Proteins and vaccines for infection by SARS-CoV-2 omicron variant XBB and its subvariates.

Abstract

This invention relates to proteins and vaccines for infection by SARS-CoV-2 omicron variant XBB and its subvariates, and belongs to the pharmaceutical field. To address the lack of effective prophylactic and therapeutic agents for infection caused by SARS-CoV-2 omicron variant XBB and its subvariates, this invention provides proteins and vaccines for infection by the variants, which are designed based on the full-length S protein, receptor-binding domain (RBD) sequence and optimized sequence of SARS-CoV-2 omicron variant XBB and its subvariate XBB.1.5, and can help the host fight coronavirus infection, and in particular has relatively good prophylactic and therapeutic effects against cross-infection caused by SARS-CoV-2 omicron variant XBB and its subvariates.

Description

[Technical Field]

[0001] This invention relates to proteins and vaccines for infection caused by SARS-CoV-2 omicron variant XBB and its submutants, and belongs to the pharmaceutical field. [Background technology]

[0002] The novel coronavirus (SARS-CoV-2) is a new beta-coronavirus named by the World Health Organization. This virus has an envelope, and its particles are round or oval in shape, often polymorphic, with a diameter of 60 nm to 140 nm. Its genetic characteristics are remarkably different from those of SARS-CoV and MERS-CoV, making it a branch of novel coronaviruses never before found in humans. Currently, there are five main types of mutated novel coronaviruses: alpha, beta, gamma, delta, and omicron. The omicron variant is further classified into several subvariates, including BA.1, BA.2, BA.2.12.1, BA.4, BA.5, BQ.1.1, and XBB.1.5.

[0003] The main structural proteins of SARS-CoV-2 include spike (Spike, S) protein, envelope (Envelope, E) protein, membrane (Membrane, M) protein and nucleocapsid (Nucleocapsid, N) protein. Among them, the S protein plays an important role in virus infection and pathogenicity and is often used as an antigen for vaccines. Since the SARS-CoV-2 variant XBB contains multiple mutation sites and the S protein of the virus also contains multiple mutation sites, the variant XBB and its sub-variants can escape from the antibodies stimulated by vaccines against the new coronavirus variants (Alpha, Beta, Gamma, Delta, and Omicron), resulting in the failure of the new coronavirus vaccine or a decrease in protection. Therefore, this has become a great pressure on the prevention and control of the new coronavirus. Therefore, for the prevention and treatment of SARS-CoV-2, it is very important to develop vaccines against the SARS-CoV-2 virus variant XBB and its sub-variants, especially broad-spectrum vaccines for various mutated SARS-CoV-2 viruses. Summary of the Invention Means for Solving the Problems

[0004] The present invention aims to solve at least one of the technical problems existing in the prior art. Therefore, the object of the present invention is to provide a protein against infection by the SARS-CoV-2 Omicron variant XBB and its sub-variants. The present invention also provides a vaccine for preventing and / or treating infections caused by the SARS-CoV-2 Omicron variant XBB and its sub-variants, including a recombinant protein vaccine and an adenovirus vector vaccine containing the described protein, and also provides a composition or combination drug containing the two vaccines.

[0005] The present invention first provides a protein for resisting infection caused by the SARS-CoV-2 Omicron variant XBB and its sub-variants, which contains an amino acid sequence selected from any one of SEQ ID NO: 1 to SEQ ID NO: 7. Preferably, the protein is selected from SEQ ID NO: 3 or SEQ ID NO: 5.

[0006] The present invention further provides a precursor of the above protein, which is linked to a signal peptide and / or a protein tag on the above protein.

[0007] Preferably, the protein tag is selected from at least one of a histidine tag (6His tag), a thioredoxin tag (Trx tag), a glutathione transferase tag, a ubiquitin-like modified protein tag, a maltose binding protein tag, a c-Myc protein tag, an Avi tag protein tag, and a nitrogen source using a protein A tag.

[0008] Furthermore, a protease recognition region for cleaving the protein tag is also linked to the protein against infection by the SARS-CoV-2 Omicron variant XBB strain and its sub-variants.

[0009] Preferably, the protease is selected from at least one of enterokinase (EK enzyme), TEV protease, thrombin, factor Xa of blood coagulation, carboxypeptidase A, and rhinovirus 3c protease.

[0010] Furthermore, the amino acid sequence of the precursor is selected from at least one of SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, and SEQ ID NO: 20. Preferably, the protein sequence is selected from SEQ ID NO: 12 or SEQ ID NO: 16.

[0011] The present invention also provides a polynucleotide encoding the described protein or precursor.

[0012] Furthermore, the nucleotide sequence is selected from at least one of SEQ ID NOs: 9, 11, 13, 15, 17, 19, and 21. Preferably, the polynucleotide sequence is selected from SEQ ID NOs: 13 or 17.

[0013] The present invention further provides a recombinant vector comprising a polynucleotide.

[0014] Furthermore, the recombinant vector uses at least one of the following: an insect baculovirus expression vector, a mammalian cell expression vector, an E. coli expression vector, and a yeast expression vector.

[0015] Preferably, the insect baculovirus expression vector is pFastBac1.

[0016] Preferably, the E. coli expression vector is pET32a.

[0017] Preferably, the yeast expression vector is pPICZaA.

[0018] Preferably, the mammalian cell expression vector is a CHO cell expression vector.

[0019] More preferably, the CHO cell expression vector is pTT5 or FTP-002.

[0020] The present invention further provides a host cell containing a recombinant vector.

[0021] Furthermore, the host cell used will be at least one of the following: insect cells, mammalian cells, E. coli, and yeast.

[0022] Preferably, the insect cells are selected from at least one of sf9 cells, sf21 cells, and Hi5 cells.

[0023] Preferably, the mammalian cells are CHO cells.

[0024] The present invention further provides a method for preparing a protein or precursor, the method comprising the steps of culturing host cells to express the protein or precursor, and then recovering the protein.

[0025] The present invention also provides a protein composition for resistance to infection caused by SARS-CoV-2 omicron variants and their submutations, comprising any two or more combinations of BA.5 sequence-1, BA.5 sequence-2, XBB.1.5 sequence-1, XBB.1.5 sequence-2, XBB.1.5 sequence-3, XBB.1.5 sequence-4, and S-XBB.1.5 protein.

[0026] Preferably, the protein compositions include one of BA.5 sequence-1 and BA.5 sequence-2, one of XBB.1.5 sequence-1, XBB.1.5 sequence-2, XBB.1.5 sequence-3, and XBB.1.5 sequence-4, and at least two combinations of S-XBB1.5 protein.

[0027] More preferably, the protein composition comprises one of BA.5 sequence-1 and BA.5 sequence-2, and one of the combinations of XBB.1.5 sequence-3 and XBB.1.5 sequence-4 with the S-XBB1.5 protein.

[0028] The present invention further provides a recombinant protein vaccine for preventing and / or treating infection by SARS-CoV-2 omicron variants and subvariants, comprising a protein, a precursor and / or a protein composition, and a pharmaceutically acceptable excipient or adjuvant component.

[0029] Furthermore, the adjuvant component is an immunoadjuvant.

[0030] Preferably, the immunoadjuvant is selected from at least one of a squalene-based oil-in-water emulsion, aluminum salts, calcium salts, plant saponins, plant polysaccharides, monophosphate tripeptide A, muramyl dipeptide, muramyl tripeptide, bacterial toxins, GM-CSF cytokines, lipids, and cationic liposome materials.

[0031] Furthermore, at least one of the following conditions is met: the squalene oil-in-water emulsion is MF59; the aluminum salt is selected from at least one of aluminum hydroxide and alum; the calcium salt is tricalcium phosphate; the plant saponin is either QS-21 or ISCOM; the plant polysaccharide is astragalus polysaccharide; the bacterial toxin is selected from at least one of recombinant cholera toxin and diphtheria toxin; and the lipid is selected from at least one of phosphatidylethanolamine, phosphatidylcholine, cholesterol, and dioleoylphosphatidylethanolamine. The cationic liposome material is selected from (2,3-dioleoyloxypropyl)trimethylammonium chloride, N-[1-(2,3-dioleoylchloride)propyl]-N,N,N-trimethylamine chloride, cationic cholesterol, dimethyl-2,3-dioleyloxypropyl-2-(2-spermidinecarboxamide)ethylammonium trifluoroacetate, trimethyldodecylammonium bromide, trimethyltetradecylammonium bromide, trimethylhexadecylammonium bromide, dimethyldioctadecylammonium bromide, and CpG ODN.

[0032] The present invention also provides an adenovirus vector vaccine against infection caused by SARS-CoV-2 omicron variant XBB and its subvariants, comprising constructing a recombinant vector comprising a polynucleotide encoding the amino acid sequence of a described protein, a described precursor, or a described protein composition.

[0033] Preferably, the nucleotide sequence of the polynucleotide is selected from at least one of SEQ ID NOs: 9, SEQ ID NOs: 11, SEQ ID NOs: 13, SEQ ID NOs: 15, SEQ ID NOs: 17, SEQ ID NOs: 19, and SEQ ID NOs: 21. More preferably, the polynucleotide sequence contained in the adenovirus vector is selected from the polynucleotides for constructing S-XBB.1.5, as shown in SEQ ID NOs: 21.

[0034] Furthermore, the adenovirus vector is selected from at least one of the following: adenovirus, Ankarawaxinia virus, and adeno-associated virus.

[0035] Preferably, it is selected from human adenovirus types 5, 35, or 26 and / or chimpanzee AdC68 or AdC7 replication-deficient adenoviruses.

[0036] More preferably, it is selected from human type 5 replication-deficient adenovirus having a combined E1 and E3 deletion.

[0037] The present invention provides a method for preparing an adenovirus in a described adenovirus vector vaccine, comprising the steps of: constructing a described polynucleotide shuttle plasmid vector; transfecting a host cell with the constructed shuttle plasmid vector together with a backbone plasmid and culturing the host cell; obtaining a replication-deficient recombinant adenovirus; and then growing and purifying the culture.

[0038] Furthermore, adenovirus vector vaccines also contain pharmaceutically acceptable adjuvants. Includes vectors, diluents, or excipients.

[0039] Furthermore, recombinant protein vaccines and adenovirus vector vaccines are available as intradermal or subcutaneous injections, intramuscular injections, intravenous injections, or oral or nasal sprays.

[0040] Preferably, the vaccine is an intramuscular injection or a nasal spray.

[0041] The present invention also provides compositions for treating and / or preventing SARS-CoV-2 omicron variants and subvariates, which are formulations comprising recombinant protein vaccines and adenovirus vector vaccines as active ingredients.

[0042] The present invention provides a combination therapy for resistance to infection caused by SARS-CoV-2 omicron variant XBB and its subvariates, comprising a recombinant protein vaccine and an adenovirus vector vaccine administered separately or simultaneously.

[0043] Preferably, the composition or combination drug is a combination or combination drug of recombinant protein vaccine 1, recombinant protein vaccine 2, and adenovirus vector vaccine.

[0044] More preferably, recombinant protein vaccine 1 comprises at least one amino acid sequence of BA.5 sequence-1, BA.5 sequence-2 protein or precursor, recombinant protein vaccine 2 comprises at least one amino acid sequence of XBB.1.5 sequence-1, XBB.1.5 sequence-2, XBB.1.5 sequence-3, XBB.1.5 sequence-4 protein or precursor, and adenovirus vector vaccine comprises a polynucleotide sequence of S-XBB.1.5.

[0045] Furthermore, the amino acid sequence contained in recombinant protein vaccine 1 is selected from at least one of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 8, and SEQ ID NO: 10.

[0046] Furthermore, the amino acid sequence contained in recombinant protein vaccine 2 is selected from at least one of SEQ ID NOs: 3 to 6, SEQ ID NOs: 12, SEQ ID NOs: 14, SEQ ID NOs: 16, and SEQ ID NOs: 18. Preferably, it is selected from at least one of SEQ ID NOs: 5, SEQ ID NOs: 6, SEQ ID NOs: 16, and SEQ ID NOs: 18.

[0047] Furthermore, the adenovirus vector vaccine contains the polynucleotide sequence shown in SEQ ID NO: 21.

[0048] Furthermore, the composition or combination drug may be an intradermal or subcutaneous injection, an intramuscular injection, an intravenous injection, or an oral or nasal spray, and preferably the vaccine may be an intramuscular injection or a nasal spray.

[0049] The present invention also provides the use of described proteins, described precursors, described protein compositions, described recombinant protein vaccines, adenovirus vector vaccines, vaccine compositions, or combination drugs in the preparation of drugs for treating and / or preventing infection or pathogenicity caused by SARS-CoV-2 omicron variants and subvariates.

[0050] The present invention also provides vaccine compositions for treating and / or preventing infection caused by SARS-CoV-2 omicron variants and subvariates, comprising a recombinant protein vaccine and an adenovirus vector vaccine, wherein the amino acid sequence of the recombinant protein vaccine is selected from at least one of SEQ ID NOs: 1 to 7 and SEQ ID NOs: 8 to 20, and the nucleotide sequence for the antigen of the adenovirus vector vaccine is selected from SEQ ID NOs: 23.

[0051] The present invention also provides a combination drug for treating and / or preventing infection caused by SARS-CoV-2 omicron variants and subvariates, wherein the recombinant protein vaccine and adenovirus vector vaccine are administered separately or simultaneously, the amino acid sequence of the recombinant protein vaccine is selected from at least one of SEQ ID NOs: 1 to SEQ ID NOs: 7 and SEQ ID NOs: 8 to SEQ ID NOs: 20, and the nucleotide sequence for the antigen of the adenovirus vector vaccine is selected from SEQ ID NOs: 23.

[0052] The following sequences were constructed by the applicant based on optimized RBD sequences in the S protein of the SARS-CoV-2 mutants Omicron mutant BA.5, XBB, and submutant XBB.1.5, and the constructed proteins or precursors are defined as BA.5 sequence-1, BA.5 sequence-2, XBB.1.5 sequence-1, XBB.1.5 sequence-2, XBB.1.5 sequence-3, XBB.1.5 sequence-4, and S-XBB.1.5 protein or precursor.

[0053] Sequence ID 1 BA.5 Sequence-1 VQPTESIVRFPNITNLCPFDEVFNATRFASVYAWNRKRISNCVADYSVLYNFAPFFAFKCYGVSPTKLNDLCFTNVYADSFVIRGNEVSQIAPGQTGNIADYNYKLPDDFTGCVIAWNSNKLDSKVGGNYNYRYRLFRKSNLKPFERDISTEIYQAGNKPCN GVAGVNCYFPLQSYGFRPTYGVGHQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKSVNFNFNGLYENQKLIANQFNSAIGKIQDSLSSTASALGKLQDVVNQNAQALNTLVKQLKNHTSPDVDLGDISGINASVVNIQKEIDRLNEVAKNLNESLIDLQEL

[0054] Sequence ID 2 BA.5 Sequence-2 VQPTESIVRFPNITNLCPFDEVFNATRFASVYAWNRKRISNCVADYSVLYNSASFSAFKCYGVSPTKLNDLCFTNVYADSFVIRGNEVSQIAPGQTGNIADYNYKLPDDFTGCVIAWNSNKLDSKVGGNYNYRYRLFRKSNLKPFERDISTEIYQAGNKPCN GVAGVNCYFPLQSYGFRPTYGVGHQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKSVNFNFNGLYENQKLIANQFNSAIGKIQDSLSSTASALGKLQDVVNQNAQALNTLVKQLKNHTSPDVDLGDISGINASVVNIQKEIDRLNEVAKNLNESLIDLQEL

[0055] Sequence ID 3 XBB.1.5 Sequence-1 VQPTESIVRFPNITNLCPFHEVFNATTFASVYAWNRKRISNCVADYSVIYNFAPFFAFKCYGVSPTKLNDLCFTNVYADSFVIRGNEVSQIAPGQTGNIADYNYKLPDDFTGC VIAWNSNKLDSKPSGNYNYLYRLFRKSKLKPFERDISTEIYQAGNKPCNGVAGPNCYSPLQSYGFRPTYGVGHQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKCVNFNFNG

[0056] Sequence ID 4 XBB.1.5 Sequence-2 VQPTESIVRFPNITNLCPFHEVFNATTFASVYAWNRKRISNCVADYSVIYNSASFSAFKCYGVSPTKLNDLCFTNVYADSFVIRGNEVSQIAPGQTGNIADYNYKLPDDFTGC VIAWNSNKLDSKPSGNYNYLYRLFRKSKLKPFERDISTEIYQAGNKPCNGVAGPNCYSPLQSYGFRPTYGVGHQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKCVNFNFNG

[0057] Sequence ID 5 XBB.1.5 Sequence-3 VQPTESIVRFPNITNLCPHEVFNATTFASVYAWNRKRISNCVADYSVIYNFAPFFAFKCYGVSPTKLNDLCFTNVYADSFVIRGNEVSQIAPGQTGNIADYNYKLPDDFTGCVIAWNSNKLDSKPSGNYNYLYRLFRKSKLKPFERDISTEIYQAGNKPCN GVAGPNCYSPLQSYGFRPTYGVGHQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKSVNFNGLYENQKLIANQFNSAIGKIQDSLSSTASALGKLQDVVNQNAQALNTLVKQLKNHTSPDVDLGDISGINASVVNIQKEIDRLNEVAKNLNESLIDLQEL

[0058] Sequence ID 6 XBB.1.5 Sequence-4 VQPTESIVRFPNITNLCPHEVFNATTFASVYAWNRKRISNCVADYSVIYNSASFSAFKCYGVSPTKLNDLCFTNVYADSFVIRGNEVSQIAPGQTGNIADYNYKLPDDFTGCVIAWNSNKLDSKPSGNYNYLYRLFRKSKLKPFERDISTEIYQAGNKPCN GVAGPNCYSPLQSYGFRPTYGVGHQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKSVNFNGLYENQKLIANQFNSAIGKIQDSLSSTASALGKLQDVVNQNAQALNTLVKQLKNHTSPDVDLGDISGINASVVNIQKEIDRLNEVAKNLNESLIDLQEL

[0059] Sequence ID 7 S-XBB.1.5

[0060] To support the secretory expression of the protein, a signal peptide was added to its amino acids during protein construction. Simultaneously, a His tag was added to the amino acid sequence of the protein to facilitate purification. The complete BA.5 sequence-1, BA.5 sequence-2, XBB.1.5 sequence-1, XBB.1.5 sequence-2, XBB.1.5 sequence-3, XBB.1.5 sequence-4, and S-XBB.1.5 amino acid sequences of the present invention are shown as SEQ ID NOs. 8, SEQ ID NOs. 10, SEQ ID NOs. 12, SEQ ID NOs. 14, SEQ ID NOs. 16, SEQ ID NOs. 18, and SEQ ID NOs. 20, respectively.

[0061] Furthermore, the corresponding nucleotide sequences encoding the amino acid sequences are shown as SEQ ID NOs. 9, SEQ ID NOs. 11, SEQ ID NOs. 13, SEQ ID NOs. 15, SEQ ID NOs. 17, SEQ ID NOs. 19, and SEQ ID NOs. 21, respectively.

[0062] Sequence ID 8: Complete BA.5 sequence - 1 signal peptide - Trx tag - 6 His tag - EK restriction site - RBD sequence - HR1 sequence - HR2 sequence MLLVNQSHQGFNKEHTSKMVSAIVLYVLLAAAAHSAFAADSIHIKDSDDLKNRLAEAGDKLVVIDFMATWCGPCKMIGPKLDEMANEMSDSIVVLKVDVDECEDIATEYNINSMPTFVFVKNS KKIEEFSGANVDKLRNTIIKLKLAGSGSGHMHHHHHHSSGDDDDKVQPTESIVRFPNITNLCPFDEVFNATRFASVYAWNRKRISNCVADYSVLYNFAPFFAFKCYGVSPTKLNDLCFTNVYA DSFVIRGNEVSQIAPGQTGNIADYNYKLPDDFTGCVIAWNSNKLDSKVGGNYNYRYRLFRKSNLKPFERDISTEIYQAGNKPCNGVAGVNCYFPLQSYGFRPTYGVGHQPYRVVVLSFELLHA PATVCGPKKSTNLVKNKSVNFNFNGLYENQKLIANQFNSAIGKIQDSLSSTASALGKLQDVVNQNAQALNTLVKQLKNHTSPDVDLGDISGINASVVNIQKEIDRLNEVAKNLNESLIDLQEL

[0063] Nucleotide sequences encoding SEQ ID NO: 9-SEQ ID NO: 8

[0064] Sequence ID 10: Complete BA.5 sequence - 2 signal peptide - Trx tag - 6 His tag - EK restriction site - RBD sequence - HR1 sequence - HR2 sequence MLLVNQSHQGFNKEHTSKMVSAIVLYVLLAAAAHSAFAADSIHIKDSDDLKNRLAEAGDKLVVIDFMATWCGPCKMIGPKLDEMANEMSDSIVVLKVDVDECEDIATEYNINSMPTFVFVKNS KKIEEFSGANVDKLRNTIIKLKLAGSGSGHMHHHHHHSSGDDDDKVQPTESIVRFPNITNLCPFDEVFNATRFASVYAWNRKRISNCVADYSVLYNSASFSAFKCYGVSPTKLNDLCFTNVYA DSFVIRGNEVSQIAPGQTGNIADYNYKLPDDFTGCVIAWNSNKLDSKVGGNYNYRYRLFRKSNLKPFERDISTEIYQAGNKPCNGVAGVNCYFPLQSYGFRPTYGVGHQPYRVVVLSFELLHA PATVCGPKKSTNLVKNKSVNFNFNGLYENQKLIANQFNSAIGKIQDSLSSTASALGKLQDVVNQNAQALNTLVKQLKNHTSPDVDLGDISGINASVVNIQKEIDRLNEVAKNLNESLIDLQEL

[0065] Nucleotide sequences encoding SEQ ID NOs: 11-10

[0066] Sequence ID 12: Complete XBB.1.5 sequence-1 signal peptide-Trx tag-6 His tag-EK restriction site-RBD sequence MLLVNQSHQGFNKEHTSKMVSAIVLYVLLAAAAHSAFAADSIHIKDSDDLKNRLAEAGDKLVVIDFMATWCGPCKMIGPKLDEMANEMSDCIVVLKVDVDECEDIATEYNINSMPTFVFVKNSKKIEEFSGANVDKLRNTIIKLKLAGSGSGHMHHHHHHSSGDDDDKVQPTESIVRFPNITNLCPFHEVFNATTFA SVYAWNRKRISNCVADYSVIYNFAPFFAFKCYGVSPTKLNDLCFTNVYADSFVIRGNEVSQIAPGQTGNIADYNYKLPDDFTGCVIAWNSNKLDSKPSGNYNYLYRLFRKSKLKPFERDISTEIYQAGNKPCNGVAGPNCYSPLQSYGFRPTYGVGHQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKCVNFNFNG

[0067] Sequence ID 13: The nucleotide sequence encoding Sequence ID 12.

[0068] Sequence ID 14: Complete XBB.1.5 sequence-2 signal peptide-Trx tag-6 His tag-EK restriction site-RBD sequence MLLVNQSHQGFNKEHTSKMVSAIVLYVLLAAAAHSAFAADSIHIKDSDDLKNRLAEAGDKLVVIDFMATWCGPCKMIGPKLDEMANEMSDCIVVLKVDVDECEDIATEYNINSMPTFVFVKNSKKIEEFSGANVDKLRNTIIKLKLAGSGSGHMHHHHHHSSGDDDDKVQPTESIVRFPNITNLCPFHEVFNATTFA SVYAWNRKRISNCVADYSVIYNSASFSAFKCYGVSPTKLNDLCFTNVYADSFVIRGNEVSQIAPGQTGNIADYNYKLPDDFTGCVIAWNSNKLDSKPSGNYNYLYRLFRKSKLKPFERDISTEIYQAGNKPCNGVAGPNCYSPLQSYGFRPTYGVGHQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKCVNFNFNG

[0069] Nucleotide sequences encoding SEQ ID NOs: 15-14

[0070] Sequence ID 16: Complete XBB.1.5 sequence - 3 signal peptide - Trx tag - 6 His tag - EK restriction site - RBD sequence - HR1 sequence - HR2 sequence MLLVNQSHQGFNKEHTSKMVSAIVLYVLLAAAAHSAFAADSIHIKDSDDLKNRLAEAGDKLVVIDFMATWCGPCKMIGPKLDEMANEMSDSIVVLKVDVDECEDIATEYNINSMPTFVFVKNS KKIEEFSGANVDKLRNTIIKLKLAGSGSGHMHHHHHHSSGDDDDKVQPTESIVRFPNITNLCPFHEVFNATTFASVYAWNRKRISNCVADYSVIYNFAPFFAFKCYGVSPTKLNDLCFTNVYA DSFVIRGNEVSQIAPGQTGNIADYNYKLPDDFTGCVIAWNSNKLDSKPSGNYNYLYRLFRKSKLKPFERDISTEIYQAGNKPCNGVAGPNCYSPLQSYGFRPTYGVGHQPYRVVVLSFELLHA PATVCGPKKSTNLVKNKSVNFNFNGLYENQKLIANQFNSAIGKIQDSLSSTASALGKLQDVVNQNAQALNTLVKQLKNHTSPDVDLGDISGINASVVNIQKEIDRLNEVAKNLNESLIDLQEL

[0071] Sequence ID 17: The nucleotide sequence encoding Sequence ID 16.

[0072] Sequence ID 18: Complete XBB.1.5 sequence - 4 signal peptide - Trx tag - 6 His tag - EK restriction site - RBD sequence - HR1 sequence - HR2 sequence MLLVNQSHQGFNKEHTSKMVSAIVLYVLLAAAAHSAFAADSIHIKDSDDLKNRLAEAGDKLVVIDFMATWCGPCKMIGPKLDEMANEMSDSIVVLKVDVDECEDIATEYNINSMPTFVFVKNS KKIEEFSGANVDKLRNTIIKLKLAGSGSGHMHHHHHHSSGDDDDKVQPTESIVRFPNITNLCPFHEVFNATTFASVYAWNRKRISNCVADYSVIYNSASFSAFKCYGVSPTKLNDLCFTNVYA DSFVIRGNEVSQIAPGQTGNIADYNYKLPDDFTGCVIAWNSNKLDSKPSGNYNYLYRLFRKSKLKPFERDISTEIYQAGNKPCNGVAGPNCYSPLQSYGFRPTYGVGHQPYRVVVLSFELLHA PATVCGPKKSTNLVKNKSVNFNFNGLYENQKLIANQFNSAIGKIQDSLSSTASALGKLQDVVNQNAQALNTLVKQLKNHTSPDVDLGDISGINASVVNIQKEIDRLNEVAKNLNESLIDLQEL

[0073] Sequence ID 19: Nucleotide sequence encoding Sequence ID 18

[0074] Sequence ID 20: Complete S-XBB.1.5 amino acid sequence, Ad5 XBB.1.5 Adenovirus vaccine antigen amino acid sequence

[0075] Sequence ID 21: The nucleotide sequence encoding Sequence ID 20, Ad5 XBB.1.5 Adenovirus vaccine antigen nucleotide sequence

[0076] The applicant prepared an adenovirus vaccine using the full-length gene of the S protein of omicron variant BA.5, as shown in Sequence ID No. 23, and then prepared a recombinant protein vaccine using the RBD sequence or RBD-HR of the S proteins of omicron variant BA.5 and XBB.1.5, and found that the combination of the adenovirus vaccine and the recombinant protein vaccine also has good prophylactic and therapeutic effects against cross-infection caused by SARS-CoV-2 or its variant viruses.

[0077] Sequence ID 22 Complete S- BA.5 Amino acid sequence, Ad5 BA.5 Adenovirus vaccine antigen amino acid sequence

[0078] Sequence ID 23 contains the nucleotide sequence encoding Sequence ID 22, Ad5 BA.5 Adenovirus vaccine antigenic nucleotide sequence

[0079] Beneficial Effects: The present invention was first used to prepare recombinant protein vaccines against SARS-CoV-2 and its variants. The main idea for constructing the recombinant protein sequence is based on the RBD sequences of the S protein of SARS-CoV-2 omicron variants BA.5 and XBB.1.5, as well as the recombinant sequence formed by the RBD sequence of the S protein of SARS-CoV-2 and the heptapeptide repeat regions HR1 and HR2 for protein construction. At the same time, after the addition of the corresponding vaccine adjuvant, it can further help the host resist cross-infection caused by omicron variant XBB and its subvariates, which is very important for the development of recombinant protein vaccines against SARS-CoV-2 omicron variant XBB and its subvariates.

[0080] Simultaneously, the present invention has also been used to construct recombinant adenovirus vectors based on the nucleotide sequences of the full-length S proteins of SARS-CoV-2 omicron variants BA.5 and XBB.1.5 to obtain adenovirus vector vaccines, and two or three prepared different recombinant adenovirus vector vaccines can be combined with different recombinant protein vaccines to obtain vaccine compositions having better prophylactic or therapeutic effects against SARS-CoV-2 and its variants, and the preparation of nasal spray reagents further promotes resistance to SARS-CoV-2 and its variants. [Brief explanation of the drawing]

[0081] [Figure 1] This is a graph showing the design of pFastBac1-GP67-Trx-His-EK-S-RBD(Omicron_XBB.1.5)-HR in Embodiment 1. [Figure 2] This is a 1% agarose gel electrophoresis graph of PCR products 1-3 in Embodiment 1. [Figure 3] This is a 1% agarose gel electrophoresis graph of recombinant bacmid identified by PCR in Embodiment 1. [Figure 4] This is a flowchart of the packaging and amplification of recombinant baculovirus in Embodiment 1. [Figure 5] This graph shows the results of WB verification during baculovirus proliferation in Embodiment 1. [Figure 6] This graph shows the results of elution supernatant after baculovirus infection using Ni-affinity chromatography in Embodiment 1. [Figure 7] This graph shows the results of verifying the enzyme cleavage effect using EK restriction enzyme cleavage samples in Embodiment 1. [Figure 8] This graph shows the packing purification by Ni-affinity chromatography and SDS-PAGE identification after EK restriction enzyme digestion in Embodiment 1. [Figure 9] This is the expression identification of XBB.1.5 adenovirus in Embodiment 2. [Figure 10] This figure shows serum IgG and washing solution IgG / IgA of the Ad5XBB.1.5+RBDXBB.1.5-HR bivalent vaccine in Embodiment 4. [Figure 11] This figure shows serum IgG and washing solution IgG / IgA of the Ad5BA.5+RBDBA.5-HR bivalent vaccine in Embodiment 4. [Figure 12] This figure shows serum IgG and washing solution IgG / IgA of the Ad5BA.5+RBDXBB.1.5-HR bivalent vaccine in Embodiment 4. [Figure 13] This figure shows serum IgG and washing solution IgG / IgA of the Ad5XBB.1.5+RBDBA.5-HR bivalent vaccine in Embodiment 4. [Figure 14] This figure shows serum IgG and washing solution IgG / IgA data for the trivalent vaccine formulation Ad5XBB.1.5+RBDXBB.1.5-HR+RBDBA.5-HR from Embodiment 4. [Figure 15] This figure shows neutralizing antibodies in mouse serum against the RBDXBB.1.5 vaccine (aluminum adjuvant) in Embodiment 5. [Figure 16] This figure shows the neutralizing antibodies in mouse serum of the RBDXBB.1.5-HR vaccine (MF59 adjuvant) in Embodiment 5. [Figure 17] This figure shows neutralizing antibodies in mouse serum of the RBDXBB.1.5 vaccine in Embodiment 5. [Figure 18] This figure shows serum neutralizing antibody data for the bivalent vaccine Ad5XBB.1.5+RBDXBB.1.5-HR from Embodiment 5. [Figure 19] This figure shows the neutralizing antibodies in the bronchoalveolar lavage fluid of the Ad5XBB.1.5+RBDXBB.1.5-HR bivalent vaccine in Embodiment 5. [Figure 20] This figure shows the serum neutralizing antibodies to the Ad5XBB.1.5+RBDXBB.1.5-HR+RBDBA.5-HR trivalent vaccine in Embodiment 5. [Figure 21] This figure shows true virus-neutralizing antibodies in serum from the Ad5XBB.1.5+RBDXBB.1.5-HR bivalent vaccine in Embodiment 6. [Figure 22] This figure shows the timetable for the mouse immune attack in Embodiment 7. [Figure 23] This figure shows the viral load in mouse pharyngeal swabs in Embodiment 7. [Figure 24] This figure shows the viral gene / subgene RNA in Embodiment 7. [Figure 25] This figure shows the histopathological changes in mouse lung tissue in Embodiment 7. [Figure 26] This figure shows the histopathological score of mouse lung tissue in Embodiment 7. [Modes for carrying out the invention]

[0082] Terms and abbreviations: Monophosphate peptide a (MPL), squalene-based oil-in-water emulsion (MF59), recombinant cholera toxin (rCTB), astragalus polysaccharide (APS), phosphatidylethanolamine (PE), phosphatidylcholine (PC), cholesterol (Chol), dioleoylphosphatidylethanolamine (DOPE), (2,3-dioleoxypropyl)trimethylammonium chloride (DOTAP), N-[1-(2,3-dioleoxy)propyl]-N,N N-trimethylammonium chloride (DOTMA), cationic cholesterol (DC-Chol), dimethyltrifluoroacetate-2,3-dioleoxypropyl-2-(2-speramide)ethylammonium (DOSPA), trimethyldodecylammonium bromide (DTAB), trimethyltetradecylammonium bromide (TTAB), trimethylcetylammonium bromide (CTAB), dimethyldidoctadecylammonium bromide (DDAB), CpG ODN (synthetic CpG with unmethylated cytosine and guanine dinucleotide as core sequences).

[0083] The present invention first designs recombinant protein vaccines against SARS-CoV-2 omicron mutant XBB and its submutations based on the amino acid sequences from positions 320 to 545 of the spike proteins derived from SARS-CoV-2 omicron mutants BA.5 and XBB.1.5, in combination with 7-amino acid repeat region 1 (HR1) and 7-amino acid repeat region 2 (HR2).

[0084] The present invention further designs recombinant adenovirus vaccines against SARS-CoV-2 omicron variant XBB and its submutations based on nucleotide sequences encoding the full-length spike proteins of SARS-CoV-2 omicron BA.5 and XBB.1.5 variants.

[0085] The present invention also provides for the preparation of bivalent and trivalent nasal sprays that combine different recombinant protein vaccines and different recombinant adenovirus vaccines to target the S protein of the SARS-CoV-2 virus, particularly blocking the ACE2 receptor binding region of the S protein, thereby inducing antibody production and other immune responses in the body, blocking the binding of the SARS-CoV-2 S protein to the ACE2 receptor on host cells, thereby helping the host resist coronavirus infection, and having good preventive and therapeutic effects against cross-infection caused by SARS-CoV-2 or its variant viruses, such as the SARS-CoV-2 omicron variant and its subvariant XBB.1.5.

[0086] The solutions of the present invention will be described with reference to embodiments. Those skilled in the art will understand that the following embodiments are not intended to limit the scope of the invention, but merely to illustrate it. Where no specific technical or condition is specified in an embodiment, the technical or condition described in the relevant art literature or product specifications shall prevail. Where the manufacturer of the reagents or equipment used is not indicated, they are all conventional products available by market purchase. [Examples]

[0087] Embodiment 1 Preparation of recombinant protein by expression using an insect baculovirus system (S-RBD XBB.1.5 (HR is given as an example.) 1. S-RBD XBB.1.5 - HR system design Since the SARS-CoV-2 S protein is a membrane-located protein, to mimic its secretion process, the GP67 signal peptide sequence was added to the N-terminus of the S-RBD(omicron_XBB.1.5)-HR protein during the construction of S-RBD(omicron_XBB.1.5)-HR protein expression in SARS-CoV-2 to assist in protein secretion and expression. However, this signal peptide would likely be spontaneously removed by insect cells during the protein secretion process. On the other hand, the thioredoxin (Trx) tag from Spodoptera fulgiperda (S. fulgiperda) was added after the GP67 signal peptide to assist in the folding of S-RBD(omicron_XBB.1.5)-HR, a 6×his tag was added to facilitate subsequent purification, and an EK restriction enzyme cleavage site was added to remove both the Trx and 6×his tags. The construction and design of protein expression would allow for the removal of all non-S-RBD(omicron_XBB.1.5)-HR redundant amino acids by EK restriction enzyme cleavage. The expression construction design patterns are shown in Figure 1.

[0088] The amino acid sequence of GP67-Trx-His-EK-S-RBD(Omicron_XBB.1.5) is shown in SEQ ID NO: 12, the amino acid sequence of GP67-Trx-His-EK-S-RBD(Omicron_XBB.1.5)-HR is shown in SEQ ID NO: 16, and the nucleotide sequences encoding SEQ ID NO: 12 and SEQ ID NO: 16 are shown in SEQ ID NO: 13 and SEQ ID NO: 17, respectively.

[0089] Similarly, the amino acid sequence of GP67-Trx-His-EK-S-RBD(Omicron_XBB.BA.5)-HR is shown in SEQ ID NO: 8, and the nucleotide sequence encoding that amino acid sequence is shown in SEQ ID NO: 9.

[0090] The above sequence was constructed based on the RBD sequence of S protein 320-545, where amino acids 52, 54, and 56 were F, P, and F, respectively.

[0091] 2. Identification for Recombinant Plasmid Construction Sequence ID 16 was used as the coding fragment, i.e., the XBB.1.5-3 sequence. The designed coding fragment was cloned into the pFastBac1 vector plasmid and identified by bacterial suspension PCR. As shown in Figure 2, the bacterial suspension PCR identification showed that, among the three selected clones, clones 2 and 3 successfully amplified the GP67-Trx-His-EK-S-RBD(Omicron_XBB.1.5)-HR fragment. 3. Identification of recombinant bacmids

[0092] We selected precisely identified pFastBac1-GP67-Trx-His-EK-S-RBD(Omicron_XBB.1.5)-HR recombinant clones, extracted the recombinant plasmids, transformed DH10B competent cells, and performed identification by bacterial suspension PCR using blue-white spots. The bacterial suspension PCR products were detected by 1% agarose gel electrophoresis, and the identification results are shown in Figure 3. White spots indicated bacmid clones that underwent recombination, while blue spots indicated bacmid clones that did not undergo recombination.

[0093] 4. Packaging of recombinant baculoviruses Recombinant bacmid was transfected into sf9 insect cells, and P0 passaged recombinant baculovirus was recovered after 5 days. The flowchart for packaging and amplification of the recombinant baculovirus is shown in Figure 4.

[0094] 5. Expression and validation of target proteins The expression of the target protein occurred simultaneously with the amplification of the baculovirus described above. Since the His tag was included before removing the target protein tag, recombinant protein expression was verified using anti-His WB experiments. The verification results showed clear stripes between the 40KD–55KD marker stripes, indicating that the size was consistent with the Trx-His-EK-S-RBD(Omicron_XBB.1.5)-HR protein, demonstrating successful baculovirus amplification and target protein expression. The test results are shown in Figure 5.

[0095] 6. Purification and identification of target proteins P0 passaged recombinant baculovirus was used to infect sf9 cells, and the cell culture medium was collected after 3 days. Protein purification was verified by Ni affinity chromatography. The results are shown in Figure 6. The target protein was mainly eluted under 40 mM and 250 mM imidazole, and it is possible that high-purity target protein was obtained after elution.

[0096] 7. Verification of tag removal after EK restriction enzyme digestion. The target protein eluate was concentrated to a concentration of 1 mg / ml, EK enzyme was added, and the protein was enzymatically digested at 18°C ​​for 14 hours. Identification was then performed by SDS-PAGE gel electrophoresis. The results are shown in Figure 7. These results demonstrate that the EK enzyme can cleave the Trx-His-EK (amino acid sequence of the EK restriction enzyme cleavage site) tag from the target protein.

[0097] 8. Verification of the removal of removed tags The sample after EK restriction enzyme digestion was back-suspended for 10 minutes using Ni-affinity chromatography packing, and then eluted using 250 mM imidazole. The Trx-His-EK-S-RBD(Omicron_XBB.1.5)-HR protein (the complete, uncleaved protein) and the S-RBD(Omicron_XBB.1.5)-HR protein were separated from the cleaved Trx-His-EK tag. The results are shown in Figure 8. These results indicate that the Trx-His-EK tag can be removed after removal by flow-through mode and subsequent purification using Ni-affinity chromatography packing, thereby obtaining the S-RBD(Omicron_XBB.1.5)-HR protein free of both tags. The amino acid sequence of the protein is shown in Sequence ID No. 5.

[0098] Using the above recombinant protein construction method, based on the S protein RBD of SARS-CoV-2 Omicron_BA.5, XBB.1.5 or the S protein RBD-HR amino acid sequence of SARS-CoV-2 variant strains, the applicants designed protein constructs as specified in SEQ ID NO: 8, SEQ ID NO: 12 and SEQ ID NO: 16, and finally the expressed protein amino acid sequences are shown in SEQ ID NO: 1, SEQ ID NO: 3, and SEQ ID NO: 5 respectively. The expressed recombinant proteins were formulated with adjuvants to obtain recombinant protein vaccines RBD XBB.1.5 -HR (SEQ ID NO: 5), RBD XBB.1.5 (SEQ ID NO: 3), and RBD BA.5 -HR (SEQ ID NO: 1) were prepared respectively, and then they were used in animal immunization tests and further studies.

[0099] Embodiment 2 Construction and Preparation of Recombinant Adenovirus Vaccine 1. Optimization and Synthesis of Nucleotide Sequences of SARS-CoV-2 Omicron_B5 and SARS-CoV-2 Omicron_B.5, Omicron_XBB.1.5 S Protein Genes Based on the mutation sites of the new coronavirus Omicron_BA.5 and Omicron_XBB.1.5 provided on the website https: / / covariants.org / , the nucleotide sequences of the S protein of each SARS-CoV-2 variant were obtained. At the same time, the signal peptides of each S protein variant were retained and the codons were optimized. Subsequently, according to the mutated and optimized sequences, the S protein genes of each variant were synthesized.

[0100] The synthesized S protein genes of SARS-CoV-2 Omicron_BA.5 and Omicron_XBB.1.5 are shown in SEQ ID NO: 23 and SEQ ID NO: 21.

[0101] 2. Packaging of Recombinant Adenovirus New Coronavirus Vaccine During the gene synthesis process, a recombinant cloning strategy was used to clone the synthesized product into the pDC316 vector, thereby obtaining a shuttle plasmid (pDC316-S). The pDC316-S, containing the SARS-CoV-2 Omicron_BA.5 and Omicron_XBB.1.5 mutant S genes constructed above, was co-transfected into HEK293 cells with the AdMax adenovirus system skeleton plasmid pBHGlox_E1,3Cre to package recombinant adenoviruses. The process was as follows:

[0102] 1) Place 8 × 10⁶ of saturates per well in a 6-well plate containing high-glucose DMEM + 10% FBS medium. 5 Each HEK293A cell was inoculated and cultured overnight in a cell culture incubator at 37°C containing 5% CO2.

[0103] 2) The following day, the culture medium was replaced with high-glucose DMEM supplemented with 2% FBS, and HEK293A cells were co-transfected with the backbone plasmid (pBHGlox_E1,3Cre) and shuttle plasmid using Lipofectamine 3000. The specific steps were as follows: 4 μg of backbone plasmid and 2 μg of shuttle plasmid were taken from each transfection well, diluted in 125 μL of Opti-MEM medium, and then 12 μL of P3000 reagent was added. In a separate 1.5 ml EP tube, 7.5 μL of Lipofectamine 3000 was diluted in 125 μL of Opti-MEM medium. The diluted plasmid and diluted Lipofectamine 3000 were mixed in a 1:1 ratio, incubated at room temperature for 10-15 minutes, then added to the cells, and the cells were allowed to continue culturing until the cells had fully grown, then incubated for 25 cm. 2 Cells were subcultured in a cell culture flask, and signs of viral cytopathic effect (CPE) were observed daily. When the cells had fully grown at the bottom of the flask, the culture was continued for 75 cm until the cells showed clear plaques. 2 They were transferred to cell culture flasks, and the virus was collected when most of the cells had become diseased and detached from the bottom.

[0104] 3) Cell cultures containing the virus were collected, centrifuged at 1200 rpm for 3 minutes, the virus-containing supernatant was aspirated, the cell pellet was resuspended in 1 / 10 of the culture volume of virus-containing supernatant, and the cell pellet was alternately placed in a -80°C refrigerator and a 37°C water bath, repeating the freezing and thawing process three times. The cell cultures were centrifuged at 3000 rpm for 20 minutes, the virus-containing supernatant was collected, and combined with the above-mentioned virus-containing supernatant, which is the virus seed for the adenovirus vaccine.

[0105] 4) 50 μL of vaccine candidate virus solution was taken, 2 μL of proteinase K was added, and digestion was carried out at 50°C for 30 minutes to release the viral genome. This was used as a template to PCR amplify the S gene sequence, and after collection of the electrophoresis gel, the PCR product was sequenced and identified. Denaturation: 95°C, 10 minutes; Denaturation: 95°C, 10 seconds; Annealing: 64°C, 30 seconds; Extension: 72°C, 2 minutes; Extension: 72°C, 5 minutes; Number of cycles: 40; The PCR amplification primers were as follows: pDC516-F1:ACACGTCAATGGGAAGTGAAA (Sequence ID 24) pDC516-R1:GCTAGACGATCCAGACATGAT(Sequence ID 25)

[0106] 3. Amplification of recombinant adenovirus novel coronavirus vaccine The identified correct recombinant adenovirus vaccine strain was stepwise amplified in 293 cells. The specific process was as follows: 293 cells at 80%–90% confluence with MOI=10 were added, and after most cells had rounded, the virus culture was collected, and a master virus seed bank and a working virus seed bank were prepared according to the repeated freeze-thaw method described above. The recombinant adenovirus vaccine was scaled up using a cell factory or bioreactor, and the virus culture was collected after most cells had mutated. The process for amplifying cells and viruses in a bioreactor was as follows: First, 3–5 g / L of Cytodex1 microcarrier was added to the bioreactor and sterilized, then cell culture medium was added to the bioreactor, and after the operating conditions stabilized at 37°C, pH 7.0, DO 50%, and 50 rpm, digestion was performed, and the amplified HEK293 cells were recovered from the cell factory, and 1.0–5.0 × 10⁶ cells were collected. 5 The cells were inoculated into a bioreactor at a seeding density of cells / ml, and the cell culture medium was replenished to a total volume of 5L. The cell culture conditions in the bioreactor were maintained at 37°C, 30-50 rpm, pH 7.15-7.25, and DO 30-50%, and samples were taken daily to monitor glucose concentration, cell density, and cell morphology on microcarriers. The cell density in the bioreactor was 1.0-5.0 × 10⁶. 6 When the culture reached a certain stage, recombinant adenovirus vaccine seed stocks were inoculated into bioreactors at an MOI of 5–30. Samples were taken daily to monitor glucose concentration after inoculation, and viral titers were measured in the culture supernatant and cell pellet. Cell morphology on microcarriers was observed. Culture was stopped when most cells had detached from the microcarriers. Virus lysis buffer was added to the bioreactor at a final concentration of 0.05%–1% (v / v) Tween 20, and lysis was carried out at 37°C for 2–4 hours, after which the virus solution was collected.

[0107] 4. Purification of recombinant adenovirus vaccine The collected viruses were purified by cesium chloride ultracentrifugation or ion exchange chromatography, and the specific process was as follows:

[0108] (1) Purification of adenovirus vaccine by cesium chloride ultracentrifugation The collected virus culture was centrifuged at 1200g for 10 minutes, the virus-containing supernatant was aspirated, the cell pellet was resuspended in 1 / 10 of the original culture volume of virus-containing supernatant, and three freeze-thaw cycles were performed using a -80°C freezer and a 37°C water bath. The cell pellet was then centrifuged at 3000rpm for 10-20 minutes, and the supernatant was collected. The virus-containing supernatant was concentrated 10-fold using a 100K-300K ultrafiltration membrane. A 1.4g / ml cesium chloride solution was prepared (by mixing 53g of cesium chloride with 87ml of 10mM Tris-HCl, pH 7.9), and a 1.2g / ml cesium chloride solution was prepared (by mixing 26.8g of cesium chloride with 92ml of 10mM Tris-HCl (pH 7.9)). 8 ml of 1.4 g / ml cesium chloride solution was slowly added to an ultracentrifuge tube, followed by the gentle addition of 6 ml of 1.2 g / ml cesium chloride solution. Finally, 20 ml of virus-containing supernatant was added onto a discontinuous gradient to equilibrate the tube, and then the mixture was centrifuged at 100,000 × g and 4°C for 90 minutes. After centrifugation, the blue virus band was extracted using a syringe, and cesium chloride was removed by dialyzing. The purified virus solution was then stored at -80°C.

[0109] (2) Purification of adenovirus by ion exchange chromatography Virus cultures were collected, lysed in 0.05%-1% Tween 20 at 37°C for 2-4 hours, the lysed cultures were clarified by filtration through 1.2 μm and 0.45 μm capsule filters, the samples were concentrated 10-fold using a tangential flow filtration membrane with a molecular weight cutoff of 100-300 kD, the concentrate was washed with 10 volumes of washing buffer (50 mM Tris-HCl, 2 mM MgCl2, 0-500 mM NaCl, pH 8.0), and the washed samples were collected. After washing the samples, nuclease was added at a final concentration of 10–50 U / ml and digested at 37°C for 1–3 hours. Anion exchange chromatography was then performed using resins such as Q Sepharose XL, Source 30Q, or Source 15Q. The detailed procedure was as follows: The columns were equilibrated with 5 column volumes of equilibration buffer at a flow rate of 20 ml / min. After equilibration, the samples were added at a flow rate of 10 ml / min, and equilibration was continued with the equilibration buffer until the conductivity stabilized. The samples were eluted over 10 column volumes using a linear gradient from 100% low-salt buffer to 100% high-salt buffer at a flow rate of 10 ml / min, and fractions corresponding to each elution peak were collected. After elution, the columns were regenerated with 5–10 column volumes of 2M NaCl buffer at a flow rate of 20 ml / min. The viral peak was collected, and then the eluted viral sample was subjected to buffer exchange by dialysis or tangential flow filtration (using a buffer consisting of 10 mM Tris, 10 mM Na-PO4, 150 mM NaCl, 2 mM MgCl2, 2% sucrose, 0.15% glycerol, and 0.02% Tween 80, pH 7.6).

[0110] The purified adenovirus was directly filled into containers, protected from light, and stored at -20°C.

[0111] 5. Identification of recombinant adenovirus vaccines The inventors first expressed the full-length spike glycoproteins of SARS-CoV-2 mutant Omicron BA.5 and Omicron XBB.1.5 using a human replication-deficient Ad5 adenovirus vector, and Ad5 XBB.1.5 and Ad5BA.5 The expression level of the spike protein in 293T cells was detected by Western blotting 48 hours after infection with recombinant adenovirus. As shown in Figure 9, adenovirus Ad5 XBB.1.5 We were able to induce high levels of spike protein expression in 293T cells after infection, but not in the empty adenovirus control Ad5 Empty, demonstrating that our recombinant adenovirus vaccine was successfully prepared.

[0112] Of these, the adenovirus vaccine prepared according to Embodiment 1 is Ad5 XBB.1.5 (amino acid sequence of SEQ ID NO: 20, creotide sequence of SEQ ID NO: 21) and Ad5 BA.5 (Amino acid sequence of SEQ ID NO: 22, nucleotide sequence of SEQ ID NO: 23) Defined as a recombinant adenovirus.

[0113] The following experiments demonstrated the efficacy of the recombinant protein vaccine and adenovirus vaccine prepared according to the present invention.

[0114] Embodiment 3: Animal preparation, mouse immunization, and sample collection 1. Female NIH mice aged 1.6–8 weeks were purchased from Charles River Laboratories and reared in a pathogen-free environment at the National Key Laboratory of Biotherapy, Sichuan University. Recombinant protein stock solutions were diluted with PBS buffer and mixed with an isovolume adjuvant, either aluminum or MF59, to a total volume of 50 μl. Mice were then immunized three times with 10 μg protein / mouse at days 0, 14, and 28. Blood was collected 7 days after the three immunizations to detect neutralizing antibodies and evaluate the immunizing effect of the vaccine.

[0115] 2.6-8 week old female BALB / c mice were purchased from Charles River Laboratories and reared in a pathogen-free environment at the National Key Laboratory of Biotherapy, Sichuan University. 2.5 × 10⁶ fertilizers were used to prepare a two-component nasal spray vaccine. 9VP (low dose) or 5 x 10 9 VP (high dose) Ad5 XBB.1.5 10 μg of RBD XBB.1.5 -HR was mixed with a total volume of 50 μl to prepare low-dose or high-dose two-component vaccines, respectively. BALB / c mice were given (1) low-dose and (2) high-dose Ad5 XBB.1.5 (3) Two components (Ad5) in single dose, low dose, and high dose. XBB.1.5 +RBD XBB.1.5 -HR) Vaccine, (5) 10μg RBD XBB.1.5 -HR protein alone or (6)RBD XBB.1.5 - 5 × 10⁶ HR protein mixed 9 Mice were immunized three times with VP's Ad5Empty, delivered intranasally at 28-day intervals. To avoid excessive fluid inflow into lung tissue, mice were immunized intranasally twice a day, at intervals of more than 3 hours, with 25 μl each time.

[0116] Other two-component vaccines Ad5 BA.5 +RBD BA.5 -HR, Ad5 BA.5 +RBD XBB.1.5 -HR, and Ad5 XBB.1.5 +RBD BA.5 -The preparation and immunization scheme of the HR was the same as described above, and the recombinant protein in the one-component, two-component, or three-component vaccine used for immunization contained an equal volume of adjuvant MF59 to the recombinant protein.

[0117] 5 x 10 9 VP's Ad5 XBB.1.5 3.3 μg of RBD BA.5 -HR and 6.6μg of RBD XBB.1.5 A three-component vaccine consisting of -HR was administered intranasally using the same immunization schedule. Serum samples were collected at weeks 3, 7, and 11 of vaccinated animals to determine binding and neutralizing antibody responses. To further evaluate vaccine-induced mucosal immunity, mice were euthanized 21 days after the third booster vaccination, and BALF and lung tissue were collected.

[0118] Embodiment 4: Detection of antibodies by enzyme-linked immunosorbent assay (ELISA) To detect RBD-specific IgG and IgA of the novel coronavirus, 96-well plates (NUNC-MaxiSorp, Thermo Fisher Scientific) were coated with 1 μg / mL recombinant RBD protein in carbonic acid-bicarbonate buffer and left overnight at 4°C. The following day, the plates were washed three times with 1×PBS containing 0.1% Tween-20 (PBST), and then sealed with PBST containing 1% BSA for 1 hour. Serially diluted serum, bronchopulmonary lavage fluid, or nasal swabs in dilution buffer were added to the wells (100 μl / well). After incubation at 37°C for 1 hour, the plates were washed three times, and then diluted horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG (1:10000, Southern Biotech, catalog number: 0107-05), HRP-conjugated goat anti-mouse IgG (1:5000), HRP-conjugated goat anti-rabbit IgG, HRP-conjugated goat anti-rabbit IgG, HRP-conjugated goat anti-human IgG (Southern Biotech, catalog number: 62-8420), or HRP-conjugated goat anti-human IgA (Southern Biotech, catalog number: 2050-05). After incubation at 37°C for 1 hour, the plates were washed three more times and developed with 3,3',5,5'-tetramethylbenzidine (TMB) at room temperature for 10 minutes. The reaction was stopped with 1 M H2SO4. Finally, absorbance at 450 nm was measured using a microwell plate reader (Spectramax ABS, Molecular Devices). Final titer was defined as the highest inverse dilution of serum with an absorbance of 2.1 times or more the negative control serum value.

[0119] The results are shown in Tables 10-14. Post-immunization preparations of recombinant adenovirus Ad5 combined with recombinant RBD induced stronger anti-RBD-specific binding antibodies in serum and bronchoalveolar lavage fluid than Ad5 adenovirus alone, indicating that adenovirus combined with recombinant subunit preparations may provide stronger humoral immune protection. Furthermore, simple intranasal immunization of RBD failed to induce strong blood-binding antibodies, but empty Ad5...Empty Alternatively, after combining it with recombinant adenovirus Ad5, its immunogenicity could be significantly improved, demonstrating that adenovirus can be used as an adjuvant for recombinant subunits to enhance the immunogenicity of protein antigens.

[0120] Embodiment 5: Neutralization test for novel coronavirus pseudovirus To detect the titer of neutralizing antibodies in serum and BALF samples, pseudovirus neutralization tests were performed as described above. Pseudoviruses with luciferase, including prototype, δ, and omicron sublineages (BA.2.75, BA.5, BF.7, BQ.1, BQ.1.1, XBB, XBB.1.5, XBB.1.16, etc.), purchased from Genomeditech, were expressed.

[0121] In short, inactivated serum and BALF samples (at 56°C for 30 minutes) were 3-fold diluted in the range of 30–65610, and then incubated with equal volumes of pseudovirus at different dilutions at 37°C for 1 hour. Subsequently, 1.2 × 10⁶ cells expressing the human ACE2 receptor were incubated. 4 Each HEK-293T cell (293T / ACE2) was added to each well and incubated at 37°C for 48 hours to express luciferase. Finally, the supernatant was removed, and then a lysis reagent (Beyotime, RG005) along with the luciferase substrate was added. The luminescence of the 293T / ACE2 cells was measured using a multimode microwell plate reader (PerkinElmer, USA). The 50% neutralization rate of pseudoviruses was measured and calculated using GraphPad Prism 8.0.2. The positive control group contained only cells and viruses, the negative control group contained only cells, and the sample group contained cells, samples, and viruses. The neutralization percentage was calculated using the following formula. Neutralization (%) = (Positive sample - Sample being measured / Positive sample - Negative sample) 100%

[0122] As shown in Figures 15-16, pure protein vaccines (recombinant RBD) XBB.1.5 -hr and recombinant RBDXBB.1.5 After three injections of ), mice produced potent serum neutralizing antibodies against WT, Delta, BA.2.75, BF.7, BA.5, BQ.1, BQ.1.1, XBB, and XBB.1.5 viruses, and the neutralizing antibodies induced by MF59 adjuvant were significantly better than those induced by aluminum adjuvant (Figure 15, RB DWT and RBD Delta -HR is a recombinant protein prepared using the protein construction method described in Embodiment 1, based on the publicly available RBD sequence of the wild-type SARS-CoV-2 S protein and the delta mutant RBD-HR sequence, respectively. Figure 17 shows the results after three intramuscular immunizations with 25 U of inactivated vaccine per dose on days 0, 14, and 42, followed by immunization with either the inactivated vaccine (manufactured by Sinopharm) or RBD. XBB.1.5 A series of booster vaccinations using either of the following methods were administered by intramuscular injection at an immunizing dose of 10 μg per dose, 84 days after the last immunization. Serum was collected 14 days after the booster and pseudovirus neutralizing antibodies were measured. XBB.1.5 Vaccinated mice produced robust serum neutralizing antibodies against WT, BA.5, XBB.1.5, XBB.1.6, XBB.1.16, XBB.1.9.1, and XBB.2.3 viruses, demonstrating particularly strong efficacy against recently dominant XBB submutants and other circulating strains.

[0123] A bivalent vaccine formulation combining adenovirus and recombinant RBD protein induced stronger neutralizing antibodies in both serum and bronchoalveolar lavage fluid compared to adenovirus alone, demonstrating that the combination of adenovirus and recombinant subunit provides stronger systemic and mucosal neutralizing protection and can effectively prevent viral infection. As shown in Figures 18-20, Ad5 XBB.1.5 +RBD XBB.1.5-HR induced high levels of serum neutralizing antibodies against WT, Delta, BA.2.75, BF.7, BA.5, BQ.1, BQ.1.1, XBB, XBB.1.5, and XBB.1.16, suggesting that the combination of the omicron S protein adenovirus vaccine and the S-RBD protein may provide stronger immune protection against omicron variants.

[0124] Embodiment 6: Neutralization test of live coronavirus of novel coronavirus Ad5 against ancestral and mutated live novel coronaviruses XBB.1.5 +RBD XBB.1.5 - Neutralizing antibodies in serum samples from mice vaccinated with the HR bivalent vaccine were measured by a true virus neutralization assay. Diluted serum from each group was mixed with live novel coronavirus at a 50% tissue culture infectious dose (TCID50). After incubation at 37°C for 1 hour, Vero E6 cells (5 × 10⁶) were used. 4 The mixture was added to a 96-well microplate pre-seed with (1 / well) and incubated for 72 hours. Cellular pathogenicity (CPF) was measured under a microscope, and the titer of the neutralizing antibody that resulted in EC50 inhibition (50% neutralization) in immunoserum was calculated.

[0125] The result is as shown in Figure 21: divalent Ad5 XBB.1.5 +RBD XBB.1.5 -Serum from mice administered the HR vaccine intranasally showed neutralizing antibody titers exceeding 1000 against live delta and omicron variants, particularly the recently circulating XBB.1.16 strain, which were significantly higher than those observed with intranasal administration of adenovirus or protein alone.

[0126] Embodiment 7: Attack by the novel coronavirus XBB.1.16 variant On days 0, 28, and 56, BALB / c mice were given three doses of Ad5, either alone or in combination with two or three components. XBB.1.5 Intranasal immunization was performed with vaccine. PBS or Ad5 EmptyMice immunized with [the specified drug] were used as controls, with n=6 mice per group. On day 21 after the last vaccination, live SARS-CoV-2 XBB.1.16 omicron variant (1 × 10⁻¹⁶) was administered. 6 All mice were challenged intranasally with PFU. The mice were euthanized, and their tissues were collected 4 days post-infection. Pathological changes in lung tissue were observed by hematoxylin eosin staining. Viral loads in nasal turbinate, tracheal, and lung tissue samples were evaluated by reverse transcription quantitative polymerase chain reaction (RT-qPCR) targeting viral genomic RNA (gRNA). The primer sequences were 5'-GACCCCAAAATCAGCGAAAT-3' (forward) (SEQ ID NO: 26) and 5'-TCTGGTTACTGCCCAGTTGAATCTG-3' (reverse) (SEQ ID NO: 27), and the probe sequence was 5'-FAM-ACGCCGCATTACGTTTGGTGGGACC-BHQ1-3' (SEQ ID NO: 28). All procedures involved in the mouse challenge using the novel coronavirus omicron variant were reviewed and approved by the Animal Experimentation Committee of the Institute of Medical and Biological Sciences, Chinese Academy of Medical Sciences, and were carried out at the ABSL-4 facility of the Kunming National High-level Biosafety Primate Research Center.

[0127] BALB / c mouse Ad5 XBB.1.5 and bivalent vaccine (Ad5 XBB.1.5 +RBD XBB.1.5 Three intranasal immunizations were performed using PBS and naked RBD. XBB.1.5 -HR, Ad5Empty+RBD XBB.1.5 Mice treated with -HR were used as controls. Immunized, then 1 × 10⁻⁶ mice were used. 6 We evaluated changes in viral load in throat swabs after SARS-CoV-2 infection using BALB / c mice challenged with live PFU SARS-CoV-2 XBB.1.16 omicron variant. Four days post-infection, nasal turbinate, tracheal, and lung tissues were collected, and gRNA and sgRNA levels were measured using RT-qPCR. Histopathological changes in lung tissue after omicron challenge were also observed.

[0128] As shown in Figures 22-26, Ad5 XBB.1.5 (5×10 9 VP / Mouse) + RBD XBB.1.5 Mice treated with immunoserum induced by the HR(10μg) combination vaccine showed nearly complete viral load clearance in throat swabs, no viral RNA was detected in the nasal turbinates, trachea, or lung tissue, lung histology appeared normal, alveolar structures were intact, and there was no significant inflammation. In contrast, PBS and other control groups showed significant viral loads in throat swab samples, elevated levels of viral RNA in the nasal turbinates, trachea, and lung tissue, and histopathological examination revealed mild pathological changes including multiple consolidation areas, slight thickening of the alveolar septa, and alveolar congestion. In addition, small inflammatory lesions consisting of macrophages, neutrophils, and lymphocytes were sometimes observed near small vessels. The results are in Ad5 XBB.1.5 +RBD XBB.1.5 - The HR bivalent vaccine demonstrated complete protection against infection by the XBB.1.16 omicron variant.

Claims

1. A protein for infection by SARS-CoV-2 omicron variant XBB and its submutants, comprising an amino acid sequence selected from any one of SEQ ID NOs: 1 to 7.

2. A precursor of the protein according to claim 1, wherein a signal peptide and / or protein tag is linked to the protein described.

3. The protein precursor according to claim 2, wherein the protein tag is selected from at least one of a histidine tag, a thioredoxin tag, a glutathione-S-transferase tag, a ubiquitin-like modifier tag, a maltose-binding protein tag, a c-Myc protein tag, an Avi protein tag, or a nitrogen assimilation A protein tag.

4. The protein precursor according to claim 3, wherein the protease recognition region for cleaving the protein tag is also linked to the protein against infection by the SARS-CoV-2 omicron variant XBB and its submutants, and preferably the protease is selected from at least one of enterokinase, TEV protease, thrombin, blood coagulation factor Xa, carboxypeptidase A, and rhinovirus 3c protease.

5. The protein precursor according to any one of claims 2 to 4, wherein the amino acid sequence of the precursor is selected from at least one of SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, and SEQ ID NO:

20.

6. A polynucleotide encoding the protein described in claim 1 or the precursor described in any one of claims 2 to 5.

7. The polynucleotide according to claim 6, wherein the nucleotide sequence is selected from at least one of SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, and SEQ ID NO:

21.

8. A recombinant vector comprising the polynucleotide described in claim 6 or 7.

9. The recombinant vector according to claim 8, wherein the recombinant vector is at least one of an insect baculovirus expression vector, a mammalian cell expression vector, an Escherichia coli expression vector, and a yeast expression vector, preferably the insect baculovirus expression vector is pFastBac1, the Escherichia coli expression vector is pET32a, the yeast expression vector is pPICZaA, the mammalian cell expression vector is a CHO cell expression vector, and more preferably the CHO cell expression vector is pTT5 or FTP-002.

10. A host cell comprising the recombinant vector according to claim 8 or 9.

11. The host cell according to claim 10, wherein the host cell is at least one of insect cells, mammalian cells, Escherichia coli, and yeast, preferably the insect cell is selected from at least one of sf9 cells, sf21 cells, and Hi5 cells, and the mammalian cell is a CHO cell.

12. A method for preparing a protein precursor according to any one of claims 2 to 5 or a protein according to claim 1, comprising the steps of: culturing a host cell according to claim 10 or 11 such that the host cell expresses the protein or precursor; and then recovering the protein to obtain the protein and precursor.

13. A protein composition for resistance to infection caused by SARS-CoV-2 omicron variants and submutants, comprising two or more of the following proteins: BA.5 sequence-1, BA.5 sequence-2, XBB.1.5 sequence-1, XBB.1.5 sequence-2, XBB.1.5 sequence-3, XBB.1.5 sequence-4, and S-XBB.1.5 protein.

14. The protein composition according to claim 13, wherein the protein composition comprises a combination of one of BA.5 sequence-1 and BA.5 sequence-2, one of XBB.1.5 sequence-1, XBB.1.5 sequence-2, XBB.1.5 sequence-3 and XBB.1.5 sequence-4, and at least two S-XBB.1.5 proteins, and preferably the protein composition comprises a combination of one of BA.5 sequence-1 and BA.5 sequence-2, one of XBB.1.5 sequence-3 and XBB.1.5 sequence-4, and an S-XBB.1.5 protein.

15. A recombinant protein vaccine for preventing and / or treating infection by SARS-CoV-2 omicron variant and subvariants, comprising the protein described in claim 1, the precursor described in any one of claims 2 to 5 and / or the protein composition described in any one of claims 13 to 14, and a pharmaceutically acceptable excipient or auxiliary component.

16. The recombinant protein vaccine according to claim 15, wherein the adjuvant component is an immunoadjuvant, preferably selected from at least one of a squalene-based oil-in-water emulsion, an aluminum salt, a calcium salt, a plant saponin, a plant polysaccharide, a monophosphate tripeptide A, a muramyl dipeptide, a muramyl tripeptide, a bacterial toxin, a GM-CSF cytokine, a lipid, and a cationic liposome material.

17. The squalene-based oil-in-water emulsion is MF59, the aluminum salt is selected from at least one of aluminum hydroxide and alum, the calcium salt is tricalcium phosphate, the plant saponin is either QS-21 or ISCOM, the plant polysaccharide is an astragalus polysaccharide, the bacterial toxin is selected from at least one of recombinant cholera toxin and diphtheria toxin, and the lipid is at least one of phosphatidylethanolamine, phosphatidylcholine, cholesterol, or dioleoylphosphatidylethanolamine. The recombinant protein vaccine according to claim 15 or 16, wherein the cationic liposome material is selected from at least one of (2,3-dioleoyloxypropyl)trimethylammonium chloride, N-[1-(2,3-dioleoylchloride)propyl]-N,N,N-trimethylamine chloride, cationic cholesterol, dimethyl-2,3-dioleyloxypropyl-2-(2-spermidinecarboxamide)ethylammonium trifluoroacetate, trimethyldodecylammonium bromide, trimethyltetradecylammonium bromide, trimethylhexadecylammonium bromide, dimethyldioctadecylammonium bromide, and CpG ODN.

18. An adenovirus vector vaccine for preventing and / or treating infection by SARS-CoV-2 omicron variant and subvariants, comprising constructing a recombinant vector comprising a polynucleotide encoding the amino acid sequence of the protein described in claim 1, the precursor described in any one of claims 2 to 5, or the protein composition described in any one of claims 13 to 14.

19. The adenovirus vector vaccine according to claim 18, characterized in that the nucleotide sequence of the polynucleotide is selected from at least one of SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, and SEQ ID NO: 21, and more preferably the polynucleotide sequence contained in the adenovirus vector is selected from polynucleotides for constructing S-XBB. 1.5 as shown in SEQ ID NO:

21.

20. The adenovirus vector vaccine according to claim 18, wherein the adenovirus vector is selected from at least one of adenovirus, Ankarawaxinia virus, and adeno-associated virus, preferably selected from human type 5, 35, or 26 and / or chimpanzee AdC68 or AdC7 replication-deficient adenovirus, and more preferably selected from human type 5 replication-deficient adenovirus having a combined E1 and E3 deletion.

21. The adenovirus vector vaccine according to any one of claims 18 to 19, also comprising a pharmaceutically acceptable adjuvant, vector, diluent or excipient.

22. A method for preparing an adenovirus in an adenovirus vector vaccine according to any one of claims 18 to 21, comprising the steps of: constructing a polynucleotide shuttle plasmid vector; transfecting a host cell with the constructed shuttle plasmid vector together with a backbone plasmid and culturing the host cell; obtaining a replication-deficient recombinant adenovirus; and then growing and purifying the culture.

23. The recombinant protein vaccine according to any one of claims 15 to 17 and the adenovirus vector vaccine according to any one of claims 18 to 21 are intradermal or subcutaneous injections, intramuscular injections, intravenous injections, or oral or nasal sprays, preferably the vaccine is an intramuscular injection or a nasal spray.

24. A vaccine composition for treating and / or preventing infection by SARS-CoV-2 omicron variant and its subvariants, comprising a formulation containing as active ingredients a recombinant protein vaccine according to any one of claims 15 to 17 and an adenovirus vector vaccine according to any one of claims 18 to 21.

25. A combination drug for treating and / or preventing infection by SARS-CoV-2 omicron variant and subvariants, comprising a recombinant protein vaccine according to any one of claims 15 to 17 and an adenovirus vector vaccine according to any one of claims 18 to 21, administered separately or simultaneously.

26. A combination or adenovirus vector vaccine comprising recombinant protein vaccine 1 and / or recombinant protein vaccine 2, preferably wherein the recombinant protein vaccine 1 comprises at least one amino acid sequence of BA.5 sequence-1, BA.5 sequence-2 protein or precursor, the recombinant protein vaccine 2 comprises at least one amino acid sequence of XBB.1.5 sequence-1, XBB.1.5 sequence-2, XBB.1.5 sequence-3, XBB.1.5 sequence-4 protein or precursor, and the adenovirus vector vaccine comprises the polynucleotide sequence of S-XBB.1.5, as described in claim 24 or the combination or adenovirus vector vaccine as described in claim 25.

27. The composition or combination drug according to claim 26, wherein the amino acid sequence contained in the recombinant protein vaccine 1 is selected from at least one of SEQ ID NOs: 1, SEQ ID NOs: 2, SEQ ID NOs: 8 and SEQ ID NOs: 10, the amino acid sequence contained in the recombinant protein vaccine 2 is selected from at least one of SEQ ID NOs: 3 to SEQ ID NOs: 6, SEQ ID NOs: 12, SEQ ID NOs: 14, SEQ ID NOs: 16 and SEQ ID NOs: 18, preferably selected from at least one of SEQ ID NOs: 5, SEQ ID NOs: 6, SEQ ID NOs: 16 and SEQ ID NOs: 18, and the adenovirus vector vaccine contains a polynucleotide sequence as shown in SEQ ID NOs:

21.

28. The composition or combination drug according to claim 27, wherein the composition or combination drug is an intradermal or subcutaneous injection, an intramuscular injection, an intravenous injection, or an oral or nasal spray, and preferably the vaccine is an intramuscular injection or a nasal spray.

29. The protein according to claim 1, the precursor according to any one of claims 2 to 5, the protein composition according to any one of claims 13 to 14, the recombinant protein vaccine according to any one of claims 15 to 17, the adenovirus vector vaccine according to any one of claims 18 to 21, and the vaccine composition or combination drug according to any one of claims 24 to 28 are applicable to the preparation of pharmaceuticals for treating and / or preventing infection or pathogenesis caused by SARS-CoV-2 omicron variants and their subvariates.

30. A vaccine composition for treating and / or preventing infection caused by SARS-CoV-2 omicron variant and subvariants, comprising a recombinant protein vaccine and an adenovirus vector vaccine according to any one of claims 15 to 17, wherein the amino acid sequence of the recombinant protein vaccine is selected from at least one of SEQ ID NOs: 1 to SEQ ID NOs: 7 and SEQ ID NOs: 8 to SEQ ID NOs: 20, and the nucleotide sequence for the antigen of the adenovirus vector vaccine is selected from SEQ ID NOs:

23.

31. A combination drug for treating and / or preventing infection by SARS-CoV-2 omicron variant and subvariants, wherein a recombinant protein vaccine and an adenovirus vector vaccine according to any one of claims 15 to 17 are administered separately or simultaneously, wherein the amino acid sequence of the recombinant protein vaccine is selected from at least one of SEQ ID NOs: 1 to SEQ ID NOs: 7 and SEQ ID NOs: 8 to SEQ ID NOs: 20, and the nucleotide sequence for the antigen of the adenovirus vector vaccine is selected from SEQ ID NOs: 23.

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