Protein, adenovirus and vaccine against infection of subtype of SARS-cov-2 omicron mutant strain xbb

Abstract

The present invention relates to the field of medicine, and in particular to a protein, adenovirus and vaccine against infection of a subtype of SARS-CoV-2 Omicron mutant strain XBB. In order to solve the problem of lack of effective prevention and treatment drugs against infection of SARS-CoV-2 Omicron mutant strain XBB and a subtype thereof, the present invention provides a protein, adenovirus and vaccine against the infection of the subtype of the SARS-CoV-2 Omicron mutant strain XBB. The vaccine is designed by optimizing the sequences of full-length S proteins of sub-lines XBB.1.16, XBB.1.5, XBB.1.16.6, BA.2.86, EG.5, JN.1, XBB.2.3 and XBB.2 of the SARS-CoV-2 Omicron mutant strain XBB, and RBD and RBD-HR sequences in the S proteins, can help a host to resist coronavirus infection, and in particular has a good prevention and treatment effect on cross infection caused by subtype viruses of the Omicron mutant strain XBB.

Description

Proteins, adenoviruses, and vaccines against SARS-CoV-2 Omicron mutant strain XBB subtype infection Technical Field

[0001] The present invention relates to proteins, adenoviruses and vaccines for resisting infection by the XBB subtype of the SARS-CoV-2 Omicron mutant strain, and belongs to the field of medicine. Background Art

[0002] The novel coronavirus (SARS-CoV-2) is a new type of beta coronavirus designated by the World Health Organization. The virus has an envelope, and particles are round or oval, often pleomorphic, with a diameter of 60-140 nm. Its genetic characteristics are distinct from those of SARS-CoV and MERS-CoV, representing a new branch of coronavirus previously undetected in humans. Currently, there are five major types of variants of the novel coronavirus: Alpha, Beta, Gamma, Delta, and Omicron. Omicron variants are further divided into sublineages: BA.1, BA.2, BA.2.12.1, BA.4 / 5, BQ.1.1, XBB.1.16, XBB.2, XBB.1.5, XBB.2.3, XBB.1.6, XBB.1.9.1, EG.5, XBB.1.16.6, JN.1, and BA.2.86.

[0003] The main structural proteins of SARS-CoV-2 include the spike protein (S), the messenger protein (E), the membrane protein (M), and the nucleocapsid protein (N). Among them, the S protein plays a key role in the infection and virulence of the virus and is often used as a vaccine antigen. Because the SARS-CoV-2 variant XBB contains multiple mutation sites, the virus's S protein also contains multiple mutation sites, which causes the variant XBB and its subtypes to escape antibodies stimulated by the vaccine against the new coronavirus mutant strains (Alpha, Beta, Gamma, Delta, Omicron) to a certain extent, causing the new coronavirus vaccine to fail or reduce its protective effect, which has brought tremendous pressure to the prevention and control of the new coronavirus epidemic. Therefore, it is very important to develop vaccines against the SARS-CoV-2 virus mutant strain XBB, especially subtypes such as XBB.1.16, XBB.2, XBB.1.5, XBB.2.3, XBB.1.6, XBB.1.9.1, EG5, XBB.1.16.6, JN.1, and BA.2.86, and especially broad-spectrum vaccines against various mutated SARS-CoV-2 viruses, for the prevention and treatment of new coronavirus pneumonia.

[0004] Summary of the Invention

[0005] The present invention aims to address at least one of the technical problems existing in the prior art. To this end, the present invention provides proteins and adenoviruses that are effective against infection with the SARS-CoV-2 Omicron mutant strain, subtype XBB. The present invention also provides vaccines for preventing and / or treating infection with the SARS-CoV-2 Omicron mutant strain, subtype XBB, including recombinant protein vaccines containing the proteins and adenovirus vector vaccines. Furthermore, multivalent vaccines comprising combinations of different proteins or vaccines, or combinations of protein vaccines and adenovirus vaccines, are provided.

[0006] In a first aspect, the present invention provides a protein for resisting infection by the SARS-CoV-2 Omicron mutant strain XBB subtype, comprising at least one amino acid sequence selected from SEQ ID No. 1 to SEQ ID No. 18, and SEQ ID No. 55. Preferably, the protein comprises at least one amino acid sequence as shown in SEQ ID No. 1 to SEQ ID No. 11, and SEQ ID No. 55.

[0007] More preferably, the protein contains at least one of the amino acid sequences shown in SEQ ID No.1, SEQ ID No.5, SEQ ID No.8 to SEQ ID No.11, and SEQ ID No.55.

[0008] The present invention also provides a precursor of the protein, which is a precursor of the protein connected to a signal peptide and / or a protein tag.

[0009] Preferably, the protein tag is selected from at least one of the following: a histidine tag, a thioredoxin tag (Trx tag), a glutathione transferase tag, a ubiquitin-like modifier protein tag, a maltose-binding protein tag, a c-Myc protein tag, an Avi tag protein tag, and a nitrogen source utilization substance A protein tag.

[0010] Furthermore, a protease recognition region for removing the protein tag is also connected to the protein that resists SARS-CoV-2 Omicron mutant strain XBB subtype infection.

[0011] Preferably, the protease is selected from at least one of the following: enterokinase (EK enzyme), TEV protease, thrombin, coagulation factor Xa, carboxypeptidase A, rhinovirus 3c protease.

[0012] Furthermore, the amino acid sequence of the precursor is selected from at least one of SEQ ID No. 19 to SEQ ID No. 36 and SEQ ID No. 56. Preferably, the amino acid sequence of the precursor is selected from at least one of SEQ ID No. 19 to SEQ ID No. 29 and SEQ ID No. 56.

[0013] More preferably, the amino acid sequence of the precursor is selected from at least one of SEQ ID No. 19, SEQ ID No. 23, SEQ ID No. 26 to SEQ ID No. 29, and SEQ ID No. 56.

[0014] The present invention also provides a polynucleotide encoding the protein or the precursor.

[0015] Furthermore, the polynucleotide sequence is selected from at least one of SEQ ID No. 37 to SEQ ID No. 54 and SEQ ID No. 57. Preferably, the polynucleotide sequence is selected from at least one of SEQ ID No. 37 to SEQ ID No. 47 and SEQ ID No. 57. More preferably, the polynucleotide sequence is selected from at least one of SEQ ID No. 37, SEQ ID No. 41, SEQ ID No. 44 to SEQ ID No. 47 and SEQ ID No. 57.

[0016] The present invention also provides a recombinant vector containing the polynucleotide.

[0017] Furthermore, the recombinant vector adopts at least one of an insect baculovirus expression vector, a mammalian cell expression vector, an Escherichia coli expression vector, and a yeast expression vector.

[0018] Preferably, the insect baculovirus expression vector is pFastBac1.

[0019] Preferably, the E. coli expression vector is pET32a.

[0020] Preferably, the yeast expression vector is pPICZaA.

[0021] Preferably, the mammalian cell expression vector is a CHO cell expression vector.

[0022] More preferably, the CHO cell expression vector is pTT5 or FTP-002.

[0023] The present invention also provides a host cell containing the recombinant vector.

[0024] Furthermore, the host cell is at least one of insect cells, mammalian cells, Escherichia coli, and yeast.

[0025] Preferably, the insect cells are selected from at least one of sf9 cells, sf21 cells, and Hi5 cells.

[0026] Preferably, the mammalian cells are CHO cells.

[0027] The present invention also provides a method for preparing the protein or the precursor, which is characterized by comprising the following steps: culturing the host cell to express the protein or the precursor, and then recovering the protein.

[0028] The present invention also provides a recombinant protein vaccine for preventing and / or treating SARS-CoV-2 Omicron mutant subtype infection, which contains the protein and / or the precursor, and pharmaceutically acceptable excipients or auxiliary ingredients.

[0029] Furthermore, the auxiliary component is an immune adjuvant.

[0030] Preferably, the immune adjuvant is selected from at least one of the following: squalene oil-in-water emulsion, aluminum salt, calcium salt, plant saponin, plant polysaccharide, monophosphoric acid lipid A, muramyl dipeptide, muramyl tripeptide, bacterial toxin, GM-CSF cytokine, lipid, cationic liposome material.

[0031] Furthermore, at least one of the following is satisfied:

[0032] The squalene oil-in-water emulsion is MF59;

[0033] The aluminum salt is selected from at least one of aluminum hydroxide and alum;

[0034] The calcium salt is tricalcium phosphate;

[0035] The plant saponin is QS-21 or ISCOM;

[0036] The plant polysaccharide is astragalus polysaccharide;

[0037] The bacterial toxin is selected from at least one of recombinant cholera toxin and diphtheria toxin;

[0038] The lipid is selected from at least one of the following: phosphatidylethanolamine, phosphatidylcholine, cholesterol, and dioleoylphosphatidylethanolamine;

[0039] The cationic liposome material is selected from at least one of the following: (2,3-dioleyloxypropyl)trimethylammonium chloride, N-[1-(2,3-dioleoyl chloride)propyl]-N,N,N-trimethylammonium chloride, cationic cholesterol, dimethyl-2,3-dioleyloxypropyl-2-(2-sperminecarboxamido)ethylammonium trifluoroacetate, trimethyldodecylammonium bromide, trimethyltetradecylammonium bromide, trimethylhexadecylammonium bromide, dimethyldioctadecylammonium bromide, and CpG ODN.

[0040] The present invention also provides a protein composition for preventing and / or treating SARS-CoV-2 Omicron mutant subtype infection, which contains any two or more proteins or any two or more protein precursors that are anti-SARS-CoV-2 Omicron mutant XBB subtype infection, wherein the protein is selected from a protein constructed by any one of the amino acid sequences of SEQ ID No.1 to SEQ ID No.18 and SEQ ID No.55; and the precursor is selected from a protein precursor constructed by any one of the amino acid sequences of SEQ ID No.19 to SEQ ID No.36 and SEQ ID No.56.

[0041] The present invention also provides a recombinant protein vaccine composition for preventing and / or treating SARS-CoV-2 Omicron mutant subtype infection, which contains any two or more different recombinant protein vaccines.

[0042] Different recombinant protein vaccines refer to vaccines containing proteins with different amino acid sequences. A recombinant protein vaccine composition refers to the preparation of recombinant protein vaccines containing different amino acid sequences, followed by the combination of two or more of these recombinant protein vaccines. The amino acid sequence is selected from at least one of SEQ ID No. 1 to SEQ ID No. 36, SEQ ID No. 55, and SEQ ID No. 56.

[0043] The present invention also provides an adenovirus vector vaccine for preventing and / or treating SARS-CoV-2 Omicron mutant subtype infection, comprising a polynucleotide encoding the spike protein of SARS-CoV-2 or its mutants. The polynucleotide sequence is obtained by codon or cell optimization.

[0044] Preferably, the nucleotide sequence of the polynucleotide is selected from at least one of SEQ ID No. 37 to SEQ ID No. 54. More preferably, the polynucleotide sequence is selected from at least one of SEQ ID No. 48 to SEQ ID No. 54. More preferably, the polynucleotide sequence is selected from at least one of SEQ ID No. 48 to SEQ ID No. 51.

[0045] Furthermore, in the adenovirus vector vaccine, the adenovirus vector is selected from at least one of the following: adenovirus, Ankara vaccinia virus, and adeno-associated virus.

[0046] Preferably, the adenovirus vector is selected from the replication-deficient adenovirus of human type 5, type 35 or type 26 and / or chimpanzee AdC68 or AdC7.

[0047] More preferably, it is selected from the human type 5 replication-deficient adenovirus in which E1 and E3 are jointly deleted.

[0048] The present invention provides a method for preparing the adenovirus in the above-mentioned adenovirus vector vaccine, comprising the following steps: constructing a shuttle plasmid vector containing a polynucleotide encoding the spike protein of SARS-CoV-2 or its mutant strains; then transfecting the constructed shuttle plasmid vector together with the backbone plasmid into host cells, and culturing the host cells; obtaining a replication-deficient recombinant adenovirus, and then expanding the culture and purifying it.

[0049] Furthermore, the polynucleotide sequence encoding the spike protein of SARS-CoV-2 or its mutant is selected from at least one of SEQ ID No. 37 to SEQ ID No. 54. Preferably, the polynucleotide sequence is selected from at least one of SEQ ID No. 48 to SEQ ID No. 54. More preferably, the polynucleotide sequence is selected from at least one of SEQ ID No. 48 to SEQ ID No. 51.

[0050] Furthermore, the shuttle plasmid vector is at least one of pDC316-S, pDC315-S, pDC516-S or pDC515-S.

[0051] Furthermore, the backbone plasmid is at least one of pBHGlox_E1,3Cre and pBHGlox_E1,3FLP.

[0052] Furthermore, the host cell is HEK293.

[0053] The present invention also provides an adenoviral vector vaccine composition for preventing and / or treating infection with the SARS-CoV-2 Omicron mutant subtype, comprising any two or more different adenoviral vector vaccines. Different adenoviral vector vaccines are recombinantly prepared containing different nucleotide sequences. An adenoviral vector vaccine composition refers to separately prepared different adenoviral vector vaccines, followed by a combination of any two or more. The nucleotide sequence is selected from at least one of SEQ ID No. 37 to SEQ ID No. 54.

[0054] Furthermore, the protein, protein precursor, protein composition, recombinant protein vaccine, recombinant protein vaccine composition, adenovirus vector vaccine, adenovirus vector vaccine composition is an intradermal or subcutaneous injection preparation, intramuscular injection preparation, intravenous injection preparation, oral or nasal spray preparation.

[0055] Preferably, the vaccine is an intramuscular injection preparation and a nasal spray preparation.

[0056] The present invention also provides a pharmaceutical composition for treating and / or preventing SARS-CoV-2 Omicron mutant subtype infection, which contains a combination of at least one of a recombinant protein, a protein composition, a recombinant protein vaccine, and a recombinant protein vaccine composition and at least one of an adenovirus vector vaccine or an adenovirus vector vaccine composition.

[0057] Preferably, the pharmaceutical composition is a multivalent vaccine composed of at least one of a recombinant protein vaccine or a recombinant protein vaccine composition and at least one of an adenovirus vector vaccine or adenovirus vector vaccine composition.

[0058] Preferably, the pharmaceutical composition is a multivalent vaccine composed of at least one of a recombinant protein or protein composition and at least one of an adenovirus vector vaccine or an adenovirus vector vaccine composition.

[0059] The present invention provides a combination drug for treating and / or preventing SARS-CoV-2 Omicron mutant subtype infection, which contains a combination of at least one of a recombinant protein, a protein composition, a recombinant protein vaccine, or a recombinant protein vaccine composition and at least one of an adenovirus vector vaccine or an adenovirus vector vaccine composition, administered separately or simultaneously.

[0060] Preferably, the combination drug is a combination of at least one of a recombinant protein vaccine or a recombinant protein vaccine composition and at least one of an adenovirus vector vaccine or an adenovirus vector vaccine composition.

[0061] Preferably, the combination drug is a combination of at least one of a recombinant protein or a protein composition and at least one of an adenovirus vector vaccine or an adenovirus vector vaccine composition.

[0062] More preferably, the recombinant protein vaccine contains the above-mentioned protein or precursor that resists infection by the SARS-CoV-2 Omicron mutant strain XBB subtype; the amino acid sequence of the protein or precursor is selected from at least one of SEQ ID No. 1 to SEQ ID No. 11, SEQ ID No. 55, SEQ ID No. 19 to SEQ ID No. 29, and SEQ ID No. 56. Preferably, the amino acid sequence of the protein or precursor is selected from at least one of SEQ ID No. 1, SEQ ID No. 5, SEQ ID No. 8 to SEQ ID No. 11, SEQ ID No. 55, or SEQ ID No. 19, SEQ ID No. 23, SEQ ID No. 26 to SEQ ID No. 29, and SEQ ID No. 56. The recombinant protein vaccine composition is a combination of any two or more recombinant protein vaccines containing different proteins.

[0063] The adenovirus vector vaccine comprises a polynucleotide sequence as shown in at least one of SEQ ID No. 48 to SEQ ID No. 54. Preferably, the adenovirus vector vaccine comprises a polynucleotide sequence as shown in at least one of SEQ ID No. 48 to SEQ ID No. 51.

[0064] The adenovirus vector vaccine composition is a combination of any two or more adenovirus vector vaccines containing different polynucleotide sequences.

[0065] Most preferably, the amino acid sequence contained in the recombinant protein vaccine is selected from at least one of SEQ ID No.1, SEQ ID No.5, SEQ ID No.8 to SEQ ID No.11, and SEQ ID No.55.

[0066] The adenovirus vector vaccine contains a polynucleotide sequence as shown in at least one of SEQ ID No. 48 to SEQ ID No. 51.

[0067] Furthermore, the pharmaceutical composition or combined drug is an intradermal or subcutaneous injection preparation, an intramuscular injection preparation, an intravenous injection preparation, an oral or nasal spray preparation; preferably, it is an intramuscular injection preparation and a nasal spray preparation.

[0068] The present invention also provides the use of the protein, the precursor, the protein composition, the recombinant protein vaccine or composition, the adenovirus vector vaccine or composition, the pharmaceutical composition or the combination drug in the preparation of a drug for treating and / or preventing SARS-CoV-2 or variant infection or pathogenicity.

[0069] Preferably, the SARS-CoV-2 variants include SARS-CoV-2 mutants Alpha, Beta, Gamma, Delta, Omicron, Omicron sublineages BA.1, BA.2, BA.2.12.1, BA.4 / 5, BQ.1.1, XBB.1.16, XBB.2, XBB.1.5, XBB.2.3, XBB.1.6, XBB.1.9.1, XBB.1.16.6, FL.1.5.1, HV.1, EG.5, EG.5.1, BA.2.86, JN.1, JN.1.13, KP.2, and KP.3.

[0070] The following sequence was constructed by the applicant based on the RBD, RBD-HR sequences and full-length S protein in the S protein of the SARS-CoV-2 mutant Omicron variant sublineage XBB.1.16, XBB.2, XBB.1.5, XBB.2.3, XBB.1.16.6, BA.2.86, EG5, and JN.1 after optimization.

[0071] SEQ ID No.1 XBB.1.16 S protein optimized RBD-HR sequence

[0072] SEQ ID No.2 XBB.2 S protein optimized RBD-HR sequence

[0073] SEQ ID No.3 XBB.1.5-1 S protein optimized RBD sequence

[0074] SEQ ID No.4 XBB.1.5-2 S protein optimized RBD sequence

[0075] SEQ ID No.5 XBB.1.5-3 S protein optimized RBD-HR sequence

[0076] SEQ ID No.6 XBB.1.5-4 S protein optimized RBD-HR sequence

[0077] SEQ ID No.7 XBB.2.3-1 S protein optimized RBD sequence

[0078] SEQ ID No.8 XBB.2.3-2 S protein optimized RBD-HR sequence

[0079] SEQ ID No.9 XBB.1.16.6 S protein optimized sequence

[0080] SEQ ID No.10 BA.2.86 S protein optimized sequence

[0081] SEQ ID No.11 Optimized sequence of S protein of EG.5

[0082] SEQ ID No.12 The optimized sequence of the full-length S protein of XBB.1.16, excluding the signal peptide

[0083] SEQ ID No.13 The optimized sequence of the full-length S protein of XBB.2, excluding the signal peptide

[0084] SEQ ID No.14 The optimized sequence of the full-length S protein of XBB.1.5, excluding the signal peptide

[0085] SEQ ID No.15 The optimized sequence of the full-length S protein of XBB.2.3, excluding the signal peptide

[0086] SEQ ID No.16 The optimized sequence of the full-length S protein of XBB.1.6, excluding the signal peptide

[0087] SEQ ID No. 16 is a variant obtained by performing sequence substitution and / or deletion and / or insertion of at least one amino acid on the amino acid sequence of the full-length S protein of XBB.1.6.

[0088] SEQ ID No.17 The optimized sequence of the full-length S protein of XBB.1.9.1, excluding the signal peptide

[0089] SEQ ID No. 17 is a variant obtained by performing sequence substitution and / or deletion and / or insertion of at least one amino acid on the amino acid sequence of the full-length S protein of XBB.1.9.1.

[0090] SEQ ID No.18 Optimized sequence of the full-length S protein of EG.5, excluding the signal peptide

[0091] SEQ ID No. 18 is a variant obtained by performing sequence substitution and / or deletion and / or insertion of at least one amino acid in the amino acid sequence of the full-length S protein of EG5.

[0092] To facilitate secretory expression of the protein, a signal peptide was added to its amino acid sequence during protein construction to facilitate secretory expression. Furthermore, a His tag was added to the amino acid sequence of the protein to facilitate purification. The designed sequences of the optimized protein expression constructs constructed in the present invention based on the S proteins of XBB.1.16, XBB.2, XBB.1.5, XBB.2.3, XBB.1.16.6, BA.2.86, and EG5 are shown in SEQ ID Nos. 19 to 29.

[0093] Furthermore, the corresponding nucleotide sequences encoding the amino acid sequences are shown as SEQ ID No. 37 to SEQ ID No. 47, respectively.

[0094] SEQ ID No.19 is an optimized design of the S protein-RBD-HR based on Omicron_XBB.1.16, including: signal peptide-Trx tag-6His tag-EK restriction site-RBD sequence-HR1 sequence-HR2 sequence

[0095] SEQ ID No. 20 is an optimized design of the S protein-RBD-HR based on Omicron_XBB.2, including: signal peptide-Trx tag-6His tag-EK restriction site-RBD sequence-HR1 sequence-HR2 sequence

[0096] SEQ ID No. 21 is based on the optimized design of S protein-RBD of Omicron_XBB.1.5-1, including: signal peptide-Trx tag-6His tag-EK restriction site-RBD sequence

[0097] SEQ ID No. 22 is based on the optimized design sequence of S protein-RBD of Omicron_XBB.1.5-2, including: signal peptide-Trx tag-6His tag-EK restriction site-RBD sequence

[0098] SEQ ID No. 23 is based on the optimized design of S protein-RBD-HR of Omicron_XBB.1.5-3, including: signal peptide-Trx tag-6His tag-EK restriction site-RBD sequence-HR1 sequence-HR2 sequence

[0099] SEQ ID No. 24 is an optimized sequence of S protein-RBD-HR based on Omicron_XBB.1.5-4, including: signal peptide-Trx tag-6His tag-EK restriction site-RBD sequence-HR1 sequence-HR2 sequence

[0100] SEQ ID No. 25 is based on the optimized design of S protein-RBD of Omicron_XBB.2.3-1, including: signal peptide-Trx tag-6His tag-EK restriction site-RBD sequence

[0101] SEQ ID No. 26 is based on the optimized design of S protein-RBD-HR of Omicron_XBB.2.3-2, including: signal peptide-Trx tag-6His tag-EK restriction site-RBD sequence-HR1 sequence-HR2 sequence

[0102] SEQ ID No. 27 is based on the optimized design sequence of S protein-RBD-HR of Omicron_XBB.1.16.6, including: signal peptide-Trx tag-6His tag-EK restriction site-RBD sequence-HR1 sequence-HR2 sequence

[0103] SEQ ID No. 28 is based on the optimized design sequence of S protein-RBD-HR of Omicron_BA.2.86, including: signal peptide-Trx tag-6His tag-EK restriction site-RBD sequence-HR1 sequence-HR2 sequence

[0104] SEQ ID No.29 is based on the optimized design sequence of S protein-RBD-HR of Omicron_EG.5, including: signal peptide-Trx tag-6His tag-EK restriction site-RBD sequence-HR1 sequence-HR2 sequence

[0105] SEQ ID No.30 is based on the optimized design of the full-length S protein of Omicron_XBB.1.16, including its own signal peptide, and is also the amino acid sequence of the S antigen of the Ad-S-XBB.1.16 vaccine

[0106] SEQ ID No.31 is an optimized sequence based on the full-length S protein of Omicron_XBB.2, including its own signal peptide, and is also the amino acid sequence of the S antigen of the Ad-S-XBB.2 vaccine

[0107] SEQ ID No.32 is based on the optimized design of the full-length S protein of Omicron_XBB.1.5, including its own signal peptide, and is also the amino acid sequence of the S antigen of the Ad-S-XBB.1.5 vaccine

[0108] SEQ ID No.33 is based on the optimized design of the full-length S protein of Omicron_XBB.2.3, including its own signal peptide, and is also the amino acid sequence of the S antigen of the Ad-S-XBB.2.3 vaccine

[0109] SEQ ID No. 34 is an optimized sequence based on the full-length S protein of Omicron_XBB.1.6, including its own signal peptide, and is also the amino acid sequence of the S antigen of the Ad-S-XBB.1.6 vaccine

[0110] SEQ ID No.35 is an optimized sequence based on the full-length S protein of Omicron_XBB.1.9.1, including its own signal peptide, and is also the amino acid sequence of the S antigen of the Ad-S-XBB.1.9.1 vaccine

[0111] SEQ ID No. 36 is an optimized sequence based on the full-length S protein of Omicron_EG5, including its own signal peptide, and is also the amino acid sequence of the S antigen of the Ad-S-EG5 vaccine

[0112] SEQ ID No.37 encodes the nucleotide sequence of the optimized design sequence of S protein-RBD-HR based on Omicron_XBB.1.16, encoding the nucleotide sequence of SEQ ID No.19

[0113] SEQ ID No.38 encodes the nucleotide sequence of the optimized design sequence of S protein-RBD-HR based on Omicron_XBB.2, encoding the nucleotide sequence of SEQ ID No.20

[0114] SEQ ID No.39 encodes the nucleotide sequence of the optimized design sequence of S protein-RBD based on Omicron_XBB.1.5-1, encoding the nucleotide sequence of SEQ ID No.21

[0115] SEQ ID No.40 encodes the nucleotide sequence of the optimized design sequence of S protein-RBD based on Omicron_XBB.1.5-2, encoding the nucleotide sequence of SEQ ID No.22

[0116] SEQ ID No.41 encodes the nucleotide sequence of the optimized design sequence of S protein-RBD-HR based on Omicron_XBB.1.5-3, encoding the nucleotide sequence of SEQ ID No.23

[0117] SEQ ID No.42 encodes the nucleotide sequence of the optimized design sequence of S protein-RBD-HR based on Omicron_XBB.1.5-4, encoding the nucleotide sequence of SEQ ID No.24

[0118] SEQ ID No.43 encodes the nucleotide sequence of the optimized design sequence of S protein-RBD based on Omicron_XBB.2.3-1, encoding the nucleotide sequence of SEQ ID No.25

[0119] SEQ ID No.44 encodes the nucleotide sequence of the optimized design sequence of S protein-RBD-HR based on Omicron_XBB.2.3-2, encoding the nucleotide sequence of SEQ ID No.26

[0120] SEQ ID No.45 encodes the nucleotide sequence of the optimized design sequence of S protein-RBD-HR based on Omicron_XBB.1.16.6, encoding the nucleotide sequence of SEQ ID No.27

[0121] SEQ ID No.46 encodes the nucleotide sequence of the optimized design sequence of S protein-RBD-HR based on Omicron_BA.2.86, encoding the nucleotide sequence of SEQ ID No.28

[0122] SEQ ID No.47 encodes the nucleotide sequence of the S protein-RBD-HR designed sequence based on Omicron_EG.5, encoding the nucleotide sequence of SEQ ID No.29

[0123] SEQ ID No.48 encodes the optimized design sequence of the full-length S protein based on Omicron_XBB.1.16, including the nucleotide sequence of its own signal peptide. It is also the nucleotide sequence of the S antigen of the Ad-S-XBB.1.16 vaccine, encoding the nucleotide sequence of SEQ ID No.30

[0124] SEQ ID No.49 encodes the optimized design of the full-length S protein based on Omicron_XBB.2, including the nucleotide sequence of its own signal peptide. It is also the nucleotide sequence of the S antigen of the Ad-S-XBB.2 vaccine, encoding the nucleotide sequence of SEQ ID No.31

[0125] SEQ ID No.50 encodes the optimized design sequence of the full-length S protein based on Omicron_XBB.1.5, including the nucleotide sequence of its own signal peptide. It is also the nucleotide sequence of the S antigen of the Ad-S-XBB.1.5 vaccine, encoding the nucleotide sequence of SEQ ID No.32

[0126] SEQ ID No.51 encodes the optimized design sequence of the full-length S protein based on Omicron_XBB.2.3, including the nucleotide sequence of its own signal peptide. It is also the nucleotide sequence of the S antigen of the Ad-S-XBB.2.3 vaccine, encoding the nucleotide sequence of SEQ ID No.33

[0127] SEQ ID No.52 encodes the optimized design sequence of the full-length S protein based on Omicron_XBB.1.6, including the nucleotide sequence of its own signal peptide, which is also the nucleotide sequence of the S antigen of the Ad-S-XBB.1.6 vaccine

[0128] SEQ ID No.53 encodes the optimized design sequence of the full-length S protein based on Omicron_XBB.1.9.1, including the nucleotide sequence of its own signal peptide, which is also the nucleotide sequence of the S antigen of the Ad-S-XBB.1.9.1 vaccine

[0129] SEQ ID No.54 encodes the optimized design sequence of the full-length S protein based on Omicron_EG.5, including the nucleotide sequence of its own signal peptide, which is also the nucleotide sequence of the S antigen of the Ad-S-EG5 vaccine

[0130] Optimized RBD-HR sequence of S protein of SEQ ID No.55JN.1

[0131] SEQ ID No.56 is an optimized sequence based on the S protein-RBD-HR of Omicron JN.1, including: signal peptide-Trx tag-6His tag-EK restriction site-RBD sequence-HR1 sequence-HR2 sequence

[0132] SEQ ID No.57 encodes the nucleotide sequence of the optimized design sequence of S protein-RBD-HR based on Omicron_JN.1, encoding the nucleotide sequence of SEQ ID No.56

[0133] Beneficial effects: The present invention first prepares a recombinant protein vaccine and an adenovirus vector vaccine. The protein antigen of the recombinant protein vaccine is based on the RBD sequence or the RBD sequence in the S protein (positions 320-545) of SARS-CoV-2 Omicron variants XBB.1.16, XBB.2, XBB.1.5, XBB.2.3, XBB.1.16.6, BA.2.86, EG5, and JN.1 and its heptapeptide repeat regions HR1 and HR2 for construction and expression. At the same time, adding the corresponding vaccine adjuvant to the prepared protein antigen can better help the host resist cross-infection caused by the Omicron variant XBB and its subtypes, which is of great significance for the development of recombinant protein vaccines against SARS-CoV-2 Omicron variant XBB and its subtype infection. Animal experiments have shown that the multivalent vaccine obtained by combining different recombinant protein antigens or vaccines has a stronger resistance to the Omicron variant XBB and its subtypes.

[0134] At the same time, the present invention also constructs a recombinant adenovirus vector based on the nucleotide sequence encoding the full-length S protein of XBB.1.16, XBB.2, XBB.1.5, and XBB.2.3 through codon optimization to obtain an adenovirus vector vaccine, and combines it with a recombinant protein vaccine to obtain a multivalent vaccine against SARS-CoV-2 and its variants with better prevention and treatment effects. The preparation of the nasal spray reagent is more conducive to resisting infection by SARS-CoV-2 and its Omicron variants XBB.1.5, XBB.1.6, XBB.1.16, XBB.1.16.6, XBB.2.3, FL.1.5.1, HV.1, EG.5, EG.5.1, BA.2.86, JN.1, JN.1.13, KP.2, KP.3 and other viruses. BRIEF DESCRIPTION OF THE DRAWINGS

[0135] Figure 1 is the recombinant protein antigen RBD of Example 1 XBB.1.16 -HR construction and characterization; including: a. design diagram of pFastBac1-GP67-Trx-His-EK-S-RBD(Omicron_XBB.1.16)-HR; b. 1% agarose gel electrophoresis of cloned PCR products; c. 1% agarose gel electrophoresis of PCR identification of recombinant bacmid; d. recombinant baculovirus packaging and amplification flow chart; e. WB verification results during baculovirus amplification.

[0136] Figure 2 is the recombinant protein antigen RBD of Example 1 XBB.1.16-HR expression and purification results; among them, a. Ni-affinity chromatography elution results of the supernatant after baculovirus infection; b. EK enzyme digestion sample verification enzymatic digestion effect result diagram; cd. Ni-affinity chromatography filler protein back-hanging purification and SDS-PAGE identification diagram after EK enzyme digestion.

[0137] Figure 3 is the recombinant protein antigen RBD of Example 1 XBB.1.5-3 -HR construction and characterization; including: a. design diagram of pFastBac1-GP67-Trx-His-EK-S-RBD(Omicron_XBB.1.5-3)-HR; b. 1% agarose gel electrophoresis of cloned PCR products; c. 1% agarose gel electrophoresis of PCR identification of blue-white bacterial solution; d. recombinant baculovirus packaging and amplification flow chart; e. WB verification results during baculovirus amplification.

[0138] Figure 4 is the recombinant protein antigen RBD of Example 1 XBB.1.5-3 -HR expression and purification results; among them, a. Ni-affinity chromatography elution results of the supernatant after baculovirus infection; b. EK enzyme digestion sample verification enzymatic digestion effect result diagram; c. Ni-affinity chromatography filler protein back-hanging purification diagram after EK enzyme digestion.

[0139] Figure 5 is the recombinant protein antigen RBD of Example 1 XBB.2.3 -HR construction and characterization; including: a. design diagram of pFastBac1-GP67-Trx-His-EK-S-RBD(Omicron_XBB.2.3)-HR; b. 1% agarose gel electrophoresis of cloned PCR products; c. 1% agarose gel electrophoresis of PCR identification of blue-white bacterial solution; d. recombinant baculovirus packaging and amplification flow chart; e. WB verification results during baculovirus amplification.

[0140] Figure 6 is the recombinant protein antigen RBD of Example 1 XBB.2.3 -HR expression and purification results; among them, a. Ni-affinity chromatography elution results of the supernatant after baculovirus infection; b. EK enzyme digestion sample verification enzymatic digestion effect result diagram; c. EK enzyme digestion and molecular exclusion chromatography purification and SDS-PAGE identification diagram.

[0141] Figure 7 is the recombinant protein antigen RBD of Example 1 XBB.1.16.6-HR construction and characterization; including: a. design diagram of pFastBac1-GP67-Trx-His-EK-S-RBD(Omicron_XBB.1.16.6)-HR; b. 1% agarose gel electrophoresis of cloned PCR products; c. 1% agarose gel electrophoresis of PCR identification of recombinant bacmid; d. recombinant baculovirus packaging and amplification flow chart; e. WB verification results during baculovirus amplification.

[0142] Figure 8 is the recombinant protein antigen RBD of Example 1 XBB.1.16.6 -HR expression and purification results; among them, a. Ni-affinity chromatography elution results of the supernatant after baculovirus infection; b. EK enzyme digestion sample verification enzymatic digestion effect result diagram; cd. EK enzyme digestion and molecular exclusion chromatography purification and SDS-PAGE identification diagram.

[0143] Figure 9 is the recombinant protein antigen RBD of Example 1 BA.2.86 -HR construction and characterization; including: a. pFastBac1-GP67-Trx-His-EK-S-RBD (BA.2.86)-HR design diagram; b. 1% agarose gel electrophoresis diagram of cloned PCR products; c. 1% agarose gel electrophoresis diagram of PCR identification of blue-white bacterial solution; d. recombinant baculovirus packaging and amplification flow chart; e. WB verification result diagram during baculovirus amplification process.

[0144] Figure 10 is the recombinant protein antigen RBD of Example 1 BA.2.86 -HR expression and purification results; among them, a. Ni-affinity chromatography elution results of the supernatant after baculovirus infection; b. EK enzyme digestion sample verification enzymatic digestion effect result diagram; cd. EK enzyme digestion and molecular exclusion chromatography purification and SDS-PAGE identification diagram.

[0145] Figure 11 is the recombinant protein antigen RBD of Example 1 EG.5 -HR construction and characterization; including: a. pFastBac1-GP67-Trx-His-EK-SS-RBD(EG.5)-HR design diagram; b. 1% agarose gel electrophoresis diagram of cloned PCR products; c. 1% agarose gel electrophoresis diagram of PCR identification of recombinant bacmid; d. recombinant baculovirus packaging and amplification flow chart; e. WB verification results during baculovirus amplification.

[0146] Figure 12 is the recombinant protein antigen RBD of Example 1 EG.5 -HR expression and purification results; among them, a. Ni-affinity chromatography elution results of the supernatant after baculovirus infection; b. EK enzyme digestion sample verification enzymatic digestion effect result diagram; cd. EK enzyme digestion and molecular exclusion chromatography purification and SDS-PAGE identification diagram.

[0147] Figure 13 is the recombinant protein antigen RBD of Example 1 JN.1 -HR construction and characterization; including: a. pFastBac1-GP67-Trx-His-EK-SS-RBD(JN.1)-HR design diagram; b. 1% agarose gel electrophoresis diagram of cloned PCR products; c. 1% agarose gel electrophoresis diagram of PCR identification of recombinant bacmid; d. recombinant baculovirus packaging and amplification flow chart; e. WB verification results during baculovirus amplification.

[0148] Figure 14 is the recombinant protein antigen RBD of Example 1 JN.1 -HR expression and purification results; among them, a. Ni-affinity chromatography elution results of the supernatant after baculovirus infection; b. EK enzyme digestion sample verification enzymatic digestion effect result diagram; c. EK enzyme digestion and molecular exclusion chromatography purification and SDS-PAGE identification diagram.

[0149] Figure 15 is the recombinant protein antigen RBD of Example 1 XBB.1.5-1 Construction and characterization of; including: a. pFastBac1-GP67-Trx-His-EK-S-RBD (XBB.1.5-1) design diagram; b. 1% agarose gel electrophoresis diagram of cloned PCR products; c. 1% agarose gel electrophoresis diagram of PCR identification of recombinant bacmid; d. recombinant baculovirus packaging and amplification flow chart; e. WB verification results during baculovirus amplification.

[0150] Figure 16 is the recombinant protein antigen RBD of Example 1 XBB.1.5-1 Expression and purification results; among them, a. Ni-affinity chromatography elution results of the supernatant after baculovirus infection; b. EK enzyme digestion sample verification enzymatic digestion effect result diagram; c. Ni-affinity chromatography filler protein back-hanging purification and SDS-PAGE identification diagram after EK enzyme digestion.

[0151] Figure 17 is the recombinant protein antigen RBD of Example 1 XBB.1.5-2 Construction and characterization of; including: a. design diagram of pFastBac1-GP67-Trx-His-EK-S-RBD (XBB.1.5-2); b. 1% agarose gel electrophoresis of cloned PCR products; c. 1% agarose gel electrophoresis of PCR identification of recombinant bacmid; d. flowchart of recombinant baculovirus packaging and amplification; e. WB verification results during baculovirus amplification.

[0152] Figure 18 is the recombinant protein antigen RBD of Example 1 XBB.1.5-2Expression and purification results; among them, a. Ni-affinity chromatography elution results of the supernatant after baculovirus infection; b. EK enzyme digestion sample verification enzymatic digestion effect result diagram; cd. Ni-affinity chromatography filler protein back-hanging purification and SDS-PAGE identification diagram after EK enzyme digestion.

[0153] Figure 19 is the recombinant protein antigen RBD of Example 1 XBB.1.5-4 -HR construction and characterization; including: a. pFastBac1-GP67-Trx-His-EK-S-RBD(XBB.1.5-4)-HR design diagram; b. 1% agarose gel electrophoresis diagram of cloned PCR products; c. 1% agarose gel electrophoresis diagram of PCR identification of recombinant bacmid; d. recombinant baculovirus packaging and amplification flow chart; e. WB verification results during baculovirus amplification.

[0154] Figure 20 is the recombinant protein antigen RBD of Example 1 XBB.1.5-4 -HR expression and purification results; among them, a. Ni-affinity chromatography elution results of the supernatant after baculovirus infection; b. EK enzyme digestion sample verification enzymatic digestion effect result diagram; cd. Ni-affinity chromatography filler back-hanging purification result diagram after EK enzyme digestion.

[0155] Figure 21 is the recombinant protein antigen RBD of Example 1 XBB.2.3 Construction and characterization of; including: a. pFastBac1-GP67-Trx-His-EK-S-RBD (XBB.2.3) design diagram; b. 1% agarose gel electrophoresis diagram of cloned PCR products; c. 1% agarose gel electrophoresis diagram of PCR identification of blue-white bacterial solution; d. recombinant baculovirus packaging and amplification flow chart; e. WB verification result diagram during baculovirus amplification process.

[0156] Figure 22 is the recombinant protein antigen RBD of Example 1 XBB.2.3 Expression and purification results; among them, a. Ni-affinity chromatography elution results of the supernatant after baculovirus infection; b. EK enzyme digestion sample verification enzymatic digestion effect result diagram; c. Ni-affinity chromatography filler back-hanging purification and SDS-PAGE identification diagram after EK enzyme digestion.

[0157] Figure 23 is the recombinant protein antigen RBD of Example 1 XBB.2 -HR construction and characterization; including: a. design diagram of pFastBac1-GP67-Trx-His-EK-S-RBD(Omicron_XBB.2)-HR; b. 1% agarose gel electrophoresis of cloned PCR products; c. 1% agarose gel electrophoresis of PCR identification of recombinant bacmid; d. recombinant baculovirus packaging and amplification flow chart; e. WB verification results during baculovirus amplification.

[0158] Figure 24 is the recombinant protein antigen RBD of Example 1 XBB.2 -HR expression and purification results; among them, a. Ni-affinity chromatography elution results of the supernatant after baculovirus infection; b. EK enzyme digestion sample verification enzymatic digestion effect result diagram; cd. EK enzyme digestion and molecular exclusion chromatography purification and SDS-PAGE identification diagram.

[0159] Figure 25 shows the RBD vaccination in Experiment 1 XBB.1.16 -HR, RBD XBB.1.5-3 -HR, RBD XBB.2.3 -HR, RBD XBB.1.16.6 -HR, RBD BA.2.86 -HR, RBD EG.5 -GMT results of neutralizing antibodies of 6 protein vaccines including HR against various pseudoviruses;

[0160] Figure 26 shows the RBD of Test Example 1 JN.1 - HR protein as an antigen neutralizing antibody GMT results against various pseudoviruses;

[0161] Figure 27 is a graph showing the neutralizing antibody GMT results of the monovalent vaccine and the quadrivalent vaccine against various pseudoviruses in Experimental Example 2;

[0162] Figure 28 shows the results of the live virus challenge experiment after mice were immunized with the quadrivalent vaccine in Experimental Example 2; a is the result of euthanizing the infected mice on the 5th day after infection to collect the nasal concha, trachea and lung tissues, and then detecting the gRNA level by RT-qPCR; b is the histopathological score of the lung tissue of mice attacked by the live XBB.1.16 real virus.

[0163] Figure 29 shows the Ad5 test case 3 XBB.1.5 Adenovirus and RBD XBB.1.5-3 -HR recombinant protein vaccine combined with intranasal administration produces neutralizing antibodies against multiple pseudoviruses in rats. DETAILED DESCRIPTION

[0164] Abbreviations:

[0165] Monophosphoryl lipid A (MPL); squalene oil-in-water emulsion (MF59), recombinant cholera toxin (rCTB); astragalus polysaccharide (APS); phosphatidylethanolamine (PE), phosphatidylcholine (PC), cholesterol (Chol), dioleoylphosphatidylethanolamine (DOPE), (2,3-dioleyloxypropyl)trimethylammonium chloride (DOTAP), N-[1-(2,3-dioleoyl chloride)propyl]-N,N,N-trimethylammonium chloride (DOTMA), cationic cholesterol (DC-Chol), dimethyl-2,3-dioleyloxypropyl-2-(2-sperminecarboxamido)ethylammonium trifluoroacetate (DOSPA), trimethyldodecylammonium bromide (DTAB), trimethyltetradecylammonium bromide (TTAB), trimethylhexadecylammonium bromide (CTAB), dimethyldioctadecylammonium bromide (DDAB), CpG ODN (a nucleotide sequence containing unmethylated cytosine and guanine dinucleotides as the core sequence, artificially synthesized CpG)

[0166] Below, the scheme of the present invention will be explained in conjunction with embodiment.It will be understood by those skilled in the art that the following examples are only used to illustrate the present invention and should not be regarded as limiting the scope of the present invention.In the embodiment, if specific technology or conditions are not indicated, the technology or conditions described in the literature in this area or the product instructions are used.The reagents or instruments used are not indicated by the manufacturer, and are all conventional products that can be obtained by commercial purchase.

[0167] Example 1 Preparation of recombinant protein and vaccine using insect baculovirus expression system

[0168] Construction of recombinant protein antigen RBD XBB.1.16 -HR:

[0169] 1. Construction and design of S-RBD (Omicron_XBB.1.16)-HR

[0170] Because the S protein of the SARS-CoV-2 coronavirus is membrane-localized, to ensure mimicking its secretion process, we constructed an expression construct for the SARS-CoV-2 S-RBD (Omicron_XBB.1.16)-HR protein. We added a GP67 signal peptide sequence to the N-terminus of the protein to facilitate secretory expression. This signal peptide is spontaneously cleaved by insect cells during secretion. Furthermore, we added a thioredoxin (Trx) tag from the fall armyworm (Spodoptera frugiperda, S. frugiperda) after the GP67 signal peptide to facilitate S-RBD (Omicron_XBB.1.16)-HR folding. We also added a 6xhis tag to facilitate subsequent purification, and an EK enzyme cleavage site to remove both the Trx and 6xhis tags. This protein expression construct design removes all redundant amino acids not associated with S-RBD (Omicron_XBB.1.16)-HR through EK enzyme cleavage. The schematic for this expression construct design is shown in Figure 1a. Its amino acid sequence is shown in SEQ ID No. 19, and its nucleotide sequence is shown in SEQ ID No. 37.

[0171] 2. Identification of recombinant plasmid construction

[0172] The coding fragment designed above was cloned into the pFastBac1 vector plasmid and identified by bacterial culture PCR. The results of bacterial culture PCR identification showed that all six selected clones successfully amplified the GP67-Trx-His-EK-S-RBD(Omicron_XBB.1.16)-HR fragment, as shown in Figure 1b.

[0173] 3. Identification of Recombinant Bacmid

[0174] The correctly identified pFastBac1-GP67-Trx-His-EK-S-RBD(Omicron_XBB.1.16)-HR recombinant clone was selected and the recombinant plasmid was extracted and transformed into DH10b competent cells. The blue-white stain was then used for PCR analysis. The PCR products were analyzed by 1% agarose gel electrophoresis. The results are shown in Figure 1c. White stains indicate bacmid clones that have undergone recombination, while blue stains indicate bacmid clones that have not undergone recombination.

[0175] 4. Packaging of Recombinant Baculovirus

[0176] The recombinant bacmid was transfected into sf9 insect cells, and the P0 recombinant baculovirus was harvested 5 days later. The flowchart of recombinant baculovirus packaging and baculovirus amplification is shown in Figure 1d.

[0177] 5. Target protein expression verification

[0178] During the aforementioned baculovirus amplification process, the target protein is simultaneously expressed. Because the target protein contains a His tag before tag removal, we used anti-His Western blotting to verify recombinant protein expression. The results showed a distinct band between the 50kD and 60kD marker bands, consistent with the size of the Trx-His-EK-S-RBD (Omicron_XBB.1.16)-HR protein, indicating successful baculovirus amplification and expression of the target protein. The results are shown in Figure 1e.

[0179] 6. Purification and identification of target protein

[0180] The recombinant baculovirus was used to infect sf9 cells. Three days later, the cell culture medium was harvested and purified using Ni-affinity chromatography. The results are shown in Figure 2a. The target protein was primarily eluted at 250 mM imidazole, resulting in a highly purified target protein.

[0181] 7. Verification of EK enzyme excision tag

[0182] The target protein eluate was concentrated and adjusted to a concentration of 1 mg / mL. EK enzyme was added and digested at 18°C ​​for 14 hours. The protein was then identified by SDS-PAGE gel electrophoresis. The results are shown in Figure 2b, demonstrating that EK enzyme can cleave the Trx-His-EK (amino acid sequence of the EK cleavage site) tag from the target protein.

[0183] 8. Verification of Removal of Excised Tags

[0184] The sample after EK enzyme digestion was subjected to Ni-affinity chromatography filler protein back-hanging purification, and the Trx-His-EK-S-RBD (Omicron_XBB.1.16) -HR protein (uncut intact protein), S-RBD (Omicron_XBB.1.16) -HR protein and the cut Trx-His-EK tag were separated by Ni-affinity chromatography filler. The results are shown in Figure 2c-d. The results show that after the Trx-His-EK tag is removed, it can be removed by subsequent purification means, thereby obtaining S-RBD (Omicron_XBB.1.16) -HR protein without any tag, whose amino acid sequence is shown in SEQ ID No. 1.

[0185] Similarly, referring to the above recombinant protein antigen RBD XBB.1.16 -HR construction method (different protein construction step 4 recombinant baculovirus packaging virus generation number is different, step 8 purification uses Ni-affinity chromatography filler or molecular exclusion chromatography column), construction of RBD XBB.1.5-3 -HR (SEQ ID No. 5), RBD XBB.2.3-HR (SEQ ID No. 8), RBD XBB.1.16.6 -HR (SEQ ID No. 9), RBD BA.2.86 -HR (SEQ ID No. 10), RBD EG.5 -HR (SEQ ID No. 11), RBD JN.1 -HR (SEQ ID No. 55) and RBD XBB.1.5-1 (SEQ ID No. 3), RBD XBB.1.5-2 (SEQ ID No. 4), RBD XBB.1.5-4 -HR (SEQ ID No. 6), RBD XBB.2.3 (SEQ ID No. 7) and RBD XBB.2 -HR (SEQ ID No. 2).

[0186] Among them, RBD XBB.1.5-3 -HR construction design, process and corresponding target protein expression data are shown in Figures 3-4; the amino acid sequence of the designed coding fragment pFastBac1-GP67-Trx-His-EK-S-RBD(XBB.1.5-3)-HR is shown in SEQ ID No.23, and its nucleotide sequence is shown in SEQ ID No.41.

[0187] Among them, RBD XBB.2.3 -HR construction design, process and corresponding target protein expression data are shown in Figures 5-6; the amino acid sequence of the designed coding fragment pFastBac1-GP67-Trx-His-EK-S-RBD(XBB.2.3)-HR is shown in SEQ ID No.26, and its nucleotide sequence is shown in SEQ ID No.44.

[0188] Among them, RBD XBB.1.16.6 -HR construction design, process and corresponding target protein expression data are shown in Figures 7-8; the designed coding fragment pFastBac1-GP67-Trx-His-EK-S-RBD(XBB.1.16.6)-HR amino acid sequence is shown in SEQ ID No.27, and its nucleotide sequence is shown in SEQ ID No.45.

[0189] Among them, RBD BA.2.86 -HR construction design, process and corresponding target protein expression data are shown in Figures 9-10; the amino acid sequence of the designed coding fragment pFastBac1-GP67-Trx-His-EK-S-RBD(BA.2.86)-HR is shown in SEQ ID No.28, and its nucleotide sequence is shown in SEQ ID No.46.

[0190] Among them, RBD EG.5 -HR construction design, process and corresponding target protein expression data are shown in Figures 11-12; the amino acid sequence of the designed coding fragment pFastBac1-GP67-Trx-His-EK-SS-RBD(EG.5)-HR is shown in SEQ ID No.29, and its nucleotide sequence is shown in SEQ ID No.47.

[0191] Among them, RBD JN.1 -HR construction design, process and corresponding target protein expression data are shown in Figures 13-14; the amino acid sequence of the designed coding fragment pFastBac1-GP67-Trx-His-EK-S-RBD(JN.1)-HR is shown in SEQ ID No.56, and its nucleotide sequence is shown in SEQ ID No.57.

[0192] Among them, RBD XBB.1.5-1 The construction design, process and corresponding target protein expression data are shown in Figures 15-16; the amino acid sequence of the designed coding fragment pFastBac1-GP67-Trx-His-EK-S-RBD (XBB.1.5-1) is shown in SEQ ID No.21, and its nucleotide sequence is shown in SEQ ID No.39.

[0193] Among them, RBD XBB.1.5-2 The construction design, process and corresponding target protein expression data are shown in Figures 17-18; the amino acid sequence of the designed coding fragment pFastBac1-GP67-Trx-His-EK-S-RBD (XBB.1.5-2) is shown in SEQ ID No.22, and its nucleotide sequence is shown in SEQ ID No.40.

[0194] Among them, RBD XBB.1.5-4 -HR construction design, process and corresponding target protein expression data are shown in Figures 19-20; the amino acid sequence of the designed coding fragment pFastBac1-GP67-Trx-His-EK-S-RBD(XBB.1.5-4)-HR is shown in SEQ ID No.24, and its nucleotide sequence is shown in SEQ ID No.42.

[0195] Among them, RBD XBB.2.3 The construction design, process and corresponding target protein expression data are shown in Figures 21-22; the amino acid sequence of the designed coding fragment pFastBac1-GP67-Trx-His-EK-S-RBD (XBB.2.3) is shown in SEQ ID No. 25, and its nucleotide sequence is shown in SEQ ID No. 43.

[0196] Among them, RBD XBB.2 -HR construction design, process and corresponding target protein expression data are shown in Figures 23-24; the amino acid sequence of the designed coding fragment pFastBac1-GP67-Trx-His-EK-S-RBD(XBB.2)-HR is shown in SEQ ID No.20, and its nucleotide sequence is shown in SEQ ID No.38.

[0197] Example 2 Preparation of recombinant adenovirus vaccine

[0198] 1. Optimization and synthesis of S protein gene sequence

[0199] The Omicron variant XBB.1.5 virus strain was used as a template to obtain the gene sequence of its S protein, while retaining the S protein's signal peptide. To ensure high protein expression, codons were optimized, and the optimized sequence was subsequently synthesized (its nucleotide sequence is shown in SEQ ID No. 50).

[0200] 2. Packaging of recombinant adenovirus COVID-19 vaccine

[0201] During gene synthesis, the S gene was cloned into the pDC316 vector using a recombinant cloning strategy, resulting in a shuttle plasmid (pDC316-S). The constructed pDC316-S vector containing the Omicron variant XBB.1.5S gene was co-transfected with the backbone plasmid pBHGlox_E1,3Cre from the AdMax adenovirus system into HEK293 cells for recombinant adenovirus packaging. The process is as follows:

[0202] 1) 8×10 5 HEK293A cells were seeded in six-well plates with high-glucose DMEM + 10% FBS medium and cultured overnight in a cell culture incubator at 37°C containing 5% CO2.

[0203] 2) The next day, the medium was replaced with high-glucose DMEM + 2% FBS, and the backbone plasmid (pBHGlox_E1,3Cre) and shuttle plasmid were co-transfected into HEK293A cells using lipofectamine 3000. The specific steps were as follows: 4 μg of backbone plasmid and 2 μg of shuttle plasmid were diluted with 125 μL Opti-MEM medium into each transfection well, and then 12 μL of P3000 reagent was added. In another 1.5 ml EP tube, 7.5 μL of lipofectamine 3000 was diluted with 125 μL 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, and then added to the cells. The cells were cultured and, after confluence, passaged on 25 cm 2 In the cell culture flask, observe the signs of cell toxicity every day, and when the cells have grown to the bottom of the flask, transfer them to 75cm 2 In the cell culture flask, the virus is collected until obvious plaques appear on the cells and most of the cells become diseased and fall off from the bottom.

[0204] 3) Collect the virus-free cell culture and centrifuge at 1200 rpm for 3 minutes. The virus-containing supernatant is aspirated and the cell pellet is resuspended in 1 / 10 the culture volume of the virus-containing supernatant. The cell pellet is placed in a -80°C refrigerator and then frozen and thawed three times in a 37°C water bath. Centrifuge at 3000 rpm for 20 minutes, collect the virus-containing supernatant, and combine it with the virus-containing supernatant above. This is the virus seed for the adenovirus vaccine.

[0205] 4) Take 50 μL of the vaccine candidate strain seed solution, add 2 μL of proteinase K, and digest at 50°C for 30 minutes to release the viral genome. Use this as a template for PCR amplification of the S gene sequence. The PCR product is recovered from the electrophoresis gel and sequenced for identification. The PCR amplification conditions are as follows:

[0206] Denaturation: 95°C for 10 min, denaturation: 95°C for 10 s, annealing: 64°C for 30 s, extension: 72°C for 2 min, extension: 72°C for 5 min, 40 cycles.

[0207] 3. Amplification of recombinant adenovirus COVID-19 vaccines

[0208] The identified recombinant adenovirus vaccine strain was amplified in 293 cells step by step. The specific process was as follows: 4.0×10 6293H cells were cultured at a concentration of 100 cells / ml for 48-72 hours, and the virus culture was collected. The master virus seed bank and the working virus seed bank were prepared by repeated freezing and thawing three times. The recombinant adenovirus vaccine was amplified on a large scale using a shake flask or bioreactor, and the virus culture was collected after most of the cells became diseased. The process of amplifying cells and viruses in a bioreactor is as follows: First, the bioreactor is sterilized after assembly, and then cell culture medium is added to the bioreactor. When the operating conditions are stable at 37°C, pH 7.0, DO 50%, and 50rpm, the cells in the cell shake flask are collected and inoculated into the bioreactor at an inoculation cell density of 1.0×10 6 Cells / ml were added to 10L of cell culture medium. The conditions for cell culture in the reactor were temperature 37°C, rotation speed 50-95rpm, pH 7.15-7.25, DO 30-50%. Glucose concentration, cell density and cell morphology were measured daily. When the cell density in the bioreactor reached 4.0×10 6 When the cell count reaches 10 cells / ml, the bioreactor is inoculated with the recombinant adenovirus vaccine seed at an MOI of 3. After inoculation, samples are taken every day to test the glucose concentration, cell viability, and virus titer. When most of the cells, 50-70%, fall off the microcarriers and die, the culture is terminated and virus lysis buffer is added to the bioreactor at a final concentration of 0.05%-1% Tween 80. The cells are lysed at 37°C for 2-4 hours, and the virus liquid is then collected.

[0209] 4. Purification of recombinant adenovirus COVID-19 vaccine

[0210] The collected virus is purified by cesium chloride ultracentrifugation or ion exchange chromatography. The specific process is as follows:

[0211] (1) Purification of adenovirus vaccine by cesium chloride ultracentrifugation

[0212] The collected virus culture was centrifuged at 1200 g for 10 minutes, the virus-containing culture supernatant was aspirated, the cell pellet was resuspended with 1 / 10 culture volume of the virus-containing supernatant, and repeatedly frozen and thawed three times in a -80°C refrigerator and a 37°C water bath, centrifuged at 3000 rpm for 10-20 minutes, and the supernatant was aspirated. The virus-containing culture supernatant was concentrated 10-fold using a 100K-300K ultrafiltration membrane pack; a 1.4 g / ml cesium chloride solution (53 g cesium chloride + 87 ml 10 mM Tris-HCl, pH 7.9) and a 1.2 g / ml cesium chloride solution (26.8 g cesium chloride + 92 ml 10 mM Tris-HCl, pH 7.9) were prepared; 8 ml of the 1.4 g / ml cesium chloride solution was slowly added to an ultracentrifuge tube, followed by a gentle addition of 6 ml of the 1.2 g / ml cesium chloride solution, and finally 20 ml of the virus-containing supernatant was added to the top of the discontinuous gradient. After balancing, the mixture was centrifuged at 100,000 × g at 4° C. for 90 minutes; after centrifugation, the blue virus band was aspirated with a syringe, dialyzed to remove cesium chloride, and stored at −80° C.

[0213] (2) Purification of adenovirus by ion exchange chromatography

[0214] The virus culture was collected and lysed with 0.05%-1% Tween 20 at 37°C for 2-4 hours. The lysed culture was clarified by filtration through 1.2μm and 0.45μm capsule filters. The sample was concentrated 5-10 times using a 100-300kD molecular weight tangential flow membrane bag, and then washed with 5-10 volumes of filtration buffer (50mM Tris-HCl, 2mM MgCl2, 0-500mM NaCl, pH 8.0). The washed sample was collected; the ultrafiltration sample was purified using Capto Q Impress, Q Sepharose XL, Source 30Q or Source Anion exchange chromatography with 15Q fillers follows the following procedure: equilibration with equilibration buffer for 5 column volumes at a flow rate of 20 ml / min. Sample is then loaded at a rate of 10 ml / min. After loading, the equilibration buffer is equilibrated to a conductivity level. The sample is then eluted using a linear gradient from 100% low-salt buffer to 100% high-salt buffer at a flow rate of 10 ml / min, with each elution peak collected. After elution, the column is regenerated with 2M NaCl buffer for 5-10 column volumes at a flow rate of 20 ml / min. The virus peak is then collected, and the eluted virus sample is then subjected to buffer exchange by dialysis or tangential flow filtration.

[0215] Ad5 prepared by the above method XBB.1.5 The amino acid sequence of the recombinant adenovirus vaccine antigen is shown in SEQ ID No.32.

[0216] The effects of the recombinant protein vaccine and adenovirus vaccine prepared by the present invention were demonstrated through the following experiments.

[0217] Animal preparation, animal immunization, and sample collection

[0218] 1. Monovalent vaccine immunization procedure: 6-8 week old SPF female NIH mice were purchased from Beijing Weitonglihua Laboratory Animal Technology Co., Ltd. (China) and housed in a special pathogen-free environment at the State Key Laboratory of Biotherapy of Sichuan University. All mouse experiments have been approved by the Institutional Animal Care and Use Committee of Sichuan University (Chengdu, Sichuan, China). The different recombinant protein antigens prepared above were mixed with an equal volume of adjuvant MF59 as monovalent vaccines. The mice were immunized three times intramuscularly on days 0, 21, and 42 with 10 μg protein per mouse. Blood was collected on the 14th day after the three immunizations to detect neutralizing antibodies and evaluate the immune effect of the vaccine.

[0219] 2. Quadrivalent Vaccine Basic Immunization Procedure: 6-8 week-old SPF female NIH mice were purchased from Beijing Weitonglihua Laboratory Animal Technology Co., Ltd. (China) and housed in a specific pathogen-free environment at the State Key Laboratory of Biotherapy at Sichuan University. Four different recombinant protein antigens were mixed with an equal volume of the adjuvant MF59 to form the quadrivalent vaccine. Mice were immunized intramuscularly three times on days 0, 21, and 42 with 10 μg of protein per mouse. Neutralizing antibody blood was collected 14 days after each immunization to evaluate the vaccine's immune efficacy.

[0220] 3. Female BALB / c rats aged 6-8 weeks were purchased from Weitonglihua Company and housed in a specific pathogen-free environment at the State Key Laboratory of Biotherapy, Sichuan University. To prepare the two-component nasal spray vaccine, 1×10 10 VP (low dose) or 2×10 10 VP (high dose) Ad5 XBB.1.5 with 20 μg RBD XBB.1.5 -HR, 40 μg RBD XBB.1.5 -HR were mixed in a total volume of 100 / 200 μL to prepare low-dose (administered volume of 100 μL) or high-dose (administered volume of 200 μL) two-component vaccines. 10 VP Ad5 Empty with 40 μg RBD XBB.1.5 -HR; (2) 1×10 10 VP Ad5 XBB.1.5 ; (3) 1×10 10 VP Ad5 XBB.1.5 with 20 μg RBD XBB.1.5 -HR; (4) 2×10 10 VP Ad5 XBB.1.5 ; (5) 2×10 10 VP Ad5 XBB.1.5with 40 μg RBD XBB.1.5 -HR was delivered intranasally to BALB / c rats immunized three times on days 0, 21, and 42. Blood samples were collected 14 days after the three immunizations to measure neutralizing antibodies and evaluate the immune efficacy of the vaccine.

[0221] Experimental Example 1: Detection of Neutralizing Antibodies in Recombinant Protein Vaccine Immune Serum by Pseudovirus Neutralization Method

[0222] SARS-CoV-2 pseudoviruses, including XBB.1.5, XBB.1.6, XBB.1.16.6, EG.5.1, FL.1.5.1, HV.1, BA.2.86, EG.5, JN.1, JN.1.13, KP.2, KP.3, XBB.1.16, and XBB.2.3, were purchased from Genomeditech.

[0223] Briefly, inactivated serum samples (56°C for 30 minutes) were diluted in three-fold factors ranging from 30 to 65610 and then incubated with an equal volume of diluted pseudovirus at 37°C for 1 hour. Then, 1.2 × 10 4 HEK-293T cells expressing human ACE2 receptors (293T / ACE2) were cultured and incubated at 37 ° C, 0.5% CO2 for 48 hours to express luciferase. Finally, the supernatant was removed, and a lysis reagent (100 μL / well) with luciferase substrate (Beyotime, RG005) was added, and the luminescence in 293T / ACE2 cells was measured using a multimode microplate reader (PerkinElmer, USA). GraphPad Prism 8.0.2 was used to determine and calculate the 50% neutralization rate of the pseudovirus. 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 to be tested / positive sample - negative sample) × 100%.

[0224] RBD XBB.1.5-3 -HR (SEQ ID No. 5), RBD XBB.1.16 -HR (SEQ ID No. 1), RBD EG.5 -HR (SEQ ID No. 11), RBD XBB.1.16.6 -HR (SEQ ID No. 9), RBD BA.2.86 -HR (SEQ ID No. 10), RBD XBB.2.3 -HR (SEQ ID No. 8), RBD JN.1 -HR (SEQ ID No. 55) and other 7 proteins and adjuvant MF59 constitute a monovalent recombinant protein vaccine, RBD XBB.1.5-3-HR, RBD XBB.1.16 -HR, RBD XBB.2.3 -HR, RBD JN.1 -HR protein antigen was mixed (mixing ratio 1:1:1:1, 1:1:2:2, 1:1:5:1) with an equal volume of MF59 adjuvant to form a quadrivalent vaccine, which was used to immunize mice, and serum was collected to determine the levels of neutralizing antibodies against pseudoviruses such as XBB.1.5, XBB.1.6, XBB.1.16.6, EG.5.1, FL.1.5.1, HV.1, BA.2.86, EG.5, JN.1, JN.1.13, KP.2, KP.3, XBB.1.16, and XBB.2.3.

[0225] As shown in Figure 25, RBD vaccination XBB.1.5-3 -HR, RBD XBB.1.16 -HR, RBD EG.5 -HR, RBD XBB.1.16.6 -HR, RBD BA.2.86 -HR, RBD XBB.2.3 -HR and other six protein vaccines have different 50% neutralization geometric mean titers (GMT) against four pseudoviruses including XBB.1.5, EG.5.1, HV.1, and BA.2.86. XBB.1.5-3 -HR and RBD XBB.2.3 -HR two protein vaccines showed excellent serum neutralizing antibody levels against different Omicron mutant viruses, especially against EG.5.1 pseudovirus, producing higher neutralizing antibody levels.

[0226] As shown in Figure 26, RBD JN.1 -HR protein was used as the antigen, and blood was collected on the 14th day after the mice were immunized with three different strains to detect neutralizing antibodies. The mice produced strong serum neutralizing antibodies against the newly discovered Omicron mutant viruses BA.2.86, JN.1, JN.1.13, KP.2 and KP.3, showing that RBD JN.1 -HR protein vaccine has a broad universal neutralizing response to Omicron mutant viruses.

[0227] As shown in Figure 27, RBD XBB.1.5-3 -HR+RBD XBB.1.16 -HR+RBD XBB.2.3 -HR+RBD JN.1The quadrivalent vaccine composed of a mixture of -HR protein antigen (mixing ratio 1:1:1:1, 1:1:2:2, 1:1:5:5) and an equal volume of MF59 adjuvant showed effective neutralization against mutant pseudoviruses of the XBB lineage currently circulating globally, such as XBB.1.5, XBB.1.6, XBB.1.16.6, BA.2.86, EG.5 and JN.1, while the pure RBD XBB.1.5-3 -HR, RBD XBB.1.16 -HR, RBD XBB.2.3 -HR, RBD JN.1 - HR protein monovalent vaccines, especially RBD JN.1 -HR protein monovalent vaccine showed weak protection against XBB lineage mutant pseudoviruses XBB.1.5, XBB.1.6, XBB.1.16.6, and EG.5, with GMTs of 190, 186, 146, and 318, respectively, but compared with RBD XBB.1.5-3 -HR, RBD XBB.1.16 -HR, RBD XBB.2.3 -HR combined use, regardless of the mixing ratio, showed better protection against all XBB lineage mutant pseudoviruses, demonstrating the unique advantages of the quadrivalent vaccine.

[0228] Test Example 2: Novel Coronavirus XBB.1.16 Variant Challenge Protection Test

[0229] Prevention of viral infection is one of the most important indicators of respiratory vaccine protection efficiency. Therefore, this study investigated whether the prepared recombinant protein quadrivalent vaccine can provide a protective immune response against the epidemic strain. NIH mice (6-8 weeks) were divided into two groups and injected intramuscularly with 10 μg protein / mouse (mixing ratio 1:1:5:5) of the quadrivalent vaccine on days 0, 21, and 42. The quadrivalent vaccine is based on RBD XBB.1.5-3 -HR+RBD XBB.1.16 -HR+RBD XBB.2.3 -HR+RBD JN.1 -HR protein antigen was mixed with an equal volume of MF59 adjuvant. Mice immunized with adjuvant alone served as controls, with n=6 mice per group. Then, on day 63, 1×10 6PFU XBB.1.16 live virus challenged mice. Changes in body weight and throat swab viral load were monitored daily. On day 5 after infection, the mice were euthanized and tissues were collected to detect viral load and pathological changes. Viral genomic RNA (gRNA) in nasal concha, trachea and lung tissue samples was detected by reverse transcription quantitative polymerase chain reaction (RT-qPCR) assay. Primer and probe sequences, including 5'-GACCCCAAAATCAGCGAAAT-3' (SEQ ID No.58 forward), 5'-TCTGGTTACTGCCAGTTGAATCTG-3' (SEQ ID No.59 reverse), 5'-FAM-ACNGCCGCATTACGTTGGTGGACC-BHQ1-3' (SEQ ID No.60 probe sequence), were used to detect gRNA levels. Pathological changes in lung tissue were assessed by hematoxylin and eosin staining (H&E).

[0230] All experiments involving animals challenged with live SARS-CoV-2 virus were thoroughly reviewed and approved by the Institutional Animal Care and Use Committee of the Institute of Medical Biology, Chinese Academy of Medical Sciences. Furthermore, these experiments were conducted in the ABSL-4 facility of the Kunming National High-Level Biosafety Primate Research Center.

[0231] As shown in Figure 28, mice were euthanized on day 5 after infection to collect tissues for assessment of viral load and histopathology. In the control group receiving adjuvant, high levels of gRNA were detected in nasal concha, trachea and lung tissues. The viral load in the nasal concha, trachea and lung tissues of mice vaccinated with the quadrivalent vaccine decreased significantly. In addition, mild pathological changes were observed in the lung tissue of the adjuvant control group, characterized by multifocal consolidation areas, thickening of alveolar septa, alveolar congestion and small plaque inflammation. As expected, the lung tissue of the vaccinated mice showed normal histological structure, with intact alveoli and no obvious inflammation. Therefore, the pathological scores of the lung tissue of vaccine-immunized mice were significantly lower than those of the adjuvant control group. These results show that the quadrivalent recombinant protein vaccine can induce protective immune responses in the upper and lower respiratory tracts and effectively prevent live virus infection.

[0232] Experimental Example 3: Pseudovirus Neutralization Experiment of Recombinant Protein Vaccine Combined with Adenovirus Vaccine

[0233] The source of pseudovirus and the neutralization method were the same as those in Experiment 1. As shown in Figure 29, 1×10 10 VP (low dose) or 2×10 10 VP (high dose) Ad5 XBB.1.5 Adenovirus with 20ug or 40ug RBD XBB.1.5-3-HR recombinant protein vaccine produced higher serum neutralizing antibodies against Omicron mutant viruses XBB.1.5, XBB.1.16, XBB.1.16.6, XBB.2.3, FL.1.5.1, HV.1, EG.5, BA.2.86, JN.1, etc. through intranasal administration. It can be seen that the adenovirus vaccine combined with the recombinant protein vaccine induced stronger serum neutralizing antibodies than the simple Ad5 adenovirus and recombinant protein vaccine, indicating that the adenovirus combined with the recombinant subunit preparation can produce stronger neutralizing protection ability in the blood and mucous membranes, preventing the infection of the new coronavirus.

Claims

1. A protein for resisting infection by the SARS-CoV-2 Omicron mutant strain XBB subtype, characterized in that: It contains at least one of the amino acid sequences selected from SEQ ID No.1 to SEQ ID No.18 and SEQ ID No.

55.

2. The protein for resisting infection of SARS-CoV-2 Omicron mutant strain XBB subtype according to claim 1, characterized in that: The protein contains at least one of the amino acid sequences shown in SEQ ID No.1 to SEQ ID No.11 and SEQ ID No.55; preferably, the protein contains at least one of the amino acid sequences shown in SEQ ID No.1, SEQ ID No.5, SEQ ID No.8 to SEQ ID No.11 and SEQ ID No.

55.

3. The precursor of the protein according to claim 1 or 2, characterized in that: The protein is connected to a signal peptide and / or a protein tag; preferably, the protein tag is selected from at least one of the following: a histidine tag, a thioredoxin 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 utilization substance A protein tag.

4. The precursor of the protein according to claim 3, characterized in that: A protease recognition region for removing the protein tag is also connected to the protein that resists SARS-CoV-2 Omicron mutant strain XBB subtype infection; preferably, the protease is selected from at least one of the following: enterokinase, TEV protease, thrombin, coagulation factor Xa, carboxypeptidase A, and rhinovirus 3c protease.

5. The precursor of the protein according to claim 3 or 4, characterized in that: The amino acid sequence of the precursor is selected from at least one of SEQ ID No.19 to SEQ ID No.36 and SEQ ID No.56; preferably, the amino acid sequence of the precursor is selected from at least one of SEQ ID No.19 to SEQ ID No.29 and SEQ ID No.56; more preferably, the amino acid sequence of the precursor is selected from at least one of SEQ ID No.19, SEQ ID No.23, SEQ ID No.26 to SEQ ID No.29 and SEQ ID No.

56.

6. A polynucleotide, characterized in that: It encodes the protein according to claim 1 or 2 or the precursor according to any one of claims 3 to 5.

7. The polynucleotide according to claim 6, characterized in that: The polynucleotide sequence is selected from at least one of SEQ ID No.37 to SEQ ID No.54 and SEQ ID No.57; preferably, the polynucleotide sequence is selected from at least one of SEQ ID No.37 to SEQ ID No.47 and SEQ ID No.57; more preferably, the polynucleotide sequence is selected from at least one of SEQ ID No.37, SEQ ID No.41, SEQ ID No.44 to SEQ ID No.47 and SEQ ID No.

57.

8. A recombinant vector, characterized in that: It contains the polynucleotide according to claim 7.

9. The recombinant vector according to claim 8, characterized in that: The recombinant vector adopts 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; further preferably, the CHO cell expression vector is pTT5 or FTP-002.

10. A host cell, characterized in that: It contains the recombinant vector according to claim 8 or 9.

11. The host cell according to claim 10, characterized in that: The host cell is at least one of insect cells, mammalian cells, Escherichia coli, and yeast; preferably, the insect cell is at least one of sf9 cells, sf21 cells, and Hi5 cells; and the mammalian cell is a CHO cell.

12. A method for preparing the protein according to claim 1 or 2 or the precursor according to any one of claims 3 to 5, characterized in that: The method comprises the following steps: culturing the host cell according to claim 10 or 11 to make it express the protein or precursor, and then recovering the protein.

13. A recombinant protein vaccine for preventing and / or treating infection with SARS-CoV-2 Omicron mutant subtype, characterized in that: It contains the protein according to claim 1 or 2 or the precursor according to any one of claims 3 to 5, and pharmaceutically acceptable auxiliary components.

14. The recombinant protein vaccine according to claim 13, characterized in that: The auxiliary component is an immune adjuvant; preferably, the immune adjuvant is selected from at least one of the following: squalene oil-in-water emulsion, aluminum salt, calcium salt, plant saponin, plant polysaccharide, monophosphoryl lipid A, muramyl dipeptide, muramyl tripeptide, bacterial toxin, GM-CSF cytokine, lipid, cationic liposome material.

15. The recombinant protein vaccine according to claim 14, characterized in that: At least one of the following 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 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; The lipid is selected from at least one of the following: phosphatidylethanolamine, phosphatidylcholine, cholesterol, dioleoylphosphatidylethanolamine; The cationic liposome material is selected from at least one of the following: (2,3-dioleyloxypropyl)trimethylammonium chloride, N-[1-(2,3-dioleoyl chloride)propyl]-N,N,N-trimethylammonium chloride, cationic cholesterol, dimethyl-2,3-dioleyloxypropyl-2-(2-sperminecarboxamido)ethylammonium trifluoroacetate, trimethyldodecylammonium bromide, trimethyltetradecylammonium bromide, trimethylhexadecylammonium bromide, dimethyldioctadecylammonium bromide, and CpG ODN.

16. A protein composition for preventing and / or treating infection with a SARS-CoV-2 Omicron mutant subtype, characterized in that: It contains any two or more of the proteins described in claim 1 or 2 or any two or more of the protein precursors described in any one of claims 3 to 5, wherein the protein is selected from a protein constructed by any one of the amino acid sequences of SEQ ID No.1 to SEQ ID No.18 and SEQ ID No.55; and the precursor is selected from a protein precursor constructed by any one of the amino acid sequences of SEQ ID No.19 to SEQ ID No.36 and SEQ ID No.

56.

17. A recombinant protein vaccine composition for preventing and / or treating infection with SARS-CoV-2 Omicron mutant subtype, characterized in that: It is a combination of any two or more different recombinant protein vaccines; the recombinant protein vaccine is as described in claim 13, 14 or 15.

18. An adenovirus vector vaccine for preventing and / or treating SARS-CoV-2 Omicron mutant subtype infection, characterized in that: It contains a polynucleotide encoding the spike protein of SARS-CoV-2 or its mutant strains; the polynucleotide sequence is obtained by codon or cell optimization.

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.37 to SEQ ID No.54; preferably, the polynucleotide sequence is selected from at least one of SEQ ID No.48 to SEQ ID No.54; more preferably, the polynucleotide sequence is selected from at least one of SEQ ID No.48 to SEQ ID No.

51.

20. The adenovirus vector vaccine according to claim 19, characterized in that: The adenovirus vector used is selected from at least one of the following: adenovirus, vaccinia Ankara virus, adeno-associated virus; preferably, the adenovirus vector is selected from human type 5, type 35 or type 26 and / or chimpanzee AdC68 or AdC7 replication-deficient adenovirus; more preferably, it is selected from human type 5 replication-deficient adenovirus with a combined deletion of E1 and E3.

21. The method for preparing the adenovirus in the adenovirus vector vaccine according to any one of claims 18 to 20, characterized in that: The following steps are involved: Construct a shuttle plasmid vector containing a polynucleotide encoding the spike protein of SARS-CoV-2 or its mutant strains; then transfect the constructed shuttle plasmid vector together with the backbone plasmid into host cells, and culture the host cells; obtain a replication-deficient recombinant adenovirus, and then expand the culture and purify it.

22. The method for preparing adenovirus according to claim 21, characterized in that: The polynucleotide sequence encoding the spike protein of SARS-CoV-2 or its mutant is selected from at least one of SEQ ID No.37 to SEQ ID No.54; preferably, the polynucleotide sequence is selected from at least one of SEQ ID No.48 to SEQ ID No.54; more preferably, the polynucleotide sequence is selected from at least one of SEQ ID No.48 to SEQ ID No.

51.

23. The method for preparing an adenovirus vector according to claim 21, characterized in that: At least one of the following is met: The shuttle plasmid vector is at least one of pDC316-S, pDC315-S, pDC516-S or pDC515-S; The backbone plasmid is at least one of pBHGlox_E1,3Cre and pBHGlox_E1,3FLP; The host cell is HEK293.

24. An adenovirus vector vaccine composition for preventing and / or treating SARS-CoV-2 Omicron mutant subtype infection, characterized in that: It is a combination of any two or more different adenovirus vector vaccines, and the adenovirus vector vaccine is as described in claim 18, 19 or 20.

25. The protein according to claim 1 or 2, the protein precursor according to any one of claims 3 to 5, the protein composition according to claim 16, the recombinant protein vaccine according to any one of claims 13 to 15, the recombinant protein vaccine composition according to claim 17, the adenovirus vector vaccine according to any one of claims 18 to 20, or the adenovirus vector vaccine composition according to claim 24, characterized in that: It is an intradermal or subcutaneous injection preparation, an intramuscular injection preparation, an intravenous injection preparation, an oral or nasal spray preparation; preferably, it is an intramuscular injection preparation and a nasal spray preparation.

26. A pharmaceutical composition for treating and / or preventing infection with a SARS-CoV-2 Omicron mutant subtype, characterized in that: It contains a combination of at least one of a recombinant protein, a protein composition, a recombinant protein vaccine, a recombinant protein vaccine composition and at least one of an adenovirus vector vaccine or an adenovirus vector vaccine composition; Preferably, the pharmaceutical composition is a multivalent vaccine composed of at least one of a recombinant protein vaccine or a recombinant protein vaccine composition and at least one of an adenovirus vector vaccine or adenovirus vector vaccine composition; Preferably, the pharmaceutical composition is a multivalent vaccine composed of at least one of a recombinant protein or a protein composition and at least one of an adenovirus vector vaccine or an adenovirus vector vaccine composition; Among them, the recombinant protein, protein composition, recombinant protein vaccine, recombinant protein vaccine composition, adenovirus vector vaccine, and adenovirus vector vaccine composition are respectively the protein according to claim 1 or 2, the protein composition according to claim 16, the recombinant protein vaccine according to any one of claims 13 to 15, the recombinant protein vaccine composition according to claim 17, the adenovirus vector vaccine according to any one of claims 18 to 20, and the adenovirus vector vaccine composition according to claim 24.

27. A combination drug for treating and / or preventing infection with a SARS-CoV-2 Omicron mutant subtype, characterized in that: It contains at least one of a recombinant protein, a protein composition, a recombinant protein vaccine, a recombinant protein vaccine composition and at least one of an adenovirus vector vaccine or an adenovirus vector vaccine composition, which are administered separately or simultaneously; Preferably, the combination drug is a combination of at least one of a recombinant protein vaccine or a recombinant protein vaccine composition and at least one of an adenovirus vector vaccine or an adenovirus vector vaccine composition; Preferably, the combined drug is a combination of at least one of a recombinant protein or a protein composition and at least one of an adenovirus vector vaccine or an adenovirus vector vaccine composition; Among them, the recombinant protein, protein composition, recombinant protein vaccine, recombinant protein vaccine composition, adenovirus vector vaccine, and adenovirus vector vaccine composition are respectively the protein according to claim 1 or 2, the protein composition according to claim 16, the recombinant protein vaccine according to any one of claims 13 to 15, the recombinant protein vaccine composition according to claim 17, the adenovirus vector vaccine according to any one of claims 18 to 20, and the adenovirus vector vaccine composition according to claim 24.

28. The composition according to claim 26 or the combined drug according to claim 27, characterized in that: The recombinant protein vaccine contains a protein or precursor that resists infection by the XBB subtype of the SARS-CoV-2 Omicron mutant strain; the amino acid sequence of the protein or precursor is selected from at least one of SEQ ID No.1 to SEQ ID No.11, SEQ ID No.55, SEQ ID No.19 to SEQ ID No.29, and SEQ ID No.56; preferably, the amino acid sequence of the protein or precursor is selected from at least one of SEQ ID No.1, SEQ ID No.5, SEQ ID No.8 to SEQ ID No.11, SEQ ID No.55 or SEQ ID No.19, SEQ ID No.23, SEQ ID No.26 to SEQ ID No.29, and SEQ ID No.56; The recombinant protein vaccine composition is a combination of any two or more recombinant protein vaccines containing different proteins; The adenovirus vector vaccine contains a polynucleotide sequence as shown in at least one of SEQ ID No.48 to SEQ ID No.54; preferably, the adenovirus vector vaccine contains a polynucleotide sequence as shown in at least one of SEQ ID No.48 to SEQ ID No.51; The adenovirus vector vaccine composition is a combination of any two or more adenovirus vector vaccines containing different polynucleotide sequences.

29. The composition or the combined drug of claim 28, characterized in that: The amino acid sequence contained in the recombinant protein vaccine is selected from at least one of SEQ ID No.1, SEQ ID No.5, SEQ ID No.8 to SEQ ID No.11, and SEQ ID No.55; the adenovirus vector vaccine contains a polynucleotide sequence shown in at least one of SEQ ID No.48 to SEQ ID No.

51.

30. The pharmaceutical composition according to any one of claims 26, 28, and 29 or the combined drug according to any one of claims 27, 28, or 29, characterized in that: It is an intradermal or subcutaneous injection preparation, an intramuscular injection preparation, an intravenous injection preparation, an oral or nasal spray preparation; preferably, it is an intramuscular injection preparation and a nasal spray preparation.

31. Use of the protein according to claim 1 or 2, the protein precursor according to any one of claims 3 to 5, the protein composition according to claim 16, the recombinant protein vaccine according to any one of claims 13 to 15, the recombinant protein vaccine composition according to claim 17, the adenovirus vector vaccine according to any one of claims 18 to 20, the adenovirus vector vaccine composition according to claim 24, the pharmaceutical composition according to any one of claims 26, 28, and 29, or the combination drug according to any one of claims 27, 28, or 29 in the preparation of a drug for treating and / or preventing SARS-CoV-2 or variant infection or pathogenicity.

32. The use according to claim 31, characterized in that: The SARS-CoV-2 variants include at least one of the SARS-CoV-2 mutant strains Alpha, Beta, Gamma, Delta, Omicron, Omicron sublineages BA.1, BA.2, BA.2.12.1, BA.4 / 5, BQ.1.1, XBB.1.16, XBB.2, XBB.1.5, XBB.2.3, XBB.1.6, XBB.1.9.1, XBB.1.16.6, FL.1.5.1, HV.1, EG.5, EG.5.1, BA.2.86, JN.1, JN.1.13, KP.2, and KP.3.

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