WO2023025287A1 - 新型冠状病毒免疫原性物质、其制备方法和应用 - Google Patents

新型冠状病毒免疫原性物质、其制备方法和应用 Download PDF

Info

Publication number
WO2023025287A1
WO2023025287A1 PCT/CN2022/115143 CN2022115143W WO2023025287A1 WO 2023025287 A1 WO2023025287 A1 WO 2023025287A1 CN 2022115143 W CN2022115143 W CN 2022115143W WO 2023025287 A1 WO2023025287 A1 WO 2023025287A1
Authority
WO
WIPO (PCT)
Prior art keywords
seq
strain
sequence shown
novel coronavirus
amino acid
Prior art date
Application number
PCT/CN2022/115143
Other languages
English (en)
French (fr)
Inventor
胡颖嵩
于佳平
张元杰
张岭
姚文荣
吴双
袁楚晓
李娇
陈健平
刘勇
Original Assignee
江苏瑞科生物技术股份有限公司
北京安百胜生物科技有限公司
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by 江苏瑞科生物技术股份有限公司, 北京安百胜生物科技有限公司 filed Critical 江苏瑞科生物技术股份有限公司
Publication of WO2023025287A1 publication Critical patent/WO2023025287A1/zh

Links

Images

Classifications

    • 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
    • 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
    • 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
    • A61K39/39—Medicinal preparations containing antigens or antibodies characterised by the immunostimulating additives, e.g. chemical adjuvants
    • 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
    • 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
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • 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
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0681—Cells of the genital tract; Non-germinal cells from gonads
    • C12N5/0682—Cells of the female genital tract, e.g. endometrium; Non-germinal cells from ovaries, e.g. ovarian follicle cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
    • A61K2039/53—DNA (RNA) vaccination
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
    • A61K2039/55511—Organic adjuvants
    • A61K2039/55566—Emulsions, e.g. Freund's adjuvant, MF59
    • 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
    • C12N2510/00—Genetically modified cells
    • 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
    • C12N2770/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses positive-sense
    • C12N2770/00011—Details
    • C12N2770/20011—Coronaviridae
    • C12N2770/20022—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
    • 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
    • C12N2770/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses positive-sense
    • C12N2770/00011—Details
    • C12N2770/20011—Coronaviridae
    • C12N2770/20034—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein
    • 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
    • C12N2800/00—Nucleic acids vectors
    • C12N2800/10—Plasmid DNA
    • C12N2800/106—Plasmid DNA for vertebrates
    • C12N2800/107—Plasmid DNA for vertebrates for mammalian
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • the invention belongs to the technical field of biomedical engineering, and relates to a novel coronavirus immunogenic substance, its preparation method and application.
  • coronavirus disease COVID-19.
  • Common signs include fever, cough, sore throat, etc.
  • infection can lead to dyspnea, hypoxemia, acute respiratory distress syndrome and even death.
  • the new coronavirus can be transmitted from person to person through the respiratory and droplet routes, and may also be transmitted through the air and digestive tract.
  • the SARS-CoV-2 virus particle contains four structural proteins, namely spike protein (S), nucleocapsid protein (N), membrane protein (M) and envelope protein (E). Studies have found that only antibodies against S protein have neutralizing activity, so all vaccines currently under development contain S protein or its components. Among them, the receptor binding region of S protein is considered to be the most important antigen target region for inducing the body to produce neutralizing antibodies. As a vaccine, the receptor binding region can focus the neutralizing antibody stimulated by the body on the receptor binding to the virus, which can improve the immunogenicity and immune efficiency of the vaccine. SARS-CoV-2 enters cells through its receptor binding domain binding to the host cell receptor hACE2.
  • the new coronavirus continues to evolve during the transmission process, and several representative mutant strains have been detected so far. Most of the new coronavirus antigens that have been developed or are currently under development can only target one strain, and cannot produce neutralizing antibodies against different strains.
  • the prototype strain RBD dimer vaccine, the Beta strain RBD dimer vaccine and the prototype strain+Beta strain chimeric RBD dimer vaccine are respectively designed. The results showed that compared with the prototype strain RBD dimer vaccine and the Beta strain RBD dimer vaccine, the prototype strain + Beta strain chimeric RBD dimer vaccine induced a more balanced antibody response.
  • the neutralizing antibody titer of the prototype strain+Beta strain chimeric RBD dimer vaccine to the Omicron mutant S protein pseudovirus decreased significantly, which shows that the The ability of vaccines to protect against currently circulating Omicron variants is poor, so it is necessary to develop vaccines with more balanced protection against different strains.
  • the purpose of the present invention is to provide a new type of coronavirus immunogenic substance, which contains the receptor binding region of the S protein S1 subunit of different strains.
  • the new type of coronavirus immunogenic substance has higher immunogenicity and can stimulate The production of neutralizing antibodies against different strains can significantly improve the immune effect.
  • a novel coronavirus immunogenic substance which comprises a first antigen derived from an immunodominant strain and a second antigen derived from an epidemic dominant strain, each antigen comprising a receptor binding region or a receptor binding region of the S protein a part of.
  • the immunodominant strain is selected from at least one of novel coronavirus WHO1 strain and Beta strain.
  • the epidemic dominant strain is selected from at least one of the novel coronavirus Delta (Delta) strain and Omicron (Omicron) strain.
  • the Omicron strains include BA.1, BA.2, BA.3, BA.4, and BA.5 variant strains.
  • the novel coronavirus immunogenic substance further comprises a third antigen derived from strains other than the immunodominant strain and the epidemic dominant strain.
  • the novel coronavirus immunogenic substance further comprises a fourth antigen derived from strains other than the immunodominant strain and the epidemic dominant strain.
  • the strains other than the immunodominant strain and the epidemic dominant strain are selected from the following strains: Alpha (Alpha) strain, Gamma (Gamma) strain, Epsilon (Epsilon), cutoff Zeta strain, Eta strain, Theta strain, Iota strain, Kappa strain, Lambda strain, Mu strain, etc.
  • each antigen constitutes a composition, or each antigen is linked directly or through an amino acid linker, for example, the amino acid linker can be GGS or multiple GGSs in series (G and S represent glycine and serine, respectively).
  • the novel coronavirus immunogenic substance comprises a first antigen and a second antigen, and the first antigen is directly connected to the second antigen or connected through an amino acid linker.
  • the novel coronavirus immunogenic substance comprises a first antigen and a second antigen, and the first antigen and the second antigen are mixed to form a composition.
  • the novel coronavirus immunogenic substance is formed by directly linking the receptor binding region of the novel coronavirus WH01 strain S protein to the receptor binding region of the Delta strain; in some embodiments , the new coronavirus immunogenic substance is formed by directly linking the receptor binding region of the S protein of the new coronavirus WH01 strain and the receptor binding region of the Omicron strain; in some embodiments, the The novel coronavirus immunogenic substance is formed by directly connecting the receptor binding region of the new coronavirus Beta (Beta) strain S protein to the receptor binding region of the Delta strain; in some embodiments, the novel coronavirus The viral immunogenic substance is formed by directly connecting the receptor binding region of the novel coronavirus Beta S protein with the receptor binding region of the Omicron strain.
  • the novel coronavirus immunogenic substance comprises a first antigen, a second antigen and a third antigen, and the first antigen, the second antigen and the third antigen are directly connected or connected through an amino acid linker.
  • the novel coronavirus immunogenic substance comprises a first antigen, a second antigen and a third antigen, the first antigen is directly connected to the second antigen or connected through an amino acid linker to form a fusion antigen, the fusion antigen Mixed with a third antigen to form a composition.
  • the novel coronavirus immunogenic substance comprises a first antigen, a second antigen and a third antigen, and the first antigen, the second antigen and the third antigen are mixed to form a composition.
  • the novel coronavirus immunogenic substance comprises a first antigen, a second antigen, a third antigen and a fourth antigen, and the first antigen, the second antigen, the third antigen and the fourth antigen are directly linked or The fusion antigens are formed by linking through amino acid linkers.
  • the novel coronavirus immunogenic substance comprises a first antigen, a second antigen, a third antigen and a fourth antigen, the first antigen is directly connected to the second antigen or connected through an amino acid linker to form a fusion antigen, The third antigen is directly connected with the fourth antigen or connected through an amino acid linker to form a fusion antigen, and the two fusion antigens are mixed to form a composition.
  • the novel coronavirus immunogenic substance comprises a first antigen, a second antigen, a third antigen and a fourth antigen, and the first antigen, the second antigen and the third antigen are connected directly or through an amino acid linker The ligation forms a fusion antigen which is mixed with a fourth antigen to form a composition.
  • the novel coronavirus immunogenic substance comprises a first antigen, a second antigen, a third antigen and a fourth antigen, and the first antigen is directly connected to the second antigen or connected through an amino acid linker to form a fusion antigen , the fusion antigen is mixed with the third antigen and the fourth antigen to form a composition.
  • the novel coronavirus immunogenic substance comprises a first antigen, a second antigen, a third antigen and a fourth antigen, and the mixture of the first antigen, the second antigen, the third antigen and the fourth antigen constitutes combination.
  • the first, second, third and fourth are only used to represent different types of antigens, and do not represent any order among the antigens.
  • the type and quantity of antigens in the present invention are not limited, and those skilled in the art can determine the appropriate type and quantity of antigens according to widely spread strains and antigen-antibody cross-reactivity.
  • each of the antigens comprises at least 8 cysteines, and the number of cysteines is an even number.
  • the receptor binding region derived from the S protein of the WHO1 strain comprises any one of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3 or SEQ ID NO: 4. amino acid sequence.
  • the receptor binding region of the S protein derived from Beta (Beta) strain comprises any one of SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7 or SEQ ID NO:8 Amino acid sequence shown.
  • the receptor binding region of the S protein derived from Delta (Delta) strain comprises any of SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11 or SEQ ID NO:12 The amino acid sequence shown in item.
  • the receptor binding region derived from the S protein of Omicron (Omicron) BA.1 variant strain comprises the amino acid sequence shown in SEQ ID NO: 42, derived from the S protein of the BA.2 variant strain
  • the receptor binding region of the protein comprises the amino acid sequence shown in SEQ ID NO:62
  • the receptor binding region of the S protein derived from the Omicron (Omicron) BA.3 variant contains the amino acid sequence shown in SEQ ID NO:63
  • the receptor binding region derived from the S protein of Omicron (Omicron) BA.4 and BA.5 variants comprises the amino acid sequence shown in SEQ ID NO:64.
  • the novel coronavirus immunogenic substance comprises the amino acid sequence shown in any one of SEQ ID NO: 19, 20, 28, 29, 47, 48, 51-55.
  • the antigen further comprises an N-terminal domain (NTD) in the S1 subunit of the S protein.
  • NTD N-terminal domain
  • the N-terminal domain is located at the N-terminus of the amino acid sequence of the first antigen and/or the second antigen.
  • the N-terminal domain of the S protein derived from WHO1 strain comprises the amino acid sequence shown in SEQ ID NO:13.
  • the N-terminal domain of the S protein derived from Beta strain comprises the amino acid sequence shown in SEQ ID NO:14.
  • the N-terminal domain of the S protein derived from the Delta strain comprises the amino acid sequence shown in SEQ ID NO: 15.
  • the N-terminal domain of the S protein derived from Omicron (Omicron) BA.1 variant comprises the amino acid sequence shown in SEQ ID NO: 43, derived from Omicron (Omicron) BA. .2
  • the N-terminal domain of the S protein of the variant strain comprises the amino acid sequence shown in SEQ ID NO: 65
  • the N-terminal domain of the S protein derived from Omicron (Omicron) BA.3 variant comprises SEQ ID NO Amino acid sequence shown in: 66
  • the N-terminal domain of the S protein that is derived from Omicron (Omicron) BA.4 and BA.5 mutant strain comprises the aminoacid sequence shown in SEQ ID NO: 67.
  • the novel coronavirus immunogenic substance comprises the amino acid sequence shown in any one of SEQ ID NO: 22, 23, 30, 49, 50, 56-59.
  • the novel coronavirus immunogenic substance further comprises an immunoglobulin Fc domain, preferably, the immunoglobulin is human IgG.
  • the Fc domain is located at the C-terminus of the amino acid sequence of the novel coronavirus immunogenic substance, preferably, the human IgG Fc domain comprises the amino acid sequence shown in SEQ ID NO:16.
  • the novel coronavirus immunogenic substance comprises the amino acid sequence shown in SEQ ID NO:24 or SEQ ID NO:25.
  • the novel coronavirus immunogenic substance further comprises a Foldon domain.
  • the Foldon domain/protein is derived from the C-terminal of T4 bacteriophage fibrin, consists of 27 amino acids, and has the function of promoting non-covalent oligomerization of the target protein to form a trimer.
  • the Foldon domain is located at the C-terminus of the amino acid sequence of the novel coronavirus immunogenic substance, preferably, the Foldon domain comprises the amino acid sequence shown in SEQ ID NO:17.
  • the novel coronavirus immunogenic substance comprises the amino acid sequence shown in any one of SEQ ID NO:26,27,60,61.
  • the present invention also provides a method for preparing the novel coronavirus immunogenic substance, comprising the following steps:
  • the cells of the expression system were transfected with the screened recombinant expression plasmid, and the supernatant was collected and purified to obtain novel coronavirus immunogenic substances after expression.
  • the cells of the expression system include mammalian cells, insect cells, yeast cells or bacterial cells, optionally, the mammalian cells include 293T cells or CHO cells, and the bacterial cells include Escherichia coli cells .
  • the present invention also provides a nucleotide sequence encoding the immunogenic substance of the novel coronavirus, a recombinant vector comprising the above nucleotide sequence, and an expression system cell carrying the above recombinant vector.
  • the present invention also provides a novel coronavirus immunogenic substance, a nucleotide sequence encoding the novel coronavirus immunogenic substance, a recombinant vector comprising the nucleotide sequence, and a recombinant vector carrying the recombinant vector.
  • the present invention also provides a novel coronavirus protein vaccine, comprising the novel coronavirus immunogenic substance and an adjuvant.
  • the adjuvant is selected from one or more of aluminum adjuvant, MF59 adjuvant, MPL adjuvant, QS-21, GLA, CpG, AS01, AS02, AS03, AS04 adjuvant, preferably AS03 adjuvant or MF59 adjuvant.
  • the present invention also provides a novel coronavirus DNA vaccine, which comprises a DNA sequence encoding the immunogenic substance of the novel coronavirus.
  • the present invention also provides a novel coronavirus mRNA vaccine, which comprises the mRNA sequence encoding the immunogenic substance of the novel coronavirus.
  • the mRNA comprises at least one of the following sequences:
  • the novel coronavirus immunogenic substance of the present invention contains at least two antigens from immunodominant strains and epidemic dominant strains, and can induce neutralizing antibodies against different strains.
  • Immunogenic substances have high immunogenicity and good cross-protection effect, and can induce balanced neutralizing antibody levels for different strains.
  • the inventors have surprisingly found that the immunodominant strain based on the WH01 strain or Beta (Beta) strain of the new coronavirus can produce outstanding immune effects by combining with the currently popular mutant strains.
  • the WH01 strain or Beta (Beta) strain As the cornerstone of the immune combination, the strain can produce antibodies against different mutant strains to ensure the stability of immunity.
  • the recombinant protein constructed by combining WH01 strain and Omicron strain, Beta strain and Delta strain can maintain a balanced neutralizing antibody GMT level for different mutant strains, At the same time, it overcomes the immune escape of the mutant strain and produces outstanding immune effects.
  • the research of the present invention also shows that the mRNA vaccine prepared by using the mRNA encoding the immunogenic substance of the present invention also has a good immune effect on different strains.
  • the present invention also studies the combined immune effect of different adjuvants and immunogenic substances, and the results show that when the oil-in-water emulsion adjuvant (especially AS03 adjuvant) is used, a higher neutralizing antibody titer can be produced.
  • oil-in-water emulsion adjuvant especially AS03 adjuvant
  • Figure 1 is a schematic diagram of the S protein domain.
  • Figure 2 is the SDS-PAGE and Western Blot results of the antigen SEQ ID NO:18.
  • Figure 3 is the SDS-PAGE and Western Blot results of the antigen SEQ ID NO:19.
  • Figure 4 is the SDS-PAGE and Western Blot results of the antigen SEQ ID NO:20.
  • Figure 5 is the SDS-PAGE and Western Blot results of the antigen SEQ ID NO:21.
  • Figure 6 is the SDS-PAGE and Western Blot results of the antigen SEQ ID NO:22.
  • Figure 7 is the SDS-PAGE and Western Blot results of the antigen SEQ ID NO:23.
  • Figure 8 is the SDS-PAGE and Western Blot results of the antigen SEQ ID NO:26.
  • Figure 9 is the SDS-PAGE and Western Blot results of the antigen SEQ ID NO:27.
  • Figure 10 is the SDS-PAGE and Western Blot results of the antigen SEQ ID NO:28.
  • Figure 11 is the SDS-PAGE and Western Blot results of the antigen SEQ ID NO:30.
  • Figure 12 is the SDS-PAGE and Western Blot results of the antigen SEQ ID NO:44.
  • Figure 13 is the SDS-PAGE and Western Blot results of the antigen SEQ ID NO:60.
  • Figure 14 is the SDS-PAGE and Western Blot results of the antigen SEQ ID NO:61.
  • the "immunodominant strain” in the present invention refers to a mutant strain whose antigen has higher immunogenicity and can provide better cross-protection to other mutant strains compared with non-immunodominant mutant strains.
  • VOC Variants of concern
  • WHO World Health Organization
  • variants of interest can refer to the working definition of the World Health Organization (WHO), that is: SARS-CoV-2 variant strains with the following characteristics:
  • the structure of the SARS-CoV-2 S protein is shown in Figure 1, where 1-13 is the signal peptide, 14-685 is the S1 subunit, and 686-1273 is the S2 subunit.
  • the S1 subunit can be divided into NTD (14-303) and CTD (334-527). 319-541 is the receptor binding region, and 788-806 is the fusion protein. 13-1213 is the extracellular domain, 1214-1234 is the transmembrane domain, and 1235-1273 is the intracellular domain.
  • the inventors designed a variety of novel coronaviruses using the receptor binding regions of the novel coronavirus WH01 strain, Beta strain, Delta strain and Omicron variant strain. Coronavirus immunogenic substances.
  • the receptor binding region of the S protein derived from WH01 strain comprises the amino acid sequence shown in any one of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3 or SEQ ID NO:4.
  • SEQ ID NO: 1 contains 8 cysteines
  • SEQ ID NO: 2 contains an extra sequence extending outward from the N-terminus of SEQ ID NO: 1
  • SEQ ID NO: 3 Contains an additional sequence extending outward from the C-terminus of SEQ ID NO:1, containing 10 cysteines
  • SEQ ID NO:4 contains an additional sequence extending outward from the N-terminus and C-terminus of SEQ ID NO:1, respectively , containing 12 cysteines.
  • the receptor binding region of the S protein derived from the Beta strain comprises the amino acid sequence shown in any one of SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7 or SEQ ID NO:8.
  • SEQ ID NO: 5 contains 8 cysteines
  • SEQ ID NO: 6 contains an extra sequence extending outward from the N-terminus of SEQ ID NO: 5
  • SEQ ID NO: 7 Comprising an additional sequence extending outward from the C-terminus of SEQ ID NO:5, containing 10 cysteines
  • SEQ ID NO:8 comprising an additional sequence extending outward from the N-terminus and C-terminus of SEQ ID NO:5, respectively , containing 12 cysteines.
  • the receptor binding region of the S protein derived from Delta (Delta) strain comprises the amino acid sequence shown in any one of SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11 or SEQ ID NO:12 .
  • SEQ ID NO: 9 contains 8 cysteines
  • SEQ ID NO: 10 contains an additional sequence extending outward from the N-terminus of SEQ ID NO: 9, containing 10 cysteines
  • SEQ ID NO: 11 Comprising an additional sequence extending outward from the C-terminus of SEQ ID NO:9, containing 10 cysteines
  • SEQ ID NO:12 comprising an additional sequence extending outward from the N-terminus and C-terminus of SEQ ID NO:9, respectively , containing 12 cysteines.
  • the receptor binding region of the S protein derived from Omicron (Omicron) BA.1 variant strain comprises the amino acid sequence shown in SEQ ID NO: 42
  • the receptor binding region of the S protein derived from the BA.2 variant strain comprises The amino acid sequence shown in SEQ ID NO:62 is derived from the receptor binding region of the S protein of Omicron (Omicron) BA.3 variant strain, which contains the amino acid sequence shown in SEQ ID NO:63, derived from Omicron
  • the receptor binding region of the S protein of Omicron (Omicron) BA.4 and BA.5 mutant strains comprises the amino acid sequence shown in SEQ ID NO:64.
  • some immunogenic substances also include the N-terminal domain (NTD) in the S1 subunit of the S protein.
  • N-terminal domain in the S1 subunit of the WH01 strain S protein comprises the amino acid sequence shown in SEQ ID NO: 13
  • the N-terminal domain in the S1 subunit of the Beta (Beta) strain S protein comprises SEQ ID NO:
  • the amino acid sequence shown in 14 the N-terminal domain in the S1 subunit of the Delta (Delta) strain comprises the amino acid sequence shown in SEQ ID NO: 15, the S1 subunit of the Omicron (Omicron) BA.1 variant strain
  • the N-terminal domain in the base comprises the amino acid sequence shown in SEQ ID NO:43
  • the N-terminal domain of the S protein of Omicron (Omicron) BA.2 mutant strain comprises the amino acid sequence shown in SEQ ID NO:65
  • the N-terminal domain of the S protein of the Omicron (Omicron) BA.3 variant strain comprises the amino acid sequence shown in S
  • some immunogenic substances also include a human IgG Fc domain to form a dimeric structure, or some immunogenic substances also include a Foldon domain to form a trimeric structure, and the human IgG Fc domain Comprising the amino acid sequence shown in SEQ ID NO: 16, the Foldon domain includes the amino acid sequence shown in SEQ ID NO: 17.
  • the inventors designed the amino acid sequences of the following antigens, and obtained the corresponding DNA sequences by codon optimization according to the host CHO cells:
  • the antigen derived from the WH01 strain is composed of two SEQ ID NO: 1 in series, and its amino acid sequence is SEQ ID NO: 18 (signal peptide SEQ ID NO: 41 is added to the 5' end, and 6 is added to the 3' end. a histidine), the corresponding DNA sequence is SEQ ID NO:31;
  • Antigen derived from WH01 strain and Delta (Delta) strain composed of SEQ ID NO: 1 and SEQ ID NO: 9 connected in series, its amino acid sequence is SEQ ID NO: 19 (signal peptide added at the 5' end, 6 histidines have been added to the 3' end), and the corresponding DNA sequence is SEQ ID NO: 32;
  • Antigen derived from Beta (Beta) strain and Delta (Delta) strain composed of SEQ ID NO: 5 and SEQ ID NO: 9 connected in series, its amino acid sequence is SEQ ID NO: 20 (5' end added Signal peptide, 6 histidines are added at the 3' end), and the corresponding DNA sequence is SEQ ID NO:33;
  • Antigen derived from Delta (Delta) strain composed of two SEQ ID NO: 9 in series, its amino acid sequence is SEQ ID NO: 21 (signal peptide added at 5' end, 6 added at 3' end Histidine), the corresponding DNA sequence is SEQ ID NO:34;
  • the antigen derived from WH01 strain and Delta strain which comprises the N-terminal domain of WH01 strain, is composed of SEQ ID NO:13, SEQ ID NO:1 and SEQ ID NO:9 in series, and its amino acid
  • the sequence is SEQ ID NO: 22 (a signal peptide is added to the 5' end, and 6 histidines are added to the 3' end), and the corresponding DNA sequence is SEQ ID NO: 35;
  • Beta (Beta) strain and Delta (Delta) strain which comprises the N-terminal domain of Beta (Beta) strain, composed of SEQ ID NO:14, SEQ ID NO:5 and SEQ ID NO: 9 in series, its amino acid sequence is SEQ ID NO: 23 (signal peptide added to the 5' end, 6 histidines added to the 3' end), and the corresponding DNA sequence is SEQ ID NO: 36;
  • the Fc dimer derived from the antigen of WH01 strain and Delta (Delta) strain is composed of SEQ ID NO: 1, SEQ ID NO: 9 and SEQ ID NO: 16 in series, and its amino acid sequence is SEQ ID NO :24 (a signal peptide is added to the 5' end, and 6 histidines are added to the 3' end);
  • Fc dimer derived from the antigen of WH01 strain and Delta (Delta) strain which comprises the N-terminal domain of WH01 strain, composed of SEQ ID NO:13, SEQ ID NO:1, SEQ ID NO:9 Constituted in tandem with SEQ ID NO: 16, its amino acid sequence is SEQ ID NO: 25 (a signal peptide is added to the 5' end, and 6 histidines are added to the 3' end);
  • the antigen derived from WH01 strain and Delta (Delta) strain is composed of SEQ ID NO: 2 and SEQ ID NO: 10 in series, and its amino acid sequence is SEQ ID NO: 28 (a signal peptide is added at the 5' end, 6 histidines are added to the 3' end), and the corresponding DNA sequence is SEQ ID NO: 39;
  • Antigen derived from WH01 strain and Delta (Delta) strain composed of SEQ ID NO: 4 and SEQ ID NO: 12 connected in series, its amino acid sequence is SEQ ID NO: 29 (signal peptide added at the 5' end, 6 histidines were added to the 3' end);
  • Antigen derived from WH01 strain and Delta strain composed of SEQ ID NO: 13, SEQ ID NO: 1, SEQ ID NO: 15 and SEQ ID NO: 9 in series, and its amino acid sequence is SEQ ID NO:30 (signal peptide added to the 5' end, 6 histidines added to the 3' end), the corresponding DNA sequence is SEQ ID NO:40.
  • the antigen derived from the WH01 strain is composed of two SEQ ID NO: 2 in series, and its amino acid sequence is SEQ ID NO: 44 (a signal peptide is added to the 5' end, and 6 histidines are added to the 3' end) ;
  • An antigen derived from the WH01 strain which comprises the N-terminal domain of the WH01 strain, composed of SEQ ID NO: 13 and two SEQ ID NO: 1 in series, and its amino acid sequence is SEQ ID NO: 45;
  • An antigen derived from the Delta strain comprising the N-terminal domain of the Delta strain, composed of SEQ ID NO:15 and two SEQ ID NO:9 in series, and its amino acid sequence is SEQ ID NO:46;
  • Antigen derived from WH01 strain and Omicron BA.1 variant strain composed of SEQ ID NO: 1 and SEQ ID NO: 42 in series, and its amino acid sequence is SEQ ID NO: 47;
  • Antigen derived from Beta strain and Omicron BA.1 variant strain composed of SEQ ID NO:5 and SEQ ID NO:42 connected in series, its amino acid sequence is SEQ ID NO:48 ;
  • Antigen derived from WH01 strain and Omicron (Omicron) BA.1 variant strain which comprises the N-terminal domain of WH01 strain, composed of SEQ ID NO: 13, SEQ ID NO: 1 and SEQ ID NO: 42 in tandem form, its amino acid sequence is SEQ ID NO: 49;
  • Beta (Beta) strain and Omicron (Omicron) BA.1 variant strain which comprises the N-terminal domain of Beta (Beta) strain, composed of SEQ ID NO: 14, SEQ ID NO: 5 and SEQ ID NO:42 are connected in series, and its amino acid sequence is SEQ ID NO:50;
  • immunogenic substances can also be designed, including but not limited to the antigens shown in Table 2.
  • an N-terminal domain (NTD) and a Foldon/Fc domain can be further added to the antigens in Table 2.
  • the N-terminal domain is located at the N-terminal of the amino acid sequence of the first antigen and/or the second antigen, for example, the N-terminal domain of the S protein derived from the WH01 strain is directly connected to the first antigen in Table 2 from WH01
  • the receptor binding region of the S protein of the Beta strain or directly link the N-terminal domain of the S protein derived from the Beta strain to the receptor binding region of the S protein from the Beta strain in Table 2.
  • the Foldon/Fc domain is located at the C-terminus of the antigen.
  • the Foldon domain includes the amino acid sequence shown in SEQ ID NO: 17 and is connected to the C-terminus of different antigens in Table 2 above.
  • Embodiment 2 Construction of expression vector
  • the DNA sequences encoding the above antigens were codon-optimized according to the host CHO cells, and then synthesized and cloned into the expression vector pWX039 to obtain the expression vector pWX039-PR-Z, which was delivered after verification of the target gene sequencing.
  • the DNA fragment of the target gene was obtained by PCR amplification and gel purification.
  • the purified DNA fragment of the target gene was cloned into the vector pWX4.1 through the SalI and NotI sites to obtain the transient expression vector pWX4.1-PR.
  • the expression vector pWX4.1-PR was verified by Sanger sequencing, and the sequence was 100% correct.
  • the expression vector pWX4.1-PR was transformed into Escherichia coli E.coli Top10 competent cells. After picking the transformants for liquid culture, draw LB agar plates (containing 100 ⁇ g/mL ampicillin). Pick a single clone from the plate and inoculate it into 300ml LB medium for expansion. NucleoBond Xtra Maxi EF kit is used to prepare a large number of plasmids, and the target gene is verified by sequencing. The pWX4.1-PR plasmid DNA can be used for transfection after being verified by sequencing.
  • ExpiCHO Expression Medium was used to subculture and expand CHO cells (EXPICHO from Thermo Company), and subculture when the cell density reached 4 ⁇ 10 6 -6 ⁇ 10 6 cells/mL.
  • Day -1 Cell expansion, expand the cultured cells to 3 ⁇ 10 6 -4 ⁇ 10 6 cells/mL, and allow the cells to grow overnight.
  • Day 0 Transfect cells, measure viable cell density and survival rate, the cell density should reach 7 ⁇ 10 6 -10 ⁇ 10 6 cells/mL, and the survival rate reaches 95-99% for transfection, fresh cell expression culture Base, preheated to 37°C, diluted the cells to a final density of 5 ⁇ 10 6 cells/mL, cultured at 37°C in a 50 mm amplitude incubator at 90 rpm, and 8% CO 2 .
  • OPti-PRO SFM medium to prepare transfection reagent and plasmid DNA complex (4°C), for example: 1ml cells prepare 40 ⁇ l OPti-PRO SFM, add 1ug plasmid and mix for 5min; prepare 40 ⁇ l OPti-PRO SFM, add 6 ⁇ g PEI reagent and mix Let it stand for 5 minutes, mix equal volumes of plasmid and transfection reagent, incubate at room temperature for 1–5 minutes, then slowly transfer the solution to a shaker flask for shaking, and shake the shaker flask gently during the addition process. Place the cells in a 50mm amplitude incubator at 90rpm for culture at 37°C, 8% CO 2 .
  • Embodiment 4 Preparation of recombinant protein vaccine
  • Each recombinant protein obtained in Example 3 was diluted to 80 ⁇ g/ml with 1 ⁇ PBS buffer solution, and fully mixed with an equal volume of AS03 adjuvant to prepare a vaccine product, wherein the AS03 adjuvant component included 10.69 mg of squalane per 0.5 ml Alkene, 11.86 mg of ⁇ -tocopherol, 4.86 mg of polysorbate 80, 3.53 mg of sodium chloride, 0.09 mg of potassium chloride, 0.51 mg of disodium hydrogen phosphate, 0.09 mg of potassium dihydrogen phosphate and water for injection .
  • AS03 adjuvant component included 10.69 mg of squalane per 0.5 ml Alkene, 11.86 mg of ⁇ -tocopherol, 4.86 mg of polysorbate 80, 3.53 mg of sodium chloride, 0.09 mg of potassium chloride, 0.51 mg of disodium hydrogen phosphate, 0.09 mg of potassium dihydrogen phosphate and water for injection .
  • Embodiment 5 Preparation of recombinant protein vaccine
  • Each recombinant protein obtained in Example 3 was diluted to 80 ⁇ g/ml with 1 ⁇ PBS buffer solution, and fully mixed with an equal volume of MF59 adjuvant to prepare a vaccine product, wherein the MF59 adjuvant contained 1% squalene, 0.5% Warm 80 and 0.5% Span 85 in citric acid buffer solution.
  • Embodiment 6 Preparation of recombinant protein vaccine
  • Each recombinant protein obtained in Example 3 was diluted to 80 ⁇ g/ml with 1 ⁇ PBS buffer, and mixed with an equal volume of aluminum adjuvant to prepare a vaccine product, wherein the aluminum adjuvant was 2 mg/ml Al(OH) 3 .
  • Each recombinant protein obtained in Example 3 was diluted to 40 ⁇ g/ml with 1 ⁇ PBS buffer solution, and fully mixed with an equal volume of AS01 adjuvant to prepare a vaccine product, wherein the components of AS01 adjuvant included 50 ⁇ g of MPL, 50 ⁇ g of MPL per 0.5 ml QS21, 1mg DOPC and 0.25mg cholesterol.
  • mice purchased from Beijing Huafukang Biotechnology Co., Ltd.
  • mice were immunized with the vaccines prepared in Examples 4-7 in groups, with 5 mice in each group.
  • the mice were immunized on day 0 and day 21 respectively, and each mouse was injected intramuscularly with 100 ⁇ l of immune samples (containing 8 ⁇ g of antigen), and blood was collected on day 0, day 21 and 14 days after the second immunization.
  • the collected blood samples were placed at 37°C for 1 hour, at 4°C for 1 hour, centrifuged at 8000r/min for 10 minutes, and the serum was collected and stored at -20°C for pseudovirus neutralization detection.
  • Example 9 The neutralization experiment of the same strain RBD combination to different types of pseudoviruses
  • the pseudovirus was constructed by using the new coronavirus S protein containing the mutation sites of each strain, and the serum isolated after immunization was detected using the new coronavirus pseudovirus neutralizing antibody detection method (chemiluminescence method) based on the VSV system.
  • the pseudovirus neutralizing antibody titers measured when the present invention uses RBD (Beta)-RBD (Beta) as antigen and combined with different adjuvants to immunize mice are shown in Table 3.
  • the present invention first investigated the neutralizing antibody titers of the same strain RBD composition candidate antigens against different strains, and the neutralizing antibody titers and geometric mean titer GMT of 5 serum samples in each group are shown in Table 4 shown.
  • the candidate antigen (SEQ ID NO: 18) formed by the RBD combination of the WH01 strain had a higher geometric mean titer against the WH01 strain and the D614G strain pseudovirus, but the geometric mean titer against the Delta strain decreased, while against the The geometric mean titer of the Omicron BA.1 strain pseudovirus then significantly decreased.
  • the candidate antigen (SEQ ID NO:21) formed by the RBD combination of the Delta strain was only higher for the geometric mean titer of the Delta strain pseudovirus, The geometric mean titer against WH01 strain, D614G strain and Omicron BA.1 strain pseudovirus then decreased obviously. This indicates that the candidate antigens have poor cross-protection ability against different strains when the RBD is from the same strain, which is consistent with published studies.
  • the present invention screened the RBD of a specific strain as a preferred antigenic component, which can increase the antibody titer to non-self strains to obtain a more balanced protective effect.
  • This strain is the one described in the present invention. immunodominant strains.
  • Embodiment 10 The neutralization experiment of different strain RBD combinations to different types of pseudoviruses
  • Example 9 the method of Example 9 was used to detect the neutralizing antibody titers of different strains of RBD combinations against different types of pseudoviruses.
  • SEQ ID NO:21 reaches 46948 for the average value of the geometric mean titer of four kinds of pseudoviruses, and the geometric mean titer for Delta strain pseudovirus is the largest, reaching 101399, about 2.2 times of the mean value, for Omicron BA
  • the geometric mean titer of 1 strain of pseudovirus is only 22267, which is about 47% of the mean value.
  • the geometric mean titer of the candidate antigen (SEQ ID NO: 20) formed by the combination of Beta strain and Delta strain RBD against Delta strain pseudovirus was slightly decreased, but against D614G strain and Omicron BA.
  • the geometric mean titer of a strain of pseudovirus increased by 2 times and 1 time respectively, the mean value of the geometric mean titer for the four pseudoviruses reached 65462, and the geometric mean titer for the D614G strain pseudovirus was the largest, reaching 92502, About 1.4 times of the average value, the geometric mean titer against Omicron BA.1 strain pseudovirus reached 50785, about 78% of the average value.
  • the geometric mean titer of the candidate antigen (SEQ ID NO: 19) formed by the RBD combination of WH01 strain and Delta strain has little change against WH01 and D614G strain pseudoviruses, but against Delta strain and Omicron
  • the geometric mean titer of BA.1 strain pseudovirus all reduces more than 50%, the mean value of the geometric mean titer of four kinds of pseudoviruses is only 29505, and the geometric mean titer of Delta strain pseudovirus is the largest, reaches 42932, About 1.5 times of the average value, the geometric mean titer for Omicron BA.1 strain pseudovirus is only 10520, about 36% of the average value.
  • the geometric mean titer of SEQ ID NO: 20 against WH01 strain, D614G strain, Delta strain and Omicron BA.1 strain pseudovirus is more balanced, and the RBD of Beta strain can improve the antigenicity of non-Beta strain mutant strains.
  • the neutralizing antibody titer, so the Beta strain can be considered as an immunodominant strain.
  • the present invention further studies the effect of the combination of RBD of different strains on the neutralizing antibody titer when forming Foldon trimer. 5 of NTD-RBD(BA.2)-RBD(BA.1)-Foldon(SEQ ID NO:60) and NTD-RBD(BA.2)-RBD(WH01)-Foldon(SEQ ID NO:61)
  • the neutralizing antibody titers and geometric mean titer GMT of serum samples against different pseudoviruses are shown in Table 6.
  • NTD-RBD(BA.2)-RBD(BA.1)-Foldon has a high neutralization titer against BA.2 pseudovirus, reaching 19802, and has a certain antibody titer against BA.4 pseudovirus , but the antibody titers against D614G and BA.1 were very low.
  • the WH01 strain can increase the neutralizing antibody titer of the antigen against different mutant strains under the condition of forming Foldon trimer, therefore, the WH01 strain can also be considered as an immunodominant virus in this case strain.
  • the further research of the present invention shows that when from Alpha (Alpha) strain, Gamma (Gamma) strain, Epsilon (epsilon), cut tower (zeta) strain, Yita (Eta) strain,
  • Alpha (Alpha) strain Gamma (Gamma) strain
  • Epsilon (epsilon) cut tower
  • Yita (Eta) strain When selecting antigenic components in the receptor binding region of Theta strain, Iota strain, Kappa strain, Lambda strain, and Mu strain, the resulting immunogenicity
  • the substance only increased the titer against the pseudoviruses of the corresponding strains, but had no significant effect on the titers of the pseudoviruses of other mutant strains, which indicated that the receptor binding region of the above strains could induce antibody responses against other strains.
  • Weaker not the immunodominant strain described in the present invention. But these can be used as additional components of antigens to further improve the balance of the protective effect of the
  • the present invention uses the Delta strain or the Omicron strain as the prevailing strain.
  • Experimental studies of the present invention show that when the immunogenic substance contains receptor binding regions derived from immunodominant strains and epidemic dominant strains, it can all show higher titers against different strains of pseudoviruses, indicating that its It can produce excellent immune effects against different strains.
  • the present invention further examines the effect of adding NTD to candidate antigens. After adding NTD, the neutralizing antibody titer and geometric mean titer GMT of the 5 serum samples of each group against different pseudoviruses are shown in Table 7.
  • the present invention further investigated the effect of adding the Foldon domain to the candidate protein to form a trimer. After adding the Foldon domain, the neutralizing antibody titers and geometric mean titers GMT of 5 serum samples in each group were directed against different pseudoviruses. As shown in Table 8.
  • the antigen formed by the combination of immunodominant strains and RBDs of epidemic dominant strains in the present invention has a more balanced immune effect on different strains, and the immune effect can be further improved after further adding NTD or Foldon domains.
  • This example prepared mRNA for the antigens shown in Table 9.
  • the tag DYKDDDDKHHHHHHHHHH was added to the C-terminus of the above antigen, and the DNA sequence encoding the antigen was codon-optimized according to human as the host, and T7 RNA polymerase binding sequence, 5'UTR (human ⁇ globin 5'UTR), kozak sequence and signal peptide, and 3'UTR (5'UTR of human ⁇ -globin) and PloyA (120) and enzyme cutting site were sequentially added at the 3' end to obtain the DNA sequence.
  • the DNA sequence was synthesized and cloned into the expression vector pUC57-kan, and the target gene was verified by sequencing.
  • the expression vector was transformed into Escherichia coli E.coli competent cells, the plasmid was extracted after expansion and cultivation, and the linearized plasmid sample was obtained by enzyme digestion.
  • the T7 High Yield RNA Transcription Kit (Novoprotein, CAT: E131-01A) was used for in vitro transcription according to the instructions.
  • Cap1 Capping System (Novoprotein, CAT: M082) was operated according to the instructions for capping mRNA, and finally purified by lithium chloride precipitation purification method to obtain mRNA.
  • the prepared mRNA sequence is SEQ ID NO:68-89, wherein,
  • the 123-2750th position of SEQ ID NO:68 is the antigen coding region
  • the 123-1436th position of SEQ ID NO:69 is the antigen coding region
  • the 123-1436th position of SEQ ID NO:70 is the antigen coding region
  • the 123-1436th position of SEQ ID NO:71 is the antigen coding region
  • the 123-2093rd of SEQ ID NO:72 is the antigen coding region
  • the 123-2093rd of SEQ ID NO:73 is the antigen coding region
  • the 123-2093rd of SEQ ID NO:74 is the antigen coding region
  • the 123-2093rd position of SEQ ID NO:75 is the antigen coding region
  • the 123-2750th position of SEQ ID NO:76 is the antigen coding region
  • the 123-2093rd of SEQ ID NO:77 is the antigen coding region
  • the 123-1436th position of SEQ ID NO:78 is the antigen coding region
  • the 123-2351st position of SEQ ID NO:79 is the antigen coding region
  • the 123-2999th position of SEQ ID NO:80 is the antigen coding region
  • the 123-2342th position of SEQ ID NO:81 is the antigen coding region
  • the 123-3008th position of SEQ ID NO:82 is the antigen coding region
  • the 123-2999th position of SEQ ID NO:83 is the antigen coding region
  • the 123-3002 position of SEQ ID NO:84 is the antigen coding region
  • the 123-2999th position of SEQ ID NO:85 is the antigen coding region
  • the 123-2999th position of SEQ ID NO:86 is the antigen coding region
  • the 123-2351th position of SEQ ID NO:87 is the antigen coding region
  • the 123-2342th position of SEQ ID NO:88 is the antigen coding region
  • the 123-2342nd of SEQ ID NO:89 is the antigen coding region.
  • Each mRNA obtained in Example 11 is mixed with lipid nanoparticles to obtain a nucleic acid-lipid nanoparticle complex, wherein the lipid nanoparticles comprise DOTMA and DOPE with a molar ratio of 1:1; the content of mRNA in the complex is 100 ⁇ g/ ml, the mass ratio of lipid nanoparticles to mRNA was 10:1.
  • Each mRNA obtained in Example 11 was mixed with lipid nanoparticles to obtain a nucleic acid-lipid nanoparticle complex, and further mixed with an equal volume of adjuvant to prepare an immune sample, wherein the lipid nanoparticles contained a molar ratio of 2:1 DOTAP and DOPE; the content of mRNA in the complex is 200 ⁇ g/ml, and the mass ratio of lipid nanoparticles to mRNA is 12:1; the adjuvant components include 10.69 mg of squalene and 11.86 mg of ⁇ - Tocopherol, 4.86 mg of polysorbate 80, 3.53 mg of sodium chloride, 0.09 mg of potassium chloride, 0.51 mg of disodium hydrogen phosphate, 0.09 mg of monopotassium phosphate and water for injection.
  • the adjuvant components include 10.69 mg of squalene and 11.86 mg of ⁇ - Tocopherol, 4.86 mg of polysorbate 80, 3.53 mg of sodium chloride, 0.09 mg of potassium chloride, 0.51 mg of diso
  • Each mRNA obtained in Example 11 was mixed with lipid nanoparticles to obtain a nucleic acid-lipid nanoparticle complex, and further mixed with an equal volume of adjuvant to prepare an immune sample, wherein the lipid nanoparticles contained a molar ratio of 1:1 DOTMA and cholesterol; the content of mRNA in the complex is 100 ⁇ g/ml, and the mass ratio of lipid nanoparticles to mRNA is 10:1; the adjuvant components include 19.5mg squalene per 0.5ml, 2.35mg Tween 80, 2.35mg Span85 and Water for Injection.
  • Each mRNA obtained in Example 11 was mixed with lipid nanoparticles to obtain a nucleic acid-lipid nanoparticle complex, and further mixed with an equal volume of adjuvant to prepare an immune sample, wherein the lipid nanoparticles contained a molar ratio of 2:1 DOTMA and DOPE; the mRNA content in the complex is 200 ⁇ g/ml, and the mass ratio of lipid nanoparticles to mRNA is 8:1; the adjuvant components include DOPC 1mg, cholesterol 0.25mg, MPL 50 ⁇ g and QS- 21 50 ⁇ g.
  • Embodiment 16 mRNA vaccine mouse immunization experiment
  • the mRNA vaccines obtained in Example 12 were used to immunize 8-10 week-old BALB/c mice (purchased from Beijing Huafukang Biotechnology Co., Ltd.) in groups, with 5 mice in each group.
  • the mice were immunized on day 0 and day 21 respectively, and each mouse was injected intramuscularly with 50 ⁇ l of immune samples (including 5 ⁇ g mRNA), and blood was collected on day 0, day 21 and 14 days after the second immunization.
  • the collected blood samples were placed at 37°C for 1 hour, at 4°C for 1 hour, centrifuged at 8000r/min for 10 minutes, and the serum was collected and stored at -20°C for pseudovirus neutralization detection.
  • the neutralizing antibody titers and geometric mean titer GMT results of 5 serum samples of each group against different pseudoviruses are shown in Table 10.
  • the average value of the neutralizing antibody titer GMT of the vaccine prepared by encoding RBD (Beta)-RBD (Beta) mRNA (SEQ ID NO:78) against different pseudoviruses is 31133, and against the Omicron strain
  • the neutralizing antibody titer was significantly lower than other strains, compared with that produced by mRNA (SEQ ID NO:68) encoding RBD(WH01)-RBD(Beta)-RBD(Delta)-RBD(BA.1)
  • the average value of the neutralizing antibody titer GMT of the vaccine against different pseudoviruses reaches 99781, which is composed of mRNA (SEQ ID NO:69) encoding RBD (WH01)-RBD (BA.1) and encoding RBD (Beta)-RBD ( Delta) mRNA (SEQ ID NO: 70) the average value of the vaccine of the neutralizing antibody titer GMT of different pseudoviruses reaches 58513, by the mRNA (
  • the mRNA vaccine prepared by using the mRNA encoding the immunogenic substance of the present invention also has a good immune effect on different strains. It can be expected that the vaccine prepared from the immunogenic substance of the present invention and the nucleic acid encoding the immunogenic substance will still have a good preventive effect against future mutant strains of the new coronavirus. Therefore, under the situation that the novel coronavirus constantly mutates, the present invention has important guiding significance.

Abstract

提供了一种新型冠状病毒免疫原性物质，其包含源自免疫优势毒株的第一抗原和源自流行优势毒株的第二抗原，所述抗原分别包含S蛋白的受体结合区或受体结合区的一部分，其中，所述免疫优势毒株选自新型冠状病毒WH01株或贝塔(Beta)株中的至少一种，所述流行优势毒株选自新型冠状病毒德尔塔(Delta)株或奥密克戎(Omicron)株中的至少一种。上述新型冠状病毒免疫原性物质具有较高的免疫原性，对于不同的毒株均能够表现出显著提高的免疫效果。

Description

新型冠状病毒免疫原性物质、其制备方法和应用 技术领域
本发明属于生物医药工程技术领域，涉及新型冠状病毒免疫原性物质、其制备方法和应用。
发明背景
新型冠状病毒(SARS-CoV-2)感染可导致冠状病毒疾病(COVID-19)，常见体征有发热、咳嗽、咽痛等，在较严重病例中，感染可导致呼吸困难、低氧血症、急性呼吸窘迫综合征，甚至死亡。新型冠状病毒可以通过呼吸道和飞沫途径在人与人之间传播，也存在通过空气和消化道传播的可能。
SARS-CoV-2病毒颗粒包含4个结构蛋白，即刺突蛋白(S)、核衣壳蛋白(N)、膜蛋白(M)和包膜蛋白(E)。研究发现，只有针对S蛋白的抗体有中和活性，因此目前研发中的疫苗均包含S蛋白或其组分。其中，S蛋白的受体结合区被认为是诱导机体产生中和抗体的最主要的抗原靶区域。受体结合区作为疫苗能够将机体刺激产生的中和抗体更加聚焦在针对病毒的受体结合，可以提高疫苗的免疫原性和免疫效率。SARS-CoV-2通过其受体结合区与宿主细胞受体hACE2结合而进入细胞。
新型冠状病毒在传播过程中不断进化，目前已检测到多个代表性突变株。目前已开发或开发中的新型冠状病毒抗原大多只能针对一种毒株，无法产生针对不同毒株的中和抗体。CN114369172A中分别设计了原型毒株RBD二聚体疫苗，Beta株RBD二聚体疫苗以及原型株+Beta株嵌合RBD二聚体疫苗。结果表明，相对于原型毒株RBD二聚体疫苗和Beta株RBD二聚体疫苗，原型株+Beta株嵌合RBD二聚体疫苗诱导了较为均衡的抗体反应。但是从其结果来看，与原型毒株和其它变异株相比，原型株+Beta株嵌合RBD二聚体疫苗对Omicron变异株S蛋白假病毒的中和抗体滴度明显下降，这说明该疫苗对当前流行的Omicron变异株的保护能力欠佳，因此，有必要开发对不同毒株具有更均衡的保护效果的疫苗。
发明简述
本发明的目的在于提供一种新型冠状病毒免疫原性物质，其包含不同毒株S蛋白S1亚基的受体结合区，该新型冠状病毒免疫原性物质具有更高的免疫原性，能够激发针对不同毒株的中和抗体的产生，因而能够显著提高免疫效果。
为实现本发明目的，本发明提供了以下技术方案：
一种新型冠状病毒免疫原性物质，其包含源自免疫优势毒株的第一抗原和源自流行优势毒株的第二抗原，各抗原分别包含S蛋白的受体结合区或受体结合区的一部分。
在一些实施方式中，所述免疫优势毒株选自新型冠状病毒WH01株和贝塔(Beta)株中的至少一种。
在一些实施方式中，所述流行优势毒株选自新型冠状病毒德尔塔(Delta)株和奥密克戎(Omicron)株中的至少一种。
在一些实施方式中，所述奥密克戎(Omicron)株包括BA.1、BA.2、BA.3、BA.4和BA.5变异株。
在一些实施方式中，所述新型冠状病毒免疫原性物质还包含源自所述免疫优势毒株和所述流行优势毒株以外毒株的第三抗原。
在一些实施方式中，所述新型冠状病毒免疫原性物质还包含源自所述免疫优势毒株和所述流行优势毒株以外毒株的第四抗原。
在一些实施方式中，所述免疫优势毒株和所述流行优势毒株以外毒株选自以下毒株：阿尔法(Alpha)株、伽马(Gamma)株、艾普希龙(Epsilon)、截塔(Zeta)株、伊塔(Eta)株、西塔(Theta)株、艾欧塔(Iota)株、喀帕(Kappa)株、拉姆达(Lambda)株、缪(Mu)株等。
在一些实施方式中，各抗原构成组合物，或者各抗原直接相连或通过氨基酸接头连接，例如所述氨基酸接头可以为GGS或多个串联的GGS(G和S分别表示甘氨酸和丝氨酸)。
例如，在一些实施方式中，所述新型冠状病毒免疫原性物质包含第一抗原和第二抗原，第一抗原与第二抗原直接相连或者通过氨基酸接头连接。在另一些实施方式中，所述新型冠状病毒免疫原性物质包含第一抗原和第二抗原，第一抗原与第二抗原混合构成组合物。在一些实施方式中，所述新型冠状病毒免疫原性物质由新型冠状病毒WH01株S蛋白的受体结合区和德尔塔(Delta)株的受体结合区直接相连而形成；在一些实施方式中，所述新型冠状病毒免疫原性物质由新型冠状病毒WH01株S蛋白的受体结合区和奥密克戎(Omicron)株的受体结合区直接相连而形成；在一些实施方式中，所述新型冠状病毒免疫原性物质由新型冠状病毒贝塔(Beta)株S蛋白的受体结合区和德尔塔(Delta)株的受体结合区直接相连而形成；在一些实施方式中，所述新型冠状病毒免疫原性物质由新型冠状病毒贝塔(Beta)S蛋白的受体结合区和奥密克戎(Omicron)株的受体结合区直接相连而形成。
在一些实施方式中，所述新型冠状病毒免疫原性物质包含第一抗原、第二抗原和第三抗原，第一抗原、第二抗原和第三抗原直接相连或者通过氨基酸接头连接。在另一些实施方式中，所述新型冠状病毒免疫原性物质包含第一抗原、第二抗原和第三抗原，第一抗原与第二抗原直接相连或者通过氨基酸接头连接形成融合抗原，该融合抗原与第三抗原混合构成组合物。在另一些实施方式中，所述新型冠状病毒免疫原性物质包含第一抗原、第二抗原和第三抗原，第一抗原、第二抗原和第三抗原混合构成组合物。
在一些实施方式中，所述新型冠状病毒免疫原性物质包含第一抗原、第二抗原、第三抗原和第四抗原，第一抗原、第二抗原、第三抗原和第四抗原直接相连或者通过氨基酸接头连接形成融合抗原。在一些实施方式中，所述新型冠状病毒免疫原性物质包含第 一抗原、第二抗原、第三抗原和第四抗原，第一抗原与第二抗原直接相连或者通过氨基酸接头连接形成融合抗原，第三抗原与第四抗原直接相连或者通过氨基酸接头连接形成融合抗原，两种融合抗原混合构成组合物。在另一些实施方式中，所述新型冠状病毒免疫原性物质包含第一抗原、第二抗原、第三抗原和第四抗原，第一抗原、第二抗原和第三抗原直接相连或者通过氨基酸接头连接形成融合抗原，该融合抗原与第四抗原混合构成组合物。在另一些实施方式中，所述新型冠状病毒免疫原性物质包含第一抗原、第二抗原、第三抗原和第四抗原，第一抗原与第二抗原直接相连或者通过氨基酸接头连接形成融合抗原，该融合抗原与第三抗原和第四抗原混合构成组合物。在另一些实施方式中，所述新型冠状病毒免疫原性物质包含第一抗原、第二抗原、第三抗原和第四抗原，第一抗原、第二抗原、第三抗原和第四抗原混合构成组合物。
在本发明中，第一、第二、第三和第四仅用于表示抗原的不同种类，并不表示各抗原之间存在任何顺序。本发明中抗原的种类和数量也没有限制，本领域技术人员可以根据广泛传播的毒株以及抗原抗体的交叉反应确定合适的抗原种类和数量。
在一些实施方式中，所述抗原各自包含至少8个半胱氨酸，且半胱氨酸的数量为偶数。
在一些实施方式中，源自WH01株的S蛋白的受体结合区包含SEQ ID NO:1、SEQ ID NO:2、SEQ ID NO:3或SEQ ID NO:4中的任一项所示的氨基酸序列。
在一些实施方式中，源自贝塔(Beta)株的S蛋白的受体结合区包含SEQ ID NO:5、SEQ ID NO:6、SEQ ID NO:7或SEQ ID NO:8中的任一项所示的氨基酸序列。
在一些实施方式中，源自德尔塔(Delta)株的S蛋白的受体结合区包含SEQ ID NO:9、SEQ ID NO:10、SEQ ID NO:11或SEQ ID NO:12中的任一项所示的氨基酸序列。
在一些实施方式中，源自奥密克戎(Omicron)BA.1变异株的S蛋白的受体结合区包含SEQ ID NO:42所示的氨基酸序列，源自BA.2变异株的S蛋白的受体结合区包含SEQ ID NO:62所示的氨基酸序列，源自奥密克戎(Omicron)BA.3变异株的S蛋白的受体结合区包含SEQ ID NO:63所示的氨基酸序列，源自奥密克戎(Omicron)BA.4和BA.5变异株的S蛋白的受体结合区包含SEQ ID NO:64所示的氨基酸序列。
在一些实施方式中，所述新型冠状病毒免疫原性物质包含SEQ ID NO:19，20，28，29，47，48，51-55中任一项所示的氨基酸序列。
在一些实施方式中，所述抗原还包含S蛋白的S1亚基中的N端结构域(NTD)。
在一些实施方式中，所述N端结构域位于所述第一抗原和/或所述第二抗原的氨基酸序列的N端。
在一些实施方式中，源自WH01株的S蛋白的N端结构域包含SEQ ID NO:13所示的氨基酸序列。
在一些实施方式中，源自贝塔(Beta)株的S蛋白的N端结构域包含SEQ ID NO:14所示的氨基酸序列。
在一些实施方式中，源自德尔塔(Delta)株的S蛋白的N端结构域包含SEQ ID NO:15 所示的氨基酸序列。
在一些实施方式中，源自奥密克戎(Omicron)BA.1变异株的S蛋白的N端结构域包含SEQ ID NO:43所示的氨基酸序列，源自奥密克戎(Omicron)BA.2变异株的S蛋白的N端结构域包含SEQ ID NO:65所示的氨基酸序列，源自奥密克戎(Omicron)BA.3变异株的S蛋白的N端结构域包含SEQ ID NO:66所示的氨基酸序列，源自奥密克戎(Omicron)BA.4和BA.5变异株的S蛋白的N端结构域包含SEQ ID NO:67所示的氨基酸序列。
在一些实施方式中，所述新型冠状病毒免疫原性物质包含SEQ ID NO:22，23，30，49，50，56-59中任一项所示的氨基酸序列。
在一些实施方式中，所述新型冠状病毒免疫原性物质还包含免疫球蛋白的Fc结构域，优选地，所述免疫球蛋白为人源IgG。
在一些实施方式中，所述Fc结构域位于所述新型冠状病毒免疫原性物质的氨基酸序列的C端，优选地，人源IgG Fc结构域包含SEQ ID NO:16所示的氨基酸序列。
在一些实施方式中，所述新型冠状病毒免疫原性物质包含SEQ ID NO:24或SEQ ID NO:25所示的氨基酸序列。
在一些实施方式中，所述新型冠状病毒免疫原性物质还包含Foldon结构域。Foldon结构域/蛋白来源于T4噬菌体纤维蛋白的C-端，由27个氨基酸组成，具有促使目标蛋白发生非共价寡聚以形成三聚体的功能。
在一些实施方式中，所述Foldon结构域位于所述新型冠状病毒免疫原性物质的氨基酸序列的C端，优选地，Foldon结构域包含SEQ ID NO:17所示的氨基酸序列。
在一些实施方式中，所述新型冠状病毒免疫原性物质包含SEQ ID NO:26，27，60，61中任一项所示的氨基酸序列。
本发明还提供了一种制备所述新型冠状病毒免疫原性物质的方法，包括以下步骤：
利用编码所述新型冠状病毒免疫原性物质的核苷酸序列构建重组表达质粒；
将构建的重组表达质粒转化宿主菌，筛选正确的重组表达质粒；
利用筛选的重组表达质粒转染表达系统的细胞，表达后收集上清并纯化得到新型冠状病毒免疫原性物质。
在一些实施方式中，所述表达系统的细胞包括哺乳动物细胞、昆虫细胞、酵母细胞或细菌细胞，可选地，所述哺乳动物细胞包括293T细胞或CHO细胞，所述细菌细胞包括大肠杆菌细胞。
本发明还提供了一种编码所述新型冠状病毒免疫原性物质的核苷酸序列、一种包含上述核苷酸序列的重组载体、一种携带有上述重组载体的表达系统细胞。
本发明还提供了一种所述新型冠状病毒免疫原性物质、编码所述新型冠状病毒免疫原性物质的核苷酸序列、包含所述核苷酸序列的重组载体、携带有所述重组载体的表达系统细胞在制备新型冠状病毒疫苗中的应用。
本发明还提供了一种新型冠状病毒蛋白疫苗，包括所述新型冠状病毒免疫原性物质 和佐剂。
在一些实施方式中，所述佐剂选自铝佐剂、MF59佐剂、MPL佐剂、QS-21、GLA、CpG、AS01、AS02、AS03、AS04佐剂中的一种或多种，优选为AS03佐剂或MF59佐剂。
本发明还提供了一种新型冠状病毒DNA疫苗，其包含编码所述新型冠状病毒免疫原性物质的DNA序列。
本发明还提供了一种新型冠状病毒mRNA疫苗，其包含编码所述新型冠状病毒免疫原性物质的mRNA序列。
在一些实施方式中，所述mRNA包含下述序列中的至少一项：
SEQ ID NO:68中第123-2750位所示的mRNA序列，
SEQ ID NO:69中第123-1436位所示的mRNA序列，
SEQ ID NO:70中第123-1436位所示的mRNA序列，
SEQ ID NO:71中第123-1436位所示的mRNA序列，
SEQ ID NO:72中第123-2093位所示的mRNA序列，
SEQ ID NO:73中第123-2093位所示的mRNA序列，
SEQ ID NO:74中第123-2093位所示的mRNA序列，
SEQ ID NO:75中第123-2093位所示的mRNA序列，
SEQ ID NO:79中第123-2351位所示的mRNA序列，
SEQ ID NO:82中第123-3008位所示的mRNA序列，
SEQ ID NO:83中第123-2999位所示的mRNA序列，
SEQ ID NO:84中第123-3002位所示的mRNA序列，
SEQ ID NO:85中第123-2999位所示的mRNA序列，
SEQ ID NO:86中第123-2999位所示的mRNA序列，
SEQ ID NO:87中第123-2351位所示的mRNA序列，
SEQ ID NO:88中第123-2342位所示的mRNA序列，
SEQ ID NO:89中第123-2342位所示的mRNA序列。
与现有技术相比，本发明的技术方案具有以下有益效果：
本发明的新型冠状病毒免疫原性物质，其包含来自免疫优势毒株和流行优势毒株的至少两种抗原，能够诱导产生针对不同毒株的中和抗体，实验表明，本发明的新型冠状病毒免疫原性物质具有较高的免疫原性和良好的交叉保护效果，对于不同的毒株均能够诱导产生均衡的中和抗体水平。
发明人惊奇地发现，以新型冠状病毒的WH01株或贝塔(Beta)株作为基础的免疫优势株，再通过与目前流行的突变株组合，能够产生突出的免疫效果，WH01株或贝塔(Beta)株作为免疫组合的基石，能够针对不同突变株产生抗体，保证免疫的稳定性。特别是在实施例中组合使用WH01株和奥密克戎(Omicron)株、贝塔(Beta)株与德尔塔(Delta)株构建的重组蛋白能够对不同突变株保持均衡的中和抗体GMT水平，同时克服突变株 的免疫逃逸，产生突出的免疫效果。本发明的研究还表明，利用编码本发明所述免疫原性物质的mRNA制备的mRNA疫苗对不同毒株同样具有很好的免疫效果。
本发明还研究了不同佐剂与免疫原性物质的组合免疫效果，结果表明当使用水包油乳剂佐剂(尤其是AS03佐剂)时，可以产生较高的中和抗体滴度。
附图简述
图1为S蛋白结构域示意图。
图2为抗原SEQ ID NO:18的SDS-PAGE和Western Blot结果。
图3为抗原SEQ ID NO:19的SDS-PAGE和Western Blot结果。
图4为抗原SEQ ID NO:20的SDS-PAGE和Western Blot结果。
图5为抗原SEQ ID NO:21的SDS-PAGE和Western Blot结果。
图6为抗原SEQ ID NO:22的SDS-PAGE和Western Blot结果。
图7为抗原SEQ ID NO:23的SDS-PAGE和Western Blot结果。
图8为抗原SEQ ID NO:26的SDS-PAGE和Western Blot结果。
图9为抗原SEQ ID NO:27的SDS-PAGE和Western Blot结果。
图10为抗原SEQ ID NO:28的SDS-PAGE和Western Blot结果。
图11为抗原SEQ ID NO:30的SDS-PAGE和Western Blot结果。
图12为抗原SEQ ID NO:44的SDS-PAGE和Western Blot结果。
图13为抗原SEQ ID NO:60的SDS-PAGE和Western Blot结果。
图14为抗原SEQ ID NO:61的SDS-PAGE和Western Blot结果。
发明详述
为使本发明的目的、技术方案和优点更加清楚明白，以下结合具体实施例，并参照附图，对本发明作进一步的详细说明。基于本发明中的实施例，本领域普通技术人员在没有作出创造性劳动前提下所获得的所有其他实施例，都属于本发明保护的范围。
本发明中的“免疫优势毒株”是指相对于非免疫优势变异株，其抗原的免疫原性较高并且能够对其他变异株提供较好的交叉保护作用的变异株。
本发明中的“流行优势毒株”是指当前流行的主要变异株，通常可以理解为当前流行的“需要关注的变异株”(Variants of concern，VOC)。VOC的定义可参考世界卫生组织(WHO)的工作定义，即：符合“需要留意的变异株”(Variants of interest(VOI))定义，并且通过比较评估，已被证明与下列一种或多种具有一定全球公共卫生意义的变化相关的SARS-CoV-2变异株：
·传播性增加或COVID-19流行病学方面的有害变化；或者
·毒性增加或临床疾病表现的变化；或者
·公共卫生和社会措施或可用的诊断方法、疫苗和治疗方法的有效性降低。
本发明中的“需要留意的变异株”(Variants of interest(VOI))的定义可参考世界卫生 组织(WHO)的工作定义，即：具有以下特征的SARS-CoV-2变异株：
具有预测或已知会影响病毒特征的基因变化，例如可传播性、疾病严重程度、免疫逃逸、诊断或治疗逃逸；以及
确认导致多个国家出现重大社区传播或多个COVID-19聚集性病例，且相对流行率上升，病例数不断增加，或其他表明全球公共卫生正面临新风险的明显流行病学影响。
SARS-CoV-2S蛋白的结构如图1所示，其中，1-13为信号肽，14-685为S1亚基，686-1273为S2亚基。其中S1亚基又可以分为NTD(14-303)和CTD(334-527)。319-541为受体结合区，788-806为融合蛋白。13-1213为细胞外结构域，1214-1234为跨膜结构域，1235-1273为细胞内结构域。
实施例1 新型冠状病毒免疫原性物质的氨基酸序列
在本发明的实施例中，本发明人利用新型冠状病毒WH01株、贝塔(Beta)株、德尔塔(Delta)株和奥密克戎(Omicron)变异株的受体结合区设计了多种新型冠状病毒免疫原性物质。
其中，源自WH01株的S蛋白的受体结合区包含SEQ ID NO:1、SEQ ID NO:2、SEQ ID NO:3或SEQ ID NO:4中的任一项所示的氨基酸序列。其中，SEQ ID NO:1含有8个半胱氨酸，SEQ ID NO:2包含自SEQ ID NO:1的N端向外延伸的额外序列，含有10个半胱氨酸，SEQ ID NO:3包含自SEQ ID NO:1的C端向外延伸的额外序列，含有10个半胱氨酸，SEQ ID NO:4包含分别自SEQ ID NO:1的N端和C端向外延伸的额外序列，含有12个半胱氨酸。
源自贝塔(Beta)株的S蛋白的受体结合区包含SEQ ID NO:5、SEQ ID NO:6、SEQ ID NO:7或SEQ ID NO:8中的任一项所示的氨基酸序列。其中，SEQ ID NO:5含有8个半胱氨酸，SEQ ID NO:6包含自SEQ ID NO:5的N端向外延伸的额外序列，含有10个半胱氨酸，SEQ ID NO:7包含自SEQ ID NO:5的C端向外延伸的额外序列，含有10个半胱氨酸，SEQ ID NO:8包含分别自SEQ ID NO:5的N端和C端向外延伸的额外序列，含有12个半胱氨酸。
源自德尔塔(Delta)株的S蛋白的受体结合区包含SEQ ID NO:9、SEQ ID NO:10、SEQ ID NO:11或SEQ ID NO:12中的任一项所示的氨基酸序列。其中，SEQ ID NO:9含有8个半胱氨酸，SEQ ID NO:10包含自SEQ ID NO:9的N端向外延伸的额外序列，含有10个半胱氨酸，SEQ ID NO:11包含自SEQ ID NO:9的C端向外延伸的额外序列，含有10个半胱氨酸，SEQ ID NO:12包含分别自SEQ ID NO:9的N端和C端向外延伸的额外序列，含有12个半胱氨酸。
源自奥密克戎(Omicron)BA.1变异株的S蛋白的受体结合区包含SEQ ID NO:42所示的氨基酸序列，源自BA.2变异株的S蛋白的受体结合区包含SEQ ID NO:62所示的氨基酸序列，源自奥密克戎(Omicron)BA.3变异株的S蛋白的受体结合区包含SEQ ID NO:63所示的氨基酸序列，源自奥密克戎(Omicron)BA.4和BA.5变异株的S蛋白的受 体结合区包含SEQ ID NO:64所示的氨基酸序列。
在该实施例中，一些免疫原性物质还包括S蛋白的S1亚基中的N端结构域(NTD)。其中，WH01株S蛋白的S1亚基中的N端结构域包含SEQ ID NO:13所示的氨基酸序列，贝塔(Beta)株S蛋白的S1亚基中的N端结构域包含SEQ ID NO:14所示的氨基酸序列，德尔塔(Delta)株的S1亚基中的N端结构域包含SEQ ID NO:15所示的氨基酸序列，奥密克戎(Omicron)BA.1变异株的S1亚基中的N端结构域包含SEQ ID NO:43所示的氨基酸序列，奥密克戎(Omicron)BA.2变异株的S蛋白的N端结构域包含SEQ ID NO:65所示的氨基酸序列，奥密克戎(Omicron)BA.3变异株的S蛋白的N端结构域包含SEQ ID NO:66所示的氨基酸序列，奥密克戎(Omicron)BA.4和BA.5变异株的S蛋白的N端结构域包含SEQ ID NO:67所示的氨基酸序列。
在该实施例中，一些免疫原性物质还包括人源IgG Fc结构域以形成二聚体结构，或者一些免疫原性物质还包括Foldon结构域以形成三聚体结构，人源IgG Fc结构域包含SEQ ID NO:16所示的氨基酸序列，Foldon结构域包含SEQ ID NO:17所示的氨基酸序列。
基于以上序列，本发明人设计了以下抗原的氨基酸序列，按照宿主CHO细胞进行密码子优化获得了相应的DNA序列：
(1)源自WH01株的抗原，由两个SEQ ID NO:1串联构成，其氨基酸序列为SEQ ID NO:18(5’端添加了信号肽SEQ ID NO:41，3’端添加了6个组氨酸)，相应的DNA序列为SEQ ID NO:31；
(2)源自WH01株与德尔塔(Delta)株的抗原，由SEQ ID NO:1与SEQ ID NO:9串联构成，其氨基酸序列为SEQ ID NO:19(5’端添加了信号肽，3’端添加了6个组氨酸)，相应的DNA序列为SEQ ID NO:32；
(3)源自贝塔(Beta)株与德尔塔(Delta)株的抗原，由SEQ ID NO:5与SEQ ID NO:9串联构成，其氨基酸序列为SEQ ID NO:20(5’端添加了信号肽，3’端添加了6个组氨酸)，相应的DNA序列为SEQ ID NO:33；
(4)源自德尔塔(Delta)株的抗原，由两个SEQ ID NO:9串联构成，其氨基酸序列为SEQ ID NO:21(5’端添加了信号肽，3’端添加了6个组氨酸)，相应的DNA序列为SEQ ID NO:34；
(5)源自WH01株与德尔塔(Delta)株的抗原，其包含WH01株的N端结构域，由SEQ ID NO:13、SEQ ID NO:1和SEQ ID NO:9串联构成，其氨基酸序列为SEQ ID NO:22(5’端添加了信号肽，3’端添加了6个组氨酸)，相应的DNA序列为SEQ ID NO:35；
(6)源自贝塔(Beta)株与德尔塔(Delta)株的抗原，其包含贝塔(Beta)株的N端结构域，由SEQ ID NO:14、SEQ ID NO:5与SEQ ID NO:9串联构成，其氨基酸序列为SEQ ID NO:23(5’端添加了信号肽，3’端添加了6个组氨酸)，相应的DNA序列为SEQ ID NO:36；
(7)源自WH01株与德尔塔(Delta)株的抗原的Fc二聚体，由SEQ ID NO:1、SEQ ID NO:9和SEQ ID NO:16串联构成，其氨基酸序列为SEQ ID NO:24(5’端添加了信号肽，3’端添加了6个组氨酸)；
(8)源自WH01株与德尔塔(Delta)株的抗原的Fc二聚体，其包含WH01株的N端结构域，由SEQ ID NO:13、SEQ ID NO:1、SEQ ID NO:9和SEQ ID NO:16串联构成，其氨基酸序列为SEQ ID NO:25(5’端添加了信号肽，3’端添加了6个组氨酸)；
(9)源自WH01株与德尔塔(Delta)株的抗原的Foldon三聚体，由SEQ ID NO:1、SEQ ID NO:9和SEQ ID NO:17串联构成，其氨基酸序列为SEQ ID NO:26(5’端添加了信号肽，3’端添加了6个组氨酸)，相应的DNA序列为SEQ ID NO:37；
(10)源自WH01株与德尔塔(Delta)株的抗原的Foldon三聚体，其包含WH01株的N端结构域，由SEQ ID NO:13、SEQ ID NO:1、SEQ ID NO:9和SEQ ID NO:17串联构成，其氨基酸序列为SEQ ID NO:27(5’端添加了信号肽，3’端添加了6个组氨酸)，相应的DNA序列为SEQ ID NO:38；
(11)源自WH01株与德尔塔(Delta)株的抗原，由SEQ ID NO:2与SEQ ID NO:10串联构成，其氨基酸序列为SEQ ID NO:28(5’端添加了信号肽，3’端添加了6个组氨酸)，相应的DNA序列为SEQ ID NO:39；
(12)源自WH01株与德尔塔(Delta)株的抗原，由SEQ ID NO:4与SEQ ID NO:12串联构成，其氨基酸序列为SEQ ID NO:29(5’端添加了信号肽，3’端添加了6个组氨酸)；
(13)源自WH01株与德尔塔(Delta)株的抗原，由SEQ ID NO:13、SEQ ID NO:1、SEQ ID NO:15与SEQ ID NO:9串联构成，其氨基酸序列为SEQ ID NO:30(5’端添加了信号肽，3’端添加了6个组氨酸)，相应的DNA序列为SEQ ID NO:40。
(14)源自WH01株的抗原，由两个SEQ ID NO:2串联构成，其氨基酸序列为SEQ ID NO:44(5’端添加了信号肽，3’端添加了6个组氨酸)；
(15)源自WH01株的抗原，其包含WH01株的N端结构域，由SEQ ID NO:13和两个SEQ ID NO:1串联构成，其氨基酸序列为SEQ ID NO:45；
(16)源自Delta株的抗原，其包含Delta株的N端结构域，由SEQ ID NO:15和两个SEQ ID NO:9串联构成，其氨基酸序列为SEQ ID NO:46；
(17)源自WH01株与奥密克戎(Omicron)BA.1变异株的抗原，由SEQ ID NO:1和SEQ ID NO:42串联构成，其氨基酸序列为SEQ ID NO:47；
(18)源自贝塔(Beta)株与奥密克戎(Omicron)BA.1变异株的抗原，由SEQ ID NO:5和SEQ ID NO:42串联构成，其氨基酸序列为SEQ ID NO:48；
(19)源自WH01株与奥密克戎(Omicron)BA.1变异株的抗原，其包含WH01株的N端结构域，由SEQ ID NO:13、SEQ ID NO:1和SEQ ID NO:42串联构成，其氨基酸序列为SEQ ID NO:49；
(20)源自贝塔(Beta)株与奥密克戎(Omicron)BA.1变异株的抗原，其包含贝塔(Beta)株的N端结构域，由SEQ ID NO:14、SEQ ID NO:5和SEQ ID NO:42串联构成，其氨基酸序列为SEQ ID NO:50；
(21)根据主要变异株，进一步设计了源自2-4种变异株的抗原，其氨基酸序列如SEQ ID NO:51-61所示。
以上抗原汇总于表1中：
表1
Figure PCTCN2022115143-appb-000001
Figure PCTCN2022115143-appb-000002
需要指出的是，根据本发明的构思，还可以设计更多的免疫原性物质，包括但不限于表2中所示的抗原。
表2
编号 目标抗原
1 RBD(WH01)-RBD(Beta)-RBD(BA.1)
2 RBD(WH01)-RBD(Beta)-RBD(BA.4/5)
3 RBD(WH01)-RBD(Beta)-RBD(Delta)
4 RBD(WH01)-RBD(Delta)-RBD(BA.1)
5 RBD(Beta)-RBD(Delta)-RBD(BA.1)
6 RBD(WH01)-RBD(Delta)-RBD(BA.4/5)
7 RBD(Beta)-RBD(Delta)-RBD(BA.4/5)
8 RBD(WH01)-RBD(BA.1)-RBD(BA.4/5)
9 RBD(Beta)-RBD(BA.1)-RBD(BA.4/5)
10 RBD(WH01)-RBD(Beta)-RBD(Delta)-RBD(BA.1)
11 RBD(WH01)-RBD(Beta)-RBD(Delta)-RBD(BA.4/5)
12 RBD(WH01)-RBD(Beta)-RBD(BA.1)-RBD(BA.4/5)
13 RBD(Beta)-RBD(WH01)-RBD(Delta)-RBD(BA.4/5)
14 RBD(Beta)-RBD(Delta)-RBD(BA.1)-RBD(BA.4/5)
15 RBD(WH01)-RBD(Delta)-RBD(BA.1)-RBD(BA.4/5)
在一些实施例中，还可以在表2中的抗原中进一步添加N端结构域(NTD)和Foldon/Fc结构域。N端结构域位于所述第一抗原和/或所述第二抗原的氨基酸序列的N端，例如将源自WH01株的S蛋白的N端结构域后直接连接表2中第一抗原来自WH01株的S蛋白的受体结合区，或者将源自Beta株的S蛋白的N端结构域后直接连接表2 中来自Beta株的S蛋白的受体结合区。Foldon/Fc结构域位于抗原的C端，例如Foldon结构域包含SEQ ID NO:17所示的氨基酸序列连接于上述表2中不同抗原的C端。
实施例2 表达载体的构建
将编码以上抗原的DNA序列按照宿主CHO细胞进行密码子优化后外送合成并克隆至表达载体pWX039中，得到表达载体pWX039-PR-Z，经目标基因测序验证后交付。
以pWX039-PR-Z载体为模板，经PCR扩增、凝胶纯化得到目的基因DNA片段。将纯化后的目的基因DNA片段通过SalI和NotI位点克隆到载体pWX4.1中，得到瞬转表达载体pWX4.1-PR。表达载体pWX4.1-PR通过Sanger测序验证，序列100％正确。
将表达载体pWX4.1-PR转化大肠杆菌E.coli Top10感受态细胞。挑取转化子液体培养后划LB琼脂平板(含100μg/mL氨苄青霉素)。从平板上挑取一个单克隆接种至300ml LB培养基中扩大培养，采用NucleoBond Xtra Maxi EF试剂盒进行质粒大量制备，并测序验证目的基因。pWX4.1-PR质粒DNA通过测序验证后可用于转染。
实施例3 蛋白表达与纯化
蛋白表达
在所有细胞操作过程中，通过温和的旋转混合细胞；避免剧烈的混合/移液。细胞健康是实现最大性能的关键。采用ExpiCHO Expression Medium继代培养和扩增CHO细胞(来自Thermo公司的EXPICHO)，细胞密度达到4×10 6–6×10 6个/mL时进行传代。
第-1天：细胞扩增，扩增培养细胞到3×10 6–4×10 6个/mL，并允许细胞过夜生长。第0天：转染细胞，测定活细胞密度和存活率，细胞的密度应该达7×10 6–10×10 6个/mL，存活率到95-99％进行转染，新鲜的细胞表达培养基，预热至37℃，将细胞稀释至最终密度为5×10 6个/mL，50mm振幅培养箱90rpm进行37℃培养，8％CO 2。使用OPti-PRO SFM培养基制备转染试剂和质粒DNA复合物(4℃)，例：1ml细胞准备40μl OPti-PRO SFM添加1ug质粒混匀放置5min；准备40μl OPti-PRO SFM添加6μg PEI试剂混匀放置5min，将质粒与转染试剂等体积混合，室温下孵育1–5分钟，然后将溶液缓慢转移至摇瓶摇晃，在添加过程中轻轻摇动摇瓶。将细胞置于50mm振幅培养箱90rpm进行37℃培养，8％CO 2。转染后18-22小时，添加补料，执行标准实验方案。例：1ml细胞添加0.2ml
Figure PCTCN2022115143-appb-000003
Advanced CHO Feed 1，将细胞置于50mm振幅培养箱90rpm进行37℃培养，8％CO 2。转染后6天收集，进行后续纯化。
蛋白纯化
培养基离心，上清添加Ni柱，振荡孵育2h，经过重力空柱管柱进行亲和层析纯化。平衡缓冲液：“PBS”，pH7.4，清洗10CV；清洗缓冲液：“PBS”，pH7.4，包含20mM咪唑，清洗10CV；洗脱缓冲液：“PBS”，pH7.4，包含500mM咪唑，洗脱1CV，重复5次。不同候选抗原的SDS-PAGE&Western Blot图谱如图2-14所示。
实施例4 重组蛋白疫苗的制备
将实施例3获得的各重组蛋白用1×PBS缓冲液稀释至80μg/ml，并与等体积AS03佐剂充分混合制得疫苗产品，其中AS03佐剂成分为每0.5ml包括10.69mg的角鲨烯、11.86mg的α-生育酚、4.86mg的聚山梨酯80、3.53mg的氯化钠、0.09mg的氯化钾、0.51mg的磷酸氢二钠、0.09mg的磷酸二氢钾和注射用水。
实施例5 重组蛋白疫苗的制备
将实施例3获得的各重组蛋白用1×PBS缓冲液稀释至80μg/ml，并与等体积MF59佐剂充分混合制得疫苗产品，其中MF59佐剂为包含1％角鲨烯、0.5％吐温80和0.5％司盘85的柠檬酸缓冲溶液。
实施例6 重组蛋白疫苗的制备
将实施例3获得的各重组蛋白用1×PBS缓冲液稀释至80μg/ml，并与等体积铝佐剂充分混合制得疫苗产品，其中铝佐剂为2mg/ml的Al(OH) 3。
实施例7 重组蛋白疫苗的制备
将实施例3获得的各重组蛋白用1×PBS缓冲液稀释至40μg/ml，并与等体积AS01佐剂充分混合制得疫苗产品，其中AS01佐剂成分为每0.5ml包括50μg的MPL、50μg的QS21、1mg DOPC和0.25mg胆固醇。
实施例8 重组蛋白疫苗小鼠免疫实验
利用实施例4-7中制备的疫苗对8～10周龄的BALB/c小鼠(购自北京华阜康生物技术有限公司)进行分组免疫，每组5只小鼠。分别于0天及21天进行小鼠免疫，每只每次肌肉注射100μl的免疫样品(包含8μg抗原)，并于0天、21天以及第二次免疫后14天采血。采集的血液样品37℃放置1小时，4℃放置1小时，8000r/min离心10分钟，收集血清，-20℃保存，用于假病毒中和检测。
实施例9 相同毒株RBD组合对不同类型假病毒的中和实验
采用包含各毒株突变位点的新冠病毒S蛋白构建假病毒，使用基于VSV系统的新冠假病毒中和抗体检测方法(化学发光法)对免疫后分离得到血清进行检测。
在佐剂方面，本发明以RBD(Beta)-RBD(Beta)为抗原并联合不同佐剂免疫小鼠时测得的假病毒中和抗体滴度如表3所示。
表3
Figure PCTCN2022115143-appb-000004
Figure PCTCN2022115143-appb-000005
结果表明，当使用AS03佐剂时，针对D614G、Beta和Omicron BA.1毒株的假病毒中和抗体滴度均显著高于使用Al(OH) 3佐剂时的中和抗体滴度。另外的实验表明，当使用MF59佐剂时，抗体滴度与使用AS03佐剂时的抗体滴度相似。在后续的实验中，本发明均采用AS03佐剂。
在抗原方面，本发明首先考察了相同毒株RBD组成的候选抗原针对不同毒株的中和抗体滴度，每组的5个血清样品的中和抗体滴度和几何平均滴度GMT如表4所示。
表4
Figure PCTCN2022115143-appb-000006
结果表明，WH01株的RBD组合形成的候选抗原(SEQ ID NO:18)针对WH01株和D614G株假病毒的几何平均滴度较高，但针对Delta株的几何平均滴度有所下降，而针对Omicron BA.1株假病毒的几何平均滴度则显著下降，与之相对，Delta株的RBD组合形成的候选抗原(SEQ ID NO:21)仅针对Delta株假病毒的几何平均滴度较高，针对WH01株、D614G株和Omicron BA.1株假病毒的几何平均滴度则明显下降。这说明当RBD来自同一毒株时，候选抗原对不同毒株的交叉保护能力较差，这一结果与已公开的研究一致。
本发明在进一步的研究中筛选了特定毒株的RBD作为优选抗原组分，其能够提高对非自身毒株的抗体滴度，以获得更均衡的保护效果，该毒株即为本发明所述的免疫优势毒株。
实施例10 不同毒株RBD组合对不同类型假病毒的中和实验
该实施例中采用实施例9的方法检测了不同毒株RBD组合针对不同类型假病毒的中和抗体滴度。
(1)免疫优势毒株RBD筛选
当把RBD(Delta)-RBD(Delta)(SEQ ID NO:21)中的一个来源于Delta变异株的RBD替换为来源于Beta变异株或WH01株的RBD时，每组的5个血清样品的中和抗体滴度和几何平均滴度GMT变化如表5所示。
表5
Figure PCTCN2022115143-appb-000007
可见，SEQ ID NO:21针对四种假病毒的几何平均滴度的平均值达到46948，且针对Delta株假病毒的几何平均滴度最大，达到101399，约为平均值的2.2倍，针对Omicron BA.1株假病毒的几何平均滴度仅为22267，约为平均值的47％。
与SEQ ID NO:21相比，Beta株与Delta株的RBD组合形成的候选抗原(SEQ ID NO:20)针对Delta株假病毒的几何平均滴度稍有下降，但针对D614G株和Omicron BA.1株假病毒的几何平均滴度分别提高了2倍和1倍，针对四种假病毒的几何平均滴度的平均 值达到65462，且针对D614G株假病毒的几何平均滴度最大，达到92502，约为平均值的1.4倍，针对Omicron BA.1株假病毒的几何平均滴度达到50785，约为平均值的78％。
与SEQ ID NO:21相比，WH01株与Delta株的RBD组合形成的候选抗原(SEQ ID NO:19)针对WH01和D614G株假病毒的几何平均滴度变化不大，但针对Delta株和Omicron BA.1株假病毒的几何平均滴度均降低50％以上，针对四种假病毒的几何平均滴度的平均值仅为29505，且针对Delta株假病毒的几何平均滴度最大，达到42932，约为平均值的1.5倍，针对Omicron BA.1株假病毒的几何平均滴度仅为10520，约为平均值的36％。
因此，总体上看，SEQ ID NO:20针对WH01株、D614G株、Delta株和Omicron BA.1株假病毒的几何平均滴度更为均衡，Beta株的RBD可以提高抗原对非Beta株变异株的中和抗体滴度，因此Beta株可认为是一种免疫优势毒株。
本发明进一步研究了在形成Foldon三聚体时不同毒株RBD的组合对中和抗体滴度的影响。NTD-RBD(BA.2)-RBD(BA.1)-Foldon(SEQ ID NO:60)和NTD-RBD(BA.2)-RBD(WH01)-Foldon(SEQ ID NO:61)的5个血清样品针对不同假病毒的中和抗体滴度和几何平均滴度GMT如表6所示。
表6
Figure PCTCN2022115143-appb-000008
可见，NTD-RBD(BA.2)-RBD(BA.1)-Foldon针对BA.2假病毒具有很高的中和滴度，达到19802，针对BA.4假病毒也具有一定的抗体滴度，但针对D614G和BA.1的抗体滴度则很低。
而把其中来源于Omicron-BA.1变异株的RBD替换为来源于WH01株的RBD时，不仅针对BA.2假病毒的中和滴度提高，针对D614G和BA.4假病毒的抗体滴度也显著提高，只是针对BA.1的抗体滴度较低，这可能是因为BA.1与BA.2和BA.4的差异较 大所致。已公开的文献也已证明，BA.1与BA.2的抗原性有着显著差别(参见Antigenic cartography of SARS-CoV-2 reveals that Omicron BA.1 and BA.2 are antigenically distinct，ANNA Z.MYKYTYN et al.SCIENCE IMMUNOLOGY，23 Jun 2022)。
上述结果表明，在形成Foldon三聚体的情况下，WH01株的RBD可以提高抗原对不同变异株的中和抗体滴度，因此，在这种情况下WH01株也可认为是一种免疫优势毒株。
与之不同的是，本发明的进一步研究表明，当从阿尔法(Alpha)株、伽马(Gamma)株、艾普希龙(epsilon)、截塔(zeta)株、伊塔(Eta)株、西塔(theta)株、艾欧塔(Iota)株、喀帕(Kappa)株、拉姆达(Lambda)株、缪(Mu)株的受体结合区中选择抗原组分时，所得免疫原性物质仅针对相应毒株假病毒的滴度有所提高，而对于其他突变毒株假病毒的滴度没有明显的提高作用，这表明上述毒株的受体结合区针对其他毒株诱导抗体反应的较弱，不是本发明所述的免疫优势毒株。但这些可作为抗原的额外组分，以进一步提高疫苗对不同毒株的保护效果的均衡性。
已有的研究表明，当免疫原性物质中存在某毒株的受体结合区时，免疫原性物质针对该毒株假病毒的滴度会明显提高。结合新型冠状病毒的流行趋势，本发明将德尔塔(Delta)株或奥密克戎(Omicron)株作为流行优势毒株。本发明的试验研究表明，当免疫原性物质同时包含源自免疫优势毒株和流行优势毒株的受体结合区时，可以针对不同毒株假病毒均表现出较高的滴度，说明其可以对不同毒株均能够产生优异的免疫效果。
(2)添加NTD的影响
本发明进一步考察了在候选抗原中添加NTD的影响。在添加NTD后，各组的5个血清样品针对不同假病毒的中和抗体滴度和几何平均滴度GMT如表7所示。
表7
Figure PCTCN2022115143-appb-000009
Figure PCTCN2022115143-appb-000010
如表7所示，当RBD来自同一毒株时，与SEQ ID NO:18和SEQ ID NO:21相比，WH01株的NTD和RBD组合形成的候选抗原SEQ ID NO:45和Delta株的NTD和RBD组合形成的候选抗原SEQ ID NO:46针对不同假病毒的几何平均滴度均不同程度地提高，针对四种假病毒的几何平均滴度的平均值分别由38492和46948上升到53563和55471。
当RBD来自不同毒株时，与SEQ ID NO:19相比，WH01株的NTD和RBD以及Delta株的RBD组合形成的候选抗原SEQ ID NO:22针对不同假病毒的几何平均滴度明显提高，针对四种假病毒的几何平均滴度的平均值由29505上升到67293；与SEQ ID NO:20相比，Beta株的NTD和RBD以及Delta株的RBD组合形成的候选抗原SEQ ID NO:23针对D614G株和Delta株假病毒的几何平均滴度有所降低，但仍维持在较高水平，同时针对WH01株和Omicron BA.1株假病毒的几何平均滴度则有所提高，尤其是针对 Omicron BA.1株假病毒的几何平均滴度达到这几种组合中的最高值。因此，总体上来看，在加入NTD可以提高交叉保护能力。
(3)添加Foldon结构域的影响
本发明进一步考察了在候选蛋白中添加Foldon结构域形成三聚体的影响，在添加Foldon结构域后，各组的5个血清样品针对不同假病毒的中和抗体滴度和几何平均滴度GMT如表8所示。
表8
Figure PCTCN2022115143-appb-000011
如表7所示，与SEQ ID NO:19相比，WH01株和Delta株的RBD组合中进一步加入Foldon结构域(SEQ ID NO:26)或同时加入NTD和Foldon结构域(SEQ ID NO:27)以形成三聚体后，针对不同假病毒的几何平均滴度均明显提高，针对四种假病毒的几何平均滴度的平均值由29505分别上升到63178和53995，说明形成三聚体可以提高交叉保护能力。
可见，本发明所述由免疫优势毒株和流行优势毒株的RBD组合形成的抗原对不同毒株具有更为均衡的免疫效果，在进一步添加NTD或Foldon结构域后，免疫效果能够进一步提高。
实施例11 mRNA的制备
该实施例制备了表9所示抗原的mRNA。
表9
Figure PCTCN2022115143-appb-000012
在以上抗原的C端添加标签DYKDDDDKHHHHHHHH，将编码所述抗原的DNA序列按照人作为宿主进行密码子优化，并在5’端依次添加T7 RNA聚合酶结合序列、5’UTR(人β珠蛋白的5’UTR)、kozak序列和信号肽，在3’端依次添加3’UTR(人β珠蛋白的5’UTR)和PloyA(120)及酶切位点，得到DNA序列。
合成DNA序列并克隆至表达载体pUC57-kan中，测序验证目标基因。
将表达载体转化大肠杆菌E.coli感受态细胞，扩大培养后提取质粒，酶切得到线性化质粒样品，使用T7 High Yield RNA Transcription Kit(Novoprotein，CAT：E131-01A)按照说明书进行体外转录，使用Cap1 Capping System(Novoprotein，CAT:M082)按照说明书操作进行mRNA加帽，最后采用氯化锂沉淀纯化法进行纯化得到mRNA。
制备的mRNA序列为SEQ ID NO:68-89，其中，
SEQ ID NO:68的第123-2750位为抗原编码区，
SEQ ID NO:69的第123-1436位为抗原编码区，
SEQ ID NO:70的第123-1436位为抗原编码区，
SEQ ID NO:71的第123-1436位为抗原编码区，
SEQ ID NO:72的第123-2093位为抗原编码区，
SEQ ID NO:73的第123-2093位为抗原编码区，
SEQ ID NO:74的第123-2093位为抗原编码区，
SEQ ID NO:75的第123-2093位为抗原编码区，
SEQ ID NO:76的第123-2750位为抗原编码区，
SEQ ID NO:77的第123-2093位为抗原编码区，
SEQ ID NO:78的第123-1436位为抗原编码区，
SEQ ID NO:79的第123-2351位为抗原编码区，
SEQ ID NO:80的第123-2999位为抗原编码区，
SEQ ID NO:81的第123-2342位为抗原编码区，
SEQ ID NO:82的第123-3008位为抗原编码区，
SEQ ID NO:83的第123-2999位为抗原编码区，
SEQ ID NO:84的第123-3002位为抗原编码区，
SEQ ID NO:85的第123-2999位为抗原编码区，
SEQ ID NO:86的第123-2999位为抗原编码区，
SEQ ID NO:87的第123-2351位为抗原编码区，
SEQ ID NO:88的第123-2342位为抗原编码区，
SEQ ID NO:89的第123-2342位为抗原编码区。
实施例12 mRNA疫苗的制备
将实施例11获得的各mRNA与脂质纳米颗粒混合得到核酸-脂质纳米颗粒复合物，其中脂质纳米颗粒包含摩尔比为1:1的DOTMA和DOPE；复合物中mRNA的含量为100μg/ml，脂质纳米颗粒与mRNA的质量比为10:1。
实施例13 mRNA疫苗的制备
将实施例11获得的各mRNA与脂质纳米颗粒混合得到核酸-脂质纳米颗粒复合物，并进一步与等体积佐剂充分混合制得免疫样品，其中脂质纳米颗粒包含摩尔比为2:1的DOTAP和DOPE；复合物中mRNA的含量为200μg/ml，脂质纳米颗粒与mRNA的质量比为12:1；佐剂成分为每0.5ml包括10.69mg的角鲨烯、11.86mg的α-生育酚、4.86mg的聚山梨酯80、3.53mg的氯化钠、0.09mg的氯化钾、0.51mg的磷酸氢二钠、0.09mg的磷酸二氢钾和注射用水。
实施例14 mRNA疫苗的制备
将实施例11获得的各mRNA与脂质纳米颗粒混合得到核酸-脂质纳米颗粒复合物，并进一步与等体积佐剂充分混合制得免疫样品，其中脂质纳米颗粒包含摩尔比为1:1的DOTMA和胆固醇；复合物中mRNA的含量为100μg/ml，脂质纳米颗粒与mRNA的质量比为10:1；佐剂成分为每0.5ml包括19.5mg的角鲨烯，2.35mg Tween 80，2.35mg Span85和注射用水。
实施例15 mRNA疫苗的制备
将实施例11获得的各mRNA与脂质纳米颗粒混合得到核酸-脂质纳米颗粒复合物，并进一步与等体积佐剂充分混合制得免疫样品，其中脂质纳米颗粒包含摩尔比为2:1的DOTMA和DOPE；复合物中mRNA的含量为200μg/ml，脂质纳米颗粒与mRNA的质量比为8:1；佐剂成分为每0.5ml包括DOPC 1mg、胆固醇0.25mg、MPL 50μg和QS-21 50μg。
实施例16 mRNA疫苗小鼠免疫实验
利用实施例12获得的各mRNA疫苗对8～10周龄的BALB/c小鼠(购自北京华阜康生物技术有限公司)进行分组免疫，每组5只小鼠。分别于0天及21天进行小鼠免疫，每只每次肌肉注射50μl的免疫样品(包含5μg mRNA)，并于0天、21天以及第二次免疫后14天采血。采集的血液样品37℃放置1小时，4℃放置1小时，8000r/min离心10分钟，收集血清，-20℃保存，用于假病毒中和检测。各组的5个血清样品针对不同假病毒的中和抗体滴度和几何平均滴度GMT结果如表10所示。
表10
Figure PCTCN2022115143-appb-000013
Figure PCTCN2022115143-appb-000014
可以看出，由编码RBD(Beta)-RBD(Beta)的mRNA(SEQ ID NO:78)制备的疫苗针对不同假病毒的中和抗体滴度GMT的平均值为31133，且针对Omicron毒株的中和抗体滴度明显低于其他毒株，与之相比，由编码RBD(WH01)-RBD(Beta)-RBD(Delta)-RBD(BA.1)的mRNA(SEQ ID NO:68)制备的疫苗针对不同假病毒的中和抗体滴度GMT的平均值达到99781，由编码RBD(WH01)-RBD(BA.1)的mRNA(SEQ ID NO:69)和编码RBD(Beta)-RBD(Delta)的mRNA(SEQ ID NO:70)制备的疫苗针对不同假病毒的中和抗体滴度GMT的平均值达到58513，由编码RBD(Beta)-RBD(BA.1)的mRNA(SEQ ID NO:71)制备的疫苗针对不同假病毒的中和抗体滴度GMT的平均值达到44561，由编码 RBD(Beta)-RBD(WH01)-RBD(Delta)的mRNA(SEQ ID NO:75)制备的疫苗针对不同假病毒的中和抗体滴度GMT的平均值达到67155，并且这些疫苗针对Omicron毒株的中和抗体滴度均有数倍的增长。
可见，利用编码本发明所述免疫原性物质的mRNA制备的mRNA疫苗对不同毒株同样具有很好的免疫效果。可以预期，由本发明的免疫原性物质和编码所述免疫原性物质的核酸制备的疫苗针对新冠病毒将来可能出现的突变株仍会具有较好的预防效果。因此，在新型冠状病毒不断发生突变的情况下，本发明具有重要的指导意义。
以上所述的具体实施例对本发明的目的、技术方案和有益效果进行了进一步详细说明，应理解的是，以上所述仅为本发明的具体实施例而已，并不用于限制本发明，凡在本发明的精神和原则之内所做的任何修改、等同替换、改进等，均应包含在本发明的保护范围之内。

Claims (24)

  1. 一种新型冠状病毒免疫原性物质，其特征在于，所述新型冠状病毒免疫原性物质包含源自免疫优势毒株的第一抗原和源自流行优势毒株的第二抗原，各抗原分别包含S蛋白的受体结合区或受体结合区的一部分，其中，所述免疫优势毒株选自新型冠状病毒WH01株和贝塔(Beta)株中的至少一种，所述流行优势毒株选自新型冠状病毒德尔塔(Delta)株、奥密克戎(Omicron)BA.1、BA.2、BA.3、BA.4和BA.5变异株中的至少一种。
  2. 根据权利要求1所述的新型冠状病毒免疫原性物质，其特征在于，源自WH01株的S蛋白的受体结合区包含SEQ ID NO:1、SEQ ID NO:2、SEQ ID NO:3或SEQ ID NO:4中的任一项所示的氨基酸序列；
    源自贝塔(Beta)株的S蛋白的受体结合区包含SEQ ID NO:5、SEQ ID NO:6、SEQ ID NO:7或SEQ ID NO:8中的任一项所示的氨基酸序列；
    源自德尔塔(Delta)株的S蛋白的受体结合区包含SEQ ID NO:9、SEQ ID NO:10、SEQ ID NO:11或SEQ ID NO:12中的任一项所示的氨基酸序列；
    源自奥密克戎(Omicron)BA.1变异株的S蛋白的受体结合区包含SEQ ID NO:42所示的氨基酸序列，源自奥密克戎(Omicron)BA.2变异株的S蛋白的受体结合区包含SEQ ID NO:62所示的氨基酸序列，源自奥密克戎(Omicron)BA.3变异株的S蛋白的受体结合区包含SEQ ID NO:63所示的氨基酸序列，源自奥密克戎(Omicron)BA.4和BA.5变异株的S蛋白的受体结合区包含SEQ ID NO:64所示的氨基酸序列。
  3. 根据权利要求1所述的新型冠状病毒免疫原性物质，其特征在于，所述新型冠状病毒免疫原性物质包含SEQ ID NO:19，20，28，29，47，48，51-55中任一项所示的氨基酸序列。
  4. 根据权利要求1所述的新型冠状病毒免疫原性物质，其特征在于，所述抗原还包含S蛋白的S1亚基中的N端结构域(NTD)，优选地，所述N端结构域位于所述第一抗原和/或所述第二抗原的氨基酸序列的N端，其中，
    源自WH01株的S蛋白的N端结构域包含SEQ ID NO:13所示的氨基酸序列，
    源自贝塔(Beta)株的S蛋白的N端结构域包含SEQ ID NO:14所示的氨基酸序列，
    源自德尔塔(Delta)株的S蛋白的N端结构域包含SEQ ID NO:15所示的氨基酸序列，
    源自奥密克戎(Omicron)BA.1变异株的S蛋白的N端结构域包含SEQ ID NO:43所示的氨基酸序列，源自奥密克戎(Omicron)BA.2变异株的S蛋白的N端结构域包含SEQ ID NO:65所示的氨基酸序列，源自奥密克戎(Omicron)BA.3变异株的S蛋白的N端结构域包含SEQ ID NO:66所示的氨基酸序列，源自奥密克戎(Omicron)BA.4和BA.5变异株的S蛋白的N端结构域包含SEQ ID NO:67所示的氨基酸序列。
  5. 根据权利要求4所述的新型冠状病毒免疫原性物质，其特征在于，所述新型冠状病毒免疫原性物质包含SEQ ID NO:22，23，30，49，50，56-59中任一项所示的氨基酸序列。
  6. 根据权利要求1所述的新型冠状病毒免疫原性物质，其特征在于，所述新型冠状病毒免疫原性物质还包含免疫球蛋白的Fc结构域，优选地，所述免疫球蛋白为人源IgG。
  7. 根据权利要求6所述的新型冠状病毒免疫原性物质，其特征在于，所述Fc结构域位于所述新型冠状病毒免疫原性物质的氨基酸序列的C端，优选地，人源IgG Fc结构域包含SEQ ID NO:16所示的氨基酸序列。
  8. 根据权利要求7所述的新型冠状病毒免疫原性物质，其特征在于，所述新型冠状病毒免疫原性物质包含SEQ ID NO:24或SEQ ID NO:25所示的氨基酸序列。
  9. 根据权利要求1所述的新型冠状病毒免疫原性物质，其特征在于，所述新型冠状病毒免疫原性物质还包含Foldon结构域。
  10. 根据权利要求9所述的新型冠状病毒免疫原性物质，其特征在于，所述Foldon结构域位于所述新型冠状病毒免疫原性物质的氨基酸序列的C端，优选地，Foldon结构域包含SEQ ID NO:17所示的氨基酸序列。
  11. 根据权利要求10所述的新型冠状病毒免疫原性物质，其特征在于，所述新型冠状病毒免疫原性物质包含SEQ ID NO:26，27，60，61中任一项所示的氨基酸序列。
  12. 根据权利要求1所述的新型冠状病毒免疫原性物质，其特征在于，所述新型冠状病毒免疫原性物质还包含源自所述免疫优势毒株和所述流行优势毒株以外毒株的一个或多个抗原。
  13. 根据权利要求12所述的新型冠状病毒免疫原性物质，其特征在于，所述免疫优势毒株和所述流行优势毒株以外毒株选自以下毒株：阿尔法(Alpha)株、伽马(Gamma)株、艾普希龙(Epsilon)、截塔(Zeta)株、伊塔(Eta)株、西塔(Theta)株、艾欧塔(Iota)株、喀帕(Kappa)株、拉姆达(Lambda)株和缪(Mu)株。
  14. 根据权利要求1-13任一项所述的新型冠状病毒免疫原性物质，其特征在于，各抗原构成组合物，或者各抗原直接相连或通过氨基酸接头连接。
  15. 一种制备权利要求1-14中任一项所述新型冠状病毒免疫原性物质的方法，其特征在于，包括以下步骤：
    利用编码所述新型冠状病毒免疫原性物质的核苷酸序列构建重组表达质粒；
    将构建的重组表达质粒转化宿主菌，筛选正确的重组表达质粒；
    利用筛选的重组表达质粒转染表达系统的细胞，表达后收集上清并纯化得到新型冠状病毒免疫原性物质。
  16. 根据权利要求15所述的方法，其特征在于，所述表达系统的细胞包括哺乳动物细胞、昆虫细胞、酵母细胞或细菌细胞，可选地，所述哺乳动物细胞包括293T细胞或CHO细胞，所述细菌细胞包括大肠杆菌细胞。
  17. 一种编码权利要求1-14中任一项所述新型冠状病毒免疫原性物质的核苷酸序列。
  18. 一种包含权利要求17所述核苷酸序列的重组载体。
  19. 一种携带有权利要求18所述重组载体的表达系统细胞。
  20. 权利要求1-14中任一项所述新型冠状病毒免疫原性物质、权利要求17所述核苷酸序列、权利要求18所述重组载体或权利要求19所述表达系统细胞在制备新型冠状病毒疫苗中的应用。
  21. 一种新型冠状病毒蛋白疫苗，其特征在于，包括权利要求1-14中任一项所述新型冠状病毒免疫原性物质和佐剂，所述佐剂选自铝佐剂、MF59佐剂、MPL佐剂、QS-21、GLA、CpG、AS01、AS02、AS03、AS04佐剂中的一种或多种，优选为AS03或MF59佐剂。
  22. 一种新型冠状病毒DNA疫苗，其特征在于，所述DNA疫苗包含编码权利要求1-14中任一项所述新型冠状病毒免疫原性物质的DNA序列。
  23. 一种新型冠状病毒mRNA疫苗，其特征在于，所述mRNA疫苗包含编码权利要求1-14中任一项所述新型冠状病毒免疫原性物质的mRNA序列。
  24. 根据权利要求23所述的新型冠状病毒mRNA疫苗，其特征在于，所述mRNA包含下述序列中的至少一项：
    SEQ ID NO:68中第123-2750位所示的mRNA序列，
    SEQ ID NO:69中第123-1436位所示的mRNA序列，
    SEQ ID NO:70中第123-1436位所示的mRNA序列，
    SEQ ID NO:71中第123-1436位所示的mRNA序列，
    SEQ ID NO:72中第123-2093位所示的mRNA序列，
    SEQ ID NO:73中第123-2093位所示的mRNA序列，
    SEQ ID NO:74中第123-2093位所示的mRNA序列，
    SEQ ID NO:75中第123-2093位所示的mRNA序列，
    SEQ ID NO:79中第123-2351位所示的mRNA序列，
    SEQ ID NO:82中第123-3008位所示的mRNA序列，
    SEQ ID NO:83中第123-2999位所示的mRNA序列，
    SEQ ID NO:84中第123-3002位所示的mRNA序列，
    SEQ ID NO:85中第123-2999位所示的mRNA序列，
    SEQ ID NO:86中第123-2999位所示的mRNA序列，
    SEQ ID NO:87中第123-2351位所示的mRNA序列，
    SEQ ID NO:88中第123-2342位所示的mRNA序列，
    SEQ ID NO:89中第123-2342位所示的mRNA序列。
PCT/CN2022/115143 2021-08-26 2022-08-26 新型冠状病毒免疫原性物质、其制备方法和应用 WO2023025287A1 (zh)

Applications Claiming Priority (8)

Application Number Priority Date Filing Date Title
CN202110988771 2021-08-26
CN202110988771.6 2021-08-26
CN202210112983.2 2022-01-29
CN202210112983 2022-01-29
CN202210387826.2 2022-04-13
CN202210387826 2022-04-13
CN202210514880.9 2022-05-11
CN202210514880 2022-05-11

Publications (1)

Publication Number Publication Date
WO2023025287A1 true WO2023025287A1 (zh) 2023-03-02

Family

ID=85322450

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2022/115143 WO2023025287A1 (zh) 2021-08-26 2022-08-26 新型冠状病毒免疫原性物质、其制备方法和应用

Country Status (2)

Country Link
CN (3) CN116041448A (zh)
WO (1) WO2023025287A1 (zh)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN116350769A (zh) * 2023-03-31 2023-06-30 北京吉诺卫生物科技有限公司 一种新冠病毒和流感病毒的联合疫苗、其制备方法与应用

Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN111944064A (zh) * 2020-08-21 2020-11-17 中国科学院微生物研究所 一种covid-19亚单位疫苗及其制备方法
CN112043825A (zh) * 2020-07-13 2020-12-08 中国医学科学院医学生物学研究所 一种基于新型冠状病毒突刺蛋白s1区域预防新型冠状病毒感染的亚单位疫苗
CN112048005A (zh) * 2020-09-04 2020-12-08 江苏省中国科学院植物研究所 新型冠状病毒s蛋白片段多倍体及其制备方法、检测试剂盒、疫苗及药物
CN112760341A (zh) * 2020-07-13 2021-05-07 中国科学院微生物研究所 重组载体、包装有该重组载体的黑猩猩腺病毒在制备2019新型冠状病毒疫苗中的应用
CN112794884A (zh) * 2020-12-28 2021-05-14 中国科学院微生物研究所 新型冠状病毒蛋白、制备方法及中和抗体检测试剂盒
WO2021160850A1 (en) * 2020-02-13 2021-08-19 Institut Pasteur Measles-vectored covid-19 immunogenic compositions and vaccines
CN113292640A (zh) * 2021-06-18 2021-08-24 国药中生生物技术研究院有限公司 一种产生广谱交叉中和活性的重组新型冠状病毒rbd三聚体蛋白疫苗、其制备方法和应用

Patent Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2021160850A1 (en) * 2020-02-13 2021-08-19 Institut Pasteur Measles-vectored covid-19 immunogenic compositions and vaccines
CN112043825A (zh) * 2020-07-13 2020-12-08 中国医学科学院医学生物学研究所 一种基于新型冠状病毒突刺蛋白s1区域预防新型冠状病毒感染的亚单位疫苗
CN112760341A (zh) * 2020-07-13 2021-05-07 中国科学院微生物研究所 重组载体、包装有该重组载体的黑猩猩腺病毒在制备2019新型冠状病毒疫苗中的应用
CN111944064A (zh) * 2020-08-21 2020-11-17 中国科学院微生物研究所 一种covid-19亚单位疫苗及其制备方法
CN112048005A (zh) * 2020-09-04 2020-12-08 江苏省中国科学院植物研究所 新型冠状病毒s蛋白片段多倍体及其制备方法、检测试剂盒、疫苗及药物
CN112794884A (zh) * 2020-12-28 2021-05-14 中国科学院微生物研究所 新型冠状病毒蛋白、制备方法及中和抗体检测试剂盒
CN113292640A (zh) * 2021-06-18 2021-08-24 国药中生生物技术研究院有限公司 一种产生广谱交叉中和活性的重组新型冠状病毒rbd三聚体蛋白疫苗、其制备方法和应用

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
COHEN ALEXANDER A, GNANAPRAGASAM PRIYANTHI, LEE YU E, HOFFMAN PAULINE R, OU SUSAN, KAKUTANI LEESA M, KEEFFE JENNIFER R, WU HUNG-JE: "Mosaic nanoparticles elicit cross-reactive immune responses to zoonotic coronaviruses in mice", SCIENCE, AMERICAN ASSOCIATION FOR THE ADVANCEMENT OF SCIENCE, US, vol. 371, no. 6530, 12 February 2021 (2021-02-12), US , pages 735 - 741, XP055846840, ISSN: 0036-8075, DOI: 10.1126/science.abf6840 *
HUAN, YU; BI, YU HAI: "Research Progress and Prospect of Vaccines for the Coronavirus Disease 2019 (COVID-19)", SCIENCE CHINA: CHINESE BULLETIN OF LIFE SCIENCE = SCIENTIA SINICA VITAE, vol. 52, no. 2, 17 May 2021 (2021-05-17), pages 237 - 248, XP009543770, ISSN: 1674-7232 *

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN116350769A (zh) * 2023-03-31 2023-06-30 北京吉诺卫生物科技有限公司 一种新冠病毒和流感病毒的联合疫苗、其制备方法与应用

Also Published As

Publication number Publication date
CN116041448A (zh) 2023-05-02
CN115960252A (zh) 2023-04-14
CN116041534A (zh) 2023-05-02

Similar Documents

Publication Publication Date Title
CN111217917B (zh) 一种新型冠状病毒SARS-CoV-2疫苗及其制备方法
CN111217918B (zh) 一种基于2,4-二氧四氢喋啶合酶的新型冠状病毒s蛋白双区域亚单位纳米疫苗
JP7250878B2 (ja) 新規多価ナノ粒子に基づくワクチン
WO2022262142A1 (zh) 一种产生广谱交叉中和活性的重组新型冠状病毒rbd三聚体蛋白疫苗、其制备方法和应用
CN111560074B (zh) 一种基于幽门螺旋杆菌铁蛋白的新型冠状病毒s蛋白单区域亚单位纳米疫苗
CN109535228B (zh) 用作诱发抗原特异性t细胞反应的免疫原性增强剂的融合蛋白
CN111217919A (zh) 一种基于火球菌铁蛋白的新型冠状病毒s蛋白双区域亚单位纳米疫苗
WO2023051850A1 (zh) 一类来源于新型冠状病毒s2蛋白hr区域的重组融合蛋白及其应用
CN111116720B (zh) 一种猪瘟病毒重组e2蛋白及其应用
WO2023138334A1 (zh) 一种重组新型冠状病毒蛋白疫苗、其制备方法和应用
CN116019906A (zh) 新型冠状病毒免疫原性组合物、其制备方法和应用
WO2023051701A1 (zh) 抗SARS-CoV-2感染的mRNA、蛋白以及抗SARS-CoV-2感染的疫苗
CN112552413B (zh) 新型冠状病毒重组蛋白亚单位疫苗
WO2023025287A1 (zh) 新型冠状病毒免疫原性物质、其制备方法和应用
KR20230107621A (ko) 메타뉴모바이러스에 대한 단백질-기반 나노입자 백신
CN109966483A (zh) 一种基于铁蛋白的多抗原通用流感疫苗及其制备方法和应用
KR20230084478A (ko) 면역원성 코로나 바이러스 융합 단백질 및 관련 방법
JP2023523423A (ja) ＳＡＲＳ－ＣｏＶ－２に対するワクチン及びその調製物
CN112142851B (zh) 一种狂犬病毒表面的亚单位融合蛋白tG及其制备方法和应用
WO2023138333A1 (zh) 一种重组新型冠状病毒蛋白疫苗、其制备方法和应用
CN114502572A (zh) 展示副粘病毒和/或肺炎病毒f蛋白的自组装蛋白纳米结构及其用途
CN115960259B (zh) 一种模块化组装双组分纳米颗粒制备方法及其应用
CN113862284B (zh) 一种编码重组禽流感病毒ha蛋白的基因、病毒样颗粒、疫苗及制备与应用
WO2021039873A1 (ja) ウイルスの非構造タンパク質が担持された複合タンパク質単量体、当該単量体の会合体、及び当該会合体を有効成分とするコンポーネントワクチン
KR20230091094A (ko) 융합 단백질 및 백신

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 22860633

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE