WO2023179514A1

WO2023179514A1 - Coronavirus vaccine composition, method, and use thereof

Info

Publication number: WO2023179514A1
Application number: PCT/CN2023/082378
Authority: WO
Inventors: 梁朋; 梁果; 宿丹梅
Original assignee: 四川三叶草生物制药有限公司
Priority date: 2022-03-24
Filing date: 2023-03-19
Publication date: 2023-09-28
Also published as: CN116836293A; CN118401565A

Abstract

Provided are an immunogenic composition comprising a recombinant peptide and a protein, the recombinant peptide and the protein comprising a coronavirus antigen and an immunogen, such as SARS-CoV -2 coronavirus delta (delta, B.1.617.2) variant strain S protein peptide, or a fragment, variant or mutant thereof, such as a chimeric antigen and immunogen containing a delta variant strain receptor binding domain and a Hu-1 or other variant strain S protein peptide sequence. In some aspects, the immunogenic composition comprises a secretory fusion protein. The secretory fusion protein comprises a soluble coronavirus antigen. The soluble coronavirus antigen protein is fused by intra-frame fusion to a C-terminal portion capable of self-trimerization, so as to form a disulfide bond-linked trimer fusion protein. In some aspects, the provided immunogenic composition may be used to produce an immune response, e.g. as a vaccine for the prevention of coronavirus infection, such as infection of SARS-CoV-2 Hu-1, alpha, beta, gamma, delta, mu, omicron, and/or other strains. In some aspects, the provided immunogenic composition can be used in a vaccine composition, for example, as part of a prophylactic and/or therapeutic vaccine. Further provided are a method for producing the recombinant peptide and the protein, a prophylactic, therapeutic and/or diagnostic method, and a related kit.

Description

Coronavirus vaccine compositions, methods and uses

Technical field

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 Delta (B.1.617.2) strain formed by disulfide bonds between trimerized ^TM tag polypeptides.

Background technique

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.

Contents of the invention

Beginning in late 2020, the emergence and spread of multiple variant SARS-CoV-2 strains carrying mutations that may lead to immune escape necessitated the rapid evaluation of second-generation vaccines with the aim of inducing optimized immunity with broad protection answer.

Despite tremendous progress and unprecedented speed in COVID-19 vaccine development, daily COVID-19 cases hit a new record in April 2021, 16 months after the SARS-CoV-2 virus outbreak first emerged. The total number of deaths caused by COVID-19 exceeded 3 million in May 2021, with a cumulative death count of 1 million in the first 3 months alone.

Most worryingly, multiple new SARS-CoV-2 variants (VOCs) began to emerge in late 2020 while COVID-19 cases were surging globally. These VOCs appear to be associated with mutations in the spike (S) protein that may increase viral transmission rates and/or immune evasion due to the first wave of COVID-19 vaccinations based on the SARS-CoV-2 Hu-1 strain. The emergence and spread of the B.1.1.7 variant in the United Kingdom (UK), the B.1.351 variant in South Africa, and the P.1 variant in Brazil have led to their classification as VOCs. These VOCs all include the N501Y mutation in the receptor-binding domain (RBD) of the S protein, which has been reported to increase transmission by 40% to 70%. 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.

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

While there is some encouraging evidence that the Hu-1 COVID-19 vaccine may prevent severe illness and death caused by VOCs, lower vaccine effectiveness against any COVID-19 disease with increased transmission rates may make Achieving herd immunity is particularly difficult, and a global shortage of COVID-19 vaccines is likely to further exacerbate the problem. If not effectively controlled, the rapid spread of SARS-CoV-2 VOCs around the world may lead to the continued emergence of new target variants or VOCs that may contain new escape mutations, such as the Indian variant (B.1.617), which is related to the 2021 This coincided with a massive surge in COVID-19 cases in the spring and has now been declared a new VOC by the WHO. The B.1.617.2 variant of this lineage is named Delta. Recently, the B.1.1.529 variant first discovered in South Africa was renamed Omicron.

In these cases, rebooster strategies must be rapidly evaluated to enhance neutralizing antibody responses to VOCs and to develop second-generation COVID-19 vaccines that may offer optimized broad protection and cross-neutralizing properties.

In some embodiments, disclosed herein are proteins comprising a plurality of recombinant polypeptides, each recombinant polypeptide comprising a coronavirus surface antigen linked to a C-terminal propeptide of collagen, wherein the C-terminal 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.

In some embodiments, disclosed herein are recombinant subunit vaccines that include an extracellular domain (e.g., no transmembrane and cytoplasmic domain), which is fused in frame to the C-propeptide of collagen capable of forming disulfide-linked homotrimers. The resulting recombinant subunit vaccines (e.g., S-trimers) can be derived from expressed and purified in transfected cells and is expected to be in the native conformation of the trimer form. This resolves a problem often encountered when viral antigens are expressed in soluble form as recombinant peptides or proteins without transmembrane and/or cytoplasmic domains Viral antigen misfolding problem. This misfolded viral antigen cannot faithfully maintain the native viral antigen conformation and often fails to produce neutralizing antibodies.

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

In any of the foregoing embodiments, 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 .

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

In any of the foregoing embodiments, 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.

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

In any of the foregoing embodiments, the surface antigen may include the S1 subunit and the S2 subunit of the S protein.

In any of the foregoing embodiments, the surface antigen may be free of transmembrane (TM) domain peptides and/or cytoplasmic (CP) domain peptides.

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

In any of the foregoing embodiments, 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.

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

In any of the foregoing embodiments, the surface antigens may be the same or different in the recombinant polypeptides of the protein.

In any of the foregoing embodiments, 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.

In any of the foregoing embodiments, the protein may be soluble or not directly bound to a lipid bilayer, such as a membrane or viral envelope.

In any of the foregoing embodiments, 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.

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

In any of the preceding embodiments, the C-terminal propeptide may be human collagen.

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

In any of the foregoing embodiments, the C-terminal propeptides may be the same or different in the recombinant polypeptides.

In any of the foregoing embodiments, the C-terminal propeptide may comprise any one of SEQ ID NOs: 67-80, or be at least 90%, 91%, 92%, 93% identical to any one of SEQ ID NOs: 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.

In any of the foregoing embodiments, 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.

In any of the foregoing embodiments, 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.

In any of the foregoing embodiments, 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.

In any of the foregoing embodiments, 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.

In any of the foregoing embodiments, 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.

In any of the foregoing embodiments, 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.

In any of the foregoing embodiments, 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.

In any of the foregoing embodiments, 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.

In any of the foregoing embodiments, 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.

In any of the foregoing embodiments, 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.

In any of the foregoing embodiments, 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.

In any of the foregoing embodiments, 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.

In any of the foregoing embodiments, 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.

In any of the foregoing embodiments, 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.

In any of the foregoing embodiments, 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.

In any of the foregoing embodiments, the surface antigen in each recombinant polypeptide can be in a prefusion conformation.

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

In any of the foregoing embodiments, 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 that is at least 80% identical thereto.

In any of the foregoing embodiments, 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.

Also provided herein are immunogens comprising proteins provided herein. Provided herein are protein nanoparticles comprising a protein provided herein linked directly or indirectly to the nanoparticle. Provided herein are virus-like particles (VLPs) comprising proteins provided herein.

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

In some embodiments, 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.

Also provided herein are vectors containing the isolated nucleic acids provided herein. In some embodiments, the vector is a viral vector.

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

Also provided herein are vaccines comprising an immunogenic composition provided herein and an optional adjuvant, wherein the vaccine is optionally a subunit vaccine. In some embodiments, the vaccine is a prophylactic and/or therapeutic vaccine. Optional adjuvants may be used in priming and/or boosting doses. Independently, 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, α-tocopherol, and Tween-80 and/or Span 85 in the form of an oil-in-water emulsion; or any combination of adjuvants.

In some aspects, 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. Provided herein are proteins produced by the methods provided herein.

Provided herein are methods 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. In some embodiments, the methods provided herein are used to treat or prevent coronavirus infection. In some embodiments, generating an immune response inhibits or reduces replication of the coronavirus in the subject. In some embodiments, 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. In some embodiments, 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. In some embodiments, administration to the subject does not result in antibody-dependent enhancement (ADE) in the subject due to prior exposure to one or more coronaviruses. In some embodiments, administration does not result in antibody-dependent enhancement (ADE) in the subject when subsequently exposed to one or more coronaviruses. In some embodiments, the method further includes a priming step and/or a boosting step. Boosting step. In some embodiments, 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. In some embodiments, 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.

Provided herein are methods 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. In some embodiments, the subject is a mammal, optionally a human or a non-human primate. In some embodiments, the method further includes isolating neutralizing antibodies or neutralizing antisera from the subject. In some embodiments, 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. In some embodiments, 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. In some embodiments, 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.

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

In some aspects, provided herein are uses of proteins, immunogens, protein nanoparticles, VLPs, isolated nucleic acids, vectors, viruses, pseudoviruses, cells, immunogenic compositions, or vaccines provided herein for inducing in a subject immune response to coronavirus, and/or used to treat or prevent coronavirus infection. In some aspects, provided herein are the use of a protein, immunogen, protein nanoparticle, VLP, isolated nucleic acid, vector, virus, pseudovirus, cell, immunogenic composition, or vaccine provided herein for the manufacture of a 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. In some embodiments, the analyte is an antibody, receptor or cell that recognizes a spike protein peptide or fragment or epitope thereof. In some embodiments, binding indicates the presence of the analyte in the sample, and/or the presence of a coronavirus infection in the subject from which the sample is derived.

Provided herein are kits comprising a protein provided herein and a matrix, pad or vial containing or immobilizing the protein, optionally wherein the kit is an ELISA or lateral flow assay kit.

Description of the drawings

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 serum-free culture highly expressing exemplary SARS-CoV-2 delta S-trimers. Reduced SDS-PAGE Coomassie Brilliant Blue staining is used to analyze secreted delta S-trimer protein. When CHO cells are cultured in serum-free and fed conditions for 13 days (D13), more than 1g/L delta can be obtained. S-trimeric protein.

Figures 3A-3B illustrate the purification and characterization of exemplary covalently linked delta S-trimers. Figure 3A shows the delta S-trimer purification process sample (loading amount 2 μg) and control substance (chimeric Hu-1 S-trimer containing beta variant RBD, loading amount 2 μg). Reduction SDS-PAGE Coomassie Brilliant Blue staining analysis, the order of loading is: Serum freee medium after depth filtration to remove cell debris (HCCF: serum freee medium after depth filtration to remove cell debris), affinity chromatography (AC-E: affinity capture chomatography elution) , Ultrafiltration (UF/DF-1-F: ultrafiltration/diafiltration 1fraction), virus inactivation (VIN: viral inactivation with low pH treatment), intermediate depth filtration (IDF: Intermediate Depth Filtration), ion exchange (AEX: Anion Exchange Chromatography), nanofiltration (NF: Nanofiltration), ultrafiltration (UF/DF-2-F: Ultrafiltration/Diafiltration 2 Fraction). Figure 3B shows SEC-HPLC purity analysis, and the purity of delta S-trimer is 97.22%.

Figures 4A-4B show the receptor binding kinetics of delta S-trimer to ACE2-Fc. Figure 4A shows that Hu-1 S-trimer binds to the receptor with a Kd of 5 nM. Figure 4B shows that delta S-trimer binds to the receptor with a Kd of 0.5 nM. Delta S-trimer has higher receptor affinity than Hu-1 S-trimer.

Figure 5 shows BALB/c mouse SARS-CoV-2 Hu-1, alpha (alpha, α, B.1.1.7), beta (beta, β, B.1.351), gamma (gamma, γ, P.1), delta (delta, δ, B.1.617.2), mu (mu, μ, B.1.621) and Omicro (Omicro, o, B.1.1.529) strain pseudoviruses and antibody _EC50 data. Hu-1 S-trimer or Delta S-trimer (n=8/group) were treated with CpG 1018 plus Alum as adjuvant on Day 0 (primer) and Day 21 (booster). Mice were immunized twice. Blood was collected from the mice on day 0, and blood and splenocytes were collected on day 35 for pseudovirus neutralizing antibodies (Figure 5) and cell-mediated immunity (CMI) testing (Figures 6A-6B). The Hu-1 S-trimer group served as the control group, and the mice were immunized twice with 150 μg CpG 1018 plus 75 μg Alum as an adjuvant and 3 μg Hu-1 S-trimer. The delta S-trimer group immunized mice twice with 3 μg of delta S-trimer in 150 μg of CpG 1018 plus 75 μg of Alum as an adjuvant. In post-booster samples of mice (day 35), delta S-trimer induced higher levels of Omicron-neutralizing antibody titers than Hu-1 S-trimer induced Omicron Chron neutralizing antibody titers were 10 times higher.

Figures 6A-6B show assessment of cell-mediated immunity (CMI) by ELISpot in mice receiving two immunizations of priming and boosting doses. The Hu-1 strain S1 peptide library or Hu-1 strain S2 peptide library was used to stimulate mouse splenocytes collected on day 35 of the Hu-1 S-trimer or Delta S-trimer group, and Th1 cytokines (IL-2 and IFNγ) or Th2 cytokines (IL-4 and IL-5) in mouse splenocytes were detected by ELISpot. Figure 6A shows the ELISpot results of Hu-1 strain S1 peptide library stimulation (n=8/group mean). Figure 6B shows the ELISpot results of Hu-1 strain S2 peptide library stimulation (n=8/group mean). The vertical axis is the number of ELISpots per 250,000 splenocytes.

Figures 7A-7G show VOC neutralizing antibodies in dose 3 vaccination boosted mice. 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 μg of Hu-1 S-trimer adjuvanted with 150 μg of CpG 1018 plus 75 μg of Alum. In research On day 57 of the study, animals were divided into three groups (n=10 per group) that received no or Hu-1 S-trimer or Delta S-trimer as the 3rd dose of vaccine. Serum was collected on study day 56 (D-1 PD3) and day 71 (D14 PD3) for pseudovirus neutralizing antibody testing. Group 1 did not receive the third dose of vaccination and served as the control group; Group 2: 3 μg Hu-1 S-trimer with 150 μg CpG 1018 plus 75 μg Alum as adjuvant was used as the third dose of vaccine; Group 3 Group: Vaccination with 150 μg CpG 1018 plus 75 μg Alum as adjuvant with 3 μg Delta S-Trimer as dose 3. Figures 7B-7G are Hu-1, Alpha, Beta, Gamma, Delta, and Omicron strain pseudovirus neutralizing antibody (EC 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 (EC ₅₀ ) is 20. The group number is marked at the bottom of each column. The fold increase in neutralizing antibody titers after dose 3 (D14 PD3) for each VOC compared to data before dose 3 is marked as an "x".

Detailed ways

Provided herein are immunogenic compositions, methods and uses of fusion peptides and proteins comprising coronavirus antigens or immunogens for the treatment (e.g., prophylactic, therapeutic) of coronavirus infections. In some embodiments, compositions and methods of using recombinant soluble surface antigens from covalently linked trimeric forms of RNA viruses are disclosed. In some embodiments, 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.

In some embodiments, 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. If adjuvants are used, optional 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.

In some embodiments, 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).

In some embodiments, 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).

In some embodiments, 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.

In some embodiments, 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 practical In 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.

In some embodiments, disclosed herein are monovalent vaccines comprising an S-trimer disclosed herein. In some embodiments, disclosed herein are bivalent vaccines comprising an S-trimer disclosed herein. In some embodiments, disclosed herein are bivalent vaccines comprising at least one S-trimer comprising a first S protein antigen and at least one S-trimer comprising a second S protein antigen. In some embodiments, the first and second S protein antigens are 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. In some embodiments, disclosed herein are trivalent vaccines comprising an S-trimer disclosed herein. In some embodiments, disclosed herein is 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. In some embodiments, the first, second and third S protein antigens 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. In some embodiments, disclosed herein are quadrivalent vaccines comprising an S-trimer disclosed herein. In some embodiments, disclosed herein is 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. - A quadrivalent vaccine of trimers and at least one S-trimer comprising a fourth S protein antigen. In some embodiments, the first, second, third and fourth S protein antigens are 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.

The proteins including coronavirus antigens and immunogens provided herein, including recombinant polypeptides and fusion proteins, can be used to effectively and safely treat (e.g., therapeutic, prophylactic) coronavirus infections. For example, 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). Furthermore, the proteins comprising coronavirus antigens and immunogens provided herein are easily produced and exhibit stability under high stress conditions such as high temperature, extreme pH, high osmolarity and low osmosis. Accordingly, the proteins and immunogenic compositions provided herein circumvent and satisfy the production, stability, safety, and efficacy issues that have hindered coronavirus vaccine development.

In some aspects, coronavirus antigens and immunogens provided herein include coronavirus spike (S) proteins or peptides, particularly SARS-CoV or SARS-CoV-2 S proteins. The spikes of SARS-CoV and SARS-CoV-2 are composed of S protein trimers, which belong to the group of viral fusion glycoproteins of class I, which also includes HIV glycoprotein 160 (Env), influenza hemagglutination protein (HA), paramyxovirus F and Ebola virus glycoprotein. 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.

In some embodiments, provided herein are recombinant S ectodomain trimers. In some embodiments, the recombinant S ectodomain trimer includes a recombinant S ectodomain protomer from an alpha-coronavirus (eg, NL63-CoV or 229E-CoV). In some embodiments, 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.

Similar to other enveloped RNA viruses (e.g., HIV, RSV, and influenza), 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. Like 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.

In some embodiments, a protein comprising a coronavirus antigen or immunogen (e.g., SARS-CoV or SARS-CoV-2 S protein peptide) is capable of generating an immune response, e.g., to SARS-CoV or SARS-CoV-2 S protein Immune responses to peptides. In some embodiments, the immune response inhibits or reduces replication of the coronavirus in a subject (eg, a patient). In some embodiments, the immune response includes the production of one or more neutralizing antibodies, such as polyclonal and/or monoclonal antibodies. In some embodiments, neutralizing antibodies inhibit or reduce replication of coronavirus in a subject (eg, a patient). In some embodiments, administration of the protein to a subject, for example as an immunogenic composition, does not result in antibody-dependent enhancement (ADE) in the subject due to prior exposure to coronavirus. In some aspects, proteins containing coronavirus antigens and immunogens are used as vaccines.

In some embodiments, coronavirus antigens and immunogens (e.g., SARS-CoV or SARS-CoV-2 S protein peptides) are linked to proteins or peptides to form fusion proteins or recombinant polypeptides. In some embodiments, the protein or peptide linked to 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.

In some embodiments, coronavirus antigens and immunogens (eg, coronavirus S protein peptide) 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. Accordingly, in some embodiments, 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. In some embodiments, the propeptide of collagen is derived from the human C-propeptide of α1 collagen and is capable of self-trimerization after expression.

In some embodiments, linking coronavirus antigens and immunogens (eg, coronavirus S protein peptide) to the propeptide of collagen (eg, at the C-terminus of the propeptide of collagen) contributes to the protein's ability to generate an immune response. For example, 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. In addition, connecting the coronavirus S protein peptide to a protein or peptide capable of self-trimerization allows the recombinant protein to aggregate, thereby mimicking the natural homotrimeric structure of the coronavirus S protein peptide on the viral envelope.

In some embodiments, linking the coronavirus S protein peptide to the C-terminal propeptide of collagen results in a self-trimerizing recombinant polypeptide. In some embodiments, the proteins provided herein include multiple self-trimerized propeptides of coronavirus S protein peptides and collagen recombinant polypeptides. In some embodiments, the trimeric nature of the recombinant protein contributes to protein stability. In some embodiments, the trimeric nature of the recombinant protein contributes to the protein's ability to generate an immune response. In some embodiments, 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.

Also provided herein are 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.

All publications, including patent documents, scientific articles and databases mentioned in this application are incorporated by reference in their entirety for all purposes to the same extent as if each individual publication was individually incorporated by reference. To the extent that a definition set forth herein is contrary or inconsistent with a definition set forth in a patent, application, published application, and other publication incorporated by reference, the definition set forth herein shall prevail over the definition set forth herein by reference. The section headings used in this article are for organizational purposes only and should not be construed as limiting the subject matter described.

I. Viral Antigens and Immunogens

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 (α, β, γ, δ), of which β-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. Non-limiting examples of 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). 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:

■ Bat coronavirus Cp/Yunnan2011

■ Bat coronavirus RaTG13

■ Bat coronavirus Rp/Shaanxi2011

○ Bat SARS coronavirus HKU3

■ Bat SARS coronavirus HKU3-1

■ Bat SARS coronavirus HKU3-10

■ Bat SARS coronavirus HKU3-11

■ Bat SARS coronavirus HKU3-12

■ Bat SARS coronavirus HKU3-13

■ Bat SARS coronavirus HKU3-2

■ Bat SARS coronavirus HKU3-3

■ Bat SARS coronavirus HKU3-4

■ Bat SARS coronavirus HKU3-5

■ Bat SARS coronavirus HKU3-6

■ Bat SARS coronavirus HKU3-7

■ Bat SARS coronavirus HKU3-8

■ Bat SARS coronavirus HKU3-9

■ Bat SARS coronavirus Rp1

■ Bat SARS coronavirus Rp2

○ Bat SARS CoV Rf1/2004

■ Bat CoV 273/2005

○ Bat SARS CoV Rm1/2004

■Bat CoV 279/2005

■ Bat SARS CoV Rp3/2004

■ Bat SARS-like coronavirus

■ Bat SARS-like coronavirus Rs3367

■ Bat SARS-like coronavirus RsSHC014

■ Bat SARS-like coronavirus WIV1

■ Bat SARS-like coronavirus YNLF_31C ■ Bat SARS-like coronavirus YNLF_34C ■ BtRf-BetaCoV/HeB2013

■ BtRf-BetaCoV/JL2012

■ BtRf-BetaCoV/SX2013

■ BtRs-BetaCoV/GX2013

■ BtRs-BetaCoV/HuB2013

■ BtRs-BetaCoV/YN2013

■ Civet SARS CoV 007/2004

■ Civet SARS CoV SZ16/2003

■ Civet SARS CoV SZ3/2003

○ Recombinant SARSr-CoV

■ SARS coronavirus ExoN1

■ SARS coronavirus MA15

■ SARS coronavirus MA15 ExoN1

■ SARS coronavirus wtic-MB

■ Rhinoceros bat coronavirus

■ SARS bat coronavirus

■ SARS coronavirus A001

■ SARS coronavirus A013

■ SARS coronavirus A021

■ SARS coronavirus A022

■ SARS coronavirus A030

■ SARS coronavirus A031

■ SARS coronavirus AS

■ SARS coronavirus B012

■ SARS coronavirus B024

■ SARS coronavirus B029

■ SARS coronavirus B033

■ SARS coronavirus B039

■ SARS coronavirus B040

■ SARS coronavirus BJ01

■ SARS coronavirus BJ02

■ SARS coronavirus BJ03

■ SARS coronavirus BJ04

■ SARS coronavirus BJ162

■ SARS coronavirus BJ182-12

■ SARS coronavirus BJ182-4

■ SARS coronavirus BJ182-8

■ SARS coronavirus BJ182a

■ SARS coronavirus BJ182b

■ SARS coronavirus BJ202

■ SARS coronavirus BJ2232

■ SARS coronavirus BJ302

■ SARS coronavirus C013

■ SARS coronavirus C014

■ SARS coronavirus C017

■ SARS coronavirus C018

■ SARS coronavirus C019

■ SARS coronavirus C025

■ SARS coronavirus C028

■ SARS coronavirus C029

■ SARS coronavirus CDC#200301157

■ SARS coronavirus civet010

■ SARS coronavirus civet014

■ SARS coronavirus civet019

■ SARS coronavirus civet020

■ SARS coronavirus CS21

■ SARS coronavirus CS24

■ SARS coronavirus CUHK-AG01

■ SARS coronavirus CUHK-AG02

■ SARS coronavirus CUHK-AG03

■ SARS coronavirus CUHK-L2

■ SARS coronavirus CUHK-Su10

■ SARS coronavirus CUHK-W1

■ SARS coronavirus cw037

■ SARS coronavirus cw049

■ SARS coronavirus ES191

■ SARS coronavirus ES260

■ SARS coronavirus FRA

○ SARS coronavirus Frankfurt 1

■ SARS coronavirus Frankfurt1-v01

■ SARS coronavirus GD01

■ SARS coronavirus GD03T0013

■ SARS coronavirus GD322

■ SARS coronavirus GD69

■ SARS coronavirus GDH-BJH01

■ SARS coronavirus GZ-A

■ SARS coronavirus GZ-B

■ SARS coronavirus GZ-C

■ SARS coronavirus GZ-D

■ SARS coronavirus GZ02

■ SARS coronavirus GZ0401

■ SARS coronavirus GZ0402

■ SARS coronavirus GZ0403

■ SARS coronavirus GZ43

■ SARS coronavirus GZ50

■ SARS coronavirus GZ60

■ SARS coronavirus HB

■ SARS coronavirus HC/SZ/61/03

■ SARS coronavirus HGZ8L1-A

■ SARS coronavirus HGZ8L1-B

■ SARS coronavirus HGZ8L2

■ SARS coronavirus HHS-2004

■ SARS coronavirus HKU-36871

■ SARS coronavirus HKU-39849

■ SARS coronavirus HKU-65806

■ SARS coronavirus HKU-66078

■ SARS coronavirusHong Kong/03/2003

■ SARS coronavirus HPZ-2003

■ SARS coronavirus HSR 1

■ SARS coronavirus HSZ-A

■ SARS coronavirus HSZ-Bb

■ SARS coronavirus HSZ-Bc

■ SARS coronavirus HSZ-Cb

■ SARS coronavirus HSZ-Cc

■ SARS coronavirus HSZ2-A

■ SARS coronavirus HZS2-Bb

■ SARS coronavirus HZS2-C

■ SARS coronavirus HZS2-D

■ SARS coronavirus HZS2-E

■ SARS coronavirus HZS2-Fb

■ SARS coronavirus HZS2-Fc

■ SARS coronavirus JMD

■ SARS coronavirus LC1

■ SARS coronavirus LC2

■ SARS coronavirus LC3

■ SARS coronavirus LC4

■ SARS coronavirus LC5

■ SARS coronavirus LLJ-2004

■ SARS coronavirus NS-1

■ SARS coronavirus P2

■ SARS coronavirus PC4-115

■ SARS coronavirus PC4-127

■ SARS coronavirus PC4-13

■ SARS coronavirus PC4-136

■ SARS coronavirus PC4-137

■ SARS coronavirus PC4-145

■ SARS coronavirus PC4-199

■ SARS coronavirus PC4-205

■ SARS coronavirus PC4-227

■ SARS coronavirus PC4-241

■ SARS coronavirus PUMC01

■ SARS coronavirus PUMC02

■ SARS coronavirus PUMC03

■ SARS coronavirus Rs_672/2006

■ SARS coronavirus sf098

■ SARS coronavirus sf099

■ SARS coronavirusShanghaiQXC1

■ SARS coronavirusShanghaiQXC2

■ SARS coronavirusShanhgai LY

■ SARS coronavirus Sin0409

■ SARS coronavirus Sin2500

■ SARS coronavirus Sin2677

■ SARS coronavirus Sin2679

■ SARS coronavirus Sin2748

■ SARS coronavirus Sin2774

■ SARS coronavirus Sin3408

■ SARS coronavirus Sin3408L

■ SARS coronavirus Sin3725V

■ SARS coronavirus Sin3765V

■ SARS coronavirus Sin842

■ SARS coronavirus Sin845

■ SARS coronavirus Sin846

■ SARS coronavirus Sin847

■ SARS coronavirus Sin848

■ SARS coronavirus Sin849

■ SARS coronavirus Sin850

■ SARS coronavirus Sin852

■ SARS coronavirus Sin_WNV

■ SARS coronavirus Sino1-11

■ SARS coronavirus Sino3-11

■ SARS coronavirus SinP1

■ SARS coronavirus SinP2

■ SARS coronavirus SinP3

■ SARS coronavirus SinP4

■ SARS coronavirus SinP5

■ SARS coronavirus SoD

■ SARS coronavirus SZ1

■ SARS coronavirus SZ13

■ SARS coronavirus Taiwan

■ SARS coronavirus Taiwan JC-2003

■ SARS coronavirus Taiwan TC1

■ SARS coronavirus Taiwan TC2

■ SARS coronavirus Taiwan TC3

■ SARS coronavirus TJ01

■ SARS coronavirus TJF

■ SARS coronavirus Tor2

○ SARS coronavirus TW

■ SARS coronavirus TW-GD1

■ SARS coronavirus TW-GD2

■ SARS coronavirus TW-GD3

■ SARS coronavirus TW-GD4

■ SARS coronavirus TW-GD5

■ SARS coronavirus TW-HP1

■ SARS coronavirus TW-HP2

■ SARS coronavirus TW-HP3

■ SARS coronavirus TW-HP4

■ SARS coronavirus TW-JC2

■ SARS coronavirus TW-KC1

■ SARS coronavirus TW-KC3

■ SARS coronavirus TW-PH1

■ SARS coronavirus TW-PH2

■ SARS coronavirus TW-YM1

■ SARS coronavirus TW-YM2

■ SARS coronavirus TW-YM3

■ SARS coronavirus TW-YM4

■ SARS coronavirus TW1

■ SARS coronavirus TW10

■ SARS coronavirus TW11

■ SARS coronavirus TW2

■ SARS coronavirus TW3

■ SARS coronavirus TW4

■ SARS coronavirus TW5

■ SARS coronavirus TW6

■ SARS coronavirus TW7

■ SARS coronavirus TW8

■ SARS coronavirus TW9

■ SARS coronavirus TWC

■ SARS coronavirus TWC2

■ SARS coronavirus TWC3

■ SARS coronavirus TWH

■ SARS coronavirus TWJ

■ SARS coronavirus TWK

■ SARS coronavirus TWS

■ SARS coronavirus TWY

■ SARS coronavirus Urbani

■ SARS coronavirusVietnam

■ SARS coronavirus WF188

■ SARS coronavirus WH20

■ SARS coronavirus WHU

■ SARS coronavirus xw002

■ SARS coronavirus ZJ01

■ SARS coronavirus ZJ02

■ SARS coronavirus ZJ0301

■ SARS coronavirus ZMY 1

■ SARS coronavirus ZS-A

■ SARS coronavirus ZS-B

■ SARS coronavirus ZS-C

■ SARS-related bat coronavirus RsSHC014

■ SARS-related beta-coronavirus Rp3/2004

■ Severe acute respiratory syndrome coronavirus 2

The table below shows exemplary SARS CoV-2 strains.

The 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. 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. 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. .

In some cases, 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 structural conformation adopted by S prior to the fusion event in which S transitions to the post-fusion conformation. The three-dimensional structure of an exemplary coronavirus S protein (HKU1-CoV) in the 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.

In some cases, 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). Furthermore, 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.

In some cases, the coronavirus antigen or immunogen is a fragment of the S protein peptide. In some embodiments, the antigen or immunogen is an epitope of 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. For example, 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. In some embodiments, the coronavirus epitope is a linear epitope. In some embodiments, the coronavirus epitope is a conformational epitope. In some embodiments, the coronavirus epitope is a neutralizing epitope site. In some embodiments, all neutralizing epitopes of the coronavirus S protein peptide or fragment thereof are present as antigens or immunogens.

In some cases, for example, 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. In some embodiments, the viral antigen or immunogen includes a signal peptide, an S1 subunit peptide, an S2 subunit peptide, or any combination thereof. In some embodiments, the viral antigen or immunogen includes a signal peptide, a receptor binding domain (RBD) peptide, a receptor binding motif (RBM) peptide, a fusion peptide (FP), a heptad repeat 1 (HR1) peptide or heptad repeat 2 (HR2) peptide or any combination thereof. In some embodiments, the viral antigen or immunogen includes the receptor binding domain (RBD) of the S protein. In some embodiments, the viral antigen or immunogen includes the S1 and S2 subunits of the S protein. In some embodiments, the viral antigen or immunogen includes the S1 subunit of the S protein, but does not include the S2 subunit. In some embodiments, the viral antigen or immunogen includes the S2 subunit of the S protein, but does not include the S1 subunit. In some embodiments, the viral antigen or immunogen does not contain transmembrane (TM) domain peptides and/or cytoplasmic (CP) domain peptides.

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

In some embodiments, 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.

In some embodiments, the viral antigen or immunogen is a SARS-CoV-2 antigen comprising at least one SARS-CoV-2 protein or fragment thereof. In some embodiments, the SARS-CoV-2 antigen is recognized by SARS-CoV-2 reactive antibodies and/or T cells. In some embodiments, the SARS-CoV-2 antigen is inactivated whole virus. In some embodiments, SARS-CoV-2 antigens include subunits of the virus. In some embodiments, the SARS-CoV-2 antigen includes a structural protein of SARS-CoV-2 or a fragment thereof. In some embodiments, 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. In some embodiments, 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, for all purposes The full text is incorporated by reference).

In some embodiments, 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:83 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.

SEQ ID NO:83：

In some embodiments, the viral antigen or immunogen includes the sequence set forth in SEQ ID NO: 55. In some embodiments, the viral antigen or immunogen includes a receptor binding motif ( RBM)) An amino acid sequence having at least or about 80%, 85%, 90%, 92%, 95% or 97% sequence identity. 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.

SEQ ID NO:55：

In some embodiments, 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 (ΔFVI-Spike) virus. In some embodiments, the viral antigen or immunogen includes the spike glycoprotein sequence of a virus in the B.1.1.7 lineage. In some embodiments, 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 immunogen includes the spike glycoprotein sequence of a virus in the B.1.351 lineage. In some embodiments, 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. In some embodiments, 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. In some embodiments, 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. Viruses in the 1.351 lineage, Any two or more viruses from the group consisting of viruses in the P.2 lineage, viruses in the B.1.1.143 lineage, viruses in the A.23.1 lineage and viruses in the B.1.617 lineage (in any appropriate combination) of the spike glycoprotein sequence.

In some embodiments, viral antigens or immunogens include T95I, G142D, Δ143-145, and/or T478K, e.g., as in B.1.617.2 Delta and/or B.1.1.529 Omicron variants .

In some embodiments, the viral antigen or immunogen includes E484K and/or S477N, for example, as in the B.1.526 variant. In some embodiments, the viral antigen or immunogen includes Δ400-402 (ΔFVI), for example, as in the Cluster 5 (ΔFVI-Spike) variant. In some embodiments, viral antigens or immunogens include Δ69-70 (ΔHV), Δ144 (ΔY), N501Y, A570D, D614G, P681H, T716I, S982A, and/or D1118H, for example, as in the B.1.1.7 variant middle. In some embodiments, the viral antigen or immunogen includes P681H, for example, as in the B.1.1.207 variant. In some embodiments, viral antigens or immunogens include L18F, T20N, P26S, D138Y, R190S, K417T, E484K, N501Y, D614G, H655Y, T1027I, and/or V1176F, for example, as in the P.1 variant. In some embodiments, the viral antigen or immunogen includes E484K, for example, as in the P.2 variant. In some embodiments, the viral antigen or immunogen includes E484K and/or N501Y, for example, as in the P.3 variant. In some embodiments, viral antigens or immunogens include L18F, D80A, D215G, Δ242-244 (ΔLAL), R246I, K417N, E484K, N501Y, D614G, and/or A701V, for example, as in the B.1.351 variant. In some embodiments, the viral antigen or immunogen includes S13I, W152C and/or L452R, for example, as in the B.1.429/CAL.20C variant. In some embodiments, the viral antigen or immunogen includes Δ69-70 (ΔHV), E484K, and/or F888L, for example, as in the B.1.525 variant. In some embodiments, 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 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. In some embodiments, 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. In some embodiments, 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. In some embodiments, a viral antigen or immunogen can include a mixture of trimers, and each trimer can include different mutations and/or different combinations of mutations.

In some embodiments, the viral antigen or immunogen includes amino acid positions 13, 18, 20, 26, 69, 70, 80, 138, 142, 144, 152, 190, 215, 242, 243, 244, 246, 400, 401, 402, 417, 440, 452, 477, 484, 501, 570, 614, 655, 681, 682, 683, 684, 685, 701, 716, 888, 982, 1027, 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. In some embodiments, 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. In some embodiments, the viral antigen or immune Epizootics include chimeric polypeptides comprising sequences from different viruses, such as one or more mutations from a first variant of a coronavirus and one or more mutations from a second variant of a coronavirus that is different from the first variant. In some embodiments, such chimeric viral antigens or immunogens (or combinations of chimeric viral antigens or immunogens) can be used to elicit a broad immune response against the first and second variants of the coronavirus.

In some embodiments, viral antigens or immunogens include selected from the group consisting of T95I, G142D, Δ143-145, T478K, S13I, L18F, T20N, P26S, Δ69-70 (ΔHV), D80A, D138Y, G142D, Δ144 (ΔY), W152C, R190S, D215G, Δ242-244(ΔLAL), R246I, Δ400-402(ΔFVI), K417T, 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 V1176F. In some embodiments, 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.

In some embodiments, the SARS-CoV-2 antigen includes a truncated S protein lacking a signal peptide, the transmembrane and cytoplasmic domains of the full-length S protein. In some embodiments, the SARS-CoV-2 antigen is a recombinant protein, while in other embodiments, the SARS-CoV-2 antigen is purified from virions. In some preferred embodiments, the SARS-CoV-2 antigen is an isolated antigen.

In some embodiments, 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, 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: 55), Deleted and/or inserted sequences. In some embodiments, 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, 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 , any one, two, three, four, five or more mutations in the group consisting of , P681R, R682G, R683S, R685G, A701V, T716I, F888L, S982A, T1027I, D1118H and V1176F.

In some embodiments, 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, 681, 682, 683, 684, 685, 701, 716, 888, 982, 1027, 1118 and 1176 (about SEQ ID NO: 55 Sequences containing substitutions, deletions and/or insertions at one or more amino acid positions in the group consisting of (amino acid positions). In some embodiments, 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, 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 , any one, two, three, four, five or more mutations in the group consisting of , P681R, R682G, R683S, R685G, A701V, T716I, F888L, S982A, T1027I, D1118H and V1176F.

In some embodiments, 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, 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: 55), Deleted and/or inserted sequences. In some embodiments, 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, 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 , any one, two, three, four, five or more mutations in the group consisting of , P681R, R682G, R683S, R685G, A701V, T716I, F888L, S982A, T1027I, D1118H and V1176F.

In some embodiments, 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, 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: 55), Deleted and/or inserted sequences. In some embodiments, 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, 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 , any one, two, three, four, five or more mutations in the group consisting of , P681R, R682G, R683S, R685G, A701V, T716I, F888L, S982A, T1027I, D1118H and V1176F.

In some embodiments, 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: 55), Deleted and/or inserted sequences. In some embodiments, 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, 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 , any one, two, three, four, five or more mutations in the group consisting of , P681R, R682G, R683S, R685G, A701V, T716I, F888L, S982A, T1027I, D1118H and V1176F.

In some embodiments, 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 %, 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: 55), Deleted and/or inserted sequences. In some embodiments, 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, 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 , any one, two, three, four, five or more mutations in the group consisting of , P681R, R682G, R683S, R685G, A701V, T716I, F888L, S982A, T1027I, D1118H and V1176F.

In some embodiments, 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, 681, 682, 683, 684, 685, Sequences containing substitutions, deletions and/or insertions at one or more amino acid positions in the group consisting of 701, 716, 888, 982, 1027, 1118 and 1176 (amino acid positions with respect to SEQ ID NO:55). In some embodiments, 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, 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 , any one, two, three, four, five or more mutations in the group consisting of , P681R, R682G, R683S, R685G, A701V, T716I, F888L, S982A, T1027I, D1118H and V1176F.

In some embodiments, 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 %, 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: 55), Deleted and/or inserted sequences. In some embodiments, 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, 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 , any one, two, three, four, five or more mutations in the group consisting of , P681R, R682G, R683S, R685G, A701V, T716I, F888L, S982A, T1027I, D1118H and V1176F.

In some embodiments, 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, 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: 55), Deleted and/or inserted sequences. In some embodiments, 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, 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 , any one, two, three, four, five or more mutations in the group consisting of , P681R, R682G, R683S, R685G, A701V, T716I, F888L, S982A, T1027I, D1118H and V1176F.

In some embodiments, 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: 55), Deleted and/or inserted sequences. In some embodiments, 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, 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 , any one, two, three, four, five or more mutations in the group consisting of , P681R, R682G, R683S, R685G, A701V, T716I, F888L, S982A, T1027I, D1118H and V1176F.

In some embodiments, 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 %, 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: 55), Deleted and/or inserted sequences. In some embodiments, the viral antigen or immunogen includes a variant of SEQ ID NO: 37, and the variant includes a variant selected from the group consisting of S13I, L18F, T20N, P26S, Δ69-70 (ΔHV), D80A, D138Y, G142D, Δ144( ΔY), W152C, R190S, D215G, Δ242-244(ΔLAL), R246I, Δ400-402(ΔFVI), K417T, K417N, N440K, L452R, S477N, S477G, E484K, E484Q, N501Y, A570D, D614G , H655Y, P681H , any one, two, three, four, five or more mutations in the group consisting of , P681R, R682G, R683S, R685G, A701V, T716I, F888L, S982A, T1027I, D1118H and V1176F.

In some embodiments, 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, 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: 55), Deleted and/or inserted sequences. In some embodiments , 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, 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, P681 H, 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 V1176F.

In some embodiments, 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: 55), Deleted and/or inserted sequences. In some embodiments, 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, 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 , any one, two, three, four, five or more mutations in the group consisting of , P681R, R682G, R683S, R685G, A701V, T716I, F888L, S982A, T1027I, D1118H and V1176F.

In some embodiments, 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 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: 55), Deleted and/or inserted sequences. In some embodiments, 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, 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 , any one, two, three, four, five or more mutations in the group consisting of , P681R, R682G, R683S, R685G, A701V, T716I, F888L, S982A, T1027I, D1118H and V1176F.

In some embodiments, 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 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 in the group consisting of NO: 55). In some embodiments, 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, 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 , any one, two, three, four, five or more mutations in the group consisting of , P681R, R682G, R683S, R685G, A701V, T716I, F888L, S982A, T1027I, D1118H and V1176F.

In some embodiments, 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, 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: 55), Deleted and/or inserted sequences. In some embodiments, 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, 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 , any one, two, three, four, five or more mutations in the group consisting of , P681R, R682G, R683S, R685G, A701V, T716I, F888L, S982A, T1027I, D1118H and V1176F.

In some embodiments, 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, 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: 55), Deleted and/or inserted sequences. In some embodiments, 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, 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, P681 R, R682G, R683S, Any one, two, three, four, five or more mutations in the group consisting of R685G, A701V, T716I, F888L, S982A, T1027I, D1118H and V1176F.

In some embodiments, 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: 55), Deleted and/or inserted sequences. In some embodiments, 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, 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 , any one, two, three, four, five or more mutations in the group consisting of , P681R, R682G, R683S, R685G, A701V, T716I, F888L, S982A, T1027I, D1118H and V1176F.

In some embodiments, 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, 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: 55), Deleted and/or inserted sequences. In some embodiments, 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, 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 , any one, two, three, four, five or more mutations in the group consisting of , P681R, R682G, R683S, R685G, A701V, T716I, F888L, S982A, T1027I, D1118H and V1176F.

In some embodiments, 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 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, Contains at one or more amino acid positions in the group consisting of 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) Substituted, deleted and/or inserted sequences. In some embodiments, 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, 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 , any one, two, three, four, five or more mutations in the group consisting of , P681R, R682G, R683S, R685G, A701V, T716I, F888L, S982A, T1027I, D1118H and V1176F.

In some embodiments, 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 %, 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: 55), Deleted and/or inserted sequences. In some embodiments, 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, 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 , any one, two, three, four, five or more mutations in the group consisting of , P681R, R682G, R683S, R685G, A701V, T716I, F888L, S982A, T1027I, D1118H and V1176F.

In some embodiments, 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, 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: 55), Deleted and/or inserted sequences. In some embodiments, 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, 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 , any one, two, three, four, five or more mutations in the group consisting of , P681R, R682G, R683S, R685G, A701V, T716I, F888L, S982A, T1027I, D1118H and V1176F.

In some embodiments, 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: 55), Deleted and/or inserted sequences. In some embodiments, 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, 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 , any one, two, three, four, five or more mutations in the group consisting of , P681R, R682G, R683S, R685G, A701V, T716I, F888L, S982A, T1027I, D1118H and V1176F.

In some embodiments, 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 %, 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: 55), Deleted and/or inserted sequences. In some embodiments, 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, 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 , any one, two, three, four, five or more mutations in the group consisting of , P681R, R682G, R683S, R685G, A701V, T716I, F888L, S982A, T1027I, D1118H and V1176F.

In some embodiments, 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, 681, 682, 683, 684, 685, Sequences containing substitutions, deletions and/or insertions at one or more amino acid positions in the group consisting of 701, 716, 888, 982, 1027, 1118 and 1176 (amino acid positions with respect to SEQ ID NO:55). In some embodiments, 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, 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 , any one, two, three, four, five or more mutations in the group consisting of , P681R, R682G, R683S, R685G, A701V, T716I, F888L, S982A, T1027I, D1118H and V1176F.

In some embodiments, 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.

In some embodiments, 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. 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: 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 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, Sequences containing substitutions, deletions and/or insertions 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. In some embodiments, 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, 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 , any one, two, three, four, five or more mutations in the group consisting of , P681R, R682G, R683S, R685G, A701V, T716I, F888L, S982A, T1027I, D1118H and V1176F.

In some embodiments, 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 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 in the group consisting of NO: 55). In some embodiments, 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, 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 , any one, two, three, four, five or more mutations in the group consisting of , P681R, R682G, R683S, R685G, A701V, T716I, F888L, S982A, T1027I, D1118H and V1176F.

In some embodiments, 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.

In some embodiments, 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.

In some embodiments, 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.

In some embodiments, 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.

In some embodiments, 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%, Amino acid sequences with 95%, 96%, 97%, 98% or 99% sequence identity include sequences containing substitutions, deletions and/or insertions at one or more amino acid positions of SEQ ID NO:62.

In some embodiments, 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.

In some embodiments, 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.

In some embodiments, 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.

In some embodiments, 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.

In some embodiments, 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.

In some embodiments, 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.

In some embodiments, 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%, Amino acid sequences with 95%, 96%, 97%, 98% or 99% sequence identity include sequences containing substitutions, deletions and/or insertions at one or more amino acid positions of SEQ ID NO:84.

In some embodiments, the viral antigen or immunogen does not include a transmembrane domain such as SEQ ID NO: 66 or a portion thereof. In some embodiments, the coronavirus antigen or immunogen includes soluble S protein peptide. In some embodiments, the soluble protein S peptide lacks the TM domain peptide and the CP domain peptide. In some embodiments, soluble S protein peptides do not bind to lipid bilayers, such as membranes or viral envelopes.

In some embodiments, 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.

In some embodiments, 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. In some cases, 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. If adjuvants are used, optional 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.

II. Recombinant Peptides and Proteins

It is contemplated that coronavirus antigens and immunogens provided herein, such as spike protein peptides (see Section I), can be combined with, e.g., linked to, other proteins or peptides to form recombinant polypeptides, including fusion peptides. In some embodiments, individual recombinant polypeptides (eg, monomers) provided herein combine to form multimers, eg, trimers, of recombinant polypeptides. In some embodiments, association of individual recombinant polypeptide monomers occurs through covalent interactions. In some embodiments, binding of individual recombinant polypeptide monomers occurs through non-covalent interactions. In some embodiments, the interaction (eg, covalent or non-covalent) is affected by a protein or peptide linked to a coronavirus antigen or immunogen (eg, spike protein peptide). In some embodiments, for example, when 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. For example, retention and/or maintenance of the native conformation of a coronavirus antigen or immunogen (eg, spike protein peptide) may improve or allow access to antigenic sites capable of generating an immune response. In some cases, a recombinant polypeptide comprising a S protein peptide described herein (eg, see Section I) is alternatively referred to herein as a recombinant S antigen, a recombinant S immunogen, or a recombinant S protein.

It is further contemplated that, in some cases, 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. For example, 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. . in some In this case, aggregation may confer structural stability to the coronavirus antigen or immunogen, which in turn may provide access to potential antigenic sites capable of promoting an immune response.

1. Fusion peptides and recombinant peptides

In some embodiments, a coronavirus antigen or immunogen can be linked to a trimerization domain at its C-terminus (C-terminal linkage) to promote trimerization of monomers. In some embodiments, 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. 1998 Protein Eng 11:329- 414), any of which can be linked to the coronavirus antigen or immunogen described herein (e.g., by linking to the C terminus of the S peptide) to promote trimerization of the recombinant viral antigen or immunogen, the above literature is based on All purposes are incorporated by reference in its entirety. See also U.S. Patent Nos. 7,268,116, 7,666,837, 7,691,815, 10,618,949, 10,906,944, and 10,960,070, and U.S. 2020/0009244, which patents are incorporated herein by reference in their entirety for all purposes.

In some embodiments, one or more peptide linkers (eg, glycine-serine linkers, eg, 10 amino acid glycine-serine peptide linkers) can be used to link the recombinant viral antigen or immunogen to the multimerization domain. As long as the recombinant viral antigen or immunogen trimer retains the desired properties (eg, prefusion conformation), the trimer may include any of the stabilizing mutations provided herein (or combinations thereof). In some embodiments, 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. In some embodiments, 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. In some embodiments, the linker includes a sequence comprising glycine-X-Y repeats.

To be therapeutically feasible, 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.

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 non-collagenous domains (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. 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 μg/mL without obvious abnormalities, indicating that C-propeptide is non-toxic. Structural studies of the trimeric C-propeptide of collagen indicate that it is a three-lobed structure with all three subunits coming together in a linking region near its N-terminus to the rest of the procollagen molecule. The geometry of this protruding protein fuses in one direction similar to that of an Fc dimer.

Collagen types I, IV, V and XI are mainly assembled into two α-1 chains and one α-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 α-1 chains. For type I collagen, the most abundant form of collagen, stable α(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. It is believed that 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) have also been fused to the N-terminus of procollagen or mature collagen to form a new collagen matrix, with the purpose of slowly releasing non-collagenous proteins from the cellular matrix. However, in both cases, C-propeptide needs to be cleaved before reorganized collagen fibers can be assembled into an insoluble cellular matrix.

Although other protein trimerization domains, such as GCN4 from the yeast fibritin of bacteriophage T4 and those from the aspartate transcarbamylase of Escherichia coli , have been previously described allowing for trimerization of heterologous proteins, polymerize, but none of these trimeric proteins are human in nature, nor are they naturally secreted proteins. Therefore, 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. Furthermore, a fatal drawback of using such non-human protein trimerization domains (e.g., from yeast, bacteriophages, and bacteria) in trimer biopharmaceutical design is their putative immunogenicity in humans, making such fusion proteins It loses effectiveness soon after being injected into the human body.

Therefore, the use of 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 Down the BMP1 The recognition site can be mutated or deleted to prevent the destruction of the trimeric fusion protein; (6) The C-propeptide domain self-trimerizes through disulfide bonds, and it provides a universal affinity tag that can be used for purification Any secreted fusion protein produced. In some embodiments, the C-propeptide of collagen combined with coronavirus antigens and immunogens (eg, spike protein peptides) can be recombinantly produced to produce soluble, covalently linked homotrimeric fusion proteins.

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

For example, 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.

In some embodiments, 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 One or more or all of -671, Cys743-749, Cys738-760, Cys840-851, Cys1032-1043 and Cys1082-1126, in any suitable combination. In some embodiments, the fusion polypeptide in the trimer can include one or more glycosylation sites (e.g., Asn-linked), 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.

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

In some embodiments, 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. In some embodiments, 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.

In some embodiments, 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. In some embodiments, 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 SEQ ID The amino acid sequence described in 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.

In some embodiments, 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 an amino acid sequence that has at least or about 85%, 90%, 92%, 95%, or 97% sequence identity to SEQ ID NO:78. In some embodiments, the C-terminal propeptide is or is the amino acid sequence set forth in SEQ ID NO:79. In some embodiments, 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.

In some embodiments, the C-terminal propeptide is or includes the amino acid sequence of a collagen trimerization domain (e.g., the C-propeptide of human α1(I) collagen) with an aspartic acid 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 . In some embodiments, the C-terminal propeptide is or includes the amino acid sequence of a collagen trimerization domain (e.g., the C-propeptide of human α1(I) collagen) 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. In some embodiments, a C-terminal propeptide herein may include a mutated BMP-1 site, e.g., RSAN instead of DDAN. In some embodiments, 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) ( For example, SEQ ID NO: 68 or 69) may be used in the fusion polypeptides disclosed herein. For example, SEQ ID NO:27 (underlined) or a fragment, variant or mutant thereof 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:

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

In some embodiments, the C-terminal propeptide can include a sequence comprising a glycine-X-Y repeat, wherein X and Y are independently any amino acid, or are at least 85%, 90%, 92%, 95%, or 97% identical thereto The amino acid sequence can form disulfide bonds between polypeptides and trimerize the recombinant polypeptide. In some embodiments, X and Y are independently proline or hydroxyproline.

In some cases where the S protein peptide is linked to the C-terminal propeptide to form a recombinant polypeptide, the recombinant polypeptide forms a trimer, thereby forming a homotrimer of the S protein peptide. In some embodiments, the S protein peptide of the trimerized recombinant polypeptide is in a prefusion conformation. In some embodiments, the S protein peptide of the trimerized recombinant polypeptide is in a post-fusion conformation. In some embodiments, the validation status allows access to different antigenic sites on the S protein peptide. In some embodiments, the antigenic site is an epitope, such as a linear epitope or a conformational epitope. The advantage of having the trimerized recombinant polypeptide is that immune responses can be carried out against a variety of potential and diverse antigenic sites.

In some embodiments, 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. In some embodiments, 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.

In some embodiments, 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.

In some embodiments, the immunogen includes recombinant SARS-CoV or SARS-CoV-2 S ectodomain trimer, such as SARS-CoV-2 Delta B.1.617.2 coronavirus S ectodomain trimer, which includes 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 Amino acid substitutions stabilize the S ectodomain trimer in its prefusion conformation. In some such embodiments, one or more (e.g., two, e.g., two consecutive) 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.

In some embodiments, one or more (e.g., two, e.g., two consecutive) proline substitutions stabilize the coronavirus (e.g., SARS-CoV or SARS-CoV-2) S cells in a prefusion conformation. Extracellular trimers, such as SARS-CoV-2 Delta B.1.617.2 coronavirus S ectodomain trimers. In some embodiments, the SARS-CoV-2 S protein peptide includes a mutation of 986K/987V to 986P/987P.

In some embodiments, 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. In some embodiments, the SARS-CoV-2 S protein peptide includes a mutation from 685R to 685A. Exemplary protease cleavage sites for various viruses are shown below:

In some embodiments, 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.

Referring to the SARS-CoV-2 S protein sequence provided as SEQ ID NO:55, 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). 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.

In some embodiments, a pre-fusion stabilized protomer of the SARS-CoV-2 S ectodomain trimer, e.g., a protomer of the SARS-CoV-2 Delta B.1.617.2 coronavirus S ectodomain trimer The body may have the C-terminal residue of NTD, RBD, S1 (at S1/S2 site 1 or S1/S2 site 2), FP, IFP, HR1, HR2 or the C-terminal residue of the extracellular domain ( It can 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. It will be appreciated that some residues (e.g., up to 10) on the N-terminus and C-terminus of any ectodomain fragment can be removed or modified in the disclosed immunogens without reducing S ectodomain trimerization. body as an immunogen.

In some embodiments, 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 SARS-CoV or SARS-CoV-2 S protein NTD peptide and RBD peptide. 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 SARS-CoV or SARS-CoV-2 S protein.

An exemplary 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.

An exemplary 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.

An exemplary SARS-CoV-2 S recombinant polypeptide without a signal peptide is provided in SEQ ID NO:85 (1507aa):

The above-mentioned SARS-CoV-2 S recombinant polypeptide may include the N-terminal signal peptide provided in SEQ ID NO: 54 (MFVFLVLLPLLVSS).

An exemplary SARS-CoV-2 S recombinant polypeptide without a signal peptide is provided in SEQ ID NO:86 (1507aa):

An exemplary SARS-CoV-2 S recombinant polypeptide without a signal peptide is provided in SEQ ID NO: 87 (1507aa):

An exemplary SARS-CoV-2 S recombinant polypeptide without a signal peptide is provided in SEQ ID NO:88 (1507aa):

In some embodiments, 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: 55) or any combination thereof) , deleted and/or inserted sequences. In some embodiments, 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, 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, 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 and V1176F, or any combination thereof.

In some embodiments, 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 a substitution 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: 55 or any combination thereof) , deleted and/or inserted sequences. In some embodiments, 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, 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, H6 55Y, 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 V1176F, or any combination thereof.

In some embodiments, 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: 55) or any combination thereof) , deleted and/or inserted sequences. In some embodiments, 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, 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, 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 and V1176F, or any combination thereof.

In some embodiments, 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: 55) or any combination thereof) , deleted and/or inserted sequences. In some embodiments, 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, 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, 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 and V1176F, or any combination thereof.

In some embodiments, the recombinant polypeptide is or includes the sequence set forth in SEQ ID NO:5. 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:5 , 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, Sequences containing substitutions, deletions and/or insertions at 684, 685, 701, 716, 888, 982, 1027, 1118 and/or 1176 (with respect to the amino acid position of SEQ ID NO: 55 or any combination thereof). In some embodiments, the recombinant polypeptide is or includes a variant of SEQ ID NO: 5, and the variant includes a variant selected from the group consisting of S13I, L18F, T20N, P26S, Δ69-70(ΔHV), D80A, D138Y, G142D, Δ144(ΔY ), W152C, R190S, D215G, Δ242-244(ΔLAL), R246I, Δ400-402(ΔFVI), K417T, K417N, N440K, L452R, S477N, S477G, E484K, E484Q, N501Y, A570D, D614G, 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 and V1176F, or any combination thereof.

In some embodiments, 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: 55) or any combination thereof) , deleted and/or inserted sequences. In some embodiments, 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, 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, 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 and V1176F, or any combination thereof.

In some embodiments, 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% of SEQ ID NO:7 , 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 amino acid position of SEQ ID NO: 55 or any combination thereof) , deleted and/or inserted sequences. In some embodiments, 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, 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, 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 and V1176F, or any combination thereof.

In some embodiments, 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%, 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: 55) or any combination thereof) , deleted and/or inserted sequences. In some embodiments, 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, 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, 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 and V1176F, or any combination thereof.

In some embodiments, 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: 55) or any combination thereof) , deleted and/or inserted sequences. In some embodiments, 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, 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, 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 and V1176F, or any combination thereof.

In some embodiments, the recombinant polypeptide is or includes the sequence set forth in SEQ ID NO:10. 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: 10 , 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 amino acid position of SEQ ID NO: 55 or any combination thereof) , deleted and/or inserted sequences. In some embodiments, recombinant The 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, 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, R6 82G, R683S, R685G Any one, two, three, four, five or more mutations in the group consisting of , A701V, T716I, F888L, S982A, T1027I, D1118H and V1176F, or any combination thereof.

In some embodiments, 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: 55) or any combination thereof) , deleted and/or inserted sequences. In some embodiments, 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, 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, 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 and V1176F, or any combination thereof.

In some embodiments, 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: 55) or any combination thereof) , deleted and/or inserted sequences. In some embodiments, 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, 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, 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 and V1176F, or any combination thereof.

In some embodiments, 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%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% Sequence identity includes amino acid sequences at one or more amino acid positions (e.g., 13, 18, 20, 26, 69, 70, 80, 138, 142, 144, 152, 190, 215, 242, 243, 244, 246, 400, 401, 402, 417, 440, 452, 477, 484, 501, 570, 614, 655, 681, 682, 683, 684, 685, 701, 716, 888, 982, 1027, 1118 and/or Sequences containing substitutions, deletions and/or insertions at 1176 (amino acid position with respect to SEQ ID NO: 55) or any combination thereof). In some embodiments, 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, 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, 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 and V1176F, or any combination thereof.

In some embodiments, 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: 55) or any combination thereof) , deleted and/or inserted sequences. In some embodiments, 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, 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, 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 and V1176F, or any combination thereof.

In some embodiments, 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 a substitution 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: 55 or any combination thereof) , deleted and/or inserted sequences. In some embodiments, 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, 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, R68 2G, R683S, R685G, Any one, two, three, four, five or more mutations of the group consisting of A701V, T716I, F888L, S982A, T1027I, D1118H and V1176F, or any combination thereof.

In some embodiments, 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: 55) or any combination thereof) , deleted and/or inserted sequences. In some embodiments, 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, 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, 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 and V1176F, or any combination thereof.

In some embodiments, 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: 55) or any combination thereof) , deleted and/or inserted sequences. In some embodiments, 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, 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, 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 and V1176F, or any combination thereof.

In some embodiments, 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 to an amino acid sequence, including at one or more amino acid positions (e.g. 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 (relating to the amino acid position of SEQ ID NO: 55) or any combination thereof) Sequences containing substitutions, deletions and/or insertions. In some embodiments, 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, 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, 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 and V1176F, or any combination thereof.

In some embodiments, 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: 55) or any combination thereof) , deleted and/or inserted sequences. In some embodiments, 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, 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, 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 and V1176F, or any combination thereof.

In some embodiments, 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 amino acid position of SEQ ID NO: 55 or any combination thereof) , deleted and/or inserted sequences. In some embodiments, 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, 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, H6 55Y, 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 V1176F, or any combination thereof.

In some embodiments, 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 position of SEQ ID NO: 55) or any combination thereof) , deleted and/or inserted sequences. In some embodiments, 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, 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, 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 and V1176F, or any combination thereof.

In some embodiments, 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: 55) or any combination thereof) , deleted and/or inserted sequences. In some embodiments, 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, 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, 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 and V1176F, or any combination thereof.

In some embodiments, 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, 570, 614, 655, 681, 682, Sequences containing substitutions, deletions and/or insertions at 683, 684, 685, 701, 716, 888, 982, 1027, 1118 and/or 1176 (with respect to the amino acid positions of SEQ ID NO: 55 or any combination thereof). In some embodiments, the recombinant polypeptide is or includes a variant of SEQ ID NO: 23, and the variant includes a variant selected from the group consisting of S13I, L18F, T20N, P26S, Δ69-70 (ΔHV), D80A, D138Y, G142D, Δ144 (ΔY ), W152C, R190S, D215G, Δ242-244(ΔLAL), R246I, Δ400-402(ΔFVI), K417T, K417N, N440K, L452R, S477N, S477G, E484K, E484Q, N501Y, A570D, D614G, 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 and V1176F, or any combination thereof.

In some embodiments, 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 amino acid position of SEQ ID NO: 55) or any combination thereof) , deleted and/or inserted sequences. In some embodiments, 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, 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, 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 and V1176F, or any combination thereof.

In some embodiments, 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% identical to SEQ ID NO:25 , 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 amino acid position of SEQ ID NO: 55 or any combination thereof) , deleted and/or inserted sequences. In some embodiments, 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, 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, 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 and V1176F, or any combination thereof.

In some embodiments, 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% 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: 26 Sequences containing substitutions, deletions and/or insertions at amino acid positions.

In some embodiments, 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.

In some embodiments, 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.

In some embodiments, 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.

In some embodiments, 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.

In some embodiments, 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.

In some embodiments, 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% identical to SEQ ID NO:90 , an amino acid sequence of 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity, including one or more of SEQ ID NO: 90 Sequences containing substitutions, deletions and/or insertions at amino acid positions.

In some embodiments, 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% with SEQ ID NO:91 , 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: 91 Sequences containing substitutions, deletions and/or insertions at amino acid positions.

In some embodiments, 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.

As noted above, in some embodiments, 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. In some embodiments, the protein formed has a macroscopic structure. In some cases, 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.

In some embodiments, trimeric recombinant polypeptides aggregate to form a protein comprising multiple trimeric recombinant polypeptides. In some embodiments, multiple trimerized recombinant polypeptides form a protein with a macroscopic structure.

In some embodiments, a protein comprising a plurality of recombinant polypeptides described herein is an immunogen. In some embodiments, a protein comprising a plurality of recombinant polypeptides described herein is comprised in a nanoparticle. For example, in some embodiments, the protein is directly attached to the nanoparticle, such as a protein nanoparticle. In some embodiments, the protein is indirectly linked to the nanoparticle. In some embodiments, a protein comprising a plurality of recombinant polypeptides described herein is contained in a virus-like particle (VLP).

In some embodiments, provided herein are complexes comprising a recombinant polypeptide selected from the group consisting of SEQ ID NOs: 1-26 and 85-92, or fragments, variants or mutants thereof, in any suitable combination. In some embodiments, provided herein are complexes comprising a recombinant polypeptide selected from the group consisting of SEQ ID NOs: 1-26 and 85-92, or a trimer of fragments, variants or mutants thereof, wherein the recombinant polypeptide Trimers are formed through trimerization of disulfide bonds between polypeptides.

In some embodiments, provided herein are fusion proteins comprising a plurality of recombinant polypeptides, each recombinant polypeptide comprising from amino to carboxyl terminus: a) a first region that includes a non-chimeric coronavirus spike located on a first coronavirus a part of the coronavirus spike protein extracellular domain preceding the coronavirus spike protein receptor binding domain (RBD) in the protein; b) a second region that includes a second coronavirus that is different from the first coronavirus coronavirus spike protein receptor binding domain (RBD); and c) C-terminal propeptide of collagen, wherein the C-terminal propeptide of the recombinant polypeptide forms an inter-polypeptide disulfide bond. In some embodiments, the fusion protein further includes a third region between the second region and the C-terminal propeptide of collagen. In some embodiments, 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. In some embodiments, 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. In some embodiments, the first region includes the N-terminal domain (NTD) of the first coronavirus. exist In some embodiments, the first region includes one or more amino acid residues that are different from the corresponding amino acid residue in the second coronavirus. In some embodiments, the second region includes one or more amino acid residues that are different from the corresponding amino acid residue in the first coronavirus. In some embodiments, the first and second coronaviruses are different variants or strains of the same coronavirus. In some embodiments, the first region includes the NTD of the first coronavirus, the second region includes the RBD of the second coronavirus, and the first and second coronaviruses are different variants of SARS-CoV-2. In some embodiments, the first coronavirus and the second coronavirus are independently selected from the group consisting of B.1.1.529, B.1.617.2, B.1.526, B.1.1.143, P.2, B.1.351, P. 1. The group consisting of SARS-CoV-2 viruses of the B.1.1.7, B.1.617 and A.23.1 lineages.

In some embodiments, provided herein are 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. In some embodiments, provided herein are 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.1.617.2 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.1.617.2 lineage or non-B.1.617.2 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. In some embodiments, provided herein are trimeric fusion proteins comprising three recombinant polypeptides, each recombinant polypeptide including from amino to carboxyl terminus: a) a first region that includes B.1.617.2 lineage or non-B. The N-terminal domain (NTD) of the coronavirus spike protein of SARS-CoV-2 of lineage 1.617.2; b) the second region, which includes the coronavirus spike of SARS-CoV-2 of lineage B.1.617.2 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.

In some embodiments, 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. In some embodiments, neutralizing antibodies are produced in the mammal against the first and second coronaviruses. In some embodiments, 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. In some embodiments, 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 three or more SARS-CoV-2 viruses. In some embodiments, methods include immunizing the mammal with two or more doses of the fusion protein. In some embodiments, 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.

In some embodiments, provided herein are engineered fusion polypeptides derived or modified from the spike (S) glycoprotein of coronaviruses, including SARS-CoV-1 and SARS-CoV-2. In some embodiments, the fusion polypeptides disclosed herein can be stabilized in a pre-fusion conformation compared to the wild-type S protein sequence of the coronavirus. In some embodiments , fusion to the trimerization domain prevents the S protein peptides in the fusion protein from forming straight helices (e.g., similar to what occurs during membrane fusion). For example, the 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. Ma et al., J Virol (2021) doi:10.1128/JVI.00194-21, incorporated by reference in full text for all purposes. In some embodiments, 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. In some embodiments, 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. In some embodiments, 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 ^™ trimerization domain.

In some of these embodiments, 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. In some embodiments, 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. In some embodiments, the immunogenic polypeptide may further comprise (a) one or more proline or glycine substitutions, and/or (b) one or more amino acid residues in the HR1 region that interacts with HR2 Base insertion. In some of these embodiments, the immunogenic polypeptide can have one or more substitutions selected from A942P, S943P, A944P, A942G, S943G, and A944G. In some of these embodiments, the insertion may be a G or GS between any residues in A942-A944.

The 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. If adjuvants are used, optional 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.

2.Polynucleotides and vectors

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

In some embodiments, polynucleotides encoding recombinant polypeptides provided herein are provided. In some aspects, a polynucleotide comprises a single nucleic acid sequence, such as a nucleic acid sequence encoding a recombinant polypeptide. In other examples, multi-core The nucleotides comprise a first nucleic acid sequence encoding a recombinant polypeptide, in particular a coronavirus antigen or immunogen, and a second nucleic acid sequence encoding a recombinant polypeptide comprising a different coronavirus antigen or immunogen.

In some embodiments, 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.

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

In some embodiments, expression of the recombinant polypeptide is inducible or conditional. Thus, in some aspects, a polynucleotide encoding a recombinant polypeptide includes a conditional promoter, enhancer, or transactivator. In some such aspects, the conditional promoter, enhancer or transactivator is an inducible promoter, enhancer or transactivator or a repressible promoter, enhancer or transactivator. For example, in some embodiments, inducible or conditional promoters can be used to restrict expression of a recombinant polypeptide to a specific microenvironment. In some embodiments, expression driven by an inducible or conditional promoter is modulated by exposure to exogenous agents (eg, heat, radiation, or drugs).

Where the polynucleotide includes more than one nucleic acid sequence encoding a recombinant polypeptide, the polynucleotide may further include a nucleic acid sequence encoding a peptide between the one or more nucleic acid sequences. In some cases, the nucleic acid located between the nucleic acid sequences encodes a peptide that separates the translation product of the nucleic acid sequence during or after translation. In some embodiments, the peptide comprises an internal ribosome entry site (IRES), a self-cleaving peptide, or a peptide that causes ribosome skipping, such as a T2A peptide.

In some embodiments, 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.

In some embodiments, 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.

Vectors or constructs containing nucleic acid molecules as described herein are also provided. In some embodiments, a vector or construct includes one or more promoters operably linked to a nucleic acid molecule encoding a recombinant polypeptide to drive expression thereof. In some embodiments, the promoter is operably linked to one or more nucleic acid molecules, such as nucleic acid molecules encoding recombinant polypeptides containing different coronavirus antigens or immunogens.

In some embodiments, the vector is a viral vector. In some embodiments, the viral vector is a retroviral vector. In some embodiments, the retroviral vector is a lentiviral vector. In some embodiments, the retroviral vector is a gamma-retroviral vector.

In some embodiments, a vector or construct includes a single promoter that drives expression of one or more nucleic acid molecules of a polynucleotide. In some embodiments, such promoters may be polycistronic (dicistronic or tricistronic, see, eg, U.S. Patent No. 6,060,273). For example, in some embodiments, the transcription unit may to be engineered to contain a bicistronic unit containing an IRES (internal ribosome entry site), which allows coexpression of gene products (e.g., encoding different recombinant polypeptides) via messages from a single promoter. In some embodiments, the vectors provided herein are bicistronic, allowing the vector to contain and express two nucleic acid sequences. In some embodiments, the vectors provided herein are tricistronic, allowing the vector to contain and express three nucleic acid sequences.

In some embodiments, 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). Thus, the ORF encodes a single polypeptide, which is processed into a single protein either during translation (in the case of 2A) or after translation. In some cases, 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).

In some embodiments, the vector is contained within a virus. In some embodiments, the virus is a pseudovirus. In some embodiments, the virus is a virus-like particle. In some embodiments, the vector is contained in the cell. In some embodiments, the virus or cell containing the vector contains a recombinant genome.

III. Immunogenic compositions and formulations

In some embodiments, provided herein are 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. In some embodiments, provided herein are 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 fragments, variants or mutants 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. Independently, the priming agent and/or any booster dose or doses may be administered without or with an adjuvant. If adjuvants are used, optional 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.

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

In some cases, it may be desirable to combine the disclosed immunogens with other pharmaceutical products (eg, vaccines) that induce protective responses to other agents. For example, 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) are administered simultaneously (usually separately) or sequentially to a group of infants (eg, infants approximately one to six months old). Accordingly, 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.

Multivalent or combination vaccines provide protection against multiple pathogens. In some aspects, polyvalent vaccines can protect against multiple strains of the same pathogen. In some cases, 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.

In some embodiments, for example, a vaccine comprising an immunogenic composition described herein is a multivalent vaccine. In some embodiments, the antigenic material for incorporation into the multivalent vaccine composition is derived from 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. In one embodiment, the antigens used for incorporation into the multivalent vaccine composition are derived from multiple strains of coronavirus. Other useful antigens include live, attenuated and inactivated viruses, such as inactivated poliovirus (Jiang et al., J. Biol. Stand., (1986) 14:103-9), 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. Med., (1984) 14:373-86) 1995) 332:1262-6) and epitopes of the pertussis virus (e.g., ACEL-IMUNE cell-free DTP, Wyeth-Lederle Vaccine, and Pediatrics), which are incorporated by reference in their entirety for all purposes.

In some respects, the vaccines presented here are universal vaccines. In some embodiments, a universal vaccine is one that protects against multiple strains of the same virus (eg, multiple strains of coronavirus). Developing an effective universal coronavirus vaccine would reduce costs and labor, such as using seasonal vaccine formulations, and allow for stronger pandemic prevention.

In some embodiments, 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.

In some aspects, a universal vaccine is a vaccine composed of multiple epitopes derived from different strains of the virus. In some aspects, a universal vaccine consists of a single epitope that is conserved across different strains of the virus. For example, 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. In some embodiments, immunogenic compositions include a trimerized recombinant polypeptide provided herein and optionally a pharmaceutically acceptable carrier. In some embodiments, 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. In some embodiments, 1.0 mL of an aqueous immunogenic composition solution includes 720 μg 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. In some embodiments, immunogenic compositions include a protein comprising a plurality of trimerized recombinant polypeptides provided herein and optionally a pharmaceutically acceptable carrier. In some embodiments, immunogenic compositions include protein nanoparticles provided herein and optionally a pharmaceutically acceptable carrier. In some embodiments, immunogenic compositions include VLPs provided herein and optionally a pharmaceutically acceptable carrier. In some embodiments, immunogenic compositions include an isolated nucleic acid provided herein and optionally a pharmaceutically acceptable carrier. In some embodiments, immunogenic compositions include a carrier provided herein and optionally a pharmaceutically acceptable carrier. In some embodiments, immunogenic compositions include a virus provided herein and optionally a pharmaceutically acceptable carrier. In some embodiments, 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. Internal, sublingual, tonsillar, oropharyngeal, or other parenteral and mucosal routes. In several embodiments, pharmaceutical 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. are described in more detail.

Therefore, immunogens described herein, such as recombinant coronavirus S antigens, such as trimers, proteins, can be formulated with pharmaceutically acceptable carriers to help maintain biological activity while also facilitating storage within an acceptable temperature range. The stability is increased. Potential 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) or other protecting agents. 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, especially liquid formulations, may contain bacteriostatic agents to prevent or minimize degradation during storage, including but not limited to benzyl alcohol, phenol, m-cresol, Chlorobutanol, methylparaben and/or propylparaben. 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. 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. Segment copolymer, MPL ^TM (3-O-deacylated monophosphate A; Corixa, Hamilton, Ind.) and IL-12 (Genetics Institute, Cambridge, Mass.) (Newman et al., 1998, Critical Reviews in Therapeutic Drug Carrier Systems 15:89-142, incorporated by reference in its entirety for all purposes). The advantage of these adjuvants is that they help stimulate the immune system in a non-specific manner, thus enhancing the immune response to the pharmaceutical product. In some embodiments, 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. In some embodiments, the immunogenic compositions of the present invention may include or be administered with various adjuvants. For example, in some cases, a vaccine such as one comprising an immunogenic composition provided herein may include or be administered in conjunction with a variety of adjuvants.

For vaccine compositions, examples of suitable adjuvants include, for example, aluminum hydroxide, lecithin, Freund's adjuvant, MPL ^™ and IL-1, one or a combination of any of them may be combined with a compound 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. In some embodiments, the vaccine compositions or nanoparticle immunogens disclosed herein (eg, SARS-COV-2 vaccine compositions) can be formulated as a controlled or sustained release formulation. This can be achieved by compositions containing slow-release polymers or by microencapsulated delivery systems or bioadhesive gels. Various pharmaceutical compositions can be prepared according to standard procedures well known in the art.

In some embodiments, 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 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). In some embodiments, 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. In some embodiments, 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. In some embodiments, 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, α- Tocopherol and polyoxyethylene sorbitan monooleate. In some embodiments, immunogenic compositions of the invention may comprise QS21, 3D-MPL and an oil-in-water emulsion, wherein the oil-in-water emulsion has the following composition: a metabolizable oil, such as keratin Squalene, alpha-tocopherol, and Tween-80 and/or Span 85. In some embodiments, the immunogenic compositions of the present invention may include an adjuvant in the form of a liposome composition.

In some embodiments, 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. In some embodiments, 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.

In some embodiments, 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. In some embodiments, immunogenic compositions of the invention may comprise a mixture of individually purified Quillaja saponaria Molina fractions, which are subsequently formulated with cholesterol and phospholipids.

In some embodiments, 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 ^™ , Matrix-A ^™ , Matrix-C ^™ , Matrix-M ^™ , 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 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) Any one or several combinations of adjuvants disclosed in the above documents This document is incorporated by reference in its entirety for all purposes.

In some embodiments, 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 The cytidine-phosphate-guanosine (also known as CpG or cytosine-phosphate-guanosine) motif, and SARS-CoV-2 antigens and oligonucleotides are required to effectively stimulate mammalian subjects (e.g., SARS- The amount of the immune response against the SARS-CoV-2 antigen in a human subject (human subject with CoV-2 antigen and oligonucleotide) is present in the immunogenic composition. 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). The best 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. In some embodiments, CpG oligonucleotides are linear. In other embodiments, the CpG oligonucleotide is circular or includes a hairpin loop. CpG oligonucleotides can be single-stranded or double-stranded. In some embodiments, 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. In some embodiments, CpG oligonucleotides include non-canonical bases. In some embodiments, CpG oligonucleotides include modified nucleosides. In some embodiments, 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. For example, in addition to phosphodiester bonds, 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. In some embodiments, the oligonucleotide includes only a phosphorothioate backbone. In some embodiments, the oligonucleotide includes only a phosphodiester backbone. In some embodiments, 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. In some embodiments, when multiple CpG oligonucleotide molecules are present in a pharmaceutical composition comprising at least one excipient, two stereoisomers of the phosphorothioate bond are present in the multiple CpG oligonucleotide molecules. in the nucleotide molecule. In some embodiments, all of the internucleotide linkages of the CpG oligonucleotide are phosphorothioate linkages, or in other words, the CpG oligonucleotide has a phosphorothioate backbone.

In the present invention, any suitable CpG oligodeoxynucleotide (ODN) or combination thereof may be used as an adjuvant. For example, K-type ODN (also known as B-type) encodes multiple CpG motifs on the phosphorothioate backbone. Type K ODN can be based on the following sequence TCCATGGA CG TTCCTGAG CG TT. 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-α, 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-α, 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 may be based on the following sequence T CG T CG TT CG AA CG A CG TTGAT. This type of ODN stimulates B cells to secrete IL-6 and pDC to produce IFN-α. P-type ODN contains two palindromic sequences, allowing them to form a higher ordered structure. P-type ODN may be based on the following sequence T CG T CG A CG AT CG G CGCGCG C CG . Compared with C-type ODN, P-type ODN activates B cells and PDC, and induce greater IFN-α production. In this paragraph, boldface letters in ODN sequences indicate self-complementary palindromes, and CpG motifs are underlined.

Exemplary 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). In some embodiments, the immunogenic composition further includes an aluminum salt adjuvant that adsorbs SARS-CoV-2 antigen. In some embodiments, 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. In some embodiments, a unit dose of the immunogenic composition includes about 0.25 to about 0.50 mg Al ³⁺ , or about 0.35 mg Al ³⁺ . In some embodiments, the immunogenic composition further includes an additional adjuvant. Other suitable adjuvants include, but are not limited to, squalene-in-water emulsions (e.g., MF59 or ASO3), TLR3 agonists (e.g., polyIC or polyICLC), TLR4 agonists (e.g., bacterial lipopolysaccharide derivatives such as Phospholipid 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 (imidazolequinoline derivatives , such as imiquimod and resiquimod) (Coffman et al., Immunity, 33:492-503, 2010, incorporated by reference in its entirety for all purposes). In some embodiments, additional adjuvants include MPL and alum (eg, AS04). For veterinary use and for the production of antibodies in non-human animals, the mitogenic components of Freund's adjuvant (intact and incomplete) can be used.

In some embodiments, a unit dose of the immunogenic composition may include from about 10 μg to about 1000 μg of one or more adjuvants, preferably from about 25 μg to about 500 μg of one or more adjuvants, preferably from about 50 μg to about 300 μg of one or more adjuvants, preferably about 100 μg to about 250 μg of one or more adjuvants, preferably about 150 μg to about 225 μg of one or more adjuvants. In some embodiments, a unit dose of the immunogenic composition may include about 10 μg to about 500 μg of aluminum-containing adjuvant, preferably about 25 μg to about 250 μg of aluminum-containing adjuvant, preferably about 50 μg to about 125 μg of aluminum-containing adjuvant, preferably about 75 μg of aluminum-containing adjuvant, such as Alum. In some embodiments, a unit dose of the immunogenic composition may include from about 10 μg to about 500 μg CpG adjuvant, preferably from about 25 μg to about 300 μg CpG adjuvant, preferably from about 50 μg to about 250 μg CpG adjuvant, preferably from about 75 μg to about 200 μg CpG adjuvant, preferably about 100 μg to about 175 μg CpG adjuvant, preferably about 150 μg CpG adjuvant adjuvant, such as CpG 1018A. In some embodiments, a unit dose of the immunogenic composition may include from about 10 μg to about 500 μg of aluminum-containing adjuvant and from about 10 μg to about 500 μg of CpG adjuvant. In some embodiments, a unit dose of the immunogenic composition may include about 25 μg to about 250 μg of aluminum-containing adjuvant and about 25 μg to about 300 μg of CpG adjuvant. In some embodiments, a unit dose of The immunogenic composition may include about 50 μg to about 125 μg of aluminum-containing adjuvant and about 50 μg to about 250 μg of CpG adjuvant. In some embodiments, a unit dose of the immunogenic composition may include about 50 μg to about 100 μg of aluminum-containing adjuvant and about 75 μg to about 200 μg of CpG adjuvant. In some embodiments, a unit dose of the immunogenic composition may include about 50 μg to about 100 μg of aluminum-containing adjuvant and about 100 μg to about 175 μg of CpG adjuvant. In some embodiments, a unit dose of the immunogenic composition can include about 75 μg to about 100 μg of an aluminum-containing adjuvant, such as Alum, and about 150 μg of a CpG adjuvant, such as CpG 1018A. In some embodiments, a unit dose of the immunogenic composition may include from about 10 μg to about 100 μg 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, for example, fusion proteins or S fusion protein trimers including SARS-CoV-2 coronavirus delta (B.1.617.2) variant S protein peptide or fragments, variants or mutants thereof. In some embodiments, a unit dose of the immunogenic composition may include about 20 μg to about 75 μg SARS-CoV-2 S fusion protein or S-trimer, preferably about 25 μg to about 60 μg SARS-CoV-2 S fusion protein or S-trimer, or about 30 μg, about 40 μg, about 50 μg SARS-CoV-2 S fusion protein or S-trimer. In some embodiments, a unit dose of the immunogenic composition can include about 3 μg of SARS-CoV-2 S fusion protein or S-trimer. In other embodiments, the dose contains 9 μg of SARS-CoV-2 S fusion protein or S-trimer. In a further embodiment, the dose contains 30 μg of SARS-CoV-2 S fusion protein or S-trimer. In some embodiments, a unit dose of the immunogenic composition may include about 30 μg of SARS-CoV-2 S-fusion protein or S-trimer, about 75 μg to about 100 μg of an aluminum-containing adjuvant, such as Alum, and about 150 μg of CpG adjuvant. agents, such as CpG 1018A.

In some embodiments, 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). In some embodiments, 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).

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

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 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.

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. In some embodiments, the filler is a protective agent that aids in freezing or spray drying and/or in Stabilizes and prevents degradation of active agents 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.

In some embodiments, 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. In some embodiments, 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. . In other embodiments, the composition further includes one or more adjuvants.

IV. Methods of inducing immune responses

In some embodiments, provided herein are methods for generating an immune response to a coronavirus surface antigen in a subject, comprising administering to the subject an effective amount of a complex comprising a 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. In some embodiments, provided herein are methods 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. In some embodiments, provided herein are methods 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 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 an initiating agent ( primary series), additional doses, and/or homologous or heterologous booster doses, such as first dose, second dose, third dose, fourth dose, and/or more doses , optionally, the initial dose, additional dose, or heterologous booster dose is combined with any one or more of other recombinant subunit vaccines, nanoparticle vaccines, mRNA vaccines, DNA vaccines, adenovirus vector vaccines, and inactivated virus vaccines. used together. In some embodiments, provided herein are methods for generating an immune response in a subject to a coronavirus surface antigen, wherein the surface antigen includes the S protein of the coronavirus or an antigen thereof Fragments, 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 includes administering to the subject an effective amount of the complex, the complex The substance includes a recombinant polypeptide including 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 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 doses, or heterologous The 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. Alternatively, 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, α-tocopherol, and Tween-80 and/or Span 85 in the form of an oil-in-water emulsion; or any combination of adjuvants.

In some embodiments, provided herein are methods 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 of complexes is used as an initial agent and/or as a booster, such as a second dose and/or a third dose booster injection. In some embodiments, 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. Alternatively, the adjuvants in any priming agent and/or booster agent 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, α-tocopherol, and Tween-80 and/or Span 85 in the form of an oil-in-water emulsion; or any combination of adjuvants.

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. In certain examples, the subject is a human. 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. To identify subjects for prevention or treatment according to the methods of the invention, an acceptable screening method is used to determine risk factors associated with the target or suspected disease or condition, or to identify an existing disease or condition in the subject status. 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. These and other conventional methods allow clinicians to select patients in need of treatment using the methods and pharmaceutical compositions of the present invention. In accordance with these methods and principles, 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.

Administration of the disclosed immunogens (eg, coronavirus S antigen, eg, trimers, proteins) may be used for prophylactic or therapeutic purposes. When provided prophylactically, 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. When provided therapeutically, 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. In certain embodiments, 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).

There can be several boosters, each of which can be a different disclosed immunogen. In some examples, a booster may be the same immunogen as another booster or priming agent. Primers and boosters can be administered as a single dose or as multiple doses, e.g., two, three, four, five, six or more doses can be administered to the subject over 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.

In some embodiments, 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 selected vaccine parameters, such as formulation, dose, regimen, etc., can be determined by taking aliquots of serum from subjects and measuring antibodies during the immunization program titer to determine. In addition, 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.

In some embodiments, 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.

For protein therapeutics, typically, each human dose will include 1-1000 μg of protein, such as from about 1 μg to about 100 μg, such as from about 1 μg to about 50 μg, such as about 1 μg, about 2 μg, about 5 μg, about 10 μg, about 15 μg, About 20 μg, about 25 μg, about 30 μg, about 40 μg, or about 50 μg.

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. An amount of a drug that is ineffective when inducing an immune response but is effective when administered in multiple doses, such as in a prime-boost regimen.

After administration of an immunogen disclosed herein, 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.

In some embodiments, 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. For example, 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, e.g., by 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% (elimination or prevention of detectably infected cells) . In additional examples, 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. For example, 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).

In some embodiments, 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.

One method of 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 liposome delivery of nucleic acids (or their own synthetic peptides) and immunostimulatory constructs or ISCOMS ^™ , 30-40 nm sized negatively charged nanoparticles that spontaneously form ^after mixing cholesterol and Quil A ^™ (saponin) Cage-like structure. The use of ISCOMS ^™ 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). Doses of antigen as low as 1 μg encapsulated in ISCOMS ^™ have been found to produce class I-mediated CTL responses (Takahashi et al., Nature 344:873, 1990, incorporated by reference in its entirety for all purposes).

In some embodiments, plasmid DNA vaccines are used to express the disclosed immunogens in a subject. For example, nucleic acid molecules encoding the disclosed immunogens can be administered to a subject to induce an immune response to the coronavirus S antigen. In some embodiments, 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).

In another method of immunization using nucleic acids, the disclosed recombinant coronavirus S antigens (e.g., trimers, proteins) can be expressed by attenuated viral hosts or vectors or bacterial vectors. Recombinant vaccinia virus, adeno-associated virus (AAV), herpesvirus, retrovirus, cytomeglovirus, or other viral vectors can be used to express peptides or proteins to elicit CTL responses. For example, vaccinia vectors and methods useful in immunization programs are described in U.S. Patent No. 4,722,848, which patent is incorporated by reference in its entirety for all purposes. 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).

In one embodiment, nucleic acid encoding the disclosed recombinant coronavirus S antigen is introduced directly into the cell. For example, 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 ^™ Gene Gun. The nucleic acid can be "naked", consisting of a plasmid under the control of a strong promoter. Typically, the DNA is injected into the muscle, but it can also be injected directly into other sites. Injectable doses are generally about 0.5 μg/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).

For example, 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 ^™ Gene Gun. The nucleic acid can be "naked", consisting of a plasmid under the control of a strong promoter. Typically, the DNA is injected into the muscle, but it can also be injected directly into other sites. Injectable doses are generally about 0.5 μg/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).

In another embodiment, mRNA-based immunization protocols can be used to deliver nucleic acids encoding the disclosed recombinant coronavirus S antigens directly into cells. In some embodiments, mRNA-based nucleic acid 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. 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 Conserved Influenza Antigens Confer Protection against Homologous and Heterosubtypic Viral Challenge," PLoS One, 11(8):e01 61193, 2016; and Brito et al., “Self-amplifying mRNA vaccines,” Adv Genet., 89:179-233, 2015). All documents in this paragraph are incorporated by reference in their entirety for all purposes.

In some embodiments, administration to a subject of a therapeutically effective amount of one or more disclosed immunogens induces a neutralizing immune response in the subject. To assess neutralizing activity, 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. In some embodiments, a panel of coronavirus pseudoviruses can be used to determine serum neutralizing activity.

In some embodiments, neutralizing immune responses induced by immunogens disclosed herein generate neutralizing antibodies against coronaviruses (eg, SARS-CoV-2). In some embodiments, the neutralizing antibodies herein bind to cellular receptors or coreceptors of a coronavirus (eg, SARS-CoV-2) or components thereof. In some embodiments, the viral receptor or co-receptor is a coronavirus receptor or co-receptor, preferably a pneumovirus receptor or co-receptor, more preferably a human coronavirus receptor, such as a SARS-CoV-2 receptor. body or co-receptor. In some embodiments, the neutralizing antibodies herein modulate, reduce, antagonize, mitigate, block, inhibit, eliminate and/or interfere with at least one coronavirus (e.g., SARS-CoV) in vitro, in situ, and/or in vivo -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 signaling, membrane SARS-CoV-2 cleavage, SARS-CoV-2 activity, SARS-CoV-2 production and/or synthesis. In some embodiments, 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).

V. Products or kits

Articles of manufacture or kits containing the provided recombinant polypeptides, proteins, and immunogenic compositions are also provided. 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. In some embodiments, 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). Alternatively, or additionally, 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.

In some embodiments, 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.

the term

Unless otherwise defined, all technical terms, symbols, and other technical and scientific terms or terminology used herein are intended to have the same meaning as commonly understood by one of ordinary skill in the art to which the claimed subject matter belongs. . In some cases, terms are defined herein to have commonly understood meanings for clarity and/or ease of reference, and such definitions contained herein do not necessarily represent a material difference from what is commonly understood in the art.

The terms "polypeptide" and "protein" are used interchangeably to refer to a polymer of amino acid residues and are not limited to a minimum length. Polypeptides (including provided receptors and other polypeptides such as linkers or peptides) 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. In some aspects, 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.

As used herein, a "subject" is a mammal, such as a human or other animal, and is typically a human. In some embodiments, the subject (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. In some embodiments, 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. In some embodiments, the subject is a non-primate mammal, such as a rodent.

As used herein, "treatment" (and its grammatical variations such as "treat" or "treating") 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.

As used herein, "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.

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

As used herein, "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.

In the context of administration, an "effective amount" of an agent (eg, pharmaceutical preparation, cell or composition) 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 (e.g., a pharmaceutical preparation or a cell) 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. In some embodiments, 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. Typically, but not necessarily, 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. Generally, this amount will be sufficient to measurably inhibit viral (eg, SARS-CoV-2) replication or infectivity. When administered to a subject, doses will typically be used that will achieve target tissue concentrations that have been shown to achieve in vitro Achieve inhibition of viral replication. In some embodiments, 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. In some embodiments, the effective amount is a therapeutically effective amount. In some embodiments, 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.

As used herein, 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. . Unless otherwise stated, the term "vaccine immunogen" is used interchangeably with "protein antigen" or "immunogenic polypeptide."

The term "conservatively modified variants" applies to both amino acid and nucleic acid sequences. With respect to a particular nucleic acid sequence, conservatively modified variants refer to 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. With respect to a polypeptide sequence, 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).

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.

Unless otherwise stated, 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 (eg, within a cell). 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. In some cases, the immune response is a protective immune response. Typically, 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. In some embodiments, 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.

Virus-like particles (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. 60:1445-1456; and Hagensee et al. (1994) J. Virol. 68:4503-4505, which are incorporated by reference in their entirety for all purposes. For example, VLPs can be isolated by density gradient centrifugation and/or identified by characteristic density bands. Alternatively, cryo-EM can be performed on vitrified aqueous samples prepared from the VLP in question and images recorded under appropriate exposure conditions.

The term "about/approximately" as used herein refers to the usual error range for each value that is readily known to those skilled in the art. References herein to "about/approximately" a value or parameter include (and describe) embodiments directed to that value or parameter itself.

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

Throughout this document, 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. this The upper and lower limits of some smaller ranges may independently be included in the smaller ranges and also within the claimed subject matter, subject to any expressly excluded limitations within the stated ranges. 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.

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

The term "vector" as used herein 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."

Exemplary Embodiment I

Embodiment 1. A protein comprising a plurality of recombinant polypeptides, each recombinant polypeptide comprising a coronavirus SARS-CoV-2 delta (B.1.617.2) surface antigen linked to a C-terminal propeptide of collagen, wherein recombinant The C-terminal propeptide of a 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.

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.

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.

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.

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.

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.

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.

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).

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 proα1(I), proα1(II), proα1(III), proα1(V), proα1(XI), proα2 (I), the C-terminal polypeptide of proα2(V), proα2(XI) or proα3(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 includes any one of SEQ ID NOs: 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 comprises SEQ ID NO: 69 or an amino acid sequence at least 90% identical thereto, which is capable of forming inter-polypeptide disulfide bonds and 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 used as a vaccine priming agent and/or as a booster, for example the second dose and/or the third dose Booster shots.

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 priming agent and/or as a booster Use, for example, a second and/or third dose booster shot.

Embodiment 36. A virus-like particle (VLP) comprising the protein of any one of embodiments 1-33, optionally used as a vaccine starter and/or as a booster, e.g. a second dose and /or a third booster shot.

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 administered as a priming dose and/or as a booster dose, such as a second and/or third booster dose.

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.

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.

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.

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).

Exemplary Embodiment II

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 comprising a soluble coronavirus SARS-CoV-2 delta ( delta, B.1.617.2) surface antigen or fragments, variants or mutants thereof, the soluble coronavirus surface antigen or fragments, variants or mutants thereof are connected to the C-terminal part of collagen by in-frame fusion to form two Sulfur-linked trimeric fusion proteins.

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

Embodiment 3. The method of embodiment 1 or 2, wherein the coronavirus surface antigen includes coronavirus spike (S) protein or a fragment or epitope thereof.

Embodiment 4. The method according to any one of embodiments 1-3, wherein the coronavirus surface antigen comprises SARS-CoV-2 spike (S) extracellular domain peptide or a fragment or epitope thereof, optionally the The S extracellular domain peptide or its fragments or epitopes include the SARS-CoV-2 delta (delta, B.1.617.2) variant S ectodomain peptide or its fragments, variants or mutants, such as those containing the delta variant affected by Chimeric sequences of body binding domain (RBD) and Hu-1 or other variant S protein peptide sequences.

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 position, optionally the NTD peptide Is the SARS-CoV-2 Hu-1, alpha, beta, gamma, delta, myon, or ometron NTD peptide or a fragment, variant, or mutant thereof.

Embodiment 6. The method according to any one of embodiments 1-5, wherein the coronavirus surface antigen comprises a SARS-CoV-2 spike (S) receptor binding domain (RBD) peptide or a fragment or expression thereof. position, optionally the RBD peptide is a SARS-CoV-2 delta (delta, B.1.617.2) variant RBD peptide or a fragment, variant or mutant thereof.

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 S1 peptide of the SARS-CoV-2 delta (delta, B.1.617.2) variant or its fragment, variant or mutant.

Embodiment 8. The method according to any one of embodiments 1-7, wherein the coronavirus surface antigen comprises SARS-CoV-2 spike (S) S2 peptide or a fragment or epitope thereof, optionally the S2 The peptide is a SARS-CoV-2 Hu-1, alpha, beta, gamma, delta, myon, or ometron S2 peptide or a fragment, variant, or mutant thereof.

Embodiment 9. The method according to any one of embodiments 1-8, wherein the coronavirus surface antigen comprises a SARS-CoV-2 spike (S) extracellular domain peptide with mutations or a fragment or epitope thereof.

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 embodiments 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 vaccine includes SEQ ID NO: 86 or is at least or about 80%, 85%, 90% with SEQ ID NO: 86 , 92%, 95%, 97%, 99% sequence identity of the amino acid sequences.

Embodiment 16. The method of any one of embodiments 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: 91 or is at least or about 80%, 85%, 90% with SEQ ID NO: 91 , 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 embodiments 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, optionally in conjunction with any one or more of other recombinant subunit vaccines, nanoparticle vaccines, mRNA vaccines, DNA vaccines, adenoviral vector vaccines, and inactivated virus vaccines. Adjuvants in the priming 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 Adjuvants, such as CpG oligodeoxynucleotide (CpG-ODN)-containing adjuvants; TLR9 agonist-containing adjuvants; metabolizable oils, alpha-tocopherol, and/or polyoxyethylene sorbitan monooleic acid ester (Tween-80), such as an adjuvant containing squalene, α-tocopherol, and Tween-80 and/or Span 85 in the form of an oil-in-water emulsion; or any combination of adjuvants.

Embodiment 26. The method of any one of embodiments 1-25, 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, Optionally, 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.

Embodiment 27. A method for detecting coronavirus antibodies from mammalian serum, the method comprising the step of contacting the serum with a soluble coronavirus SARS-CoV-2 delta (B.1.617.2) surface antigen, the Soluble coronavirus surface antigen is linked to the C-terminal portion of collagen via in-frame fusion to form a disulfide-linked trimeric fusion protein.

Embodiment 28. The method of embodiment 27, wherein the soluble coronavirus surface antigen is S protein or peptide.

Embodiment 29. A method of using a recombinant subunit vaccine comprising a soluble surface antigen from the coronavirus SARS-CoV-2 delta (B.1.617.2) linked to C of collagen by in-frame fusion - terminal portion to form a disulfide bond-linked trimeric fusion protein, the method comprising: immunizing a mammal, purifying the produced neutralizing antibody, and using the neutralizing antibody 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 a monoclonal antibody to the S protein of SARS-CoV-2.

Embodiment 34. The method of claim 29, wherein the neutralizing antibody is of SARS-CoV-2 Hu-1, alpha, beta, gamma, delta, myelin, ometron, and/or other strains Monoclonal antibody to S protein.

Embodiment 35. The method of embodiment 29, wherein the neutralizing antibody is a monoclonal antibody to the S protein of SARS-CoV-2 delta (B.1.617.2).

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: 91.

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.

Embodiment 41. 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 comprising a recombinant polypeptide selected from the group consisting of SEQ ID NOs: 85-92, optionally as a primary series, additional agents (additional dose), and/or homologous or heterologous booster dose (booster dose) use, such as a first dose, a second dose, a third dose, a fourth dose, and/or more doses, optionally the initial dose A dose, an additional dose, or a heterologous booster dose 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 42. A method for generating an immune response to a coronavirus surface antigen in a subject,

wherein the surface antigen includes a sequence selected from the group consisting of SEQ ID NOs: 27-66 and 81-84, and

Embodiment 43. A method for generating an immune response to a coronavirus surface antigen in a subject,

wherein 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 includes administering to the subject an effective amount of a complex comprising a recombinant polypeptide comprising a sequence set forth in any one of SEQ ID NOs: 85-92, optionally as a primary series ), extra dose (additional dose), 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 A dose, an additional dose, or a heterologous booster dose 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 44. A method for generating an immune response to a coronavirus surface antigen in a subject,

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 Optionally 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 embodiment 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) a first region comprising the extracellular domain of the coronavirus spike protein located before the coronavirus spike protein receptor binding domain (RBD) in the non-chimeric coronavirus spike protein of the first coronavirus part; part

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) C-terminal propeptide of collagen, wherein the C-terminal propeptide of the recombinant polypeptide forms an inter-polypeptide disulfide bond, wherein at least one of the first coronavirus and the second coronavirus is SARS-CoV-2 delta (B .1.617.2), optionally the fusion protein 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 dose, third dose, fourth dose, and/or more doses, optionally with the initial dose, additional dose, or heterologous booster dose with other recombinant subunit vaccines, nanoparticle vaccines, mRNA vaccines, DNA vaccines, adenocarcinoma vaccines, Use any one or more of viral vector vaccines and inactivated virus vaccines in combination.

Embodiment 47. The fusion protein of embodiment 46, further comprising a third region between the second region and the C-terminal propeptide of collagen.

Embodiment 48. The fusion protein of embodiment 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 embodiment 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 embodiments 46-49, wherein the first region includes the N-terminal domain (NTD) of the first coronavirus.

Embodiment 51. The fusion protein of any one of embodiments 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.

5 Embodiment 2. The fusion protein of any one of embodiments 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 embodiments 46-52, wherein the first and second coronaviruses are different variants or strains of the same coronavirus.

Embodiment 54. The fusion protein of embodiment 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 embodiment 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. The group consisting of SARS-CoV-2 viruses of the .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.

Embodiment 56. A trimeric fusion protein comprising three recombinant polypeptides, each recombinant polypeptide comprising from amino to carboxyl terminus:

a) a first region including the N-terminal domain (NTD) of the coronavirus spike protein of SARS-CoV-2 of lineage B.1.617.2;

b) a second region that includes the coronavirus spike protein receptor binding domain (RBD) of SARS-CoV-2 of lineage B.1.617.2; and

c) C-terminal propeptide of collagen, wherein the C-terminal propeptide of the recombinant polypeptide forms an inter-polypeptide disulfide bond, optionally the fusion protein 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, the additional dose, or the heterologous dose The source 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.

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 delta (B.1.617.2) 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 with SEQ The sequence described in any one of ID NOs: 81-92 has an amino acid sequence with at least or about 80%, 85%, 90%, 92%, 95%, 97%, 99% sequence identity.

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. The sequence described in any one of ID NOs: 27-66 and 81-92 has an amino acid sequence with at least or about 80%, 85%, 90%, 92%, 95%, 97%, 99% sequence identity, Optionally, 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.

Example

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

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. In one embodiment, 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 α1 collagen. Animal expression vectors to express secreted trimeric S-trimer fusion antigens, for example, as shown in Figures 1A-1B.

In order to quickly express S-trimer antigen, Protein ^{TrimerizationTM} technology was used (Liu et al., Scientific Reports, 7(1):8953, 2017). 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. After stable transfection into CHO cells, subsequent screening of high-titer production clones and extensive process optimization, a fed-batch serum-free cell culture process in a bioreactor was developed, leading to the identification of S-trimers as secreted proteins. High level expression. Liang et al., Nat. Comms., 12:1346, 2021; Richmond et al., Lancet, 397:682-694, 2021, are incorporated by reference in their entirety for all purposes.

To obtain a highly pure form of S-trimer for vaccine research, 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. Then, serum-free cell culture medium containing CHO cell-secreted S-trimers was loaded into a pre-captured Endo180-Fc on protein A column. After washing away any unbound contaminating host cell proteins (HCP) and other impurities, 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. Under non-reducing conditions, S-trimers appear in multiple high molecular weight forms, likely as a result of partial cleavage of the antigen, releasing non-covalently attached and cleaved S1 during sample processing.

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

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. High-level expression of the S-trimer fusion protein is shown in Figure 2. An 8% SDS-PAGE analysis of S-trimer expression in fed-batch serum-free CHO cell cultures was performed in a 10L bioreactor. Analyze 10 μL of cell-free conditioned medium from days 9 to 13 under reducing conditions, followed by Coomassie Brilliant Blue staining. Figure 3A shows the delta S-trimer purification process sample and reference substance reducing SDS-PAGE Coomassie brilliant blue staining analysis. Figure 3B shows the SEC-HPLC purity analysis. The purity of delta S-trimer is 97.22%.

The binding affinity of purified S-trimer antigen to the human ACE2 receptor was assessed by Fortebio BioLayer interferometry using a Protein A sensor. In Figures 4A-4B, delta S-trimer has higher receptor affinity for ACE2-Fc than Hu-1 S-trimer.

Example 2: Immunogenicity of adjuvanted SARS-CoV-2 vaccine

Assessing the immunogenicity of S-trimer in BALB/c mice. Mice were injected intramuscularly with S-trimer twice in a two-dose prime-boost regimen (day 0 and day 21). The effect of adjuvants on humoral immunogenicity is obvious, because at the corresponding antigen dose level, the S-trimer binding antibody titer, ACE2 competitive titer and neutralizing antibody titer in the adjuvant-containing group were significantly higher for unadjuvanted vaccines. S-trimers with different adjuvants elicited ACE2-competing antibody titers and pseudovirus-neutralizing antibody titers at levels similar to or higher than those observed in human convalescent serum samples. Similar results were observed in S-trimer-immunized rats.

were studied by collecting splenocytes from sacrificed immunized mice, then stimulating with S-trimer antigen and detecting Th1 (IL-2 and IFNγ) and Th2 (IL-4 and IL-5) cytokines by ELISpot S-trimer antigen-specific cell-mediated immunity (CMI). The adjuvanted group appeared to induce a stronger overall CMI response than the unadjuvanted S-trimer. Th1-biased cell-mediated immune responses were observed in the unadjuvanted and some adjuvanted S-trimer groups, whereas a mixed Th1-Th2 distribution was observed in other adjuvanted groups. CMI does not appear to be dependent on the dose of antigen.

Pseudovirus neutralization assay

SARS-CoV-2 pseudovirus neutralization tests were performed on Hu-1 strains and variant strains. To evaluate the SARS-CoV-2 pseudovirus neutralizing activity of the antisera, the samples were first heat-inactivated for 30 minutes, serially diluted (3 times), and incubated with an equal amount of 650 TCID ₅₀ pseudovirus for 1 hour at 37°C. Virus alone (positive control) and cells alone (negative control) were used simultaneously. Then, fresh trypsinized ACE2 overexpression-293 cells were added to each well at 20,000 cells/well. After incubation for 24 h at 37°C in a 5% _CO2 incubator, cells were lysed and luciferase activity was determined by the Luciferase Assay System (Beyotime) according to the manufacturer's protocol. The _EC50 neutralizing antibody titer for a given serum sample is defined as the reciprocal of the dilution where the sample shows a 50% reduction in relative light units (RLU) compared to virus alone control wells. Figure 5 shows BALB/c mouse SARS-CoV-2 Hu-1, alpha (alpha, α, B.1.1.7), beta (beta, β, B.1.351), gamma (gamma, γ, P.1), delta (delta, δ, B.1.617.2), mu (mu, μ, B.1.621) and Omicro (Omicro, o, B.1.1.529) strain pseudoviruses and antibody _EC50 data.

Splenocyte stimulation and ELISpot assay

To detect antigen-specific T-cell responses, Th1 cytokines (IFN-γ, IL-2) and Th2 cytokines (IL-4, IL-5) were measured using ELISpot kits (Mabtech) according to the manufacturer's instructions. Two weeks after the third immunization, splenocytes from immunized mice or PBMC from immunized mice were collected. 5×10 ⁵ splenocytes (96-well plate) were stimulated in vitro with 2 μg/mL SARS-CoV-2 Hu-1 S1 peptide library and Hu-1 S2 peptide library. Phorbol 12-myristate 13-acetate (PMA) and ionomycin were added to the positive control wells as non-specific stimuli, while the negative control wells received no stimuli. After incubation for 24-48 hours, add the biotinylated detection antibody in the ELISpot kit and SA-ALP/SA-HRP. Spots are formed by adding BCIP/NBT or AEC substrate solution and incubating in the dark for 5-30 minutes to produce stains. IFN-γ, IL-2, IL-4 and IL-5 spot-forming cells (SFC) were counted using an automated ELISpot reader (CTL). Figure 6A shows the ELISpot results of Hu-1 strain S1 peptide library stimulation (n=8/group mean). Figure 6B shows the ELISpot results of Hu-1 strain S2 peptide library stimulation (n=8/group mean). The vertical axis is the number of ELISpots per 250,000 spleen cells.

Example 3: Immunogenicity of Delta strain-based SARS-CoV-2 vaccine

Construction and production of fake viruses

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, α), Beta (Beta, β), gamma (gamma, γ), Delta (delta, δ), Mu (mu, μ) 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). The supernatant was harvested 24 ± 2 hours after transfection, centrifuged at 1500 rpm for 5 minutes to remove cell debris, and then stored at -80°C. live. 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 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, for all purposes its full text The form quoted is added) to calculate the TCID ₅₀ of the fake virus.

Neutralization test

Aliquots of 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. After 44 to 48 h of incubation at 37°C in a 5% CO2 incubator, cells were lysed and luciferase activity determined by the Bright-Glo Luciferase Assay System (Promega) according to the manufacturer's protocol . The _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. The detailed method is reported in 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.

Expression and purification of variant of high concern (VOC) S-trimer fusion proteins

The cDNA encoding the extracellular domain of the SARS-CoV-2 spike (S) protein from Delta 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-293 CD05 medium (OPM) and OPM-293 proFeed supplement (OPM). S-trimeric proteins were purified from conditioned media to homogeneity using a Protein Trimerizer ^™ tag-specific affinity column (Clover Biopharma).

result

A protein trimerized ^TM tag subunit vaccine based on the Delta (B.1.617.2) strain was constructed and produced and administered as a third dose booster vaccine after primary vaccination with the Hu-1 S-trimer vaccine /boosted mice, compared with other VOC vaccine candidates to observe their ability to further enhance broad coverage of neutralizing antibodies. First, two doses of Hu-1 S-trimer vaccine (3 μg) adjuvanted with CpG (150 μg) plus Alum (75 μg) were administered intramuscularly (IM) on days 0 and 21 of the study. Rats (female) were immunized. Then on day 57, the mice were divided into 3 groups (10 mice in each group) as a control group without boosting (Group 1), or boosted with 3 μg of different vaccine candidates, Including Hu-1 S-trimer (Group 2), Delta S-Trimer vaccine (Group 3), all of which are adjuvanted with CpG (150 μg) plus Alum (75 μg). Sera were collected before the third booster dose (D56, D-1 PD3) and 14 days after the third dose (D71, D14 PD3) and tested for neutralizing antibodies against multiple mutant pseudoviruses.

The Delta vaccine candidate (Group 3) significantly boosted neutralizing antibodies to Hu-1, Beta, Gamma, Delta, and Omicron VOCs, including Omicron (12.1-fold boost) (Figure 7A-7G ). 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.

In order to improve the broad spectrum of the vaccine to deal with a variety of existing viruses and viruses that mutate in the future, the inventor of the present application has also studied a series of multivalent vaccine combinations, such as bivalent, trivalent and quadrivalent. Use Hu-1 S-trimer, Omicron S-trimer, Omicron strain (BA.4/5) S-trimer, Belta S-trimer, Delta-S- Trimers are combined to obtain multivalent vaccines. The multivalent vaccine combinations and trial design are shown in Table 1 and Figure 9. Part of the test results are shown in Figure 10. 1.0μg/antigen, (+CpG1018:150ug, Alum:75ug

The invention is not intended to limit the scope to the particular disclosed embodiments, which are provided, for example, to illustrate various aspects of the invention. Various modifications to the compositions and methods will become apparent from the description and teachings herein. These changes may be made without departing from the true scope and spirit of the invention, and are intended to fall within the scope of the invention.

sequence

Claims

A method for preventing coronavirus infection in a mammal, the method comprising immunizing the mammal with an effective amount of a recombinant subunit vaccine, the recombinant subunit vaccine comprising a soluble coronavirus SARS-CoV-2 delta (delta, B. 1.617.2) Surface antigen or fragments, variants or mutants thereof, the soluble coronavirus surface antigen or fragments, variants or mutants thereof linked to the C-terminal portion of collagen by in-frame fusion to form a disulfide bond Trimeric fusion protein.
The method of claim 1, wherein the coronavirus infection is a severe acute respiratory syndrome (SARS)-coronavirus 2 (SARS-CoV-2) infection.
The method of claim 1 or 2, wherein the coronavirus surface antigen includes coronavirus spike (S) protein or a fragment or epitope thereof.
The method according to any one of claims 1-3, wherein 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 the SARS-CoV-2 delta (delta, B.1.617.2) variant S extracellular domain peptide or its fragments, variants or mutants, such as those containing the delta variant receptor binding domain ( RBD) and chimeric sequences of Hu-1 or other variant S protein peptide sequences.
The method according to any one of claims 1-4, wherein the coronavirus surface antigen includes SARS-CoV-2 spike (S) N-terminal domain (NTD) peptide or fragment or epitope 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.
The method according to any one of claims 1-5, wherein the coronavirus surface antigen includes SARS-CoV-2 spike (S) receptor binding domain (RBD) peptide or fragment or epitope thereof, optionally The RBD peptide is the SARS-CoV-2 delta (delta, B.1.617.2) variant RBD peptide or a fragment, variant or mutant thereof.
The method according to any one of claims 1-6, wherein the coronavirus surface antigen includes SARS-CoV-2 spike (S) S1 peptide or a fragment or epitope thereof, optionally the S1 peptide is SARS- CoV-2 delta (delta, B.1.617.2) variant S1 peptide or its fragment, variant or mutant.
The method according to any one of claims 1-7, wherein the coronavirus surface antigen includes SARS-CoV-2 spike (S) S2 peptide or a fragment or epitope thereof, optionally the S2 peptide is SARS- CoV-2 Hu-1, alpha, beta, gamma, delta, myon, or ometron S2 peptide or fragments, variants or mutants thereof.
The method according to any one of claims 1-8, wherein the coronavirus surface antigen includes a SARS-CoV-2 spike (S) extracellular domain peptide with mutations or a fragment or epitope thereof.
The method according to claim 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 no longer has as furin cleavage site. Lin protease cleavage site activity, optionally the mutation is located at any one or several positions 682-685, optionally the mutation includes 685R→685A.
The method according to claim 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.
The method according to any one of claims 9-11, wherein the mutation includes consecutive position amino acid substitutions, such as 986K→986P and 987V→987P.
The method according to any one of claims 1-12, wherein the recombinant subunit vaccine includes the sequence described in any one of SEQ ID NO:81-92 or is consistent with any one of SEQ ID NO:81-92 The sequences have amino acid sequences with at least or about 80%, 85%, 90%, 92%, 95%, 97%, 99% sequence identity.
The method according to any one of claims 1-13, wherein the recombinant subunit vaccine includes SEQ ID NO:85 or is at least or about 80%, 85%, 90%, 92%, Amino acid sequences with 95%, 97%, 99% sequence identity.
The method according to any one of claims 1-14, wherein the recombinant subunit vaccine includes SEQ ID NO:86 or is at least or about 80%, 85%, 90%, 92%, Amino acid sequences with 95%, 97%, 99% sequence identity.
The method according to any one of claims 1-15, wherein the recombinant subunit vaccine includes SEQ ID NO: 87 or is at least or about 80%, 85%, 90%, 92%, Amino acid sequences with 95%, 97%, 99% sequence identity.
The method according to any one of claims 1-16, wherein the recombinant subunit vaccine includes SEQ ID NO:88 or is at least or about 80%, 85%, 90%, 92%, Amino acid sequences with 95%, 97%, 99% sequence identity.
The method according to any one of claims 1-17, wherein the recombinant subunit vaccine includes the sequence described in any one of SEQ ID NO:89-92 or is consistent with any one of SEQ ID NO:89-92 The sequences have amino acid sequences with at least or about 80%, 85%, 90%, 92%, 95%, 97%, 99% sequence identity.
The method according to any one of claims 1-18, wherein the recombinant subunit vaccine includes SEQ ID NO:91 or is at least or about 80%, 85%, 90%, 92%, Amino acid sequences with 95%, 97%, 99% sequence identity.
The method according to any one of claims 1-19, wherein the recombinant subunit vaccine comprises the first sequence of any one of SEQ ID NOs: 81-84 connected to SEQ ID NOs: 67-80 Any of the second sequences, wherein the C-terminus of the first sequence is directly or indirectly connected to the N-terminus of the second sequence.
The method of any one of claims 1-20, wherein the recombinant subunit vaccine is administered by intramuscular injection.
The method of any one of claims 1-21, wherein the recombinant subunit vaccine is administered by intranasal spray.
The method of any one of claims 1-22, wherein the recombinant subunit vaccine is administered in a single dose or in a series of doses spaced weekly or monthly.
The method according to any one of claims 1 to 23, wherein the recombinant subunit vaccine is administered without an adjuvant, optionally as a primary series, an additional dose 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 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 method according to any one of claims 1 to 24, wherein the recombinant subunit vaccine is administered together with 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 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, optionally the initial dose, Adjuvants in additional 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, e.g. 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, α-tocopherol, and Tween-80 and/or Span 85 in the form of an oil-in-water emulsion; or any combination of adjuvants.
The method according to any one of claims 1-25, wherein the recombinant subunit vaccine is administered with more than one adjuvant, optionally as a primary series, an additional dose 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, adenoviral vector vaccines, and inactivated virus vaccines, optionally Adjuvants in starters, additional 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 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, α-tocopherol, and Tween-80 and/or Span 85 in the form of an oil-in-water emulsion; or any combination of adjuvants.
A method for detecting coronavirus antibodies from mammalian serum, the method comprising the step of contacting the serum with a soluble coronavirus SARS-CoV-2 delta (B.1.617.2) surface antigen, the soluble coronavirus surface antigen The antigen is linked to the C-terminal portion of collagen by in-frame fusion to form a disulfide-linked trimeric fusion protein.
The method of claim 27, wherein the soluble coronavirus surface antigen is S protein or peptide.
A method using a recombinant subunit vaccine containing a soluble surface antigen from the coronavirus SARS-CoV-2 delta (B.1.617.2) linked to the C-terminal portion of collagen by in-frame fusion to Forming a disulfide bond-linked trimeric fusion protein, the method includes: immunizing a mammal, purifying the produced neutralizing antibody, and using the neutralizing antibody to treat a patient infected with the coronavirus through passive immunotherapy.
The method of claim 29, wherein the neutralizing antibody comprises a polyclonal antibody.
The method of claim 29, wherein the neutralizing antibody is a monoclonal antibody.
The method of claim 29, wherein the neutralizing antibody is a monoclonal antibody to S protein or peptide.
The method of claim 29, wherein the neutralizing antibody is a monoclonal antibody to the S protein of SARS-CoV-2.
The method of claim 29, wherein the neutralizing antibody is S protein of SARS-CoV-2 Hu-1, Alpha, Beta, Gamma, Delta, Myriad, Omicron and/or other strains. Monoclonal antibodies.
The method of claim 29, wherein the neutralizing antibody is a monoclonal antibody of the S protein of SARS-CoV-2 delta (B.1.617.2).
A complex comprising a recombinant polypeptide selected from the group consisting of SEQ ID NOs: 85-92.
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.
An immunogenic composition comprising a trimer or a combination of any two or more trimers of a recombinant polypeptide comprising a polypeptide selected from the group consisting of SEQ ID NOs: 85-92 sequence of groups.
The immunogenic composition according to claim 38, comprising a trimer of a recombinant polypeptide having the sequence described in SEQ ID NO: 91.
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 the group consisting of SEQ ID NOs: 85-92 A set of recombinant polypeptides, optionally used as a primary series, an additional dose, and/or a homologous or heterologous booster dose, such as the first dose, the 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, adenovirus Use any one or more of vector vaccines and inactivated virus vaccines together.
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 comprising a recombinant polypeptide selected from the group consisting of SEQ ID NOs: 85-92, optionally as a primary series, additional agents (additional dose), and/or homologous or heterologous booster dose (booster dose) use, such as a first dose, a second dose, a third dose, a fourth dose, and/or more doses, optionally the initial dose A dose, an additional dose, or a heterologous booster dose 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.
A method for generating an immune response to a coronavirus surface antigen in a subject,

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 an effective amount of a complex comprising a recombinant polypeptide selected from the group consisting of SEQ ID NOs: 85-92, optionally as a primary series, additional agents (additional dose), and/or homologous or heterologous booster dose (booster dose) use, such as a first dose, a second dose, a third dose, a fourth dose, and/or more doses, optionally the initial dose A dose, an additional dose, or a heterologous booster dose 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.
A method for generating an immune response to a coronavirus surface antigen in a subject,

wherein 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 includes administering to the subject an effective amount of a complex comprising a recombinant polypeptide comprising a sequence described in any one of SEQ ID NOs: 85-92, optionally as a primary series. ), additional doses, and/or homologous or heterologous booster doses, such as a first dose, a second dose, a third dose, a fourth dose, and/or more doses, may Optionally, the initial dose, additional dose, or heterologous booster dose is combined with any one or more of other recombinant subunit vaccines, nanoparticle vaccines, mRNA vaccines, DNA vaccines, adenovirus vector vaccines, and inactivated virus vaccines. use.
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 Optionally the complex or combination of complexes is used as a primary series, additional dose, and/or homologous or heterologous booster dose, e.g. first dose, second dose, A third dose, a fourth dose, and/or more doses, optionally the initial dose, additional dose, or heterologous booster dose in combination with any one or more of other recombinant subunit vaccines, nanoparticle vaccines, mRNA vaccines, DNA vaccines, adenovirus vector vaccines, and inactivated virus vaccines.
The method of claim 44, wherein the method comprises administering to the 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.
A fusion protein containing multiple recombinant polypeptides, each recombinant polypeptide including from amino to carboxyl terminus:

a) a first region comprising the extracellular domain of the coronavirus spike protein located before the coronavirus spike protein receptor binding domain (RBD) in the non-chimeric coronavirus spike protein of the first coronavirus part; part

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) C-terminal propeptide of collagen, wherein the C-terminal propeptide of the recombinant polypeptide forms an inter-polypeptide disulfide bond, wherein at least one of the first coronavirus and the second coronavirus is SARS-CoV-2 delta (B .1.617.2), optionally the fusion protein 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 dose, third dose, fourth dose, and/or more doses, optionally with the initial dose, additional dose, or heterologous booster dose with other recombinant subunit vaccines, nanoparticle vaccines, mRNA vaccines, DNA vaccines, adenocarcinoma vaccines, Use any one or more of viral vector vaccines and inactivated virus vaccines in combination.
The fusion protein of claim 46, further comprising a third region between the second region and the C-terminal propeptide of collagen.
The fusion protein of claim 47, wherein the third region includes the S1 domain of a third coronavirus, and wherein the third coronavirus is the same as or different from the first coronavirus or the second coronavirus.
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 or different from the first, second or fourth coronavirus.
The fusion protein of any one of claims 46-49, wherein the first region includes the N-terminal domain (NTD) of the first coronavirus.
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.
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.
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.
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-CoV-2. Different variants.
The fusion protein according to 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 The group consisting of SARS-CoV-2 viruses of the .143, P.2, B.1.351, P.1, B.1.1.7, B.1.617 and A.23.1 lineages.
A trimeric fusion protein containing three recombinant polypeptides, each from amino to carboxyl terminus:

a) a first region including the N-terminal domain (NTD) of the coronavirus spike protein of SARS-CoV-2 of lineage B.1.617.2;

b) a second region that includes the coronavirus spike protein receptor binding domain (RBD) of SARS-CoV-2 of lineage B.1.617.2; and

c) C-terminal propeptide of collagen, wherein the C-terminal propeptide of the recombinant polypeptide forms an inter-polypeptide disulfide bond, optionally the fusion protein 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, the additional dose, or the heterologous dose The source 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.
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.
The method of claim 57, wherein neutralizing antibodies are produced in the mammal against the first and second coronaviruses.
The method of claim 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, B.1.617. 2.B.1.526, Two or more SARS-CoV-2 viruses of the B.1.1.143, P.2, B.1.351, P.1, B.1.1.7, B.1.617, and A.23.1 lineages.
The method of claim 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.
The method according to any one of claims 57-60, comprising immunizing the mammal with two or more doses of the fusion protein, at least one dose of the two or more doses of the fusion protein comprising SARS- CoV-2 delta (B.1.617.2) spike protein amino acid sequence, optionally the at least one dose of the fusion protein comprises the sequence described in any one of SEQ ID NO:81-92 or with SEQ ID NO:81 The sequence of any one of -92 has an amino acid sequence with at least or about 80%, 85%, 90%, 92%, 95%, 97%, 99% sequence identity.
The method of any one of claims 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 a different SARS-CoV-2 variant. The NTD and RBD spike protein peptides, optionally the one or more doses of the immunogen comprise the sequence described in any one of SEQ ID NO: 27-66 and 81-84 or are consistent with SEQ ID NO: 27 The sequence described in any one of -66 and 81-92 has an amino acid sequence with at least or about 80%, 85%, 90%, 92%, 95%, 97%, 99% sequence identity, optionally the The enhancer fusion protein comprises the sequence described in any one of SEQ ID NO:81-92 or is at least or about 80%, 85%, 90%, Amino acid sequences with 92%, 95%, 97%, 99% sequence identity.
A trimeric fusion protein comprising a soluble coronavirus SARS-CoV-2 delta (B.1.617.2) surface antigen or a fragment, variant or mutant thereof, the soluble coronavirus surface antigen or a fragment or variant thereof Or the mutant is attached to the C-terminal portion of collagen, which forms a disulfide-linked trimer, thereby forming the trimeric fusion protein.
The trimeric fusion protein according to claim 63, which comprises a recombinant polypeptide selected from the group consisting of SEQ ID NO: 85-92.
The trimeric fusion protein according to claim 63 or 64, for preventing coronavirus infection, optionally the coronavirus infection includes SARS-CoV-2 Hu-1, Alpha, Beta, Gamma, Delta, Myriad , Omicron, and other SARS-CoV-2 strains.
The trimeric fusion protein according to any one of claims 63-65, 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 a heterologous booster used in combination with any one or more of other recombinant subunit vaccines, nanoparticle vaccines, mRNA vaccines, DNA vaccines, adenovirus vector vaccines, and inactivated virus vaccines,

Optionally, the trimeric fusion protein is used as an initial agent without an adjuvant, and the initial agent includes a first shot and a second shot,

Optionally the trimeric fusion protein is administered without an adjuvant as an additional dose, including a third shot, a fourth shot, and/or more shots,

Optionally the trimeric fusion protein is used without an adjuvant as a homologous or heterologous booster including a third shot, a fourth shot, and/or more shots.
The trimeric fusion protein according to any one of claims 63-65, wherein the trimeric fusion protein is administered with an 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 Optionally, 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 oligodeoxynucleotide (CpG-ODN)-containing adjuvants; TLR9 agonist-containing adjuvants; metabolizable oils, alpha-tocopherol, and/or polyoxyethylene sorbitan monomers Adjuvants for oleate (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 of the above combinations of adjuvants,

Optionally, the trimeric fusion protein is used as an initial agent together with an adjuvant. The initial agent includes a first shot and a second shot. The adjuvant includes Alum and an adjuvant containing CpG oligodeoxynucleotide (CpG-ODN). Adjuvants containing squalene, alpha-tocopherol, and Tween-80 and/or Span 85 in the form of an agent and/or an oil-in-water emulsion,

Optionally, the trimeric fusion protein is used as an additional agent with an adjuvant including a third shot, a fourth shot, and/or more shots, the adjuvant including Alum and CpG-containing oligodeoxynucleotides (CpG-ODN) adjuvants and/or adjuvants containing squalene, alpha-tocopherol, and Tween-80 and/or Span 85 in the form of oil-in-water emulsions,

Optionally, the trimeric fusion protein is used together with an adjuvant as a homologous or heterologous booster, and the homologous or heterologous booster includes a third shot, a fourth shot, and/or more shots, the adjuvant Includes Alum and adjuvants containing CpG oligodeoxynucleotides (CpG-ODN) and/or adjuvants containing squalene, alpha-tocopherol, and Tween-80 and/or Span 85 in the form of oil-in-water emulsions .
The trimeric fusion protein according to any one of claims 63-65, wherein the trimeric fusion protein is administered with more than one adjuvant, optionally as a primary series ), additional doses, and/or homologous or heterologous booster doses, such as a first dose, a second dose, a third dose, a fourth dose, and/or more doses, may Optionally, the initial dose, additional dose, or heterologous booster dose is combined with any one or more of other recombinant subunit vaccines, nanoparticle vaccines, mRNA vaccines, DNA vaccines, adenovirus vector vaccines, and inactivated virus vaccines. Using, optionally, adjuvants in the priming agent, additional agent, and/or homologous or heterologous booster may independently include: an aluminum-containing adjuvant, such as an adjuvant containing alum and/or aluminum hydroxide; Adjuvants for oligonucleotides, such as CpG oligodeoxynucleotides (CpG-ODN); adjuvants containing TLR9 agonists; metabolizable oils, alpha-tocopherol, and/or polyoxyethylene sorbide Adjuvants for alcoholic anhydride 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 A combination of these adjuvants.
The trimeric fusion protein according to any one of claims 63 to 65 is used as an initial agent for immunizing mammals, and optionally the initial agent includes a first needle and a second needle.
According to the trimeric fusion protein of claim 69, the first shot and/or the second shot have no adjuvant.
According to the trimeric fusion protein of claim 69, the adjuvant of the first shot and/or the second shot includes: an aluminum-containing adjuvant, such as an adjuvant containing alum and/or aluminum hydroxide; and/or Oligonucleotide-containing adjuvants, such as CpG oligodeoxynucleotide-containing adjuvants (CpG-ODN).
According to the trimer fusion protein of claim 69, the adjuvant of the first shot and/or the second shot includes: a single oil containing metabolizable oil, α-tocopherol, and/or polyoxyethylene sorbitan. Adjuvants for acid esters (Tween-80), such as adjuvants containing squalene, alpha-tocopherol, and Tween-80 and/or Span 85 in the form of oil-in-water emulsions.
The trimeric fusion protein according to any one of claims 63-72 is used as a third shot, a fourth shot, and/or more additional shots for immunizing mammals.
The trimeric fusion protein according to any one of claims 63-73 is used as a third shot, a fourth shot, and/or more shot boosters for immunizing mammals,

Optionally the booster is unadjuvanted,

Optional adjuvants for the booster include: aluminum-containing adjuvants, such as alum and/or aluminum hydroxide-containing adjuvants; and/or oligonucleotide-containing adjuvants, such as CpG-containing oligodeoxynucleotides ( CpG-ODN) adjuvant,

Optional adjuvants for the booster include adjuvants containing metabolizable oils, alpha-tocopherol, and/or polyoxyethylene sorbitan monooleate (Tween-80), for example in the form of an oil-in-water emulsion. Adjuvant containing squalene, alpha-tocopherol, and Tween-80 and/or Span 85.
The trimer fusion protein according to any one of claims 63-74 is used in conjunction with one or several other vaccines, and the other one or several vaccines are independently selected from the group consisting of recombinant subunit vaccines, nanoparticle vaccines, A group consisting of mRNA vaccines, DNA vaccines, adenovirus vector vaccines, and inactivated virus vaccines.
The trimeric fusion protein according to any one of claims 77, wherein the trimeric fusion protein is used as an initial agent, and the other one or several other vaccines are used as a booster.
The trimeric fusion protein according to any one of claims 77, wherein the other one or several other vaccines are used as an initial agent, and the trimeric fusion protein is used as a booster.