkylated and/or acylated nucleotide residues. In some embodiments, the modifications are preferentially targeted to purine (especially guanine) residues, e.g. the SARS-CoV-2 inactivated particles comprise one or more modified (e.g. alkylated or acylated) guanine residues. In some cases, the inactivation step may lead to cross-linking of viral RNA with viral proteins, e.g. via guanine residues in the viral RNA. The inactivation step may also introduce nicks or strand breaks into viral RNA, e.g. resulting in fragmentation of the viral genome.

Suitable alkylating and/or acylating agents are known in the art. In one embodiment, the inactivating agent comprises beta-propiolactone, i.e. the vaccine comprises beta-propiolactone-inactivated virus particles. In any case, in a particular embodiment, beta-propiolactone (herein referred to also as “BPL”) treatment is particularly preferred according to the present invention, because it results in SARS-CoV- 2 particles, that are substantially inactive, but which retain high antigenicity and immunogenicity against neutralizing epitopes present in native SARS-CoV-2. In particular, it has been surprisingly found that beta-propiolactone can be used to inactivate SARS-CoV-2 particles with a minimum number of protein modifications. For instance, as demonstrated in Examples 6 and 7 below, inactivation of SARS-CoV-2 particles using beta-propiolactone results in a much lower number of modifications of viral proteins compared to inactivation of influenza particles by beta-propiolactone. Thus in beta- propiolactone-inactivated SARS-CoV-2 particles, a native surface conformation of the viral particles can be preserved.

In a preferred embodiment of the invention, the viral RNA is inactivated in an optimized manner, i.e. such it is just sufficiently inactivated not to be infectious anymore but not “over”-inactivated so that numerous modification at different amino acids in particular at the S-protein occur. In a further even more preferred embodiment, the BPL inactivation not only sufficiently inactivates (but not overinactivates) the SARS-CoV-2 virus but also just sufficiently inactivates viruses that might be coenriched and co-cultured in the manufacturing process (see e.g. experimental part). A particularly hard virus to inactivate that can co-culture and be co-enriched is PPV (porcine parvovirus) - see experimental part.

The concentration of beta-propiolactone in the inactivation step may be optimized to ensure complete inhibition of viral replication whilst preserving the conformation of surface proteins in the virus. For instance, the concentration of beta-propiolactone in the inactivation step may be e.g. 0.01 to 1% by weight, preferably 0.01 to 0. 1% by weight, more preferably about 0.03% by weight. A preferred amount of BPL was found to be 500ppm where the SARS-CoV-2 virus but also other concerning viruses/impurities are inactivated whilst preserving (i.e. not modifying) most of the amino acids of the S-protein (i.e. only a few amino acids were shown to be modified at low probability).

In some embodiments, the native SARS-CoV-2 particles may be contacted with beta-propiolactone for at least 5 hours, at least 10 hours, at least 24 hours or at least 4 days, e.g. 5 to 24 hours or longer such as 48 hours. The inactivation step may be performed at about 0°C to about 25°C, preferably about 4°C or about 22°C, or e.g. 18 to 24°C. In one embodiment the inactivation step (e.g. with beta- propiolactone) is performed at 2°C to 8°C for 24 hours. The inactivation step may optionally and preferably be followed by a hydrolyzation step of the inactivating agent, as is known in the art (which may be performed e.g. at about 37°C+/- 2°C for a total time of 2.5 hours +/- 0.5 hours for beta- propiolactone). Typically, longer incubation times and/or higher temperatures in the inactivation step may enhance viral inactivation, but may also lead to an increased risk of undesirable surface modifications of the viral particles, leading to reduced immunogenicity. Therefore, the inactivation step may be performed for e.g. the shortest time necessary in order to produce a fully inactivated virus particle. After completion of the hydrolysis, the inactivated viral solution was in one embodiment immediately cooled down to 5±3°C and stored there until inactivation was confirmed by large volume plaque assay and serial passaging assay. Further information on beta-propiolactone inactivation of SARS-CoV-2 may be found in WO2021/204825A3, which is incorporated herein by reference in its entirety.

Beta-propiolactone inactivation of SARS-CoV-2 particles may preferentially modify cysteine, methionine and/or histidine residues. Thus in some embodiments, the inactivated SARS-CoV-2 particle comprises one or more beta-propiolactone-modified cysteine, methionine and/or histidine residues. However, in embodiments of the present invention, the beta-propiolactone-inactivated SARS-CoV-2 particles show relatively few protein modifications. Thus, for example, an inactivated SARS-CoV-2 particle in the vaccine may comprise fewer than 200, 100, 50, 30, 20, 15, 10, 9, 8, 7 or 6 beta- propiolactone -modified amino acid residues. Preferably a spike (S) protein of the inactivated SARS- CoV-2 particle comprises fewer than 100, 50, 30, 20, 15, 10, 9, 8, 7 or 6 beta-propiolactone-modified amino acid residues. More preferably the inactivated SARS-CoV-2 particle or spike protein thereof comprises 20 or fewer, 15 or fewer, 10 or fewer, or 5 or fewer beta-propiolactone-modified amino acid residues. Most preferably the inactivated SARS-CoV-2 particle or spike protein thereof comprises 1 to 100, 2 to 70, 3 to 50, 4 to 30, 5 to 25, 5 to 20, 10 to 20 or about 15 beta-propiolactone-modified amino acid residues.

In another embodiment, fewer than 20%, 15%, 10%, 5% or 4% of SARS-CoV-2 polypeptides are beta- propiolactone-modified. For instance, 0.1 to 10%, 1 to 8%, 2 to 7% or about 3%, 4%, 5% or 6% of SARS-CoV-2 polypeptides in the particle may be beta-propiolactone-modified. Beta-propiolactone modification of residues and/or polypeptides in the vaccine may be detected by mass spectrometry, e.g. using liquid chromatography with tandem mass spectrometry (LC-MS-MS), for instance using a method as described in Examples 6 and 7. In such a method, the SARS-CoV-2 particles may be digested in order to fragment proteins into SARS-CoV-2 polypeptides for LC-MS-MS analysis. The digestion step may be performed by any suitable enzyme or combination of enzymes, e.g. by trypsin, chymotrypsin and/or PNGase F (peptide:N-glycosidase F), or by e.g. acid hydrolysis. Preferably the percentage of BPL-modified polypeptides detected by LC-MS-MS following enzymatic digestion or acid hydrolysis is: (a) trypsin digestion, 1 to 5%, 2 to 4% or about 3%; (b) trypsin + PNGase F digestion, 1 to 5%, 2 to 4% or about 3%; (c) chymotrypsin, 1 to 10%, 3 to 8% or about 6% ; (d) acid hydrolysis, 1 to 6%, 2 to 5% or about 4%. In this context, a “beta-propiolactone-modified” polypeptide means that the polypeptide comprises at least one beta-propiolactone modification, e.g. at least one beta- propiolactone-modified residue.

In some embodiments, a spike (S) protein of the inactivated SARS-CoV-2 particle comprises a beta- propiolactone modification at one or more of the following residues: 49, 146, 166, 177, 207, 245, 379, 432, 519, 625, 1029, 1032, 1058, 1083, 1088, 1101, 1159 and/or 1271, e.g. in SEQ ID NO: 5, or a corresponding position in SEQ ID NO: 2, 3, 4. Preferably the inactivated SARS-CoV-2 particle comprises a beta-propiolactone modification at one or more of the following residues: H49, H146, C166, M177, H207, H245, C432, H519, H625, M1029, H1058, H1083, H1088, Hl 101, Hl 159 and/or Hl 271, e.g. in SEQ ID NO: 5, or a corresponding position in another variant inactivated SARS-CoV-2 particle. In another embodiment, the inactivated SARS-CoV-2 particle comprises a beta-propiolactone modification at one or more of the following residues: H207, H245, C379, M1029 and/or C1032, e.g. in SEQ ID NO: 5, or a corresponding position in another variant inactivated SARS-CoV-2 particle. By “a corresponding position” it is meant a corresponding position in another variant inactivated SARS- CoV-2 particle that aligns with position H207, H245, C379, M1029 and/or C1032 in SEQ ID NO: 5, e.g. when such a corresponding sequence is aligned with SEQ ID NO: 5 using a program such as NCBI Basic Local Alignment Search Tool (BLAST).

In some embodiments, a membrane (M) glycoprotein of the inactivated SARS-CoV-2 particle comprises a beta-propiolactone modification at one or more of the following residues: 125, 154, 155, 159 and/or 210, preferably H154, H155, C159 and/or H210.

In some embodiments, a nucleocapsid (N) protein of the inactivated SARS-CoV-2 particle comprises a beta-propiolactone modification at M234.

In some embodiments, fewer than 30%, 20%, 10%, 5%, 3% or 1% of one or more of the following residues in the inactivated SARS-CoV-2 particles are beta-propiolactone modified: (i) in the spike (S) protein, e.g. in SEQ ID NO: 5, or a corresponding position in a variant: residues 49, 146, 166, 177, 207, 245, 379, 432, 519, 625, 1029, 1032, 1058, 1083, 1088, 1101, 1159 and/or 1271; preferably H49, H146, C166, M177, H207, H245, C432, H519, H625, M1029, H1058, H1083, H1088, Hl 101, Hl 159 and/or H1271; alternatively H207, H245, C379, M1029 and/or C1032; (ii) in the membrane (M) glycoprotein: residues 125, 154, 155, 159 and/or 210; preferably H154, H155, C159 and/or H210; and/or (iii) M234 of the nucleocapsid (N) protein. In preferred embodiments, fewer than 30%, 20%, 10%, 5%, 3% or 1% of at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or each of the above residues in the inactivated SARS-CoV-2 particles are beta-propiolactone modified. In this paragraph, the percentage of modified residues is intended to refer to the site occupancy, e.g. the ratio of modified to unmodified peptide for the same modification site normalized to the protein abundance as described in Examples 6 and/or 7 below. In another preferred embodiment, the proportion of beta-propiolactone-modified residues (i.e. site occupancy) at the following positions in the inactivated SARS-CoV-2 particles is:

(i) in the spike (S) protein (e.g. of SEQ ID NO: 5, or a corresponding position in a variant:

(a) H207: less than 30%, preferably 0.01 to 25%; and/or

(b) H245: less than 10%, preferably 0.1 to 5%; and/or

(c) C379: less than 5%, less than 1% or less than 0.1%; and/or

(d) M1029: less than 5%, less than 1% or less than 0.1%; and/or

(e) C1032: less than 5%, less than 1% or less than 0.1%; and/or

(ii) in the membrane (M) glycoprotein:

(f) H154: less than 5%, less than 1% or less than 0. 1%; and/or

(g) H155: less than 10%, preferably 0.1 to 5%; and/or

(h) C159: less than 5%, less than 1% or less than 0.1%; and/or

(i) H210: less than 20%, preferably 0.1 to 10%; and/or

(iii) in the nucleocapsid (N) protein:

(j) M234: less than 90%, less than 10% or less than 0. 1%.

In another preferred embodiment, the proportion of beta-propiolactone-modified residues (i.e. site occupancy) at each of the following positions in the spike (S) protein (e.g. of SEQ ID NO: 5, or a corresponding position in a variant of the inactivated SARS-CoV-2 particles is:

(a) residues H49, H146, C166, H207, H519, M1029, H1083, H1088, Hl 101, Hl 159 and/or H1271: less than 20%, preferably 0.01 to 10%, more preferably 0.1 to 5%; and/or

(b) residues M177, C432, H625: less than 30%, preferably 0.1 to 20%, more preferably 1 to 10%; and/or

(c) residues H245, H1058: less than 30%, preferably 0.1 to 20%, more preferably 5 to 15%.

In some embodiments, the proportion of beta-propiolactone-modified amino acid residues in the inactivated SARS-CoV-2 particle (or spike (S) protein thereof) may be at least 5%, 10%, 20%, 30%, 50%, 70% or 90% lower than the proportion of modified residues in a beta-propiolactone-inactivated influenza particle (or hemagglutinin (HA) or neuraminidase (NA) protein thereof), e.g. in an influenza particle that has been inactivated under similar conditions to the SARS-CoV-2 particle.

In an alternative embodiment, the viral RNA may be inactivated by treatment with ultraviolet (UV) light. UV treatment can be used to preferentially target RNA (compared to polypeptides) in the viral particles, resulting in e.g. modified nucleotides and/or fragmentation. In some embodiments, UV treatment can be combined with beta-propiolactone treatment to improve inactivation of the virus, e.g. a beta-propiolactone treatment step can be followed by a UV treatment step or vice versa, or a UV treatment step can be performed at the same time as the beta-propiolactone treatment step.

In other embodiments, the native SARS-CoV-2 particles may be inactivated using formaldehyde. However, formaldehyde inactivation is typically less preferred in the present invention, as it is less suitable for preferentially targeting viral RNA and preserving immunogenic epitopes in the viral surface proteins.

Therefore in preferred embodiments, the inactivation step(s) (especially when using formaldehyde, but also when using other inactivating agents such as e.g. beta-propiolactone) are performed under mild conditions in order to preserve surface antigen integrity, especially integrity of the S protein.

In one embodiment, such a mild inactivation method comprises contacting a liquid composition comprising native SARS-CoV-2 particles with a chemical viral inactivating agent (such as e.g. any of the chemical inactivation agents as listed above or a combination, for instance formaldehyde or preferably beta-propiolactone) in a container, mixing the chemical viral inactivating agent and the liquid composition comprising SARS-CoV-2 particles under conditions of laminar flow but not turbulent flow, and incubating the chemical viral inactivating agent and the liquid composition comprising SARS- CoV-2 particles for a time sufficient to inactivate the viral particles. The mild inactivation step is optionally performed in a flexible bioreactor bag. The mild inactivation step preferably comprises 5 or less container inversions during the period of inactivation. Preferably, the mixing of the chemical viral inactivating agent and the composition comprising native SARS-CoV-2 particles comprises subjecting the container to rocking, rotation, orbital shaking, or oscillation for not more than 10 minutes at not more than 10 rpm during the period of incubation.

Suitable mild or gentle inactivation methods are described below in the Examples. Further details of such methods are also described in WO 2021/048221, the contents of which are incorporated herein in their entirety.

Typically, the inactivation step substantially eliminates infectivity of mammalian (e.g. human) cells by the inactivated SARS-CoV-2 particle. For instance, infectivity of mammalian cells may be reduced by at least 99%, 99.99% or 99.9999% as compared to a native SARS-CoV-2 particle, or infectivity of human cells by the inactivated A SARS-CoV-2 particle may be undetectable. Standard assays may be used for determining residual infectivity and effective viral titer, e.g. plaque assays, determination of TCID50 (50% tissue culture infectious dose). For instance, the mammalian cells may be MDCK, COS or Vero cells.

In preferred embodiments of the present invention, a native surface conformation of the SARS-CoV-2 particles is preserved in the inactivated virus particles. By this it is meant that e.g. one or more or all immunogenic (neutralizing) epitopes are retained in the inactivated virus particles, such that the inactivated particles are capable of generating neutralizing antibodies against native SARS-CoV-2 particles when administered to a human subject. By “native surface conformation” it is meant to refer to the surface conformation found in native SARS-CoV-2 particles, i.e. SARS-CoV-2 particles (virions) that have not been inactivated. The property of the vaccine or inactivated SARS-CoV-2 particles in generating neutralizing antibodies in a subject may be determined using e.g. a plaque reduction neutralization test (PRNT assay), e.g. using a serum sample from the subject as known in the art.

In preferred embodiments, the present invention comprises that a native conformation of (i) spike (S) protein; (ii) nucleocapsid (N) protein; (iii) membrane (M) glycoprotein; and/or (iv) envelope (E) protein is preserved in the inactivated viral particles. Preferably, the inactivated SARS-CoV-2 particle comprises a native conformation spike (S) protein. Thus, the S (and/or N and/or M and/or E) protein in the inactivated SARS-CoV-2 particle preferably comprises one or more or all (intact) immunogenic (neutralizing) epitopes present in native SARS-CoV-2 particles. Preferably, the S (and/or N and/or M and/or E) protein in the inactivated viral particles is not modified, or not substantially modified by the inactivation step.

Preservation of the surface conformation of the viral particles can be assessed using standard techniques. For instance, methods such as X-ray crystallography, MS analysis (shift of amino acid mass by modification) and cryo-electron microscopy may be used to visualize the virus surface. The secondary and tertiary structures of proteins present on the surface of viral particles may also be analyzed by methods such as by circular dichroism (CD) spectroscopy (e.g. in the far (190-250 nm) UV or near (250-300 nm) UV range). Moreover, preservation of a native surface conformation can be confirmed by using antibodies directed against epitopes present on the native viral surface, e.g. in the S protein. Cross-reaction of anti-SARS-CoV-2 antibodies between the inactivated and native virus particles can thus be used to demonstrate retention of potentially neutralizing epitopes in the vaccine.

The surface conformation of SARS-CoV-2 virions and in particular the spike (S) protein is known, and has been published in several recent studies. See for instance Shang, J. et al. (Structural basis of receptor recognition by SARS-CoV-2. Nature https://doi.org/10.1038/s41586-020-2179-y (2020)), which describes the crystal structure of the SARS-CoV-2 receptor binding domain. In addition, Walls et al. (Structure, Function, and Antigenicity of the SARS-CoV-2 Spike Glycoprotein, Cell 180, 1-12 (2020), https://doi.Org/10.1016/j.cell.2020.02.058) provides a detailed description of the S protein surface conformation using cryo-EM, and describes cross-neutralizing antibodies that target conserved S protein epitopes. The use of antibodies from sera of infected and convalescent patients has shed further light on important S protein epitopes (Zhang B et al. Mining of epitopes on spike protein of SARS- CoV-2 from COVID-19 patients. 2020 Cell Research 30:702-704). Recent studies have also focused on the structure of the SARS-CoV-2 nucleocaspid (N) protein, which has been confirmed as an important antigen in studies using convalescent sera (Zeng W et al. Biochemical characterization of SARS-CoV- 2 nucleocapsid protein. 2020 BBRC 527(3): 618-623). Further guidance with regard to potentially important SARS-CoV-2 epitopes is available in the COVIEdb database, a compilation of information from coronavirus epitope mapping studies (http://biopharm.zju.edu.cn/coviedb/; Wu J COVIEdb: A Database for Potential Immune Epitopes of Coronaviruses. 2020 Front. Pharmacol. 11:572249; doi: 10.3389/fphar.2020.572249).

Monoclonal antibodies against SARS-CoV-2 surface epitopes (including in the S protein) are described in the literature (e.g. as mentioned above), available from commercial sources and/or can be generated using standard techniques, such as immunization of experimental animals. For example, as of September 9, 2020, at least 169 different antibodies against SARS-CoV-2 were available from MyBioSource, Inc., San Diego, CA (e.g. cat. no. MBS8574747, see www.MyBioSource.com). On the same date at least 28 different antibodies against SARS-CoV-2 were available from Sino Biological US Inc., Wayne, PA (e.g. cat. no. 40150-D006, see https://www.sinobiological.com/). Further suitable antibodies are described in Ou et al. (Characterization of spike glycoprotein of SARS-CoV-2 on virus entry and its immune cross-reactivity with SARS-CoV, Nature Communications (2020) 11: 1620; https://doi.org/10.1038/s41467-020-15562-9). In embodiments of the present invention, a skilled person can detect preservation of a native surface conformation of SARS-CoV-2 (or e.g. the S or N protein thereof) via cross-reaction of such antibodies with the inactivated particles. In other words, the inactivated particles bind specifically to one or more anti-SARS-CoV-2 antibodies directed against surface epitopes, preferably anti-S-protein antibodies, e.g. to antibodies generated against neutralizing epitopes in native SARS-CoV-2 virions.

The SARS-CoV-2 particles in the vaccine composition may be derived from any known strain of SARS- CoV-2 and one or more variants thereof. For instance, the two or more viruses may be selected from a strain as defined in Figure 2 or 8 to 11, or may comprise a nucleotide or amino acid sequence as defined therein, or a variant sequence having at least e.g. 95% sequence identity thereto. For instance, in one embodiment, the SARS-CoV-2 particle comprises an RNA sequence corresponding to a DNA sequence (i) as defined in SEQ ID NO: 1. In another embodiment, the SARS-CoV-2 particle comprises an RNA sequence corresponding to a DNA sequence (i) as defined in SEQ ID NO: 9. By “corresponding to”, it will be understood that the defined DNA sequence is an equivalent of the viral RNA sequence, i.e. is a DNA or cDNA sequence that encodes the viral RNA or a sequence complementary to the viral RNA. As described herein, the inactivation process may result in modification (e.g. alkylation or acylation) and/or fragmentation of viral RNA, and thus it will be understood that the inactivated viral particles may not comprise an intact RNA sequence as defined herein, but rather are derived from native viral particles which do comprise such a sequence.

The SARS-CoV-2 particles may also comprise variants of the known SARS-CoV-2 Wuhan-Hu-1 lineage or also referred to as the reference lineage or the INMI isolate, e.g. sequences having at least 85%, at least 90%, at least 95% or at least 99% sequence identity to SEQ ID NO: 1 and/or NCBI Reference Sequence NC_045512.2 or MN908947 or sequences having at least 85%, at least 90%, at least 95% or at least 99% sequence identity to SEQ ID NO: 9 and/or NCBI accession number MT066156. Preferably, the variant sequence encodes an infectious SARS-CoV-2 particle, e.g. a native (non-inactivated) SARS-CoV-2 particle comprising the RNA sequence that is able to pack a virulent SARS-CoV-2 virus.

Further known SARS-CoV-2 particles may also comprise variants of the known SARS-CoV-2 such as variants of concern (see e.g. SARS-CoV-2 variants of concern as of 27 January 2022 (europa.eu)): South African lineage B. 1.351 (WHO label: Beta), e.g. sequences having at least 85%, at least 90%, at least 95% or at least 99% sequence identity to NCBI Reference Sequence MW598408. Preferably, the variant sequence encodes an infectious SARS-CoV-2 particle, e.g. a native (non-inactivated) SARS- CoV-2 particle comprising the RNA sequence that is able to pack a virulent SARS-CoV-2 virus. Further examples of variants of the known SARS-CoV-2 South African lineage B.1.351 are given in Figure 2.

Further known SARS-CoV-2 particles may also comprise variants ofthe known SARS-CoV-2 Brazilian lineage P.l (WHO label: Gamma), e.g. sequences having at least 85%, at least 90%, at least 95% or at least 99% sequence identity to NCBI Reference Sequence MW520923. Preferably, the variant sequence encodes an infectious SARS-CoV-2 particle, e.g. a native (non-inactivated) SARS-CoV-2 particle comprising the RNA sequence that is able to pack a virulent SARS-CoV-2 virus. Further examples of variants of the known SARS-CoV-2 Brazilian lineage P. 1 are given in Figure 2. Further known SARS-CoV-2 particles may also comprise variants of the known SARS-CoV-2 UK lineage B.1. 1.7, e.g. sequences having at least 85%, at least 90%, at least 95% or at least 99% sequence identity to NCBI Reference Sequence MW422256. Preferably, the variant sequence encodes an infectious SARS-CoV-2 particle, e.g. a native (non-inactivated) SARS-CoV-2 particle comprising the RNA sequence that is able to pack a virulent SARS-CoV-2 virus. Further examples of variants of the known SARS-CoV-2 UK lineage B.l.1.7 are given in Figure 2.

Further known SARS-CoV-2 particles may also comprise variants of the known SARS-CoV-2 India lineages B.1.617.2 (WHO label: Delta), e.g. sequences having at least 85%, at least 90%, at least 95% or at least 99% sequence identity to SEQ ID NO: 2 or more generally a SARS-CoV-2 with a S-protein with spike mutations of interest: E452R. T478K. D6 I4G. P68 IR. Preferably, the variant sequence encodes an infectious SARS-CoV-2 particle, e.g. a native (non-inactivated) SARS-CoV-2 particle comprising the RNA sequence that is able to pack a virulent SARS-CoV-2 virus. Further examples of variants of the known SARS-CoV-2 Californian lineages are listed in Figure 2.

Further known SARS-CoV-2 particles may also comprise variants of the known SARS-CoV-2 South African/Botswana lineages B. 1.1.529 (WHO label: Omicron), e.g. sequences having at least 85%, at least 90%, at least 95% or at least 99% sequence identity to SEQ ID NOs: 3 or 4 or more generally any SARS-CoV-2 with a S-protein with spike mutations of interest: A67V, A69-70, T95I, G142D, A143- 145, N21 H, A212, ins215EPE, 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. Preferably, the variant sequence encodes an infectious SARS-CoV-2 particle, e.g. a native (non-inactivated) SARS-CoV-2 particle comprising the RNA sequence that is able to pack a virulent SARS-CoV-2 virus.

Similarly, in preferred embodiments the SARS-CoV-2 particle comprises an S protein of the Wuhan lineage comprising or consisting of (i) an amino acid sequence as defined in SEQ ID NO: 5 (see Figure 12), or (ii) an amino acid sequence having at least 95%, at least 97% or at least 99% identity to SEQ ID NO: 5.

In further preferred embodiments the SARS-CoV-2 particle comprises an S protein of the South African Bl.351 lineage comprising or consisting of (i) an amino acid sequence as defined in genebank, or (ii) an amino acid sequence having at least 95%, at least 97% or at least 99% identity to said GenBank sequence. In further preferred embodiments the SARS-CoV-2 particle comprises an S protein of the Brazilian P. 1 lineage comprising or consisting of (i) an amino acid sequence as defined in genebank, or (ii) an amino acid sequence having at least 95%, at least 97% or at least 99% identity to said GenBank sequence.

In further preferred embodiments the SARS-CoV-2 particle comprises an S protein of Delta variant comprising or consisting of (i) an amino acid sequence as defined in GenBank, or (ii) an amino acid sequence having at least 95%, at least 97% or at least 99% identity to said GenBank sequence.

In further preferred embodiments the SARS-CoV-2 particle comprises an S protein of Omicron variant comprising or consisting of (i) an amino acid sequence as defined in GenBank, or (ii) an amino acid sequence having at least 95%, at least 97% or at least 99% identity to said GenBank sequence.

In all of the above embodiments, the inactivated SARS-CoV-2 particles are combined with other inactivated SARS-CoV-2 particles in the vaccine (other = other sequence from another variant of concern).

In some embodiments, a combination of SARS-CoV-2 particles in the vaccine comprises or consists of at least two SARS-CoV-2 particles selected from the group consisting of i) the reference Wuhan_l lineage such as e.g. SEQ ID Nos: 1 or the INMI isolate provided by SEQ ID NO: 9; ii) the Delta variant such as e.g. SEQ ID NO: 2; or iii) the Omicron variant such as e.g. SEQ ID NO: 3 or SEQ ID NO: 4.

In a further embodiments, a combination of SARS-CoV-2 particles in the vaccine comprises or consists of at least three SARS-CoV-2 particles selected from the group consisting of i) the reference Wuhan_l lineage such as e.g. SEQ ID No: 1 or the INMI isolate provided by SEQ ID NO: 9; ii) the Delta variant such as e.g. SEQ ID NO: 2; or iii) the Omicron variant such as e.g. SEQ ID NO: 3 or SEQ ID NO: 4.

In a further embodiment, one or more of the SARS-CoV-2 particles of the above comprise viral RNA wherein the furin cleavage site activity is reduced or eliminated by passaging out the furin site and/or introducing mutations in the cleavage site.

The similarity between amino acid sequences and/or nucleic acid sequences is expressed in terms of the similarity between the sequences, otherwise referred to as sequence identity. Sequence identity is frequently measured in terms of percentage identity; the higher the percentage, the more similar the two sequences are. Homologs, orthologs, or variants of a polynucleotide or polypeptide will possess a relatively high degree of sequence identity when aligned using standard methods. Methods of alignment of sequences for comparison are well known in the art. Various programs and alignment algorithms are described in: Smith & Waterman, Adv. Appl. Math. 2:482, 1981; Needleman & Wunsch, Mol. Biol. 48:443, 1970; Pearson & Lipman, Proc. Natl. Acad. Sci. USA 85:2444, 1988; Higgins & Sharp, Gene, 73:237-44, 1988; Higgins & Sharp, CABIOS 5: 151-3, 1989; Corpet et al., Nuc. Acids Res. 16: 10881-90, 1988; Huang et al. Computer Appls. in the Biosciences 8, 155-65, 1992; and Pearson et al., Meth. Mol. Bio. 24:307-31, 1994. Altschul et al, J. Mol. Biol. 215:403-10, 1990, presents a detailed consideration of sequence alignment methods and homology calculations.

Once aligned, the number of matches is determined by counting the number of positions where an identical nucleotide or amino acid residue is present in both sequences. The percent sequence identity is determined by dividing the number of matches either by the length of the sequence set forth in the identified sequence, or by an articulated length (such as 100 consecutive nucleotides or amino acid residues from a sequence set forth in an identified sequence), followed by multiplying the resulting value by 100. Preferably, the percentage sequence identity is determined over the full length of the sequence. For example, a peptide sequence that has 1166 matches when aligned with a test sequence having 1554 amino acids is 75.0 percent identical to the test sequence (1166=1554* 100=75.0). The percent sequence identity value is rounded to the nearest tenth. For example, 75.11, 75.12, 75.13, and 75.14 are rounded down to 75.1, while 75.15, 75.16, 75.17, 75.18, and 75.19 are rounded up to 75.2. The length value will always be an integer.

The NCBI Basic Local Alignment Search Tool (BLAST) (Altschul et al., Mol. Biol. 215:403, 1990) is available from several sources, including the National Center for Biotechnology Information (NCBI, Bethesda, MD) and on the internet, for use in connection with the sequence analysis programs BLASTP, BLASTN, BLASTX, TBLASTN and TBLASTX. A description of how to determine sequence identity using this program is available on the NCBI website on the internet. The BLAST and the BLAST 2.0 algorithms are also described in Altschul et al., Nucleic Acids Res. 25:3389-3402, 1977. Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information (ncbi.nlm.nih.gov). The BLASTN program (for nucleotide sequences) uses as defaults a word length (W) of 11, alignments (B) of 50, expectation (E) of 10, M=5, N=-4, and a comparison of both strands. The BLASTP program (for amino acid sequences) uses as defaults a word length (W) of 3, and expectation (E) of 10, and the BLOSUM62 scoring matrix (see Henikoff & Henikoff, Proc. Natl. Acad. Sci. USA 89: 10915, 1989). Homologs and variants of a polynucleotide or polypeptide are typically characterized by possession of at least about 75%, for example at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity counted over at least 50, 100, 150, 250, 500, 1000, 2000, 5000 or 10,000 nucleotide or amino acid residues of the reference sequence, over the full length of the reference sequence or over the full length alignment with the reference amino acid sequence of interest. Polynucleotides or proteins with even greater similarity to the reference sequences will show increasing percentage identities when assessed by this method, such as at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity. For sequence comparison of amino acid or nucleic acid sequences, typically one sequence acts as a reference sequence, to which test sequences are compared. When using a sequence comparison algorithm, test and reference sequences are entered into a computer, subsequence coordinates are designated, if necessary, and sequence algorithm program parameters are designated. Default program parameters are used.

One example of a useful algorithm is PILEUP. PILEUP uses a simplification of the progressive alignment method of Feng & Doolittle, Mol. Evol. 35:351-360, 1987. The method used is similar to the method described by Higgins & Sharp, CABIOS 5: 151-153, 1989. Using PILEUP, a reference sequence is compared to other test sequences to determine the percent sequence identity relationship using the following parameters: default gap weight (3.00), default gap length weight (0.10), and weighted end gaps. PILEUP can be obtained from the GCG sequence analysis software package, e.g., version 7.0 (Devereaux et al., Nuc. Acids Res. 12:387-395, 1984).

As used herein, reference to "at least 80% identity" refers to at least 80%, 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 even 100% identity to a specified reference sequence, e.g. to at least 50, 100, 150, 250, 500, 1000, 5000 or 10,000 nucleotide or amino acid residues of the reference sequence or to the full length of the sequence. As used herein, reference to "at least 90% identity" refers to "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 even 100% identity" to a specified reference sequence, e.g. to at least 50, 100, 150, 250, 500, 1000, 5000 or 10,000 nucleotide or amino acid residues of the reference sequence or to the full length of the sequence.

In some embodiments, the inactivated SARS-CoV-2 particles are combined with an adjuvant in the vaccine. In some embodiments, the adjuvant is a Thl response-directing adjuvant (also referred to herein as “Thl adjuvant”). By this it is meant that when the vaccine is administered to a subject, the adjuvant promotes the induction of a predominantly T helper type 1 (i.e. Thl) immune response in the subject (rather than a Th2 type response). The Thl- or Th2-directing properties of commonly used vaccines are known in the art. It has surprisingly been found that using an adjuvant that promotes a predominantly Thl response can improve immunogenicity of the vaccine and thus antiviral responses, as well as reducing a risk of disadvantageous effects such as immunopathology (which may result from a predominantly Th2 type response possibly due to hypersensitivity against viral components).

In some embodiments, the adjuvant comprises 3-O-desacyl-4'-monophosphoryl lipid A (MPL), saponin QS-21, a CpG-containing oligodeoxynucleotide (CpG ODN), squalene, DL-a-tocopherol, a cationic peptide, a deoxyinosine-containing immunostimulatory oligodeoxynucleic acid molecule (I-ODN) and/or imiquimod. For instance, examples of suitable adjuvants may comprise: Adjuvant System 01 (AS01), which is a liposomal preparation comprising 3-O-desacyl-4'-monophosphoryl lipid A (MPL) and saponin QS-21; CpG 1018, a CpG ODN comprising the sequence 5’ TGACTGTGAACGTTCGAGATGA 3’ (SEQ ID NO: 8); Adjuvant System 03 (AS03), comprising squalene, DL-a-tocopherol and polysorbate 80; IC31, comprising a peptide comprising the sequence KLKL5KLK (SEQ ID NO: 7) and an I-ODN comprising oligo-d(IC)i₃ (SEQ ID NO: 6); or MF59, an oil-in-water emulsion comprising squalene, Tween 80 and Span 85.

In another embodiment, the vaccine or adjuvant does not comprise a CpG-containing oligodeoxynucleotide (CpG ODN). In another embodiment, the vaccine or adjuvant does not comprise CpG 1018, i.e. the vaccine or adjuvant does not comprise the sequence 5’ TGACTGTGAACGTTCGAGATGA 3’.

In some embodiments, the dosage of a Thl promoting adjuvant, such as especially AS01, AS03, MF59, imiquimod or CpG, will be arrived at empirically. In some embodiments, the dosage of the Thl promoting adjuvant will be determined from previous studies.

In alternative embodiments, the adjuvant may comprise an aluminium salt, e.g. aluminium oxide, aluminium hydroxide or aluminium phosphate. A preferred aluminium salt is the aluminium hydroxide with reduced Cu content, e.g. lower than 1.25 ppb based on the weight of the vaccine composition, an adjuvant described in detail in WO2013/083726 or Schlegl et al., Vaccine 33 (2015) 5989-5996. In some embodiments, an alum adjuvant is the only adjuvant in the vaccine composition. As referred to herein, the weight of the alum component refers to the weight of the Al³⁺ in the solution, regardless of what type of aluminium salt is used. For example, 0.5 mg of Al³⁺ corresponds to 1.5 mg alum. In one embodiment, the amount alum (Al³⁺) present in the SARS-CoV-2 vaccine composition is between about 0.1 and 2 mg/mL, between about 0.2 and 1.5 mg/mL, between about 0.5 and 1.3 mg/mL, especially between about 0.8 to 1.2 mg/mL, most preferably about 1 mg/mL, i.e., 0.5 mg/dose. However the use of aluminium adjuvants alone is generally less preferred in the present invention, as they tend to direct a predominantly Th2 type immune response. Therefore in embodiments where the vaccine comprises an aluminium salt, it is particularly preferred that the vaccine further comprises a Thl- directing adjuvant, e.g. as described above.

Thus in one embodiment, the adjuvant may comprise an aluminium salt and a CpG ODN, e.g. CpG 1018. CpG 1018 can be adsorbed onto alum and, when used as a combinatorial adjuvant, has been shown to induce both Thl and Th2 responses (Tian, et al. 2017 Oncotarget 8(28)45951-45964); i.e. a more “balanced” immune response. Particularly, when administered in combination with alum, CpG has been shown to increase the overall magnitude of the immune response and to reduce the Th2 bias that is induced by conventional adjuvants such as alum (X.P. loannou et al. CpG-containing oligodeoxynucleotides, in combination with conventional adjuvants, enhance the magnitude and change the bias of the immune responses to a herpesvirus glycoprotein. 2002 Vaccine 21: 127-137). The dose range for CpG in combination with alum may be anywhere between 10 pg and 1 mg per dose such as between 1 to 2 mg per dose. Further information regarding inactivated SARS-CoV-2 virus adjuvanted with CpG and alum can be found in WO2021/176434A1 and WO2021/178318A1, which are incorporated herein by reference in their entirety.

Typically, the adjuvant is combined with the inactivated SARS-CoV-2 particles during manufacture of the vaccine product, i.e. the manufactured vaccine product comprises the adjuvant and is sold/distributed in this form. In alternative embodiments the adjuvant may be combined with the inactivated SARS-CoV-2 particles at the point of use, e.g. immediately before clinical administration of the vaccine (sometimes referred to as “bedside mixing” of the components of the vaccine). Thus the present invention comprises both vaccine products comprising inactivated SARS-CoV-2 particles and an adjuvant as described herein, as well as kits comprising the individual components thereof (e.g. suitable for bedside mixing), and the combined use of the individual components of the vaccine in preventing or treating SARS-CoV-2 infection.

The SARS-CoV-2 vaccine may be produced by methods involving a step of inactivation of native SARS-CoV-2 particles, as described above. Generally, the native SARS-CoV-2 particles may be obtained by standard culture methods, e.g. by in vitro production in mammalian cells, preferably using Vero cells. For instance, the native SARS-CoV-2 particles may be produced using methods analogous to those described in e.g. WO 2017/109225 and/or WO 2019/057793, the contents of which are incorporated herein in their entirety, which describe methods for the production of Zika and Chikungunya viruses in Vero cells. The steps such as passaging, harvesting, precipitation, dialysis, filtering and purification described in those documents are equally applicable to the present process for producing SARS-CoV-2 particles.

For instance, in some embodiments, the method may comprise purifying the inactivated SARS-CoV-2 particles by one or more size exclusion methods such as (i) a sucrose density gradient centrifugation, (ii) a solid-phase matrix packed in a column comprising a ligand-activated core and an inactive shell comprising pores, wherein the molecular weight cut-off of the pores excludes the virus particles from entering the ligand-activated core, and wherein a molecule smaller than the molecular weight cut-off of the pores can enter the ligand-activated core and collecting the virus particles, and/or (iii) batch or size exclusion chromatography; to obtain purified inactivated SARS-CoV-2 particles. Preferably, in the resulting purified preparation of viral particles, (i) the concentration of residual host cell DNA is less than 100 ng/mL; (ii) the concentration of residual host cell protein is less than 1 pg/mL; and (iii) the concentration of residual aggregates of infectious virus particles is less than 1 pg/mL.

In some embodiments, the method may comprise a step of precipitating a harvested culture medium comprising SARS-CoV-2 particles, thereby producing native SARS-CoV-2 particles in a supernatant. The precipitating step may comprise contacting the culture medium with protamine sulfate or benzonase. By using such a step, both contaminating DNA derived from host cells as well as immature and otherwise non-infectious virus particles can be separated from the preparation. Moreover, protamine sulfate can be very efficiently separated from the virus fraction, e.g. using sucrose density centrifugation or a solid-phase matrix packed in a column comprising a ligand-activated core and an inactive shell comprising pores, wherein the pores comprise a molecular weight cut-off that excludes the virus particles from entering the ligand-activated core, and wherein a molecule smaller than the molecular weight cut-off of the pores (e.g. the protamine sulfate) can enter the ligand-activated core, allowing for a safer vaccine produced at high yields.

Thus the residual host cell DNA of the obtained virus preparation or vaccine may be less than 1 pg/mL, especially less than 900, 800, 700, 600, 500, 400, 300 or 200 ng/mL, preferably less than 150 or 100 ng/mL. In a preferred embodiment, the residual host cell DNA of the virus preparation or vaccine is less than 40 pg/mL. In some embodiments, the residual host cell protein of the virus preparation or vaccine is less than 10 pg/mL, especially less than 9, 8, 7, 6, 5, 4, 3 or 2 pg/mL, preferably less than 1 pg/mL. In a preferred embodiment, the residual host cell protein of the virus preparation or vaccine is less than 150 ng/mL. In some embodiments, the residual non-infectious virus particles of the virus preparation or vaccine is less than 10 pg/mL, especially less than 9, 8, 7, 6, 5, 4, 3 or 2 pg/mL, preferably less than 1 pg/mL. In a preferred embodiment, the content of residual non-infectious virus particles of the virus preparation or vaccine is less than 100 ng/mL.

In some embodiments, the vaccine and/or SARS-CoV-2 particles may comprise residual protamine (e.g. protamine sulfate), typically in trace amounts. In some embodiments, residual protamine (e.g. protamine sulfate) in the virus preparation or vaccine is less than 2 pg/mL or 1 pg/mL, especially less than 900, 800, 700, 600, 500, 400, 300 or 200 ng/mL, preferably less than 100 ng/mL, more preferably is below the detection limit of HPLC, in particular below the detection limit in the final drug substance. In some embodiments, the PS content is tested by HPLC or size exclusion chromatography (SEC). For example, HPLC is validated for PS determination in JEV sucrose gradient pool samples as a routine release assay and is very sensitive (i.e., limit of quantification (LOQ) 3 pg/mL; limit of detection (LOD) 1 pg/mL). In the current invention, PS content in SARS-CoV-2 drug substance was <LOD. In one embodiment, the HPLC assessment of PS content can be performed on a Superdex Peptide 10/300GL column (GE: 17-5176-01) using 30% Acetonitrile, 0,1% Trifluoroacetic acid as solvent with a flow rate of 0.6 ml/min at 25°C and detection at 214 nm. A more sensitive method of measurement for residual protamine in a purified virus preparation is mass spectrometry (MS). In some embodiments, the residual PS levels in a Zika virus preparation are tested by MS or other such highly sensitive method, e.g. nuclear magnetic resonance (NMR). With this method, residual PS, as well as fragments and/or break-down products of PS, can be detected at trace amounts, such as levels as low as, for example, 10⁶, 10⁷ or 10⁸ molecules per typical sample load. In some embodiments, the PS levels are tested in the drug product. In some embodiments, the PS levels are tested in the drug substance.

Preferably an amount of the inactivating agent (e.g. beta-propiolactone) in the drug product or drug substance (e.g. vaccine composition) is very low, e.g. less than 100 ppm, less than 10 ppm, or less than 1 ppm (by weight).

The SARS-CoV-2 vaccine may be administered to a subject, preferably a mammalian subject, more preferably a human subject. Typically the SARS-CoV-2 vaccine is administered to a subject at risk of SARS-CoV-2 infection, e.g. in order to prevent SARS-CoV-2 infection and/or to prevent SARS-CoV- 2 associated disease (COVID- 19), in particular to prevent severe COVID- 19 disease, hospitalization or death caused by SARS-CoV-2 infection. The subject is preferably (i) an elderly subject (e.g. older than 65 years, 70 years or 80 years) (ii) a pregnant subject (iii) an immunocompromised subject or (iv) a child (e.g. a person younger than 18 years, 16 years, 14 years, 12 years, 10 years, 8 years, 6 years, 4 years, 2 years or younger), i.e. the vaccine is particularly suitable for vulnerable subjects. The SARS- CoV-2 vaccine described herein is advantageously capable of generating robust immune responses in subjects particularly susceptible or vulnerable to SARS-CoV-2-mobidity or mortality, i.e. immunocompromised, pregnant or elderly subjects. The SARS-CoV-2 vaccine may be administered to the subject in a single dose or two or more doses, e.g. separated by intervals of about 7, 14, 21, 28 or 29 days.

In a preferred embodiment, on administration to a human subject the vaccine does not induce ADE, VAERD or ERD of SARS-CoV-2-associated disease (CO VID-19). It is an advantage of the present invention that the inactivated SARS-CoV-2 vaccine described herein shows low or no ADE, VAERD or ERD in human subjects, and can therefore be safely used for mass vaccination purposes. In particular, the vaccine described herein retains high quality immunogenic epitopes, which therefore results in high neutralizing antibody titers and diminishes the risk of ADE, VAERD or ERD on administration to subjects. The risk of ADE, VAERD or ERD development may be assessed in non-human primates (see also Luo F, et al. (2018), Virologica Sinica 33:201-204).

In another preferred embodiment, on administration to a human subject the vaccine does not result in immunopathology. It is known that under some circumstances, a vaccine (e.g. a SARS-CoV vaccine) can result in e.g. a Th2-type immunopathology, e.g. a hypersensitivity response to SARS-CoV components in animals. In embodiments of the present invention, a Thl type response is favored, e.g. by use of a Thl-directing adjuvant (e.g. AS01 or another adjuvant as described herein). Especially, a balanced Th2/Thl-type immune response is preferred, such as that induced by use of a Th2-stimulating adjuvant, e.g., alum, combined with a Thl -stimulating adjuvant. The risk of immunopathology developing may be assessed in animal models, e.g. as described in Tseng C.T. et al. (2012) PLoS ONE 7(4):e35421. In a preferred embodiment of the current invention, the vaccines of the invention show a shift in the Th2/Thl-type immune response to a Thl -type immune response compared to a vaccine adjuvanted with alum.

Any of the SARS-CoV-2 vaccines or compositions described herein may be administered to a subject in a therapeutically effective amount or a dose of a therapeutically effective amount. As used herein, a “therapeutically effective amount” of vaccine is any amount that results in a desired response or outcome in a subject, such as those described herein, including but not limited to prevention of infection, an immune response or an enhanced immune response to SARS-CoV-2, or prevention or reduction of symptoms associated with SARS-CoV-2 disease. More specifically, a therapeutic amount of the SARS- CoV-2 vaccine of the invention may be a total viral protein mass of between about 0.05 and 50 pg, more preferably between about 0.5 to 10 pg. In some embodiments, the therapeutically effective amount of a SARS-CoV-2 vaccine or composition described herein is an amount sufficient to generate antigen-specific antibodies (e.g., anti-SARS-CoV- 2 antibodies). In some embodiments, the therapeutically effective amount is sufficient to seroconvert a subject with at least 70% probability. In some embodiments, the therapeutically effective amount is sufficient to seroconvert a subject with at least 75%, 80%, 85% 90%, 95%, 96%, 97%, 98%, or at least 99% probability. Whether a subject has seroconverted can be assessed by any method known in the art, such as obtaining a serum sample from the subject and performing an assay to detect anti-SARS-CoV- 2 antibodies. In some embodiments, a subject is seroconverted if a serum sample from the subject contains an amount of anti- SARS-CoV-2 antibodies that surpasses a threshold or predetermined baseline. A subject is generally considered seroconverted if there is at least a 4-fold increase in anti- SARS-CoV-2 antibodies (i.e., anti-SARS-CoV-2 S protein IgG antibodies) present in a serum sample from the subject as compared to a serum sample previously taken from the same subject.

In one embodiment, the dose of the inactivated SARS-CoV-2 component in the vaccine composition of the current invention is between about 0.01 and 25 mAU (milli-absorption units x minutes as assessed by SEC-HPLC), preferably between about 0.05 and 10 mAU, more preferably between about 0.1 and 5 mAU, most preferably between about 0.25 and 2.5 mAU. In one embodiment, the dose of each of inactivated SARS-CoV-2 component is between about 0.05 and 50 pg total protein as measured by (p)BCA assay, between about 0.1 and 25 pg, between about 0.25 and 12.5 pg, preferably between about 0.5 and 5 pg total protein. More preferably the dose of each of the inactivated SARS-CoV-2 component in the vaccine composition is at least 2.5 pg total protein, at least 3.5 pg total protein or at least 2.5 pg total protein, e.g. the vaccine composition comprises 2.5 pg to 25 pg, 3.5 pg to 20 pg or 4 pg to 12 pg total protein/dose, preferably about 10 pg total protein/dose, e.g. 2 times 5 pg protein of each inactivated SARS-CoV-2 component. In some embodiments, the dosage is determined by the total amount of S protein in the inactivated SARS-CoV-2 formulation, as assessed by e.g. EUISA. The mass of antigen may also be estimated by assessing the SE-HPLC peak area per dose equivalent (recorded as milli- absorption units x minutes; mAU), which is estimated to be approximately 2 pg/ml total surface protein and approximately 1 pg/mL S-protein. In one embodiment, the dose is between about 0.025 and 25 pg S-protein as measured by ELISA, between about 0.05 and 12.5 pg, between about 0.125 and 6.25 pg, preferably between about 0.25 and 2.5 pg S-protein.

In a preferred embodiment, the amount of antigen in the SARS-CoV-2 vaccine is determined by ELISA. In one embodiment, the ELISA measures a SARS-CoV-2 protein or portion of a protein, e.g., nucleocapsid (N), membrane (M) or spike (S) protein; i.e., the ELISA utilizes a coating antibody specific to a SARS-CoV-2 protein or portion of a protein. In a preferred embodiment, the coating antibody is specific to the SARS-CoV-2 Spike protein SI subunit, e.g. residues 14-685 (or 14-683) of the S-protein sequence of SEQ ID NO: 5, or to the Receptor Binding Domain (RBD), e.g. residues 331 to 528 (or 319 to 541) of the S-protein sequence of SEQ ID NO: 5. In one embodiment, the ELISA readout is a mass per unit measure of the detected protein, e.g. pg/mL S-protein. In a preferred embodiment, the standard used is a spike protein trimer and the results of the SARS-CoV-2 ELISA are reported as “antigen units” (AU), corresponding to the ACE-2 binding ability of the standard protein (determined by the manufacturer).

In one embodiment, the amount of each of the SARS-CoV-2 particle administered to a subject is between about 1 to 150 AU/dose, preferably between about 2 to 75 AU/dose, preferably between about 3 and 60 AU/dose, more preferably between about 3 and 55 AU/dose, more preferably between about 33 AU/dose (if e.g. 2 different SARS-CoV-2 particles are combined in a vaccine, the total amount of the two components is about 66 AU/dose). In further preferred embodiments, the amount of each SARS- CoV-2 antigen administered to a subject is at least 10 AU/dose, at least 20 AU/dose, at least 25 AU/dose or at least 30 AU/dose, e.g. about 10 to 60 AU/dose, 20 to 50 AU/dose, 25 to 45 AU/dose or 30 to 40 AU/dose, e.g. about 35 AU/dose. The amount of each SARS-CoV-2 particle (e.g. in AU/dose) may be assessed, for example, by a SARS-CoV-2 ELISA assay as described in Example 1. It is estimated that there are about 1 to 1.5 x 10⁷ viral particles per AU, and the amounts of SARS-CoV-2 particle described above may be construed accordingly. Thus in some embodiments, the amount of each SARS-CoV-2 antigen administered to a subj ect is between about 1.5 x 10⁷ to 1.5 x 10⁹ viral particles/dose, or between about 4.5 x 10⁷ to 9.0 x 10⁸ viral particles/dose, e.g. at least 1.5 x 10⁸ viral particles/dose or at least 3.0 x 10⁸ viral particles/dose, about 1.5 x 10⁸ to 7.5 x 10⁸ viral particles/dose or about 4.5 x 10⁸ to 6.0 x 10⁸ viral particles/dose.

In a further embodiment, the ratio of the two or more different inactivated SARS-CoV-2 particles is equal, i.e. in case of two inactivated SARS-CoV-2 particles it can be 1 : 1 but also may be 1 :2 or 2: 1 or 1:3 or 3: 1. The ratio depends on the ability of one of the inactivated SARS-CoV-2 particles in the vaccine to generating more neutralizing antibodies against a native homologous and/or heterologous SARS-CoV-2 particle and/or is capable of raising more of an effective T-Cell response against a native homologous and/or heterologous SARS-CoV-2 particle in a human subject as the other SARS-CoV-2 particle. Thus depending on the ability of one vs the other SARS-CoV-2 particle to generating more neutralizing antibodies against a native homologous and/or heterologous SARS-CoV-2 particle and/or is capable of raising more of an effective T-Cell response against a native homologous and/or heterologous SARS-CoV-2 particle in a human subject the ratio may be adjusted. In some embodiments, seroconversion of a subject is assessed by performing a plaque reduction neutralization test (PRNT). Briefly, PRNT is used to determine the serum titer required to reduce the number of SARS-CoV-2 plaques by 50% (PRNT50) as compared to a control serum/antibody. The PRNT50 may be carried out using monolayers of Vero cells or any other cell type/line that can be infected with SARS-CoV-2. Sera from subjects are diluted and incubated with live, non-inactivated SARS-CoV-2. The serum/virus mixture may be applied to Vero cells and incubated for a period of time. Plaques formed on the Vero cell monolayers are counted and compared to the number of plaques formed by the SARS-CoV-2 in the absence of serum or a control antibody. A threshold of neutralizing antibodies of 1 : 10 dilution of serum in a PRNT50 is generally accepted as evidence of protection in the case of JEV (Hornbach et. al. Vaccine (2005) 23:5205-5211).

In some embodiments, the two or more SARS-CoV-2 particles may be formulated for administration in a composition, such as a pharmaceutical composition. The term “pharmaceutical composition” as used herein means a product that results from the mixing or combining of at least one active ingredient, such as an inactivated SARS-CoV-2, and one or more inactive ingredients, which may include one or more pharmaceutically acceptable excipient. A preferred pharmaceutically acceptable excipient is human serum albumin (HSA), such as, especially recombinant HSA (rHSA). In one embodiment, the SARS-CoV-2 vaccine of the invention contains about 10 to 50 pg HSA/dose, preferably about 20 to 40 pg HSA/dose, more preferably about 25 to 35 pg HSA/dose.

In some embodiments, the two or more SARS-CoV-2 particles may be not formulated for administration in the same composition, such as a pharmaceutical composition but in two different compositions and then assembled in a kit.

Pharmaceutical compositions of the invention, including vaccines, can be prepared in accordance with methods well known and routinely practiced in the art (see e.g., Remington: The Science and Practice of Pharmacy, Mack Publishing Co. 20th ed. 2000; and Ingredients of Vaccines - Fact Sheet from the Centers for Disease Control and Prevention, e.g., adjuvants and enhancers as described above to help the vaccine improve its work, preservatives and stabilizers to help the vaccine remain unchanged (e.g., albumin, such as human serum albumin (HSA) or recombinant HSA (rHSA), phenols, glycine)). As used herein, the term “vaccine” refers to an immunogenic composition, e.g. a composition capable of inducing an immune response in a (human) subject against an antigen (e.g. against a SARS-CoV-2 antigen). For instance, the vaccine or composition may be capable of generating neutralizing antibodies against SARS-CoV-2. In some embodiments, the vaccine or composition is capable of generating antibodies (e.g. IgG) against SARS-CoV-2 S (spike) protein. In some embodiments, the vaccine or composition is capable of generating a T cell response against SARS-CoV-2 proteins or peptides, for instance a T cell response against a SARS-CoV-2 S-protein, membrane (M) protein and/or nucleocapsid (N) protein or peptides derived therefrom. Typically the vaccine or immunogenic composition is capable of inducing a protective effect against a disease caused by the antigen, e.g. a protective effect against SARS-CoV-2 infection (e.g. symptomatic and/or asymptomatic infection), severe disease, hospitalization or death caused by COVID-19 disease).

Pharmaceutical compositions are preferably manufactured under GMP conditions. Typically, a therapeutically effective dose of the inactivated SARS-CoV-2 vaccine preparation is employed in the pharmaceutical composition of the invention. The inactivated SARS-CoV-2 particles are formulated into pharmaceutically acceptable dosage forms by conventional methods known to those of skill in the art. Dosage regimens are adjusted to provide the optimum desired response (e.g., the prophylactic response).

Dosages of the active ingredients in the pharmaceutical compositions of the present invention can be varied so as to obtain an amount of the active ingredient which is effective to achieve the desired pharmaceutical response for a particular subject, composition, and mode of administration, without being toxic to the subject. The selected dosage level depends upon a variety of pharmacokinetic factors, including the activity of the particular compositions of the present invention employed, the route of administration, the time of administration, the rate of excretion of the particular compound being employed, the duration of the treatment, other drugs, compounds and/or materials used in combination with the particular compositions employed, the age, sex, weight, condition, general health and prior medical history of the subject being treated, and like factors.

Primary vaccination: A physician, veterinarian or other trained practitioner, can start dosing of the inactivated SARS-CoV-2 vaccine employed in the pharmaceutical composition at levels lower than that required to achieve the desired therapeutic effect and gradually increase the dosage until the desired effect (e.g., production of anti-SARS-CoV-2 virus antibodies) is achieved. In general, effective doses of the compositions of the present invention, for the prophylactic treatment of groups of people as described herein vary depending upon many different factors, including means of administration, target site, physiological state of the patient, whether the patient is human or an animal, other medications administered, and the titer of anti-SARS-CoV-2 antibodies desired. Dosages need to be titrated to optimize safety and efficacy. In some embodiments, the dosing regimen entails subcutaneous or intramuscular administration of a dose of inactivated SARS-CoV-2 vaccine twice (primary vaccination). In some embodiments, the dosing regimen entails subcutaneous administration of a dose of inactivated SARS-CoV-2 vaccine twice, once at day 0 and once at about day 14. In some embodiments, the dosing regimen entails subcutaneous administration of a dose of inactivated SARS- CoV-2 vaccine twice, once at day 0 and once at about day 28. In some embodiments, the inactivated SARS-CoV-2 vaccine is administered to the subject once. In a preferred embodiment, the SARS-CoV- 2 vaccine is administered to the subject more than once, preferably two times. In a preferred embodiment, the vaccine is administered on day 0 and day 21. In another preferred embodiment, the vaccine is administered on day 0 and day 28.

Booster vaccination: In further embodiments, a so called booster dose of the inactivated SARS-CoV-2 vaccine of the invention is administered at least after about every 6 months, about every 7 months, about every 8 months, about every 9 months, about every 10 months, about every 11 months or about every 12 months or about every 13 months after the last dose of the SARS-CoV-2 vaccine, preferably wherein such further dose of the vaccine is the same formulation as the first. Thus in some embodiments, the booster dose of the inactivated SARS-CoV-2 vaccine is administered once after about 6 to 12 months after the primary vaccination.

In other embodiments, the inactivated SARS-CoV-2 vaccine is administered as a booster dose only, e.g. a first (prime) dose or doses of a (different) SARS-CoV-2 vaccine (e.g. vector or mRNA vaccine) is administered and then a second (boost) dose of the inactivated SARS-CoV-2 vaccine of the invention is administered, e.g. at least 180 or 360 days after the first dose. The first (prime) dose of the SARS- CoV-2 vaccine may comprise any other vaccine or immunogenic composition that stimulates an immune response and/or a protective effect in subjects against SARS-CoV-2 virus. For example, the first dose of SARS-CoV-2 vaccine may comprise a recombinant viral vector or an mRNA sequence encoding one or more SARS-CoV-2 proteins and/or fragments thereof, e.g. a SARS-CoV-2 spike (S) protein. Alternatively the first dose of SARS-CoV-2 vaccine may comprise a subunit vaccine, e.g. comprising one or more SARS-CoV-2 proteins and/or fragments thereof, e.g. a SARS-CoV-2 spike (S) protein or fragment thereof.

Also within the scope of the present disclosure are kits for use in prophylactic administration to a subject, for example to prevent or reduce the severity of SARS-CoV-2 infection. Such kits can include one or more containers comprising a composition containing two or more inactivated SARS-CoV-2, such as an inactivated SARS-CoV-2 vaccine of the invention. In some embodiments, the kit may further include one or more additional components comprising a second composition, such as a second vaccine, e.g. a second kind of SARS-CoV-2 vaccine that applies a different technology than in the first dose. In some embodiments, the second vaccine is a vaccine for an arbovirus. In some embodiments, the second vaccine is a Japanese encephalitis virus vaccine, a Zika virus vaccine, a Dengue virus vaccine and/or a Chikungunya virus vaccine.

In some embodiments, the kit can comprise instructions for use in accordance with any of the methods described herein. The included instructions can comprise a description of administration of the composition containing inactivated SARS-CoV-2 vaccine to prevent, delay the onset, or reduce the severity of SARS-CoV-2 infection. The kit may further comprise a description of selecting a subject suitable for administration based on identifying whether that subject is at risk for exposure to SARS- CoV-2 or contracting a SARS-CoV-2 infection. In still other embodiments, the instructions comprise a description of administering a composition containing inactivated SARS-CoV-2 vaccine to a subject at risk of exposure to SARS-CoV-2 or contracting SARS-CoV-2 infection.

The instructions relating to the use of the composition containing inactivated SARS-CoV-2 vaccine generally include information as to the dosage, dosing schedule, and route of administration for the intended treatment. The containers may be unit doses, bulk packages (e.g., multi-dose packages, multi- vials) or sub-unit doses. Instructions supplied in the kits of the invention are typically written instructions on a label or package insert (e.g., a paper sheet included in the kit), but machine -readable instructions are also acceptable.

The kits of the present disclosure are in suitable packaging. Suitable packaging includes, but is not limited to, vials, bottles, jars, flexible packaging, and the like. Also contemplated are packages for use in combination with a specific device, such as a syringe or an infusion device. The container may have a sterile access port, for example the container may be a vial having a stopper pierceable by a hypodermic injection needle. At least one active agent in the composition is an inactivated SARS-CoV- 2, as described herein.

This invention is not limited in its application to the details of construction and the arrangement of components set forth in the following description or illustrated in the drawings. The invention is capable of other embodiments and of being practiced or of being carried out in various ways. Also, the phraseology and terminology used herein is for the purpose of description and should not be regarded as limiting. The use of “including”, “comprising”, or “having”, “containing”, “involving”, and variations thereof herein, is meant to encompass the items listed thereafter and equivalents thereof as well as additional items. Unless otherwise defined herein, scientific and technical terms used in connection with the present disclosure shall have the meanings that are commonly understood by those of ordinary skill in the art. Further, unless otherwise required by context, singular terms shall include pluralities and plural terms shall include the singular. The methods and techniques of the present disclosure are generally performed according to conventional methods well-known in the art. Generally, nomenclatures used in connection with, and techniques of biochemistry, enzymology, molecular and cellular biology, microbiology, virology, cell or tissue culture, genetics and protein and nucleic chemistry described herein are those well-known and commonly used in the art. The methods and techniques of the present disclosure are generally performed according to conventional methods well known in the art and as described in various general and more specific references that are cited and discussed throughout the present specification unless otherwise indicated.

The present invention is further illustrated by the following examples, which in no way should be construed as further limiting. The entire contents of all of the references (including literature references, issued patents, published patent applications, and co-pending patent applications) cited throughout this application are hereby expressly incorporated by reference, in particular for the teaching that is referenced hereinabove. However, the citation of any reference is not intended to be an admission that the reference is prior art.

EXAMPLES

Example 1. Drug substance production

For the production of SARS-CoV-2, the JEV process platform (Srivastava et al., Vaccine 19 (2001) 4557-4565; US 6,309,650Bl) was used as a basis, also taking into account improvements in the process as adapted to Zika virus purification as disclosed in WO2017/109223A1 (which is incorporated herein in its entirety). Briefly, non-infectious SARS-CoV-2 particle aggregates, host cell proteins and other low molecular weight impurities are removed by protamine sulfate precipitation or benzonase treatment and the resulting preparation is optionally further purified by sucrose gradient centrifugation. See Fig. 1 for an outline of the production process.

The first SARS-CoV-2 isolate from Italy, identified and characterized at the National Institute for Infectious Diseases “Lazzaro Spallanzani” IRCCS, Rome, Italy (Accession No: MT066156), the RNA sequence thereof corresponding to the DNA sequence provided by SEQ ID NO: 9, was used in all Examples disclosed herein. Other novel coronavirus SARS-CoV-2 isolates may also be obtained from the following sources:

1. EVAg (European Virus Archive), e.g. one of the following strains: 2019-nCoV/Italy-INMIl, (Ref-SKU:008V-03893, SEQ ID NO: 9; https://www.european- virus-archive.com/virus/human-2019-ncov-strain-2019-ncovitaly-inmil) (see Fig. 8B);

2. NCBI GenBank, e.g., one of the following strains:

Severe acute respiratory syndrome coronavirus 2 isolate Wuhan-Hu-1, complete genome (Accession No: MN908947), SEQ ID NO: 1 (see Fig. 8A);

3. SARS-CoV-2 ASL517-Delta-India (B. 1.617.2), SEQ ID NO: 2, may be obtained by recombinant technology (see Fig. 9).

4. Isolates with RNA corresponding to a DNA sequence of SEQ ID NO: 4 may be obtained by KU Loewen also referred to as rega-20174.2 rega-20174 Severe acute respiratory syndrome coronavirus 2, hCoV-19/Belgium/rega-20174/2021|EPI_ISL_6794907.2|2021-l 1-24, partial genome [Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)] UTR 573' filled with n (see also Fig. 11).

5. Isolates with RNA corresponding to a DNA sequence of SEQ ID NO: 3 may be obtained by IHU Marseille: also referred to as PAC-IHU-49242.3 IHU Marseille isolate hCoV- 19/France/PAC-IHU-49242/2021|EPI_ISL_7308635|2021-12-01 plus 36n+18bp UTR5* and 21bp+45n UTR3' from virus EPI_ISL_803385 [Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)] plus N assignment with consensus of VB-2022-156-O (see also Fig. 10). Cell buildup and infection with SARS-CoV-2. The Vero cells used in the methods described herein were the VERO (WHO) cell line, obtained from the Health Protection Agency general cell collection under catalogue number 88020401, from which a master cell bank was generated. A research master seed bank (rMSB) of SARS-CoV-2 (strain used 2019-nCoV/Italy-INMIl) was prepared on Vero cells and the genomic sequence was checked by sequencing. For production of SARS-CoV-2, Vero cells were grown in Eagle's minimal essential medium (EMEM) containing 10% fetal bovine serum (FBS) and monolayers were infected with SARS-CoV-2 at a multiplicity of infection (moi) of 0.001 to 1, preferably 0.01, plaque forming units (pfu) per cell. After allowing virus adsorption, the cultures were washed 2-4 times with PBS, fed with serum-free EMEM and incubated at 35 °C with 5% CO2 until the virus titer reaches a desired level.

SARS-CoV-2 harvest. The culture medium was harvested at days 2, 3, 5 and 7 and harvests were pooled and centrifuged in a standard centrifuge. The resulting supernatant was filtered, followed by TFF ultrafiltration to remove cell culture medium components and reduce batch volume. Host cell DNA and protein reduction as well as reduction of non-infectious virus aggregates in the concentrated material was achieved by precipitation with protamine sulfate. Protamine sulfate was added to the diafiltrated SARS-CoV-2 material to a final nominal concentration of ~2 mg/mL, while stirring, followed by incubation at 2-8°C for 30 minutes. Alternatively, the diafiltrated SARS-CoV-2 material was treated with benzonase.

Optional primary inactivation. The SARS-CoV-2 virus was inactivated by treatment with betapropiolactone directly after removal of virus-containing cell culture medium from Vero cells, in order to render the virus safe to handle at BSL2. Inactivation is possible at any stage in the purification process, however, such as e.g., after centrifugation, before, during or after treatment with protamine sulfate or benzonase or before or after sucrose gradient centrifugation. Inactivation is carried out by the use of a chemical inactivation agent such as formaldehyde (formalin); enzyme; beta-propiolactone; ethanol; trifluroacetic acid; acetonitrile; bleach; urea; guanidine hydrochloride; tri-n-butyl phosphate; ethylene -imine or a derivative thereof; an organic solvent, optionally Tween, Triton, sodium deoxy cholate, or sulfobetaine; or a combination thereof. It is particularly preferred that inactivation is carried out using beta-propiolactone, which preferentially targets viral RNA whilst relatively sparing viral surface proteins and their immunogenic epitopes. Inactivation may also be achieved by pH changes (very high or very low pH), by heat treatment or by irradiation such as gamma irradiation or UV irradiation, particularly UV-C irradiation. The SARS-CoV-2 virus is optionally inactivated by two separate inactivation steps, such as, e.g. beta-propiolactone treatment and UV-C irradiation. Evaluation ofBPL starting concentration for inactivation of a highly resistant model virus (PPV). A preliminary study for evaluation of PPV virus inactivation kinetics was conducted to initially support our proposed SARS-CoV-2 BPL inactivation procedure. Porcine Parvovirus (PPV) was selected as a model virus to evaluate the inactivation capability of BPL in aqueous solution because of its high resistance to physico-chemical inactivation. Three starting concentrations of BPL were evaluated: 300 ppm (1/3333), 500 ppm (1/2000) and 700 ppm (1/1429). Virus solution was spiked with BPL at these concentrations and incubated at 5±2°C for 24 hours. Kinetic samples were taken at 0.5, 2, 6, 24h and after the BPL hydrolyzation step and analysed for remaining infectivity. The results are shown in Table A

*below limit of detection

**Note limit of detection for 500ppm BPL is lower than for 700ppm BPL

A clear effect of initial BPL concentration on the inactivation effectivity was observed with a reduction between 3.3 and 5.9 loglO after 24h incubation at 5±2°C (before hydrolysis). The following hydrolysis step further reduced the titers by on average addition 1.7 log 10 while the hold control titers remained constant throughout the whole procedure. This indicated that for highly resistant virus contaminations the hydrolysis step might serve as an additional inactivation step. With overall reduction factors of 4.84 (300 ppm), 7.43 (500 ppm) and below the limit of detection (700 ppm) the applied BPL treatment was considered effective for the inactivation of Parvoviridae at concentrations > 300ppm. Therefore, we decided to select 500ppm for SARS-CoV-2 virus inactivation in all further studies. SARS-CoV-2 virus inactivation by BP L

Based on existing data on the inactivation of model viruses by BPL (see section above on PPV inactivation) a BPL concentration of 500 ppm (1/2000) was selected for the inactivation of SARS-CoV- 2 virus harvest material. As the stability of BPL in solutions is highly temperature dependent an incubation temperature of 5±3°C and an incubation time of 24 hours were selected to ensure enough BPL present throughout the whole inactivation. After addition and mixing of BPL to the concentrated harvest, the inactivation solution is transferred to a fresh container where the inactivation takes place under controlled conditions. This transfer excludes the possibility of virus particles in potential dead- spots during initial mixing not being in contact with BPL.

To stabilize the pH of the inactivated viral solution during hydrolysis of the BPL, protamine sulfate (PS) treated concentrated harvest pre-cooled to 5±3°C is supplemented with 25 mM HEPES pH 7.4.

To reduce remaining BPL after the inactivation the solution is warmed to temperatures above 32°C for a total time of 2.5 hours ± 0.5 hours in a temperature-controlled incubator set to 37±2°C. The total time of the hydrolyzation step for the current process volume of about IL was between 5 hours 15 minutes and 6 hours 15 minutes including the warming to and the incubation above 32°C.

After completion of the hydrolysis, the inactivated viral solution (IVS) was immediately cooled down to 5±3°C in a temperature-controlled fridge and stored there until inactivation was confirmed by large volume plaque assay and serial passaging assay which currently requires 18 days in total. Recovery of virus particles throughout the inactivation process was monitored by size-exclusion chromatography.

Initial studies at lab-scale from 15 mL up to lOOOmL indicated a very fast inactivation kinetic for SARS- CoV-2 where virus titers of up to 8 log 10 pfii/mL were reduced below detectable levels within 2 hours after BPL addition. These results were confirmed for GMP production runs at a final inactivation volume of approximately IL. Taken together with the inactivation data for model viruses the applied BPL treatment can be considered efficient and includes a significant safety margin for inactivation of SARS-CoV-2 concentrated harvest material.

In a further preferred embodiment, the inactivation step(s) are particularly gentle, in order to preserve surface antigen integrity, especially integrity of the S protein. In one embodiment, the gentle inactivation method comprises contacting a liquid composition comprising SARS-CoV-2 particles with a chemical viral inactivating agent (such as e.g. any of the chemical inactivation agents as listed above or a combination thereof, preferably beta-propiolactone) in a container, mixing the chemical viral inactivating agent and the liquid composition comprising SARS-CoV-2 particles under conditions of laminar flow but not turbulent flow, and incubating the chemical viral inactivating agent and the liquid composition comprising SARS-CoV-2 particles for atime sufficient to inactivate the viruses. The gentle inactivation step is optionally performed in a flexible bioreactor bag. The gentle inactivation step preferably comprises five or less container inversions during the period of inactivation. Preferably, the mixing of the chemical viral inactivating agent and the composition comprising SARS-CoV-2 particles comprises subjecting the container to rocking, rotation, orbital shaking, or oscillation for not more than 10 minutes at not more than 10 rpm during the period of incubation.

Purification of SARS-CoV-2. Optionally, the material was immediately further processed by batch adsorption (also known herein as batch chromatography) with Capto™ Core 700 or CC400 chromatography media at a final concentration of ~1% CC700 or CC400. The material was incubated at 4°C for 15 minutes under constant agitation using a magnetic stirrer. After incubation, if used, the CC700 or CC400 solid matter was allowed to settle by gravity for 10 minutes and the SARS-CoV-2 material is removed from the top of the solution in order to avoid blockage of the filter by CaptoCore particles. Any remaining CaptoCore particles and DNA precipitate were then removed from the solution by filtration using a 0.2 pm Mini Kleenpak EKV filter capsule (Pall). The pooled filtered harvest material was adjusted to a final concentration of 25 mM Tris pH 7.5 and 10% sucrose (w/w) using stock solutions of both components. This allowed for freezing the concentrated harvest at <-65°C if required.

The resulting filtrate is further processed by sucrose density gradient centrifugation (also known herein as batch centrifugation) for final concentration and polishing of the SARS-CoV-2 material. The concentrated protamine sulfate (PS) or benzonase, preferred is PS, treated harvest was loaded on top of a solution consisting of three layers of sucrose with different densities. The volumes of individual layers for a centrifugation in 100 mb bottle scale are shown in Table la.

Table la. Volumes for sucrose density centrifugation.

The sucrose gradient bottles were prepared by stratifying the individual sucrose layers by pumping the solutions into the bottom of the bottles, starting with the SARS-CoV-2 material with the lowest sucrose density (10% sucrose (w/w)), followed by the other sucrose solutions in ascending order. The described setup is shown in Figure 3. The prepared SG bottles were transferred into a rotor pre-cooled to 4°C and centrifuged at ~11,000 RCF max at 4°C for at least 20 hours, without brake/deceleration.

After centrifugation, harvest of serial 2 mb fractions of the sucrose gradient is performed from the bottom up with a peristaltic pump. The fractions were immediately tested by SDS-PAGE / silver staining to identify virus-containing fractions with sufficiently high purity. Thus, identified fractions were pooled and further processed. The purified SARS-CoV-2 was stored at <-65°C or immediately formulated.

Formulation of SARS-CoV-2 with adjuvant. The SARS-CoV-2 particles were formulated with alum. Optionally, a Thl adjuvant was also added to the formulation or provided as a separate composition for bedside mixing.

SARS-CoV-2 ELISA Assay. Inactivated SARS-CoV-2 antigen content (i.e. content of SI as the major antigenic protein) in preparations described herein was determined (quantified) by ELISA. The SARS- CoV-2 ELISA used herein is a four-layer immuno-enzymatic assay with a SARS-CoV-2 spike antibody (AM001414; coating antibody) immobilized on a microtiter plate to which the SARS-CoV-2 sample is added. On binding of the antigen to the coating antibody, the plate was further treated with primary antibody (i.e. AbFlex® SARS-COV-2 spike antibody (rAb) (AM002414)). This was followed by addition of the secondary antibody, which is an enzyme linked conjugate antibody (i.e. Goat anti-Mouse IgG HRP Conjugate). The plates were washed between various steps using a mild detergent solution (PBS-T) to remove any unbound proteins or antibodies. The plate was developed by addition of a tetramethyl benzidine (TMB) substrate. The hydrolyzed TMB forms a stable colored conjugate that is directly proportional to the concentration of antigen content in the sample. The antigen quantification was carried out by spectrophotometric detection at X450nm (763 Onm reference) using the standard curve generated in an automated plate reader as a reference. Standards were prepared starting with a 20 antigen units (AU)/mL spike trimer working solution neat, which was further serially diluted 1 :2 for the following standard concentrations: 20 AU/mL, 10 AU/mL, 5 AU/mL, 2.5 AU/mL, 1.25 AU/mL, 0.625 AU/mL, 0.3125 AU/mL and 0.1263 AU/mL. Each dilution was tested in duplicate per plate. An “antigen unit” of the spike trimer standard, according to the supplier (R&D Systems), corresponds to its binding ability in a functional ELISA with Recombinant Human ACE-2 His-tag.

Reference Standards and Antibodies:

Coating Antibody: SARS-CoV-2 Spike Antibody (AM001414) Spike Trimer (S1+S2), His-tag (SARS-CoV-2) (e.g. BPS Lot# 200826; Cat#100728) SARS-CoV-2 QC (e.g. RSQC240920AGR)

Primary Detection Antibody AbFlex® SARS-CoV-2 Spike Antibody (rAb) (AM002414) Secondary Detection Antibody Goat anti-Mouse IgG HRP Conjugate

Coating buffer: Carbonate buffer

ELISA wash buffer: PBS + 0.05% Tween-20 (PBS-T).

Sample dilution buffer: PBS-T + 1% BSA.

The production process delivered high density and intact spike proteins (see Figure 6). Estimated were about 1 to 1.5 x 10⁷ viral particles per AU. Inactivation process by beta propiolactone provided for a fast inactivation kinetic and no detectable chemical modification of the S-protein. Key parameters and relevant process related impurities were similar to the commercial IXIARO® production process (see Table lb). SARS-CoV-2 drug substance according to the invention was highly pure (>95%) according to SDS-PAGE (silver stain, reduced) and free from aggregates (monomer virus (>95%) according to SE-HPLC (see Figure 7).

Further confirmatory studies aimed at characterizing modifications of S-protein following beta- propiolactone-inactivated SARS-CoV-2 are carried out by mass spectrometric analysis of tryptic digests of the S-protein. The modification of amino acids in important epitopes is minimal. Initial alignment of receptor binding domains (RBD) within the S protein and hACE2 interfaces and epitopes of several known (cross)-neutralizing antibodies (SARS-CoV and SARS-CoV-2) have shown no amino acids within these epitopes with potential high conversion and only few with potential lower conversion rates.

Table lb. Comparison of key parameters and relevant process related impurities of the SARS-CoV-2 drug substance and IXIARO® drug substance.

Master Seed Banks or Virus Seed Banks of SARS-CoV-2 particles comprising an RNA sequence (and/or fragments thereof, optionally comprising modified (preferably alkylated or acylated) nucleotide residues) corresponding to a DNA sequence (i) as defined by SEQ ID NO: 2; (ii) as defined by SEQ ID NO: 3; (iii) as defined by SEQ ID NO: 4 will be produced using a process to eliminate or reduce furin cleavage activity.

Example 2. In vitro and in vivo assessment of immunogenicity and protective capacity of inactivated SARS-CoV-2 virus compositions

Immunogenicity. Prior to immunization, experimental groups of 10 Balb/c mice were bled and pre- immune sera are prepared. The mice were administered a dose titration of inactivated SARS-CoV-2 formulated with alum subcutaneously (see Table 2). At two different intervals after immunization (see below), blood was collected and immune sera prepared, spleens were collected at the final bleed. All animal experiments were carried out in accordance with Austrian law (BGB1 Nr. 501/1989) and approved by “Magistratsabteilung 58”. Sera were assessed for total IgG and subclasses (IgGl/IgG2a) by ELISA and neutralizing antibodies by PRNT. Thl/Th2 responses were further assessed by IFN-y ELI Spot and intracellular cytokine staining (CD4⁺/CD8⁺).

-Schedule 1: Immunizations Day 0/Day 7, interim bleed Day 14, final bleed and spleen harvest Day 28 -Schedule 2: Immunizations Day 0/Day 21, interim bleeds Day 14/Day 28, final bleed and spleen harvest Day 35

Table 2. Design of dosing experiments, 10 mice/group: 3 dosage groups: 0.2 - 2 pg total protein; number of experiments: 3. For experimentation purposes, the Thl adjuvant is added directly to the SARS-CoV-2/alum formulation before immunization of the mice.

Plaque reduction neutralization test (PRNT). Each well of a twelve-well tissue culture plate was seeded with Vero cells and incubated 35°C with 5% CO2 for three days. Serial dilutions from pools of heat-inactivated sera from each treatment group are tested. Each serum preparation was incubated with approximately 50-80 pfu of SARS-CoV-2 at 35°C with 5% CO2 for 1 hour. The cell culture medium was aspirated from the Vero cells and the SARS-CoV-2 /serum mixtures were added to each well. The plates are gently rocked and then incubated for 2 hours at 35°C with 5% CO2. To each well, 1 mb of a 2% methylcellulose solution containing EMEM and nutrients are added, and the plates were further incubated for 4 days at 35°C with 5% CO2. The cells were then stained for 1 hour with crystal violet/5% formaldehyde and washed 3 times with deionized water. The plates were air dried and the numbers of plaques in each well manually counted. Alternatively, other methods, such as e.g. TCID50 may be applied.

Table 3. Design of schedule and longevity experiments, 10 mice/group; Immunization schedule as for Table 2, but in addition; interim bleeds 2, 6, 10, 14, 18 and 22 weeks after second immunization; end- bleed 26 weeks after second immunization; only with the preferred dose; only subcutaneous route; number of experiments: 1. For experimentation purposes, the Thl adjuvant was added directly to the SARS-CoV-2/alum formulation before immunization of the mice.

Protective capacity. The protective capacity of inactivated SARS-CoV-2 is assessed using a SARS- susceptible transgenic mouse expressing a humanized ACE2 protein (Jackson Laboratory) (Tseng, C - T.K. et al., Severe Acute Respiratory Syndrome Coronavirus Infection of Mice Transgenic for the Human Angiotensin-Converting Enzyme 2 Virus Receptor (2007) J of Virol 81: 1162-1173) or a NHP model developed for SARS-CoV-2 infection. Groups of animals are immunized subcutaneously (s.c.) with different dosages of inactivated SARS-CoV-2 with or without adjuvant or PBS as a negative control. Three weeks after the last dose, animals are challenged with SARS-CoV-2 and monitored for disease progression and survival. In addition, serum samples are taken in order to determine the neutralizing antibody titers induced by vaccination in a PRNT assay.

Table 3A. Design of dosing experiment 4743 using SARS-CoV-2 ELISA-determined dosages.

Experiment 4743 Protocol. Female Balb/c mice (10 mice/group) were immunized two times s.c. (100 pL) on days 0 and 21 with doses and adjuvants as outlined in Table 3A. The readouts from the experiment were total IgG and subclasses (IgGl/IgG2a) and virus neutralization (PRNT). Vaccine formulation used in experiment 4743 : purified inactivated SARS-CoV-2 produced from a research virus seed bank (rVSB) formulated in PBS with 17 pg Al³⁺ (alum)Zdose.

Antibody response to SARS-CoV-2 proteins. The immune responses in mice for the different doses and adjuvant formulations were assessed with a total IgG ELISA (Figure 4). Plates were coated with either the SI part (Figure 4A) or receptor binding domain (RBD) (Figure 4B) of the spike glycoprotein or the nucleoprotein (Figure 4C). Sera taken on days 28 and 35 were analyzed. Plates were coated with 2 pg/mL antigen (SI, RBD and N protein) and mouse sera were tested at a starting dilution of 1:50 in 4- fold dilutions. For detection a secondary monoclonal antibody (HRP-conjugated goat anti -mouse IgG) was used and developed with ABTS and read at absorbance 405 nm. Wells were washed with PBS-T between each step. Endpoint titers were determined with a cut-off set to 3-fold the blank.

IgG subclass immune response. Plates were coated with the SI part (Figure 4A) of spike glycoprotein and sera taken on day 35 were analyzed. Subclass specific secondary antibodies (IgGl and IgG2a) conjugated with HRP were used for detection. As standard curves (4-paramater regression) for determination of the amount of the different IgG subclasses (IgGl and IgG2a), monoclonal antibodies with different subclasses were used (IgGl mAb clone 43 and IgG2amAb clone CR3022). Bound HRP- conjugated secondary mAbs were developed with ABTS and read at absorbance 405 nm. Wells were washed with PBS-T between each step. The relative IgG subclass concentration is shown in Figure 5A and the ratio of IgG2a/IgGl in Figure 5B.

Observations from Experiment 4743. Inactivated SARS-CoV-2 formulated with alum induced antibodies in mice against SARS-CoV-2 detected by ELISA measuring antibodies to SI protein, receptor binding domain (RBD) and nucleocapsid protein (N) (Fig. 4A-C, respectively). An increase in immunogenicity was observed between bleeds on day 28 and day 35. In groups receiving the lowest dose (0.3 AU), a smaller increase not significantly above the placebo was seen for SI and RBD ELISA titers.

The alum-adjuvanted inactivated SARS-CoV-2, as expected, promoted an immune response shifted more towards a Th2 (IgGl) compared with a Thl (IgG2a) response as demonstrated by quantification of IgG subclasses by SI ELISA. The total amounts of IgG2a and IgGl measured and the ratio of IgG2a:IgGl in the treatment groups are shown in Figs. 5A and 5B, respectively. A shift in the immune response toward Thl (IgG2a) would likewise be expected by addition of a Thl -stimulating adjuvant to the SARS-CoV-2 vaccine composition.

Further immunization experiments are carried out in mice using GMP material with low doses (3, 1.2 and 0.3 AU) as a bridge between research and GMP material, as well as analyses of GMP material in mice with human doses (40, 10 and 3 AU).

Additionally, a challenge study is carried out in immunized non-human primates (NHP) (see Figure 17) and a passive transfer study is carried out in hamsters using sera from human subjects vaccinated with the SARS-CoV-2 vaccine candidate of the invention (see Table 1c).

Table 1c. Passive transfer study of the monovalent SARS-CoV-2 vaccine candidate of the invention in lamsters.

_ _ Example 3. Testing of SARS-CoV-2 vaccine for antibody-dependent enhancement (ADE) of disease and immunopathology

Although the mechanism is poorly understood, antibodies produced in response to a previous coronavirus infection or vaccination can increase the risk for 1) immunopathology and/or 2) antibodydependent enhancement of disease (ADE) during subsequent coronavirus infection(s). As such, any stimulation of antibodies to SARS-CoV-2 presents a hypothetical risk. In this regard, several approaches are undertaken to ensure safety of the current vaccine.

In vitro antibody-dependent enhancement assays. Immune sera from inactivated SARS-CoV-2- vaccinated mice are assessed for hallmarks of enhanced disease in vitro. Such assays are described by e.g. Wang, S.-F., et al. 2014 (Antibody-dependent SARS coronavirus infection is mediated by antibodies against spike proteins (2014) BBRC 451 :208-214). Briefly, susceptible cell types or cell lines are incubated with immune sera and subsequently infected with SARS-CoV-2. Cells are assessed for cytopathic effect and/or production of inflammatory markers.

Mouse model of immunopathology. The risk of vaccine-enhanced immunopathology on challenge is assessed in a Balb/c mouse model as described by Tseng C.T. et al. (Immunization with SARS Coronavirus Vaccines Leads to Pulmonary Immunopathology on Challenge with the SARS Virus (2012) PLoS ONE 7(4):e35421). Briefly, the mice are immunized twice at two-week intervals with inactivated SARS-Cov-2 formulated as described herein followed by challenge with SARS-CoV-2. SARS-CoV-2 titers and immune cell infiltration of the lung are tested.

Non-human primate model of ADE. The risk of ADE development in non-human primates is assessed as described by Luo F, et al. (Evaluation of Antibody-Dependent Enhancement of SARS-CoV Infection in Rhesus Macaques Immunized with an Inactivated SARS-CoV Vaccine (2018) Virologica Sinica 33:201-204). Briefly, NHPs are immunized with inactivated SARS-CoV-2, followed by SARS-CoV-2 challenge and evaluation of symptoms and disease pathology.

Example 4. Clinical Phase 1 study

Formulation of inactivated SARS-CoV-2 (monovalent, wild type) for Phase 1 trial. The objective of the Phase 1 trial is to assess the safety of the vaccine, along with immunogenicity, and to determine an optimal dose of the individual SARS-CoV-2 particles and adjuvant(s). As such, several antigen doses were tested in clinical phase 1 : High, Medium and Low doses are chosen to have a distance between each dose of approximately 3 -fold and a span covering about a 10-fold difference between the high and low doses. The dose range is selected in part to indicate any potential dose-sparing effect of a Thl adjuvant.

The SARS-CoV-2 virus as purified herein has a high purity of >90% as assessed by SDS-PAGE, SE- HPLC and/or SARS-CoV-2 ELISA (data not shown). Furthermore, preliminary studies have indicated that the incidence of genetic heterogeneities during passage of the virus is low and no particular individual mutations stand out (data not shown).

The SARS-CoV-2 virus as purified herein has a high purity of >90% as assessed by SDS-PAGE, SE- HPLC and/or SARS-CoV-2 ELISA (see, e.g., Fig. 7). Furthermore, preliminary studies have indicated that the incidence of genetic heterogeneities during passage of the virus is low and no particular individual mutations stand out (data not shown).

To arrive at a dose range, the SARS-CoV-2 virus was compared with JEV, specifically assessing SE- HPLC peak area per dose equivalent (recorded as milli-absorption units x minutes; mAU), the total amount of inactivated viral particles per dose and the total viral surface equivalent per dose (see Table 4). This assessment was based on the assumption of a similar surface antigen density between S (spike; SARS-CoV-2) and E (envelope; JEV) proteins. Total protein was determined by pBCA assay (Table 4). Although the assay was variable, a correspondence of 1 mAU to ~2 pg total protein per mb was observed. Another determination using an optimized SARS-CoV-2 S-protein ELISA, as outlined in Example 1, was also performed.

Table 4. Comparison of JEV and SARS-CoV-2 quantification parameters and total protein in Low, Medium and High SARS-CoV-2 dosage groups.

As SARS-CoV-2 virus particles (~92 nm diameter) are much larger than Flavivirus particles (~40 nm), corresponding to an approximately 5 -fold greater virus surface area perparticle, an equivalently higher antigen content is expected. Furthermore, other inactivated virus vaccine preparations, including JEV (IXIARO), TBE (Encepur) and HepA (VAQTA) reported antigen doses in the low pg to ng protein range. As these viruses are all formalin inactivated, the BPL-inactivated SARS-CoV-2 virus of the current invention has better preserved surface antigen proteins, i.e., a better quality antigen, and requires a lower total protein dose.

For entry into the clinic a further antigen determination assay (SARS-CoV-2 ELISA assay as described in Example 1) was developed and the doses of the vaccine formulations for entry into Phase 1 trials were determined using this assay. The Phase 1 treatment groups are set forth in Table 5.

Formulation of SARS-CoV-2 vaccine for phase 1 trial (0.5 mL/dose):

-Antigen (inactivated SARS-CoV-2) target doses:

Low: 3 AU/0.5 mL (6 AU/mL)*

Medium: 10 AU/0.5 mL (20 AU/mL)

High: 40 AU/0.5 mL (80 AU/mL)

*doses determined by the SARS-CoV-2 ELISA assay as described in Example 1

-Aluminium hydroxide (Al³⁺): 0.5 mg/dose (1 mg/mL)

-Thl adjuvant

-Recombinant Human Serum Albumin (rHSA): ~25 pg/dose (~50 pg/mL)

-Buffer: Phosphate buffered saline (PBS)

In some cases, vaccinated subjects are challenged with an infectious dose of live SARS-CoV-2 virus (Asian and/or European lineage).

Table 5. Treatment groups for Phase 1 testing of inactivated SARS-CoV-2 vaccine (low, medium and high doses are those provided in Table 4).

Formulation of inactivated SARS-CoV-2 (bivalent, wild type-Omicron or Omicron-Delta) for Phase 1 trial. The objective of the Phase 1 trial is to assess the safety of the vaccine, along with immunogenicity, and to determine an optimal dose of each of the individual SARS_CoV-2 particles and adjuvant(s). As such, several antigen doses in different ratio (i.e. 1: 1 or 1:2 or 2: 1 wild type: omicron) are tested in clinical phase 1. Other objective is to evaluate amount of adjuvant(s), e.g. it is expected that alum and a Thl adjuvant such as CpG1018 will be evaluated. Alum concentration is expected to be in the range of 0.5 mg/dose

Example 5. Testing of Sera of vaccinated organism with a neutralization assay

Sera of vaccinated mice, hamsters, non-human primates or humans can be tested in neutralization assays such as e.g. described in “Szurgot, I., Hanke, L., Sheward, D.J. et al. DNA-launched RNA replicon vaccines induce potent anti-SARS-CoV-2 immune responses in mice. Sci Rep 11, 3125 (2021). https://doi.org/10.1038/s41598-021-82498-5”.

The read-out of the test gives an indication how well sera of vaccinated subjects can neutralize new variants and thus guides in the design of the vaccine.

Example 6. Liquid chromatography with tandem mass spectrometry (LC-MS-MS) analysis of inactivated SARS-CoV-2

Methodology:

Two samples were separated using SDS-polyacrylamide gel electrophoresis and the bands were visualized by silver staining. The bands were cut and subjected to in-gel digestion with trypsin and the resulting peptides analysed with nano-liquid chromatography coupled to a high-resolution accurate mass spectrometer. Peptides were identified from raw spectra using the MaxQuant software package and the UniProt reference databases for SARS-CoV-2 and Chlorocebus sabcieus. To account for modifications the data were re-searched specifically for B-propiolactone modifications, and the obtained results were confirmed with a second independent search algorithm (Sequest in Proteome Discoverer suite). Additionally, data were searched with the FragPipe package to account for further unknown MS- detectable modifications.

Results:

Protein identification: The bands could be clearly atributed to the three main viral proteins (Spike-protein, Membrane-protein, Nucleoprotein) as well as to background proteins from the host system (see Figure 10). Traces of SARS- CoV-2 ORF9b and the replicase polyprotein could also be detected, but these proteins were probably not well resolved on the gel due to their size (data not shown). The separation patern on the gel was very similar for both samples with the exception of a host protein band (band 2.3), a slightly different S-protein patern (bands 2.10-2.13), and an expected strong band of serum albumin in one ofthe samples (sample 2) (data not shown). Additionally, a number of typical lab contaminants of human origin (e.g. keratins) were detected in the background of both samples. The processing of the Spike-protein (from full length to SI, S2, and S2’) is difficult to resolve with the applied methodology but is most likely represented by the patern in bands 9-13 in both samples (data not shown).

Modification analysis:

Based on a publication by Uitenbogaard et al. (Reactions of P-Propiolactone with Nucleobase Analogues, Nucleosides, and Peptides, Protein Structure and Folding| Volume 286, ISSUE 42, P36198- 36214, October 21, 2011), it was expected to find B-propiolactone (BPL) modifications on cysteine, methionine, and histidine. Uitenbogaard et al. studied amino acids which are subject to modification by beta-propiolactone, along with the type of modification, e.g., acylation, alkylation. They have shown that BPE can react with up to 9 different amino acids (C,H,M,D,E,Y,K,E,S) depending on actual pH. In their studies higher conversions within the relevant pH range 7 to 9 were observed for Cysteine (>95%), Histidine (15-25%) and Methionine (36%) residues. The conversion rates for Aspartic Acid, Glutamic acid and Tyrosine were much lower in the range of approximately 3-15%. It was shown that disulfide groups in Cystine residues do not react.

In BPL-inactivated SARS-CoV-2 particles, BPL modifications could be detected (mainly in the form of +72 Da) but at a low abundance. Out of 2894 (sample 1) and 3086 (sample 2) identified spectra for SARS-CoV-2 proteins only 73 and 110, respectively, carried a BPL modification, which translates to 2.5 to 3.6 % (see Table 6). This was also confirmed by the open modification search using FragPipe, which atributed a similarly low fraction of spectra to mass differences matching the BPL-modification.

Table 6. Number of identified SARS-CoV-2 peptide spectra

Spectra of all BPL-modified peptides reported for SARS-CoV-2 proteins were inspected manually of which 6 to 8 sites were confirmed for sample 1 and 2, respectively (see Table 6). For all of these validated sites also the unmodified peptides were identified suggesting that the modification with BPL never reached 100%. We estimated the degree of modification on a particular site (the so-called site occupancy) as the ratio of modified to unmodified peptide for the same modification site normalized to the protein abundance for each band. We then selected the maximum occupancy for each site as a conservative measure of the degree of site modification. As shown in Table 7 the occupancy was in general rather low for the sites identified, in agreement with the total number of identified spectra. The only exception, M234 of the nucleoprotein, has to be interpreted carefully, as that particular peptide sequence has problematic features which likely make the estimation for this particular peptide less accurate and reliable as compared to the other sites.

Table 7. BPL-modified sites identified and their occupancy

n.q. = not quantified; n.d. = not detected

* quantification uncertain, due to missed cleavages and oxidation

Apart from the expected modifications the FragPipe search revealed two other modifications (most likely acetaldehyde and acetylation) to occur in around 10% of the spectra. These modifications represent most likely artifacts introduced during gel staining and sample preparation, as they also occur on contaminant proteins.

Summary:

Based on the results described above it is concluded that the main components in these samples corresponds to SARS-CoV2 proteins. The BPL modifications were detectable but appeared to be low, i.e. around 3% on whole SARS-CoV-2 proteome level (i.e. all SARS-CoV-2 proteins identified). Only 5 amino acids of the S-protein were found to be modified and this was also only detected for a minority of the analysed S-protein (e.g. around 16% for the Spike-protein at the H207 amino acids, i.e. the probability to have a modification at H207 was around 16%). The two samples differ only slightly with respect to some background proteins and in their degree of modification, with sample 1 showing slightly lower levels of BPL-modification. Please note that only about 30% to 40% of the amino acids of the Spike protein could be tested.

Conclusion:

This data supports the view that the mild inactivation approach of the invention minimizes the modifications within the S-protein and thus the native surface of the S-protein is largely preserved. In comparison, determination of modifications by BPL inactivation of flu samples were more frequent, i.e. 83 sites on HA and 43 sites on NA for one sample flu vaccine (NIBRG-121xp) and 99 sites on HA and 39 sites on NA for another sample (NYMC-X181A) were modified, wherein HA and NA are the two major membrane glycoproteins, i.e. the primary immunogens for flu (She Yi-Min et al., Surface modifications of influenza proteins upon virus inactivation by beta-propiolactone; Proteomics 2013, 13, 3537-3547, DOI 10. 1002/pmic.201300096). Thus, BPL inactivation of influenza virus can lead to numerous protein modifications including some affecting membrane fusion.

Example 7. Further liquid chromatography with tandem mass spectrometry (LC-MSMS) analysis of inactivated SARS-CoV-2

Methodology:

A further LC-MSMS analysis of BPL-inactivated SARS-CoV-2 particles, as described in Example 6, was performed in order to obtain greater coverage of the proteins. Five aliquots of the BPL-inactivated SARS-CoV-2 sample were separated on SDS-PAGE and the bands visualized by either silver staining for visualization or Coomassie staining for processing. The Coomassie-stained bands corresponding to spike protein (based on previous analysis) were subjected to in-gel digestion with trypsin or chymotrypsin or to acid hydrolysis. Trypsin digests were performed twice, once with and once without previous PNGase F (peptide :N-glycosidase F) digestion, to identify peptides masked by glycosylation.

Digested peptides were analysed by LC-MSMS essentially as described in Example 6. In particular, the resulting peptides were analyzed with nano-liquid chromatography coupled to a high-resolution accurate mass spectrometer. Peptides were identified from raw spectra using the MaxQuant software package and the UniProt reference databases for SARS-CoV-2 and Chlorocebus sabcieus in combination with a database of common lab contaminants. To account for modifications the data were also searched specifically for B-propiolactone (BPL) modifications, and spectra of all BPL-modified peptides of the SARS-CoV-2 spike protein were manually validated. The degree of modification was globally estimated as the percentage of BPL-modified spectra identified, and on site-level by calculating site occupancies from the ratio of modified to unmodified peptides for each peptide/site separately.

Results:

The total coverage of particular SARS-CoV-2 proteins, using the combination of four digestion methods (i.e. (i) trypsin (ii) trypsin + PNGase F (iii) chymotrypsin and (iv) acid hydrolysis) was as follows: Spike (S) protein - 91.5%

Membrane (M) protein - 60.36%

Nucleoprotein (N) - 74.70%

The number of BPL-modified peptides in the inactivated SARS-CoV-2 particles, based on each digestion method, is shown in Table 8 below:

Table 8. Number of identified SARS-CoV-2 peptide spectra across all bands analyzed

As shown in Example 6, this confirms that the percentage of BPL-modified peptides is low regardless of the digestion method, e.g. less than 7%, 2% to 7% or around 2-5% on average.

Using a combination of the four digestion methods described above, a greater coverage of amino acid residues in SARS-CoV-2 proteins could be achieved. Accordingly, BPL-modifications were detected at the positions in the spike (S) and membrane (M) proteins shown in Table 9 below. The mean percentage occupancy at each site, as described in Example 6 above, is also shown in Table 9.

Table 9. BPL-modified sites identified in S protein and their occupancy

From the data in Table 9, it can be seen that up to around 16 residues in the spike (S) protein may be modified, and up to 4 residues in the membrane (M) protein. The occupancy at each site is low, e.g. less than 20%, typically less than 10%. Therefore the inactivated SARS-CoV-2 particles show a low degree of BPL-modifications.

Example 8: Outline of a next generation inactivated SARS-CoV-2 vaccine

The benefits of the approach of the invention are numerous and particularly relevant to fight the COVID- 19 pandemic:

• Breadth of protection: The whole virus presentation allows the immune system to develop antibodies against spike (S) protein and other SARS-CoV-2 structural proteins, and to create a more diverse immune response that is believed to mitigate reduced vaccine effectiveness as the S protein mutates over time.

• Established track record: COVID- 19 vaccine candidates use the same platform technology and build on over a decade of experience with FDA -approved IXIARO, increasing the probability of clinical, technical, and regulatory success.

• Adaptive manufacturing: The vaccine platform and manufacturing process allows to combine SARS-CoV-2 variants and quickly modify the formulation as needed (based on circulating strains). Therefore, the technology platform is highly suitable for a yearly booster and/or virus adaptation similar to the yearly influenza vaccinations.

• Manufacturing scale: Manufacturing production capacity can be scaled up to meet the need of its stakeholders.

• Stability: Vaccine’s platform is designed for routine use and distribution - a meaningful improvement over other COVID-19 vaccines. The vaccines of the invention can be routinely stored at 2-8°C and the anticipated minimum shelflife is 24 months. Additionally, the vaccines are expected to be stable for 24-48 hours at ambient temperature.

Clinical development

The monovalent SARS-CoV-2 vaccine (SEQ ID NO: 9) is a highly-purified, whole virus, SARS-CoV- 2 vaccine candidate produced on Vero cells and inactivated with p-propiolactone. Said vaccine is adjuvanted with a Thl adjuvant in combination with Aluminum Hydroxide.

• The monovalent SARS-CoV-2 vaccine candidate had superiority against the comparator vaccine, AstraZeneca’s AZD1222 (ChAdOxl-S), in terms of geometric mean titer (GMT) for neutralizing antibodies, as well as non-inferiority in terms of seroconversion rates (SCR above 95% in both treatment groups) at two weeks after the second vaccination.

• The monovalent SARS-CoV-2 vaccine candidate induced broad T-cell responses with antigenspecific IFN -gamma-producing T-cells against the S, M and N proteins.

• The monovalent SARS-CoV-2 vaccine candidate was generally well tolerated, demonstrating a statistically significant better tolerability profile compared to AZDI 222.

• Positive homologous booster data was showing an excellent immune response after a third dose of the monovalent SARS-CoV-2 vaccine candidate was administered seven to eight months after completion of primary vaccination with the monovalent SARS-CoV-2 vaccine candidate.

• A third dose of the monovalent SARS-CoV-2 vaccine candidate produced also neutralizing antibodies against the Omicron variants in laboratory studies.

Bivalent SARS-CoV-2 vaccine: The clinical development would be based on neutralizing antibody titer levels for one specific SARS-CoV-2 strain in a non-inferiority immunogenicity trial. In the absence of an established correlate, similar levels of neutralizing antibodies against any new SARS-CoV-2 vaccine strain would be shown through adopted and specific assays.

• The clinical trial would evaluate the safety, tolerability, and immunogenicity in healthy adults 18 to 55 years of age. The trial would have four cohorts examining different regimens of the current monovalent vaccine and bivalent vaccine.

• Similar to the annual Influenza trials one would need to show those levels of neutralizing antibodies for both strains included in the bivalent vaccine in a respective safety and immunogenicity trial.

• The Phase 3 safety and immunogenicity trial would be powered to demonstrate non-inferiority against the monovalent vaccine.

• It is believed that the bivalent vaccine may be able to induce a greater breadth of protection in seropositive individuals than the monovalent SARS-CoV-2 vaccine, but in particular compared with RNA, viral vector, and nanoparticle vaccines that are focusing solely on the S-protein. Therefore, the company believes the study can be considered as a primary two-dose and/or booster vaccine.

Clinical immunogenicity

A third dose of the monovalent SARS-CoV- 2 vaccine candidate administered 7 to 8 months after the second dose of primary vaccination increased levels of antibodies against the Wuhan virus strain 42- to 106 -fold, depending on the pre-boost antibody levels.

At the moment, the best proxy we have for the potential expected durability of the inactivated SARS- CoV-2 vaccine is IXIARO®, a vaccine against Japanese Encephalitis that uses the same or similar technology platform. At least eleven months after primary vaccination with IXIARO, we saw that 83% of subjects still had protective neutralizing antibodies (5 years in over 60% of vaccinees). Currently IXIARO is recommended to have a single booster dose at least 1 Imonths after primary immunization with IXIARO (Taucher et al. 2019, Package-Insert-and-Patient-Information-IXIARO, September 2018 [https ://www.fda.gov/media/75777/download]) .

Similar antibody persistence is expected for the bivalent vaccine candidate.

Clinical operations

The route of administration of the monovalent SARS-CoV-2 vaccine is intramuscular injection (i.m). The vaccine will be provided in both single and multi-dose vials as a liquid formulation containing Aluminium Hydroxide and a Thl adjuvant ready for use.

For the bivalent vaccine candidate (e.g. Wuhan & Omicron strains), we foresee a similar delivery and administration route as for the monovalent candidate. For example, we would use a two-dose vaccination schedule on Day 1 and Day 29. The bivalent candidate would be intended for authorization as a multi -dose vial.

Breadth of protection

Inactivated SARS-CoV-2 vaccines are a critical component of the portfolio of vaccine response to COVID- 19 since they may have the potential to offer a greater breadth of protection against variants than the mRNA, viral vector, and nanoparticle vaccines currently licensed in the U.S. and E.U. Whereas inactivated vaccines utilize all four SARS-CoV-2 structural proteins as antigens, other vaccine technologies rely on the spike (S) protein alone.

Cellular immune responses contribute to limiting the severity of COVID-19 and ultimately resolving the SARS-CoV-2 infection. The SARS-CoV-2 vaccines of the invention contain all four structural proteins of SARS-CoV-2: the spike (S) protein, the nucleocapsid (N) protein, the membrane (M) protein, and the envelope (E) protein. For the monovalent SARS-CoV-2 vaccine, we have demonstrated cellular immune responses to each of the three antigens for which cellular immune responses were evaluated.

• The monovalent SARS-CoV-2 vaccine elicited cellular responses to at least three different antigens, demonstrating the breadth of responses expected from an inactivated vaccine. o In the pivotal phase 3 trial, 74% of subjects mounted responses to S, 46% to N, and 20% to M. Responses to E were not evaluated.

• Sera from the booster extension of the monovalent SARS-CoV-2 vaccine were evaluated for cross-reactivity against Delta and Omicron. Subjects were boosted with a third dose of the monovalent SARS-CoV-2 vaccine 7-8 months after the priming schedule. o Sera from 30 individuals, collected 2 or 4 weeks after the booster, were evaluated in a pseudovirus neutralization assay. o 100% of subjects had antibodies that neutralized the ancestral strain and Delta, and 87% of subjects (26 of 30) had detectable neutralizing activity against Omicron. o As expected, the highest neutralizing antibody titers were observed for the ancestral SARS-CoV-2 virus and the lowest titers for Omicron (Figure 14). o Per individual, the mean fold reduction of neutralization relative to the ancestral virus was 2.7-fold for Delta and 16.7-fold for Omicron. These data demonstrated that subjects who received three doses of monovalent SARS-CoV-2 vaccine have crossneutralizing antibodies.

Manufacturing status According to our experience preparing viral seed banks for the Alpha, Beta, and Delta variants, the preparation of GMP viral seeds takes approximately 8 weeks upon availability of a new viral strain of SARS-CoV-2. Planned final container presentation is in both single and multi-dose vials as a liquid formulation.

Storage & Stability

For the monovalent SARS-CoV-2 vaccine candidate, the storage conditions for the product are 2-8°C. Based on the long term and accelerated stability data collected for monovalent SARS-CoV-2 vaccine to date the anticipated shelf life is at least 24 months. This would include up to 48 hour storage at ambient temperature.

Example 9: An open-label phase 3 study assessing the safety, tolerability and immunogenicity of the monovalent SARS-CoV-2 vaccine in adults aged > 56 years

Study Objectives

Primary:

• To confirm the safety and tolerability of the monovalent SARS-CoV-2 vaccine (delivered at the “standard dose” of 33 AU/0.5 mL) up to Day 43 in participants > 56 years of age.

• To assess the immunogenicity of the monovalent SARS-CoV-2 vaccine administered in a 2- dose immunization schedule 4 weeks apart, in terms of geometric mean titer (GMT) as well as the seroconversion rate of neutralizing antibodies at two weeks after the second vaccination (i.e. Day 43) in volunteers aged 56 years and older.

Secondary:

• To describe the safety of the monovalent SARS-CoV-2 vaccine up to 12 months after the first vaccination in volunteers aged 56 years and older who received 2 doses of the monovalent SARS-CoV-2 vaccine.

• To describe the safety and tolerability of the monovalent SARS-CoV-2 vaccine in adults aged 56 years and older up to 6 months after a booster dose.

• To assess immunogenicity of a 2-dose primary immunization schedule with the monovalent SARS-CoV-2 vaccine after the first vaccination in volunteers aged 56 years and older.

• To determine the immunogenicity of a single booster dose with the monovalent SARS-CoV-2 vaccine in participants aged 56 years and older.

• To evaluate cellular immune responses following administration of the monovalent SARS- CoV-2 vaccine in participants aged > 56 years old. Study design

-This is a multicentre, open label, single arm study to assess the safety, tolerability and immunogenicity of the monovalent SARS-CoV-2 vaccine in terms of GMT and seroconversion rate of SARS-CoV-2 - specific neutralizing antibodies and S-protein binding IgG levels in older adults.

-Participants aged 56 years or older and who are either generally healthy or are with a stable medical condition will be enrolled.

-Approximately 300 participants aged 56 years or older will be enrolled in a non-randomized manner to receive the monovalent SARS-CoV-2 vaccine at the recommended dose level, 28 days apart, on Days

1 and 29. The Study Overview is provided in Fig. 15.

-All visits will be conducted at the clinical site on an outpatient basis.

-Immunogenicity (neutralizing and binding antibody titers) and safety will be assessed up to month 12 after the first vaccination.

Monovalent SARS-CoV-2 vaccine Booster Group in Adults 56 years and older

-All participants, except those who already received a licensed COVID-19 vaccine outside of the study, will be offered a booster dose with the monovalent SARS-CoV-2 vaccine. All eligible and willing participants will receive a booster vaccination with the monovalent SARS-CoV-2 vaccine at Visit Bl and will have a follow-up visit 14 days (Visit B2) after the booster dose. The participants will have 1 more follow-up visit 6 months after the booster vaccination (i.e., Visit B3 which replaces Visit 7 (Day 365) for those participants who received a booster dose). This also means that Visit 6 (Day 208) will not be performed by those participants who received a booster dose.

-On the other hand, all participants who do not receive a booster dose with the monovalent SARS-CoV-

2 vaccine will continue with Visit 6 (Day 208) and Visit 7 (Day 365).

-The timing of the booster dose administration will be scheduled approximately in February 2022.

-All participants will be observed for immediate adverse events (AEs) and/or reactogenicity for at least 30 minutes after the administration of the vaccine.

Initial vaccination with the monovalent SARS-CoV-2 vaccine (2-doses, 28 days apart)

Participants will be provided with an electronic Diary (e-Diary) and will be trained to record specifically solicited systemic and local symptoms daily for 7 days following each vaccination as well as any additional AEs during follow-up period after each of both vaccinations up to the next visit to the site until Day 43 visit has been completed.

Booster vaccination with the monovalent SARS-CoV-2 vaccine Participants will be provided with an electronic Diary (e-Diary) and will be trained to record specifically solicited systemic and local symptoms daily for 7 days following booster vaccination as well as any additional AEs during follow-up period after booster vaccination up to visit B3.

The following information will be collected:

• Oral body temperature

• Solicited local (i.e. injection site) reactions

• Solicited systemic reactions

• Other AEs

• Any medication taken due to a solicited adverse event

Example 10: An open-label phase 2/3 clinical study to investigate safety and immunogenicity of a single monovalent SARS-CoV-2 vaccine booster vaccination in adult volunteers after receipt of nationally rolled out mRNA COVID-19 vaccines and/or natural SARS-CoV-2 infection

Study objectives

Primary:

• To determine the immune response in terms of GMT fold-rise for neutralizing antibodies against SARS-CoV-2 following a single booster dose with the monovalent SARS-CoV-2 vaccine

• To assess tolerability of a the monovalent SARS-CoV-2 vaccine booster vaccination

Secondary:

• To determine the immune response in terms of GMT for SARS-CoV-2-specific neutralizing antibodies following a single booster dose with the monovalent SARS-CoV-2 vaccine

• To determine the immune response in terms of GMT for IgG antibodies to SARS-CoV-2 S- protein following a single booster dose with the monovalent SARS-CoV-2 vaccine

• To determine the immune response in terms of GMT fold-rise for IgG antibodies to SARS- CoV-2 S-protein following a single booster dose with the monovalent SARS-CoV-2 vaccine

• To evaluate cellular immune responses following administration of the monovalent SARS- CoV-2 vaccine

• To describe the safety of the monovalent SARS-CoV-2 vaccine following a single booster dose with the monovalent SARS-CoV-2 vaccine

Study design

This is a multicentric, open label, phase 2/3 clinical study to investigate the safety, tolerability, and immunogenicity of a monovalent SARS-CoV-2 vaccine booster vaccination (standard dose (33 AU/0.5 mL) in adults aged >18 to <50 years or double dose (66 AU/ 1.0 mL) in volunteers aged >50 years). Volunteers who are either generally healthy or having a stable medical condition will be enrolled. In total approximately 275 participants are planned to be enrolled. It is planned to enroll approximately 25% of participants who are above 65 years into the cohorts with participants above 50 years of age. The Study Overview is provided in Fig. 16.

The monovalent SARS-CoV-2 vaccine booster (standard dose of 0.5 mL or double dose of 1.0 mL) will be applied:

Cohort 1 and Cohort 2: groups A and B: at least 6 months after vaccination with mRNA COVID-19 vaccine

Cohort 1 and Cohort 2: groups C and D: at least 6 months after vaccination with mRNA COVID-19 vaccine or at least 4 months after a documented PCR or antigen test for confirmed SARS-CoV-2 infection in case the infection occurred after the administration of the last dose of mRNA CO VID-19 vaccine Cohort 3 :

In addition, the monovalent SARS-CoV-2 vaccine booster (standard dose of 0.5 mL for participants >18 to <50 years or double dose of 1.0 mL for >50 years) will be applied at least 4 months after documented PCR or antigen test confirmation of natural SARS-CoV-2 infection.

Note: if the SARS-CoV-2 infection was present between mRNA doses, the booster dose with the monovalent SARS-CoV-2 vaccine is to be administered at least 6 months after the last mRNA dose. In case the infection occurred after the last dose of mRNA, then the booster dose with the monovalent SARS-CoV-2 vaccine is to be administered at least 4 months after the documented PCR or antigen test confirmation of the infection.

Rapid antigen test results can be considered as proof of previous COVID- 19 infection but only if the antigen test results have been officially documented or registered in an official system - as for the PCR test results, in all cases, a paper document must be available/printable to be considered sufficient proof of a previous infection prior to enrollment (in the relevant Cohorts). See Table 10 for an overview on cohorts and targeted number of participants.

Table 10. Overview on Cohorts and targeted number of participants for the Phase 2/3 clinical study described in Example 10.

Special Procedures for Certain Participants (Sentinels)

-For safety reasons, the first 10 participants aged >50 years of the Cohort 2 from any of the group (A, B, C and D) or Cohort 3 (monovalent SARS-CoV-2 vaccine double dose, i.e., 1.0 mL) will be considered sentinel participants and undergo special precautionary safety measures.

-Double dose administration of the monovalent SARS-CoV-2 vaccine for these sentinels will be done at a single site to ensure permanent oversight of safety data by one principal investigator. A second site may need to be involved in the recruitment of the sentinel participants, in this case vaccinations will be limited to one site on a specific day. Safety data exchange between the study sites will be ensured. -Sentinel participants will be observed for 60 minutes at the study site to monitor for the development of any acute reaction. Prior to discharge, vital signs will be measured and participants will be instructed to use their e-Diaries. Safety telephone calls will be performed by the study site approximately 24 and 48 hours after vaccination for safety follow-up. The information provided must be compared with the entries in the participant’s eDiary. -A Data Safety Monitoring Board (DSMB) will review the accrued safety data when all 10 sentinel participants have completed the 7-day e-diary period after vaccination. Applicable for all participants

-All visits will be conducted at the clinical site on an outpatient basis or as home visits. -Immunogenicity and safety will be assessed up to Month 12 after the booster vaccination.

-All participants will be observed for immediate AEs and/or reactogenicity for at least 30 minutes after the administration of the vaccine (60 minutes for sentinel participants, see above).

-Participants will be provided with an electronic Diary (e-Diary) and will be trained to record specifically solicited, predefined systemic and local symptoms daily for 7 days following the booster vaccination as well as any additional AEs during the follow-up period up to Day 15. The following information will be collected:

• Oral body temperature

• Solicited local (i.e. injection site) reactions

• Solicited systemic reactions

• Other AEs

• Any medication taken due to a solicited adverse event

-All unsolicited AEs (adverse events) need to be documented in the source documents throughout the study and will be captured in the respective AE section of the eCRF (Electronic Case Report Form) up to Visit 3. Serious adverse events as well as AESIs will continue to be documented in the eCRF until the end of the study.

Further preferred aspects:

Al. A SARS-CoV-2 vaccine comprising at least two or exactly two different beta-propiolactone- inactivated SARS-CoV-2 particles, wherein the vaccine is capable of generating neutralizing antibodies against a native homologous and/or heterologous SARS-CoV-2 particle and/or is capable of raising an effective T-cell response against a native homologous and/or heterologous SARS-CoV-2 particle in a human subject.

A2. A SARS-CoV-2 vaccine according to aspect Al, wherein a native surface conformation of the SARS-CoV-2 particle is preserved in the vaccine and/or wherein the activity of the furin cleavage site within the viral RNA is reduced or eliminated by passaging out the furin site and/or introducing mutations in the cleavage site.

A3. A SARS-CoV-2 vaccine according to aspect Al or A2, wherein viral RNA in the inactivated SARS-CoV-2 particle is replication-deficient, preferably wherein viral RNA in the inactivated SARS-CoV-2 particle (i) is alkylated and/or acylated (ii) comprises one or more modified purine (preferably guanine) residues and/or strand breaks and/or (iii) is cross-linked with one or more viral proteins.

A4. A SARS-CoV-2 vaccine according to any preceding aspect, wherein the SARS-CoV-2 particles are beta-propiolactone -inactivated at a concentration of 300 to 700ppm, more preferably 500ppm and inactivated for about 1 to 48h, preferably 20 to 28h, most preferred 24 hours ± 2 hours (such as also ± 1 hour or ± 0.5 hour) at 2°C to 8°C, followed optionally by a hydrolyzation for 2.5 hours ± 0.5 hours at 35°C to 39°C, preferably around 37°C.

A5. A SARS-CoV-2 vaccine according to any preceding aspect, wherein said generated neutralizing antibodies are capable to sufficiently neutralize at least one of the variants of concern such as e.g. Alpha, Beta, Gamma, Delta or Omicron.

A6. A SARS-CoV-2 vaccine according to any preceding aspect, wherein surface proteins in the inactivated SARS-CoV-2 particles comprise reduced modifications compared to viral RNA in the inactivated SARS-CoV-2 particles, preferably wherein surface proteins comprise a reduced proportion of modified residues compared to viral RNA in the inactivated SARS-CoV-2 particles; said modifications being with respect to a native SARS-CoV-2 particles, preferably wherein said modifications comprise alkylated and/or acylated nucleotide or amino acid residues.

A7. A SARS-CoV-2 vaccine according to any preceding aspect, wherein the inactivated SARS-CoV- 2 particles comprises a native conformation of (i) spike (S) protein; (ii) nucleocapsid (N) protein; (iii) membrane (M) glycoprotein; and/or (iv) envelope (E) protein; preferably wherein the inactivated SARS-CoV-2 particle comprises a native conformation spike (S) protein.

A8. A SARS-CoV-2 vaccine according to any preceding aspect, wherein the inactivated SARS-CoV- 2 particles comprises one or more beta-propiolactone-modified cysteine, methionine and/or histidine residues.

A9. A SARS-CoV-2 vaccine according to any preceding aspect, wherein an inactivated SARS-CoV- 2 particles comprises fewer than 200, 100, 50, 30, 20, 15, 10, 9, 8, 7 or 6 beta-propiolactone- modified amino acid residues; preferably wherein a spike (S) protein of the inactivated SARS- CoV-2 particle comprises fewer than 100, 50, 30, 20, 15, 10, 9, 8, 7 or 6 beta-propiolactone- modified amino acid residues; more preferably wherein the inactivated SARS-CoV-2 particles or spike proteins thereof comprises 15 or fewer beta-propiolactone-modified amino acid residues; most preferably wherein the inactivated SARS-CoV-2 particles or spike proteins thereof comprises 1 to 100, 2 to 50, 3 to 30, 5 to 20 or about 15 beta-propiolactone-modified amino acid residues.

A10. A SARS-CoV-2 vaccine according to any preceding aspect, wherein fewer than 20%, 15%, 10%, 5% or 4% of SARS-CoV-2 polypeptides in the particle are beta-propiolactone-modified; preferably wherein 0. 1 to 10%, more preferably 1 to 5%, more preferably 2 to 8% or about 3-6% of SARS-CoV-2 polypeptides in the particles, comprise at least one beta-propiolactone modification; preferably as detected in the vaccine by mass spectroscopy, optionally following enzymatic digestion with trypsin, chymotrypsin and/or PNGase F or acid hydrolysis.

Al l. A SARS-CoV-2 vaccine according to any preceding aspect, wherein a spike (S) protein of the inactivated SARS-CoV-2 particle comprises a beta-propiolactone modification at one or more of the following residues: 49, 146, 166, 177, 207, 245, 379, 432, 519, 625, 1029, 1032, 1058, 1083, 1088, 1101, 1159 and/or 1271; preferably H49, H146, C166, M177, H207, H245, C432, H519, H625, M1029, H1058, H1083, H1088, Hl 101, Hl 159 and/or H1271; or H207, H245, C379, M1029 and/or C1032, e.g. in SEQ ID NO: 5, or a corresponding position in another variant inactivated SARS-CoV-2 particle.

A12. A SARS-CoV-2 vaccine according to any preceding aspect, wherein fewer than 30%, 20%, 10%, 5%, 3% or 1% of one or more of the following residues, preferably of at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or all ofthe following residues, in the inactivated SARS- CoV-2 particles are beta-propiolactone modified in the spike (S) protein, residues 49, 146, 166, 177, 207, 245, 379, 432, 519, 625, 1029, 1032, 1058, 1083, 1088, 1101, 1159 and/or 1271; preferably H49, H146, C166, M177, H207, H245, C432, H519, H625, M1029, H1058, H1083, H1088, Hl 101, Hl 159 and/or H1271; or H207, H245, C379, M1029 and/or C1032; e.g. in SEQ ID NO: 5, or a corresponding position in another variant inactivated SARS-CoV-2 particle, wherein the variant may be the Alpha, Beta, Gamma, Delta or Omicron.

A13. A SARS-CoV-2 vaccine according to any preceding aspect, wherein infectivity of mammalian cells by the inactivated SARS-CoV-2 particles is reduced by at least 99%, 99.99% or 99.9999% compared a native SARS-CoV-2 particle, or wherein infectivity of mammalian cells by the inactivated A SARS-CoV-2 particle is undetectable. A14. A SARS-CoV-2 vaccine according to any preceding aspect, further comprising one or more pharmaceutically acceptable excipients, such as e.g., human serum albumin (HSA).

A15. A SARS-CoV-2 vaccine according to any preceding aspect, further comprising an adjuvant.

A16. A SARS-CoV-2 vaccine according to aspect A15, wherein the adjuvant comprises aluminium hydroxide or aluminium phosphate.

A17. A SARS-CoV-2 vaccine according to aspect A16, wherein aluminium hydroxide or aluminium phosphate is the only adjuvant in the vaccine.

Al 8. A SARS-CoV-2 vaccine according to aspect A16 or A 17, wherein the adjuvant comprises or further comprises a Thl response-directing adjuvant.

A19. A SARS-CoV-2 vaccine according to aspect A18, wherein the Thl response-directing adjuvant comprises 3-O-desacyl-4'-monophosphoryl lipid A (MPL), saponin QS-21, a CpG-containing oligodeoxynucleotide (CpG ODN), squalene, DL-a-tocopherol, a cationic peptide, a deoxyinosine-containing immunostimulatory oligodeoxynucleic acid molecule (I-ODN) and/or imiquimod.

A20. A SARS-CoV-2 vaccine according to aspect A15, wherein the adjuvant comprises:

(i) a liposomal preparation comprising 3-O-desacyl-4'-monophosphoryl lipid A (MPL) and saponin QS-21, preferably Adjuvant System 01;

(ii) a CpG ODN comprising the sequence 5’ TGACTGTGAACGTTCGAGATGA 3’, preferably CpG 1018 (SEQ ID NO: 8);

(iii) squalene, DL-a-tocopherol and polysorbate 80 (preferably Adjuvant System 03);

(iv) an oil-in-water emulsion comprising squalene, Tween 80 and Span 85, preferably MF59;

(v) a peptide of sequence KLKLLLLLKLK (SEQ ID NO: 7) and oligo-d(IC)i3 (SEQ ID NO: 6), preferably IC31 ; or

(vi) an aluminium salt and optionally a Thl -directing adjuvant.

A21. A SARS-CoV-2 vaccine according to aspect A15, wherein the adjuvant comprises aluminium hydroxide and a Thl -directing adjuvant. A22. The SARS-CoV-2 vaccine according to any preceding aspect, wherein the vaccine is able to seroconvert a subject that is administered the SARS-CoV-2 vaccine with at least a 70% probability.

A23. The SARS-CoV-2 vaccine according to aspect A22, wherein the SARS-CoV-2 vaccine is able to seroconvert the subject that is administered the SARS-CoV-2 vaccine with at least an 80%, 85%, 90%, or 95% probability.

A24. The SARS-CoV-2 vaccine according to any one of the preceding aspects, wherein the SARS- CoV-2 particle comprises at least two, e.g. two or three RNA sequences selected from the group consisting of

(a) an RNA sequence (and/or fragments thereof, optionally comprising modified (preferably alkylated or acylated) nucleotide residues) corresponding to a DNA sequence (i) as defined by SEQ ID NO: 1 or 9; or (ii) having at least 80%, at least 85%, at least 90%, at least 95% or at least 99% sequence identity to SEQ ID NO: 1 or 9 as provided in Figures 8A and 8B, respectively; preferably wherein a native (non-inactivated) SARS-CoV-2 particle comprising the RNA sequence is able to pack a virulent SARS-CoV-2 virus; and

(b) an RNA sequence (and/or fragments thereof, optionally comprising modified (preferably alkylated or acylated) nucleotide residues) corresponding to a DNA sequence (i) as defined by a sequence of a variant of concern; or (ii) having at least 80%, at least 85%, at least 90%, at least 95% or at least 99% sequence identity to such a sequence of a variant of concern (SEQ ID NO: 2 in Figure 9 or SEQ ID NO: 3 in Figure 10 or SEQ ID NO: 4 in Figure 11); preferably wherein a native (non-inactivated) SARS-CoV-2 particle comprising the RNA sequence is able to pack a virulent SARS-CoV-2 virus; wherein the combination of SEQ ID NO: 1 (wild-type, reference type) and SEQ ID NO: 3 or 4 (Omicron); SEQ ID NO: 9 (wild-type, INMI isolate) and SEQ ID NO: 3 or 4 (Omicron); or SEQ ID NO: 2 (Delta) and SEQ ID NO: 3 or 4 (Omicron) is preferred.

A25. The SARS-CoV-2 vaccine according to any one of the preceding aspects, wherein the said vaccine comprises an additional SARS-CoV-2 particle that comprises an RNA sequence (and/or fragments thereof, optionally comprising modified (preferably alkylated or acylated) nucleotide residues) corresponding to a DNA sequence (i) as defined by SEQ ID NO: 2; or (ii) having at least 80%, at least 85%, at least 90%, at least 95% or at least 99% sequence identity to SEQ ID NO: 2; preferably wherein a native (non-inactivated) SARS-CoV-2 particle comprising the RNA sequence is able to pack a virulent SARS-CoV-2 virus. A26. The SARS-CoV-2 vaccine according to any one of the preceding aspects, wherein the said vaccine comprises an additional SARS-CoV-2 particle that comprises an RNA sequence (and/or fragments thereof, optionally comprising modified (preferably alkylated or acylated) nucleotide residues) corresponding to a DNA sequence (i) as defined by SEQ ID NO: 3; or (ii) having at least 80%, at least 85%, at least 90%, at least 95% or at least 99% sequence identity to SEQ ID NO: 3; preferably wherein a native (non-inactivated) SARS-CoV-2 particle comprising the RNA sequence is able to pack a virulent SARS-CoV-2 virus.

A27. The SARS-CoV-2 vaccine according to any preceding aspect, wherein the vaccine is obtained or obtainable from Vero cells.

A28. The SARS-CoV-2 vaccine according to any preceding aspect, wherein, upon administration to a human subject, the vaccine (i) does not induce antibody-dependent enhancement (ADE) of SARS-CoV-2-associated disease (COVID-19); and/or (ii) does not induce immunopathology in the subject.

A29. A method of preventing or treating SARS-CoV-2 infection and/or SARS-CoV-2-associated disease (COVID-19) such as severe COVID-19 disease, hospitalization caused by COVID-19 or death caused by COVID-19, in a human subject in need thereof, comprising administering a prophy tactically or therapeutically effective amount of the SARS-CoV-2 vaccine of any preceding aspect to the subject.

A30. The method according to aspect A29, further comprising administering a second, third or further dose of a prophylactically or therapeutically effective amount of the SARS-CoV-2 vaccine, preferably wherein the second dose of the vaccine is the same formulation as the first.

A31. The method according to aspect A29 or A30, wherein said prophylactically or therapeutically effective amount of the SARS-CoV-2 vaccine per dose is defined as about 1 to 150 AU/dose per SARS-CoV-2 particle, preferably between about 2 to 75 AU/dose per SARS-CoV-2 particle, preferably between about 3 and 60 AU/dose per SARS-CoV-2 particle, more preferably between about 3 and 55 AU/dose per SARS-CoV-2 particle, more preferably between about 3 and 53 AU/dose per SARS-CoV-2 particle, as assessed by ELISA, even more preferably between about 3 and 40 AU/dose per SARS-CoV-2 particle, more preferably about 10 to 60 AU/dose per SARS- CoV-2 particle, 20 to 50 AU/dose per SARS-CoV-2 particle, 25 to 45 AU/dose per SARS-CoV- 2 particle or 30 to 40 AU/dose per SARS-CoV-2 particle, such as e.g. 33 AU/dose or similar per SARS-CoV-2 particle.

A32. The method according to aspect A29 or A30, wherein said prophylactically or therapeutically effective amount per dose of the SARS-CoV-2 variant in the vaccine is defined as about 0.05 to 50 pg total protein, about 0.1 to 25 pg, about 0.25 to 12.5 pg, preferably about 0.5 to 5 pg total protein, more preferably at least 2.5 pg total protein, at least 3.5 pg total protein or at least 2.5 pg total protein, even more preferably 2.5 pg to 25 pg, 3.5 pg to 10 pg or 4 pg to 6 pg total protein/dose, most preferably about 5 pg total protein/dose, e.g. as measured by (p)BCA.

A33. The method according to aspect A29 or A30, wherein said prophylactically or therapeutically effective amount per dose of the SARS-CoV-2 variant in the vaccine is defined as about 0.025 to 25 pg S-protein, about 0.05 to 12.5 pg, about 0.125 to 6.25 pg, preferably about 0.25 to 2.5 pg S -protein, as measured by ELISA.

A34. The method according to aspect A30, wherein

(i) a second dose of the SARS-CoV-2 vaccine is administered about 7 days, about 14 days, about 21 days, or about 28 days after a first dose of the SARS-CoV-2 vaccine, preferably wherein the second dose of the vaccine is the same formulation as the first; and/or

(ii) a further dose of the SARS-CoV-2 vaccine about every 6 months, about every 7 months, about every 8 months, about every 9 months, about every 10 months, about every 11 months or about every 12 months or about every 13 months after the last dose of the SARS-CoV-2 vaccine, preferably wherein such further dose of the vaccine is the same formulation as the first.

A35. The method according to any one of aspects A28 to A34, wherein the administering results in production of SARS-CoV-2 neutralizing antibodies.

A36. A method of producing a SARS-CoV-2 vaccine, comprising:

(a) producing at least 2 different native SARS-CoV-2 particles (e.g. wild type and Omicron or

Delta and Omicron);

(b) inactivating the two different native SARS-CoV-2 particles to obtain two different inactivated SARS-CoV-2 particles;

(c) incorporating the 2 different inactivated SARS-CoV-2 particles in a vaccine system (either in a combined formulation/vial or a kit with two different compositions/vials); wherein a native surface conformation of the SARS-CoV-2 particle is preserved in the inactivation step, such that the vaccine is capable of generating neutralizing antibodies against native SARS-CoV-2 particles in a human subject, preferably is capable of generating neutralizing antibodies against heterologous native SARS-CoV-2 particles in a human subject.

A37. The method according to aspect A36, wherein the vaccine composition comprises aluminium hydroxide.

A38. The method according to aspect A37, wherein the SARS-CoV-2 vaccine comprising aluminium hydroxide contains less than 1.25 ppb Cu.

A39. The method according to any of aspects A36 to A38, wherein the inactivation step preferentially targets viral RNA in the SARS-CoV-2 particle.

A40. The method according to aspect A36 or A39, wherein the inactivation step comprises (i) alkylating and/or acylating viral RNA (ii) modifying purine (preferably guanine) residues or introducing strand breaks into viral RNA and/or (iii) cross-linking viral RNA with one or more viral proteins.

A41. The method according to any one of aspects A36, A39 or A40, wherein the inactivation step comprises treating the native SARS-CoV-2 particles with beta-propiolactone.

A42. The method according to aspect A41, wherein the concentration of beta-propiolactone in the inactivation step is 0.01 to 1% by weight, preferably 0.05 to 0.5% by weight, more preferably about 0.1% by weight.

A43. The method according to aspect A41 or A42, wherein the native SARS-CoV-2 particles are contacted with beta-propiolactone for at least 5 hours, at least 10 hours, at least 24 hour or at least 4 days.

A44. The method according to any of aspects A36 or A39 to A43, wherein the inactivation step is performed at about 0°C to about 25°C, preferably about 4°C or about 22°C.

A45. The method according to any of aspects A36 or A39 to A44, wherein the inactivation step comprises treating the native SARS-CoV-2 particles with ultraviolet (UV) light. A46. The method according to any one of aspects A36 or A39 to A45, wherein step (a) comprises one or more of the following steps:

(i) passaging SARS-CoV-2 particles on Vero cells, thereby producing a culture medium comprising the SARS-CoV-2 particles;

(ii) passaging a second different SARS-CoV-2 virus on Vero cells, thereby producing a culture medium comprising the second SARS-CoV-2 particles;

(iii) harvesting the culture medium of (i) and (ii);

(iv) precipitating the harvested culture medium of (iii), thereby producing the two different native SARS-CoV-2 particles in a supernatant and optionally combining the two different native SARS-CoV-2 particles in 1 : 1 or 1 :2 or 2: 1 or other appropriate ratio.

A47. The method according to aspect A46, further comprising concentrating the culture medium of (ii) prior to step (iii).

A48. The method according to aspect A46 or A47, wherein the precipitating of (iii) comprises contacting the culture medium of (ii) with protamine sulfate or benzonase.

A49. The method according to any one of aspects A36 or A39 to A48, further comprising dialyzing the inactivated SARS-CoV-2 particles, thereby producing a dialyzed SARS-CoV-2.

A50. The method according to aspect A49, further comprising filtering the dialyzed SARS-CoV-2.

A51. The method according to any one of aspects A36 or A39 to A50, wherein the inactivation step comprises contacting a liquid composition comprising native SARS-CoV-2 particles with a chemical viral inactivating agent in a container, mixing the chemical viral inactivating agent and the liquid composition comprising SARS-CoV-2 particles under conditions of laminar flow but not turbulent flow, and incubating the chemical viral inactivating agent and the liquid composition comprising SARS-CoV-2 particles for a time sufficient to inactivate the viral particles.

A52. The method according to aspect A51, wherein the inactivation step is performed in a flexible bioreactor bag. A53. The method according to aspect A51 or A52, wherein the inactivation step comprises five or less container inversions during the period of inactivation.

A54. The method according to any one of aspects A51 to A53, wherein the mixing of the chemical viral inactivating agent and the composition comprising native SARS-CoV-2 particles comprises subjecting the container to rocking, rotation, orbital shaking, or oscillation for not more than 10 minutes at not more than 10 rpm during the period of incubation.

A55. The method according to any one of aspects A36 or A39 to A54, further comprising purifying the inactivated SARS-CoV-2 particles by one or more methods selected from (i) batch chromatography and/or (ii) sucrose density gradient centrifugation.

A56. The method according to any one of aspects A36 or A39 to A55, wherein step (c) comprises combining the inactivated SARS-CoV-2 particles with an adjuvant.

A57. The method according to aspect A56, wherein the adjuvant comprises a Thl response-directing adjuvant.

A58. The method according to aspect A56 or A57, wherein the adjuvant comprises 3-O-desacyl-4'- monophosphoryl lipid A (MPL), saponin QS-21, a CpG-containing oligodeoxynucleotide (CpG ODN), squalene, DL-a-tocopherol and/or imiquimod.

A59. A SARS-CoV-2 vaccine obtained or obtainable by the method of any one of aspects A36 or A39 to A58.

A60. Use of a SARS-CoV-2 vaccine of any one of aspects Al to A28 or A59 for the treatment or prevention of a SARS-CoV-2 infection in a subject.

A61. A pharmaceutical composition for use in the prevention or treatment of a SARS-CoV-2 infection in a subject, wherein said pharmaceutical composition is the inactivated SARS-CoV-2 vaccine as defined in any one of aspects Al to A28 or A59, optionally in combination with one or more pharmaceutically acceptable excipients and/or adjuvants.

A62. The SARS-CoV-2 vaccine as defined in any one of aspects Al to A28 or A59 for use as a medicament. A63. A vaccine, method, use or pharmaceutical composition according to any preceding aspect, wherein the subject is (i) an elderly subject, preferably a subject over 65, over 70 or over 80 years of age; (ii) an immunocompromised subject; or (iii) a pregnant subject.

A64. A vaccine, method, use or pharmaceutical composition according to any preceding aspect, for use in prevention or treatment of a SARS-CoV-2 infection without induction of (i) antibodydependent enhancement (ADE) of SARS-CoV-2 -associated disease (COVID- 19); and/or (ii) vaccine-associated respiratory disease (VAERD); and/or (iii) enhanced respiratory disease (ERD); and/or (iv) immunopathology in the subject.

Bl. A SARS-CoV-2 vaccine for use as a booster vaccination, wherein the vaccine comprises a betapropiolactone inactivated SARS-CoV-2 particle, wherein said SARS-CoV-2 particle comprises an RNA sequence corresponding to a DNA sequence (i) as defined by SEQ ID NO: 9; or (ii) having at least 80%, at least 85%, at least 90%, at least 95% or at least 99% sequence identity to SEQ ID NO: 9; preferably wherein a native (non-inactivated) SARS-CoV-2 particle comprising the RNA sequence is able to pack a virulent SARS-CoV-2 virus.

B2. The SARS-CoV-2 vaccine for use as a booster vaccination of aspect Bl, wherein said SARS- CoV-2 virus comprises a Spike (S) protein comprising or consisting of (i) an amino acid sequence as defined by SEQ ID NO: 5, or (ii) an amino acid sequence having at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to SEQ ID NO: 5; preferably wherein a native (non-inactivated) SARS-CoV-2 particle comprising the Spike protein is able to pack a virulent SARS-CoV-2 virus.

B3. The SARS-CoV-2 vaccine for use as a booster vaccination according to aspect Bl or B2, comprising one or more adjuvants.

B4. The SARS-CoV-2 vaccine for use as a booster vaccination according to aspect B3, wherein the adjuvant comprises a Thl response-directing adjuvant.

B5. The SARS-CoV-2 vaccine for use as a booster vaccination according to aspect B4, wherein the Thl response-directing adjuvant comprises 3-O-desacyl-4'-monophosphoryl lipid A (MPL), saponin QS-21, a CpG-containing oligodeoxynucleotide (CpG ODN), squalene, DL-a- tocopherol and/or imiquimod. B6. The SARS-CoV-2 vaccine for use as a booster vaccination according to aspect B3, wherein the adjuvant is an aluminium salt, i.e., aluminium phosphate or aluminium hydroxide.

B7. The SARS-CoV-2 vaccine for use as a booster vaccination according to aspects B3, wherein the adjuvants comprise a combination of aluminium hydroxide and a Th 1 -directing adjuvant.

B8. The SARS-CoV-2 vaccine for use as a booster vaccination according to aspects B6 or B7, wherein the aluminium hydroxide adjuvant comprises less than 1.25 ppb Cu in the final vaccine formulation.

B9. The SARS-CoV-2 vaccine for use as a booster vaccination according to any one of aspects B 1 to B8, further comprising one or more pharmaceutically acceptable excipients.

BIO. The SARS-CoV-2 vaccine for use as a booster vaccination according to aspect B9, wherein the pharmaceutically acceptable excipients comprise recombinant human serum albumin (rHSA) and/or phosphate buffered saline (PBS).

B 11. The SARS-CoV-2 vaccine for use as a booster vaccination according to any one of aspects B 1 to BIO, wherein a “standard” dose is defined as 33 AU/0.5 mb.

B 12. The SARS-CoV-2 vaccine for use as a booster vaccination according to any one of aspects B 1 to BIO, wherein a “double” dose is defined as 66 AU/1.0 mb.

B13. A method of preventing or treating SARS-CoV-2 infection and/or SARS-CoV-2-associated disease (COVID-19) in a human subject in need thereof, comprising administering as a booster vaccination a prophylactically or therapeutically effective amount of a SARS-CoV-2 vaccine comprising a beta-propiolactone inactivated SARS-CoV-2 particle, wherein said SARS-CoV-2 particle comprises an RNA sequence corresponding to a DNA sequence (i) as defined by SEQ ID NO: 9; or (ii) having at least 80%, at least 85%, at least 90%, at least 95% or at least 99% sequence identity to SEQ ID NO: 9; preferably wherein a native (non-inactivated) SARS-CoV-2 particle comprising the RNA sequence is able to pack a virulent SARS-CoV-2 virus.

B14. The method according to aspect B13, wherein said prophylactically or therapeutically effective amount of the SARS-CoV-2 vaccine per dose is defined as about 1 to 100 AU/dose, preferably between about 2 to 75 AU/dose, preferably between about 3 and 60 AU/dose, more preferably between about 3 and 55 AU/dose, more preferably between about 3 and 53 AU/dose, as assessed by EUISA, even more preferably between about 3 and 70 AU/dose, more preferably about 10 to 60 AU/dose, 20 to 50 AU/dose, 25 to 45 AU/dose or 30 to 40 AU/dose such as e.g. 33 AU/ dose, 35 AU/dose, 40 AU/dose or 66 AU/dose.

B15. The method according to aspect B13 or Bl 4, wherein said prophylactically or therapeutically effective amount of the SARS-CoV-2 vaccine per dose is defined as about 0.05 to 50 pg total protein, about 0. 1 to 25 pg, about 0.25 to 12.5 pg, preferably about 0.5 to 5 pg total protein, more preferably at least 2.5 pg total protein, at least 3.5 pg total protein or at least 2.5 pg total protein, even more preferably 2.5 pg to 25 pg, 3.5 pg to 10 pg or 4 pg to 6 pg total protein/dose, most preferably about 5 pg total protein/dose, e.g. as measured by (p)BCA.

B16. The method according to any one of aspects B13 to B15, wherein said prophylactically or therapeutically effective amount of the SARS-CoV-2 vaccine is defined as about 0.025 to 25 pg S-protein, about 0.05 to 12.5 pg, about 0.125 to 6.25 pg, preferably about 0.25 to 2.5 pg S- protein, as measured by EUISA.

B17. The method according to any one of aspects B13 to Bl 6, wherein the prophylactically or therapeutically effective amount of the SARS-CoV-2 vaccine is administered as a booster following natural COVID- 19 infection in a previously unvaccinated subject.

Bl 8. The method according to any one of aspects B13 to Bl 6, wherein the prophylactically or therapeutically effective amount of the SARS-CoV-2 vaccine is administered as a booster following vaccination with a homologous or heterologous SARS-CoV-2 vaccine.

B19. The method according to any one of aspects B13 to Bl 6, wherein the prophylactically or therapeutically effective amount of the SARS-CoV-2 vaccine is administered following both vaccination with a homologous or heterologous SARS-CoV-2 vaccine and natural COVID-19 infection.

B20. The method according to any one of aspects B13 to Bl 9, wherein the administering of the prophylactically or therapeutically effective amount of the SARS-CoV-2 vaccine results in production of SARS-CoV-2 neutralizing antibodies. B21. The method according to any one of aspects B13 to B20, wherein the booster vaccination is administered at least 2 weeks, at least 4 weeks, at least 8 weeks, at least 12 weeks, at least 16 weeks, preferably at least 6 months following the last vaccination with a homologous or heterologous SARS-CoV-2 vaccine or natural COVID-19 infection.

B22. The method according to any one of aspects B18 to B21, wherein the heterologous SARS-CoV- 2 vaccine is an mRNA SARS-CoV-2 vaccine or an adenovirus vector SARS-CoV-2 vaccine.

B23. The method according to any one of aspects B13 to B21, wherein subject is an adult, i.e., 18 years or older.

B24. The method according to any one of aspects B13 to B23, wherein subject is an older adult, i.e., 50 years or older, 55 years or older, 60 years or older, 65 years or older, 70 years or older, especially 80 years or older.

B25. The method according to aspect B23, wherein a standard dose (33 AU/0.5 mL) is administered to the adult subject.

B26. The method according to aspect B24, wherein a double dose (66 AU/1.0 mL) is administered to the older adult subject.

All publications mentioned in the above specification are herein incorporated by reference. Various modifications and variations of the described embodiments of the present invention will be apparent to those skilled in the art without departing from the scope and spirit of the present invention. Although the present invention has been described in connection with specific preferred embodiments, it should be understood that the invention as claimed should not be unduly limited to such specific embodiments.

Indeed, various modifications of the described modes for carrying out the invention which are obvious to those skilled in the art are intended to be within the scope of the following claims.

Claims

1. A SARS-CoV-2 vaccine comprising at least two or exactly two different beta-propiolactone- inactivated SARS-CoV-2 particles, wherein the vaccine is capable of generating neutralizing antibodies against a native homologous and/or heterologous SARS-CoV-2 particle and/or is capable of raising an effective T-cell response against a native homologous and/or heterologous SARS-CoV-2 particle in a human subject.

2. A SARS-CoV-2 vaccine according to claim 1, wherein a native surface conformation of the SARS-CoV-2 particle is preserved in the vaccine and/or wherein the activity of the furin cleavage site within the viral RNA is reduced or eliminated by passaging out the furin site and/or introducing mutations in the cleavage site.

3. A SARS-CoV-2 vaccine according to claim 1 or 2, wherein viral RNA in the inactivated SARS- CoV-2 particle is replication-deficient, preferably wherein viral RNA in the inactivated SARS- CoV-2 particle (i) is alkylated and/or acylated (ii) comprises one or more modified purine (preferably guanine) residues and/or strand breaks and/or (iii) is cross-linked with one or more viral proteins.

4. A SARS-CoV-2 vaccine according to any preceding claim, wherein the SARS-CoV-2 particles are beta-propiolactone -inactivated at a concentration of 300 to 700ppm, more preferably 500ppm and inactivated for about 1 to 48h, preferably 20 to 28h, most preferred 24 hours ± 2 hours (such as also ± 1 hour or ± 0.5 hour) at 2°C to 8°C, followed optionally by a hydrolyzation for 2.5 hours ± 0.5 hours at 35°C to 39°C, preferably around 37°C.

5. A SARS-CoV-2 vaccine according to any preceding claim, wherein said generated neutralizing antibodies are capable to sufficiently neutralize at least one of the variants of concern such as e.g. Alpha, Beta, Gamma, Delta or Omicron.

6. A SARS-CoV-2 vaccine according to any preceding claim, wherein surface proteins in the inactivated SARS-CoV-2 particles comprise reduced modifications compared to viral RNA in the inactivated SARS-CoV-2 particles, preferably wherein surface proteins comprise a reduced proportion of modified residues compared to viral RNA in the inactivated SARS-CoV-2 particles; said modifications being with respect to a native SARS-CoV-2 particles, preferably wherein said modifications comprise alkylated and/or acylated nucleotide or amino acid residues. A SARS-CoV-2 vaccine according to any preceding claim, wherein the inactivated SARS-CoV- 2 particles comprises a native conformation of (i) spike (S) protein; (ii) nucleocapsid (N) protein; (iii) membrane (M) glycoprotein; and/or (iv) envelope (E) protein; preferably wherein the inactivated SARS-CoV-2 particle comprises a native conformation spike (S) protein. A SARS-CoV-2 vaccine according to any preceding claim, wherein the inactivated SARS-CoV- 2 particles comprises one or more beta-propiolactone-modified cysteine, methionine and/or histidine residues. A SARS-CoV-2 vaccine according to any preceding claim, wherein an inactivated SARS-CoV- 2 particles comprises fewer than 200, 100, 50, 30, 20, 15, 10, 9, 8, 7 or 6 beta-propiolactone- modified amino acid residues; preferably wherein a spike (S) protein of the inactivated SARS- CoV-2 particle comprises fewer than 100, 50, 30, 20, 15, 10, 9, 8, 7 or 6 beta-propiolactone- modified amino acid residues; more preferably wherein the inactivated SARS-CoV-2 particles or spike proteins thereof comprises 15 or fewer beta-propiolactone-modified amino acid residues; most preferably wherein the inactivated SARS-CoV-2 particles or spike proteins thereof comprises 1 to 100, 2 to 50, 3 to 30, 5 to 20 or about 15 beta-propiolactone-modified amino acid residues. A SARS-CoV-2 vaccine according to any preceding claim, wherein fewer than 20%, 15%, 10%, 5% or 4% of SARS-CoV-2 polypeptides in the particle are beta-propiolactone-modified; preferably wherein 0. 1 to 10%, more preferably 1 to 5%, more preferably 2 to 8% or about 3-6% of SARS-CoV-2 polypeptides in the particles, comprise at least one beta-propiolactone modification; preferably as detected in the vaccine by mass spectroscopy, optionally following enzymatic digestion with trypsin, chymotrypsin and/or PNGase F or acid hydrolysis. A SARS-CoV-2 vaccine according to any preceding claim, wherein a spike (S) protein of the inactivated SARS-CoV-2 particle comprises a beta-propiolactone modification at one or more of the following residues: 49, 146, 166, 177, 207, 245, 379, 432, 519, 625, 1029, 1032, 1058, 1083, 1088, 1101, 1159 and/or 1271; preferably H49, H146, C166, M177, H207, H245, C432, H519, H625, M1029, H1058, H1083, H1088, Hl 101, Hl 159 and/or H1271; or H207, H245, C379, M1029 and/or C1032, e.g. in SEQ ID NO: 5, or a corresponding position in another variant inactivated SARS-CoV-2 particle. A SARS-CoV-2 vaccine according to any preceding claim, wherein fewer than 30%, 20%, 10%, 5%, 3% or 1% of one or more of the following residues, preferably of at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or all ofthe following residues, in the inactivated SARS- CoV-2 particles are beta-propiolactone modified in the spike (S) protein, residues 49, 146, 166, 177, 207, 245, 379, 432, 519, 625, 1029, 1032, 1058, 1083, 1088, 1101, 1159 and/or 1271; preferably H49, H146, C166, M177, H207, H245, C432, H519, H625, M1029, H1058, H1083, H1088, Hl 101, Hl 159 and/or H1271; or H207, H245, C379, M1029 and/or C1032; e.g. in SEQ ID NO: 5, or a corresponding position in another variant inactivated SARS-CoV-2 particle, wherein the variant may be the Alpha, Beta, Gamma, Delta or Omicron. A SARS-CoV-2 vaccine according to any preceding claim, wherein infectivity of mammalian cells by the inactivated SARS-CoV-2 particles is reduced by at least 99%, 99.99% or 99.9999% compared a native SARS-CoV-2 particle, or wherein infectivity of mammalian cells by the inactivated A SARS-CoV-2 particle is undetectable. A SARS-CoV-2 vaccine according to any preceding claim, further comprising one or more pharmaceutically acceptable excipients, such as e.g., human serum albumin (HSA). A SARS-CoV-2 vaccine according to any preceding claim, further comprising an adjuvant. A SARS-CoV-2 vaccine according to claim 15, wherein the adjuvant comprises aluminium hydroxide or aluminium phosphate. A SARS-CoV-2 vaccine according to claim 16, wherein aluminium hydroxide or aluminium phosphate is the only adjuvant in the vaccine. A SARS-CoV-2 vaccine according to claim 16 or 17, wherein the adjuvant comprises or further comprises a Thl response-directing adjuvant. A SARS-CoV-2 vaccine according to claim 18, wherein the Thl response -directing adjuvant comprises 3-O-desacyl-4'-monophosphoryl lipid A (MPL), saponin QS-21, a CpG-containing oligodeoxynucleotide (CpG ODN), squalene, DL-a-tocopherol, a cationic peptide, a deoxyinosine-containing immunostimulatory oligodeoxynucleic acid molecule (I-ODN) and/or imiquimod. A SARS-CoV-2 vaccine according to claim 15, wherein the adjuvant comprises:

(ii) a CpG ODN comprising the sequence 5’ TGACTGTGAACGTTCGAGATGA 3’;

(vi) an aluminium salt and optionally a Th 1 -directing adjuvant. A SARS-CoV-2 vaccine according to claim 15, wherein the adjuvant comprises aluminium hydroxide and a Th 1 -directing adjuvant. The SARS-CoV-2 vaccine according to any preceding claim, wherein the vaccine is able to seroconvert a subject that is administered the SARS-CoV-2 vaccine with at least a 70% probability. The SARS-CoV-2 vaccine according to claim 22, wherein the SARS-CoV-2 vaccine is able to seroconvert the subject that is administered the SARS-CoV-2 vaccine with at least an 80%, 85%, 90%, or 95% probability. The SARS-CoV-2 vaccine according to any one of the preceding claims, wherein the SARS- CoV-2 particle comprises at least two, e.g. two or three RNA sequences selected from the group consisting of

(i) an RNA sequence (and/or fragments thereof, optionally comprising modified (preferably alkylated or acylated) nucleotide residues) corresponding to a DNA sequence (i) as defined by SEQ ID NO: 1 or SEQ ID NO: 9; or (ii) having at least 80%, at least 85%, at least 90%, at least 95% or at least 99% sequence identity to SEQ ID NO: 1 as provided in Figure 8A or SEQ ID NO: 9 as provided in Figure 8B, respectively; preferably wherein a native (noninactivated) SARS-CoV-2 particle comprising the RNA sequence is able to pack a virulent SARS-CoV-2; and

(ii) an RNA sequence (and/or fragments thereof, optionally comprising modified (preferably alkylated or acylated) nucleotide residues) corresponding to a DNA sequence (i) as defined by a sequence of a variant of concern; or (ii) having at least 80%, at least 85%, at least 90%, at least 95% or at least 99% sequence identity to such a sequence of a variant of concern (SEQ ID NO: 2 in Figure 9 or SEQ ID NO: 3 in Figure 10 or SEQ ID NO: 4 in Figure 11); preferably wherein a native (non-inactivated) SARS-CoV-2 particle comprising the RNA sequence is able to pack a virulent SARS-CoV-2; wherein the combination of SEQ ID NO: 1 (wild type, reference type) and SEQ ID NO: 3 or 4 (Omicron); SEQ ID NO: 9 (wild type, INMI) and SEQ ID NO: 3 or 4 (Omicron); or SEQ ID NO: 2 (Delta) and SEQ ID NO: 3 or 4 (Omicron) is preferred.

25. The SARS-CoV-2 vaccine according to any one of the preceding claims, wherein the said vaccine comprises an additional SARS-CoV-2 particle that comprises an RNA sequence (and/or fragments thereof, optionally comprising modified (preferably alkylated or acylated) nucleotide residues) corresponding to a DNA sequence (i) as defined by SEQ ID NO: 2; or (ii) having at least 80%, at least 85%, at least 90%, at least 95% or at least 99% sequence identity to SEQ ID NO: 2; preferably wherein a native (non-inactivated) SARS-CoV-2 particle comprising the RNA sequence is able to pack a virulent SARS-CoV-2.

26. The SARS-CoV-2 vaccine according to any one of the preceding claims, wherein the said vaccine comprises an additional SARS-CoV-2 particle that comprises an RNA sequence (and/or fragments thereof, optionally comprising modified (preferably alkylated or acylated) nucleotide residues) corresponding to a DNA sequence (i) as defined by SEQ ID NO: 3 or SEQ ID NO: 4; or (ii) having at least 80%, at least 85%, at least 90%, at least 95% or at least 99% sequence identity to SEQ ID NO: 3 or SEQ ID NO: 4, respectively; preferably wherein a native (noninactivated) SARS-CoV-2 particle comprising the RNA sequence is able to pack a virulent SARS-CoV-2.

27. The SARS-CoV-2 vaccine according to any preceding claim, wherein the vaccine is obtained or obtainable from Vero cells.

28. The SARS-CoV-2 vaccine according to any preceding claim, wherein, upon administration to a human subject, the vaccine (i) does not induce antibody-dependent enhancement (ADE) of SARS-CoV-2-associated disease (COVID-19); and/or (ii) does not induce immunopathology in the subject.

29. A method of preventing or treating SARS-CoV-2 infection and/or SARS-CoV-2-associated disease (COVID-19) such as severe COVID-19 disease, hospitalization caused by COVID-19 or death caused by COVID-19, in a human subject in need thereof, comprising administering a prophy tactically or therapeutically effective amount of the SARS-CoV-2 vaccine of any preceding claim to the subject. The method according to claim 29, further comprising administering a second, third or further dose of a prophylactically or therapeutically effective amount of the SARS-CoV-2 vaccine, preferably wherein the second dose of the vaccine is the same formulation as the first. The method according to claim 29 or 30, wherein said prophylactically or therapeutically effective amount of the SARS-CoV-2 vaccine per dose is defined as about 1 to 150 AU/dose per SARS-CoV-2 particle, preferably between about 2 to 75 AU/dose per SARS-CoV-2 particle, preferably between about 3 and 60 AU/dose per SARS-CoV-2 particle, more preferably between about 3 and 55 AU/dose per SARS-CoV-2 particle, more preferably between about 3 and 53 AU/dose per SARS-CoV-2 particle, as assessed by EUISA, even more preferably between about 3 and 40 AU/dose per SARS-CoV-2 particle, more preferably about 10 to 60 AU/dose per SARS- CoV-2 particle, 20 to 50 AU/dose per SARS-CoV-2 particle, 25 to 45 AU/dose per SARS-CoV- 2 particle or 30 to 40 AU/dose per SARS-CoV-2 particle, such as e.g. 33 AU/dose or simitar per SARS-CoV-2 particle. The method according to claim 29 or 30, wherein said prophylactically or therapeutically effective amount per dose of the SARS-CoV-2 variant in the vaccine is defined as about 0.05 to 50 pg total protein, about 0.1 to 25 pg, about 0.25 to 12.5 pg, preferably about 0.5 to 5 pg total protein, more preferably at least 2.5 pg total protein, at least 3.5 pg total protein or at least 2.5 pg total protein, even more preferably 2.5 pg to 25 pg, 3.5 pg to 10 pg or 4 pg to 6 pg total protein/dose, most preferably about 5 pg total protein/dose, e.g. as measured by (p)BCA. The method according to claim 29 or 30, wherein said prophylactically or therapeutically effective amount per dose of the SARS-CoV-2 variant in the vaccine is defined as about 0.025 to 25 pg S-protein, about 0.05 to 12.5 pg, about 0.125 to 6.25 pg, preferably about 0.25 to 2.5 pg S -protein, as measured by EUISA. The method according to claim 30, wherein

(i) a second dose of the SARS-CoV-2 vaccine is administered about 7 days, about 14 days, about 21 days, or about 28 days after a first dose of the SARS-CoV-2 vaccine, preferably wherein the second dose of the vaccine is the same formulation as the first; and/or (ii) a further dose of the SARS-CoV-2 vaccine about every 6 months, about every 7 months, about every 8 months, about every 9 months, about every 10 months, about every 11 months or about every 12 months or about every 13 months after the last dose of the SARS-CoV-2 vaccine, preferably wherein such further dose of the vaccine is the same formulation as the first. The method according to any one of claims 28 to 34, wherein the administering results in production of SARS-CoV-2 neutralizing antibodies. A method of producing a SARS-CoV-2 vaccine, comprising:

(a) producing at least 2 different native SARS-CoV-2 particles (e.g. wild type and Omicron or Delta and Omicron);

(c) incorporating the 2 different inactivated SARS-CoV-2 particles in a vaccine system (either in a combined formulation/vial or a kit with two different compositions/vials); wherein a native surface conformation of the SARS-CoV-2 particle is preserved in the inactivation step, such that the vaccine is capable of generating neutralizing antibodies against native SARS-CoV-2 particles in a human subject, preferably is capable of generating neutralizing antibodies against heterologous native SARS-CoV-2 particles in a human subject. The method according to claim 36, wherein the vaccine composition comprises aluminium hydroxide. The method according to claim 37, wherein the SARS-CoV-2 vaccine comprising aluminium hydroxide contains less than 1.25 ppb Cu. The method according to any of claims 36 to 38, wherein the inactivation step preferentially targets viral RNA in the SARS-CoV-2 particle. The method according to claim 36 or 39, wherein the inactivation step comprises (i) alkylating and/or acylating viral RNA (ii) modifying purine (preferably guanine) residues or introducing strand breaks into viral RNA and/or (iii) cross-linking viral RNA with one or more viral proteins. The method according to any one of claims 36, 39 or 40, wherein the inactivation step comprises treating the native SARS-CoV-2 particles with beta-propiolactone. The method according to claim 41, wherein the concentration of beta-propiolactone in the inactivation step is 0.01 to 1% by weight, preferably 0.05 to 0.5% by weight, more preferably about 0.1% by weight. The method according to claim 41 or 42, wherein the native SARS-CoV-2 particles are contacted with beta-propiolactone for at least 5 hours, at least 10 hours, at least 24 hour or at least 4 days. The method according to any of claims 36 or 39 to 43, wherein the inactivation step is performed at about 0°C to about 25°C, preferably about 4°C or about 22°C. The method according to any of claims 36 or 39 to 44, wherein the inactivation step comprises treating the native SARS-CoV-2 particles with ultraviolet (UV) light. The method according to any one of claims 36 or 39 to 45, wherein step (a) comprises one or more of the following steps:

(i) passaging a SARS-CoV-2 particles on Vero cells, thereby producing a culture medium comprising the SARS-CoV-2 particles;

(iii) harvesting the culture medium of (i) and (ii);

(iv) precipitating the harvested culture mediums of (ii), thereby producing the two different native SARS-CoV-2 particles in a supernatant and optionally combining the two different native SARS-CoV-2 particles in 1: 1 or 1:2 or 2: 1 or other appropriate ratio. The method according to claim 46, further comprising concentrating the culture medium of (ii) prior to step (iii). The method according to claim 46 or 47, wherein the precipitating of (iii) comprises contacting the culture medium of (ii) with protamine sulfate or benzonase. The method according to any one of claims 36 or 39 to 48, further comprising dialyzing the inactivated SARS-CoV-2 particles, thereby producing a dialyzed SARS-CoV-2. The method according to claim 49, further comprising filtering the dialyzed SARS-CoV-2. The method according to any one of claims 36 or 39 to 50, wherein the inactivation step comprises contacting a liquid composition comprising native SARS-CoV-2 particles with a chemical viral inactivating agent in a container, mixing the chemical viral inactivating agent and the liquid composition comprising SARS-CoV-2 particles under conditions of laminar flow but not turbulent flow, and incubating the chemical viral inactivating agent and the liquid composition comprising SARS-CoV-2 particles for a time sufficient to inactivate the viral particles. The method according to claim 51, wherein the inactivation step is performed in a flexible bioreactor bag. The method according to claim 51 or 52, wherein the inactivation step comprises five or less container inversions during the period of inactivation. The method according to any one of claims 51 to 53, wherein the mixing of the chemical viral inactivating agent and the composition comprising native SARS-CoV-2 particles comprises subjecting the container to rocking, rotation, orbital shaking, or oscillation for not more than 10 minutes at not more than 10 rpm during the period of incubation. The method according to any one of claims 36 or 39 to 54, further comprising purifying the inactivated SARS-CoV-2 particles by one or more methods selected from (i) batch chromatography and/or (ii) sucrose density gradient centrifugation. The method according to any one of claims 36 or 39 to 55, wherein step (c) comprises combining the inactivated SARS-CoV-2 particles with an adjuvant. The method according to claim 56, wherein the adjuvant comprises a Thl response-directing adjuvant. The method according to claim 56 or 57, wherein the adjuvant comprises 3-O-desacyl-4'- monophosphoryl lipid A (MPL), saponin QS-21, a CpG-containing oligodeoxynucleotide (CpG ODN), squalene, DL-a-tocopherol and/or imiquimod. 59. A SARS-CoV-2 vaccine obtained or obtainable by the method of any one of claims 36 or 39 to 58.

60. Use of a SARS-CoV-2 vaccine of any one of claims 1 to 28 or 59 for the treatment or prevention of a SARS-CoV-2 infection in a subject.

61. A pharmaceutical composition for use in the prevention or treatment of a SARS-CoV-2 infection in a subject, wherein said pharmaceutical composition is the inactivated SARS-CoV-2 vaccine as defined in any one of claims 1 to 28 or 59, optionally in combination with one or more pharmaceutically acceptable excipients and/or adjuvants.

62. The SARS-CoV-2 vaccine as defined in any one of claims 1 to 28 or 59 for use as a medicament.

63. A vaccine, method, use or pharmaceutical composition according to any preceding claim, wherein the subject is (i) an elderly subject, preferably a subject over 65, over 70 or over 80 years of age; (ii) an immunocompromised subject; or (iii) a pregnant subject.

64. A vaccine, method, use or pharmaceutical composition according to any preceding claim, for use in prevention or treatment of a SARS-CoV-2 infection without induction of (i) antibodydependent enhancement (ADE) of SARS-CoV-2 -associated disease (COVID- 19); and/or (ii) Vaccine-associated respiratory disease (VAERD); and/or (iii) enhanced respiratory disease (ERD); and/or (iv) immunopathology in the subject.

65. A SARS-CoV-2 vaccine for use as a booster vaccination, wherein the vaccine comprises a betapropiolactone inactivated SARS-CoV-2 particle, wherein said SARS-CoV-2 particle comprises an RNA sequence corresponding to a DNA sequence (i) as defined by SEQ ID NO: 9; or (ii) having at least 80%, at least 85%, at least 90%, at least 95% or at least 99% sequence identity to SEQ ID NO: 9; preferably wherein a native (non-inactivated) SARS-CoV-2 particle comprising the RNA sequence is able to pack a virulent SARS-CoV-2 virus.

66. The SARS-CoV-2 vaccine for use as a booster vaccination according to claim 65, wherein said SARS-CoV-2 virus comprises a Spike (S) protein comprising or consisting of (i) an amino acid sequence as defined by SEQ ID NO: 5, or (ii) an amino acid sequence having at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to SEQ ID NO: 5; preferably wherein a native (non-inactivated) SARS-CoV-2 particle comprising the Spike protein is able to pack a virulent SARS-CoV-2 virus.

67. The SARS-CoV-2 vaccine for use as a booster vaccination according to claim 65 or 66, comprising one or more adjuvants.

68. The SARS-CoV-2 vaccine for use as a booster vaccination according to claim 67, wherein the adjuvant comprises a Thl response-directing adjuvant.

69. The SARS-CoV-2 vaccine for use as a booster vaccination according to claim 68, wherein the Thl response-directing adjuvant comprises 3-O-desacyl-4'-monophosphoryl lipid A (MPL), saponin QS-21, a CpG-containing oligodeoxynucleotide (CpG ODN), squalene, DL-a- tocopherol and/or imiquimod.

70. The SARS-CoV-2 vaccine for use as a booster vaccination according to claim 67, wherein the adjuvant is an aluminium salt, i.e., aluminium phosphate or aluminium hydroxide.

71. The SARS-CoV-2 vaccine for use as a booster vaccination according to claim 67, wherein the adjuvants comprise a combination of aluminium hydroxide and a Thl -directing adjuvant.

72. The SARS-CoV-2 vaccine for use as a booster vaccination according to claims 70 or 71, wherein the aluminium hydroxide adjuvant comprises less than 1.25 ppb Cu in the final vaccine formulation.

73. The SARS-CoV-2 vaccine for use as a booster vaccination according to any one of claims 65 to 72, further comprising one or more pharmaceutically acceptable excipients.

74. The SARS-CoV-2 vaccine for use as a booster vaccination according to claim 73, wherein the pharmaceutically acceptable excipients comprise recombinant human serum albumin (rHSA) and/or phosphate buffered saline (PBS).

75. The SARS-CoV-2 vaccine for use as a booster vaccination according to any one of claims 65 to 74, wherein a “standard” dose is defined as 33 AU/0.5 mb. The SARS-CoV-2 vaccine for use as a booster vaccination according to any one of claims 65 to 74, wherein a “double” dose is defined as 66 AU/1.0 mb. A method of preventing or treating SARS-CoV-2 infection and/or SARS-CoV-2-associated disease (COVID-19) in a human subject in need thereof, comprising administering as a booster vaccination a prophylactically or therapeutically effective amount of a SARS-CoV-2 vaccine comprising a beta-propiolactone inactivated SARS-CoV-2 particle, wherein said SARS-CoV-2 particle comprises an RNA sequence corresponding to a DNA sequence (i) as defined by SEQ ID NO: 9; or (ii) having at least 80%, at least 85%, at least 90%, at least 95% or at least 99% sequence identity to SEQ ID NO: 9; preferably wherein a native (non-inactivated) SARS-CoV-2 particle comprising the RNA sequence is able to pack a virulent SARS-CoV-2 virus. The method according to claim 77, wherein said prophylactically or therapeutically effective amount of the SARS-CoV-2 vaccine per dose is defined as about 1 to 100 AU/dose, preferably between about 2 to 75 AU/dose, preferably between about 3 and 60 AU/dose, more preferably between about 3 and 55 AU/dose, more preferably between about 3 and 53 AU/dose, as assessed by ELISA, even more preferably between about 3 and 70 AU/dose, more preferably about 10 to 60 AU/dose, 20 to 50 AU/dose, 25 to 45 AU/dose or 30 to 40 AU/dose such as e.g. 33 AU/ dose, 35 AU/dose, 40 AU/dose or 66 AU/dose. The method according to claim 77 or 78, wherein said prophylactically or therapeutically effective amount of the SARS-CoV-2 vaccine per dose is defined as about 0.05 to 50 pg total protein, about 0. 1 to 25 pg, about 0.25 to 12.5 pg, preferably about 0.5 to 5 pg total protein, more preferably at least 2.5 pg total protein, at least 3.5 pg total protein or at least 2.5 pg total protein, even more preferably 2.5 pg to 25 pg, 3.5 pg to 10 pg or 4 pg to 6 pg total protein/dose, most preferably about 5 pg total protein/dose, e.g. as measured by (p)BCA. The method according to any one of claims 77 to 79, wherein said prophylactically or therapeutically effective amount of the SARS-CoV-2 vaccine is defined as about 0.025 to 25 pg S-protein, about 0.05 to 12.5 pg, about 0.125 to 6.25 pg, preferably about 0.25 to 2.5 pg S- protein, as measured by ELISA. The method according to any one of claims 77 to 80, wherein the prophylactically or therapeutically effective amount of the SARS-CoV-2 vaccine is administered as a booster following natural COVID- 19 infection in a previously unvaccinated subject. 82. The method according to any one of claims 77 to 80, wherein the prophylactically or therapeutically effective amount of the SARS-CoV-2 vaccine is administered as a booster following vaccination with a homologous or heterologous SARS-CoV-2 vaccine.

83. The method according to any one of claims 77 to 80, wherein the prophylactically or therapeutically effective amount of the SARS-CoV-2 vaccine is administered following both vaccination with a homologous or heterologous SARS-CoV-2 vaccine and natural COVID-19 infection.

84. The method according to any one of claims 77 to 83, wherein the administering of the prophylactically or therapeutically effective amount of the SARS-CoV-2 vaccine results in production of SARS-CoV-2 neutralizing antibodies.

85. The method according to any one of claims 77 to 84, wherein the booster vaccination is administered at least 2 weeks, at least 4 weeks, at least 8 weeks, at least 12 weeks, at least 16 weeks, preferably at least 6 months following the last vaccination with a homologous or heterologous SARS-CoV-2 vaccine or natural COVID-19 infection.

86. The method according to any one of claims 82 to 85, wherein the heterologous SARS-CoV-2 vaccine is an mRNA SARS-CoV-2 vaccine or an adenovirus vector SARS-CoV-2 vaccine.

87. The method according to any one of claims 77 to 86, wherein subject is an adult, i.e., 18 years or older.

88. The method according to any one of claims 77 to 86, wherein subject is an older adult, i.e., 50 years or older, 55 years or older, 60 years or older, 65 years or older, 70 years or older, especially 80 years or older.

89. The method according to claim 87, wherein a standard dose (33 AU/0.5 mL) is administered to the adult subject.

90. The method according to claim 88, wherein a double dose (66 AU/1.0 mL) is administered to the older adult subject.