Proteins, adenoviruses, and vaccines against infection by SARS-CoV-2 Omicron XBB subvariant

Abstract

This invention relates to proteins, adenoviruses, and vaccines for infection by SARS-CoV-2 omicron XBB subvariants, belonging to the pharmaceutical field. To address the lack of effective prophylactic and therapeutic agents for preventing and / or treating infection by SARS-CoV-2 omicron XBB variants and their subvariants, the present invention provides proteins, adenoviruses, and vaccines for preventing and / or treating infection by SARS-CoV-2 omicron XBB subvariants, which are optimized and designed based on the full-length S protein and the receptor-binding domain (RBD) and receptor-binding domain-heptad repeat (RBD-HR) sequences in the S proteins of SARS-CoV-2 omicron XBB subvariants, specifically XBB.1.16, XBB.1.5, XBB.1.16.6, BA.2.86, EG.5, JN.1, XBB.2.3, and XBB.2, and which can support the host in fighting coronavirus infection and have relatively good prophylactic and therapeutic effects, in particular against cross-infection caused by SARS-CoV-2 omicron XBB subvariants.

Description

Technical Field

[0001] The present invention belongs to the field of medicine and relates to proteins, adenoviruses, and vaccines for preventing and / or treating infections caused by the SARS-CoV-2 Omicron XBB subvariant.

Background Art

[0002] The novel coronavirus (SARS-Cov-2) is a new beta coronavirus named by the World Health Organization. This virus is enveloped, its particles are circular or elliptical, often polymorphic, and have a diameter of 60 nm to 140 nm. Since its genetic characteristics are significantly different from those of SARS-CoV and MERS-CoV, the virus is a new branch species of coronavirus that has not previously been found in humans. Currently, the main variants of SARS-CoV-2 mainly include five types: alpha, beta, gamma, delta, and omicron, and the omicron variant is divided into subvariants such as 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 (S) protein, envelope (E) protein, membrane (M) protein, and nucleocapsid (N) protein, of which the S protein plays a crucial role in viral infection and pathogenicity and is often used as an antigen for vaccines. The SARS-CoV-2 XBB variant contains multiple mutation sites, and the virus's S protein contains even more mutation sites. This means that the XBB variant and its subvariants evade antibodies stimulated by vaccines against novel coronavirus variants (alpha, beta, gamma, delta, and omicron), leading to vaccine failure or reduced protection against novel coronaviruses, which puts significant pressure on the prevention and control of novel coronaviruses. Therefore, the development of vaccines against SARS-CoV-2 XBB variants, particularly vaccines against various subvariants 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 SARS-CoV-2 variants, is crucial for the prevention and control of SARS-CoV-2. [Overview of the project] [Problems that the invention aims to solve]

[0004] The present invention aims to solve at least one of the technical problems existing in the prior art. Accordingly, an object of the present invention is to provide proteins and adenoviruses for preventing and / or treating infection by SARS-CoV-2 omicron XBB subvariants. The present invention also provides vaccines for preventing and / or treating infection by SARS-CoV-2 omicron XBB subvariants, including recombinant protein vaccines and protein-containing adenovirus vector vaccines. The present invention also further provides polyvaccine combinations of different protein / vaccine combinations, protein vaccines, and adenovirus vector vaccines. [Means for solving the problem]

[0005] In a first aspect, the present invention provides a protein for preventing and / or treating infection by SARS-CoV-2 omicron XBB submutants, comprising at least one amino acid sequence shown in SEQ ID NOs: 1 to 18 and SEQ ID NO: 55. Preferably, the protein comprises at least one amino acid sequence shown in SEQ ID NOs: 1 to 11 and SEQ ID NO: 55.

[0006] More preferably, the protein comprises at least one amino acid sequence shown in SEQ ID NO: 1, SEQ ID NO: 5, SEQ ID NOs: 8-11, and SEQ ID NO: 55.

[0007] The present invention further provides protein precursors that are linked to a protein signal peptide and / or protein tag.

[0008] Preferably, the protein tag is selected from at least one of the following: histidine tag, thioredoxin tag (Trx tag), glutathione transferase tag, ubiquitin-like modifier protein tag, maltose-binding protein tag, c-Myc protein tag, Avi tag protein tag, and nitrogen source utilization substance A protein tag.

[0009] Furthermore, a protease recognition region for excising the protein tag is further ligated to a protein for preventing and / or treating infection by the SARS-CoV-2 Omicron XBB submutant.

[0010] Preferably, the protease is selected from at least one of enterokinase (EK enzyme), TEV protease, thrombin, coagulation factor Xa, carboxypeptidase A, and rhinovirus 3c protease.

[0011] Furthermore, the amino acid sequence of the precursor is selected from at least one of SEQ ID NOs: 19 to 36 and SEQ ID NO: 56. Preferably, the amino acid sequence of the precursor is selected from at least one of SEQ ID NOs: 19 to 29 and SEQ ID NO: 56.

[0012] 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 NOs: 26 to 29, and SEQ ID NO: 56.

[0013] The present invention further provides polynucleotides encoding the described proteins or precursors.

[0014] Furthermore, the polynucleotide sequence is selected from at least one of SEQ ID NOs: 37 to 54 and SEQ ID NO: 57. Preferably, the polynucleotide sequence is selected from at least one of SEQ ID NOs: 37 to 47 and SEQ ID NO: 57. More preferably, the polynucleotide sequence is selected from at least one of SEQ ID NOs: 37, SEQ ID NOs: 41, SEQ ID NOs: 44 to 47, and SEQ ID NO: 57.

[0015] The present invention further provides recombinant vectors containing polynucleotides.

[0016] Furthermore, the recombinant vector is at least one of the following: an insect baculovirus expression vector, a mammalian cell expression vector, an Escherichia coli expression vector, and a yeast expression vector.

[0017] Preferably, the insect baculovirus expression vector is pFastBac1.

[0018] Preferably, the Escherichia coli expression vector is pET32a.

[0019] Preferably, the yeast expression vector is pPICZaA.

[0020] Preferably, the mammalian cell expression vector is a CHO cell expression vector.

[0021] More preferably, the CHO cell expression vector is pTT5 or FTP-002.

[0022] The present invention further provides host cells containing a recombinant vector.

[0023] Furthermore, the host cell is at least one of the following: insect cells, mammalian cells, Escherichia coli, and yeast.

[0024] Preferably, the insect cells are selected from at least one of sf9 cells, sf21 cells, and Hi5 cells.

[0025] Preferably, the mammalian cells are CHO cells.

[0026] The present invention further provides a method for preparing a protein or precursor, comprising the steps of culturing host cells to express a protein or precursor, and then recovering the protein.

[0027] The present invention further provides recombinant protein vaccines for preventing and / or treating infection by SARS-CoV-2 omicron submutants, comprising a protein and / or precursor, as well as a pharmaceutically acceptable excipient or adjuvant component.

[0028] Furthermore, the adjuvant component is an immunoadjuvant.

[0029] Preferably, the immunoadjuvant is selected from at least one of the following: squalene-based oil-in-water emulsions, aluminum salts, calcium salts, phytosaponins, phytopolysaccharides, monophosphate tripide A, muramyl dipeptides, muramyl tripeptides, bacterial toxins, GM-CSF cytokines, lipids, and cationic liposome materials.

[0030] Furthermore, at least one of the following conditions must be met: The squalene-based oil-in-water emulsion is MF59. The aluminum salt is selected from at least one of aluminum hydroxide and arum. The calcium salt is tricalcium phosphate. Phytosaponins are either QS-21 or ISCOM. Plant polysaccharides are astragalus polysaccharides. 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 phosphatidylethanolamine, phosphatidylcholine, cholesterol, and dioleoylphosphatidylethanolamine. 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, dimethyltrifluoroacetate-2,3-dioleoxypropyl-2-(2-spermamide)ethylammonium, trimethyldodecylammonium bromide, trimethyltetradecylammonium bromide, trimethylcetylammonium bromide, dimethyldioctadecylammonium bromide, and CpG ODN.

[0031] The present invention further provides a protein composition for preventing and / or treating infection by SARS-CoV-2 omicron submutants, comprising any two or more proteins or protein precursors for preventing and / or treating infection by SARS-CoV-2 omicron XBB submutants, wherein the protein is constructed from any one amino acid sequence selected from SEQ ID NOs: 1 to 18 and SEQ ID NOs: 55, and the protein precursor is constructed from any amino acid sequence selected from SEQ ID NOs: 19 to 36 and SEQ ID NOs: 56.

[0032] The present invention further provides recombinant protein vaccine compositions for preventing and / or treating infection by SARS-CoV-2 omicron submutants, comprising any two or more different recombinant protein vaccines.

[0033] The difference between recombinant protein vaccines lies in the amino acid sequence that makes up the protein. A recombinant protein vaccine composition refers to a combination of two or more different recombinant protein vaccines prepared using different amino acid sequences. The amino acid sequence is selected from at least one of SEQ ID NOs: 1 to 36, SEQ ID NOs: 55, and SEQ ID NOs: 56.

[0034] The present invention further provides an adenovirus vector vaccine for preventing and / or treating infection by SARS-CoV-2 omicron submutants, comprising a polynucleotide encoding the SARS-CoV-2 or its variant spike protein. The polynucleotide sequence is obtained by codon optimization or cell optimization.

[0035] Preferably, the nucleotide sequence of the polynucleotide is selected from at least one of SEQ ID NOs: 37 to 54. More preferably, the polynucleotide sequence is selected from at least one of SEQ ID NOs: 48 to 54. More preferably, the polynucleotide sequence is selected from at least one of SEQ ID NOs: 48 to 51.

[0036] Furthermore, the adenovirus vector in an adenovirus vector vaccine is selected from at least one of the following: adenovirus, ankara-vaccinia virus, and adeno-associated virus.

[0037] Preferably, the adenovirus vector is selected from replication-deficient human adenovirus 5, 35, or 26, or / and replication-deficient chimpanzee adenovirus AdC68 or AdC7.

[0038] More preferably, the adenovirus vector is selected from replication-defective human adenovirus 5 having a combined E1 and E3 deletion.

[0039] The present invention provides a method for preparing an adenovirus for an adenovirus vector vaccine, which includes the steps of constructing a shuttle plasmid vector of polynucleotides encoding SARS-CoV-2 or its variant spike protein, transfecting the constructed shuttle plasmid vector and backbone plasmid into host cells and culturing the host cells, obtaining replication-deficient recombinant adenovirus, and performing large-scale culture and purification.

[0040] Furthermore, the polynucleotide sequence encoding the SARS-CoV-2 or its variant spike protein is selected from at least one of SEQ ID NOs: 37 to 54. Preferably, the polynucleotide sequence is selected from at least one of SEQ ID NOs: 48 to 54. More preferably, the polynucleotide sequence is selected from at least one of SEQ ID NOs: 48 to 51.

[0041] Furthermore, the shuttle plasmid vector is at least one of pDC316-S, pDC315-S, pDC516-S, and pDC515-S.

[0042] Furthermore, the skeletal plasmid is at least one of pBHGlox_E1,3Cre and pBHGlox_E1, and 3FLP.

[0043] Furthermore, the host cell is HEK293.

[0044] The present invention further provides adenovirus vector vaccine compositions for preventing and / or treating infection by SARS-CoV-2 omicron subvariants, comprising any two or more different adenovirus vector vaccines. Different adenovirus vector vaccines refer to adenovirus vector vaccines prepared using different nucleotide sequences. An adenovirus vector vaccine composition refers to a combination of two or more different, prepared adenovirus vector vaccines. The nucleotide sequences are selected from at least one of SEQ ID NOs: 37 to 54.

[0045] Furthermore, proteins, protein precursors, protein compositions, recombinant protein vaccines, recombinant protein vaccine compositions, adenovirus vector vaccines, and adenovirus vector vaccine compositions are formulated as intradermal or subcutaneous injection preparations, intramuscular injection preparations, intravenous injection preparations, oral preparations, or nasal spray preparations.

[0046] Preferably, the vaccine is in the form of an intramuscular injection preparation and a nasal spray preparation.

[0047] The present invention further provides pharmaceutical compositions for treating and / or preventing infection by SARS-CoV-2 omicron submutants, which are combinations of at least one recombinant protein, protein composition, recombinant protein vaccine, and recombinant protein vaccine composition, as well as at least one adenovirus vector vaccine and adenovirus vector vaccine composition.

[0048] Preferably, the pharmaceutical composition is a polyvalent vaccine combination comprising at least one recombinant protein vaccine and recombinant protein vaccine composition, and at least one adenovirus vector vaccine and adenovirus vector vaccine composition.

[0049] Preferably, the pharmaceutical composition is a polyvalent vaccine combination comprising at least one recombinant protein and protein composition, and at least one adenovirus vector vaccine and adenovirus vector vaccine composition.

[0050] The present invention provides a combination agent for treating and / or preventing infection by SARS-CoV-2 omicron subvariants, which is a combination of at least one of recombinant proteins, protein compositions, recombinant protein vaccines, recombinant protein vaccine compositions, and at least one of adenovirus vector vaccines and adenovirus vector vaccine compositions, which are administered separately or simultaneously.

[0051] Preferably, the combination drug is a combination of at least one recombinant protein vaccine and recombinant protein vaccine composition, and at least one adenovirus vector vaccine and adenovirus vector vaccine composition.

[0052] Preferably, the combination drug is a combination of at least one recombinant protein and protein composition, and at least one adenovirus vector vaccine and adenovirus vector vaccine composition.

[0053] More preferably, the recombinant protein vaccine comprises a protein or precursor for preventing and / or treating infection by the SARS-CoV-2 omicron XBB submutant. The amino acid sequence of the protein or precursor is selected from at least one of SEQ ID NOs: 1 to 11, SEQ ID NOs: 55, SEQ ID NOs: 19 to 29, and SEQ ID NOs: 56. Preferably, the amino acid sequence of the protein or precursor is selected from at least one of SEQ ID NOs: 1, SEQ ID NOs: 5, SEQ ID NOs: 8 to 11, and SEQ ID NOs: 55 or SEQ ID NOs: 19, SEQ ID NOs: 23, SEQ ID NOs: 26 to 29, and SEQ ID NOs: 56. The recombinant protein vaccine composition is a combination of any two or more recombinant protein vaccines using different proteins.

[0054] The adenovirus vector vaccine contains at least one polynucleotide sequence shown in SEQ ID NOs. 48 to 54. Preferably, the adenovirus vector vaccine contains at least one polynucleotide sequence shown in SEQ ID NOs. 48 to 51.

[0055] An adenovirus vector vaccine composition refers to a combination of two or more adenovirus vector vaccines prepared using different polynucleotide sequences.

[0056] Most preferably, the recombinant protein vaccine comprises at least one amino acid sequence shown in SEQ ID NO: 1, SEQ ID NO: 5, SEQ ID NOs: 8-11, and SEQ ID NO: 55.

[0057] The adenovirus vector vaccine contains at least one polynucleotide sequence as shown in SEQ ID NOs. 48 to 51.

[0058] Furthermore, the pharmaceutical composition or concomitant drug is formulated as an intradermal or subcutaneous injection preparation, an intramuscular injection preparation, an intravenous injection preparation, an oral preparation, or a nasal spray preparation. Preferably, the pharmaceutical composition or concomitant drug is formulated as an intramuscular injection preparation and a nasal spray preparation.

[0059] The present invention further provides the use of proteins, precursors, protein compositions, recombinant protein vaccines or compositions thereof, adenovirus vector vaccines or compositions thereof, and pharmaceutical compositions or combination drugs in the preparation of drugs for treating and / or preventing infections or pathogenicity caused by SARS-CoV-2 or its variants.

[0060] Preferably, SARS-CoV-2 variants include SARS-CoV-2 variants alpha, beta, gamma, delta, omicron, and omicron subvariants 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.

[0061] The following sequences are constructed by the applicant based on the RBD and RBD-HR sequences in the S protein and optimized full-length S protein of SARS-CoV-2 omicron submutants XBB.1.16, XBB.2, XBB.1.5, XBB.2.3, XBB.1.16.6, BA.2.86, EG5, and JN.1.

[0062] RBD-HR sequence of the optimized S protein of Sequence ID No. 1, XBB.1.16

[0063] VQPTESIVRFPNITNLCPHEVFNATTFASVYAWNRKRISNCVADYSVIYNFAPFFAFKCYGVSPTKLNDLCFTNVYADSFVIRGNEVSQIAPGQTGNIADYNYKLPDDFTGCVIAWNSNKLDSKPSGNYNYLYRLFRKSKLKPFERDISTEIYQAGNRPCN GVAGPNCYSPLQSYGFRPTYGVGHQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKSVNFNGLYENQKLIANQFNSAIGKIQDSLSSTASALGKLQDVVNQNAQALNTLVKQLKNHTSPDVDLGDISGINASVVNIQKEIDRLNEVAKNLNESLIDLQEL

[0064] Sequence ID 2, RBD-HR sequence of the optimized S protein of XBB.2

[0065] VQPTESIVRFPNITNLCPHEVFNATTFASVYAWNRKRISNCVADYSVIYNFAPFFAFKCYGVSPTKLNDLCFTNVYADSFVIRGNEVSQIAPGQTGNIADYNYKLPDDFTGCVIAWNSNKLDSKPSGNYNYLYRLFRKSKLKPFERDISTEIYQAGNKPCN GVAGSNCYSPLQSYGFRPTYGVGHQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKSVNFNGLYENQKLIANQFNSAIGKIQDSLSSTASALGKLQDVVNQNAQALNTLVKQLKNHTSPDVDLGDISGINASVVNIQKEIDRLNEVAKNLNESLIDLQEL

[0066] RBD sequence of the optimized S protein of Sequence ID No. 3, XBB.1.5-1

[0067] VQPTESIVRFPNITNLCPFHEVFNATTFASVYAWNRKRISNCVADYSVIYNFAPFFAFKCYGVSPTKLNDLCFTNVYADSFVIRGNEVSQIAPGQTGNIADYNYKLPDDFTGC VIAWNSNKLDSKPSGNYNYLYRLFRKSKLKPFERDISTEIYQAGNKPCNGVAGPNCYSPLQSYGFRPTYGVGHQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKCVNFNFNG

[0068] RBD sequence of the optimized S protein XBB.1.5-2, SEQ ID NO: 4.

[0069] VQPTESIVRFPNITNLCPFHEVFNATTFASVYAWNRKRISNCVADYSVIYNSASFSAFKCYGVSPTKLNDLCFTNVYADSFVIRGNEVSQIAPGQTGNIADYNYKLPDDFTGC VIAWNSNKLDSKPSGNYNYLYRLFRKSKLKPFERDISTEIYQAGNKPCNGVAGPNCYSPLQSYGFRPTYGVGHQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKCVNFNFNG

[0070] RBD-HR sequence of the optimized S protein XBB.1.5-3, SEQ ID NO: 5.

[0071] VQPTESIVRFPNITNLCPHEVFNATTFASVYAWNRKRISNCVADYSVIYNFAPFFAFKCYGVSPTKLNDLCFTNVYADSFVIRGNEVSQIAPGQTGNIADYNYKLPDDFTGCVIAWNSNKLDSKPSGNYNYLYRLFRKSKLKPFERDISTEIYQAGNKPCN GVAGPNCYSPLQSYGFRPTYGVGHQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKSVNFNGLYENQKLIANQFNSAIGKIQDSLSSTASALGKLQDVVNQNAQALNTLVKQLKNHTSPDVDLGDISGINASVVNIQKEIDRLNEVAKNLNESLIDLQEL

[0072] RBD-HR sequence of the optimized S protein XBB.1.5-4, SEQ ID NO: 6.

[0073] VQPTESIVRFPNITNLCPHEVFNATTFASVYAWNRKRISNCVADYSVIYNSASFSAFKCYGVSPTKLNDLCFTNVYADSFVIRGNEVSQIAPGQTGNIADYNYKLPDDFTGCVIAWNSNKLDSKPSGNYNYLYRLFRKSKLKPFERDISTEIYQAGNKPCN GVAGPNCYSPLQSYGFRPTYGVGHQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKSVNFNGLYENQKLIANQFNSAIGKIQDSLSSTASALGKLQDVVNQNAQALNTLVKQLKNHTSPDVDLGDISGINASVVNIQKEIDRLNEVAKNLNESLIDLQEL

[0074] RBD sequence of the optimized S protein of Sequence ID No. 7, XBB.2.3-1

[0075] VQPTESIVRFPNITNLCPFHEVFNATTFASVYAWNRKRISNCVADYSVIYNFAPFFAFKCYGVSPTKLNDLCFTNVYADSFVIRGNEVSQIAPGQTGNIADYNYKLPDDFTGC VIAWNSNKLDSKPSGNYNYLYRLFRKSKLKPFERDISTEIYQAGNKPCNGVAGPNCYSPLQSYGFRPTYGVGHQPYRVVVLSFELLHASATVCGPKKSTNLVKNKCVNFNFNG

[0076] RBD-HR sequence of the optimized S protein of Sequence ID No. 8, XBB.2.3-2

[0077] VQPTESIVRFPNITNLCPHEVFNATTFASVYAWNRKRISNCVADYSVIYNFAPFFAFKCYGVSPTKLNDLCFTNVYADSFVIRGNEVSQIAPGQTGNIADYNYKLPDDFTGCVIAWNSNKLDSKPSGNYNYLYRLFRKSKLKPFERDISTEIYQAGNKPCN GVAGPNCYSPLQSYGFRPTYGVGHQPYRVVVLSFELLHASATVCGPKKSTNLVKNKSVNFNGLYENQKLIANQFNSAIGKIQDSLSSTASALGKLQDVVNQNAQALNTLVKQLKNHTSPDVDLGDISGINASVVNIQKEIDRLNEVAKNLNESLIDLQEL

[0078] RBD-HR sequence of the optimized S protein of Sequence ID No. 9, XBB.1.16.6

[0079] VQPTESIVRFPNITNLCPHEVFNATTFASVYAWNRKRISNCVADYSVIYNFAPFFAFKCYGVSPTKLNDLCFTNVYADSFVIRGNEVSQIAPGQTGNIADYNYKLPDDFTGCVIAWNSNKLDSKPSGNYNYLYRLLRKSKLKPFERDISTEIYQAGNRPCN GVAGPNCYSPLQSYGFRPTYGVGHQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKSVNFNGLYENQKLIANQFNSAIGKIQDSLSSTASALGKLQDVVNQNAQALNTLVKQLKNHTSPDVDLGDISGINASVVNIQKEIDRLNEVAKNLNESLIDLQEL

[0080] RBD-HR sequence of the optimized S protein of SEQ ID NO: 10, BA.2.86

[0081] VQPTESIVRFPNVTNLCPFHEVFNATRFASVYAWNRTRISNCVADYSVLYNFAPFFAFKCYGVSPTKLNDLCFTNVYADSFVIKGNEVSQIAPGQTGNIADYNYKLPDDFTGCVIAWNSNKLDSKHSGNYDYWYRLFRKSKLKPFERDISTEIYQAGNKPC KGKGPNCYFPLQSYGFRPTYGVGHQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKSVNFNFNGLYENQKLIANQFNSAIGKIQDSLSSTASALGKLQDVVNQNAQALNTLVKQLKNHTSPDVDLGDISGINASVVNIQKEIDRLNEVAKNLNESLIDLQEL

[0082] RBD-HR sequence of the optimized S protein of SEQ ID NO: 11, EG.5

[0083] VQPTESIVRFPNITNLCPHEVFNATTFASVYAWNRKRISNCVADYSVIYNFAPFFAFKCYGVSPTKLNDLCFTNVYADSFVIRGNEVSQIAPGQTGNIADYNYKLPDDFTGCVIAWNSNKLDSKPSGNYNYLYRLLRKSKLKPFERDISTEIYQAGNKPCN GVAGPNCYSPLQSYGFRPTYGVGHQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKSVNFNGLYENQKLIANQFNSAIGKIQDSLSSTASALGKLQDVVNQNAQALNTLVKQLKNHTSPDVDLGDISGINASVVNIQKEIDRLNEVAKNLNESLIDLQEL

[0084] Sequence ID 12, optimized full-length S protein sequence of XBB.1.16, excluding the signal peptide.

[0085]

[0086] Sequence ID No. 13, optimized full-length S protein sequence of XBB.2, excluding the signal peptide.

[0087]

[0088] Sequence ID No. 14, optimized full-length S protein sequence of XBB.1.5, excluding the signal peptide.

[0089]

[0090] Sequence ID No. 15, optimized full-length S protein sequence of XBB.2.3, excluding the signal peptide.

[0091]

[0092] Sequence ID No. 16, optimized full-length S protein sequence of XBB.1.6, excluding the signal peptide.

[0093] Sequence ID No. 16 is a variant obtained by at least one amino acid sequence substitution and / or deletion and / or insertion in the amino acid sequence of the full-length S protein of XBB.1.6.

[0094] Sequence ID 17, optimized full-length S protein sequence of XBB.1.9.1, excluding the signal peptide.

[0095] Sequence ID No. 17 is a variant obtained by at least one amino acid sequence substitution and / or deletion and / or insertion in the amino acid sequence of the full-length S protein of XBB.1.9.1.

[0096] Sequence ID No. 18, optimized full-length S protein sequence of EG.5, excluding the signal peptide.

[0097] Sequence ID No. 18 is a variant obtained by at least one amino acid sequence substitution and / or deletion and / or insertion in the amino acid sequence of the full-length S protein of EG5.

[0098] To support protein secretion and expression, a signal peptide is added to the amino acids when the protein is constructed. Furthermore, a His tag can be added to the amino acid sequence of the protein to facilitate purification. The present invention relates to optimized protein expression constructed based on the S proteins XBB.1.16, XBB.2, XBB.1.5, XBB.2.3, XBB.1.16.6, BA.2.86, and EG5, the construction and design sequences of which are shown in SEQ ID NOs. 19 to 29.

[0099] Furthermore, the corresponding nucleotide sequences that encode the amino acid sequences are shown as SEQ ID NOs. 37 to 47.

[0100] Sequence ID 19, an optimized and designed S protein-RBD-HR based sequence of Omicron_XBB.1.16, including signal peptide-Trx tag-6His tag-EK digestion site-RBD sequence-HR1 sequence-HR2 sequence

[0101] MLLVNQSHQGFNKEHTSKMVSAIVLYVLLAAAAHSAFAADSIHIKDSDDLKNRLAEAGDKLVVIDFMATWCGPCKMIGPKLDEMANEMSDSIVVLKVDVDECEDIATEYNINSMPTFVFVKNS KKIEEFSGANVDKLRNTIIKLKLAGSGSGHMHHHHHHSSGDDDDKVQPTESIVRFPNITNLCPFHEVFNATTFASVYAWNRKRISNCVADYSVIYNFAPFFAFKCYGVSPTKLNDLCFTNVYA DSFVIRGNEVSQIAPGQTGNIADYNYKLPDDFTGCVIAWNSNKLDSKPSGNYNYLYRLFRKSKLKPFERDISTEIYQAGNRPCNGVAGPNCYSPLQSYGFRPTYGVGHQPYRVVVLSFELLHA PATVCGPKKSTNLVKNKSVNFNFNGLYENQKLIANQFNSAIGKIQDSLSSTASALGKLQDVVNQNAQALNTLVKQLKNHTSPDVDLGDISGINASVVNIQKEIDRLNEVAKNLNESLIDLQEL

[0102] Sequence ID 20, an optimized and designed S protein-RBD-HR based sequence of Omicron_XBB.2, including signal peptide-Trx tag-6His tag-EK digestion site-RBD sequence-HR1 sequence-HR2 sequence

[0103] MLLVNQSHQGFNKEHTSKMVSAIVLYVLLAAAAHSAFAADSIHIKDSDDLKNRLAEAGDKLVVIDFMATWCGPCKMIGPKLDEMANEMSDSIVVLKVDVDECEDIATEYNINSMPTFVFVKNS KKIEEFSGANVDKLRNTIIKLKLAGSGSGHMHHHHHHSSGDDDDKVQPTESIVRFPNITNLCPFHEVFNATTFASVYAWNRKRISNCVADYSVIYNFAPFFAFKCYGVSPTKLNDLCFTNVYA DSFVIRGNEVSQIAPGQTGNIADYNYKLPDDFTGCVIAWNSNKLDSKPSGNYNYLYRLFRKSKLKPFERDISTEIYQAGNKPCNGVAGSNCYSPLQSYGFRPTYGVGHQPYRVVVLSFELLHA PATVCGPKKSTNLVKNKSVNFNFNGLYENQKLIANQFNSAIGKIQDSLSSTASALGKLQDVVNQNAQALNTLVKQLKNHTSPDVDLGDISGINASVVNIQKEIDRLNEVAKNLNESLIDLQEL

[0104] Sequence ID 21, an optimized and designed S protein-RBD-based sequence of Omicron_XBB.1.5-1, containing a signal peptide-Trx tag-6His tag-EK digestion site-RBD sequence.

[0105] MLLVNQSHQGFNKEHTSKMVSAIVLYVLLAAAAHSAFAADSIHIKDSDDLKNRLAEAGDKLVVIDFMATWCGPCKMIGPKLDEMANEMSDCIVVLKVDVDECEDIATEYNINSMPTFVFVKNSKKIEEFSGANVDKLRNTIIKLKLAGSGSGHMHHHHHHSSGDDDDKVQPTESIVRFPNITNLCPFHEVFNATTFA SVYAWNRKRISNCVADYSVIYNFAPFFAFKCYGVSPTKLNDLCFTNVYADSFVIRGNEVSQIAPGQTGNIADYNYKLPDDFTGCVIAWNSNKLDSKPSGNYNYLYRLFRKSKLKPFERDISTEIYQAGNKPCNGVAGPNCYSPLQSYGFRPTYGVGHQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKCVNFNFNG

[0106] Sequence ID 22, an optimized and designed S protein-RBD-based sequence for Omicron_XBB.1.5-2, containing a signal peptide-Trx tag-6His tag-EK digestion site-RBD sequence.

[0107] MLLVNQSHQGFNKEHTSKMVSAIVLYVLLAAAAHSAFAADSIHIKDSDDLKNRLAEAGDKLVVIDFMATWCGPCKMIGPKLDEMANEMSDCIVVLKVDVDECEDIATEYNINSMPTFVFVKNSKKIEEFSGANVDKLRNTIIKLKLAGSGSGHMHHHHHHSSGDDDDKVQPTESIVRFPNITNLCPFHEVFNATTFA SVYAWNRKRISNCVADYSVIYNSASFSAFKCYGVSPTKLNDLCFTNVYADSFVIRGNEVSQIAPGQTGNIADYNYKLPDDFTGCVIAWNSNKLDSKPSGNYNYLYRLFRKSKLKPFERDISTEIYQAGNKPCNGVAGPNCYSPLQSYGFRPTYGVGHQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKCVNFNFNG

[0108] Sequence ID 23, an optimized and designed S protein-RBD-HR based sequence of Omicron_XBB.1.5-3, including signal peptide-Trx tag-6His tag-EK digestion site-RBD sequence-HR1 sequence-HR2 sequence.

[0109] MLLVNQSHQGFNKEHTSKMVSAIVLYVLLAAAAHSAFAADSIHIKDSDDLKNRLAEAGDKLVVIDFMATWCGPCKMIGPKLDEMANEMSDSIVVLKVDVDECEDIATEYNINSMPTFVFVKNS KKIEEFSGANVDKLRNTIIKLKLAGSGSGHMHHHHHHSSGDDDDKVQPTESIVRFPNITNLCPFHEVFNATTFASVYAWNRKRISNCVADYSVIYNFAPFFAFKCYGVSPTKLNDLCFTNVYA DSFVIRGNEVSQIAPGQTGNIADYNYKLPDDFTGCVIAWNSNKLDSKPSGNYNYLYRLFRKSKLKPFERDISTEIYQAGNKPCNGVAGPNCYSPLQSYGFRPTYGVGHQPYRVVVLSFELLHA PATVCGPKKSTNLVKNKSVNFNFNGLYENQKLIANQFNSAIGKIQDSLSSTASALGKLQDVVNQNAQALNTLVKQLKNHTSPDVDLGDISGINASVVNIQKEIDRLNEVAKNLNESLIDLQEL

[0110] Sequence ID 24, an optimized and designed S protein-RBD-HR based sequence of Omicron_XBB.1.5-4, containing signal peptide-Trx tag-6His tag-EK digestion site-RBD sequence-HR1 sequence-HR2 sequence

[0111] MLLVNQSHQGFNKEHTSKMVSAIVLYVLLAAAAHSAFAADSIHIKDSDDLKNRLAEAGDKLVVIDFMATWCGPCKMIGPKLDEMANEMSDSIVVLKVDVDECEDIATEYNINSMPTFVFVKNS KKIEEFSGANVDKLRNTIIKLKLAGSGSGHMHHHHHHSSGDDDDKVQPTESIVRFPNITNLCPFHEVFNATTFASVYAWNRKRISNCVADYSVIYNSASFSAFKCYGVSPTKLNDLCFTNVYA DSFVIRGNEVSQIAPGQTGNIADYNYKLPDDFTGCVIAWNSNKLDSKPSGNYNYLYRLFRKSKLKPFERDISTEIYQAGNKPCNGVAGPNCYSPLQSYGFRPTYGVGHQPYRVVVLSFELLHA PATVCGPKKSTNLVKNKSVNFNFNGLYENQKLIANQFNSAIGKIQDSLSSTASALGKLQDVVNQNAQALNTLVKQLKNHTSPDVDLGDISGINASVVNIQKEIDRLNEVAKNLNESLIDLQEL

[0112] Sequence ID 25, an optimized and designed S protein-RBD-based sequence of Omicron_XBB.2.3-1, containing a signal peptide-Trx tag-6His tag-EK digestion site-RBD sequence.

[0113] MLLVNQSHQGFNKEHTSKMVSAIVLYVLLAAAAHSAFAADSIHIKDSDDLKNRLAEAGDKLVVIDFMATWCGPCKMIGPKLDEMANEMSDCIVVLKVDVDECEDIATEYNINSMPTFVFVKNSKKIEEFSGANVDKLRNTIIKLKLAGSGSGHMHHHHHHSSGDDDDKVQPTESIVRFPNITNLCPFHEVFNATTFA SVYAWNRKRISNCVADYSVIYNFAPFFAFKCYGVSPTKLNDLCFTNVYADSFVIRGNEVSQIAPGQTGNIADYNYKLPDDFTGCVIAWNSNKLDSKPSGNYNYLYRLFRKSKLKPFERDISTEIYQAGNKPCNGVAGPNCYSPLQSYGFRPTYGVGHQPYRVVVLSFELLHASATVCGPKKSTNLVKNKCVNFNFNG

[0114] Sequence ID 26, an optimized and designed S protein-RBD-HR based sequence of Omicron_XBB.2.3-2, including signal peptide-Trx tag-6His tag-EK digestion site-RBD sequence-HR1 sequence-HR2 sequence

[0115] MLLVNQSHQGFNKEHTSKMVSAIVLYVLLAAAAHSAFAADSIHIKDSDDLKNRLAEAGDKLVVIDFMATWCGPCKMIGPKLDEMANEMSDSIVVLKVDVDECEDIATEYNINSMPTFVFVKNS KKIEEFSGANVDKLRNTIIKLKLAGSGSGHMHHHHHHSSGDDDDKVQPTESIVRFPNITNLCPFHEVFNATTFASVYAWNRKRISNCVADYSVIYNFAPFFAFKCYGVSPTKLNDLCFTNVYA DSFVIRGNEVSQIAPGQTGNIADYNYKLPDDFTGCVIAWNSNKLDSKPSGNYNYLYRLFRKSKLKPFERDISTEIYQAGNKPCNGVAGPNCYSPLQSYGFRPTYGVGHQPYRVVVLSFELLHA SATVCGPKKSTNLVKNKSVNFNFNGLYENQKLIANQFNSAIGKIQDSLSSTASALGKLQDVVNQNAQALNTLVKQLKNHTSPDVDLGDISGINASVVNIQKEIDRLNEVAKNLNESLIDLQEL

[0116] Sequence ID 27, an optimized and designed S protein-RBD-HR based sequence of Omicron_XBB.1.16.6, including signal peptide-Trx tag-6His tag-EK digestion site-RBD sequence-HR1 sequence-HR2 sequence

[0117] MLLVNQSHQGFNKEHTSKMVSAIVLYVLLAAAAHSAFAADSIHIKDSDDLKNRLAEAGDKLVVIDFMATWCGPCKMIGPKLDEMANEMSDSIVVLKVDVDECEDIATEYNINSMPTFVFVKNS KKIEEFSGANVDKLRNTIIKLKLAGSGSGHMHHHHHHSSGDDDDKVQPTESIVRFPNITNLCPFHEVFNATTFASVYAWNRKRISNCVADYSVIYNFAPFFAFKCYGVSPTKLNDLCFTNVYA DSFVIRGNEVSQIAPGQTGNIADYNYKLPDDFTGCVIAWNSNKLDSKPSGNYNYLYRLLRKSKLKPFERDISTEIYQAGNRPCNGVAGPNCYSPLQSYGFRPTYGVGHQPYRVVVLSFELLHA PATVCGPKKSTNLVKNKSVNFNFNGLYENQKLIANQFNSAIGKIQDSLSSTASALGKLQDVVNQNAQALNTLVKQLKNHTSPDVDLGDISGINASVVNIQKEIDRLNEVAKNLNESLIDLQEL

[0118] Sequence ID 28, an optimized and designed S protein-RBD-HR based sequence of Omicron_BA.2.86, including signal peptide-Trx tag-6His tag-EK digestion site-RBD sequence-HR1 sequence-HR2 sequence

[0119] MLLVNQSHQGFNKEHTSKMVSAIVLYVLLAAAAHSAFAADSIHIKDSDDLKNRLAEAGDKLVVIDFMATWCGPCKMIGPKLDEMANEMSDSIVVLKVDVDECEDIATEYNINSMPTFVFVKN SKKIEEFSGANVDKLRNTIIKLKLAGSGSGHMHHHHHHSSGDDDDKVQPTESIVRFPNVTNLCPFHEVFNATRFASVYAWNRTRISNCVADYSVLYNFAPFFAFKCYGVSPTKLNDLCFTNVY ADSFVIKGNEVSQIAPGQTGNIADYNYKLPDDFTGCVIAWNSNKLDSKHSGNYDYWYRLFRKSKLKPFERDISTEIYQAGNKPCKGKGPNCYFPLQSYGFRPTYGVGHQPYRVVVLSFELLHA PATVCGPKKSTNLVKNKSVNFNFNGLYENQKLIANQFNSAIGKIQDSLSSTASALGKLQDVVNQNAQALNTLVKQLKNHTSPDVDLGDISGINASVVNIQKEIDRLNEVAKNLNESLIDLQEL

[0120] Sequence ID 29, an optimized and designed S protein-RBD-HR based sequence of Omicron_EG.5, including signal peptide-Trx tag-6His tag-EK digestion site-RBD sequence-HR1 sequence-HR2 sequence.

[0121] MLLVNQSHQGFNKEHTSKMVSAIVLYVLLAAAAHSAFAADSIHIKDSDDLKNRLAEAGDKLVVIDFMATWCGPCKMIGPKLDEMANEMSDSIVVLKVDVDECEDIATEYNINSMPTFVFVKNS KKIEEFSGANVDKLRNTIIKLKLAGSGSGHMHHHHHHSSGDDDDKVQPTESIVRFPNITNLCPFHEVFNATTFASVYAWNRKRISNCVADYSVIYNFAPFFAFKCYGVSPTKLNDLCFTNVYA DSFVIRGNEVSQIAPGQTGNIADYNYKLPDDFTGCVIAWNSNKLDSKPSGNYNYLYRLLRKSKLKPFERDISTEIYQAGNKPCNGVAGPNCYSPLQSYGFRPTYGVGHQPYRVVVLSFELLHA PATVCGPKKSTNLVKNKSVNFNFNGLYENQKLIANQFNSAIGKIQDSLSSTASALGKLQDVVNQNAQALNTLVKQLKNHTSPDVDLGDISGINASVVNIQKEIDRLNEVAKNLNESLIDLQEL

[0122] Sequence ID No. 30, an optimized and designed full-length S protein-based sequence (including native signal peptide) that serves as the amino acid sequence for the S antigen in the Ad-S-XBB.1.16 vaccine.

[0123]

[0124] Sequence ID No. 31, an optimized and designed full-length S protein-based sequence (including native signal peptide) that serves as the amino acid sequence for the S antigen in the Ad-S-XBB.2 vaccine.

[0125]

[0126] Sequence ID No. 32, an optimized and designed full-length S protein-based sequence (including native signal peptide) of Omicron_XBB.1.5, which serves as the amino acid sequence for the S antigen in the Ad-S-XBB.1.5 vaccine.

[0127]

[0128] Sequence ID 33, an optimized and designed full-length S protein-based sequence (including native signal peptide) that serves as the amino acid sequence for the S antigen in the Ad-S-XBB.2.3 vaccine.

[0129]

[0130] Sequence ID No. 34, an optimized and designed full-length S protein sequence (including native signal peptide) based on Omicron_XBB.1.6, which serves as the amino acid sequence for the S antigen in the Ad-S-XBB.1.6 vaccine.

[0131] Sequence ID 35, an optimized and designed full-length S protein-based sequence (including native signal peptide) that serves as the amino acid sequence for the S antigen in the Ad-S-XBB.1.9.1 vaccine.

[0132] Sequence ID No. 36, an optimized and designed full-length S protein-based sequence (including a native signal peptide) that serves as the amino acid sequence for the S antigen in the Ad-S-EG5 vaccine.

[0133] Sequence ID 37, the nucleotide sequence encoding the optimized and designed S protein-RBD-HR based sequence of Omicron_XBB.1.16, and the nucleotide sequence encoding Sequence ID 19.

[0134]

[0135] Sequence ID 38, the nucleotide sequence encoding the optimized and designed S protein-RBD-HR based sequence of Omicron_XBB.2, and the nucleotide sequence encoding Sequence ID 20.

[0136]

[0137] Nucleotide sequences encoding sequence number 39, the optimized and designed S protein-RBD-HR based sequence of Omicron_XBB.1.5-1, and nucleotide sequences encoding sequence number 21.

[0138]

[0139] Nucleotide sequences encoding sequence number 40, the optimized and designed S protein-RBD-HR based sequence of Omicron_XBB.1.5-2, and nucleotide sequences encoding sequence number 22.

[0140]

[0141] Nucleotide sequences encoding sequence number 41, the optimized and designed S protein-RBD-HR based sequence of Omicron_XBB.1.5-3, and nucleotide sequences encoding sequence number 23.

[0142]

[0143] Nucleotide sequences encoding sequence number 42, the optimized and designed S protein-RBD-HR based sequence of Omicron_XBB.1.5-4, and nucleotide sequences encoding sequence number 24

[0144]

[0145] Nucleotide sequences encoding sequence number 43, the optimized and designed S protein-RBD-based sequence of Omicron_XBB.2.3-1, and nucleotide sequences encoding sequence number 25.

[0146]

[0147] Nucleotide sequences encoding sequence number 44, the optimized and designed S protein-RBD-HR based sequence of Omicron_XBB.2.3-2, and nucleotide sequences encoding sequence number 26.

[0148]

[0149] Nucleotide sequences encoding sequence number 45, the optimized and designed S protein-RBD-HR based sequence of Omicron_XBB.1.16.6, and nucleotide sequences encoding sequence number 27

[0150]

[0151] Nucleotide sequences encoding sequence number 46, the optimized and designed S protein-RBD-HR based sequence of Omicron_BA.2.86, and nucleotide sequences encoding sequence number 28.

[0152]

[0153] Sequence ID 47, the nucleotide sequence encoding the optimized and designed S protein-RBD-HR based sequence of Omicron_EG.5, and the nucleotide sequence encoding Sequence ID 29.

[0154]

[0155] Sequence ID 48, a nucleotide sequence encoding the sequence based on the optimized and designed full-length S protein of Omicron_XBB.1.16 (containing a natural signal peptide, which serves as the nucleotide sequence for the S antigen in the Ad-S-XBB.1.16 vaccine), and the nucleotide sequence encoding Sequence ID 30.

[0156]

[0157] Sequence ID 49, a nucleotide sequence encoding the sequence based on the optimized and designed full-length S protein of Omicron_XBB.2 (containing a natural signal peptide, which serves as the nucleotide sequence for the S antigen in the Ad-S-XBB.2 vaccine), and the nucleotide sequence encoding Sequence ID 31.

[0158]

[0159] Sequence ID 50, a nucleotide sequence encoding a sequence based on the optimized and designed full-length S protein of Omicron_XBB.1.5 (containing a natural signal peptide, which serves as the nucleotide sequence of the S antigen for the Ad-S-XBB.1.5 vaccine), and the nucleotide sequence encoding Sequence ID 32.

[0160]

[0161] Sequence ID 51, a nucleotide sequence encoding the sequence based on the optimized and designed full-length S protein of Omicron_XBB.2.3 (containing a natural signal peptide and serving as the nucleotide sequence of the S antigen for the Ad-S-XBB.2.3 vaccine), and the nucleotide sequence encoding Sequence ID 33.

[0162]

[0163] Sequence ID 52, a nucleotide sequence that includes a natural signal peptide and encodes a sequence based on the optimized and designed full-length S protein of Omicron_XBB.1.6, which serves as the nucleotide sequence for the S antigen in the Ad-S-XBB.1.6 vaccine.

[0164] Sequence ID 53, a nucleotide sequence that contains a natural signal peptide and encodes a sequence based on the optimized and designed full-length S protein of Omicron_XBB.1.9.1, which serves as the nucleotide sequence for the S antigen in the Ad-S-XBB.1.9.1 vaccine.

[0165] Sequence ID No. 54, a nucleotide sequence that contains a natural signal peptide and encodes a sequence based on the optimized and designed full-length S protein of Omicron_EG.5, which serves as the nucleotide sequence for the S antigen in the Ad-S-EG5 vaccine.

[0166] RBD-HR sequence of the optimized S protein of Sequence ID No. 55, JN.1

[0167] VQPTESIVRFPNVTNLCPFHEVFNATRFASVYAWNRTRISNCVADYSVLYNFAPFFAFKCYGVSPTKLNDLCFTNVYADSFVIKGNEVSQIAPGQTGNIADYNYKLPDDFTGCVIAWNSNKLDSKHSGNYDYWYRSFRKSKLKPFERDISTEIYQAGNKPC KGKGPNCYFPLQSYGFRPTYGVGHQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKSVNFNFNGLYENQKLIANQFNSAIGKIQDSLSSTASALGKLQDVVNQNAQALNTLVKQLKNHTSPDVDLGDISGINASVVNIQKEIDRLNEVAKNLNESLIDLQEL

[0168] Sequence ID 56, an optimized and designed S protein-RBD-HR based sequence of Omicron JN.1, including signal peptide-Trx tag-6His tag-EK digestion site-RBD sequence-HR1 sequence-HR2 sequence.

[0169] MLLVNQSHQGFNKEHTSKMVSAIVLYVLLAAAAHSAFAADSIHIKDSDDLKNRLAEAGDKLVVIDFMATWCGPCKMIGPKLDEMANEMSDSIVVLKVDVDECEDIATEYNINSMPTFVFVKN SKKIEEFSGANVDKLRNTIIKLKLAGSGSGHMHHHHHHSSGDDDDKVQPTESIVRFPNVTNLCPFHEVFNATRFASVYAWNRTRISNCVADYSVLYNFAPFFAFKCYGVSPTKLNDLCFTNVY ADSFVIKGNEVSQIAPGQTGNIADYNYKLPDDFTGCVIAWNSNKLDSKHSGNYDYWYRSFRKSKLKPFERDISTEIYQAGNKPCKGKGPNCYFPLQSYGFRPTYGVGHQPYRVVVLSFELLHA PATVCGPKKSTNLVKNKSVNFNFNGLYENQKLIANQFNSAIGKIQDSLSSTASALGKLQDVVNQNAQALNTLVKQLKNHTSPDVDLGDISGINASVVNIQKEIDRLNEVAKNLNESLIDLQEL

[0170] Sequence ID 57, the nucleotide sequence encoding the optimized and designed S protein-RBD-HR based sequence of Omicron_JN.1, and the nucleotide sequence encoding Sequence ID 56.

[0171]

[0172] Beneficial Effects: In this invention, recombinant protein vaccines and adenovirus vector vaccines are prepared, and the protein antigens of the recombinant protein vaccines are constructed and expressed based on the RBD sequence (aa320~545) of the S protein or the HR1 and HR2 sequences of RBD-HR 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. Furthermore, after adding the corresponding vaccine adjuvant to the prepared protein antigen, it can better help the host resist cross-infection caused by omicron XBB variants and their subvariants, which is crucial for the development of recombinant protein vaccines against SARS-CoV-2 omicron XBB variants and their subvariants. Animal studies have demonstrated that polyvaccines formed by combining different recombinant protein antigens or vaccines show enhanced efficacy against omicron XBB variants and their subvariants.

[0173] Furthermore, recombinant adenovirus vectors are constructed through codon optimization based on nucleotide sequences encoding the full-length S proteins of XBB.1.16, XBB.2, XBB.1.5, and XBB.2.3 to obtain adenovirus vector vaccines. These adenovirus vector vaccines are combined with recombinant protein vaccines to develop a multivalent vaccine with enhanced prophylactic and therapeutic efficacy against SARS-CoV-2 and its variants. The prepared nasal sprays exhibit superior protection against infection by SARS-CoV-2 and Omicron subvariants, including but not limited to 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, and KP.3. [Brief explanation of the drawing]

[0174] [Figure 1] The construction and expression of the recombinant protein antigen RBDXBB.1.16-HR of Embodiment 1 are shown. Hereinafter, a. schematic design diagram of pFastBac1-GP67-Trx-His-EK-S-RBD(Omicron_XBB.1.16)-HR, b. 1% agarose gel electrophoresis of the cloned PCR product, c. 1% agarose gel electrophoresis of the PCR-amplified recombinant bacmid, d. packaging and proliferation flowchart of the recombinant baculovirus, and e. WB validation results during baculovirus proliferation. [Figure 2] The expression and purification results of the recombinant protein antigen RBDXBB.1.16-HR in Embodiment 1 are shown. Here, a. Elution profile by Ni affinity chromatography from baculovirus-infected supernatant, b. Verification of EK digestion efficiency for EK digested samples, and c-d. Purification of the target protein using Ni affinity chromatography resin after EK digestion (d) and SDS-PAGE analysis (c). [Figure 3] The construction and expression of the recombinant protein antigen RBDXBB.1.5-3-HR of Embodiment 1 are shown. Hereinafter, a. schematic design diagram of pFastBac1-GP67-Trx-His-EK-S-RBD(Omicron_XBB.1.5-3)-HR, b. 1% agarose gel electrophoresis of the cloned PCR product, c. 1% agarose gel electrophoresis of PCR validation against bluish-white colony liquid, d. packaging and proliferation flowchart of the recombinant baculovirus, and e. WB validation results during baculovirus proliferation. [Figure 4] The expression and purification results of the recombinant protein antigen RBDXBB.1.5-3-HR in Embodiment 1 are shown. Here, a. Elution profile by Ni affinity chromatography from baculovirus-infected supernatant, b. Verification of EK digestion efficiency for EK-digested samples, and c. Purification of the target protein using Ni affinity chromatography resin after EK digestion. [Figure 5]The construction and expression of the recombinant protein antigen RBDXBB.2.3-HR of Embodiment 1 are shown. Hereinafter, a. schematic design diagram of pFastBac1-GP67-Trx-His-EK-S-RBD(Omicron_XBB.2.3)-HR, b. 1% agarose gel electrophoresis of the cloned PCR product, c. 1% agarose gel electrophoresis of PCR validation against bluish-white colony liquid, d. packaging and proliferation flowchart of recombinant baculovirus, and e. WB validation results during baculovirus proliferation. [Figure 6] The expression and purification results of the recombinant protein antigen RBDXBB.2.3-HR in Embodiment 1 are shown. Here, a. Elution profile by Ni affinity chromatography from baculovirus-infected supernatant, b. Verification of EK digestion efficiency for EK-digested samples, and c. Purification by size exclusion chromatography and SDS-PAGE analysis after EK digestion. [Figure 7] The construction and expression of the recombinant protein antigen RBDXBB.1.16.6-HR of Embodiment 1 are shown. Hereinafter, a. schematic design diagram of pFastBac1-GP67-Trx-His-EK-S-RBD(Omicron_XBB.1.16.6)-HR, b. 1% agarose gel electrophoresis of the cloned PCR product, c. 1% agarose gel electrophoresis of the PCR-amplified recombinant bacmid, d. packaging and proliferation flowchart of the recombinant baculovirus, and e. WB validation results during baculovirus proliferation. [Figure 8] The expression and purification results of the recombinant protein antigen RBDXBB.1.16.6-HR in Embodiment 1 are shown. Here, a. Elution profile by Ni affinity chromatography from baculovirus-infected supernatant, b. Verification of EK digestion efficiency for EK-digested samples, and c-d. Purification by size exclusion chromatography and SDS-PAGE analysis after EK digestion. [Figure 9]The construction and expression of the recombinant protein antigen RBDBA.2.86-HR of Embodiment 1 are shown. Hereinafter, a. schematic design diagram of pFastBac1-GP67-Trx-His-EK-S-RBD(Omicron_BA.2.86)-HR, b. 1% agarose gel electrophoresis of the cloned PCR product, c. 1% agarose gel electrophoresis of PCR validation against bluish-white colony liquid, d. packaging and proliferation flowchart of the recombinant baculovirus, and e. WB validation results during baculovirus proliferation. [Figure 10] The expression and purification results of the recombinant protein antigen RBDBA.2.86-HR in Embodiment 1 are shown. Here, a. Elution profile by Ni affinity chromatography from baculovirus-infected supernatant, b. Verification of EK digestion efficiency for EK digested samples, and c-d. Purification by size exclusion chromatography and SDS-PAGE analysis after EK digestion. [Figure 11] The construction and expression of the recombinant protein antigen RBDEG.5-HR of Embodiment 1 are shown. Hereinafter, a. schematic design diagram of pFastBac1-GP67-Trx-His-EK-S-RBD(EG.5)-HR, b. 1% agarose gel electrophoresis of the cloned PCR product, c. 1% agarose gel electrophoresis of the PCR-amplified recombinant bacmid, d. packaging and proliferation flowchart of the recombinant baculovirus, and e. WB validation results during baculovirus proliferation. [Figure 12] The expression and purification results of the recombinant protein antigen RBDEG.5-HR in Embodiment 1 are shown. Here, a. Elution profile by Ni affinity chromatography from baculovirus-infected supernatant, b. Verification of EK digestion efficiency for EK-digested samples, and c-d. Protein purification by size exclusion chromatography after EK digestion (d) and SDS-PAGE analysis (c). [Figure 13]The construction and expression of the recombinant protein antigen RBDJN.1-HR of Embodiment 1 are shown. Hereinafter, a. schematic design diagram of pFastBac1-GP67-Trx-His-EK-S-RBD(JN.1)-HR, b. 1% agarose gel electrophoresis of the cloned PCR product, c. 1% agarose gel electrophoresis of the PCR-amplified recombinant bacmid, d. packaging and proliferation flowchart of the recombinant baculovirus, and e. WB validation results during baculovirus proliferation. [Figure 14] The expression and purification results of the recombinant protein antigen RBDJN.1-HR in Embodiment 1 are shown. Here, a. Elution profile by Ni affinity chromatography from baculovirus-infected supernatant, b. Verification of EK digestion efficiency for EK-digested samples, and c. Purification by size exclusion chromatography and SDS-PAGE analysis after EK digestion. [Figure 15] The construction and expression of the recombinant protein antigen RBDXBB.1.5-1 of Embodiment 1 are shown. Hereinafter, a. schematic design diagram of pFastBac1-GP67-Trx-His-EK-S-RBD(XBB.1.5-1), b. 1% agarose gel electrophoresis of the cloned PCR product, c. 1% agarose gel electrophoresis of the PCR-amplified recombinant bacmid, d. packaging and proliferation flowchart of the recombinant baculovirus, and e. WB validation results during baculovirus proliferation. [Figure 16] The expression and purification results of the recombinant protein antigen RBDXBB.1.5-1 in Embodiment 1 are shown. Here, a. Elution profile by Ni affinity chromatography from baculovirus-infected supernatant, b. Verification of EK digestion efficiency for EK-digested samples, and c. Purification of the target protein using Ni affinity chromatography resin and SDS-PAGE analysis after EK digestion. [Figure 17]The construction and expression of the recombinant protein antigen RBDXBB.1.5-2 of Embodiment 1 are shown. Hereinafter, a. schematic design diagram of pFastBac1-GP67-Trx-His-EK-S-RBD(XBB.1.5-2), b. 1% agarose gel electrophoresis of the cloned PCR product, c. 1% agarose gel electrophoresis of the PCR-amplified recombinant bacmid, d. packaging and proliferation flowchart of the recombinant baculovirus, and e. WB validation results during baculovirus proliferation. [Figure 18] The expression and purification results of the recombinant protein antigen RBDXBB.1.5-2 in Embodiment 1 are shown. Here, a. Elution profile by Ni affinity chromatography from baculovirus-infected supernatant, b. Verification of EK digestion efficiency for EK-digested samples, and c-d. Purification of target protein using Ni affinity chromatography resin after EK digestion (c) and SDS-PAGE analysis (d). [Figure 19] The construction and expression of the recombinant protein antigen RBDXBB.1.5-4-HR of Embodiment 1 are shown. Hereinafter, a. schematic design diagram of pFastBac1-GP67-Trx-His-EK-S-RBD(XBB.1.5-4), b. 1% agarose gel electrophoresis of the cloned PCR product, c. 1% agarose gel electrophoresis of the PCR-amplified recombinant bacmid, d. packaging and proliferation flowchart of the recombinant baculovirus, and e. WB validation results during baculovirus proliferation. [Figure 20] The expression and purification results of the recombinant protein antigen RBDXBB.1.5-4-HR in Embodiment 1 are shown. Here, a. the elution profile of Ni affinity chromatography from baculovirus-infected supernatant, b. verification of EK digestion efficiency for EK-digested samples, and c. purification of the target protein result product of Ni affinity chromatography resin after EK digestion. [Figure 21]The construction and expression of the recombinant protein antigen RBDXBB.2.3 of Embodiment 1 are shown. Hereinafter, a. schematic design diagram of pFastBac1-GP67-Trx-His-EK-S-RBD(XBB.2.3), b. 1% agarose gel electrophoresis of the cloned PCR product, c. 1% agarose gel electrophoresis of PCR validation against bluish-white colony liquid, d. packaging and proliferation flowchart of recombinant baculovirus, and e. WB validation results during baculovirus proliferation. [Figure 22] The expression and purification results of the recombinant protein antigen RBDXBB.2.3 in Embodiment 1 are shown. Here, a. Elution profile by Ni affinity chromatography from baculovirus-infected supernatant, b. Verification of EK digestion efficiency for EK digested samples, and c. Purification of the target protein using Ni affinity chromatography resin and SDS-PAGE analysis after EK digestion. [Figure 23] The construction and expression of the recombinant protein antigen RBDXBB.2-HR of Embodiment 1 are shown. Hereinafter, a. schematic design diagram of pFastBac1-GP67-Trx-His-EK-S-RBD(Omicron_XBB.2)-HR, b. 1% agarose gel electrophoresis of the cloned PCR product, c. 1% agarose gel electrophoresis of the PCR-amplified recombinant bacmid, d. packaging and proliferation flowchart of the recombinant baculovirus, and e. WB validation results during baculovirus proliferation. [Figure 24] The expression and purification results of the recombinant protein antigen RBDXBB.2-HR in Embodiment 1 are shown. Here, a. Elution profile by Ni affinity chromatography from baculovirus-infected supernatant, b. Verification of EK digestion efficiency for EK-digested samples, and c-d. Purification by size exclusion chromatography (c) and SDS-PAGE analysis (d) after EK digestion. [Figure 25]The GMT results for serum neutralizing antibodies against multiple pseudoviruses after intramuscular immunization using six protein vaccines in Test Example 1, including RBDXBB.1.16-HR (SEQ ID NO: 1), RBDXBB.1.5-3-HR (SEQ ID NO: 5), RBDXBB.2.3-HR (SEQ ID NO: 8), RBDXBB.1.16.6-HR (SEQ ID NO: 9), RBDBA.2.86-HR (SEQ ID NO: 10), and RBDEG.5-HR (SEQ ID NO: 11), are shown. [Figure 26] The GMT results for serum neutralizing antibodies against multiple pseudoviruses, using the RBDJN.1-HR protein (SEQ ID NO: 55) as the antigen in Test Example 1, are shown. [Figure 27] The GMT results for serum neutralizing antibodies induced by intramuscular immunization against multiple pseudoviruses in Study Example 2 are shown, comparing the monovalent vaccines RBDXBB.1.5-3-HR (SEQ ID NO: 5), RBDXBB.1.16-HR (SEQ ID NO: 1), RBDXBB.2.3-HR (SEQ ID NO: 8), and RBDJN.1-HR (SEQ ID NO: 55) with a tetravalent formulation (RBDXBB.1.5-3-HR + RBDXBB.1.16-HR + RBDXBB.2.3-HR + RBDJN.1-HR, mixed in ratios of 1:1:1:1, 1:1:2:2, and 1:1:5:5). [Figure 28] The results of a live virus challenge experiment after intramuscular immunization of mice using the quadrivalent vaccine RBDXBB.1.5-3-HR+RBDXBB.1.16-HR+RBDXBB.2.3-HR+RBDJN.1-HR (mixture ratio 1:1:5:5) in Test Example 2 are shown. 'a' shows viral gRNA levels in nasal turbinates, trachea, and lung tissue collected 5 days post-infection, quantified by RT-qPCR. 'b' shows histopathological scoring of lung tissue after live XBB.1.16 virus challenge. [Figure 29] Test Example 3 shows the results of neutralizing antibodies against multiple pseudoviruses obtained by combined intranasal immunization in rats using the Ad5XBB.1.5 adenovirus vector (SEQ ID NO: 50) and the recombinant protein vaccine RBDXBB.1.5-3-HR (SEQ ID NO: 5). [Modes for carrying out the invention]

[0175] 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-spermamide)ethylammonium (DOSPA), trimethyldodecylammonium bromide (DTAB), trimethyltetradecylammonium bromide (TTAB), trimethylcetylammonium bromide (CTAB), dimethyldidoctadecylammonium bromide (DDAB), CpG ODN (synthetic CpG having unmethylated cytosine and guanine dinucleotide as core sequences).

[0176] The solutions of the present invention will be described with reference to embodiments. It will be understood by those skilled in the art that the following embodiments are not intended to limit the scope of the present invention, but merely to illustrate it. Where no specific technology or conditions are specified in the embodiments, the technology or conditions described in the literature or product specifications of the art shall prevail. Where the manufacturer of the reagents or equipment used is not indicated, they are all conventional products available by purchase on the market.

[0177] Embodiment 1: Preparation of recombinant proteins and vaccines using an insect baculovirus expression vector system

[0178] Recombinant protein antigen RBD XBB.1.16 - HR structure:

[0179] 1. Construction and design of S-RBD (Omicron_XBB.1.16)-HR

[0180] Since the SARS-CoV-2 S protein is a membrane-immobilized protein, to simulate its secretory pathway, we incorporated a GP67 signal peptide at the N-terminus of S-RBD(omicron_XBB.1.16)-HR for protein expression and construction to promote secretory expression. This signal peptide is autocatalytically cleaved by insect cells during protein secretion. Furthermore, downstream of the GP67 signal peptide, we incorporated a thioredoxin (Trx) tag from Spodoptera frugiperda (S. frugiperda) to promote proper folding of S-RBD(omicron_XBB.1.16)-HR, a 6xHis tag to enable subsequent purification, and an EK digestion site to enable removal of both the Trx and 6xHis tags. Protein expression and construction are made possible by the removal of all non-S-RBD(omicron_XBB.1.16)-HR redundant amino acids by EK digestion. The expression and construction design patterns are shown in Figure 1a. Its amino acid sequence is as shown in SEQ ID NO: 19, and its nucleotide sequence is as shown in SEQ ID NO: 37.

[0181] 2. Identification for Recombinant Plasmid Construction

[0182] The designed coding fragments were cloned into the pFastBac1 vector plasmid and validated by colony PCR. The colony PCR results, as shown in Figure 1b, indicated that all six selected clones successfully amplified the GP67-Trx-His-EK-S-RBD(Omicron_XBB.1.16)-HR fragment.

[0183] 3. Identification of recombinant bacmids

[0184] We selected correctly identified pFastBac1-GP67-Trx-His-EK-S-RBD(Omicron_XBB.1.16)-HR recombinant clones. After plasmid extraction, the recombinant plasmids were transformed into DH10b competent cells, followed by blue-white screening and colony PCR validation. Colony PCR products were detected by 1% agarose gel electrophoresis, and the identification results are shown in Figure 1c. White colonies indicated recombinant bacmid clones, and blue colonies indicated non-recombinant bacmid clones.

[0185] 4. Packaging of recombinant baculoviruses

[0186] Recombinant bacmid was transfected into sf9 insect cells, and P0 generation recombinant baculoviruses were recovered after 5 days. The packaging and replication workflow for recombinant baculoviruses is shown in Figure 1d.

[0187] 5. Expression and validation of target proteins

[0188] The expression of the target protein occurred simultaneously with the replication of the baculovirus described above. Since the target protein contained a His tag before its tag was removed, the inventors verified the expression of the recombinant protein using anti-His WB experiments. The verification results showed clear stripes between the 50KD and 60KD marker stripes, which matched the size of the Trx-His-EK-S-RBD(Omicron_XBB.1.16)-HR protein, indicating successful replication of the baculovirus and successful expression of the target protein. The detection results are shown in Figure 1e.

[0189] 6. Purification and identification of target proteins

[0190] Cell culture media of sf9 cells infected with recombinant baculovirus were collected after 3 days, and protein purification was verified using Ni affinity chromatography resin. The results are shown in Figure 2a. The target protein was mainly eluted with 250 mM imidazole, and high-purity target protein was obtained after elution.

[0191] 7. Verification of tag removal for EK consumption

[0192] The target protein eluate was concentrated and adjusted to a concentration of 1 mg / mL, and EK enzyme was added. After enzymatic digestion at 18°C ​​for 14 hours, the protein was identified by SDS-PAGE gel electrophoresis. The results are shown in Figure 2b. The results indicate that the EK enzyme was able to cleave the Trx-His-EK (amino acid sequence of the EK digestion site) tag from the target protein.

[0193] 8. Verification of the removal of removed tags

[0194] After EK digestion, the samples were subjected to further purification using Ni affinity chromatography resin to separate the Trx-His-EK-S-RBD(Omicron_XBB.1.16)-HR protein (uncleaved full-length protein), S-RBD(Omicron_XBB.1.16)-HR protein, and Trx-His-EK tag (cleaved tag fragment). The results are shown in Figures 2c to 2d. The results indicate that the Trx-His-EK tag was successfully removed and eliminated by the subsequent purification process, resulting in an untagged S-RBD(Omicron_XBB.1.16)-HR protein having the amino acid sequence shown in Sequence ID No. 1.

[0195] Similarly, recombinant protein antigen RBD XBB.1.16 -According to the method for constructing HR (involving modifications in recombinant baculovirus packaging generation in step 4 and the use of either Ni affinity chromatography resin or size exclusion chromatography for purification in step 8), the inventors constructed the following: RBD XBB.1.5-3 -HR (Sequence ID 5), RBDXBB.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), 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).

[0196] Among these, RBD XBB.1.5-3 The construction design, process, and expression data of the corresponding target protein of -HR are shown in FIGS. 3 to 4. The amino acid sequence of the designed coding fragment pFastBac1-GP67-Trx-His-EK-S-RBD(XBB.1.5-3)-HR is as shown in SEQ ID NO: 23, and the nucleotide sequence is as shown in SEQ ID NO: 41.

[0197] Among these, RBD XBB.2.3 The construction design, process, and expression data of the corresponding target protein of -HR are shown in FIGS. 5 to 6. The amino acid sequence of the designed coding fragment pFastBac1-GP67-Trx-His-EK-S-RBD(XBB.2.3)-HR is as shown in SEQ ID NO: 26, and the nucleotide sequence is as shown in SEQ ID NO: 44.

[0198] Among these, RBD XBB.1.16.6 The construction design, process, and expression data of the corresponding target protein of -HR are shown in FIGS. 7 to 8. The amino acid sequence of the designed coding fragment pFastBac1-GP67-Trx-His-EK-S-RBD(XBB.1.16.6)-HR is as shown in SEQ ID NO: 27, and the nucleotide sequence is as shown in SEQ ID NO: 45.

[0199] Among these, RBD BA.2.86The design, process, and expression data of the corresponding target protein for the HR construct 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 the nucleotide sequence is shown in SEQ ID NO: 46.

[0200] Among these, RBD EG.5 The design, process, and expression data of the corresponding target protein for the HR construct are shown in Figures 11 and 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 the nucleotide sequence is shown in SEQ ID NO: 47.

[0201] Among these, RBD JN.1 The design, process, and expression data of the corresponding target protein for the HR construct are shown in Figures 13 and 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 the nucleotide sequence is shown in SEQ ID NO: 57.

[0202] Among these, RBD XBB.1.5-1 The 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 the nucleotide sequence is shown in SEQ ID NO: 39.

[0203] Among these, RBD XBB.1.5-2 The 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 the nucleotide sequence is shown in SEQ ID NO: 40.

[0204] Among these, RBD XBB.1.5-4 The design, process, and expression data of the corresponding target protein for the HR construct 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 the nucleotide sequence is shown in SEQ ID NO: 42.

[0205] Among these, RBD XBB.2.3 The design, process, and corresponding target protein expression data are shown in Figures 21 and 22. 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: 25, and the nucleotide sequence is shown in SEQ ID NO: 43.

[0206] Among these, RBD XBB.2 The design, process, and expression data of the corresponding target protein for the HR construct 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 the nucleotide sequence is shown in SEQ ID NO: 38.

[0207] Embodiment 2: Preparation of recombinant adenovirus vaccine

[0208] 1. Optimization and synthesis of the S protein gene sequence

[0209] To obtain the gene sequence of the S protein while retaining its native signal peptide, the Omicron XBB.1.5 mutant was selected as a template. Based on this, codon optimization was performed to ensure enhanced protein expression, followed by gene synthesis of the optimized sequence (the nucleotide sequence is shown in SEQ ID NO: 50).

[0210] 2. Packaging of recombinant adenovirus vaccine against SARS-CoV-2

[0211] During the gene synthesis process, the S gene of the synthesized product was cloned into the pDC316 vector using a recombinant cloning strategy to obtain a shuttle plasmid (pDC316-S). The constructed pDC316-S, containing the S gene of the Omicron XBB.1.5 mutant, was co-transfected into HEK293 cells with the skeletal plasmid pBHGlox_E1,3Cre from the AdMax adenovirus line to package recombinant adenovirus. The process is as follows:

[0212] 1) In a 6-well plate containing high-glucose DMEM + 10% FBS medium, add 8 × 10⁶ units per well. 5 HEK293A cells were inoculated and cultured overnight in a 37°C cell culture incubator containing 5% CO2.

[0213] 2) The following day, the culture medium was replaced with high-glucose DMEM supplemented with 2% FBS, and HEK293A cells were co-transfected using the backbone plasmid (pBHGlox_E1,3Cre) and shuttle plasmid with Lipofectamine 3000. The specific procedure was 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. Another 1.5 mL EP tube was taken and 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 continued to culture 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, they were subcultured for 75 cm until the cells showed clear plaques. 2 The cells were transferred to a cell culture flask, and the virus was collected when most of the cells became diseased and fell to the bottom.

[0214] 3) The virus-containing cell culture was 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 culture was centrifuged at 3000 rpm for 20 minutes, the virus-containing supernatant was collected, and combined with the above virus-containing supernatant, this was the virus seed for the adenovirus vaccine.

[0215] 4) 50 μL of vaccine candidate virus solution was taken, 2 μL of protease 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 the PCR product after electrophoresis gel recovery was sequenced and identified. The requirements for PCT amplification are as follows:

[0216] 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.

[0217] 3. Amplification of recombinant adenovirus vaccine against SARS-CoV-2

[0218] The identified correct recombinant adenovirus vaccine strain was grown stepwise in 293 cells. The specific process was as follows: 4.0 × 10 6 293H cells at a concentration of cells / mL were added at an MOI of 3 and cultured for 48 to 72 hours. The virus culture was collected, and a master virus seed bank and a working virus seed bank were prepared by repeating the freeze-thaw cycle of the sample three times. Recombinant adenovirus vaccine was amplified in a shaking flask or bioreactor, and the virus culture was collected when a broad cytopathic effect was observed in most cells. The process for growing cells and viruses in the bioreactor was as follows: First, the assembled bioreactor was sterilized, then the cell culture medium was added to the bioreactor, and once the operating conditions stabilized at 37°C, pH 7.0, DO 50%, and 50 rpm, the cells in the shaking flask were collected and divided into 1.0 × 10⁶ cells.6 Cells were inoculated into the bioreactor at a seeding density of cells / mL, and the cell culture medium was replenished to a total volume of 10L. The bioreactor culture conditions were maintained at 37°C, 50-95 rpm, pH 7.15-7.25, and DO 30-50%. Samples were taken daily to monitor glucose concentration, cell density, and cell morphology. When the cell density in the bioreactor reached 4.0 × 10⁶ cells, 6 When the cell / mL reached the target, the bioreactor was inoculated with recombinant adenovirus vaccine seed at a MOI of 3. Samples were taken daily to monitor glucose concentration, cell viability, and viral titer after inoculation. Culture was stopped when most cells (50%–70%) had detached from the microcarrier. Virus lysis buffer was added to the bioreactor at a final concentration of 0.05%–1% Tween80, and lysis was carried out at 37°C for 2–4 hours, after which the virus solution was collected.

[0219] 4. Purification of recombinant adenovirus vaccine against SARS-CoV-2

[0220] The recovered viruses were purified by cesium chloride ultracentrifugation or ion-exchange chromatography. The specific process is as follows:

[0221] (1) Purification of adenovirus vaccine by cesium chloride ultracentrifugation The recovered virus culture was centrifuged at 1200 g 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 cells were then centrifuged at 3000 rpm for 10-20 minutes, and the supernatant was collected. The virus-containing supernatant was concentrated 10-fold using an ultrafiltration membrane at 100K to 300K to prepare 1.4 g / mL cesium chloride solution (by mixing 53 g of cesium chloride with 87 mL of 10 mM Tris-HCl (pH 7.9)) and 1.2 g / mL cesium chloride solution (by mixing 26.8 g of cesium chloride with 92 mL of 10 mM Tris-HCl (pH 7.9)). 8 mL of the 1.4 g / mL cesium chloride solution was slowly added to an ultracentrifuge tube, followed by 6 mL of the 1.2 g / mL cesium chloride solution. Finally, 20 mL of the virus-containing supernatant was added to the top of a discontinuous gradient, the tube was balanced, and then the tube 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.

[0222] (2) Purification of adenovirus by ion exchange chromatography The virus culture was collected and lysed using 0.05%–1% Tween20 at 37°C for 2–4 hours. The lysed culture was clarified by filtration through 1.2 μm and 0.45 μm capsule filters. The sample was concentrated 5–10 times using a tangential flow filtration membrane with a molecular weight cutoff of 100–300 kD. The concentrate was washed with 5–10 volumes of washing buffer (50 mM Tris-HCl, 2 mM MgCl2, 0–500 mM NaCl, pH 8.0). The washed sample was collected and subjected to anion exchange chromatography using resins such as Capto Q Impress, Q Sepharose XL, Source 30Q, or Source 15Q. The detailed procedure was as follows: The column was equilibrated with equilibration buffer at a flow rate of 20 mL / min for 5 column volumes. After equilibration, the sample was loaded at a flow rate of 10 mL / min. Once loading was complete, equilibration was continued with equilibration buffer until the conductivity stabilized. Samples were eluted using a linear gradient from 100% low-salt buffer to 100% high-salt buffer over 10 column volumes at a flow rate of 10 mL / min. Fractions corresponding to each elution peak were collected, and after elution, the columns were regenerated with 2M NaCl buffer in 5–10 column volumes at a flow rate of 20 mL / min. Virus peaks were collected, and then the eluted virus samples were subjected to buffer exchange by dialysis or tangential flow filtration.

[0223] Ad5 prepared by the method described above XBB.1.5 The amino acid sequence of the recombinant adenovirus vaccine antigen is as shown in SEQ ID NO: 32.

[0224] The efficacy of the recombinant protein vaccine and adenovirus vaccine prepared according to the present invention was demonstrated through the following experiments.

[0225] Animal preparation, animal immunization, and sample collection.

[0226] 1. Immunization Procedure for Monovalent Vaccine: Female NIH mice (6-8 weeks old, SPF grade) were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. (China) and housed under specific pathogen-free conditions at the State Key Laboratory of Biotherapy, Sichuan University. All mouse experiments were approved by the Institutional Animal Care and Use Committee (IACUC) of Sichuan University (Chengdu, Sichuan, China). The prepared recombinant protein antigen was separately mixed with an equal volume of MF59 adjuvant to formulate the monovalent vaccine. Mice were intramuscularly immunized three times on days 0, 21, and 42 (10 μg of protein per mouse). Serum was collected 14 days after the third immunization, and neutralizing antibodies were measured to evaluate the immunogenicity of the vaccine.

[0227] 2. Basic immunization procedure for the quadrivalent vaccine: Female NIH mice (6-8 weeks old, SPF grade) were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. and housed in the State Key Laboratory of Biotherapy at Sichuan University under specific pathogen-free conditions. Four different recombinant protein antigens were mixed and combined with an equal volume of MF59 adjuvant to formulate the quadrivalent vaccine. Mice were immunized intramuscularly three times on days 0, 21, and 42 (10 μg of protein per mouse). Serum was collected 14 days after the third immunization, and the neutralizing antibody response was measured to evaluate the immunogenicity of the vaccine.

[0228] 3. Female BALB / c rats aged 3.6 to 8 weeks were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. (China) and reared in a pathogen-free environment at the State Key Laboratory of Biotherapy at Sichuan University. To prepare a two-component intranasal vaccine, Ad5 XBB.1.5 (For low doses, 1 x 10 10 For VP or high doses, 2 × 10 10 VP) to RBD XBB.1.5 -HR (20 μg for low dose or 40 μg for high dose) was mixed with a total volume of 100 μL (low dose) or 200 μL (high dose). BALB / c rats were immunized intranasally three times (days 0, 21, and 42) with the following formulations: (1) 2 × 10 10 VP Ad5 空 and 40 μg of RBD XBB1.5 -HR, (2) 1 x 10 10 VP Ad5 XBB.1.5 (3) 1 × 10 10 VP Ad5 XBB.1.5 and 20 μg of RBD XBB.1.5 -HR, and (4) 2 × 10 10 VP Ad5 XBB.1.5 (5) 2 × 10 10 VP Ad5 XBB.1.5 and 40 μg of RBD XBB.1.5 -HR. Blood samples were collected 14 days after the third immunization to detect neutralizing antibodies and evaluate the immunogenicity of the vaccine.

[0229] Test Example 1: Detection of neutralizing antibodies in immunoserum for recombinant protein vaccines using pseudovirus neutralization method.

[0230] 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.

[0231] In short, inactivated serum samples (at 56°C for 30 minutes) were 3-fold diluted in the range of 30 to 65610, and then incubated with an equal volume of diluted pseudovirus at 37°C for 1 hour. Subsequently, 1.2 × 10⁶ cells expressing the human ACE2 receptor were incubated. 4 HEK-293T cells (293T / ACE2) were added to each well and incubated in 0.5% CO2 at 37°C for 48 hours to express luciferase. Finally, the supernatant was removed, and then a lysis reagent containing luciferase substrate (100 μL / well) (Beyotime, RG005) 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 control - Test sample) / (Positive control - Negative control)] × 100%.

[0232] The monovalent recombinant protein vaccine is a mixture of seven proteins: RBD and the adjuvant MF59. XBB.1.5-3 -HR (Sequence ID 5), RBD XBB.1.16 -HR (Sequence ID 1), RBD EG.5 -HR (Sequence ID 11), RBD XBB.1.16.6 -HR (Sequence ID 9), RBD BA.2.86 -HR (Sequence ID 10), RBD XBB.2.3 -HR (SEQ ID NO: 8), and RBD JN.1 - Composed of HR (SEQ ID NO: 55). The quadrivalent vaccine is the protein antigen RBD. XBB.1.5-3 -HR, RBD XBB.1.16 -HR, RBD XBB.2.3 -hr, and RBD JN.1-HR was prepared by mixing it in ratios of 1:1:1:1, 1:1:2:2, and 1:1:5:1, followed by combining it with an equal volume of MF59 adjuvant. Mice were immunized with these vaccines, and serum was collected to measure neutralizing antibody levels against 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.

[0233] As shown in Figure 25, there are six protein vaccines (RBD 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) induced various 50% neutralizing geometric mean titers (GMT) against four pseudoviruses (XBB.1.5, EG.5.1, HV.1, and BA.2.86). Among these, RBD XBB.1.5-3 -hr and RBD XBB.2.3 - The HR protein vaccine showed excellent serum neutralizing antibody responses against a variety of omicron variants, and particularly high neutralizing antibody responses against EG.5.1 pseudovirus.

[0234] As shown in Figure 26, RBD JN.1 Serum collected 14 days after the third immunization with the HR protein vaccine showed a potent neutralizing antibody response against newly emerging omicron variants, including BA.2.86, JN.1, JN.1.13, KP.2, and KP.3, demonstrating broad-spectrum neutralizing coverage against a diverse range of omicron variants.

[0235] As shown in Figure 27, RBD XBB.1.5-3 -HR, RBD XBB.1.16 -HR, RBD XBB.2.3 -hr, and RBD JN.1-HR protein antigens were mixed together (mixing ratios 1:1:1:1, 1:1:2:2, 1:1:5:5) and combined with an equal volume of MF59 adjuvant to form a quadrivalent vaccine. The quadrivalent vaccine showed effective neutralizing activity against pseudoviruses representing representative variants of the current XBB subvariants (XBB.1.5, XBB.1.6, XBB.1.16.6, BA.2.86, EG.5, and JN.1), which are representative mutations of the XBB spectrum currently spreading globally. RBD XBB.1.5-3 -HR, RBD XBB.1.16 -HR, RBD XBB.2.3 -hr, and RBD JN.1 - HR single protein vaccines, especially monovalent RBD JN.1 -HR protein vaccines showed relatively weak protection against pseudoviruses of XBB variants (XBB.1.5, XBB.1.6, XBB.1.16.6, and EG.5), with GMT values ​​of 190, 186, 146, and 318, respectively. However, RBD XBB.1.5-3 -HR, RBD XBB.1.16 -HR, RBD XBB.2.3 -hr, and RBD JN.1 The HR combination, regardless of the mixing ratio, showed better protection against all XBB variant pseudoviruses, demonstrating the unique advantages of a quadrivalent vaccine.

[0236] Study Example 2: Challenge Protection Study against SARS-CoV-2 XBB.1.16 Mutant

[0237] Prevention of viral infection is one of the most important indicators reflecting the protective efficacy of respiratory vaccines. Therefore, this study aimed to evaluate whether the prepared quadrivalent recombinant protein vaccine could provide a protective immune response to prevent and / or treat epidemic variants. NIH mice (6-8 weeks old) were divided into two groups (n=6 / group) and administered 10 μg of the quadrivalent vaccine mixed 1:1 with MF59 adjuvant (RBD in a 1:1:5:5 mixture ratio) on days 0, 21, and 42. XBB.1.5-3 -HR+RBD XBB.1.16 -HR+RBD XBB.2.3 -HR+RBDJN.1 Intramuscular immunization was performed using -HR). Mice receiving only the adjuvant served as a control. Subsequently, on day 63, mice were given 1 × 10⁶ 6 Live XBB.1.16 virus from PFU was intranasal challenged. Daily weight changes and throat swab viral loads were recorded. On day 5 post-infection, mice were euthanized, and their tissues were collected for detection of viral load and pathological changes. Viral genomic RNA (gRNA) in nasal cavity, turbinate, trachea, and lung tissue samples was detected by reverse transcription quantitative polymerase chain reaction (RT-qPCR) assay. gRNA levels were detected using primers and probe sequences including 5'-GACCCCAAAATCAGCGAAAT-3' (SEQ ID NO: 58 forward), 5'-TCTGGTTACTGCCAGTTGAATCTG-3' (SEQ ID NO: 59 reverse), and 5'-FAM-ACNGCCGCATTACGTTGGTGGACC-BHQ1-3' (SEQ ID NO: 60 probe sequence). Pathological changes in lung tissue were evaluated by hematoxylin and eosin (H&E) staining.

[0238] All experiments, including live SARS-CoV-2 virus challenges in animals, were rigorously reviewed and approved by the Animal Experimentation Committee (IACUC) of the Institute of Medical Biology, Chinese Academy of Medical Sciences. Furthermore, these experiments were conducted at ABSL-4 of the Kunming National High-Level Biosafety Primate Research Center (established under the Institute of Medical Biotechnology (IMB)).

[0239] As shown in Fig. 28, mice were euthanized on day 5 after infection for tissue collection to evaluate virus quantity and histopathology. High gRNA levels were detected in the nasal turbinate, trachea, and lung tissues of control mice with only adjuvant. In contrast, mice immunized with the quadrivalent vaccine showed a significant decrease in virus quantity in the nasal turbinate, trachea, and lung tissues. Furthermore, mild pathological changes were observed in the lung tissues of the control group with only adjuvant, characterized by multifocal sclerosis areas, thickened alveolar septa, alveolar congestion, and patchy inflammatory infiltrates. As expected, the lung tissues of vaccinated mice showed normal histological structures, had complete alveoli, and no obvious inflammation. As a result, the pathological score in the lung tissues of vaccinated mice was significantly lower compared to the control group with only adjuvant. These results demonstrated that the quadrivalent recombinant protein vaccine induced a protective immune response in both the upper and lower airways and effectively prevented infection by live virus.

[0240] Test Example 3 Pseudovirus Neutralization Experiment of Recombinant Protein Vaccine Combined with Adenovirus Vaccine

[0241] The operation of the pseudovirus source and neutralization method was the same as that described in Test Example 1. As shown in Fig. 29, 20 μg or 40 μg of RBD XBB.1.5-3 -HR recombinant protein vaccine (SEQ ID NO: 5) combined with 1×10 10 VP (low dose) or 2×10 10 VP (high dose) of either Ad5 XBB.1.5Intranasal administration of adenovirus (SEQ ID NO: 50) induced significantly higher serum neutralizing antibody titers against multiple omicron variants (XBB.1.5, XBB.1.16, XBB.1.16.6, XBB.2.3, FL.1.5.1, HV.1, EG.5, BA.2.86, and JN.1). Clearly, the combination of adenovirus vaccine and recombinant protein vaccine induced significantly stronger serum neutralizing antibodies compared to standalone Ad5 adenovirus or recombinant protein vaccines, demonstrating that adenovirus-subunit protein formulations enhance the neutralizing and protective capacity of blood and mucosal sites, thereby effectively preventing SARS-CoV-2 infection.

Claims

1. A protein for preventing and / or treating infection by SARS-CoV-2 Omicron XBB submutant, The protein is a protein having at least one amino acid sequence shown in SEQ ID NOs: 1 to 18 and SEQ ID NO:

55.

2. A protein for preventing and / or treating infection by a SARS-CoV-2 omicron XBB submutant according to claim 1, wherein the protein comprises at least one amino acid sequence shown in SEQ ID NOs: 1 to 11 and SEQ ID NOs: 55, preferably the protein comprises at least one amino acid sequence shown in SEQ ID NOs: 1, SEQ ID NOs: 5, SEQ ID NOs: 8, SEQ ID NOs: 9, SEQ ID NOs: 10, SEQ ID NOs: 11 and SEQ ID NOs:

55.

3. The precursor of the protein according to claim 1 or 2, wherein the protein is linked to a signal peptide and / or a protein tag, preferably the protein tag is selected from at least one of histidine tags, thioredoxin tags, glutathione transferase tags, ubiquitin-like modifier protein tags, maltose-binding protein tags, c-Myc protein tags, Avi-tagged protein tags, and nitrogen source utilization substance A protein tags.

4. The protein precursor according to claim 3, wherein a protease recognition region for excising the protein tag is further ligated to the protein for preventing and / or treating infection by SARS-CoV-2 omicron XBB submutant, and preferably the protease is selected from at least one of enterokinase, TEV protease, thrombin, coagulation factor Xa, carboxypeptidase A, and rhinovirus 3c protease.

5. The protein precursor according to claim 3 or 4, wherein the amino acid sequence of the precursor is selected from at least one of SEQ ID NOs: 19 to 36 and SEQ ID NOs: 56, preferably the amino acid sequence of the precursor is at least one of SEQ ID NOs: 19 to 29 and SEQ ID NOs: 56, and more preferably the amino acid sequence of the precursor is selected from at least one of SEQ ID NOs: 19, SEQ ID NOs: 23, SEQ ID NOs: 26, SEQ ID NOs: 27, SEQ ID NOs: 28, SEQ ID NOs: 29 and SEQ ID NOs:

56.

6. A polynucleotide encoding 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, wherein the sequence of the polynucleotide is selected from at least one of SEQ ID NOs: 37 to 54 and SEQ ID NOs: 57, preferably the polynucleotide sequence is selected from at least one of SEQ ID NOs: 37 to 47 and SEQ ID NOs: 57, and more preferably the polynucleotide sequence is selected from at least one of SEQ ID NOs: 37, SEQ ID NOs: 41, SEQ ID NOs: 44, SEQ ID NOs: 45, SEQ ID NOs: 46, SEQ ID NOs: 47 and SEQ ID NOs:

57.

8. A recombinant vector comprising the polynucleotide described in claim 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, preferably 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 according to claim 1 or 2 or a precursor according to any one of claims 3 to 5, comprising the steps of: culturing a host cell according to claim 10 or 11 such that the host cell expresses the protein or the precursor; and then recovering the protein to obtain the protein.

13. A recombinant protein vaccine for preventing and / or treating infection by SARS-CoV-2 omicron submutants, A recombinant protein vaccine comprising the protein described in claim 1 or 2 or the precursor described in any one of claims 3 to 5, and a pharmacologically acceptable adjuvant component.

14. The recombinant protein vaccine according to claim 13, wherein the adjuvant component is an immunoadjuvant, preferably selected from at least one of squalene-based oil-in-water emulsions, aluminum salts, calcium salts, phytosaponins, phytopolysaccharides, monophosphate tripide A, muramyl dipeptides, muramyl tripeptides, bacterial toxins, GM-CSF cytokines, lipids, and cationic liposome materials.

15. The squalene-based oil-in-water emulsion is MF59. The aluminum salt is selected from at least one of aluminum hydroxide and aram. The calcium salt is tricalcium phosphate. The phytosaponin is either QS-21 or ISCOM. Plant polysaccharides are astragalus polysaccharides. 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 phosphatidylethanolamine, phosphatidylcholine, cholesterol, and dioleoylphosphatidylethanolamine, and 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, trimethylcetylmonium bromide, dimethyldioctadecylammonium bromide, and CpG ODN. A recombinant protein vaccine according to claim 14, satisfying at least one of the following conditions.

16. A protein composition for preventing and / or treating infection by SARS-CoV-2 omicron submutants, The protein composition comprises any two or more proteins described in claim 1 or 2, or any two or more protein precursors described in any one of claims 3 to 5. The protein is constructed with any amino acid sequence selected from SEQ ID NOs: 1 to 18 and SEQ ID NO:

55. A protein composition in which the precursor is constructed with any amino acid sequence selected from SEQ ID NOs: 19 to 36 and SEQ ID NO:

56.

17. A recombinant protein vaccine composition for preventing and / or treating infection by SARS-CoV-2 omicron submutants, The recombinant protein vaccine composition is a combination of any two or more different recombinant protein vaccines, A recombinant protein vaccine composition wherein the recombinant protein vaccine is as described in claim 13, 14, or 15.

18. An adenovirus vector vaccine for preventing and / or treating infection by SARS-CoV-2 omicron subvariants, The adenovirus vector vaccine contains a polynucleotide encoding SARS-CoV-2 or its variant spike protein, An adenovirus vector vaccine in which the polynucleotide sequence is obtained by codon optimization or cell optimization.

19. The adenovirus vector vaccine according to claim 18, wherein the nucleotide sequence of the polynucleotide is selected from at least one of SEQ ID NOs: 37 to 54, preferably the polynucleotide sequence is selected from at least one of SEQ ID NOs: 48 to 54, and more preferably the polynucleotide sequence is selected from at least one of SEQ ID NOs: 48, SEQ ID NOs: 49, SEQ ID NOs: 50, and SEQ ID NOs:

51.

20. The adenovirus vector vaccine according to claim 19, wherein the adenovirus vector is selected from at least one of adenovirus, Ankarawaxinia virus, and adeno-associated virus, preferably the adenovirus vector is replication-deficient human adenovirus 5, 35, or 26 or / and replication-deficient chimpanzee adenovirus AdC68 or AdC7, and more preferably the adenovirus vector is replication-deficient human adenovirus 5 with combined E1 and E3 deletions.

21. A method for preparing an adenovirus for an adenovirus vector vaccine according to any one of claims 18 to 20, wherein the method is: A process for constructing a shuttle plasmid vector of polynucleotides encoding SARS-CoV-2 or its mutant spike protein, The process involves transfecting host cells with the constructed shuttle plasmid vector and backbone plasmid, and culturing the host cells. A process for obtaining replication-deficient recombinant adenovirus, and A method comprising the steps of performing large-scale culture and purification.

22. A method for preparing an adenovirus according to claim 21, wherein the polynucleotide sequence encoding SARS-CoV-2 or a variant spike protein is selected from at least one of SEQ ID NOs: 37 to 54, preferably the polynucleotide sequence is selected from at least one of SEQ ID NOs: 48 to 54, and more preferably the polynucleotide sequence is selected from at least one of SEQ ID NOs: 48, SEQ ID NOs: 49, SEQ ID NOs: 50, and SEQ ID NOs:

51.

23. The shuttle plasmid vector is at least one of pDC316-S, pDC315-S, pDC516-S, or pDC515-S. The aforementioned skeletal plasmid is at least one of pBHGlox_E1,3Cre and pBHGlox_E1,3FLP. The host cell is HEK293. A method for preparing an adenovirus vector according to claim 21, which satisfies at least one of the following conditions.

24. An adenovirus vector vaccine composition for preventing and / or treating infection by SARS-CoV-2 omicron submutant, wherein the adenovirus vector vaccine composition is a combination of any two or more different adenovirus vector vaccines, and the adenovirus vector vaccine is one of the adenovirus vector vaccines described in claim 18, 19, or 20.

25. A protein or protein precursor or protein composition or protein vaccine or protein vaccine composition or adenovirus vector vaccine or adenovirus vector vaccine composition, wherein these are formulated as an intradermal injection preparation or a subcutaneous injection preparation, an intramuscular injection preparation, an intravenous injection preparation, an oral preparation or a nasal spray preparation, preferably, they are formulated as an intramuscular injection preparation and a nasal spray preparation.

26. A pharmaceutical composition for treating and / or preventing infection by SARS-CoV-2 omicron submutant, The pharmaceutical composition is a combination of at least one of recombinant protein, protein composition, recombinant protein vaccine, and recombinant protein vaccine composition, and at least one of adenovirus vector vaccine and adenovirus vector vaccine composition. Preferably, the pharmaceutical composition is a combination of a polyvalent vaccine comprising at least one of the recombinant protein vaccine and the recombinant protein vaccine composition, and at least one of the adenovirus vector vaccine and the adenovirus vector vaccine composition. Preferably, the pharmaceutical composition is a combination of a polyvalent vaccine comprising at least one of the recombinant protein and the protein composition, and at least one of the adenovirus vector vaccine and the adenovirus vector vaccine composition. A pharmaceutical composition wherein the recombinant protein, the protein composition, the recombinant protein vaccine, the recombinant protein vaccine composition, the adenovirus vector vaccine, and the 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 by SARS-CoV-2 omicron submutant, The combination drug is a combination of at least one of recombinant proteins, protein compositions, recombinant protein vaccines, recombinant protein vaccine compositions, and at least one of adenovirus vector vaccines and adenovirus vector vaccine compositions, which are administered separately or simultaneously. Preferably, the combination drug is a combination of at least one of the recombinant protein vaccine and the recombinant protein vaccine composition, and at least one of the adenovirus vector vaccine and the adenovirus vector vaccine composition. Preferably, the combination drug is a combination of at least one of the recombinant protein and the protein composition, and at least one of the adenovirus vector vaccine and the adenovirus vector vaccine composition. A combination drug wherein the recombinant protein, the protein composition, the recombinant protein vaccine, the recombinant protein vaccine composition, the adenovirus vector vaccine, and the 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 recombinant protein vaccine comprises the protein or precursor for preventing and / or treating infection by the SARS-CoV-2 Omicron XBB submutant, wherein the amino acid sequence of the protein or precursor is selected from at least one of SEQ ID NOs: 1 to 11, SEQ ID NOs: 55, SEQ ID NOs: 19 to 29, and SEQ ID NOs: 56, preferably, the amino acid sequence of the protein or precursor is selected from at least one of SEQ ID NOs: 1, SEQ ID NOs: 5, SEQ ID NOs: 8, SEQ ID NOs: 9, SEQ ID NOs: 10, SEQ ID NOs: 11, SEQ ID NOs: 55, SEQ ID NOs: 19, SEQ ID NOs: 23, SEQ ID NOs: 26, SEQ ID NOs: 27, SEQ ID NOs: 28, SEQ ID NOs: 29, and SEQ ID NOs:

56. The recombinant protein vaccine composition is a combination of any two or more recombinant protein vaccines containing different proteins. The adenovirus vector vaccine comprises at least one polynucleotide sequence shown in SEQ ID NOs: 48 to 54, preferably the adenovirus vector vaccine comprises at least one polynucleotide sequence shown in SEQ ID NOs: 48, 49, 50, and 51. The composition according to claim 26 or the combination agent according to claim 27, wherein the adenovirus vector vaccine composition refers to any combination of two or more adenovirus vector vaccines prepared using different polynucleotide sequences.

29. The amino acid sequence of the recombinant protein vaccine is selected from at least one of SEQ ID NOs: 1, SEQ ID NOs: 5, SEQ ID NOs: 8, SEQ ID NOs: 9, SEQ ID NOs: 10, SEQ ID NOs: 11, and SEQ ID NOs:

55. The composition or combination agent according to claim 28, wherein the adenovirus vector vaccine comprises at least one polynucleotide sequence shown in SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO: 50, and SEQ ID NO:

51.

30. A pharmaceutical composition according to any one of claims 26, 28, and 29, formulated as an intradermal injection preparation or subcutaneous injection preparation, an intramuscular injection preparation, an intravenous injection preparation, an oral preparation, or a nasal spray preparation, preferably formulated as an intramuscular injection preparation or a nasal spray preparation, or a combination drug according to any one of claims 27, 28, and 29.

31. Use of a protein according to claim 1 or 2, a protein precursor according to any one of claims 3 to 5, a protein composition according to claim 16, a recombinant protein vaccine according to any one of claims 13 to 15, a recombinant protein vaccine composition according to claim 17, an adenovirus vector vaccine according to any one of claims 18 to 20, an adenovirus vector vaccine composition according to claim 24, a pharmaceutical composition according to any one of claims 26, 28, and 29, or a combination drug according to any one of claims 27, 28, and 29, for preparing a drug to treat and / or prevent an infection or pathogenicity caused by SARS-CoV-2 or its variants.

32. The use according to claim 31, wherein the SARS-CoV-2 variant includes at least one SARS-CoV-2 variant, specifically alpha, beta, gamma, delta, omicron, omicron subvariants 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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