Proteins and vaccines against infection with sars-cov-2 omicron variant xbb and its subtypes

Abstract

The present application relates to the protein and vaccine for resisting SARS-CoV-2 Omicron mutant XBB and its subtype infection, and belongs to the field of medicine. In order to solve the problem that there is still lack of effective prevention and treatment drugs for SARS-CoV-2 Omicron mutant XBB and its subtype infection, the present application provides the protein and vaccine for resisting SARS-CoV-2 Omicron mutant XBB and its subtype infection, which is designed based on the full-length S protein of SARS-CoV-2 Omicron mutant XBB and the RBD sequence and the optimized sequence in the S protein of the XBB.1.5 subtype, and can help the host resist coronavirus infection, especially has a good prevention and treatment effect on cross infection caused by SARS-CoV-2 Omicron mutant XBB and its subtype virus.

Description

Technical Field

[0001] This invention relates to proteins and vaccines against SARS-CoV-2 Omeprone mutant strain XBB and its subtypes, and belongs to the pharmaceutical field. Background Technology

[0002] The novel coronavirus (SARS-CoV-2) is a novel beta coronavirus named by the World Health Organization. This virus is enveloped, with round or oval particles, often pleomorphic, and a diameter of 60-140 nm. Its genetic characteristics are significantly different from SARS-CoV and MERS-CoV, representing a new branch of coronaviruses not previously found in humans. Currently, there are five main types of mutated SARS-CoV: Alpha, Beta, Gamma, Delta, and Omicron. The Omicron variant is further divided into several subspecies, including BA.1, BA.2, BA.2.12.1, BA.4, BA.5, BQ.1.1, and XBB.1.5.

[0003] The main structural proteins of SARS-CoV-2 include the spike protein (S), messenger protein (E), membrane protein (M), and nucleocapsid protein (N). Among these, the S protein plays a crucial role in viral infection and virulence and is often used as a vaccine antigen. Because the SARS-CoV-2 variant XBB contains multiple mutation sites, and the virus's S protein also contains multiple mutation sites, variant XBB and its subtypes can, to some extent, evade antibodies induced by vaccines against SARS-CoV-2 mutant strains (Alpha, Beta, Gamma, Delta, Omicron), leading to vaccine ineffectiveness or reduced protective efficacy. Therefore, developing vaccines against the SARS-CoV-2 mutant strain XBB and its subtypes, especially broad-spectrum vaccines against various variants of SARS-CoV-2, is crucial for the prevention and control of novel coronavirus infection. Summary of the Invention

[0004] This invention aims to at least solve one of the technical problems existing in the prior art. Therefore, the object of this invention is to provide a protein against SARS-CoV-2 Omeprone mutant strain XBB and its subtypes. This invention also provides vaccines for the prevention and / or treatment of SARS-CoV-2 Omeprone mutant strain XBB and its subtypes, including recombinant protein vaccines and adenovirus vector vaccines containing said protein, and also provides compositions or combinations of the two vaccines.

[0005] This invention first provides a protein resistant to SARS-CoV-2 Omeprone mutant strain XBB and its subtypes, containing an amino acid sequence selected from any one of SEQ ID No. 1 to SEQ ID No. 7. Preferably, the protein is selected from that shown in SEQ ID No. 3 or SEQ ID No. 5.

[0006] The present invention also provides a precursor of the protein, which is a protein to which a signal peptide and / or protein tag are attached.

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

[0008] Furthermore, a protease recognition region that removes the protein tag is also attached to the protein that is resistant to SARS-CoV-2 Omeprone mutant strain XBB and its subtypes.

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

[0010] Further, the amino acid sequence of the precursor is selected from at least one of SEQ ID No. 8, SEQ ID No. 10, SEQ ID No. 12, SEQ ID No. 14, SEQ ID No. 16, SEQ ID No. 18, and SEQ ID No. 20. Preferably, the protein sequence is selected from that shown in SEQ ID No. 12 or SEQ ID No. 16.

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

[0012] Further, the nucleotide sequence is selected from at least one of SEQ ID No. 9, SEQ ID No. 11, SEQ ID No. 13, SEQ ID No. 15, SEQ ID No. 17, SEQ ID No. 19, and SEQ ID No. 21. Preferably, the polynucleotide sequence is selected from that shown in SEQ ID No. 13 or SEQ ID No. 17.

[0013] The present invention also provides a recombinant vector containing the aforementioned polynucleotide.

[0014] Furthermore, the recombinant vector is at least one of insect baculovirus expression vector, mammalian cell expression vector, Escherichia coli expression vector, and yeast expression vector.

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

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

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

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

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

[0020] The present invention also provides a host cell containing the recombinant vector described above.

[0021] Furthermore, the host cell is at least one of insect cells, mammalian cells, Escherichia coli, and yeast.

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

[0023] Preferably, the mammalian cell is a CHO cell.

[0024] The present invention also provides a method for preparing the protein or the precursor, characterized by comprising the following steps: culturing the host cells to express the protein or precursor, and then recovering the protein to obtain the precursor.

[0025] The present invention also provides a protein composition for resisting infection of SARS-CoV-2 Omeprón mutant strain and its subtypes, comprising any combination of two or more of the following proteins: BA.5 sequence-1, BA.5 sequence-2, XBB.1.5 sequence-1, XBB.1.5 sequence-2, XBB.1.5 sequence-3, XBB.1.5 sequence-4, and S-XBB.1.5.

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

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

[0028] The present invention also provides a recombinant protein vaccine for the prevention and / or treatment of SARS-CoV-2 Omeprón mutant strain and its subtypes, comprising the said protein, the said precursor and / or the said protein composition, and pharmaceutically acceptable excipients or auxiliary components.

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

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

[0031] Furthermore, at least one of the following conditions must be met: the squalene oil-in-water emulsion is MF59;

[0032] The aluminum salt is selected from at least one of aluminum hydroxide and alum;

[0033] The calcium salt mentioned is tricalcium phosphate;

[0034] The plant saponins mentioned are QS-21 or ISCOM;

[0035] The plant polysaccharide mentioned is Astragalus polysaccharide;

[0036] The bacterial toxin is selected from at least one of recombinant cholera toxin and diphtheria toxin;

[0037] The lipids are selected from at least one of the following: phosphatidylethanolamine, phosphatidylcholine, cholesterol, and dioleoylphosphatidylethanolamine;

[0038] The cationic liposome material is selected from at least one of the following: (2,3-dioleoyloxypropyl)trimethylammonium chloride, N-[1-(2,3-dioleoylchloro)propyl]-N,N,N-trimethylamine chloride, cationic cholesterol, trifluoroacetic acid dimethyl-2,3-dioleenoyloxypropyl-2-(2-speramidamino)ethylammonium, trimethyl dodecylammonium bromide, trimethyl tetradecylammonium bromide, trimethyl hexadecylammonium bromide, dimethyl dioctadecylammonium bromide, and CpG ODN.

[0039] The present invention also provides an adenovirus vector vaccine against SARS-CoV-2 Omeprone mutant strain XBB and its subtypes, which is constructed as a recombinant vector containing a polynucleotide sequence encoding the protein, the precursor, or the protein composition.

[0040] Preferably, the nucleotide sequence of the polynucleotide is selected from at least one of SEQ ID No. 9, SEQ ID No. 11, SEQ ID No. 13, SEQ ID No. 15, SEQ ID No. 17, SEQ ID No. 19, and SEQ ID No. 21. More preferably, the polynucleotide sequence contained in the adenovirus vector is selected from the polynucleotides used to construct S-XBB.1.5, as shown in SEQ ID No. 21.

[0041] Furthermore, the adenovirus vector is selected from at least one of the following: adenovirus, Ankara vaccinia virus, and adeno-associated virus.

[0042] Preferably, the adenovirus is a replication-defective type selected from human type 5, 35 or 26 and / or chimpanzee type AdC68 or AdC7.

[0043] More preferably, it is selected from human type 5 replication-defective adenovirus with combined deletions of E1 and E3.

[0044] This invention provides a method for preparing adenovirus in the adenovirus vector vaccine, comprising the following steps: constructing a shuttle plasmid vector of the polynucleotide; then transfecting the constructed shuttle plasmid vector and backbone plasmid together into host cells, culturing the host cells; obtaining a replication-deficient recombinant adenovirus, and then scaling up the culture and purifying it.

[0045] Furthermore, the adenovirus vector vaccine also includes pharmaceutically acceptable adjuvants, vectors, diluents, or excipients.

[0046] Furthermore, the recombinant protein vaccine and adenovirus vector vaccine are preparations for intradermal or subcutaneous injection, intramuscular injection, intravenous injection, oral or nasal spray.

[0047] Preferably, the vaccine is an intramuscular injection preparation and a nasal spray preparation.

[0048] The present invention also provides compositions for treating and / or preventing infection with SARS-CoV-2 Omeprón mutant strains and their subtypes, which are compound preparations containing the recombinant protein vaccine and the adenovirus vector vaccine as active ingredients.

[0049] The present invention provides a combination drug for infection with SARS-CoV-2 Omeprone mutant strain XBB and its subtypes, comprising the recombinant protein vaccine and the adenovirus vector vaccine administered separately or simultaneously.

[0050] Preferably, the composition or combination of drugs is a combination or combination of recombinant protein vaccine 1, recombinant protein vaccine 2 and adenovirus vector vaccine.

[0051] More preferably, the recombinant protein vaccine 1 contains at least one amino acid sequence of BA.5 sequence-1, BA.5 sequence-2 protein or precursor; the recombinant protein vaccine 2 contains at least one amino acid sequence of XBB.1.5 sequence-1, XBB.1.5 sequence-2, XBB.1.5 sequence-3, XBB.1.5 sequence-4 protein or precursor; and the adenovirus vector vaccine contains the S-XBB.1.5 polynucleotide sequence.

[0052] Furthermore, the amino acid sequence contained in the recombinant protein vaccine 1 is selected from at least one of SEQ ID No. 1, SEQ ID No. 2, SEQ ID No. 8, and SEQ ID No. 10.

[0053] Further, the amino acid sequence contained in the recombinant protein vaccine 2 is selected from at least one of SEQ ID No. 3 to SEQ ID No. 6, SEQ ID No. 12, SEQ ID No. 14, SEQ ID No. 16, and SEQ ID No. 18. Preferably, it is selected from at least one of SEQ ID No. 5, SEQ ID No. 6, SEQ ID No. 16, and SEQ ID No. 18.

[0054] Furthermore, the adenovirus vector vaccine contains a polynucleotide sequence as shown in SEQ ID No. 21.

[0055] Further, the composition or combination of drugs is an intradermal or subcutaneous injection preparation, an intramuscular injection preparation, an intravenous injection preparation, an oral or nasal spray preparation; preferably, the vaccine is an intramuscular injection preparation and a nasal spray preparation.

[0056] The present invention also provides the use of the protein, the precursor, the protein composition, the recombinant protein vaccine, the adenovirus vector vaccine, the vaccine composition, or the combination thereof in the preparation of medicaments for treating and / or preventing infection or pathogenesis of SARS-CoV-2 Omeprón mutant strains and their subtypes.

[0057] The present invention also provides a vaccine composition for treating and / or preventing infection with SARS-CoV-2 Omeprón mutant strains and their subtypes, comprising a recombinant protein vaccine and an adenovirus vector vaccine; wherein the amino acid sequence of the recombinant protein vaccine is selected from at least one of SEQ ID No. 1 to SEQ ID No. 7, SEQ ID No. 8 to SEQ ID No. 20; and the nucleotide sequence of the antigen of the adenovirus vector vaccine is selected from SEQ ID No. 23.

[0058] The present invention also provides a combination drug for treating and / or preventing infection with SARS-CoV-2 Omeprón mutant strains and their subtypes, wherein the above-mentioned recombinant protein vaccine and adenovirus vector vaccine are administered separately or simultaneously; the amino acid sequence of the recombinant protein vaccine is selected from at least one of SEQ ID No. 1 to SEQ ID No. 7 and SEQ ID No. 8 to SEQ ID No. 20; the nucleotide sequence of the antigen of the adenovirus vector vaccine is selected from SEQ ID No. 23.

[0059] The following sequences are constructed by the applicant based on the RBD sequences of the S protein of the SARS-CoV-2 mutant Omicron strains BA.5, XBB, and subline XBB.1.5. The constructed proteins or precursors are defined as BA.5 sequence-1, BA.5 sequence-2, XBB.1.5 sequence-1, XBB.1.5 sequence-2, XBB.1.5 sequence-3, XBB.1.5 sequence-4, and S-XBB.1.5 protein or precursor, respectively.

[0060] SEQ ID No.1BA.5 sequence-1

[0061] VQPTESIVRFPNITNLCPFDEVFNATRFASVYAWNRKRISNCVADYSVLYNFAPFFAFKCYGVSPTKLNDLCFTNVYADSFVIRGNEVSQIAPGQTGNIADYNYKLPDDFTGCVIAWNSNKLDSKVGGNYNYRYRLFRKSNLKPFERDISTEIYQAGNKPCN GVAGVNCYFPLQSYGFRPTYGVGHQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKSVNFNFNGLYENQKLIANQFNSAIGKIQDSLSSTASALGKLQDVVNQNAQALNTLVKQLKNHTSPDVDLGDISGINASVVNIQKEIDRLNEVAKNLNESLIDLQEL

[0062] SEQ ID No.2BA.5 sequence-2

[0063] VQPTESIVRFPNITNLCPFDEVFNATRFASVYAWNRKRISNCVADYSVLYNSASFSAFKCYGVSPTKLNDLCFTNVYADSFVIRGNEVSQIAPGQTGNIADYNYKLPDDFTGCVIAWNSNKLDSKVGGNYNYRYRLFRKSNLKPFERDISTEIYQAGNKPCNGVAGVNCYFPLQSYGFRPTYGVGHQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKSVNFNFNGLYENQKLIANQFNSAIGKIQDSLSSTASALGKLQDVVNQNAQALNTLVKQLKNHTSPDVDLGDISGINASVVNIQKEIDRLNEVAKNLNESLIDLQEL

[0064] SEQ ID No.3 XBB.1.5 Sequence - 1

[0065] VQPTESIVRFPNITNLCPFHEVFNATTFASVYAWNRKRISNCVADYSVIYNFAPFFAFKCYGVSPTKLNDLCFTNVYADSFVIRGNEVSQIAPGQTGNIADYNYKLPDDFTGCVIAWNSNKLDSKPSGNYNYLYRLFRKSKLKPFERDISTEIYQAGNKPCNGVAGPNCYSPLQSYGFRPTYGVGHQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKCVNFNFNG

[0066] SEQ ID No.4 XBB.1.5 Sequence - 2

[0067] VQPTESIVRFPNITNLCPFHEVFNATTFASVYAWNRKRISNCVADYSVIYNSASFSAFKCYGVSPTKLNDLCFTNVYADSFVIRGNEVSQIAPGQTGNIADYNYKLPDDFTGCVIAWNSNKLDSKPSGNYNYLYRLFRKSKLKPFERDISTEIYQAGNKPCNGVAGPNCYSPLQSYGFRPTYGVGHQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKCVNFNFNG

[0068] SEQ ID No.5 XBB.1.5 Sequence - 3

[0069] VQPTESIVRFPNITNLCPFHEVFNATTFASVYAWNRKRISNCVADYSVIYNFAPFFAFKCYGVSPTKLNDLCFTNVYADSFVIRGNEVSQIAPGQTGNIADYNYKLPDDFTGCVIAWNSNKLDSKPSGNYNYLYRLFRKSKLKPFERDISTEIYQAGNKPCNGVAGPNCYSPLQSYGFRPTYGVGHQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKSVNFNFNGLYENQKLIANQFNSAIGKIQDSLSSTASALGKLQDVVNQNAQALNTLVKQLKNHTSPDVDLGDISGINASVVNIQKEIDRLNEVAKNLNESLIDLQEL

[0070] SEQ ID No.6 XBB.1.5 Sequence - 4

[0071] VQPTESIVRFPNITNLCPFHEVFNATTFASVYAWNRKRISNCVADYSVIYNSASFSAFKCYGVSPTKLNDLCFTNVYADSFVIRGNEVSQIAPGQTGNIADYNYKLPDDFTGCVIAWNSNKLDSKPSGNYNYLYRLFRKSKLKPFERDISTEIYQAGNKPCNGVAGPNCYSPLQSYGFRPTYGVGHQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKSVNFNFNGLYENQKLIANQFNSAIGKIQDSLSSTASALGKLQDVVNQNAQALNTLVKQLKNHTSPDVDLGDISGINASVVNIQKEIDRLNEVAKNLNESLIDLQEL

[0072] SEQ ID No.7 S - XBB.1.5

[0073]

[0074] To facilitate secretory expression of the protein, a signal peptide was added to its amino acids during protein construction. Additionally, a His tag was added to the protein's amino acid sequence for easier purification. The complete amino acid sequences of BA.5 sequence-1, BA.5 sequence-2, XBB.1.5 sequence-1, XBB.1.5 sequence-2, XBB.1.5 sequence-3, XBB.1.5 sequence-4, and S-XBB.1.5 are shown in SEQ ID No. 8, SEQ ID No. 10, SEQ ID No. 12, SEQ ID No. 14, SEQ ID No. 16, SEQ ID No. 18, and SEQ ID No. 20, respectively.

[0075] Furthermore, the corresponding nucleotide sequences encoding their amino acid sequences are shown in SEQ ID No. 9, SEQ ID No. 11, SEQ ID No. 13, SEQ ID No. 15, SEQ ID No. 17, SEQ ID No. 19, and SEQ ID No. 21, respectively.

[0076] SEQ ID No. 8 Complete BA.5 sequence - 1 signal peptide - Trx tag - 6 His tag - EK restriction site - RBD sequence - HR1 sequence - HR2 sequence

[0077] MLLVNQSHQGFNKEHTSKMVSAIVLYVLLAAAAHSAFAADSIHIKDSDDLKNRLAEAGDKLVVIDFMATWCGPCKMIGPKLDEMANEMSDSIVVLKVDVDECEDIATEYNINSMPTFVFVKNSKKIEEFSGANVDKLRNTIIKLKLAGSGSGHMHHHHHHSSGDDDDKVQPTESIVRFPNITNLCPFDEVFNATRFASVYAWNRKRISNCVADYSVLYNFAPFFAFKCYGVSPTKLNDLCFTNVYADSFVIRGNEVSQIAPGQTGNIADYNYKLPDDFTGCVIAWNSNKLDSKVGGNYNYRYRLFRKSNLKPFERDISTEIYQAGNKPCNGVAGVNCYFPLQSYGFRPTYGVGHQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKSVNFNFNGLYENQKLIANQFNSAIGKIQDSLSSTASALGKLQDVVNQNAQALNTLVKQLKNHTSPDVDLGDISGINASVVNIQKEIDRLNEVAKNLNESLIDLQEL

[0078] The nucleotide sequence encoding SEQ ID No.8 by SEQ ID No.9

[0079]

[0080] SEQ ID No. 10 Complete BA.5 sequence - 2-signal peptide - Trx tag - 6-His tag - EK restriction site - RBD sequence - HR1 sequence - HR2 sequence

[0081] MLLVNQSHQGFNKEHTSKMVSAIVLYVLLAAAAHSAFAADSIHIKDSDDLKNRLAEAGDKLVVIDFMATWCGPCKMIGPKLDEMANEMSDSIVVLKVDVDECEDIATEYNINSMPTFVFVKNS KKIEEFSGANVDKLRNTIIKLKLAGSGSGHMHHHHHHSSGDDDDKVQPTESIVRFPNITNLCPFDEVFNATRFASVYAWNRKRISNCVADYSVLYNSASFSAFKCYGVSPTKLNDLCFTNVYA DSFVIRGNEVSQIAPGQTGNIADYNYKLPDDFTGCVIAWNSNKLDSKVGGNYNYRYRLFRKSNLKPFERDISTEIYQAGNKPCNGVAGVNCYFPLQSYGFRPTYGVGHQPYRVVVLSFELLHA PATVCGPKKSTNLVKNKSVNFNFNGLYENQKLIANQFNSAIGKIQDSLSSTASALGKLQDVVNQNAQALNTLVKQLKNHTSPDVDLGDISGINASVVNIQKEIDRLNEVAKNLNESLIDLQEL

[0082] SEQ ID No. 11 encodes the nucleotide sequence of SEQ ID No. 10.

[0083]

[0084] SEQ ID No. 12 Complete XBB.1.5 sequence - 1 signal peptide - Trx tag - 6 His tag - EK restriction site - RBD sequence

[0085] MLLVNQSHQGFNKEHTSKMVSAIVLYVLLAAAAHSAFAADSIHIKDSDDLKNRLAEAGDKLVVIDFMATWCGPCKMIGPKLDEMANEMSDCIVVLKVDVDECEDIATEYNINSMPTFVFVKNSKKIEEFSGANVDKLRNTIIKLKLAGSGSGHMHHHHHHSSGDDDDKVQPTESIVRFPNITNLCPFHEVFNATTFA SVYAWNRKRISNCVADYSVIYNFAPFFAFKCYGVSPTKLNDLCFTNVYADSFVIRGNEVSQIAPGQTGNIADYNYKLPDDFTGCVIAWNSNKLDSKPSGNYNYLYRLFRKSKLKPFERDISTEIYQAGNKPCNGVAGPNCYSPLQSYGFRPTYGVGHQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKCVNFNFNG

[0086] SEQ ID No. 13 encodes the nucleotide sequence of SEQ ID No. 12.

[0087]

[0088] SEQ ID No. 14 Complete XBB.1.5 sequence - 2-signal peptide - Trx tag - 6-His tag - EK restriction site - RBD sequence

[0089] MLLVNQSHQGFNKEHTSKMVSAIVLYVLLAAAAHSAFAADSIHIKDSDDLKNRLAEAGDKLVVIDFMATWCGPCKMIGPKLDEMANEMSDCIVVLKVDVDECEDIATEYNINSMPTFVFVKNSKKIEEFSGANVDKLRNTIIKLKLAGSGSGHMHHHHHHSSGDDDDKVQPTESIVRFPNITNLCPFHEVFNATTFA SVYAWNRKRISNCVADYSVIYNSASFSAFKCYGVSPTKLNDLCFTNVYADSFVIRGNEVSQIAPGQTGNIADYNYKLPDDFTGCVIAWNSNKLDSKPSGNYNYLYRLFRKSKLKPFERDISTEIYQAGNKPCNGVAGPNCYSPLQSYGFRPTYGVGHQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKCVNFNFNG

[0090] SEQ ID No. 15 encodes the nucleotide sequence of SEQ ID No. 14.

[0091]

[0092] SEQ ID No. 16 Complete XBB.1.5 sequence - 3-signal peptide - Trx tag - 6-His tag - EK restriction site - RBD sequence - HR1 sequence - HR2 sequence

[0093] MLLVNQSHQGFNKEHTSKMVSAIVLYVLLAAAAHSAFAADSIHIKDSDDLKNRLAEAGDKLVVIDFMATWCGPCKMIGPKLDEMANEMSDSIVVLKVDVDECEDIATEYNINSMPTFVFVKNS KKIEEFSGANVDKLRNTIIKLKLAGSGSGHMHHHHHHSSGDDDDKVQPTESIVRFPNITNLCPFHEVFNATTFASVYAWNRKRISNCVADYSVIYNFAPFFAFKCYGVSPTKLNDLCFTNVYA DSFVIRGNEVSQIAPGQTGNIADYNYKLPDDFTGCVIAWNSNKLDSKPSGNYNYLYRLFRKSKLKPFERDISTEIYQAGNKPCNGVAGPNCYSPLQSYGFRPTYGVGHQPYRVVVLSFELLHA PATVCGPKKSTNLVKNKSVNFNFNGLYENQKLIANQFNSAIGKIQDSLSSTASALGKLQDVVNQNAQALNTLVKQLKNHTSPDVDLGDISGINASVVNIQKEIDRLNEVAKNLNESLIDLQEL

[0094] SEQ ID No. 17 encodes the nucleotide sequence of SEQ ID No. 16.

[0095]

[0096] SEQ ID No. 18 Complete XBB.1.5 sequence - 4-signal peptide - Trx tag - 6-His tag - EK restriction site - RBD sequence - HR1 sequence - HR2 sequence

[0097] MLLVNQSHQGFNKEHTSKMVSAIVLYVLLAAAAHSAFAADSIHIKDSDDLKNRLAEAGDKLVVIDFMATWCGPCKMIGPKLDEMANEMSDSIVVLKVDVDECEDIATEYNINSMPTFVFVKNS KKIEEFSGANVDKLRNTIIKLKLAGSGSGHMHHHHHHSSGDDDDKVQPTESIVRFPNITNLCPFHEVFNATTFASVYAWNRKRISNCVADYSVIYNSASFSAFKCYGVSPTKLNDLCFTNVYA DSFVIRGNEVSQIAPGQTGNIADYNYKLPDDFTGCVIAWNSNKLDSKPSGNYNYLYRLFRKSKLKPFERDISTEIYQAGNKPCNGVAGPNCYSPLQSYGFRPTYGVGHQPYRVVVLSFELLHA PATVCGPKKSTNLVKNKSVNFNFNGLYENQKLIANQFNSAIGKIQDSLSSTASALGKLQDVVNQNAQALNTLVKQLKNHTSPDVDLGDISGINASVVNIQKEIDRLNEVAKNLNESLIDLQEL

[0098] SEQ ID No. 19 encodes the nucleotide sequence of SEQ ID No. 18.

[0099]

[0100] SEQ ID No. 20: Complete S-XBB.1.5 amino acid sequence, Ad5 XBB.1.5

[0101] The nucleotide sequence encoded by SEQ ID No.21 is the nucleotide sequence of SEQ ID No.20, Ad5 XBB.1.5 Adenovirus vaccine antigen nucleotide sequence ATGTTCGTGTTCCTGGTGCTGCTGCCCCTGGTGAGCAGCCAATGCGTGAACCTGATCACAAGAACACAGAGCTACACCAACAGCTTCACAAGAGGCGTGTACTACCCCGACAAGGTGTTCAGAAGCAGCGTGCTGCATTCCACCCAAGACCTGTTCCTCCCCTTCTTTTCCAACGTGACCTGGTTCCACGCCATCCACGTGAGCGGCACCAACGGCACCAAGAGATTCGACAACCCCGCCCTGCCCTTCAACGACGGCGTGTACTTCGCTAGCACCGAGAAGAGCAACATCATCAGAGGCTGGATCTTCGGCACCACCCTGGACAGCAAGACACAGAGCCTGCTGATCGTGAACAATGCCACCAACGTGGTGATCAAGGTGTGCGAGTTTCAGTTCTGCAACGACCCC

[0102] TTCCTGGACGTGTATCAGAAGAACAACAAGAGCTGGATGGAGAGCGAGTTCAGAGTGT

[0103] ACAGCAGCGCTAACAACTGCACCTTCGAGTACGTGAGCCAACCCTTCCTGATGGACCTG

[0104] GAGGGCAAGGAGGGCAACTTCAAGAACCTCAGAGAGTTCGTGTTCAAGAACATCGACG <0​​​​​​TCAGACCCTGCTGGCCCTGCACAGAAGCTATCTGACACCTGTGGACAGCAGCAGCGGC

[0108] TGGACAGCTGGGGCCGCTGCCTACTACGTGGGCTACCTGCAGCCTAGAACCTTCCTGCT

[0109] GAAGTACAACGAGAACGGCACAATCACCGATGCCGTGGATTGCGCCCTGGACCCCCTG

[0110] AGCGAGACCAAGTGCACCCTGAAGAGCTTCACCGTGGAGAAGGGCATCTATCAGACAA

[0111] GCAACTTCAGAGTGCAGCCCACCGAGAGCATCGTGAGATTCCCCAACATCACCAACCTG

[0112] TGCCCCTTCCACGAGGTGTTCAACGCCACCACCTTCGCTAGCGTGTACGCTTGGAACAG

[0113] AAAAAGAATCAGCAACTGCGTGGCCGACTACAGCGTGATCTACAACTTCGCCCCCTTCT

[0114] TCGCCTTCAAGTGCTACGGGGTGAGCCCCACCAAGCTGAACGACCTGTGCTTCACCAAC

[0115] GTGTACGCCGACAGCTTCGTGATCAGAGGCAACGAGGTGAGCCAAATCGCCCCTGGGC

[0116] AGACCGGCAACATCGCCGACTACAACTACAAGCTGCCCGACGACTTCACCGGCTGCGT

[0117] GATCGCCTGGAACAGCAACAAGCTGGACAGCAAACCTAGCGGCAACTACAACTACCTG

[0118] TACAGACTGTTCAGAAAGAGCAAGCTGAAGCCCTTCGAGAGAGACATCAGCACCGAGA

[0119] TCTACCAAGCCGGCAACAAGCCCTGCAACGGCGTGGCCGGCCCCAACTGCTACAGCCC

[0120] CCTGCAGAGCTACGGCTTCAGACCCACCTACGGCGTGGGCCATCAGCCCTACAGAGTGG

[0121] TCGTGCTGAGCTTCGAGCTGCTGCACGCCCCCGCCACCGTGTGCGGCCCCAAGAAGAG

[0122] CACCAACCTGGTGAAGAACAAGTGCGTGAACTTCAACTTCAACGGCCTCACCGGCACC

[0123] GGCGTGCTGACCGAGAGCAACAAGAAGTTCCTGCCCTTTCAGCAGTTCGGCAGAGACA

[0124] TCGCCGACACCACCGACGCCGTGAGAGACCCTCAGACCCTGGAGATCCTGGACATCAC

[0125] CCCCTGTAGCTTCGGCGGCGTGAGCGTGATCACCCCCGGCACCAACACAAGCAACCAA

[0126] GTGGCCGTGCTGTACCAAGGCGTGAACTGCACCGAGGTGCCCGTGGCCATCCACGCCG

[0127] ATCAGCTGACCCCCACCTGGAGAGTCTACTCCACCGGCAGCAACGTGTTTCAGACAAG

[0128] AGCCGGCTGCCTGATCGGCGCCGAGTACGTGAACAACAGCTACGAGTGCGACATCCCCA

[0129] TCGGCGCCGGCATCTGCGCTAGCTATCAGACACAGACCAAGAGCCACGGCAGCGCTAG

[0130] CAGCGTGGCTAGCCAAAGCATCATCGCCTACACCATGAGCCTGGGCGCCGAGAACAGC

[0131] GTGGCCTACAGCAACAACAGCATCGCCATCCCCACCAACTTCACCATCAGCGTGACCAC

[0132] CGAGATCCTGCCCGTCAGCATGACCAAGACAAGCGTGGACTGCACCATGTACATCTGCG

[0133] GCGACAGCACCGAGTGCAGCAACCTGCTCCTGCAGTACGGCAGCTTCTGCACACAGCT

[0134] GAAGAGAGCCCTGACCGGCATCGCCGTGGAGCAAGACAAGAACACCCAAGAGGTGTT

[0135] CGCCCAAGTGAAGCAGATCTACAAGACCCCCCCCATCAAGTACTTCGGCGGCTTCAACT

[0136] TCAGCCAAATCCTCCCCGACCCCTCCAAACCTAGCAAGAGAAGCTTCATCGAGGACCTG

[0137] CTGTTCAACAAGGTGACCCTGGCCGACGCCGGCTTCATCAAGCAGTACGGCGACTGCCT

[0138] GGGCGATATCGCCGCTAGAGACCTGATCTGCGCTCAGAAGTTCAATGGCCTGACCGTGC

[0139] TGCCCCCCCTGCTGACCGACGAGATGATCGCTCAGTATACAAGCGCTCTGCTCGCTGGC

[0140] ACCATTACAAGCGGGTGGACCTTCGGCGCTGGCGCTGCCCTGCAGATCCCCTTCGCCAT

[0141] GCAGATGGCCTACAGATTCAACGGCATCGGCGTGACACAGAACGTGCTGTACGAGAATC

[0142] AGAAGCTGATCGCCAATCAGTTCAACAGCGCCATCGGCAAGATCCAAGACAGCCTGAG

[0143] CAGCACCGCTAGCGCCCTGGGCAAGCTGCAAGACGTGGTGAACCACAACGCCCAAGCC

[0144] CTGAACACCCTGGTGAAGCAGCTGAGCAGCAAGTTCGGCGCCATCAGCAGCGTGCTCA

[0145] ACGACATCCTGAGCAGACTGGACCCCCCCGAGGCCGAGGTGCAGATCGATAGACTGATC

[0146] ACCGGCAGACTGCAGAGCCTGCAGACCTACGTGACACAGCAGCTGATCAGAGCCGCCG

[0147] AGATCAGAGCTAGCGCCAACCTGGCCGCCACCAAGATGAGCGAGTGCGTGCTGGGGCA

[0148] GAGCAAGAGAGTGGACTTCTGCGGCAAGGGCTACCACCTGATGAGCTTCCCTCAGAGC

[0149] GCCCCCCACGGCGTGGTGTTCCTGCACGTGACCTACGTGCCCGCCCAAGAGAAGAACT

[0150] TCACCACAGCCCCCGCCATCTGCCACGACGGCAAGGCCCACTTCCCTAGAGAGGGCGT

[0151] GTTCGTGAGCAACGGCACCCACTGGTTCGTGACACAGAGAAACTTCTACGAGCCTCAG

[0152] ATCATCACCACCGACAACACCTTCGTGAGCGGCAACTGCGACGTGGTGATCGGCATCGT

[0153] GAATAATACCGTGTACGACCCCCTGCAGCCCGAGCTGGACAGCTTCAAGGAGGAGCTG

[0154] GACAAGTATTTCAAGAACCATACAAGCCCCGACGTGGACCTGGGCGACATCAGCGGCAT

[0155] CAACGCTAGCGTGGTGAACATTCAGAAGGAAATCGACAGACTCAACGAGGTGGCCAAG

[0156] AACCTGAACGAGAGCCTGATCGACCTGCAAGAGCTGGGCAAGTACGAGCAGTACATCA

[0157] AGTGGCCCTGGTACATCTGGCTGGGCTTCATCGCCGGCCTGATCGCCATCGGTGATGGTGA

[0158] CCATCATGCTGTGCTGCATGACAAGCTGCTGCAGCTGCCTGAAGGGCTGTTTGCAGCTGC

[0159] GGCAGCTGCTGCAAGTTCGACGAGGACGACAGCGAGCCCGTGCTGAAGGGCGTGAAG

[0160] CTGCACTACACCTGA

[0161] The applicant discovered that an adenovirus vaccine prepared using the full-length gene of the S protein of the Omicron mutant strain BA.5, as shown in SEQ ID No. 23, and then recombinant protein vaccines prepared using the RBD sequence or RBD-HR of the S protein of the Omicron mutant strains BA.5 and XBB.1.5, combined with the adenovirus vaccine, also showed good prevention and control effects against cross-infection caused by SARS-CoV-2 or its mutant viruses.

[0162] SEQ ID No. 22: Complete S-BA.5 amino acid sequence, Ad5 BA.5 Adenovirus vaccine antigen amino acid sequence

[0163]

[0164] The nucleotide sequence of SEQ ID No. 23 encodes the nucleotide sequence of SEQ ID No. 22, Ad5 BA.5 The antigen nucleotide sequence of the adenovirus vaccine ATGTTCGTGTTCCTGGTGCTGCTGCCCCTGGTGAGCAGCCAATGCGTGAACCTGATCACAAGAACACAGAGCTACACCAACAGCTTCACAAGAGGCGTGTACTACCCCGACAAGGTGTTCAGAAGCAGCGTCCTGCATTCCACCCAAGACCTGTTTCTCCCCTTCTTCAGCAACGTGACCTGGTTCCACGCCATCAGCGGCACCAACGGCACCAAGAGATTCGACAACCCCGTGCTGCCCTTCAACGACGGCGTGTACTTCGCTAGCACCGAGAAGAGCAACATCATCAGAGGCTGGATCTTCGGCACCACCCTGGACAGCAAGACACAGAGCCTGCTGATCGTGAACAACGCCACAAACGTGGTGATCAAGGTGTGCGAGTTTCAGTTCTGCAACGACCCCTTCCTGGACGTGTACTACCACAAGAACAACAAGAGCTGGATGGAGAGCGAGTTCCGGGTGTACAGCAGCGCCAACAACTGCACCTTCGAGTACGTGAGCCAACCCTTCCTGATGGACCTGGAGGGCAAGCAAGGCAACTTCAAGAACCTGAGAGAGTTCGTGTTCAAGAACATCGACGG

[0165] CTACTTCAAGATCTACAGCAAGCACACCCCCATCAACCTGGGCAGAGACCTGCCCCAAG

[0166] GCTTCAGCGCCCTGGAGCCCCTGGTGGACCTGCCCATCGGCATCAACATCACAAGATTT

[0167] CAGACACTGCTCGCCCTGCACCGGAGCTACCTCACCCCTGGCGACAGCAGCTCCGGCT

[0168] GGACCGCTGGGGCTCGCGCCTACTACGTGGGCTACCTGCAGCCTAGAACCTTCCTGCTG

[0169] AAGTAACGAGAACGGCACCATCACCGACGCTGTGGACTGTGCCCTGGACCCCCTGA

[0170] GCGAGACCAAGTGCACCCTGAAGAGCTTCACCGTGGAGAAGGGCATCTATCAGACAAG

[0171] CAACTTCAGAGTGCAGCCCACCGAGAGCATCGTGAGATTCCCCAACATCACCAACCTGT

[0172] GCCCCTTCGACGAGGTGTTCAACGCCACAAGATTCGCTAGCGTGTACGCCTGGAACAGA

[0173] AAGAGATCAGCAACTGCGTGGCCGACTACAGCGTGCTGTACAACTTCGCCCCCTTTCTT

[0174] CGCCTTCAAATGCTACGGCGTCAGCCCCACCAAGCTGAACGACCTGTGCTTCACCAACG

[0175] TGTACGCCGACAGCTTCGTGATCAGAGGCAACGAGGTGAGCCAAATCGCCCCCGGGCA

[0176] GACCGGCAACATCGCCGACTATAACTACAAACTGCCCGACGACTTCACCGGCTGCGTGA

[0177] TCGCCTGGAACTCCAACAAGCTGGACAGCAAAGTGGGCGGCAACTACAACTACAGATA

[0178] CAGACTGTTCAGAAAGAGCAACCTGAAGCCCTTCGAGAGAGACATCAGCACCGGAGATC

[0179] TACCAAGCCGGCAACAAGCCCTGCAACGGCGTGGCCGGCGTGAACTGCTACTTCCCCC

[0180] TGCAGAGCTACGGCTTCAGACCCACCTACGGCGTGGGCCATCAGCCCTACAGAGTGGTC

[0181] GTGCTGAGCTTCGAGCTGCTGCACGCCCCTGCCACAGTGTGCGGCCCCAAGAAGAGCA

[0182] CCAACCTGGTGAAGAACAAGTGCGTGAACTTCAACTTCAACGGCCTGACCGGCACCGG

[0183] CGTGCTGACCGAGAGCAACAAGAAGTTCCTGCCCTTTCAGCAGTTCGGCAGAGACATC

[0184] GCCGACACCACCGATGCTGTGAGAGACCCTCAGACCCTGGAGATCCTGGACATCACCCC

[0185] TTGCAGCTTCGGCGGCGTGAGCGTGATCACCCCCGGCACCAACACAAGCAACCAAGTG

[0186] GCCGTGCTGTACCAAGGCGTCAACTGCACAGAGGTGCCCGTGGCCATCCACGCCGATCA

[0187] GCTGACCCCCACCTGGAGAGTGTACTCCACCGGCAGCAACGTGTTTCAGACAAGAGCC

[0188] GGCTGCCTGATCGGCGCCGAGTACGTGAACAACAGCTACGAGTGCGACATCCCCATCGG

[0189] CGCCGGCATCTGCGCTAGCTATCAGACACAGACCAAGAGCCACAGAAGAGCTAGAAGC

[0190] GTGGCTAGCCAAAGCATCATCGCCTACACCATGAGCCTGGGCGCCGAGAACAGCGTGG

[0191] CCTACAGCAACAACAGCATCGCCATCCCCACCAACTTCACCATCAGCGTGACCACCGAG

[0192] ATCCTGCCTGTGAGCATGACCAAGACAAGCGTGGACTGCACCATGTACATCTGCGGCGA

[0193] CAGCACCGAGTGCAGCAACCTGCTCCTGCAGTACGGCAGCTTCTGCACACAGCTGAAG

[0194] AGAGCCCTGACCGGCATCGCCGTGGAGCAAGACAAGAACACCCAAGAGGTGTTCGCCC

[0195] AAGTGAAGCAGATCTACAAGACCCCCCCCATCAAGTACTTCGGCGGCTTCAACTTCAGC

[0196] CAAATCCTGCCCGACCCTAGCAAGCCTAGCAAGCGGAGCTTCATCGAGGACCTGCTGTT

[0197] CAACAAGGTGACCCTGGCCGACGCCGGCTTCATCAAGCAGTACGGCGACTGCCTCGGC

[0198] GACATCGCTGCTAGAGACCTGATCTGCGCTCAGAAGTTCAACGGCCTCACAGTGCTGCC

[0199] CCCCCTGCTGACCGACGAGATGATCGCTCAGTACACCTCCGCTCTGCTGGCCGGCACAA

[0200] TTACATCCGGCTGGACCTTCGGCGCCGGCGCCGCCCTGCAAATCCCCTTCGCCATGCAG

[0201] ATGGCCTACAGATTCAACGGCATCGGCGTGACACAGAACGTGCTGTACGAGAATCAGAA

[0202] GCTGATCGCCAATCAGTTCAACAGCGCCATCGGCAAGATCCAAGACAGCCTGAGCAGC

[0203] ACCGCTAGCGCCCTGGGCAAGCTGCAAGACGTGGTGAACCACAACGCCCAAGCCCTGA

[0204] ACACCCTGGTGAAGCAGCTGAGCAGCAAGTTCGGCGCCATCTCCTCCGTCCTGAACGA

[0205] CATCCTGAGCAGACTGGACCCCCCCGAGGCCGAGGTGCAGATTGACAGACTGATTACCG

[0206] GCAGACTGCAGAGCCTGCAGACCTACGTGACACAGCAGCTGATCAGAGCCGCCGAGAT

[0207] CAGAGCTAGCGCCAACCTGGCCGCCACCAAGATGAGCGAGTGCGTGCTGGGGCAGAGC

[0208] AAGAGAGTGGACTTCTGCGGCAAGGGCTACCACCTGATGAGCTTCCCTCAGAGCGCCC

[0209] CCCACGGCGTGGTGTTCCTGCACGTGACCTACGTGCCCGCCCAAGAGAAGAACTTCAC

[0210] CACCGCTCCCGCCATCTGCCACGACGGCAAGGCCCACTTCCCTAGAGAGGGCGTGTTCG

[0211] TGAGCAACGGCACCCACTGGTTCGTGACACAGAGAAACTTCTACGAGCCTCAGATCATC

[0212] ACCACCGACAACACCTTCGTGAGCGGCAACTGCGACGTGGTGATCGGCATCGTCAACA

[0213] ACACCGTGTACGACCCCCTGCAACCCGAGCTGGACAGCTTCAAGGAGGAGCTGGACAA

[0214] GTACTTTAAGAACCACACAAGCCCCGACGTGGACCTGGGGGACATCTCCGGCATCAAC

[0215] GCTAGCGTGGTGAACATTCAGAAGGAAATTGACAGACTGAATGAGGTGGCCAAGAACC

[0216] TGAACGAGAGCCTGATCGACCTGCAAGAGCTGGGCAAGTACGAGCAGTACATCAAGTG

[0217] GCCCTGGTACATCTGGCTGGGCTTCATCGCCGGCCTGATCGCCATCGTGATGGTGACCAT

[0218] CATGCTGTGCTGCATGACAAGCTGCTGCAGCTGTCTGAAGGGCTGCTGCTCCTGCGGCA

[0219] GCTGCTGCAAGTTCGACGAGGACGACAGCGAGCCCGTGCTGAAGGGCGTGAAGCTGC

[0220] ACTACACCTGA

[0221] Beneficial Effects: This invention primarily aims to prepare a recombinant protein vaccine against SARS-CoV-2 and its variants. The main recombinant protein sequence is constructed based on the RBD sequence of the S protein of SARS-CoV-2 Omicron variants BA.5 and XBB.1.5, and the recombinant sequence formed by combining the RBD sequence with the heptapeptide repeat regions HR1 and HR2 of the SARS-CoV-2 S protein. Furthermore, the addition of appropriate vaccine adjuvants enhances the host's resistance to cross-infection caused by the Omicron variant XBB and its subtypes, which is of significant importance for developing a recombinant protein vaccine against SARS-CoV-2 Omicron mutant strain XBB and its subtypes.

[0222] Meanwhile, this invention also constructs recombinant adenovirus vectors based on the nucleotide sequences of the full-length S protein of SARS-CoV-2 Omicron variants BA.5 and XBB.1.5 to obtain adenovirus vector vaccines. Different recombinant adenovirus vector vaccines are combined with two or three different recombinant protein vaccines to obtain vaccine compositions with better preventive or therapeutic effects against SARS-CoV-2 and its variants. The preparation of these compositions as nasal sprays is more conducive to resisting SARS-CoV-2 and its variants. Attached Figure Description

[0223] Figure 1This is the design diagram of pFastBac1-GP67-Trx-His-EK-S-RBD(Omicron_XBB.1.5)-HR in Example 1;

[0224] Figure 2 The images show 1% agarose gel electrophoresis images of the PCR products of clones 1-3 in Example 1.

[0225] Figure 3 The image shows a 1% agarose gel electrophoresis result of PCR identification of recombinant bacmid in Example 1.

[0226] Figure 4 This is a flowchart of the packaging and amplification process of recombinant baculovirus in Example 1;

[0227] Figure 5 This is a WB verification result diagram of the baculovirus amplification process in Example 1;

[0228] Figure 6 The results of Ni-affinity chromatography elution of the supernatant after baculovirus infection in Example 1;

[0229] Figure 7 This is a graph showing the results of EK enzyme digestion in Example 1 to verify the enzyme digestion effect.

[0230] Figure 8 The images shown are of EK enzyme digestion followed by purification using Ni-affinity chromatography and SDS-PAGE identification in Example 1.

[0231] Figure 9 Identification of XBB.1.5 adenovirus expression in Example 2;

[0232] Figure 10 Ad5 in Example 4 XBB.1.5 +RBD XBB.1.5 -HR bivalent vaccine serum IgG and irrigation fluid IgG / IgA;

[0233] Figure 11 Ad5 in Example 4 BA.5 +RBD BA.5 -HR bivalent vaccine serum IgG and irrigation fluid IgG / IgA;

[0234] Figure 12 Ad5 in Example 4 BA.5 +RBD XBB.1.5 -HR bivalent vaccine serum IgG and irrigation fluid IgG / IgA;

[0235] Figure 13 Ad5 in Example 4 XBB.1.5 +RBD BA.5 -HR bivalent vaccine serum IgG and irrigation fluid IgG / IgA;

[0236] Figure 14 Ad5 in Example 4 XBB.1.5 +RBD XBB.1.5 -HR+RBD BA.5 -HR trivalent vaccine serum IgG and lavage fluid IgG / IgA;

[0237] Figure 15 RBD in Example 5 XBB.1.5 Neutralizing antibodies in mouse serum from vaccine (aluminum adjuvant);

[0238] Figure 16 RBD in Example 5 XBB.1.5 -HR vaccine (MF59 adjuvant) mouse serum neutralizing antibodies;

[0239] Figure 17 RBD in Example 5 XBB.1.5 Neutralizing antibodies in the serum of vaccine-vaccine mice;

[0240] Figure 18 Ad5 in Example 5 XBB.1.5 +RBD XBB.1.5 -HR bivalent vaccine serum neutralizing antibodies;

[0241] Figure 19 Ad5 in Example 5 XBB.1.5 +RBD XBB.1.5 -HR bivalent vaccine bronchoalveolar lavage fluid neutralizing antibodies;

[0242] Figure 20 Ad5 in Example 5 XBB.1.5 +RBD XBB.1.5 -HR+RBD BA.5 -HR trivalent vaccine serum neutralizing antibodies;

[0243] Figure 21 The serum neutralizing antibody used in Example 6 is the true virus neutralizing antibody.

[0244] Figure 22 The timeline for mouse immunization challenge in Example 7;

[0245] Figure 23 The viral load in the mouse throat swabs in Example 7;

[0246] Figure 24 The viral gene / subgene RNA in Example 7;

[0247] Figure 25 The histopathological changes in mouse lung tissue in Example 7;

[0248] Figure 26 The histopathological score of the mouse lung tissue in Example 7. Detailed Implementation

[0249] Terminology Abbreviations:

[0250] Monophosphate lipid A (MPL); squalene oil-in-water emulsion (MF59), recombinant cholera toxin (rCTB); astragalus polysaccharide (APS); phosphatidylethanolamine (PE), phosphatidylcholine (PC), cholesterol (Chol), dioleoylphosphatidylethanolamine (DOPE), (2,3-dioleoyloxypropyl)trimethylammonium chloride (DOTAP), N-[1-(2,3-dioleoylchloro)propyl]-N,N,N-trimethylamine chloride (DOTMA), cationic cholesterol (DC-Chol), dimethyl-2,3-dioleenoyloxypropyl-2-(2-sperminecarbamoylamino)ethylammonium trifluoroacetate (DOSPA), trimethyldodecylammonium bromide (DTAB), trimethyltetradecylammonium bromide (TTAB), trimethylhexadecylammonium bromide (CTAB), dimethylbis(octadecylammonium bromide) (DDAB), CpG ODN (a nucleotide sequence containing unmethylated cytosine and guanine dinucleotides as the core sequence, artificially synthesized CpG)

[0251] This invention first designs a recombinant protein vaccine against SARS-CoV-2 Omeprone mutant strain XBB and its subtypes based on the amino acid sequence of positions 320-545 in the S protein of SARS-CoV-2 Omeprone mutant strains BA.5 and XBB.1.5, along with heptapeptide repeat region 1 (HR1) and heptapeptide repeat region 2 (HR2).

[0252] This invention also designs a recombinant adenovirus vaccine against SARS-CoV-2 Omeprone mutant strain XBB and its subtypes based on the nucleotide sequence encoding the full-length S protein of SARS-CoV-2 Omeprone mutant strains BA.5 and XBB.1.5.

[0253] This invention also combines different recombinant protein vaccines and different recombinant adenovirus vaccines to prepare bivalent and trivalent nasal spray formulations. By targeting the S protein of the SARS-CoV-2 virus, especially by blocking the ACE2 receptor binding region of the S protein, it induces the production of antibodies and other immune responses in the body, blocking the binding of the SARS-CoV-2 S protein to the ACE2 receptor of the host cell, thereby helping the host resist coronavirus infection. It has a good preventive and therapeutic effect on cross-infection caused by SARS-CoV-2 or its mutant viruses, such as cross-infection caused by the SARS-CoV-2 Omicron variant and its subtype XBB.1.5.

[0254] The present invention will be explained below with reference to embodiments. Those skilled in the art will understand that the following embodiments are for illustrative purposes only and should not be considered as limiting the scope of the invention. Where specific techniques or conditions are not specified in the embodiments, they are performed according to the techniques or conditions described in the literature in the field or according to the product instructions. Reagents or instruments whose manufacturers are not specified are all conventional products that can be obtained commercially.

[0255] Example 1: Preparation of recombinant protein (using S-RBD) by expression using an insect baculovirus system. XBB.1.5 (HR example)

[0256] 1. S-RBD XBB.1.5 -HR Construction Design

[0257] Because the S protein of SARS-CoV-2 is a membrane-bound protein, to simulate its secretion process, we added a GP67 signal peptide sequence to the N-terminus of the SARS-CoV-2 S-RBD(Omicron_XBB.1.5)-HR protein expression construct to assist in secretory expression. This signal peptide will be spontaneously cleaved by insect cells during protein secretion. Simultaneously, we added a thioredoxin (Trx) tag from the fall armyworm (S. frugiperda) after the GP67 signal peptide to assist in S-RBD(Omicron_XBB.1.5)-HR folding, a 6xhis tag to aid subsequent purification, and an EK enzyme cleavage site to cleave the Trx and 6xhis tags. This protein expression construct design, through EK cleavage, will be able to remove all redundant amino acids outside of S-RBD(Omicron_XBB.1.5)-HR. The pattern of this expression construct design is shown in [link to relevant documentation]. Figure 1 .

[0258] The amino acid sequence of GP67-Trx-His-EK-S-RBD(Omicron_XBB.1.5) is shown in SEQ ID No. 12, and the amino acid sequence of GP67-Trx-His-EK-S-RBD(Omicron_XBB.1.5)-HR is shown in SEQ ID No. 16. The nucleotide sequences encoding SEQ ID No. 12 and SEQ ID No. 16 are shown in SEQ ID No. 13 and SEQ ID No. 17, respectively.

[0259] Similarly, the amino acid sequence of GP67-Trx-His-EK-S-RBD(Omicron_XBB.BA.5)-HR is shown in SEQ ID No. 8, and the nucleotide sequence encoding its amino acid sequence is shown in SEQ ID No. 9.

[0260] The above sequences were all constructed based on the RBD sequence of the S protein from position 320 to 545, where amino acids at positions 52, 54, and 56 are F, P, and F, respectively.

[0261] 2. Identification of recombinant plasmid construction

[0262] Using SEQ ID No. 16 as the coding fragment, i.e., the XBB.1.5-3 sequence, the designed coding fragment was cloned into the pFastBac1 vector plasmid and identified by bacterial culture PCR. The bacterial culture PCR results showed that clones #2 and #3 of the three selected clones successfully amplified the GP67-Trx-His-EK-S-RBD(Omicron_XBB.1.5)-HR fragment. (See attached image) Figure 2 .

[0263] 3. Identification of recombinant bacmid

[0264] Select the correctly identified pFastBac1-GP67-Trx-His-EK-S-RBD(Omicron_XBB.1.5)-HR recombinant clone, extract the recombinant plasmid, transform DH10B competent cells, and perform blue-white bacterial culture PCR identification. The bacterial culture PCR products were detected by 1% agarose gel electrophoresis. The identification results are shown in the figure below. Figure 3 White spots represent bacmid clones that have undergone recombination, while blue spots represent bacmid clones that have not undergone recombination.

[0265] 4. Packaging of recombinant baculoviruses

[0266] Recombinant bacmid was transfected into sf9 insect cells, and P0 generation recombinant baculovirus was harvested after 5 days. A flowchart of recombinant baculovirus packaging and amplification is shown below. Figure 4 .

[0267] 5. Validation of target protein expression

[0268] During the amplification of the baculovirus described above, the target protein was expressed simultaneously. Before the tag was removed, the target protein contained a His tag; therefore, we used an anti-His Western blotting experiment to verify the expression of the recombinant protein. The verification results showed a distinct band in the 40KD–55KD marker band range, with a size consistent with the Trx-His-EK-S-RBD(Omicron_XBB.1.5)-HR protein, indicating successful baculovirus amplification and successful expression of the target protein. The detection results are shown below. Figure 5 .

[0269] 6. Purification and identification of the target protein

[0270] sf9 cells were infected with P0 generation recombinant baculovirus, and the cell culture medium was harvested after 3 days. Protein purification was then validated using Ni-affinity chromatography. Results are shown below. Figure 6 The target protein was mainly eluted with 40mM and 250mM imidazole, yielding a high-purity target protein after elution.

[0271] 7. Validation of EK restriction enzyme excision tag

[0272] The elution buffer of the target protein was concentrated and adjusted to a concentration of 1 mg / ml. EK enzyme was added, and the protein was digested at 18°C ​​for 14 hours. The results were then identified by SDS-PAGE gel electrophoresis. See below for details. Figure 7 The results show that the EK enzyme can remove the Trx-His-EK (amino acid sequence of the EK cleavage site) tag from the target protein.

[0273] 8. Verification of label removal after excision

[0274] After EK restriction enzyme digestion, the sample was back-suspended in Ni-affinity chromatography for 10 min. After sample flow-through, it was eluted with 250 mM imidazole. The Trx-His-EK-S-RBD(Omicron_XBB.1.5)-HR protein (uncleaned intact protein), S-RBD(Omicron_XBB.1.5)-HR protein, and the cleaved Trx-His-EK tag were separated. Results are shown below. Figure 8 The results showed that the Trx-His-EK tag could be removed by Ni-affinity chromatography followed by purification, thus yielding the tag-free S-RBD(Omicron_XBB.1.5)-HR protein. Its amino acid sequence is shown in SEQ ID No. 5.

[0275] Based on the amino acid sequences of the S protein RBD of SARS-CoV-2 Omicron_BA.5 and XBB.1.5 or the S protein RBD-HR of SARS-CoV-2 mutants, the applicant has provided protein construction designs as shown in SEQ ID No. 8, SEQ ID No. 12, and SEQ ID No. 16, respectively. The final expressed protein amino acid sequences are shown in SEQ ID No. 1, SEQ ID No. 3, and SEQ ID No. 5. RBDs were prepared from the expressed recombinant proteins. XBB.1.5 -HR、RBD XBB.1.5 RBD BA..5 -HR recombinant protein vaccine, for subsequent research such as animal immunization.

[0276] Example 2: Construction and preparation of recombinant adenovirus vaccine

[0277] 1. Optimization and synthesis of SARS-CoV-2 Omicron_BA.5 and Omicron_XBB.1.5S protein gene sequences.

[0278] according to https: / / covariants.org / The website provided mutation sites for the SARS-CoV-2 virus Omicron_BA.5 and Omicron_XBB.1.5. Nucleotide sequences of the S protein from various SARS-CoV-2 variants were obtained, while the signal peptides of each S protein mutant were preserved, and codons were optimized. Subsequently, the S protein genes of each variant were synthesized based on the mutated and optimized sequences.

[0279] The synthesized SARS-CoV-2 Omicron_BA.5 and Omicron_XBB.1.5S protein genes are shown in SEQ ID No. 23 and SEQ ID No. 21.

[0280] 2. Packaging of recombinant adenovirus COVID-19 vaccine

[0281] During gene synthesis, the synthesized product was cloned into the pDC316 vector using a recombinant cloning strategy, yielding the shuttle plasmid (pDC316-S). The pDC316-S vector, containing the S gene of the SARS-CoV-2 Omicron_BA.5 and Omicron_XBB.1.5 mutant strains, was then co-transfected with the AdMax adenovirus system backbone plasmid pBHGlox_E1,3Cre into HEK293 cells for recombinant adenovirus packaging. The process is as follows:

[0282] 1) 8×10 5HEK293A cells were seeded per well in six-well plates, cultured in high-glucose DMEM + 10% FBS medium, and incubated overnight at 37°C in a cell culture incubator containing 5% CO2.

[0283] 2) On the second day, change the medium with high-glucose DMEM + 2% FBS, and co-transfect HEK293A cells with the backbone plasmid (pBHGlox_E1,3Cre) and shuttle plasmid using lipofectamine 3000. The specific steps are as follows: Take 4 μg of backbone plasmid and 2 μg of shuttle plasmid from each transfection well, dilute with 125 μL of Opti-MEM medium, and then add 12 μL of P3000 reagent; In a separate 1.5 ml EP tube, dilute 7.5 μL of lipofectamine 3000 with 125 μL of Opti-MEM medium; mix the diluted plasmid and diluted lipofectamine 3000 at a 1:1 ratio, incubate at room temperature for 10-15 minutes, then add to the cells. Continue cell culture, and after the cells reach confluence, passage them in 25 cm⁻¹ cells. 2 In the cell culture flask, observe the signs of cell toxicity daily. Once the cells have filled the bottom of the flask, then transfer them to a 75cm culture medium. 2 In cell culture flasks, the cells are collected until obvious plaques appear and most of the cells become diseased and detach from the bottom.

[0284] 3) Collect the cell culture containing the virus, centrifuge at 1200 rpm for 3 minutes, and aspirate the virus-containing supernatant. Resuspend the cell pellet in 1 / 10 of the culture volume of virus-containing supernatant and freeze-thaw it three times in a -80°C freezer and a 37°C water bath. Centrifuge at 3000 rpm for 20 minutes, collect the virus-containing supernatant, and combine it with the aforementioned virus-containing supernatant. This is the seed virus for the adenovirus vaccine.

[0285] 4) Take 50 μL of vaccine candidate strain seed solution, add 2 μL of proteinase K, digest at 50℃ for 30 min to release viral genome, use this as a template to amplify the S gene sequence by PCR, and identify the PCR product by electrophoresis gel recovery and sequencing. The PCR amplification conditions are as follows:

[0286] Denaturation: 95℃, 10 min; Denaturation: 95℃, 10 s; Annealing: 64℃, 30 s; Extension: 72℃, 2 min; Extension: 72℃, 5 min; Cycle number: 40; Primers for PCR amplification are as follows:

[0287] pDC516-F1:ACACGTCAATGGGAAGTGAAA (SEQ ID No. 24)

[0288] pDC516-R1:GCTAGACGATCCAGACATGAT (SEQ ID No. 25)

[0289] 3. Amplification of recombinant adenovirus COVID-19 vaccine

[0290] The correctly identified recombinant adenovirus vaccine seed was amplified stepwise in 293 cells. The specific process is as follows: 80%-90% confluent 293 cells were added at an MOI of 10. After most cells became rounded with the virus, the virus culture was collected. Following the repeated freeze-thaw cycle described above, the master virus seed bank and working virus seed bank were prepared. The recombinant adenovirus vaccine was then amplified on a large scale using a cell factory or bioreactor. After most cells showed pathotropic effects, the virus culture was collected. The bioreactor amplification process for cells and virus is as follows: First, 3-5 g / L Lytodex1 microcarriers were added to the bioreactor and sterilized. Then, cell culture medium was added to the bioreactor. When the operating conditions stabilized at 37℃, pH 7.0, DO 50%, and 50 rpm, the HEK293 cells amplified in the cell factory were digested and collected, and then inoculated into the bioreactor at a cell density of 1.0-5.0 × 10⁶ cells / year. 5 Cells / ml were added, and cell culture medium was replenished to 5L. The cell culture conditions in the reactor were: temperature 37℃, rotation speed 30-50 rpm, pH 7.15-7.25, and DO 30-50%. Samples were taken daily to detect glucose concentration, cell density, and cell morphology on the microcarriers. When the cell density in the bioreactor reached 1.0-5.0 × 10⁶ cells / ml, the culture was continued until the desired cell density was reached. 6 When the cell / ml ratio is 1, the bioreactor is inoculated with recombinant adenovirus vaccine seed, with an MOI of 5-30. After inoculation, samples are taken daily to detect glucose concentration, virus titer in culture supernatant and cell pellet, and cell morphology on microcarriers. When most cells detach from the microcarriers, the culture is terminated, and virus lysis buffer is added to the bioreactor to a final concentration of 0.05%-1%. Lysis is performed at 37℃ for 2-4 hours with a Tween 20 reaction, and the virus solution is then collected.

[0291] 4. Purification of recombinant adenovirus vaccine

[0292] The collected virus was purified using cesium chloride ultracentrifugation or ion exchange chromatography, as detailed below:

[0293] (1) Cesium chloride ultracentrifugation purification of adenovirus vaccine

[0294] Centrifuge the collected virus culture at 1200g for 10 minutes, aspirate the virus-containing culture supernatant, resuspend the cell pellet in 1 / 10 of the culture volume of virus-containing supernatant, freeze and thaw repeatedly three times in a -80℃ freezer and a 37℃ water bath, centrifuge at 3000rpm for 10-20 minutes, and aspirate the supernatant. The virus-containing culture supernatant was concentrated 10-fold using a 100K-300K ultrafiltration membrane. Two cesium chloride solutions were prepared: 1.4 g / ml cesium chloride solution (53 g cesium chloride + 87 ml 10 mM Tris-HCl, pH 7.9) and 1.2 g / ml cesium chloride solution (26.8 g cesium chloride + 92 ml 10 mM Tris-HCl, pH 7.9). 8 ml of the 1.4 g / ml cesium chloride solution was slowly added to an ultrafiltration tube, followed by a gentle addition of 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 the discontinuous gradient. The mixture was balanced and centrifuged at 100,000 × g for 90 minutes at 4°C. After centrifugation, the blue virus band was aspirated using a syringe, dialyzed to remove cesium chloride, and then stored at -80°C.

[0295] (2) Ion exchange chromatography purification of adenovirus

[0296] Collect viral cultures and lyse them with 0.05%–1% Tween 20 at 37°C for 2–4 hours. Clarify the lysed cultures by filtering through 1.2 μm and 0.45 μm capsule filters. Concentrate the samples 10-fold using a tangential flow membrane with a molecular weight of 100–300 kDa. Then wash the samples with 10 volumes of wash buffer (50 mM Tris–HCl, 2 mM MgCl2, 0–500 mM NaCl, pH 8.0) and collect the washed samples. Add nuclease to the washed samples to a final concentration of 10–50 U / ml and digest at 37°C for 1–3 hours. Then digest the samples using Q Sepharose XL, Source 30Q, or Source... Anion exchange chromatography was performed using 15Q packing material, following these steps: Equilibrate the column with buffer at a flow rate of 20 ml / min for 5 column volumes. After equilibration, load the sample at a flow rate of 10 ml / min. After loading, equilibrate the buffer to the conductivity level. Elute the sample using a linear gradient: from 100% low-salt buffer to 100% high-salt buffer, 10V elution column volume, and a flow rate of 10 ml / min. Collect each elution peak. After elution, regenerate the column with 2M NaCl buffer for 5–10 column volumes at a flow rate of 20 ml / min. Collect the viral peak. Subsequently, the eluted viral sample is subjected to buffer replacement via dialysis or tangential flow filtration (buffer solution: 10 mM Tris, 10 mM Na-PO4, 150 mM NaCl, 2 mM MgCl2, 2% sucrose, 0.15% glycerol, 0.02% Tween 80, pH 7.6).

[0297] The purified adenovirus was directly filled into containers and stored at -20°C in the dark.

[0298] 5. Identification of recombinant adenovirus vaccines

[0299] We first expressed the full-length spike glycoproteins of the SARS-CoV-2 mutant strains OmicronBA.5 and Omicron XBB.1.5 using a human replication-defective Ad5 adenovirus vector, and then detected the Ad5 spike glycoproteins using Western blot. XBB.1.5 and Ad5 BA.5 The expression level of the spike protein Spike in 293T cells 48 hours after infection with recombinant adenovirus. Figure 9 As shown, adenovirus Ad5 XBB.1.5 Infection of 293T cells can induce high levels of Spike protein expression, while the empty adenovirus control Ad5Empty cannot, proving that our recombinant adenovirus vaccine has been successfully prepared.

[0300] The adenovirus vaccines prepared in Example 1 are defined as Ad5. XBB.1.5 and Ad5 BA.5 Recombinant adenovirus.

[0301] The following experiments demonstrate the effectiveness of the recombinant protein vaccine and adenovirus vaccine prepared according to this invention.

[0302] Example 3 Animal preparation, mouse immunization, and sample collection

[0303] 1. Female NIH mice aged 6-8 weeks were purchased from Vital River Pharmaceuticals and housed in a specific pathogen-free environment at the State Key Laboratory of Biotherapy, Sichuan University. 10 μg of recombinant protein was mixed with a certain amount of adjuvant and administered intramuscularly to the mice on days 0, 14, and 28, with 10 μg protein per mouse. Blood samples were collected 7 days after the third immunization to detect neutralizing antibodies and evaluate the vaccine's immunogenicity.

[0304] 2. Female BALB / c mice aged 6-8 weeks were purchased from Vital River Pharmaceuticals and housed in a specific pathogen-free environment at the State Key Laboratory of Biotherapy, Sichuan University. To prepare the two-component nasal spray vaccine, 2.5 × 10⁻⁶ mice were used. 9 VP (low dose) or 5×10 9 VP (high dose) of Ad5 XBB.1.5 With 10μg RBD XBB.1.5 -HR were mixed in a total volume of 50 μl to prepare low-dose or high-dose two-component vaccines, respectively. (1) Low-dose and (2) high-dose Ad5 were used. XBB.1.5 BALB / c mice were immunized three times individually, with (3) low-dose and (4) high-dose two-component (Ad5) vaccine. XBB.1.5 +RBD XBB.1.5-HR) vaccine, (5) 10 μg RBD alone XBB.1.5 -HR protein, or (6) 5×10 9 VP's Ad5 Empty With RBD XBB.1.5 -HR protein mixture was delivered intranasally 28 days later. To avoid excessive fluid inflow into the lung tissue, mice were immunized twice intranasally within one day, 25 μl each time, with an interval of more than 3 hours.

[0305] Other two-component vaccines Ad5 BA.5 +RBD BA.5 -HR, Ad5 BA.5 +RBD XBB.1.5 -HR, Ad5 XBB.1.5 +RBD BA.5 The preparation and immunization protocol for -HR are similar to those described above.

[0306] From 5×10 9 VP's Ad5 XBB.1.5 3.3 μg of RBD BA.5 -HR and 6.6μg RBD XBB.1.5 The three-component vaccine, consisting of -HR components, was administered intranasally according to the same immunization schedule. Serum samples were collected from vaccinated animals at weeks 3, 7, and 11 to determine binding and neutralizing antibody responses. To further evaluate vaccine-induced mucosal immunity, mice were euthanized on day 21 following the third booster vaccination to collect BALF and lung tissue.

[0307] Example 4: Detection of Antibodies by Enzyme-Linked Immunosorbent Assay (ELISA)

[0308] To detect RBD-specific IgG and IgA against the novel coronavirus, 96-well plates (NUNC-MaxiSorp, ThermoFisher Scientific) were coated with 1 μg / ml recombinant RBD protein in carbonate-bicarbonate buffer and incubated overnight at 4°C. The next day, the plates were washed three times with 1×PBS containing 0.1% Tween-20 (PBST) and then blocked with PBST containing 1% BSA at room temperature for 1 hour. Serially diluted serum, bronchoalveolar lavage fluid, or nasal swabs in dilution buffer were added to the wells (100 μl / well). After incubating at 37°C for 1 hour, the plates were washed three times, and then diluted horseradish peroxidase (HRP)-conjugated goat-anti-mouse IgG (1:10000, Southern Biotech, Cat: 0107-05), HRP-conjugated goat-anti-mouse IgA (1:5000), HRP-conjugated goat-anti-rabbit IgG, HRP-conjugated goat-anti-rabbit IgG, HRP-conjugated goat-anti-human IgG (Southern Biotech, Cat: 62-8420), or HRP-conjugated goat-anti-human IgA (Southern Biotech, Cat: 2050-05) were added. The plates were then incubated at 37°C for 1 hour, washed three more times, and developed with 3,3',5,5'-tetramethylbenzidine (TMB) for 10 minutes at room temperature. The reaction was stopped with 1M H₂SO₄. Finally, absorbance at 450 nm was measured using a microplate reader (SpectramaxABS, Molecular Devices). The endpoint titer was defined as the highest reciprocal dilution of serum with absorbance ≥ 2.1 times that of the negative control serum.

[0309] The results are as follows Figure 10-14 As shown, after immunization, the combination of recombinant adenovirus Ad5 and recombinant RBD induced stronger serum and bronchoalveolar lavage fluid anti-RBD-specific binding antibodies than Ad5 adenovirus alone, indicating that adenovirus combined with recombinant subunit preparations can produce stronger humoral immune protection. Furthermore, nasal immunization with RBD alone failed to induce strong blood-binding antibodies, but with empty Ad5... Empty The immunogenicity of recombinant adenovirus can be significantly enhanced upon binding to Ad5, demonstrating that adenovirus can act as an adjuvant for recombinant subunits to enhance the immunogenicity of protein antigens.

[0310] Example 5: Neutralization test of novel coronavirus pseudovirus

[0311] To detect the titers of neutralizing antibodies in serum and BALF samples, a pseudovirus neutralization assay was performed as previously described. Luciferase-expressing pseudoviruses, including the prototype, delta, and Omicron subspecies (BA.2.75, BA.5, BF.7, BQ.1, BQ.1.1, XBB, XBB.1.5, and XBB.1.16, etc.), were purchased from Genomeditech.

[0312] In short, inactivated serum and BALF samples (56°C for 30 minutes) were diluted three-fold, ranging from 30 to 65610, and then incubated with an equal volume of pseudovirus at different dilutions at 37°C for 1 hour. Then, 1.2 × 10⁻⁶ ppm of the solution was added to each well. 4 HEK-293T cells expressing the human ACE2 receptor (293T / ACE2) were incubated at 37°C for 48 hours to express luciferase. Finally, the supernatant was removed, and a lysis reagent (Beyotime, RG005) containing the luciferase substrate was added. The luminescence intensity in the 293T / ACE2 cells was measured using a multimode microplate reader (PerkinElmer, USA). The 50% neutralization rate of the pseudovirus was determined and calculated using GraphPad Prism 8.0.2. The positive control group contained only cells and virus, the negative control group contained only cells, and the sample group contained cells, sample, and virus. The neutralization percentage was calculated using the following formula:

[0313] Neutralization rate (%) = (Positive samples - Test samples / Positive samples - Negative samples) × 100%

[0314] like Figure 15-16 As shown, simple protein vaccines (recombinant RBD) XBB.1.5 -HR and Reorganized RBD XBB.1.5 After completing three doses of immunization, mice produced strong serum neutralizing antibodies against viruses such as WT, Delta, BA.2.75, BF.7, BA.5, BQ.1, BQ.1.1, XBB, and XBB.1.5, and the neutralizing antibodies induced by MF59 adjuvant were significantly better than those induced by aluminum adjuvant. Figure 17 The data shows that 25U / v / v was administered intramuscularly three times on days 0, 14, and 42, and then administered an inactivated vaccine (produced by China National Pharmaceutical Group Co., Ltd.) and RBD vaccine 84 days after the last immunization. XBB.1.5 Sequential booster, intramuscular injection, immunization dose of 10 μg / animal, serum collected 14 days after booster to detect pseudovirus neutralizing antibodies, and then inoculated with RBD. XBB.1.5 Mice vaccinated with the vaccine produced strong serum neutralizing antibodies against viruses such as WT, BA.5, XBB.1.5, XBB.1.6, XBB.1.16, XBB.1.9.1, and XBB.2.3, which were particularly effective against the recently dominant XBB subtypes and other circulating strains.

[0315] A bivalent vaccine formulation combining adenovirus and recombinant RBD protein induced stronger neutralizing antibodies in serum and bronchoalveolar lavage fluid than adenovirus alone, indicating that the combination of adenovirus and recombinant subunit formulation can produce stronger local neutralizing protection in blood and mucous membranes, preventing viral infection; Figure 18-20 As shown, Ad5 XBB.1.5 +RBD XBB.1.5 -HR produced high levels of serum neutralizing antibodies against viruses such as WT, Delta, BA.2.75, BF.7, BA.5, BQ.1, BQ.1.1, XBB, XBB.1.5, and XBB.1.16, suggesting that Omicron's S protein adenovirus vaccine combined with the S-RBD protein can produce stronger immune protection against Omicron mutant strains.

[0316] Example 6: Neutralization Test of Live Novel Coronavirus

[0317] Based on real virus neutralization tests, derived from Ad5 inoculation XBB.1.5 +RBD XBB.1.5 Neutralizing antibodies against live ancestral and mutant novel coronaviruses were obtained from mouse serum samples of the -HR bivalent vaccine. Diluted serum from each group was mixed with live novel coronavirus at 50% tissue culture infection dose (TCID50). After incubation at 37°C for 1 hour, the mixture was added to Vero E6 cells (5 × 10⁻⁶ cells). 4 Incubate in 96-well microplates (1 / well) for 72 hours. Cellular pathogenic effect (CPE) is measured under a microscope, and titers of neutralizing antibodies in immune serum that lead to EC50 (50% neutralization) inhibition are calculated.

[0318] The results show that... Figure 21 As shown, Ad5 nasal drops XBB.1.5 +RBD XBB.1.5 The neutralizing antibody titers of mouse serum from the -HR bivalent vaccine against the Delta and Omega-Jon variants of the true virus, especially the recently prevalent strain XBB.1.16, reached over 1000, and were significantly higher than those of adenovirus and protein administered via intranasal drops alone.

[0319] Example 7: Attack by the novel coronavirus XBB.1.16 variant

[0320] Use three times Ad5 on days 0, 28, and 56. XBB.1.5 BALB / c mice were intranasally immunized with a single, two-component, or three-component vaccine. The vaccine was administered with PBS or Ad5. Empty Immunized mice served as controls, with n = 6 mice in each group. Then, on day 21 after the last vaccination, mice were administered live SARS-CoV-2 XBB.1.16 Omeprone mutant (1 × 10⁻⁶). 6All mice were challenged intranasally with PFU. Four days post-infection, mice were euthanized and tissues were collected. Hematoxylin and eosin staining was used to observe pathological changes in lung tissue. Viral load in nasal turbinate, tracheal, and lung tissue samples was detected by reverse transcription quantitative polymerase chain reaction (RT-qPCR) measuring viral genomic RNA (gRNA). Primer sequences were 5′-GACCCCAAAATCAGCGAAAT-3′ (forward) (SEQ ID No. 26), 5′-TCTGGTTACTGCCCAGTTGAATCTG-3′ (reverse) (SEQ ID No. 27), and probe sequence was 5′-FAM-ACGCCGCATTACGTTTGGTGGGACC-BHQ1-3′ (SEQ ID No. 28). All procedures related to the mouse challenge with the novel coronavirus omega-3 variant were reviewed and approved by the Institutional Animal Care and Use Committee of the Institute of Medical Biology, Chinese Academy of Medical Sciences, and performed at the ABSL-4 facility of the National Advanced Biosafety Primate Research Center in Kunming.

[0321] Use Ad5 XBB.1.5 and two components (Ad5) XBB1.5 +RBD XBB.1.5 BALB / c mice were immunized three times intranasally with the -HR) vaccine. The vaccine was administered via PBS and naked RBD. XBB.1.5 -HR、Ad5Empty+RBD XBB-1.5 -HR-treated mice were used as controls. 1×10 6 BALB / c mice immunized with live PFU-treated SARS-CoV-2 XBB.1.16 Omecron virus were used to test viral load changes in pharyngeal swabs after SARS-CoV-2 infection. Nasal turbinates, trachea, and lung tissues were collected on day 4 post-infection, and gRNA and sgRNA levels were measured by RT-qPCR. Histopathological changes in lung tissues of mice after Omecron challenge were observed.

[0322] like Figure 22-26 As shown, using Ad5 XBB.1.5 (5×10 9 VP / each) + RBD XBB.1.5In mice treated with HR (10 μg) combined with vaccine-induced immune serum, the virus in pharyngeal swabs was essentially cleared. Viral RNA was undetectable in the nasal turbinates, trachea, and lung tissues. Lung histology was normal, alveolar structure was intact, and there was no obvious inflammation. In contrast, in the PBS control group, pharyngeal swab samples still showed a relatively high viral load, with high levels of viral RNA in the nasal turbinates, trachea, and lung tissues. Pathological sections showed mild pathological changes, including multifocal consolidation areas, mild alveolar septal thickening, and alveolar congestion. In addition, small patches of inflammation composed of macrophages, neutrophils, and lymphocytes were occasionally observed near small blood vessels. This indicates that Ad5... XBB.1.5 +RBD XBB.1.5 -HR bivalent vaccine provides complete protection against infection with the XBB.1.16 Omeprón variant.

Claims

1. Proteins resisting infection by the SARS-CoV-2 Omeprone mutant strain XBB and its subtypes, characterized by: It is selected from the amino acid sequence shown in SEQ ID No. 3 or SEQ ID No.

5.

2. The protein precursor of claim 1, characterized in that: It involves attaching a signal peptide and / or a protein tag to the protein.

3. The protein precursor according to claim 2, characterized in that: The protein tag is selected from at least one of the following: histidine tag, thioredoxin tag, glutathione transferase tag, ubiquitin-like modified protein tag, maltose-binding protein tag, c-Myc protein tag, Avi tag protein tag, and nitrogen-utilizing substance A protein tag.

4. The protein precursor according to claim 3, characterized in that: in The protein that resists infection by the SARS-CoV-2 Omeprone mutant strain XBB and its subtypes is also linked to a protease recognition region that removes the protein tag; the protease is selected from at least one of the following: enterokinase, TEV protease, thrombin, coagulation factor Xa, carboxypeptidase A, and rhinovirus 3c protease.

5. The protein precursor according to any one of claims 2-4, characterized in that: The amino acid sequence of the precursor is selected from SEQ ID No. 12 or SEQ ID No.

16.

6. Polynucleotides, characterized by: It encodes the protein of claim 1 or the protein precursor of any one of claims 2-5.

7. The polynucleotide according to claim 6, characterized in that: The nucleotide sequence of the polynucleotide is selected from SEQ ID No. 13 or SEQ ID No.

17.

8. A recombinant vector, characterized by: It contains the polynucleotide as described in claim 6 or 7.

9. The recombinant vector according to claim 8, characterized in that: The recombinant vector is at least one of an insect baculovirus expression vector, a mammalian cell expression vector, an Escherichia coli expression vector, or a yeast expression vector; the insect baculovirus expression vector is pFastBac1; the Escherichia coli expression vector is pET32a; the yeast expression vector is pPICZaA; and the mammalian cell expression vector is a CHO cell expression vector.

10. The recombinant vector according to claim 9, characterized in that: The CHO cell expression vector is pTT5 or FTP-002.

11. The host cell, characterized by: It contains the recombinant vector as described in any one of claims 8-10.

12. The host cell according to claim 11, characterized in that: The host cell is at least one of insect cells, mammalian cells, Escherichia coli, or yeast.

13. The host cell according to claim 12, characterized in that: The insect cells are selected from at least one of sf9 cells, sf21 cells, or Hi5 cells; the mammalian cells are CHO cells.

14. A method for preparing the protein according to claim 1 or the protein precursor according to any one of claims 2-5, characterized in that: The procedure includes the following steps: culturing the host cell according to any one of claims 11-13 to express the protein or protein precursor, and then recovering the protein.

15. A protein composition for infection with SARS-CoV-2 Omeprón mutant strain and its subtypes, characterized in that: It comprises a combination of two proteins; the amino acid sequences of the proteins are shown in SEQ ID No. 3 and SEQ ID No.

5.

16. A recombinant protein vaccine for preventing infection with SARS-CoV-2 Omeprone mutant strains and their subtypes, characterized by: It contains the protein of claim 1, the protein precursor of any one of claims 2-5 and / or the protein composition of claim 15, and pharmaceutically acceptable excipients or auxiliary ingredients.

17. The recombinant protein vaccine according to claim 16, characterized in that: The auxiliary component is an immune adjuvant; the immune adjuvant is selected from at least one of the following: squalene oil-in-water emulsion, aluminum salt, calcium salt, plant saponins, plant polysaccharides, monophosphate lipid A, muramyl dipeptide, muramyl tripeptide, bacterial toxin, GM-CSF cytokine, lipid, cationic liposome material.

18. The recombinant protein vaccine according to claim 16 or 17, characterized in that: Meet at least one of the following: The squalene oil-in-water emulsion is MF59; the aluminum salt is selected from at least one of aluminum hydroxide and alum; the calcium salt is tricalcium phosphate; the plant saponin is QS-21 or ISCOM; the plant polysaccharide is astragalus polysaccharide; the bacterial toxin is selected from at least one of recombinant cholera toxin and diphtheria toxin; the lipid is selected from at least one of the following: phosphatidylethanolamine, phosphatidylcholine, cholesterol, dioleoylphosphatidylethanolamine; the cationic liposome material is selected from at least one of the following: (2,3-dioleoyloxypropyl)trimethylammonium chloride, N-[1-(2,3-dioleoylchloro)propyl]-N,N,N-trimethylamine chloride, cationic cholesterol, trifluoroacetic acid dimethyl-2,3-dioleenoyloxypropyl-2-(2-speramidylamino)ethylammonium, trimethyl dodecylammonium bromide, trimethyl tetradecylammonium bromide, trimethyl hexadecylammonium bromide, dimethyl dioctadecylammonium bromide, CpG ODN.

19. The recombinant protein vaccine according to any one of claims 17-18, characterized in that, The recombinant protein vaccine is a formulation for intradermal or subcutaneous injection, intramuscular injection, intravenous injection, oral or nasal spray.

20. A vaccine composition for preventing infection with SARS-CoV-2 Omeprón mutant strains and their subtypes, characterized in that: A compound preparation containing the recombinant protein vaccine and adenovirus vector vaccine as active ingredients according to any one of claims 16-19; the adenovirus vector vaccine contains a polynucleotide sequence as shown in SEQ ID No. 21; the recombinant protein vaccine contains an amino acid sequence as shown in SEQ ID No. 3 or SEQ ID No.

5.

21. A combination of drugs for the prevention of infection with SARS-CoV-2 Omeprone mutant strains and their subtypes, characterized by: It contains a recombinant protein vaccine and an adenovirus vector vaccine as described in any one of claims 16-19, administered separately or simultaneously; the adenovirus vector vaccine contains a polynucleotide sequence as shown in SEQ ID No. 21; the recombinant protein vaccine contains an amino acid sequence as shown in SEQ ID No. 3 or SEQ ID No.

5.

22. The vaccine composition according to claim 20, characterized in that: It contains A combination of recombinant protein vaccine 1, recombinant protein vaccine 2, and adenovirus vector vaccine; wherein recombinant protein vaccine 1 contains an amino acid sequence as shown in SEQ ID No. 1; recombinant protein vaccine 2 contains an amino acid sequence as shown in SEQ ID No. 3 or SEQ ID No. 5; and adenovirus vector vaccine contains a polynucleotide sequence as shown in SEQ ID No.

21.

23. The combination drug according to claim 21, characterized in that: It contains a recombinant protein vaccine 1, a recombinant protein vaccine 2, and an adenovirus vector vaccine, administered separately or simultaneously; the recombinant protein vaccine 1 contains an amino acid sequence as shown in SEQ ID No. 1; the recombinant protein vaccine 2 contains an amino acid sequence as shown in SEQ ID No. 3 or SEQ ID No. 5; and the adenovirus vector vaccine contains a polynucleotide sequence as shown in SEQ ID No.

21.

24. The vaccine composition according to claim 20, characterized in that: The adenovirus vector for the adenovirus vector vaccine is selected from at least one of the following: adenovirus, Ankara vaccinia virus, and adeno-associated virus.

25. The vaccine composition according to claim 24, characterized in that: adenovirus The vectors were selected from human type 5, 35 or 26 and / or chimpanzee AdC68 or AdC7 replication-defective adenoviruses.

26. The vaccine composition according to claim 25, characterized in that: The adenovirus vector was selected from human type 5 replication-defective adenovirus with combined deletions of E1 and E3.

27. The vaccine composition according to claim 20, characterized in that: The adenovirus vector vaccine also includes pharmaceutically acceptable adjuvants, vectors, diluents, or excipients.

28. The vaccine composition according to claim 20, characterized in that: The method for preparing the adenovirus in the adenovirus vector vaccine includes the following steps: constructing the polynucleotide shuttle plasmid vector; then transfecting the constructed shuttle plasmid vector and the backbone plasmid together into host cells and culturing the host cells; obtaining the replication-deficient recombinant adenovirus, and then expanding the culture and purifying it.

29. The vaccine composition according to claim 20, characterized in that: Adenovirus vector vaccines are available as intradermal or subcutaneous injections, intramuscular injections, intravenous injections, oral or nasal sprays.

30. The combination drug according to claim 21, characterized in that: The adenovirus vector for the adenovirus vector vaccine is selected from at least one of the following: adenovirus, Ankara vaccinia virus, and adeno-associated virus.

31. The combination drug according to claim 30, characterized in that: adenovirus. The vectors were selected from human type 5, 35 or 26 and / or chimpanzee AdC68 or AdC7 replication-defective adenoviruses.

32. The combination drug according to claim 31, characterized in that: The adenovirus vector was selected from human type 5 replication-defective adenovirus with combined deletions of E1 and E3.

33. The combination drug according to claim 21, characterized in that: The adenovirus vector vaccine also includes pharmaceutically acceptable adjuvants, vectors, diluents, or excipients.

34. The combination drug according to claim 21, characterized in that: The method for preparing the adenovirus in the adenovirus vector vaccine includes the following steps: constructing the polynucleotide shuttle plasmid vector; then transfecting the constructed shuttle plasmid vector and the backbone plasmid together into host cells and culturing the host cells; obtaining the replication-deficient recombinant adenovirus, and then expanding the culture and purifying it.

35. The combination drug according to claim 21, characterized in that: Adenovirus vector vaccines are available as intradermal or subcutaneous injections, intramuscular injections, intravenous injections, oral or nasal sprays.

36. The vaccine composition according to claim 20, characterized in that: The composition is an intradermal or subcutaneous injection preparation, an intramuscular injection preparation, an intravenous injection preparation, an oral or nasal spray preparation.

37. The combination drug according to claim 21, characterized in that: The combined medications are intradermal or subcutaneous injection preparations, intramuscular injection preparations, intravenous injection preparations, oral or nasal spray preparations.

38. Use of the protein of claim 1, the protein precursor of any one of claims 2-5, the protein composition of claim 15, the recombinant protein vaccine of any one of claims 16-19, the vaccine composition of any one of claims 20, 22, 24-29, 36, or the combination drug of any one of claims 21, 23, 30-35, 37 in the preparation of a drug for preventing infection or pathogenicity of SARS-CoV-2 Omeprón mutant strains and their subtypes.

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