Coronavirus vaccine composition, method therefor and use thereof

Abstract

The present invention relates to an immunogenic composition comprising a recombinant peptide and protein, wherein the recombinant peptide and protein comprise a coronavirus antigen and immunogen, for example, a chimeric antigen and immunogen of an S protein peptide or a fragment, variant or mutant sequence thereof of SARS-CoV-2 Hu-1, SARS-CoV-2 Omicron (BA.5 and / or XBB.1.5) variant, and / or other variants. The immunogenic composition comprises a secreted fusion protein, which comprises a soluble coronavirus antigen, wherein the soluble coronavirus antigen protein is linked, by means of in-frame fusion, to a C-terminal moiety of a collagen capable of self-trimerization to form a disulfide bond-linked trimeric fusion protein. The immunogenic composition can be used for generating an immune response, and can be used in a vaccine composition. Further provided are methods for producing a recombinant peptide and protein, methods for prevention, treatment and / or diagnosis, and a related kit.

Description

Coronavirus vaccine compositions, methods, and uses thereof

[0001] This application claims priority to Chinese patent applications with application number 202310663932.3 filed on May 30, 2023, entitled “Coronavirus vaccine compositions, methods and uses thereof” and with application number 202311196430.0 filed on September 10, 2023, entitled “Coronavirus vaccine compositions, methods and uses thereof”, the entirety of which are hereby incorporated herein by reference. Technical Field

[0002] The present disclosure relates in some aspects to immunogenic compositions for treating and / or preventing coronavirus infection, comprising recombinant peptides and proteins, including coronavirus antigens and immunogens, such as coronavirus S protein peptides, including S protein peptides based on SARS-CoV-2 Hu-1, Omicron strains subtype BA.5 and XBB.1.5, and trimerized TM Subunit vaccines formed by disulfide bonds between tag polypeptides and / or their combinations. Background Art

[0003] Coronaviruses infect a wide range of birds and mammals, including humans. Coronaviruses may circulate in humans each year, usually causing mild respiratory illnesses, although they are more severe in infants, the elderly, and immunocompromised people. However, certain coronaviruses, including 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. The high pathogenicity, airborne transmission, high mortality rate, and poorly defined epidemiology of coronaviruses make effective vaccines and related therapeutic agents urgently needed. In particular, there is an urgent need for vaccines that can rapidly induce an effective immune response against SARS-CoV-2. The present invention provides methods, uses, and articles of manufacture that meet the above and other needs.

[0004] Summary of the Invention

[0005] Starting in late 2020, the emergence and spread of multiple mutant SARS-CoV-2 strains carrying mutations that may lead to immune escape necessitated the rapid evaluation of second-generation vaccines with the goal of inducing optimized immune responses that are broadly protective.

[0006] Despite tremendous progress and unprecedented speed in COVID-19 vaccine development, a new record number of daily COVID-19 cases was set in April 2021, 16 months after the SARS-CoV-2 outbreak first emerged.

[0007] Of greatest concern is the emergence of multiple new SARS-CoV-2 variants (VOCs) beginning in late 2020, coinciding with the global surge in COVID-19 cases. These VOCs appear to be associated with mutations in the spike (S) protein that could increase viral transmissibility and / or immune escape from the first wave of COVID-19 vaccinations based on the SARS-CoV-2 Hu-1 strain. The emergence and spread of the B.1.1.7 variant in the United Kingdom (UK), the B.1.351 variant in South Africa, and the P.1 variant in Brazil led to their classification as VOCs. These VOCs all include the N501Y mutation in the receptor binding domain (RBD) of the S protein, which has been reported to increase transmissibility by 40% to 70%. The B.1.351 and P.1 variants have two additional RBD mutations—E484K and K417—that could allow for immune escape from Hu-1 vaccines and antibodies induced by natural infection.

[0008] Randomized controlled clinical trials of COVID-19 vaccines have shown reduced effectiveness against VOCs compared to the SARS-CoV-2 Hu-1 strain. The adjuvant protein-based COVID-19 vaccine NVX-CoV2373 was 89% effective in the UK (where B.1.1.7 is dominant), but only 49% effective in South Africa (where B.1.351 is dominant). The adenovirus vectored COVID-19 vaccine ChAdOx1 was only 10% effective against the B.1.351 variant. Pfizer vaccine recipients were 75% effective against the B.1.351 variant, compared to 95% effective against Hu-1. Coronavac is an inactivated vaccine based on the Hu-1 strain, and no neutralizing antibody titers against P.1 were detected in subjects who received the vaccine.

[0009] While there is some encouraging evidence that the Hu-1 COVID-19 vaccine may prevent severe illness and death caused by VOCs, vaccine effectiveness against any COVID-19 disease with increasing transmission rates is low. The rapid global spread of SARS-CoV-2 VOCs is likely to result in the continued emergence of new target variants or VOCs that may contain new escape mutations, such as the Indian variant (B.1.617), which emerged in conjunction with a large surge in COVID-19 cases in the spring of 2021 and has been declared a new VOC by the WHO. The B.1.617.2 variant of this lineage was named Delta, and the B.1.1.529 variant first identified in South Africa was renamed Omicron, along with the Omicron subtypes BA.1, BA.2, BA.2.75, BA.5, BQ.1.1, BF.7, XBB, and XBB.1.5.

[0010] In these situations, it is imperative to rapidly evaluate re-boosting strategies to enhance neutralizing antibody responses to VOCs and to develop new-generation monovalent and multivalent COVID-19 vaccines that may provide optimized broad protection and cross-neutralizing properties.

[0011] In some embodiments, disclosed herein are proteins comprising a plurality of recombinant polypeptides, each recombinant polypeptide comprising a coronavirus surface antigen linked to a C-terminal propeptide of collagen, wherein the C-terminal propeptides of the recombinant polypeptides form an inter-polypeptide disulfide bond. The recombinant polypeptides or proteins can be used as immunogens, such as vaccines.

[0012] In some embodiments, disclosed herein are recombinant subunit vaccines comprising an extracellular domain (e.g., without transmembrane and cytoplasmic domains) of the subtype XBB.1.5 S protein or a fragment thereof from a coronavirus (e.g., SARS-CoV-2 Omicron (B.1.1.529) fused in frame to the C-propeptide of collagen capable of forming disulfide-linked homotrimers. The recombinant subunit vaccine (e.g., S-trimer) thus produced can be expressed and purified from transfected cells and is expected to be in its native conformation as a trimer. This solves the problem of viral antigen misfolding that is often encountered when viral antigens are expressed in a soluble form as recombinant peptides or proteins without transmembrane and / or cytoplasmic domains. Such misfolded viral antigens cannot faithfully maintain the native viral antigen conformation and often fail to generate neutralizing antibodies.

[0013] In some embodiments, the coronavirus is severe acute respiratory syndrome (SARS) coronavirus (SARS-CoV-1), SARS coronavirus 2 (SARS-CoV-2), a SARS-like coronavirus, Middle East respiratory syndrome (MERS) coronavirus (MERS-CoV), a MERS-like coronavirus, NL63-CoV, 229E-CoV, OC43-CoV, HKU1-CoV, WIV1-CoV, MHV, HKU9-CoV, PEDV-CoV, or SDCV.

[0014] In some embodiments, disclosed herein is a multivalent vaccine comprising a coronavirus (S) protein surface antigen or a fragment, variant or mutant thereof, wherein the coronavirus (S) protein surface antigen or a fragment, variant or mutant thereof is freely selected from soluble SARS-CoV-2 Hu-1, SARS-CoV-2 Omicron (B.1.1.529) and its subtypes, alpha, beta, gamma, delta, muon spike (S) protein surface antigen or a fragment, variant or mutant thereof, which coronavirus (S) protein surface antigen or a fragment, variant or mutant thereof is linked to the C-terminal propeptide portion of collagen to form a disulfide-linked trimeric fusion protein.

[0015] The multivalent vaccine described in any of the preceding embodiments, wherein the SARS-CoV-2 Omicron subtype spike (S) protein surface antigen or its fragment, variant or mutant includes BA.1, BA.2, BA.2.75, BA.5, BQ.1.1, BF.7, XBB, XBB.1.5, BQ.1.1 spike (S) protein surface antigen or its fragment, variant or mutant.

[0016] The multivalent vaccine described in any of the preceding embodiments comprises the soluble SARS-CoV-2 Hu-1(S) protein surface antigen or a fragment, variant or mutant thereof.

[0017] The multivalent vaccine described in any of the foregoing embodiments comprises a soluble SARS-CoV-2 Omicron BA.5(S) protein surface antigen or a fragment, variant or mutant thereof.

[0018] The multivalent vaccine described in any of the foregoing embodiments comprises a soluble SARS-CoV-2 Omicron XBB.1.5(S) protein surface antigen or a fragment, variant or mutant thereof.

[0019] The multivalent vaccine described in any of the foregoing embodiments is a trivalent vaccine and includes soluble SARS-CoV-2 Hu-1, Omicron strains BA.5 and XBB.1.5 and spike (S) protein surface antigen or its fragments, variants or mutants, in a weight ratio of 0.5-4:0.5-4:0.5-4.

[0020] The multivalent vaccine described in any of the foregoing embodiments is a trivalent vaccine and comprises soluble SARS-CoV-2 Hu-1, Omicron strains BA.5 and XBB.1.5 and spike (S) protein surface antigen or its fragments, variants or mutants in a weight ratio of 1:1:1 or 1:2:2.

[0021] The multivalent vaccine described in any of the preceding embodiments, wherein the coronavirus (S) protein surface antigen or a fragment, variant or mutant thereof comprises a sequence described in any one of SEQ ID NOs: 27-66 or an amino acid sequence that has at least or about 80%, 85%, 90%, 92%, 95%, 97%, or 99% sequence identity with the sequence described in any one of SEQ ID NOs: 27-66.

[0022] The multivalent vaccine described in any of the preceding embodiments, wherein the coronavirus (S) protein surface antigen or fragment thereof or variant or mutant thereof comprises the sequence described in any one of SEQ ID NOs: 81-84 or an amino acid sequence that has at least or about 80%, 85%, 90%, 92%, 95%, 97%, or 99% sequence identity with the sequence described in any one of SEQ ID NOs: 81-84.

[0023] The multivalent vaccine described in any of the preceding embodiments, wherein the coronavirus (S) protein surface antigen or fragment thereof or variant or mutant thereof comprises the sequence of SEQ ID NO: 126 or an amino acid sequence that has at least or about 80%, 85%, 90%, 92%, 95%, 97%, or 99% sequence identity to the sequence of SEQ ID NO: 126.

[0024] The multivalent vaccine described in any of the preceding embodiments, wherein the coronavirus (S) protein surface antigen or fragment thereof or variant or mutant thereof comprises the sequence of SEQ ID NO: 122 or an amino acid sequence that has at least or about 80%, 85%, 90%, 92%, 95%, 97%, or 99% sequence identity to the sequence of SEQ ID NO: 122.

[0025] In some embodiments, the multivalent vaccine is a bivalent vaccine and can be freely selected from SARS-CoV-2 Hu-1, Alpha, Beta, Gamma, Delta, Mu Spike, Omicron (B.1.1.529), BA.1, BA.2, BA.2.75, BA.5, BQ.1.1, BF.7, XBB, XBB.1.5, BQ.1.1 spike (S) protein surface antigen or its fragment, variant or mutant, wherein the weight ratio of the two coronavirus (S) protein surface antigens or its fragment or variant or mutant included in the bivalent vaccine is 0.5-4:0.5-4.

[0026] The bivalent vaccine described in the aforementioned embodiment includes the BA.5 and XBB.1.5 in a weight ratio of 1:1 or 1:2; or the bivalent vaccine includes the Hu-1 and BA.5 in a weight ratio of 1:1 or 1:2; or the bivalent vaccine includes the Hu-1 and XBB.1.5 in a weight ratio of 1:1 or 1:2.

[0027] The multivalent vaccine described in any of the preceding embodiments, wherein the multivalent vaccine is administered without an adjuvant, optionally, the multivalent vaccine is used as a primary series, additional dose, and / or homologous or heterologous booster dose, such as the first dose, second dose, third dose, fourth dose, and / or more doses, optionally, the primary dose, additional dose or heterologous booster is used in combination with any one or more of other recombinant subunit vaccines, nanoparticle vaccines, mRNA vaccines, DNA vaccines, adenovirus vector vaccines and inactivated virus vaccines.

[0028] In some embodiments, the multivalent vaccine is administered with one or more adjuvants, optionally used as a primary series, additional dose, and / or homologous or heterologous booster dose, such as the first dose, second dose, third dose, fourth dose, and / or more doses, optionally used in combination with any one or more of other recombinant subunit vaccines, nanoparticle vaccines, mRNA vaccines, DNA vaccines, adenovirus vector vaccines and inactivated virus vaccines.

[0029] In the multivalent vaccine of any of the aforementioned embodiments, the adjuvants in the initial dose, additional dose and / or homologous or heterologous booster may independently include: aluminum-containing adjuvants, such as adjuvants containing alum and / or aluminum hydroxide; oligonucleotide-containing adjuvants, such as adjuvants containing CpG oligodeoxynucleotides (CpG-ODN); adjuvants containing TLR9 agonists; adjuvants containing metabolizable oils, α-tocopherol, and / or polyoxyethylene sorbitan monooleate (Tween-80), such as adjuvants containing squalene, α-tocopherol, and Tween-80 and / or Span 85 in the form of an oil-in-water emulsion; or combinations of any of said adjuvants.

[0030] In the multivalent vaccine of any of the aforementioned embodiments, the adjuvant in the initial dose, additional dose and / or homologous or heterologous booster may include an adjuvant of CpG oligodeoxynucleotide (CpG-ODN) and / or an adjuvant containing alum and / or aluminum hydroxide.

[0031] In any of the preceding embodiments, the multivalent vaccine of any one of claims is administered by intramuscular injection.

[0032] In any of the preceding embodiments, the multivalent vaccine of any one of claims is administered by intranasal spray.

[0033] In any of the preceding embodiments, the multivalent vaccine of any one of claims is administered as a single dose or as a series of doses spaced apart by weeks or months.

[0034] In any of the foregoing embodiments, any of the multivalent vaccines described can prevent or treat coronavirus infection, such as severe acute respiratory syndrome (SARS)-coronavirus 2 (SARS-CoV-2) infection.

[0035] In any of the foregoing embodiments, any of the multivalent vaccines, wherein the C-terminal propeptide comprises any one of SEQ ID NOs: 67-80 or an amino acid sequence that is at least 80%, 85%, 90%, 92%, 95%, 97%, or 99% identical thereto.

[0036] Also provided herein are immunogens comprising proteins provided herein.Protein nanoparticles comprising proteins provided herein directly or indirectly attached to nanoparticles are provided herein.Virus-like particles (VLPs) comprising proteins provided herein are provided herein.

[0037] Also provided herein are isolated nucleic acids 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 a polypeptide encoding the C-terminal propeptide of collagen. In some embodiments, the isolated nucleic acids provided herein are operably linked to a promoter.

[0038] 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, optionally an mRNA molecule, such as a nucleoside-modified mRNA, a non-amplified mRNA, a self-amplified mRNA, or a trans-amplified mRNA.

[0039] Also provided herein are vectors comprising the isolated nucleic acids provided herein.In some embodiments, the vector is a viral vector.

[0040] In some aspects, provided herein are viruses, pseudoviruses or cells comprising a vector provided herein, optionally wherein the virus or cell has 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 and a pharmaceutically acceptable carrier provided herein.

[0041] Also provided herein are vaccines comprising the immunogenic compositions provided herein and optional adjuvants, wherein the vaccine is optionally a subunit vaccine. In some embodiments, the vaccine is a preventive and / or therapeutic vaccine. Optional adjuvants can be used in primes and / or boosters. Independently, the adjuvants of primes and / or any one or more boosters may include: aluminum-containing adjuvants, such as adjuvants containing alum and / or aluminum hydroxide; adjuvants containing oligonucleotides, such as adjuvants containing CpG oligodeoxynucleotides (CpG-ODN); adjuvants containing TLR9 agonists; adjuvants containing metabolizable oils, α-tocopherol, and / or polyoxyethylene sorbitan monooleate (Tween-80), such as adjuvants containing squalene, α-tocopherol, and Tween-80 and / or Span 85 in the form of an oil-in-water emulsion; or any combination of adjuvants.

[0042] In some aspects, provided herein are methods for producing a protein, 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.

[0043] Provided herein are methods for generating an immune response to a coronavirus S protein peptide or fragment or epitope thereof in a subject, 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 as provided herein to generate an immune response. In some embodiments, the methods provided herein are used to treat or prevent coronavirus infection. In some embodiments, generating an immune response inhibits or reduces the replication of coronavirus in the subject. In some embodiments, the immune response comprises a cell-mediated response and / or a humoral response, optionally comprising the production of one or more neutralizing antibodies, such as polyclonal antibodies or monoclonal antibodies. In some embodiments, the immune response is directed against a coronavirus S protein peptide or fragment or epitope thereof, but not against a C-terminal propeptide. In some embodiments, due to prior exposure to one or more coronaviruses, administration to the subject does not result in antibody-dependent enhancement (ADE) in the subject. In some embodiments, when subsequently exposed to one or more coronaviruses, administration does not result in antibody-dependent enhancement (ADE) in the subject. In some embodiments, the method further comprises a priming step and / or a boosting step. In some embodiments, the administering step is performed topically, transdermally, subcutaneously, intradermally, orally, intranasally (e.g., intranasal spray), intratracheally, sublingually, orally, rectally, vaginally, by inhalation, intravenously (e.g., intravenous injection), intraarterially, intramuscularly (e.g., intramuscular injection), intracardially, intraosseously, intraperitoneally, transmucosally, intravitreally, subretinally, intraarticularly, periarticularly, topically, or by 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 the use of an adjuvant. In some embodiments, the effective amount is administered with an adjuvant.

[0044] Provided herein is a method comprising administering to a subject an effective amount of a protein provided herein to produce neutralizing antibodies or neutralizing antiserum 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 neutralizing antibodies or neutralizing antiserum from the subject. In some embodiments, the method further comprises administering to a human subject an effective amount of isolated neutralizing antibodies or neutralizing antiserum by passive immunization to prevent or treat coronavirus infection. In some embodiments, neutralizing antibodies or neutralizing antiserum against coronavirus include polyclonal antibodies against coronavirus S protein peptides or fragments or epitopes thereof, optionally, wherein the neutralizing antibodies or neutralizing antiserum do not contain or are substantially free of antibodies against the C-terminal propeptide of collagen. In some embodiments, neutralizing antibodies include monoclonal antibodies against coronavirus S protein peptides or fragments or epitopes thereof, optionally, wherein the neutralizing antibodies do not contain or are substantially free of antibodies against the C-terminal propeptide of collagen.

[0045] In some aspects, the proteins, immunogens, protein nanoparticles, VLPs, isolated nucleic acids, vectors, viruses, pseudoviruses, cells, immunogenic compositions, or vaccines provided herein are used to induce an immune response to a coronavirus in a subject, and / or to treat or prevent a coronavirus infection.

[0046] In some aspects, provided herein are uses of proteins, immunogens, protein nanoparticles, VLPs, isolated nucleic acids, vectors, viruses, pseudoviruses, cells, immunogenic compositions, or vaccines provided herein for inducing an immune response to coronavirus in a subject, and / or for treating or preventing coronavirus infection. In some aspects, provided herein are uses of proteins, immunogens, protein nanoparticles, VLPs, isolated nucleic acids, vectors, viruses, pseudoviruses, cells, immunogenic compositions, or vaccines provided herein for manufacturing a medicine or prophylactic for inducing an immune response to coronavirus in a subject, and / or for treating or preventing coronavirus infection.

[0047] Also provided herein are methods for analyzing a sample, comprising contacting the sample with a protein provided herein, and detecting binding between the protein and an analyte that specifically binds 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 the presence of a coronavirus infection in the subject from whom the sample was derived.

[0048] Provided herein are kits comprising a protein provided herein and a matrix, pad, or vial containing or immobilizing the protein, optionally wherein the kit is an ELISA or lateral flow assay kit. BRIEF DESCRIPTION OF THE DRAWINGS

[0049] Figure 1 shows a schematic diagram of the cross-neutralization immunogenicity assay design in mice using two doses of monovalent, bivalent, or trivalent S-trimer antigen (prime dose, homologous boost dose, homologous boost dose). Mice were immunized twice (n=20 / group) with S-trimer adjuvanted with CpG 1018 and Alum on day 0 (prime dose) and day 21 (boost dose). In the Hu-1 homologous boost group, both the prime and boost doses were Hu-1 S-trimer; in the Omicron Ba.5 or XBB.1.5 homologous boost groups, both the prime and boost doses were Omicron S-trimer Ba.5 or XBB.1.5, respectively. Adjuvants were CpG 1018: 150 μg, Alum: 75 μg; total S-trimer antigen dosage per dose: 3 μg. Intramuscular injection (single point), 0.05mL / mouse / time. After the injection, observe the animal's condition and return it to the cage for free movement. On D0 (before immunization), blood was collected from the orbital venous plexus using a capillary glass blood collection tube, with a collection volume of approximately 200μL. On D35, the mice were euthanized and all blood was collected from the mice. All serum samples were allowed to stand at room temperature for 0.5 to 1 hour, then centrifuged (×2500g) for 10 minutes, separated, and aliquoted. Storage conditions: frozen at ≤-20°C. The titer of neutralizing antibodies against the new coronavirus pseudovirus was tested on D0 and D35 sera.

[0050] Figure 2 shows the neutralizing antibody IC values ​​of pseudoviruses of SARS-CoV-2 Hu-1, beta (beta, β, B.1.351), delta (delta, δ, B.1.617.2), and Omicron (O, B.1.1.529) subtypes BA.1, BA.2, BA.2.75, BA.5, BQ.1.1, BF.7, XBB, XBB.1.5 after mice received two doses of S-trimer antigen (first dose, primary immunization - second dose, booster) according to the experimental design of Figure 1 and the vaccine dose of Table 2 50 Data. After two immunizations with 150 μg CpG 1018 and 75 μg Alum as adjuvant, mice were bled on day 35 (D4 PD2) for pseudovirus neutralizing antibody testing. A. is the IC of Hu-1 pseudovirus neutralizing antibody. 50 Data, Hu-1 ((Prototype)) novel coronavirus pseudovirus neutralizing antibody titer (IC 50) GMT are 6392, 326, 87, 5661, 1825, 3227, 1740, 461, 207, 2236; B. is the IC of the neutralizing antibody against the beta strain pseudovirus 50 Data, the titer of neutralizing antibodies to the new coronavirus pseudovirus (Beta) in the serum of immunized mice on day 35 (IC 50 ) GMT are 659, 42, 32, 722, 135, 224, 117, 91, 102, 334; C. is the Delta strain pseudovirus neutralizing antibody IC 50 Data, the neutralizing antibody titer (IC) of the new coronavirus pseudovirus (Delta) in the serum of immunized mice on D35 in groups 1-10 (n=20) 50 ) GMT are 1367, 53, 26, 1386, 392, 1095, 353, 131, 58, 541; D. is the IC of the neutralizing antibody against the BA.1 strain pseudovirus 50 Data, the titer of neutralizing antibodies to the new coronavirus pseudovirus (BA.1) in the serum of immunized mice on day 35 (IC 50 ) GMT are 60, 269, 57, 450, 381, 111, 51, 377, 442, and 288; E. is the IC of the neutralizing antibody against the BA.2 pseudovirus 50 Data, the titer of neutralizing antibodies to the new coronavirus pseudovirus (BA.2) in the serum of immunized mice on day 35 (IC 50 ) GMT are 102, 4703, 843, 5523, 6239, 887, 1093, 5114, 8091, 5647; F. is the IC of the neutralizing antibody against the BA.2.75 strain pseudovirus 50 Data, the titer of neutralizing antibodies to the new coronavirus pseudovirus (BA.2.75) in the serum of immunized mice on day 35 (IC 50 )DUGMT are 110, 1614, 979, 2187, 1996, 1035, 1008, 3374, 3883, and 2798 respectively; G. is the IC of the neutralizing antibody against the BA.5 pseudovirus. 50 Data, the titer of neutralizing antibodies to the new coronavirus pseudovirus (BA.5) in the serum of immunized mice on day 35 (IC 50 ) GMT are 79, 22438, 4685, 27151, 33923, 1529, 2318, 19379, 19086, and 17767; H. is the IC of the neutralizing antibody against the BQ.1.1 pseudovirus. 50 Data, the neutralizing antibody titer (IC 50) GMT are 41, 18599, 3093, 13337, 21384, 1320, 1015, 9356, 10120; I. is the IC of the BF.7 pseudovirus neutralizing antibody 50 Data, the titer of neutralizing antibodies to the new coronavirus pseudovirus (BF.7) in the serum of immunized mice on day 35 (IC 50 ) GMT are 51, 21514, 3849, 21797, 31887, 2100, 1998, 21410, 21798, 19817; J. is the IC of the neutralizing antibody against the XBB strain pseudovirus 50 Data, the titer of neutralizing antibodies against the new coronavirus pseudovirus (XBB) in the serum of immunized mice on day 35 (IC 50 ) GMT are 22, 1396, 4934, 355, 1035, 3149, 3119, 4430, 5345, 3307; K. is the IC of the neutralizing antibody against the XBB.1.5 strain pseudovirus 50 Data, the titer of neutralizing antibodies to the new coronavirus pseudovirus (XBB.1.5) in the serum of immunized mice on day 35 (IC 50 ) GMT are 22, 1045, 15420, 555, 769, 7632, 9772, 6841, 9255, and 5834. GMT is the geometric mean titer.

[0051] Figure 3 shows a statistical analysis of serum neutralizing antibody titers against the COVID-19 pseudovirus in patients receiving the trivalent S-trimer vaccine versus the monovalent or bivalent S-trimer vaccines. A. Spider plot of the trivalent vaccine versus the bivalent vaccine with a 1:1 antigen ratio; B. Spider plot of the trivalent vaccine versus the bivalent vaccine with a 1:2 antigen ratio; C. Spider plot of the trivalent vaccine versus the monovalent vaccine.

[0052] Figure 4 shows a spider web diagram of the statistical analysis of the ratio of antigens contained in the multivalent vaccine to the neutralization titer of the pseudovirus. A. Bivalent vaccine Hu-1 + BA.5 (1:1 VS. 1:2); B. Bivalent vaccine Hu-1 + XBB.1.5 (1:1 VS. 1:2); C. Bivalent vaccine BA.5 + XBB.1.5 (1:1 VS. 1:2). Overall, there is not much difference between the 1:1 formulation and the 1:2 formulation of the three vaccines. However, reducing the proportion of Hu-1 may reduce the immunogenicity against the earlier virus Hu-1, beta, and delta variants. Adding BA.5 and XBB.1.5 antigens to the three vaccine formulations did not show any benefit.

[0053] Figure 5 shows the design of a heterologous booster trial with Hu-1 S-trimer as the primary dose and bivalent or trivalent vaccine as the booster. Freshly infected BALB / c mice were immunized twice with 3 μg of Hu-1 S-trimer adjuvanted with 150 μg CpG 1018 and 75 μg Alum on day 0 (dose 1) and day 21 (dose 2). On day 156 of the study, blood was drawn for serum collection and the mice received the third dose of vaccination: immunization with 3 μg Hu-1 S-trimer vaccine (control) or bivalent or trivalent S-trimer vaccine adjuvanted with 150 μg CpG 1018 plus 75 μg Alum; blood was drawn again for serum collection on day 170 of the study (D14, pd 3), blood was drawn for serum collection on day 184 of the study (D28 PD3) and the mice were immunized again, and finally blood was drawn for serum collection on day 198 (D14, pd 4) and day 212 (D28, pd 4).

[0054] Figure 6 shows the neutralizing antibody IC of pseudoviruses of SARS-CoV-2 Hu-1, Beta (beta, β, B.1.351), Delta (delta, δ, B.1.617.2), and Omicron (O, B.1.1.529) subtypes BA.1, BA.2, BA.2.75, BA.5, BQ.1.1, BF.7, XBB, and XBB.1.5 strains after newly infected mice received 4 doses of heterologous booster immunization with S-trimer antigen according to the vaccine dose in Figure 5. 50 Data. A. Neutralizing antibody titer against Hu-1 pseudovirus in sera of immunized mice; B. Neutralizing antibody titer against Beta pseudovirus in sera of immunized mice; C. Neutralizing antibody titer against Delta pseudovirus in sera of immunized mice; D. Neutralizing antibody titer against BA.1 pseudovirus in sera of immunized mice; E. Neutralizing antibody titer against BA.2 pseudovirus in sera of immunized mice; F. Neutralizing antibody titer against BA.2.75 pseudovirus in sera of immunized mice; G. Neutralizing antibody titer against BA.5 pseudovirus in sera of immunized mice; H. Neutralizing antibody titer against BF.7 pseudovirus in sera of immunized mice; I. Neutralizing antibody titer against BQ.1.1 pseudovirus in sera of immunized mice; J. Neutralizing antibody titer against XBB pseudovirus in sera of immunized mice; K. Neutralizing antibody titer against XBB.1.5 pseudovirus in sera of immunized mice. GMT is the geometric mean titer.

[0055] FIG7 shows a spider graph of neutralizing antibody titers on D184 (D28 after the first booster immunization).

[0056] Figure 8 is a schematic diagram of the cross-neutralization immunogenicity test design based on pseudovirus neutralization titers in immunized BALB / c mice using Hu-1 S-trimer as the initial dose and the second dose and monovalent or multivalent S-trimer antigen as the third dose for heterologous boosting. After vaccination with Hu-1 as the basic immunization program, the S-Trimer recombinant subunit vaccine (multivalent or monovalent new crown vaccine) under different combination conditions was selected for the third dose booster. Pre-existing immunization was performed on D0 and D7 using Hu-1 S-trimer antigen, and the pre-existing immunization ended after two doses. Booster immunization was performed on D108 using intramuscular injection (single point), 0.05 mL / animal / time. After the injection, the animal's condition was observed and returned to the cage for free movement. A total of three doses were administered during the whole process, and the day of the first dose of pre-existing immunization was defined as D0. Blood was collected from the orbital venous plexus using capillary glass blood collection tubes on D107 (before immunization), D122, and D136, with a collection volume of approximately 400 μL. All serum samples were allowed to stand at room temperature for 0.5 to 1 hour, then centrifuged (×2500g) for 10 minutes. Serum was separated and aliquoted for storage at ≤ -20°C. SARS-CoV-2 Hu-1, Beta, Delta, and Omicron (Omicron, B.1.1.529) subtypes BA.1, BA.2, BA.2.75, BA.5, BF.7, XBB, XBB.1.5, BQ.1.1, XBB.1.16.1, and EG.5.1 were used to determine pseudovirus neutralizing antibody titers. SARS-CoV-2 Hu-1, Beta, and Delta are pre-Omicron strains.

[0057] Figure 9 shows the neutralizing antibody titers of the new coronavirus pseudovirus (Hu-1 / Beta / Delta / BA.1 / BA.2 / BA.2.75 / BA.5 / BF.7 / BQ.1.1 / XBB / XBB.1.5 / XBB.1.16.1 / EG.5.1) measured after immunization according to the experimental design of Figure 8. A. Neutralizing antibody titers of the new coronavirus pseudovirus Hu-1 in the serum of immunized mice (IC 50 ); B. Neutralizing antibody titer of novel coronavirus pseudovirus beta in sera of immunized mice (IC 50 ); C. Neutralizing antibody titer of novel coronavirus pseudovirus delta in sera of immunized mice (IC 50 ); D. Neutralizing antibody titer of SARS-CoV-2 pseudovirus BA.1 in sera of immunized mice (IC 50 ); E. Neutralizing antibody titer of SARS-CoV-2 pseudovirus in sera of immunized mice (IC 50 ); F. Neutralizing antibody titer of SARS-CoV-2 pseudovirus BA.2.75 in sera of immunized mice (IC 50 ); G. Neutralizing antibody titer of SARS-CoV-2 pseudovirus BA.5 in sera of immunized mice (IC 50); H. Neutralizing antibody titer of SARS-CoV-2 pseudovirus BF.7 in sera of immunized mice (IC 50 ); I. Neutralizing antibody titer of novel coronavirus pseudovirus BQ.1.1 in sera of immunized mice (IC 50 ); J. Neutralizing antibody titer of novel coronavirus pseudovirus XBB in sera of immunized mice (IC 50 ); K. Neutralizing antibody titer of novel coronavirus pseudovirus XBB1.5 in sera of immunized mice (IC 50 ); L. Neutralizing antibody titer of novel coronavirus pseudovirus XBB.1.16.1 in sera of immunized mice (IC 50 ); M. Neutralizing antibody titer of novel coronavirus pseudovirus EG.5.1 in sera of immunized mice (IC 50 ). GMT is the geometric mean titer.

[0058] FIG10 shows a spider graph of neutralizing antibody titers on D122 (D14 after booster immunization).

[0059] FIG11 shows a spider graph of neutralizing antibody titers on D135 (D28 after booster immunization). DETAILED DESCRIPTION

[0060] Provided herein are immunogenic compositions, methods, and uses of fusion peptides and proteins comprising coronavirus antigens or immunogens for the treatment (e.g., prophylactic, therapeutic) of coronavirus infections. In some embodiments, compositions and methods of use of recombinant soluble surface antigens from RNA viruses in the form of covalently linked trimers are disclosed. In some embodiments, the fusion proteins produced are secreted as disulfide-linked homotrimers, which are more structurally stable while retaining the conformation of native trimeric viral antigens, and therefore can be used as more effective vaccines against these dangerous pathogens.

[0061] In some embodiments, disclosed herein is a method for preventing viral infection using a viral antigen trimer as a vaccine or as part of a multivalent vaccine, without or with an adjuvant, or with more than one adjuvant, optionally administered by intramuscular injection or intranasal administration. The viral antigen trimer can be used in an initial dose, an additional dose, and / or a booster. Independently, the initial dose, the additional dose, and / or any one or more boosters may be administered without or with an adjuvant. If an adjuvant is used, the optional adjuvant may include: an aluminum-containing adjuvant, such as an adjuvant containing alum and / or aluminum hydroxide; an adjuvant containing an oligonucleotide, such as an adjuvant containing CpG oligodeoxynucleotide (CpG-ODN); an adjuvant containing a TLR9 agonist; an adjuvant containing a metabolizable oil, α-tocopherol, and / or polyoxyethylene sorbitan monooleate (Tween-80), such as an adjuvant containing squalene, α-tocopherol, and Tween-80 and / or Span 85 in the form of an oil-in-water emulsion; or any combination of adjuvants.

[0062] In some embodiments, disclosed herein are methods of using viral antigen trimers as antigens for diagnosing viral infection by detecting antibodies (eg, IgM or IgG) that recognize viral antigens (eg, 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 infection).

[0064] In some embodiments, disclosed herein are viral antigen trimers as a vaccine or as part of a multivalent vaccine, wherein the vaccine comprises a plurality of trimeric subunit vaccines comprising viral antigens of the same protein of a virus, or comprising viral antigens of two or more different proteins of one or more viruses or viral antigens of 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 is a monovalent vaccine comprising an S-trimer disclosed herein. In some embodiments, disclosed herein is a bivalent vaccine comprising an S-trimer disclosed herein. In some embodiments, disclosed herein is a bivalent vaccine 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 from the same S protein of one or more viral species or strains / subtypes, or two or more different S proteins from one or more viral species or one or more strains / subtypes of the same viral species. In some embodiments, disclosed herein is a trivalent vaccine comprising an S-trimer disclosed herein. In some embodiments, disclosed herein is a trivalent vaccine 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 is a tetravalent vaccine comprising an S-trimer disclosed herein. In some embodiments, disclosed herein is a tetravalent vaccine 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] In some embodiments, disclosed herein is a multivalent vaccine, such as a bivalent vaccine, comprising: a first trimeric fusion protein comprising a soluble coronavirus SARS-CoV-2 Omicron (B.1.1.529) spike (S) subtype protein surface antigen or a fragment, variant or mutant thereof, the soluble coronavirus surface antigen or a fragment, variant or mutant thereof being linked to the C-terminal portion of collagen, the C-terminal portion of the collagen forming a disulfide bond-linked trimer, thereby forming the first trimeric fusion protein; a second and / or more trimeric fusion proteins, the second and / or more trimeric fusion proteins comprising a soluble coronavirus SARS-CoV-2 Hu-1, alpha, beta, gamma, delta, Omicron (B.1.1.529) and subtypes thereof or mu spike (S) protein surface antigen or fragments, variants or mutants thereof, the soluble coronavirus surface antigen or fragments, variants or mutants thereof are linked to the C-terminal portion of collagen, and the C-terminal portion of the collagen forms a disulfide-linked trimer, thereby forming the second and / or more trimeric fusion proteins. In some embodiments, the Omicron subtype spike (S) protein surface antigen or fragments, variants or mutants thereof include BA.1, BA.2, BA.2.75, BA.5, BQ.1.1, BF.7, XBB, XBB.1.5, BQ.1.1 spike (S) protein surface antigen or fragments, variants or mutants thereof. In some embodiments, the first trimeric fusion protein includes a soluble coronavirus SARS-CoV-2 BA.2 or XBB.1.5 spike (S) protein surface antigen or fragments, variants or mutants thereof. In some embodiments, the second trimeric fusion protein comprises a soluble coronavirus SARS-CoV-2 Hu-1 or Omicron subtype BA.2 or XBB.1.5 spike (S) protein surface antigen or a fragment, variant or mutant thereof. In some embodiments, disclosed herein is a multivalent vaccine, such as a bivalent vaccine, comprising: a soluble coronavirus SARS-CoV-2 Hu-1 and an Omicron subtype BA.2 spike (S) protein surface antigen or a fragment thereof, in a weight ratio of 0.5-4:0.5-4, such as 4:1, 3:1, 2:1, 1:1, 1:2, 1:3 or 1:4. In some embodiments, disclosed herein is a multivalent vaccine, such as a bivalent vaccine, comprising: soluble coronavirus SARS-CoV-2 Omicron subtype BA.5 and XBB.1.5 spike (S) protein surface antigens or fragments thereof, in a weight ratio of 0.5-4:0.5-4, for example, 4:1, 3:1, 2:1, 1:1, 1:2, 1:3 or 1:4.In some embodiments, disclosed herein is a multivalent vaccine, such as a bivalent vaccine, comprising: soluble coronavirus SARS-CoV-2 Hu-1 and Omicron subtype XBB.1.5 spike (S) protein surface antigens or fragments thereof, in a weight ratio of 0.5-4:0.5-4, such as 4:1, 3:1, 2:1, 1:1, 1:2, 1:3 or 1:4. In some embodiments, the multivalent vaccine is administered without an adjuvant, and optionally the multivalent vaccine is used as a primary series, additional dose and / or homologous or heterologous booster dose, such as the first dose, the second dose, the third dose, the fourth dose, and / or more doses, and optionally the primary dose, additional dose or heterologous booster is used in combination with any one or more of other recombinant subunit vaccines, nanoparticle vaccines, mRNA vaccines, DNA vaccines, adenoviral vector vaccines, and inactivated virus vaccines. In some embodiments, the multivalent vaccine is administered with one or more adjuvants, optionally as a primary series, additional dose, and / or homologous or heterologous booster. dose), for example, the first dose, the second dose, the third dose, the fourth dose and / or more doses, optionally, the initial dose, the additional dose or the heterologous booster is used in combination with any one or more of other recombinant subunit vaccines, nanoparticle vaccines, mRNA vaccines, DNA vaccines, adenovirus vector vaccines, and inactivated virus vaccines, optionally, the adjuvant in the initial dose, the additional dose and / or the homologous or heterologous booster may independently include: an aluminum-containing adjuvant, for example, an adjuvant containing alum and / or aluminum hydroxide; an oligonucleotide-containing adjuvant, for example, an adjuvant containing CpG oligodeoxynucleotide (CpG-ODN); an adjuvant containing a TLR9 agonist; an adjuvant containing a metabolizable oil, α-tocopherol, and / or polyoxyethylene sorbitan monooleate (Tween-80), for example, an adjuvant containing squalene, α-tocopherol, and Tween-80 and / or Span 85 in the form of an oil-in-water emulsion; or a combination of any one of the adjuvants. In some embodiments, the first trimeric fusion protein in the multivalent vaccine comprises a sequence described in any one of SEQ ID NOs: 81-84, 122, and 126, or an amino acid sequence that has at least or about 80%, 85%, 90%, 92%, 95%, 97%, or 99% sequence identity to any one of SEQ ID NOs: 81-84, 122, and 126.In some embodiments, the second and / or more trimeric fusion proteins in the multivalent vaccine include a sequence described in any one of SEQ ID NOs: 27-66, or an amino acid sequence that has at least or about 80%, 85%, 90%, 92%, 95%, 97%, or 99% sequence identity to any one of SEQ ID NOs: 27-66.

[0068] In some embodiments, disclosed herein is a multivalent vaccine, such as a trivalent vaccine, comprising: a first trimeric fusion protein comprising a soluble coronavirus SARS-CoV-2 Omicron (B.1.1.529) spike (S) subtype protein surface antigen or a fragment, variant or mutant thereof, the soluble coronavirus surface antigen or a fragment, variant or mutant thereof being linked to the C-terminal portion of collagen, the C-terminal portion of the collagen forming a disulfide-linked trimer, thereby forming the first trimeric fusion protein; a second and third trimeric fusion protein and / or more trimeric fusion proteins, the second and third or more trimeric fusion proteins comprising a soluble coronavirus SARS-CoV-2 Hu-1, alpha, beta, gamma, delta, and a subtype of the Omicron (B.1.1.529) spike (S) protein that is different from the first trimeric fusion protein, or a mu spike (S) protein surface antigen or a fragment, variant or mutant thereof, the soluble coronavirus surface antigen or a fragment, variant or mutant thereof is linked to the C-terminal portion of collagen, and the C-terminal portion of the collagen forms a disulfide-linked trimer, thereby forming the second, third and / or more trimeric fusion proteins. In some embodiments, the Omicron subtype spike (S) protein surface antigen or a fragment, variant or mutant thereof includes BA.1, BA.2, BA.2.75, BA.5, BQ.1.1, BF.7, XBB, XBB.1.5, BQ.1.1 spike (S) protein surface antigen or a fragment, variant or mutant thereof. In some embodiments, the first and third trimeric fusion proteins comprise soluble coronavirus SARS-CoV-2 Omicron subtype BA.5 and XBB.1.5 spike (S) protein surface antigens or fragments, variants or mutants thereof. In some embodiments, the second trimeric fusion protein comprises soluble coronavirus SARS-CoV-2 Hu-1 spike (S) protein surface antigens or fragments, variants or mutants thereof. In some embodiments, the multivalent vaccine is a trivalent vaccine comprising: soluble coronavirus SARS-CoV-2 Hu-1, BA.5 and XBB.1.5 spike (S) protein surface antigens or fragments, variants or mutants thereof, in a weight ratio of 0.5-4:0.5-4:0.5-4, for example, 1:1:1 or 1:2:2, 1:3:3, 1:4:4, 2:1:1, 3:1:1, or 4:1:1.In some embodiments, the multivalent vaccine is administered without an adjuvant, optionally as a primary series, additional dose, and / or homologous or heterologous booster dose, for example, the first dose, the second dose, the third dose, the fourth dose, and / or more doses, optionally as a primary series, additional dose, or heterologous booster dose in combination with any one or more of other recombinant subunit vaccines, nanoparticle vaccines, mRNA vaccines, DNA vaccines, adenovirus vector vaccines, and inactivated virus vaccines. In some embodiments, the multivalent vaccine is administered with more than one adjuvant, optionally as a primary series, additional dose, and / or homologous or heterologous booster dose. dose), for example, the first dose, the second dose, the third dose, the fourth dose and / or more doses, optionally, the initial dose, the additional dose or the heterologous booster is used in combination with any one or more of other recombinant subunit vaccines, nanoparticle vaccines, mRNA vaccines, DNA vaccines, adenovirus vector vaccines, and inactivated virus vaccines, optionally, the adjuvant in the initial dose, the additional dose and / or the homologous or heterologous booster may independently include: an aluminum-containing adjuvant, for example, an adjuvant containing alum and / or aluminum hydroxide; an oligonucleotide-containing adjuvant, for example, an adjuvant containing CpG oligodeoxynucleotide (CpG-ODN); an adjuvant containing a TLR9 agonist; an adjuvant containing a metabolizable oil, α-tocopherol, and / or polyoxyethylene sorbitan monooleate (Tween-80), for example, an adjuvant containing squalene, α-tocopherol, and Tween-80 and / or Span 85 in the form of an oil-in-water emulsion; or a combination of any one of the adjuvants. In some embodiments, the first and third trimeric fusion proteins in the multivalent vaccine comprise the sequence of any one of SEQ ID NOs: 81-84, 122, and 126, or an amino acid sequence that has at least or about 80%, 85%, 90%, 92%, 95%, 97%, or 99% sequence identity to any one of SEQ ID NOs: 81-84, 122, and 126. In some embodiments, the second and / or more trimeric fusion proteins in the multivalent vaccine comprise the sequence of any one of SEQ ID NOs: 27-66, or an amino acid sequence that has at least or about 80%, 85%, 90%, 92%, 95%, 97%, or 99% sequence identity to any one of SEQ ID NOs: 27-66.

[0069] The proteins comprising coronavirus antigens and immunogens provided herein, including recombinant polypeptides and fusion proteins, can be used to effectively and safely treat (e.g., therapeutic, preventive) coronavirus infections. For example, the protein treatment coronavirus infections comprising coronavirus antigens and immunogens provided herein, without mediated vaccine-induced disease enhancement (VED) and / or antibody-dependent enhancement (ADE). In addition, the proteins comprising coronavirus antigens and immunogens provided herein are easy to produce and exhibit stability under high stress conditions such as high temperature, extreme pH, hyperosmotic pressure and hypoosmotic pressure. Therefore, the proteins and immunogenic compositions provided herein circumvent and meet the production, stability, safety and effectiveness issues that hinder the development of coronavirus vaccines.

[0070] In some aspects, the coronavirus antigens and immunogens provided herein include coronavirus spike (S) proteins or peptides, particularly SARS-CoV or SARS-CoV-2 S proteins. The spikes of SARS-CoV and SARS-CoV-2 are composed of S protein trimers, which belong to the group of class I viral fusion glycoproteins, which also includes HIV glycoprotein 160 (Env), influenza hemagglutinin (HA), paramyxovirus F, and Ebola virus glycoproteins. SARS-CoV and SARS-CoV-2 S proteins each encode a surface glycoprotein precursor, and the amino terminus and most of the protein are predicted to be located on the cell surface or outside of the virus particle. The S protein includes a signal peptide, an extracellular domain, a transmembrane domain, and an intracellular domain at the N-terminus. Similar to other coronaviruses, the S protein of SARS-CoV and SARS-CoV-2 can be cleaved by proteases into S1 and S2 subunits. In particular, SARS-CoV-2 contains a furin-like cleavage site, which is lacking in other SARS-like CoVs.

[0071] In some embodiments, provided herein are recombinant S ectodomain trimers. In some embodiments, the recombinant S ectodomain trimers include recombinant S ectodomain protomers from α-coronaviruses (e.g., NL63-CoV or 229E-CoV). In some embodiments, the recombinant S ectodomain trimers include S ectodomain protomers from β-coronaviruses (e.g., OC43-CoV, SARS-CoV, SARS-CoV-2, MERS-CoV, HKU1-CoV, WIV1-CoV, mouse hepatitis virus (MHV), or HKU9-CoV).

[0072] Similar to other enveloped RNA viruses (such as HIV, RSV and influenza), coronaviruses including SARS-CoV-2 have trimeric surface antigens on their viral envelopes to enter different host cells through specific cell surface receptors during infection. Like SARS-CoV-1, SARS-CoV-2 uses its trimeric viral surface antigen S protein to bind to its specific cell surface receptor ACE2 to enter host cells in the mammalian respiratory system. The prerequisite for the production of an effective recombinant subunit vaccine is the ability to produce a natural-like viral S antigen, especially to maintain its trimeric conformation to elicit a sufficient number of antibodies that can bind to the receptor binding domain (RBD) of the viral S protein, thereby preventing the virus from binding to the ACE2 receptor and eliminating viral infection.

[0073] In some embodiments, a protein comprising a coronavirus antigen or immunogen (e.g., SARS-CoV or SARS-CoV-2 S protein peptide) is capable of generating an immune response, e.g., an immune response to SARS-CoV or SARS-CoV-2 S protein peptide. In some embodiments, the immune response suppresses or reduces the replication of coronavirus in a subject (e.g., a patient). In some embodiments, the immune response comprises producing one or more neutralizing antibodies, e.g., polyclonal and / or monoclonal antibodies. In some embodiments, the neutralizing antibodies suppress or reduce the replication of coronavirus in a subject (e.g., a patient). In some embodiments, administering a protein to a subject, e.g., as an immunogenic composition, will not result in antibody-dependent enhancement (ADE) in the subject due to prior exposure to coronavirus. In some aspects, a protein comprising a coronavirus antigen and an immunogen is used as a vaccine.

[0074] In some embodiments, coronavirus antigens and immunogens (e.g., SARS-CoV or SARS-CoV-2 S protein peptides) are linked to proteins or peptides to form fusion proteins or recombinant polypeptides. In some embodiments, the protein or peptide linked to a coronavirus antigen or immunogen is capable of binding to a protein or peptide (e.g., a protein or peptide of a fusion protein or recombinant polypeptide), such as by covalent or non-covalent linkage. Thus, in some cases, the protein or peptide linked to a coronavirus antigen or immunogen is a multimeric domain.

[0075] In some embodiments, coronavirus antigens and immunogens (e.g., coronavirus S protein peptides) are linked to the propeptide of collagen (e.g., at the C-terminus of the propeptide of collagen) to form a fusion peptide or recombinant polypeptide. Thus, in some embodiments, the proteins provided herein include recombinant polypeptides containing coronavirus antigens and immunogens (e.g., coronavirus S protein peptides or fragments or epitopes thereof), linked to the C-terminal propeptide of collagen. In some embodiments, the propeptide of collagen is derived from the human C-propeptide of α1 collagen and is capable of self-trimerization after expression.

[0076] In some embodiments, linking coronavirus antigens and immunogens (e.g., coronavirus S protein peptides) to the propeptide of collagen (e.g., at the C-terminus of the propeptide of collagen) contributes to the ability of the protein to produce an immune response. For example, the production of recombinant proteins can retain the tertiary and quaternary structures of the coronavirus S protein peptide, which may be important for the stability of the native conformation of the coronavirus S protein peptide, and the availability of antigenic sites on the protein surface can in turn elicit an immune response, such as neutralizing antibodies. In addition, linking the coronavirus S protein peptide to a protein or peptide capable of self-trimerization allows the recombinant protein to aggregate, thereby mimicking the natural homotrimeric structure of the coronavirus S protein peptide on the viral envelope.

[0077] In some embodiments, the coronavirus S protein peptide is linked to the C-terminal propeptide of collagen to form a self-trimerized recombinant polypeptide. In some embodiments, the proteins provided herein include a plurality of self-trimerized coronavirus S protein peptides and propeptides of collagen recombinant polypeptides. In some embodiments, the trimeric nature of the recombinant protein contributes to the stability of the protein. In some embodiments, the trimeric nature of the recombinant protein contributes to the ability of the protein to produce an immune response. In some embodiments, the trimeric nature of the recombinant protein and / or the macroscopic structure of the plurality of self-trimerized recombinant proteins contribute to the ability of the protein to produce an immune response.

[0078] 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.

[0079] All publications, including patent documents, scientific articles, and databases, mentioned in this application are incorporated by reference in their entirety for all purposes to the same extent as if each individual publication were incorporated by reference individually. To the extent that definitions set forth herein contradict or are inconsistent with definitions set forth in patents, applications, published applications, and other publications incorporated by reference, the definitions set forth herein take precedence over the definitions incorporated by reference. The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described.

[0080] I. Viral Antigens and Immunogens

[0081] Proteins provided herein include coronavirus antigens and immunogens. The coronavirus antigens and immunogens contemplated herein can promote or stimulate a cell-mediated response and / or a humoral response. In some embodiments, the response (e.g., a cell-mediated or humoral response) includes the production of antibodies (e.g., neutralizing antibodies). In some embodiments, the coronavirus antigen or immunogen is a coronavirus spike protein peptide.

[0082] Coronaviruses are a family of positive-sense, single-stranded RNA viruses known to cause severe respiratory illness. They possess the largest genomes of any known RNA virus (26-32 kb) and are phylogenetically divided into four genera (α, β, γ, and δ), with the β-coronaviruses further divided into four lineages (A, B, C, and D). Currently, viruses from the coronavirus family known to infect humans are from the α-coronavirus and β-coronavirus genera. Furthermore, it is believed that the γ-coronavirus and δ-coronavirus genera may infect humans in the future. Non-limiting examples of β-coronaviruses 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), Murine Hepatitis Virus (MHV-CoV), Bat SARS-like Coronavirus WIV1 (WIV1-CoV), and Human Coronavirus HKU9 (HKU9-CoV). Non-limiting examples of alpha coronaviruses 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 delta coronavirus is porcine delta coronavirus (SDCV).

[0083] This article discloses a list of severe acute respiratory syndrome-related coronaviruses:

[0084] 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-like coronavirus 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 SARS CoV 007 / 2004; Civet SARS CoV SZ16 / 2003; Civet SARS CoV SZ3 / 2003; recombinant SARSr-CoV, SARS coronavirus ExoN1, SARS coronavirus MA15, SARS coronavirus MA15 ExoN1, SARS coronavirus wtic-MB; Chinese 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#2003011 57; SARS coronavirus civet010; SARS coronavirus civet014; SARS coronavirus civet019; SARS coronavirus civet020; 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 Frankfurt1-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 HSR 1; 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; SAR S 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 ShanhgaiLY; SARS coronavirus Sin0409; SARS coronavirus Sin2500; SARS coronavirus Sin2677; SARS coronavirus Sin2679; SARS coronavirus Sin2748; SARS coronavirus Sin2774; S ARS 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 TW 10; 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 Urbani; 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 ZMY 1; SARS coronavirus ZS-A; SARS coronavirus ZS-B; SARS coronavirus ZS-C; SARS-related bat coronavirus RsSHC014; SARS-related beta-coronavirus Rp3 / 2004; severe acute respiratory syndrome coronavirus 2. ;

[0085] The table below shows exemplary SARS CoV-2 strains.

[0086] The coronavirus genome is capped, polyadenylated, and covered with a nucleocapsid protein. The coronavirus particle comprises a viral envelope containing a type I fusion glycoprotein called the spike (S) protein. Most coronaviruses share a common genomic structure, with the replicase gene contained in the 5' portion of the genome and the structural genes in the 3' portion of the genome.

[0087] The coronavirus spike (S) protein is a class I fusion glycoprotein initially synthesized as a precursor protein. The single precursor S polypeptide forms a homotrimer and is glycosylated and processed within the Golgi apparatus to remove the signal peptide. It is then cleaved by cellular proteases to produce the separate S1 and S2 polypeptide chains, which remain associated within the homotrimer as the S1 / S2 protomer, thus forming a heterodimer. The S1 subunit, located distal to the viral membrane, contains the receptor-binding domain (RBD), which mediates attachment of the virus to its host receptor. The S2 subunit comprises the fusion protein machinery, including the fusion peptide, two heptad repeats (HR1 and HR2) characteristic of fusion glycoproteins, and the central helix, transmembrane domain, and cytosolic tail domain.

[0088] In some cases, the coronavirus antigen or immunogen is a coronavirus S protein peptide in a prefusion conformation, which is the structural conformation adopted by the extracellular domain of the coronavirus S protein after being processed into the mature coronavirus S protein in the secretory system and before triggering the fusion event that causes the coronavirus S to transition to the postfusion conformation. The three-dimensional structure of an exemplary coronavirus S protein (HKU1-CoV) in a prefusion conformation is provided in Kirchdoerfer et al., "Pre-fusion structure of a human coronavirus spike protein," Nature, 531: 118-121, 2016, which is incorporated by reference in its entirety for all purposes.

[0089] In some cases, coronavirus antigen or immunogen include one or more amino acid replacements, deletions or insertions compared with natural coronavirus S sequence, compared with the coronavirus S extracellular domain trimer formed by the corresponding natural coronavirus S sequence, and it provides the pre-fusion conformation retention of increase. " stabile " of the pre-fusion conformation by one or more amino acid replacements, deletions or insertions can be, for example, energy stabilization (for example, reducing the energy of the pre-fusion conformation relative to the open conformation after fusion) and / or kinetic stabilization (for example, reducing the conversion rate of the conformation from pre-fusion to the post-fusion conformation). In addition, compared with the corresponding natural coronavirus S sequence, the stabilization of the coronavirus S extracellular domain trimer in the pre-fusion conformation can include increasing resistance to denaturation. Provided herein is a method for determining whether the coronavirus S extracellular domain trimer is in the pre-fusion conformation, including, but not limited to, negative staining electron microscopy and antibody binding analysis using pre-fusion conformation-specific antibodies.

[0090] In some cases, the coronavirus antigen or immunogen is a fragment of the S protein peptide. In some embodiments, the antigen or immunogen is an epitope of the S protein peptide. Epitopes include antigenic determinant chemical groups or peptide sequences on a molecule that are antigenic, thereby triggering a specific immune response. For example, an epitope is an antigenic region to which B cells and / or T cells respond. Antibodies can bind to specific antigenic epitopes, such as epitopes on the extracellular domain of coronavirus S. Epitopes can be formed by continuous amino acids or by non-continuous amino acids juxtaposed by tertiary folding of the protein. In some embodiments, coronavirus epitopes are linear epitopes. In some embodiments, coronavirus epitopes are conformational epitopes. In some embodiments, coronavirus epitopes are neutralizing epitope sites. In some embodiments, all neutralizing epitopes of the coronavirus S protein peptide or its fragments are present as antigens or immunogens.

[0091] In some cases, for example, when the viral antigen or immunogen is a fragment of the S protein peptide, only a single subunit of the S protein peptide is present, and the single subunit of the S protein peptide is trimerized. In some embodiments, the viral antigen or immunogen includes a signal peptide, an S1 subunit peptide, an S2 subunit peptide, or any combination thereof. In some embodiments, the viral antigen or immunogen includes 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 includes the receptor binding domain (RBD) of the S protein. In some embodiments, the viral antigen or immunogen includes the S1 subunit and the S2 subunit of the S protein. In some embodiments, the viral antigen or immunogen includes the S1 subunit of the S protein, but does not include the S2 subunit. In some embodiments, the viral antigen or immunogen includes the S2 subunit of the S protein, but does not include the S1 subunit. In some embodiments, the viral antigen or immunogen does not contain a transmembrane (TM) domain peptide and / or a cytoplasmic (CP) domain peptide.

[0092] In some embodiments, the viral antigen or immunogen includes a protease cleavage site, wherein the protease is optionally furin, trypsin, Factor Xa, or cathepsin L.

[0093] In some embodiments, the viral antigen or immunogen does not contain a protease cleavage site, where the protease is optionally furin, trypsin, Factor Xa, or cathepsin L, or comprises a mutated protease cleavage site that is not cleavable by the protease.

[0094] 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 subunit of the virus. In some embodiments, the SARS-CoV-2 antigen comprises a structural protein of SARS-CoV-2 or a fragment thereof. In some embodiments, the structural protein of SARS-CoV-2 comprises one or more of the group consisting of a spike (S) protein, a membrane (M) protein, a nucleocapsid (N) protein, and an envelope (E) protein. In some embodiments, the SARS-CoV-2 antigen comprises or further comprises a non-structural protein of SARS-CoV-2 or a fragment thereof. The nucleotide sequence of a representative SARS-CoV-2 isolate (Hu-1) is recorded in GenBank No. MN908947.3 (Wu et al., Nature, 579:265-269, 2020), which is incorporated by reference in its entirety for all purposes.

[0095] In some embodiments, the viral antigen or immunogen comprises a sequence described in any one of SEQ ID NOs: 81-84. In some embodiments, the viral antigen or immunogen comprises an amino acid sequence having at least or about 80%, 85%, 90%, 92%, 95%, 97%, or 99% sequence identity to SEQ ID NO: 82 as shown below. In some embodiments, the viral antigen or immunogen comprises an RBD trimer, for example, a SARS-CoV-2 RBD sequence linked to any one of SEQ ID NOs: 67-80.

[0096] In some embodiments, the viral antigen or immunogen comprises a sequence described in SEQ ID NO: 55. In some embodiments, the viral antigen or immunogen comprises an amino acid sequence having at least or about 80%, 85%, 90%, 92%, 95%, or 97% sequence identity to SEQ ID NO: 55 as shown below (the underlined sequence represents a receptor binding motif (RBM) within the receptor binding domain (RBD) of Thr333-Gly526 (bold)). In some embodiments, the viral antigen or immunogen comprises an RBD trimer, for example, a SARS-CoV-2 RBD sequence linked to any one of SEQ ID NOs: 67-80.

[0097] In some embodiments, the viral antigens or immunogens include the spike glycoprotein sequence of the Hu-1 coronavirus (e.g., NC_045512). In some embodiments, the viral antigens or immunogens include the spike glycoprotein sequence of the virus in the B.1.526 lineage. In some embodiments, the viral antigens or immunogens include the spike glycoprotein sequence of the virus in Cluster 5 (ΔFVI-spike). In some embodiments, the viral antigens or immunogens include the spike glycoprotein sequence of the virus in the B.1.1.529 lineage. In some embodiments, the viral antigens or immunogens include the spike glycoprotein sequence of the virus in the B.1.1.7 lineage. In some embodiments, the viral antigens or immunogens include the spike glycoprotein sequence of the virus in the B.1.1.207 lineage. In some embodiments, the viral antigens or immunogens include the spike glycoprotein sequence of the virus in the B.1.1.317 lineage. In some embodiments, the viral antigens or immunogens include the spike glycoprotein sequence of the virus in the B.1.1.318 lineage. In some embodiments, the viral antigens or immunogens include the spike glycoprotein sequence of a virus in the P.1 lineage. In some embodiments, the viral antigens or immunogens include the spike glycoprotein sequence of a virus in the B.1.351 lineage. In some embodiments, the viral antigens or immunogens include the spike glycoprotein sequence of a virus in the B.1.429 / CAL.20C lineage. In some embodiments, the viral antigens or immunogens include the spike glycoprotein sequence of a virus in the B.1.525 lineage. In some embodiments, the viral antigens or immunogens include the spike glycoprotein sequence of a virus in the B.1.526 lineage. In some embodiments, the viral antigens or immunogens include the spike glycoprotein sequence of a virus in the B.1.617 lineage. In some embodiments, the viral antigens or immunogens include the spike glycoprotein sequence of a virus in the B.1.617.2 lineage. In some embodiments, the viral antigens or immunogens include the spike glycoprotein sequence of a virus in the B.1.618 lineage. In some embodiments, the viral antigens or immunogens include the spike glycoprotein sequence of a virus in the B.1.620 lineage. In some embodiments, the viral antigens or immunogens include the spike glycoprotein sequence of a virus in the P.2 lineage. In some embodiments, the viral antigens or immunogens include the spike glycoprotein sequence of a virus in the P.3 lineage. In some embodiments, the viral antigens or immunogens include the spike glycoprotein sequence of a virus in the B.1.1.143 lineage. In some embodiments, the viral antigens or immunogens include the spike glycoprotein sequence of a virus in the A.23.1 lineage. In some embodiments, the viral antigens or immunogens include the spike glycoprotein sequence of a virus in the B.1.617 lineage.In some embodiments, the viral antigen or immunogen includes sequences of spike glycoproteins derived from any two or more viruses (in any suitable combination) selected from the group consisting of 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.

[0098] In some embodiments, the viral antigens or immunogens include T95I, G142D, Δ143-145, and / or T478K, e.g., as in the B.1.1.529 Omicron variant.

[0099] In some embodiments, the viral antigens or immunogens include E484K and / or S477N, for example, as in the B.1.526 variant. In some embodiments, the viral antigens or immunogens include Δ400-402 (ΔFVI), for example, as in the Cluster 5 (ΔFVI-spike) variant. In some embodiments, the viral antigens or immunogens include Δ69-70 (ΔHV), Δ144 (ΔY), N501Y, A570D, D614G, P681H, T716I, S982A and / or D1118H, for example, as in the B.1.1.7 variant. In some embodiments, the viral antigens or immunogens include P681H, for example, as in the B.1.1.207 variant. In some embodiments, the viral antigens or immunogens include L18F, T20N, P26S, D138Y, R190S, K417T, E484K, N501Y, D614G, H655Y, T1027I, and / or V1176F, for example, as in the P.1 variant. In some embodiments, the viral antigens or immunogens include E484K, for example, as in the P.2 variant. In some embodiments, the viral antigens or immunogens include E484K and / or N501Y, for example, as in the P.3 variant. In some embodiments, the viral antigens or immunogens include L18F, D80A, D215G, Δ242-244 (ΔLAL), R246I, K417N, E484K, N501Y, D614G and / or A701V, for example, as in the B.1.351 variant. In some embodiments, the viral antigens or immunogens include S13I, W152C and / or L452R, for example, as in the B.1.429 / CAL.20C variant. In some embodiments, the viral antigens or immunogens include Δ69-70 (ΔHV), E484K and / or F888L, for example, as in the B.1.525 variant. In some embodiments, the viral antigens or immunogens include G142D, L452R, E484Q and / or P681R, for example, as in the B.1.617 variant. In some embodiments, the viral antigen or immunogen includes G142D, L452R and / or P681R, for example, as in the B.1.617.2 variant. In some embodiments, the viral antigen or immunogen includes E484K, for example, as in the B.1.618 variant. In some embodiments, the viral antigen or immunogen may include a fusion polypeptide (protomer) comprising any one or more of the aforementioned mutations in any appropriate combination. In some embodiments, the viral antigen or immunogen may include a trimer of three fusion polypeptides, and any one of the three protomer fusion polypeptides may include any one or more of the aforementioned mutations in any appropriate combination.In some embodiments, two or all three of the three protomer fusion polypeptides that form a trimer can include different mutations and / or different combinations of mutations in each protomer. In some embodiments, a viral antigen or immunogen can include a mixture of trimers, and each trimer can include different mutations and / or different combinations of mutations.

[0100] In some embodiments, the viral antigen or immunogen includes any one, two, three, four, five or more mutations selected from the group consisting of mutations (e.g., substitutions, deletions and / or insertions) at amino acid 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 of SEQ ID NO: 55. In some embodiments, the viral antigen or immunogen includes any one, two, three, four, five, six, seven, eight or all mutations selected from the group consisting of mutations (e.g., substitutions, deletions and / or insertions) at amino acid positions 440, 452, 477, 484, 501, 614, 655, 681 and 701. In some embodiments, the viral antigen or immunogen includes a chimeric polypeptide comprising sequences from different viruses, such as one or more mutations from a first variant of a coronavirus and one or more mutations from a second variant of a coronavirus that is different from the first variant. In some embodiments, such chimeric viral antigens or immunogens (or a combination of chimeric viral antigens or immunogens) can be used to elicit a broad immune response to the first and second variants of a coronavirus.

[0101] In some embodiments, the viral antigen or immunogen comprises an antigen selected from the group consisting of T95I, G142D, Δ143-145, T478K, S13I, L18F, T20N, P26S, Δ69-70 (ΔHV), D80A, D138Y, G142D, Δ144 (ΔY), W152C, R190S, D215G, Δ242-244 (ΔLAL), R246I, Δ400-402 (ΔFVI), K417T, K any one, two, three, four, five or more mutations from the group consisting of 417N, 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 includes 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.

[0102] In some embodiments, the SARS-CoV-2 antigen includes a truncated S protein lacking a signal peptide, the transmembrane and cytoplasmic domains 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 viral particles. In some preferred embodiments, the SARS-CoV-2 antigen is an isolated antigen.

[0103] In some embodiments, the viral antigen or immunogen comprises a sequence as set forth in any one of SEQ ID NOs: 27-51. 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 a sequence as set forth in any one of SEQ ID NOs: 27-51, including an amino acid sequence selected from the group consisting of 13, 18, 20, 26, 69, 70, 80, 1 82, 1027, 1118, and 1176 (with respect to amino acid positions of SEQ ID NO: 55) at one or more amino acid positions. In some embodiments, the viral antigen or immunogen comprises a variant of the sequence described in any one of SEQ ID NOs: 27-51, and the variant comprises a variant selected from the group consisting of 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, Any one, two, three, four, five or more mutations from the group consisting of 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: 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 SEQ ID NO: 52.

[0105] 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 that is 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% identical to SEQ ID NO: 53.

[0106] 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 that has 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 SEQ ID NO: 54.

[0107] 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 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 SEQ ID NO: 55, including an amino acid sequence selected from the group consisting of 13, 18, 20, 26, 69, 70, 80, 138, 142, 144, 152, 190, 2 15, 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. In some embodiments, the viral antigen or immunogen comprises SEQ ID NO: NO: 55 variants, and variants include those selected from 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 R, 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: 56. 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 SEQ ID NO: 56, including an amino acid sequence selected from the group consisting of 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 (with respect to amino acid positions of SEQ ID NO: 55) The sequence comprises a substitution, deletion and / or insertion at one or more amino acid positions in the group consisting of. In some embodiments, the viral antigen or immunogen comprises a variant of SEQ ID NO: 56, and the variant comprises a member selected from the group consisting of 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, Any one, two, three, four, five or more mutations from the group consisting of 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 a sequence as set forth in any one of SEQ ID NOs: 57-58. 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 a sequence as set forth in any one of SEQ ID NOs: 57-58, including sequences comprising substitutions, deletions and / or insertions at one or more amino acid positions of a sequence as set forth in any one of SEQ ID NOs: 57-58.

[0110] In some embodiments, the viral antigen or immunogen comprises the sequence set forth in SEQ ID NO: 59. In some embodiments, the viral antigen or immunogen comprises a sequence comprising a substitution, deletion, and / or insertion at one or more amino acid positions of SEQ ID NO: 59. In some embodiments, the viral antigen or immunogen comprises the sequence set forth in SEQ ID NO: 60. In some embodiments, the viral antigen or immunogen comprises a sequence comprising a substitution, deletion, and / or insertion at one or more amino acid positions of SEQ ID NO: 60.

[0111] In some embodiments, the viral antigen or immunogen comprises the sequence of any one of SEQ ID NOs: 61-65 and 81-84. 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 any one of SEQ ID NOs: 61-65 and 81-84, including sequences comprising substitutions, deletions and / or insertions at one or more amino acid positions of the sequence of any one of SEQ ID NOs: 61-65 and 81-84.

[0112] In some embodiments, the viral antigen or immunogen comprises a sequence as described in any one of SEQ ID NOs: 122-123 and 126-127. 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 SEQ ID NO: 122, including sequences comprising substitutions, deletions and / or insertions at one or more amino acid positions of the sequence as described in any one of SEQ ID NOs: 122-123 and 126-127. In some embodiments, the viral antigen or immunogen comprises an RBD trimer, for example, a SARS-CoV-2 RBD sequence linked to any one of SEQ ID NOs: 67-80.

[0113] In some embodiments, the viral antigen or immunogen does not include a transmembrane domain, such as SEQ ID NO: 66 or a portion thereof. In some embodiments, the coronavirus antigen or immunogen includes a soluble S protein peptide. In some embodiments, the soluble S protein peptide lacks a TM domain peptide and a CP domain peptide. In some embodiments, the soluble S protein peptide does not bind to a lipid bilayer, such as a membrane or viral envelope.

[0114] In some embodiments, the S protein peptide is produced from a codon-optimized nucleic acid sequence. In some embodiments, the S protein peptide is produced from a non-codon-optimized nucleic acid sequence.

[0115] In some embodiments, the viral antigens or immunogens mentioned herein may include recombinant polypeptides or fusion peptides comprising the viral antigens or immunogens. The term viral antigen or immunogen may be used to refer to proteins comprising coronavirus antigens or immunogens. In some cases, the coronavirus antigens or immunogens are coronavirus protein peptides provided herein. The viral antigens or immunogens mentioned herein may be used in an initial dose, an additional dose, and / or a booster dose. Independently, the initial dose, the additional dose, and / or any one or more boosters may be used without or with an adjuvant. If an adjuvant is used, optional adjuvants may include: aluminum-containing adjuvants, such as adjuvants containing alum and / or aluminum hydroxide; oligonucleotide-containing adjuvants, such as adjuvants containing CpG oligodeoxynucleotides (CpG-ODN); adjuvants containing TLR9 agonists; adjuvants containing metabolizable oils, α-tocopherol, and / or polyoxyethylene sorbitan monooleate (Tween-80), such as adjuvants containing squalene, α-tocopherol, and Tween-80 and / or Span 85 in the form of an oil-in-water emulsion; or any combination of adjuvants.

[0116] II. Recombinant Peptides and Proteins

[0117] It is contemplated that the coronavirus antigens and immunogens provided herein, such as S protein peptides (see Section 1), can be combined with other proteins or peptides, such as by linking, to form recombinant polypeptides, including fusion peptides. In some embodiments, the single recombinant polypeptides provided herein (e.g., monomers) combine to form a multimer, such as a trimer, of the recombinant polypeptides. In some embodiments, the binding of single recombinant polypeptide monomers occurs through covalent interactions. In some embodiments, the binding of single recombinant polypeptide monomers occurs through non-covalent interactions. In some embodiments, the interaction (e.g., covalent or non-covalent) is affected by the protein or peptide to which the coronavirus antigen or immunogen (e.g., S protein peptide) is connected. In some embodiments, for example, when the coronavirus antigen or immunogen is an S protein peptide as described herein, the protein or peptide to which it is connected can be selected so as to retain the natural homotrimeric structure of the glycoprotein. This can be beneficial for stimulating a strong and effective immunogenic response to the S protein peptide. For example, the retention and / or maintenance of the natural conformation of the coronavirus antigen or immunogen (e.g., S protein peptide) can improve or allow access to antigenic sites that can produce an immune response. In some cases, a recombinant polypeptide comprising an S protein peptide described herein (e.g., see Section 1) is alternatively referred to herein as a recombinant S antigen, a recombinant S immunogen, or a recombinant S protein.

[0118] It is further expected that, in some cases, recombinant polypeptide or its multimeric recombinant polypeptide aggregation or can be gathered to form a protein or complex comprising multiple coronavirus antigens and / or immunogen recombinant polypeptide.The formation of this protein can be conducive to the strong and effective immunogenic response to coronavirus antigen and / or immunogen.For example, the protein comprising multiple recombinant polypeptides is formed, thereby forming multiple coronavirus antigens, such as coronavirus S protein peptides, can retain the tertiary and / or quaternary structure of viral antigens, allow to trigger (mounted) immune response for native structure.In some cases, aggregation can give the structural stability of coronavirus antigen or immunogen, which in turn can provide entry to the potential antigenic site that can promote immune response.

[0119] 1. Fusion peptides and recombinant peptides

[0120] In some embodiments, the coronavirus antigen or immunogen can be connected to a trimerization domain at its C-terminus (C-terminal connection) to promote trimerization of the monomer. In some embodiments, trimerization stabilizes the membrane proximal aspect of the coronavirus antigen or immunogen (e.g., coronavirus S protein peptide) in a trimeric configuration.

[0121] Non-limiting examples of exogenous multimerization domains that promote stable trimers of soluble recombinant proteins include: GCN4 leucine zipper (Harbury et al. 1993 Science 262: 1401-1407), the trimerization motif of pulmonary surfactant protein (Hoppe et al. 1994 FEBS Lett 344: 191-195), collagen (McAlinden et al. 2003 J Biol Chem 278: 42200-42207), and bacteriophage T4 fibritin Foldon (Miroshnikov et al. 1998 Protein Eng 11: 329-414), any of which can be linked to the coronavirus antigens or immunogens described herein (e.g., by linking to the C-terminus of the S peptide) to promote trimerization of the recombinant viral antigens or immunogens, which are incorporated by reference in their entirety for all purposes. 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 US 2020 / 0009244, which are incorporated herein by reference in their entirety for all purposes.

[0122] In some embodiments, one or more peptide linkers (e.g., glycine-serine linkers, e.g., 10 amino acid glycine-serine peptide linkers) can be used to connect recombinant viral antigens or immunogens to a multimerization domain. As long as the recombinant viral antigen or immunogen trimer maintains the desired properties (e.g., pre-fusion conformation), the trimer may include any stabilizing mutations (or combinations thereof) provided herein. In some embodiments, the recombinant polypeptide or fusion protein includes a first sequence described in any one of SEQ ID NOs: 27-66 and 81-84, the first sequence being connected to a second sequence described in any one of SEQ ID NOs: 67-80, wherein the C-terminus of the first sequence is directly connected to the N-terminus of the second sequence. In some embodiments, the recombinant polypeptide or fusion protein includes a first sequence described in any one of SEQ ID NOs: 27-66 and 81-84, the first sequence being connected to a second sequence described in any one of SEQ ID NOs: 67-80, wherein the C-terminus of the first sequence is indirectly connected to the N-terminus of the second sequence, e.g., through a linker. In some embodiments, the linker includes a sequence comprising a glycine-XY repeat sequence.

[0123] To be therapeutically viable, the trimerizing protein portion required for biopharmaceutical design should meet the following criteria. Ideally, it should be a portion of a naturally secreted protein, such as immunoglobulin Fc, which is abundant in the circulation (non-toxic), derived from humans (lack of immunogenicity), relatively stable (long half-life) and capable of efficient self-trimerization, which is strengthened by interchain covalent disulfide bonds, so that the trimerized coronavirus antigen or immunogen is structurally stable.

[0124] Collagen is a member of the fibrous protein family, the primary component of the extracellular matrix. It is the most abundant protein in mammals, comprising nearly 25% of the body's total protein. Collagen plays an important structural role in the formation of bones, tendons, skin, cornea, cartilage, blood vessels, and teeth. The fibrillar forms of collagens I, II, III, IV, V, and XI are synthesized as large, trimeric precursors called procollagens. A central, uninterrupted triple-helical domain composed of hundreds of "GXY" repeats (or glycine repeats) is flanked by a non-collagenous domain (NC), an N-propeptide, and a C-propeptide. Both the C-terminal and N-terminal extensions undergo proteolytic processing after procollagen secretion, triggering the assembly of the mature protein into collagen fibrils, forming an 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 removing the propeptide. Concentrations of shed trimeric C-propeptide of type I collagen are found in normal adult human serum at levels ranging from 50 to 300 ng / mL, with higher levels in children, indicating active bone formation. In individuals with familial high serum concentrations of type I collagen C-propeptide, levels can reach as high as 1 to 6 μg / mL without apparent abnormalities, suggesting that the C-propeptide is nontoxic. Structural studies of the trimeric C-propeptide of collagen have revealed a trilobal structure, with all three subunits brought together at a linker region near its N-terminus, where they connect to the rest of the procollagen molecule. The geometry of this protruding protein resembles that of an Fc dimer when fused in one orientation.

[0125] Type I, IV, V, and XI collagens primarily assemble into heterotrimeric forms composed of two α-1 chains and one α-2 chain (for types I, IV, and V) or three different chains (for type XI), which are highly homologous in sequence. Type II and III collagens are both homotrimers of α-1 chains. Type I collagen (the most abundant collagen form) also forms stable α(I) homotrimers and is present at variable levels in different tissues. When overexpressed individually in cells, most of these collagen C-propeptide chains can self-assemble into homotrimers. Although the N-propeptide domain is synthesized first, molecular assembly of trimeric collagen begins with the registered binding of the C-propeptide. The C-propeptide complex is believed to be stabilized by the formation of interchain disulfide bonds, but the necessity of disulfide bond formation for proper chain alignment is unclear. The triple helical repeats of glycine then propagate from the associated C-terminus to the N-terminus in a zipper-like manner. This knowledge has been used to create non-natural collagen matrices 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-termini of procollagen or mature collagen to form new collagen matrices, with the goal of slowly releasing the non-collagenous proteins from the cell matrix. However, in both cases, the C-propeptide needs to be cleaved before the recombinant collagen fibers can assemble into an insoluble cell matrix.

[0126] Although other protein trimerization domains (e.g., GCN4 from yeast fibritin from bacteriophage T4 and aspartate transcarbamylase from Escherichia coli) have been previously described to allow trimerization of heterologous proteins, none of these trimeric proteins are of human nature, nor are they naturally secreted proteins. Therefore, any trimeric fusion protein must be synthesized intracellularly, which not only may lead to misfolding of naturally secreted proteins (e.g., soluble receptors), but also makes it difficult to purify such fusion proteins from thousands of other intracellular proteins. Furthermore, a fatal drawback of using such non-human protein trimerization domains (e.g., from yeast, bacteriophage, and bacteria) in the design of trimeric biopharmaceuticals is their presumed immunogenicity in humans, rendering such fusion proteins ineffective shortly after injection into humans.

[0127] Therefore, the use of collagen in the recombinant polypeptides described herein has many advantages, including: (1) collagen is the most abundant protein secreted by mammals, accounting for nearly 25% of the total protein in the body; (2) the major form of collagen occurs naturally as a trimeric helical form, with the globular C-propeptide responsible for the initiation of trimerization; (3) the trimeric C-propeptide of collagen, released from the hydrolysis of mature collagen proteins, is naturally present in mammalian blood at submicrogram / ml levels and is not known to be toxic to the body; (4) the linear triple helical region of collagen can be included as a linker, with the predicted spacing of each residue being or excluded as part of the fusion protein so that the distance between the protein to be trimerized and the C-propeptide of collagen can be precisely adjusted to achieve optimal biological activity; (5) the recognition site of BMP1 that cleaves the C-propeptide from the original collagen can be mutated or deleted to prevent the destruction of the trimeric fusion protein; (6) the C-propeptide domain self-trimerizes through disulfide bonds, and it provides a universal affinity tag that can be used to purify any secreted fusion protein produced. In some embodiments, the C-propeptide of collagen combined with coronavirus antigens and immunogens (such as S protein peptides) can be recombinantly produced as a soluble, covalently linked homotrimeric fusion protein.

[0128] In some embodiments, a coronavirus antigen or immunogen is linked to the 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 antigen or immunogen is a S protein peptide as described in Section 1.

[0129] For example, a fusion polypeptide comprising the signal peptide MFVFLVLLPLVSS (SEQ ID NO: 54) at the N-terminus of the fusion polypeptide in SEQ ID NO: 1 can be generated and trimerized via inter-polypeptide disulfide bonds (Cys residues that can form inter-polypeptide disulfide bonds are shown in bold).

[0130] In some embodiments, the inter-polypeptide 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 can include one or more glycosylation sites (e.g., Asn-linked), for example, at one or more or all of the Asn residues at 17, 61, 122, 149, 165, 234, 282, 331, 343, 603, 616, 657, 709, 717, 801, 1074, 1098, and 1134, in any suitable combination.

[0131] In some embodiments, the C-terminal propeptide is human collagen. In some embodiments, the C-terminal propeptide includes the C-terminal polypeptide 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).

[0132] In some embodiments, the C-terminal propeptide is or includes the amino acid sequence of any one of SEQ ID NOs: 67-80. In some embodiments, the C-terminal propeptide is an amino acid sequence that has at least or about 85%, 90%, 92%, 95%, or 97% sequence identity to the amino acid sequence of any one of SEQ ID NOs: 67-80.

[0133] 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 in the BMP-1 site, for example, as shown in SEQ ID NO: 68, wherein RAD is mutated to RAN. 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 alanine (A) to asparagine (N) substitution in the BMP-1 site, for example, as shown in SEQ ID NO: 69, wherein RAD is mutated to RND. In some embodiments, the C-terminal propeptide herein may include a mutated BMP-1 site, for example, RSAN instead of DDAN. In some embodiments, the C-terminal propeptide herein may include a BMP-1 site, for example, a sequence comprising a RAD (e.g., RADDAN) sequence rather than a RAN (e.g., RANDAN) or RND (e.g., RNDDAN) sequence (e.g., SEQ ID NO: 68 or 69) 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 linked directly or indirectly to SEQ ID NO: 67 (italics) or a fragment, variant, or mutant thereof, for example, to form the following fusion protein:

[0134] In some embodiments, the C-terminal propeptide is or includes an amino acid sequence that is a fragment of any one of SEQ ID NOs: 67-80.

[0135] In some embodiments, the C-terminal propeptide may comprise a sequence comprising a glycine-XY repeat sequence, wherein X and Y are independently any amino acid, or an amino acid sequence at least 85%, 90%, 92%, 95%, or 97% identical thereto, that is capable of forming an interpolypeptide disulfide bond and allowing trimerization of the recombinant polypeptide. In some embodiments, X and Y are independently proline or hydroxyproline.

[0136] In some cases where an S protein peptide is linked to a C-terminal propeptide to form a recombinant polypeptide, the recombinant polypeptide forms a trimer, thereby forming 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, the confirmation state allows access to different antigenic sites on the S protein peptide. In some embodiments, the antigenic site is an epitope, such as a linear epitope or a conformational epitope. The advantage of having such a trimerized recombinant polypeptide is that an immune response can be directed against a variety of potential and diverse antigenic sites.

[0137] In some embodiments, the trimerized recombinant polypeptide comprises a single recombinant polypeptide comprising the same viral antigen or immunogen. In some embodiments, the trimerized recombinant polypeptide comprises a single recombinant polypeptide, each recombinant polypeptide comprising a viral antigen or immunogen different from the other recombinant polypeptides. In some embodiments, the trimerized recombinant polypeptide comprises a single recombinant polypeptide, wherein one of the single recombinant polypeptides comprises a viral antigen or immunogen different from the other recombinant polypeptides. In some embodiments, the trimerized recombinant polypeptide comprises a single recombinant polypeptide, wherein two of the single recombinant polypeptides comprise 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.

[0138] In some embodiments, the recombinant polypeptide comprises any of the coronavirus antigens or immunogens described in Section 1. In some embodiments, the recombinant polypeptide comprises any of the coronavirus antigens or immunogens described in Section 1 as described herein linked to a C-terminal propeptide of collagen as described herein.

[0139] In some embodiments, the immunogen comprises a recombinant SARS-CoV or SARS-CoV-2 S ectodomain trimer, such as a SARS-CoV-2 Omicron (B.1.1.529) coronavirus S ectodomain trimer, comprising a protomer comprising one or more (e.g., two, e.g., two consecutive) proline substitutions at or near the boundary between the HR1 domain and the central helical domain, the proline substitutions stabilizing the S ectodomain trimer in a prefusion conformation. In some such embodiments, the one or more (e.g., two, e.g., two consecutive) proline substitutions stabilizing the S ectodomain in a prefusion conformation are located between amino acid position 15 N-terminal to the C-terminal residue of HR1 and amino acid position 5 C-terminal to the N-terminal residue of the central helix.

[0140] In some embodiments, one or more (e.g., two, e.g., two consecutive) proline substitutions stabilize the coronavirus (e.g., SARS-CoV or SARS-CoV-2) S extracellular domain trimer in the prefusion conformation, such as the SARS-CoV-2 Omicron (B.1.1.529) coronavirus S extracellular domain trimer. In some embodiments, the SARS-CoV-2 S protein peptide includes a mutation from 986K / 987V to 986P / 987P.

[0141] In some embodiments, the stabilized recombinant coronavirus (e.g., SARS-CoV or SARS-CoV-2) S ectodomain trimer in the prefusion conformation includes a single-chain S ectodomain protomer comprising a mutation in the S1 / S2 and / or S2′ protease cleavage sites to prevent protease cleavage at these sites. In some embodiments, the SARS-CoV-2 S protein peptide comprises a mutation from 685R to 685A. Exemplary protease cleavage sites for various viruses are shown below:

[0142] In some embodiments, the protomer of a recombinant coronavirus (e.g., SARS-CoV or SARS-CoV-2) S ectodomain trimer stabilized in a prefusion conformation by one or more proline substitutions (e.g., 986P / 987P substitutions) includes additional modifications for stabilization of the prefusion conformation, such as mutations at protease cleavage sites to prevent protease cleavage.

[0143] With reference to the SARS-CoV-2 S protein sequence provided as SEQ ID NO: 55, the extracellular domain 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); a heptad repeat sequence 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, ncbi.nlm.nih.gov / nuccore / MN908947.2. Additional sequences can be found at ncbi.nlm.nih.gov / genbank / sars-cov-2-seqs / , including the complete genome of the pneumonia virus isolate Hu-1.

[0144] In some embodiments, the protomer of a pre-fusion stabilized SARS-CoV-2 S ectodomain trimer, such as a protomer of a SARS-CoV-2 Omicron (B.1.1.529) coronavirus S ectodomain trimer, may have an NTD, RBD, S1 (at S1 / S2 site 1 or S1 / S2 site 2), FP, IFP, HR1, HR2, or the C-terminal residue of the C-terminal residue of the ectodomain (which may be connected to, for example, a trimerization domain or a transmembrane domain). The position numbering of the S protein may vary between SARS-CoV strains, but the sequences can be aligned to determine the relevant domains and cleavage sites. It will be appreciated that some residues (e.g., up to 10) at the N-terminus and C-terminus of any ectodomain fragment may be removed or modified in the disclosed immunogens without reducing the utility of the S ectodomain trimer as an immunogen.

[0145] In some embodiments, the recombinant polypeptide is or includes an NTD peptide of the SARS-CoV or SARS-CoV-2 S protein. In some embodiments, the recombinant polypeptide is or includes an RBD peptide of the SARS-CoV or SARS-CoV-2 S protein. In some embodiments, the recombinant polypeptide is or includes an NTD peptide and an RBD peptide of the SARS-CoV or SARS-CoV-2 S protein. In some embodiments, the recombinant polypeptide is or includes an S1 domain peptide of the SARS-CoV or SARS-CoV-2 S protein. In some embodiments, the recombinant polypeptide is or includes an S2 domain peptide of the SARS-CoV or SARS-CoV-2 S protein.

[0146] An exemplary SARS-CoV-1 S recombinant polypeptide without the signal peptide is provided in SEQ ID NO: 26 (1491 aa).

[0147] The above-mentioned SARS-CoV-1 S recombinant polypeptide may include an N-terminal signal peptide provided in SEQ ID NO:53.

[0148] An exemplary SARS-CoV-2 S recombinant polypeptide without the signal peptide is provided in SEQ ID NO: 1.

[0149] The above-mentioned SARS-CoV-2 S recombinant polypeptide may include an N-terminal signal peptide provided in SEQ ID NO:54.

[0150] An exemplary SARS-CoV-2 S recombinant polypeptide without a signal peptide is provided in SEQ ID NO: 81 (1506aa):

[0151] The above-mentioned SARS-CoV-2 S recombinant polypeptide may include an N-terminal signal peptide provided in SEQ ID NO: 54 (MFVFLVLLPLVSS).

[0152] An exemplary SARS-CoV-2 S recombinant polypeptide without the signal peptide is provided in SEQ ID NO: 82 (1506aa).

[0153] The above-mentioned SARS-CoV-2 S recombinant polypeptide may include an N-terminal signal peptide provided in SEQ ID NO: 54 (MFVFLVLLPLVSS).

[0154] An exemplary SARS-CoV-2 S recombinant polypeptide without a signal peptide is provided in SEQ ID NO: 83 (1506aa):

[0155] The above-mentioned SARS-CoV-2 S recombinant polypeptide may include an N-terminal signal peptide provided in SEQ ID NO: 54 (MFVFLVLLPLVSS).

[0156] An exemplary SARS-CoV-2 S recombinant polypeptide without a signal peptide is provided in SEQ ID NO: 84 (1506aa):

[0157] The above-mentioned SARS-CoV-2 S recombinant polypeptide may include an N-terminal signal peptide provided in SEQ ID NO: 54 (MFVFLVLLPLVSS).

[0158] In some embodiments, the recombinant polypeptide is or includes the sequence of any one of SEQ ID NOs: 1-25. In some embodiments, the recombinant polypeptide is or includes an amino acid sequence that has 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 any one of SEQ ID NOs: 1-25, including at one or more amino acid positions (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, 716, 888, 982, 1027, 1118 and / or 1176 (with respect to the amino acid positions of SEQ ID NO: 55), or any combination thereof). In some embodiments, the recombinant polypeptide is or includes SEQ ID NO: NO: 1-25 of any one of the variants of the sequence, and the variant includes selected from 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 any one, two, three, four, five or more mutations 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.

[0159] In some embodiments, the recombinant polypeptide is or includes the sequence of any one of SEQ ID NOs: 26, 85-92, 120-121, and 124-125. In some embodiments, the recombinant polypeptide is or 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 any one of SEQ ID NOs: 26, 85-92, 120-121, and 124-125, including sequences comprising substitutions, deletions, and / or insertions at one or more amino acid positions of the sequence of any one of SEQ ID NOs: 26, 85-92, 120-121, and 124-125.

[0160] As described above, in some embodiments, the recombinant polypeptides provided herein not only combine to form trimers, but can also aggregate or be aggregated to produce proteins comprising multiple recombinant polypeptides. In some embodiments, the formed protein has a macrostructure. In some cases, the macrostructure can confer structural stability to the coronavirus antigen or immunogen recombinant polypeptide, which in turn can provide access to potential antigenic sites that can promote an immune response.

[0161] In some embodiments, the trimerized recombinant polypeptides aggregate to form a protein comprising multiple trimerized recombinant polypeptides. In some embodiments, the multiple trimerized recombinant polypeptides form a protein having a macrostructure.

[0162] In some embodiments, the proteins comprising multiple recombinant polypeptides described herein are immunogens. In some embodiments, the proteins comprising multiple recombinant polypeptides described herein are contained in nanoparticles. For example, in some embodiments, the protein is directly attached to the nanoparticles, such as protein nanoparticles. In some embodiments, the protein is indirectly attached to the nanoparticles. In some embodiments, the proteins comprising multiple recombinant polypeptides described herein are contained in virus-like particles (VLPs).

[0163] In some embodiments, provided herein are complexes comprising a recombinant polypeptide selected from the group consisting of SEQ ID NOs: 1-26 and 85-92, or a fragment, variant, or mutant thereof, in any suitable combination. In some embodiments, provided herein are complexes comprising a trimer of a recombinant polypeptide selected from the group consisting of SEQ ID NOs: 1-26 and 85-92, or a fragment, variant, or mutant thereof, wherein the recombinant polypeptides are trimerized to form trimers via inter-polypeptide disulfide bonds.

[0164] In some embodiments, provided herein are fusion proteins comprising a plurality of recombinant polypeptides, each recombinant polypeptide comprising, from amino to carboxyl terminus, a) a first region comprising a portion of a coronavirus spike protein ectodomain preceding the coronavirus spike protein receptor binding domain (RBD) in a non-chimeric coronavirus spike protein of a first coronavirus; b) a second region comprising a coronavirus spike protein receptor binding domain (RBD) of a second coronavirus different from the first coronavirus; and c) a C-terminal propeptide of collagen, wherein 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 the S1 domain of a third coronavirus, wherein the third coronavirus is the same or different from the first coronavirus or the second coronavirus. In some embodiments, the third region comprises the S2 domain of a fourth coronavirus, wherein the fourth coronavirus is the same or different from the first, second, or 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 are different from the corresponding amino acid residues in the second coronavirus. In some embodiments, the second region includes one or more amino acid residues that are different from the corresponding amino acid residues in the first coronavirus. In some embodiments, the first and second coronaviruses are different variants or strains of the same coronavirus. In some embodiments, the first region includes the NTD of the first coronavirus, the second region includes the RBD of the second coronavirus, and the first and second coronaviruses 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.1.1.529, B.1.617.2, 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.

[0165] In some embodiments, provided herein are trimeric fusion proteins comprising three recombinant polypeptides, each recombinant polypeptide comprising, from amino to carboxyl terminus: a) a first region comprising the N-terminal domain (NTD) of the coronavirus spike protein of SARS-CoV-2 of the B.1.526 lineage; b) a second region comprising the receptor binding domain (RBD) of the coronavirus spike protein of SARS-CoV-2 of the B.1.351 lineage; and c) a C-terminal propeptide of collagen, wherein the C-terminal propeptide of the recombinant polypeptide forms an inter-polypeptide disulfide bond. In some embodiments, provided herein are trimeric fusion proteins comprising three recombinant polypeptides, each recombinant polypeptide comprising, from amino to carboxyl terminus: a) a first region comprising the N-terminal domain (NTD) of the coronavirus spike protein of SARS-CoV-2 of the B.1.1.529 lineage; b) a second region comprising the receptor binding domain (RBD) of the coronavirus spike protein of SARS-CoV-2 of the B.1.1.529 lineage or non-B.1.1.529 lineage; and c) a C-terminal propeptide of collagen, wherein the C-terminal propeptide of the recombinant polypeptide forms an inter-polypeptide disulfide bond. In some embodiments, provided herein are trimeric fusion proteins comprising three recombinant polypeptides, each recombinant polypeptide comprising, from amino to carboxyl terminus: a) a first region comprising the N-terminal domain (NTD) of the coronavirus spike protein of SARS-CoV-2 of the B.1.1.529 lineage or non-B.1.1.529 lineage; b) a second region comprising the receptor binding domain (RBD) of the coronavirus spike protein of SARS-CoV-2 of the B.1.1.529 lineage; and c) a C-terminal propeptide of collagen, wherein the C-terminal propeptide of the recombinant polypeptide forms an inter-polypeptide disulfide bond.

[0166] In some embodiments, provided herein are methods for preventing coronavirus infection in mammals, comprising immunizing a mammal with an effective amount of a fusion protein disclosed herein. In some embodiments, neutralizing antibodies against the first and second coronaviruses are produced in a mammal. In some embodiments, the first and second coronaviruses are different variants of SARS-CoV-2, and the neutralizing antibodies produced in the mammal neutralize two or more SARS-CoV-2 viruses of the B.1.1.529, B.1.617.2, 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 produced in the mammal neutralize three or more SARS-CoV-2 viruses of the B.1.1.529, B.1.617.2, 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 comprises immunizing the mammal with two or more doses of the fusion protein. In some embodiments, after one or more doses of the immunogen (including a spike protein peptide comprising an NTD and an RBD from the same SARS-CoV-2 variant), the fusion protein is administered as a booster.

[0167] In some embodiments, provided herein are engineered fusion polypeptides derived or modified from spike (S) glycoproteins of coronaviruses including SARS-CoV-1 and SARS-CoV-2. In some embodiments, the fusion polypeptides disclosed herein can be stabilized in a pre-fusion conformation compared to the wild-type S protein sequence of the coronavirus. In some embodiments, fusion with the trimerization domain prevents the S protein peptide in the fusion protein from forming a straight helix (e.g., similar to what occurs during membrane fusion). For example, the cryoEM structure of the S-trimer subunit candidate vaccine shows that it primarily adopts a tightly closed pre-fusion state, unlike the full-length wild-type spike protein, which forms a pre-fusion and post-fusion state in the presence of a detergent. Ma et al., J Virol (2021) doi: 10.1128 / JVI.00194-21, which is incorporated by reference in its entirety for all purposes. In some embodiments, the fusion protein may include an altered soluble S sequence with a modification that inactivates the S1 / S2 cleavage site; a mutation in the turn region between the heptad repeat 1 (HR1) region and the central helix (CH) region that prevents HR1 and CH from forming a straight helix; and / or a truncation of the heptad repeat 2 region (HR2) in addition to stabilizing mutations. 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 useful for stabilizing the spike protein disclosed herein comprising a trimerization of the protein. TM Fusion of the trimerization domain to the polypeptide is not required.

[0168] In some of these embodiments, the mutation that inactivates the S1 / S2 cleavage site may comprise a substitution of RRAR (682-685 in SEQ ID NO: 55) with GSAG (SEQ ID NO: 60), and the mutation in the turn region may comprise the double mutation K986G / V987G, K986P / V987P, K986G / V987P, or K986P / V987G. In some embodiments, truncation of HR2 entails deleting one or more residues shown in SEQ ID NO: 65 at the C-terminus of the wild-type soluble S sequence. In some embodiments, the immunogenic polypeptide may further comprise (a) one or more proline or glycine substitutions, and / or (b) one or more amino acid residue insertions, in the region of HR1 that interacts with HR2. In some of these embodiments, the immunogenic polypeptide may 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 between any of residues A942-A944.

[0169] The recombinant polypeptides or fragments, variants or mutants thereof mentioned herein, in any suitable combination, including trimers of recombinant polypeptides or fragments, variants or mutants thereof selected from the group consisting of SEQ ID NOs: 1-26 and 85-92, wherein the recombinant polypeptides are trimerized to form trimers through disulfide bonds between polypeptides, and can be used in the initial dose and / or booster. Independently, the initial dose and / or any one or more boosters may be used without or with an adjuvant. If an adjuvant is used, the optional adjuvant may include: an aluminum-containing adjuvant, such as an adjuvant containing alum and / or aluminum hydroxide; an adjuvant containing an oligonucleotide, such as an adjuvant containing CpG oligodeoxynucleotide (CpG-ODN); an adjuvant containing a TLR9 agonist; an adjuvant containing a metabolizable oil, α-tocopherol, and / or polyoxyethylene sorbitan monooleate (Tween-80), such as an adjuvant containing squalene, α-tocopherol, and Tween-80 and / or Span 85 in the form of an oil-in-water emulsion; or any combination of adjuvants.

[0170] 2. Polynucleotides and Vectors

[0171] 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.

[0172] In some embodiments, polynucleotides encoding recombinant polypeptides provided herein are provided. In some aspects, the polynucleotide comprises a single nucleic acid sequence, such as a nucleic acid sequence encoding a recombinant polypeptide. In other examples, the polynucleotide comprises a first nucleic acid sequence encoding a recombinant polypeptide, particularly a coronavirus antigen or immunogen, and a second nucleic acid sequence encoding a recombinant polypeptide comprising a different coronavirus antigen or immunogen.

[0173] In some embodiments, the polynucleotide encoding the recombinant polypeptide comprises at least one promoter operably linked to control the expression of the recombinant polypeptide. In some embodiments, the polynucleotide comprises two, three or more promoters operably linked to control the expression of the recombinant polypeptide.

[0174] In some embodiments, for example, when the polynucleotide comprises two or more nucleic acid coding sequences, such as sequences encoding recombinant polypeptides comprising different coronavirus antigens or immunogens, at least one promoter is operably linked to control the expression of the two or more nucleic acid sequences. In some embodiments, the polynucleotide comprises two, three, or more promoters that are operably linked to control the expression of the recombinant polypeptide.

[0175] In some embodiments, the expression of the recombinant polypeptide is inducible or conditional. Therefore, in some aspects, the polynucleotide encoding the recombinant polypeptide comprises a conditional promoter, an enhancer or a transactivator. In some such aspects, the conditional promoter, the enhancer or the transactivator is an inducible promoter, an enhancer or a transactivator or is a repressible promoter, an enhancer or a transactivator. For example, in some embodiments, an inducible or conditional promoter can be used for limiting the expression of the recombinant polypeptide to a specific microenvironment. In some embodiments, the expression driven by an inducible or conditional promoter is regulated by exposure to an exogenous agent (such as heat, radiation or a drug).

[0176] In the case where the polynucleotide comprises more than one nucleic acid sequence encoding a recombinant polypeptide, the polynucleotide may further include a nucleic acid sequence encoding a peptide between one or more nucleic acid sequences. In some cases, the nucleic acid encoding peptide located between the nucleic acid sequences separates the translation product of the nucleic acid sequence 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.

[0177] In some embodiments, a polynucleotide encoding a recombinant polypeptide is introduced into a composition containing cultured cells (e.g., host cells), e.g., by retroviral transduction, transfection, or transformation. In some embodiments, this allows expression (e.g., production) of the recombinant polypeptide. In some embodiments, the expressed recombinant polypeptide is purified.

[0178] In some embodiments, provided herein are polynucleotides (nucleic acid molecules) encoding coronavirus antigens or immunogens as described herein. In some embodiments, provided herein are polynucleotides (nucleic acid molecules) encoding recombinant polypeptides comprising coronavirus antigens or immunogens (e.g., coronavirus S protein peptides) as described herein.

[0179] Also provided are vectors or constructs containing nucleic acid molecules as 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 promoter is operably linked to one or more nucleic acid molecules, such as nucleic acid molecules encoding recombinant polypeptides containing different coronavirus antigens or immunogens.

[0180] 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.

[0181] In some embodiments, carrier or construct include single promoter, which drives the expression of one or more nucleic acid molecules of polynucleotide. In some embodiments, such promoter can be polycistronic (bicistronic or tricistronic, referring to, for example, U.S. Patent number 6,060,273). For example, in some embodiments, transcription unit can be engineered to include a bicistronic unit of IRES (internal ribosome entry site), which allows coexpression of gene products (for example, encoding different recombinant polypeptides) by the message from a single promoter. In some embodiments, provided herein is a bicistronic, which allows carrier to include and express two nucleotide sequences. In some embodiments, provided herein is a tricistronic, which allows carrier to include and express three nucleotide sequences.

[0182] In some embodiments, a single promoter directs the expression of RNA that contains two or three genes (e.g., encoding a chimeric signaling receptor and encoding a recombinant receptor) in a single open reading frame (ORF) that are separated from each other by sequences encoding self-cleaving peptides (e.g., 2A sequences) or protease recognition sites (e.g., furin). Thus, the ORF encodes a single polypeptide that is processed into a single protein during translation (for 2A) or after translation. In some cases, a peptide (e.g., T2A) can cause the ribosome to skip (ribosome skipping) the synthesis of the C-terminal peptide bond of the 2A element, 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), incorporated by reference in their entirety for all purposes). 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 Publication No. 20070116690.

[0183] In some embodiments, the vector is contained 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 contained in a cell. In some embodiments, the virus or cell containing the vector contains a recombinant genome.

[0184] III. Immunogenic Compositions and Formulations

[0185] In some embodiments, provided herein are immunogenic compositions comprising trimers of recombinant polypeptides comprising a sequence selected from the group consisting of SEQ ID NOs: 1-26, 85-92, 120, and 124, or a combination of any two or more trimers. In some embodiments, provided herein are immunogenic compositions comprising trimers of recombinant polypeptides having a sequence described in SEQ ID NO: 1. The immunogenic compositions mentioned herein, comprising trimers of recombinant polypeptides selected from the group consisting of SEQ ID NOs: 85-92, or fragments, variants, or mutants thereof, wherein the recombinant polypeptides are trimerized to form trimers through inter-polypeptide disulfide bonds, can be used in initial doses and / or boosters. The immunogenic compositions mentioned herein, comprising trimers of recombinant polypeptides selected from the group consisting of SEQ ID NOs: 120, or fragments, variants, or mutants thereof, wherein the recombinant polypeptides are trimerized to form trimers through inter-polypeptide disulfide bonds, can be used in initial doses and / or boosters. The immunogenic composition mentioned herein, which includes a trimer selected from the recombinant polypeptide of SEQ ID NO: 124 or a fragment, variant or mutant thereof, wherein the recombinant polypeptide is trimerized to form a trimer through disulfide bonds between polypeptides, can be used in the initial dose and / or booster. Independently, the initial dose and / or any one or more boosters may be used without or with an adjuvant. If an adjuvant is used, the optional adjuvant may include: an aluminum-containing adjuvant, such as an adjuvant containing alum and / or aluminum hydroxide; an adjuvant containing oligonucleotides, such as an adjuvant containing CpG oligodeoxynucleotides (CpG-ODN); an adjuvant containing a TLR9 agonist; an adjuvant containing metabolizable oil, α-tocopherol, and / or polyoxyethylene sorbitan monooleate (Tween-80), such as an adjuvant containing squalene, α-tocopherol, and Tween-80 and / or Span 85 in the form of an oil-in-water emulsion; or any combination of adjuvants.

[0186] In some embodiments, a unit dose of the immunogenic composition may include 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 comprises 3 μg of SARS-CoV-2 antigen. In other embodiments, the dose comprises 9 μg of SARS-CoV-2 antigen. In further embodiments, the dose comprises 30 μg of SARS-CoV-2 antigen.

[0187] In some cases, it may be desirable to combine the disclosed immunogens with other pharmaceutical products (e.g., vaccines) that induce protective responses to other agents. For example, compositions comprising a recombinant coronavirus S antigen (e.g., a trimer or protein) as described herein can be administered simultaneously (usually alone) or sequentially with other vaccines (e.g., influenza vaccine or varicella zoster vaccine) recommended by the Advisory Committee on Immunization Practices (ACIP; cdc.gov / vaccines / acip / index.html) for target age groups (e.g., infants approximately one to six months old). Thus, disclosed immunogens comprising the recombinant coronavirus S antigens described herein can be administered simultaneously or sequentially with vaccines for, for example, hepatitis B (HepB), diphtheria, tetanus, and pertussis (DTaP), pneumococcus (PCV), Haemophilus influenzae type b (Hib), polio, influenza, and rotavirus.

[0188] Multivalent or combination vaccines provide protection against multiple pathogens. In some aspects, multivalent vaccines can protect against multiple strains of the same pathogen. In some aspects, multivalent vaccines protect against multiple pathogens, such as the combination vaccine Tdap, which protects against strains of tetanus, pertussis, and diphtheria. Multivalent vaccines are essential for minimizing the number of immunizations required to confer protection against multiple pathogens or strains of viruses, in order to reduce administrative costs and increase coverage. This can be particularly useful, for example, when vaccinating infants or children.

[0189] In some embodiments, for example, a vaccine comprising an immunogenic composition as described herein is a multivalent vaccine. In some embodiments, the antigenic material for incorporation into the multivalent vaccine composition is derived from a coronavirus strain or type, for example as described herein (see, for example, Section 1). The antigens for incorporation into the multivalent vaccine composition can be derived from one or more strains of coronavirus (e.g., between two and five strains) to provide a wider range 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 vaccine, and Pediatrics), all of which are incorporated by reference in their entirety for all purposes.

[0190] In some aspects, the vaccines provided herein are universal vaccines. In some embodiments, universal vaccines are vaccines that protect against multiple strains of the same virus (e.g., multiple strains of coronavirus). Developing an effective universal coronavirus vaccine would reduce costs and labor, such as using seasonal vaccine formulations, and allow for more robust pandemic prevention.

[0191] In some embodiments, the immunogens described herein can be used as a primary series, additional doses, and / or homologous or heterologous booster doses, such as the first, second, third, fourth, and / or more doses. In some embodiments, the immunogens described herein can be used as seasonal vaccines. In some embodiments, the immunogens described herein can be used as the first, second, third, fourth, and / or more doses within five years, four years, three years, two years, one year, and / or six months.

[0192] In some aspects, a universal vaccine is a vaccine composed of multiple epitopes derived from different virus strains. In some aspects, a universal vaccine is composed of a single epitope that is conserved among different virus strains. For example, a universal vaccine can be based on a relatively conserved domain of the S protein.

[0193] Also provided are immunogenic compositions comprising the disclosed immunogens (e.g., the disclosed recombinant coronavirus S antigens or nucleic acid molecules encoding the protomers of the disclosed recombinant coronavirus S antigens) and pharmaceutically acceptable carriers. In some embodiments, the immunogenic composition includes a trimerized recombinant polypeptide provided herein and an optional pharmaceutically acceptable carrier. In some embodiments, the immunogenic composition includes a trimerized recombinant polypeptide and disodium hydrogen phosphate, such as disodium hydrogen phosphate dihydrate, sodium dihydrogen phosphate, such as disodium hydrogen phosphate monohydrate, sodium chloride, and Tween 80 provided herein. In some embodiments, 1.0 mL of an immunogenic composition aqueous solution includes 720 μg of a trimerized recombinant polypeptide provided herein and 0.62 mg of disodium hydrogen phosphate dihydrate, 0.62 mg of disodium hydrogen phosphate monohydrate, 9.0 mg of sodium chloride, and 0.2 mg of Tween 80. In some embodiments, the immunogenic composition includes a protein comprising a plurality of trimerized recombinant polypeptides provided herein and an optional pharmaceutically acceptable carrier. In some embodiments, the immunogenic composition comprises a protein nanoparticle as provided herein and an optional pharmaceutically acceptable carrier. In some embodiments, the immunogenic composition comprises a VLP as provided herein and an optional pharmaceutically acceptable carrier. In some embodiments, the immunogenic composition comprises an isolated nucleic acid as provided herein and an optional pharmaceutically acceptable carrier. In some embodiments, the immunogenic composition comprises a carrier as provided herein and an optional pharmaceutically acceptable carrier. In some embodiments, the immunogenic composition comprises a virus as provided herein and an optional pharmaceutically acceptable carrier. In some embodiments, the immunogenic composition comprises a pseudovirus as provided herein and an optional pharmaceutically acceptable carrier. In some embodiments, the immunogenic composition comprises a cell as provided herein and an optional pharmaceutically acceptable carrier. In some embodiments, the immunogenic composition as 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 vaccine and a therapeutic vaccine. Such pharmaceutical compositions can be administered to a subject by a variety of modes of administration known to those of ordinary skill in the art, for example, intramuscularly, intradermally, subcutaneously, intravenously, intraarterially, intraarticularly, intraperitoneally, intranasally, sublingually, tonsilarily, oropharyngeally, or other parenteral and mucosal routes. In several embodiments, the pharmaceutical composition comprising one or more disclosed immunogens is an immunogenic composition. Actual methods for preparing administrable compositions are known or apparent to those skilled in the art and are described in more detail in publications such as Remingtons Pharmaceutical Sciences, 19th Ed., Mack Publishing Company, Easton, Pa., 1995.

[0194] Therefore, immunogens as described herein, such as recombinant coronavirus S antigens, such as trimers, proteins, can be formulated with pharmaceutically acceptable carriers to help maintain biological activity, while also promoting increased stability during storage within an acceptable temperature range. Potential carriers include, but are not limited to, physiologically balanced culture medium, phosphate buffered saline, water, emulsions (such as, oil / water or water / oil emulsions), various types of wetting agents, antifreeze additives or stabilizers, such as proteins, peptides or hydrolysates (such as, albumin, gelatin), sugars (such as, sucrose, lactose, sorbitol), amino acids (such as, sodium glutamate) or other protective agents. The resulting aqueous solution can be packaged for use or lyophilized as is. The lyophilized preparation is mixed with a sterile solution before single or multiple dose administration.

[0195] Formulated compositions, especially liquid formulations, may contain bacteriostatic agents to prevent or minimize degradation during storage, including but not limited to benzyl alcohol, phenol, m-cresol, chlorobutanol, methylparaben, and / or propylparaben at effective concentrations (typically 1% w / v). Some patients may be contraindicated for the use of bacteriostatic agents; therefore, lyophilized formulations can be reconstituted in solutions with or without such ingredients.

[0196] The immunogenic composition of the present invention may contain pharmaceutically acceptable carrier substances required for approximate physiological conditions, such as pH regulators and buffers, tension regulators, wetting agents, etc., such as sodium acetate, sodium lactate, sodium chloride, potassium chloride, calcium chloride, sorbitan monolaurate and triethanolamine oleate, polysorbate 80. The immunogenic composition may optionally include an adjuvant to enhance the host's immune response. Suitable adjuvants are, for example, toll-like receptor (TLR) agonists, alum, AlPO4, hydrogels (alhydrogel), lipid-A and its derivatives or variants, oil emulsions, saponins, neutral liposomes, liposomes containing vaccines and cytokines, non-ionic block copolymers and chemokines. Non-ionic block polymers containing polyoxyethylene (POE) and polyoxypropylene (POP) among many other suitable adjuvants well known in the art can be used as adjuvants, such as POE-POP-POE block copolymers, MPL TM(3-O-deacylated monophospholipid A; Corixa, Hamilton, Ind.) and IL-12 (Genetics Institute, Cambridge, Mass.) (Newman et al., 1998, Critical Reviews in Therapeutic Drug Carrier Systems 15:89-142, incorporated herein by reference in its entirety for all purposes). The advantage of these adjuvants is that they help stimulate the immune system in a non-specific manner, thereby enhancing the immune response to the drug product. In some embodiments, the immunogenic compositions of the present invention may include or be administered with more than one adjuvant. In some embodiments, the immunogenic compositions of the present invention may include or be administered with two adjuvants. In some embodiments, the immunogenic compositions of the present invention may include or be administered with multiple adjuvants. For example, in some cases, a vaccine, such as one comprising the immunogenic compositions provided herein, may include or be administered in combination with multiple adjuvants.

[0197] For vaccine compositions, examples of suitable adjuvants include, for example, aluminum hydroxide, lecithin, Freund's adjuvant, MPL TM and IL-1, one or any combination thereof can be used in conjunction with a trimer comprising a recombinant polypeptide selected from the group consisting of SEQ ID NOs: 85-92 or a fragment, variant or mutant thereof. In some embodiments, the vaccine compositions or nanoparticle immunogens disclosed herein (e.g., SARS-COV-2 vaccine compositions) can be formulated as controlled-release or sustained-release formulations. This can be achieved by compositions containing sustained-release polymers or by microencapsulated delivery systems or bioadhesive gels. Various pharmaceutical compositions can be prepared according to standard procedures well known in the art.

[0198] In some embodiments, the immunogenic composition of the present invention, comprising a trimer of a recombinant polypeptide selected from the group consisting of SEQ ID NOs: 85-92 or a fragment, variant, or mutant thereof, wherein the recombinant polypeptide is trimerized to form a trimer through inter-polypeptide disulfide bonds, may comprise an adjuvant formulation comprising a metabolizable oil (e.g., squalene) and α-tocopherol (e.g., DL-α-tocopherol) in the form of an oil-in-water emulsion, and polyoxyethylene sorbitan monooleate (Tween-80). In some embodiments, the adjuvant formulation may comprise about 2% to about 10% squalene, about 2% to about 10% α-tocopherol (e.g., D-α-tocopherol), and about 0.3% to about 3% polyoxyethylene sorbitan monooleate. In some embodiments, the adjuvant formulation may comprise about 5% squalene, about 5% tocopherol, and about 0.4% polyoxyethylene sorbitan monooleate. In some embodiments, the immunogenic compositions of the present invention may comprise triple des-O-acylated monophospholipid A (3D-MPL) and an adjuvant in the form of an oil-in-water emulsion comprising a metabolizable oil, α-tocopherol, and polyoxyethylene sorbitan monooleate. In some embodiments, the immunogenic compositions of the present invention may comprise QS21 (Quillaja saponaria Molina extract: Fraction 21), 3D-MPL, and an oil-in-water emulsion, wherein the oil-in-water emulsion comprises a metabolizable oil, α-tocopherol, and polyoxyethylene sorbitan monooleate. In some embodiments, the immunogenic compositions of the present invention may comprise QS21, 3D-MPL, and an oil-in-water emulsion, wherein the oil-in-water emulsion has the following composition: a metabolizable oil, such as squalene, α-tocopherol, and Tween-80 and / or Span 85. In some embodiments, the immunogenic compositions of the present invention may comprise an adjuvant in the form of a liposomal composition.

[0199] In some embodiments, the immunogenic composition of the present invention, comprising a trimer of a recombinant polypeptide selected from the group consisting of SEQ ID NOs: 120 and 124, or a fragment, variant, or mutant thereof, wherein the recombinant polypeptide is trimerized to form a trimer via inter-polypeptide disulfide bonds, may comprise an adjuvant formulation comprising a metabolizable oil (e.g., squalene), α-tocopherol, polyoxyethylene sorbitan monooleate (Tween-80), and / or Span 85. In some embodiments, the adjuvant formulation may comprise about 5% (w / v) squalene, about 5% (w / v) α-tocopherol, about 0.5% (w / v) polyoxyethylene sorbitan monooleate, and / or about 0.5% (w / v) Span 85.

[0200] In some embodiments, the immunogenic compositions of the invention, comprising trimers of recombinant polypeptides selected from the group consisting of SEQ ID NOs: 85-92, or fragments, variants, or mutants thereof, wherein the recombinant polypeptides are trimerized to form trimers via inter-polypeptide disulfide bonds, may comprise an adjuvant formulation comprising Quillaja saponins, cholesterol, and a phospholipid, for example, in the form of a nanoparticle composition. In some embodiments, the immunogenic compositions of the invention may comprise a mixture of individually purified Quillaja saponaria Molina fractions, which are subsequently formulated with cholesterol and a phospholipid.

[0201] In some embodiments, the immunogenic composition of the present invention comprises a trimer of a recombinant polypeptide selected from the group consisting of SEQ ID NOs: 85-92, 120 and 124, or a fragment, variant or mutant thereof, wherein the recombinant polypeptide is trimerized to form a trimer through disulfide bonds between polypeptides, and may comprise a trimer selected from the group consisting of MF59 TM Matrix-A TM Matrix-C TM 、Matrix-M TM , AS01, AS02, AS03 and AS04. Optional adjuvants include O'Hagan et al, The history of adjuvant: a phoenix that arose from the ashes, Expert Review of Vaccines, DOI: 10.1586 / ERV.12.140 (2013); et al, Development and evaluation of AS03, an Adjuvant System containing α-tocopherol and squalene in an oil-in-water emulsion, Expert Review of Vaccines, 11(3), 349-366(2012); Morel et al., Adjuvant System AS03 containing α-tocopherol modulates innate immune response and leads to improved adaptive immunity, Vaccine, doi: 10.1016 / j.vaccine.2011.01.011(2011), any one or a combination of the adjuvants disclosed in, are incorporated herein by reference in their entirety for all purposes.

[0202] In some embodiments, the immunogenic composition of the present invention comprises a trimer of a recombinant polypeptide selected from the group consisting of SEQ ID NOs: 85-92, 120, and 124, or a fragment, variant, or mutant thereof, wherein the recombinant polypeptide forms a trimer by trimerization via inter-polypeptide disulfide bonds, and may comprise a toll-like receptor 9 (TLR9) agonist, wherein the TLR9 agonist is an oligonucleotide having a length of 8 to 35 nucleotides comprising an unmethylated cytidine-phosphate-guanosine (also known as CpG or cytosine-phosphate-guanosine) motif, and the SARS-CoV-2 antigen and the oligonucleotide are present in the immunogenic composition in an amount effective to stimulate an immune response against the SARS-CoV-2 antigen in a mammalian subject (e.g., a human subject in need of the SARS-CoV-2 antigen and the oligonucleotide). TLR9 (CD289) recognizes unmethylated cytidine-phosphate-guanosine (CpG) motifs found in microbial DNA, which can be mimicked using synthetic CpG-containing oligodeoxynucleotides (CpG-ODNs). It is known that CpG-ODN can enhance antibody production and stimulate T helper cell 1 (Th1) cell response (Coffman et al., Immunity, 33:492-503, 2010, which is incorporated by reference in its entirety for all purposes). The best oligonucleotide TLR9 agonists generally contain a palindromic sequence of the following 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 by reference in its entirety herein. In some embodiments, the CpG oligonucleotide is linear. In other embodiments, the CpG oligonucleotide is circular or includes a hairpin loop. The CpG oligonucleotide can be single-stranded or double-stranded. In some embodiments, the CpG oligonucleotide may include modifications. Modifications include, but are not limited to, modifications of 3'OH or 5'OH groups, modifications of nucleotide bases, modifications of sugar components, and modifications of phosphate groups. As long as the modified base maintains the same specificity to its natural complement by Watson-Crick base pairing (e.g., the palindrome remains self-complementary), the modified base may be included in the palindromic sequence of the CpG oligonucleotide. In some embodiments, the CpG oligonucleotide includes non-classical bases. In some embodiments, the CpG oligonucleotide includes modified nucleosides. In some embodiments, the modified nucleosides are selected from 2'-deoxy-7-deazaguanosine, 2'-deoxy-6-thioguanosine, arabinoguanosine, 2'-deoxy-2' substituted-arabinoguanosine, and 2'-O-substituted-arabinoguanosine. The CpG oligonucleotide may include modifications of the phosphate group.For example, in addition to phosphodiester bonds, phosphoric acid modification includes but is not limited to methyl phosphonate, phosphorothioate, phosphoramide (bridged or non-bridged), phosphotriester and phosphorodithioate, and can be used in any combination. Other non-phosphate bonds can also be used. In some embodiments, oligonucleotides only include thiophosphate backbones. In some embodiments, oligonucleotides only include phosphodiester backbones. In some embodiments, oligonucleotides include a combination of the phosphate bonds in the phosphate backbone, such as a combination of phosphodiester bonds and thiophosphate bonds. Oligonucleotides with thiophosphate backbones can have higher immunogenicity than oligonucleotides with phosphodiester backbones, and seem to be more resistant to degradation after being injected into the host (Braun et al., J Immunol, 141: 2084-2089, 1988; and Latimer et al., Mol Immunol, 32: 1057-1064, 1995, cited in its entirety for all purposes). The CpG oligonucleotides of the present invention include at least one, two, or three internucleotide phosphorothioate bonds. In some embodiments, when a plurality of CpG oligonucleotide molecules are present in a pharmaceutical composition comprising at least one excipient, two stereoisomers of the phosphorothioate bonds are present in the plurality of CpG oligonucleotide molecules. In some embodiments, all internucleotide bonds of the CpG oligonucleotide are phosphorothioate bonds, or in other words, the CpG oligonucleotide has a phosphorothioate backbone.

[0203] In the present invention, any suitable CpG oligodeoxynucleotide (ODN) or combination thereof can be used as an adjuvant. For example, K-type ODN (also known as B-type) encodes multiple CpG motifs on a phosphorothioate backbone. K-type ODN can be based on the following sequence: The use of phosphorothioate nucleotides enhances resistance to nuclease digestion compared to natural phosphodiester nucleotides, resulting in a significantly prolonged half-life in vivo. K-type ODN triggers pDC differentiation and TNF-α production, and triggers B cell proliferation and IgM secretion. D-type ODN (also known as A-type) is composed of a mixed phosphodiester / phosphorothioate backbone, contains a single CpG motif, flanked by palindromic sequences, and has poly-G tails (structural motifs that promote concatemer formation) at the 3' and 5' ends. D-type ODN can be based on the following sequence GGTGCATCGATGCAGGGGGG. D-type ODN triggers pDC maturation and IFN-α secretion, but has no effect on B cells. C-type ODN is similar to K-type and is composed entirely of phosphorothioate nucleotides, but similar to D-type ODN, contains a palindromic CpG motif. C-type ODN can be based on the following sequence This type of ODN stimulates B cells to secrete IL-6 and pDC to produce IFN-α. P-type ODN contains two palindromic sequences, which enable them to form a higher order structure. P-type ODN can be based on the following sequence P-type ODN activates B cells and PDCs and induces greater IFN-α production compared to C-type ODN. In this paragraph, boldfaced text within the ODN sequence indicates a self-complementary palindrome, and the CpG motif is underlined.

[0204] Exemplary CpG ODNs, such as CpG 7909 (5′-TCGTCGTTTTGTCGTTTTGTCGTT-3′) and CpG 1018 (5′-TGACTGTGAACGTTCGAGATGA-3′), are known and disclosed in U.S. Pat. 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; 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.

[0205] 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 microparticles, such as poly(lactide-co-glycolide) microparticles (Shah et al., Methods Mol Biol, 1494: 1-14, 2017, incorporated by reference in its entirety for all purposes). In some embodiments, the immunogenic composition further comprises an aluminum salt adjuvant that adsorbs SARS-CoV-2 antigens. 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 0.01 mg to about 0.8 mg Al 3+ ; or about 0.05 mg to about 0.7 mg Al 3+ ; or about 0.06 mg to about 0.6 mg Al 3+ ; or about 0.07 mg Al 3+ , about 0.08 mg to about 0.45 mg Al 3+ ; or about 0.08 mg to about 0.30 mg, about 0.35 mg Al 3+ , about 0.40mg Al3+ or about 0.25 mg to about 0.50 mg Al 3+ ; or about 0.068 mg Al 3+ , about 0.078mg Al 3+ , about 0.13mg Al 3+ , about 0.19mg Al 3+ , about 0.22mg Al 3+ , about 0.26mg, about 0.35mg Al 3+ In some embodiments, the immunogenic composition further comprises an additional adjuvant. Other suitable adjuvants include, but are not limited to, squalene in water emulsion (e.g., MF59 or AS03), TLR3 agonists (e.g., poly IC or poly ICLC), TLR4 agonists (e.g., bacterial lipopolysaccharide derivatives, such as monophospholipid A (MPL), and / or saponins, such as QuilA or QS-21, such as AS01 or AS02), TLR5 agonists (bacterial flagellin) and TLR7, TLR8 and / or TLR9 agonists (imidazole quinoline derivatives, such as imiquimod and resiquimod) (Coffman et al., Immunity, 33:492-503, 2010, incorporated by reference in its entirety for all purposes). In some embodiments, additional adjuvants include MPL and aluminum hydroxide / alum (e.g., AS04). For veterinary use and for the production of antibodies in non-human animals, the mitogenic component (complete and incomplete) of Freund's adjuvant may be used.

[0206] In some embodiments, a unit dose of the immunogenic composition may include from about 3 μg to about 1000 μg of one or more adjuvants, from about 8 μg to about 1000 μg of one or more adjuvants, preferably from about 25 μg to about 500 μg of one or more adjuvants, preferably from about 9 μg to about 500 μg of one or more adjuvants, preferably from about 50 μg to about 300 μg of one or more adjuvants, preferably from about 100 μg to about 250 μg of one or more adjuvants, preferably from about 150 μg to about 225 μg of one or more adjuvants. In some embodiments, a unit dose of the immunogenic composition may include about 3 μg to about 700 μg, 750 μg, 800 μg of aluminum-containing adjuvant, about 3 μg to about 500 μg of aluminum-containing adjuvant, about 8 μg to about 250 μg of aluminum-containing adjuvant, about 9 μg to about 240 μg of aluminum-containing adjuvant, preferably about 25 μg to about 230 μg of aluminum-containing adjuvant, preferably about 200 μg to about 800 μg of aluminum-containing adjuvant, preferably about 700 μg to about 800 μg of aluminum-containing adjuvant, preferably about 740 μg to about 780 μg of aluminum-containing adjuvant, preferably about 750, 760, 770 μg to about 780 μg of aluminum-containing adjuvant, preferably about 260 μg to about 280 μg of aluminum-containing adjuvant, preferably about 200 μg to about 240 μg of aluminum-containing adjuvant. 0 μg aluminum-containing adjuvant, preferably about 210 μg to about 230 μg aluminum-containing adjuvant, preferably about 220 μg to about 230 μg aluminum-containing adjuvant, preferably about 224 μg to about 228 μg aluminum-containing adjuvant, preferably about 50 μg to about 125 μg aluminum-containing adjuvant, preferably about 750 μg aluminum-containing adjuvant, such as Alum, about 50 μg aluminum-containing adjuvant, about 45 μg aluminum-containing adjuvant, about 40 μg aluminum-containing adjuvant, about 35 μg aluminum-containing adjuvant, about 25 μg aluminum-containing adjuvant, about 24 μg, about 23 μg, about 22 μg, about 20 μg aluminum-containing adjuvant, about 15 μg aluminum-containing adjuvant, about 10 μg aluminum-containing adjuvant, about 9 μg aluminum-containing adjuvant, or about 8, about 7, about 6, about 5, about 4, about 3 μg aluminum-containing adjuvant.In some embodiments, a unit dose of an immunogenic composition may include about 10 μg to about 2000 μg of CpG adjuvant, or about 20 μg to about 1800, 1700, or 1600 μg of CpG adjuvant, or about 40 μg to about 1500 μg of CpG adjuvant, or about 1000 μg to about 1900 μg of CpG adjuvant, or about 1200 μg to about 1800 μg of CpG adjuvant, or about 1300 μg to about 1700 μg of CpG adjuvant, or about 1400 μg to about 1600 μg of CpG adjuvant, or about 10 μg to about 500 μg of CpG adjuvant, or about 460 μg to about 470 μg of CpG adjuvant, or about 410 μg, 420 μg, 430 μg, 440 μg to about 490 μg of CpG adjuvant. CpG adjuvant, or about 10 μg to about 500 μg CpG adjuvant, or preferably about 25 μg to about 300 μg CpG adjuvant, preferably about 50 μg to about 250 μg CpG adjuvant, preferably about 75 μg to about 200 μg CpG adjuvant, preferably about 100 μg to about 175 μg CpG adjuvant, or preferably about 1500 μg CpG adjuvant, or preferably about 1600 μg CpG adjuvant, or preferably about 1400 μg, about 1450 μg, about 1455 μg CpG adjuvant, preferably about 150 μg CpG adjuvant, such as CpG 1018A. In some embodiments, a unit dose of the immunogenic composition may include about 10 μg to about 500 μg of aluminum-containing adjuvant and about 10 μg to about 500 μg of CpG adjuvant. In some embodiments, a unit dose of the immunogenic composition may include about 10 μg to about 800 μg of aluminum-containing adjuvant and about 10 μg to about 1800 μg of CpG adjuvant, and in some embodiments, a unit dose of the immunogenic composition may include about 25 μg to about 250 μg of aluminum-containing adjuvant and about 25 μg to about 300 μg of CpG adjuvant. In some embodiments, a unit dose of the immunogenic composition may include about 200 μg to about 800 μg of aluminum-containing adjuvant and about 500 μg to about 1800 μg of CpG adjuvant. In some embodiments, a unit dose of the immunogenic composition may include about 200 μg to about 250 μg of aluminum-containing adjuvant and about 400 μg to about 500 μg of CpG adjuvant. In some embodiments, a unit dose of the immunogenic composition may include about 210 μg to about 240 μg of aluminum-containing adjuvant and about 420 μg to about 490 μg of CpG adjuvant. In some embodiments, a unit dose of the immunogenic composition may include about 220 μg to about 230 μg of aluminum-containing adjuvant and about 430 μg to about 470 μg of CpG adjuvant. In some embodiments, a unit dose of the immunogenic composition may include about 221 μg to about 227 μg of aluminum-containing adjuvant and about 440 μg to about 460 μg of CpG adjuvant.In some embodiments, a unit dose of the immunogenic composition may include about 600 μg to about 900 μg of aluminum-containing adjuvant and about 1000 μg to about 2000 μg of CpG adjuvant. In some embodiments, a unit dose of the immunogenic composition may include about 700 μg to about 800 μg of aluminum-containing adjuvant and about 1100 μg to about 1900 or 1800 μg of CpG adjuvant. In some embodiments, a unit dose of the immunogenic composition may include about 720 μg to about 790 μg of aluminum-containing adjuvant and about 1200 or 1300 μg to about 1700 μg of CpG adjuvant. In some embodiments, a unit dose of the immunogenic composition may include about 730 or 740 μg to about 760 μg of aluminum-containing adjuvant and about 1400 μg to about 1600 μg of CpG adjuvant. In some embodiments, a unit dose of the immunogenic composition may include about 50 μg to about 125 μg of aluminum-containing adjuvant and about 50 μg to about 250 μg of CpG adjuvant. In some embodiments, a unit dose of the immunogenic composition may include about 50 μg to about 100 μg of aluminum-containing adjuvant and about 75 μg to about 200 μg of CpG adjuvant. In some embodiments, a unit dose of the immunogenic composition may include about 50 μg to about 100 μg of aluminum-containing adjuvant and about 100 μg to about 175 μg of CpG adjuvant. In some embodiments, a unit dose of the immunogenic composition may include about 75 μg to about 100 μg of aluminum-containing adjuvant, such as Alum, and about 150 μg of CpG adjuvant, such as CpG 1018A. In some embodiments, a unit dose of an immunogenic composition may include about 200 μg to about 250 μg of an aluminum-containing adjuvant, such as Alum, and about 400 μg to about 500 μg of a CpG adjuvant, such as CpG 1018A. In some embodiments, a unit dose of an immunogenic composition may include about 210 μg to about 240 μg of an aluminum-containing adjuvant, such as Alum, and about 420 μg to about 490 μg of a CpG adjuvant, such as CpG 1018A. In some embodiments, a unit dose of an immunogenic composition may include about 220 μg to about 230 μg of an aluminum-containing adjuvant, such as Alum, and about 430 μg to about 470 μg of a CpG adjuvant, such as CpG 1018A. In some embodiments, a unit dose of the immunogenic composition may include about 221 μg to about 227 μg of an aluminum-containing adjuvant, such as Alum, and about 440 μg to about 460 μg of a CpG adjuvant, such as CpG 1018A. In some embodiments, a unit dose of the immunogenic composition may include about 600 μg to about 900 μg of an aluminum-containing adjuvant, such as Alum, and about 1000 μg to about 2000 μg of a CpG adjuvant, such as CpG 1018A.In some embodiments, a unit dose of an immunogenic composition may include about 700 μg to about 800 μg of an aluminum-containing adjuvant, such as Alum, and about 1100 μg to about 1900 or 1800 μg of a CpG adjuvant, such as CpG 1018A. In some embodiments, a unit dose of an immunogenic composition may include about 720 μg to about 790 μg of an aluminum-containing adjuvant, such as Alum, and about 1200 or 1300 μg to about 1700 μg of a CpG adjuvant, such as CpG 1018A. In some embodiments, a unit dose of an immunogenic composition may include about 730 or 740 μg to about 760 μg of an aluminum-containing adjuvant, such as Alum, and about 1400 μg to about 1600 μg of a CpG adjuvant, such as CpG 1018A. In some embodiments, a unit dose of an immunogenic composition may include about 10 μg to about 100 μg of a SARS-CoV-2 antigen, such as any one or more of the SARS-CoV-2 S fusion proteins or S-trimers of the present invention, such as a fusion protein or S fusion protein trimer comprising a SARS-CoV-2 coronavirus Omicron (B.1.1.529) variant S protein peptide or a fragment, variant, or mutant thereof. In some embodiments, a unit dose of an immunogenic composition may include about 20 μg to about 75 μg of a SARS-CoV-2 S fusion protein or S-trimer, preferably about 25 μg to about 60 μg of a SARS-CoV-2 S fusion protein or S-trimer, or about 30 μg, about 40 μg, or about 50 μg of a SARS-CoV-2 S fusion protein or S-trimer. In some embodiments, a unit dose of an immunogenic composition may include about 3 μg of a SARS-CoV-2 S fusion protein or S-trimer. In other embodiments, the dose comprises 15 μg of SARS-CoV-2 S fusion protein or S-trimer. In other embodiments, the dose comprises 9 μg of SARS-CoV-2 S fusion protein or S-trimer. In further embodiments, the dose comprises 30 μg of SARS-CoV-2 S fusion protein or S-trimer. In some embodiments, a unit dose of the immunogenic composition may include about 5 μg to about 20 μg of a SARS-CoV-2 antigen, such as any one or more of the SARS-CoV-2 S fusion proteins or S-trimers of the present invention, such as a fusion protein or S fusion protein trimer comprising a SARS-CoV-2 coronavirus Omicron (B.1.1.529) variant S protein peptide or a fragment, variant, or mutant thereof, and may include about 200 μg to about 250 μg of an aluminum-containing adjuvant, such as Alum, and about 400 μg to about 500 μg of a CpG adjuvant, such as CpG 1018A.In some embodiments, a unit dose of an immunogenic composition may include about 6 μg to about 17, 18, or 19 μg of a SARS-CoV-2 antigen, such as any one or more of the SARS-CoV-2 S fusion proteins or S-trimers of the present invention, such as a fusion protein or S fusion protein trimer comprising the S protein peptide of the SARS-CoV-2 coronavirus Omicron (B.1.1.529) variant or a fragment, variant, or mutant thereof, and may include about 210 μg to about 240 μg of an aluminum-containing adjuvant, such as Alum, and about 420 μg to about 490 μg of a CpG adjuvant, such as CpG 1018A. In some embodiments, a unit dose of an immunogenic composition may include about 8 μg to about 12, 13, 14, 15, or 16 μg of a SARS-CoV-2 antigen, such as any one or more of the SARS-CoV-2 S fusion proteins or S-trimers of the present invention, such as a fusion protein or S fusion protein trimer comprising the S protein peptide of the SARS-CoV-2 coronavirus Omicron (B.1.1.529) variant or a fragment, variant, or mutant thereof, and may include about 220 μg to about 230 μg of an aluminum-containing adjuvant, such as Alum, and about 430 μg to about 470 μg of a CpG adjuvant, such as CpG 1018A. In some embodiments, a unit dose of an immunogenic composition may include about 8 μg to about 11 μg of a SARS-CoV-2 antigen, such as any one or more of the SARS-CoV-2 S fusion proteins or S-trimers of the present invention, such as a fusion protein or S fusion protein trimer comprising the SARS-CoV-2 coronavirus Omicron (B.1.1.529) variant S protein peptide or a fragment, variant or mutant thereof, and may include about 221 μg to about 227 μg of an aluminum-containing adjuvant, such as Alum, and about 440 μg to about 460 μg of a CpG adjuvant, such as CpG 1018A. In some embodiments, a unit dose of an immunogenic composition may include about 8 μg to about 12, 13, 14, 15, or 16 μg of a SARS-CoV-2 antigen, such as any one or more of the SARS-CoV-2 S fusion proteins or S-trimers of the present invention, such as a fusion protein or S fusion protein trimer comprising a SARS-CoV-2 coronavirus Omicron (B.1.1.529) variant S protein peptide or a fragment, variant, or mutant thereof, and may include about 600 μg to about 900 μg of an aluminum-containing adjuvant, such as Alum, and about 1000 μg to about 2000 μg of a CpG adjuvant, such as CpG 1018A.In some embodiments, a unit dose of an immunogenic composition may include about 10 μg to about 80 μg of a SARS-CoV-2 antigen, such as any one or more of the SARS-CoV-2 S fusion proteins or S-trimers of the present invention, such as a fusion protein or S fusion protein trimer comprising the SARS-CoV-2 coronavirus Omicron (B.1.1.529) variant S protein peptide or a fragment, variant or mutant thereof, and may include about 700 μg to about 800 μg of an aluminum-containing adjuvant, such as Alum, and about 1100 μg to about 1900 or 1800 μg of a CpG adjuvant, such as CpG 1018A. In some embodiments, a unit dose of an immunogenic composition may include about 15 μg to about 60, 70 μg of a SARS-CoV-2 antigen, such as any one or more of the SARS-CoV-2 S fusion proteins or S-trimers of the present invention, such as a fusion protein or S fusion protein trimer comprising the S protein peptide of the SARS-CoV-2 coronavirus Omicron (B.1.1.529) variant or a fragment, variant or mutant thereof, and may include about 720 μg to about 790 μg of an aluminum-containing adjuvant, such as Alum, and about 1200 or 1300 μg to about 1700 μg of a CpG adjuvant, such as CpG 1018A. In some embodiments, a unit dose of an immunogenic composition may include about 25 μg to about 40, 50 μg of a SARS-CoV-2 antigen, such as any one or more of the SARS-CoV-2 S fusion proteins or S-trimers of the present invention, such as a fusion protein or S fusion protein trimer comprising a SARS-CoV-2 coronavirus Omicron (B.1.1.529) variant S protein peptide or a fragment, variant, or mutant thereof, and may include about 730 or 740 μg to about 760 μg of an aluminum-containing adjuvant, such as Alum, and about 1400 μg to about 1600 μg of a CpG adjuvant, such as CpG 1018A. In some embodiments, a unit dose of an immunogenic composition may include about 30 μg of a SARS-CoV-2 S fusion protein or S-trimer, about 75 μg to about 100 μg of an aluminum-containing adjuvant, such as Alum, and about 150 μg of a CpG adjuvant, such as CpG 1018A. In some embodiments, a unit dose of an immunogenic composition may include about 9 μg of a SARS-CoV-2 antigen, such as any one or more of the SARS-CoV-2 S fusion proteins or S-trimers of the present invention, such as a fusion protein or S fusion protein trimer comprising the S protein peptide of the SARS-CoV-2 coronavirus Omicron (B.1.1.529) variant or a fragment, variant or mutant thereof, and may include about 225 μg of an aluminum-containing adjuvant, such as Alum, and about 450 μg of a CpG adjuvant, such as CpG 1018A.In some embodiments, a unit dose of an immunogenic composition may include about 30 μg of a SARS-CoV-2 antigen, such as any one or more of the SARS-CoV-2 S fusion proteins or S-trimers of the present invention, such as a fusion protein or S fusion protein trimer comprising the S protein peptide of the SARS-CoV-2 coronavirus Omicron (B.1.1.529) variant or a fragment, variant or mutant thereof, and may include about 750 μg of an aluminum-containing adjuvant, such as Alum, and about 1500 μg of a CpG adjuvant, such as CpG 1018A.

[0207] In some embodiments, the immunogenic composition includes a pharmaceutically acceptable excipient, including, for example, a solvent, a filler, a buffer, a tonicity adjuster, and a preservative (Pramanick et al., Pharma Times, 45:65-77, 2013, incorporated by reference in its entirety for all purposes). In some embodiments, the immunogenic composition may include an excipient that acts as one or more of a solvent, a filler, a buffer, and a tonicity adjuster (e.g., sodium chloride in saline can serve as both an aqueous carrier and a tonicity adjuster).

[0208] In some embodiments, the immunogenic composition includes an aqueous carrier as a solvent. Suitable carriers include, for example, sterile water, saline, phosphate-buffered saline, and Ringer's solution. In some embodiments, the composition is isotonic.

[0209] The immunogenic composition may include a buffer. The buffer controls pH to suppress degradation of the active agent during processing, storage, and alternatively reconstruction. Suitable buffers include, for example, salts comprising acetate, citrate, phosphate (e.g., sodium dihydrogen phosphate monohydrate, sodium hydrogen phosphate dihydrate) or sulfate. For example, other suitable buffers include, for example, amino acids, such as arginine, glycine, histidine, and lysine. The buffer may further include hydrochloric acid or sodium hydroxide. In some embodiments, the buffer maintains the pH of the composition within the range of 6 to 9. In some embodiments, the pH is greater than (lower limit) 6, 7, or 8. In some embodiments, the pH is less than (upper limit) 9, 8, or 7. That is, the pH is in the range of about 6 to 9, wherein the lower limit is less than the upper limit. In some embodiments, the immunogenic composition may include 0.01-1 or 0.02-0.09 or 0.03-0.08 or 0.04-0.07 or 0.04-0.06 mg / ml sodium phosphate monobasic monohydrate, 0.01-1 or 0.02-0.09 or 0.03-0.08 or 0.04-0.07 or 0.04-0.06 mg / ml sodium phosphate dibasic dihydrate. In some embodiments, the immunogenic composition may include 0.052 mg / ml sodium phosphate monobasic monohydrate, 0.052 mg / ml sodium phosphate dibasic dihydrate. In some embodiments, the immunogenic composition may include 0.08-1 or 0.09-0.9 or 0.1-0.8 or 0.2-0.7 or 0.3-0.65 or 0.35-0.55 or 0.38-0.45 hydrochloric acid (37%). In some embodiments, the immunogenic composition can include 0.406 mg / ml hydrochloric acid (37%).

[0210] The immunogenic composition may include a tension regulator. Suitable tension regulators include, for example, glucose, glycerol, sodium chloride, glycerin, and mannitol. In some embodiments, the immunogenic composition may include 1.00-12.00, 2.00-11.00, 3.00-10.00, 4.00-9.00, 5.00-8.00, or 6.00-7.00 mg / ml sodium chloride. In some embodiments, the immunogenic composition may include 6.860 mg / ml sodium chloride.

[0211] In some embodiments, the immunogenic composition may include a filler. When the pharmaceutical composition is lyophilized before administration, the filler is particularly useful. In some embodiments, the filler is a protective agent that helps to stabilize and prevent the active agent from degrading during freezing or spray drying and / or during storage. Suitable fillers are sugars (single, double and polysaccharides), such as sucrose, lactose, trehalose, mannitol, sorbitol, tromethamine, polysorbate 80, glucose and raffinose. In some embodiments, the immunogenic composition may include 0.08-1 or 0.09-0.9 or 0.1-0.8 or 0.2-0.7 or 0.3-0.65 or 0.4-0.64 or 0.5-0.61mg / ml tromethamine. In some embodiments, the immunogenic composition may include 0.605mg / ml tromethamine. In some embodiments, the immunogenic composition may include 0.001-0.8 or 0.002-0.7 or 0.003-0.6 or 0.004-0.7 or 0.005-0.6 or 0.008-0.4 or 0.01-0.2 or 0.02-0.09 or 0.021-0.07 or 0.022-0.05 or 0.023-0.03 mg / ml polysorbate 80. In some embodiments, the immunogenic composition may include 0.025 mg / ml polysorbate 80.

[0212] The immunogenic composition may include a preservative. Suitable preservatives include, for example, antioxidants and antimicrobial agents. However, in a preferred embodiment, the immunogenic composition is prepared under sterile conditions and in a disposable container and therefore does not need to include a preservative.

[0213] In some embodiments, the immunogenic composition may include 0.001-0.1 mg / ml SARS-CoV-2 Hu-1 spike (S) protein surface antigen or a fragment, variant or mutant thereof, 0.001-0.1 mg / ml BA.5 spike (S) protein surface antigen or a fragment, variant or mutant thereof, 0.001-0.1 mg / ml XBB.1.5 spike (S) protein surface antigen or a fragment, variant or mutant thereof, 0.5-5 mg / ml Alum, 0.5-6 mg / ml CpG1018, 0.02-0.09 mg / ml sodium dihydrogen phosphate monohydrate, 0.02-0.09 mg / ml sodium dihydrogen phosphate dihydrate, 3.00-10.00 mg / ml sodium chloride, 0.003-0.6 mg / ml polysorbate 80, tromethamine, 0.09-0.9 mg / ml hydrochloric acid (37%), and an appropriate amount of water for injection (WFI). In some embodiments, the immunogenic composition may include 0.012 mg / ml SARS-CoV-2 Hu-1 spike (S) protein surface antigen or a fragment, variant or mutant thereof, 0.024 mg / ml BA.5 spike (S) protein surface antigen or a fragment, variant or mutant thereof, 0.024 mg / ml XBB.1.5 spike (S) protein surface antigen or a fragment, variant or mutant thereof, 1.500 mg / ml Alum, 3.00 mg / ml CpG1018, 0.052 mg / ml sodium dihydrogen phosphate monohydrate, 0.052 mg / ml sodium dihydrogen phosphate dihydrate, 6.86 mg / ml sodium chloride, 0.025 mg / ml polysorbate 80, 0.605 mg / ml tromethamine, 0.406 mg / ml hydrochloric acid (37%), and an appropriate amount of WFI.

[0214] In some embodiments, the composition can be provided as a sterile composition. Pharmaceutical compositions generally comprise an effective amount of the disclosed immunogen and can be prepared by conventional techniques. Typically, the amount of the immunogen in each dose of the immunogenic composition is selected as an amount that induces an immune response without significant adverse side effects. In some embodiments, the composition can be provided in unit dosage form for inducing an immune response in a subject. The unit dosage form comprises a suitable single preselected dose for administration to the subject, or a suitably labeled or measured multiple of two or more preselected unit doses, and / or a metering mechanism for administering a unit dose or its multiple. In other embodiments, the composition further comprises one or more adjuvants.

[0215] IV. Methods of Inducing an Immune Response

[0216] In some embodiments, provided herein are methods for generating an immune response to a coronavirus surface antigen in a subject, 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, 85-92, 120 and 124, optionally wherein the complex is used as a primary series, additional dose and / or homologous or heterologous booster dose, such as the first dose, second dose, third dose, fourth dose and / or more doses, optionally wherein the primary dose, additional dose or heterologous booster dose is used in combination with any one or more of other recombinant subunit vaccines, nanoparticle vaccines, mRNA vaccines, DNA vaccines, adenovirus vector vaccines, and inactivated virus vaccines. In some embodiments, provided herein are methods for generating an immune response to a coronavirus surface antigen in a subject, wherein the surface antigen comprises an S protein or an antigenic fragment thereof, and the method comprises 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 and 85-92, optionally the complex is used as a primary series, additional dose and / or homologous or heterologous booster dose, such as the first dose, second dose, third dose, fourth dose and / or more doses, optionally the primary dose, additional dose or heterologous booster is used in combination with any one or more of other recombinant subunit vaccines, nanoparticle vaccines, mRNA vaccines, DNA vaccines, adenovirus vector vaccines, and inactivated virus vaccines. In some embodiments, provided herein are methods for generating an immune response to a coronavirus surface antigen in a subject, wherein the surface antigen comprises a sequence selected from the group consisting of SEQ ID NOs: 27-66 and 81-84, and the method comprises 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 and 85-92, optionally the complex is used as a primary series, additional dose and / or homologous or heterologous booster dose, such as the first dose, second dose, third dose, fourth dose and / or more doses, optionally the primary dose, additional dose or heterologous booster is used in combination with any one or more of other recombinant subunit vaccines, nanoparticle vaccines, mRNA vaccines, DNA vaccines, adenoviral vector vaccines, and inactivated virus vaccines.In some embodiments, provided herein are methods for generating an immune response to a coronavirus surface antigen in a subject, wherein the surface antigen comprises an S protein of a coronavirus or an antigenic fragment thereof, and optionally, the surface antigen comprises a sequence selected from the group consisting of SEQ ID NOs: 27-66 and 81-84 or an antigenic fragment thereof, and the method comprises administering to the subject an effective amount of a complex comprising a recombinant polypeptide comprising a sequence of any one of SEQ ID NOs: 85-92, optionally the complex is used as a primary series, additional dose and / or homologous or heterologous booster dose, such as a first dose, a second dose, a third dose, a fourth dose and / or more doses, optionally the primary dose, additional dose or heterologous booster is used in combination with any one or more of other recombinant subunit vaccines, nanoparticle vaccines, mRNA vaccines, DNA vaccines, adenoviral vector vaccines, and inactivated virus vaccines. Alternatively, the adjuvant in any initial dose, additional dose and / or booster dose may independently include: an aluminum-containing adjuvant, such as an adjuvant containing alum and / or aluminum hydroxide; an oligonucleotide-containing adjuvant, such as an adjuvant containing CpG oligodeoxynucleotide (CpG-ODN); an adjuvant containing a TLR9 agonist; an adjuvant containing a metabolizable oil, α-tocopherol, and / or polyoxyethylene sorbitan monooleate (Tween-80), such as an adjuvant containing squalene, α-tocopherol, and Tween-80 and / or Span 85 in the form of an oil-in-water emulsion; or any combination of adjuvants.

[0217] In some embodiments, provided herein are methods for generating an immune response to a coronavirus surface antigen in a subject, wherein the surface antigen comprises an S protein or an antigenic fragment thereof, and the method comprises administering to the subject an effective amount of a complex or a combination of any two or more of the complexes, the complex comprising a recombinant polypeptide comprising a sequence selected from the group consisting of SEQ ID NOs: 1-26 and 85-92, optionally the complex or combination as an initial dose and / or as a booster, e.g., a second and / or third booster injection. 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: 85, SEQ ID NO: 86, SEQ ID NO: 87, and / or SEQ ID NO: 88. Alternatively, the adjuvant in any initial dose, additional dose and / or booster dose may independently include: an aluminum-containing adjuvant, such as an adjuvant containing alum and / or aluminum hydroxide; an oligonucleotide-containing adjuvant, such as an adjuvant containing CpG oligodeoxynucleotide (CpG-ODN); an adjuvant containing a TLR9 agonist; an adjuvant containing a metabolizable oil, α-tocopherol, and / or polyoxyethylene sorbitan monooleate (Tween-80), such as an adjuvant containing squalene, α-tocopherol, and Tween-80 and / or Span 85 in the form of an oil-in-water emulsion; or any combination of adjuvants.

[0218] 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 to the corresponding coronavirus S antigen in the subject. In a specific example, the subject is a human. The immune response can be a protective immune response, such as a response that inhibits subsequent infection with the corresponding coronavirus. The stimulation of an immune response can also be used to treat or inhibit infections and diseases associated with the corresponding coronavirus.

[0219] A subject can be selected for treatment who has or is at risk of infection with a coronavirus, for example because of exposure or potential exposure to a coronavirus. Following administration of the disclosed immunogens, the subject can be monitored for infection or symptoms associated with the coronavirus, or both.

[0220] Typical subjects intended to be treated with the therapy and methods of the present invention include humans and non-human primates and other animals. In order to identify the subject for prevention or treatment according to the method of the present invention, an acceptable screening method is adopted to determine the risk factors associated with the target or suspected disease or condition, or to determine the state of the existing disease or condition in the subject. These screening methods include, for example, routine examinations, to determine the environment, family, occupation and other such risk factors that may be associated with the target or suspected disease or condition, and diagnostic methods, such as various ELISAs and other immunoassays for detecting and / or characterizing coronavirus infection. These and other conventional methods allow clinicians to use the method and pharmaceutical composition of the present invention to select patients in need of treatment. According to these methods and principles, compositions can be applied according to the teachings of this article or other conventional methods, as an independent prevention or treatment regimen, or as a follow-up, auxiliary or coordinated treatment regimen for other treatments.

[0221] Administration of the disclosed immunogens (e.g., coronavirus S antigens, e.g., trimers, proteins) can be used for preventive or therapeutic purposes. When provided prophylactically, the disclosed therapeutic agent is provided before any symptoms, e.g., before infection. The prophylactic administration of the disclosed therapeutic agent is used to prevent or improve any subsequent infection. When provided therapeutically, the disclosed therapeutic agent is provided at the onset of disease or infection symptoms or after, e.g., after the development of coronavirus infection symptoms corresponding to the coronavirus S antigen, or after diagnosis of coronavirus infection. Therefore, a therapeutic agent can be provided before expected exposure to the coronavirus so as to reduce the expected severity, duration or extent of infection and / or related disease symptoms after exposure or suspected exposure to the virus, or after the actual onset of infection.

[0222] The immunogens and immunogenic compositions described herein are provided to the subject in an amount that effectively induces or enhances the immune response of the subject (preferably human) to the coronavirus S antigen. The actual dosage of the disclosed immunogen will vary according to factors such as the subject's disease signs and specific state (e.g., the subject's age, size, health status, symptom degree, susceptibility factors, etc.), the time and route of administration, other drugs or treatments administered simultaneously, and the specific pharmacology of the composition to stimulate the desired activity or biological response in the subject. The dosage regimen can be adjusted to provide the best prevention or therapeutic response.

[0223] Immunogenic compositions comprising one or more of the disclosed immunogens can be used in coordinated (or prime-boost) vaccination regimens or combined formulations. In certain embodiments, the new combined immunogenic compositions and coordinated immunization regimens use separate immunogens or formulations, each of which is intended to elicit an antiviral immune response, such as an immune response to a coronavirus S antigen. Separate immunogenic compositions that elicit an antiviral immune response can be combined in 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 regimen.

[0224] There can be several boosters, each of which can be a different disclosed immunogen. In some examples, the booster can be the same immunogen as another booster or the primary immunization. The primary immunization and booster can be administered as a single dose or multiple doses, for example, two, three, four, five, six or more doses can be administered to the subject over a few days, weeks or months. Multiple boosters can also be given, for example, one to five (e.g., 1, 2, 3, 4 or 5 boosters) or more. Different doses can be used for a series of continuous immunizations. For example, a relatively large dose is used in the primary immunization, and then a relatively small dose is used for enhancement.

[0225] In some embodiments, can be after the first immunization about two weeks, about three to eight weeks or about four weeks or first immunization about several months after administration of booster.In some embodiments, can be after the first immunization about 5, about 6, about 7, about 8, about 10, about 12, about 18, about 24 months or the first immunization more or less time administration of booster.Also can regularly use other booster at appropriate time point, to strengthen experimenter's " immune memory ".The suitability of the vaccine parameter of selection, such as preparation, dosage, scheme etc., can be by taking out aliquots of serum from experimenter and measuring antibody titer during immunization program and determine.In addition, experimenter's clinical condition can be monitored to obtain expected effect, such as preventing infection or improving disease state (such as reducing viral load).If this type of monitoring shows that vaccination is suboptimal, then the immunogenic composition of extra dose can be used to make experimenter be strengthened, and the mode of expection enhancing immune response can be improved vaccination parameter.

[0226] In some embodiments, a prime-boost method can include a DNA-primer and protein-boost vaccination regimen for a subject. The method can include two or more administrations of a nucleic acid molecule or protein.

[0227] For protein therapeutics, typically, each human dose will include 1-1000 μg of protein, e.g., from about 1 μg to about 100 μg, such as from 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.

[0228] The amount used in the immunogenic composition is selected based on the subject population (e.g., infants or the elderly). The optimal dosage of a particular component can be determined by standard studies involving observation of antibody titers and other reactions in the subject. It should be understood that the therapeutically effective amount of a disclosed immunogen (e.g., a disclosed recombinant coronavirus S antigen, such as a trimer, protein, viral vector, or nucleic acid molecule in an immunogenic composition) can include an amount that is ineffective when an immune response is induced by a single dose but effective when multiple doses are administered, such as in a primary-boost regimen.

[0229] After administration of the immunogens disclosed herein, the subject's immune system typically responds to the immunogenic composition by producing antibodies specific for the coronavirus S protein peptides contained in the immunogen. This response means that an immunologically effective dose has been delivered to the subject.

[0230] In some embodiments, the antibody response of the experimenter will be determined in the case of an effective dose / immunization regimen. In most cases, it is sufficient to assess the antibody titer in the serum or plasma obtained from the experimenter. The decision on whether to give a booster vaccination and / or change the amount of the therapeutic agent given to an individual can be based at least in part on antibody titer levels. Antibody titer levels can be based on, for example, an immune binding assay, which measures the concentration of the antibody in serum bound to an antigen (including, for example, a recombinant coronavirus S antigen, such as an S-trimer).

[0231] No need to completely eliminate, reduce or prevent coronavirus infection, method can be effective.For example, compared with the coronavirus infection in the absence of an immunogen, stimulating an immune response to coronavirus with one or more disclosed immunogens can reduce or suppress the coronavirus infection of the desired amount, for example, 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%, even at least 100% (eliminating or preventing detectable infected cells). In another example, coronavirus replication can be reduced or suppressed by the disclosed method. No need to completely eliminate coronavirus replication, method can be effective. For example, compared with the coronavirus replication in the absence of an immune response, stimulating an immune response with one or more disclosed immunogens can reduce the corresponding coronavirus replication of the desired amount, for example, 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%, even at least 100% (eliminating or preventing detectable coronavirus replication).

[0232] In some embodiments, the disclosed immunogens are administered to a subject concurrently with administration of an adjuvant. In other embodiments, the disclosed immunogens are administered to a subject after administration of an adjuvant and within a sufficient time to induce an immune response.

[0233] A method of nucleic acid administration is direct immunization with plasmid DNA, for example, with mammalian expression plasmids. Immunization by nucleic acid constructs is well known in the art and is disclosed in, for example, U.S. Patent No. 5,643,578 (which describes methods for immunizing vertebrates by introducing DNA encoding the desired antigen to induce cell-mediated or humoral responses) and U.S. Patent Nos. 5,593,972 and 5,817,637 (which describe methods for operably linking nucleic acid sequences encoding antigens to regulatory sequences capable of expression). U.S. Patent No. 5,880,103 describes several methods for delivering nucleic acids encoding immunogenic peptides or other antigens to organisms. Methods include nucleic acids (or their own synthetic peptides) and immunostimulatory constructs or ISCOMS. TM Liposomal delivery of ISCOMS TM Cholesterol and QuilA TM (saponin) spontaneously formed a 30-40nm negatively charged cage structure after mixing. Using ISCOMS TM As a delivery vehicle for antigens, protective immunity has been generated in experimental models of various infections, including toxoplasmosis and Epstein-Barr virus-induced tumors (Mowat and Donachie, Immunol. Today 12:383, 1991). TMAntigen doses in the range of 1:1 to 2:1 produce class I-mediated CTL responses (Takahashi et al., Nature 344:873, 1990, incorporated by reference in its entirety for all purposes).

[0234] In some embodiments, plasmid DNA vaccines are used to express the disclosed immunogens in a subject. For example, nucleic acid molecules encoding the disclosed immunogens can be administered to a subject to induce an immune response to a coronavirus S antigen. In some embodiments, the nucleic acid molecules can be included in a plasmid vector for DNA immunization, such as the pVRC8400 vector (as described in Barouch et al., J. Virol, 79, 8828-8834, 2005, which is incorporated by reference in its entirety for all purposes).

[0235] In another method for immunizing using nucleic acid, disclosed recombinant coronavirus S antigen (such as trimer, protein) can be expressed by attenuated viral host or vector or bacterial vector. Recombinant vaccinia virus, adeno-associated virus (AAV), herpes virus, retrovirus, cytomegalovirus (cytogmeglo virus) or other viral vectors can be used to express peptides or proteins, thereby initiating CTL reactions. For example, U.S. Patent number 4,722,848 describes vaccinia vectors and methods useful in immunization planning, which are cited in its full form for all purposes. Bacillus Calmette-Guerin (Bacillus Calmette-Guerin) provides another vector for expressing peptides (see Stover, Nature 351:456-460,1991, which is cited in its full form for all purposes).

[0236] In one embodiment, nucleic acids encoding the disclosed recombinant coronavirus S antigens are introduced directly into cells. For example, nucleic acids can be loaded onto gold microspheres by standard methods and expressed by a HELIOS® microsphere, such as that from Bio-Rad. TM The nucleic acid can be introduced into the skin using a device such as a gene gun. The nucleic acid can be "naked," consisting of a plasmid under the control of a strong promoter. Typically, the DNA is injected into the muscle, but direct injection into other sites is also possible. The injection dose is generally about 0.5 μg / kg to about 50 mg / kg, and typically about 0.005 mg / kg to about 5 mg / kg (see, e.g., U.S. Patent No. 5,589,466).

[0237] In another embodiment, an mRNA-based immunization scheme can be used to deliver nucleic acids encoding the disclosed recombinant coronavirus S antigen directly into cells. In some embodiments, mRNA-based nucleic acid vaccines can provide an effective alternative to the aforementioned methods. mRNA vaccines eliminate the safety issues of DNA integration into the host genome and can be translated directly in the host cell cytoplasm. In addition, the simple cell-free in vitro synthesis of RNA avoids the manufacturing complications associated with viral vectors. Two exemplary forms of RNA-based vaccines that can be used to deliver nucleic acids encoding the disclosed recombinant coronavirus S antigens include conventional non-amplified 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). All references in this section are incorporated by reference in their entirety for all purposes.

[0238] In some embodiments, administering a therapeutically effective amount of one or more disclosed immunogens to a subject can induce a neutralizing immune response in the subject. To assess neutralizing activity, serum can be collected from the subject at an appropriate time point after immunization, frozen and stored for neutralization testing. Methods for determining neutralizing activity are known to those of ordinary skill 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, a panel of coronavirus pseudoviruses can be used to determine serum neutralizing activity.

[0239] In some embodiments, the neutralizing immune response induced by the immunogen disclosed herein produces neutralizing antibodies against coronavirus (e.g., SARS-CoV-2). In some embodiments, the neutralizing antibodies herein bind to cellular receptors or co-receptors of coronavirus (e.g., SARS-CoV-2) or its components. In some embodiments, the viral receptor or co-receptor is a coronavirus receptor or co-receptor, preferably a pneumonia virus receptor or co-receptor, more preferably a human coronavirus receptor, such as a SARS-CoV-2 receptor or co-receptor. In some embodiments, the neutralizing antibodies herein regulate, reduce, antagonize, alleviate, block, inhibit, eliminate and / or interfere with at least one coronavirus (e.g., SARS-CoV-2) activity or binding, or coronavirus (e.g., SARS-CoV-2) receptor activity or binding, such as 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, mitigate, block, inhibit, abrogate, and / or interfere with the binding of SARS-CoV-2 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-grasping non-integrin (DC-SIGN), and / or liver / lymph node-SIGN (L-SIGN).

[0240] V. Products or Kits

[0241] Also provided are articles or kits containing the provided recombinant polypeptides, proteins, and immunogenic compositions. The articles can 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 container can be formed from various materials (e.g., glass or plastic). In some embodiments, the container has a sterile access port. Exemplary containers include intravenous solution bags, vials, including containers with stoppers that can be pierced by injection needles. The article or kit can further include a package insert indicating that the composition can be used to treat a specific condition, such as the conditions described herein (e.g., coronavirus infection). Alternatively, or additionally, the article or kit can further include another or the same container containing a pharmaceutically acceptable buffer. It can further include other materials, such as other buffers, diluents, filters, needles, and / or syringes.

[0242] The label or package insert may indicate that the composition is for treating a coronavirus infection in a subject. The label or package insert on or associated with the container may indicate instructions for reconstitution and / or use of the formulation. The label or package insert may further indicate that the formulation is for or intended for subcutaneous, intravenous, or other administration to treat or prevent a coronavirus infection in a subject.

[0243] In some embodiments, the container holds a composition that is effective, alone or in combination with another composition, for treating, preventing, and / or diagnosing a condition. An article of manufacture or kit can include (a) a first container having a composition contained therein (i.e., a first agent), wherein the composition comprises an immunogenic composition or a protein or recombinant polypeptide thereof; and (b) a second container having a composition contained therein (i.e., a second agent), wherein the composition comprises another agent, such as an adjuvant or other therapeutic agent, and the article or kit further includes instructions on a label or package insert for treating a subject with the second agent in an effective amount.

[0244] the term

[0245] Unless otherwise defined, all special terms, symbols, 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 belongs. In some cases, terms with commonly understood meanings are defined herein for clarity and / or ease of reference, and such definitions included herein do not necessarily represent a substantial difference from what is generally understood in the art.

[0246] The terms "polypeptide" and "protein" are used interchangeably to refer to polymers of amino acid residues and are not limited to a minimum length. Polypeptides (including the provided receptors and other polypeptides, such as linkers or peptides) can include amino acid residues, including natural and / or non-natural amino acid residues. The term also includes post-expression modifications of the polypeptide, such as glycosylation, sialylation, acetylation, and phosphorylation. In some aspects, a polypeptide may contain modifications to the native or natural sequence, as long as the protein retains the desired activity. These modifications can be intentional, such as through site-directed mutagenesis, or accidental, such as through mutations in the host that produces the protein or errors caused by PCR amplification.

[0247] As used herein, a "subject" is a mammal, such as a human or other animal, and is typically a human. In some embodiments, the subject (e.g., patient) to which one or more agents, cells, cell populations, or compositions are administered is a mammal, typically a primate, such as a human. In some embodiments, the primate is a monkey or ape. The subject can be male or female and can be of any suitable age, including infants, teenagers, adolescents, adults, and elderly subjects. In some embodiments, the subject is a non-primate mammal, such as a rodent.

[0248] As used herein, "treatment" (and grammatical variations thereof, such as "treat" or "treating") refers to the complete or partial improvement or reduction of a disease, condition, or disorder, or a symptom, adverse reaction, or outcome, or phenotype associated therewith. Desirable effects of treatment include, but are not limited to, preventing the occurrence or recurrence of the disease, alleviating symptoms, alleviating any direct or indirect pathological consequences of the disease, preventing metastasis, reducing the rate of disease progression, ameliorating or relieving the disease state, and alleviating or improving prognosis. The term does not imply a complete cure of the disease or the complete elimination of any symptom or the effect on all symptoms or outcomes.

[0249] As used herein, "delaying the progression of a disease" means delaying, hindering, slowing, slowing, stabilizing, inhibiting, and / or postponing the progression of a disease (e.g., cancer). The length of time for the delay may vary depending on the history of the disease and / or the individual being treated. In some embodiments, sufficient or significant delay may actually encompass prevention, in that the individual will not develop the disease. For example, the development of advanced cancer, such as metastasis, may be delayed.

[0250] As used herein, "prevention" includes providing prevention against the occurrence or recurrence of a disease in a subject who may be susceptible to the disease but has not yet been diagnosed with the disease. In some embodiments, provided cells and compositions are used to delay the development of a disease or slow the progression of a disease.

[0251] As used herein, "inhibiting" a function or activity means reducing the function or activity when compared to other identical conditions except for the condition or parameter of interest or compared to another condition. For example, cells that inhibit tumor growth will reduce the growth rate of the tumor compared to the growth rate of the tumor in the absence of the cells.

[0252] In the context of administration, an "effective amount" of an agent (eg, a pharmaceutical preparation, cell, or composition) refers to an amount effective at the dosage / amount and for the period of time necessary to achieve the desired effect (eg, a therapeutic or prophylactic effect).

[0253] A "therapeutically effective amount" of an agent (e.g., a pharmaceutical formulation or cell) refers to an effective amount at the dosage and time period required to achieve the desired therapeutic effect (e.g., for treating a disease, condition, or disorder) and / or the pharmacokinetic or pharmacodynamic effect of the treatment. The 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 being administered. In some embodiments, provided methods involve administering cells and / or compositions in an effective amount (e.g., a therapeutically effective amount).

[0254] A "prophylactically effective amount" refers to an effective amount for the dosage and time period required to achieve the desired preventive effect. Typically, but not necessarily, because a prophylactic dose is used for subjects before or in the early stages of the disease, the prophylactic effective amount will be less than the therapeutically effective amount. In cases where the tumor burden is low, the prophylactic effective amount in some aspects will be higher than the therapeutically effective amount. An effective amount of a vaccine or other agent is sufficient to produce the 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 change the external symptoms of a viral infection. Generally speaking, this amount will be sufficient to measurably inhibit viral (e.g., SARS-CoV-2) replication or infectivity. When administered to a subject, the dose typically used will achieve a target tissue concentration that has been shown to inhibit viral replication in vitro. In some embodiments, an "effective amount" is an amount that treats (including prevents) one or more symptoms and / or potential causes of any condition or disease, such as for the treatment of coronavirus infection. In some embodiments, the effective amount is a therapeutically effective amount. In some embodiments, the effective amount is an amount that prevents the development of one or more signs or symptoms of a particular disease or condition, such as one or more signs or symptoms associated with coronavirus infection.

[0255] As used herein, the terms "antigen" or "immunogen" are used interchangeably to refer to a substance, typically a protein, that is capable of inducing an immune response in a subject. The term also refers to an immunologically active protein, i.e., one that, upon administration to a subject (directly or by administering a nucleotide sequence or vector encoding the protein to the subject), is capable of eliciting a humoral and / or cellular immune response against the protein. Unless otherwise indicated, the term "vaccine immunogen" is used interchangeably with "protein antigen" or "immunogenic polypeptide."

[0256] The term "conservatively modified variants" applies to both amino acid and nucleic acid sequences. With respect to specific nucleic acid sequences, conservatively modified variants refer to those nucleic acids that encode identical or essentially identical amino acid sequences, or, where 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" refer to variants having conservative amino acid substitutions, in which an amino acid residue is replaced by another amino acid residue having a side chain with a similar charge. Families of amino acid residues having 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 chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine).

[0257] Epitopes are antigenic determinants. These are specific chemical groups or peptide sequences on an antigenic molecule that trigger a specific immune response. For example, an epitope is a region of an antigen that responds to B cells and / or T cells. Epitopes can be formed by either contiguous amino acids or non-contiguous amino acids juxtaposed by the tertiary folding of a protein.

[0258] Unless otherwise indicated, a fusion protein is a recombinant protein containing the amino acid sequences of at least two unrelated proteins that are linked together by peptide bonds to form a single protein. Therefore, it does not contain naturally occurring coronavirus surface antigens, i.e., fusion (F) proteins as described herein. Unrelated amino acid sequences can be directly linked to each other, or they can be connected using a linker sequence. As used herein, if the amino acid sequence of a protein is not typically linked together by peptide bonds in its natural environment (e.g., within a cell), the protein is unrelated. For example, the amino acid sequence of a viral antigen and the amino acid sequence of a collagen or procollagen are not typically linked together by peptide bonds.

[0259] An immunogen is a protein or portion thereof that is capable of inducing an immune response in a mammal, such as a mammal infected with or at risk of infection by a pathogen. Administration of an immunogen can result in protective and / or active immunity against the target pathogen.

[0260] 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 induces a measurable B cell response (eg, production of antibodies) against the immunogenic polypeptide.

[0261] The 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 according to percentage identity; The higher the percentage, the more identical the sequence.When compared and aligned by a comparison window or using one of the following sequence comparison algorithms or by manually comparing and visually inspecting the measured designated region to obtain maximum correspondence, if the two sequences have the same amino acid residue or nucleotide of a specified percentage, the two sequences are "substantially identical" (i.e., on a specified region, or in the case of unspecified, on the entire sequence, 60% identity, preferably 65%, 70%, 75%, 80%, 85%, 90%, 95% or 99% identity). Alternatively, identity is present in a region of at least about 50 nucleotides (or 10 amino acids) in length, or more preferably in a region of 100 to 500 or 1000 or more nucleotides (or 20, 50, 200 or more amino acids) in length.

[0262] Vaccine refers to a pharmaceutical composition that elicits a prophylactic or therapeutic immune response in a subject. In some cases, the immune response is a protective immune response. Typically, vaccines induce an antigen-specific immune response against an antigen of a pathogen (e.g., a viral pathogen) or a cellular component associated with a pathological condition. Vaccines may include polynucleotides (e.g., nucleic acids encoding disclosed antigens), peptides or polypeptides (e.g., disclosed antigens), viruses, cells, or one or more cellular components. In some embodiments, a vaccine or vaccine immunogen or vaccine composition is expressed from a fusion construct and self-assembles into nanoparticles that display an immunogenic polypeptide or protein on the surface.

[0263] Virus-like particles (VLPs) refer to non-replicable viral coats, derived from any of several viruses. VLPs are typically composed of one or more viral proteins, such as, but not limited to, proteins called capsids, coats, shells, surfaces, and / or envelope proteins, or particle-forming polypeptides derived from these proteins. After recombinant expression of proteins in an appropriate expression system, VLPs can form spontaneously. 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, for example, Baker et al. (1991) Biophys. J. 60: 1445-1456; and Hagensee et al. (1994) J. Virol. 68: 4503-4505, which are incorporated by reference in their entirety for all purposes. For example, VLPs can be separated and / or identified by characteristic density bands by density gradient centrifugation. Alternatively, cryogenic electron microscopy can be performed on a vitrified aqueous sample prepared from the VLP in question, and images are recorded under appropriate exposure conditions.

[0264] The term "about" as used herein refers to the usual error range of each value that is readily known to those skilled in the art. References to "about" values ​​or parameters herein include (and describe) embodiments for the value or parameter itself.

[0265] Herein, various aspects of the claimed subject matter are presented in range format. It should be understood that descriptions in range format are merely for convenience and brevity and should not be construed as inflexible limitations on the scope of the claimed subject matter. Therefore, the description of a range should be considered to have specifically disclosed all possible subranges and individual values ​​within that range. For example, where a range of values ​​is provided, it should be understood that each intervening value between the upper and lower limits of that range and any other stated or intervening value within that 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 included in the claimed subject matter, subject to any explicitly excluded limitations within the stated ranges. If the stated range includes one or both limits, then ranges excluding one or both of those included limits are also included in the claimed subject matter. This applies to the width of any range.

[0266] As used herein, a composition refers to any mixture of two or more products, substances or compounds (including cells). It can be a solution, suspension, liquid, powder, paste, aqueous, non-aqueous or any combination thereof.

[0267] 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 that are self-replicating nucleic acid structures as well as vectors that are incorporated into the genome of a host cell into which the vector has been introduced. Certain vectors are capable of directing the expression of nucleic acids to which they are operatively linked. Such vectors are referred to herein as "expression vectors."

[0268] Exemplary embodiment 1

[0269] Embodiment 1. A protein comprising multiple recombinant polypeptides, each recombinant polypeptide comprising the XBB.1.5 surface antigen of the coronavirus SARS-CoV-2 Omicron (B.1.1.529) connected to the C-terminal propeptide of collagen, wherein the C-terminal propeptide of the recombinant polypeptide forms an inter-polypeptide disulfide bond.

[0270] Embodiment 2. The protein according to embodiment 1, wherein the coronavirus infection is a SARS-coronavirus 2 (SARS-CoV-2) infection.

[0271] Embodiment 3. A protein according to 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.

[0272] Embodiment 4. The protein according to embodiment 3, wherein the surface antigen comprises a signal peptide, an S1 subunit peptide, an S2 subunit peptide, or any combination thereof.

[0273] Embodiment 5. The protein according to 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.

[0274] Embodiment 6. A protein according to any one of embodiments 3-5, wherein the surface antigen comprises the receptor binding domain (RBD) of the S protein.

[0275] Embodiment 7. The protein according to any one of embodiments 3-6, wherein the surface antigen comprises the S1 subunit and the S2 subunit of the S protein.

[0276] Embodiment 8. The protein according to any one of embodiments 3-7, wherein the surface antigen does not include a transmembrane (TM) domain peptide and / or a cytoplasmic (CP) domain peptide.

[0277] Embodiment 9. The protein according to any one of embodiments 3-8, wherein the surface antigen comprises a protease cleavage site, wherein the protease is optionally furin, trypsin, factor Xa, thrombin or cathepsin L.

[0278] Embodiment 10. The protein according to any one of embodiments 3-8, wherein the surface antigen does not comprise a protease cleavage site, wherein the protease is optionally furin, trypsin, factor Xa, thrombin or cathepsin L.

[0279] Embodiment 11. The protein according to any one of embodiments 1-10, wherein the surface antigen is soluble or not directly bound to a lipid bilayer, such as a membrane or viral envelope.

[0280] Embodiment 12. The protein according to any one of embodiments 1-11, wherein the surface antigens are the same or different in the recombinant polypeptides of the protein.

[0281] Embodiment 13. The protein according to any one of embodiments 1-12, wherein the surface antigen is fused directly to the C-terminal propeptide, or is linked to the C-terminal propeptide via a linker (e.g., a linker comprising a glycine-XY repeat sequence), wherein X and Y are independently any amino acids, and are optionally proline or hydroxyproline.

[0282] Embodiment 14. The protein according to any one of embodiments 1-13, which is soluble or not directly bound to a lipid bilayer, such as a membrane or viral envelope.

[0283] Embodiment 15. The protein according to any one of embodiments 1-14, wherein the protein is capable of binding to a cell surface receptor in a subject, optionally wherein the subject is a mammal, such as a primate, such as a human.

[0284] Embodiment 16. The protein according to embodiment 15, wherein the cell surface receptor is angiotensin-converting enzyme 2 (ACE2), dipeptidyl peptidase 4 (DPP4), dendritic cell-specific intercellular adhesion molecule-3-grasping non-integrin (DC-SIGN) or liver / lymph node-SIGN (L-SIGN).

[0285] Embodiment 17. The protein according to any one of embodiments 1-16, wherein the C-terminal propeptide is human collagen.

[0286] Embodiment 18. The protein according to any one of embodiments 1-17, wherein the C-terminal propeptide comprises the C-terminal polypeptide of proα1(I), proα1(II), proα1(III), proα1(V), proα1(XI), proα2(I), proα2(V), proα2(XI) or proα3(XI) or a fragment thereof.

[0287] Embodiment 19. The protein according to any one of embodiments 1-18, wherein the C-terminal propeptides are identical or different in the recombinant polypeptides.

[0288] Embodiment 20. The protein according to any one of embodiments 1-19, wherein the C-terminal propeptide comprises any one of SEQ ID NOs: 67-80 or an amino acid sequence at least 90% identical thereto, which is capable of forming inter-polypeptide disulfide bonds and trimerizing the recombinant polypeptide.

[0289] Embodiment 21. The protein according to any one of embodiments 1-20, wherein the C-terminal propeptide comprises SEQ ID NO: 67 or an amino acid sequence at least 90% identical thereto, which is capable of forming inter-polypeptide disulfide bonds and trimerizing the recombinant polypeptide.

[0290] Embodiment 22. The protein according to any one of embodiments 1-20, wherein the C-terminal propeptide comprises SEQ ID NO: 68 or an amino acid sequence at least 90% identical thereto, which is capable of forming inter-polypeptide disulfide bonds and trimerizing the recombinant polypeptide.

[0291] Embodiment 23. The protein according to any one of embodiments 1-20, wherein the C-terminal propeptide comprises SEQ ID NO: 69 or an amino acid sequence at least 90% identical thereto, which is capable of forming inter-polypeptide disulfide bonds and trimerizing the recombinant polypeptide.

[0292] Embodiment 24. The protein according to any one of embodiments 1-20, wherein the C-terminal propeptide comprises SEQ ID NO: 70 or an amino acid sequence at least 90% identical thereto, which is capable of forming inter-polypeptide disulfide bonds and trimerizing the recombinant polypeptide.

[0293] Embodiment 25. The protein according to any one of embodiments 1-20, wherein the C-terminal propeptide comprises SEQ ID NO: 71 or an amino acid sequence at least 90% identical thereto, which is capable of forming inter-polypeptide disulfide bonds and trimerizing the recombinant polypeptide.

[0294] Embodiment 26. The protein according to any one of embodiments 1-20, wherein the C-terminal propeptide comprises SEQ ID NO: 72 or an amino acid sequence at least 90% identical thereto, which is capable of forming inter-polypeptide disulfide bonds and trimerizing the recombinant polypeptide.

[0295] Embodiment 27. The protein according to any one of embodiments 1-20, wherein the C-terminal propeptide comprises SEQ ID NO: 73 or an amino acid sequence at least 90% identical thereto, which is capable of forming inter-polypeptide disulfide bonds and trimerizing the recombinant polypeptide.

[0296] Embodiment 28. The protein according to any one of embodiments 1-20, wherein the C-terminal propeptide comprises SEQ ID NO: 74 or an amino acid sequence at least 90% identical thereto, which is capable of forming inter-polypeptide disulfide bonds and trimerizing the recombinant polypeptide.

[0297] Embodiment 29. The protein according to any one of embodiments 1-20, 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, which is capable of forming inter-polypeptide disulfide bonds and trimerizing the recombinant polypeptide.

[0298] Embodiment 30. The protein according to any one of embodiments 1-29, wherein the C-terminal propeptide comprises a sequence comprising glycine-XY repeats linked to the N-terminus of any one of SEQ ID NOs: 67-80, wherein X and Y are independently any amino acids, and are optionally proline or hydroxyproline, or an amino acid sequence at least 90% identical thereto, which is capable of forming an inter-polypeptide disulfide bond and trimerizing the recombinant polypeptide.

[0299] Embodiment 31. The protein according to any one of embodiments 1-30, wherein the surface antigen in each recombinant polypeptide is in a pre-fusion conformation or a post-fusion conformation.

[0300] Embodiment 32. The protein according to any one of embodiments 1-31, wherein the surface antigen in each recombinant polypeptide comprises any one of SEQ ID NOs: 27-66 and 81-84, 122 and 126, or an amino acid sequence at least 80% identical thereto.

[0301] Embodiment 33. The protein according to any one of embodiments 1-32, wherein the recombinant polypeptide comprises any one of SEQ ID NOs: 1-26 and 85-92, 120 and 124, or an amino acid sequence at least 80% identical thereto.

[0302] Embodiment 34. An immunogen comprising a protein according to any one of embodiments 1 to 33, optionally as a vaccine primary series, additional dose and / or homologous or heterologous booster. dose), for example, the first dose, the second dose, the third dose, the fourth dose and / or more doses, optionally, the initial dose, the additional dose or the heterologous booster is used in combination with any one or more of other recombinant subunit vaccines, nanoparticle vaccines, mRNA vaccines, DNA vaccines, adenovirus vector vaccines, and inactivated virus vaccines, optionally, the adjuvant in the initial dose, the additional dose and / or the homologous or heterologous booster may independently include: an aluminum-containing adjuvant, for example, an adjuvant containing alum and / or aluminum hydroxide; an oligonucleotide-containing adjuvant, for example, an adjuvant containing CpG oligodeoxynucleotide (CpG-ODN); an adjuvant containing a TLR9 agonist; an adjuvant containing a metabolizable oil, α-tocopherol, and / or polyoxyethylene sorbitan monooleate (Tween-80), for example, an adjuvant containing squalene, α-tocopherol, and Tween-80 and / or Span 85 in the form of an oil-in-water emulsion; or a combination of any one of the adjuvants.

[0303] Embodiment 35. A protein nanoparticle comprising a protein according to any one of embodiments 1-33, said protein being directly or indirectly attached to the nanoparticle, optionally as a vaccine primary series, additional dose and / or homologous or heterologous booster. dose), for example, the first dose, the second dose, the third dose, the fourth dose and / or more doses, optionally, the initial dose, the additional dose or the heterologous booster is used in combination with any one or more of other recombinant subunit vaccines, nanoparticle vaccines, mRNA vaccines, DNA vaccines, adenovirus vector vaccines, and inactivated virus vaccines, optionally, the adjuvant in the initial dose, the additional dose and / or the homologous or heterologous booster may independently include: an aluminum-containing adjuvant, for example, an adjuvant containing alum and / or aluminum hydroxide; an oligonucleotide-containing adjuvant, for example, an adjuvant containing CpG oligodeoxynucleotide (CpG-ODN); an adjuvant containing a TLR9 agonist; an adjuvant containing a metabolizable oil, α-tocopherol, and / or polyoxyethylene sorbitan monooleate (Tween-80), for example, an adjuvant containing squalene, α-tocopherol, and Tween-80 and / or Span 85 in the form of an oil-in-water emulsion; or a combination of any one of the adjuvants.

[0304] Embodiment 36. A virus-like particle (VLP) comprising a protein according to any one of embodiments 1-33, optionally as a vaccine primary series, additional dose and / or homologous or heterologous booster. dose), for example, the first dose, the second dose, the third dose, the fourth dose and / or more doses, optionally, the initial dose, the additional dose or the heterologous booster is used in combination with any one or more of other recombinant subunit vaccines, nanoparticle vaccines, mRNA vaccines, DNA vaccines, adenovirus vector vaccines, and inactivated virus vaccines, optionally, the adjuvant in the initial dose, the additional dose and / or the homologous or heterologous booster may independently include: an aluminum-containing adjuvant, for example, an adjuvant containing alum and / or aluminum hydroxide; an oligonucleotide-containing adjuvant, for example, an adjuvant containing CpG oligodeoxynucleotide (CpG-ODN); an adjuvant containing a TLR9 agonist; an adjuvant containing a metabolizable oil, α-tocopherol, and / or polyoxyethylene sorbitan monooleate (Tween-80), for example, an adjuvant containing squalene, α-tocopherol, and Tween-80 and / or Span 85 in the form of an oil-in-water emulsion; or a combination of any one of the adjuvants.

[0305] Embodiment 37. An isolated nucleic acid encoding one, two, three or more recombinant polypeptides of the protein according to any one of embodiments 1-33.

[0306] 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.

[0307] Embodiment 39. The isolated nucleic acid according to embodiment 37 or 38, which is operably linked to a promoter.

[0308] Embodiment 40. The isolated nucleic acid according to any one of embodiments 37-39, which is a DNA molecule.

[0309] Embodiment 41. The isolated nucleic acid according to any one of embodiments 37-39, which is an RNA molecule, optionally an mRNA molecule, such as a nucleoside-modified mRNA, a non-amplified mRNA, a self-amplified mRNA, or a trans-amplified mRNA.

[0310] Embodiment 42. A vector comprising the isolated nucleic acid according to any one of embodiments 37-41.

[0311] Embodiment 43. The vector according to embodiment 42, which is a viral vector.

[0312] Embodiment 44. A virus, pseudovirus or cell comprising a vector according to embodiment 42 or 43, optionally wherein the virus or cell has a recombinant genome.

[0313] Embodiment 45. An immunogenic composition comprising the protein, immunogen, protein nanoparticle, VLP, isolated nucleic acid, vector, virus, pseudovirus or cell according to any one of embodiments 1-44 and a pharmaceutically acceptable carrier.

[0314] Embodiment 46. A vaccine comprising the immunogenic composition according to embodiment 45 and optionally an adjuvant, wherein the vaccine is optionally a subunit vaccine, and / or optionally wherein the vaccine is a prophylactic and / or therapeutic vaccine, optionally the vaccine is administered as a primary series, additional dose and / or homologous or heterologous booster. dose), for example, the first dose, the second dose, the third dose, the fourth dose and / or more doses, optionally, the initial dose, the additional dose or the heterologous booster is used in combination with any one or more of other recombinant subunit vaccines, nanoparticle vaccines, mRNA vaccines, DNA vaccines, adenovirus vector vaccines, and inactivated virus vaccines, optionally, the adjuvant in the initial dose, the additional dose and / or the homologous or heterologous booster may independently include: an aluminum-containing adjuvant, for example, an adjuvant containing alum and / or aluminum hydroxide; an oligonucleotide-containing adjuvant, for example, an adjuvant containing CpG oligodeoxynucleotide (CpG-ODN); an adjuvant containing a TLR9 agonist; an adjuvant containing a metabolizable oil, α-tocopherol, and / or polyoxyethylene sorbitan monooleate (Tween-80), for example, an adjuvant containing squalene, α-tocopherol, and Tween-80 and / or Span 85 in the form of an oil-in-water emulsion; or a combination of any one of the adjuvants.

[0315] Embodiment 47. The vaccine according to embodiment 46, wherein the vaccine comprises a plurality of different adjuvants.

[0316] Embodiment 48. A method of producing a protein, comprising: expressing the isolated nucleic acid or vector according to any one of embodiments 37-43 in a host cell to produce the protein according to any one of embodiments 1-33; and purifying the protein.

[0317] Embodiment 49. A protein produced by the method of embodiment 48.

[0318] Embodiment 50. A method for generating an immune response to a coronavirus surface antigen in a subject, comprising administering to the subject an effective amount of the protein, immunogen, protein nanoparticle, VLP, isolated nucleic acid, vector, virus, pseudovirus, cell, immunogenic composition or vaccine of any one of embodiments 1-47 and 49 to generate an immune response.

[0319] Embodiment 51. The method according to embodiment 50, for treating or preventing a coronavirus infection.

[0320] Embodiment 52. The method of embodiment 50 or 51, wherein generating an immune response inhibits or reduces coronavirus replication in the subject.

[0321] Embodiment 53. The method according to any one of embodiments 50-52, wherein the immune response comprises a cell-mediated response and / or a humoral response, optionally comprising the production of one or more neutralizing antibodies, such as polyclonal antibodies or monoclonal antibodies.

[0322] Embodiment 54. The method according to any one of embodiments 50-53, wherein the immune response is directed against the surface antigen of the coronavirus but not against the C-terminal propeptide.

[0323] Embodiment 55. The method according to any one of embodiments 50-54, wherein administration does not result in antibody-dependent enhancement (ADE) in the subject due to prior exposure to one or more coronaviruses.

[0324] Embodiment 56. The method according to any one of embodiments 50-55, wherein the administration does not result in antibody-dependent enhancement (ADE) in the subject upon subsequent exposure to one or more coronaviruses.

[0325] Embodiment 57. The method according to any one of embodiments 50-56, further comprising a priming step and / or a boosting step.

[0326] Embodiment 58. The method according to any one of embodiments 50-57, wherein the administering step is performed topically, transdermally, subcutaneously, intradermally, orally, intranasally (e.g., intranasal spray), intratracheally, sublingually, buccally, rectally, vaginally, by inhalation, intravenously (e.g., intravenous injection), intraarterially, intramuscularly (e.g., intramuscular injection), intracardially, intraosseously, intraperitoneally, transmucosally, intravitreally, subretinally, intraarticularly, periarticularly, topically, or epicutaneously.

[0327] Embodiment 59. The method according to any one of embodiments 50-58, wherein the effective amount is administered in a single dose or in a series of doses with one or more intervals.

[0328] Embodiment 60. The method according to any one of embodiments 50-59, wherein the effective amount is administered in the absence of an adjuvant.

[0329] Embodiment 61. The method according to any one of embodiments 50-59, wherein the effective amount is administered with an adjuvant or adjuvants.

[0330] Embodiment 62. A method comprising administering to a subject an effective amount of a protein according to any one of embodiments 1-33 to produce neutralizing antibodies or neutralizing antiserum against a coronavirus in the subject.

[0331] Embodiment 63. The method according to embodiment 62, wherein the subject is a mammal, optionally a human or a non-human primate.

[0332] Embodiment 64. The method according to embodiment 62 or 63, further comprising isolating neutralizing antibodies or neutralizing antiserum from the subject.

[0333] Embodiment 65. The method according to embodiment 64, further comprising administering an effective amount of the isolated neutralizing antibody or neutralizing antiserum to a human subject by passive immunization to prevent or treat coronavirus infection.

[0334] Embodiment 66. A method according to any one of embodiments 62-65, wherein the neutralizing antibodies or neutralizing antiserum comprise polyclonal antibodies against a coronavirus surface antigen, optionally wherein the neutralizing antibodies or neutralizing antiserum do not contain or are substantially free of antibodies against a C-terminal propeptide of collagen.

[0335] Embodiment 67. A method according to any one of embodiments 62-65, wherein the neutralizing antibodies comprise monoclonal antibodies directed against a coronavirus surface antigen, optionally wherein the neutralizing antibodies are free of or substantially free of antibodies directed against a C-terminal propeptide of collagen.

[0336] Embodiment 68. A protein, immunogen, protein nanoparticle, VLP, isolated nucleic acid, vector, virus, pseudovirus, cell, immunogenic composition or vaccine according to any one of embodiments 1-47 and 49 for inducing an immune response to a coronavirus in a subject, and / or treating or preventing a coronavirus infection.

[0337] Embodiment 69. Use of a protein, immunogen, protein nanoparticle, VLP, isolated nucleic acid, vector, virus, pseudovirus, cell, immunogenic composition or vaccine according to any one of embodiments 1-47 and 49 for inducing an immune response to a coronavirus in a subject, and / or for treating or preventing a coronavirus infection.

[0338] Embodiment 70. Use of a protein, immunogen, protein nanoparticle, VLP, isolated nucleic acid, vector, virus, pseudovirus, cell, immunogenic composition or vaccine according to any one of embodiments 1-47 and 49 for the manufacture of a medicament or prophylactic agent for inducing an immune response to a coronavirus in a subject, and / or for treating or preventing a coronavirus infection.

[0339] Embodiment 71. A method for analyzing a sample, comprising: contacting the sample with the protein of any one of embodiments 1-33, and detecting binding between the protein and an analyte that can specifically bind to a coronavirus surface antigen.

[0340] Embodiment 72. The method according to embodiment 71, wherein the analyte is an antibody, a receptor or a cell that recognizes a surface antigen.

[0341] Embodiment 73. The method according to embodiment 71 or 72, wherein the binding indicates the presence of the analyte in the sample, and / or the presence of a coronavirus infection in the subject from which the sample was derived.

[0342] Embodiment 74. A kit comprising the protein of any one of embodiments 1-33 and a substrate, plate or vial containing or immobilizing the protein, optionally wherein the kit is an ELISA or lateral flow assay kit.

[0343] Exemplary embodiment II

[0344] Embodiment 1 A method for preventing coronavirus infection in a mammal, the method comprising immunizing the mammal with an effective amount of a recombinant subunit vaccine, the recombinant subunit vaccine comprising a soluble surface antigen of the coronavirus SARS-CoV-2 Omicron (B.1.1.529), subtype XBB.1.5, or a fragment, variant, or mutant thereof, linked in-frame to the C-terminal portion of collagen to form a disulfide-linked trimeric fusion protein.

[0345] Embodiment 2. The method of embodiment 1, wherein the coronavirus infection is severe acute respiratory syndrome (SARS)-coronavirus 2 (SARS-CoV-2) infection.

[0346] Embodiment 3. The method according to embodiment 1 or 2, wherein the coronavirus surface antigen comprises a coronavirus spike (S) protein or a fragment or epitope thereof.

[0347] The embodiment described in any one of embodiments 4.1-3, wherein the coronavirus surface antigen comprises a SARS-CoV-2 spike (S) extracellular domain peptide or a fragment or epitope thereof, optionally, the S extracellular domain peptide or a fragment or epitope thereof comprises a SARS-CoV-2 Omicron (B.1.1.529) subtype XBB.1.5 variant S extracellular domain peptide or a fragment, variant or mutant thereof, such as a chimeric sequence containing the Omicron variant receptor binding domain (RBD) and the Hu-1 or other variant S protein peptide sequence.

[0348] Embodiment 5. The method of any one of embodiments 1-4, wherein the coronavirus surface antigen comprises a SARS-CoV-2 spike (S) N-terminal domain (NTD) peptide or a fragment or epitope thereof, optionally the NTD peptide is a SARS-CoV-2 Hu-1, alpha, beta, gamma, delta, muon, or omicron and subtype NTD peptides or fragments, variants or mutants thereof.

[0349] Embodiment 6. A method according to any one of embodiments 1-5, wherein the coronavirus surface antigen comprises a SARS-CoV-2 spike (S) receptor binding domain (RBD) peptide or a fragment or epitope thereof, optionally the RBD peptide is a SARS-CoV-2 Omicron (B.1.1.529) subtype XBB.1.5 variant RBD peptide or a fragment, variant or mutant thereof.

[0350] Embodiment 7. A method according to any one of embodiments 1-6, wherein the coronavirus surface antigen comprises the SARS-CoV-2 spike (S) S1 peptide or a fragment or epitope thereof, optionally the S1 peptide is the S1 peptide of the subtype XBB.1.5 variant of SARS-CoV-2 Omicron (B.1.1.529) or a fragment, variant or mutant thereof.

[0351] Embodiment 8. The method of any one of claims 1-7, wherein the coronavirus surface antigen comprises a SARS-CoV-2 spike (S) S2 peptide or a fragment or epitope thereof, optionally the S2 peptide is a SARS-CoV-2 Hu-1, alpha, beta, gamma, delta, mu, or omicron and subtype S2 peptides or fragments, variants or mutants thereof.

[0352] Embodiment 1.9. The method of any one of Embodiments 1-8, wherein the coronavirus epiantigen comprises a SARS-CoV-2 spike (S) ectodomain peptide or fragment or epitope thereof having a mutation.

[0353] Embodiment 10. The method according to embodiment 9, wherein the mutation comprises a furin cleavage site mutation, optionally the mutation is a deletion, substitution or addition of one or more amino acids such that the furin cleavage site no longer has activity as a furin cleavage site, optionally the mutation is located at any one or more sites among 682-685, optionally the mutation comprises 685R→685A.

[0354] Embodiment 11. A method according to embodiment 9 or 10, wherein the mutation comprises a mutation located at or adjacent to the junction of the heptad repeat sequence HR1 and the central helix, optionally the mutation comprises a proline substitution, such as 986K→986P and / or 987V→987P.

[0355] Embodiment 12. The method according to any one of embodiments 9-11, wherein the mutation comprises consecutive site amino acid substitutions, such as 986K→986P and 987V→987P.

[0356] Embodiment 13. The method of any one of embodiments 1-12, wherein the recombinant subunit vaccine comprises a sequence of any one of SEQ ID NOs: 81-92, and 124-127, or an amino acid sequence that has at least or about 80%, 85%, 90%, 92%, 95%, 97%, or 99% sequence identity to any one of SEQ ID NOs: 81-92, 122, and 126.

[0357] Embodiment 14. The method of any one of claims 1-13, wherein the recombinant subunit vaccine comprises SEQ ID NO: 124 or an amino acid sequence that is at least or about 80%, 85%, 90%, 92%, 95%, 97%, or 99% identical to SEQ ID NO: 124.

[0358] Embodiment 15. The method of any one of embodiments 1-14, wherein the recombinant subunit vaccine comprises SEQ ID NO: 125 or an amino acid sequence that is at least or about 80%, 85%, 90%, 92%, 95%, 97%, or 99% identical to SEQ ID NO: 125.

[0359] Embodiment 16. The method of any one of claims 1-15, wherein the recombinant subunit vaccine comprises SEQ ID NO: 126 or an amino acid sequence that is at least or about 80%, 85%, 90%, 92%, 95%, 97%, or 99% identical to SEQ ID NO: 126.

[0360] Embodiment 17. The method of any one of embodiments 1-16, wherein the recombinant subunit vaccine comprises SEQ ID NO: 127 or an amino acid sequence that is at least or about 80%, 85%, 90%, 92%, 95%, 97%, or 99% identical to SEQ ID NO: 127.

[0361] Embodiment 18. The method of any one of embodiments 1-17, wherein the recombinant subunit vaccine comprises a first sequence of any one of SEQ ID NOs: 81-84 linked to a second sequence of any one of SEQ ID NOs: 67-80, wherein the C-terminus of the first sequence is directly or indirectly linked to the N-terminus of the second sequence.

[0362] Embodiment 19. The method of any one of claims 1-18, wherein the recombinant subunit vaccine is administered by intramuscular injection.

[0363] Embodiment 20. The method of any one of embodiments 1-19, wherein the recombinant subunit vaccine is administered by intranasal spray.

[0364] Embodiment 21. The method according to any one of embodiments 1-20, wherein the recombinant subunit vaccine is administered as a single dose or as a series of doses spaced apart by weeks or months.

[0365] Embodiment 22. The method of any one of embodiments 1-21, wherein the recombinant subunit vaccine is administered without an adjuvant, optionally the recombinant subunit vaccine is used as a primary series, additional dose and / or homologous or heterologous booster dose, such as the first dose, second dose, third dose, fourth dose and / or more doses, optionally the primary dose, additional dose or heterologous booster dose is used in combination with any one or more of other recombinant subunit vaccines, nanoparticle vaccines, mRNA vaccines, DNA vaccines, adenoviral vector vaccines, and inactivated virus vaccines.

[0366] Embodiment 23. The method according to any one of embodiments 1-22, wherein the recombinant subunit vaccine is administered with an adjuvant, optionally as a primary series, additional dose and / or homologous or heterologous booster. dose), for example, the first dose, the second dose, the third dose, the fourth dose and / or more doses, optionally, the initial dose, the additional dose or the heterologous booster is used in combination with any one or more of other recombinant subunit vaccines, nanoparticle vaccines, mRNA vaccines, DNA vaccines, adenovirus vector vaccines, and inactivated virus vaccines, optionally, the adjuvant in the initial dose, the additional dose and / or the homologous or heterologous booster may independently include: an aluminum-containing adjuvant, for example, an adjuvant containing alum and / or aluminum hydroxide; an oligonucleotide-containing adjuvant, for example, an adjuvant containing CpG oligodeoxynucleotide (CpG-ODN); an adjuvant containing a TLR9 agonist; an adjuvant containing a metabolizable oil, α-tocopherol, and / or polyoxyethylene sorbitan monooleate (Tween-80), for example, an adjuvant containing squalene, α-tocopherol, and Tween-80 and / or Span 85 in the form of an oil-in-water emulsion; or any combination of adjuvants.

[0367] Embodiment 24. The method according to any one of Embodiments 1 to 23, wherein the recombinant subunit vaccine is administered with one or more adjuvants, optionally as a primary series, additional dose and / or homologous or heterologous booster. dose), for example, the first dose, the second dose, the third dose, the fourth dose and / or more doses, optionally, the initial dose, the additional dose or the heterologous booster is used in combination with any one or more of other recombinant subunit vaccines, nanoparticle vaccines, mRNA vaccines, DNA vaccines, adenovirus vector vaccines, and inactivated virus vaccines, optionally, the adjuvant in the initial dose, the additional dose and / or the homologous or heterologous booster may independently include: an aluminum-containing adjuvant, for example, an adjuvant containing alum and / or aluminum hydroxide; an oligonucleotide-containing adjuvant, for example, an adjuvant containing CpG oligodeoxynucleotide (CpG-ODN); an adjuvant containing a TLR9 agonist; an adjuvant containing a metabolizable oil, α-tocopherol, and / or polyoxyethylene sorbitan monooleate (Tween-80), for example, an adjuvant containing squalene, α-tocopherol, and Tween-80 and / or Span 85 in the form of an oil-in-water emulsion; or any combination of adjuvants.

[0368] Embodiment 25. A method for detecting coronavirus antibodies from mammalian serum, the method comprising the step of contacting the serum with a soluble surface antigen of the coronavirus SARS-CoV-2 Omicron (B.1.1.529), subtype XBB.1.5, which is linked to the C-terminal portion of collagen by in-frame fusion to form a disulfide-linked trimeric fusion protein.

[0369] Embodiment 26. The method of embodiment 25, wherein the soluble coronavirus surface antigen is an S protein or peptide.

[0370] Embodiment 27. A method of using a recombinant subunit vaccine comprising a soluble surface antigen of subtype XBB.1.5 from the coronavirus SARS-CoV-2 Omicron (B.1.1.529), which is linked to the C-terminal portion of collagen by in-frame fusion to form a disulfide-linked trimeric fusion protein, the method comprising: immunizing a mammal, purifying the neutralizing antibodies produced, and using the neutralizing antibodies to passively immunize a patient infected with the coronavirus.

[0371] Embodiment 28. The method of embodiment 29, wherein the neutralizing antibodies comprise polyclonal antibodies.

[0372] Embodiment 29. The method according to embodiment 29, wherein the neutralizing antibody is a monoclonal antibody.

[0373] Embodiment 30. A method according to embodiment 27, wherein the neutralizing antibody is a monoclonal antibody to the S protein or peptide.

[0374] Embodiment 31. The method of embodiment 27, wherein the neutralizing antibody is a monoclonal antibody against the S protein of SARS-CoV-2.

[0375] Embodiment 32. The method of embodiment 27, wherein the neutralizing antibody is a monoclonal antibody against the S protein of SARS-CoV-2 Hu-1, Alpha, Beta, Gamma, Delta, Mu, Omicron, and subtypes thereof, and other strains.

[0376] Embodiment 33. The method of embodiment 27, wherein the neutralizing antibody is a monoclonal antibody against the subtype XBB.1.5 S protein of SARS-CoV-2 Omicron (B.1.1.529).

[0377] Embodiment 34. A complex comprising a recombinant polypeptide selected from the group consisting of SEQ ID NOs: 124-127.

[0378] Embodiment 35. A complex comprising a trimer of recombinant polypeptides selected from the group consisting of SEQ ID NOs: 124-127, wherein the recombinant polypeptides are trimerized via inter-polypeptide disulfide bonds to form the trimer.

[0379] Embodiment 36. An immunogenic composition comprising a trimer or a combination of any two or more trimers of a recombinant polypeptide comprising a sequence selected from the group consisting of SEQ ID NOs: 124-127.

[0380] Embodiment 37. The immunogenic composition of embodiment 38, comprising a trimer of the recombinant polypeptide having the sequence set forth in SEQ ID NO: 125.

[0381] Embodiment 38. A method for generating an immune response to a coronavirus surface antigen in a subject, 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: 85-92, optionally the complex is used as a primary series, additional dose and / or homologous or heterologous booster dose, such as the first dose, second dose, third dose, fourth dose and / or more doses, optionally the primary dose, additional dose or heterologous booster is used in combination with any one or more of other recombinant subunit vaccines, nanoparticle vaccines, mRNA vaccines, DNA vaccines, adenoviral vector vaccines, and inactivated virus vaccines.

[0382] Embodiment 39. A method for generating an immune response to a coronavirus surface antigen in a subject.

[0383] The surface antigen comprises an S protein or an antigenic fragment thereof, and the method comprises administering to the subject an effective amount of a complex comprising a recombinant polypeptide selected from the group consisting of SEQ ID NOs: 124-127, optionally the complex is used as a primary series, an additional dose and / or a homologous or heterologous booster dose, such as the first dose, the second dose, the third dose, the fourth dose and / or more doses, optionally the primary dose, the additional dose or the heterologous booster is used in combination with any one or more of other recombinant subunit vaccines, nanoparticle vaccines, mRNA vaccines, DNA vaccines, adenovirus vector vaccines, and inactivated virus vaccines.

[0384] Embodiment 40. A method for generating an immune response to a coronavirus surface antigen in a subject.

[0385] The surface antigen comprises a sequence selected from the group consisting of SEQ ID NOs: 27-66, 81-84, 122 and 124, and the method comprises administering to the subject an effective amount of a complex comprising a recombinant polypeptide selected from the group consisting of SEQ ID NOs: 85-92, optionally the complex is used as a primary series, additional dose and / or homologous or heterologous booster dose, such as the first dose, second dose, third dose, fourth dose and / or more doses, optionally the primary dose, additional dose or heterologous booster is used in combination with any one or more of other recombinant subunit vaccines, nanoparticle vaccines, mRNA vaccines, DNA vaccines, adenovirus vector vaccines, and inactivated virus vaccines.

[0386] Embodiment 41. A method for generating an immune response to a coronavirus surface antigen in a subject.

[0387] The surface antigen comprises the S protein of a coronavirus or an antigenic fragment thereof, and optionally, the surface antigen comprises a sequence selected from the group consisting of SEQ ID NOs: 27-6681-84, 122 and 124 or an antigenic fragment thereof, and the method comprises administering to the subject an effective amount of a complex comprising a recombinant polypeptide comprising a sequence described in any one of SEQ ID NOs: 85-92, optionally the complex is used as a primary series, additional dose and / or homologous or heterologous booster dose, such as the first dose, second dose, third dose, fourth dose and / or more doses, optionally the primary dose, additional dose or heterologous booster is used in combination with any one or more of other recombinant subunit vaccines, nanoparticle vaccines, mRNA vaccines, DNA vaccines, adenovirus vector vaccines, and inactivated virus vaccines.

[0388] Embodiment 42. A method for generating an immune response to a coronavirus surface antigen in a subject.

[0389] Wherein the surface antigen includes S protein or its antigenic fragment, and

[0390] The method comprises administering to the subject an effective amount of a complex or a combination of any two or more complexes, wherein the complex comprises a recombinant polypeptide comprising a sequence selected from the group consisting of SEQ ID NOs: 85-92, 120 and 125, optionally wherein the complex or combination of complexes is used as a primary series, additional dose and / or homologous or heterologous booster dose, such as the first dose, second dose, third dose, fourth dose and / or more doses, optionally wherein the primary dose, additional dose or heterologous booster is used in conjunction with any one or more of other recombinant subunit vaccines, nanoparticle vaccines, mRNA vaccines, DNA vaccines, adenoviral vector vaccines, and inactivated virus vaccines.

[0391] Embodiment 43. The method of claim 44, wherein the method comprises administering to the subject an effective amount of a complex comprising a recombinant polypeptide comprising the sequence set forth in SEQ ID NO: 125.

[0392] Embodiment 44. A fusion protein comprising a plurality of recombinant polypeptides, each recombinant polypeptide comprising, from amino to carboxyl terminus:

[0393] a) a first region comprising a portion of the coronavirus spike protein ectodomain preceding the coronavirus spike protein receptor binding domain (RBD) in a non-chimeric coronavirus spike protein of a first coronavirus;

[0394] b) a second region comprising a coronavirus spike protein receptor binding domain (RBD) of a second coronavirus different from the first coronavirus; and

[0395] c) a C-terminal propeptide of collagen, wherein the C-terminal propeptide of the recombinant polypeptide forms an inter-polypeptide disulfide bond, wherein at least one of the first coronavirus and the second coronavirus is SARS-CoV-2 Omicron (B.1.1.529) subtype XBB.1.5, and the fusion protein is optionally used as a primary series, additional dose and / or homologous or heterologous booster dose, such as the first dose, the second dose, the third dose, the fourth dose and / or more doses, and optionally the primary dose, additional dose or heterologous booster is used in combination with any one or more of other recombinant subunit vaccines, nanoparticle vaccines, mRNA vaccines, DNA vaccines, adenovirus vector vaccines, and inactivated virus vaccines.

[0396] Embodiment 45. The fusion protein of claim 44, further comprising a third region between the second region and the C-terminal propeptide of the collagen.

[0397] Embodiment 46. The fusion protein of claim 45, wherein the third region comprises the S1 domain of a third coronavirus, wherein the third coronavirus is the same as or different from the first coronavirus or the second coronavirus.

[0398] Embodiment 47. The fusion protein of claim 45 or 46, wherein the third region comprises the S2 domain of a fourth coronavirus, wherein the fourth coronavirus is the same as or different from the first, second, or fourth coronavirus.

[0399] Embodiment 48. The fusion protein of any one of claims 44-47, wherein the first region comprises the N-terminal domain (NTD) of a first coronavirus.

[0400] Embodiment 49. The fusion protein of any one of claims 44-48, wherein the first region comprises one or more amino acid residues that are different from the corresponding amino acid residues in the second coronavirus.

[0401] Embodiment 50. The fusion protein of any one of claims 44-49, wherein the second region comprises one or more amino acid residues that are different from the corresponding amino acid residues in the first coronavirus.

[0402] Embodiment 51. The fusion protein of any one of claims 44-50, wherein the first and second coronaviruses are different variants or strains of the same coronavirus.

[0403] Embodiment 52. The fusion protein of claim 51 , wherein the first region comprises the NTD of a first coronavirus, the second region comprises the RBD of a second coronavirus, and the first and second coronaviruses are different variants of SARS-CoV-2.

[0404] Embodiment 53. A fusion protein according to any one of claims 44-52, wherein the first coronavirus and the second coronavirus are independently selected from the group consisting of SARS-CoV-2 viruses of the B.1.1.529, B.1.617.2, B.1.526, B.1.1.143, P.2, B.1.351, P.1, B.1.1.7, B.1.617, A.23.1, BA.1, BA.2, BA.2.75, BA.5, BQ.1.1, BF.7, XBB, XBB.1.5, and BQ.1.1 lineages.

[0405] Embodiment 54. A trimeric fusion protein comprising three recombinant polypeptides, each recombinant polypeptide comprising, from amino to carboxyl terminus:

[0406] a) a first region comprising the N-terminal domain (NTD) of the spike protein of SARS-CoV-2 of the XBB.1.5 lineage;

[0407] b) a second region comprising the receptor binding domain (RBD) of the spike protein of SARS-CoV-2, a coronavirus of the XBB.1.5 lineage; and

[0408] c) a C-terminal propeptide of collagen, wherein the C-terminal propeptide of the recombinant polypeptide forms an inter-polypeptide disulfide bond, optionally the trimeric fusion protein is used as a primary series, additional dose and / or homologous or heterologous booster dose, such as the first dose, the second dose, the third dose, the fourth dose and / or more doses, optionally the primary dose, additional dose or heterologous booster is used in combination with any one or more of other recombinant subunit vaccines, nanoparticle vaccines, mRNA vaccines, DNA vaccines, adenovirus vector vaccines, and inactivated virus vaccines.

[0409] Embodiment 55. A method for preventing coronavirus infection in a mammal, the method comprising immunizing the mammal with an effective amount of the fusion protein according to any one of claims 44-54.

[0410] Embodiment 56. The method of embodiment 55, wherein neutralizing antibodies against the first and second coronaviruses are produced in the mammal.

[0411] Embodiment 57. A method according to embodiment 56, wherein the first and second coronaviruses are different variants of SARS-CoV-2, and the neutralizing antibodies produced in the mammal neutralize two or more SARS-CoV-2 viruses of the B.1.1.529, B.1.617.2, B.1.526, B.1.1.143, P.2, B.1.351, P.1, B.1.1.7, B.1.617, A.23.1, BA.1, BA.2, BA.2.75, BA.5, BQ.1.1, BF.7, XBB and BQ.1.1 lineages.

[0412] Embodiment 58. A method according to embodiment 57, wherein the neutralizing antibodies produced in the mammal neutralize three or more SARS-CoV-2 viruses of the B.1.1.529, B.1.617.2, B.1.526, B.1.1.143, P.2, B.1.351, P.1, B.1.1.7, B.1.617, A.23.1, BA.1, BA.2, BA.2.75, BA.5, BQ.1.1, BF.7, XBB and BQ.1.1 lineages.

[0413] Embodiment 59. The method of any one of embodiments 55-58, comprising immunizing a mammal with two or more doses of a fusion protein, wherein at least one of the two or more doses of the fusion protein comprises the amino acid sequence of the SARS-CoV-2 Omicron XBB.1.5 spike protein, optionally comprising an amino acid sequence of any one of SEQ ID NOs: 24-27 or an amino acid sequence that has at least or about 80%, 85%, 90%, 92%, 95%, 97%, or 99% sequence identity to any one of SEQ ID NOs: 24-27.

[0414] Embodiment 60. The method of any one of embodiments 55-59, wherein the fusion protein is administered as a second booster dose after one or more doses of an immunogen comprising a spike protein peptide comprising an NTD and an RBD from the same or different SARS-CoV-2 variant, optionally wherein the one or more doses of the immunogen comprise a sequence of any one of SEQ ID NOs: 27-66, 81-84, 120-127, or an amino acid sequence that has at least or about 80%, 85%, 90%, 92%, 95%, 97%, or 99% sequence identity to any one of SEQ ID NOs: 27-66, 81-84, 120-127, optionally wherein the booster fusion protein comprises a sequence of any one of SEQ ID NOs: 24-27, or an amino acid sequence that has at least or about 80%, 85%, 90%, 92%, 95%, 97%, or 99% sequence identity to any one of SEQ ID NOs: 24-27.

[0415] Example

[0416] The following examples are included for illustrative purposes only and are not intended to limit the scope of the present invention.

[0417] Example 1: Production of recombinant disulfide-linked SARS-CoV-2 S-trimer fusion protein

[0418] Secreted recombinant disulfide-linked polypeptides comprising SARS-CoV-2 protein peptides fused to a trimerization domain are generated as candidate protein subunit vaccines. In one embodiment, the extracellular domain of the spike protein from SARS-CoV2, including its signal peptide (SP), S1, and S2 domains, is fused in frame at the C-terminus to a mammalian expression vector encoding the human C-propeptide of α1 collagen to enable expression of the secreted trimeric S-trimer fusion antigen.

[0419] In order to rapidly express the S-trimer antigen, protein trimerization was used TM The cDNA encoding the extracellular domain of the SARS-CoV-2 spike (S) protein was subcloned into the pTRIMER mammalian expression vector to allow in-frame fusion to the protein trimerization protein. TM Tag, protein trimerization TMThe tag is capable of self-trimerization via disulfide bonds. After stable transfection into CHO cells, subsequent screening for high-titer producing clones, and extensive process optimization, a fed-batch, serum-free cell culture process in a bioreactor was developed, resulting in high-level expression of the S-trimer as a secreted protein. Liang et al., Nat. Comms., 12:1346, 2021; Richmond et al., Lancet, 397:682-694, 2021, are incorporated by reference in their entirety for all purposes.

[0420] To obtain a highly purified form of S-trimer for vaccine research, protein trimerization was employed. TM An affinity purification protocol was developed based on the high binding affinity between the tag and Endo180, a collagen receptor that binds to the C-terminal region of type 1 procollagen and matures the collagen. The Endo180-Fc fusion protein was loaded onto a Protein A column and captured by the resin through high-affinity binding between Protein A and the IgG1 Fc domain of Endo180-Fc. Serum-free cell culture medium containing S-trimer secreted by CHO cells was then loaded onto the Protein A column with pre-captured Endo180-Fc. After washing away any unbound contaminating host cell proteins (HCPs) and other impurities, the bound S-trimer was purified to near homogeneity using a mild salt elution step without dissociating Endo180-Fc from the Protein A column. The S-trimer was further purified by low pH for preventive viral inactivation (VI), anion exchange chromatography to remove host cell DNA and any residual endotoxins, nanofiltration as a preventive viral removal (VR) step, and finally UF / DF to concentrate the S-trimer to the desired concentration in the formulation buffer and sterile filtration to obtain the active drug substance stock (DS) of the S-trimer subunit vaccine candidate. Stability analysis of the purified S-trimer showed that the S-trimer was stable in liquid solution formulation at 2-8°C.

[0421] SDS-PAGE analysis under non-reducing and reducing conditions confirmed that the purified S-trimer is a disulfide-linked trimer that is partially cleaved at the S1 / S2 boundary by furin produced in CHO cells. Under non-reducing conditions, the S-trimer appears as multiple high-molecular-weight forms, likely the result of partial cleavage of the antigen, releasing non-covalently linked and cleaved S1 during sample processing.

[0422] Production and characterization of spike antigens based on Hu-1 and VOC strains

[0423] Utilizing protein trimerization TMThe technology (Liang et al., Nat. Comms., 12:1346, 2021) produces spike antigen covalent trimers based on Hu-1 strains and VOC strains, such as BA.5 and XBB. 1.5. The S-trimer expression in serum-free fed-batch CHO cell culture in a 10L bioreactor was analyzed by 8% SDS-PAGE. The cell-free conditioned medium was analyzed under reducing conditions and then stained with Coomassie Brilliant Blue.

[0424] The binding affinity of purified S-trimer antigen to the human ACE2 receptor was assessed by Fortebio BioLayer interferometry using a Protein A sensor.

[0425] Example 2: Immunogenicity of Adjuvanted SARS-CoV-2 Vaccines

[0426] The immunogenicity of S-trimer was evaluated in BALB / c mice. In a two-dose prime-boost regimen (day 0 and day 21), mice were injected intramuscularly with S-trimer twice. The effect of adjuvant on humoral immunogenicity was significant because the S-trimer binding antibody titer, ACE2 competitive titer and neutralizing antibody titer of the adjuvanted group were significantly higher than those of the unadjuvanted vaccine at the corresponding antigen dose level. S-trimers with different adjuvants elicited ACE2 competitive antibody titers and pseudovirus neutralizing antibody titers similar to or higher than those observed in human convalescent serum samples. Similar results were observed in rats immunized with S-trimer. PCT / CN2021 / 087066PCT / CN2021 / 093895 and PCT / CN2021 / 099285 are incorporated by reference in their entirety for all purposes.

[0427] S-Trimer antigen-specific cell-mediated immunity (CMI) was investigated by harvesting splenocytes from sacrificed, immunized mice, stimulating them with S-Trimer antigen, and measuring Th1 (IL-2 and IFNγ) and Th2 (IL-4 and IL-5) cytokines by ELISpot. Adjuvanted groups appeared to induce a stronger overall CMI response than unadjuvanted S-Trimer. A Th1-biased cell-mediated immune response was observed in unadjuvanted and some adjuvanted S-Trimer groups, while a mixed Th1-Th2 distribution was observed in other adjuvanted groups. CMI appeared to be independent of antigen dose.

[0428] Example 3: Immunogenicity of SARS-CoV-2 vaccines based on the Omicron strain

[0429] method

[0430] Construction and production of pseudoviruses

[0431] The spike protein (S) genes of the original SARS-CoV-2 strain and high-concern variants were optimized using mammalian codons, synthesized by GenScript, and cloned into the pcDNA3.1(+) eukaryotic expression vector. Plasmids encoding the S glycoproteins of the Hu-1, Beta (β), Delta (δ), Omicron (ο), BA.1, BA.2, BA.2.75, BA.5, BQ.1.1, BF.7, XBB, XBB.1.5, BQ.1.1, XBB.1.16.1, and EG.5.1 SARS-CoV-2 variants were constructed. Specifically, the full-length S gene sequence was deleted from the C-terminal 18 amino acids and constructed into the pcDNA3.1(+) vector via Hind III and Xba I sites. The reporter gene was pLVX-AcGFP-N1-Fluc from Honorgene, and the lentiviral packaging plasmid was psPAX2 from Honorgene. Lentiviral packaging was performed using a Thermo Lipo3000. The three plasmids were transfected into 293T cells at a mass ratio of 5:3:4 for viral packaging, and the viral supernatant was harvested 46 h ± 2 h after viral titer determination and pseudovirus detection experiments.

[0432] Lentiviral packaging plasmid psPAX2 and pLVX-AcGFP-N1-Fluc lentiviral reporter plasmid expressing GFP and luciferase are from HonorGene (Aonuo Gene, China). PsPAX2, pLVX-AcGFP-N1-Fluc and plasmids encoding various S genes were co-transfected into HEK 293T cells using Lipofectamine 3000 (Invitrogen, L3000-015) to produce pseudovirus. The supernatant was harvested 24 ± 2 hours after transfection, centrifuged at 1500rpm for 5 minutes to remove cell debris, and then stored at -80°C. The pseudovirus reservoir was titrated by infecting 293T-ACE2 cells, and luciferase activity was measured using a microplate reader (TECAN, Spark) after a 44 to 48 hour incubation period at 37°C and 5% CO2 by adding the Bright-Glo luciferase detection system (Promega, E2650). The TCID of the pseudovirus was then calculated according to the Reed-Muench method (Quantification of SARS-CoV-2 neutralizing antibody by a pseudotyped virus based assay. Nie J. et al. DOI: 10.21203 / rs.3.pex-941 / v11) 50 .

[0433] Neutralization test

[0434] Aliquots of the test serum samples were first heat inactivated at 56°C for 30 minutes and then clarified by centrifugation at 10,000 rcf for 5 minutes. The samples were serially diluted (3-fold) with the assay medium (100 ml) and diluted with 650 TCID 50 The pseudovirus (50 ml, 50 μL / well) was incubated at 37 ° C for 1 hour, along with untreated controls (virus alone) and cells alone (background control) infected with the virus. Fresh trypsinized 293T-ACE2 cells were then added to each well at 20,000 cells / well in 100 mL (100 μL / well). After incubation at 37 ° C in a 5% CO2 incubator for 44 to 48 hours, the cells were lysed according to the manufacturer's protocol and luciferase activity was determined by the Bright-GlovLuciferase Luciferase Assay System (Promega). The IC of a given serum sample was 24 h. 50 Neutralizing antibody titers were defined as the serum dilution at which the sample showed a 50% reduction in relative light units (RLU) compared to virus-infected control wells. The detailed method was based on the Quantification of SARS-CoV-2 neutralizing antibody by a pseudotyped virus-based assay reported by Nie J. et al. DOI: 10.21203 / rs.3.pex-941 / v11.

[0435] Expression and purification of variant of high concern (VOC) S-trimer fusion protein

[0436] cDNA encoding the ectodomain of the SARS-CoV-2 spike (S) protein from Beta, Delta, Omicron BA.5, and XBB.1.5 was synthesized by GenScript using codons preferred by Cricetulus griseus (Chinese hamster). The cDNA was subcloned into the pTRIMER expression vector (GenHunter) at the Hind III and Bgl II sites to allow in-frame fusion of the soluble S protein to the protein trimerization vector. TM The tag (amino acid residues 1156-1406 from human type I (α) collagen) was expressed as described previously. The expression construct was transiently transfected into the HEK-293F cell line (Clover Biopharma) using PEI (Polyscience) and grown in OPM-293 CD05 medium (OPM) and OPM-293 proFeed supplement (OPM). Protein trimerization was used. TMThe S-trimer protein was purified to homogeneity from the conditioned medium using a tag-specific affinity column (Clover Biopharma).

[0437] Pseudovirus neutralizing antibody IC 50 Titer data processing

[0438] Pseudovirus experimental data processing (IC 50 Calculation): 50% inhibition dilution (IC 50 ) means: after deducting the blank control RLU value, the serum sample RLU value is the dilution factor corresponding to the RLU value of the virus well (single virus infected cell) decreasing by 50%. GraphPad Prism software was used to perform four-parameter fitting and IC 50 Value calculation.

[0439] Result judgment: If the inhibition rate of the first well of the sample is ≤50%, the titer result is judged to be negative, and the dilution multiple of the first well is used as the titer value; if the inhibition rate of the first well of the test sample is higher than 50%, the result of the four-parameter fitting is used as the IC 50 Titer.

[0440] Acceptance criteria: The inhibition rate of the negative control first well should be <50%, and the inhibition rate of the positive control SCB-719 first well should be ≥90%.

[0441] Statistical Analysis and Software: This study used R (version 4.2.1) and SAS (version 9) for statistical analysis. The immunogenicity titers of the trivalent vaccine were tested for noninferiority to those of the monovalent and bivalent vaccines, respectively. The lower limit of the one-sided 90% confidence interval for the GMT ratio between the two groups was >0.5, which was used as the noninferiority test threshold. The main computerized systems used in this study include (but are not limited to):

[0442] Table 1 Data processing software

[0443] Test product information

[0444] Table 2 lists the composition of the test samples and the corresponding animal groups.

[0445] Table 2 Test article information and corresponding animal groups (homologous boosting)

[0446] Example 4: Vaccine preparation and formulation

[0447] The antigen stock solution and the CpG1018 adjuvant stock solution are thawed separately, and then the antigen stock solution, the CpG1018 adjuvant intermediate and the aluminum adjuvant are mixed to prepare a semi-finished product, which is then filled, stoppered, capped, visually inspected and stored.

[0448] Table 3. List of formulation compositions

[0449] Example 5: Clinical trial

[0450] Trial vaccine: Trivalent vaccine: includes 30 μg of antigen (including 12 μg of Omicron BA.5, 12 μg of Omicron XBB1.5 and 6 μg of Hu-1 prototype strain Hu-1) and a CpG 1018 / alum adjuvant system (1.5 mg of CpG 1018 adjuvant and 0.75 mg of aluminum hydroxide) in a 0.5 mL dose.

[0451] Control vaccine: Hu-1 vaccine (monovalent): includes 30 μg Hu-1 antigen (prototype strain) and CpG 1018 / alum adjuvant system (1.5 mg CpG 1018 adjuvant and 0.75 mg aluminum hydroxide) in a 0.5 mL dose.

[0452] Trivalent vaccine clinical trial study design: The subjects were those who had received three doses of Hu-1 S-trimer vaccine, with an interval of ≥6 months (randomization 2:1, N=600). Screening and vaccination were performed on the first day for eligible subjects. Blood tests were drawn on the 15th day. Follow-up calls were made on the 29th and 90th days, blood tests were drawn again on the 180th day, and a final follow-up call was made on the 180th day.

[0453] Route of administration: intramuscular injection (im).

[0454] result

[0455] Pseudovirus neutralizing antibody titer results

[0456] Constructed and produced subtype BA.5 and XBB.1.5 protein trimers based on Hu-1 and Omicron strains TM Tagged subunit monovalent vaccines, and bivalent or trivalent vaccines composed of subtype BA.5 and XBB.1.5 antigens selected from Hu-1 and Omicron strains, are administered to mice as priming and / or booster vaccines for homologous boosting, or used in combination with Hu-1 S-trimer for heterologous boosting.

[0457] Homologous reinforcement

[0458] Homologous boost D35 pseudovirus neutralizing antibody titer results: Figure 2 shows the neutralizing antibody IC of Hu-1, Beta, Delta, and Omicron subtypes BA.1, BA.2, BA.2.75, BA.5, BQ.1.1, BF.7, XBB, and XBB.1.5 pseudovirus strains in naive mice after receiving two doses of S-trimer antigen (first dose primary immunization - second dose boost) according to Figure 1 and Table 2. 50 Figure 2 shows the experimental design of Figure 1 and the vaccine doses in Table 2 after mice received two doses of S-trimer antigen (first dose primary immunization - second dose booster) immunization with SARS-CoV-2 Hu-1), beta (beta, β, B.1.351), delta (delta, δ, B.1.617.2), and Omicron (O, B.1.1.529) subtypes BA.1, BA.2, BA.2.75, BA.5, BQ.1.1, BF.7, XBB, XBB.1.5, strain pseudovirus neutralizing antibody IC 50 Data. After two immunizations with 150 μg CpG 1018 and 75 μg Alum as adjuvant, mice were bled on day 35 (D4 PD2) for pseudovirus neutralizing antibody testing. A. is the Hu-1 strain pseudovirus neutralizing antibody IC 50 Data; B. is the IC of the neutralizing antibody against the Beta strain pseudovirus 50 Data; C. is the IC of the Delta strain pseudovirus neutralizing antibody 50 Data; D. is the IC of the neutralizing antibody against the BA.1 strain pseudovirus 50 Data; E. is the IC value of neutralizing antibody against BA.2 strain pseudovirus 50 Data; F. is the IC value of neutralizing antibody against BA.2.75 strain pseudovirus 50 Data; G. is the IC value of neutralizing antibody against BA.5 strain pseudovirus 50 Data; H. is the BQ.1.1 strain pseudovirus neutralizing antibody IC 50 Data; I. is the BF.7 strain pseudovirus neutralizing antibody IC 50 Data; J. is the IC of the neutralizing antibody against XBB strain pseudovirus 50 Data; K. is the IC value of neutralizing antibody against XBB.1.5 pseudovirus 50 data.

[0459] To further understand the advantages of the trivalent vaccine over the monovalent and bivalent vaccines, this study compiled neutralizing antibody titers against 11 pseudoviruses in mice from each immunization group and created a spider diagram. The data comparing the trivalent and monovalent vaccines (Figure 3, C) show that the trivalent vaccine has the highest broad-spectrum activity. While the Hu-1 (Prototype) S-trimer and XBB.1.5 S-trimer monovalent vaccines have the highest neutralizing titers against their own pseudovirus strains, they are less effective than the trivalent vaccine against broad-spectrum strains outside their own clade.

[0460] In terms of statistics, this article conducted a non-inferiority test analysis on the GMT (geometric mean titer) ratios of the neutralizing antibody titers of 11 pseudoviruses corresponding to the trivalent vaccine and the monovalent vaccines Hu-1 S-trimer, BA.5 S-trimer, and XBB.1.5 S-trimer (the threshold was set: the 90% CI lower limit (LowCI) corresponding to the GMT ratio > 0.5 was comparable). It was found that for the early COVID-19 strains Hu-1, Beta and Delta, except for the Hu-1 S-trimer monovalent vaccine, the trivalent vaccines were non-inferior to the BA.5 S-trimer and XBB.1.5 S-trimer monovalent vaccines; and for the 8 Omicron series branch strains, although the monovalent vaccine XBB.1.5 S-trimer showed advantages against XBB and XBB.1.5, BA.5 S-trimer showed advantages against BA.1 and BQ.1.1, but compared with the monovalent vaccine, the trivalent vaccine was non-inferior to the monovalent vaccine in 6 of the 8 strains in the Omicron series, indicating that the trivalent vaccine has better broad spectrum among the Omicron series strains. The specific non-inferiority test results are shown in Table 4 and Figure 3C.

[0461] Table 4. Non-inferiority test results of "trivalent vaccine" vs "monovalent vaccine"

[0462] Table 4 (continued). Non-inferiority test results of “trivalent vaccine” vs “monovalent vaccine” Note: **: superior; *: non-inferior; others: ratio > 0.5 / LCI < 0.5.

[0463] From A and B of spider web diagram 3 of "trivalent" versus "bivalent", when the ratio of bivalent antigens is 1:1 or 1:2, the comprehensive data of 11 pseudoviruses show that the trivalent vaccine has a higher spectrum.

[0464] In terms of statistics, the GMT ratios of the neutralizing antibody titers of mice in the "trivalent" and "bivalent" immunization groups corresponding to 11 pseudoviruses were subjected to a non-inferiority test (the threshold was set: the 90% CI lower limit (LowCI) corresponding to the GMT ratio was >0.5, which was comparable). In the exploration of the bivalent vaccine antigen ratio, the three combinations of Hu-1+BA.5, Hu-1+XBB.1.5, and BA.5+XBB.1.5, when the bivalent antigen ratio was 1:1, showed an advantage in the early COVID-19 strains Hu-1 / Beta / Delta with the bivalent combination containing Hu-1 (Figure 3A); when the bivalent antigen ratio was 1:2, none of the three "bivalent" antigen combinations showed a significant advantage (Figure 3B). For specific data, see Table 5 for the non-inferiority statistics of the "trivalent" versus "bivalent" 11 pseudovirus strains.

[0465] Table 5. Non-inferiority test results of trivalent vaccine vs bivalent vaccine (1:1 or 1:2) Note: **: superior; *: non-inferior; others: ratio > 0.5 / LCI < 0.5.

[0466] In this experiment (Figures 5 and 6), serum samples were collected on D14 and D28 after the third and fourth immunizations (D156: D0 of the third immunization; D170: D14 after the third immunization; D184: D28 after the third immunization and D0 of the fourth immunization; D198: D14 after the fourth immunization; D212: D28 after the fourth immunization), and the neutralizing antibody titer against the new coronavirus pseudovirus (Hu-1, Beta, Delta, and Omicron subtypes BA.1, BA.2, BA.2.75, BA.5, BQ.1.1, BF.7, XBB, XBB.1.5) in the mouse serum was detected through a pseudovirus experiment.

[0467] To further compare the advantages and disadvantages of three heterologous booster shots after HU-1 S-trimer as basic immunization, that is, the advantages and disadvantages of one trivalent vaccine and two bivalent vaccines compared with the monovalent vaccine as a booster shot, this article summarized the neutralizing antibody titer levels of mice in each immunization group corresponding to 11 pseudoviruses and drew a spider web diagram (Figure 7). It can be seen from the figure that both the trivalent vaccine and the bivalent vaccine have higher broad-spectrum than the monovalent vaccine. Among them, the trivalent vaccine and the bivalent vaccine (BA.5+XBB.1.5) have similar and highest broad-spectrum types, while the bivalent vaccine (Hu-1+XBB.1.5) is second. When the monovalent vaccine is used as a booster shot, the neutralizing antibody titer level against the Omicron series is relatively weak. In terms of statistics, this article conducted a non-inferiority test on the GMT ratios of neutralizing antibody titers of 11 pseudoviruses corresponding to the broad-spectrum heterologous booster shot, i.e., the trivalent vaccine, with those of the bivalent vaccine and the monovalent vaccine (set threshold: the 90% CI lower limit (LowCI) corresponding to the GMT ratio was >0.5, which is comparable). The results showed that the neutralizing antibody level of the trivalent vaccine was compared with the monovalent vaccine and the bivalent vaccine (Hu-1+XBB.1.5). In more than half of the pseudovirus strains, the statistical analysis results were "non-inferior", indicating that the trivalent vaccine was better than the monovalent and bivalent (Hu-1+XBB.1.5); the neutralizing antibody level of the trivalent vaccine was compared with the bivalent vaccine (BA.5+XBB.1.5). Although the statistical analysis results showed "inferiority" for 8 strains, the GMT ratios were all greater than 0.5, and some GMT ratios were greater than 1, resulting in an "inferiority" result, which may be due to the small number of samples in each group.

[0468] Pre-existing heterologous booster immunization

[0469] Animal grouping and dosage design This study is divided into two parts, namely, pre-existing immunization in the early stage and booster immunization in the later stage. The pre-existing immunization scheme is shown in Table 6:

[0470] Table 6. Pre-existing immune experiment design and dosing regimen

[0471] For booster immunization, a total of 80 female BALB / c mice were randomly divided into four groups based on body weight, with 20 mice in each group. The experimental design and dosing schedule are shown in Table 7.

[0472] Table 7. Booster Immunization Experiment Design & Dosage Scheme Note: “1vial:” antigen, aluminum agent and CpG1018 are in the same bottle; “2vial:” antigen and aluminum agent are in the same bottle.

[0473] In this experiment, serum samples from mice were collected 1 day before booster immunization (D107), 14 days (D122), and 28 days (D136) after booster immunization. The neutralizing antibody titer against the new coronavirus pseudovirus (Hu-1 / Beta / Delta / BA.1 / BA.2 / BA.2.75 / BA.5 / BF.7 / BQ.1.1 / XBB / XBB.1.5 / XBB.1.16.1 / EG.5.1) in mouse serum was detected by pseudovirus experiment ( Figure 9 ).

[0474] In order to further compare the advantages and disadvantages of two heterologous booster shots after two doses of HU-1 S-trimer as basic immunization, that is, the second group of monovalent vaccine XBB.1.5 S-trimer and the third group of S-trimer trivalent vaccine (1:2:2) compared with the first group of first-generation new crown monovalent vaccine HU-1 S-trimer as a booster shot, this article summarized the geometric mean titer (GMT) levels of neutralizing antibodies corresponding to 12 pseudoviruses in mice in each immunization group and drew a spider web diagram (Figures 10 and 11). From the figure, we can see that both the s-trimer trivalent vaccine (1:2:2) and the XBB.1.5 S-trimer monovalent vaccine show higher broad spectrum than the first-generation monovalent vaccine HU-1 S-trimer. Among them, the advantage of the s-trimer trivalent vaccine (1:2:2) is that it has the highest broad spectrum of neutralizing antibodies among the 13 pseudoviruses detected in the D122 and D136 sera; and when the XBB.1.5 S-trimer monovalent vaccine is used as a booster shot, the neutralizing antibody titers against all pseudoviruses are significantly improved, especially showing higher protective efficacy against XBB-related branches.

[0475] In addition, in the two groups, the Group 2 XBB.1.5 S-trimer full-dose group (3μg XBB.1.5) and the Group 4 XBB.1.5 S-trimer 2 / 5-dose group (the same dose as the XBB.1.5 antigen in the trivalent vaccine, i.e., 1.2μg XBB.1.5), it was observed that the titer levels of the two groups were relatively similar at D14 of immunization (Figure 10), but at D28 of immunization, the Group 2 XBB.1.5 S-trimer full-dose group showed an advantage in the neutralizing antibody level against 8 pseudoviruses from BA.2 to XBB.1.16.1. We also observed that the antibody levels against the XBB series strains produced by the Group 4 group and the trivalent vaccine group using the same dose of XBB.1.5 antigen in the trivalent vaccine were very similar (Figure 11), but no advantage was shown against strains outside the XBB series, indicating that the dose in the trivalent vaccine is positively correlated with the antibody produced by it, and the additional Hu-1 S-trimer and BA.5 The two antigens of S-trimer played a beneficial role in neutralizing strains other than XBB.

[0476] Statistical analysis of antibody titers of heterologous booster shots: In terms of statistics, we performed non-inferiority tests on the GMT ratios of neutralizing antibody titers of 13 pseudoviruses corresponding to the monovalent vaccine XBB.1.5 S-trimer, the first-generation monovalent vaccine HU-1 S-trimer, the S-trimer trivalent vaccine (1:2:2), and 2 / 5 doses of XBB.1.5 S-trimer (the non-inferiority standard was set as the lower limit of the 90% CI of the GMT ratio (LowCI>0.5). The results of the non-inferiority test of neutralizing antibody titers of D136 pseudoviruses showed (Table 8) that the monovalent vaccine XBB.1.5 Comparing neutralizing antibody levels with the first-generation monovalent vaccine HU-1S-trimer, S-trimer demonstrated "non-inferiority" against all strains, with the exception of pre-Omicron-related strains Beta and Delta, and BA.1, where it showed "inferiority." Furthermore, GMT ratios for XBB-related strains were significantly superior. Comparing the monovalent XBB.1.5 S-trimer with the trivalent XBB.1.5 S-trimer, the monovalent vaccine demonstrated "non-inferiority" in neutralizing antibody levels against XBB-...

Claims

1. A multivalent vaccine comprising a coronavirus (S) protein surface antigen or a fragment, variant or mutant thereof, wherein the coronavirus (S) protein surface antigen or a fragment, variant or mutant thereof is freely selected from soluble SARS-CoV-2 Hu-1, SARS-CoV-2 Omicron (B.1.1.529) and its subtypes, alpha, beta, gamma, delta, mu spike (S) protein surface antigen or a fragment, variant or mutant thereof, and the coronavirus (S) protein surface antigen or a fragment, variant or mutant thereof is connected to the C-terminal propeptide portion of collagen to form a disulfide-linked trimeric fusion protein.

2. A multivalent vaccine according to claim 1, wherein the SARS-CoV-2 Omicron subtype spike (S) protein surface antigen or its fragment, variant or mutant comprises BA.1, BA.2, BA.2.75, BA.5, BQ.1.1, BF.7, XBB, XBB.1.5, BQ.1.1 spike (S) protein surface antigen or its fragment, variant or mutant.

3. The multivalent vaccine according to claim 1 or 2 comprises the soluble SARS-CoV-2 Hu-1 (S) protein surface antigen or a fragment, variant or mutant thereof.

4. The multivalent vaccine according to any one of claims 1-3 comprises the soluble SARS-CoV-2 Omicron BA.5 (S) protein surface antigen or a fragment, variant or mutant thereof.

5. The multivalent vaccine according to any one of claims 1-4 comprises the soluble SARS-CoV-2 Omicron XBB.1.5 (S) protein surface antigen or a fragment, variant or mutant thereof.

6. The multivalent vaccine according to claim 5, which is a trivalent vaccine and comprises the soluble SARS-CoV-2Hu-1, Omicron strain BA.5 and XBB.1.5 spike (S) protein surface antigens or fragments, variants or mutants thereof in a weight ratio of 0.5-4:0.5-4:0.5-4.

7. A multivalent vaccine according to claim 6, wherein the weight ratio of the soluble SARS-CoV-2Hu-1, Omicron strains BA.5 and XBB.1.5 and the spike (S) protein surface antigen or its fragment, variant or mutant is 1:1:1 or 1:2:

2.

8. The multivalent vaccine according to any one of claims 1-5, wherein the coronavirus (S) protein surface antigen or a fragment, variant or mutant thereof comprises a sequence described in any one of SEQ ID NOs: 27-66 or an amino acid sequence having at least or about 80%, 85%, 90%, 92%, 95%, 97%, or 99% sequence identity with the sequence described in any one of SEQ ID NOs: 27-66.

9. The multivalent vaccine according to any one of claims 1-8, wherein the coronavirus (S) protein surface antigen or a fragment, variant or mutant thereof comprises a sequence described in any one of SEQ ID NOs: 27-34 or an amino acid sequence having at least or about 80%, 85%, 90%, 92%, 95%, 97%, 99% sequence identity with any one of SEQ ID NOs: 27-34.

10. The multivalent vaccine according to any one of claims 1 to 9, in, The coronavirus (S) protein surface antigen or a fragment thereof or a variant or mutant thereof comprises a sequence described in any one of SEQ ID NOs: 81-84 or an amino acid sequence having at least or about 80%, 85%, 90%, 92%, 95%, 97%, or 99% sequence identity with the sequence described in any one of SEQ ID NOs: 81-84.

11. The multivalent vaccine according to any one of claims 1 to 10, in, The coronavirus (S) protein surface antigen or a fragment thereof or a variant or mutant thereof comprises the sequence of SEQ ID NO: 126 or an amino acid sequence having at least or about 80%, 85%, 90%, 92%, 95%, 97%, 99% sequence identity with the sequence in SEQ ID NO:

126.

12. The multivalent vaccine according to any one of claims 1 to 11, in, The coronavirus (S) protein surface antigen or a fragment thereof or a variant or mutant thereof comprises the sequence of SEQ ID NO: 122 or an amino acid sequence having at least or about 80%, 85%, 90%, 92%, 95%, 97%, or 99% sequence identity with the sequence in SEQ ID NO:

122.

13. A multivalent vaccine according to any one of claims 2 or 3, which is a bivalent vaccine, can be freely selected from SARS-CoV-2Hu-1, Alpha, Beta, Gamma, Delta, Mu Spike, Omicron (B.1.1.529), BA.1, BA.2, BA.2.75, BA.5, BQ.1.1, BF.7, XBB, XBB.1.5, BQ.1.1, XBB.1.16.1, EG.5.1 spike (S) protein surface antigen or its fragment, variant or mutant, wherein the weight ratio of the two coronavirus (S) protein surface antigens or their fragments or variants or mutants included in the bivalent vaccine is 0.5-4:0.5-4.

14. The multivalent vaccine according to claim 13, wherein the bivalent vaccine comprises the BA.5 and XBB.1.5 in a weight ratio of 1:1 or 1:2; or the bivalent vaccine comprises the Hu-1 and BA.5 in a weight ratio of 1:1 or 1:2; or the bivalent vaccine comprises the Hu-1 and XBB.1.5 in a weight ratio of 1:1 or 1:

2.

15. A multivalent vaccine according to any one of claims 1-14, which is administered without an adjuvant. Optionally, the multivalent vaccine is used as a primary series, an additional dose and / or a homologous or heterologous booster dose, such as the first dose, the second dose, the third dose, the fourth dose and / or more doses. Optionally, the primary series, the additional dose or the heterologous booster is used in combination with any one or more of other recombinant subunit vaccines, nanoparticle vaccines, mRNA vaccines, DNA vaccines, adenovirus vector vaccines and inactivated virus vaccines.

16. A multivalent vaccine according to any one of claims 1-14, which is administered together with one or more adjuvants, and optionally used as a primary series, additional dose and / or homologous or heterologous booster dose, such as the first dose, second dose, third dose, fourth dose and / or more doses, and optionally used in combination with any one or more of other recombinant subunit vaccines, nanoparticle vaccines, mRNA vaccines, DNA vaccines, adenovirus vector vaccines and inactivated virus vaccines.

17. The multivalent vaccine according to any one of claims 1 to 14 and 16, wherein the adjuvant in the initial dose, the additional dose, and / or the homologous or heterologous booster dose can be independently include: Aluminum-containing adjuvants, such as adjuvants containing alum and / or aluminum hydroxide; oligonucleotide-containing adjuvants, such as adjuvants containing CpG oligodeoxynucleotides (CpG-ODN); adjuvants containing TLR9 agonists; adjuvants containing metabolizable oils, α-tocopherol, and / or polyoxyethylene sorbitan monooleate (Tween-80), such as adjuvants containing squalene, α-tocopherol, and Tween-80 and / or Span 85 in the form of an oil-in-water emulsion; or a combination of any of the adjuvants.

18. The multivalent vaccine according to any one of claims 1-14 and 16-17, wherein the adjuvant in the initial dose, the additional dose, and / or the homologous or heterologous booster may include an adjuvant of CpG oligodeoxynucleotide (CpG-ODN) and / or an adjuvant containing alum and / or aluminum hydroxide.

19. The multivalent vaccine according to any one of claims 1 to 18 is administered by intramuscular injection.

20. The multivalent vaccine according to any one of claims 1 to 18 is administered by intranasal spray.

21. The multivalent vaccine according to any one of claims 1 to 20 is administered in a single dose or in a series of doses spaced apart by weeks or months.

22. The multivalent vaccine according to any one of claims 1 to 21 can prevent or treat coronavirus infection, such as severe acute respiratory syndrome (SARS)-coronavirus 2 (SARS-CoV-2) infection.

23. The multivalent vaccine according to any one of claims 1-21, wherein the C-terminal propeptide comprises any one of SEQ ID NOs: 67-80 or an amino acid sequence at least 80%, 85%, 90%, 92%, 95%, 97%, 99% identical thereto.

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