Coronavirus vaccine compositions, methods, and uses thereof

Abstract

In some embodiments, the present disclosure relates to immunogenic compositions, such as coronavirus virus antigens and immunogens, e.g., recombinant peptides and proteins comprising coronavirus S protein peptides. In some embodiments, the immunogenic compositions comprise secreted fusion proteins comprising a soluble coronavirus virus antigen linked by in-frame fusion to the C-terminal portion of a collagen capable of self-trimerizing to form a disulfide-linked trimeric fusion protein. In some embodiments, the immunogenic compositions provided herein are useful for generating an immune response, e.g., for treating or preventing coronavirus infection. In some embodiments, the immunogenic compositions provided herein can be used in vaccine compositions, e.g., as part of a prophylactic and / or therapeutic vaccine. Also provided herein are methods for producing the recombinant peptides and proteins, prophylactic, therapeutic, and / or diagnostic methods, and related kits.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to and the benefit of PCT / CN2020 / 095269, filed June 10, 2020, PCT / CN2021 / 087066, filed April 13, 2021, and PCT / CN2021 / 093895, filed May 14, 2021, the disclosures of which are incorporated herein by reference in their entireties for all purposes.

[0002] Submission of sequence listing as an ASCII text file The contents of the ASCII text file submission: Computer-readable format sequence listing (CRF) (Filename: 165762000143SEQLIST.TXT, Recorded: June 9, 2021, Size: 575 KB) are incorporated herein by reference in their entirety.

[0003] The present disclosure, in some embodiments, relates to immunogenic compositions, such as coronavirus viral antigens and immunogens, for example, recombinant peptides and proteins, including coronavirus S protein peptides, for treating and / or preventing coronavirus infections. [Background technology]

[0004] Coronaviruses infect a wide range of birds and mammals, including humans. Coronaviruses can circulate in humans annually, generally causing mild respiratory illness, although the severity can be greater in infants, the elderly, and the immunocompromised. However, certain coronaviruses, such as Middle East Respiratory Syndrome Coronavirus (MERS-CoV), Severe Acute Respiratory Syndrome Coronavirus (SARS-CoV-1), and Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), are highly pathogenic. Due to their high pathogenicity, airborne transmission, high mortality rate, and poorly defined epidemiology, effective vaccines and related therapeutics are urgently needed. In particular, there is an urgent need for a vaccine that can rapidly induce an effective immune response against SARS-CoV-2. Summary of the Invention [Problem to be solved by the invention]

[0005] Methods, uses, and articles of manufacture that meet these and other needs are provided. [Means for solving the problem]

[0006] In some embodiments, disclosed herein are proteins comprising a plurality of recombinant polypeptides, each recombinant polypeptide comprising a surface antigen of a coronavirus linked to a C-terminal propeptide of collagen, wherein the C-terminal propeptides of the recombinant polypeptides form an inter-polypeptide disulfide bond.

[0007] In some embodiments, disclosed herein are recombinant subunit vaccines that include the ectodomain (e.g., excluding the transmembrane and cytoplasmic domains) of the S protein or a fragment thereof from a coronavirus, such as SARS-CoV-2, fused in-frame to the C-propeptide of collagen, which can form a disulfide-linked homotrimer. The resulting recombinant subunit vaccine, such as the S-trimer, can be expressed and purified from transfected cells and is predicted to be in a native-like conformation in its trimeric form. This overcomes the problem of misfolding of viral antigens, which is often encountered when viral antigens are expressed as soluble recombinant peptides or proteins that do not contain the transmembrane and / or cytoplasmic domains. Such misfolded viral antigens often fail to elicit neutralizing antibodies because they do not faithfully retain the conformation of the native viral antigen.

[0008] In some embodiments, the coronavirus is severe acute respiratory syndrome (SARS)-coronavirus (SARS-CoV-1), SARS-coronavirus 2 (SARS-CoV-2), SARS-like coronavirus, Middle East respiratory syndrome (MERS)-coronavirus (MERS-CoV), MERS-like coronavirus, NL63-CoV, 229E-CoV, OC43-CoV, HKU1-CoV, WIV1-CoV, MHV, HKU9-CoV, PEDV-CoV, or SDCV.

[0009] In any of the foregoing embodiments, the surface antigen can comprise a coronavirus spike (S) protein or a fragment or epitope thereof, the epitope optionally being a linear epitope or a conformational epitope, and the protein comprises three recombinant polypeptides.

[0010] In any of the foregoing embodiments, the surface antigen may comprise a signal peptide, an S1 subunit peptide, an S2 subunit peptide, or any combination thereof.

[0011] In any of the foregoing embodiments, the surface antigen may comprise a signal peptide, a receptor binding domain (RBD) peptide, a receptor binding motif (RBM) peptide, a fusion peptide (FP), a heptad repeat 1 (HR1) peptide, or a heptad repeat 2 (HR2) peptide, or any combination thereof.

[0012] In any of the foregoing embodiments, the surface antigen may comprise the receptor binding domain (RBD) of the S protein.

[0013] In any of the foregoing embodiments, the surface antigen may include the S1 and S2 subunits of the S protein.

[0014] In any of the foregoing embodiments, the surface antigen may not include a transmembrane (TM) domain peptide and / or a cytoplasmic (CP) domain peptide.

[0015] In any of the foregoing embodiments, the surface antigen may comprise a protease cleavage site, and the protease is optionally furin, trypsin, factor Xa, thrombin, or cathepsin L.

[0016] In any of the foregoing embodiments, the surface antigen may not contain a protease cleavage site, and the protease may optionally be furin, trypsin, Factor Xa, thrombin, or cathepsin L, or may contain a mutant protease cleavage site that is not cleaved by the protease.

[0017] In any of the foregoing embodiments, the surface antigen may be soluble or not directly bound to a lipid bilayer, eg, a membrane or viral envelope.

[0018] In any of the foregoing embodiments, the surface antigens may be the same or different between the recombinant polypeptides of the protein.

[0019] In any of the foregoing embodiments, the surface antigen can be fused directly to the C-terminal propeptide or can be linked to the C-terminal propeptide via a linker, such as a linker comprising a glycine-XY repeat, where X and Y are independently any amino acid, optionally proline or hydroxyproline.

[0020] In any of the foregoing embodiments, the protein may be soluble or not directly bound to a lipid bilayer, eg, a membrane or viral envelope.

[0021] In any of the foregoing embodiments, the protein is capable of binding to a cell surface receptor of a subject, and optionally the subject is a mammal, eg, a primate, eg, a human.

[0022] In any of the foregoing embodiments, the cell surface receptor may be angiotensin-converting enzyme 2 (ACE2), dipeptidyl peptidase 4 (DPP4), dendritic cell-specific intercellular adhesion molecule-3-grabbing non-integrin (DC-SIGN), or liver / lymph node-SIGN (L-SIGN).

[0023] In any of the foregoing embodiments, the C-terminal propeptide may be that of human collagen.

[0024] In any of the foregoing embodiments, the C-terminal propeptide may include the C-terminal propeptide of proα1(I), proα1(II), proα1(III), proα1(V), proα1(XI), proα2(I), proα2(V), proα2(XI), or proα3(XI), or a fragment thereof.

[0025] In any of the foregoing embodiments, the C-terminal propeptides may be the same or different between the recombinant polypeptides.

[0026] In any of the foregoing embodiments, the C-terminal propeptide can comprise any of SEQ ID NOs: 67-80, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.5% sequence identity to any of SEQ ID NOs: 67-80, and can form inter-polypeptide disulfide bonds to trimerize the recombinant polypeptide.

[0027] In any of the foregoing embodiments, the C-terminal propeptide can comprise SEQ ID NO: 67 or an amino acid sequence at least 95% identical to SEQ ID NO: 67 and can form inter-polypeptide disulfide bonds and trimerize the recombinant polypeptide.

[0028] In any of the foregoing embodiments, the C-terminal propeptide can comprise SEQ ID NO: 68 or an amino acid sequence at least 95% identical to SEQ ID NO: 68 and can form inter-polypeptide disulfide bonds and trimerize the recombinant polypeptide.

[0029] In any of the foregoing embodiments, the C-terminal propeptide can comprise SEQ ID NO:69 or an amino acid sequence at least 95% identical to SEQ ID NO:69 and can form inter-polypeptide disulfide bonds and trimerize the recombinant polypeptide.

[0030] In any of the foregoing embodiments, the C-terminal propeptide can comprise SEQ ID NO: 70 or an amino acid sequence at least 95% identical to SEQ ID NO: 70 and can form inter-polypeptide disulfide bonds and trimerize the recombinant polypeptide.

[0031] In any of the foregoing embodiments, the C-terminal propeptide can comprise SEQ ID NO: 71 or an amino acid sequence at least 95% identical to SEQ ID NO: 71 and can form inter-polypeptide disulfide bonds and trimerize the recombinant polypeptide.

[0032] In any of the foregoing embodiments, the C-terminal propeptide can comprise SEQ ID NO: 72 or an amino acid sequence at least 95% identical to SEQ ID NO: 72 and can form inter-polypeptide disulfide bonds and trimerize the recombinant polypeptide.

[0033] In any of the foregoing embodiments, the C-terminal propeptide can comprise SEQ ID NO: 73 or an amino acid sequence at least 95% identical to SEQ ID NO: 73 and can form inter-polypeptide disulfide bonds and trimerize the recombinant polypeptide.

[0034] In any of the foregoing embodiments, the C-terminal propeptide can comprise SEQ ID NO: 74 or an amino acid sequence at least 95% identical to SEQ ID NO: 74 and can form inter-polypeptide disulfide bonds and trimerize the recombinant polypeptide.

[0035] In any of the foregoing embodiments, the C-terminal propeptide can comprise SEQ ID NO: 75 or an amino acid sequence at least 95% identical to SEQ ID NO: 75 and can form inter-polypeptide disulfide bonds and trimerize the recombinant polypeptide.

[0036] In any of the foregoing embodiments, the C-terminal propeptide can comprise SEQ ID NO: 76 or an amino acid sequence at least 95% identical to SEQ ID NO: 76 and can form inter-polypeptide disulfide bonds and trimerize the recombinant polypeptide.

[0037] In any of the foregoing embodiments, the C-terminal propeptide can comprise SEQ ID NO: 77 or an amino acid sequence at least 95% identical to SEQ ID NO: 77 and can form inter-polypeptide disulfide bonds and trimerize the recombinant polypeptide.

[0038] In any of the foregoing embodiments, the C-terminal propeptide can comprise SEQ ID NO: 78 or an amino acid sequence at least 95% identical to SEQ ID NO: 78 and can form inter-polypeptide disulfide bonds and trimerize the recombinant polypeptide.

[0039] In any of the foregoing embodiments, the C-terminal propeptide can comprise SEQ ID NO: 79 or an amino acid sequence at least 95% identical to SEQ ID NO: 79 and can form inter-polypeptide disulfide bonds and trimerize the recombinant polypeptide.

[0040] In any of the foregoing embodiments, the C-terminal propeptide can comprise SEQ ID NO: 80 or an amino acid sequence at least 95% identical to SEQ ID NO: 80 and can form inter-polypeptide disulfide bonds and trimerize the recombinant polypeptide.

[0041] In any of the foregoing embodiments, the C-terminal propeptide comprises a sequence comprising a glycine-XY repeat linked to the N-terminus of any of SEQ ID NOs: 67-80, where X and Y are independently any amino acid and optionally proline or hydroxyproline, or an amino acid sequence at least 90% identical to SEQ ID NOs: 67-80, capable of forming an inter-polypeptide disulfide bond and trimerizing the recombinant polypeptide.

[0042] In any of the foregoing embodiments, the surface antigen in each recombinant polypeptide may be in a pre-fusion conformation.

[0043] In any of the foregoing embodiments, the surface antigen in each recombinant polypeptide may be in a post-fusion conformation.

[0044] In any of the foregoing embodiments, the surface antigen in each recombinant polypeptide may comprise any of SEQ ID NOs: 27-66 or an amino acid sequence at least 80% identical to SEQ ID NOs: 27-66.

[0045] In any of the foregoing embodiments, the recombinant polypeptide may comprise any of SEQ ID NOs: 1-26 or an amino acid sequence at least 80% identical to SEQ ID NOs: 1-26.

[0046] Also provided herein is an immunogen comprising the protein provided herein. Provided herein is a protein nanoparticle comprising the protein provided herein, directly or indirectly linked to a nanoparticle. Provided herein is a virus-like particle (VLP) comprising the protein provided herein.

[0047] Also provided herein is an isolated nucleic acid encoding one, two, three or more recombinant polypeptides of the proteins provided herein. In some embodiments, the polypeptide encoding the S protein peptide is fused in-frame to the polypeptide encoding the C-terminal propeptide of collagen. In some embodiments, the isolated nucleic acid provided herein is operably linked to a promoter.

[0048] In some embodiments, the isolated nucleic acid provided herein is a DNA molecule. In some embodiments, the isolated nucleic acid provided herein is an RNA molecule, and optionally an mRNA molecule, such as a nucleoside-modified mRNA, a non-amplified mRNA, a self-amplified mRNA, or a trans-amplified mRNA.

[0049] Also provided herein are vectors comprising the isolated nucleic acids provided herein. In some embodiments, the vector is a viral vector.

[0050] In some aspects, provided herein are cells comprising a virus, pseudovirus, or vector provided herein, optionally the virus or cell having a recombinant genome. In some aspects, provided herein are immunogenic compositions comprising a protein, immunogen, protein nanoparticle, VLP, isolated nucleic acid, vector, virus, pseudovirus, or cell provided herein, and a pharmaceutically acceptable carrier.

[0051] Also provided herein is a vaccine comprising the immunogenic compositions provided herein and, optionally, an adjuvant, wherein the vaccine is optionally a subunit vaccine. In some embodiments, the vaccine is a prophylactic and / or therapeutic vaccine.

[0052] In some aspects, provided herein are methods of producing a protein, the methods comprising expressing an isolated nucleic acid or vector provided herein in a host cell to produce a protein provided herein; and purifying the protein. Provided herein are proteins produced by the methods provided herein.

[0053] Provided herein are methods for generating an immune response in a subject against a coronavirus S protein peptide, or a fragment or epitope thereof, the method comprising administering to the subject an effective amount of a protein, immunogen, protein nanoparticle, VLP, isolated nucleic acid, vector, virus, pseudovirus, cell, immunogenic composition, or vaccine provided herein to generate an immune response. In some embodiments, the methods provided herein are for treating or preventing infection by a coronavirus. In some embodiments, the generated immune response inhibits or reduces coronavirus replication in the subject. In some embodiments, the immune response comprises a cellular response and / or a humoral response, and optionally includes the production of one or more neutralizing antibodies, such as polyclonal or monoclonal antibodies. In some embodiments, the immune response is against the coronavirus S protein peptide, or a fragment or epitope thereof, but not against the C-terminal propeptide. In some embodiments, administration to a subject does not result in antibody-dependent enhancement (ADE) in the subject due to previous exposure to one or more coronaviruses. In some embodiments, administration does not result in antibody-dependent enhancement (ADE) in the subject upon subsequent exposure to one or more coronaviruses. In some embodiments, the method further comprises a priming step and / or a boosting step. In some embodiments, the administering step is carried out via topical, transdermal, subcutaneous, intradermal, oral, intranasal (e.g., intranasal spray), intratracheal, sublingual, buccal, rectal, intravaginal, inhalation, intravenous (e.g., intravenous injection), intraarterial, intramuscular (e.g., intramuscular injection), intracardiac, intraosseous, intraperitoneal, transmucosal, intravitreal, subretinal, intraarticular, periarticular, topical, or epicutaneous administration. In some embodiments, the effective amount is administered in a single dose or in a series of doses separated by one or more intervals. In some embodiments, the effective amount is administered without an adjuvant. In some embodiments, the effective amount is administered with an adjuvant.

[0054] Provided herein are methods comprising administering an effective amount of a protein provided herein to a subject to generate neutralizing antibodies or neutralizing antisera against coronavirus in the subject. In some embodiments, the subject is a mammal, optionally a human or non-human primate. In some embodiments, the method further comprises isolating the neutralizing antibodies or neutralizing antisera from the subject. In some embodiments, the method further comprises administering an effective amount of the isolated neutralizing antibodies or neutralizing antisera to a human subject via passive immunization to prevent or treat infection with coronavirus. In some embodiments, the neutralizing antibodies or neutralizing antisera against coronavirus comprise polyclonal antibodies against a coronavirus S protein peptide, or a fragment or epitope thereof, optionally, the neutralizing antibodies or neutralizing antisera are free or substantially free of antibodies against the C-terminal propeptide of collagen. In some embodiments, the neutralizing antibodies comprise monoclonal antibodies against a coronavirus S protein peptide, or a fragment or epitope thereof, optionally, the neutralizing antibodies are free or substantially free of antibodies against the C-terminal propeptide of collagen.

[0055] In some aspects, the proteins, immunogens, protein nanoparticles, VLPs, isolated nucleic acids, vectors, viruses, pseudoviruses, cells, immunogenic compositions, or vaccines provided herein are for use in inducing an immune response to coronavirus in a subject and / or treating or preventing infection by coronavirus.

[0056] In some aspects, provided herein is the use of a protein, immunogen, protein nanoparticle, VLP, isolated nucleic acid, vector, virus, pseudovirus, cell, immunogenic composition, or vaccine provided herein for inducing an immune response against coronavirus and / or treating or preventing infection by coronavirus in a subject. In some aspects, provided herein is the use of a protein, immunogen, protein nanoparticle, VLP, isolated nucleic acid, vector, virus, pseudovirus, cell, immunogenic composition, or vaccine provided herein for the manufacture of a medicament or prophylactic for inducing an immune response against coronavirus and / or treating or preventing infection by coronavirus in a subject.

[0057] Also provided herein are methods for analyzing a sample, the methods comprising contacting the sample with a protein provided herein and detecting binding of the protein to an analyte capable of specifically binding to a coronavirus S protein peptide, or a fragment or epitope thereof. In some embodiments, the analyte is an antibody, receptor, or cell that recognizes the S protein peptide, or a fragment or epitope thereof. In some embodiments, binding indicates the presence of the analyte in the sample and / or infection with a coronavirus in the subject from which the sample was derived.

[0058] Provided herein are kits comprising a protein provided herein and a substrate, pad, or vial containing or immobilizing the protein, optionally wherein the kit is an ELISA or lateral flow assay kit. [Brief explanation of the drawings]

[0059] [Figure 1](A) is a diagram showing the structural features of an exemplary soluble S-trimeric subunit vaccine for SARS-CoV-2, depicting a schematic of the S-trimer structural domains. (B) is a diagram showing the structural features of an exemplary soluble S-trimeric subunit vaccine for SARS-CoV-2, depicting its trimer and covalently linked three-dimensional conformation. [Figure 2] Figure 1 shows high-level expression of exemplary S-trimers: full-length S-trimer and forms partially truncated at the S1 / S2 furin site, i.e., S2-trimer and truncated S1 fragment. [Figure 3A] Figure 1 shows the purification and characterization of an exemplary covalently linked S-trimer. The S-trimer was purified from clear cell culture media via Protein A (PA) affinity chromatography and an anion exchange column (Q), followed by ultrafiltration and diafiltration (UF / DF) to yield the drug substance (DS). [Figure 3B] 1 shows the purification and characterization of an exemplary covalently linked S-trimer, which is a disulfide-linked trimer. [Figure 3C]

[0023] Figure 1 shows the purification and characterization of an exemplary covalently linked S-trimer. The S-trimer was purified to near homogeneity as judged by SEC-HPLC analysis, and a portion of the cleaved S1 was isolated during size exclusion chromatography. [Figure 3D] Figure 1 shows the purification and characterization of an exemplary covalently linked S-trimer. The receptor binding kinetics of the S-trimer to ACE2-Fc was assessed. [Figure 4]

[0023] Figure 1 shows an exemplary S-trimer that is hyperglycosylated with N-linked glycans. Full-length S-trimer, S2-trimer, and truncated S1 are shown before and after deglycosylation. [Figure 5] Electron micrograph (EM) of an exemplary S-trimer and the predicted conformation of the S-trimer (right panel). [Figure 6]FIG. 1 shows detection of S-specific and neutralizing antibodies from convalescent sera using an exemplary S-trimer as antigen. [Figure 7A] FIG. 1 shows the induction of antigen-specific antibodies in rats with an exemplary S-trimer alone and without any adjuvant. [Figure 7B] FIG. 1 shows the induction of neutralizing antibodies in rats with an exemplary S-trimer alone and without any adjuvant. [Figure 7C] FIG. 1 shows the induction of antigen-specific antibodies by an exemplary S-trimer vaccine in mice without adjuvant or with alum (aluminum hydroxide) as the adjuvant. [Figure 7D] FIG. 1 shows the induction of antigen-specific antibodies in rats by an exemplary S-trimer adjuvanted with a squalene-based adjuvant. [Figure 7E] FIG. 1 shows the induction of neutralizing antibodies in rats by an exemplary S-trimer adjuvanted with a squalene-based adjuvant. [Figure 8] FIG. 1 shows the structure and specific sequences of exemplary coronaviruses. [Figure 9A] Detection of SARS-CoV-2 neutralizing antibody titers and ACE2 competitive titers in human COVID-19 convalescent serum. Eight human convalescent serum (HCS) samples with moderate to high antibody titers collected from COVID-19 patients infected with the original strain (Wuhan-Hu-1) were analyzed for SARS-CoV-2 pseudovirus neutralization titers. Data are shown comparing variant strain titers to original strain titers. Dots represent data for individual HCS samples, and bars represent geometric mean titers (GMT) of half-maximal effective concentration (EC50) values. Error bars represent 95% confidence intervals (95% CI). [Figure 9B]Detection of SARS-CoV-2 neutralizing antibody titers and ACE2 competitive titers in human COVID-19 convalescent serum. Eight human convalescent serum (HCS) samples with moderate to high antibody titers collected from COVID-19 patients infected with the original strain (Wuhan-Hu-1) were analyzed for ACE2 competitive ELISA titers based on the original strain (Wuhan-Hu-1), UK (B.1.1.7), South African (B.1.351), and Brazilian (P.1) strains. Data comparing variant strain titers to original strain titers are shown. Dots represent data for individual HCS samples, and bars represent geometric mean titers (GMT) of half-maximal effective concentration (EC50) values. Error bars represent 95% confidence intervals (95% CI). [Figure 10A] Figure 1 shows the prototype and modified B.1.351 S-trimer antigen and mouse immunogenicity study design. Figure 2 shows a schematic diagram of the prototype S-trimer and modified B.1.351 S-trimer containing three RBD mutations, and the D614G mutation in the B.1.351 variant. [Figure 10B] Figure 1 shows the prototype and modified B.1.351 S-trimer antigens and mouse immunogenicity study design. Figure 2 shows the determination of binding affinity (KD) of S-trimers (prototype and B.1.351) with human ACE2-Fc by ForteBioBioLayer interferometry. [Figure 10C]Figure 1 shows prototype and modified B.1.351 S-trimer antigens and mouse immunogenicity study design. BALB / c mice (n=16-32 / group) were immunized in Phase 1 of the study with either two doses of prototype S-trimer (3 μg), a heterologous prime-boost (1st dose prototype S-trimer; 2nd dose B.1.351 S-trimer; 3 μg of each antigen); two doses of B.1.351 S-trimer (3 μg), or two doses of bivalent vaccine (3 μg prototype S-trimer and 3 μg B.1.351 S-trimer mixed). All animals in Phase 1 received an adjuvanted prime dose (1st dose) containing alum (aluminum hydroxide). For the second booster dose, half of the animals in each group received an adjuvanted booster containing alum (aluminum hydroxide), and the other half received an unadjuvanted booster (antigen only). In the second phase of the study, animals in Group 1 were randomly assigned to receive a booster dose (third dose) of 3 μg of prototype S-trimer or 3 μg of B.1.351S-trimer (half adjuvanted, half unadjuvanted). Animals in Groups 2-3 were randomly assigned to receive a booster dose (third dose) of either 3 μg of unadjuvanted prototype or B.1.351S-trimer. [Figure 10D] Figure 1 shows the prototype and modified B.1.351 S-trimer antigen and mouse immunogenicity study design. BALB / c mice were immunized in the first stage, primed on day 0 (first dose) and boosted on day 21 (second dose), with primary analysis of humoral immunogenicity performed on blood samples on day 35. In the second stage, a booster dose was administered on day 35 (third dose), with primary analysis of humoral and cellular immune responses performed on blood samples on day 49. [Figure 11A]This figure shows humoral immune responses in mice following two doses of prototype and / or B.1.351 spike trimer antigens. Humoral immune responses in the first phase of this study were assessed on day 35 (2 weeks after the second dose) based on SARS-CoV-2 pseudovirus neutralization assays against the original strain, UK strain (B.1.1.7), South African strain (B.1.351), and Brazilian strain (P.1). Dots represent individual animals; bars represent geometric mean titers (GMTs) of EC50 values, and error bars represent 95% confidence intervals (95% CI). The fold difference (Δ) in GMTs for groups 2-4 compared to group 1 (prototype S-trimer) is shown; statistically significant differences are indicated in black. A P value of <0.05 was considered significant. [Figure 11B] This figure shows humoral immune responses in mice following two doses of prototype and / or B.1.351 spike trimer antigens. Humoral immune responses in the first phase of this study were assessed on day 35 (2 weeks after the second dose) using an ACE2 competition ELISA to detect competition of vaccine-induced antibodies for binding to ACE2 by S-trimers based on SARS-CoV-2 pseudovirus neutralization assays against the original strain (Wuhan-Hu-1), UK strain (B.1.1.7), South African strain (B.1.351), and Brazilian strain (P.1). Dots represent individual animals; bars represent geometric mean titers (GMTs) of EC50 values, and error bars represent 95% confidence intervals (95% CI). The fold difference (Δ) in GMTs for groups 2-4 compared to group 1 (prototype S-trimer) is shown, with statistically significant differences indicated in black. A P value of <0.05 was considered significant. [Figure 12A]Figure 1 shows the effect of adjuvants on humoral immune responses for the booster (second dose) and booster (third dose). Humoral immune responses in this study were assessed based on SARS-CoV-2 pseudovirus neutralization assays against the original strain, UK (B.1.1.7), South Africa (B.1.351), and Brazil (P.1) strains. Pseudovirus neutralization titer results were based on factorial analysis and are shown here for the following: in the first phase, all animals received an adjuvanted prime dose (first dose) with alum (aluminum hydroxide), half of the animals received an adjuvanted boost (second dose) with alum (aluminum hydroxide), and the other half of the animals received an unadjuvanted boost (antigen only). [Figure 12B] This figure shows the effect of adjuvants on humoral immune responses for the booster (second dose) and booster (third dose). Humoral immune responses in this study were evaluated based on SARS-CoV-2 pseudovirus neutralization assays against the original strain, UK (B.1.1.7), South Africa (B.1.351), and Brazil (P.1) strains. Pseudovirus neutralization titer results were based on factor analysis and are shown here: in the second phase, half of the animals in Group 1 received a third dose of an adjuvanted booster containing alum (aluminum hydroxide), and the other half of the animals received an unadjuvanted booster (antigen only). Dots represent individual animals; bars represent geometric mean titers (GMTs) of EC50 values, and error bars represent 95% confidence intervals (95% CI). A P value of <0.05 was considered significant. [Figure 13A]This figure shows the humoral immune response of mice to three doses of prototype and / or B.1.351 spike trimer antigens. Humoral immune responses in the second phase of this study were assessed in animals receiving a non-adjuvanted booster (third dose) on day 49 (two weeks after the third dose) based on a SARS-CoV-2 pseudovirus neutralization assay against the original strain (Wuhan-Hu-1). Neutralization titers on day 35 (two weeks after the second dose) are also shown. The fold difference in titers (days 49 and 35) for each booster group is shown. Dots represent individual animals; bars represent geometric mean titers (GMTs) of half-maximal effective concentration (EC50) values, and error bars represent 95% confidence intervals (95% CI). [Figure 13B] This figure shows humoral immune responses in mice following three doses of prototype and / or B.1.351 spike trimer antigens. Humoral immune responses in the second phase of this study were assessed in animals receiving a non-adjuvanted booster (third dose) on day 49 (two weeks after the third dose) based on a SARS-CoV-2 pseudovirus neutralization assay against the UK (B.1.1.7) variant. Neutralization titers on day 35 (two weeks after the second dose) are also shown. The fold difference in titers (days 49 and 35) for each booster group is shown. Dots represent individual animals; bars represent geometric mean titers (GMTs) of half maximally effective concentration (EC50) values, and error bars represent 95% confidence intervals (95% CI). [Figure 13C] This figure shows humoral immune responses in mice to three doses of prototype and / or B.1.351 spike trimer antigens. Humoral immune responses in the second phase of this study were assessed in animals receiving a non-adjuvanted booster (third dose) on day 49 (two weeks after the third dose) based on a SARS-CoV-2 pseudovirus neutralization assay against the South African (B.1.351) variant. Neutralization titers on day 35 (two weeks after the second dose) are also shown. The fold difference in titers (days 49 and 35) for each booster group is shown. Dots represent individual animals; bars represent geometric mean titers (GMTs) of half maximally effective concentration (EC50) values, and error bars represent 95% confidence intervals (95% CI). [Figure 13D] This figure shows humoral immune responses in mice following three doses of prototype and / or B.1.351 spike trimer antigens. Humoral immune responses in the second phase of this study were assessed in animals receiving a non-adjuvanted booster (third dose) on day 49 (two weeks after the third dose) based on a SARS-CoV-2 pseudovirus neutralization assay against the Brazil (P.1) variant pseudovirus. Neutralization titers on day 35 (two weeks after the second dose) are also shown. The fold difference in titers (days 49 and 35) for each booster group is shown. Dots represent individual animals; bars represent geometric mean titers (GMTs) of half maximally effective concentration (EC50) values, and error bars represent 95% confidence intervals (95% CI). [Figure 14A] Figure 1 shows the cellular immune responses of mice to the third dose of prototype or B.1.351 spike trimer antigen. Cellular immune responses in the second phase of this study were assessed on day 49 (2 weeks after the third dose) based on ELISpot, which detects Th1 cytokines (IFNγ and IL-2) or Th2 cytokines (IL-5) in splenocytes harvested after stimulation with an S1 peptide pool derived from the original (Wuhan-Hu-1) strain. Dots represent individual animals; bars represent group means, and error bars represent 95% confidence intervals (95% CI). [Figure 14B] Figure 1 shows the cellular immune response to the third dose of prototype or B.1.351 spike trimer antigen in mice. Cellular immune responses in the second phase of the study were assessed on day 49 (2 weeks after the third dose) based on ELISpot, which detects Th1 cytokines (IFNγ and IL-2) or Th2 cytokines (IL-5) in splenocytes harvested after stimulation with a SARS-CoV-derived S1 peptide pool. Dots represent individual animals; bars represent group means, and error bars represent 95% confidence intervals (95% CI). [Figure 14C]Figure 1 shows the cellular immune response following the third dose of prototype or B.1.351 spike trimer antigen in mice. Cellular immune responses in the second phase of this study were assessed on day 49 (2 weeks after the third dose) based on ELISpot, which detects Th1 cytokines (IFNγ and IL-2) or Th2 cytokine (IL-5) in harvested splenocytes stimulated with a pool of RBD peptides derived from B.1.351 variants. Dots represent individual animals; bars represent group means, and error bars represent 95% confidence intervals (95% CI). [Figure 14D] Figure 1 shows the cellular immune response to the third dose of prototype or B.1.351 spike trimer antigen in mice. Cellular immune responses in the second phase of this study were assessed on day 49 (2 weeks after the third dose) based on ELISpot, which detects Th1 cytokines (IFNγ and IL-2) or Th2 cytokines (IL-5) in harvested splenocytes stimulated with a pool of RBD peptides derived from the P.1 variant. Dots represent individual animals; bars represent group means, and error bars represent 95% confidence intervals (95% CI). [Figure 15A] Figure 1 shows cross-neutralization based on pseudovirus neutralization titers after a second dose of prototype and / or B.1.351S-trimer antigens in mice. Humoral immune responses in the first phase of this study were assessed on day 35 (2 weeks after the second dose) based on SARS-CoV-2 pseudovirus neutralization assays against the original strain, UK variant (B.1.1.7), South African variant (B.1.351), and Brazil variant (P.1) strains. Results are shown for Group 1 (two doses of prototype S-trimer). Individual animal results are represented by dots in each figure, with lines connecting the original and variant neutralization titers. Geometric mean titers (GMTs) of EC50 values ​​are shown, and error bars represent the 95% confidence interval (95% CI). A P value of <0.05 was considered significant (ns, not significant). [Figure 15B]Figure 1 shows cross-neutralization based on pseudovirus neutralization titers after a second dose of prototype and / or B.1.351S-trimer antigens in mice. Humoral immune responses in the first phase of this study were assessed on day 35 (2 weeks after the second dose) based on SARS-CoV-2 pseudovirus neutralization assays against the original strain, UK variant (B.1.1.7), South African variant (B.1.351), and Brazil variant (P.1) strains. Results are shown for Group 2 (heterologous prime-boost). Individual animal results are represented by dots in each figure, with lines connecting the original and variant neutralization titers. Geometric mean titers (GMTs) of EC50 values ​​are shown, and error bars represent the 95% confidence interval (95% CI). A P value of <0.05 was considered significant (ns, not significant). [Figure 15C] Figure 1 shows cross-neutralization based on pseudovirus neutralization titers after a second dose of prototype and / or B.1.351S-trimer antigens in mice. Humoral immune responses in the first phase of this study were evaluated on day 35 (2 weeks after the second dose) based on SARS-CoV-2 pseudovirus neutralization assays against the original strain, UK variant (B.1.1.7), South African variant (B.1.351), and Brazil variant (P.1) strains. Results are shown for Group 3 (two doses of B.1.351S-trimer). Individual animal results are represented by dots in each figure, with lines connecting the original and variant neutralization titers. Geometric mean titers (GMTs) of EC50 values ​​are shown, and error bars represent the 95% confidence interval (95% CI). A P value of <0.05 was considered significant (ns, not significant). [Figure 15D]Figure 1 shows cross-neutralization based on pseudovirus neutralization titers after a second dose of prototype and / or B.1.351S-trimeric antigens in mice. Humoral immune responses in the first phase of this study were evaluated on day 35 (2 weeks after the second dose) based on SARS-CoV-2 pseudovirus neutralization assays against the original strain, UK variant (B.1.1.7), South African variant (B.1.351), and Brazil variant (P.1) strains. Results are shown for two doses of the bivalent vaccine. Individual animal results are represented by dots in each figure, with lines connecting the original and variant neutralization titers. Geometric mean titers (GMTs) of EC50 values ​​are shown, and error bars represent the 95% confidence interval (95% CI). A P value of <0.05 was considered significant (ns, not significant). [Figure 16A] Figure 1 shows cross-neutralization based on ACE2 competitive titers in mice after a second dose of prototype and / or B.1.351S-trimer antigens. Humoral immune responses in the first phase of this study were assessed on day 35 (2 weeks after the second dose) based on ACE2 competitive ELISA assays against the original strain, UK variant (B.1.1.7), South African variant (B.1.351), and Brazil variant (P.1) strains. Results are shown for Group 1 (two doses of prototype S-trimer). Individual animal results are represented by dots in each figure, with lines connecting the original and variant neutralization titers. Geometric mean titers (GMTs) of EC50 values ​​are shown, and error bars represent the 95% confidence interval (95% CI). A P value of <0.05 was considered significant (ns, not significant). [Figure 16B]Figure 1 shows cross-neutralization based on ACE2 competitive titers after a second dose of prototype and / or B.1.351S-trimeric antigens in mice. Humoral immune responses in the first phase of this study were assessed on day 35 (2 weeks after the second dose) based on ACE2 competitive ELISA assays against the original strain, the UK variant (B.1.1.7), the South African variant (B.1.351), and the Brazil variant (P.1) strains. Results are shown for Group 2 (heterologous prime-boost). Individual animal results are represented by dots in each figure, with lines connecting the original and variant neutralization titers. Geometric mean titers (GMTs) of EC50 values ​​are shown, and error bars represent the 95% confidence interval (95% CI). A P value of <0.05 was considered significant (ns, not significant). [Figure 16C] Figure 1 shows cross-neutralization based on ACE2 competitive titers in mice after a second dose of prototype and / or B.1.351S-trimer antigens. Humoral immune responses in the first phase of this study were assessed on day 35 (2 weeks after the second dose) based on ACE2 competitive ELISA assays against the original strain, UK variant (B.1.1.7), South African variant (B.1.351), and Brazil variant (P.1) strains. Results are shown for Group 3 (two doses of B.1.351S-trimer). Individual animal results are represented by dots in each figure, with lines connecting the original and variant neutralization titers. Geometric mean titers (GMTs) of EC50 values ​​are shown, and error bars represent the 95% confidence interval (95% CI). A P value of <0.05 was considered significant (ns, not significant). [Figure 16D]Figure 1 shows cross-neutralization based on ACE2 competitive titers after a second dose of prototype and / or B.1.351S-trimeric antigens in mice. Humoral immune responses in the first phase of this study were assessed on day 35 (2 weeks after the second dose) based on ACE2 competitive ELISA assays against the original strain, UK variant (B.1.1.7), South African variant (B.1.351), and Brazil variant (P.1) strains. Results are shown for two doses of the bivalent vaccine. Individual animal results are represented by dots in each figure, with lines connecting the original and variant neutralization titers. Geometric mean titers (GMTs) of EC50 values ​​are shown, and error bars represent the 95% confidence interval (95% CI). A P value of <0.05 was considered significant (ns, not significant). [Figure 17A] This figure shows humoral immune responses in mice after two doses of prototype and / or B.1.351S-trimeric antigens. Humoral immune responses in the first phase of this study were assessed on day 35 (2 weeks after the second dose) based on a SARS-CoV-2 pseudovirus neutralization assay against the original strain. All animals in the first phase received an alum-adjuvanted (aluminum hydroxide) prime dose (first dose), half of the animals in each group received an alum-adjuvanted (aluminum hydroxide) boost (second dose), and the other half received an unadjuvanted boost (antigen only). Results are shown for subgroups of animals that received either the adjuvanted or unadjuvanted boost (second dose). Dots represent individual animals; bars represent geometric mean titers (GMTs) of EC50 values, and error bars represent 95% confidence intervals (95% CI). A P value <0.05 was considered significant. [Figure 17B]This figure shows humoral immune responses in mice after two doses of prototype and / or B.1.351S-trimeric antigen. Humoral immune responses in the first phase of this study were assessed on day 35 (two weeks after the second dose) based on a SARS-CoV-2 pseudovirus neutralization assay against the UK variant (B.1.1.7). All animals in the first phase received an alum (aluminum hydroxide)-adjuvanted prime dose (first dose), half of the animals in each group received an alum (aluminum hydroxide)-adjuvanted boost (second dose), and the other half received an unadjuvanted boost (antigen only). Results are shown for subgroups of animals that received either the adjuvanted or unadjuvanted boost (second dose). Dots represent individual animals; bars represent geometric mean titers (GMT) of EC50 values, and error bars represent 95% confidence intervals (95% CI). A P value <0.05 was considered significant. [Figure 17C] This figure shows humoral immune responses in mice after two doses of prototype and / or B.1.351S-trimeric antigens. Humoral immune responses in the first phase of this study were assessed on day 35 (two weeks after the second dose) based on a SARS-CoV-2 pseudovirus neutralization assay against the South African variant (B.1.351). All animals in the first phase received an alum (aluminum hydroxide)-adjuvanted prime dose (first dose), half of the animals in each group received an alum (aluminum hydroxide)-adjuvanted boost (second dose), and the other half received an unadjuvanted boost (antigen only). Results are shown for subgroups of animals that received either the adjuvanted or unadjuvanted boost (second dose). Dots represent individual animals; bars represent geometric mean titers (GMT) of EC50 values, and error bars represent 95% confidence intervals (95% CI). A P value <0.05 was considered significant. [Figure 17D]This figure shows humoral immune responses in mice after two doses of prototype and / or B.1.351S-trimeric antigens. Humoral immune responses in the first phase of this study were assessed on day 35 (2 weeks after the second dose) based on a SARS-CoV-2 pseudovirus neutralization assay against the Brazil variant (P.1) strain. All animals in the first phase received an alum-adjuvanted (aluminum hydroxide) prime dose (first dose), half of the animals in each group received an alum-adjuvanted (aluminum hydroxide) boost (second dose), and the other half received an unadjuvanted boost (antigen only). Results are shown for subgroups of animals that received either the adjuvanted or unadjuvanted boost (second dose). Dots represent individual animals; bars represent geometric mean titers (GMTs) of EC50 values, and error bars represent 95% confidence intervals (95% CI). A P value <0.05 was considered significant. [Figure 18A] This figure shows humoral immune responses in mice after three doses of prototype and / or B.1.351S-trimeric antigens. Humoral immune responses in the second phase of this study were assessed on day 49 (2 weeks after the third dose) based on a SARS-CoV-2 pseudovirus neutralization assay against the original strain (Wuhan-Hu-1). Results are shown for all subgroups (representing all vaccination combinations) in this study (n=4 / subgroup). Dots represent individual animals; bars represent geometric mean titers (GMTs) of EC50 values, and error bars represent 95% confidence intervals (95% CI). [Figure 18B]This figure shows humoral immune responses in mice after three doses of prototype and / or B.1.351S-trimeric antigens. Humoral immune responses in the second phase of the study were assessed on day 49 (2 weeks after the third dose) based on a SARS-CoV-2 pseudovirus neutralization assay against the UK (B.1.1.7) variant pseudovirus. Results are shown for all subgroups (representing all vaccination combinations) in the study (n=4 / subgroup). Dots represent individual animals; bars represent geometric mean titers (GMTs) of EC50 values, and error bars represent 95% confidence intervals (95% CI). [Figure 18C] This figure shows humoral immune responses in mice after three doses of prototype and / or B.1.351S-trimeric antigens. Humoral immune responses in the second phase of the study were assessed on day 49 (2 weeks after the third dose) based on a SARS-CoV-2 pseudovirus neutralization assay against the South African (B.1.351) variant pseudovirus. Results are shown for all subgroups (representing all vaccination combinations) in the study (n=4 / subgroup). Dots represent individual animals; bars represent geometric mean titers (GMTs) of EC50 values, and error bars represent 95% confidence intervals (95% CI). [Figure 18D] This figure shows humoral immune responses in mice after three doses of prototype and / or B.1.351S-trimeric antigens. Humoral immune responses in the second phase of this study were assessed on day 49 (2 weeks after the third dose) based on a SARS-CoV-2 pseudovirus neutralization assay against the Brazilian (P.1) variant pseudovirus. Results are shown for all subgroups (representing all vaccination combinations) in this study (n=4 / subgroup). Dots represent individual animals; bars represent geometric mean titers (GMTs) of EC50 values, and error bars represent 95% confidence intervals (95% CI). DETAILED DESCRIPTION OF THE INVENTION

[0060] Provided herein are immunogenic compositions, methods, and uses of fusion peptides and proteins comprising coronavirus antigens or immunogens for the treatment, e.g., prevention, and treatment, of coronavirus infections. In some embodiments, compositions and methods of use of recombinant soluble surface antigens derived from RNA viruses in covalently linked trimeric forms are disclosed. In some embodiments, the resulting fusion proteins are secreted as disulfide-linked homotrimers, which are structurally more stable while retaining the conformation of native-like trimeric viral antigens, allowing them to be used as more effective vaccines against these dangerous pathogens.

[0061] In some embodiments, disclosed herein are methods for using viral antigen trimers as vaccines or as part of multivalent vaccines to prevent viral infection, either without an adjuvant, with an adjuvant, or with two or more adjuvants, optionally via either intramuscular injection or intranasal administration.

[0062] In some embodiments, disclosed herein are methods for using viral antigen trimers as antigens to diagnose viral infections by detection of antibodies, e.g., IgM or IgG, that recognize viral antigens, such as neutralizing antibodies.

[0063] In some embodiments, disclosed herein are methods for using viral antigen trimers as antigens to generate polyclonal or monoclonal antibodies that can be used for passive immunization, e.g., neutralizing mAbs for treating coronavirus infections.

[0064] In some embodiments, disclosed herein are viral antigen trimers as vaccines or as part of multivalent vaccines, which include multiple trimeric subunit vaccines that include viral antigens of the same protein from a virus, or viral antigens of two or more different proteins from one or more viruses, or one or more strains of the same virus.

[0065] In some embodiments, disclosed herein are monovalent vaccines comprising the viral antigen trimers disclosed herein. In some embodiments, disclosed herein are bivalent vaccines comprising the viral antigen trimers disclosed herein. In some embodiments, disclosed herein are trivalent vaccines comprising the viral antigen trimers disclosed herein. In some embodiments, disclosed herein are tetravalent vaccines comprising the viral antigen trimers disclosed herein.

[0066] In some embodiments, disclosed herein are monovalent vaccines comprising the S-trimers disclosed herein. In some embodiments, disclosed herein are bivalent vaccines comprising the S-trimers disclosed herein. In some embodiments, disclosed herein are bivalent vaccines comprising at least one S-trimer comprising a first S protein antigen and at least one S-trimer comprising a second S protein antigen. In some embodiments, the first and second S protein antigens are derived from the same S protein of one or more viral species or strains / subtypes, or from two or more different S proteins of one or more viral species or one or more strains / subtypes of the same viral species. In some embodiments, disclosed herein are trivalent vaccines comprising the S-trimers disclosed herein. In some embodiments, disclosed herein are trivalent vaccines comprising at least one S-trimer comprising a first S protein antigen, at least one S-trimer comprising a second S protein antigen, and at least one S-trimer comprising a third S protein antigen. In some embodiments, the first, second, and third S protein antigens are derived from the same S protein of one or more viral species or strains / subtypes, or from two, three, or more different S proteins of one or more viral species or one or more strains / subtypes of the same viral species. In some embodiments, disclosed herein are tetravalent vaccines comprising the S-trimers disclosed herein. In some embodiments, disclosed herein are tetravalent vaccines comprising at least one S-trimer comprising a first S protein antigen, at least one S-trimer comprising a second S protein antigen, at least one S-trimer comprising a third S protein antigen, and at least one S-trimer comprising a fourth S protein antigen. In some embodiments, the first, second, third, and fourth S protein antigens are derived from the same S protein of one or more viral species or strains / subtypes, or from two, three, four, or more different S proteins of one or more viral species or one or more strains / subtypes of the same viral species.

[0067] Proteins, such as recombinant polypeptides and fusion proteins, comprising coronavirus virus antigens and immunogens provided herein are useful for effectively and safely treating coronavirus infections (e.g., therapeutically and prophylactically). For example, proteins comprising coronavirus virus antigens and immunogens provided herein treat coronavirus infections without mediated vaccine-induced disease enhancement (VED) and / or antibody-dependent enhancement (ADE). Furthermore, proteins comprising coronavirus virus antigens and immunogens provided herein are easily produced and exhibit stability under high-stress conditions, such as high temperature, extreme pH, and hyperosmolarity and hypoosmolarity. Thus, the proteins and immunogenic compositions provided herein avoid or satisfy the production, stability, safety, and efficacy issues that have hindered the development of coronavirus vaccines.

[0068] In some embodiments, the coronavirus virus antigens and immunogens provided herein include coronavirus spike (S) proteins or peptides, particularly SARS-CoV or SARS-CoV-2 S proteins. The SARS-CoV and SARS-CoV-2 spikes are composed of a trimer of S protein, which belongs to the class I viral fusion glycoprotein group, which also includes HIV glycoprotein 160 (Env), influenza hemagglutinin (HA), paramyxovirus F, and Ebola virus glycoproteins. The SARS-CoV and SARS-CoV-2 S proteins, respectively, encode surface glycoprotein precursors, with the amino-terminal and most protein portions predicted to be located on the cell surface or outside of the virus particle. The S protein contains an N-terminal signal peptide, an extracellular domain, a transmembrane domain, and an intracellular domain. As with other coronaviruses, the S proteins of SARS-CoV and SARS-CoV-2 are cleaved into S1 and S2 subunits by proteases. Notably, SARS-CoV-2 contains a furin-like cleavage site that is absent in other SARS-like CoVs.

[0069] In some embodiments, provided herein are recombinant S ectodomain trimers. In some embodiments, the recombinant S ectodomain trimers comprise recombinant S ectodomain protomers from an alphacoronavirus, such as NL63-CoV or 229E-CoV. In some embodiments, the recombinant S ectodomain trimers comprise S ectodomain protomers from a betacoronavirus, such as OC43-CoV, SARS-CoV, SARS-CoV-2, MERS-CoV, HKU1-CoV, WIV1-CoV, mouse hepatitis virus (MHV), or HKU9-CoV.

[0070] Like other enveloped RNA viruses such as HIV, RSV, and influenza, coronaviruses such as SARS-CoV-2 each possess a trimeric surface antigen on their viral envelope to gain entry into different host cells via specific cell surface receptors during infection. Similar to SARS-CoV-1, SARS-CoV-2 uses its trimeric viral surface antigen S protein to enter host cells of the mammalian respiratory system upon binding to its specific cell surface receptor, ACE2. A prerequisite for generating an effective recombinant subunit vaccine is the ability to create a native-like viral S antigen, particularly one that maintains its trimeric structure, to elicit sufficient antibodies capable of binding to the receptor-binding domain (RBD) of the viral S protein, thereby preventing the virus from binding to the ACE2 receptor and thus eliminating viral infection.

[0071] In some embodiments, proteins comprising coronavirus virus antigens or immunogens, e.g., SARS-CoV or SARS-CoV-2 S protein peptides, can generate an immune response, e.g., an immune response to a SARS-CoV or SARS-CoV-2 S protein peptide. In some embodiments, the immune response inhibits or reduces coronavirus replication in a subject, e.g., a patient. In some embodiments, the immune response comprises producing one or more neutralizing antibodies, such as polyclonal and / or monoclonal antibodies. In some embodiments, the neutralizing antibodies inhibit or reduce coronavirus replication in a subject, e.g., a patient. In some embodiments, administering the proteins to a subject, e.g., as an immunogenic composition, does not result in antibody-dependent enhancement (ADE) in the subject due to previous exposure to coronavirus. In some aspects, proteins comprising coronavirus virus antigens and immunogens are used as vaccines.

[0072] In some embodiments, coronavirus virus antigens and immunogens, e.g., SARS-CoV or SARS-CoV-2 S protein peptides, are linked to a protein or peptide to form a fusion protein or recombinant polypeptide. In some embodiments, the protein or peptide to which the coronavirus virus antigen or immunogen is linked is associated, e.g., covalently or non-covalently linked, with the protein or peptide, e.g., the protein or peptide of the fusion protein or recombinant polypeptide. Thus, in some cases, the protein or peptide to which the coronavirus virus antigen or immunogen is linked is a multimerization domain.

[0073] In some embodiments, coronavirus virus antigens and immunogens, e.g., coronavirus S protein peptides, are linked to a collagen propeptide, e.g., at the C-terminus of the collagen propeptide, to form a fusion peptide or recombinant polypeptide. Thus, in some embodiments, the proteins provided herein comprise a recombinant polypeptide comprising a coronavirus virus antigen and immunogen, e.g., a coronavirus S protein peptide, or a fragment or epitope thereof, linked to a collagen C-terminal propeptide. In some embodiments, the collagen propeptide is derived from the human C-propeptide of α1 collagen and is capable of self-trimerization upon expression.

[0074] In some embodiments, coronavirus viral antigens and immunogens, such as coronavirus S protein peptides, are linked to collagen propeptides, e.g., at the C-terminus of the collagen propeptide, to aid in the protein's ability to generate an immune response. For example, recombinant proteins can be produced to preserve the tertiary and quaternary structure of the coronavirus S protein peptide, which can be important for the stability of the coronavirus S protein peptide's native conformation and the availability of antigenic sites on the surface of the protein that can elicit an immune response, e.g., neutralizing antibodies. Furthermore, coronavirus S protein peptides can be linked to proteins or peptides capable of self-trimerization, allowing the recombinant protein to aggregate, thus mimicking the native homotrimeric structure of the coronavirus S protein peptide on the viral envelope.

[0075] In some embodiments, a coronavirus S protein peptide is linked to the C-terminal propeptide of collagen, resulting in a self-trimerizing recombinant polypeptide. In some embodiments, the proteins provided herein comprise multiple self-trimerizing coronavirus S protein peptides and a propeptide of a collagen recombinant polypeptide. In some embodiments, the trimeric nature of the recombinant protein aids in the stability of the protein. In some embodiments, the trimeric nature of the recombinant protein aids in the ability of the protein to generate an immune response. In some embodiments, the trimeric nature of the recombinant protein and / or the macrostructure of multiple self-trimerizing recombinant proteins aids in the ability of the protein to generate an immune response.

[0076] Also provided herein are immunogenic compositions comprising the proteins provided herein, methods of producing the proteins provided herein, methods of treating subjects with the proteins and compositions provided herein, and kits.

[0077] All publications, including patent documents, scientific articles, and databases, referenced in this application are incorporated by reference in their entirety for all purposes to the same extent as if each individual publication were individually incorporated by reference. To the extent that a definition set forth herein is contrary to or inconsistent with a definition set forth in a patent, application, published application, or other publication incorporated herein by reference, the definition set forth herein shall take precedence over the definition incorporated herein by reference. The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described. I. Viral Antigens and Immunogens

[0078] The proteins provided herein include coronavirus virus antigens and immunogens. The coronavirus virus antigens and immunogens contemplated herein can promote or stimulate a cellular and / or humoral response. In some embodiments, the response, e.g., a cellular or humoral response, includes the production of antibodies, e.g., neutralizing antibodies. In some embodiments, the coronavirus virus antigen or immunogen is a coronavirus spike (S) protein peptide.

[0079] Coronaviruses are a family of positive-sense, single-stranded RNA viruses known to cause severe respiratory illness. These viruses have the largest genomes (26–32 kb) of any known RNA virus and are phylogenetically divided into four genera (α, β, γ, and δ), with betacoronaviruses further subdivided into four lineages (A, B, C, and D). Currently, viruses from the coronavirus family known to infect humans originate from the alphacoronavirus and betacoronavirus genera. Additionally, the gammacoronavirus and deltacoronavirus genera are thought to be capable of infecting humans in the future. Non-limiting examples of betacoronaviruses include Middle East respiratory syndrome coronavirus (MERS-CoV), severe acute respiratory syndrome coronavirus (SARS-CoV), human coronavirus HKU1 (HKU1-CoV), human coronavirus OC43 (OC43-CoV), mouse hepatitis virus (MHV-CoV), bat SARS-like coronavirus WIV1 (WIV1-CoV), and human coronavirus HKU9 (HKU9-CoV). Non-limiting examples of alphacoronaviruses include human coronavirus 229E (229E-CoV), human coronavirus NL63 (NL63-CoV), porcine epidemic diarrhea virus (PEDV), and transmissible gastroenteritis coronavirus (TGEV). A non-limiting example of a deltacoronavirus is porcine deltacoronavirus (SDCV).

[0080] A list of coronaviruses associated with severe acute respiratory syndrome is provided here: Bat coronavirus Cp / Yunnan2011 Bat coronavirus RaTG13 Bat coronavirus Rp / Shaanxi2011 - Bat SARS coronavirus HKU3 Bat SARS coronavirus HKU3-1 Bat SARS coronavirus HKU3-10 Bat SARS coronavirus HKU3-11 Bat SARS coronavirus HKU3-12 Bat SARS coronavirus HKU3-13 Bat SARS coronavirus HKU3-2 Bat SARS coronavirus HKU3-3 Bat SARS coronavirus HKU3-4 Bat SARS coronavirus HKU3-5 Bat SARS coronavirus HKU3-6 Bat SARS coronavirus HKU3-7 Bat SARS coronavirus HKU3-8 Bat SARS coronavirus HKU3-9 Bat SARS coronavirus Rp1 Bat SARS coronavirus Rp2 - Bat SARS CoV Rf1 / 2004 Bat CoV 273 / 2005 - Bat SARS CoV Rm1 / 2004 Bat CoV 279 / 2005 Bat SARS CoV Rp3 / 2004 Bat SARS-like coronavirus Bat SARS-like coronavirus Rs3367 Bat SARS-like coronavirus RsSHC014 Bat SARS-like coronavirus WIV1 Bat SARS-like coronavirus YNLF_31C Bat SARS-like coronavirus YNLF_34C BtRf-BetaCoV / HeB2013 BtRf-BetaCoV / JL2012 BtRf-BetaCoV / SX2013 BtRs-BetaCoV / GX2013 BtRs-BetaCoV / HuB2013 BtRs-BetaCoV / YN2013 Civet cat SARS CoV 007 / 2004 Civet cat SARS CoV SZ16 / 2003 Civet cat SARS CoV SZ3 / 2003 - Recombinant SARS-CoV SARS coronavirus ExoN1 SARS coronavirus MA15 SARS coronavirus MA15 ExoN1 SARS coronavirus wtic-MB Horseshoe bat coronavirus SARS bat coronavirus SARS coronavirus A001 SARS coronavirus A013 SARS coronavirus A021 SARS coronavirus A022 SARS coronavirus A030 SARS coronavirus A031 SARS Coronavirus AS SARS coronavirus B012 SARS coronavirus B024 SARS coronavirus B029 SARS coronavirus B033 SARS coronavirus B039 SARS coronavirus B040 SARS coronavirus BJ01 SARS coronavirus BJ02 SARS coronavirus BJ03 SARS coronavirus BJ04 SARS coronavirus BJ162 SARS coronavirus BJ182-12 SARS coronavirus BJ182-4 SARS coronavirus BJ182-8 SARS coronavirus BJ182a SARS coronavirus BJ182b SARS coronavirus BJ202 SARS coronavirus BJ2232 SARS coronavirus BJ302 SARS coronavirus C013 SARS coronavirus C014 SARS coronavirus C017 SARS Coronavirus C018 SARS Coronavirus C019 SARS coronavirus C025 SARS coronavirus C028 SARS coronavirus C029 SARS Coronavirus CDC #200301157 SARS coronavirus civet cat 010 SARS coronavirus civet cat 014 SARS coronavirus civet cat 019 SARS coronavirus civet cat 020 SARS coronavirus CS21 SARS Coronavirus CS24 SARS coronavirus CUHK-AG01 SARS coronavirus CUHK-AG02 SARS coronavirus CUHK-AG03 SARS coronavirus CUHK-L2 SARS coronavirus CUHK-Su10 SARS coronavirus CUHK-W1 SARS coronavirus cw037 SARS coronavirus cw049 SARS coronavirus ES191 SARS coronavirus ES260 SARS Coronavirus FRA -SARS Coronavirus Frankfurt 1 SARS Coronavirus Frankfurt 1-v01 SARS coronavirus GD01 SARS coronavirus GD03T0013 SARS coronavirus GD322 SARS coronavirus GD69 SARS coronavirus GDH-BJH01 SARS coronavirus GZ-A SARS coronavirus GZ-B SARS coronavirus GZ-C SARS coronavirus GZ-D SARS coronavirus GZ02 SARS coronavirus GZ0401 SARS coronavirus GZ0402 SARS coronavirus GZ0403 SARS coronavirus GZ43 SARS coronavirus GZ50 SARS coronavirus GZ60 SARS coronavirus HB SARS coronavirus HC / SZ / 61 / 03 SARS coronavirus HGZ8L1-A SARS coronavirus HGZ8L1-B SARS coronavirus HGZ8L2 SARS Coronavirus HHS-2004 SARS coronavirus HKU-36871 SARS coronavirus HKU-39849 SARS coronavirus HKU-65806 SARS coronavirus HKU-66078 SARS Coronavirus Hong Kong / 03 / 2003 SARS coronavirus HPZ-2003 SARS coronavirus HSR1 SARS coronavirus HSZ-A SARS coronavirus HSZ-Bb SARS coronavirus HSZ-Bc SARS coronavirus HSZ-Cb SARS coronavirus HSZ-Cc SARS coronavirus HSZ2-A SARS coronavirus HZS2-Bb SARS coronavirus HZS2-C SARS coronavirus HZS2-D SARS coronavirus HZS2-E SARS coronavirus HZS2-Fb SARS coronavirus HZS2-Fc SARS Coronavirus JMD SARS coronavirus LC1 SARS coronavirus LC2 SARS coronavirus LC3 SARS coronavirus LC4 SARS coronavirus LC5 SARS coronavirus LLJ-2004 SARS coronavirus NS-1 SARS coronavirus P2 SARS coronavirus PC4-115 SARS coronavirus PC4-127 SARS coronavirus PC4-13 SARS coronavirus PC4-136 SARS coronavirus PC4-137 SARS coronavirus PC4-145 SARS coronavirus PC4-199 SARS coronavirus PC4-205 SARS coronavirus PC4-227 SARS coronavirus PC4-241 SARS coronavirus PUMC01 SARS coronavirus PUMC02 SARS coronavirus PUMC03 SARS Coronavirus Rs_672 / 2006 SARS coronavirus sf098 SARS coronavirus sf099 SARS coronavirus ShanghaiQXC1 SARS coronavirus ShanghaiQXC2 SARS Coronavirus Shanhgai LY SARS coronavirus Sin0409 SARS Coronavirus Sin2500 SARS coronavirus Sin2677 SARS coronavirus Sin2679 SARS coronavirus Sin2748 SARS coronavirus Sin2774 SARS coronavirus Sin3408 SARS coronavirus Sin3408L SARS coronavirus Sin3725V SARS coronavirus Sin3765V SARS coronavirus Sin842 SARS coronavirus Sin845 SARS coronavirus Sin846 SARS coronavirus Sin847 SARS coronavirus Sin848 SARS coronavirus Sin849 SARS coronavirus Sin850 SARS coronavirus Sin852 SARS Coronavirus Sin_WNV SARS Coronavirus Sino1-11 SARS Coronavirus Sino3-11 SARS coronavirus SinP1 SARS coronavirus SinP2 SARS coronavirus SinP3 SARS coronavirus SinP4 SARS coronavirus SinP5 SARS Coronavirus SoD SARS coronavirus SZ1 SARS coronavirus SZ13 SARS Coronavirus Taiwan SARS coronavirus Taiwan JC-2003 SARS coronavirus Taiwan TC1 SARS Coronavirus Taiwan TC2 SARS Coronavirus Taiwan TC3 SARS coronavirus TJ01 SARS Coronavirus TJF SARS Coronavirus Tor2 -SARS Coronavirus TW SARS coronavirus TW-GD1 SARS coronavirus TW-GD2 SARS coronavirus TW-GD3 SARS coronavirus TW-GD4 SARS coronavirus TW-GD5 SARS coronavirus TW-HP1 SARS coronavirus TW-HP2 SARS coronavirus TW-HP3 SARS coronavirus TW-HP4 SARS coronavirus TW-JC2 SARS coronavirus TW-KC1 SARS coronavirus TW-KC3 SARS coronavirus TW-PH1 SARS coronavirus TW-PH2 SARS coronavirus TW-YM1 SARS coronavirus TW-YM2 SARS coronavirus TW-YM3 SARS coronavirus TW-YM4 SARS coronavirus TW1 SARS coronavirus TW10 SARS coronavirus TW11 SARS coronavirus TW2 SARS coronavirus TW3 SARS coronavirus TW4 SARS coronavirus TW5 SARS coronavirus TW6 SARS coronavirus TW7 SARS coronavirus TW8 SARS coronavirus TW9 SARS Coronavirus TWC SARS coronavirus TWC2 SARS coronavirus TWC3 SARS Coronavirus TWH SARS Coronavirus TWJ SARS Coronavirus TWK SARS Coronavirus TWS SARS Coronavirus TWY SARS Coronavirus Urban SARS Coronavirus Vietnam SARS coronavirus WF188 SARS Coronavirus WH20 SARS coronavirus WHU SARS coronavirus xw002 SARS coronavirus ZJ01 SARS coronavirus ZJ02 SARS coronavirus ZJ0301 SARS coronavirus ZMY1 SARS coronavirus ZS-A SARS coronavirus ZS-B SARS coronavirus ZS-C SARS-associated bat coronavirus RsSHC014 SARS-associated betacoronavirus Rp3 / 2004 Severe acute respiratory syndrome coronavirus 2

[0081] Exemplary SARS CoV-2 strains are shown in the table below.

[0082] [Table 1]

[0083] The coronavirus viral genome is capped, polyadenylated, and surrounded by nucleocapsid proteins. Coronavirus virions contain a viral envelope containing a type I fusion glycoprotein called the spike (S) protein. Most coronaviruses share a common genome organization, with the replicase gene contained in the 5' portion of the genome and the structural genes contained in the 3' portion of the genome.

[0084] The coronavirus spike (S) protein is a class I fusion glycoprotein initially synthesized as a precursor protein. Individual precursor S polypeptides form homotrimers, undergo glycosylation in the Golgi apparatus, and undergo processing to remove the signal peptide and cleavage by cellular proteases. Within the homotrimer, they still associate as S1 / S2 protomers, thereby generating distinct S1 and S2 polypeptide chains, which are trimers of heterodimers. The S1 subunit is distal to the viral membrane and contains the receptor-binding domain (RBD) that mediates viral attachment to host receptors. The S2 subunit contains fusion protein machinery, including a fusion peptide, two heptad repeat sequences (HR1 and HR2), a central helix typical of fusion glycoproteins, a transmembrane domain, and a cytosolic tail domain.

[0085] In some cases, the coronavirus viral antigen or immunogen is a coronavirus S protein peptide in the pre-fusion conformation, which is the structural conformation adopted by the ectodomain of the coronavirus S protein after it has been processed into the mature coronavirus S protein in the secretory system and before undergoing a fusion gene event that results in the transition of coronavirus S to the post-fusion conformation. The three-dimensional structure of an exemplary coronavirus S protein (HKU1-CoV) in the pre-fusion conformation is described in Kirchdoerfer et al., "Pre-fusion structure of a human coronavirus spike protein," Nature, 531:118-121, 2016.

[0086] In some cases, the coronavirus virus antigen or immunogen contains one or more amino acid substitutions, deletions, or insertions compared to the native coronavirus S sequence that result in increased retention of the pre-fusion conformation compared to a coronavirus S ectodomain trimer formed from the corresponding native coronavirus S sequence. "Stabilization" of the pre-fusion conformation by one or more amino acid substitutions, deletions, or insertions can be, for example, energetic stabilization (e.g., a decrease in the energy of the pre-fusion conformation compared to the post-fusion open conformation) and / or kinetic stabilization (e.g., a decrease in the rate of transition from the pre-fusion conformation to the post-fusion conformation). Furthermore, stabilization of the coronavirus S ectodomain trimer in the pre-fusion conformation can include increased resistance to denaturation compared to the corresponding native coronavirus S sequence. Methods for determining whether a coronavirus ectodomain trimer is in the pre-fusion conformation are provided herein, including (but not limited to) negative stain electron microscopy and antibody binding assays using a pre-fusion conformation-specific antibody.

[0087] In some cases, the coronavirus viral antigen or immunogen is a fragment of an S protein peptide. In some embodiments, the antigen or immunogen is an epitope of an S protein peptide. An epitope comprises an antigenic determinant chemical group or peptide sequence on a molecule that is antigenic so as to elicit a specific immune response. For example, an epitope is a region of an antigen to which B cells and / or T cells respond. Antibodies can bind to specific antigenic epitopes, such as epitopes on the coronavirus S ectodomain. Epitopes can be formed from both contiguous and non-contiguous amino acids juxtaposed by tertiary folding of a protein. In some embodiments, the coronavirus epitope is a linear epitope. In some embodiments, the coronavirus epitope is a conformational epitope. In some embodiments, the coronavirus epitope is a neutralizing epitope site. In some embodiments, all neutralizing epitopes of a coronavirus S protein peptide or fragment thereof are present as the antigen or immunogen.

[0088] In some cases, for example, when the viral antigen or immunogen is a fragment of an S protein peptide, only a single subunit of the S protein peptide is present, and this single subunit of the S protein peptide is trimerized. In some embodiments, the viral antigen or immunogen comprises a signal peptide, an S1 subunit peptide, an S2 subunit peptide, or any combination thereof. In some embodiments, the viral antigen or immunogen may comprise a signal peptide, a receptor binding domain (RBD) peptide, a receptor binding motif (RBM) peptide, a fusion peptide (FP), a heptad repeat 1 (HR1) peptide, or a heptad repeat 2 (HR2) peptide, or any combination thereof. In some embodiments, the viral antigen or immunogen comprises the receptor binding domain (RBD) of the S protein. In some embodiments, the viral antigen or immunogen comprises the S1 and S2 subunits of the S protein. In some embodiments, the viral antigen or immunogen comprises the S1 subunit of the S protein, but not the S2 subunit. In some embodiments, the viral antigen or immunogen comprises the S2 subunit of the S protein but not the S1 subunit, hi some embodiments, the viral antigen or immunogen does not comprise a transmembrane (TM) domain peptide and / or a cytoplasmic (CP) domain peptide.

[0089] In some embodiments, the viral antigen or immunogen comprises a protease cleavage site, and the protease is optionally furin, trypsin, factor Xa, or cathepsin L.

[0090] In some embodiments, the viral antigen or immunogen does not comprise a protease cleavage site, and the protease is optionally furin, trypsin, factor Xa, or cathepsin L, or comprises a mutant protease cleavage site that cannot be cleaved by the protease.

[0091] In some embodiments, the viral antigen or immunogen is a SARS-CoV-2 antigen comprising at least one SARS-CoV-2 protein or fragment thereof. In some embodiments, the SARS-CoV-2 antigen is recognized by SARS-CoV-2-reactive antibodies and / or T cells. In some embodiments, the SARS-CoV-2 antigen is an inactivated whole virus. In some embodiments, the SARS-CoV-2 antigen comprises a viral subunit. In some embodiments, the SARS-CoV-2 antigen comprises a SARS-CoV-2 structural protein or fragment thereof. In some embodiments, the SARS-CoV-2 structural protein comprises one or more of the group consisting of spike (S) protein, membrane (M) protein, nucleocapsid (N) protein, and envelope (E) protein. In some embodiments, the SARS-CoV-2 antigen comprises or further comprises a SARS-CoV-2 nonstructural protein or fragment thereof. The nucleotide sequence of a representative SARS-CoV-2 isolate (Wuhan-Hu-1) is represented as GenBank No. MN908947.3 (Wu et al., Nature, 579:265-269, 2020).

[0092] In some embodiments, the viral antigen or immunogen comprises the sequence set forth in SEQ ID NO: 55. In some embodiments, the viral antigen or immunogen has an amino acid sequence having at least or about 80%, 85%, 90%, 92%, 95%, or 97% sequence identity to SEQ ID NO: 55, shown below (the sequence representing the receptor binding motif (RBM) within the receptor binding domain (RBD) is underlined). In some embodiments, the viral antigen or immunogen comprises an RBD-trimer, e.g., a SARS-CoV-2 RBD sequence set forth in any of SEQ ID NOs: 67-80. 10 20 30 40 50 60 MFVFLVLLPLVSSQCVNLTTRTQLPPAYTNSFTRGVYYPDKVFRSSVLHSTQDLFLPFFS 70 80 90 100 110 120 NVTWFHAIHVSGTNGTKRFDNPVLPFNDGVYFASTEKSNIIRGWIFGTTLDSKTQSLLIV 130 140 150 160 170 180 NNATNVVIKVCEFQFCNDPFLGVYYHKNNKSWMESEFRVYSSANNCTFEYVSQPFLMDLE 190 200 210 220 230 240 GKQGNFKNLREFVFKNIDGYFKIYSKHTPINLVRDLPQGFSALEPLVDLPIGINITRFQT 250 260 270 280 290 300 LLALHRSYLTPGDSSSGWTAGAAAYYVGYLQPRTFLLKYNENGTITDAVDCALDPLSETK 310 320 330 340 350 360 CTLKSFTVEKGIYQTSNFRVQPTESIVRFPNITNLCPFGEVFNATRFASVYAWNRKRISN 370 380 390 400 410 420 CVADYSVLYNSASFSTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGKIAD 430 440 450 460 470 480 YNYKLPDDFTGCVIAW NSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPC 490 500 510 520 530 540 NGVEGFNCYFPLQSYGFQPTNGVGYQPYR VVVLSFELLHAPATVCGPKKSTNLVKNKCVN 550 560 570 580 590 600 FNFNGLTGTGVLTESNKKFLPFQQFGRDIADTTDAVRDPQTLEILDITPCSFGGVSVITP 610 620 630 640 650 660 GTNTSNQVAVLYQDVNCTEVPVAIHADQLTPTWRVYSTGSNVFQTRAGCLIGAEHVNNSY 670 680 690 700 710 720 ECDIPIGAGICASYQTQTNSPRRARSVASQSIIAYTMSLGAENSVAYSNNSIAIPTNFTI 730 740 750 760 770 780 SVTTEILPVSMTKTSVDCTMYICGDSTECSNLLLQYGSFCTQLNRALTGIAVEQDKNTQE 790 800 810 820 830 840 VFAQVKQIYKTPPIKDFGGFNFSQILPDPSKPSKRSFIEDLLFNKVTLADAGFIKQYGDC 850 860 870 880 890 900 LGDIAARDLICAQKFNGLTVLPPLLTDEMIAQYTSALLAGTITSGWTFGAGAALQIPFAM 910 920 930 940 950 960 QMAYRFNGIGVTQNVLYENQKLIANQFNSAIGKIQDSLSSTASALGKLQDVVNQNAQALN 970 980 990 1000 1010 1020 TLVKQLSSNFGAISSVLNDILSRLDKVEAEVQIDRLITGRLQSLQTYVTQQLIRAAEIRA 1030 1040 1050 1060 1070 1080 SANLAATKMSECVLGQSKRVDFCGKGYHLMSFPQSAPHGVVFLHVTYVPAQEKNFTTAPA 1090 1100 1110 1120 1130 1140 ICHDGKAHFPREGVFVSNGTHWFVTQRNFYEPQIITTDNTFVSGNCDVVIGIVNNTVYDP 1150 1160 1170 1180 1190 1200 LQPELDSFKEELDKYFKNHTSPDVDLGDISGINASVVNIQKEIDRLNEVAKNLNESLIDL 1210 1220 1230 1240 1250 1260 QELGKYEQYIKWPWYIWLGFIAGLIAIVMVTIMLCCMTSCCSCLKGCCSCGSCCKFDEDD 1270 SEPVLKGVKLHYT

[0093] In some embodiments, the viral antigen or immunogen comprises the spike glycoprotein sequence of the original Wuhan-Hu-1 coronavirus (e.g., NC_045512). In some embodiments, the viral antigen or immunogen comprises the spike glycoprotein sequence of a virus within the B.1.526 lineage. In some embodiments, the viral antigen or immunogen comprises the spike glycoprotein sequence of a cluster-5 (ΔFVI spike) virus. In some embodiments, the viral antigen or immunogen comprises the spike glycoprotein sequence of a virus within the B.1.1.7 lineage. In some embodiments, the viral antigen or immunogen comprises the spike glycoprotein sequence of a virus within the B.1.1.207 lineage. In some embodiments, the viral antigen or immunogen comprises the spike glycoprotein sequence of a virus within the B.1.1.317 lineage. In some embodiments, the viral antigen or immunogen comprises the spike glycoprotein sequence of a virus within the B.1.1.318 lineage. In some embodiments, the viral antigen or immunogen comprises the sequence of the spike glycoprotein of a virus within the P.1 lineage. In some embodiments, the viral antigen or immunogen comprises the sequence of the spike glycoprotein of a virus within the B.1.351 lineage. In some embodiments, the viral antigen or immunogen comprises the sequence of the spike glycoprotein of a virus within the B.1.429 / CAL.20C lineage. In some embodiments, the viral antigen or immunogen comprises the sequence of the spike glycoprotein of a virus within the B.1.525 lineage. In some embodiments, the viral antigen or immunogen comprises the sequence of the spike glycoprotein of a virus within the B.1.526 lineage. In some embodiments, the viral antigen or immunogen comprises the sequence of the spike glycoprotein of a virus within the B.1.617 lineage. In some embodiments, the viral antigen or immunogen comprises the sequence of the spike glycoprotein of a virus within the B.1.617.2 lineage. In some embodiments, the viral antigen or immunogen comprises the sequence of the spike glycoprotein of a virus within the B.1.618 lineage. In some embodiments, the viral antigen or immunogen comprises the sequence of the spike glycoprotein of a virus within the B.1.620 lineage.In some embodiments, the viral antigen or immunogen comprises the sequence of the spike glycoprotein of a virus within the P.2 lineage. In some embodiments, the viral antigen or immunogen comprises the sequence of the spike glycoprotein of a virus within the P.3 lineage. In some embodiments, the viral antigen or immunogen comprises the sequence of the spike glycoprotein of a virus within the B.1.1.143 lineage. In some embodiments, the viral antigen or immunogen comprises the sequence of the spike glycoprotein of a virus within the A.23.1 lineage. In some embodiments, the viral antigen or immunogen comprises the sequence of the spike glycoprotein of a virus within the B.1.617 lineage. In some embodiments, the viral antigen or immunogen comprises a sequence derived from the spike glycoprotein of any two or more viruses, in any suitable combination, selected from the group consisting of Wuhan-Hu-1, viruses in the B.1.526 lineage, viruses in the B.1.1.7 lineage, viruses in the P.1 lineage, viruses in the B.1.351 lineage, viruses in the P.2 lineage, viruses in the B.1.1.143 lineage, viruses in the A.23.1 lineage, and viruses in the B.1.617 lineage.

[0094] In some embodiments, the viral antigen or immunogen comprises E484K and / or S477N, e.g., as in the B.1.526 variant. In some embodiments, the viral antigen or immunogen comprises Δ400-402 (ΔFVI), e.g., as in the Cluster 5 (ΔFVI spike) variant. In some embodiments, the viral antigen or immunogen comprises Δ69-70 (ΔHV), Δ144 (ΔY), N501Y, A570D, D614G, P681H, T716I, S982A, and / or D1118H, e.g., as in the B.1.1.7 variant. In some embodiments, the viral antigen or immunogen comprises P681H, e.g., as in the B.1.1.207 variant. In some embodiments, the viral antigen or immunogen comprises L18F, T20N, P26S, D138Y, R190S, K417T, E484K, N501Y, D614G, H655Y, T1027I, and / or V1176F, e.g., as in the P.1 variant. In some embodiments, the viral antigen or immunogen comprises E484K, e.g., as in the P.2 variant. In some embodiments, the viral antigen or immunogen comprises E484K and / or N501Y, e.g., as in the P.3 variant. In some embodiments, the viral antigen or immunogen comprises L18F, D80A, D215G, Δ242-244 (ΔLAL), R246I, K417N, E484K, N501Y, D614G, and / or A701V, e.g., as in the B.1.351 variant. In some embodiments, the viral antigen or immunogen comprises S13I, W152C, and / or L452R, e.g., as in the B.1.429 / CAL.20C variant. In some embodiments, the viral antigen or immunogen comprises Δ69-70 (ΔHV), E484K, and / or F888L, e.g., as in the B.1.525 variant. In some embodiments, the viral antigen or immunogen comprises G142D, L452R, E484Q, and / or P681R, for example, as in the B.1.617 variant.In some embodiments, the viral antigen or immunogen comprises G142D, L452R, and / or P681R, e.g., as in the B.1.617.2 variant. In some embodiments, the viral antigen or immunogen comprises E484K, e.g., as in the B.1.618 variant. In some embodiments, the viral antigen or immunogen may comprise a fusion polypeptide (protomer) comprising any one or more of the aforementioned mutations in any suitable combination. In some embodiments, the viral antigen or immunogen may comprise a trimer of three fusion polypeptides, where any of the three protomer fusion polypeptides may comprise any one or more of the aforementioned mutations in any suitable combination. In some embodiments, two or all three of the three protomer fusion polypeptides forming the trimer may comprise different mutations and / or different combinations of mutations in each protomer. In some embodiments, the viral antigen or immunogen may comprise a mixture of trimers, where each trimer may comprise a different mutation and / or different combinations of mutations.

[0095] In some embodiments, the viral antigen or immunogen is selected from the group consisting of amino acids 13, 18, 20, 26, 69, 70, 80, 138, 142, 144, 152, 190, 215, 242, 243, 244, 246, 400, 401, 402, 417, 440, 452, 477, 484, 501, 570, 580, 582, 584, 586, 588, 589, 590, 600, 601, 602, 603, 604, 605, 606, 607, 608, 609, 610, 611, 612, 613, 614, 615, 616, 617, 618, 619, 620, 621, 622, 623, 624, 625, 626, 627, 628, 629, 630, 631, 632, 633, 634, 635, 636, 637, 638, 639, 640, 641, 642, 643, 644, 646, 647, 648, 649, 650, 651, 652, 653, 654, 655, 656, 657, 658, 659, 660, 661, 662, 663, 664, 665, 666 , 614, 655, 681, 682, 683, 684, 685, 701, 716, 888, 982, 1027, 1118, and 1176. In some embodiments, the viral antigen or immunogen comprises any one, two, three, four, five, six, seven, eight, or all of the mutations selected from the group consisting of mutations (e.g., substitution(s), deletion(s), and / or insertion(s)) at amino acid positions 440, 452, 477, 484, 501, 614, 655, 681, and 701. In some embodiments, the viral antigen or immunogen comprises a chimeric polypeptide that includes sequences from different viruses, such as one or more mutations from a first variant of the coronavirus and one or more mutations from a second variant of the coronavirus that differs from the first variant. In some embodiments, such chimeric viral antigens or immunogens (or combinations of chimeric viral antigens or immunogens) can be used to elicit a broad immune response against both the first and second variants of the coronavirus.

[0096] In some embodiments, the viral antigen or immunogen is S13I, L18F, T20N, P26S, Δ69-70(ΔHV), D80A, D138Y, G142D, Δ144(ΔY), W152C, R190S, D215G, Δ242-244(ΔLAL), R246I, Δ400-402(ΔFVI), K417T, K417N, N440K, L452R, S477N , S477G, E484K, E484Q, N501Y, A570D, D614G, H655Y, P681H, P681R, R682G, R683S, R685G, A701V, T716I, F888L, S982A, T1027I, D1118H, and V1176F. In some embodiments, the viral antigen or immunogen comprises any one, two, three, four, five or more mutations selected from the group consisting of N440K, L452R, S477G, S477N, E484K, E484Q, N501Y, D614G, H655Y, P681H, P681R, and A701V.

[0097] In some embodiments, the SARS-CoV-2 antigen comprises a truncated S protein that does not include the signal peptide, transmembrane domain, and cytoplasmic domain of the full-length S protein. In some embodiments, the SARS-CoV-2 antigen is a recombinant protein, while in other embodiments, the SARS-CoV-2 antigen is purified from virions. In some preferred embodiments, the SARS-CoV-2 antigen is an isolated antigen.

[0098] In some embodiments, the viral antigen or immunogen comprises the sequence set forth in SEQ ID NO: 27. In some embodiments, the viral antigen or immunogen comprises the sequence set forth in SEQ ID NO: 27. In some embodiments, the viral antigen or immunogen comprises the sequence set forth in SEQ ID NO: 27 at positions 13, 18, 20, 26, 69, 70, 80, 138, 142, 144, 152, 190, 215, 242, 243, 244, 246, 400, 401, 402, 417, 440, 452, 477, 484, 501, 570, 614, 655, 681, 682, 683, 684, 685, 701, 716, 888, 982, 990, 1000, 1010, 1020, 1030, 1040, 1050, 1060, 1070, 1080, 1090, 1100, 1111, 1120, 1130, 1140, 1152, 1163, 1174, 1180, 1190, 1200, 1210, 1220, 1230, 1240, 1250, 1260, 1270, 1280, 1290, 1300, 1310, 1320, 1330, 1340, 1350, 1360, 1370, 1380, 1390, 1410, 27, including amino acid sequences having at least or about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the sequence of SEQ ID NO:27, such as sequences containing substitutions, deletions, and / or insertions at one or more amino acid positions selected from the group consisting of positions 1027, 1118, and 1176 (amino acid positions relative to SEQ ID NO:55). In some embodiments, the viral antigen or immunogen comprises a variant of SEQ ID NO:27, the variants being S13I, L18F, T20N, P26S, Δ69-70(ΔHV), D80A, D138Y, G142D, Δ144(ΔY), W152C, R190S, D215G, Δ242-244(ΔLAL), R246I, Δ400-402(ΔFVI), K417T, K417N, N440K, and any one, two, three, four, five or more of the mutations selected from the group consisting of L452R, S477N, S477G, E484K, E484Q, N501Y, A570D, D614G, H655Y, P681H, P681R, R682G, R683S, R685G, A701V, T716I, F888L, S982A, T1027I, D1118H, and V1176F.

[0099] In some embodiments, the viral antigen or immunogen comprises the sequence set forth in SEQ ID NO: 28. In some embodiments, the viral antigen or immunogen comprises the sequence set forth in SEQ ID NO: 28. In some embodiments, the viral antigen or immunogen comprises the sequence set forth in SEQ ID NO: 28 at positions 13, 18, 20, 26, 69, 70, 80, 138, 142, 144, 152, 190, 215, 242, 243, 244, 246, 400, 401, 402, 417, 440, 452, 477, 484, 501, 570, 614, 655, 681, 682, 683, 684, 685, 701, 716, 888, 982, 990, 1000, 1010, 1020, 1030, 1040, 1050, 1060, 1070, 1080, 1090, 1100, 1111, 1120, 1130, 1140, 1152, 1163, 1170, 1180, 1190, 1200, 1210, 1220, 1230, 1240, 1250, 1260, 1270, 1280, 1290, 1300, 1310, 1320, 1330, 1340, 1350, 1360, 1370, 1380, 1390, 1410, 28, including amino acid sequences having at least or about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the sequence of SEQ ID NO:28, such as sequences containing substitutions, deletions, and / or insertions at one or more amino acid positions selected from the group consisting of positions 1027, 1118, and 1176 (amino acid positions relative to SEQ ID NO:55). In some embodiments, the viral antigen or immunogen comprises a variant of SEQ ID NO:28, the variants being S13I, L18F, T20N, P26S, Δ69-70(ΔHV), D80A, D138Y, G142D, Δ144(ΔY), W152C, R190S, D215G, Δ242-244(ΔLAL), R246I, Δ400-402(ΔFVI), K417T, K417N, N440K, and any one, two, three, four, five or more of the mutations selected from the group consisting of L452R, S477N, S477G, E484K, E484Q, N501Y, A570D, D614G, H655Y, P681H, P681R, R682G, R683S, R685G, A701V, T716I, F888L, S982A, T1027I, D1118H, and V1176F.

[0100] In some embodiments, the viral antigen or immunogen comprises the sequence set forth in SEQ ID NO: 29. In some embodiments, the viral antigen or immunogen comprises the sequence set forth in SEQ ID NO: 29. In some embodiments, the viral antigen or immunogen comprises the sequence set forth in SEQ ID NO: 13, 18, 20, 26, 69, 70, 80, 138, 142, 144, 152, 190, 215, 242, 243, 244, 246, 400, 401, 402, 417, 440, 452, 477, 484, 501, 570, 614, 655, 681, 682, 683, 684, 685, 701, 716, 888, 982 29, including sequences containing substitutions, deletions, and / or insertions at one or more amino acid positions selected from the group consisting of positions 1027, 1118, and 1176 (amino acid positions relative to SEQ ID NO: 55). In some embodiments, the viral antigen or immunogen comprises a variant of SEQ ID NO:29, the variants being S13I, L18F, T20N, P26S, Δ69-70(ΔHV), D80A, D138Y, G142D, Δ144(ΔY), W152C, R190S, D215G, Δ242-244(ΔLAL), R246I, Δ400-402(ΔFVI), K417T, K417N, N440K, and any one, two, three, four, five or more of the mutations selected from the group consisting of L452R, S477N, S477G, E484K, E484Q, N501Y, A570D, D614G, H655Y, P681H, P681R, R682G, R683S, R685G, A701V, T716I, F888L, S982A, T1027I, D1118H, and V1176F.

[0101] In some embodiments, the viral antigen or immunogen comprises the sequence set forth in SEQ ID NO: 30. In some embodiments, the viral antigen or immunogen comprises the sequence set forth in SEQ ID NO: 30. In some embodiments, the viral antigen or immunogen comprises the sequence set forth in SEQ ID NO: 13, 18, 20, 26, 69, 70, 80, 138, 142, 144, 152, 190, 215, 242, 243, 244, 246, 400, 401, 402, 417, 440, 452, 477, 484, 501, 570, 614, 655, 681, 682, 683, 684, 685, 701, 716, 888, 982 30, including sequences containing substitutions, deletions, and / or insertions at one or more amino acid positions selected from the group consisting of positions 1027, 1118, and 1176 (amino acid positions relative to SEQ ID NO:55). In some embodiments, the viral antigen or immunogen comprises a variant of SEQ ID NO: 30, the variants being S13I, L18F, T20N, P26S, Δ69-70(ΔHV), D80A, D138Y, G142D, Δ144(ΔY), W152C, R190S, D215G, Δ242-244(ΔLAL), R246I, Δ400-402(ΔFVI), K417T, K417N, N440K, and any one, two, three, four, five or more of the mutations selected from the group consisting of L452R, S477N, S477G, E484K, E484Q, N501Y, A570D, D614G, H655Y, P681H, P681R, R682G, R683S, R685G, A701V, T716I, F888L, S982A, T1027I, D1118H, and V1176F.

[0102] In some embodiments, the viral antigen or immunogen comprises the sequence set forth in SEQ ID NO: 31. In some embodiments, the viral antigen or immunogen comprises the sequence set forth in SEQ ID NO: 31. In some embodiments, the viral antigen or immunogen comprises the sequence set forth in SEQ ID NO: 13, 18, 20, 26, 69, 70, 80, 138, 142, 144, 152, 190, 215, 242, 243, 244, 246, 400, 401, 402, 417, 440, 452, 477, 484, 501, 570, 614, 655, 681, 682, 683, 684, 685, 701, 716, 888, 982 31, including amino acid sequences having at least or about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the sequence of SEQ ID NO: 31, such as sequences containing substitutions, deletions, and / or insertions at one or more amino acid positions selected from the group consisting of positions 1027, 1118, and 1176 (amino acid positions relative to SEQ ID NO: 55). In some embodiments, the viral antigen or immunogen comprises a variant of SEQ ID NO: 31, the variants being S13I, L18F, T20N, P26S, Δ69-70(ΔHV), D80A, D138Y, G142D, Δ144(ΔY), W152C, R190S, D215G, Δ242-244(ΔLAL), R246I, Δ400-402(ΔFVI), K417T, K417N, N440K, and any one, two, three, four, five or more of the mutations selected from the group consisting of L452R, S477N, S477G, E484K, E484Q, N501Y, A570D, D614G, H655Y, P681H, P681R, R682G, R683S, R685G, A701V, T716I, F888L, S982A, T1027I, D1118H, and V1176F.

[0103] In some embodiments, the viral antigen or immunogen comprises the sequence set forth in SEQ ID NO: 32. In some embodiments, the viral antigen or immunogen comprises the sequence set forth in SEQ ID NO: 32. In some embodiments, the viral antigen or immunogen comprises the sequence set forth in SEQ ID NO: 32 at positions 13, 18, 20, 26, 69, 70, 80, 138, 142, 144, 152, 190, 215, 242, 243, 244, 246, 400, 401, 402, 417, 440, 452, 477, 484, 501, 570, 614, 655, 681, 682, 683, 684, 685, 701, 716, 888, 982, 983, 984, 985, 986, 987, 988, 989, 990, 1000, 1001, 1002, 1003, 1004, 1005, 1006, 1007, 1008, 1009, 1010, 1011, 1012, 1013, 1014, 1015, 1016, 1017, 1018, 1019, 1020, 1021, 1022, 1023, 1024, 1025, 1026, 1027, 1028, 1029, 1030, 1031, 1032, 1033, 1034, 1 32, including sequences containing substitutions, deletions, and / or insertions at one or more amino acid positions selected from the group consisting of positions 1027, 1118, and 1176 (amino acid positions relative to SEQ ID NO: 55). In some embodiments, the viral antigen or immunogen comprises a variant of SEQ ID NO: 32, the variants being S13I, L18F, T20N, P26S, Δ69-70(ΔHV), D80A, D138Y, G142D, Δ144(ΔY), W152C, R190S, D215G, Δ242-244(ΔLAL), R246I, Δ400-402(ΔFVI), K417T, K417N, N440K, and any one, two, three, four, five or more of the mutations selected from the group consisting of L452R, S477N, S477G, E484K, E484Q, N501Y, A570D, D614G, H655Y, P681H, P681R, R682G, R683S, R685G, A701V, T716I, F888L, S982A, T1027I, D1118H, and V1176F.

[0104] In some embodiments, the viral antigen or immunogen comprises the sequence set forth in SEQ ID NO: 33. In some embodiments, the viral antigen or immunogen comprises the sequence set forth in SEQ ID NO: 33. In some embodiments, the viral antigen or immunogen comprises the sequence set forth in SEQ ID NO: 13, 18, 20, 26, 69, 70, 80, 138, 142, 144, 152, 190, 215, 242, 243, 244, 246, 400, 401, 402, 417, 440, 452, 477, 484, 501, 570, 614, 655, 681, 682, 683, 684, 685, 701, 716, 888, 982 33, including sequences containing substitutions, deletions, and / or insertions at one or more amino acid positions selected from the group consisting of positions 1027, 1118, and 1176 (amino acid positions relative to SEQ ID NO: 55). In some embodiments, the viral antigen or immunogen comprises a variant of SEQ ID NO: 33, the variants being S13I, L18F, T20N, P26S, Δ69-70(ΔHV), D80A, D138Y, G142D, Δ144(ΔY), W152C, R190S, D215G, Δ242-244(ΔLAL), R246I, Δ400-402(ΔFVI), K417T, K417N, N440K, and any one, two, three, four, five or more of the mutations selected from the group consisting of L452R, S477N, S477G, E484K, E484Q, N501Y, A570D, D614G, H655Y, P681H, P681R, R682G, R683S, R685G, A701V, T716I, F888L, S982A, T1027I, D1118H, and V1176F.

[0105] In some embodiments, the viral antigen or immunogen comprises the sequence set forth in SEQ ID NO: 34. In some embodiments, the viral antigen or immunogen comprises the sequence set forth in SEQ ID NO: 34. In some embodiments, the viral antigen or immunogen comprises the sequence set forth in SEQ ID NO: 13, 18, 20, 26, 69, 70, 80, 138, 142, 144, 152, 190, 215, 242, 243, 244, 246, 400, 401, 402, 417, 440, 452, 477, 484, 501, 570, 614, 655, 681, 682, 683, 684, 685, 701, 716, 888, 982 55。 55. The amino acid sequence of SEQ ID NO: 34 includes an amino acid sequence having at least or about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the sequence of SEQ ID NO: 34, such as a sequence containing a substitution, deletion, and / or insertion at one or more amino acid positions selected from the group consisting of positions 1027, 1118, and 1176 (amino acid positions relative to SEQ ID NO: 55). In some embodiments, the viral antigen or immunogen comprises a variant of SEQ ID NO: 34, the variants being S13I, L18F, T20N, P26S, Δ69-70(ΔHV), D80A, D138Y, G142D, Δ144(ΔY), W152C, R190S, D215G, Δ242-244(ΔLAL), R246I, Δ400-402(ΔFVI), K417T, K417N, N440K, and any one, two, three, four, five or more of the mutations selected from the group consisting of L452R, S477N, S477G, E484K, E484Q, N501Y, A570D, D614G, H655Y, P681H, P681R, R682G, R683S, R685G, A701V, T716I, F888L, S982A, T1027I, D1118H, and V1176F.

[0106] In some embodiments, the viral antigen or immunogen comprises the sequence set forth in SEQ ID NO: 35. In some embodiments, the viral antigen or immunogen comprises the sequence set forth in SEQ ID NO: 35. In some embodiments, the viral antigen or immunogen comprises the sequence set forth in SEQ ID NO: 13, 18, 20, 26, 69, 70, 80, 138, 142, 144, 152, 190, 215, 242, 243, 244, 246, 400, 401, 402, 417, 440, 452, 477, 484, 501, 570, 614, 655, 681, 682, 683, 684, 685, 701, 716, 888, 982 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the sequence of SEQ ID NO: 35, such as sequences containing substitutions, deletions, and / or insertions at one or more amino acid positions selected from the group consisting of positions 1027, 1118, and 1176 (amino acid positions relative to SEQ ID NO: 55). In some embodiments, the viral antigen or immunogen comprises a variant of SEQ ID NO: 35, the variants being S13I, L18F, T20N, P26S, Δ69-70(ΔHV), D80A, D138Y, G142D, Δ144(ΔY), W152C, R190S, D215G, Δ242-244(ΔLAL), R246I, Δ400-402(ΔFVI), K417T, K417N, N440K, and any one, two, three, four, five or more of the mutations selected from the group consisting of L452R, S477N, S477G, E484K, E484Q, N501Y, A570D, D614G, H655Y, P681H, P681R, R682G, R683S, R685G, A701V, T716I, F888L, S982A, T1027I, D1118H, and V1176F.

[0107] In some embodiments, the viral antigen or immunogen comprises the sequence set forth in SEQ ID NO: 36. In some embodiments, the viral antigen or immunogen comprises the sequence set forth in SEQ ID NO: 36. In some embodiments, the viral antigen or immunogen comprises the sequence set forth in SEQ ID NO: 13, 18, 20, 26, 69, 70, 80, 138, 142, 144, 152, 190, 215, 242, 243, 244, 246, 400, 401, 402, 417, 440, 452, 477, 484, 501, 570, 614, 655, 681, 682, 683, 684, 685, 701, 716, 888, 982 36, including sequences that contain substitutions, deletions, and / or insertions at one or more amino acid positions selected from the group consisting of positions 1027, 1118, and 1176 (amino acid positions relative to SEQ ID NO: 55). In some embodiments, the viral antigen or immunogen comprises a variant of SEQ ID NO: 36, the variants being S13I, L18F, T20N, P26S, Δ69-70(ΔHV), D80A, D138Y, G142D, Δ144(ΔY), W152C, R190S, D215G, Δ242-244(ΔLAL), R246I, Δ400-402(ΔFVI), K417T, K417N, N440K, and any one, two, three, four, five or more of the mutations selected from the group consisting of L452R, S477N, S477G, E484K, E484Q, N501Y, A570D, D614G, H655Y, P681H, P681R, R682G, R683S, R685G, A701V, T716I, F888L, S982A, T1027I, D1118H, and V1176F.

[0108] In some embodiments, the viral antigen or immunogen comprises the sequence set forth in SEQ ID NO: 37. In some embodiments, the viral antigen or immunogen comprises the sequence set forth in SEQ ID NO: 37. In some embodiments, the viral antigen or immunogen comprises the sequence set forth in SEQ ID NO: 37 at positions 13, 18, 20, 26, 69, 70, 80, 138, 142, 144, 152, 190, 215, 242, 243, 244, 246, 400, 401, 402, 417, 440, 452, 477, 484, 501, 570, 614, 655, 681, 682, 683, 684, 685, 701, 716, 888, 982, 983, 984, 985, 986, 987, 988, 989, 990, 1000, 1001, 1002, 1003, 1004, 1005, 1006, 1007, 1008, 1009, 1010, 1011, 1012, 1013, 1014, 1015, 1016, 1017, 1018, 1019, 1020, 1021, 1022, 1023, 1024, 1025, 1026, 1027, 1028, 1029, 1030, 1031, 1032, 1033, 1034, 1 37, including sequences containing substitutions, deletions, and / or insertions at one or more amino acid positions selected from the group consisting of positions 1027, 1118, and 1176 (amino acid positions relative to SEQ ID NO: 55). In some embodiments, the viral antigen or immunogen comprises a variant of SEQ ID NO: 37, the variants being S13I, L18F, T20N, P26S, Δ69-70(ΔHV), D80A, D138Y, G142D, Δ144(ΔY), W152C, R190S, D215G, Δ242-244(ΔLAL), R246I, Δ400-402(ΔFVI), K417T, K417N, N440K, and any one, two, three, four, five or more of the mutations selected from the group consisting of L452R, S477N, S477G, E484K, E484Q, N501Y, A570D, D614G, H655Y, P681H, P681R, R682G, R683S, R685G, A701V, T716I, F888L, S982A, T1027I, D1118H, and V1176F.

[0109] In some embodiments, the viral antigen or immunogen comprises the sequence set forth in SEQ ID NO: 38. In some embodiments, the viral antigen or immunogen comprises the sequence set forth in SEQ ID NO: 38. In some embodiments, the viral antigen or immunogen comprises the sequence set forth in SEQ ID NO: 13, 18, 20, 26, 69, 70, 80, 138, 142, 144, 152, 190, 215, 242, 243, 244, 246, 400, 401, 402, 417, 440, 452, 477, 484, 501, 570, 614, 655, 681, 682, 683, 684, 685, 701, 716, 888, 982 38, including sequences containing substitutions, deletions, and / or insertions at one or more amino acid positions selected from the group consisting of positions 1027, 1118, and 1176 (amino acid positions relative to SEQ ID NO: 55). In some embodiments, the viral antigen or immunogen comprises a variant of SEQ ID NO: 38, the variants being S13I, L18F, T20N, P26S, Δ69-70(ΔHV), D80A, D138Y, G142D, Δ144(ΔY), W152C, R190S, D215G, Δ242-244(ΔLAL), R246I, Δ400-402(ΔFVI), K417T, K417N, N440K, and any one, two, three, four, five or more of the mutations selected from the group consisting of L452R, S477N, S477G, E484K, E484Q, N501Y, A570D, D614G, H655Y, P681H, P681R, R682G, R683S, R685G, A701V, T716I, F888L, S982A, T1027I, D1118H, and V1176F.

[0110] In some embodiments, the viral antigen or immunogen comprises the sequence set forth in SEQ ID NO: 39. In some embodiments, the viral antigen or immunogen comprises the sequence set forth in SEQ ID NO: 39. In some embodiments, the viral antigen or immunogen comprises the sequence set forth in SEQ ID NO: 13, 18, 20, 26, 69, 70, 80, 138, 142, 144, 152, 190, 215, 242, 243, 244, 246, 400, 401, 402, 417, 440, 452, 477, 484, 501, 570, 614, 655, 681, 682, 683, 684, 685, 701, 716, 888, 982 39, including sequences containing substitutions, deletions, and / or insertions at one or more amino acid positions selected from the group consisting of positions 1027, 1118, and 1176 (amino acid positions relative to SEQ ID NO: 55). In some embodiments, the viral antigen or immunogen comprises a variant of SEQ ID NO: 39, the variants being S13I, L18F, T20N, P26S, Δ69-70(ΔHV), D80A, D138Y, G142D, Δ144(ΔY), W152C, R190S, D215G, Δ242-244(ΔLAL), R246I, Δ400-402(ΔFVI), K417T, K417N, N440K, and any one, two, three, four, five or more of the mutations selected from the group consisting of L452R, S477N, S477G, E484K, E484Q, N501Y, A570D, D614G, H655Y, P681H, P681R, R682G, R683S, R685G, A701V, T716I, F888L, S982A, T1027I, D1118H, and V1176F.

[0111] In some embodiments, the viral antigen or immunogen comprises the sequence set forth in SEQ ID NO: 40. In some embodiments, the viral antigen or immunogen comprises the sequence set forth in SEQ ID NO: 40. In some embodiments, the viral antigen or immunogen comprises the sequence set forth in SEQ ID NO: 13, 18, 20, 26, 69, 70, 80, 138, 142, 144, 152, 190, 215, 242, 243, 244, 246, 400, 401, 402, 417, 440, 452, 477, 484, 501, 570, 614, 655, 681, 682, 683, 684, 685, 701, 716, 888, 982 55。 The present invention also includes amino acid sequences having at least or about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the sequence of SEQ ID NO: 40, such as sequences containing substitutions, deletions, and / or insertions at one or more amino acid positions selected from the group consisting of positions 1027, 1118, and 1176 (amino acid positions relative to SEQ ID NO: 55). In some embodiments, the viral antigen or immunogen comprises a variant of SEQ ID NO: 40, the variants being S13I, L18F, T20N, P26S, Δ69-70(ΔHV), D80A, D138Y, G142D, Δ144(ΔY), W152C, R190S, D215G, Δ242-244(ΔLAL), R246I, Δ400-402(ΔFVI), K417T, K417N, N440K, and any one, two, three, four, five or more of the mutations selected from the group consisting of L452R, S477N, S477G, E484K, E484Q, N501Y, A570D, D614G, H655Y, P681H, P681R, R682G, R683S, R685G, A701V, T716I, F888L, S982A, T1027I, D1118H, and V1176F.

[0112] In some embodiments, the viral antigen or immunogen comprises the sequence set forth in SEQ ID NO: 41. In some embodiments, the viral antigen or immunogen comprises the sequence set forth in SEQ ID NO: 41. In some embodiments, the viral antigen or immunogen comprises the sequence set forth in SEQ ID NO: 41 at positions 13, 18, 20, 26, 69, 70, 80, 138, 142, 144, 152, 190, 215, 242, 243, 244, 246, 400, 401, 402, 417, 440, 452, 477, 484, 501, 570, 614, 655, 681, 682, 683, 684, 685, 701, 716, 888, 982, 983, 984, 985, 986, 987, 988, 989, 990, 1000, 1001, 1002, 1003, 1004, 1005, 1006, 1007, 1008, 1009, 1010, 1011, 1012, 1013, 1014, 1015, 1016, 1017, 1018, 1019, 1020, 1021, 1022, 1023, 1024, 1025, 1026, 1027, 1028, 1029, 1030, 1031, 1032, 1033, 1034, 1 55。 The present invention also includes amino acid sequences having at least or about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the sequence of SEQ ID NO: 41, such as sequences containing substitutions, deletions, and / or insertions at one or more amino acid positions selected from the group consisting of positions 1027, 1118, and 1176 (amino acid positions relative to SEQ ID NO: 55). In some embodiments, the viral antigen or immunogen comprises a variant of SEQ ID NO: 41, the variants being S13I, L18F, T20N, P26S, Δ69-70(ΔHV), D80A, D138Y, G142D, Δ144(ΔY), W152C, R190S, D215G, Δ242-244(ΔLAL), R246I, Δ400-402(ΔFVI), K417T, K417N, N440K, and any one, two, three, four, five or more of the mutations selected from the group consisting of L452R, S477N, S477G, E484K, E484Q, N501Y, A570D, D614G, H655Y, P681H, P681R, R682G, R683S, R685G, A701V, T716I, F888L, S982A, T1027I, D1118H, and V1176F.

[0113] In some embodiments, the viral antigen or immunogen comprises the sequence set forth in SEQ ID NO: 42. In some embodiments, the viral antigen or immunogen comprises the sequence set forth in SEQ ID NO: 42. In some embodiments, the viral antigen or immunogen comprises the sequence set forth in SEQ ID NO: 42 at positions 13, 18, 20, 26, 69, 70, 80, 138, 142, 144, 152, 190, 215, 242, 243, 244, 246, 400, 401, 402, 417, 440, 452, 477, 484, 501, 570, 614, 655, 681, 682, 683, 684, 685, 701, 716, 888, 982, 983, 984, 985, 986, 987, 988, 989, 990, 1000, 1001, 1002, 1003, 1004, 1005, 1006, 1007, 1008, 1009, 1010, 1011, 1012, 1013, 1014, 1015, 1016, 1017, 1018, 1019, 1020, 1021, 1022, 1023, 1024, 1025, 1026, 1027, 1028, 1029, 1030, 1031, 1032, 1033, 1034, 1 55。 The present invention also includes amino acid sequences having at least or about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the sequence of SEQ ID NO: 42, such as sequences containing substitutions, deletions, and / or insertions at one or more amino acid positions selected from the group consisting of positions 1027, 1118, and 1176 (amino acid positions relative to SEQ ID NO: 55). In some embodiments, the viral antigen or immunogen comprises a variant of SEQ ID NO: 42, the variants being S13I, L18F, T20N, P26S, Δ69-70(ΔHV), D80A, D138Y, G142D, Δ144(ΔY), W152C, R190S, D215G, Δ242-244(ΔLAL), R246I, Δ400-402(ΔFVI), K417T, K417N, N440K, and any one, two, three, four, five or more of the mutations selected from the group consisting of L452R, S477N, S477G, E484K, E484Q, N501Y, A570D, D614G, H655Y, P681H, P681R, R682G, R683S, R685G, A701V, T716I, F888L, S982A, T1027I, D1118H, and V1176F.

[0114] In some embodiments, the viral antigen or immunogen comprises the sequence set forth in SEQ ID NO: 43. In some embodiments, the viral antigen or immunogen comprises the sequence set forth in SEQ ID NO: 43. In some embodiments, the viral antigen or immunogen comprises the sequence set forth in SEQ ID NO: 43 at positions 13, 18, 20, 26, 69, 70, 80, 138, 142, 144, 152, 190, 215, 242, 243, 244, 246, 400, 401, 402, 417, 440, 452, 477, 484, 501, 570, 614, 655, 681, 682, 683, 684, 685, 701, 716, 888, 982, 983, 984, 985, 986, 987, 988, 989, 990, 1000, 1001, 1002, 1003, 1004, 1005, 1006, 1007, 1008, 1009, 1010, 1011, 1012, 1013, 1014, 1015, 1016, 1017, 1018, 1019, 1020, 1021, 1022, 1023, 1024, 1025, 1026, 1027, 1028, 1029, 1030, 1031, 1032, 1033, 1034, 1 55。 The present invention also includes amino acid sequences having at least or about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the sequence of SEQ ID NO: 43, such as sequences containing substitutions, deletions, and / or insertions at one or more amino acid positions selected from the group consisting of positions 1027, 1118, and 1176 (amino acid positions relative to SEQ ID NO: 55). In some embodiments, the viral antigen or immunogen comprises a variant of SEQ ID NO: 43, the variants being S13I, L18F, T20N, P26S, Δ69-70(ΔHV), D80A, D138Y, G142D, Δ144(ΔY), W152C, R190S, D215G, Δ242-244(ΔLAL), R246I, Δ400-402(ΔFVI), K417T, K417N, N440K, and any one, two, three, four, five or more of the mutations selected from the group consisting of L452R, S477N, S477G, E484K, E484Q, N501Y, A570D, D614G, H655Y, P681H, P681R, R682G, R683S, R685G, A701V, T716I, F888L, S982A, T1027I, D1118H, and V1176F.

[0115] In some embodiments, the viral antigen or immunogen comprises the sequence set forth in SEQ ID NO: 44. In some embodiments, the viral antigen or immunogen comprises the sequence set forth in SEQ ID NO: 44. In some embodiments, the viral antigen or immunogen comprises the sequence set forth in SEQ ID NO: 13, 18, 20, 26, 69, 70, 80, 138, 142, 144, 152, 190, 215, 242, 243, 244, 246, 400, 401, 402, 417, 440, 452, 477, 484, 501, 570, 614, 655, 681, 682, 683, 684, 685, 701, 716, 888, 982 55。 The present invention also includes amino acid sequences having at least or about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the sequence of SEQ ID NO: 44, such as sequences containing substitutions, deletions, and / or insertions at one or more amino acid positions selected from the group consisting of positions 1027, 1118, and 1176 (amino acid positions relative to SEQ ID NO: 55). In some embodiments, the viral antigen or immunogen comprises a variant of SEQ ID NO:44, the variants being S13I, L18F, T20N, P26S, Δ69-70(ΔHV), D80A, D138Y, G142D, Δ144(ΔY), W152C, R190S, D215G, Δ242-244(ΔLAL), R246I, Δ400-402(ΔFVI), K417T, K417N, N440K, and any one, two, three, four, five or more of the mutations selected from the group consisting of L452R, S477N, S477G, E484K, E484Q, N501Y, A570D, D614G, H655Y, P681H, P681R, R682G, R683S, R685G, A701V, T716I, F888L, S982A, T1027I, D1118H, and V1176F.

[0116] In some embodiments, the viral antigen or immunogen comprises the sequence set forth in SEQ ID NO: 45. In some embodiments, the viral antigen or immunogen comprises the sequence set forth in SEQ ID NO: 45. In some embodiments, the viral antigen or immunogen comprises the sequence set forth in SEQ ID NO: 45 at positions 13, 18, 20, 26, 69, 70, 80, 138, 142, 144, 152, 190, 215, 242, 243, 244, 246, 400, 401, 402, 417, 440, 452, 477, 484, 501, 570, 614, 655, 681, 682, 683, 684, 685, 701, 716, 888, 982, 983, 984, 985, 986, 987, 988, 989, 990, 1000, 1001, 1002, 1003, 1004, 1005, 1006, 1007, 1008, 1009, 1010, 1011, 1012, 1013, 1014, 1015, 1016, 1017, 1018, 1019, 1020, 1021, 1022, 1023, 1024, 1025, 1026, 1027, 1028, 1029, 1030, 1031, 1032, 1033, 1034, 1 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the sequence of SEQ ID NO:45, such as sequences containing substitutions, deletions, and / or insertions at one or more amino acid positions selected from the group consisting of positions 1027, 1118, and 1176 (amino acid positions relative to SEQ ID NO:55). In some embodiments, the viral antigen or immunogen comprises a variant of SEQ ID NO: 45, the variants being S13I, L18F, T20N, P26S, Δ69-70(ΔHV), D80A, D138Y, G142D, Δ144(ΔY), W152C, R190S, D215G, Δ242-244(ΔLAL), R246I, Δ400-402(ΔFVI), K417T, K417N, N440K, and any one, two, three, four, five or more of the mutations selected from the group consisting of L452R, S477N, S477G, E484K, E484Q, N501Y, A570D, D614G, H655Y, P681H, P681R, R682G, R683S, R685G, A701V, T716I, F888L, S982A, T1027I, D1118H, and V1176F.

[0117] In some embodiments, the viral antigen or immunogen comprises the sequence set forth in SEQ ID NO: 46. In some embodiments, the viral antigen or immunogen comprises the sequence set forth in SEQ ID NO: 46. In some embodiments, the viral antigen or immunogen comprises the sequence set forth in SEQ ID NO: 13, 18, 20, 26, 69, 70, 80, 138, 142, 144, 152, 190, 215, 242, 243, 244, 246, 400, 401, 402, 417, 440, 452, 477, 484, 501, 570, 614, 655, 681, 682, 683, 684, 685, 701, 716, 888, 982 55。 The present invention also includes amino acid sequences having at least or about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the sequence of SEQ ID NO: 46, such as sequences containing substitutions, deletions, and / or insertions at one or more amino acid positions selected from the group consisting of positions 1027, 1118, and 1176 (amino acid positions relative to SEQ ID NO: 55). In some embodiments, the viral antigen or immunogen comprises a variant of SEQ ID NO: 46, the variants being S13I, L18F, T20N, P26S, Δ69-70(ΔHV), D80A, D138Y, G142D, Δ144(ΔY), W152C, R190S, D215G, Δ242-244(ΔLAL), R246I, Δ400-402(ΔFVI), K417T, K417N, N440K, and any one, two, three, four, five or more of the mutations selected from the group consisting of L452R, S477N, S477G, E484K, E484Q, N501Y, A570D, D614G, H655Y, P681H, P681R, R682G, R683S, R685G, A701V, T716I, F888L, S982A, T1027I, D1118H, and V1176F.

[0118] In some embodiments, the viral antigen or immunogen comprises the sequence set forth in SEQ ID NO: 47. In some embodiments, the viral antigen or immunogen comprises the sequence set forth in SEQ ID NO: 47. In some embodiments, the viral antigen or immunogen comprises the sequence set forth in SEQ ID NO: 47 at positions 13, 18, 20, 26, 69, 70, 80, 138, 142, 144, 152, 190, 215, 242, 243, 244, 246, 400, 401, 402, 417, 440, 452, 477, 484, 501, 570, 614, 655, 681, 682, 683, 684, 685, 701, 716, 888, 982, 983, 984, 985, 986, 987, 988, 989, 990, 1000, 1001, 1002, 1003, 1004, 1005, 1006, 1007, 1008, 1009, 1010, 1011, 1012, 1013, 1014, 1015, 1016, 1017, 1018, 1019, 1020, 1021, 1022, 1023, 1024, 1025, 1026, 1027, 1028, 1029, 1030, 1031, 1032, 1033, 1034, 1 55。 The present invention also includes amino acid sequences having at least or about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the sequence of SEQ ID NO: 47, such as sequences containing substitutions, deletions, and / or insertions at one or more amino acid positions selected from the group consisting of positions 1027, 1118, and 1176 (amino acid positions relative to SEQ ID NO: 55). In some embodiments, the viral antigen or immunogen comprises a variant of SEQ ID NO:47, the variants being S13I, L18F, T20N, P26S, Δ69-70(ΔHV), D80A, D138Y, G142D, Δ144(ΔY), W152C, R190S, D215G, Δ242-244(ΔLAL), R246I, Δ400-402(ΔFVI), K417T, K417N, N440K, and any one, two, three, four, five or more of the mutations selected from the group consisting of L452R, S477N, S477G, E484K, E484Q, N501Y, A570D, D614G, H655Y, P681H, P681R, R682G, R683S, R685G, A701V, T716I, F888L, S982A, T1027I, D1118H, and V1176F.

[0119] In some embodiments, the viral antigen or immunogen comprises the sequence set forth in SEQ ID NO: 48. In some embodiments, the viral antigen or immunogen comprises the sequence set forth in SEQ ID NO: 48. In some embodiments, the viral antigen or immunogen comprises the sequence set forth in SEQ ID NO: 13, 18, 20, 26, 69, 70, 80, 138, 142, 144, 152, 190, 215, 242, 243, 244, 246, 400, 401, 402, 417, 440, 452, 477, 484, 501, 570, 614, 655, 681, 682, 683, 684, 685, 701, 716, 888, 982 55。 The present invention also includes amino acid sequences having at least or about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the sequence of SEQ ID NO: 48, such as sequences containing substitutions, deletions, and / or insertions at one or more amino acid positions selected from the group consisting of positions 1027, 1118, and 1176 (amino acid positions relative to SEQ ID NO: 55). In some embodiments, the viral antigen or immunogen comprises a variant of SEQ ID NO:48, the variants being S13I, L18F, T20N, P26S, Δ69-70(ΔHV), D80A, D138Y, G142D, Δ144(ΔY), W152C, R190S, D215G, Δ242-244(ΔLAL), R246I, Δ400-402(ΔFVI), K417T, K417N, N440K, and any one, two, three, four, five or more of the mutations selected from the group consisting of L452R, S477N, S477G, E484K, E484Q, N501Y, A570D, D614G, H655Y, P681H, P681R, R682G, R683S, R685G, A701V, T716I, F888L, S982A, T1027I, D1118H, and V1176F.

[0120] In some embodiments, the viral antigen or immunogen comprises the sequence set forth in SEQ ID NO: 49. In some embodiments, the viral antigen or immunogen comprises the sequence set forth in SEQ ID NO: 49. In some embodiments, the viral antigen or immunogen comprises the sequence set forth in SEQ ID NO: 13, 18, 20, 26, 69, 70, 80, 138, 142, 144, 152, 190, 215, 242, 243, 244, 246, 400, 401, 402, 417, 440, 452, 477, 484, 501, 570, 614, 655, 681, 682, 683, 684, 685, 701, 716, 888, 982 55。 55. The amino acid sequence of SEQ ID NO: 49 includes an amino acid sequence having at least or about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the sequence of SEQ ID NO: 49, such as a sequence containing a substitution, deletion, and / or insertion at one or more amino acid positions selected from the group consisting of positions 1027, 1118, and 1176 (amino acid positions relative to SEQ ID NO: 55). In some embodiments, the viral antigen or immunogen comprises a variant of SEQ ID NO:49, the variants being S13I, L18F, T20N, P26S, Δ69-70(ΔHV), D80A, D138Y, G142D, Δ144(ΔY), W152C, R190S, D215G, Δ242-244(ΔLAL), R246I, Δ400-402(ΔFVI), K417T, K417N, N440K, and any one, two, three, four, five or more of the mutations selected from the group consisting of L452R, S477N, S477G, E484K, E484Q, N501Y, A570D, D614G, H655Y, P681H, P681R, R682G, R683S, R685G, A701V, T716I, F888L, S982A, T1027I, D1118H, and V1176F.

[0121] In some embodiments, the viral antigen or immunogen comprises the sequence set forth in SEQ ID NO: 50. In some embodiments, the viral antigen or immunogen comprises the sequence set forth in SEQ ID NO: 50. In some embodiments, the viral antigen or immunogen comprises the sequence set forth in SEQ ID NO: 13, 18, 20, 26, 69, 70, 80, 138, 142, 144, 152, 190, 215, 242, 243, 244, 246, 400, 401, 402, 417, 440, 452, 477, 484, 501, 570, 614, 655, 681, 682, 683, 684, 685, 701, 716, 888, 982 50, including sequences containing substitutions, deletions, and / or insertions at one or more amino acid positions selected from the group consisting of positions 1027, 1118, and 1176 (amino acid positions relative to SEQ ID NO: 55). In some embodiments, the viral antigen or immunogen comprises a variant of SEQ ID NO: 50, the variants being S13I, L18F, T20N, P26S, Δ69-70(ΔHV), D80A, D138Y, G142D, Δ144(ΔY), W152C, R190S, D215G, Δ242-244(ΔLAL), R246I, Δ400-402(ΔFVI), K417T, K417N, N440K, and any one, two, three, four, five or more of the mutations selected from the group consisting of L452R, S477N, S477G, E484K, E484Q, N501Y, A570D, D614G, H655Y, P681H, P681R, R682G, R683S, R685G, A701V, T716I, F888L, S982A, T1027I, D1118H, and V1176F.

[0122] In some embodiments, the viral antigen or immunogen comprises the sequence set forth in SEQ ID NO: 51. In some embodiments, the viral antigen or immunogen comprises the sequence set forth in SEQ ID NO: 51. In some embodiments, the viral antigen or immunogen comprises the sequence set forth in SEQ ID NO: 51 at positions 13, 18, 20, 26, 69, 70, 80, 138, 142, 144, 152, 190, 215, 242, 243, 244, 246, 400, 401, 402, 417, 440, 452, 477, 484, 501, 570, 614, 655, 681, 682, 683, 684, 685, 701, 716, 888, 982 51, including amino acid sequences having at least or about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the sequence of SEQ ID NO:51, such as sequences containing substitutions, deletions, and / or insertions at one or more amino acid positions selected from the group consisting of positions 1027, 1118, and 1176 (amino acid positions relative to SEQ ID NO:55). In some embodiments, the viral antigen or immunogen comprises a variant of SEQ ID NO:51, the variants being S13I, L18F, T20N, P26S, Δ69-70(ΔHV), D80A, D138Y, G142D, Δ144(ΔY), W152C, R190S, D215G, Δ242-244(ΔLAL), R246I, Δ400-402(ΔFVI), K417T, K417N, N440K, and any one, two, three, four, five or more of the mutations selected from the group consisting of L452R, S477N, S477G, E484K, E484Q, N501Y, A570D, D614G, H655Y, P681H, P681R, R682G, R683S, R685G, A701V, T716I, F888L, S982A, T1027I, D1118H, and V1176F.

[0123] In some embodiments, the viral antigen or immunogen comprises the sequence set forth in SEQ ID NO: 52. In some embodiments, the viral antigen or immunogen comprises an amino acid sequence having at least or about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the sequence of SEQ ID NO: 52.

[0124] In some embodiments, the viral antigen or immunogen comprises a signal peptide. In some embodiments, the viral antigen or immunogen comprises the sequence set forth in SEQ ID NO: 53. In some embodiments, the viral antigen or immunogen comprises an amino acid sequence having at least or about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the sequence of SEQ ID NO: 53. In some embodiments, the viral antigen or immunogen comprises the sequence set forth in SEQ ID NO: 54. In some embodiments, the viral antigen or immunogen comprises an amino acid sequence having at least or about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the sequence of SEQ ID NO:54.

[0125] In some embodiments, the viral antigen or immunogen comprises the sequence set forth in SEQ ID NO: 55. In some embodiments, the viral antigen or immunogen comprises the sequence set forth in SEQ ID NO: 55. In some embodiments, the viral antigen or immunogen comprises the sequence set forth in SEQ ID NO: 55 at positions 13, 18, 20, 26, 69, 70, 80, 138, 142, 144, 152, 190, 215, 242, 243, 244, 246, 400, 401, 402, 417, 440, 452, 477, 484, 501, 570, 614, 655, 681, 682, 683, 684, 685, 701, 716 , 888, 982, 1027, 1118, and 1176, and the like. In some embodiments, the viral antigen or immunogen comprises a variant of SEQ ID NO: 55, the variants being S13I, L18F, T20N, P26S, Δ69-70(ΔHV), D80A, D138Y, G142D, Δ144(ΔY), W152C, R190S, D215G, Δ242-244(ΔLAL), R246I, Δ400-402(ΔFVI), K417T, K417N, N440K, and any one, two, three, four, five or more of the mutations selected from the group consisting of L452R, S477N, S477G, E484K, E484Q, N501Y, A570D, D614G, H655Y, P681H, P681R, R682G, R683S, R685G, A701V, T716I, F888L, S982A, T1027I, D1118H, and V1176F.

[0126] In some embodiments, the viral antigen or immunogen comprises the sequence set forth in SEQ ID NO: 56. In some embodiments, the viral antigen or immunogen comprises the sequence set forth in SEQ ID NO: 56. In some embodiments, the viral antigen or immunogen comprises the sequence set forth in SEQ ID NO: 56. In some embodiments, the viral antigen or immunogen comprises the sequence set forth in SEQ ID NO: 56. In some embodiments, the viral antigen or immunogen comprises the sequence set forth in SEQ ID NO: 56. In some embodiments, the viral antigen or immunogen comprises the sequence set forth in SEQ ID NO: 56. 56, including amino acid sequences having at least or about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the sequence of SEQ ID NO: 56, such as sequences containing substitutions, deletions, and / or insertions at one or more amino acid positions selected from the group consisting of positions 1027, 1118, and 1176 (amino acid positions relative to SEQ ID NO: 55). In some embodiments, the viral antigen or immunogen comprises a variant of SEQ ID NO: 56, the variants being S13I, L18F, T20N, P26S, Δ69-70(ΔHV), D80A, D138Y, G142D, Δ144(ΔY), W152C, R190S, D215G, Δ242-244(ΔLAL), R246I, Δ400-402(ΔFVI), K417T, K417N, N440K, and any one, two, three, four, five or more of the mutations selected from the group consisting of L452R, S477N, S477G, E484K, E484Q, N501Y, A570D, D614G, H655Y, P681H, P681R, R682G, R683S, R685G, A701V, T716I, F888L, S982A, T1027I, D1118H, and V1176F.

[0127] In some embodiments, the viral antigen or immunogen comprises the sequence set forth in SEQ ID NO: 57. In some embodiments, the viral antigen or immunogen comprises an amino acid sequence having at least or about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the sequence of SEQ ID NO: 57, including sequences containing substitutions, deletions, and / or insertions at one or more amino acid positions of SEQ ID NO: 57.

[0128] In some embodiments, the viral antigen or immunogen comprises the sequence set forth in SEQ ID NO: 58. In some embodiments, the viral antigen or immunogen comprises an amino acid sequence having at least or about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the sequence of SEQ ID NO: 58, including sequences containing substitutions, deletions, and / or insertions at one or more amino acid positions of SEQ ID NO: 58.

[0129] In some embodiments, the viral antigen or immunogen comprises the sequence represented by SEQ ID NO: 59. In some embodiments, the viral antigen or immunogen comprises an amino acid sequence comprising substitutions, deletions, and / or insertions at one or more amino acid positions of SEQ ID NO: 59. In some embodiments, the viral antigen or immunogen comprises the sequence represented by SEQ ID NO: 60. In some embodiments, the viral antigen or immunogen comprises an amino acid sequence comprising substitutions, deletions, and / or insertions at one or more amino acid positions of SEQ ID NO: 60.

[0130] In some embodiments, the viral antigen or immunogen comprises the sequence set forth in SEQ ID NO: 61. In some embodiments, the viral antigen or immunogen comprises an amino acid sequence having at least or about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the sequence of SEQ ID NO: 61, including sequences containing substitutions, deletions, and / or insertions at one or more amino acid positions of SEQ ID NO: 61.

[0131] In some embodiments, the viral antigen or immunogen comprises the sequence set forth in SEQ ID NO: 62. In some embodiments, the viral antigen or immunogen comprises an amino acid sequence having at least or about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the sequence of SEQ ID NO: 62, including sequences containing substitutions, deletions, and / or insertions at one or more amino acid positions of SEQ ID NO: 62.

[0132] In some embodiments, the viral antigen or immunogen comprises the sequence set forth in SEQ ID NO: 63. In some embodiments, the viral antigen or immunogen comprises an amino acid sequence having at least or about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the sequence of SEQ ID NO: 63, including sequences containing substitutions, deletions, and / or insertions at one or more amino acid positions of SEQ ID NO: 63.

[0133] In some embodiments, the viral antigen or immunogen comprises the sequence set forth in SEQ ID NO: 64. In some embodiments, the viral antigen or immunogen comprises an amino acid sequence having at least or about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the sequence of SEQ ID NO: 64, including sequences containing substitutions, deletions, and / or insertions at one or more amino acid positions of SEQ ID NO: 64.

[0134] In some embodiments, the viral antigen or immunogen comprises the sequence set forth in SEQ ID NO: 65. In some embodiments, the viral antigen or immunogen comprises an amino acid sequence having at least or about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the sequence of SEQ ID NO: 65, including sequences containing substitutions, deletions, and / or insertions at one or more amino acid positions of SEQ ID NO: 65.

[0135] In some embodiments, the viral antigen or immunogen does not comprise a transmembrane domain, such as SEQ ID NO: 66 or a portion thereof. In some embodiments, the coronavirus viral antigen or immunogen comprises a soluble S protein peptide. In some embodiments, the soluble S protein peptide does not comprise a TM domain peptide or a CP domain peptide. In some embodiments, the soluble S protein peptide does not bind to a membrane or lipid bilayer, such as a viral envelope.

[0136] In some embodiments, the S protein peptides are generated from a nucleic acid sequence that is codon optimized. In some embodiments, the S protein peptides are generated from a nucleic acid sequence that is not codon optimized.

[0137] In some embodiments, the viral antigens or immunogens referred to herein can include recombinant polypeptides or fusion peptides comprising the viral antigens or immunogens. The term viral antigen or immunogen can be used to refer to proteins comprising coronavirus viral antigens or immunogens. In certain cases, the coronavirus viral antigens or immunogens are coronavirus protein peptides provided herein.

[0138] II. Recombinant Peptides and Proteins It is contemplated that the coronavirus virus antigens and immunogens provided herein, e.g., S protein peptides (see Section I), can be combined, e.g., linked, with other proteins or peptides to form recombinant polypeptides, such as fusion peptides. In some embodiments, individual recombinant polypeptides (e.g., monomers) provided herein associate to form recombinant polypeptide multimers, e.g., trimers. In some embodiments, the association of individual recombinant polypeptide monomers occurs through covalent interactions. In some embodiments, the association of individual recombinant polypeptide monomers occurs through non-covalent interactions. In some embodiments, the interaction, e.g., covalent or non-covalent, is mediated by a protein or peptide to which the coronavirus virus antigen or immunogen, e.g., the S protein peptide, is linked. In some embodiments, for example, when the coronavirus virus antigen or immunogen is an S protein peptide described herein, the protein or peptide to which it is linked can be selected such that the native homotrimeric structure of the glycoprotein is maintained. This can be advantageous in eliciting a strong and effective immunogenic response to the S protein peptide. For example, preserving and / or maintaining the native conformation of a coronavirus viral antigen or immunogen (e.g., an S protein peptide) can improve or allow access to antigenic sites to which an immune response can be generated. In some cases, recombinant polypeptides comprising the S protein peptides described herein (see, e.g., Section I) are alternatively referred to herein as recombinant S antigens, recombinant S immunogens, or recombinant S proteins.

[0139] It is further contemplated that, in some cases, the recombinant polypeptides or multimerized recombinant polypeptides thereof will or can aggregate to form a protein or complex containing multiple coronavirus virus antigens and / or immunogens. Formation of such a protein can be advantageous for generating a strong and effective immunogenic response to the coronavirus virus antigens and / or immunogens. For example, forming a protein containing multiple recombinant polypeptides, and thus multiple coronavirus virus antigens, such as coronavirus S protein peptides, can preserve the tertiary and / or quaternary structure of the viral antigen, enhancing the immune response relative to the native structure. In some cases, aggregation can provide structural stability to the coronavirus virus antigen or immunogen, thereby allowing access to potential antigenic sites that can enhance the immune response.

[0140] 1. Fusion peptides and recombinant polypeptides In some embodiments, coronavirus virus antigens or immunogens can be linked at their C-termini (C-terminal linked) to a trimerization domain to promote trimerization of the monomers, hi some embodiments, trimerization stabilizes the membrane-proximal face of the coronavirus virus antigen or immunogen, e.g., a coronavirus S protein peptide, in a trimeric configuration.

[0141] Non-limiting examples of exogenous multimerization domains that promote stable trimers of soluble recombinant proteins include the GCN4 leucine zipper (Harbury et al. 1993 Science 262:1401-1407), trimerization motifs from pulmonary surfactant proteins (Hoppe et al. 1994 FEBS Lett 344:191-195), collagen (McAlinden et al. 2003 J Biol Chem 278:42200-42207), and phage T4 fibritin foldon (Miroshnikov et al. 1998 Protein Eng 11:329-414), any of which can be linked to the coronavirus virus antigens or immunogens described herein (e.g., by linkage to the C-terminus of the S peptide) to promote trimerization of the recombinant viral antigens or immunogens. See also U.S. Patent Nos. 7,268,116, 7,666,837, 7,691,815, 10,618,949, 10,906,944, and 10,960,070, and U.S. Patent No. 2020 / 0009244, which are incorporated by reference in their entireties for all purposes.

[0142] In some embodiments, one or more peptide linkers (such as gly-ser linkers, e.g., 10-amino acid glycine-serine peptide linkers) can be used to link the recombinant viral antigen or immunogen to the multimerization domain. The trimer can include any of the stabilizing mutations provided herein (or a combination thereof), so long as the recombinant viral antigen or immunogen trimer retains the desired properties (e.g., pre-fusion conformation). In some embodiments, the recombinant polypeptide or fusion protein comprises a first sequence represented by any of SEQ ID NOs: 27-66 linked to a second sequence represented by any of SEQ ID NOs: 67-80, wherein the C-terminus of the first sequence is directly linked to the N-terminus of the second sequence. In some embodiments, the recombinant polypeptide or fusion protein comprises a first sequence represented by any of SEQ ID NOs: 27-66 linked to a second sequence represented by any of SEQ ID NOs: 67-80, wherein the C-terminus of the first sequence is indirectly linked to the N-terminus of the second sequence, e.g., via a linker. In some embodiments, the linker comprises a sequence comprising a glycine-XY repeat.

[0143] To be therapeutically feasible, a desirable trimerizing protein moiety for biologics design should meet the following criteria: ideally, it should be part of a naturally secreted protein such as immunoglobulin Fc, be abundant in the circulation (non-toxic), be human in origin (non-immunogenic), be relatively stable (long half-life), be capable of efficient self-trimerization, and be reinforced by covalent disulfide bonds between the chains so that the trimerized coronavirus virus antigen or immunogen is structurally stable.

[0144] Collagen is a family of fibrous proteins that are major components of the extracellular matrix. It is the most abundant protein in mammals, accounting for approximately 25% of the total protein in the body. Collagen plays a major structural role in the formation of bone, tendon, skin, cornea, cartilage, blood vessels, and teeth. Fibrillar collagens types I, II, III, IV, V, and XI are all synthesized as larger trimeric precursors called procollagens, in which a central, uninterrupted triple-helical domain consisting of hundreds of "GXY" repeats (or glycine repeats) is flanked by noncollagenous domains (NC), N-propeptides, and C-propeptides. Both the C- and N-terminal extensions are proteolytically processed during procollagen secretion, an event that triggers the assembly of the mature protein into collagen fibers that form the insoluble cellular matrix. BMP-1 is a protease that recognizes a specific peptide sequence in procollagen near the junction between the glycine repeats and the collagen C-prodomain and is responsible for the removal of the propeptide. The unshelled trimeric C-propeptide of type I collagen is found in normal adult human serum at concentrations ranging from 50 to 300 ng / mL, with children having much higher levels indicative of active bone formation. In humans with familial elevated serum concentrations of type I collagen C-propeptide, levels can reach as low as 1 to 6 μg / mL without apparent abnormalities, suggesting that C-propeptide is not toxic. Structural studies of the trimeric C-propeptide of collagen suggest a three-lobed structure in which all three subunits assemble at a junction region near the N-terminus to connect to the rest of the procollagen molecule. This shape, when projected in one direction, resembles that of an Fc dimer.

[0145] Types I, IV, V, and XI collagens primarily assemble into heterotrimeric forms consisting of either two α-1 chains and one α-2 chain (types I, IV, and V) or three distinct α chains (type XI), which share high sequence homology. Both types II and III collagens are homotrimers of α-1 chains. In the case of type I collagen, the most abundant form of collagen, a stable α(I) homotrimer also forms, present at various levels in various tissues. Most of these collagen C-propeptide chains self-assemble into homotrimers when overexpressed alone in cells. While the N-propeptide domain is synthesized first, molecular assembly into trimeric collagen begins with the registered association of the C-propeptides. The C-propeptide complex is thought to be stabilized by interchain disulfide bond formation, although the necessity of disulfide bond formation for proper chain registration is unclear. The glycine triple helix extends from the repeating, associated C-terminus to the N-terminus in a zipper-like fashion. With this knowledge, non-native collagen matrices have been created by exchanging the C-propeptides of different collagen chains using recombinant DNA technology. Non-collagenous proteins, such as cytokines and growth factors, have also been fused to the N-terminus of either procollagen or mature collagen, allowing the formation of new collagen matrices, with the intention of allowing the sustained release of non-collagenous proteins from the cell matrix. However, in both situations, the C-propeptide must be cleaved before the recombinant collagen fibers can assemble into an insoluble cell matrix.

[0146] Other protein trimerization domains, such as those derived from yeast fibritin GCN4 from bacterial phage T4 and aspartate transcarbamoylase from Escherichia coli, have been described to enable the trimerization of heterologous proteins. However, none of these trimerization proteins are inherently human or naturally secreted proteins. Therefore, trimeric fusion proteins must be produced intracellularly. This not only raises the risk of misfolding relative to naturally secreted proteins such as soluble receptors, but also makes the purification of such fusion proteins difficult from thousands of other intracellular proteins. Furthermore, a critical drawback of using such non-human protein trimerization domains (e.g., derived from yeast, bacteriophages, and bacteria) to design trimeric biologics is their potential immunogenicity in the human body, rendering such fusion proteins ineffective immediately after injection.

[0147] Therefore, the use of collagen in the recombinant polypeptides described herein has many advantages, including: (1) collagen is the most abundant protein secreted in the mammalian body, accounting for nearly 25% of all proteins in the body; (2) the major form of collagen occurs naturally as a trimeric helix, the globular C-propeptide of which is responsible for initiating trimerization; (3) the trimeric C-propeptide of collagen, which is proteolytically released from mature collagen, is naturally found in mammalian blood at submicrogram / mL levels and is not known to be toxic to the body; (4 (5) The linear triple-helical region of collagen can be included as a linker at a predicted spacing of 2.9 A per residue or excluded as part of the fusion protein, allowing the distance between the protein to be trimerized and the collagen C-propeptide to be precisely adjusted to achieve optimal biological activity; (6) The recognition site for BMP1, which cleaves the C-propeptide from procollagen, can be mutated or deleted to prevent destruction of the trimeric fusion protein; and (7) the C-propeptide domain self-trimerizes via disulfide bonds, providing a universal affinity tag that can be used for purification of any secreted fusion protein produced. In some embodiments, the collagen C-propeptide, to which coronavirus virus antigens and immunogens, such as the S protein peptide, bind, enables the recombinant production of soluble, covalently linked homotrimeric fusion proteins.

[0148] In some embodiments, the coronavirus virus antigen or immunogen is linked to a C-terminal propeptide of collagen to form a recombinant polypeptide. In some embodiments, the C-terminal propeptide of the recombinant polypeptide forms an inter-polypeptide disulfide bond. In some embodiments, the recombinant protein forms a trimer. In some embodiments, the coronavirus virus antigen or immunogen is an S protein peptide described in Section I.

[0149] For example, a fusion polypeptide comprising the signal peptide MFVFLVLLPLVSS (SEQ ID NO: 54) on the N-terminus of the fusion polypeptide in SEQ ID NO: 1 can be produced and trimerized via interpolypeptide disulfide bonds (Cys residues capable of forming interpolypeptide disulfide bonds are shown in bold). 10 20 30 40 50 60 MFVFLVLLPLVSSQCVNLTTRTQLPPAYTNSFTRGVYYPDKVFRSSVLHSTQDLFLPFFS 70 80 90 100 110 120 NVTWFHAIHVSGTNGTKRFDNPVLPFNDGVYFASTEKSNIIRGWIFGTTLDSKTQSLLIV 130 140 150 160 170 180 NNATNVVIKVCEFQFCNDPFLGVYYHKNNKSWMESEFRVYSSANNCTFEYVSQPFLMDLE 190 200 210 220 230 240 GKQGNFKNLREFVFKNIDGYFKIYSKHTPINLVRDLPQGFSALEPLVDLPIGINITRFQT 250 260 270 280 290 300 LLALHRSYLTPGDSSSGWTAGAAAYYVGYLQPRTFLLKYNENGTITDAVDCALDPLSETK 310 320 330 340 350 360 CTLKSFTVEKGIYQTSNFRVQPTESIVRFPNITNLCPFGEVFNATRFASVYAWNRKRISN 370 380 390 400 410 420 CVADYSVLYNSASFSTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGKIAD 430 440 450 460 470 480 YNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPC 490 500 510 520 530 540 NGVEGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKCVN 550 560 570 580 590 600 FNFNGLTGTGVLTESNKKFLPFQQFGRDIADTTDAVRDPQTLEILDITPCSFGGVSVITP 610 620 630 640 650 660 GTNTSNQVAVLYQDVNCTEVPVAIHADQLTPTWRVYSTGSNVFQTRAGCLIGAEHVNNSY 670 680 690 700 710 720 ECDIPIGAGICASYQTQTNSPRRARSVASQSIIAYTMSLGAENSVAYSNNSIAIPTNFTI 730 740 750 760 770 780 SVTTEILPVSMTKTSVDCTMYICGDSTECSNLLLQYGSFCTQLNRALTGIAVEQDKNTQE 790 800 810 820 830 840 VFAQVKQIYKTPPIKDFGGFNFSQILPDPSKPSKRSFIEDLLFNKVTLADAGFIKQYGDC 850 860 870 880 890 900 LGDIAARDLICAQKFNGLTVLPPLLTDEMIAQYTSALLAGTITSGWTFGAGAALQIPFAM 910 920 930 940 950 960 QMAYRFNGIGVTQNVLYENQKLIANQFNSAIGKIQDSLSSTASALGKLQDVVNQNAQALN 970 980 990 1000 1010 1020 TLVKQLSSNFGAISSVLNDILSRLDKVEAEVQIDRLITGRLQSLQTYVTQQLIRAAEIRA 1030 1040 1050 1060 1070 1080 SANLAATKMSECVLGQSKRVDFCGKGYHLMSFPQSAPHGVVFLHVTYVPAQEKNFTTAPA 1090 1100 1110 1120 1130 1140 ICHDGKAHFPREGVFVSNGTHWFVTQRNFYEPQIITTDNTFVSGNCDVVIGIVNNTVYDP 1150 1160 1170 1180 1190 1200 LQPELDSFKEELDKYFKNHTSPDVDLGDISGINASVVNIQKEIDRLNEVAKNLNESLIDL 1210 1220 1230 1240 1250 1260 QELGKYEQYIKRSNGLPGPIGPPGPRGRTGDAGPVGPPGPPGPPGPPGPPSAGFDFSFLP 1270 1280 1290 1300 1310 1320 QPPQEKAHDGGRYYRANDANVVRDRDLEVDTTLKSLSQQIENIRSPEGSRKNPARTCRDL 1330 1340 1350 1360 1370 1380 KMCHSDWKSGEYWIDPNQGCNLDAIKVFCNMETGETCVYPTQPSVAQKNWYISKNPKDKR 1390 1400 1410 1420 1430 1440 HVWFGESMTDGFQFEYGGQGSDPADVAIQLTFLRLMSTEASQNITYHCKNSVAYMDQQTG 1450 1460 1470 1480 1490 1500 NLKKALLLQGSNEIEIRAEGNSRFTYSVTVDGCTSHTGAWGKTVIEYKTTKTSRLPIIDV 1510 1520 APLDVGAPDQEFGFDVGPVCFL

[0150] In some embodiments, the interpolypeptide disulfide bonds may include one or more or all of Cys15-136, Cys131-166, Cys291-301, Cys379-432, Cys336-361, Cys391-525, Cys480-488, Cys538-590, Cys617-649, Cys662-671, Cys743-749, Cys738-760, Cys840-851, Cys1032-1043, and Cys1082-1126, in any suitable combination. In some embodiments, the fusion polypeptide in the trimer may include one or more glycosylation sites (e.g., Asn-linked) at one or more or all of the Asn residues at positions 17, 61, 122, 149, 165, 234, 282, 331, 343, 603, 616, 657, 709, 717, 801, 1074, 1098, and 1134, in any suitable combination, for example.

[0151] In some embodiments, the C-terminal propeptide is that of human collagen. In some embodiments, the C-terminal propeptide can include the C-terminal propeptide of proα1(I), proα1(II), proα1(III), proα1(V), proα1(XI), proα2(I), proα2(V), proα2(XI), or proα3(XI), or a fragment thereof. In some embodiments, the C-terminal propeptide is or includes the C-terminal polypeptide of proα1(I).

[0152] In some embodiments, the C-terminal propeptide is or comprises the amino acid sequence represented by SEQ ID NO: 67. In some embodiments, the C-terminal propeptide is an amino acid sequence having at least or about 85%, 90%, 92%, 95%, or 97% sequence identity to the sequence of SEQ ID NO: 67. In some embodiments, the C-terminal propeptide is or comprises the amino acid sequence represented by SEQ ID NO: 68. In some embodiments, the C-terminal propeptide is an amino acid sequence having at least or about 85%, 90%, 92%, 95%, or 97% sequence identity to the sequence of SEQ ID NO: 68. In some embodiments, the C-terminal propeptide comprises the amino acid sequence represented by SEQ ID NO: 69. In some embodiments, the C-terminal propeptide exhibits an amino acid sequence having at least or about 85%, 90%, 92%, 95%, or 97% sequence identity to the sequence of SEQ ID NO: 69. In some embodiments, the C-terminal propeptide comprises the amino acid sequence represented by SEQ ID NO: 70. In some embodiments, the C-terminal propeptide is an amino acid sequence having at least or about 85%, 90%, 92%, 95%, or 97% sequence identity to the sequence of SEQ ID NO: 70. In some embodiments, the C-terminal propeptide is or comprises the amino acid sequence represented by SEQ ID NO: 71. In some embodiments, the C-terminal propeptide is an amino acid sequence having at least or about 85%, 90%, 92%, 95%, or 97% sequence identity to the sequence of SEQ ID NO: 71.

[0153] In some embodiments, the C-terminal propeptide is or comprises the amino acid sequence represented by SEQ ID NO: 72. In some embodiments, the C-terminal propeptide is an amino acid sequence having at least or about 85%, 90%, 92%, 95%, or 97% sequence identity to the sequence of SEQ ID NO: 72. In some embodiments, the C-terminal propeptide is or comprises the amino acid sequence represented by SEQ ID NO: 73. In some embodiments, the C-terminal propeptide is an amino acid sequence having at least or about 85%, 90%, 92%, 95%, or 97% sequence identity to the sequence of SEQ ID NO: 73. In some embodiments, the C-terminal propeptide comprises the amino acid sequence represented by SEQ ID NO: 74. In some embodiments, the C-terminal propeptide exhibits an amino acid sequence having at least or about 85%, 90%, 92%, 95%, or 97% sequence identity to the sequence of SEQ ID NO: 74. In some embodiments, the C-terminal propeptide is or comprises the amino acid sequence represented by SEQ ID NO: 75. In some embodiments, the C-terminal propeptide is an amino acid sequence having at least or about 85%, 90%, 92%, 95%, or 97% sequence identity to the sequence of SEQ ID NO: 75. In some embodiments, the C-terminal propeptide is or comprises the amino acid sequence represented by SEQ ID NO: 76. In some embodiments, the C-terminal propeptide is an amino acid sequence having at least or about 85%, 90%, 92%, 95%, or 97% sequence identity to the sequence of SEQ ID NO: 76.

[0154] In some embodiments, the C-terminal propeptide is or comprises the amino acid sequence represented by SEQ ID NO: 77. In some embodiments, the C-terminal propeptide is an amino acid sequence having at least or about 85%, 90%, 92%, 95%, or 97% sequence identity to the sequence of SEQ ID NO: 77. In some embodiments, the C-terminal propeptide is or comprises the amino acid sequence represented by SEQ ID NO: 78. In some embodiments, the C-terminal propeptide is an amino acid sequence having at least or about 85%, 90%, 92%, 95%, or 97% sequence identity to the sequence of SEQ ID NO: 78. In some embodiments, the C-terminal propeptide comprises the amino acid sequence represented by SEQ ID NO: 79. In some embodiments, the C-terminal propeptide exhibits an amino acid sequence having at least or about 85%, 90%, 92%, 95%, or 97% sequence identity to the sequence of SEQ ID NO: 79. In some embodiments, the C-terminal propeptide is or comprises the amino acid sequence represented by SEQ ID NO: 80. In some embodiments, the C-terminal propeptide is an amino acid sequence having at least or about 85%, 90%, 92%, 95%, or 97% sequence identity to the sequence of SEQ ID NO:80.

[0155] In some embodiments, the C-terminal propeptide is, for example, as shown in SEQ ID NO: 68 (wherein RA D However, RA N In some embodiments, the C-terminal propeptide is or includes the amino acid sequence of a collagen trimerization domain (e.g., the C-propeptide of human α1(I) collagen) with an aspartic acid (D) to asparagine (N) substitution at the BMP-1 site (mutated to α1(I)). In some embodiments, the C-terminal propeptide is, for example, as set forth in SEQ ID NO: 69 (wherein R A D is R NIn some embodiments, the C-terminal propeptide herein may comprise a mutated BMP-1 site, e.g., RSAN instead of DDAN. In some embodiments, the C-terminal propeptide herein may comprise a BMP-1 site, e.g., a sequence comprising a RAD (e.g., RADDAN) sequence (e.g., SEQ ID NO: 68 or 69), instead of RAN (e.g., RANDAN) or RND (e.g., RNDDAN) that may be used in the fusion polypeptides disclosed herein. For example, SEQ ID NO: 27 (underlined), or a fragment, variant, or mutant thereof, may be directly or indirectly linked to SEQ ID NO: 67 (italicized), or a fragment, variant, or mutant thereof, to form, for example, the following fusion protein:

[0156] [Table 2]

[0157] In some embodiments, the C-terminal propeptide is or comprises an amino acid sequence that is a fragment of any of SEQ ID NOs: 67-80.

[0158] In some embodiments, the C-terminal propeptide can comprise a sequence comprising a glycine-XY repeat (where X and Y are independently any amino acid or amino acid sequences that are at least 85%, 90%, 92%, 95%, or 97% identical), which can form inter-polypeptide disulfide bonds and trimerize the recombinant polypeptide. In some embodiments, X and Y are independently proline or hydroxyproline.

[0159] In some cases where the S protein peptide is linked to a C-terminal propeptide to form a recombinant polypeptide, the recombinant polypeptide forms a trimer, resulting in a homotrimer of the S protein peptide. In some embodiments, the S protein peptide of the trimerized recombinant polypeptide is in a pre-fusion conformation. In some embodiments, the S protein peptide of the trimerized recombinant polypeptide is in a post-fusion conformation. In some embodiments, this conformational state allows access to different antigenic sites on the S protein peptide. In some embodiments, the antigenic sites are epitopes, such as linear or conformational epitopes. An advantage of having the described trimerized recombinant polypeptide is the ability to enhance immune responses against a variety of potential and diverse antigenic sites.

[0160] In some embodiments, trimerizing recombinant polypeptides include individual recombinant polypeptides that contain the same viral antigen or immunogen. In some embodiments, trimerizing recombinant polypeptides include individual recombinant polypeptides that each contain a viral antigen or immunogen that is different from the other recombinant polypeptides. In some embodiments, trimerizing recombinant polypeptides include individual recombinant polypeptides where one of the individual recombinant polypeptides contains a viral antigen or immunogen that is different from the other recombinant polypeptides. In some embodiments, trimerizing recombinant polypeptides include individual recombinant polypeptides where two of the individual recombinant polypeptides contain the same viral antigen or immunogen, and the viral antigen or immunogen is different from the viral antigen or immunogen contained in the remaining recombinant polypeptide.

[0161] In some embodiments, the recombinant polypeptide comprises any coronavirus virus antigen or immunogen described in Section I. In some embodiments, the recombinant polypeptide comprises any coronavirus virus antigen or immunogen described in Section I linked as described herein to a C-terminal propeptide of a collagen described herein.

[0162] In some embodiments, the immunogen comprises a recombinant SARS-CoV or SARS-CoV-2 S ectodomain trimer comprising a promoter comprising one or more (such as two, e.g., two consecutive) proline substitutions at or near the boundary between the HR1 domain and the central helix domain that stabilize the S ectodomain trimer in the pre-fusion conformation. In some such embodiments, the one or more (such as two, e.g., two consecutive) proline substitutions that stabilize the S ectodomain in the pre-fusion conformation are between amino acid 15 N-terminal to the C-terminal residue of HR1 and amino acid 5 C-terminal to the N-terminal residue of the central helix.

[0163] In some embodiments, one or more (such as two, e.g., two consecutive) proline substitutions stabilize the coronavirus (e.g., SARS-CoV or SARS-CoV-2) S ectodomain trimer in the pre-fusion conformation. In some embodiments, the SARS-CoV-2 S protein peptide comprises mutations 986K / 987V to 986P / 987P.

[0164] In some embodiments, a recombinant coronavirus (e.g., SARS-CoV or SARS-CoV-2) S ectodomain trimer stabilized in a pre-fusion conformation comprises a single S ectodomain protomer containing mutations to the S1 / S2 and / or S2' protease cleavage sites, thereby preventing protease cleavage at these sites. In some embodiments, a SARS-CoV-2 S protein peptide contains a 685R to 685A mutation. Examples of protease cleavage sites for various viruses are shown below:

[0165] [Table 3]

[0166] In some embodiments, the recombinant coronavirus (e.g., SARS-CoV or SARS-CoV-2) S ectodomain trimer promoter stabilized in the pre-fusion conformation by one or more proline substitutions (e.g., 986P / 987P substitutions) contains additional modifications to stabilize it in the pre-fusion conformation, such as mutations at the protease cleavage site, thereby preventing protease cleavage.

[0167] For the SARS-CoV-2S protein sequence provided as SEQ ID NO: 55, the ectodomain includes a signal peptide (SP), which is removed during cellular processing; an N-terminal domain (NTD); a receptor-binding domain (RBD); one or more S1 / S2 cleavage sites; a fusion peptide (FP); an internal fusion peptide (IFP); heptad repeats 1 / 2 (HR1 / 2), and a transmembrane domain (TM). Exemplary sources of sequences can be found at ncbi.nlm.nih.gov / nuccore / MN908947.3, ncbi.nlm.nih.gov / nuccore / MN908947, and ncbi.nlm.nih.gov / nuccore / MN908947.2. Additional sequences, including the pneumovirus isolate Wuhan-Hu-1 and the complete genome, can be found at ncbi.nlm.nih.gov / genbank / sars-cov-2-seqs / .

[0168] In some embodiments, a protomer of a prefusion-stabilized SARS-CoV-2 S ectodomain trimer can have the C-terminal residue of the NTD, RBD, S1 (either S1 / S2 site 1 or S1 / S2 site 2), FP, IFP, HR1, HR2, or the C-terminal residue of the ectodomain (e.g., can be linked to the trimerization domain or transmembrane domain). The numbering of positions in the S protein can vary between SARS-CoV strains, but sequences can be aligned to determine relevant structural domains and cleavage sites. It will be understood that several residues (e.g., up to 10) on the N- and C-termini of any of the ectodomain fragments can be removed or modified in the disclosed immunogens without reducing the utility of the S ectodomain trimer as an immunogen.

[0169] In some embodiments, the recombinant polypeptide is or comprises an NTD peptide of a SARS-CoV or SARS-CoV-2 S protein. In some embodiments, the recombinant polypeptide is or comprises an RBD peptide of a SARS-CoV or SARS-CoV-2 S protein. In some embodiments, the recombinant polypeptide is or comprises an NTD peptide and an RBD peptide of a SARS-CoV or SARS-CoV-2 S protein. In some embodiments, the recombinant polypeptide is or comprises an S1 domain peptide of a SARS-CoV or SARS-CoV-2 S protein. In some embodiments, the recombinant polypeptide is or comprises an S2 domain peptide of a SARS-CoV or SARS-CoV-2 S protein.

[0170] An exemplary SARS-CoV-1 S recombinant polypeptide without the signal peptide is provided in SEQ ID NO: 26 (1491 aa): 1 0 2 0 3 0 4 0 5 0 6 0 SDLDRCTTFD DVQAPNYTQH TSSMRGVYYP DEIFRSDTLY LTQDLFLPFY SNVTGFHTIN 7 0 8 0 9 0 10 0 11 0 12 0 HTFDNPVIPF KDGIYFAATE KSNVVRGWVF GSTMNNKSQS VIIINNSTNV VIRACNFELC 13 0 14 0 15 0 16 0 17 0 18 0 DNPFFAVSKP MGTQTHTMIF DNAFNCTFEY ISDAFSLDVS EKSGNFKHLR EFVFKNKDGF 19 0 20 0 21 0 22 0 23 0 24 0 LYVYKGYQPI DVVRDLPSGF NTLKPIFKLP LGINITNFRA ILTAFLPAQD TWGTSAAAYF 25 0 26 0 27 0 28 0 29 0 30 0 VGYLKPTTFM LKYDENGTIT DAVDCSQNPL AELKCSVKSF EIDKGIYQTS NFRVVPSRDV 31 0 32 0 33 0 34 0 35 0 36 0 VRFPNITNLC PFGEVFNATK FPSVYAWERK RISNCVADYS VLYNSTFFST FKCYGVSATK 37 0 38 0 39 0 40 0 41 0 42 0 LNDLCFSNVY ADSFVVKGDD VRQIAPGQTG VIADYNYKLP DDFMGCVLAW NTRNIDATST 43 0 44 0 45 0 46 0 47 0 48 0 GNYNYKYRYL RHGKLRPFER DISNVPFSPD GKPCTPPALN CYWPLNDYGF YTTTGIGYQP 49 0 500 51 0 52 0 53 0 54 0 YRVVVLSFEL LNAPATVCGP KLSTDLIKNQ CVNFNFNGLT GTGVLTPSSK RFQPFQQFGR 55 0 56 0 57 0 58 0 59 0 60 0 DVSDFTDSVR DPKTSEILDI SPCSFGGVSV ITPGTNASSE VAVLYQDVNC TDVSTAIHAD 61 0 62 0 63 0 64 0 65 0 66 0 QLTPAWRIYS TGNNVFQTQA GCLIGAEHVD TSYECDIPIG AGICASYHTV SLLRSTSQKS 67 0 68 0 69 0 70 0 71 0 72 0 IVAYTMSLGA DSSIAYSNNT IAIPTNFSIS ITTEVMPVSM AKTSVDCNMY ICGDSTECAN 73 0 74 0 75 0 76 0 77 0 78 0 LLLQYGSFCT QLNRALSGIA AEQDRNTREV FAQVKQMYKT PTLKDFGGFN FSQILPDPLK 79 0 80 0 81 0 82 0 83 0 84 0 PTKRSFIEDL LFNKVTLADA GFMKQYGECL GDINARDLIC AQKFNGLTVL PPLLTDDMIA 85 0 86 0 87 0 88 0 89 0 90 0 AYTAALVSGT ATAGWTFGAG AALQIPFAMQ MAYRFNGIGV TQNVLYENQK QIANQFNKAI 91 0 92 0 93 0 94 0 95 0 96 0 SQIQESLTTT STALGKLQDV VNQNAQALNT LVKQLSSNFG AISSVLNDIL SRLDKVEAEV 97 0 98 0 99 0 100 0 101 0 102 0 QIDRLITGRL QSLQTYVTQQ LIRAAEIRAS ANLAATKMSE CVLGQSKRVD FCGKGYHLMS 103 0 104 0 105 0 106 0 107 0 108 0 FPQAAPHGVV FLHVTYVPSQ ERNFTTAPAI CHEGKAYFPR EGVFVFNGTS WFITQRNFFS 109 0 110 0 111 0 112 0 113 0 114 0 PQIITTDNTF VSGNCDVVIG IINNTVYDPL QPELDSFKEE LDKYFKNHTS PDVDLGDISG 1150 116 0 117 0 118 0 119 0 120 0 INASVVNIQE EIDRLNEVAK NLNESLIDLQ ELGKYEQYIK RSNGLPGPIG PPGPRGRTGD 121 0 122 0 123 0 124 0 125 0 126 0 AGPVGPPGPP GPPGPPGPPS AGFDFSFLPQ PPQEKAHDGG RYYRANDANV VRDRDLEVDT 127 0 128 0 129 0 130 0 131 0 132 0 TLKSLSQQIE NIRSPEGSRK NPARTCRDLK MCHSDWKSGE YWIDPNQGCN LDAIKVFCNM 133 0 134 0 135 0 136 0 137 0 138 0 ETGETCVYPT QPSVAQKNWY ISKNPKDKRH VWFGESMTDG FQFEYGGQGS DPADVAIQLT 139 0 140 0 141 0 142 0 143 0 144 0 FLRLMSTEAS QNITYHCKNS VAYMDQQTGN LKKALLLQGS NEIEIRAEGN SRFTYSVTVD 145 0 146 0 147 0 148 0 1490 GCTSHTGAWG KTVIEYKTTK TSRLPIIDVA PLDVGAPDQE FGFDVGPVCF L

[0171] The SARS-CoV-1 S recombinant polypeptide described above may include an N-terminal signal peptide as provided in SEQ ID NO:53.

[0172] An exemplary SARS-CoV-2 S recombinant polypeptide without the signal peptide is provided in SEQ ID NO:1 (1509 aa): 10 20 30 40 50 60 QCVNLTTRTQ LPPAYTNSFT RGVYYPDKVF RSSVLHSTQD LFLPFFSNVT WFHAIHVSGT 70 80 90 100 110 120 NGTKRFDNPV LPFNDGVYFA STEKSNIIRG WIFGTTLDSK TQSLLIVNNA TNVVIKVCEF 130 140 150 160 170 180 QFCNDPFLGV YYHKNNKSWM ESEFRVYSSA NNCTFEYVSQ PFLMDLEGKQ GNFKNLREFV 190 200 210 220 230 240 FKNIDGYFKI YSKHTPINLV RDLPQGFSAL EPLVDLPIGI NITRFQTLLA LHRSYLTPGD 250 260 270 280 290 300 SSSGWTAGAA AYYVGYLQPR TFLLKYNENG TITDAVDCAL DPLSETKCTL KSFTVEKGIY 310 320 330 340 350 360 QTSNFRVQPT ESIVRFPNIT NLCPFGEVFN ATRFASVYAW NRKRISNCVA DYSVLYNSAS 370 380 390 400 410 420 FSTFKCYGVS PTKLNDLCFT NVYADSFVIR GDEVRQIAPG QTGKIADYNY KLPDDFTGCV 430 440 450 460 470 480 IAWNSNNLDS KVGGNYNYLY RLFRKSNLKP FERDISTEIY QAGSTPCNGV EGFNCYFPLQ 490 500 510 520 530 540 SYGFQPTNGV GYQPYRVVVL SFELLHAPAT VCGPKKSTNL VKNKCVNFNF NGLTGTGVLT 550 560 570 580 590 600 ESNKKFLPFQ QFGRDIADTT DAVRDPQTLE ILDITPCSFG GVSVITPGTN TSNQVAVLYQ 610 620 630 640 650 660 DVNCTEVPVA IHADQLTTPW RVYSTGSNVF QTRAGCLIGA EHVNNSYECD IPIGAGICAS 670 680 690 700 710 720 YQTQTNSPRR ARSVASQSII AYTMSLGAEN SVAYSNNSIA IPTNFTISVT TEILPVSMTK 730 740 750 760 770 780 TSVDCTMYIC GDSTECSNLL LQYGSFCTQL NRALTGIAVE QDKNTQEVFA QVKQIYKTPP 790 800 810 820 830 840 IKDFGGFNFS QILPDPSKPS KRSFIEDLLF NKVTLADAGF IKQYGDCLGD IAARDLICAQ 850 860 870 880 890 900 KFNGLTVLPP LLTDEMIAQY TSALLAGTIT SGWTFGAGAA LQIPFAMQMA YRFNGIGVTQ 910 920 930 940 950 960 NVLYENQKLI ANQFNSAIGK IQDSLSSTAS ALGKLQDVVN QNAQALNTLV KQLSSNFGAI 970 980 990 1000 1010 1020 SSVLNDILSR LDKVEAEVQI DRLITGRLQS LQTYVTQQLI RAAEIRASAN LAATKMSECV 1030 1040 1050 1060 1070 1080 LGQSKRWDFC GKGYHLMSFP QSAPHGVVFL HVTYVPAQEK NFTTAPAICH DGKAHFPREG 1090 1100 1110 1120 1130 1140 VFVSNGTHWF VTQRNFYEPQ IITTDNTFVS GNCDVVIGIV NNTVYDPLQP ELDSFKEELD 1150 1160 1170 1180 1190 1200 KYFKNHTSPD VDLGDISGIN ASVVNIQKEI DRLNEVAKNL NESLIDLQEL GKYEQYIKRS 1210 1220 1230 1240 1250 1260 NGLPGPIGPP GPRGRTGDAG PVGPPPGPGP PGPPGPPSAG FDFSFLPQPP QEKAHDGGRY 1270 1280 1290 1300 1310 1320 IRANDANVVR DRDLEVDTTL KSLSQQIENI RSPEGSRKNP ARTCRDLKMC HSDWKSGEYW 1330 1340 1350 1360 1370 1380 IDPNQGCNLD AIKVFCNMET GETCVYPTQP SVAQKNWYIS KNPKDKRHVW FGESMTDGFQ 1390 1400 1410 1420 1430 1440 FEYGGQGSDP ADVAIQLTFL RLMSTEASQN ITYHCKNSVA YMDQQTGNLK KALLLQGSNE 1450 1460 1470 1480 1490 1500 IEIRAEGNSR FTYSVTVDGC TSHTGAWGKT VIEYKTTKTS RLPIIDVAPL DVGAPDQEFG 1509 FDVGPVCFL

[0173] The SARS-CoV-2 S recombinant polypeptide described above may include an N-terminal signal peptide as provided in SEQ ID NO:54.

[0174] In some embodiments, the recombinant polypeptide is or comprises the sequence set forth in SEQ ID NO: 1. In some embodiments, the recombinant polypeptide comprises a sequence at one or more amino acid positions (amino acid positions relative to SEQ ID NO: 55), e.g., 13, 18, 20, 26, 69, 70, 80, 138, 142, 144, 152, 190, 215, 242, 243, 244, 246, 400, 401, 402, 417, 440, 452, 477, 484, 501, 570, 614, 655, 681, 682, 683, 684, 685, 700, 701, 702, 703, 704, 705, 706, 707, 708, 709, 710, 711, 712, 713, 714, 715, 716, 717, 718, 719, 720, 721, 722, 723, 724, 725, 726, 727, 728, 729, 730, 731, 732, 733, 734, 735, 736, 737, 738, 739, 740, 741, 742, 743, 744, 745, 746, 747, 748, 749, 750, 751, 752, 753, 754, 755, 756, 75 or comprises an amino acid sequence having at least or about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the sequence of SEQ ID NO: 1, such as a sequence containing substitutions, deletions, and / or insertions at positions 01, 716, 888, 982, 1027, 1118, and / or 1176, or any combination thereof. In some embodiments, the recombinant polypeptide comprises or consists of a variant of SEQ ID NO: 1, the variants being S13I, L18F, T20N, P26S, Δ69-70(ΔHV), D80A, D138Y, G142D, Δ144(ΔY), W152C, R190S, D215G, Δ242-244(ΔLAL), R246I, Δ400-402(ΔFVI), K417T, K417N, N440K, L and any one, two, three, four, five or more of the mutations selected from the group consisting of 452R, S477N, S477G, E484K, E484Q, N501Y, A570D, D614G, H655Y, P681H, P681R, R682G, R683S, R685G, A701V, T716I, F888L, S982A, T1027I, D1118H, and V1176F, or any combination thereof.

[0175] In some embodiments, the recombinant polypeptide is or comprises the sequence set forth in SEQ ID NO: 2. In some embodiments, the recombinant polypeptide comprises a sequence at one or more amino acid positions (amino acid positions relative to SEQ ID NO: 55), e.g., 13, 18, 20, 26, 69, 70, 80, 138, 142, 144, 152, 190, 215, 242, 243, 244, 246, 400, 401, 402, 417, 440, 452, 477, 484, 501, 570, 614, 655, 681, 682, 683, 684, 685, 700, 701, 702, 703, 704, 705, 706, 707, 708, 709, 710, 711, 712, 713, 714, 715, 716, 717, 718, 719, 720, 721, 722, 723, 724, 725, 726, 727, 728, 729, 730, 731, 732, 733, 734, 735, 736, 737, 738, 739, 740, 741, 742, 743, 744, 745, 746, 747, 748, 749, 750, 751, 752, 753, 754, 755, 756, 75 or comprises an amino acid sequence having at least or about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the sequence of SEQ ID NO:2, such as a sequence containing substitutions, deletions, and / or insertions at positions 01, 716, 888, 982, 1027, 1118, and / or 1176, or any combination thereof. In some embodiments, the recombinant polypeptide is or comprises a variant of SEQ ID NO:2, wherein the variant is S13I, L18F, T20N, P26S, Δ69-70(ΔHV), D80A, D138Y, G142D, Δ144(ΔY), W152C, R190S, D215G, Δ242-244(ΔLAL), R246I, Δ400-402(ΔFVI), K417T, K417N, N440K, L452 and any one, two, three, four, five or more of the mutations selected from the group consisting of R, S477N, S477G, E484K, E484Q, N501Y, A570D, D614G, H655Y, P681H, P681R, R682G, R683S, R685G, A701V, T716I, F888L, S982A, T1027I, D1118H, and V1176F, or any combination thereof.

[0176] In some embodiments, the recombinant polypeptide is or comprises the sequence set forth in SEQ ID NO: 3. In some embodiments, the recombinant polypeptide comprises a sequence at one or more amino acid positions (amino acid positions relative to SEQ ID NO: 55), e.g., 13, 18, 20, 26, 69, 70, 80, 138, 142, 144, 152, 190, 215, 242, 243, 244, 246, 400, 401, 402, 417, 440, 452, 477, 484, 501, 570, 614, 655, 681, 682, 683, 684, 685, 700, 701, 702, 703, 704, 705, 706, 707, 708, 709, 710, 711, 712, 713, 714, 715, 716, 717, 718, 719, 720, 721, 722, 723, 724, 725, 726, 727, 728, 729, 730, 731, 732, 733, 734, 735, 736, 737, 738, 739, 740, 741, 742, 743, 744, 745, 746, 747, 748, 749, 750, 751, 752, 753, 754, 755, 756, 75 or comprises an amino acid sequence having at least or about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the sequence of SEQ ID NO: 3, such as a sequence containing substitutions, deletions, and / or insertions at positions 01, 716, 888, 982, 1027, 1118, and / or 1176, or any combination thereof. In some embodiments, the recombinant polypeptide is or comprises a variant of SEQ ID NO: 3, the variants being S13I, L18F, T20N, P26S, Δ69-70(ΔHV), D80A, D138Y, G142D, Δ144(ΔY), W152C, R190S, D215G, Δ242-244(ΔLAL), R246I, Δ400-402(ΔFVI), K417T, K417N, N440K, L452 and any one, two, three, four, five or more of the mutations selected from the group consisting of R, S477N, S477G, E484K, E484Q, N501Y, A570D, D614G, H655Y, P681H, P681R, R682G, R683S, R685G, A701V, T716I, F888L, S982A, T1027I, D1118H, and V1176F, or any combination thereof.

[0177] In some embodiments, the recombinant polypeptide is or comprises the sequence set forth in SEQ ID NO: 4. In some embodiments, the recombinant polypeptide comprises a sequence at one or more amino acid positions (amino acid positions relative to SEQ ID NO: 55), e.g., 13, 18, 20, 26, 69, 70, 80, 138, 142, 144, 152, 190, 215, 242, 243, 244, 246, 400, 401, 402, 417, 440, 452, 477, 484, 501, 570, 614, 655, 681, 682, 683, 684, 685, 700, 701, 702, 703, 704, 705, 706, 707, 708, 709, 710, 711, 712, 713, 714, 715, 716, 717, 718, 719, 720, 721, 722, 723, 724, 725, 726, 727, 728, 729, 730, 731, 732, 733, 734, 735, 736, 737, 738, 739, 740, 741, 742, 743, 744, 745, 746, 747, 748, 749, 750, 751, 752, 753, 754, 755, 756, 75 or comprises an amino acid sequence having at least or about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the sequence of SEQ ID NO:4, such as a sequence containing substitutions, deletions, and / or insertions at positions 01, 716, 888, 982, 1027, 1118, and / or 1176, or any combination thereof. In some embodiments, the recombinant polypeptide is or comprises a variant of SEQ ID NO: 4, the variants being S13I, L18F, T20N, P26S, Δ69-70(ΔHV), D80A, D138Y, G142D, Δ144(ΔY), W152C, R190S, D215G, Δ242-244(ΔLAL), R246I, Δ400-402(ΔFVI), K417T, K417N, N440K, L45 2R, S477N, S477G, E484K, E484Q, N501Y, A570D, D614G, H655Y, P681H, P681R, R682G, R683S, R685G, A701V, T716I, F888L, S982A, T1027I, D1118H, and V1176F, or any combination thereof.

[0178] In some embodiments, the recombinant polypeptide is or comprises the sequence set forth in SEQ ID NO: 5. In some embodiments, the recombinant polypeptide comprises a sequence at one or more amino acid positions (amino acid positions relative to SEQ ID NO: 55), e.g., 13, 18, 20, 26, 69, 70, 80, 138, 142, 144, 152, 190, 215, 242, 243, 244, 246, 400, 401, 402, 417, 440, 452, 477, 484, 501, 570, 614, 655, 681, 682, 683, 684, 685, 700, 701, 702, 703, 704, 705, 706, 707, 708, 709, 710, 711, 712, 713, 714, 715, 716, 717, 718, 719, 720, 721, 722, 723, 724, 725, 726, 727, 728, 729, 730, 731, 732, 733, 734, 735, 736, 737, 738, 739, 740, 741, 742, 743, 744, 745, 746, 747, 748, 749, 750, 751, 752, 753, 754, 755, 756, 75 or comprises an amino acid sequence having at least or about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the sequence of SEQ ID NO: 5, such as a sequence containing substitutions, deletions, and / or insertions at positions 01, 716, 888, 982, 1027, 1118, and / or 1176, or any combination thereof. In some embodiments, the recombinant polypeptide is or comprises a variant of SEQ ID NO:5, the variants being S13I, L18F, T20N, P26S, Δ69-70(ΔHV), D80A, D138Y, G142D, Δ144(ΔY), W152C, R190S, D215G, Δ242-244(ΔLAL), R246I, Δ400-402(ΔFVI), K417T, K417N, N440K, L4 and any one, two, three, four, five or more of the mutations selected from the group consisting of 52R, S477N, S477G, E484K, E484Q, N501Y, A570D, D614G, H655Y, P681H, P681R, R682G, R683S, R685G, A701V, T716I, F888L, S982A, T1027I, D1118H, and V1176F, or any combination thereof.

[0179] In some embodiments, the recombinant polypeptide is or comprises the sequence set forth in SEQ ID NO: 6. In some embodiments, the recombinant polypeptide comprises a sequence at one or more amino acid positions (amino acid positions relative to SEQ ID NO: 55), e.g., 13, 18, 20, 26, 69, 70, 80, 138, 142, 144, 152, 190, 215, 242, 243, 244, 246, 400, 401, 402, 417, 440, 452, 477, 484, 501, 570, 614, 655, 681, 682, 683, 684, 685, 700, 701, 702, 703, 704, 705, 706, 707, 708, 709, 710, 711, 712, 713, 714, 715, 716, 717, 718, 719, 720, 721, 722, 723, 724, 725, 726, 727, 728, 729, 730, 731, 732, 733, 734, 735, 736, 737, 738, 739, 740, 741, 742, 743, 744, 745, 746, 747, 748, 749, 750, 751, 752, 753, 754, 755, 756, 75 or comprises an amino acid sequence having at least or about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the sequence of SEQ ID NO: 6, such as a sequence containing substitutions, deletions, and / or insertions at positions 01, 716, 888, 982, 1027, 1118, and / or 1176, or any combination thereof. In some embodiments, the recombinant polypeptide is or comprises a variant of SEQ ID NO: 6, wherein the variants are S13I, L18F, T20N, P26S, Δ69-70(ΔHV), D80A, D138Y, G142D, Δ144(ΔY), W152C, R190S, D215G, Δ242-244(ΔLAL), R246I, Δ400-402(ΔFVI), K417T, K417N, N440K, L4 and any one, two, three, four, five or more of the mutations selected from the group consisting of 52R, S477N, S477G, E484K, E484Q, N501Y, A570D, D614G, H655Y, P681H, P681R, R682G, R683S, R685G, A701V, T716I, F888L, S982A, T1027I, D1118H, and V1176F, or any combination thereof.

[0180] In some embodiments, the recombinant polypeptide is or comprises the sequence set forth in SEQ ID NO: 7. In some embodiments, the recombinant polypeptide comprises a sequence at one or more amino acid positions (amino acid positions relative to SEQ ID NO: 55), e.g., 13, 18, 20, 26, 69, 70, 80, 138, 142, 144, 152, 190, 215, 242, 243, 244, 246, 400, 401, 402, 417, 440, 452, 477, 484, 501, 570, 614, 655, 681, 682, 683, 684, 685, 701, 702, 703, 704, 705, 706, 707, 708, 709, 710, 711, 712, 713, 714, 715, 716, 717, 718, 719, 720, 721, 722, 723, 724, 725, 726, 727, 728, 729, 730, 731, 732, 733, 734, 735, 736, 737, 738, 739, 740, 741, 742, 743, 744, 745, 746, 747, 748, 749, 750, 751, 752, 753, 754, 755, 756, 757, 75 or comprises an amino acid sequence having at least or about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the sequence of SEQ ID NO: 7, such as a sequence containing substitutions, deletions, and / or insertions at positions 01, 716, 888, 982, 1027, 1118, and / or 1176, or any combination thereof. In some embodiments, the recombinant polypeptide is or comprises a variant of SEQ ID NO: 7, the variants being S13I, L18F, T20N, P26S, Δ69-70(ΔHV), D80A, D138Y, G142D, Δ144(ΔY), W152C, R190S, D215G, Δ242-244(ΔLAL), R246I, Δ400-402(ΔFVI), K417T, K417N, N440K, L4 and any one, two, three, four, five or more of the mutations selected from the group consisting of 52R, S477N, S477G, E484K, E484Q, N501Y, A570D, D614G, H655Y, P681H, P681R, R682G, R683S, R685G, A701V, T716I, F888L, S982A, T1027I, D1118H, and V1176F, or any combination thereof.

[0181] In some embodiments, the recombinant polypeptide is or comprises the sequence set forth in SEQ ID NO: 8. In some embodiments, the recombinant polypeptide comprises a sequence at one or more amino acid positions (amino acid positions relative to SEQ ID NO: 55), e.g., 13, 18, 20, 26, 69, 70, 80, 138, 142, 144, 152, 190, 215, 242, 243, 244, 246, 400, 401, 402, 417, 440, 452, 477, 484, 501, 570, 614, 655, 681, 682, 683, 684, 685, 700, 701, 702, 703, 704, 705, 706, 707, 708, 709, 710, 711, 712, 713, 714, 715, 716, 717, 718, 719, 720, 721, 722, 723, 724, 725, 726, 727, 728, 729, 730, 731, 732, 733, 734, 735, 736, 737, 738, 739, 740, 741, 742, 743, 744, 745, 746, 747, 748, 749, 750, 751, 752, 753, 754, 755, 756, 75 or comprises an amino acid sequence having at least or about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the sequence of SEQ ID NO: 8, such as a sequence containing substitutions, deletions, and / or insertions at positions 01, 716, 888, 982, 1027, 1118, and / or 1176, or any combination thereof. In some embodiments, the recombinant polypeptide is or comprises a variant of SEQ ID NO: 8, the variants being S13I, L18F, T20N, P26S, Δ69-70(ΔHV), D80A, D138Y, G142D, Δ144(ΔY), W152C, R190S, D215G, Δ242-244(ΔLAL), R246I, Δ400-402(ΔFVI), K417T, K417N, N440K, L4 and any one, two, three, four, five or more of the mutations selected from the group consisting of 52R, S477N, S477G, E484K, E484Q, N501Y, A570D, D614G, H655Y, P681H, P681R, R682G, R683S, R685G, A701V, T716I, F888L, S982A, T1027I, D1118H, and V1176F, or any combination thereof.

[0182] In some embodiments, the recombinant polypeptide is or comprises the sequence set forth in SEQ ID NO: 9. In some embodiments, the recombinant polypeptide comprises a sequence at one or more amino acid positions (amino acid positions relative to SEQ ID NO: 55), e.g., 13, 18, 20, 26, 69, 70, 80, 138, 142, 144, 152, 190, 215, 242, 243, 244, 246, 400, 401, 402, 417, 440, 452, 477, 484, 501, 570, 614, 655, 681, 682, 683, 684, 685, 700, 701, 702, 703, 704, 705, 706, 707, 708, 709, 710, 711, 712, 713, 714, 715, 716, 717, 718, 719, 720, 721, 722, 723, 724, 725, 726, 727, 728, 729, 730, 731, 732, 733, 734, 735, 736, 737, 738, 739, 740, 741, 742, 743, 744, 745, 746, 747, 748, 749, 750, 751, 752, 753, 754, 755, 756, 75 or comprises an amino acid sequence having at least or about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the sequence of SEQ ID NO:9, such as a sequence containing substitutions, deletions, and / or insertions at positions 01, 716, 888, 982, 1027, 1118, and / or 1176, or any combination thereof. In some embodiments, the recombinant polypeptide is or comprises a variant of SEQ ID NO:9, wherein the variants are S13I, L18F, T20N, P26S, Δ69-70(ΔHV), D80A, D138Y, G142D, Δ144(ΔY), W152C, R190S, D215G, Δ242-244(ΔLAL), R246I, Δ400-402(ΔFVI), K417T, K417N, N440K, L4 and any one, two, three, four, five or more of the mutations selected from the group consisting of 52R, S477N, S477G, E484K, E484Q, N501Y, A570D, D614G, H655Y, P681H, P681R, R682G, R683S, R685G, A701V, T716I, F888L, S982A, T1027I, D1118H, and V1176F, or any combination thereof.

[0183] In some embodiments, the recombinant polypeptide is or comprises the sequence set forth in SEQ ID NO: 10. In some embodiments, the recombinant polypeptide comprises a sequence at one or more amino acid positions (amino acid positions relative to SEQ ID NO: 55), e.g., 13, 18, 20, 26, 69, 70, 80, 138, 142, 144, 152, 190, 215, 242, 243, 244, 246, 400, 401, 402, 417, 440, 452, 477, 484, 501, 570, 614, 655, 681, 682, 683, 684, 685, 70 or comprises an amino acid sequence having at least or about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the sequence of SEQ ID NO: 10, such as a sequence containing substitutions, deletions, and / or insertions at positions 1, 716, 888, 982, 1027, 1118, and / or 1176, or any combination thereof. In some embodiments, the recombinant polypeptide is or comprises a variant of SEQ ID NO: 10, the variants being S13I, L18F, T20N, P26S, Δ69-70(ΔHV), D80A, D138Y, G142D, Δ144(ΔY), W152C, R190S, D215G, Δ242-244(ΔLAL), R246I, Δ400-402(ΔFVI), K417T, K417N, N440K, L and any one, two, three, four, five or more of the mutations selected from the group consisting of 452R, S477N, S477G, E484K, E484Q, N501Y, A570D, D614G, H655Y, P681H, P681R, R682G, R683S, R685G, A701V, T716I, F888L, S982A, T1027I, D1118H, and V1176F, or any combination thereof.

[0184] In some embodiments, the recombinant polypeptide is or comprises the sequence set forth in SEQ ID NO: 11. In some embodiments, the recombinant polypeptide comprises a sequence at one or more amino acid positions (amino acid positions relative to SEQ ID NO: 55), e.g., 13, 18, 20, 26, 69, 70, 80, 138, 142, 144, 152, 190, 215, 242, 243, 244, 246, 400, 401, 402, 417, 440, 452, 477, 484, 501, 570, 614, 655, 681, 682, 683, 684, 685, 70 or comprises an amino acid sequence having at least or about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the sequence of SEQ ID NO: 11, such as a sequence containing substitutions, deletions, and / or insertions at positions 1, 716, 888, 982, 1027, 1118, and / or 1176, or any combination thereof. In some embodiments, the recombinant polypeptide is or comprises a variant of SEQ ID NO: 11, the variants being S13I, L18F, T20N, P26S, Δ69-70(ΔHV), D80A, D138Y, G142D, Δ144(ΔY), W152C, R190S, D215G, Δ242-244(ΔLAL), R246I, Δ400-402(ΔFVI), K417T, K417N, N440K, L and any one, two, three, four, five or more of the mutations selected from the group consisting of 452R, S477N, S477G, E484K, E484Q, N501Y, A570D, D614G, H655Y, P681H, P681R, R682G, R683S, R685G, A701V, T716I, F888L, S982A, T1027I, D1118H, and V1176F, or any combination thereof.

[0185] In some embodiments, the recombinant polypeptide is or comprises the sequence set forth in SEQ ID NO: 12. In some embodiments, the recombinant polypeptide comprises a sequence at one or more amino acid positions (amino acid positions relative to SEQ ID NO: 55), e.g., 13, 18, 20, 26, 69, 70, 80, 138, 142, 144, 152, 190, 215, 242, 243, 244, 246, 400, 401, 402, 417, 440, 452, 477, 484, 501, 570, 614, 655, 681, 682, 683, 684, 685, 70 or comprises an amino acid sequence having at least or about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the sequence of SEQ ID NO: 12, such as a sequence containing substitutions, deletions, and / or insertions at positions 1, 716, 888, 982, 1027, 1118, and / or 1176, or any combination thereof. In some embodiments, the recombinant polypeptide is or comprises a variant of SEQ ID NO: 12, the variants being S13I, L18F, T20N, P26S, Δ69-70(ΔHV), D80A, D138Y, G142D, Δ144(ΔY), W152C, R190S, D215G, Δ242-244(ΔLAL), R246I, Δ400-402(ΔFVI), K417T, K417N, N440K, L and any one, two, three, four, five or more of the mutations selected from the group consisting of 452R, S477N, S477G, E484K, E484Q, N501Y, A570D, D614G, H655Y, P681H, P681R, R682G, R683S, R685G, A701V, T716I, F888L, S982A, T1027I, D1118H, and V1176F, or any combination thereof.

[0186] In some embodiments, the recombinant polypeptide is or comprises the sequence set forth in SEQ ID NO: 13. In some embodiments, the recombinant polypeptide comprises a sequence at one or more amino acid positions (amino acid positions relative to SEQ ID NO: 55), e.g., 13, 18, 20, 26, 69, 70, 80, 138, 142, 144, 152, 190, 215, 242, 243, 244, 246, 400, 401, 402, 417, 440, 452, 477, 484, 501, 570, 614, 655, 681, 682, 683, 684, 685, 70 or comprises an amino acid sequence having at least or about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the sequence of SEQ ID NO: 13, such as a sequence containing substitutions, deletions, and / or insertions at positions 1, 716, 888, 982, 1027, 1118, and / or 1176, or any combination thereof. In some embodiments, the recombinant polypeptide is or comprises a variant of SEQ ID NO: 13, the variants being S13I, L18F, T20N, P26S, Δ69-70(ΔHV), D80A, D138Y, G142D, Δ144(ΔY), W152C, R190S, D215G, Δ242-244(ΔLAL), R246I, Δ400-402(ΔFVI), K417T, K417N, N440K, L and any one, two, three, four, five or more of the mutations selected from the group consisting of 452R, S477N, S477G, E484K, E484Q, N501Y, A570D, D614G, H655Y, P681H, P681R, R682G, R683S, R685G, A701V, T716I, F888L, S982A, T1027I, D1118H, and V1176F, or any combination thereof.

[0187] In some embodiments, the recombinant polypeptide is or comprises the sequence set forth in SEQ ID NO: 14. In some embodiments, the recombinant polypeptide comprises a sequence at one or more amino acid positions (amino acid positions relative to SEQ ID NO: 55), e.g., 13, 18, 20, 26, 69, 70, 80, 138, 142, 144, 152, 190, 215, 242, 243, 244, 246, 400, 401, 402, 417, 440, 452, 477, 484, 501, 570, 614, 655, 681, 682, 683, 684, 685, 70 or comprises an amino acid sequence having at least or about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the sequence of SEQ ID NO: 14, such as a sequence containing substitutions, deletions, and / or insertions at positions 1, 716, 888, 982, 1027, 1118, and / or 1176, or any combination thereof. In some embodiments, the recombinant polypeptide is or comprises a variant of SEQ ID NO: 14, the variants being S13I, L18F, T20N, P26S, Δ69-70(ΔHV), D80A, D138Y, G142D, Δ144(ΔY), W152C, R190S, D215G, Δ242-244(ΔLAL), R246I, Δ400-402(ΔFVI), K417T, K417N, N440K, L and any one, two, three, four, five or more of the mutations selected from the group consisting of 452R, S477N, S477G, E484K, E484Q, N501Y, A570D, D614G, H655Y, P681H, P681R, R682G, R683S, R685G, A701V, T716I, F888L, S982A, T1027I, D1118H, and V1176F, or any combination thereof.

[0188] In some embodiments, the recombinant polypeptide is or comprises the sequence set forth in SEQ ID NO: 15. In some embodiments, the recombinant polypeptide comprises a sequence at one or more amino acid positions (amino acid positions relative to SEQ ID NO: 55), e.g., 13, 18, 20, 26, 69, 70, 80, 138, 142, 144, 152, 190, 215, 242, 243, 244, 246, 400, 401, 402, 417, 440, 452, 477, 484, 501, 570, 614, 655, 681, 682, 683, 684, 685, 701, 710, 720, 730, 740, 750, 760, 770, 781, 790, 801, 812, 814, 820, 822, 824, 826, 828, 829, 830, 831, 832, 833, 834, 835, 836, 837, 838, 839, 840, 841, 842, 843, 844, 845, 846, 847, 848, 849, 850, 851, 852, 853, 854, 855, 856, 857, 858, 859, 860, 861, 862, 863, 864, 865, 866, 867, 8 15, such as a sequence comprising substitutions, deletions, and / or insertions at positions 716, 888, 982, 1027, 1118, and / or 1176, or any combination thereof. In some embodiments, the recombinant polypeptide is or comprises a variant of SEQ ID NO: 15, the variants being S13I, L18F, T20N, P26S, Δ69-70(ΔHV), D80A, D138Y, G142D, Δ144(ΔY), W152C, R190S, D215G, Δ242-244(ΔLAL), R246I, Δ400-402(ΔFVI), K417T, K417N, N440K, L and any one, two, three, four, five or more of the mutations selected from the group consisting of 452R, S477N, S477G, E484K, E484Q, N501Y, A570D, D614G, H655Y, P681H, P681R, R682G, R683S, R685G, A701V, T716I, F888L, S982A, T1027I, D1118H, and V1176F, or any combination thereof.

[0189] In some embodiments, the recombinant polypeptide is or comprises the sequence set forth in SEQ ID NO: 16. In some embodiments, the recombinant polypeptide comprises a sequence at one or more amino acid positions (amino acid positions relative to SEQ ID NO: 55), e.g., 13, 18, 20, 26, 69, 70, 80, 138, 142, 144, 152, 190, 215, 242, 243, 244, 246, 400, 401, 402, 417, 440, 452, 477, 484, 501, 570, 614, 655, 681, 682, 683, 684, 685, 70 or comprises an amino acid sequence having at least or about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the sequence of SEQ ID NO: 16, such as a sequence containing substitutions, deletions, and / or insertions at positions 1, 716, 888, 982, 1027, 1118, and / or 1176, or any combination thereof. In some embodiments, the recombinant polypeptide is or comprises a variant of SEQ ID NO: 16, the variants being S13I, L18F, T20N, P26S, Δ69-70(ΔHV), D80A, D138Y, G142D, Δ144(ΔY), W152C, R190S, D215G, Δ242-244(ΔLAL), R246I, Δ400-402(ΔFVI), K417T, K417N, N440K, L and any one, two, three, four, five or more of the mutations selected from the group consisting of 452R, S477N, S477G, E484K, E484Q, N501Y, A570D, D614G, H655Y, P681H, P681R, R682G, R683S, R685G, A701V, T716I, F888L, S982A, T1027I, D1118H, and V1176F, or any combination thereof.

[0190] In some embodiments, the recombinant polypeptide is or comprises the sequence set forth in SEQ ID NO: 17. In some embodiments, the recombinant polypeptide comprises a sequence at one or more amino acid positions (amino acid positions relative to SEQ ID NO: 55), e.g., 13, 18, 20, 26, 69, 70, 80, 138, 142, 144, 152, 190, 215, 242, 243, 244, 246, 400, 401, 402, 417, 440, 452, 477, 484, 501, 570, 614, 655, 681, 682, 683, 684, 685, 70 or comprises an amino acid sequence having at least or about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the sequence of SEQ ID NO: 17, such as a sequence containing substitutions, deletions, and / or insertions at positions 1, 716, 888, 982, 1027, 1118, and / or 1176, or any combination thereof. In some embodiments, the recombinant polypeptide is or comprises a variant of SEQ ID NO: 17, the variants being S13I, L18F, T20N, P26S, Δ69-70(ΔHV), D80A, D138Y, G142D, Δ144(ΔY), W152C, R190S, D215G, Δ242-244(ΔLAL), R246I, Δ400-402(ΔFVI), K417T, K417N, N440K, L and any one, two, three, four, five or more of the mutations selected from the group consisting of 452R, S477N, S477G, E484K, E484Q, N501Y, A570D, D614G, H655Y, P681H, P681R, R682G, R683S, R685G, A701V, T716I, F888L, S982A, T1027I, D1118H, and V1176F, or any combination thereof.

[0191] In some embodiments, the recombinant polypeptide is or comprises the sequence set forth in SEQ ID NO: 18. In some embodiments, the recombinant polypeptide comprises a sequence at one or more amino acid positions (amino acid positions relative to SEQ ID NO: 55), e.g., 13, 18, 20, 26, 69, 70, 80, 138, 142, 144, 152, 190, 215, 242, 243, 244, 246, 400, 401, 402, 417, 440, 452, 477, 484, 501, 570, 614, 655, 681, 682, 683, 684, 685, 70 or comprises an amino acid sequence having at least or about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the sequence of SEQ ID NO: 18, such as a sequence containing substitutions, deletions, and / or insertions at positions 1, 716, 888, 982, 1027, 1118, and / or 1176, or any combination thereof. In some embodiments, the recombinant polypeptide is or comprises a variant of SEQ ID NO: 18, the variants being S13I, L18F, T20N, P26S, Δ69-70(ΔHV), D80A, D138Y, G142D, Δ144(ΔY), W152C, R190S, D215G, Δ242-244(ΔLAL), R246I, Δ400-402(ΔFVI), K417T, K417N, N440K, L and any one, two, three, four, five or more of the mutations selected from the group consisting of 452R, S477N, S477G, E484K, E484Q, N501Y, A570D, D614G, H655Y, P681H, P681R, R682G, R683S, R685G, A701V, T716I, F888L, S982A, T1027I, D1118H, and V1176F, or any combination thereof.

[0192] In some embodiments, the recombinant polypeptide is or comprises the sequence set forth in SEQ ID NO: 19. In some embodiments, the recombinant polypeptide comprises a sequence at one or more amino acid positions (amino acid positions relative to SEQ ID NO: 55), e.g., 13, 18, 20, 26, 69, 70, 80, 138, 142, 144, 152, 190, 215, 242, 243, 244, 246, 400, 401, 402, 417, 440, 452, 477, 484, 501, 570, 614, 655, 681, 682, 683, 684, 685, 70 or comprises an amino acid sequence having at least or about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the sequence of SEQ ID NO: 19, such as a sequence containing substitutions, deletions, and / or insertions at positions 1, 716, 888, 982, 1027, 1118, and / or 1176, or any combination thereof. In some embodiments, the recombinant polypeptide is or comprises a variant of SEQ ID NO: 19, the variants being S13I, L18F, T20N, P26S, Δ69-70(ΔHV), D80A, D138Y, G142D, Δ144(ΔY), W152C, R190S, D215G, Δ242-244(ΔLAL), R246I, Δ400-402(ΔFVI), K417T, K417N, N440K, L and any one, two, three, four, five or more of the mutations selected from the group consisting of 452R, S477N, S477G, E484K, E484Q, N501Y, A570D, D614G, H655Y, P681H, P681R, R682G, R683S, R685G, A701V, T716I, F888L, S982A, T1027I, D1118H, and V1176F, or any combination thereof.

[0193] In some embodiments, the recombinant polypeptide is or comprises the sequence set forth in SEQ ID NO: 20. In some embodiments, the recombinant polypeptide comprises a sequence at one or more amino acid positions (amino acid positions relative to SEQ ID NO: 55), e.g., 13, 18, 20, 26, 69, 70, 80, 138, 142, 144, 152, 190, 215, 242, 243, 244, 246, 400, 401, 402, 417, 440, 452, 477, 484, 501, 570, 614, 655, 681, 682, 683, 684, 685, 70 or comprises an amino acid sequence having at least or about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the sequence of SEQ ID NO: 20, such as a sequence containing substitutions, deletions, and / or insertions at positions 1, 716, 888, 982, 1027, 1118, and / or 1176, or any combination thereof. In some embodiments, the recombinant polypeptide is or comprises a variant of SEQ ID NO: 20, the variants being S13I, L18F, T20N, P26S, Δ69-70(ΔHV), D80A, D138Y, G142D, Δ144(ΔY), W152C, R190S, D215G, Δ242-244(ΔLAL), R246I, Δ400-402(ΔFVI), K417T, K417N, N440K, L and any one, two, three, four, five or more of the mutations selected from the group consisting of 452R, S477N, S477G, E484K, E484Q, N501Y, A570D, D614G, H655Y, P681H, P681R, R682G, R683S, R685G, A701V, T716I, F888L, S982A, T1027I, D1118H, and V1176F, or any combination thereof.

[0194] In some embodiments, the recombinant polypeptide is or comprises the sequence set forth in SEQ ID NO: 21. In some embodiments, the recombinant polypeptide comprises a sequence at one or more amino acid positions (amino acid positions relative to SEQ ID NO: 55), e.g., 13, 18, 20, 26, 69, 70, 80, 138, 142, 144, 152, 190, 215, 242, 243, 244, 246, 400, 401, 402, 417, 440, 452, 477, 484, 501, 570, 614, 655, 681, 682, 683, 684, 685, 70 or comprises an amino acid sequence having at least or about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the sequence of SEQ ID NO: 21, such as a sequence containing substitutions, deletions, and / or insertions at positions 1, 716, 888, 982, 1027, 1118, and / or 1176, or any combination thereof. In some embodiments, the recombinant polypeptide is or comprises a variant of SEQ ID NO: 21, the variants being S13I, L18F, T20N, P26S, Δ69-70(ΔHV), D80A, D138Y, G142D, Δ144(ΔY), W152C, R190S, D215G, Δ242-244(ΔLAL), R246I, Δ400-402(ΔFVI), K417T, K417N, N440K, L and any one, two, three, four, five or more of the mutations selected from the group consisting of 452R, S477N, S477G, E484K, E484Q, N501Y, A570D, D614G, H655Y, P681H, P681R, R682G, R683S, R685G, A701V, T716I, F888L, S982A, T1027I, D1118H, and V1176F, or any combination thereof.

[0195] In some embodiments, the recombinant polypeptide is or comprises the sequence set forth in SEQ ID NO: 22. In some embodiments, the recombinant polypeptide comprises a sequence at one or more amino acid positions (amino acid positions relative to SEQ ID NO: 55), e.g., 13, 18, 20, 26, 69, 70, 80, 138, 142, 144, 152, 190, 215, 242, 243, 244, 246, 400, 401, 402, 417, 440, 452, 477, 484, 501, 570, 614, 655, 681, 682, 683, 684, 685, 70 or comprises an amino acid sequence having at least or about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the sequence of SEQ ID NO: 22, such as a sequence containing substitutions, deletions, and / or insertions at positions 1, 716, 888, 982, 1027, 1118, and / or 1176, or any combination thereof. In some embodiments, the recombinant polypeptide is or comprises a variant of SEQ ID NO: 22, the variants being S13I, L18F, T20N, P26S, Δ69-70(ΔHV), D80A, D138Y, G142D, Δ144(ΔY), W152C, R190S, D215G, Δ242-244(ΔLAL), R246I, Δ400-402(ΔFVI), K417T, K417N, N440K, L and any one, two, three, four, five or more of the mutations selected from the group consisting of 452R, S477N, S477G, E484K, E484Q, N501Y, A570D, D614G, H655Y, P681H, P681R, R682G, R683S, R685G, A701V, T716I, F888L, S982A, T1027I, D1118H, and V1176F, or any combination thereof.

[0196] In some embodiments, the recombinant polypeptide is or comprises the sequence set forth in SEQ ID NO: 23. In some embodiments, the recombinant polypeptide comprises a sequence at one or more amino acid positions (amino acid positions relative to SEQ ID NO: 55), e.g., 13, 18, 20, 26, 69, 70, 80, 138, 142, 144, 152, 190, 215, 242, 243, 244, 246, 400, 401, 402, 417, 440, 452, 477, 484, 501, 570, 614, 655, 681, 682, 683, 684, 685, 70 or comprises an amino acid sequence having at least or about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the sequence of SEQ ID NO: 23, such as a sequence containing substitutions, deletions, and / or insertions at positions 1, 716, 888, 982, 1027, 1118, and / or 1176, or any combination thereof. In some embodiments, the recombinant polypeptide is or comprises a variant of SEQ ID NO: 23, the variants being S13I, L18F, T20N, P26S, Δ69-70(ΔHV), D80A, D138Y, G142D, Δ144(ΔY), W152C, R190S, D215G, Δ242-244(ΔLAL), R246I, Δ400-402(ΔFVI), K417T, K417N, N440K, L and any one, two, three, four, five or more of the mutations selected from the group consisting of 452R, S477N, S477G, E484K, E484Q, N501Y, A570D, D614G, H655Y, P681H, P681R, R682G, R683S, R685G, A701V, T716I, F888L, S982A, T1027I, D1118H, and V1176F, or any combination thereof.

[0197] In some embodiments, the recombinant polypeptide is or comprises the sequence set forth in SEQ ID NO: 24. In some embodiments, the recombinant polypeptide comprises a sequence at one or more amino acid positions (amino acid positions relative to SEQ ID NO: 55), e.g., 13, 18, 20, 26, 69, 70, 80, 138, 142, 144, 152, 190, 215, 242, 243, 244, 246, 400, 401, 402, 417, 440, 452, 477, 484, 501, 570, 614, 655, 681, 682, 683, 684, 685, 70 or comprises an amino acid sequence having at least or about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the sequence of SEQ ID NO: 24, such as a sequence containing substitutions, deletions, and / or insertions at positions 1, 716, 888, 982, 1027, 1118, and / or 1176, or any combination thereof. In some embodiments, the recombinant polypeptide is or comprises a variant of SEQ ID NO:24, wherein the variants are S13I, L18F, T20N, P26S, Δ69-70(ΔHV), D80A, D138Y, G142D, Δ144(ΔY), W152C, R190S, D215G, Δ242-244(ΔLAL), R246I, Δ400-402(ΔFVI), K417T, K417N, N440K, L and any one, two, three, four, five or more of the mutations selected from the group consisting of 452R, S477N, S477G, E484K, E484Q, N501Y, A570D, D614G, H655Y, P681H, P681R, R682G, R683S, R685G, A701V, T716I, F888L, S982A, T1027I, D1118H, and V1176F, or any combination thereof.

[0198] In some embodiments, the recombinant polypeptide is or comprises the sequence set forth in SEQ ID NO: 25. In some embodiments, the recombinant polypeptide comprises a sequence at one or more amino acid positions (amino acid positions relative to SEQ ID NO: 55), e.g., 13, 18, 20, 26, 69, 70, 80, 138, 142, 144, 152, 190, 215, 242, 243, 244, 246, 400, 401, 402, 417, 440, 452, 477, 484, 501, 570, 614, 655, 681, 682, 683, 684, 685, 70 or comprises an amino acid sequence having at least or about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the sequence of SEQ ID NO: 25, such as a sequence containing substitutions, deletions, and / or insertions at positions 1, 716, 888, 982, 1027, 1118, and / or 1176, or any combination thereof. In some embodiments, the recombinant polypeptide is or comprises a variant of SEQ ID NO: 25, the variants being S13I, L18F, T20N, P26S, Δ69-70(ΔHV), D80A, D138Y, G142D, Δ144(ΔY), W152C, R190S, D215G, Δ242-244(ΔLAL), R246I, Δ400-402(ΔFVI), K417T, K417N, N440K, L and any one, two, three, four, five or more of the mutations selected from the group consisting of 452R, S477N, S477G, E484K, E484Q, N501Y, A570D, D614G, H655Y, P681H, P681R, R682G, R683S, R685G, A701V, T716I, F888L, S982A, T1027I, D1118H, and V1176F, or any combination thereof.

[0199] In some embodiments, the recombinant polypeptide is or comprises the sequence set forth in SEQ ID NO: 26. In some embodiments, the recombinant polypeptide is or comprises an amino acid sequence having at least or about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the sequence of SEQ ID NO: 26, including sequences containing substitutions, deletions, and / or insertions at one or more amino acid positions of SEQ ID NO: 26.

[0200] As noted above, in some embodiments, the recombinant polypeptides provided herein can not only associate to form trimers, but also aggregate or aggregate to produce proteins comprising multiple recombinant polypeptides. In some embodiments, the proteins formed have a macrostructure. In some cases, the macrostructure can provide structural stability to the coronavirus virus antigen or immunogenic recombinant polypeptide, thereby allowing access to potential antigenic sites that can enhance an immune response.

[0201] In some embodiments, the trimerized recombinant polypeptides aggregate to form a protein comprising a plurality of trimerized recombinant polypeptides, hi some embodiments, the plurality of trimerized recombinant polypeptides forms a protein having a macrostructure.

[0202] In some embodiments, a protein described herein comprising multiple recombinant polypeptides is an immunogen. In some embodiments, a protein described herein comprising multiple recombinant polypeptides is comprised in a nanoparticle. For example, in some embodiments, the protein is directly linked to a nanoparticle, e.g., a protein nanoparticle. In some embodiments, the protein is indirectly linked to a nanoparticle. In some embodiments, a protein described herein comprising multiple recombinant polypeptides is comprised in a virus-like particle (VLP).

[0203] In some embodiments, provided herein are complexes comprising recombinant polypeptides selected from the group consisting of SEQ ID NOs: 1-26, or fragments, variants, or mutants thereof, in any suitable combination. In some embodiments, provided are complexes comprising trimers of recombinant polypeptides selected from the group consisting of SEQ ID NOs: 1-26, or fragments, variants, or mutants thereof, wherein the recombinant polypeptides are trimerized via inter-polypeptide disulfide bonds to form the trimers.

[0204] In some embodiments, provided herein are fusion proteins comprising multiple recombinant polypeptides, each recombinant polypeptide comprising, from amino terminus to carboxy terminus, a) a first region comprising a portion of a coronavirus spike protein ectodomain, the region preceding the coronavirus spike protein receptor-binding domain (RBD) of a first coronavirus in the non-chimeric coronavirus spike protein; b) a second region comprising the coronavirus spike protein receptor-binding domain (RBD) of a second coronavirus different from the first coronavirus; and c) a C-terminal propeptide of collagen with which the C-terminal propeptide of the recombinant polypeptide forms an inter-polypeptide disulfide bond. In some embodiments, the fusion protein further comprises a third region between the second region and the C-terminal propeptide of collagen. In some embodiments, the third region comprises an S1 domain of a third coronavirus, which is the same or different from the first coronavirus or the second coronavirus. In some embodiments, the third region comprises an S2 domain of a fourth coronavirus, which is the same or different from the first coronavirus, the second coronavirus, or the fourth coronavirus. In some embodiments, the first region comprises the N-terminal domain (NTD) of the first coronavirus. In some embodiments, the first region comprises one or more amino acid residues that differ from corresponding amino acid residue(s) in the second coronavirus. In some embodiments, the second region comprises one or more amino acid residues that differ from corresponding amino acid residue(s) in the first coronavirus. In some embodiments, the first coronavirus and the second coronavirus are different variants or strains of the same coronavirus. In some embodiments, the first region comprises the NTD of the first coronavirus and the second region comprises the RBD of the second coronavirus, and the first coronavirus and the second coronavirus are different variants of SARS-CoV-2.In some embodiments, the first coronavirus and the second coronavirus are independently selected from the group consisting of SARS-CoV-2 viruses of the B.1.526, B.1.1.143, P.2, B.1.351, P.1, B.1.1.7, B.1.617, and A.23.1 lineages.

[0205] In some embodiments, provided herein are trimeric fusion proteins comprising three recombinant polypeptides, each recombinant polypeptide comprising, from amino terminus to carboxy terminus: a) a first region comprising the coronavirus spike protein N-terminal domain (NTD) of SARS-CoV-2 of the B.1.526 lineage; b) a second region comprising the coronavirus spike protein receptor-binding domain (RBD) of SARS-CoV-2 of the B.1.351 lineage; and c) a C-terminal propeptide of collagen, wherein the C-terminal propeptides of the recombinant polypeptides form inter-polypeptide disulfide bonds.

[0206] In some embodiments, provided herein are methods for preventing coronavirus infection in a mammal, the methods comprising immunizing the mammal with an effective amount of a fusion protein disclosed herein. In some embodiments, neutralizing antibodies against a first coronavirus and a second coronavirus are generated in the mammal. In some embodiments, the first coronavirus and the second coronavirus are different variants of SARS-CoV-2, and the neutralizing antibodies generated in the mammal neutralize two or more of SARS-CoV-2 viruses of the B.1.526, B.1.1.143, P.2, B.1.351, P.1, B.1.1.7, B.1.617, and A.23.1 lineages. In some embodiments, the neutralizing antibodies generated in the mammal neutralize three or more of SARS-CoV-2 viruses of the B.1.526, B.1.1.143, P.2, B.1.351, P.1, B.1.1.7, B.1.617, and A.23.1 lineages. In some embodiments, the method includes immunizing the mammal with two or more administrations of the fusion protein. In some embodiments, the fusion protein is administered as a booster dose following one or more administrations of an immunogen comprising a spike protein peptide comprising the NTD and RBD from the same SARS-CoV-2 variant.

[0207] In some embodiments, provided herein are engineered fusion polypeptides derived from or modified from the spike (S) glycoprotein of coronaviruses, such as SARS-CoV-1 and SARS-CoV-2. In some embodiments, compared to the wild-type S protein sequence of a coronavirus, the fusion polypeptides disclosed herein may be stabilized in a pre-fusion conformation. In some embodiments, fusion to a trimerization domain may prevent the S protein peptide from forming a linear helix in the fusion protein (e.g., similar to that which occurs during the membrane fusion process). For example, the cryo-EM structure of an S-trimer subunit vaccine candidate shows that it adopts a predominantly sealed pre-fusion state, unlike the full-length wild-type spike protein, which forms both pre- and post-fusion states in the presence of detergent. (Ma et al., J Virol (2021) doi:10.1128 / JVI.00194-21). In some embodiments, the fusion protein may include a modified soluble S sequence with a truncation of the heptad repeat 2 region (HR2), in addition to a modification(s) that inactivates the S1 / S2 cleavage site; a mutation(s) in the turn region between the heptad repeat 1 (HR1) and central helix (CH) regions that prevents HR1 and CH from forming a linear helix; and / or a stabilizing mutation. In some embodiments, the fusion protein herein may, but need not, include one or more mutations, such as K986G / V987G, K986P / V987P, K986G / V987P, or K986P / V987G, which are believed to stabilize the spike protein in the pre-fusion state. In some embodiments, mutations such as K986G / V987G, K986P / V987P, K986G / V987P or K986P / V987G are not required to stabilize the fusion polypeptides disclosed herein that include a Trimer-Tag® trimerization domain.

[0208] In some of these embodiments, the mutation that inactivates the S1 / S2 cleavage site can include a substitution of RRAR (682-685 of SEQ ID NO:55) with GSAG (SEQ ID NO:60), and the mutation in the turn region can include a double mutation K986G / V987G, K986P / V987P, K986G / V987P, or K986P / V987G. In some embodiments, the truncation of HR2 involves a deletion of one or more of the residues set forth in SEQ ID NO:65 at the C-terminus of the wild-type soluble S sequence. In some embodiments, the immunogenic polypeptide can further include (a) one or more proline or glycine substitutions and / or (b) an insertion of one or more amino acid residues within the region of HR1 that interacts with HR2. In some of these embodiments, the immunogenic polypeptide can have one or more substitutions selected from A942P, S943P, A944P, A942G, S943G, and A944G. In some of these embodiments, the insertion may be a G or GS insertion between any residues A942 to A944.

[0209] 2. Polynucleotides and Vectors Also provided are polynucleotides (nucleic acid molecules) encoding the coronavirus antigens or immunogens and recombinant polypeptides provided herein, as well as vectors for genetically engineering cells to express such coronavirus antigens or immunogens and recombinant polypeptides.

[0210] In some embodiments, polynucleotides are provided that encode the recombinant polypeptides provided herein. In some aspects, the polynucleotides comprise a single nucleic acid sequence, such as a nucleic acid sequence encoding the recombinant polypeptide. In other examples, the polynucleotides comprise a first nucleic acid sequence that encodes a recombinant polypeptide of a particular coronavirus virus antigen or immunogen and a second nucleic acid sequence that encodes a recombinant polypeptide comprising a different coronavirus virus antigen or immunogen.

[0211] In some embodiments, the polynucleotide encoding the recombinant polypeptide comprises at least one promoter operably linked to control expression of the recombinant polypeptide, hi some embodiments, the polynucleotide comprises two, three, or more promoters operably linked to control expression of the recombinant polypeptide.

[0212] In some embodiments, for example, when a polynucleotide comprises two or more nucleic acid coding sequences, such as sequences encoding recombinant polypeptides comprising different coronavirus virus antigens or immunogens, at least one promoter is operably linked to control expression of the two or more nucleic acid sequences, hi some embodiments, the polynucleotide comprises two, three, or more promoters operably linked to control expression of the recombinant polypeptides.

[0213] In some embodiments, expression of the recombinant polypeptide(s) is inducible or conditional. Thus, in some aspects, the polynucleotide encoding the recombinant polypeptide(s) comprises a conditional promoter, enhancer, or transactivator. In some such aspects, the conditional promoter, enhancer, or transactivator is an inducible promoter, enhancer, or transactivator, or a repressible promoter, enhancer, or transactivator. For example, in some embodiments, an inducible or conditional promoter can be used to restrict expression of the recombinant polypeptide to a particular microenvironment. In some embodiments, expression driven by an inducible or conditional promoter is regulated by exposure to an exogenous factor, such as heat, radiation, or a drug.

[0214] When a polynucleotide comprises multiple nucleic acid sequences encoding recombinant polypeptides, the polynucleotide may further comprise a nucleic acid sequence encoding a peptide between one or more nucleic acid sequences. In some cases, the nucleic acid located between the nucleic acid sequences encodes a peptide that separates the translation products of the nucleic acid sequences during or after translation. In some embodiments, the peptide comprises an internal ribosome entry site (IRES), a self-cleaving peptide, or a peptide that causes ribosome skipping, such as a T2A peptide.

[0215] In some embodiments, a polynucleotide encoding a recombinant polypeptide(s) is introduced into a composition comprising cultured cells (e.g., host cells), such as by retroviral transduction, transfection, or transformation. In some embodiments, this may allow for expression (e.g., production) of the recombinant polypeptide. In some embodiments, the expressed recombinant polypeptide is purified.

[0216] In some embodiments, the polynucleotides (nucleic acid molecules) provided herein encode a coronavirus virus antigen or immunogen described herein. In some embodiments, the polynucleotides (nucleic acid molecules) provided herein encode a coronavirus virus antigen or immunogen described herein, such as a recombinant polypeptide comprising a coronavirus S protein peptide.

[0217] Also provided are vectors or constructs comprising the nucleic acid molecules described herein. In some embodiments, the vectors or constructs comprise one or more promoters operably linked to nucleic acid molecules encoding recombinant polypeptides to drive their expression. In some embodiments, the promoters are operably linked to one or more nucleic acid molecules, for example, nucleic acid molecules encoding recombinant polypeptides comprising different coronavirus virus antigens or immunogens.

[0218] In some embodiments, the vector is a viral vector. In some embodiments, the viral vector is a retroviral vector. In some embodiments, the retroviral vector is a lentiviral vector. In some embodiments, the retroviral vector is a gamma retroviral vector.

[0219] In some embodiments, a vector or construct comprises a single promoter that drives expression of one or more nucleic acid molecules of a polynucleotide. In some embodiments, such a promoter can be multicistronic (bicistronic or tricistronic; see, e.g., U.S. Pat. No. 6,060,273). For example, in some embodiments, a transcription unit can be engineered as a bicistronic unit containing an IRES (internal ribosome entry site), thereby allowing co-expression of gene products (e.g., encoding different recombinant polypeptides) by messages from a single promoter. In some embodiments, the vectors provided herein are bicistronic, allowing the vector to contain and express two nucleic acid sequences. In some embodiments, the vectors provided herein are tricistronic, allowing the vector to contain and express three nucleic acid sequences.

[0220] In some embodiments, a single promoter directs the expression of RNA containing two or three genes (e.g., encoding a chimeric signaling receptor and encoding a recombinant receptor) within a single open reading frame (ORF), separated from each other by a sequence encoding a self-cleaving peptide (e.g., a 2A sequence) or a protease recognition site (e.g., furin). Thus, the ORF encodes a single polypeptide that is processed into individual proteins during translation (in the case of 2A) or post-translation. In some cases, a peptide such as T2A allows the ribosome to skip synthesis of the peptide attached at the C-terminus of the 2A element (ribosomal skipping), resulting in a separation between the end of the 2A sequence and the next peptide downstream (see, e.g., de Felipe. Genetic Vaccines and Ther. 2:13 (2004) and de Felipe et al. Traffic 5:616-626 (2004)). Many 2A elements are known in the art. Examples of 2A sequences that can be used in the methods and nucleic acids disclosed herein include, but are not limited to, 2A sequences from foot and mouth disease virus (F2A), equine rhinitis A virus (E2A), Thosea asigna virus (T2A), and porcine teschovirus-1 (P2A), as described in U.S. Patent Application Publication No. 20070116690.

[0221] In some embodiments, the vector is comprised in a virus. In some embodiments, the virus is a pseudovirus. In some embodiments, the virus is a virus-like particle. In some embodiments, the vector is comprised in a cell. In some embodiments, the virus or cell containing the vector comprises a recombinant genome.

[0222] III. Immunogenic Compositions and Formulations In some embodiments, provided herein is an immunogenic composition comprising a trimer of a recombinant polypeptide comprising a sequence selected from the group consisting of SEQ ID NOs: 1-26, or a combination of any two or more of the trimers. In some embodiments, provided herein is an immunogenic composition comprising a trimer of a recombinant polypeptide having the sequence set forth in SEQ ID NO: 1.

[0223] In some embodiments, a unit dose of the immunogenic composition may contain about 10 μg to about 100 μg of SARS-CoV-2 antigen, preferably about 25 μg to about 75 μg of SARS-CoV-2 antigen, preferably about 40 μg to about 60 μg of SARS-CoV-2 antigen, or about 50 μg of SARS-CoV-2 antigen. In some embodiments, the dose contains 3 μg of SARS-CoV-2 antigen. In other embodiments, the dose contains 9 μg of SARS-CoV-2 antigen. In further embodiments, the dose contains 30 μg of SARS-CoV-2 antigen.

[0224] In some cases, it may be desirable to combine the disclosed immunogens with other pharmaceutical agents (e.g., vaccines) that induce protective responses against other agents. For example, compositions containing the recombinant coronavirus S antigens described herein, e.g., trimers or proteins, can be administered to a target age group (e.g., infants approximately 1-6 months of age) simultaneously (typically separately) or sequentially with other vaccines recommended by the Advisory Committee on Immunization Practices (ACIP; cdc.gov / vaccines / acip / index.html), such as influenza or varicella-zoster vaccines. Thus, the disclosed immunogens, including the recombinant coronavirus S antigens described herein, can be administered simultaneously or sequentially with vaccines against, for example, hepatitis B (HepB), diphtheria, tetanus, and pertussis (DTaP), pneumococcal virus (PCV), Haemophilus influenzae type b (Hib), polio, influenza, and rotavirus.

[0225] Multivalent or combination vaccines provide protection against multiple pathogens. In some embodiments, multivalent vaccines can protect against multiple strains of the same pathogen. In some embodiments, multivalent vaccines protect against multiple pathogens, such as the combination vaccine Tdap, which protects against strains of tetanus, pertussis, and diphtheria. Multivalent vaccines are highly desirable because they minimize the number of immunizations required to confer protection against multiple pathogens or pathogenic strains, thereby reducing administration costs and increasing reach. This can be particularly useful, for example, when vaccinating newborns and children.

[0226] In some embodiments, a vaccine, e.g., comprising an immunogenic composition described herein, is a multivalent vaccine. In some embodiments, the antigenic material for incorporation into the multivalent vaccine composition is derived from, e.g., a coronavirus strain or type described herein (see, e.g., Section I). The antigens for incorporation into the multivalent vaccine composition can be derived from one or multiple strains of coronavirus, e.g., 2-5 strains, to confer a broader spectrum of protection. In one embodiment, the antigens for incorporation into the multivalent vaccine composition are derived from multiple strains of coronavirus. Other useful antigens include live, attenuated, and inactivated viruses such as inactivated poliovirus (Jiang et al., J. Biol. Stand., (1986) 14:103-9), attenuated strains of hepatitis A virus (Bradley et al., J. Med. Virol., (1984) 14:373-86), attenuated measles virus (James et al., N. Engl. J. Med., (1995) 332:1262-6), and epitopes of pertussis virus (e.g., ACEL-IMUNE acellular DTP, Wyeth-Lederle Vaccines and Pediatrics).

[0227] In some aspects, the vaccines provided herein are universal vaccines. In some embodiments, a universal vaccine is a vaccine that protects against multiple strains of the same virus, such as multiple strains of coronavirus. The development of an effective universal coronavirus vaccine would reduce the cost and effort required, for example, with seasonal vaccine formulations, and enable stronger pandemic preparedness.

[0228] In some embodiments, a universal vaccine is a vaccine that is composed of multiple epitopes from different virus strains. In some embodiments, a universal vaccine is composed of a single epitope that is conserved in different virus strains. For example, a universal vaccine can be based on a relatively conserved domain(s) of the S protein.

[0229] Immunogenic compositions comprising the disclosed immunogens (e.g., nucleic acid molecules encoding the disclosed recombinant coronavirus S antigens or protomers of the disclosed recombinant coronavirus S antigens) and a pharmaceutically acceptable carrier are also provided. In some embodiments, the immunogenic compositions comprise a trimerized recombinant polypeptide provided herein and, optionally, a pharmaceutically acceptable carrier. In some embodiments, the immunogenic compositions comprise a protein comprising a plurality of trimerized recombinant polypeptides provided herein and, optionally, a pharmaceutically acceptable carrier. In some embodiments, the immunogenic compositions are protein nanoparticles provided herein and, optionally, a pharmaceutically acceptable carrier. In some embodiments, the immunogenic compositions comprise a VLP provided herein and, optionally, a pharmaceutically acceptable carrier. In some embodiments, the immunogenic compositions comprise an isolated nucleic acid provided herein and, optionally, a pharmaceutically acceptable carrier. In some embodiments, the immunogenic compositions comprise a vector provided herein and, optionally, a pharmaceutically acceptable carrier. In some embodiments, the immunogenic compositions comprise a virus provided herein and, optionally, a pharmaceutically acceptable carrier. In some embodiments, the immunogenic composition comprises a pseudovirus provided herein and, optionally, a pharmaceutically acceptable carrier. In some embodiments, the immunogenic composition comprises a cell provided herein and, optionally, a pharmaceutically acceptable carrier. In some embodiments, an immunogenic composition such as those described herein is a vaccine. In some embodiments, the vaccine is a prophylactic vaccine. In some embodiments, the vaccine is a therapeutic vaccine. In some embodiments, the vaccine is a prophylactic and therapeutic vaccine. Such pharmaceutical compositions can be administered to a subject by various modes of administration known to those skilled in the art, for example, intramuscular, intradermal, subcutaneous, intravenous, intraarterial, intraarticular, intraperitoneal, intranasal, sublingual, tonsillar, oropharyngeal, or other parenteral and mucosal routes. In some embodiments, a pharmaceutical composition comprising one or more of the disclosed immunogens is an immunogenic composition.Actual methods for preparing administrable compositions will be known or apparent to those skilled in the art and are described in more detail in publications such as Remington's Pharmaceutical Sciences, 19th Ed., Mack Publishing Company, Easton, Pa., 1995.

[0230] Thus, the immunogens described herein, such as recombinant coronavirus S antigens, e.g., trimers, proteins, can be formulated with pharmaceutically acceptable carriers to help retain biological activity while promoting improved stability during storage within acceptable temperature ranges. Potential carriers include, but are not limited to, physiologically balanced culture media, phosphate-buffered saline, water, emulsions (e.g., oil / water or water / oil emulsions), various types of humectants, cryoprotective additives, or stabilizers, such as proteins, peptides, or hydrolysates (e.g., albumin, gelatin), sugars (e.g., sucrose, lactose, sorbitol), amino acids (e.g., monosodium glutamate), or other protective agents. The resulting aqueous solutions can be packaged for immediate use or lyophilized. Lyophilized preparations are mixed with a sterile solution prior to administration, either in single or multiple doses.

[0231] Formulated compositions, particularly liquid formulations, may contain bacteriostatic agents to prevent or minimize degradation during storage, such as, but not limited to, effective concentrations (typically 1% w / v) of benzyl alcohol, phenol, m-cresol, chlorobutanol, methylparaben, and / or propylparaben. Bacteriostatic agents may be contraindicated for some patients. Thus, lyophilized formulations may be reconstituted in solutions with or without such ingredients.

[0232] The immunogenic compositions of the present disclosure may contain pharmaceutically acceptable vehicle substances necessary to approximate physiological conditions, such as pH adjusting and buffering agents, tonicity adjusting agents, and wetting agents, such as sodium acetate, sodium lactate, sodium chloride, potassium chloride, calcium chloride, sorbitan monolaurate, and triethanolamine oleate. The immunogenic compositions may optionally contain an adjuvant to enhance the host's immune response. Suitable adjuvants include, for example, toll-like receptor (TLR) agonists, alum, AlPO4, alhydrogel, lipid-A and its derivatives or variants, oil emulsions, saponins, neutral liposomes, vaccine and cytokine-containing liposomes, non-ionic block copolymers, and chemokines. Among many other suitable adjuvants known in the art, nonionic block polymers containing polyoxyethylene (POE) and polyxylpropylene (POP), such as POE-POP-POE block copolymer, MPL™ (3-O-deacylated monophosphoryl lipid A; Corixa, Hamilton, Ind.), and IL-12 (Genetics Institute, Cambridge, Mass.) can be used as adjuvants (Newman et al., 1998, Critical Reviews in Therapeutic Drug Carrier Systems 15:89-142). These adjuvants serve to stimulate the immune system in a nonspecific manner, thus providing the advantage of enhancing the immune response to pharmaceuticals. In some embodiments, the immunogenic compositions of the present disclosure can include or be administered with two or more adjuvants. In some embodiments, the immunogenic compositions of the present disclosure can include or be administered with two adjuvants. In some embodiments, the immunogenic compositions of the present disclosure may include or be administered in conjunction with multiple adjuvants, e.g., in some cases, vaccines, including, for example, the immunogenic compositions provided herein, may include or be administered in combination with multiple adjuvants.

[0233] For vaccine compositions, examples of suitable adjuvants include, for example, aluminum hydroxide, lecithin, Freund's adjuvant, MPL™, and IL-12. In some embodiments, the vaccine compositions or nanoparticle immunogens (e.g., SARS-COV-2 vaccine compositions) disclosed herein can be formulated as controlled- or sustained-release formulations. This can be achieved in compositions comprising slow-release polymers, or via microencapsulated delivery systems or bioadhesive gels. Various pharmaceutical compositions can be prepared according to standard procedures well known in the art.

[0234] In some embodiments, the immunogenic compositions of the present disclosure can include an adjuvant formulation comprising a metabolizable oil (e.g., squalene) and alpha-tocopherol in the form of an oil-in-water emulsion, and polyoxyethylene sorbitan monooleate (Tween-80). In some embodiments, the adjuvant formulation can comprise about 2% to about 10% squalene, about 2% to about 10% alpha-tocopherol (e.g., D-α-tocopherol), and about 0.3% to about 3% polyoxyethylene sorbitan monooleate. In some embodiments, the adjuvant formulation can comprise about 5% squalene, about 5% tocopherol, and about 0.4% polyoxyethylene sorbitan monooleate. In some embodiments, immunogenic compositions of the disclosure can include 3 De-O-acylated monophosphoryl lipid A (3D-MPL) and an adjuvant in the form of an oil-in-water emulsion, wherein the adjuvant comprises a metabolizable oil, alpha-tocopherol, and polyoxyethylene sorbitan monooleate. In some embodiments, immunogenic compositions of the disclosure can include QS21 (extract of Quillaja saponaria Molina: fraction 21), 3D-MPL, and an oil-in-water emulsion, wherein the oil-in-water emulsion comprises a metabolizable oil, alpha-tocopherol, and polyoxyethylene sorbitan monooleate. In some embodiments, immunogenic compositions of the disclosure can include QS21, 3D-MPL, and an oil-in-water emulsion, wherein the oil-in-water emulsion has the following composition: squalene, alpha-tocopherol, and a metabolizable oil such as Tween-80. In some embodiments, the immunogenic compositions of the present disclosure may include an adjuvant in the form of a liposomal composition.

[0235] In some embodiments, the immunogenic compositions of the disclosure can include an adjuvant formulation comprising a metabolizable oil (e.g., squalene), polyoxyethylene sorbitan monooleate (Tween-80), and Span 85. In some embodiments, the adjuvant formulation can include about 5% (w / v) squalene, about 0.5% (w / v) polyoxyethylene sorbitan monooleate, and about 0.5% (w / v) Span 85.

[0236] In some embodiments, the immunogenic compositions of the disclosure can include an adjuvant formulation comprising Quillaja saponin, cholesterol, and phospholipids, e.g., in the form of a nanoparticle composition. In some embodiments, the immunogenic compositions of the disclosure can include a mixture of separately purified fractions of Quillaja saponaria Molina, which are then formulated with cholesterol and phospholipids.

[0237] In some embodiments, the immunogenic compositions of the present disclosure may comprise an adjuvant selected from the group consisting of MF59™, Matrix-A™, Matrix-C™, Matrix-M™, AS01, AS02, AS03, and AS04.

[0238] In some embodiments, the immunogenic compositions of the present disclosure can include a Toll-like receptor 9 (TLR9) agonist, which is an 8-35 nucleotide oligonucleotide containing an unmethylated cytidine-phospho-guanosine (CpG or cytosine-phosphate-guanosine) motif. The SARS-CoV-2 antigen and the oligonucleotide are present in the immunogenic composition in an amount effective to stimulate an immune response to the SARS-CoV-2 antigen in a mammalian subject, such as a human subject, in need thereof. TLR9 (CD289) recognizes unmethylated cytidine-phospho-guanosine (CpG) motifs found in microbial DNA, which can be mimicked using synthetic CpG-containing oligodeoxynucleotides (CpG-ODNs). CpG-ODNs are known to enhance antibody production and stimulate T helper 1 (Th1) cell responses (Coffman et al., Immunity, 33:492-503, 2010). Optimal oligonucleotide TLR9 agonists often contain a palindromic sequence following the general formula: 5'-purine-purine-CG-pyrimidine-pyrimidine-3', or 5'-purine-purine-CG-pyrimidine-pyrimidine-CG-3'. U.S. Patent No. 6,589,940, which is incorporated herein by reference in its entirety, is incorporated by reference. In some embodiments, the CpG oligonucleotide is linear. In other embodiments, the CpG oligonucleotide is circular or contains hairpin loop(s). The CpG oligonucleotide can be single-stranded or double-stranded. In some embodiments, the CpG oligonucleotide can contain modifications, including, but not limited to, modifications of the 3'OH or 5'OH group, modifications of the nucleotide base, modifications of the sugar moiety, and modifications of the phosphate group. Modified base(s) can be included in the palindromic sequence of a CpG oligonucleotide so long as the modified base(s) maintain the same specificity relative to their natural complement through Watson-Crick base pairing (e.g., the palindromic portion is still self-complementary). In some embodiments, the CpG oligonucleotide includes a non-standard base. In some embodiments, the CpG oligonucleotide includes a modified nucleoside.In some embodiments, the modified nucleoside is selected from the group consisting of 2'-deoxy-7-deazaguanosine, 2'-deoxy-6-thioguanosine, arabinoguanosine, 2'-deoxy-2'-substituted-arabinoguanosine, and 2'-O-substituted-arabinoguanosine. The CpG oligonucleotide may contain modifications of the phosphate group. For example, in addition to phosphodiester linkages, phosphate modifications include, but are not limited to, methylphosphonates, phosphorothioates, phosphoramidates (bridged or unbridged), phosphotriesters, and phosphorodithioates, which may be used in any combination. Other non-phosphate linkages may also be used. In some embodiments, the oligonucleotide contains only a phosphorothioate backbone. In some embodiments, the oligonucleotide contains only a phosphodiester backbone. In some embodiments, the oligonucleotide contains a combination of phosphate linkages in the phosphate backbone, such as a combination of phosphodiester and phosphorothioate linkages. Oligonucleotides with phosphorothioate backbones may be more immunogenic than oligonucleotides with phosphodiester backbones and are thought to be more resistant to degradation after injection into a host (Braun et al., J Immunol, 141:2084-2089, 1988; and Latimer et al., Mol Immunol, 32:1057-1064, 1995). The CpG oligonucleotides of the present disclosure contain at least one, two, or three internucleotide phosphorothioate ester linkages. In some embodiments, when a plurality of CpG oligonucleotide molecules are present in a pharmaceutical composition containing at least one excipient, both stereoisomers of the phosphorothioate ester linkage are present in the plurality of CpG oligonucleotide molecules. In some embodiments, all of the internucleotide linkages of the CpG oligonucleotide are phosphorothioate linkages; in other methods, the CpG oligonucleotide has a phosphorothioate backbone.

[0239] Any suitable CpG oligodeoxynucleotide (ODN) or their combination can be used as adjuvant in the present disclosure.For example, K-type ODN (also called B-type) encodes multiple CpG motifs on phosphorothioate backbone.K-type ODN has the following sequence:TCCATGGA CG TTCCTGAG CG The use of phosphorothioate nucleotides results in higher resistance to nuclease digestion and significantly longer in vivo half-life compared to native phosphodiester nucleotides. K-type ODNs induce pDC differentiation and TNF-α production, and B cell proliferation and IgM secretion. D-type ODNs (also called A-type) are constructed with a mixed phosphodiester / phosphorothioate backbone, contain a single CpG motif flanked by palindromic sequences, and have poly-G tails (structural motifs that facilitate the formation of concatemers) at the 3' and 5' ends. D-type ODNs have the following sequence: GGTGCAT CG ATGCAGGGGGG. D-type ODN induces pDC maturation and IFN-α secretion, but has no effect on B cells. C-type ODN is similar to K-type in that it is completely composed of phosphorothioate nucleotides, but similar to D-type in that it contains a palindromic CpG motif. C-type ODN has the following sequence: T CG T CG TT CG AA CG A CG It can be based on TTGAT. This class of ODN stimulates B cells to secrete IL-6 and stimulates pDC to produce IFN-α. P-type ODN contains two palindromic sequences, which can form higher-order structures. P-type ODN is composed of the following sequence T CG T CG A CG AT CG G CGCGCG C CGP-type ODNs activate B cells and pDCs and induce significantly more IFN-α production compared to C-type ODNs. In this paragraph, bold in the ODN sequence indicates a self-complementary palindrome, and CpG motifs are underlined.

[0240] Exemplary CpG ODNs include, for example, CpG7909 (5'-TCGTCGTTTTGTCGTTTTGTCGTT-3') and CpG 1018 (5'-TGACTGTGAACGTTCGAGATGA-3') is known and is described in U.S. Patent Nos. 7,255,868, 7,491,706, 7,479,285, 7,745,598, 7,785,610, 8,003,115, 8,133,874, 8,114,418, 8,222,398, 8,333,980, 8,597,665, 8,669,237, 9,028,845, and 10,052,378; U.S. Patent Application Publication No. 2020 / 0002704; and Bode et al., "CpG DNA as a vaccine adjuvant,” Expert Rev Vaccines (2011), 10(4):499-511, all of which are incorporated herein by reference in their entirety for all purposes.

[0241] One or more adjuvants may be used in combination, including, but not limited to, alum (aluminum salts), oil-in-water emulsions, water-in-oil emulsions, liposomes, and poly(lactide-co-glycolide) microparticles (Shah et al., Methods Mol Biol, 1494:1-14, 2017). In some embodiments, the immunogenic composition further comprises an aluminum salt adjuvant to which the SARS-CoV-2 antigen is adsorbed. In some embodiments, the aluminum salt adjuvant comprises one or more of the group consisting of amorphous aluminum hydroxyphosphate sulfate, aluminum hydroxide, aluminum phosphate, and potassium aluminum sulfate. In some embodiments, the aluminum salt adjuvant comprises one or both of aluminum hydroxide and aluminum phosphate. In some embodiments, the aluminum salt adjuvant comprises aluminum hydroxide. In some embodiments, a unit dose of the immunogenic composition comprises about 0.25 to about 0.50 mg of Al. 3+ , or approximately 0.35 mg of Al 3+ In some embodiments, the immunogenic composition further comprises an additional adjuvant. Other suitable adjuvants include, but are not limited to, a squalene-in-water emulsion (e.g., MF59 or AS03), a TLR3 agonist (e.g., poly-IC or poly-ICLC), a TLR4 agonist (e.g., a bacterial lipopolysaccharide derivative such as monophosphoryl lipid A) (MPL), and / or a saponin such as Quil A or QS-21, AS01 or AS02), a TLR5 agonist (bacterial flagellin), and a TLR7, TLR8, and / or TLR9 agonist (an imidazoquinoline derivative such as imiquimod, and resiquimod) (Coffman et al., Immunity, 33:492-503, 2010). In some embodiments, the additional adjuvant comprises MPL and alum (e.g., AS04). For veterinary use and antibody production in non-human animals, the mitogenic components of Freund's adjuvant (both complete and incomplete) can be used.

[0242] In some embodiments, the immunogenic composition comprises a pharmaceutically acceptable excipient, such as, for example, a solvent, a bulking agent, a buffer, a tonicity adjuster, and a preservative (Pramanick et al., Pharma Times, 45:65-77, 2013). In some embodiments, the immunogenic composition may comprise an excipient that functions as one or more of a solvent, a bulking agent, a buffer, and a tonicity adjuster (e.g., sodium chloride in saline may function as both an aqueous vehicle and a tonicity adjuster).

[0243] In some embodiments, the immunogenic composition comprises an aqueous vehicle as a solvent. Suitable excipients include, for example, sterile water, saline, phosphate-buffered saline, and Ringer's solution. In some embodiments, the composition is isotonic.

[0244] The immunogenic composition may contain a buffering agent. The buffering agent controls the pH and inhibits degradation of the active agent during processing, storage, and, optionally, reconstitution. Suitable buffers include salts, including, for example, acetate, citrate, phosphate, or sulfate. Other suitable buffers include amino acids, such as arginine, glycine, histidine, and lysine. The buffering agent may further comprise hydrochloric acid or sodium hydroxide. In some embodiments, the buffering agent maintains the pH of the composition within a range of 6 to 9. In some embodiments, the pH is greater than 6 (lower limit), 7, or 8. In some embodiments, the pH is less than 9 (upper limit), 8, or 7. That is, the pH is in the range of about 6 to 9, where the lower limit is less than the upper limit.

[0245] The immunogenic composition may include a tonicity adjusting agent. Suitable tonicity adjusting agents include, for example, dextrose, glycerol, sodium chloride, glycerin, and mannitol.

[0246] The immunogenic composition may include a bulking agent. Bulking agents are particularly useful when the pharmaceutical composition is lyophilized prior to administration. In some embodiments, the bulking agent is a protective agent that helps stabilize and prevent degradation of the active agent during freeze- or spray-drying and / or storage. Suitable bulking agents are sugars (monosaccharides, disaccharides, and polysaccharides), such as sucrose, lactose, trehalose, mannitol, sorbitol, glucose, and raffinose.

[0247] The immunogenic composition may contain a preservative. Suitable preservatives include, for example, antioxidants and antimicrobial agents. However, in preferred embodiments, the immunogenic composition is prepared under sterile conditions and is in a single-use container, so it is not necessary to include a preservative.

[0248] In some embodiments, the composition can be provided as a sterile composition. Pharmaceutical compositions typically contain an effective amount of the disclosed immunogens and can be prepared by conventional techniques. Typically, the amount of immunogen in each dose of the immunogenic composition is selected to induce an immune response without significant adverse side effects. In some embodiments, the composition can be provided in a unit dosage form for use in inducing an immune response in a subject. The unit dosage form includes a single preselected dose suitable for administration to a subject, a suitable marked or measured multiple of two or more preselected unit dosages, and / or a metering mechanism for administering a unit dose or multiples thereof. In other embodiments, the composition further includes an adjuvant.

[0249] IV. Methods of Inducing an Immune Response In some embodiments, provided herein are methods for generating an immune response in a subject against a surface antigen of a coronavirus, the method comprising administering to the subject an effective amount of a complex comprising a recombinant polypeptide selected from the group consisting of SEQ ID NOs: 1-26. In some embodiments, provided herein are methods for generating an immune response in a subject against a surface antigen of a coronavirus, wherein the surface antigen comprises an S protein or an antigenic fragment thereof, the method comprising administering to the subject an effective amount of a complex comprising a recombinant polypeptide selected from the group consisting of SEQ ID NOs: 1-26. In some embodiments, provided herein are methods for generating an immune response in a subject against a surface antigen of a coronavirus, wherein the surface antigen comprises a sequence selected from the group consisting of SEQ ID NOs: 27-66, the method comprising administering to the subject an effective amount of a complex comprising a recombinant polypeptide selected from the group consisting of SEQ ID NOs: 1-26. In some embodiments, provided herein are methods for generating an immune response in a subject against a surface antigen of a coronavirus, wherein the surface antigen comprises a coronavirus S protein or an antigenic fragment thereof, and optionally, the surface antigen comprises a sequence set forth in SEQ ID NO: 55 or an antigenic fragment thereof, the method comprising administering to the subject an effective amount of a complex comprising a recombinant polypeptide comprising a sequence set forth in any one of SEQ ID NOs: 10-25.

[0250] In some embodiments, provided herein are methods for generating an immune response to a surface antigen of a coronavirus in a subject, wherein the surface antigen comprises an S protein or an antigenic fragment thereof, the method comprising administering to the subject an effective amount of a complex, or a combination of any two or more of the complexes, comprising a recombinant polypeptide comprising a sequence selected from the group consisting of SEQ ID NOs: 1-26. In some embodiments, the method comprises administering to the subject an effective amount of a complex comprising a recombinant polypeptide comprising a sequence set forth in SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, and / or SEQ ID NO: 13.

[0251] The disclosed immunogens (e.g., recombinant coronavirus S antigens, e.g., S-trimers or S proteins described herein, nucleic acid molecules (e.g., RNA molecules), or vectors encoding protomers of the disclosed recombinant coronavirus S antigens, or protein nanoparticles or virus-like particles comprising the disclosed recombinant coronavirus S antigens) can be administered to a subject to induce an immune response in the subject against the corresponding coronavirus S antigen. In particular examples, the subject is a human. The immune response can be a protective immune response, e.g., a response that inhibits subsequent infection by the corresponding coronavirus. Eliciting an immune response can also be used to treat or inhibit infections and diseases associated with the corresponding coronavirus.

[0252] For example, a subject who has or is at risk of developing an infection with a coronavirus due to exposure or potential exposure to the coronavirus can be selected for treatment. After administration of the disclosed immunogens, the subject can be monitored for infection or symptoms, or both, associated with the coronavirus.

[0253] Typical subjects intended for treatment with the disclosed therapeutic methods include humans, as well as non-human primates and other animals. To identify subjects for prevention or treatment with the disclosed methods, accepted screening methods are used to determine risk factors associated with the target or suspected disease or condition, or to determine the status of an existing disease or condition in the subject. These screening methods include, for example, conventional screening to determine environmental, familial, occupational, and other such risk factors that may be associated with the target or suspected disease or condition, as well as diagnostic methods such as various ELISAs and other immunoassays for detecting and / or characterizing coronavirus infections. These and other routine methods enable clinicians to select patients in need of treatment using the disclosed methods and pharmaceutical compositions. In accordance with these methods and principles, the compositions can be administered as a stand-alone preventative or therapeutic program, or as a follow-up, adjunctive, or coordinated treatment regimen to other treatments, according to the teachings herein or other conventional methods.

[0254] Administration of the disclosed immunogens, e.g., coronavirus S antigens, e.g., trimers, proteins, can be prophylactic or therapeutic. When provided prophylactically, the disclosed therapeutic agents are provided prior to any symptoms, e.g., prior to infection. Prophylactic administration of the disclosed therapeutic agents serves to prevent or ameliorate any subsequent infection. When provided therapeutically, the disclosed therapeutic agents are provided at or after the onset of symptoms of disease or infection, e.g., after the onset of symptoms of infection with a coronavirus corresponding to a coronavirus S antigen, or after a diagnosis of coronavirus infection. Thus, a therapeutic agent can be provided after exposure or suspected exposure to the virus, or after the actual onset of infection, prior to anticipated exposure to the coronavirus, to reduce the anticipated severity, duration, or extent of infection and / or associated disease symptoms.

[0255] The immunogens and immunogenic compositions thereof described herein are provided to a subject, preferably a human, in an amount effective to induce or enhance an immune response to coronavirus S antigen in the subject. The actual amount of the disclosed immunogens administered will vary depending on factors such as the disease symptoms and specific condition of the subject (e.g., the subject's age, size, health, severity of symptoms, susceptibility factors, etc.), the time and route of administration, other drugs or treatments administered concomitantly, and the specific pharmacology of the composition to elicit the desired activity or biological response in the subject. Dosage regimens can be adjusted to provide an optimal prophylactic or therapeutic response.

[0256] Immunogenic compositions comprising one or more of the disclosed immunogens can be used in coordinated (or prime-boost) vaccination protocols or combination formulations. In certain embodiments, the novel combination immunogenic compositions and coordinated immunization protocols use separate immunogens or formulations, each aimed at eliciting an antiviral immune response, such as an immune response against the coronavirus S antigen. Separate immunogenic compositions that elicit antiviral immune responses can be combined into a multivalent immunogenic composition administered to a subject in a single immunization step, or they can be administered separately (in a monovalent immunogenic composition) in a coordinated (or prime-boost) immunization protocol.

[0257] There may be several boosts, and each boost may be a different disclosed immunogen. In some examples, a boost may be the same immunogen as another boost or prime. Primes and boosts may be administered as a single dose or multiple doses, e.g., two, three, four, five, six, or more, administered to a subject over the course of several days, weeks, or months. Multiple boosts may also be given, such as one to five (e.g., one, two, three, four, or five boosts) or more. Different dosages may be used in a series of sequential immunizations, e.g., a relatively large dose for the primary immunization, followed by a relatively smaller boost dose.

[0258] In some embodiments, the booster can be administered about 2 weeks, about 3 to 8 weeks, or about 4 weeks after the priming, or about several months after the priming. In some embodiments, the booster can be administered about 5 months, about 6 months, about 7 months, about 8 months, about 10 months, about 12 months, about 18 months, about 24 months, or some time after the priming. Periodic additional boosters can also be used at appropriate time points to strengthen the subject's "immunological memory." The appropriateness of selected vaccination parameters, such as formulation, dose, and schedule, can be determined by taking aliquots of serum from the subject and assaying antibody titers over the course of the immunization program. Additionally, the subject's clinical status can be monitored for the desired effect, such as prevention of infection or amelioration of disease (e.g., reduction in viral load). If such monitoring indicates that vaccination is suboptimal, the subject can be boosted with additional doses of the immunogenic composition, and vaccination parameters can be modified in a manner expected to enhance the immune response.

[0259] In some embodiments, a prime-boost regimen can include administering a DNA primer and protein boost vaccination protocol to a subject, which can include two or more administrations of the nucleic acid molecule or protein.

[0260] For protein therapeutics, typically each human dose contains 1 to 1000 μg of protein, e.g., about 1 μg to about 100 μg, e.g., about 1 μg to about 50 μg, e.g., about 1 μg, about 2 μg, about 5 μg, about 10 μg, about 15 μg, about 20 μg, about 25 μg, about 30 μg, about 40 μg, or about 50 μg.

[0261] The amount utilized in the immunogenic composition is selected based on the subject population (e.g., infants or the elderly). Optimal amounts for a particular composition can be ascertained by standard testing involving observation of antibody titers and other responses in subjects. It is understood that a therapeutically effective amount of a disclosed immunogen, such as a disclosed recombinant coronavirus S antigen, e.g., a trimer, protein, viral vector, or nucleic acid molecule, in an immunogenic composition may comprise an amount that is not effective to elicit an immune response by administration of a single dose, but is effective when administered in multiple doses, e.g., in a prime-boost administration protocol.

[0262] Upon administration of the disclosed immunogens of the present disclosure, the subject's immune system typically responds to the immunogenic composition by producing antibodies specific to the coronavirus S protein peptides contained in the immunogen. Such a response indicates that an immunologically effective dose has been delivered to the subject.

[0263] In some embodiments, the subject's antibody response is determined in the context of evaluating an effective dosage / immunization protocol. In most cases, assessing the antibody titer in serum or plasma obtained from the subject is sufficient. Decisions about whether to administer a booster vaccination and / or modify the amount of therapeutic agent administered to an individual can be based, at least in part, on the antibody titer level. The antibody titer level can be based, for example, on an immunobinding assay that measures the concentration of antibodies in the serum that bind to an antigen, such as a recombinant coronavirus S antigen, e.g., an S-trimer.

[0264] These methods do not require complete elimination, reduction, or prevention of coronavirus infection to be effective. For example, eliciting an immune response against coronavirus with one or more of the disclosed immunogens can reduce or inhibit (eliminate or prevent detectable infected cells) coronavirus infection by a desired amount, e.g., at least 10%, at least 20%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, or even at least 100%, compared to coronavirus infection in the absence of the immunogen. In a further example, coronavirus replication can be reduced or inhibited by the disclosed methods. Coronavirus replication need not be completely eliminated for the method to be effective. For example, an immune response elicited using one or more of the disclosed immunogens can reduce replication of the corresponding coronavirus (eliminate or prevent detectable replication of the coronavirus) by a desired amount, e.g., at least 10%, at least 20%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, or even at least 100%, compared to replication of the coronavirus in the absence of an immune response.

[0265] In some embodiments, the disclosed immunogens are administered to a subject simultaneously with the administration of an adjuvant, hi other embodiments, the disclosed immunogens are administered to a subject within a time sufficient to induce an immune response after the administration of an adjuvant.

[0266] One approach to administering nucleic acids is direct immunization with plasmid DNA, such as via a mammalian expression plasmid. Immunization with nucleic acid constructs is well known in the art and is taught, for example, in U.S. Pat. No. 5,643,578 (which describes a method for immunizing vertebrates by introducing DNA encoding a desired antigen to elicit a cellular or humoral response), and U.S. Pat. Nos. 5,593,972 and 5,817,637 (which describe operably linking an antigen-encoding nucleic acid sequence to a regulatory sequence enabling expression). U.S. Pat. No. 5,880,103 describes several methods for delivering nucleic acids encoding immunogenic peptides or other antigens to organisms. These methods include liposomal delivery of nucleic acids (or synthetic peptides themselves) and immunostimulatory constructs, or ISCOMS™, negatively charged cage-like structures 30-40 nm in size that spontaneously form upon mixing cholesterol and Quil A™ (saponin). Protective immunity has been generated using ISCOMS™ as antigen delivery vehicles in various experimental models of infection, including toxoplasmosis and Epstein-Barr virus-induced tumors (Mowat and Donachie, Immunol. Today 12:383, 1991). Doses as low as 1 μg of antigen encapsulated in ISCOMS™ have been found to generate class I-mediated CTL responses (Takahashi et al., Nature 344:873, 1990).

[0267] In some embodiments, a plasmid DNA vaccine is used to express the disclosed immunogens in a subject. For example, a nucleic acid molecule encoding the disclosed immunogen can be administered to a subject to induce an immune response against the coronavirus S antigen. In some embodiments, the nucleic acid molecule can be included in a plasmid vector for DNA immunization, such as the pVRC8400 vector (described in Barouch et al., J. Virol. 79, 8828-8834, 2005, incorporated herein by reference).

[0268] In another approach using nucleic acids for immunization, the disclosed recombinant coronavirus S antigens, e.g., trimers, proteins, can be expressed by attenuated viral hosts or vectors or bacterial vectors. Recombinant vaccinia virus, adeno-associated virus (AAV), herpesvirus, retrovirus, cytogluvirus, or other viral vectors can be used to express peptides or proteins, thereby eliciting CTL responses. For example, vaccinia vectors and methods useful for immunization protocols are described in U.S. Patent No. 4,722,848. BCG (Bacillus Calmette-Guerin) provides another vector for peptide expression (see Stover, Nature 351:456-460, 1991).

[0269] In one embodiment, nucleic acids encoding the disclosed recombinant coronavirus S antigens are directly introduced into cells. For example, the nucleic acids are loaded into gold microspheres by standard methods and then introduced into the skin using a device such as Bio-Rad's HELIOS™ Gene Gun. The nucleic acids can be "naked," consisting of a plasmid under the control of a strong promoter. Typically, DNA is injected into muscle, but it can also be injected directly into other sites. The dosage for injection is usually about 0.5 μg / kg to about 50 mg / kg, typically about 0.005 mg / kg to about 5 mg / kg (see, e.g., U.S. Patent No. 5,589,466).

[0270] For example, nucleic acids can be loaded into gold microspheres by standard methods and introduced into the skin using a device such as Bio-Rad's HELIOS™ Gene Gun. The nucleic acid can be "naked," consisting of a plasmid under the control of a strong promoter. Typically, DNA is injected into muscle, but it can also be injected directly into other sites. The injection dosage is usually about 0.5 μg / kg to about 50 mg / kg, typically about 0.005 mg / kg to about 5 mg / kg (see, e.g., U.S. Patent No. 5,589,466).

[0271] In another embodiment, mRNA-based immunization protocols can be used to deliver nucleic acids encoding the disclosed recombinant coronavirus S antigens directly into cells. In some embodiments, mRNA-based nucleic acid-based vaccines may offer a powerful alternative to the aforementioned approaches. mRNA vaccines eliminate safety concerns related to DNA integration into the host genome and can be directly translated in the cytoplasm of host cells. Furthermore, simple cell-free in vitro synthesis of RNA avoids the manufacturing complexities associated with viral vectors. Two exemplary forms of RNA-based vaccination that can be used to deliver nucleic acids encoding the disclosed recombinant coronavirus S antigens include conventional non-amplifying mRNA immunization (see, e.g., Petsch et al., "Protective efficacy of in vitro synthesized, specific mRNA vaccines against influenza A virus infection," Nature biotechnology, 30(12):1210-6, 2012), and self-amplifying mRNA immunization (see, e.g., Geall et al., "Nonviral delivery of self-amplifying RNA vaccines," PNAS, 109(36):14604-14609, 2012; Magini et al., "Self-Amplifying mRNA Vaccines Expressing Multiple Conserved Influenza Antigens Confer Protection against Homologous and Heterosubtypic Viral Challenge," PLoS One, 11(8):e0161193, 2016; and Brito et al., "Self-amplifying mRNA vaccines," Adv. Genet., 89:179-233, 2015).

[0272] In some embodiments, a neutralizing immune response is induced in a subject by administering a therapeutically effective amount of one or more of the disclosed immunogens to the subject. To assess neutralizing activity, serum can be collected from the subject at appropriate time points after immunization, frozen, and stored for neutralization testing. Methods for assaying neutralizing activity are known to those skilled in the art and are further described herein, including, but not limited to, plaque reduction neutralization (PRNT) assays, microneutralization assays, flow cytometry-based assays, and single-cycle infection assays. In some embodiments, serum neutralizing activity can be assayed using a panel of coronavirus pseudoviruses.

[0273] In some embodiments, a neutralizing immune response is induced in a subject by administering a therapeutically effective amount of one or more of the disclosed immunogens to the subject. To assess neutralizing activity, serum can be collected from the subject at appropriate time points after immunization, frozen, and stored for neutralization testing. Methods for assaying neutralizing activity are known to those skilled in the art and are further described herein, including, but not limited to, plaque reduction neutralization (PRNT) assays, microneutralization assays, flow cytometry-based assays, and single-cycle infection assays. In some embodiments, serum neutralizing activity can be assayed using a panel of coronavirus pseudoviruses.

[0274] In some embodiments, the neutralizing immune response induced by the immunogens disclosed herein generates neutralizing antibodies against a coronavirus, such as SARS-CoV-2. In some embodiments, the neutralizing antibodies herein bind to a cellular receptor or co-receptor of a coronavirus, such as SARS-CoV-2 or a component thereof. In some embodiments, the viral receptor or co-receptor is a coronavirus receptor or co-receptor, preferably a pneumovirus receptor or co-receptor, more preferably a human coronavirus receptor, e.g., a SARS-CoV-2 receptor or co-receptor. In some embodiments, the neutralizing antibodies herein modulate, decrease, antagonize, reduce, block, inhibit, prevent, and / or interfere with the activity or binding of at least one coronavirus, e.g., SARS-CoV-2, the activity or binding of at least one coronavirus, e.g., SARS-CoV-2 receptor, e.g., SARS-CoV-2 release, SARS-CoV-2 receptor signaling, membrane SARS-CoV-2 cleavage, SARS-CoV-2 activity, SARS-CoV-2 production and / or synthesis in vitro, in situ, and / or in vivo. In some embodiments, the immunogens disclosed herein induce neutralizing antibodies against SARS-CoV-2 that modulate, reduce, antagonize, attenuate, block, inhibit, prevent, and / or interfere with SARS-CoV-2 binding to a SARS-CoV-2 receptor or co-receptor, such as angiotensin-converting enzyme 2 (ACE2), dipeptidyl peptidase 4 (DPP4), dendritic cell-specific intercellular adhesion molecule-3-grabbing nonintegrin (DC-SIGN), and / or liver / lymph node-SIGN (L-SIGN).

[0275] V. Manufactured Articles or Kits Also provided are articles of manufacture or kits containing the provided recombinant polypeptides, proteins, and immunogenic compositions. The articles of manufacture may include a container and a label or package insert on or associated with the container. Suitable containers include, for example, bottles, vials, syringes, test tubes, IV solution bags, and the like. The containers may be formed from a variety of materials, such as glass or plastic. In some embodiments, the containers have a sterile access port. Exemplary containers include intravenous solution bags and vials, including those with stoppers pierceable by an injection needle. The articles of manufacture or kits may further include a package insert indicating that the compositions can be used to treat a particular condition, such as a condition described herein (e.g., coronavirus infection). Alternatively or additionally, the articles of manufacture or kits may further include another or the same container containing a pharmaceutically acceptable buffer. These may further include other materials, such as other buffers, diluents, filters, needles, and / or syringes.

[0276] The label or package insert may indicate that the composition is used to treat a coronavirus infection in an individual. The label or package insert on or associated with the container may indicate instructions for reconstituting and / or using the formulation. The label or package insert may further indicate that the formulation is useful or intended for subcutaneous, intravenous, or other mode of administration for treating or preventing a coronavirus infection in an individual.

[0277] In some embodiments, the container holds a composition by itself or in combination with another composition that is effective for treating, preventing, and / or diagnosing a condition. The article of manufacture or kit may include (a) a first container containing a composition therein (i.e., a first pharmaceutical agent), where the composition comprises an immunogenic composition or a protein or recombinant polypeptide; and (b) a second container containing a composition therein (i.e., a second pharmaceutical agent), where the composition comprises an additional agent, such as an adjuvant or other therapeutic agent, wherein the article of manufacture or kit further includes instructions on a label or package insert for treating a subject with an effective amount of the second pharmaceutical agent.

[0278] term Unless otherwise defined, all technical terms, notations, and other technical and scientific terms or terminology used herein are intended to have the same meaning as commonly understood by one of ordinary skill in the art to which the claimed subject matter pertains. In some instances, terms having a commonly understood meaning are defined herein for clarity and / or ease of reference, and the inclusion of such definitions herein should not necessarily be construed as representing a substantial difference from what is commonly understood in the art.

[0279] The terms "polypeptide" and "protein" are used interchangeably to refer to a polymer of amino acid residues and are not limited to a minimum length. Polypeptides, such as the receptor and other polypeptides provided, e.g., linkers or peptides, can contain amino acid residues, including natural and / or unnatural amino acid residues. The term also includes post-expression modifications of the polypeptide, e.g., glycosylation, sialylation, acetylation, and phosphorylation. In some embodiments, a polypeptide can contain modifications relative to the native or naturally occurring sequence, so long as the protein maintains the desired activity. These modifications can be deliberate, such as through site-directed mutagenesis, or can be accidental, such as through host mutations that produce errors in the protein or PCR amplification.

[0280] As used herein, a "subject" is a mammal, such as a human or other animal, typically a human. In some embodiments, the subject, e.g., a patient, to which one or more agents, cells, cell populations, or compositions are administered is a mammal, typically a primate, e.g., a human. In some embodiments, the primate is a monkey or ape. The subject may be male or female and of any suitable age, including infants, juveniles, adolescents, adults, and geriatric subjects. In some embodiments, the subject is a non-primate mammal, e.g., a rodent.

[0281] As used herein, "treatment" (and its grammatical variations, e.g., "treat" or "treating") refers to the complete or partial improvement or alleviation of a disease or condition or disorder or symptom, side effect or outcome, or phenotype associated therewith. Desirable effects of treatment include, but are not limited to, prevention of disease onset or recurrence, alleviation of symptoms, reduction of any direct or indirect pathological consequences of disease, prevention of metastasis, slowing the rate of disease progression, amelioration or palliation of the condition, and remission or improved prognosis. These terms do not imply complete cure of a disease or complete elimination of any symptoms or effect(s) on all symptoms or outcomes.

[0282] As used herein, "delaying the onset of disease" means to postpone, prevent, slow, retard, stabilize, inhibit, and / or postpone the onset of a disease (such as cancer). This delay can be for various lengths of time, depending on the history of the disease and / or the individual undergoing treatment. In some embodiments, a sufficient or significant delay can, in effect, encompass prevention, in that the individual does not develop the disease. For example, late-stage cancer, such as the development of metastases, can be delayed.

[0283] As used herein, "preventing" includes providing prophylaxis against the occurrence or recurrence of a disease in a subject who may be predisposed to the disease but has not yet been diagnosed with the disease. In some embodiments, the provided cells and compositions are used to delay the onset of the disease or slow the progression of the disease.

[0284] As used herein, "inhibiting" a function or activity means reducing the function or activity when compared to an otherwise identical condition or when compared to another condition, except for the condition or parameter of interest. For example, a cell that inhibits tumor growth reduces the rate of tumor growth compared to the rate of tumor growth in the absence of the cell.

[0285] An "effective amount" of an agent, e.g., a pharmaceutical formulation, cell, or composition, refers to an amount effective, in the context of administration, at dosages / amounts and for periods of time necessary to achieve a desired result, e.g., a therapeutic or prophylactic result.

[0286] A "therapeutically effective amount" of an agent, e.g., a pharmaceutical formulation or cells, refers to an amount effective, at dosages and for periods of time necessary, to achieve a desired therapeutic result and / or the pharmacokinetic or pharmacodynamic effect of treatment, such as for treating a disease, condition, or disorder. A therapeutically effective amount may vary depending on factors such as the disease state, age, sex, and weight of the subject, as well as the cell population administered. In some embodiments, the methods provided include administering cells and / or compositions in an effective amount, e.g., a therapeutically effective amount.

[0287] A "prophylactically effective amount" refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired prophylactic result. Typically, but not necessarily, a prophylactic dose is used in subjects prior to or at an early stage of disease, such that the prophylactically effective amount will be less than the therapeutically effective amount. In situations with a low tumor burden, the prophylactically effective amount will, in some embodiments, be greater than the therapeutically effective amount. This is an effective amount of a vaccine or other agent sufficient to produce a desired response, such as reducing or eliminating the signs or symptoms of a condition or disease, such as pneumonia. For example, this may be the amount necessary to inhibit viral replication or measurably alter the outward symptoms of a viral infection. Generally, this amount is sufficient to measurably inhibit the replication or infectivity of a virus (e.g., SARS-CoV-2). When administered to a subject, a dosage that achieves a target tissue concentration shown to achieve in vitro inhibition of viral replication is generally used. In some embodiments, an "effective amount" is one that treats (including prevents) one or more symptoms and / or underlying causes of any of the disorders or diseases, e.g., to treat a coronavirus infection. In some embodiments, the effective amount is a therapeutically effective amount. In some embodiments, an effective amount is an amount that prevents the onset of one or more signs or symptoms of a particular disease or condition, such as one or more signs or symptoms associated with a coronavirus infection.

[0288] As used herein, the terms "antigen" or "immunogen" are used interchangeably and refer to a substance, typically a protein, that is capable of inducing an immune response in a subject. The terms also refer to a protein that is immunologically active, in the sense that it is capable of eliciting a humoral and / or cellular immune response against the protein once administered to a subject (either directly or by administering to the subject a nucleotide sequence or vector encoding the protein). Unless otherwise specified, the term "vaccine immunogen" is used interchangeably with "protein antigen" or "immunogenic polypeptide."

[0289] The term "conservatively modified variants" applies to both amino acid and nucleic acid sequences. With respect to a particular nucleic acid sequence, conservatively modified variants refer to nucleic acids that encode the same or essentially identical amino acid sequences, or, if the nucleic acid does not encode an amino acid sequence, to essentially identical sequences. Due to the degeneracy of the genetic code, a large number of functionally identical nucleic acids encode any given protein. With respect to polypeptide sequences, "conservatively modified variants" refers to variants with conservative amino acid substitutions, i.e., variants that have amino acid residues substituted with other amino acid residues having side chains with similar charges. Families of amino acid residues with side chains with similar charges have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine).

[0290] Epitope refers to an antigenic determinant. These include chemical groups or peptide sequences on a molecule that are antigenic, particularly those that elicit a specific immune response; for example, an epitope is a region of an antigen to which B cells and / or T cells respond. Epitopes can be formed from both contiguous or non-contiguous amino acids juxtaposed by tertiary folding of a protein.

[0291] Unless otherwise specified, a fusion protein is a recombinant protein containing the amino acid sequences of at least two unrelated proteins linked together via peptide bonds to form a single protein. Therefore, it does not encompass the naturally occurring coronavirus surface antigens referred to herein as fusion (F) proteins. Unrelated amino acid sequences can be linked directly to each other or can be linked using a linker sequence. As used herein, proteins are unrelated if their amino acid sequences are not normally found linked together via peptide bonds in their natural environment (e.g., within a cell). For example, the amino acid sequence of a viral antigen and the amino acid sequence of collagen or procollagen are not normally found linked together via peptide bonds.

[0292] An immunogen is a protein or portion thereof that can induce an immune response in a mammal, such as a mammal infected with or at risk of infection by a pathogen. Administration of the immunogen can result in protective and / or prophylactic immunity against the pathogen of interest.

[0293] An immunogenic composition refers to a composition comprising an immunogenic polypeptide that induces a measurable CTL response against a virus expressing the immunogenic polypeptide or that induces a measurable B cell response (such as the production of antibodies) against the immunogenic polypeptide.

[0294] Sequence identity or similarity between two or more nucleic acid sequences, or two or more amino acid sequences, is expressed in terms of the identity or similarity between the sequences. Sequence identity can be measured in percentage identity; the higher the percentage, the more identical the sequences. Two sequences are "substantially identical" if, when compared and aligned for maximum correspondence over a comparison window or designated region as measured using one of the following sequence comparison algorithms or by manual alignment and visual inspection, the two sequences have a specified percentage of amino acid residues or nucleotides that are the same (i.e., 60% identity, optionally 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% identity over a designated region, or, if not specified, over the entire sequence). Optionally, identity exists over a region that is at least about 50 nucleotides (or 10 amino acids) in length, or more preferably over a region that is 100 to 500 or 1000 or more nucleotides in length (or 20, 50, 200 or more amino acids).

[0295] A vaccine refers to a pharmaceutical composition that induces a preventative or therapeutic immune response in a subject. In some cases, the immune response is a protective immune response. Typically, a vaccine induces an antigen-specific immune response against an antigen of a pathogen, such as a viral pathogen, or a cellular component correlated with a pathological condition. A vaccine may comprise a polynucleotide (such as a nucleic acid encoding a disclosed antigen), a peptide or polypeptide (such as a disclosed antigen), a virus, a cell, or one or more cellular components. In some embodiments, the vaccine or vaccine immunogen or vaccine composition is expressed from a fusion construct and self-assembles into nanoparticles that display the immunogenic polypeptide or protein on their surface.

[0296] Virus-like particles (VLPs) refer to the shells of non-replicating viruses derived from any of several viruses. VLPs are generally composed of one or more viral proteins, including, but not limited to, proteins called capsid, coat, shell, surface, and / or envelope proteins, or particle-forming polypeptides derived from these proteins. VLPs can form naturally upon recombinant expression of proteins in an appropriate expression system. Methods for producing specific VLPs are known in the art. The presence of VLPs after recombinant expression of viral proteins can be detected using conventional techniques known in the art, such as by electron microscopy, biophysical characterization, etc. See, e.g., Baker et al. (1991) Biophys. J. 60:1445-1456; and Hagensee et al. (1994) J. Virol. 68:4503-4505. For example, VLPs can be isolated by density gradient centrifugation and / or identified by characteristic density banding. Alternatively, cryo-electron microscopy is performed on a vitrified aqueous sample of the VLP preparation in question, and images are recorded under appropriate exposure conditions.

[0297] The term "about" as used herein refers to the normal error range for the respective value, which is readily known to one of ordinary skill in the art. Reference herein to "about" a value or parameter includes (and describes) embodiments directed to the value or parameter itself.

[0298] As used herein, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. For example, "a" or "an" means "at least one" or "one or more."

[0299] Throughout this disclosure, various aspects of the claimed subject matter are presented in a range format. It should be understood that the description in range format is for convenience and brevity only and should not be construed as an inflexible limitation on the scope of the claimed subject matter. Accordingly, the description of a range should be considered to specifically disclose not only each individual numerical value within that range, but also all possible subranges. For example, when a range of values ​​is provided, it is understood that each intervening value between the upper and lower limits of that range, and any other stated or intervening value within that stated range, is included in the claimed subject matter. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges, and are also encompassed within the scope of the claimed subject matter, subject to any limits specifically excluded in the stated ranges. When a stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the claimed subject matter. This applies regardless of the broadness of the range.

[0300] As used herein, a composition refers to any mixture of two or more products, substances, or compounds, such as cells, which may be a solution, suspension, liquid, powder, paste, aqueous, non-aqueous, or any combination thereof.

[0301] As used herein, the term "vector" refers to a nucleic acid molecule capable of propagating another nucleic acid to which it is linked. The term includes vectors as self-replicating nucleic acid structures and vectors that integrate into the genome of a host cell into which they are introduced. Certain vectors are capable of directing the expression of nucleic acids to which they are operably linked. Such vectors are referred to herein as "expression vectors."

[0302] Exemplary Embodiments Embodiment 1. A protein comprising a plurality of recombinant polypeptides, each recombinant polypeptide comprising a surface antigen of a coronavirus linked to a C-terminal propeptide of collagen, wherein the C-terminal propeptides of the recombinant polypeptides form an inter-polypeptide disulfide bond.

[0303] Embodiment 2. The protein of embodiment 1, wherein the coronavirus is Severe Acute Respiratory Syndrome (SARS)-coronavirus (SARS-CoV), SARS-coronavirus 2 (SARS-CoV-2), SARS-like coronavirus, Middle East Respiratory Syndrome (MERS)-coronavirus (MERS-CoV), MERS-like coronavirus, NL63-CoV, 229E-CoV, OC43-CoV, HKU1-CoV, WIV1-CoV, MHV, HKU9-CoV, PEDV-CoV, or SDCV.

[0304] Embodiment 3. The protein of embodiment 1 or 2, wherein the surface antigen comprises a coronavirus spike (S) protein or a fragment or epitope thereof, wherein the epitope is optionally a linear epitope or a conformational epitope, and wherein the protein comprises three recombinant polypeptides.

[0305] Embodiment 4. The protein of embodiment 3, wherein the surface antigen comprises a signal peptide, an S1 subunit peptide, an S2 subunit peptide, or any combination thereof.

[0306] Embodiment 5. The protein of embodiment 3, wherein the surface antigen comprises a signal peptide, a receptor binding domain (RBD) peptide, a receptor binding motif (RBM) peptide, a fusion peptide (FP), a heptad repeat 1 (HR1) peptide, or a heptad repeat 2 (HR2) peptide, or any combination thereof.

[0307] Embodiment 6. The protein of any one of embodiments 3 to 5, wherein the surface antigen comprises the receptor binding domain (RBD) of the S protein.

[0308] Embodiment 7. The protein of any one of embodiments 3 to 6, wherein the surface antigen comprises the S1 and S2 subunits of the S protein.

[0309] Embodiment 8. The protein of any one of embodiments 3 to 7, wherein the surface antigen does not comprise a transmembrane (TM) domain peptide and / or a cytoplasmic (CP) domain peptide.

[0310] Embodiment 9. The protein of any of embodiments 3 to 8, wherein the surface antigen comprises a protease cleavage site, and the protease is optionally furin, trypsin, factor Xa, thrombin, or cathepsin L.

[0311] Embodiment 10. The protein of any of embodiments 3 to 8, wherein the surface antigen does not comprise a protease cleavage site, and the protease is optionally furin, trypsin, factor Xa, thrombin, or cathepsin L.

[0312] Embodiment 11. The protein of any of embodiments 1 to 10, wherein the surface antigen is soluble or does not directly bind to a lipid bilayer, such as a membrane or viral envelope.

[0313] Embodiment 12. The protein of any one of embodiments 1 to 11, wherein the surface antigens are identical or different between the recombinant polypeptides of the protein.

[0314] Embodiment 13. The protein of any of embodiments 1-12, wherein the surface antigen is fused directly to the C-terminal propeptide or linked to the C-terminal propeptide via a linker, such as a linker comprising a glycine-XY repeat, where X and Y are independently any amino acid, optionally proline or hydroxyproline.

[0315] Embodiment 14. The protein of any of embodiments 1 to 13, which is soluble or does not directly bind to a lipid bilayer, such as a membrane or viral envelope.

[0316] Embodiment 15. A protein according to any of embodiments 1 to 14, wherein the protein is capable of binding to a cell surface receptor of a subject, and optionally the subject is a mammal, such as a primate, such as a human.

[0317] Embodiment 16. The protein of embodiment 15, wherein the cell surface receptor is angiotensin-converting enzyme 2 (ACE2), dipeptidyl peptidase 4 (DPP4), dendritic cell-specific intercellular adhesion molecule-3-grabbing nonintegrin (DC-SIGN), or liver / lymph node-SIGN (L-SIGN).

[0318] Embodiment 17. The protein of any one of embodiments 1 to 16, wherein the C-terminal propeptide is that of human collagen.

[0319] Embodiment 18. The protein of any one of embodiments 1 to 17, wherein the C-terminal propeptide comprises the C-terminal propeptide of proα1(I), proα1(II), proα1(III), proα1(V), proα1(XI), proα2(I), proα2(V), proα2(XI), or proα3(XI), or a fragment thereof.

[0320] Embodiment 19. The protein of any of embodiments 1 to 18, wherein the C-terminal propeptides are identical or different between the recombinant polypeptides.

[0321] Embodiment 20. The protein of any of embodiments 1 to 19, wherein the C-terminal propeptide can comprise any of SEQ ID NOs: 67-80, or an amino acid sequence at least 90% identical thereto, and can form inter-polypeptide disulfide bonds and trimerize the recombinant polypeptide.

[0322] Embodiment 21. The protein of any of embodiments 1 to 20, wherein the C-terminal propeptide comprises SEQ ID NO: 67, or an amino acid sequence at least 90% identical thereto, and is capable of forming inter-polypeptide disulfide bonds and trimerizing the recombinant polypeptide.

[0323] Embodiment 22. The protein of any of embodiments 1 to 20, wherein the C-terminal propeptide comprises SEQ ID NO: 68, or an amino acid sequence at least 90% identical thereto, and is capable of forming inter-polypeptide disulfide bonds and trimerizing the recombinant polypeptide.

[0324] Embodiment 23. The protein of any of embodiments 1 to 20, wherein the C-terminal propeptide comprises SEQ ID NO: 69, or an amino acid sequence at least 90% identical thereto, and is capable of forming inter-polypeptide disulfide bonds and trimerizing the recombinant polypeptide.

[0325] Embodiment 24. The protein of any of embodiments 1 to 20, wherein the C-terminal propeptide comprises SEQ ID NO: 70, or an amino acid sequence at least 90% identical thereto, and is capable of forming inter-polypeptide disulfide bonds and trimerizing the recombinant polypeptide.

[0326] Embodiment 25. The protein of any of embodiments 1 to 20, wherein the C-terminal propeptide comprises SEQ ID NO: 71, or an amino acid sequence at least 90% identical thereto, and is capable of forming inter-polypeptide disulfide bonds and trimerizing the recombinant polypeptide.

[0327] Embodiment 26. The protein of any of embodiments 1 to 20, wherein the C-terminal propeptide comprises SEQ ID NO: 72, or an amino acid sequence at least 90% identical thereto, and is capable of forming inter-polypeptide disulfide bonds and trimerizing the recombinant polypeptide.

[0328] Embodiment 27. The protein of any of embodiments 1 to 20, wherein the C-terminal propeptide comprises SEQ ID NO: 73, or an amino acid sequence at least 90% identical thereto, and is capable of forming inter-polypeptide disulfide bonds and trimerizing the recombinant polypeptide.

[0329] Embodiment 28. The protein of any of embodiments 1 to 20, wherein the C-terminal propeptide comprises SEQ ID NO: 74, or an amino acid sequence at least 90% identical thereto, and is capable of forming inter-polypeptide disulfide bonds and trimerizing the recombinant polypeptide.

[0330] Embodiment 29. The protein of any of embodiments 1 to 20, wherein the C-terminal propeptide comprises SEQ ID NO: 75 or SEQ ID NO: 76, or an amino acid sequence at least 90% identical thereto, and is capable of forming inter-polypeptide disulfide bonds and trimerizing the recombinant polypeptide.

[0331] Embodiment 30. The protein of any of embodiments 1-29, wherein the C-terminal propeptide comprises a sequence comprising a glycine-XY repeat linked to the N-terminus of any of SEQ ID NOs: 67-80, where X and Y are independently any amino acid and optionally proline or hydroxyproline, or an amino acid sequence at least 90% identical to SEQ ID NOs: 67-80, and is capable of forming inter-polypeptide disulfide bonds and trimerizing the recombinant polypeptide.

[0332] Embodiment 31. The protein of any one of embodiments 1 to 30, wherein the surface antigen in each recombinant polypeptide is in the pre-fusion conformation or the post-fusion conformation.

[0333] Embodiment 32. A protein according to any one of embodiments 1 to 31, wherein the surface antigen in each recombinant polypeptide comprises any one of SEQ ID NOs: 27 to 66 or an amino acid sequence at least 80% identical to SEQ ID NOs: 27 to 66.

[0334] Embodiment 33. A protein according to any one of embodiments 1 to 32, wherein the surface antigen in each recombinant polypeptide comprises any one of SEQ ID NOs: 1 to 26 or an amino acid sequence at least 80% identical to SEQ ID NOs: 1 to 26.

[0335] Embodiment 34. An immunogen comprising the protein according to any one of embodiments 1 to 33.

[0336] Embodiment 35. A protein nanoparticle comprising the protein according to any one of embodiments 1 to 33 directly or indirectly linked to the nanoparticle.

[0337] Embodiment 36. A virus-like particle (VLP) comprising a protein according to any one of embodiments 1 to 33.

[0338] Embodiment 37. An isolated nucleic acid encoding one, two, three or more recombinant polypeptides of a protein according to any one of embodiments 1 to 33.

[0339] Embodiment 38. The isolated nucleic acid of embodiment 37, wherein the polypeptide encoding the surface antigen is fused in frame to a polypeptide encoding the C-terminal propeptide of collagen.

[0340] Embodiment 39. The isolated nucleic acid of embodiment 37 or 38, operably linked to a promoter.

[0341] Embodiment 40. The isolated nucleic acid of any one of embodiments 37 to 39, which is a DNA molecule.

[0342] Embodiment 41. The isolated nucleic acid of any of embodiments 37 to 39, which is an RNA molecule, optionally an mRNA molecule such as a nucleoside-modified mRNA, a non-amplified mRNA, an auto-amplified mRNA, or a trans-amplified mRNA.

[0343] Embodiment 42. A vector comprising the isolated nucleic acid of any one of embodiments 37 to 41.

[0344] Embodiment 43. The vector of embodiment 42, which is a viral vector.

[0345] Embodiment 44. A virus, pseudovirus, or cell comprising the vector of embodiment 42 or 43, optionally wherein the virus or cell has a recombinant genome.

[0346] Embodiment 45. An immunogenic composition comprising the protein, immunogen, protein nanoparticle, VLP, isolated nucleic acid, vector, virus, pseudovirus, or cell of any one of embodiments 1 to 44, and a pharmaceutically acceptable carrier.

[0347] Embodiment 46. A vaccine comprising the immunogenic composition of embodiment 45 and optionally an adjuvant, optionally a subunit vaccine, and / or optionally the vaccine is a prophylactic and / or therapeutic vaccine.

[0348] Embodiment 47. The vaccine of embodiment 46, wherein the vaccine comprises multiple different adjuvants.

[0349] Embodiment 48. A method for producing a protein, comprising expressing in a host cell an isolated nucleic acid or vector according to any one of embodiments 37 to 43 to produce a protein according to any one of embodiments 1 to 33; and purifying the protein.

[0350] Embodiment 49. A protein produced by the method of embodiment 48.

[0351] Embodiment 50. A method for generating an immune response in a subject against a surface antigen of a coronavirus, the method comprising administering to the subject an effective amount of a protein, immunogen, protein nanoparticle, VLP, isolated nucleic acid, vector, virus, pseudovirus, cell, immunogenic composition, or vaccine described in any one of embodiments 1 to 47 or 49 to generate an immune response.

[0352] Embodiment 51. The method of embodiment 50 for treating or preventing infection by a coronavirus.

[0353] Embodiment 52. The method of embodiment 50 or 51, wherein an immune response is generated, thereby inhibiting or reducing coronavirus replication in the subject.

[0354] Embodiment 53. The method of any of embodiments 50-52, wherein the immune response comprises a cellular response and / or a humoral response, and optionally includes producing one or more neutralizing antibodies, such as polyclonal or monoclonal antibodies.

[0355] Embodiment 54. The method of any one of embodiments 50 to 53, wherein the immune response is directed against a surface antigen of the coronavirus, but not against the C-terminal propeptide.

[0356] Embodiment 55. The method of any of embodiments 50-54, wherein administering does not result in antibody-dependent enhancement (ADE) in the subject due to previous exposure to one or more coronaviruses.

[0357] Embodiment 56. The method of any of embodiments 50-55, wherein administering does not result in antibody-dependent enhancement (ADE) in the subject upon subsequent exposure to one or more coronaviruses.

[0358] Embodiment 57. The method of any one of embodiments 50 to 56, further comprising a priming step and / or a boosting step.

[0359] Embodiment 58. The method of any of embodiments 50-57, wherein the administering step is carried out via topical, transdermal, subcutaneous, intradermal, oral, intranasal (e.g., intranasal spray), intratracheal, sublingual, buccal, rectal, intravaginal, inhalation, intravenous (e.g., intravenous injection), intraarterial, intramuscular (e.g., intramuscular injection), intracardiac, intraosseous, intraperitoneal, transmucosal, intravitreal, subretinal, intraarticular, periarticular, topical, or epicutaneous administration.

[0360] Embodiment 59. The method of any of embodiments 50-58, wherein the effective amount is administered in a single dose or in a series of doses separated by one or more intervals.

[0361] Embodiment 60. The method of any one of embodiments 50-59, wherein the effective amount is administered without an adjuvant.

[0362] Embodiment 61. The method of any one of embodiments 50 to 59, wherein the effective amount is administered together with an adjuvant or multiple adjuvants.

[0363] Embodiment 62. A method comprising administering to a subject an effective amount of a protein described in any one of embodiments 1 to 33, to produce neutralizing antibodies or neutralizing antisera against coronavirus in the subject.

[0364] Embodiment 63. The method of embodiment 62, wherein the subject is a mammal, optionally a human or non-human primate.

[0365] Embodiment 64. The method of embodiment 62 or 63, further comprising isolating neutralizing antibodies or neutralizing antisera from the subject.

[0366] Embodiment 65. The method of embodiment 64, further comprising administering an effective amount of an isolated neutralizing antibody or neutralizing antiserum to a human subject via passive immunization to prevent or treat infection with a coronavirus.

[0367] Embodiment 66. The method of any one of embodiments 62-65, wherein the neutralizing antibody or neutralizing antiserum comp...

Claims

1. A recombinant subunit vaccine for use in preventing infection with a coronavirus in a mammal, said recombinant subunit vaccine comprising a soluble coronavirus viral surface antigen attached by in-frame fusion to a C-terminal portion of procollagen to form a disulfide-linked trimeric fusion protein, said soluble coronavirus viral surface antigen comprising the spike (S) extracellular domain of SARS-CoV-2 virus Wuhan-Hu-1 or B.1.351 strain.

2. 2. The recombinant subunit vaccine of claim 1, wherein the coronavirus viral surface antigen comprises a SARS-CoV-2 spike (S) ectodomain peptide having a mutation.

3. 3. The recombinant subunit vaccine of claim 2, wherein the mutation comprises 685R->685A.

4. The recombinant subunit vaccine of claim 2 or 3, wherein the mutation comprises 986K->986P.

5. The recombinant subunit vaccine of any one of claims 2 to 4, wherein the mutation comprises 987V->987P.

6. The recombinant subunit vaccine of any one of claims 1 to 5, wherein the recombinant subunit vaccine comprises a sequence represented by any one of SEQ ID NOs: 27 to 65.

7. The recombinant subunit vaccine of any one of claims 1 to 6, wherein the recombinant subunit vaccine comprises a sequence represented by any one of SEQ ID NOs: 1 to 9.

8. The recombinant subunit vaccine according to any one of claims 1 to 7, wherein the recombinant subunit vaccine comprises a sequence represented by any one of SEQ ID NOs: 10 to 13.

9. The recombinant subunit vaccine of any one of claims 1 to 8, wherein the recombinant subunit vaccine comprises the sequence represented by SEQ ID NO:

26.

10. 10. The recombinant subunit vaccine of any one of claims 1 to 9, wherein the recombinant subunit vaccine comprises a first sequence represented by any of SEQ ID NOs: 27 to 56 linked to a second sequence represented by any of SEQ ID NOs: 67 to 80, wherein the C-terminus of the first sequence is linked directly or indirectly to the N-terminus of the second sequence.

11. The recombinant subunit vaccine of any one of claims 1 to 10, wherein the recombinant subunit vaccine is administered by intramuscular injection.

12. The recombinant subunit vaccine of any one of claims 1 to 11, wherein the recombinant subunit vaccine is administered by nasal spray.

13. The recombinant subunit vaccine of any one of claims 1 to 12, wherein the recombinant subunit vaccine is administered in a single dose or in a series of doses separated by weekly or monthly intervals.

14. The recombinant subunit vaccine of any one of claims 1 to 13, wherein the recombinant subunit vaccine is administered without an adjuvant.

15. The recombinant subunit vaccine of any one of claims 1 to 13, wherein the recombinant subunit vaccine is administered with an adjuvant.

16. The recombinant subunit vaccine of any one of claims 1 to 13, wherein the recombinant subunit vaccine is administered with two or more adjuvants.

17. 1. A method for detecting antibodies to coronavirus from mammalian serum, comprising contacting the serum with a soluble coronavirus viral surface antigen linked by in-frame fusion to the C-terminal portion of procollagen to form a disulfide-linked trimeric fusion protein, wherein the soluble coronavirus viral surface antigen comprises the spike (S) extracellular domain of SARS-CoV-2 virus Wuhan-Hu-1 or B.1.351 strain.

18. 18. The method of claim 17, wherein the soluble coronavirus virus surface antigen is an S protein or peptide.

19. A recombinant subunit vaccine for use in a method for treating a patient infected with a coronavirus, the method comprising immunizing a mammal, purifying the neutralizing antibodies produced, and treating the patient infected with the coronavirus by passive immunization with the neutralizing antibodies, wherein the recombinant subunit vaccine comprises a soluble surface antigen from a coronavirus, the soluble surface antigen being linked by in-frame fusion to a C-terminal portion of procollagen to form a disulfide-linked trimeric fusion protein, the soluble coronavirus viral surface antigen comprising the spike (S) extracellular domain of SARS-CoV-2 virus Wuhan-Hu-1 or B.1.351 strain.

20. 20. The recombinant subunit vaccine of claim 19, wherein the neutralizing antibody comprises a polyclonal antibody.

21. 20. The recombinant subunit vaccine of claim 19, wherein the neutralizing antibody comprises a monoclonal antibody.

22. 20. The recombinant subunit vaccine of claim 19, wherein the neutralizing antibody is a monoclonal antibody against the S protein or peptide.

23. 20. The recombinant subunit vaccine of claim 19, wherein the neutralizing antibody is a monoclonal antibody against the S protein or SARS-CoV-2.

24. 20. The recombinant subunit vaccine of claim 19, wherein the neutralizing antibody is a monoclonal antibody against the S protein or SARS-CoV-1.

25. 20. The recombinant subunit vaccine of claim 19, wherein the neutralizing antibody is a monoclonal antibody against the S protein of MERS.

26. A complex comprising a recombinant polypeptide selected from the group consisting of SEQ ID NOs: 1-26.

27. A complex comprising a trimer of a recombinant polypeptide selected from the group consisting of SEQ ID NOs: 1-26, wherein said recombinant polypeptide is trimerized via an inter-polypeptide disulfide bond to form said trimer.

28. An immunogenic composition comprising a trimer of a recombinant polypeptide comprising a sequence selected from the group consisting of SEQ ID NOs: 1 to 26, or a combination of any two or more of said trimers.

29. 29. The immunogenic composition of claim 28, comprising said trimer of a recombinant polypeptide having the sequence set forth in SEQ ID NO:

1.

30. A complex comprising a recombinant polypeptide selected from the group consisting of SEQ ID NOs: 1 to 26 for use in generating an immune response to a surface antigen of a coronavirus in a subject.

31. A complex comprising a recombinant polypeptide selected from the group consisting of SEQ ID NOs: 1-26 for use in generating an immune response in a subject against a surface antigen of a coronavirus, wherein the surface antigen comprises an S protein.

32. A complex comprising a recombinant polypeptide selected from the group consisting of SEQ ID NOs: 1-26 for use in generating an immune response in a subject against a surface antigen of a coronavirus, wherein the surface antigen comprises a sequence selected from the group consisting of SEQ ID NOs: 27-66.

33. A complex comprising a recombinant polypeptide comprising a sequence set forth in any one of SEQ ID NOs: 10 to 25, for use in generating an immune response in a subject against a surface antigen of a coronavirus, wherein the surface antigen comprises the S protein of the coronavirus.

34. A complex as described in claim 33, wherein the surface antigen comprises an array represented by sequence number 55.

35. A complex or combination of any two or more of said complexes, for use in generating an immune response in a subject against a surface antigen of a coronavirus, said complex or combination comprising a recombinant polypeptide comprising a sequence selected from the group consisting of SEQ ID NOs: 1-26, wherein said surface antigen comprises an S protein or an antigenic fragment thereof.

36. 36. The complex or combination thereof of claim 35, comprising the recombinant polypeptide comprising the sequence represented by SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, and / or SEQ ID NO:

13.

37. A fusion protein comprising multiple recombinant polypeptides, each recombinant polypeptide comprising, from amino terminus to carboxy terminus: a) a soluble coronavirus surface antigen containing the spike (S) extracellular domain of the SARS-CoV-2 virus Wuhan-Hu-1 or B.1.351 strain; b) A fusion protein comprising the C-terminal propeptide of a procollagen, wherein the C-terminal propeptide of said recombinant polypeptide forms an inter-polypeptide disulfide bond.

38. A trimeric fusion protein comprising three recombinant polypeptides, each recombinant polypeptide comprising, from amino terminus to carboxy terminus: a) a soluble coronavirus surface antigen comprising the spike (S) extracellular domain of SARS-CoV-2 virus Wuhan-Hu-1 or B.1.351 strain; b) a C-terminal propeptide of a procollagen, wherein the C-terminal propeptide of the recombinant polypeptide forms an inter-polypeptide disulfide bond.

39. A fusion protein described in claim 37 or 38, for use in preventing infection by coronavirus in mammals.

40. The fusion protein of claim 39, wherein the coronavirus is selected from the group consisting of Wuhan-Hu-1, B. 1.1.7, B. 1.351, and P. 1 strains.

41. 41. The fusion protein of claim 39 or 40, comprising immunizing the mammal by administering the fusion protein two or more times.

42. 42. The fusion protein of any one of claims 39-41, wherein the fusion protein is administered as a booster dose following one or more doses of an immunogen comprising a spike protein peptide comprising the NTD and RBD from the same SARS-CoV-2 variant.

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