Coronavirus glycoprotein, nucleic acid, expression vector, nanoparticle, cell, vaccine composition and immune response stimulation method, immunogenic composition and immune response stimulation method

Abstract

FIELD: virology.SUBSTANCE: this document discloses coronavirus spike (S) proteins and nanoparticles containing them that are suitable for use in vaccines. Nanoparticles present antigens from pathogens surrounding and bound to a detergent core, resulting in high stability and good immunogenicity. Dosages, compositions and methods for producing vaccines and nanoparticles are also disclosed.EFFECT: development of stable and highly immunogenic nanoparticles containing coronavirus spike protein for use in vaccines.64 cl, 67 dwg, 13 tbl, 11 ex

Description

AREA OF TECHNOLOGY

[0003] The present invention generally relates to non-naturally occurring coronavirus (CoV) spike (S) protein polypeptides and nanoparticles containing them, and vaccines that are useful for stimulating immune responses. The nanoparticles comprise antigens, such as glycoprotein antigens, optionally linked to a detergent-presented core, and are typically produced using recombinant approaches. The nanoparticles are characterized by improved stability and an increased level of epitope presentation. The present invention also provides compositions containing the nanoparticles, methods for producing them, and methods for stimulating immune responses. BACKGROUND

[0004] Infectious diseases continue to pose a challenge worldwide. Despite progress in developing vaccines against some pathogens, many continue to pose a threat to human health. The outbreak of sudden acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has infected more than 79 million people in the United States alone, with at least 960,000 deaths. The global death toll has exceeded 6 million. SARS-CoV-2 belongs to the same family of viruses as severe acute respiratory syndrome coronavirus (SARS-CoV) and Middle East respiratory syndrome coronavirus (MERS-CoV), which have killed hundreds of people over the past 17 years. SARS-CoV-2 causes the disease COVID-19. In March 2020, the World Health Organization classified COVID-19 as a global pandemic. This pandemic is still ongoing.Efforts to control it have been complicated by the emergence of SARS-CoV-2 variants.

[0005] Development of vaccines that prevent or reduce the severity of life-threatening infectious diseases caused by SARS-CoV-2 and its variants is needed. However, developing vaccines for humans remains challenging due to the highly complex evasion mechanisms of the pathogens and the difficulties in stabilizing vaccines. Optimally, a vaccine should both induce the production of antibodies that block or neutralize infectious agents and remain stable under a variety of environmental conditions, including those that do not allow refrigeration. SUMMARY OF THE INVENTION

[0006] The present invention provides unnaturally occurring CoV S protein polypeptides suitable for inducing immune responses against SARS-CoV-2 and SARS-CoV-2 variants. The present invention also provides nanoparticles containing glycoproteins, as well as methods for stimulating immune responses.

[0007] The present invention also provides CoV S protein polypeptides suitable for inducing immune responses against multiple coronaviruses, including SARS-CoV-2 and its variants, Middle East respiratory syndrome (MERS) virus, and severe acute respiratory syndrome (SARS) virus.

[0008] Provided herein are CoV S protein polypeptides comprising(i) an S1 subunit with an inactivated furin cleavage site, wherein the S1 subunit comprises an N-terminal domain (NTD), a receptor binding domain (RBD), subdomains 1 and 2 (SD1 / 2), wherein the inactivated furin cleavage site is characterized by the amino acid sequence QQAQ;wherein the NTD optionally provides one or more modifications selected from the group consisting of(a) a deletion of one or more amino acids selected from the group consisting of amino acids 56, 57, 131, 132, 144, 145, 228, 229, 230, 231, 234, 235, 236, 237, 238, 239, 240 and combinations;(b) insertions of 1, 2, 3, or 4 amino acids after amino acid 132; and(c) a mutation affecting one or more amino acids selected from the group consisting of amino acids 5, 6, 7, 13, 39, 51, 53, 54, 56, 57, 62, 63, 67, 82, 125, 129, 131, 132, 133, 139, 143, 144, 145, 177, 200, 201, 202, 209, 229, 233, 240, 245, and combinations thereof;wherein the RBD optionally provides a mutation affecting one or more amino acids selected from the group consisting of amino acids 333, 404, 419, 426, 439, 440, 464, 465, 471, 477, 481, 488, and combinations thereof; wherein the SD1 / 2 domain optionally provides a mutation affecting one or more amino acids selected from the group consisting of 557, 600, 601, 642, 664, 668, and combinations thereof; and(ii) an S2 subunit, wherein amino acids 973 and 974 are proline, wherein the S2 subunit optionally comprises one or more modifications selected from the group consisting of(a) a deletion of one or more amino acids from amino acids 676-685, 676-702, 702-711, 775-793, 806-815, and combinations thereof;(b) a mutation affecting one or more amino acids selected from the group consisting of 688, 703, 846, 875, 937, 969, 973, 974, 1014, 1058, 1105, and 1163, and combinations thereof; and(c) a deletion of one or more amino acids from TMCT;wherein the amino acids of the S CoV glycoprotein are numbered relative to the polypeptide characterized by the sequence under SEQ ID NO: 2.;

[0009] In embodiments, the coronavirus S glycoprotein provides a deletion of amino acids 676-685. In embodiments, the coronavirus S glycoprotein provides a deletion of amino acids 702-711. In embodiments, the coronavirus S glycoprotein provides a deletion of amino acids 806-815. In embodiments, the coronavirus S glycoprotein provides a deletion of amino acids 775-793. In embodiments, the coronavirus S glycoprotein provides a deletion of amino acids 1-292 NTD. In embodiments, the coronavirus S glycoprotein provides a deletion of amino acids 1201-1260 TMCT.In embodiments, the coronavirus S glycoprotein comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 85-89, 105, 106 and 112-115, 164-168, or an amino acid sequence characterized by at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity with any of SEQ ID NOs: 85-89, 105, 106 and 112-115, 164-168, or consists of it. In embodiments, the coronavirus S glycoprotein comprises a signal peptide, wherein the signal peptide optionally comprises the amino acid sequence of SEQ ID NO: 5 or SEQ ID NO: 117. In embodiments, the coronavirus S glycoprotein comprises a C-terminal fusion protein. In embodiments, the C-terminal fusion protein is a hexahistidine tag.In embodiments, the C-terminal fusion protein is a foldon. In embodiments, the foldon is characterized by an amino acid sequence corresponding to SEQ ID NO: 68. In embodiments, the coronavirus S glycoprotein is characterized by a ΔHcal that is at least 2-fold higher than the ΔHcal of the wild-type CoV S glycoprotein (SEQ ID NO: 2). In embodiments, the present document provides a coronavirus S glycoprotein comprising an S2 subunit, an NTD, an RBD, and an SD1 / 2 that are at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% identical to the corresponding subunit or domain of the CoV S glycoprotein characterized by the amino acid sequence of SEQ ID NO: 2.

[0010] This document provides an isolated nucleic acid encoding the CoV S glycoprotein described herein.

[0011] This document provides a vector containing an isolated nucleic acid encoding the CoV S glycoprotein described herein.

[0012] This document provides a nanoparticle comprising the CoV S glycoprotein described herein. In embodiments, the nanoparticle has a Z-average diameter of from about 20 nm to about 35 nm. In embodiments, the nanoparticle has a polydispersity index of from about 0.2 to about 0.45. This document provides a cell expressing the CoV S glycoprotein described herein.

[0013] This document provides a vaccine composition comprising a nanoparticle comprising the CoV S glycoprotein described herein. In embodiments, the vaccine composition comprises an adjuvant. In embodiments, the adjuvant comprises at least two iscom particles, wherein the first iscom particle comprises the AQuillaja Saponaria Molina fraction and does not comprise the CQuillaja Saponaria Molina fraction; and the second iscom particle comprises the CQuillaja Saponaria Molina fraction and does not comprise the AQuillaja Saponaria Molina fraction. In embodiments, the AQuillaja Saponaria Molina fraction and the CQuillaja Saponaria Molina fraction constitute approximately 85% by weight and approximately 15% by weight, respectively, of the sum of the weights of the AQuillaja Saponaria Molina fraction and the CQuillaja Saponaria Molina fraction in the adjuvant.In embodiments, the AQuillaja Saponaria Molina fraction and the CQuillaja Saponaria Molina fraction constitute approximately 92% by weight and approximately 8% by weight, respectively, of the sum of the weights of the AQuillaja Saponaria Molina fraction and the CQuillaja Saponaria Molina fraction in the adjuvant. In embodiments, the vaccine composition comprises approximately 50 μg of adjuvant.

[0014] This document provides a method for stimulating an immune response against SARS-CoV-2 in a subject, comprising administering the vaccine composition described herein. In embodiments, the subject is administered a first dose on day 0 and a booster dose on day 21. In embodiments, the subject is administered from about 3 μg to about 25 μg of coronavirus S glycoprotein. In embodiments, the subject is administered about 5 μg of coronavirus S glycoprotein. In embodiments, the vaccine composition is administered intramuscularly. In embodiments, a single dose of the vaccine composition is administered. In embodiments, multiple doses of the vaccine composition are administered. In embodiments, the vaccine composition is co-administered with an influenza virus glycoprotein.

[0015] Provided herein is an immunogenic composition comprising (i) a nanoparticle comprising the CoV S glycoprotein described herein and a core represented by a non-ionic detergent; (ii) a pharmaceutically acceptable buffer; and (iii) a saponin adjuvant. In embodiments, the immunogenic composition comprises from about 3 μg to about 25 μg of the CoV S glycoprotein. In embodiments, the immunogenic composition comprises about 5 μg of the CoV S glycoprotein. In embodiments, the saponin adjuvant comprises at least two iscom particles, wherein the first iscom particle comprises an AQuillaja Saponaria Molina fraction and does not comprise a CQuillaja Saponaria Molina fraction; and the second iscom particle comprises a CQuillaja Saponaria Molina fraction and does not comprise an AQuillaja Saponaria Molina fraction.In embodiments, the AQuillaja Saponaria Molina fraction accounts for 50-96% by weight, and the CQuillaja Saponaria Molina fraction accounts for the remaining amount, respectively, of the sum of the weights of the AQuillaja Saponaria Molina fraction and the CQuillaja Saponaria Molina fraction in the adjuvant. In embodiments, the AQuillaja Saponaria Molina fraction and the CQuillaja Saponaria Molina fraction account for approximately 85% by weight and approximately 15% by weight, respectively, of the sum of the weights of the AQuillaja Saponaria Molina fraction and the CQuillaja Saponaria Molina fraction in the adjuvant. In embodiments, the immunogenic composition comprises approximately 50 μg of saponin adjuvant. In embodiments, the nonionic detergent is selected from the group consisting of polysorbate-20 (PS20), polysorbate-40 (PS40), polysorbate-60 (PS60), polysorbate-65 (PS65), and polysorbate-80 (PS80).

[0016] In embodiments, provided herein is a method for stimulating an immune response against SARS-CoV-2 or a heterogeneous strain of SARS-CoV-2 in a subject, comprising administering an immunogenic composition or a vaccine composition provided herein. In embodiments, the method comprises administering from about 3 μg to about 25 μg of CoV S glycoprotein. In embodiments, the method comprises administering about 5 μg of CoV S glycoprotein. In embodiments, the saponin adjuvant comprises at least two iscom particles, wherein the first iscom particle comprises an AQuillaja Saponaria Molina fraction and does not comprise a CQuillaja Saponaria Molina fraction; and the second iscom particle comprises a CQuillaja Saponaria Molina fraction and does not comprise an AQuillaja Saponaria Molina fraction. In embodiments, the AQuillaja Saponaria Molina fraction is 50-96% by weight,and the CQuillaja Saponaria Molina fraction constitutes the remaining amount, respectively, of the sum of the weights of the AQuillaja Saponaria Molina fraction and the CQuillaja Saponaria Molina fraction in the adjuvant. In embodiments, the AQuillaja Saponaria Molina fraction and the CQuillaja Saponaria Molina fraction constitute approximately 85% by weight and approximately 15% by weight, respectively, of the sum of the weights of the AQuillaja Saponaria Molina fraction and the CQuillaja Saponaria fraction. In embodiments, the method comprises administering approximately 50 μg of a saponin adjuvant. In embodiments, the non-ionic detergent is selected from the group consisting of polysorbate-20 (PS20), polysorbate-40 (PS40), polysorbate-60 (PS60),polysorbate-65 (PS65) and polysorbate-80 (PS80). In embodiments, the subject is administered a first dose on day 0 and a booster dose on day 21. In embodiments, a single dose of the immunogenic composition is administered. In embodiments, the method includes administering a second immunogenic composition. In embodiments, the second immunogenic composition comprises mRNA encoding the SARS-CoV-2 spike glycoprotein, plasmid DNA encoding the SARS-CoV-2 spike glycoprotein, a viral vector encoding the SARS-CoV-2 spike glycoprotein, or an inactivated SARS-CoV-2 virus. In embodiments, the heterogeneous strain of SARS-CoV-2 is selected from the group consisting of SARS-CoV-2 strain B.1.1.7, SARS-CoV-2 strain B.1.351, SARS-CoV-2 strain P.1, SARS-CoV-2 strain B.1.617.2, SARS-CoV-2 strain B.1.525, SARS-CoV-2 strain B.1.526, SARS-CoV-2 strain B.1.617.1, SARS-CoV-2 strain C.37,strain B.1.621 SARS-CoV-2 and strain Cal.20C SARS-CoV-2. In embodiments, the efficacy of the immunogenic composition for preventing coronavirus disease 19 (COVID-19) is at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% for a period of up to about 2 months, up to about 2.5 months, up to about 3 months,up to about 3.5 months, up to about 4 months, up to about 4.5 months, up to about 5 months, up to about 5.5 months, up to about 6 months, up to about 6.5 months, up to about 7 months, up to about 7.5 months, up to about 8 months, up to about 8.5 months, up to about 9 months, up to about 9.5 months, up to about 10 months, up to about 10.5 months, up to about 11 months, up to about 11.5 months, or up to about 12 months after administration of the immunogenic composition. In embodiments, the efficacy of the immunogenic composition for preventing coronavirus disease 19 (COVID-19) is from about 50% to about 99%, from about 50% to about 95%, from about 50% to about 90%, from about 50% to about 85%, from about 50% to about 80%, from about 60% to about 99%,from about 60% to about 95%, from about 60% to about 90%, from about 60% to about 85%, from about 60% to about 80%, from about 40% to about 99%, from about 40% to about 95%, from about 40% to about 90%, from about 40% to about 85%, from about 40% to about 80%, from about 40% to about 75%, from about 40% to about 70%, from about 40% to about 65%, from about 40% to about 55%, or from about 40% to about 50% over a period of up to about 2 months, up to about 2.5 months, up to about 3 months, up to about 3.5 months, up to about 4 months, up to about 4.5 months, up to about 5 months, up to about 5.5 months, up to approximately 6 months, up to approximately 6.5 months, up to approximately 7 months,up to about 7.5 months, up to about 8 months, up to about 8.5 months, up to about 9 months, up to about 9.5 months, up to about 10 months, up to about 10.5 months, up to about 11 months, up to about 11.5 months, or up to about 12 months after administration of the immunogenic composition. In embodiments, the COVID-19 is mild COVID-19. In embodiments, the COVID-19 is moderate COVID-19. In embodiments, the COVID-19 is severe COVID-19. In embodiments, provided herein is a method for inducing a protective immune response against a heterogeneous strain of SARS-CoV-2, comprising administering to a subject a nanoparticle comprising a coronavirus (S CoV) S glycoprotein characterized by the amino acid sequence of SEQ ID NO: 87, and a core represented by a non-ionic detergent,a pharmaceutically acceptable buffer and (iii) a saponin adjuvant, wherein the heterogeneous strain of SARS-CoV-2 is characterized by a SARS-CoV-2 S glycoprotein characterized by having from about 1 to about 60 modifications compared to the SARS-CoV-2 glycoprotein of SEQ ID NO: 1. In embodiments, the heterogeneous strain of SARS-CoV-2 is characterized by a SARS-CoV-2 S glycoprotein characterized by having from about 1 to about 20 modifications, from about 1 to about 10 modifications, from about 10 to about 20 modifications, from 10 to about 30 modifications, from about 10 to about 40 modifications, from 10 to about 50 modifications, from 10 to about 60 modifications, from 20 to about 60 modifications, from 20 to about 50 modifications, from approximately 20 to approximately 40 modifications,from about 5 to about 15 modifications or from about 5 to about 10 modifications compared to the SARS-CoV-2 glycoprotein of SEQ ID NO: 1. BRIEF DESCRIPTION OF THE DRAWINGS,

[0017] The patent or application file contains at least one illustration in color. Copies of this patent or patent application publication containing the color illustration(s) will be provided by the Office upon request and payment of the required fee.

[0018] Fig. 1 shows the amino acid sequence of the wild-type SARS-CoV-2 spike (S) protein (SEQ ID NO: 1). The furin cleavage site of RRAR (SEQ ID NO: 6) is shown in bold, and the signal peptide is underlined.

[0019] Fig. 2 shows the primary structure of the SARS-CoV-2 S protein polypeptide, which is characterized by an inactive furin cleavage site, a deletion of the fusion peptide, and the K986P and V987P mutations. Domain positions are numbered relative to the amino acid sequence of the wild-type SARS-CoV-2 S protein polypeptide containing the signal peptide (SEQ ID NO: 1).

[0020] Fig. 3 shows the primary structure of the CoV BV2378 S protein polypeptide, which is characterized by an inactive furin cleavage site, a deletion of amino acids 819-828 of the fusion peptide, and the K986P and V987P mutations. Domain positions are numbered relative to the amino acid sequence of the wild-type CoV S protein polypeptide from SARS-CoV-2 containing the signal peptide (SEQ ID NO: 1).

[0021] Fig. 4 shows the purification of CoV S protein polypeptides BV2364, BV2365, BV2366, BV2367, BV2368, BV2369, BV2373, BV2374, and BV2375. The data show that BV2365 (SEQ ID NO: 4) and BV2373 (SEQ ID NO: 87), which has an inactive furin cleavage site characterized by the amino acid sequence QQAQ (SEQ ID NO: 7), are expressed as a single chain (S0). In contrast, CoV S protein polypeptides containing an intact furin cleavage site (e.g., BV2364, BV2366, and BV2374) undergo cleavage, as evidenced by the presence of the S2 cleavage product.

[0022] Fig. 5 shows that CoV S protein polypeptides BV2361, BV2365, BV2369, BV2365, BV2373, and BV2374 bind to the human angiotensin-converting enzyme 2 (hACE2) precursor by biolayer interferometry.

[0023] Fig. 6 shows that BV2361 from SARS-CoV-2 does not bind to the MERS-CoV receptor, dipeptidyl peptidase IV (DPP4), and MERS S protein does not bind to the human angiotensin-converting enzyme precursor 2 (hACE2) according to biolayer interferometry results.

[0024] Fig. 7 shows that BV2361 binds to hACE2 as determined by enzyme-linked immunosorbent assay (ELISA).

[0025] Fig. 8 shows the primary structure of the CoV BV2373 S protein polypeptide and modifications of the furin cleavage site K986P and V987P.

[0026] Fig. 9 shows the purification of the wild-type CoV S protein polypeptide and the CoV BV2365 and BV2373 S protein polypeptides.

[0027] Fig. 10 shows the cryoelectron microscopy (cryoEM) structure of the S protein polypeptide of CoV BV2373 superimposed on the cryoEM structure of the SARS-CoV-2 spike protein (EMB ID: 21374).

[0028] Figures 11A-F show that the CoV S spike protein polypeptides BV2365 and BV2373 bind to hACE2. Biolayer interferometry shows that BV2365 (Figure 11B) and BV2373 (Figure 11C) bind to hACE2 with dissociation kinetics similar to that of the wild-type CoV S protein polypeptide (Figure 11A). ELISA shows that the wild-type CoV S protein polypeptide (Figure 11D) and BV2365 (Figure 11E) bind to hACE2 with similar affinity, while BV2373 binds to hACE2 with higher affinity (Figure 11F).

[0029] Fig. 12A-B show the effect of stress conditions such as temperature, two freeze / thaw cycles, oxidation, agitation, and extreme pH values ​​on the binding of CoV BV2373 (Fig. 12A) and BV2365 (Fig. 12B) S protein polypeptides to hACE2.

[0030] Figures 13A-B show the IgG antibody titers to the CoV S protein polypeptide 13 days, 21 days, and 28 days after immunization of mice with two doses (Figure 13A) and one dose of 0.1 μg to 10 μg of BV2373 with or without the iscom matrix based on fraction A and fraction C (i.e., MATRIX-M™) (Figure 13B).

[0031] Fig. 14 shows the induction of antibody production that blocks interaction with hACE2 in mice immunized with one or two doses of 0.1 μg to 10 μg BV2373 with or without MATRIX-M™.

[0032] Fig. 15 shows virus-neutralizing antibodies detected in mice immunized with one dose or two doses of 0.1 to 10 μg BV2373 with or without MATRIX-M™.

[0033] Fig. 16 shows the viral load (SARS-CoV-2) in the lungs of Ad / CMV / hACE2 mice immunized with either a single dose of BV2373 or two doses of BV2373 14 days apart with or without MATRIX-M™.

[0034] Fig. 17A-C show the weight loss exhibited by mice after immunization with BV2373. Fig. 17A shows the effect of immunization on weight loss when a single dose of 0.01 μg, 0.1 μg, 1 μg, or 10 μg of BV2373 plus MATRIX-M™ was administered. Fig. 17B shows the effect of immunization on weight loss when two doses of BV2373 (0.01 μg, 0.1 μg, 1 μg) plus MATRIX-M™ were administered. Fig. 17C shows the effect of immunization on weight loss when two doses of BV2373 (10 μg) were administered in the presence or absence of MATRIX-M™.

[0035] Fig. 18A-B show the effect of BV2373 on the histopathological characteristics of the lungs of mice four days (Fig. 18A) or seven days (Fig. 18B) after SARS-CoV-2 infection.

[0036] Fig. 19 shows the number of IFN-γ-secreting cells after ex vivo stimulation in the spleen of mice immunized with BV2373 in the absence of adjuvant, compared with mice immunized with BV2373 in the presence of MATRIX-M™.

[0037] Figures 20A-E show the frequency of cytokine-secreting CD4+ T cells in the spleen of mice immunized with BV2373 in the presence or absence of MATRIX-M™. Figure 20A shows the frequency of IFN-γ-secreting CD4+ T cells. Figure 20B shows the frequency of TNF-α-secreting CD4+ T cells. Figure 20C shows the frequency of IL-2-secreting CD4+ T cells. Figure 20D shows the frequency of CD4+ T cells that secrete two cytokines selected from IFN-γ, TNF-α, and IL-2. Figure 20E shows the frequency of CD4+ T cells that express IFN-γ, TNF-α, and IL-2.

[0038] Fig. 21A-E shows the frequency of cytokine-secreting CD8 + T cells in the spleen of mice immunized with BV2373 in the presence or absence of MATRIX-M™. Fig. 21A shows the frequency of IFN-γ-secreting CD8 + T cells. Fig. 21B shows the frequency of occurrence of TNF-α-secreting CD8 +T cells. Fig. 21C shows the frequency of IL-2-secreting CD8 + T cells. Fig. 20D shows the frequency of CD8 + T cells that secrete two cytokines selected from IFN-γ, TNF-α, and IL-2. Fig. 21E shows the frequency of CD8 + T cells that express IFN-γ, TNF-α and IL-2.

[0039] Fig. 22 shows the CD4 frequency + or CD8 + cells that express one (single status), two (dual status) or three (triple status) cytokines selected from IFN-γ, TNF-α and IL-2 in the spleen of mice immunized with BV2373 in the presence or absence of MATRIX-M™.

[0040] Fig. 23A-C show the effect of immunization with BV2373 in the presence or absence of MATRIX-M™ on the secretion of type 2 cytokines from CD4 + T cells. Fig. 23A shows the frequency of IL-4-secreting cells. Fig. 23B shows the frequency of IL-5-secreting CD4 +cells. Fig. 23C shows the ratio of IFN-γ-secreting and IL-4-secreting CD4 cells. + T cells.

[0041] Fig. 24A-B shows the effect of immunization of mice with BV2373 in the presence or absence of MATRIX-M™) on germinal center formation by assessing the presence of CD4 + follicular helper T cells (TFH). Fig. 24A shows the CD4 frequency + follicular helper T cells in the spleen, but fig. 24B shows the phenotype (for example, CD4 + CXCR5 + PD-1 + ) CD4 + follicular helper T cells.

[0042] Figure 25A-B shows the effect of immunization of mice with BV2373 in the presence or absence of MATRIX-M™ on germinal center formation by assessing the presence of germinal center (GC) B cells. Figure 25A shows the frequency of GC B cells in the spleen, and Figure 25B shows the phenotype (e.g., CD19 + GL7 + CD-95 + ) CD4 +follicular helper T cells.

[0043] Figures 26A-C show the effect of immunization with BV2373 in the presence or absence of MATRIX-M™ on the antibody response in olive baboons. Figure 26A shows the IgG antibody titer to the SARS-CoV-2 protein S polypeptide in baboons after immunization with BV2373. Figure 26B shows the presence of hACE2 receptor blocking antibodies in baboons after a single immunization with 5 μg or 25 μg BV2373 in the presence of MATRIX-M™. Figure 26C shows the virus-neutralizing antibody titer after a single immunization with BV2373 and MATRIX-M™.

[0044] Fig. 27 shows a significant correlation between the titers of IgG antibodies to the SARS-CoV-2 protein S polypeptide and neutralizing antibodies in olive baboons after immunization with BV2373.

[0045] Fig. 28 shows the frequency of IFN-γ-secreting cells in peripheral blood mononuclear cells (PBMCs) of olive baboons immunized with BV2373 in the presence or absence of MATRIX-M™.

[0046] Figures 29A-E show the frequency of cytokine-secreting CD4+ T cells in PBMCs from olive baboons immunized with BV2373 in the presence or absence of MATRIX-M™. Figure 29A shows the frequency of IFN-γ-secreting CD4+ T cells. Figure 29B shows the frequency of IL-2-secreting CD4+ T cells. Figure 29C shows the frequency of TNF-α-secreting CD4+ T cells. Figure 29D shows the frequency of CD4+ T cells that secrete two cytokines selected from IFN-γ, TNF-α, and IL-2. Figure 29E shows the frequency of CD4+ T cells that express IFN-γ, TNF-α, and IL-2.

[0047] Fig. 30 shows a schematic of the spike (S) protein of coronavirus (SEQ ID NO: 109) (BV2384). The furin cleavage site of GSAS (SEQ ID NO: 97) is underlined with a single line, and the K986P and V987P mutations are underlined with two lines.

[0048] Fig. 31 shows a schematic of the spike (S) protein of coronavirus (SEQ ID NO: 86) (BV2373). The furin cleavage site QQAQ (SEQ ID NO: 7) is underlined with one line, and the K986P and V987P mutations are underlined with two lines.

[0049] Fig. 32 shows the purification of CoV S protein polypeptides BV2373 (SEQ ID NO: 87) and BV2384 (SEQ ID NO: 109).

[0050] Fig. 33 shows the scanning densitometry results for the purity of BV2384 (SEQ ID NO: 109) after purification.

[0051] Fig. 34 shows a graph of the scanning densitometry results for the purity of BV2373 (SEQ ID NO: 87) after purification.

[0052] Figures 35A-B show the induction of anti-S (Figure 35A) and neutralizing antibody (Figure 35B) responses to BV2373 and MATRIX-M™. Cynomolgus monkeys were administered one or two doses (day 0 and day 21) of 2.5 μg, 5 μg, or 25 μg BV2373 with 25 μg or 50 μg MATRIX-M™ adjuvant. Controls received neither BV2373 nor MATRIX-M™. Antibodies were measured on days 21 and 33.

[0053] Figures 36A-B show the reduction in SARS-CoV-2 viral replication by the vaccine formulations disclosed herein, based on bronchoalveolar lavage (BAL) assessment in cynomolgus macaques. Cynomolgus macaques were administered BV2373 and MATRIX-M™ as indicated. Subjects were immunized on day 0 and in two-dose groups on day 0 and day 21. On day 37, subject animals were challenged with 1x10 4SARS-CoV-2 virus pfu. Levels of viral RNA (Figure 36A, corresponding to the total amount of RNA present) and viral subgenomic RNA (Figure 36B, corresponding to replicating virus) were assessed in bronchiolar lavage (BAL) at 2 and 4 days post-infectious virus challenge (d2pi and d4pi). Most subjects demonstrated undetectable viral RNA. On day 2, small amounts of RNA were measurable in some subjects. By day 4, RNA levels were undetectable except in two subjects receiving the lowest dose of 2.5 μg. Subgenomic RNA was undetectable at both days 2 and 4, except in one subject, also receiving the lowest dose.

[0054] Figures 37A-B show the reduction in SARS-CoV-2 virus replication by the vaccine formulations disclosed herein, based on nasal swab assessment in cynomolgus macaques. Cynomolgus macaques were administered BV2373 with MATRIX-M™ as indicated. Subjects were immunized on day 0 and in two-dose groups on day 0 and day 21. On day 37, subject animals were challenged with 1 x 10 SARS-CoV-2 virus. 4Viral RNA (Fig. 37A) and viral subgenomic (sg) RNA (Fig. 37B) were assessed by nasal swab at 2 days and 4 days postinfection (d2pi and d4pi). Most subjects demonstrated undetectable viral RNA. On day 2 and day 4, small amounts of RNA were measured in some subjects. Subgenomic RNA was undetectable at neither day 2 nor day 4. Subjects were immunized on day 0 and in two-dose groups on day 0 and day 21. These data demonstrate that the vaccine results in a 100- to 1,000-fold reduction in nasal total viral RNA and undetectable sgRNA levels and confirm that the immune response to the vaccine will block viral replication and prevent viral dissemination.

[0055] Figures 38A-B show the IgG antibody titers to the CoV S protein polypeptide 21 days and 35 days after immunization of cynomolgus monkeys with one dose (Figure 38A) or two doses of BV2373 and 25 μg or 50 μg MATRIX-M™ (Figure 38B).

[0056] Figs. 38C-38D show the hACE2 inhibition titer in cynomolgus monkeys 21 days and 35 days after immunization of cynomolgus monkeys with one dose (Fig. 38C) or two doses of BV2373 (5 μg) and MATRIX-M™ (25 μg or 50 μg) (Fig. 38D).

[0057] Fig. 38E shows a significant correlation between the IgG antibody titer to the CoV S protein polypeptide and the hACE2 inhibitory titer in cynomolgus macaques after administration of BV2373 and MATRIX-M™. Data are shown for groups 2–6 from Table 4.

[0058] Fig. 39 shows the CoV S protein polypeptide antibody titers and hACE2 inhibition titers in cynomolgus macaques 35 days after immunization with two doses of BV2373 and MATRIX-M™ or after immunization with human convalescent serum (Groups 2, 4, and 6) from Table 4. These data demonstrate that the CoV S protein polypeptide antibody titers and hACE2 inhibition titers in cynomolgus macaques immunized with BV2373 and MATRIX-M™ are higher than those in cynomolgus macaques immunized with human convalescent serum.

[0059] Figures 40A-B show the neutralizing titers for SARS-CoV-2 in cynomolgus macaques immunized with BV2373 and MATRIX-M™, as determined by the cytopathic effect (CPE) (Figure 40A) and the plaque inhibition neutralization test (PRNT) (Figure 40B).

[0060] Fig. 41 shows the timeframe of administration during a clinical trial evaluating the safety and efficacy of a vaccine containing BV2373 and optionally MATRIX-M™. AESI stands for adverse event of special concern, MAEE stands for adverse event requiring medical intervention, and SAE stands for serious adverse event.

[0061] Figures 42A-B show local (Figure 42A) and systemic adverse events (Figure 42B) experienced by patients in a clinical trial evaluating a vaccine containing BV2373 and MATRIX-M™. The AE groups are defined in Table 5. The data demonstrate that the vaccine was well tolerated and safe.

[0062] Figures 43A-B show IgG antibody titers to the CoV S protein polypeptide (Figure 43A) and neutralization titers (Figure 43B) 21 days and 35 days after immunization in participants in a clinical trial evaluating a vaccine containing BV2373 and MATRIX-M™. Horizontal bars represent the interquartile range (IRQ) and median area under the curve, respectively. Whisker endpoints are equal to the maximum and minimum values ​​below or above the median ± 1.5 times the IQR. The convalescent serum panel included samples from participants with PCR-confirmed COVID-19 from Baylor College of Medicine (29 ELISA samples and 32 microneutralization (MN IC> 99). The severity of COVID-19 is indicated by a red flag for hospitalized patients (including intensive care unit), a blue flag for outpatients (specimen collected in the emergency department), and a green flag for asymptomatic (exposed) patients (specimen collected based on contact / exposure assessment).

[0063] Figures 44A-C show the correlation between IgG antibody titers to the CoV S protein polypeptide and neutralizing antibody titers in patients treated with convalescent serum samples (Figure 44A), two 25 μg doses of BV2373 (Figure 44B), and two doses (5 μg and 25 μg) of BV2373 with MATRIX-M™ (Figure 44C). A strong correlation was observed between neutralizing antibody titers and IgG antibody titers to CoV-S in patients treated with convalescent serum samples or BV2373 with adjuvant, but not in patients treated with BV2373 without adjuvant.

[0064] Fig. 45A-D shows the frequency values ​​of antigen-specific CD4 +T cells producing the helper T cell type 1 (Th1) cytokines interferon-gamma (IFN-γ), tumor necrosis factor-alpha (TNF-α), and interleukin (IL)-2, and the helper T cell type 2 (Th2) cytokines IL-5 and IL-13, wherein these cytokines are shown for participants in groups A (placebo, Fig. 45A), B (25 μg BV2373, Fig. 45B), C (5 μg BV2373 and 50 μg MATRIX-M™), Fig. 45C), and D (25 μg BV2373 and 50 μg MATRIX-M™), Fig. 45D), following stimulation with BV2373. The designation "any 2" on the Th1 cytokine panel stands for CD4 + T cells that can produce two types of Th1 cytokines simultaneously. The designation "all 3" refers to CD4 + T cells that simultaneously produce IFN-γ, TNF-α, and IL-2. The designation “both” in the Th2 panel refers to CD4 + T cells that can produce Th2 cytokines IL-5 and IL-13 simultaneously.

[0065] Fig. 46A shows the primary structure of the wild-type SARS-CoV-2 S protein polypeptide containing the signal peptide, numbered relative to SEQ ID NO: 1. Fig. 46B shows the primary structure of the wild-type SARS-CoV-2 S protein polypeptide without the signal peptide, numbered relative to SEQ ID NO: 2.

[0066] Fig. 47 shows the randomization of subjects in a Phase 3 clinical trial evaluating the efficacy, immunogenicity, and safety of BV2373 in combination with an adjuvant containing iscom matrix based on fraction A and fraction C (MATRIX-M™).

[0067] Fig. 48 shows the Kaplan-Meier plot showing the incidence of symptomatic COVID-19 (cumulative event rate (%)) observed in subjects after vaccination with BV2373 in combination with iscom matrix-based fraction A and fraction C (MATRIX-M™) or placebo.

[0068] Fig. 49 shows the vaccine efficacy of BV2373 in combination with iscom matrix based on fraction A and fraction C (MATRIX-M™) against SARS-CoV-2 containing the CoV S protein polypeptide characterized by the amino acid sequence of SEQ ID NO: 1, or the heterogeneous SARS-CoV-2 strain B.1.1.7, which contains the CoV S protein polypeptide characterized by deletions of amino acids 69, 70 and 144 and mutations N501Y, A570D, D614G, P681H, T716I, S982A and D1118H.

[0069] Fig. 50 is a graph showing the adverse events observed in subjects after the first dose of a vaccination (labeled "Vaccination 1") and the second dose of a vaccination (labeled "Vaccination 2") using BV2373 in combination with the iscom matrix based on fraction A and fraction C (MATRIX-M™) (labeled "A") or placebo (labeled "B").

[0070] Fig. 51 shows a schematic of the BV2438 CoV S protein polypeptide. The structural elements include the cleavable signal peptide (SP), N-terminal domain (NTD), receptor binding domain (RBD), subdomains 1 and 2 (SD1 and SD2), S2 cleavage site (S2'), fusion peptide (FP), heptad repeat 1 (HR1), central helix (CH), heptad repeat 2 (HR2), transmembrane domain (TM), and cytoplasmic tail (CT). Amino acid substitutions of the CoV S protein polypeptide characterized by the amino acid sequence of SEQ ID NO: 1 are shown in black text below the line diagram.

[0071] Fig. 52A shows a reducing SDS-PAGE gel with Coomassie blue staining of purified full-length BV2438, showing the major protein product with the expected molecular mass of ~170 kDa. Fig. 52B shows a graph of the scanning densitometry results. Fig. 52C shows negative stain transmission electron microscopy micrographs of BV2438. BV2438 forms a well-defined light-bulb-shaped particle with a length of 15 nm and a width of 11 nm (left panel). Trimers were characterized by an 8 nm flexible linker attached to PS-80 micelles (left panel). Class-averaged images showed high agreement for the rS-B.1.351 trimer with the determined cryo-EM structure of the SARS-CoV-2 trimeric spike protein ectodomain in the pre-fusion conformation (PDB ID 6VXX) overlaid on a 2D image (middle panel). The right panel shows two BV2438 trimers anchored in a PS-80 micelle.

[0072] Screw it. 53 shows the design of the mouse study in Example 10. Groups of mice (n = 20 / group) were immunized in a prime / boost schedule on days 0 and 14 of the study with different combinations of recombinant protein S (rS) of BV2438 (SA or BV2373 (WU). Mice were either primed and boosted with BV2438, primed and boosted with BV2373, primed with BV2373 and boosted with BV2438, or primed and boosted via bivalent immunization with BV2373+BV2438. Antigen doses were 1 μg rS for each monovalent immunization procedure or 1 μg rS for each construct in the bivalent immunization (in (a total of 2 μg rS). All antigen doses were administered with 5 μg saponin adjuvant. The control group received formulation buffer (placebo). Serum and tissue samples were collected at the time points listed in the diagram.

[0073] No way. 54A-B show the IgG antibody titers to SARS-CoV-2 S in serum samples collected on day 21 of the mouse study in Example 10. ELISA was used to measure antibody titers to the Wuhan-Hu-1 spike protein (Figure 54A) or B.1.351 spike protein (Figure 54B). Bars represent the geometric mean titer (GMT), and error bars represent the 95% confidence interval (CI) for each group. Titers for individual animals are indicated by colored symbols. Figures 54C-D show the titers of functional antibodies (measured by ELISA) in serum samples collected on day 21 that are capable of disrupting binding between the SARS-CoV-2 hACE2 receptor and the Wuhan-Hu-1 spike protein. (Fig. 54C or B.1.351 spike protein (Fig. 54D). Bars represent the geometric mean titer (GMT) and error bars represent the 95% confidence interval (CI) for each group. Titers for individual animals are indicated by colored symbols. Fig.Figure 54E shows SARS-CoV-2 neutralizing antibody titers in serum samples collected on day 32 from n = 5 animals / group, determined using the PRNT assay. Serum samples were assessed for their ability to neutralize USA-WA1, the B.1.351 variant, or the B.1.1.7 variant of SARS-CoV-2. Bars represent the geometric mean titer (GMT), and error bars represent the 95% confidence interval (CI) for each group. Titers for individual animals are indicated by symbols. Statistical significance was calculated by performing a one-way ANOVA with Tukey's post hoc test on log. 10 -transformed data.

[0074] Figures 55A-F show the protective efficacy of immunization with Wuhan-Hu-1- or B.1.351-based SARS-CoV-2 rS against B.1.351 or B.1.1.7 live SARS-CoV-2 virus challenge. The study design is described in Fig. 53. Immunized mice (n = 10 / group) were infected with B.1.351 (left panels) or B.1.1.7 (right panels) of live SARS-CoV-2. For four days after infection, mice were weighed daily, and the percentage of weight loss relative to their body weight on the day of infection was calculated. In Fig. 55A, Fig. 55B, symbols show the mean percentage of body weight loss. Error bars represent the standard error of the mean. Half of the mice were sacrificed 2 days after infection, and lung tissue was subjected to plaque assay to determine viral titers in the lungs (Fig. 55C, Fig. 55D). The remaining mice were sacrificed 4 days after infection. Fig. 55E, Fig.55F shows SARS-CoV-2 subgenomic RNA levels in lung tissue, expressed as fold change in RNA levels relative to the mean in the corresponding placebo group on day 2 post-infection. Horizontal bars represent the group mean fold change for n = 5 mice at each time point, and error bars represent standard deviation.

[0075] Figures 56A-H show cell-mediated immunity induced by immunization of mice with BV2373- or BV2438-based regimens. Figure 56A shows the design of the mouse study. Groups of mice (n = 8 / group) were immunized with different combinations of BV2373- or BV2438-based rS SARS-CoV-2 based on a prime / boost schedule on days 0 and 21. Mice were primed and boosted with BV2438, primed and boosted with BV2373, primed with BV2373 and boosted with BV2438, or primed and boosted with bivalent immunization using BV2373 and BV2438. Antigen doses were 1 μg rS for each monovalent immunization procedure or 1 μg rS for each construct in the bivalent immunization (a total of 2 μg rS). All immunization administration procedures were performed using 5 μg Matrix-M1 adjuvant.The control group received formulation buffer (placebo, n = 5). Spleens were collected on day 28 for cell harvesting. Splenocytes were stimulated with BV2373 or BV2438, then subjected to ELISA to determine IFN-γ-positive cells as secreting a representative Th1 cytokine (Fig. 56B) and IL-5-positive cells as secreting a representative Th2 cytokine (Fig. 56C). Data from Figs. 56B and 56C were used to calculate the Th1 / Th2 balance of responses to immunization (Fig. 56D). In Fig. 56E shows the number of multifunctional CD4+ T cells that were positive for three Th1 cytokines (IFN-γ, IL-2, and TNF-α) using intracellular cytokine staining, which was quantified and expressed as the number of cells positive for the three cytokines per 10. 6CD4+ T cells. Figure 56F shows quantification of follicular helper T cells. Follicular helper T cells were quantified by determining the percentage of PD-1+CXCR5+ cells among all CD4+ T cells. Figure 56G shows germinal center formation, where germinal center formation was assessed by determining the percentage of GL7+CD95+ cells among CD19+ B cells using flow cytometry. Gray bars represent mean values, and error bars represent standard deviation. Data for individual animals are shown using colored symbols. An example of the gating strategy is shown in Figure 56H. Differences between experimental groups were assessed by one-way ANOVA with Tukey's post hoc test (data in Figure 56B were subjected to log 10 -transformation before analysis). P-values ​​< 0.05 were considered statistically significant; **** = p < 0.0001.

[0076] Fig. 57A-E show the CD4+ and CD8+ T cell-mediated response to immunization with BV2373 or BV2438. Groups of mice (n = 8 / group) were immunized according to a prime / boost schedule on days 0 and 21 with different combinations of BV2373 or BV2438. Mice were subjected to either prime and boost immunization with BV2438, prime and boost immunization with BV2373, prime immunization with BV2373 and boost immunization with BV2438, or prime and boost immunization with bivalent immunization with BV2373 and BV2438. Antigen doses were 1 μg rS for each monovalent immunization procedure or 1 μg rS for each construct during bivalent immunization (a total of 2 μg rS). All antigen doses were administered with 5 μg saponin adjuvant. The control group received formulation buffer (placebo, n = 5). Spleens were collected on day 28 for cell harvesting.Isolated splenocytes were stimulated with either rS-WU1 or rS-B.1.351 and then subjected to intracellular cytokine staining to determine whether CD4+ T cells were positive for IFN-γ (Fig. 57A), IL-2 (Fig. 57B), TNF-α (Fig. 57C), or IL-4 (Fig. 57D). To examine CD8+ T cell-mediated responses, cells were stimulated with a pool of peptides corresponding to the entire Wuhan-Hu-1 spike protein sequence and then subjected to ICS for IFN-γ, IL-2, and TNF-α (Fig. 57E).

[0077] Figures 58A-G show the immunogenicity of one or two booster doses of BV2438 approximately one year after immunization of baboons with BV2373. Figure 58A shows the study design. A small cohort of baboons (n ​​= 2-3 / group) were initially immunized with 1 μg, 5 μg, or 25 μg of BV2373 with saponin adjuvant or 25 μg of BV2373 without adjuvant on days 0 and 21 (weeks 0 and 3, respectively). Approximately 1 year later, all animals were boosted with one or two doses of 3 μg BV2438 with 50 μg saponin adjuvant on days 318 and 339 (weeks 45 and 48, respectively). Figure 58B shows the IgG antibody titers to S-CoV over the course of the study. Titers for individual animals are shown over time, with symbols and lines of different colors representing different dose groups for the initial series of immunizations using rS-WU1.Serum samples collected before BV2438 boost (day 303) and at days 7, 21, 35, and 81 after boost were analyzed for IgG antibody titers to rS-WU1 (Fig. 58C) and rS-B.1.351 (Fig. 58D) by ELISA (horizontal lines represent mean values), antibody titers capable of disrupting the interaction between rS-WU1 or rS-B.1.351 and the hACE2 receptor by ELISA (Fig. 58E, horizontal lines represent mean values), and antibody titers capable of neutralizing the USA-WA1, B.1.351, and B.1.1.7 SARS-CoV-2 strains by PRNT assay (Fig. 58F, gray bars represent geometric means, and error bars represent 95% confidence intervals).The presence of multifunctional CD4+ T cells positive for three Th1 cytokines (IFN-γ, IL-2, and TNF-α) was assessed by intracellular cytokine staining after stimulation with BV2373 or BV2438 (Fig. 58G). Gray bars represent mean values, and colored symbols represent data from individual animals.

[0078] Figures 59A-G show responses for individual cytokines to booster immunization with BV2438 in baboons. A small cohort of baboons (n ​​= 2-3 / group) was immunized with 1 μg, 5 μg, or 25 μg BV2373 with 50 μg saponin adjuvant or 25 μg BV2373 without adjuvant on days 0 and 21 (weeks 0 and 3, respectively). Approximately 1 year later, all animals were boosted with one or two doses of 3 μg BV2438 with 50 μg saponin adjuvant on days 318 and 339 (weeks 45 and 48, respectively). PBMCs were collected before the booster (day 303; week 43), 7 days after the first booster with rS-B.1.351 (day 325; week 46), and 35 days after the first booster with rS-B.1.351 (day 353; week 50). PBMCs were stimulated with BV2373 or BV2438 and subjected to ELISA to measure the number (Fig. 59A) of IFN-γ-producing cells as Th1 cytokine-secreting cells and (Fig.59B) IL-4-producing cells as Th2 cytokine secreting cells. CD4+ T cells were also stimulated with BV2373 or BV2438 and then subjected to ICS to determine cells producing IFN-γ (Fig. 59C), IL-2 (Fig. 59D), TNF-α (Fig. 59E), IL-5 (Fig. 59F), and IL-13 (Fig. 59G).

[0079] Figures 60A-B show neutralizing titers for SARS-CoV-2 variants in human subjects immunized with BV2373. Serum samples from clinical trial participants (n = 30) were subjected to PRNT analysis to determine the presence of neutralizing antibodies directed against USA-WA1 compared to those directed against B.1.1.7 (Figure 60A) and B.1.351 (Figure 60B). Titers for individual subjects are shown using black circles; lines connect the individuals' anti-USA-WA1 antibody titers with their antibody titers directed against the corresponding variant.

[0080] Figures 61A-B show the anti-S protein IgG titers before and after booster immunization with BV2373 and saponin adjuvant for the following SARS-CoV-2 variants: (i) the SARS-CoV-2 virus characterized by the CoV S protein polypeptide with the D614G mutation compared to the protein characterized by the amino acid sequence of SEQ ID NO: 1; (ii) SARS-CoV-2 alpha, SARS-CoV-2 beta, and SARS-CoV-2 delta. Figure 61A shows the fold increase from day 35 to day 217. Figure 61B shows the fold increase from day 189 to day 217.

[0081] Figures 62A-B show the functional inhibition of hACE2 before and after booster immunization with BV2373 and saponin adjuvant for the following SARS-CoV-2 variants: (i) SARS-CoV-2 virus characterized by the CoV S protein polypeptide with the D614G mutation compared to the protein characterized by the amino acid sequence of SEQ ID NO: 1; (ii) SARS-CoV-2 alpha, SARS-CoV-2 beta, and SARS-CoV-2 delta. Figure 62A shows the fold increase from day 35 to day 217. Figure 62B shows the fold increase from day 189 to day 217.

[0082] Fuck it. 63shows a diagram of booster dose administration for participants in the trial described in Example 11.

[0083] Fig. 64A-B show local (Fig. 64A) and systemic (Fig. 64B) reactogenicity in patients in Group B2 of the trial described in Example 11.

[0084] Fig. 65 shows serum IgG titers to the ancestral SARS-CoV-2 strain according to the day of the study for the patients described in Example 11.

[0085] Fig. 66 shows the activity of neutralizing antibodies to the ancestral SARS-CoV-2 strain according to the day of the study for the patients described in Example 11.

[0086] Fig. 67 shows the neutralizing antibody 99 (neut99) levels for the immunogenic composition containing BV2373 and the saponin adjuvant of Example 11, directed against the SARS-CoV-2 strain containing the D614G mutation and B.1.617.2 (delta variant). DETAILED DESCRIPTION OF THE INVENTION Definitions

[0087] As used herein and in the appended claims, the singular form "a" includes reference to the plural forms unless the context clearly dictates otherwise. Thus, for example, a reference to "a protein" may refer to a single protein or to mixtures based on such a protein, and a reference to "a method" includes reference to equivalent steps and / or methods known to those skilled in the art, etc.

[0088] As used herein, the term "adjuvant" refers to a compound that, when used in combination with an immunogen, enhances or otherwise alters or modifies the immune response induced against the immunogen. Modification of the immune response may involve increasing the intensity or broadening the specificity of both antibody-mediated and cellular immune responses.

[0089] When used in this document, the term “approximately” or “approximately” preceding a numerical value means the value plus or minus a range of 10%. For example, “approximately 100” covers 90 and 110.

[0090] As used in this document, the terms “immunogen,” “antigen,” and “epitope” refer to substances such as proteins, including glycoproteins, and peptides that are capable of eliciting an immune response.

[0091] As used herein, the term "immunogenic composition" means a composition that contains an antigen, wherein administration of the composition to a subject results in the development of a humoral and / or cellular immune response to the antigen in the subject.

[0092] As used herein, the term "subunit" composition, for example, a vaccine, comprises one or more selected antigens, but not all antigens, of a pathogen. Such a composition does not substantially contain intact virus or a lysate of such cells or particles and is typically derived from at least partially purified, often substantially purified, immunogenic polypeptides of the pathogen. The antigens in the subunit composition disclosed herein are typically produced recombinantly, often using a baculovirus system.

[0093] As used herein, "substantially" refers to the isolation of a substance (e.g., a compound, polynucleotide, or polypeptide) such that the substance constitutes a large percentage of the sample in which it is contained. For example, in a sample, the substantially purified component constitutes 85%, preferably 85-90%, more preferably at least 95-99.5%, and most preferably at least 99% of the sample. If the component is substantially substituted, the amount thereof remaining in the sample is less than or equal to an amount of from about 0.5% to about 10%, preferably less than an amount of from about 0.5% to about 1.0%.

[0094] The terms "treat," "treatment," and "treating" as used herein refer to an approach for obtaining beneficial or desired results, such as clinical results. For the purposes of the present invention, beneficial or desired results may include inhibiting or suppressing the onset or progression of an infection or disease; reducing the intensity of manifestations or reducing the intensity of the development of symptoms of an infection or disease; or a combination thereof.

[0095] "Prevention" as used herein is used interchangeably with the expression "prophylaxis" and may mean the complete prevention of an infection or disease or the prevention of the development of symptoms of that infection or disease; delaying the onset of the development of an infection or disease or its symptoms; or reducing the severity of a subsequently developed infection or disease or its symptoms.

[0096] As used herein, "effective dose" or "effective amount" refers to an amount of an immunogen sufficient to induce an immune response that reduces the severity of at least one symptom of a pathogenic infection. An effective dose or effective amount can be determined, for example, by measuring the amount of neutralizing secretory and / or serum antibodies, such as by plaque neutralization, complement fixation, enzyme-linked immunosorbent assay (ELISA), or microneutralization assay.

[0097] As used herein, the term "vaccine" refers to an immunogenic composition, such as one based on an immunogen derived from a pathogen, that is used to induce an immune response against the pathogen. The immune response may include the formation of antibodies and / or a cell-mediated response. Depending on the context, the term "vaccine" may also refer to a suspension or solution of the immunogen that is administered to a subject to elicit an immune response. Preferably, vaccines induce an immune response that is effective in preventing infection with SARS-CoV-2 or its variant.

[0098] As used herein, the term "subject" includes humans and other animals. Typically, a subject is a human. For example, a subject may be an adult, an adolescent, a child (2 years to 14 years of age), an infant (birth to 2 years of age), or a newborn (up to 2 months of age). In particular aspects, the subject is up to 4 months of age or up to 6 months of age. In some aspects, adults are elderly humans, approximately 65 years of age or older, or approximately 60 years of age or older. In some aspects, a subject is a pregnant woman or a woman intending to become pregnant. In other aspects, the subject is not a human; for example, a non-human primate; for example, a baboon, a chimpanzee, a gorilla, or a macaque. In certain aspects, a subject may be a pet, such as a dog or a cat.

[0099] In some aspects, the subject is immunocompromised. In embodiments, the immunocompromised subject is administered a drug that causes immunosuppression. Non-limiting examples of drugs that cause immunosuppression include corticosteroids (e.g., prednisolone), alkylating agents (e.g., cyclophosphamide), antimetabolites (e.g., azathioprine or 6-mercaptopurine), transplant-related immunosuppressive drugs (e.g., cyclosporine, tacrolimus, sirolimus, or mycophenolate mofetil), mitoxantrone, chemotherapeutic agents, methotrexate, tumor necrosis factor (TNF) blocking agents (e.g., etanercept, adalimumab, infliximab). In embodiments, the immunocompromised subject is infected with a virus (e.g., human immunodeficiency virus or Epstein-Barr virus). In embodiments, the virus is a respiratory virus, such as respiratory syncytial virus,influenza virus, parainfluenza virus, adenovirus, or picornavirus. In embodiments, the immunocompromised subject has acquired immunodeficiency syndrome (AIDS). In embodiments, the immunocompromised subject is a person living with the human immunodeficiency virus (HIV). In embodiments, the immunocompromised subject is immunocompromised due to a treatment regimen designed to prevent inflammation or to prevent transplant rejection. In embodiments, the immunocompromised subject is a subject who has received a transplant. In embodiments, the immunocompromised subject has undergone radiation therapy or a splenectomy. In embodiments, the immunocompromised subject has been diagnosed with cancer, an autoimmune disease, tuberculosis, a substance use disorder (e.g., an alcohol use disorder,opioids or cocaine), stroke or cerebrovascular disease, solid organ or stem cell transplant, sickle cell disease, thalassemia, autoimmune lymphoproliferative syndrome (ALPS), autoimmune polyglandular syndrome type 1 (APS-1), B-cell expansion disorder with NF-κB and T-cell anergy (BENTA), caspase-8 deficiency (CEDS), chronic granulomatous disease (CGD), common variable immunodeficiency (CVID), congenital neutropenia syndromes, cytotoxic T lymphocyte-associated antigen 4 (CTLA-4) deficiency, DOCK8 deficiency, GATA2 deficiency, glycosylation disorder with immunodeficiency, hyperimmunoglobulinemia E syndrome (HIES), hyperimmunoglobulinemia M syndrome, diabetes, Type 1 diabetes, type 2 diabetes, gamma interferon deficiency, interleukin 12 deficiency, interleukin 23 deficiency, leukocyte adhesion deficiency, lipopolysaccharide-dependent beige anchor (LRBA) deficiency, PI3 kinase-mediated disease,PLCG2-associated antibody deficiency and immune dysregulation (PLAID), severe combined immunodeficiency (SCID), dominant-negative STAT3 disease, STAT3 gain-of-function disease, warts, hypogammaglobulinemia, infections and myelokathexis syndrome (WHIM), Wiskott-Aldrich syndrome (WAS), X-linked agammaglobulinemia (XLA), X-linked lymphoproliferative disorder (XLP), uremia, nutritional deficiency, or XMEN disease. In embodiments, the immunocompromised subject is a smoker or former smoker. In embodiments, the immunocompromised subject has a B cell defect, a T cell defect, a macrophage defect, a cytokine defect, a phagocyte deficiency, a phagocyte dysfunction, a complement system deficiency, or a combination thereof.

[0100] In embodiments, the subject is overweight or obese. In embodiments, the overweight subject has a body mass index (BMI) that is equal to or greater than 25 kg / m 2 and less than 30 kg / m 2 In embodiments, the obese subject has a BMI that is equal to or greater than 30 kg / m 2 In some embodiments, the subject has a mental disorder. In some embodiments, the mental disorder is depression, schizophrenia, or anxiety.

[0101] As used herein, the term "pharmaceutically acceptable" means approved by a regulatory agency of the U.S. federal or state government or listed in the United States Pharmacopoeia, the European Pharmacopoeia, or other generally recognized pharmacopeia for use in mammals, and in particular in humans. These compositions may be useful as vaccine and / or antigen compositions for inducing a protective immune response in a vertebrate.

[0102] The term “approximately” used in this document means plus or minus 10% of the stated numerical value.

[0103] As used herein, the term “NVX-CoV2373” refers to a vaccine composition comprising the spike glycoprotein BV2373 (SEQ ID NO: 87) and an iscom matrix based on fraction A and fraction C (e.g., MATRIX-M™).

[0104] As used herein, the term "modification" with respect to a CoV S protein polypeptide refers to a mutation, deletion, or addition of one or more amino acids of a CoV S protein polypeptide. The location of the modification in a CoV S protein polypeptide can be determined based on the alignment of the polypeptide sequence with SEQ ID NO: 1 (CoV S protein polypeptide containing the signal peptide) or SEQ ID NO: 2 (mature CoV S protein polypeptide lacking the signal peptide).

[0105] The term "SARS-CoV-2 variant" as used herein interchangeably with the term "heterogeneous SARS-CoV-2 strain" is a SARS-CoV-2 virus comprising a CoV S protein polypeptide characterized by the presence of at least about 2, at least about 3, at least about 4, at least about 5, at least about 6, at least about 7, at least about 8, at least about 9, at least about 10, at least about 11, at least about 12, at least about 13, at least about 14, at least about 15, at least about 16, at least about 17, at least about 18, at least about 19, at least about 20, at least about 21, at least about 22, at least about 23,at least about 24, at least about 25, at least about 26, at least about 27, at least about 28, at least about 29, at least about 30, at least about 31, at least about 32, at least about 33, at least about 34, or at least about 35 modifications, from about 2 to about 35 modifications, from about 5 to about 10 modifications, from about 5 to about 20 modifications, from about 10 to about 20 modifications, from about 15 to about 25 modifications, from about 20 to about 30 modifications, from about 20 to about 40 modifications, from about 25 to about 45 modifications compared to the CoV S protein polypeptide,characterized by the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2. In embodiments, the heterogeneous SARS-CoV-2 strain is a SARS-CoV-2 virus comprising a CoV S protein polypeptide characterized by at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity to a CoV S protein polypeptide characterized by the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2. In embodiments, the heterogeneous SARS-CoV-2 strain is a SARS-CoV-2 virus comprising a CoV S protein polypeptide characterized by at least about 70% to at least about 99.9% identity to CoV S protein polypeptide,characterized by the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2. In embodiments, the heterogeneous strain of SARS-CoV-2 is a SARS-CoV-2 virus comprising a CoV S protein polypeptide characterized by at least about 70% to at least about 99.5% identity to a CoV S protein polypeptide characterized by the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2. In embodiments, the heterogeneous strain of SARS-CoV-2 is a SARS-CoV-2 virus comprising a CoV S protein polypeptide characterized by at least about 90% to at least about 99.9% identity to a CoV S protein polypeptide characterized by the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2. In embodiments, the heterogeneous strain of SARS-CoV-2 is a SARS-CoV-2 virus comprising a CoV S protein polypeptide,characterized by at least about 90% to at least about 99.8% identity to a CoV S protein polypeptide characterized by the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2. In embodiments, the heterogeneous SARS-CoV-2 strain is a SARS-CoV-2 virus comprising a CoV S protein polypeptide characterized by at least about 95% to at least about 99.9% identity to a CoV S protein polypeptide characterized by the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2. In embodiments, the heterogeneous SARS-CoV-2 strain is a SARS-CoV-2 virus comprising a CoV S protein polypeptide characterized by at least about 95% to at least about 99.8% identity to a CoV S protein polypeptide,characterized by the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2. In embodiments, the heterogeneous strain of SARS-CoV-2 is a SARS-CoV-2 virus comprising a CoV S protein polypeptide characterized by at least about 95% to at least about 99% identity to a CoV S protein polypeptide characterized by the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2.,

[0106] The term "SARS-CoV-2 strain B.1.1.7" (also referred to as the "alpha" strain) refers to a heterogeneous SARS-CoV-2 strain comprising a CoV S protein polypeptide comprising deletions at amino acids 69, 70, and 144 and mutations N501Y, A570D, D614G, P681H or P681R, T716I, S982A, and D1118H, wherein the CoV S protein polypeptide is numbered relative to the wild-type SARS-CoV-2 S protein polypeptide characterized by the amino acid sequence of SEQ ID NO: 1. The SARS-CoV-2 strain B.1.1.7 S CoV protein polypeptide may optionally comprise a deletion at amino acid 145, an E484K, L432R, or S494P mutation, or a combination thereof.

[0107] The term "SARS-CoV-2 strain B.1.351" (also referred to as the "beta" strain) refers to a heterogeneous SARS-CoV-2 strain comprising a CoV S protein polypeptide comprising the mutations D80A, K417N, E484K, N501Y, D614G, and A701V, wherein the CoV S protein polypeptide is numbered relative to the wild-type SARS-CoV-2 S protein polypeptide characterized by the amino acid sequence of SEQ ID NO: 1. The SARS-CoV-2 strain B.1.617.2 CoV S protein polypeptide may optionally comprise one or more of the following mutations: D215G; L242H; R246I or a deletion of 1, 2 or 3 amino acids from 241-243, wherein the CoV S protein polypeptide is numbered relative to the wild-type SARS-CoV-2 S protein polypeptide characterized by the amino acid sequence of SEQ ID NO: 1.In embodiments, the beta strain CoV S protein polypeptide comprises the mutations D80A, D215G, L242H, K417N, E484K, N501Y, D614G, and A701V, wherein the CoV S protein polypeptide is numbered relative to the wild-type SARS-CoV-2 S protein polypeptide having the amino acid sequence of SEQ ID NO: 1. In embodiments, the beta strain CoV S protein polypeptide comprises the mutations D80A, D215G, a deletion of 1, 2, or 3 amino acids from amino acids 241-243, K417N, E484K, N501Y, D614G, and A701V, wherein the CoV S protein polypeptide is numbered relative to the wild-type SARS-CoV-2 S protein polypeptide having the amino acid sequence of SEQ ID NO: 1. In embodiments, the beta strain CoV S protein polypeptide comprises the mutations D80A, L242H, R246I, N501Y, K417N, E484K, D614G and A701V, wherein the CoV S protein polypeptide is numbered relative to the wild-type SARS-CoV-2 S protein polypeptide having the amino acid sequence of SEQ ID NO: 1.

[0108] The term "SARS-CoV-2 P.1 strain" (also referred to as the "gamma" strain) refers to a heterogeneous strain of SARS-CoV-2 comprising a CoV S protein polypeptide comprising the mutations L18F, T20N, P26S, D138Y, R190S, K417T, E484K, N501Y, D614G, H655Y, T1027I, and V1176F, wherein the CoV S protein polypeptide is numbered relative to the wild-type SARS-CoV-2 S protein polypeptide characterized by the amino acid sequence of SEQ ID NO: 1.

[0109] The term "SARS-CoV-2 strain Cal.20C" refers to a heterogeneous strain of SARS-CoV-2 comprising a CoV S protein polypeptide containing the mutations S13I, W152C, and L452R, wherein the CoV S protein polypeptide is numbered relative to the wild-type SARS-CoV-2 S protein polypeptide characterized by the amino acid sequence of SEQ ID NO: 1.

[0110] The term "B.1.617.2 strain" (also referred to as "delta" strain) refers to a heterogeneous strain of SARS-CoV-2 comprising a CoV S protein polypeptide comprising deletions of amino acids 157 and 158 and mutations T19R, E156G, L452R, T478K, D614G, P681R, and D950N, wherein the CoV S protein polypeptide is numbered relative to the wild-type SARS-CoV-2 S protein polypeptide characterized by the amino acid sequence of SEQ ID NO: 1. The SARS-CoV-2 strain B.1.617.2 CoV S protein polypeptide may optionally comprise one or more of the following mutations: G142D; W64H; H66W; V70F; T95I; Y145H; D213V; L214R; A222V; W258I or W258L; K417N; N439K; E484K or E484Q; N501Y and Q613H, wherein the CoV S protein polypeptide is numbered relative to the wild-type SARS-CoV-2 S protein polypeptide characterized by the amino acid sequence of SEQ ID NO: 1.In embodiments, the delta strain comprises a CoV S protein polypeptide that includes deletions of amino acids 157 and 158 and mutations T19R, G142D, E156G, L452R, T478K, D614G, P681R, and D950N. In embodiments, the delta strain comprises a CoV S protein polypeptide having deletions at amino acids 157 and 158 and the mutations T19R, T95I, G142D, Y145H, E156G, A222V, K417N, L452R, T478K, D614G, P681R, and D950N, wherein the S CoV polypeptide is numbered relative to the wild-type SARS-CoV-2 S protein polypeptide having the amino acid sequence of SEQ ID NO: 1. In embodiments, the delta strain comprises a CoV S protein polypeptide having deletions at amino acids 157 and 158 and the mutations T19R, G142D, E156G, W258I, K417N, N439K, L452R, T478K, E484K, N501Y, D614G, P681R and D950N, wherein the CoV S protein polypeptide is numbered relative to the wild-type SARS-CoV-2 S protein polypeptide having the amino acid sequence of SEQ ID NO: 1.In embodiments, the delta strain comprises a CoV S protein polypeptide having deletions at amino acids 157 and 158 and the mutations T19R, W64H, H66W, G142D, E156G, D213V, L214R, W258I, K417N, N439K, L452R, T478K, E484K, N501Y, D614G, P681R, and D950N, wherein the S CoV polypeptide is numbered relative to the wild-type SARS-CoV-2 S protein polypeptide having the amino acid sequence of SEQ ID NO: 1. In embodiments, the delta strain comprises a CoV S protein polypeptide having deletions at amino acids 157 and 158 and the mutations T19R, G142D, E156G, K417N, L452R, T478K, E484Q, D614G, P681R and D950N, wherein the S CoV polypeptide is numbered relative to the wild-type SARS-CoV-2 S protein polypeptide having the amino acid sequence of SEQ ID NO: 1.

[0111] The term "B.1.525 strain" (also referred to as "eta" strain) refers to a heterogeneous strain of SARS-CoV-2 comprising a CoV S protein polypeptide comprising the mutations Q52R; A67V; E484K; D614G; Q677H; F888L and a deletion of 1, 2, 3, or 4 amino acids 69, 70, 144, 145, wherein the CoV S protein polypeptide is numbered relative to the wild-type SARS-CoV-2 S protein polypeptide characterized by the amino acid sequence of SEQ ID NO: 1.

[0112] The term "B.1.526 strain" (also referred to as the "iota" strain) refers to a heterogeneous strain of SARS-CoV-2 comprising a CoV S protein polypeptide containing the mutations L5F; T95I; D253G; E484K; D614G and A701V, wherein the CoV S protein polypeptide is numbered relative to the wild-type SARS-CoV-2 S protein polypeptide characterized by the amino acid sequence of SEQ ID NO: 1.

[0113] The term "B.1.617.1 strain" (also referred to as "kappa" strain) refers to a heterogeneous strain of SARS-CoV-2 comprising a CoV S protein polypeptide containing the mutations L452R; E484Q; D614G; P681R and Q1071H, wherein the CoV S protein polypeptide is numbered relative to the wild-type SARS-CoV-2 S protein polypeptide characterized by the amino acid sequence of SEQ ID NO: 1.

[0114] The term "C.37 strain" (also referred to as "lambda" strain) refers to a heterogeneous strain of SARS-CoV-2 comprising a CoV S protein polypeptide comprising the mutations G75V; T76I; R246N; L452Q; F490S; D614G; T859N and a deletion of 1, 2, 3, 4, 5, or 6 amino acids 247-253, wherein the CoV S protein polypeptide is numbered relative to the wild-type SARS-CoV-2 S protein polypeptide characterized by the amino acid sequence of SEQ ID NO: 1.

[0115] The term "B.1.621 strain" (also referred to as the "mu" strain) refers to a heterogeneous strain of SARS-CoV-2 comprising a CoV S protein polypeptide containing the mutations T95I; Y144S; Y145N; R346K; E484K; N501Y; D614G; P681H, and D950N, wherein the CoV S protein polypeptide is numbered relative to the wild-type SARS-CoV-2 S protein polypeptide characterized by the amino acid sequence of SEQ ID NO: 1.

[0116] The term "efficacy" of an immunogenic composition or vaccine composition described herein refers to the percentage reduction in the severity of a disease (e.g., COVID-19) in a group administered the immunogenic composition compared to a group that was not administered the immunogenic composition. In embodiments, efficacy (E) is calculated using the following equation: E (%) = (1 - RR) × 100, where RR = the relative risk with respect to the incidence rates between the group administered the immunogenic composition and the group that was not administered the immunogenic composition. In embodiments, the immunogenic compositions described herein are characterized by an efficacy against the SARS-CoV-2 virus or a heterogeneous strain of SARS-CoV-2 that is at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%,at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, from about 50% to about 99%, from about 50% to about 98%, from about 60% to about 99%, from about 60% to about 98%, from about 70% to about 98%, from about 70% to about 95%, from about 70% to about 99%, from about 80% to about 99%, from about 80% to about 98%, from about 80% to about 95%, from approximately 85% to approximately 99%,from about 85% to about 98%, from about 85% to about 95%, from about 90% to about 95%, from about 90% to 98%, or from about 90% to about 99%.

[0117] A subject who is "positive" for SARS-CoV-2 or its variant has a positive PCR or serologic test for SARS-CoV-2 or its variant. A positive PCR test is characterized by the detection of genetic material from SARS-CoV-2 or its variant. A positive serologic test is characterized by the presence of antibodies to a SARS-CoV-2 protein, usually the nucleocapsid protein of SARS-CoV-2 or its variant.

[0118] The term “asymptomatic” refers to a subject who is positive for SARS-CoV-2 or its SARS-CoV-2 variant but does not experience any symptoms of COVID-19.

[0119] The term “mild” as applied to COVID-19 refers to a subject who has a positive PCR or serological test result for SARS-CoV-2 or its variant and who has one or more of the following symptoms: (i) fever; (ii) onset of a new episode of cough (iii) or two additional symptoms of COVID-19 selected from the onset of a new episode or worsening of shortness of breath or difficulty breathing; fatigue; generalized muscle or body aches; headache; loss of taste or smell; sore throat, nasal congestion or runny nose; or nausea, vomiting or diarrhea.

[0120] The term “moderate severity” when applied to COVID-19 refers to a subject who has a positive PCR or serological test result for SARS-CoV-2 or its variant and one or more of the following symptoms: (i) high fever with a temperature equal to or greater than 38.4°C for three or more days; (ii) any evidence of significant lower respiratory tract infection (LRTI), where the evidence is selected from: (a) dyspnea with or without exertion; (b) tachypnea (24-29 breaths per minute at rest); (c) SpO2 between 94% and 95%; (d) abnormal chest radiography or computed tomography (CT) scan results consistent with pneumonia or LRTI; or (e) adventitious sounds on auscultation of the lungs (e.g., crackles / crackles, whistles, dry crackles, pleural friction rubs, stridor).

[0121] The term “severe” when applied to COVID-19 refers to a subject who has a positive PCR or serological test result for SARS-CoV-2 or its variant and one or more of the following symptoms: (i) tachypnea, which is equal to or greater than 30 breaths per minute at rest; (ii) resting heart rate, which is equal to or greater than 125 beats per minute; (iii) SpO2, which is equal to or less than 93%, or PaO2 / FiO2, which is less than 300 mmHg.; (iv) the need for high-flow oxygen therapy or non-invasive ventilation, non-invasive positive pressure ventilation (e.g., continuous positive airway pressure (CPAP) or bilevel positive airway pressure (BiPAP); (v) the need for mechanical ventilation or extracorporeal membrane oxygenation (ECMO); (vi) dysfunction or failure of one or more major organ systems selected from (a) acute respiratory failure, including acute respiratory distress syndrome (ARDS); (b) acute renal failure; (c) acute liver failure; (d) acute right-sided or left-sided heart failure; (e) septic or cardiogenic shock (with shock being defined as a systolic blood pressure (SBP) that is equal to or less than 90 mmHg or a diastolic blood pressure (DBP) that is less than 60 mmHg).); (f) acute stroke (ischemic or hemorrhagic); (g) acute thrombotic event such as acute myocardial infarction (AMI), deep vein thrombosis (DVT) or pulmonary embolism (PE); (h) requirement for vasopressors, systemic corticosteroids or hemodialysis; (vii) admission to an intensive care unit; or (viii) death. Vaccine compositions containing coronavirus (CoV) spike (S) proteins.

[0122] The present invention provides non-naturally occurring coronavirus (CoV) spike (S) protein polypeptides, nanoparticles comprising CoV S protein polypeptides, and immunogenic compositions and vaccine compositions comprising either non-naturally occurring CoV S protein polypeptides or nanoparticles comprising CoV S protein polypeptides. In embodiments, methods of using CoV S protein polypeptides, nanoparticles, immunogenic compositions, and vaccine compositions to stimulate an immune response are provided herein.

[0123] This document also provides methods for producing nanoparticles and vaccine compositions. Advantageously, the methods involve producing nanoparticles that are substantially free of contaminants represented by other proteins, such as proteins associated with recombinant expression of proteins in insect cells. In embodiments, expression occurs in baculovirus / Sf9-based systems. Antigens represented by the CoV S protein polypeptide

[0124] The vaccine compositions of the present invention comprise non-naturally occurring CoV S protein polypeptides. The CoV S protein polypeptides can be derived from coronaviruses, including but not limited to SARS-CoV-2, such as SARS-CoV-2, MERS CoV, and SARS CoV. In some embodiments, the CoV S protein polypeptide is derived from a SARS-CoV-2 variant. In embodiments, the SARS-CoV-2 variant is VUI 202012 / 01, B.1.1.7 (also referred to as "501Y.V1" and "alpha"), B.1.351 (also referred to as "501Y.V2" and "beta"), B.1.617.2 (also referred to as "delta"), Cal.20C (also referred to as "epsilon"), or P.1 (also referred to as "gamma") SARS-CoV-2. The SARS-CoV-2 variant is designated by its World Health Organization (WHO) designation (e.g., alpha, beta, gamma, delta, etc.), by its phylogenetic attribution to named global epidemic lineages (PANGO), by its clade according to GISAID, or by its clade according to Nextstrain.

[0125] The table below shows a list of SARS-CoV-2 strain variants:

[0126] In embodiments, the SARS-CoV-2 virus comprises a CoV S protein polypeptide characterized by the amino acid sequence of SEQ ID NO: 1, and the SARS-CoV-2 variant comprises a CoV S protein polypeptide characterized by having at least about 1, at least about 2, at least about 3, at least about 4, at least about 5, at least about 6, at least about 7, at least about 8, at least about 9, at least about 10, at least about 11, at least about 12, at least about 13, at least about 14, at least about 15, at least about 16, at least about 17, at least about 18, at least about 19, at least about 20, at least about 21, at least about 22,at least about 23, at least about 24, at least about 25, at least about 26, at least about 27, at least about 28, at least about 29, at least about 30, at least about 31, at least about 32, at least about 33, at least about 34, at least about 35 modifications, at least about 36, at least about 37, at least about 38, at least about 39, at least about 40, at least about 41, at least about 42, at least about 43, at least about 44, at least about 45, at least about 46, at least about 47, at least about 48, at least about 49, at least about 50,at least about 51, at least about 52, at least about 53, at least about 54, at least about 55, at least about 56, at least about 57, at least about 58, at least about 59, or at least about 60 modifications compared to SEQ ID NO: 1.,

[0127] Unlike the SARS-CoV S protein, the SARS-CoV-2 S protein contains a four-amino acid insertion at the S1 / S2 cleavage site, resulting in the formation of a polybasic furin-like RRAR cleavage motif. The SARS-CoV-2 S protein is synthesized as an inactive precursor (S0), which is proteolytically cleaved at the furin cleavage site into S1 and S2 subunits, which remain non-covalently associated to form trimers in a pre-fusion conformation. The S2 domain of the SARS-CoV-2 S protein contains a fusion peptide (FP), two heptad repeats (HR1 and HR2), a transmembrane (™) domain, and a cytoplasmic tail (CT). The S1 domain of the SARS-CoV-2 S protein folds into four distinct domains: the N-terminal domain (NTD) and the C-terminal domain, which contains the receptor-binding domain (RBD) and two subdomains, SD1 and SD2.SARS-CoV-2 S protein trimers, which are in the pre-fusion conformation, undergo a structural rearrangement from the pre-fusion conformation to the post-fusion conformation upon binding and cleavage of the S protein receptor.

[0128] In embodiments, the CoV S protein polypeptides are glycoproteins formed by post-translational glycosylation. The glycoproteins comprise one or more of a signal peptide, an S1 subunit, an S2 subunit, an NTD, an RBD, two subdomains (SD1 and SD2, indicated as SD1 / 2 in Fig. 46A-B and referred to as "SD1 / 2" herein), an intact or modified fusion peptide, an HR1 domain, an HR2 domain, TM, and CD. In embodiments, the amino acids for each domain are shown in Fig. 2, Fig. 46A (shown according to SEQ ID NO: 1), Fig. 46B (shown according to SEQ ID NO: 2), and Fig. 3 (shown according to SEQ ID NO: 1). In embodiments, each domain may be characterized by at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99,5% sequence identity with the sequences for each domain as shown in SEQ ID NO: 1 or SEQ ID NO: 2. Each domain may be characterized by a deletion, insertion, or mutation affecting up to about 1, to about 2, to about 3, to about 4, to about 5, to about 10, to about 20, to about 30, to about 35, to about 40, to about 45, to about 50, to about 55, to about 60, to about 65, or to about 70 amino acids, as compared to those shown in SEQ ID NO: 1 or SEQ ID NO: 2. Each domain may be characterized by a deletion, insertion, or mutation affecting from about 1 to about 5 amino acids, from about 3 to about 10 amino acids, from about 5 to about 10 amino acids, from about 8 to about 12 amino acids, from about 10 to about 15 amino acids,from about 12 to about 17 amino acids, from about 15 to about 20 amino acids, from about 18 to about 23 amino acids, from about 20 to about 25 amino acids, from about 22 to about 27 amino acids, from about 25 to about 30 amino acids, from about 30 to about 35 amino acids, from about 35 to about 40 amino acids, from about 40 to about 45 amino acids, from about 45 to about 50 amino acids, from about 50 to about 55 amino acids, or from about 55 to about 60 amino acids, as compared to those shown in SEQ ID NO: 1 or SEQ ID NO: 2. It should be noted that Figs. 2 and 3 illustrate a 13 amino acid N-terminal signal peptide,which is absent in the mature peptide. CoV S protein polypeptides can be used to stimulate immune responses against the native CoV spike (S) protein polypeptide.

[0129] In embodiments, the native CoV spike (S) protein polypeptide (SEQ ID NO: 2) is modified, resulting in non-naturally occurring CoV spike (S) protein polypeptides (Fig. 1). In embodiments, the CoV spike (S) glycoproteins comprise an S1 subunit and an S2 subunit, wherein the S1 subunit comprises an NTD, an RBD, an SD1 / 2, and an inactive furin cleavage site (amino acids 669-672), and wherein the S2 subunit provides mutations affecting amino acids 973 and 974; wherein the NTD (amino acids 1-318) optionally provides one or more modifications selected from the group consisting of (a) a deletion of one or more amino acids selected from the group consisting of amino acids 56, 57, 131, 132, 144, 145, 228, 229, 230, 231, 234, 235, 236, 237, 238, 239, 240 and their combinations;(b) insertions of 1, 2, 3, or 4 amino acids after amino acid 132, and(c) a mutation affecting one or more amino acids selected from the group consisting of amino acids 5, 6, 7, 13, 39, 51, 53, 54, 56, 57, 62, 63, 67, 82, 125, 129, 131, 132, 133, 139, 143, 144, 145, 177, 200, 201, 202, 209, 229, 233, 240, 245, and combinations thereof; wherein the RBD optionally provides for a mutation affecting one or more amino acids selected from the group consisting of amino acids 333, 404, 419, 426, 439, 440, 464, 465, 471, 477, 481, 488, and combinations thereof; wherein the SD1 / 2 domain optionally comprises a mutation affecting one or more amino acids selected from the group consisting of 557, 600, 601, 642, 664, 668, and combinations thereof; and wherein the S2 subunit optionally comprises one or more modifications selected from the group consisting of (a) a deletion of one or more amino acids from amino acids 676-685, 676-702, 702-711, 775-793, 806-815, and combinations thereof;(b) a mutation affecting one or more amino acids selected from the group consisting of 688, 703, 846, 875, 937, 969, 973, 974, 1014, 1058, 1105, and 1163, and combinations thereof; and (c) a deletion of one or more amino acids from the transmembrane and cytoplasmic domain (TMCT) (amino acids 1201-1260), wherein the amino acids of the CoV S glycoprotein are numbered relative to SEQ ID NO: 2.

[0130] Fig. 3 shows a CoV S protein polypeptide designated BV2378, which is characterized by an inactive furin cleavage site, a deleted fusion peptide (e.g., deletion of amino acids 819-828), a K986P mutation, and V987, where the amino acids are numbered relative to SEQ ID NO: 1. The mature BV2378 polypeptide lacks one or more amino acids of the signal peptide, which are amino acids 1-13 of SEQ ID NO: 1.

[0131] In embodiments, the CoV S protein polypeptides described herein are in a pre-fusion conformation. In embodiments, the CoV S protein polypeptides described herein comprise a flexible HR2 domain. Unless otherwise noted, domain flexibility is determined by transmission electron microscopy (TEM) and 2D class averaging. A decrease in electron density corresponds to a flexible domain. Antigens presented by the CoV S protein polypeptide are modifications of the S1 subunit.

[0132] In embodiments, the CoV S protein polypeptides comprise one or more modifications of the S1 subunit characterized by the amino acid sequence of SEQ ID NO: 121.

[0133] The amino acid sequence of subunit S1 (SEQ ID NO: 121) is shown below.QCVN L T T RTQLP P AYTNSFTGVYYPDKVFRSSVLHSTQDLFLPFFSNVTWFHAI HV SGTNGTKRF D NPVLPFNDGVYFAS T EKSNIIRGWIFGTTLDSKTQSLLIVNNATNVVIKVCEFQFCND PFLGV YY HKNNKS W MESEFRVYSSANNCTFEYVSQPFLMDLEGKQGNFKNL R EFVFKNIDGYFKIYSKHTPINLVR D LPQGFSALEPLVDLPIGINITRFQTL L ALH R SYLTPG D S SSG WTAGAAAYYVGYLQPRTFLLKYNENGTITDAVDCALDPLSETKCTLKSFTVEKGIYQTSNFRVQPTESIVRFPNITNLCPFGEVFNATRFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTG K IADYNYKLPDDFTGCVIAWNS N NLDSKVGGNYNY LY RLFRKSNLKPFERDISTEIYQAG S TPCNGV E GFNCYFPLQSYGFQPT N GVGYQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKCVNFNFNGLTGTGVLTESNKKFLPFQQFGRDI A DTTDAVRDPQTLEILDITPCSFGGVSVITPGTNTSNQVAVLYQ D VNCTEVPVAIHADQLTPTWRVYSTGSNVFQTRAGCLIGAE H VNNSYECDIPIGAGICASYQTQTNS PRRAR

[0134] The underlined regions of SEQ ID NO: 121 represent amino acids within the S1 subunit that may be modified.

[0135] In embodiments, the CoV S protein polypeptides described herein comprise an S1 subunit having at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% identity to the S1 subunit of SEQ ID NO: 1 or SEQ ID NO: 2. The S1 subunit may have a deletion, insertion, or mutation affecting up to about 1, up to about 2, up to about 3, up to about 4, up to about 5, up to about 10, up to about 15, up to about 20, up to about 25, or up to about 30 amino acids, compared to the amino acid sequence of the S1 subunit of SEQ ID NO: 1 or SEQ ID NO: 2.The S1 subunit may be characterized by a deletion, insertion, or mutation affecting from about 1 to about 5 amino acids, from about 3 to about 10 amino acids, from about 5 to 10 amino acids, from about 8 to 12 amino acids, from about 10 to 15 amino acids, from about 12 to 17 amino acids, from about 15 to 20 amino acids, from about 18 to 23 amino acids, from about 20 to 25 amino acids, from about 22 to about 27 amino acids, or from about 25 to 30 amino acids, compared to the S1 subunit of SEQ ID NO: 1 or SEQ ID NO: 2.

[0136] In embodiments, the S1 subunit may comprise any combination of the modifications shown in Table 1A.Table 1A Antigens represented by the CoV S protein polypeptide - modifications of the S1 subunit - NTD

[0137] In embodiments, the CoV S protein polypeptides comprise one or more NTD modifications. In embodiments, the NTD is characterized by the amino acid sequence of SEQ ID NO: 118, which corresponds to amino acids 14-305 of SEQ ID NO: 1 or amino acids 1-292 of SEQ ID NO: 2.

[0138] The amino acid sequence of NTD (SEQ ID NO: 118) is shown below.QCVNLTTRTQLPPAYTNSFTRGVYYPDKVFRSSVLHSTQDLFLPFFSNVTWFHAIHVSGTNGTKRFDNPVLPFNDGVYFASTEKSNIIRGWIFGTTLDSKTQSLLIVNNATNVVIKVCEFQFCNDPFLGVYYHKNNKSWMESEFRVYSSANNCTFEYVSQPFLMDLEGKQGNFKNLREFVFKNIDGYFKIYSKHTPINLVRDLPQGFSALEPLVDLPIGINITRFQTLLALHRSYLTPGDSSSGWTAGAAAYYVGYLQPRTFLLKYNENGTITDAVDCALDPLSETKCTLKS

[0139] In embodiments, the NTD is characterized by the amino acid sequence of SEQ ID NO: 45, which corresponds to amino acids 14-331 of SEQ ID NO: 1 or amino acids 1-318 of SEQ ID NO: 2. The amino acid sequence of the NTD (SEQ ID NO: 45) is shown below.

[0140] QCVNLTTRTQLPPAYTNSFTRGVYYPDKVFRSSVLHSTQDLFLPFFSNVTWFHAIHVSGTNGTKRFDNPVLPFNDGVYFASTEKSNIIRGWIFGTTLDSKTQSLLIVNNATNVVIKVCEFQFCNDPFLGVYYHKNNKSWMESEFRVYSSANNCTFEYVS QPFLMDLEGKQGNFKNLREFVFKNIDGYFKIYSKHTPINLVRDLPQGFSALEPLVDLPIGINITRFQTLLALHRSYLTPGDSSSGWTAGAAAYYVGYLQPRTFLLKYNENGTITDAVDCALDPLSETKCTLKSFTVEKGIYQTSNFRVQPTESIVRFPN

[0141] In embodiments, the NTD and RBD overlap by up to about 1 amino acid, up to about 5 amino acids, up to about 10 amino acids, or up to about 20 amino acids.

[0142] In embodiments, the NTD as provided herein may be extended at the C-terminus by up to 5, up to 10, up to 15, up to 20, up to 25, or up to 30 amino acids.

[0143] In embodiments, the CoV S protein polypeptides described herein comprise an NTD having at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% identity to the NTD of SEQ ID NO: 1 or SEQ ID NO: 2. The NTD may have a deletion, insertion, or mutation affecting up to about 1, up to about 2, up to about 3, up to about 4, up to about 5, up to about 10, up to about 15, up to about 20, up to about 25, or up to about 30 amino acids, compared to the amino acid sequence of the NTD of SEQ ID NO: 1 or SEQ ID NO: 2.The NTD may be characterized by a deletion, insertion, or mutation affecting from about 1 to about 5 amino acids, from about 3 to about 10 amino acids, from about 5 to 10 amino acids, from about 8 to 12 amino acids, from about 10 to 15 amino acids, from about 12 to 17 amino acids, from about 15 to 20 amino acids, from about 18 to 23 amino acids, from about 20 to 25 amino acids, from about 22 to about 27 amino acids, or from about 25 to 30 amino acids, compared to the NTD of SEQ ID NO: 1 or SEQ ID NO: 2.

[0144] In embodiments, the CoV S protein polypeptides comprise a deletion of one or more amino acids from the N-terminal domain (NTD) (corresponding to amino acids 1-292 of SEQ ID NO: 2. In embodiments, the CoV S protein polypeptides comprise a deletion of up to about 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, or 292 amino acids of the NTD.

[0145] In embodiments, the CoV S protein polypeptides comprise a deletion of one or more amino acids from the NTD (corresponding to amino acids 1-318 of SEQ ID NO: 2). In embodiments, the CoV S protein polypeptides comprise a deletion of amino acids 1-318 of the NTD of SEQ ID NO: 2. In embodiments, the NTD deletion increases protein expression of the CoV spike (S) protein polypeptide. In embodiments, CoV S protein polypeptides that are characterized by an NTD deletion are characterized by the amino acid sequences presented under SEQ ID NOs: 46, 48, 49, 51, 52, and 54. In embodiments, CoV S protein polypeptides that are characterized by an NTD deletion are encoded by an isolated nucleic acid sequence selected from the group consisting of SEQ ID NO: 47, SEQ ID NO: 50, and SEQ ID NO: 53.

[0146] In embodiments, the NTD may comprise any combination of the modifications shown in Table 1B. The modifications are shown relative to SEQ ID NO: 2, the mature protein S polypeptide sequence, for comparison.Table 1B Antigens represented by the CoV S protein polypeptide are modifications of the S1 subunit - RBD

[0147] In embodiments, the CoV S protein polypeptides comprise one or more RBD modifications.

[0148] In embodiments, the RBD is characterized by the amino acid sequence of SEQ ID NO: 126, which corresponds to amino acids 331-527 of SEQ ID NO: 1 or amino acids 318-514 of SEQ ID NO: 2.

[0149] The amino acid sequence of the RBD (SEQ ID NO: 126) is shown below: NITNLCPFGEVFNATRFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNGVEGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGP In embodiments, the RBD is characterized by the amino acid sequence under SEQ ID NO: 116, which corresponds to amino acids 335-530 of SEQ ID NO: 1 or amino acids 322-517 of SEQ ID NO: 2.

[0150] The amino acid sequence of RBD (SEQ ID NO: 116) is shown below.LCPFGEVFNATRFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNGVEGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKS

[0151] In embodiments, the RBD as provided herein may be extended at the N-terminus or C-terminus by up to 1 amino acid, up to 5 amino acids, up to 10 amino acids, up to 15 amino acids, up to 20 amino acids, up to 25 amino acids, or up to 30 amino acids.

[0152] In embodiments, the CoV S protein polypeptides described herein comprise an RBD having at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% identity to the RBD of SEQ ID NO: 1 or SEQ ID NO: 2. The RBD may have a deletion, insertion, or mutation affecting up to about 1, up to about 2, up to about 3, up to about 4, up to about 5, up to about 10, up to about 15, up to about 20, up to about 25, or up to about 30 amino acids, compared to the amino acid sequence of the RBD of SEQ ID NO: 1 or SEQ ID NO: 2.The RBD may be characterized by a deletion, insertion, or mutation affecting from about 1 to about 5 amino acids, from about 3 to about 10 amino acids, from about 5 to 10 amino acids, from about 8 to 12 amino acids, from about 10 to 15 amino acids, from about 12 to 17 amino acids, from about 15 to 20 amino acids, from about 18 to 23 amino acids, from about 20 to 25 amino acids, from about 22 to about 27 amino acids, or from about 25 to 30 amino acids, compared to the RBD of SEQ ID NO: 1 or SEQ ID NO: 2.

[0153] In embodiments, the CoV S protein polypeptide is characterized by having at least one, at least two, at least three, at least four, at least four, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, or at least 20 mutations in the RBD. In embodiments, the RBD may comprise any combination of the modifications shown in Table 1C.Table 1C Antigens represented by the CoV S protein polypeptide - SD1 / 2 modifications

[0154] ​​In embodiments, the CoV S protein polypeptides comprise one or more modifications of the SD1 / 2 domain characterized by the amino acid sequence of SEQ ID NO: 122, which corresponds to amino acids 542-681 of SEQ ID NO: 1 or amino acids 529-668 of SEQ ID NO: 2.

[0155] The amino acid sequence of the SD1 / 2 domain (SEQ ID NO: 122) is shown below.NFNGLTGTGVLTESNKKFLPFQQFGRDIADTTDAVRDPQTLEILDITPCSFGGVSVITPGTNTSNQVAVLYQDVNCTEVPVAIHADQLTPTWRVYSTGSNVFQTRAGCLIGAEHVNNSYECDIPIGAGICASYQTQTNSP

[0156] In embodiments, the CoV S protein polypeptides described herein comprise an SD1 / 2 domain having at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% identity to the SD1 / 2 domain of SEQ ID NO: 1 or SEQ ID NO: 2. The SD1 / 2 domain may have a deletion, insertion, or mutation affecting up to about 1, up to about 2, up to about 3, up to about 4, up to about 5, up to about 10, up to about 15, up to about 20, up to about 25, or up to about 30 amino acids, compared to the SD1 / 2 amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2.The SD1 / 2 domain may be characterized by a deletion, insertion, or mutation affecting from about 1 to about 5 amino acids, from about 3 to about 10 amino acids, from about 5 to 10 amino acids, from about 8 to 12 amino acids, from about 10 to 15 amino acids, from about 12 to 17 amino acids, from about 15 to 20 amino acids, from about 18 to 23 amino acids, from about 20 to 25 amino acids, from about 22 to about 27 amino acids, or from about 25 to 30 amino acids, compared to the SD1 / 2 domain of SEQ ID NO: 1 or SEQ ID NO: 2.

[0157] In embodiments, the CoV S protein polypeptide is characterized by having at least one, at least two, at least three, at least four, at least four, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, or at least 20 mutations in the SD1 / 2 domain. In embodiments, the SD1 / 2 domain may comprise any combination of the modifications shown in Table 1D.Table 1D Antigens presented by the CoV S protein polypeptide - furin cleavage site modifications

[0158] In embodiments, the CoV S protein polypeptides comprise a furin cleavage site (RRAR) corresponding to amino acids 682-685 of SEQ ID NO: 1 or amino acids 669-672 of SEQ ID NO: 2, which is inactivated by one or more mutations. Inactivation of the furin cleavage site prevents furin cleavage of the CoV S protein polypeptide. In embodiments, the CoV S protein polypeptides described herein that comprise an inactivated furin cleavage site are expressed as a single chain.

[0159] In embodiments, one or more amino acids constituting the native furin cleavage site are mutated to any naturally occurring amino acid. In embodiments, the amino acids are L-amino acids. Non-limiting examples of amino acids include alanine, arginine, glycine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, serine, threonine, histidine, lysine, methionine, proline, valine, isoleucine, leucine, tyrosine, tryptophan, and phenylalanine.

[0160] In embodiments, one or more amino acids constituting the native furin cleavage site are mutated to glutamine. In embodiments, 1, 2, 3, or 4 amino acids can be mutated to glutamine. In embodiments, one of the arginines constituting the native furin cleavage site is mutated to glutamine. In embodiments, two of the arginines constituting the native furin cleavage site are mutated to glutamine. In embodiments, three of the arginines constituting the native furin cleavage site are mutated to glutamine.

[0161] In embodiments, one or more amino acids constituting the native furin cleavage site are mutated to alanine. In embodiments, 1, 2, 3, or 4 amino acids can be mutated to alanine. In embodiments, one of the arginines constituting the native furin cleavage site is mutated to alanine. In embodiments, two of the arginines constituting the native furin cleavage site are mutated to alanine. In embodiments, three of the arginines constituting the native furin cleavage site are mutated to alanine.

[0162] In embodiments, one or more amino acids in the native furin cleavage site are mutated to glycine. In embodiments, 1, 2, 3, or 4 amino acids can be mutated to glycine. In embodiments, one of the arginines in the native furin cleavage site is mutated to glycine. In embodiments, two of the arginines in the native furin cleavage site are mutated to glycine. In embodiments, three of the arginines constituting the native furin cleavage site are mutated to glycine.

[0163] In embodiments, one or more amino acids in the native furin cleavage site are mutated to asparagine. For example, 1, 2, 3, or 4 amino acids can be mutated to asparagine. In embodiments, one of the arginines in the native furin cleavage site is mutated to asparagine. In embodiments, two of the arginines in the native furin cleavage site are mutated to asparagine. In embodiments, three of the arginines in the native furin cleavage site are mutated to asparagine.

[0164] Non-limiting examples of amino acid sequences of inactivated furin sites contained within CoV S polypeptides are provided in Table 1E.Table 1E

[0165] In embodiments, instead of an active furin cleavage site (SEQ ID NO: 6), the CoV S protein polypeptides described herein comprise an inactivated furin cleavage site. In embodiments, the amino acid sequence of the inactivated furin cleavage site is presented as any of SEQ ID NOs: 7-34 or SEQ ID NO: 97. In embodiments, the amino acid sequence of the inactivated furin cleavage site is QQAQ (SEQ ID NO: 7). In embodiments, the amino acid sequence of the inactivated furin cleavage site is GSAS (SEQ ID NO: 97). In embodiments, the amino acid sequence of the inactivated furin cleavage site is GSGA (SEQ ID NO: 111). In embodiments, the amino acid sequence of the inactivated furin cleavage site is GG, GGG (SEQ ID NO: 127), GGGG (SEQ ID NO: 128, or GGGGG (SEQ ID NO: 129).Antigens represented by the CoV S protein polypeptide are modifications of the S2 subunit.

[0166] In embodiments, the CoV S protein polypeptides comprise one or more modifications of the S2 subunit characterized by the amino acid sequence of SEQ ID NO: 120, which corresponds to amino acids 686-1273 of SEQ ID NO: 1 or amino acids 673-1260 of SEQ ID NO: 2.

[0167] The amino acid sequence of the S2 subunit (SEQ ID NO: 120) is shown below.SVASQSIIAYTMSLGAENSVAYSNNSIAIPTNFTISVTTEILPVSMTKTSVDC™YICGDSTECSNLLQYGSFCTQLNRALTGIAVEQDKNTQEVFAQVKQIYKTPPIKDFGGFNFSQILPDPSKPSKRSFIEDLLFNKVTL ADAGFIKQYGDCLGDIAARDLICAQKFNGLTVLPPLLTDEMIAQYTSALLAGTITSGWTFGAGAALQIPFAMQMAYRFNGIGVTQNVLYENQKLIANQFNSAIGKIQDSLSSTASALGKLQDVVNQALNTLVKQLSNFGAISSV LNDILSRLDKVEAEVQIDRLITGRLQSLQTYVTQQLIRAAEIRASANLAATKMSECVLGQSKRVDFCGKGYHLMSFPQSAPHGVVFLHVTYVPAQEKNFTTAPAICHDGKAHFPREGVFVSNGTHWFVTQRNFYEPQITDNTFVSGIT NCDVVIGIVNNTVYDPLQPELDSFKEELDKYFKNHTSPDVDLGDISGINASSVVNIQKEIDRLNEVAKNLNESLIDLQELGKYEQYIKWPWYIWLGFIAGLIAIVMVTIMLCCMTSCCSCLKGCCSCGFDEDDSEPVLKGVKLHYT

[0168] In embodiments, the CoV S protein polypeptides described herein comprise an S2 subunit having at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% identity to the S2 subunit of SEQ ID NO: 1 or SEQ ID NO: 2. The S2 subunit may have a deletion, insertion, or mutation affecting up to about 1, up to about 2, up to about 3, up to about 4, up to about 5, up to about 10, up to about 15, up to about 20, up to about 25, or up to about 30 amino acids, compared to the amino acid sequence of the S2 subunit of SEQ ID NO: 1 or SEQ ID NO: 2.The S2 subunit may be characterized by a deletion, insertion, or mutation affecting from about 1 to about 5 amino acids, from about 3 to about 10 amino acids, from about 5 to 10 amino acids, from about 8 to 12 amino acids, from about 10 to 15 amino acids, from about 12 to 17 amino acids, from about 15 to 20 amino acids, from about 18 to 23 amino acids, from about 20 to 25 amino acids, from about 22 to about 27 amino acids, or from about 25 to 30 amino acids, compared to the S2 subunit of SEQ ID NO: 1 or SEQ ID NO: 2.

[0169] In embodiments, the S2 subunit may comprise any combination of the modifications shown in Table 1F.Table 1F

[0170] In embodiments, the CoV S protein polypeptides comprise a deletion corresponding to one or more deletions within amino acids 676-685 of the native CoV spike (S) protein polypeptide (SEQ ID NO: 2). In embodiments, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids from amino acids 676-685 of the native CoV spike (S) protein polypeptide (SEQ ID NO: 2) are deleted. In embodiments, the amino acid deletions within amino acids 676-685 are consecutive, such as amino acids 676 and 677 are deleted, or amino acids 680 and 681 are deleted. In embodiments, the amino acid deletions within amino acids 676-685 are non-consecutive, such as amino acids 676 and 680 are deleted, or amino acids 677 and 682 are deleted.In embodiments, the CoV S protein polypeptides comprising a deletion corresponding to one or more deletions within amino acids 676-685 are characterized by an amino acid sequence selected from the group consisting of SEQ ID NO: 62 and SEQ ID NO: 63.

[0171] In embodiments, the CoV S protein polypeptides comprise a deletion corresponding to one or more deletions within amino acids 702-711 of the native CoV spike (S) protein polypeptide (SEQ ID NO: 2). In embodiments, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids from amino acids 702-711 of the native SARS-CoV-2 spike (S) protein polypeptide (SEQ ID NO: 2) are deleted. In embodiments, one or more amino acid deletions within amino acids 702-711 are consecutive, such as amino acids 702 and 703 are deleted, or amino acids 708 and 709 are deleted. In embodiments, amino acid deletions within amino acids 702-711 are non-consecutive, such as amino acids 702 and 704 are deleted, or amino acids 707 and 710 are deleted.In embodiments, the CoV S protein polypeptides comprising a deletion corresponding to one or more deletions within amino acids 702-711 are characterized by an amino acid sequence selected from the group consisting of SEQ ID NO: 64 and SEQ ID NO: 65.

[0172] In embodiments, the CoV S protein polypeptides comprise a deletion corresponding to one or more deletions within amino acids 775-793 of the native CoV S protein polypeptide (SEQ ID NO: 2). In embodiments, up to about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, or 19 amino acids from amino acids 775-793 of the native SARS-CoV-2 spike (S) protein polypeptide (SEQ ID NO: 2) are deleted. In embodiments, one or more amino acid deletions within amino acids 775-793 are consecutive, such as amino acids 776 and 777 are deleted, or amino acids 780 and 781 are deleted. In embodiments, the amino acid deletions within amino acids 775-793 are non-consecutive, such as amino acids 775 and 790 are deleted, or amino acids 777 and 781 are deleted.

[0173] In embodiments, the CoV S protein polypeptides comprise a deletion of the fusion peptide (SEQ ID NO: 104) that corresponds to amino acids 806-815 of SEQ ID NO: 2. In embodiments, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids of the fusion peptide from the CoV spike (S) protein polypeptide (SEQ ID NO: 2) are deleted. In embodiments, the amino acid deletions within the fusion peptide are consecutive, such as amino acids 806 and 807 are deleted, or amino acids 809 and 810 are deleted. In embodiments, the amino acid deletions within the fusion peptide are non-consecutive, such as amino acids 806 and 808 are deleted, or amino acids 810 and 813 are deleted. In embodiments, CoV S protein polypeptides comprising a deletion corresponding to one or more amino acids of the fusion peptide are characterized by an amino acid sequence selected from SEQ ID NOs: 66, 77, and 105-108.

[0174] In embodiments, the CoV S protein polypeptides comprise a mutation at Lys-973 of the native CoV spike (S) protein polypeptide (SEQ ID NO: 2). In embodiments, Lys-973 is mutated to any naturally occurring amino acid. In embodiments, Lys-973 is mutated to proline. In embodiments, Lys-973 is mutated to glycine. In embodiments, the CoV spike (S) protein polypeptides comprising a mutation at amino acid 973 are selected from the group consisting of SEQ ID NOs: 84-89, 105-106, and 109-110.

[0175] In embodiments, the CoV S protein polypeptides comprise a mutation at Val-974 of the native CoV spike (S) protein polypeptide (SEQ ID NO: 2). In embodiments, Val-974 is mutated to any naturally occurring amino acid. In embodiments, Val-974 is mutated to proline. In embodiments, Val-974 is mutated to glycine. In embodiments, the CoV spike (S) protein polypeptides comprising a mutation at amino acid 974 are selected from the group consisting of SEQ ID NOs: 84-89, 105-106, and 109-110.

[0176] In embodiments, the CoV S protein polypeptides comprise a mutation at Lys-973 and Val-974 of the native CoV spike (S) protein polypeptide (SEQ ID NO: 2). In embodiments, Lys-973 and Val-974 are mutated to any naturally occurring amino acid. In embodiments, Lys-973 and Val-974 are mutated to proline. In embodiments, the CoV spike (S) protein polypeptides comprising a mutation at amino acids 973 and 974 are selected from SEQ ID NOs: 84-89, 105-106, and 109-110. Antigens represented by the CoV S protein polypeptide - modifications of the S2 subunit - HR1 domain

[0177] In embodiments, the CoV S protein polypeptides comprise one or more modifications of the HR1 domain characterized by the amino acid sequence of SEQ ID NO: 119, which corresponds to amino acids 912-984 of SEQ ID NO: 1 or amino acids 889-971 of SEQ ID NO: 2.

[0178] The amino acid sequence of the HR1 domain (SEQ ID NO: 119) is shown below.MAYRFNGIGVTQNVLYENQKLIANQFNSAIGKIQDSLSSTASALGKLQDVVNQNAQALNTLVKQLSSNFGAISSVLNDILSRL

[0179] In embodiments, the CoV S protein polypeptides described herein comprise an HR1 domain having at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% identity to the HR1 domain of SEQ ID NO: 1 or SEQ ID NO: 2. The HR1 domain may have a deletion, insertion, or mutation affecting up to about 1, up to about 2, up to about 3, up to about 4, up to about 5, up to about 10, up to about 15, up to about 20, up to about 25, or up to about 30 amino acids, compared to the amino acid sequence of the HR1 domain of SEQ ID NO: 1 or SEQ ID NO: 2.The HR1 domain may be characterized by a deletion, insertion, or mutation affecting from about 1 to about 5 amino acids, from about 3 to about 10 amino acids, from about 5 to 10 amino acids, from about 8 to 12 amino acids, from about 10 to 15 amino acids, from about 12 to 17 amino acids, from about 15 to 20 amino acids, from about 18 to 23 amino acids, from about 20 to 25 amino acids, from about 22 to about 27 amino acids, or from about 25 to 30 amino acids, compared to the HR1 domain of SEQ ID NO: 1 or SEQ ID NO: 2.

[0180] In embodiments, the HR1 domain may comprise any combination of the modifications shown in Table 1G.Table 1G Antigens represented by the CoV S protein polypeptide are modifications of the S2 subunit - the HR2 domain

[0181] In embodiments, the CoV S protein polypeptides comprise one or more modifications of the HR2 domain characterized by the amino acid sequence of SEQ ID NO: 125, which corresponds to amino acids 1163-1213 of SEQ ID NO: 1 or amino acids 1150-1200 of SEQ ID NO: 2.

[0182] The amino acid sequence of the HR2 domain (SEQ ID NO: 125) is shown below.DVDLGDISGINASVVNIQKEIDRLNEVAKNLNESLIDLQELGKYEQYIKWP

[0183] In embodiments, the CoV S protein polypeptides described herein comprise an HR2 domain having at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% identity to the HR2 domain of SEQ ID NO: 1 or SEQ ID NO: 2. The HR2 domain may have a deletion, insertion, or mutation affecting up to about 1, up to about 2, up to about 3, up to about 4, up to about 5, up to about 10, up to about 15, up to about 20, up to about 25, or up to about 30 amino acids, compared to the amino acid sequence of the HR2 domain of SEQ ID NO: 1 or SEQ ID NO: 2.The HR2 domain may be characterized by a deletion, insertion, or mutation affecting from about 1 to about 5 amino acids, from about 3 to about 10 amino acids, from about 5 to 10 amino acids, from about 8 to 12 amino acids, from about 10 to 15 amino acids, from about 12 to 17 amino acids, from about 15 to 20 amino acids, from about 18 to 23 amino acids, from about 20 to 25 amino acids, from about 22 to about 27 amino acids, or from about 25 to 30 amino acids, compared to the HR2 domain of SEQ ID NO: 1 or SEQ ID NO: 2. Antigens represented by the CoV S protein polypeptide - domain modifications TM.

[0184] In embodiments, the CoV S protein polypeptides comprise one or more modifications of the TM domain characterized by the amino acid sequence of SEQ ID NO: 123, which corresponds to amino acids 1214-1237 of SEQ ID NO: 1 or amino acids 1201-1224 of SEQ ID NO: 2.

[0185] The amino acid sequence of the TM domain (SEQ ID NO: 123) is shown below.WYIWLGFIAGLIAIVMVTIMLCCM

[0186] In embodiments, the CoV S protein polypeptides described herein comprise a TM domain having at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% identity to the TM domain of SEQ ID NO: 1 or SEQ ID NO: 2. The TM domain may have a deletion, insertion, or mutation affecting up to about 1, up to about 2, up to about 3, up to about 4, up to about 5, up to about 10, up to about 15, up to about 20, up to about 25, or up to about 30 amino acids, compared to the amino acid sequence of the TM domain) of SEQ ID NO: 1 or SEQ ID NO: 2.The TM domain may be characterized by a deletion, insertion, or mutation affecting from about 1 to about 5 amino acids, from about 3 to about 10 amino acids, from about 5 to 10 amino acids, from about 8 to 12 amino acids, from about 10 to 15 amino acids, from about 12 to 17 amino acids, from about 15 to 20 amino acids, from about 18 to 23 amino acids, from about 20 to 25 amino acids, from about 22 to about 27 amino acids, or from about 25 to 30 amino acids, compared to the TM domain of SEQ ID NO: 1 or SEQ ID NO: 2.

[0187] In embodiments, the CoV S protein polypeptides described herein do not comprise the entire TM domain. In embodiments, the CoV S protein polypeptides described herein comprise the TM domain. Antigens presented by the CoV S protein polypeptide are CT modifications.

[0188] In embodiments, the CoV S protein polypeptides comprise one or more CT modifications characterized by the amino acid sequence of SEQ ID NO: 124, which corresponds to amino acids 1238-1273 of SEQ ID NO: 1 or amino acids 1225-1260 of SEQ ID NO: 2.

[0189] The amino acid sequence of CT (SEQ ID NO: 124) is shown below:TSCCSCLKGCCSCGSCCKFDEDDSEPVLKGVKLHYT

[0190] In embodiments, the CoV S protein polypeptides described herein comprise a CT having at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% identity to the CT of SEQ ID NO: 1 or SEQ ID NO: 2. The CT may have a deletion, insertion, or mutation affecting up to about 1, up to about 2, up to about 3, up to about 4, up to about 5, up to about 10, up to about 15, up to about 20, up to about 25, or up to about 30 amino acids, compared to the amino acid sequence of the CT of SEQ ID NO: 1 or SEQ ID NO: 2.The CT may be characterized by a deletion, insertion, or mutation affecting from about 1 to about 5 amino acids, from about 3 to about 10 amino acids, from about 5 to 10 amino acids, from about 8 to 12 amino acids, from about 10 to 15 amino acids, from about 12 to 17 amino acids, from about 15 to 20 amino acids, from about 18 to 23 amino acids, from about 20 to 25 amino acids, from about 22 to about 27 amino acids, or from about 25 to 30 amino acids, compared to the CT of SEQ ID NO: 1 or SEQ ID NO: 2.

[0191] In embodiments, the CoV S protein polypeptides described herein do not contain CT. In embodiments, the CoV S protein polypeptides contain CT.

[0192] In embodiments, the CoV S protein polypeptides comprise TM and CT. In embodiments, the CoV spike (S) protein polypeptides comprise a deletion of one or more amino acids from the transmembrane region and cytoplasmic tail (TMCT) (corresponding to amino acids 1201-1260). The amino acid sequence of TMCT is presented under SEQ ID NO: 39. In embodiments, CoV S protein polypeptides that are characterized by a deletion of one or more residues from TMCT are characterized by increased protein expression. In embodiments, the CoV spike (S) protein polypeptides that are characterized by one or more deletions in TMCT are characterized by an amino acid sequence selected from the group consisting of SEQ ID NO: 40, 41, 42, 52, 54, 59, 61, 88, and 89.In embodiments, CoV S protein polypeptides that are characterized by one or more deletions in the TM-CD are encoded by an isolated nucleic acid sequence selected from the group consisting of SEQ ID NO: 39, 43, 53, and 60. The antigens represented by the CoV S protein polypeptide are non-limiting combinations of mutations.

[0193] In embodiments, the CoV S protein polypeptides comprise a deletion of amino acids 56 and 57 of the native CoV spike (S) protein polypeptide (SEQ ID NO: 2).

[0194] In embodiments, the CoV S protein polypeptides comprise deletions of amino acids 131 and 132 of the native CoV spike (S) protein polypeptide (SEQ ID NO: 2).

[0195] In embodiments, the CoV S protein polypeptides comprise a deletion of amino acids 56 and 131 of the native CoV spike (S) protein polypeptide (SEQ ID NO: 2). In embodiments, the CoV S protein polypeptides comprise a deletion of amino acids 57 and 131 of the native CoV spike (S) protein polypeptide (SEQ ID NO: 2).

[0196] In embodiments, the CoV S protein polypeptides comprise a deletion of amino acids 56, 57, and 131 of the native CoV spike (S) protein polypeptide (SEQ ID NO: 2).

[0197] In embodiments, the CoV S protein polypeptides comprise a deletion of amino acids 56 and 132 of the native CoV spike (S) protein polypeptide (SEQ ID NO: 2).

[0198] In embodiments, the CoV S protein polypeptides comprise a deletion of amino acids 57 and 132 of the native CoV spike (S) protein polypeptide (SEQ ID NO: 2).

[0199] In embodiments, the CoV S protein polypeptides comprise a deletion of amino acids 56, 57, and 132 of the native CoV spike (S) protein polypeptide (SEQ ID NO: 2).

[0200] In embodiments, the CoV S protein polypeptides comprise a deletion of amino acids 56, 57, 131, and 132 of the native CoV spike (S) protein polypeptide (SEQ ID NO: 2).

[0201] In embodiments, the CoV S protein polypeptides comprise mutations that stabilize the pre-fusion conformation of the CoV S protein polypeptide. In embodiments, the CoV S protein polypeptides comprise proline or glycine substitutions that stabilize the pre-fusion conformation. This strategy was used to develop a MERS-CoV S protein stabilized in the pre-fusion conformation as described in the following documents, each of which is incorporated herein by reference in its entirety: Proc Natl Acad Sci USA. 2017 Aug 29;114(35):E7348-E7357; Sci Rep. 2018 Oct 24;8(1):15701; US ​​Patent Application Publication No. 2020 / 0061185 and PCT Application No. PCT / US2017 / 058370.

[0202] In embodiments, the CoV S protein polypeptides comprise a mutation at Lys-973 and Val-974 and an inactivated furin cleavage site. In embodiments, the CoV S protein polypeptides comprise mutations of Lys-973 and Val-974 to proline and an inactivated furin cleavage site characterized by the amino acid sequence QQAQ (SEQ ID NO: 7) or GSAS (SEQ ID NO: 96). An exemplary CoV S protein polypeptide comprising a mutation of Lys-973 and Val-974 and an inactivated furin cleavage site is depicted in Fig. 8. In embodiments, the CoV S protein polypeptides comprising mutations of Lys-973 and Val-974 to proline and an inactivated furin cleavage site are characterized by the amino acid sequences of SEQ ID NO: 86 or 87 and the nucleic acid sequence of SEQ ID NO: 96.

[0203] In embodiments, the CoV S protein polypeptides comprise a mutation at Lys-973 and Val-974, an inactivated furin cleavage site, and a deletion of one or more amino acids of the fusion peptide. In embodiments, the CoV S protein polypeptides comprise mutations of Lys-973 and Val-974 to proline and an inactivated furin cleavage site characterized by the amino acid sequence QQAQ (SEQ ID NO: 7 or GSAS (SEQ ID NO: 96), and a deletion of one or more amino acids of the fusion peptide. In embodiments, the CoV S protein polypeptides comprising mutations of Lys-973 and Val-974 to proline, an inactivated furin cleavage site, and a deletion of one or more amino acids of the fusion peptide are characterized by the amino acid sequence of SEQ ID NO: 105 or 106.In embodiments, the CoV S protein polypeptide comprises a Leu-5 to phenylalanine mutation, a Thr-7 to asparagine mutation, a Pro-13 to serine mutation, an Asp-125 to tyrosine mutation, an Arg-177 to serine mutation, a Lys-404 to threonine mutation, a Glu-471 to lysine mutation, an Asn-488 to tyrosine mutation, a His-642 to tyrosine mutation, a Thr-1014 to isoleucine mutation, Lys-973 and Val-974 to proline mutations, and an inactivated furin cleavage site characterized by the amino acid sequence QQAQ (SEQ ID NO: 7) or GSAS (SEQ ID NO: 96), relative to the native CoV spike (S) protein polypeptide (SEQ ID NO: 2).

[0204] In embodiments, the CoV S protein polypeptides comprise a Trp-139 mutation to cysteine, a Leu-439 mutation to arginine, Lys-973 and Val-974 mutations to proline, and an inactivated furin cleavage site characterized by the amino acid sequence QQAQ (SEQ ID NO: 7 or GSAS (SEQ ID NO: 96), relative to the native CoV spike (S) protein polypeptide (SEQ ID NO: 2). In embodiments, the CoV S protein polypeptides comprise a Trp-152 mutation to cysteine, a Leu-452 mutation to arginine, a Ser-13 mutation to isoleucine, Lys-986 and Val-987 mutations to proline, and an inactivated furin cleavage site characterized by the amino acid sequence QQAQ (SEQ ID NO: 7 or GSAS (SEQ ID NO: 96), relative to the native CoV spike (S) protein polypeptide (SEQ ID NO: 1).

[0205] In embodiments, the CoV S protein polypeptide comprises a Lys-404 mutation to threonine or asparagine, a Glu-471 mutation to lysine, an Asn-488 mutation to tyrosine, a Leu-5 mutation to phenylalanine, an Asp-67 mutation to alanine, an Asp-202 mutation to glycine, a deletion of one or more amino acids 229-231, an Arg-233 ​​mutation to isoleucine, Lys-973 and Val-974 mutations to proline, and an inactivated furin cleavage site characterized by the amino acid sequence QQAQ (SEQ ID NO: 7) or GSAS (SEQ ID NO: 96), relative to the native CoV spike (S) protein polypeptide (SEQ ID NO: 2).

[0206] In embodiments, the CoV S protein polypeptide comprises an Asn-488 mutation to tyrosine, Lys-973 and Val-974 mutations to proline, and an inactivated furin cleavage site characterized by the amino acid sequence QQAQ (SEQ ID NO: 7 or GSAS (SEQ ID NO: 96), relative to the native CoV spike (S) protein polypeptide (SEQ ID NO: 2). In embodiments, the CoV S protein polypeptide characterized by an Asn-488 mutation to tyrosine, Lys-973 and Val-974 mutations to proline, and an inactivated furin cleavage site characterized by the amino acid sequence QQAQ (SEQ ID NO: 7 or GSAS (SEQ ID NO: 96) comprises the amino acid sequence of SEQ ID NO: 112.

[0207] In embodiments, the CoV S protein polypeptides comprise an Asp-601 to glycine mutation, an Asn-488 to tyrosine mutation, Lys-973 and Val-974 to proline mutations, and an inactivated furin cleavage site characterized by the amino acid sequence QQAQ (SEQ ID NO: 7 or GSAS (SEQ ID NO: 96), relative to the native CoV spike (S) protein polypeptide (SEQ ID NO: 2). In embodiments, the CoV S protein polypeptide characterized by an Asn-488 to tyrosine mutation, Lys-973 and Val-974 to proline mutations, and an inactivated furin cleavage site characterized by the amino acid sequence QQAQ (SEQ ID NO: 7 or GSAS (SEQ ID NO: 96) comprises the amino acid sequence under SEQ ID NO: 113.

[0208] In embodiments, the CoV S protein polypeptide comprises a deletion of amino acids 56, 57, and 131, an Asn-488 to tyrosine mutation, an Ala-557 to aspartate mutation, an Asp-601 to glycine mutation, a Pro-668 to histidine mutation, a Thr-703 to isoleucine mutation, a Ser-969 to alanine mutation, an Asp-1105 to histidine mutation, Lys-973 and Val-974 to proline mutations, and an inactivated furin cleavage site characterized by the amino acid sequence QQAQ (SEQ ID NO: 7), GSAS (SEQ ID NO: 96, or GG, relative to the native CoV spike (S) protein polypeptide (SEQ ID NO: 2).In embodiments, the CoV S protein polypeptide characterized by a deletion of amino acids 56, 57, and 131, an Asn-488 mutation to tyrosine, an Ala-557 mutation to aspartate, an Asp-601 mutation to glycine, a Pro-668 mutation to histidine, a Thr-703 mutation to isoleucine, a Ser-969 mutation to alanine, an Asp-1105 mutation to histidine, Lys-973 and Val-974 mutations to proline, and an inactivated furin cleavage site characterized by the amino acid sequence QQAQ (SEQ ID NO: 7 or GSAS (SEQ ID NO: 96) comprises the amino acid sequence of SEQ ID NO: 114.In embodiments, the CoV S protein polypeptide is characterized by a deletion of amino acids 56, 57, and 131, an Asn-488 to tyrosine mutation, an Ala-557 to aspartate mutation, an Asp-601 to glycine mutation, a Pro-668 to histidine mutation, a Thr-703 to isoleucine mutation, a Ser-969 to alanine mutation, an Asp-1105 to histidine mutation, Lys-973 and Val-974 to proline mutations, and an inactivated furin cleavage site characterized by the amino acid sequence QQAQ (SEQ ID NO: 7) or GSAS (SEQ ID NO: 96 or GG, comprises the amino acid sequence of SEQ ID NO: 136.In embodiments, a CoV S protein polypeptide characterized by a deletion of amino acids 56, 57, and 131, an Asn-488 to tyrosine mutation, an Ala-557 to aspartate mutation, an Asp-601 to glycine mutation, a Pro-668 to histidine mutation, a Thr-703 to isoleucine mutation, a Ser-969 to alanine mutation, an Asp-1105 to histidine mutation, Lys-973 and Val-974 to proline mutations, and an inactivated furin cleavage site characterized by the amino acid sequence GG comprises the amino acid sequence of SEQ ID NO: 137 or SEQ ID NO: 138. In some embodiments, a CoV S protein polypeptide characterized by the amino acid sequence of SEQ ID NO: 114 or SEQ ID NO: 136 is encoded by a nucleic acid characterized by the sequence nucleic acid under SEQ ID NO: 135.In some embodiments, the CoV S protein polypeptide having the amino acid sequence of SEQ ID NO: 137 or SEQ ID NO: 138 is encoded by a nucleic acid having the sequence of SEQ ID NO: 139.

[0209] In embodiments, the CoV S protein polypeptide comprises a deletion of amino acids 56, 57, and 132, an Asn-488 to tyrosine mutation, an Ala-557 to aspartate mutation, an Asp-601 to glycine mutation, a Pro-668 to histidine mutation, a Thr-703 to isoleucine mutation, a Ser-969 to alanine mutation, an Asp-1105 to histidine mutation, Lys-973 and Val-974 to proline mutations, and an inactivated furin cleavage site characterized by the amino acid sequence QQAQ (SEQ ID NO: 7) or GSAS (SEQ ID NO: 96), relative to the native CoV spike (S) protein polypeptide (SEQ ID NO: 2).In embodiments, the CoV S protein polypeptide characterized by a deletion of amino acids 56, 57, and 132, an Asn-488 to tyrosine mutation, an Ala-557 to aspartate mutation, an Asp-601 to glycine mutation, a Pro-668 to histidine mutation, a Thr-703 to isoleucine mutation, a Ser-969 to alanine mutation, an Asp-1105 to histidine mutation, Lys-973 and Val-974 to proline mutations, and an inactivated furin cleavage site characterized by the amino acid sequence QQAQ (SEQ ID NO: 7 or GSAS (SEQ ID NO: 96) comprises the amino acid sequence of SEQ ID NO: 114.

[0210] In embodiments, the CoV S protein polypeptide comprises an Asp-488 to tyrosine mutation, an Asp-67 to alanine mutation, a Leu-229 to histidine mutation, an Asp-202 to glycine mutation, a Lys-404 to asparagine mutation, a Glu-471 to lysine mutation, an Ala-688 to valine mutation, an Asp-601 to glycine mutation, Lys-973 and Val-974 to proline mutations, and an inactivated furin cleavage site characterized by the amino acid sequence QQAQ (SEQ ID NO: 7) or GSAS (SEQ ID NO: 96), relative to the native CoV spike (S) protein polypeptide (SEQ ID NO: 2).In embodiments, the CoV S protein polypeptide characterized by an Asp-488 to tyrosine mutation, an Asp-67 to alanine mutation, a Leu-229 to histidine mutation, an Asp-202 to glycine mutation, a Lys-404 to asparagine mutation, a Glu-471 to lysine mutation, an Ala-688 to valine mutation, an Asp-601 to glycine mutation, Lys-973 and Val-974 to proline mutations, and an inactivated furin cleavage site characterized by the amino acid sequence QQAQ (SEQ ID NO: 7 or GSAS (SEQ ID NO: 96) comprises the amino acid sequence of SEQ ID NO: 115.

[0211] In embodiments, the CoV S protein polypeptide comprises one or more modifications selected from K973P, V974P, an inactivated furin cleavage site, deletions of amino acid 56, deletion of amino acid 57, deletion of amino acid 131, N488Y, A557D, D601G, P668H, T703I, S969A and D1105H, wherein the amino acids are numbered relative to the CoV S protein polypeptide characterized by the amino acid sequence of SEQ ID NO: 2. In embodiments, the inactivated furin cleavage site is characterized by the amino acid sequence QQAQ (SEQ ID NO: 7). In embodiments, the inactivated furin cleavage site is characterized by the amino acid sequence GG.

[0212] In embodiments, the CoV S protein polypeptide comprises one or more modifications selected from K973P, V974P, an inactivated furin cleavage site, D67A, D202G, L229H, K404N, E471K, N488Y, D601G, and A688V, where the amino acids are numbered relative to the CoV S protein polypeptide characterized by the amino acid sequence of SEQ ID NO: 2. In embodiments, the inactivated furin cleavage site is characterized by the amino acid sequence QQAQ (SEQ ID NO: 7). In embodiments, the inactivated furin cleavage site is characterized by the amino acid sequence GG.

[0213] In embodiments, the CoV S protein polypeptide comprises one or more modifications selected from K973P, V974P, an inactivated furin cleavage site, a deletion of amino acids 229-231, D67A, D202G, K404N, E471K, N488Y, D601G, and A688V, wherein the amino acids are numbered relative to the CoV S protein polypeptide characterized by the amino acid sequence of SEQ ID NO: 2.

[0214] In embodiments, the CoV S protein polypeptide comprises one or more modifications selected from K973P, V974P, an inactivated furin cleavage site characterized by the amino acid sequence QQAQ (SEQ ID NO: 7), a deletion of amino acids 229-231, L5F, D67A, D202G, K404N, E471K, N488Y, D601G, and A688V, wherein the amino acids are numbered relative to the CoV S protein polypeptide characterized by the amino acid sequence of SEQ ID NO: 2. In embodiments, the CoV S protein polypeptide characterized by one or more modifications selected from K973P, V974P, an inactivated furin cleavage site characterized by the amino acid sequence QQAQ (SEQ ID NO: 7), a deletion of amino acids 229-231, L5F, D67A, D202G, K404N, E471K, N488Y, D601G and A688V, where the amino acids are numbered relative to the CoV S protein polypeptide characterized by the amino acid sequence under SEQ ID NO: 2, contains the amino acid sequence under SEQ ID NO: 144.In embodiments, the CoV S protein polypeptide having the amino acid sequence of SEQ ID NO: 144 is encoded by a nucleic acid having the sequence of SEQ ID NO: 145.

[0215] In embodiments, the CoV S protein polypeptide comprises one or more modifications selected from K973P, V974P, an inactivated furin cleavage site characterized by the amino acid sequence GG, a deletion of amino acids 229-231, L5F, D67A, D202G, K404N, E471K, N488Y, D601G, and A688V, wherein the amino acids are numbered relative to the CoV S protein polypeptide characterized by the amino acid sequence of SEQ ID NO: 2. In embodiments, the CoV S protein polypeptide characterized by one or more modifications selected from K973P, V974P, an inactivated furin cleavage site characterized by the amino acid sequence GG, a deletion of amino acids 229-231, L5F, D67A, D202G, K404N, E471K, N488Y, D601G and A688V, where the amino acids are numbered relative to the CoV S protein polypeptide characterized by the amino acid sequence of SEQ ID NO: 2, comprises the amino acid sequence of SEQ ID NO: 144.In embodiments, the CoV S protein polypeptide having the amino acid sequence of SEQ ID NO: 144 is encoded by a nucleic acid having the sequence of SEQ ID NO: 145.

[0216] In embodiments, the CoV S protein polypeptide comprises one or more modifications selected from K973P, V974P, an inactivated furin cleavage site, L5F, T7N, P13S, D125Y, R177S, K404T, E471K, N488Y, D601G, H642Y, T1014I, and V1163F, wherein the amino acids are numbered relative to the CoV S protein polypeptide characterized by the amino acid sequence of SEQ ID NO: 2. In embodiments, the CoV S protein polypeptide comprising one or more modifications selected from K973P, V974P, an inactivated furin cleavage site, L5F, T7N, P13S, D125Y, R177S, K404T, E471K, N488Y, D601G, H642Y, T1014I and V1163F, where the amino acids are numbered relative to the CoV S protein polypeptide having the amino acid sequence of SEQ ID NO: 2, having the amino acid sequence of SEQ ID NO: 151.In embodiments, the CoV S protein polypeptide having the amino acid sequence of SEQ ID NO: 151 is encoded by a nucleic acid having the sequence of SEQ ID NO: 150.

[0217] In embodiments, the CoV S protein polypeptide comprises one or more modifications selected from K973P, V974P, an inactivated furin cleavage site, a deletion of amino acids 229-231, L5F, D67A, D202G, L229H, K404N, E471K, N488Y, D601G, and A688V, wherein the amino acids are numbered relative to the CoV S protein polypeptide characterized by the amino acid sequence of SEQ ID NO: 2.

[0218] In embodiments, the CoV S protein polypeptide comprises one or more modifications selected from K973P, V974P, an inactivated furin cleavage site, K404N, E471K, N488Y, L5F, D67A, D202G, L229H, D601G, A688V, and a deletion of amino acids 229-231, where the amino acids are numbered relative to the CoV S protein polypeptide characterized by the amino acid sequence of SEQ ID NO: 2. In embodiments, the inactivated furin cleavage site is characterized by the amino acid sequence QQAQ (SEQ ID NO: 7). In embodiments, the inactivated furin cleavage site is characterized by the amino acid sequence GG.

[0219] In embodiments, the CoV S protein polypeptide comprises one or more modifications selected from K973P, V974P, an inactivated furin cleavage site, K404N, E471K, and N488K, where the amino acids are numbered relative to the CoV S protein polypeptide characterized by the amino acid sequence of SEQ ID NO: 2. In embodiments, the CoV S protein polypeptide comprises one or more modifications selected from K973P, V974P, an inactivated furin cleavage site, K404N, E471K, and N488Y. In embodiments, the CoV S protein polypeptide is an RBD of a CoV S protein polypeptide characterized by one or more modifications selected from K973P, V974P, an inactivated furin cleavage site, K404N, E471K, and N488K, wherein the amino acids are numbered relative to the CoV S protein polypeptide characterized by the amino acid sequence of SEQ ID NO: 2.In embodiments, the CoV S protein polypeptide is an RBD of a CoV S protein polypeptide characterized by one or more modifications selected from K973P, V974P, an inactivated furin cleavage site, K404N, E471K, and N488Y, wherein the amino acids are numbered relative to the CoV S protein polypeptide characterized by the amino acid sequence of SEQ ID NO: 2.

[0220] In embodiments, the CoV S protein polypeptide comprises one or more modifications selected from K973P, V974P, an inactivated furin cleavage site characterized by the amino acid sequence GG, D601G, E404N, E471K, and N488Y. In embodiments, the CoV S protein polypeptide comprises one or more modifications selected from K973P, V974P, an inactivated furin cleavage site characterized by the amino acid sequence GG, and the D601G mutation, wherein the amino acids are numbered relative to the CoV S protein polypeptide characterized by the amino acid sequence of SEQ ID NO: 2. In embodiments, the CoV S protein polypeptide comprising modifications selected from K973P, V974P, an inactivated furin cleavage site characterized by the amino acid sequence GG, and the D601G mutation, wherein the amino acids are numbered relative to the CoV S protein polypeptide, is characterized by the amino acid sequence of SEQ ID NO: 133.

[0221] In embodiments, the CoV S protein polypeptide comprises one or more modifications selected from K973P, V974P, an inactivated furin cleavage site, optionally wherein the inactivated furin cleavage site is QQAQ (SEQ ID NO: 7 or GG, K404N, E471K, N488K, D67A, D202G, L229H, D601G and A688V, wherein the amino acids are numbered relative to the CoV S protein polypeptide characterized by the amino acid sequence of SEQ ID NO: 2. In embodiments, the CoV S protein polypeptide comprising one or more modifications selected from K973P, V974P, an inactivated furin cleavage site, optionally wherein the inactivated furin cleavage site is QQAQ (SEQ ID NO: 7 or GG, K404N, E471K, N488K, D67A, D202G, L229H, D601G and A688V, characterized by the amino acid sequence under SEQ ID NO: 132 or SEQ ID NO: 141.In embodiments, the CoV S protein polypeptide having the amino acid sequence of SEQ ID NO: 132 is encoded by a nucleic acid having the nucleic acid sequence of SEQ ID NO: 131. In embodiments, the CoV S protein polypeptide having the amino acid sequence of SEQ ID NO: 132 is encoded by a nucleic acid having the nucleic acid sequence of SEQ ID NO: 142.

[0222] In embodiments, the CoV S protein polypeptide comprises one or more modifications selected from K973P, V974P, an inactivated furin cleavage site, W139C, and L439R, wherein the amino acids are numbered relative to the CoV S protein polypeptide having the amino acid sequence of SEQ ID NO: 2. In embodiments, the CoV S protein polypeptide comprising K973P, V974P, an inactivated furin cleavage site, the modifications W139C, and L439R is expressed with a signal peptide having the amino acid sequence of SEQ ID NO: 117 or SEQ ID NO: 5. In embodiments, the CoV S protein polypeptide provides one or more modifications selected from K973P, V974P, an inactivated furin cleavage site, D601G, W139C, and L439R, wherein the amino acids are numbered relative to the CoV S protein polypeptide having the amino acid sequence of SEQ ID NO: 2.In embodiments, the CoV S protein polypeptide comprises K973P, V974P, an inactivated furin cleavage site, D601G, W139C, and L439R modifications, and is expressed with a signal peptide characterized by the amino acid sequence of SEQ ID NO: 117 or SEQ ID NO: 5.

[0223] In embodiments, the CoV S protein polypeptide comprises one or more modifications selected from K973P, V974P, an inactivated furin cleavage site, D601G, L5F, D67A, D202G, deletions of amino acids 229-231, R233I, K404N, E471K, N488Y, and A688V, wherein the amino acids are numbered relative to the CoV S protein polypeptide characterized by the amino acid sequence of SEQ ID NO: 2.

[0224] In embodiments, the CoV S protein polypeptide comprises one or more modifications selected from K973P, V974P, an inactivated furin cleavage site, optionally wherein the inactivated furin cleavage site is QQAQ (SEQ ID NO: 7), W139C, S481P, D601G, and L439R, wherein the amino acids are numbered relative to the CoV S protein polypeptide having the amino acid sequence of SEQ ID NO: 2. In embodiments, the CoV S protein polypeptide comprises one or more modifications selected from K973P, V974P, an inactivated furin cleavage site, optionally wherein the inactivated furin cleavage site is QQAQ (SEQ ID NO: 7), W139C, D601G, and L439R, wherein the amino acids are numbered relative to the CoV S protein polypeptide characterized by the amino acid sequence under SEQ ID NO: 2.In embodiments, the CoV S protein polypeptide comprises one or more modifications selected from K973P, V974P, an inactivated furin cleavage site, optionally wherein the inactivated furin cleavage site is QQAQ (SEQ ID NO: 7), W139C, S481P, and D601G, wherein the amino acids are numbered relative to the CoV S protein polypeptide characterized by the amino acid sequence of SEQ ID NO: 2. In embodiments, the CoV S protein polypeptide comprising one or more modifications selected from K973P, V974P, an inactivated furin cleavage site, optionally wherein the inactivated furin cleavage site is QQAQ (SEQ ID NO: 7), W139C, S481P, D601G, and L439R, is characterized by the amino acid sequence of SEQ ID NO: 153.In embodiments, the CoV S protein polypeptide having the amino acid sequence of SEQ ID NO: 153 comprises a signal peptide having the amino acid sequence of SEQ ID NO: 117. In embodiments, the CoV S protein polypeptide having the amino acid sequence of SEQ ID NO: 153 comprises a signal peptide having the amino acid sequence of SEQ ID NO: 5.

[0225] In embodiments, the CoV S protein polypeptide comprises one or more modifications selected from K973P, V974P, an inactivated furin cleavage site, optionally wherein the inactivated furin cleavage site is QQAQ (SEQ ID NO: 7), T82I, D240G, E471K, D601G, and A688V, wherein the amino acids are numbered relative to the CoV S protein polypeptide having the amino acid sequence of SEQ ID NO: 2. In embodiments, the CoV S protein polypeptide comprising one or more modifications selected from K973P, V974P, an inactivated furin cleavage site, optionally wherein the inactivated furin cleavage site is QQAQ (SEQ ID NO: 7), T82I, D240G, E471K, D601G and A688V, where the amino acids are numbered relative to the CoV S protein polypeptide having the amino acid sequence of SEQ ID NO: 2, is characterized by the amino acid sequence of SEQ ID NO: 156.In embodiments, the CoV S protein polypeptide comprising one or more modifications selected from K973P, V974P, an inactivated furin cleavage site, optionally wherein the inactivated furin cleavage site is QQAQ (SEQ ID NO: 7), T82I, D240G, E471K, D601G and A688V, wherein the amino acids are numbered relative to the CoV S protein polypeptide having the amino acid sequence of SEQ ID NO: 2, comprises a signal peptide having the amino acid sequence of SEQ ID NO: 154 or SEQ ID NO: 5.

[0226] In embodiments, the CoV S protein polypeptide comprises one or more modifications selected from K973P, V974P, an inactivated furin cleavage site, optionally wherein the inactivated furin cleavage site is QQAQ (SEQ ID NO: 7), T82I, D240G, S464N, D601G, and A688V, wherein the amino acids are numbered relative to the CoV S protein polypeptide having the amino acid sequence of SEQ ID NO: 2. In embodiments, the CoV S protein polypeptide comprising one or more modifications selected from K973P, V974P, an inactivated furin cleavage site, optionally wherein the inactivated furin cleavage site is QQAQ (SEQ ID NO: 7), T82I, D240G, S464N, D601G and A688V, where the amino acids are numbered relative to the CoV S protein polypeptide having the amino acid sequence of SEQ ID NO: 2, is characterized by the amino acid sequence of SEQ ID NO: 158.In embodiments, the CoV S protein polypeptide comprising one or more modifications selected from K973P, V974P, an inactivated furin cleavage site, optionally wherein the inactivated furin cleavage site is QQAQ (SEQ ID NO: 7), T82I, D240G, S464N, D601G and A688V, wherein the amino acids are numbered relative to the CoV S protein polypeptide characterized by the amino acid sequence under SEQ ID NO: 2, comprises a signal peptide under SEQ ID NO: 154.

[0227] In embodiments, the CoV S protein polypeptide comprises one or more modifications selected from K973P, V974P, an inactivated furin cleavage site, optionally wherein the inactivated furin cleavage site is QQAQ (SEQ ID NO: 7), a deletion of amino acid 56, a deletion of amino acid 57, a deletion of amino acid 131, a N488Y mutation, an A557D mutation, a D601G mutation, a P668H mutation, a T703I mutation, an S969A mutation, and a D1105H mutation, wherein the CoV S protein polypeptide is numbered relative to a wild-type SARS-CoV-2 S protein polypeptide characterized by the amino acid sequence of SEQ ID NO: 2.In embodiments, the CoV S protein polypeptide comprises one or more modifications selected from K973P, V974P, an inactivated furin cleavage site, optionally wherein the inactivated furin cleavage site is QQAQ (SEQ ID NO: 7), deletion of amino acid 56, deletion of amino acid 57, deletion of amino acid 132, N488Y mutation, A557D mutation, D601G mutation, P668H mutation, T703I mutation, S969A mutation, and D1105H mutation, wherein the CoV S protein polypeptide is numbered relative to the wild-type SARS-CoV-2 S protein polypeptide characterized by the amino acid sequence of SEQ ID NO: 2.

[0228] In embodiments, the CoV S protein polypeptide comprises one or more modifications selected from K973P, V974P, an inactivated furin cleavage site, optionally wherein the inactivated furin cleavage site is QQAQ (SEQ ID NO: 7), a D67A mutation, a L229H mutation, a R233I mutation, an A688V mutation, a N488Y mutation, a K404N mutation, an E471K mutation, and a D601G mutation, wherein the CoV S protein polypeptide is numbered relative to a wild-type SARS-CoV-2 S protein polypeptide characterized by the amino acid sequence of SEQ ID NO: 2.

[0229] In embodiments, the CoV S protein polypeptide comprises one or more modifications selected from K973P, V974P, an inactivated furin cleavage site, optionally wherein the inactivated furin cleavage site is QQAQ (SEQ ID NO: 7), an L5F mutation, a T7N mutation, a P13S mutation, a D125Y mutation, a R177S mutation, a K404T mutation, an E471K mutation, a N488Y mutation, a D601G mutation, an H642Y mutation, a T1014I mutation, and a T1163F mutation, wherein the CoV S protein polypeptide is numbered relative to a wild-type SARS-CoV-2 S protein polypeptide characterized by the amino acid sequence of SEQ ID NO: 2.

[0230] In embodiments, the CoV S protein polypeptide comprises one or more modifications selected from K986P, V987P, an inactivated furin cleavage site, optionally wherein the inactivated furin cleavage site is QQAQ (SEQ ID NO: 7), the S13I mutation, the W152C mutation, and the L452R mutation, wherein the CoV S protein polypeptide is numbered relative to the wild-type SARS-CoV-2 S protein polypeptide characterized by the amino acid sequence of SEQ ID NO: 1.In embodiments, the CoV S protein polypeptide comprises one or more modifications selected from K986P, V987P, an inactivated furin cleavage site, optionally wherein the inactivated furin cleavage site is QQAQ (SEQ ID NO: 7), the S13I mutation, the W152C mutation, and the L452R mutation, wherein the CoV S protein polypeptide is numbered relative to the wild-type SARS-CoV-2 S protein polypeptide characterized by the amino acid sequence of SEQ ID NO: 1, does not comprise an N-terminal signal peptide.

[0231] In embodiments, the CoV spike (S) protein polypeptides comprise a polypeptide linker. In embodiments, the polypeptide linker comprises glycine and serine. In embodiments, the linker comprises about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or about 100% glycine.

[0232] In embodiments, the polypeptide linker comprises a repeat (SGGG) n (SEQ ID NO: 91), wherein n is an integer from 1 to 50 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50). In embodiments, the polypeptide linker has an amino acid sequence corresponding to SEQ ID NO: 90.

[0233] In embodiments, the polypeptide linker comprises a repeat (GGGGS) n (SEQ ID NO: 93), wherein n is an integer from 1 to 50 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50).

[0234] In embodiments, the polypeptide linker comprises a repeat (GGGS) n(SEQ ID NO: 92), wherein n is an integer from 1 to 50 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50).

[0235] In some aspects, the polypeptide linker is a poly-(Gly)n linker, wherein n is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 16, 17, 18, 19, or 20. In other embodiments, the linker is selected from the group consisting of dipeptides, tripeptides, and quadripeptides. In embodiments, the linker is a dipeptide selected from the group consisting of alanine-serine (AS), leucine-glutamic acid (LE), and serine-arginine (SR).

[0236] In embodiments, the polypeptide linker comprises 1 to 100 contiguous amino acids of a naturally occurring CoV S protein polypeptide or a CoV S protein polypeptide disclosed herein. In embodiments, the polypeptide linker is characterized by an amino acid sequence corresponding to SEQ ID NO: 94.

[0237] In embodiments, the CoV spike (S) protein polypeptides comprise a foldon. In embodiments, TMCT is replaced with foldon. In embodiments, the foldon causes trimerization of the CoV spike (S) protein polypeptide. In embodiments, the foldon is an amino acid sequence known in the art. In embodiments, the foldon is characterized by the amino acid sequence of SEQ ID NO: 68. In embodiments, the foldon is a T4 fibritin trimerization motif. In embodiments, the T4 fibritin trimerization domain is characterized by the amino acid sequence of SEQ ID NO: 103. In embodiments, the foldon is separated in amino acid sequence from the CoV spike (S) protein polypeptide by a polypeptide linker. Non-limiting examples of polypeptide linkers are provided throughout the present description.

[0238] In embodiments, the present invention provides CoV S protein polypeptides comprising a fragment of the coronavirus S protein and nanoparticles and vaccines containing them.In embodiments, the coronavirus S protein fragment is between 10 and 1500 amino acids in length (e.g., about 10, about 20, about 30, about 40, about 50, about 60, about 70, about 80, about 90, about 100, about 150, about 200, about 250, about 300, about 350, about 400, about 450, about 500, about 550, about 600, about 650, about 700, about 750, about 800, about 850, about 900, about 950, about 1000, about 1050, about 1100, about 1150, about 1200, about 1250, about 1300, about 1350, about 1400, approximately 1450, or approximately 1500 amino acids).In embodiments, the coronavirus S protein fragment is selected from the group consisting of the receptor binding domain (RBD), subdomain 1, subdomain 2, upper helix, fusion peptide, connecting region, heptad repeat 1, central helix, heptad repeat 2, NTD, and TMCT.

[0239] In embodiments, the CoV S protein polypeptide comprises an RBD and subdomain 1. In embodiments, the CoV S protein polypeptide comprising an RBD and subdomain 1 is amino acids 319-591 of SEQ ID NO: 1.

[0240] In embodiments, the CoV S protein polypeptide comprises a fragment of a coronavirus S protein, wherein the fragment of a coronavirus S protein is an RBD. Non-limiting examples of RBDs include the SARS-CoV-2 RBD (amino acid sequence = SEQ ID NO: 69), the SARS RBD (amino acid sequence = SEQ ID NO: 70), and the MERS RBD (amino acid sequence = SEQ ID NO: 71).

[0241] In embodiments, the CoV S protein polypeptide comprises two or more RBDs that are connected by a polypeptide linker. In embodiments, the polypeptide linker is characterized by the amino acid sequence of SEQ ID NO: 90 or SEQ ID NO: 94.

[0242] In embodiments, the CoV S protein polypeptide comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 RBDs.

[0243] In some embodiments, the CoV S protein polypeptide comprises two or more SARS-CoV-2 RBDs connected by a polypeptide linker. In embodiments, the antigen comprising two or more SARS-CoV-2 RBDs is characterized by an amino acid sequence corresponding to one of SEQ ID NOs: 72-75.

[0244] In embodiments, the CoV S protein polypeptide comprises a SARS-CoV-2 RBD and a SARS RBD. In embodiments, the CoV S protein polypeptide comprises a SARS-CoV-2 RBD and a SARS RBD, wherein each RBD is separated by a polypeptide linker. In embodiments, the CoV S protein polypeptide comprising a SARS-CoV-2 RBD and a SARS RBD is characterized by an amino acid sequence selected from the group consisting of SEQ ID NOs: 76-79.

[0245] In embodiments, the CoV S protein polypeptide comprises the SARS-CoV-2 RBD and the MERS RBD. In embodiments, the CoV S protein polypeptide comprises the SARS-CoV-2 RBD and the MERS RBD, wherein each RBD is separated by a polypeptide linker.

[0246] In embodiments, the CoV S protein polypeptide comprises a SARS RBD and a MERS RBD. In embodiments, the CoV S protein polypeptide comprises a SARS RBD and a MERS RBD, wherein each RBD is separated by a polypeptide linker.

[0247] In embodiments, the CoV S protein polypeptide comprises the SARS-CoV-2 RBD, the SARS RBD, and the MERS RBD. In embodiments, the CoV S protein polypeptide comprises the SARS-CoV-2 RBD, the SARS RBD, and the MERS RBD, wherein each RBD is separated by a polypeptide linker. In embodiments, the CoV S protein polypeptide comprising the SARS-CoV-2 RBD, the SARS RBD, and the MERS RBD is characterized by an amino acid sequence selected from the group consisting of SEQ ID NOs: 80-83.

[0248] In embodiments, the CoV S protein polypeptides described herein are expressed with an N-terminal signal peptide. In embodiments, the N-terminal signal peptide is characterized by the amino acid sequence of SEQ ID NO: 5 (MFVFLVLLPLVSS). In embodiments, the N-terminal signal peptide is characterized by the amino acid sequence of SEQ ID NO: 117 (MFVFLVLLPLVSI). In embodiments, the N-terminal signal peptide is characterized by the amino acid sequence of SEQ ID NO: 154 (MFVFFVLLPLVSS). In embodiments, the signal peptide can be replaced with any signal peptide that ensures expression of the CoV S protein. In embodiments, one or more amino acids of the CoV S protein signal peptide can be deleted or mutated. The initiating methionine residue is retained to ensure initiation of expression.In embodiments, the CoV S protein polypeptides are encoded by a nucleic acid sequence selected from the group consisting of SEQ ID NO: 35, SEQ ID NO: 37, SEQ ID NO: 95, SEQ ID NO: 43, SEQ ID NO: 47, SEQ ID NO: 50, SEQ ID NO: 53, SEQ ID NO: 55, SEQ ID NO: 57, SEQ ID NO: 96, SEQ ID NO: 60, SEQ ID NO: 131, SEQ ID NO: 135, SEQ ID NO: 142, SEQ ID NO: 145, SEQ ID NO: 148, and SEQ ID NO: 150. In embodiments, the N-terminal signal peptide of the CoV S protein polypeptide comprises a mutation at Ser-13 relative to the native CoV spike (S) protein signal polypeptide (SEQ ID NO: 5). In embodiments, Ser-13 is mutated to any naturally occurring amino acid. In embodiments, Ser-13 is mutated to alanine, methionine, isoleucine, leucine, threonine, or valine. In embodiments, Ser-13 is mutated to isoleucine.

[0249] After expression of the CoV S protein in the host cell, the N-terminal signal peptide is cleaved to form the mature CoV protein sequence (SEQ ID NO: 2, 4, 38, 41, 44, 48, 51, 54, 58, 61, 63, 65, 67, 73, 75, 78, 79, 82, 83, 85, 87, 89, 106, 110, 132, 133, 114, 138, 141, 144, 147, 151, 153, 156 and 158, 164-168). In embodiments, the signal peptide is cleaved by host cell proteases. In some aspects, the full-length protein can be isolated from the host cell and the signal peptide then cleaved.

[0250] After cleaving the signal peptide from the CoV spike (S) protein polypeptide with the amino acid sequence corresponding to SEQ ID NO: 1, 3, 36, 40, 42, 46, 49, 52, 56, 59, 62, 64, 66, 72, 74, 76, 77, 80, 81, 84, 86, 87, 105, 107, 88, 109, 130, 134, 136, 137, 140, 143, 146, 149, 152, 155, 157, 159-163, during expression and purification, a mature polypeptide characterized by an amino acid sequence selected from the group, consisting of SEQ ID NO: 2, 4, 38, 41, 44, 48, 51, 54, 58, 61, 63, 65, 67, 73, 75, 78, 79, 82, 83, 85, 106, 108, 89 and 110, 112-115, 132, 133, 114, 138, 141, 144, 147, 151, 153, 156 and 158, 164-168, are obtained and used to obtain a vaccine based on S CoV protein nanoparticles or S CoV protein nanoparticles.

[0251] The advantageously disclosed CoV S protein polypeptides may be characterized by enhanced protein expression and stability compared to the native CoV spike (S) protein.

[0252] In embodiments, the CoV S protein polypeptides described herein comprise additional modifications relative to the native coronavirus S protein (SEQ ID NO: 2). In embodiments, the coronavirus S proteins described herein are characterized by at least 80%, or at least 90%, or at least 95%, or at least 97%, or at least 99% identity with the native coronavirus S protein. One skilled in the art can use known techniques to calculate the percentage identity of the recombinant coronavirus S protein with the native protein or any of the CoV S protein polypeptides described herein. For example, the percentage identity can be calculated using the CLUSTALW2 or the Basic Local Alignment Search (BLAST) tools, which are available online.The following default parameters can be used for pairwise alignment using CLUSTALW2: protein weight matrix = Gonnet; gap opening = 10; gap extension = 0.1.

[0253] In embodiments, the CoV S protein polypeptides described herein are at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% identical to a CoV S protein polypeptide characterized by the amino acid sequence of SEQ ID NO: 87. The CoV S protein polypeptide may be characterized by a deletion, insertion, or mutation affecting up to about 1, up to about 2, up to about 3, up to about 4, up to about 5, up to about 10, up to about 15, up to about 20, up to about 25, up to about 30, up to about 35, up to about 40, up to about 45, or up to about 50 amino acids, compared to the amino acid sequence of the CoV S protein polypeptide characterized by the amino acid sequence of SEQ ID NO: 87.The CoV S protein polypeptide may be characterized by a deletion, insertion, or mutation affecting from about 1 to about 5 amino acids, from about 3 to about 10 amino acids, from about 5 to 10 amino acids, from about 8 to 12 amino acids, from about 10 to 15 amino acids, from about 12 to 17 amino acids, from about 15 to 20 amino acids, from about 18 to 23 amino acids, from about 20 to 25 amino acids, from about 22 to 27 amino acids, from about 25 to 30 amino acids, from about 30 to 35 amino acids, from about 35 to 40 amino acids, from about 40 to 45 amino acids, or from about 45 to 50 amino acids, compared to the CoV S protein polypeptide characterized by the amino acid sequence under SEQ ID NO: 87.In embodiments, the CoV S protein polypeptides described herein comprise about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, about 20, about 21, about 22, about 23, about 24, or about 25 substitutions compared to the coronavirus S protein (SEQ ID NO: 87).

[0254] In embodiments, the CoV S protein polypeptides described herein are at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% identical to a CoV S protein polypeptide having an amino acid sequence selected from any of SEQ ID NOs: 2, 4, 38, 41, 44, 48, 51, 54, 58, 61, 63, 65, 67, 73, 75, 78, 79, 82, 83, 85, 106, 108, 89 and 110, 112-115, 132, 133, 114, 138, 141, 144, 147, 151, 153, 156 and 158, 164-168. The CoV S protein polypeptide may be characterized by a deletion, insertion, or mutation affecting up to about 1, up to about 2, up to about 3, up to about 4, up to about 5, up to about 10, up to about 15, up to about 20, up to about 25, up to about 30, up to about 35, up to about 40, up to about 45, or up to about 50 amino acids,compared to the amino acid sequence of a CoV S protein polypeptide characterized by an amino acid sequence selected from any of SEQ ID NOs: 2, 4, 38, 41, 44, 48, 51, 54, 58, 61, 63, 65, 67, 73, 75, 78, 79, 82, 83, 85, 106, 108, 89 and 110, 112-115, 132, 133, 114, 138, 141, 144, 147, 151, 153, 156 and 158, 164-168. The CoV S protein polypeptide may be characterized by a deletion, insertion, or mutation affecting from about 1 to about 5 amino acids, from about 3 to about 10 amino acids, from about 5 to 10 amino acids, from about 8 to 12 amino acids, from about 10 to 15 amino acids, from about 12 to 17 amino acids, from about 15 to 20 amino acids, from about 18 to 23 amino acids, from about 20 to 25 amino acids, from about 22 to 27 amino acids, from about 25 to 30 amino acids, from about 30 to 35 amino acids, from about 35 to 40 amino acids,from about 40 to 45 amino acids or from about 45 to 50 amino acids, compared to a CoV S protein polypeptide characterized by an amino acid sequence selected from any of SEQ ID NOs: 2, 4, 38, 41, 44, 48, 51, 54, 58, 61, 63, 65, 67, 73, 75, 78, 79, 82, 83, 85, 106, 108, 89 and 110, 112-115, 132, 133, 114, 138, 141, 144, 147, 151, 153, 156 and 158, 164-168. In embodiments, the CoV S protein polypeptides described herein provide about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, about 20, about 21, about 22, about 23,approximately 24 or approximately 25 substitutions compared to the coronavirus S protein (SEQ ID NO: 87).,

[0255] In some embodiments, the coronavirus S protein polypeptide is extended at the N-terminus, the C-terminus, or both the N-terminus and the C-terminus. In some aspects, the extension is a tag useful for a function such as purification or detection. In some aspects, the tag comprises an epitope. For example, the tag may be a polyglutamate tag, a FLAG tag, an HA tag, a poly-His tag (characterized by approximately 5-10 histidine molecules) (SEQ ID NO: 101), a hexahistidine tag (SEQ ID NO: 100), an 8X-His tag (characterized by eight histidine molecules) (SEQ ID NO: 102), a Myc tag, a glutathione S-transferase tag, a green fluorescent protein tag, a maltose-binding protein tag, a thioredoxin tag, or an Fc tag. In other aspects, the extension may be an N-terminal signal peptide fused to the protein to enhance expression.Although such signal peptides are often cleaved during cellular expression, some nanoparticles may contain an antigen with an intact signal peptide. Thus, when a nanoparticle contains an antigen, the antigen may contain an extension and thus may constitute a fusion protein when incorporated into the nanoparticle. For the purposes of calculating sequence identity, the extensions are not included. In some embodiments, the tag is a protease cleavage site. Non-limiting examples of protease cleavage sites include a protease cleavage site represented by HRV3C, chymotrypsin, trypsin, elastase, endopeptidase, caspase-1, caspase-2, caspase-3, caspase-4, caspase-5, caspase-6, caspase-7, caspase-8, caspase-9, caspase-10, enterokinase, factor Xa, granzyme B, TEV protease, and thrombin. In embodiments, the protease cleavage site is a protease cleavage site represented by HRV3C.In embodiments, the protease cleavage site comprises the amino acid sequence of SEQ ID NO: 98.

[0256] In embodiments, the CoV S glycoprotein comprises a fusion protein. In embodiments, the CoV S glycoprotein comprises an N-terminal fusion protein. In embodiments, the Cov S glycoprotein comprises a C-terminal fusion protein. In embodiments, the fusion protein encompasses a tag useful for expression, purification, or detection of the protein. In embodiments, the tag is a poly-His tag (characterized by about 5-10 histidine molecules), a Myc tag, a tag represented by glutathione S-transferase, a tag represented by green fluorescent protein, a tag represented by maltose-binding protein, a tag represented by thioredoxin, a Strep tag, a Twin-Strep tag, or an Fc tag. In embodiments, the tag is an Fc tag. In embodiments, the Fc tag is monomeric, dimeric, or trimeric.In some embodiments, the tag is a hexahistidine tag, such as a polyhistidine tag, which contains six histidine molecules (SEQ ID NO: 100). In other embodiments, the tag is a Twin-Strep tag with the amino acid sequence of SEQ ID NO: 99.

[0257] In some embodiments, the CoV S protein polypeptide is a fusion protein comprising another coronavirus protein. In some embodiments, the other coronavirus protein is derived from the same coronavirus. In some embodiments, the other coronavirus protein is derived from a different coronavirus.

[0258] In some aspects, the CoV S protein may be truncated. For example, the N-terminus may be truncated by about 10 amino acids, about 30 amino acids, about 50 amino acids, about 75 amino acids, about 100 amino acids, or about 200 amino acids. Instead of the N-terminus or in addition to it, the C-terminus may be truncated. For example, the C-terminus may be truncated by about 10 amino acids, about 30 amino acids, about 50 amino acids, about 75 amino acids, about 100 amino acids, or about 200 amino acids. For the purpose of calculating the identity of a protein characterized by the presence of truncations, the identity is measured based on the remaining portion of the protein. Nanoparticles comprising CoV spike (S) protein polypeptides

[0259] In embodiments, antigens represented by a mature CoV S protein polypeptide are used to produce a vaccine comprising nanoparticles with the coronavirus S protein. In embodiments, the nanoparticles of the present invention comprise the CoV S protein polypeptides described herein. In embodiments, the nanoparticles of the present invention comprise the CoV S protein polypeptides linked to a core represented by a detergent. The presence of the detergent promotes the formation of nanoparticles by forming a core that organizes and presents antigens. In embodiments, the nanoparticles may comprise CoV S protein polypeptides assembled into multi-oligomeric glycoprotein-detergent (e.g., PS80) nanoparticles with protruding head regions and hydrophobic regions, and wherein the detergent PS80 forms the central core surrounded by the glycoprotein.In embodiments, the CoV S protein polypeptide initially comprises, or is adapted to contain, a transmembrane domain that facilitates the association of the protein with a core represented by a detergent. In embodiments, the CoV S protein polypeptide comprises a head domain. Fig. 10 shows an exemplary structure of a CoV S protein polypeptide of the present invention. The transmembrane domains of the CoV S protein polypeptide trimer primarily bind to the detergent; however, other portions of the polypeptide may also interact. Advantageously, the nanoparticles are characterized by increased resistance to environmental stress factors, as a result of which they provide increased stability and / or an improved level of presentation to the immune system due to the organization of multiple copies of the protein around the detergent.

[0260] In embodiments, the core represented by a detergent is a core represented by a non-ionic detergent. In embodiments, the CoV S protein polypeptide is linked to a core represented by a non-ionic detergent. In embodiments, the detergent is selected from the group consisting of polysorbate-20 (PS20), polysorbate-40 (PS40), polysorbate-60 (PS60), polysorbate-65 (PS65), and polysorbate-80 (PS80).

[0261] In embodiments, the detergent is PS80.

[0262] In embodiments, the CoV S protein polypeptide forms a trimer. In embodiments, the CoV S protein polypeptide-based nanoparticles consist of several polypeptide trimers surrounding a core represented by a non-ionic detergent. In embodiments, the nanoparticles comprise at least about 1 trimer or more. In embodiments, the nanoparticles comprise from at least about 5 trimers to about 30 trimers of the spike protein. In embodiments, each nanoparticle can comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or 15, 20, 25 or 30 trimers, including all values ​​​​and ranges therebetween. The compositions disclosed herein can comprise nanoparticles characterized by a different number of trimers. For example, the composition may comprise nanoparticles wherein the number of trimers is in the range of 2-9; in embodiments, the nanoparticles in the composition may comprise 2-6 trimers.In embodiments, the compositions comprise a heterogeneous population of nanoparticles characterized by the presence of 2 to 6 trimers per nanoparticle or 2 to 9 trimers per nanoparticle. In embodiments, the compositions may comprise a substantially homogeneous population of nanoparticles. For example, the population may comprise approximately 95% of the nanoparticles characterized by the presence of 5 trimers.

[0263] The nanoparticles disclosed herein vary in particle size. In embodiments, the nanoparticles disclosed herein are characterized by a particle size range in terms of Z-average size from about 20 nm to about 60 nm, from about 20 nm to about 50 nm, from about 20 nm to about 45 nm, from about 20 nm to about 35 nm, from about 20 nm to about 30 nm, from about 25 nm to about 35 nm, or from about 25 nm to about 45 nm. Particle size (Z-average) is measured by dynamic light scattering (DLS) using a Zetasizer NanoZS (Malvern, United Kingdom), unless otherwise noted.

[0264] In embodiments, the nanoparticles comprising the CoV S protein polypeptides disclosed herein are characterized by a reduced particle size compared to nanoparticles comprising a wild-type CoV S protein polypeptide. In embodiments, the CoV S protein polypeptides are characterized by a particle size that is at least about 40% smaller, such as a particle size that is at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, or at least about 85% smaller.

[0265] The nanoparticles comprising the CoV S protein polypeptides disclosed herein are more uniform in size, shape, and mass than the nanoparticles comprising the wild-type CoV S protein polypeptide. The polydispersity index (PDI), which is an indicator of heterogeneity, is measured by dynamic light scattering using a Malvern Setasizer, unless otherwise noted. In embodiments, the particles measured herein are characterized by a PDI of from about 0.2 to about 0.45, such as about 0.2, about 0.25, about 0.29, about 0.3, about 0.35, about 0.40, or about 0.45.In embodiments, the nanoparticles measured herein have a PDI that is at least about 25% less than the PDI of nanoparticles comprising a wild-type CoV S protein polypeptide, such as at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, or at least about 60% less.

[0266] CoV S protein polypeptides and nanoparticles comprising them are characterized by increased thermal stability compared to a wild-type CoV S protein polypeptide or a nanoparticle comprising it. The thermal stability of CoV S protein polypeptides is measured using differential scanning calorimetry (DSC), unless otherwise indicated. The transition enthalpy (ΔHcal) is the energy required to unfold the CoV S protein polypeptide. In embodiments, the CoV S protein polypeptides are characterized by an increased ΔHcal compared to a wild-type CoV S protein polypeptide. In embodiments, the ΔHcal of the CoV S protein polypeptide is about 2 times greater, about 3 times greater, about 4 times greater, about 5 times greater, about 6 times greater, about 7 times greater, about 8 times greater, about 9 times greater, or about 10 times greater than the ΔHcal of the wild-type CoV S protein polypeptide.

[0267] Several types of nanoparticles can be included in the vaccine compositions disclosed herein. In some aspects, the nanoparticle type is characterized by an anisotropic rod shape, which can be a dimer or a monomer. In other aspects, the nanoparticle type is a spherical oligomer. In still other aspects, the nanoparticle can be described as an intermediate nanoparticle, characterized by sedimentation properties that are intermediate between those of the first two types. The formation of the nanoparticle types can be controlled by controlling the concentration of detergent and protein during manufacture. The nanoparticle type can be determined by measuring the sedimentation coefficient. Production of nanoparticles containing antigens presented by the CoV S protein polypeptide

[0268] The nanoparticles of the present invention are non-naturally occurring products whose components do not co-occur in nature. Typically, the methods disclosed herein utilize a detergent-exchange approach, wherein a first detergent is used to isolate the protein and then this first detergent is exchanged for a second detergent to form the nanoparticles.

[0269] The antigens contained in the nanoparticles are typically produced by recombinant expression in host cells. Standard recombinant techniques can be used. In embodiments, the CoV S protein polypeptides are expressed in insect host cells using a baculovirus system. In embodiments, the baculovirus is a cathepsin-L knockout baculovirus or a chitinase knockout baculovirus. Optionally, the baculovirus is characterized by a double knockout of both cathepsin-L and chitinase. High-level expression can be obtained in insect cell-based expression systems. Non-limiting examples of insect cells include Spodoptera frugiperda (Sf) cells, such as Sf9, Sf21, Trichoplusiani cells, such as High Five cells, and Drosophila S2 cells. In embodiments, the CoV S protein polypeptide described herein is produced in any suitable host cell.In some embodiments, the host cell is an insect cell. In some embodiments, the insect cell is an Sf9 cell.

[0270] Typical methods for transfection and cell growth can be used for cell culture. Vectors, such as vectors containing polynucleotides encoding fusion proteins, can be transfected into host cells using methods well known in the art. For example, the introduction of nucleic acids into eukaryotic cells can be achieved by calcium phosphate coprecipitation, electroporation, microinjection, lipofection, and transfection using polyamine-based transfection reagents. In one embodiment, the vector is a recombinant baculovirus.

[0271] Methods for culturing host cells include, but are not limited to, batch, fed-batch, continuous, and perfusion cell culture techniques. Cell culturing refers to the growth and reproduction of cells in a bioreactor (fermentation chamber) where the cells proliferate and express protein (e.g., recombinant proteins) for purification and isolation. Typically, cell culturing is carried out under sterile conditions with controlled temperature and atmosphere in a bioreactor. A bioreactor is a chamber used for culturing cells in which environmental conditions such as temperature, atmosphere, agitation, and / or pH can be monitored. In one embodiment, the bioreactor is a stainless steel chamber. In another embodiment, the bioreactor is a pre-sterilized plastic bag (e.g., Cellbag®, Wave Biotech, Bridgewater, NJ).In another embodiment, the pre-sterilized plastic bags are bags with a volume of from about 50 L to 3500 L. Extraction and purification of nanoparticles containing antigens presented by the spike (S) protein of CoV.

[0272] After allowing the host cells to grow, the protein can be collected from the host cells using detergents and purification protocols. After allowing the host cells to grow for 48-96 hours, the cells are isolated from the media and a detergent-containing solution is added to solubilize the cell membrane, releasing the protein in a detergent extract. Triton X-100 and TERGITOL® nonylphenol ethoxylate, also known as NP-9, are each preferred detergents for extraction. The detergent can be added to a final concentration of between approximately 0.1% and approximately 1.0%. For example, the concentration can be approximately 0.1%, approximately 0.2%, approximately 0.3%, approximately 0.5%, approximately 0.7%, approximately 0.8%, or approximately 1.0%. The range may be from approximately 0.1% to approximately 0.3%. In some aspects, the concentration is approximately 0.5%.

[0273] In other aspects, various first detergents can be used to isolate a protein from a host cell. For example, the first detergent may be bis(polyethylene glycol bis[imidazoylcarbonyl]), nonoxynol-9, bis(polyethylene glycol bis[imidazoylcarbonyl]), polyethylene glycol dodecyl ether 35 BRIJ®, polyethylene glycol (3) cetyl ether 56 BRIJ®, alcohol ethoxylate 72 BRIJ®, polyoxyl-2-stearyl ether 76 BRIJ®, polyethylene glycol monooleyl ether 92V BRIJ®, polyoxyethylene (10) oleyl ether 97 BRIJ®, polyethylene glycol hexadecyl ether 58P BRIJ®, macrogolglycerol ricinoleate CREMOPHOR® EL, decaethylene glycol monododecyl ether, N-decanoyl-N-methylglucamine, n-decyl alpha-D-glucopyranoside, decyl beta-D-maltopyranoside, n-dodecanoyl-N-methylglucamide, n-dodecyl alpha-D-maltoside, n-dodecyl beta-D-maltoside, n-dodecyl beta-D-maltoside, heptaethylene glycol monodecyl ether,Heptaethylene glycol monododecyl ether, Heptaethylene glycol monotetradecyl ether, N-hexadecyl beta-D-maltoside, Hexaethylene glycol monododecyl ether, Hexaethylene glycol monohexadecyl ether, Hexaethylene glycol monooctadecyl ether, Hexaethylene glycol monotetradecyl ether, Igepal CA-630, Igepal CA-630, Methyl 6-0-(N-heptylcarbamoyl)-alpha-D-glucopyranoside, Nonaethylene glycol monododecyl ether, N-nonanoyl-N-methylglucamine, N-nonanoyl-N-methylglucamine, Octaethylene glycol monodecyl ether, Octaethylene glycol monododecyl ether, Monohexadecyl ether Octaethylene glycol, Octaethylene glycol monooctadecyl ether, Octaethylene glycol monotetradecyl ether, Octyl beta-D-glucopyranoside, Pentaethylene glycol monodecyl ether, Pentaethylene glycol monododecyl ether, Pentaethylene glycol monohexadecyl ether, Pentaethylene glycol monohexyl ether,Pentaethylene Glycol Monooctadecyl Ether, Pentaethylene Glycol Monooctyl Ether, Polyethylene Glycol Diglycidyl Ether, Polyethylene Glycol W-1 Ether, Polyoxyethylene 10 Tridecyl Ether, Polyoxyethylene 100 Stearate, Polyoxyethylene 20 Isohexadecyl Ether, Polyoxyethylene 20 Oleyl Ether, Polyoxyethylene 40 Stearate, Polyoxyethylene 50 Stearate, Polyoxyethylene 8 Stearate, Polyoxyethylene Bis(imidazolylcarbonyl), Polyoxyethylene 25 Propylene Glycol Stearate, Quillaja Bark Saponin, Sorbitan Laurate SPAN® 20, Sorbitan Monopalmitate SPAN® 40, Sorbitan Stearate SPAN® 60, Sorbitan Tristearate SPAN® 65, SPAN® 80 Sorbitan Monooleate, SPAN® 85 Sorbitan Trioleate, TERGITOL® Secondary Alcohol Ethoxylate Type 15-S-12, TERGITOL® Secondary Alcohol Ethoxylate Type 15-S-30, TERGITOL® Secondary Alcohol Ethoxylate Type 15-S-5, TERGITOL® Secondary Alcohol Ethoxylate Type 15-S-7, TERGITOL® Nonylphenol Ethoxylate Type 15-S-9,TERGITOL® nonylphenol ethoxylate type NP-10, TERGITOL® nonylphenol ethoxylate type NP-4, TERGITOL® nonylphenol ethoxylate type NP-40, TERGITOL® nonylphenol ethoxylate type NP-7, TERGITOL® nonylphenol ethoxylate type NP-9, TERGITOL® branched secondary alcohol ethoxylate type TMN-10, TERGITOL® branched secondary alcohol ethoxylate type TMN-6, polyethyleneglycol tert-octylphenyl ether TRITON™) X-100, or combinations thereof.

[0274] The nanoparticles can then be isolated from the cellular debris using centrifugation. In some embodiments, gradient centrifugation can be used, such as with cesium chloride, sucrose, and iodixanol. Alternatively, or in addition, other techniques can be used, such as standard purification techniques, including, for example, ion exchange, affinity, and gel filtration chromatography.

[0275] For example, the first column may include an ion exchange chromatography resin such as FRACTOGEL® EMD methacrylate-based TMAE polymer beads (EMD Millipore), the second column may include a lentil (Lens culinaris) lectin-based affinity resin, and the third column may include a cation exchange column such as FRACTOGEL® EMD methacrylate-based SO3 polymer beads resin (EMD Millipore). In other aspects, the cation exchange column may be an MMC column or a Nuvia C Prime column (Bio-Rad Laboratories, Inc). Preferably, the methods disclosed herein do not use a detergent extraction column; for example, a hydrophobic interaction column. Such a column is often used to remove detergent during purification, but this may negatively impact the methods disclosed in this document. Detergent replacement for nanoparticles containing antigens presented by the CoV S protein polypeptide

[0276] To form nanoparticles, the first detergent used to extract protein from the host cell is essentially replaced by a second detergent to achieve nanoparticle structure formation. NP-9 is the preferred detergent for extraction. Nanoparticles typically do not contain detectable NP-9 when measured by HPLC. The second detergent is typically selected from the group consisting of PS20, PS40, PS60, PS65, and PS80. Preferably, the second detergent is PS80.

[0277] In certain aspects, the detergent exchange is accomplished using affinity chromatography to bind glycoproteins via their carbohydrate moiety. For example, a legume lectin column can be used in affinity chromatography. Legume lectins are proteins originally identified in plants and found to have a specific and reversible interaction with carbohydrate residues. See, e.g., Sharon and Lis, “Legume lectins—a large family of homologous proteins,” FASEB J. 1990 Nov;4(14):3198–208; Liener, “The Lectins: Properties, Functions, and Applications in Biology and Medicine,” Elsevier, 2012. Suitable lectins include concanavalin A (con A), pea lectin, sainfoin lectin, and lentil lectin. Lentil lectin is preferred for detergent replacement columns due to its binding properties. Lectin-based columns are commercially available; for example, Capto Lentil Lectin is available from GE Healthcare.In certain aspects, a lentil lectin-based column may utilize a recombinant lectin. It is believed that, at the molecular level, carbohydrate moieties bind to the lentil lectin, freeing the protein's amino acids to coalesce around the detergent, resulting in the formation of a detergent-represented core, providing nanoparticles containing multiple copies of the antigen, such as glycoprotein oligomers, which may be dimers, trimers, or tetramers, anchored in the detergent. In embodiments, the CoV S protein polypeptides form trimers. In embodiments, the trimers of the CoV S protein polypeptide are anchored in the detergent. In embodiments, each CoV S protein polypeptide-based nanoparticle comprises at least one trimer associated with a non-ionic core.

[0278] The detergent in the incubation with the protein to form nanoparticles during detergent exchange may be present at a concentration of up to approximately 0.1% (w / v) in the early stages of purification, and this amount is reduced to obtain final nanoparticles characterized by optimal stability. For example, the nonionic detergent (e.g., PS80) can be from about 0.005% (v / v) to about 0.1% (v / v), such as about 0.005% (v / v), about 0.006% (v / v), about 0.007% (v / v), about 0.008% (v / v), about 0.009% (v / v), about 0.01% (v / v), about 0.015% (v / v), about 0.02% (v / v), about 0.025% (v / v), about 0.03% (v / v), about 0.035% (v / v), about 0.04% (v / v), about 0.045% (v / v), about 0.0 ...0.55% (v / v), about 0.06% (v / v), about 0.065% (v / v), about 0.07% (v / v), about 0.075% (v / v), about 0.08% (v / v), about 0.085% (v / v), about 0.09% (v / v), about 0.095% (v / v, or about 0.1% (v / v) PS80. In embodiments, the nanoparticle comprises from about 0.03% to about 0.05% PS80. In embodiments, the nanoparticle comprises about 0.01% (v / v) PS80.

[0279] In embodiments, purified CoV S protein polypeptides are dialyzed. In embodiments, dialysis is performed after purification. In embodiments, the CoV S protein polypeptides are dialyzed in a solution containing sodium phosphate, NaCl, and PS80. In embodiments, the dialysis solution containing sodium phosphate comprises from about 5 mM to about 100 mM sodium phosphate, such as about 5 mM, about 10 mM, about 15 mM, about 20 mM, about 25 mM, about 30 mM, about 35 mM, about 40 mM, about 45 mM, about 50 mM, about 55 mM, about 60 mM, about 65 mM, about 70 mM, about 75 mM, about 80 mM, about 85 mM, about 90 mM, about 95 mM, or about 100 mM sodium phosphate. In embodiments, the pH of the solution containing sodium phosphate is about 6.5, about 6,6, approximately 6.7, approximately 6.8, approximately 6.9, approximately 7.0, approximately 7.1, approximately 7.2, approximately 7.3, approximately 7.4, or approximately 7.5. In embodiments, the dialysis solution comprising sodium chloride comprises from about 50 mM NaCl to about 500 mM NaCl, such as about 50 mM, about 60 mM, about 70 mM, about 80 mM, about 90 mM, about 100 mM, about 110 mM, about 120 mM, about 130 mM, about 140 mM, about 150 mM, about 160 mM, about 170 mM, about 180 mM, about 190 mM, about 200 mM, about 210 mM, about 220 mM, about 230 mM, about 240 mM, about 250 mM, about 260 mM, about 270 mM, about 280 mM, approximately 290 mM, approximately 300 mM, approximately 310 mM, approximately 320 mM, approximately 330 mM,about 340 mM, about 350 mM, about 360 mM, about 370 mM, about 380 mM, about 390 mM, about 400 mM, about 410 mM, about 420 mM, about 430 mM, about 440 mM, about 450 mM, about 460 mM, about 470 mM, about 480 mM, about 490 mM or about 500 mM NaCl. In embodiments, the dialysis solution comprising PS80 comprises about 0.005% (v / v), about 0.006% (v / v), about 0.007% (v / v), about 0.008% (v / v), about 0.009% (v / v), about 0.01% (v / v), about 0.015% (v / v), about 0.02% (v / v), about 0.025% (v / v), about 0.03% (v / v), about 0.035% (v / v), about 0.04% (v / v), about 0.045% (v / v), about 0.05% (v / v),about 0.055% (v / v), about 0.06% (v / v), about 0.065% (v / v), about 0.07% (v / v), about 0.075% (v / v), about 0.08% (v / v), about 0.085% (v / v), about 0.09% (v / v), about 0.095% (v / v, or about 0.1% (v / v) PS80. In embodiments, the dialysis solution comprises about 25 mM sodium phosphate (pH 7.2), about 300 mM NaCl, and about 0.01% (v / v) PS80.

[0280] Detergent replacement can be accomplished using purified proteins as discussed above and purified, frozen for storage, and then thawed to perform detergent replacement.

[0281] The stability of the compositions disclosed herein can be measured in a number of different ways. In one approach, a peptide map can be generated to determine the integrity of the antigenic protein after various treatments designed to stress the nanoparticles by simulating harsh storage conditions. Thus, a quantitative measure of stability is the relative abundance of antigenic peptides in a stressed sample compared to a control sample. For example, the stability of nanoparticles containing CoV S protein polypeptides can be assessed by exposing the nanoparticles to various pH values, proteases, salt, oxidizing agents, including but not limited to hydrogen peroxide, various temperatures, freeze / thaw cycles, and agitation. Fig. 12A-B shows that BV2373 (SEQ ID NO: 87) and BV2365 (SEQ ID NO: 4) retain their ability to bind to hACE2 under a number of different stress conditions.The position of the glycoprotein anchored in the detergent core is believed to provide increased stability by reducing undesired interactions. For example, increased protection against protease-induced degradation may be achieved through a shielding effect, in which anchoring the glycoproteins in the core at the molar ratios disclosed herein results in steric hindrance that blocks protease access. Stability can also be measured by monitoring intact proteins. Fig. 33 and Fig. 34 compare nanoparticles containing CoV polypeptides characterized by the amino acid sequences of SEQ ID NO: 109 and 87, respectively. Fig. 34 shows that the CoV polypeptides characterized by the amino acid sequence of SEQ ID NO: 87 exhibit particularly high stability during purification. The polypeptide shown in Fig.34, contains a furin cleavage site characterized by the amino acid sequence QQAQ (SEQ ID NO: 7). Vaccine compositions containing antigens presented by the CoV S protein polypeptide.

[0282] The present invention provides vaccine compositions comprising CoV S protein polypeptides, for example, in a nanoparticle. In some aspects, the vaccine composition may comprise nanoparticles with antigens from more than one viral strain of the same viral species. In another embodiment, the present invention provides a pharmaceutical package or kit comprising one or more containers filled with one or more components of the vaccine compositions.

[0283] The compositions disclosed herein can be used either prophylactically or therapeutically, but will typically be prophylactic. Accordingly, the present invention provides methods for treating or preventing an infection. The methods comprise administering to a subject a therapeutic or prophylactic amount of the immunogenic compositions of the present invention. Preferably, the pharmaceutical composition is a vaccine composition that provides a protective effect. In other aspects, the protective effect may include a reduction in the intensity of the manifestations of a symptom associated with an infection in a certain percentage of the exposed population.For example, the composition may prevent or reduce one or more symptoms of a viral disease selected from fever, fatigue, muscle pain, headache, sore throat, vomiting, diarrhea, rash, symptoms of kidney and liver dysfunction, internal bleeding, and external bleeding, compared to an untreated subject.

[0284] Nanoparticles can be formulated for administration as vaccines in the presence of various excipients, buffers, etc. For example, vaccine compositions can contain sodium phosphate, sodium chloride, and / or histidine. Sodium phosphate can be present at a concentration of from about 10 mM to about 50 mM, from about 15 mM to about 25 mM, or about 25 mM; in particular cases, approximately 22 mM sodium phosphate is present. Histidine may be present in an amount of about 0.1% (w / v), about 0.5% (w / v), about 0.7% (w / v), about 1% (w / v), about 1.5% (w / v), about 2% (w / v), or about 2.5% (w / v). Sodium chloride, if present, may provide about 150 mM. In certain compositions, sodium chloride may be present in higher concentrations, such as from about 200 mM to about 500 mM.In embodiments, sodium chloride is present at a high concentration, including, but not limited to, about 200 mM, about 250 mM, about 300 mM, about 350 mM, about 400 mM, about 450 mM, or about 500 mM.

[0285] In embodiments, the nanoparticles described herein are characterized by increased stability at certain pH levels. In embodiments, the nanoparticles are stable at slightly acidic pH levels. For example, the nanoparticles are stable at a slightly acidic pH, such as from pH 5.8 to pH 7.0. In embodiments, the nanoparticles and compositions comprising the nanoparticles can be stable at pH values ​​in the range from about pH 5.8 to about pH 7.0, including from about pH 5.9 to about pH 6.8, from about pH 6.0 to about pH 6.5, from about pH 6.1 to about pH 6.4, from about pH 6.1 to about pH 6.3, or about pH 6.2. In embodiments, the nanoparticles and compositions described herein are stable at neutral pH values, including from about pH 7.0 to about pH 7.4.In embodiments, the nanoparticles and compositions described herein are stable at slightly alkaline pH values, such as from about pH 7.0 to about pH 8.5, from about pH 7.0 to about pH 8.0, or from about pH 7.0 to about pH 7.5, including all values ​​and ranges therebetween. Adjuvants.

[0286] In certain embodiments, the compositions disclosed herein can be combined with one or more adjuvants to enhance the immune response. In other embodiments, the compositions are prepared without adjuvants and are thus available for administration as adjuvant-free compositions. The predominantly adjuvant-free compositions described herein can provide protective immune responses when administered as a single dose. Alum-free compositions that induce strong immune responses are particularly useful for adults aged approximately 60 years and older. Aluminum-based adjuvants

[0287] In embodiments, the adjuvant can be alum (e.g., AlPO4 or Al(OH)3). Typically, the nanoparticle is substantially bound to the alum. For example, the nanoparticle can be at least 80% bound, at least 85% bound, at least 90% bound, or at least 95% bound to the alum. Often, the nanoparticles are 92-97% bound to the alum in the composition. The amount of alum present per dose typically ranges from about 400 μg to about 1250 μg. For example, alum may be present in an amount per dose of from about 300 μg to about 900 μg, from about 400 μg to about 800 μg, from about 500 μg to about 700 μg, from about 400 μg to about 600 μg, or from about 400 μg to about 500 μg. Typically, alum is present in an amount of about 400 μg per dose providing 120 μg of protein nanoparticle. Saponin adjuvants

[0288] Saponin-containing adjuvants can also be combined with the immunogens disclosed herein. Saponins are glycosides obtained from the bark of the Quillaja saponaria Molina tree. Saponin is typically obtained using a multi-step purification procedure, resulting in several fractions. As used herein, the term "Quillaja saponaria Molina saponin fraction" is used generally to describe a semi-purified or defined fraction of Quillaja saponaria saponins or a substantially pure fraction thereof. Saponin fractions

[0289] Several approaches to obtaining saponin fractions are suitable. Fractions A, B, and C are described in U.S. Patent No. 6,352,697 and can be prepared as follows. The lipophilic fraction of Quil A, a crude aqueous extract of Quillaja saponaria Molina, is separated by chromatography and eluted with 70% acetonitrile in water to recover the lipophilic fraction. This lipophilic fraction is then separated by semi-preparative HPLC eluting with a gradient of 25% to 60% acetonitrile in acidic water. The fraction referred to herein as “fraction A” or “QH-A” is or corresponds to the fraction that elutes at approximately 39% acetonitrile. The fraction referred to in this document as "fraction B" or "QH-B" is or corresponds to the fraction that elutes at approximately 47% acetonitrile.The fraction referred to herein as "fraction C" or "QH-C" is the fraction that elutes at or near 49% acetonitrile. Additional information regarding the purification of the fractions is contained in U.S. Patent No. 5,057,540. When prepared as described herein, each of fractions A, B, and C of Quillaja saponaria Molina represents a group or family of chemically closely related molecules with distinct properties. The chromatographic conditions under which they are prepared are such that batch-to-batch reproducibility in terms of elution profile and biological activity is highly consistent.

[0290] Other saponin fractions have been described. Fractions B3, B4, and B4b are described in EP 0436620. Fractions QA1-QA22, which are described in EP03632279 B2, Q-VAC (Nor-Feed, AS Denmark), spicoside Quillaja saponaria Molina (lsconova AB, 2B, 756 51, Uppsala, Sweden). Fractions QA-1, QA-2, QA-3, QA-4, QA-5, QA-6, QA-7, QA-8, QA-9, QA-10, QA-11, QA-12, QA-13, QA-14, QA-15, QA-16, QA-17, QA-18, QA-19, QA-20, QA-21 and QA-22 from EP03632279 B2, especially QA-7, QA-17, QA-18 and QA-21, can be used. They are prepared as described in EP03632279 B2, especially on page 6 and in example 1 on pages 8 and 9.

[0291] The saponin fractions described herein and used to form adjuvants are often substantially pure fractions; that is, the fractions are substantially free of contaminants from other materials. In particular aspects, a substantially pure saponin fraction may contain other compounds, such as other saponins or other adjuvant materials, in an amount of up to 40% by weight, up to 30% by weight, up to 25% by weight, up to 20% by weight, up to 15% by weight, up to 10% by weight, up to 7% by weight, up to 5% by weight, up to 2% by weight, up to 1% by weight, up to 0.5% by weight, or up to 0.1% by weight. ISCOM Structures

[0292] Saponin fractions can be administered in the form of scaffold particles referred to as ISCOMs (immunostimulating complex). ISCOMs can be prepared as described in EP0109942B1, EP0242380B1 and EP0180546 B1. In specific embodiments, a transport and / or passenger antigen can be used, as described in EP 9600647-3 (PCT / SE97 / 00289). Matrix adjuvants

[0293] In embodiments, the ISCOM is an ISCOM-matrix complex. The ISCOM-matrix complex comprises at least one saponin fraction and a lipid. The lipid is at least a sterol, such as cholesterol. In certain aspects, the ISCOM-matrix complex also comprises a phospholipid. ISCOM-matrix complexes may also contain one or more other immunomodulatory (adjuvant-active) substances, not necessarily a glycoside, and can be prepared as described in EP0436620B1, which is incorporated herein by reference in its entirety.

[0294] In other aspects, the ISCOM is an ISCOM complex. The ISCOM complex comprises at least one saponin, at least one lipid, and at least one type of antigen or epitope. The ISCOM complex comprises an antigen bound by treatment with a detergent such that a portion of the antigen is integrated into the particle. In contrast, the ISCOM matrix is ​​formulated as a mixture with the antigen, and the association between the ISCOM matrix-based particles and the antigen is mediated by electrostatic and / or hydrophobic interactions.

[0295] According to one embodiment, the saponin fraction integrated into the ISCOM matrix complex or the ISCOM complex, or at least one additional adjuvant that is also integrated into the ISCOM or ISCOM matrix complex or mixed with them, is selected from fraction A, fraction B, or fraction C of Quillaja saponaria, a semi-purified preparation of Quillaja saponaria, a purified preparation of Quillaja saponaria, or any purified subfraction, such as QA 1-21.

[0296] In certain aspects, each ISCOM particle may comprise at least two saponin fractions. Any combination of weight percent values ​​of different saponin fractions may be used. Any combination of weight percent values ​​of any two fractions may be used. For example, a particle may comprise any weight percent of fraction A and any weight percent of another saponin fraction, such as a crude saponin fraction or fraction C, respectively.Accordingly, in particular aspects, each ISCOM matrix-based particle or each ISCOM complex-based particle may comprise from 0.1 to 99.9% by weight, from 5 to 95% by weight, from 10 to 90% by weight, from 15 to 85% by weight, from 20 to 80% by weight, from 25 to 75% by weight, from 30 to 70% by weight, from 35 to 65% by weight, from 40 to 60% by weight, from 45 to 55% by weight, from 40 to 60% by weight, or 50% by weight of one saponin fraction, such as Fraction A, and the remaining amount up to 100% in each case is represented by another saponin, such as any crude fraction or any other fraction, such as Fraction C. The weight is calculated as the total weight of saponin fractions. Examples of adjuvants based on the ISCOM matrix complex and ISCOM complex are disclosed in Published U.S. Patent Application No. 2013 / 0129770, which is incorporated herein by reference in its entirety.

[0297] In certain embodiments, the ISCOM matrix or ISCOM complex comprises one fraction, such as fraction A, in an amount of 5 to 99% by weight, and the remaining amount required to reach 100% by weight is represented by another fraction, such as a crude saponin fraction or fraction C. The weight is calculated as the total weight of the saponin fractions.

[0298] In another embodiment, the ISCOM matrix or ISCOM complex comprises one fraction, such as fraction A, in an amount of 40 to 99% by weight, and another fraction, such as a crude saponin fraction or fraction C, in an amount of 1% to 60% by weight. The weight is calculated as the total weight of the saponin fractions.

[0299] In yet another embodiment, the ISCOM matrix or ISCOM complex comprises one fraction, for example, fraction A, in an amount of 70% to 95% by weight, and another fraction, for example, a crude saponin fraction or fraction C, in an amount of 30% to 5% by weight. The weight is calculated as the total weight of the saponin fractions. In other embodiments, the saponin fraction of Quillaja saponaria Molina is selected from any of QA 1-21.

[0300] In addition to particles containing mixtures of saponin fractions, both ISCOM matrix-based particles and ISCOM complex-based particles can be formed using only one saponin fraction. The compositions disclosed herein can contain multiple particles, wherein each particle contains only one saponin fraction. That is, certain compositions can contain one or more different types of ISCOM matrix complex-based particles and / or one or more different types of ISCOM complex-based particles, wherein each individual particle contains one saponin fraction from Quillaja saponaria Molina, wherein the saponin fraction in one complex differs from the saponin fraction in another particle complex.

[0301] In certain aspects, one type of saponin fraction or a crude saponin fraction can be integrated into one ISCOM matrix complex or particle, and another type of substantially pure saponin fraction or a crude saponin fraction can be integrated into another ISCOM matrix complex or particle. The composition or vaccine can comprise at least two types of complexes or particles, wherein each type is characterized by one type of saponin integrated into physically different particles.

[0302] The compositions may use mixtures of ISCOM matrix complex-based particles and / or ISCOM complex-based particles in which one fraction of Quillaja saponaria Molina saponins and another fraction of Quillaja saponaria Molina saponins are separately included in different ISCOM matrix complex-based particles and / or ISCOM complex-based particles.

[0303] ISCOM matrix or ISCOM complex based particles, each characterized by one fraction of saponins, may be present in the composition in any combination of weight %. In particular aspects, the composition may comprise an ISCOM matrix or complex comprising a first fraction of saponins in an amount of from 0.1% to 99.9% by weight, from 5% to 95% by weight, from 10% to 90% by weight, from 15% to 85% by weight, from 20% to 80% by weight, from 25% to 75% by weight, from 30% to 70% by weight, from 35% to 65% by weight, from 40% to 60% by weight, from 45% to 55% by weight, from 40 to 60% by weight, or 50% by weight, wherein the remainder consists of an ISCOM matrix or complex comprising a different fraction of saponins. In some aspects, the remaining portion comprises one or more ISCOM matrices or complexes, wherein each matrix-based or complex-based particle contains only one saponin fraction. In other aspects, ISCOM-based matrix-based or complex-based particles may contain more than one saponin fraction.

[0304] In the specific compositions, the only fraction of saponins in the first particle based on the ISCOM matrix or ISCOM complex is fraction A, and the only fraction of saponins in the second particle based on the ISCOM matrix or ISCOM complex is fraction C.

[0305] Preferred compositions provide a first ISCOM matrix containing fraction A and a second ISCOM matrix containing fraction C, wherein the ISCOM matrix with fraction A constitutes approximately 70% by weight of the total amount of saponin adjuvant, and the ISCOM matrix with fraction C constitutes approximately 30% by weight of the total amount of saponin adjuvant. In another preferred composition, the ISCOM matrix with fraction A constitutes approximately 85% by weight of the total amount of saponin adjuvant, and the ISCOM matrix with fraction C constitutes approximately 15% by weight of the total amount of saponin adjuvant. Thus, in some compositions, the ISCOM matrix with fraction A is present in an amount in the range of from about 70% to about 85%, and the ISCOM matrix with fraction C is present in an amount in the range of from about 15% to about 30% of the total weight amount of saponin adjuvant in the composition.In embodiments, the ISCOM matrix fraction A constitutes 50-96% by weight, and the ISCOM matrix fraction C constitutes the remainder of the sum of the weights of the ISCOM matrix fraction A and the ISCOM matrix fraction C, respectively, in the adjuvant. In a particularly preferred composition, referred to herein as MATRIX-M™, the ISCOM matrix fraction A is present in an amount of approximately 85%, and the ISCOM matrix fraction C is present in an amount of approximately 15%, of the total weight of the saponin adjuvant in the composition. MATRIX-M™ may be referred to interchangeably as Matrix-M1.

[0306] Illustrative fractions QS-7 and QS-21, their preparation and their use are described in U.S. Patent Nos. 5,057,540; 6,231,859; 6,352,697; 6,524,584; 6,846,489; 7,776,343 and 8,173,141, which are incorporated herein by reference.

[0307] In embodiments, other adjuvants can be used in addition to or as an alternative. Inclusion of any adjuvant described in Vogel et al., "A Compendium of Vaccine Adjuvants and Excipients (2nd Edition)," incorporated herein by reference in its entirety for all purposes, is within the scope of the present invention. Other adjuvants include complete Freund's adjuvant (a non-specific immune response stimulator containing destroyed Mycobacterium tuberculosis), incomplete Freund's adjuvants, and aluminum hydroxide adjuvant. Other adjuvants include GMCSP, BCG, MDP compounds such as thur-MDP and nor-MDP, CGP (MTP-PE), lipid A and monophosphoryl lipid A (MPL), MF-59, RIBI, which contains three components extracted from bacteria, MPL, trehalose dimycolate (TDM) and cell wall skeleton (CWS) in a 2% squalene / polysorbate 80 TWEEN® emulsion.In embodiments, the adjuvant may be a low-lamellar lipid vesicle; for example, NOVASOMES®. NOVASOMES® are low-lamellar non-phospholipid vesicles ranging in size from about 100 nm to about 500 nm. They contain BRIJ® 72 alcohol ethoxylate, cholesterol, oleic acid, and squalene. NOVASOMES® has been shown to be an effective adjuvant (see U.S. Pat. Nos. 5,629,021, 6,387,373, and 4,911,928). Administration and Dosage

[0308] In embodiments, the present invention provides a method for inducing an immune response against one or more coronaviruses. In embodiments, the response is directed against one or more of the SARS-CoV-2 virus, MERS, and SARS. In embodiments, the response is directed against a heterogeneous strain of SARS-CoV-2. Non-limiting examples of heterogeneous strains of SARS-CoV-2 include the Cal.20C SARS-CoV-2 strain, the P.1 SARS-CoV-2 strain, the B.1.351 SARS-CoV-2 strain, and the B.1.1.7 SARS-CoV-2 strain. The method comprises administering to a subject an immunologically effective amount of a composition comprising a nanoparticle or comprising a recombinant CoV spike (S) protein polypeptide. Advantageously, the proteins disclosed herein induce one or more particularly useful anti-coronavirus responses.

[0309] In embodiments, the CoV S protein nanoparticles or polypeptides are administered with an adjuvant. In some aspects, the CoV S protein nanoparticles or polypeptides are administered without an adjuvant. In some aspects, the adjuvant can be associated with the nanoparticle, for example, through a non-covalent interaction. In other aspects, the adjuvant is co-administered with the nanoparticle, but the adjuvant and nanoparticle do not substantially interact.

[0310] In embodiments, the CoV S protein nanoparticles or polypeptides can be used to prevent and / or treat one or more of SARS-CoV-2 infection, a heterogeneous SARS-CoV-2 strain infection, a SARS infection, or a MERS infection. Thus, the present invention provides a method for inducing the development of an immune response against one or more of the SARS-CoV-2 virus, a heterogeneous SARS-CoV-2 virus, MERS, and SARS. The method comprises administering to a subject an immunologically effective amount of a composition comprising a CoV S protein nanoparticle or polypeptide. Advantageously, the proteins disclosed herein induce particularly useful anti-coronavirus responses.

[0311] In embodiments, the compositions comprising the CoV S protein nanoparticles or polypeptides described herein induce a protective response against SARS-CoV-2 or a heterogeneous strain of SARS-CoV-2 in a subject for a period of up to about 3 months, up to about 4 months, up to about 5 months, up to about 6 months, up to about 7 months, up to about 8 months, up to about 9 months, up to about 10 months, up to about 11 months, up to about 12 months, up to about 13 months, up to about 14 months, up to about 15 months, up to about 16 months, up to about 17 months, up to about 18 months, up to about 19 months, up to about 20 months, up to about 21 months, up to about 22 months, up to about 23 months, up to about 24 months, up to about 2.5 years, up to about 3 years, up to about 3.5 years,up to about 4 years, up to about 4.5 years, up to about 5 years after the last dose of the CoV S protein nanoparticle or polypeptide. In embodiments, the CoV S protein nanoparticles or polypeptides described herein induce a protective response in a subject for at least 6 months.

[0312] In embodiments, the protective response is directed against an asymptomatic infection caused by SARS-CoV-2 or a heterogeneous strain of SARS-CoV-2. In embodiments, the protective response is directed against a symptomatic infection caused by SARS-CoV-2 or a heterogeneous strain of SARS-CoV-2.

[0313] In embodiments, the compositions comprising the CoV S protein nanoparticles or polypeptides described herein are effective in preventing coronavirus disease 19 (COVID-19) due to the SARS-CoV-2 virus or a heterogeneous strain of SARS-CoV-2 (e.g., SARS-CoV-2 strain B.1.1.7, SARS-CoV-2 strain B.1.351, SARS-CoV-2 strain P.1, SARS-CoV-2 strain B.1.617.2, SARS-CoV-2 strain B.1.525, SARS-CoV-2 strain B.1.526, SARS-CoV-2 strain B.1.617.1, SARS-CoV-2 strain C.37, SARS-CoV-2 strain B.1.621, or Cal.20C strain SARS-CoV-2), comprising from about 50% to about 99%, from about 80% to about 99%, from about 75% to about 99%, from about 80% to about 95%, from about 90% to about 98%, from about 75% to about 95%, from about 80% to about 90%, from about 85% to about 95%, from about 80% to about 95%,at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99%, over a period of up to about 1 month, up to about 2 months, up to about 2.5 months, up to about 3 months, up to about 3.5 months, up to about 4 months, up to about 4.5 months, up to about 5 months, up to about 5.5 months, up to about 6 months, up to about 6,5 months, to about 7 months, to about 7.5 months, to about 8 months, to about 8.5 months, to about 9 months, to about 9.5 months, to about 10 months, to about 10.5 months, to about 11 months, to about 11.5 months, to about 12 months, to about 12.5 months, to about 13 months, to about 13.5 months, to about 14 months, to about 14.5 months, to about 15 months, to about 15.5 months, to about 16 months, to about 16.5 months, to about 17 months, to about 17.5 months, to about 18 months, to about 18.5 months, to about 19 months, to about 19.5 months, to about 20 months, to about 20.5 months, to about 21 months, to about 21.5 months, to about 22 months, up to approximately 22.5 months,up to about 23 months, up to about 23.5 months, up to about 24 months, up to about 2.1 years, up to about 2.2 years, up to about 2.3 years, up to about 2.4 years, up to about 2.5 years, up to about 2.6 years, up to about 2.7 years, up to about 2.8 years, up to about 2.9 years, up to about 3 years, or longer after administration of the last dose of the CoV S protein nanoparticles or polypeptides described herein. In embodiments, the COVID-19 is mild COVID-19. In embodiments, the COVID-19 is moderate COVID-19. In embodiments, the COVID-19 is severe COVID-19. In embodiments, the COVID-19 is asymptomatic COVID-19.

[0314] In embodiments, the compositions comprising the CoV S protein nanoparticles or polypeptides described herein are characterized by an efficacy against the SARS-CoV-2 virus or a heterogeneous strain of SARS-CoV-2 of at least 82% for a period of up to about 7.5 months after administration of the last dose of the CoV S protein nanoparticles or polypeptides described herein. In embodiments, the compositions comprising the CoV S protein nanoparticles or polypeptides described herein are characterized by an efficacy against the SARS-CoV-2 virus or a heterogeneous strain of SARS-CoV-2 of from 80% to about 90% for a period of up to about 7.5 months after administration of the last dose of the CoV S protein nanoparticles or polypeptides described herein.

[0315] In embodiments, compositions comprising nanoparticles or polypeptides of the CoV S protein are characterized by an efficacy of at least 75% against asymptomatic disease. In embodiments, the CoV S protein nanoparticles or polypeptides have an efficacy of 80% to 90%, 80% to 99%, 82% to 99%, 82% to 95%, 85% to 95%, 85% to 99%, 85% to 97%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%, against symptomatic COVID-19 for a period of up to about 1 month, up to about 2 months, up to about 2.5 months,up to about 3 months, up to about 3.5 months, up to about 4 months, up to about 4.5 months, up to about 5 months, up to about 5.5 months, up to about 6 months, up to about 6.5 months, up to about 7 months, up to about 7.5 months, up to about 8 months, up to about 8.5 months, up to about 9 months, up to about 9.5 months, up to about 10 months, up to about 10.5 months, up to about 11 months, up to about 11.5 months, up to about 12 months, up to about 12.5 months, up to about 13 months, up to about 13.5 months, up to about 14 months, up to about 14.5 months, up to about 15 months, up to about 15.5 months, up to about 16 months, up to about 16.5 months, up to about 17 months, up to about 17.5 months, up to about 18 months, up to about 18.5 months, up approximately 19 months,up to about 19.5 months, up to about 20 months, up to about 20.5 months, up to about 21 months, up to about 21.5 months, up to about 22 months, up to about 22.5 months, up to about 23 months, up to about 23.5 months, or up to about 24 months or more.,

[0316] In embodiments, compositions comprising the CoV S protein nanoparticles or polypeptides are characterized by an efficacy of from 95% to 97%, from 95% to 99%, from 95% to 98%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%, against severe COVID-19 for a period of up to about 1 month, up to about 2 months, up to about 2.5 months, up to about 3 months, up to about 3.5 months, up to about 4 months, up to about 4.5 months, up to about 5 months, up to about 5.5 months, up to about 6 months, up to about 6.5 months, up to about 7 months, up to about 7.5 months, up to about 8 months, up to about 8.5 months, up to about 9 months, up to approximately 9.5 months, up to approximately 10 months, up to approximately 10.5 months, up to approximately 11 months, up to approximately 11.5 months,up to about 12 months, up to about 12.5 months, up to about 13 months, up to about 13.5 months, up to about 14 months, up to about 14.5 months, up to about 15 months, up to about 15.5 months, up to about 16 months, up to about 16.5 months, up to about 17 months, up to about 17.5 months, up to about 18 months, up to about 18.5 months, up to about 19 months, up to about 19.5 months, up to about 20 months, up to about 20.5 months, up to about 21 months, up to about 21.5 months, up to about 22 months, up to about 22.5 months, up to about 23 months, up to about 23.5 months, or up to about 24 months or more.,

[0317] In embodiments, the compositions comprising the CoV S protein nanoparticles or polypeptides have an efficacy of 75% to 95%, 75% to 90%, 75% to 85%, 75% to 98%, 80% to 98%, 80% to 95%, 80% to 90%, 85% to 98%, 85% to 95%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%, against moderate COVID-19 for a period of up to about 1 month, up to about 2 months, up to about 2.5 months, up to about 3 months,up to about 3.5 months, up to about 4 months, up to about 4.5 months, up to about 5 months, up to about 5.5 months, up to about 6 months, up to about 6.5 months, up to about 7 months, up to about 7.5 months, up to about 8 months, up to about 8.5 months, up to about 9 months, up to about 9.5 months, up to about 10 months, up to about 10.5 months, up to about 11 months, up to about 11.5 months, up to about 12 months, up to about 12.5 months, up to about 13 months, up to about 13.5 months, up to about 14 months, up to about 14.5 months, up to about 15 months, up to about 15.5 months, up to about 16 months, up to about 16.5 months, up to about 17 months, up to about 17.5 months, up to about 18 months, up to about 18.5 months, up to about 19 months, up to approximately 19.5 months,up to about 20 months, up to about 20.5 months, up to about 21 months, up to about 21.5 months, up to about 22 months, up to about 22.5 months, up to about 23 months, up to about 23.5 months, or up to about 24 months or more.,

[0318] In embodiments, the compositions comprising the CoV S protein nanoparticles or polypeptides have an efficacy of 40% to 95%, 40% to 90%, 40% to 85%, 40% to 80%, 40% to 75%, 40% to 70%, 40% to 65%, 40% to 60%, 50% to 95%, 50% to 90%, 50% to 85%, 50% to 80%, 50% to 75%, 50% to 70%, 50% to 65%, 50% to 60%, at least 40%, at least 41%, at least 42%, at least 43%, at least 44%, at least 45%, at least 46%, at least 47%, at least 48%, at least 49%, at least 50%, at least 51%, at least 52%, at least 53%, at least 54%, at least 55%, at least 56%, at least 57%, at least 58%, at least 59%, at least 60%, at least 61%, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%,at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100%, against mild COVID-19 for a period of up to about 1 month, up to about 2 months, up to about 2.5 months, up to about 3 months, up to about 3.5 months, up to about 4 months, up to about 4.5 months, up to about 5 months, up to about 5.5 months,Up to about 6 months, Up to about 6.5 months, Up to about 7 months, Up to about 7.5 months, Up to about 8 months, Up to about 8.5 months, Up to about 9 months, Up to about 9.5 months, Up to about 10 months, Up to about 10.5 months, Up to about 11 months, Up to about 11.5 months, Up to about 12 months, Up to about 12.5 months, Up to about 13 months, Up to about 13.5 months, Up to about 14 months, Up to about 14.5 months, Up to about 15 months, Up to about 15.5 months, Up to about 16 months, Up to about 16.5 months, Up to about 17 months, Up to about 17.5 months, Up to about 18 months, Up to about 18.5 months, Up to about 19 months, Up to about 19.5 months, Up to about 20 months, Up to about 20.5 months, Up to about 21 months, Up to about 21.5 months, up to approximately 22 months,up to about 22.5 months, up to about 23 months, up to about 23.5 months, or up to about 24 months or more.,

[0319] The compositions disclosed herein can be administered by a systemic route, or by a mucosal route, or by a transdermal route, or directly into a specific tissue. The term "systemic administration" as used herein includes parenteral routes of administration. In particular, parenteral administration includes subcutaneous, intraperitoneal, intravenous, intraarterial, intramuscular or intrasternal injection techniques, intravenous or renal dialytic infusion. Typically, systemic parenteral administration is intramuscular injection. As used herein, the term "mucosal administration" includes oral, intranasal, intravaginal, intrarectal, intratracheal, intestinal and ophthalmic administration. Preferably, the administration is intramuscular.

[0320] The compositions can be administered in a single dose regimen or in a multiple dose regimen. Multiple doses can be used in a priming immunization regimen or in a booster immunization regimen. In embodiments, about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 doses are administered. In a multiple-dose regimen, different doses may be administered via the same or different routes, such as parenteral priming and mucosal booster, mucosal priming and parenteral booster, etc.In some aspects, the booster dose is administered at about 2 weeks, about 3 weeks, about 4 weeks, about 5 weeks, about 6 weeks, about 2 months, about 3 months, about 4 months, about 5 months, about 6 months, about 7 months, about 8 months, about 9 months, about 10 months, about 11 months, about 12 months (1 year), about 2 years, about 3 years, about 4 years, about 5 years, about 6 years, about 7 years, about 8 years, about 9 years, or about 10 years after the first dose. In embodiments, the booster dose is administered every year after the initial dose. In embodiments, the subsequent booster dose is administered 3 weeks or 4 weeks after the previous dose. In embodiments, the first dose is administered on day 0 and the booster dose is administered on day 21.In embodiments, the first dose is administered on day 0, and the booster dose is administered on day 28. In embodiments, the first dose is administered on day 0, the booster dose is administered on day 21, and the second booster dose is administered approximately six months after the first dose is administered. In embodiments, the first dose is administered on day 0, the booster dose is administered on day 28, and the second booster dose is administered approximately six months after the first dose is administered. In embodiments, the first dose is administered on day 0, the booster dose is administered on day 21, and the second booster dose is administered approximately six months after the second dose is administered. In embodiments, the first dose is administered on day 0, the booster dose is administered on day 28, and the second booster dose is administered approximately six months after the second dose is administered.

[0321] In embodiments, the booster dose comprises the same immunological composition as the initial dose. In embodiments, the booster dose comprises an immunological composition different from that of the initial dose. In embodiments, the different immunological composition is SARS-CoV-2 spike glycoprotein, mRNA encoding SARS-CoV-2 spike glycoprotein, plasmid DNA encoding SARS-CoV-2 spike glycoprotein, a viral vector encoding SARS-CoV-2 spike glycoprotein, or an inactivated SARS-CoV-2 virus. In embodiments, the booster dose comprises the initial composition.In embodiments, the initial dose comprises a SARS-CoV-2 S glycoprotein (e.g., a SARS-CoV-2 S glycoprotein having the amino acid sequence of SEQ ID NO: 87) and the booster dose comprises the same SARS-CoV-2 S glycoprotein (e.g., a SARS-CoV-2 S glycoprotein having the amino acid sequence of SEQ ID NO: 87). In embodiments, the initial dose comprises a SARS-CoV-2 S glycoprotein (e.g., a SARS-CoV-2 S glycoprotein having the amino acid sequence of SEQ ID NO: 87) and the booster dose comprises a different SARS-CoV-2 S glycoprotein (e.g., a SARS-CoV-2 S glycoprotein having the amino acid sequence of SEQ ID NO: 132).In embodiments, the initial dose comprises a combination of SARS-CoV-2 S glycoproteins (e.g., SARS CoV-2 S glycoprotein having the amino acid sequence of SEQ ID NO: 87 and SARS CoV-2 S glycoprotein having the amino acid sequence of SEQ ID NO: 132). In embodiments, the booster dose comprises a combination of SARS-CoV-2 S glycoproteins (e.g., SARS CoV-2 S glycoprotein having the amino acid sequence of SEQ ID NO: 87 and SARS CoV-2 S glycoprotein having the amino acid sequence of SEQ ID NO: 132). In embodiments, the initial dose comprises SARS-CoV-2 S glycoprotein, plasmid DNA encoding SARS-CoV-2 S glycoprotein, a viral vector encoding SARS-CoV-2 spike glycoprotein, or inactivated SARS-CoV-2 virus.In embodiments, the initial dose comprises a SARS-CoV-2 spike glycoprotein, a plasmid DNA encoding a SARS-CoV-2 spike glycoprotein, a viral vector encoding a SARS-CoV-2 spike glycoprotein, or an inactivated SARS-CoV-2 virus, and the booster dose comprises one or more SARS-CoV-2 S glycoproteins.

[0322] In embodiments, the dose measured in μg may be represented by the total weight of the dose, including the solute, or the weight of the nanoparticles with the CoV S protein polypeptide, or the weight of the CoV S protein polypeptide. The dose is measured using an assay for determining protein concentration by A280 or ELISA.

[0323] The dose of the antigen, including for administration to pediatric subjects, can be in the range of from about 5 μg to about 25 μg, from about 1 μg to about 300 μg, from about 90 μg to about 270 μg, from about 100 μg to about 160 μg, from about 110 μg to about 150 μg, from about 120 μg to about 140 μg, or from about 140 μg to about 160 μg. In embodiments, the dose is about 120 μg, administered with alum. In some aspects, the dose for pediatric subjects can be in the range of from about 1 μg to about 90 μg. In embodiments, the dose of the CoV spike (S) protein polypeptide is about 1 μg, about 2 μg, about 3 μg, about 4 μg, about 5 μg, about 6 μg, about 7 μg, about 8 μg, about 9 μg, about 10 μg,approximately 11 mcg, approximately 12 mcg, approximately 13 mcg, approximately 14 mcg, approximately 15 mcg, approximately 16 mcg, approximately 17 mcg, approximately 18 mcg, approximately 19 mcg, approximately 20 mcg, approximately 21, approximately 22, approximately 23, approximately 24, approximately 25 mcg, approximately 26 mcg, approximately 27 mcg, approximately 28 mcg, approximately 29 mcg, approximately 30 mcg, approximately 40 mcg, approximately 50, approximately 60, approximately 70, approximately 80, approximately 90 mcg, approximately 100 mcg, approximately 110 mcg, approximately 120 mcg, approximately 130 mcg, approximately 140 mcg, approximately 150 mcg, approximately 160 mcg, approximately 170 mcg, approximately 180 mcg, approximately 190 mcg, approximately 200 mcg, approximately 210 mcg, approximately 220 mcg, approximately 230 mcg, approximately 240 mcg, approximately 250 mcg, approximately 260 mcg, approximately 270 mcg,approximately 280 μg, approximately 290 μg, or approximately 300 μg, including all values ​​and ranges therebetween. In embodiments, the dose of the CoV protein S polypeptide is 5 μg. In embodiments, the dose of the CoV protein S polypeptide is 25 μg. In embodiments, the dose of the CoV protein S polypeptide is the same for the initial dose and for the booster doses. In embodiments, the dose of the CoV protein S polypeptide is different for the initial dose and for the booster doses.

[0324] Administration to certain populations may be performed with or without adjuvants. In certain aspects, the compositions may not contain an added adjuvant. In such cases, the dose may be increased by approximately 10%.

[0325] In embodiments, the dose of adjuvant administered with the non-naturally occurring CoV S protein polypeptide is from about 1 μg to about 100 μg, such as about 1 μg, about 2 μg, about 3 μg, about 4 μg, about 5 μg, about 6 μg, about 7 μg, about 8 μg, about 9 μg, about 10 μg, about 11 μg, about 12 μg, about 13 μg, about 14 μg, about 15 μg, about 16 μg, about 17 μg, about 18 μg, about 19 μg, about 20 μg, about 21 μg, about 22 μg, about 23 μg, about 24 μg, about 25 mcg, approximately 26 mcg, approximately 27 mcg, approximately 28 mcg, approximately 29 mcg, approximately 30 mcg, approximately 31 mcg, approximately 32 mcg, approximately 33 mcg, approximately 34 mcg, approximately 35 mcg,approximately 36 mcg, approximately 37 mcg, approximately 38 mcg, approximately 39 mcg, approximately 40 mcg, approximately 41 mcg, approximately 42 mcg, approximately 43 mcg, approximately 44 mcg, approximately 45 mcg, approximately 46 mcg, approximately 47 mcg, approximately 48 mcg, approximately 49 mcg, approximately 50 mcg, approximately 51 mcg, approximately 52 mcg, approximately 53 mcg, approximately 54 mcg, approximately 55 mcg, approximately 56 mcg, approximately 57 mcg, approximately 58 mcg, approximately 59 mcg, approximately 60 mcg, approximately 61 mcg, approximately 62 mcg, approximately 63 mcg, approximately 64 mcg, approximately 65 mcg, approximately 66 mcg, approximately 67 mcg, approximately 68 mcg, approximately 69 mcg, approximately 70 mcg, approximately 71 mcg, approximately 72 mcg, approximately 73 mcg, approximately 74 mcg, approximately 75 mcg, approximately 76 mcg, approximately 77 mcg, approximately 78 mcg,about 79 μg, about 80 μg, about 81 μg, about 82 μg, about 83 μg, about 84 μg, about 85 μg, about 86 μg, about 87 μg, about 88 μg, about 89 μg, about 90 μg, about 91 μg, about 92 μg, about 93 μg, about 94 μg, about 95 μg, about 96 μg, about 97 μg, about 98 μg, about 99 μg, or about 100 μg of adjuvant. In embodiments, the dose of adjuvant is about 50 μg. In embodiments, the adjuvant is a saponin adjuvant, such as MATRIX-M™.

[0326] In embodiments, the dose is administered in a volume of from about 0.1 ml to about 1.5 ml, such as about 0.1 ml, about 0.2 ml, about 0.25 ml, about 0.3 ml, about 0.4 ml, about 0.5 ml, about 0.6 ml, about 0.7 ml, about 0.8 ml, about 0.9 ml, about 1.0 ml, about 1.1 ml, about 1.2 ml, about 1.3 ml, about 1.4 ml, or about 1.5 ml. In embodiments, the dose is administered in a volume of 0.25 ml. In embodiments, the dose is administered in a volume of 0.5 ml. In embodiments, the dose is administered in a volume of 0.6 ml.

[0327] In specific embodiments, for a vaccine against MERS, SARS, or SARS-CoV-2 coronavirus, the dose may comprise a CoV S protein polypeptide concentration of from about 1 μg / ml to about 50 μg / ml, from about 10 μg / ml to about 100 μg / ml, from about 10 μg / ml to about 50 μg / ml, from about 175 μg / ml to about 325 μg / ml, from about 200 μg / ml to about 300 μg / ml, from about 220 μg / ml to about 280 μg / ml, or from about 240 μg / ml to about 260 μg / ml.

[0328] In another embodiment, the present invention provides a method for formulating a vaccine composition that induces immunity against an infection or at least one symptom of a disease caused by it in a mammal, comprising adding to the composition an effective dose of a nanoparticle or a CoV S protein polypeptide. The disclosed CoV S protein polypeptides and nanoparticles based on them are useful for producing compositions that stimulate an immune response that confers immunity or substantial immunity against infectious agents. Thus, in one embodiment, the present invention provides a method for inducing immunity against infections or at least one symptom of a disease caused by them in a subject, comprising administering at least one effective dose of a nanoparticle and / or a CoV S protein polypeptide.

[0329] In embodiments, the CoV S protein polypeptides or nanoparticles comprising them are administered in combination with an additional immunogenic composition. In embodiments, the additional immunogenic composition induces an immune response against SARS-CoV-2. In embodiments, the additional immunogenic composition is administered for about 1 minute, about 5 minutes, about 10 minutes, about 20 minutes, about 30 minutes, about 40 minutes, about 50 minutes, about 1 hour, about 2 hours, about 3 hours, about 4 hours, about 5 hours, about 6 hours, about 7 hours, about 8 hours, about 9 hours, about 10 hours, about 11 hours, about 12 hours, about 13 hours, about 14 hours, about 15 hours, about 16 hours, about 17 hours, about 18 hours, about 19 hours,approximately 20 hours, approximately 21 hours, approximately 22 hours, approximately 23 hours, approximately 1 day, approximately 2 days, approximately 3 days, approximately 4 days, approximately 5 days, approximately 6 days, approximately 7 days, approximately 8 days, approximately 9 days, approximately 10 days, approximately 11 days, approximately 12 days, approximately 13 days, approximately 14 days, approximately 15 days, approximately 16 days, approximately 17 days, approximately 18 days, approximately 19 days, approximately 20 days, approximately 21 days, approximately 22 days, approximately 23 days, approximately 24 days, approximately 25 days, approximately 26 days, approximately 27 days, approximately 28 days, approximately 29 days,approximately 30 days or approximately 31 days relative to the time of administration of the disclosed CoV S protein polypeptides or nanoparticles containing them. In embodiments, the additional composition is administered with a first dose of a composition comprising a CoV S protein polypeptide or a nanoparticle containing the same. In embodiments, the additional composition is administered with a booster dose of a composition comprising a CoV S protein polypeptide or a nanoparticle containing the same.

[0330] In embodiments, the additional immunogenic composition comprises mRNA encoding the SARS-Cov-2 spike glycoprotein, plasmid DNA encoding the SARS-Cov-2 spike glycoprotein, a viral vector encoding the SARS-Cov-2 spike glycoprotein, or an inactivated SARS-CoV-2 virus.

[0331] In embodiments, the additional immunogenic composition comprises mRNA that encodes a CoV S protein polypeptide. In embodiments, the mRNA encodes a CoV S protein polypeptide having proline substitutions at positions 986 and 987 of SEQ ID NO: 1. In embodiments, the mRNA encodes a CoV S protein polypeptide having an intact furin cleavage site. In embodiments, the mRNA encodes a CoV S protein polypeptide having proline substitutions at positions 986 and 987 of SEQ ID NO: 1 and an intact furin cleavage site. In embodiments, the mRNA encodes a CoV S protein polypeptide having proline substitutions at positions 986 and 987 of SEQ ID NO: 1 and an inactive furin cleavage site. In embodiments, the mRNA encodes a CoV S protein polypeptide characterized by the amino acid sequence of SEQ ID NO: 87. In embodiments, the mRNA encoding the CoV S protein polypeptide is encapsulated in a lipid nanoparticle.An exemplary immunogenic composition comprising mRNA encoding a CoV S protein polypeptide is described in Jackson et al. N. Eng. J. Med. 2020. An mRNA Vaccine against SARS-CoV-2 - preliminary report, which is incorporated herein by reference in its entirety. In embodiments, the composition comprising mRNA encoding a CoV S protein polypeptide is administered at a dose of 25 μg, 100 μg, or 250 μg.

[0332] In embodiments, the additional immunogenic composition comprises an adenoviral vector encoding a CoV S protein polypeptide. In embodiments, the AAV-based vector encodes a wild-type CoV S protein polypeptide. In embodiments, the AAV-based vector encodes a CoV S protein polypeptide having proline substitutions at positions 986 and 987 of SEQ ID NO: 1 and an intact furin cleavage site. In embodiments, the AAV-based vector encodes a CoV S protein polypeptide having proline substitutions at positions 986 and 987 of SEQ ID NO: 1 and an inactive furin cleavage site. In embodiments, the AAV-based vector encodes a CoV S protein polypeptide characterized by the amino acid sequence of SEQ ID NO: 87. The following publications describe immunogenic compositions comprising an adenoviral vector encoding a CoV S protein polypeptide, each of which is incorporated herein by reference in its entirety: van Doremalen N. et al.A single dose of ChAdOx1 MERS provides protective immunity in rhesus macaques. Science Advances, 2020; van Doremalen N. et al. ChAdOx1 nCoV-19 vaccination prevents SARS-CoV-2 pneumonia in rhesus macaques. bioRxiv, (2020).

[0333] In embodiments, the additional immunogenic composition comprises deoxyribonucleic acid (DNA). In embodiments, the additional immunogenic composition comprises plasmid DNA. In embodiments, the plasmid DNA encodes a CoV S protein polypeptide. In embodiments, the DNA encodes a CoV S protein polypeptide having proline substitutions at positions 986 and 987 of SEQ ID NO: 1 and an intact furin cleavage site. In embodiments, the DNA encodes a CoV S protein polypeptide having proline substitutions at positions 986 and 987 of SEQ ID NO: 1 and an inactive furin cleavage site. In embodiments, the DNA encodes a CoV S protein polypeptide having the amino acid sequence of SEQ ID NO: 87.

[0334] In embodiments, the additional immunogenic composition comprises an inactivated viral vaccine.

[0335] In embodiments, CoV S protein polypeptides or nanoparticles comprising CoV S protein polypeptides are administered to a patient who has or has previously had a confirmed infection with SARS-CoV-2 or a heterogeneous strain of SARS-CoV-2. Infection with SARS-CoV-2 or a heterogeneous strain of SARS-CoV-2 can be confirmed by a nucleic acid amplification test (e.g., polymerase chain reaction) or serological testing (e.g., testing for antibodies to a SARS-CoV-2 viral antigen). In embodiments, CoV S protein polypeptides or nanoparticles comprising CoV S protein polypeptides are administered to a patient at least about 3 days, at least about 1 week, at least about 2 weeks, at least about 3 weeks, at least about 4 weeks after the patient has been diagnosed with COVID-19. In embodiments, CoV S protein polypeptides or nanoparticles,containing CoV S protein polypeptides are administered to the patient within 1 week to 1 year after the patient is diagnosed with COVID-19, such as about 1 week, about 2 weeks, about 3 weeks, about 4 weeks, about 5 weeks, about 6 weeks, about 1 month, about 2 months, about 3 months, about 4 months, about 5 months, about 6 months, about 7 months, about 8 months, about 9 months, about 10 months, about 11 months, or about 1 year. In embodiments, the CoV S protein polypeptides or nanoparticles comprising the CoV S protein polypeptides are administered to the patient within 1 week to 20 years after the patient is diagnosed with COVID-19, such as about 1 week, about 2 weeks, about 3 weeks, about 4 weeks, about 5 weeks, about 6 weeks, about 1 month,approximately 2 months, approximately 3 months, approximately 4 months, approximately 5 months, approximately 6 months, approximately 7 months, approximately 8 months, approximately 9 months, approximately 10 months, approximately 11 months, approximately 1 year, approximately 2 years, approximately 3 years, approximately 4 years, approximately 5 years, approximately 6 years, approximately 7 years, approximately 8 years, approximately 9 years, approximately 10 years, approximately 11 years, approximately 12 years, approximately 13 years, approximately 14 years, approximately 15 years, approximately 16 years, approximately 17 years, approximately 18 years, approximately 19 years, or approximately 20 years.

[0336] In embodiments, the CoV S protein polypeptides or nanoparticles comprising them are administered after the first immunogenic composition has been administered to the patient. Non-limiting examples of the first immunogenic compositions include the SARS-CoV-2 spike glycoprotein, mRNA encoding the SARS-CoV-2 spike glycoprotein, plasmid DNA encoding the SARS-CoV-2 spike glycoprotein, a viral vector encoding the SARS-CoV-2 spike glycoprotein, or an inactivated SARS-CoV-2 virus.In embodiments, the CoV S protein polypeptides or nanoparticles comprising them are administered within a period of from about 1 week to about 1 year, from about 1 week to 1 month, from about 3 weeks to 4 weeks, from about 1 week to 5 years, from about 1 year to about 5 years, from about 1 year to about 3 years, from about 3 years to about 5 years, from about 5 years to about 10 years, from about 1 year to about 10 years, or from about 1 year to about 2 years after administration of the first immunogenic composition.In embodiments, the CoV S protein polypeptides or nanoparticles comprising them are administered in a period of from about 1 week to about 1 year after the administration of the first immunogenic composition, such as about 1 week, about 2 weeks, about 3 weeks, about 4 weeks, about 5 weeks, about 6 weeks, about 7 weeks, about 8 weeks, about 9 weeks, about 10 weeks, about 1 month, about 2 months, about 3 months, about 4 months, about 5 months, about 6 months, about 7 months, about 8 months, about 9 months, about 10 months, about 11 months, or about 1 year after the administration of the first immunogenic composition.

[0337] In embodiments, CoV S proteins or nanoparticles comprising CoV S proteins are useful for producing immunogenic compositions for stimulating an immune response that confers immunity or substantial immunity against one or more of MERS, SARS, SARS-CoV-2, and a heterogeneous strain of SARS-CoV-2. Both mucosal and cellular immunity can contribute to immunity against infections and disease. Antibodies secreted locally in the upper respiratory tract are a major factor in resistance to natural infection. Secretory immunoglobulin A (sIgA) is involved in protecting the upper respiratory tract, and serum IgG is involved in protecting the lower respiratory tract. The immune response induced by infection protects against reinfection with the same virus or an antigenically similar viral strain.Antibodies produced in a host following immunization with the nanoparticles disclosed herein can also be administered to other individuals, thereby providing passive administration to the subject.

[0338] In embodiments, CoV S proteins or nanoparticles comprising CoV S proteins induce cross-neutralizing antibodies against SARS-CoV-2 viruses comprising S proteins with one or more modifications selected from (a) a deletion of one or more amino acids of the NTD, wherein the one or more amino acids are selected from the group consisting of amino acids 56, 57, 131, 132, 229, 230, 231, or combinations thereof; and(b) a mutation affecting one or more amino acids of the NTD, wherein the one or more amino acids affected by the mutation are selected from the group consisting of amino acids 67, 82, 133, 229, 202, 209, 240, 139, 5, 233, 7, 13, 125, 177, or combinations thereof;(c) a mutation affecting one or more amino acids of the RBD, wherein the one or more amino acids affected by the mutation are selected from the group consisting of amino acids 488, 404, 471, 464, 439, 481, 426, 440, and combinations thereof;(d) a mutation affecting one or more amino acids of SD1 / 2, wherein the one or more amino acids are selected from the group consisting of amino acids 601, 557, 668, 642, and combinations thereof; (e) an inactive furin cleavage site (corresponding to one or more mutations affecting amino acids 669-672); (f) a deletion of one or more amino acids of the S2 subunit, wherein the amino acids are selected from the group consisting of 676-702, 702-711, 775-793, 806-815, and combinations thereof; (g) a mutation affecting one or more amino acids of the S2 subunit, wherein the amino acids are selected from the group consisting of 973, 974, 703, 1105, 688, 969, 1014, and 1163, and combinations thereof; (h) deletions of one or more amino acids from TMCT (amino acids 1201-1260), where the amino acids of the CoV S glycoprotein are numbered relative to SEQ ID NO: 2.;

[0339] In embodiments, CoV S proteins or nanoparticles comprising CoV S proteins induce cross-neutralizing antibodies against SARS-CoV-2 viruses comprising S proteins with one or more modifications selected from a deletion of amino acid 56, a deletion of amino acid 57, a deletion of amino acid 131, N488Y, A557D, D601G, P668H, T703I, S969A, D1105H, N426K and Y440F, wherein the amino acids are numbered relative to the CoV S protein polypeptide characterized by the amino acid sequence under SEQ ID NO: 2.

[0340] In embodiments, the CoV S protein or a nanoparticle comprising the CoV S protein induces cross-neutralizing antibodies against SARS-CoV-2 viruses comprising S proteins with one or more modifications selected from a deletion of amino acid 56, a deletion of amino acid 57, a deletion of amino acid 131, N488Y, A557D, D601G, P668H, T703I, S969A, and D1105H, wherein the amino acids are numbered relative to the CoV S protein polypeptide characterized by the amino acid sequence of SEQ ID NO: 2.

[0341] In embodiments, the CoV S protein or a nanoparticle comprising the CoV S protein induces cross-neutralizing antibodies against SARS-CoV-2 viruses comprising S proteins with one or more modifications selected from D67A, D202G, L229H, K404N, E471K, N488Y, D601G and A688V, wherein the amino acids are numbered relative to the CoV S protein polypeptide characterized by the amino acid sequence under SEQ ID NO: 2.

[0342] In embodiments, the CoV S protein or a nanoparticle comprising the CoV S protein induces cross-neutralizing antibodies against SARS-CoV-2 viruses comprising S proteins with one or more modifications selected from a deletion of amino acids 229-231, D67A, D202G, K404N, E471K, N488Y, D601G and A688V, where the amino acids are numbered relative to the CoV S protein polypeptide characterized by the amino acid sequence under SEQ ID NO: 2.

[0343] In embodiments, the CoV S protein or a nanoparticle comprising the CoV S protein induces cross-neutralizing antibodies against SARS-CoV-2 viruses comprising S proteins with one or more modifications selected from deletion of amino acids 229-231, L5F D67A, D202G, K404N, E471K, N488Y, D601G and A688V, wherein the amino acids are numbered relative to the CoV S protein polypeptide characterized by the amino acid sequence under SEQ ID NO: 2.

[0344] In embodiments, the CoV S protein or a nanoparticle comprising the CoV S protein induces cross-neutralizing antibodies against SARS-CoV-2 viruses comprising S proteins with one or more modifications selected from L5F, T7N, P13S, D125Y, R177S, K404T, E471K, N488Y, D601G, H642Y, T1014I and V1163F, wherein the amino acids are numbered relative to the CoV S protein polypeptide characterized by the amino acid sequence under SEQ ID NO: 2.

[0345] In embodiments, the CoV S protein or a nanoparticle comprising the CoV S protein induces cross-neutralizing antibodies against SARS-CoV-2 viruses with the S protein comprising one or more modifications selected from W139C and L439R, wherein the amino acids are numbered relative to the CoV S protein polypeptide characterized by the amino acid sequence of SEQ ID NO: 2. In embodiments, the CoV S protein polypeptide comprising the modifications W139C and L439R is expressed with a signal peptide characterized by the amino acid sequence of SEQ ID NO: 117 or SEQ ID NO: 5. In embodiments, the CoV S protein or a nanoparticle comprising the CoV S protein induces cross-neutralizing antibodies against SARS-CoV-2 viruses with one or more modifications selected from D601G, W139C and L439R, wherein the amino acids are numbered relative to the CoV S protein polypeptide having the amino acid sequence of SEQ ID NO: 2.In embodiments, the CoV S protein polypeptide or nanoparticle comprising the modifications D601G, W139C and L439R is expressed with a signal peptide characterized by the amino acid sequence of SEQ ID NO: 117 or SEQ ID NO: 5.

[0346] In embodiments, the CoV S protein or a nanoparticle comprising the CoV S protein induces cross-neutralizing antibodies against SARS-CoV-2 viruses with one or more modifications selected from D601G, L5F, D67A, D202G, deletions of amino acids 229-231, R233I, K404N, E471K, N488Y, and A688V, wherein the amino acids are numbered relative to the CoV S protein polypeptide characterized by the amino acid sequence of SEQ ID NO: 2. In embodiments, the CoV S protein or a nanoparticle comprising the CoV S protein induces cross-neutralizing antibodies against SARS-CoV-2 viruses with one or more modifications selected from L5F, D67A, D202G, deletions of amino acids 229-231, R233I, K404N, E471K, N488Y and A688V, where the amino acids are numbered relative to the CoV S protein polypeptide having the amino acid sequence of SEQ ID NO: 2.

[0347] In embodiments, the present invention provides a method for producing one or more high-affinity antibodies to MERS-CoV, to SARS-CoV, and to the SARS-CoV-2 virus. High-affinity antibodies obtained by immunization with the nanoparticles described herein are obtained by administering to an animal an immunogenic composition comprising a CoV S protein polypeptide or nanoparticles comprising a CoV S protein polypeptide, collecting blood serum and / or blood plasma from the animal, and purifying the antibody from the blood serum and / or blood plasma. In one embodiment, the animal is a human. In embodiments, the animal is a chicken, a mouse, a guinea pig, a rat, a rabbit, a goat, a human, a horse, a sheep, or a cow. In one embodiment, the animal is a bovine animal or a horse. In another embodiment, the bovine animal or horse is transgenic.In yet another embodiment, transgenic bovine or equine representatives produce human antibodies. In some embodiments, the animal produces monoclonal antibodies. In other embodiments, the animal produces polyclonal antibodies. In one embodiment, the method further comprises administering an adjuvant or an immunostimulatory compound. In a further embodiment, the purified high-affinity antibody is administered to a human subject. In one embodiment, the human subject is at risk of infection with one or more of MERS, SARS, and SARS-CoV-2.

[0348] In embodiments, CoV S proteins or nanoparticles are co-administered with an influenza virus glycoprotein or a nanoparticle comprising an influenza virus glycoprotein. Suitable glycoproteins and nanoparticles are described in U.S. Patent Application Publication No. 2018 / 0133308 and U.S. Patent Application Publication No. 2019 / 0314487, each of which is incorporated herein by reference in its entirety. In embodiments, the CoV S protein or nanoparticle is administered together with (a) a nanoparticle with a core represented by a detergent, wherein the nanoparticle with a core represented by a detergent comprises a recombinant influenza virus hemagglutinin (HA) glycoprotein from an influenza virus strain of type B and (b) a hemagglutinin-containing nanoparticle with a saponin matrix (HaSMaN), wherein HaSMaN comprises a recombinant influenza virus HA glycoprotein from an influenza virus strain of type A and an adjuvant based on an ISCOM matrix.In embodiments, the CoV S protein or nanoparticle is administered together with a nanoparticle comprising a core represented by a non-ionic detergent and an influenza virus HA glycoprotein, wherein the influenza virus HA glycoprotein comprises a head region that protrudes outward from the core represented by a non-ionic detergent and a transmembrane domain that is associated with the core represented by a non-ionic detergent, wherein the influenza virus HA glycoprotein is an HA0 glycoprotein, wherein the amino acid sequence of the influenza virus HA glycoprotein is characterized by 100% identity to the amino acid sequence of the native influenza virus HA protein. In embodiments, the influenza virus glycoprotein or nanoparticle is formulated together with the CoV S protein or nanoparticle.

[0349] All patents, patent applications, references, and journal articles cited in this disclosure are expressly incorporated herein by reference in their entirety for all purposes. EXAMPLES Example 1 Expression and Purification of Nanoparticles Based on the Coronavirus Spike (S) Protein Polypeptide

[0350] Native coronavirus spike (S) protein polypeptide (SEQ ID NO: 1 and SEQ ID NO: 2) and CoV spike protein polypeptides that comprise amino acid sequences corresponding to the sequences of SEQ ID NOs: 3, 4, 38, 41, 44, 48, 51, 54, 58, 61, 63, 65, 67, 73, 75, 78, 79, 82, 83, 85, 87, 106, 108, 89, 112-115, 132, 133, 114, 138, 141, 144, 147, 151, 153, 156, 158, 164-168 were expressed in The baculovirus-based expression system, and recombinant plaques expressing coronavirus spike (S) protein polypeptides were selected and confirmed. In each case, the signal peptide is the sequence of SEQ ID NO: 5. Fig. 4 and Fig. 9 show the successful purification of CoV spike protein polypeptides BV2364, BV2365, BV2366, BV2367, BV2368, BV2369, BV2373, BV2374, and BV2375. Table 2 shows the sequence characteristics of the aforementioned CoV spike protein polypeptides. Table 2. Individual CoV spike protein polypeptides

[0351] The wild-type BV2361 protein (SEQ ID NO: 2) binds to the human angiotensin-converting enzyme 2 (hACE2) precursor. Biolayer interferometry and ELISA were used to assess the binding of CoV S protein polypeptides. Biolayer interferometry (BLI)

[0352] BLI-based experiments were performed using the Octet QK384 system (Pall Fremont, California). Human ACE2 with a His-tag attached (2 μg mL -1), were immobilized on nickel-loaded Ni-NTA biosensor tips. After baseline determination, samples containing the SARS-CoV-2 S protein were serially diluted 2-fold and allowed to associate for 600 seconds, followed by dissociation for an additional 900 seconds. Data were analyzed using Octet HT 10 software with a 1:1 global curve fit.

[0353] CoV S protein polypeptides BV2361, BV2365, BV2369, BV2365, BV2373, BV2374 retained the ability to bind to hACE2 (Fig. 5, Fig. 11A-C). Dissociation kinetics showed that the S proteins remained tightly bound, as evidenced by minimal or no dissociation over 900 seconds of observation in the absence of liquid phase S protein (Fig. 11A-C).

[0354] Moreover, the binding is specific. The wild-type CoV S protein, BV2361, and the CoV S protein polypeptides BV2365 and BV2373 do not bind to the MERS-CoV receptor, dipeptidyl peptidase IV (DPP4). In addition, the MERS S protein does not bind to the human angiotensin-converting enzyme 2 (hACE2) precursor (Fig. 6, Fig. 11D-F). ELISA

[0355] The specificity of CoV S protein polypeptides for hACE2 was confirmed by ELISA. Ninety-six-well plates were coated with 100 μL of SARS-CoV-2 spike protein (2 μg / mL) overnight at 4°C. The plates were washed with phosphate-buffered saline containing 0.05% Tween buffer (PBS-T) and blocked with TBS Startblock blocking buffer (ThermoFisher, Scientific). His-tagged hACE2 and hDPP4 receptors were serially diluted 3-fold (5-0.0001 μg mL -1) and added to coated wells for 2 h at room temperature. Plates were washed with PBS-T. Optimally diluted horseradish peroxidase (HRP)-conjugated anti-histidine antibody was added and color development was achieved by adding 3,3',5,5'-tetramethylbenzidine peroxidase (™)B substrate (T0440-IL, Sigma, St. Louis, MO, USA). Plates were read at OD 450 nm using a SpectraMax Plus plate reader (Molecular Devices, Sunnyvale, CA, USA) and data analyzed using SoftMax software. EC50 values ​​were calculated by 4-parameter fitting using GraphPad Prism 7.05 software.

[0356] ELISA results demonstrated that the S protein polypeptides of wild-type CoV (BV2361), BV2365, and BV2373 specifically bound hACE2 but did not bind the hDPP-4 receptor used by MERS-CoV (IC 50 >5000 ng ml -1 ). The wild-type CoV S protein polypeptide and BV2365 bound to hACE2 with similar affinity (IC50 = 36-38 ng / ml), whereas BV2373 achieved 50% saturation of binding to hACE2 at a concentration that was 2-fold lower (IC 50 = 18 ng / ml) (Fig. 7, Fig. 11D-F). Preparation of proteins and nanoparticles

[0357] The recombinant virus is amplified by infecting Sf9 insect cells. The insect cell culture is infected with baculovirus at approximately 3 MOI (multiplicity of infection = ffu or pfu virus / cell). The culture and supernatant are collected 48-72 hours after infection. The collected crude cells (approximately 30 ml) are clarified by centrifugation for 15 minutes at approximately 800 × g. The resulting collected crude cells, containing the coronavirus spike (S) protein, are purified into nanoparticles as described below.

[0358] The membrane protein extraction protocol utilizes the nonionic surfactant TERGITOL® NP-9, a nonylphenol ethoxylate, to produce nanoparticles. The crude extract is further purified using anion exchange chromatography, lentil lectin affinity chromatography / HIC, and cation exchange chromatography. Washed cells are lysed with detergent and then subjected to low pH treatment, which precipitates BV DNA and protein and Sf9 host cells. The neutralized low pH-treated lysate is clarified and further purified using anion exchange and affinity chromatography before a second low pH treatment.

[0359] Affinity chromatography is used to remove Sf9 / BV proteins, DNA, and NP-9, and to concentrate the coronavirus spike (S) protein. Briefly, lentil lectin is a calcium-manganese metalloprotein that reversibly binds polysaccharides and glycosylated proteins containing glucose or mannose. The flow-through fraction containing the coronavirus spike (S) protein obtained by anion exchange chromatography is loaded onto lentil lectin affinity chromatography resin (Capto Lentil Lectin, GE Healthcare). Glycosylated coronavirus spike (S) protein selectively binds to the resin, while non-glycosylated proteins and DNA are removed by flow-through. Loosely bound glycoproteins are removed using buffers containing a high concentration of salt and a low molar concentration of methyl alpha-D-mannopyranoside (MMP).

[0360] Column washes are also used to replace the detergent NP-9 with a surfactant polysorbate 80 (PS80). Coronavirus spike (S) protein polypeptides are eluted as nanoparticle structures from the lentil lectin column using MMP at high concentrations. After elution, the coronavirus spike (S) protein trimers assemble into nanoparticles consisting of trimers of the coronavirus spike (S) protein and PS80 contained in a detergent core. Example 2 Immunogenicity of coronavirus spike (S) protein polypeptide-based nanoparticle vaccines in mice

[0361] A coronavirus spike (S) protein composition comprising the CoV S protein polypeptide of SEQ ID NO: 87 (also referred to as "BV2373" as described in Example 1) was evaluated for immunogenicity and toxicity in a mouse model using female BALB / c mice (7-9 weeks old; Harlan Laboratories Inc., Frederick, MD). The compositions were evaluated in the presence and absence of a saponin adjuvant, such as MATRIX-M™. The compositions containing MATRIX-M™ contained 5 μg MATRIX-M™. Vaccines containing the coronavirus spike (S) protein polypeptide at various doses, including 0.01 μg, 0.1 μg, 1 μg, and 10 μg, were administered intramuscularly as a single dose (also referred to as a single prime dose) (study day 14) or as two doses (also referred to as a prime / booster schedule) 14 days apart (study days 0 and 14). The placebo group served as a non-immunized control.Blood serum for analysis was collected on study days 1, 13, 21, and 28. Vaccinated and control animals were intranasally infected with SARS-CoV-2 42 days after one (single dose) or two (two doses) immunizations. Immunogenicity of vaccines.

[0362] Animals immunized with a single priming dose of 0.1-10 μg BV2373 and MATRIX-M™) had elevated titers of IgG antibodies to the S protein, which were detectable 21-28 days after a single immunization (Fig. 13B). Mice immunized with a 10 μg dose of BV2373 and MATRIX-M™) produced antibodies that blocked the binding of the hACE2 receptor to the CoV S protein and virus-neutralizing antibodies, which were detectable 21-28 days after a single priming dose (Fig. 14Fig. 15). Animals immunized with the prime / booster schedule (two doses) had significantly elevated IgG antibody titers to protein S, which were detectable 7–16 days after the booster at all dose levels (Fig. 13A). Animals immunized with BV2373 (1 μg and 10 μg) and MATRIX-M™ had similarly high IgG antibody titers to protein S after immunization (GMT = 139,000 and 84,000, respectively).Mice immunized with BV2373 (0.1 μg, 1 μg, or 10 μg) and MATRIX-M™ had significantly (p ≤ 0.05 and p ≤ 0.0001) higher IgG antibody titers to protein S compared to mice immunized with 10 μg BV2373 without adjuvant (Fig. 13A). These results indicate that the MATRIX-M™ adjuvant provides a 10- to 100-fold reduction in the required dose. Moreover, immunization with two doses of BV2373 and MATRIX-M™ induced high-titer antibodies that blocked hACE2 receptor binding to protein S (IC50 = 218 - 1642) and neutralized the cytopathic effect (CPE) of SARS-CoV-2 against Vero E6 cells (100% blockade CPE = 7680 - 20000) at all dose levels (Fig. 14Fig. 15). SARS CoV-2 challenge.

[0363] To assess the induction of protective immunity, immunized mice were challenged with SARS-CoV-2. Since mice do not support wild-type SARS-CoV-2 virus replication, mice were intranasally infected with hACE2-expressing adenovirus (Ad / hACE2) on day 52 after the initial vaccination to render them susceptible. Mice were inoculated intranasally with 1.5 x 10 5 SARS-CoV-2 pfu in 50 µl, divided between the nostrils. Infected mice were weighed on the day of infection and daily for up to 7 days post-infection. Four and seven days post-infection, five mice from each vaccination group and the control group were sacrificed, and the lungs were collected and prepared for histological analysis.

[0364] Viral titer was quantified using a plaque assay. Briefly, collected lungs were homogenized in PBS using 1.0 mm glass beads (Sigma Aldrich) and a Beadruptor (Omini International Inc.). Homogenates were added to nearly confluent Vero E6 cultures, and SARS-CoV-2 viral titers were determined by counting plaque-forming units (pfu) using a 6-point dilution curve.

[0365] Four days after infection, placebo-treated mice showed 10 4 SARS-CoV-2 pfu / lung, whereas mice immunized with BV2363 without MATRIX-M™) were characterized by 10 3pfu / lung (Fig. 16). A dose-dependent reduction in viral titer was observed in groups of mice that received only prime immunization with BV2373 with MATRIX-M™, with recipients of the 10 μg dose of BV2373 having no detectable amounts of virus at day 4 post-infection. All mice receiving doses of BV2373 of 1 μg, 0.1 μg, and 0.01 μg showed a significant reduction in titer compared to mice vaccinated with placebo. In the prime / booster groups, mice immunized with doses of 10 μg, 1 μg, and 0.1 μg had nearly undetectable viral loads in the lungs, while the 0.01 μg group showed a 1 log reduction compared to placebo-treated animals.

[0366] Weight loss data and viral load determination results were compared. Animals receiving a single dose of BV2373 (0.1 μg, 1 μg, and 10 μg) and MATRIX-M™ showed significant protection against weight loss compared to unvaccinated animals receiving placebo (Fig. 17A). Mice receiving a primer and booster dose with adjuvant also showed significant protection against weight loss at all dose levels (Figs. 17B-C). The effect of the presence of adjuvant on protection against weight loss was assessed. Mice receiving the prime / booster immunization (two doses) plus adjuvant showed significant protection against weight loss compared to placebo, while the group immunized without adjuvant did not (Fig. 17C).These results showed that BV2373 confers protection against SARS-CoV-2 and that low vaccine doses associated with weaker serologic responses do not exacerbate weight loss or demonstrate significant disease.

[0367] The histopathology of the lungs was assessed at days 4 and 7 post-infection (Fig. 18A, Fig. 18B). At day 4 post-infection, placebo-immunized mice showed denuded epithelial cells in the large airways with thickened alveolar septa surrounded by a mixed population of inflammatory cells. Periarteriolar cuffs with inflammatory cells consisting mainly of neutrophils and macrophages were observed throughout the lungs. At day 7 post-infection, placebo-treated mice showed peribronchiolar inflammation with an enlarged periarteriolar cuff. Thickened alveolar septa persisted with increased diffuse interstitial inflammation throughout the alveolar septum (Fig. 18B).

[0368] Mice immunized with BV2373 showed a significant reduction in lung pathological changes at both days 4 and 7 post-infection in a dose-dependent manner. In the primer-only group, inflammation was reduced at doses of 10 μg and 1 μg, as well as inflammation around the bronchi and arterioles, compared with placebo-treated mice. In the primer-only groups, lung inflammation at lower doses resembled that in the placebo groups, which correlated with weight loss and viral titers in the lungs. The primer / booster groups showed a significant reduction in lung inflammation at all doses tested, which also correlated with viral titers in the lungs and weight loss data. Epithelial cells in the large and small bronchi on days 4 and 7 were largely preserved with minimal bronchiolar rejection and signs of viral infection.Arterioles from animals immunized with doses of 10 μg, 1 μg, and 0.1 μg showed minimal inflammation, with only moderate cuff tightening similar to placebo observed at the 0.01 μg dose. Alveolar inflammation was reduced in animals receiving higher doses, with only the lower dose of 0.01 μg associated with inflammation (Figs. 18A-18B). These data demonstrate that BV2373 reduces lung inflammation after challenge and that even doses and schedules of BV2373 that produce minimal or no detectable neutralizing activity are not associated with an exacerbated inflammatory response to the virus. Moreover, the vaccine does not induce vaccine-associated enhanced respiratory disease (VAERD) in infected mice.T cell response.

[0369] The effect of a vaccine composition containing the CoV S protein polypeptide under SEQ ID NO: 87 on the T-cell response was evaluated. BALB / c mice (N = 6 per group) were immunized intramuscularly with 10 μg BV2373 with or without 5 μg MATRIX-M™ in two doses at an interval of 21 days. Spleen samples were collected 7 days after the second immunization (study day 28). The unvaccinated group (N = 3) served as a control.

[0370] Antigen-specific T cell responses were measured using ELISPOT™ enzyme-linked immunosorbent assay and intracellular cytokine staining (ICCS) in spleen samples collected 7 days after the second immunization (study day 28). The number of IFN-γ-secreting cells after ex vivo stimulation increased 20-fold (p = 0.002) in spleen samples from mice immunized with BV2373 and MATRIX-M™) compared with that after immunization with BV2373 alone, as measured by the ELISPOT™ assay (Fig. 19). To examine CD4+ and CD8+ T cell responses separately, ICCS assays were performed in combination with surface marker staining. Data shown are gated on CD44hiCD62L, a population of effector memory T cells.The frequencies of CD4+ and CD8+ T cells secreting IFN-γ+, TNF-α+, and IL-2+ cytokines were significantly higher (p<0.0001) in spleen samples from mice immunized with BV2373 compared to mice immunized without adjuvant (Figs. 20A-C). Furthermore, the frequencies of multifunctional CD4+ and CD8+ T cells that simultaneously produced at least two or three cytokines were also significantly increased (p<0.0001) in spleen samples from mice immunized with BV2373 / MATRIX-M™ compared to mice immunized in the absence of adjuvant (Figs. 20D-E). Immunization with BV2373 / MATRIX-M™ resulted in increased proportions of multifunctional phenotypes (e.g., T cells that secrete more than one of IFN-γ, TNF-α, and IL-2) in both CD4+ and CD8+ T cell populations. The proportions of multifunctional phenotypes detected in memory CD4+ T cells were higher than those in CD8+ T cells (Fig. 22).

[0371] Secretion of type 2 cytokines IL-4 and IL-5 from CD4+ T cells was also determined by ICCS and ELISPOT™, respectively. Immunization with BV2373 / MATRIX-M™ also resulted in an increase in the secretion of type 2 cytokines IL-4 and IL-5 (2-fold) compared to immunization with BV2373 alone, but to a lesser extent than the increase in type 1 cytokine production (e.g., IFN-γ levels were increased 20-fold) (Fig. 23A-C). These results indicate that administration of the MATRIX-M™ adjuvant biased CD4+ T cell development towards Th1-type responses.

[0372] The effect of immunization on germinal center formation was assessed by measuring the frequency of CD4+ follicular helper T cells (TFH) and germinal center B cells (GC) in spleen samples. Administration of MATRIX-M™ significantly increased the frequency of TFH cells (CD4+ CXCR5+ PD-1+) (p = 0.01) and GC B cells (CD19+ GL7+ CD95+) (p = 0.0002) in spleen samples (Fig. 24A-B Fig. 25A-B). Example 3 Immunogenicity of coronavirus spike (S) protein polypeptide-based nanoparticle vaccines in olive baboons

[0373] The immunogenicity of a vaccine composition containing BV2373 was evaluated in baboons. Adult olive baboons were immunized with a range of doses (1 μg, 5 μg, and 25 μg) of BV2373 and 50 μg of MATRIX-M™ adjuvant administered by intramuscular (IM) injection in two doses 21 days apart. To evaluate the adjuvant activity of MATRIX-M™ in non-human primates, another group of animals was immunized with 25 μg of BV2373 without MATRIX-M™. IgG antibody titers to protein S were detectable within 21 days after a single prime in animals immunized with BV2373 / MATRIX-M™ at all dose levels (GMT = 1249-19000). IgG antibody titers to protein S increased more than logarithmically (GMT = 33000-174000) within 1-2 weeks after booster immunization (days 28 and 35) at all dose levels (Fig. 26A).

[0374] Low levels of hACE2 receptor blocking antibodies were detected in animals after a single immunization with BV2373 (5 μg or 25 μg) and MATRIX-M™ (GMT = 22-37). Receptor blocking antibody titers increased significantly within one to two weeks after booster immunization in all BV2373 / MATRIX-M™ immunized groups (GMT = 150-600) (Fig. 26B). Virus neutralizing antibodies increased (GMT = 190-446) in all dose groups after a single immunization with BV2373 / MATRIX-M™. Animals immunized with 25 μg BV2373 alone produced no detectable antibodies that block S protein binding to hACE2 (Fig. 26C). Neutralizing titers increased 6-8-fold one week after the booster immunization (GMT = 1160-3846). Neutralizing titers increased an additional 25-38-fold after the second immunization (GMT = 6400-17000) (Fig. 26C).A significant correlation (p < 0.0001) was observed between IgG antibody levels to protein S and neutralizing antibody titers (Fig. 27). The immunogenicity of the adjuvanted vaccine in non-human primates is consistent with the results of Example 2 and further supports the role of MATRIX-M™ in promoting neutralizing antibody production and reducing the required dose.

[0375] PBMCs were collected 7 days after the second immunization (day 28) and the T cell response was measured using an ELISPOT assay. PBMCs from animals immunized with BV2373 (5 μg or 25 μg) and MATRIX-M™) had the highest number of IFN-γ-secreting cells, which was 5-fold higher than that of animals immunized with 25 μg BV2373 or BV2373 alone (1 μg) and MATRIX-M™) (Fig. 28). According to the ICCS analysis, immunization with BV2373 (5 μg) and MATRIX-M™ had the highest frequency of IFN-γ+, IL-2+, and TNF-α+ CD4+ T cells (Fig. 29A-C). This trend was also true for multifunctional CD4+ T cells that simultaneously produced at least two or three type 1 cytokines (Fig. 29D-E). Example 4 Structural characterization of nanoparticle vaccines based on the spike (S) protein polypeptide of the coronavirus

[0376] Transmission electron microscopy (TEM) and two-dimensional (2D) class averaging were used to determine the ultrastructure of BV2373. TEM images of negatively stained BV2373 at high magnification (67,000x and 100,000x) were characterized by the presence of particles corresponding to protein S homotrimers.

[0377] The automatic selection protocol (Lander GC et al. J Struct Biol. 166, 95–102 (2009); Sorzano CO et al., J Struct Biol. 148, 194–204 (2004)) was used to construct images using 2D class averaging. Two rounds of 2D class averaging of homotrimeric structures revealed triangular-shaped particles with a length of 15 nm and a width of 13 nm (Fig. 10, top left). Overlay of the recently discovered cryoEM structure of the SARS-CoV-2 spike protein (EMD ID: 21374) on the 2D image of BV2373 showed high agreement with the crown subunit S1 (NTD and RBD) and the stalk subunit S2 (Fig. 10, bottom left). The 2D images also showed a small protrusion protruding from the tip of the trimeric structure opposite the NTD / RBD crown (Fig. 10, top right). 2D class averaging using a larger rectangle revealed that these small protrusions form a connection between the S trimer and the amorphous structure (Fig. 10, bottom right).

[0378] Dynamic light scattering (DLS) showed that the wild-type CoV S protein had a Z-mean particle diameter of 69.53 nm, compared with the 2-fold smaller particle size of BV2365 (33.4 nm) and BV2373 (27.2 nm). The polydispersity index (PDI) indicated that the BV2365 and BV2373 particles were generally uniform in size, shape, and mass (PDI = 0.25-0.29) compared with the wild-type spike protein (PDI = 0.46) (Table 3). Table 3. Particle size and thermal stability of trimeric SARS-CoV-2 spike proteins

[0379] The thermal stability of S trimers was determined by differential scanning calorimetry (DSC). The thermal transition temperature of the wild-type CoV S protein (T max = 58.6°C) was similar to that of BV2365 and BV2373, for which T maxwere 61.3°C and 60.4°C, respectively (Table 3). Of greater significance was the 3- to 5-fold increase in the transition enthalpy required for unfolding of the BV2365 and BV2373 variants (ΔHcal = 466 kJ / mol and 732 kJ / mol, respectively) compared to the lower enthalpy required for unfolding the WT spike protein (ΔHcal = 153 kJ / mol). These results are consistent with the increased thermal stability of BV2365 and BV2373 compared to the WT spike protein (Table 3).

[0380] The stability of the CoV spike (S) protein polypeptide-based nanoparticle vaccines was evaluated using dynamic light scattering. Different pH, temperature, salt concentration, and protease concentration were used to compare the stability of the CoV spike (S) protein polypeptide-based nanoparticle vaccines with that of the native CoV spike (S) protein polypeptide-based nanoparticle vaccines. Example 5. Stability of Coronavirus Spike (S) Protein Polypeptide-based Nanoparticle Vaccines

[0381] The stability of nanoparticle vaccines based on the CoV spike (S) protein polypeptide was assessed using dynamic light scattering. To compare the stability of nanoparticle vaccines based on the CoV spike (S) protein polypeptide with that of nanoparticle vaccines containing the native CoV spike (S) protein polypeptide, different pH, temperature, salt, and protease concentrations were used. The stability of BV2365 without two proline substitutions and BV2373 with two proline substitutions were assessed under various environmental stress conditions using an hACE2-captured ELISA. Incubation of BV2373 at extreme pH (48 hours at pH 4 and pH 9), prolonged agitation (48 hours), and freezing / thawing (2 cycles) and elevated temperature (48 hours at 25°C and 37°C) had no effect on hACE2 receptor binding (IC50 = 14.0-18.3 ng ml -1 ).

[0382] Hydrogen peroxide oxidation conditions resulted in a decrease in binding, with hACE2 binding to BV2373 being 8-fold (IC50 = 120 ng ml -1 ) (Fig. 12A). BV2365 without the 2 proline substitutions was less stable, as determined by a significant loss of hACE2 binding under several conditions (Fig. 12B).

[0383] The stability of BV2384 (SEQ ID NO: 110) and BV2373 (SEQ ID NO: 87) was compared. BV2384 contains the furin cleavage site sequence GSAS (SEQ ID NO: 97), while BV2373 contains the furin cleavage site QQAQ (SEQ ID NO: 7). As demonstrated by SDS-PAGE and Western blot, BV2384 exhibited a significant level of cleavage compared with BV2373 (Fig. 32). Furthermore, scanning densitometry and recovery data demonstrate an unexpected loss of full-length S protein of CoV, BV2384, lower purity and recovery (Fig. 33) compared to BV2373 (Fig. 34).Example 6 Immune response in cynomolgus monkeys

[0384] The present inventors evaluated the immune response induced by BV2373 in a cynomolgus macaque model of SARS-CoV-2 infection. Groups 1-6 were treated as shown in Table 4. Table 4 Groups 1-6 from the cynomolgus macaque study

[0385] Administration of the vaccine containing BV2373 resulted in the induction of the production of antibodies to CoV-S (Fig. 35A), including neutralizing antibodies (Fig. 35B). Anti-CoV-S antibodies were induced after administration of one (Fig. 38A) or two doses (Fig. 38B) of BV2373. Administration of the BV2373-containing vaccine also resulted in the production of antibodies that blocked the binding of the CoV S protein to hACE2 (Figs. 38C, 38D). There was a significant correlation between the IgG antibody titer to the CoV S protein polypeptide and the titer providing hACE2 inhibition in cynomolgus monkeys after administration of BV2373 (Fig. 38E). The ability of BV2373 to induce neutralizing antibodies was assessed by the cytopathic effect (CPE) (Fig. 40A) and the plaque inhibition neutralization test (PRNT) (Fig. 40B). The data showed that the vaccine formulations from Table 4 resulted in to the formation of neutralizing titers of SARS-CoV-2, in contrast to the control.

[0386] The ability of the BV2373-containing vaccine to induce the production of antibodies to CoV-S and antibodies that block hACE2 binding to the CoV S protein in cynomolgus macaques was compared with that of the serum of convalescent individuals. The data showed that the BV2373-based vaccine formulation induced superior titers of antibodies to the CoV S protein polypeptide and titers that inhibit hACE2, compared with the serum of convalescent individuals (Fig. 39).

[0387] The BV2373-based vaccine formulation also reduced SARS-CoV-2 viral replication (Fig. 36A-B). Levels of viral RNA (Fig. 36A, corresponding to the total amount of RNA present) and viral subgenomic RNA (sgRNA) (Fig. 36B, corresponding to replicating virus) were assessed in bronchiolar lavage (BAL) at 2 days and 4 days after infection with infective virus (d2pi and d4pi). Most subjects demonstrated undetectable viral RNA. On day 2, small amounts of RNA were measurable in some subjects. By day 4, RNA levels were undetectable except in two subjects receiving the lowest dose of 2.5 μg. Subgenomic RNA was not detected at either 2 or 4 days, except in 1 subject, also receiving the lowest dose. Viral RNA (Fig. 37A) and viral subgenomic (sg) RNA (Fig. 37B) were assessed by nasal swab at 2 and 4 days postinfection (d2pi and d4pi).Viral RNA was undetectable in most subjects. Small amounts of RNA were measured in some subjects on days 2 and 4. Subgenomic RNA was undetectable after neither day 2 nor day 4. Subjects were immunized on day 0 and in two-dose groups on day 0 and day 21. These data demonstrate that the vaccine reduces total viral RNA levels in the nasal cavity by 100- to 1,000-fold and sgRNA to undetectable levels, and confirm that the immune response to the vaccine will block viral replication and prevent viral spread. Example 7: Evaluation of nanoparticle vaccines based on the CoV S protein polypeptide in humans.

[0388] The present inventors evaluated the safety and efficacy of a vaccine containing BV2373 in a randomized, placebo-controlled, observer-blinded Phase 1 clinical trial in 131 healthy participants aged 18-59 years. Participants were immunized with two intramuscular injections 21 days apart. Participants received BV2373 with or without MATRIX-M™ (n=106) or placebo (n=25). Groups A-E were treated as shown in Table 5. Figure 41 shows the clinical endpoint assessment graph. Table 5. Groups AE of the Phase 1 Human Study

[0389] Overall reactogenicity was weak, vaccinations were well tolerated. Local reactogenicity was more common in patients receiving BV2373 and MATRIX-M™ (Fig. 42A-B).

[0390] The immunogenicity of BV2373 with and without MATRIX-M™ was assessed. At 21 days post-vaccination, antibodies to CoV-S were detectable in all vaccination schedules (Fig. 43A). Geometric mean fold increases (GMFRs) in the MATRIX-M™-containing schedules were higher than those induced by BV2373 without adjuvant. Seven days post-second vaccination (day 28), anti-CoV-S antibody titers increased an additional eight-fold compared to the responses observed with the first vaccination, and by 14 days (day 35), responses had increased again by more than twofold, reaching GMFRs that were approximately 100-fold higher than those observed with BV2373 alone. A single vaccination with BV2373 / MATRIX-M™ achieved antibody titers to CoV-S similar to those in asymptomatic (exposed) COVID-19 patients.The second vaccination resulted in GMEU levels that were sixfold higher than those in the serum of recovering COVID-19 patients undergoing outpatient treatment, similar to those in the serum of recovering COVID-19 patients hospitalized with the disease, and nearly sixfold higher than the total antibody levels to CoV-S in the serum of recovering patients. Responses were similar in the two-dose regimens of 5 mcg and 25 mcg BV2373 / MATRIX-M™. This underscores the ability of the adjuvant (MATRIX-M™) to reduce the required dose.

[0391] Neutralizing antibody production was induced in all BV2373-treated groups (Fig. 43B). GMFRs were approximately fivefold higher in BV2373- and MATRIX-M™-based regimens than in BV2373-treated groups alone (Fig. 43B). Second vaccinations with adjuvant significantly impacted neutralizing antibody titers, inducing a >100-fold increase compared to single vaccinations without adjuvant. Compared with convalescent serum, second vaccinations with BV2373 / MATRIX-M™ resulted in GMT levels that were four times higher than those seen in outpatient COVID-19 patients, levels that overlapped those seen in hospitalized COVID-19 patients, and four times higher than the overall GMT level in convalescent serum.

[0392] Convalescent serum from COVID-19 patients with clinical symptoms requiring medical attention showed proportional IgG antibody titers to CoV-S and neutralization titers that increased with disease severity (Fig. 43A-B).

[0393] A significant correlation was observed between neutralizing antibody titers and IgG antibodies to CoV-S in patients treated with BV2373 and MATRIX-M™ (r=0.9466, Fig. 44C), similar to that observed in patients treated with convalescent serum (r=0.958) (Fig. 44A). This correlation was not observed in subjects administered BV2373 without adjuvant (r=0.7616) (Fig. 44B). The groups receiving both 5 mcg and 25 mcg BV2373 / MATRIX-M™ (CE groups Table 5) demonstrated similar magnitudes of response to the two doses, and with the two-dose regimen, every participant seroconverted using either assay.T cell responses in 16 participants (four participants from each group A through D) showed that BV2373 / MATRIX-M™ regimens induced antigen-specific polyfunctional CD4+ T cell responses in terms of IFN-γ, IL-2, and TNF-α production after stimulation with BV2373. There was a significant bias toward Th1 cytokine production (Fig. 45A-D). Example 8 Expression, Purification, and Evaluation of Next-Generation CoV S Protein Polypeptide-Based Nanoparticles.

[0394] CoV S protein polypeptides having the amino acid sequence shown in SEQ ID NO: 112, SEQ ID NO: 113, SEQ ID NO: 114, or SEQ ID NO: 115 are expressed in a baculovirus-based expression system, and recombinant plaques expressing coronavirus spike (S) protein polypeptides are selected and confirmed. CoV S protein polypeptides having the sequence shown in SEQ ID NO: 112, SEQ ID NO: 113, SEQ ID NO: 114, and SEQ ID NO: 115 are expressed using an N-terminal signal peptide having the amino acid sequence shown in SEQ ID NO: 5.

[0395] The CoV S protein polypeptide having the sequence of SEQ ID NO: 112 contains a mutation resulting in the substitution of Asn-488 with tyrosine, mutations resulting in the substitution of Lys-973 and Val-974 with proline, and an inactivated furin cleavage site having the amino acid sequence QQAQ (SEQ ID NO: 7).

[0396] The CoV S protein polypeptide having the sequence of SEQ ID NO: 113 has a mutation where Asp-601 is replaced by glycine, a mutation where Asn-488 is replaced by tyrosine, mutations where Lys-973 and Val-974 are replaced by proline, and an inactivated furin cleavage site having the amino acid sequence QQAQ (SEQ ID NO: 7).

[0397] The CoV S protein polypeptide having the sequence of SEQ ID NO: 114 comprises a deletion of amino acids 56, 57, and 131, a mutation causing Asn-488 to be replaced by tyrosine, a mutation causing Ala-557 to be replaced by aspartate, a mutation causing Asp-601 to be replaced by glycine, a mutation causing Pro-668 to be replaced by histidine, a mutation causing Thr-703 to be replaced by isoleucine, a mutation causing Ser-969 to be replaced by alanine, a mutation causing Asp-1105 to be replaced by histidine, mutations causing Lys-973 and Val-974 to be replaced by proline, and an inactivated furin cleavage site having the amino acid sequence QQAQ (SEQ ID NO: 7).

[0398] The CoV S protein polypeptide having the sequence of SEQ ID NO: 115 comprises a mutation where Asp-488 is replaced by tyrosine, a mutation where Asp-67 is replaced by alanine, a mutation where Leu-229 is replaced by histidine, a mutation where Asp-202 is replaced by glycine, a mutation where Lys-404 is replaced by asparagine, a mutation where Glu-471 is replaced by lysine, a mutation where Ala-688 is replaced by valine, a mutation where Asp-601 is replaced by glycine, mutations where Lys-973 and Val-974 are replaced by proline, and an inactivated furin cleavage site having the amino acid sequence QQAQ.

[0399] Nanoparticles based on the CoV S protein polypeptide were obtained as in Example 1. The stability and immunogenicity of the CoV S protein polypeptides characterized by the amino acid sequence of SEQ ID NO: 112, SEQ ID NO: 112, SEQ ID NO: 113, and SEQ ID NO: 115 are evaluated in Examples 2-7. Example 9BV2373 and a saponin adjuvant induce protective immune responses against heterogeneous SARS-CoV-2 strains

[0400] Aim. We conducted a phase 3, randomized, placebo-controlled, observer-blinded study in adults aged 18-84 years who received two intramuscular doses of 5 μg BV2373 and a saponin adjuvant (iscom-Matrix with Fraction A and Fraction C, also referred to in this example as MATRIX-M™) or placebo (1:1) 21 days apart at 33 study sites in the United Kingdom. The primary efficacy endpoint was virologically confirmed mild, moderate, or severe COVID-19 with disease onset 7 days after the second vaccination.

[0401] A total of 15,187 participants were randomized, of whom 7,569 received BV2373 and MATRIX-M™ and 7,570 received placebo; 27.8% were 65 years or older, and 4% had serological evidence of SARS-CoV-2 infection at baseline. There were 10 cases of COVID-19 among BV2373 and MATRIX-M™ recipients and 96 cases among placebo recipients, with symptom onset at least 7 days after the second vaccination; BV2373 and MATRIX-M™ had an efficacy of 89.7% (95% CI 80.2-94.6) in preventing COVID-19. There were five cases of severe COVID-19, all of which occurred in the placebo group. Post-hoc analyses revealed efficacy rates against the prototype SARS-CoV-2 strain and the B.1.1.7 variant of 96.4% (73.8%–99.5%) and 86.3% (71.3%–93.5%), respectively.The prototype SARS-CoV-2 strain contains the CoV S protein with the amino acid sequence SEQ ID NO: 2. Variant B.1.1.7 contains the CoV S protein with deletions at amino acids 56, 57, and 131 and mutations N488Y, A557D, D601G, P668H, T703I, S969A, and D1105H, where the CoV S protein polypeptide is numbered relative to the wild-type SARS-CoV-2 S protein polypeptide with the amino acid sequence SEQ ID NO: 2. Vaccine efficacy was similar across all subgroups, including participants with comorbidities and individuals aged ≥65 years. Reactogenicity was generally mild and transient and was observed more frequently in the BV2373 and MATRIX-M™ group. The incidence of serious adverse events was low and similar in both groups. The two-dose regimen using BV2373 and MATRIX-M™ demonstrated efficacy against a mixture of the prototype and the B.1.1 variant.7, accounting for 89.7%, with a safety profile similar to that of other registered COVID-19 vaccines.

[0402] Methods. Trial Design and Participants. We evaluated the safety and efficacy of two 5 mcg doses of BV2373 and MATRIX-M™ or placebo administered intramuscularly 21 days apart. This Phase 3 study was conducted at 33 recruiting sites in the United Kingdom. Eligible participants were men and non-pregnant women aged 18 to 84 years (inclusive) who were healthy or had stable chronic medical conditions, including but not limited to human immunodeficiency virus infection and cardiac and respiratory disease. Health status assessed at screening was based on medical history, vital signs, and physical examination. Key exclusion criteria included a confirmed history of COVID-19, treatment with immunosuppressive therapy, or a diagnosis of an immunocompromised state.

[0403] Participants were randomly assigned in a 1:1 ratio using block randomization to receive two doses of BV2373 and MATRIX-M™ or placebo (saline) 21 days apart using an interactive response system according to predefined randomization schedules. Randomization was stratified by study center and age ≥65 years. In an additional 400-person study, participants received a concomitant dose of seasonal influenza vaccine with the first dose. This was an observer-blinded study.

[0404] After each vaccination, participants remained under observation at the study center for at least 30 minutes to monitor for any acute reactions. Data on reportable local and systemic adverse events were collected via an electronic diary for 7 days after each dose in a subset of participants (reportable adverse event analysis subset). All participants were assessed for reportable adverse events other than reportable ones from the first dose until 28 days after the second dose; serious adverse events, adverse events of special interest, and adverse events requiring medical intervention were assessed from the first dose until 1 year after the second dose. Safety data are presented for all participants who received at least one dose of vaccine or placebo.

[0405] Safety and Efficacy. The primary endpoint was the efficacy of BV2373 and MATRIX-M™ against the first case of virologically confirmed symptomatic COVID-19 (mild, moderate, or severe) with onset at least 7 days after the second vaccination in participants who were seronegative at baseline. Symptomatic COVID-19 was defined according to the U.S. Food and Drug Administration (FDA) criteria.

[0406] Suspected COVID-19 symptoms were monitored throughout the study and collected using an electronic COVID-19 symptom diary (InFLUenza Patient-Reported Outcomes Questionnaire [FLU-PRO©]) for at least 10 days. At the onset of suspected COVID-19 symptoms, nasal and pharyngeal specimens were collected daily for 3 days to confirm SARS-CoV-2 infection. Virological confirmation was performed using a polymerase chain reaction (PCR) test (UK DHSC laboratories) using the Thermo TaqPath™ system (Thermo Fisher Scientific, Waltham, MA, USA).

[0407] Safety was analyzed in all participants who received at least one dose of BV2373 and MATRIX-M™ or placebo, and summarized data were presented narratively. Summary data on local and systemic adverse events requested for reporting were also presented using FDA toxicity classification criteria and with the duration after each injection. Adverse events other than those requested for reporting were coded based on the preferred term and system organ class using the Medical Dictionary for Regulatory Activities (MedDRA) version 23.1, and summarized data were presented as severity and relationship to the study vaccine.

[0408] The study was designed and based on the number of events expected to achieve statistical significance for the primary endpoint, corresponding to a target of 100 cases of mild, moderate, or severe Covid-19. The target number of 100 cases for the final analysis was chosen to provide a power of greater than 95% with a vaccine efficacy of 70% or higher. A single interim efficacy analysis was performed based on accumulating approximately 50% (50 events) of the total number of expected primary endpoints using Pocock boundary conditions. The main (hypothesis testing) event-driven analysis for the interim and final analyses of the primary objective was performed with an overall one-sided Type I error rate of 0.025 for the primary endpoint.The primary endpoint was analyzed in participants who were seronegative at baseline, received both doses of the study vaccine or placebo, had no major protocol deviations affecting the primary endpoint, and had no confirmed cases of symptomatic Covid-19 within 6 days of the second injection (the per-protocol efficacy population). Vaccine efficacy was defined as E (%) = (1 - RR) × 100, where RR = relative risk for the incidence rate when comparing the two study groups (BV2373 and MATRIX-M™ or placebo). The mean incidence rate was expressed as the incidence rate per year per 1,000 people. The estimated RR and its confidence interval (CI) were obtained using Poisson regression with robust error variance.Hypothesis testing for the primary endpoint was conducted against the null hypothesis: H0 - vaccine efficacy ≤30%. The success criterion required rejection of the null hypothesis to demonstrate statistically significant vaccine efficacy.

[0409] From September 28 to November 28, 2020, a total of 16,645 participants were screened and 15,187 participants were randomized (Fig. 47). A total of 15,139 participants received at least one dose of BV2373 and MATRIX-M™ (7,569) or placebo (7,570), with 14,039 participants (7,020 in the BV2373 and MATRIX-M™ group and 7,019 in the placebo group) meeting criteria for the per-protocol efficacy population. Demographics at baseline were well distributed between the BV2373 and MATRIX-M™ and placebo groups in the per-protocol efficacy population, with 48.4% being female, 94.5% being Caucasian, 0.4% being Black or African American, 0.8% being Hispanic or Latino, and 44.6% having at least one comorbid condition (as defined by the Centers for Disease Control and Prevention diseases, [CDC]).The median age of these participants was 56 years, and 27.9% were ≥65 years old. Table 6 provides a summary of the demographic characteristics of the clinical trial participants at baseline.Table 6. Demographic data and characteristics at baseline of clinical trial participants.

[0410] SD - standard deviation; body mass index (BMI) is calculated as weight (kg) divided by height squared (m). Percentages are based on per-protocol analysis of efficacy data within each treatment and overall. *Subjects with comorbidities are identified individuals with a history of at least one comorbidity or a BMI at screening greater than 30 kg / m 2 .

[0411] The subset for the analysis of reportable adverse events included 2714 participants. Overall, BV2373 and MATRIX-M™ recipients had a higher rate of reportable local adverse events than placebo recipients after both the first dose (59.4% and 20.9%) and the second dose (80.2% and 17.0%) (Fig. 50).

[0412] Among BV2373 and MATRIX-M™ recipients, the most commonly reported local adverse events were injection site tenderness and pain after both the first dose (54.9% and 30.7%) and the second dose (76.6% and 51.9%), with most events rated as Grade 1 (mild) or Grade 2 (moderate) in severity and having a short median duration (2.3 and 1.7 days after the first dose and 2.8 and 2.2 days after the second dose). Local adverse events requested for reporting were more frequently reported among younger BV2373 and MATRIX-M™ recipients (18 to 64 years) than among older BV2373 and MATRIX-M™ recipients (≥65 years).

[0413] Overall, BV2373 and MATRIX-M™ recipients had a higher rate of systemic adverse events requested for reporting than placebo recipients after both the first dose (47.6% and 37.9%) and the second dose (64.6% and 30.8%) (Fig. 50). Among BV2373 and MATRIX-M™ recipients, the most commonly reported systemic adverse events were headache, muscle pain, and fatigue after both the first dose (24.5%, 22.3%, and 20.5%) and the second dose (40.7%, 41.1%, and 41.0%), with most events being Grade 1 or Grade 2 in severity and having a short median duration (1.6, 1.5, and 1.9 days after the first dose and 1.9, 1.8, and 1.9 days after the second dose). Grade 4 systemic adverse events were reported in two participants in the BV2373 and MATRIX-M™ group after the first dose and in one participant in the BV2373 and MATRIX-M™ group after the second dose.Systemic adverse events were reported more frequently in younger vaccine recipients than in older recipients and more frequently after Dose 2 than after Dose 1. Notably, fever (temperature ≥38°C) was reported in 2.3% and 5.1% of participants in the BV2373 and MATRIX-M™ groups after the first and second doses, with Grade 3 fever (39-40°C) occurring in 0.4% and 0.6% of participants after the first and second doses, respectively; one case of Grade 4 fever (>40°C) was reported after each vaccine dose.

[0414] All 15,139 participants who received at least one dose of vaccine or placebo before the data cutoff date for the final efficacy analysis were assessed for reportable adverse events. The incidence of non-reportable adverse events was higher in BV2373 and MATRIX-M™ recipients than in placebo recipients (25.3% and 20.5%), with similar rates of severe adverse events (1.0% and 0.8%), serious adverse events (0.5% and 0.5%), adverse events requiring medical intervention (3.8% and 3.9%), adverse events leading to discontinuation of vaccination (0.3% and 0.3% or study discontinuation (0.2% and 0.2%), potential immune-mediated m...

Claims

1. A coronavirus (CoV) spike (S) glycoprotein for stimulating an immune response against SARS-CoV-2 or a heterogeneous strain of SARS-CoV-2 in a subject, comprising (i) an S1 subunit with an inactivated furin cleavage site, wherein the S1 subunit comprises an N-terminal domain (NTD), a receptor binding domain (RBD), subdomains 1 and 2 (SD1 / 2), and the inactivated furin cleavage site is characterized by the amino acid sequence QQAQ; wherein the NTD optionally provides for one or more modifications selected from the group consisting of (a) deletions of one or more amino acids selected from the group consisting of amino acids 56, 57, 131, 132, 144, 145, 228, 229, 230, 231, 234, 235, 236, 237, 238, 239, 240, and combinations thereof; (b) insertions of 1, 2, 3, or 4 amino acids after amino acid 132 and (c) a mutation affecting one or more amino acids selected from the group consisting of amino acids 5, 6, 7, 13, 51, 53, 56, 57, 62, 63, 67, 82, 125, 129, 131, 132, 133, 139, 143, 144, 145, 177, 200, 201, 202, 209, 229, 233, 240, 245, and combinations thereof; wherein the RBD optionally provides for a mutation affecting one or more amino acids selected from the group consisting of amino acids 333, 404, 419, 426, 439, 440, 464, 465, 471, 477, 481, 488 and combinations thereof; wherein the SD1 / 2 domain optionally comprises a mutation affecting one or more amino acids selected from the group consisting of 557, 600, 601, 642, 664, 668 and combinations thereof; and (ii) the S2 subunit, where amino acids 973 and 974 are represented by proline, wherein the S2 subunit optionally comprises one or more modifications selected from the group consisting of (a) deletions of one or more amino acids from amino acids 676-685, 676-702, 702-711, 775-793, 806-815 and combinations thereof; (b) a mutation affecting one or more amino acids selected from the group consisting of 688, 703, 846, 875, 937, 969, 1014, 1058, 1105 and 1163 and combinations thereof; and (c) deletions of one or more amino acids from TMCT; wherein the amino acids of the S CoV glycoprotein are numbered relative to the polypeptide characterized by the sequence under SEQ ID NO:

2.

2. The S glycoprotein of coronavirus according to paragraph 1, which provides for the deletion of amino acids 676-685.

3. The S glycoprotein of coronavirus according to paragraph 1, which provides for the deletion of amino acids 702-711.

4. The S glycoprotein of coronavirus according to paragraph 1, which provides for the deletion of amino acids 806-815.

5. The S glycoprotein of coronavirus according to paragraph 1, which provides for the deletion of amino acids 775-793.

6. The S glycoprotein of coronavirus according to claim 1, which provides for the deletion of amino acids 1-292 NTD.

7. The S glycoprotein of coronavirus according to claim 1, which provides for the deletion of amino acids 1201-1260 of the transmembrane region and the cytoplasmic “tail” (TMCT).

8. The coronavirus S glycoprotein according to claim 1, comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 85-89, 105, 106 and 112-115, 164-168, or consisting of it.

9. The S glycoprotein of coronavirus according to any one of claims 1-8, comprising a signal peptide, wherein optionally the signal peptide comprises the amino acid sequence of SEQ ID NO: 5 or SEQ ID NO:

117.

10. The S glycoprotein of coronavirus according to any one of claims 1 to 9, comprising a C-terminal fusion protein.

11. The coronavirus S glycoprotein according to claim 10, wherein the C-terminal fusion protein is a hexahistidine tag.

12. The coronavirus S glycoprotein according to claim 10, wherein the C-terminal fusion protein is a foldon.

13. The coronavirus S glycoprotein according to claim 12, wherein the foldon is characterized by an amino acid sequence corresponding to SEQ ID NO:

68.

14. The coronavirus S glycoprotein according to any one of claims 1-13, wherein the ΔHcal is at least 2 times higher than the ΔHcal of the wild-type CoV S glycoprotein (SEQ ID NO: 2).

15. The S glycoprotein of coronavirus according to any one of paragraphs 1-14, where each of subunits S2, NTD, RBD and SD1 / 2 is 95% identical to the corresponding subunit or domain of the CoV S glycoprotein, characterized by the amino acid sequence under SEQ ID NO:

2.

16. The S glycoprotein of coronavirus according to any one of paragraphs 1-14, where each of subunits S2, NTD, RBD and SD1 / 2 is 97% identical to the corresponding subunit or domain of the CoV S glycoprotein, characterized by the amino acid sequence under SEQ ID NO:

2.

17. The S glycoprotein of coronavirus according to any one of paragraphs 1-14, where each of subunits S2, NTD, RBD and SD1 / 2 is 99% identical to the corresponding subunit or domain of the CoV S glycoprotein, characterized by the amino acid sequence under SEQ ID NO:

2.

18. The S glycoprotein of coronavirus according to any one of paragraphs 1-14, where each of subunits S2, NTD, RBD and SD1 / 2 is 99.5% identical to the corresponding subunit or domain of the CoV S glycoprotein, characterized by the amino acid sequence under SEQ ID NO:

2.

19. An isolated nucleic acid encoding the S glycoprotein according to any one of claims 1-18.

20. An expression vector containing the nucleic acid of claim 19.

21. A nanoparticle for stimulating an immune response against SARS-CoV-2 or a heterogeneous strain of SARS-CoV-2 in a subject, comprising the S glycoprotein of the coronavirus according to any one of claims 1-18.

22. The nanoparticle of claim 21, wherein the nanoparticle is characterized by a Z-average diameter of from 18 nm to 38.5 nm.

23. A nanoparticle according to claim 21, wherein the nanoparticle is characterized by a polydispersity index ranging from 0.18 to 0.

495.

24. A cell containing the expression vector of claim 20 and expressing the S glycoprotein of the coronavirus of any one of claims 1-18.

25. A vaccine composition comprising the S glycoprotein of the coronavirus according to any one of claims 1-18 and a pharmaceutically acceptable buffer.

26. A vaccine composition according to claim 25, containing an adjuvant.

27. The vaccine composition according to claim 26, wherein the adjuvant comprises at least two iscom particles, wherein the first iscom particle contains the fraction AQuillaja SaponariaMolina and does not contain the fraction CQuillaja SaponariaMolina; and The second iscom particle contains the fraction CQuillaja SaponariaMolina and does not contain the fraction AQuillaja SaponariaMolina.

28. The vaccine composition according to claim 27, wherein the fraction AQuillaja Saponaria Molina and the fraction CQuillaja Saponaria Molina constitute 76.5-93.5% by weight and 13.5-16.5% by weight, respectively, of the sum of the weight values ​​of the fraction AQuillaja Saponaria Molina and the fraction CQuillaja Saponaria Molina in the adjuvant.

29. The vaccine composition according to claim 27, wherein the fraction AQuillaja Saponaria Molina and the fraction CQuillaja Saponaria Molina constitute 92% by weight and 8% by weight, respectively, of the sum of the weight values ​​of the fraction AQuillaja Saponaria Molina and the fraction CQuillaja Saponaria Molina in the adjuvant.

30. The vaccine composition according to claim 26, wherein the adjuvant is administered in a dose of 45-55 mcg.

31. A method for stimulating an immune response against SARS-CoV-2 in a subject, comprising administering a vaccine composition according to any one of claims 25-30.

32. The method of claim 31, wherein the subject is administered a first dose on day 0 and a booster dose on day 21.

33. The method according to claim 31, wherein the subject is administered from 2.7 μg to 27.5 μg of the S glycoprotein of the coronavirus.

34. The method according to claim 31, wherein the subject is administered 4.5-5.5 μg of coronavirus S glycoprotein.

35. The method according to any one of paragraphs 31-34, wherein the vaccine composition is administered intramuscularly.

36. The method according to any one of claims 31, 33, 34 and 35, wherein a single dose of the vaccine composition is administered.

37. The method according to any one of paragraphs 31-35, wherein several doses of the vaccine composition are administered.

38. The method according to any one of claims 31-37, wherein the vaccine composition is administered together with an influenza virus glycoprotein.

39. An immunogenic composition containing (i) a nanoparticle comprising the S glycoprotein of coronavirus (S CoV) according to any one of claims 1-18 and a core represented by a non-ionic detergent; (ii) a pharmaceutically acceptable buffer and (iii) saponin adjuvant.

40. An immunogenic composition according to claim 39, containing from 2.7 μg to 27.5 μg of CoV S glycoprotein.

41. An immunogenic composition according to claim 40, containing 4.5-5.5 μg of CoV S glycoprotein.

42. The immunogenic composition according to claim 39, wherein the saponin adjuvant comprises at least two iscom particles, wherein the first iscom particle contains the fraction AQuillaja SaponariaMolina and does not contain the fraction CQuillaja SaponariaMolina; and The second iscom particle contains the fraction CQuillaja SaponariaMolina and does not contain the fraction AQuillaja SaponariaMolina.

43. The immunogenic composition according to claim 42, wherein the fraction AQuillaja Saponaria Molina constitutes 50-96% by weight and the fraction CQuillaja Saponaria Molina constitutes the remaining amount, respectively, of the sum of the weight values ​​of the fraction AQuillaja Saponaria Molina and the fraction CQuillaja Saponaria Molina in the adjuvant.

44. The immunogenic composition according to claim 42, wherein the AQuillaja Saponaria Molina fraction and the CQuillaja Saponaria Molina fraction constitute 76.5-93.5% by weight and 13.5-16.5% by weight, respectively, of the sum of the weight values ​​of the AQuillaja Saponaria Molina fraction and the CQuillaja Saponaria Molina fraction in the adjuvant.

45. An immunogenic composition according to claim 39, containing 45-55 μg of a saponin adjuvant.

46. ​​The immunogenic composition of claim 39, wherein the non-ionic detergent is selected from the group consisting of polysorbate-20 (PS20), polysorbate-40 (PS40), polysorbate-60 (PS60), polysorbate-65 (PS65) and polysorbate-80 (PS80).

47. A method for stimulating an immune response against SARS-CoV-2 or a heterogeneous strain of SARS-CoV-2 in a subject, comprising administering an immunogenic composition according to any one of paragraphs 39-46.

48. The method according to claim 47, wherein from 2.7 μg to 27.5 μg of CoV S glycoprotein is administered.

49. The method according to claim 47 or 48, wherein 4.5-5.5 μg of CoV S glycoprotein is administered.

50. The method according to any one of claims 47-49, wherein the saponin adjuvant comprises at least two iscom particles, wherein the first iscom particle contains the fraction AQuillaja SaponariaMolina and does not contain the fraction CQuillaja SaponariaMolina; and The second iscom particle contains the fraction CQuillaja SaponariaMolina and does not contain the fraction AQuillaja SaponariaMolina.

51. The method according to claim 50, wherein the fraction AQuillaja Saponaria Molina constitutes 50-96% by weight and the fraction CQuillaja Saponaria Molina constitutes the remaining amount, respectively, of the sum of the weight values ​​of the fraction AQuillaja Saponaria Molina and the fraction CQuillaja Saponaria Molina in the adjuvant.

52. The method according to claim 50 or 51, wherein the fraction AQuillaja SaponariaMolina and the fraction CQuillaja SaponariaMolina constitute 76.5-93.5% by weight and 13.5-16.5% by weight, respectively, of the sum of the weight values ​​of the fraction AQuillaja SaponariaMolina and the fraction CQuillaja SaponariaMolina in the adjuvant.

53. The method according to any one of paragraphs 47-52, comprising administering 45-55 μg of a saponin adjuvant.

54. The method according to any one of claims 47-53, wherein the non-ionic detergent is selected from the group consisting of polysorbate-20 (PS20), polysorbate-40 (PS40), polysorbate-60 (PS60), polysorbate-65 (PS65) and polysorbate-80 (PS80).

55. The method of any one of paragraphs 47-54, wherein the subject is administered the first dose on day 0 and a booster dose on day 21.

56. The method according to any one of paragraphs 47-55, wherein a single dose of the immunogenic composition is administered.

57. The method of any one of claims 47-56, further comprising administering a second immunogenic composition different from the immunogenic composition of claims 39-46.

58. The method according to claim 57, wherein the second immunogenic composition comprises mRNA encoding the SARS-Cov-2 spike glycoprotein, plasmid DNA encoding the SARS-Cov-2 spike glycoprotein, a viral vector encoding the SARS-Cov-2 spike glycoprotein, or an inactivated SARS-CoV-2 virus.

59. The method according to any one of paragraphs. 47-58, wherein the heterogeneous strain of SARS-CoV-2 is selected from the group consisting of the SARS-CoV-2 strain B.1.1.7, the SARS-CoV-2 strain B.1.351, the SARS-CoV-2 strain P.1, the SARS-CoV-2 strain B.1.617.2, the SARS-CoV-2 strain B.1.525, the SARS-CoV-2 strain B.1.526, the SARS-CoV-2 strain B.1.617.1, the SARS-CoV-2 strain C.37, the SARS-CoV-2 strain B.1.621, and the SARS-CoV-2 strain Cal.20C.

60. The method according to any of paragraphs. 47-59, wherein the efficacy of the immunogenic composition for preventing coronavirus disease 19 (COVID-19) is 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 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% over a period of up to 2 months, up to 2.5 months, up to 3 months, up to 3.5 months, up to 4 months, up to 4.5 months, up to 5 months, up to 5.5 months, up to 6 months, up to 6.5 months, up to 7 months, up to 7.5 months, up to 8 months, up to 8.5 months, up to 9 months, up to 9.5 months, up to 10 months, up to 10.5 months, up to 11 months, up to 11.5 months or up to 12 months after administration of the immunogenic composition.

61. The method according to any of paragraphs. 47-60, wherein the efficacy of the immunogenic composition for preventing coronavirus disease 19 (COVID-19) is from 50% to 99%, from 50% to 95%, from 50% to 90%, from 50% to 85%, from 50% to 80%, from 60% to 99%, from 60% to 95%, from 60% to 90%, from 60% to 85%, from 60% to 80%, from 40% to 99%, from 40% to 95%, from 40% to 90%, from 40% to 85%, from 40% to 80%, from 40% to 75%, from 40% to 70%, from 40% to 65%, from 40% to 55%, or from 40% to 50% over a period of up to 2 months, up to 2.5 months, up to 3 months, up to 3.5 months, up to 4 months, up to 4.5 months, up to 5 months, up to 5.5 months, up to 6 months, up to 6.5 months, up to 7 months, up to 7.5 months, up to 8 months, up to 8.5 months, up to 9 months, up to 9.5 months, up to 10 months, up to 10.5 months, up to 11 months, up to 11.5 months, or up to 12 months after administration of the immunogenic composition.

62. The method according to any one of paragraphs 47-61, wherein COVID-19 is mild COVID-19.

63. The method according to any one of paragraphs 47-61, wherein COVID-19 is moderate COVID-19.

64. The method according to any one of paragraphs 47-61, wherein COVID-19 is severe COVID-19.

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