WO2023138648A1 - Sars-cov-2 spike protein variant, pharmaceutical composition and use thereof - Google Patents

Sars-cov-2 spike protein variant, pharmaceutical composition and use thereof Download PDF

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Publication number
WO2023138648A1
WO2023138648A1 PCT/CN2023/073084 CN2023073084W WO2023138648A1 WO 2023138648 A1 WO2023138648 A1 WO 2023138648A1 CN 2023073084 W CN2023073084 W CN 2023073084W WO 2023138648 A1 WO2023138648 A1 WO 2023138648A1
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seq
strain
polypeptide
polypeptide component
amino acid
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PCT/CN2023/073084
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French (fr)
Chinese (zh)
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袁权
巫洋涛
王邵娟
张雅丽
魏敏
王楷
王子康
肖瑾
张天英
张军
夏宁邵
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厦门大学
厦门万泰沧海生物技术有限公司
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Publication of WO2023138648A1 publication Critical patent/WO2023138648A1/en

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    • 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
    • A61K39/12—Viral antigens
    • A61K39/215—Coronaviridae, e.g. avian infectious bronchitis virus
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
    • C07K14/08—RNA viruses
    • C07K14/165—Coronaviridae, e.g. avian infectious bronchitis virus
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K19/00—Hybrid peptides, i.e. peptides covalently bound to nucleic acids, or non-covalently bound protein-protein complexes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/10—Cells modified by introduction of foreign genetic material

Definitions

  • the invention belongs to the field of biomedicine, and relates to a variant of SARS-CoV-2 spike protein, its pharmaceutical composition and application.
  • the COVID-19 pandemic caused by SARS-CoV-2 is still raging around the world, causing a significant public health burden.
  • a variety of new crown vaccines have been approved for marketing or emergency use, which play a preventive role in combating the pathogenicity and spread of SARS-CoV-2 infection.
  • the Spike protein (S protein for short) of SARS-CoV-2 is a key component of the new crown vaccine and consists of three main domains: (1) N-terminal domain NTD; (2) receptor binding domain (RBD); (3) S2 subunit; all three can induce targeted neutralizing antibodies.
  • the current first-generation COVID-19 vaccine developed based on the Spike protein sequence of the prototype strain may have reduced vaccine protection efficacy against some immune escape variants, such as Beta strain (B.1.351) and Omicron (B.1.1.529). In view of this, it is necessary to develop new immunogenic molecules that can stimulate broad-spectrum neutralizing antibody responses against mutant strains.
  • the invention discloses a method and technology for using a C-terminus truncated and mutated recombinant recombinant protein of the extramembrane domain of the new coronavirus as a new coronavirus vaccine immunogen.
  • Animal tests have proved that the modified antigen can stimulate broad-spectrum neutralizing antibodies against different existing novel coronavirus variants in a monovalent or bivalent combination, and the results of animal challenge protection tests show that the antigen of the present invention has a good protective effect on both the prototype and variants of SARS-CoV-2.
  • One aspect of the present invention relates to an isolated polypeptide having an amino acid sequence as shown in any one of SEQ ID NOs: 1-27.
  • Another aspect of the invention relates to an isolated fusion protein comprising at least one polypeptide of the invention.
  • the fusion protein consists of at least one polypeptide of the present invention and a screening tag such as a his tag.
  • Yet another aspect of the invention relates to an isolated polynucleotide encoding a polypeptide of the invention.
  • nucleic acid construct which contains the polynucleotide of the present invention; preferably, the nucleic acid construct is a recombinant vector; preferably, the nucleic acid construct is a recombinant expression vector.
  • a further aspect of the invention relates to a transformed cell comprising a polynucleotide of the invention, or a nucleic acid construct of the invention.
  • compositions which contains at least one polypeptide of the present invention; optionally, it also contains pharmaceutically acceptable excipients;
  • the pharmaceutical composition is a vaccine preparation
  • the unit dose of the pharmaceutical composition is 0.01-100 ⁇ g, preferably 0.1-50 ⁇ g, more preferably 5-30 ⁇ g, 5-20 ⁇ g or 5-15 ⁇ g, especially preferably 10 ⁇ g;
  • the adjuvant is a vaccine adjuvant; preferably, the vaccine adjuvant is FH002C adjuvant or aluminum adjuvant.
  • the pharmaceutical composition comprises the first polypeptide component and/or the second polypeptide component, wherein:
  • the first polypeptide component is selected from one or more of the polypeptides shown in any sequence of SEQ ID NOs: 1-14, and/or the second polypeptide component is selected from one or more of the polypeptides shown in any sequence of SEQ ID NOs: 15-27;
  • the first polypeptide component is selected from one or more of the polypeptides shown in any sequence of SEQ ID NOs:9-14, and/or the second polypeptide component is selected from one or more of the polypeptides shown in any sequence of SEQ ID NOs:24-27;
  • amino acid sequence of the first polypeptide component is shown in SEQ ID NO: 9, and/or the second polypeptide component is selected from one, two, three or four of the polypeptides shown in any sequence of SEQ ID NOs: 24-27;
  • amino acid sequence of the first polypeptide component is shown in SEQ ID NO: 10
  • the second polypeptide component is selected from one, two, three or four of the polypeptides shown in any sequence of SEQ ID NOs: 24-27;
  • amino acid sequence of the first polypeptide component is shown in SEQ ID NO: 11, and/or the second polypeptide component is selected from one, two, three or four of the polypeptides shown in any sequence of SEQ ID NOs: 24-27;
  • amino acid sequence of the first polypeptide component is shown in SEQ ID NO: 12, and/or the second polypeptide component is selected from one, two, three or four of the polypeptides shown in any sequence of SEQ ID NOs: 24-27;
  • amino acid sequence of the first polypeptide component is shown in SEQ ID NO: 13, and/or the second polypeptide component is selected from one, two, three or four of the polypeptides shown in any sequence of SEQ ID NOs: 24-27;
  • amino acid sequence of the first polypeptide component is shown in SEQ ID NO: 14, and/or the second polypeptide component is selected from one, two, three or four of the polypeptides shown in any sequence of SEQ ID NOs: 24-27;
  • amino acid sequence of the second polypeptide component is shown in SEQ ID NO:24, and/or the first polypeptide component is selected from one, two, three, four, five or six of the polypeptides shown in any sequence of SEQ ID NOs:9-14;
  • amino acid sequence of the second polypeptide component is shown in SEQ ID NO:25, and/or the first polypeptide component is selected from one, two, three, four, five or six of the polypeptides shown in any sequence of SEQ ID NOs:9-14;
  • amino acid sequence of the second polypeptide component is shown in SEQ ID NO:26, and/or the first polypeptide component is selected from one, two, three, four, five or six of the polypeptides shown in any sequence of SEQ ID NOs:9-14;
  • amino acid sequence of the second polypeptide component is shown in SEQ ID NO:27, and/or the first polypeptide component is selected from one, two, three, four, five or six of the polypeptides shown in any sequence of SEQ ID NOs:9-14.
  • the pharmaceutical composition wherein the amino acid sequence of the first polypeptide component is shown in SEQ ID NO:14, and/or the amino acid sequence of the second polypeptide component is shown in SEQ ID NO:24.
  • the pharmaceutical composition wherein the amino acid sequence of the first polypeptide component is shown in SEQ ID NO: 14, and/or the amino acid sequence of the second polypeptide component is shown in SEQ ID NO: 25.
  • the pharmaceutical composition wherein the amino acid sequence of the first polypeptide component is shown in SEQ ID NO:14, and/or the amino acid sequence of the second polypeptide component is shown in SEQ ID NO:26.
  • the pharmaceutical composition wherein the amino acid sequence of the first polypeptide component is shown in SEQ ID NO:14, and/or the amino acid sequence of the second polypeptide component is shown in SEQ ID NO:27.
  • the pharmaceutical composition wherein the amino acid sequence of the first polypeptide component is shown in SEQ ID NO: 14, and/or the second polypeptide component is the polypeptide shown in SEQ ID NO: 24 and SEQ ID NO: 25.
  • the pharmaceutical composition wherein the amino acid sequence of the first polypeptide component is shown in SEQ ID NO: 14, and/or the second polypeptide component is the polypeptide shown in SEQ ID NO: 24 and SEQ ID NO: 26.
  • the pharmaceutical composition wherein the amino acid sequence of the first polypeptide component is shown in SEQ ID NO: 14, and/or the second polypeptide component is the polypeptide shown in SEQ ID NO: 24 and SEQ ID NO: 27.
  • the pharmaceutical composition wherein the amino acid sequence of the first polypeptide component is shown in SEQ ID NO: 14, and/or the second polypeptide component is the polypeptide shown in SEQ ID NO: 25 and SEQ ID NO: 26.
  • the pharmaceutical composition wherein the amino acid sequence of the first polypeptide component is shown in SEQ ID NO: 14, and/or the second polypeptide component is the polypeptide shown in SEQ ID NO: 25 and SEQ ID NO: 27.
  • the pharmaceutical composition wherein the amino acid sequence of the first polypeptide component is shown in SEQ ID NO: 14, and/or the second polypeptide component is the polypeptide shown in SEQ ID NO: 26 and SEQ ID NO: 27.
  • the pharmaceutical composition wherein the amino acid sequence of the first polypeptide component is shown in SEQ ID NO: 14, and/or the second polypeptide component is the polypeptide shown in SEQ ID NO: 24, SEQ ID NO: 25 and SEQ ID NO: 26.
  • the pharmaceutical composition wherein the amino acid sequence of the first polypeptide component is shown in SEQ ID NO: 14, and/or the second polypeptide component is the polypeptide shown in SEQ ID NO: 24, SEQ ID NO: 25 and SEQ ID NO: 27.
  • the pharmaceutical composition wherein the amino acid sequence of the first polypeptide component is shown in SEQ ID NO: 14, and/or the second polypeptide component is the polypeptide shown in SEQ ID NO: 24, SEQ ID NO: 26 and SEQ ID NO: 27.
  • the pharmaceutical composition wherein the amino acid sequence of the first polypeptide component is shown in SEQ ID NO: 14, and/or the second polypeptide component is the polypeptide shown in SEQ ID NO: 25, SEQ ID NO: 26 and SEQ ID NO: 27.
  • the pharmaceutical composition wherein, the amino acid sequence of the first polypeptide component is shown in SEQ ID NO: 14, and/or the second polypeptide component is the polypeptide shown in SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26 and SEQ ID NO: 27.
  • the pharmaceutical composition wherein, when the first polypeptide component contains more than one polypeptide, the mass ratio between each polypeptide is 1:1; and/or
  • the pharmaceutical composition wherein, when the second polypeptide component contains more than one polypeptide, the mass ratio between two polypeptides is 1:1.
  • the pharmaceutical composition wherein, when the second polypeptide component is selected from two, three or four of the polypeptides shown in any sequence of SEQ ID NOs: 24-27, the mass ratio between each polypeptide is 1:1.
  • the pharmaceutical composition wherein, when the first polypeptide component is selected from two, three, four, five or six of the polypeptides shown in any sequence of SEQ ID NOs: 9-14, the mass ratio between each polypeptide is 1:1.
  • the mass ratio of the first polypeptide component to the second polypeptide component is (1:10) to (10:1), (1:5) to (5:1), (1:3) to (3:1), (1:2) to (2:1), (1:1.5) to (1.5:1) or 1:1;
  • the active ingredient of the pharmaceutical composition consists of the first polypeptide component and the second polypeptide component.
  • Another aspect of the present invention relates to the use of the polypeptide, fusion protein, polynucleotide or nucleic acid construct of the present invention in the preparation of anti-SARS-CoV-2 medicaments;
  • the SARS-CoV-2 is selected from a prototype strain of SARS-CoV-2 or a variant strain of SARS-CoV-2; preferably, the variant strain is selected from one or more of the following:
  • the Omicron strain is one or more selected from the following:
  • the drug is any one of the pharmaceutical compositions of the present invention.
  • Another aspect of the present invention relates to the use of the polypeptide of the present invention, the polynucleotide of the present invention or the nucleic acid construct of the present invention in the preparation of medicines (such as vaccine preparations) for the treatment or prevention of COVID-19 or the symptoms caused by it;
  • the drug is any one of the pharmaceutical compositions of the present invention.
  • the use, wherein the unit dose of the drug or vaccine preparation The amount is 0.01-100 ⁇ g, preferably 0.1-50 ⁇ g, more preferably 5-30 ⁇ g, 5-20 ⁇ g or 5-15 ⁇ g, particularly preferably 10 ⁇ g.
  • polypeptide, fusion protein, polynucleotide or nucleic acid construct of the present invention it is used for anti-SARS-CoV-2;
  • the SARS-CoV-2 is selected from a prototype strain of SARS-CoV-2 or a variant strain of SARS-CoV-2; preferably, the variant strain is selected from one or more of the following:
  • the Omicron strain is one or more selected from the following:
  • polypeptide, fusion protein, polynucleotide or nucleic acid construct of the present invention it is used for treating or preventing COVID-19 or the symptoms caused by it.
  • Another aspect of the present invention relates to an anti-SARS-CoV-2 method, comprising the step of administering an effective amount of the polypeptide, fusion protein, polynucleotide or nucleic acid construct of the present invention to a subject in need;
  • the SARS-CoV-2 is selected from a prototype strain of SARS-CoV-2 or a variant strain of SARS-CoV-2; preferably, the variant strain is selected from one or more of the following:
  • the Omicron strain is one or more selected from the following:
  • the administration is by a pharmaceutical composition according to any one of the present invention.
  • Another aspect of the present invention relates to a method for treating or preventing COVID-19 or the symptoms caused by it, comprising the step of administering an effective amount of the polypeptide, fusion protein, polynucleotide or nucleic acid construct of the present invention to a subject in need;
  • the administration is by a pharmaceutical composition according to any one of the present invention.
  • the vaccine formulations of the present invention may be therapeutic vaccine formulations or prophylactic vaccine formulations.
  • the vaccine formulation of the present invention can be administered by intramuscular injection or subcutaneous injection.
  • first polypeptide component and “second polypeptide component” may refer to one polypeptide or a mixture of several polypeptides.
  • first and second are just to distinguish or express clearly, not without typical The meaning of the order of the type.
  • unit dose is calculated according to the mass of the antigen contained therein (for example, the first polypeptide component, or the first polypeptide component + the second polypeptide component).
  • Figure 1 SDS-PAGE analysis of cell supernatants of STFK-a to STFK-h eukaryotic expression for 6 days.
  • FIG. 1 SDS-PAGE analysis results of purified proteins from STFK-a to STFK-h.
  • Figure 3 Curve analysis of the binding ability of S trimer and monomer S purified protein to Ace2 and EC 50 results.
  • Figure 4A SDS-PAGE detection of protein expression in cell supernatant 6 days after C-terminal mutation.
  • Figure 4B Native-PAGE detection of protein expression in cell supernatant 6 days after C-terminal mutation.
  • Figure 5A Binding curves of STFK-HR to STFK-MR to RBD mAb 85F7.
  • Figure 5B Binding curves of STFK-HR to STFK-MR to RBD mAb 81H10.
  • Figure 6A Neutralizing antibody titers induced by 1 ⁇ g antigen doses of monomeric and trimeric vaccines. Note: when the neutralizing antibody detection ID50 ⁇ 30, it will be included in the drawing and statistical analysis according to 30.
  • Figure 6B Neutralizing antibody titers induced by 10 ⁇ g antigen doses of monomeric and trimeric vaccines. Note: when the neutralizing antibody detection ID50 ⁇ 30, it will be included in the drawing and statistical analysis according to 30.
  • Figure 7A Anti-RBD antibody titers in serum of mice immunized with different doses of STFK vaccine.
  • Figure 7B Anti-Spike antibody titers in serum of mice immunized with different doses of STFK vaccine.
  • Figure 8A Serum pseudovirus neutralizing antibody titers after mice were immunized with different doses of STFK vaccine.
  • Figure 8B Serum euvirus neutralizing antibody titers after mice were immunized with different doses of STFK vaccine.
  • Figure 8C Correlation of serum pseudovirus and true virus neutralizing antibodies after mice were immunized with different doses of STFK vaccine.
  • Figure 9 Analysis of cellular immune response induced by STFK vaccine in mice by IFN- ⁇ ELISPOT assay.
  • Fig. 10A Anti-RBD antibody titer in serum after immunizing rhesus macaques with 1 ⁇ g antigen dose of STFK vaccine.
  • Figure 10B Anti-spike antibody titer in serum after immunizing rhesus macaques with 1 ⁇ g antigen dose of STFK vaccine.
  • Figure 10C Anti-RBD antibody titer in serum after immunizing rhesus monkeys with 15 ⁇ g antigen dose of STFK vaccine.
  • FIG. 10D Anti-spike antibody titers in serum after immunizing rhesus monkeys with 15 ⁇ g antigen dose of STFK vaccine.
  • Figure 11A Pseudovirus neutralizing antibody titers in serum after immunizing rhesus monkeys with 1 ⁇ g antigen dose of STFK vaccine.
  • Figure 11B Pseudovirus neutralizing antibody titers in serum after immunizing rhesus monkeys with 15 ⁇ g antigen dose of STFK vaccine.
  • Fig. 11C The titer of true virus neutralizing antibody in serum after rhesus macaques were immunized with 1 ⁇ g antigen dose of STFK vaccine.
  • Figure 11D The titer of true virus neutralizing antibody in serum after immunizing rhesus macaques with 15 ⁇ g antigen dose of STFK vaccine.
  • Figure 11E Correlation of neutralizing antibody titers between pseudoviruses and true viruses in the serum of rhesus monkeys immunized with STFK vaccine.
  • Figure 12A STFK vaccine 1 ⁇ g antigen dose immunized rhesus macaque serum for comparison of neutralizing antibodies against various new coronavirus variants.
  • Figure 12B STFK vaccine 15 ⁇ g antigen dose immunized rhesus macaque serum for comparison of neutralizing antibodies against various new coronavirus variants.
  • Figure 13 Schematic diagram of the construction of STFK molecules based on SARS-CoV2 mutant strains.
  • Figure 14A SDS-PAGE analysis of ExpiCHO expressed purified STFK1351. Lane 1: Cell supernatant; Lane 2: Q-FF column flow-through; Lane 3: Fraction 1 eluted with 100mM NaCl; Lane 4: Fraction 2 eluted with 100mM NaCl; the arrow indicates the target protein.
  • Figure 14B SDS-PAGE analysis of ExpiCHO expression of purified STFK1128.
  • Lane 1 Cell supernatant
  • Lane 2 Q-FF column flow-through
  • Lane 3 Fraction 1 eluted with 100mM NaCl
  • Lane 4 Fraction 2 eluted with 100mM NaCl
  • the arrow indicates the target protein.
  • Figure 14C SDS-PAGE analysis of ExpiCHO expressing purified STFK1620.
  • Lane 1 Cell supernatant
  • Lane 2 Q-FF column flow-through
  • Lane 3 Fraction 1 eluted with 100mM NaCl
  • Lane 4 Fraction 2 eluted with 100mM NaCl
  • the arrow indicates the target protein.
  • Figure 15A Neutralizing antibodies induced by STFK1351 in hamsters. Note: The broken line and histogram represent the neutralizing antibody titers induced by STFK of the prototype strain against the corresponding mutant strains.
  • Figure 15B Neutralizing antibodies induced by STFK1128 in hamsters. Note: The broken line and histogram represent the neutralizing antibody titers induced by STFK of the prototype strain against the corresponding mutant strains.
  • Figure 15C Neutralizing antibodies induced by STFK1620 in hamsters. Note: The broken line and histogram represent the neutralizing antibody titers induced by STFK of the prototype strain against the corresponding mutant strains.
  • Figure 16A SDS-PAGE analysis of ExpiCHO expression of purified STFK1128b.
  • Lane 1 Cell supernatant;
  • Lane 2 Q-FF column flow-through;
  • Lane 3 Fraction 1 eluted with 100mM NaCl;
  • Lane 4 Fraction 2 eluted with 100mM NaCl; the arrow indicates the target protein.
  • Figure 16B SDS-PAGE analysis of ExpiCHO expressed purified STFK1128c.
  • Lane 1 Cell supernatant;
  • Lane 2 Q-FF column flow-through;
  • Lane 3 Fraction 1 eluted with 100mM NaCl;
  • Lane 4 Fraction 2 eluted with 100mM NaCl; the arrow indicates the target protein.
  • Figure 16C SDS-PAGE analysis of ExpiCHO expression of purified STFK1128d.
  • Lane 1 cell supernatant
  • lane 2 Q-FF chromatography column flow-through
  • lane 3 fraction 1 eluted with 100 mM NaCl
  • lane 4 fraction 2 eluted with 100 mM NaCl
  • the arrow indicates the target protein.
  • Figure 16D SDS-PAGE analysis of ExpiCHO expression of purified STFK1128e.
  • Lane 1 Cell supernatant;
  • Lane 2 Q-FF column flow-through;
  • Lane 3 Fraction 1 eluted with 100mM NaCl;
  • Lane 4 Fraction 2 eluted with 100mM NaCl; the arrow indicates the target protein.
  • Figure 16E SDS-PAGE analysis of ExpiCHO expression of purified STFK1128f.
  • Lane 1 Cell supernatant;
  • Lane 2 Q-FF column flow-through;
  • Lane 3 Fraction 1 eluted with 100mM NaCl;
  • Lane 4 Fraction 2 eluted with 100mM NaCl; the arrow indicates the target protein.
  • Figure 16F SDS-PAGE analysis of ExpiCHO expression of purified STFK1128g.
  • Lane 1 Cell supernatant; Lane 2: Q-FF column flow-through; Lane 3: Fraction 1 eluted with 100mM NaCl; Lane 4: Fraction 2 eluted with 100mM NaCl; the arrow indicates the target protein.
  • Figure 17A Neutralizing antibodies induced by STFK1128b in hamsters. Note: The broken line and histogram represent the neutralizing antibody titers induced by STFK of the prototype strain against the corresponding mutant strains.
  • Figure 17B Neutralizing antibodies induced by STFK1128c in hamsters. Note: The broken line and histogram represent the neutralizing antibody titers induced by STFK of the prototype strain against the corresponding mutant strains.
  • Figure 17C Neutralizing antibodies induced by STFK1128d in hamsters. Note: The broken line and histogram represent the neutralizing antibody titers induced by STFK of the prototype strain against the corresponding mutant strains.
  • Figure 17D Neutralizing antibodies induced by STFK1128e in hamsters. Note: The broken line and histogram represent the neutralizing antibody titers induced by STFK of the prototype strain against the corresponding mutant strains.
  • Figure 17E Neutralizing antibodies induced by STFK1128f in hamsters. Note: The broken line and histogram represent the neutralizing antibody titers induced by STFK of the prototype strain against the corresponding mutant strains.
  • Figure 17F Neutralizing antibodies induced by STFK1128g in hamsters. Note: The broken line and histogram represent the neutralizing antibody titers induced by STFK of the prototype strain against the corresponding mutant strains.
  • Figure 18A SDS-PAGE analysis of ExpiCHO expression of purified STFK1628x.
  • Lane 1 Cell supernatant;
  • Lane 2 Q-FF column flow-through;
  • Lane 3 Fraction 1 eluted with 100mM NaCl;
  • Lane 4 Fraction 2 eluted with 100mM NaCl;
  • Lane 5 Fraction 2 eluted with 2M NaCl; the arrow indicates the target protein.
  • Figure 18B SDS-PAGE analysis of ExpiCHO expressed purified STFK1628x and STFK1328x.
  • Lane 1 Cell supernatant
  • Lane 2 Q-FF column flow-through
  • Lane 3 Fraction 1 eluted with 100mM NaCl
  • Lane 4 Fraction 2 eluted with 100mM NaCl
  • Lane 5 Fraction 2 eluted with 2M NaCl
  • the arrow indicates the target protein.
  • Figure 18C SDS-PAGE analysis of ExpiCHO expressed purified STFK1628z.
  • Lane 1 Cell supernatant;
  • Lane 2 Q-FF column flow-through;
  • Lane 3 Fraction 1 eluted with 100mM NaCl;
  • Lane 4 100mM NaCl Elution fraction 2;
  • lane 5 2M NaCl elution fraction 2; the arrow indicates the target protein.
  • Figure 19A Neutralizing antibodies induced by STFK1328x in hamsters. Note: The broken line and histogram represent the neutralizing antibody titers induced by STFK of the prototype strain against the corresponding mutant strains.
  • Figure 19B Neutralizing antibodies induced by STFK1628x in hamsters. Note: The broken line and histogram represent the neutralizing antibody titers induced by STFK of the prototype strain against the corresponding mutant strains.
  • Figure 19C Neutralizing antibodies induced by STFK1628y in hamsters. Note: The broken line and histogram represent the neutralizing antibody titers induced by STFK of the prototype strain against the corresponding mutant strains.
  • Figure 19D Neutralizing antibodies induced by STFK1628z in hamsters. Note: The broken line and histogram represent the neutralizing antibody titers induced by STFK of the prototype strain against the corresponding mutant strains.
  • Figure 20A Neutralizing antibodies induced by STFK1628x in hamsters. Note: The horizontal short line represents the neutralizing antibody titer induced by STFK of the prototype strain against the corresponding mutant strain.
  • Figure 20B Neutralizing antibodies induced by STFK1628y in hamsters. Note: The horizontal short line represents the neutralizing antibody titer induced by STFK of the prototype strain against the corresponding mutant strain.
  • Figure 20C STFK1628x-induced neutralizing antibodies to Omicron variants in hamsters. Note: The broken line represents the neutralizing antibody titer induced by the prototype strain STFK against the corresponding mutant strain, and the value at the top of the bar graph represents the fold increase of the neutralizing titer of STFK1628x compared to STFK.
  • FIG. 20D STFK1628y-induced neutralizing antibodies to Omicron variants in hamsters. Note: The broken line represents the neutralizing antibody titer induced by the prototype strain STFK against the corresponding mutant strain, and the value at the top of the bar graph represents the fold increase of the neutralizing titer of STFK1628y compared to STFK.
  • Figure 20E Neutralizing antibodies induced by bivalent vaccine in hamsters. Note: The horizontal short line represents the neutralizing antibody titer induced by STFK of the prototype strain against the corresponding mutant strain.
  • Figure 20F Neutralizing antibodies against Omicron variants induced by bivalent vaccine in hamsters. Note: The broken line represents the neutralizing antibody titer induced by the prototype strain STFK against the corresponding mutant strain, and the value at the top of the bar graph represents the increase in the neutralizing titer of the bivalent vaccine compared to STFK.
  • Figure 21A Schematic diagram of the in vivo protection experiment of STFK, STFK1628x and bivalent vaccinated hamsters against SARS-CoV-2 prototype strain and Beta strain challenge.
  • Figure 21B STFK, STFK1628x and bivalent vaccine hamster body weight changes after challenge with SARS-CoV-2 prototype strain. Asterisks indicate statistical significance (****P ⁇ 0.0001; ***P ⁇ 0.001; **P ⁇ 0.01; *P ⁇ 0.05; ns, not significant.
  • Figure 21C STFK, STFK1628x and bivalent vaccine hamsters challenged with SARS-CoV-2 prototype strain Save the curve.
  • Figure 21D STFK, STFK1628x and bivalent vaccine hamster SARS-CoV-2 prototype strain challenged viral RNA load in lung tissue.
  • Figure 21E STFK, STFK1628x and bivalent vaccine hamster SARS-CoV-2 prototype strain challenged lung tissue sections H&E staining pathological score.
  • Fig. 21F STFK, STFK1628x and bivalent vaccine hamster body weight changes after challenge with SARS-CoV-2 Beta strain.
  • Figure 21G Survival curves after challenge with STFK, STFK1628x and bivalent vaccinated hamsters with SARS-CoV-2 Beta strain.
  • Figure 21H STFK, STFK1628x and bivalent vaccine hamster SARS-CoV-2 Beta strain challenged lung tissue virus RNA load.
  • Figure 21I STFK, STFK1628x and bivalent vaccine hamster SARS-CoV-2 Beta strain challenged lung tissue sections H&E staining pathological score.
  • Figure 21J STFK, STFK1628x, STFK1628y and bivalent vaccine hamster body weight changes after SARS-CoV-2 Omicron BA.1 challenge.
  • Figure 21K Survival curves after challenge with STFK, STFK1628x, STFK1628y and bivalent vaccine hamsters SARS-CoV-2 Omicron BA.1.
  • Figure 21L STFK, STFK1628x, STFK1628y and SARS-CoV-2 Omicron BA.1 challenged lung tissue viral RNA load of hamsters vaccinated with bivalent vaccine.
  • Figure 21M STFK, STFK1628x, STFK1628y and bivalent vaccine hamster SARS-CoV-2 Omicron BA.1 challenged lung tissue sections H&E staining pathological score.
  • Partial sequences involved in the present invention are as follows:
  • aa1-1192 also referred to as aa1192, STFK-c or STFK-NR in the examples.
  • Example 1 Construction of a C-terminally truncated monomeric novel coronavirus spike protein STFK
  • the inventors designed a novel coronavirus recombinant subunit vaccine based on highly active trimeric antigen StriFK and novel adjuvant FH002C (Wu, Y., et al., A recombinant spike protein subunit vaccine confers protective immunity against SARS-CoV-2 infection and transmission in hamsters. Science Tanslational Medicine.20 Jul 2021.Vol 13, Issue 606), the vaccine can induce potent humoral and cellular immune responses, and has good immunogenicity in mice, rats, hamsters and non-human primates.
  • the exogenously introduced trimerization domain sequence of the trimer protein StriFK may cause the body to produce antibodies against the trimerization domain sequence, and may depolymerize into a heterogeneous form during the expression preparation process; therefore, the inventors designed to remove the foreign sequence without affecting the immunogenicity, and obtain a protein that can improve the expression and stability of the protein. monomeric form of the recombinant spike protein Spike.
  • EIRBsMie-StriFK expression plasmid On the basis of the EIRBsMie-StriFK expression plasmid, the C-terminal T4 fibrin trimerization domain sequence was removed by PCR cloning but the His tag was retained.
  • the end of the extramembrane region of the spike protein Spike started from aa1208 (counting from the N-terminus, the 1208th amino acid is the C-terminus, because the total amino acid length of the S protein extramembrane region is 1208 amino acids; SEQ ID NO: 1), every 8 amino acids were gradually reduced to aa1200(
  • ExpiCHO cells (Thermo Scientific Company) were cultured at a density of 3 ⁇ 10 6 cells/mL in Erlenmeyer shaker flasks with an appropriate amount of medium ExpiCHO TM Expression Medium (Thermo Scientific Company), and placed in a constant temperature shaker at 37°C, 8% CO 2 , and an appropriate speed for 24 hours until the cell density reached 6 ⁇ 10 6 cells/mL.
  • the kit use the ExpiFectamine TM CHO Transfection Kit (Thermo Scientific Company) to transfect the expression vectors EIRBsMie-STFK-a to EIRBsMie-STFK-h obtained in step 1.1 into ExpiCHO cells with his-tagged STFK-a to STFK-h, respectively, continue to culture under the same conditions for 17-24h, and then add the feed and expression enhancer provided in the kit.
  • the cells were replaced to 32° C., 5% CO 2 , in a constant temperature shaker with an appropriate rotation speed, and continued to culture for 6 days.
  • Sample reaction Take a microtiter plate coated with 45C3 antibody, add 100 ⁇ L of diluted sample to each well, and place in a 25°C incubator for 60 minutes to react.
  • Ace2 binding reaction After completing the sample reaction step, wash the ELISA plate 5 times with PBST washing solution (20mM PB7.4, 150mM NaCl, 0.1% Tween20), add 100 ⁇ L 1ng/mL Ace2-huFc (Sino Biological 10108-H02H) protein to each well, and place it in an incubator at 25°C for 60 minutes.
  • PBST washing solution (20mM PB7.4, 150mM NaCl, 0.1% Tween20)
  • Termination reaction and reading value measurement After completing the color reaction step, add 50 ⁇ L of stop solution (purchased from Beijing Wantai Bio-Pharmaceutical Co., Ltd.) to each well of the reacted microplate plate, and detect the OD450/630 value of each well on a microplate reader.
  • stop solution purchased from Beijing Wantai Bio-Pharmaceutical Co., Ltd.
  • STFK-c (aa1192, SEQ ID NO: 3) was used as a candidate molecule for subsequent screening experiments.
  • the inventors used bioinformatics methods and conventional dynamics simulations to scan the amino acid substitution of the last amino acid aa1192, and selected several mutations that can reduce the helical stability (weaken the tendency of intermolecular aggregation) to construct CHO expression clones, including mutant molecules in which the terminal amino acid aa1192 was replaced from the original N to H, A, Y, G, D, and M.
  • STFK-NR (aa1192 is N, SEQ ID NO:3), STFK-HR (aa1192 is H, SEQ ID NO:9), STFK-AR (aa1192 is A, SEQ ID NO:10), STFK-YR (aa1192 is Y, SEQ ID NO:11), STFK-GR (aa1192 is G, SEQ ID NO:12), STFK-DR (aa1192 is D, SEQ ID NO:13), STFK-MR (aa1192 is M, SEQ ID NO:14).
  • step 1.2 for protein expression SDS-PAGE electrophoresis (Fig. 4A) and native-PAGE electrophoresis (Fig. 4B) of the supernatant of ExpiCHO cells transiently transfected for 6 days showed that the mutation of amino acid 1192 at the C-terminus did not affect the protein expression.
  • the band at the position of the target protein (the protein band at 130kD-250kD in Fig. 4A) showed that the protein expression of each mutant strain was basically equivalent.
  • STFK-HR to STFK-MR protein purification Collect the ExpiCHO expression cell suspension in 1.4.1, centrifuge at 12000 rpm for 30 min at room temperature, and filter the supernatant through a 0.22 ⁇ m filter membrane. The supernatant sample was then purified using a Q-FF chromatography column (Q Sepharose Fast Flow, Cytiva, 17051001), and the target protein was eluted with 100 mM NaCl.
  • Q-FF chromatography column Q Sepharose Fast Flow, Cytiva, 17051001
  • ELISA binding activity detection see step 1.3.1 for reaction plate preparation. Take the purified STFK-HR to STFK-MR proteins and dilute them to 10 ⁇ g/mL in PBS solution containing 20% newborn bovine serum, and carry out the 5-fold gradient downward Dilution, the last concentration gradient is 0.128ng/mL, and ELISA detection is performed as follows:
  • Sample reaction Take a microtiter plate coated with 45C3 antibody, add 100 ⁇ L of diluted sample to each well, and place it in a 37°C incubator for 60 minutes to react.
  • Termination reaction and reading value measurement After completing the color reaction step, add 50 ⁇ L of stop solution (purchased from Beijing Wantai Bio-Pharmaceutical Co., Ltd.) to each well of the reacted microplate plate, and detect the OD450/630 value of each well on a microplate reader.
  • stop solution purchased from Beijing Wantai Bio-Pharmaceutical Co., Ltd.
  • Table 1 EC 50 results of STFK-HR to STFK-MR combined with 2 strains of RBD monoclonal antibodies
  • the substitution of the amino acid at position 1192 of aa1 has no significant effect on the expression of STFK protein.
  • the binding activity of the variant protein to the neutralizing antibody 85F7 is improved to a certain extent, and the substitution of M is relatively better.
  • the inventors studied the antigen STFK-MR (SEQ ID NO: 14) with aa1192 replaced by N to M as the prototype vaccine immunogen STFK.
  • STFK, the STFK of the prototype strain, or "STFK" in the STFK vaccine refer to STFK-MR unless otherwise specified.
  • VSV vesicular stomatitis virus
  • VSVpp new coronavirus
  • SARS-CoV-2 neutralizing antibody titer produced by the mice was drawn using GraphPad Prism software to draw the histogram/scatter coexistence graph of the group, and the results are shown in Figure 6A and Figure 6B.
  • the vaccine based on the monomeric STFK antigen has certain advantages compared with the trimeric antigen in terms of recombinant protein expression, neutralizing monoclonal antibody binding activity, Ace2 binding ability and mouse immunogenicity, and can be used as the immunogen of the new crown vaccine.
  • STFK vaccine By immunizing different doses of STFK vaccine (same as the STFK vaccine in step 1.5) in BALB/c mice, use an enzyme-linked immunoassay kit to detect (mouse anti-new coronavirus (2019-nCoV) S protein IgG antibody detection kit, Beijing Wantai Biological Pharmaceutical Co., Ltd., batch number: NCOmG20200902B-8022E; mouse anti-new coronavirus (2019-nCoV) S-RBD protein IgG antibody detection Kit, Beijing Wantai Bio-Pharmaceutical Co., Ltd., batch number: NCOmsG20201201B-8011E) Anti-new coronavirus S protein-specific IgG antibody (Anti-Spike IgG), anti-new coronavirus S protein RBD domain-specific IgG antibody (Anti-RBD IgG) and pseudovirus neutralizing antibody levels in the serum of mice at different time points after vaccination, to evaluate the humoral immune response induced by STFK vaccine in mouse models
  • mice with SPF grade 6-8 weeks old female BALB/c mice with SPF grade were randomly divided into 4 groups: 0.01 ⁇ g group, 0.1 ⁇ g group, 1 ⁇ g group and 10 ⁇ g group (the dose was calculated according to the antigen). Orbital venous blood was collected at week 0, week 2 and week 4.
  • the GMT levels of the induced Anti-Spike IgG antibodies were 7992, 77469, 123541, and 267035, respectively, while the GMT levels of the induced Anti-RBD IgG antibodies were 2411, 24465, 31213, and 61901, respectively, which also gradually increased with the increase of the antigen content.
  • all 4 doses of STFK vaccine immunized mice had two antibody index positive conversion rates all reached 100% (6/6).
  • VSV vesicular stomatitis virus
  • VSVpp new coronavirus pseudovirus pseudovirus
  • SARS-CoV-2 true virus neutralization test were used to detect the neutralizing antibody titer of the sera of immunized mice.
  • the results are shown in Figure 8A, Figure 8B and Figure 8C.
  • the antigen content was 0.01 ⁇ g, 0.1 ⁇ g, 1 ⁇ g and 10 ⁇ g.
  • the GMT levels of neutralizing antibodies induced were ⁇ 30, 46, 133, and 507, respectively.
  • the neutralizing antibody titer GMT after immunizing BALB/c mice with the antigen content of 0.01 ⁇ g, 0.1 ⁇ g, 1 ⁇ g and 10 ⁇ g STFK vaccine was 32, 362, 575, and 1625, respectively.
  • Neutralizing antibody titers showed a significant dose-dependent effect.
  • a single dose of STFK vaccine with antigen content ⁇ 0.1 ⁇ g per dose can successfully induce Anti-Spike IgG antibody and RBD-IgG antibody seroconversion in all tested animals 2 weeks after immunization in BALB/c mice, and the seroconversion rate is 100%.
  • STFK vaccine with an antigen content ⁇ 1 ⁇ g per dose a single dose of BALB/c mice can successfully induce neutralizing antibody seroconversion in all tested animals 2 weeks after immunization, and the seroconversion rate is 100%.
  • the STFK vaccine with an antigen content of 0.01 ⁇ g per dose can successfully induce Anti-Spike IgG, Anti-RBD IgG antibody and new coronavirus neutralizing antibody seroconversion in all tested animals one week after two doses (0/3 weeks) of immunization, and the seroconversion rate is 100%. It shows that STFK vaccine has good immunogenicity.
  • STFK vaccine has good immunogenicity.
  • BALB/c mice were immunized with STFK vaccine with an antigen content of 0.01 ⁇ g-10 ⁇ g, no matter whether it was a single dose or two doses (0/3 weeks), the levels of Anti-Spike IgG, Anti-RBD IgG antibodies and SARS-CoV-2 neutralizing antibodies showed obvious antigen dose-dependent effects.
  • the inventor used the STFK-Al vaccine of the traditional aluminum adjuvant (the preparation method can refer to Wu, Y., et al., A recombinant spike protein subunit vaccine confers protective immunity against SARS-CoV-2 infection and transmission in hamsters. Science Tanslational Medicine.20 Jul 2021.Vol 13, Issue 606) is a control vaccine, and ELISPOT technology is used to analyze the ability of the vaccine to induce lymphocytes to produce IFN- ⁇ after immunizing mice.
  • the preparation method can refer to Wu, Y., et al., A recombinant spike protein subunit vaccine confers protective immunity against SARS-CoV-2 infection and transmission in hamsters. Science Tanslational Medicine.20 Jul 2021.Vol 13, Issue 606
  • ELISPOT technology is used to analyze the ability of the vaccine to induce lymphocytes to produce IFN- ⁇ after immunizing mice.
  • C57BL/6 mice were randomly divided into 8 groups, and divided into 3 groups: 2 groups received one dose of STFK vaccine (named STFK Vac group), STFK-Al vaccine (using STFK immunogen (STFK-MR) and control aluminum adjuvant, named STFK-Al Vac group), and the other group was not immunized (named Unvaccinated group), the immunization dose was 10 ⁇ g, and the mice were sacrificed on the 7th day after immunization.
  • STFK Vac group STFK-Al vaccine (using STFK immunogen (STFK-MR) and control aluminum adjuvant, named STFK-Al Vac group)
  • Unvaccinated group the immunization dose was 10 ⁇ g, and the mice were sacrificed on the 7th day after immunization.
  • Spleen lymphocytes of mice were isolated after dissection, and T cells that responded to antigen-specific stimulation and secreted IFN- ⁇ in spleen lymphocytes of immunized mice were analyzed by enzyme-linked immunospot assay (ELISPOT), so as to analyze the cellular immune response of STFK vaccine in mice.
  • ELISPOT enzyme-linked immunospot assay
  • the average number of spots in the STFK vaccine immunized group was 441
  • the average number of spots in the STFK-Al vaccine immunized group was 186
  • the average number of spots in the non-immunized group was 40.
  • the GMTs of Anti-Spike IgG and Anti-RBD IgG antibodies induced by STFK vaccine were 18547 and 15320, and the GMTs of Anti-Spike IgG and Anti-RBD IgG antibodies induced by STFK-Al vaccine were 11558 and 4352, respectively.
  • the antibody titers of STFK vaccine were slightly higher than those of STFK-Al vaccine, but the difference was not statistically significant (P>0.05, Fig. 10C and Figure 10D).
  • the GMTs of Anti-Spike IgG and Anti-RBD IgG antibodies induced by STFK vaccine were 1,088,930 and 640,036, respectively, and the GMTs of Anti-Spike IgG and Anti-RBD IgG antibodies induced by STFK-Al vaccine were 1,818,584 and 552,812, respectively. 0D).
  • the neutralizing antibody titers of immunized rhesus macaque sera were detected by vesicular stomatitis virus (VSV) new coronavirus pseudovirus (VSVpp)-cell neutralization assay and SARS-CoV-2 true virus neutralization assay.
  • VSV vesicular stomatitis virus
  • VSVpp new coronavirus pseudovirus pseudovirus
  • SARS-CoV-2 true virus neutralization assay The results are shown in Figures 11A to 11E. From the perspective of vaccine-induced pseudovirus neutralizing antibody levels, after immunizing rhesus monkeys for 1 dose, the STFK vaccine-induced neutralizing antibody positive conversion rate was 100% at 1 ⁇ g and 15 ⁇ g doses, and the GMT was 70 and 134 respectively. In the same period, the neutralizing antibody positive conversion rate induced by the STFK-Al vaccine was 60% and 75%, and the GMT was 45 and 68 respectively.
  • the antibody titer of the STFK vaccine was about 2 times higher than that of the STFK-Al vaccine. times, but the difference is not yet statistically significant (P>0.05, Figure 11A and Figure 11B).
  • the GMT of neutralizing antibody induced by STFK vaccine at 1 ⁇ g dose was 12,570
  • the GMT of neutralizing antibody induced by STFK-Al vaccine was 1037 in the same period.
  • the neutralizing antibody titer of STFK vaccine was 12 times higher than that of STFK-Al vaccine, and the difference was statistically significant (P ⁇ 0.05).
  • the GMT of neutralizing antibody induced by STFK vaccine was 29556, and the GMT of neutralizing antibody induced by STFK-Al vaccine was 19171.
  • the neutralizing antibody titer of STFK vaccine was 1.5 times higher than that of STFK-Al vaccine, but the difference was not statistically significant (P>0.05).
  • the GMT of neutralizing antibody induced by STFK vaccine at 1 ⁇ g dose was 588, and the GMT of neutralizing antibody induced by STFK-Al vaccine was 42 during the same period.
  • the neutralizing antibody titer of STFK vaccine was 14 times higher than that of STFK-Al vaccine, and the difference was statistically significant (P ⁇ 0.01).
  • the GMT of neutralizing antibody induced by STFK vaccine was 1,351, and the GMT of neutralizing antibody induced by STFK-Al vaccine was 431 at the same time.
  • the neutralizing antibody titer of STFK vaccine was 3 times higher than that of STFK-Al vaccine, and the difference was statistically significant (P ⁇ 0.01).
  • SARS-CoV-2 can undergo natural mutations during the virus evolution process, and some mutations can lead to changes in the virus's transmission ability, pathogenicity and other characteristics, thereby affecting the protective efficacy of vaccines, the therapeutic effect of drugs, the sensitivity and specificity of diagnostic tools, etc., and even cause serious consequences such as continuous outbreaks.
  • SARS-CoV-2 D614G mutant strain was the first to appear and quickly became a global mainstream strain.
  • the inventors evaluated the neutralization effect of various main new coronavirus variants on the serum of rhesus monkeys immunized with STFK vaccine.
  • the detection of neutralizing antibodies of serum mutant strains was carried out using a lentiviral (LV) vector-based detection method for neutralizing antibodies to novel coronavirus pseudoviruses.
  • the pseudoviruses of the main mutant strains of the new coronavirus evaluated are shown in Table 3.
  • Table 3 Pseudoviruses of the main mutant strains of the new coronavirus based on lentiviral vectors
  • the average neutralizing antibody titer (GMT) of the immune serum to the D614G prototype strain virus was 7817, and the neutralizing antibody titers to different mutant strains were 6403 (B.1.1.7, British/Alpha strain), 2861 (P.1, Brazil/Gamma strain) ), 1174 (B.1.351, South Africa/Beta strain), 4787 (B.1.429, California/Epsilon strain), 2746 (B.1.525, Nigeria/Eta strain), 3364 (B.1.526a, New York/Iota_S477N strain), 3553 (B.1.526b, New York/Iota_E484K strain), 400 6 (B.1.617.1, India/Kappa strain), 6109 (B.
  • the relative neutralizing titer of the monkey immune serum to the B.1.351/Beta strain (dropped to 19%) was significantly reduced, including the B.1.617.2/Delta strain,
  • the neutralizing titers of other mutant strains including the C.37/Lambda strain had a relatively small decrease ( ⁇ 3 times, and the relative neutralizing titers remained above 33%).
  • the average neutralizing antibody titer (GMT) of the immune serum to the mainstream strain of SARS-CoV-2 D614G was 16794, and the GMT of neutralizing antibody titers to different mutant strains were 13743 (B.1.1.7, British/Alpha strain), 3690 (P.1, Brazil/Gamma strain), 1499 (B.1.3 51, South Africa/Beta strain), 12387 (B.1.429, California/Epsilon strain), 3886 (B.1.525, Nigeria/Eta strain), 6439 (New York/Iota_S477N strain), 3723 (B.1.526b, New York/Iota_E484K strain), 6335 (B.1.617.1, India/Kappa strain), 9904 (B.1.617.2, Indian/Delta strain), 8168 (C.37, Peruvian/Lambda strain).
  • monkey immune serum is to B.1.351/Beta strain (down to 12%), P.1/Gamma strain (down to 31%), B.1.525/Eta strain (down to 26%), B.1.526/Iota_E484K strain (down to 24%), to including B.1.617.2/Delta strain, C.37
  • the neutralizing titers of other mutant strains including the /Lambda strain had a relatively small decrease ( ⁇ 3 times, and the relative neutralizing titers remained above 33%).
  • the serum neutralizing antibody of the rhesus monkeys immunized with the mutant strain was compared with the D614G prototype strain virus neutralizing antibody.
  • the relative neutralization titer of the STFK vaccine immunized rhesus monkey serum to the B.1.1.7/Alpha strain is 89% (no significant reduction) of the D614G prototype strain on average, 34% (the decline is 2-3 times) of the D614G prototype strain to the P.1/Gamma strain, and 15% (the decline is 6.5 times) of the D614G prototype strain to the B.1.351/Beta strain on average.
  • the B.1.429/Epsilon strain is 76% of the D614G prototype strain on average (no significant reduction)
  • the B.1.525/Eta strain is an average of 32% of the D614G prototype strain (the decline is 3.2 times)
  • the B.1.526a/Iota_S477N strain is an average of 41% of the D614G prototype strain (the decline is 2.4 times)
  • the B.1.526b/Iota _E484K strain is on average 35% of D614G prototype strain (decrease rate is 2.8 times)
  • B.1.617.1/Kappa strain it is 49% of D614G prototype strain on average (decrease rate is 2.0 times)
  • B.1.617.2/Delta strain it is 76% of D614G prototype strain on average (no obvious decrease)
  • C.37/Lambda strain it is 5% of D614G prototype strain on average 5% (no significant reduction).
  • step 2.3 the inventors found that although the STFK vaccine immune serum has good neutralizing activity against a variety of new coronavirus variants, it still has a certain degree of reduction compared to the prototype strain. Therefore, it is particularly important to modify the Spike protein for mutant strains to obtain vaccine molecules that can complement STFK.
  • the overall design idea is shown in Figure 13.
  • mutant STFK molecular skeleton such as Beta strain (B.1.351), Gamma strain (B.1.1.28) and B.1.620 strain, named STFK1351, STFK1128 and STFK1620 respectively. Then based on these three mutant strains, the STFK molecules were chiseled and the mutations in the RBD region of other mutant strains were introduced.
  • STFK1128 STFK1351
  • STFK1620 SEQ ID NO: 17
  • the sequences encoding these three candidate backbone molecules were constructed into the expression vector pGS01, followed by eukaryotic expression referring to step 1.2. After culturing for 6 days, the ExpiCHO expression cell suspension was collected, centrifuged at 12000 rpm, room temperature for 30 min, and the supernatant was collected and filtered through a 0.22 ⁇ m filter membrane.
  • the supernatant sample was then purified using a Q-FF chromatography column (Q Sepharose Fast Flow, Cytiva, 17051001), and the target protein was eluted with 100 mM NaCl. SDS-PAGE gel pictures are shown in Figure 14A to Figure 14C. The obtained target protein was dialyzed into PBS buffer and stored at -20°C.
  • SPF grade male and female hamsters (body weight range: 100-130 g) aged 8-14 weeks were used, with equal numbers of females and males per group in all experiments.
  • the immunization method is intramuscular injection, and the injection volume is 200 ⁇ L, that is, 100 ⁇ L is injected into the gastrocnemius muscle of both hind limbs of the animal.
  • the immunization program was 0/3 week immunization respectively, and the injection dose was 10 ⁇ g. Two weeks after the second dose of immunization, orbital venous blood was collected for antibody titer detection.
  • the neutralizing antibody titers produced by hamsters against various mutant strains of SARS-CoV-2 are shown in Figures 15A to 15C.
  • the neutralizing antibodies induced by STFK1351, STFK1128 and STFK1620 in hamsters were all complementary to the prototype strain of STFK vaccine, but in terms of immunogenicity, the antibody titers induced by STFK1128 and STFK1620 were higher than those of STFK1351. Therefore, the inventors selected STFK1128 and STFK1620 as the antigen backbone for further modification.
  • E484K also known as escape mutation, which can improve the ability of the virus to escape host immunity and affect the immunogenicity of the virus, mainly appearing in Gamma mutants, Zeta mutants and Beta mutants
  • E484Q which can enhance the binding ability of the virus to the receptor Ace2, and can also reduce the ability of antibodies produced by vaccine immunization to bind to the mutant spike protein, mainly appearing in Kappa mutants
  • S477N In a number of studies, the researchers used bioinformatics and statistical methods to determine that aa475-aa485 is a highly flexible region in the receptor binding domain RBD of SARS-CoV2.
  • the S477 amino acid has the highest flexibility and is the residue with the highest mutation frequency in the RBD so far.
  • the S477N mutation can enhance the binding ability of the virus spike protein and the receptor Ace2; Expanded to all over the world. This mutation can enhance the binding ability of the virus to the receptor Ace2, and can also reduce the ability of the antibody produced by vaccine immunization to bind to the mutant spike protein. Some studies have shown that this mutation can enhance the tolerance of the virus to T cells; (5) T478K, the 478th residue is also the site of interaction between the viral spike protein and the receptor Ace2.
  • the transformed proteins were named STFK1128b, STFK1128c, STFK1128d, STFK1128e, STFK1128f, STFK1128g, respectively, and the amino acid sequences were shown in SEQ ID NOs: 18-23.
  • the transformed STFK1128b to STFK1128g protein particles were eukaryotically expressed according to step 1.2. After 6 days of culture, the ExpiCHO expression cell suspension was collected, centrifuged at 12,000 rpm for 30 min at room temperature, and the supernatant was collected and filtered through a 0.22 ⁇ m filter membrane. The supernatant sample was then purified using a Q-FF chromatography column (Q Sepharose Fast Flow, Cytiva, 17051001), and the target protein was eluted with 100 mM NaCl. SDS-PAGE gel map is shown in Fig. 16A to Fig. 16F. The obtained target protein was dialyzed into PBS buffer and stored at -20°C.
  • STFK1628x consists of NTD of STFK1620, RBD and S2 of STFK1128, and introduces L452R and T478K.
  • STFK1628y consists of the introduction of N440K in STFK1628x.
  • STFK1628z consists of introducing G446V in STFK1628x.
  • STFK1328x consists of the NTD of STFK1351, the RBD and S2 of STFK1128, and introduces K417N, L452R and T478K.
  • the modified protein expression plasmid was expressed and purified according to the method in step 3.1. SDS-PAGE gel pictures are shown in Figure 18A to Figure 18C. The obtained target protein was dialyzed into PBS buffer and stored at -20°C.
  • the modified antigens above were immunized with hamsters respectively, and the results of immune neutralizing antibody analysis of the prototype strain molecule STFK in hamsters are shown in Figure 19A to Figure 19D, and Figure 20A to Figure 20B.
  • the modified molecules STFK1328x (SEQ ID NO: 24), STFK1628x (SEQ ID NO: 25), STFK1628y (SEQ ID NO: 26) and STFK1628z (SEQ ID NO: 27) still maintain high-titer neutralizing antibodies against Beta strains and Gamma strains, while enhancing the anti-B.1.6 20 and the neutralizing antibody titer of A.VOI.V2.
  • the neutralizing antibody induced by the modified antigen can well cover the mutant virus that escapes significantly from the STFK of the prototype strain.
  • STFK1628y also induced high titers of neutralizing antibodies against various Omicron strains (including BA.1, BA.2, BA.2.12.1, BA.2.75, and BA.4/5), and these mutant viruses showed great immune escape against the antibodies induced by the prototype strain vaccine (Fig. 20D).
  • STFK1628x also induced neutralizing antibodies against the Omicron strain, but the titer was lower than that of STFK1628y and significantly higher than that of the prototype strain (Fig. 20C and Fig. 20D).
  • STFK1628x and STFK1628y have great advantages in the immune prototype and in the complementarity between the induced neutralizing antibody against the mutant strain and the STFK (SEQ ID NO: 14) of the prototype strain.
  • the bivalent vaccine with STFK1628x and STFK (STFK-MR) as the antigen is likely to be able to effectively induce all current VOC and VOI mutant strains.
  • step 3.1 STFK1628x and STFK were mixed at a ratio of 1:1 to prepare a bivalent vaccine for immunization of hamsters.
  • the immunization dose was 10 ⁇ g (including 5 ⁇ g STFK1628x and 5 ⁇ g STFK).
  • the results of the immune neutralizing antibody binding analysis of the prototype strain molecule STFK in hamsters were shown in Figure 20E.
  • the bivalent vaccine induced a high level of neutralizing antibodies against 14 major variants including all VOCs and VOIs, which can effectively cover the current major variants of the new coronavirus.
  • the neutralizing antibody induced by the bivalent vaccine had high-titer neutralizing activity against various Omicron variants, including BA.1, BA.2, BA.2.12.1, BA.2.75, and BA.4/5, which were 15-27 times higher than the neutralizing antibody titer induced by the prototype strain vaccine (Fig. 20F).
  • hamsters after intranasal challenge vaccination were administered with SARS-CoV-2 prototype strain, Beta strain and Omicron BA.1 (Fig. 21A).
  • Hamsters were inoculated with two doses of STFK, STFK1628x and bivalent vaccine at week 0 and week 3, respectively.
  • Two weeks after the booster immunization the hamsters were challenged nasally with 10 4 PFU of SARS-CoV-2 prototype or Beta strain.
  • the viral RNA load in the lung tissue of hamsters immunized with the bivalent vaccine was reduced by more than 100,000 times, and the median viral RNA load was 1.76log 10 copies mL -1 .
  • Hamsters vaccinated with either STFK or STFK 1628x vaccines also showed similar reductions in viral RNA load. Consistent with the results of viral RNA load reduction, STFK, STFK1628x and bivalent vaccines completely protected hamsters from lung disease caused by prototype virus infection, and the pathological score was significantly lower than that of control hamsters (P ⁇ 0.01, Figure 21E).
  • Unvaccinated control animals developed severe disease in the lungs, including (i) alveolar septum thickening and consolidation; (ii) hemorrhage, exudation, pulmonary edema, and mucus; and (iii) recruitment and infiltration of inflammatory immune cells.
  • the above experimental results prove that both STFK1628x and the bivalent vaccine can significantly reduce the viral load in respiratory tissues, completely protect the disease caused by the prototype strain of the new coronavirus, and the protective effect is equivalent to that of animals vaccinated with the prototype strain.
  • the median viral RNA load in the lung tissue of unvaccinated hamsters was 6.25log 10 copies mL -1
  • the viral RNA load in the lung tissues of hamsters vaccinated with STFK1628x, STFK1628y and bivalent vaccine were 1.93log 10 copies mL -1 , 1.46log 10 copies mL -1 , and 1.55log 10 copies mL -1 respectively , significantly decreased compared to the unvaccinated control group and the STFK vaccine (Fig. 21L).

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Abstract

The present invention belongs to the field of biomedicine, and relates to a SARS-CoV-2 spike protein variant, a pharmaceutical composition and the use thereof. Specifically, the present invention relates to an isolated polypeptide having an amino acid sequence as shown in any one of SEQ ID NOs: 1-27. The polypeptide of the present invention can simulate broad-spectrum neutralizing antibodies against existing different COVID-19 variant strains, and has a good protection effect on both a SARS-CoV-2 prototype strain and variant strain.

Description

SARS-CoV-2刺突蛋白变体、其药物组合物及用途SARS-CoV-2 spike protein variant, its pharmaceutical composition and use 技术领域technical field
本发明属于生物医药领域，涉及一种SARS-CoV-2刺突蛋白变体、其药物组合物及用途。The invention belongs to the field of biomedicine, and relates to a variant of SARS-CoV-2 spike protein, its pharmaceutical composition and application.
背景技术Background technique
SARS-CoV-2造成席卷全球的COVID-19大流行仍在肆虐全球，造成重大公共卫生负担。当前已有多种新冠疫苗获批上市或应急使用，为对抗SARS-CoV-2的感染致病和传播起到预防作用。SARS-CoV-2的刺突(Spike)蛋白(简称为S蛋白)是新冠疫苗的关键成分，包含三个主要结构域：(1)N端结构域NTD；(2)受体结合结构域(RBD)；(3)S2亚基；三者均能诱导针对性的中和抗体。由于SARS-CoV-2的高度变异性，当前基于原型株病毒Spike蛋白序列发展的第一代新冠疫苗对一些免疫逃逸变异株，如Beta株(B.1.351)、Omicron(B.1.1.529)等可能存在疫苗保护效力降低的问题。有鉴于此，有必要发展针对变异株且能激发广谱中和抗体应答的新的免疫原分子。The COVID-19 pandemic caused by SARS-CoV-2 is still raging around the world, causing a significant public health burden. At present, a variety of new crown vaccines have been approved for marketing or emergency use, which play a preventive role in combating the pathogenicity and spread of SARS-CoV-2 infection. The Spike protein (S protein for short) of SARS-CoV-2 is a key component of the new crown vaccine and consists of three main domains: (1) N-terminal domain NTD; (2) receptor binding domain (RBD); (3) S2 subunit; all three can induce targeted neutralizing antibodies. Due to the high variability of SARS-CoV-2, the current first-generation COVID-19 vaccine developed based on the Spike protein sequence of the prototype strain may have reduced vaccine protection efficacy against some immune escape variants, such as Beta strain (B.1.351) and Omicron (B.1.1.529). In view of this, it is necessary to develop new immunogenic molecules that can stimulate broad-spectrum neutralizing antibody responses against mutant strains.
发明内容Contents of the invention
本发明公开了使用C末端截短和突变改造型的重组新冠病毒膜外区结构域重组蛋白作为新冠疫苗免疫原的方法和技术。动物试验测试证明，改造的抗原以单价或者二价联合的方式，能激发针对现有的不同新冠变异株的广谱中和抗体，并且动物攻毒保护试验结果显示，本发明的抗原对SARS-CoV-2原型毒株和变异毒株均具有良好的保护效果。由此提供了下述发明：The invention discloses a method and technology for using a C-terminus truncated and mutated recombinant recombinant protein of the extramembrane domain of the new coronavirus as a new coronavirus vaccine immunogen. Animal tests have proved that the modified antigen can stimulate broad-spectrum neutralizing antibodies against different existing novel coronavirus variants in a monovalent or bivalent combination, and the results of animal challenge protection tests show that the antigen of the present invention has a good protective effect on both the prototype and variants of SARS-CoV-2. The following inventions are thus provided:
本发明的一个方面涉及一种分离的多肽，其氨基酸序列如SEQ ID NOs:1-27中的任一序列所示。One aspect of the present invention relates to an isolated polypeptide having an amino acid sequence as shown in any one of SEQ ID NOs: 1-27.
本发明的另一方面涉及一种分离的融合蛋白，其包含至少一种本发明的多肽。Another aspect of the invention relates to an isolated fusion protein comprising at least one polypeptide of the invention.
在本发明的一些实施方式中，所述融合蛋白由至少一种本发明的多肽与筛选标签例如his标签组成。In some embodiments of the present invention, the fusion protein consists of at least one polypeptide of the present invention and a screening tag such as a his tag.
本发明的再一方面涉及一种分离的多核苷酸，其编码本发明的多肽。 Yet another aspect of the invention relates to an isolated polynucleotide encoding a polypeptide of the invention.
本发明的再一方面涉及一种核酸构建体，其含有本发明的多核苷酸；优选地，所述核酸构建体为重组载体；优选地，所述核酸构建体为重组表达载体。Another aspect of the present invention relates to a nucleic acid construct, which contains the polynucleotide of the present invention; preferably, the nucleic acid construct is a recombinant vector; preferably, the nucleic acid construct is a recombinant expression vector.
本发明的再一方面涉及一种转化的细胞，其含有本发明的多核苷酸，或者本发明的核酸构建体。A further aspect of the invention relates to a transformed cell comprising a polynucleotide of the invention, or a nucleic acid construct of the invention.
本发明的再一方面涉及一种药物组合物，其含有至少一种本发明的多肽；可选地，其还包含药学上可接受的辅料；Another aspect of the present invention relates to a pharmaceutical composition, which contains at least one polypeptide of the present invention; optionally, it also contains pharmaceutically acceptable excipients;
优选地，所述药物组合物为疫苗制剂；Preferably, the pharmaceutical composition is a vaccine preparation;
优选地，所述药物组合物的单位剂量为0.01-100μg，优选0.1-50μg，更优选为5-30μg、5-20μg或5-15μg，特别优选为10μg；Preferably, the unit dose of the pharmaceutical composition is 0.01-100 μg, preferably 0.1-50 μg, more preferably 5-30 μg, 5-20 μg or 5-15 μg, especially preferably 10 μg;
优选地，所述辅料为疫苗佐剂；优选地，所述疫苗佐剂为FH002C佐剂或铝佐剂。Preferably, the adjuvant is a vaccine adjuvant; preferably, the vaccine adjuvant is FH002C adjuvant or aluminum adjuvant.
在本发明的一些实施方式中，所述的药物组合物，其包含第一多肽成分和/或第二多肽成分，其中：In some embodiments of the present invention, the pharmaceutical composition comprises the first polypeptide component and/or the second polypeptide component, wherein:
所述第一多肽成分选自SEQ ID NOs:1-14中的任一序列所示多肽中的一种或多种，和/或所述第二多肽成分选自SEQ ID NOs:15-27中的任一序列所示多肽中的一种或多种；The first polypeptide component is selected from one or more of the polypeptides shown in any sequence of SEQ ID NOs: 1-14, and/or the second polypeptide component is selected from one or more of the polypeptides shown in any sequence of SEQ ID NOs: 15-27;
优选地，所述第一多肽成分选自SEQ ID NOs:9-14中的任一序列所示多肽中的一种或多种，和/或所述第二多肽成分选自SEQ ID NOs:24-27中的任一序列所示多肽中的一种或多种；Preferably, the first polypeptide component is selected from one or more of the polypeptides shown in any sequence of SEQ ID NOs:9-14, and/or the second polypeptide component is selected from one or more of the polypeptides shown in any sequence of SEQ ID NOs:24-27;
优选地，所述第一多肽成分的氨基酸序列如SEQ ID NO:9所示，和/或所述第二多肽成分选自SEQ ID NOs:24-27中的任一序列所示多肽中的一种、两种、三种或者四种；Preferably, the amino acid sequence of the first polypeptide component is shown in SEQ ID NO: 9, and/or the second polypeptide component is selected from one, two, three or four of the polypeptides shown in any sequence of SEQ ID NOs: 24-27;
优选地，所述第一多肽成分的氨基酸序列如SEQ ID NO:10所示，和/或所述第二多肽成分选自SEQ ID NOs:24-27中的任一序列所示多肽中的一种、两种、三种或者四种；Preferably, the amino acid sequence of the first polypeptide component is shown in SEQ ID NO: 10, and/or the second polypeptide component is selected from one, two, three or four of the polypeptides shown in any sequence of SEQ ID NOs: 24-27;
优选地，所述第一多肽成分的氨基酸序列如SEQ ID NO:11所示，和/或所述第二多肽成分选自SEQ ID NOs:24-27中的任一序列所示多肽中的一种、两种、三种或者四种；Preferably, the amino acid sequence of the first polypeptide component is shown in SEQ ID NO: 11, and/or the second polypeptide component is selected from one, two, three or four of the polypeptides shown in any sequence of SEQ ID NOs: 24-27;
优选地，所述第一多肽成分的氨基酸序列如SEQ ID NO:12所示，和/或所述第二多肽成分选自SEQ ID NOs:24-27中的任一序列所示多肽中的一种、两种、三种或者四种； Preferably, the amino acid sequence of the first polypeptide component is shown in SEQ ID NO: 12, and/or the second polypeptide component is selected from one, two, three or four of the polypeptides shown in any sequence of SEQ ID NOs: 24-27;
优选地，所述第一多肽成分的氨基酸序列如SEQ ID NO:13所示，和/或所述第二多肽成分选自SEQ ID NOs:24-27中的任一序列所示多肽中的一种、两种、三种或者四种；Preferably, the amino acid sequence of the first polypeptide component is shown in SEQ ID NO: 13, and/or the second polypeptide component is selected from one, two, three or four of the polypeptides shown in any sequence of SEQ ID NOs: 24-27;
优选地，所述第一多肽成分的氨基酸序列如SEQ ID NO:14所示，和/或所述第二多肽成分选自SEQ ID NOs:24-27中的任一序列所示多肽中的一种、两种、三种或者四种；Preferably, the amino acid sequence of the first polypeptide component is shown in SEQ ID NO: 14, and/or the second polypeptide component is selected from one, two, three or four of the polypeptides shown in any sequence of SEQ ID NOs: 24-27;
优选地，所述第二多肽成分的氨基酸序列如SEQ ID NO:24所示，和/或所述第一多肽成分选自SEQ ID NOs:9-14中的任一序列所示多肽中的一种、两种、三种、四种、五种或六种；Preferably, the amino acid sequence of the second polypeptide component is shown in SEQ ID NO:24, and/or the first polypeptide component is selected from one, two, three, four, five or six of the polypeptides shown in any sequence of SEQ ID NOs:9-14;
优选地，所述第二多肽成分的氨基酸序列如SEQ ID NO:25所示，和/或所述第一多肽成分选自SEQ ID NOs:9-14中的任一序列所示多肽中的一种、两种、三种、四种、五种或六种；Preferably, the amino acid sequence of the second polypeptide component is shown in SEQ ID NO:25, and/or the first polypeptide component is selected from one, two, three, four, five or six of the polypeptides shown in any sequence of SEQ ID NOs:9-14;
优选地，所述第二多肽成分的氨基酸序列如SEQ ID NO:26所示，和/或所述第一多肽成分选自SEQ ID NOs:9-14中的任一序列所示多肽中的一种、两种、三种、四种、五种或六种；Preferably, the amino acid sequence of the second polypeptide component is shown in SEQ ID NO:26, and/or the first polypeptide component is selected from one, two, three, four, five or six of the polypeptides shown in any sequence of SEQ ID NOs:9-14;
优选地，所述第二多肽成分的氨基酸序列如SEQ ID NO:27所示，和/或所述第一多肽成分选自SEQ ID NOs:9-14中的任一序列所示多肽中的一种、两种、三种、四种、五种或六种。Preferably, the amino acid sequence of the second polypeptide component is shown in SEQ ID NO:27, and/or the first polypeptide component is selected from one, two, three, four, five or six of the polypeptides shown in any sequence of SEQ ID NOs:9-14.
在本发明的一些实施方式中，所述的药物组合物，其中，所述第一多肽成分的氨基酸序列如SEQ ID NO:14所示，和/或所述第二多肽成分的氨基酸序列如SEQ ID NO:24所示。In some embodiments of the present invention, the pharmaceutical composition, wherein the amino acid sequence of the first polypeptide component is shown in SEQ ID NO:14, and/or the amino acid sequence of the second polypeptide component is shown in SEQ ID NO:24.
在本发明的一些实施方式中，所述的药物组合物，其中，所述第一多肽成分的氨基酸序列如SEQ ID NO:14所示，和/或所述第二多肽成分的氨基酸序列如SEQ ID NO:25所示。In some embodiments of the present invention, the pharmaceutical composition, wherein the amino acid sequence of the first polypeptide component is shown in SEQ ID NO: 14, and/or the amino acid sequence of the second polypeptide component is shown in SEQ ID NO: 25.
在本发明的一些实施方式中，所述的药物组合物，其中，所述第一多肽成分的氨基酸序列如SEQ ID NO:14所示，和/或所述第二多肽成分的氨基酸序列如SEQ ID NO:26所示。In some embodiments of the present invention, the pharmaceutical composition, wherein the amino acid sequence of the first polypeptide component is shown in SEQ ID NO:14, and/or the amino acid sequence of the second polypeptide component is shown in SEQ ID NO:26.
在本发明的一些实施方式中，所述的药物组合物，其中，所述第一多肽成分的氨基酸序列如SEQ ID NO:14所示，和/或所述第二多肽成分的氨基酸序列如SEQ ID NO:27所示。 In some embodiments of the present invention, the pharmaceutical composition, wherein the amino acid sequence of the first polypeptide component is shown in SEQ ID NO:14, and/or the amino acid sequence of the second polypeptide component is shown in SEQ ID NO:27.
在本发明的一些实施方式中，所述的药物组合物，其中，所述第一多肽成分的氨基酸序列如SEQ ID NO:14所示，和/或所述第二多肽成分为SEQ ID NO:24和SEQ ID NO:25所示的多肽。In some embodiments of the present invention, the pharmaceutical composition, wherein the amino acid sequence of the first polypeptide component is shown in SEQ ID NO: 14, and/or the second polypeptide component is the polypeptide shown in SEQ ID NO: 24 and SEQ ID NO: 25.
在本发明的一些实施方式中，所述的药物组合物，其中，所述第一多肽成分的氨基酸序列如SEQ ID NO:14所示，和/或所述第二多肽成分为SEQ ID NO:24和SEQ ID NO:26所示的多肽。In some embodiments of the present invention, the pharmaceutical composition, wherein the amino acid sequence of the first polypeptide component is shown in SEQ ID NO: 14, and/or the second polypeptide component is the polypeptide shown in SEQ ID NO: 24 and SEQ ID NO: 26.
在本发明的一些实施方式中，所述的药物组合物，其中，所述第一多肽成分的氨基酸序列如SEQ ID NO:14所示，和/或所述第二多肽成分为SEQ ID NO:24和SEQ ID NO:27所示的多肽。In some embodiments of the present invention, the pharmaceutical composition, wherein the amino acid sequence of the first polypeptide component is shown in SEQ ID NO: 14, and/or the second polypeptide component is the polypeptide shown in SEQ ID NO: 24 and SEQ ID NO: 27.
在本发明的一些实施方式中，所述的药物组合物，其中，所述第一多肽成分的氨基酸序列如SEQ ID NO:14所示，和/或所述第二多肽成分为SEQ ID NO:25和SEQ ID NO:26所示的多肽。In some embodiments of the present invention, the pharmaceutical composition, wherein the amino acid sequence of the first polypeptide component is shown in SEQ ID NO: 14, and/or the second polypeptide component is the polypeptide shown in SEQ ID NO: 25 and SEQ ID NO: 26.
在本发明的一些实施方式中，所述的药物组合物，其中，所述第一多肽成分的氨基酸序列如SEQ ID NO:14所示，和/或所述第二多肽成分为SEQ ID NO:25和SEQ ID NO:27所示的多肽。In some embodiments of the present invention, the pharmaceutical composition, wherein the amino acid sequence of the first polypeptide component is shown in SEQ ID NO: 14, and/or the second polypeptide component is the polypeptide shown in SEQ ID NO: 25 and SEQ ID NO: 27.
在本发明的一些实施方式中，所述的药物组合物，其中，所述第一多肽成分的氨基酸序列如SEQ ID NO:14所示，和/或所述第二多肽成分为SEQ ID NO:26和SEQ ID NO:27所示的多肽。In some embodiments of the present invention, the pharmaceutical composition, wherein the amino acid sequence of the first polypeptide component is shown in SEQ ID NO: 14, and/or the second polypeptide component is the polypeptide shown in SEQ ID NO: 26 and SEQ ID NO: 27.
在本发明的一些实施方式中，所述的药物组合物，其中，所述第一多肽成分的氨基酸序列如SEQ ID NO:14所示，和/或所述第二多肽成分为SEQ ID NO:24、SEQ ID NO:25和SEQ ID NO:26所示的多肽。In some embodiments of the present invention, the pharmaceutical composition, wherein the amino acid sequence of the first polypeptide component is shown in SEQ ID NO: 14, and/or the second polypeptide component is the polypeptide shown in SEQ ID NO: 24, SEQ ID NO: 25 and SEQ ID NO: 26.
在本发明的一些实施方式中，所述的药物组合物，其中，所述第一多肽成分的氨基酸序列如SEQ ID NO:14所示，和/或所述第二多肽成分为SEQ ID NO:24、SEQ ID NO:25和SEQ ID NO:27所示的多肽。In some embodiments of the present invention, the pharmaceutical composition, wherein the amino acid sequence of the first polypeptide component is shown in SEQ ID NO: 14, and/or the second polypeptide component is the polypeptide shown in SEQ ID NO: 24, SEQ ID NO: 25 and SEQ ID NO: 27.
在本发明的一些实施方式中，所述的药物组合物，其中，所述第一多肽成分的氨基酸序列如SEQ ID NO:14所示，和/或所述第二多肽成分为SEQ ID NO:24、SEQ ID NO:26和SEQ ID NO:27所示的多肽。In some embodiments of the present invention, the pharmaceutical composition, wherein the amino acid sequence of the first polypeptide component is shown in SEQ ID NO: 14, and/or the second polypeptide component is the polypeptide shown in SEQ ID NO: 24, SEQ ID NO: 26 and SEQ ID NO: 27.
在本发明的一些实施方式中，所述的药物组合物，其中，所述第一多肽成分的氨基酸序列如SEQ ID NO:14所示，和/或所述第二多肽成分为SEQ ID NO:25、SEQ ID NO:26和SEQ ID NO:27所示的多肽。 In some embodiments of the present invention, the pharmaceutical composition, wherein the amino acid sequence of the first polypeptide component is shown in SEQ ID NO: 14, and/or the second polypeptide component is the polypeptide shown in SEQ ID NO: 25, SEQ ID NO: 26 and SEQ ID NO: 27.
在本发明的一些实施方式中，所述的药物组合物，其中，所述第一多肽成分的氨基酸序列如SEQ ID NO:14所示，和/或所述第二多肽成分为SEQ ID NO:24、SEQ ID NO:25、SEQ ID NO:26和SEQ ID NO:27所示的多肽。In some embodiments of the present invention, the pharmaceutical composition, wherein, the amino acid sequence of the first polypeptide component is shown in SEQ ID NO: 14, and/or the second polypeptide component is the polypeptide shown in SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26 and SEQ ID NO: 27.
在本发明的一些实施方式中，所述的药物组合物，其中，当所述第一多肽成分包含一种以上多肽时，各多肽两两之间的质量比为1：1；和/或In some embodiments of the present invention, the pharmaceutical composition, wherein, when the first polypeptide component contains more than one polypeptide, the mass ratio between each polypeptide is 1:1; and/or
在本发明的一些实施方式中，所述的药物组合物，其中，当所述第二多肽成分包含一种以上多肽时，各多肽两两之间的质量比为1：1。In some embodiments of the present invention, the pharmaceutical composition, wherein, when the second polypeptide component contains more than one polypeptide, the mass ratio between two polypeptides is 1:1.
在本发明的一些实施方式中，所述的药物组合物，其中，当所述第二多肽成分选自SEQ ID NOs:24-27中的任一序列所示多肽中的两种、三种或者四种时，各多肽两两之间的质量比为1：1。In some embodiments of the present invention, the pharmaceutical composition, wherein, when the second polypeptide component is selected from two, three or four of the polypeptides shown in any sequence of SEQ ID NOs: 24-27, the mass ratio between each polypeptide is 1:1.
在本发明的一些实施方式中，所述的药物组合物，其中，当所述第一多肽成分选自SEQ ID NOs:9-14中的任一序列所示多肽中的两种、三种、四种、五种或六种时，各多肽两两之间的质量比为1：1。In some embodiments of the present invention, the pharmaceutical composition, wherein, when the first polypeptide component is selected from two, three, four, five or six of the polypeptides shown in any sequence of SEQ ID NOs: 9-14, the mass ratio between each polypeptide is 1:1.
在本发明的一些实施方式中，所述的药物组合物，第一多肽成分和第二多肽成分的质量比为(1：10)至(10：1)、(1：5)至(5：1)、(1：3)至(3：1)、(1：2)至(2：1)、(1：1.5)至(1.5：1)或者1：1；In some embodiments of the present invention, in the pharmaceutical composition, the mass ratio of the first polypeptide component to the second polypeptide component is (1:10) to (10:1), (1:5) to (5:1), (1:3) to (3:1), (1:2) to (2:1), (1:1.5) to (1.5:1) or 1:1;
优选地，所述药物组合物的活性成分由第一多肽成分和第二多肽成分组成。Preferably, the active ingredient of the pharmaceutical composition consists of the first polypeptide component and the second polypeptide component.
本发明的再一方面涉及本发明的多肽、融合蛋白、多核苷酸或者核酸构建体在制备抗SARS-CoV-2的药物中的用途；Another aspect of the present invention relates to the use of the polypeptide, fusion protein, polynucleotide or nucleic acid construct of the present invention in the preparation of anti-SARS-CoV-2 medicaments;
优选地，所述SARS-CoV-2选自SARS-CoV-2原型毒株或SARS-CoV-2变异毒株；优选地，所述变异毒株选自如下的一种或多种：Preferably, the SARS-CoV-2 is selected from a prototype strain of SARS-CoV-2 or a variant strain of SARS-CoV-2; preferably, the variant strain is selected from one or more of the following:
Alpha毒株、Gamma毒株、Beta毒株、Iota_S477N毒株、Iota_E484K毒株、Epsilon毒株、Eta毒株、Kappa毒株、Delta毒株、Lambda毒株和Omicron毒株；Alpha strain, Gamma strain, Beta strain, Iota_S477N strain, Iota_E484K strain, Epsilon strain, Eta strain, Kappa strain, Delta strain, Lambda strain and Omicron strain;
优选地，所述Omicron毒株为选自如下的一种或多种：Preferably, the Omicron strain is one or more selected from the following:
BA.1、BA.2、BA.2.12.1、BA.2.75、BA.4和BA.5；BA.1, BA.2, BA.2.12.1, BA.2.75, BA.4 and BA.5;
优选地，所述药物为本发明中任一项所述的药物组合物。Preferably, the drug is any one of the pharmaceutical compositions of the present invention.
本发明的再一方面涉及本发明的多肽、本发明的多核苷酸或者本发明的核酸构建体在制备治疗或预防COVID-19或其所致症状的药物(例如疫苗制剂)中的用途；Another aspect of the present invention relates to the use of the polypeptide of the present invention, the polynucleotide of the present invention or the nucleic acid construct of the present invention in the preparation of medicines (such as vaccine preparations) for the treatment or prevention of COVID-19 or the symptoms caused by it;
优选地，所述药物为本发明中任一项所述的药物组合物。Preferably, the drug is any one of the pharmaceutical compositions of the present invention.
在本发明的一些实施方式中，所述的用途，其中，所述药物或疫苗制剂的单位剂 量为0.01-100μg，优选0.1-50μg，更优选为5-30μg、5-20μg或5-15μg，特别优选为10μg。In some embodiments of the present invention, the use, wherein the unit dose of the drug or vaccine preparation The amount is 0.01-100 μg, preferably 0.1-50 μg, more preferably 5-30 μg, 5-20 μg or 5-15 μg, particularly preferably 10 μg.
根据本发明的多肽、融合蛋白、多核苷酸或者核酸构建体，其用于抗SARS-CoV-2；According to the polypeptide, fusion protein, polynucleotide or nucleic acid construct of the present invention, it is used for anti-SARS-CoV-2;
优选地，所述SARS-CoV-2选自SARS-CoV-2原型毒株或SARS-CoV-2变异毒株；优选地，所述变异毒株选自如下的一种或多种：Preferably, the SARS-CoV-2 is selected from a prototype strain of SARS-CoV-2 or a variant strain of SARS-CoV-2; preferably, the variant strain is selected from one or more of the following:
Alpha毒株、Gamma毒株、Beta毒株、Iota_S477N毒株、Iota_E484K毒株、Epsilon毒株、Eta毒株、Kappa毒株、Delta毒株、Lambda毒株和Omicron毒株；Alpha strain, Gamma strain, Beta strain, Iota_S477N strain, Iota_E484K strain, Epsilon strain, Eta strain, Kappa strain, Delta strain, Lambda strain and Omicron strain;
优选地，所述Omicron毒株为选自如下的一种或多种：Preferably, the Omicron strain is one or more selected from the following:
BA.1、BA.2、BA.2.12.1、BA.2.75、BA.4和BA.5。BA.1, BA.2, BA.2.12.1, BA.2.75, BA.4, and BA.5.
根据本发明的多肽、融合蛋白、多核苷酸或者核酸构建体，其用于治疗或预防COVID-19或其所致症状。According to the polypeptide, fusion protein, polynucleotide or nucleic acid construct of the present invention, it is used for treating or preventing COVID-19 or the symptoms caused by it.
本发明的再一方面涉及一种抗SARS-CoV-2的方法，包括给予有需求的受试者以有效量的本发明的多肽、融合蛋白、多核苷酸或者核酸构建体的步骤；Another aspect of the present invention relates to an anti-SARS-CoV-2 method, comprising the step of administering an effective amount of the polypeptide, fusion protein, polynucleotide or nucleic acid construct of the present invention to a subject in need;
优选地，所述SARS-CoV-2选自SARS-CoV-2原型毒株或SARS-CoV-2变异毒株；优选地，所述变异毒株选自如下的一种或多种：Preferably, the SARS-CoV-2 is selected from a prototype strain of SARS-CoV-2 or a variant strain of SARS-CoV-2; preferably, the variant strain is selected from one or more of the following:
Alpha毒株、Gamma毒株、Beta毒株、Iota_S477N毒株、Iota_E484K毒株、Epsilon毒株、Eta毒株、Kappa毒株、Delta毒株、Lambda毒株和Omicron毒株；Alpha strain, Gamma strain, Beta strain, Iota_S477N strain, Iota_E484K strain, Epsilon strain, Eta strain, Kappa strain, Delta strain, Lambda strain and Omicron strain;
优选地，所述Omicron毒株为选自如下的一种或多种：Preferably, the Omicron strain is one or more selected from the following:
BA.1、BA.2、BA.2.12.1、BA.2.75、BA.4和BA.5；BA.1, BA.2, BA.2.12.1, BA.2.75, BA.4 and BA.5;
优选地，通过本发明中任一项所述的药物组合物进行给药。Preferably, the administration is by a pharmaceutical composition according to any one of the present invention.
本发明的再一方面涉及一种治疗或预防COVID-19或其所致症状的方法，包括给予有需求的受试者以有效量的本发明的多肽、融合蛋白、多核苷酸或者核酸构建体的步骤；Another aspect of the present invention relates to a method for treating or preventing COVID-19 or the symptoms caused by it, comprising the step of administering an effective amount of the polypeptide, fusion protein, polynucleotide or nucleic acid construct of the present invention to a subject in need;
优选地，通过本发明中任一项所述的药物组合物进行给药。Preferably, the administration is by a pharmaceutical composition according to any one of the present invention.
本发明中的疫苗制剂可以是治疗性疫苗制剂或者预防性疫苗制剂。The vaccine formulations of the present invention may be therapeutic vaccine formulations or prophylactic vaccine formulations.
本发明中的疫苗制剂可以通过肌肉注射或者皮下注射给药。The vaccine formulation of the present invention can be administered by intramuscular injection or subcutaneous injection.
本发明中，术语“第一多肽成分”和“第二多肽成分”分别可以是一种多肽，或者几种多肽的混合物。其中的“第一”和“第二”仅仅是为了区分或者表述清楚，并不没有典 型的次序上的含义。In the present invention, the terms "first polypeptide component" and "second polypeptide component" may refer to one polypeptide or a mixture of several polypeptides. The "first" and "second" are just to distinguish or express clearly, not without typical The meaning of the order of the type.
本发明中，术语“单位剂量”按照其中包含的抗原(例如第一多肽成分，或者第一多肽成分+第二多肽成分)的质量计算。In the present invention, the term "unit dose" is calculated according to the mass of the antigen contained therein (for example, the first polypeptide component, or the first polypeptide component + the second polypeptide component).
附图说明Description of drawings
图1：STFK-a至STFK-h真核表达6天细胞上清SDS-PAGE分析。Figure 1: SDS-PAGE analysis of cell supernatants of STFK-a to STFK-h eukaryotic expression for 6 days.
图2：STFK-a至STFK-h纯化蛋白SDS-PAGE分析结果。Figure 2: SDS-PAGE analysis results of purified proteins from STFK-a to STFK-h.
图3：S三聚体和单体S纯化蛋白与Ace2结合能力曲线分析及EC50结果。Figure 3: Curve analysis of the binding ability of S trimer and monomer S purified protein to Ace2 and EC 50 results.
图4A：C末端突变后蛋白表达6天细胞上清SDS-PAGE检测。Figure 4A: SDS-PAGE detection of protein expression in cell supernatant 6 days after C-terminal mutation.
图4B：C末端突变后蛋白表达6天细胞上清Native-PAGE检测。Figure 4B: Native-PAGE detection of protein expression in cell supernatant 6 days after C-terminal mutation.
图5A：STFK-HR至STFK-MR与RBD单抗85F7的结合曲线。Figure 5A: Binding curves of STFK-HR to STFK-MR to RBD mAb 85F7.
图5B：STFK-HR至STFK-MR与RBD单抗81H10的结合曲线。Figure 5B: Binding curves of STFK-HR to STFK-MR to RBD mAb 81H10.
图6A：单体和三聚体疫苗1μg抗原剂量所诱导中和抗体滴度。注：中和抗体检测ID50<30时均按照30计入作图和统计分析。Figure 6A: Neutralizing antibody titers induced by 1 μg antigen doses of monomeric and trimeric vaccines. Note: when the neutralizing antibody detection ID50<30, it will be included in the drawing and statistical analysis according to 30.
图6B：单体和三聚体疫苗10μg抗原剂量所诱导中和抗体滴度。注：中和抗体检测ID50<30时均按照30计入作图和统计分析。Figure 6B: Neutralizing antibody titers induced by 10 μg antigen doses of monomeric and trimeric vaccines. Note: when the neutralizing antibody detection ID50<30, it will be included in the drawing and statistical analysis according to 30.
图7A：不同剂量STFK疫苗免疫小鼠后血清中Anti-RBD抗体滴度。Figure 7A: Anti-RBD antibody titers in serum of mice immunized with different doses of STFK vaccine.
图7B：不同剂量STFK疫苗免疫小鼠后血清中Anti-Spike抗体滴度。Figure 7B: Anti-Spike antibody titers in serum of mice immunized with different doses of STFK vaccine.
图8A：不同剂量STFK疫苗免疫小鼠后血清假病毒中和抗体滴度。Figure 8A: Serum pseudovirus neutralizing antibody titers after mice were immunized with different doses of STFK vaccine.
图8B：不同剂量STFK疫苗免疫小鼠后血清真病毒中和抗体滴度。Figure 8B: Serum euvirus neutralizing antibody titers after mice were immunized with different doses of STFK vaccine.
图8C：不同剂量STFK疫苗免疫小鼠后血清假病毒与真病毒中和抗体相关性。Figure 8C: Correlation of serum pseudovirus and true virus neutralizing antibodies after mice were immunized with different doses of STFK vaccine.
图9：IFN-γELISPOT实验分析STFK疫苗在小鼠中诱导的细胞免疫反应。Figure 9: Analysis of cellular immune response induced by STFK vaccine in mice by IFN-γ ELISPOT assay.
图10A：STFK疫苗1μg抗原剂量免疫恒河猴后血清中Anti-RBD抗体滴度。Fig. 10A: Anti-RBD antibody titer in serum after immunizing rhesus macaques with 1 μg antigen dose of STFK vaccine.
图10B：STFK疫苗1μg抗原剂量免疫恒河猴后血清中Anti-spike抗体滴度。Figure 10B: Anti-spike antibody titer in serum after immunizing rhesus macaques with 1 μg antigen dose of STFK vaccine.
图10C：STFK疫苗15μg抗原剂量免疫恒河猴后血清中Anti-RBD抗体滴度。Figure 10C: Anti-RBD antibody titer in serum after immunizing rhesus monkeys with 15 μg antigen dose of STFK vaccine.
图10D：STFK疫苗15μg抗原剂量免疫恒河猴后血清中Anti-spike抗体滴度。FIG. 10D : Anti-spike antibody titers in serum after immunizing rhesus monkeys with 15 μg antigen dose of STFK vaccine.
图11A：STFK疫苗1μg抗原剂量免疫恒河猴后血清中假病毒中和抗体滴度。Figure 11A: Pseudovirus neutralizing antibody titers in serum after immunizing rhesus monkeys with 1 μg antigen dose of STFK vaccine.
图11B：STFK疫苗15μg抗原剂量免疫恒河猴后血清中假病毒中和抗体滴度。Figure 11B: Pseudovirus neutralizing antibody titers in serum after immunizing rhesus monkeys with 15 μg antigen dose of STFK vaccine.
图11C：STFK疫苗1μg抗原剂量免疫恒河猴后血清中真病毒中和抗体滴度。Fig. 11C: The titer of true virus neutralizing antibody in serum after rhesus macaques were immunized with 1 μg antigen dose of STFK vaccine.
图11D：STFK疫苗15μg抗原剂量免疫恒河猴后血清中真病毒中和抗体滴度。 Figure 11D: The titer of true virus neutralizing antibody in serum after immunizing rhesus macaques with 15 μg antigen dose of STFK vaccine.
图11E：STFK疫苗免疫恒河猴后血清中假病毒与真病毒中和抗体滴度相关性。Figure 11E: Correlation of neutralizing antibody titers between pseudoviruses and true viruses in the serum of rhesus monkeys immunized with STFK vaccine.
图12A：STFK疫苗1μg抗原剂量免疫恒河猴血清对多种新冠病毒变异株中和抗体比较。Figure 12A: STFK vaccine 1 μg antigen dose immunized rhesus macaque serum for comparison of neutralizing antibodies against various new coronavirus variants.
图12B：STFK疫苗15μg抗原剂量免疫恒河猴血清对多种新冠病毒变异株中和抗体比较。Figure 12B: STFK vaccine 15 μg antigen dose immunized rhesus macaque serum for comparison of neutralizing antibodies against various new coronavirus variants.
图13：基于SARS-CoV2变异株的STFK分子构建思路示意图。Figure 13: Schematic diagram of the construction of STFK molecules based on SARS-CoV2 mutant strains.
图14A：SDS-PAGE分析ExpiCHO表达纯化的STFK1351。泳道1：细胞上清；泳道2：Q-FF层析柱流穿液；泳道3：100mM NaCl洗脱组分1；泳道4：100mM NaCl洗脱组分2；箭头指示目的蛋白。Figure 14A: SDS-PAGE analysis of ExpiCHO expressed purified STFK1351. Lane 1: Cell supernatant; Lane 2: Q-FF column flow-through; Lane 3: Fraction 1 eluted with 100mM NaCl; Lane 4: Fraction 2 eluted with 100mM NaCl; the arrow indicates the target protein.
图14B：SDS-PAGE分析ExpiCHO表达纯化的STFK1128。泳道1：细胞上清；泳道2：Q-FF层析柱流穿液；泳道3：100mM NaCl洗脱组分1；泳道4：100mM NaCl洗脱组分2；箭头指示目的蛋白。Figure 14B: SDS-PAGE analysis of ExpiCHO expression of purified STFK1128. Lane 1: Cell supernatant; Lane 2: Q-FF column flow-through; Lane 3: Fraction 1 eluted with 100mM NaCl; Lane 4: Fraction 2 eluted with 100mM NaCl; the arrow indicates the target protein.
图14C：SDS-PAGE分析ExpiCHO表达纯化的STFK1620。泳道1：细胞上清；泳道2：Q-FF层析柱流穿液；泳道3：100mM NaCl洗脱组分1；泳道4：100mM NaCl洗脱组分2；箭头指示目的蛋白。Figure 14C: SDS-PAGE analysis of ExpiCHO expressing purified STFK1620. Lane 1: Cell supernatant; Lane 2: Q-FF column flow-through; Lane 3: Fraction 1 eluted with 100mM NaCl; Lane 4: Fraction 2 eluted with 100mM NaCl; the arrow indicates the target protein.
图15A：STFK1351在仓鼠中诱导的中和抗体。注：其中折线和柱状图表示原型株的STFK诱导的针对相应变异株的中和抗体滴度。Figure 15A: Neutralizing antibodies induced by STFK1351 in hamsters. Note: The broken line and histogram represent the neutralizing antibody titers induced by STFK of the prototype strain against the corresponding mutant strains.
图15B：STFK1128在仓鼠中诱导的中和抗体。注：其中折线和柱状图表示原型株的STFK诱导的针对相应变异株的中和抗体滴度。Figure 15B: Neutralizing antibodies induced by STFK1128 in hamsters. Note: The broken line and histogram represent the neutralizing antibody titers induced by STFK of the prototype strain against the corresponding mutant strains.
图15C：STFK1620在仓鼠中诱导的中和抗体。注：其中折线和柱状图表示原型株的STFK诱导的针对相应变异株的中和抗体滴度。Figure 15C: Neutralizing antibodies induced by STFK1620 in hamsters. Note: The broken line and histogram represent the neutralizing antibody titers induced by STFK of the prototype strain against the corresponding mutant strains.
图16A：SDS-PAGE分析ExpiCHO表达纯化的STFK1128b。泳道1：细胞上清；泳道2：Q-FF层析柱流穿液；泳道3：100mM NaCl洗脱组分1；泳道4：100mM NaCl洗脱组分2；箭头指示目的蛋白。Figure 16A: SDS-PAGE analysis of ExpiCHO expression of purified STFK1128b. Lane 1: Cell supernatant; Lane 2: Q-FF column flow-through; Lane 3: Fraction 1 eluted with 100mM NaCl; Lane 4: Fraction 2 eluted with 100mM NaCl; the arrow indicates the target protein.
图16B：SDS-PAGE分析ExpiCHO表达纯化的STFK1128c。泳道1：细胞上清；泳道2：Q-FF层析柱流穿液；泳道3：100mM NaCl洗脱组分1；泳道4：100mM NaCl洗脱组分2；箭头指示目的蛋白。Figure 16B: SDS-PAGE analysis of ExpiCHO expressed purified STFK1128c. Lane 1: Cell supernatant; Lane 2: Q-FF column flow-through; Lane 3: Fraction 1 eluted with 100mM NaCl; Lane 4: Fraction 2 eluted with 100mM NaCl; the arrow indicates the target protein.
图16C：SDS-PAGE分析ExpiCHO表达纯化的STFK1128d。泳道1：细胞上清；泳道2：Q-FF层析柱流穿液；泳道3：100mM NaCl洗脱组分1；泳道4：100mM NaCl洗脱组分2；箭头指示目的蛋白。 Figure 16C: SDS-PAGE analysis of ExpiCHO expression of purified STFK1128d. Lane 1: cell supernatant; lane 2: Q-FF chromatography column flow-through; lane 3: fraction 1 eluted with 100 mM NaCl; lane 4: fraction 2 eluted with 100 mM NaCl; the arrow indicates the target protein.
图16D：SDS-PAGE分析ExpiCHO表达纯化的STFK1128e。泳道1：细胞上清；泳道2：Q-FF层析柱流穿液；泳道3：100mM NaCl洗脱组分1；泳道4：100mM NaCl洗脱组分2；箭头指示目的蛋白。Figure 16D: SDS-PAGE analysis of ExpiCHO expression of purified STFK1128e. Lane 1: Cell supernatant; Lane 2: Q-FF column flow-through; Lane 3: Fraction 1 eluted with 100mM NaCl; Lane 4: Fraction 2 eluted with 100mM NaCl; the arrow indicates the target protein.
图16E：SDS-PAGE分析ExpiCHO表达纯化的STFK1128f。泳道1：细胞上清；泳道2：Q-FF层析柱流穿液；泳道3：100mM NaCl洗脱组分1；泳道4：100mM NaCl洗脱组分2；箭头指示目的蛋白。Figure 16E: SDS-PAGE analysis of ExpiCHO expression of purified STFK1128f. Lane 1: Cell supernatant; Lane 2: Q-FF column flow-through; Lane 3: Fraction 1 eluted with 100mM NaCl; Lane 4: Fraction 2 eluted with 100mM NaCl; the arrow indicates the target protein.
图16F：SDS-PAGE分析ExpiCHO表达纯化的STFK1128g。泳道1：细胞上清；泳道2：Q-FF层析柱流穿液；泳道3：100mM NaCl洗脱组分1；泳道4：100mM NaCl洗脱组分2；箭头指示目的蛋白。Figure 16F: SDS-PAGE analysis of ExpiCHO expression of purified STFK1128g. Lane 1: Cell supernatant; Lane 2: Q-FF column flow-through; Lane 3: Fraction 1 eluted with 100mM NaCl; Lane 4: Fraction 2 eluted with 100mM NaCl; the arrow indicates the target protein.
图17A：STFK1128b在仓鼠中诱导的中和抗体。注：其中折线和柱状图表示原型株的STFK诱导的针对相应变异株的中和抗体滴度。Figure 17A: Neutralizing antibodies induced by STFK1128b in hamsters. Note: The broken line and histogram represent the neutralizing antibody titers induced by STFK of the prototype strain against the corresponding mutant strains.
图17B：STFK1128c在仓鼠中诱导的中和抗体。注：其中折线和柱状图表示原型株的STFK诱导的针对相应变异株的中和抗体滴度。Figure 17B: Neutralizing antibodies induced by STFK1128c in hamsters. Note: The broken line and histogram represent the neutralizing antibody titers induced by STFK of the prototype strain against the corresponding mutant strains.
图17C：STFK1128d在仓鼠中诱导的中和抗体。注：其中折线和柱状图表示原型株的STFK诱导的针对相应变异株的中和抗体滴度。Figure 17C: Neutralizing antibodies induced by STFK1128d in hamsters. Note: The broken line and histogram represent the neutralizing antibody titers induced by STFK of the prototype strain against the corresponding mutant strains.
图17D：STFK1128e在仓鼠中诱导的中和抗体。注：其中折线和柱状图表示原型株的STFK诱导的针对相应变异株的中和抗体滴度。Figure 17D: Neutralizing antibodies induced by STFK1128e in hamsters. Note: The broken line and histogram represent the neutralizing antibody titers induced by STFK of the prototype strain against the corresponding mutant strains.
图17E：STFK1128f在仓鼠中诱导的中和抗体。注：其中折线和柱状图表示原型株的STFK诱导的针对相应变异株的中和抗体滴度。Figure 17E: Neutralizing antibodies induced by STFK1128f in hamsters. Note: The broken line and histogram represent the neutralizing antibody titers induced by STFK of the prototype strain against the corresponding mutant strains.
图17F：STFK1128g在仓鼠中诱导的中和抗体。注：其中折线和柱状图表示原型株的STFK诱导的针对相应变异株的中和抗体滴度。Figure 17F: Neutralizing antibodies induced by STFK1128g in hamsters. Note: The broken line and histogram represent the neutralizing antibody titers induced by STFK of the prototype strain against the corresponding mutant strains.
图18A：SDS-PAGE分析ExpiCHO表达纯化的STFK1628x。泳道1：细胞上清；泳道2：Q-FF层析柱流穿液；泳道3：100mM NaCl洗脱组分1；泳道4：100mM NaCl洗脱组分2；泳道5：2M NaCl洗脱组分2；箭头指示目的蛋白。Figure 18A: SDS-PAGE analysis of ExpiCHO expression of purified STFK1628x. Lane 1: Cell supernatant; Lane 2: Q-FF column flow-through; Lane 3: Fraction 1 eluted with 100mM NaCl; Lane 4: Fraction 2 eluted with 100mM NaCl; Lane 5: Fraction 2 eluted with 2M NaCl; the arrow indicates the target protein.
图18B：SDS-PAGE分析ExpiCHO表达纯化的STFK1628x和STFK1328x。泳道1：细胞上清；泳道2：Q-FF层析柱流穿液；泳道3：100mM NaCl洗脱组分1；泳道4：100mM NaCl洗脱组分2；泳道5：2M NaCl洗脱组分2；箭头指示目的蛋白。Figure 18B: SDS-PAGE analysis of ExpiCHO expressed purified STFK1628x and STFK1328x. Lane 1: Cell supernatant; Lane 2: Q-FF column flow-through; Lane 3: Fraction 1 eluted with 100mM NaCl; Lane 4: Fraction 2 eluted with 100mM NaCl; Lane 5: Fraction 2 eluted with 2M NaCl; the arrow indicates the target protein.
图18C：SDS-PAGE分析ExpiCHO表达纯化的STFK1628z。泳道1：细胞上清；泳道2：Q-FF层析柱流穿液；泳道3：100mM NaCl洗脱组分1；泳道4：100mM NaCl 洗脱组分2；泳道5：2M NaCl洗脱组分2；箭头指示目的蛋白。Figure 18C: SDS-PAGE analysis of ExpiCHO expressed purified STFK1628z. Lane 1: Cell supernatant; Lane 2: Q-FF column flow-through; Lane 3: Fraction 1 eluted with 100mM NaCl; Lane 4: 100mM NaCl Elution fraction 2; lane 5: 2M NaCl elution fraction 2; the arrow indicates the target protein.
图19A：STFK1328x在仓鼠中诱导的中和抗体。注：其中折线和柱状图表示原型株的STFK诱导的针对相应变异株的中和抗体滴度。Figure 19A: Neutralizing antibodies induced by STFK1328x in hamsters. Note: The broken line and histogram represent the neutralizing antibody titers induced by STFK of the prototype strain against the corresponding mutant strains.
图19B：STFK1628x在仓鼠中诱导的中和抗体。注：其中折线和柱状图表示原型株的STFK诱导的针对相应变异株的中和抗体滴度。Figure 19B: Neutralizing antibodies induced by STFK1628x in hamsters. Note: The broken line and histogram represent the neutralizing antibody titers induced by STFK of the prototype strain against the corresponding mutant strains.
图19C：STFK1628y在仓鼠中诱导的中和抗体。注：其中折线和柱状图表示原型株的STFK诱导的针对相应变异株的中和抗体滴度。Figure 19C: Neutralizing antibodies induced by STFK1628y in hamsters. Note: The broken line and histogram represent the neutralizing antibody titers induced by STFK of the prototype strain against the corresponding mutant strains.
图19D：STFK1628z在仓鼠中诱导的中和抗体。注：其中折线和柱状图表示原型株的STFK诱导的针对相应变异株的中和抗体滴度。Figure 19D: Neutralizing antibodies induced by STFK1628z in hamsters. Note: The broken line and histogram represent the neutralizing antibody titers induced by STFK of the prototype strain against the corresponding mutant strains.
图20A：STFK1628x在仓鼠中诱导的中和抗体。注：其中水平短线表示原型株的STFK诱导的针对相应变异株的中和抗体滴度。Figure 20A: Neutralizing antibodies induced by STFK1628x in hamsters. Note: The horizontal short line represents the neutralizing antibody titer induced by STFK of the prototype strain against the corresponding mutant strain.
图20B：STFK1628y在仓鼠中诱导的中和抗体。注：其中水平短线表示原型株的STFK诱导的针对相应变异株的中和抗体滴度。Figure 20B: Neutralizing antibodies induced by STFK1628y in hamsters. Note: The horizontal short line represents the neutralizing antibody titer induced by STFK of the prototype strain against the corresponding mutant strain.
图20C：STFK1628x在仓鼠中诱导的对Omicron变异株中和抗体。注：其中折线表示原型株STFK诱导的针对相应变异株的中和抗体滴度，柱状图顶部的数值表示STFK1628x相比STFK的中和滴度增加倍数。Figure 20C: STFK1628x-induced neutralizing antibodies to Omicron variants in hamsters. Note: The broken line represents the neutralizing antibody titer induced by the prototype strain STFK against the corresponding mutant strain, and the value at the top of the bar graph represents the fold increase of the neutralizing titer of STFK1628x compared to STFK.
图20D：STFK1628y在仓鼠中诱导的对Omicron变异株中和抗体。注：其中折线表示原型株STFK诱导的针对相应变异株的中和抗体滴度，柱状图顶部的数值表示STFK1628y相比STFK的中和滴度增加倍数。FIG. 20D : STFK1628y-induced neutralizing antibodies to Omicron variants in hamsters. Note: The broken line represents the neutralizing antibody titer induced by the prototype strain STFK against the corresponding mutant strain, and the value at the top of the bar graph represents the fold increase of the neutralizing titer of STFK1628y compared to STFK.
图20E：二价疫苗在仓鼠中诱导的中和抗体。注：其中水平短线表示原型株的STFK诱导的针对相应变异株的中和抗体滴度。Figure 20E: Neutralizing antibodies induced by bivalent vaccine in hamsters. Note: The horizontal short line represents the neutralizing antibody titer induced by STFK of the prototype strain against the corresponding mutant strain.
图20F：二价疫苗在仓鼠中诱导的对Omicron变异株中和抗体。注：其中折线表示原型株STFK诱导的针对相应变异株的中和抗体滴度，柱状图顶部的数值表示二价疫苗相比STFK的中和滴度增加倍数。Figure 20F: Neutralizing antibodies against Omicron variants induced by bivalent vaccine in hamsters. Note: The broken line represents the neutralizing antibody titer induced by the prototype strain STFK against the corresponding mutant strain, and the value at the top of the bar graph represents the increase in the neutralizing titer of the bivalent vaccine compared to STFK.
图21A：STFK、STFK1628x和二价疫苗接种仓鼠对SARS-CoV-2原型株和Beta株攻毒的体内保护实验示意图。Figure 21A: Schematic diagram of the in vivo protection experiment of STFK, STFK1628x and bivalent vaccinated hamsters against SARS-CoV-2 prototype strain and Beta strain challenge.
图21B：STFK、STFK1628x和二价疫苗接种仓鼠SARS-CoV-2原型株攻毒后体重变化。星号表示统计学分析显著性(****P<0.0001；***P<0.001；**P<0.01；*P<0.05；ns，不显著。Figure 21B: STFK, STFK1628x and bivalent vaccine hamster body weight changes after challenge with SARS-CoV-2 prototype strain. Asterisks indicate statistical significance (****P<0.0001; ***P<0.001; **P<0.01; *P<0.05; ns, not significant.
图21C：STFK、STFK1628x和二价疫苗接种仓鼠SARS-CoV-2原型株攻毒后生 存曲线。Figure 21C: STFK, STFK1628x and bivalent vaccine hamsters challenged with SARS-CoV-2 prototype strain Save the curve.
图21D：STFK、STFK1628x和二价疫苗接种仓鼠SARS-CoV-2原型株攻毒后肺组织病毒RNA载量。Figure 21D: STFK, STFK1628x and bivalent vaccine hamster SARS-CoV-2 prototype strain challenged viral RNA load in lung tissue.
图21E：STFK、STFK1628x和二价疫苗接种仓鼠SARS-CoV-2原型株攻毒后肺组织切片H&E染色病理评分。Figure 21E: STFK, STFK1628x and bivalent vaccine hamster SARS-CoV-2 prototype strain challenged lung tissue sections H&E staining pathological score.
图21F：STFK、STFK1628x和二价疫苗接种仓鼠SARS-CoV-2 Beta株攻毒后体重变化。Fig. 21F: STFK, STFK1628x and bivalent vaccine hamster body weight changes after challenge with SARS-CoV-2 Beta strain.
图21G：STFK、STFK1628x和二价疫苗接种仓鼠SARS-CoV-2 Beta株攻毒后生存曲线。Figure 21G: Survival curves after challenge with STFK, STFK1628x and bivalent vaccinated hamsters with SARS-CoV-2 Beta strain.
图21H：STFK、STFK1628x和二价疫苗接种仓鼠SARS-CoV-2 Beta株攻毒后肺组织病毒RNA载量。Figure 21H: STFK, STFK1628x and bivalent vaccine hamster SARS-CoV-2 Beta strain challenged lung tissue virus RNA load.
图21I：STFK、STFK1628x和二价疫苗接种仓鼠SARS-CoV-2 Beta株攻毒后肺组织切片H&E染色病理评分。Figure 21I: STFK, STFK1628x and bivalent vaccine hamster SARS-CoV-2 Beta strain challenged lung tissue sections H&E staining pathological score.
图21J：STFK、STFK1628x、STFK1628y和二价疫苗接种仓鼠SARS-CoV-2 Omicron BA.1攻毒后体重变化。Figure 21J: STFK, STFK1628x, STFK1628y and bivalent vaccine hamster body weight changes after SARS-CoV-2 Omicron BA.1 challenge.
图21K：STFK、STFK1628x、STFK1628y和二价疫苗接种仓鼠SARS-CoV-2 Omicron BA.1攻毒后生存曲线。Figure 21K: Survival curves after challenge with STFK, STFK1628x, STFK1628y and bivalent vaccine hamsters SARS-CoV-2 Omicron BA.1.
图21L：STFK、STFK1628x、STFK1628y和二价疫苗接种仓鼠SARS-CoV-2 Omicron BA.1攻毒后肺组织病毒RNA载量。Figure 21L: STFK, STFK1628x, STFK1628y and SARS-CoV-2 Omicron BA.1 challenged lung tissue viral RNA load of hamsters vaccinated with bivalent vaccine.
图21M：STFK、STFK1628x、STFK1628y和二价疫苗接种仓鼠SARS-CoV-2 Omicron BA.1攻毒后肺组织切片H&E染色病理评分。Figure 21M: STFK, STFK1628x, STFK1628y and bivalent vaccine hamster SARS-CoV-2 Omicron BA.1 challenged lung tissue sections H&E staining pathological score.
上面图21A至图21M中，星号表示统计学分析显著性，****P<0.0001；***P<0.001；**P<0.01；*P<0.05；ns，不显著。In Figure 21A to Figure 21M above, asterisks indicate statistical significance, ****P<0.0001; ***P<0.001; **P<0.01; *P<0.05; ns, not significant.
本发明涉及的部分序列如下：Partial sequences involved in the present invention are as follows:
表A：序列的编号和对应的命名


Table A: Sequence numbers and corresponding names


aa1-1208
aa1-1208
aa1-1200

aa1-1200

aa1-1192(实施例中又称为aa1192、STFK-c或STFK-NR)

aa1-1192 (also referred to as aa1192, STFK-c or STFK-NR in the examples)

aa1-1184
aa1-1184
aa1-1176

aa1-1176

aa1-1168

aa1-1168

aa1-1160
aa1-1160
aa1-1152

aa1-1152

STFK-HR

STFK-HR

STFK-AR
STFK-AR
STFK-YR

STFK-YR

STFK-GR

STFK-GR

STFK-DR
STFK-DR
STFK-MR
STFK-MR
STFK1128

STFK1128

STFK1351

STFK1351

STFK1620
STFK1620
STFK1128b

STFK1128b

STFK1128c

STFK1128c

STFK1128d
STFK1128d
STFK1128e

STFK1128e

STFK1128f

STFK1128f

STFK1128g
STFK1128g
STFK1328x

STFK1328x

STFK1628x

STFK1628x

STFK1628y
STFK1628y
STFK1628z

STFK1628z

45C3 VH
45C3 VH
45C3 VL
45C3 VL
85F7 VH
85F7 VH
85F7 VL
85F7 VL
81H10 VH
81H10 VH
81H10 VL
81H10 VL
具体实施方式Detailed ways
下面将结合实施例对本发明的实施方案进行详细描述，但是本领域技术人员将会理解，下列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条件者，按照常规条件或制造商建议的条件进行。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。Embodiments of the present invention will be described in detail below in conjunction with examples, but those skilled in the art will understand that the following examples are only used to illustrate the present invention, and should not be considered as limiting the scope of the present invention. Those who do not indicate the specific conditions in the examples are carried out according to the conventional conditions or the conditions suggested by the manufacturer. The reagents or instruments used were not indicated by the manufacturer, and they were all commercially available conventional products.
实施例1：构建C末端截短的单体新冠刺突蛋白STFKExample 1: Construction of a C-terminally truncated monomeric novel coronavirus spike protein STFK
在前期研究中，本发明人设计了基于高活性的三聚体抗原StriFK和新型佐剂FH002C的新型冠状病毒重组亚单位疫苗(Wu,Y.,et al.,A recombinant spike protein subunit vaccine confers protective immunity against SARS-CoV-2 infection and transmission in hamsters.Science Tanslational Medicine.20 Jul 2021.Vol 13,Issue 606)，该疫苗能够诱导强效的体液免疫应答和细胞免疫应答，在小鼠、大鼠、仓鼠和非人灵长类动物中均具有良好的免疫原性。In previous studies, the inventors designed a novel coronavirus recombinant subunit vaccine based on highly active trimeric antigen StriFK and novel adjuvant FH002C (Wu, Y., et al., A recombinant spike protein subunit vaccine confers protective immunity against SARS-CoV-2 infection and transmission in hamsters. Science Tanslational Medicine.20 Jul 2021.Vol 13, Issue 606), the vaccine can induce potent humoral and cellular immune responses, and has good immunogenicity in mice, rats, hamsters and non-human primates.
然而，三聚体蛋白StriFK外源引入的三聚化结构域序列，可能引起机体产生针对三聚化结构域序列的抗体，且在表达制备过程中可能解聚为不均一形式；因此本发明人设计在不影响免疫原性的基础上去除外源序列，获得可提升蛋白表达量和稳定性 的单体形式的重组刺突蛋白Spike。However, the exogenously introduced trimerization domain sequence of the trimer protein StriFK may cause the body to produce antibodies against the trimerization domain sequence, and may depolymerize into a heterogeneous form during the expression preparation process; therefore, the inventors designed to remove the foreign sequence without affecting the immunogenicity, and obtain a protein that can improve the expression and stability of the protein. monomeric form of the recombinant spike protein Spike.
1.1 SARS-CoV2的Spike蛋白C末端截短型蛋白的重组制备1.1 Recombinant preparation of the C-terminal truncated protein of the Spike protein of SARS-CoV2
在EIRBsMie-StriFK表达质粒的基础上，通过PCR克隆去除C端T4纤维蛋白三聚化结构域序列但保留His标签，刺突蛋白Spike膜外区结尾出从aa1208(从N端开始数，第1208位氨基酸即C末端，因为S蛋白膜外区氨基酸全长为1208个氨基酸；SEQ ID NO:1)开始，每8个氨基酸往N端逐步缩减至aa1200(SEQ ID NO:2)、aa1192(SEQ ID NO:3)、aa1184(SEQ ID NO:4)、aa1176(SEQ ID NO:5)、aa1168(SEQ ID NO:6)、aa1160(SEQ ID NO:7)和aa1152(SEQ ID NO:8)，分别构建表达载体EIRBsMie-STFK-a(表达aa1-1208)、EIRBsMie-STFK-b(表达aa1-1200)、EIRBsMie-STFK-c(表达aa1-1192)、EIRBsMie-STFK-d(表达aa1-1184)、EIRBsMie-STFK-e(表达aa1-1176)、EIRBsMie-STFK-f(表达aa1-1168)、EIRBsMie-STFK-g(表达aa1-1160)和EIRBsMie-STFK-h(表达aa1-1152)。构建成功后，经过DNA测序证明上述重组载体携带的表达框序列与设计完全一致。On the basis of the EIRBsMie-StriFK expression plasmid, the C-terminal T4 fibrin trimerization domain sequence was removed by PCR cloning but the His tag was retained. The end of the extramembrane region of the spike protein Spike started from aa1208 (counting from the N-terminus, the 1208th amino acid is the C-terminus, because the total amino acid length of the S protein extramembrane region is 1208 amino acids; SEQ ID NO: 1), every 8 amino acids were gradually reduced to aa1200( The expression vectors EIRBsMie- STFK-a (express aa1-1208), EIRBsMie-STFK-b (express aa1-1200), EIRBsMie-STFK-c (express aa1-1192), EIRBsMie-STFK-d (express aa1-1184), EIRBsMie-STFK-e (express aa1-1176), EIRBsMie-STFK -f (expresses aa1-1168), EIRBsMie-STFK-g (expresses aa1-1160) and EIRBsMie-STFK-h (expresses aa1-1152). After successful construction, DNA sequencing proved that the sequence of the expression cassette carried by the recombinant vector was completely consistent with the design.
1.2 SARS-CoV2的Spike蛋白C末端截短蛋白的表达与纯化1.2 Expression and purification of the C-terminal truncated protein of Spike protein of SARS-CoV2
1.2.1表达细胞准备1.2.1 Expression cell preparation
以3×106个细胞/mL的密度将ExpiCHO细胞(Thermo Scientific公司)以适量的培养基ExpiCHOTM Expression Medium(Thermo Scientific公司)培养于三角摇瓶中，置于37℃、8％CO2、转速适当的恒温摇床中培养24h，待细胞密度达到6×106细胞/mL的密度。ExpiCHO cells (Thermo Scientific Company) were cultured at a density of 3×10 6 cells/mL in Erlenmeyer shaker flasks with an appropriate amount of medium ExpiCHO TM Expression Medium (Thermo Scientific Company), and placed in a constant temperature shaker at 37°C, 8% CO 2 , and an appropriate speed for 24 hours until the cell density reached 6×10 6 cells/mL.
1.2.2抗原表达1.2.2 Antigen expression
按照试剂盒的说明书使用ExpiFectamineTM CHO Transfection Kit(Thermo Scientific公司)将步骤1.1中得到的带his标签的STFK-a至STFK-h的表达载体EIRBsMie-STFK-a至EIRBsMie-STFK-h分别转染至ExpiCHO细胞中，继续以相同的条件培养17-24h后添加试剂盒中提供的补料和表达增强剂，将细胞更换至32℃、5％CO2，转速适当的恒温摇床中，继续培养6天。According to the instructions of the kit, use the ExpiFectamine TM CHO Transfection Kit (Thermo Scientific Company) to transfect the expression vectors EIRBsMie-STFK-a to EIRBsMie-STFK-h obtained in step 1.1 into ExpiCHO cells with his-tagged STFK-a to STFK-h, respectively, continue to culture under the same conditions for 17-24h, and then add the feed and expression enhancer provided in the kit. The cells were replaced to 32° C., 5% CO 2 , in a constant temperature shaker with an appropriate rotation speed, and continued to culture for 6 days.
培养的第六天的细胞培养上清的SDS-PAGE电泳分析如图1，结果显示，随着S蛋白膜外区C末端氨基酸截短，目的蛋白表达量呈增大趋势。The SDS-PAGE electrophoresis analysis of the cell culture supernatant on the sixth day of culture is shown in Figure 1. The results show that with the truncation of the C-terminal amino acid in the outer region of the S protein, the expression of the target protein tends to increase.
1.2.3蛋白纯化1.2.3 Protein purification
培养6天后，收集ExpiCHO表达细胞悬液，12000rpm，室温离心30min，留取 上清并透析至PBS中，后经0.22μm滤膜过滤。将透析至PBS中的上清样品经中压Ni-excel层析(GE介质)纯化，30mM咪唑去除杂蛋白，250mM咪唑洗脱目的蛋白。SDS-PAGE胶图如图2，结果显示，纯化获得的STFK-a至STFK-h纯度均在90％以上。从纯化蛋白的产出情看，S蛋白膜外区截短至aa1192(STFK-c)及截短更多的STFK-d、STFK-e、STFK-f、STFK-g、STFK-h总体得率高于STFK-a和STFK-b。上述结果表明，C末端截短有利于重组S蛋白在CHO细胞中的表达和纯化得率的提升。将得到的目的蛋白透析至PBS缓冲液并置于-20℃保存。After culturing for 6 days, collect the ExpiCHO expression cell suspension, centrifuge at 12000rpm for 30min at room temperature, and set aside The supernatant was dialyzed into PBS and then filtered through a 0.22 μm membrane filter. The supernatant sample dialyzed into PBS was purified by medium-pressure Ni-excel chromatography (GE medium), 30mM imidazole removed impurity proteins, and 250mM imidazole eluted the target protein. The SDS-PAGE gel picture is shown in Figure 2, and the results show that the purity of the purified STFK-a to STFK-h are all above 90%. From the yield of purified protein, the overall yield of S protein ectoregion truncated to aa1192 (STFK-c) and more truncated STFK-d, STFK-e, STFK-f, STFK-g, STFK-h was higher than that of STFK-a and STFK-b. The above results indicated that C-terminal truncation is beneficial to the expression and purification yield of recombinant S protein in CHO cells. The obtained target protein was dialyzed into PBS buffer and stored at -20°C.
1.3 STFK-a至STFK-h蛋白和三聚体StriFK蛋白与受体Ace2蛋白的结合活性1.3 Binding activity of STFK-a to STFK-h protein and trimeric StriFK protein and receptor Ace2 protein
1.3.1反应板的制备1.3.1 Preparation of reaction plate
将特异性识别SARS-CoV-2 spike蛋白S2结构域的单克隆抗体(命名为45C3)用pH7.4的20mM PB缓冲液(Na2HPO4/NaH2PO4溶液)稀释，终浓度为2μg/mL；在96孔酶标板每孔中加入100μL的包被液，2-8℃包被16-24小时；用PBST洗涤液(20mM PB7.4，150mM NaCl，0.1％Tween20)洗涤1次；然后每孔加入200μL的封闭液(含有20％小牛血清及1％酪蛋白的pH值为7.4的20mM Na2HPO4/NaH2PO4溶液)，放入37℃封闭2小时；弃去封闭液。干燥后装入铝箔袋2-8℃保存备用。将特异性识别SARS-CoV-2 spike蛋白S2结构域的单克隆抗体(命名为45C3)用pH7.4的20mM PB缓冲液(Na 2 HPO 4 /NaH 2 PO 4溶液)稀释，终浓度为2μg/mL；在96孔酶标板每孔中加入100μL的包被液，2-8℃包被16-24小时；用PBST洗涤液(20mM PB7.4，150mM NaCl，0.1％Tween20)洗涤1次；然后每孔加入200μL的封闭液(含有20％小牛血清及1％酪蛋白的pH值为7.4的20mM Na 2 HPO 4 /NaH 2 PO 4溶液)，放入37℃封闭2小时；弃去封闭液。 After drying, put it into an aluminum foil bag and store it at 2-8°C for later use.
1.3.2 ELISA结合活性检测1.3.2 ELISA binding activity detection
将步骤1.2.3中制得的STFK-a至STFK-h蛋白以及三聚体StriFK蛋白分别以含20％新生牛血清的PBS溶液稀梯度释至10μg/mL并两倍梯度往下进行稀释，最后一个浓度梯度为9.76ng/mL，按如下步骤进行ELISA检测：Dilute the STFK-a to STFK-h proteins and the trimeric StriFK protein prepared in step 1.2.3 to 10 μg/mL in PBS solution containing 20% newborn bovine serum, and then dilute the two-fold downward gradient. The final concentration gradient is 9.76 ng/mL, and ELISA detection is performed as follows:
(1)样品反应：取已包被45C3抗体的酶标板，每孔加入100μL已稀释的样品，置于25℃温箱反应60分钟。(1) Sample reaction: Take a microtiter plate coated with 45C3 antibody, add 100 μL of diluted sample to each well, and place in a 25°C incubator for 60 minutes to react.
(2)Ace2结合反应：完成样品反应步骤后，将酶标板用PBST洗液(20mM PB7.4，150mM NaCl，0.1％Tween20)洗涤5遍，每孔加入100μL 1ng/mL Ace2-huFc(Sino Biological 10108-H02H)蛋白，置于25℃温箱反应60分钟。(2) Ace2 binding reaction: After completing the sample reaction step, wash the ELISA plate 5 times with PBST washing solution (20mM PB7.4, 150mM NaCl, 0.1% Tween20), add 100μL 1ng/mL Ace2-huFc (Sino Biological 10108-H02H) protein to each well, and place it in an incubator at 25°C for 60 minutes.
(3)酶标记物反应：完成Ace2结合反应步骤后，将酶标板用PBST洗液(20mM PB7.4，150mM NaCl，0.1％Tween20)洗涤5遍，每孔加入100μL HRP标记的羊抗人IgG(GAH)反应液，置于25℃温箱反应60分钟。(3) Enzyme marker reaction: After completing the Ace2 binding reaction step, wash the ELISA plate with PBST washing solution (20mM PB7.4, 150mM NaCl, 0.1% Tween20) 5 times, add 100 μL of HRP-labeled goat anti-human IgG (GAH) reaction solution to each well, and place it in a 25°C incubator for 60 minutes.
(4)显色反应：完成酶标记物反应步骤后，将酶标板用PBST洗液(20mM PB7.4，150mM NaCl，0.1％Tween20)洗涤5遍，每孔加入TMB显色剂(购自北京万泰生物药业股份有限公司)各50μL，置于25℃温箱反应10分钟。 (4) Color reaction: After the enzyme marker reaction step was completed, the ELISA plate was washed 5 times with PBST washing solution (20mM PB7.4, 150mM NaCl, 0.1% Tween20), and 50 μL of TMB chromogenic reagent (purchased from Beijing Wantai Bio-Pharmaceutical Co., Ltd.) was added to each well, and placed in an incubator at 25°C for 10 minutes.
(5)终止反应及读值测量：完成显色反应步骤后，在反应完的酶标板中每孔加入终止液(购自北京万泰生物药业股份有限公司)50μL，并于酶标仪上检测各孔的OD450/630值。(5) Termination reaction and reading value measurement: After completing the color reaction step, add 50 μL of stop solution (purchased from Beijing Wantai Bio-Pharmaceutical Co., Ltd.) to each well of the reacted microplate plate, and detect the OD450/630 value of each well on a microplate reader.
(6)结果分析：结果显示如图3所示。三聚体StriFK与单体分子与受体Ace2结合活性对比结果显示：相较三聚体StriFK蛋白，STFK-a、STFK-b及STFK-c蛋白与受体Ace2结合的EC50浓度有近1倍左右的降低，提示此三种蛋白对SARS-CoV-2细胞受体ACE2的结合能力有所提高。(6) Result analysis: the results are shown in Figure 3. The comparison results of the binding activity between the trimeric StriFK and the monomeric molecule and the receptor Ace2 showed that compared with the trimeric StriFK protein, the EC 50 concentration of the STFK-a, STFK-b and STFK-c proteins binding to the receptor Ace2 was nearly 1-fold lower, suggesting that the binding ability of these three proteins to the SARS-CoV-2 cell receptor ACE2 has improved.
综合蛋白表达量、Ace2结合能力分析，将STFK-c(aa1192，SEQ ID NO:3)作为候选分子进行后续筛选实验。Based on the analysis of protein expression and Ace2 binding ability, STFK-c (aa1192, SEQ ID NO: 3) was used as a candidate molecule for subsequent screening experiments.
1.4单体S末端氨基酸的替换改造1.4 Substitution of S-terminal amino acids of monomers
1.4.1 STFK末端氨基酸(aa1192)的替换变体的构建和表达1.4.1 Construction and expression of substitution variants of STFK terminal amino acid (aa1192)
出于降低处于单体状态的S蛋白分子间形成聚体造成不均一状态的考虑，本发明人采用生物信息学的方法，使用常规动力学模拟对最后一位氨基酸aa1192进行氨基酸替换扫描，挑选了其中可降低螺旋稳定性(减弱分子间聚集倾向)的几个突变进行CHO表达克隆构建，包括末端氨基酸aa1192从原来的N替换为H、A、Y、G、D、和M的突变分子。分别命名为STFK-NR(aa1192为N，SEQ ID NO:3)、STFK-HR(aa1192为H，SEQ ID NO:9)、STFK-AR(aa1192为A，SEQ ID NO:10)、STFK-YR(aa1192为Y，SEQ ID NO:11)、STFK-GR(aa1192为G，SEQ ID NO:12)、STFK-DR(aa1192为D，SEQ ID NO:13)、STFK-MR(aa1192为M，SEQ ID NO:14)。In order to reduce the inhomogeneous state caused by the formation of aggregates between the S protein molecules in the monomer state, the inventors used bioinformatics methods and conventional dynamics simulations to scan the amino acid substitution of the last amino acid aa1192, and selected several mutations that can reduce the helical stability (weaken the tendency of intermolecular aggregation) to construct CHO expression clones, including mutant molecules in which the terminal amino acid aa1192 was replaced from the original N to H, A, Y, G, D, and M. Respectively named as STFK-NR (aa1192 is N, SEQ ID NO:3), STFK-HR (aa1192 is H, SEQ ID NO:9), STFK-AR (aa1192 is A, SEQ ID NO:10), STFK-YR (aa1192 is Y, SEQ ID NO:11), STFK-GR (aa1192 is G, SEQ ID NO:12), STFK-DR (aa1192 is D, SEQ ID NO:13), STFK-MR (aa1192 is M, SEQ ID NO:14).
蛋白表达参照步骤1.2，ExpiCHO细胞瞬时转染6天表达上清SDS-PAGE电泳(图4A)和native-PAGE电泳(图4B)显示，C末端1192位氨基酸突变并不影响蛋白表达量，在目的蛋白位置(图4A的130kD-250kD处蛋白条带)条带显示各突变株蛋白表达量基本相当。Referring to step 1.2 for protein expression, SDS-PAGE electrophoresis (Fig. 4A) and native-PAGE electrophoresis (Fig. 4B) of the supernatant of ExpiCHO cells transiently transfected for 6 days showed that the mutation of amino acid 1192 at the C-terminus did not affect the protein expression. The band at the position of the target protein (the protein band at 130kD-250kD in Fig. 4A) showed that the protein expression of each mutant strain was basically equivalent.
1.4.3 1192位氨基酸替换蛋白与SARS-CoV-2中和抗体的结合活性1.4.3 Binding activity of 1192 amino acid substitution protein to SARS-CoV-2 neutralizing antibody
STFK-HR至STFK-MR蛋白纯化：收集1.4.1中ExpiCHO表达细胞悬液，12000rpm，室温离心30min，留取上清经0.22μm滤膜过滤。将上清样品后使用Q-FF层析柱(Q Sepharose Fast Flow，Cytiva，17051001)进行纯化，100mM NaCl洗脱目的蛋白。STFK-HR to STFK-MR protein purification: Collect the ExpiCHO expression cell suspension in 1.4.1, centrifuge at 12000 rpm for 30 min at room temperature, and filter the supernatant through a 0.22 μm filter membrane. The supernatant sample was then purified using a Q-FF chromatography column (Q Sepharose Fast Flow, Cytiva, 17051001), and the target protein was eluted with 100 mM NaCl.
ELISA结合活性检测：反应板制备见步骤1.3.1。取纯化的STFK-HR至STFK-MR蛋白分别以含20％新生牛血清的PBS溶液稀梯度释至10μg/mL并5倍梯度往下进行 稀释，最后一个浓度梯度为0.128ng/mL，按如下步骤进行ELISA检测：ELISA binding activity detection: see step 1.3.1 for reaction plate preparation. Take the purified STFK-HR to STFK-MR proteins and dilute them to 10 μg/mL in PBS solution containing 20% newborn bovine serum, and carry out the 5-fold gradient downward Dilution, the last concentration gradient is 0.128ng/mL, and ELISA detection is performed as follows:
(1)样品反应：取已包被45C3抗体的酶标板，每孔加入100μL已稀释的样品，置于37℃温箱反应60分钟。(1) Sample reaction: Take a microtiter plate coated with 45C3 antibody, add 100 μL of diluted sample to each well, and place it in a 37°C incubator for 60 minutes to react.
(2)酶标记物反应：完成样品反应步骤后，将酶标板用PBST洗液(20mM PB7.4，150mM NaCl，0.1％Tween20)洗涤5遍，每孔加入100μL HRP标记的RBD单抗85F7或81H10(本室前期筛选的两株SARS-CoV-2 RBD蛋白的鼠单克隆抗体，具有强中和活性)反应液，置于37℃温箱反应30分钟。(2) Enzyme marker reaction: After completing the sample reaction step, wash the ELISA plate 5 times with PBST washing solution (20mM PB7.4, 150mM NaCl, 0.1% Tween20), add 100 μL of HRP-labeled RBD monoclonal antibody 85F7 or 81H10 (mouse monoclonal antibody of two strains of SARS-CoV-2 RBD protein screened in the previous stage of our laboratory, which has strong neutralizing activity) reaction solution, and place in 3 React in an incubator at 7°C for 30 minutes.
(3)显色反应：完成酶标记物反应步骤后，将酶标板用PBST洗液(20mM PB7.4，150mM NaCl，0.1％Tween20)洗涤5遍，每孔加入TMB显色剂(购自北京万泰生物药业股份有限公司)各50μL，置于37℃温箱反应15分钟。(3) Color reaction: After the enzyme marker reaction step was completed, the ELISA plate was washed 5 times with PBST washing solution (20mM PB7.4, 150mM NaCl, 0.1% Tween20), and 50 μL of TMB chromogenic reagent (purchased from Beijing Wantai Bio-Pharmaceutical Co., Ltd.) was added to each well, and placed in a 37°C incubator for 15 minutes.
(4)终止反应及读值测量：完成显色反应步骤后，在反应完的酶标板中每孔加入终止液(购自北京万泰生物药业股份有限公司)50μL，并于酶标仪上检测各孔的OD450/630值。(4) Termination reaction and reading value measurement: After completing the color reaction step, add 50 μL of stop solution (purchased from Beijing Wantai Bio-Pharmaceutical Co., Ltd.) to each well of the reacted microplate plate, and detect the OD450/630 value of each well on a microplate reader.
结果分析：结果如图5A、图5B和表1所示，七个C末端氨基酸突变改造分子与81H10抗体结合能力未见显著差异。但针对85F7中和单抗，该单抗对多数SARS-CoV-2变异株均有广谱高中和活性，提示该抗体识别表位为广谱中和表位。在上述7个改造分子中，aa1192替换为M的变体对85F7的结合能力相对更优，其结合的EC50相较对照STFK-NR(STFK-c)可提升近1倍。Analysis of the results: As shown in Figure 5A, Figure 5B and Table 1, there was no significant difference in the ability of the seven C-terminal amino acid mutation-modified molecules to bind to the 81H10 antibody. However, for the 85F7 neutralizing monoclonal antibody, the monoclonal antibody has broad-spectrum high neutralizing activity against most SARS-CoV-2 variants, suggesting that the antibody recognizes a broad-spectrum neutralizing epitope. Among the above seven modified molecules, the variant in which aa1192 was replaced by M had a relatively better binding ability to 85F7, and its binding EC 50 was nearly double that of the control STFK-NR (STFK-c).
表1：STFK-HR至STFK-MR与2株RBD单抗结合的EC50结果
Table 1: EC 50 results of STFK-HR to STFK-MR combined with 2 strains of RBD monoclonal antibodies
综上，aa1192位氨基酸的替换对STFK蛋白的表达量未产生明显影响，将其改变为M、Y、D、A、或G时，变体蛋白对中和抗体85F7的结合活性有一定程度的提高，其中以替换为M时相对更优。在后面的实验中，本发明人将带有aa1192由N替换为M的抗原STFK-MR(SEQ ID NO:14)作为原型株疫苗免疫原STFK进行研究。后文中，提到STFK、原型株的STFK或者STFK疫苗中的“STFK”，如果没有特别说明，均是指STFK-MR。In summary, the substitution of the amino acid at position 1192 of aa1 has no significant effect on the expression of STFK protein. When it is changed to M, Y, D, A, or G, the binding activity of the variant protein to the neutralizing antibody 85F7 is improved to a certain extent, and the substitution of M is relatively better. In subsequent experiments, the inventors studied the antigen STFK-MR (SEQ ID NO: 14) with aa1192 replaced by N to M as the prototype vaccine immunogen STFK. In the following, STFK, the STFK of the prototype strain, or "STFK" in the STFK vaccine refer to STFK-MR unless otherwise specified.
1.5单体STFK与三聚体StriFK免疫原性比较 1.5 Comparison of immunogenicity between monomeric STFK and trimeric StriFK
通过在BALB/c小鼠中分别免疫接种单体STFK抗原(STFK-MR)制备的疫苗、三聚体StriFK抗原制备的疫苗(制备方法可以参考Wu,Y.,et al.,A recombinant spike protein subunit vaccine confers protective immunity against SARS-CoV-2 infection and transmission in hamsters.Science Tanslational Medicine.20 Jul 2021.Vol 13,Issue 606)，检测小鼠接种后不同时间点的血清中新冠病毒中和抗体水平，评估单体和三聚体形式的免疫原性(STFK疫苗每组6只，StriFK疫苗每组5只)：小鼠分为STFK疫苗(单体)10μg组，STFK疫苗1μg组，StriFK(三聚体)疫苗10μg组，StriFK疫苗1μg组(疫苗剂量按照抗原计算，下同)，按0，2，4周3针接种的免疫程序进行。免疫后血清使用水泡性口炎病毒(VSV)的新冠假病毒(VSVpp)-细胞中和试验检测免疫小鼠血清的中和抗体滴度，具体实验步骤和检测方法参考新冠假病毒(VSVpp)-细胞中和试验检测方法报告进行(Emerg Microbes Infect.2020；9(1):2105-2113.)。小鼠产生SARS-CoV-2中和抗体滴度采用GraphPad Prism软件绘制成分组柱状/散点共存图，结果如图6A和图6B所示。By immunizing the vaccine prepared by monomeric STFK antigen (STFK-MR) and the vaccine prepared by trimeric StriFK antigen respectively in BALB/c mice (the preparation method can refer to Wu, Y., et al., A recombinant spike protein subunit vaccine confers protective immunity against SARS-CoV-2 infection and transmission in hamsters.S Science Tanslational Medicine.20 Jul 2021.Vol 13, Issue 606), detect the level of neutralizing antibody to the new coronavirus in the serum of mice at different time points after vaccination, and evaluate the immunogenicity of the monomer and trimer forms (6 for each group of STFK vaccine, 5 for each group of StriFK vaccine): Mice were divided into STFK vaccine (monomer) 10 μg group, STFK vaccine 1 μg group, StriFK (trimer) Vaccine 10 μg group, StriFK vaccine 1 μg group (vaccine dose is calculated according to the antigen, the same below), according to the immunization schedule of 0, 2, 4 weeks 3 injections. After immunization, the vesicular stomatitis virus (VSV) new coronavirus (VSVpp)-cell neutralization test was used to detect the neutralizing antibody titer of the immunized mouse serum. The specific experimental steps and detection methods were carried out according to the new coronavirus (VSVpp)-cell neutralization test method report (Emerg Microbes Infect.2020; 9(1):2105-2113.). The SARS-CoV-2 neutralizing antibody titer produced by the mice was drawn using GraphPad Prism software to draw the histogram/scatter coexistence graph of the group, and the results are shown in Figure 6A and Figure 6B.
由图6A和图6B的结果可知，三聚体和单体STFK抗原(STFK-MR)配制的疫苗以1μg剂量与10μg剂量在小鼠体内均能诱导动物产生高水平新冠病毒中和抗体。在1μg抗原剂量时，STFK疫苗(单体)和StriFK疫苗(三聚体)在第2周时中和抗体GMT分别为541和309(p＝0.03)，第2针免疫后2周中和抗体GMT分别为16912和8320(p＝0.08)，第3针免疫后2周中和抗体GMT分别为34826和18097(p＝0.05)；在10μg抗原剂量时，STFK疫苗(单体)和StriFK疫苗(三聚体)在第2周时中和抗体GMT分别为635和616(p＝0.79)，第2针免疫后2周中和抗体GMT分别为27681和11306(p＝0.05)，第3针免疫后2周中和抗体GMT分别为77870和33459(p＝0.02)。以上结果均显示STFK疫苗(单体)诱导小鼠产生的中和抗体滴度高于StriFK疫苗(三聚体)。From the results in Figure 6A and Figure 6B, it can be seen that the vaccines formulated with trimeric and monomeric STFK antigens (STFK-MR) can induce high levels of neutralizing antibodies to the new coronavirus in mice at both 1 μg and 10 μg doses. At the dose of 1 μg antigen, the neutralizing antibody GMTs of STFK vaccine (monomer) and StriFK vaccine (trimer) at the second week were 541 and 309 (p=0.03), the neutralizing antibody GMTs were 16912 and 8320 (p=0.08) two weeks after the second immunization, and the neutralizing antibody GMTs were 34826 and 18097 (p=0.05) two weeks after the third immunization; When the antigen dose was 0 μg, the neutralizing antibody GMTs of the STFK vaccine (monomer) and StriFK vaccine (trimer) at the second week were 635 and 616 (p=0.79), the neutralizing antibody GMTs were 27681 and 11306 (p=0.05) two weeks after the second immunization, and the neutralizing antibody GMTs were 77870 and 33459 (p=0.02) two weeks after the third immunization. The above results all showed that the neutralizing antibody titer induced by STFK vaccine (monomer) was higher than that of StriFK vaccine (trimer).
综合实施例1的结果可见：基于单体STFK抗原的疫苗在重组蛋白表达量、中和单抗结合活性、Ace2结合能力及小鼠免疫原性上相较于三聚体抗原均有一定优势，能够作为新冠疫苗的免疫原。Based on the results of Example 1, it can be seen that the vaccine based on the monomeric STFK antigen has certain advantages compared with the trimeric antigen in terms of recombinant protein expression, neutralizing monoclonal antibody binding activity, Ace2 binding ability and mouse immunogenicity, and can be used as the immunogen of the new crown vaccine.
实施例2：STFK疫苗的免疫原性Example 2: Immunogenicity of STFK Vaccines
2.1 STFK在小鼠中的免疫原性2.1 Immunogenicity of STFK in mice
2.1.1 STFK在小鼠中的抗体应答 2.1.1 Antibody response to STFK in mice
通过在BALB/c小鼠中免疫接种不同剂量的STFK疫苗(与步骤1.5中的STFK疫苗相同)，使用酶联免疫试剂盒检测(小鼠抗新型冠状病毒(2019-nCoV)S蛋白IgG抗体检测试剂盒，北京万泰生物药业股份有限公司，批号：NCOmG20200902B-8022E；小鼠抗新型冠状病毒(2019-nCoV)S-RBD蛋白IgG抗体检测试剂盒，北京万泰生物药业股份有限公司，批号：NCOmsG20201201B-8011E)小鼠接种后不同时间点的血清中抗新冠病毒S蛋白特异性IgG抗体(Anti-Spike IgG)、抗新冠病毒S蛋白RBD结构域特异性IgG抗体(Anti-RBD IgG)和假病毒中和抗体水平，评估STFK疫苗在小鼠模型中诱导体液免疫应答。将6-8周龄SPF级雌性BALB/c小鼠随机分组，每组6只，共分成4组：0.01μg组，0.1μg组，1μg组和10μg组(剂量按照抗原计算)，按0、3周2剂接种的免疫程序进行。并于第0周、第2周和第4周采眼眶静脉血。By immunizing different doses of STFK vaccine (same as the STFK vaccine in step 1.5) in BALB/c mice, use an enzyme-linked immunoassay kit to detect (mouse anti-new coronavirus (2019-nCoV) S protein IgG antibody detection kit, Beijing Wantai Biological Pharmaceutical Co., Ltd., batch number: NCOmG20200902B-8022E; mouse anti-new coronavirus (2019-nCoV) S-RBD protein IgG antibody detection Kit, Beijing Wantai Bio-Pharmaceutical Co., Ltd., batch number: NCOmsG20201201B-8011E) Anti-new coronavirus S protein-specific IgG antibody (Anti-Spike IgG), anti-new coronavirus S protein RBD domain-specific IgG antibody (Anti-RBD IgG) and pseudovirus neutralizing antibody levels in the serum of mice at different time points after vaccination, to evaluate the humoral immune response induced by STFK vaccine in mouse models. 6-8 weeks old female BALB/c mice with SPF grade were randomly divided into 4 groups: 0.01 μg group, 0.1 μg group, 1 μg group and 10 μg group (the dose was calculated according to the antigen). Orbital venous blood was collected at week 0, week 2 and week 4.
Anti-RBD IgG和Anti-Spike IgG结果如图7A和图7B所示，抗原含量为0.01μg、0.1μg、1μg和10μg的STFK疫苗免疫BALB/c小鼠第1剂后2周，诱导产生的Anti-Spike IgG抗体GMT水平分别为<100、1033、5062、11255，而诱导产生的Anti-RBD IgG抗体GMT水平分别为<100、360、1767、2885，两个指标均随抗原含量的增加而梯度升高。分析4个组的S-IgG抗体和RBD-IgG抗体的阳转率，可知在第1剂后2周，抗原含量为0.01μg的STFK疫苗免疫组两个抗体指标阳转率均为17％(1/6)，而抗原含量在0.1μg及以上的3个STFK疫苗免疫组两个抗体指标阳转率全部为100％(6/6)。在第2剂免疫后1周，诱导产生的Anti-Spike IgG抗体GMT水平分别为7992、77469、123541、267035，而诱导产生的Anti-RBD IgG抗体GMT水平分别为2411、24465、31213、61901，也随抗原含量的增加而梯度升高。2剂疫苗免疫后，所有4个剂量的STFK疫苗免疫小鼠的两个抗体指标阳转率全部达到100％(6/6)。The results of Anti-RBD IgG and Anti-Spike IgG are shown in Figure 7A and Figure 7B. Two weeks after the first dose of BALB/c mice were immunized with STFK vaccines with antigen contents of 0.01 μg, 0.1 μg, 1 μg and 10 μg, the GMT levels of the induced Anti-Spike IgG antibodies were <100, 1033, 5062 and 11255, respectively, while the induced Anti-RBD I The GMT levels of gG antibody were <100, 360, 1767, and 2885, respectively, and the two indexes increased gradually with the increase of antigen content. Analysis of the positive conversion rates of S-IgG antibodies and RBD-IgG antibodies in the four groups showed that two weeks after the first dose, the positive conversion rates of the two antibody indicators in the STFK vaccine immunization group with an antigen content of 0.01 μg were both 17% (1/6), while the positive conversion rates of the two antibody indicators in the three STFK vaccine immunization groups with an antigen content of 0.1 μg and above were all 100% (6/6). One week after the second dose of immunization, the GMT levels of the induced Anti-Spike IgG antibodies were 7992, 77469, 123541, and 267035, respectively, while the GMT levels of the induced Anti-RBD IgG antibodies were 2411, 24465, 31213, and 61901, respectively, which also gradually increased with the increase of the antigen content. After 2 doses of vaccine immunization, all 4 doses of STFK vaccine immunized mice had two antibody index positive conversion rates all reached 100% (6/6).
使用水疱性口炎病毒(VSV)的新冠假病毒(VSVpp)-细胞中和试验和SARS-CoV-2真病毒中和实验检测免疫小鼠血清的中和抗体滴度。结果如图8A、图8B和图8C所示，根据假病毒中和抗体检测结果可知(图8A)，抗原含量为0.01μg、0.1μg、1μg和10μg STFK疫苗免疫BALB/c小鼠第1剂后2周，诱导产生的中和抗体GMT水平分别为<30、46、133、507。分析4个组的中和抗体阳转率，可知在第1剂后2周，抗原含量为0.01μg的STFK疫苗免疫组尚无动物实现中和抗体阳转，抗原含量为0.1μg的STFK疫苗中和抗体阳转率为83％(5/6)，抗原含量为1μg及以上的STFK疫 苗免疫组中和抗体阳转率全部为100％(6/6)。在第2剂免疫后1周，4个不同剂量疫苗免疫小鼠诱导产生的中和抗体GMT水平分别为592、8839、10633、32029；全部小鼠血清中和抗体均实现100％阳转。The vesicular stomatitis virus (VSV) new coronavirus pseudovirus (VSVpp)-cell neutralization test and the SARS-CoV-2 true virus neutralization test were used to detect the neutralizing antibody titer of the sera of immunized mice. The results are shown in Figure 8A, Figure 8B and Figure 8C. According to the detection results of pseudovirus neutralizing antibodies (Figure 8A), the antigen content was 0.01 μg, 0.1 μg, 1 μg and 10 μg. Two weeks after the first dose of STFK vaccine immunization of BALB/c mice, the GMT levels of neutralizing antibodies induced were <30, 46, 133, and 507, respectively. Analysis of the positive conversion rate of neutralizing antibodies in the four groups showed that 2 weeks after the first dose, no animals in the STFK vaccine immunization group with an antigen content of 0.01 μg achieved neutralizing antibody positive conversion. The neutralizing antibody positive conversion rate of the STFK vaccine with an antigen content of 0.1 μg was 83% (5/6). The positive conversion rate of neutralizing antibody in the vaccine immunized group was all 100% (6/6). One week after the second dose of immunization, the GMT levels of neutralizing antibodies induced by the four different doses of vaccine immunization mice were 592, 8839, 10633, and 32029 respectively; all mice serum neutralizing antibodies achieved 100% seroconversion.
根据对第4周的真病毒中和抗体检测可知(图8B)，抗原含量为0.01μg、0.1μg、1μg和10μg STFK疫苗免疫BALB/c小鼠后的中和抗体滴度GMT分别为32、362、575、1625。中和抗体滴度呈明显的剂量依赖效应。According to the detection of true virus neutralizing antibody at the 4th week (Figure 8B), the neutralizing antibody titer GMT after immunizing BALB/c mice with the antigen content of 0.01 μg, 0.1 μg, 1 μg and 10 μg STFK vaccine was 32, 362, 575, and 1625, respectively. Neutralizing antibody titers showed a significant dose-dependent effect.
STFK疫苗免疫小鼠后血清新冠真病毒与假病毒中和抗体滴度相关性如图8C所示，两种中和抗体检测方法的相关系数达到0.94，表明假病毒中和抗体与真病毒中和抗体具有很好的相关性。The correlation between the neutralizing antibody titers between the true virus and the pseudovirus in the serum of mice immunized with the STFK vaccine is shown in Figure 8C. The correlation coefficient of the two neutralizing antibody detection methods reached 0.94, indicating that the neutralizing antibody of the pseudovirus and the neutralizing antibody of the true virus have a good correlation.
综上实验结果，抗原含量≥0.1μg每剂的STFK疫苗，单剂免疫BALB/c小鼠即可在免疫后2周成功诱导所有受试动物的Anti-Spike IgG抗体和RBD-IgG抗体阳转，阳转率为100％。抗原含量≥1μg每剂的STFK疫苗，单剂免疫BALB/c小鼠即可在免疫后2周成功诱导所有受试动物的中和抗体阳转，阳转率为100％。抗原含量0.01μg每剂的STFK疫苗，两剂(0/3周)免疫后1周可成功诱导所有受试动物的Anti-Spike IgG、Anti-RBD IgG抗体和新冠病毒中和抗体阳转，阳转率为100％。表明STFK疫苗具有良好的免疫原性。抗原含量在0.01μg-10μg的STFK疫苗免疫BALB/c小鼠，无论是单剂还是两剂(0/3周)免疫，诱导的Anti-Spike IgG、Anti-RBD IgG抗体和新冠病毒中和抗体水平均呈现明显的抗原剂量依赖效应。To sum up the experimental results, a single dose of STFK vaccine with antigen content ≥ 0.1 μg per dose can successfully induce Anti-Spike IgG antibody and RBD-IgG antibody seroconversion in all tested animals 2 weeks after immunization in BALB/c mice, and the seroconversion rate is 100%. STFK vaccine with an antigen content ≥ 1 μg per dose, a single dose of BALB/c mice can successfully induce neutralizing antibody seroconversion in all tested animals 2 weeks after immunization, and the seroconversion rate is 100%. The STFK vaccine with an antigen content of 0.01 μg per dose can successfully induce Anti-Spike IgG, Anti-RBD IgG antibody and new coronavirus neutralizing antibody seroconversion in all tested animals one week after two doses (0/3 weeks) of immunization, and the seroconversion rate is 100%. It shows that STFK vaccine has good immunogenicity. When BALB/c mice were immunized with STFK vaccine with an antigen content of 0.01 μg-10 μg, no matter whether it was a single dose or two doses (0/3 weeks), the levels of Anti-Spike IgG, Anti-RBD IgG antibodies and SARS-CoV-2 neutralizing antibodies showed obvious antigen dose-dependent effects.
2.1.2 STFK在小鼠中的细胞免疫应答2.1.2 Cellular immune response of STFK in mice
同样地，为考察STFK疫苗诱导细胞免疫的能力，本发明人以使用传统铝佐剂的STFK-Al疫苗(制备方法可以参考Wu,Y.,et al.,A recombinant spike protein subunit vaccine confers protective immunity against SARS-CoV-2 infection and transmission in hamsters.Science Tanslational Medicine.20 Jul 2021.Vol 13,Issue 606)为对照疫苗，采用ELISPOT技术分析疫苗免疫小鼠后诱导淋巴细胞产生的IFN-γ的能力。Similarly, in order to investigate the ability of the STFK vaccine to induce cellular immunity, the inventor used the STFK-Al vaccine of the traditional aluminum adjuvant (the preparation method can refer to Wu, Y., et al., A recombinant spike protein subunit vaccine confers protective immunity against SARS-CoV-2 infection and transmission in hamsters. Science Tanslational Medicine.20 Jul 2021.Vol 13, Issue 606) is a control vaccine, and ELISPOT technology is used to analyze the ability of the vaccine to induce lymphocytes to produce IFN-γ after immunizing mice.
将C57BL/6小鼠随机分组，每组8只，共分成3组：其中2组分别接受1剂STFK疫苗(命名为STFK Vac组)、STFK-Al疫苗(使用STFK免疫原(STFK-MR)和对照铝佐剂，命名为STFK-Al Vac组)，另外1组未进行免疫接种(命名为Unvaccinated组)，免疫剂量为10μg，免疫后第7天牺牲小鼠。解剖后分离小鼠脾脏淋巴细胞，使用酶联免疫斑点试验(ELISPOT)分析免疫后小鼠脾脏淋巴细胞中响应抗原特异性刺激并分泌IFN-γ的T细胞，以对STFK疫苗在小鼠体内的细胞免疫应答进行分析。 C57BL/6 mice were randomly divided into 8 groups, and divided into 3 groups: 2 groups received one dose of STFK vaccine (named STFK Vac group), STFK-Al vaccine (using STFK immunogen (STFK-MR) and control aluminum adjuvant, named STFK-Al Vac group), and the other group was not immunized (named Unvaccinated group), the immunization dose was 10 μg, and the mice were sacrificed on the 7th day after immunization. Spleen lymphocytes of mice were isolated after dissection, and T cells that responded to antigen-specific stimulation and secreted IFN-γ in spleen lymphocytes of immunized mice were analyzed by enzyme-linked immunospot assay (ELISPOT), so as to analyze the cellular immune response of STFK vaccine in mice.
结果如图9所示，STFK疫苗免疫组的平均斑点数为441，STFK-Al疫苗免疫组的平均斑点数为186，未免疫组平均斑点数为40。STFK疫苗免疫组斑点数显著高于STFK-Al疫苗组(P＝0.047)和未免疫组(P<0.0001)，分别高2.4倍和11倍。表明STFK疫苗免疫小鼠脾脏细胞中能检测到较强的特异性T淋巴细胞免疫应答，且显著强于铝佐剂疫苗STFK-Al。The results are shown in Figure 9, the average number of spots in the STFK vaccine immunized group was 441, the average number of spots in the STFK-Al vaccine immunized group was 186, and the average number of spots in the non-immunized group was 40. The number of spots in the STFK vaccine immunized group was significantly higher than that in the STFK-Al vaccine group (P=0.047) and the unimmunized group (P<0.0001), which were 2.4 times and 11 times higher, respectively. It shows that a strong specific T lymphocyte immune response can be detected in the spleen cells of mice immunized with STFK vaccine, which is significantly stronger than that of the aluminum adjuvant vaccine STFK-Al.
2.2 STFK疫苗在恒河猴中免疫原性2.2 Immunogenicity of STFK vaccine in rhesus monkeys
为进一步考察STFK疫苗在灵长类动物中的免疫原性，在恒河猴模型中分别免疫接种不同抗原剂量的含FH002C佐剂的疫苗(STFK疫苗)和含对照铝佐剂的疫苗(STFK-Al疫苗)，检测接种后不同时间点的血清中Anti-Spike IgG抗体、Anti-RBD IgG抗体和中和抗体水平，比较不同佐剂配制的疫苗在恒河猴体内的免疫原性。根据恒河猴免疫前Anti-Spike IgG抗体和Anti-RBD IgG抗体检验情况，结合试验动物其他信息，初始入组20只恒河猴随机分为4组，A1-A5计划免疫低剂量对照疫苗(1μg/剂，STFK-Al疫苗)、B1-B5计划免疫高剂量对照疫苗(15μg/剂，STFK-Al疫苗)、C1-C5计划免疫低剂量STFK疫苗(1μg/剂STFK疫苗)、D1-D5计划免疫高剂量STFK疫苗(15μg/剂STFK疫苗)(剂量均按照抗原计算)。首剂免疫前，因B1猴咳嗽且体温偏高，不符合受试标准，未进行免疫。其余入组动物按0、4周2针接种的免疫程序进行接种。依照临床拟用给药方式，于上臂三角肌注射150μL，单点注射。于第0周、第3周和第6周采手臂静脉血用于抗体分析。In order to further investigate the immunogenicity of STFK vaccine in primates, rhesus monkey models were immunized with different antigen doses of vaccines containing FH002C adjuvant (STFK vaccine) and the vaccine containing control aluminum adjuvant (STFK-Al vaccine). The levels of Anti-Spike IgG antibody, Anti-RBD IgG antibody and neutralizing antibody in serum at different time points after vaccination were detected, and the immunogenicity of vaccines formulated with different adjuvants in rhesus monkeys was compared. According to the detection of Anti-Spike IgG antibody and Anti-RBD IgG antibody before rhesus macaque immunization, combined with other information of experimental animals, 20 rhesus macaques initially enrolled were randomly divided into 4 groups, A1-A5 planned immunization low-dose control vaccine (1 μg/dose, STFK-Al vaccine), B1-B5 planned immunization high-dose control vaccine (15 μg/dose, STFK-Al vaccine), C1-C5 planned immunization low-dose STFK vaccine (1 μg/dose STFK vaccine), D1-D5 planned immunization with high-dose STFK vaccine (15 μg/dose of STFK vaccine) (the dose is calculated according to the antigen). Before the first dose of immunization, because the B1 monkey had a cough and a high body temperature, it did not meet the test criteria and was not immunized. The rest of the animals in the group were vaccinated according to the immunization procedure of 2 injections at 0 and 4 weeks. Inject 150 μL into the deltoid muscle of the upper arm according to the intended clinical administration method, single-point injection. Arm venous blood was collected at week 0, week 3 and week 6 for antibody analysis.
Anti-RBD IgG和Anti-Spike IgG结果如图10A至图10D所示，免疫恒河猴1剂后，在1μg剂量时STFK疫苗诱导产生Anti-Spike IgG和Anti-RBD IgG抗体GMT分别为11782和6788，STFK-Al疫苗诱导产生Anti-Spike IgG和Anti-RBD IgG抗体GMT分别为1386和976，STFK疫苗抗体滴度比STFK-Al疫苗分别高8.5倍和7.0倍(图10A和图10B)。在15μg剂量时STFK疫苗诱导产生Anti-Spike IgG和Anti-RBD IgG抗体GMT分别为18547和15320，STFK-Al疫苗诱导产生Anti-Spike IgG和Anti-RBD IgG抗体GMT分别为11558和4352，STFK疫苗抗体滴度略高于STFK-Al疫苗，但差异无统计学意义(P>0.05，图10C和图10D)。The results of Anti-RBD IgG and Anti-Spike IgG are shown in Figure 10A to Figure 10D. After immunizing rhesus monkeys for 1 dose, the GMT of Anti-Spike IgG and Anti-RBD IgG antibodies induced by STFK vaccine at a dose of 1 μg were 11782 and 6788, respectively, and the GMT of Anti-Spike IgG and Anti-RBD IgG antibodies induced by STFK-Al vaccine were 1386 and 976, the antibody titer of STFK vaccine was 8.5 times and 7.0 times higher than that of STFK-Al vaccine respectively (Fig. 10A and Fig. 10B). At 15 μg dose, the GMTs of Anti-Spike IgG and Anti-RBD IgG antibodies induced by STFK vaccine were 18547 and 15320, and the GMTs of Anti-Spike IgG and Anti-RBD IgG antibodies induced by STFK-Al vaccine were 11558 and 4352, respectively. The antibody titers of STFK vaccine were slightly higher than those of STFK-Al vaccine, but the difference was not statistically significant (P>0.05, Fig. 10C and Figure 10D).
免疫恒河猴2剂后2周(第6周)，在1μg剂量时STFK疫苗诱导产生Anti-Spike IgG和Anti-RBD IgG抗体GMT分别为1,558,853和634,202，STFK-Al疫苗诱导产生Anti-Spike IgG和Anti-RBD IgG抗体GMT分别为86,823和38,097，STFK疫苗抗体滴度比STFK-Al疫苗分别高18倍和17倍(图10A和图10B)。在15μg剂量时 STFK疫苗诱导产生Anti-Spike IgG和Anti-RBD IgG抗体GMT分别为1,088,930和640,036，STFK-Al疫苗诱导产生Anti-Spike IgG和Anti-RBD IgG抗体GMT分别为1,818,584和552,812，STFK疫苗抗体与STFK-Al疫苗相当(P>0.05，图10C和图10D)。Two weeks after immunizing rhesus monkeys with 2 doses (week 6), the GMTs of Anti-Spike IgG and Anti-RBD IgG antibodies induced by STFK vaccine at 1 μg dose were 1,558,853 and 634,202, respectively, and the GMTs of Anti-Spike IgG and Anti-RBD IgG antibodies induced by STFK-Al vaccine were 86,823 and 38,097, respectively. The K-Al vaccine was 18-fold and 17-fold higher, respectively (Fig. 10A and Fig. 10B). At 15 μg dose The GMTs of Anti-Spike IgG and Anti-RBD IgG antibodies induced by STFK vaccine were 1,088,930 and 640,036, respectively, and the GMTs of Anti-Spike IgG and Anti-RBD IgG antibodies induced by STFK-Al vaccine were 1,818,584 and 552,812, respectively. 0D).
以上结果显示，在1μg剂量免疫时STFK疫苗在恒河猴体内诱导的Anti-Spike IgG和Anti-RBD IgG抗体滴度比STFK-Al疫苗高7-18倍，表明STFK疫苗相比STFK-Al疫苗能诱导更强抗体应答。The above results showed that the Anti-Spike IgG and Anti-RBD IgG antibody titers induced by STFK vaccine in rhesus macaques were 7-18 times higher than that of STFK-Al vaccine when immunized at a dose of 1 μg, indicating that STFK vaccine can induce stronger antibody responses than STFK-Al vaccine.
使用水疱性口炎病毒(VSV)的新冠假病毒(VSVpp)-细胞中和试验和SARS-CoV-2真病毒中和实验检测免疫恒河猴血清的中和抗体滴度。结果如图11A至图11E所示，从疫苗诱导的假病毒中和抗体水平来看，免疫恒河猴1剂后，在1μg和15μg剂量时STFK疫苗诱导产生中和抗体阳转率均为100％，GMT分别为70和134，同期STFK-Al疫苗诱导产生中和抗体阳转率为60％和75％，GMT分别为45和68，STFK疫苗抗体滴度比STFK-Al疫苗高约2倍，但差异尚不具备统计学意义(P>0.05，图11A和图11B)。免疫恒河猴2剂后2周(第6周)，在1μg剂量时STFK疫苗诱导产生中和抗体GMT为12,570，同期STFK-Al疫苗诱导产生中和抗体GMT为1037，STFK疫苗中和抗体滴度比STFK-Al疫苗高12倍，差异具备统计学意义(P<0.05)。在15μg剂量时STFK疫苗诱导产生中和抗体GMT为29556，STFK-Al疫苗诱导产生中和抗体GMT为19171，STFK疫苗中和抗体滴度高于STFK-Al疫苗1.5倍，但差异无统计学意义(P>0.05)。The neutralizing antibody titers of immunized rhesus macaque sera were detected by vesicular stomatitis virus (VSV) new coronavirus pseudovirus (VSVpp)-cell neutralization assay and SARS-CoV-2 true virus neutralization assay. The results are shown in Figures 11A to 11E. From the perspective of vaccine-induced pseudovirus neutralizing antibody levels, after immunizing rhesus monkeys for 1 dose, the STFK vaccine-induced neutralizing antibody positive conversion rate was 100% at 1 μg and 15 μg doses, and the GMT was 70 and 134 respectively. In the same period, the neutralizing antibody positive conversion rate induced by the STFK-Al vaccine was 60% and 75%, and the GMT was 45 and 68 respectively. The antibody titer of the STFK vaccine was about 2 times higher than that of the STFK-Al vaccine. times, but the difference is not yet statistically significant (P>0.05, Figure 11A and Figure 11B). Two weeks after the rhesus monkeys were immunized with 2 doses (week 6), the GMT of neutralizing antibody induced by STFK vaccine at 1 μg dose was 12,570, and the GMT of neutralizing antibody induced by STFK-Al vaccine was 1037 in the same period. The neutralizing antibody titer of STFK vaccine was 12 times higher than that of STFK-Al vaccine, and the difference was statistically significant (P<0.05). At 15 μg dose, the GMT of neutralizing antibody induced by STFK vaccine was 29556, and the GMT of neutralizing antibody induced by STFK-Al vaccine was 19171. The neutralizing antibody titer of STFK vaccine was 1.5 times higher than that of STFK-Al vaccine, but the difference was not statistically significant (P>0.05).
从疫苗诱导的真病毒中和抗体水平来看(图11C和图11D)，免疫恒河猴1剂后，在1μg和15μg剂量时STFK疫苗诱导产生中和抗体阳转率分别为40％和80％，GMT分别为<4和8,同期STFK-Al疫苗诱导产生中和抗体阳转率为20％和无阳转，GMT均小于4。免疫恒河猴2剂后(第6周)，在1μg剂量时STFK疫苗诱导产生中和抗体GMT为588，同期STFK-Al疫苗诱导产生中和抗体GMT为42，STFK疫苗中和抗体滴度比STFK-Al疫苗高14倍，差异具备统计学意义(P<0.01)。在15μg剂量时STFK疫苗诱导产生中和抗体GMT为1,351，同期STFK-Al疫苗诱导产生中和抗体GMT为431，STFK疫苗中和抗体滴度比STFK-Al疫苗高3倍，差异具备统计学意义(P<0.01)。From the perspective of the level of neutralizing antibodies induced by the vaccine (Figure 11C and Figure 11D), after 1 dose of immunization of rhesus macaques, the seroconversion rates of neutralizing antibodies induced by the STFK vaccine at 1 μg and 15 μg doses were 40% and 80%, respectively, and the GMTs were <4 and 8, respectively. In the same period, the seroconversion rate of neutralizing antibodies induced by the STFK-Al vaccine was 20% and there was no seroconversion, and the GMTs were all less than 4. After immunizing rhesus monkeys for 2 doses (week 6), the GMT of neutralizing antibody induced by STFK vaccine at 1 μg dose was 588, and the GMT of neutralizing antibody induced by STFK-Al vaccine was 42 during the same period. The neutralizing antibody titer of STFK vaccine was 14 times higher than that of STFK-Al vaccine, and the difference was statistically significant (P<0.01). At a dose of 15 μg, the GMT of neutralizing antibody induced by STFK vaccine was 1,351, and the GMT of neutralizing antibody induced by STFK-Al vaccine was 431 at the same time. The neutralizing antibody titer of STFK vaccine was 3 times higher than that of STFK-Al vaccine, and the difference was statistically significant (P<0.01).
STFK疫苗免疫恒河猴后血清新冠真病毒与假病毒中和抗体滴度相关性如图11E所示，两种中和抗体检测方法的相关系数达到0.98，表明恒河猴血清中假病毒中和抗体与真病毒中和抗体具有良好相关性。 The correlation between the neutralizing antibody titers of the true virus and the pseudovirus in the serum of rhesus monkeys immunized with the STFK vaccine is shown in Figure 11E. The correlation coefficient of the two neutralizing antibody detection methods reached 0.98, indicating that the neutralizing antibodies of the pseudovirus in the serum of rhesus monkeys have a good correlation with the neutralizing antibodies of the true virus.
综合以上实验结果，在恒河猴体内以1μg和15μg剂量STFK疫苗免疫接种均能诱导高水平Anti-RBD IgG和Anti-Spike IgG抗体和中和抗体滴度，表明STFK疫苗在恒河猴体内具有良好的免疫原性。在1μg免疫剂量下，两剂免后2周，STFK疫苗诱导产生的Anti-RBD IgG、Anti-Spike IgG和中和抗体(真病毒和假病毒)GMT水平较STFK-Al疫苗高12-18倍，表明STFK疫苗相比STFK-Al疫苗能诱导更强的抗体应答。Based on the above experimental results, immunization with 1 μg and 15 μg doses of STFK vaccine in rhesus monkeys can induce high levels of Anti-RBD IgG and Anti-Spike IgG antibodies and neutralizing antibody titers, indicating that STFK vaccine has good immunogenicity in rhesus monkeys. At 1 μg immunization dose, 2 weeks after two doses of immunization, the GMT levels of Anti-RBD IgG, Anti-Spike IgG and neutralizing antibodies (true virus and pseudovirus) induced by STFK vaccine were 12-18 times higher than those of STFK-Al vaccine, indicating that STFK vaccine can induce a stronger antibody response than STFK-Al vaccine.
2.3 STFK疫苗对主要变异株的中和效果2.3 Neutralizing effect of STFK vaccine on main mutant strains
SARS-CoV-2在病毒进化过程中可发生自然变异，部分变异可导致病毒的传播能力、致病能力等特性发生改变，从而影响疫苗的保护效力、药物的治疗效果、诊断工具的灵敏度和特异度等，甚至造成疫情持续暴发等严重后果。在英国、南非、巴西、印度等地区出现了多种新冠病毒变异株的流行，对于变异株对目前的预防措施的影响受到全世界的广泛关注。多个近期刚完成三期临床试验的疫苗都出现了保护效果的下降。SARS-CoV-2 D614G突变株是最早出现并迅速成为全球主流毒株的变异株。本发明人对STFK疫苗免疫恒河猴后的血清进行了多种主要新冠病毒变异株的中和效果评估。血清变异株中和抗体的检测采用基于慢病毒(LV)载体的新型冠状病毒假病毒中和抗体检测方法进行。评估的新冠病毒主要变异株假病毒见表3。SARS-CoV-2 can undergo natural mutations during the virus evolution process, and some mutations can lead to changes in the virus's transmission ability, pathogenicity and other characteristics, thereby affecting the protective efficacy of vaccines, the therapeutic effect of drugs, the sensitivity and specificity of diagnostic tools, etc., and even cause serious consequences such as continuous outbreaks. In the United Kingdom, South Africa, Brazil, India and other regions, a variety of new coronavirus variants have emerged, and the impact of the variants on current preventive measures has received widespread attention from all over the world. Several vaccines that have recently completed phase III clinical trials have shown a decline in protective effect. The SARS-CoV-2 D614G mutant strain was the first to appear and quickly became a global mainstream strain. The inventors evaluated the neutralization effect of various main new coronavirus variants on the serum of rhesus monkeys immunized with STFK vaccine. The detection of neutralizing antibodies of serum mutant strains was carried out using a lentiviral (LV) vector-based detection method for neutralizing antibodies to novel coronavirus pseudoviruses. The pseudoviruses of the main mutant strains of the new coronavirus evaluated are shown in Table 3.
表3：基于慢病毒载体的新冠病毒主要变异株假病毒

Table 3: Pseudoviruses of the main mutant strains of the new coronavirus based on lentiviral vectors

根据STFK疫苗免疫恒河猴血清对新冠病毒变异株中和抗体检测结果可知，在1μg剂量STFK疫苗免疫恒河猴后(图12A)，免疫血清对D614G原型株病毒的平均中和抗体滴度(GMT)为7817，对不同变异株的中和抗体滴度GMT分别为6403(B.1.1.7，英国/Alpha株)，2861(P.1，巴西/Gamma株)，1174(B.1.351，南非/Beta株)，4787(B.1.429，加州/Epsilon株)，2746(B.1.525，尼日利亚/Eta株)，3364(B.1.526a，纽约/Iota_S477N株)，3553(B.1.526b，纽约/Iota_E484K株)，4006(B.1.617.1，印度/Kappa株)，6109(B.1.617.2，印度/Delta株),4419(C.37，秘鲁/Lambda株)。与其对D614G原型株的中和抗体滴度相比，猴免疫血清对B.1.351/Beta株(下降至19％)的相对中和滴度降低较为明显，对包括B.1.617.2/Delta株、 C.37/Lambda株在内的其他变异株的中和滴度降幅相对较小(降低<3倍，相对中和滴度保持在33％以上)。According to the test results of neutralizing antibodies against the new coronavirus mutant strains in rhesus monkeys immunized with STFK vaccine, after immunizing rhesus monkeys with 1 μg dose of STFK vaccine (Figure 12A), the average neutralizing antibody titer (GMT) of the immune serum to the D614G prototype strain virus was 7817, and the neutralizing antibody titers to different mutant strains were 6403 (B.1.1.7, British/Alpha strain), 2861 (P.1, Brazil/Gamma strain) ), 1174 (B.1.351, South Africa/Beta strain), 4787 (B.1.429, California/Epsilon strain), 2746 (B.1.525, Nigeria/Eta strain), 3364 (B.1.526a, New York/Iota_S477N strain), 3553 (B.1.526b, New York/Iota_E484K strain), 400 6 (B.1.617.1, India/Kappa strain), 6109 (B.1.617.2, India/Delta strain), 4419 (C.37, Peru/Lambda strain). Compared with the neutralizing antibody titer of the D614G prototype strain, the relative neutralizing titer of the monkey immune serum to the B.1.351/Beta strain (dropped to 19%) was significantly reduced, including the B.1.617.2/Delta strain, The neutralizing titers of other mutant strains including the C.37/Lambda strain had a relatively small decrease (<3 times, and the relative neutralizing titers remained above 33%).
在15μg剂量STFK疫苗免疫恒河猴后(图12B)，免疫血清对主流株病毒SARS-CoV-2 D614G的平均中和抗体滴度(GMT)为16794，对不同变异株的中和抗体滴度GMT分别为13743(B.1.1.7，英国/Alpha株)，3690(P.1，巴西/Gamma株)，1499(B.1.351，南非/Beta株)，12387(B.1.429，加州/Epsilon株)，3886(B.1.525，尼日利亚/Eta株)，6439(纽约/Iota_S477N株)，3723(B.1.526b，纽约/Iota_E484K株)，6335(B.1.617.1，印度/Kappa株)，9904(B.1.617.2，印度/Delta株)，8168(C.37，秘鲁/Lambda株)。与其对D614G原型株中和抗体滴度对比，猴免疫血清对B.1.351/Beta株(下降至12％)、P.1/Gamma株(下降至31％)、B.1.525/Eta株(下降至26％)、B.1.526/Iota_E484K株(下降至24％)，对包括B.1.617.2/Delta株、C.37/Lambda株在内的其他变异株的中和滴度降幅相对较小(降低<3倍，相对中和滴度保持在33％以上)。After immunizing rhesus monkeys with a dose of STFK vaccine of 15 μg (Figure 12B), the average neutralizing antibody titer (GMT) of the immune serum to the mainstream strain of SARS-CoV-2 D614G was 16794, and the GMT of neutralizing antibody titers to different mutant strains were 13743 (B.1.1.7, British/Alpha strain), 3690 (P.1, Brazil/Gamma strain), 1499 (B.1.3 51, South Africa/Beta strain), 12387 (B.1.429, California/Epsilon strain), 3886 (B.1.525, Nigeria/Eta strain), 6439 (New York/Iota_S477N strain), 3723 (B.1.526b, New York/Iota_E484K strain), 6335 (B.1.617.1, India/Kappa strain), 9904 (B.1.617.2, Indian/Delta strain), 8168 (C.37, Peruvian/Lambda strain). Compared with its neutralizing antibody titer to the D614G prototype strain, monkey immune serum is to B.1.351/Beta strain (down to 12%), P.1/Gamma strain (down to 31%), B.1.525/Eta strain (down to 26%), B.1.526/Iota_E484K strain (down to 24%), to including B.1.617.2/Delta strain, C.37 The neutralizing titers of other mutant strains including the /Lambda strain had a relatively small decrease (<3 times, and the relative neutralizing titers remained above 33%).
对比1μg和15μg剂量STFK疫苗(剂量均按照抗原计算)免疫恒河猴血清变异株中和抗体相对D614G原型株病毒中和抗体，1μg剂量STFK疫苗免疫恒河猴后血清相对变异株中和抗体相对D614G原型株病毒中和抗体下降幅度略小于15μg剂量STFK疫苗，但两组整体趋势上较为一致。根据两组合并分析的结果，STFK疫苗免疫恒河猴血清对B.1.1.7/Alpha株的相对中和滴度平均为D614G原型株的89％(无明显降低)、对P.1/Gamma株平均为D614G原型株的34％(降幅为2-3倍)、对B.1.351/Beta株平均为D614G原型株的15％(降幅为6.5倍)、对B.1.429/Epsilon株平均为D614G原型株的76％(无明显降低)、对B.1.525/Eta株平均为D614G原型株的32％(降幅为3.2倍)、对B.1.526a/Iota_S477N株平均为D614G原型株的41％(降幅为2.4倍)、对B.1.526b/Iota_E484K株平均为D614G原型株的35％(降幅为2.8倍)、对B.1.617.1/Kappa株平均为D614G原型株的49％(降幅为2.0倍)、对B.1.617.2/Delta株平均为D614G原型株的76％(无明显降低)、对C.37/Lambda株平均为D614G原型株的55％(无明显降低)。Compared with 1 μg and 15 μg doses of STFK vaccine (the doses are calculated according to the antigen), the serum neutralizing antibody of the rhesus monkeys immunized with the mutant strain was compared with the D614G prototype strain virus neutralizing antibody. According to the results of the combined analysis of the two groups, the relative neutralization titer of the STFK vaccine immunized rhesus monkey serum to the B.1.1.7/Alpha strain is 89% (no significant reduction) of the D614G prototype strain on average, 34% (the decline is 2-3 times) of the D614G prototype strain to the P.1/Gamma strain, and 15% (the decline is 6.5 times) of the D614G prototype strain to the B.1.351/Beta strain on average. The B.1.429/Epsilon strain is 76% of the D614G prototype strain on average (no significant reduction), the B.1.525/Eta strain is an average of 32% of the D614G prototype strain (the decline is 3.2 times), the B.1.526a/Iota_S477N strain is an average of 41% of the D614G prototype strain (the decline is 2.4 times), and the B.1.526b/Iota _E484K strain is on average 35% of D614G prototype strain (decrease rate is 2.8 times), to B.1.617.1/Kappa strain, it is 49% of D614G prototype strain on average (decrease rate is 2.0 times), to B.1.617.2/Delta strain, it is 76% of D614G prototype strain on average (no obvious decrease), to C.37/Lambda strain, it is 5% of D614G prototype strain on average 5% (no significant reduction).
以上结果表明，STFK疫苗免疫恒河猴后血清对多种新冠病毒变异株均具有良好的中和活性，中和抗体阳性率均为100％。1μg和15μg剂量STFK疫苗免疫恒河猴血清对不同新冠变异株的交叉中和模式整体一致，对本评估中测试的10种VOC/VOI变异株假病毒的中和滴度几何平均值GMT均>1000。STFK疫苗免疫恒河猴血清对 B.1.1.7/Alpha株(89％)、B.1.429/Epsilon株(76％)、B.1.617.2/Delta株(76％)和C.37/Lambda株(55％)的相对中和滴度与D614G原型株相比变化较小，降幅小于2倍；对P.1/Gamma株(34％)、对B.1.525/Eta株(32％)、B.1.526a/Iota_S477N株(41％)、B.1.526b/Iota_E484K株(35％)、B.1.617.1/Kappa株(49％)的相对中和滴度与D614G原型株相比降幅在2-4倍左右；受B.1.351/Beta变异株的影响较为明显，相对中和滴度与D614G原型株相比平均降幅为6.5倍。The above results show that the serum of rhesus monkeys immunized with STFK vaccine has good neutralizing activity against a variety of new coronavirus variants, and the positive rate of neutralizing antibodies is 100%. The cross-neutralization patterns of rhesus macaque sera immunized with STFK vaccine at 1 μg and 15 μg doses were generally consistent with different new coronavirus variants, and the geometric mean neutralization titers GMT of the 10 VOC/VOI variant pseudoviruses tested in this evaluation were all >1000. STFK vaccine immune rhesus monkey serum The relative neutralization titers of B.1.1.7/Alpha strain (89%), B.1.429/Epsilon strain (76%), B.1.617.2/Delta strain (76%) and C.37/Lambda strain (55%) had little change compared with the D614G prototype strain, and the decline was less than 2 times; for P.1/Gamma strain (34%), for B.1.525/Eta strain (32%), B.1.526a/Iota_S477N strain (41%), B.1.526b/Iota_E484K strain (35%), the relative neutralization titer of B.1.617.1/Kappa strain (49%) compared with the D614G prototype strain, the decline rate is about 2-4 times; affected by the B.1.351/Beta mutant strain is more obvious, and the relative neutralization titer and Compared with the D614G prototype strain, the average reduction rate was 6.5 times.
实施例3：基于SARS-CoV2变异株的STFK分子构建Example 3: Construction of STFK molecules based on SARS-CoV2 mutant strains
由于SARS-CoV2病毒在进化过程中不断发生变异，对现有疫苗的保护作用也产生了巨大影响，步骤2.3中，本发明人发现，尽管STFK疫苗免疫血清对多种新冠变异株具有良好的中和活性，但相较原型株仍具有一定程度的降低。因此，针对变异株进行Spike蛋白改造以获得能够和STFK形成互补的疫苗分子尤为重要。总体设计思路如图13所示，首先选择几个免疫逃逸较为显著的变异株病毒的Spike进行变异株STFK分子骨架筛选，如Beta株(B.1.351)、Gamma株(B.1.1.28)和B.1.620株，分别命名为STFK1351、STFK1128和STFK1620。随后基于这3种变异株STFK分子进行嵌合并引入其他变异株病毒RBD区的突变。Since the SARS-CoV2 virus is constantly mutating during the evolution process, it has also had a huge impact on the protective effect of existing vaccines. In step 2.3, the inventors found that although the STFK vaccine immune serum has good neutralizing activity against a variety of new coronavirus variants, it still has a certain degree of reduction compared to the prototype strain. Therefore, it is particularly important to modify the Spike protein for mutant strains to obtain vaccine molecules that can complement STFK. The overall design idea is shown in Figure 13. First, select Spike of several mutant strains with significant immune escape to screen the mutant STFK molecular skeleton, such as Beta strain (B.1.351), Gamma strain (B.1.1.28) and B.1.620 strain, named STFK1351, STFK1128 and STFK1620 respectively. Then based on these three mutant strains, the STFK molecules were chiseled and the mutations in the RBD region of other mutant strains were introduced.
3.1基于变异株分子进行STFK分子框架设计和选择3.1 Design and selection of STFK molecular framework based on mutant strain molecules
候选骨架分子STFK1128(SEQ ID NO:15)、STFK1351(SEQ ID NO:16)和STFK1620(SEQ ID NO:17)的氨基酸突变位点如图13所示。将编码这三种候选骨架分子的序列构建至表达载体pGS01中，随后参照步骤1.2进行真核表达。培养6天后，收集ExpiCHO表达细胞悬液，12000rpm，室温离心30min，留取上清经0.22μm滤膜过滤。将上清样品后使用Q-FF层析柱(Q Sepharose Fast Flow，Cytiva，17051001)进行纯化，100mM NaCl洗脱目的蛋白。SDS-PAGE胶图如图14A至图14C。将得到的目的蛋白透析至PBS缓冲液并置于-20℃保存。The amino acid mutation sites of candidate backbone molecules STFK1128 (SEQ ID NO: 15), STFK1351 (SEQ ID NO: 16) and STFK1620 (SEQ ID NO: 17) are shown in Figure 13. The sequences encoding these three candidate backbone molecules were constructed into the expression vector pGS01, followed by eukaryotic expression referring to step 1.2. After culturing for 6 days, the ExpiCHO expression cell suspension was collected, centrifuged at 12000 rpm, room temperature for 30 min, and the supernatant was collected and filtered through a 0.22 μm filter membrane. The supernatant sample was then purified using a Q-FF chromatography column (Q Sepharose Fast Flow, Cytiva, 17051001), and the target protein was eluted with 100 mM NaCl. SDS-PAGE gel pictures are shown in Figure 14A to Figure 14C. The obtained target protein was dialyzed into PBS buffer and stored at -20°C.
使用8-14周龄的SPF级雄性和雌性仓鼠(体重范围：100-130g)，所有实验中每组仓鼠雌性和雄性数量均相等。免疫方式为肌肉注射，注射体积为200μL，即于动物双后肢腓肠肌各注射100μL。免疫程序分别为0/3周免疫，注射剂量为10μg。在第二剂免疫两周后采集眼眶静脉血用于抗体滴度检测。分别在仓鼠中接种三种候选变异株STFK骨架抗原制备的疫苗，检测仓鼠接种后第二针免疫后2周对多种新冠病毒变异株的中和抗体水平，评估三种候选骨架分子分子的免疫原性。免疫后血清使用慢 病毒(Lentivirus)的新冠假病毒(Lvpp)-细胞中和试验检测免疫小鼠血清的中和抗体滴度，具体实验步骤和检测方法参考新冠假病毒(Lvpp)-细胞中和试验检测方法报告进行(Small Methods.2021 Feb 15；5(2):2001031.)。仓鼠产生的针对SARS-CoV-2多种变异株(包括：D614G、Alpha株、Delta株、Epsilon株、Lambda株、Kappa株、Iota_477N株、Iota_484K株、Eta株、Gamma株、A.VOI.V2株、Beta株、B.1.620株)的中和抗体滴度如图15A至图15C所示。STFK1351、STFK1128和STFK1620在仓鼠中诱导的中和抗体与原型株的STFK疫苗相比均具有一定的互补性，但从免疫原性上来看，STFK1128与STFK1620诱导的抗体滴度相比STFK1351更高。因此，本发明人选择STFK1128和STFK1620作为抗原骨架进行进一步改造。SPF grade male and female hamsters (body weight range: 100-130 g) aged 8-14 weeks were used, with equal numbers of females and males per group in all experiments. The immunization method is intramuscular injection, and the injection volume is 200 μL, that is, 100 μL is injected into the gastrocnemius muscle of both hind limbs of the animal. The immunization program was 0/3 week immunization respectively, and the injection dose was 10μg. Two weeks after the second dose of immunization, orbital venous blood was collected for antibody titer detection. Hamsters were inoculated with vaccines prepared from three candidate variant strains of STFK backbone antigens, and the level of neutralizing antibodies against various new coronavirus variant strains was detected 2 weeks after the second dose of immunization in hamsters, and the immunogenicity of the three candidate backbone molecules was evaluated. Slow use of serum after immunization The new coronavirus (Lvpp)-cell neutralization test of the virus (Lentivirus) detects the neutralizing antibody titer of the immunized mouse serum, and the specific experimental steps and detection methods refer to the new coronavirus (Lvpp)-cell neutralization test method report (Small Methods.2021 Feb 15; 5(2):2001031.). The neutralizing antibody titers produced by hamsters against various mutant strains of SARS-CoV-2 (including: D614G, Alpha strain, Delta strain, Epsilon strain, Lambda strain, Kappa strain, Iota_477N strain, Iota_484K strain, Eta strain, Gamma strain, A.VOI.V2 strain, Beta strain, and B.1.620 strain) are shown in Figures 15A to 15C. The neutralizing antibodies induced by STFK1351, STFK1128 and STFK1620 in hamsters were all complementary to the prototype strain of STFK vaccine, but in terms of immunogenicity, the antibody titers induced by STFK1128 and STFK1620 were higher than those of STFK1351. Therefore, the inventors selected STFK1128 and STFK1620 as the antigen backbone for further modification.
3.2基于STFK1128改造的变异株STFK分子构建3.2 Molecular construction of mutant strain STFK based on STFK1128 transformation
以STFK1128为框架，结合陆续出现的变异株，将其中RBD区的重要突变引入STFK1128中。其中重要的氨基酸突变如下：(1)E484K，也被称为逃逸突变，该突变能够提高病毒逃逸宿主免疫的能力，对病毒的免疫原性产生影响，主要出现在Gamma变异株、Zeta变异株和Beta变异株中；(2)E484Q，能够增强病毒与受体Ace2的结合能力，也可降低疫苗免疫产生的抗体结合该突变刺突蛋白的能力，主要出现在Kappa变异株中；(3)S477N，多项研究中，研究者们利用生物信息学和统计学方法确定了aa475-aa485是SARS-CoV2的受体结合域RBD中高度灵活的区域，其中，S477位的氨基酸灵活性最高，且是迄今为止RBD中突变频率最高的残基，S477N突变能够增强病毒刺突蛋白和受体Ace2的结合能力；(4)L452R，早期主要出现在印度流行的Delta变异株和Kappa变异株中，随后传播范围扩大至世界各地。该突变能够增强病毒与受体Ace2的结合能力，也可降低疫苗免疫产生的抗体结合该突变刺突蛋白的能力，部分研究表明该突变可增强病毒对T细胞的耐受性；(5)T478K，478位残基也是病毒spike蛋白与受体Ace2相互作用的位点，该突变可增强病毒与受体Ace2的结合能力进而提高病毒感染能力。改造后的蛋白分别命名为STFK1128b、STFK1128c、STFK1128d、STFK1128e、STFK1128f、STFK1128g，氨基酸序列分别如SEQ ID NOs:18-23所示。Using STFK1128 as the framework, combined with the successively emerging mutant strains, the important mutations in the RBD region were introduced into STFK1128. The important amino acid mutations are as follows: (1) E484K, also known as escape mutation, which can improve the ability of the virus to escape host immunity and affect the immunogenicity of the virus, mainly appearing in Gamma mutants, Zeta mutants and Beta mutants; (2) E484Q, which can enhance the binding ability of the virus to the receptor Ace2, and can also reduce the ability of antibodies produced by vaccine immunization to bind to the mutant spike protein, mainly appearing in Kappa mutants; (3) S477N, In a number of studies, the researchers used bioinformatics and statistical methods to determine that aa475-aa485 is a highly flexible region in the receptor binding domain RBD of SARS-CoV2. Among them, the S477 amino acid has the highest flexibility and is the residue with the highest mutation frequency in the RBD so far. The S477N mutation can enhance the binding ability of the virus spike protein and the receptor Ace2; Expanded to all over the world. This mutation can enhance the binding ability of the virus to the receptor Ace2, and can also reduce the ability of the antibody produced by vaccine immunization to bind to the mutant spike protein. Some studies have shown that this mutation can enhance the tolerance of the virus to T cells; (5) T478K, the 478th residue is also the site of interaction between the viral spike protein and the receptor Ace2. The transformed proteins were named STFK1128b, STFK1128c, STFK1128d, STFK1128e, STFK1128f, STFK1128g, respectively, and the amino acid sequences were shown in SEQ ID NOs: 18-23.
改造后的STFK1128b至STFK1128g蛋白质粒参照步骤1.2进行真核表达，培养6天后，收集ExpiCHO表达细胞悬液，12000rpm，室温离心30min，留取上清经0.22μm滤膜过滤。将上清样品后使用Q-FF层析柱(Q Sepharose Fast Flow，Cytiva，17051001)进行纯化，100mM NaCl洗脱目的蛋白。SDS-PAGE胶图如图16A至图 16F。将得到的目的蛋白透析至PBS缓冲液并置于-20℃保存。The transformed STFK1128b to STFK1128g protein particles were eukaryotically expressed according to step 1.2. After 6 days of culture, the ExpiCHO expression cell suspension was collected, centrifuged at 12,000 rpm for 30 min at room temperature, and the supernatant was collected and filtered through a 0.22 μm filter membrane. The supernatant sample was then purified using a Q-FF chromatography column (Q Sepharose Fast Flow, Cytiva, 17051001), and the target protein was eluted with 100 mM NaCl. SDS-PAGE gel map is shown in Fig. 16A to Fig. 16F. The obtained target protein was dialyzed into PBS buffer and stored at -20°C.
对6种改造后的STFK1128b至STFK1128g抗原在仓鼠中进行免疫和抗体分析。仓鼠的免疫和中和抗体检测按步骤3.1中所述进行操作。中和抗体结果如图17A至图17F所示：改造后抗原在仓鼠中均诱导出高水平的对Gamma株和Beta株的中和抗体，相比原型株的STFK(STFK-MR)显著更高。但是，仍然对A.VOI.V2株和B.1.620株仅有低水平的中和活性，与原型株的STFK相当。因此，可考虑进一步在STFK1128的基础上嵌合B.1.620株spike中的相关突变位点。Immunization and antibody analysis of the six modified STFK1128b to STFK1128g antigens were performed in hamsters. Immunization of hamsters and detection of neutralizing antibodies were performed as described in step 3.1. The results of neutralizing antibodies are shown in Figures 17A to 17F: the modified antigens induced high levels of neutralizing antibodies to Gamma and Beta strains in hamsters, which were significantly higher than the STFK (STFK-MR) of the prototype strain. However, there is still only a low level of neutralizing activity against A.VOI.V2 strain and B.1.620 strain, which is comparable to the STFK of the prototype strain. Therefore, it may be considered to further chimerize relevant mutation sites in B.1.620 strain spike on the basis of STFK1128.
3.3基于STFK1128、STFK1620和STFK1351改造的变异株STFK分子构建3.3 Molecular construction of mutant strain STFK based on STFK1128, STFK1620 and STFK1351 transformation
以STFK1128、STFK1620和STFK1351为骨架进一步构建STFK变异株分子。STFK1628x由STFK1620的NTD、STFK1128的RBD和S2、并引入L452R和T478K组成。STFK1628y由在STFK1628x中引入N440K组成。STFK1628z由在STFK1628x中引入G446V组成。STFK1328x由STFK1351的NTD、STFK1128的RBD和S2、并引入K417N、L452R和T478K组成。改造后的蛋白表达质粒参照步骤3.1中的方法进行表达和纯化。SDS-PAGE胶图如图18A至图18C。将得到的目的蛋白透析至PBS缓冲液并置于-20℃保存。Using STFK1128, STFK1620 and STFK1351 as the backbone to further construct the STFK mutant strain molecule. STFK1628x consists of NTD of STFK1620, RBD and S2 of STFK1128, and introduces L452R and T478K. STFK1628y consists of the introduction of N440K in STFK1628x. STFK1628z consists of introducing G446V in STFK1628x. STFK1328x consists of the NTD of STFK1351, the RBD and S2 of STFK1128, and introduces K417N, L452R and T478K. The modified protein expression plasmid was expressed and purified according to the method in step 3.1. SDS-PAGE gel pictures are shown in Figure 18A to Figure 18C. The obtained target protein was dialyzed into PBS buffer and stored at -20°C.
参考步骤3.1中所述的方法将上述改造后的抗原分别进行仓鼠免疫，和原型株分子STFK在仓鼠中的免疫中和抗体分析结果如图19A至图19D，图20A至图20B所示。与STFK1128相比，改造后的分子STFK1328x(SEQ ID NO:24)、STFK1628x(SEQ ID NO:25)、STFK1628y(SEQ ID NO:26)和STFK1628z(SEQ ID NO:27)仍然保持对Beta株和Gamma株的高滴度中和抗体，同时增强了对B.1.620和A.VOI.V2的中和抗体滴度。与原型株的STFK相比，改造后的抗原诱导的中和抗体能够很好的覆盖对原型株的STFK逃逸显著的变异株病毒。Referring to the method described in step 3.1, the modified antigens above were immunized with hamsters respectively, and the results of immune neutralizing antibody analysis of the prototype strain molecule STFK in hamsters are shown in Figure 19A to Figure 19D, and Figure 20A to Figure 20B. Compared with STFK1128, the modified molecules STFK1328x (SEQ ID NO: 24), STFK1628x (SEQ ID NO: 25), STFK1628y (SEQ ID NO: 26) and STFK1628z (SEQ ID NO: 27) still maintain high-titer neutralizing antibodies against Beta strains and Gamma strains, while enhancing the anti-B.1.6 20 and the neutralizing antibody titer of A.VOI.V2. Compared with the STFK of the prototype strain, the neutralizing antibody induced by the modified antigen can well cover the mutant virus that escapes significantly from the STFK of the prototype strain.
此外，STFK1628y还诱导产生了高滴度针对多种Omicron株(包括BA.1、BA.2、BA.2.12.1、BA.2.75以及BA.4/5)的中和抗体，这些变异株病毒对原型株疫苗诱导的抗体表现出极大的免疫逃逸(图20D)。STFK1628x也诱导产生了针对Omicron株的中和抗体，但滴度低于STFK1628y，显著高于原型株的STFK(图20C和图20D)。In addition, STFK1628y also induced high titers of neutralizing antibodies against various Omicron strains (including BA.1, BA.2, BA.2.12.1, BA.2.75, and BA.4/5), and these mutant viruses showed great immune escape against the antibodies induced by the prototype strain vaccine (Fig. 20D). STFK1628x also induced neutralizing antibodies against the Omicron strain, but the titer was lower than that of STFK1628y and significantly higher than that of the prototype strain (Fig. 20C and Fig. 20D).
以上结果提示，STFK1628x和STFK1628y在免疫原型上和诱导的针对变异株的中和抗体与原型株的STFK(SEQ ID NO:14)的互补性上均具有较大优势，以STFK1628x和STFK(STFK-MR)为抗原的二价疫苗很可能能够有效诱导针对所有当前的VOC和VOI变异毒株。 The above results suggest that STFK1628x and STFK1628y have great advantages in the immune prototype and in the complementarity between the induced neutralizing antibody against the mutant strain and the STFK (SEQ ID NO: 14) of the prototype strain. The bivalent vaccine with STFK1628x and STFK (STFK-MR) as the antigen is likely to be able to effectively induce all current VOC and VOI mutant strains.
参考步骤3.1中所述的方法将STFK1628x与STFK按1:1混合制备二价疫苗进行仓鼠免疫，免疫剂量为10μg(含5μg STFK1628x和5μg STFK)，对比原型株分子STFK在仓鼠中的免疫中和抗体结合分析结果如图20E所示。二价疫苗诱导了高水平的针对包括所有VOC和VOI在内的14种主要变异株的中和抗体，能够有效覆盖当前主要的新冠病毒变异株。此外，二价疫苗诱导的中和抗体对多种Omicron变异株具有高滴度中和活性，包括BA.1、BA.2、BA.2.12.1、BA.2.75、BA.4/5，相比原型株疫苗诱导的中和抗体滴度高15-27倍(图20F)。Referring to the method described in step 3.1, STFK1628x and STFK were mixed at a ratio of 1:1 to prepare a bivalent vaccine for immunization of hamsters. The immunization dose was 10 μg (including 5 μg STFK1628x and 5 μg STFK). The results of the immune neutralizing antibody binding analysis of the prototype strain molecule STFK in hamsters were shown in Figure 20E. The bivalent vaccine induced a high level of neutralizing antibodies against 14 major variants including all VOCs and VOIs, which can effectively cover the current major variants of the new coronavirus. In addition, the neutralizing antibody induced by the bivalent vaccine had high-titer neutralizing activity against various Omicron variants, including BA.1, BA.2, BA.2.12.1, BA.2.75, and BA.4/5, which were 15-27 times higher than the neutralizing antibody titer induced by the prototype strain vaccine (Fig. 20F).
3.4 STFK1628x和二价疫苗在仓鼠攻毒模型中的保护效果3.4 Protective effect of STFK1628x and bivalent vaccine in hamster challenge model
为了评估STFK1628x和二价疫苗在仓鼠攻毒模型中的保护效果，用SARS-CoV-2原型株、Beta株和Omicron BA.1进行滴鼻攻毒疫苗接种后的仓鼠(图21A)。在第0周和第3周仓鼠分别接种STFK、STFK1628x和二价疫苗两剂。在加强免疫后两周，对仓鼠进行滴鼻攻毒104PFU SARS-CoV-2原型株或Beta株。To evaluate the protective effect of STFK1628x and bivalent vaccine in hamster challenge model, hamsters after intranasal challenge vaccination were administered with SARS-CoV-2 prototype strain, Beta strain and Omicron BA.1 (Fig. 21A). Hamsters were inoculated with two doses of STFK, STFK1628x and bivalent vaccine at week 0 and week 3, respectively. Two weeks after the booster immunization, the hamsters were challenged nasally with 10 4 PFU of SARS-CoV-2 prototype or Beta strain.
在原型株攻毒实验中，所有接种疫苗的仓鼠在攻毒后的7天内均未出现体重减轻(图21B)，并且没有出现动物死亡(图21C)。同时，未接种疫苗的对照仓鼠体重最大下降了14.8％，并且其中一只动物在感染后第五天死亡。对所有仓鼠在感染后第7天安乐死，并收集肺组织用于病毒RNA定量分析和病理检测。结果显示，对照仓鼠肺中病毒RNA载量的中位数为7.26log10 copies mL-1(图21D)。与对照组相比，二价疫苗免疫的仓鼠肺组织中病毒RNA载量降低倍数超过100000倍，病毒RNA载量的中位数为1.76log10 copies mL-1。接种STFK或STFK 1628x疫苗的仓鼠也显示出类似的病毒RNA载量下降。与病毒RNA载量减少的结果一致，STFK、STFK1628x和二价疫苗完全保护仓鼠免受原型病毒感染引起的肺部疾病，病理评分显著低于对照组仓鼠(P<0.01，图21E)。而未接种疫苗的对照动物在肺部出现严重疾病，包括(i)肺泡隔膜增厚和实变；(ii)出血、渗出、肺水肿和粘液；(iii)炎症免疫细胞的募集和浸润。以上实验结果证明了STFK1628x和二价疫苗均能够显著降低呼吸道组织中的病毒载量，完全保护新冠病毒原型株感染导致的疾病，保护效果与原型株疫苗接种的动物相当。In the prototype strain challenge experiment, all vaccinated hamsters showed no weight loss within 7 days after challenge ( FIG. 21B ), and no animals died ( FIG. 21C ). Meanwhile, unvaccinated control hamsters lost the most weight of 14.8 percent, and one of the animals died by day five after infection. All hamsters were euthanized on day 7 after infection, and lung tissues were collected for quantitative analysis of viral RNA and pathological detection. The results showed that the median viral RNA load in the lungs of control hamsters was 7.26 log 10 copies mL −1 ( FIG. 21D ). Compared with the control group, the viral RNA load in the lung tissue of hamsters immunized with the bivalent vaccine was reduced by more than 100,000 times, and the median viral RNA load was 1.76log 10 copies mL -1 . Hamsters vaccinated with either STFK or STFK 1628x vaccines also showed similar reductions in viral RNA load. Consistent with the results of viral RNA load reduction, STFK, STFK1628x and bivalent vaccines completely protected hamsters from lung disease caused by prototype virus infection, and the pathological score was significantly lower than that of control hamsters (P<0.01, Figure 21E). Unvaccinated control animals developed severe disease in the lungs, including (i) alveolar septum thickening and consolidation; (ii) hemorrhage, exudation, pulmonary edema, and mucus; and (iii) recruitment and infiltration of inflammatory immune cells. The above experimental results prove that both STFK1628x and the bivalent vaccine can significantly reduce the viral load in respiratory tissues, completely protect the disease caused by the prototype strain of the new coronavirus, and the protective effect is equivalent to that of animals vaccinated with the prototype strain.
在Beta株攻毒实验中，未接种疫苗的对照组在感染后第7天时体重下降了13.8％(图21F)，并且超过一半的仓鼠死亡(5/8，图21G)。而接种STFK1628x或二价疫苗的仓鼠则完全未出现体重下降，且所有动物均存活(图21G)。与对照相比，接种STFK1628x或二价疫苗的仓鼠肺组织中病毒RNA载量减少了6-7log10(图21H)。 更为重要的是，STFK1628x和二价疫苗均完全保护所有仓鼠免受Beta株感染引起的肺部损伤(图21I)。尽管基于原型株Spike的STFK疫苗接种也可以显著降低仓鼠肺部的病毒RNA载量，但载量仍显著高于接种STFK1628x和二价疫苗的仓鼠(图21H)。而且，STFK疫苗接种组中的一只仓鼠在肺部出现中度病理损伤(图21I)。这些证据表明，基于原型株的STKF疫苗无法完全保护Beta株的攻毒，而STFK1628x和二价疫苗可以在仓鼠模型中完全保护SARS-CoV-2原型株和Beta株的攻毒。In the Beta strain challenge experiment, the unvaccinated control group lost 13.8% of body weight on day 7 after infection (Fig. 21F), and more than half of the hamsters died (5/8, Fig. 21G). In contrast, hamsters vaccinated with STFK1628x or the bivalent vaccine showed no weight loss at all, and all animals survived ( FIG. 21G ). Viral RNA load was reduced by 6-7 log 10 in lung tissue of hamsters vaccinated with STFK1628x or the bivalent vaccine compared to controls ( FIG. 21H ). More importantly, both STFK1628x and the bivalent vaccine completely protected all hamsters from lung injury induced by Beta strain infection (Fig. 21I). Although STFK vaccination based on the prototype strain Spike could also significantly reduce the viral RNA load in the lungs of hamsters, the load was still significantly higher than that of hamsters vaccinated with STFK1628x and bivalent vaccine ( FIG. 21H ). Also, one hamster in the STFK vaccinated group developed moderate pathological lesions in the lungs (Fig. 21I). These evidences suggest that the prototype-based STKF vaccine cannot fully protect against Beta strain challenge, whereas STFK1628x and bivalent vaccines can fully protect both SARS-CoV-2 prototype and Beta strain challenges in a hamster model.
在Omciron BA.1攻毒实验中，未接种疫苗的对照组仓鼠显示出轻度体重下降，在感染后第5天平均体重下降5.5％(图21J)，所有动物均存活(图21K)。而接种STFK1628x、STFK1628y或二价疫苗的仓鼠在感染后第5天平均体重增加1.6-2.4％(图21J)，所有动物均存活(图21K)。感染后第5天，未接种疫苗仓鼠肺组织病毒RNA载量中位数为6.25log10 copies mL-1，接种STFK1628x、STFK1628y和二价疫苗的仓鼠肺组织中病毒RNA载量分别为1.93log10 copies mL-1、1.46log10 copies mL-1、1.55log10 copies mL-1，相比未接种疫苗对照组和STFK疫苗均显著下降(图21L)。实验终点仓鼠的肺组织切片HE染色结果显示，接种疫苗的仓鼠的病理严重程度评分显著低于对照组(图21M)。这些证据表明，STFK1628x、STFK1628y和二价疫苗可以在仓鼠模型中高效保护SARS-CoV-2 Omicron BA.1的攻毒，且保护效果优于基于原型株的STFK。In the Omciron BA.1 challenge experiment, unvaccinated control hamsters showed mild weight loss, with an average weight loss of 5.5% on day 5 after infection (FIG. 21J), and all animals survived (FIG. 21K). While hamsters vaccinated with STFK1628x, STFK1628y or bivalent vaccines had an average body weight gain of 1.6-2.4% on day 5 post infection (FIG. 21J), all animals survived (FIG. 21K). On the 5th day after infection, the median viral RNA load in the lung tissue of unvaccinated hamsters was 6.25log 10 copies mL -1 , and the viral RNA load in the lung tissues of hamsters vaccinated with STFK1628x, STFK1628y and bivalent vaccine were 1.93log 10 copies mL -1 , 1.46log 10 copies mL -1 , and 1.55log 10 copies mL -1 respectively , significantly decreased compared to the unvaccinated control group and the STFK vaccine (Fig. 21L). The HE staining results of the lung tissue sections of the hamsters at the end of the experiment showed that the pathological severity score of the vaccinated hamsters was significantly lower than that of the control group ( FIG. 21M ). These evidences suggest that STFK1628x, STFK1628y and bivalent vaccines can efficiently protect against SARS-CoV-2 Omicron BA.1 challenge in the hamster model, and the protective effect is better than STFK based on the prototype strain.
尽管本发明的具体实施方式已经得到详细的描述，本领域技术人员将会理解。根据已经公开的所有教导，可以对那些细节进行各种修改和替换，这些改变均在本发明的保护范围之内。本发明的全部范围由所附权利要求及其任何等同物给出。 Although specific embodiments of the present invention have been described in detail, those skilled in the art will understand. Based on all the teachings that have been disclosed, various modifications and substitutions can be made to those details, and these changes are all within the scope of the invention. The full scope of the invention is given by the appended claims and any equivalents thereof.

Claims (15)

  1. 一种分离的多肽，其氨基酸序列如SEQ ID NOs:1-27中的任一序列所示。An isolated polypeptide whose amino acid sequence is shown in any one of SEQ ID NOs: 1-27.
  2. 一种分离的融合蛋白，其包含至少一种权利要求1所述的多肽。An isolated fusion protein comprising at least one polypeptide of claim 1.
  3. 一种分离的多核苷酸，其编码权利要求1所述的多肽。An isolated polynucleotide encoding the polypeptide of claim 1.
  4. 一种核酸构建体，其含有权利要求3所述的多核苷酸；优选地，所述核酸构建体为重组载体；优选地，所述核酸构建体为重组表达载体。A nucleic acid construct comprising the polynucleotide according to claim 3; preferably, the nucleic acid construct is a recombinant vector; preferably, the nucleic acid construct is a recombinant expression vector.
  5. 一种转化的细胞，其含有权利要求3所述的多核苷酸，或者权利要求4所述的核酸构建体。A transformed cell comprising the polynucleotide of claim 3, or the nucleic acid construct of claim 4.
  6. 一种药物组合物，其含有至少一种权利要求1所述的多肽；可选地，其还包含药学上可接受的辅料；A pharmaceutical composition, which contains at least one polypeptide according to claim 1; optionally, it also includes pharmaceutically acceptable excipients;
    优选地，所述药物组合物为疫苗制剂；Preferably, the pharmaceutical composition is a vaccine preparation;
    优选地，所述药物组合物的单位剂量为0.01-100μg，优选0.1-50μg，更优选为5-30μg、5-20μg或5-15μg，特别优选为10μg；Preferably, the unit dose of the pharmaceutical composition is 0.01-100 μg, preferably 0.1-50 μg, more preferably 5-30 μg, 5-20 μg or 5-15 μg, especially preferably 10 μg;
    优选地，所述辅料为疫苗佐剂；优选地，所述疫苗佐剂为FH002C佐剂或铝佐剂。Preferably, the adjuvant is a vaccine adjuvant; preferably, the vaccine adjuvant is FH002C adjuvant or aluminum adjuvant.
  7. 根据权利要求6所述的药物组合物，其包含第一多肽成分和/或第二多肽成分，其中：The pharmaceutical composition according to claim 6, comprising the first polypeptide component and/or the second polypeptide component, wherein:
    所述第一多肽成分选自SEQ ID NOs:1-14中的任一序列所示多肽中的一种或多种，和/或所述第二多肽成分选自SEQ ID NOs:15-27中的任一序列所示多肽中的一种或多种；The first polypeptide component is selected from one or more of the polypeptides shown in any sequence of SEQ ID NOs: 1-14, and/or the second polypeptide component is selected from one or more of the polypeptides shown in any sequence of SEQ ID NOs: 15-27;
    优选地，所述第一多肽成分选自SEQ ID NOs:9-14中的任一序列所示多肽中的一种或多种，和/或所述第二多肽成分选自SEQ ID NOs:24-27中的任一序列所示多肽中的一种或多种；Preferably, the first polypeptide component is selected from one or more of the polypeptides shown in any sequence of SEQ ID NOs:9-14, and/or the second polypeptide component is selected from one or more of the polypeptides shown in any sequence of SEQ ID NOs:24-27;
    优选地，所述第一多肽成分的氨基酸序列如SEQ ID NO:9所示，和/或所述第二 多肽成分选自SEQ ID NOs:24-27中的任一序列所示多肽中的一种、两种、三种或者四种；Preferably, the amino acid sequence of the first polypeptide component is shown in SEQ ID NO: 9, and/or the second The polypeptide component is selected from one, two, three or four of the polypeptides shown in any sequence of SEQ ID NOs:24-27;
    优选地，所述第一多肽成分的氨基酸序列如SEQ ID NO:10所示，和/或所述第二多肽成分选自SEQ ID NOs:24-27中的任一序列所示多肽中的一种、两种、三种或者四种；Preferably, the amino acid sequence of the first polypeptide component is shown in SEQ ID NO: 10, and/or the second polypeptide component is selected from one, two, three or four of the polypeptides shown in any sequence of SEQ ID NOs: 24-27;
    优选地，所述第一多肽成分的氨基酸序列如SEQ ID NO:11所示，和/或所述第二多肽成分选自SEQ ID NOs:24-27中的任一序列所示多肽中的一种、两种、三种或者四种；Preferably, the amino acid sequence of the first polypeptide component is shown in SEQ ID NO: 11, and/or the second polypeptide component is selected from one, two, three or four of the polypeptides shown in any sequence of SEQ ID NOs: 24-27;
    优选地，所述第一多肽成分的氨基酸序列如SEQ ID NO:12所示，和/或所述第二多肽成分选自SEQ ID NOs:24-27中的任一序列所示多肽中的一种、两种、三种或者四种；Preferably, the amino acid sequence of the first polypeptide component is shown in SEQ ID NO: 12, and/or the second polypeptide component is selected from one, two, three or four of the polypeptides shown in any sequence of SEQ ID NOs: 24-27;
    优选地，所述第一多肽成分的氨基酸序列如SEQ ID NO:13所示，和/或所述第二多肽成分选自SEQ ID NOs:24-27中的任一序列所示多肽中的一种、两种、三种或者四种；Preferably, the amino acid sequence of the first polypeptide component is shown in SEQ ID NO: 13, and/or the second polypeptide component is selected from one, two, three or four of the polypeptides shown in any sequence of SEQ ID NOs: 24-27;
    优选地，所述第一多肽成分的氨基酸序列如SEQ ID NO:14所示，和/或所述第二多肽成分选自SEQ ID NOs:24-27中的任一序列所示多肽中的一种、两种、三种或者四种；Preferably, the amino acid sequence of the first polypeptide component is shown in SEQ ID NO: 14, and/or the second polypeptide component is selected from one, two, three or four of the polypeptides shown in any sequence of SEQ ID NOs: 24-27;
    优选地，所述第二多肽成分的氨基酸序列如SEQ ID NO:24所示，和/或所述第一多肽成分选自SEQ ID NOs:9-14中的任一序列所示多肽中的一种、两种、三种、四种、五种或六种；Preferably, the amino acid sequence of the second polypeptide component is shown in SEQ ID NO:24, and/or the first polypeptide component is selected from one, two, three, four, five or six of the polypeptides shown in any sequence of SEQ ID NOs:9-14;
    优选地，所述第二多肽成分的氨基酸序列如SEQ ID NO:25所示，和/或所述第一多肽成分选自SEQ ID NOs:9-14中的任一序列所示多肽中的一种、两种、三种、四种、五种或六种；Preferably, the amino acid sequence of the second polypeptide component is shown in SEQ ID NO:25, and/or the first polypeptide component is selected from one, two, three, four, five or six of the polypeptides shown in any sequence of SEQ ID NOs:9-14;
    优选地，所述第二多肽成分的氨基酸序列如SEQ ID NO:26所示，和/或所述第一多肽成分选自SEQ ID NOs:9-14中的任一序列所示多肽中的一种、两种、三种、四种、五种或六种；Preferably, the amino acid sequence of the second polypeptide component is shown in SEQ ID NO:26, and/or the first polypeptide component is selected from one, two, three, four, five or six of the polypeptides shown in any sequence of SEQ ID NOs:9-14;
    优选地，所述第二多肽成分的氨基酸序列如SEQ ID NO:27所示，和/或所述第一多肽成分选自SEQ ID NOs:9-14中的任一序列所示多肽中的一种、两种、三种、四种、五种或六种；Preferably, the amino acid sequence of the second polypeptide component is shown in SEQ ID NO:27, and/or the first polypeptide component is selected from one, two, three, four, five or six of the polypeptides shown in any sequence of SEQ ID NOs:9-14;
    优选地，所述第一多肽成分的氨基酸序列如SEQ ID NO:14所示，和/或所述第二 多肽成分的氨基酸序列如SEQ ID NO:24所示；Preferably, the amino acid sequence of the first polypeptide component is shown in SEQ ID NO: 14, and/or the second The amino acid sequence of the polypeptide component is shown in SEQ ID NO: 24;
    优选地，所述第一多肽成分的氨基酸序列如SEQ ID NO:14所示，和/或所述第二多肽成分的氨基酸序列如SEQ ID NO:25所示；Preferably, the amino acid sequence of the first polypeptide component is shown in SEQ ID NO: 14, and/or the amino acid sequence of the second polypeptide component is shown in SEQ ID NO: 25;
    优选地，所述第一多肽成分的氨基酸序列如SEQ ID NO:14所示，和/或所述第二多肽成分的氨基酸序列如SEQ ID NO:26所示；Preferably, the amino acid sequence of the first polypeptide component is shown in SEQ ID NO: 14, and/or the amino acid sequence of the second polypeptide component is shown in SEQ ID NO: 26;
    优选地，所述第一多肽成分的氨基酸序列如SEQ ID NO:14所示，和/或所述第二多肽成分的氨基酸序列如SEQ ID NO:27所示；Preferably, the amino acid sequence of the first polypeptide component is shown in SEQ ID NO: 14, and/or the amino acid sequence of the second polypeptide component is shown in SEQ ID NO: 27;
    优选地，所述第一多肽成分的氨基酸序列如SEQ ID NO:14所示，和/或所述第二多肽成分为SEQ ID NO:24和SEQ ID NO:25所示的多肽；Preferably, the amino acid sequence of the first polypeptide component is shown in SEQ ID NO: 14, and/or the second polypeptide component is the polypeptide shown in SEQ ID NO: 24 and SEQ ID NO: 25;
    优选地，所述第一多肽成分的氨基酸序列如SEQ ID NO:14所示，和/或所述第二多肽成分为SEQ ID NO:24和SEQ ID NO:26所示的多肽；Preferably, the amino acid sequence of the first polypeptide component is shown in SEQ ID NO: 14, and/or the second polypeptide component is the polypeptide shown in SEQ ID NO: 24 and SEQ ID NO: 26;
    优选地，所述第一多肽成分的氨基酸序列如SEQ ID NO:14所示，和/或所述第二多肽成分为SEQ ID NO:24和SEQ ID NO:27所示的多肽；Preferably, the amino acid sequence of the first polypeptide component is shown in SEQ ID NO: 14, and/or the second polypeptide component is the polypeptide shown in SEQ ID NO: 24 and SEQ ID NO: 27;
    优选地，所述第一多肽成分的氨基酸序列如SEQ ID NO:14所示，和/或所述第二多肽成分为SEQ ID NO:25和SEQ ID NO:26所示的多肽；Preferably, the amino acid sequence of the first polypeptide component is shown in SEQ ID NO: 14, and/or the second polypeptide component is the polypeptide shown in SEQ ID NO: 25 and SEQ ID NO: 26;
    优选地，所述第一多肽成分的氨基酸序列如SEQ ID NO:14所示，和/或所述第二多肽成分为SEQ ID NO:25和SEQ ID NO:27所示的多肽；Preferably, the amino acid sequence of the first polypeptide component is shown in SEQ ID NO: 14, and/or the second polypeptide component is the polypeptide shown in SEQ ID NO: 25 and SEQ ID NO: 27;
    优选地，所述第一多肽成分的氨基酸序列如SEQ ID NO:14所示，和/或所述第二多肽成分为SEQ ID NO:26和SEQ ID NO:27所示的多肽；Preferably, the amino acid sequence of the first polypeptide component is shown in SEQ ID NO: 14, and/or the second polypeptide component is the polypeptide shown in SEQ ID NO: 26 and SEQ ID NO: 27;
    优选地，所述第一多肽成分的氨基酸序列如SEQ ID NO:14所示，和/或所述第二多肽成分为SEQ ID NO:24、SEQ ID NO:25和SEQ ID NO:26所示的多肽；Preferably, the amino acid sequence of the first polypeptide component is shown in SEQ ID NO: 14, and/or the second polypeptide component is the polypeptide shown in SEQ ID NO: 24, SEQ ID NO: 25 and SEQ ID NO: 26;
    优选地，所述第一多肽成分的氨基酸序列如SEQ ID NO:14所示，和/或所述第二多肽成分为SEQ ID NO:24、SEQ ID NO:25和SEQ ID NO:27所示的多肽；Preferably, the amino acid sequence of the first polypeptide component is shown in SEQ ID NO: 14, and/or the second polypeptide component is the polypeptide shown in SEQ ID NO: 24, SEQ ID NO: 25 and SEQ ID NO: 27;
    优选地，所述第一多肽成分的氨基酸序列如SEQ ID NO:14所示，和/或所述第二多肽成分为SEQ ID NO:24、SEQ ID NO:26和SEQ ID NO:27所示的多肽；Preferably, the amino acid sequence of the first polypeptide component is shown in SEQ ID NO: 14, and/or the second polypeptide component is the polypeptide shown in SEQ ID NO: 24, SEQ ID NO: 26 and SEQ ID NO: 27;
    优选地，所述第一多肽成分的氨基酸序列如SEQ ID NO:14所示，和/或所述第二多肽成分为SEQ ID NO:25、SEQ ID NO:26和SEQ ID NO:27所示的多肽；Preferably, the amino acid sequence of the first polypeptide component is shown in SEQ ID NO: 14, and/or the second polypeptide component is the polypeptide shown in SEQ ID NO: 25, SEQ ID NO: 26 and SEQ ID NO: 27;
    优选地，所述第一多肽成分的氨基酸序列如SEQ ID NO:14所示，和/或所述第二多肽成分为SEQ ID NO:24、SEQ ID NO:25、SEQ ID NO:26和SEQ ID NO:27所示的多肽。 Preferably, the amino acid sequence of the first polypeptide component is shown in SEQ ID NO:14, and/or the second polypeptide component is the polypeptides shown in SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26 and SEQ ID NO:27.
  8. 根据权利要求7所述的药物组合物，其中，第一多肽成分和第二多肽成分的质量比为(1：10)至(10：1)、(1：5)至(5：1)、(1：3)至(3：1)、(1：2)至(2：1)、(1：1.5)至(1.5：1)或者1：1；The pharmaceutical composition according to claim 7, wherein the mass ratio of the first polypeptide component to the second polypeptide component is (1:10) to (10:1), (1:5) to (5:1), (1:3) to (3:1), (1:2) to (2:1), (1:1.5) to (1.5:1) or 1:1;
    优选地，所述药物组合物的活性成分由第一多肽成分和第二多肽成分组成。Preferably, the active ingredient of the pharmaceutical composition consists of the first polypeptide component and the second polypeptide component.
  9. 根据权利要求7至8中任一权利要求所述的药物组合物，其中：The pharmaceutical composition according to any one of claims 7 to 8, wherein:
    当所述第一多肽成分包含一种以上多肽时，各多肽两两之间的质量比为1：1；和/或When the first polypeptide component comprises more than one polypeptide, the mass ratio between each polypeptide is 1:1; and/or
    当所述第二多肽成分包含一种以上多肽时，各多肽两两之间的质量比为1：1。When the second polypeptide component comprises more than one polypeptide, the mass ratio between two polypeptides is 1:1.
  10. 权利要求1所述的多肽、权利要求2所述的融合蛋白、权利要求3所述的多核苷酸或者权利要求4所述的核酸构建体在制备抗SARS-CoV-2的药物中的用途；Use of the polypeptide according to claim 1, the fusion protein according to claim 2, the polynucleotide according to claim 3 or the nucleic acid construct according to claim 4 in the preparation of anti-SARS-CoV-2 medicaments;
    优选地，所述SARS-CoV-2选自SARS-CoV-2原型毒株或SARS-CoV-2变异毒株；优选地，所述变异毒株选自如下的一种或多种：Preferably, the SARS-CoV-2 is selected from a prototype strain of SARS-CoV-2 or a variant strain of SARS-CoV-2; preferably, the variant strain is selected from one or more of the following:
    Alpha毒株、Gamma毒株、Beta毒株、Iota_S477N毒株、Iota_E484K毒株、Epsilon毒株、Eta毒株、Kappa毒株、Delta毒株、Lambda毒株和Omicron毒株；Alpha strain, Gamma strain, Beta strain, Iota_S477N strain, Iota_E484K strain, Epsilon strain, Eta strain, Kappa strain, Delta strain, Lambda strain and Omicron strain;
    优选地，所述Omicron毒株为选自如下的一种或多种：Preferably, the Omicron strain is one or more selected from the following:
    BA.1、BA.2、BA.2.12.1、BA.2.75、BA.4和BA.5；BA.1, BA.2, BA.2.12.1, BA.2.75, BA.4 and BA.5;
    优选地，所述药物为权利要求6至9中任一权利要求所述的药物组合物。Preferably, the drug is the pharmaceutical composition according to any one of claims 6-9.
  11. 权利要求1所述的多肽、权利要求2所述的融合蛋白、权利要求3所述的多核苷酸或者权利要求4所述的核酸构建体在制备治疗或预防COVID-19或其所致症状的药物中的用途；Use of the polypeptide according to claim 1, the fusion protein according to claim 2, the polynucleotide according to claim 3 or the nucleic acid construct according to claim 4 in the preparation of medicines for the treatment or prevention of COVID-19 or the symptoms caused by it;
    优选地，所述药物为权利要求6至9中任一权利要求所述的药物组合物。Preferably, the drug is the pharmaceutical composition according to any one of claims 6-9.
  12. 根据权利要求1所述的多肽、权利要求2所述的融合蛋白、权利要求3所述的多核苷酸或者权利要求4所述的核酸构建体，其用于抗SARS-CoV-2；The polypeptide according to claim 1, the fusion protein according to claim 2, the polynucleotide according to claim 3 or the nucleic acid construct according to claim 4, which are used for anti-SARS-CoV-2;
    优选地，所述SARS-CoV-2选自SARS-CoV-2原型毒株或SARS-CoV-2变异毒株；优选地，所述变异毒株选自如下的一种或多种： Preferably, the SARS-CoV-2 is selected from a prototype strain of SARS-CoV-2 or a variant strain of SARS-CoV-2; preferably, the variant strain is selected from one or more of the following:
    Alpha毒株、Gamma毒株、Beta毒株、Iota_S477N毒株、Iota_E484K毒株、Epsilon毒株、Eta毒株、Kappa毒株、Delta毒株、Lambda毒株和Omicron毒株；Alpha strain, Gamma strain, Beta strain, Iota_S477N strain, Iota_E484K strain, Epsilon strain, Eta strain, Kappa strain, Delta strain, Lambda strain and Omicron strain;
    优选地，所述Omicron毒株为选自如下的一种或多种：Preferably, the Omicron strain is one or more selected from the following:
    BA.1、BA.2、BA.2.12.1、BA.2.75、BA.4和BA.5。BA.1, BA.2, BA.2.12.1, BA.2.75, BA.4, and BA.5.
  13. 根据权利要求1所述的多肽、权利要求2所述的融合蛋白、权利要求3所述的多核苷酸或者权利要求4所述的核酸构建体，其用于治疗或预防COVID-19或其所致症状。The polypeptide according to claim 1, the fusion protein according to claim 2, the polynucleotide according to claim 3 or the nucleic acid construct according to claim 4, which are used for treating or preventing COVID-19 or the symptoms caused by it.
  14. 一种抗SARS-CoV-2的方法，包括给予有需求的受试者以有效量的权利要求1所述的多肽、权利要求2所述的融合蛋白、权利要求3所述的多核苷酸或者权利要求4所述的核酸构建体的步骤；An anti-SARS-CoV-2 method, comprising the step of administering an effective amount of the polypeptide according to claim 1, the fusion protein according to claim 2, the polynucleotide according to claim 3 or the nucleic acid construct according to claim 4 to a subject in need;
    优选地，所述SARS-CoV-2选自SARS-CoV-2原型毒株或SARS-CoV-2变异毒株；优选地，所述变异毒株选自如下的一种或多种：Preferably, the SARS-CoV-2 is selected from a prototype strain of SARS-CoV-2 or a variant strain of SARS-CoV-2; preferably, the variant strain is selected from one or more of the following:
    Alpha毒株、Gamma毒株、Beta毒株、Iota_S477N毒株、Iota_E484K毒株、Epsilon毒株、Eta毒株、Kappa毒株、Delta毒株、Lambda毒株和Omicron毒株；Alpha strain, Gamma strain, Beta strain, Iota_S477N strain, Iota_E484K strain, Epsilon strain, Eta strain, Kappa strain, Delta strain, Lambda strain and Omicron strain;
    优选地，所述Omicron毒株为选自如下的一种或多种：Preferably, the Omicron strain is one or more selected from the following:
    BA.1、BA.2、BA.2.12.1、BA.2.75、BA.4和BA.5。BA.1, BA.2, BA.2.12.1, BA.2.75, BA.4, and BA.5.
  15. 一种治疗或预防COVID-19或其所致症状的方法，包括给予有需求的受试者以有效量的权利要求1所述的多肽、权利要求2所述的融合蛋白、权利要求3所述的多核苷酸或者权利要求4所述的核酸构建体的步骤。 A method for treating or preventing COVID-19 or the symptoms caused by it, comprising the step of administering an effective amount of the polypeptide according to claim 1, the fusion protein according to claim 2, the polynucleotide according to claim 3 or the nucleic acid construct according to claim 4 to a subject in need.
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US10953089B1 (en) * 2020-01-27 2021-03-23 Novavax, Inc. Coronavirus vaccine formulations
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CN113336832A (en) * 2020-03-02 2021-09-03 成都威斯克生物医药有限公司 Protein for resisting SARS-CoV-2 infection and vaccine containing the protein

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