WO2023179513A1 - 冠状病毒疫苗组合物、方法及其使用 - Google Patents

冠状病毒疫苗组合物、方法及其使用 Download PDF

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WO2023179513A1
WO2023179513A1 PCT/CN2023/082377 CN2023082377W WO2023179513A1 WO 2023179513 A1 WO2023179513 A1 WO 2023179513A1 CN 2023082377 W CN2023082377 W CN 2023082377W WO 2023179513 A1 WO2023179513 A1 WO 2023179513A1
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dose
coronavirus
vaccines
sars
seq
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PCT/CN2023/082377
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English (en)
French (fr)
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梁朋
梁果
宿丹梅
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四川三叶草生物制药有限公司
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Priority claimed from CN202210546036.4A external-priority patent/CN116836294A/zh
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Publication of WO2023179513A1 publication Critical patent/WO2023179513A1/zh

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K19/00—Hybrid peptides, i.e. peptides covalently bound to nucleic acids, or non-covalently bound protein-protein complexes

Definitions

  • the present disclosure in some aspects relates to immunogenic compositions for treating and/or preventing coronavirus infections, including recombinant peptides and proteins, including coronavirus antigens and immunogens , such as coronavirus S protein peptides, including subunit vaccines based on the S protein peptide of the SARS-CoV-2 Omicron (B.1.1.529) strain formed by disulfide bonds between trimerized TM tag polypeptides. .
  • Coronaviruses infect a wide range of birds and mammals, including humans. Coronaviruses may circulate through the human body annually, often causing mild respiratory illness, although severity is higher in infants, young children, the elderly and the immunocompromised. 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) , highly pathogenic. The high pathogenicity, airborne transmissibility, high case fatality rate, and ambiguously defined epidemiology of coronaviruses create an urgent need for effective vaccines and related therapeutic agents. In particular, vaccines that can rapidly induce effective immune responses against SARS-CoV-2 are urgently needed.
  • the present invention provides methods, uses, and articles of manufacture that satisfy these and other needs.
  • VOCs SARS-CoV-2 variants
  • S spike
  • P.1 variant in Brazil 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 have led to their classification as VOCs.
  • VOCs all include the N501Y mutation in the receptor-binding domain (RBD) of the S protein, which has been reported to increase transmission by 40% to 70%.
  • RBD receptor-binding domain
  • the B.1.351 and P.1 variants have two additional RBD mutations—E484K and K417—that may allow immune escape from antibodies induced by the Hu-1 vaccine and natural infection.
  • 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 propeptide of the recombinant polypeptide forms an inter-polypeptide peptide. Sulfur bonds.
  • the recombinant polypeptide or protein can be used as an immunogen, such as a vaccine.
  • recombinant subunit vaccines that include an extracellular domain (e.g., without transmembrane and cytoplasmic domains), which is fused in frame to the C-propeptide of collagen capable of forming disulfide-linked homotrimers.
  • the resulting recombinant subunit vaccine e.g., S-trimer
  • S-trimer Can be expressed and purified from transfected cells and is expected to be in the native conformation as a trimer.
  • the misfolding problem of viral antigens arises. This misfolded viral antigen cannot faithfully maintain the native viral antigen conformation and often fails to produce neutralizing antibodies.
  • the coronavirus is severe acute respiratory syndrome (SARS) coronavirus (SARS-CoV-1), SARS coronavirus 2 (SARS-CoV-2), SARS-like coronavirus, Middle East respiratory syndrome (MERS ) coronavirus (MERS-CoV), MERS-like coronavirus, NL63-CoV, 229E-CoV, OC43-CoV, HKU1-CoV, WIV1-CoV, MHV, HKU9-CoV, PEDV-CoV or SDCV.
  • SARS severe acute respiratory syndrome
  • SARS-CoV-1 SARS coronavirus
  • SARS-CoV-2 SARS coronavirus 2
  • MERS-like coronavirus Middle East respiratory syndrome coronavirus
  • NL63-CoV 229E-CoV
  • OC43-CoV HKU1-CoV
  • WIV1-CoV WIV1-CoV
  • MHV HKU9-CoV
  • PEDV-CoV or SDCV.
  • the surface antigen may include the 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 includes three recombinant polypeptides .
  • S coronavirus spike
  • the surface antigen may include a signal peptide, an S1 subunit peptide, an S2 subunit peptide, or any combination thereof.
  • the surface antigen may include a signal peptide, a receptor binding domain (RBD) peptide, a receptor binding motif (RBM) peptide, a fusion peptide (FP), heptapeptide repeat 1 (HR1), or Heptapeptide repeat 2 (HR2) or any combination thereof.
  • RBD receptor binding domain
  • RBM receptor binding motif
  • FP fusion peptide
  • HR1 heptapeptide repeat 1
  • HR2 Heptapeptide repeat 2
  • the surface antigen may comprise the receptor binding domain (RBD) of the S protein.
  • the surface antigen may include the S1 subunit and the S2 subunit of the S protein.
  • the surface antigen may be free of transmembrane (TM) domain peptides and/or cytoplasmic (CP) domain peptides.
  • the surface antigen may comprise a protease cleavage site, wherein the protease is optionally furin, trypsin, factor Xa, thrombin or cathepsin L.
  • the surface antigen may be free of protease cleavage sites, wherein the protease is optionally furin, trypsin, factor Xa, thrombin, or cathepsin L, or may be non-protease-resistant Cleaved mutated protease cleavage site.
  • the surface antigen may be soluble or not directly bound to a lipid bilayer, such as a membrane or viral envelope.
  • the surface antigens may be the same or different in the recombinant polypeptides of the protein.
  • the surface antigen can be fused directly to the C-terminal propeptide, or can be fused via a linker (e.g., a linker containing glycine-X-Y repeats, where X and Y are independently any amino acid and optionally prolyl amino acid or hydroxyproline) linked to the C-terminal propeptide.
  • a linker e.g., a linker containing glycine-X-Y repeats, where X and Y are independently any amino acid and optionally prolyl amino acid or hydroxyproline
  • the protein may be soluble or not directly bound to a lipid bilayer, such as a membrane or viral envelope.
  • the protein may bind to a cell surface receptor in a subject, optionally wherein the subject is a mammal, such as a primate, such as a human.
  • the cell surface receptor may be angiotensin-converting enzyme 2 (ACE2), dipeptidyl peptidase 4 (DPP4), dendritic cell-specific intercellular adhesion molecule-3-grasp Non-integrin (DC-SIGN) or liver/lymph node-SIGN (L-SIGN).
  • ACE2 angiotensin-converting enzyme 2
  • DPP4 dipeptidyl peptidase 4
  • DC-SIGN dendritic cell-specific intercellular adhesion molecule-3-grasp Non-integrin
  • L-SIGN liver/lymph node-SIGN
  • the C-terminal propeptide may be human collagen.
  • the C-terminal propeptide may include pro ⁇ 1(I), pro ⁇ 1(II), pro ⁇ 1(III), pro ⁇ 1(V), pro ⁇ 1(XI), pro ⁇ 2(I), pro ⁇ 2(V), The C-terminal propeptide of pro ⁇ 2(XI) or pro ⁇ 3(XI) or a fragment thereof.
  • the C-terminal propeptides may be the same or different in the recombinant polypeptides.
  • the C-terminal propeptide may comprise any one of SEQ ID NO: 67-80, or be at least 90%, 91%, 92%, 93% identical to any one of SEQ ID NO: 67-80.
  • Amino acid sequences with %, 94%, 95%, 96%, 97%, 98%, 99% or 99.5% sequence homology are capable of forming inter-polypeptide disulfide bonds and trimerizing the recombinant polypeptide.
  • the C-terminal propeptide may comprise SEQ ID NO: 67 or an amino acid sequence at least 95% identical thereto, capable of forming inter-polypeptide disulfide bonds and trimerizing the recombinant polypeptide.
  • the C-terminal propeptide may comprise SEQ ID NO: 68 or an amino acid sequence at least 95% identical thereto, capable of forming inter-polypeptide disulfide bonds and trimerizing the recombinant polypeptide.
  • the C-terminal propeptide may comprise SEQ ID NO: 69 or an amino acid sequence at least 95% identical thereto, capable of forming inter-polypeptide disulfide bonds and trimerizing the recombinant polypeptide.
  • the C-terminal propeptide may comprise SEQ ID NO: 70 or an amino acid sequence at least 95% identical thereto, capable of forming inter-polypeptide disulfide bonds and trimerizing the recombinant polypeptide.
  • the C-terminal propeptide may comprise SEQ ID NO: 71 or an amino acid sequence at least 95% identical thereto, capable of forming inter-polypeptide disulfide bonds and trimerizing the recombinant polypeptide.
  • the C-terminal propeptide may comprise SEQ ID NO: 72 or an amino acid sequence at least 95% identical thereto, capable of forming inter-polypeptide disulfide bonds and trimerizing the recombinant polypeptide.
  • the C-terminal propeptide may comprise SEQ ID NO: 73 or an amino acid sequence at least 95% identical thereto, capable of forming inter-polypeptide disulfide bonds and trimerizing the recombinant polypeptide.
  • the C-terminal propeptide may comprise SEQ ID NO: 74 or an amino acid sequence at least 95% identical thereto, capable of forming inter-polypeptide disulfide bonds and trimerizing the recombinant polypeptide.
  • the C-terminal propeptide may comprise SEQ ID NO: 75 or an amino acid sequence at least 95% identical thereto, capable of forming inter-polypeptide disulfide bonds and trimerizing the recombinant polypeptide.
  • the C-terminal propeptide may comprise SEQ ID NO: 76 or an amino acid sequence at least 95% identical thereto, capable of forming inter-polypeptide disulfide bonds and trimerizing the recombinant polypeptide.
  • the C-terminal propeptide may comprise SEQ ID NO: 77 or an amino acid sequence at least 95% identical thereto, capable of forming inter-polypeptide disulfide bonds and trimerizing the recombinant polypeptide.
  • the C-terminal propeptide may comprise SEQ ID NO: 78 or an amino acid sequence at least 95% identical thereto, capable of forming inter-polypeptide disulfide bonds and trimerizing the recombinant polypeptide.
  • the C-terminal propeptide may comprise SEQ ID NO: 79 or an amino acid sequence at least 95% identical thereto, capable of forming inter-polypeptide disulfide bonds and trimerizing the recombinant polypeptide.
  • the C-terminal propeptide may comprise SEQ ID NO: 80 or an amino acid sequence at least 95% identical thereto, capable of forming inter-polypeptide disulfide bonds and trimerizing the recombinant polypeptide.
  • the C-terminal propeptide may include a sequence comprising a glycine-X-Y repeat linked to the N-terminus of any one of SEQ ID NOs: 67-80, wherein X and Y are independently any amino acid and Alternatively proline or hydroxyproline, or an amino acid sequence at least 90% identical thereto, capable of forming inter-polypeptide disulfide bonds and trimerizing the recombinant polypeptide.
  • the surface antigen in each recombinant polypeptide can be in a prefusion conformation.
  • the surface antigen in each recombinant polypeptide can be in a post-fusion conformation.
  • the surface antigen in each recombinant polypeptide may comprise any one of SEQ ID NOs: 27-66 and 81-84 or an amino acid sequence at least 80% identical thereto.
  • the recombinant polypeptide may comprise any one of SEQ ID NOs: 1-26 and 85-92 or an amino acid sequence that is at least 80% identical thereto.
  • immunogens comprising proteins provided herein.
  • protein nanoparticles comprising a protein provided herein linked directly or indirectly to the nanoparticle.
  • VLPs virus-like particles
  • isolated nucleic acids encoding one, two, three or more recombinant polypeptides of the proteins provided herein.
  • a polypeptide encoding a spike protein peptide is fused in frame to a polypeptide encoding a C-terminal propeptide of collagen.
  • the isolated nucleic acids provided herein are operably linked to a promoter.
  • the isolated nucleic acids provided herein are DNA molecules. In some embodiments, the isolated nucleic acids provided herein are RNA molecules, optionally mRNA molecules, such as nucleoside-modified mRNA, non-amplified mRNA, self-amplified mRNA, or trans-amplified mRNA.
  • vectors containing the isolated nucleic acids provided herein are also provided herein.
  • the vector is a viral vector.
  • viruses, pseudoviruses, or cells comprising a vector provided herein, optionally, wherein the virus or cell has a recombinant genome.
  • immunogenic compositions comprising a protein, immunogen, protein nanoparticle, VLP, isolated nucleic acid, vector, virus, pseudovirus or cell provided herein and a pharmaceutically acceptable carrier.
  • vaccines comprising an immunogenic composition provided herein and an optional adjuvant, wherein the vaccine is optionally a subunit vaccine.
  • the vaccine is a prophylactic and/or therapeutic vaccine.
  • Optional adjuvants may be used in priming and/or boosting doses.
  • adjuvants for the priming agent and/or any booster agent or doses may include: aluminum-containing adjuvants, such as alum and/or aluminum hydroxide-containing adjuvants; oligonucleotide-containing adjuvants, such as Adjuvants containing CpG oligodeoxynucleotides (CpG-ODN); adjuvants containing TLR9 agonists; adjuvants containing metabolizable oil, alpha-tocopherol, and/or polyoxyethylene sorbitan monooleate (Tween -80), such as an adjuvant containing squalene, ⁇ -tocopherol, and Tween-80 and/or Span 85 in the form of an oil-in-water emulsion; or any combination of adjuvants.
  • aluminum-containing adjuvants such as alum and/or aluminum hydroxide-containing adjuvants
  • oligonucleotide-containing adjuvants such as Adju
  • provided herein are methods of 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.
  • proteins produced by the methods provided herein comprising: expressing an isolated nucleic acid or vector provided herein in a host cell to produce a protein provided herein; and purifying the protein.
  • kits for generating an immune response to a S protein peptide of a coronavirus or a fragment or epitope thereof in a subject comprising administering to the subject an effective amount of a protein, immunogen, protein nanoparticle as provided herein Particles, VLPs, isolated nucleic acids, vectors, viruses, pseudoviruses, cells, immunogenic compositions or vaccines, to generate an immune response.
  • the methods provided herein are used to treat or prevent coronavirus infection.
  • generating an immune response inhibits or reduces replication of the coronavirus in the subject.
  • the immune response includes a cell-mediated response and/or a humoral response, optionally including the production of one or more neutralizing antibodies, such as polyclonal antibodies or monoclonal antibodies.
  • the immune response is directed against the S protein peptide of the coronavirus, or a fragment or epitope thereof, but not against the C-terminal propeptide.
  • administration to the subject does not result in an antibody-dependent increase in the subject due to prior exposure to one or more coronaviruses. Strong (ADE).
  • administration does not result in antibody-dependent enhancement (ADE) in the subject when subsequently exposed to one or more coronaviruses.
  • the method further includes a priming step and/or a boosting step.
  • a priming step by topical, transdermal, subcutaneous, intradermal, oral, intranasal (e.g., intranasal spray), intratracheal, sublingual, oral, rectal, vaginal, inhalation, intravenous (e.g., intravenous injection), intraarterial, intramuscular (e.g., intramuscular injection), intracardiac, intraosseous, intraperitoneal, transmucosal, intravitreal, subretinal, intraarticular, periarticular, topical or epicutaneous administration Administration steps.
  • the effective amount is administered as a single dose or as a series of doses with one or more intervals. In some embodiments, the effective amount is administered without the use of adjuvants. In some embodiments, an effective amount is administered with an adjuvant.
  • kits for treating coronavirus infection comprising administering to a subject an effective amount of a protein provided herein to produce neutralizing antibodies or neutralizing antisera against coronavirus in the subject.
  • the subject is a mammal, optionally a human or a non-human primate.
  • the method further includes isolating neutralizing antibodies or neutralizing antisera from the subject.
  • the methods further comprise administering to a human subject an effective amount of an isolated neutralizing antibody or neutralizing antisera by passive immunization to prevent or treat coronavirus infection.
  • the neutralizing antibodies or neutralizing antisera against coronavirus include polyclonal antibodies against coronavirus S protein peptides or fragments or epitopes thereof, optionally, wherein the neutralizing antibodies or neutralizing antisera do not Contains or essentially contains no antibodies directed against the C-terminal propeptide of collagen.
  • the neutralizing antibodies include monoclonal antibodies directed against coronavirus S protein peptides or fragments or epitopes thereof, optionally, wherein the neutralizing antibodies do not contain or substantially contain no antibodies directed against the C-terminal propeptide of collagen. Antibody.
  • 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 for treating or preventing coronavirus infection.
  • 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 in a subject immune response to coronavirus, and/or used to treat or prevent coronavirus infection.
  • a protein, immunogen, protein nanoparticle, VLP, isolated nucleic acid, vector, virus, pseudovirus, cell, immunogenic composition, or vaccine provided herein for the manufacture of a method for inducing a subject Drugs or prophylactics that target the immune response to coronavirus in subjects, and/or are used to treat or prevent coronavirus infection.
  • This article also provides a method for analyzing a sample, including: contacting the sample with a protein provided herein, and detecting binding between the protein and an analyte capable of specifically binding to the S protein peptide of the coronavirus or a fragment or epitope thereof.
  • the analyte is an antibody, receptor or cell that recognizes a spike protein peptide or fragment or epitope thereof.
  • 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 is derived.
  • 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.
  • Figures 1A-1B illustrate the structural features of an exemplary soluble S-trimer subunit vaccine for SARS-CoV-2.
  • Figure 1A is a schematic diagram of the S-trimer domain, and Figure 1B shows its trimer and covalently linked three-dimensional conformation.
  • Figure 2 shows an exemplary characterization of the S-trimer based on Omicron strains, staining with Coomassie brilliant blue showing the full-length S-trimer and the S1/S2 furin sites. Partially cleaved forms of point cleavage, including S2-trimers and cleaved S1 fragments.
  • Lane A shows wild-type Omicron S-trimer.
  • Lane B shows the 685R ⁇ 685A mutant S-trimer of Omicron S1/S2 furin cleavage site.
  • Lane C shows the cleavage site 685R ⁇ 685A of Omicron S1/S2 furin and the S-trimer of proline 986K/987V ⁇ 986P/987P mutant.
  • Lane D is Hu-1 S-trimer. The Omicron S-trimer S1 was cleaved to a greater extent than the Hu-S-trimer (compare lanes A and D).
  • Figures 3A-3B show the receptor binding kinetics of exemplary Omicron S-trimers to ACE2-Fc.
  • Figure 3A shows that Hu-1 S-trimer binds to the receptor with a Kd of 5 nM.
  • Figure 4B shows that Omicron S-trimer binds to the receptor with a Kd of 2 pM.
  • the Omicron S-trimer has a higher receptor affinity than the Hu-1 S-trimer.
  • bivalent vaccines include Hu-1 S-trimer and Omicron S-trimer ).
  • Figures 5A-5B show the SARS-CoV-2 Hu-1, Alpha ( alpha, ⁇ ,B.1.1.7), beta (beta, ⁇ ,B.1.351), gamma (gamma, ⁇ ,P.1), delta (delta, ⁇ ,B.1.617.2), and o IC 50 data of neutralizing antibodies against Omicron (Omicron,o,B.1.1.529) strain pseudovirus.
  • Omicron,o,B.1.1.529 strain pseudovirus.
  • Figure 5A shows the homologous enhancement group of Hu-1 S-trimer
  • Figure 5B shows the homology enhancement group of Omicron S-trimer
  • Figure 5C shows Hu-1 S-trimer as the initial agent and Omi Heterologous boosting group with Cron S-trimer as the enhancer
  • Figure 5D shows the heterologous boosting group with Omicron S-trimer as the initiator and Hu-1 S-trimer as the enhancer
  • Figure 5D 5E is a bivalent vaccine including Hu-1 S-trimer and Omicron S-trimer as both the initial dose and the booster dose.
  • Hu-1 S-trimer as the priming agent and Omicron S-trimer as the booster produced generally higher neutralizing antibody titers than Omicron S- trimer as The heterologous booster group (Fig. 5D) with the initiator and Hu-1 S-trimer as booster was good.
  • Using the bivalent vaccine as the initial dose and booster dose as shown in Figure 5E, can both increase the neutralizing antibody titer against the Omicron strain pseudovirus (compared with two doses of Hu-1 S-trimer immunization ), and can maintain or increase neutralizing antibody titers against other strains (compared to two doses of Hu-1S-trimer immunization).
  • Figures 6A-6B show VOC neutralizing antibodies in mice boosted with the 3rd dose of Omicron S-trimer vaccination.
  • Figure 6A is a schematic diagram of three immunizations and pseudovirus neutralizing antibody testing.
  • BALB/c mice were immunized twice on day 0 (dose 1) and day 21 (dose 2) with 3 ⁇ g of Hu-1 S-trimer adjuvanted with 150 ⁇ g of CpG 1018 plus 75 ⁇ g of Alum.
  • the limit of detection (LOD) titer is 20.
  • the group GMP number is marked at the bottom of each column. Mice that received the third dose of the Omicron S-trimer vaccine had significantly increased neutralizing antibody titers against the Omicron strain pseudovirus compared with the data before the third dose of vaccination.
  • Figures 7A-7G show VOC neutralizing antibodies in mice boosted with different dose 3 vaccines.
  • Figure 7A is a schematic diagram of three immunizations and pseudovirus neutralizing antibody testing.
  • BALB/c mice were immunized twice on day 0 (dose 1) and day 21 (dose 2) with 3 ⁇ g of Hu-1 S-trimer adjuvanted with 150 ⁇ g of CpG 1018 plus 75 ⁇ g of Alum.
  • dose 1 dose 1
  • dose 2 day 21
  • Sera were collected on study day 56 (D-1PD3) and day 71 (D14PD3) for pseudovirus neutralizing antibody testing.
  • Group 1 did not receive the third dose of vaccination and served as the control group;
  • Group 2 3 ⁇ g Hu-1 S-trimer with 150 ⁇ g CpG 1018 plus 75 ⁇ g Alum as adjuvant was used as the third dose of vaccine;
  • Group 3 3 ⁇ g Omicron S-trimer with 150 ⁇ g CpG 1018 plus 75 ⁇ g Alum as adjuvant as the third dose of vaccination;
  • Group 4 Bivalent vaccine with 150 ⁇ g CpG 1018 plus 75 ⁇ g Alum as adjuvant ( Includes 1.5 ⁇ g Hu-1 S-trimer and 1.5 ⁇ g Omicron S-trimer) as the 3rd dose of vaccination.
  • Figures 7B-7G show Hu-1, Alpha, Beta, Gamma, Delta, and Omicron strain pseudovirus neutralizing antibody (IC 50 ) data respectively.
  • Dots represent data from individual animals; horizontal lines represent geometric mean titers (GMT) ⁇ SEM for each group.
  • the limit of detection (LOD) titer is 20.
  • the group number is marked at the bottom of each column.
  • the fold increase in neutralizing antibody titers after dose 3 (D14PD3) for each VOC compared to data before dose 3 is marked as an "x".
  • FIGS 8A-8C Hu-1 S-trimer (ancestral) vaccine, Omicron S-trimer vaccine and bivalent vaccine (Hu-1 S-trimer + Omicron S-trimer ) as the third dose in mice that have been primed (1st dose)/boosted (2nd dose) with Hu-1S-trimer and boosted again (3rd dose) to induce persistence
  • Hu-1 S-trimer equal to 50%
  • Omicron S-trimer vaccine bivalent vaccine
  • Figure 8B Day 85 serum samples studied for neutralizing antibodies to VOCs analyzed by PsV neutralization assay.
  • Data points represent pseudovirus neutralizing antibody titers (IC50) for individual animals; horizontal bars represent geometric mean titers (GMT) ⁇ 95% CI for each group.
  • the limit of detection (LOD) titer (IC50) is 20. The number marked in each bar is the GMT for each test group.
  • Figure 8C Kinetic analysis of 6 pseudovirus neutralizing antibodies on sera from D0, D35, D56, D85 (1M after 3rd dose), D113 (2M after 3rd dose) and D141 (3M after 3rd dose) curve.
  • Light yellow bars/boxes represent samples from control mice that did not receive further immunization; gray bars/boxes represent samples from Hu-1S-trimer immunized mice; green bars/boxes represent samples from Omicron S-trimer Samples from aggregate-immunized mice. Pink bars/boxes represent samples from mice immunized with the bivalent vaccine. For statistical analysis, comparisons were made using a two-tailed Mann-Whitney t test. P values ⁇ 0.05 were considered significant. *: P ⁇ 0.05, **: P ⁇ 0.01, ***: P ⁇ 0.001.
  • Figure 10 shows mice receiving two doses (first dose prime - second dose boost) of Omicron strain (BA.5) S-trimer and two doses containing the same according to the experimental design of Figure 9
  • the trivalent vaccine is immunized against SARS-CoV-2 Hu-1, Alpha, Beta, Delta and Omicron BA.1, BA.2, BA.5, BA.2.75, BF.7, BQ.1.1, XBB strain pseudovirus neutralizing antibody IC 50 number.
  • mice were bled on day 35 (D4PD2) for pseudovirus neutralizing antibody testing.
  • A S-trimer of Omicron strain (BA.5); B: Hu-1 S-trimer + S-trimer of Omicron strain (BA.5), C: Omicron Micron S-trimer vaccine + Omicron strain (BA.5) S-trimer; D: Belta S-trimer + Delta-S-trimer + Omicron strain (BA.5)S-trimer.
  • compositions, methods and uses of fusion peptides and proteins comprising coronavirus antigens or immunogens for the treatment (e.g., prophylactic, therapeutic) of coronavirus infections.
  • compositions and methods of using recombinant soluble surface antigens from covalently linked trimeric forms of RNA viruses are disclosed.
  • the resulting fusion protein is secreted as a disulfide-linked homotrimer, which is more structurally stable while retaining a conformation similar to that of native trimeric viral antigens, and thus can be used as a more advanced therapeutic agent against these dangerous pathogens. Effective vaccines.
  • disclosed herein are methods of preventing viral infection using viral antigen trimers as a vaccine or as part of a multivalent vaccine, without or with an adjuvant, or with more than one adjuvant, optionally by Intramuscular injection or intranasal administration.
  • Viral antigen trimers can be used in priming doses, additional doses, and/or boosting doses. Independently, the initial agent, additional agent, and/or any one or more boosters may be administered without or with an adjuvant.
  • adjuvants may include: aluminum-containing adjuvants, such as alum and/or aluminum hydroxide-containing adjuvants; oligonucleotide-containing adjuvants, such as CpG-containing oligodeoxynucleotides (CpG- ODN); adjuvants containing TLR9 agonists; adjuvants containing metabolizable oils, alpha-tocopherol, and/or polyoxyethylene sorbitan monooleate (Tween-80), e.g., in water Adjuvant containing squalene, alpha-tocopherol, and Tween-80 and/or Span 85 in the form of an oil emulsion; or any combination of adjuvants.
  • aluminum-containing adjuvants such as alum and/or aluminum hydroxide-containing adjuvants
  • oligonucleotide-containing adjuvants such as CpG-containing oligodeoxynucleotides (CpG
  • disclosed herein are methods of using viral antigen trimers as antigens for diagnosing viral infections by detecting antibodies (eg, IgM or IgG) that recognize viral antigens (eg, neutralizing antibodies).
  • antibodies eg, IgM or IgG
  • viral antigens eg, neutralizing antibodies
  • disclosed herein are methods of using viral antigen trimers as antigens to generate polyclonal or monoclonal antibodies useful for passive immunization (eg, neutralizing mAbs for treating coronavirus infections).
  • viral antigen trimers are disclosed herein as a vaccine or as part of a multivalent vaccine, wherein the vaccine includes multiple trimer subunit vaccines that include the same protein of the virus A viral antigen, or a viral antigen that includes two or more different proteins of one or more viruses or a viral antigen of one or more strains of the same virus.
  • disclosed herein are monovalent vaccines comprising trimers of viral antigens disclosed herein. In some embodiments, disclosed herein are bivalent vaccines comprising trimers of viral antigens disclosed herein. In some embodiments, disclosed herein are trivalent vaccines comprising trimers of viral antigens disclosed herein. In some embodiments, disclosed herein are quadrivalent vaccines comprising trimers of viral antigens disclosed herein.
  • disclosed herein are monovalent vaccines comprising an S-trimer disclosed herein.
  • bivalent vaccines comprising an S-trimer disclosed herein.
  • bivalent vaccines comprising at least one S-trimer comprising a first S protein antigen and at least one S-trimer comprising a second S protein antigen.
  • the first and second S protein antigens are from the same S protein from one or more viral species or strains/subtypes, or from one or more viral species or from the same S protein species. Two or more different S proteins for one or more strains/subtypes.
  • disclosed herein are trivalent vaccines comprising an S-trimer disclosed herein.
  • a method 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.
  • - Trivalent vaccine of trimers are from the same S protein of one or more viral species or strains/subtypes, or are from one or more viral species or the same S protein Two, three or more different S proteins for one or more strains/subtypes of the species.
  • disclosed herein are quadrivalent vaccines comprising an S-trimer disclosed herein.
  • a method 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.
  • the first, second, third and fourth S protein antigens are from the same S protein of one or more viral species or strains/subtypes, or from one or more viral species or two, three, four or more different S proteins of one or more strains/subtypes of the same virus species.
  • a multivalent vaccine such as a bivalent vaccine, comprising: a first trimeric fusion protein comprising a soluble coronavirus SARS-CoV-2 Omic Rong (B.1.1.529) 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 connected to the C-terminal portion of collagen, The C-terminal portion of the collagen forms a disulfide-linked trimer, thereby forming the first trimer fusion protein; the second and/or more trimer fusion proteins, the second or more trimers
  • the fusion protein includes a soluble coronavirus SARS-CoV-2 Hu-1, alpha, beta, gamma, delta, or spike (S) protein surface antigen or a fragment, variant or mutant thereof, the soluble coronavirus surface The antigen or fragment, variant or mutant thereof is attached to a C-terminal portion of collagen that forms a disulfide-linked trim
  • the second and/or trimeric fusion protein includes a soluble coronavirus SARS-CoV-2 Hu-1 spike (S) protein surface antigen or a fragment, variant or mutant thereof.
  • the multivalent vaccine is administered without an adjuvant, optionally as a primary series, an additional dose, and/or a homologous or heterologous booster A booster dose is used, such as a first dose, a second dose, a third dose, a fourth dose, and/or more doses, optionally with the initial dose, additional dose, or heterologous booster dose with other recombinant sub-doses.
  • the multivalent vaccine is administered with more than one adjuvant, optionally as a primary series, an additional dose, and/or a homologous or heterologous booster
  • a booster dose is used, such as a first dose, a second dose, a third dose, a fourth dose, and/or more doses, optionally with the initial dose, additional dose, or heterologous booster dose with other recombinant sub-doses.
  • any one or more of unit vaccines, nanoparticle vaccines, mRNA vaccines, DNA vaccines, adenovirus vector vaccines, and inactivated virus vaccines are used in combination, optionally the initial dose, additional doses, and/or homologous or Adjuvants in the heterologous booster may independently include: aluminum-containing adjuvants, such as alum and/or aluminum hydroxide-containing adjuvants; oligonucleotide-containing adjuvants, such as CpG-containing oligodeoxynucleotides (CpG -ODN); adjuvants containing TLR9 agonists; adjuvants containing metabolizable oils, alpha-tocopherol, and/or polyoxyethylene sorbitan monooleate (Tween-80), such as water An adjuvant containing squalene, alpha-tocopherol, and Tween-80 and/or Span 85 in the form of an oil-in-oil emulsion; or
  • the first trimeric fusion protein in the multivalent vaccine includes the sequence set forth in any one of SEQ ID NOs: 81-84 or is consistent with the sequence set forth in any one of SEQ ID NOs: 81-84 The sequence has at least or about 80%, 85%, 90%, 92%, 95%, 97%, 99% sequence identity to the amino acid sequence.
  • the second and/or more trimeric fusion proteins in the multivalent vaccine comprise the sequence set forth in any one of SEQ ID NOs: 27-66 or are identical to those in SEQ ID NOs: 27-66 Any of the sequences described has at least or about 80%, 85%, 90%, 92%, 95%, 97%, 99% sequence identity to the amino acid sequence.
  • the proteins including coronavirus antigens and immunogens provided herein can be used to effectively and safely treat (e.g., therapeutic, prophylactic) coronavirus infections.
  • proteins comprising coronavirus antigens and immunogens provided herein treat coronavirus infections without mediated vaccine-induced enhancement of disease (VED) and/or antibody-dependent enhancement (ADE).
  • VED vaccine-induced enhancement of disease
  • AD antibody-dependent enhancement
  • the proteins containing coronavirus antigens and immunogens provided herein are easy to produce and perform well under conditions such as high temperatures, extreme pH, high and low osmotic pressures, etc. Demonstrates stability under high stress conditions. Accordingly, the proteins and immunogenic compositions provided herein circumvent and satisfy the production, stability, safety, and efficacy issues that have hindered coronavirus vaccine development.
  • coronavirus antigens and immunogens provided herein include coronavirus spike (S) proteins or peptides, particularly SARS-CoV or SARS-CoV-2 S proteins.
  • S coronavirus spike
  • SARS-CoV and SARS-CoV-2 S proteins 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 hemagglutination protein (HA), paramyxovirus F and Ebola virus glycoprotein.
  • the SARS-CoV and SARS-CoV-2 S proteins each encode surface glycoprotein precursors, and the amino terminus and most of the protein are predicted to be located on the cell surface or on the outside of the virus particle.
  • the S protein includes a signal peptide located at the N terminus, an extracellular domain, a transmembrane domain and an intracellular domain. 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 that is lacking in other SARS-like CoVs.
  • the recombinant S ectodomain trimer includes a recombinant S ectodomain protomer from an alpha-coronavirus (eg, NL63-CoV or 229E-CoV).
  • the recombinant S ectodomain trimers include those from beta-coronavirus (e.g., OC43-CoV, SARS-CoV, SARS-CoV-2, MERS-CoV, HKU1-CoV, WIV1-CoV, mouse hepatitis Virus (MHV) or HKU9-CoV) S ectodomain protomer.
  • beta-coronavirus e.g., OC43-CoV, SARS-CoV, SARS-CoV-2, MERS-CoV, HKU1-CoV, WIV1-CoV, mouse hepatitis Virus (MHV) or HKU9-CoV
  • coronaviruses including SARS-CoV-2 have trimeric surface antigens on their viral envelopes to target specific cell surface receptors during infection. enter different host cells.
  • SARS-CoV-1 SARS-CoV-2 utilizes 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.
  • a prerequisite for the generation of effective recombinant subunit vaccines is the ability to produce viral S antigens similar to those of the native virus, specifically maintaining its trimer 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, thus eliminating the viral infection.
  • RBD receptor binding domain
  • a protein comprising a coronavirus antigen or immunogen is capable of generating an immune response, e.g., to SARS-CoV or SARS-CoV-2 S protein Immune responses to peptides.
  • the immune response inhibits or reduces replication of the coronavirus in a subject (eg, a patient).
  • the immune response includes the production of one or more neutralizing antibodies, such as polyclonal and/or monoclonal antibodies.
  • neutralizing antibodies inhibit or reduce replication of coronavirus in a subject (eg, a patient).
  • administration of the protein to a subject does not result in antibody-dependent enhancement (ADE) in the subject due to prior exposure to coronavirus.
  • ADE antibody-dependent enhancement
  • proteins containing coronavirus antigens and immunogens are used as vaccines.
  • coronavirus antigens and immunogens are linked to proteins or peptides to form fusion proteins or recombinant polypeptides.
  • the protein or peptide linked to the coronavirus antigen or immunogen is capable of being conjugated, eg, covalently or non-covalently linked, to the protein or peptide (eg, a protein or peptide of a fusion protein or recombinant polypeptide). Therefore, in some cases, the protein or peptide linked to the coronavirus antigen or immunogen is a multimeric domain.
  • coronavirus antigens and immunogens are linked to the propeptide of collagen (eg, at the C-terminus of the propeptide of collagen) to form a fusion peptide or recombinant polypeptide.
  • proteins provided herein include recombinant polypeptides containing coronavirus antigens and immunogens (eg, coronavirus S protein peptides or fragments or epitopes thereof) linked to the C-terminal propeptide of collagen.
  • the propeptide of collagen is derived from the human C-propeptide of ⁇ 1 collagen and is capable of self-trimerization after expression.
  • linking coronavirus antigens and immunogens eg, coronavirus S protein peptide
  • immunogens eg, coronavirus S protein peptide
  • the propeptide of collagen eg, at the C-terminus of the propeptide of collagen
  • the production of recombinant proteins can preserve 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, while the availability of antigenic sites on the protein surface can in turn Trigger an immune response, such as neutralizing antibodies.
  • 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.
  • linking the coronavirus S protein peptide to the C-terminal propeptide of collagen results in a self-trimerizing recombinant polypeptide.
  • the proteins provided herein include multiple self-trimerized propeptides of coronavirus S protein peptides and collagen recombinant polypeptides.
  • the trimeric nature of the recombinant protein contributes to protein stability.
  • the trimeric nature of the recombinant protein contributes to the protein's ability to generate an immune response.
  • the trimeric nature of the recombinant protein and/or the macrostructure of the plurality of self-trimerized recombinant proteins contributes to the protein's ability to generate an immune response.
  • immunogenic compositions comprising proteins provided herein, methods of producing the proteins provided herein, methods of treating a subject with the proteins and compositions provided herein, and kits.
  • Proteins provided herein include coronavirus antigens and immunogens. Coronavirus antigens and immunogens contemplated herein are capable of promoting or stimulating cell-mediated responses and/or humoral responses. In some embodiments, the response (eg, cell-mediated or humoral response) includes the production of antibodies (eg, neutralizing antibodies). In some embodiments, the coronavirus antigen or immunogen is a coronavirus spike protein peptide.
  • Coronaviruses are a family of positive-sense single-stranded RNA viruses known to cause severe respiratory disease. They possess the largest genomes (26-32 kb) of known RNA viruses and are phylogenetically divided into four genera ( ⁇ , ⁇ , ⁇ , ⁇ ), of which ⁇ -coronaviruses are further subdivided into four lineages (A, B, C, D).
  • the viruses currently known to infect humans in the coronavirus family come from the alpha-coronavirus and beta-coronavirus genera. Additionally, it is believed that gamma- and delta-coronavirus genera may infect humans in the future.
  • beta-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).
  • MERS-CoV Middle East respiratory syndrome coronavirus
  • SARS-CoV severe acute respiratory syndrome coronavirus
  • HKU1-CoV human coronavirus HKU1
  • OC43-CoV human coronavirus OC43
  • MHV murine hepatitis virus
  • WIV1-CoV bat SARS-like coronavirus WIV1
  • HKU9-CoV human coronavirus HKU9
  • 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 swine delta-coronavirus (SDCV).
  • This article discloses a list of severe acute respiratory syndrome-related coronaviruses:
  • Taiwan TC1 SARS coronavirus Taiwan TC1
  • Taiwan TC2 SARS coronavirus Taiwan TC2
  • Taiwan TC3 SARS coronavirus Taiwan TC3
  • coronavirus genome is capped, polyadenylated, and covered with nucleocapsid proteins.
  • Coronavirus particles include a viral envelope containing a type I fusion glycoprotein called the spike (S) protein.
  • S spike
  • Most coronaviruses have a common genome structure in which the replicase gene is contained in the 5′ part of the genome and the structural genes are contained in the 3′ part of the genome.
  • 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 in the Golgi to remove the signal peptide and cleaved by cellular proteases to produce separate S1 and S2 polypeptide chains, which remain in the homotrimer Binds as an S1/S2 protomer and is therefore a trimer of heterodimers.
  • the S1 subunit is located at the distal end of the viral membrane and contains a receptor-binding domain (RBD) that mediates attachment of the virus to its host receptor.
  • RBD receptor-binding domain
  • the S2 subunit contains the fusion protein machinery, such as the fusion peptide, the two heptapeptide repeats (HR1 and HR2) unique to the fusion glycoprotein, and the central helix, transmembrane domain, and cytosolic tail domain. .
  • the coronavirus antigen or immunogen is a coronavirus S protein peptide in a prefusion conformation, which is the extracellular domain of the coronavirus S protein that is processed into the mature coronavirus S protein in the secretion system and triggers the formation of the coronavirus S protein.
  • the three-dimensional structure of an exemplary coronavirus spike protein (HKU1-CoV) in a pre-fusion conformation is provided in Kirchdoerfer et al., "Pre-fusion structure of a human coronavirus spike protein," Nature, 531:118-121, 2016, Incorporated by reference in its entirety for all purposes.
  • the coronavirus antigen or immunogen includes one or more amino acid substitutions, deletions, or insertions compared to the native coronavirus S sequence, compared to the coronavirus S ectodomain formed by the corresponding native coronavirus S sequence. It provides increased prefusion conformational retention compared to polymers. "Stabilization" of a prefusion conformation by one or more amino acid substitutions, deletions, or insertions can be, for example, energetic stabilization (e.g., reducing the energy of the prefusion conformation relative to the open postfusion conformation) and/or kinetic stabilization ( For example, reduce the conversion rate from the prefusion conformation to the postfusion conformation).
  • energetic stabilization e.g., reducing the energy of the prefusion conformation relative to the open postfusion conformation
  • kinetic stabilization For example, reduce the conversion rate from the prefusion conformation to the postfusion conformation.
  • stabilization of the coronavirus S ectodomain trimer in the prefusion conformation may include increased resistance to denaturation compared to the corresponding native coronavirus S sequence.
  • This article provides methods to determine whether the coronavirus S ectodomain trimer is in a prefusion conformation, including (but not limited to) negative stain electron microscopy and antibody binding assays using prefusion conformation-specific antibodies.
  • the coronavirus antigen or immunogen is a fragment of the S protein peptide.
  • the antigen or immunogen is an epitope of a S protein peptide.
  • Epitopes include antigenic determinant chemical groups or peptide sequences on a molecule that are antigenic and thereby elicit a specific immune response.
  • an epitope is an antigenic region to which B cells and/or T cells respond.
  • Antibodies can bind to specific epitopes, such as those on the S extracellular domain of the coronavirus.
  • Epitopes can be formed either from contiguous amino acids or from non-contiguous amino acids juxtaposed by the tertiary folding of the protein.
  • the coronavirus epitope is a linear epitope. In some embodiments, the coronavirus epitope is a conformational epitope. In some embodiments, the coronavirus epitope is a neutralizing epitope site. In some embodiments, all neutralizing epitopes of the coronavirus S protein peptide or fragment thereof are present as antigens or immunogens.
  • the viral antigen or immunogen when the viral antigen or immunogen is a fragment of the spike protein peptide, only a single subunit of the spike protein peptide is present, and the single subunit of the spike protein peptide is trimerized.
  • the viral antigen or immunogen includes a signal peptide, an S1 subunit peptide, an S2 subunit peptide, or any combination thereof.
  • 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 heptad repeat 2 (HR2) peptide or any combination thereof.
  • the viral antigen or immunogen includes the receptor binding domain (RBD) of the S protein.
  • the viral antigen or immunogen includes the S1 and S2 subunits of the S protein.
  • the viral antigen or immunogen includes the S1 subunit of the S protein, but does not include the S2 subunit.
  • 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 transmembrane (TM) domain peptides and/or cytoplasmic (CP) domain peptides.
  • TM transmembrane
  • CP cytoplasmic
  • the viral antigen or immunogen includes a protease cleavage site, wherein the protease is optionally furin, trypsin, factor Xa, or cathepsin L.
  • the viral antigen or immunogen does not contain a protease cleavage site, wherein the protease is optionally furin, trypsin, factor Xa, or cathepsin L, or contains a mutation that is not cleavable by a protease Protease cleavage site.
  • the viral antigen or immunogen is a SARS-CoV-2 antigen comprising at least one SARS-CoV-2 protein or fragment thereof.
  • the SARS-CoV-2 antigen is recognized by SARS-CoV-2 reactive antibodies and/or T cells.
  • the SARS-CoV-2 antigen is inactivated whole virus.
  • SARS-CoV-2 antigens include subunits of the virus.
  • SARS- CoV-2 antigens include structural proteins of SARS-CoV-2 or fragments thereof.
  • the structural proteins of SARS-CoV-2 include one from the group consisting of spike (S) protein, membrane (M) protein, nucleocapsid (N) protein, and envelope (E) protein or more.
  • the SARS-CoV-2 antigen includes or further includes 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 as GenBank number MN908947.3 (Wu et al., Nature, 579:265-269, 2020) and for all purposes Incorporated by reference in its entirety.
  • the viral antigen or immunogen includes the sequence set forth in any one of SEQ ID NOs: 81-84. In some embodiments, the viral antigen or immunogen includes at least or about 80%, 85%, 90%, 92%, 95%, 97%, or 99% sequence identity to SEQ ID NO:82 shown below sexual amino acid sequence. In some embodiments, the viral antigen or immunogen includes an RBD trimer, e.g., a SARS-CoV-2 RBD sequence linked to any one of SEQ ID Nos: 67-80.
  • RBD trimer e.g., a SARS-CoV-2 RBD sequence linked to any one of SEQ ID Nos: 67-80.
  • the viral antigen or immunogen includes the sequence set forth in SEQ ID NO:55.
  • the viral antigen or immunogen includes a receptor binding motif within the receptor binding domain (RBD) of SEQ ID NO: 55 shown below (the underlined sequence indicates Thr333-Gly526 (bold) RBM)) has at least or an amino acid sequence that has about 80%, 85%, 90%, 92%, 95% or 97% sequence identity.
  • the viral antigen or immunogen includes an RBD trimer, e.g., a SARS-CoV-2 RBD sequence linked to any one of SEQ ID Nos: 67-80.
  • the viral antigen or immunogen includes the spike glycoprotein sequence of Hu-1 coronavirus (eg, NC_045512). In some embodiments, the viral antigen or immunogen includes the spike glycoprotein sequence of a virus in the B.1.526 lineage. In some embodiments, the viral antigen or immunogen includes the spike glycoprotein sequence of Cluster 5 ( ⁇ FVI-Spike) virus. In some embodiments, the viral antigen or immunogen includes the spike glycoprotein sequence of a virus in the B.1.1.529 lineage. In some embodiments, the viral antigen or immunogen includes the spike glycoprotein sequence of a virus in the B.1.1.7 lineage.
  • Hu-1 coronavirus eg, NC_045512
  • the viral antigen or immunogen includes the spike glycoprotein sequence of a virus in the B.1.526 lineage. In some embodiments, the viral antigen or immunogen includes the spike glycoprotein sequence of Cluster 5 ( ⁇ FVI-Spike) virus. In some embodiments, the viral antigen or immunogen includes the spike glycoprotein sequence of a
  • the viral antigen or immunogen includes the spike glycoprotein sequence of a virus in the B.1.1.207 lineage. In some embodiments, the viral antigen or immunogen includes the spike glycoprotein sequence of a virus in the B.1.1.317 lineage. In some embodiments, the viral antigen or immunogen includes the spike glycoprotein sequence of a virus in the B.1.1.318 lineage. In some embodiments, the viral antigen or immunogen includes the spike glycoprotein sequence of a virus in the P.1 lineage. In some embodiments, the viral antigen Or the immunogen includes the spike glycoprotein sequence of a virus in the B.1.351 lineage.
  • the viral antigen or immunogen includes the spike glycoprotein sequence of a virus in the B.1.429/CAL.20C lineage. In some embodiments, the viral antigen or immunogen includes the spike glycoprotein sequence of a virus in the B.1.525 lineage. In some embodiments, the viral antigen or immunogen includes the spike glycoprotein sequence of a virus in the B.1.526 lineage. In some embodiments, the viral antigen or immunogen includes the spike glycoprotein sequence of a virus in the B.1.617 lineage. In some embodiments, the viral antigen or immunogen includes the spike glycoprotein sequence of a virus in the B.1.617.2 lineage. In some embodiments, the viral antigen or immunogen includes the spike glycoprotein sequence of a virus in the B.1.618 lineage.
  • the viral antigen or immunogen includes the spike glycoprotein sequence of a virus in the B.1.620 lineage. In some embodiments, the viral antigen or immunogen includes the spike glycoprotein sequence of a virus in the P.2 lineage. In some embodiments, the viral antigen or immunogen includes the spike glycoprotein sequence of a virus in the P.3 lineage. In some embodiments, the viral antigen or immunogen includes the spike glycoprotein sequence of a virus in the B.1.1.143 lineage. In some embodiments, the viral antigen or immunogen includes the spike glycoprotein sequence of a virus in the A.23.1 lineage. In some embodiments, the viral antigen or immunogen includes the spike glycoprotein sequence of a virus in the B.1.617 lineage.
  • the viral antigen or immunogen includes a virus derived from a virus selected from the group consisting of Wuhan-Hu-1, a virus in the B.1.526 lineage, a virus in the B.1.1.7 lineage, a virus in the P.1 lineage, B. Any two or more of the group consisting of viruses of lineage 1.351, viruses of lineage P.2, viruses of lineage B.1.1.143, viruses of lineage A.23.1 and viruses of lineage B.1.617 Sequences of the virus's spike glycoprotein (in any suitable combination).
  • viral antigens or immunogens include T95I, G142D, ⁇ 143-145, and/or T478K, for example, as in B.1.1.529 Omicron variant.
  • the viral antigen or immunogen includes E484K and/or S477N, for example, as in the B.1.526 variant.
  • the viral antigen or immunogen includes ⁇ 400-402 ( ⁇ FVI), for example, as in the Cluster 5 ( ⁇ FVI-Spike) variant.
  • viral antigens or immunogens include ⁇ 69-70 ( ⁇ HV), ⁇ 144 ( ⁇ Y), N501Y, A570D, D614G, P681H, T716I, S982A, and/or D1118H, for example, as in the B.1.1.7 variant middle.
  • the viral antigen or immunogen includes P681H, for example, as in the B.1.1.207 variant.
  • 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.
  • the viral antigen or immunogen includes E484K, for example, as in the P.2 variant.
  • the viral antigen or immunogen includes E484K and/or N501Y, for example, as in the P.3 variant.
  • viral antigens or immunogens include L18F, D80A, D215G, ⁇ 242-244 ( ⁇ LAL), R246I, K417N, E484K, N501Y, D614G, and/or A701V, for example, as in the B.1.351 variant.
  • the viral antigen or immunogen includes S13I, W152C and/or L452R, for example, as in the B.1.429/CAL.20C variant.
  • the viral antigen or immunogen includes ⁇ 69-70 ( ⁇ HV), E484K, and/or F888L, for example, as in the B.1.525 variant.
  • the viral antigen or immunogen includes 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 In the B.1.618 variant. In some embodiments, a viral antigen or immunogen can comprise a fusion polypeptide (protomer) comprising any one or more of the aforementioned mutations in any suitable combination.
  • a fusion polypeptide protomer
  • a viral antigen or immunogen can comprise a trimer of three fusion polypeptides, and any of the three protomeric fusion polypeptides can comprise any one or more of the foregoing in any suitable combination. mutation.
  • two or all three of the three protomeric fusion polypeptides forming a trimer may include different mutations and/or different combinations of mutations in each protomer.
  • a viral antigen or immunogen can include a mixture of trimers, and each trimer can include different mutations and/or different combinations of mutations.
  • the viral antigen or immunogen includes amino acid positions 13, 18, 20, 26, 69, 70, 80, 138, 142, 144, 152, 190, 215, 242, selected from SEQ ID NO: 55. 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, Any one, two, three, four, five or more mutations from the group consisting of mutations (eg, substitutions, deletions, and/or insertions) at 1118 and 1176.
  • mutations eg, substitutions, deletions, and/or insertions
  • the viral antigen or immunogen includes a protein 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 Any one, two, three, four, five, six, seven, eight or all mutations in the group.
  • viral antigens or immunogens include chimeric polypeptides comprising sequences from different viruses, such as one or more mutations from a first variant of a coronavirus and from a second variant of a coronavirus that is different from the first variant one or more mutations.
  • such chimeric viral antigens or immunogens can be used to elicit a broad immune response against the first and second variants of the coronavirus.
  • viral antigens or immunogens include selected from the group consisting of T95I, G142D, ⁇ 143-145, T478K, S13I, L18F, T20N, P26S, ⁇ 69-70 ( ⁇ HV), D80A, D138Y, G142D, ⁇ 144 ( ⁇ Y), W152C, R190S, D215G, ⁇ 242-244( ⁇ LAL), R246I, ⁇ 400-402( ⁇ FVI), K417T, K417N, N440K, L452R, S477N, S477G, E484K, E484Q, N501Y, A570D, D614G, H655 Y, P681H, P681R, Any one, two, three, four, five or more mutations in the group consisting of R682G, R683S, R685G, A701V, T716I, F888L, S982A, T1027I, D1118H and
  • the viral antigen or immunogen includes any one, two, or three selected from the group consisting of N440K, L452R, S477G, S477N, E484K, E484Q, N501Y, D614G, H655Y, P681H, P681R, and A701V , four, five or more mutations.
  • 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.
  • the SARS-CoV-2 antigen is a recombinant protein, while in other embodiments, the SARS-CoV-2 antigen is purified from virions. In some preferred embodiments, the SARS-CoV-2 antigen is an isolated antigen.
  • the viral antigen or immunogen includes the sequence set forth in SEQ ID NO:27. In some embodiments, the viral antigen or immunogen includes 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 of the amino acid sequence included in 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 the amino acid position of SEQ ID NO: 55) at one or more amino acid positions in the group consisting of substitutions, deletions and/or or inserted
  • the viral antigen or immunogen includes a variant of SEQ ID NO: 27, and the variant includes a variant selected from the group consisting of S13I, L18F, T20N, P26S, ⁇ 69-70 ( ⁇ HV), D80A, D138Y, G142D, ⁇ 144( ⁇ Y), W152C, R190S, D215G, ⁇ 242-244( ⁇ LAL), R246I, ⁇ 400-402( ⁇ FVI), K417T, K417N, N440K, L452R, S477N, S477G, E484K, E484Q, N501Y, A570D, D614G , H655Y, P681H , any one, two, three, four, five or more mutations in the group consisting of , P681R, R682G, R683S, R685G, A701V, T716I, F888L, S982A, T1027I, D1118H
  • the viral antigen or immunogen includes the sequence set forth in SEQ ID NO: 28. In some embodiments, the viral antigen or immunogen includes 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 of the amino acid sequence included in 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, Contains a substitution at one or more amino acid positions in the group consisting of 681, 682, 683, 684, 685, 701, 716, 888, 982, 1027, 1118 and 1176 (with respect to the amino acid position of SEQ ID NO: 28.
  • the viral antigen or immunogen includes a variant of SEQ ID NO: 28, and the variant includes a variant selected from the group consisting of S13I, L18F, T20N, P26S, ⁇ 69-70 ( ⁇ HV), D80A, D138Y, G142D, ⁇ 144( ⁇ Y), W152C, R190S, D215G, ⁇ 242-244( ⁇ LAL), R246I, ⁇ 400-402( ⁇ FVI), K417T, K417N, N440K, L452R, S477N, S477G, E484K, E484Q, N501Y, A570D, D614G , H655Y, P681H , any one, two, three, four, five or more mutations in the group consisting of , P681R, R682G, R683S, R685G, A701V, T716I, F888L, S982A, T1027I, D1118H
  • the viral antigen or immunogen includes the sequence set forth in SEQ ID NO:29. In some embodiments, the viral antigen or immunogen includes 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 of the amino acid sequence included in 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, Comprises a substitution at one or more amino acid positions in the group consisting of 681, 682, 683, 684, 685, 701, 716, 888, 982, 1027, 1118 and 1176 (with respect to the amino acid position of SEQ ID NO:29
  • the viral antigen or immunogen includes a variant of SEQ ID NO: 29, and the variant includes a variant selected from the group consisting of S13I, L18F, T20N, P26S, ⁇ 69-70 ( ⁇ HV), D80A, D138Y, G142D, ⁇ 144( ⁇ Y), W152C, R190S, D215G, ⁇ 242-244( ⁇ LAL), R246I, ⁇ 400-402( ⁇ FVI), K417T, K417N, N440K, L452R, S477N, S477G, E484K, E484Q, N501Y, A570D, D614G , H655Y, P681H ,P681R,R682G, Any one, two, three, four, five or more mutations in the group consisting of R683S, R685G, A701V, T716I, F888L, S982A, T1027I, D1118H and V
  • the viral antigen or immunogen includes the sequence set forth in SEQ ID NO: 30. In some embodiments, the viral antigen or immunogen includes 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 of the amino acid sequence included in 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, Contains a substitution at one or more amino acid positions in the group consisting of 681, 682, 683, 684, 685, 701, 716, 888, 982, 1027, 1118 and 1176 (with respect to the amino acid position of SEQ ID NO: 30.
  • the viral antigen or immunogen includes a variant of SEQ ID NO: 30, and the variant includes a variant selected from the group consisting of S13I, L18F, T20N, P26S, ⁇ 69-70 ( ⁇ HV), D80A, D138Y, G142D, ⁇ 144( ⁇ Y), W152C, R190S, D215G, ⁇ 242-244( ⁇ LAL), R246I, ⁇ 400-402( ⁇ FVI), K417T, K417N, N440K, L452R, S477N, S477G, E484K, E484Q, N501Y, A570D, D614G , H655Y, P681H , any one, two, three, four, five or more mutations in the group consisting of , P681R, R682G, R683S, R685G, A701V, T716I, F888L, S982A, T1027I, D1118H
  • the viral antigen or immunogen includes the sequence set forth in SEQ ID NO: 31. In some embodiments, the viral antigen or immunogen includes 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 of the amino acid sequence included in 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, Contains a substitution at one or more amino acid positions in the group consisting of 681, 682, 683, 684, 685, 701, 716, 888, 982, 1027, 1118 and 1176 (with respect to the amino acid position of SEQ ID NO: 31.
  • the viral antigen or immunogen includes a variant of SEQ ID NO: 31, and the variant includes a variant selected from the group consisting of S13I, L18F, T20N, P26S, ⁇ 69-70 ( ⁇ HV), D80A, D138Y, G142D, ⁇ 144( ⁇ Y), W152C, R190S, D215G, ⁇ 242-244( ⁇ LAL), R246I, ⁇ 400-402( ⁇ FVI), K417T, K417N, N440K, L452R, S477N, S477G, E484K, E484Q, N501Y, A570D, D614G , H655Y, P681H , any one, two, three, four, five or more mutations in the group consisting of , P681R, R682G, R683S, R685G, A701V, T716I, F888L, S982A, T1027I, D1118H
  • the viral antigen or immunogen includes the sequence set forth in SEQ ID NO:32. In some embodiments, the viral antigen or immunogen includes at least or about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89 Amino acid sequences with %, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity, including amino acid sequences 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 Sequences containing substitutions, deletions and/or insertions at one or more amino acid positions in the group consisting of , 888, 982, 1027, 1118 and
  • the viral antigen or immunogen includes a variant of SEQ ID NO: 32, and the variant includes a variant selected from the group consisting of S13I, L18F, T20N, P26S, ⁇ 69-70 ( ⁇ HV), D80A, D138Y, G142D, ⁇ 144( ⁇ Y), W152C, R190S, D215G, ⁇ 242-244( ⁇ LAL), R246I, ⁇ 400-402( ⁇ FVI), K417T, K417N, N440K, L452R, S477N, S477G, E484K, E484Q, N501Y, A570D, D614G , H655Y, P681H , any one, two, three, four, five or more mutations in the group consisting of , P681R, R682G, R683S, R685G, A701V, T716I, F888L, S982A, T1027I, D1118
  • the viral antigen or immunogen includes the sequence set forth in SEQ ID NO: 33. In some embodiments, the viral antigen or immunogen includes 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 of the amino acid sequence included in 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, Contains a substitution at one or more amino acid positions in the group consisting of 681, 682, 683, 684, 685, 701, 716, 888, 982, 1027, 1118 and 1176 (with respect to the amino acid position of SEQ ID NO: 33.
  • the viral antigen or immunogen includes a variant of SEQ ID NO: 33, and the variant includes a variant selected from the group consisting of S13I, L18F, T20N, P26S, ⁇ 69-70 ( ⁇ HV), D80A, D138Y, G142D, ⁇ 144( ⁇ Y), W152C, R190S, D215G, ⁇ 242-244( ⁇ LAL), R246I, ⁇ 400-402( ⁇ FVI), K417T, K417N, N440K, L452R, S477N, S477G, E484K, E484Q, N501Y, A570D, D614G , H655Y, P681H , any one, two, three, four, five or more mutations in the group consisting of , P681R, R682G, R683S, R685G, A701V, T716I, F888L, S982A, T1027I, D1118
  • the viral antigen or immunogen includes the sequence set forth in SEQ ID NO:34. In some embodiments, the viral antigen or immunogen includes at least or about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89 Amino acid sequences with %, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity, including amino acid sequences 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, Comprises a substitution at one or more amino acid positions in the group consisting of 681, 682, 683, 684, 685, 701, 716, 888, 982, 1027, 1118 and 1176 (with respect to the amino acid position of
  • the viral antigen or immunogen includes a variant of SEQ ID NO: 34, and the variant includes a variant selected from the group consisting of S13I, L18F, T20N, P26S, ⁇ 69-70 ( ⁇ HV), D80A, D138Y, G142D, ⁇ 144( ⁇ Y), W152C, R190S, D215G, ⁇ 242-244( ⁇ LAL), R246I, ⁇ 400-402( ⁇ FVI), K417T, K417N, N440K, L452R, S477N, S477G, E484K, E484Q, N501Y, A570D, D614G , H655Y, P681H , any one, two, three, four, five or more mutations in the group consisting of , P681R, R682G, R683S, R685G, A701V, T716I, F888L, S982A, T1027I, D1118
  • the viral antigen or immunogen includes the sequence set forth in SEQ ID NO: 35. In some embodiments, the viral antigen or immunogen includes 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 of the amino acid sequence included in 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, Contains a substitution at one or more amino acid positions in the group consisting of 681, 682, 683, 684, 685, 701, 716, 888, 982, 1027, 1118 and 1176 (with respect to the amino acid position of SEQ ID NO: 35.
  • the viral antigen or immunogen includes a variant of SEQ ID NO: 35, and the variant includes a variant selected from the group consisting of S13I, L18F, T20N, P26S, ⁇ 69-70 ( ⁇ HV), D80A, D138Y, G142D, ⁇ 144( ⁇ Y), W152C, R190S, D215G, ⁇ 242-244( ⁇ LAL), R246I, ⁇ 400-402( ⁇ FVI), K417T, K417N, N440K, L452R, S477N, S477G, E484K, E484Q, N501Y, A570D, D614G , H655Y, P681H , any one, two, three, four, five or more mutations in the group consisting of , P681R, R682G, R683S, R685G, A701V, T716I, F888L, S982A, T1027I, D1118
  • the viral antigen or immunogen includes the sequence set forth in SEQ ID NO: 36. In some embodiments, the viral antigen or immunogen includes 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 of the amino acid sequence included in 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, Contains a substitution at one or more amino acid positions in the group consisting of 681, 682, 683, 684, 685, 701, 716, 888, 982, 1027, 1118 and 1176 (with respect to the amino acid position of SEQ ID NO: 36.
  • the viral antigen or immunogen includes a variant of SEQ ID NO: 36, and the variant includes a variant selected from the group consisting of S13I, L18F, T20N, P26S, ⁇ 69-70 ( ⁇ HV), D80A, D138Y, G142D, ⁇ 144( ⁇ Y), W152C, R190S, D215G, ⁇ 242-244( ⁇ LAL), R246I, ⁇ 400-402( ⁇ FVI), K417T, K417N, N440K, L452R, S477N, S477G, E484K, E484Q, N501Y, A570D, D614G , H655Y, P681H , any one, two, three, four, five or more mutations in the group consisting of , P681R, R682G, R683S, R685G, A701V, T716I, F888L, S982A, T1027I, D1118
  • the viral antigen or immunogen includes the sequence set forth in SEQ ID NO:37. In some embodiments, the viral antigen or immunogen includes at least or about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89 Amino acid sequences with %, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity, including amino acid sequences 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, Comprises a substitution at one or more amino acid positions in the group consisting of 681, 682, 683, 684, 685, 701, 716, 888, 982, 1027, 1118 and 1176 (with respect to the amino acid position of
  • the disease Toxic antigens or immunogens include variants of SEQ ID NO: 37, and variants include selected from the group consisting of S13I, L18F, T20N, P26S, ⁇ 69-70 ( ⁇ HV), D80A, D138Y, G142D, ⁇ 144 ( ⁇ Y), W152C, R190S , D215G, ⁇ 242-244( ⁇ LAL), R246I, ⁇ 400-402( ⁇ FVI), K417T, K417N, N440K, L452R, S477N, S477G, E484K, E484Q, N501Y, A570D, D614G, H655Y, P681H, P6 81R, R682G, R683S , any one, two, three, four, five or more mutations in the group consisting of , R685G, A701V, T716I, F888L, S982A, T1027I, D11
  • the viral antigen or immunogen includes the sequence set forth in SEQ ID NO: 38. In some embodiments, the viral antigen or immunogen includes 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 of the amino acid sequence included in 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, Contains a substitution at one or more amino acid positions in the group consisting of 681, 682, 683, 684, 685, 701, 716, 888, 982, 1027, 1118 and 1176 (with respect to the amino acid position of SEQ ID NO: 38.
  • the viral antigen or immunogen includes a variant of SEQ ID NO: 38, and the variant includes a variant selected from the group consisting of S13I, L18F, T20N, P26S, ⁇ 69-70 ( ⁇ HV), D80A, D138Y, G142D, ⁇ 144( ⁇ Y), W152C, R190S, D215G, ⁇ 242-244( ⁇ LAL), R246I, ⁇ 400-402( ⁇ FVI), K417T, K417N, N440K, L452R, S477N, S477G, E484K, E484Q, N501Y, A570D, D614G , H655Y, P681H , any one, two, three, four, five or more mutations in the group consisting of , P681R, R682G, R683S, R685G, A701V, T716I, F888L, S982A, T1027I, D1118
  • the viral antigen or immunogen includes the sequence set forth in SEQ ID NO: 39. In some embodiments, the viral antigen or immunogen includes 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 of the amino acid sequence included in 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, Contains a substitution at one or more amino acid positions in the group consisting of 681, 682, 683, 684, 685, 701, 716, 888, 982, 1027, 1118 and 1176 (with respect to the amino acid position of SEQ ID NO: 39.
  • the viral antigen or immunogen includes a variant of SEQ ID NO: 39, and the variant includes a variant selected from the group consisting of S13I, L18F, T20N, P26S, ⁇ 69-70 ( ⁇ HV), D80A, D138Y, G142D, ⁇ 144( ⁇ Y), W152C, R190S, D215G, ⁇ 242-244( ⁇ LAL), R246I, ⁇ 400-402( ⁇ FVI), K417T, K417N, N440K, L452R, S477N, S477G, E484K, E484Q, N501Y, A570D, D614G , H655Y, P681H , any one, two, three, four, five or more mutations in the group consisting of , P681R, R682G, R683S, R685G, A701V, T716I, F888L, S982A, T1027I, D1118
  • the viral antigen or immunogen includes the sequence set forth in SEQ ID NO:40. In some embodiments, the viral antigen or immunogen includes 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 of the amino acid sequence included in the 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 (about SEQ ID Sequences containing substitutions, deletions and/or insertions at one or more amino acid positions
  • the viral antigen or immunogen includes a variant of SEQ ID NO: 40, and the variant includes a variant selected from the group consisting of S13I, L18F, T20N, P26S, ⁇ 69-70 ( ⁇ HV), D80A, D138Y, G142D, ⁇ 144( ⁇ Y), W152C, R190S, D215G, ⁇ 242-244( ⁇ LAL), R246I, ⁇ 400-402( ⁇ FVI), K417T, K417N, N440K, L452R, S477N, S477G, E484K, E484Q, N501Y, A570D, D614G , H655Y, P681H , any one, two, three, four, five or more mutations in the group consisting of , P681R, R682G, R683S, R685G, A701V, T716I, F888L, S982A, T1027I, D1118
  • the viral antigen or immunogen includes the sequence set forth in SEQ ID NO: 41. In some embodiments, the viral antigen or immunogen includes 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 of the amino acid sequence included in 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, Contains a substitution at one or more amino acid positions in the group consisting of 681, 682, 683, 684, 685, 701, 716, 888, 982, 1027, 1118 and 1176 (with respect to the amino acid position of SEQ ID NO: 41.
  • the viral antigen or immunogen includes a variant of SEQ ID NO: 41, and the variant includes a variant selected from the group consisting of S13I, L18F, T20N, P26S, ⁇ 69-70 ( ⁇ HV), D80A, D138Y, G142D, ⁇ 144( ⁇ Y), W152C, R190S, D215G, ⁇ 242-244( ⁇ LAL), R246I, ⁇ 400-402( ⁇ FVI), K417T, K417N, N440K, L452R, S477N, S477G, E484K, E484Q, N501Y, A570D, D614G , H655Y, P681H , any one, two, three, four, five or more mutations in the group consisting of , P681R, R682G, R683S, R685G, A701V, T716I, F888L, S982A, T1027I, D1118
  • the viral antigen or immunogen includes the sequence set forth in SEQ ID NO:42. In some embodiments, the viral antigen or immunogen includes 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 of the amino acid sequence included in 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, Comprises a substitution at one or more amino acid positions in the group consisting of 681, 682, 683, 684, 685, 701, 716, 888, 982, 1027, 1118 and 1176 (with respect to the amino acid position of SEQ ID NO:42
  • the viral antigen or immunogen includes a variant of SEQ ID NO: 42, and the variant includes a variant selected from the group consisting of S13I, L18F, T20N, P26S, ⁇ 69-70 ( ⁇ HV), D80A, D138Y, G142D, ⁇ 144( ⁇ Y), W152C, R190S, D215G, ⁇ 242-244( ⁇ LAL), R246I, ⁇ 400-402( ⁇ FVI), K417T, K417N, N440K, L452R, S477N, S477G, E484K, E484Q, N501Y, A570D, D614G, H655Y, P681H, P681R, R68 2G, R683S, R685G, Any one, two, three, four, five or more mutations in the group consisting of A701V, T716I, F888L, S982A, T1027I, D1118H and V11
  • the viral antigen or immunogen includes the sequence set forth in SEQ ID NO: 43. In some embodiments, the viral antigen or immunogen includes 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 of the amino acid sequence included in 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, Contains a substitution at one or more amino acid positions in the group consisting of 681, 682, 683, 684, 685, 701, 716, 888, 982, 1027, 1118 and 1176 (with respect to the amino acid position of SEQ ID NO: 43.
  • the viral antigen or immunogen includes a variant of SEQ ID NO: 43, and the variant includes a variant selected from the group consisting of S13I, L18F, T20N, P26S, ⁇ 69-70 ( ⁇ HV), D80A, D138Y, G142D, ⁇ 144( ⁇ Y), W152C, R190S, D215G, ⁇ 242-244( ⁇ LAL), R246I, ⁇ 400-402( ⁇ FVI), K417T, K417N, N440K, L452R, S477N, S477G, E484K, E484Q, N501Y, A570D, D614G , H655Y, P681H , any one, two, three, four, five or more mutations in the group consisting of , P681R, R682G, R683S, R685G, A701V, T716I, F888L, S982A, T1027I, D1118
  • the viral antigen or immunogen includes the sequence set forth in SEQ ID NO: 44. In some embodiments, the viral antigen or immunogen includes 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 of the amino acid sequence included in 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, Contains a substitution at one or more amino acid positions in the group consisting of 681, 682, 683, 684, 685, 701, 716, 888, 982, 1027, 1118 and 1176 (with respect to the amino acid position of SEQ ID NO: 44.
  • the viral antigen or immunogen includes a variant of SEQ ID NO: 44, and the variant includes a variant selected from the group consisting of S13I, L18F, T20N, P26S, ⁇ 69-70 ( ⁇ HV), D80A, D138Y, G142D, ⁇ 144( ⁇ Y), W152C, R190S, D215G, ⁇ 242-244( ⁇ LAL), R246I, ⁇ 400-402( ⁇ FVI), K417T, K417N, N440K, L452R, S477N, S477G, E484K, E484Q, N501Y, A570D, D614G , H655Y, P681H , any one, two, three, four, five or more mutations in the group consisting of , P681R, R682G, R683S, R685G, A701V, T716I, F888L, S982A, T1027I, D1118
  • the viral antigen or immunogen includes the sequence set forth in SEQ ID NO:45. In some embodiments, the viral antigen or immunogen includes 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 of the amino acid sequence included in 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 the amino acid position of SEQ ID NO: 55) at one or more amino acid positions in the group consisting of substitutions, deletions and/or or inserted
  • the viral antigen or immunogen includes a variant of SEQ ID NO: 45, and the variant includes a variant selected from the group consisting of S13I, L18F, T20N, P26S, ⁇ 69-70 ( ⁇ HV), D80A, D138Y, G142D, ⁇ 144( ⁇ Y), W152C, R190S, D215G, ⁇ 242-244( ⁇ LAL), R246I, ⁇ 400-402( ⁇ FVI), K417T, K417N, N440K, L452R, S477N, S477G, E484K, E484Q, N501Y, A570D, D614G , H655Y, P681H , any one, two, three, four, five or more mutations in the group consisting of , P681R, R682G, R683S, R685G, A701V, T716I, F888L, S982A, T1027I, D1118
  • the viral antigen or immunogen includes the sequence set forth in SEQ ID NO: 46. In some embodiments, the viral antigen or immunogen includes 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 of the amino acid sequence included in 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, Contains a substitution at one or more amino acid positions in the group consisting of 681, 682, 683, 684, 685, 701, 716, 888, 982, 1027, 1118 and 1176 (with respect to the amino acid position of SEQ ID NO: 46.
  • the viral antigen or immunogen includes a variant of SEQ ID NO: 46, and the variant includes a variant selected from the group consisting of S13I, L18F, T20N, P26S, ⁇ 69-70 ( ⁇ HV), D80A, D138Y, G142D, ⁇ 144( ⁇ Y), W152C, R190S, D215G, ⁇ 242-244( ⁇ LAL), R246I, ⁇ 400-402( ⁇ FVI), K417T, K417N, N440K, L452R, S477N, S477G, E484K, E484Q, N501Y, A570D, D614G , H655Y, P681H , any one, two, three, four, five or more mutations in the group consisting of , P681R, R682G, R683S, R685G, A701V, T716I, F888L, S982A, T1027I, D1118
  • the viral antigen or immunogen includes the sequence set forth in SEQ ID NO:47. In some embodiments, the viral antigen or immunogen includes at least or about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89 Amino acid sequences with %, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity, including amino acid sequences 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, Comprises a substitution at one or more amino acid positions in the group consisting of 681, 682, 683, 684, 685, 701, 716, 888, 982, 1027, 1118 and 1176 (with respect to the amino acid position of
  • the viral antigen or immunogen includes a variant of SEQ ID NO: 47, and the variant includes a variant selected from the group consisting of S13I, L18F, T20N, P26S, ⁇ 69-70 ( ⁇ HV), D80A, D138Y, G142D, ⁇ 144( ⁇ Y), W152C, R190S, D215G, ⁇ 242-244( ⁇ LAL), R246I, ⁇ 400-402( ⁇ FVI), K417T, K417N, N440K, L452R, S477N, S477G, E484K, E484Q, N501Y, A570D, D614G , H655Y, P681H ,P681R,R682G, Any one, two, three, four, five or more mutations in the group consisting of R683S, R685G, A701V, T716I, F888L, S982A, T1027I, D1118H and
  • the viral antigen or immunogen includes the sequence set forth in SEQ ID NO: 48. In some embodiments, the viral antigen or immunogen includes 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 of the amino acid sequence included in 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, Contains a substitution at one or more amino acid positions in the group consisting of 681, 682, 683, 684, 685, 701, 716, 888, 982, 1027, 1118 and 1176 (with respect to the amino acid position of SEQ ID NO: 48.
  • the viral antigen or immunogen includes a variant of SEQ ID NO: 48, and the variant includes a variant selected from the group consisting of S13I, L18F, T20N, P26S, ⁇ 69-70 ( ⁇ HV), D80A, D138Y, G142D, ⁇ 144( ⁇ Y), W152C, R190S, D215G, ⁇ 242-244( ⁇ LAL), R246I, ⁇ 400-402( ⁇ FVI), K417T, K417N, N440K, L452R, S477N, S477G, E484K, E484Q, N501Y, A570D, D614G , H655Y, P681H , any one, two, three, four, five or more mutations in the group consisting of , P681R, R682G, R683S, R685G, A701V, T716I, F888L, S982A, T1027I, D1118
  • the viral antigen or immunogen includes the sequence set forth in SEQ ID NO: 49. In some embodiments, the viral antigen or immunogen includes 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 of the amino acid sequence included in 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, Contains a substitution at one or more amino acid positions in the group consisting of 681, 682, 683, 684, 685, 701, 716, 888, 982, 1027, 1118 and 1176 (with respect to the amino acid position of SEQ ID NO: 49.
  • the viral antigen or immunogen includes a variant of SEQ ID NO: 49, and the variant includes a variant selected from the group consisting of S13I, L18F, T20N, P26S, ⁇ 69-70 ( ⁇ HV), D80A, D138Y, G142D, ⁇ 144( ⁇ Y), W152C, R190S, D215G, ⁇ 242-244( ⁇ LAL), R246I, ⁇ 400-402( ⁇ FVI), K417T, K417N, N440K, L452R, S477N, S477G, E484K, E484Q, N501Y, A570D, D614G , H655Y, P681H , any one, two, three, four, five or more mutations in the group consisting of , P681R, R682G, R683S, R685G, A701V, T716I, F888L, S982A, T1027I, D1118
  • the viral antigen or immunogen includes the sequence set forth in SEQ ID NO:50. In some embodiments, the viral antigen or immunogen includes at least or about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89 Amino acid sequences with %, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity, including amino acid sequences 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 Sequences containing substitutions, deletions and/or insertions at one or more amino acid positions in the group consisting of , 888, 982, 1027, 1118 and
  • the viral antigen or immunogen includes a variant of SEQ ID NO: 50, and the variant includes a variant selected from the group consisting of S13I, L18F, T20N, P26S, ⁇ 69-70 ( ⁇ HV), D80A, D138Y, G142D, ⁇ 144( ⁇ Y), W152C, R190S, D215G, ⁇ 242-244( ⁇ LAL), R246I, ⁇ 400-402( ⁇ FVI), K417T, K417N, N440K, L452R, S477N, S477G, E484K, E484Q, N501Y, A570D, D614G , H655Y, P681H , any one, two, three, four, five or more mutations in the group consisting of , P681R, R682G, R683S, R685G, A701V, T716I, F888L, S982A, T1027I, D1118
  • the viral antigen or immunogen includes the sequence set forth in SEQ ID NO: 51. In some embodiments, the viral antigen or immunogen includes 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 of the amino acid sequence included in 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, Contains a substitution at one or more amino acid positions in the group consisting of 681, 682, 683, 684, 685, 701, 716, 888, 982, 1027, 1118 and 1176 (with respect to the amino acid position of SEQ ID NO: 51.
  • the viral antigen or immunogen includes a variant of SEQ ID NO: 51, and the variant includes a variant selected from the group consisting of S13I, L18F, T20N, P26S, ⁇ 69-70 ( ⁇ HV), D80A, D138Y, G142D, ⁇ 144( ⁇ Y), W152C, R190S, D215G, ⁇ 242-244( ⁇ LAL), R246I, ⁇ 400-402( ⁇ FVI), K417T, K417N, N440K, L452R, S477N, S477G, E484K, E484Q, N501Y, A570D, D614G , H655Y, P681H , any one, two, three, four, five or more mutations in the group consisting of , P681R, R682G, R683S, R685G, A701V, T716I, F888L, S982A, T1027I, D1118
  • the viral antigen or immunogen includes the sequence set forth in SEQ ID NO: 52. In some embodiments, the viral antigen or immunogen includes at least or about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89 Amino acid sequences with %, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity.
  • the viral antigen or immunogen includes a signal peptide. In some embodiments, the viral antigen or immunogen includes the sequence set forth in SEQ ID NO: 53. In some embodiments, the viral antigen or immunogen includes at least or about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89 Amino acid sequences with %, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity. In some embodiments, the viral antigen or immunogen includes the sequence set forth in SEQ ID NO: 54.
  • the viral antigen or immunogen includes at least or about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89 Amino acid sequences with %, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity.
  • the viral antigen or immunogen includes the sequence set forth in SEQ ID NO:55. In some embodiments, the viral antigen or immunogen includes 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 of the amino acid sequence included in the 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 Sequences containing substitutions, deletions and/or insertions at one or more amino acid positions.
  • the viral antigen or immunogen includes a variant of SEQ ID NO: 55, and the variant includes a variant selected from the group consisting of S13I, L18F, T20N, P26S, ⁇ 69-70 ( ⁇ HV), D80A, D138Y, G142D, ⁇ 144( ⁇ Y), W152C, R190S, D215G, ⁇ 242-244( ⁇ LAL), R246I, ⁇ 400-402( ⁇ FVI), K417T, K417N, N440K, L452R, S477N, S477G, E484K, E484Q, N501Y, A570D, D614G , H655Y, P681H , any one, two, three, four, five or more mutations in the group consisting of , P681R, R682G, R683S, R685G, A701V, T716I, F888L, S982A, T1027I, D1118
  • the viral antigen or immunogen includes the sequence set forth in SEQ ID NO: 56. In some embodiments, the viral antigen or immunogen includes 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 of the amino acid sequence included in 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, Contains a substitution at one or more amino acid positions in the group consisting of 681, 682, 683, 684, 685, 701, 716, 888, 982, 1027, 1118 and 1176 (with respect to the amino acid position of SEQ ID NO: 56.
  • the viral antigen or immunogen includes a variant of SEQ ID NO: 56, and the variant includes a variant selected from the group consisting of S13I, L18F, T20N, P26S, ⁇ 69-70 ( ⁇ HV), D80A, D138Y, G142D, ⁇ 144( ⁇ Y), W152C, R190S, D215G, ⁇ 242-244( ⁇ LAL), R246I, ⁇ 400-402( ⁇ FVI), K417T, K417N, N440K, L452R, S477N, S477G, E484K, E484Q, N501Y, A570D, D614G , H655Y, P681H , any one, two, three, four, five or more mutations in the group consisting of , P681R, R682G, R683S, R685G, A701V, T716I, F888L, S982A, T1027I, D1118
  • the viral antigen or immunogen includes the sequence set forth in SEQ ID NO: 57. In some embodiments, the viral antigen or immunogen includes 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 an amino acid sequence including one or more of SEQ ID NO:57 Sequences containing substitutions, deletions and/or insertions at amino acid positions.
  • the viral antigen or immunogen includes the sequence set forth in SEQ ID NO:58. In some embodiments, the viral antigen or immunogen includes 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 an amino acid sequence including one or more of SEQ ID NO: 58 Sequences containing substitutions, deletions and/or insertions at amino acid positions.
  • the viral antigen or immunogen includes the sequence set forth in SEQ ID NO: 59. In some embodiments, a viral antigen or immunogen includes a sequence comprising substitutions, deletions, and/or insertions at one or more amino acid positions of SEQ ID NO: 59. In some embodiments, the viral antigen or immunogen includes the sequence set forth in SEQ ID NO: 60. In some embodiments, a viral antigen or immunogen includes a sequence comprising substitutions, deletions, and/or insertions at one or more amino acid positions of SEQ ID NO: 60.
  • the viral antigen or immunogen includes the sequence set forth in SEQ ID NO: 61. In some embodiments, the viral antigen or immunogen includes 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 an amino acid sequence including one or more of SEQ ID NO: 61 Sequences containing substitutions, deletions and/or insertions at amino acid positions.
  • the viral antigen or immunogen includes the sequence set forth in SEQ ID NO: 62. In some embodiments, the viral antigen or immunogen includes 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 an amino acid sequence including one or more of SEQ ID NO: 62 Sequences containing substitutions, deletions and/or insertions at amino acid positions.
  • the viral antigen or immunogen includes the sequence set forth in SEQ ID NO: 63. In some embodiments, the viral antigen or immunogen includes 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 an amino acid sequence including one or more of SEQ ID NO:63 Sequences containing substitutions, deletions and/or insertions at amino acid positions.
  • the viral antigen or immunogen includes the sequence set forth in SEQ ID NO: 64. In some embodiments, the viral antigen or immunogen includes 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 an amino acid sequence including one or more of SEQ ID NO:64 Sequences containing substitutions, deletions and/or insertions at amino acid positions.
  • the viral antigen or immunogen includes the sequence set forth in SEQ ID NO: 65. In some embodiments, the viral antigen or immunogen includes 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 an amino acid sequence including one or more of SEQ ID NO: 65 Sequences containing substitutions, deletions and/or insertions at amino acid positions.
  • the viral antigen or immunogen includes the sequence set forth in SEQ ID NO:81. In some embodiments, the viral antigen or immunogen includes 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 an amino acid sequence including one or more of SEQ ID NO:81 Sequences containing substitutions, deletions and/or insertions at amino acid positions.
  • the viral antigen or immunogen includes the sequence set forth in SEQ ID NO: 82. In some embodiments, the viral antigen or immunogen includes 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 an amino acid sequence including one or more of SEQ ID NO:82 Sequences containing substitutions, deletions and/or insertions at amino acid positions.
  • the viral antigen or immunogen includes the sequence set forth in SEQ ID NO: 83. In some embodiments, the viral antigen or immunogen includes 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 an amino acid sequence including one or more of SEQ ID NO:83 Sequences containing substitutions, deletions and/or insertions at amino acid positions.
  • the viral antigen or immunogen includes the sequence set forth in SEQ ID NO: 84. In some embodiments, the viral antigen or immunogen includes 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 an amino acid sequence including one or more of SEQ ID NO:84 Sequences containing substitutions, deletions and/or insertions at amino acid positions.
  • the viral antigen or immunogen includes the sequence set forth in SEQ ID NO: 122. In some embodiments, the viral antigen or immunogen includes 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 an amino acid sequence including one or more of SEQ ID NO: 122 Sequences containing substitutions, deletions and/or insertions at amino acid positions. In some embodiments, the viral antigen or immunogen includes an RBD trimer, e.g., a SARS-CoV-2 RBD sequence linked to any one of SEQ ID Nos: 67-80.
  • RBD trimer e.g., a SARS-CoV-2 RBD sequence linked to any one of SEQ ID Nos: 67-80.
  • the viral antigen or immunogen includes the sequence set forth in SEQ ID NO: 123. In some embodiments, the viral antigen or immunogen includes 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 an amino acid sequence including one or more of SEQ ID NO: 123 Sequences containing substitutions, deletions and/or insertions at amino acid positions. In some embodiments, the viral antigen or immunogen includes an RBD trimer, e.g., a SARS-CoV-2 RBD sequence linked to any one of SEQ ID Nos: 67-80.
  • RBD trimer e.g., a SARS-CoV-2 RBD sequence linked to any one of SEQ ID Nos: 67-80.
  • the viral antigen or immunogen does not include a transmembrane domain such as SEQ ID NO: 66 or a portion thereof.
  • the coronavirus antigen or immunogen includes soluble S protein peptide.
  • the soluble protein S peptide lacks the TM domain peptide and the CP domain peptide.
  • soluble S protein peptides do not bind to lipid bilayers, such as membranes or viral envelopes.
  • the spike protein peptide is produced from a codon-optimized nucleic acid sequence. In some embodiments, spike protein peptides are produced from nucleic acid sequences that are not codon optimized.
  • a viral antigen or immunogen referred to herein may include a recombinant polypeptide or fusion peptide comprising the viral antigen or immunogen.
  • the term viral antigen or immunogen may be used to refer to the protein that contains the coronavirus antigen or immunogen.
  • the coronavirus antigen or immunogen is a coronavirus protein peptide provided herein.
  • the viral antigens or immunogens mentioned herein can be used in initial doses, additional doses, and/or booster doses. Independently, the initial agent, additional agent, and/or any one or more boosters may be administered without or with an adjuvant.
  • adjuvants may include: aluminum-containing adjuvants, such as alum and/or aluminum hydroxide-containing adjuvants; oligonucleotide-containing adjuvants, such as CpG-containing oligodeoxynucleotides (CpG- ODN); adjuvants containing TLR9 agonists; adjuvants containing metabolizable oils, alpha-tocopherol, and/or polyoxyethylene sorbitan monooleate (Tween-80), e.g., in water Adjuvant containing squalene, alpha-tocopherol, and Tween-80 and/or Span 85 in the form of an oil emulsion; or any combination of adjuvants.
  • aluminum-containing adjuvants such as alum and/or aluminum hydroxide-containing adjuvants
  • oligonucleotide-containing adjuvants such as CpG-containing oligodeoxynucleotides (CpG
  • coronavirus antigens and immunogens provided herein can be combined with, e.g., linked to, other proteins or peptides to form recombinant polypeptides, including fusion peptides.
  • individual recombinant polypeptides (eg, monomers) provided herein combine to form multimers, eg, trimers, of recombinant polypeptides.
  • association of individual recombinant polypeptide monomers occurs through covalent interactions.
  • binding of individual recombinant polypeptide monomers occurs through non-covalent interactions.
  • the interaction is affected by a protein or peptide linked to a coronavirus antigen or immunogen (eg, spike protein peptide).
  • a coronavirus antigen or immunogen eg, spike protein peptide
  • the coronavirus antigen or immunogen is a S protein peptide as described herein
  • the protein or peptide to which it is linked can be selected such that the native homotrimeric structure of the glycoprotein is retained. This can facilitate eliciting a strong and effective immunogenic response to the S protein peptide.
  • retention and/or maintenance of the native conformation of a coronavirus antigen or immunogen may improve or allow access to antigenic sites capable of generating an immune response.
  • a recombinant polypeptide comprising a S protein peptide described herein is alternatively referred to herein as a recombinant S antigen, a recombinant S immunogen, or a recombinant S protein.
  • recombinant polypeptides or multimeric recombinant polypeptides thereof aggregate or can aggregate to form proteins or complexes comprising multiple coronavirus antigens and/or immunogenic recombinant polypeptides.
  • the formation of this protein may facilitate the generation of strong and effective immunogenic responses to coronavirus antigens and/or immunogens.
  • forming a protein containing multiple recombinant polypeptides, thereby forming multiple coronavirus antigens, such as coronavirus S protein peptides can retain the tertiary and/or quaternary structure of the viral antigen, allowing for the mounting of an immune response against the native structure.
  • aggregation can confer structural stability to coronavirus antigens or immunogens, which in turn can provide access to potential antigenic sites capable of promoting immune responses.
  • a coronavirus antigen or immunogen can be linked to a trimerization domain at its C-terminus (C-terminal linkage) to promote trimerization of monomers.
  • trimerization stabilizes the membrane-proximal aspect of a coronavirus antigen or immunogen (eg, coronavirus spike protein peptide) in a trimer configuration.
  • 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), trimerization of pulmonary surfactant protein Motif (Hoppe et al. 1994 FEBS Lett 344:191-195), collagen (McAlinden et al. 2003 J Biol Chem 278:42200-42207) and phage T4 fibritin Foldon (Miroshnikov et al.
  • one or more peptide linkers can be used to link the recombinant viral antigen or immunogen to the multimerization domain.
  • the trimer may include any of the stabilizing mutations provided herein (or combinations thereof).
  • the recombinant polypeptide or fusion protein includes the first sequence of any one of SEQ ID NOs: 27-66 and 81-84 linked to any one of SEQ ID NOs: 67-80 The second sequence, wherein the C-terminus of the first sequence is directly connected to the N-terminus of the second sequence.
  • the recombinant polypeptide or fusion protein includes the first sequence of any one of SEQ ID NOs: 27-66 and 81-84 linked to any one of SEQ ID NOs: 67-80
  • the second sequence wherein the C-terminus of the first sequence is indirectly connected to the N-terminus of the second sequence, such as through a linker.
  • the linker includes a sequence comprising glycine-X-Y repeats.
  • the trimeric protein moieties required for biopharmaceutical design should meet the following criteria. Ideally, it should be part of a naturally occurring secreted protein, such as an immunoglobulin Fc, that is also abundant in circulation (non-toxic), of human origin (lack of immunogenicity), relatively stable (long half-life), and capable of efficacious autoimmune Trimerization, the self-trimerization is enhanced by inter-chain covalent disulfide bonds, so the trimerized coronavirus antigen or immunogen is structurally stable.
  • a naturally occurring secreted protein such as an immunoglobulin Fc
  • an immunoglobulin Fc that is also abundant in circulation (non-toxic), of human origin (lack of immunogenicity), relatively stable (long half-life), and capable of efficacious autoimmune Trimerization
  • the self-trimerization is enhanced by inter-chain covalent disulfide bonds, so the trimerized coronavirus antigen or immunogen is structurally stable.
  • Collagen is a member of the fibrin family, which is a major component of the extracellular matrix. It is the most abundant protein in mammals, accounting for nearly 25% of total body protein. Collagen plays an important structural role in the formation of bones, tendons, skin, cornea, cartilage, blood vessels, and teeth.
  • the fibrillar types of collagens I, II, III, IV, V, and The central uninterrupted triple helical domain is flanked by the noncollagen domain (NC), N-propeptide, and C-propeptide. Both C-terminal and N-terminal extensions undergo proteolytic processing following procollagen secretion, an event that triggers the assembly of mature proteins into collagen fibrils, forming an insoluble cellular matrix.
  • NC noncollagen domain
  • N-propeptide N-propeptide
  • C-propeptide C-propeptide
  • BMP-1 is a protease that recognizes a specific peptide sequence of procollagen near the junction between the glycine repeats and the collagen C-prodomain and is responsible for removing the propeptide.
  • Concentrations of the exfoliated trimeric C-propeptide of type I collagen are found in the human serum of normal adults in the range of 50-300 ng/mL, with higher levels in children, indicating active bone formation. In people with familial high serum concentrations of C-propeptide of type I collagen, its levels can be as high as 1-6 ⁇ g/mL without obvious abnormalities, indicating that C-propeptide is non-toxic.
  • Collagen types I, IV, V and XI are mainly assembled into two ⁇ -1 chains and one ⁇ -2 chain (for types I, IV and V) or three different chains (for type XI). Heterotrimeric forms, which are highly homologous in sequence. Both type II and type III collagen are homotrimers of ⁇ -1 chains. For type I collagen, the most abundant form of collagen, stable ⁇ (I) homotrimers are also formed and are 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, the molecular assembly of trimeric collagen begins with the registered binding of the C-propeptide.
  • C-propeptide complexes are stabilized by the formation of interchain disulfide bonds, but the necessity of disulfide bond formation for proper chain registration is unclear.
  • the triple helical repeat of glycine then spreads from the associated C-terminus to the N-terminus in a zipper-like manner.
  • This knowledge creates a non-natural type of collagen matrix by using recombinant DNA technology to exchange the C-propeptides of different collagen chains.
  • Non-collagenous proteins such as cytokines and growth factors
  • C-propeptide needs to be cleaved before reorganized collagen fibers can be assembled into an insoluble cellular matrix.
  • any trimeric fusion protein must be synthesized intracellularly, which not only may lead to misfolding of native secreted proteins (e.g., soluble receptors) but also makes purification of such fusion proteins from thousands of other intracellular proteins cumbersome. Difficulty.
  • trimerization domains e.g., from yeast, bacteriophages, and bacteria
  • trimer biopharmaceutical design is their putative immunogenicity in humans, making such fusion proteins It loses effectiveness soon after being injected into the human body.
  • collagen in the recombinant peptides described herein has many advantages, including: (1) collagen is the most abundant protein secreted in mammals, accounting for nearly 25% of total protein in the body; (2) the primary form of collagen is naturally occurring Ground appears in the form of a trimer helix, and its globular C-propeptide is responsible for the initiation of trimerization; (3) The trimeric C-propeptide of collagen released from hydrolysis of mature collagen proteins is present in mammalian blood at submicrogram/ Milliliter levels occur naturally and are 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 between 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) Cleavage of the C-propeptide from procollagen The recognition site of BMP1 can be mutated or deleted to prevent the destruction of the trimeric fusion protein;
  • the coronavirus antigen or immunogen is linked to the C-terminal propeptide of collagen to form a recombinant polypeptide.
  • the C-terminal propeptide of the recombinant polypeptide forms an inter-polypeptide disulfide bond.
  • the recombinant proteins form trimers.
  • the coronavirus antigen or immunogen is a S protein peptide as described in Section I.
  • a fusion polypeptide including the fusion polypeptide in SEQ ID NO: 1 can be generated and trimerized through inter-polypeptide disulfide bonds (Cys residues that can form inter-polypeptide disulfide bonds are shown in bold).
  • the signal peptide MFVFLVLLPLVSS (SEQ ID NO: 54) on the N-terminus of .
  • inter-polypeptide disulfide bonds may include 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- One or more or all of 1126, in any suitable combination.
  • the fusion polypeptide in the trimer can include one or more glycosylation sites (e.g., Asn-linked), e.g., at 17, 61, 122, 149, 165, 234, 282, One or more or all Asn residues at 331, 343, 603, 616, 657, 709, 717, 801, 1074, 1098 and 1134, in any suitable combination.
  • one or more glycosylation sites e.g., Asn-linked
  • Asn-linked e.g., Asn-linked
  • the C-terminal propeptide is human collagen. In some embodiments, the C-terminal propeptide includes pro ⁇ 1(I), pro ⁇ 1(II), pro ⁇ 1(III), pro ⁇ 1(V), pro ⁇ 1(XI), pro ⁇ 2(I), pro ⁇ 2(V), pro ⁇ 2(XI ) or the C-terminal polypeptide of pro ⁇ 3(XI) or a fragment thereof. In some embodiments, the C-terminal propeptide is or includes the C-terminal polypeptide of proal(I).
  • the C-terminal propeptide is or includes the amino acid sequence set forth in SEQ ID NO: 67. 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 SEQ ID NO: 67. In some embodiments, the C-terminal propeptide is or includes the amino acid sequence set forth in SEQ ID NO: 68. 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 SEQ ID NO: 68. In some embodiments, the C-terminal propeptide is or is the amino acid sequence set forth in SEQ ID NO: 69.
  • the C-terminal propeptide exhibits an amino acid sequence that has at least or about 85%, 90%, 92%, 95%, or 97% sequence identity to SEQ ID NO: 69. In some embodiments, the C-terminal propeptide is or includes the amino acid sequence set forth in SEQ ID NO: 70. 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 SEQ ID NO:70. In some embodiments, the C-terminal propeptide is or includes the amino acid sequence set forth in SEQ ID NO: 71. 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 SEQ ID NO:71.
  • the C-terminal propeptide is or includes the amino acid sequence set forth in SEQ ID NO: 72. 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 SEQ ID NO:72. In some embodiments, the C-terminal propeptide is or includes the amino acid sequence set forth in SEQ ID NO: 73. 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 SEQ ID NO:73. In some embodiments, the C-terminal propeptide is or is the amino acid sequence set forth in SEQ ID NO:74.
  • the C-terminal propeptide exhibits an amino acid sequence that has at least or about 85%, 90%, 92%, 95%, or 97% sequence identity to SEQ ID NO:74. In some embodiments, the C-terminal propeptide is or includes the amino acid sequence set forth in SEQ ID NO: 75. 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 SEQ ID NO:75. In some embodiments, the C-terminal propeptide is or includes the amino acid sequence set forth in SEQ ID NO: 76. 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 SEQ ID NO:76.
  • the C-terminal propeptide is or includes the amino acid sequence set forth in SEQ ID NO:77. 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 SEQ ID NO:77. In some embodiments, the C-terminal propeptide is or includes the amino acid sequence set forth in SEQ ID NO:78. In some embodiments, the C-terminal propeptide is the same as SEQ ID NO:78 Amino acid sequences that are at least or about 85%, 90%, 92%, 95% or 97% sequence identity. In some embodiments, the C-terminal propeptide is or is the amino acid sequence set forth in SEQ ID NO:79.
  • the C-terminal propeptide exhibits an amino acid sequence that has at least or about 85%, 90%, 92%, 95%, or 97% sequence identity to SEQ ID NO:79. In some embodiments, the C-terminal propeptide is or includes the amino acid sequence set forth in SEQ ID NO: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 SEQ ID NO:80.
  • the C-terminal propeptide is or includes the amino acid sequence of a collagen trimerization domain (e.g., the C-propeptide of human ⁇ 1(I) collagen) with an aspartic acid in the BMP-1 site (D) Substitution to asparagine (N), for example, as shown in SEQ ID NO:68, where RAD is mutated to RAN .
  • a collagen trimerization domain e.g., the C-propeptide of human ⁇ 1(I) collagen
  • D aspartic acid in the BMP-1 site
  • N Substitution to asparagine
  • the C-terminal propeptide is or includes the amino acid sequence of a collagen trimerization domain (e.g., the C-propeptide of human ⁇ 1(I) collagen) having an alanine in the BMP-1 site Substitution of (A) to asparagine (N), for example, as shown in SEQ ID NO:69, where R A D is mutated to R N D.
  • a C-terminal propeptide herein may include a mutated BMP-1 site, e.g., RSAN instead of DDAN.
  • a C-terminal propeptide herein may include a BMP-1 site, e.g., a sequence that includes a RAD (e.g., RADDAN) sequence instead of a RAN (e.g., RANDAN) or RND (e.g., RNDDAN) (
  • RAD e.g., RADDAN
  • RND e.g., RNDDAN
  • SEQ ID NO: 68 or 69 may be used in the fusion polypeptides disclosed herein.
  • SEQ ID NO:27 (underlined) or a fragment, variant or mutant thereof can be directly or indirectly linked to SEQ ID NO:67 (italics) or a fragment, variant or mutant thereof, for example, to form the following fusion protein:
  • the C-terminal propeptide is or includes an amino acid sequence that is a fragment of any one of SEQ ID NOs: 67-80.
  • the C-terminal propeptide can include a sequence comprising a glycine-XY repeat, wherein X and Y are independently any amino acid, or are at least 85%, 90%, 92%, 95%, or 97% identical thereto of amino acids Sequences capable of forming inter-polypeptide disulfide bonds and trimerizing recombinant polypeptides.
  • X and Y are independently proline or hydroxyproline.
  • the recombinant polypeptide forms a trimer, thereby forming a homotrimer of the S protein peptide.
  • the S protein peptide of the trimerized recombinant polypeptide is in a prefusion conformation.
  • the S protein peptide of the trimerized recombinant polypeptide is in a post-fusion conformation.
  • the validation status allows access to different antigenic sites on the S protein peptide.
  • the antigenic site is an epitope, such as a linear epitope or a conformational epitope.
  • a trimerized recombinant polypeptide includes a single recombinant polypeptide comprising the same viral antigen or immunogen. In some embodiments, a trimerized recombinant polypeptide includes a single recombinant polypeptide, each recombinant polypeptide comprising a different viral antigen or immunogen than the other recombinant polypeptides. In some embodiments, a trimerized recombinant polypeptide includes a single recombinant polypeptide, wherein one of the single recombinant polypeptides includes a different viral antigen or immunogen than the other recombinant polypeptides.
  • a trimerized recombinant polypeptide includes a single recombinant polypeptide, wherein two of the single recombinant polypeptides include the same viral antigen or immunogen, and the viral antigen or immunogen is different from the virus contained in the remaining recombinant polypeptide. Antigen or immunogen.
  • the recombinant polypeptide includes any coronavirus antigen or immunogen described in Section I. In some embodiments, the recombinant polypeptide includes any coronavirus antigen or immunogen described in Section I linked to a C-terminal propeptide of collagen as described herein.
  • the immunogen includes recombinant SARS-CoV or SARS-CoV-2 S ectodomain trimer, e.g., SARS-CoV-2 Omicron (B.1.1.529) coronavirus S ectodomain trimer
  • SARS-CoV-2 S ectodomain trimer e.g., SARS-CoV-2 Omicron (B.1.1.529) coronavirus S ectodomain trimer
  • a body 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 substitution stabilizes the S ectodomain trimer in the prefusion conformation.
  • one or more proline substitutions at position 15 of the C-terminal residue of HR1 stabilize the S ectodomain in the prefusion conformation. Between the N-terminus of the amino acid and the C-terminus of amino acid 5 of the N-terminal residue of the central helix.
  • one or more proline substitutions stabilize the coronavirus (e.g., SARS-CoV or SARS-CoV-2) S cells in a prefusion conformation.
  • Extracellular domain trimers such as SARS-CoV-2 Omicron (B.1.1.529) coronavirus S ectodomain trimers.
  • the SARS-CoV-2 S protein peptide includes a mutation of 986K/987V to 986P/987P.
  • a stabilized recombinant coronavirus e.g., SARS-CoV or SARS-CoV-2
  • S ectodomain trimer in a prefusion conformation includes a single-chain S ectodomain protomer comprising S1 /Mutation of S2 and/or S2′ protease cleavage sites to prevent protease cleavage at these sites.
  • the SARS-CoV-2 S protein peptide includes a mutation from 685R to 685A. Exemplary protease cleavage sites for various viruses are shown below:
  • the S ectodomain III of a recombinant coronavirus (e.g., SARS-CoV or SARS-CoV-2) in a prefusion conformation is stabilized by one or more proline substitutions (e.g., 986P/987P substitutions).
  • the protomer of the polymer includes additional modifications for stabilization in the prefusion conformation, such as mutations at the protease cleavage site to prevent protease cleavage.
  • the extracellular domain includes the signal peptide (SP), which is removed during cellular processing; the N-terminal domain (NTD); the receptor binding domain (RBD); one or more S1/S2 cleavage sites; fusion peptide (FP); internal fusion peptide (IFP); heptad repeat 1/2 (HR1/2) and transmembrane domain (TM).
  • SP signal peptide
  • NTD N-terminal domain
  • RBD receptor binding domain
  • FP fusion peptide
  • IFP internal fusion peptide
  • HR1/2 heptad repeat 1/2
  • TM transmembrane domain
  • 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 pneumonia virus isolate Hu-1.
  • protomers of prefusion stabilized SARS-CoV-2 S ectodomain trimers such as SARS-CoV-2 Omicron (B.1.1.529) coronavirus S ectodomain trimers
  • Protomers of the body may have NTD, RBD, S1 (at S1/S2 site 1 or S1/S2 site 2), FP, IFP, HR1, HR2 or C-terminal residues of the extracellular domain. Terminal residues (which may be linked 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 identify relevant domains and cleavage sites.
  • the recombinant polypeptide is or includes an NTD peptide of SARS-CoV or SARS-CoV-2 S protein. In some embodiments, the recombinant polypeptide is or includes the RBD peptide of SARS-CoV or SARS-CoV-2 S protein. In some embodiments, the recombinant polypeptide is or includes the NTD peptide and RBD peptide of 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.
  • SARS-CoV-1 S recombinant polypeptide without a signal peptide is provided in SEQ ID NO: 26 (1491aa):
  • the above-mentioned SARS-CoV-1 S recombinant polypeptide may include the N-terminal signal peptide provided in SEQ ID NO:53.
  • SARS-CoV-2 S recombinant polypeptide without a signal peptide is provided in SEQ ID NO: 1:
  • the above-mentioned SARS-CoV-2 S recombinant polypeptide may include the N-terminal signal peptide provided in SEQ ID NO:54.
  • SARS-CoV-2 S recombinant polypeptide without a signal peptide is provided in SEQ ID NO: 81 (1506aa):
  • the above-mentioned SARS-CoV-2 S recombinant polypeptide may include the N-terminal signal peptide provided in SEQ ID NO: 54 (MFVFLVLLPLLVSS).
  • SARS-CoV-2 S recombinant polypeptide without a signal peptide is provided in SEQ ID NO:82 (1506aa):
  • the above-mentioned SARS-CoV-2 S recombinant polypeptide may include the N-terminal signal peptide provided in SEQ ID NO: 54 (MFVFLVLLPLLVSS).
  • SARS-CoV-2 S recombinant polypeptide without a signal peptide is provided in SEQ ID NO:83 (1506aa):
  • the above-mentioned SARS-CoV-2 S recombinant polypeptide may include the N-terminal signal peptide provided in SEQ ID NO: 54 (MFVFLVLLPLLVSS).
  • SARS-CoV-2 S recombinant polypeptide without a signal peptide is provided in SEQ ID NO:84 (1506aa):
  • the above-mentioned SARS-CoV-2 S recombinant polypeptide may include the N-terminal signal peptide provided in SEQ ID NO: 54 (MFVFLVLLPLLVSS).
  • the recombinant polypeptide is or includes the sequence set forth in SEQ ID NO: 1. In some embodiments, the recombinant polypeptide is or includes 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 of the amino acid sequence, 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, Comprising substitutions at 570, 614, 655, 681, 682, 683, 684, 685, 701, 716, 888, 982, 1027, 1118 and/or 1176 (with respect to the amino acid position of SEQ ID NO:
  • the recombinant polypeptide is or includes a variant of SEQ ID NO: 1, and the variant includes a variant selected from the group consisting of S13I, L18F, T20N, P26S, ⁇ 69-70 ( ⁇ HV), D80A, D138Y, G142D, ⁇ 144 ( ⁇ Y ), W152C, R190S, D215G, ⁇ 242-244( ⁇ LAL), R246I, ⁇ 400-402( ⁇ FVI), K417T, K417N, N440K, L452R, S477N, S477G, E484K, E484Q, N501Y, A570D, D614G, H6 55Y, P681H, Any one, two, three, four, five or more mutations in the group consisting of P681R, R682G, R683S, R685G, A701V, T716I, F888L, S982A, T1027I, D1118H
  • the recombinant polypeptide is or includes the sequence set forth in SEQ ID NO:2. In some embodiments, the recombinant polypeptide is or includes at least or about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89% of SEQ ID NO:2 , 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity of the amino acid sequence, 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, Comprising substitutions at 570, 614, 655, 681, 682, 683, 684, 685, 701, 716, 888, 982, 1027, 1118 and/or 1176 (with respect to the amino acid
  • the recombinant polypeptide is or includes a variant of SEQ ID NO:2, and the variant includes a variant selected from the group consisting of S13I, L18F, T20N, P26S, ⁇ 69-70( ⁇ HV), D80A, D138Y, G142D, ⁇ 144( ⁇ Y ), W152C, R190S, D215G, ⁇ 242-244( ⁇ LAL), R246I, ⁇ 400-402( ⁇ FVI), K417T, K417N, N440K, L452R, S477N, S477G, E484K, E484Q, N501Y, A570D, D614G, H6 55Y, P681H, Any one, two, three, four, five or more mutations in the group consisting of P681R, R682G, R683S, R685G, A701V, T716I, F888L, S982A, T1027I, D1118H
  • the recombinant polypeptide is or includes the sequence set forth in SEQ ID NO:3. In some embodiments, the recombinant polypeptide is or includes 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 of the amino acid sequence, 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, Comprising substitutions at 570, 614, 655, 681, 682, 683, 684, 685, 701, 716, 888, 982, 1027, 1118 and/or 1176 (with respect to the amino acid position of SEQ ID NO:
  • the recombinant polypeptide is or includes a variant of SEQ ID NO:3, and the variant includes a variant selected from the group consisting of S13I, L18F, T20N, P26S, ⁇ 69-70 ( ⁇ HV), D80A, D138Y, G142D, ⁇ 144 ( ⁇ Y ), W152C, R190S, D215G, ⁇ 242-244( ⁇ LAL), R246I, ⁇ 400-402( ⁇ FVI), K417T, K417N, N440K, L452R, S477N, S477G, E484K, E484Q, N501Y, A570D, D614G, H6 55Y, P681H, Any one, two, three, four, five or more mutations in the group consisting of P681R, R682G, R683S, R685G, A701V, T716I, F888L, S982A, T1027I, D1118H
  • the recombinant polypeptide is or includes the sequence set forth in SEQ ID NO:4. In some embodiments, the recombinant polypeptide is or includes 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 of the amino acid sequence, 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, Comprising substitutions at 570, 614, 655, 681, 682, 683, 684, 685, 701, 716, 888, 982, 1027, 1118 and/or 1176 (with respect to the amino acid position of SEQ ID NO:
  • the recombinant polypeptide is or includes a variant of SEQ ID NO: 4, and the variant includes a variant selected from the group consisting of S13I, L18F, T20N, P26S, ⁇ 69-70 ( ⁇ HV), D80A, D138Y, G142D, ⁇ 144 ( ⁇ Y ), W152C, R190S, D215G, ⁇ 242-244( ⁇ LAL), R246I, ⁇ 400-402( ⁇ FVI), K417T, K417N, N440K, L452R, S477N, S477G, E484K, E484Q, N501Y, A570D, D614G, H6 55Y, P681H, Any one, two, three, four, five or more mutations in the group consisting of P681R, R682G, R683S, R685G, A701V, T716I, F888L, S982A, T1027I, D1118H
  • the recombinant polypeptide is Or include a variant of SEQ ID NO: 5, and the variant includes a variant selected from the group consisting of S13I, L18F, T20N, P26S, ⁇ 69-70 ( ⁇ HV), D80A, D138Y, G142D, ⁇ 144 ( ⁇ Y), W152C, R190S, D215G, ⁇ 242 -244( ⁇ LAL), R246I, ⁇ 400-402( ⁇ FVI), K417T, K417N, N440K, L452R, S477N, S477G, E484K, E484Q, N501Y, A570D, D614G, H655Y, P681H, P681R, R682G, R6 83S, R685G, A701V Any one, two, three, four, five or more mutations in the group consisting of , T716I, F888L, S982A, T1027I, D
  • the recombinant polypeptide is or includes the sequence set forth in SEQ ID NO: 6. In some embodiments, the recombinant polypeptide is or includes 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 of the amino acid sequence, 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, Comprising substitutions at 570, 614, 655, 681, 682, 683, 684, 685, 701, 716, 888, 982, 1027, 1118 and/or 1176 (with respect to the amino acid position of SEQ ID NO:
  • the recombinant polypeptide is or includes a variant of SEQ ID NO: 6, and the variant includes a variant selected from the group consisting of S13I, L18F, T20N, P26S, ⁇ 69-70 ( ⁇ HV), D80A, D138Y, G142D, ⁇ 144 ( ⁇ Y ), W152C, R190S, D215G, ⁇ 242-244( ⁇ LAL), R246I, ⁇ 400-402( ⁇ FVI), K417T, K417N, N440K, L452R, S477N, S477G, E484K, E484Q, N501Y, A570D, D614G, H6 55Y, P681H, Any one, two, three, four, five or more mutations in the group consisting of P681R, R682G, R683S, R685G, A701V, T716I, F888L, S982A, T1027I, D1118H
  • the recombinant polypeptide is or includes the sequence set forth in SEQ ID NO:7. In some embodiments, the recombinant polypeptide is or includes 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 of the amino acid sequence, 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, Comprising substitutions at 570, 614, 655, 681, 682, 683, 684, 685, 701, 716, 888, 982, 1027, 1118 and/or 1176 (with respect to the amino acid position of SEQ ID NO:7. In
  • the recombinant polypeptide is or includes a variant of SEQ ID NO:7, and the variant includes a variant selected from the group consisting of S13I, L18F, T20N, P26S, ⁇ 69-70 ( ⁇ HV), D80A, D138Y, G142D, ⁇ 144 ( ⁇ Y ), W152C, R190S, D215G, ⁇ 242-244( ⁇ LAL), R246I, ⁇ 400-402( ⁇ FVI), K417T, K417N, N440K, L452R, S477N, S477G, E484K, E484Q, N501Y, A570D, D614G, H6 55Y, P681H, Any one, two, three, four, five or more mutations in the group consisting of P681R, R682G, R683S, R685G, A701V, T716I, F888L, S982A, T1027I, D1118
  • the recombinant polypeptide is or includes the sequence set forth in SEQ ID NO:8. In some embodiments, the recombinant polypeptide is or includes at least or about 80%, 81%, 82%, 83% SEQ ID NO:8 , 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity Sexual amino acid sequence, including at one or more amino acid positions (such as 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( Sequences containing substitutions, deletions and/or insertions at the amino acid position (amino acid position) of SEQ ID NO:
  • the recombinant polypeptide is or includes a variant of SEQ ID NO: 8, and the variant includes a variant selected from the group consisting of S13I, L18F, T20N, P26S, ⁇ 69-70( ⁇ HV), D80A, D138Y, G142D, ⁇ 144( ⁇ Y ), W152C, R190S, D215G, ⁇ 242-244( ⁇ LAL), R246I, ⁇ 400-402( ⁇ FVI), K417T, K417N, N440K, L452R, S477N, S477G, E484K, E484Q, N501Y, A570D, D614G, H6 55Y, P681H, Any one, two, three, four, five or more mutations in the group consisting of P681R, R682G, R683S, R685G, A701V, T716I, F888L, S982A, T1027I, D1118H
  • the recombinant polypeptide is or includes the sequence set forth in SEQ ID NO: 9. In some embodiments, the recombinant polypeptide is or includes 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 of the amino acid sequence, 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, Comprising substitutions at 570, 614, 655, 681, 682, 683, 684, 685, 701, 716, 888, 982, 1027, 1118 and/or 1176 (with respect to the amino acid position of SEQ ID NO:
  • the recombinant polypeptide is or includes a variant of SEQ ID NO: 9, and the variant includes a variant selected from the group consisting of S13I, L18F, T20N, P26S, ⁇ 69-70 ( ⁇ HV), D80A, D138Y, G142D, ⁇ 144 ( ⁇ Y ), W152C, R190S, D215G, ⁇ 242-244( ⁇ LAL), R246I, ⁇ 400-402( ⁇ FVI), K417T, K417N, N440K, L452R, S477N, S477G, E484K, E484Q, N501Y, A570D, D614G, H6 55Y, P681H, Any one, two, three, four, five or more mutations in the group consisting of P681R, R682G, R683S, R685G, A701V, T716I, F888L, S982A, T1027I, D1118H
  • the recombinant polypeptide is or includes a variant of SEQ ID NO: 10, and the variant includes a variant selected from the group consisting of S13I, L18F, T20N, P26S, ⁇ 69-70 ( ⁇ HV), D80A, D138Y, G142D, ⁇ 144 ( ⁇ Y ), W152C, R190S, D215G, ⁇ 242-244 ( ⁇ LAL), R246I, ⁇ 400-402( ⁇ FVI), K417T, K417N, N440K, L452R, S477N, S477G, E484K, E484Q, N501Y, A570D, D614G, H655Y, P681H, P681R, R682G, R683S, R685G, A701V, T716I Any one, two, three, four, five or more mutations in the group consisting of , F888L, S982A, T1027I, D1118
  • the recombinant polypeptide is or includes the sequence set forth in SEQ ID NO: 11. In some embodiments, the recombinant polypeptide is or includes 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 of the amino acid sequence, 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, Comprising substitutions at 570, 614, 655, 681, 682, 683, 684, 685, 701, 716, 888, 982, 1027, 1118 and/or 1176 (with respect to the amino acid position of SEQ ID NO:
  • the recombinant polypeptide is or includes a variant of SEQ ID NO: 11, and the variant includes a variant selected from the group consisting of S13I, L18F, T20N, P26S, ⁇ 69-70 ( ⁇ HV), D80A, D138Y, G142D, ⁇ 144 ( ⁇ Y ), W152C, R190S, D215G, ⁇ 242-244( ⁇ LAL), R246I, ⁇ 400-402( ⁇ FVI), K417T, K417N, N440K, L452R, S477N, S477G, E484K, E484Q, N501Y, A570D, D614G, H6 55Y, P681H, Any one, two, three, four, five or more mutations in the group consisting of P681R, R682G, R683S, R685G, A701V, T716I, F888L, S982A, T1027I, D1118H
  • the recombinant polypeptide is or includes the sequence set forth in SEQ ID NO: 12. In some embodiments, the recombinant polypeptide is or includes 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 of the amino acid sequence, 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, Comprising substitutions at 570, 614, 655, 681, 682, 683, 684, 685, 701, 716, 888, 982, 1027, 1118 and/or 1176 (with respect to the amino acid position of SEQ ID NO:
  • the recombinant polypeptide is or includes a variant of SEQ ID NO: 12, and the variant includes a variant selected from the group consisting of S13I, L18F, T20N, P26S, ⁇ 69-70 ( ⁇ HV), D80A, D138Y, G142D, ⁇ 144 ( ⁇ Y ), W152C, R190S, D215G, ⁇ 242-244( ⁇ LAL), R246I, ⁇ 400-402( ⁇ FVI), K417T, K417N, N440K, L452R, S477N, S477G, E484K, E484Q, N501Y, A570D, D614G, H6 55Y, P681H, Any one, two, three, four, five or more mutations in the group consisting of P681R, R682G, R683S, R685G, A701V, T716I, F888L, S982A, T1027I, D1118H
  • the recombinant polypeptide is or includes the sequence set forth in SEQ ID NO:13. In some embodiments, the recombinant polypeptide is or includes at least or about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89% identical to SEQ ID NO: 13 , 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity of the amino acid sequence, including at one or more amino acid positions (e.g., 13, 18.
  • the recombinant polypeptide is or includes a variant of SEQ ID NO: 13, and the variant includes a variant selected from the group consisting of S13I, L18F, T20N, P26S, ⁇ 69-70 ( ⁇ HV), D80A, D138Y, G142D, ⁇ 144 ( ⁇ Y ), W152C, R190S, D215G, ⁇ 242-244( ⁇ LAL), R246I, ⁇ 400-402( ⁇ FVI), K417T, K417N, N440K, L452R, S477N, S477G, E484K, E484Q, N501Y, A570D, D614G, H6 55Y, P681H, Any one, two, three, four, five or more mutations in the group consisting of P681R, R682G, R683S, R685G, A701V, T716I, F888L, S982A, T1027I, D1118H
  • the recombinant polypeptide is or includes the sequence set forth in SEQ ID NO: 14. In some embodiments, the recombinant polypeptide is or includes 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 of the amino acid sequence, 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, Comprising substitutions at 570, 614, 655, 681, 682, 683, 684, 685, 701, 716, 888, 982, 1027, 1118 and/or 1176 (with respect to the amino acid position of SEQ ID NO:
  • the recombinant polypeptide is or includes a variant of SEQ ID NO: 14, and the variant includes a variant selected from the group consisting of S13I, L18F, T20N, P26S, ⁇ 69-70 ( ⁇ HV), D80A, D138Y, G142D, ⁇ 144 ( ⁇ Y ), W152C, R190S, D215G, ⁇ 242-244( ⁇ LAL), R246I, ⁇ 400-402( ⁇ FVI), K417T, K417N, N440K, L452R, S477N, S477G, E484K, E484Q, N501Y, A570D, D614G, H6 55Y, P681H, Any one, two, three, four, five or more mutations in the group consisting of P681R, R682G, R683S, R685G, A701V, T716I, F888L, S982A, T1027I, D1118H
  • the recombinant polypeptide is or includes the sequence set forth in SEQ ID NO:15. In some embodiments, the recombinant polypeptide is or includes at least or about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89% identical to SEQ ID NO: 15 , 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity of the amino acid sequence, 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, Comprising substitutions at 570, 614, 655, 681, 682, 683, 684, 685, 701, 716, 888, 982, 1027, 1118 and/or 1176 (with respect to the
  • the recombinant polypeptide is or includes a variant of SEQ ID NO: 15, and the variant includes a variant selected from the group consisting of S13I, L18F, T20N, P26S, ⁇ 69-70 ( ⁇ HV), D80A, D138Y, G142D, ⁇ 144 ( ⁇ Y ), W152C, R190S, D215G, ⁇ 242-244( ⁇ LAL), R246I, ⁇ 400-402( ⁇ FVI), K417T, K417N, N440K, L452R, S477N, S477G, E484K, E484Q, N501Y, A570D, D614G, H6 55Y, P681H, P681R, R682G, R683S, R685G Any one, two, three, four, five or more mutations in the group consisting of , A701V, T716I, F888L, S982A, T1027I, D1118
  • the recombinant polypeptide is or includes the sequence set forth in SEQ ID NO: 16. In some embodiments, the recombinant polypeptide is or includes 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 of the amino acid sequence, 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, Comprising substitutions at 570, 614, 655, 681, 682, 683, 684, 685, 701, 716, 888, 982, 1027, 1118 and/or 1176 (with respect to the amino acid position of SEQ ID NO:
  • the recombinant polypeptide is or includes a variant of SEQ ID NO: 16, and the variant includes a variant selected from the group consisting of S13I, L18F, T20N, P26S, ⁇ 69-70 ( ⁇ HV), D80A, D138Y, G142D, ⁇ 144 ( ⁇ Y ), W152C, R190S, D215G, ⁇ 242-244( ⁇ LAL), R246I, ⁇ 400-402( ⁇ FVI), K417T, K417N, N440K, L452R, S477N, S477G, E484K, E484Q, N501Y, A570D, D614G, H6 55Y, P681H, Any one, two, three, four, five or more mutations in the group consisting of P681R, R682G, R683S, R685G, A701V, T716I, F888L, S982A, T1027I, D1118H
  • the recombinant polypeptide is or includes the sequence set forth in SEQ ID NO: 17. In some embodiments, the recombinant polypeptide is or includes 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 of the amino acid sequence, 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, Comprising substitutions at 570, 614, 655, 681, 682, 683, 684, 685, 701, 716, 888, 982, 1027, 1118 and/or 1176 (with respect to the amino acid position of SEQ ID NO:
  • the recombinant polypeptide is or includes a variant of SEQ ID NO: 17, and the variant includes a variant selected from the group consisting of S13I, L18F, T20N, P26S, ⁇ 69-70 ( ⁇ HV), D80A, D138Y, G142D, ⁇ 144 ( ⁇ Y ), W152C, R190S, D215G, ⁇ 242-244( ⁇ LAL), R246I, ⁇ 400-402( ⁇ FVI), K417T, K417N, N440K, L452R, S477N, S477G, E484K, E484Q, N501Y, A570D, D614G, H6 55Y, P681H, Any one, two, three, four, five or more mutations in the group consisting of P681R, R682G, R683S, R685G, A701V, T716I, F888L, S982A, T1027I, D1118H
  • the recombinant polypeptide is or includes the sequence set forth in SEQ ID NO:18. In some embodiments, the recombinant polypeptide is or includes at least or about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89% identical to SEQ ID NO: 18 , 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity of the amino acid sequence, 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, Sequences containing substitutions, deletions and/or insertions at 685, 701, 716, 888, 982, 1027, 11
  • the recombinant polypeptide is or includes a variant of SEQ ID NO: 18, and the variant includes a variant selected from the group consisting of S13I, L18F, T20N, P26S, ⁇ 69-70 ( ⁇ HV), D80A, D138Y, G142D, ⁇ 144 ( ⁇ Y ), W152C, R190S, D215G, ⁇ 242-244( ⁇ LAL), R246I, ⁇ 400-402( ⁇ FVI), K417T, K417N, N440K, L452R, S477N, S477G, E484K, E484Q, N501Y, A570D, D614G, H6 55Y, P681H, Any one, two, three, four, five or more mutations in the group consisting of P681R, R682G, R683S, R685G, A701V, T716I, F888L, S982A, T1027I, D1118H
  • the recombinant polypeptide is or includes the sequence set forth in SEQ ID NO: 19. In some embodiments, the recombinant polypeptide is or includes 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 of the amino acid sequence, 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, Comprising substitutions at 570, 614, 655, 681, 682, 683, 684, 685, 701, 716, 888, 982, 1027, 1118 and/or 1176 (with respect to the amino acid position of SEQ ID NO:
  • the recombinant polypeptide is or includes a variant of SEQ ID NO: 19, and the variant includes a variant selected from the group consisting of S13I, L18F, T20N, P26S, ⁇ 69-70 ( ⁇ HV), D80A, D138Y, G142D, ⁇ 144 ( ⁇ Y ), W152C, R190S, D215G, ⁇ 242-244( ⁇ LAL), R246I, ⁇ 400-402( ⁇ FVI), K417T, K417N, N440K, L452R, S477N, S477G, E484K, E484Q, N501Y, A570D, D614G, H6 55Y, P681H, Any one, two, three, four, five or more mutations in the group consisting of P681R, R682G, R683S, R685G, A701V, T716I, F888L, S982A, T1027I, D1118H
  • the recombinant polypeptide is or includes the sequence set forth in SEQ ID NO:20. In some embodiments, the recombinant polypeptide is or includes at least or about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89% identical to SEQ ID NO:20 , 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity of the amino acid sequence, 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, Comprising a substitution at 570, 614, 655, 681, 682, 683, 684, 685, 701, 716, 888, 982, 1027, 1118 and/or 1176 (with respect to
  • the recombinant polypeptide is or includes a variant of SEQ ID NO: 20, and the variant includes a variant selected from the group consisting of S13I, L18F, T20N, P26S, ⁇ 69-70 ( ⁇ HV), D80A, D138Y, G142D, ⁇ 144 ( ⁇ Y ), W152C, R190S, D215G, ⁇ 242-244( ⁇ LAL), R246I, ⁇ 400-402( ⁇ FVI), K417T, K417N, N440K, L452R, S477N, S477G, E484K, E484Q, N501Y, A570D, D614G, H6 55Y, P681H, Any one, two, three, four, five or more mutations in the group consisting of P681R, R682G, R683S, R685G, A701V, T716I, F888L, S982A, T1027I, D1118H
  • the recombinant polypeptide is or includes the sequence set forth in SEQ ID NO: 21. In some embodiments, the recombinant polypeptide is or includes at least or about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89% with SEQ ID NO:21 , 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity of the amino acid sequence, 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, Comprising substitutions at 570, 614, 655, 681, 682, 683, 684, 685, 701, 716, 888, 982, 1027, 1118 and/or 1176 (with respect to the amino acid sequence, including
  • the recombinant polypeptide is or includes a variant of SEQ ID NO: 21, and the variant includes a variant selected from the group consisting of S13I, L18F, T20N, P26S, ⁇ 69-70 ( ⁇ HV), D80A, D138Y, G142D, ⁇ 144 ( ⁇ Y ), W152C, R190S, D215G, ⁇ 242-244( ⁇ LAL), R246I, ⁇ 400-402( ⁇ FVI), K417T, K417N, N440K, L452R, S477N, S477G, E484K, E484Q, N501Y, A570D, D614G, H6 55Y, P681H, Any one, two, three, four, five or more mutations in the group consisting of P681R, R682G, R683S, R685G, A701V, T716I, F888L, S982A, T1027I, D1118H
  • the recombinant polypeptide is or includes the sequence set forth in SEQ ID NO: 22. In some embodiments, the recombinant polypeptide is or includes 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 of the amino acid sequence, 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, Comprising substitutions at 570, 614, 655, 681, 682, 683, 684, 685, 701, 716, 888, 982, 1027, 1118 and/or 1176 (with respect to the amino acid position of SEQ ID NO: 22. In
  • the recombinant polypeptide is or includes a variant of SEQ ID NO: 22, and the variant includes a variant selected from the group consisting of S13I, L18F, T20N, P26S, ⁇ 69-70 ( ⁇ HV), D80A, D138Y, G142D, ⁇ 144 ( ⁇ Y ), W152C, R190S, D215G, ⁇ 242-244( ⁇ LAL), R246I, ⁇ 400-402( ⁇ FVI), K417T, K417N, N440K, L452R, S477N, S477G, E484K, E484Q, N501Y, A570D, D614G, H6 55Y, P681H, Any one, two, three, four, five or more mutations in the group consisting of P681R, R682G, R683S, R685G, A701V, T716I, F888L, S982A, T1027I, D1118H
  • the recombinant polypeptide is or includes the sequence set forth in SEQ ID NO:23. In some embodiments, the recombinant polypeptide is or includes at least or about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89% identical to SEQ ID NO:23 , 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity of the amino acid sequence, 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, Comprising a substitution at 570, 614, 655, 681, 682, 683, 684, 685, 701, 716, 888, 982, 1027, 1118 and/or 1176 (with respect to
  • the recombinant polypeptide is or includes the sequence set forth in SEQ ID NO: 24. In some embodiments, the recombinant polypeptide is or includes at least or about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89% identical to SEQ ID NO:24 , 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity of the amino acid sequence, 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, Comprising substitutions at 570, 614, 655, 681, 682, 683, 684, 685, 701, 716, 888, 982, 1027, 1118 and/or 1176 (with respect to the
  • the recombinant polypeptide is or includes a variant of SEQ ID NO: 24, and the variant includes a variant selected from the group consisting of S13I, L18F, T20N, P26S, ⁇ 69-70 ( ⁇ HV), D80A, D138Y, G142D, ⁇ 144 ( ⁇ Y ), W152C, R190S, D215G, ⁇ 242-244( ⁇ LAL), R246I, ⁇ 400-402( ⁇ FVI), K417T, K417N, N440K, L452R, S477N, S477G, E484K, E484Q, N501Y, A570D, D614G, H6 55Y, P681H, Any one, two, three, four, five or more mutations in the group consisting of P681R, R682G, R683S, R685G, A701V, T716I, F888L, S982A, T1027I, D1118H
  • the recombinant polypeptide is or includes the sequence set forth in SEQ ID NO: 25. In some embodiments, the recombinant polypeptide is or includes 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 of the amino acid sequence, 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, Comprising substitutions at 570, 614, 655, 681, 682, 683, 684, 685, 701, 716, 888, 982, 1027, 1118 and/or 1176 (with respect to the amino acid position of SEQ ID NO: 25. In
  • the recombinant polypeptide is or includes a variant of SEQ ID NO: 25, and the variant includes a variant selected from the group consisting of S13I, L18F, T20N, P26S, ⁇ 69-70 ( ⁇ HV), D80A, D138Y, G142D, ⁇ 144 ( ⁇ Y ), W152C, R190S, D215G, ⁇ 242-244( ⁇ LAL), R246I, ⁇ 400-402( ⁇ FVI), K417T, K417N, N440K, L452R, S477N, S477G, E484K, E484Q, N501Y, A570D, D614G, H6 55Y, P681H, Any one, two, three, four, five or more mutations in the group consisting of P681R, R682G, R683S, R685G, A701V, T716I, F888L, S982A, T1027I, D1118H
  • the recombinant polypeptide is or includes the sequence set forth in SEQ ID NO:26. In some embodiments, the recombinant polypeptide is or includes 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% Amino acid sequences of sequence identity include sequences containing substitutions, deletions and/or insertions at one or more amino acid positions of SEQ ID NO:26.
  • the recombinant polypeptide is or includes the sequence set forth in SEQ ID NO:85. In some embodiments, the recombinant polypeptide is or includes at least or about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89% with SEQ ID NO:26 , 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to an amino acid sequence including one or more of SEQ ID NO:85 Sequences containing substitutions, deletions and/or insertions at amino acid positions.
  • the recombinant polypeptide is or includes the sequence set forth in SEQ ID NO:86. In some embodiments, the recombinant polypeptide is or includes 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 an amino acid sequence including one or more of SEQ ID NO:86 Sequences containing substitutions, deletions and/or insertions at amino acid positions.
  • the recombinant polypeptide is or includes the sequence set forth in SEQ ID NO: 87. In some embodiments, the recombinant polypeptide is or includes at least or about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89% identical to SEQ ID NO:87 , 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to an amino acid sequence including one or more of SEQ ID NO:87 Sequences containing substitutions, deletions and/or insertions at amino acid positions.
  • the recombinant polypeptide is or includes the sequence set forth in SEQ ID NO:88. In some embodiments, the recombinant polypeptide is or includes 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 an amino acid sequence including one or more of SEQ ID NO:88 Sequences containing substitutions, deletions and/or insertions at amino acid positions.
  • the recombinant polypeptide is or includes the sequence set forth in SEQ ID NO:89. In some embodiments, the recombinant polypeptide is or includes 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 an amino acid sequence including one or more of SEQ ID NO:89 Sequences containing substitutions, deletions and/or insertions at amino acid positions.
  • the recombinant polypeptide is or includes the sequence set forth in SEQ ID NO:90. In some embodiments, the recombinant polypeptide is or includes 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 of the amino acid sequence, including one or more of SEQ ID NO: 90 Sequences containing substitutions, deletions and/or insertions at amino acid positions.
  • the recombinant polypeptide is or includes the sequence set forth in SEQ ID NO:91. In some embodiments, the recombinant polypeptide is or includes at least or about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89% identical to SEQ ID NO:91 ,90%,91%,92%,93%,94%,95%,96%,97%, Amino acid sequences with 98% or 99% sequence identity include sequences containing substitutions, deletions, and/or insertions at one or more amino acid positions of SEQ ID NO:91.
  • the recombinant polypeptide is or includes the sequence set forth in SEQ ID NO: 92. In some embodiments, the recombinant polypeptide is or includes at least or about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89% with SEQ ID NO:92 , 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to an amino acid sequence including one or more of SEQ ID NO: 92 Sequences containing substitutions, deletions and/or insertions at amino acid positions.
  • the recombinant polypeptide is or includes a sequence described in SEQ ID NO: 120. In some embodiments, the recombinant polypeptide is or includes a sequence that is at least or about 80%, 81%, 82% identical to SEQ ID NO: 120. , 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99 Amino acid sequences with % sequence identity include sequences containing substitutions, deletions, and/or insertions at one or more amino acid positions of SEQ ID NO: 120.
  • the recombinant polypeptide is or includes a sequence described in SEQ ID NO: 121. In some embodiments, the recombinant polypeptide is or includes a sequence that is at least or about 80%, 81%, 82% identical to SEQ ID NO: 121. , 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99 Amino acid sequences with % sequence identity include sequences containing substitutions, deletions and/or insertions at one or more amino acid positions of SEQ ID NO: 121.
  • the recombinant polypeptides provided herein not only combine to form trimers, but can also aggregate or be aggregated to produce a protein comprising multiple recombinant polypeptides.
  • the protein formed has a macroscopic structure.
  • macrostructure can confer structural stability to coronavirus antigens or immunogenic recombinant polypeptides, which in turn can provide access to potential antigenic sites capable of promoting immune responses.
  • trimeric recombinant polypeptides aggregate to form a protein comprising multiple trimeric recombinant polypeptides.
  • multiple trimerized recombinant polypeptides form a protein with a macroscopic structure.
  • a protein comprising a plurality of recombinant polypeptides described herein is an immunogen.
  • a protein comprising a plurality of recombinant polypeptides described herein is comprised in a nanoparticle.
  • the protein is directly attached to the nanoparticle, such as a protein nanoparticle.
  • the protein is indirectly linked to the nanoparticle.
  • a protein comprising a plurality of recombinant polypeptides described herein is contained in a virus-like particle (VLP).
  • VLP virus-like particle
  • complexes comprising a recombinant polypeptide selected from the group consisting of SEQ ID NOs: 1-26 and 85-92, or fragments, variants or mutants thereof, in any suitable combination.
  • complexes comprising a recombinant polypeptide selected from the group consisting of SEQ ID NOs: 1-26 and 85-92, or a trimer of fragments, variants or mutants thereof, wherein the recombinant polypeptide Trimers are formed through trimerization of disulfide bonds between polypeptides.
  • fusion proteins comprising a plurality of recombinant polypeptides, each recombinant polypeptide comprising from amino to carboxyl terminus: a) a first region that includes a non-chimeric coronavirus spike located on a first coronavirus The coronavirus spike protein extracellular domain that precedes the coronavirus spike protein receptor-binding domain (RBD) in the protein a part of; b) a second region comprising a coronavirus spike protein receptor binding domain (RBD) of a second coronavirus that is different from said first coronavirus; and c) a C-terminal propeptide of collagen, The C-terminal propeptide of the recombinant polypeptide forms an inter-polypeptide disulfide bond.
  • the fusion protein further includes a third region between the second region and the C-terminal propeptide of collagen.
  • the third region includes 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.
  • the third region includes the S2 domain of a fourth coronavirus, wherein the fourth coronavirus is the same as or different from the first, second, or fourth coronavirus.
  • the first region includes the N-terminal domain (NTD) of the first coronavirus.
  • the first region includes one or more amino acid residues that are different from the corresponding amino acid residue in the second coronavirus.
  • the second region includes one or more amino acid residues that are different from the corresponding amino acid residue in the first coronavirus.
  • the first and second coronaviruses are different variants or strains of the same coronavirus.
  • the first region includes the NTD of the first coronavirus
  • the second region includes the RBD of the second coronavirus
  • the first and second coronaviruses are different variants of SARS-CoV-2.
  • the first coronavirus and the second coronavirus are independently selected from the group consisting of B.1.1.529, B.1.617.2, B.1.526, B.1.1.143, P.2, B.1.351, P. 1.
  • trimeric fusion proteins comprising three recombinant polypeptides, each recombinant polypeptide including from amino to carboxyl terminus: a) a first region that includes SARS-CoV-2 of the B.1.526 lineage the coronavirus spike protein N-terminal domain (NTD); b) a second region that includes the coronavirus spike protein receptor binding domain (RBD) of SARS-CoV-2 of the B.1.351 lineage; and c ) C-terminal propeptide of collagen, wherein the C-terminal propeptide of the recombinant polypeptide forms an inter-polypeptide disulfide bond.
  • NTD coronavirus spike protein N-terminal domain
  • RBD coronavirus spike protein receptor binding domain
  • trimeric fusion proteins comprising three recombinant polypeptides, each recombinant polypeptide including from amino to carboxyl terminus: a) a first region that includes SARS-CoV of the B.1.1.529 lineage The N-terminal domain (NTD) of the coronavirus spike protein of -2; b) the second region, which includes the coronavirus spike of SARS-CoV-2 of the B.1.1.529 lineage or non-B.1.1.529 lineage a protein receptor binding domain (RBD); and c) a C-terminal propeptide of collagen, wherein the C-terminal propeptide of the recombinant polypeptide forms an inter-polypeptide disulfide bond.
  • NTD N-terminal domain
  • RBD protein receptor binding domain
  • trimeric fusion proteins comprising three recombinant polypeptides, each recombinant polypeptide including from amino to carboxyl terminus: a) a first region that includes B.1.1.529 lineage or non-B. 1.1.529 lineage of SARS-CoV-2 coronavirus spike protein N-terminal domain (NTD); b) the second region, which includes the B.1.1.529 lineage of SARS-CoV-2 coronavirus spike a protein receptor binding domain (RBD); and c) a C-terminal propeptide of collagen, wherein the C-terminal propeptide of the recombinant polypeptide forms an inter-polypeptide disulfide bond
  • provided herein are methods for preventing coronavirus infection in a mammal, comprising immunizing the mammal with an effective amount of a fusion protein disclosed herein.
  • neutralizing antibodies are produced in the mammal against the first and second coronaviruses.
  • the first and second coronaviruses are different variants of SARS-CoV-2, and the neutralizing antibodies produced in the mammal neutralize 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 of two or more SARS-CoV-2 viruses.
  • the neutralizing antibodies produced in the mammal neutralize B.1.1.529, B.1.617.2, B.1.526, B.1.1.143, P.2, B.1.351, P.1 , Three or more SARS-CoV-2 viruses of the B.1.1.7, B.1.617, and A.23.1 lineages.
  • methods include immunizing the mammal with two or more doses of the fusion protein.
  • the fusion protein is administered as a booster following one or more doses of immunogen, including a spike protein peptide comprising an NTD and an RBD from the same SARS-CoV-2 variant.
  • fusion polypeptides derived or modified from the spike (S) glycoprotein of coronaviruses, including SARS-CoV-1 and SARS-CoV-2.
  • the fusion polypeptides disclosed herein can be stabilized in a pre-fusion conformation compared to the wild-type S protein sequence of the coronavirus.
  • fusion to a trimerization domain prevents the spike protein peptides in the fusion protein from forming straight helices (e.g., similar to what occurs during membrane fusion).
  • cryo-EM structure of the S-trimer subunit vaccine candidate shows that it predominantly adopts a tightly closed prefusion state, unlike the full-length wild-type spike protein, which forms both prefusion and fusion in the presence of detergents post state.
  • the fusion protein can include an altered soluble S sequence with modifications that inactivate the S1/S2 cleavage site; the turn region between the heptad repeat 1 (HR1) region and the central helix (CH) region mutations in that prevent HR1 and CH from forming a straight helix; and/or truncation of the heptad repeat 2 region (HR2) in addition to stabilizing mutations.
  • the fusion proteins 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 in the prefusion state. spike protein.
  • mutations such as K986G/V987G, K986P/V987P, K986G/V987P or K986P/V987G are not necessary to stabilize the fusion polypeptides disclosed herein comprising the Protein Trimerization TM trimerization domain.
  • the mutation that inactivates the S1/S2 cleavage site can comprise replacing RRAR (682-685 in SEQ ID NO:55) with GSAG (SEQ ID NO:60), and turning Mutations in the region may include the double mutations K986G/V987G, K986P/V987P, K986G/V987P or K986P/V987G.
  • the truncation of HR2 entails deleting one or more residues set forth in SEQ ID NO: 65 at the C-terminus of the wild-type soluble S sequence.
  • the immunogenic polypeptide may further comprise (a) one or more proline or glycine substitutions, and/or (b) one or more amino acid residues in the HR1 region that interacts with HR2 Base insertion.
  • the immunogenic polypeptide can have one or more substitutions selected from A942P, S943P, A944P, A942G, S943G, and A944G.
  • the insertion may be a G or GS between any residues in A942-A944.
  • recombinant polypeptides mentioned herein, or fragments, variants or mutants thereof, in any suitable combination including recombinant polypeptides selected from the group consisting of SEQ ID NOs: 1-26 and 85-92 or fragments, variants or Trimers of mutants, in which the recombinant polypeptide forms trimers through trimerization of inter-polypeptide disulfide bonds, may be used in starters and/or boosters. Independently, the priming agent and/or any booster dose or doses may be administered without or with an adjuvant.
  • adjuvants may include: aluminum-containing adjuvants, such as alum and/or aluminum hydroxide-containing adjuvants; oligonucleotide-containing adjuvants, such as CpG-containing oligodeoxynucleotides (CpG- ODN) adjuvant; adjuvant containing TLR9 agonist; containing metabolizable oil, Adjuvants for alpha-tocopherol, and/or polyoxyethylene sorbitan monooleate (Tween-80), such as oil-in-water emulsions containing squalene, alpha-tocopherol, and Tween-80 and/or Span 85 adjuvant; or any combination of adjuvants.
  • aluminum-containing adjuvants such as alum and/or aluminum hydroxide-containing adjuvants
  • oligonucleotide-containing adjuvants such as CpG-containing oligodeoxynucleotides (CpG- ODN
  • polynucleotides 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.
  • polynucleotides encoding recombinant polypeptides provided herein are provided.
  • a polynucleotide comprises a single nucleic acid sequence, such as a nucleic acid sequence encoding a recombinant polypeptide.
  • the polynucleotides comprise 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.
  • a polynucleotide encoding a recombinant polypeptide includes at least one promoter operably linked to control expression of the recombinant polypeptide. In some embodiments, the polynucleotide includes two, three, or more promoters operably linked to control expression of the recombinant polypeptide.
  • 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 both Expression of one or more nucleic acid sequences.
  • the polynucleotide includes two, three, or more promoters operably linked to control expression of the recombinant polypeptide.
  • expression of the recombinant polypeptide is inducible or conditional.
  • a polynucleotide encoding a recombinant polypeptide includes a conditional promoter, enhancer, or transactivator.
  • the conditional promoter, enhancer or transactivator is an inducible promoter, enhancer or transactivator or a repressible promoter, enhancer or transactivator.
  • inducible or conditional promoters can be used to restrict expression of a recombinant polypeptide to a specific microenvironment.
  • expression driven by an inducible or conditional promoter is modulated by exposure to exogenous agents (eg, heat, radiation, or drugs).
  • the polynucleotide may further include a nucleic acid sequence encoding a peptide between the one or more nucleic acid sequences.
  • the nucleic acid located between the nucleic acid sequences encodes a peptide that separates the translation product of the nucleic acid sequence during or after translation.
  • 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.
  • a polynucleotide encoding a recombinant polypeptide is introduced into a composition containing a cultured cell (eg, a host cell), such as by retroviral transduction, transfection, or transformation. In some embodiments, this may allow expression (eg, production) of recombinant polypeptides. In some embodiments, the expressed recombinant polypeptide is purified.
  • the polynucleotides (nucleic acid molecules) provided herein encode a coronavirus antigen or immunogen as described herein. In some embodiments, the polynucleotides (nucleic acid molecules) provided herein encode a recombinant polypeptide comprising a coronavirus antigen or immunogen (eg, coronavirus S protein peptide) as described herein.
  • a coronavirus antigen or immunogen eg, coronavirus S protein peptide
  • a vector or construct includes one or more promoters operably linked to a nucleic acid molecule encoding a recombinant polypeptide to drive expression thereof.
  • 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.
  • the vector is a viral vector.
  • the viral vector is a retroviral vector.
  • the retroviral vector is a lentiviral vector.
  • the retroviral vector is a gamma-retroviral vector.
  • a vector or construct includes a single promoter that drives expression of one or more nucleic acid molecules of a polynucleotide.
  • promoters may be polycistronic (dicistronic or tricistronic, see, eg, U.S. Patent No. 6,060,273).
  • the transcription unit can be engineered as a bicistronic unit containing an IRES (internal ribosome entry site), which allows for coexpression of gene products (e.g., encoding different recombinant peptides).
  • the vectors provided herein are bicistronic, allowing the vector to contain and express two nucleic acid sequences.
  • the vectors provided herein are tricistronic, allowing the vector to contain and express three nucleic acid sequences.
  • a single promoter directs the expression of an RNA containing two or three genes (e.g., encoding a chimeric signaling receptor and encoding a recombinant receptor) in a single open reading frame (ORF) that Genes are separated from each other by sequences encoding self-cleaving peptides (eg, 2A sequences) or protease recognition sites (eg, furin).
  • ORF open reading frame
  • the ORF encodes a single polypeptide, which is processed into a single protein either during translation (in the case of 2A) or after translation.
  • peptides such as T2A can cause ribosomes to skip (ribosome hopping) the synthesis of the C-terminal peptide bond of the 2A element, resulting in dissociation 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 deFelipe et al. Traffic 5:616-626 (2004), which are incorporated by reference in their entirety for all purposes).
  • Many 2A components are known in the art.
  • Examples of 2A sequences useful in the methods and nucleic acids disclosed herein include, but are not limited to, those from foot-and-mouth disease virus (F2A), equine rhinitis A virus (E2A), Thosea asigna virus (T2A), and 2A sequence of porcine teschovirus-1 (P2A).
  • F2A foot-and-mouth disease virus
  • E2A equine rhinitis A virus
  • T2A Thosea asigna virus
  • P2A sequence of porcine teschovirus-1 P2A
  • the vector is contained within a virus.
  • the virus is a pseudovirus.
  • the virus is a virus-like particle.
  • the vector is contained in the cell.
  • the virus or cell containing the vector contains a recombinant genome.
  • immunogenic compositions comprising trimers of recombinant polypeptides comprising sequences selected from the group consisting of SEQ ID NOs: 1-26 and 85-92, or any A combination of two or more trimers.
  • immunogenic compositions comprising trimers of recombinant polypeptides having the sequence set forth in SEQ ID NO: 1.
  • the immunogenic composition mentioned herein includes a recombinant polypeptide selected from the group consisting of SEQ ID NO: 85-92, or a trimer of a fragment, variant or mutant thereof, wherein the recombinant polypeptide is formed by an inter-polypeptide disulfide bond.
  • trimerization forms trimers that can be used in starters and/or boosters. alone Alternatively, the priming agent and/or any booster dose or doses may be administered without or with an adjuvant.
  • adjuvants may include: aluminum-containing adjuvants, such as alum and/or aluminum hydroxide-containing adjuvants; oligonucleotide-containing adjuvants, such as CpG-containing oligodeoxynucleotides (CpG- ODN); adjuvants containing TLR9 agonists; adjuvants containing metabolizable oils, alpha-tocopherol, and/or polyoxyethylene sorbitan monooleate (Tween-80), e.g., in water Adjuvant containing squalene, alpha-tocopherol, and Tween-80 and/or Span 85 in the form of an oil emulsion; or any combination of adjuvants.
  • aluminum-containing adjuvants such as alum and/
  • a unit dose of the immunogenic composition may include from about 10 ⁇ g to about 100 ⁇ g SARS-CoV-2 antigen, preferably from about 25 ⁇ g to about 75 ⁇ g SARS-CoV-2 antigen, preferably from about 40 ⁇ g to about 60 ⁇ g SARS-CoV. -2 antigen or approximately 50 ⁇ g SARS-CoV-2 antigen.
  • the dose contains 3 ⁇ g of SARS-CoV-2 antigen.
  • the dose contains 9 ⁇ g of SARS-CoV-2 antigen.
  • the dose contains 30 ⁇ g of SARS-CoV-2 antigen.
  • compositions including recombinant coronavirus S antigens (e.g., trimers or proteins) as described herein can be combined with those recommended by the Advisory Committee on Immunization Practices (ACIP; cdc.gov/vaccines/acip/index.html) for target ages.
  • Other vaccines eg, influenza vaccine or varicella-zoster vaccine
  • infants eg, infants approximately one to six months old.
  • the disclosed immunogens including the recombinant coronavirus S antigens described herein may be used against, for example, hepatitis B (HepB), diphtheria, tetanus and pertussis (DTaP), pneumococci (PCV), Haemophilus influenzae type b (Hib), polio, influenza, and rotavirus vaccines are administered simultaneously or sequentially.
  • HepB hepatitis B
  • DTaP diphtheria
  • PCV pneumococci
  • Hib Haemophilus influenzae type b
  • polio influenza
  • rotavirus vaccines are administered simultaneously or sequentially.
  • Multivalent or combination vaccines provide protection against multiple pathogens.
  • polyvalent vaccines can protect against multiple strains of the same pathogen.
  • polyvalent vaccines protect against multiple pathogens, such as the combination vaccine Tdap, which protects against strains of tetanus, pertussis, and diphtheria.
  • Multivalent vaccines are necessary to minimize the number of immunizations required to confer protection against multiple pathogens or virulent strains, in order to reduce administration costs and improve coverage. This may be particularly useful when vaccinating infants or children, for example.
  • a vaccine comprising an immunogenic composition described herein is a multivalent vaccine.
  • 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).
  • Antigens for incorporation into multivalent vaccine compositions can be derived from one strain or multiple strains (eg, between two and five strains) of coronavirus to provide a broader spectrum of protection.
  • the antigens used for incorporation into the multivalent vaccine composition are derived from multiple strains of coronavirus.
  • antigens include live, attenuated and inactivated viruses, such as inactivated poliovirus (Jiang et al., J. Biol. Stand., (1986) 14:103-9), influenza A Attenuated strains of hepatitis virus (Bradley et al., J. Med. Virol., (1984) 14:373-86), attenuated measles virus (James et al., N. Engl. J.
  • viruses such as inactivated poliovirus (Jiang et al., J. Biol. Stand., (1986) 14:103-9), influenza A Attenuated strains of hepatitis virus (Bradley et al., J. Med. Virol., (1984) 14:373-86), attenuated measles virus (James et al., N. Engl. J.
  • the vaccines presented here are universal vaccines.
  • a universal vaccine is one that protects against multiple strains of the same virus (eg, multiple strains of coronavirus).
  • Develop effective communication A coronavirus vaccine would reduce costs and labor, such as using seasonal vaccine formulations, and allow for stronger pandemic prevention.
  • the immunogens described herein can be used as a primary series, an additional dose, and/or a homologous or heterologous booster dose, e.g., a first dose, a second dose, or a booster dose. Second dose, third dose, fourth dose, 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 dose, second dose, third dose, third dose within five years, within four years, within three years, within two years, within one year, and/or within six months. Four doses, and/or more doses are used.
  • a universal vaccine is a vaccine composed of multiple epitopes derived from different strains of the virus.
  • a universal vaccine consists of a single epitope that is conserved across different strains of the virus.
  • a universal vaccine could be based on relatively conserved domains of the S protein.
  • Immunogenic combinations comprising a disclosed immunogen (e.g., a disclosed recombinant coronavirus S antigen or a nucleic acid molecule encoding a protomer of a disclosed recombinant coronavirus S antigen) and a pharmaceutically acceptable carrier are also provided. things.
  • immunogenic compositions include a trimerized recombinant polypeptide provided herein and optionally a pharmaceutically acceptable carrier.
  • an immunogenic composition includes a trimerized recombinant polypeptide provided herein and disodium hydrogen phosphate, e.g., disodium hydrogen phosphate dihydrate, sodium phosphate dibasic, e.g., disodium hydrogen phosphate monohydrate, sodium chloride , and Twain 80.
  • 1.0 mL of an aqueous immunogenic composition solution includes 720 ⁇ g of a trimerized recombinant polypeptide provided herein and 0.62 mg disodium hydrogen phosphate dihydrate, 0.62 mg disodium hydrogen phosphate monohydrate, 9.0 mg sodium chloride, and 0.2mg Tween 80.
  • immunogenic compositions include a protein comprising a plurality of trimerized recombinant polypeptides provided herein and optionally a pharmaceutically acceptable carrier.
  • immunogenic compositions include protein nanoparticles provided herein and optionally a pharmaceutically acceptable carrier.
  • immunogenic compositions include VLPs provided herein and optionally a pharmaceutically acceptable carrier.
  • immunogenic compositions include an isolated nucleic acid provided herein and optionally a pharmaceutically acceptable carrier.
  • immunogenic compositions include a carrier provided herein and optionally a pharmaceutically acceptable carrier.
  • immunogenic compositions include a virus provided herein and optionally a pharmaceutically acceptable carrier.
  • immunogenic compositions include pseudoviruses provided herein and optionally a pharmaceutically acceptable carrier. In some embodiments, immunogenic compositions include cells provided herein and optionally a pharmaceutically acceptable carrier. In some embodiments, an 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 vaccines are prophylactic and therapeutic vaccines. Such pharmaceutical compositions may be administered to a subject via a variety of modes of administration known to those of ordinary skill in the art, for example, intramuscular, intradermal, subcutaneous, intravenous, intraarterial, intraarticular, intraperitoneal, nasal.
  • compositions including one or more disclosed immunogens are immunogenic compositions.
  • Actual methods for preparing administrable compositions are known or apparent to those skilled in the art and are described, for example, in Remingtons Pharmaceutical Sciences, 19th Ed., Mack Publishing Company, Easton, Pa., 1995, is described in more detail in this publication.
  • immunogens described herein such as recombinant coronavirus S antigens, such as trimers, proteins
  • pharmaceutically acceptable carriers include, but are not limited to, physiologically balanced media, phosphate buffered saline, water, emulsions (e.g., oil/water or water/oil emulsions), various types of wetting agents, antifreeze additives or stabilizers, e.g.
  • Proteins, peptides or hydrolysates eg, albumin, gelatin
  • sugars eg, sucrose, lactose, sorbitol
  • amino acids eg, sodium glutamate
  • the resulting aqueous solution can be packaged and used as is or lyophilized. Lyophilized formulations are mixed with sterile solutions prior to single or multiple dose administration.
  • Formulated compositions 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.
  • Bacteriostatic agents may be contraindicated in some patients; therefore, lyophilized preparations may be reconstituted in solutions with or without such ingredients.
  • the immunogenic composition of the present invention may contain pharmaceutically acceptable carrier substances required to approximate physiological conditions, such as pH adjusters and buffers, tension adjusters, wetting agents, etc., such as sodium acetate, sodium lactate, sodium chloride , potassium chloride, calcium chloride, sorbitan monolaurate and triethanolamine oleate.
  • Immunogenic compositions may optionally include adjuvants to enhance the host's immune response.
  • Suitable adjuvants are, for example, toll-like receptor (TLR) agonists, alum, AlPO 4 , alhydrogel, lipid-A and its derivatives or variants, oil emulsions, saponins, neutral liposomes , liposomes containing vaccines and cytokines, non-ionic block copolymers and chemokines.
  • TLR toll-like receptor
  • Nonionic block polymers containing polyoxyethylene (POE) and polyoxypropylene (POP) may be used as adjuvants, among many other suitable adjuvants well known in the art, such as POE-POP-POE blocks.
  • immunogenic compositions of the invention may include or be administered with more than one adjuvant. In some embodiments, immunogenic compositions of the invention may include or be administered with two adjuvants.
  • the immunogenic compositions of the present invention may include or be administered with various adjuvants.
  • a vaccine such as one comprising an immunogenic composition provided herein may include or be administered in conjunction with a variety of adjuvants.
  • suitable adjuvants include, for example, aluminum hydroxide, lecithin, Freund's adjuvant, MPL TM and IL-1, one or a combination of any of them may be combined with a adjuvant selected from the group consisting of SEQ ID NO: 85 - Trimers of recombinant polypeptides or fragments, variants or mutants of the group consisting of -92 are used together.
  • the vaccine compositions or nanoparticle immunogens disclosed herein eg, SARS-COV-2 vaccine compositions
  • Various pharmaceutical compositions can be prepared according to standard procedures well known in the art.
  • the immunogenic composition of the present invention includes a recombinant polypeptide selected from the group consisting of SEQ ID NO: 85-92, or a trimer of a fragment, variant or mutant thereof, wherein the recombinant polypeptide is by polypeptide Trimerization of metadisulfide bonds to form trimers may include an adjuvant formulation containing a metabolizable oil (e.g., squalene) and alpha-tocopherol (e.g., DL-alpha- Tocopherol), and polyoxyethylene sorbitan monooleate (Tween-80).
  • a metabolizable oil e.g., squalene
  • alpha-tocopherol e.g., DL-alpha- Tocopherol
  • Tween-80 polyoxyethylene sorbitan monooleate
  • the adjuvant formulation may include about 2% to about 10% squalene, about 2% to about 10% alpha-tocopherol (eg, D-alpha-tocopherol), and about 0.3% to about 3% Polyoxyethylene sorbitan monooleate. In some embodiments, the adjuvant formulation can include about 5% squalene, about 5% tocopherol, and about 0.4% polyoxyethylene sorbitan monooleate.
  • immunogenic compositions of the invention may comprise 3 deO-acylated monophosphate lipid A (3D-MPL) and an adjuvant in the form of an oil-in-water emulsion, the adjuvant comprising a metabolizable oil, Alpha-tocopherol and polyoxyethylene sorbitan monooleate.
  • the immunogenic composition of the present invention may comprise QS21 (Quillaja saponaria Molina extract: component 21), 3D-MPL and an oil-in-water emulsion, wherein the oil-in-water emulsion includes metabolizable oil, ⁇ - Tocopherol and polyoxyethylene sorbitan monooleate.
  • the immunogenic composition of the invention may comprise QS21, 3D-MPL and an oil-in-water emulsion, wherein the oil-in-water emulsion has the following composition: metabolizable oil, such as squalene, alpha-tocopherol , and Tween-80 and/or Span 85.
  • the immunogenic compositions of the present invention may include an adjuvant in the form of a liposome composition.
  • the immunogenic composition of the present invention includes a recombinant polypeptide selected from the group consisting of SEQ ID NO: 85-92, or a trimer of a fragment, variant or mutant thereof, wherein the recombinant polypeptide is by polypeptide Trimerization of disulfide bonds to form trimers may include adjuvant formulations including metabolizable oils (e.g., squalene), alpha-tocopherol, polyoxyethylene sorbitan monooleic acid ester (Tween-80), and/or Span 85.
  • metabolizable oils e.g., squalene
  • alpha-tocopherol alpha-tocopherol
  • polyoxyethylene sorbitan monooleic acid ester Tween-80
  • Span 85 polyoxyethylene sorbitan monooleic acid ester
  • the adjuvant formulation can include about 5% (w/v) squalene, about 5% (w/v) alpha-tocopherol, about 0.5% (w/v) polyoxyethylene sorbitan monooleate, and/or about 0.5% (w/v) Span 85.
  • the immunogenic composition of the present invention includes a recombinant polypeptide selected from the group consisting of SEQ ID NO: 85-92, or a trimer of a fragment, variant or mutant thereof, wherein the recombinant polypeptide is by polypeptide Trimerization of disulfide bonds to form trimers may include an adjuvant formulation comprising Quillaja saponin, cholesterol and phospholipids, for example, in the form of a nanoparticle composition.
  • immunogenic compositions of the invention may comprise a mixture of individually purified Quillaja saponaria Molina fractions, which are subsequently formulated with cholesterol and phospholipids.
  • the immunogenic composition of the present invention includes a recombinant polypeptide selected from the group consisting of SEQ ID NO: 85-92, or a trimer of fragments, variants or mutants thereof, wherein the recombinant polypeptide is by polypeptide
  • the inter-disulfide bonds are trimerized to form a trimer, which may contain an adjuvant 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 ⁇ -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 ⁇ -tocopherol modulates innate immune response and leads to improved adaptive immunity, Vaccine, doi:10.1016/j.vaccine.2011.01.011 (2011), any one or several combinations of adjuvants disclosed in this document, the full text of which is used for all purposes. The form is incorporated into this article by reference.
  • the immunogenic composition of the present invention includes a recombinant polypeptide selected from the group consisting of SEQ ID NO: 85-92, or a trimer of fragments, variants or mutants thereof, wherein the recombinant polypeptide is by polypeptide
  • the disulfide bonds trimerize to form a trimer, which may contain a toll-like receptor 9 (TLR9) agonist, where the TLR9 agonist is an oligonucleotide with a length of 8 to 35 nucleotides and contains unmethylated
  • TLR9 agonist is an oligonucleotide with a length of 8 to 35 nucleotides and contains unmethylated
  • TLR9 agonist is an oligonucleotide with a length of 8 to 35 nucleotides and contains unmethylated
  • TLR9 (CD289) recognizes the unmethylated cytidine-phosphate-guanosine (CpG) motif found in microbial DNA, which can be mimicked using synthetic CpG-containing oligodeoxynucleotides (CpG-ODN).
  • CpG-ODN is known to enhance antibody production and stimulate T helper 1 (Th1) cell responses (Coffman et al., Immunity, 33:492-503, 2010, incorporated by reference in its entirety for all purposes).
  • oligonucleotide TLR9 agonists typically contain palindromic sequences of the general formula: 5'-purine-purine-CG-pyrimidine-pyrimidine-3' or 5'-purine-purine-CG-pyrimidine-pyrimidine-CG- 3'.
  • U.S. Patent No. 6,589,940 which is incorporated by reference in its entirety.
  • CpG oligonucleotides are linear.
  • the CpG oligonucleotide is circular or includes a hairpin loop.
  • CpG oligonucleotides can be single-stranded or double-stranded.
  • CpG oligonucleotides can contain 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.
  • Modified bases can be included at the back of a CpG oligonucleotide as long as they retain the same specificity for their native complement via Watson-Crick base pairing (e.g., the palindrome remains self-complementary). in text sequence.
  • CpG oligonucleotides include non-canonical bases.
  • CpG oligonucleotides include modified nucleosides.
  • the modified nucleoside is selected from the group consisting of 2'-deoxy-7-deazaguanosine, 2'-deoxy-6-thioguanosine, arabinosine glycosides (arabinoguanosine), 2'-deoxy-2'-substituted-arabinoguanosine and 2'-O-substituted-arabinoguanosine.
  • CpG oligonucleotides may contain modifications to the phosphate group.
  • phosphate modifications include, but are not limited to, methylphosphonate, phosphorothioate, phosphoramide (bridged or non-bridged), phosphotriester, and phosphorodithioate, and may be in any combination use. Other non-phosphate linkages may also be used.
  • the oligonucleotide includes only a phosphorothioate backbone. In some embodiments, the oligonucleotide includes only a phosphodiester backbone.
  • the oligonucleotide includes a combination of phosphate linkages in the phosphate backbone, such as a combination of phosphodiester linkages and phosphorothioate linkages.
  • Oligonucleotides with a phosphorothioate backbone can be more immunogenic than oligonucleotides with a phosphodiester backbone and appear to be more resistant to degradation after injection into the host (Braun et al., J Immunol, 141:2084-2089, 1988; and Latimer et al., Mol Immunol, 32:1057-1064, 1995, are incorporated by reference in their entirety for all purposes).
  • CpG oligonucleotides of the invention include at least one, two or three internucleotide phosphorothioate linkages.
  • two stereoisomers of the phosphorothioate bond are present in the multiple CpG oligonucleotide molecules. in the nucleotide molecule.
  • All internucleotide linkages of CpG oligonucleotides are phosphorothioate linkages, or in other words, CpG oligonucleotides have a phosphorothioate backbone.
  • any suitable CpG oligodeoxynucleotide (ODN) or combination thereof may be used as an adjuvant.
  • K-type ODN also known as B-type
  • Type K ODN can be based on the following sequence The use of phosphorothioate nucleotides increases resistance to nuclease digestion compared to natural phosphodiester nucleotides, resulting in a significantly longer half-life in vivo.
  • K-type ODN triggers pDC differentiation and production of TNF- ⁇ , and triggers B cell proliferation and secretion of IgM.
  • Type D ODN (also known as type A) consists of a mixed phosphodiester/phosphorothioate backbone containing a single CpG motif flanked by palindromic sequences and with poly-G at the 3' and 5' ends Tail (structural motif that promotes concatemer formation).
  • Type D ODN can be based on the following sequence GGTGCAT CG ATGCAGGGGGG.
  • Type D ODN triggers pDC maturation and secretion of IFN- ⁇ , but has no effect on B cells.
  • Type C ODN is similar to type K in being composed entirely of phosphorothioate nucleotides, but similar to type D ODN, contains a palindromic CpG motif.
  • Type C ODN can be based on the following sequence This type of ODN stimulates B cells to secrete IL-6 and pDC to produce IFN- ⁇ .
  • P-type ODN contains two palindromic sequences, allowing them to form a higher ordered structure.
  • P-type ODN can be based on the following sequence Compared with C-type ODN, P-type ODN activates B cells and PDCs and induces greater IFN- ⁇ production.
  • boldface letters in ODN sequences indicate self-complementary palindromes, and CpG motifs are underlined.
  • CpG ODNs such as CpG 7909 (5′-TCGTCGTTTTGTCGTTTTGTCGTT-3′) and CpG 1018 (5′-TGACTGTGAACGTTCGAGATGA-3′), are known and disclosed in U.S. Patent Nos. 7,255,868, 7,491,706, 7,479,285, 7,745,598, 7,785 ,610, and Bode et al., “CpG DNA as a vaccine adjuvant”, Exper t Rev Vaccines(2011),10(4):499-511, All of which are incorporated by reference in their entirety for all purposes.
  • One or more adjuvants may be used in combination, including but not limited to alum (aluminum salt), oil-in-water emulsions, water-in-oil emulsions, liposomes, and microparticles, such as poly(lactide-co-glycolide) ) particles (Shah et al., Methods Mol Biol, 1494:1-14, 2017, incorporated by reference in its entirety for all purposes).
  • the immunogenic composition further includes an aluminum salt adjuvant that adsorbs SARS-CoV-2 antigen.
  • the aluminum salt adjuvant includes one or more from the group consisting of amorphous aluminum hydroxyphosphate sulfate, aluminum hydroxide, aluminum phosphate, and aluminum potassium sulfate. In some embodiments, the aluminum salt adjuvant includes one or both of aluminum hydroxide and aluminum phosphate. In some embodiments, the aluminum salt adjuvant includes aluminum hydroxide.
  • a unit dose of the immunogenic composition includes 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 0.40 mg Al 3+ ; or about 0.25 mg to about 0.50 mg Al 3+ ; or about 0.068 mg Al 3+ , about 0.078 mg Al 3+ , about 0.13 mg Al 3+ , about 0.19 mg Al 3+ , about 0.22 mg Al 3+ , about 0.26 mg, about 0.35 mg Al 3+ .
  • the immunogenic composition further includes an additional adjuvant.
  • suitable adjuvants include, but are not limited to, squalene-in-water emulsions (e.g., MF59 or AS03), TLR3 agonists (e.g., polyIC or polyICLC), TLR4 agonists (e.g., bacterial lipopolysaccharide derivatives such as monophosphate lipid A (MPL), and/or saponins such as Quil A or QS- 21, such as AS01 or AS02), TLR5 agonists (bacterial flagellin) and TLR7, TLR8 and/or TLR9 agonists (imidazoquinoline derivatives, such as imiquimod and resiquimod) (Coffman et al., Immunity, 33:492-503, 2010, incorporated by reference in its entirety for all purposes).
  • additional adjuvants include MPL and aluminum hydroxide/alum (eg, AS04).
  • MPL aluminum hydroxide/alum
  • AS04 aluminum hydroxide/alum
  • a unit dose of the immunogenic composition may include from about 3 ⁇ g to about 1000 ⁇ g of one or more adjuvants, from about 8 ⁇ g to about 1000 ⁇ g of one or more adjuvants, preferably from about 25 ⁇ g to about 500 ⁇ g.
  • One or more adjuvants preferably about 9 ⁇ g to about 500 ⁇ g of one or more adjuvants, preferably about 50 ⁇ g to about 300 ⁇ g of one or more adjuvants, preferably about 100 ⁇ g to about 250 ⁇ g of one or more adjuvants
  • Multiple adjuvants preferably from about 150 ⁇ g to about 225 ⁇ g of one or more adjuvants.
  • a unit dose of the immunogenic composition may include about 3 ⁇ g to about 700 ⁇ g, 750 ⁇ g, 800 ⁇ g of aluminum-containing adjuvant, about 3 ⁇ g to about 500 ⁇ g of aluminum-containing adjuvant, about 8 ⁇ g to about 250 ⁇ g of aluminum-containing adjuvant, About 9 ⁇ g to about 240 ⁇ g of aluminum-containing adjuvant, preferably about 25 ⁇ g to about 230 ⁇ g of aluminum-containing adjuvant, preferably about 200 ⁇ g to about 800 ⁇ g of aluminum-containing adjuvant, preferably about 700 ⁇ g to about 800 ⁇ g of aluminum-containing adjuvant, preferably about 740 ⁇ g to about 780 ⁇ g of aluminum-containing adjuvant.
  • Aluminum adjuvant preferably about 750, 760, 770 ⁇ g to about 780 ⁇ g aluminum-containing adjuvant, preferably about 260 ⁇ g to about 280 ⁇ g aluminum-containing adjuvant, preferably about 200 ⁇ g to about 240 ⁇ g aluminum-containing adjuvant, preferably about 210 ⁇ g to about 230 ⁇ g aluminum-containing adjuvant agent, preferably about 220 ⁇ g to about 230 ⁇ g of aluminum-containing adjuvant, preferably about 224 ⁇ g to about 228 ⁇ g of aluminum-containing adjuvant, preferably about 50 ⁇ g to about 125 ⁇ g of aluminum-containing adjuvant, preferably about 750 ⁇ g of aluminum-containing adjuvant, such as Alum, about 50 ⁇ g of aluminum-containing adjuvant Adjuvant, about 45 ⁇ g of aluminum-containing adjuvant, about 40 ⁇ g of aluminum-containing adjuvant, about 35 ⁇ g of aluminum-containing adjuvant, about 25 ⁇ g of aluminum-containing adjuvant
  • a unit dose of the immunogenic composition may include from about 10 ⁇ g to about 2000 ⁇ g CpG adjuvant, or from about 20 ⁇ g to about 1800, 1700, 1600 ⁇ g CpG adjuvant, or from about 40 ⁇ g to about 1500 ⁇ g CpG adjuvant, or About 1000 ⁇ g to about 1900 ⁇ g CpG adjuvant, or about 1200 ⁇ g to about 1800 ⁇ g CpG adjuvant, or about 1300 ⁇ g to about 1700 ⁇ g CpG adjuvant, or about 1400 ⁇ g to about 1600 ⁇ g CpG adjuvant, or about 10 ⁇ g to about 500 ⁇ g CpG adjuvant, or About 460 ⁇ g to about 470 ⁇ g CpG adjuvant, or about 410 ⁇ g, 420 ⁇ g, 430 ⁇ g, 440 ⁇ g to about 490 ⁇ g CpG adjuvant, or about 10 ⁇ g to about 500 ⁇ g CpG adjuvant, or
  • a unit dose of the immunogenic composition may include from about 10 ⁇ g to about 500 ⁇ g of aluminum-containing adjuvant and from about 10 ⁇ g to about 500 ⁇ g of CpG adjuvant. In some embodiments, a unit dose of the immunogenic composition may include from about 10 ⁇ g to about 800 ⁇ g of aluminum-containing adjuvant and from about 10 ⁇ g to about 1800 ⁇ g of CpG adjuvant. In some embodiments, the unit dose of the immunogenic composition may include About 25 ⁇ g to about 250 ⁇ g aluminum-containing adjuvant and about 25 ⁇ g to Approximately 300 ⁇ g CpG adjuvant.
  • a unit dose of the immunogenic composition may include about 200 ⁇ g to about 800 ⁇ g of aluminum-containing adjuvant and about 500 ⁇ g to about 1800 ⁇ g of CpG adjuvant. In some embodiments, a unit dose of the immunogenic composition may include about 200 ⁇ g to about 250 ⁇ g of aluminum-containing adjuvant and about 400 ⁇ g to about 500 ⁇ g of CpG adjuvant. In some embodiments, a unit dose of the immunogenic composition may include about 210 ⁇ g to about 240 ⁇ g of aluminum-containing adjuvant and about 420 ⁇ g to about 490 ⁇ g of CpG adjuvant.
  • a unit dose of the immunogenic composition may include about 220 ⁇ g to about 230 ⁇ g of aluminum-containing adjuvant and about 430 ⁇ g to about 470 ⁇ g of CpG adjuvant. In some embodiments, a unit dose of the immunogenic composition may include about 221 ⁇ g to about 227 ⁇ g of aluminum-containing adjuvant and about 440 ⁇ g to about 460 ⁇ g of CpG adjuvant. In some embodiments, a unit dose of the immunogenic composition may include about 600 ⁇ g to about 900 ⁇ g of aluminum-containing adjuvant and about 1000 ⁇ g to about 2000 ⁇ g of CpG adjuvant.
  • a unit dose of the immunogenic composition may include about 700 ⁇ g to about 800 ⁇ g of aluminum-containing adjuvant and about 1100 ⁇ g to about 1900 or 1800 ⁇ g of CpG adjuvant. In some embodiments, a unit dose of the immunogenic composition may include about 720 ⁇ g to about 790 ⁇ g of aluminum-containing adjuvant and about 1200 or 1300 ⁇ g to about 1700 ⁇ g of CpG adjuvant. In some embodiments, a unit dose of the immunogenic composition may include from about 730 or 740 ⁇ g to about 760 ⁇ g of aluminum-containing adjuvant and from about 1400 ⁇ g to about 1600 ⁇ g of CpG adjuvant.
  • a unit dose of the immunogenic composition may include about 50 ⁇ g to about 125 ⁇ g of aluminum-containing adjuvant and about 50 ⁇ g to about 250 ⁇ g of CpG adjuvant. In some embodiments, a unit dose of the immunogenic composition may include about 50 ⁇ g to about 100 ⁇ g of aluminum-containing adjuvant and about 75 ⁇ g to about 200 ⁇ g of CpG adjuvant. In some embodiments, a unit dose of the immunogenic composition may include about 50 ⁇ g to about 100 ⁇ g of aluminum-containing adjuvant and about 100 ⁇ g to about 175 ⁇ g of CpG adjuvant.
  • a unit dose of the immunogenic composition can include about 75 ⁇ g to about 100 ⁇ g of an aluminum-containing adjuvant, such as Alum, and about 150 ⁇ g of a CpG adjuvant, such as CpG 1018A.
  • a unit dose of the immunogenic composition may include about 200 ⁇ g to about 250 ⁇ g of an aluminum-containing adjuvant, such as Alum, and about 400 ⁇ g to about 500 ⁇ g of a CpG adjuvant, such as CpG 1018A.
  • a unit dose of the immunogenic composition can include about 210 ⁇ g to about 240 ⁇ g of an aluminum-containing adjuvant, such as Alum, and about 420 ⁇ g to about 490 ⁇ g of a CpG adjuvant, such as CpG 1018A.
  • a unit dose of the immunogenic composition may include about 220 ⁇ g to about 230 ⁇ g of an aluminum-containing adjuvant, such as Alum, and about 430 ⁇ g to about 470 ⁇ g of a CpG adjuvant, such as CpG 1018A.
  • a unit dose of the immunogenic composition can include about 221 ⁇ g to about 227 ⁇ g of an aluminum-containing adjuvant, such as Alum, and about 440 ⁇ g to about 460 ⁇ g of a CpG adjuvant, such as CpG 1018A.
  • a unit dose of the immunogenic composition can include about 600 ⁇ g to about 900 ⁇ g of an aluminum-containing adjuvant, such as Alum, and about 1000 ⁇ g to about 2000 ⁇ g of a CpG adjuvant, such as CpG 1018A.
  • a unit dose of the immunogenic composition may include about 700 ⁇ g to about 800 ⁇ g of an aluminum-containing adjuvant, such as Alum, and about 1100 ⁇ g to about 1900 or 1800 ⁇ g of a CpG adjuvant, such as CpG 1018A.
  • a unit dose of the immunogenic composition may include about 720 ⁇ g to about 790 ⁇ g of an aluminum-containing adjuvant, such as Alum, and about 1200 or 1300 ⁇ g to about 1700 ⁇ g of a CpG adjuvant, such as CpG 1018A.
  • a unit dose of the immunogenic composition may include from about 730 or 740 ⁇ g to about 760 ⁇ g of an aluminum-containing adjuvant, such as Alum, and from about 1400 ⁇ g to about 1600 ⁇ g of a CpG adjuvant, such as CpG 1018A.
  • a unit dose of the immunogenic composition may include about 10 ⁇ g to about 100 ⁇ g of SARS-CoV-2 antigen, such as any one or more SARS-CoV-2 antigens of the present invention.
  • CoV-2 S fusion protein or S-trimer for example, a fusion protein including SARS-CoV-2 coronavirus Omicron (B.1.1.529) variant S protein peptide or fragments, variants or mutants thereof or S fusion protein trimer.
  • a unit dose of the immunogenic composition may include about 20 ⁇ g to about 75 ⁇ g SARS-CoV-2 S fusion protein or S-trimer, preferably about 25 ⁇ g to about 60 ⁇ g SARS-CoV-2 S fusion protein or S-trimer, or about 30 ⁇ g, about 40 ⁇ g, about 50 ⁇ g SARS-CoV-2 S fusion protein or S-trimer.
  • a unit dose of the immunogenic composition can include about 3 ⁇ g of SARS-CoV-2 S fusion protein or S-trimer. In other embodiments, the dose contains 15 ⁇ g of SARS-CoV-2 S fusion protein or S-trimer. In other embodiments, the dose contains 9 ⁇ g of SARS-CoV-2 S fusion protein or S-trimer. In a further embodiment, the dose contains 30 ⁇ g of SARS-CoV-2 S fusion protein or S-trimer. In some embodiments, a unit dose of the immunogenic composition may include about 5 ⁇ g to about 20 ⁇ g of SARS-CoV-2 antigen, such as any one or several SARS-CoV-2 S fusion proteins or S-III of the present invention.
  • Polymers such as fusion proteins or S fusion protein trimers including SARS-CoV-2 coronavirus Omicron (B.1.1.529) variant S protein peptide or fragments, variants or mutants thereof, may include About 200 ⁇ g to about 250 ⁇ g of an aluminum-containing adjuvant, such as Alum, and about 400 ⁇ g to about 500 ⁇ g of a CpG adjuvant, such as CpG 1018A.
  • a unit dose of the immunogenic composition may include about 6 ⁇ g to about 17, 18, 19 ⁇ g of SARS-CoV-2 antigen, such as any one or several SARS-CoV-2 S fusion proteins of the invention.
  • S-trimer for example, a fusion protein or S-fusion protein trimer including SARS-CoV-2 coronavirus Omicron (B.1.1.529) variant S protein peptide or fragments, variants or mutants thereof
  • the body may include about 210 ⁇ g to about 240 ⁇ g of an aluminum-containing adjuvant, such as Alum, and about 420 ⁇ g to about 490 ⁇ g of a CpG adjuvant, such as CpG 1018A.
  • a unit dose of the immunogenic composition may include about 8 ⁇ g to about 12, 13, 14, 15, 16 ⁇ g of SARS-CoV-2 antigen, such as any one or several SARS-CoV-2 antigens of the present invention.
  • 2 S fusion protein or S-trimer for example, a fusion protein or S including SARS-CoV-2 coronavirus Omicron (B.1.1.529) variant S protein peptide or fragments, variants or mutants thereof
  • the fusion protein trimer may include about 220 ⁇ g to about 230 ⁇ g of an aluminum-containing adjuvant, such as Alum, and about 430 ⁇ g to about 470 ⁇ g of a CpG adjuvant, such as CpG 1018A.
  • a unit dose of the immunogenic composition may include about 8 ⁇ g to about 11 ⁇ g of a SARS-CoV-2 antigen, such as any one or several SARS-CoV-2 S fusion proteins or S-III of the invention.
  • Polymers such as fusion proteins or S fusion protein trimers including SARS-CoV-2 coronavirus Omicron (B.1.1.529) variant S protein peptide or fragments, variants or mutants thereof, may include About 221 ⁇ g to about 227 ⁇ g of an aluminum-containing adjuvant, such as Alum, and about 440 ⁇ g to about 460 ⁇ g of a CpG adjuvant, such as CpG 1018A.
  • a unit dose of the immunogenic composition may include about 8 ⁇ g to about 12, 13, 14, 15, 16 ⁇ g of SARS-CoV-2 antigen, such as any one or several SARS-CoV-2 antigens of the present invention.
  • the fusion protein trimer may include about 600 ⁇ g to about 900 ⁇ g of an aluminum-containing adjuvant, such as Alum, and about 1000 ⁇ g to about 2000 ⁇ g of a CpG adjuvant, such as CpG 1018A.
  • a unit dose of the immunogenic composition may include from about 10 ⁇ g to about 80 ⁇ g of SARS-CoV-2 antigen, such as any one or several SARS-CoV-2 S fusion proteins or S-III of the invention.
  • Polymers such as fusion proteins or S fusion protein trimers including SARS-CoV-2 coronavirus Omicron (B.1.1.529) variant S protein peptide or fragments, variants or mutants thereof, may include about 700 ⁇ g to about 800 ⁇ g of an aluminum-containing adjuvant, such as Alum, and about 1100 ⁇ g to about 1900 or 1800 ⁇ g of a CpG adjuvant, such as CpG 1018A.
  • a unit dose of the immunogenic composition may include about 15 ⁇ g to about 60, 70 ⁇ g of SARS-CoV-2 antigen, such as any one or several SARS-CoV-2 S fusion proteins or S of the present invention.
  • fusion proteins or S fusion protein trimers including SARS-CoV-2 coronavirus Omicron (B.1.1.529) variant S protein peptide or fragments, variants or mutants thereof,
  • About 720 ⁇ g to about 790 ⁇ g of an aluminum-containing adjuvant, such as Alum, and about 1200 or 1300 ⁇ g to about 1700 ⁇ g of a CpG adjuvant, such as CpG1018A, may be included.
  • a unit dose of the immunogenic composition may include about 25 ⁇ g to about 40, 50 ⁇ g of SARS-CoV-2 antigen, such as any one or several SARS-CoV-2 S fusion proteins or S of the present invention.
  • fusion proteins or S fusion protein trimers including SARS-CoV-2 coronavirus Omicron (B.1.1.529) variant S protein peptide or fragments, variants or mutants thereof, about 730 or 740 ⁇ g to about 760 ⁇ g of an aluminum-containing adjuvant, such as Alum, and about 1400 ⁇ g to about 1600 ⁇ g of a CpG adjuvant, such as CpG 1018A, may be included.
  • a unit dose of the immunogenic composition may include about 30 ⁇ g of SARS-CoV-2 S-fusion protein or S-trimer, about 75 ⁇ g to about 100 ⁇ g of an aluminum-containing adjuvant, such as Alum, and about 150 ⁇ g of CpG adjuvant. agents, such as CpG 1018A.
  • a unit dose of the immunogenic composition may include about 9 ⁇ g of SARS-CoV-2 antigen, such as any one or several SARS-CoV-2 S fusion proteins or S-trimers of the invention,
  • SARS-CoV-2 antigen such as any one or several SARS-CoV-2 S fusion proteins or S-trimers of the invention
  • a fusion protein or S fusion protein trimer including the S protein peptide of the SARS-CoV-2 coronavirus Omicron (B.1.1.529) variant strain or its fragment, variant or mutant may include about 225 ⁇ g.
  • Aluminum adjuvant such as Alum
  • CpG adjuvant such as CpG 1018A.
  • a unit dose of the immunogenic composition may include about 30 ⁇ g of SARS-CoV-2 antigen, such as any one or several SARS-CoV-2 S fusion proteins or S-trimers of the invention,
  • SARS-CoV-2 antigen such as any one or several SARS-CoV-2 S fusion proteins or S-trimers of the invention
  • a fusion protein or S fusion protein trimer including the S protein peptide of the SARS-CoV-2 coronavirus Omicron (B.1.1.529) variant strain or its fragment, variant or mutant may include about 750 ⁇ g containing Aluminum adjuvant, such as Alum, and approximately 1500 ⁇ g of CpG adjuvant, such as CpG 1018A.
  • immunogenic compositions include pharmaceutically acceptable excipients, including, for example, solvents, fillers, buffers, tonicity adjusting agents, and preservatives (Pramanick et al., Pharma Times, 45:65- 77, 2013, incorporated by reference in its entirety for all purposes).
  • immunogenic compositions can include excipients that serve as one or more of solvents, fillers, buffers, and tonicity adjusters (e.g., sodium chloride in saline can be used simultaneously As aqueous carrier and tension regulator).
  • 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.
  • the composition is isotonic.
  • Immunogenic compositions may include buffering agents. Buffers control pH to inhibit degradation of the active agent during handling, storage and optional reconstitution. Suitable buffers include, for example, salts containing acetates, citrates, phosphates or sulfates. Other suitable buffering agents 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 in the range of 6 to 9. In some embodiments, the pH is greater than (lower limit) 6, 7, or 8. in some In 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, with the lower limit being smaller than the upper limit.
  • buffering agents control pH to inhibit degradation of the active agent during handling, storage and optional reconstitution.
  • Suitable buffers include, for example, salts containing acetates, citrates, phosphates or sulfates
  • the immunogenic composition may include a tonicity adjusting agent.
  • Suitable tonicity adjusting agents include, for example, glucose, glycerol, sodium chloride, glycerin and mannitol.
  • Immunogenic compositions may include fillers. Fillers are particularly useful when the pharmaceutical composition is lyophilized prior to administration.
  • the filler is a protective agent that helps stabilize and prevent degradation of the active agent during freeze or spray drying and/or during storage. Suitable fillers are sugars (mono-, di- and polysaccharides), such as sucrose, lactose, trehalose, mannitol, sorbitol, glucose and raffinose.
  • Immunogenic compositions may include preservatives. Suitable preservatives include, for example, antioxidants and antibacterial agents. However, in preferred embodiments, the immunogenic composition is prepared under sterile conditions and in disposable containers and therefore does not need to contain a preservative.
  • the compositions may be provided as sterile compositions.
  • Pharmaceutical compositions generally contain an effective amount of the disclosed immunogens and can be prepared by conventional techniques. Generally, the amount of immunogen per dose of the immunogenic composition is selected to induce an immune response without significant adverse side effects.
  • the compositions may be provided in unit dosage form for inducing an immune response in a subject. Unit dosage forms contain a single preselected dose, or suitably labeled or measured multiples of two or more preselected unit doses, for administration to a subject, and/or a metering mechanism for administration of unit doses or multiples thereof. .
  • the composition further includes one or more adjuvants.
  • kits 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 complex selected from the group consisting of SEQ ID NO. : Recombinant polypeptides of the group consisting of 1-26 and 85-92, optionally the complex serves as a primary series, an additional dose, and/or a homologous or heterologous booster dose Using, for example, a first dose, a second dose, a third dose, a fourth dose, and/or more doses, optionally the initial dose, additional doses, or heterologous boosters with other recombinant subunit vaccines, nanoparticles Any one or more of vaccines, mRNA vaccines, DNA vaccines, adenovirus vector vaccines, and inactivated virus vaccines can be used together.
  • kits for generating an immune response to a coronavirus surface antigen in a subject wherein the surface antigen includes S protein or an antigenic fragment thereof, and the method includes 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 as a primary series, an additional dose, and /or the use of a 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 initial dose, additional doses, or
  • the heterologous booster is used in conjunction with any one or more of other recombinant subunit vaccines, nanoparticle vaccines, mRNA vaccines, DNA vaccines, adenovirus vector vaccines, and inactivated virus vaccines.
  • kits for generating an immune response in a subject to a coronavirus surface antigen wherein the surface antigen includes a sequence selected from the group consisting of SEQ ID NOs: 27-66 and 81-84 , and the method includes administering to the subject Use an effective amount of a complex including a recombinant polypeptide selected from the group consisting of SEQ ID NOs: 1-26 and 85-92, optionally as a primary series, an additional dose), and/or the use of a 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 initial dose, Additional doses, or heterologous boosters, are used in conjunction with any one or more of other recombinant subunit vaccines, nanoparticle vaccines, mRNA vaccines, DNA vaccines, adenovirus vector vaccines, and inactivated virus vaccines.
  • a homologous or heterologous booster dose such as a first dose, a
  • a coronavirus surface antigen includes the S protein of the coronavirus or an antigenic fragment thereof, and optionally, the surface antigen includes A sequence selected from the group consisting of SEQ ID NOs: 27-66 and 81-84, or an antigenic fragment thereof
  • the method includes administering to the subject an effective amount of a complex comprising a recombinant polypeptide comprising The sequence described in any one of SEQ ID NO:85-92, optionally the complex serves as a primary series, an additional dose, and/or a homologous or heterologous booster dose ) using, for example, a first dose, a second dose, a third dose, a fourth dose, and/or more doses, optionally with the initial dose, additional doses, or heterologous boosters with other recombinant subunit vaccines, nano Any one or more of particle vaccines, mRNA vaccines, DNA vaccines, aden
  • adjuvants in any priming agent, additional agent, and/or booster agent may independently include: aluminum-containing adjuvants, such as alum and/or aluminum hydroxide-containing adjuvants; oligonucleotide-containing adjuvants.
  • adjuvants such as adjuvants containing CpG oligodeoxynucleotides (CpG-ODN); adjuvants containing TLR9 agonists; adjuvants containing metabolizable oils, alpha-tocopherol, and/or polyoxyethylene sorbitan monooleate (Tween-80) adjuvant, such as an adjuvant containing squalene, ⁇ -tocopherol, and Tween-80 and/or Span 85 in the form of an oil-in-water emulsion; or any combination of adjuvants.
  • CpG-ODN CpG oligodeoxynucleotides
  • TLR9 agonists such as an adjuvant containing metabolizable oils, alpha-tocopherol, and/or polyoxyethylene sorbitan monooleate (Tween-80) adjuvant, such as an adjuvant containing squalene, ⁇ -tocopherol, and Twe
  • kits for generating an immune response to a coronavirus surface antigen in a subject wherein the surface antigen includes S protein or an antigenic fragment thereof, and the method includes administering to the subject an effective amount of A complex or a combination of any two or more in a 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 is used as an initial agent and/or as a booster, such as a second dose and/or a third dose booster injection.
  • methods include administering to a subject an effective amount of a complex comprising a recombinant polypeptide comprising SEQ ID NO:85, SEQ ID NO:86, SEQ ID NO:87, and /or the sequence described in SEQ ID NO:88.
  • adjuvants in any priming agent, additional agent, and/or booster agent may independently include: aluminum-containing adjuvants, such as alum and/or aluminum hydroxide-containing adjuvants; oligonucleotide-containing adjuvants.
  • adjuvants such as adjuvants containing CpG oligodeoxynucleotides (CpG-ODN); adjuvants containing TLR9 agonists; adjuvants containing metabolizable oils, alpha-tocopherol, and/or polyoxyethylene sorbitan monooleate (Tween-80) adjuvant, such as an adjuvant containing squalene, ⁇ -tocopherol, and Tween-80 and/or Span 85 in the form of an oil-in-water emulsion; or any combination of adjuvants.
  • CpG-ODN CpG oligodeoxynucleotides
  • TLR9 agonists such as an adjuvant containing metabolizable oils, alpha-tocopherol, and/or polyoxyethylene sorbitan monooleate (Tween-80) adjuvant, such as an adjuvant containing squalene, ⁇ -tocopherol, and Twe
  • a subject may be administered a disclosed immunogen (e.g., a recombinant coronavirus S antigen, e.g., an S-trimer or S protein described herein, a nucleic acid encoding a protomer of a disclosed recombinant coronavirus S antigen molecules (e.g., RNA molecules) or vectors, or protein nanoparticles or virus-like particles containing the disclosed recombinant coronavirus S antigen) to induce an immune response to the corresponding coronavirus S antigen in a subject.
  • subject to The tester is a human being.
  • the immune response may be a protective immune response, such as a response that inhibits subsequent infection by the corresponding coronavirus.
  • the stimulation of immune responses can also be used to treat or suppress infections and diseases associated with the corresponding coronavirus.
  • Subjects may be selected for treatment who have or are at risk of contracting coronavirus, for example because of exposure or potential exposure to coronavirus. Following administration of the disclosed immunogens, the subject can be monitored for infection or coronavirus-related symptoms, or both.
  • Exemplary subjects to be treated with the therapies and methods of the present invention include humans as well as non-human primates and other animals.
  • an acceptable screening method is used to determine risk factors associated with the target or suspected disease or condition, or to determine the status of an existing disease or condition in the subject .
  • These screening methods include, for example, routine examinations to identify environmental, familial, occupational, and other such risk factors that may be associated with the target or suspected disease or condition, as well as diagnostic methods, such as those used to detect and/or characterize coronavirus infection.
  • Various ELISA and other immunoassay methods allow clinicians to select patients in need of treatment using the methods and pharmaceutical compositions of the present invention.
  • the compositions may be administered in accordance with the teachings herein or other conventional methods, as a stand-alone preventive or therapeutic regimen, or as a follow-up, adjunctive or coordinated treatment regimen to other treatments.
  • the disclosed immunogens may be used for prophylactic or therapeutic purposes.
  • the disclosed therapeutic agents are provided prior to any symptoms, such as prior to infection.
  • Prophylactic administration of the disclosed therapeutic agents serves to prevent or ameliorate any subsequent infection.
  • the disclosed therapeutic agents are provided at or after the onset of symptoms of disease or infection, for example, after the development of symptoms of coronavirus infection corresponding to the coronavirus S antigen, or after diagnosis of coronavirus infection. Accordingly, therapeutic agents may be provided prior to anticipated exposure to the coronavirus in order to attenuate the expected severity, duration, or extent of infection and/or associated disease symptoms following exposure or suspected exposure to the virus, or after actual onset of infection.
  • the immunogens and immunogenic compositions thereof described herein are provided to a subject in an amount effective to induce or enhance the immune response of the subject (preferably a human) against the coronavirus S antigen.
  • the actual dose of the disclosed immunogen will depend, for example, on the subject's disease signs and specific status (e.g., subject's age, size, health, severity of symptoms, predisposing factors, etc.), time and route of administration, concurrent Factors such as other drugs or treatments administered will vary as well as the specific pharmacology of the composition to elicit the desired activity or biological response in the subject. Dosage regimens can be adjusted to provide optimal prophylactic or therapeutic response.
  • Immunogenic compositions including one or more of the disclosed immunogens can be used in coordinated (or prime-boost) vaccination regimens or combination formulations.
  • novel combination immunogenic compositions and coordinated immunization regimens use separate immunogens or formulations, each designed to elicit an antiviral immune response, such as immunity to the coronavirus S antigen answer.
  • Separate immunogenic compositions that elicit an antiviral immune response can be combined in a multivalent immunogenic composition administered to the subject in a single immunization step, or they can be in a coordinated (or prime-boost) immunization regimen. Administered alone (in monovalent immunogenic compositions).
  • boosters there can be several boosters, each of which can be a different disclosed immunogen.
  • a booster may be the same immunogen as another booster or priming agent.
  • Primer and booster available as single doses
  • One or more doses may be administered, for example, two, three, four, five, six or more doses may be administered to the subject over several days, weeks or months. Multiple boosters may also be administered, such as one to five (eg, 1, 2, 3, 4 or 5 boosters) or more.
  • Different doses are available for a series of sequential immunizations. For example, a relatively large dose is used in primary immunization followed by a relatively small dose for boosting.
  • the booster may be administered about two weeks, about three to eight weeks, or about four weeks after the priming dose, or about several months after the priming dose. In some embodiments, the booster can be administered about 5, about 6, about 7, about 8, about 10, about 12, about 18, about 24 months after the priming dose, or more or less time after the priming dose . Additional boosters may also be administered periodically at appropriate time points to enhance the subject's "immune memory.”
  • the suitability of the selected vaccine parameters, such as formulation, dosage, regimen, etc. can be determined by withdrawing aliquots of serum from subjects and determining antibody titers during the immunization program.
  • the subject's clinical status can be monitored for desired effects, such as prevention of infection or improvement of disease status (e.g., reduction of viral load). If such monitoring indicates that vaccination is suboptimal, the subject can be boosted with additional doses of the immunogenic composition and vaccination parameters can be improved in a manner that is expected to enhance the immune response.
  • desired effects such as prevention of infection or improvement of disease status (e.g., reduction of viral load). If such monitoring indicates that vaccination is suboptimal, the subject can be boosted with additional doses of the immunogenic composition and vaccination parameters can be improved in a manner that is expected to enhance the immune response.
  • a prime-boost approach may include DNA-primer and protein-boost vaccination regimens for the subject.
  • the method may include two or more administrations of the nucleic acid molecule or protein.
  • each human dose will include 1-1000 ⁇ g of protein, such as from about 1 ⁇ g to about 100 ⁇ g, such as from about 1 ⁇ g to about 50 ⁇ g, such as about 1 ⁇ g, about 2 ⁇ g, about 5 ⁇ g, about 10 ⁇ g, about 15 ⁇ g, About 20 ⁇ g, about 25 ⁇ g, about 30 ⁇ g, about 40 ⁇ g, or about 50 ⁇ g.
  • the amounts used in the immunogenic composition are selected based on the subject population (eg, infants or the elderly).
  • the optimal dosage of a particular ingredient can be determined through standard studies involving observation of antibody titers and other responses in subjects. It will be appreciated that a therapeutically effective amount of a disclosed immunogen (e.g., a disclosed recombinant coronavirus S antigen, e.g., a trimer, protein, viral vector, or nucleic acid molecule in an immunogenic composition) may be included in the dose administered by a single dose.
  • the subject's immune system typically responds to the immunogenic composition by producing antibodies specific for the coronavirus S protein peptide contained in the immunogen. This response means that an immunologically effective dose was delivered to the subject.
  • the subject's antibody response will be determined with an assessment of effective doses/immunization regimens. In most cases, assessment of antibody titers in serum or plasma obtained from subjects is sufficient.
  • the decision as to whether to administer a booster vaccination and/or to change the amount of therapeutic agent administered to an individual may be based, at least in part, on the antibody titer level.
  • Antibody titer levels may be based, for example, on immunobinding assays that measure the concentration of antibodies in serum that bind to an antigen, including, for example, recombinant coronavirus S antigen, such as S-trimer.
  • Methods do not need to completely eliminate, reduce or prevent coronavirus infections to be effective.
  • priming an immune response to a coronavirus with one or more of the disclosed immunogens can reduce or inhibit a coronavirus infection by a desired amount, such as 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% (Eliminate or prevent detectable infected cells).
  • coronavirus replication can be reduced or inhibited by the disclosed methods. Complete elimination of coronavirus replication is not required for the approach to be effective.
  • priming an immune response with one or more of the disclosed immunogens can reduce corresponding coronavirus replication by a desired amount, e.g., at least 10%, at least 20%, compared to coronavirus replication in the absence of an immune response. %, 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% (eliminate or prevent detectable coronavirus replication).
  • the disclosed immunogen is administered to the subject simultaneously with the administration of the adjuvant. In other embodiments, the disclosed immunogens are administered to the subject after administration of the adjuvant and within a sufficient time to induce an immune response.
  • nucleic acid delivery is direct immunization with plasmid DNA, such as a mammalian expression plasmid.
  • Immunization by nucleic acid constructs is well known in the art and is disclosed, for example, in U.S. Patent No. 5,643,578, which describes methods of immunizing vertebrates by introducing DNA encoding a desired antigen to elicit a cell-mediated or humoral response, and U.S. Patent Nos. 5,593,972 and 5,817,637 (which describe operably linking a nucleic acid sequence encoding an antigen to a regulatory sequence capable of expression).
  • US Patent No. 5,880,103 describes several methods of delivering nucleic acids encoding immunogenic peptides or other antigens to organisms.
  • Methods include liposomal delivery of nucleic acids (or their own synthetic peptides) and immunostimulatory constructs or ISCOMS TM , which are 30-40 nm sized negatively charged nanoparticles that spontaneously form after mixing cholesterol and Quil A TM (saponin). Cage-like structure.
  • ISCOMS TM immunostimulatory constructs
  • the use of ISCOMS TM as a delivery vehicle for antigen has produced protective immunity in experimental models of various infections, including toxoplasmosis and Epstein-Barr virus-induced tumors (Mowat and Donachie, Immunol. Today 12:383, 1991).
  • plasmid DNA vaccines are used to express the disclosed immunogens in a subject.
  • nucleic acid molecules encoding the disclosed immunogens can be administered to a subject to induce an immune response to the coronavirus S antigen.
  • the nucleic acid molecule can be included on a plasmid vector for DNA immunization, such as the pVRC8400 vector (as described in Barouch et al., J. Virol, 79, 8828-8834, 2005, for all purposes The full text is incorporated by reference).
  • the disclosed recombinant coronavirus S antigens can be expressed by attenuated viral hosts or vectors or bacterial vectors.
  • Recombinant vaccinia virus, adeno-associated virus (AAV), herpesvirus, retrovirus, cytomeglovirus, or other viral vectors can be used to express peptides or proteins to elicit CTL responses.
  • AAV adeno-associated virus
  • U.S. Patent No. 4,722,848 which is incorporated by reference in its entirety for all purposes, describes vaccinia vectors and methods useful in immunization programs.
  • Bacillus Calmette Guerin provides another vector for expressing peptides (see Stover, Nature 351:456-460, 1991, incorporated by reference in its entirety for all purposes).
  • nucleic acid encoding the disclosed recombinant coronavirus S antigen is introduced directly into the cell.
  • nucleic acids can be loaded onto gold microspheres by standard methods and introduced into the skin via a device such as Bio-Rad's HELIOS TM Gene Gun. Nucleic acids can be "naked", under the control of a strong promoter Plasmid composition. Typically, the DNA is injected into the muscle, but it can also be injected directly into other sites. Injectable doses are generally about 0.5 ⁇ g/kg to about 50 mg/kg, and typically about 0.005 mg/kg to about 5 mg/kg (see, eg, U.S. Patent No. 5,589,466).
  • mRNA-based immunization protocols can be used to deliver nucleic acids encoding the disclosed recombinant coronavirus S antigens directly into cells.
  • mRNA-based nucleic acid vaccines may provide an effective alternative to the aforementioned approaches. mRNA vaccines eliminate the safety issue of DNA integration into the host genome and can be translated directly in the host cell cytoplasm. Furthermore, simple cell-free in vitro synthesis of RNA avoids the manufacturing complications associated with viral vectors.
  • RNA-based vaccines Two exemplary formats 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 immunity (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 conserveed Influenza Antigens Confer Protection against Homologous and Heterosubtypic Viral Challenge,” PLoS One, 11(8):e01 61193, 2016; and Brito
  • administration to a subject of a therapeutically effective amount of one or more disclosed immunogens induces a neutralizing immune response in the subject.
  • serum can be collected from the subject at appropriate time points after immunization, frozen, and stored for neutralization assays.
  • Methods for measuring 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, single Cycle infection assay.
  • PRNT plaque reduction neutralization
  • microneutralization assays microneutralization assays
  • flow cytometry-based assays single Cycle infection assay.
  • a panel of coronavirus pseudoviruses can be used to determine serum neutralizing activity.
  • administration to a subject of a therapeutically effective amount of one or more disclosed immunogens induces a neutralizing immune response in the subject.
  • serum can be collected from the subject at appropriate time points after immunization, frozen, and stored for neutralization assays.
  • Methods for measuring 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, single Cycle infection assay.
  • PRNT plaque reduction neutralization
  • microneutralization assays microneutralization assays
  • flow cytometry-based assays single Cycle infection assay.
  • a panel of coronavirus pseudoviruses can be used to determine serum neutralizing activity.
  • neutralizing immune responses induced by immunogens disclosed herein generate neutralizing antibodies against coronaviruses (eg, SARS-CoV-2).
  • the neutralizing antibodies herein bind to cellular receptors or coreceptors of a coronavirus (eg, SARS-CoV-2) or components thereof.
  • the viral receptor or co-receptor is a coronavirus receptor or co-receptor, preferably a pneumovirus receptor or co-receptor, more preferably a human coronavirus receptor, such as a SARS-CoV-2 receptor. body or co-receptor.
  • the neutralizing antibodies herein modulate, reduce, antagonize, alleviate, block, inhibit, eliminate and/or in vitro, in situ and/or in vivo or interfere with at least one coronavirus (such as SARS-CoV-2) activity or binding, or coronavirus (such as SARS-CoV-2) receptor activity or binding, such as SARS-CoV-2 release, SARS-CoV-2 receptor In vivo signaling, membrane SARS-CoV-2 cleavage, SARS-CoV-2 activity, SARS-CoV-2 production and/or synthesis.
  • coronavirus such as SARS-CoV-2
  • coronavirus such as SARS-CoV-2 receptor activity or binding
  • the immunogens disclosed herein induce neutralizing antibodies against SARS-CoV-2 that modulate, reduce, antagonize, mitigate, block, inhibit, eliminate, and/or interfere with the interaction between SARS-CoV-2 and SARS- Binding of CoV-2 receptors or coreceptors, such as angiotensin-converting enzyme 2 (ACE2), dipeptidyl peptidase 4 (DPP4), dendritic cell-specific intercellular adhesion molecule-3-grabbing non- Integrin (DC-SIGN) and/or liver/lymph node-SIGN (L-SIGN).
  • ACE2 angiotensin-converting enzyme 2
  • DPP4 dipeptidyl peptidase 4
  • DC-SIGN dendritic cell-specific intercellular adhesion molecule-3-grabbing non- Integrin
  • L-SIGN liver/lymph node-SIGN
  • the article of manufacture may include a container and a label or package insert on or associated with the container.
  • Suitable containers include, for example, bottles, vials, syringes, test tubes, IV solution bags, and the like.
  • Containers can be formed from a variety of materials, such as glass or plastic.
  • the container has a sterile access port.
  • Exemplary containers include intravenous solution bags, vials, including containers with stoppers pierceable by an injection needle.
  • the article of manufacture or kit may further include package insert indicating that the composition can be used to treat a specific condition, such as a condition described herein (eg, coronavirus infection).
  • the article of manufacture or kit may further comprise another or the same container containing a pharmaceutically acceptable buffer. It may further include other materials such as other buffers, diluents, filters, needles and/or syringes.
  • the label or package insert may indicate that the composition is used to treat a coronavirus infection in an individual.
  • a label or package insert on or associated with the container may indicate instructions for the reconstitution and/or use of the preparation.
  • 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 coronavirus infection in an individual.
  • the container contains a composition, alone or in combination with another composition effective in treating, preventing, and/or diagnosing a condition.
  • the article of manufacture or kit may include (a) a first container having a composition contained therein (i.e., a first agent), wherein the composition includes an immunogenic composition or a protein or recombinant polypeptide thereof; and (b) a second container A container having a composition contained therein (i.e., a second agent), wherein the composition includes another agent, such as an adjuvant or other therapeutic agent, and the article or kit further includes a label or package insert with the second agent A description of the agent in an effective amount to treat the subject.
  • polypeptide and “protein” are used interchangeably to refer to a polymer of amino acid residues and are not limited to a minimum length.
  • Polypeptides may include amino acid residues, including natural and/or unnatural amino acid residues.
  • the term also includes post-expression modifications of the polypeptide, such as glycosylation, sialylation, acetylation, and phosphorylation.
  • the 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 in which the protein is produced or due to errors in PCR amplification.
  • a "subject" is a mammal, such as a human or other animal, and is typically a human.
  • the subject (eg, patient) to which one or more agents, cells, cell populations, or compositions is administered is a mammal, typically a primate, such as a human.
  • the primate is a monkey or ape.
  • Subjects may be male or female and of any suitable age group, including infants, juveniles, adolescents, adults, and geriatric subjects.
  • the subject is a non-primate mammal, such as a rodent.
  • treatment means the amelioration or reduction, in whole or in part, of a disease, condition or disorder, or associated with related symptoms, adverse reactions or results, or phenotypes. Desirable effects of treatment include, but are not limited to, preventing the occurrence or recurrence of disease, alleviating symptoms, alleviating any direct or indirect pathological consequences of the disease, preventing metastasis, reducing the rate of disease progression, improving or alleviating the disease state, and alleviating or improving prognosis. The term does not imply complete cure of a disease or complete elimination of any symptoms or effects on all symptoms or outcomes.
  • delaying the development of a disease means retarding, hindering, slowing down, slowing down, stabilizing, inhibiting and/or delaying the development of a disease (eg, cancer).
  • the length of delay may vary depending on the disease history and/or the individual receiving treatment. In some embodiments, sufficient or significant delay may actually involve prevention, as the individual will not develop the disease. For example, the development of advanced cancer, such as metastasis, may be delayed.
  • 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.
  • provided cells and compositions are used to delay the development of a disease or slow the progression of a disease.
  • inhibiting function or activity means reducing function or activity when compared to otherwise identical conditions other than the condition or parameter of interest, or when compared to another condition. For example, cells that inhibit tumor growth slow down the growth of a tumor compared to how fast it would grow without the cells.
  • an “effective amount” of an agent refers to the effective amount at the dosage/amount and time period required to achieve the desired effect (eg, therapeutic or prophylactic effect).
  • a “therapeutically effective amount” of an agent is the dose and amount necessary 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 effective amount of time period.
  • 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 administered.
  • provided methods involve administering cells and/or compositions in an effective amount (eg, a therapeutically effective amount).
  • Preventatively effective amount refers to the effective amount at the dosage and time period required to achieve the desired preventive effect.
  • the prophylactically effective amount will be less than the therapeutically effective amount because the prophylactic dose is administered to a subject prior to or in an early stage of the disease. In cases where tumor burden is low, the prophylactically effective dose in some aspects will be higher than the therapeutically effective dose.
  • An effective amount of a vaccine or other agent sufficient to produce a desired response such as reduction or elimination of signs or symptoms of a condition or disease, such as pneumonia. For example, this may be an amount necessary to inhibit viral replication or measurably alter the external symptoms of a viral infection.
  • an "effective amount" is an amount that treats (including prevents) one or more symptoms and/or underlying causes of any condition or disease, such as for treating coronavirus infection.
  • the effective amount is a therapeutically effective amount.
  • an effective amount is an amount that prevents the development of one or more signs or symptoms of a particular disease or disorder, such as one or more signs or symptoms associated with a coronavirus infection.
  • the terms "antigen” or “immunogen” are used interchangeably to refer to a substance, typically a protein, capable of inducing an immune response in a subject.
  • the term also refers to an immunologically active protein that, upon administration to a subject (either directly or by administering to the subject a nucleotide sequence or vector encoding the protein), is capable of eliciting a humoral and/or cellular immune response against the protein.
  • vaccine immunogen is used interchangeably with "protein antigen” or "immunogenic polypeptide.”
  • conservatively modified variants refer to those nucleic acids that encode the same or essentially the same amino acid sequence, or in the case of a nucleic acid that does not encode an amino acid sequence, to essentially the same sequence. Due to the degeneracy of the genetic code, a large number of functionally identical nucleic acids encode any given protein.
  • a “conservatively modified variant” refers to a variant having conservative amino acid substitutions, whereby an amino acid residue is replaced by another amino acid residue having a similarly charged side chain. Families of amino acid residues having side chains with similar charges have been defined in the art.
  • These families include those 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), non-polar side chains (e.g., alanine, valine, leucine, isoleucine acid, proline, phenylalanine, methionine, tryptophan), beta branched chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine acid, phenylalanine, tryptophan, histidine).
  • basic side chains e.g., lysine, arginine, histidine
  • acidic side chains e.g., aspartic acid, glutamic acid
  • An epitope refers to an antigenic determinant. These are specific chemical groups or peptide sequences on the antigen molecule so that they trigger a specific immune response, for example, an epitope is an antigenic region that responds to B cells and/or T cells. Epitopes can be formed either from contiguous amino acids or from non-contiguous amino acids juxtaposed by the tertiary folding of the protein.
  • fusion proteins are recombinant proteins containing the amino acid sequences of at least two unrelated proteins linked together by peptide bonds to form a single protein. Therefore, it does not contain the naturally occurring coronavirus surface antigen, the fusion (F) protein described herein. Unrelated amino acid sequences can be linked directly to each other, or they can be linked using linker sequences. As used herein, a protein is unrelated if its amino acid sequence is not normally linked together by peptide bonds in its natural environment (e.g., within a cell) of. For example, the amino acid sequence of a viral antigen and the amino acid sequence of collagen or procollagen are not typically linked together by peptide bonds.
  • An immunogen is a protein, or a portion thereof, capable of inducing an immune response in a mammal, such as a mammal infected by or at risk of infection by a pathogen. Administration of the immunogen can result in protective immunity and/or active immunity against the target pathogen.
  • 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 against the immunogenic polypeptide. response (e.g. production of antibodies).
  • 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 as percent identity; the higher the percent, the more identical the sequences are. Two sequences have a specified percentage of identical amino acid residues when compared and aligned for maximum correspondence through a comparison window or specified regions measured using one of the following sequence comparison algorithms or by manual alignment and visual inspection or nucleotides, the two sequences are "substantially identical" (i.e., 60% identity, preferably 65%, 70%, 75%, 80%, 85%, 90%, 95% or 99% identity). Alternatively, identity exists over a region of at least about 50 nucleotides (or 10 amino acids) in length, or more preferably 100 to 500 or 1000 or more nucleotides (or 20 amino acids) in length. , 50, 200 or more amino acids).
  • a vaccine refers to a pharmaceutical composition that elicits a preventive or therapeutic immune response in a subject.
  • the immune response is a protective immune response.
  • vaccines elicit antigen-specific immune responses against antigens of pathogens (eg, viral pathogens) or cellular components associated with pathological conditions.
  • Vaccines may include polynucleotides (eg, nucleic acids encoding the disclosed antigens), peptides or polypeptides (eg, the disclosed antigens), viruses, cells, or one or more cellular components.
  • a vaccine or vaccine immunogen or vaccine composition is expressed from a fusion construct and self-assembles into nanoparticles displaying the immunogenic polypeptide or protein on the surface.
  • VLPs refer to non-replicatable viral shells derived from any of several viruses.
  • VLPs typically consist of one or more viral proteins, such as, but not limited to, proteins known as capsid, coat, coat, surface and/or envelope proteins, or particle-forming polypeptides derived from these proteins.
  • VLPs can form spontaneously after recombinantly expressing proteins in an appropriate expression system. Methods for producing specific VLPs are known in the art.
  • the presence of VLPs following recombinant expression of viral proteins can be detected using conventional techniques known in the art, such as by electron microscopy, biophysical characterization, and the like. See, e.g., Baker et al. (1991) Biophys. J.
  • VLPs can be isolated by density gradient centrifugation and/or identified by characteristic density bands.
  • cryo-EM can be performed on vitrified aqueous samples prepared from the VLP in question and images recorded under appropriate exposure conditions.
  • range format various aspects of the claimed subject matter are presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the claimed subject matter. Accordingly, descriptions of ranges should be considered to have specifically disclosed all possible subranges and individual values within such ranges. For example, where a range of values is provided, it is to be understood that every intervening value between the upper and lower limits of the range and any other statement or intervening value within the range is included within the claimed subject matter. The upper and lower limits of these smaller ranges may independently be included in the smaller range and also within the claimed subject matter, subject to any expressly excluded limitations within the stated range. If the stated range includes one or both of those limitations, ranges excluding one or both of those included limitations are also included within the claimed subject matter. This works for any range width.
  • 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.
  • vector refers to a nucleic acid molecule capable of transmitting 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 operably linked. Such vectors are referred to herein as "expression vectors.”
  • Embodiment 1 A protein comprising a plurality of recombinant polypeptides, each recombinant polypeptide comprising a coronavirus SARS-CoV-2 Omicron (B.1.1.529) surface antigen linked to a C-terminal propeptide of collagen, The C-terminal propeptide of the recombinant polypeptide forms an inter-polypeptide disulfide bond.
  • Embodiment 2 The protein according to embodiment 1, wherein the coronavirus infection is a SARS-coronavirus 2 (SARS-CoV-2) infection.
  • SARS-CoV-2 SARS-coronavirus 2
  • Embodiment 3 The protein according to embodiment 1 or 2, wherein the surface antigen comprises 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 Includes three recombinant peptides.
  • S coronavirus spike
  • the epitope is optionally a linear epitope or a conformational epitope
  • the protein Includes three recombinant peptides.
  • 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.
  • 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 heptapeptide repeat 1 ( HR1) peptide or heptad repeat 2 (HR2) peptide or any combination thereof.
  • RBD receptor binding domain
  • RBM receptor binding motif
  • FP fusion peptide
  • HR1 heptapeptide repeat 1
  • HR2 heptad repeat 2
  • Embodiment 6 The protein according to any one of embodiments 3-5, wherein the surface antigen comprises the receptor binding domain (RBD) of the S protein.
  • RBD receptor binding domain
  • 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.
  • Embodiment 8 The protein according to any one of embodiments 3-7, wherein the surface antigen does not comprise a transmembrane (TM) domain peptide and/or a cytoplasmic (CP) domain peptide.
  • TM transmembrane
  • CP cytoplasmic
  • 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.
  • 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 tissue Protease L.
  • 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.
  • 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.
  • 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 through a linker (eg a linker comprising a glycine-X-Y repeat sequence) , where X and Y are independently any amino acid, and optionally proline or hydroxyproline.
  • a linker eg a linker comprising a glycine-X-Y repeat sequence
  • 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.
  • a lipid bilayer such as a membrane or viral envelope.
  • Embodiment 15 The protein according to any one of embodiments 1-14, wherein the protein is capable of binding to a cell surface receptor of a subject, optionally wherein the subject is a mammal, such as a primate, such as a human .
  • 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 - Grab non-integrin (DC-SIGN) or liver/lymph node-SIGN (L-SIGN).
  • ACE2 angiotensin-converting enzyme 2
  • DPP4 dipeptidyl peptidase 4
  • DC-SIGN dendritic cell-specific intercellular adhesion molecule-3 - Grab non-integrin
  • L-SIGN liver/lymph node-SIGN
  • Embodiment 17 The protein according to any one of embodiments 1-16, wherein the C-terminal propeptide is human collagen.
  • Embodiment 18 The protein according to any one of embodiments 1-17, wherein the C-terminal propeptide includes pro ⁇ 1(I), pro ⁇ 1(II), pro ⁇ 1(III), pro ⁇ 1(V), pro ⁇ 1(XI), pro ⁇ 2 (I), the C-terminal polypeptide of pro ⁇ 2(V), pro ⁇ 2(XI) or pro ⁇ 3(XI) or a fragment thereof.
  • Embodiment 19 The protein according to any one of embodiments 1-18, wherein the C-terminal propeptides are the same or different in the recombinant polypeptide.
  • Embodiment 20 The protein according to any one of embodiments 1-19, wherein the C-terminal propeptide comprises any one of SEQ ID NO: 67-80 or an amino acid sequence at least 90% identical thereto, which is capable of forming an inter-polypeptide Disulfide bonds and trimerization of recombinant polypeptides.
  • Embodiment 21 The protein according to any one of embodiments 1-20, wherein the C-terminal propeptide includes SEQ ID NO: 67 or an amino acid sequence at least 90% identical thereto, which is capable of forming inter-polypeptide disulfide bonds and enabling recombination. Peptide trimerization.
  • Embodiment 22 The protein according to any one of embodiments 1-20, wherein the C-terminal propeptide includes SEQ ID NO: 68 or an amino acid sequence at least 90% identical thereto, which is capable of forming inter-polypeptide disulfide bonds and enabling recombination. Peptide trimerization.
  • Embodiment 23 The protein according to any one of embodiments 1-20, wherein the C-terminal propeptide includes SEQ ID NO: 69 or an amino acid sequence at least 90% identical thereto, which is capable of forming inter-polypeptide disulfide bonds and enabling recombination. Peptide trimerization.
  • Embodiment 24 The protein according to any one of embodiments 1-20, wherein the C-terminal propeptide includes SEQ ID NO: 70 or an amino acid sequence at least 90% identical thereto, which is capable of forming inter-polypeptide disulfide bonds and enabling recombination. Peptide trimerization.
  • Embodiment 25 The protein according to any one of embodiments 1-20, wherein the C-terminal propeptide includes SEQ ID NO: 71 or an amino acid sequence at least 90% identical thereto, which is capable of forming inter-polypeptide disulfide bonds and enabling recombination. Peptide trimerization.
  • Embodiment 26 The protein according to any one of embodiments 1-20, wherein the C-terminal propeptide includes SEQ ID NO: 72 or an amino acid sequence at least 90% identical thereto, which is capable of forming inter-polypeptide disulfide bonds and enabling recombination. Peptide trimerization.
  • Embodiment 27 The protein according to any one of embodiments 1-20, wherein the C-terminal propeptide includes SEQ ID NO: 73 or an amino acid sequence at least 90% identical thereto, which is capable of forming inter-polypeptide disulfide bonds and enabling recombination. Peptide trimerization.
  • Embodiment 28 The protein according to any one of embodiments 1-20, wherein the C-terminal propeptide includes SEQ ID NO: 74 or an amino acid sequence at least 90% identical thereto, which is capable of forming inter-polypeptide disulfide bonds and enabling recombination. Peptide trimerization.
  • 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 an inter-polypeptide Disulfide bonds and trimerization of recombinant polypeptides.
  • Embodiment 30 The protein according to any one of embodiments 1-29, wherein the C-terminal propeptide comprises a sequence comprising a glycine-X-Y repeat linked to the N-terminus of any one of SEQ ID NOs: 67-80 , wherein .
  • 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.
  • Embodiment 32 The protein according to any one of embodiments 1-31, wherein the surface antigen in each recombinant polypeptide includes any one of SEQ ID NOs: 27-66 and 81-84 or an amino acid that is at least 80% identical thereto. sequence.
  • 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 or an amino acid sequence at least 80% identical thereto.
  • Embodiment 34 An immunogen comprising the protein of any one of embodiments 1-33, optionally as a vaccine primary series, additional dose, and/or homologous or A heterologous booster dose is used, such as a first dose, a second dose, a third dose, a fourth dose, and/or more doses, optionally with the initial dose, additional dose, or heterologous booster dose.
  • any one or more of other recombinant subunit vaccines, nanoparticle vaccines, mRNA vaccines, DNA vaccines, adenovirus vector vaccines, and inactivated virus vaccines are used together, optionally the initial dose, additional dose, and/or Adjuvants in homologous or heterologous boosters may independently include: aluminum-containing adjuvants, such as alum and/or aluminum hydroxide-containing adjuvants; oligonucleotide-containing adjuvants, such as CpG oligodeoxynucleoside-containing adjuvants Adjuvants containing acid (CpG-ODN); adjuvants containing TLR9 agonists; adjuvants containing metabolizable oil, alpha-tocopherol, and/or polyoxyethylene sorbitan monooleate (Tween-80) , for example, an adjuvant containing squalene, ⁇ -tocopherol, and Tween-80 and/or Span 85 in the
  • Embodiment 35 A protein nanoparticle comprising the protein of any one of embodiments 1-33, directly or indirectly linked to the nanoparticle, optionally as a vaccine primary series, additionally An additional dose, and/or a homologous or heterologous booster dose is used, such as a first dose, a second dose, a third dose, a fourth dose, and/or more doses, optionally the
  • the initial dose, additional dose, or heterologous booster dose can be used in conjunction with any one or more of other recombinant subunit vaccines, nanoparticle vaccines, mRNA vaccines, DNA vaccines, adenovirus vector vaccines, and inactivated virus vaccines, and can be used
  • the adjuvants in the initial agent, additional agent, and/or homologous or heterologous booster may independently include: aluminum-containing adjuvants, such as alum and/or aluminum hydroxide-containing adjuvants; oligonucleotide-containing adjuvants Acid-containing adjuvants, such as CpG
  • Embodiment 36 A virus-like particle (VLP) comprising the protein of any one of embodiments 1-33, optionally as a vaccine primary series, an additional dose, and /or the use of a 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 initial dose, additional doses, or
  • the 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.
  • the initial dose, additional Adjuvants in agents, and/or homologous or heterologous boosters may independently include: aluminum-containing adjuvants, such as alum and/or aluminum hydroxide-containing adjuvants; oligonucleotide-containing adjuvants, such as those containing Adjuvants containing CpG oligodeoxynucleotides (CpG-ODN); adjuvants containing TLR9 agonists; adjuvants containing metabolizable oil, alpha-tocopherol, and/or polyoxyethylene sorbitan monooleate (Tween- 80), such as an adjuvant containing squalene, ⁇ -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.
  • aluminum-containing adjuvants such as alum and/or aluminum hydroxide-containing adjuvants
  • Embodiment 37 An isolated nucleic acid encoding one, two, three or more of the recombinant polypeptides of the protein according to any one of embodiments 1-33.
  • Embodiment 38 The isolated nucleic acid according to embodiment 37, wherein the polypeptide encoding a surface antigen is fused in frame to a polypeptide encoding the C-terminal propeptide of collagen.
  • Embodiment 39 The isolated nucleic acid according to embodiment 37 or 38, which is operably linked to a promoter.
  • Embodiment 40 The isolated nucleic acid according to any one of embodiments 37-39, which is a DNA molecule.
  • 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 nucleoside-modified mRNA, non-amplified mRNA, self-amplified mRNA or trans-amplified mRNA. Increase mRNA.
  • Embodiment 42 A vector comprising an isolated nucleic acid according to any one of embodiments 37-41.
  • Embodiment 43 The vector according to embodiment 42, which is a viral vector.
  • 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.
  • Embodiment 45 An immunogenic composition comprising a protein, immunogen, protein nanoparticle, VLP, isolated nucleic acid, vector, virus, pseudovirus or cell according to any one of embodiments 1-44, and a pharmaceutical Acceptable carrier.
  • Embodiment 46 A vaccine comprising an 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 prophylactic and/or Or a therapeutic vaccine, optionally used as a primary series, an additional dose, and/or a homologous or heterologous booster dose, such as a first dose, a second dose, Third dose, fourth dose, and/or more doses, optionally the initial dose, additional dose, or heterologous booster dose with other recombinant subunit vaccines, nanoparticle vaccines, mRNA vaccines, DNA vaccines, adenoviral vectors Vaccine, and any one or more of the inactivated virus vaccines are used together.
  • a therapeutic vaccine optionally used as a primary series, an additional dose, and/or a homologous or heterologous booster dose, such as a first dose, a second dose, Third dose, fourth dose, and/or more doses, optionally the initial dose, additional dose, or hetero
  • the adjuvants in the initial dose, additional dose, and/or homologous or heterologous booster may independently include: aluminum-containing adjuvant Adjuvants, such as alum and/or aluminum hydroxide-containing adjuvants; oligonucleotide-containing adjuvants, such as CpG oligodeoxynucleotide (CpG-ODN)-containing adjuvants; TLR9 agonist-containing adjuvants; Adjuvants for metabolizable oils, alpha-tocopherol, and/or polyoxyethylene sorbitan monooleate (Tween-80), such as oil-in-water emulsions containing squalene, alpha-tocopherol, and Adjuvant of Tween-80 and/or Span 85; or a combination of any of the adjuvants.
  • Adjuvants such as alum and/or aluminum hydroxide-containing adjuvants
  • Embodiment 47 The vaccine according to embodiment 46, wherein the vaccine includes a plurality of different adjuvants.
  • Embodiment 48 A method of producing a protein, comprising: expressing an isolated nucleic acid or vector according to any one of embodiments 37-43 in a host cell to produce a protein according to any one of embodiments 1-33; and Purified protein.
  • Embodiment 49 Protein produced by the method of Embodiment 48.
  • 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 of any one of embodiments 1-47 and 49 , protein nanoparticles, VLPs, isolated nucleic acids, vectors, viruses, pseudoviruses, cells, immunogenic compositions or vaccines to generate an immune response.
  • Embodiment 51 The method according to embodiment 50 for treating or preventing coronavirus infection.
  • Embodiment 52 The method according to embodiment 50 or 51, wherein generating an immune response inhibits or reduces coronavirus replication in the subject.
  • Embodiment 53 The method according to any one of embodiments 50-52, wherein the immune response includes a cell-mediated response and/or a humoral response, optionally including the production of one or more neutralizing antibodies, e.g., polyclonal Antibodies or monoclonal antibodies.
  • the immune response includes a cell-mediated response and/or a humoral response, optionally including the production of one or more neutralizing antibodies, e.g., polyclonal Antibodies or monoclonal antibodies.
  • Embodiment 54 The method according to any one of embodiments 50-53, wherein the immune response is directed against a surface antigen of the coronavirus but not against the C-terminal propeptide.
  • 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.
  • AD antibody-dependent enhancement
  • Embodiment 56 The method according to any one of embodiments 50-55, wherein administration does not result in antibody-dependent enhancement (ADE) in the subject when subsequently exposed to one or more coronaviruses.
  • ADE antibody-dependent enhancement
  • Embodiment 57 The method according to any one of embodiments 50-56, further comprising a priming step and/or a boosting step.
  • Embodiment 58 The method according to any one of embodiments 50-57, wherein by topical, transdermal, subcutaneous, intradermal, oral, intranasal (e.g., intranasal spray), intratracheal, sublingual, buccal, Rectal, vaginal, inhalation, intravenous (e.g., intravenous injection), intraarterial, intramuscular (e.g., intramuscular injection), intracardiac, intraosseous, intraperitoneal, transmucosal, intravitreal, subretinal, intraarticular, articular
  • the administration step is performed by peripheral, topical or epicutaneous administration.
  • Embodiment 59 The method according to any one of embodiments 50-58, wherein the effective amount is administered as a single dose or as a series of doses with one or more intervals.
  • Embodiment 60 The method according to any one of embodiments 50-59, wherein the effective amount is administered without an adjuvant.
  • Embodiment 61 The method according to any one of embodiments 50-59, wherein the effective amount is administered with the adjuvant or adjuvants.
  • 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 antisera against coronavirus in the subject.
  • Embodiment 63 The method according to embodiment 62, wherein the subject is a mammal, optionally a human or a non-human primate.
  • Embodiment 64 The method of embodiment 62 or 63, further comprising isolating the neutralizing antibody or neutralizing antisera from the subject.
  • Embodiment 65 The method according to embodiment 64, further comprising administering to a human subject an effective amount of an isolated neutralizing antibody or neutralizing antisera by passive immunization to prevent or treat coronavirus infection.
  • Embodiment 66 The method according to any one of embodiments 62-65, wherein the neutralizing antibody or neutralizing antisera comprises a polyclonal antibody directed against a coronavirus surface antigen, optionally wherein the neutralizing antibody or neutralizing antisera does not Contains or essentially contains no antibodies directed against the C-terminal propeptide of collagen.
  • Embodiment 67 The method according to any one of embodiments 62-65, wherein the neutralizing antibody comprises a monoclonal antibody directed against a coronavirus surface antigen, optionally wherein the neutralizing antibody does not contain or substantially contains no C-specific antibodies against collagen. Antibodies to terminal propeptides.
  • 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 use Inducing an immune response to coronavirus in a subject, and/or treating or preventing coronavirus infection.
  • 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 coronavirus in a subject, and/or for treating or preventing coronavirus infection.
  • 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 medicaments or prophylactic agents for inducing an immune response to coronavirus in a subject, and/or for treating or preventing coronavirus infection.
  • Embodiment 71 A method for analyzing a sample, comprising contacting the sample with the protein of any one of embodiments 1-33, and detecting the interaction between the protein and an analyte capable of specifically binding to a coronavirus surface antigen. combine.
  • Embodiment 72 The method according to embodiment 71, wherein the analyte is an antibody, receptor or cell that recognizes a surface antigen.
  • 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 is derived.
  • 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 detection kit ( lateral flow assay kit).
  • 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 soluble coronavirus SARS-CoV-2 Omicron (B.1.1.529) Surface antigen, or a fragment, variant or mutant thereof, which is a soluble coronavirus surface antigen or a fragment, variant or mutant thereof linked to the C-terminal portion of collagen by in-frame fusion to form a disulfide bonded trimeric fusion protein.
  • soluble coronavirus SARS-CoV-2 Omicron B.1.1.529)
  • Surface antigen, or a fragment, variant or mutant thereof which is a soluble coronavirus surface antigen or a fragment, variant or mutant thereof linked to the C-terminal portion of collagen by in-frame fusion to form a disulfide bonded trimeric fusion protein.
  • Embodiment 2 The method of embodiment 1, wherein the coronavirus infection is a severe acute respiratory syndrome (SARS)-coronavirus 2 (SARS-CoV-2) infection.
  • SARS severe acute respiratory syndrome
  • SARS-CoV-2 severe acute respiratory syndrome-2
  • Embodiment 3 The method according to embodiment 1 or 2, wherein the coronavirus surface antigen encompasses coronavirus spike (S) protein or a fragment or epitope thereof.
  • S coronavirus spike
  • the coronavirus surface antigen includes the SARS-CoV-2 spike (S) extracellular domain peptide or a fragment or epitope thereof, optionally the S extracellular domain peptide or Its fragments or epitopes include SARS-CoV-2 Omicron (B.1.1.529) variant S extracellular domain peptide or its fragments, variants or mutants, for example Such as chimeric sequences containing the receptor-binding domain (RBD) of the Omicron variant and the S protein peptide sequence of Hu-1 or other variants.
  • S SARS-CoV-2 spike
  • B.1.1.529 S extracellular domain peptide or its fragments, variants or mutants
  • Embodiment 5 The method according to any one of embodiments 1-4, wherein the coronavirus surface antigen comprises SARS-CoV-2 spike (S) N-terminal domain (NTD) peptide or fragment or expression thereof , optionally the NTD peptide is a SARS-CoV-2 Hu-1, alpha, beta, gamma, delta, murine, or ometron NTD peptide or a fragment, variant or mutant thereof.
  • S S
  • NTD N-terminal domain
  • Embodiment 6 The method according to any one of embodiments 1-5, wherein the coronavirus surface protein includes a SARS-CoV-2 spike (S) receptor binding domain (RBD) peptide or a fragment or expression thereof. position, optionally the RBD peptide is the SARS-CoV-2 Omicron (B.1.1.529) variant RBD peptide or a fragment, variant or mutant thereof.
  • S SARS-CoV-2 spike
  • RBD receptor binding domain
  • Embodiment 7 The 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 The peptide is the SARS-CoV-2 Omicron (B.1.1.529) variant S1 peptide or its fragment, variant or mutant.
  • S SARS-CoV-2 spike
  • B.1.1.529 SARS-CoV-2 Omicron
  • Embodiment 8 The method of any one of claims 1-7, wherein the coronavirus surface antigen comprises SARS-CoV-2 spike (S) S2 peptide or fragment or epitope thereof, optionally the S2 The peptide is the SARS-CoV-2 Hu-1, alpha, beta, gamma, delta, myon, or ometron S2 peptide or a fragment, variant, or mutant thereof.
  • S SARS-CoV-2 spike
  • Embodiment 1.9 The method according to any one of embodiments 1-8, wherein the coronavirus epitope comprises a SARS-CoV-2 spike (S) extracellular domain peptide with mutations or a fragment or epitope thereof.
  • S SARS-CoV-2 spike
  • Embodiment 10 The method of embodiment 9, wherein the mutation comprises a furin cleavage site mutation, optionally the mutation is by deletion, substitution or addition of one or several amino acids such that the furin cleavage site is not It also has the activity as a furin cleavage site, optionally the mutation is located at any one or several positions from 682 to 685, optionally the mutation includes 685R ⁇ 685A.
  • Embodiment 11 The method of embodiment 9 or 10, wherein the mutation includes a mutation located at or adjacent to the junction of the heptad repeat sequence HR1 and the central helix, optionally the mutation includes a proline substitution, such as 986K ⁇ 986P and/or 987V ⁇ 987P.
  • Embodiment 12 The method of any one of embodiments 9-11, wherein the mutation comprises consecutive site amino acid substitutions, such as 986K ⁇ 986P and 987V ⁇ 987P.
  • Embodiment 13 The method according to any one of embodiments 1-12, wherein the recombinant subunit vaccine includes the sequence described in any one of SEQ ID NO:81-92 or is identical to SEQ ID NO:81-92 Any one of the sequences has an amino acid sequence identity of at least or about 80%, 85%, 90%, 92%, 95%, 97%, or 99%.
  • Embodiment 14 The method of any one of claims 1-13, wherein the recombinant subunit vaccine includes or is at least or about 80%, 85%, 90% SEQ ID NO: 85 , 92%, 95%, 97%, 99% sequence identity of the amino acid sequences.
  • Embodiment 15 The method according to any one of embodiments 1-14, wherein the recombinant subunit The unit vaccine includes SEQ ID NO:86 or an amino acid sequence having at least or about 80%, 85%, 90%, 92%, 95%, 97%, 99% sequence identity to SEQ ID NO:86.
  • Embodiment 16 The method of any one of claims 1-15, wherein the recombinant subunit vaccine includes or is at least or about 80%, 85%, 90% SEQ ID NO: 87 , 92%, 95%, 97%, 99% sequence identity of the amino acid sequences.
  • Embodiment 17 The method of any one of embodiments 1-16, wherein the recombinant subunit vaccine includes SEQ ID NO: 88 or is at least or about 80%, 85%, 90% SEQ ID NO: 88 , 92%, 95%, 97%, 99% sequence identity of the amino acid sequences.
  • Embodiment 18 The method according to any one of embodiments 1-17, wherein the recombinant subunit vaccine comprises the sequence of any one of SEQ ID NO:89-92 or is identical to SEQ ID NO:89-92 Any one of the sequences has an amino acid sequence identity of at least or about 80%, 85%, 90%, 92%, 95%, 97%, or 99%.
  • Embodiment 19 The method according to any one of embodiments 1-18, wherein the recombinant subunit vaccine includes SEQ ID NO:90 or is at least or about 80%, 85%, 90% with SEQ ID NO:90 , 92%, 95%, 97%, 99% sequence identity of the amino acid sequences.
  • Embodiment 20 The method according to any one of embodiments 1-19, wherein the recombinant subunit vaccine comprises the first sequence of any one of SEQ ID NOs: 81-84 connected to SEQ ID NO: 67 The second sequence of any one of -80, wherein the C-terminus of the first sequence is directly or indirectly connected to the N-terminus of the second sequence.
  • Embodiment 21 The method of any one of claims 1-20, wherein the recombinant subunit vaccine is administered by intramuscular injection.
  • Embodiment 22 The method of any one of embodiments 1-21, wherein the recombinant subunit vaccine is administered by intranasal spray.
  • Embodiment 23 The method of any one of embodiments 1-22, wherein the recombinant subunit vaccine is administered in a single dose or in a series of doses spaced weekly or monthly.
  • Embodiment 24 The method of any one of embodiments 1-23, wherein the recombinant subunit vaccine is administered without an adjuvant, optionally as a primary series, Additional doses, and/or homologous or heterologous booster doses are used, such as a first dose, a second dose, a third dose, a fourth dose, and/or more doses, optionally
  • the initial 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.
  • Embodiment 25 The method of any one of embodiments 1-24, wherein the recombinant subunit vaccine is administered with an adjuvant, optionally as a primary series, additional doses ( additional doses), and/or the use of homologous or heterologous booster doses, such as a first dose, a second dose, a third dose, a fourth dose, and/or more doses, optionally the initial dose , additional doses, or heterologous boosters with other recombinant subunit vaccines, nanoparticle vaccines, mRNA vaccines, DNA vaccines, adenoviral vector vaccines, Used in conjunction with any one or more of the inactivated virus vaccines, optionally the adjuvants in the initial dose, additional dose, and/or homologous or heterologous booster may independently include: aluminum-containing adjuvants, For example, adjuvants containing alum and/or aluminum hydroxide; adjuvants containing oligonucleotides, such as adjuvants containing CpG oli
  • Embodiment 26 The method according to any one of 1 to 25, wherein the recombinant subunit vaccine is administered with more than one adjuvant, optionally as a primary series, Additional doses, and/or homologous or heterologous booster doses are used, such as a first dose, a second dose, a third dose, a fourth dose, and/or more doses, optionally
  • the initial dose, additional dose, or heterologous booster dose is used in conjunction with any one or more of other recombinant subunit vaccines, nanoparticle vaccines, mRNA vaccines, DNA vaccines, adenovirus vector vaccines, and inactivated virus vaccines
  • the adjuvants in the initial agent, additional agent, and/or homologous or heterologous booster may independently include: aluminum-containing adjuvants, such as alum and/or aluminum hydroxide-containing adjuvants; oligonuclear-containing adjuvants.
  • Adjuvants containing glycosides such as CpG oligodeoxynucleotides (CpG-ODN); adjuvants containing TLR9 agonists; metabolizable oils, alpha-tocopherol, and/or polyoxyethylene sorbitan Adjuvants to monooleate (Tween-80), such as adjuvants containing squalene, alpha-tocopherol, and Tween-80 and/or Span 85 in the form of an oil-in-water emulsion; or any adjuvant agent combination.
  • CpG-ODN CpG oligodeoxynucleotides
  • TLR9 agonists metabolizable oils, alpha-tocopherol, and/or polyoxyethylene sorbitan Adjuvants to monooleate (Tween-80), such as adjuvants containing squalene, alpha-tocopherol, and Tween-80 and/or Span 85 in the form of an
  • Embodiment 27 A method for detecting coronavirus antibodies from mammalian serum
  • the method includes the step of contacting serum with a soluble coronavirus SARS-CoV-2 Omicron (B.1.1.529) surface antigen linked to the C-terminal portion of collagen via in-frame fusion to Formation of disulfide-linked trimeric fusion proteins.
  • a soluble coronavirus SARS-CoV-2 Omicron B.1.1.529
  • Embodiment 28 The method of embodiment 27, wherein the soluble coronavirus surface antigen is S protein or peptide.
  • Embodiment 29 A method for use containing Omicron from the coronavirus SARS-CoV-2
  • Methods for recombinant subunit vaccines of (B.1.1.529) a soluble surface antigen linked to the C-terminal portion of collagen by in-frame fusion to form a disulfide-linked trimer fusion protein the method Including: immunizing mammals, purifying the produced neutralizing antibodies, and using the neutralizing antibodies to treat patients infected with the coronavirus through passive immunotherapy.
  • Embodiment 30 The method of embodiment 29, wherein the neutralizing antibody comprises a polyclonal antibody.
  • Embodiment 31 The method of embodiment 29, wherein the neutralizing antibody is a monoclonal antibody.
  • Embodiment 32 The method of embodiment 29, wherein the neutralizing antibody is a monoclonal antibody to S protein or peptide.
  • Embodiment 33 The method of embodiment 29, wherein the neutralizing antibody is SARS-
  • Embodiment 34 The method of embodiment 29, wherein the neutralizing antibody is SARS-
  • Monoclonal antibodies to the S protein of CoV-2 Hu-1, alpha, beta, gamma, delta, mu, Omicron, or other strains Monoclonal antibodies to the S protein of CoV-2 Hu-1, alpha, beta, gamma, delta, mu, Omicron, or other strains.
  • Embodiment 35 The method of embodiment 29, wherein the neutralizing antibody is SARS-
  • Embodiment 36 A complex comprising a recombinant polypeptide selected from the group consisting of SEQ ID NOs: 85-92.
  • Embodiment 37 A complex comprising a trimer of a recombinant polypeptide selected from the group consisting of SEQ ID NOs: 85-92, wherein the recombinant polypeptide trimers through inter-polypeptide disulfide bonds to form a trimer.
  • Embodiment 38 An immunogenic composition comprising a trimer of a recombinant polypeptide or a combination of any two or more trimers, the recombinant polypeptide comprising a trimer selected from the group consisting of SEQ ID NO: 85 - A sequence of groups of 92.
  • Embodiment 39 The immunogenic composition of embodiment 38, comprising a trimer of a recombinant polypeptide having the sequence set forth in SEQ ID NO: 90.
  • Embodiment 40 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 compound selected from SEQ ID NO: 85 - Recombinant polypeptides of the group consisting of 92, 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 , second dose, third dose, fourth dose, and/or more doses, optionally the initial dose, additional dose, or heterologous booster dose with other recombinant subunit vaccines, nanoparticle vaccines, mRNA vaccines, DNA Any one or more of vaccines, adenovirus vector vaccines, and inactivated virus vaccines can be used in combination.
  • a complex comprising a compound selected from SEQ ID NO: 85 - Recombinant polypeptides of the group consisting of 92, optionally the complex is used as a primary series, an additional dose, and
  • Embodiment 41 A method for generating an immune response to a coronavirus surface antigen in a subject
  • the surface antigen comprises S protein or an antigenic fragment thereof
  • 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, an additional dose, and/or a homologous or heterologous booster dose, such as the first dose, the second dose, the third dose, and the fourth dose.
  • a homologous or heterologous booster dose such as the first dose, the second dose, the third dose, and the fourth dose.
  • additional dose optionally with the initial dose, additional dose, or heterologous booster dose with other recombinant subunit vaccines, nanoparticle vaccines, mRNA vaccines, DNA vaccines, adenoviral vector vaccines, and inactivated virus vaccines Use any one or more combinations of them.
  • Embodiment 42 A method for generating an immune response to a coronavirus surface antigen in a subject
  • the surface antigen comprises a sequence selected from the group consisting of SEQ ID NO: 27-66 and 81-84
  • the method comprises administering to the subject an effective amount of a complex comprising a sequence selected from the group consisting of SEQ ID NO: 85 - Recombinant polypeptides of the group consisting of 92, optionally the complex serves as a primary series, an additional dose), and/or the use of a 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 initial dose, Additional doses, or heterologous boosters, may be 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.
  • Embodiment 43 A method for generating an immune response to a coronavirus surface antigen in a subject
  • the surface antigen includes the S protein of coronavirus or an antigenic fragment thereof, and optionally, the surface antigen includes a sequence selected from the group consisting of SEQ ID NOs: 27-66 and 81-84 or an antigenic fragment thereof, and the method Comprised of administering to a subject an effective amount of a complex comprising a recombinant polypeptide comprising the sequence described in any one of SEQ ID NOs: 85-92, optionally as a primary series, Additional doses, and/or homologous or heterologous booster doses are used, such as a first dose, a second dose, a third dose, a fourth dose, and/or more doses, optionally The initial 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.
  • Embodiment 44 A method for generating an immune response to a coronavirus surface antigen in a subject, wherein the surface antigen includes S protein or an antigenic fragment thereof, and
  • the method includes administering to the subject an effective amount of a complex or a combination of any two or more complexes comprising a recombinant polypeptide comprising a sequence selected from the group consisting of SEQ ID NOs: 85-92, which may
  • the complex or combination of complexes is used as a primary series, an additional dose, and/or a homologous or heterologous booster dose, e.g., a first dose, a second dose, Third dose, fourth dose, and/or more doses, optionally the initial dose, additional dose, or heterologous booster dose with other recombinant subunit vaccines, nanoparticle vaccines, mRNA vaccines, DNA vaccines, adenoviral vectors Vaccine, and any one or more of the inactivated virus vaccines are used together.
  • Embodiment 45 The method of claim 44, wherein the method comprises administering to the subject an effective amount of a complex comprising a recombinant polypeptide, the recombinant polypeptide comprising SEQ ID NO: 85, SEQ ID NO: 86, SEQ ID The sequence described in NO:87 and/or SEQ ID NO:88.
  • Embodiment 46 A fusion protein comprising a plurality of recombinant polypeptides, each recombinant polypeptide comprising from the amino to the carboxyl terminus:
  • a second region comprising a coronavirus spike protein receptor binding domain (RBD) of a second coronavirus that is different from said first coronavirus; and
  • RBD coronavirus spike protein receptor binding domain
  • the fusion protein is used as a primary series, an additional dose, and/or a homologous or heterologous booster dose, e.g., the first dose, Second dose, third dose, fourth dose, and/or more dose, optionally the initial dose, additional dose, or heterologous booster dose with any one of other recombinant subunit vaccines, nanoparticle vaccines, mRNA vaccines, DNA vaccines, adenoviral vector vaccines, and inactivated virus vaccines, or Use several combinations.
  • Embodiment 47 The fusion protein of claim 46, further comprising a third region between the second region and the C-terminal propeptide of collagen.
  • Embodiment 48 The fusion protein of claim 47, wherein the third region includes 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.
  • Embodiment 49 The fusion protein of claim 47 or 48, wherein the third region includes the S2 domain of a fourth coronavirus, wherein the fourth coronavirus is the same as the first, second or fourth coronavirus, or different.
  • Embodiment 50 The fusion protein of any one of claims 46-49, wherein the first region includes the N-terminal domain (NTD) of the first coronavirus.
  • NTD N-terminal domain
  • Embodiment 51 The fusion protein of any one of claims 46-50, wherein the first region includes one or more amino acid residues that are different from the corresponding amino acid residues in the second coronavirus.
  • Embodiment 52 The fusion protein of any one of claims 46-51, wherein the second region includes one or more amino acid residues that are different from the corresponding amino acid residues in the first coronavirus.
  • Embodiment 53 The fusion protein of any one of claims 46-52, wherein the first and second coronaviruses are different variants or strains of the same coronavirus.
  • Embodiment 54 The fusion protein of claim 53, wherein 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 SARS- Different variants of CoV-2.
  • Embodiment 55 The fusion protein of any one of claims 46-54, wherein the first coronavirus and the second coronavirus are independently selected from the group consisting of 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 of SARS-CoV-2 viruses.
  • Embodiment 56 A trimeric fusion protein comprising three recombinant polypeptides, each recombinant polypeptide comprising from amino to carboxyl terminus:
  • NTD N-terminal domain
  • the trimer fusion protein serves as a primary series, additional dose , and/or the use of a 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 initial dose, additional doses , or heterologous boosters with other recombinant subunit vaccines, nanoparticles
  • a 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 initial dose, additional doses , or heterologous boosters with other recombinant subunit vaccines, nanoparticles
  • Any one or more of vaccines, mRNA vaccines, DNA vaccines, adenovirus vector vaccines, and inactivated virus vaccines can be used together.
  • Embodiment 57 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 46-56.
  • Embodiment 58 The method of embodiment 57, wherein neutralizing antibodies are produced in the mammal to the first and second coronaviruses.
  • Embodiment 59 The method of embodiment 58, wherein the first and second coronaviruses are different variants of SARS-CoV-2, and the neutralizing antibodies produced in the mammal neutralize B.1.1.529, Two or more SARS of the 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 -CoV-2 virus.
  • Embodiment 60 The method of embodiment 59, wherein the neutralizing antibody produced in the mammal neutralizes 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 of three or more SARS-CoV-2 viruses.
  • Embodiment 61 The method of any one of embodiments 57-60, comprising immunizing the mammal with two or more doses of the fusion protein, at least one of the two or more doses of the fusion protein
  • the protein includes the SARS-CoV-2 Omicron (B.1.1.529) spike protein amino acid sequence, and optionally the at least one dose of the fusion protein includes the sequence described in any one of SEQ ID NOs: 81-92 or An amino acid sequence that has at least or about 80%, 85%, 90%, 92%, 95%, 97%, 99% sequence identity with the sequence described in any one of SEQ ID NOs: 81-92.
  • Embodiment 62 The method of any one of embodiments 57-61, wherein the fusion protein is administered as a booster after one or more doses of an immunogen comprising a protein from the same or different SARS- Spike protein peptides of NTD and RBD of CoV-2 variants, optionally the one or more doses of the immunogen comprise the sequence described in any one of SEQ ID NOs: 27-66 and 81-84 or are consistent with SEQ ID NOs: 27-66 and 81-84.
  • sequence described in any one of ID NOs: 27-66 and 81-84 has an amino acid sequence with at least or about 80%, 85%, 90%, 92%, 95%, 97%, 99% sequence identity
  • the enhancer fusion protein comprises the sequence described in any one of SEQ ID NO:81-92 or has at least or about 80% or 85% of the sequence described in any one of SEQ ID NO:81-92. , 90%, 92%, 95%, 97%, 99% sequence identity of the amino acid sequences.
  • Embodiment 80 A bivalent vaccine, comprising:
  • the first trimeric fusion protein includes the soluble coronavirus SARS-CoV-2 Omicron (B.1.1.529) spike (S) protein surface antigen or fragments or variants thereof Or a mutant, the soluble coronavirus surface antigen or a fragment, variant or mutant thereof is connected to the C-terminal portion of the collagen, the C-terminal portion of the collagen forms a disulfide-linked trimer, thereby forming the first Trimeric fusion proteins;
  • a second trimeric fusion protein comprising a soluble coronavirus SARS-CoV-2 Hu-1, alpha, beta, gamma, delta, or Myus 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 a C-terminal portion of collagen, the C-terminal portion of collagen forming a disulfide-linked trimer,
  • S Myus spike
  • Embodiment 81 The bivalent vaccine according to claim 80, the second trimer fusion protein includes soluble coronavirus SARS-CoV-2 Hu-1 spike (S) protein surface antigen or fragments, variants or mutant.
  • S SARS-CoV-2 Hu-1 spike
  • Embodiment 82 The bivalent vaccine according to embodiment 80 or 81, which bivalent vaccine is administered without an adjuvant, optionally as a primary series, an additional dose ), and/or the use of a 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 initial dose, additional dose, or heterologous booster dose, used in conjunction with any one or more of other recombinant subunit vaccines, nanoparticle vaccines, mRNA vaccines, DNA vaccines, adenovirus vector vaccines, and inactivated virus vaccines.
  • a 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 initial dose, additional dose, or heterologous booster dose, used in conjunction with any one or more of other recombinant subunit vaccines, nanoparticle vaccines, mRNA vaccines, DNA vaccines,
  • Embodiment 83 The bivalent vaccine according to any one of embodiments 80-82, administered with more than one adjuvant, optionally as a primary series, additionally An additional dose, and/or a homologous or heterologous booster dose is used, such as a first dose, a second dose, a third dose, a fourth dose, and/or more doses, optionally the
  • the initial dose, additional dose, or heterologous booster dose can be used in conjunction with any one or more of other recombinant subunit vaccines, nanoparticle vaccines, mRNA vaccines, DNA vaccines, adenovirus vector vaccines, and inactivated virus vaccines, and can be used
  • the adjuvants in the initial agent, additional agent, and/or homologous or heterologous booster may independently include: aluminum-containing adjuvants, such as alum and/or aluminum hydroxide-containing adjuvants; oligonucleotide-containing adjuvants Acid-containing adjuvants, such as CpG oligode
  • Embodiment 84 The bivalent vaccine according to any one of embodiments 80-83, wherein the first trimer fusion protein in the bivalent vaccine includes 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%, 99% sequence identity with the sequence described in any one of SEQ ID NOs: 81-84.
  • Embodiment 85 The bivalent vaccine according to any one of embodiments 80-84, the second trimer fusion protein in the bivalent vaccine includes the sequence described in any one of SEQ ID NO: 27-66 Or an amino acid sequence that has at least or about 80%, 85%, 90%, 92%, 95%, 97%, 99% sequence identity with the sequence described in any one of SEQ ID NOs: 27-66.
  • Embodiment 86 A multivalent vaccine, which includes soluble coronavirus Hu-1, SARS-CoV-2 Omicron (B.1.1.529), Omicron strain (BA.5) freely selected from the group consisting of , alpha, beta, gamma, delta, or Myotis spike (S) protein surface antigen or a fragment, variant or mutant thereof, the soluble coronavirus surface antigen or a fragment, variant or mutant thereof connected to collagen The C-terminal portion of the collagen forms a disulfide-linked trimer, thereby forming the first trimer fusion protein.
  • soluble coronavirus Hu-1, SARS-CoV-2 Omicron (B.1.1.529), Omicron strain (BA.5) freely selected from the group consisting of , alpha, beta, gamma, delta, or Myotis spike (S) protein surface antigen or a fragment, variant or mutant thereof, the soluble coronavirus surface antigen or a fragment, variant or mutant thereof connected to collagen
  • S Myot
  • Embodiment 87 The multivalent vaccine according to embodiment 86 comprising the soluble coronavirus Hu-1 and Omicron strain (BA.5) spike (S) protein surface antigen or fragments, variants thereof or mutants.
  • BA.5 soluble coronavirus Hu-1 and Omicron strain
  • S spike
  • Embodiment 88 The multivalent vaccine according to embodiment 86 comprising said soluble coronavirus Viruses, SARS-CoV-2 Omicron (B.1.1.529) and Omicron strain (BA.5) spike (S) protein surface antigen or fragments, variants or mutants thereof.
  • Embodiment 89 The multivalent vaccine according to embodiment 86 comprising the soluble coronavirus Beta, Delta and Omicron strain (BA.5) spike (S) protein surface antigen or fragments, variants thereof. body or mutant.
  • BA.5 Delta and Omicron strain
  • S spike
  • Example 1 Generation of recombinant disulfide-linked SARS-CoV-2 S-trimer fusion protein
  • a secreted recombinant disulfide-linked polypeptide containing a SARS-CoV-2 protein peptide fused to a trimerization domain was generated as a candidate protein subunit vaccine.
  • the ectodomain of the spike protein from SARS-CoV2, including its signal peptide (SP), S1 and S2 domains is C-terminally fused in-frame to a lactating protein encoding the human C-propeptide of ⁇ 1 collagen.
  • Animal expression vectors to express secreted trimeric S-trimer fusion antigens for example, as shown in Figures 1A-1B.
  • Protein Trimerization TM technology was used (Liu et al., Scientific Reports, 7(1):8953, 2017, incorporated by reference in its entirety for all purposes).
  • S SARS-CoV-2 spike
  • 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 TrimerizationTM tag, Protein TrimerizationTM tag Able to self-trimerize via disulfide bonds.
  • an affinity purification protocol was developed taking advantage of the high binding affinity between the Protein TrimerizerTM tag and Endo180, which is capable of binding to type 1 pro- Collagen receptors in the C-terminal region of collagen and mature it.
  • the Endo180-Fc fusion protein is loaded onto a Protein A column and captured by the resin through high-affinity binding between Protein A and the human IgG1 Fc domain of Endo180-Fc.
  • Serum-free cell culture medium containing CHO cell-secreted S-trimers was then loaded onto a protein A column with precaptured Endo180-Fc.
  • the bound S-trimer is purified using a mild salt elution step without causing separation of Endo180-Fc from the Protein A column. to close to uniformity.
  • the S-trimer is further purified by low pH for prophylactic virus inactivation (VI), anion exchange chromatography to remove host cell DNA and any residual endotoxin, and nanofiltration as a prophylactic virus removal (VR) step , and finally UF/DF to the concentration required to concentrate the S-trimer into the formulation buffer, thereby obtaining the active drug (DS) of the S-trimer subunit vaccine candidate.
  • Stability analysis of the purified S-trimer showed that the S-trimer is stable in liquid solution formulations at 2-8°C.
  • SDS-PAGE analysis under non-reducing and reducing conditions confirmed that the purified S-trimer is a disulfide-linked trimer and is partially cleaved by CHO cell-produced furin at the S1/S2 boundary.
  • S-trimers occur in multiple high molecular weight forms, possibly as a result of partial cleavage of the antigen, releasing non-covalently attached and cleaved S1 during sample processing.
  • Protein TrimerizationTM technology (Liang et al., Nat. Comms., 12:1346, 2021) was used to generate covalent trimers of spike antigens based on Hu-1 strains and VOC strains.
  • An 8% SDS-PAGE analysis of S-trimer expression in fed-batch serum-free CHO cell cultures was performed in a 10L bioreactor. Cell-free conditioned media were analyzed under reducing conditions followed by Coomassie brilliant blue staining.
  • CMI ELISpot S-trimer antigen-specific cell-mediated immunity
  • Example 3 Immunogenicity of SARS-CoV-2 vaccine based on Omicron strain
  • the spike protein (S) gene of SARS-CoV-2 variants of high concern was optimized using mammalian codons, synthesized by Genscript, and then cloned into the pcDNA3.1(+) eukaryotic expression vector. Constructed to encode Hu-1, Alpha (Alpha, ⁇ ), Beta (Beta, ⁇ ), gamma (gamma, ⁇ ), Delta (delta, ⁇ ), Miu (miu, ⁇ ) and Omicron , o) Plasmid of S glycoprotein of SARS-CoV-2 variant strain.
  • the lentiviral packaging plasmid psPAX2 and the pLVX-AcGFP-N1-Fluc lentiviral reporter plasmid expressing GFP and luciferase were from HonorGene (China).
  • Pseudoviruses were generated by co-transfecting psPAX2, pLVX-AcGFP-N1-Fluc and plasmids encoding various S genes into HEK 293T cells using Lipofectamine 3000 (Invitrogen, L3000-015). Supernatants were harvested 24 ⁇ 2 h after transfection, centrifuged at 1500 rpm for 5 min to remove cell debris, and stored at -80°C.
  • Pseudoviral reservoirs were titrated by infecting 293T-ACE2 cells by adding the Bright-Glo Luciferase Assay System (Promega, E2650) after an incubation period of 44 to 48 h at 37°C and 5% CO using microbiome. Luciferase activity was measured with a plate reader (TECAN, Spark). Then the TCID 50 of the pseudovirus is 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).
  • test serum samples were first heat inactivated at 56°C for 30 min and then clarified by centrifugation at 10,000 rcf for 5 min.
  • Samples were serially diluted (3-fold) in assay medium (100 ml) and incubated with 650 TCID 50 of pseudovirus (50 ml) for 1 hour at 37°C, while virus-infected untreated controls (virus alone) and cells alone ( background control). Then, add fresh trypsinized 293T-ACE2 cells to each well at 100 mcL of 20,000 cells/well.
  • IC50 neutralizing antibody titer for a given serum sample is defined as the serum dilution at which the sample shows a 50% reduction in relative light units (RLU) compared to virus-infected control wells.
  • RLU relative light units
  • VOC high concern
  • the cDNA encoding the extracellular domain of the SARS-CoV-2 spike (S) protein from Omicron was gene synthesized by GenScript using Cricetulus griseus (Chinese hamster) preferred codons. This cDNA was subcloned into the pTRIMER expression vector (GenHunter Corporation) at the Hind III and Bgl II sites to allow in-frame fusion of the soluble S protein to the Protein TrimerizationTM tag (from human type I (alpha) collagen). Amino acid residues 1156-1406) as described above.
  • the expression vector was transiently transfected into the HEK-293F cell line (Clover Biopharma) using PEI (Polyscience) and grown in OPM-293CD05 medium (OPM) and OPM-293proFeed supplement (OPM). S-trimeric proteins were purified from conditioned media to homogeneity using a Protein Trimerizer TM tag-specific affinity column (Clover Biopharma).
  • a protein trimerized TM tag subunit vaccine based on the Omicron strain was constructed and produced and administered to mice as a priming agent and/or booster vaccine for homologous boosting or with Hu-1 S- Used together with trimers Perform heterologous reinforcement.
  • a bivalent vaccine containing Hu-1 S-trimer and Omicron S-trimer was constructed and produced as the initial dose and booster dose, as shown in Figure 4.
  • Figures 5A-5B show the SARS-CoV-2 Hu-1, alpha (alpha, ⁇ ,B.1.1.7), beta (beta, ⁇ ,B.1.351), gamma (gamma, ⁇ ,P.1), delta (delta, ⁇ ,B.1.617.2), and Omicron (Omicro,o,B.1.1.529) strain pseudovirus neutralizing antibody IC 50 data.
  • Hu-1 S-trimer as the priming agent and Omicron S-trimer as the booster produced generally higher neutralizing antibody titers than Omicron S-
  • the heterologous booster group with trimer as initiator and Hu-1 S-trimer as booster was good.
  • Using the bivalent vaccine as the initial dose and booster dose as shown in Figure 5E, can both increase the neutralizing antibody titer against the Omicron strain pseudovirus (compared to two doses of Hu-1S-trimer immunization) , and can maintain or increase neutralizing antibody titers against other strains (compared to two doses of Hu-1 S-trimer immunization).
  • Omicron S-trimer was administered as the third booster dose to mice primed/boosted with the Hu-1 S-trimer vaccine to observe its further enhanced neutralization compared with other VOC vaccine candidates.
  • Figures 6A-6B show VOC neutralizing antibodies in mice boosted with the 3rd dose of Omicron S-trimer vaccination.
  • BALB/c mice were immunized twice on day 0 (dose 1) and day 21 (dose 2) with 3 ⁇ g of Hu-1 S-trimer adjuvanted with 150 ⁇ g of CpG 1018 plus 75 ⁇ g of Alum.
  • animals received a third dose of vaccination: 3 ⁇ g of Omicron S1/S2 furin cleavage site mutant S-trimer adjuvanted with 150 ⁇ g of CpG 1018 plus 75 ⁇ g of Alum; Serum was collected on study day 56 (D35PD2 or D-1PD3) and day 64 (D7PD3) for pseudovirus neutralizing antibody testing.
  • Figures 7A-7G show VOC neutralizing antibodies in mice boosted with different dose 3 vaccines.
  • BALB/c mice were immunized twice on day 0 (dose 1) and day 21 (dose 2) with 3 ⁇ g of Hu-1 S-trimer adjuvanted with 150 ⁇ g of CpG 1018 plus 75 ⁇ g of Alum.
  • dose 1 dose 1
  • dose 2 dose 2
  • dose 3 dose 3
  • Hu-1 S-trimer adjuvanted 150 ⁇ g of CpG 1018 plus 75 ⁇ g of Alum.
  • Group 1 did not receive the third dose of vaccination and served as the control group;
  • Group 2 3 ⁇ g Hu-1 S-trimer with 150 ⁇ g CpG1018 plus 75 ⁇ g Alum as adjuvant was used as the third dose of vaccination;
  • Group 3 Vaccination with 3 ⁇ g Omicron S-trimer adjuvanted with 150 ⁇ g CpG 1018 plus 75 ⁇ g Alum as the third dose;
  • Group 4 Bivalent vaccine with 150 ⁇ g CpG 1018 plus 75 ⁇ g Alum as adjuvant (including 1.5 ⁇ g Hu-1 S-trimer and 1.5 ⁇ g Omicron S1/S2 furin cleavage site mutant S-trimer) as the third dose of vaccination.
  • Serum was collected on study day 56 (D-1PD3) and day 71 (D14PD3) and tested for neutralizing antibodies against multiple mutant pseudoviruses.
  • the Omicron vaccine candidate (Group 3) significantly potentiated Omicron-specific neutralizing antibodies (51.9-fold) compared with the Hu-1 S-trimer and Omicron S-trimer bivalent vaccines A similar but smaller degree of boosting (28.5-fold) was observed in the vaccine group (Group 4). Enhancement of neutralizing antibody breadth was also observed in the Hu-1 S-trimer group with Alum/CpG as adjuvant (Group 2).
  • the potentiation of certain VOC (Hu-1, Alpha, Delta and Omicron) neutralizing antibodies by the fully adjuvanted Hu-1 S-trimer (group 2) was not significant. This may be due to the short interval between the last two doses of the vaccine (36 days), which did not allow enough time for more memory responses to develop.
  • mice vaccinated with a second booster vaccination with the bivalent vaccine further significantly enhanced their response to the addition of Already strong neutralizing antibody responses to all VOCs except beta variants (Fig. 8B); the Hu-1S-trimer monovalent vaccine significantly enhanced neutralizing antibodies against beta, gamma, and omikon strains. and antibodies, whereas the Omicron S-trimer monovalent vaccine significantly enhanced responses against Delta and Omicron.
  • Serum antibody neutralizing responses were monitored for three months after the third booster dose to assess durability of vaccine protection (Figure 8C).
  • Sera from the control group (not vaccinated with dose 3) showed strong neutralizing responses to all VOCs except Omicron, while the Omicron group showed low GMT49 (12-1197) (95% CI) ;
  • the third dose of Hu-1S-trimer significantly improved the antibody neutralizing response of vaccinated mice to Hu-1, alpha, beta, gamma, and delta strains.
  • the improvement of antibody neutralization level of Hu-1S-trimer dose 3 against Omicron was lower than that of other variants, with a GMT of 202 (129-2508) (95% CI), it also improved the level of antibody neutralization against Omicron.
  • the third dose of Omicron S-trimer significantly improved the antibody neutralizing response to Omicron compared with the control group (without a second booster vaccination), with a GMT of 1349 (1324-2112) (95 %CI); showed a trend toward lower responses to other VOCs and was comparable to the control group.
  • the secondary booster vaccination of the bivalent vaccine significantly improved the antibody neutralization response to all VOCs, and the response trend was higher than that of the Omicron S-trimer monovalent booster vaccination; its GMT against Omicron virus was 799 ( 762-1973) (95% CI)), which is comparable to the results elicited by Omicron S-trimer alone.
  • the BA.1, BA.2, BA.5, BA.2.75, BF.7, and BQ.1.1 pseudoviruses of Alpha, Beta, Delta, and Omicron all produce strong neutralizing antibodies.
  • C of Figure 10 shows that the bivalent vaccine including Omicron S-trimer + Omicron strain (BA.5) S-trimer is effective against Omicron BA.1, BA. 2, BA.5, BA.2.75, BF.7, and BQ.1.1 pseudoviruses all induced strong neutralizing antibodies.
  • B of Figure 10 shows that the bivalent vaccine including Hu-1 S-trimer + Omicron strain (BA.5) S-trimer is more effective against SARS than pseudoviruses tested in other trials.
  • -CoV-2 Hu-1 and Omicron BA.5 pseudoviruses both induced the strongest neutralizing antibodies.

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Abstract

涉及包括重组肽和蛋白质的免疫原性组合物，所述重组肽和蛋白质包含冠状病毒抗原和免疫原，例如SARS-CoV-2冠状病毒奥密克戎(omicron,B.1.1.529或BA.5)变异株S蛋白肽或其片段、变体或突变体，变体或突变体例如含奥密克戎变异株受体结合域及Hu-1或其它变异株S蛋白肽序列的嵌合抗原和免疫原。在一些方面，所述免疫原性组合物包括分泌性融合蛋白，所述分泌性融合蛋白包括可溶性冠状病毒抗原，所述可溶性冠状病毒抗原蛋白通过框内融合连接到能够自三聚化的胶原的C-末端部分以形成二硫键连接的三聚体融合蛋白。在一些方面，提供的免疫原性组合物可用于产生免疫应答，例如作为疫苗用于预防冠状病毒感染，例如SARS-CoV-2 Hu-1、阿尔法、贝塔、伽马、奥密克戎、缪、奥密克戎和/或其它毒株的感染。在一些方面，提供的免疫原性组合物可用于疫苗组合物中，例如，作为预防性和/或治疗性疫苗的一部分。还提供用于产生重组肽和蛋白质的方法、预防、治疗和/或诊断方法以及相关试剂盒。

Description

冠状病毒疫苗组合物、方法及其使用 技术领域
本公开内容在一些方面涉及用于治疗和/或预防冠状病毒感染的免疫原性组合物，所述免疫原性组合物包括重组肽和蛋白质，所述重组肽和蛋白质包括冠状病毒抗原和免疫原，例如冠状病毒S蛋白肽，包括基于SARS-CoV-2奥密克戎(B.1.1.529)毒株的S蛋白肽由三聚体化TM标签多肽间二硫键连接形成的亚单位疫苗。
背景技术
冠状病毒感染广泛范围的鸟类和哺乳动物，包括人类。冠状病毒可能每年在人体内传播，通常会引起轻微的呼吸系统疾病，尽管在婴幼儿、老年人和免疫功能低下的人群中严重程度更高。但是，某些冠状病毒，包括中东呼吸综合征冠状病毒(MERS-CoV)、严重急性呼吸综合征冠状病毒(SARS-CoV-1)和严重急性呼吸综合征冠状病毒2(SARS-CoV-2)，具有高致病性。冠状病毒的高致病性、空气传播性、高病死率和模糊确定的流行病学使得迫切需要有效的疫苗和相关的治疗药剂。特别是，迫切需要能够迅速诱导针对SARS-CoV-2的有效免疫应答的疫苗。本发明提供了满足上述和其它需要的方法、用途和制品(article of manufacture)。
发明内容
从2020年末开始，携带有突变的可能导致免疫逃逸的多重变异SARS-CoV-2毒株的出现和传播使得对第二代疫苗进行快速评估成为必要，目的是诱导具有广泛保护性的优化的免疫应答。
尽管COVID-19疫苗开发的巨大进展和空前速度，但在SARS-CoV-2病毒爆发首次出现16个月后的2021年4月，COVID-19每日病例创下了新的纪录。COVID-19导致的总死亡人数在2021年5月超过300万人，仅在前3个月就累计死亡100万人。
最令人担忧的是，在全球COVID-19病例激增的同时，从2020年底开始出现多种新的SARS-CoV-2变异株(VOC)。这些VOC似乎与刺突(S)蛋白的突变有关，突变可能会增加病毒传播率和/或基于SARS-CoV-2 Hu-1毒株的第一波COVID-19疫苗接种引起的免疫逃逸。B.1.1.7变种在英国(UK)、B.1.351变种在南非和P.1变种在巴西的出现和传播导致其被归类为VOC。这些VOC都包括S蛋白的受体结合结构域(RBD)中的N501Y突变，据报道，该突变可使传播增加40％至70％。B.1.351和P.1变种还有两个另外的RBD突变——E484K和K417——这可能允许从Hu-1疫苗和自然感染诱导的抗体中免疫逃逸。
COVID-19疫苗的随机对照临床试验表明，与SARS-CoV-2 Hu-1毒株相比，疫苗对VOC的有效性降低。基于佐剂蛋白的COVID-19疫苗NVX-CoV2373在英国(B.1.1.7占主导地位)的有效性为89％，但在南非(B.1.351占主导地位)的有效性仅为49％。腺病毒载体COVID-19疫苗ChAdOx1对B.1.351变种的有效性仅为10％。辉瑞疫苗接种者对B.1.351变种的有效性为75％，而对Hu-1的有效性为95％。Coronavac是基于Hu-1毒株的灭活疫苗，接种该疫苗的受试者未检测到针对P.1的中和抗体滴度。
虽然有一些令人鼓舞的证据表明，Hu-1 COVID-19疫苗可能会预防由VOC引起的严重疾病和死亡，但对任何传播率上升的COVID-19疾病的疫苗有效性较低，可能会使实现群体免疫特别困难，全球COVID-19疫苗短缺可能会进一步加剧这一问题。如果不能得到有效控制，SARS-CoV-2 VOC在全球的迅速传播可能会导致新的目标变种或可能包含新的逃逸突变的VOC的继续出现，例如印度变种(B.1.617)，它与2021年春季COVID-19病例的大规模激增同时出现，现已被世卫组织宣布为新的VOC。该谱系的B.1.617.2变种被命名为Delta。最近，在南非首次发现的B.1.1.529变种被重新命名为Omicron(奥密克戎)。
在这些情况下，必须迅速评估再次加强剂策略，以增强对VOC的中和抗体应答，并开发可能提供优化的广泛保护和交叉中和特性的第二代COVID-19疫苗。
在一些实施方案中，本文公开了包含多种重组多肽的蛋白质，每种重组多肽包括连接到胶原的C-末端前肽的冠状病毒表面抗原，其中重组多肽的C-末端前肽形成多肽间二硫键。该重组多肽或蛋白质可作为免疫原例如疫苗使用。
在一些实施方案中，本文公开了重组亚单位疫苗，其包括来自冠状病毒(例如SARS-CoV-2奥密克戎(B.1.1.529)的S蛋白或其片段的胞外域(例如，没有跨膜和细胞质结构域)，其在框内与能够形成二硫键连接的同源三聚体的胶原的C-前肽融合。由此产生的重组亚单位疫苗(例如S-三聚体)可以从转染的细胞中表达和纯化，并且预期为三聚体形式的天然构象。这解决了当病毒抗原以可溶性形式表达为重组肽或蛋白质而没有跨膜和/或细胞质结构域时经常遇到的病毒抗原错误折叠问题。这种错误折叠的病毒抗原不能忠实地保持天然的病毒抗原构象，并且常常不能产生中和抗体。
在一些实施方案中，冠状病毒是严重急性呼吸综合征(SARS)冠状病毒(SARS-CoV-1)、SARS冠状病毒2(SARS-CoV-2)、SARS样冠状病毒、中东呼吸综合征(MERS)冠状病毒(MERS-CoV)、MERS样冠状病毒、NL63-CoV、229E-CoV、OC43-CoV、HKU1-CoV、WIV1-CoV、MHV、HKU9-CoV、PEDV-CoV或SDCV。
在前述任一实施方案中，表面抗原可以包括冠状病毒刺突(S)蛋白或其片段或表位，其中表位可选地是线性表位或构象表位，并且其中蛋白质包括三种重组多肽。
在前述任一实施方案中，表面抗原可以包括信号肽、S1亚单位肽、S2亚单位肽或其任意组合。
在前述任一实施方案中，表面抗原可以包括信号肽、受体结合结构域(RBD)肽、受体结合基序(RBM)肽、融合肽(FP)、七肽重复序列1(HR1)或七肽重复序列2(HR2)或其任意组合。
在前述任一实施方案中，表面抗原可以包括S蛋白的受体结合结构域(RBD)。
在前述任一实施方案中，表面抗原可以包括S蛋白的S1亚单位和S2亚单位。
在前述任一实施方案中，表面抗原可以不含跨膜(TM)结构域肽和/或细胞质(CP)结构域肽。
在前述任一实施方案中，表面抗原可以包括蛋白酶裂解位点，其中蛋白酶可选地为弗林蛋白酶(furin)、胰蛋白酶、因子Xa、凝血酶或组织蛋白酶L。
在前述任一实施方案中，表面抗原可以不含蛋白酶裂解位点，其中蛋白酶可选地为弗林蛋白酶(furin)、胰蛋白酶、因子Xa、凝血酶或组织蛋白酶L，或者可以包含不可被蛋白酶裂解的突变的蛋白酶裂解位点。
在前述任一实施方案中，表面抗原可以是可溶解的或不直接结合到脂质双层，例如膜或病毒包膜。
在前述任一实施方案中，在蛋白质的重组多肽中表面抗原可以相同或不同。
在前述任一实施方案中，表面抗原可以直接融合到C-末端前肽，或可以通过接头(例如包含甘氨酸-X-Y重复序列的接头，其中X和Y独立地是任何氨基酸并且可选地是脯氨酸或羟脯氨酸)连接到C-末端前肽。
在前述任一实施方案中，蛋白质可以是可溶解的或不直接结合到脂质双层，例如膜或病毒包膜。
在前述任一实施方案中，蛋白质可以结合到受试者的细胞表面受体，可选地其中受试者是哺乳动物，例如灵长类动物，例如人类。
在前述任一实施方案中，细胞表面受体可以是血管紧张素转换酶2(ACE2)、二肽基肽酶4(DPP4)、树突状细胞特异性细胞间粘附分子-3-抓取非整合素(DC-SIGN)或肝脏/淋巴结-SIGN(L-SIGN)。
在前述任一实施方案中，C-末端前肽可以是人胶原。
在前述任一实施方案中，C-末端前肽可以包括proα1(I)、proα1(II)、proα1(III)、proα1(V)、proα1(XI)、proα2(I)、proα2(V)、proα2(XI)或proα3(XI)的C-末端前肽或其片段。
在前述任一实施方案中，在重组多肽中C-末端前肽可以相同或不同。
在前述任一实施方案中，C-末端前肽可以包括SEQ ID NO:67-80中任一个，或与SEQ ID NO:67-80中任一个具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或99.5％序列同源性的氨基酸序列，能够形成多肽间二硫键并使重组多肽三聚化。
在前述任一实施方案中，C-末端前肽可以包括SEQ ID NO:67或与其至少95％相同的氨基酸序列，能够形成多肽间二硫键并使重组多肽三聚化。
在前述任一实施方案中，C-末端前肽可以包括SEQ ID NO:68或与其至少95％相同的氨基酸序列，能够形成多肽间二硫键并使重组多肽三聚化。
在前述任一实施方案中，C-末端前肽可以包括SEQ ID NO:69或与其至少95％相同的氨基酸序列，能够形成多肽间二硫键并使重组多肽三聚化。
在前述任一实施方案中，C-末端前肽可以包括SEQ ID NO:70或与其至少95％相同的氨基酸序列，能够形成多肽间二硫键并使重组多肽三聚化。
在前述任一实施方案中，C-末端前肽可以包括SEQ ID NO:71或与其至少95％相同的氨基酸序列，能够形成多肽间二硫键并使重组多肽三聚化。
在前述任一实施方案中，C-末端前肽可以包括SEQ ID NO:72或与其至少95％相同的氨基酸序列，能够形成多肽间二硫键并使重组多肽三聚化。
在前述任一实施方案中，C-末端前肽可以包括SEQ ID NO:73或与其至少95％相同的氨基酸序列，能够形成多肽间二硫键并使重组多肽三聚化。
在前述任一实施方案中，C-末端前肽可以包括SEQ ID NO:74或与其至少95％相同的氨基酸序列，能够形成多肽间二硫键并使重组多肽三聚化。
在前述任一实施方案中，C-末端前肽可以包括SEQ ID NO:75或与其至少95％相同的氨基酸序列，能够形成多肽间二硫键并使重组多肽三聚化。
在前述任一实施方案中，C-末端前肽可以包括SEQ ID NO:76或与其至少95％相同的氨基酸序列，能够形成多肽间二硫键并使重组多肽三聚化。
在前述任一实施方案中，C-末端前肽可以包括SEQ ID NO:77或与其至少95％相同的氨基酸序列，能够形成多肽间二硫键并使重组多肽三聚化。
在前述任一实施方案中，C-末端前肽可以包括SEQ ID NO:78或与其至少95％相同的氨基酸序列，能够形成多肽间二硫键并使重组多肽三聚化。
在前述任一实施方案中，C-末端前肽可以包括SEQ ID NO:79或与其至少95％相同的氨基酸序列，能够形成多肽间二硫键并使重组多肽三聚化。
在前述任一实施方案中，C-末端前肽可以包括SEQ ID NO:80或与其至少95％相同的氨基酸序列，能够形成多肽间二硫键并使重组多肽三聚化。
在前述任一实施方案中，C-末端前肽可以包括包含甘氨酸-X-Y重复序列连接到SEQ ID NO:67-80中任一个的N-末端的序列，其中X和Y独立地是任何氨基酸并且可选地是脯氨酸或羟脯氨酸，或与其至少90％相同的氨基酸序列，能够形成多肽间二硫键并使重组多肽三聚化。
在前述任一实施方案中，每种重组多肽中的表面抗原可以为融合前构象。
在前述任一实施方案中，每种重组多肽中的表面抗原可以为融合后构象。
在前述任一实施方案中，每种重组多肽中的表面抗原可以包括SEQ ID NO:27-66和81-84中任一个或与其至少80％相同的氨基酸序列。
在前述任一实施方案中，重组多肽可以包括SEQ ID NO:1-26和85-92中任一个或与其至少80％相同的氨基酸序列。
本文还提供了包含本文提供的蛋白质的免疫原。本文提供了包含直接或间接连接到纳米颗粒的本文提供的蛋白质的蛋白质纳米颗粒。本文提供了包含本文提供的蛋白质的类病毒颗粒(VLP)。
本文还提供了编码本文提供的蛋白质的一种、两种、三种或更多种重组多肽的分离核酸。在一些实施方案中，编码S蛋白肽的多肽在框内与编码胶原的C-末端前肽的多肽融合。在一些实施方案中，本文提供的分离核酸可操作地连接到启动子。
在一些实施方案中，本文提供的分离核酸是DNA分子。在一些实施方案中，本文提供的分离核酸是RNA分子，可选地是mRNA分子，例如核苷修饰的mRNA、非扩增mRNA、自扩增mRNA或反式扩增mRNA。
本文还提供了包含本文提供的分离核酸的载体。在一些实施方案中，载体是病毒载体。
在一些方面，本文提供了包含本文提供的载体的病毒、假病毒或细胞，可选地，其中病毒或细胞具有重组基因组。在一些方面，本文提供了免疫原性组合物，其包含本文提供的蛋白质、免疫原、蛋白质纳米颗粒、VLP、分离核酸、载体、病毒、假病毒或细胞以及药学上可接受的载体。
本文还提供了包含本文提供的免疫原性组合物和可选佐剂的疫苗，其中疫苗可选地是亚单位疫苗。在一些实施方案中，疫苗是预防性和/或治疗性疫苗。可选佐剂可在初始剂和/或加强剂中使用。独立地，初始剂和/或任意一剂或多剂加强剂的佐剂可包括：含铝佐剂，例如含明矾和/或氢氧化铝的佐剂；含寡核苷酸的佐剂，例如含CpG寡脱氧核苷酸(CpG-ODN)的佐剂；含TLR9激动剂的佐剂；含可代谢油、α-生育酚、和/或聚氧乙烯山梨醇酐单油酸酯(吐温-80)的佐剂，例如水包油乳液形式的含角鲨烯、α-生育酚、和吐温-80和/或Span 85的佐剂；或任意一种佐剂组合。
在一些方面，本文提供了产生蛋白质的方法，包括：在宿主细胞中表达本文提供的分离核酸或载体，以产生本文提供的蛋白质；并纯化蛋白质。本文提供了通过本文提供的方法产生的蛋白质。
本文提供了用于在受试者中产生对冠状病毒的S蛋白肽或其片段或表位的免疫应答的方法，包括向受试者施用有效量的如本文提供的蛋白质、免疫原、蛋白质纳米颗粒、VLP、分离核酸、载体、病毒、假病毒、细胞、免疫原性组合物或疫苗，以产生免疫应答。在一些实施方案中，本文提供的方法用于治疗或预防冠状病毒感染。在一些实施方案中，产生免疫应答抑制或减少了受试者中冠状病毒的复制。在一些实施方案中，免疫应答包括细胞介导的应答和/或体液应答，可选地包括产生一种或更多种中和抗体，例如多克隆抗体或单克隆抗体。在一些实施方案中，免疫应答针对冠状病毒的S蛋白肽或其片段或表位，但不针对C-末端前肽。在一些实施方案中，由于先前暴露于一种或更多种冠状病毒，对受试者的施用不会导致受试者中的抗体依赖性增 强(ADE)。在一些实施方案中，当随后暴露于一种或更多种冠状病毒时，施用不会导致受试者中的抗体依赖性增强(ADE)。在一些实施方案中，方法进一步包括初免步骤(priming step)和/或加强步骤(boosting step)。在一些实施方案中，通过局部、经皮、皮下、皮内、口服、鼻内(例如，鼻内喷雾剂)、气管内、舌下、口腔、直肠、阴道、吸入、静脉内(例如，静脉注射)、动脉内、肌肉内(例如，肌肉注射)、心内、骨内、腹腔内、跨粘膜、玻璃体内、视网膜下、关节内、关节周围、局部或应用皮肤(epicutaneous)给药来进行给药步骤。在一些实施方案中，以单剂量或具有一个间隔或更多个间隔的一系列剂量施用有效量。在一些实施方案中，在不使用佐剂的情况下施用有效量。在一些实施方案中，有效量与佐剂一起施用。
本文提供了方法，该方法包括向受试者施用有效量的本文提供的蛋白质，以在受试者中产生针对冠状病毒的中和抗体或中和抗血清。在一些实施方案中，受试者是哺乳动物，可选地是人类或非人类灵长类动物。在一些实施方案中，方法进一步包括从受试者分离中和抗体或中和抗血清。在一些实施方案中，方法进一步包括通过被动免疫向人类受试者施用有效量的分离中和抗体或中和抗血清，以预防或治疗冠状病毒感染。在一些实施方案中，针对冠状病毒的中和抗体或中和抗血清包括针对冠状病毒S蛋白肽或其片段或表位的多克隆抗体，可选地，其中中和抗体或中和抗血清不含或基本不含针对胶原的C-末端前肽的抗体。在一些实施方案中，中和抗体包括针对冠状病毒S蛋白肽或其片段或表位的单克隆抗体，可选地，其中中和抗体不含或基本不含针对胶原的C-末端前肽的抗体。
在一些方面，本文提供的蛋白质、免疫原、蛋白质纳米颗粒、VLP、分离核酸、载体、病毒、假病毒、细胞、免疫原性组合物或疫苗用于诱导受试者中对冠状病毒的免疫应答，和/或用于治疗或预防冠状病毒感染。
在一些方面，本文提供了本文提供的蛋白质、免疫原、蛋白质纳米颗粒、VLP、分离核酸、载体、病毒、假病毒、细胞、免疫原性组合物或疫苗的用途，用于诱导受试者中对冠状病毒的免疫应答，和/或用于治疗或预防冠状病毒感染。在一些方面，本文提供了本文提供的蛋白质、免疫原、蛋白质纳米颗粒、VLP、分离核酸、载体、病毒、假病毒、细胞、免疫原性组合物或疫苗的用途，用于制造用于诱导受试者中对冠状病毒的免疫应答的药物或预防剂，和/或用于治疗或预防冠状病毒感染。
本文还提供了用于分析样品的方法，包括：将样品与本文提供的蛋白质接触，并检测蛋白质与能够特异性结合冠状病毒的S蛋白肽或其片段或表位的分析物之间的结合。在一些实施方案中，分析物是识别S蛋白肽或其片段或表位的抗体、受体或细胞。在一些实施方案中，结合指示样品中存在分析物，和/或样品来源于的受试者中存在冠状病毒感染。
本文提供了试剂盒，其包括本文提供的蛋白质和含有或固定蛋白质的基质、垫或小瓶，可选地其中试剂盒是ELISA或侧向流动检测试剂盒(lateral flow assay kit)。
附图说明
图1A-1B示出了用于SARS-CoV-2的示例性可溶性S-三聚体亚单位疫苗的结构特征。图1A为S-三聚体结构域的示意图，图1B为其三聚体和共价连接的三维构象。
图2示出了示例性的基于奥密克戎毒株的S-三聚体的表征，用考马斯亮蓝染色显示了全长S-三聚体和在S1/S2弗林蛋白酶(furin)位点裂解的部分裂解形式，包括S2-三聚体和裂解的S1片段。A道为野生型奥密克戎S-三聚体。B道为奥密克戎S1/S2弗林蛋白酶裂解位点685R→685A突变体S-三聚体。C道为奥密克戎S1/S2弗林蛋白酶裂解位点685R→685A和脯氨酸986K/987V→986P/987P突变体S-三聚体。D道为Hu-1 S-三聚体。与Hu-S-三聚体相比，奥密克戎S-三聚体S1裂解的程度更高(对比A道和D道)。
图3A-3B示出了示例性奥密克戎S-三聚体与ACE2-Fc的受体结合动力学。图3A示出了与Hu-1 S-三聚体与受体结合Kd为5nM。图4B示出了奥密克戎S-三聚体与受体结合Kd为2pM。与Hu-1 S-三聚体相比，奥密克戎S-三聚体受体亲和力更高。
图4为小鼠中使用两剂S-三聚体抗原(第一剂初免-第二剂加强)后基于假病毒中和滴度的交叉中和免疫原性试验设计示意图。在第0天(初始剂)和第21天(加强剂)用CpG 1018加Alum作为佐剂的S-三聚体(n＝10/组)对小鼠进行两次免疫。在Hu-1同源加强组中，初始剂和加强剂均为Hu-1 S-三聚体；在奥密克戎同源加强组中，初始剂和加强剂均为奥密克戎S-三聚体；在异源加强组中，Hu-1 S-三聚体作为初始剂和奥密克戎S-三聚体作为加强剂，或奥密克戎S-三聚体作为初始剂和Hu-1 S-三聚体作为加强剂；在二价加强组中，初始剂和加强剂均为二价疫苗(二价疫苗包括Hu-1 S-三聚体和奥密克戎S-三聚体)。
图5A-5B示出了根据图4的试验设计小鼠接受两剂S-三聚体抗原(第一剂初免-第二剂加强)免疫后的SARS-CoV-2 Hu-1、阿尔法(alpha,α,B.1.1.7)、贝塔(beta,β,B.1.351)、伽马(gamma,γ,P.1)、德尔塔(delta,δ,B.1.617.2)、和奥密克戎(Omicron,o,B.1.1.529)毒株假病毒中和抗体IC50数据。在采用150μg CpG1018加75μg Alum作为佐剂的两次免疫后，第35天(D4PD2)对小鼠采血，进行假病毒中和抗体测试。图5A为Hu-1 S-三聚体同源加强组；图5B为奥密克戎S-三聚体同源加强组；图5C为Hu-1 S-三聚体作为初始剂和奥密克戎S-三聚体作为加强剂的异源加强组；图5D为奥密克戎S-三聚体作为初始剂和Hu-1 S-三聚体作为加强剂的异源加强组；图5E为初始剂和加强剂均为包括Hu-1 S-三聚体和奥密克戎S-三聚体的二价疫苗。对比图5A和图5B中对奥密克戎毒株假病毒的中和抗体滴度，两剂奥密克戎S-三聚体免疫后的小鼠针对奥密克戎毒株假病毒的中和抗体滴度比两剂Hu-1 S-三聚体免疫后的小鼠显著增加，但对其它某些毒株的中和抗体滴度有所降低。两剂Hu-1 S-三聚体免疫对其它毒株产生良好的中和抗体滴度，但对奥密克戎毒株产生的中和抗体滴度显著低于对其它毒株产生的中和抗体滴度。在异源加强组中，Hu-1 S-三聚体作为初始剂和奥密克戎S-三聚体作为加强剂(图5C)产生的中和抗体滴度总体较奥密克戎S-三聚体作为 初始剂和Hu-1 S-三聚体作为加强剂的异源加强组(图5D)好。使用二价疫苗为初始剂和加强剂，如图5E所示，既能提高针对奥密克戎毒株假病毒的中和抗体滴度(与两剂Hu-1 S-三聚体免疫相比)，又能保持或提高针对其它毒株的中和抗体滴度(与两剂Hu-1S-三聚体免疫相比)。
图6A-6B示出了第3剂奥密克戎S-三聚体疫苗接种加强小鼠中的VOC中和抗体。图6A为三次免疫及假病毒中和抗体测试示意图。在第0天(第1剂)和第21天(第2剂)用150μg CpG 1018加75μg Alum作为佐剂的3μg Hu-1 S-三聚体对BALB/c小鼠进行两次免疫。在研究的第57天，动物接受第3剂的疫苗接种：用150μg CpG1018加75μg Alum作为佐剂的3μg奥密克戎S-三聚体；在研究第56天(D35PD2或D-1PD3)和第64天(D7PD3)收集血清，进行假病毒中和抗体测试。图6B为在第35天(D14PD2)，第56天(D35PD2)和第64天(D7PD3)的SARS-CoV-2 Hu-1、阿尔法、贝塔、伽马、德尔塔、和奥密克戎毒株假病毒中和抗体(IC50)数据。点代表个体动物的数据；横线表示几何平均滴度(GMT)±SEM。检测限(LOD)滴度为20。每一列的底部都标注了组别GMP的编号。接受第3剂奥密克戎S-三聚体疫苗的小鼠与第3剂疫苗接种前的数据相比较，显著增加对奥密克戎毒株假病毒中和抗体的滴度。
图7A-7G示出了不同第3剂疫苗接种加强小鼠中的VOC中和抗体。图7A为三次免疫及假病毒中和抗体测试示意图。在第0天(第1剂)和第21天(第2剂)用150μg CpG 1018加75μg Alum作为佐剂的3μg Hu-1 S-三聚体对BALB/c小鼠进行两次免疫。在研究的第57天，将动物分为四组(每组n＝10)，不接受第3剂的疫苗接种，或接受Hu-1 S-三聚体，奥密克戎S-三聚体，或二价疫苗作为第3剂的疫苗接种。在研究第56天(D-1PD3)和第71天(D14PD3)收集血清，进行假病毒中和抗体测试。第1组不接受第3剂的疫苗接种，作为对照组；第2组：用150μg CpG 1018加75μg Alum作为佐剂的3μg Hu-1 S-三聚体作为第3剂的疫苗接种；第3组：用150μg CpG 1018加75μg Alum作为佐剂的3μg奥密克戎S-三聚体作为第3剂的疫苗接种；第4组：用150μg CpG 1018加75μg Alum作为佐剂的二价疫苗(包括1.5μg Hu-1 S-三聚体和1.5μg奥密克戎S-三聚体)作为第3剂的疫苗接种。图7B-7G分别为Hu-1、阿尔法、贝塔、伽马、德尔塔、和奥密克戎毒株假病毒中和抗体(IC50)数据。点代表个体动物的数据；横线表示每组的几何平均滴度(GMT)±SEM。检测限(LOD)滴度为20。每一列的底部都标注了组别的编号。与第3剂疫苗接种前的数据相比较，每个VOC的第3剂后(D14PD3)中和抗体滴度增加的倍数被标为"x"。
图8A-8C：Hu-1S-三聚体(祖先)疫苗、奥密克戎S-三聚体疫苗和二价疫苗(Hu-1 S-三聚体+奥密克戎S-三聚体)作为第3剂对已用Hu-1S-三聚体进行了初免(第1剂)/加强(第2剂)的免疫小鼠的再次(第3剂)进行加强免疫以诱导产生持久性广泛性的中和抗体研究结果。图8A：在第0天和第21天，对Balb/c小鼠(n＝10/组)用3μg Hu-1S-三聚体与150μg CpG 1018加75μg铝剂一起进行初免和加强免疫，然后该免疫后的小鼠作为没有第三剂的对照或用Hu-1S-三聚体(3μ g)，奥密克戎S-三聚体(3μg)或二价疫苗(1.5μg Hu-1 S-三聚体+1.5μg奥密克戎S-三聚体)与150μg CpG 1018加75μg铝剂一起配制，在第57天再次(第3剂)进行加强免疫。图8B；通过PsV中和试验分析研究第85天血清样品的对VOC的中和抗体。数据点代表个体动物的假病毒中和抗体滴度(IC50)；条形水平线表示每组的几何平均滴度(GMT)±95％CI。检测限(LOD)滴度(IC50)为20。每个条中标记的数字是每个测试组的GMT。图8C：对来自D0、D35、D56、D85(第3剂后1M)、D113(第3剂后2M)和D141(第3剂后3M)的血清的6种假病毒中和抗体动力学分析曲线。浅黄色条/框代表来自未接受进一步免疫的对照小鼠的样品；灰色条/框代表来自Hu-1S-三聚体免疫小鼠的样本；绿色条/框代表来自奥密克戎S-三聚体免疫小鼠的样品。粉红色条/框代表来自使用二价疫苗免疫的小鼠的样品。对于统计分析，使用双尾Mann-Whitney t检验进行比较。P值<0.05被认为是显着的。*：P<0.05，**：P<0.01，***：P<0.001。
图9为小鼠中使用两剂S-三聚体抗原(第一剂初免-第二剂加强)后基于假病毒中和滴度的交叉中和免疫原性试验设计示意图。在第0天(初始剂)和第21天(加强剂)用CpG 1018加Alum作为佐剂的S-三聚体(n＝10/组)对小鼠进行两次免疫，在第35天(D4PD2)对小鼠采血，进行假病毒中和抗体测试。
图10示出了根据图9的试验设计的小鼠接受两剂(第一剂初免-第二剂加强)奥密克戎毒株(BA.5)S-三聚体和包含其的二价，三价的疫苗免疫后的对SARS-CoV-2Hu-1、阿尔法、贝塔、德尔塔、奥密克戎的BA.1,BA.2,BA.5,BA.2.75,BF.7,BQ.1.1,XBB毒株假病毒中和抗体IC50数。在采用3μg抗原加上150μg CpG 1018和75μg Alum作为佐剂的两次免疫后，第35天(D4PD2)对小鼠采血，进行假病毒中和抗体测试。A:奥密克戎毒株(BA.5)S-三聚体；B:Hu-1 S-三聚体+奥密克戎毒株(BA.5)S-三聚体,C:奥密克戎S-三聚体疫苗+奥密克戎毒株(BA.5)S-三聚体；D:Belta S-三聚体+Delta-S-三聚体+奥密克戎毒株(BA.5)S-三聚体。
具体实施方式
本文提供了包含冠状病毒抗原或免疫原的融合肽和蛋白质的免疫原性组合物、方法和用途，用于冠状病毒感染的治疗(例如预防性、治疗性)。在一些实施方案中，公开了来自共价连接三聚体形式的RNA病毒的重组可溶性表面抗原的组合物和使用方法。在一些实施方案中，产生的融合蛋白作为二硫键连接的同源三聚体分泌，其结构更稳定，同时保留类天然三聚体病毒抗原的构象，因此可以用作对抗这些危险病原体的更有效疫苗。
在一些实施方案中，本文公开了使用病毒抗原三聚体作为疫苗或作为多价疫苗的一部分来预防病毒感染的方法，不使用或使用佐剂，或使用一种以上佐剂，可选地通过肌肉内注射或鼻内施用。病毒抗原三聚体可在初始剂，额外剂，和/或加强剂中使用。独立地，初始剂，额外剂，和/或任意一剂或多剂加强剂可不使用或使用佐剂。 如果使用佐剂，可选佐剂可包括：含铝佐剂，例如含明矾和/或氢氧化铝的佐剂；含寡核苷酸的佐剂，例如含CpG寡脱氧核苷酸(CpG-ODN)的佐剂；含TLR9激动剂的佐剂；含可代谢油、α-生育酚、和/或聚氧乙烯山梨醇酐单油酸酯(吐温-80)的佐剂，例如水包油乳液形式的含角鲨烯、α-生育酚、和吐温-80和/或Span 85的佐剂；或任意一种佐剂组合。
在一些实施方案中，本文公开了使用病毒抗原三聚体作为抗原用于通过检测识别病毒抗原(例如中和抗体)的抗体(例如IgM或IgG)来诊断病毒感染的方法。
在一些实施方案中，本文公开了使用病毒抗原三聚体作为抗原来产生可用于被动免疫的多克隆或单克隆抗体(例如，用于治疗冠状病毒感染的中和mAb)的方法。
在一些实施方案中，本文公开了作为疫苗或作为多价疫苗的一部分的病毒抗原三聚体，其中疫苗包括多种三聚体亚单位疫苗，所述三聚体亚单位疫苗包括病毒的相同蛋白质的病毒抗原，或包括一种或更多种病毒的两种或更多种不同蛋白质的病毒抗原或相同病毒的一种或更多种毒株的病毒抗原。
在一些实施方案中，本文公开了包含本文公开的病毒抗原三聚体的单价疫苗。在一些实施方案中，本文公开了包含本文公开的病毒抗原三聚体的二价疫苗。在一些实施方案中，本文公开了包含本文公开的病毒抗原三聚体的三价疫苗。在一些实施方案中，本文公开了包含本文公开的病毒抗原三聚体的四价疫苗。
在一些实施方案中，本文公开了包含本文公开的S-三聚体的单价疫苗。在一些实施方案中，本文公开了包含本文公开的S-三聚体的二价疫苗。在一些实施方案中，本文公开了包含至少一个包含第一S蛋白抗原的S-三聚体和至少一个包含第二S蛋白抗原的S-三聚体的二价疫苗。在一些实施方案中，第一和第二S蛋白抗原来自一种或更多种病毒物种或毒株/亚型的相同S蛋白，或来自一种或更多种病毒物种或相同病毒物种的一种或更多种毒株/亚型的两种或更多种不同S蛋白。在一些实施方案中，本文公开了包含本文公开的S-三聚体的三价疫苗。在一些实施方案中，本文公开了包含至少一个包含第一S蛋白抗原的S-三聚体、至少一个包含第二S蛋白抗原的S-三聚体和至少一个包含第三S蛋白抗原的S-三聚体的三价疫苗。在一些实施方案中，第一、第二和第三S蛋白抗原来自一种或更多种病毒物种或毒株/亚型的相同S蛋白，或来自一种或更多种病毒物种或相同病毒物种的一种或更多种毒株/亚型的两种、三种或更多种不同S蛋白。在一些实施方案中，本文公开了包含本文公开的S-三聚体的四价疫苗。在一些实施方案中，本文公开了包含至少一个包含第一S蛋白抗原的S-三聚体、至少一个包含第二S蛋白抗原的S-三聚体、至少一个包含第三S蛋白抗原的S-三聚体和至少一个包含第四S蛋白抗原的S-三聚体的四价疫苗。在一些实施方案中，第一、第二、第三和第四S蛋白抗原来自一种或更多种病毒物种或毒株/亚型的相同S蛋白，或来自一种或更多种病毒物种或相同病毒物种的一种或更多种毒株/亚型的两种、三种、四种或更多种不同S蛋白。
在一些实施方案中，本文公开了一种多价疫苗，例如二价疫苗，包括：第一三聚体融合蛋白，该第一三聚体融合蛋白包括可溶性冠状病毒SARS-CoV-2奥密克戎(B.1.1.529)刺突(S)蛋白表面抗原或其片段、变体或突变体，该可溶性冠状病毒表面抗原或其片段、变体或突变体连接到胶原的C-末端部分，该胶原的C-末端部分形成二硫键连接的三聚体，从而形成该第一三聚体融合蛋白；第二和/或更多三聚体融合蛋白，该第二或更多三聚体融合蛋白包括可溶性冠状病毒SARS-CoV-2 Hu-1、阿尔法、贝塔、伽马、德尔塔、或缪刺突(S)蛋白表面抗原或其片段、变体或突变体，该可溶性冠状病毒表面抗原或其片段、变体或突变体连接到胶原的C-末端部分，该胶原的C-末端部分形成二硫键连接的三聚体，从而形成该第二和/或更多三聚体融合蛋白。在一些实施方案中，该第二和/或三聚体融合蛋白包括可溶性冠状病毒SARS-CoV-2 Hu-1刺突(S)蛋白表面抗原或其片段、变体或突变体。在一些实施方案中，该多价疫苗在没有佐剂的情况下施用，可选地该多价疫苗作为初始剂(primary series)，额外剂(additional dose)，和/或同源或异源加强剂(booster dose)使用，例如第一剂，第二剂，第三剂，第四剂，和/或更多剂，可选地该初始剂，额外剂，或异源加强剂与其它重组亚单位疫苗、纳米颗粒疫苗、mRNA疫苗、DNA疫苗、腺病毒载体疫苗、和灭活病毒疫苗中的任意一种或几种配合使用。在一些实施方案中，该多价疫苗与一种以上佐剂一起施用，可选地该多价疫苗作为初始剂(primary series)，额外剂(additional dose)，和/或同源或异源加强剂(booster dose)使用，例如第一剂，第二剂，第三剂，第四剂，和/或更多剂，可选地该初始剂，额外剂，或异源加强剂与其它重组亚单位疫苗、纳米颗粒疫苗、mRNA疫苗、DNA疫苗、腺病毒载体疫苗、和灭活病毒疫苗中的任意一种或几种配合使用，可选地该初始剂，额外剂，和/或同源或异源加强剂中的佐剂可独立地包括：含铝佐剂，例如含明矾和/或氢氧化铝的佐剂；含寡核苷酸的佐剂，例如含CpG寡脱氧核苷酸(CpG-ODN)的佐剂；含TLR9激动剂的佐剂；含可代谢油、α-生育酚、和/或聚氧乙烯山梨醇酐单油酸酯(吐温-80)的佐剂，例如水包油乳液形式的含角鲨烯、α-生育酚、和吐温-80和/或Span 85的佐剂；或任意一种所述佐剂的组合。在一些实施方案中，该多价疫苗中的第一三聚体融合蛋白包括SEQ ID NO:81-84中的任一个所述的序列或与SEQ ID NO:81-84中的任一个所述的序列有至少或约80％、85％、90％、92％、95％、97％、99％的序列同一性的氨基酸序列。在一些实施方案中，该多价疫苗中的第二和/或更多三聚体融合蛋白包括SEQ ID NO:27-66中的任一个所述的序列或与SEQ ID NO:27-66中的任一个所述的序列有至少或约80％、85％、90％、92％、95％、97％、99％的序列同一性的氨基酸序列。
本文提供的包含冠状病毒抗原和免疫原的蛋白质，包括重组多肽和融合蛋白，可用于有效且安全地治疗(例如，治疗性、预防性)冠状病毒感染。例如，本文提供的包含冠状病毒抗原和免疫原的蛋白质治疗冠状病毒感染，而没有介导的(mediated)疫苗诱导的疾病增强(VED)和/或抗体依赖性增强(ADE)。此外，本文提供的包含冠状病毒抗原和免疫原的蛋白质容易产生，并且在例如高温、极端pH、高渗透压和低渗透压等 高应激条件下表现出稳定性。因此，本文提供的蛋白质和免疫原性组合物规避并满足了阻碍冠状病毒疫苗开发的生产、稳定性、安全性和有效性问题。
在一些方面，本文提供的冠状病毒抗原和免疫原包括冠状病毒刺突(S)蛋白或肽，尤其是SARS-CoV或SARS-CoV-2 S蛋白。SARS-CoV和SARS-CoV-2的刺突由S蛋白三聚体组成，S蛋白三聚体属于I类病毒融合糖蛋白的组，该组还包括HIV糖蛋白160(Env)、流感血凝素(HA)、副粘病毒F和埃博拉病毒糖蛋白。SARS-CoV和SARS-CoV-2 S蛋白各自编码表面糖蛋白前体，并且预测氨基端和大部分蛋白质位于细胞表面或病毒颗粒的外侧。S蛋白包括位于N末端的信号肽、细胞外结构域、跨膜结构域和细胞内结构域。与其它冠状病毒相似，SARS-CoV和SARS-CoV-2的S蛋白可以被蛋白酶裂解成S1和S2亚基。特别是，SARS-CoV-2包含弗林蛋白酶(furin)样裂解位点，这是其它SARS样CoV所缺乏的。
在一些实施方案中，本文提供了重组S胞外域三聚体。在一些实施方案中，重组S胞外域三聚体包括来自α-冠状病毒(例如，NL63-CoV或229E-CoV)的重组S胞外域原聚体。在一些实施方案中，重组S胞外域三聚体包括来自β-冠状病毒(例如OC43-CoV、SARS-CoV、SARS-CoV-2、MERS-CoV、HKU1-CoV、WIV1-CoV、小鼠肝炎病毒(MHV)或HKU9-CoV)的S胞外域原聚体。
与其它包膜RNA病毒(例如HIV、RSV和流感)类似，包括SARS-CoV-2在内的冠状病毒在其病毒包膜上都有三聚体表面抗原，以便在感染期间通过特定的细胞表面受体进入不同的宿主细胞。与SARS-CoV-1一样，SARS-CoV-2利用其三聚体病毒表面抗原S蛋白与其特异性细胞表面受体ACE2结合后进入哺乳动物呼吸系统的宿主细胞。产生有效的重组亚单位疫苗的先决条件是能够产生类似天然的病毒S抗原，特别是保持其三聚体构象，以引起足够数量的抗体可以结合到病毒S蛋白的受体结合结构域(RBD)，从而阻止病毒与ACE2受体结合，从而消除病毒感染。
在一些实施方案中，包含冠状病毒抗原或免疫原(例如，SARS-CoV或SARS-CoV-2 S蛋白肽)的蛋白质能够产生免疫应答，例如，对SARS-CoV或SARS-CoV-2 S蛋白肽的免疫应答。在一些实施方案中，免疫应答抑制或减少受试者(例如患者)中冠状病毒的复制。在一些实施方案中，免疫应答包括产生一种或更多种中和抗体，例如多克隆和/或单克隆抗体。在一些实施方案中，中和抗体抑制或减少受试者(例如患者)中冠状病毒的复制。在一些实施方案中，例如作为免疫原性组合物向受试者施用蛋白质不会由于先前暴露于冠状病毒而导致受试者中的抗体依赖性增强(ADE)。在一些方面，包含冠状病毒抗原和免疫原的蛋白质用作疫苗。
在一些实施方案中，冠状病毒抗原和免疫原(例如，SARS-CoV或SARS-CoV-2S蛋白肽)与蛋白质或肽连接以形成融合蛋白或重组多肽。在一些实施方案中，与冠状病毒抗原或免疫原连接的蛋白质或肽能够与蛋白质或肽(例如，融合蛋白或重组多肽的蛋白质或肽)结合，例如共价或非共价连接。因此，在一些情况下，与冠状病毒抗原或免疫原连接的蛋白质或肽是多聚结构域。
在一些实施方案中，冠状病毒抗原和免疫原(例如冠状病毒S蛋白肽)连接到胶原的前肽(例如在胶原的前肽的C-末端)，以形成融合肽或重组多肽。因此，在一些实施方案中，本文提供的蛋白质包括含有冠状病毒抗原和免疫原(例如冠状病毒S蛋白肽或其片段或表位)的重组多肽，连接到胶原的C-末端前肽。在一些实施方案中，胶原的前肽来源于α1胶原的人类C-前肽，表达后能够自三聚体化。
在一些实施方案中，将冠状病毒抗原和免疫原(例如冠状病毒S蛋白肽)连接到胶原的前肽(例如在胶原的前肽的C-末端)有助于蛋白质产生免疫应答的能力。例如，重组蛋白的产生可保留冠状病毒S蛋白肽的三级和四级结构，这可能对冠状病毒S蛋白肽的天然构象的稳定性很重要，而蛋白质表面上抗原位点的可用性反过来能够引发免疫应答，例如中和抗体。此外，将冠状病毒S蛋白肽连接到能够自三聚体化的蛋白质或肽使得重组蛋白聚集，从而模仿病毒包膜上冠状病毒S蛋白肽的天然同源三聚体结构。
在一些实施方案中，将冠状病毒S蛋白肽连接到胶原的C-末端前肽导致自三聚体化的重组多肽。在一些实施方案中，本文提供的蛋白质包括多种自三聚体化的冠状病毒S蛋白肽和胶原重组多肽的前肽。在一些实施方案中，重组蛋白的三聚体性质有助于蛋白质的稳定性。在一些实施方案中，重组蛋白的三聚体性质有助于蛋白质产生免疫应答的能力。在一些实施方案中，重组蛋白的三聚体性质和/或多种自三聚体化的重组蛋白的宏观结构有助于蛋白质产生免疫应答的能力。
本文还提供了包含本文提供的蛋白质的免疫原性组合物、生产本文提供的蛋白质的方法、用本文提供的蛋白质和组合物治疗受试者的方法以及试剂盒。
本申请中提及的所有出版物，包括专利文件、科学文章和数据库，均出于所有目的以其全文形式被援引加入，其程度与各单独出版物被单独援引加入的程度相同。如果本文所述定义与被援引加入本文的专利、申请、公开申请和其它出版物中所述定义相反或不一致，则本文所述定义优先于被援引加入本文的定义。本文使用的章节标题仅出于组织的目的，不得解释为限制所描述的主题。
I.病毒抗原和免疫原
本文提供的蛋白质包括冠状病毒抗原和免疫原。本文所预期的冠状病毒抗原和免疫原能够促进或刺激细胞介导的应答和/或体液应答。在一些实施方案中，应答(例如，细胞介导的或体液应答)包括抗体(例如，中和抗体)的产生。在一些实施方案中，冠状病毒抗原或免疫原是冠状病毒刺突蛋白肽。
冠状病毒是正义单链RNA病毒家族，正义单链RNA病毒已知可引起严重呼吸道疾病。它们拥有已知RNA病毒中最大的基因组(26-32kb)，在系统发生上分为四个属(α、β、γ、δ)，其中β-冠状病毒进一步细分为四个谱系(A、B、C、D)。目前已知冠状病毒家族感染人类的病毒来自α-冠状病毒和β-冠状病毒属。此外，人们认为，γ-冠状病毒和δ-冠状病毒属将来可能会感染人类。β-冠状病毒的非限制性示例包括 中东呼吸综合征冠状病毒(MERS-CoV)、严重急性呼吸综合征冠状病毒(SARS-CoV)、人类冠状病毒HKU1(HKU1-CoV)、人类冠状病毒OC43(OC43-CoV)、鼠肝炎病毒(MHV-CoV)、蝙蝠SARS样冠状病毒WIV1(WIV1-CoV)和人类冠状病毒HKU9(HKU9-CoV)。α-冠状病毒的非限制性示例包括人类冠状病毒229E(229E-CoV)、人类冠状病毒NL63(NL63-CoV)、猪流行性腹泻病毒(PEDV)和传染性胃肠炎冠状病毒(TGEV)。δ-冠状病毒的非限制性示例是猪δ-冠状病毒(SDCV)。
本文公开了严重急性呼吸综合征相关冠状病毒的列表：
■蝙蝠冠状病毒Cp/Yunnan2011
■蝙蝠冠状病毒RaTG13
■蝙蝠冠状病毒Rp/Shaanxi2011
○蝙蝠SARS冠状病毒HKU3
■蝙蝠SARS冠状病毒HKU3-1
■蝙蝠SARS冠状病毒HKU3-10
■蝙蝠SARS冠状病毒HKU3-11
■蝙蝠SARS冠状病毒HKU3-12
■蝙蝠SARS冠状病毒HKU3-13
■蝙蝠SARS冠状病毒HKU3-2
■蝙蝠SARS冠状病毒HKU3-3
■蝙蝠SARS冠状病毒HKU3-4
■蝙蝠SARS冠状病毒HKU3-5
■蝙蝠SARS冠状病毒HKU3-6
■蝙蝠SARS冠状病毒HKU3-7
■蝙蝠SARS冠状病毒HKU3-8
■蝙蝠SARS冠状病毒HKU3-9
■蝙蝠SARS冠状病毒Rp1
■蝙蝠SARS冠状病毒Rp2
○蝙蝠SARS CoV Rf1/2004
■蝙蝠CoV 273/2005
○蝙蝠SARS CoV Rm1/2004
■蝙蝠CoV 279/2005
■蝙蝠SARS CoV Rp3/2004
■蝙蝠SARS样冠状病毒
■蝙蝠SARS样冠状病毒Rs3367
■蝙蝠SARS样冠状病毒RsSHC014
■蝙蝠SARS样冠状病毒WIV1
■蝙蝠SARS样冠状病毒YNLF_31C
■蝙蝠SARS样冠状病毒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
○重组SARSr-CoV
■SARS冠状病毒ExoN1
■SARS冠状病毒MA15
■SARS冠状病毒MA15ExoN1
■SARS冠状病毒wtic-MB
■中菊头蝠冠状病毒
■SARS蝙蝠冠状病毒
■SARS冠状病毒A001
■SARS冠状病毒A013
■SARS冠状病毒A021
■SARS冠状病毒A022
■SARS冠状病毒A030
■SARS冠状病毒A031
■SARS冠状病毒AS
■SARS冠状病毒B012
■SARS冠状病毒B024
■SARS冠状病毒B029
■SARS冠状病毒B033
■SARS冠状病毒B039
■SARS冠状病毒B040
■SARS冠状病毒BJ01
■SARS冠状病毒BJ02
■SARS冠状病毒BJ03
■SARS冠状病毒BJ04
■SARS冠状病毒BJ162
■SARS冠状病毒BJ182-12
■SARS冠状病毒BJ182-4
■SARS冠状病毒BJ182-8
■SARS冠状病毒BJ182a
■SARS冠状病毒BJ182b
■SARS冠状病毒BJ202
■SARS冠状病毒BJ2232
■SARS冠状病毒BJ302
■SARS冠状病毒C013
■SARS冠状病毒C014
■SARS冠状病毒C017
■SARS冠状病毒C018
■SARS冠状病毒C019
■SARS冠状病毒C025
■SARS冠状病毒C028
■SARS冠状病毒C029
■SARS冠状病毒CDC#200301157
■SARS冠状病毒civet010
■SARS冠状病毒civet014
■SARS冠状病毒civet019
■SARS冠状病毒civet020
■SARS冠状病毒CS21
■SARS冠状病毒CS24
■SARS冠状病毒CUHK-AG01
■SARS冠状病毒CUHK-AG02
■SARS冠状病毒CUHK-AG03
■SARS冠状病毒CUHK-L2
■SARS冠状病毒CUHK-Su10
■SARS冠状病毒CUHK-W1
■SARS冠状病毒cw037
■SARS冠状病毒cw049
■SARS冠状病毒ES191
■SARS冠状病毒ES260
■SARS冠状病毒FRA
○SARS冠状病毒Frankfurt 1
■SARS冠状病毒Frankfurt1-v01
■SARS冠状病毒GD01
■SARS冠状病毒GD03T0013
■SARS冠状病毒GD322
■SARS冠状病毒GD69
■SARS冠状病毒GDH-BJH01
■SARS冠状病毒GZ-A
■SARS冠状病毒GZ-B
■SARS冠状病毒GZ-C
■SARS冠状病毒GZ-D
■SARS冠状病毒GZ02
■SARS冠状病毒GZ0401
■SARS冠状病毒GZ0402
■SARS冠状病毒GZ0403
■SARS冠状病毒GZ43
■SARS冠状病毒GZ50
■SARS冠状病毒GZ60
■SARS冠状病毒HB
■SARS冠状病毒HC/SZ/61/03
■SARS冠状病毒HGZ8L1-A
■SARS冠状病毒HGZ8L1-B
■SARS冠状病毒HGZ8L2
■SARS冠状病毒HHS-2004
■SARS冠状病毒HKU-36871
■SARS冠状病毒HKU-39849
■SARS冠状病毒HKU-65806
■SARS冠状病毒HKU-66078
■SARS冠状病毒Hong Kong/03/2003
■SARS冠状病毒HPZ-2003
■SARS冠状病毒HSR 1
■SARS冠状病毒HSZ-A
■SARS冠状病毒HSZ-Bb
■SARS冠状病毒HSZ-Bc
■SARS冠状病毒HSZ-Cb
■SARS冠状病毒HSZ-Cc
■SARS冠状病毒HSZ2-A
■SARS冠状病毒HZS2-Bb
■SARS冠状病毒HZS2-C
■SARS冠状病毒HZS2-D
■SARS冠状病毒HZS2-E
■SARS冠状病毒HZS2-Fb
■SARS冠状病毒HZS2-Fc
■SARS冠状病毒JMD
■SARS冠状病毒LC1
■SARS冠状病毒LC2
■SARS冠状病毒LC3
■SARS冠状病毒LC4
■SARS冠状病毒LC5
■SARS冠状病毒LLJ-2004
■SARS冠状病毒NS-1
■SARS冠状病毒P2
■SARS冠状病毒PC4-115
■SARS冠状病毒PC4-127
■SARS冠状病毒PC4-13
■SARS冠状病毒PC4-136
■SARS冠状病毒PC4-137
■SARS冠状病毒PC4-145
■SARS冠状病毒PC4-199
■SARS冠状病毒PC4-205
■SARS冠状病毒PC4-227
■SARS冠状病毒PC4-241
■SARS冠状病毒PUMC01
■SARS冠状病毒PUMC02
■SARS冠状病毒PUMC03
■SARS冠状病毒Rs_672/2006
■SARS冠状病毒sf098
■SARS冠状病毒sf099
■SARS冠状病毒ShanghaiQXC1
■SARS冠状病毒ShanghaiQXC2
■SARS冠状病毒Shanhgai LY
■SARS冠状病毒Sin0409
■SARS冠状病毒Sin2500
■SARS冠状病毒Sin2677
■SARS冠状病毒Sin2679
■SARS冠状病毒Sin2748
■SARS冠状病毒Sin2774
■SARS冠状病毒Sin3408
■SARS冠状病毒Sin3408L
■SARS冠状病毒Sin3725V
■SARS冠状病毒Sin3765V
■SARS冠状病毒Sin842
■SARS冠状病毒Sin845
■SARS冠状病毒Sin846
■SARS冠状病毒Sin847
■SARS冠状病毒Sin848
■SARS冠状病毒Sin849
■SARS冠状病毒Sin850
■SARS冠状病毒Sin852
■SARS冠状病毒Sin_WNV
■SARS冠状病毒Sino1-11
■SARS冠状病毒Sino3-11
■SARS冠状病毒SinP1
■SARS冠状病毒SinP2
■SARS冠状病毒SinP3
■SARS冠状病毒SinP4
■SARS冠状病毒SinP5
■SARS冠状病毒SoD
■SARS冠状病毒SZ1
■SARS冠状病毒SZ13
■SARS冠状病毒Taiwan
■SARS冠状病毒Taiwan JC-2003
■SARS冠状病毒Taiwan TC1
■SARS冠状病毒Taiwan TC2
■SARS冠状病毒Taiwan TC3
■SARS冠状病毒TJ01
■SARS冠状病毒TJF
■SARS冠状病毒Tor2
○SARS冠状病毒TW
■SARS冠状病毒TW-GD1
■SARS冠状病毒TW-GD2
■SARS冠状病毒TW-GD3
■SARS冠状病毒TW-GD4
■SARS冠状病毒TW-GD5
■SARS冠状病毒TW-HP1
■SARS冠状病毒TW-HP2
■SARS冠状病毒TW-HP3
■SARS冠状病毒TW-HP4
■SARS冠状病毒TW-JC2
■SARS冠状病毒TW-KC1
■SARS冠状病毒TW-KC3
■SARS冠状病毒TW-PH1
■SARS冠状病毒TW-PH2
■SARS冠状病毒TW-YM1
■SARS冠状病毒TW-YM2
■SARS冠状病毒TW-YM3
■SARS冠状病毒TW-YM4
■SARS冠状病毒TW1
■SARS冠状病毒TW10
■SARS冠状病毒TW11
■SARS冠状病毒TW2
■SARS冠状病毒TW3
■SARS冠状病毒TW4
■SARS冠状病毒TW5
■SARS冠状病毒TW6
■SARS冠状病毒TW7
■SARS冠状病毒TW8
■SARS冠状病毒TW9
■SARS冠状病毒TWC
■SARS冠状病毒TWC2
■SARS冠状病毒TWC3
■SARS冠状病毒TWH
■SARS冠状病毒TWJ
■SARS冠状病毒TWK
■SARS冠状病毒TWS
■SARS冠状病毒TWY
■SARS冠状病毒Urbani
■SARS冠状病毒Vietnam
■SARS冠状病毒WF188
■SARS冠状病毒WH20
■SARS冠状病毒WHU
■SARS冠状病毒xw002
■SARS冠状病毒ZJ01
■SARS冠状病毒ZJ02
■SARS冠状病毒ZJ0301
■SARS冠状病毒ZMY 1
■SARS冠状病毒ZS-A
■SARS冠状病毒ZS-B
■SARS冠状病毒ZS-C
■SARS相关蝙蝠冠状病毒RsSHC014
■SARS相关β-冠状病毒Rp3/2004
■严重急性呼吸综合征冠状病毒2
下表示出了示例性SARS CoV-2毒株。
冠状病毒基因组被加帽(capped)、多聚腺苷酸化并被核衣壳蛋白覆盖。冠状病毒粒子包括包含称为刺突(S)蛋白的I型融合糖蛋白的病毒包膜。大多数冠状病毒具有共同的基因组结构，其中复制酶基因包含在基因组的5′部分，而结构基因包含在基因组的3′部分。
冠状病毒刺突(S)蛋白是最初作为前体蛋白合成的I类融合糖蛋白。单个前体S多肽形成同源三聚体，并在高尔基体内进行糖基化并加工以去除信号肽，并被细胞蛋白酶裂解以产生单独的S1和S2多肽链，在同源三聚体中仍然作为S1/S2原聚体结合，因此是异二聚体的三聚体。S1亚单位位于病毒膜的远端，包含受体结合结构域(RBD)，该受体结合结构域介导病毒与其宿主受体的附着(attachment)。S2亚单位包含融合蛋白机制(machinery)，例如融合肽、融合糖蛋白特有的两个七肽重复序列(HR1和HR2)和中心螺旋、跨膜结构域和细胞溶质尾部结构域(cytosolic tail domain)。
在一些情况下，冠状病毒抗原或免疫原是处于融合前构象的冠状病毒S蛋白肽，它是冠状病毒S蛋白的胞外域在分泌系统中加工成成熟冠状病毒S蛋白之后并且在触发导致冠状病毒S向融合后构象转变的融合事件之前采用的结构构象。Kirchdoerfer et al.,“Pre-fusion structure of a human coronavirus spike protein,”Nature,531:118-121,2016中提供了处于融合前构象的示例性冠状病毒S蛋白(HKU1-CoV)的三维结构，出于所有目的以其全文形式被援引加入。
在一些情况下，冠状病毒抗原或免疫原包括一个或更多个与天然冠状病毒S序列相比的氨基酸取代、缺失或插入，与由相应的天然冠状病毒S序列形成的冠状病毒S胞外域三聚体相比，其提供了增加的融合前构象保留。通过一个或更多个氨基酸取代、缺失或插入的融合前构象的“稳定”可以是，例如，能量稳定(例如，降低融合前构象相对于融合后开放构象的能量)和/或动力学稳定(例如，降低从融合前构象到融合后构象的转化率)。此外，与相应的天然冠状病毒S序列相比，处于融合前构象的冠状病毒S胞外域三聚体的稳定可以包括增加对变性的抗性。本文提供了确定冠状病毒S胞外域三聚体是否处于融合前构象的方法，包括(但不限于)使用融合前构象特异性抗体的负染色电子显微镜和抗体结合分析。
在一些情况下，冠状病毒抗原或免疫原是S蛋白肽的片段。在一些实施方案中，抗原或免疫原是S蛋白肽的表位。表位包括分子上的抗原决定簇化学基团或肽序列，它们是抗原性的，从而引发特定的免疫应答，例如，表位是B细胞和/或T细胞作出应答的抗原区域。抗体可以结合到特定的抗原表位，例如冠状病毒S胞外域上的表位。表位既可以由连续氨基酸形成，也可以由通过蛋白质三级折叠而并列的非连续氨基酸形成。在一些实施方案中，冠状病毒表位是线性表位。在一些实施方案中，冠状病毒表位是构象表位。在一些实施方案中，冠状病毒表位是中和表位位点。在一些实施方案中，冠状病毒S蛋白肽或其片段的所有中和表位作为抗原或免疫原存在。
在一些情况下，例如，当病毒抗原或免疫原是S蛋白肽的片段时，仅存在S蛋白肽的单个亚单位，并且S蛋白肽的单个亚单位是三聚化的。在一些实施方案中，病毒抗原或免疫原包括信号肽、S1亚单位肽、S2亚单位肽或其任意组合。在一些实施方案中，病毒抗原或免疫原包括信号肽、受体结合结构域(RBD)肽、受体结合基序(RBM)肽、融合肽(FP)、七肽重复序列1(HR1)肽或七肽重复序列2(HR2)肽或其任意组合。在一些实施方案中，病毒抗原或免疫原包括S蛋白的受体结合结构域(RBD)。在一些实施方案中，病毒抗原或免疫原包括S蛋白的S1亚单位和S2亚单位。在一些实施方案中，病毒抗原或免疫原包括S蛋白的S1亚单位，但不包括S2亚单位。在一些实施方案中，病毒抗原或免疫原包括S蛋白的S2亚单位，但不包括S1亚单位。在一些实施方案中，病毒抗原或免疫原不含跨膜(TM)结构域肽和/或细胞质(CP)结构域肽。
在一些实施方案中，病毒抗原或免疫原包括蛋白酶裂解位点，其中蛋白酶可选地为弗林蛋白酶(furin)、胰蛋白酶、因子Xa或组织蛋白酶L。
在一些实施方案中，病毒抗原或免疫原不含蛋白酶裂解位点，其中蛋白酶可选地为弗林蛋白酶(furin)、胰蛋白酶、因子Xa或组织蛋白酶L，或者包含不可被蛋白酶裂解的突变的蛋白酶裂解位点。
在一些实施方案中，病毒抗原或免疫原是包含至少一种SARS-CoV-2蛋白质或其片段的SARS-CoV-2抗原。在一些实施方案中，SARS-CoV-2抗原被SARS-CoV-2反应性抗体和/或T细胞识别。在一些实施方案中，SARS-CoV-2抗原是灭活的全病毒。在一些实施方案中，SARS-CoV-2抗原包括病毒的亚单位。在一些实施方案中，SARS- CoV-2抗原包括SARS-CoV-2的结构蛋白或其片段。在一些实施方案中，SARS-CoV-2的结构蛋白包括由刺突(S)蛋白、膜(M)蛋白、核衣壳(N)蛋白和包膜(E)蛋白组成的组中的一种或更多种。在一些实施方案中，SARS-CoV-2抗原包括或进一步包括SARS-CoV-2的非结构蛋白或其片段。代表性SARS-CoV-2分离菌(isolate)(Hu-1)的核苷酸序列记载为GenBank号MN908947.3(Wu et al.,Nature,579:265-269,2020)，出于所有目的以其全文形式被援引加入。
在一些实施方案中，病毒抗原或免疫原包括SEQ ID NO:81-84中任意一个所述的序列。在一些实施方案中，病毒抗原或免疫原包括与如下所示的SEQ ID NO:82具有至少或约80％、85％、90％、92％、95％、97％、或99％的序列同一性的氨基酸序列。在一些实施方案中，病毒抗原或免疫原包括RBD三聚体，例如，与SEQ ID No:67-80中的任何一个连接的SARS-CoV-2 RBD序列。
在一些实施方案中，病毒抗原或免疫原包括SEQ ID NO:55中所述的序列。在一些实施方案中，病毒抗原或免疫原包括与如下所示的SEQ ID NO:55(下划线序列表示Thr333-Gly526(粗体)的受体结合结构域(RBD)内的受体结合基序(RBM))具有至少 或约80％、85％、90％、92％、95％或97％的序列同一性的氨基酸序列。在一些实施方案中，病毒抗原或免疫原包括RBD三聚体，例如，与SEQ ID No:67-80中的任何一个连接的SARS-CoV-2 RBD序列。
在一些实施方案中，病毒抗原或免疫原包括Hu-1冠状病毒的刺突糖蛋白序列(例如NC_045512)。在一些实施方案中，病毒抗原或免疫原包括B.1.526谱系中的病毒的刺突糖蛋白序列。在一些实施方案中，病毒抗原或免疫原包括Cluster 5(ΔFVI-刺突)病毒的刺突糖蛋白序列。在一些实施方案中，病毒抗原或免疫原包括B.1.1.529谱系中的病毒的刺突糖蛋白序列。在一些实施方案中，病毒抗原或免疫原包括B.1.1.7谱系中的病毒的刺突糖蛋白序列。在一些实施方案中，病毒抗原或免疫原包括B.1.1.207谱系中的病毒的刺突糖蛋白序列。在一些实施方案中，病毒抗原或免疫原包括B.1.1.317谱系中的病毒的刺突糖蛋白序列。在一些实施方案中，病毒抗原或免疫原包括B.1.1.318谱系中的病毒的刺突糖蛋白序列。在一些实施方案中，病毒抗原或免疫原包括P.1谱系中的病毒的刺突糖蛋白序列。在一些实施方案中，病毒抗原 或免疫原包括B.1.351谱系中的病毒的刺突糖蛋白序列。在一些实施方案中，病毒抗原或免疫原包括B.1.429/CAL.20C谱系中的病毒的刺突糖蛋白序列。在一些实施方案中，病毒抗原或免疫原包括B.1.525谱系中的病毒的刺突糖蛋白序列。在一些实施方案中，病毒抗原或免疫原包括B.1.526谱系中的病毒的刺突糖蛋白序列。在一些实施方案中，病毒抗原或免疫原包括B.1.617谱系中的病毒的刺突糖蛋白序列。在一些实施方案中，病毒抗原或免疫原包括B.1.617.2谱系中的病毒的刺突糖蛋白序列。在一些实施方案中，病毒抗原或免疫原包括B.1.618谱系中的病毒的刺突糖蛋白序列。在一些实施方案中，病毒抗原或免疫原包括B.1.620谱系中的病毒的刺突糖蛋白序列。在一些实施方案中，病毒抗原或免疫原包括P.2谱系中的病毒的刺突糖蛋白序列。在一些实施方案中，病毒抗原或免疫原包括P.3谱系中的病毒的刺突糖蛋白序列。在一些实施方案中，病毒抗原或免疫原包括B.1.1.143谱系中的病毒的刺突糖蛋白序列。在一些实施方案中，病毒抗原或免疫原包括A.23.1谱系中的病毒的刺突糖蛋白序列。在一些实施方案中，病毒抗原或免疫原包括B.1.617谱系中的病毒的刺突糖蛋白序列。在一些实施方案中，病毒抗原或免疫原包括来源于选自由Wuhan-Hu-1、B.1.526谱系中的病毒、B.1.1.7谱系中的病毒、P.1谱系中的病毒、B.1.351谱系中的病毒、P.2谱系中的病毒、B.1.1.143谱系中的病毒、A.23.1谱系中的病毒和B.1.617谱系中的病毒组成的组的任何两种或更多种病毒(以任何合适的组合)的刺突糖蛋白的序列。
在一些实施方案中，病毒抗原或免疫原包括T95I，G142D，Δ143-145，和/或T478K，例如，如在B.1.1.529奥密克戎变种中。
在一些实施方案中，病毒抗原或免疫原包括E484K和/或S477N，例如，如在B.1.526变种中。在一些实施方案中，病毒抗原或免疫原包括Δ400-402(ΔFVI)，例如，如在Cluster 5(ΔFVI-刺突)变体中。在一些实施方案中，病毒抗原或免疫原包括Δ69-70(ΔHV)、Δ144(ΔY)、N501Y、A570D、D614G、P681H、T716I、S982A和/或D1118H，例如，如在B.1.1.7变种中。在一些实施方案中，病毒抗原或免疫原包括P681H，例如，如在B.1.1.207变种中。在一些实施方案中，病毒抗原或免疫原包括L18F、T20N、P26S、D138Y、R190S、K417T、E484K、N501Y、D614G、H655Y、T1027I和/或V1176F，例如，如在P.1变种中。在一些实施方案中，病毒抗原或免疫原包括E484K，例如，如在P.2变种中。在一些实施方案中，病毒抗原或免疫原包括E484K和/或N501Y，例如，如在P.3变种中。在一些实施方案中，病毒抗原或免疫原包括L18F、D80A、D215G，Δ242-244(ΔLAL)、R246I、K417N、E484K、N501Y、D614G和/或A701V，例如，如在B.1.351变种中。在一些实施方案中，病毒抗原或免疫原包括S13I、W152C和/或L452R，例如，如在B.1.429/CAL.20C变种中。在一些实施方案中，病毒抗原或免疫原包括Δ69-70(ΔHV)、E484K和/或F888L，例如，如在B.1.525变种中。在一些实施方案中，病毒抗原或免疫原包括G142D、L452R、E484Q和/或P681R，例如，如在B.1.617变种中。在一些实施方案中，病毒抗原或免疫原包括G142D、L452R和/或P681R，例如，如在B.1.617.2变种中。在一些实施方案中，病毒抗原或免疫原包括E484K，例如，如在 B.1.618变种中。在一些实施方案中，病毒抗原或免疫原可以包括融合多肽(原聚体)，所述融合多肽(原聚体)包括以任何适当组合的任何一种或更多种前述突变。在一些实施方案中，病毒抗原或免疫原可以包括三种融合多肽的三聚体，并且三种原聚体融合多肽中的任何一种可以包括以任何适当组合的任何一种或更多种前述突变。在一些实施方案中，形成三聚体的三种原聚体融合多肽中的两种或全部三种可以在每个原聚体中包括不同的突变和/或不同的突变组合。在一些实施方案中，病毒抗原或免疫原可以包括三聚体的混合物，并且每个三聚体可以包括不同的突变和/或不同的突变组合。
在一些实施方案中，病毒抗原或免疫原包括选自由SEQ ID NO:55的氨基酸位置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和1176处的突变(例如，取代、缺失和/或插入)组成的组中的任何一个、两个、三个、四个、五个或更多个突变。在一些实施方案中，病毒抗原或免疫原包括选自由在氨基酸位置440、452、477、484、501、614、655、681和701处的突变(例如，取代、缺失和/或插入)组成的组中的任何一个、两个、三个、四个、五个、六个、七个、八个或所有突变。在一些实施方案中，病毒抗原或免疫原包括包含来自不同病毒的序列的嵌合多肽，例如来自冠状病毒第一变种的一个或更多个突变以及来自不同于第一变种的冠状病毒第二变种的一个或更多个突变。在一些实施方案中，此类嵌合病毒抗原或免疫原(或嵌合病毒抗原或免疫原的组合)可用于引发针对冠状病毒的第一和第二变种的广泛免疫应答。
在一些实施方案中，病毒抗原或免疫原包括选自由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、K417N、N440K、L452R、S477N、S477G、E484K、E484Q、N501Y、A570D、D614G、H655Y、P681H、P681R、R682G、R683S、R685G、A701V、T716I、F888L、S982A、T1027I、D1118H和V1176F组成的组中的任何一个、两个、三个、四个、五个或更多个突变。在一些实施方案中，病毒抗原或免疫原包括选自由N440K、L452R、S477G、S477N、E484K、E484Q、N501Y、D614G、H655Y、P681H、P681R和A701V组成的组中的任何一个、两个、三个、四个、五个或更多个突变。
在一些实施方案中，SARS-CoV-2抗原包括缺乏信号肽的截短的S蛋白、全长S蛋白的跨膜和细胞质结构域。在一些实施方案中，SARS-CoV-2抗原是重组蛋白，而在其它实施方案中，SARS-CoV-2抗原从病毒粒子中纯化。在一些优选的实施方案中，SARS-CoV-2抗原是分离抗原。
在一些实施方案中，病毒抗原或免疫原包括SEQ ID NO:27中所述的序列。在一些实施方案中，病毒抗原或免疫原包括与SEQ ID NO:27具有至少或约80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％序列同一性的氨基酸序列，包括在选自由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和1176(关于SEQ ID NO:55的氨基酸位置)组成的组中的一个或更多个氨基酸位置处包含取代、缺失和/或插入的序列。在一些实施方案中，病毒抗原或免疫原包括SEQ ID NO:27的变体，并且变体包括选自由S13I、L18F、T20N、P26S、Δ69-70(ΔHV)、D80A、D138Y、G142D、Δ144(ΔY)、W152C、R190S、D215G、Δ242-244(ΔLAL)、R246I、Δ400-402(ΔFVI)、K417T、K417N、N440K、L452R、S477N、S477G、E484K、E484Q、N501Y、A570D、D614G、H655Y、P681H、P681R、R682G、R683S、R685G、A701V、T716I、F888L、S982A、T1027I、D1118H和V1176F组成的组中的任何一个、两个、三个、四个、五个或更多个突变。
在一些实施方案中，病毒抗原或免疫原包括SEQ ID NO:28中所述的序列。在一些实施方案中，病毒抗原或免疫原包括与SEQ ID NO:28具有至少或约80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％序列同一性的氨基酸序列，包括在选自由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和1176(关于SEQ ID NO:55的氨基酸位置)组成的组中的一个或更多个氨基酸位置处包含取代、缺失和/或插入的序列。在一些实施方案中，病毒抗原或免疫原包括SEQ ID NO:28的变体，并且变体包括选自由S13I、L18F、T20N、P26S、Δ69-70(ΔHV)、D80A、D138Y、G142D、Δ144(ΔY)、W152C、R190S、D215G、Δ242-244(ΔLAL)、R246I、Δ400-402(ΔFVI)、K417T、K417N、N440K、L452R、S477N、S477G、E484K、E484Q、N501Y、A570D、D614G、H655Y、P681H、P681R、R682G、R683S、R685G、A701V、T716I、F888L、S982A、T1027I、D1118H和V1176F组成的组中的任何一个、两个、三个、四个、五个或更多个突变。
在一些实施方案中，病毒抗原或免疫原包括SEQ ID NO:29中所述的序列。在一些实施方案中，病毒抗原或免疫原包括与SEQ ID NO:29具有至少或约80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％序列同一性的氨基酸序列，包括在选自由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和1176(关于SEQ ID NO:55的氨基酸位置)组成的组中的一个或更多个氨基酸位置处包含取代、缺失和/或插入的序列。在一些实施方案中，病毒抗原或免疫原包括SEQ ID NO:29的变体，并且变体包括选自由S13I、L18F、T20N、P26S、Δ69-70(ΔHV)、D80A、D138Y、G142D、Δ144(ΔY)、W152C、R190S、D215G、Δ242-244(ΔLAL)、R246I、Δ400-402(ΔFVI)、K417T、K417N、N440K、L452R、S477N、S477G、E484K、E484Q、N501Y、A570D、D614G、H655Y、P681H、P681R、R682G、 R683S、R685G、A701V、T716I、F888L、S982A、T1027I、D1118H和V1176F组成的组中的任何一个、两个、三个、四个、五个或更多个突变。
在一些实施方案中，病毒抗原或免疫原包括SEQ ID NO:30中所述的序列。在一些实施方案中，病毒抗原或免疫原包括与SEQ ID NO:30具有至少或约80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％序列同一性的氨基酸序列，包括在选自由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和1176(关于SEQ ID NO:55的氨基酸位置)组成的组中的一个或更多个氨基酸位置处包含取代、缺失和/或插入的序列。在一些实施方案中，病毒抗原或免疫原包括SEQ ID NO:30的变体，并且变体包括选自由S13I、L18F、T20N、P26S、Δ69-70(ΔHV)、D80A、D138Y、G142D、Δ144(ΔY)、W152C、R190S、D215G、Δ242-244(ΔLAL)、R246I、Δ400-402(ΔFVI)、K417T、K417N、N440K、L452R、S477N、S477G、E484K、E484Q、N501Y、A570D、D614G、H655Y、P681H、P681R、R682G、R683S、R685G、A701V、T716I、F888L、S982A、T1027I、D1118H和V1176F组成的组中的任何一个、两个、三个、四个、五个或更多个突变。
在一些实施方案中，病毒抗原或免疫原包括SEQ ID NO:31中所述的序列。在一些实施方案中，病毒抗原或免疫原包括与SEQ ID NO:31具有至少或约80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％序列同一性的氨基酸序列，包括在选自由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和1176(关于SEQ ID NO:55的氨基酸位置)组成的组中的一个或更多个氨基酸位置处包含取代、缺失和/或插入的序列。在一些实施方案中，病毒抗原或免疫原包括SEQ ID NO:31的变体，并且变体包括选自由S13I、L18F、T20N、P26S、Δ69-70(ΔHV)、D80A、D138Y、G142D、Δ144(ΔY)、W152C、R190S、D215G、Δ242-244(ΔLAL)、R246I、Δ400-402(ΔFVI)、K417T、K417N、N440K、L452R、S477N、S477G、E484K、E484Q、N501Y、A570D、D614G、H655Y、P681H、P681R、R682G、R683S、R685G、A701V、T716I、F888L、S982A、T1027I、D1118H和V1176F组成的组中的任何一个、两个、三个、四个、五个或更多个突变。
在一些实施方案中，病毒抗原或免疫原包括SEQ ID NO:32中所述的序列。在一些实施方案中，病毒抗原或免疫原包括与SEQ ID NO:32具有至少或约80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％序列同一性的氨基酸序列，包括在选自由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和1176(关于SEQ ID NO:55的氨基酸位置)组成的组中的一个或更多个氨基酸位置处包含取代、缺失和/或插入的序列。在一些实施方案中，病毒抗原或免疫原包括SEQ ID NO:32的变体，并且变体包括选自由S13I、L18F、T20N、P26S、Δ69-70(ΔHV)、D80A、D138Y、G142D、Δ144(ΔY)、W152C、R190S、D215G、Δ242-244(ΔLAL)、R246I、Δ400-402(ΔFVI)、K417T、K417N、N440K、L452R、S477N、S477G、E484K、E484Q、N501Y、A570D、D614G、H655Y、P681H、P681R、R682G、R683S、R685G、A701V、T716I、F888L、S982A、T1027I、D1118H和V1176F组成的组中的任何一个、两个、三个、四个、五个或更多个突变。
在一些实施方案中，病毒抗原或免疫原包括SEQ ID NO:33中所述的序列。在一些实施方案中，病毒抗原或免疫原包括与SEQ ID NO:33具有至少或约80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％序列同一性的氨基酸序列，包括在选自由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和1176(关于SEQ ID NO:55的氨基酸位置)组成的组中的一个或更多个氨基酸位置处包含取代、缺失和/或插入的序列。在一些实施方案中，病毒抗原或免疫原包括SEQ ID NO:33的变体，并且变体包括选自由S13I、L18F、T20N、P26S、Δ69-70(ΔHV)、D80A、D138Y、G142D、Δ144(ΔY)、W152C、R190S、D215G、Δ242-244(ΔLAL)、R246I、Δ400-402(ΔFVI)、K417T、K417N、N440K、L452R、S477N、S477G、E484K、E484Q、N501Y、A570D、D614G、H655Y、P681H、P681R、R682G、R683S、R685G、A701V、T716I、F888L、S982A、T1027I、D1118H和V1176F组成的组中的任何一个、两个、三个、四个、五个或更多个突变。
在一些实施方案中，病毒抗原或免疫原包括SEQ ID NO:34中所述的序列。在一些实施方案中，病毒抗原或免疫原包括与SEQ ID NO:34具有至少或约80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％序列同一性的氨基酸序列，包括在选自由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和1176(关于SEQ ID NO:55的氨基酸位置)组成的组中的一个或更多个氨基酸位置处包含取代、缺失和/或插入的序列。在一些实施方案中，病毒抗原或免疫原包括SEQ ID NO:34的变体，并且变体包括选自由S13I、L18F、T20N、P26S、Δ69-70(ΔHV)、D80A、D138Y、G142D、Δ144(ΔY)、W152C、R190S、D215G、Δ242-244(ΔLAL)、R246I、Δ400-402(ΔFVI)、K417T、K417N、N440K、L452R、S477N、S477G、E484K、E484Q、N501Y、A570D、D614G、H655Y、P681H、P681R、R682G、R683S、R685G、A701V、T716I、F888L、S982A、T1027I、D1118H和V1176F组成的组中的任何一个、两个、三个、四个、五个或更多个突变。
在一些实施方案中，病毒抗原或免疫原包括SEQ ID NO:35中所述的序列。在一些实施方案中，病毒抗原或免疫原包括与SEQ ID NO:35具有至少或约80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％序列同一性的氨基酸序列，包括在选自由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和1176(关于SEQ ID NO:55的氨基酸位置)组成的组中的一个或更多个氨基酸位置处包含取代、缺失和/或插入的序列。在一些实施方案中，病毒抗原或免疫原包括SEQ ID NO:35的变体，并且变体包括选自由S13I、L18F、T20N、P26S、Δ69-70(ΔHV)、D80A、D138Y、G142D、Δ144(ΔY)、W152C、R190S、D215G、Δ242-244(ΔLAL)、R246I、Δ400-402(ΔFVI)、K417T、K417N、N440K、L452R、S477N、S477G、E484K、E484Q、N501Y、A570D、D614G、H655Y、P681H、P681R、R682G、R683S、R685G、A701V、T716I、F888L、S982A、T1027I、D1118H和V1176F组成的组中的任何一个、两个、三个、四个、五个或更多个突变。
在一些实施方案中，病毒抗原或免疫原包括SEQ ID NO:36中所述的序列。在一些实施方案中，病毒抗原或免疫原包括与SEQ ID NO:36具有至少或约80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％序列同一性的氨基酸序列，包括在选自由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和1176(关于SEQ ID NO:55的氨基酸位置)组成的组中的一个或更多个氨基酸位置处包含取代、缺失和/或插入的序列。在一些实施方案中，病毒抗原或免疫原包括SEQ ID NO:36的变体，并且变体包括选自由S13I、L18F、T20N、P26S、Δ69-70(ΔHV)、D80A、D138Y、G142D、Δ144(ΔY)、W152C、R190S、D215G、Δ242-244(ΔLAL)、R246I、Δ400-402(ΔFVI)、K417T、K417N、N440K、L452R、S477N、S477G、E484K、E484Q、N501Y、A570D、D614G、H655Y、P681H、P681R、R682G、R683S、R685G、A701V、T716I、F888L、S982A、T1027I、D1118H和V1176F组成的组中的任何一个、两个、三个、四个、五个或更多个突变。
在一些实施方案中，病毒抗原或免疫原包括SEQ ID NO:37中所述的序列。在一些实施方案中，病毒抗原或免疫原包括与SEQ ID NO:37具有至少或约80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％序列同一性的氨基酸序列，包括在选自由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和1176(关于SEQ ID NO:55的氨基酸位置)组成的组中的一个或更多个氨基酸位置处包含取代、缺失和/或插入的序列。在一些实施方案中，病 毒抗原或免疫原包括SEQ ID NO:37的变体，并且变体包括选自由S13I、L18F、T20N、P26S、Δ69-70(ΔHV)、D80A、D138Y、G142D、Δ144(ΔY)、W152C、R190S、D215G、Δ242-244(ΔLAL)、R246I、Δ400-402(ΔFVI)、K417T、K417N、N440K、L452R、S477N、S477G、E484K、E484Q、N501Y、A570D、D614G、H655Y、P681H、P681R、R682G、R683S、R685G、A701V、T716I、F888L、S982A、T1027I、D1118H和V1176F组成的组中的任何一个、两个、三个、四个、五个或更多个突变。
在一些实施方案中，病毒抗原或免疫原包括SEQ ID NO:38中所述的序列。在一些实施方案中，病毒抗原或免疫原包括与SEQ ID NO:38具有至少或约80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％序列同一性的氨基酸序列，包括在选自由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和1176(关于SEQ ID NO:55的氨基酸位置)组成的组中的一个或更多个氨基酸位置处包含取代、缺失和/或插入的序列。在一些实施方案中，病毒抗原或免疫原包括SEQ ID NO:38的变体，并且变体包括选自由S13I、L18F、T20N、P26S、Δ69-70(ΔHV)、D80A、D138Y、G142D、Δ144(ΔY)、W152C、R190S、D215G、Δ242-244(ΔLAL)、R246I、Δ400-402(ΔFVI)、K417T、K417N、N440K、L452R、S477N、S477G、E484K、E484Q、N501Y、A570D、D614G、H655Y、P681H、P681R、R682G、R683S、R685G、A701V、T716I、F888L、S982A、T1027I、D1118H和V1176F组成的组中的任何一个、两个、三个、四个、五个或更多个突变。
在一些实施方案中，病毒抗原或免疫原包括SEQ ID NO:39中所述的序列。在一些实施方案中，病毒抗原或免疫原包括与SEQ ID NO:39具有至少或约80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％序列同一性的氨基酸序列，包括在选自由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和1176(关于SEQ ID NO:55的氨基酸位置)组成的组中的一个或更多个氨基酸位置处包含取代、缺失和/或插入的序列。在一些实施方案中，病毒抗原或免疫原包括SEQ ID NO:39的变体，并且变体包括选自由S13I、L18F、T20N、P26S、Δ69-70(ΔHV)、D80A、D138Y、G142D、Δ144(ΔY)、W152C、R190S、D215G、Δ242-244(ΔLAL)、R246I、Δ400-402(ΔFVI)、K417T、K417N、N440K、L452R、S477N、S477G、E484K、E484Q、N501Y、A570D、D614G、H655Y、P681H、P681R、R682G、R683S、R685G、A701V、T716I、F888L、S982A、T1027I、D1118H和V1176F组成的组中的任何一个、两个、三个、四个、五个或更多个突变。
在一些实施方案中，病毒抗原或免疫原包括SEQ ID NO:40中所述的序列。在一些实施方案中，病毒抗原或免疫原包括与SEQ ID NO:40具有至少或约80％、81％ 、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％序列同一性的氨基酸序列，包括在选自由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和1176(关于SEQ ID NO:55的氨基酸位置)组成的组中的一个或更多个氨基酸位置处包含取代、缺失和/或插入的序列。在一些实施方案中，病毒抗原或免疫原包括SEQ ID NO:40的变体，并且变体包括选自由S13I、L18F、T20N、P26S、Δ69-70(ΔHV)、D80A、D138Y、G142D、Δ144(ΔY)、W152C、R190S、D215G、Δ242-244(ΔLAL)、R246I、Δ400-402(ΔFVI)、K417T、K417N、N440K、L452R、S477N、S477G、E484K、E484Q、N501Y、A570D、D614G、H655Y、P681H、P681R、R682G、R683S、R685G、A701V、T716I、F888L、S982A、T1027I、D1118H和V1176F组成的组中的任何一个、两个、三个、四个、五个或更多个突变。
在一些实施方案中，病毒抗原或免疫原包括SEQ ID NO:41中所述的序列。在一些实施方案中，病毒抗原或免疫原包括与SEQ ID NO:41具有至少或约80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％序列同一性的氨基酸序列，包括在选自由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和1176(关于SEQ ID NO:55的氨基酸位置)组成的组中的一个或更多个氨基酸位置处包含取代、缺失和/或插入的序列。在一些实施方案中，病毒抗原或免疫原包括SEQ ID NO:41的变体，并且变体包括选自由S13I、L18F、T20N、P26S、Δ69-70(ΔHV)、D80A、D138Y、G142D、Δ144(ΔY)、W152C、R190S、D215G、Δ242-244(ΔLAL)、R246I、Δ400-402(ΔFVI)、K417T、K417N、N440K、L452R、S477N、S477G、E484K、E484Q、N501Y、A570D、D614G、H655Y、P681H、P681R、R682G、R683S、R685G、A701V、T716I、F888L、S982A、T1027I、D1118H和V1176F组成的组中的任何一个、两个、三个、四个、五个或更多个突变。
在一些实施方案中，病毒抗原或免疫原包括SEQ ID NO:42中所述的序列。在一些实施方案中，病毒抗原或免疫原包括与SEQ ID NO:42具有至少或约80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％序列同一性的氨基酸序列，包括在选自由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和1176(关于SEQ ID NO:55的氨基酸位置)组成的组中的一个或更多个氨基酸位置处包含取代、缺失和/或插入的序列。在一些实施方案中，病毒抗原或免疫原包括SEQ ID NO:42的变体，并且变体包括选自由S13I、L18F、T20N、P26S、Δ69-70(ΔHV)、D80A、D138Y、G142D、Δ144(ΔY)、W152C、R190S、D215G、 Δ242-244(ΔLAL)、R246I、Δ400-402(ΔFVI)、K417T、K417N、N440K、L452R、S477N、S477G、E484K、E484Q、N501Y、A570D、D614G、H655Y、P681H、P681R、R682G、R683S、R685G、A701V、T716I、F888L、S982A、T1027I、D1118H和V1176F组成的组中的任何一个、两个、三个、四个、五个或更多个突变。
在一些实施方案中，病毒抗原或免疫原包括SEQ ID NO:43中所述的序列。在一些实施方案中，病毒抗原或免疫原包括与SEQ ID NO:43具有至少或约80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％序列同一性的氨基酸序列，包括在选自由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和1176(关于SEQ ID NO:55的氨基酸位置)组成的组中的一个或更多个氨基酸位置处包含取代、缺失和/或插入的序列。在一些实施方案中，病毒抗原或免疫原包括SEQ ID NO:43的变体，并且变体包括选自由S13I、L18F、T20N、P26S、Δ69-70(ΔHV)、D80A、D138Y、G142D、Δ144(ΔY)、W152C、R190S、D215G、Δ242-244(ΔLAL)、R246I、Δ400-402(ΔFVI)、K417T、K417N、N440K、L452R、S477N、S477G、E484K、E484Q、N501Y、A570D、D614G、H655Y、P681H、P681R、R682G、R683S、R685G、A701V、T716I、F888L、S982A、T1027I、D1118H和V1176F组成的组中的任何一个、两个、三个、四个、五个或更多个突变。
在一些实施方案中，病毒抗原或免疫原包括SEQ ID NO:44中所述的序列。在一些实施方案中，病毒抗原或免疫原包括与SEQ ID NO:44具有至少或约80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％序列同一性的氨基酸序列，包括在选自由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和1176(关于SEQ ID NO:55的氨基酸位置)组成的组中的一个或更多个氨基酸位置处包含取代、缺失和/或插入的序列。在一些实施方案中，病毒抗原或免疫原包括SEQ ID NO:44的变体，并且变体包括选自由S13I、L18F、T20N、P26S、Δ69-70(ΔHV)、D80A、D138Y、G142D、Δ144(ΔY)、W152C、R190S、D215G、Δ242-244(ΔLAL)、R246I、Δ400-402(ΔFVI)、K417T、K417N、N440K、L452R、S477N、S477G、E484K、E484Q、N501Y、A570D、D614G、H655Y、P681H、P681R、R682G、R683S、R685G、A701V、T716I、F888L、S982A、T1027I、D1118H和V1176F组成的组中的任何一个、两个、三个、四个、五个或更多个突变。
在一些实施方案中，病毒抗原或免疫原包括SEQ ID NO:45中所述的序列。在一些实施方案中，病毒抗原或免疫原包括与SEQ ID NO:45具有至少或约80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％序列同一性的氨基酸序列，包括在选自由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和1176(关于SEQ ID NO:55的氨基酸位置)组成的组中的一个或更多个氨基酸位置处包含取代、缺失和/或插入的序列。在一些实施方案中，病毒抗原或免疫原包括SEQ ID NO:45的变体，并且变体包括选自由S13I、L18F、T20N、P26S、Δ69-70(ΔHV)、D80A、D138Y、G142D、Δ144(ΔY)、W152C、R190S、D215G、Δ242-244(ΔLAL)、R246I、Δ400-402(ΔFVI)、K417T、K417N、N440K、L452R、S477N、S477G、E484K、E484Q、N501Y、A570D、D614G、H655Y、P681H、P681R、R682G、R683S、R685G、A701V、T716I、F888L、S982A、T1027I、D1118H和V1176F组成的组中的任何一个、两个、三个、四个、五个或更多个突变。
在一些实施方案中，病毒抗原或免疫原包括SEQ ID NO:46中所述的序列。在一些实施方案中，病毒抗原或免疫原包括与SEQ ID NO:46具有至少或约80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％序列同一性的氨基酸序列，包括在选自由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和1176(关于SEQ ID NO:55的氨基酸位置)组成的组中的一个或更多个氨基酸位置处包含取代、缺失和/或插入的序列。在一些实施方案中，病毒抗原或免疫原包括SEQ ID NO:46的变体，并且变体包括选自由S13I、L18F、T20N、P26S、Δ69-70(ΔHV)、D80A、D138Y、G142D、Δ144(ΔY)、W152C、R190S、D215G、Δ242-244(ΔLAL)、R246I、Δ400-402(ΔFVI)、K417T、K417N、N440K、L452R、S477N、S477G、E484K、E484Q、N501Y、A570D、D614G、H655Y、P681H、P681R、R682G、R683S、R685G、A701V、T716I、F888L、S982A、T1027I、D1118H和V1176F组成的组中的任何一个、两个、三个、四个、五个或更多个突变。
在一些实施方案中，病毒抗原或免疫原包括SEQ ID NO:47中所述的序列。在一些实施方案中，病毒抗原或免疫原包括与SEQ ID NO:47具有至少或约80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％序列同一性的氨基酸序列，包括在选自由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和1176(关于SEQ ID NO:55的氨基酸位置)组成的组中的一个或更多个氨基酸位置处包含取代、缺失和/或插入的序列。在一些实施方案中，病毒抗原或免疫原包括SEQ ID NO:47的变体，并且变体包括选自由S13I、L18F、T20N、P26S、Δ69-70(ΔHV)、D80A、D138Y、G142D、Δ144(ΔY)、W152C、R190S、D215G、Δ242-244(ΔLAL)、R246I、Δ400-402(ΔFVI)、K417T、K417N、N440K、L452R、S477N、S477G、E484K、E484Q、N501Y、A570D、D614G、H655Y、P681H、P681R、R682G、 R683S、R685G、A701V、T716I、F888L、S982A、T1027I、D1118H和V1176F组成的组中的任何一个、两个、三个、四个、五个或更多个突变。
在一些实施方案中，病毒抗原或免疫原包括SEQ ID NO:48中所述的序列。在一些实施方案中，病毒抗原或免疫原包括与SEQ ID NO:48具有至少或约80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％序列同一性的氨基酸序列，包括在选自由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和1176(关于SEQ ID NO:55的氨基酸位置)组成的组中的一个或更多个氨基酸位置处包含取代、缺失和/或插入的序列。在一些实施方案中，病毒抗原或免疫原包括SEQ ID NO:48的变体，并且变体包括选自由S13I、L18F、T20N、P26S、Δ69-70(ΔHV)、D80A、D138Y、G142D、Δ144(ΔY)、W152C、R190S、D215G、Δ242-244(ΔLAL)、R246I、Δ400-402(ΔFVI)、K417T、K417N、N440K、L452R、S477N、S477G、E484K、E484Q、N501Y、A570D、D614G、H655Y、P681H、P681R、R682G、R683S、R685G、A701V、T716I、F888L、S982A、T1027I、D1118H和V1176F组成的组中的任何一个、两个、三个、四个、五个或更多个突变。
在一些实施方案中，病毒抗原或免疫原包括SEQ ID NO:49中所述的序列。在一些实施方案中，病毒抗原或免疫原包括与SEQ ID NO:49具有至少或约80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％序列同一性的氨基酸序列，包括在选自由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和1176(关于SEQ ID NO:55的氨基酸位置)组成的组中的一个或更多个氨基酸位置处包含取代、缺失和/或插入的序列。在一些实施方案中，病毒抗原或免疫原包括SEQ ID NO:49的变体，并且变体包括选自由S13I、L18F、T20N、P26S、Δ69-70(ΔHV)、D80A、D138Y、G142D、Δ144(ΔY)、W152C、R190S、D215G、Δ242-244(ΔLAL)、R246I、Δ400-402(ΔFVI)、K417T、K417N、N440K、L452R、S477N、S477G、E484K、E484Q、N501Y、A570D、D614G、H655Y、P681H、P681R、R682G、R683S、R685G、A701V、T716I、F888L、S982A、T1027I、D1118H和V1176F组成的组中的任何一个、两个、三个、四个、五个或更多个突变。
在一些实施方案中，病毒抗原或免疫原包括SEQ ID NO:50中所述的序列。在一些实施方案中，病毒抗原或免疫原包括与SEQ ID NO:50具有至少或约80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％序列同一性的氨基酸序列，包括在选自由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和1176(关于SEQ ID NO:55的氨基酸位置)组成的组中的一个或更多个氨基酸位置处包含取代、缺失和/或插入的序列。在一些实施方案中，病毒抗原或免疫原包括SEQ ID NO:50的变体，并且变体包括选自由S13I、L18F、T20N、P26S、Δ69-70(ΔHV)、D80A、D138Y、G142D、Δ144(ΔY)、W152C、R190S、D215G、Δ242-244(ΔLAL)、R246I、Δ400-402(ΔFVI)、K417T、K417N、N440K、L452R、S477N、S477G、E484K、E484Q、N501Y、A570D、D614G、H655Y、P681H、P681R、R682G、R683S、R685G、A701V、T716I、F888L、S982A、T1027I、D1118H和V1176F组成的组中的任何一个、两个、三个、四个、五个或更多个突变。
在一些实施方案中，病毒抗原或免疫原包括SEQ ID NO:51中所述的序列。在一些实施方案中，病毒抗原或免疫原包括与SEQ ID NO:51具有至少或约80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％序列同一性的氨基酸序列，包括在选自由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和1176(关于SEQ ID NO:55的氨基酸位置)组成的组中的一个或更多个氨基酸位置处包含取代、缺失和/或插入的序列。在一些实施方案中，病毒抗原或免疫原包括SEQ ID NO:51的变体，并且变体包括选自由S13I、L18F、T20N、P26S、Δ69-70(ΔHV)、D80A、D138Y、G142D、Δ144(ΔY)、W152C、R190S、D215G、Δ242-244(ΔLAL)、R246I、Δ400-402(ΔFVI)、K417T、K417N、N440K、L452R、S477N、S477G、E484K、E484Q、N501Y、A570D、D614G、H655Y、P681H、P681R、R682G、R683S、R685G、A701V、T716I、F888L、S982A、T1027I、D1118H和V1176F组成的组中的任何一个、两个、三个、四个、五个或更多个突变。
在一些实施方案中，病毒抗原或免疫原包括SEQ ID NO:52中所述的序列。在一些实施方案中，病毒抗原或免疫原包括与SEQ ID NO:52具有至少或约80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％序列同一性的氨基酸序列。
在一些实施方案中，病毒抗原或免疫原包括信号肽。在一些实施方案中，病毒抗原或免疫原包括SEQ ID NO:53中所述的序列。在一些实施方案中，病毒抗原或免疫原包括与SEQ ID NO:53具有至少或约80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％序列同一性的氨基酸序列。在一些实施方案中，病毒抗原或免疫原包括SEQ ID NO:54中所述的序列。在一些实施方案中，病毒抗原或免疫原包括与SEQ ID NO:54具有至少或约80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％序列同一性的氨基酸序列。
在一些实施方案中，病毒抗原或免疫原包括SEQ ID NO:55中所述的序列。在一些实施方案中，病毒抗原或免疫原包括与SEQ ID NO:55具有至少或约80％、81％ 、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％序列同一性的氨基酸序列，包括在选自由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和1176组成的组中的一个或更多个氨基酸位置处包含取代、缺失和/或插入的序列。在一些实施方案中，病毒抗原或免疫原包括SEQ ID NO:55的变体，并且变体包括选自由S13I、L18F、T20N、P26S、Δ69-70(ΔHV)、D80A、D138Y、G142D、Δ144(ΔY)、W152C、R190S、D215G、Δ242-244(ΔLAL)、R246I、Δ400-402(ΔFVI)、K417T、K417N、N440K、L452R、S477N、S477G、E484K、E484Q、N501Y、A570D、D614G、H655Y、P681H、P681R、R682G、R683S、R685G、A701V、T716I、F888L、S982A、T1027I、D1118H和V1176F组成的组中的任何一个、两个、三个、四个、五个或更多个突变。
在一些实施方案中，病毒抗原或免疫原包括SEQ ID NO:56中所述的序列。在一些实施方案中，病毒抗原或免疫原包括与SEQ ID NO:56具有至少或约80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％序列同一性的氨基酸序列，包括在选自由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和1176(关于SEQ ID NO:55的氨基酸位置)组成的组中的一个或更多个氨基酸位置处包含取代、缺失和/或插入的序列。在一些实施方案中，病毒抗原或免疫原包括SEQ ID NO:56的变体，并且变体包括选自由S13I、L18F、T20N、P26S、Δ69-70(ΔHV)、D80A、D138Y、G142D、Δ144(ΔY)、W152C、R190S、D215G、Δ242-244(ΔLAL)、R246I、Δ400-402(ΔFVI)、K417T、K417N、N440K、L452R、S477N、S477G、E484K、E484Q、N501Y、A570D、D614G、H655Y、P681H、P681R、R682G、R683S、R685G、A701V、T716I、F888L、S982A、T1027I、D1118H和V1176F组成的组中的任何一个、两个、三个、四个、五个或更多个突变。
在一些实施方案中，病毒抗原或免疫原包括SEQ ID NO:57中所述的序列。在一些实施方案中，病毒抗原或免疫原包括与SEQ ID NO:57具有至少或约80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％序列同一性的氨基酸序列，包括在SEQ ID NO:57的一个或更多个氨基酸位置处包含取代、缺失和/或插入的序列。
在一些实施方案中，病毒抗原或免疫原包括SEQ ID NO:58中所述的序列。在一些实施方案中，病毒抗原或免疫原包括与SEQ ID NO:58具有至少或约80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％序列同一性的氨基酸序列，包括在SEQ ID NO:58的一个或更多个氨基酸位置处包含取代、缺失和/或插入的序列。
在一些实施方案中，病毒抗原或免疫原包括SEQ ID NO:59中所述的序列。在一些实施方案中，病毒抗原或免疫原包括在SEQ ID NO:59的一个或更多个氨基酸位置处包含取代、缺失和/或插入的序列。在一些实施方案中，病毒抗原或免疫原包括SEQ ID NO:60中所述的序列。在一些实施方案中，病毒抗原或免疫原包括在SEQ ID NO:60的一个或更多个氨基酸位置处包含取代、缺失和/或插入的序列。
在一些实施方案中，病毒抗原或免疫原包括SEQ ID NO:61中所述的序列。在一些实施方案中，病毒抗原或免疫原包括与SEQ ID NO:61具有至少或约80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％序列同一性的氨基酸序列，包括在SEQ ID NO:61的一个或更多个氨基酸位置处包含取代、缺失和/或插入的序列。
在一些实施方案中，病毒抗原或免疫原包括SEQ ID NO:62中所述的序列。在一些实施方案中，病毒抗原或免疫原包括与SEQ ID NO:62具有至少或约80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％序列同一性的氨基酸序列，包括在SEQ ID NO:62的一个或更多个氨基酸位置处包含取代、缺失和/或插入的序列。
在一些实施方案中，病毒抗原或免疫原包括SEQ ID NO:63中所述的序列。在一些实施方案中，病毒抗原或免疫原包括与SEQ ID NO:63具有至少或约80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％序列同一性的氨基酸序列，包括在SEQ ID NO:63的一个或更多个氨基酸位置处包含取代、缺失和/或插入的序列。
在一些实施方案中，病毒抗原或免疫原包括SEQ ID NO:64中所述的序列。在一些实施方案中，病毒抗原或免疫原包括与SEQ ID NO:64具有至少或约80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％序列同一性的氨基酸序列，包括在SEQ ID NO:64的一个或更多个氨基酸位置处包含取代、缺失和/或插入的序列。
在一些实施方案中，病毒抗原或免疫原包括SEQ ID NO:65中所述的序列。在一些实施方案中，病毒抗原或免疫原包括与SEQ ID NO:65具有至少或约80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％序列同一性的氨基酸序列，包括在SEQ ID NO:65的一个或更多个氨基酸位置处包含取代、缺失和/或插入的序列。
在一些实施方案中，病毒抗原或免疫原包括SEQ ID NO:81中所述的序列。在一些实施方案中，病毒抗原或免疫原包括与SEQ ID NO:81具有至少或约80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％序列同一性的氨基酸序列，包括在SEQ ID NO:81的一个或更多个氨基酸位置处包含取代、缺失和/或插入的序列。
在一些实施方案中，病毒抗原或免疫原包括SEQ ID NO:82中所述的序列。在一些实施方案中，病毒抗原或免疫原包括与SEQ ID NO:82具有至少或约80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％序列同一性的氨基酸序列，包括在SEQ ID NO:82的一个或更多个氨基酸位置处包含取代、缺失和/或插入的序列。
在一些实施方案中，病毒抗原或免疫原包括SEQ ID NO:83中所述的序列。在一些实施方案中，病毒抗原或免疫原包括与SEQ ID NO:83具有至少或约80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％序列同一性的氨基酸序列，包括在SEQ ID NO:83的一个或更多个氨基酸位置处包含取代、缺失和/或插入的序列。
在一些实施方案中，病毒抗原或免疫原包括SEQ ID NO:84中所述的序列。在一些实施方案中，病毒抗原或免疫原包括与SEQ ID NO:84具有至少或约80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％序列同一性的氨基酸序列，包括在SEQ ID NO:84的一个或更多个氨基酸位置处包含取代、缺失和/或插入的序列。
在一些实施方案中，病毒抗原或免疫原包括SEQ ID NO:122中所述的序列。在一些实施方案中，病毒抗原或免疫原包括与SEQ ID NO:122具有至少或约80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％序列同一性的氨基酸序列，包括在SEQ ID NO:122的一个或更多个氨基酸位置处包含取代、缺失和/或插入的序列。在一些实施方案中，病毒抗原或免疫原包括RBD三聚体，例如，与SEQ ID No:67-80中的任何一个连接的SARS-CoV-2RBD序列。
在一些实施方案中，病毒抗原或免疫原包括SEQ ID NO:123中所述的序列。在一些实施方案中，病毒抗原或免疫原包括与SEQ ID NO:123具有至少或约80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％序列同一性的氨基酸序列，包括在SEQ ID NO:123的一个或更多个氨基酸位置处包含取代、缺失和/或插入的序列。在一些实施方案中，病毒抗原或免疫原包括RBD三聚体，例如，与SEQ ID No:67-80中的任何一个连接的SARS-CoV-2RBD序列。
一些实施方案中，病毒抗原或免疫原不包括例如SEQ ID NO:66或其一部分的跨膜结构域。在一些实施方案中，冠状病毒抗原或免疫原包括可溶性S蛋白肽。在一些实施方案中，可溶性S蛋白肽缺少TM结构域肽和CP结构域肽。在一些实施方案中，可溶性S蛋白肽不结合到脂质双层，例如膜或病毒包膜。
在一些实施方案中，S蛋白肽由经密码子优化的核酸序列产生。在一些实施方案中，S蛋白肽由未经密码子优化的核酸序列产生。
在一些实施方案中，本文提及的病毒抗原或免疫原可包括包含所述病毒抗原或免疫原的重组多肽或融合肽。术语病毒抗原或免疫原可用于指包含冠状病毒抗原或免疫原的蛋白质。在某些情况下，冠状病毒抗原或免疫原是本文提供的冠状病毒蛋白肽。本文提及的病毒抗原或免疫原可在初始剂，额外剂，和/或加强剂中使用。独立地，初始剂，额外剂，和/或任意一剂或多剂加强剂可不使用或使用佐剂。如果使用佐剂，可选佐剂可包括：含铝佐剂，例如含明矾和/或氢氧化铝的佐剂；含寡核苷酸的佐剂，例如含CpG寡脱氧核苷酸(CpG-ODN)的佐剂；含TLR9激动剂的佐剂；含可代谢油、α-生育酚、和/或聚氧乙烯山梨醇酐单油酸酯(吐温-80)的佐剂，例如水包油乳液形式的含角鲨烯、α-生育酚、和吐温-80和/或Span 85的佐剂；或任意一种佐剂组合。
II.重组肽和蛋白质
可以预期，本文提供的冠状病毒抗原和免疫原，例如S蛋白肽(参见第I节)，可以与其它蛋白质或肽结合，例如连接，以形成重组多肽，包括融合肽。在一些实施方案中，本文提供的单个重组多肽(例如单体)结合形成重组多肽的多聚体，例如三聚体。在一些实施方案中，单个重组多肽单体的结合通过共价相互作用发生。在一些实施方案中，单个重组多肽单体的结合通过非共价相互作用发生。在一些实施方案中，相互作用(例如共价或非共价)受连接冠状病毒抗原或免疫原(例如S蛋白肽)的蛋白质或肽影响。在一些实施方案中，例如，当冠状病毒抗原或免疫原是如本文所述的S蛋白肽时，可选择将其连接到的蛋白质或肽，从而保留糖蛋白的天然同源三聚体结构。这可以有利于激发对S蛋白肽的强而有效的免疫原性应答。例如，冠状病毒抗原或免疫原(例如S蛋白肽)的天然构象的保留和/或维持可改善或允许进入能够产生免疫应答的抗原位点。在一些情况下，包含本文所述S蛋白肽的重组多肽(例如，参见第I节)在本文中可选地被称为重组S抗原、重组S免疫原或重组S蛋白。
进一步预期，在一些情况下，重组多肽或其多聚重组多肽聚集或可聚集以形成包含多个冠状病毒抗原和/或免疫原重组多肽的蛋白质或复合物。这种蛋白质的形成可有利于产生对冠状病毒抗原和/或免疫原的强而有效的免疫原性应答。例如，形成包含多个重组多肽的蛋白质，从而形成多个冠状病毒抗原，例如冠状病毒S蛋白肽，可保留病毒抗原的三级和/或四级结构，允许针对天然结构引发(mounted)免疫应答。在一些情况下，聚集可赋予冠状病毒抗原或免疫原的结构稳定性，这反过来又可提供对能够促进免疫应答的潜在抗原位点的进入。
1.融合肽和重组多肽
在一些实施方案中，冠状病毒抗原或免疫原可在其C-末端(C-末端连接)连接至三聚化结构域，以促进单体的三聚化。在一些实施方案中，三聚化以三聚体构型稳定冠状病毒抗原或免疫原(例如冠状病毒S蛋白肽)的膜近端方面。
促进可溶性重组蛋白稳定三聚体的外源性多聚结构域的非限制性示例包括：GCN4亮氨酸拉链(Harbury et al.1993 Science 262:1401-1407)、肺表面活性物质蛋白的三聚基序(Hoppe et al.1994FEBS Lett 344:191-195)、胶原(McAlinden et al.2003 J Biol Chem 278:42200-42207)和噬菌体T4 fibritin Foldon(Miroshnikov et al.1998 Protein Eng 11:329-414)，其中任何一种都可连接到本文所述的冠状病毒抗原或免疫原(例如，通过连接到S肽的C末端)以促进重组病毒抗原或免疫原的三聚化，以上文献出于所有目的以其全文形式被援引加入。另请参见美国专利号7,268,116、7,666,837、7,691,815、10,618,949、10,906,944和10,960,070，以及US 2020/0009244，这些专利出于所有目的以其全文形式被援引加入本文。
在一些实施方案中，一个或更多个肽接头(例如，甘氨酸-丝氨酸接头，例如，10个氨基酸的甘氨酸-丝氨酸肽接头)可用于将重组病毒抗原或免疫原连接到多聚化结构域。只要重组病毒抗原或免疫原三聚体保持所需性质(例如，融合前构象)，三聚体可包括本文提供的任何稳定突变(或其组合)。在一些实施方案中，重组多肽或融合蛋白包括SEQ ID NO:27-66和81-84中任一个所述的第一序列，所述第一序列连接到SEQ ID NO:67-80中任一个所述的第二序列，其中第一序列的C末端直接连接到第二序列的N末端。在一些实施方案中，重组多肽或融合蛋白包括SEQ ID NO:27-66和81-84中任一个所述的第一序列，所述第一序列连接到SEQ ID NO:67-80中任一个所述的第二序列，其中第一序列的C末端间接连接到第二序列的N末端，例如通过接头。在一些实施方案中，接头包括包含甘氨酸-X-Y重复序列的序列。
为了在治疗上可行，生物药物设计所需的三聚化蛋白部分应满足以下标准。理想情况下，它应该是天然分泌蛋白的一部分，如免疫球蛋白Fc，其还是在循环中丰富(无毒)、来源于人类(缺乏免疫原性)、相对稳定(半衰期长)并且能够有效自三聚化，所述自三聚化通过链间共价二硫键加强，因此三聚化的冠状病毒抗原或免疫原在结构上是稳定的。
胶原是纤维蛋白家族，纤维蛋白家族是细胞外基质的主要成分。它是哺乳动物中含量最丰富的蛋白质，占身体总蛋白质的近25％。胶原在骨骼、肌腱、皮肤、角膜、软骨、血管和牙齿的形成中起着重要的结构作用。胶原I、II、III、IV、V和XI的纤维状类型都合成为较大的三聚体前体，称为前胶原，其中由数百个“G-X-Y”重复序列(或甘氨酸重复序列)组成的中心不间断的三螺旋结构域两侧是非胶原结构域(NC)、N-前肽和C-前肽。C-末端和N-末端延伸都在前胶原分泌后进行蛋白质水解处理，这一事件触发成熟蛋白质组装成胶原原纤维，形成不溶性细胞基质。BMP-1是识别甘氨酸重复序列和胶原C-前结构域之间的连接处附近的前胶原的特定肽序列并负责去除前肽的蛋白酶。在正常成人的人类血清中发现I型胶原的脱落三聚体C-前肽的浓度在50-300ng/mL范围内，儿童具有更高的水平，这表明活跃的骨形成。在I型胶原的C-前肽的家族性高血清浓度人群中，其水平可高达1-6μg/mL而无明显异常，表明C-前肽是无毒的。对胶原的三聚体C-前肽的结构研究表明，它是三叶结构，所有三个亚单位 在靠近其N-末端的连接区域聚集在一起，连接到前胶原分子的其余部分。这种突出蛋白质的几何形状在一个方向上的融合与Fc二聚体相似。
I型、Ⅳ型、V型和XI型胶原主要组装成由两个α-1链和一个α-2链(对于Ⅰ、Ⅳ、Ⅴ型)或三个不同的链(对于XI型)组成的异源三聚体形式，它们在序列上高度同源。Ⅱ型和Ⅲ型胶原均为α-1链的同源三聚体。对于I型胶原(最丰富的胶原形式)，也形成稳定的α(I)同源三聚体，并且在不同组织中以可变水平存在。当单独在细胞中过表达时，这些胶原C-前肽链中的大多数可以自组装成同源三聚体。虽然N-前肽结构域是首先合成的，但三聚体胶原的分子组装始于C-前肽的配准结合。据信，C-前肽复合物通过链间二硫键的形成而稳定，但二硫键形成对于适当的链配准的必要性尚不清楚。甘氨酸的三螺旋重复，然后以类似拉链的方式从相关的C-末端传播到N-末端。通过使用重组DNA技术交换不同胶原链的C-前肽，这一知识创造了非天然类型的胶原基质。非胶原蛋白质(例如细胞因子和生长因子)也已融合到前胶原或成熟胶原的N-末端，以形成新的胶原基质，其目的是使非胶原蛋白质从细胞基质中缓慢释放。然而，在这两种情况下，在重组胶原纤维组装成不溶性细胞基质之前，C-前肽都需要被裂解。
尽管其它蛋白质三聚化结构域(例如来自细菌噬菌体T4的酵母原纤蛋白(fibritin)的GCN4和大肠杆菌的天冬氨酸转氨基甲酰酶的那些)先前已经描述过允许异源蛋白质的三聚化，但这些三聚体蛋白均不是人类性质的，它们也不是天然分泌的蛋白质。因此，任何三聚体融合蛋白都必须在细胞内合成，这不仅可能导致天然分泌蛋白(例如可溶性受体)的错误折叠，而且也使得从数千种其它细胞内蛋白中纯化此类融合蛋白变得困难。此外，在三聚体生物药物设计中使用此类非人类蛋白质三聚化结构域(例如来自酵母、细菌噬菌体和细菌)的致命缺点是其在人体内的假定免疫原性，使得此类融合蛋白在注射到人体后不久就失效。
因此，在本文所述的重组多肽中使用胶原具有许多优点，包括：(1)胶原是哺乳动物体内分泌的最丰富的蛋白质，占体内总蛋白质的近25％；(2)胶原的主要形式天然地以三聚体螺旋形式出现，其球状C-前肽负责三聚化的启动；(3)从成熟胶原蛋白质水解释放的胶原的三聚体C-前肽在哺乳动物血液中以亚微克/毫升的水平天然存在，并且不知道对身体有毒；(4)胶原的线性三螺旋区域可以作为接头包含在内，预测每个残基的间距为或排除为融合蛋白的一部分，以便可精确调整待三聚化的蛋白质和胶原的C-前肽之间的距离，以实现最佳生物活性；(5)将C-前肽从原胶原上裂解下来的BMP1的识别位点可以被突变或删除，以防止三聚体融合蛋白的破坏；(6)C-前肽结构域通过二硫键自三聚化，并且它提供通用的亲和标签，可用于纯化产生的任何分泌性融合蛋白。在一些实施方案中，与冠状病毒抗原和免疫原(例如S蛋白肽)结合的胶原的C-前肽能够重组产生可溶性的、共价连接的同源三聚体融合蛋白。
在一些实施方案中，冠状病毒抗原或免疫原连接到胶原的C-末端前肽以形成重组多肽。在一些实施方案中，重组多肽的C-末端前肽形成多肽间二硫键。在一些实 施方案中，重组蛋白形成三聚体。在一些实施方案中，冠状病毒抗原或免疫原是如第I节中所述的S蛋白肽。
例如，可通过多肽间二硫键(可形成多肽间二硫键的Cys残基用粗体显示)产生融合多肽并使融合多肽三聚化，所述融合多肽包括SEQ ID NO:1中融合多肽的N-末端上的信号肽MFVFLVLLPLVSS(SEQ ID NO:54)。
在一些实施方案中，多肽间二硫键可包括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和Cys1082- 1126中的一个或多个或全部，以任何合适的组合。在一些实施方案中，三聚体中的融合多肽可包括一个或多个糖基化位点(例如，Asn-连接的)，例如，在17、61、122、149、165、234、282、331、343、603、616、657、709、717、801、1074、1098和1134处的一个或多个或全部Asn残基处，以任何合适的组合。
在一些实施方案中，C-末端前肽为人胶原。在一些实施方案中，C-末端前肽包括proα1(I)、proα1(II)、proα1(III)、proα1(V)、proα1(XI)、proα2(I)、proα2(V)、proα2(XI)或proα3(XI)的C-末端多肽或其片段。在一些实施方案中，C-末端前肽是或包括proα1(I)的C-末端多肽。
在一些实施方案中，C-端前肽是或包括SEQ ID NO:67所述的氨基酸序列。在一些实施方案中，C-末端前肽是与SEQ ID NO:67具有至少或约85％、90％、92％、95％或97％序列同一性的氨基酸序列。在一些实施方案中，C-末端前肽是或包括SEQ ID NO:68所述的氨基酸序列。在一些实施方案中，C-末端前肽是与SEQ ID NO:68具有至少或约85％、90％、92％、95％或97％序列同一性的氨基酸序列。在一些实施方案中，C-末端前肽是或是SEQ ID NO:69所述的氨基酸序列。在一些实施方案中，C-末端前肽显示与SEQ ID NO:69具有至少或约85％、90％、92％、95％或97％序列同一性的氨基酸序列。在一些实施方案中，C-末端前肽是或包括SEQ ID NO:70所述的氨基酸序列。在一些实施方案中，C-末端前肽是与SEQ ID NO:70具有至少或约85％、90％、92％、95％或97％序列同一性的氨基酸序列。在一些实施方案中，C-末端前肽是或包括SEQ ID NO:71所述的氨基酸序列。在一些实施方案中，C-末端前肽是与SEQ ID NO:71具有至少或约85％、90％、92％、95％或97％序列同一性的氨基酸序列。
在一些实施方案中，C-末端前肽是或包括SEQ ID NO:72所述的氨基酸序列。在一些实施方案中，C-末端前肽是与SEQ ID NO:72具有至少或约85％、90％、92％、95％或97％序列同一性的氨基酸序列。在一些实施方案中，C-末端前肽是或包括SEQ ID NO:73所述的氨基酸序列。在一些实施方案中，C-末端前肽是与SEQ ID NO:73具有至少或约85％、90％、92％、95％或97％序列同一性的氨基酸序列。在一些实施方案中，C-末端前肽是或是SEQ ID NO:74所述的氨基酸序列。在一些实施方案中，C-末端前肽显示与SEQ ID NO:74具有至少或约85％、90％、92％、95％或97％序列同一性的氨基酸序列。在一些实施方案中，C-末端前肽是或包括SEQ ID NO:75所述的氨基酸序列。在一些实施方案中，C-末端前肽是与SEQ ID NO:75具有至少或约85％、90％、92％、95％或97％序列同一性的氨基酸序列。在一些实施方案中，C-末端前肽是或包括SEQ ID NO:76所述的氨基酸序列。在一些实施方案中，C-末端前肽是与SEQ ID NO:76具有至少或约85％、90％、92％、95％或97％序列同一性的氨基酸序列。
在一些实施方案中，C-末端前肽是或包括SEQ ID NO:77所述的氨基酸序列。在一些实施方案中，C-末端前肽是与SEQ ID NO:77具有至少或约85％、90％、92％、95％或97％序列同一性的氨基酸序列。在一些实施方案中，C-末端前肽是或包括SEQ ID NO:78所述的氨基酸序列。在一些实施方案中，C-末端前肽是与SEQ ID NO:78具有 至少或约85％、90％、92％、95％或97％序列同一性的氨基酸序列。在一些实施方案中，C-末端前肽是或是SEQ ID NO:79所述的氨基酸序列。在一些实施方案中，C-末端前肽显示与SEQ ID NO:79具有至少或约85％、90％、92％、95％或97％序列同一性的氨基酸序列。在一些实施方案中，C-末端前肽是或包括SEQ ID NO:80所述的氨基酸序列。在一些实施方案中，C-末端前肽是与SEQ ID NO:80具有至少或约85％、90％、92％、95％或97％序列同一性的氨基酸序列。
在一些实施方案中，C-末端前肽是或包括胶原三聚结构域(例如，人α1(I)胶原的C-前肽)的氨基酸序列，在BMP-1位点中具有天冬氨酸(D)到天冬酰胺(N)的取代，例如，如SEQ ID NO:68中所示，其中RAD突变为RAN。在一些实施方案中，C-末端前肽是或包括胶原三聚化结构域(例如，人α1(I)胶原的C-前肽)的氨基酸序列，在BMP-1位点中具有丙氨酸(A)到天冬酰胺(N)的取代，例如，如SEQ ID NO:69中所示，其中RAD突变为RND。在一些实施方案中，本文中的C-末端前肽可包括突变的BMP-1位点，例如，RSAN而不是DDAN。在一些实施方案中，本文中的C-末端前肽可包括BMP-1位点，例如，包含RAD(例如，RADDAN)序列而非RAN(例如，RANDAN)或RND(例如，RNDDAN)的序列(例如，SEQ ID NO:68或69)可用于本文中公开的融合多肽中。例如，SEQ ID NO:27(带下划线)或其片段、变体或突变体可直接或间接连接到SEQ ID NO:67(斜体)或其片段、变体或突变体，例如，以形成以下融合蛋白：
在一些实施方案中，C-末端前肽是或包括氨基酸序列，所述氨基酸序列是SEQ ID NO:67-80中任何一个的片段。
在一些实施方案中，C-末端前肽可包括包含甘氨酸-X-Y重复序列的序列，其中X和Y独立地为任何氨基酸，或与其至少85％、90％、92％、95％或97％相同的氨基酸 序列，其能够形成多肽间二硫键并使重组多肽三聚化。在一些实施方案中，X和Y独立地为脯氨酸或羟脯氨酸。
在S蛋白肽连接到C-末端前肽以形成重组多肽的一些情况下，重组多肽形成三聚体，从而形成S蛋白肽的同源三聚体。在一些实施方案中，三聚化重组多肽的S蛋白肽处于融合前构象。在一些实施方案中，三聚化重组多肽的S蛋白肽处于融合后构象。在一些实施方案中，确认状态允许进入S蛋白肽上的不同抗原位点。在一些实施方案中，抗原位点是表位，例如线性表位或构象表位。具有所述三聚化重组多肽的优点是可以针对多种潜在和多样的抗原位点进行免疫应答。
在一些实施方案中，三聚化重组多肽包括包含相同的病毒抗原或免疫原的单个重组多肽。在一些实施方案中，三聚化重组多肽包括单个重组多肽，每个重组多肽包含不同于其它重组多肽的病毒抗原或免疫原。在一些实施方案中，三聚化重组多肽包括单个重组多肽，其中单个重组多肽中的一个包括不同于其它重组多肽的病毒抗原或免疫原。在一些实施方案中，三聚化重组多肽包括单个重组多肽，其中单个重组多肽中的两个包括相同的病毒抗原或免疫原，并且该病毒抗原或免疫原不同于包含在剩余重组多肽中的病毒抗原或免疫原。
在一些实施方案中，重组多肽包括第I节中所述的任何冠状病毒抗原或免疫原。在一些实施方案中，重组多肽包括如本文所述连接到如本文所述的胶原的C-末端前肽的第I节中所述的任何冠状病毒抗原或免疫原。
在一些实施方案中，免疫原包括重组SARS-CoV或SARS-CoV-2 S胞外域三聚体，例如SARS-CoV-2奥密克戎(B.1.1.529)冠状病毒S胞外域三聚体，其包括原聚体，所述原聚体在HR1结构域和中心螺旋结构域之间的边界处或附近包含一个或更多个(例如两个，例如两个连续的)脯氨酸取代，所述脯氨酸取代稳定了处于融合前构象的S胞外域三聚体。在一些这样的实施方案中，稳定了处于融合前构象的S胞外域的一个或更多个(例如两个，例如两个连续的)脯氨酸取代位于HR1的C-末端残基的15位氨基酸N-末端和中心螺旋的N-末端残基的5位氨基酸C-末端之间。
在一些实施方案中，一个或更多个(例如两个，例如两个连续的)脯氨酸取代稳定了处于融合前构象的冠状病毒(例如，SARS-CoV或SARS-CoV-2)S胞外域三聚体，例如SARS-CoV-2奥密克戎(B.1.1.529)冠状病毒S胞外域三聚体。在一些实施方案中，SARS-CoV-2 S蛋白肽包括986K/987V向986P/987P的突变。
在一些实施方案中，处于融合前构象的被稳定了的重组冠状病毒(例如，SARS-CoV或SARS-CoV-2)S胞外域三聚体包括单链S胞外域原聚体，其包含S1/S2和/或S2′蛋白酶裂解位点的突变，以防止这些位点处的蛋白酶裂解。在一些实施方案中，SARS-CoV-2 S蛋白肽包括685R向685A的突变。各种病毒的示例性蛋白酶裂解位点如下所示：
在一些实施方案中，通过一个或更多个脯氨酸取代(例如986P/987P取代)稳定了处于融合前构象的重组冠状病毒(例如，SARS-CoV或SARS-CoV-2)S胞外域三聚体的原聚体包括另外的修饰，用于处于融合前构象的稳定，例如在蛋白酶裂解位点处发生突变以阻止蛋白酶裂解。
参考作为SEQ ID NO:55提供的SARS-CoV-2 S蛋白质序列，胞外域包括信号肽(SP)，其在细胞加工过程中被去除；N-末端结构域(NTD)；受体结合结构域(RBD)；一个或更多个S1/S2裂解位点；融合肽(FP)；内部融合肽(IFP)；七肽重复序列1/2(HR1/2)和跨膜结构域(TM)。序列的示例性来源可在ncbi.nlm.nih.gov/nuccore/MN908947.3、ncbi.nlm.nih.gov/nuccore/MN908947、ncbi.nlm.nih.gov/nuccore/MN908947.2上找到。另外的序列可在ncbi.nlm.nih.gov/genbank/sars-cov-2-seqs/上找到，包括肺炎病毒分离株Hu-1的完整基因组。
在一些实施方案中，融合前稳定的SARS-CoV-2 S胞外域三聚体的原聚体，例如SARS-CoV-2奥密克戎(B.1.1.529)冠状病毒S胞外域三聚体的原聚体，可具有NTD、RBD、S1(在S1/S2位点1或S1/S2位点2处)、FP、IFP、HR1、HR2或胞外域的C-末端残基的C-末端残基(其可连接到例如三聚化结构域或跨膜结构域)。S蛋白的位置编号可能在SARS-CoV毒株之间变化，但序列可以对齐以确定相关的结构域和裂解位点。应了解，任何胞外域片段的N-末端和C-末端上的一些残基(例如多达10个)可在所公开的免疫原中被移除或修饰，而不会降低S胞外域三聚体作为免疫原的效用。
在一些实施方案中，重组多肽是或包括SARS-CoV或SARS-CoV-2 S蛋白的NTD肽。在一些实施方案中，重组多肽是或包括SARS-CoV或SARS-CoV-2 S蛋白的RBD肽。在一些实施方案中，重组多肽是或包括SARS-CoV或SARS-CoV-2 S蛋白的NTD肽和RBD肽。在一些实施方案中，重组多肽是或包括SARS-CoV或SARS-CoV-2 S蛋白的S1结构域肽。在一些实施方案中，重组多肽是或包括SARS-CoV或SARS-CoV-2 S蛋白的S2结构域肽。
SEQ ID NO:26(1491aa)中提供了不含信号肽的示例性SARS-CoV-1 S重组多肽：
上述SARS-CoV-1 S重组多肽可包括SEQ ID NO:53中提供的N-末端信号肽。
SEQ ID NO:1中提供了不含信号肽的示例性SARS-CoV-2 S重组多肽：

上述SARS-CoV-2 S重组多肽可包括SEQ ID NO:54中提供的N-末端信号肽。
SEQ ID NO:81(1506aa)中提供了不含信号肽的示例性SARS-CoV-2 S重组多肽：

上述SARS-CoV-2 S重组多肽可包括SEQ ID NO:54(MFVFLVLLPLVSS)中提供的N-末端信号肽。
SEQ ID NO:82(1506aa)中提供了不含信号肽的示例性SARS-CoV-2 S重组多肽：

上述SARS-CoV-2 S重组多肽可包括SEQ ID NO:54(MFVFLVLLPLVSS)中提供的N-末端信号肽。
SEQ ID NO:83(1506aa)中提供了不含信号肽的示例性SARS-CoV-2 S重组多肽：

上述SARS-CoV-2 S重组多肽可包括SEQ ID NO:54(MFVFLVLLPLVSS)中提供的N-末端信号肽。
SEQ ID NO:84(1506aa)中提供了不含信号肽的示例性SARS-CoV-2 S重组多肽：

上述SARS-CoV-2 S重组多肽可包括SEQ ID NO:54(MFVFLVLLPLVSS)中提供的N-末端信号肽。
在一些实施方案中，重组多肽是或包括SEQ ID NO:1中所述的序列。在一些实施方案中，重组多肽是或包括与SEQ ID NO:1具有至少或约80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％序列同一性的氨基酸序列，包括在一个或更多个氨基酸位置(例如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和/或1176(关于SEQ ID NO:55的氨基酸位置)或其任何组合)处包含取代、缺失和/或插入的序列。在一些实施方案中，重组多肽是或包括SEQ ID NO:1的变体，并且变体包括选自由S13I、L18F、T20N、P26S、Δ69-70(ΔHV)、D80A、D138Y、G142D、Δ144(ΔY)、W152C、R190S、D215G、Δ242-244(ΔLAL)、R246I、Δ400-402(ΔFVI)、K417T、K417N、N440K、L452R、S477N、S477G、E484K、E484Q、N501Y、A570D、D614G、H655Y、P681H、P681R、R682G、R683S、R685G、A701V、T716I、F888L、S982A、T1027I、D1118H和V1176F或其任何组合组成的组中的任何一个、两个、三个、四个、五个或更多个突变。
在一些实施方案中，重组多肽是或包括SEQ ID NO:2中所述的序列。在一些实施方案中，重组多肽是或包括与SEQ ID NO:2具有至少或约80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％序列同一性的氨基酸序列，包括在一个或更多个氨基酸位置(例如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和/或1176(关于SEQ ID NO:55的氨基酸位置)或其任何组合)处包含取代、缺失和/或插入的序列。在一些实施方案中，重组多肽是或包括SEQ ID NO:2的变体，并且变体包括选自由S13I、L18F、T20N、P26S、Δ69-70(ΔHV)、D80A、D138Y、G142D、Δ144(ΔY)、W152C、R190S、D215G、Δ242-244(ΔLAL)、R246I、Δ400-402(ΔFVI)、K417T、K417N、N440K、L452R、S477N、S477G、E484K、E484Q、N501Y、A570D、D614G、H655Y、P681H、P681R、R682G、R683S、R685G、A701V、T716I、F888L、S982A、T1027I、D1118H和V1176F或其任何组合组成的组中的任何一个、两个、三个、四个、五个或更多个突变。
在一些实施方案中，重组多肽是或包括SEQ ID NO:3中所述的序列。在一些实施方案中，重组多肽是或包括与SEQ ID NO:3具有至少或约80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％序列同一性的氨基酸序列，包括在一个或更多个氨基酸位置(例如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和/或1176(关于SEQ ID NO:55的氨基酸位置)或其任何组合)处包含取代、缺失和/或插入的序列。在一些实施方案中，重组多肽是或包括SEQ ID NO:3的变体，并且变体包括选自由S13I、L18F、T20N、P26S、Δ69-70(ΔHV)、D80A、D138Y、G142D、Δ144(ΔY)、W152C、R190S、D215G、Δ242-244(ΔLAL)、R246I、Δ400-402(ΔFVI)、K417T、K417N、N440K、L452R、S477N、S477G、E484K、E484Q、N501Y、A570D、D614G、H655Y、P681H、P681R、R682G、R683S、R685G、A701V、T716I、F888L、S982A、T1027I、D1118H和V1176F或其任何组合组成的组中的任何一个、两个、三个、四个、五个或更多个突变。
在一些实施方案中，重组多肽是或包括SEQ ID NO:4中所述的序列。在一些实施方案中，重组多肽是或包括与SEQ ID NO:4具有至少或约80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％序列同一性的氨基酸序列，包括在一个或更多个氨基酸位置(例如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和/或1176(关于SEQ ID NO:55的氨基酸位置)或其任何组合)处包含取代、缺失和/或插入的序列。在一些实施方案中，重组多肽是或包括SEQ ID NO:4的变体，并且变体包括选自由S13I、L18F、T20N、P26S、Δ69-70(ΔHV)、D80A、D138Y、G142D、Δ144(ΔY)、W152C、R190S、D215G、Δ242-244(ΔLAL)、R246I、Δ400-402(ΔFVI)、K417T、K417N、N440K、L452R、S477N、S477G、E484K、E484Q、N501Y、A570D、D614G、H655Y、P681H、P681R、R682G、R683S、R685G、A701V、T716I、F888L、S982A、T1027I、D1118H和V1176F或其任何组合组成的组中的任何一个、两个、三个、四个、五个或更多个突变。
在一些实施方案中，重组多肽是或包括SEQ ID NO:5中所述的序列。在一些实施方案中，重组多肽是或包括与SEQ ID NO:5具有至少或约80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％序列同一性的氨基酸序列，包括在一个或更多个氨基酸位置(例如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和/或1176(关于SEQ ID NO:55的氨基酸位置)或其任何组合)处包含取代、缺失和/或插入的序列。在一些实施方案中，重组多肽是 或包括SEQ ID NO:5的变体，并且变体包括选自由S13I、L18F、T20N、P26S、Δ69-70(ΔHV)、D80A、D138Y、G142D、Δ144(ΔY)、W152C、R190S、D215G、Δ242-244(ΔLAL)、R246I、Δ400-402(ΔFVI)、K417T、K417N、N440K、L452R、S477N、S477G、E484K、E484Q、N501Y、A570D、D614G、H655Y、P681H、P681R、R682G、R683S、R685G、A701V、T716I、F888L、S982A、T1027I、D1118H和V1176F或其任何组合组成的组中的任何一个、两个、三个、四个、五个或更多个突变。
在一些实施方案中，重组多肽是或包括SEQ ID NO:6中所述的序列。在一些实施方案中，重组多肽是或包括与SEQ ID NO:6具有至少或约80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％序列同一性的氨基酸序列，包括在一个或更多个氨基酸位置(例如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和/或1176(关于SEQ ID NO:55的氨基酸位置)或其任何组合)处包含取代、缺失和/或插入的序列。在一些实施方案中，重组多肽是或包括SEQ ID NO:6的变体，并且变体包括选自由S13I、L18F、T20N、P26S、Δ69-70(ΔHV)、D80A、D138Y、G142D、Δ144(ΔY)、W152C、R190S、D215G、Δ242-244(ΔLAL)、R246I、Δ400-402(ΔFVI)、K417T、K417N、N440K、L452R、S477N、S477G、E484K、E484Q、N501Y、A570D、D614G、H655Y、P681H、P681R、R682G、R683S、R685G、A701V、T716I、F888L、S982A、T1027I、D1118H和V1176F或其任何组合组成的组中的任何一个、两个、三个、四个、五个或更多个突变。
在一些实施方案中，重组多肽是或包括SEQ ID NO:7中所述的序列。在一些实施方案中，重组多肽是或包括与SEQ ID NO:7具有至少或约80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％序列同一性的氨基酸序列，包括在一个或更多个氨基酸位置(例如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和/或1176(关于SEQ ID NO:55的氨基酸位置)或其任何组合)处包含取代、缺失和/或插入的序列。在一些实施方案中，重组多肽是或包括SEQ ID NO:7的变体，并且变体包括选自由S13I、L18F、T20N、P26S、Δ69-70(ΔHV)、D80A、D138Y、G142D、Δ144(ΔY)、W152C、R190S、D215G、Δ242-244(ΔLAL)、R246I、Δ400-402(ΔFVI)、K417T、K417N、N440K、L452R、S477N、S477G、E484K、E484Q、N501Y、A570D、D614G、H655Y、P681H、P681R、R682G、R683S、R685G、A701V、T716I、F888L、S982A、T1027I、D1118H和V1176F或其任何组合组成的组中的任何一个、两个、三个、四个、五个或更多个突变。
在一些实施方案中，重组多肽是或包括SEQ ID NO:8中所述的序列。在一些实施方案中，重组多肽是或包括与SEQ ID NO:8具有至少或约80％、81％、82％、83％ 、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％序列同一性的氨基酸序列，包括在一个或更多个氨基酸位置(例如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和/或1176(关于SEQ ID NO:55的氨基酸位置)或其任何组合)处包含取代、缺失和/或插入的序列。在一些实施方案中，重组多肽是或包括SEQ ID NO:8的变体，并且变体包括选自由S13I、L18F、T20N、P26S、Δ69-70(ΔHV)、D80A、D138Y、G142D、Δ144(ΔY)、W152C、R190S、D215G、Δ242-244(ΔLAL)、R246I、Δ400-402(ΔFVI)、K417T、K417N、N440K、L452R、S477N、S477G、E484K、E484Q、N501Y、A570D、D614G、H655Y、P681H、P681R、R682G、R683S、R685G、A701V、T716I、F888L、S982A、T1027I、D1118H和V1176F或其任何组合组成的组中的任何一个、两个、三个、四个、五个或更多个突变。
在一些实施方案中，重组多肽是或包括SEQ ID NO:9中所述的序列。在一些实施方案中，重组多肽是或包括与SEQ ID NO:9具有至少或约80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％序列同一性的氨基酸序列，包括在一个或更多个氨基酸位置(例如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和/或1176(关于SEQ ID NO:55的氨基酸位置)或其任何组合)处包含取代、缺失和/或插入的序列。在一些实施方案中，重组多肽是或包括SEQ ID NO:9的变体，并且变体包括选自由S13I、L18F、T20N、P26S、Δ69-70(ΔHV)、D80A、D138Y、G142D、Δ144(ΔY)、W152C、R190S、D215G、Δ242-244(ΔLAL)、R246I、Δ400-402(ΔFVI)、K417T、K417N、N440K、L452R、S477N、S477G、E484K、E484Q、N501Y、A570D、D614G、H655Y、P681H、P681R、R682G、R683S、R685G、A701V、T716I、F888L、S982A、T1027I、D1118H和V1176F或其任何组合组成的组中的任何一个、两个、三个、四个、五个或更多个突变。
在一些实施方案中，重组多肽是或包括SEQ ID NO:10中所述的序列。在一些实施方案中，重组多肽是或包括与SEQ ID NO:10具有至少或约80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％序列同一性的氨基酸序列，包括在一个或更多个氨基酸位置(例如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和/或1176(关于SEQ ID NO:55的氨基酸位置)或其任何组合)处包含取代、缺失和/或插入的序列。在一些实施方案中，重组多肽是或包括SEQ ID NO:10的变体，并且变体包括选自由S13I、L18F、T20N、P26S、Δ69-70(ΔHV)、D80A、D138Y、G142D、Δ144(ΔY)、W152C、R190S、D215G、Δ242-244 (ΔLAL)、R246I、Δ400-402(ΔFVI)、K417T、K417N、N440K、L452R、S477N、S477G、E484K、E484Q、N501Y、A570D、D614G、H655Y、P681H、P681R、R682G、R683S、R685G、A701V、T716I、F888L、S982A、T1027I、D1118H和V1176F或其任何组合组成的组中的任何一个、两个、三个、四个、五个或更多个突变。
在一些实施方案中，重组多肽是或包括SEQ ID NO:11中所述的序列。在一些实施方案中，重组多肽是或包括与SEQ ID NO:11具有至少或约80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％序列同一性的氨基酸序列，包括在一个或更多个氨基酸位置(例如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和/或1176(关于SEQ ID NO:55的氨基酸位置)或其任何组合)处包含取代、缺失和/或插入的序列。在一些实施方案中，重组多肽是或包括SEQ ID NO:11的变体，并且变体包括选自由S13I、L18F、T20N、P26S、Δ69-70(ΔHV)、D80A、D138Y、G142D、Δ144(ΔY)、W152C、R190S、D215G、Δ242-244(ΔLAL)、R246I、Δ400-402(ΔFVI)、K417T、K417N、N440K、L452R、S477N、S477G、E484K、E484Q、N501Y、A570D、D614G、H655Y、P681H、P681R、R682G、R683S、R685G、A701V、T716I、F888L、S982A、T1027I、D1118H和V1176F或其任何组合组成的组中的任何一个、两个、三个、四个、五个或更多个突变。
在一些实施方案中，重组多肽是或包括SEQ ID NO:12中所述的序列。在一些实施方案中，重组多肽是或包括与SEQ ID NO:12具有至少或约80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％序列同一性的氨基酸序列，包括在一个或更多个氨基酸位置(例如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和/或1176(关于SEQ ID NO:55的氨基酸位置)或其任何组合)处包含取代、缺失和/或插入的序列。在一些实施方案中，重组多肽是或包括SEQ ID NO:12的变体，并且变体包括选自由S13I、L18F、T20N、P26S、Δ69-70(ΔHV)、D80A、D138Y、G142D、Δ144(ΔY)、W152C、R190S、D215G、Δ242-244(ΔLAL)、R246I、Δ400-402(ΔFVI)、K417T、K417N、N440K、L452R、S477N、S477G、E484K、E484Q、N501Y、A570D、D614G、H655Y、P681H、P681R、R682G、R683S、R685G、A701V、T716I、F888L、S982A、T1027I、D1118H和V1176F或其任何组合组成的组中的任何一个、两个、三个、四个、五个或更多个突变。
在一些实施方案中，重组多肽是或包括SEQ ID NO:13中所述的序列。在一些实施方案中，重组多肽是或包括与SEQ ID NO:13具有至少或约80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％序列同一性的氨基酸序列，包括在一个或更多个氨基酸位置(例如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和/或1176(关于SEQ ID NO:55的氨基酸位置)或其任何组合)处包含取代、缺失和/或插入的序列。在一些实施方案中，重组多肽是或包括SEQ ID NO:13的变体，并且变体包括选自由S13I、L18F、T20N、P26S、Δ69-70(ΔHV)、D80A、D138Y、G142D、Δ144(ΔY)、W152C、R190S、D215G、Δ242-244(ΔLAL)、R246I、Δ400-402(ΔFVI)、K417T、K417N、N440K、L452R、S477N、S477G、E484K、E484Q、N501Y、A570D、D614G、H655Y、P681H、P681R、R682G、R683S、R685G、A701V、T716I、F888L、S982A、T1027I、D1118H和V1176F或其任何组合组成的组中的任何一个、两个、三个、四个、五个或更多个突变。
在一些实施方案中，重组多肽是或包括SEQ ID NO:14中所述的序列。在一些实施方案中，重组多肽是或包括与SEQ ID NO:14具有至少或约80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％序列同一性的氨基酸序列，包括在一个或更多个氨基酸位置(例如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和/或1176(关于SEQ ID NO:55的氨基酸位置)或其任何组合)处包含取代、缺失和/或插入的序列。在一些实施方案中，重组多肽是或包括SEQ ID NO:14的变体，并且变体包括选自由S13I、L18F、T20N、P26S、Δ69-70(ΔHV)、D80A、D138Y、G142D、Δ144(ΔY)、W152C、R190S、D215G、Δ242-244(ΔLAL)、R246I、Δ400-402(ΔFVI)、K417T、K417N、N440K、L452R、S477N、S477G、E484K、E484Q、N501Y、A570D、D614G、H655Y、P681H、P681R、R682G、R683S、R685G、A701V、T716I、F888L、S982A、T1027I、D1118H和V1176F或其任何组合组成的组中的任何一个、两个、三个、四个、五个或更多个突变。
在一些实施方案中，重组多肽是或包括SEQ ID NO:15中所述的序列。在一些实施方案中，重组多肽是或包括与SEQ ID NO:15具有至少或约80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％序列同一性的氨基酸序列，包括在一个或更多个氨基酸位置(例如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和/或1176(关于SEQ ID NO:55的氨基酸位置)或其任何组合)处包含取代、缺失和/或插入的序列。在一些实施方案中，重组多肽是或包括SEQ ID NO:15的变体，并且变体包括选自由S13I、L18F、T20N、P26S、Δ69-70(ΔHV)、D80A、D138Y、G142D、Δ144(ΔY)、W152C、R190S、D215G、Δ242-244(ΔLAL)、R246I、Δ400-402(ΔFVI)、K417T、K417N、N440K、L452R、S477N、S477G、E484K、E484Q、N501Y、A570D、D614G、H655Y、P681H、P681R、R682G、R683S、R685G 、A701V、T716I、F888L、S982A、T1027I、D1118H和V1176F或其任何组合组成的组中的任何一个、两个、三个、四个、五个或更多个突变。
在一些实施方案中，重组多肽是或包括SEQ ID NO:16中所述的序列。在一些实施方案中，重组多肽是或包括与SEQ ID NO:16具有至少或约80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％序列同一性的氨基酸序列，包括在一个或更多个氨基酸位置(例如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和/或1176(关于SEQ ID NO:55的氨基酸位置)或其任何组合)处包含取代、缺失和/或插入的序列。在一些实施方案中，重组多肽是或包括SEQ ID NO:16的变体，并且变体包括选自由S13I、L18F、T20N、P26S、Δ69-70(ΔHV)、D80A、D138Y、G142D、Δ144(ΔY)、W152C、R190S、D215G、Δ242-244(ΔLAL)、R246I、Δ400-402(ΔFVI)、K417T、K417N、N440K、L452R、S477N、S477G、E484K、E484Q、N501Y、A570D、D614G、H655Y、P681H、P681R、R682G、R683S、R685G、A701V、T716I、F888L、S982A、T1027I、D1118H和V1176F或其任何组合组成的组中的任何一个、两个、三个、四个、五个或更多个突变。
在一些实施方案中，重组多肽是或包括SEQ ID NO:17中所述的序列。在一些实施方案中，重组多肽是或包括与SEQ ID NO:17具有至少或约80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％序列同一性的氨基酸序列，包括在一个或更多个氨基酸位置(例如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和/或1176(关于SEQ ID NO:55的氨基酸位置)或其任何组合)处包含取代、缺失和/或插入的序列。在一些实施方案中，重组多肽是或包括SEQ ID NO:17的变体，并且变体包括选自由S13I、L18F、T20N、P26S、Δ69-70(ΔHV)、D80A、D138Y、G142D、Δ144(ΔY)、W152C、R190S、D215G、Δ242-244(ΔLAL)、R246I、Δ400-402(ΔFVI)、K417T、K417N、N440K、L452R、S477N、S477G、E484K、E484Q、N501Y、A570D、D614G、H655Y、P681H、P681R、R682G、R683S、R685G、A701V、T716I、F888L、S982A、T1027I、D1118H和V1176F或其任何组合组成的组中的任何一个、两个、三个、四个、五个或更多个突变。
在一些实施方案中，重组多肽是或包括SEQ ID NO:18中所述的序列。在一些实施方案中，重组多肽是或包括与SEQ ID NO:18具有至少或约80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％序列同一性的氨基酸序列，包括在一个或更多个氨基酸位置(例如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和/或1176(关于SEQ ID NO:55的氨基酸位置)或其任何组合)处包含取代、缺失和/或插入的序列。在一些实施方案中，重组多肽是或包括SEQ ID NO:18的变体，并且变体包括选自由S13I、L18F、T20N、P26S、Δ69-70(ΔHV)、D80A、D138Y、G142D、Δ144(ΔY)、W152C、R190S、D215G、Δ242-244(ΔLAL)、R246I、Δ400-402(ΔFVI)、K417T、K417N、N440K、L452R、S477N、S477G、E484K、E484Q、N501Y、A570D、D614G、H655Y、P681H、P681R、R682G、R683S、R685G、A701V、T716I、F888L、S982A、T1027I、D1118H和V1176F或其任何组合组成的组中的任何一个、两个、三个、四个、五个或更多个突变。
在一些实施方案中，重组多肽是或包括SEQ ID NO:19中所述的序列。在一些实施方案中，重组多肽是或包括与SEQ ID NO:19具有至少或约80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％序列同一性的氨基酸序列，包括在一个或更多个氨基酸位置(例如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和/或1176(关于SEQ ID NO:55的氨基酸位置)或其任何组合)处包含取代、缺失和/或插入的序列。在一些实施方案中，重组多肽是或包括SEQ ID NO:19的变体，并且变体包括选自由S13I、L18F、T20N、P26S、Δ69-70(ΔHV)、D80A、D138Y、G142D、Δ144(ΔY)、W152C、R190S、D215G、Δ242-244(ΔLAL)、R246I、Δ400-402(ΔFVI)、K417T、K417N、N440K、L452R、S477N、S477G、E484K、E484Q、N501Y、A570D、D614G、H655Y、P681H、P681R、R682G、R683S、R685G、A701V、T716I、F888L、S982A、T1027I、D1118H和V1176F或其任何组合组成的组中的任何一个、两个、三个、四个、五个或更多个突变。
在一些实施方案中，重组多肽是或包括SEQ ID NO:20中所述的序列。在一些实施方案中，重组多肽是或包括与SEQ ID NO:20具有至少或约80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％序列同一性的氨基酸序列，包括在一个或更多个氨基酸位置(例如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和/或1176(关于SEQ ID NO:55的氨基酸位置)或其任何组合)处包含取代、缺失和/或插入的序列。在一些实施方案中，重组多肽是或包括SEQ ID NO:20的变体，并且变体包括选自由S13I、L18F、T20N、P26S、Δ69-70(ΔHV)、D80A、D138Y、G142D、Δ144(ΔY)、W152C、R190S、D215G、Δ242-244(ΔLAL)、R246I、Δ400-402(ΔFVI)、K417T、K417N、N440K、L452R、S477N、S477G、E484K、E484Q、N501Y、A570D、D614G、H655Y、P681H、P681R、R682G、R683S、R685G、A701V、T716I、F888L、S982A、T1027I、D1118H和V1176F或其任何组合组成的组中的任何一个、两个、三个、四个、五个或更多个突变。
在一些实施方案中，重组多肽是或包括SEQ ID NO:21中所述的序列。在一些实施方案中，重组多肽是或包括与SEQ ID NO:21具有至少或约80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％序列同一性的氨基酸序列，包括在一个或更多个氨基酸位置(例如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和/或1176(关于SEQ ID NO:55的氨基酸位置)或其任何组合)处包含取代、缺失和/或插入的序列。在一些实施方案中，重组多肽是或包括SEQ ID NO:21的变体，并且变体包括选自由S13I、L18F、T20N、P26S、Δ69-70(ΔHV)、D80A、D138Y、G142D、Δ144(ΔY)、W152C、R190S、D215G、Δ242-244(ΔLAL)、R246I、Δ400-402(ΔFVI)、K417T、K417N、N440K、L452R、S477N、S477G、E484K、E484Q、N501Y、A570D、D614G、H655Y、P681H、P681R、R682G、R683S、R685G、A701V、T716I、F888L、S982A、T1027I、D1118H和V1176F或其任何组合组成的组中的任何一个、两个、三个、四个、五个或更多个突变。
在一些实施方案中，重组多肽是或包括SEQ ID NO:22中所述的序列。在一些实施方案中，重组多肽是或包括与SEQ ID NO:22具有至少或约80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％序列同一性的氨基酸序列，包括在一个或更多个氨基酸位置(例如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和/或1176(关于SEQ ID NO:55的氨基酸位置)或其任何组合)处包含取代、缺失和/或插入的序列。在一些实施方案中，重组多肽是或包括SEQ ID NO:22的变体，并且变体包括选自由S13I、L18F、T20N、P26S、Δ69-70(ΔHV)、D80A、D138Y、G142D、Δ144(ΔY)、W152C、R190S、D215G、Δ242-244(ΔLAL)、R246I、Δ400-402(ΔFVI)、K417T、K417N、N440K、L452R、S477N、S477G、E484K、E484Q、N501Y、A570D、D614G、H655Y、P681H、P681R、R682G、R683S、R685G、A701V、T716I、F888L、S982A、T1027I、D1118H和V1176F或其任何组合组成的组中的任何一个、两个、三个、四个、五个或更多个突变。
在一些实施方案中，重组多肽是或包括SEQ ID NO:23中所述的序列。在一些实施方案中，重组多肽是或包括与SEQ ID NO:23具有至少或约80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％序列同一性的氨基酸序列，包括在一个或更多个氨基酸位置(例如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和/或1176(关于SEQ ID NO:55的氨基酸位置)或其任何组合)处包含取代、缺失和/或插入的序列。在一些实施方案中，重组多肽 是或包括SEQ ID NO:23的变体，并且变体包括选自由S13I、L18F、T20N、P26S、Δ69-70(ΔHV)、D80A、D138Y、G142D、Δ144(ΔY)、W152C、R190S、D215G、Δ242-244(ΔLAL)、R246I、Δ400-402(ΔFVI)、K417T、K417N、N440K、L452R、S477N、S477G、E484K、E484Q、N501Y、A570D、D614G、H655Y、P681H、P681R、R682G、R683S、R685G、A701V、T716I、F888L、S982A、T1027I、D1118H和V1176F或其任何组合组成的组中的任何一个、两个、三个、四个、五个或更多个突变。
在一些实施方案中，重组多肽是或包括SEQ ID NO:24中所述的序列。在一些实施方案中，重组多肽是或包括与SEQ ID NO:24具有至少或约80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％序列同一性的氨基酸序列，包括在一个或更多个氨基酸位置(例如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和/或1176(关于SEQ ID NO:55的氨基酸位置)或其任何组合)处包含取代、缺失和/或插入的序列。在一些实施方案中，重组多肽是或包括SEQ ID NO:24的变体，并且变体包括选自由S13I、L18F、T20N、P26S、Δ69-70(ΔHV)、D80A、D138Y、G142D、Δ144(ΔY)、W152C、R190S、D215G、Δ242-244(ΔLAL)、R246I、Δ400-402(ΔFVI)、K417T、K417N、N440K、L452R、S477N、S477G、E484K、E484Q、N501Y、A570D、D614G、H655Y、P681H、P681R、R682G、R683S、R685G、A701V、T716I、F888L、S982A、T1027I、D1118H和V1176F或其任何组合组成的组中的任何一个、两个、三个、四个、五个或更多个突变。
在一些实施方案中，重组多肽是或包括SEQ ID NO:25中所述的序列。在一些实施方案中，重组多肽是或包括与SEQ ID NO:25具有至少或约80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％序列同一性的氨基酸序列，包括在一个或更多个氨基酸位置(例如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和/或1176(关于SEQ ID NO:55的氨基酸位置)或其任何组合)处包含取代、缺失和/或插入的序列。在一些实施方案中，重组多肽是或包括SEQ ID NO:25的变体，并且变体包括选自由S13I、L18F、T20N、P26S、Δ69-70(ΔHV)、D80A、D138Y、G142D、Δ144(ΔY)、W152C、R190S、D215G、Δ242-244(ΔLAL)、R246I、Δ400-402(ΔFVI)、K417T、K417N、N440K、L452R、S477N、S477G、E484K、E484Q、N501Y、A570D、D614G、H655Y、P681H、P681R、R682G、R683S、R685G、A701V、T716I、F888L、S982A、T1027I、D1118H和V1176F或其任何组合组成的组中的任何一个、两个、三个、四个、五个或更多个突变。
在一些实施方案中，重组多肽是或包括SEQ ID NO:26中所述的序列。在一些实施方案中，重组多肽是或包括与SEQ ID NO:26具有至少或约80％、81％、82％、 83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％序列同一性的氨基酸序列，包括在SEQ ID NO:26的一个或更多个氨基酸位置处包含取代、缺失和/或插入的序列。
在一些实施方案中，重组多肽是或包括SEQ ID NO:85中所述的序列。在一些实施方案中，重组多肽是或包括与SEQ ID NO:26具有至少或约80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％序列同一性的氨基酸序列，包括在SEQ ID NO:85的一个或更多个氨基酸位置处包含取代、缺失和/或插入的序列。
在一些实施方案中，重组多肽是或包括SEQ ID NO:86中所述的序列。在一些实施方案中，重组多肽是或包括与SEQ ID NO:86具有至少或约80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％序列同一性的氨基酸序列，包括在SEQ ID NO:86的一个或更多个氨基酸位置处包含取代、缺失和/或插入的序列。
在一些实施方案中，重组多肽是或包括SEQ ID NO:87中所述的序列。在一些实施方案中，重组多肽是或包括与SEQ ID NO:87具有至少或约80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％序列同一性的氨基酸序列，包括在SEQ ID NO:87的一个或更多个氨基酸位置处包含取代、缺失和/或插入的序列。
在一些实施方案中，重组多肽是或包括SEQ ID NO:88中所述的序列。在一些实施方案中，重组多肽是或包括与SEQ ID NO:88具有至少或约80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％序列同一性的氨基酸序列，包括在SEQ ID NO:88的一个或更多个氨基酸位置处包含取代、缺失和/或插入的序列。
在一些实施方案中，重组多肽是或包括SEQ ID NO:89中所述的序列。在一些实施方案中，重组多肽是或包括与SEQ ID NO:89具有至少或约80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％序列同一性的氨基酸序列，包括在SEQ ID NO:89的一个或更多个氨基酸位置处包含取代、缺失和/或插入的序列。
在一些实施方案中，重组多肽是或包括SEQ ID NO:90中所述的序列。在一些实施方案中，重组多肽是或包括与SEQ ID NO:90具有至少或约80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％序列同一性的氨基酸序列，包括在SEQ ID NO:90的一个或更多个氨基酸位置处包含取代、缺失和/或插入的序列。
在一些实施方案中，重组多肽是或包括SEQ ID NO:91中所述的序列。在一些实施方案中，重组多肽是或包括与SEQ ID NO:91具有至少或约80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、 98％或99％序列同一性的氨基酸序列，包括在SEQ ID NO:91的一个或更多个氨基酸位置处包含取代、缺失和/或插入的序列。
在一些实施方案中，重组多肽是或包括SEQ ID NO:92中所述的序列。在一些实施方案中，重组多肽是或包括与SEQ ID NO:92具有至少或约80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％序列同一性的氨基酸序列，包括在SEQ ID NO:92的一个或更多个氨基酸位置处包含取代、缺失和/或插入的序列。
在一些实施方案中，重组多肽是或包括SEQ ID NO:120中所述的序列在一些实施方案中，重组多肽是或包括与SEQ ID NO:120具有至少或约80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％序列同一性的氨基酸序列，包括在SEQ ID NO:120的一个或更多个氨基酸位置处包含取代、缺失和/或插入的序列。
在一些实施方案中，重组多肽是或包括SEQ ID NO:121中所述的序列在一些实施方案中，重组多肽是或包括与SEQ ID NO:121具有至少或约80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％序列同一性的氨基酸序列，包括在SEQ ID NO:121的一个或更多个氨基酸位置处包含取代、缺失和/或插入的序列。
如上所述，在一些实施方案中，本文提供的重组多肽不仅结合形成三聚体，而且还可以聚集或被聚集以产生包含多种重组多肽的蛋白质。在一些实施方案中，形成的蛋白质具有宏观结构。在一些情况下，宏观结构可赋予冠状病毒抗原或免疫原重组多肽的结构稳定性，其反过来又可提供进入能够促进免疫应答的潜在抗原位点。
在一些实施方案中，三聚化重组多肽聚集形成包含多种三聚化重组多肽的蛋白质。在一些实施方案中，多种三聚化重组多肽形成具有宏观结构的蛋白质。
在一些实施方案中，本文所述的包含多种重组多肽的蛋白质是免疫原。在一些实施方案中，本文所述的包含多种重组多肽的蛋白质包含在纳米颗粒中。例如，在一些实施方案中，蛋白质直接连接到纳米颗粒，例如蛋白质纳米颗粒。在一些实施方案中，蛋白质间接连接到纳米颗粒。在一些实施方案中，本文所述的包含多种重组多肽的蛋白质包含在病毒样颗粒(VLP)中。
在一些实施方案中，本文提供了复合物，其包括选自由SEQ ID NO:1-26和85-92组成的组的重组多肽或其片段、变体或突变体，以任何合适的组合。在一些实施方案中，本文提供了复合物，其包括选自由SEQ ID NO:1-26和85-92组成的组的重组多肽或其片段、变体或突变体的三聚体，其中重组多肽通过多肽间二硫键三聚化形成三聚体。
在一些实施方案中，本文提供了包含多个重组多肽的融合蛋白，每个重组多肽从氨基到羧基末端包括：a)第一区域，其包括位于第一冠状病毒的非嵌合冠状病毒刺突蛋白中的冠状病毒刺突蛋白受体结合结构域(RBD)之前的冠状病毒刺突蛋白胞外域 的一部分；b)第二区域，其包括不同于所述第一冠状病毒的第二冠状病毒的冠状病毒刺突蛋白受体结合结构域(RBD)；和c)胶原的C-末端前肽，其中重组多肽的C-末端前肽形成多肽间二硫键。在一些实施方案中，融合蛋白进一步包括第二区域和胶原的C-末端前肽之间的第三区域。在一些实施方案中，第三区域包括第三冠状病毒的S1结构域，其中第三冠状病毒与第一冠状病毒或第二冠状病毒相同或不同。在一些实施方案中，第三区域包括第四冠状病毒的S2结构域，其中第四冠状病毒与第一、第二或第四冠状病毒相同或不同。在一些实施方案中，第一区域包括第一冠状病毒的N-末端结构域(NTD)。在一些实施方案中，第一区域包括与第二冠状病毒中的相应氨基酸残基不同的一个或更多个氨基酸残基。在一些实施方案中，第二区域包括与第一冠状病毒中的相应氨基酸残基不同的一个或更多个氨基酸残基。在一些实施方案中，第一和第二冠状病毒是相同冠状病毒的不同变种或毒株。在一些实施方案中，第一区域包括第一冠状病毒的NTD，第二区域包括第二冠状病毒的RBD，并且第一和第二冠状病毒是SARS-CoV-2的不同变种。在一些实施方案中，第一冠状病毒和第二冠状病毒独立选自由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和A.23.1谱系的SARS-CoV-2病毒组成的组。
在一些实施方案中，本文提供了包含三个重组多肽的三聚体融合蛋白，每个重组多肽从氨基到羧基末端包括：a)第一区域，其包括B.1.526谱系的SARS-CoV-2的冠状病毒刺突蛋白N-末端结构域(NTD)；b)第二区域，其包括B.1.351谱系的SARS-CoV-2的冠状病毒刺突蛋白受体结合结构域(RBD)；和c)胶原的C-末端前肽，其中重组多肽的C-末端前肽形成多肽间二硫键。在一些实施方案中，本文提供了包含三个重组多肽的三聚体融合蛋白，每个重组多肽从氨基到羧基末端包括：a)第一区域，其包括B.1.1.529谱系的SARS-CoV-2的冠状病毒刺突蛋白N-末端结构域(NTD)；b)第二区域，其包括B.1.1.529谱系或非B.1.1.529谱系的SARS-CoV-2的冠状病毒刺突蛋白受体结合结构域(RBD)；和c)胶原的C-末端前肽，其中重组多肽的C-末端前肽形成多肽间二硫键。在一些实施方案中，本文提供了包含三个重组多肽的三聚体融合蛋白，每个重组多肽从氨基到羧基末端包括：a)第一区域，其包括B.1.1.529谱系或非B.1.1.529谱系的SARS-CoV-2的冠状病毒刺突蛋白N-末端结构域(NTD)；b)第二区域，其包括B.1.1.529谱系的SARS-CoV-2的冠状病毒刺突蛋白受体结合结构域(RBD)；和c)胶原的C-末端前肽，其中重组多肽的C-末端前肽形成多肽间二硫键
在一些实施方案中，本文提供了用于预防哺乳动物中冠状病毒感染的方法，包括用本文公开的有效量的融合蛋白来免疫哺乳动物。在一些实施方案中，在哺乳动物中产生针对第一和第二冠状病毒的中和抗体。在一些实施方案中，第一和第二冠状病毒是SARS-CoV-2的不同变种，并且在哺乳动物中产生的中和抗体中和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和A.23.1谱系的两种或更多种SARS-CoV-2病毒。在一些实施方案中，在哺乳动物中产生的中和抗体中和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和A.23.1谱系的三种或更多种SARS-CoV-2病毒。在一些实施方案中，方法包括用两剂或更多剂的融合蛋白免疫哺乳动物。在一些实施方案中，在一剂或更多剂的免疫原(包括包含来自相同SARS-CoV-2变种的NTD和RBD的刺突蛋白肽)之后，以再次加强剂施用融合蛋白。
在一些实施方案中，本文提供了从包括SARS-CoV-1和SARS-CoV-2的冠状病毒的刺突(S)糖蛋白衍生或修饰的工程化融合多肽。在一些实施方案中，与冠状病毒的野生型S蛋白序列相比，本文公开的融合多肽可稳定在融合前构象中。在一些实施方案中，与三聚化结构域的融合可防止融合蛋白中的S蛋白肽形成直螺旋(例如，类似于膜融合过程中发生的情况)。例如，S-三聚体亚单位候选疫苗的冷冻EM结构表明，它主要采用紧密闭合的融合前状态，与全长野生型刺突蛋白不同，后者在去污剂存在下形成融合前和融合后状态。Ma et al.,J Virol(2021)doi:10.1128/JVI.00194-21，出于所有目的以其全文形式被援引加入。在一些实施方案中，融合蛋白可以包括改变的可溶性S序列，具有使S1/S2裂解位点失活的修饰；七肽重复序列1(HR1)区域和中心螺旋(CH)区域之间的转角区域中的突变，阻止HR1和CH形成直螺旋；和/或除稳定突变外，七肽重复序列2区域(HR2)的截断。在一些实施方案中，本文中的融合蛋白可以但不必须包括一个或更多个突变，例如K986G/V987G、K986P/V987P、K986G/V987P或K986P/V987G，它们被认为稳定处于融合前状态的刺突蛋白。在一些实施方案中，突变例如K986G/V987G、K986P/V987P、K986G/V987P或K986P/V987G对于稳定本文公开的包含蛋白质三聚体化TM三聚体化结构域的融合多肽不是必需的。
在这些实施方案中的一些实施方案中，使得S1/S2裂解位点失活的突变可以包含用GSAG(SEQ ID NO:60)取代RRAR(SEQ ID NO:55中的682-685)，并且转角区域中的突变可以包含双突变K986G/V987G、K986P/V987P、K986G/V987P或K986P/V987G。在一些实施方案中，HR2的截断必须在野生型可溶性S序列的C-末端删除SEQ ID NO:65中所示的一个或更多个残基。在一些实施方案中，免疫原多肽可以进一步包括在与HR2相互作用的HR1区域中的(a)一个或更多个脯氨酸或甘氨酸取代，和/或(b)一个或更多个氨基酸残基的插入。在这些实施方案中的一些实施方案中，免疫原多肽可以具有选自A942P、S943P、A944P、A942G、S943G和A944G的一个或更多个取代。在这些实施方案中的一些实施方案中，插入可以是在A942-A944中的任何残基之间插入G或GS。
本文提及的重组多肽或其片段、变体或突变体，以任何合适的组合，其包括选自由SEQ ID NO:1-26和85-92组成的组的重组多肽或其片段、变体或突变体的三聚体，其中重组多肽通过多肽间二硫键三聚化形成三聚体，可在初始剂和/或加强剂中使用。独立地，初始剂和/或任意一剂或多剂加强剂可不使用或使用佐剂。如果使用佐剂，可选佐剂可包括：含铝佐剂，例如含明矾和/或氢氧化铝的佐剂；含寡核苷酸的佐剂，例如含CpG寡脱氧核苷酸(CpG-ODN)的佐剂；含TLR9激动剂的佐剂；含可代谢油、 α-生育酚、和/或聚氧乙烯山梨醇酐单油酸酯(吐温-80)的佐剂，例如水包油乳液形式的含角鲨烯、α-生育酚、和吐温-80和/或Span 85的佐剂；或任意一种佐剂组合。
2.多核苷酸和载体
还提供了编码本文提供的冠状病毒抗原或免疫原和重组多肽的多核苷酸(核酸分子)，以及用于基因工程化细胞以表达这种冠状病毒抗原或免疫原和重组多肽的载体。
在一些实施方案中，提供了编码本文提供的重组多肽的多核苷酸。在一些方面，多核苷酸包含单个核酸序列，例如编码重组多肽的核酸序列。在其它实例中，多核苷酸包含编码重组多肽特别是冠状病毒抗原或免疫原的第一核酸序列和编码包含不同冠状病毒抗原或免疫原的重组多肽的第二核酸序列。
在一些实施方案中，编码重组多肽的多核苷酸包含至少一个启动子，所述启动子可操作地连接以控制重组多肽的表达。在一些实施方案中，多核苷酸包含两个、三个或更多个启动子，所述启动子可操作地连接以控制重组多肽的表达。
在一些实施方案中，例如当多核苷酸包含两个或更多个核酸编码序列，例如编码包含不同冠状病毒抗原或免疫原的重组多肽的序列时，至少一个启动子可操作地连接以控制两个或更多个核酸序列的表达。在一些实施方案中，多核苷酸包含两个、三个或更多个启动子，所述启动子可操作地连接以控制重组多肽的表达。
在一些实施方案中，重组多肽的表达是可诱导的或有条件的。因此，在一些方面，编码重组多肽的多核苷酸包含条件启动子、增强子或反式激活子。在一些这样的方面中，条件启动子、增强子或反式激活子是诱导型启动子、增强子或反式激活子或者是可抑制启动子、增强子或反式激活子。例如，在一些实施方案中，诱导型或条件启动子可用于将重组多肽的表达限制在特定微环境中。在一些实施方案中，由诱导型或条件启动子驱动的表达通过暴露于外源剂(例如热、辐射或药物)来调节。
在多核苷酸包含一个以上编码重组多肽的核酸序列的情况下，多核苷酸可以进一步包括编码一个或更多个核酸序列之间的肽的核酸序列。在一些情况下，位于核酸序列之间的核酸编码肽，所述肽在翻译期间或之后分离核酸序列的翻译产物。在一些实施方案中，肽包含内部核糖体进入位点(IRES)、自裂解肽或引起核糖体跳跃(skipping)的肽，例如T2A肽。
在一些实施方案中，编码重组多肽的多核苷酸被引入含有培养细胞(例如宿主细胞)的组合物中，例如通过逆转录病毒转导、转染或转化。在一些实施方案中，这可允许重组多肽的表达(例如产生)。在一些实施方案中，对表达的重组多肽进行纯化。
在一些实施方案中，本文提供的多核苷酸(核酸分子)编码如本文所述的冠状病毒抗原或免疫原。在一些实施方案中，本文提供的多核苷酸(核酸分子)编码如本文所述的包含冠状病毒抗原或免疫原(例如冠状病毒S蛋白肽)的重组多肽。
还提供了含有如本文所述的核酸分子的载体或构建体。在一些实施方案中，载体或构建体包含一个或更多个启动子，所述启动子可操作地连接到编码重组多肽的核酸分子以驱动其表达。在一些实施方案中，启动子可操作地连接到一个或一个以上核酸分子，例如编码含有不同冠状病毒抗原或免疫原的重组多肽的核酸分子。
在一些实施方案中，载体是病毒载体。在一些实施方案中，病毒载体是逆转录病毒载体。在一些实施方案中，逆转录病毒载体是慢病毒载体。在一些实施方案中，逆转录病毒载体是γ-逆转录病毒载体。
在一些实施方案中，载体或构建体包括单个启动子，其驱动多核苷酸的一个或更多个核酸分子的表达。在一些实施方案中，此类启动子可以是多顺反子(双顺反子或三顺反子，参见例如美国专利号6,060,273)。例如，在一些实施方案中，转录单元可以被工程化为包含IRES(内部核糖体进入位点)的双顺反子单元，其允许通过来自单个启动子的消息来共表达基因产物(例如，编码不同的重组多肽)。在一些实施方案中，本文提供的载体为双顺反子，允许载体包含并表达两个核酸序列。在一些实施方案中，本文提供的载体为三顺反子，允许载体包含并表达三个核酸序列。
在一些实施方案中，单个启动子指导RNA的表达，所述RNA在单个开放阅读框(ORF)中包含两个或三个基因(例如编码嵌合信号传导受体和编码重组受体)，这些基因通过编码自裂解肽的序列(例如2A序列)或蛋白酶识别位点(例如弗林蛋白酶)彼此分离。因此，ORF编码单个多肽，在翻译过程中(对于2A而言)或翻译后，该多肽被加工成单个蛋白质。在一些情况下，肽(例如T2A)可导致核糖体跳过(核糖体跳跃)2A元件C-末端肽键的合成，导致2A序列末端与下游下一个肽之间的分离(参见，例如，de Felipe.Genetic Vaccines and Ther.2:13(2004)和deFelipe et al.Traffic 5:616-626(2004)，出于所有目的以其全文形式被援引加入)。许多2A元件是本领域已知的。可用于本文公开的方法和核酸中的2A序列的实例包括但不限于如美国专利公开号20070116690中所述的来自口蹄疫病毒(F2A)、马鼻炎A病毒(E2A)、Thosea asigna病毒(T2A)和猪捷申病毒(porcine teschovirus)-1(P2A)的2A序列。
在一些实施方案中，载体包含在病毒中。在一些实施方案中，病毒为假病毒。在一些实施方案中，病毒为病毒样颗粒。在一些实施方案中，载体包含在细胞中。在一些实施方案中，包含载体的病毒或细胞包含重组基因组。
III.免疫原性组合物和制剂
在一些实施方案中，本文提供了免疫原性组合物，其包括重组多肽的三聚体，所述重组多肽包括选自由SEQ ID NO:1-26和85-92组成的组的序列，或任何两种或更多种三聚体的组合。在一些实施方案中，本文提供了包含具有SEQ ID NO:1中所述的序列的重组多肽的三聚体的免疫原性组合物。本文提及的免疫原性组合物，其包括选自由SEQ ID NO:85-92组成的组的重组多肽或其片段、变体或突变体的三聚体，其中重组多肽通过多肽间二硫键三聚化形成三聚体，可在初始剂和/或加强剂中使用。独 立地，初始剂和/或任意一剂或多剂加强剂可不使用或使用佐剂。如果使用佐剂，可选佐剂可包括：含铝佐剂，例如含明矾和/或氢氧化铝的佐剂；含寡核苷酸的佐剂，例如含CpG寡脱氧核苷酸(CpG-ODN)的佐剂；含TLR9激动剂的佐剂；含可代谢油、α-生育酚、和/或聚氧乙烯山梨醇酐单油酸酯(吐温-80)的佐剂，例如水包油乳液形式的含角鲨烯、α-生育酚、和吐温-80和/或Span 85的佐剂；或任意一种佐剂组合。
在一些实施方案中，单位剂量的免疫原性组合物可包括约10μg至约100μg SARS-CoV-2抗原、优选约25μg至约75μg SARS-CoV-2抗原、优选约40μg至约60μg SARS-CoV-2抗原或约50μg SARS-CoV-2抗原。在一些实施方案中，剂量包含3μg SARS-CoV-2抗原。在其它实施方案中，剂量包含9μg SARS-CoV-2抗原。在进一步的实施方案中，剂量包含30μg SARS-CoV-2抗原。
在一些情况下，可能需要将所公开的免疫原与诱导对其它试剂的保护性应答的其它制药产品(例如疫苗)联合。例如，包括如本文所述的重组冠状病毒S抗原(例如三聚体或蛋白质)的组合物可以与免疫实践咨询委员会(ACIP；cdc.gov/vaccines/acip/index.html)推荐的针对目标年龄组(例如，大约一到六个月大的婴儿)的其它疫苗(例如流感疫苗或水痘带状疱疹疫苗)同时(通常单独)或顺序施用。因此，包括本文所述的重组冠状病毒S抗原的公开的免疫原可以与针对例如乙型肝炎(HepB)、白喉、破伤风和百日咳(DTaP)、肺炎球菌(PCV)、b型流感嗜血杆菌(Hib)、脊髓灰质炎、流感和轮状病毒的疫苗同时或顺序施用。
多价或联合疫苗提供对多种病原体的抵御。在一些方面，多价疫苗可以抵御同一病原体的多种毒株。在一些方面，多价疫苗抵御多种病原体，例如联合疫苗Tdap，它抵御破伤风、百日咳和白喉的毒株。多价疫苗对于使对多种病原体或致病毒株赋予抵御所需的免疫接种的数量最小化是非常必要的，以降低管理成本，并提高覆盖率。例如，在为婴儿或儿童接种疫苗时，这可能特别有用。
在一些实施方案中，例如包含本文所述的免疫原性组合物的疫苗是多价疫苗。在一些实施方案中，用于并入多价疫苗组合物的抗原材料来源于冠状病毒毒株或类型，例如如本文所述(参见，例如，第一节)。用于并入多价疫苗组合物的抗原可以来源于冠状病毒的一个毒株或多个毒株(例如，两个和五个毒株之间)，以提供更广泛的保护范围。在一个实施方案中，用于并入多价疫苗组合物的抗原来源于冠状病毒的多个毒株。其它有用的抗原包括活的、减毒的和灭活的病毒，例如灭活的脊髓灰质炎病毒(Jiang et al.,J.Biol.Stand.,(1986)14:103-9)、甲型肝炎病毒的减毒株(Bradley et al.,J.Med.Virol.,(1984)14:373-86)、减毒的麻疹病毒(James et al.,N.Engl.J.Med.,(1995)332:1262-6)以及百日咳病毒的表位(例如，ACEL-IMUNE无细胞DTP、Wyeth-Lederle疫苗和Pediatrics)，以上文献出于所有目的以其全文形式被援引加入。
在一些方面，本文提供的疫苗是通用疫苗。在一些实施方案中，通用疫苗是针对同一病毒的多种毒株(例如冠状病毒的多种毒株)进行保护的疫苗。开发有效的通 用冠状病毒疫苗将降低成本和劳动力，例如使用季节性疫苗配方，并允许更强有力的大流行预防。
在一些实施方案中，本文所述的免疫原可作为初始剂(primary series)，额外剂(additional dose)，和/或同源或异源加强剂(booster dose)使用，例如第一剂，第二剂，第三剂，第四剂，和/或更多剂。在一些实施方案中，本文所述的免疫原可作为季节性疫苗使用。在一些实施方案中，本文所述的免疫原可作为五年内，四年内，三年内，两年内，一年内，和/或六个月内的第一剂，第二剂，第三剂，第四剂，和/或更多剂使用。
在一些方面，通用疫苗是由来源于不同病毒株的多个表位组成的疫苗。在一些方面，通用疫苗由在不同病毒株中保守的单个表位组成。例如，通用疫苗可以基于S蛋白的相对保守的结构域。
还提供了包含所公开的免疫原(例如，所公开的重组冠状病毒S抗原或编码所公开的重组冠状病毒S抗原的原聚体的核酸分子)和药学上可接受的载体的免疫原性组合物。在一些实施方案中，免疫原性组合物包括本文提供的三聚化重组多肽和可选的药学上可接受的载体。在一些实施方案中，免疫原性组合物包括本文提供的三聚化重组多肽和磷酸氢二钠，例如二水合磷酸氢二钠，磷酸二氢钠，例如一水合磷酸氢二钠，氯化钠，和吐温80。在一些实施方案中，1.0mL免疫原性组合物水溶液包括720μg本文提供的三聚化重组多肽和0.62mg二水合磷酸氢二钠，0.62mg一水合磷酸氢二钠，9.0mg氯化钠，和0.2mg吐温80。在一些实施方案中，免疫原性组合物包括包含本文提供的多个三聚化重组多肽的蛋白质以及可选的药学上可接受的载体。在一些实施方案中，免疫原性组合物包括本文提供的蛋白质纳米颗粒和可选的药学上可接受的载体。在一些实施方案中，免疫原性组合物包括本文提供的VLP和可选的药学上可接受的载体。在一些实施方案中，免疫原性组合物包括本文提供的分离核酸和可选的药学上可接受的载体。在一些实施方案中，免疫原性组合物包括本文提供的载体和可选的药学上可接受的载体。在一些实施方案中，免疫原性组合物包括本文提供的病毒和可选的药学上可接受的载体。在一些实施方案中，免疫原性组合物包括本文提供的假病毒和可选的药学上可接受的载体。在一些实施方案中，免疫原性组合物包括本文提供的细胞和可选的药学上可接受的载体。在一些实施方案中，如本文所述的免疫原性组合物是疫苗。在一些实施方案中，疫苗是预防性疫苗。在一些实施方案中，疫苗是治疗性疫苗。在一些实施方案中，疫苗是预防性疫苗和治疗性疫苗。此类药物组合物可通过本领域普通技术人员已知的多种给药模式向受试者给药，例如，肌肉内、皮内、皮下、静脉内、动脉内、关节内、腹腔内、鼻内、舌下、扁桃体、口咽或其它肠胃外和粘膜途径。在几个实施方案中，包括一个或更多个所公开的免疫原的药物组合物是免疫原性组合物。用于制备可给药组合物的实际方法对于本领域技术人员来说是已知的或显而易见的，并且在例如Remingtons Pharmaceutical Sciences,19th  Ed.,Mack Publishing Company,Easton,Pa.,1995这种出版物中进行了更详细的描述。
因此，本文所述的免疫原，例如重组冠状病毒S抗原，例如三聚体、蛋白质，可与药学上可接受的载体调配，以帮助保持生物活性，同时也促进在可接受温度范围内储存期间的稳定性增加。潜在的载体包括但不限于生理平衡培养基、磷酸盐缓冲盐溶液、水、乳液(例如，油/水或水/油乳液)、各种类型的润湿剂、防冷冻添加剂或稳定剂，例如蛋白质、肽或水解产物(例如，白蛋白、明胶)、糖(例如，蔗糖、乳糖、山梨醇)、氨基酸(例如，谷氨酸钠)或其它保护剂。所得的水溶液可按原样包装使用或冻干。冻干制剂在单剂量或多剂量给药前与无菌溶液混合。
配方组合物，尤其是液体制剂，可含有抑菌剂，以防止或最小化储存期间的降解，包括但不限于有效浓度(通常为1％w/v)的苯甲醇、苯酚、间甲酚、氯丁醇、对羟基苯甲酸甲酯和/或对羟基苯甲酸丙酯。一些患者可能禁用抑菌剂；因此，冻干制剂可在含有或不含有此类成分的溶液中重新组合。
本发明的免疫原性组合物可包含近似生理条件所需的药学上可接受的载体物质，例如pH调节剂和缓冲剂、张力调节剂、润湿剂等，例如醋酸钠、乳酸钠、氯化钠、氯化钾，氯化钙、山梨醇酐单月桂酸酯和油酸三乙醇胺。免疫原性组合物可以可选地包括佐剂以增强宿主的免疫应答。合适的佐剂例如为toll样受体(TLR)激动剂、明矾、AlPO4、水凝胶(alhydrogel)、脂质-A及其衍生物或变体、油乳剂、皂甙、中性脂质体、含有疫苗和细胞因子的脂质体、非离子嵌段共聚物和趋化因子。本领域众所周知的许多其它合适的佐剂中的含有聚氧乙烯(polyoxyethylene,POE)和聚氧丙烯(polyxylpropylene,POP)的非离子嵌段聚合物可作为佐剂使用，例如POE-POP-POE嵌段共聚物、MPLTM(3-O-脱酰基单磷酸脂A；Corixa,Hamilton,Ind.)和IL-12(Genetics Institute,Cambridge,Mass.)(Newman et al.,1998,Critical Reviews in Therapeutic Drug Carrier Systems 15:89-142，出于所有目的以其全文形式被援引加入)。这些佐剂的优点在于，它们有助于以非特异性的方式刺激免疫系统，从而增强对药物产品的免疫应答。在一些实施方案中，本发明的免疫原性组合物可包括或与一种以上佐剂一起施用。在一些实施方案中，本发明的免疫原性组合物可包括或与两种佐剂一起施用。在一些实施方案中，本发明的免疫原性组合物可包括或与多种佐剂一起施用。例如，在一些情况下，例如包含本文提供的免疫原性组合物的疫苗可包括或与多种佐剂联合施用。
对于疫苗组合物，合适佐剂的实例包括，例如氢氧化铝、卵磷脂、Freund’s佐剂、MPLTM和IL-1，其一种或任意几种的组合可与包括选自由SEQ ID NO:85-92组成的组的重组多肽或其片段、变体或突变体的三聚体配合使用。在一些实施方案中，本文公开的疫苗组合物或纳米颗粒免疫原(例如，SARS-COV-2疫苗组合物)可配制为控释或缓释制剂。这可以通过含有缓释聚合物的组合物或通过微胶囊递送系统或生物粘附凝胶来实现。各种药物组合物可根据本领域众所周知的标准程序制备。
在一些实施方案中，本发明的免疫原性组合物，包括选自由SEQ ID NO:85-92组成的组的重组多肽或其片段、变体或突变体的三聚体，其中重组多肽通过多肽间二硫键三聚化形成三聚体，可包含佐剂制剂，所述佐剂制剂包含水包油乳液形式的可代谢油(例如角鲨烯)和α-生育酚(例如DL-α-生育酚)，以及聚氧乙烯山梨醇酐单油酸酯(吐温-80)。在一些实施方案中，佐剂制剂可包括约2％至约10％角鲨烯、约2％至约10％α-生育酚(例如，D-α-生育酚)和约0.3％至约3％聚氧乙烯山梨醇酐单油酸酯。在一些实施方案中，佐剂制剂可包括约5％角鲨烯、约5％生育酚和约0.4％聚氧乙烯山梨醇酐单油酸酯。在一些实施方案中，本发明的免疫原性组合物可包含3个脱O-酰化单磷酸脂A(3D-MPL)和水包油乳液形式的佐剂，该佐剂包含可代谢油、α-生育酚和聚氧乙烯山梨醇酐单油酸酯。在一些实施方案中，本发明的免疫原性组合物可包含QS21(Quillaja saponaria Molina提取物：组分21)、3D-MPL和水包油乳液，其中水包油乳液包括可代谢油、α-生育酚和聚氧乙烯山梨醇酐单油酸酯。在一些实施方案中，本发明的免疫原性组合物可包含QS21、3D-MPL和水包油乳液，其中水包油乳液具有以下组合物：可代谢油，例如角鲨烯、α-生育酚、和吐温-80和/或Span 85。在一些实施方案中，本发明的免疫原性组合物可包含脂质体组合物形式的佐剂。
在一些实施方案中，本发明的免疫原性组合物，包括选自由SEQ ID NO:85-92组成的组的重组多肽或其片段、变体或突变体的三聚体，其中重组多肽通过多肽间二硫键三聚化形成三聚体，可包含佐剂制剂，所述佐剂制剂包含可代谢的油(例如，角鲨烯)、α-生育酚、聚氧乙烯山梨醇酐单油酸酯(吐温-80)、和/或Span 85。在一些实施方案中，佐剂制剂可包括约5％(w/v)角鲨烯、约5％(w/v)α-生育酚、约0.5％(w/v)聚氧乙烯山梨醇酐单油酸酯、和/或约0.5％(w/v)Span 85。
在一些实施方案中，本发明的免疫原性组合物，包括选自由SEQ ID NO:85-92组成的组的重组多肽或其片段、变体或突变体的三聚体，其中重组多肽通过多肽间二硫键三聚化形成三聚体，可包含佐剂制剂，所述佐剂制剂包含皂树(Quillaja)皂苷、胆固醇和磷脂，例如，以纳米颗粒组合物的形式。在一些实施方案中，本发明的免疫原性组合物可包含单独纯化的Quillaja saponaria Molina部分的混合物，其中随后与胆固醇和磷脂调配。
在一些实施方案中，本发明的免疫原性组合物，包括选自由SEQ ID NO:85-92组成的组的重组多肽或其片段、变体或突变体的三聚体，其中重组多肽通过多肽间二硫键三聚化形成三聚体，可包含选自由MF59TM、Matrix-ATM、Matrix-CTM、Matrix-MTM、AS01、AS02、AS03和AS04组成的组的佐剂。可选的佐剂包含O’Hagan et al,The history ofadjuvant: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 containingα-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)所披露的佐剂的任意一种或几种的组合，以上文献均出于所有目的以其全文形式被援引加入本文。
在一些实施方案中，本发明的免疫原性组合物，包括选自由SEQ ID NO:85-92组成的组的重组多肽或其片段、变体或突变体的三聚体，其中重组多肽通过多肽间二硫键三聚化形成三聚体，可包含toll样受体9(TLR9)激动剂，其中TLR9激动剂为长度为8至35个核苷酸的寡核苷酸，包含未甲基化的胞苷-磷酸-鸟苷(也称为CpG或胞嘧啶-磷酸-鸟苷)基序，并且SARS-CoV-2抗原和寡核苷酸以有效刺激哺乳动物受试者(例如需要SARS-CoV-2抗原和寡核苷酸的人类受试者)中针对SARS-CoV-2抗原的免疫应答的量存在于免疫原性组合物中。TLR9(CD289)识别微生物DNA中发现的未甲基化的胞苷-磷酸-鸟苷(CpG)基序，可使用合成的含CpG的寡脱氧核苷酸(CpG-ODN)对其进行模仿。已知CpG-ODN可增强抗体产生并刺激T辅助细胞1(Th1)细胞应答(Coffman et al.,Immunity,33:492-503,2010，出于所有目的以其全文形式被援引加入)。最佳寡核苷酸TLR9激动剂通常包含以下通式的回文序列：5’-嘌呤-嘌呤-CG-嘧啶-嘧啶-3’或5’-嘌呤-嘌呤-CG-嘧啶-嘧啶-CG-3’。美国专利号6,589,940，其以其全文形式被援引加入本文。在一些实施方案中，CpG寡核苷酸是线性的。在其它实施方案中，CpG寡核苷酸是环状的或包括发夹环。CpG寡核苷酸可以是单链的或双链的。在一些实施方案中，CpG寡核苷酸可包含修饰。修饰包括但不限于3’OH或5’OH基团的修饰、核苷酸碱基的修饰、糖组分的修饰和磷酸基团的修饰。只要经修饰的碱基通过Watson-Crick碱基配对对其天然补体保持相同的特异性(例如，回文部分仍然是自我互补的)，经修饰的碱基可以包括在CpG寡核苷酸的回文序列中。在一些实施方案中，CpG寡核苷酸包括非经典碱基。在一些实施方案中，CpG寡核苷酸包括经修饰的核苷。在一些实施方案中，经修饰的核苷选自由2’-脱氧-7-脱氮鸟苷(2’-deoxy-7-deazaguanosine)、2’-脱氧-6-硫代鸟苷、阿糖鸟苷(arabinoguanosine)、2’-脱氧-2’取代-阿糖鸟苷和2’-O-取代-阿糖鸟苷。CpG寡核苷酸可能包含磷酸基团的修饰。例如，除了磷酸二酯键之外，磷酸修饰包括但不限于膦酸甲酯、硫代磷酸酯、磷酰胺(桥接或非桥接)、磷酸三酯和二硫代磷酸酯，并且可以以任何组合使用。也可使用其它非磷酸键。在一些实施方案中，寡核苷酸仅包括硫代磷酸酯主链。在一些实施方案中，寡核苷酸仅包括磷酸二酯主链。在一些实施方案中，寡核苷酸包括磷酸主链中的磷酸键的组合，例如磷酸二酯键和硫代磷酸酯键的组合。具有硫代磷酸酯主链的寡核苷酸可以比具有磷酸二酯主链的寡核苷酸具有更高的免疫原性，并且在注射到宿主中后似乎更耐降解(Braun et al.,J Immunol,141:2084-2089,1988；和Latimer et al.,Mol Immunol,32:1057-1064,1995，出于所有目的以其全文形式被援引加入)。本发明的CpG寡核苷酸包括至少一个、两个或三个核苷酸间硫代磷酸酯键。在一些实施方案中，当多个CpG寡核苷酸分子存在于包含至少一种赋形剂的药物组合物中时，硫代磷酸酯键的两个立体异构体存在于多个CpG寡核苷酸分子中。在一些实施方案中， CpG寡核苷酸的所有核苷酸间键均为硫代磷酸酯键，或者换句话说，CpG寡核苷酸具有硫代磷酸酯主链。
在本发明中，任何合适的CpG寡脱氧核苷酸(ODN)或其组合可用作佐剂。例如，K型ODN(也称为B型)编码硫代磷酸酯主链上的多个CpG基序。K型ODN可基于以下序列与天然磷酸二酯核苷酸相比，硫代磷酸酯核苷酸的使用增强了对核酸酶消化的抵抗力，从而导致体内半衰期显著延长。K型ODN引发pDC分化并产生TNF-α，并引发B细胞增殖并分泌IgM。D型ODN(也称为A型)由混合的磷酸二酯/硫代磷酸酯主链构成，包含单个CpG基序，两侧为回文序列，并在3’和5’端具有多聚G尾巴(促进串联体形成的结构基序)。D型ODN可基于以下序列GGTGCATCGATGCAGGGGGG。D型ODN引发pDC成熟并分泌IFN-α，但对B细胞无影响。C型ODN与K型相似，完全由硫代磷酸酯核苷酸组成，但与D型ODN相似，包含回文CpG基序。C型ODN可基于以下序列这类ODN刺激B细胞分泌IL-6和pDC以产生IFN-α。P型ODN包含两个回文序列，使得它们能够形成更高的有序结构。P型ODN可基于以下序列与C型ODN相比，P型ODN激活B细胞和PDC，并诱导更大量IFN-α产生。在本段中，ODN序列中的粗体字表示自我互补回文，CpG基序加下划线。
示例性CpG ODN，例如CpG 7909(5′-TCGTCGTTTTGTCGTTTTGTCGTT-3’)和CpG 1018(5’-TGACTGTGAACGTTCGAGATGA-3’)，是已知的并且公开于美国专利号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和10,052,378；和Bode et al.,“CpG DNA as a vaccine adjuvant”,Expert Rev Vaccines(2011),10(4):499-511中，所有这些均出于所有目的以其全文形式被援引加入本文。
一种或更多种佐剂可组合使用，包括但不限于明矾(铝盐)、水包油乳液、油包水乳液、脂质体和微粒，例如聚(丙交酯-共-乙交酯)微粒(Shah et al.,Methods Mol Biol,1494:1-14,2017，出于所有目的以其全文形式被援引加入)。在一些实施方案中，免疫原性组合物进一步包括吸附SARS-CoV-2抗原的铝盐佐剂。在一些实施方案中，铝盐佐剂包括由无定形羟基磷酸铝硫酸盐、氢氧化铝、磷酸铝和硫酸铝钾组成的组中的一种或更多种。在一些实施方案中，铝盐佐剂包括氢氧化铝和磷酸铝中的一种或两者。在一些实施方案中，铝盐佐剂包括氢氧化铝。在一些实施方案中，单位剂量的免疫原性组合物包括约0.01mg至约0.8mg Al3+；或约0.05mg至约0.7mg Al3+；或约0.06mg至约0.6mg Al3+；或约0.07mg Al3+、约0.08mg至约0.45mg Al3+；或约0.08mg至约0.30mg、约0.35mg Al3+、约0.40mg Al3+；或约0.25mg至约0.50mg Al3+；或约0.068mg Al3+、约0.078mg Al3+、约0.13mg Al3+、约0.19mg Al3+、约0.22mg Al3+、约0.26mg、约0.35mg Al3+。在一些实施方案中，免疫原性组合物进一步包括另外的佐剂。其它合适的佐剂包括但不限于水包角鲨烯乳液(例如， MF59或AS03)、TLR3激动剂(例如，聚IC或聚ICLC)、TLR4激动剂(例如，细菌脂多糖衍生物，如单磷酸脂A(MPL)，和/或皂甙，如Quil A或QS-21，如AS01或AS02)、TLR5激动剂(细菌鞭毛蛋白)和TLR7、TLR8和/或TLR9激动剂(咪唑喹啉衍生物，例如咪喹莫特和瑞喹莫特)(Coffman et al.,Immunity,33:492-503,2010，出于所有目的以其全文形式被援引加入)。在一些实施方案中，另外的佐剂包括MPL和氢氧化铝/明矾(例如AS04)。对于兽医用途和对于非人类动物抗体的生产，可以使用Freund’s佐剂的促有丝分裂成分(完整的和不完整的)。
在一些实施方案中，单位剂量的免疫原性组合物可包括约3μg至约1000μg一种或更多种佐剂、约8μg至约1000μg一种或更多种佐剂,优选约25μg至约500μg一种或更多种佐剂、优选约9μg至约500μg一种或更多种佐剂优,优选约50μg至约300μg一种或更多种佐剂、优选约100μg至约250μg一种或更多种佐剂、优选约150μg至约225μg一种或更多种佐剂。在一些实施方案中，单位剂量的免疫原性组合物可包括约3μg至约700μg、750μg、800μg含铝佐剂，约3μg至约500μg含铝佐剂，约8μg至约250μg含铝佐剂,约9μg至约240μg含铝佐剂,优选约25μg至约230μg含铝佐剂，优选约200μg至约800μg含铝佐剂，优选约700μg至约800μg含铝佐剂，优选约740μg至约780μg含铝佐剂，优选约750、760、770μg至约780μg含铝佐剂，优选约260μg至约280μg含铝佐剂、优选约200μg至约240μg含铝佐剂、优选约210μg至约230μg含铝佐剂、优选约220μg至约230μg含铝佐剂、优选约224μg至约228μg含铝佐剂、优选约50μg至约125μg含铝佐剂、优选约750μg含铝佐剂，例如Alum,约50μg含铝佐剂,约45μg含铝佐剂,约40μg含铝佐剂,约35μg含铝佐剂,约25μg含铝佐剂,约24μg，约23μg，约22μg，约20μg含铝佐剂,约15μg含铝佐剂,约10μg含铝佐剂,约9μg含铝佐剂,或约8、约7、约6、约5、约4、约3μg含铝佐剂。在一些实施方案中，单位剂量的免疫原性组合物可包括约10μg至约2000μg CpG佐剂，或约20μg至约1800、1700，1600μg CpG佐剂，或约40μg至约1500μg CpG佐剂，或约1000μg至约1900μg CpG佐剂，或约1200μg至约1800μg CpG佐剂，或约1300μg至约1700μg CpG佐剂，或约1400μg至约1600μg CpG佐剂，或约10μg至约500μg CpG佐剂，或约460μg至约470μg CpG佐剂，或约410μg、420μg、430μg、440μg至约490μg CpG佐剂，或约10μg至约500μg CpG佐剂，或优选约25μg至约300μg CpG佐剂、优选约50μg至约250μg CpG佐剂、优选约75μg至约200μg CpG佐剂、优选约100μg至约175μg CpG佐剂，或优选约1500μg CpG佐剂，或优选约1600μg CpG佐剂，或优选约1400μg、约1450μg、约1455μg CpG佐剂，优选约150μg CpG佐剂，例如CpG 1018A。在一些实施方案中，单位剂量的免疫原性组合物可包括约10μg至约500μg含铝佐剂和约10μg至约500μg CpG佐剂。在一些实施方案中，单位剂量的免疫原性组合物可包括约10μg至约800μg含铝佐剂和约10μg至约1800μg CpG佐剂，在一些实施方案中，单位剂量的免疫原性组合物可包括约25μg至约250μg含铝佐剂和约25μg至 约300μg CpG佐剂。在一些实施方案中，单位剂量的免疫原性组合物可包括约200μg至约800μg含铝佐剂和约500μg至约1800μg CpG佐剂。在一些实施方案中，单位剂量的免疫原性组合物可包括约200μg至约250μg含铝佐剂和约400μg至约500μg CpG佐剂。在一些实施方案中，单位剂量的免疫原性组合物可包括约210μg至约240μg含铝佐剂和约420μg至约490μg CpG佐剂。在一些实施方案中，单位剂量的免疫原性组合物可包括约220μg至约230μg含铝佐剂和约430μg至约470μg CpG佐剂。在一些实施方案中，单位剂量的免疫原性组合物可包括约221μg至约227μg含铝佐剂和约440μg至约460μg CpG佐剂。在一些实施方案中，单位剂量的免疫原性组合物可包括约600μg至约900μg含铝佐剂和约1000μg至约2000μg CpG佐剂。在一些实施方案中，单位剂量的免疫原性组合物可包括约700μg至约800μg含铝佐剂和约1100μg至约1900或1800μg CpG佐剂。在一些实施方案中，单位剂量的免疫原性组合物可包括约720μg至约790μg含铝佐剂和约1200或1300μg至约1700μg CpG佐剂。在一些实施方案中，单位剂量的免疫原性组合物可包括约730或740μg至约760μg含铝佐剂和约1400μg至约1600μg CpG佐剂。在一些实施方案中，单位剂量的免疫原性组合物可包括约50μg至约125μg含铝佐剂和约50μg至约250μg CpG佐剂。在一些实施方案中，单位剂量的免疫原性组合物可包括约50μg至约100μg含铝佐剂和约75μg至约200μg CpG佐剂。在一些实施方案中，单位剂量的免疫原性组合物可包括约50μg至约100μg含铝佐剂和约100μg至约175μg CpG佐剂。在一些实施方案中，单位剂量的免疫原性组合物可包括约75μg至约100μg含铝佐剂，例如Alum，和约150μg CpG佐剂，例如CpG 1018A。在一些实施方案中，单位剂量的免疫原性组合物可包括约200μg至约250μg含铝佐剂，例如Alum，和约400μg至约500μg CpG佐剂，例如CpG 1018A。在一些实施方案中，单位剂量的免疫原性组合物可包括约210μg至约240μg含铝佐剂，例如Alum，和约420μg至约490μg CpG佐剂，例如CpG 1018A。在一些实施方案中，单位剂量的免疫原性组合物可包括约220μg至约230μg含铝佐剂，例如Alum，和约430μg至约470μg CpG佐剂，例如CpG 1018A。在一些实施方案中，单位剂量的免疫原性组合物可包括约221μg至约227μg含铝佐剂，例如Alum，和约440μg至约460μg CpG佐剂，例如CpG 1018A。在一些实施方案中，单位剂量的免疫原性组合物可包括约600μg至约900μg含铝佐剂，例如Alum，和约1000μg至约2000μg CpG佐剂，例如CpG 1018A。在一些实施方案中，单位剂量的免疫原性组合物可包括约700μg至约800μg含铝佐剂，例如Alum，和约1100μg至约1900或1800μg CpG佐剂，例如CpG 1018A。在一些实施方案中，单位剂量的免疫原性组合物可包括约720μg至约790μg含铝佐剂，例如Alum，和约1200或1300μg至约1700μg CpG佐剂，例如CpG 1018A。在一些实施方案中，单位剂量的免疫原性组合物可包括约730或740μg至约760μg含铝佐剂，例如Alum，和约1400μg至约1600μg CpG佐剂，例如CpG 1018A。在一些实施方案中，单位剂量的免疫原性组合物可包括约10μg至约100μg SARS-CoV-2抗原，例如本发明中任意一种或几种SARS- CoV-2 S融合蛋白或S-三聚体、例如包括SARS-CoV-2冠状病毒奥密克戎(B.1.1.529)变异株S蛋白肽或其片段、变体或突变体的融合蛋白或S融合蛋白三聚体。在一些实施方案中，单位剂量的免疫原性组合物可包括约20μg至约75μg SARS-CoV-2 S融合蛋白或S-三聚体、优选约25μg至约60μg SARS-CoV-2 S融合蛋白或S-三聚体，或约30μg，约40μg，约50μg SARS-CoV-2 S融合蛋白或S-三聚体。在一些实施方案中，单位剂量的免疫原性组合物可包括约3μg SARS-CoV-2 S融合蛋白或S-三聚体。在其它实施方案中，剂量包含15μg SARS-CoV-2 S融合蛋白或S-三聚体。在其它实施方案中，剂量包含9μg SARS-CoV-2 S融合蛋白或S-三聚体。在进一步的实施方案中，剂量包含30μg SARS-CoV-2 S融合蛋白或S-三聚体。在一些实施方案中，单位剂量的免疫原性组合物可包括约5μg至约20μg SARS-CoV-2抗原，例如本发明中任意一种或几种SARS-CoV-2 S融合蛋白或S-三聚体、例如包括SARS-CoV-2冠状病毒奥密克戎(B.1.1.529)变异株S蛋白肽或其片段、变体或突变体的融合蛋白或S融合蛋白三聚体，可包括约200μg至约250μg含铝佐剂，例如Alum，和约400μg至约500μg CpG佐剂，例如CpG 1018A。在一些实施方案中，单位剂量的免疫原性组合物可包括约6μg至约17、18、19μg SARS-CoV-2抗原，例如本发明中任意一种或几种SARS-CoV-2 S融合蛋白或S-三聚体、例如包括SARS-CoV-2冠状病毒奥密克戎(B.1.1.529)变异株S蛋白肽或其片段、变体或突变体的融合蛋白或S融合蛋白三聚体，可包括约210μg至约240μg含铝佐剂，例如Alum，和约420μg至约490μg CpG佐剂，例如CpG 1018A。在一些实施方案中，单位剂量的免疫原性组合物可包括约8μg至约12、13、14、15、16μg SARS-CoV-2抗原，例如本发明中任意一种或几种SARS-CoV-2 S融合蛋白或S-三聚体、例如包括SARS-CoV-2冠状病毒奥密克戎(B.1.1.529)变异株S蛋白肽或其片段、变体或突变体的融合蛋白或S融合蛋白三聚体，可包括约220μg至约230μg含铝佐剂，例如Alum，和约430μg至约470μg CpG佐剂，例如CpG 1018A。在一些实施方案中，单位剂量的免疫原性组合物可包括约8μg至约11μg SARS-CoV-2抗原，例如本发明中任意一种或几种SARS-CoV-2 S融合蛋白或S-三聚体、例如包括SARS-CoV-2冠状病毒奥密克戎(B.1.1.529)变异株S蛋白肽或其片段、变体或突变体的融合蛋白或S融合蛋白三聚体，可包括约221μg至约227μg含铝佐剂，例如Alum，和约440μg至约460μg CpG佐剂，例如CpG 1018A。在一些实施方案中，单位剂量的免疫原性组合物可包括约8μg至约12、13、14、15、16μg SARS-CoV-2抗原，例如本发明中任意一种或几种SARS-CoV-2 S融合蛋白或S-三聚体、例如包括SARS-CoV-2冠状病毒奥密克戎(B.1.1.529)变异株S蛋白肽或其片段、变体或突变体的融合蛋白或S融合蛋白三聚体，可包括约600μg至约900μg含铝佐剂，例如Alum，和约1000μg至约2000μg CpG佐剂，例如CpG 1018A。在一些实施方案中，单位剂量的免疫原性组合物可包括约10μg至约80μg SARS-CoV-2抗原，例如本发明中任意一种或几种SARS-CoV-2 S融合蛋白或S-三聚体、例如包括SARS-CoV-2冠状病毒奥密克戎(B.1.1.529)变异株S蛋白肽或其片段、变体或突变体的融合蛋白或S融合蛋白三聚体，可包括约 700μg至约800μg含铝佐剂，例如Alum，和约1100μg至约1900或1800μg CpG佐剂，例如CpG 1018A。在一些实施方案中，单位剂量的免疫原性组合物可包括约15μg至约60、70μg SARS-CoV-2抗原，例如本发明中任意一种或几种SARS-CoV-2 S融合蛋白或S-三聚体、例如包括SARS-CoV-2冠状病毒奥密克戎(B.1.1.529)变异株S蛋白肽或其片段、变体或突变体的融合蛋白或S融合蛋白三聚体，可包括约720μg至约790μg含铝佐剂，例如Alum，和约1200或1300μg至约1700μg CpG佐剂，例如CpG1018A。在一些实施方案中，单位剂量的免疫原性组合物可包括约25μg至约40、50μg SARS-CoV-2抗原，例如本发明中任意一种或几种SARS-CoV-2 S融合蛋白或S-三聚体、例如包括SARS-CoV-2冠状病毒奥密克戎(B.1.1.529)变异株S蛋白肽或其片段、变体或突变体的融合蛋白或S融合蛋白三聚体，可包括约730或740μg至约760μg含铝佐剂，例如Alum，和约1400μg至约1600μg CpG佐剂，例如CpG 1018A。在一些实施方案中，单位剂量的免疫原性组合物可包括约30μg SARS-CoV-2 S融合蛋白或S-三聚体，约75μg至约100μg含铝佐剂，例如Alum，和约150μg CpG佐剂，例如CpG 1018A。在一些实施方案中，单位剂量的免疫原性组合物可包括约9μg SARS-CoV-2抗原，例如本发明中任意一种或几种SARS-CoV-2 S融合蛋白或S-三聚体、例如包括SARS-CoV-2冠状病毒奥密克戎(B.1.1.529)变异株S蛋白肽或其片段、变体或突变体的融合蛋白或S融合蛋白三聚体，可包括约225μg含铝佐剂，例如Alum，和约450μg CpG佐剂，例如CpG 1018A。在一些实施方案中，单位剂量的免疫原性组合物可包括约30μg SARS-CoV-2抗原，例如本发明中任意一种或几种SARS-CoV-2 S融合蛋白或S-三聚体、例如包括SARS-CoV-2冠状病毒奥密克戎(B.1.1.529)变异株S蛋白肽或其片段、变体或突变体的融合蛋白或S融合蛋白三聚体，可包括约750μg含铝佐剂，例如Alum，和约1500μg CpG佐剂，例如CpG 1018A。
在一些实施方案中，免疫原性组合物包括药学上可接受的赋形剂，包括例如溶剂、填充剂、缓冲剂、张力调节剂和防腐剂(Pramanick et al.,Pharma Times,45:65-77,2013，出于所有目的以其全文形式被援引加入)。在一些实施方案中，免疫原性组合物可包括赋形剂，其充当溶剂、填充剂、缓冲剂和张力调节剂中的一种或更多种(例如，盐水中的氯化钠可同时用作水性载体和张力调节剂)。
在一些实施方案中，免疫原性组合物包括作为溶剂的水性载体。合适的载体包括例如无菌水、盐水、磷酸盐缓冲盐水和Ringer’s溶液。在一些实施方案中，组合物是等渗的。
免疫原性组合物可包括缓冲剂。缓冲剂控制pH，以抑制在处理、储存和可选地重构期间活性剂的降解。合适的缓冲剂包括例如包含醋酸盐、柠檬酸盐、磷酸盐或硫酸盐的盐。其它合适的缓冲剂包括例如氨基酸，例如精氨酸、甘氨酸、组氨酸和赖氨酸。缓冲剂可以进一步包括盐酸或氢氧化钠。在一些实施方案中，缓冲剂将组合物的pH保持在6到9的范围内。在一些实施方案中，pH大于(下限)6、7或8。在一些 实施方案中，pH小于(上限)9、8或7。也就是说，pH在约6到9的范围内，其中下限小于上限。
免疫原性组合物可包括张力调节剂。合适的张力调节剂包括例如葡萄糖、甘油(glycerol)、氯化钠、甘油(glycerin)和甘露醇。
免疫原性组合物可包括填充剂。当在给药前将药物组合物冻干时，填充剂特别有用。在一些实施方案中，填充剂是保护剂，有助于在冷冻或喷雾干燥期间和/或在储存期间稳定和防止活性剂降解。合适的填充剂为糖(单、双和多糖)，例如蔗糖、乳糖、海藻糖、甘露醇、山梨醇、葡萄糖和棉子糖。
免疫原性组合物可包括防腐剂。合适的防腐剂包括例如抗氧化剂和抗菌剂。然而，在优选实施方案中，免疫原性组合物在无菌条件下制备并在一次性容器中，因此不需要包含防腐剂。
在一些实施方案中，组合物可作为无菌组合物提供。药物组合物通常包含有效量的所公开的免疫原，并且可以通过常规技术制备。通常，选择每剂量免疫原性组合物中免疫原的量作为诱导免疫应答而无显著不良副作用的量。在一些实施方案中，组合物可以单位剂型提供，用于诱导受试者的免疫应答。单位剂型包含用于给受试者施用的合适的单个预选剂量，或两个或更多个预选单位剂量的合适标记的或测量的倍数，和/或用于施用单位剂量或其倍数的计量机构。在其它实施方案中，组合物进一步包括一种或几种佐剂。
IV.诱导免疫应答的方法
在一些实施方案中，本文提供了用于在受试者中产生对冠状病毒表面抗原的免疫应答的方法，包括向受试者施用有效量的复合物，所述复合物包括选自由SEQ ID NO:1-26和85-92组成的组的重组多肽，可选地该复合物作为初始剂(primary series)，额外剂(additional dose)，和/或同源或异源加强剂(booster dose)使用，例如第一剂，第二剂，第三剂，第四剂，和/或更多剂，可选地该初始剂，额外剂，或异源加强剂与其它重组亚单位疫苗、纳米颗粒疫苗、mRNA疫苗、DNA疫苗、腺病毒载体疫苗、和灭活病毒疫苗中的任意一种或几种配合使用。在一些实施方案中，本文提供了用于在受试者中产生对冠状病毒表面抗原的免疫应答的方法，其中表面抗原包括S蛋白或其抗原片段，并且方法包括向受试者施用有效量的复合物，所述复合物包括选自由SEQ ID NO:1-26和85-92组成的组的重组多肽，可选地该复合物作为初始剂(primary series)，额外剂(additional dose)，和/或同源或异源加强剂(booster dose)使用，例如第一剂，第二剂，第三剂，第四剂，和/或更多剂，可选地该初始剂，额外剂，或异源加强剂与其它重组亚单位疫苗、纳米颗粒疫苗、mRNA疫苗、DNA疫苗、腺病毒载体疫苗、和灭活病毒疫苗中的任意一种或几种配合使用。在一些实施方案中，本文提供了用于在受试者中产生对冠状病毒表面抗原的免疫应答的方法，其中表面抗原包括选自由SEQ ID NO:27-66和81-84组成的组的序列，并且方法包括向受试者施 用有效量的复合物，所述复合物包括选自由SEQ ID NO:1-26和85-92组成的组的重组多肽，可选地该复合物作为初始剂(primary series)，额外剂(additional dose)，和/或同源或异源加强剂(booster dose)使用，例如第一剂，第二剂，第三剂，第四剂，和/或更多剂，可选地该初始剂，额外剂，或异源加强剂与其它重组亚单位疫苗、纳米颗粒疫苗、mRNA疫苗、DNA疫苗、腺病毒载体疫苗、和灭活病毒疫苗中的任意一种或几种配合使用。在一些实施方案中，本文提供了用于在受试者中产生对冠状病毒表面抗原的免疫应答的方法，其中表面抗原包括冠状病毒的S蛋白或其抗原片段，并且可选地，表面抗原包括选自由SEQ ID NO:27-66和81-84组成的组的序列或其抗原片段，并且方法包括向受试者施用有效量的复合物，所述复合物包括重组多肽，所述重组多肽包括SEQ ID NO:85-92中任一项所述的序列，可选地该复合物作为初始剂(primary series)，额外剂(additional dose)，和/或同源或异源加强剂(booster dose)使用，例如第一剂，第二剂，第三剂，第四剂，和/或更多剂，可选地该初始剂，额外剂，或异源加强剂与其它重组亚单位疫苗、纳米颗粒疫苗、mRNA疫苗、DNA疫苗、腺病毒载体疫苗、和灭活病毒疫苗中的任意一种或几种配合使用。可选地，任意初始剂，额外剂，和/或加强剂中的佐剂可独立地包括：含铝佐剂，例如含明矾和/或氢氧化铝的佐剂；含寡核苷酸的佐剂，例如含CpG寡脱氧核苷酸(CpG-ODN)的佐剂；含TLR9激动剂的佐剂；含可代谢油、α-生育酚、和/或聚氧乙烯山梨醇酐单油酸酯(吐温-80)的佐剂，例如水包油乳液形式的含角鲨烯、α-生育酚、和吐温-80和/或Span 85的佐剂；或任意一种佐剂组合。
在一些实施方案中，本文提供了用于在受试者中产生对冠状病毒表面抗原的免疫应答的方法，其中表面抗原包括S蛋白或其抗原片段，并且方法包括向受试者施用有效量的复合物或复合物中任何两种或更多种的组合，所述复合物包括重组多肽，所述重组多肽包括选自由SEQ ID NO:1-26和85-92组成的组的序列，可选地该复合物或复组合作为初始剂和/或作为加强剂使用，例如第二剂和/或第三剂加强针剂。在一些实施方案中，方法包括向受试者施用有效量的复合物，所述复合物包括重组多肽，所述重组多肽包括SEQ ID NO:85、SEQ ID NO:86、SEQ ID NO:87和/或SEQ ID NO:88中所述的序列。可选地，任意初始剂，额外剂，和/或加强剂中的佐剂可独立地包括：含铝佐剂，例如含明矾和/或氢氧化铝的佐剂；含寡核苷酸的佐剂，例如含CpG寡脱氧核苷酸(CpG-ODN)的佐剂；含TLR9激动剂的佐剂；含可代谢油、α-生育酚、和/或聚氧乙烯山梨醇酐单油酸酯(吐温-80)的佐剂，例如水包油乳液形式的含角鲨烯、α-生育酚、和吐温-80和/或Span 85的佐剂；或任意一种佐剂组合。
可以向受试者施用所公开的免疫原(例如，重组冠状病毒S抗原，例如，本文所述的S-三聚体或S蛋白，编码所公开的重组冠状病毒S抗原的原聚体的核酸分子(例如RNA分子)或载体，或包含所公开的重组冠状病毒S抗原的蛋白质纳米颗粒或病毒样颗粒)，以诱导对受试者中相应冠状病毒S抗原的免疫应答。在特定的示例中，受 试者是人。免疫应答可以是保护性免疫应答，例如抑制随后相应冠状病毒感染的应答。免疫应答的激发也可用于治疗或抑制与相应冠状病毒相关的感染和疾病。
可以选择受试者进行具有或处于感染冠状病毒的风险中的治疗，例如因为暴露于或可能暴露于冠状病毒。在施用所公开的免疫原后，可以监测受试者的感染或与冠状病毒相关的症状，或两者。
拟用本发明的疗法和方法进行治疗的典型受试者包括人类以及非人类灵长类和其它动物。为了鉴定根据本发明的方法进行预防或治疗的受试者，采用可接受的筛查方法来确定与目标或疑似疾病或病症相关的风险因素，或确定受试者中现有疾病或病症的状态。这些筛查方法包括，例如常规检查，以确定可能与目标或疑似疾病或病症相关的环境、家族、职业和其它此类风险因素，以及诊断方法，例如用于检测和/或表征冠状病毒感染的各种ELISA和其它免疫分析方法。这些和其它常规方法允许临床医生使用本发明的方法和药物组合物选择需要治疗的患者。根据这些方法和原则，可以根据本文的教导或其它常规方法施用组合物，作为独立的预防或治疗方案，或作为其它治疗的后续、辅助或协调治疗方案。
施用所公开的免疫原(例如，冠状病毒S抗原，例如，三聚体、蛋白质)可用于预防性或治疗性目的。当预防性地提供时，所公开的治疗剂在任何症状之前提供，例如在感染之前。所公开的治疗剂的预防性施用用于预防或改善任何后续感染。当治疗性地提供时，所公开的治疗剂在疾病或感染症状开始时或之后提供，例如，在与冠状病毒S抗原对应的冠状病毒感染症状发展之后，或在诊断冠状病毒感染之后。因此，可以在预期暴露于冠状病毒之前提供治疗剂，以便在暴露于或怀疑暴露于病毒之后，或在实际开始感染之后，减弱感染和/或相关疾病症状的预期严重程度、持续时间或程度。
本文所述的免疫原及其免疫原性组合物以有效诱导或增强受试者(优选为人类)针对冠状病毒S抗原的免疫应答的量提供给受试者。所公开的免疫原的实际剂量将根据例如受试者的疾病迹象和特定状态(例如，受试者的年龄、大小、健康状况、症状程度、易感因素等)、给药时间和途径、同时给药的其它药物或治疗等因素以及组合物在受试者中激发所需活性或生物反应的特定药理学而变化。剂量方案可以调整，以提供最佳的预防或治疗反应。
包括一种或更多种所公开的免疫原的免疫原性组合物可用于协调(或初免-加强)疫苗接种方案或组合制剂中。在某些实施方案中，新的组合免疫原性组合物和协调免疫方案使用单独的免疫原或制剂，每种免疫原或制剂均旨在引发抗病毒免疫应答，例如对冠状病毒S抗原的免疫应答。引发抗病毒免疫应答的单独免疫原性组合物可以在单个免疫步骤中组合在向受试者施用的多价免疫原性组合物中，或者它们可以在协调(或初免-加强)免疫方案中单独施用(在单价免疫原性组合物中)。
可以有几个加强剂，每个加强剂可以是不同的所公开的免疫原。在一些示例中，加强剂可以是与另一加强剂或初免剂相同的免疫原。初免剂和加强剂可作为单剂 量或多剂量施用，例如，可在几天、几周或几个月内向受试者施用两剂、三剂、四剂、五剂、六剂或更多。也可以进行多次加强剂，例如一到五次(例如，1、2、3、4或5次加强剂)或更多次。不同剂量可用于一系列连续免疫。例如，在初级免疫中使用相对较大的剂量，然后使用相对较小的剂量进行增强。
在一些实施方案中，可在初免剂后约两周、约三到八周或约四周或者初免后约几个月施用加强剂。在一些实施方案中，可在初免剂后约5、约6、约7、约8、约10、约12、约18、约24个月或者初免剂后或多或少时间施用加强剂。也可以在适当的时间点定期使用另外的加强剂，以增强受试者的“免疫记忆”。选择的疫苗参数的适合性，例如制剂、剂量、方案等，可通过从受试者中取出等份血清并在免疫程序期间测定抗体滴度来确定。此外，可监测受试者的临床状况以获得预期效果，例如预防感染或改善疾病状态(例如降低病毒载量)。如果此类监测表明疫苗接种是次优的，则可以使用额外剂量的免疫原性组合物使受试者得到加强，并且可以以预期增强免疫应答的方式改进疫苗接种参数。
在一些实施方案中，初免-加强方法可以包括针对受试者的DNA-引物和蛋白质-加强疫苗接种方案。该方法可以包括两次或更多次施用核酸分子或蛋白质。
对于蛋白质疗法，通常，每个人体剂量将包括1-1000μg的蛋白质，例如从约1μg到约100μg，例如从约1μg到约50μg，例如约1μg、约2μg、约5μg、约10μg、约15μg、约20μg、约25μg、约30μg、约40μg或约50μg。
免疫原性组合物中使用的量基于受试人群(例如，婴儿或老年人)进行选择。通过涉及观察受试者中的抗体滴度和其它反应的标准研究，可以确定特定成分的最佳用量。应理解，所公开的免疫原(例如所公开的重组冠状病毒S抗原，例如免疫原性组合物中的三聚体、蛋白质、病毒载体或核酸分子)的治疗有效量可以包括在通过单剂量给药诱导免疫应答时无效但在多剂量给药时有效的量，例如在初免-加强给药方案中。
在施用本发明所公开的免疫原后，受试者的免疫系统通常通过产生对免疫原中包含的冠状病毒S蛋白肽具有特异性的抗体来对免疫原性组合物作出反应。这种反应意味着向受试者递送了免疫有效剂量。
在一些实施方案中，将在评估有效剂量/免疫方案的情况下确定受试者的抗体反应。在大多数情况下，评估从受试者获得的血清或血浆中的抗体滴度就足够了。关于是否给予再次加强接种和/或改变给予个体的治疗剂的量的决定可以至少部分地基于抗体滴度水平。抗体滴度水平可以基于例如免疫结合分析，该免疫结合分析测量血清中结合抗原(包括例如重组冠状病毒S抗原，例如S-三聚体)的抗体的浓度。
无需完全消除、减少或防止冠状病毒感染，方法即可有效。例如，与在没有免疫原的情况下的冠状病毒感染相比，用一种或更多种所公开的免疫原激发对冠状病毒的免疫应答可以减少或抑制希望量的冠状病毒感染，例如至少10％、至少20％、至少50％、至少60％、至少70％、至少80％、至少90％、至少95％、至少98％、甚至至少100％ (消除或防止可检测到的感染的细胞)。在另外的示例中，可以通过所公开的方法减少或抑制冠状病毒复制。不需要完全消除冠状病毒复制，方法即可有效。例如，与在没有免疫应答的情况下的冠状病毒复制相比，用一种或更多种所公开的免疫原激发免疫应答可以减少希望量的相应冠状病毒复制，例如，至少10％、至少20％、至少50％、至少60％、至少70％、至少80％、至少90％、至少95％、至少98％、甚至至少100％(消除或防止可检测到的冠状病毒复制)。
在一些实施方案中，在施用佐剂的同时向受试者施用所公开的免疫原。在其它实施方案中，在施用佐剂之后并在足够的时间内向受试者施用所公开的免疫原以诱导免疫应答。
核酸给药的一种方法是用质粒DNA直接免疫，例如用哺乳动物表达质粒。通过核酸构建体进行免疫是本领域众所周知的，并且披露于例如美国专利号5,643,578(其描述了通过引入编码所需抗原的DNA以引发细胞介导的或体液应答来免疫脊椎动物的方法)以及美国专利号5,593,972和5,817,637(其描述了将编码抗原的核酸序列可操作地连接到能够表达的调控序列)。美国专利号5,880,103描述了几种将编码免疫原性肽或其它抗原的核酸递送至生物体的方法。方法包括核酸(或它们自己的合成肽)和免疫刺激构建体或ISCOMSTM的脂质体递送，ISCOMSTM是胆固醇和Quil ATM(皂甙)混合后自发形成的30-40nm大小的带负电荷的笼状结构。使用ISCOMSTM作为抗原的递送载体已经在各种感染(包括弓形虫病和EB病毒诱导的肿瘤)的实验模型中产生了保护性免疫(Mowat and Donachie,Immunol.Today 12:383,1991)。已发现低至1μg的封装在ISCOMSTM中的抗原剂量产生I类介导的CTL反应(Takahashi et al.,Nature 344:873,1990，出于所有目的以其全文形式被援引加入)。
在一些实施方案中，质粒DNA疫苗用于在受试者中表达所公开的免疫原。例如，可将编码所公开的免疫原的核酸分子施用给受试者以诱导对冠状病毒S抗原的免疫应答。在一些实施方案中，核酸分子可以包括在用于DNA免疫的质粒载体上，例如pVRC8400载体(如Barouch et al.,J.Virol,79,8828-8834,2005中所述，出于所有目的以其全文形式被援引加入)。
在使用核酸进行免疫的另一种方法中，所公开的重组冠状病毒S抗原(例如三聚体、蛋白质)可以通过减毒的病毒宿主或载体或细菌载体来表达。重组牛痘病毒、腺病毒相关病毒(AAV)、疱疹病毒、逆转录病毒、巨细胞病毒(cytogmeglo virus)或其它病毒载体可用于表达肽或蛋白质，从而引发CTL反应。例如，美国专利号4,722,848中描述了在免疫规划中有用的牛痘载体和方法，出于所有目的以其全文形式被援引加入。卡介苗(Bacillus Calmette Guerin)提供了另一种用于表达肽的载体(参见Stover,Nature 351:456-460,1991，出于所有目的以其全文形式被援引加入)。
在一个实施方案中，将编码所公开的重组冠状病毒S抗原的核酸直接引入细胞中。例如，可以通过标准方法将核酸装载到金微球上，并通过诸如Bio-Rad的HELIOSTM基因枪等设备将其引入皮肤中。核酸可以是“裸露的”，由强启动子控制下的 质粒组成。通常，DNA被注射到肌肉中，但也可以直接注射到其它部位。注射剂量通常约为0.5μg/kg至约50mg/kg，并且典型地约为0.005mg/kg至约5mg/kg(参见，例如，美国专利号5,589,466)。
在另一实施方案中，基于mRNA的免疫方案可用于将编码所公开的重组冠状病毒S抗原的核酸直接递送到细胞中。在一些实施方案中，基于mRNA的核酸疫苗可提供对前述方法的有效替代。mRNA疫苗排除了DNA整合到宿主基因组的安全性问题，并且可以直接在宿主细胞细胞质中翻译。此外，RNA的简单无细胞的体外合成避免了与病毒载体相关的制造并发症。可用于递送编码所公开的重组冠状病毒S抗原的核酸的两种基于RNA的疫苗的示例性形式包括常规非扩增mRNA免疫(参见，例如，Petsch et al.,“Protective efficacy of in vitro synthesized,specific mRNA vaccines against influenza A virus infection,”Nature biotechnology,30(12):1210-6,2012)和自扩增mRNA免疫(参见，例如，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；和Brito et al.,“Self-amplifying mRNA vaccines,”Adv Genet.,89:179-233,2015)。本段所有文献出于所有目的以其全文形式被援引加入。
在一些实施方案中，向受试者施用治疗有效量的一种或更多种所公开的免疫原可诱导受试者中的中和免疫应答。为评估中和活性，在受试者免疫后，可在适当的时间点从受试者收集血清，冷冻并储存以进行中和试验。中和活性的测定方法是本领域普通技术人员已知的，并在此进一步描述，包括但不限于蚀斑减少中和(PRNT)测定、微量中和测定、基于流式细胞术的测定、单周期感染测定。在一些实施方案中，可使用一组冠状病毒假病毒来测定血清中和活性。
在一些实施方案中，向受试者施用治疗有效量的一种或更多种所公开的免疫原可诱导受试者中的中和免疫应答。为评估中和活性，在受试者免疫后，可在适当的时间点从受试者收集血清，冷冻并储存以进行中和试验。中和活性的测定方法是本领域普通技术人员已知的，并在此进一步描述，包括但不限于蚀斑减少中和(PRNT)测定、微量中和测定、基于流式细胞术的测定、单周期感染测定。在一些实施方案中，可使用一组冠状病毒假病毒来测定血清中和活性。
在一些实施方案中，由本文公开的免疫原诱导的中和免疫应答产生针对冠状病毒(例如SARS-CoV-2)的中和抗体。在一些实施方案中，本文的中和抗体结合到冠状病毒(例如SARS-CoV-2)或其组分的细胞受体或共同受体。在一些实施方案中，病毒受体或共同受体是冠状病毒受体或共同受体，优选是肺炎病毒受体或共同受体，更优选是人冠状病毒受体，例如SARS-CoV-2受体或共同受体。在一些实施方案中，本文的中和抗体在体外、在原位和/或在体内调节、减少、拮抗、减轻、阻断、抑制、消除和/ 或干扰至少一种冠状病毒(例如SARS-CoV-2)活性或结合，或冠状病毒(例如SARS-CoV-2)受体活性或结合，例如SARS-CoV-2释放、SARS-CoV-2受体信号传导、膜SARS-CoV-2裂解、SARS-CoV-2活性、SARS-CoV-2产生和/或合成。在一些实施方案中，本文公开的免疫原诱导针对SARS-CoV-2的中和抗体，其调节、减少、拮抗、减轻、阻断、抑制、消除和/或干扰SARS-CoV-2与SARS-CoV-2受体或共同受体的结合，例如血管紧张素转换酶2(ACE2)、二肽基肽酶4(DPP4)、树突状细胞特异性细胞间粘附分子-3-抓取非整合素(DC-SIGN)和/或肝脏/淋巴结-SIGN(L-SIGN)。
V.制品或试剂盒
还提供了含有所提供的重组多肽、蛋白质和免疫原性组合物的制品或试剂盒。制品可以包括容器以及容器上或与容器相关的标签或包装说明书。合适的容器包括，例如瓶子、小瓶、注射器、试管、IV溶液袋等。容器可以由各种材料(例如玻璃或塑料)形成。在一些实施方案中，容器具有无菌进入口。示例性容器包括静脉注射溶液袋、小瓶，包括带有可被注射用针刺穿的塞子的容器。制品或试剂盒可以进一步包括包装说明书，其指示组合物可用于治疗特定病症，例如本文所述的病症(例如冠状病毒感染)。或者，或者另外地，制品或试剂盒可以进一步包括包含药学上可接受的缓冲液的另一或相同容器。它可以进一步包括其它材料，例如其它缓冲液、稀释剂、过滤器、针头和/或注射器。
标签或包装说明书可以表明组合物用于治疗个体的冠状病毒感染。容器上或与容器相关的标签或包装说明书可以表明制剂的重组和/或使用说明。标签或包装说明书可以进一步表明制剂用于或目的在于皮下、静脉或其它给药方式，以治疗或预防个体中的冠状病毒感染。
在一些实施方案中，容器容纳组合物，该组合物是单独地或与另一种有效治疗、预防和/或诊断病症的组合物组合。制品或试剂盒可以包括(a)第一容器，具有包含在其中的组合物(即，第一药剂)，其中组合物包括免疫原性组合物或其蛋白质或重组多肽；和(b)第二容器，具有包含在其中的组合物(即，第二药剂)，其中组合物包括另一种药剂，例如佐剂或其它治疗剂，并且该物品或试剂盒进一步包括标签或包装说明书上用第二药剂以有效量治疗受试者的说明。
术语
除非另有定义，否则本文中使用的所有专用术语、符号和其它技术和科学术语(term)或术语(terminology)的含义旨在与所要求保护的主题所属领域的普通技术人员通常理解的含义相同。在一些情况下，为了清楚和/或便于参考，本文定义了具有通常理解的含义的术语，并且本文中包含的此类定义不一定表示与本领域中通常理解的内容的实质性差异。
术语“多肽”和“蛋白质”可互换地用于指代氨基酸残基的聚合物，且不限于最小长度。多肽(包括所提供的受体和其它多肽，例如接头或肽)可包括氨基酸残基，包括天然和/或非天然氨基酸残基。术语还包括多肽的表达后修饰，例如糖基化、唾液酸化、乙酰化和磷酸化。在一些方面，只要蛋白质保持所需的活性，多肽可包含关于天然(native)或天然(natural)序列的修饰。这些修饰可以是故意的，如通过定点突变，也可以是偶然的，如通过产生蛋白质的宿主的突变或由于PCR扩增产生的错误。
如本文所使用的，“受试者”是哺乳动物，例如人类或其它动物，并且通常是人类。在一些实施方案中，向其施用一种或多种药剂、细胞、细胞群或组合物的受试者(例如患者)是哺乳动物，通常是灵长类动物，例如人类。在一些实施方案中，灵长类动物是猴子或猿。受试者可以是男性或女性，并且可以是任何合适的年龄段，包括婴儿、少年、青少年、成人和老年受试者。在一些实施方案中，受试者是非灵长类哺乳动物，例如啮齿动物。
如本文所使用的，“治疗(treatment)”(及其语法变体，如“治疗(treat)”或“治疗(treating)”)是指完全或部分改善或减少疾病、病症或紊乱，或与之相关的症状、不良反应或结果、或表型。治疗的理想效果包括但不限于预防疾病的发生或复发、缓解症状、减轻疾病的任何直接或间接病理后果、预防转移、降低疾病进展速度、改善或缓解疾病状态以及缓解或改善预后。该术语并不意味着完全治愈疾病或完全消除任何症状或对所有症状或结果的影响。
如本文所使用的，“延缓疾病的发展”是指延缓、阻碍、减缓、减慢、稳定、抑制和/或延迟疾病(例如癌症)的发展。取决于疾病史和/或接受治疗的个体，延缓的时间长度可能不同。在一些实施方案中，充分或显著的延缓实际上可以包含预防，因为个体不会发生疾病。例如，晚期癌症，如转移的发展，可能会延缓。
如本文所使用的，“预防”包括针对可能易患疾病但尚未被诊断患有疾病的受试者的疾病发生或复发提供预防。在一些实施方案中，所提供的细胞和组合物用于延缓疾病的发展或减缓疾病的进展。
如本文所使用的，“抑制”功能或活性是指当与除感兴趣的条件或参数之外的其它相同条件相比或者与另一条件相比时，降低功能或活性。例如，与没有细胞时的肿瘤生长速度相比，抑制肿瘤生长的细胞会降低肿瘤的生长速度。
在给药的情况下，药剂(例如药物制剂、细胞或组合物)的“有效量”是指达到预期效果(例如治疗或预防效果)所需的剂量/量和时间段的有效量。
药剂(例如药物制剂或细胞)的“治疗有效量”是指达到预期治疗效果(例如用于治疗疾病、病症或紊乱)和/或治疗的药代动力学或药效学效果所需的剂量和时间段的有效量。治疗有效量可根据例如受试者的疾病状态、年龄、性别和体重以及施用的细胞群等因素而变化。在一些实施方案中，所提供的方法涉及以有效量(例如，治疗有效量)施用细胞和/或组合物。
“预防有效量”是指达到预期预防效果所需的剂量和时间段的有效量。通常但不一定的是，因为预防剂量用于疾病之前或早期阶段的受试者，因此预防有效量将小于治疗有效量。在肿瘤负担较低的情况下，一些方面的预防有效量将高于治疗有效量。疫苗或其它药剂的有效量，足以产生期望的反应，如减少或消除病症或疾病的病征或症状，如肺炎。例如，这可能是抑制病毒复制或可测定地改变病毒感染的外部症状所必需的量。一般来说，这一数量将足以可测定地抑制病毒(例如，SARS-CoV-2)复制或传染性。当向受试者给药时，通常使用的剂量将达到目标组织浓度，该浓度已被证明能够在体外实现病毒复制的抑制。在一些实施方案中，“有效量”是治疗(包括预防)任何病症或疾病的一个或更多个症状和/或潜在原因的量，例如用于治疗冠状病毒感染。在一些实施方案中，有效量是治疗有效量。在一些实施方案中，有效量是防止特定疾病或病症的一个或更多个病征或症状发展的量，例如与冠状病毒感染相关的一个或更多个病征或症状。
如本文所使用的，术语“抗原”或“免疫原”可互换地用于指代能够在受试者中诱导免疫应答的物质，通常为蛋白质。该术语还指免疫活性蛋白质，即一旦施用给受试者(直接或通过向受试者施用编码蛋白质的核苷酸序列或载体)，能够引发针对该蛋白质的体液和/或细胞类型的免疫应答。除非另有说明，否则术语“疫苗免疫原”可与“蛋白质抗原”或“免疫原多肽”互换使用。
术语“保守修饰的变体”适用于氨基酸和核酸序列。关于特定核酸序列，保守修饰的变体是指编码相同或基本相同的氨基酸序列的那些核酸，或在核酸不编码氨基酸序列的情况下，指基本相同的序列。由于遗传密码的简并性，大量功能相同的核酸编码任何给定的蛋白质。对于多肽序列，“保守修饰的变体”是指具有保守氨基酸取代的变体，氨基酸残基被具有有着类似电荷的侧链的其它氨基酸残基替换。本领域已经定义了具有有着类似电荷的侧链的氨基酸残基家族。这些家族包括具有碱性侧链(例如，赖氨酸、精氨酸、组氨酸)、酸性侧链(例如，天冬氨酸、谷氨酸)、不带电荷的极性侧链(例如，甘氨酸、天冬酰胺、谷氨酰胺、丝氨酸、苏氨酸、酪氨酸、半胱氨酸)、非极性侧链(例如，丙氨酸、缬氨酸、亮氨酸、异亮氨酸、脯氨酸、苯丙氨酸、甲硫氨酸、色氨酸)、β支链(例如，苏氨酸、缬氨酸、异亮氨酸)和芳香族侧链(例如，酪氨酸、苯丙氨酸、色氨酸、组氨酸)。
表位是指抗原决定簇。这些是抗原分子上的特定化学基团或肽序列，因此它们引发特异性免疫应答，例如，表位是B细胞和/或T细胞应答的抗原区域。表位既可以由连续氨基酸形成，也可以由通过蛋白质的三级折叠而并列的非连续氨基酸形成。
除非另有说明，否则融合蛋白是重组蛋白，含有至少两种不相关蛋白质的氨基酸序列，这些蛋白质通过肽键连接在一起形成单个蛋白质。因此，它不包含天然存在的冠状病毒表面抗原，即本文所述的融合(F)蛋白。不相关的氨基酸序列可以直接相互连接，或者它们可以使用接头序列连接。如本文所使用的，如果蛋白质的氨基酸序列在其自然环境中(例如，在细胞内)通常不通过肽键连接在一起，则蛋白质是不相关 的。例如，病毒抗原的氨基酸序列和胶原或前胶原的氨基酸序列通常不会通过肽键连接在一起。
免疫原是蛋白质或其一部分，能够在哺乳动物中诱导免疫应答，例如被病原体感染或有病原体感染风险的哺乳动物。免疫原的施用可导致针对目标病原体的保护性免疫和/或主动免疫。
免疫原性组合物是指包含免疫原性多肽的组合物，该免疫原性多肽诱导针对表达免疫原性多肽的病毒的可测量的CTL反应，或诱导针对免疫原性多肽的可测量的B细胞应答(例如产生抗体)。
两个或更多个核酸序列或两个或更多个氨基酸序列之间的序列同一性或相似性按照序列之间的同一性或相似性表示。序列一致性可以按照百分比一致性来衡量；百分比越高，序列越相同。当通过比较窗口或使用以下序列比较算法之一或通过手动比对和目视检查所测量的指定区域进行比较和比对以获得最大对应时，如果两个序列具有指定百分比的相同的氨基酸残基或核苷酸，则两个序列是“基本相同的”(即，在指定区域上，或者在未指定的情况下在整个序列上，60％同一性，优选65％、70％、75％、80％、85％、90％、95％或99％同一性)。可选地，同一性存在于长度至少约为50个核苷酸(或10个氨基酸)的区域上，或更优选地存在于长度为100至500或1000或更多个核苷酸(或20、50、200或更多个氨基酸)的区域上。
疫苗是指在受试者中引发预防性或治疗性免疫应答的药物组合物。在一些情况下，免疫应答是保护性免疫应答。通常，疫苗针对病原体的抗原(例如病毒病原体)或与病理状况相关的细胞成分诱发抗原特异性的免疫应答。疫苗可包括多核苷酸(例如编码所公开的抗原的核酸)、肽或多肽(例如所公开的抗原)、病毒、细胞或一种或更多种细胞成分。在一些实施方案中，疫苗或疫苗免疫原或疫苗组合物从融合构建体表达并自组装成在表面上显示免疫原多肽或蛋白质的纳米颗粒。
类病毒颗粒(VLP)是指不可复制的病毒外壳，来源于几种病毒中的任何一种。VLP通常由一种或更多种病毒蛋白质组成，例如但不限于称为衣壳、包被、外壳、表面和/或包膜蛋白的蛋白质，或来源于这些蛋白质的颗粒形成多肽。在适当的表达系统中重组表达蛋白质后，VLP可以自发形成。用于生产特定VLP的方法是本领域已知的。在病毒蛋白质的重组表达之后的VLP的存在可以使用本领域已知的常规技术进行检测，例如通过电子显微镜、生物物理表征等。参见例如Baker et al.(1991)Biophys.J.60:1445-1456；和Hagensee et al.(1994)J.Virol.68:4503-4505，出于所有目的以其全文形式被援引加入。例如，VLP可以通过密度梯度离心进行分离和/或通过特征密度带进行鉴定。或者，低温电子显微镜可以在讨论中的VLP制备的玻璃化含水样品上进行，并在适当的曝光条件下记录图像。
本文使用的术语“大约/约”是指本技术领域的技术人员容易知道的各个值的通常误差范围。本文中对“大约/约”值或参数的提及包括(并描述)针对该值或参数本身的实施方案。
如本文所使用的，单数形式“一个/一种(a)”、“一个/一种(an)”和“所述/该(the)”包括复数指代，除非上下文另有明确规定。例如，“一个/一种(a)”或“一个/一种(an)”表示“至少一个/一种”或“一个或更多个/一种或更多种”。
在本文中，所要求保护的主题的各个方面以范围格式呈现。应当理解，范围格式的描述仅仅是为了方便和简洁，不应当被解释为对所要求保护的主题的范围的不灵活的限制。因此，应当认为范围的描述已经具体地公开了所有可能的子范围以及该范围内的各个数值。例如，在提供值的范围的情况下，应当理解，在该范围的上限和下限之间与该范围内的任何其它陈述或介入值的每个介入值都包含在所要求保护的主题内。这些较小范围的上限和下限可以独立地包括在较小范围内，并且也包括在所要求保护的主题内，受制于所述范围内的任何明确排除的限制。如果所述范围包括一个或两个限制，则不包括其中一个或两个那些包括在内的限制的范围也包括在所要求保护的主题内。这适用于任何范围的宽度。
如本文所使用的，组合物指两种或更多种产品、物质或化合物(包括细胞)的任何混合物。它可以是溶液、悬浮液、液体、粉末、糊状物、水性的、非水性的或其任何组合。
本文使用的术语“载体”是指能够传播与其连接的另一种核酸的核酸分子。该术语包括作为自我复制核酸结构的载体以及并入已引入载体的宿主细胞基因组中的载体。某些载体能够引导与它们可操作地连接的核酸的表达。此类载体在本文中称为“表达载体”。
示例性实施方案I
实施方案1.一种包含多个重组多肽的蛋白质，每个重组多肽包含连接到胶原的C-末端前肽的冠状病毒SARS-CoV-2奥密克戎(B.1.1.529)表面抗原，其中重组多肽的C-末端前肽形成多肽间二硫键。
实施方案2.根据实施方案1的蛋白质，其中冠状病毒感染为SARS-冠状病毒2(SARS-CoV-2)感染。
实施方案3.根据实施方案1或2的蛋白质，其中表面抗原包括冠状病毒刺突(S)蛋白或其片段或表位，其中表位可选地为线性表位或构象表位，并且其中蛋白质包括三个重组多肽。
实施方案4.根据实施方案3的蛋白质，其中表面抗原包括信号肽、S1亚单位肽、S2亚单位肽或其任何组合。
实施方案5.根据实施方案3的蛋白质，其中表面抗原包括信号肽、受体结合结构域(RBD)肽、受体结合基序(RBM)肽、融合肽(FP)、七肽重复序列1(HR1)肽或七肽重复序列2(HR2)肽或其任何组合。
实施方案6.根据实施方案3-5中任一项的蛋白质，其中表面抗原包括S蛋白的受体结合结构域(RBD)。
实施方案7.根据实施方案3-6中任一项的蛋白质，其中表面抗原包括S蛋白的S1亚单位和S2亚单位。
实施方案8.根据实施方案3-7中任一项的蛋白质，其中表面抗原不包括跨膜(TM)结构域肽和/或细胞质(CP)结构域肽。
实施方案9.根据实施方案3-8中任一项的蛋白质，其中表面抗原包括蛋白酶裂解位点，其中蛋白酶可选地为弗林蛋白酶(furin)、胰蛋白酶、因子Xa、凝血酶或组织蛋白酶L。
实施方案10.根据实施方案3-8中任一项的蛋白质，其中表面抗原不包括蛋白酶裂解位点，其中蛋白酶可选地为弗林蛋白酶(furin)、胰蛋白酶、因子Xa、凝血酶或组织蛋白酶L。
实施方案11.根据实施方案1-10中任一项的蛋白质，其中表面抗原是可溶的或不直接结合到脂质双层，例如膜或病毒包膜。
实施方案12.根据实施方案1-11中任一项的蛋白质，其中在蛋白质的重组多肽中表面抗原相同或不同。
实施方案13.根据实施方案1-12中任一项的蛋白质，其中表面抗原直接融合到C-末端前肽，或通过接头(例如包含甘氨酸-X-Y重复序列的接头)连接到C-末端前肽，其中X和Y独立地是任何氨基酸，并且可选地是脯氨酸或羟脯氨酸。
实施方案14.根据实施方案1-13中任一项的蛋白质，其是可溶的或不直接结合到脂质双层，例如膜或病毒包膜。
实施方案15.根据实施方案1-14中任一项的蛋白质，其中蛋白质能够结合到受试者的细胞表面受体，可选地其中受试者是哺乳动物，例如灵长类动物，例如人类。
实施方案16.根据实施方案15的蛋白质，其中细胞表面受体为血管紧张素转换酶2(ACE2)、二肽基肽酶4(DPP4)、树突状细胞特异性细胞间粘附分子-3-抓取非整合素(DC-SIGN)或肝脏/淋巴结-SIGN(L-SIGN)。
实施方案17.根据实施方案1-16中任一项的蛋白质，其中C-末端前肽是人胶原。
实施方案18.根据实施方案1-17中任一项的蛋白质，其中C-末端前肽包括proα1(I)、proα1(II)、proα1(III)、proα1(V)、proα1(XI)、proα2(I)、proα2(V)、proα2(XI)或proα3(XI)的C-末端多肽或其片段。
实施方案19.根据实施方案1-18中任一项的蛋白质，其中在重组多肽中C-末端前肽相同或不同。
实施方案20.根据实施方案1-19中任一项的蛋白质，其中C-末端前肽包括SEQ ID NO:67-80中的任一个或与其至少90％相同的氨基酸序列，其能够形成多肽间二硫键并使重组多肽三聚化。
实施方案21.根据实施方案1-20中任一项的蛋白质，其中C-末端前肽包括SEQ ID NO:67或与其至少90％相同的氨基酸序列，其能够形成多肽间二硫键并使重组多肽三聚化。
实施方案22.根据实施方案1-20中任一项的蛋白质，其中C-末端前肽包括SEQ ID NO:68或与其至少90％相同的氨基酸序列，其能够形成多肽间二硫键并使重组多肽三聚化。
实施方案23.根据实施方案1-20中任一项的蛋白质，其中C-末端前肽包括SEQ ID NO:69或与其至少90％相同的氨基酸序列，其能够形成多肽间二硫键并使重组多肽三聚化。
实施方案24.根据实施方案1-20中任一项的蛋白质，其中C-末端前肽包括SEQ ID NO:70或与其至少90％相同的氨基酸序列，其能够形成多肽间二硫键并使重组多肽三聚化。
实施方案25.根据实施方案1-20中任一项的蛋白质，其中C-末端前肽包括SEQ ID NO:71或与其至少90％相同的氨基酸序列，其能够形成多肽间二硫键并使重组多肽三聚化。
实施方案26.根据实施方案1-20中任一项的蛋白质，其中C-末端前肽包括SEQ ID NO:72或与其至少90％相同的氨基酸序列，其能够形成多肽间二硫键并使重组多肽三聚化。
实施方案27.根据实施方案1-20中任一项的蛋白质，其中C-末端前肽包括SEQ ID NO:73或与其至少90％相同的氨基酸序列，其能够形成多肽间二硫键并使重组多肽三聚化。
实施方案28.根据实施方案1-20中任一项的蛋白质，其中C-末端前肽包括SEQ ID NO:74或与其至少90％相同的氨基酸序列，其能够形成多肽间二硫键并使重组多肽三聚化。
实施方案29.根据实施方案1-20中任一项的蛋白质，其中C-末端前肽包括SEQ ID NO:75或SEQ ID NO:76或与其至少90％相同的氨基酸序列，其能够形成多肽间二硫键并使重组多肽三聚化。
实施方案30.根据实施方案1-29中任一项的蛋白质，其中C-端前肽包括包含连接到SEQ ID NO:67-80中任一种的N-末端的甘氨酸-X-Y重复序列的序列，其中X和Y独立地是任何氨基酸，并且可选地是脯氨酸或羟脯氨酸，或与其至少90％相同的氨基酸序列，其能够形成多肽间二硫键并使重组多肽三聚化。
实施方案31.根据实施方案1-30中任一项的蛋白质，其中每个重组多肽中的表面抗原为融合前构象或融合后构象。
实施方案32.根据实施方案1-31中任一项的蛋白质，其中每个重组多肽中的表面抗原包括SEQ ID NO:27-66和81-84中的任一个或与其至少80％相同的氨基酸序列。
实施方案33.根据实施方案1-32中任一项的蛋白质，其中重组多肽包括SEQ ID NO:1-26和85-92中的任一个或与其至少80％相同的氨基酸序列。
实施方案34.一种包含实施方案1-33中任一项的蛋白质的免疫原，可选地该免疫原作为疫苗初始剂(primary series)，额外剂(additional dose)，和/或同源或异源加强剂(booster dose)使用，例如第一剂，第二剂，第三剂，第四剂，和/或更多剂，可选地该初始剂，额外剂，或异源加强剂与其它重组亚单位疫苗、纳米颗粒疫苗、mRNA疫苗、DNA疫苗、腺病毒载体疫苗、和灭活病毒疫苗中的任意一种或几种配合使用，可选地该初始剂，额外剂，和/或同源或异源加强剂中的佐剂可独立地包括：含铝佐剂，例如含明矾和/或氢氧化铝的佐剂；含寡核苷酸的佐剂，例如含CpG寡脱氧核苷酸(CpG-ODN)的佐剂；含TLR9激动剂的佐剂；含可代谢油、α-生育酚、和/或聚氧乙烯山梨醇酐单油酸酯(吐温-80)的佐剂，例如水包油乳液形式的含角鲨烯、α-生育酚、和吐温-80和/或Span 85的佐剂；或任意一种所述佐剂的组合。
实施方案35.一种包含实施方案1-33中任一项的蛋白质的蛋白质纳米颗粒，所述蛋白质直接或间接连接到纳米颗粒，可选地该纳米颗粒作为疫苗初始剂(primary series)，额外剂(additional dose)，和/或同源或异源加强剂(booster dose)使用，例如第一剂，第二剂，第三剂，第四剂，和/或更多剂，可选地该初始剂，额外剂，或异源加强剂与其它重组亚单位疫苗、纳米颗粒疫苗、mRNA疫苗、DNA疫苗、腺病毒载体疫苗、和灭活病毒疫苗中的任意一种或几种配合使用，可选地该初始剂，额外剂，和/或同源或异源加强剂中的佐剂可独立地包括：含铝佐剂，例如含明矾和/或氢氧化铝的佐剂；含寡核苷酸的佐剂，例如含CpG寡脱氧核苷酸(CpG-ODN)的佐剂；含TLR9激动剂的佐剂；含可代谢油、α-生育酚、和/或聚氧乙烯山梨醇酐单油酸酯(吐温-80)的佐剂，例如水包油乳液形式的含角鲨烯、α-生育酚、和吐温-80和/或Span 85的佐剂；或任意一种所述佐剂的组合。
实施方案36.一种包含实施方案1-33中任一项的蛋白质的病毒样颗粒(VLP)，可选地该病毒样颗粒作为疫苗初始剂(primary series)，额外剂(additional dose)，和/或同源或异源加强剂(booster dose)使用，例如第一剂，第二剂，第三剂，第四剂，和/或更多剂，可选地该初始剂，额外剂，或异源加强剂与其它重组亚单位疫苗、纳米颗粒疫苗、mRNA疫苗、DNA疫苗、腺病毒载体疫苗、和灭活病毒疫苗中的任意一种或几种配合使用，可选地该初始剂，额外剂，和/或同源或异源加强剂中的佐剂可独立地包括：含铝佐剂，例如含明矾和/或氢氧化铝的佐剂；含寡核苷酸的佐剂，例如含CpG寡脱氧核苷酸(CpG-ODN)的佐剂；含TLR9激动剂的佐剂；含可代谢油、α-生育酚、和/或聚氧乙烯山梨醇酐单油酸酯(吐温-80)的佐剂，例如水包油乳液形式的含角鲨烯、α-生育酚、和吐温-80和/或Span 85的佐剂；或任意一种所述佐剂的组合。
实施方案37.一种分离核酸，其编码根据实施方案1-33中任一项的蛋白质的重组多肽中的一个、两个、三个或更多个。
实施方案38.根据实施方案37的分离核酸，其中编码表面抗原的多肽在框架内融合到编码胶原的C-末端前肽的多肽。
实施方案39.根据实施方案37或38的分离核酸，其可操作地连接到启动子。
实施方案40.根据实施方案37-39中任一项的分离核酸，其是DNA分子。
实施方案41.根据实施方案37-39中任一项的分离核酸，其为RNA分子，可选地为mRNA分子，例如核苷修饰的mRNA、非扩增mRNA、自扩增mRNA或反式扩增mRNA。
实施方案42.一种包含根据实施方案37-41中任一项的分离核酸的载体。
实施方案43.根据实施方案42的载体，其为病毒载体。
实施方案44.一种包含根据实施方案42或43的载体的病毒、假病毒或细胞，可选地其中病毒或细胞具有重组基因组。
实施方案45.一种免疫原性组合物，其包含根据实施方案1-44中任一项的蛋白质、免疫原、蛋白质纳米颗粒、VLP、分离核酸、载体、病毒、假病毒或细胞以及药学上可接受的载体。
实施方案46.一种疫苗，其包含根据实施方案45的免疫原性组合物和可选的佐剂，其中疫苗可选地为亚单位疫苗，和/或可选地其中疫苗为预防性和/或治疗性疫苗，可选地该疫苗作为初始剂(primary series)，额外剂(additional dose)，和/或同源或异源加强剂(booster dose)使用，例如第一剂，第二剂，第三剂，第四剂，和/或更多剂，可选地该初始剂，额外剂，或异源加强剂与其它重组亚单位疫苗、纳米颗粒疫苗、mRNA疫苗、DNA疫苗、腺病毒载体疫苗、和灭活病毒疫苗中的任意一种或几种配合使用，可选地该初始剂，额外剂，和/或同源或异源加强剂中的佐剂可独立地包括：含铝佐剂，例如含明矾和/或氢氧化铝的佐剂；含寡核苷酸的佐剂，例如含CpG寡脱氧核苷酸(CpG-ODN)的佐剂；含TLR9激动剂的佐剂；含可代谢油、α-生育酚、和/或聚氧乙烯山梨醇酐单油酸酯(吐温-80)的佐剂，例如水包油乳液形式的含角鲨烯、α-生育酚、和吐温-80和/或Span 85的佐剂；或任意一种所述佐剂的组合。
实施方案47.根据实施方案46的疫苗，其中疫苗包括多种不同佐剂。
实施方案48.一种产生蛋白质的方法，包括：在宿主细胞中表达根据实施方案37-43中任一项的分离核酸或载体，以产生根据实施方案1-33中任一项的蛋白质；和纯化蛋白质。
实施方案49.通过实施方案48的方法产生的蛋白质。
实施方案50.一种用于在受试者中产生对冠状病毒表面抗原的免疫应答的方法，包括向受试者施用有效量的实施方案1-47和49中任一项的蛋白质、免疫原、蛋白质纳米颗粒、VLP、分离核酸、载体、病毒、假病毒、细胞、免疫原性组合物或疫苗，以产生免疫应答。
实施方案51.根据实施方案50的方法，用于治疗或预防冠状病毒感染。
实施方案52.根据实施方案50或51的方法，其中产生免疫应答抑制或减少受试者中的冠状病毒复制。
实施方案53.根据实施方案50-52中任一项的方法，其中免疫应答包括细胞介导的应答和/或体液应答，可选地包括产生一种或更多种中和抗体，例如多克隆抗体或单克隆抗体。
实施方案54.根据实施方案50-53中任一项的方法，其中免疫应答针对冠状病毒的表面抗原，但不针对C-末端前肽。
实施方案55.根据实施方案50-54中任一项的方法，其中施用不会由于先前暴露于一种或更多种冠状病毒而导致受试者中的抗体依赖性增强(ADE)。
实施方案56.根据实施方案50-55中任一项的方法，其中当随后暴露于一种或更多种冠状病毒时，施用不会导致受试者中的抗体依赖性增强(ADE)。
实施方案57.根据实施方案50-56中任一项的方法，进一步包括初免步骤和/或加强步骤。
实施方案58.根据实施方案50-57中任一项的方法，其中通过局部、经皮、皮下、皮内、口服、鼻内(例如，鼻内喷雾剂)、气管内、舌下、口腔、直肠、阴道、吸入、静脉内(例如，静脉注射)、动脉内、肌肉内(例如，肌内注射)、心内、骨内、腹腔内、跨粘膜、玻璃体内、视网膜下、关节内、关节周围、局部或应用皮肤(epicutaneous)给药来进行给药步骤。
实施方案59.根据实施方案50-58中任一项的方法，其中以单剂量或具有一个间隔或更多个间隔的一系列剂量施用有效量。
实施方案60.根据实施方案50-59中任一项的方法，其中在没有佐剂的情况下施用有效量。
实施方案61.根据实施方案50-59中任一项的方法，其中有效量与佐剂或多种佐剂一起施用。
实施方案62.一种方法，包括向受试者施用有效量的根据实施方案1-33中任一项的蛋白质，以在受试者中产生针对冠状病毒的中和抗体或中和抗血清。
实施方案63.根据实施方案62的方法，其中受试者为哺乳动物，可选地为人类或非人类灵长类。
实施方案64.根据实施方案62或63的方法，进一步包括从受试者分离中和抗体或中和抗血清。
实施方案65.根据实施方案64的方法，进一步包括通过被动免疫向人类受试者施用有效量的分离的中和抗体或中和抗血清，以预防或治疗冠状病毒感染。
实施方案66.根据实施方案62-65中任一项的方法，其中中和抗体或中和抗血清包括针对冠状病毒表面抗原的多克隆抗体，可选地其中中和抗体或中和抗血清不含或基本不含针对胶原的C-末端前肽的抗体。
实施方案67.根据实施方案62-65中任一项的方法，其中中和抗体包括针对冠状病毒表面抗原的单克隆抗体，可选地其中中和抗体不含或基本不含针对胶原的C-末端前肽的抗体。
实施方案68.根据实施方案1-47和49中任一项的蛋白质、免疫原、蛋白质纳米颗粒、VLP、分离核酸、载体、病毒、假病毒、细胞、免疫原性组合物或疫苗，用于诱导受试者中对冠状病毒的免疫应答，和/或治疗或预防冠状病毒感染。
实施方案69.根据实施方案1-47和49中任一项的蛋白质、免疫原、蛋白质纳米颗粒、VLP、分离核酸、载体、病毒、假病毒、细胞、免疫原性组合物或疫苗的用途，用于诱导受试者中对冠状病毒的免疫应答，和/或用于治疗或预防冠状病毒感染。
实施方案70.根据实施方案1-47和49中任一项的蛋白质、免疫原、蛋白质纳米颗粒、VLP、分离核酸、载体、病毒、假病毒、细胞、免疫原性组合物或疫苗的用途，用于制造用于诱导受试者中对冠状病毒的免疫应答的药物或预防剂，和/或用于治疗或预防冠状病毒感染。
实施方案71.一种用于分析样品的方法，包括：将样品与实施方案1-33中任一项的蛋白质接触，并检测蛋白质与能够特异性结合到冠状病毒表面抗原的分析物之间的结合。
实施方案72.根据实施方案71的方法，其中分析物为识别表面抗原的抗体、受体或细胞。
实施方案73.根据实施方案71或72的方法，其中结合表明样品中存在分析物，和/或样品来源于的受试者中存在冠状病毒感染。
实施方案74.一种试剂盒，包括实施方案1-33中任一项的蛋白质和含有或固定蛋白质的底物、板或小瓶，可选地其中试剂盒是ELISA或侧向流动检测试剂盒(lateral flow assay kit)。
示例性实施方案II
实施方案1一种用于预防哺乳动物中冠状病毒感染的方法，该方法包括用有效量的重组亚单位疫苗免疫哺乳动物，该重组亚单位疫苗包括可溶性冠状病毒SARS-CoV-2奥密克戎(B.1.1.529)表面抗原或其片段、变体或突变体，该可溶性冠状病毒表面抗原或其片段、变体或突变体通过框内融合连接到胶原的C-末端部分以形成二硫键连接的三聚体融合蛋白。
实施方案2.根据实施方案1所述的方法，其中冠状病毒感染是严重急性呼综合征(SARS)-冠状病毒2(SARS-CoV-2)感染。
实施方案3.根据实施方案1或2所述的方法，其中该冠状病毒表面抗原包冠状病毒刺突(S)蛋白或其片段或表位。
实施方案4.1-3中任一项所述实施方案，其中该冠状病毒表面抗原包SARS-CoV-2刺突(S)胞外域肽或其片段或表位，可选地该S胞外域肽或其片段或表位包括SARS-CoV-2奥密克戎(B.1.1.529)变异株S胞外域肽或其片段、变体或突变体，例 如含奥密克戎变异株受体结合域(RBD)及Hu-1或其它变异株S蛋白肽序列的嵌合序列。
实施方案5.根据实施方案1-4中任一项所述的方法，其中该冠状病毒表面抗原包括SARS-CoV-2刺突(S)N-末端结构域(NTD)肽或其片段或表位，可选地该NTD肽是SARS-CoV-2 Hu-1、阿尔法、贝塔、伽马、德尔塔、缪、或奥密克戎NTD肽或其片段、变体或突变体。
实施方案6.根据实施方案1-5中任一项所述的方法，其中该冠状病毒表面原包括SARS-CoV-2刺突(S)受体结合结构域(RBD)肽或其片段或表位，可选地该RBD肽是SARS-CoV-2奥密克戎(B.1.1.529)变异株RBD肽或其片段、变体或突变体。
实施方案7.根据实施方案1-6中任一项所述的方法，其中该冠状病毒表面抗原包括SARS-CoV-2刺突(S)S1肽或其片段或表位，可选地该S1肽是SARS-CoV-2奥密克戎(B.1.1.529)变异株S1肽或其片段、变体或突变体。
实施方案8.根据权利要求1-7中任一项所述的方法，其中该冠状病毒表面抗原包括SARS-CoV-2刺突(S)S2肽或其片段或表位，可选地该S2肽是SARS-CoV-2 Hu-1、阿尔法、贝塔、伽马、德尔塔、缪、或奥密克戎S2肽或其片段、变体或突变体。
实施方案1.9.根据实施方案1-8中任一项所述的方法，其中该冠状病毒表抗原包括具有突变的SARS-CoV-2刺突(S)胞外域肽或其片段或表位。
实施方案10.根据实施方案9所述的方法，其中该突变包括弗林蛋白酶裂解位点突变，可选地该突变是经缺失、取代或添加一个或几个氨基酸使得弗林蛋白酶切位点不再具有作为弗林蛋白酶切位点的活性，可选地该突变位于682-685中任意一个或几个位点，可选地该突变包括685R→685A。
实施方案11.根据实施方案9或10所述的方法，其中该突变包括位于或邻近七肽重复序列HR1和中心螺旋交界处的突变，可选地该突变包括脯氨酸替换，例如986K→986P和/或987V→987P。
实施方案12.根据实施方案9-11中任一项所述的方法，其中该突变包括连续位点氨基酸替换，例如986K→986P和987V→987P。
实施方案13.根据实施方案1-12中任一项所述的方法，其中该重组亚单位疫苗包括SEQ ID NO:81-92中的任一个所述的序列或与SEQ ID NO:81-92中的任一个所述的序列有至少或约80％、85％、90％、92％、95％、97％、99％的序列同一性的氨基酸序列。
实施方案14.根据权利要求1-13中任一项所述的方法，其中该重组亚单位疫苗包括SEQ ID NO:85或与SEQ ID NO:85有至少或约80％、85％、90％、92％、95％、97％、99％的序列同一性的氨基酸序列。
实施方案15.根据实施方案1-14中任一项所述的方法，其中该重组亚 单位疫苗包括SEQ ID NO:86或与SEQ ID NO:86有至少或约80％、85％、90％、92％、95％、97％、99％的序列同一性的氨基酸序列。
实施方案16.根据权利要求1-15中任一项所述的方法，其中该重组亚单位疫苗包括SEQ ID NO:87或与SEQ ID NO:87有至少或约80％、85％、90％、92％、95％、97％、99％的序列同一性的氨基酸序列。
实施方案17.根据实施方案1-16中任一项所述的方法，其中该重组亚单位疫苗包括SEQ ID NO:88或与SEQ ID NO:88有至少或约80％、85％、90％、92％、95％、97％、99％的序列同一性的氨基酸序列。
实施方案18.根据实施方案1-17中任一项所述的方法，其中该重组亚单位疫苗包括SEQ ID NO:89-92中的任一个所述的序列或与SEQ ID NO:89-92中的任一个所述的序列有至少或约80％、85％、90％、92％、95％、97％、99％的序列同一性的氨基酸序列。
实施方案19.根据实施方案1-18中任一项所述的方法，其中该重组亚单位疫苗包括SEQ ID NO:90或与SEQ ID NO:90有至少或约80％、85％、90％、92％、95％、97％、99％的序列同一性的氨基酸序列。
实施方案20.根据实施方案1-19中任一项所述的方法，其中该重组亚单位疫苗包括SEQ ID NO:81-84中的任一个所述的第一序列连接到SEQ ID NO:67-80中的任一个所述的第二序列，其中该第一序列的C末端直接或间接连接到该第二序列的N末端。
实施方案21.根据权利要求1-20中任一项所述的方法，其中该重组亚单位疫苗通过肌肉内注射施用。
实施方案22.根据实施方案1-21中任一项所述的方法，其中该重组亚单位疫苗通过鼻内喷雾剂施用。
实施方案23.根据实施方案1-22中任一项所述的方法，其中该重组亚单位疫苗以单剂量施用或以周或月间隔开的一系列剂量施用。
实施方案24.根据实施方案1-23中任一项所述的方法，其中该重组亚单位疫苗在没有佐剂的情况下施用，可选地该重组亚单位疫苗作为初始剂(primary series)，额外剂(additional dose)，和/或同源或异源加强剂(booster dose)使用，例如第一剂，第二剂，第三剂，第四剂，和/或更多剂，可选地该初始剂，额外剂，或异源加强剂与其它重组亚单位疫苗、纳米颗粒疫苗、mRNA疫苗、DNA疫苗、腺病毒载体疫苗、和灭活病毒疫苗中的任意一种或几种配合使用。
实施方案25.根据实施方案1-24中任一项所述的方法，其中该重组亚单位疫苗与佐剂一起施用，可选地该重组亚单位疫苗作为初始剂(primary series)，额外剂(additional dose)，和/或同源或异源加强剂(booster dose)使用，例如第一剂，第二剂，第三剂，第四剂，和/或更多剂，可选地该初始剂，额外剂，或异源加强剂与其它重组亚单位疫苗、纳米颗粒疫苗、mRNA疫苗、DNA疫苗、腺病毒载体疫苗、 和灭活病毒疫苗中的任意一种或几种配合使用，可选地该初始剂，额外剂，和/或同源或异源加强剂中的佐剂可独立地包括：含铝佐剂，例如含明矾和/或氢氧化铝的佐剂；含寡核苷酸的佐剂，例如含CpG寡脱氧核苷酸(CpG-ODN)的佐剂；含TLR9激动剂的佐剂；含可代谢油、α-生育酚、和/或聚氧乙烯山梨醇酐单油酸酯(吐温-80)的佐剂，例如水包油乳液形式的含角鲨烯、α-生育酚、和吐温-80和/或Span 85的佐剂；或任意一种佐剂组合。
实施方案26.根据1-实施方案25中任一项所述的方法，其中该重组亚单位疫苗与一种以上佐剂一起施用，可选地该重组亚单位疫苗作为初始剂(primary series)，额外剂(additional dose)，和/或同源或异源加强剂(booster dose)使用，例如第一剂，第二剂，第三剂，第四剂，和/或更多剂，可选地该初始剂，额外剂，或异源加强剂与其它重组亚单位疫苗、纳米颗粒疫苗、mRNA疫苗、DNA疫苗、腺病毒载体疫苗、和灭活病毒疫苗中的任意一种或几种配合使用，可选地该初始剂，额外剂，和/或同源或异源加强剂中的佐剂可独立地包括：含铝佐剂，例如含明矾和/或氢氧化铝的佐剂；含寡核苷酸的佐剂，例如含CpG寡脱氧核苷酸(CpG-ODN)的佐剂；含TLR9激动剂的佐剂；含可代谢油、α-生育酚、和/或聚氧乙烯山梨醇酐单油酸酯(吐温-80)的佐剂，例如水包油乳液形式的含角鲨烯、α-生育酚、和吐温-80和/或Span 85的佐剂；或任意一种佐剂组合。
实施方案27.一种用于检测来自哺乳动物血清的冠状病毒抗体的方法，
该方法包括将血清与可溶性冠状病毒SARS-CoV-2奥密克戎(B.1.1.529)表面抗原接触的步骤，该可溶性冠状病毒表面抗原通过框内融合连接到胶原的C-末端部分以形成二硫键连接的三聚体融合蛋白。
实施方案28.根据实施方案27所述的方法，其中该可溶性冠状病毒表面抗原是S蛋白或肽。
实施方案29.一种使用包含来自冠状病毒SARS-CoV-2奥密克戎
(B.1.1.529)的可溶性表面抗原的重组亚单位疫苗的方法，该可溶性表面抗原通过框内融合连接到胶原的C-末端部分以形成二硫键连接的三聚体融合蛋白，该方法包括：免疫哺乳动物，纯化产生的中和抗体，以及使用所述中和抗体通过被动免疫治疗感染所述冠状病毒的患者。
实施方案30.根据实施方案29所述的方法，其中该中和抗体包括多克隆抗体。
实施方案31.根据实施方案29所述的方法，其中该中和抗体是单克隆抗体。
实施方案32.根据实施方案29所述的方法，其中该中和抗体是S蛋白或肽的单克隆抗体。
实施方案33.根据实施方案29所述的方法，其中该中和抗体是SARS-
CoV-2的S蛋白的单克隆抗体。
实施方案34.根据实施方案29所述的方法，其中该中和抗体是SARS-
CoV-2 Hu-1、阿尔法、贝塔、伽马、德尔塔、缪、奥密克戎和或其它毒株的S蛋白的单克隆抗体。
实施方案35.根据实施方案29所述的方法，其中该中和抗体是SARS-
CoV-2奥密克戎(B.1.1.529)的S蛋白的单克隆抗体。
实施方案36.一种包括选自由SEQ ID NO:85-92组成的组的重组多肽的复合物。
实施方案37.一种包含选自由SEQ ID NO:85-92组成的组的重组多肽的三聚体的复合物，其中该重组多肽通过多肽间二硫键三聚化以形成三聚体。
实施方案38.一种免疫原性组合物，该免疫原性组合物包括重组多肽的三聚体或任何两个或更多个三聚体的组合，该重组多肽包括选自由SEQ ID NO:85-92组成的组的序列。
实施方案39.根据实施方案38所述的免疫原性组合物，包括具有SEQ ID NO:90中所述的序列的重组多肽的三聚体。
实施方案40.一种用于在受试者中产生对冠状病毒表面抗原的免疫应答的方法，该方法包括向受试者施用有效量的复合物，该复合物包括选自由SEQ ID NO:85-92组成的组的重组多肽，可选地该复合物作为初始剂(primary series)，额外剂(additional dose)，和/或同源或异源加强剂(booster dose)使用，例如第一剂，第二剂，第三剂，第四剂，和/或更多剂，可选地该初始剂，额外剂，或异源加强剂与其它重组亚单位疫苗、纳米颗粒疫苗、mRNA疫苗、DNA疫苗、腺病毒载体疫苗、和灭活病毒疫苗中的任意一种或几种配合使用。
实施方案41.一种用于在受试者中产生对冠状病毒表面抗原的免疫应答的方法，
其中该表面抗原包括S蛋白或其抗原片段，以及该方法包括向受试者施用有效量的复合物，该复合物包括选自由SEQ ID NO:85-92组成的组的重组多肽，可选地该复合物作为初始剂(primary series)，额外剂(additional dose)，和/或同源或异源加强剂(booster dose)使用，例如第一剂，第二剂，第三剂，第四剂，和/或更多剂，可选地该初始剂，额外剂，或异源加强剂与其它重组亚单位疫苗、纳米颗粒疫苗、mRNA疫苗、DNA疫苗、腺病毒载体疫苗、和灭活病毒疫苗中的任意一种或几种配合使。
实施方案42.一种用于在受试者中产生对冠状病毒表面抗原的免疫应答的方法，
其中该表面抗原包括选自由SEQ ID NO:27-66和81-84组成的组的序列，以及该方法包括向受试者施用有效量的复合物，该复合物包括选自由SEQ ID NO:85-92组成的组的重组多肽，可选地该复合物作为初始剂(primary series)，额外剂(additional  dose)，和/或同源或异源加强剂(booster dose)使用，例如第一剂，第二剂，第三剂，第四剂，和/或更多剂，可选地该初始剂，额外剂，或异源加强剂与其它重组亚单位疫苗、纳米颗粒疫苗、mRNA疫苗、DNA疫苗、腺病毒载体疫苗、和灭活病毒疫苗中的任意一种或几种配合使。
实施方案43.一种用于在受试者中产生对冠状病毒表面抗原的免疫应答的方法，
其中该表面抗原包括冠状病毒的S蛋白或其抗原片段，并且可选地，该表面抗原包括选自由SEQ ID NO:27-66和81-84组成的组的序列或其抗原片段，以及该方法包括向受试者施用有效量的复合物，该复合物包括包含SEQ ID NO:85-92中的任一个所述的序列的重组多肽，可选地该复合物作为初始剂(primary series)，额外剂(additional dose)，和/或同源或异源加强剂(booster dose)使用，例如第一剂，第二剂，第三剂，第四剂，和/或更多剂，可选地该初始剂，额外剂，或异源加强剂与其它重组亚单位疫苗、纳米颗粒疫苗、mRNA疫苗、DNA疫苗、腺病毒载体疫苗、和灭活病毒疫苗中的任意一种或几种配合使用。
实施方案44.一种用于在受试者中产生对冠状病毒表面抗原的免疫应答的方法，其中该表面抗原包括S蛋白或其抗原片段，以及
该方法包括向受试者施用有效量的复合物或任何两种或更多种复合物的组合，该复合物包括包含选自由SEQ ID NO:85-92组成的组的序列的重组多肽，可选地该复合物或复合物的组合作为初始剂(primary series)，额外剂(additional dose)，和/或同源或异源加强剂(booster dose)使用，例如第一剂，第二剂，第三剂，第四剂，和/或更多剂，可选地该初始剂，额外剂，或异源加强剂与其它重组亚单位疫苗、纳米颗粒疫苗、mRNA疫苗、DNA疫苗、腺病毒载体疫苗、和灭活病毒疫苗中的任意一种或几种配合使用。
实施方案45.根据权利要求44所述的方法，其中该方法包括向受试者施用有效量的包括重组多肽的复合物，该重组多肽包括SEQ ID NO:85、SEQ ID NO:86、SEQ ID NO:87和/或SEQ ID NO:88中所述的序列。
实施方案46.一种包含多个重组多肽的融合蛋白，每个重组多肽从氨基到羧基末端包括：
a)第一区域，该第一区域包括位于第一冠状病毒的非嵌合冠状病毒刺突蛋白中的冠状病毒刺突蛋白受体结合结构域(RBD)之前的冠状病毒刺突蛋白胞外域的一部分；
b)第二区域，该第二区域包括不同于所述第一冠状病毒的第二冠状病毒的冠状病毒刺突蛋白受体结合结构域(RBD)；和
c)胶原的C-末端前肽，其中该重组多肽的C-末端前肽形成多肽间二硫键，其中第一冠状病毒和第二冠状病毒至少有一个是SARS-CoV-2奥密克戎(B.1.1.529)，可选地该融合蛋白作为初始剂(primary series)，额外剂(additional dose)，和/或同源或异源加强剂(booster dose)使用，例如第一剂，第二剂，第三剂，第四剂，和/或更多 剂，可选地该初始剂，额外剂，或异源加强剂与其它重组亚单位疫苗、纳米颗粒疫苗、mRNA疫苗、DNA疫苗、腺病毒载体疫苗、和灭活病毒疫苗中的任意一种或几种配合使用。
实施方案47.根据权利要求46所述的融合蛋白，还包括该第二区域与该胶原的C-末端前肽之间的第三区域。
实施方案48.根据权利要求47所述的融合蛋白，其中该第三区域包括第三冠状病毒的S1结构域，其中该第三冠状病毒与第一冠状病毒或第二冠状病毒相同或不同。
实施方案49.根据权利要求47或48所述的融合蛋白，其中该第三区域包括第四冠状病毒的S2结构域，其中该第四冠状病毒与第一、第二或第四冠状病毒相同或不同。
实施方案50.根据权利要求46-49中任一项所述的融合蛋白，其中该第一区域包括第一冠状病毒的N-末端结构域(NTD)。
实施方案51.根据权利要求46-50中任一项所述的融合蛋白，其中该第一区域包括与第二冠状病毒中的相应氨基酸残基不同的一个或更多个氨基酸残基。
实施方案52.根据权利要求46-51中任一项所述的融合蛋白，其中该第二区域包括与第一冠状病毒中的相应氨基酸残基不同的一个或更多个氨基酸残基。
实施方案53.根据权利要求46-52中任一项所述的融合蛋白，其中该第一和第二冠状病毒是相同冠状病毒的不同变种或毒株。
实施方案54.根据权利要求53所述的融合蛋白，其中该第一区域包括第一冠状病毒的NTD，该第二区域包括第二冠状病毒的RBD，并且第一和第二冠状病毒是SARS-CoV-2的不同变种。
实施方案55.根据权利要求46-54中任一项所述的融合蛋白，其中该第一冠状病毒和第二冠状病毒独立地选自由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和A.23.1谱系的SARS-CoV-2病毒组成的组。
实施方案56.一种包含三个重组多肽的三聚体融合蛋白，每个重组多肽从氨基到羧基末端包括：
a)第一区域，该第一区域包括B.1.1.529谱系的SARS-CoV-2的冠状病毒刺突蛋白N-末端结构域(NTD)；
b)第二区域，该第二区域包括B.1.1.529谱系的SARS-CoV-2的冠状病毒刺突蛋白受体结合结构域(RBD)；和
c)胶原的C-末端前肽，其中该重组多肽的C-末端前肽形成多肽间二硫键，可选地该三聚体融合蛋白作为初始剂(primary series)，额外剂(additional dose)，和/或同源或异源加强剂(booster dose)使用，例如第一剂，第二剂，第三剂，第四剂，和/或更多剂，可选地该初始剂，额外剂，或异源加强剂与其它重组亚单位疫苗、纳米颗粒 疫苗、mRNA疫苗、DNA疫苗、腺病毒载体疫苗、和灭活病毒疫苗中的任意一种或几种配合使用。
实施方案57.一种用于预防哺乳动物中冠状病毒感染的方法，该方法包括用有效量的根据权利要求46-56中任一项所述的融合蛋白免疫哺乳动物。
实施方案58.根据实施方案57所述的方法，其中在哺乳动物中产生针对该第一和第二冠状病毒的中和抗体。
实施方案59.根据实施方案58所述的方法，其中该第一和第二冠状病毒是SARS-CoV-2的不同变种，并且在哺乳动物中产生的中和抗体中和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和A.23.1谱系的两种或更多种SARS-CoV-2病毒。
实施方案60.根据实施方案59所述的方法，其中在哺乳动物中产生的中和抗体中和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和A.23.1谱系的三种或更多种SARS-CoV-2病毒。
实施方案61.根据实施方案57-60中任一项所述的方法，包括用两剂或更多剂的融合蛋白免疫哺乳动物，该两剂或更多剂的融合蛋白中至少有一剂的融合蛋白包含SARS-CoV-2奥密克戎(B.1.1.529)刺突蛋白氨基酸序列，可选地该至少一剂融合蛋白包含SEQ ID NO:81-92中的任一个所述的序列或与SEQ ID NO:81-92中的任一个所述的序列有至少或约80％、85％、90％、92％、95％、97％、99％的序列同一性的氨基酸序列。
实施方案62.根据实施方案57-61中任一项所述的方法，其中在一剂或更多剂的免疫原之后以再次加强剂施用融合蛋白，该免疫原包括包含来自相同或不同SARS-CoV-2变种的NTD和RBD的刺突蛋白肽，可选地该一剂或更多剂的免疫原包含SEQ ID NO:27-66和81-84中的任一个所述的序列或与SEQ ID NO:27-66和81-84中的任一个所述的序列有至少或约80％、85％、90％、92％、95％、97％、99％的序列同一性的氨基酸序列，可选地该加强剂融合蛋白包含SEQ ID NO:81-92中的任一个所述的序列或与SEQ ID NO:81-92中的任一个所述的序列有至少或约80％、85％、90％、92％、95％、97％、99％的序列同一性的氨基酸序列。
实施方案80.一种二价疫苗，包括：
第一三聚体融合蛋白，该第一三聚体融合蛋白包括可溶性冠状病毒SARS-CoV-2奥密克戎(B.1.1.529)刺突(S)蛋白表面抗原或其片段、变体或突变体，该可溶性冠状病毒表面抗原或其片段、变体或突变体连接到胶原的C-末端部分，该胶原的C-末端部分形成二硫键连接的三聚体，从而形成该第一三聚体融合蛋白；
第二三聚体融合蛋白，该第二三聚体融合蛋白包括可溶性冠状病毒SARS-CoV-2 Hu-1、阿尔法、贝塔、伽马、德尔塔、或缪刺突(S)蛋白表面抗原或其片段、变体或突变体，该可溶性冠状病毒表面抗原或其片段、变体或突变体连接到胶原的C-末端部分，该胶原的C-末端部分形成二硫键连接的三聚体，从而形成该第二三聚体融合蛋白。
实施方案81.根据权利要求80所述的二价疫苗，该第二三聚体融合蛋白包括可溶性冠状病毒SARS-CoV-2 Hu-1刺突(S)蛋白表面抗原或其片段、变体或突变体。
实施方案82.根据实施方案80或81所述的二价疫苗，该二价疫苗在没有佐剂的情况下施用，可选地该二价疫苗作为初始剂(primary series)，额外剂(additional dose)，和/或同源或异源加强剂(booster dose)使用，例如第一剂，第二剂，第三剂，第四剂，和/或更多剂，可选地该初始剂，额外剂，或异源加强剂与其它重组亚单位疫苗、纳米颗粒疫苗、mRNA疫苗、DNA疫苗、腺病毒载体疫苗、和灭活病毒疫苗中的任意一种或几种配合使用。
实施方案83.根据实施方案80-82中任一项所述的二价疫苗，该二价疫苗与一种以上佐剂一起施用，可选地该二价疫苗作为初始剂(primary series)，额外剂(additional dose)，和/或同源或异源加强剂(booster dose)使用，例如第一剂，第二剂，第三剂，第四剂，和/或更多剂，可选地该初始剂，额外剂，或异源加强剂与其它重组亚单位疫苗、纳米颗粒疫苗、mRNA疫苗、DNA疫苗、腺病毒载体疫苗、和灭活病毒疫苗中的任意一种或几种配合使用，可选地该初始剂，额外剂，和/或同源或异源加强剂中的佐剂可独立地包括：含铝佐剂，例如含明矾和/或氢氧化铝的佐剂；含寡核苷酸的佐剂，例如含CpG寡脱氧核苷酸(CpG-ODN)的佐剂；含TLR9激动剂的佐剂；含可代谢油、α-生育酚、和/或聚氧乙烯山梨醇酐单油酸酯(吐温-80)的佐剂，例如水包油乳液形式的含角鲨烯、α-生育酚、和吐温-80和/或Span 85的佐剂；或任意一种所述佐剂的组合。
实施方案84.根据实施方案80-83中任一项所述的二价疫苗，该二价疫苗中的第一三聚体融合蛋白包括SEQ ID NO:81-84中的任一个所述的序列或与SEQ ID NO:81-84中的任一个所述的序列有至少或约80％、85％、90％、92％、95％、97％、99％的序列同一性的氨基酸序列。
实施方案85.根据实施方案80-84中任一项所述的二价疫苗，该二价疫苗中的第二三聚体融合蛋白包括SEQ ID NO:27-66中的任一个所述的序列或与SEQ ID NO:27-66中的任一个所述的序列有至少或约80％、85％、90％、92％、95％、97％、99％的序列同一性的氨基酸序列。
实施方案86.一种多价疫苗，其包括自由选自可溶性冠状病毒Hu-1，SARS-CoV-2奥密克戎(B.1.1.529)，奥密克戎毒株(BA.5)，阿尔法、贝塔、伽马、德尔塔、或缪刺突(S)蛋白表面抗原或其片段、变体或突变体，该可溶性冠状病毒表面抗原或其片段、变体或突变体连接到胶原的C-末端部分，该胶原的C-末端部分形成二硫键连接的三聚体，从而形成该第一三聚体融合蛋白。
实施方案87.根据实施方案86中所述的多价疫苗包括所述可溶性冠状病毒Hu-1和奥密克戎毒株(BA.5)刺突(S)蛋白表面抗原或其片段、变体或突变体。
实施方案88.根据实施方案86中所述的多价疫苗包括所述可溶性冠状 病毒，SARS-CoV-2奥密克戎(B.1.1.529)和奥密克戎毒株(BA.5)刺突(S)蛋白表面抗原或其片段、变体或突变体。
实施方案89.根据实施方案86中所述的多价疫苗包括所述可溶性冠状病毒贝塔，德尔塔和奥密克戎毒株(BA.5)刺突(S)蛋白表面抗原或其片段、变体或突变体。
实施例
包括以下实施例仅用于说明目的，并不旨在限制本发明的范围。
实施例1：产生重组二硫键连接的SARS-CoV-2 S-三聚体融合蛋白
产生分泌型重组二硫键连接的多肽，其包含融合到三聚化结构域的SARS-CoV-2蛋白肽，作为候选蛋白亚单位疫苗。在一个实施例中，来自SARS-CoV2的包括其信号肽(SP)、S1和S2结构域的刺突蛋白的胞外域在框内在C-末端融合到编码α1胶原的人类C-前肽的哺乳动物表达载体，以使分泌的三聚体S-三聚体融合抗原得以表达，例如如图1A-1B所示。
为了快速表达S-三聚体抗原，采用了蛋白质三聚体化TM技术(Liu et al.,Scientific Reports,7(1):8953,2017，出于所有目的以其全文形式被援引加入)。编码SARS-CoV-2刺突(S)蛋白的胞外域的cDNA被亚克隆到pTRIMER哺乳动物表达载体中，以允许在框内融合到蛋白质三聚体化TM标签，蛋白质三聚体化TM标签能够通过二硫键自我三聚化。在稳定转染到CHO细胞中、随后筛选高滴度生产克隆和广泛的工艺优化后，开发了生物反应器中的补料分批无血清细胞培养工艺，导致S-三聚体作为分泌蛋白的高水平表达。Liang et al.,Nat.Comms.,12:1346,2021；Richmond et al.,Lancet,397:682-694,2021，出于所有目的以其全文形式被援引加入。
为了获得用于疫苗研究的高纯度形式的S-三聚体，利用蛋白质三聚体化TM标签和Endo180之间的高结合亲和力的优势开发了亲和纯化方案，Endo180是能够结合到1型前胶原的C-末端区域并使胶原成熟的胶原受体。将Endo180-Fc融合蛋白装载到蛋白A柱上，并通过蛋白A与Endo180-Fc的人IgG1Fc结构域之间的高亲和力结合被树脂捕获。然后，将含有CHO细胞分泌的S-三聚体的无血清细胞培养基装载到具有预捕获的Endo180-Fc的蛋白A柱上。在冲洗掉任何未结合的污染宿主细胞蛋白质(HCP)和其它杂质后，在不使Endo180-Fc从蛋白A柱分离的条件下，使用温和的盐洗脱一步将结合的S-三聚体纯化至接近均一性。进一步纯化S-三聚体，通过低pH值用于预防性病毒灭活(VI)，阴离子交换层析以去除宿主细胞DNA和任何残留的内毒素，纳滤作为预防性病毒去除(VR)步骤，以及最终UF/DF以将S-三聚体浓缩到制剂缓冲液中所需的浓度，从而获得S-三聚体亚单位候选疫苗的活性药物(DS)。纯化的S-三聚体的稳定性分析表明，S-三聚体在2-8℃的液体溶液制剂中是稳定的。
非还原和还原条件下的SDS-PAGE分析证实，纯化的S-三聚体是二硫键连接的三聚体，并在S1/S2边界被CHO细胞产生的弗林蛋白酶部分裂解。在非还原条件下， S-三聚体以多种高分子量形式出现，可能是抗原的部分裂解的结果，在样品处理期间释放非共价连接的和裂解的S1。
基于Hu-1毒株和VOC毒株的刺突抗原的生产和表征
利用蛋白质三聚体化TM技术(Liang et al.,Nat.Comms.,12:1346,2021)产生基于Hu-1毒株和VOC毒株的刺突抗原共价三聚体。在10L生物反应器中对补料分批无血清CHO细胞培养物中的S-三聚体表达进行了8％SDS-PAGE分析。在还原条件下分析无细胞条件培养基，然后进行考马斯亮蓝染色。
使用蛋白质A传感器通过Fortebio BioLayer干涉测量法评估纯化的S-三聚体抗原与人类ACE2受体的结合亲和力。在图3A-3B中，与Hu-1 S-三聚体相比，奥密克戎S-三聚体与ACE2-Fc的受体亲和力更高。
实施例2：含佐剂SARS-CoV-2疫苗的免疫原性
评估BALB/c小鼠中S-三聚体的免疫原性。在两剂初免-加强方案(第0天和第21天)中，小鼠被两次肌肉注射S-三聚体。佐剂对体液免疫原性的影响是明显的，因为在相应的抗原剂量水平下，含佐剂组的S-三聚体结合抗体滴度、ACE2竞争性滴度和中和抗体滴度显著高于无佐剂疫苗。具有不同佐剂的S-三聚体引发与在人类康复期血清样品中观察到的水平相似或更高的ACE2竞争性抗体滴度和假病毒中和抗体滴度。在S-三聚体免疫的大鼠中观察到类似的结果。
通过收集被处死的免疫的小鼠的脾细胞，然后用S-三聚体抗原刺激并通过ELISpot检测Th1(IL-2和IFNγ)和Th2(IL-4和IL-5)细胞因子，来研究S-三聚体抗原特异性的细胞介导的免疫(CMI)。含佐剂组似乎比无佐剂S-三聚体诱导更强的整体CMI应答。在无佐剂组和某些含佐剂S-三聚体组中观察到Th1偏好的细胞介导的免疫应答，而在其它含佐剂组中观察到混合的Th1-Th2分布。CMI似乎不依赖于抗原的剂量。
实施例3：基于奥密克戎毒株的SARS-CoV-2疫苗的免疫原性
方法
假病毒的构建和生产
SARS-CoV-2高关注变异株刺突蛋白(spike,S)基因采用哺乳动物密码子进行优化，并由Genscript合成，然后克隆到pcDNA3.1(+)真核表达载体中。构建了编码Hu-1、阿尔法(Alpha，α)、贝塔(Beta，β)、伽马(gamma，γ)、德尔塔(delta，δ)、缪(miu，μ)和奥密克戎(Omicron，ο)SARS-CoV-2变异株S糖蛋白的质粒。并且在wild-type Omicron S-Trimer中引入S1/S2切割位点的单点突变R685A以产生奥密克戎S-三聚体S-Trimer或引入S1/S2切割位点的单点突变R685A和另外的单点突变K986P和/或V987P。
慢病毒包装质粒psPAX2和表达GFP和荧光素酶的pLVX-AcGFP-N1-Fluc慢病毒报告质粒来自HonorGene(奥诺基因，中国)。通过使用Lipofectamine 3000(Invitrogen，L3000-015)将psPAX2、pLVX-AcGFP-N1-Fluc和编码各种S基因的质粒共同转染HEK 293T细胞，产生假病毒。在转染后24±2小时收获上清液，在1500rpm下离心5分钟以去除细胞碎片，然后在-80℃储存。通过感染293T-ACE2细胞对假病毒储库进行滴定，通过添加Bright-Glo荧光素酶检测系统(Promega，E2650)，在37℃和5％CO2条件下44至48小时的温育期后，使用微板阅读器(TECAN，Spark)测定荧光素酶活性。然后根据Reed-Muench方法(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)计算假病毒的TCID50。
中和试验
试验血清样品的等分试样首先在56℃下热灭活30分钟，然后在10,000rcf下离心5分钟进行澄清。样品用检测介质(100毫升)连续稀释(3倍)，与650TCID50的假病毒(50毫升)在37℃下温育1小时，同时使用病毒感染的未处理对照(单独病毒)和单独细胞(背景对照)。然后，将新鲜的胰蛋白酶化的293T-ACE2细胞以100mcL的20000个细胞/孔添加到的每个孔中。在37℃、在5％CO2培养箱中在37℃下温育44至48小时后，根据制造商的方案，裂解细胞，并通过Bright-Glo荧光素酶检测系统(Promega)测定荧光素酶活性。给定血清样品的IC50中和抗体滴度被定义为血清稀释度，其中样品显示与病毒感染对照孔相比，相对光单位(RLU)减少50％。详细方法根据Quantification of SARS-CoV-2neutralizing antibody by a pseudotyped  virus based assay.Nie J.et al.DOI:10.21203/rs.3.pex-941/v11所报导。
高关注变异体(VOC)S-三聚体融合蛋白的表达和纯化
使用Cricetulus griseus(中国仓鼠)偏好的密码子，由GenScript公司对编码来自奥密克戎的SARS-CoV-2刺突(S)蛋白胞外域的cDNA进行基因合成。该cDNA在Hind III和Bgl II位点被亚克隆到pTRIMER表达载体(GenHunter公司)，以允许可溶性S蛋白在框内融合到蛋白质三聚体化TM标签(来自人类I(α)型胶原蛋白的氨基酸残基1156-1406)，如前所述。使用PEI(Polyscience)将表达载体瞬时转染到HEK-293F细胞系(Clover Biopharma)，并在OPM-293CD05培养基(OPM)和OPM-293proFeed补充剂(OPM)中生长。使用蛋白质三聚体化TM标签特异性亲和柱(Clover Biopharma)将S-三聚体蛋白从条件培养基中纯化至均一性。
结果
构建并生产了基于奥密克戎毒株的蛋白质三聚体化TM标签亚单位疫苗，并将其作为初始剂和/或加强疫苗给予小鼠，用于同源加强或者与Hu-1 S-三聚体配合使用 进行异源加强。另外构建并生产了含有Hu-1 S-三聚体和奥密克戎S-三聚体的二价疫苗作为初始剂和加强剂，如图4所示。图5A-5B示出了根据图4小鼠接受两剂S-三聚体抗原(第一剂初免-第二剂加强)免疫后的SARS-CoV-2 Hu-1、阿尔法(alpha,α,B.1.1.7)、贝塔(beta,β,B.1.351)、伽马(gamma,γ,P.1)、德尔塔(delta,δ,B.1.617.2)、和奥密克戎(Omicro,o,B.1.1.529)毒株假病毒中和抗体IC50数据。对比图5A和图5B中对奥密克戎毒株假病毒的中和抗体滴度，两剂奥密克戎S-三聚体免疫后的小鼠针对奥密克戎毒株假病毒的中和抗体滴度比两剂Hu-1 S-三聚体免疫后的小鼠显著增加，但对其它某些毒株的中和抗体滴度有所降低。两剂Hu-1 S-三聚体免疫对其它毒株产生良好的中和抗体滴度，但对奥密克戎毒株产生的中和抗体滴度显著低于对其它毒株产生的中和抗体滴度。在异源加强组中，Hu-1 S-三聚体作为初始剂和奥密克戎S-三聚体作为加强剂(图5C)产生的中和抗体滴度总体较奥密克戎S-三聚体作为初始剂和Hu-1 S-三聚体作为加强剂的异源加强组(图5D)好。使用二价疫苗为初始剂和加强剂，如图5E所示，既能提高针对奥密克戎毒株假病毒的中和抗体滴度(与两剂Hu-1S-三聚体免疫相比)，又能保持或提高针对其它毒株的中和抗体滴度(与两剂Hu-1 S-三聚体免疫相比)。
另外将奥密克戎S-三聚体作为第三剂加强疫苗给予经Hu-1 S-三聚体疫苗初免/加强的小鼠，与其他VOC候选疫苗相比较以观察其进一步加强中和抗体广泛覆盖的能力。
图6A-6B示出了第3剂奥密克戎S-三聚体疫苗接种加强小鼠中的VOC中和抗体。在第0天(第1剂)和第21天(第2剂)用150μg CpG 1018加75μg Alum作为佐剂的3μg Hu-1 S-三聚体对BALB/c小鼠进行两次免疫。在研究的第57天，动物接受第3剂的疫苗接种：用150μg CpG 1018加75μg Alum作为佐剂的3μg奥密克戎S1/S2弗林蛋白酶裂解位点突变体S-三聚体；在研究第56天(D35PD2或D-1PD3)和第64天(D7PD3)收集血清，进行假病毒中和抗体测试。在第35天(D14PD2)，第56天(D35PD2)和第64天(D7PD3)的SARS-CoV-2 Hu-1、阿尔法、贝塔、伽马、德尔塔、和奥密克戎毒株假病毒中和抗体(IC50)数据。与第3剂疫苗接种前的数据相比较，接受第3剂奥密克戎S-三聚体疫苗的小鼠对奥密克戎毒株假病毒中和抗体的滴度显著增加。
图7A-7G示出了不同第3剂疫苗接种加强小鼠中的VOC中和抗体。在第0天(第1剂)和第21天(第2剂)用150μg CpG 1018加75μg Alum作为佐剂的3μg Hu-1 S-三聚体对BALB/c小鼠进行两次免疫。在研究的第57天，将动物分为四组(每组n＝10)。第1组不接受第3剂的疫苗接种，作为对照组；第2组：用150μg CpG1018加75μg Alum作为佐剂的3μg Hu-1 S-三聚体作为第3剂的疫苗接种；第3组：用150μg CpG 1018加75μg Alum作为佐剂的3μg奥密克戎S-三聚体作为第3剂的疫苗接种；第4组：用150μg CpG 1018加75μg Alum作为佐剂的二价疫苗(包括1.5μg Hu-1 S-三聚体和1.5μg奥密克戎S1/S2弗林蛋白酶裂解位点突变体 S-三聚体)作为第3剂的疫苗接种。在研究第56天(D-1PD3)和第71天(D14PD3)收集血清，测试其对多重变异假病毒的中和抗体。
奥密克戎候选疫苗(第3组)显著加强了奥密克戎特异性中和抗体(51.9倍)，在Hu-1 S-三聚体和奥密克戎S-三聚体疫苗二价疫苗组(第4组)也观察到类似但较少的加强程度(28.5倍)。在用Alum/CpG作为佐剂的Hu-1 S-三聚体组(第2组)中也观察到中和抗体滴度和宽度(neutralizing antibody breadth)的加强。全佐剂Hu-1S-三聚体(第2组)对某些VOC(Hu-1、阿尔法、德尔塔和奥密克戎)中和抗体的加强并不明显。这可能是由于最后两剂疫苗接种的时间间隔较短(36天)，导致没有足够的时间来产生更多的记忆应答。
为了模拟许多人已经使用祖先疫苗免疫和/或已经感染病毒的人类现状，我们进一步评估了Hu-1S-三聚体预免疫动物中二价候选疫苗的免疫原性的持久性。Balb/c小鼠(雌性，N＝10)在第0天和第21天分别用CpG 1018/铝剂配制的Hu-1S-三聚体对小鼠进行免疫接种，然后用Hu-1S-三聚体或奥密克戎S-三聚体或二价疫苗并配制CpG 1018/Alum在第57天进行二次(第3剂)加强免疫。在D35(14天PD2)、D56(第3次加强免疫日)、D85(第3剂后1个月、1MPD3)、D113(第3剂后2个月、2MPD3)和D141(第3剂后3个月，3MPD3)采集血清用假病毒中和抗体试验测试小鼠对VOC的免疫力(图8A)。第85天(1MPD3)血清样本的的分析结果表明，与对照组(无第3剂加强免疫接种)相比，用二价疫苗进行的二次加强免疫接种的小鼠进一步显著地增强了针对除贝塔变体之外的所有VOC的原本已经很强的中和抗体反应(图8B)；Hu-1S-三聚体单价疫苗显著地增强了针对贝塔、伽马和奥密克戎毒株的中和抗体，而奥密克戎S-三聚体单价疫苗显着增强了针对德尔塔和奥密克戎的反应。
在第3剂加强剂接种后三个月内监测血清抗体中和反应，以评估疫苗保护的持久性(图8C)。对照组(未接种第3剂)的血清显示出对除奥密克戎之外的所有VOC的强中和反应，奥密克戎组则显示了低GMT49(12-1197)(95％CI)；Hu-1S-三聚体第3剂显著改善了接种小鼠对Hu-1、阿尔法、贝塔、伽马、德尔塔菌株的抗体中和反应。同时，尽管Hu-1S-三聚体第3剂对奥密克戎的抗体中和水平的提高低于其他变体，GMT为202(129-2508)(95％CI)，但是也改善了对奥密克戎的抗体中和水平。奥密克戎S-三聚体第3剂与对照组(没有二次加强接种)相比显著地改善了对奥密克戎的抗体中和反应，其GMT为1349(1324-2112)(95％CI)；显示具有对其他VOC的较低反应趋势，并与对照组可比。二价疫苗的二次加强接种显著地改善了对所有VOC的抗体中和反应，反应趋势高于奥密克戎S-三聚体单价加强接种；其对奥密克戎病毒的GMT为799(762-1973)(95％CI))，与奥密克戎S-三聚体单独引发的结果相当。这些高奥密克戎特异性抗体滴度在延长的观察期内得以保持(图8C)。
为了提高疫苗的广谱性以应对多种现有病毒和将来变异的病毒，本申请的发明人也研究了一系列的多价疫苗组合，例如二价，三价和四价疫苗。使用Hu-1 S- 三聚体，奥密克戎S-三聚体，奥密克戎毒株(BA.5)S-三聚体，Belta S-三聚体，Delta-S-三聚体，进行组合获得多价疫苗。多价疫苗组合和试验设计见表1和图9。部分试验结果显示在图10。图10的D表明，包括了Delta-S-三聚体+奥密克戎毒株(BA.5)S-三聚体+Belta S-三聚体的三价疫苗对SARS-CoV-2 Hu-1、阿尔法、贝塔、德尔塔、奥密克戎的BA.1,BA.2,BA.5,BA.2.75,BF.7,和BQ.1.1假病毒均产生较强中和抗体。图10的C表明，包括了奥密克戎S-三聚体+奥密克戎毒株(BA.5)S-三聚体的二价疫苗对奥密克戎的BA.1,BA.2,BA.5,BA.2.75,BF.7,和BQ.1.1假病毒均诱导处出了强中和抗体。图10的B表明，包括了Hu-1 S-三聚体+奥密克戎毒株(BA.5)S-三聚体的二价疫苗，相对于其它试验的假病毒来说，对SARS-CoV-2 Hu-1和奥密克戎BA.5假病毒均诱导出最强中和抗体。
表1.多价疫苗组合试验设计

本发明并不旨在将范围限于特定的公开实施方案，这些实施方案被提供例如用于阐明本发明的各个方面。通过本文的描述和教导，对所述组合物和方法的各种改动将变得显而易见。可以在不偏离本发明的真实范围和精神的情况下实施这些变化，并且这些变化旨在落入本发明的范围。
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Claims (87)

  1. 一种用于预防哺乳动物中冠状病毒感染的方法，该方法包括用有效量的重组亚单位疫苗免疫哺乳动物，该重组亚单位疫苗包括可溶性冠状病毒SARS-CoV-2奥密克戎(B.1.1.529)表面抗原或其片段、变体或突变体，该可溶性冠状病毒表面抗原或其片段、变体或突变体通过框内融合连接到胶原的C-末端部分以形成二硫键连接的三聚体融合蛋白。
  2. 根据权利要求1所述的方法，其中冠状病毒感染是严重急性呼吸综合征(SARS)-冠状病毒2(SARS-CoV-2)感染。
  3. 根据权利要求1或2所述的方法，其中该冠状病毒表面抗原包括冠状病毒刺突(S)蛋白或其片段或表位。
  4. 根据权利要求1-3中任一项所述的方法，其中该冠状病毒表面抗原包括SARS-CoV-2刺突(S)胞外域肽或其片段或表位，可选地该S胞外域肽或其片段或表位包括SARS-CoV-2奥密克戎(B.1.1.529)变异株S胞外域肽或其片段、变体或突变体，例如含奥密克戎变异株受体结合域(RBD)及Hu-1或其它变异株S蛋白肽序列的嵌合序列。
  5. 根据权利要求1-4中任一项所述的方法，其中该冠状病毒表面抗原包括SARS-CoV-2刺突(S)N-末端结构域(NTD)肽或其片段或表位，可选地该NTD肽是SARS-CoV-2Hu-1、阿尔法、贝塔、伽马、德尔塔、缪、或奥密克戎NTD肽或其片段、变体或突变体。
  6. 根据权利要求1-5中任一项所述的方法，其中该冠状病毒表面抗原包括SARS-CoV-2刺突(S)受体结合结构域(RBD)肽或其片段或表位，可选地该RBD肽是SARS-CoV-2奥密克戎(B.1.1.529)变异株RBD肽或其片段、变体或突变体。
  7. 根据权利要求1-6中任一项所述的方法，其中该冠状病毒表面抗原包括SARS-CoV-2刺突(S)S1肽或其片段或表位，可选地该S1肽是SARS-CoV-2奥密克戎(B.1.1.529)变异株S1肽或其片段、变体或突变体。
  8. 根据权利要求1-7中任一项所述的方法，其中该冠状病毒表面抗原包括SARS-CoV-2刺突(S)S2肽或其片段或表位，可选地该S2肽是SARS-CoV-2 Hu-1、阿尔法、贝塔、伽马、德尔塔、缪、或奥密克戎S2肽或其片段、变体或突变体。
  9. 根据权利要求1-8中任一项所述的方法，其中该冠状病毒表面抗原包括具有突变的SARS-CoV-2刺突(S)胞外域肽或其片段或表位。
  10. 根据权利要求9所述的方法，其中该突变包括弗林蛋白酶裂解位点突变，可选地该突变是经缺失、取代或添加一个或几个氨基酸使得弗林蛋白酶切位点不再具有作为弗林蛋白酶切位点的活性，可选地该突变位于682-685中任意一个或几个位点，可选地该突变包括685R→685A。
  11. 根据权利要求9或10所述的方法，其中该突变包括位于或邻近七肽重复序列HR1和中心螺旋交界处的突变，可选地该突变包括脯氨酸替换，例如986K→986P和/或987V→987P。
  12. 根据权利要求9-11中任一项所述的方法，其中该突变包括连续位点氨基酸替换，例如986K→986P和987V→987P。
  13. 根据权利要求1-12中任一项所述的方法，其中该重组亚单位疫苗包括SEQ ID NO:81-92中的任一个所述的序列或与SEQ ID NO:81-92中的任一个所述的序列有至少或约80％、85％、90％、92％、95％、97％、99％的序列同一性的氨基酸序列。
  14. 根据权利要求1-13中任一项所述的方法，其中该重组亚单位疫苗包括SEQ ID NO:85或与SEQ ID NO:85有至少或约80％、85％、90％、92％、95％、97％、99％的序列同一性的氨基酸序列。
  15. 根据权利要求1-14中任一项所述的方法，其中该重组亚单位疫苗包括SEQ ID NO:86或与SEQ ID NO:86有至少或约80％、85％、90％、92％、95％、97％、99％的序列同一性的氨基酸序列。
  16. 根据权利要求1-15中任一项所述的方法，其中该重组亚单位疫苗包括SEQ ID NO:87或与SEQ ID NO:87有至少或约80％、85％、90％、92％、95％、97％、99％的序列同一性的氨基酸序列。
  17. 根据权利要求1-16中任一项所述的方法，其中该重组亚单位疫苗包括SEQ ID NO:88或与SEQ ID NO:88有至少或约80％、85％、90％、92％、95％、97％、99％的序列同一性的氨基酸序列。
  18. 根据权利要求1-17中任一项所述的方法，其中该重组亚单位疫苗包括SEQ ID NO:89-92中的任一个所述的序列或与SEQ ID NO:89-92中的任一个所述的序列有至少或约80％、85％、90％、92％、95％、97％、99％的序列同一性的氨基酸序列。
  19. 根据权利要求1-18中任一项所述的方法，其中该重组亚单位疫苗包括SEQ ID NO:90或与SEQ ID NO:90有至少或约80％、85％、90％、92％、95％、97％、99％的序列同一性的氨基酸序列。
  20. 根据权利要求1-19中任一项所述的方法，其中该重组亚单位疫苗包括SEQ ID NO:81-84中的任一个所述的第一序列连接到SEQ ID NO:67-80中的任一个所述的第二序列，其中该第一序列的C末端直接或间接连接到该第二序列的N末端。
  21. 根据权利要求1-20中任一项所述的方法，其中该重组亚单位疫苗通过肌肉内注射施用。
  22. 根据权利要求1-21中任一项所述的方法，其中该重组亚单位疫苗通过鼻内喷雾剂施用。
  23. 根据权利要求1-22中任一项所述的方法，其中该重组亚单位疫苗以单剂量施用或以周或月间隔开的一系列剂量施用。
  24. 根据权利要求1-23中任一项所述的方法，其中该重组亚单位疫苗在没有佐剂的情况下施用，可选地该重组亚单位疫苗作为初始剂(primary series)，额外剂(additional dose)，和/或同源或异源加强剂(booster dose)使用，例如第一剂，第二剂，第三剂，第四剂，和/或更多剂，可选地该初始剂，额外剂，或异源加强剂与其它重组亚单位疫苗、纳米颗粒疫苗、mRNA疫苗、DNA疫苗、腺病毒载体疫苗、和灭活病毒疫苗中的任意一种或几种配合使用。
  25. 根据权利要求1-24中任一项所述的方法，其中该重组亚单位疫苗与佐剂一起施用，可选地该重组亚单位疫苗作为初始剂(primary series)，额外剂(additional dose)，和/或同源或异源加强剂(booster dose)使用，例如第一剂，第二剂，第三剂，第四剂，和/或更多剂，可选地该初始剂，额外剂，或异源加强剂与其它重组亚单位疫苗、纳米颗粒疫苗、mRNA疫苗、DNA疫苗、腺病毒载体疫苗、和灭活病毒疫苗中的任意一种或几种配合使用，可选地该初始剂，额外剂，和/或同源或异源加强剂中的佐剂可独立地包括：含铝佐剂，例如含明矾和/或氢氧化铝的佐剂；含寡核苷酸的佐剂，例如含CpG寡脱氧核苷酸(CpG-ODN)的佐剂；含TLR9激动剂的佐剂；含可代谢油、α-生育酚、和/或聚氧 乙烯山梨醇酐单油酸酯(吐温-80)的佐剂，例如水包油乳液形式的含角鲨烯、α-生育酚、和吐温-80和/或Span 85的佐剂；或任意一种佐剂组合。
  26. 根据权利要求1-25中任一项所述的方法，其中该重组亚单位疫苗与一种以上佐剂一起施用，可选地该重组亚单位疫苗作为初始剂(primary series)，额外剂(additional dose)，和/或同源或异源加强剂(booster dose)使用，例如第一剂，第二剂，第三剂，第四剂，和/或更多剂，可选地该初始剂，额外剂，或异源加强剂与其它重组亚单位疫苗、纳米颗粒疫苗、mRNA疫苗、DNA疫苗、腺病毒载体疫苗、和灭活病毒疫苗中的任意一种或几种配合使用，可选地该初始剂，额外剂，和/或同源或异源加强剂中的佐剂可独立地包括：含铝佐剂，例如含明矾和/或氢氧化铝的佐剂；含寡核苷酸的佐剂，例如含CpG寡脱氧核苷酸(CpG-ODN)的佐剂；含TLR9激动剂的佐剂；含可代谢油、α-生育酚、和/或聚氧乙烯山梨醇酐单油酸酯(吐温-80)的佐剂，例如水包油乳液形式的含角鲨烯、α-生育酚、和吐温-80和/或Span 85的佐剂；或任意一种佐剂组合。
  27. 一种用于检测来自哺乳动物血清的冠状病毒抗体的方法，该方法包括将血清与可溶性冠状病毒SARS-CoV-2奥密克戎(B.1.1.529)表面抗原接触的步骤，该可溶性冠状病毒表面抗原通过框内融合连接到胶原的C-末端部分以形成二硫键连接的三聚体融合蛋白。
  28. 根据权利要求27所述的方法，其中该可溶性冠状病毒表面抗原是S蛋白或肽。
  29. 一种使用包含来自冠状病毒SARS-CoV-2奥密克戎(B.1.1.529)的可溶性表面抗原的重组亚单位疫苗的方法，该可溶性表面抗原通过框内融合连接到胶原的C-末端部分以形成二硫键连接的三聚体融合蛋白，该方法包括：免疫哺乳动物，纯化产生的中和抗体，以及使用所述中和抗体通过被动免疫治疗感染所述冠状病毒的患者。
  30. 根据权利要求29所述的方法，其中该中和抗体包括多克隆抗体。
  31. 根据权利要求29所述的方法，其中该中和抗体是单克隆抗体。
  32. 根据权利要求29所述的方法，其中该中和抗体是S蛋白或肽的单克隆抗体。
  33. 根据权利要求29所述的方法，其中该中和抗体是SARS-CoV-2的S蛋白的单克隆抗体。
  34. 根据权利要求29所述的方法，其中该中和抗体是SARS-CoV-2 Hu-1、阿尔法、贝塔、伽马、德尔塔、缪、奥密克戎和或其它毒株的S蛋白的单克隆抗体。
  35. 根据权利要求29所述的方法，其中该中和抗体是SARS-CoV-2奥密克戎(B.1.1.529)的S蛋白的单克隆抗体。
  36. 一种包括选自由SEQ ID NO:85-92组成的组的重组多肽的复合物。
  37. 一种包含选自由SEQ ID NO:85-92组成的组的重组多肽的三聚体的复合物，其中该重组多肽通过多肽间二硫键三聚化以形成三聚体。
  38. 一种免疫原性组合物，该免疫原性组合物包括重组多肽的三聚体或任何两个或更多个三聚体的组合，该重组多肽包括选自由SEQ ID NO:85-92组成的组的序列。
  39. 根据权利要求38所述的免疫原性组合物，包括具有SEQ ID NO:90中所述的序列的重组多肽的三聚体。
  40. 一种用于在受试者中产生对冠状病毒表面抗原的免疫应答的方法，该方法包括向受试者施用有效量的复合物，该复合物包括选自由SEQ ID NO:85-92组成的组的重组多肽，可选地该复合物作为初始剂(primary series)，额外剂(additional dose)，和/或同源或异源加强剂(booster dose)使用，例如第一剂，第二剂，第三剂，第四剂，和/或更多剂，可选地该初始剂，额外剂，或异源加强剂与其它重组亚单位疫苗、纳米颗粒疫苗、mRNA疫苗、DNA疫苗、腺病毒载体疫苗、和灭活病毒疫苗中的任意一种或几种配合使用。
  41. 一种用于在受试者中产生对冠状病毒表面抗原的免疫应答的方法，
    其中该表面抗原包括S蛋白或其抗原片段，以及
    该方法包括向受试者施用有效量的复合物，该复合物包括选自由SEQ ID NO:85-92组成的组的重组多肽，可选地该复合物作为初始剂(primary series)，额外剂(additional dose)，和/或同源或异源加强剂(booster dose)使用，例如第一剂，第二剂，第三剂，第四剂，和/或更多剂，可选地该初始剂，额外剂，或异源加强剂与其它重组亚单位疫苗、纳米颗粒疫苗、mRNA疫苗、DNA疫苗、腺病毒载体疫苗、和灭活病毒疫苗中的任意一种或几种配合使用。
  42. 一种用于在受试者中产生对冠状病毒表面抗原的免疫应答的方法，
    其中该表面抗原包括选自由SEQ ID NO:27-66和81-84组成的组的序列，以及
    该方法包括向受试者施用有效量的复合物，该复合物包括选自由SEQ ID NO:85-92组成的组的重组多肽，可选地该复合物作为初始剂(primary series)，额外剂(additional dose)，和/或同源或异源加强剂(booster dose)使用，例如第一剂，第二剂，第三剂，第四 剂，和/或更多剂，可选地该初始剂，额外剂，或异源加强剂与其它重组亚单位疫苗、纳米颗粒疫苗、mRNA疫苗、DNA疫苗、腺病毒载体疫苗、和灭活病毒疫苗中的任意一种或几种配合使用。
  43. 一种用于在受试者中产生对冠状病毒表面抗原的免疫应答的方法，
    其中该表面抗原包括冠状病毒的S蛋白或其抗原片段，并且可选地，该表面抗原包括选自由SEQ ID NO:27-66和81-84组成的组的序列或其抗原片段，以及
    该方法包括向受试者施用有效量的复合物，该复合物包括包含SEQ ID NO:85-92中的任一个所述的序列的重组多肽，可选地该复合物作为初始剂(primary series)，额外剂(additional dose)，和/或同源或异源加强剂(booster dose)使用，例如第一剂，第二剂，第三剂，第四剂，和/或更多剂，可选地该初始剂，额外剂，或异源加强剂与其它重组亚单位疫苗、纳米颗粒疫苗、mRNA疫苗、DNA疫苗、腺病毒载体疫苗、和灭活病毒疫苗中的任意一种或几种配合使用。
  44. 一种用于在受试者中产生对冠状病毒表面抗原的免疫应答的方法，
    其中该表面抗原包括S蛋白或其抗原片段，以及
    该方法包括向受试者施用有效量的复合物或任何两种或更多种复合物的组合，该复合物包括包含选自由SEQ ID NO:85-92组成的组的序列的重组多肽，可选地该复合物或复合物的组合作为初始剂(primary series)，额外剂(additional dose)，和/或同源或异源加强剂(booster dose)使用，例如第一剂，第二剂，第三剂，第四剂，和/或更多剂，可选地该初始剂，额外剂，或异源加强剂与其它重组亚单位疫苗、纳米颗粒疫苗、mRNA疫苗、DNA疫苗、腺病毒载体疫苗、和灭活病毒疫苗中的任意一种或几种配合使用。
  45. 根据权利要求44所述的方法，其中该方法包括向受试者施用有效量的包括重组多肽的复合物，该重组多肽包括SEQ ID NO:85、SEQ ID NO:86、SEQ ID NO:87和/或SEQ ID NO:88中所述的序列。
  46. 一种包含多个重组多肽的融合蛋白，每个重组多肽从氨基到羧基末端包括：
    a)第一区域，该第一区域包括位于第一冠状病毒的非嵌合冠状病毒刺突蛋白中的冠状病毒刺突蛋白受体结合结构域(RBD)之前的冠状病毒刺突蛋白胞外域的一部分；
    b)第二区域，该第二区域包括不同于所述第一冠状病毒的第二冠状病毒的冠状病毒刺突蛋白受体结合结构域(RBD)；和
    c)胶原的C-末端前肽，其中该重组多肽的C-末端前肽形成多肽间二硫键，其中第一冠状病毒和第二冠状病毒至少有一个是SARS-CoV-2奥密克戎(B.1.1.529)，可选地该融合蛋白作为初始剂(primary series)，额外剂(additional dose)，和/或同源或异源加强剂(booster dose)使用，例如第一剂，第二剂，第三剂，第四剂，和/或更多剂，可选地该初 始剂，额外剂，或异源加强剂与其它重组亚单位疫苗、纳米颗粒疫苗、mRNA疫苗、DNA疫苗、腺病毒载体疫苗、和灭活病毒疫苗中的任意一种或几种配合使用。
  47. 根据权利要求46所述的融合蛋白，还包括该第二区域与该胶原的C-末端前肽之间的第三区域。
  48. 根据权利要求47所述的融合蛋白，其中该第三区域包括第三冠状病毒的S1结构域，其中该第三冠状病毒与第一冠状病毒或第二冠状病毒相同或不同。
  49. 根据权利要求47或48所述的融合蛋白，其中该第三区域包括第四冠状病毒的S2结构域，其中该第四冠状病毒与第一、第二或第四冠状病毒相同或不同。
  50. 根据权利要求46-49中任一项所述的融合蛋白，其中该第一区域包括第一冠状病毒的N-末端结构域(NTD)。
  51. 根据权利要求46-50中任一项所述的融合蛋白，其中该第一区域包括与第二冠状病毒中的相应氨基酸残基不同的一个或更多个氨基酸残基。
  52. 根据权利要求46-51中任一项所述的融合蛋白，其中该第二区域包括与第一冠状病毒中的相应氨基酸残基不同的一个或更多个氨基酸残基。
  53. 根据权利要求46-52中任一项所述的融合蛋白，其中该第一和第二冠状病毒是相同冠状病毒的不同变种或毒株。
  54. 根据权利要求53所述的融合蛋白，其中该第一区域包括第一冠状病毒的NTD，该第二区域包括第二冠状病毒的RBD，并且第一和第二冠状病毒是SARS-CoV-2的不同变种。
  55. 根据权利要求46-54中任一项所述的融合蛋白，其中该第一冠状病毒和第二冠状病毒独立地选自由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和A.23.1谱系的SARS-CoV-2病毒组成的组。
  56. 一种包含三个重组多肽的三聚体融合蛋白，每个重组多肽从氨基到羧基末端包括：
    a)第一区域，该第一区域包括B.1.1.529谱系的SARS-CoV-2的冠状病毒刺突蛋白N-末端结构域(NTD)；
    b)第二区域，该第二区域包括B.1.1.529谱系的SARS-CoV-2的冠状病毒刺突蛋白受体结合结构域(RBD)；和
    c)胶原的C-末端前肽，其中该重组多肽的C-末端前肽形成多肽间二硫键，可选地该三聚体融合蛋白作为初始剂(primary series)，额外剂(additional dose)，和/或同源或异源加强剂(booster dose)使用，例如第一剂，第二剂，第三剂，第四剂，和/或更多剂，可选地该初始剂，额外剂，或异源加强剂与其它重组亚单位疫苗、纳米颗粒疫苗、mRNA疫苗、DNA疫苗、腺病毒载体疫苗、和灭活病毒疫苗中的任意一种或几种配合使用。
  57. 一种用于预防哺乳动物中冠状病毒感染的方法，该方法包括用有效量的根据权利要求46-56中任一项所述的融合蛋白免疫哺乳动物。
  58. 根据权利要求57所述的方法，其中在哺乳动物中产生针对该第一和第二冠状病毒的中和抗体。
  59. 根据权利要求58所述的方法，其中该第一和第二冠状病毒是SARS-CoV-2的不同变种，并且在哺乳动物中产生的中和抗体中和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和A.23.1谱系的两种或更多种SARS-CoV-2病毒。
  60. 根据权利要求59所述的方法，其中在哺乳动物中产生的中和抗体中和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和A.23.1谱系的三种或更多种SARS-CoV-2病毒。
  61. 根据权利要求57-60中任一项所述的方法，包括用两剂或更多剂的融合蛋白免疫哺乳动物，该两剂或更多剂的融合蛋白中至少有一剂的融合蛋白包含SARS-CoV-2奥密克戎(B.1.1.529)刺突蛋白氨基酸序列，可选地该至少一剂融合蛋白包含SEQ ID NO:81-92中的任一个所述的序列或与SEQ ID NO:81-92中的任一个所述的序列有至少或约80％、85％、90％、92％、95％、97％、99％的序列同一性的氨基酸序列。
  62. 根据权利要求57-61中任一项所述的方法，其中在一剂或更多剂的免疫原之后以再次加强剂施用融合蛋白，该免疫原包括包含来自相同或不同SARS-CoV-2变种的NTD和RBD的刺突蛋白肽，可选地该一剂或更多剂的免疫原包含SEQ ID NO:27-66和81-84中的任一个所述的序列或与SEQ ID NO:27-66和81-84中的任一个所述的序列有至少或约80％、85％、90％、92％、95％、97％、99％的序列同一性的氨基酸序列，可选地该加强剂融合蛋白包含SEQ ID NO:81-92中的任一个所述的序列或与SEQ ID NO:81-92中的任一个所述的序列有至少或约80％、85％、90％、92％、95％、97％、99％的序列同一性的氨基酸序列。
  63. 一种三聚体融合蛋白，包括可溶性冠状病毒SARS-CoV-2奥密克戎(B.1.1.529)表面抗原或其片段、变体或突变体，该可溶性冠状病毒表面抗原或其片段、变体或突变体连接到胶原的C-末端部分，该胶原的C-末端部分形成二硫键连接的三聚体，从而形成该三聚体融合蛋白。
  64. 根据权利要求63所述的三聚体融合蛋白，该三聚体融合蛋白包括选自由SEQ ID NO:85-92组成的组的重组多肽。
  65. 根据权利要求63或64所述的三聚体融合蛋白，用于预防冠状病毒感染，可选地该冠状病毒感染包括SARS-CoV-2 Hu-1、阿尔法、贝塔、伽马、德尔塔、缪、奥密克戎和其它SARS-CoV-2毒株中的任意一种或几种感染。
  66. 根据权利要求63-65中任一项所述的三聚体融合蛋白，在没有佐剂的情况下施用，可选地该三聚体融合蛋白作为初始剂(primary series)，额外剂(additional dose)，和/或同源或异源加强剂(booster dose)使用，例如第一剂，第二剂，第三剂，第四剂，和/或更多剂，可选地该初始剂，额外剂，或异源加强剂与其它重组亚单位疫苗、纳米颗粒疫苗、mRNA疫苗、DNA疫苗、腺病毒载体疫苗、和灭活病毒疫苗中的任意一种或几种配合使用，
    可选地该三聚体融合蛋白在没有佐剂的情况下作为初始剂使用，该初始剂包括第一针和第二针,
    可选地该三聚体融合蛋白在没有佐剂的情况下作为额外剂使用，该额外剂包括第三针，第四针，和/或更多针,
    可选地该三聚体融合蛋白在没有佐剂的情况下作为同源或异源加强剂使用，该同源或异源加强剂包括第三针，第四针，和/或更多针。
  67. 根据权利要求63-65中任一项所述的三聚体融合蛋白，其中该三聚体融合蛋白与佐剂一起施用，可选地该三聚体融合蛋白作为初始剂(primary series)，额外剂(additional dose)，和/或同源或异源加强剂(booster dose)使用，例如第一剂，第二剂，第三剂，第四剂，和/或更多剂，可选地该初始剂，额外剂，或异源加强剂与其它重组亚单位疫苗、纳米颗粒疫苗、mRNA疫苗、DNA疫苗、腺病毒载体疫苗、和灭活病毒疫苗中的任意一种或几种配合使用，可选地该初始剂，额外剂，和/或同源或异源加强剂中的佐剂可独立地包括：含铝佐剂，例如含明矾和/或氢氧化铝的佐剂；含寡核苷酸的佐剂，例如含CpG寡脱氧核苷酸(CpG-ODN)的佐剂；含TLR9激动剂的佐剂；含可代谢油、α-生育酚、和/或聚氧乙烯山梨醇酐单油酸酯(吐温-80)的佐剂，例如水包油乳液形式的含角鲨烯、α-生育酚、和吐温-80和/或Span 85的佐剂；或任意一种所述佐剂的组合，
    可选地该三聚体融合蛋白与佐剂一起作为初始剂使用，该初始剂包括第一针和第二针，该佐剂包括Alum和含CpG寡脱氧核苷酸(CpG-ODN)的佐剂和/或水包油乳液形式的含角鲨烯、α-生育酚、和吐温-80和/或Span 85的佐剂,
    可选地该三聚体融合蛋白与佐剂一起作为额外剂使用，该额外剂包括第三针，第四针，和/或更多针，该佐剂包括Alum和含CpG寡脱氧核苷酸(CpG-ODN)的佐剂和/或水包油乳液形式的含角鲨烯、α-生育酚、和吐温-80和/或Span 85的佐剂,
    可选地该三聚体融合蛋白与佐剂一起作为同源或异源加强剂使用，该同源或异源加强剂包括第三针，第四针，和/或更多针，该佐剂包括Alum和含CpG寡脱氧核苷酸(CpG-ODN)的佐剂和/或水包油乳液形式的含角鲨烯、α-生育酚、和吐温-80和/或Span 85的佐剂。
  68. 根据权利要求63-65中任一项所述的三聚体融合蛋白，其中该三聚体融合蛋白与一种以上佐剂一起施用，可选地该三聚体融合蛋白作为初始剂(primary series)，额外剂(additional dose)，和/或同源或异源加强剂(booster dose)使用，例如第一剂，第二剂，第三剂，第四剂，和/或更多剂，可选地该初始剂，额外剂，或异源加强剂与其它重组亚单位疫苗、纳米颗粒疫苗、mRNA疫苗、DNA疫苗、腺病毒载体疫苗、和灭活病毒疫苗中的任意一种或几种配合使用，可选地该初始剂，额外剂，和/或同源或异源加强剂中的佐剂可独立地包括：含铝佐剂，例如含明矾和/或氢氧化铝的佐剂；含寡核苷酸的佐剂，例如含CpG寡脱氧核苷酸(CpG-ODN)的佐剂；含TLR9激动剂的佐剂；含可代谢油、α-生育酚、和/或聚氧乙烯山梨醇酐单油酸酯(吐温-80)的佐剂，例如水包油乳液形式的含角鲨烯、α-生育酚、和吐温-80和/或Span 85的佐剂；或任意一种所述佐剂的组合。
  69. 根据权利要求63-65中任一项所述的三聚体融合蛋白，作为初始剂用于免疫哺乳动物，可选地该初始剂包括第一针和第二针。
  70. 根据权利要求69中所述的三聚体融合蛋白，该第一针和/或第二针没有佐剂。
  71. 根据权利要求69中所述的三聚体融合蛋白，该第一针和/或第二针的佐剂包括：含铝佐剂，例如含明矾和/或氢氧化铝的佐剂；和/或含寡核苷酸的佐剂，例如含CpG寡脱氧核苷酸(CpG-ODN)的佐剂。
  72. 根据权利要求69中所述的三聚体融合蛋白，该第一针和/或第二针的佐剂包括：含可代谢油、α-生育酚、和/或聚氧乙烯山梨醇酐单油酸酯(吐温-80)的佐剂，例如水包油乳液形式的含角鲨烯、α-生育酚、和吐温-80和/或Span 85的佐剂。
  73. 根据权利要求63-72中任一项所述的三聚体融合蛋白，作为第三针，第四针，和/或更多针额外剂用于免疫哺乳动物。
  74. 根据权利要求63-73中任一项所述的三聚体融合蛋白，作为第三针，第四针，和/或更多针加强剂用于免疫哺乳动物,
    可选地该加强剂没有佐剂，
    可选地该加强剂的佐剂包括：含铝佐剂，例如含明矾和/或氢氧化铝的佐剂；和/或含寡核苷酸的佐剂，例如含CpG寡脱氧核苷酸(CpG-ODN)的佐剂，
    可选地该加强剂的佐剂包括含可代谢油、α-生育酚、和/或聚氧乙烯山梨醇酐单油酸酯(吐温-80)的佐剂，例如水包油乳液形式的含角鲨烯、α-生育酚、和吐温-80和/或Span 85的佐剂。
  75. 根据权利要求63-74中任一项所述的三聚体融合蛋白，与其它一种或几种疫苗配合使用，该其它一种或几种疫苗独立选自由重组亚单位疫苗、纳米颗粒疫苗、mRNA疫苗、DNA疫苗、腺病毒载体疫苗、和灭活病毒疫苗所组成的组。
  76. 根据权利要求77中所述的三聚体融合蛋白，其中该三聚体融合蛋白作为初始剂使用，该其它一种或几种疫苗作为加强剂使用。
  77. 根据权利要求77中所述的三聚体融合蛋白，其中该其它一种或几种疫苗作为初始剂使用，该三聚体融合蛋白作为加强剂使用。
  78. 一种二价疫苗，包括：
    第一三聚体融合蛋白，该第一三聚体融合蛋白包括可溶性冠状病毒SARS-CoV-2奥密克戎(B.1.1.529)刺突(S)蛋白表面抗原或其片段、变体或突变体，该可溶性冠状病毒表面抗原或其片段、变体或突变体连接到胶原的C-末端部分，该胶原的C-末端部分形成二硫键连接的三聚体，从而形成该第一三聚体融合蛋白；
    第二三聚体融合蛋白，该第二三聚体融合蛋白包括可溶性冠状病毒SARS-CoV-2 Hu-1、阿尔法、贝塔、伽马、德尔塔、或缪刺突(S)蛋白表面抗原或其片段、变体或突变体，该可溶性冠状病毒表面抗原或其片段、变体或突变体连接到胶原的C-末端部分，该胶原的C-末端部分形成二硫键连接的三聚体，从而形成该第二三聚体融合蛋白。
  79. 根据权利要求80所述的二价疫苗，该第二三聚体融合蛋白包括可溶性冠状病毒SARS-CoV-2 Hu-1刺突(S)蛋白表面抗原或其片段、变体或突变体。
  80. 根据权利要求80或81所述的二价疫苗，该二价疫苗在没有佐剂的情况下施用，可选地该二价疫苗作为初始剂(primary series)，额外剂(additional dose)，和/或同源或异源加强剂(booster dose)使用，例如第一剂，第二剂，第三剂，第四剂，和/或更多剂，可选 地该初始剂，额外剂，或异源加强剂与其它重组亚单位疫苗、纳米颗粒疫苗、mRNA疫苗、DNA疫苗、腺病毒载体疫苗、和灭活病毒疫苗中的任意一种或几种配合使用。
  81. 根据权利要求80-82中任一项所述的二价疫苗，该二价疫苗与一种以上佐剂一起施用，可选地该二价疫苗作为初始剂(primary series)，额外剂(additional dose)，和/或同源或异源加强剂(booster dose)使用，例如第一剂，第二剂，第三剂，第四剂，和/或更多剂，可选地该初始剂，额外剂，或异源加强剂与其它重组亚单位疫苗、纳米颗粒疫苗、mRNA疫苗、DNA疫苗、腺病毒载体疫苗、和灭活病毒疫苗中的任意一种或几种配合使用，可选地该初始剂，额外剂，和/或同源或异源加强剂中的佐剂可独立地包括：含铝佐剂，例如含明矾和/或氢氧化铝的佐剂；含寡核苷酸的佐剂，例如含CpG寡脱氧核苷酸(CpG-ODN)的佐剂；含TLR9激动剂的佐剂；含可代谢油、α-生育酚、和/或聚氧乙烯山梨醇酐单油酸酯(吐温-80)的佐剂，例如水包油乳液形式的含角鲨烯、α-生育酚、和吐温-80和/或Span 85的佐剂；或任意一种所述佐剂的组合。
  82. 根据权利要求80-83中任一项所述的二价疫苗，该二价疫苗中的第一三聚体融合蛋白包括SEQ ID NO:81-84中的任一个所述的序列或与SEQ ID NO:81-84中的任一个所述的序列有至少或约80％、85％、90％、92％、95％、97％、99％的序列同一性的氨基酸序列。
  83. 根据权利要求80-84中任一项所述的二价疫苗，该二价疫苗中的第二三聚体融合蛋白包括SEQ ID NO:27-66中的任一个所述的序列或与SEQ ID NO:27-66中的任一个所述的序列有至少或约80％、85％、90％、92％、95％、97％、99％的序列同一性的氨基酸序列。
  84. 一种多价疫苗，其包括自由选自可溶性冠状病毒Hu-1，SARS-CoV-2奥密克戎(B.1.1.529)，奥密克戎毒株(BA.5)，阿尔法、贝塔、伽马、德尔塔、或缪刺突(S)蛋白表面抗原或其片段、变体或突变体，该可溶性冠状病毒表面抗原或其片段、变体或突变体连接到胶原的C-末端部分形成二硫键连接的三聚体融合蛋白。
  85. 根据权利要求86中所述的多价疫苗包括所述可溶性冠状病毒Hu-1和奥密克戎毒株(BA.5)刺突(S)蛋白表面抗原或其片段、变体或突变体。
  86. 根据权利要求86中所述的多价疫苗包括所述可溶性冠状病毒SARS-CoV-2奥密克戎(B.1.1.529)和奥密克戎毒株(BA.5)刺突(S)蛋白表面抗原或其片段、变体或突变体。
  87. 根据权利要求86中所述的多价疫苗包括所述可溶性冠状病毒贝塔，德尔塔和奥密克戎毒株(BA.5)刺突(S)蛋白表面抗原或其片段、变体或突变体。
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