CN115960252A - Novel coronavirus immunogenic substance, preparation method and application thereof - Google Patents

Novel coronavirus immunogenic substance, preparation method and application thereof Download PDF

Info

Publication number
CN115960252A
CN115960252A CN202211033865.9A CN202211033865A CN115960252A CN 115960252 A CN115960252 A CN 115960252A CN 202211033865 A CN202211033865 A CN 202211033865A CN 115960252 A CN115960252 A CN 115960252A
Authority
CN
China
Prior art keywords
strain
seq
amino acid
antigen
acid sequence
Prior art date
Legal status (The legal status 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 status listed.)
Pending
Application number
CN202211033865.9A
Other languages
Chinese (zh)
Inventor
胡颖嵩
张元杰
张岭
姚文荣
吴双
袁楚晓
李娇
陈健平
于佳平
刘勇
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Abzymo Biosciences Co ltd
Jiangsu Ruike Biotechnology Co ltd
Original Assignee
Abzymo Biosciences Co ltd
Jiangsu Ruike Biotechnology Co ltd
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 Abzymo Biosciences Co ltd, Jiangsu Ruike Biotechnology Co ltd filed Critical Abzymo Biosciences Co ltd
Publication of CN115960252A publication Critical patent/CN115960252A/en
Pending legal-status Critical Current

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

Abstract

The present invention provides a novel coronavirus immunogenic material comprising a first antigen from an immunodominant strain and a second antigen from an epidemic dominant strain, said antigens comprising a receptor binding region or a part of a receptor binding region and an N-terminal domain (NTD) or a part of an N-terminal domain of the S protein, respectively, wherein the immunodominant strain is selected from at least one of a novel coronavirus WH01 strain and a Beta (Beta) strain, and the epidemic dominant strain is selected from at least one of a novel coronavirus Delta (Delta) strain and an ormekron (omitron) strain. The novel coronavirus immunogenic substance has higher immunogenicity, and can show remarkably improved immune effect on different strains.

Description

Novel coronavirus immunogenic substance, preparation method and application thereof
Technical Field
The invention belongs to the technical field of biomedical engineering, and relates to a novel coronavirus immunogenic substance, a preparation method and application thereof.
Background
Infection with the novel coronavirus (SARS-CoV-2) can lead to coronavirus disease (COVID-19), with the common signs of fever, cough, sore throat, etc., and in more severe cases, the infection can lead to dyspnea, hypoxemia, acute respiratory distress syndrome, and even death. The novel coronaviruses can be transmitted from person to person via respiratory and droplet routes, and there is also the possibility of transmission through the air and digestive tracts.
The SARS-CoV-2 virus particle comprises 4 structural proteins, namely spike protein (S), nucleocapsid protein (N), membrane protein (M) and envelope protein (E). It has been found that only antibodies directed against the S protein have neutralizing activity and therefore vaccines currently under development all comprise the S protein or a component thereof. Among them, the receptor binding region of the S protein is considered to be the most important antigen target region for inducing the body to produce neutralizing antibodies. The receptor binding region as a vaccine can focus the neutralizing antibody generated by the stimulation of an organism on the receptor binding of the virus, and can improve the immunogenicity and the immune efficiency of the vaccine. SARS-CoV-2 enters the cell by binding to the host cell receptor hACE2 through its receptor binding region.
New coronaviruses evolve continuously during transmission, and a plurality of representative mutants have been detected at present. Most of the novel coronavirus antigens developed or developed at present can only be directed to one strain, and neutralizing antibodies directed to different strains cannot be generated. CN114369172A designs prototype strain RBD dimer vaccine, beta strain RBD dimer vaccine and prototype strain + Beta strain chimeric RBD dimer vaccine respectively. The result shows that compared with the prototype strain RBD dimer vaccine and the Beta strain RBD dimer vaccine, the prototype strain and Beta strain chimeric RBD dimer vaccine induces more balanced antibody reaction. However, from the results thereof, the neutralizing antibody titer of the prototype strain + Beta strain chimeric RBD dimer vaccine against the Omicron variant S protein pseudovirus was significantly reduced as compared with the prototype strain and other variants, which indicates that the vaccine is not excellent in the protective ability against the currently circulating Omicron variant, and therefore, it is necessary to develop a vaccine having a more balanced protective effect against different strains.
Disclosure of Invention
The invention aims to provide a novel coronavirus immunogenic substance, which comprises receptor binding regions and N-terminal domains (NTD) of S protein S1 subunits of different strains, has higher immunogenicity, can stimulate the generation of neutralizing antibodies aiming at different strains, and can obviously improve the immune effect.
In order to realize the purpose of the invention, the invention provides the following technical scheme:
a novel coronavirus immunogenic substance comprising 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 part of a receptor binding region and an N-terminal domain (NTD) or part of an N-terminal domain of an S protein, respectively.
In some embodiments, the immunodominant strain is selected from at least one of the novel coronavirus WH01 strain and the Beta (Beta) strain.
In some embodiments, the pandemic dominant strain is selected from at least one of the novel coronavirus Delta (Delta) strain and the ormekron (Omicron) strain.
In some embodiments, the Ormckron (Omicron) strains include BA.1, BA.2, BA.3, BA.4, and BA.5 variant strains.
In some embodiments, the novel coronavirus immunogenic agent further comprises a third antigen derived from the immunodominant strain and a strain other than the circulating dominant strain.
In some embodiments, the novel coronavirus immunogenic material further comprises a fourth antigen derived from the immunodominant strain and a strain other than the pandemic dominant strain.
In some embodiments, the immunodominant strain and the strain other than the pandemic dominant strain are selected from the following strains: alpha (Alpha) strain, gamma (Gamma) strain, epsilon (Epsilon), zeta (Zeta) strain, eta (Eta) strain, theta (Theta) strain, eatota (Iota) strain, kappa (Kappa) strain, lambda (Lambda) strain, mur (Mu) strain, etc.
In some embodiments, each antigen constitutes a composition, or each antigen is linked directly or through an amino acid linker, for example the amino acid linker may be a GGS or multiple GGSs in series (G and S represent glycine and serine, respectively).
For example, in some embodiments, the novel coronavirus immunogenic material comprises a first antigen and a second antigen, the first antigen being linked to the second antigen either directly or through an amino acid linker. In other embodiments, the novel coronavirus immunogenic material comprises a first antigen and a second antigen, wherein the first antigen is mixed with the second antigen to form a composition. In some embodiments, the novel immunogenic agent of coronavirus is formed by directly linking the receptor binding domain of the S protein of novel coronavirus strain WH01 and the receptor binding domain of Delta (Delta) strain; in some embodiments, the novel immunogenic agent of coronavirus is formed by direct linkage of the receptor binding domain of the S protein of novel coronavirus WH01 strain and the receptor binding domain of the ormekron (omitron) strain; in some embodiments, the novel coronavirus immunogenic agent is formed by direct linkage of the receptor binding domain of the S protein of the novel coronavirus Beta (Beta) strain and the receptor binding domain of a Delta (Delta) strain; in some embodiments, the novel immunogenic coronavirus material is formed by directly linking the receptor binding domain of the S protein of the novel coronavirus Beta (Beta) strain to the receptor binding domain of the Ormck Ron (Omicron) strain.
In some embodiments, the novel coronavirus immunogenic material comprises a first antigen, a second antigen, and a third antigen, the first antigen, the second antigen, and the third antigen being linked directly or through an amino acid linker. In other embodiments, the novel coronavirus immunogenic material comprises a first antigen, a second antigen and a third antigen, wherein the first antigen is directly linked to the second antigen or linked through an amino acid linker to form a fusion antigen, and the fusion antigen is mixed with the third antigen to form a composition. In other embodiments, the novel coronavirus immunogenic material comprises a first antigen, a second antigen and a third antigen, wherein the first antigen, the second antigen and the third antigen are mixed to form a composition.
In some embodiments, the novel coronavirus immunogenic material comprises a first antigen, a second antigen, a third antigen, and a fourth antigen, wherein the first antigen, the second antigen, the third antigen, and the fourth antigen are linked directly or via an amino acid linker to form a fusion antigen. In some embodiments, the novel coronavirus immunogenic material comprises a first antigen, a second antigen, a third antigen and a fourth antigen, wherein the first antigen is directly connected with 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 the composition. In other embodiments, the novel coronavirus immunogenic material comprises a first antigen, a second antigen, a third antigen, and a fourth antigen, wherein the first antigen, the second antigen, and the third antigen are linked directly or via an amino acid linker to form a fusion antigen, and the fusion antigen is mixed with the fourth antigen to form a composition. In other embodiments, the novel coronavirus immunogenic material comprises a first antigen, a second antigen, a third antigen, and a fourth antigen, wherein the first antigen is linked to the second antigen directly or via an amino acid linker to form a fusion antigen, and the fusion antigen is mixed with the third antigen and the fourth antigen to form a composition. In other embodiments, the novel coronavirus immunogenic material comprises a first antigen, a second antigen, a third antigen, and a fourth antigen, wherein the first antigen, the second antigen, the third antigen, and the fourth antigen are mixed to form a composition.
In the present invention, the first, second, third and fourth are used only to indicate different kinds of antigens, and do not indicate any order between the antigens. The kind and amount of the antigen in the present invention are not limited, and those skilled in the art can determine the appropriate kind and amount of the antigen according to the cross-reactivity of the widely spread strains and the antigen-antibody.
In some embodiments, the antigens each comprise at least 8 cysteines, and the number of cysteines is an even number.
In some embodiments, the receptor binding region of the S protein derived from the WH01 strain comprises the amino acid sequence set forth in any one of SEQ ID NO 1, SEQ ID NO 2, SEQ ID NO 3, or SEQ ID NO 4.
In some embodiments, the receptor binding region of the S protein derived from a Beta (Beta) strain comprises an amino acid sequence set forth in any one of SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, or SEQ ID NO: 8.
In some embodiments, the receptor binding region of the S protein derived from the Delta strain comprises the amino acid sequence set forth in any one of SEQ ID NO 9, SEQ ID NO 10, SEQ ID NO 11, or SEQ ID NO 12.
In some embodiments, the receptor binding region of the S protein from the Ormcknon (Omicron) BA.1 variant comprises the amino acid sequence shown in SEQ ID NO:42, the receptor binding region of the S protein from the BA.2 variant comprises the amino acid sequence shown in SEQ ID NO:62, the receptor binding region of the S protein from the Ormcknon (Omicron) BA.3 variant comprises the amino acid sequence shown in SEQ ID NO:63, and the receptor binding regions of the S proteins from the Ormcknon (Omicron) BA.4 and BA.5 variants comprise the amino acid sequence shown in SEQ ID NO: 64.
In some embodiments, the N-terminal domain is N-terminal to the amino acid sequence of the first antigen and/or the second antigen.
In some embodiments, the N-terminal domain of the S protein derived from the WH01 strain comprises the amino acid sequence set forth in SEQ ID NO 13.
In some embodiments, the N-terminal domain of the S protein derived from the Beta (Beta) strain comprises the amino acid sequence set forth in SEQ ID NO 14.
In some embodiments, the N-terminal domain of the S protein derived from the Delta strain comprises the amino acid sequence set forth in SEQ ID NO. 15.
In some embodiments, the N-terminal domain of the S protein from the Oromkron (Omicron) BA.1 variant comprises the amino acid sequence shown by SEQ ID NO:43, the N-terminal domain of the S protein from the Oromkron (Omicron) BA.2 variant comprises the amino acid sequence shown by SEQ ID NO:65, the N-terminal domain of the S protein from the Oromkron (Omicron) BA.3 variant comprises the amino acid sequence shown by SEQ ID NO:66, and the N-terminal domains of the S proteins from the Oomkron (Omicron) BA.4 and BA.5 variants comprise the amino acid sequence shown by SEQ ID NO: 67.
In some embodiments, the novel coronavirus immunogenic material comprises an amino acid sequence set forth in any one of SEQ ID NOs 22,23,30,49,50, 56-59.
In some embodiments, the novel coronavirus immunogenic agent further comprises an Fc domain of an immunoglobulin, preferably the immunoglobulin is human IgG.
In some embodiments, the Fc domain is C-terminal to the amino acid sequence of the novel coronavirus immunogenic agent, preferably, the human IgG Fc domain comprises the amino acid sequence set forth in SEQ ID No. 16.
In some embodiments, the novel coronavirus immunogenic material comprises the amino acid sequence set forth in SEQ ID NO. 24 or SEQ ID NO. 25.
In some embodiments, the novel coronavirus immunogenic material further comprises a Foldon domain. The Foldon domain/protein is derived from the C-terminal of the fibrous protein of the T4 bacteriophage, consists of 27 amino acids, and has the function of promoting the non-covalent oligomerization of the target protein to form a trimer.
In some embodiments, the Foldon domain is C-terminal to the amino acid sequence of the novel coronavirus immunogenic agent, preferably the Foldon domain comprises the amino acid sequence shown in SEQ ID No. 17.
In some embodiments, the novel coronavirus immunogenic material comprises an amino acid sequence set forth in any one of SEQ ID NOs 26,27,60, 61.
The invention also provides a method for preparing the novel coronavirus immunogenic material, which comprises the following steps:
constructing a recombinant expression plasmid by using a nucleotide sequence for coding the novel coronavirus immunogenic substance;
transforming the constructed recombinant expression plasmid into host bacteria, and screening correct recombinant expression plasmid;
and transfecting cells of the expression system by using the screened recombinant expression plasmid, and collecting and purifying supernatant after expression to obtain the novel coronavirus immunogenic substance.
In some embodiments, the cell of the expression system comprises a mammalian cell, optionally a 293T cell or a CHO cell, an insect cell, a yeast cell or a bacterial cell, optionally a 293T cell or a CHO cell, and a bacterial cell, optionally a bacterial cell, comprising an e.
The invention also provides a nucleotide sequence for coding the novel coronavirus immunogenic substance, a recombinant vector containing the nucleotide sequence and an expression system cell carrying the recombinant vector.
The invention also provides the novel coronavirus immunogenic substance, a nucleotide sequence for coding the novel coronavirus immunogenic substance, a recombinant vector containing the nucleotide sequence and application of an expression system cell carrying the recombinant vector in preparation of a novel coronavirus vaccine.
The invention also provides a novel coronavirus protein vaccine which comprises the novel coronavirus immunogenic substance and an adjuvant.
In some embodiments, the adjuvant is selected from one or more of aluminium adjuvant, MF59 adjuvant, MPL adjuvant, QS-21, GLA, cpG, AS01, AS02, AS03, AS04 adjuvant, preferably AS03 adjuvant or MF59 adjuvant.
The invention also provides a novel coronavirus DNA vaccine comprising a DNA sequence encoding said novel coronavirus immunogenic material.
The invention also provides a novel coronavirus mRNA vaccine comprising an mRNA sequence encoding the novel coronavirus immunogenic substance.
Compared with the prior art, the technical scheme of the invention has the following beneficial effects:
the novel coronavirus immunogenic substance comprises at least two antigens from immunodominant strains and epidemic dominant strains, and can induce and generate neutralizing antibodies aiming at different strains.
The inventor surprisingly finds that the immune dominant strain based on the WH01 strain or the Beta (Beta) strain of the novel coronavirus can generate remarkable immune effect by combining with the currently popular mutant strain, and the WH01 strain or the Beta (Beta) strain can generate antibodies aiming at different mutant strains by using the stone of immune combination to ensure the stability of immunity. Particularly, in the examples, recombinant proteins constructed by using WH01 strain and Ormckron (Omicron) strain, beta (Beta) strain and Delta (Delta) strain in combination can maintain balanced GMT level of neutralizing antibody for different mutant strains, and overcome the immune escape of the mutant strains to produce outstanding immune effect. The research of the invention also shows that the mRNA vaccine prepared by the mRNA for encoding the immunogenic substance has good immune effect on different strains.
The invention also researches the combined immune effect of different adjuvants and immunogenic substances, and the result shows that when an oil-in-water emulsion adjuvant (especially AS03 adjuvant) is used, higher neutralizing antibody titer can be generated.
Drawings
FIG. 1 is a schematic diagram of the S protein domain.
FIG. 2 shows the results of SDS-PAGE and Western Blot for the antigen SEQ ID NO. 18.
FIG. 3 shows the results of SDS-PAGE and Western Blot for antigen SEQ ID NO 19.
FIG. 4 shows the results of SDS-PAGE and Western Blot for the antigen SEQ ID NO. 20.
FIG. 5 shows the results of SDS-PAGE and Western Blot for antigen SEQ ID NO: 21.
FIG. 6 shows the results of SDS-PAGE and Western Blot for the antigen SEQ ID NO. 22.
FIG. 7 shows the results of SDS-PAGE and Western Blot for antigen SEQ ID NO: 23.
FIG. 8 shows the results of SDS-PAGE and Western Blot of antigen SEQ ID NO: 26.
FIG. 9 shows the results of SDS-PAGE and Western Blot of antigen SEQ ID NO: 27.
FIG. 10 shows the results of SDS-PAGE and Western Blot of antigen SEQ ID NO: 28.
FIG. 11 shows the results of SDS-PAGE and Western Blot of antigen SEQ ID NO: 30.
FIG. 12 shows the results of SDS-PAGE and Western Blot for antigen SEQ ID NO: 44.
FIG. 13 shows the results of SDS-PAGE and Western Blot for the antigen SEQ ID NO: 60.
FIG. 14 shows the results of SDS-PAGE and Western Blot for the antigen SEQ ID NO 61.
Detailed Description
In order that the objects, technical solutions and advantages of the present invention will become more apparent, the present invention will be further described in detail with reference to the accompanying drawings in conjunction with the following specific embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
The "immunodominant strain" in the present invention refers to a variant strain which has higher immunogenicity of antigen than a non-immunodominant variant strain and can provide better cross-protection for other variants.
The "epidemic dominant strain" in the present invention refers to the main variant strain that is currently epidemic, and can be generally understood as the "variant strain that needs attention" (VOC) that is currently epidemic. The definition of VOC can be referred to the work definition of the World Health Organization (WHO), namely: variants of SARS-CoV-2 that meet the definition of "Variants in need of attention" (VOI), and have been shown to be associated with one or more of the following changes of some global public health significance, as assessed by comparison:
increased transmissibility or deleterious changes in the epidemiology of COVID-19; or alternatively
Increased toxicity or changes in clinical disease manifestations; or alternatively
Reduced effectiveness of public health and social measures or available diagnostic, vaccine and therapeutic methods.
The definition of "Variants to be noted" (VOI) in the present invention can be defined by reference to the work of the World Health Organization (WHO), namely: a SARS-CoV-2 variant having the following characteristics:
have genetic changes predicted or known to affect viral characteristics, such as transmissibility, disease severity, immune escape, diagnostic or therapeutic escape; and
confirmation results in significant community spread or multiple COVID-19 aggregate cases across multiple countries, with increasing relative prevalence, increasing numbers of cases, or other significant epidemiological impact that indicates global public health is facing new risks.
The SARS-CoV-2S protein structure is shown in figure 1, wherein 1-13 is signal peptide, 14-685 is S1 subunit, 686-1273 is S2 subunit. Wherein the S1 subunit can be further divided into NTD (14-303) and CTD (334-527). 319-541 are receptor binding regions, 788-806 are fusion proteins. 13-1213 are extracellular domains, 1214-1234 are transmembrane domains, and 1235-1273 are intracellular domains.
EXAMPLE 1 amino acid sequence of novel coronavirus immunogenic substances
In the examples of the present invention, the present inventors designed a variety of novel coronavirus immunogenic substances using the receptor binding regions of the novel coronavirus WH01 strain, beta (Beta) strain, delta (Delta) strain, and Ormck Ron (Omicron) variant strain.
Wherein the receptor binding region of the S protein derived from the WH01 strain comprises an amino acid sequence shown by any one of SEQ ID NO. 1, SEQ ID NO. 2, SEQ ID NO. 3 or SEQ ID NO. 4. Wherein SEQ ID NO 1 contains 8 cysteines, SEQ ID NO 2 contains an additional sequence extending out from the N-terminus of SEQ ID NO 1 containing 10 cysteines, SEQ ID NO 3 contains an additional sequence extending out from the C-terminus of SEQ ID NO 1 containing 10 cysteines, and SEQ ID NO 4 contains an additional sequence extending out from the N-terminus and the 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. Wherein SEQ ID NO 5 contains 8 cysteines, SEQ ID NO 6 contains an additional sequence extending out from the N-terminus of SEQ ID NO 5 containing 10 cysteines, SEQ ID NO 7 contains an additional sequence extending out from the C-terminus of SEQ ID NO 5 containing 10 cysteines, and SEQ ID NO 8 contains additional sequences extending out 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 the 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. Wherein SEQ ID NO 9 contains 8 cysteines, SEQ ID NO 10 contains an additional sequence extending out from the N-terminus of SEQ ID NO 9 containing 10 cysteines, SEQ ID NO 11 contains an additional sequence extending out from the C-terminus of SEQ ID NO 9 containing 10 cysteines, and SEQ ID NO 12 contains an additional sequence extending out from the N-terminus and the C-terminus of SEQ ID NO 9, respectively, containing 12 cysteines.
The receptor binding region of the S protein derived from the Ornkendon (Omicron) BA.1 variant 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 comprises the amino acid sequence shown in SEQ ID NO:62, the receptor binding region of the S protein derived from the Ornkendon (Omicron) BA.3 variant comprises the amino acid sequence shown in SEQ ID NO:63, and the receptor binding regions of the S proteins derived from the Ornkendon (Omicron) BA.4 and BA.5 variants comprise the amino acid sequence shown in SEQ ID NO: 64.
In this example, some immunogenic agents also include an N-terminal domain (NTD) in the S1 subunit of the S protein. Wherein the N-terminal domain of the S1 subunit of the WH01 strain S protein comprises the amino acid sequence shown by SEQ ID NO:13, the N-terminal domain of the S1 subunit of the Beta (Beta) strain S protein comprises the amino acid sequence shown by SEQ ID NO:14, the N-terminal domain of the S1 subunit of the Delta (Delta) strain comprises the amino acid sequence shown by SEQ ID NO:15, the N-terminal domain of the S1 subunit of the Ormck-Kan (Omicron) BA.1 variant strain comprises the amino acid sequence shown by SEQ ID NO:43, the N-terminal domain of the S protein of the Ormck-Kan (Omicron) BA.2 variant strain comprises the amino acid sequence shown by SEQ ID NO:65, the N-terminal domain of the S protein of the Ormck-Kan (Omicron) BA.3 variant strain comprises the amino acid sequence shown by SEQ ID NO:66, the N-terminal domains of the S proteins of the Ormck-Kan (Omicron) BA.4 and S5 strains comprise the amino acid sequences shown by SEQ ID NO: 67.
In this example, some immunogenic agents further include a human IgG Fc domain to form a dimeric structure, or some immunogenic agents further include a Foldon domain to form a trimeric structure, the human IgG Fc domain comprising the amino acid sequence set forth in SEQ ID No. 16 and the Foldon domain comprising the amino acid sequence set forth in SEQ ID No. 17.
Based on the above sequences, the present inventors designed the amino acid sequences of the following antigens, and obtained the corresponding DNA sequences according to codon optimization of host CHO cells:
(1) The antigen derived from WH01 strain is composed of two SEQ ID NO 1 connected in series, the amino acid sequence of the antigen is SEQ ID NO 18 (the 5 'end is added with a signal peptide SEQ ID NO 41, the 3' end is added with 6 histidines), and the corresponding DNA sequence is SEQ ID NO 31;
(2) An antigen derived from WH01 strain and Delta strain, which is composed of SEQ ID NO 1 and SEQ ID NO 9 connected in series, and has an amino acid sequence of SEQ ID NO 19 (a signal peptide is added to the 5 'end and 6 histidines are added to the 3' end), and a corresponding DNA sequence of SEQ ID NO 32;
(3) The antigen derived from Beta and Delta strains, which is composed of 5 and 9 SEQ ID NO in series, has an amino acid sequence of 20 (signal peptide added to 5 'end and 6 histidine added to 3' end) and a corresponding DNA sequence of 33 SEQ ID NO;
(4) An antigen derived from a Delta strain consisting of two SEQ ID NO's 9 connected in series, having the amino acid sequence of SEQ ID NO ' 21 (signal peptide added to the 5' end and 6 histidines added to the 3' end) and the corresponding DNA sequence of SEQ ID NO ' 34;
(5) An antigen derived from the WH01 strain and a Delta (Delta) strain, which comprises an N-terminal domain of the WH01 strain and is composed of SEQ ID NO 13, SEQ ID NO 1 and SEQ ID NO 9 in series, and has an amino acid sequence of SEQ ID NO 22 (a signal peptide is added at the 5 'end and 6 histidines are added at the 3' end) and a corresponding DNA sequence of SEQ ID NO 35;
(6) An antigen derived from a Beta (Beta) strain and a Delta (Delta) strain, which comprises an N-terminal domain of the Beta (Beta) strain, is composed of SEQ ID NO 14, SEQ ID NO 5 and SEQ ID NO 9 in series, has an amino acid sequence of SEQ ID NO 23 (a signal peptide is added at the 5 'end, 6 histidines are added at the 3' end), and has a corresponding DNA sequence of SEQ ID NO 36;
(7) An Fc dimer of antigens derived from WH01 strain and Delta (Delta) strain, which is composed of SEQ ID NO:1, SEQ ID NO:9 and SEQ ID NO:16 in tandem, has an amino acid sequence of SEQ ID NO:24 (signal peptide added to the 5 'end and 6 histidines added to the 3' end);
(8) An Fc dimer of an antigen derived from the WH01 strain and the Delta (Delta) strain, comprising the N-terminal domain of the WH01 strain, consisting of SEQ ID NO 13, SEQ ID NO 1, SEQ ID NO 9 and SEQ ID NO 16 in tandem, and having an amino acid sequence of SEQ ID NO 25 (with a signal peptide added to the 5 'end and 6 histidines added to the 3' end);
(9) A Foldon trimer derived from antigens of WH01 strain and Delta strain is composed of SEQ ID NO 1, SEQ ID NO 9 and SEQ ID NO 17 connected in series, the amino acid sequence thereof is SEQ ID NO 26 (signal peptide is added at the 5 'end and 6 histidines are added at the 3' end), and the corresponding DNA sequence thereof is SEQ ID NO 37;
(10) A Foldon trimer derived from the antigen of the WH01 strain and the Delta strain, which comprises the N-terminal domain of the WH01 strain and is composed of SEQ ID NO 13, SEQ ID NO 1, SEQ ID NO 9 and SEQ ID NO 17 in series, the amino acid sequence of the Foldon trimer is SEQ ID NO 27 (a signal peptide is added at the 5 'end and 6 histidines are added at the 3' end), and the corresponding DNA sequence is SEQ ID NO 38;
(11) An antigen derived from WH01 strain and Delta strain consisting of SEQ ID NO 2 and SEQ ID NO 10 in tandem, having an amino acid sequence of SEQ ID NO 28 (signal peptide added to the 5 'end and 6 histidines added to the 3' end) and a corresponding DNA sequence of SEQ ID NO 39;
(12) An antigen derived from WH01 strain and Delta strain, consisting of SEQ ID NO. 4 and SEQ ID NO. 12 connected in series, and having an amino acid sequence of SEQ ID NO. 29 (with a signal peptide added to the 5 'end and 6 histidines added to the 3' end);
(13) The antigen derived from WH01 strain and Delta strain is composed of SEQ ID NO 13, SEQ ID NO 1, SEQ ID NO 15 and SEQ ID NO 9 in series, the amino acid sequence thereof is SEQ ID NO 30 (signal peptide is added at the 5 'end, 6 histidines are added at the 3' end), and the corresponding DNA sequence thereof is SEQ ID NO 40.
(14) An antigen derived from the WH01 strain, which is composed of two SEQ ID NOS 2 connected in series, and has an amino acid sequence of SEQ ID NO 44 (a signal peptide is added to the 5 'end, and 6 histidines are added to the 3' end);
(15) An antigen derived from the WH01 strain, which comprises an N-terminal domain of the WH01 strain and is composed of SEQ ID NO 13 and two SEQ ID NO 1 which are connected in series, and the amino acid sequence of the antigen is SEQ ID NO 45;
(16) An antigen derived from the Delta strain, which comprises the N-terminal domain of the Delta strain, is composed of SEQ ID NO. 15 and two SEQ ID NO. 9 connected in series, and has an amino acid sequence of SEQ ID NO. 46;
(17) An antigen derived from the WH01 strain and Ormckren (Omicron) BA.1 variant consisting of SEQ ID NO 1 and SEQ ID NO 42 in tandem, having the amino acid sequence SEQ ID NO 47;
(18) An antigen derived from a Beta (Beta) strain and an ormekron (omitron) ba.1 variant, consisting of SEQ ID NO 5 and SEQ ID NO 42 in tandem, having the amino acid sequence SEQ ID NO 48;
(19) An antigen derived from the WH01 strain and Oromkren (Omicron) BA.1 variant, comprising the N-terminal domain of the WH01 strain consisting of SEQ ID NO 13, SEQ ID NO 1 and SEQ ID NO 42 in tandem, having the amino acid sequence of SEQ ID NO 49;
(20) An antigen derived from a Beta (Beta) strain and an Ormckron (Omicron) BA.1 variant strain comprising the N-terminal domain of the Beta (Beta) strain consisting of SEQ ID NO 14, SEQ ID NO 5 and SEQ ID NO 42 in tandem and having the amino acid sequence of SEQ ID NO 50;
(21) According to the main variant strains, antigens derived from 2-4 variant strains are further designed, and the amino acid sequences of the antigens are shown as SEQ ID NO. 51-61.
The above antigens are summarized in table 1:
TABLE 1
Figure BDA0003818163870000111
/>
Figure BDA0003818163870000121
EXAMPLE 2 construction of expression vectors
The DNA sequence coding the antigen is sent out, synthesized and cloned into an expression vector pWX039 after codon optimization according to a host CHO cell to obtain the expression vector pWX039-PR-Z, and the delivery is carried out after sequencing verification of a target gene.
The pWX039-PR-Z vector is used as template and through PCR amplification and gel purification, target gene DNA segment is obtained. The purified target gene DNA fragment was cloned into the vector pWX4.1 through SalI and NotI sites to obtain the transient expression vector pWX4.1-PR. The expression vector pWX4.1-PR was verified by Sanger sequencing to have a 100% correct sequence.
Coli E.coli Top10 competent cells were transformed with the expression vector pWX4.1-PR. Transformants were picked and streaked onto LB agar plates (containing 100. Mu.g/mL ampicillin) after liquid culture. One single clone is selected from the plate and inoculated into 300ml LB culture medium for amplification culture, a NucleoBond Xtra Maxi EF kit is adopted for mass preparation of plasmids, and sequencing is carried out to verify the target gene. The pWX4.1-PR plasmid DNA was verified by sequencing and used for transfection.
Example 3 protein expression and purification
Protein expression
During all cell manipulations, the cells were mixed by gentle rotation; vigorous mixing/pipetting is avoided. Cell health is the key to achieving maximum performance. CHO cells (EXPICHO from Thermo) were subcultured and amplified using ExpicHO Expression Medium (cell density 4X 10) 6 –6×10 6 Passage was performed at one/mL.
Day-1: thin and thinAmplifying the cells, and amplifying the cultured cells to 3 × 10 6 –4×10 6 one/mL, and cells were allowed to grow overnight. Day 0: transfecting cells, determining viable cell density and viability, wherein the cell density should reach 7 × 10 6 –10×10 6 Transfection at cell/mL, 95-99% viability, fresh cell expression medium, pre-warmed to 37 ℃, and cell dilution to final density of 5X 10 6 seed/mL, cultured at 37 ℃ in a 50mm amplitude incubator at 90rpm, 8% 2 . Transfection reagent and plasmid DNA complexes were prepared using OPti-PRO SFM medium (4 ℃), examples: 1ml cell preparation 40 u l OPti-PRO SFM adding 1ug plasmid mixing evenly and standing for 5min; prepare 40 μ l of OPti-PRO SFM and add 6 μ g PEI reagent mix well and stand for 5min, mix the plasmid with equal volume of transfection reagent, incubate at room temperature for 1-5 min, then slowly transfer the solution to shake flask and shake flask gently during addition. Culturing the cells at 37 ℃ in a 50mm amplitude incubator at 90rpm, 8% 2 . At 18-22 hours post-transfection, feed was added and standard protocols were performed. Example (c): 0.2ml EX-
Figure BDA0003818163870000131
Advanced CHO Feed 1, culturing the cells at 37 ℃ in a 50mm amplitude incubator at 90rpm, 8% CO 2 . Collected 6 days after transfection for subsequent purification.
Protein purification
Centrifuging the culture medium, adding a Ni column into the supernatant, oscillating and incubating for 2h, and performing affinity chromatography purification by a gravity empty column. And (3) an equilibrium buffer: "PBS", pH7.4, washing 10CV; washing buffer solution: "PBS", pH7.4, containing 20mM imidazole, washed 10CV; elution buffer: "PBS", pH7.4, containing 500mM imidazole, eluted 1CV, repeated 5 times. SDS-PAGE & Western Blot profiles of different candidate antigens are shown in FIGS. 2-14.
EXAMPLE 4 preparation of recombinant protein vaccine
Each of the recombinant proteins obtained in example 3 was diluted to 80 μ g/ml with 1 × PBS buffer, and thoroughly mixed with an equal volume of AS03 adjuvant to prepare a vaccine product, wherein the AS03 adjuvant component included 10.69mg of squalene, 11.86mg of α -tocopherol, 4.86mg of polysorbate 80, 3.53mg of sodium chloride, 0.09mg of potassium chloride, 0.51mg of disodium hydrogen phosphate, 0.09mg of potassium dihydrogen phosphate, and water for injection per 0.5 ml.
EXAMPLE 5 preparation of recombinant protein vaccine
Each of the recombinant proteins obtained in example 3 was diluted to 80. Mu.g/ml with 1 XPBS buffer and mixed well with an equal volume of MF59 adjuvant to prepare a vaccine product, wherein the MF59 adjuvant was a citric acid buffer solution containing 1% squalene, 0.5% Tween 80 and 0.5% span 85.
EXAMPLE 6 preparation of recombinant protein vaccine
Each of the recombinant proteins obtained in example 3 was diluted to 80. Mu.g/ml with 1 XPBS buffer and mixed well with an equal volume of aluminum adjuvant, wherein the aluminum adjuvant was 2mg/ml Al (OH) 3 。
EXAMPLE 7 preparation of recombinant protein vaccine
Each of the recombinant proteins obtained in example 3 was diluted to 40. Mu.g/ml with 1 XPBS buffer and mixed well with an equal volume of AS01 adjuvant to produce a vaccine product, wherein the AS01 adjuvant component included 50. Mu.g MPL, 50. Mu.g QS21, 1mg DOPC and 0.25mg cholesterol per 0.5 ml.
Example 8 recombinant protein vaccine mouse immunization experiment
BALB/c mice (purchased from Beijing Wakuukang Biotechnology Co., ltd.) aged 8-10 weeks were immunized in groups of 5 mice each using the vaccines prepared in examples 4-7. Mice were immunized on days 0 and 21, respectively, each intramuscular with 100 μ l of an immune sample (containing 8 μ g of antigen) and blood was collected on days 0, 21, and 14 days after the second immunization. The collected blood samples are placed for 1 hour at 37 ℃,1 hour at 4 ℃, centrifuged for 10 minutes at 8000r/min, and serum is collected and stored at-20 ℃ for neutralization detection of the pseudoviruses.
EXAMPLE 9 neutralization of different types of pseudoviruses with RBD combinations of the same strains
The new coronavirus S protein containing mutation sites of each strain is adopted to construct pseudoviruses, and serum separated after immunization is detected by using a detection method (chemiluminescence method) for neutralizing antibodies of the new coronavirus based on a VSV system.
In terms of adjuvant, the titer of pseudovirus neutralizing antibodies measured when mice were immunized with RBD (Beta) -RBD (Beta) according to the present invention in combination with different adjuvants is shown in Table 2.
TABLE 2
Figure BDA0003818163870000151
The results show that when AS03 adjuvant was used, the pseudovirus neutralizing antibody titers against the D614G, beta and Omicron BA.1 strains were all significantly higher than with Al (OH) 3 Neutralizing antibody titers in adjuvant. Additional experiments showed that the antibody titers when MF59 adjuvant was used were similar to those when AS03 adjuvant was used. In subsequent experiments, the AS03 adjuvant was used in the present invention.
In terms of antigens, the present invention first examined the neutralizing antibody titers of candidate antigens consisting of the same strain RBD against different strains, the neutralizing antibody titers and the geometric mean titer GMT of 5 serum samples of each group being as shown in table 3.
TABLE 3
Figure BDA0003818163870000152
The results showed that the candidate antigen formed by the RBD combination of strain WH01 (SEQ ID NO: 18) had a higher geometric mean titer against both strain WH01 and strain D614G pseudoviruses, but a lower geometric mean titer against strain Delta, while the geometric mean titer against Omicron BA.1 pseudovirus was significantly lower, in contrast to the candidate antigen formed by the RBD combination of strain Delta (SEQ ID NO: 21) which had a higher geometric mean titer against only strain Delta pseudovirus and a significantly lower geometric mean titer against strain WH01, strain D614G and Omicron BA.1 pseudovirus. This suggests that candidate antigens have poor cross-protection against different strains when the RBDs are from the same strain, a result consistent with published studies.
In the further research, RBD of a specific strain is screened as a preferable antigen component, the antibody titer to a non-self strain can be improved, so that a more balanced protection effect can be obtained, and the strain is the immunodominant strain.
Example 10 neutralization of different strains of RBD combinations against different types of pseudoviruses
In this example, neutralizing antibody titers against different types of pseudoviruses were tested for different strain RBD combinations using the method of example 9.
(1) Immunodominant strain RBD screening
When one RBD from the Delta variant was replaced with one from among RBD (Delta) -RBD (Delta) (SEQ ID NO: 21), from either the Beta variant or the WH01 strain, the neutralizing antibody titer and the geometric mean titer GMT change for the 5 serum samples of each group are shown in Table 4.
TABLE 4
Figure BDA0003818163870000161
As can be seen, the mean value of the geometric mean titres of SEQ ID NO 21 against the four pseudoviruses reached 46948 and the geometric mean titre against the Delta strain pseudovirus reached 101399, about 2.2 times the mean value, the geometric mean titre against the Omicron BA.1 strain pseudovirus being only 22267, about 47% of the mean value.
Compared with SEQ ID NO:21, the geometric mean titer of the candidate antigen (SEQ ID NO: 20) formed by the Beta strain and the RBD of the Delta strain in combination is slightly reduced for the Delta strain pseudovirus, but the geometric mean titer of the D614G strain and the Omicron BA.1 strain pseudovirus is respectively improved by 2 times and 1 time, the geometric mean titer of the four pseudoviruses reaches 65462, the geometric mean titer of the D614G strain pseudovirus is the maximum and reaches 92502 which is about 1.4 times of the mean value, and the geometric mean titer of the Omicron BA.1 strain pseudovirus reaches 50785 which is about 78 percent of the mean value.
Compared with SEQ ID NO:21, the geometry mean titer of the candidate antigen (SEQ ID NO: 19) formed by the RBD combination of the WH01 strain and the Delta strain is not changed greatly against WH01 and D614G pseudoviruses, but the geometry mean titer of both the Delta strain and the Omicron BA.1 pseudovirus is reduced by more than 50%, the mean value of the geometry mean titer of the four pseudoviruses is only 29505, the geometry mean titer of the Delta strain pseudovirus is maximal and reaches 42932 which is about 1.5 times of the mean value, and the geometry mean titer of the Omicron BA.1 pseudovirus is only 10520 which is about 36% of the mean value.
Thus, in general, the geometric mean titers of SEQ ID NO 20 against WH01, D614G, delta and Omicron BA.1 pseudoviruses were more balanced, and the RBD of Beta strain increased the neutralizing antibody titer of antigen against non-Beta variant strains, so that Beta strain was considered an immunodominant strain.
The present invention further investigated the effect of the combination of different strains of RBD on neutralizing antibody titers in the formation of Foldon trimers. The neutralizing antibody titers and the geometric mean titers GMT against different pseudoviruses for 5 serum samples of NTD-RBD (BA.2) -RBD (BA.1) -Foldon (SEQ ID NO: 60) and NTD-RBD (BA.2) -RBD (WH 01) -Foldon (SEQ ID NO: 61) are shown in Table 5.
TABLE 5
Figure BDA0003818163870000171
It can be seen that NTD-RBD (BA.2) -RBD (BA.1) -Foldon has a high neutralizing titer against BA.2 pseudovirus, up to 19802, and a certain antibody titer against BA.4 pseudovirus, but low antibody titers against D614G and BA.1.
When the RBD derived from the Omicron-BA.1 variant was replaced with the RBD derived from the WH01 strain, the neutralizing titer against not only the BA.2 pseudovirus but also the antibody titers against D614G and BA.4 pseudovirus were significantly increased, except for the lower antibody titer against BA.1, which is probably due to the greater difference between BA.1 and BA.2 and BA.4. Published literature has also demonstrated significant differences in the antigenicity of BA.1 and BA.2 (see antibiotic cartography of SARS-CoV-2 vitamins at Omicron BA.1 and BA.2 area Antigenic disorders, ANNA Z.MYKYTYN et al.SCIENCE IMMUNOLOGY,23 Jun 2022).
The above results show that the RBD of WH01 strain can increase the neutralizing antibody titer of antigen against different variants in the case of the formation of Foldon trimer, and therefore, the WH01 strain can also be considered as an immunodominant strain in this case.
In contrast, further studies of the present invention have shown that when antigen components are selected from receptor binding regions of Alpha (Alpha) strain, gamma (Gamma) strain, eplerenone (epsilon), zeta potential (zeta) strain, eta (Eta) strain, theta (theta) strain, elta (Iota) strain, kappa (Kappa) strain, lambda (Lambda) strain, and Mu (Mu) strain, the resulting immunogenic substance is increased only in titer against pseudoviruses of the corresponding strains, but is not significantly increased in titer against pseudoviruses of other mutant strains, which indicates that the receptor binding regions of the above strains are less reactive to antibodies induced by other strains, and are not immunodominant strains as described in the present invention. However, these may serve as additional components of the antigen to further improve the balance of protective effects of the vaccine against different strains.
It has been shown in the prior art that when a receptor binding region of a certain strain is present in an immunogenic substance, the titer of the immunogenic substance against the pseudovirus of the strain is significantly increased. In combination with the prevalence of new coronaviruses, the present invention uses the Delta (Delta) strain or the Ormck Ron (Omicron) strain as the prevalent dominant strain. The experimental research of the invention shows that when the immunogenic material simultaneously comprises receptor binding regions from immunodominant strains and epidemic dominant strains, the immunogenic material can show higher titer aiming at pseudoviruses of different strains, which indicates that the immunogenic material can generate excellent immune effect on different strains.
(2) Effect of NTD addition
The present invention further investigates the effect of adding NTD to a candidate antigen. After addition of NTD, the neutralizing antibody titers and the geometric mean titer GMT against different pseudoviruses for 5 serum samples of each group are shown in table 6.
TABLE 6
Figure BDA0003818163870000181
Figure BDA0003818163870000191
/>
As shown in Table 6, when RBDs are from the same strain, the geometric mean titers against different pseudoviruses of the candidate antigens formed by the combination of NTD and RBD of strain WH01, SEQ ID NO:45 and NTD and RBD of strain Delta, SEQ ID NO:46, were all increased to different degrees compared to those of SEQ ID NO:18 and SEQ ID NO:21, and the mean values of the geometric mean titers against the four pseudoviruses were increased from 38492 and 46948 to 53563 and 55471, respectively.
When RBDs are from different strains, compared with SEQ ID NO:19, the geometric mean titer of the candidate antigen SEQ ID NO:22 formed by combining NTD and RBD of WH01 strain and RBD of Delta strain is obviously improved for different pseudoviruses, and the mean value of the geometric mean titer for four pseudoviruses is increased from 29505 to 67293; the geometric mean titer of the candidate antigen SEQ ID NO. 23 formed by the combination of NTD and RBD of Beta strain and RBD of Delta strain was reduced but still maintained at a higher level against D614G strain and Delta strain pseudoviruses than that of SEQ ID NO. 20, while the geometric mean titer against WH01 strain and Omicron BA.1 strain pseudoviruses was increased, especially against Omicron BA.1 strain pseudovirus to the highest value among these combinations. Thus, in general, cross-protection capability can be improved upon addition of NTD.
(3) Effect of addition of Foldon Domain
The present invention further examined the effect of trimer formation by adding the Foldon domain to candidate proteins, and the neutralizing antibody titers and geometric mean titer GMTs against different pseudoviruses for 5 serum samples of each group after addition of the Foldon domain are shown in table 7.
TABLE 7
Figure BDA0003818163870000201
As shown in Table 7, compared with SEQ ID NO:19, when the Foldon domain (SEQ ID NO: 26) or the NTD and Foldon domains (SEQ ID NO: 27) are added into the RBD combination of the WH01 strain and the Delta strain to form the trimer, the geometric mean titer of different pseudoviruses is obviously improved, and the mean value of the geometric mean titer of four pseudoviruses is respectively increased from 29505 to 63178 and 53995, which shows that the cross protection capability can be improved by forming the trimer.
Therefore, the antigen formed by RBD combination of the immunodominant strain and the epidemic dominant strain has more balanced immune effect on different strains, and the immune effect can be further improved after an NTD or Foldon structural domain is further added.
Example 11 preparation of mRNA
This example prepares mRNA for the antigens shown in Table 8.
TABLE 8
Figure BDA0003818163870000211
The above antigen was labeled with DYKDDDDKKHHHHHHHHHHHHHHHH at the C-terminus, the DNA sequence encoding the antigen was codon-optimized for human host, and the T7 RNA polymerase binding sequence, 5'UTR (5' UTR of human β -globin), kozak sequence and signal peptide were sequentially added to the 5 '-terminus, and 3' UTR (5 'UTR of human β -globin), ployA (120) and enzyme cleavage site were sequentially added to the 3' -terminus to obtain a DNA sequence.
A DNA sequence was synthesized and cloned into an expression vector pUC57-kan, and the target gene was verified by sequencing.
Coli E.coli competent cells were transformed with the expression vector, amplified, and plasmid was extracted, and the linearized plasmid sample was obtained by digestion, in vitro Transcription was performed using T7 High Yield RNA Transcription Kit (Novoprotein, CAT: E131-01A) according to the instructions, mRNA Capping was performed using Cap1 Capping System (Novoprotein, CAT: M082) according to the instructions, and finally mRNA was purified by lithium chloride precipitation purification.
The sequence of the prepared mRNA is SEQ ID NO:68-73, wherein,
68 is an antigen coding region from position 123-2750 of SEQ ID NO,
the 123-1436 position of SEQ ID NO:69 is the antigen coding region,
the 123-1436 position of SEQ ID NO. 70 is the antigen coding region,
71 from position 123 to 1436 is the antigen coding region,
72 is an antigen coding region from position 123-2093 of SEQ ID NO,
the 123-1436 position of SEQ ID NO. 73 is the antigen coding region.
Example 12 preparation of mRNA vaccine
Mixing each mRNA obtained in example 11 with lipid nanoparticles to obtain a nucleic acid-lipid nanoparticle complex, wherein the lipid nanoparticles comprise DOTMA and DOPE in a molar ratio of 1; the content of mRNA in the compound is 100 mu g/ml, and the mass ratio of the lipid nanoparticles to the mRNA is 10.
Example 13mRNA vaccine mouse immunization experiments
BALB/c mice (purchased from Beijing Huafukang Biotechnology Co., ltd.) aged 8 to 10 weeks were immunized in groups of 5 mice each with each mRNA vaccine obtained in example 12. Mice were immunized on days 0 and 21, respectively, and each was given an intramuscular injection of 50. Mu.l of each immune sample (containing 5. Mu.g of mRNA) and blood was collected on days 0, 21 and 14 days after the second immunization. The collected blood samples are placed for 1 hour at 37 ℃,1 hour at 4 ℃, centrifuged for 10 minutes at 8000r/min, and serum is collected and stored at-20 ℃ for pseudovirus neutralization detection. The neutralizing antibody titers and geometric mean titer GMT results for 5 serum samples of each group against different pseudoviruses are shown in table 9.
TABLE 9
Figure BDA0003818163870000221
It can be seen that the mean value of the neutralizing antibody titers GMT against the different pseudoviruses of the vaccine prepared from the mRNA (SEQ ID NO: 73) encoding RBD (Beta) -RBD (Beta) was 31133, and the neutralizing antibody titers against the Omicron strain were significantly lower than those against the other strains, compared to that of the vaccine prepared from the mRNA (SEQ ID NO: 68) encoding RBD (WH 01) -RBD (Beta) -RBD (Delta) -RBD (BA.1) reaching 99781, the mean value of the neutralizing antibody titers GMT against the different pseudoviruses of the vaccine prepared from the mRNA (SEQ ID NO: 69) encoding RBD (WH 01) -RBD (BA.1) and the mRNA (SEQ ID NO: 70) encoding RBD (Beta) -RBD (Delta) reaching 58513, the mean value of the neutralizing antibody titers against the different pseudoviruses of the vaccine prepared from the mRNA (SEQ ID NO: 71) encoding RBD (Beta) -RBD (BA.1) and the mean value of the neutralizing antibody titers against the different pseudoviruses of the mRNA (SEQ ID NO: 4471) and the neutralizing antibody titers against the Omicron strain reached 5872, and the neutralizing antibody titers against the different pseudoviruses of the mRNA (SEQ ID NO: 72).
Therefore, the mRNA vaccine prepared by the mRNA for encoding the immunogenic substance has good immune effect on different strains. It is expected that vaccines prepared from the immunogenic agent of the invention and nucleic acids encoding the immunogenic agent will still have a superior prophylactic effect against mutants of the new coronavirus that may appear in the future. Therefore, the invention has important guiding significance under the condition that the novel coronavirus is continuously mutated.
The above-mentioned embodiments are further described in detail for illustrating the purpose, technical solutions and advantages of the present invention, and it should be understood that the above-mentioned embodiments are only examples of the present invention and are not intended to limit the present invention, and any modifications, equivalents, improvements and the like made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims (15)

1. A novel coronavirus immunogenic material comprising 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 part of a receptor binding region and an N-terminal domain (NTD) or part of an N-terminal domain of the S protein, wherein the immunodominant strain is selected from at least one of a novel coronavirus WH01 strain and a Beta (Beta) strain, and the epidemic dominant strain is selected from at least one of a novel coronavirus Delta (Delta) strain, ormekron (Omicron) ba.1, ba.2, ba.3, ba.4 and ba.5 variant strains.
2. The novel coronavirus immunogenic material according to claim 1, wherein the receptor binding domain of the S protein derived from the WH01 strain comprises an amino acid sequence represented by any one of SEQ ID NO 1, SEQ ID NO 2, SEQ ID NO 3 or SEQ ID NO 4, and the N-terminal domain comprises an amino acid sequence represented by SEQ ID NO 13;
the receptor binding region of the S protein derived from the Beta (Beta) strain comprises an amino acid sequence shown by any one of SEQ ID NO 5, SEQ ID NO 6, SEQ ID NO 7 or SEQ ID NO 8, and the N-terminal domain comprises an amino acid sequence shown by SEQ ID NO 14;
the receptor binding region of the S protein derived from the Delta strain comprises the amino acid sequence shown in any one of SEQ ID NO 9, 10, 11 or 12, and the N-terminal domain comprises the amino acid sequence shown in SEQ ID NO 15;
the receptor binding region of the S protein derived from the Ormck Ron (Omicron) BA.1 variant comprises the amino acid sequence shown by SEQ ID NO:42, the N-terminal domain comprises the amino acid sequence shown by SEQ ID NO:43, the receptor binding region of the S protein derived from the Ormck Ron (Omicron) BA.2 variant comprises the amino acid sequence shown by SEQ ID NO:62, the N-terminal domain comprises the amino acid sequence shown by SEQ ID NO:65, the receptor binding region of the S protein derived from the Ormck Ron (Omicron) BA.3 variant comprises the amino acid sequence shown by SEQ ID NO:63, the N-terminal domain comprises the amino acid sequence shown by SEQ ID NO:66, and the receptor binding regions of the S proteins derived from the Ormck Ron (Omicron) BA.4 and BA.5 variants comprise the amino acid sequence shown by SEQ ID NO:64, and the N-terminal domain comprises the amino acid sequence shown by SEQ ID NO: 67.
3. The novel immunogenic substance for coronavirus according to claim 1, wherein the novel immunogenic substance for coronavirus comprises an amino acid sequence represented by any one of SEQ ID NOs 22,23,30,49,50,56 to 59.
4. The novel immunogenic agent for coronavirus according to claim 1, wherein the novel immunogenic agent for coronavirus further comprises one or more antigens derived from said immunodominant strain and a strain other than said pandemic dominant strain.
5. The novel immunogenic substance for coronavirus according to claim 4, wherein said immunodominant strain and said strains other than the pandemic dominant strain are selected from the following strains: alpha (Alpha) strain, gamma (Gamma) strain, epsilon (Epsilon), secatetra (Zeta) strain, eta (Eta) strain, sita (Theta) strain, eatota (Iota) strain, eicota (Iota) strain, kappa (Kappa) strain, lambda (Lambda) strain, and Mu (Mu) strain.
6. The novel coronavirus immunogenic material according to any one of claims 1 to 5, wherein each antigen constitutes a composition, or each antigen is directly linked or linked through an amino acid linker.
7. A process for preparing a novel coronavirus immunogenic material according to any one of claims 1 to 6, comprising the steps of:
constructing a recombinant expression plasmid by using a nucleotide sequence for coding the novel coronavirus immunogenic substance;
transforming the constructed recombinant expression plasmid into host bacteria, screening correct recombinant expression plasmid,
and transfecting cells of the expression system by using the screened recombinant expression plasmid, and collecting and purifying supernatant after expression to obtain the novel coronavirus immunogenic substance.
8. The method of claim 7, wherein the cell of the expression system comprises a mammalian cell, an insect cell, a yeast cell, or a bacterial cell, optionally; the mammalian cells include 293T cells or CHO cells, and the bacterial cells include E.coli cells.
9. A nucleotide sequence encoding the novel coronavirus immunogenic agent according to any one of claims 1 to 6.
10. A recombinant vector comprising the nucleotide sequence of claim 9.
11. An expression system cell carrying the recombinant vector of claim 10.
12. Use of a novel coronavirus immunogenic material according to any one of claims 1 to 6, a nucleotide sequence according to claim 9, a recombinant vector according to claim 10 or an expression system cell according to claim 11 for the preparation of a novel coronavirus vaccine.
13. A novel coronavirus protein vaccine comprising a novel coronavirus immunogenic substance according to any one of claims 1-6 and an adjuvant selected from one or more of the group consisting of aluminium adjuvant, MF59 adjuvant, MPL adjuvant, QS-21, GLA, cpG, AS01, AS02, AS03, AS04 adjuvant, preferably AS03 or MF59 adjuvant.
14. A novel coronavirus DNA vaccine comprising a DNA sequence encoding the novel coronavirus immunogenic agent of any one of claims 1-6.
15. A novel coronavirus mRNA vaccine comprising an mRNA sequence encoding the novel coronavirus immunogenic agent of any one of claims 1-6.
CN202211033865.9A 2021-08-26 2022-08-26 Novel coronavirus immunogenic substance, preparation method and application thereof Pending CN115960252A (en)

Applications Claiming Priority (8)

Application Number Priority Date Filing Date Title
CN2021109887716 2021-08-26
CN202110988771 2021-08-26
CN2022101129832 2022-01-29
CN202210112983 2022-01-29
CN202210387826 2022-04-13
CN2022103878262 2022-04-13
CN202210514880 2022-05-11
CN2022105148809 2022-05-11

Publications (1)

Publication Number Publication Date
CN115960252A true CN115960252A (en) 2023-04-14

Family

ID=85322450

Family Applications (3)

Application Number Title Priority Date Filing Date
CN202211032251.9A Pending CN116041534A (en) 2021-08-26 2022-08-26 Novel coronavirus immunogenic substance, preparation method and application thereof
CN202211032243.4A Pending CN116041448A (en) 2021-08-26 2022-08-26 Novel coronavirus immunogenic substance, preparation method and application thereof
CN202211033865.9A Pending CN115960252A (en) 2021-08-26 2022-08-26 Novel coronavirus immunogenic substance, preparation method and application thereof

Family Applications Before (2)

Application Number Title Priority Date Filing Date
CN202211032251.9A Pending CN116041534A (en) 2021-08-26 2022-08-26 Novel coronavirus immunogenic substance, preparation method and application thereof
CN202211032243.4A Pending CN116041448A (en) 2021-08-26 2022-08-26 Novel coronavirus immunogenic substance, preparation method and application thereof

Country Status (2)

Country Link
CN (3) CN116041534A (en)
WO (1) WO2023025287A1 (en)

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN116350769A (en) * 2023-03-31 2023-06-30 北京吉诺卫生物科技有限公司 Combined vaccine of novel coronavirus and influenza virus, preparation method and application thereof

Family Cites Families (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20230021583A1 (en) * 2020-02-13 2023-01-26 Institut Pasteur Measles-vectored covid-19 immunogenic compositions and vaccines
CN112760341B (en) * 2020-07-13 2022-12-09 中国科学院微生物研究所 Recombinant vector and application of chimpanzee adenovirus packaged with recombinant vector in preparation of 2019 novel coronavirus vaccine
CN112043825B (en) * 2020-07-13 2023-12-05 中国医学科学院医学生物学研究所 Subunit vaccine for preventing novel coronavirus infection based on novel coronavirus spike protein S1 region
CN111944064B (en) * 2020-08-21 2021-10-15 中国科学院微生物研究所 COVID-19 subunit vaccine and preparation method thereof
CN112048005B (en) * 2020-09-04 2022-09-09 江苏省中国科学院植物研究所 Novel coronavirus S protein fragment polyploid, preparation method thereof, detection kit, vaccine and medicament
CN112794884B (en) * 2020-12-28 2022-02-18 中国科学院微生物研究所 Novel coronavirus protein, preparation method and neutralizing antibody detection kit
CN114057894B (en) * 2021-06-18 2022-08-05 国药中生生物技术研究院有限公司 Novel recombinant coronavirus RBD trimer protein vaccine capable of generating broad-spectrum cross-neutralization activity, and preparation method and application thereof

Also Published As

Publication number Publication date
CN116041534A (en) 2023-05-02
WO2023025287A1 (en) 2023-03-02
CN116041448A (en) 2023-05-02

Similar Documents

Publication Publication Date Title
CN111217919B (en) Novel coronavirus S protein double-region subunit nano vaccine based on pyrococcus ferritin
CN111217918B (en) Novel coronavirus S protein double-region subunit nano vaccine based on 2, 4-dioxotetrahydropteridine synthase
CN113861278B (en) Novel recombinant coronavirus RBD trimer protein vaccine capable of generating broad-spectrum cross-neutralization activity, and preparation method and application thereof
WO2021184415A1 (en) Helicobacter pylori ferritin-based novel coronavirus s protein single-region subunit nanovaccine
CN111607002B (en) Novel coronavirus S protein double-region subunit nano vaccine based on helicobacter pylori ferritin
JP7088841B2 (en) Stabilized soluble pre-fusion RSVF protein
CN113164586B (en) Immune composition and preparation method and application thereof
CN116121277B (en) Nucleic acid molecules encoding structural proteins of novel coronaviruses and uses thereof
Singh et al. Expression of rabies glycoprotein and ricin toxin B chain (RGP–RTB) fusion protein in tomato hairy roots: a step towards Oral vaccination for rabies
CN113736801B (en) mRNA and novel coronavirus mRNA vaccine comprising same
CN115838433A (en) Beta coronavirus polymer antigen, preparation method and application thereof
WO2023051701A1 (en) Mrna, protein and vaccine against sars-cov-2 infection
WO2023138334A1 (en) Recombinant novel coronavirus protein vaccine, and preparation method and use thereof
CN114480441B (en) Nucleotide sequence and application of recombinant protein nanoparticles expressed by nucleotide sequence in canine distemper virus vaccine
CN115960252A (en) Novel coronavirus immunogenic substance, preparation method and application thereof
CN112142851B (en) Subunit fusion protein tG on rabies virus surface and preparation method and application thereof
WO2023138333A1 (en) Recombinant sars-cov-2 protein vaccine, and preparation method therefor and use thereof
WO2022116528A1 (en) Circular rna, vaccine containing circular rna, and kit for detecting novel coronavirus neutralizing antibody
AU2021389014A1 (en) Virus-like particles and methods of production thereof
CN114437236A (en) Recombinant African swine fever virus multi-epitope fusion protein, preparation and application thereof
CN111732667B (en) Peste des petits ruminants virus genetic engineering subunit vaccine
CN113801206A (en) Method for inducing anti-neocoronavirus neutralizing antibody by using receptor recognition domain
CN115043915B (en) Method for enhancing immunogenicity of novel coronavirus variant strain and application thereof
CN106337038B (en) Method for preparing vaccine by transpeptidase shearing and application thereof
CN114292314B (en) Flagellin mutant and application thereof in preparation of African swine fever antigen fusion protein

Legal Events

Date Code Title Description
PB01 Publication
PB01 Publication
SE01 Entry into force of request for substantive examination
SE01 Entry into force of request for substantive examination