CN117999089A

CN117999089A - SARS-CoV-2 subunit vaccine

Info

Publication number: CN117999089A
Application number: CN202280048191.1A
Authority: CN
Inventors: 安东尼奥·巴雷罗·巴斯克斯; 安东尼奥·普雷那菲塔·阿马戈斯; 劳拉·费雷尔·索勒; 路易斯·冈萨雷斯·冈萨雷斯; 艾斯特·普伊格维特·莫拉斯; 乔迪·帕尔马达·科洛默; 玛利亚·特蕾莎·普拉特·卡巴尼亚斯; 卡门·加里加·阿尔西纳
Original assignee: Haibolai Laboratory Co ltd; Laboratorios Hipra SA
Current assignee: Haibolai Laboratory Co ltd; Laboratorios Hipra SA
Priority date: 2021-05-06
Filing date: 2022-04-25
Publication date: 2024-05-07
Also published as: CN118284431A

Abstract

The present invention relates to a protein subunit vaccine comprising at least one antigen, characterized in that it comprises at least one monomer from at least one variant of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), wherein the at least one monomer is selected from the group consisting of the S1 subunit of spike protein or the Receptor Binding Domain (RBD) of spike protein. In one aspect of the invention, the protein subunit vaccine comprises at least one antigen, characterized in that it comprises two monomers from at least one variant of SARS-CoV-2, wherein each monomer is selected from the group consisting of an S1 subunit or an RBD protein, and wherein the monomers are chemically bound to each other, optionally through a linker, thereby forming a fusion dimer or a non-fusion dimer. The protein subunit vaccine may further comprise at least an adjuvant and at least an immunostimulant.

Description

SARS-CoV-2 subunit vaccine

Technical Field

The present invention relates to a protein subunit vaccine comprising at least one antigen from at least one variant of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), and optionally at least one adjuvant and at least one immunostimulant. The invention also relates to the use of said vaccine for generating an immunogenic and/or protective immune response against at least one variant of SARS-CoV-2, and to a kit comprising one or more doses of said vaccine.

Background

SARS-CoV-2 is an enveloped virus carrying a single-stranded positive-sense RNA genome (about 30 kb), belonging to the genus Corona of the family Coronaviridae. The viral RNA encodes 4 structural proteins (including spike protein (S), envelope protein (E), membrane protein (M) and nucleocapsid protein (N)), 16 non-structural proteins and 9 accessory proteins. S glycoproteins consist of an extracellular domain (processable into S1 and S2 subunits), a transmembrane domain, and an intracellular domain. Like SARS-CoV, SARS-CoV-2 binds to human angiotensin converting enzyme 2 (ACE 2) through the Receptor Binding Domain (RBD) within the S1 subunit to facilitate entry into host cells, and then mediates membrane fusion through the S2 subunit.

Developing a safe and effective COVID-19 vaccine is not easy, but the manufacture, distribution and management of the vaccine can also face significant challenges, especially in developing countries and if it is necessary to inject the vaccine, because of the need for a cold chain to maintain its stability and activity. Various vaccine strategies against COVID-19 are also under intense investigation, with spike proteins being the primary target. These vaccines are produced by different platforms: RNA, DNA, recombinant proteins, viral carrier-based, virus-like particles (VLPs), live attenuated viruses, and inactivated viruses. These different types of vaccine candidates face various challenges associated with the development, manufacture, storage and distribution of large-scale vaccination.

Subunit vaccines or recombinant protein vaccines use whole proteins (e.g., spike proteins) or protein fragments (e.g., S1, RBD, or fusion proteins) as antigens. Subunit vaccines have several advantages over other types of vaccines, for example they are inexpensive and easy to produce, and are more stable than other types of vaccines (e.g. mRNA-based or vaccines containing whole viruses or bacteria).

However, a major disadvantage of subunit vaccines is that the antigens used to elicit an immune response may lack a molecular structure known as a pathogen-associated molecular pattern common to a class of pathogens. These structures can be read and recognized by immune cells as dangerous signals, so their absence may result in a weaker immune response. Furthermore, subunit vaccines are primarily dedicated to triggering antibody-mediated immune responses, as these types of antigens do not infect cells. Again, this means that the immune response may be weaker than other types of vaccines. To overcome this problem, subunit vaccines are sometimes delivered with adjuvants. Thus, subunit vaccines often require adjuvants in the formulation to increase immunogenicity.

Several adjuvants and immunostimulants have been developed or studied, such AS aluminium salts, oil-in-water emulsions (MF 59, AS03 and AF 03), virosomes and AS04 AS adjuvants, and QS-21 or other saponins, monophosphoryl lipid a (MPLA), cpG (ODN) AS immunostimulants. However, it is critical, but not simple, that the selection of an appropriate adjuvant helps to promote an appropriate immune response against the pathogen of interest on a prior daily and adaptive level, thereby eliciting protective immunity while maintaining safety. The wrong choice of adjuvant may result in the failure of a particular vaccine antigen. Thus, the choice of vaccine antigen must take into account the choice of adjuvant to avoid discarding potentially effective candidate vaccine antigens. Development of a safe vaccine while achieving proper efficacy remains a paramount need.

Thus, there is a need for new safe and effective vaccines against SARS-CoV-2, particularly vaccines that enhance immunogenicity.

Drawings

Fig. 1: quantification of SARS-CoV-2 neutralizing antibody titers for each treatment group by a pseudovirus neutralization assay (PBNA). The abscissa represents each group (a to I), and the ordinate represents log10 of dilutions of each group corresponding to IC ₅₀.

Fig. 2A-H: cytokine concentration in spleen cell cultures stimulated with the corresponding vaccine antigen (RBD or S1) for each group. In FIG. 2A, the abscissa is from groups A to E and the ordinate is IFN-gamma concentration (pg/ml) from groups A to E. In FIG. 2B, the abscissa represents groups A to E and the ordinate represents IL-4 concentration (pg/ml) from group A to group E. In FIG. 2C, the abscissa represents groups A to E, and the ordinate represents IL-6 concentration (pg/ml) from group A to group E. In FIG. 2D, the abscissa represents groups A to E and the ordinate represents IL-10 concentration (pg/ml) from group A to group E. In FIG. 2E, the abscissa is for group A, and groups F to I, and the ordinate is for IFN-gamma concentrations (pg/ml) for groups A, and groups F to I. In FIG. 2F, the abscissa represents group A and groups F to I, and the ordinate represents the IL-4 concentration (pg/ml) of groups A and F to I. In FIG. 2G, the abscissa indicates the IL-6 concentrations (pg/ml) of group A, and of group F to group I, and the ordinate indicates the IL-6 concentrations of group A, and of group F to group I. In FIG. 2H, the abscissa indicates the IL-10 concentration (pg/ml) of group A, and groups F to I, and the ordinate indicates the IL-10 concentration of group A, and groups F to I.

Fig. 3A-B: comparison of total anti-SARS-CoV-2 RBD IgG antibody titers (log 10 EC ₅₀) in convalescent human serum samples and negative human serum samples quantified by ELISA. Error bars represent geometric mean and geometric standard deviation (geometric SD). Figure 3A shows IgG antibody titers against RBD produced in HEK293 cells. FIG. 3B shows IgG antibody titers against RBD produced in CHO cells.

Fig. 4: group comparison of anti-SARS-CoV-2 RBD (produced in CHO cells) and anti-SARS-CoV-2 RBD (produced in HEK293 cells) total IgG antibody titers (log 10 EC ₅₀) in convalescent human serum samples quantified by ELISA. Error bars represent geometric mean and geometric standard deviation (geometric SD).

Fig. 5: correlation between anti-SARS-CoV-2 RBD total IgG antibody titer (log 10 EC ₅₀) indicated on the ordinate and the number of days elapsed between the first positive PCR and serum donation indicated on the abscissa. Black square: igG antibody titer against RBD produced in CHO cells. Grey triangles: igG antibody titer against RBD generated in HEK293 cells.

Fig. 6: paired comparison of anti-SARS-CoV-2 RBD (produced in CHO cells) and anti-SARS-CoV-2 RBD (produced in HEK293 cells) total IgG antibody titers (log 10 EC ₅₀) (ordinate) in each convalescent human serum sample (abscissa) quantified by ELISA. Each dot represents a single serum sample. Gray point: igG antibody titer against RBD produced in CHO cells. Black dot: igG antibody titer against RBD generated in HEK293 cells.

Fig. 7: the abscissa represents each treatment group (A to E), and the ordinate represents the anti-SARS-CoV-2 RBD IgG antibody titer (Log 10 EC ₅₀) of each treatment group (A to E), wherein the anti-SARS-CoV-2 RBD IgG antibody titer (Log 10 EC ₅₀) on day 18 of the study (A) and the anti-SARS-CoV-2 RBD IgG antibody titer (Log 10 EC ₅₀) on day 30 of the study (B).

Fig. 8: the abscissa represents the treatment groups (a to I) and the ordinate depicts the anti-SARS-CoV-2 RBD IgG antibody titer (Log 10 EC ₅₀) after administration of one dose of the different vaccine formulations to each treatment group (a to I).

Fig. 9: the abscissa represents each treatment group (a to I) and the ordinate depicts anti-SARS-CoV-2 RBD IgG antibody titers (Log 10 EC ₅₀) after administration of the second dose of the different vaccine formulations for each treatment group (a to I).

Fig. 10: the abscissa represents each treatment group (A to D), and the ordinate depicts the anti-SARS-CoV-2 RBD IgG antibody titer (Log 10 endpoint titer) of each treatment group (A to D). (A) anti-SARS-CoV-2 RBD IgG antibody titer at study day 21 (Log 10 endpoint titer), (B) anti-SARS-CoV-2 RBD IgG antibody titer between study day 35 and day 37 (Log 10 endpoint titer).

Fig. 11: neutralizing antibody response against SARS-CoV-2 Wuhan-Hu-1 variant measured by PBNA. The abscissa represents each treatment group (a to D), and the ordinate depicts the neutralizing antibody titer (Log 10 IC ₅₀) between day 35 and day 37 of the study for each treatment group (a to D).

Fig. 12: neutralizing antibody responses against various SARS-CoV-2 variants as measured by PBNA. The abscissa plots the different SARS-CoV-2 pseudovirus variants and the ordinate plots the neutralizing antibody titres (Log 10 IC ₅₀) for the different SARS-CoV-2 pseudovirus variants. The SARS-CoV-2 variants evaluated were Wu-1 (Wuhan-Hu-1 original sequence), alpha variants (UK; B.1.1.7), beta variants (south Africa; B.1.351), gamma variants (Brazil; P.1) and delta variants (India; B.1.617.2). (a) neutralizing antibody titer obtained from animals of group D, (B) neutralizing antibody titer obtained from animals of group E, and (C) neutralizing antibody titer obtained from animals of group F. The broken line LD represents the limit of detection of the assay. The neutralizing antibody titres of the indian variants (delta; b.1.617.2) were determined only for group D (fig. 12A).

Fig. 13: the abscissa represents the respective groups (a to C), and the ordinate depicts the average rectal temperature (°c) of the respective groups (a to C) on different days of the study. (A) Average rectal temperatures on the day before administration of the first dose (day-1), on administration of the first dose (day 0), and 4 hours, 6 hours, 1 day, 2 days, and 3 days after the first inoculation (day 0+4 hours, day 0+6 hours, day 1, day 2, and day 3). (B) Mean rectal temperatures on day before (day 20), at (day 21), and 4 hours, 6 hours, 1 day, 2 days, and 3 days (day 21+4 hours, 21+6 hours, 22 days, and 23 days) after administration of the second dose.

Fig. 14: the abscissa represents each treatment group (A to C), and the ordinate depicts the anti-SARS-CoV-2 neutralizing antibody titer (Log 10 IC ₅₀) of each treatment group (A to C). Neutralizing antibody titers for the following different variants are depicted: alpha variants (uk; b.1.1.7), beta variants (south africa; b.1.351), gamma variants (brazil; p.1) and delta variants (india; b.1.617.2).

Fig. 15: the daily survival after experimental infection of the different treatment groups (a to C) is depicted. The abscissa indicates the number of days elapsed after experimental infection, and the ordinate indicates the survival rate (%). Group A animals received a vaccine composition comprising 20 μg of recombinant fusion dimeric RBD variant SARS-CoV-2 antigen; group B animals received a vaccine composition comprising 10 μg of recombinant fusion dimeric RBD variant SARS-CoV-2 antigen; animals of group C received a mock vaccine comprising PBS.

Disclosure of Invention

General definition

It must be noted that, as used herein, the singular forms "a", "an" and "the" include plural referents unless the context clearly dictates otherwise. Furthermore, unless otherwise indicated, the term "at least" preceding a series of elements should be understood to refer to each element in the series. Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the present invention.

When referring to a given amount or quantity, the term "about" means that the number may vary between ±20% around its specified value. Preferably, "about" means about ±15% around its value, more preferably, "about" means about ±10%, 8%, 6%, 5%, 4%, 3%, 2%, even "about" means about ±1% around its value, in this order of preference.

As used herein, the connection term "and/or" between multiple referenced elements is understood to encompass both individual and combined options. For example, when two elements are connected by an "and/or," a first option refers to the first element being applicable without the second element. The second option refers to the second element being applicable without the first element. The third option means that the first element and the second element are applicable together. Any of these options is understood to fall within this meaning and thus meet the requirements of the term "and/or" as used herein. Simultaneous adaptation of more than one option is also understood to fall within this meaning and thus fulfill the requirements of the term "and/or".

Throughout the specification and claims, unless the context requires otherwise, the word "comprise", and variations such as "comprises" or "comprising", will be understood to imply the inclusion of a stated integer or step or group of integers or steps but not the exclusion of any other integer or step or group of integers or steps. The term "comprising" as used herein may be replaced by the term "containing" or "including" or sometimes by the term "having" as used herein. Any of the foregoing terms (including, containing, comprising, having) may be substituted with the term "consisting of whenever used in the context of aspects or embodiments of the present invention, although less preferred.

When used herein, "consisting of" and "consisting of" are excluded any element, step, or ingredient not specified in the claim elements. As used herein, "consisting essentially of" does not exclude materials or steps that do not materially affect the basic and novel characteristics of the claims.

The term "subtype" in this context may be replaced by "species". It includes strains, isolates, clades, lineages (lineage), lines (linage) and/or variants of any severe acute respiratory syndrome coronavirus (i.e., SARS-CoV-2). The terms "strain", "clade", "lineage or line", "isolate" and/or "variant" are technical terms well known to the person skilled in the art and relate to the taxonomy of microorganisms, i.e. to all microorganisms characterized by the order of the family, genus, species, strain. The criteria for members of the family are their phylogenetic relationships, which include all members that share common characteristics, while species are defined as a multi-principle taxonomy that constitutes a replication lineage and occupies a particular niche. The term "strain" or "clade" describes a microorganism (i.e., virus in the present invention) that has common characteristics (e.g., basic morphology or genomic structure and organization) with other microorganisms but differs in biological properties (e.g., host range, tissue tropism, geographical distribution, attenuation, or pathogenicity). In the present invention, the term "variant" describes a microorganism (i.e. virus in the present invention) that replicates and introduces one or more new mutations into its genome, resulting in a difference from the original virus. The term "pedigree" or "lineage" describes a set of viral sequences derived from a common ancestor that are associated with an epidemiological event, e.g., the introduction of a virus into different geographical areas with evidence of further spread. The pedigree is intended to capture the imminent pandemic and has fine-grained resolution suitable for genomic epidemiological monitoring and outbreak investigation. SARS-CoV-2 lineage nomenclature is described, for example, in ,Rambaut A.et al.A dynamic nomenclature proposal for SARS-CoV-2lineages to assist genomic epidemiology.Nat Microbiol.2020;5(11):1403-1407. Thus, "at least one variant of Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)" refers to at least one variant, strain, isolate, lineage or line, and/or clade of SARS-CoV-2 virus. Preferably, the term "variant" is consistent with the previous definition, and also with the WHO website, particularly different SARS-CoV-2 virus sequences with one or more mutations derived from the same ancestral or etiologic virus (i.e., SARS-CoV-2 virus in this case). Thus, in this particular context, the term "SARS-CoV-2 variant" or "SARS-CoV-2 lineage or pedigree" is preferably understood to exclude viral genomes from other viruses (e.g. SARS or MERS viruses) nor viral genomes derived from said other viruses.

More preferably, the term "variant" or "line" includes all SARS-CoV-2 viral sequences encoding a spike protein having at least 90%, 91%, 92%, 93%, 94%, preferably at least 95%, 96%, 97%, 98% or 99% percent amino acid sequence identity to the spike protein of reference strain SARS-CoV-2 Wuhan-Hu-1 (GenBank accession number QHD43416.1 or UniprotID: P0DTC 2) (when the two spike proteins are aligned locally, for example by using a Basic Local Alignment Search Tool (BLAST)).

Still more preferably, the term "variant" or "line" includes all SARS-CoV-2 viral sequences encoding RBDs of spike protein having at least 85%, 86%, 87%, 88% or 89%, preferably at least 90%, 91%, 92%, 93%, 94%, most preferably at least 95%, 96%, 97%, 98% or 99% percent amino acid sequence identity to the RBD of spike protein of reference strain SARS-CoV-2 Wuhan-Hu-1 (when both RBD proteins are locally aligned, such as by local alignment using Basic Local Alignment Search Tools (BLAST)), for example, by using GenBank accession numbers QHD43416.1 or UniprotID: P0DTC2, amino acid residues 319 to 541.

Different variants of SARS-CoV-2 can be found in databases, e.g., of the Emma B.Hodcroft.2021."CoVariants:SARS-CoV-2Mutations and Variations of Interest"(covariants.org/variants) or O'Toole A.et al.,2020"A dynamic nomenclature proposal for SARS-CoV-2lineages to assist genomic epidemiology",PANGO lineages (CoV-lineages. Org /).

In the context of two or more nucleotide sequences, polypeptide sequences, or protein sequences, the term "sequence identity" or "percent identity" refers to a particular percent of the same nucleotide or amino acid residues ("percent identity") when compared and aligned to the greatest correspondence of a second molecule using a sequence comparison algorithm (e.g., by BLAST alignment or any other algorithm known to those of skill in the art) or by visual inspection. "sequence identity" or "percent identity" can be determined by counting the number of identical nucleotides or amino acids at the same position in a nucleic acid, polypeptide or protein. Calculation of the percent identity includes determining an optimal alignment between two or more sequences. Alignment may take into account insertions and deletions (i.e. "gaps") in each sequence to be tested, such as, but not limited to, non-coding regions of nucleic acids and truncations or extensions of polypeptide sequences. Computer programs and algorithms such as Basic Local Alignment Search Tools (BLAST) can be used to determine percent identity. BLAST is one of the many sources offered by the national center for biotechnology information. Because the genetic code is degenerate and more than one codon can encode a given amino acid, the coding regions of nucleic acids are considered identical if the nucleic acids encode the same polypeptide. Thus, percent identity may also be calculated based on the polypeptide encoded by the nucleic acid. The percent identity may be calculated based on the full-length consensus genomic sequence or a portion of the genomic sequence, such as, but not limited to, an Open Reading Frame (ORF) alone.

The protein or peptide of the invention is substantially identical to another protein or peptide if, optimally aligned, it has an amino acid sequence identity of at least about 60% identity, typically at least about 70% identity, more typically at least about 80% identity, preferably at least about 90% identity, more preferably at least about 95% identity, and most preferably at least about 98% or 100% identity to the synthetic or naturally occurring protein or peptide derived therefrom. Identity refers to the degree of sequence relatedness between two polypeptide or two polynucleotide sequences, as determined by the identity of a match between two strings of such sequences, e.g., all and complete sequences. Identity can be easily calculated. Although there are a variety of methods for measuring identity between polypeptide sequences, the term "identity" is well known to those skilled in the art.

"Percent (%) amino acid sequence identity" with respect to proteins, polypeptides, antigen protein fragments, antigens, and epitopes described herein is defined as the percentage of amino acid residues in a candidate sequence that are identical to amino acid residues in a reference sequence (i.e., the protein, polypeptide, antigen protein fragment, antigen, or epitope from which it is derived) after alignment of the sequences and, if necessary, introduction of gaps (to achieve the maximum percent sequence identity), and without regard to any conservative substitutions as part of sequence identity. Alignment for the purpose of determining the percent amino acid sequence identity can be accomplished in a variety of ways within the skill of the art, for example, using publicly available computer software such as BLAST, ALIGN, or Megalign (DNASTAR) software. One skilled in the art can determine appropriate parameters for measuring the alignment, including any algorithms needed to achieve maximum alignment over the full length of the sequences compared.

The term "subject" or "host" as used herein is a living multicellular vertebrate organism, including for example humans and non-human mammals, including (non-human) primates, companion animals such as dogs and cats, and domestic animals such as horses, bovine species such as cattle and sheep, ferrets, porcine species such as pigs, piglets, sows or backup sows, and zoo mammals such as cats, dogs and cows. Thus, the term "subject" or "host" may be used interchangeably herein with the term "animal" or "human". Typically, the "object" is a person. For example, the person may be a newborn (less than 2 months), an infant (birth to 2 years), a child (2 years to 14 years), a teenager (15 years to 18 years), an adult (over 18 years), or an elderly person (about 65 years or over).

An "immunological response" or "immune response" to an antigen or composition is an innate, humoral, and/or cellular immune response of a subject to an antigen present in the composition of interest. When the term "boost" is used in reference to an immune response against a SARS-CoV-2 antigen, such as an antibody response (e.g., a neutralizing antigen specific antibody response), a cytokine response, a CD 8T cell response (e.g., an immunodominant CD 8T cell response), or a CD 4T cell response, refers to an increase in the immune response in a subject administered with a vaccine comprising at least one SARS-CoV-2 antigen relative to the corresponding immune response observed from administration of a vaccine that does not comprise any SARS-CoV-2 antigen.

In the context of the present invention, the term "monomer" is preferably used to refer to, but is not limited to, the Receptor Binding Domain (RBD) or S1 subunit of spike protein from any variant of SARS-CoV-2 virus. In particular, the term "monomer" as used herein refers to any protein comprising, consisting of, or consisting essentially of: SEQ ID NO: 1.2, 3 or 4, or over its entire length with the sequence SEQ ID NO: 1.2, 3 or 4, has a sequence of at least 50%, 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity. The monomer has the ability to form a chemical bond with at least one other monomer molecule to form a multimer, i.e., dimer, trimer, tetramer, pentamer, etc. Dimers are polymers formed from two monomers, which may be identical or different in sequence.

An "antigen" or "immunogen" refers to a substance that induces a specific immune response in a host animal. The antigen or immunogen may comprise: a whole organism, which is inactivated, attenuated or live; subunits or partial fragments of an organism; a recombinant carrier comprising an insert having immunogenic properties; a DNA fragment capable of inducing an immune response upon presentation to a host animal; a polypeptide, a protein or fragment thereof, an epitope, or any combination thereof. In the context of the present invention, an "antigen" refers to a protein comprising or consisting of at least one monomer. In the context of the present invention, an "antigen" refers to a protein comprising or consisting of at least one multimer. The multimer or antigen may comprise two monomers (dimer antigen or dimer antigen), three monomers (trimer antigen or trimer antigen), four monomers (tetramer antigen or tetramer antigen), or more monomers. The term "multimeric antigen" or "multimeric antigen" is synonymous. In the specific case where the antigen consists of two monomers (understood as two RBDs, two S1, or one each), then the antigen is understood as a dimer. It should be noted that "dimeric antigen" and "dimeric form of antigen" are synonymous and are used interchangeably herein. In the context of the present invention, the two monomers of the dimeric antigen are optionally chemically linked or bound to each other by a linker. "coupled" means that the monomers of the dimer are chemically linked by very weak, strong or very strong bonds, such as covalent, non-covalent, disulfide, or peptide bonds.

In the present invention, two dimeric forms of antigen are described: "non-fused dimers" and "fused dimers". By "non-fusion dimer" is herein understood an antigen formed from two monomers, wherein the two monomers are bound to each other by a reversible bond, e.g. by an intermolecular disulfide bond formed between their cysteines, thereby forming a non-fusion dimeric antigen. For example, reference herein to a "non-fusion dimer" is to two soluble RBD monomers that are produced intracellularly after transfection with a nucleic acid encoding the RBD monomers, and which interact with each other to form disulfide bonds, e.g., through their free (unbound) cysteines, when the RBD monomers are released into the cell supernatant, thereby forming what is referred to herein as a "non-fusion dimer". Importantly, the two monomers in the non-fusion dimer are not linked by peptide bonds, nor are they part of a single polypeptide.

"Fusion dimer" in this context refers to an antigen formed from two monomers, where the two monomers are linked one to the other such that they are synthesized or translated into a single unit, and thus the two monomers of the fusion dimer are part of a single polypeptide. Thus, in contrast to the monomers of the non-fused dimer, the two monomers contained in the fused dimer are linked by peptide bonds, optionally by linkers.

Furthermore, in the present invention, when referring to "dimeric antigen" or "dimeric form of antigen" it is understood to include the above-described non-fusion dimeric antigen and fusion dimeric antigen. "monomeric RBD antigen" or "RBD-monomer" refers herein to an antigen comprising or consisting of one monomer, wherein the monomer is RBD. "dimeric RBD antigen" or "RBD-dimer" refers herein to an antigen comprising or consisting of two monomers that bind to each other, wherein the monomers are RBDs. If the "dimeric RBD antigen" is a non-fusion dimer, it is referred to herein as a "non-fusion dimeric RBD antigen" or "non-fusion RBD-dimer". If the "dimeric RBD antigen" is a fusion dimer, it is referred to as a "fusion dimeric RBD antigen" or "fusion RBD-dimer". Unless specified as a "dimeric RBD antigen" is a "non-fusion dimeric RBD antigen" or a "fusion dimeric RBD antigen," it is understood that "dimeric RBD antigen" encompasses both types, i.e., fusion dimers and non-fusion dimers of RBD. "monomeric S1 antigen" or "S1-monomer" refers herein to an antigen comprising or consisting of one monomer, wherein the monomer is S1. "dimeric S1 antigen" or "S1-dimer" refers herein to an antigen comprising or consisting of two monomers that bind to each other, wherein the monomers are S1. If the "dimeric S1 antigen" is a non-fused dimer, it is referred to herein as a "non-fused dimeric S1 antigen" or "non-fused S1-dimer". If the "dimeric S1 antigen" is a fusion dimer, it is referred to as a "fusion dimeric S1 antigen" or "fusion S1-dimer". Unless the "dimeric S1 antigen" is designated as a "non-fusion dimeric S1 antigen" or a "fusion dimeric S1 antigen," it is understood that "dimeric S1 antigen" encompasses both types, i.e., fusion dimers and non-fusion dimers of S1.

The presence of antigens in the body will normally elicit an immune response. Thus, the antigen is "targeted" by the antibody. An "epitope" refers to a specific antigenic determinant of an antigen. An epitope may comprise three amino acids in a spatial conformation that is unique to the epitope. Typically, an epitope consists of at least five such amino acids, more typically at least 8-10 such amino acids. Methods for determining the spatial conformation of such amino acids are known in the art.

Subunit vaccines are vaccines that present one or more antigens to the immune system without introducing whole or other forms of pathogen particles. "protein subunit vaccine" refers herein to a specific isolated antigen from a viral or bacterial pathogen. "protein subunit vaccine" is also referred to herein as a specific recombinant antigen from a viral pathogen.

The "SARS-CoV-2 spike (S) protein" refers to one of the four structural proteins of SARS-CoV-2 virus (spike (S) protein, nucleocapsid (N) protein, envelope (E) protein and membrane (M) protein). The S protein is about 180-200kDa in size and consists of an extracellular N-terminal, a Transmembrane (TM) domain anchored to the viral membrane, and an intracellular C-terminal short fragment. The total length of SARS-CoV-2S protein is about 1273 amino acids, and consists of signal peptide (amino acids 1-13), S1 subunit (13-685 residues) and S2 subunit (686-1273 residues) at N-terminal end; the last two regions are responsible for receptor binding and membrane fusion, respectively. In the S1 subunit, there is an N-terminal domain (residues 14-305) and a receptor binding domain (residues RBD, 319-541); fusion Peptide (FP) (residues 788-806), heptad repeat 1 (HR 1) (residues 912-984), HR2 (residues 1163-1213), TM domain (residues 1213-1237) and cytoplasmic domain (residues 1237-1273) comprise the S2 subunit. Thus, "S1" or "S1 subunit" or "S1 antigen" refers to the S1 subunit located on the spike protein of a coronavirus (CoV), and "RBD" or "RBD antigen" refers to the receptor binding domain located on the spike protein of a coronavirus (CoV).

An "immunogenic fragment" of an antigen according to the invention is a partial amino acid sequence of the antigen or a functional equivalent of such a fragment that also serves as an antigen, which is detected and bound by an antigen-specific antibody or B cell receptor. The immunogenic fragment of the antigen is shorter than the intact antigen and is preferably between about 10, 50 or 100 and about 1000 amino acids in length, more preferably between about 10, 50 or 30 and about 500 amino acids in length, even more preferably between about 50 and about 250 amino acids in length. Fragments of RBD or S1 antigen comprise amino acids having at least 15, 20 or 65 consecutive amino acid residues, which correspond to SEQ ID NOs: 1. 3, 4 or SEQ ID NO:2, at least about 15, 20 or 65 consecutive amino acid residues have at least about 70%, at least about 80%, at least about 90%, preferably at least about 95%, more preferably at least about 98%. Depending on the expression system chosen, the protein fragments may or may not be expressed in a native glycosylated form.

A protein or fragment that "substantially corresponds to" a protein or fragment of SARS-CoV-2 virus is a protein or fragment that has substantially the same amino acid sequence and substantially the same function as the specified protein or fragment of SARS-CoV-2 virus.

Proteins or fragments having a "substantially identical amino acid sequence" to a protein or fragment of the SARS-CoV-2 virus typically have more than 90% amino acid identity to the protein or fragment. Conservative amino acid substitutions are included in this definition.

As used herein, an "antibody" is a polyclonal and/or monoclonal antibody or fragment thereof, including recombinant antibody fragments and immunological binding equivalents thereof, which are capable of specifically binding to SARS-CoV-2 protein and/or fragments thereof. The term "antibody" is used to refer to a homogeneous molecular entity or mixture, such as a serum product composed of a plurality of different molecular entities. Recombinant antibody fragments may, for example, be derived from monoclonal antibodies or may be isolated from libraries constructed from immunized non-human animals.

An "adjuvant" as used herein is a substance used to enhance an immune response. The term adjuvant is derived from latin: adjuve, meaning "help". Many classes of compounds have been described as adjuvants, including mineral salts, microbial products, emulsions, saponins, cytokines, polymers, microparticles and liposomes. There are a number of compounds with adjuvant properties that function by different mechanisms of action. The action of an immunostimulant is to activate an innate response or to directly (i.e., cytokine) activate an innate response through a Pattern Recognition Receptor (PRR).

"EC ₅₀" or "half maximal effective concentration" or "50% effective dilution" refers herein to the concentration of antibody in serum that provides half maximal binding (50% of its maximal effect observed). EC ₅₀ can be determined by direct and saturable binding of the dilution series to the target antigen. EC ₅₀ in the pseudovirus-based neutralization assay is the dilution at which the Relative Light Units (RLU) are reduced by 50% compared to the virus control wells after subtraction of the background RLU in the control group. Methods for determining EC ₅₀ in a pseudovirus-based neutralization assay are known to those skilled in the art, such as those described in Nie J.et al.Establishment and validation of a pseudovirus neutralization assay for SARS-CoV-2.Emerg Microbes Infect.2020Dec;9(1):680-686、Nie J.et al.Quantification of SARS-CoV-2neutralizing antibody by a pseudotyped virus-based assay.Nat Protoc.2020Nov;15(11):3699-3715、 or Hu J.et al.Development of cell-based pseudovirus entry assay to identify potential viral entry inhibitors and neutralizing antibodies against SARS-CoV-2.Genes Dis.2020Dec;7(4):551-557.

"IC ₅₀" or "half maximal inhibitory concentration" or "50% inhibitory dilution" refers herein to the concentration of antibody in serum required to inhibit 50% of infection. IC ₅₀ can be determined by direct and saturable binding of the dilution series to the target antigen. IC50 in the pseudovirus-based neutralization assay is the dilution at which Relative Light Units (RLU) are reduced by 50% compared to virus control wells after subtraction of background RLU in control. Methods for assaying IC ₅₀ in pseudovirus neutralization assays are known to those skilled in the art, for example, the methods described in Nie J.et al.Establishment and validation of a pseudovirus neutralization assay for SARS-CoV-2.Emerg Microbes Infect.2020Dec;9(1):680-686、Nie J.et al.Quantification of SARS-CoV-2neutralizing antibody by a pseudotyped virus-based assay.Nat Protoc.2020Nov;15(11):3699-3715、 or Hu J.et al.Development of cell-based pseudovirus entry assay to identify potential viral entry inhibitors and neutralizing antibodies against SARS-CoV-2.Genes Dis.2020Dec;7(4):551-557.

By "endpoint titer" is meant herein the reciprocal of the highest dilution giving a reading above the cutoff. The cut-off value is preferably two to three times the negative control reading or average background, more preferably three times the negative control reading or average background. Endpoint titer can be determined by direct and saturable binding of the dilution series to the target antigen in an ELISA assay. Methods for determining endpoint titer in an ELISA assay are known to those skilled in the art, such as the method described in Frey A.et al.A statistically defined endpoint titre determination method for immunoassays.J Immunol Methods.1998Dec 1;221(1-2):35-41.

As used herein, a "linker peptide" is a short peptide sequence located between two monomers of a fusion dimer. The linker peptide is placed to provide flexibility of movement to the two monomers contained in the fusion dimer. In the context of the present invention, a linker peptide has at least one amino acid residue, preferably at least two consecutive amino acid residues, optionally 3,4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more amino acid residues. The linker peptides include flexible linkers, rigid linkers, and in vivo cleavable linkers.

Detailed Description

It is not straightforward to design and optimize the antigens contained in a vaccine that promotes an appropriate immune response to the pathogen of interest at both the innate and adaptive levels. Although the most antigenic epitopes or proteins of pathogens may be known, the generation of vaccines, in particular protein subunit vaccines, still requires fine tuning of the antigens to enhance their immunogenicity and avoid their misfolded or hypoimmunogenic forms, which may push the immune response in the wrong direction. The selection of the wrong antigen may lead to inefficiency in the particular vaccine. Thus, antigen selection must be carefully considered to avoid discarding potentially effective candidate vaccines and to aid vaccine development and to provide new solutions to combat pandemics, such as COVID-19.

In the present invention, the inventors have shown in FIGS. 1 and 2 and example 2 herein that RBD and S1 subunits of SARS-CoV-2 virus have the ability to elicit potent neutralizing antibodies and cellular immune responses, indicating that they are good candidates as starting points for the generation of protein subunit vaccines against SARS-CoV-2 virus.

Thus, in a first aspect, the present invention relates to a protein subunit vaccine comprising or consisting of at least one antigen, characterized in that the at least one antigen comprises or consists of at least one monomer from at least one variant of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), wherein the at least one monomer is selected from the group consisting of the S1 subunit of spike protein or the Receptor Binding Domain (RBD) of spike protein, or any immunogenic fragment thereof.

In an embodiment, the at least one monomer comprised in the at least one antigen is the Receptor Binding Domain (RBD) of a spike protein or an immunogenic fragment thereof. Preferably, the at least one monomer comprised in or consisting of the antigen is a recombinant Receptor Binding Domain (RBD) of a spike protein or an immunogenic fragment thereof.

Preferably, the Receptor Binding Domain (RBD) of the spike protein substantially corresponds to amino acid residues 319 to 541 of SARS-CoV-2 spike protein. Preferably, said Receptor Binding Domain (RBD) of a spike protein comprises, consists of, or consists essentially of: SEQ ID NO:1 or a sequence which hybridizes over its entire length to SEQ ID NO:1 has at least 50%, 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity. In some embodiments, the Receptor Binding Domain (RBD) of the spike protein comprises the amino acid sequence of SEQ ID NO: 1. consists of SEQ ID NO:1 or consists essentially of SEQ ID NO: 1.

Preferably, the Receptor Binding Domain (RBD) of the spike protein substantially corresponds to amino acid residues 319 to 537 of SARS-CoV-2 spike protein. Preferably, said Receptor Binding Domain (RBD) of a spike protein comprises, consists of, or consists essentially of: SEQ ID NO:3 or a sequence which hybridizes over its entire length to SEQ ID NO:3 has at least 50%, 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity. In some embodiments, the Receptor Binding Domain (RBD) of the spike protein comprises the amino acid sequence of SEQ ID NO: 3. consists of SEQ ID NO:3 or consists essentially of SEQ ID NO: 3.

Preferably, said Receptor Binding Domain (RBD) of a spike protein comprises, consists of, or consists essentially of: SEQ ID NO:4 or a sequence which hybridizes over its entire length to SEQ ID NO:4 has at least 50%, 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity. In some embodiments, the Receptor Binding Domain (RBD) of the spike protein comprises the amino acid sequence of SEQ ID NO: 4. consists of SEQ ID NO:4 or consists essentially of SEQ ID NO: 4.

In embodiments, at least one monomer comprised in at least one antigen comprises or consists of a Receptor Binding Domain (RBD) and is substantially identical to SEQ ID NO: 1. SEQ ID NO:3 or SEQ ID NO:4, has at least 50%, 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity.

In another embodiment, the at least one monomer comprised in the at least one antigen comprises or consists of the S1 subunit of spike protein or an immunogenic fragment thereof. Preferably, at least one of the monomers is a recombinant S1 subunit of a spike protein or an immunogenic fragment thereof. Preferably, the S1 subunit corresponds to amino acid residues 13 to 685 of SARS-CoV-2 spike protein. More preferably, the S1 subunit corresponds to amino acid residues 16 to 682 of SARS-CoV-2 spike protein. Preferably, said S1 subunit of spike protein comprises, consists of, or consists essentially of: SEQ ID NO:2 or a sequence which hybridizes over its entire length to SEQ ID NO:2 has at least 50%, 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity. In some embodiments, the S1 subunit of spike protein comprises SEQ ID NO: 2. consists of SEQ ID NO:2 or consists essentially of SEQ ID NO: 2.

In an embodiment, the at least one monomer comprised in the at least one antigen according to the first aspect or any embodiment thereof is derived from a SARS-CoV-2 Wuhan-Hu-1 pneumovirus isolate. Wuhan-Hu-1 consists of the spike protein of SEQ ID NO:9 (UniProt No. P0DTC 2). In another embodiment, the Wuhan-Hu-1 variant comprises the mutation D614G in the spike protein.

In another embodiment, at least one monomer from at least one variant of SARS-CoV-2 comprised in at least one antigen according to the first aspect or any embodiment thereof is derived from the american center for disease control and prevention (CDC) "classification and definition of SARS-CoV-2 variants" defined variant of concern (variant of concern, VOC). It was observed that SARS-CoV-2 was mutated, with some combinations of specific site-directed mutations proving to be more alarming than others. These mutations are responsible for the increased transmissibility, increased virulence and the possible occurrence of escape mutations in the new variants. The term "variant of concern" (VOC) refers to a newly emerging variant of SARS-CoV-2, the mutation of which increases transmissibility and/or morbidity and/or mortality and/or decreases sensitivity to antiviral or therapeutic agents and/or has the ability to evade immunity and/or the ability to infect vaccinated individuals, etc. As described above, the term "variant" is preferably understood to mean "lineage" or "line", i.e., different viral sequences derived from the same SARS-CoV-2 common ancestor. Thus, preferably, the different "variant of concern" referred to herein does not include viral sequences derived from other viruses such as SARS or MERS.

In another embodiment, at least one monomer from at least one variant of SARS-CoV-2 comprised in at least one antigen according to the first aspect or any of its embodiments is derived from the british SARS-CoV-2 variant VOC 202012/01 (linear b.1.1.7). The british authorities reported to WHO a variant called SARS-CoV-2voc 202012/01 in the united kingdom (variant of concern, month 2020, variant 01), also known as linear b.1.1.7 or 501y.v1, on day 14 of WHO's name. This variant is described in the scientific literature, see for example Wise, J.Covid-19:New coronavirus variant is identified in UK.BMJ 2020,371,m4857. The variant comprises 23 nucleotide substitutions and is phylogenetically unrelated to the SARS-CoV-2 virus that was prevalent in the United kingdom when the variant was detected. Among the several mutations of this variant, one mutation in the Receptor Binding Domain (RBD) of spike protein alters asparagine at position 501 to tyrosine (N501Y). Another mutation in the VOC 202012/01 variant, a deletion at the 69/70del position, was found to affect the performance of some diagnostic PCR assays against the S gene target. By 12 months 30 days, 31 other countries/regions of 5 out of 6 WHO regions have reported VOC-202012/01 variants.

In another embodiment, at least one monomer from at least one variant of SARS-CoV-2 comprised in at least one antigen according to the first aspect or any embodiment thereof is derived from a south africa SARS-CoV-2 variant (lineage b.1.351). On day 12 and 18, the south Africa national authorities announced that a new variant of SARS-CoV-2 was detected that was rapidly spreading in three provinces in south Africa. This variant was named linear b.1.351, also denoted 501y.v2, in south africa. This variant is described in the scientific literature, see, for example, ,Tegally et al.Emergence and rapid spread of a new severe acute respiratory syndrome-related coronavirus 2(SARS-CoV-2)lineage with multiple spike mutations in South Africa.medRxiv 2020.SARS-CoV-2 south africa variant characterized by the presence of three mutations K417N, E484K and N501Y in RBD. Although SARS-CoV-2VOC 202012/01 from the United kingdom also has the N501Y mutation, phylogenetic analysis indicated that viruses from south Africa are different viral variants.

In another embodiment, at least one monomer from at least one variant of SARS-CoV-2 comprised in at least one antigen according to the first aspect or any of its embodiments is derived from the brazilian SARS-CoV-2 variant VOC-202101/02 (Linage b.1.1.28). Brazil variants are also known as Linear P.1, also known as 20J/501Y.V3, related variants 202101/02 (VOC-202101/02). This variant is described in the scientific literature, see for example Faria,et al.Genomic Characterisation of an Emergent SARS-CoV-2Lineage in Manaus:Preliminary Findings.SARS-CoV-2 for such variants with 17 unique amino acid changes, 10 of which are located in their spike proteins, including three designated as particularly interesting: N501Y, E484K and K417T. This variant of SARS-CoV-2 was first detected by the Japanese National Institute of Infectious Diseases (NIID) in four people arriving at Tokyo on day 1 and 6 of 2021, who had visited Amazon, brazil four days ago. It is then announced to circulate in brazil and spread throughout the world.

Recently, california variant is also known as the cut-off variant cal.20c. Thus, in another embodiment, at least one monomer from at least one variant of SARS-CoV-2 comprised in at least one antigen according to the first aspect or any embodiment thereof is derived from california SARS-CoV-2 (Linage b.1.427 or b.1.429). The variant is characterized by mutations S131, W152C in the N-terminal domain (NTD) of the spike protein and the L452R mutation in the RBD of the spike protein. This variant was originally detected in california (Linage b.1.427 or b.1.429).

Many other variants have been described, for example, the b.1.207 variant of nigeria, which has a mutation in the spike protein (P681H), which is also found in the VOC 202012/01 variant; b.1.617 variants of india (Linage b.1.617, india variants) with mutations P681R, E484Q and L425R in the spike protein; or a danish variant, called "Cluster 5" variant by the Danish authorities, and having a combination of mutations that have not been observed before. The skilled artisan can readily find an ever-increasing number of variants worldwide in the website databases disclosed, for example, in Emma B.Hodcroft.2021."CoVariants:SARS-CoV-2Mutations and Variations of Interest."(covariants.org/variants) or O' Toole a.et al, 2020"PANGO lineages" (cov-lineges.org /). Thus, it is to be understood that the present invention encompasses a protein subunit vaccine comprising at least one antigen, characterized in that the at least one antigen comprises or consists of at least one monomer from at least one variant of SARS-CoV-2, wherein the monomer from at least one variant of SARS-CoV-2 comprised in the at least one antigen is derived from any strain or clade or variant or lineage or isolate of SARS-CoV-2.

Olmicin Rong Bianti (linear b.1.1.529 or GR/484A, unless specified otherwise, is considered to include all BA lineages (ba.1, ba.2, ba.3, ba.4, ba.5 and offspring lineages)) was first reported by south africa to the WHO on month 11, 2021, 24 and was then categorized as VOC. This variant has a large number of spike protein substitutions, including A67V、del69-70、T95I、del142-144、Y145D、del211、L212I、ins214EPE、G339D、S371L、S373P、S375F、K417N、N440K、G446S、S477N、T478K、E484A、Q493R、G496S、Q498R、N501Y、Y505H、T547K、D614G、H655Y、N679K、P681H、N764K、D796Y、N856K、Q954H、N969K、L981F. the amikatone variants mentioned herein also include circulating recombinant variants, such as the ba.1/ba.2 lineage, known as XE. XE combines the genetic material of the amikates ba.1 and ba.2 lineages with three new mutations that are not present in both existing strains.

Delta variants (linear b.1.617.2 or G/478k.v1 and AY lineages) carry the spike protein substitutions T19R, (V70F), T95I, G142D, E-, F157-, R158G, (a 222V), (W258L), (K417N), L452R, T478K, D614G, P681R, D950N. It was first discovered in india and categorized as VOC. The delta variants mentioned herein also include cyclic recombinant variants, such as delta variants having the ba.1 lineage, referred to as XD and XF. Both XD and XF are recombinants of genetic material from the delta and ba.1 lineages.

Importantly, given the evolving SARS-CoV-2 virus and our understanding of the effects of variants, the work definitions and terms used to refer to the different variants may be regularly adjusted. The naming system used in current scientific research for naming and tracking the SARS-CoV-2 genetic lineage is formulated by GISAID, nextstrain and Pango. Thus, since the names of variants may change over time, we provide a table retrieved from the WHO website, where by 2021, 8-6, the following variant nomenclature represents the nomenclature established so far, and thus the nomenclature used in drafting the application:

Currently named variants of interest:

* Significant spike protein (S) amino acid changes under monitoring are currently reported in a few sequencing samples.

From month 4 of 2022, 12, the WHO website also contains the following variant nomenclature:

In view of these tables, and by way of example, the uk variant may also be referred to as variant b.1.1.7 or alpha variant; south african variants may also be referred to as variant b.1.351 or beta variants; brazil variants may also be referred to as variants P.1 or gamma variants; indian variants may also be referred to as variant b.1.617.2 or delta. The different names of each variant are considered synonymous and are used interchangeably herein. The different names and specific point mutations used to designate the different SARS-CoV-2 variants can be easily retrieved and updated by the skilled person, see for example the WHO website: who.int/en/activites/tracking-SARS-CoV-2-variants/.

In a specific embodiment, the at least one monomer from at least one variant of SARS-CoV-2 comprised in the at least one antigen according to the first aspect or any embodiment thereof is derived from a SASR-CoV-2 variant selected from the group including, but not limited to: wuhan-Hu-1 pneumovirus isolates (GenBank accession number: MN 908947), linage B.1.1.28 (Brazil variant), linage B.1.351 (south Africa variant), linage B.1.427 or Linage B.1.429 (California variant), linage B.1.617 (Indian variant), linage B.1.1.7 (British variant), linage B.1.617.2 or G/478K.V1 (Deltavariant) or Linage B.1.1.529 or GR/484A (Omik Rong Bianti) or any combination thereof. In a preferred embodiment, the at least one monomer from at least one variant of SARS-CoV-2 comprised in the at least one antigen according to the first aspect or any embodiment thereof is derived from a SASR-CoV-2 variant selected from the group consisting of: wuhan-Hu-1 pneumovirus isolates (GenBank accession number: MN 908947), linage B.1.1.28 (Brazil variant), linage B.1.351 (south Africa variant), linage B.1.427 or Linage B.1.429 (California variant), linage B.1.617 (Indian variant), linage B.1.1.7 (British variant), linage B.1.617.2 or G/478K.V1 (Deltavariant) or Linage B.1.1.529 or GR/484A (Omik Rong Bianti) or any combination thereof.

In embodiments, the at least one antigen may be in the form of a monomer or a multimer, such as a dimer, trimer, tetramer, pentamer, hexamer, heptamer, octamer, nonamer, or decamer, or any combination thereof. In embodiments, at least one antigen consists of two monomers, and is in the form of a dimer (dimeric antigen). In another embodiment, the protein subunit vaccine comprises one antigen or a mixture of more than one antigen in different forms (e.g., monomer and dimer presence). In embodiments, the protein subunit vaccine comprises at least one antigen in a different form (e.g., monomeric and dimeric forms), wherein the monomeric and dimeric forms of the monomer are RBDs (hereinafter referred to as monomeric RBD antigen or RBD-monomeric antigen and dimeric RBD antigen or RBD-dimeric antigen, respectively, as defined in the definition section). In embodiments, the protein subunit vaccine comprises a mixture of at least one monomeric RBD antigen and at least one dimeric RBD antigen. In embodiments, the protein subunit vaccine comprises a higher proportion of dimeric RBD antigen than monomeric RBD antigen. In embodiments, the proportion of one or more antigens included in the protein subunit vaccine is at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% dimeric RBD antigen. The calculation of the percentages of monomeric RBD antigen and dimeric RBD antigen can be determined by using standard methods, such as Size Exclusion Chromatography (SEC) or High Performance Liquid Chromatography (HPLC). In size exclusion chromatography, the area under the peak of the dimer peak and monomer peak identified represents the relative amounts of RBD monomer and RBD dimer. Obtaining a specific ratio of dimeric RBD antigen to monomeric RBD antigen by mixing, for example, different volumes of dimeric RBD antigen and monomeric RBD antigen is known to those skilled in the art. In embodiments, the protein subunit vaccine comprises a higher proportion of non-fusion dimeric RBD antigen than monomeric RBD antigen. In embodiments, the protein subunit vaccine comprises at least 51%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% percent of the total antigen contained in the protein subunit vaccine.

Furthermore, the inventors of the present invention tested different vaccine formulations comprising different proportions of monomers and non-fusion dimers. The results are shown in fig. 7, where it is shown that the humoral response is significantly increased when the vaccine formulation has a higher ratio of dimeric RBD antigen (56%) to monomeric RBD antigen (44%) (group E). That is, even though the vaccine composition did not contain any immunostimulant and even though the vaccine contained half the dose of RBD antigen (10 μg/dose), the humoral response was higher compared to the other groups (see fig. 7B). The immunogenicity of this RBD dimer-enriched non-fusion dimer vaccine was also compared to the immunogenicity of a commercially available mRNA vaccine (Spikevax, COVID-19mRNA vaccine (Moderna Biotech Spain, s.l.). Figure 10 shows that high levels of IgG antibody titers were elicited in all groups after the second dose, with higher levels in group C, demonstrating the enhanced immunogenicity elicited by vaccines comprising non-fusion RBD dimers: monomeric non-variant SARS-CoV-2 antigen (with a high proportion of dimeric RBD as antigen). This trend is also shown in fig. 11 when measuring neutralizing antibodies.

The inventors have also designed a new dimeric RBD antigen by fusing two RBD monomers of two different SARS-CoV-2 variants (uk variant and south africa variant), thereby generating a candidate vaccine comprising the fused dimeric RBD antigen. The ability of the fusion dimeric RBD antigen to induce antibodies against SARS-CoV-2 virus as compared to the non-fusion dimer of Wuhan-Hu-1 variant was tested and the results are shown in FIG. 8. The results indicate that animals were able to produce higher anti-SARS-CoV-2 RBD IgG antibody titers even when they received a low dose of vaccine comprising the fusion dimeric RBD antigen and no immunostimulant (groups B and C) compared to the group receiving a vaccine formulation comprising 20 μg dose of non-fusion Wuhan-Hu-1 RBD dimer and adjuvant alone (group G) or the group comprising 20 μg dose of non-fusion Wuhan-Hu-1 RBD dimer and adjuvant together with QS-21 immunostimulant (group I). Furthermore, at the same dose of antigen (20 μg), the fusion dimeric RBD antigen elicited an enhanced response compared to the group (group H) receiving the non-fusion Wuhan-Hu-1 RBD antigen plus adjuvant and MPLA immunostimulant, even though the composition did not contain any immunostimulant (group D). It is also shown herein that immunization of animals with the novel recombinant fusion dimeric RBD SARS-CoV-2 antigen elicited pseudovirus neutralizing antibody titers against different SARS-CoV-2 variants (Wuhan-Hu-1, uk, south africa, india and brazil variants) in all groups, which also indicated that the neutralizing antibody titers generated by vaccinating mice with the fusion dimeric RBD antigen remained high regardless of the variant tested and the presence or absence of an immunostimulant such as MPLA (group E) or qs.21 (group F) in the vaccine formulation (fig. 12). Next, studies were performed using a more human animal model, such as pigs. The results indicate that fusion dimeric RBD antigen induced significantly higher titers against south african variants compared to commercial vaccines, and vaccines comprising fusion dimeric RBD antigen were also safer than commercial mRNA-based vaccines, as measured by temperature measurement of pigs after vaccination (see fig. 14 and 13). These results indicate that the fusion dimeric RBD antigen exhibits an excellent balance between safety and immunogenicity compared to commercially available vaccines, as it is capable of inducing neutralizing antibodies without increasing body temperature or causing adverse reactions.

Further studies, in particular human clinical trials, have been carried out. The results show that the fusion dimeric RBD antigens of the invention elicit high levels of neutralizing antibodies against different SARS-CoV-2 variants, such as beta, delta and armyworm variants. Furthermore, an increased and better immunogenic response against SARS-CoV-2-related variants was also observed for the fusion dimeric RBD antigen of the invention when compared to mRNA-based vaccines (Comirnaty, bioNTech), see example 11.

Taken together, these results unexpectedly demonstrate that formulations comprising fused dimeric RBD antigen and formulations comprising non-fused RBD antigen (wherein the ratio of dimeric RBD antigen to monomeric RBD antigen is increased by more than 50%) have a powerful ability to produce anti-SARS-CoV-2 RBD antibodies. Furthermore, the potential for immunogenicity and safety of novel recombinant fusion dimeric RBD antigens based on the two different SARS-CoV-2 variants produced herein has been shown to be increased.

Thus, in a second aspect of the invention, a protein subunit vaccine comprises at least one antigen, characterized in that the at least one antigen comprises at least two monomers from at least one variant of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) or at least two monomers from at least one variant of SARS-CoV-2, wherein each monomer is selected from the group consisting of the S1 subunit of spike protein, or the Receptor Binding Domain (RBD) of spike protein, or any immunogenic fragment thereof, and wherein the two monomers are chemically bound to each other, optionally via a linker, thereby forming a dimer, preferably a fusion dimer or a non-fusion dimer. It should be noted that the terms "fused dimer", "non-fused dimer" and "in conjunction with each other" are defined in the definition section above. In a preferred embodiment, the two monomers are different. By "different monomers" is meant that each monomer of the dimer has a different amino acid sequence, e.g., a mixture of RBD antigens derived from different variants, or a mixture of RBD and S1 antigens derived from the same or different variants. Preferably, the amino acid sequence of each monomer in the fusion dimer corresponds to a different SARS-CoV-2 amino acid sequence.

Notably, the dimeric antigen defined above is more preferably formed from two monomers. Thus, in a preferred embodiment, the protein subunit vaccine comprises at least one antigen comprising or consisting of at least two monomers from at least one variant of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), wherein each of said monomers is selected from the group consisting of the S1 subunit of spike protein, or the Receptor Binding Domain (RBD) of spike protein, or any immunogenic fragment thereof, and wherein the two monomers are chemically bound to each other, optionally via a linker, thereby forming a dimer.

In an embodiment, the dimeric antigen is a homodimer characterized by comprising, consisting of, or consisting essentially of two monomers, wherein each monomer is selected from the group consisting of the S1 subunit of spike protein or the receptor binding monomer (RBD) of spike protein of at least one variant of SARS-CoV-2, or any immunogenic fragment thereof. In embodiments, the dimeric antigen is a homodimer comprising, consisting of, or consisting essentially of two monomers of the RBD of the selected SARS-CoV-2 variant. In another embodiment, at least one antigen is a homodimer comprising, consisting of, or consisting essentially of two monomers of the S1 subunit of the selected SARS-CoV-2 variant. In embodiments, each monomer contained in the homodimeric antigen is derived from the same SARS-CoV-2 variant. In a preferred embodiment, both monomers contained in the dimeric antigen are RBDs of spike proteins from at least one variant of SARS-CoV-2 virus.

In another embodiment, the amino acid sequences of the monomers forming the dimeric antigen are different (also referred to as heterodimers). Their amino acid sequence differences may be due to monomers derived from different SARS-CoV-2 variants, or due to their being different antigens from selected SARS-CoV-2 variants. In an embodiment, at least one antigen is a heterodimer characterized by consisting of two monomers, wherein a first monomer is selected from the group consisting of the S1 subunit of a spike protein, or the Receptor Binding Domain (RBD) of a spike protein, or any immunogenic fragment thereof, of a first SARS-CoV-2 variant, and a second monomer is selected from the group consisting of the S1 subunit of a spike protein, or the Receptor Binding Domain (RBD) of a spike protein, or any immunogenic fragment thereof, of a second SARS-CoV-2 variant, wherein the first and second SARS-CoV-2 variants are the same or different. In another embodiment, the heterodimeric antigen is composed of two monomers, one of which is the S1 subunit and the other of which is RBD.

In a preferred embodiment, the dimeric antigen comprises or consists of first and second monomers that are bound to each other. As defined above, "bonded to each other" means that they are chemically linked to each other by very weak, strong or very strong bonds. In a preferred embodiment, the dimeric antigen is a non-fused dimer, wherein the two monomers of the non-fused dimer are bound by a reversible bond, preferably a disulfide bond. In embodiments, the amino acid sequences of the two monomers of the non-fusion dimer antigen are identical. In another embodiment, the amino acid sequences of the two monomers of the non-fusion dimeric antigen are different.

In another embodiment, the dimeric antigen is a fusion dimer comprising or consisting of two monomers, wherein the two monomers are part of a single polypeptide. In a preferred embodiment, the two monomers fused to the dimeric antigen are part of a single polypeptide, as they are linked at least by peptide bonds. In the case of fusion dimeric antigens, the two monomers are synthesized as part of the same polypeptide chain by the same translational complex. Thus, two monomers fused to a dimeric antigen are contained within the same molecule, which means that one antigen is formed. In embodiments, the fusion dimer comprises at least two monomers positioned in any order in tandem or tandem repeat and optionally linked by a linker peptide. In embodiments, the amino acid sequences of the two monomers of the fusion dimer are identical. In another embodiment, the amino acid sequences of the two monomers fused to the dimeric antigen are different.

In another embodiment, the protein subunit vaccine according to the second aspect or any of its embodiments comprises a mixture of antigens present in different forms, e.g. monomers and dimers, wherein the dimers may be non-fusions and/or fusions, as defined above. In embodiments, the protein subunit vaccine preferably comprises a mixture of monomeric and dimeric antigens, wherein the dimers may be non-fusions and/or fusions, wherein the antigens comprise RBD monomers. In embodiments, the protein subunit vaccine comprises a higher proportion of dimeric antigen (non-fusions or fusions) than monomeric antigen. In embodiments, the protein subunit vaccine comprises at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% of non-fused or fused dimers comprising RBD monomers. The calculation of the percentage of monomeric antigen comprising one RBD monomer and dimeric antigen comprising two RBD monomers can be determined by using standard methods, such as Size Exclusion Chromatography (SEC) or High Performance Liquid Chromatography (HPLC). In size exclusion chromatography, the area under the peak of the dimer peak and monomer peak identified represents the relative amounts of RBD monomer and RBD dimer. Obtaining a specific ratio of dimeric RBD antigen to monomeric RBD antigen by mixing, for example, different volumes of dimeric RBD antigen and monomeric RBD antigen is known to those skilled in the art. In embodiments, the protein subunit vaccine comprises a higher proportion of non-fusion dimeric RBD antigen than monomeric RBD antigen. In one embodiment, the protein subunit vaccine comprises at least 35%, 40%, 45%, 50%, 51%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% percent of the non-fusion dimeric RBD antigen. In a preferred embodiment, the protein subunit vaccine comprises a mixture of at least monomeric RBD antigen and at least dimeric RBD antigen, wherein at least 35%, 40%, 45%, 50%, 51%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% of the total antigen comprised in the protein subunit vaccine is dimeric RBD antigen.

In an embodiment of the second aspect, each monomer comprised in the non-fusion or fusion dimeric antigen is derived from the same SARS-CoV-2 variant. In an embodiment of the second aspect, the non-fusion dimeric antigen or each monomer comprised in the fusion dimeric antigen is derived from the same SARS-CoV-2 variant, wherein the SARS-CoV-2 variant is selected from the group consisting of the american centers for disease control and prevention (CDC) "classification and definition of SARS-CoV-2 variants" defined variant of interest (VOC). In an embodiment of the second aspect, each monomer comprised in the non-fusion or fusion dimeric antigen is derived from the same SARS-CoV-2 variant, wherein the variant is selected from the group comprising, but not limited to: wuhan-Hu-1 pneumovirus isolates (GenBank accession number: MN 908947), linage B.1.1.28 (Brazil variant), linage B.1.351 (south Africa variant), linage B.1.427 or Linage B.1.429 (California variant), linage B.1.617 (Indian variant), linage B.1.1.7 (British variant), linage B.1.617.2 or G/478K.V1 (Deltavariant) or Linage B.1.1.529 or GR/484A (Omik Rong Bianti) or any combination thereof. In an embodiment of the second aspect, each monomer comprised in the non-fusion or fusion dimeric antigen is derived from the same SARS-CoV-2 variant, wherein the variant is selected from the group comprising or consisting of: wuhan-Hu-1 pneumovirus isolates (GenBank accession number: MN 908947), linage B.1.1.28 (Brazil variant), linage B.1.351 (south Africa variant), linage B.1.427 or Linage B.1.429 (California variant), linage B.1.617 (Indian variant), linage B.1.1.7 (British variant), linage B.1.617.2 or G/478K.V1 (Deltavariant) or Linage B.1.1.529 or GR/484A (Omik Rong Bianti) or any combination thereof.

In another embodiment, each of the two monomers comprised in the non-fusion or fusion dimeric antigen is derived from a different SARS-CoV-2 variant. In embodiments, the non-fusion dimeric antigen or each monomer comprised in the fusion dimeric antigen is derived from a different SARS-CoV-2 variant, wherein each SARS-CoV-2 variant is selected from the group consisting of the american centers for disease control and prevention (CDC) "classification and definition of SARS-CoV-2 variants" defined care Variants (VOCs). In embodiments, each monomer comprised in the non-fusion or fusion dimeric antigen is derived from a different SARS-CoV-2 variant, wherein each SARS-CoV-2 variant is selected from the group comprising, but not limited to: wuhan-Hu-1 pneumovirus isolates (GenBank accession number: MN 908947), linage B.1.1.28 (Brazil variant), linage B.1.351 (south Africa variant), linage B.1.427 or Linage B.1.429 (California variant), linage B.1.617 (Indian variant), linage B.1.1.7 (British variant), linage B.1.617.2 or G/478K.V1 (Deltavariant) or Linage B.1.1.529 or GR/484A (Omik Rong Bianti) or any combination thereof. In embodiments, each monomer comprised in the non-fusion or fusion dimeric antigen is derived from a different SARS-CoV-2 variant, wherein each SARS-CoV-2 variant is selected from the group consisting of: wuhan-Hu-1 pneumovirus isolates (GenBank accession number: MN 908947), linage B.1.1.28 (Brazil variant), linage B.1.351 (south Africa variant), linage B.1.427 or Linage B.1.429 (California variant), linage B.1.617 (Indian variant), linage B.1.1.7 (British variant), linage B.1.617.2 or G/478K.V1 (Deltavariant) or Linage B.1.1.529 or GR/484A (Omik Rong Bianti) or any combination thereof. It is understood that any combination of different SARS-CoV-2 variants in each monomer that is not a fusion or fusion dimeric antigen is included within the scope of the invention.

In an embodiment of the second aspect, one or both of the non-fusion dimeric antigen and the two monomers of the fusion dimeric antigen is a Receptor Binding Domain (RBD) of spike protein from at least one variant of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). In an embodiment of the second aspect, one or both of the non-fusion dimeric antigen and the two monomers of the fusion dimeric antigen is a Receptor Binding Domain (RBD) of a spike protein comprising, consisting of, or consisting essentially of amino acid residues 319 to 537 of SARS-CoV-2. In an embodiment of the second aspect, one or both of the non-fusion dimeric antigen and the two monomers of the fusion dimeric antigen is a Receptor Binding Domain (RBD) of a spike protein comprising, consisting of, or consisting essentially of amino acid residues 319 to 541 of SARS-CoV-2. In a preferred embodiment, both the non-fusion dimeric antigen and the two monomers of the fusion dimeric antigen are Receptor Binding Domains (RBDs) of spike proteins from at least one variant of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), wherein the RBD monomers are identical in their full length sequence to SEQ ID NO: 1. SEQ ID NO: 3. or SEQ ID NO:4 or any combination thereof has at least 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% sequence identity.

In a preferred embodiment, the antigen is a non-fused or fused dimer comprising two RBD monomers from at least one variant of SARS-CoV-2, wherein the at least one variant of SARS-CoV-2 is selected from the group consisting of a variant of interest (VOC).

In a preferred embodiment, the antigen is a non-fused or fused dimer comprising two RBD monomers from at least one variant of SARS-CoV-2, wherein the variant is selected from the group including, but not limited to: wuhan-Hu-1 pneumovirus isolates (GenBank accession number: MN 908947), linage B.1.1.28 (Brazil variant), linage B.1.351 (south Africa variant), linage B.1.427 or Linage B.1.429 (California variant), linage B.1.617 (Indian variant), linage B.1.1.7 (British variant), linage B.1.617.2 or G/478K.V1 (Deltavariant) or Linage B.1.1.529 or GR/484A (Omik Rong Bianti) or any combination thereof.

In a preferred embodiment, the antigen is a non-fused or fused dimer comprising two RBD monomers from at least one variant of SARS-CoV-2, wherein the at least one variant of SARS-CoV-2 is selected from the group consisting of: wuhan-Hu-1 pneumovirus isolates (GenBank accession number: MN 908947), linage B.1.1.28 (Brazil variant), linage B.1.351 (south Africa variant), linage B.1.427 or Linage B.1.429 (California variant), linage B.1.617 (Indian variant), linage B.1.1.7 (British variant), linage B.1.617.2 or G/478K.V1 (Deltavariant) or Linage B.1.1.529 or GR/484A (Omik Rong Bianti) or any combination thereof.

In an embodiment, the protein subunit vaccine comprises or consists of at least one non-fusion dimer and the non-fusion dimer comprises or consists of a first monomer and a second monomer, both derived from Wuhan-Hu-1 pneumovirus isolate (GenBank accession number: MN 908947), and wherein the two monomers of the non-fusion dimer are bound by a reversible bond. In another embodiment, the first monomer and/or the second monomer of the non-fusion dimer comprises, consists of, or consists essentially of a protein that hybridizes to the sequence of SEQ ID NO: 1. SEQ ID NO:3 or SEQ ID NO:4 or any combination thereof has at least 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% sequence identity.

In an embodiment of the second aspect, the fusion dimer consists of a first RBD monomer from a first SARS-CoV-2 variant and a second RBD monomer from a second, different SARS-CoV-2 variant. Preferably, the protein subunit vaccine comprises at least one antigen, wherein the at least one antigen is a fusion dimer, and wherein the fusion dimer comprises, consists of, or consists essentially of: a first monomer derived from Linage b.1.351 (south african SARS-CoV-2 variant) and a second monomer derived from Linage b.1.1.7 (uk SARS-CoV-2 variant), and wherein the two monomers of the fusion dimer are part of a single polypeptide. Preferably, the fusion dimer comprises two RBD monomers (hereinafter referred to as fusion dimeric RBD antigens).

In embodiments, the fusion dimeric RBD antigen comprises a first monomer derived from a b.1.351 variant and a second monomer derived from a b.1.1.7 variant. More preferably, the fusion dimeric RBD antigen comprises a first RBD monomer and a second RBD monomer, said first RBD monomer comprising, consisting of, or consisting essentially of: SEQ ID NO:4 or a sequence which hybridizes over its entire length to SEQ ID NO:4, the second RBD monomer comprises, consists of, or consists essentially of a sequence having at least 50%, 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity: SEQ ID NO:3 or a sequence which hybridizes over its entire length to SEQ ID NO:3 has at least 50%, 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity. In some embodiments, the fusion dimeric RBD antigen comprises a first RBD monomer comprising the amino acid sequence of SEQ ID NO: 4. consists of SEQ ID NO:4 or consists essentially of SEQ ID NO:4, the second RBD monomer comprises SEQ ID NO: 3. consists of SEQ ID NO:3 or consists essentially of SEQ ID NO: 3. More preferably, the fusion dimeric RBD antigen comprises, consists of, or consists essentially of a protein which hybridizes over its entire length to the amino acid sequence of SEQ ID NO:5 has at least 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% sequence identity. In some embodiments, the fusion dimeric RBD antigen comprises SEQ ID NO:5 (fusion dimeric RBD antigen sequence), consisting of SEQ ID NO:5 (fusion dimeric RBD antigen sequence) or consists essentially of SEQ ID NO:5 (fusion dimeric RBD antigen sequence).

In another embodiment, the fusion dimeric RBD antigen consists of a sequence which hybridizes over its entire length to SEQ ID NO:7 has at least 50%, 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity. In some embodiments, the fusion dimeric RBD antigen consists of a nucleic acid sequence comprising SEQ ID NO:7 (fusion dimeric RBD nucleotide sequence), consisting of SEQ ID NO:7 (fusion dimeric RBD nucleotide sequence) or consists essentially of SEQ ID NO:7 (fusion dimeric RBD nucleotide sequence). In another embodiment, the fusion dimeric RBD antigen consists of a sequence which hybridizes over its entire length to SEQ ID NO:8 has at least 50%, 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity. In some embodiments, the fusion dimeric RBD antigen consists of a nucleic acid sequence comprising SEQ ID NO:8 (fusion dimeric RBD nucleotide sequence), consisting of SEQ ID NO:8 (fusion dimeric RBD nucleotide sequence) or consists essentially of SEQ ID NO:8 (fusion dimeric RBD nucleotide sequence).

In embodiments, a protein subunit vaccine, preferably a fusion dimeric RBD antigen, is capable of inducing an immunogenic and/or protective immune response without increasing or altering the basal body temperature of a subject immunized with the vaccine, an increase in basal body temperature being understood as an increase in basal body temperature of 0.5 ℃, 0.6 ℃, 0.7 ℃, 0.8 ℃, 0.9 ℃,1 ℃, 1.2 ℃, 1.4 ℃, 1.6 ℃, 1.8 ℃,2 ℃ or more than 2 ℃ after immunization. In embodiments, the protein subunit vaccine, preferably a fusion dimeric RBD antigen, is capable of inducing an immunogenic and/or protective immune response without significant side effects. Preferably, the protein subunit vaccine, preferably the fusion dimeric RBD antigen, is capable of inducing an immunogenic and/or protective immune response without producing significant side effects such as fatigue, injection site pain, or tenderness, as shown in example 11.

In an embodiment, any monomer comprised in the antigen of the first or second aspect of the invention or from any embodiment thereof may be selected from the group consisting of the S1 subunit of a spike protein or a receptor binding monomer (RBD) of a spike protein or any immunogenic fragment thereof, comprising in their amino acid sequence a tag sequence or a signal peptide sequence or both. In embodiments, the RBD monomers comprise a signal peptide sequence at the N-terminus. In embodiments, the signal peptide is located at the N-terminus and is selected from the group consisting of SEQ ID NO:6 or SEQ ID NO: 10. In alternative embodiments, the signal peptide may be replaced by any signal peptide that enables expression of at least one antigen. In an alternative embodiment, the processed antigen does not comprise a signal peptide. After expression of at least one monomer, the N-terminal signal peptide is cleaved. In an embodiment, the monomer comprises a tag sequence, preferably a His tag sequence. The monomers and antigens described herein may also include additional modifications to the native sequence, such as additional internal deletions, additions, and substitutions. These modifications may be deliberate, such as by site-directed mutagenesis, or may be occasional, such as by naturally occurring mutational events.

In another embodiment, the antigen of the first or second aspect of the invention or any antigen from any embodiment thereof is a recombinant expression product. Methods for producing recombinant antigens are known in the art and they generally comprise cloning at least one antigen into an expression vector, preferably a plasmid, transfecting eukaryotic or prokaryotic cells with the plasmid vector, expressing the antigen in the cells and purifying the at least one antigen from the cells or supernatant thereof.

In embodiments, the plasmid vector is a mammalian expression vector. More preferably, the expression vector backbone for expressing the at least one antigen is selected from the group consisting of pcdna3.4 (GENSCRIPT) or pD2610-v10 (ATUM). In a preferred embodiment, the DNA sequence for expressing the antigen of the invention is codon optimized and inserted into a carrier selected from the group consisting of pcDNA3.4 or the carriers pD2610-v10 (ATUM). In an embodiment, the cell for expressing the at least one antigen is a eukaryotic cell, preferably a CHO cell or a HEK293 mammalian cell. In a preferred embodiment, at least one antigen is collected and purified from the culture supernatant.

It will be appreciated that the one or more antigens contained in the protein subunit vaccines provided herein are produced and maintained under suitable media conditions that allow for proper folding of the one or more antigens. The skilled artisan will know the physical and chemical conditions to maintain and preserve the desired structure of the antigen (including monomeric and multimeric forms thereof) during all stages of production. In embodiments, the media conditions are selected such that the dimeric form of the antigen is superior to the monomeric form. Spontaneous dimerization may be controlled by very weak, strong or very strong keys, and may be covalent (e.g., disulfide bridges) or non-covalent. The skilled person will know how to optimise the culture medium conditions to obtain the desired ratio of dimeric and/or monomeric antigen. For example, the use of high temperatures (above the melting temperature of the dimer) or ionic detergents (e.g., SDS) to self-form the dimer is not suggested.

In embodiments, the antigen is produced in the presence of an oxidizing agent, such as glutathione. In embodiments, a reducing agent such as dithiothreitol is not present in the antigen-producing medium. In embodiments, the one or more antigens of the protein subunit vaccine are produced at a temperature suitable to preserve their structure or to facilitate dimer formation. The person skilled in the art is also aware of adjusting the temperature. In embodiments, the antigen production temperature ranges from 30 ℃ to 40 ℃, preferably from 33 ℃ to 37 ℃, most preferably 33 ℃.

In embodiments, the pH of the protein subunit vaccine and/or antigen-producing medium is maintained at pH7 or less. In embodiments, the pH of the protein subunit vaccine and/or antigen-producing medium is maintained at pH7 or above. In embodiments, the pH is an acidic pH (below 7). In an embodiment, the pH is an alkaline pH (7 or more). In an embodiment, the pH is neutral (about 7). In embodiments, the pH of the protein subunit vaccine is about 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14. In preferred embodiments, the pH range of the protein subunit vaccine is 4 to 9, 5 to 8, 5 to 7.5, 5 to 7, 5 to 6.5, 5.5 to 6.5 or any value comprised within these ranges. In embodiments, the pH ranges from 5 to 9, 5.5 to 9, 6 to 9, 6.5 to 9, 7 to 8, or any value contained within these ranges.

The choice of an appropriate adjuvant helps to promote an appropriate immune response against the pathogen of interest on a prior day and level of fitness, thus eliciting protective immunity while maintaining safety, which is not straightforward. The wrong choice of adjuvant may result in the failure of a particular vaccine antigen. Thus, the choice of vaccine antigen must be carefully considered with respect to which adjuvant or combination of adjuvants and/or immunostimulants is used, to avoid discarding potentially effective candidate vaccine antigens and to aid in vaccine development. The invention disclosed herein also shows that squalene or an oil-in-water adjuvant formulation of squalene is a suitable adjuvant comprised in a protein subunit SARS-CoV-2 vaccine, in particular in a vaccine comprising at least one antigen selected from the group consisting of the S1 subunit of spike protein or the Receptor Binding Domain (RBD) of spike protein. In particular we show here a protein subunit vaccine comprising at least one antigen, characterised in that it has squalene or an oil-in-water adjuvant formulation of squalene as adjuvant and comprises S1 subunits or RBD monomers, which is capable of eliciting high neutralizing antibody titers against SARS-CoV-2 virus as shown in figure 1 and producing release of cytokines, thereby indicating the presence of a cellular immune response as shown in figure 2. The combination of the S1 subunit or RBD monomer with squalene or squalene oil-in-water adjuvant formulation and immunostimulant (e.g. MPLA) also elicits high neutralizing antibody titers and even higher cellular immune responses (fig. 2, panels c and G).

Thus, in another embodiment of the first or second aspect, the protein subunit vaccine as defined in the first or second aspect or any embodiment thereof, further comprises at least one adjuvant, preferably mf59c.1. In a further embodiment, the at least one adjuvant is preferably squalene or an oil-in-water adjuvant formulation of squalene. In addition, in another embodiment, the protein subunit vaccine as defined in the first or second aspect or any embodiment thereof, further comprises at least one immunostimulant. Possible adjuvants and immunostimulants are defined as follows.

At least one adjuvant

As mentioned above, the protein subunit vaccine according to the first or second aspect may further comprise at least one adjuvant. The at least one adjuvant may include, but is not limited to: aluminum salts (alum) (e.g., aluminum hydroxide, aluminum phosphate, aluminum sulfate, etc.), oil-in-water or water-in-oil emulsion formulations (e.g., complete Freund's Adjuvant (CFA) and Incomplete Freund's Adjuvant (IFA)), mineral adjuvants, block copolymers, adjuvants formed from bacterial cell wall components such as adjuvants including liposaccharides (e.g., lipid A or monophosphoryl lipid A (MPLA), trehalose Dimycolate (TDB)) and adjuvants formed from components of the Cell Wall Skeleton (CWS), heat shock proteins or derivatives thereof, adjuvants derived from ADP-ribosylated bacterial toxins, among these are Diphtheria Toxin (DT), pertussis Toxin (PT), cholera Toxin (CT), escherichia coli heat labile toxins (LT 1 and LT 2), pseudomonas endotoxin A and exotoxin, bacillus cereus exoenzyme B, bacillus sphaericus toxin, clostridium botulinum toxin C2 and C3, clostridium calx exoenzyme and Clostridium perfringens, clostridium spiralis (Clostridium spiriforma) and Clostridium difficile, mutant of Staphylococcus aureus toxin, EDIM and mutant toxins, mutant toxins such as CRM-197, diphtheria toxin, chemokines and cytokines (e.g., interleukin-2, interleukin-7, interleukin-12, granulocyte-macrophage colony stimulating factor (GM-CSF), interferon-y, interleukin-1 (IL-1 p) and IL-1 (3 peptide or Sclavo)), non-toxic mutants, cytokine-containing liposomes, triterpene glycosides or saponins (e.g., ISCOM, quilla and QS-21), squalene or squalene oil-in-water adjuvant formulations, squalane or squalane oil-in-water adjuvant formulations (e.g., SAF, MF59 and mf59 c.1), muramyl Dipeptide (MDP) derivatives (e.g., N-acetyl-muramyl-L-threonyl-D-isoglutamine (threonyl-MDP), GMDP, N-acetyl-N-muramyl-L-alanyl-D-isoglutamine, N-acetylmuramyl-L-alanyl-D-isoglutamine-L-alanine 2- (1 '-2' -dipalmitoyl-sn-glycerol-3-hydroxyphosphoryloxy) -ethylamine, muramyl tripeptide phosphatidylethanolamine (MTP-PE)), unmethylated CpG dinucleotides and oligonucleotides (e.g., bacterial DNA and fragments thereof), oligodeoxynucleotides (ODN) and polyphosphazenes.

Other suitable mineral adjuvants include, but are not limited to: aluminum hydroxide gel (ALHYDROGEL, REHYDRAGEL), aluminum phosphate gel (including aluminum hydroxy phosphate gel (AlPO ₄; adju-Phos CRODA)), calcium phosphate, N-acetyl-muramyl-L-threonyl-D-isoglutamine (thr-MDP) -acetyl-N-muramyl-L-alanyl-D-isoglutamine, N-acetylmuramyl-L-alanyl-D-isoglutaminyl-L-alanine-2- (1 '-2' -dipalmitoyl-sn-glycero-3-hydroxyphosphoryloxy) -ethylamine.

In another embodiment, a particulate adjuvant is used. Particulate adjuvants include, but are not limited to: biodegradable and biocompatible polyesters, homopolymers and copolymers of lactic acid (PLA) and glycolic acid (PGA), poly (lactide-co-glycolide) (PLGA) microparticles, self-assembling polymers (poloxamer particles), soluble polymers (polyphosphazenes), and virus-like particles (VLPs), such as recombinant protein particles, e.g., hepatitis b surface antigen (HbsAg).

Another type of adjuvant that may also be used includes mucosal adjuvants including, but not limited to, thermolabile enterotoxins (LT) from E.coli, cholera holotoxin (CT) and cholera toxin B subunits (CTB) from Vibrio cholera, mutant toxins (e.g., LTK63 and LTR 72), microparticles, and polymerized liposomes. Other examples of mucus targeting adjuvants are e.coli mutant thermolabile toxins LT with reduced toxicity, live attenuated organisms that bind to gastrointestinal M cells (e.g. vibrio cholerae and salmonella typhi, mycobacterium Bovis (BCG)), and mucosal targeting particulate carriers such as phospholipid artificial membrane vesicles, copolymer microspheres, lipophilic immunostimulatory complexes, and bacterial outer membrane protein preparations (proteasomes).

Other adjuvants known in the art are also included within the scope of the invention (see, e.g., VACCINE DESIGN: the Subunit and Adjuvant Approach, chap.7, michael F.).

Preferably, the at least one adjuvant is selected from the list consisting of: an aluminium phosphate gel adjuvant, preferably an AlPO ₄ gel; adju-Phos CRODA, or squalene or an oil-in-water adjuvant formulation of squalene, preferably MF59C.1 or a derivative thereof. More preferably, the at least one adjuvant is mf59c.1. More preferably, the at least one adjuvant is squalene or an oil-in-water adjuvant formulation of squalene.

The MF59 adjuvant is an oil-in-water emulsion consisting of squalene (2,6,10,15,23-hexamethyl-2, 6,10,14,18, 22-tetracosane) (4.3%) and two nonionic surfactants polysorbate 80 (also known as Tween 80) (0.5%) and sorbitan trioleate 85 (also known as Span 85) (0.5%). The emulsion is a milky oil-in-water emulsion stabilized by two nonionic surfactants (polysorbate 80 and sorbitan trioleate). The basic process involves dispersing sorbitan trioleate in squalene, dispersing polysorbate 80 in an aqueous buffer, and then mixing at high speed to form a macroemulsion. The macroemulsion is then repeatedly passed through a microfluidizer to produce an o/w emulsion of uniform droplet size, which can be sterile filtered and filled into vials for later use. This process is well described in The art, for example, O' Hagan D.T.et al, the history of The inventionAdjuvant a phoenix that arose from the ashes. Expert Rev vaccines.2013Jan;12 (1):13-30. Mf59c.1 adjuvant is an optimized version of the MF59 original adjuvant, consisting of identical components, and further comprising an aqueous buffer of citrate for injection (citric acid monohydrate, and sodium citrate dihydrate) to provide higher stability than the original MF59 adjuvant.

Methods of preparing mf59c.1 adjuvants are also known to those skilled in the art (see O' Hagan d.t.et al, supra or in u.s.app.no. 2009/0208523).

In embodiments, the adjuvant may be formulated with the copolymer, viral particles, liposomes (snail shell (cochleated)) as an emulsion, an oil-in-water formulation, or with an immunostimulant. In embodiments, the at least one adjuvant may be mixed with (prior to or concurrent with) other components of the protein subunit vaccine, or alternatively, the at least one adjuvant is not mixed with other components of the protein subunit vaccine but is co-administered with them separately. In a preferred embodiment, the adjuvant mf59c.1 is mixed with at least one antigen. In a preferred embodiment, the adjuvant is squalene or an oil-in-water adjuvant formulation of squalene and which is admixed with at least one antigen.

Preferably, in the context of the present invention, when referring to "squalene or squalene oil-in-water adjuvant formulation" it is meant in particular to make up an oil-in-water emulsion consisting of squalene (2,6,10,15,23-hexamethyl-2, 6,10,14,18, 22-tetracosane) (4.3%) and two non-ionic surfactants polysorbate 80 (also known as Tween 80) (0.5%) and sorbitan trioleate 85 (also known as Span 85) (0.5%). The emulsion is a milky oil-in-water emulsion stabilized by two nonionic surfactants, polysorbate 80 and sorbitan trioleate. Preferably, the process comprises dispersing sorbitan trioleate in squalene, dispersing polysorbate 80 in an aqueous buffer and then mixing at high speed to form a macroemulsion. The macroemulsion is then repeatedly passed through a microfluidizer to produce an o/w emulsion of uniform droplet size, which can be sterile filtered and filled into vials for later use. More preferably, it is noted that in the context of the present invention the following squalene or squalene oil-in-water adjuvant formulation is particularly preferred and is selected from the following list, hereinafter referred to as "specific squalene or squalene oil-in-water adjuvant formulation":

Preferably, the specific squalene or squalene oil-in-water adjuvant formulation comprises or preferably consists of: about 1 to 15mg squalene per dose, 0.1 to 2mg polysorbate 80 per dose, 0.1 to 2mg sorbitan trioleate per dose, 0.08 to 1mg sodium citrate per dose, and 0.004 to 0.05 citric acid per dose.

Preferably, the specific squalene or squalene oil-in-water adjuvant formulation comprises or preferably consists of the following per 0.1ml dose: 1.46mg squalene, 0.18mg polysorbate 80, 0.18mg sorbitan trioleate, 0.099mg sodium citrate and 0.006mg citric acid.

Preferably, the specific squalene or squalene oil-in-water adjuvant formulation comprises or preferably consists of the following per 0.1ml dose: 1.95mg squalene, 0.235mg polysorbate 80, 0.235mg sorbitan trioleate, 0.132mg sodium citrate and 0.008mg citric acid.

Preferably, the specific squalene or squalene oil-in-water adjuvant formulation comprises or preferably consists of the following per 0.5ml dose: 9.75mg squalene, 1.175mg polysorbate 80, 1.175mg sorbitan trioleate, 0.66mg sodium citrate and 0.04mg citric acid.

Preferably, the specific squalene or squalene oil-in-water adjuvant formulation comprises or preferably consists of: 10to 60mg/ml squalene, 1to 6mg/ml polysorbate 80, 1to 6mg/ml sorbitan trioleate, 0.5 to 6mg/ml sodium citrate, and 0.01 to 0.5mg/ml citric acid.

Preferably, the specific squalene or squalene oil-in-water adjuvant formulation comprises or preferably consists of: about 19.5mg/ml squalene, 2.35mg/ml polysorbate 80, 2.35mg/ml sorbitan trioleate, 1.32mg/ml sodium citrate, and 0.08mg/ml citric acid.

Preferably, the specific squalene or squalene oil-in-water adjuvant formulation comprises or preferably consists of: about 39mg/ml squalene, 4.7mg/ml polysorbate 80, 4.7mg/ml sorbitan trioleate, 2.64mg/ml sodium citrate, and 0.16mg/ml citric acid.

In embodiments, mf59c.1 is formulated as about 1 to 15mg squalene per dose, 0.1 to 2mg polysorbate 80 per dose, 0.1 to 2mg sorbitan trioleate per dose, 0.08 to 1mg sodium citrate per dose, and 0.004 to 0.05 citric acid per dose. In an embodiment, mf59c.1 is formulated to be about 1.46mg squalene, 0.18mg polysorbate 80, 0.18mg sorbitan trioleate, 0.099mg sodium citrate, and 0.006mg citric acid per 0.1ml dose. In an embodiment, mf59c.1 is formulated to be about 1.95mg squalene, 0.235mg polysorbate 80, 0.235mg sorbitan trioleate, 0.132mg sodium citrate, and 0.008mg citric acid per 0.1ml dose. In a preferred embodiment, mf59c.1 is formulated to be about 9.75mg squalene, 1.175mg polysorbate 80, 1.175mg sorbitan trioleate, 0.66mg sodium citrate and 0.04mg citric acid per 0.5ml dose.

In embodiments, mf59c.1 is formulated as about 10 to 60mg/ml squalene, 1 to 6mg/ml polysorbate 80, 1 to 6mg/ml sorbitan trioleate, 0.5 to 6mg/ml sodium citrate, and 0.01 to 0.5mg/ml citric acid. In embodiments, MF59C.1 is formulated as about 19.5mg/ml squalene, 2.35mg/ml polysorbate 80, 2.35mg/ml sorbitan trioleate, 1.32mg/ml sodium citrate, and 0.08mg/ml citric acid. In a preferred embodiment, MF59C.1 is formulated as about 39mg/ml squalene, 4.7mg/ml polysorbate 80, 4.7mg/ml sorbitan trioleate, 2.64mg/ml sodium citrate, and 0.16mg/ml citric acid.

In other embodiments, any of the above classes of adjuvants may be used in combination with each other or with other adjuvants, antigens or immunostimulants.

At least one immunostimulant

As described above, the protein subunit vaccine according to the first or second aspect or any embodiment thereof may optionally further comprise at least one immunostimulant. The at least one immunostimulant may include, but is not limited to, a Toll-like receptor (TLR) agonist, a NOD-like receptor agonist, or a cytokine. Toll-like receptors are expressed primarily in immune cells, play a key role in immune activity, and are known to promote dendritic cell maturation by stimulation of active pharmaceutical ingredients. Toll-like receptor agonists may comprise a compound selected from the group consisting of: for example, but not limited to, a TLR-1 agonist, a TLR-2 agonist, a TLR-3 agonist, a TLR-4 agonist, a TLR-5 agonist, a TLR-6 agonist, a TLR-7 agonist, a TLR-8 agonist, and a TLR-9 agonist. NOD-like receptors are intracellular sensors of pathogen-associated molecular patterns (PAMPs) and damage-associated molecular pattern molecules (DAMPS) associated with cellular stress entering cells through phagocytosis or pores, are part of pattern recognition receptors and play an important role in innate immune responses. NOD-like receptor agonists may include, for example, NLRA agonists, NLRB agonists, NLRC agonists, or NLRP agonists, but are not limited thereto. Cytokines are a generic term for proteins secreted by immune cells and are known to induce proliferation or promote differentiation of macrophages and lymphocytes. Cytokines may include, for example ,IL-1α、IL-113、IL-1、IL-2、IL-3、IL-4、IL-5、IL-6、IL-7、IL-8、IL-9、IL-10、IL-11、IL-12、IL-13、IL-15、IL-16、IL-17、IL-18、IL-19、IL-20、IL-21、IL-22、IL-23、IL-24、IL-25、GM-CSF、G-CSF、M-CSF、TNF-α、TNF-β、IFNα or ifnβ. The immunostimulants included in the present invention may also be poly (I: CU), cpG, imiquimod, racemoset, dSLIM, toll-like receptor agonists such as monophosphoryl lipid A (MPLA), flagellin, plasmid DNA double stranded DNA, single stranded DNA, saponins such as QS-21 and interleukin cytokines, but are not limited thereto.

In a preferred embodiment, the at least one immunostimulant is selected from the group consisting of Toll-like receptor agonists such as monophosphoryl lipid a (MPLA) or saponins such as C ₉₂O₄₆H₁₄₈ (QS-21). In a preferred embodiment, the protein subunit vaccine comprises at least one immunostimulant, wherein the immunostimulant is selected from the group consisting of monophosphoryl lipid a (MPLA) and/or C ₉₂O₄₆H₁₄₈ (QS-21).

QS-21 is an acylated 3, 28-double stranded triterpene glycoside (3, 28-bisdesmodic triterpene glycoside), molecular formula C ₉₂O₄₆H₁₄₈, molecular weight 1990Da. It was originally designated as a specific fraction in complex RP-HPLC traces, particularly active fraction 21 (RP-HPLC peak) of quillaja saponaria (Quillaja saponaria), as described in Kensil C.et al.Separation and characterization of saponins with adjuvant activity from Quillaja saponaria Molina cortex.J Immunol 1991;146:431e7 and Ragupathi et al.Natural and synthetic saponin adjuvant QS-21 for vaccines against cancer.Exp Rev Vaccin 2011;10:463e70. The QS-21 fraction exhibits excellent immunostimulatory and adjuvant properties to a range of antigens. It can enhance the clinically significant response of antibodies and T cells to vaccine antigens against a variety of infectious diseases, degenerative diseases and cancers.

Monophosphoryl lipid A (MPLA or MPL) is a known immunostimulant obtained from bacterial lipopolysaccharides, typically from Salmonella minnesota (Salmonella Minnesota) lipopolysaccharide, e.g., an immunostimulant commercially available from SIGMA under the trade designation "Monophosphoryl lipid A, re 595 (Re mutant) from Salmonella minnesota" (product L6895). In the context of the present invention, monophosphoryl lipid A also includes derivatives and synthetic analogues thereof which are also suitable as immunostimulants, such as 3-deacylated derivatives (3D-MPL or 3D-MPLA), such as those commercially available under the name MPL ^TM by the company SIGMA. Synthetic analogues of monophosphoryl lipid A may also be used, such as those described in patent application WO2008/153541-A1 or those commercially available through Avanti Polar Lipids company (product PHAD ^TM) or AdipoGen (product AG-CU 1-0002).

Methods of preparing immunostimulants are known to those skilled in the art.

In embodiments, the at least one immunostimulant may be mixed with (prior to or concurrent with) other components of the protein subunit vaccine, or alternatively, the at least one immunostimulant is not mixed with other components of the protein subunit vaccine but is co-administered separately therewith. In a preferred embodiment, the immunostimulant MPLA is mixed with at least one antigen and with at least one adjuvant. In a preferred embodiment, the immunostimulant QS-21 is admixed with at least one antigen and with at least one adjuvant.

In another embodiment, any of the above classes of immunostimulants may be used in combination with each other or with other adjuvants, antigens or immunostimulants.

Dosage of

The amount of each component of a protein subunit vaccine as defined above can be readily determined by the skilled person, for example by determining the dose effective to elicit a prophylactic or therapeutic immune response, for example by measuring the serum titer of vaccine specific immunoglobulins or by measuring the inhibition rate of a serum sample compared to a control not receiving the component. Furthermore, the person skilled in the art is also able to adapt the dose of each component of the protein subunit vaccine to the subject to whom the protein subunit vaccine is administered. For example, the dose tested in the mouse model can be extrapolated to humans by including the same dose tested in the mouse model or multiplying the dose tested in the mouse model by 2,3, 4, 5,6,7, or 8 times. Preferably, the adjuvant and immunostimulant modulator dosages for humans are obtained by multiplying the amount tested in the mouse model by 5.

In an embodiment, the protein subunit vaccine according to the first or second aspect or any embodiment thereof comprises a therapeutically effective amount of at least one or more antigens as desired. "therapeutically effective amount" refers to the amount that induces an immunogenic and protective immune response in an uninfected, infected or unexposed individual to which the vaccine is administered. By "therapeutically effective amount" is meant an amount of antigen sufficient to induce an immune response that alleviates at least one symptom or clinical sign associated with a SARS-CoV-2 infection or related disease. The terms "immunogenic and protective immune response", "protective immunity" or "protective immune response" as used herein refer to the ability of a subject vaccinated against an infection or disease, to prevent the development of symptoms or clinical signs of the infection or disease, to delay the onset of an infection or disease or symptoms or clinical signs thereof, or to reduce the severity of a subsequent infection or disease or symptoms or clinical signs. Such a response typically results in the subject producing a secretion-mediated immune response, a cell-mediated immune response, and/or an antibody-mediated immune response against the vaccine. Cell-mediated immune responses include cd4+ T helper cell responses, cytotoxic T lymphocyte cd8+, cellular antiviral responses, and antiviral chemokine responses. Antibody-mediated immune responses include immune responses measured by serological assays (e.g., virus neutralization assays, ADCC assays, ELISA, immunoblot assays, and other known assays). Thus, protective immune responses include, but are not limited to, one or more of the following effects: producing antibodies of any immunological class, such as immunoglobulin A, D, E, G or M; proliferation of B and T lymphocytes; providing activation, growth and differentiation signals to immune cells; expansion of helper T cells, suppressor T cells and/or cytotoxic T cells. Several methods are known in the art for studying protective immunity generated by candidate vaccines in preclinical and clinical trials. Protective immunity can be analyzed at preclinical levels, for example, by calculating the percent survival of vaccinated animals after infection with a lethal or sublethal dose of SARS-CoV-2, identifying the progression of symptoms indicative of disease (weight loss, fever), or quantifying viral load in the infected organ.

As shown in example 10 and fig. 15, a fully protective immune response (100% survival and no weight loss) was achieved in immunized mice challenged with a lethal dose of Wuhan-Hu-1-like isolate comprising the D614G mutation using a vaccine comprising the fusion dimeric RBD variant SARS-CoV-2 antigen (subunit vaccine comprising a fusion protein consisting of a first monomer comprising RBD derived from the b.1.351 (south africa) variant and a second monomer comprising RBD derived from the b.1.1.7 (uk) variant). The control group had a weight loss and a mortality rate of 100%. It is important that this complete protection is achieved even at the lowest test dose (10 μg) of subunit vaccine.

Thus, in an embodiment, the subunit vaccine according to the first or second aspect or any embodiment thereof is capable of preventing a SARS-CoV-2 virus infection. "preventing" includes, but is not limited to, reducing, or ameliorating the risk or severity of a symptom, disorder, condition, or disease, as well as protecting an animal from the symptom, disorder, condition, or disease. In embodiments, subunit vaccines provided herein are applied or administered prophylactically and/or therapeutically. In a preferred embodiment, a subunit vaccine comprising at least one antigen, preferably comprising at least one fusion dimer, as defined in the first or second aspect or any embodiment thereof, is capable of inducing an immunogenic and/or protective immune response capable of preventing a SARS-CoV-2 viral infection and/or clinical signs or manifestations associated with a SARS-CoV-2 infection caused by at least one or any of the SARS-CoV-2 variants. "clinical signs associated with SARS-CoV-2 infection" include, but are not limited to: fever, cold tremor, fatigue, dry cough, loss of taste or smell, rash, chest pain, weight loss, anorexia, headache, myalgia, diarrhea, phlegm, sore throat, nasal obstruction, dyspnea, runny nose, lymphocyte depletion, and other symptoms and death. Preferably, the subunit vaccine provided herein is capable of inducing an immunogenic and/or protective immune response capable of preventing a SARS-CoV-2 viral infection and/or clinical signs or manifestations associated with a SARS-CoV-2 infection caused by at least one variant of SARS-CoV-2, wherein the variant is selected from the group comprising, but not limited to: wuhan-Hu-1 pneumovirus isolates (GenBank accession number: MN 908947), linage B.1.1.28 (Brazil variant), linage B.1.351 (south Africa variant), linage B.1.427 or Linage B.1.429 (California variant), linage B.1.617 (Indian variant), linage B.1.1.7 (British variant), linage B.1.617.2 or G/478K.V1 (Deltavariant) or Linage B.1.1.529 or GR/484A (Omik Rong Bianti) or any combination thereof. More preferably, the fusion dimer is capable of inducing an immunogenic and/or protective immune response capable of preventing SARS-CoV-2 infection and/or clinical signs or manifestations associated with SARS-CoV-2 infection by at least one or any one of the SARS-CoV-2 variants, the fusion dimer being comprising a sequence that is identical to SEQ ID NO:5, consisting of, or consisting essentially of a protein having at least 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% sequence identity over its full length to SEQ ID NO:5, or a fusion dimeric RBD antigen consisting of a protein having at least 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% sequence identity over its entire length to SEQ ID NO:7 have at least 50%, 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% sequence identity.

In an embodiment, the subunit vaccine according to the first or second aspect or any embodiment thereof is capable of preventing death and weight loss caused by SARS-CoV-2 infection. In a preferred embodiment, a subunit vaccine comprising at least one antigen, preferably comprising at least one fusion dimer, as defined in the first or second aspect or any embodiment thereof, is capable of inducing an immunogenic and/or protective immune response capable of preventing mortality and weight loss caused by infection with SARS-CoV-2 virus of at least one or any SARS-CoV-2 variant. Preferably, the subunit vaccine provided herein is capable of inducing an immunogenic and/or protective immune response capable of preventing death and weight loss caused by infection with SARS-CoV-2 virus of at least one variant of SARS-CoV-2, wherein the variant is selected from the group comprising, but not limited to: wuhan-Hu-1 pneumovirus isolates (GenBank accession number: MN 908947), linage B.1.1.28 (Brazil variant), linage B.1.351 (south Africa variant), linage B.1.427 or Linage B.1.429 (California variant), linage B.1.617 (Indian variant), linage B.1.1.7 (British variant), linage B.1.617.2 or G/478K.V1 (Deltavariant) or Linage B.1.1.529 or GR/484A (Omik Rong Bianti) or any combination thereof. More preferably, the fusion dimer is capable of inducing an immunogenic and/or protective immune response capable of preventing death and weight loss caused by infection with SARS-CoV-2 virus of at least one or any one of the SARS-CoV-2 variants, which fusion dimer is comprised of a sequence that hybridizes over its entire length to SEQ ID NO:5, consisting of, or consisting essentially of a protein having at least 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% sequence identity over its full length to SEQ ID NO:5, or a fusion dimeric RBD antigen consisting of a protein having at least 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% sequence identity over its entire length to SEQ ID NO:7 have at least 50%, 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% sequence identity.

In embodiments, the subunit vaccines provided herein are capable of inducing an immunogenic and/or protective immune response capable of preventing infection by a SARS-CoV-2 virus, wherein such immunogenic and/or protective immune response may be homoimmunogenic and/or heteroimmunogenic. "homologous immunogenicity" and "homologous protective immune response" refer herein to the generation of an immunity or protective immunity against one pathogen or variant after exposure of the host to the same pathogen or antigen. "heterologous immunogenicity" and "heterologous protective immune response" refer herein to the generation of an immunity or protective immunity against a different pathogen or variant after exposure of the host to one pathogen or antigen.

Example 10 shows that a fusion dimeric RBD variant SARS-CoV-2 subunit vaccine comprising two monomers derived from ZA and UK variants, respectively, is capable of inducing an immunogenic and protective immune response against heterologous challenge, i.e. against SARS-CoV-2 variants (Wuhan-Hu-1-like variants comprising the D614G mutation) that are not used in the vaccine. This supports the following viewpoints: subunit vaccines, preferably dimeric subunit vaccines, more preferably fusion dimers, provided herein are capable of inducing heterologous immunogenicity and/or heterologous protective immune responses.

In an embodiment, the protein subunit vaccine according to the first or second aspect or any embodiment thereof is administered in an amount effective to cause treatment or prevention of a symptom of the disease. One skilled in the art can readily determine a suitable effective amount based on the age, sex, weight and other physical and/or metabolic conditions of the subject in need thereof. The "therapeutically effective amount" may fall within a relatively broad range that can be determined by routine experimentation.

More specifically, the possible amounts of the different components of the protein subunit vaccine according to the first or second aspect or any embodiment thereof are detailed below:

With respect to the amount of one or more antigens, in embodiments, the total amount of antigen contained in the protein subunit vaccine is about 1 μg/dose, 2 μg/dose, 3 μg/dose, 4 μg/dose, 5 μg/dose, 6 μg/dose, 7 μg/dose, 8 μg/dose, 9 μg/dose, 10 μg/dose, 11 μg/dose, 12 μg/dose, 13 μg/dose, 14 μg/dose, 15 μg/dose, 16 μg/dose, 17 μg/dose, 18 μg/dose, 19 μg/dose, 20 μg/dose, 25 μg/dose, 30 μg/dose, 35 μg/dose, 40 μg/dose, 45 μg/dose, 50 μg/dose, 60 μg/dose, 70 μg/dose, 80 μg/dose, 90 μg/dose or more than 100 μg/dose. Preferably, the total amount of the one or more antigens comprised in the protein subunit vaccine according to the first or second aspect or any embodiment thereof is about 10 μg total antigen, 15 μg total antigen, 20 μg total antigen, 25 μg total antigen, 30 μg total antigen, 35 μg total antigen, 40 μg total antigen, 45 μg total antigen, 50 μg total antigen, 55 μg total antigen, 60 μg total antigen, 70 μg total antigen, 80 μg total antigen, 90 μg total antigen or 100 μg total antigen. Preferably, the total amount of the one or more antigens is from 5 to 50 μg per dose, most preferably 10 μg per dose, 20 μg per dose or 40 μg per dose of antigen.

With respect to the amount of adjuvant, in a preferred embodiment, mf59c.1 adjuvant is present in the protein subunit vaccine according to the first or second aspect or any embodiment thereof in a relative percentage of about 10%/90%, 20%/80%, 30%/70%, 40%/60%, 50%/50%, 60%/40%, 70%/30%, 80%/20%,90%/10% adjuvant/antigen (v/v) per dose. Preferably, the amount of adjuvant present in the protein subunit vaccine is 60-40%/40-60%, preferably 75%/25%, more preferably 50%/50% relative to the amount of antigen(s) (% adjuvant/% antigen) (v/v).

In a further preferred embodiment, the adjuvant is a specific squalene or an oil-in-water adjuvant formulation of squalene and is present in the protein subunit vaccine according to the first or second aspect or any embodiment thereof in a relative percentage of adjuvant/antigen (v/v) of about 10%/90%, 20%/80%, 30%/70%, 40%/60%, 50%/50%, 60%/40%, 70%/30%, 80%/20%,90%/10% per dose. Preferably, the amount of said adjuvant present in the protein subunit vaccine is 60-40%/40-60%, preferably 75%/25%, more preferably 50%/50% relative to the amount of antigen(s) (% adjuvant/% antigen) (v/v).

In another embodiment, the aluminium phosphate adjuvant, preferably AlPO ₄ gel, is present in a protein subunit vaccine according to the first or second aspect or any embodiment thereof at a dose of at least 5mg per dose, 10mg per dose, 20mg per dose, 30mg per dose, 40mg per dose, 50mg per dose, 60mg per dose, 70mg per dose, 80mg per dose, 90mg per dose, 100mg per dose or more than 100mg per dose. In embodiments, the aluminium phosphate adjuvant, preferably an AlPO ₄ gel, is present in the protein subunit vaccine according to the first or second aspect or any embodiment thereof at a dose of about 1-10 mg/dose, 5-15 mg/dose, 5-20 mg/dose, 10-20 mg/dose, 20-30 mg/dose, 30-40 mg/dose, 40-50 mg/dose, 50-60 mg/dose, 60-70 mg/dose, or 70-80 mg/dose. Preferably, the aluminium phosphate adjuvant, more preferably the AlPO ₄ gel, is formulated at a dose of 10 to 60 mg/dose, preferably about 10 mg/dose or 50 mg/dose.

With respect to immunostimulants, in embodiments, the total amount of at least one immunostimulant optionally comprised in the protein subunit vaccine according to the first or second aspect or any embodiment thereof is about 5 μg/dose, 10 μg/dose, 15 μg/dose, 20 μg/dose, 25 μg/dose, 30 μg/dose, 35 μg/dose, 40 μg/dose, 45 μg/dose, 50 μg/dose, 60 μg/dose, 70 μg/dose, 80/dose, 90/dose, 100/dose. In a preferred embodiment, the total amount of immunostimulant per dose ranges from 1 to 100 μg, from 10 to 90 μg, from 20 to 80 μg, from 20 to 70 μg, preferably from 5 to 60 μg, or any value contained within these ranges. More preferably, the total amount of immunostimulant is 10 μg or 50 μg. In another preferred embodiment, the immunostimulant is selected from the group consisting of MPLA or QS-21, present in an amount of about 1 μg per dose, 2 μg per dose, 3 μg per dose, 4 μg per dose, 5 μg per dose, 6 μg per dose, 7 μg per dose, 8 μg per dose, 9 μg per dose, 10 μg per dose, 15 μg per dose, 20 μg per dose, 25 μg per dose, 30 μg per dose, 35 μg per dose, 40 μg per dose, 45 μg per dose, 50 μg per dose, 60 μg per dose, 70 μg per dose, 80 μg per dose, 90 μg per dose, 100 μg per dose. Preferably, the at least one immunostimulant is MPLA or QS-21 in a dose range of 1 to 100 μg per dose, 5 to 60 μg per dose, 10 to 90 μg per dose, 20 to 80 μg per dose, 20 to 70 μg per dose, preferably 5 to 60 μg per dose, or any value comprised in these ranges. More preferably, the total amount of MPLA or QS-21 is 10 μg per dose or 50 μg per dose.

In other embodiments, any of the above classes of immunostimulants may be used in combination with each other or with other adjuvants and antigens. In embodiments, the protein subunit vaccine comprises at least two immunostimulants. Preferably, the protein subunit vaccine comprises two immunostimulants MPLA and QS-21, which are mixed together and administered simultaneously, or separately in different containers but simultaneously or sequentially. In a preferred embodiment, the total amount of each dose of MPLA and QS-21 is about 1 μg, preferably 2μg、3μg、4μg、5μg、6μg、7μg、8μg、9μg、10μg、15μg、20μg、25μg、30μg、35μg、40μg、45μg、50μg、60μg、70μg、80μg、90μg、100μg, wherein the ratio of the two immunostimulants is 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9 or 1:10 in any order (i.e. independently, such as QS-21:MPLA or MPLA:QS-21). Preferably, the ratio between the two immunostimulants is 1:1. In embodiments, their total amounts range from 5 to 30 μg per dose, preferably from 5 to 25 μg per dose. More preferably, they are each present in a total amount of 10 μg per dose, 5 μg from QS-21 and 5 μg from MPLA. In another embodiment, the total amount is a 50 μg dose, where 25 μg is from QS-21 and the other 25 μg is from MPLA.

In the following paragraphs, we will point out a non-exhaustive list of further preferred combinations of at least one antigen, at least one adjuvant and optionally at least one immunostimulant in a protein subunit vaccine according to the first or second aspect or any embodiment thereof. From the following, when referring to the term "RBD antigen", it is understood that the term encompasses any "monomeric RBD antigen" or "dimeric RBD antigen", including "non-fusion RBD antigen" and "fusion RBD antigen". From the following, when referring to the term "S1 antigen", it is understood that the term encompasses any "monomeric S1 antigen" or "dimeric S1 antigen", including "non-fused S1 antigen" and "fused S1 antigen".

In an embodiment, the protein subunit vaccine according to the first aspect or any embodiment thereof comprises or consists of at least RBD antigen and at least one adjuvant, wherein at least one adjuvant is mf59c.1. In a further embodiment, the protein subunit vaccine comprises or consists of at least the S1 subunit antigen and at least one adjuvant, wherein the at least one adjuvant is mf59c.1. In embodiments, the protein subunit vaccine consists of at least RBD antigen and mf59c.1 as an adjuvant. In another embodiment, the protein subunit vaccine consists of at least the S1 subunit antigen and mf59c.1 as an adjuvant.

In an embodiment, the protein subunit vaccine according to the first aspect or any embodiment thereof comprises or at least consists of RBD antigen and at least one adjuvant, wherein at least one adjuvant is a specific squalene or an oil-in-water squalene adjuvant formulation. In a further embodiment, the protein subunit vaccine comprises or consists of at least the S1 subunit antigen and at least one adjuvant, wherein at least one adjuvant is a specific squalene or an oil-in-water adjuvant formulation of squalene. In embodiments, the protein subunit vaccine consists of at least the RBD antigen and a specific squalene or squalene oil-in-water adjuvant formulation. In another embodiment, the protein subunit vaccine consists of at least the S1 subunit antigen and a specific squalene or squalene oil-in-water adjuvant formulation.

In an embodiment, the protein subunit vaccine according to the first aspect or any embodiment thereof comprises or consists of at least RBD antigen and at least one adjuvant, wherein at least one adjuvant is AlPO ₄ gel. In a further embodiment, the protein subunit vaccine comprises or consists of at least the S1 subunit antigen and at least one adjuvant, wherein the at least one adjuvant is AlPO ₄ gel. In an embodiment, the protein subunit vaccine consists of at least RBD antigen and AlPO ₄ gel as an adjuvant. In another embodiment, the protein subunit vaccine consists of at least the S1 subunit antigen and AlPO ₄ gel as an adjuvant.

In a preferred embodiment, the protein subunit vaccine according to the first aspect comprises or consists of: at least one S1 subunit of spike protein of variant SARS-CoV-2, 5 to 50 μg per dose, preferably 10 μg per dose, 20 μg per dose or 40 μg per dose, and

I) MF59C.1 as adjuvant in a ratio (v/v) of 40-60% adjuvant and 60-40% antigen, preferably 50% adjuvant and 50% antigen, or

Ii) AlPO ₄ gel as adjuvant, 10-60mg per dose, preferably 10mg per dose or 50mg per dose.

I) Specific squalene or squalene oil-in-water adjuvant formulation, in a ratio (v/v) of 40-60% adjuvant and 60-40% antigen, preferably 50% adjuvant and 50% antigen, or

In a preferred embodiment, the protein subunit vaccine according to the first aspect or any embodiment thereof comprises: at least one Receptor Binding Domain (RBD) antigen of spike protein of variant SARS-CoV-2, 5 to 50 μg per dose, preferably 10 μg per dose, 20 μg per dose or 40 μg per dose, and

I) Adjuvants comprising squalene 10 to 60mg/ml per dose, polysorbate 80 1 to 6mg/ml per dose, sorbitan trioleate 1 to 6mg/ml per dose, sodium citrate 0.5 to 6mg/ml per dose, and citric acid 0.01 to 0.5mg/ml per dose, or

According to the first aspect or any embodiment thereof, the at least one immunostimulant may be combined with at least one adjuvant as described above. In a preferred embodiment of the first aspect or any embodiment thereof, the protein subunit vaccine comprises or consists of mf59c.1 as an adjuvant and MPLA as an immunostimulant. In a preferred embodiment, the protein subunit vaccine comprises or consists of at least one RBD antigen, mf59c.1 and MPLA. In another preferred embodiment, the protein subunit vaccine comprises or consists of at least one S1 subunit antigen, mf59c.1 and MPLA. In a preferred embodiment, the protein subunit vaccine comprises or consists of at least one RBD antigen, MF59C.1 and QS-21. In another preferred embodiment, the protein subunit vaccine comprises or consists of at least one S1 subunit antigen, MF59C.1 and QS-21.

According to the first aspect or any embodiment thereof, the at least one immunostimulant may be combined with at least one adjuvant as described above. In a preferred embodiment of the first aspect or any embodiment thereof, the protein subunit vaccine comprises or consists of a specific squalene or squalene oil-in-water adjuvant formulation and MPLA as an immunostimulant. In a preferred embodiment, the protein subunit vaccine comprises or consists of at least one RBD antigen, a specific squalene or squalene oil-in-water adjuvant formulation and MPLA. In another preferred embodiment, the protein subunit vaccine comprises or consists of at least one S1 subunit antigen, a specific squalene or squalene oil-in-water adjuvant formulation and MPLA. In a preferred embodiment, the protein subunit vaccine comprises or consists of at least one RBD antigen, a specific squalene or an oil-in-water adjuvant formulation of squalene and QS-21. In another preferred embodiment, the protein subunit vaccine comprises or consists of at least one S1 subunit antigen, a specific squalene or squalene oil-in-water adjuvant formulation and QS-21.

In a preferred embodiment of the first aspect or any embodiment thereof, the protein subunit vaccine comprises or consists of AlPO ₄ gel as an adjuvant and MPLA as an immunostimulant. In a preferred embodiment, the protein subunit vaccine comprises or consists of at least one RBD antigen, alPO ₄ gel and MPLA. In another preferred embodiment, the protein subunit vaccine comprises or consists of at least one S1 subunit antigen, alPO ₄ gel and MPLA. In a preferred embodiment, the protein subunit vaccine comprises or consists of at least one RBD antigen, alPO ₄ gel and QS-21. In another preferred embodiment, the protein subunit vaccine comprises or consists of at least one S1 subunit antigen, A1PO ₄ gel and QS-21.

In a preferred embodiment of the first aspect or any embodiment thereof, the protein subunit vaccine comprises or consists of at least one RBD antigen, mf59c.1, MPLA and QS-21. In another preferred embodiment, the protein subunit vaccine comprises or consists of at least one S1 subunit antigen, MF59C.1, MPLA and QS-21. In a preferred embodiment, the protein subunit vaccine comprises or consists of at least one RBD antigen, alPO ₄ gel, MPLA and QS-21. In another preferred embodiment, the protein subunit vaccine comprises or consists of at least one S1 subunit antigen, alPO ₄ gel, MPLA and QS-21.

In a preferred embodiment of the first aspect or any embodiment thereof, the protein subunit vaccine comprises or consists of at least one RBD antigen, a specific squalene or squalene oil-in-water adjuvant formulation, MPLA and QS-21. In another preferred embodiment, the protein subunit vaccine comprises or consists of at least one S1 subunit antigen, a specific squalene or squalene oil-in-water adjuvant formulation, MPLA and QS-21. In a preferred embodiment, the protein subunit vaccine comprises or consists of at least one RBD antigen, alPO ₄ gel, MPLA and QS-21. In another preferred embodiment, the protein subunit vaccine comprises or consists of at least one S1 subunit antigen, alPO ₄ gel, MPLA and QS-21.

In a preferred embodiment of the first aspect or any embodiment thereof, the protein subunit vaccine comprises: at least one S1 subunit of spike protein of variant SARS-CoV-2, 5 to 50 μg per dose, preferably 10 μg per dose, 20 μg per dose or 40 μg per dose, and

And wherein the protein subunit vaccine further comprises at least one immunostimulant, wherein the at least one immunostimulant consists of:

a) 5-60. Mu.g/dose, preferably 10. Mu.g/dose or 50. Mu.g/dose of MPLA, or

B) 5-60. Mu.g/dose, preferably 10. Mu.g/dose or 50. Mu.g/dose QS-21, or,

C) 5-30. Mu.g/dose, preferably 5. Mu.g/dose or 25. Mu.g/dose of MPLA, and 5-30. Mu.g/dose, preferably 5. Mu.g/dose or 25. Mu.g/dose of QS-21.

Ii) AlPO ₄ gel as adjuvant, 10-60mg per dose, preferably 10mg per dose or 50mg per dose,

a) 5-60. Mu.g/dose, preferably 10. Mu.g/dose or 50. Mu.g/dose of MPLA, or

B) 5-60. Mu.g/dose, preferably 10. Mu.g/dose or 50. Mu.g/dose QS-21, or,

In a preferred embodiment of the first aspect or any embodiment thereof, the protein subunit vaccine comprises: at least one RBD antigen of spike protein of variant SARS-CoV-2, 5 to 50 μg per dose, preferably 10 μg per dose, 20 μg per dose or 40 μg per dose, and

a) 5-60. Mu.g/dose, preferably 10. Mu.g/dose or 50. Mu.g/dose of MPLA, or

B) 5-60. Mu.g/dose, preferably 10. Mu.g/dose or 50. Mu.g/dose QS-21, or,

a) 5-60. Mu.g/dose, preferably 10. Mu.g/dose or 50. Mu.g/dose of MPLA, or

B) 5-60. Mu.g/dose, preferably 10. Mu.g/dose or 50. Mu.g/dose QS-21, or,

I) Adjuvants comprising about 10 to 60mg/ml squalene per dose, 1 to 6mg/ml polysorbate 80 per dose, 1 to 6mg/ml sorbitan trioleate per dose, 0.5 to 6mg/ml sodium citrate per dose, and 0.01 to 0.5mg/ml citric acid per dose, or

a) 5-60. Mu.g/dose, preferably 10. Mu.g/dose or 50. Mu.g/dose of MPLA, or

B) 5-60. Mu.g/dose, preferably 10. Mu.g/dose or 50. Mu.g/dose QS-21, or,

a) 5-60. Mu.g/dose, preferably 10. Mu.g/dose or 50. Mu.g/dose of MPLA, or

B) 5-60. Mu.g/dose, preferably 10. Mu.g/dose or 50. Mu.g/dose QS-21, or,

In an embodiment, the protein subunit vaccine according to the second aspect or any embodiment thereof comprises or consists of at least RBD antigen and at least one adjuvant, wherein at least one adjuvant is mf59c.1. In a further embodiment, the protein subunit vaccine comprises or consists of at least the S1 subunit antigen and at least one adjuvant, wherein the at least one adjuvant is mf59c.1. In embodiments, the protein subunit vaccine consists of at least RBD antigen and mf59c.1 as an adjuvant. In another embodiment, the protein subunit vaccine consists of at least the S1 subunit antigen and mf59c.1 as an adjuvant.

In an embodiment, the protein subunit vaccine according to the second aspect or any embodiment thereof comprises or consists of at least RBD antigen and at least one adjuvant, wherein at least one adjuvant is a specific squalene or an oil-in-water squalene adjuvant formulation. In a further embodiment, the protein subunit vaccine comprises or consists of at least the S1 subunit antigen and at least one adjuvant, wherein at least one adjuvant is a specific squalene or an oil-in-water adjuvant formulation of squalene. In embodiments, the protein subunit vaccine consists of at least the RBD antigen and a specific squalene or squalene oil-in-water adjuvant formulation. In another embodiment, the protein subunit vaccine consists of at least the S1 subunit antigen and a specific squalene or squalene oil-in-water adjuvant formulation.

In an embodiment, the protein subunit vaccine according to the second aspect or any embodiment thereof comprises or consists of at least RBD antigen and at least one adjuvant, wherein at least one adjuvant is AlPO ₄ gel. In a further embodiment, the protein subunit vaccine comprises or consists of at least the S1 subunit antigen and at least one adjuvant, wherein the at least one adjuvant is AlPO ₄ gel. In an embodiment, the protein subunit vaccine consists of at least RBD antigen and AlPO ₄ gel as an adjuvant. In another embodiment, the protein subunit vaccine consists of at least the S1 subunit antigen and AlPO ₄ gel as an adjuvant.

In a preferred embodiment, the protein subunit vaccine according to the second aspect comprises or consists of: at least one S1 subunit of spike protein of variant SARS-CoV-2, 5 to 50 μg per dose, preferably 10 μg per dose, 20 μg per dose or 40 μg per dose, and

In a preferred embodiment, the protein subunit vaccine according to the second aspect or any embodiment thereof comprises: at least one Receptor Binding Domain (RBD) antigen of spike protein of variant SARS-CoV-2, 5 to 50 μg per dose, preferably 10 μg per dose, 20 μg per dose or 40 μg per dose, and

According to the second aspect or any embodiment thereof, the at least one immunostimulant may be combined with at least one adjuvant as described above. In a preferred embodiment of the second aspect or any embodiment thereof, the protein subunit vaccine comprises or consists of mf59c.1 as an adjuvant and MPLA as an immunostimulant. In a preferred embodiment, the protein subunit vaccine comprises or consists of at least one RBD antigen, mf59c.1 and MPLA. In another preferred embodiment, the protein subunit vaccine comprises or consists of at least one S1 subunit antigen, mf59c.1 and MPLA. In a preferred embodiment, the protein subunit vaccine comprises or consists of at least one RBD antigen, MF59C.1 and QS-21. In another preferred embodiment, the protein subunit vaccine comprises or consists of at least one S1 subunit antigen, MF59C.1 and QS-21.

According to the second aspect or any embodiment thereof, the at least one immunostimulant may be combined with at least one adjuvant as described above. In a preferred embodiment of the second aspect or any embodiment thereof, the protein subunit vaccine comprises or consists of a specific squalene or squalene oil-in-water adjuvant formulation and MPLA as an immunostimulant. In a preferred embodiment, the protein subunit vaccine comprises or consists of at least one RBD antigen, a specific squalene or squalene oil-in-water adjuvant formulation and MPLA. In another preferred embodiment, the protein subunit vaccine comprises or consists of at least one S1 subunit antigen, a specific squalene or squalene oil-in-water adjuvant formulation and MPLA. In a preferred embodiment, the protein subunit vaccine comprises or consists of at least one RBD antigen, a specific squalene or an oil-in-water adjuvant formulation of squalene and QS-21. In another preferred embodiment, the protein subunit vaccine comprises or consists of at least one S1 subunit antigen, a specific squalene or squalene oil-in-water adjuvant formulation and QS-21.

In a preferred embodiment of the first aspect or any embodiment thereof, the protein subunit vaccine comprises or consists of AlPO ₄ gel as an adjuvant and MPLA as an immunostimulant. In a preferred embodiment, the protein subunit vaccine comprises or consists of at least one RBD antigen, alPO ₄₄ gel and MPLA. In another preferred embodiment, the protein subunit vaccine comprises or consists of at least one S1 subunit antigen, alPO ₄ gel and MPLA. In a preferred embodiment, the protein subunit vaccine comprises or consists of at least one RBD antigen, alPO ₄ gel and QS-21. In another preferred embodiment, the protein subunit vaccine comprises or consists of at least one S1 subunit antigen, A1PO ₄ gel and QS-21.

In a preferred embodiment of the second aspect or any embodiment thereof, the protein subunit vaccine comprises or consists of at least one RBD antigen, mf59c.1, MPLA and QS-21. In another preferred embodiment, the protein subunit vaccine comprises or consists of at least one S1 subunit antigen, MF59C.1, MPLA and QS-21. In a preferred embodiment, the protein subunit vaccine comprises or consists of at least one RBD antigen, alPO ₄ gel, MPLA and QS-21. In another preferred embodiment, the protein subunit vaccine comprises or consists of at least one S1 subunit antigen, alPO ₄ gel, MPLA and QS-21.

In a preferred embodiment of the second aspect or any embodiment thereof, the protein subunit vaccine comprises or consists of at least one RBD antigen, a specific squalene or squalene oil-in-water adjuvant formulation, MPLA and QS-21. In another preferred embodiment, the protein subunit vaccine comprises or consists of at least one S1 subunit antigen, a specific squalene or squalene oil-in-water adjuvant formulation, MPLA and QS-21. In a preferred embodiment, the protein subunit vaccine comprises or consists of at least one RBD antigen, alPO ₄ gel, MPLA and QS-21. In another preferred embodiment, the protein subunit vaccine comprises or consists of at least one S1 subunit antigen, alPO ₄ gel, MPLA and QS-21.

In a preferred embodiment of the second aspect or any embodiment thereof, the protein subunit vaccine comprises: at least one S1 subunit of spike protein of variant SARS-CoV-2, 5 to 50 μg per dose, preferably 10 μg per dose, 20 μg per dose or 40 μg per dose, and

a) 5-60. Mu.g/dose, preferably 10. Mu.g/dose or 50. Mu.g/dose of MPLA, or

B) 5-60. Mu.g/dose, preferably 10. Mu.g/dose or 50. Mu.g/dose QS-21, or,

a) 5-60. Mu.g/dose, preferably 10. Mu.g/dose or 50. Mu.g/dose of MPLA, or

B) 5-60. Mu.g/dose, preferably 10. Mu.g/dose or 50. Mu.g/dose QS-21, or,

In a preferred embodiment of the second aspect or any embodiment thereof, the protein subunit vaccine comprises: at least one RBD antigen of spike protein of variant SARS-CoV-2, 5 to 50 μg per dose, preferably 10 μg per dose, 20 μg per dose or 40 μg per dose, and

a) 5-60. Mu.g/dose, preferably 10. Mu.g/dose or 50. Mu.g/dose of MPLA, or

B) 5-60. Mu.g/dose, preferably 10. Mu.g/dose or 50. Mu.g/dose QS-21, or,

a) 5-60. Mu.g/dose, preferably 10. Mu.g/dose or 50. Mu.g/dose of MPLA, or

B) 5-60. Mu.g/dose, preferably 10. Mu.g/dose or 50. Mu.g/dose QS-21, or,

a) 5-60. Mu.g/dose, preferably 10. Mu.g/dose or 50. Mu.g/dose of MPLA, or

B) 5-60. Mu.g/dose, preferably 10. Mu.g/dose or 50. Mu.g/dose QS-21, or,

a) 5-60. Mu.g/dose, preferably 10. Mu.g/dose or 50. Mu.g/dose of MPLA, or

B) 5-60. Mu.g/dose, preferably 10. Mu.g/dose or 50. Mu.g/dose QS-21, or,

The vaccine described herein in the first or second aspect or any embodiment thereof may generally comprise one or more "pharmaceutically acceptable excipients or vehicles", such as water, saline, glycerol, ethanol, and the like. In addition, auxiliary substances, such as wetting or emulsifying agents, pH buffering substances, and the like, may be present in such vehicles. Typically, protein subunit vaccines are prepared as injectables, either as liquid solutions or suspensions; solid forms suitable for dissolution in or suspension in a liquid vehicle prior to injection may also be prepared. Optionally present is a carrier, which is a molecule that does not itself induce the production of antibodies harmful to the individual receiving the composition. Suitable carriers are typically large, slowly metabolizing macromolecules such as proteins, polysaccharides, polylactic acids, polyglycolic acids, polymeric amino acids, amino acid copolymers, lipid aggregates (e.g. oil droplets or liposomes) and inactivated virus particles. Such vectors are well known to those of ordinary skill in the art. In another embodiment, the composition may be delivered in vesicles, for example in liposomes. Methods of preparing pharmaceutical formulations are well known to those skilled in the art, for example, as described in the leimington manual The Science and Practice of Pharmacy,20^th Ed.,Lippincott Williams&Wilkins,Philadelphia,2000[ISBN:0-683-306472].

Administration protocol

The route of administration and schedule can be selected and optimized in a known manner by the person skilled in the art.

The route of administration may be systemic or local. Many methods of administration may be used, including but not limited to: oral, parenteral (e.g., intradermal, intramuscular, intravenous, and subcutaneous), transdermal, transmucosal (e.g., intranasal and oral or pulmonary routes or by pessary), pulmonary delivery, suppository, scarification (laceration of the skin surface, e.g., using a bifurcated needle). In particular embodiments, the protein subunit vaccine of the invention is administered parenterally via an intramuscular, intravenous, intradermal, or subcutaneous route, or via a transdermal route. Preferably, the protein subunit vaccine is administered by intramuscular or subcutaneous routes. More preferably, the protein subunit vaccine is administered intramuscularly in a volume of about 0.10ml to 10ml, or 0.10ml to 1 ml. Preferably, the protein subunit vaccine is administered in a volume of 0.25ml to 1.0 ml. More preferably, the protein subunit vaccine is administered in a volume of about 0.1 ml. Even more preferably, the protein subunit vaccine is administered in a volume of about 0.5 ml.

In certain embodiments, the protein subunit vaccine provided in the first or second aspect or any embodiment thereof is administered to a subject according to a vaccine regimen or schedule comprising a single dose or alternatively multiple (i.e., 2,3, 4, etc.) doses. Preferably, the protein subunit vaccine is administered to a subject in need thereof in two doses. In certain embodiments, the protein subunit vaccine is administered to a subject in need thereof according to a schedule comprising a first dose (priming) and a second dose (boosting).

As used herein, priming refers to any method by which the first administration of a protein subunit vaccine described herein allows for the generation of an immune response to a target antigen. Once the subject is primed, a second administration of a second vaccine induces a second immune response that is stronger or longer in duration than that achieved by the first immunization. Prime encompasses regimens that include a first single dose or multiple doses. In embodiments, the first infection with SARS-CoV-2 can be considered priming and the first administration of a single dose of the protein subunit vaccine as a booster.

The time interval between priming and boosting administration may be hours, days, weeks, months or years. In other embodiments, the protein subunit vaccines described herein can be administered as a booster to increase the immune response achieved after priming of a subject. The boosting composition is typically administered one or more times several weeks or months after administration of the priming composition, e.g., about 1 or 2 weeks or 3 weeks, or 4 weeks, or 6 weeks, or 8 weeks, or 16 weeks, or 20 weeks, or 24 weeks, or 28 weeks or 32 weeks, or one to two years. Preferably, the booster vaccination is administered 1-12 weeks or 2-12 weeks after priming, more preferably 1,2, 3 or 4 weeks after priming. In a preferred embodiment, the second or booster dose is administered one week, preferably two weeks, three weeks or four weeks after the first dose or priming. In further embodiments, the second dose is performed at least 2 weeks or at least 4 weeks after priming. In another preferred embodiment, the second dose is administered about 4-12 weeks or 4-8 weeks after priming.

In addition, the third or subsequent booster dose may be administered after the second dose and within three months to two years after the initial administration, or even longer, preferably 4 to 6 months, or 6 months to one year. The third dose may optionally be administered when no specific immunoglobulin or low levels of specific immunoglobulin are detected in the serum and/or urine or mucosal secretions of the subject after the second dose.

In embodiments, the protein subunit vaccines provided herein can be administered as a priming agent and as a subsequent booster. In other embodiments, the protein subunit vaccine may be used in combination with other vaccines for priming and/or boosting, such as mRNA vaccines, plasmid vaccines, carrier vaccines, other protein subunit vaccines, or combinations thereof.

In embodiments, the protein subunit vaccine provided in the first or second aspect or any embodiment thereof may be administered as a booster in a single dose to a subject that has been previously vaccinated with the protein subunit vaccine provided herein or other vaccine. In this case, the protein subunit vaccine provided herein is administered one, two, three, four, five, six, seven, eight, nine, ten, or more than ten weeks, months, or years after the previous vaccine has been administered to the subject.

In some embodiments, the protein subunit vaccine provided herein is administered to a subject at any dose, route, or schedule defined herein prior to exposure to the SARS-CoV-2 virus, e.g., 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 13 hours, 14 hours, 15 hours, 16 hours, 17 hours, 18 hours, 19 hours, 20 hours, 21 hours, 22 hours, 23 hours, or 24 hours or 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, or 7 days prior to exposure to the SARS-CoV-2 virus. In certain embodiments, the protein subunit vaccine provided herein is administered to a subject at any dose, route or schedule defined herein after exposure to SARS-CoV-2 virus, e.g., 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 13 hours, 14 hours, 15 hours, 16 hours, 17 hours, 18 hours, 19 hours, 20 hours, 21 hours, 22 hours, 23 hours or 24 hours or 1 day, 2 days, 3 days, 4 days, 5 days, 6 days or 7 days, or 1 week, 2 weeks, 3 weeks, or 4 weeks, or 6 weeks, or 8 weeks, or 16 weeks, or 20 weeks, or 24 weeks, or 28 weeks, or 32 weeks after exposure to SARS-CoV-2 virus.

Kit for detecting a substance in a sample

Also provided herein are kits comprising the protein subunit vaccines of the first or second aspects or any embodiment thereof. Thus, a third aspect of the invention relates to a kit comprising one, preferably two or more doses of a protein subunit vaccine as defined in the first or second aspect of the invention or any embodiment thereof. Thus, the kit may comprise at least one antigen, at least one adjuvant and optionally at least one immunostimulant as defined in the first or second aspect or any embodiment thereof. The components of the protein subunit vaccine may be provided separately, i.e. in separate containers, or mixed, i.e. all together in one or more containers.

Thus, in embodiments, the kit may comprise one or more containers or vials of the protein subunit vaccine of the invention, or one or more containers or vials of the protein subunit vaccine, and instructions for administration to a subject at risk of SARS-CoV-2 infection. In certain embodiments, the instructions instruct to administer the protein subunit vaccine of the invention to a subject in a single dose or in multiple doses (i.e., 2,3, 4, etc.) doses, as defined in the administration schedule section above. In certain embodiments, the instructions indicate that the protein subunit vaccine of the invention is administered to a subject for primary or non-primary administration in a first (priming) and subsequent (boosting) administration. Preferably, the kit comprises at least two vials for priming/boosting comprising a first vaccination or dose ("priming") of the protein subunit vaccine of the invention in a first vial/container and at least a second and/or third and/or further vaccination or dose ("boosting") of the protein subunit vaccine of the invention in a second and/or further vial/container.

Preferably, the kit comprises an immunologically effective amount of a protein subunit vaccine according to the first or second aspect of the invention or any embodiment thereof in a first vial or container for a first administration or first dose (priming) and in a second vial or container for a second administration or second dose (boosting).

In another embodiment of the second aspect of the invention, any of the kits mentioned herein may comprise a third, fourth or further vial or container for a third, fourth or further administration, said vial or container comprising a protein subunit vaccine specified throughout the present invention.

In a further preferred embodiment, the protein subunit vaccine and kit provided in any of the preceding aspects is used to generate an immune response against at least one variant of the SARS-CoV-2 virus.

In another preferred embodiment, the protein subunit vaccine and kit provided in any of the preceding aspects are used to generate a protective immune response against at least one variant of the SARS-CoV-2 virus.

Method and use of protein subunit vaccine

In a fourth aspect, the invention also provides a method for immunizing a subject against at least one variant of the SARS-CoV-2 virus using the protein subunit vaccine and kit as described in the first, second and third aspects of the invention or any embodiment thereof. The fourth aspect also relates to the use of the protein subunit vaccine and kit as described in the first, second and third aspects or any embodiment thereof for generating an immunogenic and/or protective immune response against at least one variant of SARS-CoV-2 virus in a subject in need thereof. Preferably, the fourth aspect relates to the use of a protein subunit vaccine and kit as described in the first, second and third aspects or any embodiment thereof, wherein the variants are selected from the group of related Variants (VOCs) described in the center for disease control and prevention (CDC), for generating an immunogenic and/or protective immune response against at least one different variant of the SARS-CoV-2 virus in a subject in need thereof. Preferably, the fourth aspect relates to the use of a protein subunit vaccine and kit as described in the first, second and third aspects or any embodiment thereof, for generating an immunogenic and/or protective immune response against at least one different variant of SARS-CoV-2 virus in a subject in need thereof, wherein the variant is selected from the group comprising or consisting of: wuhan-Hu-1 pneumovirus isolates (GenBank accession number: MN 908947), linage B.1.1.28 (Brazil variant), linage B.1.351 (south Africa variant), linage B.1.427 or Linage B.1.429 (California variant), linage B.1.617 (Indian variant), linage B.1.1.7 (British variant), linage B.1.617.2 or G/478K.V1 (Deltavariant) or Linage B.1.1.529 or GR/484A (Omik Rong Bianti) or any combination thereof. Preferably, the fourth aspect relates to the use of a protein subunit vaccine and kit as described in the first, second and third aspects or any embodiment thereof, for generating an immunogenic and/or protective immune response against at least two different variants of SARS-CoV-2 virus in a subject in need thereof. Preferably, the fourth aspect relates to the use of a protein subunit vaccine and kit as described in the first, second and third aspects or any embodiment thereof, wherein the variants are selected from the group of related Variants (VOCs) described in the center of disease control and prevention (CDC), for generating an immunogenic and/or protective immune response against at least two different variants of SARS-CoV-2 virus in a subject in need thereof. Preferably, the fourth aspect relates to the use of a protein subunit vaccine and kit as described in the first, second and third aspects or any embodiment thereof, for generating an immunogenic and/or protective immune response against at least two different variants of SARS-CoV-2 virus in a subject in need thereof, wherein the variants are selected from the group comprising or consisting of: wuhan-Hu-1 pneumovirus isolates (GenBank accession number: MN 908947), linage B.1.1.28 (Brazil variant), linage B.1.351 (south Africa variant), linage B.1.427 or Linage B.1.429 (California variant), linage B.1.617 (Indian variant), linage B.1.1.7 (British variant), linage B.1.617.2 or G/478K.V1 (Deltavariant) or Linage B.1.1.529 or GR/484A (Omik Rong Bianti) or any combination thereof.

Also included is the use of the protein subunit vaccine and kit as described above for the manufacture of a medicament or protein subunit vaccine for immunizing a subject, in particular for the manufacture of a medicament or vaccine for treating and/or preventing a disease caused by SARS-CoV-2 in a subject, wherein the SARS-CoV-2 disease is caused by at least one variant of the SARS-CoV-2 virus. Also provided herein are the protein subunit vaccines and kits according to any embodiment herein for eliciting or enhancing an immune response against SARS-CoV-2 infection, wherein the protein subunit vaccine is administered once, twice, three times or four times. Preferably, the protein subunit vaccine is administered twice. Also provided herein are the protein subunit vaccines and kits according to any of the embodiments herein for boosting an immune response against SARS-CoV-2 infection in a subject that has been previously vaccinated against SARS-CoV-2, wherein the protein subunit vaccine is administered in a single dose.

Thus, the fourth aspect of the invention also provides a method of generating an immunogenic and/or protective immune response against at least one variant of the SARS-CoV-2 virus in a subject in need thereof, preferably a human subject, the method comprising administering to the subject a protein subunit vaccine as described in the first or second aspect of the invention or any embodiment thereof. The terms "immunogenic and protective immune response", "protective immunity" or "protective immune response" have been defined above.

In certain embodiments, the subject is a mammalian or avian species. The subject may be humans, companion animals such as dogs and cats, domestic animals such as chickens and geese, horses, cattle and sheep, ferrets, porcine species such as pigs, piglets, sows or backup sows, and zoo mammals such as non-human primates, felines, canines and bovids.

In embodiments, the method comprises administering at least one dose of the protein subunit vaccine of the invention to a subject, preferably the subject is a human.

In certain embodiments of the fourth aspect of the invention, the subject is a human. In certain embodiments, the subject is a neonate (up to 2 months), infant (birth to 2 years), child (2 years to 14 years), adolescent (15 years to 18 years), adult (over 18 years), or elderly (about 65 years or over). In certain embodiments, the adult is immunocompromised.

In a further embodiment, the protein subunit vaccine or kit as defined in the first, second or third aspect of the invention or any embodiment thereof is used to generate an immunogenic and/or protective immune response against at least one variant of SARS-CoV-2 in a subject.

In a further embodiment, the protein subunit vaccine or kit as defined in the first, second or third aspect of the invention or any embodiment thereof is used to generate an immunogenic and/or protective immune response against at least two different variants of SARS-CoV-2 in a subject.

Sequence listing

SEQ ID NO:1: RBD monomer: amino acid residues 319 to 541 of SARS-CoV-2 spike protein (Wuhan-Hu-1 variant):

RVQPTESIVRFPNITNLCPFGEVFNATRFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNGVEGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKCVNF

SEQ ID NO:2: s1 subunit monomer: amino acid residues 13 to 685 of SARS-CoV-2 spike protein (Wuhan-Hu-1 variant):

SQCVNLTTRTQLPPAYTNSFTRGVYYPDKVFRSSVLHSTQDLFLPFFSNVTWFHAIHVSGTNGTKRFDNPVLPFNDGVYFASTEKSNIIRGWIFGTTLDSKTQSLLIVNNATNVVIKVCEFQFCNDPFLGVYYHKNNKSWMESEFRVYSSANNCTFEYVSQPFLMDLEGKQGNFKNLREFVFKNIDGYFKIYSKHTPINLVRDLPQGFSALEPLVDLPIGINITRFQTLLALHRSYLTPGDSSSGWTAGAAAYYVGYLQPRTFLLKYNENGTITDAVDCALDPLSETKCTLKSFTVEKGIYQTSNFRVQPTESIVRFPNITNLCPFGEVFNATRFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNGVEGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKCVNFNFNGLTGTGVLTESNKKFLPFQQFGRDIADTTDAVRDPQTLEILDITPCSFGGVSVITPGTNTSNQVAVLYQDVNCTEVPVAIHADQLTPTWRVYSTGSNVFQTRAGCLIGAEHVNNSYECDIPIGAGICASYQTQTNSPRRAR

SEQ ID NO:3: amino acid residues 319 to 537 of the SARS-CoV-2 spike protein RBD monomer of the B1.1.7 variant:

RVQPTESIVRFPNITNLCPFGEVFNATRFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNGVEGFNCYFPLQSYGFQPTYGVGYQPYRVVVLSFELLHAPATVCGPKKSTNLVKNK

SEQ ID NO:4: amino acid residues 319 to 537 of the SARS-CoV-2 spike protein RBD monomer of the variant b.1.351:

RVQPTESIVRFPNITNLCPFGEVFNATRFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGNIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNGVKGFNCYFPLQSYGFQPTYGVGYQPYRVVVLSFELLHAPATVCGPKKSTNLVKNK

SEQ ID NO:5: fusion dimeric RBD variant SARS-CoV-2 antigen comprising an amino acid sequence comprising a first monomer derived from the B.1.351 variant (positions 319 to 537 of the RBD of the SARS-CoV-2 spike protein) and a second monomer derived from the B.1.1.7 variant (positions 319 to 537 of the RBD of the SARS-CoV-2 spike protein) )：RVQPTESIVRFPNITNLCPFGEVFNATRFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGNIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNGVKGFNCYFPLQSYGFQPTYGVGYQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKRVQPTESIVRFPNITNLCPFGEVFNATRFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNGVEGFNCYFPLQSYGFQPTYGVGYQPYRVVVLSFELLHAPATVCGPKKSTNLVKNK

SEQ ID NO:6: signal peptide:

MGWSCIILFLVATATGVHS

SEQ ID NO:7: a DNA sequence encoding a Kozak sequence, a signal peptide, a fusion dimeric RBD variant SARS-CoV-2 antigen (which is a tandem of nucleotide sequences encoding amino acids 319 to 537 of the RBD monomer of the b.1.351 variant and amino acids 319 to 537 of the RBD monomer of the b.1.1.7 variant), a histidine tag and a stop codon:

GCCACCATGGGCTGGTCCTGCATCATCCTGTTTCTGGTGGCTACCGCTACCGGCGTGCACAGTAGAGTGCAGCCTACCGAGTCTATCGTGCGGTTCCCCAACATCACCAACCTGTGTCCTTTCGGCGAGGTGTTCAACGCCACCAGATTCGCCTCTGTGTACGCCTGGAACCGGAAGCGGATCTCTAACTGCGTGGCCGACTACTCCGTGCTGTACAACTCCGCCTCCTTCAGCACCTTCAAGTGCTACGGCGTGTCCCCTACCAAGCTGAACGACCTGTGCTTCACCAACGTGTACGCCGACTCCTTCGTGATCAGAGGCGACGAAGTGCGGCAGATCGCTCCTGGACAGACCGGCAATATCGCCGACTACAACTACAAGCTGCCCGACGACTTCACCGGCTGTGTGATCGCTTGGAACTCCAACAACCTGGACTCCAAAGTCGGCGGCAACTACAATTACCTGTACCGGCTGTTCCGGAAGTCCAACCTGAAGCCTTTCGAGCGGGACATCTCCACCGAGATCTACCAGGCTGGCAGCACCCCTTGTAATGGCGTGAAGGGCTTCAACTGCTACTTCCCACTGCAGTCCTACGGCTTCCAGCCTACCTATGGCGTGGGCTACCAGCCTTACAGAGTGGTGGTGCTGTCCTTCGAGCTGCTGCATGCTCCTGCTACCGTGTGCGGCCCTAAGAAATCTACCAACCTGGTCAAGAACAAGCGGGTGCAGCCCACTGAGAGCATTGTGCGCTTCCCTAATATCACAAATCTGTGCCCCTTCGGGGAAGTCTTTAATGCTACCCGCTTCGCTTCCGTGTATGCTTGGAATAGAAAGCGGATCAGCAATTGCGTCGCCGATTACAGCGTCCTGTACAATAGCGCCAGCTTCTCCACCTTTAAGTGTTATGGCGTCAGCCCCACAAAGCTCAACGATCTCTGTTTTACCAATGTCTACGCCGATAGCTTTGTGATTCGCGGAGATGAAGTCCGCCAGATCGCACCAGGCCAGACTGGAAAGATCGCTGATTACAATTATAAGCTCCCTGATGATTTCACAGGATGCGTTATCGCCTGGAATAGCAACAACCTCGACAGCAAAGTTGGAGGGAATTACAACTACCTCTACCGCCTCTTCAGAAAGAGCAACCTCAAGCCATTTGAGAGAGACATCAGTACAGAAATCTATCAGGCCGGCTCTACCCCTTGCAACGGCGTCGAGGGGTTTAACTGTTACTTTCCCCTGCAATCTTATGGGTTTCAGCCCACATACGGCGTGGGGTATCAACCCTATCGCGTGGTGGTTCTGAGTTTCGAACTCCTGCACGCCCCAGCCACAGTGTGTGGCCCAAAAAAGAGCACCAATCTCGTTAAGAACAAGCACCATCACCATCACCATTAG

SEQ ID NO:8: DNA sequence encoding the fusion dimeric RBD variant SARS-CoV-2 antigen (which is a tandem of nucleotide sequences encoding amino acids 319 to 537 of the RBD monomer of the b.1.351 variant and amino acids 391 to 537 of the RBD monomer of the b.1.1.7 variant):

AGAGTGCAGCCTACCGAGTCTATCGTGCGGTTCCCCAACATCACCAACCTGTGTCCTTTCGGCGAGGTGTTCAACGCCACCAGATTCGCCTCTGTGTACGCCTGGAACCGGAAGCGGATCTCTAACTGCGTGGCCGACTACTCCGTGCTGTACAACTCCGCCTCCTTCAGCACCTTCAAGTGCTACGGCGTGTCCCCTACCAAGCTGAACGACCTGTGCTTCACCAACGTGTACGCCGACTCCTTCGTGATCAGAGGCGACGAAGTGCGGCAGATCGCTCCTGGACAGACCGGCAATATCGCCGACTACAACTACAAGCTGCCCGACGACTTCACCGGCTGTGTGATCGCTTGGAACTCCAACAACCTGGACTCCAAAGTCGGCGGCAACTACAATTACCTGTACCGGCTGTTCCGGAAGTCCAACCTGAAGCCTTTCGAGCGGGACATCTCCACCGAGATCTACCAGGCTGGCAGCACCCCTTGTAATGGCGTGAAGGGCTTCAACTGCTACTTCCCACTGCAGTCCTACGGCTTCCAGCCTACCTATGGCGTGGGCTACCAGCCTTACAGAGTGGTGGTGCTGTCCTTCGAGCTGCTGCATGCTCCTGCTACCGTGTGCGGCCCTAAGAAATCTACCAACCTGGTCAAGAACAAGCGGGTGCAGCCCACTGAGAGCATTGTGCGCTTCCCTAATATCACAAATCTGTGCCCCTTCGGGGAAGTCTTTAATGCTACCCGCTTCGCTTCCGTGTATGCTTGGAATAGAAAGCGGATCAGCAATTGCGTCGCCGATTACAGCGTCCTGTACAATAGCGCCAGCTTCTCCACCTTTAAGTGTTATGGCGTCAGCCCCACAAAGCTCAACGATCTCTGTTTTACCAATGTCTACGCCGATAGCTTTGTGATTCGCGGAGATGAAGTCCGCCAGATCGCACCAGGCCAGACTGGAAAGATCGCTGATTACAATTATAAGCTCCCTGATGATTTCACAGGATGCGTTATCGCCTGGAATAGCAACAACCTCGACAGCAAAGTTGGAGGGAATTACAACTACCTCTACCGCCTCTTCAGAAAGAGCAACCTCAAGCCATTTGAGAGAGACATCAGTACAGAAATCTATCAGGCCGGCTCTACCCCTTGCAACGGCGTCGAGGGGTTTAACTGTTACTTTCCCCTGCAATCTTATGGGTTTCAGCCCACATACGGCGTGGGGTATCAACCCTATCGCGTGGTGGTTCTGAGTTTCGAACTCCTGCACGCCCCAGCCACAGTGTGTGGCCCAAAAAAGAGCACCAATCTCGTTAAGAACAAG

SEQ ID NO:9: wuhan-Hu-1 SARS-CoV-2 spike protein sequence (UniProt No. P0DTC2):

MFVFLVLLPLVSSQCVNLTTRTQLPPAYTNSFTRGVYYPDKVFRSSVLHSTQDLFLPFFSNVTWFHAIHVSGTNGTKRFDNPVLPFNDGVYFASTEKSNIIRGWIFGTTLDSKTQSLLIVNNATNVVIKVCEFQFCNDPFLGVYYHKNNKSWMESEFRVYSSANNCTFEYVSQPFLMDLEGKQGNFKNLREFVFKNIDGYFKIYSKHTPINLVRDLPQGFSALEPLVDLPIGINITRFQTLLALHRSYLTPGDSSSGWTAGAAAYYVGYLQPRTFLLKYNENGTITDAVDCALDPLSETKCTLKSFTVEKGIYQTSNFRVQPTESIVRFPNITNLCPFGEVFNATRFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNGVEGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKCVNFNFNGLTGTGVLTESNKKFLPFQQFGRDIADTTDAVRDPQTLEILDITPCSFGGVSVITPGTNTSNQVAVLYQDVNCTEVPVAIHADQLTPTWRVYSTGSNVFQTRAGCLIGAEHVNNSYECDIPIGAGICASYQTQTNSPRRARSVASQSIIAYTMSLGAENSVAYSNNSIAIPTNFTISVTTEILPVSMTKTSVDCTMYICGDSTECSNLLLQYGSFCTQLNRALTGIAVEQDKNTQEVFAQVKQIYKTPPIKDFGGFNFSQILPDPSKPSKRSFIEDLLFNKVTLADAGFIKQYGDCLGDIAARDLICAQKFNGLTVLPPLLTDEMIAQYTSALLAGTITSGWTFGAGAALQIPFAMQMAYRFNGIGVTQNVLYENQKLIANQFNSAIGKIQDSLSSTASALGKLQDVVNQNAQALNTLVKQLSSNFGAISSVLNDILSRLDKVEAEVQIDRLITGRLQSLQTYVTQQLIRAAEIRASANLAATKMSECVLGQSKRVDFCGKGYHLMSFPQSAPHGVVFLHVTYVPAQEKNFTTAPAICHDGKAHFPREGVFVSNGTHWFVTQRNFYEPQIITTDNTFVSGNCDVVIGIVNNTVYDPLQPELDSFKEELDKYFKNHTSPDVDLGDISGINASVVNIQKEIDRLNEVAKNLNESLIDLQELGKYEQYIKWPWYIWLGFIAGLIAIVMVTIMLCCMTSCCSCLKGCCSCGSCCKFDEDDSEPVLKGVKLHYT

SEQ ID NO:10: a signal peptide.

MGWSLILLFLVAVATRVLS

Other embodiments of the invention will be apparent to those skilled in the art from consideration of the specification and practice of the invention disclosed herein. It is intended that the specification and examples be considered as exemplary only, with a true scope and spirit of the invention being indicated by the following claims.

Examples

Example 1: in vivo immunization studies in mice were tested for combinations of 2 different candidate antigens with 4 different adjuvants.

This study evaluated different protein subunit candidate vaccines against SARS-CoV-2. The study evaluated the immunogenicity ability of different recombinant subunit antigens of SARS-CoV-2 in different vaccine formulations, and different protein subunit vaccine formulations in mice.

A total of 86 BALB/c mice 6-7 weeks old were selected for this study. Mice were assigned to 9 different groups, each receiving a different vaccine formulation. Except that the control group (group a) included 6 mice, 10 mice were assigned to each group. According to the treatment group, animals received two doses of 0.1ml of the following vaccine formulation administered subcutaneously. These vaccines are formulated as "ready-to-use" vaccines. The first dose was administered to the animals on day 0 and the second dose was 3 weeks apart (day 21).

-Group a: the group was a control group. Animals in this group received a mock vaccine comprising PBS.

-Group B: animals in this group received a vaccine containing 20 μg of recombinant RBD antigen of SARS-CoV-2 per dose. The vaccine was formulated with 0.1ml dose of oil-in-water adjuvant in a v/v ratio of 75% adjuvant and 25% antigen. The oil-in-water adjuvant was formulated as follows: 19.5mg/ml squalene, 2.35mg/ml polysorbate 80, 2.35mg/ml sorbitan trioleate, 1.32mg/ml sodium citrate and 0.08mg/ml citric acid. Thus, the 0.1ml dose of vaccine administered contained 1.46mg squalene, 0.18mg polysorbate 80, 0.18mg sorbitan trioleate, 0.099mg sodium citrate and 0.006mg citric acid.

Group-C: animals in this group received a vaccine containing 20 μg of recombinant RBD antigen of SARS-CoV-2 per dose. The vaccine was formulated with the same adjuvant as group B, with v/v 75% adjuvant and 25% antigen in the vaccine composition at a dose of 0.1ml, and a 10 μg/dose of MPLA immunostimulant (L6895, SIGMA) added to the antigen phase.

-Group D: animals in this group received a vaccine containing 20 μg of recombinant RBD antigen of SARS-CoV-2 per dose. The vaccine was formulated with 10mg of adjuvant AlPO4 gel (Adju-Phos CRODA) per dose, with a ratio of v/v 75% adjuvant and 25% antigen in the vaccine composition at the dose of 0.1ml, and 10 μg/dose of MPLA immunostimulant (L6895, SIGMA) added to the antigen phase.

Group E: animals in this group received a vaccine containing 20 μg of recombinant RBD antigen of SARS-CoV-2 per dose. The vaccine was formulated with 10mg of adjuvant AlPO4 gel (Adju-Phos CRODA) per dose, with a ratio of v/v 75% adjuvant and 25% antigen in a 0.1ml dose vaccine composition, and 5. Mu.g/dose of immunostimulant MPLA immunostimulant (L6895, SIGMA) and 5. Mu.g/dose of immunostimulant QS-21 (QS-21, DESERT KING) added to the antigen phase.

-Group F: animals in this group received a vaccine containing 20 μg of recombinant S1 antigen of SARS-CoV-2 per dose. The vaccine was formulated with the same adjuvant as group B, with a ratio of v/v 75% adjuvant and 25% antigen in the vaccine composition at the 0.1ml dose.

Group G: animals in this group received a vaccine containing 20 μg of recombinant S1 antigen of SARS-CoV-2 per dose. The vaccine was formulated with the same adjuvant as group B, with v/v 75% adjuvant and 25% antigen in the vaccine composition at a dose of 0.1ml, and a 10 μg/dose of MPLA immunostimulant (L6895, SIGMA) added to the antigen phase.

-Group H: animals in this group received a vaccine containing 20 μg of recombinant S1 antigen of SARS-CoV-2 per dose. The vaccine was formulated with 10mg of adjuvant AlPO4 gel (Adju-Phos CRODA) per dose, with a ratio of v/v 75% adjuvant and 25% antigen in the vaccine composition at the dose of 0.1ml, and 10 μg/dose of MPLA immunostimulant (L6895, SIGMA) added to the antigen phase.

-Group I: animals in this group received a vaccine containing 20 μg of recombinant S1 antigen of SARS-CoV-2 per dose. The vaccine was formulated with 10mg of adjuvant AlPO4 gel (Adju-Phos CRODA) per dose, with a ratio of v/v 75% adjuvant and 25% antigen in a 0.1ml dose vaccine composition, and 5. Mu.g/dose of immunostimulant MPLA immunostimulant (L6895, SIGMA) and 5. Mu.g/dose of immunostimulant QS-21 (QS-21, DESERT KING) added to the antigen phase.

The SARS-CoV-2 RBD sequence used in this study to produce the RBD subunit antigen consists of SARS-CoV-2 spike glycoprotein (UniProt No. P0DTC 2) at positions 319 to 541.

The SARS-CoV-2 S1 sequence used to produce the S1 subunit antigen in this study consisted of the SARS-CoV-2 spike glycoprotein (UniProt No. P0DTC2) at positions 16 to 682.

To prepare the vaccine, the RBD and S1 encoding genes were codon optimized for CHO cell expression and further cloned into the expression plasmid pD2610-v10 (transient expression vector, from ATUM) along with the N-terminal signal peptide sequence MGWSLILLFLVAVATRVLS (SEQ ID NO: 10) and the C-terminal hexahistidine tag for transient expression.

The oil-in-water adjuvant is prepared by dispersing sorbitan trioleate in squalene to obtain an oil phase. The aqueous phase is then obtained by mixing polysorbate 80 with an aqueous buffer of sodium citrate in citric acid. Both the oil and water phases were filtered and then mixed at high speed to form an oil-in-water emulsion of uniform droplets of less than 1 μm in size.

In this example, an oil-in-water adjuvant was produced, yielding an oil-in-water adjuvant formulation of 19.5mg/ml squalene, 2.35mg/ml polysorbate 80, 2.35mg/ml sorbitan trioleate, 1.32mg/ml sodium citrate, and 0.08mg/ml citric acid.

SARS-CoV-2 RBD and S1 antigens were produced in the ExpiCHO-S cell line (ThermoFisher). ExpiCHO-S cell lines were cultured in ExpiCHO expression medium (ThermoFisher) at 37℃under 80% humidity and 8% CO ₂ and stirred at 125rpm for expansion. Cells with a density of 6x10 ⁶ cells/ml were then transiently transfected with 1 μg/ml expression plasmid pre-mixed with ExpiFectamine CHO reagent (ThermoFisher) in OptiPRO SFM complex medium (ThermoFisher). On day 5 of culture, cells were removed by centrifugation at 300g for 5 minutes at room temperature, and the supernatant was retained.

The supernatant was then purified by immobilized metal affinity chromatography using a 5ml HiScreen Ni FF (Cytiva) column. The target protein was eluted with a pH7.2 buffer containing 20mM sodium phosphate, 500mM NaCl, 500mM imidazole. The purified target protein was dialyzed against PBS (10 kDa RC membrane), concentrated to 1mg/ml, and filtered using a PES filter (Millex-GP) with a pore size of 0.22 μm, and stored at-80℃for use.

To evaluate the immune response against SARS-CoV-2 after vaccination of animals, two different parameters were analyzed: (i) Neutralizing antibodies in serum, and (ii) cellular responses (cytokines and active lymphocytes). The results are provided in example 2.

Example 2: assessment of neutralizing antibodies and immune cell responses.

Neutralizing antibodies in serum were evaluated. For this purpose, serum samples were taken from all vaccinated animals 20 to 21 days (days 40-41) after the second dose of vaccine.

The neutralizing antibodies in serum were determined by a pseudovirus neutralization assay (PBNA) using SARS-CoV-2 pseudovirus, as described in Nie J.et al.Quantification of SARS-CoV-2 neutralizing antibody by a pseudotyped virus-based assay.Nat Protoc.2020 Nov;15(11):3699-3715. For this purpose, pseudoviruses expressing the SARS-CoV-2S protein and luciferase were generated.

For neutralization assays, 200TCID ₅₀ pseudovirus supernatants were pre-incubated with serial dilutions of heat-inactivated serum samples at 37 ℃ for 1 hour and then added to human ACE-2 overexpressing HEK293T cells. After 48 hours, the cells were lysed with Britelite Plus luciferase reagent (PERKIN ELMER, waltham, MA, USA). Luminescence was measured using EnSight multimode microplate reader (PERKIN ELMER) for 0.2 seconds. All assays were performed in duplicate wells. The neutralization capacity of plasma samples was calculated by comparing the experimental RLU (relative light units) calculated from infected cells treated with each plasma with the maximum RLU (maximum infectivity calculated from untreated infected cells) and the minimum RLU (minimum infectivity calculated from untreated cells) and expressed as percent neutralization:

Normalized dose response neutralization curves were fitted to four parameter curves with variable slope using GRAPH PAD PRISM (v8.3.0). All IC ₅₀ values are expressed as the reciprocal dilution (concentration required to inhibit 50% of infection).

Samples were tested at the following dilutions: 1/60, 1/180, 1/540, 1/1620, 1/4860 and 1/14580. Neutralization titers between 60 and 14580 can be quantified. Lower and higher titers below or above the quantification limit are indicated as < 60 and > 14580, respectively.

To assess cellular immune responses, animals were euthanized 21 days after receiving the second dose of vaccine. Then, the spleen was extracted to obtain spleen cells. The splenocytes were stimulated in vitro with an antigen corresponding to the antigen (RBD or S1) present in the vaccine formulation, depending on the treatment group from which the splenocytes were derived. Splenocytes were cultured for 66 to 72 hours after stimulation. Then, the culture concentrations of cytokines INF-gamma, IL-4, IL-10 and IL-6 obtained in the supernatant were determined. Cytokine concentrations (pg/ml) in cell culture supernatants were determined by standard ELISA techniques.

The results of neutralizing antibodies in serum showed higher titers of neutralizing antibodies in all vaccinated groups compared to the control group (group a). The results indicate that the vaccine comprising RBD antigen induced a higher humoral response with higher neutralizing antibodies than the vaccine comprising S1 antigen (fig. 1). The humoral response of most animals in the group vaccinated with the vaccine comprising RBD antigen (groups B to E) was above the limit of quantitation determined by PBNA (> 14580) over the dilution range tested. This result surprisingly shows the strong ability of all tested vaccines, in particular vaccines comprising RBD antigens, to generate neutralizing antibodies.

Regarding the cellular immune response, it can clearly be observed that the choice of adjuvant plays a critical role in obtaining a strong immune response (FIGS. 2A-H). In the group (groups C and G) receiving the vaccine comprising the oil-in-water adjuvant formulated as 19.5mg/ml squalene, 2.35mg/ml polysorbate 80, 2.35mg/ml sorbitan trioleate, 1.32mg/ml sodium citrate and 0.08mg/ml citric acid and MPLA as immunostimulant, it was surprisingly shown that the cytokine production in cultures of splenocytes stimulated with the corresponding antigen was higher compared to the remaining group, whether treated with RBD antigen or S1 antigen. The results also show that further inclusion of an immunostimulant, in particular MPLA (groups C and G), in the oil-in-water adjuvant significantly increased the production of cytokines (IFN- γ, IL-4 and IL-6) compared to the vaccine comprising only the oil-in-water adjuvant (groups B and F). It was also demonstrated that the addition of the immunostimulant QS-21 to the formulations comprising AlPO ₄ gel and MPLA (groups E and I) also increased the production of cytokines (IFN-. Gamma., IL-4 and IL-6) compared to the formulations comprising AlPO ₄ gel and MPLA alone (groups D and H), whether animals were treated with RBD antigen or S1 antigen.

It is described that "antibody-dependent enhancement" (ADE) in SARS-CoV-2 infection is associated with increased production of IL-6 by macrophages and decreased production of IL-10 (IWASAKI AND YANG, 2020). Thus, the study also showed that the oil-in-water adjuvant and MPLA formulation would be suitable for reducing the ADE risk of vaccinated persons who may be exposed to virus, as it showed an increased IFN- γ/IL-6 cytokine ratio and high IL-10 yield after splenocyte stimulation.

Overall, the results indicate that subunit vaccines against SARS-CoV-2 comprising the subunit RBD antigen or S1 antigen of SARS-CoV-2 and an adjuvant and further comprising an immunostimulant provide a higher immune response to the subject.

As previously described, two oil-in-water adjuvants (formulated as 19.5mg/ml squalene, 2.35mg/ml polysorbate 80, 2.35mg/ml sorbitan trioleate, 1.32mg/ml sodium citrate and 0.08mg/ml citric acid) and MPLA were used for human vaccine. From the results, it can be seen that the oil-in-water adjuvant is sufficient to induce the vaccinated subject to mount an immune response against SARS-CoV-2. Due to its known safety, the adjuvant is also suitable for use in vaccine compositions. Furthermore, when an immunostimulant is further included in the oil-in-water adjuvant vaccine formulation, the immune response is significantly enhanced. Thus, the use of an immunostimulant in a vaccine composition further enhances the immune response of a subject receiving said vaccine composition.

Example 3: antigen production and analysis of convalescent human serum collections

The SARS-CoV-2 RBD sequence used to produce the RBD subunit antigen consists of SARS-CoV-2 spike glycoprotein (UniProt No. P0DTC2) at positions 319-541.

To prepare the vaccine, the RBD encoding gene was codon optimized for CHO and HEK293 cell expression and further cloned into the expression plasmid pD2610-v10 (transient expression vector, from ATUM) along with the N-terminal signal peptide sequence MGWSLILLFLVAVATRVLS (SEQ ID NO: 10) and the C-terminal hexahistidine tag for transient expression.

SARS-CoV-2 RBD antigen was produced in the ExpiCHO-S cell line (ThermoFisher). ExpiCHO-S cell lines were cultured in ExpiCHO expression medium (ThermoFisher) at 37℃under 80% humidity and 8% CO ₂ and stirred at 125rpm for expansion. Cells with a density of 6x10 ⁶ cells/ml were then transiently transfected with 1 μg/ml expression plasmid pre-mixed with ExpiFectamine CHO reagent (ThermoFisher) in OptiPRO SFM complex medium (ThermoFisher). On day 5 of culture, cells were removed by centrifugation at 300g for 5 minutes at room temperature, and the supernatant was retained.

For expression of the RBD and S1 antigens of SARS-CoV-2 in HEK293 cell lines, the same method as described for CHO cell expression was used. The components used for HEK293 expression were the Expi293F (ThermoFisher) cell line, the Expi293 expression medium (ThermoFisher), expiFectamine 293 reagent (ThermoFisher) and Opti-MEM complex medium (ThermoFisher).

Two different serum collections were collected: one purchased from Ray Biotech (ref. CoV-PosSet-S1), the other obtained from convalescence hospitalized patients in the herona region (spanish gartania), both with different levels of anti-SARS-CoV-2 antibodies. Thirty (30) sera obtained from PCR-diagnosed positive patients were tested simultaneously with candidate RBD antigens produced in HEK293 or CHO. In addition, the study included 10 negative serum samples obtained prior to the pandemic outbreak. The total antibody titer of human SARS-CoV-2 RBD IgG (log 10 EC ₅₀) was determined for each sample, and the results indicated that RBD antibody levels were significantly higher for all convalescent serum samples than for negative samples, regardless of antigen source (FIG. 3). Consistent responses to RBD in convalescent samples indicate the importance of RBD as a COVID-19 vaccine candidate antigen.

Human SARS-CoV-2 RBD IgG total antibody titer was determined by ELISA. Nunc Maxisorp ELISA plates (ThermoFisher, ref. 10547781) were coated with 100ng SARS-CoV-2 RBD protein (positions 319 to 541 of SARS-CoV-2 spike glycoprotein (UniProt No. P0DTC2)) per well overnight at 4 ℃. Plates were washed with phosphate buffered saline containing 0.05% Tween buffer and blocked with Stabilblock immunoassay stabilizing buffer (Surmotics IVD, ref. ST 01-1000). Serum samples obtained from mice were serially diluted 4-fold and added to the coated wells for 1 hour at 37 ℃ under 5% co ₂ and humid atmosphere. The plates were washed with PBS. Next, diluted horseradish peroxidase (HRP) conjugated to anti-mouse (Jackson ImmunoResearch, ref.115-035-003) was added and developed by adding 2,2' -azino-bis- (3-ethylbenzothiazoline sulfonic acid) peroxidase substrate (ABTS, civest). Plates were read at an OD of 405nm using a Gene5 microplate reader (Synergy HTX, multimode microplate reader) and data were analyzed using SoftMax software. Half maximal binding antibody concentrations (EC ₅₀ values) were calculated by 4-parameter fitting using GRAPHPAD PRISM software.

There is a high degree of equivalence between the two expression systems in terms of IgG antibody titer. No significant difference was detected by comparing the titer of the grouped IgG antibodies against SARS-CoV-2 RBD produced in HEK293 cells or those against SARS-CoV-2 RBD produced in CHO cells (fig. 4). In addition, the paired IgG antibody titres against SARS-CoV-2 RBD produced in HEK293 cells and SARS-CoV-2 RBD produced in CHO cells showed very good similarity (fig. 6). Together, these results support the equivalence between antigen production in both mammalian expression systems.

The IgG antibody titer against SARS-CoV-2 RBD was plotted against the number of days elapsed between the first PCR positive result and each serum sample donation (fig. 5). During the sample collection period (36 to 105 days), no obvious correlation was found between the two factors. This result suggests that antibodies raised against SARS-CoV-2 RBD specifically in convalescent patients can last for at least several months.

Example 4: immunogenicity studies in mice using different candidate vaccines.

This study evaluated different RBD subunit candidate vaccines against SARS-CoV-2. The study also evaluated different RBD subunit vaccine formulations. Furthermore, the present study assessed the immunogenicity potential of different protein subunit candidate vaccines.

A total of 46 BALB/c mice 5-6 weeks old were selected for this study. Mice were divided into 5 different groups and received different vaccine formulations, respectively. Except that the control group (group a) included 6 mice, 10 mice were assigned to each group. The animals were administered intramuscularly one of the following protein subunit vaccine formulations received two doses of 0.1 ml. These vaccines are formulated as "ready-to-use" vaccines. The first dose (priming) was administered to the animals on day 0 and the second dose (boosting) was administered 18 days after the first dose (day 18).

The different vaccine formulations administered to mice were as follows:

-group a: control group. Animals in this group received a mock vaccine comprising PBS.

-Group B: animals in this group received a vaccine containing 20 μg of recombinant RBD antigen of SARS-CoV-2. The recombinant antigen is based on a ratio of 12% non-fusion dimeric RBD antigen to 88% monomeric RBD antigen. The proportion of non-fused dimer to monomer was determined by size exclusion chromatography HPLC (Agilent Technologies). The vaccine was formulated with an oil-in-water adjuvant formulated as 19.5mg/ml squalene, 2.35mg/ml polysorbate 80, 2.35mg/ml sorbitan trioleate, 1.32mg/ml sodium citrate and 0.08mg/ml citric acid in a ratio of v/v 75% adjuvant and 25% antigen.

Group-C: animals in this group received a vaccine containing 20 μg of recombinant RBD antigen of SARS-CoV-2. Recombinant antigen is based on the ratio of 12% non-fusion dimeric RBD antigen to 88% monomeric RBD antigen (non-fusion dimer: monomer ratio as determined in group B). The vaccine was formulated with the same adjuvant as group B, in a ratio of v/v 75% adjuvant and 25% antigen, and 10 μg/dose of MPLA (L6895, SIGMA) was added to the antigen phase.

-Group D: animals in this group received a vaccine containing 20 μg of recombinant RBD antigen of SARS-CoV-2. Recombinant antigen is based on the ratio of 12% non-fusion dimeric RBD antigen to 88% monomeric RBD antigen (non-fusion dimer: monomer ratio as determined in group B). The vaccine was formulated with the same adjuvant as group B, in a ratio of v/v 75% adjuvant and 25% antigen, and with the addition of 10 μg/dose of QS-21 (QS-21, DESERT kit), QS-21 was added to the antigen phase.

Group E: animals in this group received a vaccine containing 10 μg of recombinant RBD antigen of SARS-CoV-2. The recombinant RBD used in this group was tailored to contain a higher proportion of non-fusion dimeric RBD antigen. The recombinant antigens used in this group contained 56% non-fusion dimeric RBD antigen and 44% monomeric RBD antigen. To adjust the ratio of dimeric SARS-CoV-2 RBD antigen, the dimeric RBD antigen fraction and the monomeric RBD antigen fraction were separated by size exclusion chromatography (HiPrep 26/60Sephacryl S-100HR,Cytiva ref.17119401). The final antigen concentration of each fraction was determined by reading with a microplate reader (Synergy HTX, multimode microplate reader) at OD 280 nm. Finally, fractions containing monomeric RBD antigen and non-fusion dimeric RBD antigen are mixed in different volumes to obtain specific ratios of dimeric RBD antigen. The vaccine was formulated with the same adjuvant as group B in the ratio v/v 75% adjuvant and 25% antigen.

As described above, the oil-in-water adjuvants used in this study were formulated as follows: 19.5mg/ml squalene, 2.35mg/ml polysorbate 80, 2.35mg/ml sorbitan trioleate, 1.32mg/ml sodium citrate and 0.08mg/ml citric acid. Thus, when mixed in a ratio of 75% adjuvant to 25% antigen, each 0.1ml dose of the vaccine administered contained 1.46mg squalene, 0.18mg polysorbate 80, 0.18mg sorbitan trioleate, 0.099mg sodium citrate and 0.006mg citric acid.

The preparation of the oil-in-water adjuvant was the same as in example 1.

Recombinant RBD antigen was produced in ExpiCHO-S cell line as follows: the SARS-CoV-2 RBD sequence used in this study to produce the RBD subunit antigen consists of SARS-CoV-2 spike glycoprotein (UniProt No. P0DTC 2) at positions 319 to 541. For vaccine preparation, the RBD gene was codon optimized for CHO cell expression and further cloned into the expression plasmid pD2610-v10 (transient expression vector, from ATUM) along with the N-terminal signal peptide sequence MGWSLILLFLVAVATRVLS (SEQ ID NO: 10) and the C-terminal hexahistidine tag for transient expression.

To assess immune responses against SARS-CoV-2 following the vaccination regimen, serum samples were taken from each mouse on day 18 (prior to the second dose of vaccine) and day 30 (fig. 7B) of the study. Serum samples were analyzed for anti-SARS-CoV-2 RBD IgG antibody titer by ELISA. The log10 EC ₅₀ titers of each set of anti-SARS-CoV-2 RBD IgG antibodies are shown in FIG. 7.

Nunc Maxisorp ELISA plates (ThermoFisher, ref. 10547781) were coated with 100ng SARS-CoV-2 RBD protein (positions 319 to 541 of SARS-CoV-2 spike glycoprotein (UniProt No. P0DTC2)) per well overnight at 4 ℃. Plates were washed with phosphate buffered saline containing 0.05% Tween buffer and blocked with Stabilblock immunoassay stabilizing buffer (Surmotics IVD, ref. ST 01-1000). Serum samples obtained from mice were serially diluted 4-fold and added to the coated wells for 1 hour at 37 ℃ under 5% co ₂ and humid atmosphere. The plates were washed with PBS. Next, diluted horseradish peroxidase (HRP) conjugated to anti-mouse (Jackson ImmunoResearch, ref.115-035-003) was added and developed by adding 2,2' -azino-bis- (3-ethylbenzothiazoline sulfonic acid) peroxidase substrate (ABTS, civest). Plates were read at an OD of 405nm using a Gene5 microplate reader (Synergy HTX, multimode microplate reader) and data were analyzed using SoftMax software. EC ₅₀ values were calculated by 4-parameter fitting using GRAPHPAD PRISM software.

The results clearly demonstrate that all animals immunized with the candidate vaccine according to the two dose regimen (groups B to E) were significantly higher against SARS-CoV-2 RBD antibodies on day 30 compared to the control group (group a). Thus, the results demonstrate the ability of a composition comprising RBD antigen to mount an immune response. The results also demonstrate the applicability of using an oil-in-water adjuvant in subunit SARS-CoV-2 vaccine comprising RBD antigen. Furthermore, it is clearly observed that when vaccine formulations are combined with immunostimulants, in particular MPLA (group C), a higher humoral response is obtained. Surprisingly, group C animals achieved a high immune response at day 18 after the first dose. The anti-SARS-CoV-2 RBD antibody titer was also highest on day 30 compared to the other groups.

The results also show that when the non-fusion dimeric RBD antigen of the RBD antigen present in the vaccine formulation has a high proportion (group E) relative to monomeric RBD antigen, the humoral response is significantly enhanced even though the vaccine composition does not contain an immunostimulant. It was observed that a vaccine comprising half dose RBD antigen (10 μg/dose), formulated with a high proportion of non-fusion dimeric RBD antigen and with an oil-in-water adjuvant provided an enhanced humoral response at day 30 (group E) when compared to the group receiving a formulated vaccine comprising a low proportion of non-fusion dimeric RBD antigen and no immunostimulant (group B), and the group comprising a low proportion of non-fusion dimeric RBD antigen and formulation vaccine comprising immunostimulant QS-21 (group D). It was also observed that the vaccine administered to group E animals (recombinant RBD comprising 10 μg of SARS-CoV-2 antigen, with an increased proportion of non-fusion dimeric RBD antigen and no immunostimulant) produced anti-SARS-CoV-2 RBD antibody titres comparable to those obtained in group C animals (vaccine receiving recombinant RBD comprising 20 μg of SARS-CoV-2 antigen, with a reduced proportion of non-fusion dimeric RBD antigen to monomeric RBD antigen and also with MPLA as immunostimulant).

The results surprisingly demonstrate that formulations based on non-fusion dimeric RBD antigen, and formulations based on increased proportions of non-fusion dimeric RBD antigen relative to monomeric RBD antigen in RBD antigen, have a strong ability to produce anti-SARS-CoV-2 RBD antibodies.

Example 5: immunogenicity studies in mice with fusion dimeric RBD antigen.

This study evaluated a novel recombinant subunit antigen for SARS-CoV-2. The novel recombinant subunit antigen is a fusion dimeric RBD antigen comprising two monomers, a first monomer comprising an RBD derived from the b.1.351 (south africa) variant and a second monomer comprising an RBD derived from the b.1.1.7 (uk) variant. This novel recombinant subunit antigen of SARS-CoV-2 is designated herein as a fusion dimeric RBD variant antigen. This study evaluated the immunogenicity ability of different subunit vaccine formulations and such recombinant fusion dimeric RBD variant antigens in mice. The study also included a comparison between the fusion dimeric RBD variant antigen and the recombinant non-fusion dimeric SARS-CoV-2 as previously described, monomeric RBD antigen (consisting of a combination of non-fusion dimeric RBD antigen and monomeric RBD antigen derived from Wuhan-Hu-1 variants and formulated in different dimer to monomer ratios). The latter protein subunit vaccine is designated herein as non-fusion dimeric monomeric RBD non-variant SARS-CoV-2 antigen.

The recombinant fusion dimeric RBD variant SARS-CoV-2 antigen used in this study is a concatamer comprising the amino acid sequence at positions 319 to 537 (as defined in SEQ ID NO: 4) of the SARS-CoV-2 spike protein RBD monomer derived from the B.1.351 variant as a first monomer followed by the amino acid sequence at positions 319 to 537 (as defined in SEQ ID NO: 3) of the SARS-CoV-2 spike protein RBD monomer derived from the B.1.1.7 variant as a second monomer. The amino acid sequence of the recombinant fusion dimeric RBD variant antigen as a tandem fusion antigen is defined in SEQ ID NO: 5.

For vaccine preparation, the fusion dimeric RBD variant SARS-CoV-2 antigen was codon optimized for CHO cell expression (SEQ ID NO: 8) and further cloned into the expression plasmid pcDNA3.4 (GENSCRIPT) along with the N-terminal signal peptide sequence MGWSCIILFLVATATGVHS (SEQ ID NO: 6) and the C-terminal hexahistidine tag for transient expression. A DNA construct comprising a signal peptide, a codon optimized SARS-CoV-2 RBD dimeric variant and a C-terminal histidine tag is set forth in SEQ ID NO: defined in 7.

The recombinant non-fusion dimeric monomer RBD non-variant SARS-CoV-2 antigen used in this study consisted of positions 319 to 541 of the SARS-CoV-2 spike glycoprotein (UniProt No. P0DTC2) that is a Wuhan-Hu-1 variant. To prepare recombinant non-fusion dimeric monomeric RBD non-variant SARS-CoV-2 antigen, the RBD gene was codon optimized for CHO cell expression and further cloned into expression plasmid pD2610-v10 (ATUM) along with N-terminal signal peptide sequence MGWSLILLFLVAVATRVLS (SEQ ID NO: 10) and C-terminal hexahistidine tag for transient expression.

Recombinant fusion dimeric RBD variant SARS-CoV-2 antigen and recombinant non-fusion dimeric monomeric RBD non-variant SARS-CoV-2 antigen were produced in ExpiCHO-S cell line (ThermoFisher). ExpiCHO-S cell lines were cultured in ExpiCHO expression medium (ThermoFisher) at 37℃under 80% humidity and 8% CO ₂ and stirred at 125rpm for expansion. Cells with a density of 6x10 ⁶ cells/ml were then transiently transfected with 1 μg/ml expression plasmid pre-mixed with ExpiFectamine CHO reagent (ThermoFisher) in OptiPRO SFM complex medium (ThermoFisher). On day 5 of culture, cells were removed by centrifugation at 300g for 5 minutes at room temperature, and the supernatant was retained.

The supernatant was then purified by immobilized metal affinity chromatography using a 5ml HiScreen Ni FF (Cytiva) column. The target protein was eluted with a pH7.2 buffer containing 20mM sodium phosphate, 500mM NaCl, 500mM imidazole. The purified target protein was dialyzed against PBS-0.01% Tween 80 (30 kDa RC membrane), concentrated to 1mg/ml, and filtered using a PES filter (Millex-GP) with a pore size of 0.22 μm and stored at-80℃for use.

A total of 86 BALB/c mice 5-6 weeks old were selected for this study. Mice were assigned to 9 different groups. Each group received a different vaccine formulation as described below. Each group included 10 mice, except the control group (group a) included only 6 mice. All animals were administered a dose of 0.1ml of the following vaccine formulation by the intramuscular route. These vaccines are formulated as "ready-to-use" vaccines. The animals were administered a vaccine on day 0 of the study.

The different vaccine formulations administered to mice were as follows:

Group B (fusion dimeric RBD variant SARS-CoV-2 antigen, 1-fold dose): animals of this group received a vaccine formulation comprising 1 μg of recombinant fusion dimeric RBD variant SARS-CoV-2 antigen. The vaccine was formulated with an oil-in-water adjuvant in the ratio v/v 50% adjuvant and 50% antigen.

Group C (fusion dimeric RBD variant SARS-CoV-2 antigen, 5-fold dose): animals of this group received a vaccine formulation comprising 5 μg of recombinant fusion dimeric RBD variant SARS-CoV-2 antigen. The vaccine was formulated with an oil-in-water adjuvant in the ratio v/v 50% adjuvant and 50% antigen.

Group D (fusion dimeric RBD variant SARS-CoV-2 antigen, 20-fold dose): animals of this group received a vaccine formulation comprising 20 μg of recombinant fusion dimeric RBD variant SARS-CoV-2 antigen. The vaccine was formulated with an oil-in-water adjuvant in the ratio v/v 50% adjuvant and 50% antigen.

Group E (fusion dimeric RBD variant SARS-CoV-2 antigen, 20-fold dose, plus immunostimulant 1): animals of this group received a vaccine formulation comprising 20 μg of recombinant fusion dimeric RBD variant SARS-CoV-2 antigen. The vaccine was formulated as follows: oil-in-water adjuvants, in a ratio of v/v50% adjuvant and 50% antigen, and 10 μg/dose of MPLA (L6895, SIGMA) added to the antigen phase.

Group F (fusion dimeric RBD variant SARS-CoV-2 antigen, 20-fold dose, plus immunostimulant 2): animals of this group received a vaccine formulation comprising 20 μg of recombinant fusion dimeric RBD variant SARS-CoV-2 antigen. The vaccine was formulated as follows: an oil-in-water adjuvant, in a ratio of v/v50% adjuvant and 50% antigen, and 10 μg/dose of QS-21 (QS-21, DESERT kit), QS-21 being added to the antigen phase.

Group G (non-fusion dimeric: monomeric RBD non-variant SARS-CoV-2 antigen, 20-fold dose): animals of this group received a vaccine formulation comprising 20 μg of recombinant non-fusion dimeric monomeric RBD non-variant SARS-CoV-2 antigen. The recombinant antigen is based on recombinant SARS-CoV-2 RBD antigen in non-fusion dimeric form, and the ratio of the recombinant antigen to the recombinant SARS-CoV-2 RBD antigen is 80% to 20% of non-fusion RBD dimer to RBD monomer. To adjust the ratio of non-fusion dimeric SARS-CoV-2 RBD antigen, the non-fusion dimeric RBD antigen fraction and monomeric RBD antigen fraction were separated by size exclusion chromatography (HiPrep 26/60Sephacryl S-100HR,Cytiva ref.17119401). The final antigen concentration of each fraction was determined by reading with a microplate reader (Synergy HTX, multimode microplate reader) at OD 280 nm. Finally, the fractions containing monomeric RBD antigen and non-fusion dimeric RBD antigen are mixed in different volumes to obtain a specific ratio of non-fusion dimeric RBD antigen. The vaccine was formulated with an oil-in-water adjuvant in the ratio v/v 50% adjuvant and 50% antigen.

Group H (non-fusion dimeric: monomeric RBD non-variant SARS-CoV-2 antigen, 20-fold dose, plus immunostimulant 1): animals of this group received a vaccine formulation comprising 20 μg of the non-fusion dimeric monomeric RBD non-variant SARS-CoV-2 antigen. The recombinant antigen is based on the recombinant SARS-CoV-2 RBD antigen in a non-fusion dimeric form, the ratio is 80% to 20% of non-fusion RBD dimer to RBD monomer (the ratio of dimer to monomer is adjusted in the same way as in group G). The vaccine was formulated as follows: oil-in-water adjuvants, in a ratio of v/v 50% adjuvant and 50% antigen, and 10 μg/dose of MPLA (L6895, SIGMA) added to the antigen phase.

Group I (non-fusion dimeric: monomeric RBD non-variant SARS-CoV-2 antigen, 20-fold dose, plus immunostimulant 2): animals of this group received a vaccine formulation comprising 20 μg of the non-fusion dimeric monomeric RBD non-variant SARS-CoV-2 antigen. The recombinant antigen is based on the recombinant SARS-CoV-2 RBD antigen in a non-fusion dimeric form, the ratio is 80% to 20% of non-fusion RBD dimer to RBD monomer (the ratio of dimer to monomer is adjusted in the same way as in group G). The vaccine was formulated as follows: an oil-in-water adjuvant, in a ratio of v/v 50% adjuvant and 50% antigen, and 10 μg/dose of QS-21 (QS-21, DESERT kit), QS-21 being added to the antigen phase.

The oil-in-water adjuvants used in this study were formulated as follows: 39mg/ml squalene, 4.7mg/ml polysorbate 80, 4.7mg/ml sorbitan trioleate, 2.64mg/ml sodium citrate and 0.16mg/ml citric acid. Thus, when mixed in a ratio of 50% adjuvant to 50% antigen, the 0.1ml dose of the administered vaccine contained 1.95mg squalene, 0.235mg polysorbate 80, 0.235mg sorbitan trioleate, 0.132mg sodium citrate and 0.008mg citric acid. The formulation was comparable to the standard concentration of known oil-in-water adjuvants administered to humans at a dose of 0.5ml, 9.75mg squalene, 1.175mg polysorbate 80, 1.175mg sorbitan trioleate, 0.66mg sodium citrate and 0.04mg citric acid.

To assess immune responses against SARS-CoV-2 following vaccination with the different vaccine formulations of the present study, serum samples of each mouse were taken on day 21 and analyzed for anti-SARS-CoV-2 RBD IgG antibody titers by ELISA. Log10EC ₅₀ values for anti-SARS-CoV-2 RBD IgG antibody titers are shown in figure 8.

Anti-SARS-CoV-2 IgG antibody titers were determined by ELISA as follows: nunc Maxisorp ELISA plates (ThermoFisher, ref. 10547781) were coated with 100ng SARS-CoV-2 RBD protein per well overnight at 4 ℃. To analyze serum extracted from animals of groups A through F, ELISA plates were coated with recombinant fusion dimeric RBD variant SARS-CoV-2 antigen as described above, and to analyze serum extracted from animals of groups G through I, ELISA plates were coated with recombinant non-fusion 80% dimeric 20% monomeric RBD non-variant SARS-CoV-2 RBD antigen as described above. Plates were washed with phosphate buffered saline containing 0.05% Tween buffer and blocked with Stabilblock immunoassay stabilizing buffer (Surmotics IVD, ref. ST 01-1000). Serum samples obtained from mice were serially diluted 4-fold and added to the coated wells for 1 hour at 37 ℃ under 5% co ₂ and humid atmosphere. The plates were washed with PBS. Next, diluted horseradish peroxidase (HRP) conjugated to anti-mouse (Jackson ImmunoResearch, ref.115-035-003) was added and developed by adding 2,2' -azino-bis- (3-ethylbenzothiazoline sulfonic acid) peroxidase substrate (ABTS, civest). Plates were read at an OD of 405nm using a Gene5 microplate reader (Synergy HTX, multimode microplate reader) and data were analyzed using SoftMax software. EC ₅₀ values were calculated by 4-parameter fitting using GRAPHPAD PRISM software.

From the results of the study, it was surprising that vaccinated animals exhibited an enhanced immune response after a single administration of a single dose of recombinant fusion dimeric RBD variant SARS-CoV-2 antigen. Even animals receiving vaccine formulations containing low doses of the fusion dimeric RBD variant SARS-CoV-2 antigen and without any immunostimulant (groups B and C) produced higher anti-SARS-CoV-2 RBD IgG antibody titers than the group receiving vaccine formulations comprising: comprising a 20 μg dose of non-fusion dimeric monomeric RBD non-variant SARS-CoV-2 antigen plus an oil-in-water adjuvant alone (group G), or plus an oil-in-water adjuvant further comprising a QS-21 immunostimulant (group I). In the presence of the same dose (e.g., 20 μg) of total antigen in the vaccine composition, it was also observed that the response of those groups (D-F groups) receiving the recombinant fusion dimeric RBD variant SARS-CoV-2 antigen was enhanced when compared to the group (H group) receiving the vaccine composition comprising the non-fusion dimeric: monomeric RBD non-variant SARS-CoV-2 antigen formulated with an oil-in-water adjuvant and a MPLA immunostimulant, even though the composition did not comprise any immunostimulant (D group). Overall, the results unexpectedly demonstrate the increased potential of recombinant dimeric RBD antigens (fused and unfused dimeric RBD) to produce anti-SARS-CoV-2 RBD IgG antibodies against SARS-CoV-2.

In addition, the results confirm the suitability of using an oil-in-water adjuvant formulated in the final vaccine formulation as 39mg/ml squalene, 4.7mg/ml polysorbate 80, 4.7mg/ml sorbitan trioleate, 2.64mg/ml sodium citrate and 0.16mg/ml citric acid. Finally, animals vaccinated with a vaccine composition comprising an oil-in-water adjuvant in combination with an immunostimulant, in particular with MPLA, showed higher antibody titers than animals immunized with an oil-in-water adjuvant without an immunostimulant, indicating that the vaccinated subjects had better immune responses when they received both adjuvant and immunostimulant together with either a fusion dimeric RBD variant antigen or a non-fusion dimeric monomeric RBD non-variant SARS-CoV-2 antigen.

Example 6: immunogenicity studies in mice with two doses of fusion dimeric RBD antigen.

Considering the unexpectedly good results obtained after vaccinating animals with a dose comprising the fusion dimeric RBD variant SARS-CoV-2 antigen as described in example 5, the present study assessed the novel recombinant fusion dimeric RBD variant SARS-CoV-2 antigen in a two dose regimen.

In this study, animals belonging to the different groups described in example 5 received a second dose of vaccine. On day 21, 21 days after the first dose, the animals received 0.1ml of the corresponding vaccine formulation per group (groups a to I, as described in example 5) of the second dose by the intramuscular route.

To evaluate the immune response against SARS-CoV-2 following the second dose of the different vaccine formulations of the present study, serum samples of each mouse were taken on day 35 (14 days after the second dose) and analyzed for anti-SARS-CoV-2 RBD IgG antibody titers by ELISA, as described in example 5. Log10 EC ₅₀ values of anti-SARS-CoV-2 RBD IgG antibody titers present in animal serum 14 days after the second dose administration are shown in figure 9.

After the second dose of protein subunit vaccine, an increase in anti-SARS-CoV-2 RBD IgG antibody titer was observed in all vaccinated groups compared to anti-SARS-CoV-2 RBD IgG antibody titer after a single dose. Thus, priming/boosting or two dose regimens increase the immunogenic response. In addition, animals receiving vaccines comprising fusion dimeric RBD variant SARS-CoV-2 antigen (panels B through F) exhibited increased anti-SARS-CoV-2 RBD IgG antibody titers compared to animals receiving vaccines comprising non-fusion dimeric monomeric RBD non-variant SARS-CoV-2 antigen (panels G through I). Even animals that received vaccines formulated with low doses of the fusion dimeric RBD variant SARS-CoV-2 antigen and that did not contain any immunostimulant (groups B through C) exhibited an increase in anti-SARS-CoV-2 RBD IgG antibody titer as compared to animals that received vaccines comprising the non-fusion dimeric monomeric RBD non-variant SARS-CoV-2 antigen (groups G through I). Overall, these results demonstrate the unexpected potential to generate dimeric RBD antigens obtained in example 5, in particular anti-SARS-CoV-2 IgG antibodies fused to dimeric RBD variant SARS-CoV-2 antigens.

From the results it can be concluded that protein subunit vaccines, in particular based on dimeric RBD antigens, are also suitable for use as booster vaccines in combination with other vaccines or for yearly re-vaccination, as it significantly increases the anti-SARS-CoV-2 RBD IgG antibody titre.

Example 7: immunogenicity studies in mice with non-fusion dimeric RBD antigen compared to commercially available vaccines.

The present study evaluates the immunogenicity of subunit candidate vaccines based on the non-variant SARS-CoV-2 RBD antigen, which are formulated to contain a high proportion of non-fusion dimeric RBD antigen relative to monomeric RBD antigen. Candidate vaccines were formulated with an oil-in-water adjuvant formulated as 39mg/ml squalene, 4.7mg/ml polysorbate 80, 4.7mg/ml sorbitan trioleate, 2.64mg/ml sodium citrate and 0.16mg/ml citric acid, with or without MPLA as immunostimulant, and the immunogenicity was compared with the commercial SARS-CoV-2 vaccine Spikevax, COVID-19mRNA vaccine (Moderna Biotech Spain, s.l.).

A total of 46 BALB/c mice, 6-7 weeks old, were divided into 4 different groups. Each group received a different vaccine formulation as described below. Animals received two doses of 0.1ml vaccine by the intramuscular route, three weeks apart, with the first dose injected (priming) on day 0 and the second dose injected (boosting) on day 21.

Group-a (control group, 10 animals): animals in this group received a mock vaccine comprising PBS.

Group B (non-fusion dimeric: monomeric RBD non-variant SARS-CoV-2 antigen, 12 animals): animals in this group received vaccine formulations containing 20 μg of recombinant non-fusion dimeric: monomeric RBD non-variant antigen (non-fusion RBD dimer: ratio of RBD monomers 80%: 20%). Preparation of recombinant non-fusion dimeric monomer RBD non-variant SARS-CoV-2 antigen and non-fusion dimeric dimer of SARS-CoV-2 RBD antigen the ratio of monomers was performed as described in example 5. The vaccine was formulated with an oil-in-water adjuvant in the ratio v/v 50% adjuvant and 50% antigen. Thus, when mixed in a ratio of 50% adjuvant to 50% antigen, a 0.1ml dose of vaccine contains 1.95mg squalene, 0.235mg polysorbate 80, 0.235mg sorbitan trioleate, 0.132mg sodium citrate and 0.008mg citric acid.

Group C (non-fusion dimeric: monomeric RBD non-variant SARS-CoV-2 antigen, plus immunostimulant, 12 animals): animals in this group received vaccine formulations (antigen preparation in this study and dimer: monomer ratio were performed as described in example 5) containing 20 μg of recombinant non-fusion dimeric: monomeric RBD non-variant antigen (non-fusion RBD dimeric: RBD monomer ratio 80%: 20%). The vaccine was formulated with the same adjuvant as group B, in a ratio of v/v 50% adjuvant and 50% antigen, and 10 μg/dose of MPLA was added as immunostimulant (L6895, SIGMA), MPLA being added to the antigen phase.

Group D (commercial vaccine, 12 animals): animals of this group received a vaccine formulation comprising 1 μg mRNA (embedded SM-102 lipid nanoparticle) of Spikevax vaccine, COVID-19 mRNA vaccine (Moderna Biotech Spain, s.l.). The doses of commercial vaccine injected into mice in this study were obtained from the well-preserved residual volume of vials provided by the public health authorities after vaccination of the human population. The dose (1 μg mRNA) was selected according to paper Corbett K.S.,et al.SARS-CoV-2 mRNAvaccine design enabled by prototype pathogen preparedness.Nature,2020,vol.586,no 7830,p.567-571, where it was shown in a dose response study to be near the saturation limit of mice.

To assess the immunogenic response against SARS-CoV-2 following vaccination with the different vaccine formulations of the present study, serum samples of all animals were taken on day 21 (before the second dose) and on day 35 (14 days after the second dose) and analyzed for anti-SARS-CoV-2 RBD IgG antibody titers by ELISA. In addition, the serum samples taken on day 35 were also analyzed to determine neutralizing antibodies against SARS-CoV-2 isolate Wuhan-1 (Wuhan-Hu-1) by a pseudovirus-based neutralization assay (PBNA). Due to laboratory limitations, some samples were not analyzed at day 35, so half of the serum samples were eventually taken and tested for anti-SARS-CoV-2 RBD IgG titer and neutralizing antibodies against SARS-CoV-2 at day 37.

Anti-SARS-CoV-2 RBD IgG antibody titers were determined by ELISA as follows:

Nunc Maxisorp ELISA plates (ThermoFisher, ref.10547781) were coated with 100ng recombinant SARS-CoV-2 RBD (Sino Biologicals, ref.40592-V08B) per well and blocked with 5% nonfat dry milk (Sigma) in PBS. Plates were washed with phosphate buffered saline containing 0.05% Tween buffer and blocked with Stabilblock immunoassay stabilizing buffer (Surmotics IVD, ref. ST 01-1000). Wells were incubated with serial dilutions of serum samples obtained from mice for 1 hour at 37 ℃, 5% co2 and a humid atmosphere. The plates were then washed with PBS. Next, peroxidase conjugated goat anti-mouse IgG (Sigma, ref. Ap308 p) was added. Finally, wells were incubated with K-Blue Advanced Substrate (Neogen, ref.379175) and absorbance at 450nm was measured using a Gene5 microplate reader (Synergy HTX, multimode microplate reader) and the data analyzed using softMax software. The average of absorbance for each dilution of serum samples was calculated in duplicate. The endpoint titer of SARS-CoV-2 RBD specific total IgG binding antibody was determined as the inverse of the last serum dilution, which is 3 times the average optical density of the technical negative control (wells without added serum).

PBNA assay the use of HIV reporter pseudoviruses based on the S protein expressing SARS-CoV-2 and luciferase production, as described in Nie J.et al.Quantification of SARS-CoV-2neutralizing antibody by a pseudotyped virus-based assay.Nat Protoc.2020Nov;15(11):3699-563715. An HIV reporter pseudovirus was generated that expressed SARS-CoV-2S protein from Wuhan-1 (Wuhan-Hu-1) and luciferase.

For neutralization assays, 200TCID ₅₀ pseudovirus supernatants were pre-incubated with serial dilutions of heat-inactivated serum samples at 37 ℃ for 1 hour before addition to HEK293T cells over-expressed by ACE 2. After 48 hours, the cells were lysed with Britelite Plus luciferase reagent (PERKIN ELMER, waltham, MA, USA). Luminescence was measured for 0.2 seconds using EnSight multimode microplate reader (PERKIN ELMER). The neutralization capacity of the serum samples was calculated by comparing the experimental RLU calculated from the infected cells treated with each serum with the maximum RLU (maximum infectivity calculated from the untreated infected cells) and the minimum RLU (minimum infectivity calculated from the untreated cells) and expressed as percent neutralization:

% neutralization= (RLUmax-RLU experiment)/(RLUmax-RLUmin) ×100.

The results of the study showed that vaccinated groups B-D induced significantly higher anti-SARS-CoV-2 RBD IgG antibody responses than the unvaccinated control group (group a). In addition, vaccine formulations administered to groups C and D induced similar antibody titers on day 21. It was also observed that all vaccinated groups (including group B without MPLA) induced similar antibody titers between day 35 and day 37 after receiving the second dose (14-16 days after receiving the booster). Notably, vaccinated group C exhibited a higher trend of IgG antibody responses than group D (fig. 10B).

This result demonstrates that non-fusion RBD dimerization of monomeric non-variant SARS-CoV-2 antigen (formulated with a high proportion of dimeric RBD as antigen) is capable of generating an immune response against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), thereby demonstrating the applicability for the preparation of vaccines against SARS-CoV-2 infection.

Furthermore, the results of the neutralizing antibody responses obtained for the different vaccine formulations of the present study showed that after the second dose, all vaccinated groups (groups B to D) obtained equivalent neutralizing antibody levels, again demonstrating the applicability of the non-fusion RBD dimerization of the monomeric non-variant SARS-CoV-2 antigen (with a high proportion of dimeric RBD as antigen) for preparing vaccine compositions against SARS-CoV-2 infection. It was further observed that the addition of an immunostimulant, such as MPLA in group C, in the vaccine formulation positively influences the ability to generate neutralizing antibody titres (fig. 11), confirming the trend mentioned in fig. 10B above.

Example 8: serum neutralization assay for fusion dimeric RBD antigen against SARS-CoV-2 variant

This study assessed the neutralizing capacity of the novel recombinant fusion dimeric RBD variant SARS-CoV-2 antigen against different SARS-CoV-2 gateway variants based on a first monomer comprising RBD derived from the b.1.351 (south africa) variant and a second monomer comprising RBD derived from the b.1.1.7 (uk) variant. This novel SARS-CoV-2 recombinant subunit antigen is designated as a fusion dimeric RBD variant antigen. The recombinant fusion dimeric RBD variant SARS-CoV-2 antigen is the same as described in groups B-F of example 5.

Groups D, E and F of examples 5 and 6 were further selected for this study. These groups included 5-6 week old BALB/c mice vaccinated with 20 μg doses of fusion dimeric RBD variant SARS-CoV-2 antigen. Group D was formulated with the oil-in-water adjuvant used in example 5, group E was formulated with the same adjuvant as group D plus 10 μg/dose of MPLA as the immunostimulant, and group F was formulated with the same adjuvant as group D plus 10 μg/dose of QS-21 as the immunostimulant, according to the description in example 5.

In this study, serum samples of each mouse in groups D, E and F of examples 5 and 6 were taken on day 45 (24 days after receiving the second dose) and analyzed for neutralization capacity against different SARS-CoV-2 variants: wuhan-Hu-1, british (alpha; B.1.1.7), south Africa (beta; B.1.351), brazil (gamma; P.1) and Indian (delta; B.1.617.2) variants.

Neutralizing antibodies in serum against the SARS-CoV-2 Wuhan-Hu-1 isolate, british (alpha; B.1.1.7), south Africa (beta; B.1.351), brazil (gamma; P.1) and India (delta; B.1.617.2) variants were determined by a pseudovirus-based neutralization assay (PBNA). Neutralizing antibodies against the indian variant (delta; b.1.617.2) were determined only in the serum of group D (without immunostimulant).

The neutralizing antibodies in serum were determined by a pseudovirus neutralization assay (PBNA) using SARS-CoV-2 pseudovirus, as described in Nie J.et al.Quantification of SARS-CoV-2neutralizing antibody by a pseudotyped virus-based assay.Nat Protoc.2020Nov;15(11):3699-563715. For this assay, five pseudoviruses were generated that expressed the SARS-CoV-2S protein and luciferase, each pseudovirus expressing a different variant (i.e., wuhan-Hu-1 isolate, british (alpha; B.1.1.7) variant, south Africa (beta; B.1.351) variant, brazil (gamma; P1) variant, and Indian (delta; B.1.617.2) variant) of the corresponding SARS-CoV-2S protein. Differences in spike proteins between variants are known and are well defined in the "classification and definition of SARS-CoV-2 variants" by the american center for disease control and prevention (CDC).

For neutralization assays, each variant pseudovirus supernatant of 200TCID ₅₀ was pre-incubated with serial dilutions of heat-inactivated serum samples of D, E and group F at 37 ℃ for 1 hour and then added to HEK293T cells over-expressed in ACE 2. After 48 hours, the cells were lysed with Britelite Plus luciferase reagent (PERKIN ELMER, waltham, MA, USA). Luminescence was measured for 0.2 seconds using EnSight multimode microplate reader (PERKIN ELMER). The neutralization capacity of the serum samples was calculated by comparing the experimental RLU calculated from the infected cells treated with each serum with the maximum RLU (maximum infectivity calculated from the untreated infected cells) and the minimum RLU (minimum infectivity calculated from the untreated cells) and expressed as percent neutralization:

% neutralization= (RLUmax-RLU experiment)/(RLUmax-RLUmin) ×100.

The results of this study surprisingly demonstrate that immunization of animals with recombinant fusion dimeric RBD SARS-CoV-2 antigen elicited comparable pseudovirus neutralizing antibody titers against four different SARS-CoV-2 variants (e.g., wuhan-Hu-1, british, south Africa and Brazilian variants) in all groups. No significant differences were observed between them (fig. 12). This demonstrates that the titre of neutralising antibodies produced by vaccinating mice with the fusion dimeric RBD antigen remains high, regardless of the variant tested and whether or not an immunostimulant such as MPLA (group E) or qs.21 (group F) is present in the vaccine formulation.

Regarding the pseudovirus neutralizing antibody titers obtained for the indian variants (delta) in group D, the results shown in fig. 12A also indicate that high levels of neutralizing antibody titers were also produced for this variant.

In summary, the results demonstrate that recombinant fusion dimeric RBD variant SARS-CoV-2 antigen is capable of inducing similar levels of antibody responses without the need for immunostimulants. Thus, the increased potential of recombinant fusion dimeric RBD variant SARS-CoV-2 antigen in inducing an immune response was again demonstrated, as has been shown in the previous examples.

Overall, the results demonstrate that vaccine compositions based on the novel recombinant fusion dimeric RBD variant SARS-CoV-2 antigen induce a high level immune response against different SARS-CoV-2 variants (including novel delta variants).

Example 9: safety and immunogenicity studies of fusion dimeric RBD antigens against different SARS-CoV-2 variants were performed in pigs compared to commercially available vaccines.

This study evaluated a novel recombinant subunit antigen for SARS-CoV-2. The novel recombinant subunit antigen is a fusion dimeric RBD antigen comprising two monomers, a first monomer comprising an RBD derived from the b.1.351 (south africa) variant and a second monomer comprising an RBD derived from the b.1.1.7 (uk) variant. This novel SARS-CoV-2 recombinant subunit antigen is designated as a fusion dimeric RBD variant antigen. The recombinant fusion dimeric RBD variant SARS-CoV-2 antigen is the same as described in groups B-F of example 5.

This study assessed the immunogenicity and safety of the recombinant fusion dimeric RBD variant antigen in pigs. Pigs have proven to be more suitable animal models than small animal models for accurately predicting vaccine outcome in humans.

A total of 13 large white and long white pigs (LARGE WHITE-landrace cross-breeding pig) of 8-9 weeks of age were assigned to 3 different groups. Each group received a different vaccine formulation as described below. Group a included 5 pigs and groups B and C included 4 pigs each. Animals received two doses of vaccine at 21 days apart on day 0 and day 21. Each animal received 0.5ml of the following vaccine formulation per dose by the intramuscular route.

The different vaccine formulations administered to pigs were as follows:

Group a (fusion dimeric RBD variant SARS-CoV-2 antigen): animals of this group received a vaccine formulation comprising 20 μg of recombinant fusion dimeric RBD variant SARS-CoV-2 antigen. The vaccine was formulated as follows: an oil-in-water adjuvant formulated as 39mg/ml squalene, 4.7mg/ml polysorbate 80, 4.7mg/ml sorbitan trioleate, 2.64mg/ml sodium citrate and 0.16mg/ml citric acid in a ratio of v/v 50% adjuvant and 50% antigen. Thus, when mixed in a ratio of 50% adjuvant to 50% antigen, a 0.5ml dose of vaccine contains 9.75mg squalene, 1.175mg polysorbate 80, 1.175mg sorbitan trioleate, 0.66mg sodium citrate and 0.04mg citric acid.

Group-B (commercial vaccine): animals in this group received a commercial vaccine Spikevax, COVID-19mRNA vaccine (Moderna Biotech Spain, s.l.). Each dose of Spikevax ml contains 100 μg of mRNA encoding the viral spike protein of SARS-CoV-2 (embedded within SM-102 lipid nanoparticle) at 0.5 ml. The doses of commercial vaccine injected into pigs in this study were obtained from the well-preserved residual volume of vials provided by the public health authorities after vaccination of the human population.

Group-C (control): animals in this group received a mock vaccine comprising PBS.

To evaluate the safety after vaccination with the different vaccine formulations of the present study, the rectal temperature was recorded daily for each dose of day before vaccination (day-1 and day 20), at the time of vaccination (day 0), 4 and 6 hours after vaccination, and three days after the first and second vaccination. The average rectal temperature for each group was calculated (fig. 13).

Animals of group B (commercial vaccine) showed an average rise in body temperature after receiving the first administration, and 6 hours after inoculation were considered to be abnormally elevated, although the animals recovered after one day. After the first dose, statistically significant differences were observed between group a (fusion dimeric RBD variant SARS-CoV-2 antigen) and group B (commercial vaccine) 6 hours post-inoculation. The average body temperature of group B was considered abnormally high and had clinical symptoms in the normal state of the animals (fig. 13). Similar average temperature rise results were observed after the second dose. In contrast, the average body temperatures of group a (fusion dimeric RBD variant SARS-CoV-2 antigen) and group C (control) were considered to be within basal values during all studies, and no clinically relevant differences were observed between the two groups. Thus, the results of this study demonstrate that subunit vaccines based on fusion dimeric RBD variant SARS-CoV-2 antigen have better safety than mRNA vaccines (fig. 13).

To evaluate the immunogenic response of the different vaccine formulations of the present study against different SARS-CoV-2 variants, SARS-CoV-2 neutralizing antibodies in porcine serum were analyzed. Neutralizing antibodies in serum against the SARS-CoV-2 British (alpha; B.1.1.7), south Africa (beta; B.1.351), brazil (gamma; P.1) and India (delta; B.1.617.2) variants were determined by a pseudovirus-based neutralization assay (PBNA).

To conduct this analysis, blood samples of all animals in the different groups were taken for serum collection on day 35 (14 days post vaccination after the second administration).

PBNA used was based on the generation of HIV reporter pseudoviruses expressing the S protein of SARS-CoV-2 and luciferase as described in Nie J.et al.Quantification of SARS-CoV-2 neutralizing antibody by a pseudotyped virus-based assay.Nat Protoc.2020Nov;15(11):3699-563715. Four pseudoviruses were produced that expressed the SARS-CoV-2S protein and luciferase, each pseudovirus expressing a different variant (i.e., the British (alpha; B.1.1.7) variant, the south Africa (beta; B.1.351) variant, the Brazil (gamma; P1) variant and the Indian (delta; B.1.617.2) variant) corresponding SARS-CoV-2S protein. Differences in spike proteins between variants are known and are well defined in the "classification and definition of SARS-CoV-2 variants" by the american center for disease control and prevention (CDC).

For neutralization assays, each variant pseudovirus supernatant of 200TCID ₅₀ was pre-incubated with serial dilutions of heat-inactivated serum samples of groups a to C for 1 hour at 37 ℃ and then added to HEK293T cells over-expressed in ACE 2. After 48 hours, the cells were lysed with Britelite Plus luciferase reagent (PERKIN ELMER, waltham, MA, USA). Luminescence was measured for 0.2 seconds using EnSight multimode microplate reader (PERKIN ELMER). The neutralization capacity of the serum samples was calculated by comparing the experimental RLU calculated from the infected cells treated with each serum with the maximum RLU (maximum infectivity calculated from the untreated infected cells) and the minimum RLU (minimum infectivity calculated from the untreated cells) and expressed as percent neutralization:

% neutralization= (RLUmax-RLU experiment)/(RLUmax-RLUmin) ×100.

The results indicate that the prime-boost regimens performed on both groups A and B induced high neutralizing antibody titers against pseudoviruses containing the SARS-CoV-2 variants of British (alpha; B.1.1.7), south Africa (beta; B.1.351), brazil (gamma; P.1) and India (delta; B.1.617.2) (FIG. 14). Both groups induced comparable neutralizing antibody titers against uk (p=0.190), brazil (p=0.412) and india (p=0.111) variants, but group a based on the fusion dimeric RBD variant SARS-CoV-2 antigen induced significantly higher titers against south africa variant (p=0.015) compared to group B (commercial vaccine). Pair wise comparisons were made using the mannite test; p < 0.05.

Thus, the results clearly demonstrate that vaccines based on fusion dimeric RBD variant SARS-CoV-2 antigen induce neutralizing antibodies against different variants, in particular against uk (alpha), south africa (beta), brazil (gamma) and indian (delta) variants. In vaccinated animals (group A), fusion of dimeric RBD variant SARS-CoV-2 antigen resulted in neutralizing antibody titers comparable to those of the commercial vaccine group (group B), even higher in the neutralization assay against the south Africa (Beta) variant.

This study shows that the fusion dimeric RBD variant SARS-CoV-2 antigen formulated in an oil-in-water adjuvant achieves an optimal balance between immunogenicity and safety, and that it performs even better against certain VOCs (e.g. south africa variants) than existing commercial vaccines.

Example 10: the protective efficacy of the fusion dimeric RBD antigen against heterologous SARS-CoV-2 infection was evaluated in mice.

This study evaluated the protective effect of the recombinant fusion dimeric RBD variant antigen on COVID-19 disease and the pathogenic consequences derived from heterologous SARS-CoV-2 infection in mice. To assess efficacy, the study used an challenge model based on K18-hACE2 transgenic mice.

Since the announcement of a pandemic, several challenge models of small mammalian species have been described. Due to the transgenic expression of the ACE2 human receptor, K18-hACE2 transgenic mice are susceptible to infection by the SARS-CoV-2 virus, as described in Winkler E.S.et al.SARS-CoV-2 infection of human ACE2-transgenic mice causes severe lung inflammation and impaired function.Nature immunology,2020,vol.21,no 11,p.1327-1335 and Yinda C.K.et al.K18-hACE2 mice develop respiratory disease resembling severe COVID-19.PLoS pathogens,2021,vol.17,no 1,p.e1009195. The challenge model was established in K18-hACE2 mice, based on clinical disease, characterized by moderate clinical, pathological and virologic outcomes following SARS-CoV-2 infection.

A total of 18K 18-hACE2 transgenic mice (Jackson laboratories, ref.034860) 4-5 weeks old were assigned to 3 different groups. Each group received a different vaccine formulation as described below. Groups a to C each included 6 mice. Animals received two doses of vaccine at 21 days apart on day 0 and day 21. Each animal received 0.1ml of the following vaccine formulation per dose by the intramuscular route.

The different vaccine formulations administered to mice were as follows:

Group A (fusion dimeric RBD variant SARS-CoV-2 antigen, 20 μg): animals of this group received a vaccine formulation comprising 20 μg of recombinant fusion dimeric RBD variant SARS-CoV-2 antigen. The vaccine was formulated with an oil-in-water adjuvant in the ratio v/v 50% adjuvant and 50% antigen. The oil-in-water adjuvant was formulated as about 39mg/ml squalene, 4.7mg/ml polysorbate 80, 4.7mg/ml sorbitan trioleate, 2.64mg/ml sodium citrate and 0.16mg/ml citric acid. Thus, when mixed in a ratio of 50% adjuvant to 50% antigen, a 0.1ml dose of vaccine contains 1.95mg squalene, 0.235mg polysorbate 80, 0.235mg sorbitan trioleate, 0.132mg sodium citrate and 0.008mg citric acid.

Group B (fusion dimeric RBD variant SARS-CoV-2 antigen, 10 μg): animals of this group received a vaccine formulation comprising 10 μg of recombinant fusion dimeric RBD variant SARS-CoV-2 antigen. The vaccine was formulated with the same adjuvant as group a in the ratio v/v 50% adjuvant and 50% antigen.

Animals were then challenged by intranasal infection on day 35 (2 weeks after the second dose). Each nostril of the animals was subjected to 25. Mu.l of a solution containing SARS-CoV-2 virus at a titer of 10 ⁶TCID₅₀/ml using a micropipette. Thus, each animal received a dose of 10 ³TCID₅₀ SARS-CoV-2 virus. The SARS-CoV-2 isolate used for challenge was Wuhan/Hu-1/2019-like isolate, i.e., hCoV-19/Spain/CT-IrsiCaixa-JP/2020 (GISAID ID EPI _ISL_ 471472), designated Cat02, which was isolated from human patients from Spain in month 3 of 2020. The spike protein of Cat02 isolate has a D614G point mutation compared to Wuhan/Hu-1/2019 strain.

The primary endpoint reporting the protective capacity of the candidate vaccine is weight loss and/or death following challenge.

Thus, to evaluate the protective efficacy following challenge with the different vaccine formulations of the present study, body weight and mortality were monitored during one week (day 42) following challenge. Unprotected animals susceptible to SARS-CoV-2 virus infection are expected to experience weight loss at the end of the study. Thus, body weight is monitored daily during the challenge phase.

Secondary endpoints, including the spread of virus throughout the organism (assessed by RT-PCR and virus titration), are also monitored, especially in organs and tissues belonging to the respiratory system, which are the primary targets for viral infection and replication.

The results surprisingly show that all animals receiving a vaccine (10 or 20 μg/dose) comprising recombinant fusion dimeric RBD variant SARS-CoV-2 antigen survived 7 days after experimental infection (groups a and B). On the other hand, the mortality rate of the control group (group C) was 100%. All animals in the control group died within 5-6 days after challenge (fig. 15).

In addition, animals receiving a vaccine comprising recombinant fusion dimeric RBD variant SARS-CoV-2 antigen (either 10 μg/dose or 20 μg/dose) did not develop weight loss after experimental infection (groups A and B). In contrast, all animals in the control group (PBS mock vaccinated, group C) observed significant weight loss after experimental infection.

Thus, the results clearly demonstrate that recombinant fusion dimeric RBD variant SARS-CoV-2 antigen is capable of protecting against heterologous SARS-CoV-2 infection and is also capable of preventing clinical symptoms of SARS-CoV-2 infection, such as weight loss and death.

Example 11: immunogenicity and safety of booster vaccinations against SARS-CoV-2 in adult subjects using recombinant protein vaccine compositions based on fusion dimeric RBD variants were evaluated.

The study summarises that in use two doses of reference vaccine, for example(BioNTech Manufacturing GmbH) clinical data obtained after evaluating the immunogenicity and safety of a booster dose of the novel fusion heterodimeric RBD variant SARS-CoV-2 antigen composition (named PHH-1V) in healthy adult subjects subjected to complete vaccination against COVID-19. The study was a phase 2, double blind, randomized, active control, multicenter, non-inferior efficacy trial to determine and compare the immunogenicity and safety of PHH-1V at baseline (day 0) and day 14 for subjects who had been fully vaccinated with Pfizer-BioNTech vaccine for at least 182 days and up to 365 days prior to booster vaccination. Approximately 602 adults 18 years old and older are randomly assigned to PHH-1V group or/>A group.

Overall, efficacy studies ultimately evaluate 752 subjects. They were randomly assigned to two different treatment groups in a 2:1 ratio. Cohort 1 (n=504) received a single booster dose of 0.5ml vaccine (PHH-1V) by the intramuscular route on day 0. A dose (0.5 ml) of PHH-1V vaccine comprising 40 μg of the novel fusion heterodimeric RBD variant SARS-CoV-2 antigen, based on a first monomer comprising RBD derived from the B.1.351 SARS-CoV-2 variant and a second monomer comprising RBD derived from the B.1.1.7SARS-CoV-2 variant, as described in example 3, was produced by recombinant DNA technology using a plasmid expression vector in a CHO cell line optimized for stable production. PHH-1V was also adjuvanted with 0.25ml of adjuvant, containing per 0.5ml dose: squalene (9.75 mg), polysorbate 80 (1.175 mg), sorbitan trioleate (1.175 mg), sodium citrate (0.66 mg) and citric acid (0.04 mg). The recombinant fusion heterodimeric RBD variant SARS-CoV-2 antigen is the same as described in example 5 (panels B to F). Cohort 2 (n=248) received a single booster dose of 0.3ml by intramuscular route on day 0Vaccine (BioNTech Manufacturing GmbH).

Accordingly, the subject received a single booster dose on day 0 according to treatment allocation.

Following booster vaccination on day 0, each subject was followed up for 52 weeks (364 days). The total clinical study duration for each subject was up to 56 weeks.

Immunogenicity of booster vaccinations with both vaccines was assessed at baseline and day 14 post booster vaccination. Neutralizing antibody titers against VOC variants such as beta (b.1.351), delta (b.1.617.2) and omnikow (b.1.1.529) variants of SARS-CoV-2 were measured as half maximal inhibitory concentration (IC 50) by a pseudovirus-based neutralization assay (PBNA), as described in example 2, and reported as Geometric Mean Titers (GMT) of the treatment group (table 1). Geometric mean fold increases in binding neutralizing antibody titers relative to baseline (day 0) and day 14 were also determined (GMFR). The percentage of subjects whose bound antibody titer changed by > 4-fold relative to baseline (day 0) and day 14 after boost dose was also calculated.

The GMT and geometric mean fold increase (GMFR) ratio of the treatment mean was estimated from a fitted model MMRM (mixed model repeat measurement) on the log10 scale using LS mean (least squares mean) and reverse transformed.

Table 1: geometric Mean Titer (GMT) of neutralizing antibodies against the relevant Variants (VOCs) at baseline (day 0) and day 14: queue 1 and queue 2.

With PHH-1V orAfter 14 days of treatment, the following results were obtained:

SARS-CoV-2 beta variant (b.1.351): at baseline, log10 transformed geometric mean neutralizing antibody levels between cohort 1 and cohort 2 were similar (66.92 and 60.76, respectively). On day 14, the neutralizing antibody levels of both cohorts increased, with a greater increase in PHH-1V vaccine group (4352.89) Vaccine group (2665.33).

SARS-CoV-2 delta variant (b.1.617.2): at baseline, log10 transformed geometric mean neutralizing antibody levels between cohort 1 and cohort 2 were similar (44.88 and 41.17, respectively). On day 14, the neutralizing antibody levels of both cohorts increased to similar levels: PHH1-V vaccine group (1471.78),Vaccine group (1487.11).

SARS-CoV-2 omimetic Rong Bianti (B.1.1.529): at baseline, log10 transformed geometric mean neutralizing antibody levels between cohort 1 and cohort 2 were similar (32.87 and 29.06, respectively). On day 14, the neutralizing antibody levels of both cohorts increased, with a greater increase in PHH-1V vaccine group (2063.44)Vaccine group (1222.00).

The neutralizing antibody titer results at day 14 post boost inoculation clearly demonstrate that vaccine boost doses based on the novel fusion heterodimeric RBD variant SARS-CoV-2 antigen of PHH-1V induced high levels of neutralizing antibodies against different SARS-CoV-2 related Variants (VOCs).

Surprisingly, the novel fusion heterodimeric RBD antigen of PHH-1V induces a higher neutralizing antibody titer (GMT) than the reference Covid-19 vaccine for the beta (1.351) variant and the armstrong (b.1.1.529) variantsInduced neutralizing antibodies, and for delta variants, resulted in similar high levels of neutralizing antibody titer (GMT). Likewise, PBNA assay for PHH-1V of Wuhan SARS-CoV-2 also demonstrated high neutralizing antibody titers against this variant. Overall, the results indicate that with acceptance/>The immunogenic response of PHH-1V against SARS-CoV-2 variant of interest is enhanced and better compared to the comparative set of (C).

Likewise, the fold increase in neutralizing antibody titer over baseline on day 14 confirms the previous data.

Thus, the geometric mean fold increase (GMFR) ratio of neutralizing antibody titers for beta and amikatone variants SARS-CoV-2 demonstrated that cohort 1/PHH-1V was superior to cohort 2/comparison vaccineThe GMFR ratio was 0.69 (p-value 0.0003) for the beta SARS-CoV-2 variant and the GMFR ratio was 0.68 (p-value 0.0001) for the omimetic SARS-CoV-2 variant. For delta SARS-CoV-2 variants, the results of the doubling of neutralizing antibody titers indicate that queue 1/PHH-1V is not inferior to queue/>GMFR ratio was 1.11 (p-value 0.2446).

The mean fold increase in titer indicated that the increase in the number of cells in the sample was demonstrated for the new SARS-CoV-2 variant versus the control (cohort 2,) In contrast, PHH-1V vaccines comprising the novel fusion heterodimeric RBD variant SARS-CoV-2 antigen have higher and better immunogenicity.

To assess SARS-CoV-2 specific T cell responses, different peptide pools of overlapping SARS-CoV-2 peptides were used, each pool covering the SARS-CoV-2 region S (both pools), RBD, nucleoprotein, membrane and envelope.

Baseline and day 14 presence or absence of T cell responses were analyzed and reported as the number and proportion of subjects responding to each peptide pool at each time point. The total ELISpot response was described as the sum of all positive responses of SFC/106PBMC (peripheral blood mononuclear cells) per peptide pool (after background subtraction). For each subject, if at least one of the pools of peptides is positive for any SARS-CoV-2 at any time, the subject is classified as a responder; if the ELISpot responses were negative, they were classified as no responders.

In addition, T cell assays based on Intracellular Cytokine Staining (ICS) were determined at different time points. ICS assays include the use of flow cytometry to determine Th1/Th2 pathways (e.g., IL-2, IL-4, INFγ) CD4+ and CD8+ T cells. Cd4+ and cd8+ T cell responses at baseline were measured on day 14.

ICS is considered positive if the percentage of cytokine-positive cells in the stimulated sample is three times the value obtained in the unstimulated control and the amplitude after background subtraction is higher than 0.02%. For each subject, if there is at least one positive IFN- γics reaction for any SARS-CoV-2 peptide pool at a determined time point, the subject is classified as a responder; if the responses at these time points were negative, they were classified as no responders.

Following in vitro peptide stimulation of Peripheral Blood Mononuclear Cells (PBMC) by a group of subjects randomized in cohorts 1 and 2, followed by IFN-gamma ELISA spots (IFN-gamma ELISPots), T cell mediated immune responses to SARS-CoV-2 were assessed, wherein the subjects of both cohorts were previously vaccinated with two dosesThen use one dose of PHH-1V (queue 1) or one dose/>(Queue 2) reinforcement.

Different peptide pools of overlapping SARS-CoV-2 spike proteins were used, namely spike protein SA and spike protein SB pools, RBD alpha, RBD beta and RBD delta variant pools. Specifically, peptides used to stimulate PBMCs were: SPIKE SA (194 peptides overlapping the S1-2016 to S1-2196 region of SPIKE protein), SPIKE SB (168 peptides overlapping the S1-2197 to S2-2377 region of SPIKE protein), RBD alpha variant (84 peptides overlapping the RBD region of SARS-CoV-2 alpha variant) and RBD beta variant (84 peptides overlapping the RBD region of SARS-CoV-2 beta variant), and RBD delta variant (84 peptides overlapping the RBD region of SARS-CoV-2 delta variant).

T cell response results showed a significant increase in IFN- γ producing lymphocytes upon in vitro re-stimulation with peptide pools 2 weeks after boosting, compared to the levels observed at baseline. Interestingly, after restimulation of the RBD peptide pool from alpha, beta and delta variant, PHH-1V vaccine boost (cohort 1) induced significant activation of IFN- γ expressing cd4+ T cells. In addition, and those usesThis reaction is significantly stronger compared to the reinforced object (queue 2). For the PHH-1V booster vaccine, no IL-4 expression was detected in activated CD4+ T cells after in vitro restimulation, indicating that the vaccine induced a Th1 biased T cell response. Furthermore, heterologous boosting of the PHH-1V (cohort 1) vaccine was demonstrated to induce activation of IFN-gamma expressing CD8+ T cells.

To assess the tolerability and safety of PHH-1V, the number, percentage and characteristics of local and systemic events elicited from day 0 to day 7 post vaccination were assessed. Overall, acceptThe frequency of local and systemic adverse events reported was higher for the subjects of the control vaccine (cohort 2), cohort 2/>The percentage was higher than in queue 1 (PHH-1V vaccine) in all cases.

From day 0 to day 7, the most commonly reported local reactions that are caused are pain and tenderness. 51.1% of subjects in cohort 1 reported pain, and 68.8% of subjects in cohort 2 reported pain. 48.5% of subjects in queue 1 reported tenderness and 63.5% of subjects in queue 2 reported tenderness. The most commonly reported systemic adverse event that resulted from day 0, 12 hours to the next day was fatigue. Compared to cohort 1 (PHH-1V vaccine) (16.0%, 16.0% and 7.6%), cohort 2Fatigue was reported more frequently at 12 hours (18.7%), day 1 (35.3%) and day 2 (13.1%).

Overall, PHH-1V consistently showed a good safety profile in adult subjects, and the level of neutralizing antibodies against different SARS-CoV-2 related Variants (VOCs) was high and increased. Notably, PHH-1V also exhibited higher and increased neutralization titers against the Omnkorn SARS-CoV-2 variant than the comparative vaccine, even though highly mutated spike proteins were observed in this novel B.1.1.529 variant. These results support that PHH-1V candidate vaccines based on the novel fusion heterodimeric SARS-CoV-2 variant antigen have higher and better immunogenicity than the comparative vaccine. Thus, the results support PHH-1V to be effective against different SARS-CoV-2 variants and potentially provide protection against future SARS-CoV-2-related variants.

Example 12: the natural-like structure was evaluated by surface plasmon resonance.

As shown and discussed in the previous examples, PHH-1 candidate vaccines elicit strong humoral responses through high titers of neutralizing antibodies. The generation of a natural-like protein subunit vaccine is critical because the natural structure is a powerful marker that better elicits neutralizing antibodies with higher affinity to antigens present in wild-type viruses. To confirm that the fusion heterodimeric RBD variant SARS-CoV-2 antigen has a natural-like structure, surface Plasmon Resonance (SPR) analysis was performed with human ACE2 by ACROBiosystems. Fc-labeled ACE2 (AC 2-H5257, ACROBiosystems) was immobilized in S-series sensor chip CM5 (Cytiva) on Biacore T200 (Cytiva) using a human antibody capture kit (Cytiva). Affinity measurements were obtained using 8 different RBD heterodimer concentrations. Antigen structural simulations were performed using UCSF ChimeraX.

The specific materials and the method are as follows:

human running buffer:

IxHEPES (10mM HEPES,150mM NaCl,3mMEDTA) containing 0.005% Tween-20, pH7.4. Human antibody Capture kit (BR-1008-39, cytiva): anti-human IgG (Fc) antibody (500. Mu.g/mL), immobilization buffer (10 mM sodium acetate, pH 5.0). Regeneration buffer (3M magnesium chloride).

Chip preparation

Anti-human IgG (Fc) antibodies were diluted to 25. Mu.g/mL in immobilization buffer 10mM sodium acetate (pH 5.0) (50. Mu.L of anti-human IgG (Fc) antibodies were added to 950. Mu.L of immobilization buffer for 8 channels). The activator was prepared by mixing 400mM EDC and 100mM NHS (GE) prior to injection. The CM5 sensor chip was activated using a mixture with a flow rate of 10 μl/min for 420 seconds. 25 μg/mL of anti-human IgG (Fc) antibody in an immobilization buffer of 10mM sodium acetate (pH 5.0) was then injected into the FC2 sample channel at a flow rate of 10 μL/min for 420 seconds, which typically resulted in an immobilization level of 9000 to 14000 RU. The chip was deactivated using 3M magnesium chloride (Cytiva) at a flow rate of 20. Mu.L/min for 30 seconds. The preparation method of the reference surface FC1 channel should be the same as that of the active surface FC2 channel.

(See GE human antibody capture kit Specification 29237227 AB).

Ligand protein reconstitution

Human ACE2/ACEH protein was reconstituted according to COA. To avoid surface adsorption losses and inactivation, the split-up amount of reconstituted protein should not be less than 10 μg per vial, see table 2.

Table 2: reconstruction information of ligand proteins

Name of the name	Cat.No.	Concentration (mg/mL)
			Human ACE2/ACEH protein, fc Tag	AC2-H5257	0.355

Captured ligands

Human ACE2 was diluted to 10 μg/mL with running buffer and then injected into the sample channel (FC 2) at a flow rate of 10 μl/min to reach a capture level of about 300 RU. The reference channel (FC 1) does not require a ligand capture step.

Analyte analysis by a multi-cycle method

The customer samples were diluted to the corresponding concentrations with running buffer (table 6). The diluted sample was injected into channels FC1-FC2 at a flow rate of 30pL/min, subjected to an association phase of 90 seconds, and then subjected to dissociation of 210 seconds.

The association and dissociation processes were all performed in running buffer. After each interaction cycle analysis, the sensor chip surface should be completely regenerated for 30s using 3M magnesium chloride as injection buffer at a flow rate of 20pL/min to remove the ligand and any bound analyte.

Table 3: affinity test parameters for binding of antibody samples to human ACE2 protein.

Other details

The whole process is carried out in running buffer. Other buffers used in the SPR assay were the same as the injection buffer and were placed in the tray of the sample chamber.

Results

Kinetic affinity (SPR)

Affinity was analyzed by Biacore Insight Evaluation in Biacore T200. The reference channel (FC 1) is used for background subtraction.

Table 4: summary of affinity assays between antibody samples and human ACE2 protein.

Conclusion:

As shown in Table 4, the fusion heterodimeric RBD variant SARS-CoV-2 antigen has an affinity constant of 98.5pM for hACE2, indicating excellent binding affinity to its natural ligand, a clear indicator of a natural-like structure, which explains the potent neutralizing antibodies raised against the different SARS-CoV-2 virus variants.

Clause of (b)

The invention also includes the following clauses:

1. A protein subunit vaccine comprising at least one antigen comprising two monomers from at least one variant of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), wherein each monomer is selected from the group consisting of the S1 subunit of spike protein, or the Receptor Binding Domain (RBD) of spike protein, or any immunogenic fragment thereof, and wherein the two monomers are chemically bound to each other, optionally through a linker, thereby forming a dimer.

2. The protein subunit vaccine of clause 1, wherein the antigen comprises or consists of two monomers, wherein the two monomers comprise or consist of a Receptor Binding Domain (RBD) of spike protein from at least one variant of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2).

3. A protein subunit vaccine according to clause 2, wherein said Receptor Binding Domain (RBD) of the spike protein hybridizes over its entire length to SEQ ID NO: 1. SEQ ID NO:3 or SEQ ID NO:4, has at least 90% sequence identity.

4. The protein subunit vaccine of any one of clauses 1-3, wherein at least one variant of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is selected from the group consisting of: wuhan-Hu-1 pneumovirus isolates (GenBank accession number: MN 908947), linage B.1.1.28 (Brazil variant), linage B.1.351 (south Africa variant), linage B.1.427 or Linage B.1.429 (California variant), linage B.1.617 (Indian variant) or Linage B.1.1.7 (British variant) or any combination thereof.

5. The protein subunit vaccine of any one of clauses 1-4, wherein the dimer is a fusion dimer comprising or consisting of two monomers, wherein the two monomers are part of a single polypeptide.

6. The protein subunit vaccine of clause 5, wherein the fusion dimer consists of a first RBD monomer from a first SARS-CoV-2 variant and a second RBD monomer from a second, different SARS-CoV-2 variant.

7. The protein subunit vaccine of clause 6, wherein the fusion dimer consists of a first monomer derived from Linage b.1.351 (south africa SARS-CoV-2 variant) and a second monomer derived from Linage b.1.1.7 (uk SARS-CoV-2 variant).

8. The protein subunit vaccine of clause 7, wherein the fusion dimer hybridizes over its entire length to SEQ ID NO:5 has at least 90% sequence identity.

9. The protein subunit vaccine of clause 8, wherein the fusion dimer comprises the amino acid sequence of SEQ ID NO:5 or consists of SEQ ID NO: 5.

10. The protein subunit vaccine of any one of clauses 1-4, wherein the dimer consists of a non-fused dimer comprising or consisting of two monomers, wherein the two monomers are bound by a reversible bond.

11. The protein subunit vaccine of clause 10, wherein the non-fusion dimer consists of a first monomer and a second monomer, both of which are derived from Wuhan-Hu-1 pneumovirus isolate (GenBank accession number: MN 908947).

12. The protein subunit vaccine of any one of clauses 10 and 11, wherein the first monomer and the second monomer are identical to SEQ ID NO:1 has at least 90% sequence identity.

13. The protein subunit vaccine of clause 12, wherein the non-fusion dimer comprises the amino acid sequence of SEQ ID NO:1 or consists of SEQ ID NO: 1.

14. The protein subunit vaccine of any one of clauses 1 to 13, wherein the protein subunit vaccine comprises a total amount of 5 to 50 μg of antigen per dose.

15. The protein subunit vaccine of any one of clauses 1 to 14, further comprising at least an adjuvant.

16. The protein subunit vaccine of clause 15, wherein the adjuvant comprises about 10 to 60mg/ml squalene, 1 to 6mg/ml polysorbate 80, 1 to 6mg/ml sorbitan trioleate, 0.5 to 6mg/ml sodium citrate, and 0.01 to 0.5mg/ml citric acid.

17. The protein subunit vaccine of any one of clauses 1 to 16, wherein the protein subunit vaccine further comprises monophosphoryl lipid a (MPLA) and/or C ₉₂O₄₆H₁₄₈ (QS-21) as an immunostimulant.

18. A protein subunit vaccine according to any one of clauses 1 to 17 for use in generating an immunogenic and/or protective immune response against at least one variant of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virus in a subject in need thereof.

19. The protein subunit vaccine for use according to clause 18, wherein the protein subunit vaccine is administered to a subject in need thereof in a single dose or in multiple doses, preferably in two doses.

20. The protein subunit vaccine for use according to any one of clauses 18 and 19, wherein the protein subunit vaccine is administered to a subject in need thereof at a schedule comprising a first dose or prime and a second dose or boost.

21. The protein subunit vaccine for use according to any one of clauses 18 to 20, wherein the second dose is administered one week, preferably two weeks, three weeks or four weeks after the first dose.

22. The protein subunit vaccine for use according to any one of clauses 18 to 21, wherein the protein subunit vaccine is administered intramuscularly or subcutaneously.

23. A kit comprising at least one dose, preferably two or more doses, of a protein subunit vaccine according to any one of clauses 1 to 17.

24. A protein subunit vaccine comprising at least one antigen, characterized in that the at least one antigen comprises at least one monomer from at least one variant of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), wherein the at least one monomer is selected from the group consisting of the S1 subunit of spike protein, or the Receptor Binding Domain (RBD) of spike protein, or any immunogenic fragment thereof, and wherein the protein subunit vaccine further comprises at least one adjuvant, wherein the adjuvant comprises about 10 to 60mg/ml squalene, 1 to 6mg/ml polysorbate 80, 1 to 6mg/ml sorbitan trioleate, 0.5 to 6mg/ml sodium citrate, and 0.01 to 0.5mg/ml citric acid.

25. The protein subunit vaccine of clause 24, wherein at least one monomer comprises or consists of a recombinant Receptor Binding Domain (RBD) of a spike protein or an immunogenic fragment thereof.

26. The protein subunit vaccine of clause 25, wherein the Receptor Binding Domain (RBD) hybridizes over its entire length to SEQ ID NO: 1. SEQ ID NO:3 or SEQ ID NO:4, has at least 90% sequence identity.

27. The protein subunit vaccine of clause 24, wherein at least one monomer is a recombinant S1 subunit of spike protein or an immunogenic fragment thereof.

28. The protein subunit vaccine of clause 27, wherein the S1 subunit is identical to SEQ ID NO:2 has at least 90% sequence identity.

29. The protein subunit vaccine of any one of clauses 24 to 28, wherein the protein subunit vaccine further comprises monophosphoryl lipid a (MPLA) and/or C ₉₂O₄₆H₁₄₈ (QS-21) as an immunostimulant.

30. The protein subunit vaccine of any one of clauses 25 and 26, wherein at least one monomer is RBD of spike protein and the immunostimulatory agent is monophosphoryl lipid a (MPLA).

31. The protein subunit vaccine of any one of clauses 27 and 28, wherein at least one monomer is the S1 subunit of spike protein and the immunostimulatory agent is monophosphoryl lipid a (MPLA).

32. The protein subunit vaccine of any one of clauses 24 to 31, wherein at least one antigen is a monomer or a multimer, preferably a dimer.

33. The protein subunit vaccine of clause 24, wherein the protein subunit vaccine comprises at least one antigen comprising two monomers from at least one variant of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) or two monomers from at least one variant of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), wherein each of the monomers is selected from the group consisting of the S1 subunit of spike protein, or the Receptor Binding Domain (RBD) of spike protein, or any immunogenic fragment thereof, and wherein the two monomers are optionally chemically bound to each other by a linker, thereby forming a dimer.

34. The protein subunit vaccine of clause 33, wherein the two monomers are RBDs of spike proteins from at least one variant of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2).

35. The protein subunit vaccine of any one of clauses 33 and 34, wherein the first monomer is from a first SARS-CoV-2 variant and the second monomer is from a second, different SARS-CoV-2 variant.

36. The protein subunit vaccine of any one of clauses 24 to 35, wherein at least one variant of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is selected from the group consisting of: wuhan-Hu-1 pneumovirus isolates (GenBank accession number: MN 908947), linage B.1.1.28 (Brazil variant), linage B.1.351 (south Africa variant), linage B.1.427 or Linage B.1.429 (California variant), linage B.1.617 (Indian variant) or Linage B.1.1.7 (British variant) or any combination thereof.

37. The protein subunit vaccine of any one of clauses 33 to 35, wherein the dimer is a fusion dimer comprising or consisting of: a first monomer derived from Linage b.1.351 (south african SARS-CoV-2 variant) and a second monomer derived from Linage b.1.1.7 (uk SARS-CoV-2 variant), wherein the two monomers of the fusion dimer are part of a single polypeptide.

38. A protein subunit vaccine according to clause 37, wherein the fusion dimer hybridizes over its entire length to SEQ ID NO:5 has at least 90% sequence identity.

39. The protein subunit vaccine of any one of clauses 33 to 35, wherein the dimer is a non-fusion dimer comprising or consisting of a first monomer and a second monomer, each derived from Wuhan-Hu-1 pneumovirus isolate (GenBank accession number: MN 908947), wherein the two monomers of the non-fusion dimer are bound by a reversible bond.

40. The protein subunit vaccine of clause 39, wherein the first monomer and the second monomer are identical to SEQ ID NO:1 has at least 90% sequence identity.

41. The protein subunit vaccine of clauses 24-36, 39, and 40, wherein the protein subunit vaccine comprises a mixture of at least monomeric RBD antigen and at least dimeric RBD antigen, wherein at least 45% of the total antigen comprised in the protein subunit vaccine is dimeric RBD antigen.

42. The protein subunit vaccine of any one of clauses 33 to 41, wherein the protein subunit vaccine further comprises monophosphoryl lipid a (MPLA) and/or C ₉₂O₄₆H₁₄₈ (QS-21) as an immunostimulant.

43. The protein subunit vaccine of any one of clauses 24 to 42, wherein the protein subunit vaccine comprises a total amount of 5 to 50 μg of antigen per dose.

44. The protein subunit vaccine of any one of clauses 24 to 43, wherein the adjuvant comprises about 39mg/ml squalene, 4.7mg/ml polysorbate 80, 4.7mg/ml sorbitan trioleate, 2.64mg/ml sodium citrate, and 0.16mg/ml citric acid.

45. A protein subunit vaccine according to any one of clauses 24 to 44 for use in generating an immunogenic and/or protective immune response against at least one variant of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virus in a subject in need thereof.

46. The protein subunit vaccine for use according to clause 45, wherein the protein subunit vaccine is administered to a subject in need thereof in a single dose or in multiple doses, preferably in two doses.

47. The protein subunit vaccine for use of clause 46, wherein the protein subunit vaccine is administered to a subject in need thereof on a schedule comprising a first dose or prime and a second dose or boost.

48. The protein subunit vaccine for use according to any one of clauses 45 to 47, wherein the second dose is administered one week, preferably two weeks, three weeks or four weeks after the first dose.

49. The protein subunit vaccine for use according to any one of clauses 45 to 48, wherein the protein subunit vaccine is administered intramuscularly or subcutaneously.

50. A kit comprising at least one dose, preferably two or more doses, of a protein subunit vaccine according to any one of clauses 24 to 45.

Sequence listing

<110> Haibles science Co., ltd

Haibles laboratory Co Ltd

<120> SARS-CoV-2 subunit vaccine

<130> 907 117

<150> EP21 382 750.4

<151> 2021-08-09

<150> EP21 382 410.5

<151> 2021-05-06

<160> 10

<170> BiSSAP 1.3.6

<210> 1

<211> 223

<212> PRT

<213> Artificial sequence

<220>

<223> RBD monomer amino acid residues 319-541 of SARS-CoV-2 spike protein (Whan variant)

<400> 1

Arg Val Gln Pro Thr Glu Ser Ile Val Arg Phe Pro Asn Ile Thr Asn

1 5 10 15

Leu Cys Pro Phe Gly Glu Val Phe Asn Ala Thr Arg Phe Ala Ser Val

20 25 30

Tyr Ala Trp Asn Arg Lys Arg Ile Ser Asn Cys Val Ala Asp Tyr Ser

35 40 45

Val Leu Tyr Asn Ser Ala Ser Phe Ser Thr Phe Lys Cys Tyr Gly Val

50 55 60

Ser Pro Thr Lys Leu Asn Asp Leu Cys Phe Thr Asn Val Tyr Ala Asp

65 70 75 80

Ser Phe Val Ile Arg Gly Asp Glu Val Arg Gln Ile Ala Pro Gly Gln

85 90 95

Thr Gly Lys Ile Ala Asp Tyr Asn Tyr Lys Leu Pro Asp Asp Phe Thr

100 105 110

Gly Cys Val Ile Ala Trp Asn Ser Asn Asn Leu Asp Ser Lys Val Gly

115 120 125

Gly Asn Tyr Asn Tyr Leu Tyr Arg Leu Phe Arg Lys Ser Asn Leu Lys

130 135 140

Pro Phe Glu Arg Asp Ile Ser Thr Glu Ile Tyr Gln Ala Gly Ser Thr

145 150 155 160

Pro Cys Asn Gly Val Glu Gly Phe Asn Cys Tyr Phe Pro Leu Gln Ser

165 170 175

Tyr Gly Phe Gln Pro Thr Asn Gly Val Gly Tyr Gln Pro Tyr Arg Val

180 185 190

Val Val Leu Ser Phe Glu Leu Leu His Ala Pro Ala Thr Val Cys Gly

195 200 205

Pro Lys Lys Ser Thr Asn Leu Val Lys Asn Lys Cys Val Asn Phe

210 215 220

<210> 2

<211> 673

<212> PRT

<213> Artificial sequence

<220>

<223> S1 subunit monomer amino acid residues 13-685 of SARS-CoV-2 spike protein (Wohan variant)

<400> 2

Ser Gln Cys Val Asn Leu Thr Thr Arg Thr Gln Leu Pro Pro Ala Tyr

1 5 10 15

Thr Asn Ser Phe Thr Arg Gly Val Tyr Tyr Pro Asp Lys Val Phe Arg

20 25 30

Ser Ser Val Leu His Ser Thr Gln Asp Leu Phe Leu Pro Phe Phe Ser

35 40 45

Asn Val Thr Trp Phe His Ala Ile His Val Ser Gly Thr Asn Gly Thr

50 55 60

Lys Arg Phe Asp Asn Pro Val Leu Pro Phe Asn Asp Gly Val Tyr Phe

65 70 75 80

Ala Ser Thr Glu Lys Ser Asn Ile Ile Arg Gly Trp Ile Phe Gly Thr

85 90 95

Thr Leu Asp Ser Lys Thr Gln Ser Leu Leu Ile Val Asn Asn Ala Thr

100 105 110

Asn Val Val Ile Lys Val Cys Glu Phe Gln Phe Cys Asn Asp Pro Phe

115 120 125

Leu Gly Val Tyr Tyr His Lys Asn Asn Lys Ser Trp Met Glu Ser Glu

130 135 140

Phe Arg Val Tyr Ser Ser Ala Asn Asn Cys Thr Phe Glu Tyr Val Ser

145 150 155 160

Gln Pro Phe Leu Met Asp Leu Glu Gly Lys Gln Gly Asn Phe Lys Asn

165 170 175

Leu Arg Glu Phe Val Phe Lys Asn Ile Asp Gly Tyr Phe Lys Ile Tyr

180 185 190

Ser Lys His Thr Pro Ile Asn Leu Val Arg Asp Leu Pro Gln Gly Phe

195 200 205

Ser Ala Leu Glu Pro Leu Val Asp Leu Pro Ile Gly Ile Asn Ile Thr

210 215 220

Arg Phe Gln Thr Leu Leu Ala Leu His Arg Ser Tyr Leu Thr Pro Gly

225 230 235 240

Asp Ser Ser Ser Gly Trp Thr Ala Gly Ala Ala Ala Tyr Tyr Val Gly

245 250 255

Tyr Leu Gln Pro Arg Thr Phe Leu Leu Lys Tyr Asn Glu Asn Gly Thr

260 265 270

Ile Thr Asp Ala Val Asp Cys Ala Leu Asp Pro Leu Ser Glu Thr Lys

275 280 285

Cys Thr Leu Lys Ser Phe Thr Val Glu Lys Gly Ile Tyr Gln Thr Ser

290 295 300

Asn Phe Arg Val Gln Pro Thr Glu Ser Ile Val Arg Phe Pro Asn Ile

305 310 315 320

Thr Asn Leu Cys Pro Phe Gly Glu Val Phe Asn Ala Thr Arg Phe Ala

325 330 335

Ser Val Tyr Ala Trp Asn Arg Lys Arg Ile Ser Asn Cys Val Ala Asp

340 345 350

Tyr Ser Val Leu Tyr Asn Ser Ala Ser Phe Ser Thr Phe Lys Cys Tyr

355 360 365

Gly Val Ser Pro Thr Lys Leu Asn Asp Leu Cys Phe Thr Asn Val Tyr

370 375 380

Ala Asp Ser Phe Val Ile Arg Gly Asp Glu Val Arg Gln Ile Ala Pro

385 390 395 400

Gly Gln Thr Gly Lys Ile Ala Asp Tyr Asn Tyr Lys Leu Pro Asp Asp

405 410 415

Phe Thr Gly Cys Val Ile Ala Trp Asn Ser Asn Asn Leu Asp Ser Lys

420 425 430

Val Gly Gly Asn Tyr Asn Tyr Leu Tyr Arg Leu Phe Arg Lys Ser Asn

435 440 445

Leu Lys Pro Phe Glu Arg Asp Ile Ser Thr Glu Ile Tyr Gln Ala Gly

450 455 460

Ser Thr Pro Cys Asn Gly Val Glu Gly Phe Asn Cys Tyr Phe Pro Leu

465 470 475 480

Gln Ser Tyr Gly Phe Gln Pro Thr Asn Gly Val Gly Tyr Gln Pro Tyr

485 490 495

Arg Val Val Val Leu Ser Phe Glu Leu Leu His Ala Pro Ala Thr Val

500 505 510

Cys Gly Pro Lys Lys Ser Thr Asn Leu Val Lys Asn Lys Cys Val Asn

515 520 525

Phe Asn Phe Asn Gly Leu Thr Gly Thr Gly Val Leu Thr Glu Ser Asn

530 535 540

Lys Lys Phe Leu Pro Phe Gln Gln Phe Gly Arg Asp Ile Ala Asp Thr

545 550 555 560

Thr Asp Ala Val Arg Asp Pro Gln Thr Leu Glu Ile Leu Asp Ile Thr

565 570 575

Pro Cys Ser Phe Gly Gly Val Ser Val Ile Thr Pro Gly Thr Asn Thr

580 585 590

Ser Asn Gln Val Ala Val Leu Tyr Gln Asp Val Asn Cys Thr Glu Val

595 600 605

Pro Val Ala Ile His Ala Asp Gln Leu Thr Pro Thr Trp Arg Val Tyr

610 615 620

Ser Thr Gly Ser Asn Val Phe Gln Thr Arg Ala Gly Cys Leu Ile Gly

625 630 635 640

Ala Glu His Val Asn Asn Ser Tyr Glu Cys Asp Ile Pro Ile Gly Ala

645 650 655

Gly Ile Cys Ala Ser Tyr Gln Thr Gln Thr Asn Ser Pro Arg Arg Ala

660 665 670

Arg

<210> 3

<211> 219

<212> PRT

<213> Artificial sequence

<220>

<223> B1.1.7 amino acid residues 319-537 of SARS-CoV-2 spike protein RBD monomer of variant

<400> 3

Arg Val Gln Pro Thr Glu Ser Ile Val Arg Phe Pro Asn Ile Thr Asn

1 5 10 15

Leu Cys Pro Phe Gly Glu Val Phe Asn Ala Thr Arg Phe Ala Ser Val

20 25 30

Tyr Ala Trp Asn Arg Lys Arg Ile Ser Asn Cys Val Ala Asp Tyr Ser

35 40 45

Val Leu Tyr Asn Ser Ala Ser Phe Ser Thr Phe Lys Cys Tyr Gly Val

50 55 60

Ser Pro Thr Lys Leu Asn Asp Leu Cys Phe Thr Asn Val Tyr Ala Asp

65 70 75 80

Ser Phe Val Ile Arg Gly Asp Glu Val Arg Gln Ile Ala Pro Gly Gln

85 90 95

Thr Gly Lys Ile Ala Asp Tyr Asn Tyr Lys Leu Pro Asp Asp Phe Thr

100 105 110

Gly Cys Val Ile Ala Trp Asn Ser Asn Asn Leu Asp Ser Lys Val Gly

115 120 125

Gly Asn Tyr Asn Tyr Leu Tyr Arg Leu Phe Arg Lys Ser Asn Leu Lys

130 135 140

Pro Phe Glu Arg Asp Ile Ser Thr Glu Ile Tyr Gln Ala Gly Ser Thr

145 150 155 160

Pro Cys Asn Gly Val Glu Gly Phe Asn Cys Tyr Phe Pro Leu Gln Ser

165 170 175

Tyr Gly Phe Gln Pro Thr Tyr Gly Val Gly Tyr Gln Pro Tyr Arg Val

180 185 190

Val Val Leu Ser Phe Glu Leu Leu His Ala Pro Ala Thr Val Cys Gly

195 200 205

Pro Lys Lys Ser Thr Asn Leu Val Lys Asn Lys

210 215

<210> 4

<211> 219

<212> PRT

<213> Artificial sequence

<220>

<223> Amino acid residues 319-537 of SARS-CoV-2 spike protein RBD monomer of B.1.351 variant

<400> 4

Arg Val Gln Pro Thr Glu Ser Ile Val Arg Phe Pro Asn Ile Thr Asn

1 5 10 15

Leu Cys Pro Phe Gly Glu Val Phe Asn Ala Thr Arg Phe Ala Ser Val

20 25 30

Tyr Ala Trp Asn Arg Lys Arg Ile Ser Asn Cys Val Ala Asp Tyr Ser

35 40 45

Val Leu Tyr Asn Ser Ala Ser Phe Ser Thr Phe Lys Cys Tyr Gly Val

50 55 60

Ser Pro Thr Lys Leu Asn Asp Leu Cys Phe Thr Asn Val Tyr Ala Asp

65 70 75 80

Ser Phe Val Ile Arg Gly Asp Glu Val Arg Gln Ile Ala Pro Gly Gln

85 90 95

Thr Gly Asn Ile Ala Asp Tyr Asn Tyr Lys Leu Pro Asp Asp Phe Thr

100 105 110

Gly Cys Val Ile Ala Trp Asn Ser Asn Asn Leu Asp Ser Lys Val Gly

115 120 125

Gly Asn Tyr Asn Tyr Leu Tyr Arg Leu Phe Arg Lys Ser Asn Leu Lys

130 135 140

Pro Phe Glu Arg Asp Ile Ser Thr Glu Ile Tyr Gln Ala Gly Ser Thr

145 150 155 160

Pro Cys Asn Gly Val Lys Gly Phe Asn Cys Tyr Phe Pro Leu Gln Ser

165 170 175

Tyr Gly Phe Gln Pro Thr Tyr Gly Val Gly Tyr Gln Pro Tyr Arg Val

180 185 190

Val Val Leu Ser Phe Glu Leu Leu His Ala Pro Ala Thr Val Cys Gly

195 200 205

Pro Lys Lys Ser Thr Asn Leu Val Lys Asn Lys

210 215

<210> 5

<211> 438

<212> PRT

<213> Artificial sequence

<220>

<223> Fusion dimeric RBD variant SARS-CoV-2 antigen

<400> 5

Arg Val Gln Pro Thr Glu Ser Ile Val Arg Phe Pro Asn Ile Thr Asn

1 5 10 15

Leu Cys Pro Phe Gly Glu Val Phe Asn Ala Thr Arg Phe Ala Ser Val

20 25 30

Tyr Ala Trp Asn Arg Lys Arg Ile Ser Asn Cys Val Ala Asp Tyr Ser

35 40 45

Val Leu Tyr Asn Ser Ala Ser Phe Ser Thr Phe Lys Cys Tyr Gly Val

50 55 60

Ser Pro Thr Lys Leu Asn Asp Leu Cys Phe Thr Asn Val Tyr Ala Asp

65 70 75 80

Ser Phe Val Ile Arg Gly Asp Glu Val Arg Gln Ile Ala Pro Gly Gln

85 90 95

Thr Gly Asn Ile Ala Asp Tyr Asn Tyr Lys Leu Pro Asp Asp Phe Thr

100 105 110

Gly Cys Val Ile Ala Trp Asn Ser Asn Asn Leu Asp Ser Lys Val Gly

115 120 125

Gly Asn Tyr Asn Tyr Leu Tyr Arg Leu Phe Arg Lys Ser Asn Leu Lys

130 135 140

Pro Phe Glu Arg Asp Ile Ser Thr Glu Ile Tyr Gln Ala Gly Ser Thr

145 150 155 160

Pro Cys Asn Gly Val Lys Gly Phe Asn Cys Tyr Phe Pro Leu Gln Ser

165 170 175

Tyr Gly Phe Gln Pro Thr Tyr Gly Val Gly Tyr Gln Pro Tyr Arg Val

180 185 190

Val Val Leu Ser Phe Glu Leu Leu His Ala Pro Ala Thr Val Cys Gly

195 200 205

Pro Lys Lys Ser Thr Asn Leu Val Lys Asn Lys Arg Val Gln Pro Thr

210 215 220

Glu Ser Ile Val Arg Phe Pro Asn Ile Thr Asn Leu Cys Pro Phe Gly

225 230 235 240

Glu Val Phe Asn Ala Thr Arg Phe Ala Ser Val Tyr Ala Trp Asn Arg

245 250 255

Lys Arg Ile Ser Asn Cys Val Ala Asp Tyr Ser Val Leu Tyr Asn Ser

260 265 270

Ala Ser Phe Ser Thr Phe Lys Cys Tyr Gly Val Ser Pro Thr Lys Leu

275 280 285

Asn Asp Leu Cys Phe Thr Asn Val Tyr Ala Asp Ser Phe Val Ile Arg

290 295 300

Gly Asp Glu Val Arg Gln Ile Ala Pro Gly Gln Thr Gly Lys Ile Ala

305 310 315 320

Asp Tyr Asn Tyr Lys Leu Pro Asp Asp Phe Thr Gly Cys Val Ile Ala

325 330 335

Trp Asn Ser Asn Asn Leu Asp Ser Lys Val Gly Gly Asn Tyr Asn Tyr

340 345 350

Leu Tyr Arg Leu Phe Arg Lys Ser Asn Leu Lys Pro Phe Glu Arg Asp

355 360 365

Ile Ser Thr Glu Ile Tyr Gln Ala Gly Ser Thr Pro Cys Asn Gly Val

370 375 380

Glu Gly Phe Asn Cys Tyr Phe Pro Leu Gln Ser Tyr Gly Phe Gln Pro

385 390 395 400

Thr Tyr Gly Val Gly Tyr Gln Pro Tyr Arg Val Val Val Leu Ser Phe

405 410 415

Glu Leu Leu His Ala Pro Ala Thr Val Cys Gly Pro Lys Lys Ser Thr

420 425 430

Asn Leu Val Lys Asn Lys

435

<210> 6

<211> 19

<212> PRT

<213> Artificial sequence

<220>

<223> Signal peptide

<400> 6

Met Gly Trp Ser Cys Ile Ile Leu Phe Leu Val Ala Thr Ala Thr Gly

1 5 10 15

Val His Ser

<210> 7

<211> 1398

<212> DNA

<213> Artificial sequence

<220>

<223> Fusion dimeric RBD variant SARS-CoV-2 antigen with Kozak sequence, signal peptide, histidine tag and stop codon

<400> 7

gccaccatgg gctggtcctg catcatcctg tttctggtgg ctaccgctac cggcgtgcac 60

agtagagtgc agcctaccga gtctatcgtg cggttcccca acatcaccaa cctgtgtcct 120

ttcggcgagg tgttcaacgc caccagattc gcctctgtgt acgcctggaa ccggaagcgg 180

atctctaact gcgtggccga ctactccgtg ctgtacaact ccgcctcctt cagcaccttc 240

aagtgctacg gcgtgtcccc taccaagctg aacgacctgt gcttcaccaa cgtgtacgcc 300

gactccttcg tgatcagagg cgacgaagtg cggcagatcg ctcctggaca gaccggcaat 360

atcgccgact acaactacaa gctgcccgac gacttcaccg gctgtgtgat cgcttggaac 420

tccaacaacc tggactccaa agtcggcggc aactacaatt acctgtaccg gctgttccgg 480

aagtccaacc tgaagccttt cgagcgggac atctccaccg agatctacca ggctggcagc 540

accccttgta atggcgtgaa gggcttcaac tgctacttcc cactgcagtc ctacggcttc 600

cagcctacct atggcgtggg ctaccagcct tacagagtgg tggtgctgtc cttcgagctg 660

ctgcatgctc ctgctaccgt gtgcggccct aagaaatcta ccaacctggt caagaacaag 720

cgggtgcagc ccactgagag cattgtgcgc ttccctaata tcacaaatct gtgccccttc 780

ggggaagtct ttaatgctac ccgcttcgct tccgtgtatg cttggaatag aaagcggatc 840

agcaattgcg tcgccgatta cagcgtcctg tacaatagcg ccagcttctc cacctttaag 900

tgttatggcg tcagccccac aaagctcaac gatctctgtt ttaccaatgt ctacgccgat 960

agctttgtga ttcgcggaga tgaagtccgc cagatcgcac caggccagac tggaaagatc 1020

gctgattaca attataagct ccctgatgat ttcacaggat gcgttatcgc ctggaatagc 1080

aacaacctcg acagcaaagt tggagggaat tacaactacc tctaccgcct cttcagaaag 1140

agcaacctca agccatttga gagagacatc agtacagaaa tctatcaggc cggctctacc 1200

ccttgcaacg gcgtcgaggg gtttaactgt tactttcccc tgcaatctta tgggtttcag 1260

cccacatacg gcgtggggta tcaaccctat cgcgtggtgg ttctgagttt cgaactcctg 1320

cacgccccag ccacagtgtg tggcccaaaa aagagcacca atctcgttaa gaacaagcac 1380

catcaccatc accattag 1398

<210> 8

<211> 1314

<212> DNA

<213> Artificial sequence

<220>

<223> Fusion dimeric RBD variant SARS-CoV-2 antigen

<400> 8

agagtgcagc ctaccgagtc tatcgtgcgg ttccccaaca tcaccaacct gtgtcctttc 60

ggcgaggtgt tcaacgccac cagattcgcc tctgtgtacg cctggaaccg gaagcggatc 120

tctaactgcg tggccgacta ctccgtgctg tacaactccg cctccttcag caccttcaag 180

tgctacggcg tgtcccctac caagctgaac gacctgtgct tcaccaacgt gtacgccgac 240

tccttcgtga tcagaggcga cgaagtgcgg cagatcgctc ctggacagac cggcaatatc 300

gccgactaca actacaagct gcccgacgac ttcaccggct gtgtgatcgc ttggaactcc 360

aacaacctgg actccaaagt cggcggcaac tacaattacc tgtaccggct gttccggaag 420

tccaacctga agcctttcga gcgggacatc tccaccgaga tctaccaggc tggcagcacc 480

ccttgtaatg gcgtgaaggg cttcaactgc tacttcccac tgcagtccta cggcttccag 540

cctacctatg gcgtgggcta ccagccttac agagtggtgg tgctgtcctt cgagctgctg 600

catgctcctg ctaccgtgtg cggccctaag aaatctacca acctggtcaa gaacaagcgg 660

gtgcagccca ctgagagcat tgtgcgcttc cctaatatca caaatctgtg ccccttcggg 720

gaagtcttta atgctacccg cttcgcttcc gtgtatgctt ggaatagaaa gcggatcagc 780

aattgcgtcg ccgattacag cgtcctgtac aatagcgcca gcttctccac ctttaagtgt 840

tatggcgtca gccccacaaa gctcaacgat ctctgtttta ccaatgtcta cgccgatagc 900

tttgtgattc gcggagatga agtccgccag atcgcaccag gccagactgg aaagatcgct 960

gattacaatt ataagctccc tgatgatttc acaggatgcg ttatcgcctg gaatagcaac 1020

aacctcgaca gcaaagttgg agggaattac aactacctct accgcctctt cagaaagagc 1080

aacctcaagc catttgagag agacatcagt acagaaatct atcaggccgg ctctacccct 1140

tgcaacggcg tcgaggggtt taactgttac tttcccctgc aatcttatgg gtttcagccc 1200

acatacggcg tggggtatca accctatcgc gtggtggttc tgagtttcga actcctgcac 1260

gccccagcca cagtgtgtgg cccaaaaaag agcaccaatc tcgttaagaa caag 1314

<210> 9

<211> 1273

<212> PRT

<213> Coronaviridae

<220>

<223> Wuhan-Hu-1 SARS-CoV-2 spike protein sequence (UniProt No. P0DTC 2):

<400> 9

Met Phe Val Phe Leu Val Leu Leu Pro Leu Val Ser Ser Gln Cys Val

1 5 10 15

Asn Leu Thr Thr Arg Thr Gln Leu Pro Pro Ala Tyr Thr Asn Ser Phe

20 25 30

Thr Arg Gly Val Tyr Tyr Pro Asp Lys Val Phe Arg Ser Ser Val Leu

35 40 45

His Ser Thr Gln Asp Leu Phe Leu Pro Phe Phe Ser Asn Val Thr Trp

50 55 60

Phe His Ala Ile His Val Ser Gly Thr Asn Gly Thr Lys Arg Phe Asp

65 70 75 80

Asn Pro Val Leu Pro Phe Asn Asp Gly Val Tyr Phe Ala Ser Thr Glu

85 90 95

Lys Ser Asn Ile Ile Arg Gly Trp Ile Phe Gly Thr Thr Leu Asp Ser

100 105 110

Lys Thr Gln Ser Leu Leu Ile Val Asn Asn Ala Thr Asn Val Val Ile

115 120 125

Lys Val Cys Glu Phe Gln Phe Cys Asn Asp Pro Phe Leu Gly Val Tyr

130 135 140

Tyr His Lys Asn Asn Lys Ser Trp Met Glu Ser Glu Phe Arg Val Tyr

145 150 155 160

Ser Ser Ala Asn Asn Cys Thr Phe Glu Tyr Val Ser Gln Pro Phe Leu

165 170 175

Met Asp Leu Glu Gly Lys Gln Gly Asn Phe Lys Asn Leu Arg Glu Phe

180 185 190

Val Phe Lys Asn Ile Asp Gly Tyr Phe Lys Ile Tyr Ser Lys His Thr

195 200 205

Pro Ile Asn Leu Val Arg Asp Leu Pro Gln Gly Phe Ser Ala Leu Glu

210 215 220

Pro Leu Val Asp Leu Pro Ile Gly Ile Asn Ile Thr Arg Phe Gln Thr

225 230 235 240

Leu Leu Ala Leu His Arg Ser Tyr Leu Thr Pro Gly Asp Ser Ser Ser

245 250 255

Gly Trp Thr Ala Gly Ala Ala Ala Tyr Tyr Val Gly Tyr Leu Gln Pro

260 265 270

Arg Thr Phe Leu Leu Lys Tyr Asn Glu Asn Gly Thr Ile Thr Asp Ala

275 280 285

Val Asp Cys Ala Leu Asp Pro Leu Ser Glu Thr Lys Cys Thr Leu Lys

290 295 300

Ser Phe Thr Val Glu Lys Gly Ile Tyr Gln Thr Ser Asn Phe Arg Val

305 310 315 320

Gln Pro Thr Glu Ser Ile Val Arg Phe Pro Asn Ile Thr Asn Leu Cys

325 330 335

Pro Phe Gly Glu Val Phe Asn Ala Thr Arg Phe Ala Ser Val Tyr Ala

340 345 350

Trp Asn Arg Lys Arg Ile Ser Asn Cys Val Ala Asp Tyr Ser Val Leu

355 360 365

Tyr Asn Ser Ala Ser Phe Ser Thr Phe Lys Cys Tyr Gly Val Ser Pro

370 375 380

Thr Lys Leu Asn Asp Leu Cys Phe Thr Asn Val Tyr Ala Asp Ser Phe

385 390 395 400

Val Ile Arg Gly Asp Glu Val Arg Gln Ile Ala Pro Gly Gln Thr Gly

405 410 415

Lys Ile Ala Asp Tyr Asn Tyr Lys Leu Pro Asp Asp Phe Thr Gly Cys

420 425 430

Val Ile Ala Trp Asn Ser Asn Asn Leu Asp Ser Lys Val Gly Gly Asn

435 440 445

Tyr Asn Tyr Leu Tyr Arg Leu Phe Arg Lys Ser Asn Leu Lys Pro Phe

450 455 460

Glu Arg Asp Ile Ser Thr Glu Ile Tyr Gln Ala Gly Ser Thr Pro Cys

465 470 475 480

Asn Gly Val Glu Gly Phe Asn Cys Tyr Phe Pro Leu Gln Ser Tyr Gly

485 490 495

Phe Gln Pro Thr Asn Gly Val Gly Tyr Gln Pro Tyr Arg Val Val Val

500 505 510

Leu Ser Phe Glu Leu Leu His Ala Pro Ala Thr Val Cys Gly Pro Lys

515 520 525

Lys Ser Thr Asn Leu Val Lys Asn Lys Cys Val Asn Phe Asn Phe Asn

530 535 540

Gly Leu Thr Gly Thr Gly Val Leu Thr Glu Ser Asn Lys Lys Phe Leu

545 550 555 560

Pro Phe Gln Gln Phe Gly Arg Asp Ile Ala Asp Thr Thr Asp Ala Val

565 570 575

Arg Asp Pro Gln Thr Leu Glu Ile Leu Asp Ile Thr Pro Cys Ser Phe

580 585 590

Gly Gly Val Ser Val Ile Thr Pro Gly Thr Asn Thr Ser Asn Gln Val

595 600 605

Ala Val Leu Tyr Gln Asp Val Asn Cys Thr Glu Val Pro Val Ala Ile

610 615 620

His Ala Asp Gln Leu Thr Pro Thr Trp Arg Val Tyr Ser Thr Gly Ser

625 630 635 640

Asn Val Phe Gln Thr Arg Ala Gly Cys Leu Ile Gly Ala Glu His Val

645 650 655

Asn Asn Ser Tyr Glu Cys Asp Ile Pro Ile Gly Ala Gly Ile Cys Ala

660 665 670

Ser Tyr Gln Thr Gln Thr Asn Ser Pro Arg Arg Ala Arg Ser Val Ala

675 680 685

Ser Gln Ser Ile Ile Ala Tyr Thr Met Ser Leu Gly Ala Glu Asn Ser

690 695 700

Val Ala Tyr Ser Asn Asn Ser Ile Ala Ile Pro Thr Asn Phe Thr Ile

705 710 715 720

Ser Val Thr Thr Glu Ile Leu Pro Val Ser Met Thr Lys Thr Ser Val

725 730 735

Asp Cys Thr Met Tyr Ile Cys Gly Asp Ser Thr Glu Cys Ser Asn Leu

740 745 750

Leu Leu Gln Tyr Gly Ser Phe Cys Thr Gln Leu Asn Arg Ala Leu Thr

755 760 765

Gly Ile Ala Val Glu Gln Asp Lys Asn Thr Gln Glu Val Phe Ala Gln

770 775 780

Val Lys Gln Ile Tyr Lys Thr Pro Pro Ile Lys Asp Phe Gly Gly Phe

785 790 795 800

Asn Phe Ser Gln Ile Leu Pro Asp Pro Ser Lys Pro Ser Lys Arg Ser

805 810 815

Phe Ile Glu Asp Leu Leu Phe Asn Lys Val Thr Leu Ala Asp Ala Gly

820 825 830

Phe Ile Lys Gln Tyr Gly Asp Cys Leu Gly Asp Ile Ala Ala Arg Asp

835 840 845

Leu Ile Cys Ala Gln Lys Phe Asn Gly Leu Thr Val Leu Pro Pro Leu

850 855 860

Leu Thr Asp Glu Met Ile Ala Gln Tyr Thr Ser Ala Leu Leu Ala Gly

865 870 875 880

Thr Ile Thr Ser Gly Trp Thr Phe Gly Ala Gly Ala Ala Leu Gln Ile

885 890 895

Pro Phe Ala Met Gln Met Ala Tyr Arg Phe Asn Gly Ile Gly Val Thr

900 905 910

Gln Asn Val Leu Tyr Glu Asn Gln Lys Leu Ile Ala Asn Gln Phe Asn

915 920 925

Ser Ala Ile Gly Lys Ile Gln Asp Ser Leu Ser Ser Thr Ala Ser Ala

930 935 940

Leu Gly Lys Leu Gln Asp Val Val Asn Gln Asn Ala Gln Ala Leu Asn

945 950 955 960

Thr Leu Val Lys Gln Leu Ser Ser Asn Phe Gly Ala Ile Ser Ser Val

965 970 975

Leu Asn Asp Ile Leu Ser Arg Leu Asp Lys Val Glu Ala Glu Val Gln

980 985 990

Ile Asp Arg Leu Ile Thr Gly Arg Leu Gln Ser Leu Gln Thr Tyr Val

995 1000 1005

Thr Gln Gln Leu Ile Arg Ala Ala Glu Ile Arg Ala Ser Ala Asn Leu

1010 1015 1020

Ala Ala Thr Lys Met Ser Glu Cys Val Leu Gly Gln Ser Lys Arg Val

1025 1030 1035 1040

Asp Phe Cys Gly Lys Gly Tyr His Leu Met Ser Phe Pro Gln Ser Ala

1045 1050 1055

Pro His Gly Val Val Phe Leu His Val Thr Tyr Val Pro Ala Gln Glu

1060 1065 1070

Lys Asn Phe Thr Thr Ala Pro Ala Ile Cys His Asp Gly Lys Ala His

1075 1080 1085

Phe Pro Arg Glu Gly Val Phe Val Ser Asn Gly Thr His Trp Phe Val

1090 1095 1100

Thr Gln Arg Asn Phe Tyr Glu Pro Gln Ile Ile Thr Thr Asp Asn Thr

1105 1110 1115 1120

Phe Val Ser Gly Asn Cys Asp Val Val Ile Gly Ile Val Asn Asn Thr

1125 1130 1135

Val Tyr Asp Pro Leu Gln Pro Glu Leu Asp Ser Phe Lys Glu Glu Leu

1140 1145 1150

Asp Lys Tyr Phe Lys Asn His Thr Ser Pro Asp Val Asp Leu Gly Asp

1155 1160 1165

Ile Ser Gly Ile Asn Ala Ser Val Val Asn Ile Gln Lys Glu Ile Asp

1170 1175 1180

Arg Leu Asn Glu Val Ala Lys Asn Leu Asn Glu Ser Leu Ile Asp Leu

1185 1190 1195 1200

Gln Glu Leu Gly Lys Tyr Glu Gln Tyr Ile Lys Trp Pro Trp Tyr Ile

1205 1210 1215

Trp Leu Gly Phe Ile Ala Gly Leu Ile Ala Ile Val Met Val Thr Ile

1220 1225 1230

Met Leu Cys Cys Met Thr Ser Cys Cys Ser Cys Leu Lys Gly Cys Cys

1235 1240 1245

Ser Cys Gly Ser Cys Cys Lys Phe Asp Glu Asp Asp Ser Glu Pro Val

1250 1255 1260

Leu Lys Gly Val Lys Leu His Tyr Thr

1265 1270

<210> 10

<211> 19

<212> PRT

<213> artificial sequence

<220>

<223> Signal peptide.

<400> 10

Met Gly Trp Ser Leu Ile Leu Leu Phe Leu Val Ala Val Ala Thr Arg

1 5 10 15

Val Leu Ser

Claims

1. A protein subunit vaccine comprising at least one antigen consisting of two monomers, wherein both monomers comprise a Receptor Binding Domain (RBD) of spike protein from severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), wherein the two monomers are part of a single polypeptide and chemically bound to each other, optionally through a linker, to each other, thereby forming a fusion dimer, wherein a first RBD monomer is derived from a first SARS-CoV-2 line and a second RBD monomer is derived from a second, different SARS-CoV-2 line.

2. The protein subunit vaccine of claim 1, wherein the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is selected from the group consisting of: wuhan-Hu-1 pneumovirus isolates (GenBank accession number: MN 908947), linage B.1.1.28 (Brazil variant), linage B.1.351 (south Africa variant), linage B.1.427 or Linage B.1.429 (California variant), linage B.1.617 (Indian variant), linage B.1.1.7 (British variant), linage B.1.617.2 or G/478K.V1 (Deltavariant) or Linage B.1.1.529 or GR/484A (Omik Rong Bianti) or any combination thereof.

3. The protein subunit vaccine of claim 1 or 2, wherein the first monomer of the fusion dimer is derived from Linage b.1.351 (south african SARS-CoV-2 variant) and the second monomer of the fusion dimer is derived from Linage b.1.1.7 (uk SARS-CoV-2 variant).

4. A protein subunit vaccine of claim 3, wherein the fusion dimer consists of SEQ ID NO: 5.

5. The protein subunit vaccine of any one of claims 1 to 4, wherein the protein subunit vaccine comprises a total amount of antigen of 5 to 50 μg per dose.

6. The protein subunit vaccine of any one of claims 1 to 5, further comprising at least an adjuvant.

7. The protein subunit vaccine of claim 6, wherein the adjuvant comprises about 10 to 60mg/ml squalene, 1 to 6mg/ml polysorbate 80, 1 to 6mg/ml sorbitan trioleate, 0.5 to 6mg/ml sodium citrate, and 0.01 to 0.5mg/ml citric acid.

8. The protein subunit vaccine of any one of claims 1 to 7, wherein the protein subunit vaccine further comprises monophosphoryl lipid a (MPLA) and/or C ₉₂O₄₆H₁₄₈ (QS-21) as an immunostimulant.

9. A protein subunit vaccine according to any one of claims 1 to 8 for use in generating an immunogenic and/or protective immune response against at least one lineage of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virus in a subject in need thereof.

10. The protein subunit vaccine for use according to claim 9, wherein the protein subunit vaccine is administered to the subject in need thereof in a single dose or in multiple doses, preferably in two doses.

11. The protein subunit vaccine for use according to claim 9 or 10, wherein the protein subunit vaccine is administered as a booster to the subject.

12. A kit comprising at least one dose, preferably two or more doses, of a protein subunit vaccine according to any one of claims 1 to 8.