Coronavirus vaccine formulations

Abstract

To provide non-naturally occurring coronavirus (CoV) spike (S) polypeptides and nanoparticles, and vaccines comprising the same, which are useful for stimulating an immune response.SOLUTION: Disclosed herein are coronavirus spike (S) proteins and nanoparticles comprising the same that are suitable for use in vaccines. The nanoparticles surround the surfactant core and present antigens from the pathogen associated with it, resulting in improved stability and better immunogenicity. Doses, formulations and methods for preparing the vaccines and nanoparticles are also disclosed.SELECTED DRAWING: None

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application is related to U.S. Provisional Patent Application No. 62 / 966,271, filed January 27, 2020; U.S. Provisional Patent Application No. 62 / 976,858, filed February 14, 2020; U.S. Provisional Patent Application No. 62 / 983,180, filed February 28, 2020; U.S. Provisional Patent Application No. 62 / 983,180, filed July 7, 2020; and U.S. Provisional Patent Application No. 62 / 983,180, filed July 7, 2020, each of which is incorporated by reference in its entirety for all purposes. This application claims priority to U.S. Provisional Patent Application No. 3 / 048,945, filed July 14, 2020; U.S. Provisional Patent Application No. 63 / 051,706, filed July 20, 2020; U.S. Provisional Patent Application No. 63 / 054,182, filed July 20, 2020; U.S. Provisional Patent Application No. 63 / 129,392, filed December 22, 2020; and U.S. Provisional Patent Application No. 16 / 997,001, filed August 19, 2020. Instructions for electronically submitted text files

[0002] The contents of the text file submitted electronically herewith are incorporated herein by reference in their entirety: Computer-readable copy of the Sequence Listing (Filename: NOVV_088_08WO_SeqList_ST25.txt, Recording Date: January 26, 2021, File Size: 577 kilobytes). Field

[0003] The present disclosure generally relates to non-naturally occurring coronavirus (CoV) spike (S) polypeptides and nanoparticles, and vaccines comprising the same, that are useful for stimulating an immune response. The nanoparticles, optionally associated with a surfactant core, provide antigens, e.g., glycoprotein antigens, typically produced using recombinant approaches. The nanoparticles have improved stability and enhanced epitope presentation. The disclosure also provides compositions comprising the nanoparticles, methods for making them, and methods for stimulating an immune response. [Background technology]

[0002] Background of the Invention

[0004] Infectious diseases remain a problem worldwide. While progress has been made in developing vaccines against some pathogens, many remain a threat to human health. The outbreak of sudden acute respiratory syndrome coronavirus 2 (SARS-CoV-2) (also known as Wuhan coronavirus and SARS-CoV-2) has infected over 2,000 people in China and killed at least 17. Recently, the SARS-CoV-2 coronavirus has spread to the United States, Thailand, South Korea, Taiwan, and Japan. SARS-CoV-2 coronavirus belongs to the same viral family 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. Summary of the Invention [Problem to be solved by the invention]

[0003]

[0005] The development of vaccines to prevent or reduce the severity of life-threatening infectious diseases such as the SARS-CoV-2 coronavirus is desirable. However, human vaccine development remains a challenge due to the highly sophisticated evasion mechanisms of pathogens and the difficulty of stabilizing vaccines. Optionally, vaccines must induce antibodies that block or neutralize the infectious agent and must remain stable in a variety of environments, including those where refrigeration is not possible. [Means for solving the problem]

[0004] Summary of the Invention

[0006] The present disclosure provides non-naturally occurring CoV S polypeptides suitable for eliciting an immune response against SARS-CoV-2 (also known as Wuhan CoV and 2019-nCoV). The present disclosure also provides nanoparticles comprising the glycoprotein and methods for stimulating an immune response.

[0005]

[0007] The present disclosure also provides CoV S polypeptides suitable for eliciting an immune response against multiple coronaviruses, including SARS-CoV-2, Middle East Respiratory Syndrome (MERS), and Severe Acute Respiratory Syndrome (SARS). BRIEF DESCRIPTION OF THE DRAWINGS

[0008] The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee. [Brief explanation of the drawings]

[0006] [Figure 1]

[0009] A schematic diagram of the wild-type amino acid sequence of the SARS-CoV-2 spike (S) protein (SEQ ID NO: 1) is shown. The furin cleavage site RRAR (SEQ ID NO: 6) is highlighted in bold and the signal peptide is underlined. [Figure 2]

[0010] 1 shows the primary structure of the SARS-CoV-2 S polypeptide, which contains 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 CoV S polypeptide from SARS-CoV-2, including the signal peptide (SEQ ID NO: 1). [Figure 3]

[0011] Figure 1 shows the primary structure of the BV2378 CoV S polypeptide, which contains an inactive furin cleavage site, a deletion of the fusion peptide from amino acids 819 to 828, and the K986P and V987P mutations. Domain positions are numbered relative to the amino acid sequence of the wild-type CoV S polypeptide from SARS-CoV-2, including the signal peptide (SEQ ID NO: 1). [Figure 4]

[0012] Figure 1 shows the purification of CoV S polypeptides BV2364, BV2365, BV2366, BV2367, BV2368, BV2369, BV2373, BV2374, and BV2375. The data demonstrate that BV2365 (SEQ ID NO: 4) and BV2373 (SEQ ID NO: 87), which contains an inactive furin cleavage site with the amino acid sequence QQAQ (SEQ ID NO: 7), are expressed as single chains (S0). In contrast, CoV S polypeptides containing an intact furin cleavage site (e.g., BV2364, BV2366, and BV2374) are cleaved as evidenced by the presence of cleavage product S2. [Figure 5]

[0013] We demonstrate by biolayer interferometry that CoV S polypeptides BV2361, BV2365, BV2369, BV2365, BV2373 and BV2374 bind to human angiotensin-converting enzyme 2 precursor (hACE2). [Figure 6]

[0014] Biolayer interferometry demonstrates that SARS-CoV-2-derived BV2361 does not bind to the MERS-CoV receptor dipeptidyl peptidase IV (DPP4), and that the MERS S protein does not bind to human angiotensin-converting enzyme 2 precursor (hACE2). [Figure 7]

[0015] We demonstrate that BV2361 binds to hACE2 by enzyme-linked immunosorbent assay (ELISA). [Figure 8]

[0016] The primary structure of the BV2373 CoV S polypeptide and modifications to the furin cleavage site, K986P and V987P, are shown. [Figure 9]

[0017] Purification of wild-type CoV S polypeptide and CoV S polypeptides BV2365 and BV2373 is shown. [Figure 10]

[0018] Shown is the cryo-electron microscopy (cryoEM) structure of the BV2373CoV S polypeptide superimposed on the cryoEM structure of the SARS-CoV-2 spike protein (EMB ID: 21374). [Figure 11A]

[0019] Figure 11 shows that CoV S spike polypeptides BV2365 and BV2373 bind to hACE2. Biolayer interferometry reveals that BV2365 (Figure 11B) and BV2373 (Figure 11C) bind to hACE2 with dissociation rates similar to those of the wild-type CoV S polypeptide (Figure 11A). [Figure 11B]

[0019] We show that the CoV S spike polypeptide BV2365 binds to hACE2. Biolayer interferometry reveals that BV2365 binds to hACE2 with a dissociation rate similar to that of the wild-type CoV S polypeptide (Fig. 11A). [Figure 11C]

[0019] We show that the CoV S spike polypeptide BV2373 binds to hACE2. Biolayer interferometry reveals that BV2373 binds to hACE2 with a dissociation rate similar to that of the wild-type CoV S polypeptide (Fig. 11A). [Figure 11D]

[0019] CoV S spike polypeptides BV2365 and BV2373 bind to hACE2. ELISA shows that wild-type CoV S polypeptide (Fig. 11D) and BV2365 (Fig. 11E) bind to hACE2 with similar affinity, but BV2373 (Fig. 11F) binds to hACE2 with higher affinity. [Figure 11E]

[0019] CoV S spike polypeptides BV2365 and BV2373 bind to hACE2. ELISA shows that wild-type CoV S polypeptide (Fig. 11D) and BV2365 (Fig. 11E) bind to hACE2 with similar affinity, but BV2373 (Fig. 11F) binds to hACE2 with higher affinity. [Figure 11F]

[0019] CoV S spike polypeptides BV2365 and BV2373 bind to hACE2. ELISA shows that wild-type CoV S polypeptide (Fig. 11D) and BV2365 (Fig. 11E) bind to hACE2 with similar affinity, but BV2373 (Fig. 11F) binds to hACE2 with higher affinity. [Figure 12A]

[0020] Figure 1 shows the effect of stress conditions such as temperature, two freeze / thaw cycles, oxidation, agitation and extreme pH on the binding of CoV S polypeptide BV2373 to hACE2. [Figure 12B]

[0020] Figure 1 shows the effect of stress conditions such as temperature, two freeze / thaw cycles, oxidation, agitation and extreme pH on the binding of CoV S polypeptide BV2365 to hACE2. [Figure 13A]

[0021] Figure 1 shows anti-CoV S polypeptide IgG titers 13, 21, and 28 days after immunization of mice with two doses of 0.1 μg to 10 μg of BV2373 with or without Fraction A and Fraction C iscom matrix (e.g., MATRIX-M™). [Figure 13B]

[0021] Figure 1 shows anti-CoV S polypeptide IgG titers 13, 21 and 28 days after immunization of mice with a single dose of 0.1 μg to 10 μg of BV2373 with or without fraction A and fraction C iscom matrix (e.g., MATRIX-M™). [Figure 14]

[0022] 1 shows the induction of antibodies that block hACE2 interactions in mice immunized with one or two doses of 0.1 μg to 10 μg of BV2373, with or without MATRIX-M™. [Figure 15]

[0023] 1 shows virus-neutralizing antibodies detected in mice immunized with one or two doses of 0.1 μg to 10 μg of BV2373, with or without MATRIX-M™. [Figure 16]

[0024] 1 shows viral load (SARS-CoV-2) in the lungs of Ad / CMV / hACE2 mice immunized with either a single dose of BV2373 with or without MATRIX-M™ or two doses of BV2373 spaced 14 days apart. [Figure 17A]

[0025] Figure 1 shows the weight loss exhibited by mice after immunization with BV2373. Figure 2 shows the effect of immunization with a single 0.01 μg, 0.1 μg, 1 μg, or 10 μg dose of BV2373 + MATRIX-M™ on weight loss. [Figure 17B]

[0025] Figure 1 shows the weight loss exhibited by mice after immunization with BV2373. Figure 2 shows the effect of immunization on weight loss with two doses of BV2373 (0.01 μg, 0.1 μg, 1 μg) + MATRIX-M™. [Figure 17C]

[0025] Figure 1 shows the weight loss exhibited by mice after immunization with BV2373. Figure 2 shows the effect of immunization on weight loss with two doses of BV2373 (10 μg) in the presence or absence of MATRIX-M™. [Figure 18A]

[0026] Shows the effect of BV2373 on lung histopathology in mice 4 days after infection with SARS-CoV-2. [Figure 18B]

[0026] Shows the effect of BV2373 on lung histopathology in mice 7 days after infection with SARS-CoV-2. [Figure 19]

[0027] 1 shows the number of IFN-γ secreting cells after ex vivo stimulation in the spleens of mice immunized with BV2373 in the absence of adjuvant compared to mice immunized with BV2373 in the presence of MATRIX-M™. [Figure 20A]

[0028] Figure 1 shows the frequency of cytokine-secreting CD4+ T cells in the spleens of mice immunized with BV2373 in the presence or absence of MATRIX-M™. Figure 2 shows the frequency of IFN-γ-secreting CD4+ T cells. [Figure 20B]

[0028] Figure 1 shows the frequency of cytokine-secreting CD4+ T cells in the spleens of mice immunized with BV2373 in the presence or absence of MATRIX-M™. Figure 2 shows the frequency of TNF-α-secreting CD4+ T cells. [Figure 20C]

[0028] Figure 1 shows the frequency of cytokine-secreting CD4+ T cells in the spleens of mice immunized with BV2373 in the presence or absence of MATRIX-M™. Figure 2 shows the frequency of IL-2-secreting CD4+ T cells. [Figure 20D]

[0028] Figure 1 shows the frequency of cytokine-secreting CD4+ T cells in the spleens of mice immunized with BV2373 in the presence or absence of MATRIX-M™. Figure 2 shows the frequency of CD4+ T cells secreting two cytokines selected from IFN-γ, TNF-α, and IL-2. [Figure 20E]

[0028] Figure 1 shows the frequency of cytokine-secreting CD4+ T cells in the spleens of mice immunized with BV2373 in the presence or absence of MATRIX-M™. Figure 2 shows the frequency of CD4+ T cells expressing IFN-γ, TNF-α, and IL-2. [Figure 21A]

[0029] Figure 1 shows the frequency of cytokine-secreting CD8+ T cells in the spleens of mice immunized with BV2373 in the presence or absence of MATRIX-M™. Figure 2 shows the frequency of IFN-γ-secreting CD8+ T cells. [Figure 21B]

[0029] Figure 1 shows the frequency of cytokine-secreting CD8+ T cells in the spleens of mice immunized with BV2373 in the presence or absence of MATRIX-M™. Figure 2 shows the frequency of TNF-α-secreting CD8+ T cells. [Figure 21C]

[0029] Figure 1 shows the frequency of cytokine-secreting CD8+ T cells in the spleens of mice immunized with BV2373 in the presence or absence of MATRIX-M™. Figure 2 shows the frequency of IL-2-secreting CD8+ T cells. [Figure 21D]

[0029] Figure 1 shows the frequency of cytokine-secreting CD8+ T cells in the spleens of mice immunized with BV2373 in the presence or absence of MATRIX-M™. Figure 2 shows the frequency of CD8+ T cells secreting two cytokines selected from IFN-γ, TNF-α, and IL-2. [Figure 21E]

[0029] Figure 1 shows the frequency of cytokine-secreting CD8+ T cells in the spleens of mice immunized with BV2373 in the presence or absence of MATRIX-M™. Figure 2 shows the frequency of CD8+ T cells expressing IFN-γ, TNF-α, and IL-2. [Figure 22]

[0030] 1 illustrates the frequency of CD4+ or CD8+ cells expressing one (single), two (double), or three (triple) cytokines selected from IFN-γ, TNF-α, and IL-2 in the spleens of mice immunized with BV2373 in the presence or absence of MATRIX-M™. [Figure 23A]

[0031] 1 illustrates the effect of immunization with BV2373 in the presence or absence of MATRIX-M™ on type 2 cytokine secretion from CD4+ T cells. The frequency of IL-4 secreting cells is shown. [Figure 23B]

[0031] Figure 1 illustrates the effect of immunization with BV2373 in the presence or absence of MATRIX-M™ on type 2 cytokine secretion from CD4+ T cells. The frequency of IL-5 CD4+ secreting cells is shown. [Figure 23C]

[0031] Figure 1 illustrates the effect of immunization with BV2373 in the presence or absence of MATRIX-M™ on type 2 cytokine secretion from CD4+ T cells. The ratio of IFN-γ-secreting CD4+ T cells to IL-4-secreting CD4+ T cells is shown. [Figure 24A]

[0032] Figure 1 illustrates the effect of immunizing mice with BV2373 in the presence or absence of MATRIX-M™ on germinal center formation by assessing the presence of CD4+ T follicular helper cells (TFH). The frequency of CD4+ T follicular helper cells in the spleen is shown. [Figure 24B]

[0032] The effect of immunizing mice with BV2373 in the presence or absence of MATRIX-M™ on germinal center formation is illustrated by assessing the presence of CD4+ T follicular helper cells (TFH), which exhibit a CD4+ T follicular helper cell phenotype (e.g., CD4+ CXCR5+ PD-1+). [Figure 25A]

[0033] Figure 1 shows the effect of immunizing mice with BV2373 in the presence or absence of MATRIX-M™ on germinal center (GC) B cell formation by assessing the presence of GC B cells. Figure 2 shows the frequency of GC B cells in the spleen. [Figure 25B]

[0033] We show the effect of immunizing mice with BV2373 in the presence or absence of MATRIX-M™ on germinal center (GC) B cell formation by assessing the presence of CD4+ T follicular helper cell phenotype (e.g., CD19+ GL7+ CD-95+). [Figure 26A]

[0034] Figure 1 shows the effect of immunization with BV2373 in the presence or absence of MATRIX-M™ on antibody responses in Anubis baboons. Figure 2 shows anti-SARS-CoV-2 S polypeptide IgG titers in baboons after immunization with BV2373. [Figure 26B]

[0034] Figure 1 shows the effect of immunization with BV2373 in the presence or absence of MATRIX-M™ on antibody responses in Anubis baboons. Figure 2 shows the presence of hACE2 receptor-blocking antibodies in baboons after a single immunization with 5 μg or 25 μg of BV2373 in the presence of MATRIX-M™. [Figure 26C]

[0034] Figure 1 shows the effect of immunization with BV2373 in the presence or absence of MATRIX-M™ on antibody responses in Anubis baboons. Virus-neutralizing antibody titers after a single immunization with BV2373 and MATRIX-M™ are shown. [Figure 27]

[0035] Figure 1 shows a significant correlation between anti-SARS-CoV-2 S polypeptide IgG and neutralizing antibody titers in Anubis baboons after immunization with BV2373. [Figure 28]

[0036] 1 shows the frequency of IFN-γ secreting cells in peripheral blood mononuclear cells (PBMCs) of baboons immunized with BV2373 in the presence or absence of MATRIX-M™. [Figure 29A]

[0037] 1 shows the frequency of cytokine-secreting CD4+ T cells in PBMCs of baboons immunized with BV2373 in the presence or absence of MATRIX-M™. 1 shows the frequency of IFN-γ-secreting CD4+ T cells. [Figure 29B]

[0037] Figure 1 shows the frequency of cytokine-secreting CD4+ T cells in PBMCs of baboons immunized with BV2373 in the presence or absence of MATRIX-M™. Figure 2 shows the frequency of IL-2-secreting CD4+ T cells. [Figure 29C]

[0037] Figure 1 shows the frequency of cytokine-secreting CD4+ T cells in PBMCs of baboons immunized with BV2373 in the presence or absence of MATRIX-M™. Figure 2 shows the frequency of TNF-α-secreting CD4+ T cells. [Figure 29D]

[0037] Figure 1 shows the frequency of cytokine-secreting CD4+ T cells in PBMCs from baboons immunized with BV2373 in the presence or absence of MATRIX-M™. Figure 2 shows the frequency of CD4+ T cells secreting two cytokines selected from IFN-γ, TNF-α, and IL-2. [Figure 29E]

[0037] Figure 1 shows the frequency of cytokine-secreting CD4+ T cells in PBMCs of baboons immunized with BV2373 in the presence or absence of MATRIX-M™. Figure 2 shows the frequency of CD4+ T cells expressing IFN-γ, TNF-α, and IL-2. [Figure 30]

[0038] 1 shows a schematic diagram of the coronavirus spike (S) protein (SEQ ID NO: 109) (BV2384), with the furin cleavage site GSAS (SEQ ID NO: 97) underlined once and the K986P and V987P mutations underlined twice. [Figure 31]

[0039] 1 shows a schematic diagram of the coronavirus spike (S) protein (SEQ ID NO: 86) (BV2373), with the furin cleavage site QQAQ (SEQ ID NO: 7) underlined once and the K986P and V987P mutations underlined twice. [Figure 32]

[0040] 1 shows the purification of CoV S polypeptides BV2373 (SEQ ID NO: 87) and BV2384 (SEQ ID NO: 109). [Figure 33]

[0041] 1 shows a scanning densitometry plot of BV2384 (SEQ ID NO: 109) purity after purification. [Figure 34]

[0042] 1 shows a scanning densitometry plot of BV2373 (SEQ ID NO: 87) purity after purification. [Figure 35A]

[0043] This example illustrates the induction of anti-S antibodies in response to administration of BV2373 and MATRIX-M™. Cynomolgus macaques were administered 2.5 μg, 5 μg, or 25 μg of BV2373 once or twice (days 0 and 21) with 25 μg or 50 μg of MATRIX-M™ adjuvant. Controls received neither BV2373 nor MATRIX-M™. Antibodies were measured on days 21 and 33. [Figure 35B]

[0043] The induction of neutralizing antibodies in response to administration of BV2373 and MATRIX-M™ is illustrated. Cynomolgus macaques were administered 2.5 μg, 5 μg, or 25 μg of BV2373 once or twice (days 0 and 21) with 25 μg or 50 μg of MATRIX-M™ adjuvant. Controls received neither BV2373 nor MATRIX-M™. Antibodies were measured on days 21 and 33. [Figure 36A]

[0044] Figure 1 shows the reduction of SARS-CoV-2 viral replication by vaccine formulations disclosed herein in Cynomolgus macaques as assessed by bronchoalveolar lavage (BAL). Cynomolgus macaques were administered BV2373 and MATRIX-M™ as indicated. Subjects were immunized on day 0, and in the two-dose group, on days 0 and 21. Subjects were challenged with 1 x 10 pfu of SARS-CoV-2 virus on day 37. Viral RNA (corresponding to total RNA present) levels were assessed by bronchial lavage (BAL) 2 and 4 days after challenge with infectious virus (d2pi and d4pi). Most subjects showed no viral RNA. Small amounts of RNA were measured in some subjects on day 2. By day 4, no RNA was measured except in two subjects receiving the lowest dose of 2.5 μg. Subgenomic RNA was again not detected at the lowest dose on days 2 or 4 in all but one subject. [Figure 36B]

[0044] Figure 1 shows the reduction of SARS-CoV-2 viral replication by vaccine formulations disclosed herein in Cynomolgus macaques as assessed by bronchoalveolar lavage (BAL). Cynomolgus macaques were administered BV2373 and MATRIX-M™ as indicated. Subjects were immunized on day 0, and in the two-dose group, on days 0 and 21. Subjects were challenged with 1 x 10 pfu of SARS-CoV-2 virus on day 37. Viral subgenomic RNA (corresponding to replicating virus) levels were assessed by bronchial lavage (BAL) 2 and 4 days after challenge with infectious virus (d2pi and d4pi). Most subjects showed no viral RNA. Low amounts of RNA were measured in some subjects on day 2. By day 4, no RNA was measured except in two subjects at the lowest dose of 2.5 μg. Subgenomic RNA was not detected on days 2 or 4 except in one subject, again at the lowest dose. [Figure 37A]

[0045] Figure 1 illustrates the reduction of SARS-CoV-2 viral replication by vaccine formulations disclosed herein, as assessed in nasal swabs from Cynomolgus macaques. Cynomolgus macaques were administered BV2373 with MATRIX-M™ as indicated. Subjects were immunized on day 0, and in the two-dose group, on days 0 and 21. Subjects were challenged with 1 x 10 SARS-CoV-2 virus on day 37. Viral RNA was assessed by nasal swab 2 and 4 days post-infection (d2pi and d4pi). Most subjects showed no viral RNA. Small amounts of RNA were measured in some subjects on days 2 and 4. Subgenomic RNA was not detected on either day 2 or 4. Subjects were immunized on day 0, and in the two-dose group, on days 0 and 21. These data demonstrate that the vaccine reduces total viral RNA in the nose by 100-1000-fold, reduces sgRNA to undetectable levels, and that the immune response to the vaccine effectively blocks viral replication and prevents the spread of the virus. [Figure 37B]

[0045] Figure 1 illustrates the reduction of SARS-CoV-2 viral replication by vaccine formulations disclosed herein, as assessed in nasal swabs of Cynomolgus macaques. Cynomolgus macaques were administered BV2373 with MATRIX-M™ as indicated. Subjects were immunized on day 0, and in the two-dose group, on days 0 and 21. Subjects were challenged with 1 x 104 SARS-CoV-2 virus on day 37. Viral subgenomic (sg) RNA was assessed by nasal swabs 2 and 4 days post-infection (d2pi and d4pi). Most subjects showed no viral RNA. Small amounts of RNA were measured in some subjects on days 2 and 4. Subgenomic RNA was not detected on either day 2 or day 4. Subjects were immunized on day 0, and in the two-dose group, on days 0 and 21. These data demonstrate that the vaccine reduces total nasal viral RNA by 100-1000-fold, reduces sgRNA to undetectable levels, and that the immune response to the vaccine effectively blocks viral replication and prevents viral spread. [Figure 38A]

[0046] 1 shows anti-CoV S polypeptide IgG titers 21 and 35 days after immunization of Cynomolgus macaques with a single dose of BV2373 and 25 μg or 50 μg of MATRIX-M™. [Figure 38B]

[0046] Figure 1 shows anti-CoV S polypeptide IgG titers 21 and 35 days after immunization of Cynomolgus macaques with two doses of BV2373 and 25 μg or 50 μg of MATRIX-M™. [Figure 38C]

[0047] 1 shows hACE2 inhibitory titers in Cynomolgus macaques 21 and 35 days after immunization of Cynomolgus macaques with a single dose of BV2373 (5 μg) and MATRIX-M™ (25 μg or 50 μg). [Figure 38D]

[0047] Figure 1 shows the hACE2 inhibitory titers in Cynomolgus macaques 21 and 35 days after immunization of Cynomolgus macaques with two doses of BV2373 (5 μg) and MATRIX-M™ (25 μg or 50 μg). [Figure 38E]

[0048] Figure 1 shows a significant correlation between anti-CoV S polypeptide IgG titers and hACE2 inhibitory titers in Cynomolgus macaques after administration of BV2373 and MATRIX-M™. Data for Groups 2-6 from Table 4 are shown. [Figure 39]

[0049] Table 4 shows the anti-CoV S polypeptide titers and hACE2 inhibitory titers in Cynomolgus macaques 35 days after immunization with two doses of BV2373 and MATRIX-M™ or immunization with convalescent human serum (Groups 2, 4, and 6). These data demonstrate that the anti-CoV S polypeptide titers and hACE2 inhibitory titers of Cynomolgus macaques immunized with BV2373 and MATRIX-M™ are superior to those of Cynomolgus macaques immunized with convalescent serum. [Figure 40A]

[0050] 1 shows the SARS-CoV-2 neutralization titers of Cynomolgus macaques immunized with BV2373 and MATRIX-M™, as determined by cytopathic effect (CPE). [Figure 40B]

[0050] Shows the SARS-CoV-2 neutralization titers of Cynomolgus macaques immunized with BV2373 and MATRIX-M™ as determined by plaque reduction neutralization test (PRNT). [Figure 41]

[0051] The timing of administration for clinical trials evaluating the safety and efficacy of vaccines containing BV2373 and optionally MATRIX-M™ is shown. AESI indicates adverse events of particular interest. MAEE stands for medically-accompanied adverse events, and SAE stands for serious adverse events. [Figure 42A]

[0052] Local adverse events experienced by patients in clinical trials evaluating a vaccine containing BV2373 and MATRIX-M™ are shown in Table 5. Groups A through E are shown in Table 5. The data indicate that the vaccine was well tolerated and safe. [Figure 42B]

[0052] Systemic adverse events experienced by patients in clinical trials evaluating a vaccine containing BV2373 and MATRIX-M™ are shown in Table 5. Groups A through E are shown in Table 5. The data indicate that the vaccine was well tolerated and safe. [Figure 43A]

[0053] Figure 1 shows anti-CoV S polypeptide IgG 21 and 35 days after immunization in participants in a clinical trial evaluating vaccines containing BV2373 and MATRIX-M™. Horizontal bars represent the interquartile range (IRQ) and median area under the curve, respectively. The whisker endpoints equal the maximum and minimum values ​​lower or higher than the median ± 1.5 × IQR. The convalescent serum panel includes specimens from participants with PCR-confirmed COVID-19 at Baylor College of Medicine (29 specimens for ELISA and 32 specimens for microneutralization (MN IC > 99)). COVID-19 severity is indicated by a red mark for hospitalized patients (including intensive care units), a blue mark for outpatients (specimens collected in the emergency department), and a green mark for asymptomatic (exposed) patients (specimens collected during contact / exposure assessment). [Figure 43B]Neutralization titers 21 and 35 days after immunization in participants in a clinical trial evaluating a vaccine containing BV2373 and MATRIX-M™ are shown. Horizontal bars represent the interquartile range (IRQ) and median area under the curve, respectively. The whisker endpoints equal the maximum and minimum values ​​lower or higher than the median ± 1.5 × IQR. The convalescent serum panel includes samples from participants with PCR-confirmed COVID-19 at Baylor College of Medicine (29 samples for ELISA and 32 samples for microneutralization (MN IC>99)). COVID-19 severity is indicated by a red mark for hospitalized patients (including intensive care units), a blue mark for outpatients (samples collected in the emergency department), and a green mark for asymptomatic (exposed) patients (samples collected during contact / exposure assessment). [Figure 44A]

[0054] Figure 1 shows the correlation between anti-CoV S polypeptide IgG and neutralizing antibody titers in patients receiving convalescent serum. A strong correlation between neutralizing antibody titers and anti-CoV-S IgG titers was observed in patients treated with convalescent serum or the adjuvant BV2373, but not in patients treated with BV2373 in the absence of adjuvant. [Figure 44B]

[0054] Figure 1 shows the correlation between anti-CoV S polypeptide IgG and neutralizing antibody titers in patients receiving two 25 μg doses of BV2373. A strong correlation between neutralizing antibody titers and anti-CoV-S IgG titers was observed in patients treated with convalescent serum or adjuvanted BV2373, but not in patients treated with BV2373 in the absence of adjuvant. [Figure 44C]

[0054] Figure 1 shows the correlation between anti-CoV S polypeptide IgG and neutralizing antibody titers in patients receiving two doses (5 μg and 25 μg) of BV2373 combined with MATRIX-M™. A strong correlation between neutralizing antibody titers and anti-CoV-S IgG titers was observed in patients treated with convalescent serum or adjuvanted BV2373, but not in patients treated with BV2373 in the absence of adjuvant. [Figure 45A]

[0055] Figure 1 shows the frequency of antigen-specific CD4+ T cells producing the T helper 1 (Th1) cytokines interferon-γ (IFN-γ), tumor necrosis factor-α (TNF-α), and interleukin (IL)-2, as well as the T helper 2 (Th2) cytokines IL-5 and IL-13, from participants in Group A (placebo) after stimulation with BV2373. "Any two" in the Th1 cytokine panel refers to CD4+ T cells capable of simultaneously producing two Th1 cytokines. "All three" refers to CD4+ T cells simultaneously producing IFN-γ, TNF-α, and IL-2. "Both" in the Th2 panel refers to CD4+ T cells simultaneously producing the Th2 cytokines IL-5 and IL-13. [Figure 45B] Cytokines from participants in Group B (25 μg BV2373) after stimulation with BV2373 were shown. The frequency of antigen-specific CD4+ T cells producing the T helper 1 (Th1) cytokines interferon-γ (IFN-γ), tumor necrosis factor-α (TNF-α), and interleukin (IL)-2, and the T helper 2 (Th2) cytokines IL-5 and IL-13, are shown. "Any two" in the Th1 cytokine panel refers to CD4+ T cells capable of simultaneously producing two Th1 cytokines. "All three" refers to CD4+ T cells simultaneously producing IFN-γ, TNF-α, and IL-2. "Both" in the Th2 panel refers to CD4+ T cells simultaneously producing the Th2 cytokines IL-5 and IL-13. [Figure 45C]Cytokines from participants in Group C (5 μg BV2373 and 50 μg MATRIX-M™) after stimulation with BV2373 are shown. The frequency of antigen-specific CD4+ T cells producing the T helper 1 (Th1) cytokines interferon-γ (IFN-γ), tumor necrosis factor-α (TNF-α), and interleukin (IL)-2, and the T helper 2 (Th2) cytokines IL-5 and IL-13, are shown. "Any two" in the Th1 cytokine panel refers to CD4+ T cells capable of simultaneously producing two Th1 cytokines. "All three" refers to CD4+ T cells simultaneously producing IFN-γ, TNF-α, and IL-2. "Both" in the Th2 panel refers to CD4+ T cells simultaneously producing the Th2 cytokines IL-5 and IL-13. [Figure 45D] Cytokines from participants in Group D (25 μg BV2373 and 50 μg MATRIX-M™) after stimulation with BV2373 are shown. The frequency of antigen-specific CD4+ T cells producing the T helper 1 (Th1) cytokines interferon-γ (IFN-γ), tumor necrosis factor-α (TNF-α), and interleukin (IL)-2, and the T helper 2 (Th2) cytokines IL-5 and IL-13, are shown. "Any two" in the Th1 cytokine panel refers to CD4+ T cells capable of simultaneously producing two Th1 cytokines. "All three" refers to CD4+ T cells simultaneously producing IFN-γ, TNF-α, and IL-2. "Both" in the Th2 panel refers to CD4+ T cells simultaneously producing the Th2 cytokines IL-5 and IL-13. [Figure 46A]

[0056] 1 shows the primary structure of the wild-type SARS-CoV-2 S polypeptide, including the signal peptide, numbered relative to SEQ ID NO:1. [Figure 46B]

[0056] The primary structure of the wild-type SARS-CoV-2 S polypeptide without the signal peptide is shown, numbered relative to SEQ ID NO: 2. DETAILED DESCRIPTION OF THE INVENTION

[0007] Detailed Description of the Invention definition

[0057] As used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to a "protein" refers to one protein or a mixture of such proteins, reference to a "process" includes reference to equivalent steps and / or methods known to those skilled in the art, and so forth.

[0008]

[0058] 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 elicited against the immunogen. Modification of the immune response can include enhancing or broadening the specificity of either or both the antibody and cellular immune responses.

[0009]

[0059] As used herein, the term "about" or "approximately" before a numerical value indicates a range of plus or minus 10% of the value. For example, "about 100" includes 90 and 110.

[0010]

[0060] As used herein, the terms "immunogen," "antigen," and "epitope" refer to substances such as proteins, including glycoproteins, and peptides that are capable of eliciting an immune response.

[0011]

[0061] As used herein, an "immunogenic composition" is a composition comprising an antigen, the administration of which to a subject results in the subject developing a humoral and / or cellular immune response to the antigen.

[0012]

[0062] As used herein, a "subunit" composition, e.g., a vaccine, comprises one or more selected antigens, rather than all antigens, from a pathogen. Such compositions are substantially free of intact virus or lysates of such cells or particles, and are typically prepared from at least partially purified, and often substantially purified, immunogenic polypeptides from the pathogen. The antigens in the subunit compositions disclosed herein are typically prepared recombinantly, often using a baculovirus system.

[0013]

[0063] As used herein, "substantially" refers to the isolation of a substance (e.g., a compound, polynucleotide, or polypeptide) such that the substance constitutes a majority percentage of the sample in which it is contained. For example, in a sample, a substantially purified component comprises 85% of the sample, preferably 85% to 90%, more preferably at least 95% to 99.5%, and most preferably at least 99%. When a component is substantially replaced, the amount remaining in the sample is about 0.5% to about 10% or less, preferably about 0.5% to about 1.0% or less.

[0014]

[0064] As used herein, the terms "treat," "treatment," and "treating" refer to an approach for obtaining beneficial or desired results, e.g., clinical results. For purposes of this disclosure, beneficial or desired results may include inhibiting or suppressing the onset or progression of an infection or disease; ameliorating or alleviating the development or symptoms of an infection or disease; or a combination thereof.

[0015]

[0065] As used herein, "prevention" is used interchangeably with "prophylaxis" and can mean preventing an infection or disease altogether or preventing the onset of symptoms of that infection or disease; delaying the onset of an infection or disease or its symptoms; or reducing the severity of an infection or disease or its symptoms that subsequently occurs.

[0016]

[0066] As used herein, "effective dose" or "effective amount" refers to an amount of an immunogen sufficient to elicit an immune response that alleviates at least one symptom of a pathogen infection. An effective dose or amount can be determined, for example, by measuring the amount of neutralizing secretory and / or serum antibodies, e.g., by plaque neutralization, complement fixation, enzyme-linked immunosorbent (ELISA), or microneutralization assays.

[0017]

[0067] As used herein, the term "vaccine" refers to an immunogenic composition, such as an immunogen derived from a pathogen, used to elicit an immune response against a pathogen that provides protective immunity (e.g., immunity that protects a subject from infection by the pathogen and / or reduces the severity of a disease or condition caused by infection with the pathogen). A protective immune response can include the formation of antibodies and / or a cellular response. Depending on the context, the term "vaccine" can also refer to a suspension or solution of immunogens that is administered to a subject to provide protective immunity.

[0018]

[0068] As used herein, the term "subject" includes humans and other animals. Typically, the subject is a human. For example, the subject can be an adult, a teenager, a child (ages 2-14), an infant (birth-2 years), or a newborn (up to 2 months). In certain embodiments, the subject is up to 4 months old or 6 months old. In some embodiments, the adult is about 65 years old or older, or about 60 years old or older. In some embodiments, the subject is a pregnant woman or a woman who wishes to become pregnant. In other embodiments, the subject is not a human; for example, a non-human primate; for example, a baboon, chimpanzee, gorilla, or macaque. In certain embodiments, the subject can be a pet, such as a dog or cat.

[0019]

[0069] 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 U.S. Pharmacopoeia, the European Pharmacopoeia, or other pharmacopoeias generally recognized for use in mammals, more particularly humans. These compositions may be useful as vaccine and / or antigen compositions for eliciting a protective immune response in vertebrates.

[0020]

[0070] As used herein, the term "about" means plus or minus 10% of the indicated numerical value.

[0021]

[0071] As used herein, the term "NVX-CoV2373" refers to a vaccine composition comprising the BV2373 spike glycoprotein (SEQ ID NO: 87) and Fraction A and Fraction C iscom matrix (e.g., MATRIX-M™).

[0022]

[0072] As used herein, the term "modification" when referring to a CoV S polypeptide refers to a mutation, deletion, or addition of one or more amino acids in the CoV S polypeptide. The location of the modification within the CoV S polypeptide can be determined based on alignment of the sequence of the polypeptide to SEQ ID NO: 1 (CoV S polypeptide including the signal peptide) or SEQ ID NO: 2 (mature CoV S polypeptide lacking the signal peptide). Vaccine compositions containing coronavirus (CoV) spike (S) proteins

[0073] The present disclosure provides non-naturally occurring coronavirus (CoV) spike (S) polypeptides, nanoparticles comprising CoV S polypeptides, and immunogenic and vaccine compositions comprising either non-naturally occurring CoV S polypeptides or nanoparticles comprising CoV S polypeptides. In embodiments, provided herein are methods of stimulating an immune response using the CoV S polypeptides, nanoparticles, immunogenic and vaccine compositions.

[0023]

[0074] Also provided herein are methods for producing nanoparticles and vaccine compositions. Advantageously, the methods provide nanoparticles that are substantially free of contamination with other proteins, such as proteins associated with recombinant expression of proteins in insect cells. In embodiments, expression occurs in the baculovirus / Sf9 system. CoV S polypeptide antigen

[0075] The vaccine compositions of the present disclosure include a non-naturally occurring CoV S polypeptide. The CoV S polypeptide can be derived from a coronavirus, including, but not limited to, SARS-CoV-2, such as SARS-CoV-2, MERS-CoV, and SARS-CoV. In embodiments, the CoV S polypeptide is derived from a SARS-CoV-2 variant. In embodiments, the SARS-CoV-2 variant is SARS-CoV-2 VUI 202012 / 01, B.1.1.7, 501Y.V2, Cal.20C, or P.1. In contrast to the SARS-CoV S protein, the SARS-CoV-2 S protein contains a four-amino acid insertion at the S1 / S2 cleavage site that forms a polybasic RRAR furin-like cleavage motif. The SARS-CoV-2 S protein is synthesized as an inactive precursor (S0), which is proteolytically cleaved at a furin cleavage site into S1 and S2 subunits, which remain noncovalently associated to form a prefusion trimer. The S2 domain of the SARS-CoV-2 S protein contains a fusion peptide (FP), two heptad repeats (HR1 and HR2), a transmembrane (TM) domain, and a cytoplasmic tail (CT). The S1 domain of the SARS-CoV-2 S protein folds into four distinct domains: an N-terminal domain (NTD), a C-terminal domain containing a receptor-binding domain (RBD), and two subdomains, SD1 and SD2. The prefusion SARS-CoV-2 S protein trimer undergoes structural rearrangement from the prefusion conformation to the postfusion conformation upon S protein receptor binding and cleavage.

[0024]

[0076] In embodiments, the CoV S polypeptide is a glycoprotein that is post-translationally glycosylated. The glycoprotein includes one or more of a signal peptide, an S1 subunit, an S2 subunit, an NTD, an RBD, two subdomains (SD1 and SD2, designated SD1 / 2 in Figures 46A and 46B and referred to herein as "SD1 / 2"), an intact or modified fusion peptide, an HR1 domain, an HR2 domain, a TM, and a CD. In embodiments, the amino acids of each domain are shown in Figures 2 and 46A (shown according to SEQ ID NO:1), 46B (shown according to SEQ ID NO:2), and 3 (shown corresponding to SEQ ID NO:1). In embodiments, each domain may have at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% identity to the sequence of the respective domain in SEQ ID NO:1 or SEQ ID NO:2. Each domain may have deletions, insertions, or mutations of 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 20, or up to about 30 amino acids compared to the respective domains shown in SEQ ID NO: 1 or SEQ ID NO: 2. Each domain may have deletions, insertions, or mutations of about 1 to about 5 amino acids, about 3 to about 10 amino acids, about 5 to 10 amino acids, about 8 to 12 amino acids, about 10 to 15 amino acids, about 12 to 17 amino acids, about 15 to 20 amino acids, about 18 to 23 amino acids, about 20 to 25 amino acids, about 22 to about 27 amino acids, or about 25 to 30 amino acids compared to the respective domains shown in SEQ ID NO: 1 or SEQ ID NO: 2. Note that Figures 2 and 3 illustrate a 13-amino acid N-terminal signal peptide that is not present in the mature peptide. CoV S polypeptides can be used to stimulate an immune response to native CoV spike (S) polypeptides.

[0025]

[0077] In embodiments, a native CoV spike (S) polypeptide (SEQ ID NO: 2) is modified to obtain a non-naturally occurring CoV spike (S) polypeptide (Figure 1). In embodiments, the CoV spike (S) glycoprotein comprises an S1 subunit and an S2 subunit, where the S1 subunit comprises the NTD, RBD, SD1 / 2, and an inactive furin cleavage site (amino acids 669-672), and the S2 subunit comprises mutations at amino acids 973 and 974; The NTD (amino acids 1-318) optionally comprises: (a) a deletion of one or more amino acids from the NTD, optionally wherein the one or more modifications are selected from the group consisting of amino acids 56, 57, 131, 132, 229, 230, 231, and combinations thereof; and (b) mutation of one or more amino acids selected from the group consisting of amino acids 67, 229, 202, 139, 5, 233, 7, 13, 125, 177, or a combination thereof comprising one or more modifications selected from the group consisting of: the RBD optionally comprises a mutation of one or more amino acids selected from the group consisting of amino acids 488, 404, 471, 439, 426, 440, and combinations thereof; SD1 / 2 optionally comprises one or more amino acid mutations selected from the group consisting of 601, 557, 668, 642, and combinations thereof; and The S2 subunit optionally comprises: (a) deletion of one or more amino acids from 676 to 702, 702 to 711, 775 to 793; (b) deletion of one or more amino acids from the fusion peptide (amino acids 806-815); (c) mutation of one or more amino acids selected from the group consisting of 973, 974, 703, 1105, 688, 969, and 1014; and (d) deletion of one or more amino acids from the transmembrane and cytoplasmic domain (TMCT) (amino acids 1201 to 1260) and comprising one or more modifications selected from the group consisting of: The amino acids of the CoV S glycoprotein are numbered relative to SEQ ID NO:2. Figure 3 shows a CoV S polypeptide designated BV2378, which has an inactive furin cleavage site, a deleted fusion peptide (e.g., deletion of amino acids 819-828), and K986P and V987 mutations, with these amino acids numbered relative to SEQ ID NO:1. The mature BV2378 polypeptide lacks one or more amino acids of the signal peptide, amino acids 1-13 of SEQ ID NO:1. In embodiments, the CoV S polypeptides described herein exist in a pre-fusion conformation. In embodiments, the CoV S polypeptides described herein comprise a flexible HR2 domain. Unless otherwise specified, domain flexibility is determined by transmission electron microscopy (TEM) and 2D class averaging. Decreased electron density corresponds to flexible domains. CoV S polypeptide antigen - modifications to the S1 subunit

[0078] In embodiments, the CoV S polypeptide comprises one or more modifications to the S1 subunit having the amino acid sequence of SEQ ID NO:121.

[0026]

[0079] The amino acid sequence of the S1 subunit (SEQ ID NO: 121) is shown below.

[0027] [ka]

[0028]

[0080] The underlined region of SEQ ID NO: 121 represents amino acids within the S1 subunit that may be modified.

[0029]

[0081] In embodiments, the CoV S 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 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 acid deletions, insertions, or mutations compared to the amino acid sequence of the S1 subunit of SEQ ID NO: 1 or SEQ ID NO: 2. The S1 subunit may have deletions, insertions, or mutations of about 1 to about 5 amino acids, about 3 to about 10 amino acids, about 5 to 10 amino acids, about 8 to 12 amino acids, about 10 to 15 amino acids, about 12 to 17 amino acids, about 15 to 20 amino acids, about 18 to 23 amino acids, about 20 to 25 amino acids, about 22 to about 27 amino acids, or about 25 to 30 amino acids, compared to the S1 subunit of SEQ ID NO: 1 or SEQ ID NO: 2.

[0030]

[0082] In embodiments, the S1 subunit may include any combination of the modifications shown in Table 1A.

[0031] [Table 1]

[0032] [Table 2]

[0033] [Table 3]

[0034] CoV S polypeptide antigen - Modifications to the S1 subunit - NTD

[0083] In embodiments, the CoV S polypeptide comprises one or more modifications to the NTD. In embodiments, the NTD has the amino acid sequence of SEQ ID NO: 118, which corresponds to amino acids 14 to 305 of SEQ ID NO: 1 or amino acids 1 to 292 of SEQ ID NO: 2.

[0035]

[0084] The amino acid sequence of the NTD (SEQ ID NO: 118) is shown below.

[0036] [ka]

[0037]

[0085] The underlined region of SEQ ID NO: 118 represents amino acids within the NTD that may be modified.

[0038]

[0086] In embodiments, the NTD has the amino acid sequence of SEQ ID NO: 45, which corresponds to amino acids 14 to 331 of SEQ ID NO: 1 or amino acids 1 to 318 of SEQ ID NO: 2. The amino acid sequence of the NTD (SEQ ID NO: 45) is shown below.

[0039]

[0087]

[0040] [ka]

[0041]

[0088] In embodiments, the NTD and RBD overlap by at most about 1 amino acid, at most about 5 amino acids, at most about 10 amino acids, or at most about 20 amino acids.

[0042]

[0089] In embodiments, the NTDs 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.

[0043]

[0090] In embodiments, a CoV S polypeptide described herein comprises 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 can have 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 acid deletions, insertions, or mutations compared to the amino acid sequence of the NTD of SEQ ID NO: 1 or SEQ ID NO: 2. The NTD may have deletions, insertions, or mutations of about 1 to about 5 amino acids, about 3 to about 10 amino acids, about 5 to 10 amino acids, about 8 to 12 amino acids, about 10 to 15 amino acids, about 12 to 17 amino acids, about 15 to 20 amino acids, about 18 to 23 amino acids, about 20 to 25 amino acids, about 22 to about 27 amino acids, or about 25 to 30 amino acids, compared to the NTD of SEQ ID NO: 1 or SEQ ID NO: 2.

[0044]

[0091] In embodiments, the CoV S polypeptide comprises 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 some embodiments, the CoV S polypeptide comprises a deletion of up to about 10, up to about 20, up to about 30, up to about 40, up to about 50, up to about 60, up to about 70, up to about 80, up to about 90, up to about 100, up to about 110, up to about 120, up to about 130, up to about 140, up to about 150, up to about 160, up to about 170, up to about 180, up to about 190, up to about 200, up to about 210, up to about 220, up to about 230, up to about 240, up to about 250, up to about 260, up to about 270, up to about 280, up to about 290, or up to about 292 amino acids of the NTD.

[0045]

[0092] In some embodiments, the CoV S polypeptide comprises 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 polypeptide comprises a deletion of amino acids 1-318 of the NTD of SEQ ID NO:2. In embodiments, the deletion of the NTD facilitates protein expression of a CoV spike (S) polypeptide. In embodiments, the CoV S polypeptide having a deletion of the NTD has an amino acid sequence represented by SEQ ID NOs:46, 48, 49, 51, 52, and 54. In embodiments, the CoV S polypeptide having a deletion of the NTD is 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.

[0046]

[0093] In embodiments, the NTD may include any combination of modifications shown in Table 1B. The modifications are shown with respect to SEQ ID NO:2, the mature S polypeptide sequence for reference.

[0047] [Table 4]

[0048] CoV S polypeptide antigen - Modifications to the S1 subunit - RBD

[0094] In embodiments, the CoV S polypeptide comprises one or more modifications to the RBD.

[0049]

[0095] In embodiments, the RBD has the amino acid sequence of SEQ ID NO: 126, which corresponds to amino acids 331 to 527 of SEQ ID NO: 1 or amino acids 318 to 514 of SEQ ID NO: 2.

[0050]

[0096] The amino acid sequence of the RBD (SEQ ID NO: 126) is shown below.

[0051] [ka]

[0052]

[0097] The underlined region of SEQ ID NO: 126 represents amino acids within the RBD subunit that may be modified.

[0053]

[0098] In embodiments, the RBD has the amino acid sequence of SEQ ID NO: 116, which corresponds to amino acids 335 to 530 of SEQ ID NO: 1 or amino acids 322 to 517 of SEQ ID NO: 2.

[0054]

[0099] The amino acid sequence of the RBD (SEQ ID NO: 116) is shown below.

[0055] [ka]

[0056]

[0100] The underlined region of SEQ ID NO: 116 represents amino acids within the RBD subunit that may be modified.

[0057]

[0101] In embodiments, the RBDs provided herein may be extended at the N- 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.

[0058]

[0102] In embodiments, the CoV S polypeptides described herein comprise an RBD that has 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 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 acid deletions, insertions, or mutations compared to the amino acid sequence of the RBD of SEQ ID NO: 1 or SEQ ID NO: 2. The RBD may have deletions, insertions, or mutations of about 1 to about 5 amino acids, about 3 to about 10 amino acids, about 5 to 10 amino acids, about 8 to 12 amino acids, about 10 to 15 amino acids, about 12 to 17 amino acids, about 15 to 20 amino acids, about 18 to 23 amino acids, about 20 to 25 amino acids, about 22 to about 27 amino acids, or about 25 to 30 amino acids compared to the RBD of SEQ ID NO: 1 or SEQ ID NO: 2.

[0059]

[0103] In embodiments, the CoV S polypeptide comprises at least 1, at least 2, at least 3, at least 4, at least 4, 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 modifications shown in Table 1C.

[0060] [Table 5]

[0061] CoV S polypeptide antigen - modifications to SD1 / 2

[0104] In embodiments, the CoV S polypeptide comprises one or more modifications to SD1 / 2 having the amino acid sequence of SEQ ID NO: 122, which corresponds to amino acids 542 to 681 of SEQ ID NO: 1 or amino acids 529 to 668 of SEQ ID NO: 2.

[0062]

[0105] The amino acid sequence of SD1 / 2 (SEQ ID NO: 122) is shown below.

[0063] [ka]

[0064]

[0106] The underlined region of SEQ ID NO: 122 represents amino acids within SD1 / 2 that may be modified.

[0065]

[0107] In embodiments, the CoV S polypeptides described herein comprise an SD1 / 2 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 of SEQ ID NO: 1 or SEQ ID NO: 2. The SD1 / 2 may have 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 acid deletions, insertions, or mutations compared to the amino acid sequence of SD1 / 2 of SEQ ID NO: 1 or SEQ ID NO: 2. Compared to SD1 / 2 of SEQ ID NO: 1 or SEQ ID NO: 2, SD1 / 2 may have deletions, insertions, or mutations of about 1 to about 5 amino acids, about 3 to about 10 amino acids, about 5 to 10 amino acids, about 8 to 12 amino acids, about 10 to 15 amino acids, about 12 to 17 amino acids, about 15 to 20 amino acids, about 18 to 23 amino acids, about 20 to 25 amino acids, about 22 to about 27 amino acids, or about 25 to 30 amino acids.

[0066]

[0108] In embodiments, the CoV S polypeptide has at least 1, at least 2, at least 3, at least 4, at least 4, 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 SD1 / 2. In embodiments, SD1 / 2 can include any combination of modifications shown in Table 1D.

[0067] [Table 6]

[0068] CoV S polypeptide antigen - modifications to the furin cleavage site

[0109] In embodiments, the CoV S polypeptide contains a furin site (RRAR) corresponding to amino acids 682-685 of SEQ ID NO: 1 or amino acids 669-672 of SEQ ID NO: 2 that is inactivated by one or more mutations. Inactivation of the furin cleavage site prevents furin from cleaving the CoV S polypeptide. In embodiments, the CoV S polypeptide described herein containing an inactivated furin cleavage site is expressed as a single chain.

[0069]

[0110] In embodiments, one or more of the amino acids that make up the natural furin cleavage site are mutated to any natural 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.

[0070]

[0111] In embodiments, one or more of the amino acids comprising the native furin cleavage site are mutated to glutamine. In embodiments, one, two, three, or four amino acids may be mutated to glutamine. In embodiments, one of the arginines comprising the native furin cleavage site is mutated to glutamine. In embodiments, two of the arginines comprising the native furin cleavage site are mutated to glutamine. In embodiments, three of the arginines comprising the native furin cleavage site are mutated to glutamine.

[0071]

[0112] In embodiments, one or more of the amino acids comprising the native furin cleavage site are mutated to alanine. In embodiments, one, two, three, or four amino acids may be mutated to alanine. In embodiments, one of the arginines comprising the native furin cleavage site is mutated to alanine. In embodiments, two of the arginines comprising the native furin cleavage site are mutated to alanine. In embodiments, three of the arginines comprising the native furin cleavage site are mutated to alanine.

[0072]

[0113] In embodiments, one or more of the amino acids comprising the native furin cleavage site are mutated to glycine. In embodiments, one, two, three or four amino acids may be mutated to glycine. In embodiments, one of the arginines comprising the native furin cleavage site is mutated to glycine. In embodiments, two of the arginines comprising the native furin cleavage site are mutated to glycine. In embodiments, three of the arginines comprising the native furin cleavage site are mutated to glycine.

[0073]

[0114] In embodiments, one or more of the amino acids constituting the natural furin cleavage site are mutated to asparagine. For example, one, two, three, or four amino acids may be mutated to asparagine. In embodiments, one of the arginines constituting the natural furin cleavage site is mutated to asparagine. In embodiments, two of the arginines constituting the natural furin cleavage site are mutated to asparagine. In embodiments, three of the arginines constituting the natural furin cleavage site are mutated to asparagine.

[0074]

[0115] Non-limiting examples of amino acid sequences of inactivating furin sites contained within CoV S polypeptides are shown in Table 1E.

[0075] [Table 7]

[0076]

[0116] In embodiments, instead of an active furin cleavage site (SEQ ID NO: 6), a CoV S polypeptide described herein comprises an inactivated furin cleavage site. In embodiments, the amino acid sequence of the inactivated furin cleavage site is represented by any one 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). CoV S polypeptide antigen - modifications to the S2 subunit

[0117] In embodiments, the CoV S polypeptide comprises one or more modifications to the S2 subunit having the amino acid sequence of SEQ ID NO: 120, which corresponds to amino acids 686 to 1273 of SEQ ID NO: 1 or amino acids 673 to 1260 of SEQ ID NO: 2.

[0077]

[0118] The amino acid sequence of the S2 subunit (SEQ ID NO: 120) is shown below.

[0078] [ka]

[0079]

[0119] The underlined region of SEQ ID NO: 120 represents amino acids within the S2 subunit that may be modified.

[0080]

[0120] In embodiments, the CoV S 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 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 acid deletions, insertions, or mutations compared to the amino acid sequence of the S2 subunit of SEQ ID NO: 1 or SEQ ID NO: 2. The S2 subunit may have deletions, insertions, or mutations of about 1 to about 5 amino acids, about 3 to about 10 amino acids, about 5 to 10 amino acids, about 8 to 12 amino acids, about 10 to 15 amino acids, about 12 to 17 amino acids, about 15 to 20 amino acids, about 18 to 23 amino acids, about 20 to 25 amino acids, about 22 to about 27 amino acids, or about 25 to 30 amino acids, compared to the S2 subunit of SEQ ID NO: 1 or SEQ ID NO: 2.

[0081]

[0121] In embodiments, the S2 subunit may include any combination of modifications shown in Table 1F.

[0082] [Table 8]

[0083] [Table 9]

[0084]

[0122] In embodiments, the CoV S polypeptide comprises a deletion corresponding to one or more deletions within amino acids 676-685 of a native CoV spike (S) polypeptide (SEQ ID NO: 2). In embodiments, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids are deleted within amino acids 676-685 of a native CoV spike (S) polypeptide (SEQ ID NO: 2). In embodiments, the deletion of amino acids within amino acids 676-685 is contiguous, e.g., amino acids 676 and 677 are deleted, or amino acids 680 and 681 are deleted. In embodiments, the deletion of amino acids within amino acids 676-685 is non-contiguous, e.g., amino acids 676 and 680 are deleted, or amino acids 677 and 682 are deleted. In embodiments, the CoV S polypeptide comprising a deletion corresponding to one or more deletions within amino acids 676-685 has an amino acid sequence selected from the group consisting of SEQ ID NO:62 and SEQ ID NO:63.

[0085]

[0123] In embodiments, the CoV S polypeptide comprises a deletion corresponding to one or more deletions within amino acids 702-711 of a native CoV spike (S) polypeptide (SEQ ID NO: 2). In embodiments, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids are deleted within amino acids 702-711 of a native SARS-CoV-2 spike (S) polypeptide (SEQ ID NO: 2). In embodiments, the deletion of one or more amino acids within amino acids 702-711 is contiguous, e.g., amino acids 702 and 703 are deleted, or amino acids 708 and 709 are deleted. In embodiments, the deletion of amino acids within amino acids 702-711 is non-contiguous, e.g., amino acids 702 and 704 are deleted, or amino acids 707 and 710 are deleted. In embodiments, the CoV S polypeptide including a deletion corresponding to one or more deletions within amino acids 702-711 has an amino acid sequence selected from the group consisting of SEQ ID NO:64 and SEQ ID NO:65.

[0086]

[0124] In embodiments, the CoV S polypeptide comprises a deletion corresponding to one or more deletions within amino acids 775-793 of a native CoV S polypeptide (SEQ ID NO: 2). In embodiments, up to about 1, up to about 2, up to about 3, up to about 4, up to about 5, up to about 6, up to about 7, up to about 8, up to about 9, up to about 10, up to about 11, up to about 12, up to about 13, up to about 14, up to about 15, up to about 16, up to about 17, up to about 18, or up to about 19 amino acids are deleted within amino acids 775-793 of a native SARS-CoV-2 spike (S) polypeptide (SEQ ID NO: 2). In embodiments, the deletion(s) of one or more amino acids within amino acids 775-793 is contiguous, e.g., amino acids 776 and 777 are deleted, or amino acids 780 and 781 are deleted. In an embodiment, the deletion of amino acids within amino acids 775 to 793 is non-contiguous, for example, amino acids 775 and 790 are deleted, or amino acids 777 and 781 are deleted.

[0087]

[0125] In embodiments, the CoV S polypeptide comprises a deletion of the fusion peptide corresponding to amino acids 806-815 of SEQ ID NO:2 (SEQ ID NO:104). In embodiments, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids are deleted from the fusion peptide of the CoV spike (S) polypeptide (SEQ ID NO:2). In embodiments, the deletion of amino acids within the fusion peptide is contiguous, e.g., amino acids 806 and 807 are deleted, or amino acids 809 and 810 are deleted. In embodiments, the deletion of amino acids within the fusion peptide is non-contiguous, e.g., amino acids 806 and 808 are deleted, or amino acids 810 and 813 are deleted. In embodiments, the CoV S polypeptide comprising a deletion corresponding to one or more amino acids of the fusion peptide has an amino acid sequence selected from SEQ ID NOs:66, 77, and 105-108.

[0088]

[0126] In embodiments, the CoV S polypeptide comprises a mutation at Lys-973 of a native CoV spike (S) polypeptide (SEQ ID NO: 2). In embodiments, Lys-973 is mutated to any native amino acid. In embodiments, Lys-973 is mutated to proline. In embodiments, Lys-973 is mutated to glycine. In embodiments, the CoV S polypeptide comprising a mutation at amino acid 973 is selected from the group consisting of SEQ ID NOs: 84-89, 105-106, and 109-110.

[0089]

[0127] In embodiments, the CoV S polypeptide comprises a mutation at Val-974 of a native CoV spike (S) polypeptide (SEQ ID NO: 2). In embodiments, Val-974 is mutated to any native amino acid. In embodiments, Val-974 is mutated to proline. In embodiments, Val-974 is mutated to glycine. In embodiments, the CoV S polypeptide comprising a mutation at amino acid 974 is selected from the group consisting of SEQ ID NOs: 84-89, 105-106, and 109-110.

[0090]

[0128] In embodiments, the CoV S polypeptide comprises mutations at Lys-973 and Val-974 of a native CoV spike (S) polypeptide (SEQ ID NO: 2). In embodiments, Lys-973 and Val-974 are mutated to any native amino acid. In embodiments, Lys-973 and Val-974 are mutated to proline. In embodiments, the CoV S polypeptide comprising mutations at amino acids 973 and 974 is selected from SEQ ID NOs: 84-89, 105-106, and 109-110. CoV S polypeptide antigen - Modifications to the S2 subunit - HR1 domain

[0129] In embodiments, the CoV S polypeptide comprises one or more modifications to the HR1 domain having the amino acid sequence of SEQ ID NO: 119, which corresponds to amino acids 912 to 984 of SEQ ID NO: 1 or amino acids 889 to 971 of SEQ ID NO: 2.

[0091]

[0130] The amino acid sequence of the HR1 domain (SEQ ID NO: 119) is shown below.

[0092] [ka]

[0093]

[0131] The underlined region of SEQ ID NO: 119 represents amino acids within the HR1 domain that may be modified.

[0094]

[0132] In embodiments, the CoV S 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 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 acid deletions, insertions, or mutations compared to the amino acid sequence of the HR1 domain of SEQ ID NO: 1 or SEQ ID NO: 2. The HR1 domain may have deletions, insertions, or mutations of about 1 to about 5 amino acids, about 3 to about 10 amino acids, about 5 to 10 amino acids, about 8 to 12 amino acids, about 10 to 15 amino acids, about 12 to 17 amino acids, about 15 to 20 amino acids, about 18 to 23 amino acids, about 20 to 25 amino acids, about 22 to about 27 amino acids, or about 25 to 30 amino acids, compared to the HR1 domain of SEQ ID NO: 1 or SEQ ID NO: 2.

[0095]

[0133] In embodiments, the HR1 domain may include any combination of modifications shown in Table 1G.

[0096] [Table 10]

[0097] CoV S polypeptide antigen - Modifications to the S2 subunit - HR2 domain

[0134] In embodiments, the CoV S polypeptide comprises one or more modifications to the HR2 domain having the amino acid sequence of SEQ ID NO: 125, which corresponds to amino acids 1163 to 1213 of SEQ ID NO: 1 or amino acids 1150 to 1200 of SEQ ID NO: 2.

[0098]

[0135] The amino acid sequence of the HR2 domain (SEQ ID NO: 125) is shown below. DVDLGDISGINASVVNIQKEIDRLNEVAKNLNESLIDLQELGKYEQYIKWP

[0136] In embodiments, the CoV S 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 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 acid deletions, insertions, or mutations compared to the amino acid sequence of the HR2 domain of SEQ ID NO: 1 or SEQ ID NO: 2. The HR2 domain may have deletions, insertions, or mutations of about 1 to about 5 amino acids, about 3 to about 10 amino acids, about 5 to 10 amino acids, about 8 to 12 amino acids, about 10 to 15 amino acids, about 12 to 17 amino acids, about 15 to 20 amino acids, about 18 to 23 amino acids, about 20 to 25 amino acids, about 22 to about 27 amino acids, or about 25 to 30 amino acids, compared to the HR2 domain of SEQ ID NO: 1 or SEQ ID NO: 2. CoV S polypeptide antigen - modifications to the TM domain

[0137] In embodiments, the CoV S polypeptide comprises one or more modifications to the TM domain having the amino acid sequence of SEQ ID NO: 123, which corresponds to amino acids 1214 to 1237 of SEQ ID NO: 1 or amino acids 1201 to 1224 of SEQ ID NO: 2.

[0099]

[0138] The amino acid sequence of the TM domain (SEQ ID NO: 123) is shown below. WYIWLGFIAGLIAIVMVTIMLCCM

[0139] In embodiments, the CoV S 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 can have 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 acid deletions, insertions, or mutations compared to the amino acid sequence of the TM domain of SEQ ID NO: 1 or SEQ ID NO: 2. The TM domain may have deletions, insertions, or mutations of about 1 to about 5 amino acids, about 3 to about 10 amino acids, about 5 to 10 amino acids, about 8 to 12 amino acids, about 10 to 15 amino acids, about 12 to 17 amino acids, about 15 to 20 amino acids, about 18 to 23 amino acids, about 20 to 25 amino acids, about 22 to about 27 amino acids, or about 25 to 30 amino acids, compared to the TM domain of SEQ ID NO: 1 or SEQ ID NO: 2.

[0100]

[0140] In embodiments, the CoV S polypeptides described herein lack the entire TM domain. In embodiments, the CoV S polypeptides comprise a TM domain. CoV S polypeptide antigen - modifications to CT

[0141] In embodiments, the CoV S polypeptide comprises one or more modifications to a CT having the amino acid sequence of SEQ ID NO: 124, which corresponds to amino acids 1238 to 1273 of SEQ ID NO: 1 or amino acids 1225 to 1260 of SEQ ID NO: 2.

[0101]

[0142] The amino acid sequence of CT (SEQ ID NO: 124) is shown below. TSCCSCLKGCCSCGSCCKFDEDDSEPVLKGVKLHYT

[0143] In embodiments, the CoV S 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 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 acid deletions, insertions, or mutations compared to the amino acid sequence of the CT of SEQ ID NO: 1 or SEQ ID NO: 2. Compared to the CT of SEQ ID NO: 1 or SEQ ID NO: 2, the CT may have deletions, insertions, or mutations of about 1 to about 5 amino acids, about 3 to about 10 amino acids, about 5 to 10 amino acids, about 8 to 12 amino acids, about 10 to 15 amino acids, about 12 to 17 amino acids, about 15 to 20 amino acids, about 18 to 23 amino acids, about 20 to 25 amino acids, about 22 to about 27 amino acids, or about 25 to 30 amino acids.

[0102]

[0144] In embodiments, the CoV S polypeptides described herein lack a CT. In embodiments, the CoV S polypeptides comprise a CT.

[0103]

[0145] In embodiments, the CoV S polypeptide comprises a TM and a CT. In embodiments, the CoV spike (S) polypeptide comprises a deletion of one or more amino acids from the transmembrane and cytoplasmic tail (TMCT) (corresponding to amino acids 1201-1260). The amino acid sequence of the TMCT is represented by SEQ ID NO: 39. In embodiments, CoV S polypeptides having deletions of one or more residues of the TMCT have enhanced protein expression. In embodiments, the CoV spike (S) polypeptides having one or more deletions from the TMCT have an amino acid sequence selected from the group consisting of SEQ ID NOs: 40, 41, 42, 52, 54, 59, 61, 88, and 89. In embodiments, the CoV S polypeptides having one or more deletions from the TM-CD are encoded by an isolated nucleic acid sequence selected from the group consisting of SEQ ID NOs: 39, 43, 53, and 60. CoV S polypeptide antigen - non-restrictive mutation combinations

[0146] In embodiments, the CoV S polypeptide comprises a deletion of amino acids 56 and 57 of the native CoV spike (S) polypeptide (SEQ ID NO: 2).

[0104]

[0147] In embodiments, the CoV S polypeptide comprises a deletion of amino acids 131 and 132 of the native CoV spike (S) polypeptide (SEQ ID NO: 2).

[0105]

[0148] In embodiments, the CoV S polypeptide comprises a deletion of amino acids 56 and 131 of the naturally occurring CoV spike (S) polypeptide (SEQ ID NO: 2). In embodiments, the CoV S polypeptide comprises a deletion of amino acids 57 and 131 of the naturally occurring CoV spike (S) polypeptide (SEQ ID NO: 2).

[0106]

[0149] In embodiments, the CoV S polypeptide comprises a deletion of amino acids 56, 57 and 131 of the native CoV spike (S) polypeptide (SEQ ID NO: 2).

[0107]

[0150] In embodiments, the CoV S polypeptide comprises a deletion of amino acids 56 and 132 of the native CoV spike (S) polypeptide (SEQ ID NO: 2).

[0108]

[0151] In embodiments, the CoV S polypeptide comprises a deletion of amino acids 57 and 132 of the native CoV spike (S) polypeptide (SEQ ID NO: 2).

[0109]

[0152] In embodiments, the CoV S polypeptide comprises a deletion of amino acids 56, 57 and 132 of the native CoV spike (S) polypeptide (SEQ ID NO: 2).

[0110]

[0153] In embodiments, the CoV S polypeptide comprises a deletion of amino acids 56, 57, 131 and 132 of the native CoV spike (S) polypeptide (SEQ ID NO: 2).

[0111]

[0154] In embodiments, the CoV S polypeptide comprises a mutation that stabilizes the pre-fusion conformation of the CoV S polypeptide. In embodiments, the CoV S polypeptide comprises a proline or glycine substitution that stabilizes the pre-fusion conformation. This strategy has been utilized to develop a pre-fusion stabilized MERS-CoV S protein, as described in Proc Natl Acad Sci USA. 2017 Aug 29; 114 (35): E7348-E7357; Sci Rep. 2018 Oct 24; 8 (1): 15701; U.S. Patent Application Publication No. 2020 / 0061185; and PCT Application No. PCT / US2017 / 058370, each of which is incorporated herein by reference in its entirety.

[0112]

[0155] In embodiments, the CoV S polypeptide comprises mutations at Lys-973 and Val-974 and an inactivated furin cleavage site. In embodiments, the CoV S polypeptide comprises mutations at Lys-973 and Val-974 to proline and an inactivated furin cleavage site having the amino acid sequence of QQAQ (SEQ ID NO:7) or GSAS (SEQ ID NO:96). Exemplary CoV S polypeptides comprising mutations at Lys-973 and Val-974 and an inactivated furin cleavage site are shown in Figure 8. In embodiments, the CoV S polypeptide comprising mutations at Lys-973 and Val-974 to proline and an inactivated furin cleavage site has the amino acid sequence of SEQ ID NO:86 or 87 and the nucleic acid sequence of SEQ ID NO:96.

[0113]

[0156] In embodiments, the CoV S polypeptide comprises mutations at Lys-973 and Val-974, an inactivated furin cleavage site, and a deletion of one or more amino acids in the fusion peptide. In embodiments, the CoV S polypeptide comprises mutations at Lys-973 and Val-974 to proline, an inactivated furin cleavage site, and a deletion of one or more amino acids in the fusion peptide having the amino acid sequence of QQAQ (SEQ ID NO:7) or GSAS (SEQ ID NO:96). In embodiments, the CoV S polypeptide comprising mutations at Lys-973 and Val-974 to proline, an inactivated furin cleavage site, and a deletion of one or more amino acids in the fusion peptide has the amino acid sequence of SEQ ID NO:105 or SEQ ID NO:106. In embodiments, the CoV S polypeptide comprises, relative to a native CoV spike (S) polypeptide (SEQ ID NO: 2), 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 having the amino acid sequence of QQAQ (SEQ ID NO: 7) or GSAS (SEQ ID NO: 96).

[0114]

[0157] In embodiments, the CoV S polypeptide comprises, relative to a native CoV spike (S) polypeptide (SEQ ID NO: 2), a mutation of Trp-139 to cysteine, a mutation of Leu-439 to arginine, a mutation of Lys-973 and Val-974 to proline, and an inactivated furin cleavage site having the amino acid sequence of QQAQ (SEQ ID NO: 7) or GSAS (SEQ ID NO: 96). In embodiments, the CoV S polypeptide comprises, relative to a native CoV spike (S) polypeptide (SEQ ID NO: 1), a mutation of Trp-152 to cysteine, a mutation of Leu-452 to arginine, a mutation of Ser-13 to isoleucine, a mutation of Lys-986 and Val-987 to proline, and an inactivated furin cleavage site having the amino acid sequence of QQAQ (SEQ ID NO: 7) or GSAS (SEQ ID NO: 96).

[0115]

[0158] In embodiments, the CoV S polypeptide comprises, relative to a native CoV spike (S) polypeptide (SEQ ID NO: 2), a mutation of Lys-404 to threonine or asparagine, a mutation of Glu-471 to lysine, a mutation of Asn-488 to tyrosine, a mutation of Leu-5 to phenylalanine, a mutation of Asp-67 to alanine, a mutation of Asp-202 to glycine, one or more deletions of amino acids 229-231, a mutation of Arg-233 ​​to isoleucine, mutations of Lys-973 and Val-974 to proline, and an inactivated furin cleavage site having the amino acid sequence of QQAQ (SEQ ID NO: 7) or GSAS (SEQ ID NO: 96).

[0116]

[0159] In embodiments, the CoV S polypeptide comprises, relative to a native CoV spike (S) polypeptide (SEQ ID NO: 2), a mutation of Asn-488 to tyrosine, a mutation of Lys-973 and Val-974 to proline, and an inactivated furin cleavage site having the amino acid sequence of QQAQ (SEQ ID NO: 7) or GSAS (SEQ ID NO: 96). In embodiments, the CoV S polypeptide with a mutation of Asn-488 to tyrosine, a mutation of Lys-973 and Val-974 to proline, and an inactivated furin cleavage site having the amino acid sequence of QQAQ (SEQ ID NO: 7) or GSAS (SEQ ID NO: 96) comprises the amino acid sequence of SEQ ID NO: 112.

[0117]

[0160] In embodiments, the CoV S polypeptide comprises, relative to a native CoV spike (S) polypeptide (SEQ ID NO: 2), a mutation of Asn-488 to tyrosine, a mutation of Lys-973 and Val-974 to proline, and an inactivated furin cleavage site having the amino acid sequence of QQAQ (SEQ ID NO: 7) or GSAS (SEQ ID NO: 96). In embodiments, the CoV S polypeptide having a mutation of Asn-488 to tyrosine, a mutation of Lys-973 and Val-974 to proline, and an inactivated furin cleavage site having the amino acid sequence of QQAQ (SEQ ID NO: 7) or GSAS (SEQ ID NO: 96) comprises the amino acid sequence of SEQ ID NO: 113.

[0118]

[0161] In embodiments, the CoV S polypeptide comprises, relative to a native CoV spike (S) polypeptide (SEQ ID NO: 2), a deletion of amino acids 56, 57, and 131, a mutation of Asn-488 to tyrosine, a mutation of Ala-557 to aspartic acid, a mutation of Asp-601 to glycine, a mutation of Pro-668 to histidine, a mutation of Thr-703 to isoleucine, a mutation of Ser-969 to alanine, a mutation of Asp-1105 to histidine, mutations of Lys-973 and Val-974 to proline, and an inactivated furin cleavage site having the amino acid sequence of QQAQ (SEQ ID NO: 7) or GSAS (SEQ ID NO: 96). In embodiments, the CoV S polypeptide having a deletion of amino acids 56, 57, and 131, a mutation of Asn-488 to tyrosine, a mutation of Ala-557 to aspartic acid, a mutation of Asp-601 to glycine, a mutation of Pro-668 to histidine, a mutation of Thr-703 to isoleucine, a mutation of Ser-969 to alanine, a mutation of Asp-1105 to histidine, a mutation of Lys-973 and Val-974 to proline, and an inactivated furin cleavage site having the amino acid sequence of QQAQ (SEQ ID NO: 7) or GSAS (SEQ ID NO: 96), comprises the amino acid sequence of SEQ ID NO: 114.

[0119]

[0162] In embodiments, the CoV S polypeptide has, compared to a native CoV spike (S) polypeptide (SEQ ID NO: 2), a deletion of amino acids 56, 57, and 132, a mutation of Asn-488 to tyrosine, a mutation of Ala-557 to aspartic acid, a mutation of Asp-601 to glycine, a mutation of Pro-668 to histidine, a mutation of Thr-703 to isoleucine, a mutation of Ser-969 to alanine, a mutation of Asp-1105 to histidine, a mutation of Lys-973 and Val-974 to proline, and an inactive CoV S polypeptide having the amino acid sequence of QQAQ (SEQ ID NO: 7) or GSAS (SEQ ID NO: 96). In embodiments, the CoV S polypeptide having a deletion of amino acids 56, 57, and 132, a mutation of Asn-488 to tyrosine, a mutation of Ala-557 to aspartic acid, a mutation of Asp-601 to glycine, a mutation of Pro-668 to histidine, a mutation of Thr-703 to isoleucine, a mutation of Ser-969 to alanine, a mutation of Asp-1105 to histidine, a mutation of Lys-973 and Val-974 to proline, and an inactivated furin cleavage site having the amino acid sequence of QQAQ (SEQ ID NO: 7) or GSAS (SEQ ID NO: 96), comprises the amino acid sequence of SEQ ID NO: 114.

[0120]

[0163] In embodiments, the CoV S polypeptide comprises, relative to a native CoV spike (S) polypeptide (SEQ ID NO: 2), an Asn-488 mutation to tyrosine, an Asp-67 mutation to alanine, a Leu-229 mutation to histidine, an Asp-202 mutation to glycine, a Lys-404 mutation to asparagine, a Glu-471 mutation to lysine, an Ala-688 mutation to valine, an Asp-601 mutation to glycine, mutations of Lys-973 and Val-974 to proline, and an inactivated furin cleavage site having the amino acid sequence of QQAQ (SEQ ID NO: 7) or GSAS (SEQ ID NO: 96). In embodiments, the CoV S polypeptide having an Asn-488 mutation to tyrosine, an Asp-67 mutation to alanine, a Leu-229 mutation to histidine, an Asp-202 mutation to glycine, a Lys-404 mutation to asparagine, a Glu-471 mutation to lysine, an Ala-688 mutation to valine, an Asp-601 mutation to glycine, a Lys-973 and a Val-974 mutation to proline, and an inactivated furin cleavage site having the amino acid sequence of QQAQ (SEQ ID NO: 7) or GSAS (SEQ ID NO: 96) comprises the amino acid sequence of SEQ ID NO: 115.

[0121]

[0164] In embodiments, the CoV spike (S) polypeptide comprises a polypeptide linker. In embodiments, the polypeptide linker comprises glycine and serine. In embodiments, the linker has about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or about 100% glycines.

[0122]

[0165] In embodiments, the polypeptide linker is (SGGG) n(SEQ ID NO:91), where n is an integer between 1 and 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.

[0123]

[0166] In embodiments, the polypeptide linker is (GGGGS) n (SEQ ID NO: 93), where n is an integer between 1 and 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).

[0124]

[0167] In embodiments, the polypeptide linker is (GGGS) n (SEQ ID NO: 92), where n is an integer between 1 and 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).

[0125]

[0168] In some aspects, the polypeptide linker is a poly-(Gly)n linker, where 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 a dipeptide, a tripeptide, and a quadripeptide. In embodiments, the linker is a dipeptide selected from the group consisting of alanine-serine (AS), leucine-glutamic acid (LE), and serine-arginine (SR).

[0126]

[0169] In embodiments, the polypeptide linker comprises 1 to 100 contiguous amino acids of a naturally occurring CoV S polypeptide or a CoV S polypeptide disclosed herein. In embodiments, the polypeptide linker has an amino acid sequence corresponding to SEQ ID NO:94.

[0127]

[0170] In embodiments, the CoV spike (S) polypeptide comprises a foldon. In embodiments, TMCT is replaced by a foldon. In embodiments, the foldon causes trimerization of the CoV spike (S) polypeptide. In embodiments, the foldon is an amino acid sequence known in the art. In embodiments, the foldon has 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 has the amino acid sequence of SEQ ID NO: 103. In embodiments, the foldon is separated in amino acid sequence from the CoV spike (S) polypeptide by a polypeptide linker. Non-limiting examples of polypeptide linkers are found throughout this disclosure.

[0128]

[0171] In embodiments, the present disclosure provides CoV S polypeptides, including fragments of coronavirus S protein and nanoparticles, and vaccines comprising the same. In embodiments, the coronavirus S protein fragment is 10 to 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, about 1450, or about 1500 amino acids in length). In embodiments, the fragment of a coronavirus S protein is selected from the group consisting of a receptor binding domain (RBD), subdomain 1, subdomain 2, upper helix, fusion peptide, binding region, heptad repeat 1, central helix, heptad repeat 2, NTD, and TMCT.

[0129]

[0172] In embodiments, the CoV S polypeptide comprises an RBD and subdomain 1. In embodiments, the CoV S polypeptide comprising an RBD and subdomain 1 is amino acids 319 to 591 of SEQ ID NO:1.

[0130]

[0173] In embodiments, the CoV S polypeptide comprises a fragment of a coronavirus S protein, wherein the fragment of the coronavirus S protein is an RBD. Non-limiting examples of RBDs include the RBD of SARS-CoV-2 (amino acid sequence = SEQ ID NO: 69), the RBD of SARS (amino acid sequence = SEQ ID NO: 70), and the RBD of MERS (amino acid sequence = SEQ ID NO: 71).

[0131]

[0174] In embodiments, the CoV S polypeptide comprises two or more RBDs linked by a polypeptide linker, hi embodiments, the polypeptide linker has the amino acid sequence of SEQ ID NO:90 or SEQ ID NO:94.

[0132]

[0175] In embodiments, the CoV S polypeptide comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 RBDs.

[0133]

[0176] In some embodiments, the CoV S polypeptide comprises two or more SARS-CoV-2 RBDs linked by a polypeptide linker. In embodiments, the antigen comprising two or more SARS-CoV-2 RBDs has an amino acid sequence corresponding to one of SEQ ID NOs: 72-75.

[0134]

[0177] In embodiments, the CoV S polypeptide comprises a SARS-CoV-2 RBD and a SARS RBD. In embodiments, the CoV S polypeptide comprises a SARS-CoV-2 RBD and a SARS RBD, each RBD separated by a polypeptide linker. In embodiments, the CoV S polypeptide comprising a SARS-CoV-2 RBD and a SARS RBD has an amino acid sequence selected from the group consisting of SEQ ID NOs: 76-79.

[0135]

[0178] In embodiments, the CoV S polypeptide comprises a SARS-CoV-2 RBD and a MERS RBD. In embodiments, the CoV S polypeptide comprises a SARS-CoV-2 RBD and a MERS RBD, each RBD separated by a polypeptide linker.

[0136]

[0179] In embodiments, the CoV S polypeptide comprises a SARS RBD and a MERS RBD. In embodiments, the CoV S polypeptide comprises a SARS RBD and a MERS RBD, each RBD separated by a polypeptide linker.

[0137]

[0180] In embodiments, the CoV S polypeptide comprises a SARS-CoV-2 RBD, a SARS RBD, and a MERS RBD. In embodiments, the CoV S polypeptide comprises a SARS-CoV-2 RBD, a SARS RBD, and a MERS RBD, each RBD being separated by a polypeptide linker. In embodiments, the CoV S polypeptide comprising a SARS-CoV-2 RBD, a SARS RBD, and a MERS RBD has an amino acid sequence selected from the group consisting of SEQ ID NOs: 80-83.

[0138]

[0181] In embodiments, the CoV S polypeptides described herein are expressed with an N-terminal signal peptide. In embodiments, the N-terminal signal peptide has the amino acid sequence of SEQ ID NO: 5 (MFVFLVLLPLVSS). In embodiments, the N-terminal signal peptide has the amino acid sequence of SEQ ID NO: 117 (MFVFLVLLPLVSI). In embodiments, the signal peptide can be replaced with any signal peptide that allows for expression of the CoV S protein. In embodiments, one or more of the amino acids of the CoV S protein signal peptide can be deleted or mutated. The initiation methionine residue is maintained to initiate expression. In embodiments, the CoV S polypeptide is 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, and SEQ ID NO: 60. In embodiments, the N-terminal signal peptide of the CoV S polypeptide comprises a mutation at Ser-13 compared to the native CoV spike (S) 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.

[0139]

[0182] Following expression of the CoV S protein in a host cell, the N-terminal signal peptide is cleaved to yield the mature CoV protein sequence (SEQ ID NOs: 2, 4, 38, 41, 44, 48, 51, 54, 58, 61, 63, 65, 67, 73, 75, 78, 79, 82, 83, 85, 87, 89, 106, and 110). In embodiments, the signal peptide is cleaved by a host cell protease. In aspects, the full-length protein can be isolated from the host cell, followed by cleavage of the signal peptide.

[0140]

[0183] After cleavage of the signal peptide from a CoV spike (S) polypeptide having an amino acid sequence corresponding to SEQ ID NOs: 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, and 109 during expression and purification, a mature polypeptide having an amino acid sequence selected from the group consisting 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 or 112-115 is obtained, and this mature polypeptide is used to produce a CoV S nanoparticle vaccine or CoV S nanoparticles.

[0141]

[0184] Advantageously, the disclosed CoV S polypeptides may exhibit enhanced protein expression and stability compared to native CoV spike (S) proteins.

[0142]

[0185] In embodiments, the CoV S polypeptides described herein contain further modifications from the native coronavirus S protein (SEQ ID NO: 2). In embodiments, the coronavirus S proteins described herein exhibit at least 80%, or at least 90%, or at least 95%, or at least 97%, or at least 99% identity to the native coronavirus S protein. One of skill in the art would use known techniques to calculate the percent identity of a recombinant coronavirus S protein to a native protein or any of the CoV S polypeptides described herein. For example, percentage identity can be calculated using the tools CLUSTALW2 or Basic Local Alignment Search Tool (BLAST), available online. For CLUSTALW2 pairwise alignments, the following default parameters can be used: Protein Weight Matrix=Gonnet; Gap Open=10; Gap Extension=0.1.

[0143]

[0186] In embodiments, the CoV S 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 polypeptide having the amino acid sequence of SEQ ID NO: 87. The CoV S polypeptide may have 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 acid deletions, insertions, or mutations compared to the amino acid sequence of a CoV S polypeptide having the amino acid sequence of SEQ ID NO: 87. The CoV S polypeptide may have deletions, insertions, or mutations of about 1 to about 5 amino acids, about 3 to about 10 amino acids, about 5 to 10 amino acids, about 8 to 12 amino acids, about 10 to 15 amino acids, about 12 to 17 amino acids, about 15 to 20 amino acids, about 18 to 23 amino acids, about 20 to 25 amino acids, about 22 to about 27 amino acids, about 25 to 30 amino acids, about 30 to 35 amino acids, about 35 to 40 amino acids, about 40 to 45 amino acids, or about 45 to 50 amino acids, compared to the CoV S polypeptide having the amino acid sequence of SEQ ID NO: 87. In embodiments, the CoV S 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).

[0144]

[0187] In embodiments, the coronavirus S polypeptide is extended at the N-terminus, C-terminus, or both the N-terminus and C-terminus. In some aspects, the extension is a tag useful for functions such as purification or detection. In some aspects, the tag comprises an epitope. For example, the tag can be a polyglutamic acid tag, a FLAG tag, an HA tag, a polyHis tag (having approximately 5-10 histidines) (SEQ ID NO: 101), a hexahistidine tag (SEQ ID NO: 100), an 8X-His tag (having 8 histidines) (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 can be an N-terminal signal peptide fused to the protein to facilitate expression. Such signal peptides are often cleaved during expression in cells, but some nanoparticles may contain antigens containing an intact signal peptide. Thus, if the nanoparticle contains an antigen, the antigen may include an extension, and thus, when incorporated into the nanoparticle, may be a fusion protein. For purposes of calculating sequence identity, the extension is not included. In embodiments, the tag is a protease cleavage site. Non-limiting examples of protease cleavage sites include HRV3C protease cleavage site, 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 an HRV3C protease cleavage site. In embodiments, the protease cleavage site comprises the amino acid sequence of SEQ ID NO:98.

[0145]

[0188] 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 includes a tag useful for protein expression, purification, or detection. In embodiments, the tag is a poly-His tag (having about 5-10 histidines), a Myc tag, a glutathione-S-transferase tag, a green fluorescent protein tag, a maltose binding protein tag, a thioredoxin tag, 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 a monomer, a dimer, or a trimer. In embodiments, the tag is a hexahistidine tag, e.g., a poly-His tag containing six histidines (SEQ ID NO: 100). In embodiments, the tag is a Twin-Strep tag having the amino acid sequence of SEQ ID NO: 99.

[0146]

[0189] In embodiments, the CoV S polypeptide is a fusion protein that includes another coronavirus protein. In embodiments, the other coronavirus protein is from the same coronavirus. In embodiments, the other coronavirus protein is from a different coronavirus.

[0147]

[0190] In some embodiments, the CoV S protein can be truncated. For example, the N-terminus can 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. The C-terminus can be truncated instead of or in addition to the N-terminus. For example, the C-terminus can 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 purposes of calculating identity to a protein having a truncation, identity is measured for the remainder of the protein. Nanoparticles containing CoV spike (S) polypeptide

[0191] In embodiments, mature CoV S polypeptide antigens are used to generate vaccines comprising coronavirus S nanoparticles. In embodiments, nanoparticles of the present disclosure comprise a CoV S polypeptide described herein. In embodiments, nanoparticles of the present disclosure comprise a CoV S polypeptide associated with a surfactant core. The presence of the surfactant promotes nanoparticle formation by forming a core that collectively presents the antigen. In embodiments, nanoparticles may comprise a CoV S polypeptide incorporated into a multi-oligomeric glycoprotein-surfactant (e.g., PS80) nanoparticle, in which the head regions protrude outward and the hydrophobic regions and PS80 surfactant form a central core surrounded by glycoproteins. In embodiments, the CoV S polypeptide inherently comprises or is adapted to comprise a transmembrane domain that promotes protein association into the surfactant core. In embodiments, the CoV S polypeptide comprises a head domain. Figure 10 shows an exemplary structure of a CoV S polypeptide of the present disclosure. Primarily, the transmembrane domain of the CoV S polypeptide trimer is associated with the surfactant; however, other portions of the polypeptide may also interact. Advantageously, the nanoparticles have improved resistance to environmental stresses, as multiple copies of the protein are packaged around the surfactant, improving stability and / or improving presentation to the immune system.

[0148]

[0192] In embodiments, the surfactant core is a non-ionic surfactant core. In embodiments, the CoV S polypeptide is associated with the non-ionic surfactant core. In embodiments, the surfactant is selected from the group consisting of polysorbate-20 (PS20), polysorbate-40 (PS40), polysorbate-60 (PS60), polysorbate-65 (PS65), and polysorbate-80 (PS80).

[0149]

[0193] In an embodiment, the surfactant is PS80.

[0150]

[0194] In embodiments, CoV S polypeptides form trimers. In embodiments, CoV S polypeptide nanoparticles are composed of multiple polypeptide trimers surrounding a non-ionic surfactant core. In embodiments, the nanoparticles comprise at least about one trimer or more. In embodiments, the nanoparticles comprise at least about 5 to about 30 trimers of spike protein. In embodiments, each nanoparticle may 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). Compositions disclosed herein may comprise nanoparticles having different numbers of trimers. For example, a composition may comprise nanoparticles in which the number of trimers ranges from 2 to 9; in embodiments, nanoparticles in a composition may comprise 2 to 6 trimers. In embodiments, a composition comprises a heterogeneous population of nanoparticles having 2 to 6 trimers per nanoparticle or 2 to 9 trimers per nanoparticle. In embodiments, the composition may include a substantially homogeneous population of nanoparticles, for example, the population may include about 95% of nanoparticles having 5 trimers.

[0151]

[0195] The nanoparticles disclosed herein vary in particle size. In embodiments, the nanoparticles disclosed herein have a Z-ave size ranging from about 20 nm to about 60 nm, about 20 nm to about 50 nm, about 20 nm to about 45 nm, about 20 nm to about 35 nm, about 20 nm to about 30 nm, about 25 nm to about 35 nm, or about 25 nm to about 45 nm. Unless otherwise specified, particle size (Z-ave) is measured by dynamic light scattering (DLS) using a Zetasizer NanoZS (Malvern, UK).

[0152]

[0196] In embodiments, nanoparticles comprising a CoV S polypeptide disclosed herein have a reduced particle size compared to nanoparticles comprising a wild-type CoV S polypeptide, in embodiments, the CoV S polypeptide has a particle size that is at least about 40% smaller, e.g., 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.

[0153]

[0197] Nanoparticles comprising CoV S polypeptides disclosed herein are more uniform in size, shape, and mass than nanoparticles comprising wild-type CoV S polypeptides. The polydispersity index (PDI), a measure of heterogeneity, is measured by dynamic light scattering using a Malvern Setasizer unless otherwise specified. In embodiments, the particles measured herein have a PDI of about 0.2 to about 0.45, e.g., 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 wild-type CoV S polypeptides, e.g., 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.

[0154]

[0198] CoV S polypeptides and nanoparticles comprising the same have improved thermal stability compared to wild-type CoV S polypeptides or nanoparticles thereof. The thermal stability of CoV S polypeptides is measured using differential scanning calorimetry (DSC) unless otherwise specified. The transition enthalpy (ΔHcal) is the energy required to unfold a CoV S polypeptide. In embodiments, the CoV S polypeptide has an increased ΔHcal compared to a wild-type CoV S polypeptide. In embodiments, the ΔHcal of the CoV S polypeptide is about 2-fold, about 3-fold, about 4-fold, about 5-fold, about 6-fold, about 7-fold, about 8-fold, about 9-fold, or about 10-fold greater than the ΔHcal of the wild-type CoV S polypeptide.

[0155]

[0199] The vaccine compositions disclosed herein may contain several nanoparticle types. In some embodiments, the nanoparticle types are in the form of anisotropic rods that may be dimeric or monomeric. In other embodiments, the nanoparticle types are spherical oligomers. In still other embodiments, the nanoparticles can be described as intermediate nanoparticles with sedimentation properties intermediate between the first two types. The formation of nanoparticle types can be regulated by controlling the concentrations of surfactants and proteins during the manufacturing process. The nanoparticle type can be determined by measuring the sedimentation coefficient. Production of nanoparticles containing CoV S polypeptide antigens

[0200] The nanoparticles of the present disclosure are non-naturally occurring products, and their components do not occur together in nature. Generally, the methods disclosed herein use a detergent exchange approach, in which a first detergent is used to isolate proteins, and then the first detergent is exchanged with a second detergent to form nanoparticles.

[0156]

[0201] Antigens contained in nanoparticles are typically produced by recombinant expression in host cells. Standard recombinant techniques can be used. In embodiments, CoV S polypeptides are expressed in insect host cells using a baculovirus system. In embodiments, the baculovirus is a cathepsin L knockout baculovirus and a chitinase knockout baculovirus. Optionally, the baculovirus is a double knockout baculovirus for both cathepsin L and chitinase. High-level expression can be achieved in insect cell expression systems. Non-limiting examples of insect cells are Spodoptera frugiperda (Sf) cells, e.g., Sf9 and Sf21, Trichoplusia ni cells, e.g., High Five cells, and Drosophila S2 cells. In embodiments, the CoV S polypeptides described herein are produced in any suitable host cell. In embodiments, the host cell is an insect cell. In embodiments, the insect cell is an Sf9 cell.

[0157]

[0202] Typical transfection and cell growth methods can be used to culture the cells. A vector, such as a vector containing a polynucleotide encoding a fusion protein, can be transfected into a host cell according to methods well known in the art. For example, introduction of a nucleic acid into a eukaryotic cell can be achieved by calcium phosphate co-precipitation, electroporation, microinjection, lipofection, and transfection using polyamine transfection reagents. In one embodiment, the vector is a recombinant baculovirus.

[0158]

[0203] Methods for growing host cells include, but are not limited to, batch, fed-batch, continuous, and perfusion cell culture techniques. Cell culture refers to the growth and propagation of cells in a bioreactor (fermentation chamber) where the cells grow and express proteins (e.g., recombinant proteins) for purification and isolation. Typically, cell culture is carried out in a bioreactor that is sterile and under controlled temperature and atmospheric conditions. A bioreactor is a chamber used for cell culture, and 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 sterilized plastic bag (e.g., Cellbag®, Wave Biotech, Bridgewater, NJ). In another embodiment, the sterilized plastic bag is a bag of approximately 50 L to 3500 L. Extraction and purification of nanoparticles containing CoV spike (S) protein antigen

[0204] After host cell growth, proteins can be recovered from the host cells using detergents and purification protocols. After growing the host cells for 48 to 96 hours, the cells are isolated from the culture medium and a detergent-containing solution is added to solubilize the cell membrane and release the protein into a detergent extract. Triton X-100 and TERGITOL® nonylphenol ethoxylate (also known as NP-9) are suitable detergents for extraction. The detergent can be added to a final concentration of about 0.1% to about 1.0%. For example, the concentration can be about 0.1%, about 0.2%, about 0.3%, about 0.5%, about 0.7%, about 0.8%, or about 1.0%. The concentration range can be about 0.1% to about 0.3%. In some embodiments, the concentration is about 0.5%.

[0159] [

[0205] In other embodiments, proteins can be isolated from host cells using a different first detergent. For example, the first detergent can be bis(polyethylene glycol bis[imidazoylcarbonyl]), nonoxynol-9, bis(polyethylene glycol bis[imidazoylcarbonyl]), BRIJ® 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, CREMOPHOR® EL Macrogol Glycerol Lysate, or CREMOPHOR® EL Macrogol Glycerol Lysate. Cinoleate, Decaethylene Glycol Monododecyl Ether, N-Decanoyl-N-Methylglucamine, n-Decyl α-D-Glucopyranoside, Decyl β-D-Maltopyranoside, n-Dodecanoyl-N-Methylglucamide, n-Dodecyl α-D-Maltoside, n-Dodecyl β-D-Maltoside, n-Dodecyl β-D-Maltoside, Heptaethylene Glycol Monodecyl Ether, Heptaethylene Glycol Monododecyl Ether, Heptaethylene Glycol Monotetradecyl Ether, n-Hexadecyl β-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)-α-D-glucopyranoside, nonaethylene glycol monododecyl ether, N-nonanoyl-N-methylglucamine, N-nonanoyl-N-methylglucamine, octaethylene glycol monodecyl ether, octaethylene glycol monododecyl ether, octaethylene glycol monohexadecyl ether, octaethylene glycol monooctadecyl ether, octaethylene glycol monotetradecyl ether, octyl-β-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 ether W-1, 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, quillaia bark saponin, SPAN® 20 sorbitan laurate, SPAN® 40 sorbitan monopalmitate, SPAN® 60 sorbitan stearate, SPAN® 65 sorbitan tristearate, SPAN (R) 80 Sorbitan Monooleate, SPAN (R) 85 Sorbitan Trioleate, TERGITOL (R) Secondary Alcohol Ethoxylate Type 15-S-12, TERGITOL (R) Secondary Alcohol Ethoxylate Type 15-S-30, TERGITOL (R) Secondary Alcohol Ethoxylate Type 15-S-5, TERGITOL (R) Secondary Alcohol Ethoxylate Type 15-S-7, TERGITOL (R) Secondary Alcohol Ethoxylate Type 15-S-9, TERGITOL (R) TERGITOL® Nonylphenol Ethoxylate NP-10 Type, TERGITOL® Nonylphenol Ethoxylate NP-4 Type, TERGITOL® Nonylphenol Ethoxylate NP-40 Type, TERGITOL® Nonylphenol Ethoxylate NP-7 Type, TERGITOL® Nonylphenol Ethoxylate NP-9 Type, TERGITOL® Branched Secondary Alcohol Ethoxylate TMN-10 Type, TERGITOL® Branched Secondary Alcohol Ethoxylate TMN-6 Type,TRITON™ X-100 polyethylene glycol tert-octylphenyl ether, or a combination thereof.

[0160]

[0206] The nanoparticles can then be isolated from the cellular debris using centrifugation. In embodiments, gradient centrifugation, such as using cesium chloride, sucrose, and iodixanol, can be used. Other techniques can alternatively or additionally be used, such as standard purification techniques including, for example, ion exchange chromatography, affinity chromatography, and gel filtration chromatography.

[0161]

[0207] For example, the first column can be an ion exchange chromatography resin such as FRACTOGEL® EMD methacrylate-based polymer bead TMAE (EMD Millipore), the second column can be a lentil (Lens culinaris) lectin affinity resin, and the third column can be a cation exchange column such as FRACTOGEL® EMD methacrylate-based polymer bead SO3 (EMD Millipore) resin. In other embodiments, the cation exchange column can be an MMC column or a Nuvia C Prime column (Bio-Rad Laboratories, Inc.). Preferably, the methods disclosed herein do not use detergent extraction columns; e.g., hydrophobic interaction columns. Such columns are often used to remove detergents during purification, but can adversely affect the methods disclosed herein. Surfactant exchange of nanoparticles containing CoV S polypeptide antigens

[0208] To form nanoparticles, the first detergent used to extract proteins from host cells is essentially replaced with a second detergent to arrive at a nanoparticle structure. NP-9 is a preferred extraction detergent. Typically, the nanoparticles contain no detectable NP-9 as 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.

[0162]

[0209] In certain embodiments, detergent exchange is performed using affinity chromatography to bind glycoproteins via their carbohydrate moieties. For example, affinity chromatography can use a legume lectin column. Legume lectins are proteins originally identified in plants and have been shown to interact specifically and reversibly with carbohydrate residues. See, for example, 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 a preferred column for detergent exchange due to its binding properties. Lectin columns are commercially available; for example, Capto Lentil Lectin is available from GE Healthcare. In some embodiments, the lentil lectin column can use recombinant lectin. At the molecular level, it is believed that carbohydrate moieties bind to the lentil lectin, freeing amino acids from the protein to coalesce around the surfactant, resulting in the formation of a surfactant core that provides nanoparticles bearing multiple copies of the antigen, e.g., glycoprotein oligomers that may be dimers, trimers, or tetramers immobilized on the surfactant. In embodiments, the CoV S polypeptide forms a trimer. In embodiments, the CoV S polypeptide trimer is immobilized on the surfactant. In embodiments, each CoV S polypeptide nanoparticle comprises at least one trimer associated with a non-ionic core.

[0163]

[0210] Detergents can be present at up to about 0.1% (w / v) during the initial purification steps when incubated with the protein to form nanoparticles during detergent exchange; this amount is reduced to achieve final nanoparticles with optimal stability. For example, nonionic detergents (e.g., PS80) can be present at concentrations of 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), and about 0.04% (v / v). The PS80 may be 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). In embodiments, the nanoparticles comprise about 0.03% to about 0.05% PS80. In embodiments, the nanoparticles comprise about 0.01% (v / v) PS80.

[0164]

[0211] In embodiments, the purified CoV S polypeptide is dialyzed. In embodiments, dialysis is performed after purification. In embodiments, the CoV S polypeptide is dialyzed in a solution comprising sodium phosphate, NaCl, and PS80. In embodiments, the sodium phosphate-containing dialysis solution comprises about 5 mM to about 100 mM sodium phosphate, for example, 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 an embodiment, the pH of the solution containing sodium phosphate is about 6.5, about 6.6, about 6.7, about 6.8, about 6.9, about 7.0, about 7.1, about 7.2, about 7.3, about 7.4, or about 7.5. In an embodiment, the dialysis solution containing sodium chloride has about 50 mM to about 500 mM NaCl, for example, 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, about 290 mM, about 300 mM, about 310 mM, about 320 mM, about 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 containing PS80 is 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 0.05% (v / v), about 0.06% (v / v), about 0.07% (v / v), about 0.08% (v / v), about 0.09% (v / v), about 0.10% (v / v), about 0.11% (v / v), about 0.12% (v / v), about 0.13% (v / v), about 0.14% (v / v), about 0.15% (v / v), about 0.16% (v / v), about 0.17% (v / v), about 0.18% (v / v), about 0.19% (v / v), about 0.20% (v / v), about 0.21% (v / v), about 0.22% (v / v), about 0.23% (v / v), about 0.24% (v / v), about 0.25% (v / v), about 0.26% (v / v), about 0.27% (v / v), about 0.28% (v / v), about 0.29% (v / v), about 0.30% (v / v), about 0.31% (v / v), about 0.32% (v / v), about 0.33% (v / % (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) of 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) of PS80.

[0165]

[0212] Detergent exchange can be performed on proteins that have been purified as described above, purified, frozen for storage, and then thawed for detergent exchange.

[0166]

[0213] The stability of the compositions disclosed herein can be measured in a variety of ways. In one approach, peptide maps can be generated to determine the integrity of antigenic proteins after various treatments designed to stress nanoparticles by mimicking harsh storage conditions. Thus, a measure of stability is the relative abundance of antigenic peptides in stressed samples compared to control samples. For example, the stability of nanoparticles containing CoV S polypeptides can be assessed by exposing them to various pHs, proteases, salts, oxidizing agents, including but not limited to, hydrogen peroxide, various temperatures, freeze / thaw cycles, and agitation. Figures 12A and 12B show that BV2373 (SEQ ID NO: 87) and BV2365 (SEQ ID NO: 4) maintain binding to hACE2 under various stress conditions. The location of the glycoprotein anchored to the surfactant core is believed to enhance stability by reducing undesirable interactions. For example, improved protection against protease-based degradation can be achieved by anchoring the glycoprotein to the core at the molar ratios disclosed herein, resulting in a shielding effect where steric hindrance blocks protease access. Stability can also be measured by monitoring intact protein. Figures 33 and 34 compare nanoparticles containing CoV polypeptides having the amino acid sequences of SEQ ID NOs: 109 and 87, respectively. Figure 34 shows that the CoV polypeptide having the amino acid sequence of SEQ ID NO: 87 exhibits particularly good stability during purification. The polypeptide in Figure 34 contains a furin cleavage site with the amino acid sequence of QQAQ (SEQ ID NO: 7). Vaccine compositions comprising CoV S polypeptide antigens

[0214] The present disclosure provides vaccine compositions comprising CoV S polypeptides, e.g., in nanoparticles. In some aspects, the vaccine compositions may comprise nanoparticles comprising antigens from two or more viral strains from the same virus species. In another embodiment, the present disclosure provides a pharmaceutical pack or kit comprising one or more containers filled with one or more of the components of the vaccine composition.

[0167]

[0215] The compositions disclosed herein can be used either prophylactically or therapeutically, typically prophylactically. Accordingly, the present disclosure includes a method for treating or preventing an infection. The method comprises administering a therapeutic or prophylactic amount of an immunogenic composition of the present disclosure to a subject. Preferably, the pharmaceutical composition is a vaccine composition that provides a protective effect. In other aspects, the protective effect may include an improvement in symptoms associated with the infection in a proportion of an exposed population. For example, the composition may prevent or reduce one or more symptoms of a viral disease selected from heat exhaustion, muscle pain, headache, sore throat, vomiting, diarrhea, rash, symptoms of kidney and liver dysfunction, internal bleeding, and external bleeding, compared to untreated subjects.

[0168]

[0216] The nanoparticles can be formulated for administration as a vaccine in the presence of various excipients, buffers, and the like. For example, a vaccine composition can include sodium phosphate, sodium chloride, and / or histidine. Sodium phosphate can be present at about 10 mM to about 50 mM, about 15 mM to about 25 mM, or about 25 mM; in a particular example, about 22 mM sodium phosphate is present. Histidine can be present at 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, can be about 150 mM. In certain compositions, sodium chloride can be present at higher concentrations, for example, 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.

[0169]

[0217] In embodiments, the nanoparticles described herein have improved stability at specific pH levels. In embodiments, the nanoparticles are stable at slightly acidic pH levels. For example, the nanoparticles are stable at slightly acidic pHs, such as pH 5.8 to pH 7.0. In embodiments, the nanoparticles and compositions comprising the nanoparticles may be stable at a pH range of about pH 5.8 to about pH 7.0, including about pH 5.9 to about pH 6.8, about pH 6.0 to about pH 6.5, about pH 6.1 to about pH 6.4, 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 pHs, including about pH 7.0 to about pH 7.4. In embodiments, the nanoparticles and compositions described herein are stable at slightly alkaline pHs, such as about pH 7.0 to about pH 8.5, about pH 7.0 to about pH 8.0, or about pH 7.0 to about pH 7.5, including all values ​​and ranges therebetween. Adjuvants

[0218] 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 can be prepared without an adjuvant and thus administered as adjuvant-free compositions. Advantageously, the adjuvant-free compositions disclosed herein can provide a protective immune response when administered as a single dose. Alum-free compositions that induce a strong immune response are particularly useful in adults over the age of about 60. Aluminum-based adjuvants

[0219] In embodiments, the adjuvant can be alum (e.g., AlPO4 or Al(OH)3). Typically, the nanoparticles are substantially bound to the alum. For example, the nanoparticles 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% to 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 can be present in an amount of about 300 μg to about 900 μg, about 400 μg to about 800 μg, about 500 μg to about 700 μg, about 400 μg to about 600 μg, or about 400 μg to about 500 μg per dose. Typically, about 400 μg of alum is present in a 120 μg dose of protein nanoparticles. Saponin adjuvants

[0220] Adjuvants containing saponins can also be combined with the immunogens disclosed herein. Saponins are glycosides derived from the bark of the Quillaja saponaria Molina tree. Typically, saponins are prepared using a multi-step purification process to obtain multiple fractions. As used herein, the term "saponin fraction from Quillaja saponaria Molina" is used generally to refer to a semi-purified or defined saponin fraction of Quillaja saponaria, or a substantially pure fraction thereof. Saponin fraction

[0221] Several approaches are suitable for producing saponin fractions. Fractions A, B, and C are described in U.S. Patent No. 6,352,697 and can be prepared as follows: The lipophilic fraction from Quil A, a crude aqueous Quillaja saponaria Molina extract, 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 using a gradient of 25% to 60% acetonitrile in acidic water. The fraction referred to herein as "Fraction A" or "QH-A" corresponds to or is eluted at approximately 39% acetonitrile. The fraction referred to herein as "Fraction B" or "QH-B" corresponds to or is eluted at approximately 47% acetonitrile. The fraction referred to herein as "Fraction C" or "QH-C" is or corresponds to the fraction eluted at approximately 49% acetonitrile. Additional information regarding fraction purification can be found in U.S. Pat. No. 5,057,540. When prepared as described herein, Quillaja saponaria Molina fractions A, B, and C each represent a group or family of closely chemically related molecules with definable properties. The chromatographic conditions under which fractions A, B, and C are obtained are such that batch-to-batch reproducibility with respect to elution profile and biological activity is highly consistent.

[0170]

[0222] Other saponin fractions have also been described: fractions B3, B4, and B4b are described in EP 0436620. Fractions QA1 to QA22 are described in EP 03632279B2, Q-VAC (Nor-Feed, AS Denmark), and Quillaja saponaria Molina Spikoside (Isconova AB, Ultunaallen 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 of EP 03632279B2 can be used, in particular QA-7, QA-17, QA-18 and QA-21, which can be obtained as described in Example 1 of EP 03632279B2, in particular pages 6 and 8 and 9.

[0171]

[0223] The saponin fractions described herein and used to form adjuvants are often substantially pure fractions; i.e., the fractions are substantially free of contaminating other substances. In certain embodiments, a substantially pure saponin fraction may contain up to 40%, up to 30%, up to 25%, up to 20%, up to 15%, up to 10%, up to 7%, up to 5%, up to 2%, up to 1%, up to 0.5%, or up to 0.1% by weight of other compounds, such as other saponins or other adjuvant substances. ISCOM Structure

[0224] Saponin fractions can be administered in the form of cage-like particles called ISCOMs (immunostimulating complexes). ISCOMs can be prepared as described in European Patent Nos. 0109942B1, 0242380B1, and 0180546B1. In certain embodiments, transport and / or passenger antigens can be used, as described in European Patent No. 9600647-3 (PCT / Swedish Patent No. 97 / 00289). Matrix adjuvants

[0225] 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. The ISCOM matrix complex may also comprise one or more other immunomodulatory (adjuvant-active) substances that are not necessarily glycosides, and may be produced as described in EP 0 436 620 B1, which is incorporated herein by reference in its entirety.

[0172]

[0226] In another embodiment, the ISCOM is an ISCOM complex. An ISCOM complex comprises at least one saponin, at least one lipid, and at least one antigen or epitope. The ISCOM complex comprises an antigen associated with the particle by detergent treatment, such that a portion of the antigen is incorporated into the particle. In contrast, an ISCOM matrix is ​​formulated as a mixture with the antigen, and the association between the ISCOM matrix particle and the antigen is mediated by electrostatic and / or hydrophobic interactions.

[0173]

[0227] According to one embodiment, the ISCOM matrix complex or the saponin fraction incorporated into the ISCOM complex, or at least one additional adjuvant also incorporated into or mixed with the ISCOM or ISCOM matrix complex, is selected from Quillaja saponaria, a semi-purified preparation of Quillaja saponaria, fraction A, fraction B or fraction C of a purified preparation of Quillaja saponaria, or any purified subfraction, such as QA1 to 21.

[0174]

[0228] In certain embodiments, each ISCOM particle may contain at least two saponin fractions. Any combination of weight percents of different saponin fractions may be used. Any combination of weight percents of any two fractions may be used. For example, a particle may contain any weight percent of Fraction A and any weight percent of another saponin fraction, such as a crude saponin fraction or Fraction C, respectively. Thus, in certain embodiments, each ISCOM matrix particle or each ISCOM complex particle may contain 0.1-99.9%, 5-95%, 10-90%, 15-85%, 20-80%, 25-75%, 30-70%, 35-65%, 40-60%, 45-55%, 40-60%, or 50% by weight of one saponin fraction, e.g., fraction A, and the remainder, in each case up to 100%, of another fraction, e.g., any coarse fraction, or any other fraction, e.g., fraction C. Weights are calculated as the total weight of the saponin fractions. Examples of ISCOM matrix complex and ISCOM complex adjuvants are disclosed in U.S. Patent Application Publication No. 2013 / 0129770, which is incorporated herein by reference in its entirety.

[0175]

[0229] In certain embodiments, the ISCOM matrix or ISCOM complex comprises 5-99% by weight of one fraction, e.g., fraction A, and the remainder up to 100% by weight, e.g., crude saponin fraction or fraction C. Weights are calculated as the total weight of the saponin fractions.

[0176]

[0230] In another embodiment, the ISCOM matrix or ISCOM complex comprises 40-99% by weight of one fraction, such as fraction A, and 1-60% by weight of another fraction, such as the crude saponin fraction or fraction C. Weights are calculated as the total weight of the saponin fractions.

[0177]

[0231] In yet another embodiment, the ISCOM matrix or ISCOM complex comprises 70-95% by weight of one fraction, such as fraction A, and 30-5% by weight of another fraction, such as the crude saponin fraction or fraction C. Weights are calculated as the total weight of the saponin fractions. In another embodiment, the saponin fraction from Quillaja saponaria Molina is selected from any one of QA1-21.

[0178]

[0232] In addition to particles containing a mixture of saponin fractions, ISCOM matrix particles and ISCOM complex particles can each be formed using only one saponin fraction. The compositions disclosed herein can include multiple particles, each containing only one saponin fraction. That is, a particular composition can include one or more different types of ISCOM matrix complex particles and / or one or more different types of ISCOM complex particles, each containing a single saponin fraction from Quillaja saponaria Molina, and the saponin fraction in one complex particle being different from the saponin fraction in another complex particle.

[0179]

[0233] In certain embodiments, one type of saponin fraction or crude saponin fraction can be incorporated into one ISCOM matrix complex or particle, and another type of substantially pure saponin fraction or crude saponin fraction can be incorporated into another ISCOM matrix complex or particle. A composition or vaccine can comprise at least two types of complexes or particles, each type having one type of saponin incorporated into a physically distinct particle.

[0180]

[0234] The compositions may use mixtures of ISCOM matrix complex particles and / or ISCOM complex particles in which one saponin fraction of Quillaja saponaria Molina and another saponin fraction of Quillaja saponaria Molina are separately incorporated into different ISCOM matrix complex particles and / or ISCOM complex particles.

[0181]

[0235] ISCOM matrices or ISCOM complex particles, each containing one saponin fraction, can be present in the composition in any combination of weight percents. In certain embodiments, the composition can contain 0.1-99.9%, 5-95%, 10-90%, 15-85%, 20-80%, 25-75%, 30-70%, 35-65%, 40-60%, 45-55%, 40-60%, or 50% by weight of an ISCOM matrix or complex containing a first saponin fraction, with the remainder being made up of ISCOM matrices or complexes containing a different saponin fraction. In some embodiments, the remainder is one or more ISCOM matrices or complexes, each matrix or complex particle containing only one saponin fraction. In other embodiments, an ISCOM matrix or complex particle can contain two or more saponin fractions.

[0182]

[0236] In certain compositions, the only saponin fraction in the first ISCOM matrix or ISCOM complex particle is fraction A, and the only saponin fraction in the second ISCOM matrix or ISCOM complex particle is fraction C.

[0183]

[0237] A preferred composition comprises a first ISCOM matrix comprising Fraction A and a second ISCOM matrix comprising Fraction C, with the Fraction A ISCOM matrix comprising about 70% by weight of the total saponin adjuvant and the Fraction C ISCOM matrix comprising about 30% by weight of the total saponin adjuvant. In another preferred composition, the Fraction A ISCOM matrix comprises about 85% by weight of the total saponin adjuvant and the Fraction C ISCOM matrix comprises about 15% by weight of the total saponin adjuvant. Thus, in certain compositions, the Fraction A ISCOM matrix will be present in an amount ranging from about 70% to about 85% by weight of the total saponin adjuvant in the composition, and the Fraction C ISCOM matrix will be present in an amount ranging from about 15% to about 30% by weight of the total saponin adjuvant in the composition. In embodiments, the Fraction A ISCOM matrix comprises 50-96% by weight of the combined weight of the Fraction A ISCOM matrix and Fraction C ISCOMs in the adjuvant, with the Fraction C ISCOM matrix comprising the remainder. In a particularly preferred composition herein referred to as MATRIX-M™, the Fraction A ISCOM matrix is ​​present at about 85% of the total weight of the saponin adjuvant in the composition, and the Fraction C ISCOM matrix is ​​present at about 15% of the total weight of the saponin adjuvant in the composition. MATRIX-M™ may be referred to interchangeably as Matrix-M1.

[0184]

[0238] Exemplary QS-7 and QS-21 fractions, their preparation and their uses 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.

[0185]

[0239] Other adjuvants may be used in addition to or instead of in some compositions. It is contemplated within the scope of this disclosure to include any of the adjuvants described in Vogel et al., "A Compendium of Vaccine Adjuvants and Excipients (2nd Edition)," which is incorporated herein by reference in its entirety for all purposes. Other adjuvants include complete Freund's adjuvant (a non-specific stimulator of the immune response containing killed Mycobacterium tuberculosis), incomplete Freund's adjuvant, 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 (containing three components extracted from bacteria), MPL, trehalose dimycolate (TDM), and cell wall skeleton (CWS) (in a 2% squalene / TWEEN® polysorbate 80 emulsion). In embodiments, the adjuvant can be paucilamellar lipid vesicles, such as NOVASOMES®. NOVASOMES® are paucilamellar lipid vesicles ranging from about 100 nm to about 500 nm in size. NOVASOMES® contains BRIJ® alcohol ethoxylate 72, cholesterol, oleic acid, and squalene. NOVASOMES® has been shown to be an effective adjuvant (see US Pat. Nos. 5,629,021, 6,387,373, and 4,911,928). Administration and Dosage

[0240] In embodiments, the present disclosure provides methods for eliciting an immune response against one or more coronaviruses. In embodiments, the immune response is against one or more of the SARS-CoV-2 virus, MERS, and SARS. The method comprises administering to a subject an immunologically effective amount of a composition comprising nanoparticles or a recombinant CoV spike (S) polypeptide. Advantageously, the proteins disclosed herein elicit one or more particularly useful anti-coronavirus responses.

[0186]

[0241] In embodiments, the nanoparticles or CoV S polypeptide are administered with an adjuvant. In other aspects, the nanoparticles or CoV S polypeptide are administered without an adjuvant. In some aspects, the adjuvant can be bound to the nanoparticles, such as by a non-covalent interaction. In other aspects, the adjuvant is co-administered with the nanoparticles, but the adjuvant and the nanoparticles do not substantially interact.

[0187]

[0242] In embodiments, the nanoparticles may be used for the prevention and / or treatment of one or more of SARS-CoV-2 infection, SARS infection, or MERS infection. Accordingly, the present disclosure provides a method of eliciting an immune response against one or more of the SARS-CoV-2 virus, MERS, and SARS. The method comprises administering to a subject an immunologically effective amount of a composition comprising nanoparticles or a CoV S polypeptide. Advantageously, the proteins disclosed herein elicit a particularly useful anti-coronavirus response.

[0188]

[0243] The compositions disclosed herein can be administered via a systemic route, a mucosal route, a transdermal route, or directly to a specific tissue. As used herein, the term "systemic administration" includes parenteral administration routes. In particular, parenteral administration includes subcutaneous, intraperitoneal, intravenous, intraarterial, intramuscular, or intrasternal injection, intravenous, or kidney dialysis infusion techniques. Typically, systemic parenteral administration is intramuscular injection. As used herein, the term "mucosal administration" includes oral, intranasal, intravaginal, intrarectal, intratracheal, intraintestinal, and intraocular administration. Preferably, administration is intramuscular.

[0189]

[0244] The compositions can be administered in a single dose schedule or a multiple dose schedule. Multiple doses can be used in a primary immunization schedule or a booster schedule. In a multiple dose schedule, various doses can be administered by the same or different routes, e.g., a parenteral prime and a mucosal boost, a mucosal prime and a parenteral boost, etc. In some aspects, subsequent boost doses are administered about 2 weeks, about 3 weeks, about 4 weeks, about 5 weeks, or about 6 weeks after the previous dose. In embodiments, subsequent boost doses are administered 3 weeks after the previous dose. In embodiments, a primary dose is administered on day 0 and a boost dose is administered on day 21. In embodiments, a primary dose is administered on day 0 and a boost dose is administered on day 28.

[0190]

[0245] In embodiments, the dose measured in μg can be the total weight of the dose including solute, or the weight of the CoV S polypeptide nanoparticles, or the weight of the CoV S polypeptide. The dose is measured using a protein concentration assay, either A280 or ELISA.

[0191]

[0246] The dose of antigen, including for pediatric administration, can range from about 5 μg to about 25 μg, about 1 μg to about 300 μg, about 90 μg to about 270 μg, about 100 μg to about 160 μg, about 110 μg to about 150 μg, about 120 μg to about 140 μg, or about 140 μg to about 160 μg. In embodiments, the dose is about 120 μg and is administered with alum. In some aspects, the pediatric dose can range from about 1 μg to about 90 μg. In embodiments, the dose of CoV spike (S) 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, 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, about 22, about 23, about 24, about 25 μg, about 26 μg, about 27 μg, about 28 μg, about 29 μg, about 30 μg, about 40 μg, about 41 μg, about 42 μg, about 43 μg, about 44 μg, about 45 μg, about 46 μg, about 47 μg, about 48 μg, about 49 μg, about 50 μg, about 51 μg, about 52 μg, about 53 μg, about 54 μg, about 55 μg, about 56 μg, about 57 μg, about 58 μg, about 59 μg, about 60 μg, about 61 μg, about 62 μg, about 63 μg, about 64 μg, about 65 μg, about 66 μg, about 67 μg, about 68 μg, about 69 μg, about 70 μg, about 71 μg, about 72 μg, about 73 μg, about 74 μg, about 75 μg, about 76 μg, about 77 μg, about 78 μg, about 79 μg, about 80 μg, about 81 μg, about 82 μg, about 8 In some embodiments, the dose of CoV S polypeptide is about 5 μg. In some embodiments, the dose of CoV S polypeptide is about 2 ...

[0192]

[0247] Certain populations can be administered with or without an adjuvant. In certain embodiments, the composition may not include an added adjuvant. In such cases, the dose may be increased by about 10%.

[0193]

[0248] In embodiments, the dose of adjuvant administered with the non-naturally occurring CoV S polypeptide is between about 1 μg and about 100 μg, e.g., about 1 μg, about 2 μg, about 3 μg, about 4 μg, about 5 μg, about 6 μg, about 7 μg, about 8 μg, about 9 μg, about 10 μg, about 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 μg, about 26 μg, about 27 μg, about 28 μg, about 29 μg, about 30 μg, about 31 μg, about 32 μg, about 33 μg, about 34 μg, about 35 μg, about 36 μg, about 37 μg, about 38 μg, about 39 μg, about 40 μg, about 41 μg, about 42 μg, about 43 μg, about 44 μg, about 45 μg, about 46 μg, about 47 μg, about 48 μg, about 49 μg, about 50 μg, about 51 μg, about 52 μg, about 53 μg, about 54 μg, about 55 μg, about 56 μg, about 57 μg, about 58 μg, about 59 μg, about 60 μg, about 61 μg, about 62 μg, about 63 μg, about 64 μg, about 65 μg, about 66 μg, about 67 μg, about 68 μg, about , about 23, about 24, about 25μg, about 26μg, about 27μg, about 28μg, about 29μg, about 30μg, about 31μg, about 32μg, about 33μg, about 34μg, about 35μg, about 36μ g, about 37μg, about 38μg, about 39μg, about 40μg, about 41μg, about 42μg, about 43μg, about 44μg, about 45μg, about 46μg, about 47μg, about 48μg, about 49μg, Approximately 50μg, approximately 51μg, approximately 52μg, approximately 53μg, approximately 54μg, approximately 55μg, approximately 56μg, approximately 57μg, approximately 58μg, approximately 59μg, approximately 60μg, approximately 61μg, approximately 62μg, approximately 63μg, about 64μg, about 65μg, about 66μg, about 67μg, about 68μg, about 69μg, about 70μg, about 71μg, about 72μg, about 73μg, about 74μg, about 75μg, about 76 In some embodiments, the adjuvant is administered in an amount of about 77 μg, about 78 μg, 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™.

[0194]

[0249] In embodiments, the dose is administered in a volume of about 0.1 mL to about 1.5 mL, e.g., 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.

[0195]

[0250] In certain embodiments of vaccines against MERS, SARS or SARS-CoV-2 coronaviruses, a dose may comprise a CoV S polypeptide concentration of about 1 μg / mL to about 50 μg / mL, 10 μg / mL to about 100 μg / mL, about 10 μg / mL to about 50 μg / mL, about 175 μg / mL to about 325 μg / mL, about 200 μg / mL to about 300 μg / mL, about 220 μg / mL to about 280 μg / mL, or about 240 μg / mL to about 260 μg / mL.

[0196]

[0251] In another embodiment, the present disclosure provides a method for formulating a vaccine composition for inducing immunity to an infection or at least one disease symptom thereof in a mammal, comprising adding an effective dose of nanoparticles or a CoV S polypeptide to the composition. The disclosed CoV S polypeptides and nanoparticles are useful in preparing compositions that stimulate an immune response that confers immunity or substantial immunity to an infectious pathogen. Thus, in one embodiment, the present disclosure provides a method for inducing immunity to an infection or at least one disease symptom thereof in a subject, comprising administering at least one effective dose of nanoparticles and / or a CoV S polypeptide.

[0197]

[0252] In embodiments, the CoV S polypeptide or nanoparticles comprising same are administered in combination with an additional immunogenic composition. In embodiments, the additional immunogenic composition elicits an immune response against SARS-CoV-2. In embodiments, the additional immunogenic composition is administered within 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, about 20 hours, about 21 hours, about 22 hours, or about 24 hours of the disclosed CoV S polypeptide or nanoparticles comprising same. In embodiments, the additional composition is administered within about 23 hours, about 1 day, about 2 days, about 3 days, about 4 days, about 5 days, about 6 days, about 7 days, about 8 days, about 9 days, about 10 days, about 11 days, about 12 days, about 13 days, about 14 days, about 15 days, about 16 days, about 17 days, about 18 days, about 19 days, about 20 days, about 21 days, about 22 days, about 23 days, about 24 days, about 25 days, about 26 days, about 27 days, about 28 days, about 29 days, about 30 days, or about 31 days. In embodiments, the additional composition is administered with an initial dose of a composition comprising a CoV S polypeptide or nanoparticles comprising same. In embodiments, the additional composition is administered with a booster dose of a composition comprising a CoV S polypeptide or nanoparticles comprising same.

[0198]

[0253] 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.

[0199]

[0254] In embodiments, additional immunogenic compositions comprise an mRNA encoding a CoV S polypeptide. In embodiments, the mRNA encodes a CoV S polypeptide comprising proline substitutions at positions 986 and 987 of SEQ ID NO: 1. In embodiments, the mRNA encodes a CoV S polypeptide comprising an intact furin cleavage site. In embodiments, the mRNA encodes a CoV S polypeptide comprising 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 polypeptide comprising 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 polypeptide having the amino acid sequence of SEQ ID NO: 87. The mRNA encoding the CoV S polypeptide is encapsulated within a lipid nanoparticle. An example of an immunogenic composition comprising an mRNA encoding a CoV S 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 polypeptide is administered in a dose of 25 μg, 100 μg or 250 μg.

[0200]

[0255] In embodiments, additional immunogenic compositions comprise an adenoviral vector encoding a CoV S polypeptide. In embodiments, the AAV vector encodes a wild-type CoV S polypeptide. In embodiments, the AAV vector encodes a CoV S polypeptide comprising a proline substitution at positions 986 and 987 of SEQ ID NO: 1 and an intact furin cleavage site. In embodiments, the AAV vector encodes a CoV S polypeptide comprising a proline substitution at positions 986 and 987 of SEQ ID NO: 1 and an inactive furin cleavage site. In embodiments, the AAV vector encodes a CoV S polypeptide having the amino acid sequence of SEQ ID NO: 87. The following publications, each of which is incorporated by reference in its entirety, describe immunogenic compositions comprising adenoviral vectors encoding CoV S polypeptides: 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).

[0201]

[0256] 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 polypeptide. In embodiments, the DNA encodes a CoV S polypeptide comprising 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 polypeptide comprising 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 polypeptide having the amino acid sequence of SEQ ID NO:87.

[0202]

[0257] In embodiments, the additional immunogenic composition includes an inactivated virus vaccine.

[0203]

[0258] In embodiments, CoV S protein or nanoparticles comprising CoV S protein are useful for preparing immunogenic compositions that stimulate immune responses that confer immunity or substantial immunity to one or more of MERS, SARS, and SARS-CoV-2. Both mucosal and cellular immunity can contribute to immunity against infection 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, while serum IgG is involved in protecting the lower respiratory tract. Immune responses elicited by infection protect against reinfection with the same virus or antigenically similar virus strains. Antibodies produced in a host after immunization with the nanoparticles disclosed herein can be administered to others, thereby providing passive administration to a subject.

[0204]

[0259] In embodiments, the CoV S protein or nanoparticles comprising the CoV S protein are (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 a combination thereof; and (b) a mutation of one or more amino acids in the NTD, wherein the one or more mutations are selected from the group consisting of amino acids 67, 229, 202, 139, 5, 233, 7, 13, 125, 177, or a combination thereof; (c) a mutation of one or more amino acids in the RBD, wherein the one or more mutations are selected from the group consisting of amino acids 488, 404, 471, 439, 426, 440, and combinations thereof; (d) a mutation to one or more amino acids of SD1 / 2, wherein the one or more amino acids are selected from the group consisting of 601, 557, 668, 642, and combinations thereof; (e) an inactive furin cleavage site (corresponding to one or more mutations in amino acids 669 to 672); (f) a deletion of one or more amino acids in 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) mutation of 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, and 1014; and combinations thereof; (h) A deletion 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. The present invention induces cross-neutralizing antibodies against SARS-CoV-2 viruses containing S proteins having one or more modifications selected from the group consisting of:

[0205]

[0260] In embodiments, the CoV S protein or nanoparticles comprising the CoV S protein induce cross-neutralizing antibodies against SARS-CoV-2 viruses comprising an S protein having 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, where these amino acids are numbered relative to the CoV S polypeptide having the amino acid sequence of SEQ ID NO: 2.

[0206]

[0261] In embodiments, the present disclosure provides methods for producing one or more high-affinity anti-MERS-CoV, anti-SARS-CoV, and anti-SARS-CoV-2 virus antibodies. High-affinity antibodies produced by immunization with the nanoparticles disclosed herein are produced by administering to an animal an immunogenic composition comprising an S CoV polypeptide or nanoparticles comprising an S CoV polypeptide, collecting serum and / or plasma from the animal, and purifying the antibodies from the serum and / or plasma. In one embodiment, the animal is a human. In an embodiment, the animal is a chicken, mouse, guinea pig, rat, rabbit, goat, human, horse, sheep, or cow. In one embodiment, the animal is a bovine or equine animal. In another embodiment, the bovine or equine animal is transgenic. In yet another embodiment, the transgenic bovine or equine animal produces human antibodies. In an embodiment, the animal produces monoclonal antibodies. In an embodiment, the animal produces polyclonal antibodies. In one embodiment, the method further comprises administering an adjuvant or immunostimulatory compound. In a further embodiment, the purified high affinity antibodies are 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.

[0207]

[0262] In embodiments, the CoV S protein or nanoparticles are co-administered with influenza glycoproteins or nanoparticles comprising influenza glycoproteins. Suitable glycoproteins and nanoparticles are described in U.S. Patent Application Publication Nos. 2018 / 0133308 and 2019 / 0314487, respectively, which are incorporated by reference in their entireties. In embodiments, the CoV S protein or nanoparticles are co-administered with (a) surfactant-core nanoparticles comprising a recombinant influenza hemagglutinin (HA) glycoprotein from an influenza B strain; and (b) hemagglutinin saponin matrix nanoparticles (HaSMaN) comprising a recombinant influenza HA glycoprotein from an influenza A strain and an ISCOM matrix adjuvant. In embodiments, the CoV S protein or nanoparticles are co-administered with nanoparticles comprising a non-ionic surfactant core and an influenza HA glycoprotein, the influenza HA glycoprotein comprising a head region protruding outward from the non-ionic surfactant core and a transmembrane domain associated with the non-ionic surfactant core, the influenza HA glycoprotein being an HA0 glycoprotein, and the amino acid sequence of the influenza HA glycoprotein having 100% identity to the amino acid sequence of a native influenza HA protein. In embodiments, the influenza glycoprotein or nanoparticles are co-formulated with the CoV S protein or nanoparticles.

[0208]

[0263] All patents, patent applications, references and journal articles cited in this disclosure are expressly incorporated herein by reference in their entirety for all purposes. [Example]

[0209] Example Example 1 Expression and purification of coronavirus spike (S) polypeptide nanoparticles

[0264] CoV spike polypeptides having amino acid sequences corresponding to the native coronavirus spike (S) polypeptide (SEQ ID NO: 1 and SEQ ID NO: 2) and 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, and 89 were expressed in a baculovirus expression system, and recombinant plaques expressing the coronavirus spike (S) polypeptides were picked and characterized. In each case, the signal peptide is SEQ ID NO: 5. Figures 4 and 9 show the successful purification of CoV spike polypeptides BV2364, BV2365, BV2366, BV2367, BV2368, BV2369, BV2373, BV2374, and BV2375. Table 2 shows the sequence characteristics of the aforementioned CoV spike polypeptides.

[0210] [Table 11]

[0211]

[0265] The wild-type BV2361 protein (SEQ ID NO: 2) binds to human angiotensin-converting enzyme 2 precursor (hACE2). Biolayer interferometry and ELISA were performed to assess binding of CoV S polypeptides. Biolayer Interferometry (BLI):

[0266] BLI experiments were performed using an Octet QK384 system (Pall Forte Bio, Fremont, CA). His-tagged human ACE2 (2 μg mL) was immobilized on a nickel-charged Ni-NTA biosensor chip. After baseline analysis, samples containing 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 the Octet software HT 101:1 global curve fit.

[0212]

[0267] CoV S polypeptides BV2361, BV2365, BV2369, BV2365, BV2373, and BV2374 retain the ability to bind to hACE2 (Fig. 5 and Figs. 11A-C). Dissociation rates showed that in the absence of solution-phase S protein, the S protein remained tightly bound, as evidenced by little or no dissociation over a 900-second observation period (Figs. 11A-C).

[0213]

[0268] Furthermore, the binding is specific: wild-type CoV S protein BV2361 and CoV S 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 human angiotensin-converting enzyme 2 precursor (hACE2) (Figures 6 and 11D-11F). ELISA

[0269] The specificity of CoV S polypeptide for hACE2 was confirmed by ELISA. A 96-well plate was coated with 100 μL of SARS-CoV-2 spike protein (2 μg / mL) overnight at 4°C. The plate was washed with phosphate-buffered saline containing 0.05% Tween (PBS-T) buffer and blocked with TBS Startblock blocking buffer (ThermoFisher Scientific). His-tagged hACE2 and hDPP4 receptor were serially diluted threefold (5 to 0.0001 μg mL) and added to the coated wells for 2 hours at room temperature. The plate was then washed with PBS-T. An optimally diluted horseradish peroxidase (HRP)-conjugated antihistidine was added, and color development was achieved by the addition of 3,3',5,5'-tetramethylbenzidine peroxidase substrate (TMB, 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 were analyzed using SoftMax software. EC50 values ​​were calculated by four-parameter fitting using GraphPad Prism 7.05 software.

[0214]

[0270] ELISA results showed that the wild-type CoV S polypeptide (BV2361), BV2365, and BV2373 proteins specifically bind to hACE2 but are unable to bind to the hDPP-4 receptor used by MERS-CoV (IC 50 >5000ng mL-1). The wild-type CoV S polypeptide and BV2365 had similar affinities (IC 50 = 36–38 ng / mL), whereas BV2373 bound to hACE2 at half the concentration (IC 50 = 18 ng / mL) achieved 50% saturation of hACE2 binding (Figure 7, Figures 11D-11F). Protein and nanoparticle production

[0271] The recombinant virus is amplified by infection of Sf9 insect cells. Insect cell cultures are infected with baculovirus at approximately 3 MOI (multiplicity of infection = virus ffu or pfu / cell). The culture and supernatant are harvested 48-72 hours post-infection. Approximately 30 mL of crude cell harvest is clarified by centrifugation at approximately 800 x g for 15 minutes. The resulting crude cell harvest, containing the coronavirus spike (S) protein, is purified as nanoparticles as described below.

[0215]

[0272] To produce nanoparticles, the nonionic surfactant TERGITOL® nonylphenol ethoxylate NP-9 is used in the membrane protein extraction protocol. The crude extract is further purified by passing it through anion exchange chromatography, lentil lectin affinity / HIC, and cation exchange chromatography. Washed cells are lysed by detergent treatment and subjected to a low pH treatment, which results in precipitation of BV and Sf9 host cell DNA and proteins. The neutralized low pH-treated lysate is clarified and further purified by anion exchange and affinity chromatography before a second low pH treatment.

[0216]

[0273] Affinity chromatography was used to remove Sf9 / BV proteins, DNA, and NP-9, and further enrich for coronavirus spike (S) protein. Briefly, lentil lectin is a calcium- and manganese-containing metalloprotein that reversibly binds to polysaccharides and glycosylated proteins containing glucose or mannose. The anion-exchange flow-through fraction containing coronavirus spike (S) protein was loaded onto a lentil lectin affinity chromatography resin (Capto Lentil Lectin, GE Healthcare). Glycosylated coronavirus spike (S) protein selectively bound to the resin, while nonglycosylated proteins and DNA were removed in the column flow-through. Weakly bound glycoproteins were removed using a buffer containing high salt and low molar concentration of methyl α-D-mannopyranoside (MMP).

[0217]

[0274] The column wash is also used to detergent exchange the NP-9 detergent with the surfactant polysorbate 80 (PS80). Coronavirus spike (S) polypeptides are eluted in nanoparticle structures from the lentil lectin column containing a high concentration of MMPs. After elution, coronavirus spike (S) protein trimers are incorporated into nanoparticles composed of coronavirus spike (S) protein trimers and PS80 contained within a surfactant core. Example 2 Immunogenicity of a coronavirus spike (S) polypeptide nanoparticle vaccine in mice

[0275] A coronavirus spike (S) protein composition containing the CoV S polypeptide of SEQ ID NO:87 (also referred to as "BV2373") 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 composition was evaluated in the presence and absence of a saponin adjuvant, e.g., MATRIX-M™. Compositions containing MATRIX-M™ contained 5 μg of MATRIX-M™. Vaccines containing various doses of coronavirus spike (S) polypeptide, 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 14 days apart (also referred to as a prime / boost regimen) (Study Days 0 and 14). A placebo group served as a non-immunized control. Serum was collected for analysis on study days 1, 13, 21, and 28. Vaccinated and control animals were challenged intranasally with SARS-CoV-2 42 days after one (single-dose) or two (dual-dose) immunizations. Vaccine immunogenicity

[0276] Animals immunized with a single prime dose of 0.1-10 μg BV2373 and MATRIX-M™ had elevated anti-S IgG titers detected 21-28 days after the single immunization (Figure 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 detected 21-28 days after the single prime dose (Figures 14 and 15). Animals immunized with a prime / boost regimen (two doses) had significantly elevated anti-S IgG titers detected 7-16 days after the boost at all dose levels (Figure 13A). Animals immunized with BV2373 (1 μg and 10 μg) and MATRIX-M™ had similarly high anti-S IgG titers 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 higher anti-S IgG titers compared with mice immunized with 10 μg of BV2373 without adjuvant (p≦0.05 and p≦0.0001, respectively) (FIG. 13A). These results suggest that the MATRIX-M™ adjuvant may result in a 10- to 100-fold dose savings. Furthermore, immunization with two doses of BV2373 and MATRIX-M™ induced highly potent antibodies (IC50 = 218-1642) that blocked binding of the hACE2 receptor to the S protein and neutralized the cytopathic effect (CPE) of SARS-CoV-2 on Vero E6 cells at all dose levels (100% blocking of CPE = 7,680-20,000) (Figures 14 and 15). SARS Co V-2 exposure

[0277] To assess the induction of protective immunity, immunized mice were challenged with SARS-CoV-2. Because mice do not support wild-type SARS-CoV-2 virus replication, 52 days after the first vaccination, mice were intranasally infected with an adenovirus expressing hACE2 (Ad / hACE2) to render them permissive. Mice were challenged with 1.5 × 10 Adenoviruses in 50 μL divided between the nostrils. 5Mice were inoculated intranasally with pfu of SARS-CoV-2. Exposed mice were weighed on the day of infection and daily for up to 7 days post-infection. At 4 and 7 days post-infection, five mice from each vaccinated and control group were sacrificed, and lungs were harvested and prepared for lung histology.

[0218]

[0278] Viral titers were quantified by plaque assay. Briefly, harvested lungs were homogenized in PBS using 1.0 mm glass beads (Sigma-Aldrich) and a Beadruptor (Omini International Inc.). The homogenate was added to near-confluent Vero E6 cultures, and SARS-CoV-2 viral titers were determined by counting plaque-forming units (pfu) using a six-point dilution curve.

[0219]

[0279] Four days after infection, placebo-treated mice were treated with 10 4 Mice immunized with BV2363, but without MATRIX-M™, had 10 SARS-CoV-2 pfu / lung. 3 pfu / lung (Figure 16). The BV2373 with MATRIX-M™ prime-only group of mice showed a dose-dependent reduction in viral titers, with recipients of the 10 μg BV2373 dose having no detectable virus 4 days after infection. Mice receiving the 1 μg, 0.1 μg, and 0.01 μg BV2373 doses all showed significant reductions in titers compared to placebo-vaccinated mice. In the prime / boost groups, mice immunized with the 10 μg, 1 μg, and 0.1 μg doses had barely detectable lung viral loads, while the 0.01 μg group saw a 1-log reduction compared to placebo animals.

[0220]

[0280] Weight loss was consistent with the viral load findings. Animals receiving a single dose of BV2373 (0.1 μg, 1 μg, and 10 μg) and MATRIX-M™ demonstrated significant prevention of weight loss compared to unvaccinated placebo animals (Figure 17A). Mice receiving prime and boost doses with adjuvant also demonstrated significant prevention of weight loss at all dose levels (Figures 17B and 17C). The effect of the presence of adjuvant on preventing weight loss was evaluated. Mice receiving prime / boost (two doses) and adjuvant significantly prevented weight loss compared to placebo, whereas the group immunized without adjuvant did not (Figure 17C). These results demonstrated that BV2373 protects against SARS-CoV-2 and that low vaccine doses associated with low serological responses did not exacerbate weight loss or demonstrate exacerbated disease.

[0221]

[0281] Lung histopathology was evaluated 4 and 7 days after infection (Figures 18A and 18B). Four days after infection, placebo-immunized mice exhibited denuded epithelial cells in the large airways with thickened alveolar septa surrounded by mixed inflammatory cell populations. Periarteriolar cellular infiltration by inflammatory cells, primarily neutrophils and macrophages, was observed throughout the lungs. By 7 days after infection, placebo-treated mice exhibited peribronchiolar inflammation and increased periarteriolar cellular infiltration. The thickened alveolar septa remained consistent with increased diffuse interstitial inflammation throughout the alveolar septa (Figure 18B).

[0222]

[0282] Mice immunized with BV2373 showed a significant dose-dependent reduction in lung lesions both 4 and 7 days after infection. The prime-only group showed reduced inflammation at the 10 μg and 1 μg doses compared to placebo mice, with reduced inflammation around the bronchi and arterioles. In the low-dose prime-only group, lung inflammation resembled that of the placebo group and correlated with weight loss and lung virus titers. The prime / boost group showed a significant reduction in lung inflammation at all doses tested, which also correlated with lung virus titers and weight loss data. Epithelial cells in the large and small bronchi were substantially preserved on days 4 and 7, with minimal bronchiolar desquamation and signs of viral infection. The arterioles of animals immunized with the 10 μg, 1 μg, and 0.1 μg doses had minimal inflammation, similar to placebo, with only moderate cellular infiltration seen at the 0.01 μg dose. Alveolar inflammation was reduced in animals receiving the high dose, with inflammation-associated doses as low as 0.01 μg (Figures 18A and 18B). These data demonstrate that BV2373 reduces lung inflammation after challenge and that doses and dosing regimens of BV2373 that elicit little or no detectable neutralizing activity are not associated with an exacerbated inflammatory response to the virus. Furthermore, the vaccine does not promote vaccine-associated respiratory disease (VAERD) in challenged mice. T cell response

[0283] The effect of a vaccine composition containing the CoV S polypeptide of SEQ ID NO: 87 on T cell responses was evaluated. BALB / c mice (N=6 per group) were immunized intramuscularly with 10 μg of BV2373 with or without 5 μg of MATRIX-M™ in two doses 21 days apart. Spleens were harvested 7 days after the second immunization (study day 28). An unvaccinated group (N=3) served as a control.

[0223]

[0284] Antigen-specific T cell responses were measured by ELISPOT™ enzyme-linked immunosorbent assay and intracellular cytokine staining (ICCS) from spleens harvested 7 days after the second immunization (study day 28). The number of IFN-γ-secreting cells after ex vivo stimulation was increased 20-fold in the spleens of mice immunized with BV2373 and MATRIX-M™ compared to BV2373 alone, as measured by ELISPOT™ assay (p=0.002) (Figure 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 the CD44hi CD62L- effector memory T cell population. The frequencies of IFN-γ+, TNF-α+, and IL-2+ cytokine-secreting CD4+ and CD8+ T cells were significantly higher in the spleens of mice immunized with BV2373 compared to mice immunized without adjuvant (p<0.0001) (Figures 20A-C and 21A-C). Furthermore, the frequencies of polyfunctional CD4+ and CD8+ T cells, simultaneously producing at least two or three cytokines, were also significantly increased in the spleens of mice immunized with BV2373 / MATRIX-M™ compared to mice immunized in the absence of adjuvant (p<0.0001) (Figures 20D-E and 21D-E). Immunization with BV2373 / MATRIX-M™ increased the proportion of polyfunctional phenotypes (e.g., T cells secreting two or more of IFN-γ, TNF-α, and IL-2) in both CD4+ and CD8+ T cell populations. The proportion of polyfunctional phenotypes detected in memory CD4+ T cells was higher than that in CD8+ T cells (Figure 22).

[0224]

[0285] Secretion of type 2 cytokines IL-4 and IL-5 from CD4+ T cells was also measured by ICCS and ELISPOT™, respectively. Immunization with BV2373 / MATRIX-M™ also increased secretion of type 2 cytokines IL-4 and IL-5 (2-fold) compared to immunization with BV2373 alone, but not to the same extent as enhanced type 1 cytokine production (e.g., IFN-γ increased 20-fold) (Figures 23A-C). These results indicate that administration of MATRIX-M™ adjuvant biased CD4+ T cell development toward a Th1 response.

[0225]

[0286] The effect of immunization on germinal center formation was assessed by measuring the frequency of CD4+ T follicular helper (TFH) cells and germinal center (GC) B cells in the spleen. MATRIX-M™ administration significantly increased the frequency of TFH cells (CD4+ CXCR5+ PD-1+) (p=0.01) and GC B cells (CD19+GL7+CD95+) in the spleen (p=0.0002) (Figures 24A and 24B and 25A and 25B). Example 3 Immunogenicity of a coronavirus spike (S) polypeptide nanoparticle vaccine in Anubis baboons

[0287] The immunogenicity of a vaccine composition containing BY2373 was evaluated in baboons. Adult Anubis baboons were immunized with a range of doses (1 μg, 5 μg, and 25 μg) of BV2373 and 50 μg of MATRIX-M™ adjuvant via intramuscular (IM) injection, administered twice, 21 days apart. To evaluate the adjuvant activity of MATRIX-M™ in nonhuman primates, another group of animals was immunized with 25 μg of BV2373 without MATRIX-M™. In animals immunized with BV2373 / MATRIX-M™, anti-S protein IgG titers were detected at all dose levels within 21 days of a single primary immunization (GMT = 1,249-19,000). Anti-S protein IgG titers increased logarithmically (GMT = 33,000-174,000) at all dose levels within 1-2 weeks (days 28 and 35) after the booster immunization (Figure 26A).

[0226]

[0288] After a single immunization with BV2373 (5 μg or 25 μg) and MATRIX-M™, low levels of hACE2 receptor-blocking antibodies were detected in animals (GMT = 22-37). Receptor-blocking antibody titers significantly increased in all groups immunized with BV2373 / MATRIX-M™ within 1-2 weeks of the booster immunization (GMT = 150-600) (Figure 26B). Virus-neutralizing antibodies increased in all dose groups after a single immunization with BV2373 / MATRIX-M™ (GMT = 190-446). Animals immunized with 25 μg of BV2373 alone failed to produce detectable antibodies that blocked S protein binding to hACE2 (Figure 26C). Neutralizing titers increased 6- to 8-fold 1 week after the booster immunization (GMT = 1,160-3,846). Neutralizing titers increased further 25- to 38-fold after the second immunization (GMT = 6,400-17,000) (Figure 26C). There was a significant correlation between anti-S IgG levels and neutralizing antibody titers (p<0.0001) (Figure 27). The immunogenicity of the adjuvanted vaccine in non-human primates is consistent with the results of Example 2, further supporting the role of MATRIX-M™ in generating neutralizing antibodies and promoting dose sparing.

[0227]

[0289] PBMCs were collected 7 days after the second immunization (day 28), and T cell responses were measured by ELISPOT assay. PBMCs from animals immunized with BV2373 (5 μg or 25 μg) and MATRIX-M™ contained the highest number of IFN-γ-secreting cells, fivefold higher than animals immunized with 25 μg of BV2373 alone or BV2373 (1 μg) and MATRIX-M™ (Figure 28). ICCS analysis showed that immunization with BV2373 (5 μg) and MATRIX-M™ resulted in the highest frequencies of IFN-γ+, IL-2+, and TNF-α+ CD4+ T cells (Figures 29A-C). This trend also held true for polyfunctional CD4+ T cells, which simultaneously produced at least two or three type 1 cytokines (Figures 29D and 29E). Example 4 Structural characterization of coronavirus spike (S) polypeptide nanoparticle vaccines

[0290] The ultrastructure of BV2373 was determined using transmission electron microscopy (TEM) and two-dimensional (2D) class averaging. High-magnification (67,000x and 100,000x) TEM images of negatively stained BV2373 showed particles corresponding to S protein homotrimers.

[0228]

[0291] An automated picking protocol was used to construct 2D class-averaged images (Lander GC et al. J Struct Biol. 166, 95-102 (2009); Sorzano CO et al., J Struct Biol. 148, 194-204 (2004)). Two rounds of 2D class-averaging of the homotrimeric structure revealed the appearance of a triangular particle 15 nm long and 13 nm wide (Figure 10, top left). Overlaying the recently solved cryoEM structure of the SARS-CoV-2 spike protein (EMD ID: 21374) onto the 2D BV2373 image showed a good fit to the crown-shaped S1 (NTD and RBD) and S2 stems (Figure 10, bottom left). The 2D image also revealed a faint protrusion protruding from the tip of the trimeric structure on the opposite side of the NTD / RBD crown (Figure 10, top right). 2D class averaging using larger box sizes showed that these faint protrusions form links between the S trimers and the amorphous structure (Figure 10, bottom right).

[0229]

[0292] Dynamic light scattering (DLS) showed that the wild-type CoV S protein had a Z-avg particle size of 69.53 nm, compared with half the size of BV2365 (33.4 nm) and BV2373 (27.2 nm). Polydispersity index (PDI) showed that 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).

[0230] [Table 12]

[0231]

[0293] The thermal stability of the S trimer was determined by differential scanning calorimetry (DSC). The thermal transition temperature (T max =58.6℃) are T max The thermal stability of BV2365 and BV2373 was similar to that of BV2365 and BV2373, with ΔH = 61.3 °C and 60.4 °C, respectively (Table 3). Crucially, the transition enthalpy required to unfold the BV2365 and BV2373 mutants (ΔH = 466 and 732 kJ / mol, respectively) was increased 3-5 fold compared to the low enthalpy required to unfold the WT spike protein (ΔH = 153 kJ / mol). These results are consistent with the improved thermal stability of BV2365 and BV2373 compared to the WT spike protein (Table 3).

[0232]

[0294] The stability of the CoV spike (S) polypeptide nanoparticle vaccine was assessed by dynamic light scattering and compared to that of a nanoparticle vaccine containing the native CoV spike (S) polypeptide using various pH, temperature, salt concentrations, and proteases. Example 5 Stability of coronavirus spike (S) polypeptide nanoparticle vaccines

[0295] The stability of CoV spike (S) polypeptide nanoparticle vaccines was assessed by dynamic light scattering. The stability of the CoV spike (S) polypeptide nanoparticle vaccine was compared with that of a nanoparticle vaccine containing the native CoV spike (S) polypeptide using various pH, temperature, salt concentrations, and proteases. The stability of BV2365 without the two-proline substitution and BV2373 with two proline substitutions was assessed under various environmental stress conditions using hACE2 capture ELISA. Prolonged agitation (48 hours), two freeze / thaw cycles, elevated temperatures (25°C and 37°C for 48 hours), and incubation of BV2373 at extreme pHs (pH 4 and pH 9 for 48 hours) did not affect hACE2 receptor binding (IC50 = 14.0-18.3 ng mL-1).

[0233]

[0296] Oxidative conditions with hydrogen peroxide reduced hACE2 binding to BV2373 by 8-fold (IC50 = 120 ng mL-1) (Figure 12A). BV2365, lacking the 2-proline substitution, was less stable as determined by a significant decrease in hACE2 binding under multiple conditions (Figure 12B).

[0234]

[0297] The stability of BV2384 (SEQ ID NO: 110) and BV2373 (SEQ ID NO: 87) was compared. BV2384 contains a furin cleavage site sequence of GSAS (SEQ ID NO: 97), whereas BV2373 contains a furin cleavage site sequence of QQAQ (SEQ ID NO: 7). As demonstrated by SDS-PAGE and Western blot, BV2384 showed extensive degradation compared to BV2373 (Figure 32). Furthermore, scanning densitometry and recovery data demonstrate an unexpected decrease in full-length CoV S protein, lower purity, and recovery (Figure 33) of BV2384 compared to BV2373 (Figure 34). Example 6 Immune responses in cynomolgus macaques

[0298] We evaluated the immune response induced by BV2373 in a Cynomolgus macaque model of SARS-CoV-2 infection. Groups 1 to 6 were treated as shown in Table 4.

[0235] [Table 13]

[0236]

[0299] Administration of a vaccine containing BV2373 induced anti-CoV-S antibodies (Figure 35A), including neutralizing antibodies (Figure 35B). Anti-CoV-S antibodies were induced after one (Figure 38A) or two (Figure 38B) doses of BV2373. Administration of a vaccine containing BV2373 also produced antibodies that blocked the binding of CoV S protein to hACE2 (Figures 38C and 38D). There was a significant correlation between anti-CoV S polypeptide IgG titers and hACE2 inhibitory titers in cynomolgus macaques after BV2373 administration (Figure 38E). The ability of BV2373 to induce neutralizing antibody production was assessed by cytopathic effect (CPE) (Figure 40A) and plaque reduction neutralization test (PRNT) (Figure 40B). The data revealed that the vaccine formulations in Table 4 produced SARS-CoV-2 neutralizing titers compared to the control.

[0237]

[0300] The ability of vaccine containing BV2373 to induce anti-CoV-S antibodies and antibodies that block the binding of hACE2 to the CoV S protein in cynomolgus macaques was compared with human convalescent serum. The data revealed that the BV2373 vaccine formulation induced superior anti-CoV S polypeptide and hACE2 inhibitory titers compared to human convalescent serum (Figure 39).

[0238]

[0301] The BV2373 vaccine formulation also reduced SARS-CoV-2 viral replication (Figures 36A and 36B). Viral RNA (Figure 36A, corresponding to total RNA present) and viral subgenomic RNA (sgRNA) (Figure 36B, corresponding to replicating virus) levels were assessed in bronchial lavage (BAL) samples 2 and 4 days after infection (d2pi and d4pi). Most subjects showed no viral RNA. Small amounts of RNA were measured in some subjects on day 2. By day 4, no RNA was measured except in two subjects receiving the lowest dose of 2.5 μg. Subgenomic RNA was not detected at the lowest dose on days 2 or 4, except in one subject. Viral RNA (Figure 37A) and viral subgenomic (sg) RNA (Figure 37B) were assessed by nasal swab 2 and 4 days after infection (d2pi and d4pi). Most subjects showed no viral RNA. Small amounts of RNA were measured in some subjects on days 2 and 4. Subgenomic RNA was not detected on either days 2 or 4. Subjects were immunized on day 0, and in the two-dose group, on days 0 and 21. These data demonstrate that the vaccine reduces total nasal viral RNA by 100- to 1000-fold and sgRNA to undetectable levels, demonstrating that the immune response to the vaccine blocks viral replication and prevents viral spread. Example 7 Evaluation of CoV S polypeptide nanoparticle vaccines in humans

[0302] We evaluated the safety and efficacy of a vaccine containing BV2373 in a randomized, observer-blinded, placebo-controlled 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 timeline of clinical endpoint assessments.

[0239] [Table 14]

[0240]

[0303] Overall reactogenicity was mild and vaccination was well tolerated. Site reactogenicity was more frequent in patients treated with BV2373 and MATRIX-M™ (Figures 42A and 42B).

[0241]

[0304] The immunogenicity of BV2373 with and without MATRIX-M™ was evaluated. Twenty-one days after vaccination, anti-CoV-S antibodies were detected with all vaccine regimens (Figure 43A). The geometric mean fold increase (GMFR) in the vaccine regimens containing MATRIX-M™ exceeded that induced by unadjuvanted BV2373. Seven days after the second vaccination (day 28), anti-CoV-S titers increased more than 8-fold over the response seen with the first vaccination relative to that observed with BV2373 alone, and within 14 days (day 35), the response increased more than 2-fold, achieving a GMFR of approximately 100-fold. A single vaccination with BV2373 / MATRIX-M™ achieved anti-CoV-S titer levels similar to those observed in asymptomatic (exposed) COVID-19 patients. The second vaccination achieved GMEU levels six-fold greater than convalescent sera from outpatient-treated COVID-19 patients, similar levels to convalescent sera from hospitalized COVID-19 patients, and nearly six-fold greater than overall convalescent serum anti-CoV-S antibodies. Responses to two doses of 5 μg and 25 μg BV2373 / MATRIX-M™ were similar, highlighting the ability of the dose-sparing adjuvant (MATRIX-M™).

[0242]

[0305] Neutralizing antibodies were induced in all groups treated with BV2373 (Figure 43B). Groups treated with the BV2373 and MATRIX-M™ regimen demonstrated approximately 5-fold higher GMFR than groups treated with BV2373 alone (Figure 43B). A second vaccination with adjuvant significantly impacted neutralizing antibody titers, increasing them by more than 100-fold relative to a single vaccination without adjuvant. Compared to convalescent sera, a second vaccination with BV2373 / MATRIX-M™ achieved GMT levels 4-fold higher than those of outpatient-treated COVID-19 patients, levels approaching those of hospitalized COVID-19 patients, and an overall convalescent serum GMT of more than 4-fold.

[0243]

[0306] Convalescent sera obtained from clinically symptomatic COVID-19 patients seeking medical care demonstrated anti-CoV-S IgG and neutralizing titers that increased in proportion to disease severity (Figures 43A and 43B).

[0244]

[0307] A strong correlation was observed between neutralizing antibody titers and anti-CoV-S IgG in patients treated with BV2373 and MATRIX-M™ (r=0.9466, Figure 44C), similar to that observed in patients treated with convalescent serum (r=0.958) (Figure 44A). This correlation was not observed in subjects receiving unadjuvanted BV2373 (r=0.7616) (Figure 44B). Both the 5 μg and 25 μg BV2373 / MATRIX-M™ groups (Groups C-E in Table 5) demonstrated a similar two-dose response, with all participants seroconverting using either assay measure when the two-dose regimen was utilized. T cell responses in 16 participants (4 participants from each of Groups A-D) showed that the BV2373 / MATRIX-M™ regimen induced antigen-specific polyfunctional CD4+ T cell responses with respect to the production of IFN-γ, IL-2, and TNF-α upon stimulation with BV2373. There was a strong bias toward Th1 cytokine production (Figures 45A-D). Example 8 Expression, purification and evaluation of next-generation CoV S polypeptide nanoparticles

[0308] CoV S polypeptides having the amino acid sequence of SEQ ID NO:112, SEQ ID NO:113, SEQ ID NO:114, or SEQ ID NO:115 are expressed in a baculovirus expression system, and recombinant plaques expressing coronavirus spike (S) polypeptides are selected and confirmed. CoV S polypeptides having the sequences of 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 of SEQ ID NO:5.

[0245]

[0309] The CoV S polypeptide having the sequence of SEQ ID NO: 112 contains a mutation of Asn-488 to tyrosine, mutations of Lys-973 and Val-974 to proline, and an inactivated furin cleavage site having the amino acid sequence QQAQ (SEQ ID NO: 7).

[0246]

[0310] The CoV S polypeptide having the sequence of SEQ ID NO: 113 contains a mutation of Asp-601 to glycine, a mutation of Asn-488 to tyrosine, a mutation of Lys-973 and Val-974 to proline, and an inactivated furin cleavage site having the amino acid sequence QQAQ (SEQ ID NO: 7).

[0247]

[0311] The CoV S polypeptide having the sequence of SEQ ID NO: 114 contains a deletion of amino acids 56, 57, and 131, a mutation of Asn-488 to tyrosine, a mutation of Ala-557 to aspartic acid, a mutation of Asp-601 to glycine, a mutation of Pro-668 to histidine, a mutation of Thr-703 to isoleucine, a mutation of Ser-969 to alanine, a mutation of Asp-1105 to histidine, a mutation of Lys-973 and Val-974 to proline, and an inactivated furin cleavage site having the amino acid sequence QQAQ (SEQ ID NO: 7).

[0248]

[0312] The CoV S polypeptide having the sequence of SEQ ID NO: 115 comprises an Asn-488 mutation to tyrosine, an Asp-67 mutation to alanine, a Leu-229 mutation to histidine, an Asp-202 mutation to glycine, a Lys-404 mutation to asparagine, a Glu-471 mutation to lysine, an Ala-688 mutation to valine, an Asp-601 mutation to glycine, mutations of Lys-973 and Val-974 to proline, and an inactivated furin cleavage site having the amino acid sequence QQAQ.

[0249]

[0313] CoV S polypeptide nanoparticles are prepared as in Example 1. The stability and immunogenicity of CoV S polypeptides having the amino acid sequences of SEQ ID NO: 112, SEQ ID NO: 112, SEQ ID NO: 113, and SEQ ID NO: 115 are evaluated as in Examples 2-7. Numbered Embodiments 1. An immunogenic composition comprising: (i) nanoparticles comprising a coronavirus S (CoV S) glycoprotein having the amino acid sequence of SEQ ID NO: 87 and a non-ionic surfactant core; (ii) a pharmaceutically acceptable buffer; and (iii) a saponin adjuvant; An immunogenic composition comprising: 2. The immunogenic composition of embodiment 1, comprising about 5 μg to about 25 μg of CoV S glycoprotein. 3. The immunogenic composition of embodiment 2, comprising about 5 μg of CoV S glycoprotein. 4. The saponin adjuvant comprises at least two iscom particles, The first iscom particle contains fraction A of Quillaja Saponaria Molina and does not contain fraction C of Quillaja Saponaria Molina; and 2. The immunogenic composition of embodiment 1, wherein the second iscom particles comprise Fraction C of Quillaja Saponaria Molina and do not comprise Fraction A of Quillaja Saponaria Molina. 5. The immunogenic composition of embodiment 4, wherein Quillaja Saponaria Molina Fraction A comprises 50 to 96% by weight of the sum of the weights of Quillaja Saponaria Molina Fraction A and Quillaja Saponaria Molina Fraction C in the adjuvant, and Quillaja Saponaria Molina Fraction C comprises the remaining weight of the sum, respectively. 6. The immunogenic composition of embodiment 4, wherein Quillaja Saponaria Molina Fraction A and Quillaja Saponaria Molina Fraction C account for about 85% by weight and about 15% by weight, respectively, of the combined weight of Quillaja Saponaria Molina Fraction A and Quillaja Saponaria Molina Fraction C in the adjuvant. 7. The immunogenic composition of embodiment 1, comprising about 50 μg of a saponin adjuvant. 8. The immunogenic composition of embodiment 1, wherein the nonionic surfactant core is selected from the group consisting of polysorbate-20 (PS20), polysorbate-40 (PS40), polysorbate-60 (PS60), polysorbate-65 (PS65) and polysorbate-80 (PS80). 9. A method of stimulating an immune response to SARS-CoV-2 in a subject, comprising administering the immunogenic composition of embodiment 1. 10. The method of embodiment 9, comprising about 5 μg to about 25 μg of CoV S glycoprotein. 11. The method of embodiment 10, comprising 11.5 μg of CoV S glycoprotein. 12. The saponin adjuvant comprises at least two iscom particles, The first iscom particle contains fraction A of Quillaja Saponaria Molina and does not contain fraction C of Quillaja Saponaria Molina; and 10. The method of embodiment 9, wherein the second iscom particles comprise Fraction C of Quillaja Saponaria Molina and do not comprise Fraction A of Quillaja Saponaria Molina. 13. The method of embodiment 12, wherein Quillaja Saponaria Molina Fraction A comprises 50-96% by weight of the sum of the weights of Quillaja Saponaria Molina Fraction A and Quillaja Saponaria Molina Fraction C in the adjuvant, and Quillaja Saponaria Molina Fraction C comprises the remaining weight of the sum, respectively. 14. The method of embodiment 12, wherein Quillaja Saponaria Molina Fraction A and Quillaja Saponaria Molina Fraction C account for about 85% by weight and about 15% by weight, respectively, of the combined weight of Quillaja Saponaria Molina Fraction A and Quillaja Saponaria Molina Fraction C in the adjuvant. 15. The method of embodiment 9, comprising about 50 μg of a saponin adjuvant. 16. The method of embodiment 9, wherein the nonionic surfactant core is selected from the group consisting of polysorbate-20 (PS20), polysorbate-40 (PS40), polysorbate-60 (PS60), polysorbate-65 (PS65) and polysorbate-80 (PS80). 17. The method of embodiment 9, wherein the subject receives an initial dose on day 0 and a booster dose on day 21. 18. The method of embodiment 9, wherein a single dose of the immunogenic composition is administered. 19. The method of embodiment 9, comprising administering a second immunogenic composition that is different from the first immunogenic composition. 20. The method of embodiment 19, 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. Incorporation by Reference

[0314] All references, articles, publications, patents, patent publications, and patent applications cited herein are incorporated by reference in their entirety for all purposes. However, the mention of any reference, article, publication, patent, patent publication, or patent application cited herein is not, and should not be construed as, an acknowledgment or any form of suggestion that they constitute valid prior art or form part of the common general knowledge in any country in the world.

Claims

Claim 1: A nanoparticle comprising a non-ionic surfactant core and a severe acute respiratory syndrome coronavirus 2 spike (CoV S) glycoprotein having at least 90% identity to SEQ ID NO:2, wherein the CoV S glycoprotein is: an inactivated furin cleavage site at amino acids 669-672 comprising the amino acid sequence of QQAQ (SEQ ID NO: 7); Prolines at amino acids 973 and 974; Including, The nanoparticles, wherein the amino acids are numbered according to SEQ ID NO:

2.

2. The CoV S glycoprotein comprises an S1 subunit and an S2 subunit; the S1 subunit has at least 95% identity to the SD1 / 2 domain located at amino acids 529-668 (SEQ ID NO: 122); and the S2 domain has at least 95% identity to amino acids 673-1260 (SEQ ID NO: 120); The nanoparticle of claim 1 , wherein the amino acids are numbered according to SEQ ID NO:

2.

3. The nanoparticle of claim 1, wherein the CoV S glycoprotein is at least 95%, at least 97%, or at least 99% identical to a CoV S glycoprotein comprising the sequence of SEQ ID NO:

2.

4. The nanoparticles of claim 1, wherein the CoV S glycoprotein is at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% identical to a CoV S glycoprotein comprising the sequence of SEQ ID NO:

87.

5. Nanoparticles described in any one of claims 1 to 4, wherein the nonionic surfactant is selected from the group consisting of polysorbate-20 (PS20), polysorbate-40 (PS40), polysorbate-60 (PS60), polysorbate-65 (PS65), and polysorbate-80 (PS80).

6. Nanoparticles described in claim 5, wherein the nonionic surfactant is PS80.

7. A nanoparticle described in any one of claims 1 to 4, wherein the nanoparticle has a Zavg diameter of 20 nm to 35 nm.

8. Nanoparticles described in any one of claims 1 to 4, wherein the nanoparticles have a polydispersity index of 0.2 to 0.

45. (a) a nanoparticle according to any one of claims 1 to 4; and (b) a saponin adjuvant; and An immunogenic composition comprising:

10. The saponin adjuvant comprising at least two iscom particles: the first iscom particles comprise fraction A of Quillaja Saponaria Molina and do not comprise fraction C of Quillaja Saponaria Molina; and 10. The immunogenic composition of claim 9, wherein the second iscom particles comprise fraction C of Quillaja Saponaria Molina and do not comprise fraction A of Quillaja Saponaria Molina.

11. The immunogenic composition of claim 9, wherein Fraction A of Quillaja Saponaria Molina and Fraction C of Quillaja Saponaria Molina constitute 50 to 96% by weight of the total amount of Fraction A of Quillaja Saponaria Molina and Fraction C of Quillaja Saponaria Molina in the adjuvant, and the remainder, respectively.

12. The immunogenic composition of claim 10, wherein fraction A of Quillaja Saponaria Molina and fraction C of Quillaja Saponaria Molina constitute approximately 85% by weight and approximately 15% by weight, respectively, of the total amount of fraction A of Quillaja Saponaria Molina and fraction C of Quillaja Saponaria Molina in the adjuvant.

13. A pharmaceutical composition for stimulating an immune response to SARS-CoV-2 in humans, the pharmaceutical composition comprising nanoparticles according to any one of claims 1 to 4 or the immunogenic composition according to claim 9, the pharmaceutical composition being in the form of a vaccine formulation.

14. The pharmaceutical composition of claim 13, wherein a single dose of the vaccine formulation is administered to the human.

15. The pharmaceutical composition of claim 13, wherein the human is administered an initial dose of the vaccine formulation and an additional dose of the vaccine formulation.

16. The pharmaceutical composition of claim 13, wherein the stimulation comprises administering a second immunogenic composition to the human, the second immunogenic composition being different from the vaccine formulation.

17. The pharmaceutical composition of claim 16, 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 CoV S glycoprotein, an inactivated SARS-CoV-2 virus, or the SARS-CoV-2 S glycoprotein.

18. The pharmaceutical composition of claim 16, wherein the stimulation comprises administering the second immunogenic composition prior to the vaccine formulation.

19. The pharmaceutical composition of claim 16, wherein the stimulation comprises administering the second immunogenic composition after the vaccine formulation.

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