WO2024082795A1 - 抗SARS-CoV-2奥密克戎突变株XBB及其亚型感染的蛋白及疫苗 - Google Patents

抗SARS-CoV-2奥密克戎突变株XBB及其亚型感染的蛋白及疫苗 Download PDF

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WO2024082795A1
WO2024082795A1 PCT/CN2023/113170 CN2023113170W WO2024082795A1 WO 2024082795 A1 WO2024082795 A1 WO 2024082795A1 CN 2023113170 W CN2023113170 W CN 2023113170W WO 2024082795 A1 WO2024082795 A1 WO 2024082795A1
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seq
protein
xbb
vaccine
sequence
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PCT/CN2023/113170
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French (fr)
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魏霞蔚
逯光文
程平
杨莉
李炯
王玮
杨静云
魏于全
王震玲
沈国波
杨金亮
赵志伟
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成都威斯克生物医药有限公司
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    • C07—ORGANIC CHEMISTRY
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    • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
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    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C12N15/86—Viral vectors
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
    • A61K2039/53—DNA (RNA) vaccination
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/54—Medicinal preparations containing antigens or antibodies characterised by the route of administration
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    • C07—ORGANIC CHEMISTRY
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    • C07K2319/00—Fusion polypeptide
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    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
    • C07K2319/02—Fusion polypeptide containing a localisation/targetting motif containing a signal sequence
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/20—Fusion polypeptide containing a tag with affinity for a non-protein ligand
    • C07K2319/21—Fusion polypeptide containing a tag with affinity for a non-protein ligand containing a His-tag
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/35—Fusion polypeptide containing a fusion for enhanced stability/folding during expression, e.g. fusions with chaperones or thioredoxin
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    • C12N2710/10011—Adenoviridae
    • C12N2710/10311—Mastadenovirus, e.g. human or simian adenoviruses
    • C12N2710/10341—Use of virus, viral particle or viral elements as a vector
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    • C12N2770/00011—Details
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    • C12N2770/00011—Details
    • C12N2770/20011—Coronaviridae
    • C12N2770/20034—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein
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    • C12N2800/00—Nucleic acids vectors
    • C12N2800/10—Plasmid DNA
    • C12N2800/106—Plasmid DNA for vertebrates
    • C12N2800/107—Plasmid DNA for vertebrates for mammalian
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • the present invention relates to proteins and vaccines for resisting infection of SARS-CoV-2 Omicron mutant strain XBB and its subtypes, belonging to the field of medicine.
  • the new coronavirus (SARS-Cov-2) is a new type of beta coronavirus named by the World Health Organization.
  • the virus has an envelope, and the particles are round or oval, often polymorphic, with a diameter of 60-140nm. Its genetic characteristics are significantly different from those of SARS-CoV and MERS-CoV, and it is a new coronavirus branch that has not been found in humans before.
  • the Omicron variants are further divided into several sub-lineages such as BA.1, BA.2, BA.2.12.1, BA.4, BA.5, BQ.1.1, and XBB.1.5.
  • SARS-CoV-2 spike protein
  • E messenger protein
  • M membrane protein
  • N nucleocapsid protein
  • S protein plays a key role in the infection and virulence of the virus and is often used as a vaccine antigen.
  • SARS-CoV-2 variant XBB contains multiple mutation sites
  • the virus's S protein also contains multiple mutation sites, which causes the variant XBB and its subtypes to escape antibodies stimulated by the new coronavirus mutant strain (Alpha, Beta, Gamma, Delta, Omicron) vaccine to a certain extent, resulting in the failure of the new coronavirus vaccine or reduced protection, which brings great pressure to the prevention and control of the new coronavirus epidemic. Therefore, it is very important to develop vaccines against the SARS-CoV-2 virus mutant strain XBB and its subtypes, especially broad-spectrum vaccines for various variants of SARS-CoV-2 viruses, for the prevention and treatment of new coronavirus pneumonia.
  • the present invention aims to solve at least one of the technical problems existing in the prior art.
  • the purpose of the present invention is to provide a protein that resists infection by SARS-CoV-2 Omicron mutant strain XBB and its subtypes.
  • the present invention also provides a vaccine for preventing and/or treating infection by SARS-CoV-2 Omicron mutant strain XBB and its subtypes, including a recombinant protein vaccine and an adenovirus vector vaccine containing the protein, and also provides a composition or a combined drug containing the two vaccines.
  • the present invention first provides a protein for resisting infection by SARS-CoV-2 Omicron mutant strain XBB and its subtypes, which contains an amino acid sequence selected from any one of SEQ ID No. 1 to SEQ ID No. 7.
  • the protein is selected from SEQ ID No. 3 or SEQ ID No. 5.
  • the present invention also provides a precursor of the protein, which is a precursor of the protein to which a signal peptide and/or a protein tag is connected.
  • 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, nitrogen source utilization substance A protein tag.
  • protease recognition region for removing the protein tag is also connected to the protein that resists infection of the SARS-CoV-2 Omicron mutant strain XBB and its subtypes.
  • the protease is selected from at least one of the following: enterokinase (EK enzyme), TEV protease, thrombin, coagulation factor Xa, carboxypeptidase A, rhinovirus 3c protease.
  • EK enzyme enterokinase
  • TEV protease TEV protease
  • thrombin thrombin
  • coagulation factor Xa carboxypeptidase A
  • rhinovirus 3c protease e.
  • 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.
  • the protein sequence is selected from SEQ ID No. 12 or SEQ ID No. 16.
  • the present invention also provides a polynucleotide encoding the protein or the precursor.
  • 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.
  • polynucleotide sequence is selected from SEQ ID No. 13 or SEQ ID No. 17.
  • the present invention also provides a recombinant vector containing the polynucleotide.
  • the recombinant vector adopts at least one of an insect baculovirus expression vector, a mammalian cell expression vector, an Escherichia coli expression vector, and a yeast expression vector.
  • the insect baculovirus expression vector is pFastBac1.
  • the E. coli expression vector is pET32a.
  • the yeast expression vector is pPICZaA.
  • the mammalian cell expression vector is a CHO cell expression vector.
  • the CHO cell expression vector is pTT5 or FTP-002.
  • the present invention also provides a host cell, which contains the recombinant vector.
  • the host cell is at least one of insect cells, mammalian cells, Escherichia coli, and yeast.
  • the insect cells are selected from at least one of sf9 cells, sf21 cells and Hi5 cells.
  • the mammalian cells are CHO cells.
  • the present invention also provides a method for preparing the protein or the precursor, which is characterized in that it comprises the following steps: culturing the host cell to express the protein or the precursor, and then recovering the protein.
  • the present invention also provides a protein composition for resisting infection by SARS-CoV-2 Omicron mutant strains and their subtypes, which includes a combination of any two or more 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 proteins.
  • 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 proteins.
  • 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.
  • the present invention also provides a recombinant protein vaccine for preventing and/or treating infection by SARS-CoV-2 Omicron mutant strains and their subtypes, which contains the protein, the precursor and/or the protein composition, and pharmaceutically acceptable excipients or auxiliary ingredients.
  • 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 saponin, plant polysaccharide, monophosphoryl lipid A, muramyl dipeptide, muramyl tripeptide, bacterial toxin, GM-CSF cytokine, lipid, cationic liposome material.
  • the squalene oil-in-water emulsion is MF59;
  • the aluminum salt is selected from at least one of aluminum hydroxide and alum;
  • the calcium salt is tricalcium phosphate
  • the plant saponin is QS-21 or ISCOM;
  • the plant polysaccharide is astragalus polysaccharide
  • the bacterial toxin is selected from at least one of recombinant cholera toxin and diphtheria toxin;
  • the lipid is selected from at least one of the following: phosphatidylethanolamine, phosphatidylcholine, cholesterol, dioleoylphosphatidylethanolamine;
  • the cationic liposome material is selected from at least one of the following: (2,3-dioleyloxypropyl)trimethylammonium chloride, N-[1-(2,3-dioleoyl chloride)propyl]-N,N,N-trimethylammonium chloride, cationic cholesterol, dimethyl-2,3-dioleyloxypropyl-2-(2-sperminecarboxamido)ethylammonium trifluoroacetate, trimethyldodecylammonium bromide, trimethyltetradecylammonium bromide, trimethylhexadecylammonium bromide, dimethyldioctadecylammonium bromide, and CpG ODN.
  • the present invention also provides an adenovirus vector vaccine for infection with SARS-CoV-2 Omicron mutant strain XBB and its subtypes, which constructs a recombinant vector containing a polynucleotide encoding the amino acid sequence of the protein, the precursor or the protein composition.
  • 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 adenoviral vector is selected from the polynucleotide for constructing S-XBB.1.5, as shown in SEQ ID No. 21.
  • the adenovirus vector is selected from at least one of the following: adenovirus, vaccinia Ankara virus, and adeno-associated virus.
  • the replication-deficient adenopathy is selected from human type 5, type 35 or type 26 and/or chimpanzee AdC68 or AdC7. poison.
  • it is selected from the human type 5 replication-deficient adenovirus in which E1 and E3 are jointly deleted.
  • the present invention provides a method for preparing the 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 together with the backbone plasmid into host cells, and culturing the host cells; obtaining replication-deficient recombinant adenovirus, and then expanding the culture and purifying.
  • the adenovirus vector vaccine also includes a pharmaceutically acceptable adjuvant, carrier, diluent or excipient.
  • the recombinant protein vaccine and adenovirus vector vaccine are intradermal or subcutaneous injection preparations, intramuscular injection preparations, intravenous injection preparations, oral or nasal spray preparations.
  • the vaccine is an intramuscular injection preparation and a nasal spray preparation.
  • the present invention also provides a composition for treating and/or preventing infection by SARS-CoV-2 Omicron mutant strains and their subtypes, which is a compound preparation containing the recombinant protein vaccine and the adenovirus vector vaccine as active ingredients.
  • the present invention provides a combined drug for resisting infection by SARS-CoV-2 Omicron mutant strain XBB and its subtypes, which contains the recombinant protein vaccine and the adenovirus vector vaccine administered separately or simultaneously.
  • the composition or combination drug is a combination or combination drug of recombinant protein vaccine 1, recombinant protein vaccine 2 and adenovirus vector vaccine.
  • the recombinant protein vaccine 1 contains at least one amino acid sequence of BA.5 sequence-1, BA.5 sequence-2 proteins or precursors; 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 proteins or precursors; and the adenovirus vector vaccine contains the polynucleotide sequence of S-XBB.1.5.
  • 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.
  • 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.
  • the adenovirus vector vaccine contains a polynucleotide sequence as shown in SEQ ID No.21.
  • composition or combined drug is an intradermal or subcutaneous injection preparation, an intramuscular injection preparation, an intravenous injection preparation, an oral or nasal spray preparation; preferably, the vaccine is an intramuscular injection preparation and a nasal spray preparation.
  • 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 combined drug in the preparation of a drug for treating and/or preventing infection or pathogenicity of SARS-CoV-2 Omicron mutant strains and their subtypes.
  • the present invention also provides a vaccine composition for treating and/or preventing infection by SARS-CoV-2 Omicron mutant strains and their subtypes, which contains a recombinant protein vaccine and an adenovirus vector vaccine; 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.
  • the present invention also provides a combination drug for treating and/or preventing infection with SARS-CoV-2 Omicron mutant strains and their subtypes, which comprises administering the above-mentioned recombinant protein vaccine and adenovirus vector vaccine 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.
  • the following sequences are constructed by the applicant after optimizing the RBD sequences in the S proteins of the SARS-CoV-2 mutant Omicron variants BA.5, XBB and sublineage 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.
  • a signal peptide was added to its amino acid when the protein was constructed to assist the secretory expression of the protein.
  • a His tag was also added to the amino acid sequence of the protein.
  • SEQ ID No.8 The complete BA.5 sequence-1, BA.5 sequence-2, XBB.1.5 sequence-1, XBB.1.5 sequence-2, XBB.1.5 sequence-3, XBB.1.5 sequence-4, and S-XBB.1.5 amino acid sequences of the present invention are shown 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.
  • nucleotide sequences encoding their amino acid sequences are shown as 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.
  • SEQ ID No.21 encodes the nucleotide sequence of SEQ ID No.20, Ad5 XBB.1.5 adenovirus vaccine antigen nucleotide sequence
  • SEQ ID No.22 Complete S-BA.5 amino acid sequence, amino acid sequence of the antigen of Ad5 BA.5 adenovirus vaccine
  • SEQ ID No.23 encodes the nucleotide sequence of SEQ ID No.22, the antigen nucleotide sequence of Ad5 BA.5 adenovirus vaccine
  • the present invention first prepares a recombinant protein vaccine against infection with SARS-CoV-2 and its variants.
  • the main idea of constructing the recombinant protein sequence is to construct the protein based on the RBD sequence in the S protein of the SARS-CoV-2 Omicron variants BA.5 and XBB.1.5, and the recombinant sequence formed by the RBD sequence and the heptapeptide repeat regions HR1 and HR2 of the S protein of SARS-CoV-2.
  • adding the corresponding vaccine adjuvant can better help the host resist the cross infection caused by the Omicron variant XBB and its subtypes, which is of great significance for the development of a recombinant protein vaccine against infection with the SARS-CoV-2 Omicron variant XBB and its subtypes.
  • the present invention also constructs a recombinant adenovirus vector based on the nucleotide sequence of the full-length S protein of SARS-CoV-2 Omicron variants BA.5 and XBB.1.5 to obtain an adenovirus vector vaccine, and prepares different recombinant adenovirus vectors.
  • a vaccine composition with better prevention or treatment effect against SARS-CoV-2 and its variants infection is obtained by combining two or three of the somatic vaccine with different recombinant protein vaccines.
  • the preparation of the vaccine composition as a nasal spray is more conducive to resisting SARS-CoV-2 and its variants infection.
  • FIG1 is a design diagram of pFastBac1-GP67-Trx-His-EK-S-RBD (Omicron_XBB.1.5)-HR in Example 1;
  • FIG2 is a 1% agarose gel electrophoresis diagram of PCR products of clones 1 to 3# in Example 1;
  • FIG3 is a 1% agarose gel electrophoresis diagram of PCR identification of the recombinant bacmid in Example 1;
  • FIG4 is a flow chart of recombinant baculovirus packaging and amplification in Example 1;
  • FIG5 is a diagram showing the WB verification results during the baculovirus amplification process in Example 1;
  • FIG6 is the result of Ni-affinity chromatography elution of the supernatant after baculovirus infection in Example 1;
  • FIG7 is a diagram showing the results of the EK digestion sample verification of the digestion effect in Example 1;
  • FIG8 is a diagram showing the purification of EK after enzyme digestion by Ni-affinity chromatography and SDS-PAGE identification in Example 1;
  • Figure 9 is the expression identification of XBB.1.5 adenovirus in Example 2.
  • Figure 10 shows serum IgG and lavage fluid IgG ⁇ IgA of the Ad5 XBB.1.5 +RBD XBB.1.5 -HR bivalent vaccine in Example 4;
  • Figure 11 shows serum IgG and lavage fluid IgG ⁇ IgA of the Ad5 BA.5 +RBD BA.5 -HR bivalent vaccine in Example 4;
  • Figure 12 shows serum IgG and lavage fluid IgG ⁇ IgA of the Ad5 BA.5 +RBD XBB.1.5 -HR bivalent vaccine in Example 4;
  • Figure 13 shows serum IgG and lavage fluid IgG ⁇ IgA of the Ad5 XBB.1.5 +RBD BA.5 -HR bivalent vaccine in Example 4;
  • Figure 14 shows serum IgG and lavage fluid IgG ⁇ IgA of the Ad5 XBB.1.5 +RBD XBB.1.5 -HR + RBD BA.5 -HR trivalent vaccine in Example 4;
  • Figure 15 shows the neutralizing antibodies in mouse serum of the RBD XBB.1.5 vaccine (aluminum adjuvant) in Example 5;
  • Figure 16 shows the neutralizing antibodies in the serum of mice of the RBD XBB.1.5 -HR vaccine (MF59 adjuvant) in Example 5;
  • Figure 17 shows the neutralizing antibodies in serum of mice with the RBD XBB.1.5 vaccine in Example 5;
  • Figure 18 shows the serum neutralizing antibodies of the Ad5 XBB.1.5 +RBD XBB.1.5 -HR bivalent vaccine in Example 5;
  • Figure 19 shows neutralizing antibodies in bronchoalveolar lavage fluid of the Ad5 XBB.1.5 +RBD XBB.1.5 -HR bivalent vaccine in Example 5;
  • Figure 20 shows the serum neutralizing antibodies of the trivalent vaccine Ad5 XBB.1.5 +RBD XBB.1.5 -HR + RBD BA.5 -HR in Example 5;
  • Figure 21 shows serum true virus neutralizing antibodies in Example 6
  • Figure 22 is a timetable for immune challenge of mice in Example 7.
  • Figure 23 shows the viral load of mouse throat swabs in Example 7.
  • Figure 24 is the viral gene/subgene RNA in Example 7.
  • Figure 25 shows the histopathological changes of mouse lung tissue in Example 7.
  • FIG. 26 shows the histopathological scores of mouse lung tissues in Example 7.
  • Monophosphoryl lipid A MPL
  • squalane oil-in-water emulsion MF59
  • rCTB recombinant cholera toxin
  • astragalus polysaccharide APS
  • PE phosphatidylethanolamine
  • PC phosphatidylcholine
  • cholesterol Chol
  • DOPE dioleoylphosphatidylethanolamine
  • DC-Chol cationic cholesterol
  • DOSPA dimethyl-2,3-dioleyloxypropyl-2-(2-sperminecarboxamido)ethylammonium trifluoroacetate
  • DOSPA trimethyldodecylammonium bromide
  • TTAB trimethyltetradecylammonium bromide
  • the present invention first designs a recombinant protein vaccine against infection by SARS-CoV-2 Omicron mutant strain XBB and its subtypes based on the amino acid sequence at positions 320-545 in the S protein of SARS-CoV-2 Omicron mutant strains BA.5 and XBB.1.5, and the heptapeptide repeat region 1 (HR1) and the heptapeptide repeat region 2 (HR2).
  • the present invention also designs a recombinant adenovirus vaccine for preventing infection with SARS-CoV-2 Omicron mutant strain XBB and its subtypes based on the nucleotide sequence encoding the full-length S protein of SARS-CoV-2 Omicron mutant strains BA.5 and XBB.1.5.
  • the present invention also combines different recombinant protein vaccines and different recombinant adenovirus vaccines to prepare bivalent and trivalent nasal spray preparations, which target the S protein of the SARS-CoV-2 virus, especially block the ACE2 receptor binding region of the S protein, thereby inducing the production of antibodies and other immune responses in the body, blocking the binding of the S protein of SARS-CoV-2 to the ACE2 receptor of the host cell, thereby helping the host resist coronavirus infection, especially having 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 sublineage XBB.1.5 parasitic subtype.
  • Example 1 Preparation of recombinant protein by expression using insect baculovirus system (taking S-RBD XBB.1.5 -HR as an example)
  • the S protein of the new coronavirus SARS-CoV-2 is located on the membrane, in order to simulate its secretion process, we added the signal peptide sequence of GP67 to the N-terminus of the protein when constructing the new coronavirus S-RBD (Omicron_XBB.1.5)-HR protein expression construct to assist the secretion expression of the protein.
  • This signal peptide will spontaneously secrete the protein during secretion.
  • Trx thioredoxin
  • amino acid sequence of GP67-Trx-His-EK-S-RBD(Omicron_XBB.1.5) is shown as SEQ ID No.12
  • amino acid sequence of GP67-Trx-His-EK-S-RBD(Omicron_XBB.1.5)-HR is shown as SEQ ID No.16.
  • the nucleotide sequences encoding SEQ ID No.12 and SEQ ID No.16 are shown as SEQ ID No.13 and SEQ ID No.17, respectively.
  • 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.
  • the construction of the above sequences is based on the RBD sequence at positions 320-545 of the S protein, in which the amino acids at positions 52, 54, and 56 are F, P, and F, respectively.
  • the designed coding fragment was cloned into the pFastBac1 vector plasmid using SEQ ID No.16, i.e., the XBB.1.5-3 sequence, and identified by bacterial liquid PCR.
  • the results of bacterial liquid PCR identification showed that the GP67-Trx-His-EK-S-RBD (Omicron_XBB.1.5)-HR fragment was successfully amplified in clones 2# and 3# of the three selected clones, as shown in Figure 2.
  • the correct pFastBac1-GP67-Trx-His-EK-S-RBD(Omicron_XBB.1.5)-HR recombinant clone was selected and identified. After extracting the recombinant plasmid, it was transformed into DH10B competent cells and identified by blue-white bacmid PCR. The PCR product of the bacmid was detected by 1% agarose gel electrophoresis, and the identification results are shown in Figure 3. White spots are bacmid clones that have undergone recombination, and blue spots are bacmid clones that have not undergone recombination.
  • the recombinant bacmid was transfected into sf9 insect cells, and the P0 generation recombinant baculovirus was harvested 5 days later.
  • the flow chart of recombinant baculovirus packaging and baculovirus amplification is shown in Figure 4.
  • the target protein was expressed at the same time. Before the target protein was excised, it contained a His tag, so we used the Anti-His WB experiment to verify the expression of the recombinant protein. The verification results showed that an obvious band was observed between the 40KD and 55KD Marker bands, and the size was consistent with the size of the Trx-His-EK-S-RBD (Omicron_XBB.1.5)-HR protein, indicating that the baculovirus was successfully amplified and the target protein was successfully expressed. The test results are shown in Figure 5.
  • the sf9 cells were infected with the P0 recombinant baculovirus, and the cell culture fluid was harvested 3 days later, and the protein purification was verified by Ni-affinity chromatography filler. The results are shown in Figure 6.
  • the target protein was mainly eluted at 40mM and 250mM imidazole, and a high-purity target protein was obtained after elution.
  • the target protein eluate was concentrated and adjusted to a concentration of 1 mg/ml, and EK enzyme was added. After enzyme digestion at 18°C for 14 hours, it was identified by SDS-PAGE gel electrophoresis. The results are shown in Figure 7. It shows that EK enzyme can remove the Trx-His-EK (amino acid sequence of EK cleavage site) tag from the target protein.
  • the applicant respectively provided protein construction designs such as SEQ ID No.8, SEQ ID No.12, and SEQ ID No.16.
  • the amino acid sequences of the finally expressed proteins are shown in SEQ ID No.1, SEQ ID No.3, and SEQ ID No.5.
  • the expressed recombinant proteins were used to prepare RBD XBB.1.5 -HR, RBD XBB.1.5 , and RBD BA..5 -HR recombinant protein vaccines, respectively, for subsequent studies such as animal immunization.
  • the synthetic 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.
  • the synthesized product was cloned into the pDC316 vector using a recombinant cloning strategy to obtain a shuttle plasmid (pDC316-S).
  • the pDC316-S containing the S gene of the SARS-CoV-2 Omicron_BA.5 and Omicron_XBB.1.5 variants constructed above was co-transfected with the backbone plasmid pBHGlox_E1,3Cre of the AdMax adenovirus system into HEK293 cells for packaging of the recombinant adenovirus.
  • the process is as follows:
  • the specific steps are as follows: take 4 ⁇ g of backbone plasmid and 2 ⁇ g of shuttle plasmid from each transfection well, dilute with 125 ⁇ L Opti-MEM medium, and then add 12 ⁇ L P3000 reagent; take another 1.5ml EP tube, dilute 7.5 ⁇ L lipofectamine3000 with 125 ⁇ L Opti-MEM medium; mix the diluted plasmid and diluted lipofectamine3000 in a 1:1 ratio, incubate at room temperature for 10-15 minutes, and then add to the cells.
  • the cells continue to be cultured, and after the cells are full, they are subcultured in 25cm2 cell culture flasks. The signs of cell poisoning are observed every day. When the cells have grown to the bottom of the flask, they are transferred to 75cm2 cell culture flasks until obvious plaques appear on the cells, and the poison is collected when most of the cells are diseased and fall off from the bottom.
  • PCR amplification conditions are as follows:
  • pDC516-R1 GCTAGACGATCCAGACATGAT (SEQ ID No. 25)
  • the identified correct recombinant adenovirus vaccine strains are amplified step by step in 293 cells.
  • the process of amplifying cells and viruses in the bioreactor is as follows: first, 3-5 g/L Cytodex1 microcarriers are added to the bioreactor and sterilized, and then cell culture fluid is added to the bioreactor.
  • the operating conditions are stable at 37°C, pH 7.0, DO 50%, and 50 rpm
  • the HEK293 cells amplified by the cell factory are digested and collected, inoculated in the bioreactor, and the inoculation cell density is 1.0-5.0 ⁇ 10 5 cells/ml.
  • the cell culture fluid is supplemented to 5L.
  • the conditions for the cell culture in the reactor are temperature 37°C, rotation speed 30-50 rpm, pH between 7.15 and 7.25, and DO 30-50%.
  • Samples are taken every day to detect the glucose concentration, cell density, and the morphology of cells on the microcarriers; when the cell density in the bioreactor reaches 1.0-5.0 ⁇ 10 6 cells/ml, the bioreactor is inoculated with the recombinant adenovirus vaccine virus with an MOI of 5-30.
  • samples are taken every day to detect the glucose concentration, the virus titer in the culture supernatant and the cell precipitate, and the morphology of the cells on the microcarrier is observed; when most of the cells fall off the microcarrier, the culture is terminated, and a virus lysis solution is added to the bioreactor with a final concentration of 0.05%-1% Tween 20 for lysis at 37°C for 2-4 hours, and then the virus solution is collected.
  • the collected virus is purified by cesium chloride ultracentrifugation or ion exchange chromatography.
  • the specific process is as follows:
  • the collected virus culture was centrifuged at 1200g for 10 minutes, the virus-containing culture supernatant was aspirated, the cell pellet was resuspended with 1/10 culture volume of the virus-containing supernatant, repeatedly frozen and thawed three times in a -80°C refrigerator and a 37°C water bath, centrifuged at 3000rpm for 10-20 minutes, and the supernatant was aspirated.
  • the virus-containing culture supernatant was concentrated 10 times using a 100K-300K ultrafiltration membrane pack; 1.4g/ml cesium chloride solution (53g cesium chloride + 87ml 10mM Tris-HCl, PH 7.9) and 1.2g/ml cesium chloride solution (cesium chloride 26.8 + 92ml 10mM Tris-HCl, PH 7.9) were prepared; 8ml 1.4g/ml cesium chloride solution was slowly added to the ultracentrifuge tube, and then 6ml 1.2g/ml cesium chloride solution was gently added, and finally 20ml of the virus-containing supernatant was added to the top of the discontinuous gradient, and the mixture was balanced and centrifuged at 100000 ⁇ g, 4°C for 90 minutes; after centrifugation, the blue virus band was aspirated with a syringe, and the cesium chloride was removed by dialyzing and stored at -80°C.
  • the virus culture was collected and lysed with 0.05%-1% Tween 20 at 37°C for 2-4 hours.
  • the lysed culture was clarified by filtration through 1.2 ⁇ m and 0.45 ⁇ m capsule filters.
  • the sample was concentrated 10 times using a 100-300kD molecular weight tangential flow membrane package, and then filtered with 10 volumes of filtration buffer (50mM Tris-HCl, 2mM MgCl2, 0-500mM NaCl, pH 8.0) and the filtration sample was collected; nuclease was added to the filtered sample at a final concentration of 10-50U/ml.
  • the sample was digested using Q Sepharose XL, Source 30Q or Source 15Q and other fillers are subjected to anion exchange chromatography, and the specific process is as follows: 5 column volumes are balanced with a flow rate of 20 ml/min of the balance buffer, and the sample is loaded at a flow rate of 10 ml/min after the balance is completed.
  • the balance buffer is balanced to the conductivity level; the sample is eluted with a linear gradient, and the elution conditions are 100% low salt buffer to 100% high salt buffer, 10V elution column volume, and the flow rate is 10 ml/min, and each elution peak is collected; after the elution is completed, the column is regenerated with a 2M NaCl buffer for 5 to 10 column volumes at a speed of 20 ml/min.
  • the virus peak is collected, and then the eluted virus sample is dialyzed or tangential flow filtration for buffer replacement (the buffer is 10mM Tris, 10mM Na- PO4 , 150mM NaCl, 2mM MgCl2, 2% sucrose, 0.15% glycerol, 0.02% Tween 80, pH7.6).
  • the buffer is 10mM Tris, 10mM Na- PO4 , 150mM NaCl, 2mM MgCl2, 2% sucrose, 0.15% glycerol, 0.02% Tween 80, pH7.6.
  • the purified adenovirus was directly bottled and stored at -20°C away from light.
  • the adenovirus vaccines prepared by Example 1 are respectively defined as Ad5 XBB.1.5 and Ad5 BA.5 recombinant adenoviruses.
  • mice Female NIH mice aged 6-8 weeks were purchased from Vital River Company and were raised in a special pathogen-free environment at the State Key Laboratory of Biotherapy of Sichuan University. 10 ⁇ g of recombinant protein was mixed with a certain amount of adjuvant and immunized three times intramuscularly on days 0, 14, and 28 of the mice, with 10 ⁇ g protein per mouse. Blood was collected 7 days after the three immunizations to detect neutralizing antibodies and evaluate the immune effect of the vaccine.
  • mice Female BALB/c mice aged 6-8 weeks were purchased from Vital River Company and were housed in a special pathogen-free environment at the State Key Laboratory of Biotherapy, Sichuan University.
  • To prepare the two-component nasal spray vaccine 2.5 ⁇ 10 9 VP (low dose) or 5 ⁇ 10 9 VP (high dose) of Ad5 XBB.1.5 was mixed with 10 ⁇ g of RBD XBB.1.5 -HR in a total volume of 50 ⁇ l to prepare low-dose or high-dose two-component vaccines, respectively.
  • mice were immunized three times with (1) low-dose and (2) high-dose Ad5 XBB.1.5 alone, (3) low-dose and (4) high-dose two-component (Ad5 XBB.1.5 + RBD XBB.1.5 -HR) vaccines, (5) 10 ⁇ g of RBD XBB.1.5 -HR protein alone, or (6) 5 ⁇ 10 9 VP of Ad5 Empty mixed with RBD XBB.1.5 -HR protein, delivered intranasally at intervals of 28 days. To avoid excessive fluid influx into lung tissue, mice received two intranasal immunizations of 25 ⁇ l each, more than 3 hours apart, on one day.
  • Ad5 BA.5 +RBD BA.5 -HR Ad5 BA.5 +RBD XBB.1.5 -HR
  • Ad5 XBB.1.5 +RBD BA.5 -HR Ad5 BA.5 +RBD BA.5 -HR
  • Ad5 BA.5 +RBD XBB.1.5 +RBD BA.5 -HR Ad5 BA.5 +RBD XBB.1.5 +RBD BA.5 -HR
  • the three-component vaccine consisting of 5 ⁇ 10 9 VP of Ad5 XBB.1.5 , 3.3 ⁇ g of RBD BA.5 -HR, and 6.6 ⁇ g of RBD XBB.1.5 -HR, was administered intranasally with the same immunization schedule. Serum samples were collected at weeks 3, 7, and 11 from vaccinated animals to determine binding and neutralizing antibody responses. To further evaluate vaccine-induced mucosal immunity, mice were euthanized on day 21 after the third booster vaccination to collect BALF and lung tissues.
  • 96-well plates (NUNC-MaxiSorp, Thermo Fisher Scientific) were coated with 1 ⁇ g/ml recombinant RBD protein in carbonate-bicarbonate buffer at 4°C overnight. 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 for 1 hour at room temperature. Serial dilutions of serum, tracheal-lung washes, or nasal swabs in dilution buffer were added to the wells (100 ⁇ l/well).
  • PBST PBS containing 0.1% Tween-20
  • HRP horseradish peroxidase
  • pseudovirus neutralization assays were performed as described above.
  • Pseudoviruses expressing luciferase including prototype, delta, and Omicron sublines (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.
  • inactivated serum and BALF samples were diluted three times, ranging from 30-65610, and then incubated with an equal volume of pseudoviruses of different dilutions at 37°C for 1 hour.
  • 1.2 ⁇ 10 4 HEK-293T cells expressing human ACE2 receptor (293T/ACE2) were added to each well and incubated at 37°C for 48 hours to express luciferase.
  • the supernatant was removed, and a lysis reagent (Beyotime, RG005) with a luciferase substrate was added, and the luminescence in 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 Prism8.0.2.
  • the positive control group contained only cells and viruses
  • the negative control group contained only cells
  • the sample group contained cells, samples, and viruses.
  • the neutralization percentage was calculated using the following formula:
  • Neutralization degree (%) (positive sample - sample to be tested / positive sample - negative sample) ⁇ 100%
  • mice after three injections of pure protein vaccine (recombinant RBD XBB.1.5 -HR and recombinant RBD XBB.1.5 ), 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 shows that three intramuscular immunizations of inactivated vaccine 25U/mouse were performed on days 0, 14, and 42, and inactivated vaccine (produced by China National Pharmaceutical Group Co., Ltd.) and RBD XBB.1.5 were sequentially boosted 84 days after the last immunization, with intramuscular injection, and the immunization dose was 10 ⁇ g/mouse.
  • the bivalent vaccine preparation of adenovirus combined with recombinant RBD protein induced stronger neutralizing antibodies in serum and bronchoalveolar lavage fluid than simple adenovirus, indicating that adenovirus combined with recombinant subunit preparation can produce stronger neutralizing protection ability in blood and mucosa to prevent viral infection; as shown in Figures 18-20, Ad5 XBB.1.5 +RBD XBB.1.5 -HR produced higher levels of serum neutralizing antibodies against WT, Delta, BA.2.75, BF.7, BA.5, BQ.1, BQ.1.1, XBB, XBB.1.5, XBB.1.16 and other viruses, indicating that Omicron's S protein adenovirus vaccine combined with S-RBD protein can produce stronger neutralizing antibodies against Omicron mutant strains. Immune protection.
  • mice were immunized intranasally with three doses of Ad5 XBB.1.5 alone or with two-component or three-component vaccines on days 0, 28, and 56.
  • mice were euthanized and tissues were collected. Pathological changes in lung tissues were observed by hematoxylin and eosin staining.
  • RT-qPCR reverse transcription quantitative polymerase chain reaction
  • mice with the novel coronavirus omiclonal variant All procedures related to the challenge of mice with the novel coronavirus omiclonal 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 in the ABSL-4 facility of the National Advanced Biosafety Primate Research Center in Kunming.
  • mice were intranasally immunized three times with Ad5 XBB.1.5 and two-component (Ad5 XBB1.5 +RBD XBB.1.5 -HR) vaccines. Mice treated with PBS, naked RBD XBB.1.5 -HR, and Ad5Empty+RBD XBB-1.5 -HR were used as controls.
  • Immunized BALB/c mice were challenged with 1 ⁇ 10 6 PFU of live severe acute respiratory syndrome coronavirus type 2 XBB.1.16 Omicron virus, and the changes in the viral load of throat swabs after severe acute respiratory syndrome coronavirus type 2 infection were determined; nasal concha, trachea, and lung tissues were collected on day 4 after infection, and the levels of gRNA and sgRNA were determined by RT-qPCR; the histopathological changes of mouse lung tissues after Omicron challenge were observed.

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Abstract

涉及抗SARS-CoV-2奥密克戎突变株XBB及其亚型感染的蛋白及疫苗，属于医药领域。为解决针对SARS-CoV-2奥密克戎突变株XBB及其亚型感染，尚缺乏有效预防和治疗药物的问题，提供了抗SARS-CoV-2奥密克戎突变株XBB及其亚型感染的蛋白及疫苗，该疫苗是基于SARS-CoV-2奥密克戎突变株XBB及亚系XBB.1.5的全长S蛋白和S蛋白中RBD序列及其优化序列设计的，能够帮助宿主抵抗冠状病毒感染，特别是对SARS-CoV-2奥密克戎突变株XBB及其亚型病毒引起的交叉感染有较好的防治效果。

Description

抗SARS-CoV-2奥密克戎突变株XBB及其亚型感染的蛋白及疫苗 技术领域
本发明涉及抗SARS-CoV-2奥密克戎突变株XBB及其亚型感染的蛋白及疫苗，属于医药领域。
背景技术
新型冠状病毒(SARS-Cov-2)是由世界卫生组织命名的一种新型的β属的冠状病毒。该病毒有包膜，颗粒呈圆形或椭圆形，常为多形性，直径60-140nm。其基因特征与SARS-CoV和MERS-CoV有明显区别，是一种以前尚未在人类中发现的新冠状病毒分支。目前，变异新冠病毒主要有五大种类：Alpha、Beta、Gamma、Delta、Omicron，Omicron变异株又分为BA.1、BA.2、BA.2.12.1、BA.4、BA.5、BQ.1.1、XBB.1.5等几种亚系。
SARS-CoV-2的主要结构蛋白包括刺突蛋白(Spike，S)、信使蛋白(Envelop，E)、膜蛋白(Membrane，M)和核衣壳蛋白(Nucleocapsid，N)，其中，S蛋白在病毒的感染和毒力中起着关键作用，常被用来作为疫苗的抗原。由于SARS-CoV-2变异株XBB含有多个突变位点，病毒的S蛋白也含有多个突变位点，导致变异株XBB及其亚型在一定程度上能够逃逸新冠病毒突变株(Alpha、Beta、Gamma、Delta、Omicron)疫苗激发的抗体，导致新冠疫苗失效或保护力降低，为新冠疫情防控工作带来巨大压力。因此，开发针对SARS-CoV-2病毒突变株XBB及其亚型的疫苗，特别对各变异的SARS-CoV-2病毒的广谱疫苗，对于新型冠状病毒肺炎的防治非常重要。
发明内容
本发明旨在至少解决现有技术中存在的技术问题之一。为此，本发明的目的在于提供抗SARS-CoV-2奥密克戎突变株XBB及其亚型感染的蛋白。本发明还提供用于预防和/或治疗SARS-CoV-2奥密克戎突变株XBB及其亚型感染的疫苗，包括含有所述蛋白的重组蛋白疫苗和腺病毒载体疫苗，还提供了含有两种疫苗的组合物或联合用药物。
本发明首先提供抗SARS-CoV-2奥密克戎突变株XBB及其亚型感染的蛋白，其含有选自SEQ ID No.1～SEQ ID No.7中任意一项所示的氨基酸序列。优选地，所述蛋白选自如SEQ ID No.3或SEQ ID No.5所示。
本发明还提供了所述蛋白的前体，其是在所述蛋白上连接了信号肽和/或蛋白标签。
优选地，所述的蛋白标签选自如下至少一种：组氨酸标签(6His标签)、硫氧还蛋白标签(Trx标签)、谷胱甘肽转移酶标签、泛素样修饰蛋白标签、麦芽糖结合蛋白标签、c-Myc蛋白标签、Avi tag蛋白标签、氮源利用物质A蛋白标签。
进一步地，在所述抗SARS-CoV-2奥密克戎突变株XBB及其亚型感染的蛋白上还连接了切除蛋白标签的蛋白酶识别区。
优选地，所述的蛋白酶选自如下至少一种：肠激酶(EK酶)、TEV蛋白酶、凝血酶、凝血因子Xa、羧肽酶A、鼻病毒3c蛋白酶。
进一步地，所述前体的氨基酸序列选自SEQ ID No.8、SEQ ID No.10、SEQ ID No.12、SEQ ID No.14、SEQ ID No.16、SEQ ID No.18、SEQ ID No.20中至少一种。优选地，所述蛋白序列选自如SEQ ID No.12或SEQ ID No.16所示。
本发明还提供了多核苷酸，其编码所述的蛋白或所述的前体。
进一步地，所述核苷酸序列选自SEQ ID No.9、SEQ ID No.11、SEQ ID No.13、SEQ ID No.15、SEQ ID No.17、SEQ ID No.19、SEQ ID No.21中至少一种。优选地，所述多核苷酸序列选自如SEQ ID No.13或SEQ ID No.17所示。
本发明还提供了重组载体，其含有所述的多核苷酸。
进一步地，所述重组载体采用昆虫杆状病毒表达载体、哺乳动物细胞表达载体、大肠杆菌表达载体、酵母表达载体中至少一种。
优选地，所述的昆虫杆状病毒表达载体为pFastBac1。
优选地，所述的大肠杆菌表达载体为pET32a。
优选地，所述的酵母表达载体为pPICZaA。
优选地，所述的哺乳动物细胞表达载体为CHO细胞表达载体。
进一步优选地，所述的CHO细胞表达载体为pTT5或FTP-002。
本发明还提供了宿主细胞，其含有所述的重组载体。
进一步地，所述的宿主细胞采用昆虫细胞、哺乳动物细胞、大肠杆菌、酵母中至少一种。
优选地，所述的昆虫细胞选自sf9细胞、sf21细胞、Hi5细胞中至少一种。
优选地，所述的哺乳动物细胞为CHO细胞。
本发明还提供了所述的蛋白或所述的前体的制备方法，其特征是：包括如下步骤：培养所述的宿主细胞，使其表达所述的蛋白或前体，然后回收所述的蛋白，即得。
本发明还提供了抗SARS-CoV-2奥密克戎突变株及其亚型感染的蛋白组合物，其包括BA.5序列-1、BA.5序列-2、XBB.1.5序列-1、XBB.1.5序列-2、XBB.1.5序列-3、XBB.1.5序列-4、S-XBB.1.5蛋白中任意两种及以上的组合。
优选地，所述蛋白组合物包括BA.5序列-1、BA.5序列-2中的一种、XBB.1.5序列-1、XBB.1.5序列-2、XBB.1.5序列-3、XBB.1.5序列-4中的任意一种、S-XBB.1.5蛋白至少两种组合。
更优选地，所述蛋白组合物包括BA.5序列-1、BA.5序列-2中的一种、XBB.1.5序列-3、XBB.1.5序列-4中的任意一种与S-XBB.1.5蛋白进行的组合。
本发明还提供了预防和/或治疗SARS-CoV-2奥密克戎突变株及其亚型感染的重组蛋白疫苗，其含有所述的蛋白、所述的前体和/或所述的蛋白组合物，以及药学上可接受的辅料或者辅助性成分。
进一步地，所述的辅助性成分为免疫佐剂。
优选地，所述的免疫佐剂选自如下至少一种：鲨烯水包油乳剂、铝盐、钙盐、植物皂苷、植物多糖、单磷酸脂质A、胞壁酰二肽、胞壁酰三肽、细菌毒素、GM-CSF细胞因子、脂质、阳离子脂质体材料。
进一步地，满足以下至少一项：所述的鲨烯水包油乳剂为MF59；
所述的铝盐选自氢氧化铝、明矾中至少一种；
所述的钙盐为磷酸三钙；
所述的植物皂苷为QS-21或ISCOM；
所述的植物多糖为黄芪多糖；
所述的细菌毒素选自重组霍乱毒素、白喉毒素中至少一种；
所述的脂质选自如下至少一种：磷脂酰乙醇胺、磷脂酰胆碱、胆固醇、二油酰基磷脂酰乙醇胺；
所述的阳离子脂质体材料选自如下至少一种：(2,3-二油氧基丙基)三甲基氯化铵、N-[1-(2,3-二油酰氯)丙基]-N,N,N-氯化三甲胺、阳离子胆固醇、三氟乙酸二甲基-2,3-二油烯氧基丙基-2-(2-精胺甲酰氨基)乙基铵、溴化三甲基十二烷基铵、溴化三甲基十四烷基铵、溴化三甲基十六烷基铵、溴化二甲基双十八烷基铵、CpG ODN。
本发明还提供了抗SARS-CoV-2奥密克戎突变株XBB及其亚型感染的腺病毒载体疫苗，其构建含有编码所述蛋白、所述前体或所述蛋白组合物的氨基酸序列的多核苷酸的重组载体。
优选地，所述多核苷酸的核苷酸序列选自SEQ ID No.9、SEQ ID No.11、SEQ ID No.13、SEQ ID No.15、SEQ ID No.17、SEQ ID No.19、SEQ ID No.21中至少一种。更优选地，所述腺病毒载体含有的多核苷酸序列选自构建S-XBB.1.5的多核苷酸，如SEQ ID No.21所示。
进一步地，所述腺病毒载体选自如下至少一种：腺病毒、安卡拉痘苗病毒、腺相关病毒。
优选地，选自人5型、35型或26型或/和黑猩猩AdC68型或AdC7型的复制缺陷型腺病 毒。
更优选地，选自E1、E3联合缺失的人5型复制缺陷型腺病毒。
本发明提供了所述腺病毒载体疫苗中的腺病毒的制备方法，包括以下步骤：构建所述多核苷酸的穿梭质粒载体；然后将构建的穿梭质粒载体与骨架质粒一起转染入宿主细胞，培养宿主细胞；获得复制缺陷重组腺病毒，然后扩大培养、纯化，即可。
进一步的，所述腺病毒载体疫苗还包括药学上可接受的佐剂、载体、稀释剂或赋形剂。
进一步地，所述重组蛋白疫苗、腺病毒载体疫苗为皮内或皮下注射制剂、肌肉注射制剂、静脉注射制剂、口服或鼻喷制剂。
优选地，所述的疫苗为肌肉注射制剂和鼻喷制剂。
本发明还提供了治疗和/或预防SARS-CoV-2奥密克戎突变株及其亚型感染的组合物，是含有所述的重组蛋白疫苗和所述的腺病毒载体疫苗为活性成分的复方制剂。
本发明提供了抗SARS-CoV-2奥密克戎突变株XBB及其亚型感染的联合用药物，其含有分别或同时给药的所述的重组蛋白疫苗和所述的腺病毒载体疫苗。
优选地，所述组合物或联合用药物是重组蛋白疫苗1、重组蛋白疫苗2和腺病毒载体疫苗的组合或联合用药。
更优选地，所述重组蛋白疫苗1含有BA.5序列-1、BA.5序列-2蛋白或前体中至少一种的氨基酸序列；所述重组蛋白疫苗2含有XBB.1.5序列-1、XBB.1.5序列-2、XBB.1.5序列-3、XBB.1.5序列-4蛋白或前体中的至少一种氨基酸序列；所述腺病毒载体疫苗含有S-XBB.1.5的多核苷酸序列。
进一步地，所述重组蛋白疫苗1含有的氨基酸序列选自SEQ ID No.1、SEQ ID No.2、SEQ ID No.8、SEQ ID No.10中至少一种。
进一步地，重组蛋白疫苗2含有的氨基酸序列选自SEQ ID No.3～SEQ ID No.6、SEQ ID No.12、SEQ ID No.14、SEQ ID No.16、SEQ ID No.18中至少一种。优选地，选自SEQ ID No.5、SEQ ID No.6、SEQ ID No.16、SEQ ID No.18中至少一种。
进一步地，所述腺病毒载体疫苗含有如SEQ ID No.21所示的多核苷酸序列。
进一步地，所述组合物或联合用药物为皮内或皮下注射制剂、肌肉注射制剂、静脉注射制剂、口服或鼻喷制剂；优选地，所述的疫苗为肌肉注射制剂和鼻喷制剂。
本发明还提供了所述的蛋白、所述的前体、所述的蛋白组合物、所述的重组蛋白疫苗、腺病毒载体疫苗、所述的疫苗组合物或联合用药物在制备治疗和/或预防SARS-CoV-2奥密克戎突变株及其亚型感染或致病的药物中的用途。
本发明还提供了治疗和/或预防SARS-CoV-2奥密克戎突变株及其亚型感染的疫苗组合物，其含有重组蛋白疫苗和腺病毒载体疫苗；所述重组蛋白疫苗的氨基酸序列选自SEQ ID No.1～SEQ ID No.7、SEQ ID No.8～SEQ ID No.20中至少一种；所述腺病毒载体疫苗的抗原的核苷酸序列选自SEQ ID No.23。
本发明还提供了治疗和/或预防SARS-CoV-2奥密克戎突变株及其亚型感染的联合用药物，其分别或同时给药上述的重组蛋白疫苗和腺病毒载体疫苗；所述重组蛋白疫苗的氨基酸序列选自SEQ ID No.1～SEQ ID No.7、SEQ ID No.8～SEQ ID No.20中至少一种；所述腺病毒载体疫苗的抗原的核苷酸序列选自SEQ ID No.23。
以下序列是申请人基于SARS-CoV-2突变体Omicron变异株BA.5、XBB及亚系XBB.1.5的S蛋白中的RBD序列优化后构建的序列，其构建的蛋白或前体分别定义为BA.5序列-1、BA.5序列-2、XBB.1.5序列-1、XBB.1.5序列-2、XBB.1.5序列-3、XBB.1.5序列-4、S-XBB.1.5蛋白或前体。
SEQ ID No.1 BA.5序列-1
SEQ ID No.2 BA.5序列-2
SEQ ID No.3 XBB.1.5序列-1

SEQ ID No.4 XBB.1.5序列-2
SEQ ID No.5 XBB.1.5序列-3
SEQ ID No.6 XBB.1.5序列-4
SEQ ID No.7 S-XBB.1.5

为了能够辅助蛋白的分泌表达，在构建蛋白时，在其氨基酸上添加了信号肽，以辅助蛋白的分泌表达。同时，为了方便纯化，还在蛋白的氨基酸序列添加了His标签。本发明完整的BA.5序列-1、BA.5序列-2、XBB.1.5序列-1、XBB.1.5序列-2、XBB.1.5序列-3、XBB.1.5序列-4、S-XBB.1.5氨基酸序列分别如SEQ ID No.8、SEQ ID No.10、SEQ ID No.12、SEQ ID No.14、SEQ ID No.16、SEQ ID No.18、SEQ ID No.20所示。
进一步地，其对应的编码其氨基酸序列的核苷酸序列分别如SEQ ID No.9、SEQ ID No.11、SEQ ID No.13、SEQ ID No.15、SEQ ID No.17、SEQ ID No.19、SEQ ID No.21所示。
SEQ ID No.8完整的BA.5序列-1信号肽-Trx标签-6His标签-EK酶切位点-RBD序列-HR1序列-HR2序列

SEQ ID No.9编码SEQ ID No.8的核苷酸序列
SEQ ID No.10完整的BA.5序列-2信号肽-Trx标签-6His标签-EK酶切位点-RBD序列-HR1序列-HR2序列
SEQ ID No.11编码SEQ ID No.10的核苷酸序列

SEQ ID No.12完整的XBB.1.5序列-1信号肽-Trx标签-6His标签-EK酶切位点-RBD序列
SEQ ID No.13编码SEQ ID No.12的核苷酸序列

SEQ ID No.14完整的XBB.1.5序列-2信号肽-Trx标签-6His标签-EK酶切位点-RBD序列
SEQ ID No.15编码SEQ ID No.14的核苷酸序列

SEQ ID No.16完整的XBB.1.5序列-3信号肽-Trx标签-6His标签-EK酶切位点-RBD序列-HR1序列-HR2序列
SEQ ID No.17编码SEQ ID No.16的核苷酸序列

SEQ ID No.18完整的XBB.1.5序列-4信号肽-Trx标签-6His标签-EK酶切位点-RBD序列-HR1序列-HR2序列

SEQ ID No.19编码SEQ ID No.18的核苷酸序列

SEQ ID No.20完整的S-XBB.1.5氨基酸序列，Ad5XBB.1.5腺病毒疫苗抗原氨基酸序列
SEQ ID No.21编码SEQ ID No.20的核苷酸序列，Ad5XBB.1.5腺病毒疫苗抗原核苷酸序列


申请人发现，以Omicron变异株BA.5的S蛋白的全长基因，如SEQ ID No.23所示制备出的腺病毒疫苗，然后以Omicron变异株BA.5、XBB.1.5的S蛋白中的RBD序列或RBD-HR分别制备重组蛋白疫苗，将腺病毒疫苗和重组蛋白疫苗联用，对于SARS-CoV-2或其突变病毒引起的交叉感染也有较好的防治效果。
SEQ ID No.22完整的S-BA.5氨基酸序列，Ad5BA.5腺病毒疫苗的抗原氨基酸序列
SEQ ID No.23编码SEQ ID No.22的核苷酸序列，Ad5BA.5腺病毒疫苗的抗原核苷酸序列


有益效果：本发明首先是制备抗SARS-CoV-2及其变异株感染的重组蛋白疫苗，起主要作用的重组蛋白序列构建思路是基于SARS-CoV-2 Omicron变异株BA.5、XBB.1.5的S蛋白中的RBD序列、RBD序列与SARS-CoV-2的S蛋白的七肽重复区HR1和HR2形成的重组序列进行蛋白构建的。同时，再添加相应的疫苗佐剂，能更好地帮助宿主抵抗Omicron变异株XBB及其亚型引起的交叉感染，对开发抗SARS-CoV-2奥密克戎突变株XBB及其亚型感染的重组蛋白疫苗具有重要的意义。
同时，本发明还基于SARS-CoV-2 Omicron变异株BA.5、XBB.1.5的全长S蛋白的核苷酸序列进行重组腺病毒载体的构建，获得腺病毒载体疫苗，并将制备的不同的重组腺病毒载 体疫苗与不同的重组蛋白疫苗两种或三种结合获得预防或治疗效果更好的抗SARS-CoV-2及其变异株感染的疫苗组合物，其制备为鼻喷试剂更利于抵抗SARS-CoV-2及其变异株感染。
附图说明
图1为实施例1中pFastBac1-GP67-Trx-His-EK-S-RBD(Omicron_XBB.1.5)-HR的设计图；
图2为实施例1中1～3#克隆PCR产物的1％琼脂糖凝胶电泳图；
图3为实施例1中重组bacmid的PCR鉴定1％琼脂糖凝胶电泳图；
图4为实施例1中重组杆状病毒包装及扩增流程图；
图5为实施例1中杆状病毒扩毒过程中的WB验证结果图；
图6为实施例1中杆状病毒感染后上清经Ni-亲和层析洗脱结果；
图7为实施例1中EK酶切样品验证酶切效果结果图；
图8为实施例1中EK酶切后经Ni-亲和层析填料纯化及SDS-PAGE鉴定图；
图9为实施例2中XBB.1.5腺病毒表达鉴定；
图10为实施例4中Ad5XBB.1.5+RBDXBB.1.5-HR双价苗血清IgG和灌洗液IgG\IgA；
图11为实施例4中Ad5BA.5+RBDBA.5-HR双价苗血清IgG和灌洗液IgG\IgA；
图12为实施例4中Ad5BA.5+RBDXBB.1.5-HR双价苗血清IgG和灌洗液IgG\IgA；
图13为实施例4中Ad5XBB.1.5+RBDBA.5-HR双价苗血清IgG和灌洗液IgG\IgA；
图14为实施例4中Ad5XBB.1.5+RBDXBB.1.5-HR+RBDBA.5-HR三价苗血清IgG和灌洗液IgG\IgA；
图15为实施例5中RBDXBB.1.5疫苗(铝佐剂)小鼠血清中和抗体；
图16为实施例5中RBDXBB.1.5-HR疫苗(MF59佐剂)小鼠血清中和抗体；
图17为实施例5中RBDXBB.1.5疫苗小鼠血清中和抗体；
图18为实施例5中Ad5XBB.1.5+RBDXBB.1.5-HR双价苗血清中和抗体；
图19为实施例5中Ad5XBB.1.5+RBDXBB.1.5-HR双价苗肺泡灌洗液中和抗体；
图20为实施例5中Ad5XBB.1.5+RBDXBB.1.5-HR+RBDBA.5-HR三价苗血清中和抗体；
图21为实施例6中血清真病毒中和抗体；
图22为实施例7中小鼠免疫攻毒时间表；
图23为实施例7中小鼠咽拭子病毒载量；
图24为实施例7中病毒基因/亚基因RNA；
图25为实施例7中小鼠肺组织的组织病理学变化；
图26为实施例7中小鼠肺组织的组织病理学评分。
具体实施方式
术语缩写：
单磷酸脂质A(MPL)；鲨烯水包油乳剂(MF59)、重组霍乱毒素(rCTB)；黄芪多糖(APS)；磷脂酰乙醇胺(PE)、磷脂酰胆碱(PC)、胆固醇(Chol)、二油酰基磷脂酰乙醇胺(DOPE)(2,3-二油氧基丙基)三甲基氯化铵(DOTAP)、N-[1-(2,3-二油酰氯)丙基]-N,N,N-氯化三甲胺(DOTMA)、阳离子胆固醇(DC-Chol)、三氟乙酸二甲基-2,3-二油烯氧基丙基-2-(2-精胺甲酰氨基)乙基铵(DOSPA)、溴化三甲基十二烷基铵(DTAB)、溴化三甲基十四烷基铵(TTAB)、溴化三甲基十六烷基铵(CTAB)、溴化二甲基双十八烷基铵(DDAB)、CpG ODN(含有非甲基化的胞嘧啶和鸟嘌呤二核苷酸为核心序列的核苷酸序列,人工合成的CpG)
本发明先基于SARS-CoV-2奥密克戎突变株BA.5、XBB.1.5的S蛋白中320-545位氨基酸序列，与七肽重复区1(HR1)、七肽重复区2(HR2)来设计抗SARS-CoV-2奥密克戎突变株XBB及其亚型感染的重组蛋白疫苗。
本发明还基于编码SARS-CoV-2奥密克戎突变株BA.5、XBB.1.5的全长S蛋白的核苷酸序列设计抗SARS-CoV-2奥密克戎突变株XBB及其亚型感染的重组腺病毒疫苗。
本发明还将制备的不同的重组蛋白疫苗和不同的重组腺病毒疫苗进行两者和三者组合，制备成二价和三价鼻喷制剂，通过针对SARS-CoV-2病毒的S蛋白，特别是通过阻断S蛋白的ACE2受体结合区，诱导体内产生抗体等免疫反应，阻断SARS-CoV-2的S蛋白与宿主细胞ACE2受体的结合，从而帮助宿主抵抗冠状病毒感染，尤其是对SARS-CoV-2或其突变病毒引起的交叉感染有较好的防治效果，例如SARS-CoV-2 Omicron变异株及其亚系XBB.1.5寄亚型等引起的交叉感染。
下面将结合实施例对本发明的方案进行解释。本领域技术人员将会理解，下面的实施例仅用于说明本发明，而不应视为限定本发明的范围。实施例中未注明具体技术或条件的，按照本领域内的文献所描述的技术或条件或者按照产品说明书进行。所用试剂或仪器未注明生产厂商者，均为可以通过市购获得的常规产品。
实施例1用昆虫杆状病毒系统表达制备重组蛋白(以S-RBDXBB.1.5-HR为例)
1.S-RBDXBB.1.5-HR的构建设计
因新冠病毒SARS-CoV-2的S蛋白为定位于膜上的蛋白，因此，为了保证模拟其分泌过程，我们在构建新冠病毒S-RBD(Omicron_XBB.1.5)-HR蛋白表达构建时，在蛋白的N-端加入了GP67的信号肽序列，以辅助蛋白的分泌表达，这一信号肽将在蛋白分泌的过程中自发 被昆虫细胞所切除；同时，我们在GP67信号肽后加入了草地贪夜蛾(Spodoptera frugiperda，S.frugiperda)的硫氧还蛋白(Trx)标签用以辅助S-RBD(Omicron_XBB.1.5)-HR折叠，加入了6xhis标签用以帮助后续纯化，加入了EK酶酶切位点，用以切除Trx和6xhis标签。此蛋白表达构建设计，通过EK酶切，将能够移除所有非S-RBD(Omicron_XBB.1.5)-HR的冗余氨基酸。该表达构建设计的模式见图1。
GP67-Trx-His-EK-S-RBD(Omicron_XBB.1.5)的氨基酸序列如SEQ ID No.12所示，GP67-Trx-His-EK-S-RBD(Omicron_XBB.1.5)-HR的氨基酸序列如SEQ ID No.16所示，编码SEQ ID No.12、SEQ ID No.16的核苷酸序列分别如SEQ ID No.13、SEQ ID No.17所示。
同样地，构建GP67-Trx-His-EK-S-RBD(Omicron_XBB.BA.5)-HR的氨基酸序列如SEQ ID No.8所示，编码其氨基酸序列的核苷酸序列如SEQ ID No.9所示。
上述序列的构建均是以S蛋白320-545位的RBD序列为基础，其中第52、54、56位氨基酸均分别为F、P、F。
2.重组质粒构建的鉴定
以SEQ ID No.16为编码片段，即XBB.1.5-3序列，将设计的编码片段克隆入pFastBac1载体质粒中，并通过菌液PCR鉴定。菌液PCR鉴定结果显示，所挑选的3个克隆中2#、3#克隆成功扩出GP67-Trx-His-EK-S-RBD(Omicron_XBB.1.5)-HR片段，见图2。
3.重组bacmid的鉴定
选择鉴定正确的pFastBac1-GP67-Trx-His-EK-S-RBD(Omicron_XBB.1.5)-HR重组克隆，提取重组质粒后，转化DH10B感受态细胞，并进行蓝白斑菌液PCR鉴定。菌液PCR产物经过1％琼脂糖凝胶电泳检测，鉴定结果见图3。白斑为发生重组的bacmid克隆，蓝斑为未发生重组的bacmid克隆。
4.重组杆状病毒的包装
将重组bacmid转染入sf9昆虫细胞中，5天后收获P0代重组杆状病毒。重组杆状病毒包装及杆状病毒扩增流程图见图4。
5.目的蛋白的表达验证
在上述杆状病毒的扩增过程中，同时伴随着目的蛋白的表达。而目的蛋白切除标签前，含有His标签，因此我们用Anti-His的WB实验，验证重组蛋白的表达情况。验证结果显示，在40KD～55KD Marker条带间均观察到一条明显的条带，大小与Trx-His-EK-S-RBD(Omicron_XBB.1.5)-HR蛋白大小吻合，表明杆状病毒扩增成功，目的蛋白也能够成功表达。检测结果见图5。
6.目的蛋白的纯化鉴定
以P0代重组杆状病毒感染sf9细胞，3天后收获细胞培养液，并通过Ni-亲和层析填料进行蛋白纯化的验证工作。结果见图6。目的蛋白主要在40mM和250mM咪唑下被洗脱，洗脱后可得到高纯度的目的蛋白。
7.EK酶切切除标签的验证
将目的蛋白洗脱液浓缩，并调整浓度为1mg/ml，加入EK酶，18℃酶切14h后，通过SDS-PAGE凝胶电泳鉴定。结果见图7。显示EK酶能够将Trx-His-EK(EK酶切位点的氨基酸序列)标签从目的蛋白上切除。
8.切除的标签的去除验证
将EK酶切之后的样品，使用Ni-亲和层析填料反挂10min，样品流穿后，再使用250mM咪唑洗脱。将Trx-His-EK-S-RBD(Omicron_XBB.1.5)-HR蛋白(未切开的完整蛋白)、S-RBD(Omicron_XBB.1.5)-HR蛋白和被切下的Trx-His-EK标签分离开。结果见图8。结果显示，Trx-His-EK标签切除后，能够通过Ni-亲和层析填料反挂后续纯化手段去除，由此获得不含任何标签的S-RBD(Omicron_XBB.1.5)-HR蛋白。其蛋白氨基酸序列如SEQ ID No.5所示。
通过上述重组蛋白的构建方法基于SARS-CoV-2 Omicron_BA.5、XBB.1.5的S蛋白RBD或SARS-CoV-2突变体的S蛋白RBD-HR氨基酸序列，申请人分别给出如SEQ ID No.8、SEQ ID No.12、SEQ ID No.16的蛋白构建设计，最终表达的蛋白氨基酸序列如SEQ ID No.1、SEQ ID No.3、SEQ ID No.5所示，并以表达的重组蛋白分别制备RBDXBB.1.5-HR、RBDXBB.1.5、RBD BA..5-HR重组蛋白疫苗，用于动物免疫等后续研究。
实施例2重组腺病毒疫苗的构建及制备
1.SARS-CoV-2 Omicron_BA.5、Omicron_XBB.1.5S蛋白基因序列的优化和合成
根据https://covariants.org/网站上提供的新冠病毒Omicron_BA.5、Omicron_XBB.1.5的突变位点，获得SARS-CoV-2各变异株S蛋白的核苷酸序列，同时保留了各S蛋白突变体自身的信号肽，对密码子进行优化。随后根据突变和优化的序列合成各变异株S蛋白基因。
所述合成的SARS-CoV-2 Omicron_BA.5、Omicron_XBB.1.5S蛋白基因如SEQ ID No.23、SEQ ID No.21所示。
2.重组腺病毒新冠疫苗的包装
在基因合成过程中，将合成后的产物利用重组的克隆策略将S基因克隆至pDC316载体中，得穿梭质粒(pDC316-S)。将以上构建好的包含SARS-CoV-2 Omicron_BA.5、Omicron_XBB.1.5变异株S基因的pDC316-S分别与AdMax腺病毒系统的骨架质粒pBHGlox_E1,3Cre共转染HEK293细胞进行重组腺病毒的包装。过程如下：
1)将8×105个/孔HEK293A细胞接种于六孔板中，高糖DMEM+10％FBS培养基，置于 37℃含5％CO2细胞培养箱中培养过夜。
2)第二天用高糖DMEM+2％FBS换液，将骨架质粒(pBHGlox_E1,3Cre)和穿梭质粒利用lipofectamine3000共转染HEK293A细胞。具体步骤为：每个转染孔取骨架质粒4μg，穿梭质粒2μg，用125μL Opti-MEM培养基进行稀释，然后加入12μL P3000试剂；另取一支1.5ml EP管，用125μL Opti-MEM培养基进行稀释7.5μL lipofectamine3000；将稀释的质粒和稀释的lipofectamine3000按1：1比例混合，室温孵育10-15分钟后加入细胞中，细胞继续培养，细胞长满后传代于25cm 2细胞培养瓶中，每天观察细胞出毒迹象，待细胞长满瓶底时，再传入75cm2细胞培养瓶中，直到细胞出现明显噬斑，待细胞大部分病变并从底部脱落时进行收毒。
3)收集出毒的细胞培养物，1200rpm离心3分钟，吸取含病毒的上清，细胞沉淀用1/10培养体积的含病毒上清重悬，置于-80℃冰箱和37℃水浴锅中反复冻融三次。3000rpm离心20分钟，收集含病毒的上清液并与前述的含病毒上清合并，此即为腺病毒疫苗的毒种。
4)取50μL疫苗候选株毒种液，加入2μL蛋白酶K，50℃消化30min释放病毒基因组，以此为模版PCR扩增S基因序列，PCR产物电泳胶回收后测序鉴定，PCR扩增的条件如下：
变性：95℃，10min；变性：95℃，10s；退火：64℃，30s；延伸：72℃，2min；延伸：72℃，5min；循环数，40次；PCR扩增的引物如下：
pDC516-F1：ACACGTCAATGGGAAGTGAAA(SEQ ID No.24)
pDC516-R1：GCTAGACGATCCAGACATGAT(SEQ ID No.25)
3.重组腺病毒新冠疫苗的扩增
经鉴定正确的重组腺病毒疫苗毒种在293细胞中逐级扩增。具体过程如下：按MOI＝10加入80％-90％长满的293细胞，待大部分细胞病毒变圆后，收集病毒培养物，按照上述反复冻融的方法，制备主病毒种子库和工作病毒种子库。利用细胞工厂或生物反应器规模化扩增重组腺病毒疫苗，细胞大部分病变后收集病毒培养物。生物反应器扩增细胞和病毒的过程如下：首先在生物反应器中加入3-5g/LCytodex1微载体后灭菌，然后生物反应器中加入细胞培养液，当运行条件稳定在37℃，pH 7.0，DO 50％，50rpm时，消化收集细胞工厂扩增的HEK293细胞，接种于生物反应器内，接种细胞密度1.0-5.0×105个细胞/ml，补充细胞培养液至5L，反应器细胞培养的条件为温度37℃、转速为30-50rpm、pH7.15～7.25之间、DO 30～50％，每天取样检测葡萄糖浓度、细胞密度和微载体上细胞的形态；当生物反应器内的细胞密度达到1.0～5.0×106个细胞/ml时，生物反应器接种重组腺病毒疫苗毒种，MOI为5-30，接种后每天取样检测葡萄糖浓度、培养上清和细胞沉淀中的病毒滴度和观察微载体上细胞的形态；当大部分细胞重微载体上脱落时，终止培养，向生物反应器中加入病毒裂解液，终浓度为0.05％-1％Tween 20 37℃裂解2-4小时，后收集病毒液。
4.重组腺病毒疫苗的纯化
收集的病毒利用氯化铯超速离心或离子交换层析的的方法纯化，具体过程如下：
(1)氯化铯超速离心纯化腺病毒疫苗
收集的病毒培养物1200g离心10分钟，吸取含病毒的培养上清，细胞沉淀用1/10培养体积的含病毒上清重悬，-80℃冰箱和37℃水浴锅中反复冻融三次，3000rpm离心10-20分钟，吸取上清。含病毒的培养上清用100K-300K超滤膜包浓缩10倍；配制1.4g/ml氯化铯溶液(53g氯化铯+87ml 10mM Tris-HCl，PH 7.9)和1.2g/ml氯化铯溶液(氯化铯26.8+92ml10mM Tris-HCl，PH 7.9)；在超速管中缓慢加入8ml 1.4g/ml氯化铯溶液，然后轻缓地加入6ml 1.2g/ml氯化铯溶液，最后在不连续梯度顶部加入20ml含病毒上清液，配平后100000×g，4℃离心90分钟；离心结束后注射器抽吸出蓝色的病毒带，透析去除氯化铯后保存在-80℃。
(2)离子交换层析纯化腺病毒
收集病毒培养物，用0.05％-1％Tween 20 37℃裂解2-4小时，裂解的培养物经1.2μm和0.45μm囊式滤器过滤澄清，使用100-300kD分子量的切向流膜包将样品浓缩10倍，随后用10体积的滤洗缓冲液(50mM Tris–HCl，2mM MgCl2，0–500mM NaCl，pH 8.0)进行滤洗，收集滤洗样品；滤洗的样品中加加入核酸酶，终浓度为10-50U/ml，37℃消化1-3小时后，样品利用Q Sepharose XL、Source 30Q或Source 15Q等填料进行阴离子交换层析，具体过程如下：平衡缓冲液20ml/min流速平衡5个柱体积，平衡结束后10ml/min的流速上样，上样结束后，平衡缓冲液平衡至电导水平；线性梯度洗脱样品，洗脱的条件为100％低盐缓冲液到100％高盐缓冲液，10V洗脱柱体积，流速为10ml/min，收集各个洗脱峰；洗脱结束后用2M NaCl缓冲液对柱子进行再生5～10个柱体积，速度为20ml/min。收集病毒峰，随后洗脱的病毒样品经透析或切向流过滤进行缓冲液置换(缓冲液成10mM Tris、10mM Na-PO4、150mM NaCl、2mM MgCl2、2％蔗糖、0.15％甘油、0.02％吐温80，pH7.6)。
纯化后的腺病毒直接灌装，-20℃避光保存。
5.重组腺病毒疫苗的鉴定
我们首先通过人复制缺陷Ad5腺病毒载体分别表达SARS-CoV-2突变株Omicron BA.5和Omicron XBB.1.5的全长刺突糖蛋白，通过免疫印迹Western blot的方法检测Ad5XBB.1.5和Ad5BA.5重组腺病毒感染293T细胞48小时后刺突蛋白Spike的表达水平。如图9所示，腺病毒Ad5XBB.1.5感染293T细胞后可以诱导产生高水平的Spike蛋白表达，而空载腺病毒对照Ad5Empty则不能，证明我们的重组腺病毒疫苗制备成功。
其中，通过实施例1制备的腺病毒疫苗分别定义为Ad5XBB.1.5和Ad5BA.5重组腺病毒。
通过以下试验证明本发明制备的重组蛋白疫苗和腺病毒疫苗的效果。
实施例3动物准备及小鼠免疫和样品收集
1、6-8周龄的雌性NIH小鼠购买自维通利华公司，小鼠饲养在四川大学生物治疗国家重点实验室的无特殊病原体的环境中。将10μg重组蛋白和一定量的佐剂混合，于小鼠第0、14、28天肌肉免疫三次，10μg蛋白/只小鼠，三免后7天取血检测中和抗体，评价疫苗的免疫效果。
2、6-8周龄的雌性BALB/c小鼠购买自维通利华公司，小鼠饲养在四川大学生物治疗国家重点实验室的无特殊病原体的环境中。为了制备双组分鼻喷疫苗，将2.5×109VP(低剂量)或5×109VP(高剂量)的Ad5XBB.1.5与10μg RBDXBB.1.5-HR以50μl的总体积混合，分别配制低剂量或高剂量的双组分疫苗。用(1)低剂量和(2)高剂量的Ad5XBB.1.5单独免疫三次BALB/c小鼠，(3)低剂量和(4)高剂量的双组分(Ad5XBB.1.5+RBDXBB.1.5-HR)疫苗，(5)10μg单独的RBDXBB.1.5-HR蛋白，或(6)5×109VP的Ad5Empty与RBDXBB.1.5-HR蛋白混合经鼻内递送，间隔28天。为了避免过多的液体流入肺组织，小鼠在一天内接受两次鼻内免疫，每次25μl，间隔3小时以上。
其他双组分疫苗Ad5BA.5+RBDBA.5-HR、Ad5BA.5+RBDXBB.1.5-HR、Ad5XBB.1.5+RBDBA.5-HR的制备和免疫方案与上述相似。
由5×109VP的Ad5XBB.1.5，3.3μg的RBDBA.5-HR和6.6μg的RBDXBB.1.5-HR组成的三组分疫苗，以相同的免疫程序经鼻给药。在接种动物的第3、7和11周收集血清样本，以测定结合和中和抗体应答。为了进一步评估疫苗诱导的粘膜免疫，在第三次加强疫苗接种后的第21天对小鼠实施安乐死，以收集BALF和肺组织。
实施例4酶联免疫吸附试验(ELISA)检测抗体
为了检测抗新型冠状病毒RBD特异性IgG和IgA，96孔板(NUNC-MaxiSorp，Thermo Fisher Scientific)在碳酸盐-碳酸氢盐缓冲液中涂覆1μg/ml重组RBD蛋白，在4℃过夜。第二天，用含有0.1％吐温-20(PBST)的1×PBS洗涤平板三次，然后用含有1％BSA的PBST在室温下封闭1小时。将连续稀释的血清、气管-肺洗液或稀释缓冲液中的鼻拭子加入孔中(100μl/孔)。在37℃孵育1小时后，将平板洗涤三次，然后加入稀释的辣根过氧化物酶(HRP)缀合的山羊-抗小鼠IgG(1:10000，southern biotech，Cat：0107-05)、HRP-缀合的山羊-抗小鼠IgA(1:5000)、HRP-缀合的山羊-抗兔IgG、HRP-缀合的山羊-抗兔IgG、HRP-缀合的山羊-抗人IgG(southern biotech，Cat：62-8420)或HRP-缀合的山羊-抗人IgA(southern biotech，Cat：2050-05)。然后，在37℃孵育1小时，进一步洗涤平板三次，并在室温下用3，3’，5，5’-四甲基联苯二胺(TMB)显影10分钟。用1M H2SO4停止反应。最后，在微孔板阅读器(Spectramax ABS，Molecular Devices)上测量450nm处的吸光度。终点滴度定义为吸光度≥2.1 倍阴性对照血清值的血清的最高倒数稀释度。
结果如图10-14所示，在完成免疫后，重组腺病毒Ad5联合重组RBD的制剂诱导出比单纯的Ad5腺病毒更强的血清和肺泡灌洗液抗RBD特异性的结合抗体，说明腺病毒联合重组亚单位制剂可以产生更强的体液免疫保护能力。此外，单纯的RBD滴鼻免疫无法诱导出强烈的血液结合抗体，但是与空载Ad5Empty或重组的腺病毒Ad5结合后，可以显著提高其免疫原性，这证明腺病毒可以作为重组亚单位的佐剂增强蛋白抗原的免疫原性。
实施例5新型冠状病毒假病毒中和试验
为了检测血清和BALF样品中中和抗体的滴度，如前所述进行假病毒中和试验。表达荧光素酶的假病毒，包括原型、δ和Omicron亚系(BA.2.75、BA.5、BF.7、BQ.1、BQ.1.1、XBB、XBB.1.5和XBB.1.16等)，购自Genomeditech公司。
简言之，将灭活的血清和BALF样本(56℃30分钟)稀释三倍，范围为30-65610，然后与等体积不同稀释度的假病毒在37℃孵育1小时。然后，在每个孔中加入1.2×104个表达人ACE2受体(293T/ACE2)的HEK-293T细胞，并在37℃下孵育48小时以表达荧光素酶。最后，除去上清液，然后加入具有荧光素酶底物的裂解试剂(Beyotime，RG005)，使用多模式微孔板阅读器(PerkinElmer，USA)测量293T/ACE2细胞中的发光量。用GraphPad Prism8.0.2测定和计算假病毒的50％中和率。阳性对照组仅包含细胞和病毒，阴性对照组仅包含细胞，样品组包含细胞、样品和病毒。使用以下公式计算中和百分比：
中和度(％)＝(阳性样本-待测样本/阳性样本-阴性样本)×100％
如图15-16所示，单纯的蛋白疫苗(重组RBDXBB.1.5-HR和重组RBDXBB.1.5)在完成三针免疫后，小鼠产生了较强的针对WT、Delta、BA.2.75、BF.7、BA.5、BQ.1、BQ.1.1、XBB和XBB.1.5等病毒的血清中和抗体，且MF59佐剂诱导的中和抗体明显优于铝佐剂；图17显示，在第0、14、42天三次肌肉免疫灭活疫苗25U/只，于末次免疫后84天分别进行灭活苗(中国医药集团有限公司生产)、RBDXBB.1.5序贯加强，肌肉注射，免疫剂量为10μg/只，加强后14天采集血清检测假病毒中和抗体，接种RBDXBB.1.5疫苗的小鼠，产生了较强的对WT、BA.5、XBB.1.5、XBB.1.6、XBB.1.16、XBB.1.9.1和XBB.2.3等病毒的血清中和抗体，其中对最近占优势的XBB等亚型流行株尤其有效。
腺病毒联合重组RBD蛋白的双价苗制剂诱导出比单纯的腺病毒更强的血清和肺泡灌洗液中和抗体，说明腺病毒联合重组亚单位制剂可以产生更强的血液和黏膜局部的中和保护能力，阻止病毒的感染；如图18-20所示，Ad5XBB.1.5+RBDXBB.1.5-HR对WT、Delta、BA.2.75、BF.7、BA.5、BQ.1、BQ.1.1、XBB、XBB.1.5、XBB.1.16等病毒产生了较高水平的血清中和抗体，提示Omicron的S蛋白腺病毒疫苗联合S-RBD蛋白，能产生对Omicron突变株更强的 免疫保护。
实施例6新型冠状病毒活病毒中和试验
通过真实的病毒中和试验进行来自接种Ad5XBB.1.5+RBDXBB.1.5-HR双价苗的小鼠血清样品中针对活的祖先和突变的新型冠状病毒的中和抗体。将每组的稀释血清以50％的组织培养感染剂量(TCID50)与活新型冠状病毒混合。37℃孵育1小时后，将混合物加入覆盖有Vero E6细胞(5×104/孔)的96孔微孔板中孵育72小时。用显微镜测量细胞致病效应(CPE)，并计算免疫血清中导致EC50(50％中和)抑制的中和抗体的滴度。
结果显示如图21所示，滴鼻Ad5XBB.1.5+RBDXBB.1.5-HR双价苗的小鼠血清对真病毒Delta、奥米克戎变异株，特别是近期流行株XBB.1.16的中和抗体滴度达到1000以上，且显著高于滴鼻单独的腺病毒和蛋白。
实施例7新型冠状病毒XBB.1.16变异体的攻击
在第0、28和56天，用三倍的Ad5XBB.1.5单独或双组分或三组分疫苗鼻内免疫BALB/c小鼠。用PBS或Ad5Empty免疫的小鼠作为对照，每组n＝6只小鼠。然后在最后一次疫苗接种后的第21天，用活的新型冠状病毒XBB.1.16奥密克戎突变体(1×106PFU)对所有小鼠进行鼻内攻击。感染后四天，对小鼠实施安乐死并收集组织。苏木精伊红染色观察肺组织病理改变。通过测量病毒基因组RNA(gRNA)的逆转录定量聚合酶链反应(RT-qPCR)检测鼻甲、气管和肺组织样品中的病毒载量，引物序列为5′-GACCCCAAAATCAGCGAAAT-3′(正向)(SEQ ID No.26)，5′-TCTGGTTACTGCCCAGTTGAATCTG-3′(反向)(SEQ ID No.27)，探针序列为5′-FAM-ACGCCGCATTACGTTTGGTGGGACC-BHQ1-3′(SEQ ID No.28)。与新型冠状病毒奥米克隆变异体小鼠攻击相关的所有程序均由中国医学科学院医学生物学研究所机构动物护理和使用委员会审查和批准，并在昆明国家高级生物安全灵长类动物研究中心的ABSL-4设施中进行。
用Ad5XBB.1.5和双组分(Ad5XBB1.5+RBDXBB.1.5-HR)疫苗对BALB/c小鼠进行三次鼻内免疫。用PBS、裸RBDXBB.1.5-HR、Ad5Empty+RBDXBB-1.5-HR处理的小鼠用作对照。用1×106PFU的活的严重急性呼吸系统综合征冠状病毒2型XBB.1.16奥密克戎病毒攻击免疫的BALB/c小鼠，测定严重急性呼吸系统综合征冠状病毒2型感染后咽拭子病毒载量的变化；在感染后第4天收集鼻甲、气管和肺组织，通过RT-qPCR测定gRNA和sgRNA的水平；观察奥密克戎攻击后小鼠肺组织的组织病理学变化。
如图22-26所示，用Ad5XBB.1.5(5×109VP/只)+RBDXBB.1.5-HR(10μg)联合疫苗诱导的免疫血清处理小鼠后，咽拭子病毒基本被清除干净，鼻甲、气管和肺组织中的病毒RNA均未检测到，肺组织学正常，肺泡结构完整，没有明显炎症；而PBS等对照组，咽拭子样本仍 有较多的病毒载量，鼻甲、气管和肺组织中的病毒RNA水平较高，病理切片可观察到轻微的病理变化，包括多灶性实变区和轻度肺泡间隔增厚和肺泡充血；此外，在小血管附近偶尔观察到由巨噬细胞、中性粒细胞和淋巴细胞组成的小块炎症。显示出Ad5XBB.1.5+RBDXBB.1.5-HR双价疫苗对XBB.1.16奥密克戎变异株的感染具有完全的保护作用。

Claims (31)

  1. 抗SARS-CoV-2奥密克戎突变株XBB及其亚型感染的蛋白，其特征是：其含有选自SEQ ID No.1～SEQ ID No.7中任意一项所示的氨基酸序列。
  2. 权利要求1所述蛋白的前体，其特征是：其是在所述蛋白上连接了信号肽和/或蛋白标签。
  3. 根据权利要求2所述的蛋白前体，其特征是：所述的蛋白标签选自如下至少一种：组氨酸标签、硫氧还蛋白标签、谷胱甘肽转移酶标签、泛素样修饰蛋白标签、麦芽糖结合蛋白标签、c-Myc蛋白标签、Avi tag蛋白标签、氮源利用物质A蛋白标签。
  4. 根据权利要求3所述的蛋白前体，其特征是：在所述抗SARS-CoV-2奥密克戎突变株XBB及其亚型感染的蛋白上还连接了切除蛋白标签的蛋白酶识别区；优选地，所述的蛋白酶选自如下至少一种：肠激酶、TEV蛋白酶、凝血酶、凝血因子Xa、羧肽酶A、鼻病毒3c蛋白酶。
  5. 根据权利要求2-4任一项所述的蛋白前体，其特征是：所述前体的氨基酸序列选自SEQ ID No.8、SEQ ID No.10、SEQ ID No.12、SEQ ID No.14、SEQ ID No.16、SEQ ID No.18、SEQ ID No.20中至少一种。
  6. 多核苷酸，其特征是：其编码权利要求1所述的蛋白或权利要求2-5任一项所述的前体。
  7. 根据权利要求6所述的多核苷酸，其特征是：所述核苷酸序列选自SEQ ID No.9、SEQ ID No.11、SEQ ID No.13、SEQ ID No.15、SEQ ID No.17、SEQ ID No.19、SEQ ID No.21中至少一种。
  8. 重组载体，其特征是：其含有权利要求6或7所述的多核苷酸。
  9. 根据权利要求8所述的重组载体，其特征是：所述重组载体采用昆虫杆状病毒表达载体、哺乳动物细胞表达载体、大肠杆菌表达载体、酵母表达载体中至少一种；所述的昆虫杆状病毒表达载体为pFastBac1；所述的大肠杆菌表达载体为pET32a；所述的酵母表达载体为pPICZaA；所述的哺乳动物细胞表达载体为CHO细胞表达载体；进一步优选地，所述的CHO细胞表达载体为pTT5或FTP-002。
  10. 宿主细胞，其特征是：其含有权利要求8或9所述的重组载体。
  11. 根据权利要求10所述的宿主细胞，其特征是：所述的宿主细胞采用昆虫细胞、哺乳动物细胞、大肠杆菌、酵母中至少一种；优选地，所述的昆虫细胞选自sf9细胞、sf21细胞、Hi5细胞中至少一种；所述的哺乳动物细胞为CHO细胞。
  12. 权利要求1所述的蛋白或权利要求2-5任一项所述的前体的制备方法，其特征是：包括如下步骤：培养权利要求10或11所述的宿主细胞，使其表达所述的蛋白或前体，然后回收所述的蛋白，即得。
  13. 抗SARS-CoV-2奥密克戎突变株及其亚型感染的蛋白组合物，其特征是：其包括BA.5序列-1、BA.5序列-2、XBB.1.5序列-1、XBB.1.5序列-2、XBB.1.5序列-3、XBB.1.5序列-4、S-XBB.1.5蛋白中任意两种及以上的组合。
  14. 根据权利要求13所述的蛋白组合物，其特征是：所述蛋白组合物包括BA.5序列-1、BA.5序列-2中的一种、XBB.1.5序列-1、XBB.1.5序列-2、XBB.1.5序列-3、XBB.1.5序列-4中的任意一种、S-XBB.1.5蛋白至少两种组合；优选地，所述蛋白组合物包括BA.5序列-1、BA.5序列-2中的一种、XBB.1.5序列-3、XBB.1.5序列-4中的任意一种与S-XBB.1.5蛋白进行的组合。
  15. 预防和/或治疗SARS-CoV-2奥密克戎突变株及其亚型感染的重组蛋白疫苗，其特征是：其含有权利要求1所述的蛋白、权利要求2-5任一项所述的前体和/或权利要求13-14任一项所述的蛋白组合物，以及药学上可接受的辅料或者辅助性成分。
  16. 根据权利要求15所述的重组蛋白疫苗，其特征是：所述的辅助性成分为免疫佐剂；优选地，所述的免疫佐剂选自如下至少一种：鲨烯水包油乳剂、铝盐、钙盐、植物皂苷、植物多糖、单磷酸脂质A、胞壁酰二肽、胞壁酰三肽、细菌毒素、GM-CSF细胞因子、脂质、阳离子脂质体材料。
  17. 根据权利要求15或16所述的重组蛋白疫苗，其特征是：满足以下至少一项：
    所述的鲨烯水包油乳剂为MF59；所述的铝盐选自氢氧化铝、明矾中至少一种；所述的钙盐为磷酸三钙；所述的植物皂苷为QS-21或ISCOM；所述的植物多糖为黄芪多糖；所述的细菌毒素选自重组霍乱毒素、白喉毒素中至少一种；所述的脂质选自如下至少一种：磷脂酰乙醇胺、磷脂酰胆碱、胆固醇、二油酰基磷脂酰乙醇胺；所述的阳离子脂质体材料选自如下至少一种：(2,3-二油氧基丙基) 三甲基氯化铵、N-[1-(2,3-二油酰氯)丙基]-N,N,N-氯化三甲胺、阳离子胆固醇、三氟乙酸二甲基-2,3-二油烯氧基丙基-2-(2-精胺甲酰氨基)乙基铵、溴化三甲基十二烷基铵、溴化三甲基十四烷基铵、溴化三甲基十六烷基铵、溴化二甲基双十八烷基铵、CpG ODN。
  18. 预防和/或治疗SARS-CoV-2奥密克戎突变株及其亚型感染的腺病毒载体疫苗，其特征是：其构建含有编码权利要求1所述蛋白、权利要求2-5任一项所述前体或权利要求13-14任一项所述蛋白组合物的氨基酸序列的多核苷酸的重组载体。
  19. 根据权利要求18所述的腺病毒载体疫苗，其特征是：所述多核苷酸的核苷酸序列选自SEQ ID No.9、SEQ ID No.11、SEQ ID No.13、SEQ ID No.15、SEQ ID No.17、SEQ ID No.19、SEQ ID No.21中至少一种；更优选地，所述腺病毒载体含有的多核苷酸序列选自构建S-XBB.1.5的多核苷酸，如SEQ ID No.21所示。
  20. 根据权利要求18所述的腺病毒载体疫苗，其特征是：所述腺病毒载体选自如下至少一种：腺病毒、安卡拉痘苗病毒、腺相关病毒；优选地，选自人5型、35型或26型或/和黑猩猩AdC68型或AdC7型的复制缺陷型腺病毒；更优选地，选自E1、E3联合缺失的人5型复制缺陷型腺病毒。
  21. 根据权利要求18-19任一项所述腺病毒载体疫苗，其特征是：还包括药学上可接受的佐剂、载体、稀释剂或赋形剂。
  22. 权利要求18-21任一项所述腺病毒载体疫苗中的腺病毒的制备方法，其特征是：包括以下步骤：构建所述多核苷酸的穿梭质粒载体；然后将构建的穿梭质粒载体与骨架质粒一起转染入宿主细胞，培养宿主细胞；获得复制缺陷重组腺病毒，然后扩大培养、纯化，即可。
  23. 权利要求15-17任一项所述重组蛋白疫苗、权利要求18-21任一项所述腺病毒载体疫苗为皮内或皮下注射制剂、肌肉注射制剂、静脉注射制剂、口服或鼻喷制剂；优选地，所述的疫苗为肌肉注射制剂和鼻喷制剂。
  24. 治疗和/或预防SARS-CoV-2奥密克戎突变株及其亚型感染的疫苗组合物，其特征是：是含有权利要求15-17任一项所述的重组蛋白疫苗和权利要求18-21任一项所述的腺病毒载体疫苗为活性成分的复方制剂。
  25. 治疗和/或预防SARS-CoV-2奥密克戎突变株及其亚型感染的联合用药物，其特征是：其含有分别或同时给药的权利要求15-17任一项所述的重组蛋白疫苗和权利要求18-21任一项所述的腺病毒载体疫苗。
  26. 根据权利要求24所述组合物或权利要求25所述的联合用药物，其特征是：其含有重组蛋白疫苗1、重组蛋白疫苗2和腺病毒载体疫苗的组合或联合用药；优选地，所述重组蛋白疫苗1含有BA.5序列-1、BA.5序列-2蛋白或前体中至少一种的氨基酸序列；所述重组蛋白疫苗2含有XBB.1.5序列-1、XBB.1.5序列-2、XBB.1.5序列-3、XBB.1.5序列-4蛋白或前体中的至少一种氨基酸序列；所述腺病毒载体疫苗含有S-XBB.1.5的多核苷酸序列。
  27. 根据权利要求26所述的组合物或联合用药物，其特征是：所述重组蛋白疫苗1含有的氨基酸序列选自SEQ ID No.1、SEQ ID No.2、SEQ ID No.8、SEQ ID No.10中至少一种；所述重组蛋白疫苗2含有的氨基酸序列选自SEQ ID No.3～SEQ ID No.6、SEQ ID No.12、SEQ ID No.14、SEQ ID No.16、SEQ ID No.18中至少一种；优选地，选自SEQ ID No.5、SEQ ID No.6、SEQ ID No.16、SEQ ID No.18中至少一种；所述腺病毒载体疫苗含有如SEQ ID No.21所示的多核苷酸序列。
  28. 根据权利要求27所述的组合物或联合用药物，其特征是：所述组合物或联合用药物为皮内或皮下注射制剂、肌肉注射制剂、静脉注射制剂、口服或鼻喷制剂；优选地，所述的疫苗为肌肉注射制剂和鼻喷制剂。
  29. 权利要求1所述的蛋白、权利要求2-5任一项所述的前体、13-14任一项所述的蛋白组合物、15-17任一项所述的重组蛋白疫苗、权利要求18-21任一项所述的腺病毒载体疫苗、24-28任一项所述的疫苗组合物或联合用药物在制备治疗和/或预防SARS-CoV-2奥密克戎突变株及其亚型感染或致病的药物中的用途。
  30. 治疗和/或预防SARS-CoV-2奥密克戎突变株及其亚型感染的疫苗组合物，其特征是：其含有权利要求15-17任一项所述重组蛋白疫苗和腺病毒载体疫苗；所述重组蛋白疫苗的氨基酸序列选自SEQ ID No.1～SEQ ID No.7、SEQ ID No.8～SEQ ID No.20中至少一种；所述腺病毒载体疫苗的抗原的核苷酸序列选自SEQ ID No.23。
  31. 治疗和/或预防SARS-CoV-2奥密克戎突变株及其亚型感染的联合用药物，其特征是：其分别或同时给药权利要求15-17任一项所述的重组蛋白疫苗和 腺病毒载体疫苗；所述重组蛋白疫苗的氨基酸序列选自SEQ ID No.1～SEQ ID No.7、SEQ ID No.8～SEQ ID No.20中至少一种；所述腺病毒载体疫苗的抗原的核苷酸序列选自SEQ ID No.23。
PCT/CN2023/113170 2023-03-17 2023-08-15 抗SARS-CoV-2奥密克戎突变株XBB及其亚型感染的蛋白及疫苗 WO2024082795A1 (zh)

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