WO2022184027A1 - Nouvel antigène multivalent de coronavirus, son procédé de préparation et son utilisation - Google Patents

Nouvel antigène multivalent de coronavirus, son procédé de préparation et son utilisation Download PDF

Info

Publication number
WO2022184027A1
WO2022184027A1 PCT/CN2022/078426 CN2022078426W WO2022184027A1 WO 2022184027 A1 WO2022184027 A1 WO 2022184027A1 CN 2022078426 W CN2022078426 W CN 2022078426W WO 2022184027 A1 WO2022184027 A1 WO 2022184027A1
Authority
WO
WIPO (PCT)
Prior art keywords
novel coronavirus
amino acid
rbd
acid sequence
protein
Prior art date
Application number
PCT/CN2022/078426
Other languages
English (en)
Chinese (zh)
Inventor
高福
戴连攀
徐坤
高萍
安亚玲
Original Assignee
中国科学院微生物研究所
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 中国科学院微生物研究所 filed Critical 中国科学院微生物研究所
Publication of WO2022184027A1 publication Critical patent/WO2022184027A1/fr

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
    • 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
    • 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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • 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

Definitions

  • the present application relates to the field of biomedicine, in particular to a novel coronavirus multivalent antigen, its preparation method and application.
  • Novel coronavirus pneumonia (new coronary pneumonia, COVID-19) is caused by the infection of the new coronavirus (new coronavirus, SARS-CoV-2).
  • the new coronavirus belongs to the genus ⁇ -coronavirus of the family Coronaviridae, has an envelope, and is a positive-strand RNA virus.
  • the spike (S) protein on the surface of the virus is responsible for the recognition, binding and membrane fusion with the receptor ACE2 (Angiotensin-converting enzyme 2) protein of the host cell, and mediates virus invasion.
  • S spike
  • ACE2 Angiotensin-converting enzyme 2
  • RBD receptor binding domain
  • the purpose of this application is to provide a novel coronavirus multivalent antigen, its preparation method and application.
  • the partial or full-length amino acid sequences of two monomeric RBD proteins are concatenated, wherein the partial or full-length amino acid sequence of one monomeric RBD protein is the original strain sequence, and the amino acid sequence of the other monomeric RBD protein is introduced
  • One, two or three point mutations, the point mutations are K417N, E484K and N501Y respectively.
  • the multivalent antigen of the novel coronavirus of the present application can activate a broad-spectrum protective antibody, which plays a good role in both the original strain and the current epidemic strain. preventive effect.
  • a novel coronavirus antigen its amino acid sequence comprises: the amino acid sequence arranged according to (A-B)-(A-B') pattern or the amino acid sequence arranged according to (A-B)-C-(A-B') pattern, wherein: A-B represent the partial amino acid sequence or the full-length amino acid sequence of the receptor binding region of the surface spike protein of the novel coronavirus, and the partial amino acid sequence of the receptor binding region of the surface spike protein of the novel coronavirus includes at least K417, E484 or One or more amino acids in N501, C represents the connecting amino acid sequence, A-B' represents the amino acid sequence obtained by substituting the amino acid sequence of A-B with one or more amino acids in K417N, E484K or N501Y.
  • the partial amino acid sequence of the receptor binding region of the novel coronavirus surface spike protein is at least 50% or more of the full-length amino acid sequence of the receptor binding region of the novel coronavirus surface spike protein , 60% or more, 70% or more, 80% or more, 90% or more, 95% or more, or 99% or more.
  • the partial amino acid sequence or full-length amino acid sequence of the receptor binding region of the surface spike protein of the novel coronavirus is SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 3.
  • SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3 sequences are all derived from part of the S protein sequence of the WH01 strain of the new coronavirus (GenBank on NCBI: QHR63250), which are respectively the new coronavirus The R319-S530 region, the R319-K537 region, and the R319-F541 region of the RBD of the S protein.
  • A-B' represents the amino acid sequence obtained by simultaneously substituting the amino acid sequence of A-B with K417N, E484K and N501Y.
  • amino acid sequence of the novel coronavirus antigen is SEQ ID NO:4.
  • the linking amino acid sequence includes: (GGS) n linking sequence, where n represents the number of GGS, and n is an integer ⁇ 1; optionally, n is selected from 1-10 Integer; further optionally, n is an integer selected from 1-5.
  • the three letters GGS represent the amino acids G, G, and S, respectively.
  • the application also provides a method for preparing the above-mentioned novel coronavirus antigen, comprising the following steps: adding a sequence encoding a signal peptide to the 5' end of the nucleotide sequence encoding the above-mentioned novel coronavirus antigen, adding histidine to the 3' end acid and stop codon, clone and express, screen the correct recombinant, and then transfect cells of the expression system for expression, collect the cell supernatant after expression, and purify to obtain the novel coronavirus antigen.
  • the cells of the expression system include mammalian cells, insect cells, yeast cells or bacterial cells, optionally; the mammalian cells include HEK293T cells, HEK293F cells or CHO cells, wherein The bacterial cells include E. coli cells.
  • the present application also provides a nucleotide sequence encoding the above novel coronavirus antigen.
  • nucleotide sequence is SEQ ID NO:5.
  • the present application also provides a recombinant vector comprising the above-mentioned nucleotide sequence.
  • the present application also provides an expression system cell comprising the above-mentioned recombinant vector.
  • the application also provides an application of the above-mentioned novel coronavirus antigen, the above-mentioned nucleotide sequence, the above-mentioned recombinant vector, and the above-mentioned expression system cell in the preparation of a novel coronavirus vaccine.
  • the present application also provides a novel coronavirus vaccine, comprising the above novel coronavirus antigen and adjuvant.
  • the adjuvant is selected from aluminum adjuvant, MF59 adjuvant or MF59-like adjuvant.
  • the present application also provides a novel coronavirus DNA vaccine, comprising: a recombinant vector comprising the DNA sequence encoding the novel coronavirus antigen.
  • the application also provides a novel coronavirus mRNA vaccine, comprising: a recombinant vector comprising the mRNA sequence encoding the novel coronavirus antigen.
  • the application also provides a novel coronavirus virus vector vaccine, including: a recombinant virus vector comprising a nucleotide sequence encoding the above-mentioned novel coronavirus antigen; optionally, the virus vector is selected from one or more of the following: Adenovirus vector, poxvirus vector, influenza virus vector, adeno-associated virus vector.
  • a novel coronavirus RBD partial or full-length amino acid sequence tandem dimer antigen is designed in this application, and it can not introduce any exogenous linking sequence, wherein the partial or full-length amino acid sequence of a monomeric RBD protein is The original strain sequence (GenBank on NCBI: QHR63250), and the amino acid sequence of another monomeric RBD protein introduced three point mutations based on the original strain sequence, namely K417N, E484K and N501Y.
  • the novel coronavirus antigen can efficiently induce an immune response against the original virus strain, and can also efficiently induce an immune response against the mutant virus strain.
  • the novel coronavirus antigen can enhance the immune response that activates the conserved epitopes of the two RBD proteins, thus providing broad-spectrum protection.
  • Figure 1 shows that in Example 2 of the present application, the plasmids expressing RBD-tr2-WTV2 and RBD-tr2 were transfected in HEK293T cells, and the supernatant and cells were collected 72 hours later, and the expression of the target protein was detected by Western blot experiment.
  • Figure 2 shows in Example 3 of the present application, the supernatant of RBD-tr2-WTV2 protein expressed by HEK293F cells was used to purify the target protein through a nickel affinity column, and the RBD-tr2-WTV2 protein was detected by Coomassie brilliant blue staining.
  • Figure 3 shows that in Example 3 of the present application, the RBD-tr2-WTV2 protein was purified by a nickel affinity column, and the target protein was purified by molecular sieve chromatography, and the RBD-tr2-WTV2 protein was detected by Coomassie brilliant blue staining.
  • Fig. 4 shows that in Example 4 of the present application, after the purified RBD-tr2-WTV2 protein is combined with the new coronavirus receptor human ACE2 protein, molecular sieve chromatography is performed to purify the complex of RBD-tr2-WTV2 and human ACE2 protein, and Protein detection was performed by Coomassie brilliant blue staining.
  • Figure 5 shows the complex of RBD-tr2-WTV2 and CB6 antibody protein purified by molecular sieve chromatography in Example 5 of the present application, and protein detection by Coomassie brilliant blue staining.
  • Figure 6 shows that in Example 5 of the present application, the complex of RBD-tr2-WTV2 and CB6 antibody was continuously mixed with human ACE2 protein to form a complex of RBD-tr2-WTV2, CB06 antibody and human ACE2 protein, purified by molecular sieve chromatography , and protein detection by Coomassie brilliant blue staining.
  • RBD-tr2 is the RBD of two prototype virus strains in series to form a dimer
  • RBD-tr2-V2 is the RBD of two Beta variant strains Concatenated into a dimer
  • RBD-tr2-WTV2 is a chimeric dimer of the RBD of a prototype virus strain and the RBD of a Beta variant strain.
  • Figure 8 is the supernatant of RBD-tr2, RBD-tr2-V2 and RBD-tr2-WTV2 proteins expressed by 293F cells in Example 6 of the present application, purified by molecular sieve chromatography, and purified by Coomassie brilliant blue stain to detect the protein of interest.
  • Figure 9 shows the use of the novel coronavirus receptor protein hACE2 and representative antibodies of 5 different antibody epitopes to identify antigenic protein epitopes in Example 7 of the present application; wherein, prototype RBD-monomer represents the prototype virus strain monomer RBD protein, Beta RBD-monomer represents the monomeric RBD protein of the Beta variant, RBD-tr2 represents the dimer RBD protein of the prototype virus strain, RBD-tr2-V2 represents the dimer RBD protein of the Beta variant strain, and RBD-tr2-WTV2 represents the prototype virus strain RBD is a tandem chimeric dimer protein with Beta variant RBD protein, and N/B means no binding.
  • Example 10 is a schematic diagram of the S protein mutation site of each new coronavirus variant strain in Example 8 of the present application.
  • Figure 11 shows the neutralization results of sera collected from mice 14 days after the second immunization of the new coronavirus prototype strain and pseudoviruses of each new coronavirus variant strain in Example 8 of the present application, wherein Sham is the negative control immunization group , RBD-tr2 is the prototype strain dimer RBD protein immune group, RBD-tr2-V2 is the Beta mutant dimer RBD protein immune group, RBD-tr2-WTV2 is the prototype strain RBD and the Beta mutant RBD protein in series Chimeric Dimeric Protein Immunogroup.
  • Figure 12 shows the neutralizing antibody titers of the sera of mice immunized with RBD-tr2, RBD-tr2-V2 and RBD-tr2-WTV2 to the prototype strain of the new coronavirus and the pseudoviruses of each new coronavirus variant in Example 8 of the application Radar chart plotted with geometric mean (GMT) of degrees to analyze the effect of broad-spectrum neutralization.
  • GCT geometric mean
  • Figure 13 shows the results of the live virus challenge experiment of the prototype strain of the new coronavirus or the Beta variant of each immunized mouse group in Example 9 of the present application; wherein, Sham is the negative control immunization group, and RBD-tr2 is the prototype virus Strain dimer RBD protein immunization group, RBD-tr2-V2 is the Beta variant strain dimer RBD protein immunization group, RBD-tr2-WTV2 is the prototype strain-Beta variant chimeric RBD dimer protein immunization group; and , where A-C is the challenge result of the prototype strain of SARS-CoV-2, D-F is the challenge result of the SARS-CoV-2 Beta variant strain, A and D are the genomic RNA detected in mouse lung tissue on the 5th day after challenge (gRNA) load, B and E are the subgenomic RNA (sgRNA) load of mouse lung tissue detected on the 5th day after challenge, C and F are the neutralizing antibody titer after immunization and the Cor
  • Example 1 Design of novel coronavirus broad-spectrum protective vaccine RBD-tr2-WTV2
  • N501Y N501Y
  • 69-70del deletion
  • P681H P681H
  • N501Y mutation enhances the binding ability of S protein to receptor ACE2 protein
  • 501Y.V1 has stronger transmission ability, but does not enhance pathogenicity .
  • a new mutant strain 501Y.V2 B.1.351 lineage was reported in South Africa. In South Africa, the 501Y.V2 mutant strain quickly replaced other strains and was called the main epidemic strain. The study found that 501Y.V2 infection will cause resulting in higher viral loads, suggesting a possible increase in transmissibility.
  • the mutations of 501Y.V2 on the S protein include: D80A, L242del, A243del, L244del, R246I, K417N, E484K, N501Y, D614G, A701V. 501Y.V2 was also named after Beta variant.
  • RBD-tr2 is two identical new coronavirus RBD tandem repeat dimers (R319-K537-R319-K537). Based on this, we mutated one of the RBD sequences K417N, E484K, N501Y, and the other RBD The sequence remained unchanged, and the resulting dimer was named RBD-tr2-WTV2.
  • RBD-tr2-WTV2 can efficiently induce both an immune response against the original virus strain and an immune response against the mutant virus strain.
  • the RBD-tr2-WTV2 protein can also enhance the activation of two of the conserved RBD proteins. specific antibody response, thus providing a broader spectrum of protection.
  • the amino acid sequence of the designed RBD-tr2-WTV2 antigen is SEQ ID NO: 4 (signal peptide and histidine sequence are not given in the sequence), and the nucleotide sequence for expressing the antigen is SEQ ID NO: 5, and the nucleotide sequence is SEQ ID NO: 5.
  • the elements of the sequence from N-terminal to C-terminal are (1) Kozak sequence gccacc; (2) the sequence encoding the signal peptide of MERS-S protein: MIHSVFLLMFLLTPTES (SEQ ID NO: 6); (3) New coronavirus (GenBank on NCBI: QHR63250) S protein RBD (R319-K537); (4) New coronavirus (GenBank on NCBI: QHR63250) S protein RBD (R319-K537), which contains three point mutations K417N, E484K, N501Y; (5) 6 histidines; (6) stop codon.
  • the gene sequence was synthesized in Suzhou Jinweizhi Company, and cloned into pCAGGS plasmid through EcoRI and XhoI restriction sites to obtain pCAGGS-RBD-tr2-WTV2 plasmid.
  • the pCAGGS plasmid expressing RBD-tr2-WTV2 was transfected into HEK293T cells, and at the same time, the pCAGGS plasmid expressing RBD-tr2 was transfected as a positive control, and only the transfection reagent was added as a negative control. After 72 hours, the cells and the culture supernatant were harvested, and the expression of the target protein was detected by Western blot. In the early stage, the research group immunized rabbits with the new coronavirus RBD protein to prepare a rabbit anti-new crown RBD polyclonal antibody.
  • HEK293F cells suitable for large-scale protein expression to express RBD-tr2-WTV2 antigen Transfect the plasmid pCAGGS-RBD-tr2-WTV2 into HEK293F cells, collect the supernatant after 5 days, remove the precipitate by centrifugation, and filter it through a 0.22 ⁇ m filter. Remove impurities. The cell supernatant was adsorbed through a nickel affinity column (Histrap, GE Healthcare) at 4°C and washed with buffer A (20 mM Tris, 150 mM NaCl, pH 8.0) to remove non-specifically bound proteins.
  • buffer A (20 mM Tris, 150 mM NaCl, pH 8.0
  • buffer B (20mM Tris, 150mM NaCl, pH 8.0, 1000mM imidazole) to gradient elute the target protein from the Histrap, and set the ratio of buffer B to 2%, 5%, 10%, 20% in turn , 30%, 50%, 100%, the results are shown in Figure 2.
  • the target protein was further purified by molecular sieve chromatography on Hiload TM 16/600 Superdex TM 200pg column (GE Healthcare).
  • the molecular sieve chromatography buffer was PBS buffer (8 mM Na2HPO4, 136 mM NaCl, 2 mM KH2PO4, 2.6 mM KCl, pH 7.2).
  • RBD-tr2-WTV2 has an elution peak at about 80ml ( Figure 3).
  • SDS-PAGE analysis shows that the protein is 62KD under both non-reducing (without DTT) and reducing (with DTT) conditions Left and right, it is a single-chain dimer (the size of the monomer is 31KD). This shows that RBD-tr2-WTV2 can be folded and secreted correctly, and high-purity antigen protein can be obtained by purification.
  • RBD-tr2-WTV2 protein was mixed with human ACE2 protein and incubated at 4°C for 8 hours.
  • the complex of RBD-tr2-WTV2 with human ACE2 protein was then purified by molecular sieve chromatography on a Superdex TM 200Increase 10/300GL column (GE Healthcare). It can be seen from Figure 4 that the corresponding elution peak at about 11ml is the RBD-tr2-WTV2 binding human ACE2 protein complex, and the corresponding elution peak at about 15ml is RBD-tr2-WTV2 protein, indicating that RBD-tr2-WTV2 can interact with human ACE2 protein. ACE2 protein binding, proving that the conformation of RBD-tr2-WTV2 is correct.
  • CB6 antibody can bind to the original Wuhan isolated new coronavirus RBD (PMID: 32454512), so we used CB6 antibody and RBD- tr2-WTV2 binding experiments were performed.
  • 0.5 mg of RBD-tr2-WTV2 protein at a concentration of 5.9 mg/ml was mixed with 1 mg of CB6 antibody protein at a concentration of 15.4 mg/ml, and incubated at 4°C for 4 hours.
  • RBD-WTV2 can be used as a vaccine to activate the generation of a broad-spectrum immune response.
  • the inventors designed three dimer proteins, which are: (1) RBD-tr2, which is a dimer formed by concatenating the RBDs of two prototype virus strains; (2) RBD-tr2-V2, which is a dimer formed by concatenating the RBDs of two Beta variant strains; ( 3) RBD-tr2-WTV2, which is a chimeric dimer composed of the RBD of a prototype virus strain and the RBD of a Beta variant strain in series; the schematic diagram of the structural design of each dimer protein is shown in FIG. 7 .
  • the RBD-tr2 protein, RBD-tr2-V2 protein and RBD-tr2-WTV2 protein were all expressed using 293F cells.
  • the specific procedure is as follows: 293F cells were transfected with plasmid pCAGGS-RBD-tr2, plasmid pCAGGS-RBD-tr2-V2 and plasmid pCAGGS-RBD-tr2-WTV2 respectively. After 5 days, the supernatant was collected, centrifuged to remove the precipitate, and then passed through 0.22 ⁇ m filter membrane to further remove impurities.
  • Cell supernatants were adsorbed through a nickel affinity column (Histrap, GE Healthcare) at 4°C and washed with buffer A (20 mM Tris, 150 mM NaCl, pH 8.0) to remove non-specifically bound proteins; then buffer B ( 20mM Tris, 150mM NaCl, pH 8.0, 1000mM imidazole) to wash the column to elute the target protein from the Histrap gradient, the corresponding eluted protein solution was concentrated with a 10kDa concentrator tube, and the solution was changed more than 30 times to buffer A.
  • buffer A (20 mM Tris, 150 mM NaCl, pH 8.0
  • buffer B 20mM Tris, 150mM NaCl, pH 8.0, 1000mM imidazole
  • the volume is less than 1 ml; then molecular sieve chromatography is performed through a Hiload TM 16/600 SuperdexTM 200pg column (GE Healthcare) to further purify the target protein.
  • the molecular sieve chromatography buffer was PBS buffer (8 mM Na 2 HPO 4 , 136 mM NaCl, 2 mM KH 2 PO 4 , 2.6 mM KCl, pH 7.2). After molecular sieve chromatography, the protein solution corresponding to the elution peak was collected and analyzed by SDS-PAGE. The results are shown in Figure 8; The size is about 31kDa). Thus, purified RBD-tr2 protein, RBD-tr2-V2 protein and RBD-tr2-WTV2 protein were obtained.
  • the inventors identified the RBD binding motif (RBM) of the antigenic protein and the exposure of the main neutralizing antibody epitopes by Surface Plasmon Resonance (SPR), and detected the human receptor of the antigenic protein for the new coronavirus - human Affinity of angiotensin-converting enzyme (hACE2), and a representative monoclonal antibody CB6 targeting 5 different epitopes in the SARS-CoV-2 RBD (for specific information on this antibody, see A human neutralizing antibody targets the receptor-binding site of SARS-CoV-2.Nature, 2020, PMID: 32454512), CV07-270 (for specific information of this antibody, please refer to A Therapeutic Non-self-reactive SARS-CoV-2 Antibody Protects from Lung Pathology in a COVID-19 Hamster Model.
  • SPR Surface Plasmon Resonance
  • SARS-CoV-2 neutralizing antibody structures inform therapeutic strategies. Nature, 2020, PMID: 33045718), S309 (this antibody For specific information, please refer to Cross-neutralization of SARS-CoV-2 by a human monoclonal SARS-CoV antibody.
  • Affinity test method This test was performed using a BIAcore 8000 (GE Healthcare) instrument. Before the test, the target antigen protein was exchanged into PBS-T buffer (10 mM Na 2 HPO 4 , 2 mM KH 2 PO 4 , pH 7.4, 137 mM NaCl, 2.7 mM KCl, 0.005% Tween 20). First, the antigen protein was immobilized on the CM5 chip by amino-coupling method, and the target response value was about 1000RU; then, the antibody Fab protein was diluted in multiples, and the diluent was used as the mobile phase to flow through the immobilized Fab protein at a speed of 30mL/min. Different real-time binding response signals are obtained for antigen protein, and the collected data is calculated using BIAevaluation Version 4.1 (GE Healthcare) software according to the 1:1 binding model, and finally the binding affinity of antigen protein and antibody is obtained.
  • PBS-T buffer 10 mM Na 2 HPO 4 , 2 mM KH
  • the chimeric RBD dimer antigen RBD-tr2-WTV2 maintained affinity for all tested mAbs (see bottom panel in Figure 9), indicating that the chimeric antigen was designed to expose the receptor binding site well and Major neutralizing antibody epitope conformations are shown.
  • Example 8 Detection of humoral immune responses induced by RBD-tr2-WTV2 vaccine
  • the inventors used the above-mentioned dimeric RBD antigen vaccines to immunize BALB/c mice, and the experimental groups were as follows: (1) The prototype strain RBD dimer RBD-tr2 immunized group (2) Beta RBD dimer RBD-tr2-V2 immune group; (3) Prototype strain+Beta strain chimeric RBD dimer RBD-tr2-WTV2 immune group; (4) Sham group, PBS solution.
  • each dimeric antigen protein was mixed with Addavax adjuvant to prepare a vaccine; for the Sham group, PBS solution was mixed with Addavax adjuvant to prepare a vaccine control.
  • the experimental mice were immunized twice, with an interval of 21 days between the two immunizations. Each dose contained 0.5 ⁇ g of antigenic protein and was injected intramuscularly in the hind legs. On the 14th day after the second immunization, the blood of mice was collected, and the pseudovirus of the new coronavirus was used to detect the 50% pseudovirus neutralization titers (pVNT 50 ) of the mouse serum to the pseudovirus of the prototype strain and the mutant strain of the new coronavirus, respectively.
  • pVNT 50 pseudovirus neutralization titers
  • the variant strains include Alpha (B.1.1.7), Beta (B.1.351), Gamma (P.1), Delta (B.1.617.2), Delta plus (S protein mutation site is the same as B.1.617.2) , plus K417N), Kappa (B.1.617.1), Lambda (C.37) and Omicron (B.1.1.529) variant strains, the mutation sites of each variant strain relative to the prototype strain are shown in Figure 10.
  • the new coronavirus pseudovirus used in this example is a pseudovirus that displays the new coronavirus S protein prepared based on the vesicular stomatitis virus (VSV) skeleton. Booster Interval on Neutralization of Omicron Variant, N Engl J Med, 2022, PMID:35081296).
  • the method for detecting the neutralizing antibody titer of the new coronavirus pseudovirus (hereinafter referred to as pseudovirus) is as follows: in a 96-well plate, the immunized mouse serum is diluted by a 2-fold gradient, and then mixed with the pseudovirus, and the blank medium is also Mixed with pseudovirus as a control, incubated at 37°C for 1 hour. The immune serum-pseudovirus mixture was transferred to a 96-well plate plated with Vero cells.
  • the geometric mean (GMT) of the neutralizing antibody titers to the pseudovirus displaying the prototype strain S protein is 1779, but the neutralization effect on some variant strains is slightly higher.
  • the declines, including Beta (GMT, 487), Gamma (GMT, 699), etc. are 1100 and 122 for the currently popular Delta and Omicron mutant strains, and the fold reduction for Omicron mutant strains is higher.
  • Beta RBD dimer vaccine RBD-tr2-V2 its neutralizing antibody titers to the S protein pseudovirus of the Beta and Gamma variant strains were higher, 809 and 1650, respectively, but the titers to the prototype strain and other variant strains were higher.
  • the neutralizing activity of the rhizome is not high, and the GMT range is 104-385, among which the GMTs for the currently popular Delta and Omicron variants are 104 and 136.
  • Example 9 New coronavirus live virus challenge experiment to verify the effect of RBD-tr2-WTV2 vaccine
  • BALB/c mice Due to the low affinity of BALB/c mouse ACE2 to the S protein of the prototype strain, BALB/c mice are not susceptible to the prototype new coronavirus, but the N501Y mutation of the S protein can improve the affinity of the S protein to the mouse ACE2.
  • the Beta variant contains the N501Y mutation, so BALB/c mice are susceptible to the Beta variant.
  • mice in each experimental group were challenged with the prototype strain of the new coronavirus, while the other four mice in each experimental group were challenged with the Beta variant of the new coronavirus.
  • the challenge experiment method of the prototype strain of 2019-nCoV is as follows: 8 ⁇ 10 9 vp adenovirus Ad5-hACE2 was transduced by intranasal, and the hACE2 model was transiently expressed. Five days after transduction of Ad5-hACE2, 5 ⁇ 10 were infected by intranasal injection. 5 TCID 50 prototype strain of novel coronavirus (hCoV-19/China/CAS-B001/2020 strain).
  • Beta variant strain (GDPCC-nCoV84 strain) was as follows: Mice were directly infected with 1 ⁇ 10 6 TCID 50 new coronavirus Beta variant strain (GDPCC-nCoV84 strain) by intranasal instillation.
  • mice On the 5th day after infection with the new coronavirus prototype strain or Beta variant strain, the mice were euthanized and dissected; the lungs of each mouse were taken out and divided into 2 parts: one part was added to DMEM medium and then homogenized and ground to extract the virus Nucleic acid, using the qRT-PCR method to quantify the viral genomic gRNA and subgenomic sgRNA of the virus, gRNA represents the entire viral nucleic acid, and sgRNA represents the viral nucleic acid in the replication process, which is an indicator of the level of virus replication; After formaldehyde fixation, hematoxylin and eosin (H&E) staining was performed to observe histopathology.
  • H&E hematoxylin and eosin
  • the method for detecting viral gRNA and sgRNA is as follows: after the mouse lung tissue is homogenized, take 140 ⁇ L of the tissue homogenate supernatant and use the viral RNA extraction kit QIAamp Viral RNA Mini Kit (GIAGEN company, cat.no.52906) to extract viral RNA . Using FastKing One-Step Probe RT-qPCR Kit (Tiangen Bio, China) on CFX96Touch real-time PCR detection system (Bio-Rad, USA), following the kit instructions for SARS-CoV-2-specific quantitative reversal PCR (qRT-PCR) detection. Two sets of primers and probes were used to detect SARS-CoV-2 genome gRNA and sgRNA, respectively.
  • the primer probe sequences for detecting SARS-CoV-2 virus gRNA are as follows:
  • the primer probe sequences for detecting SARS-CoV-2 virus sgRNA are as follows:
  • sgRNA-F CGATCCTTGTAGATCTGTTCTC (SEQ ID NO: 10);
  • sgRNA-R ATATTGCAGCAGTACGCACACA (SEQ ID NO: 11);
  • sgRNA-probe FAM-ACACTAGCCATCCTTACTGCGCTTCG (SEQ ID NO: 12)-TAMRA.
  • FIG. 13 The detection results of the challenge experiment are shown in Figure 13; it can be seen from Figure 13 that for the mice challenged with the new coronavirus prototype strain, the control group mice detected a high level of gRNA (mean: 1.72 ⁇ 10 9 copies/g ) and sgRNA (mean: 1.9 x 108 copies/g) ( Figures 13A and 13B), by contrast, the viral loads (both gRNA and sgRNA) detected in vaccine-immunized mice were significantly reduced, Among them, the mean values of gRNA in lung tissue of RBD-tr2, RBD-tr2-V2 and RBD-tr2-WTV2 immunized groups were 4.61 ⁇ 10 5 copies/g, 2.58 ⁇ 10 6 copies/g and 2.66 ⁇ 10 5 copies/g, respectively.
  • RBD-tr2-WTV2 was the most effective at inhibiting the prototype strain of the new coronavirus, which was significantly different from the RBD-tr2-V2 group.
  • no lung tissue viral sgRNAs were detected in all vaccine groups, indicating that they completely inhibited viral replication (Figure 13B). Correlation analysis was performed based on the linear model based on the correlation analysis between the neutralizing antibody titer of each mouse against the pseudovirus of the prototype strain of the new coronavirus and the corresponding lung tissue virus gRNA after challenge.
  • mice challenged with the 2019-nCoV Beta variant high levels of gRNA (mean: 4.01 ⁇ 10 8 copies/g) and sgRNA (mean: 3.03 ⁇ 10 7 copies/g) were detected in control mice (Fig. 13D). and 13E), in contrast, significantly lower viral loads, including both gRNA and sgRNA, were detected in mice immunized with vaccines with RBD-tr2, RBD-tr2-V2, and RBD-tr2-WTV2 immunized
  • the average value of lung tissue gRNA in the group was 6.34 ⁇ 10 6 copies/g, 5.46 ⁇ 10 5 copies/g copies/g and 9.81 ⁇ 10 4 copies/g, respectively, indicating that the three vaccines have the effect of inhibiting the new coronavirus Beta variant strain.
  • RBD-tr2-WTV2 The best was RBD-tr2-WTV2, which was significantly different from the RBD-tr2 group; neither RBD-tr2-V2 nor RBD-tr2-WTV2 vaccine groups detected viral sgRNAs in lung tissue, indicating that they completely inhibited viral replication ; sgRNA was still detected in mice in the RBD-tr2 group, and the mean value of viral sgRNA in the lung tissue of the RBD-tr2 group was 4.51 ⁇ 10 5 copies/g ( FIG. 13E ).
  • Correlation analysis was performed based on the linear model based on the correlation analysis between the neutralizing antibody titer of each mouse against the pseudovirus of the new coronavirus beta variant strain and the corresponding lung tissue virus gRNA after challenge with the new coronavirus beta variant strain, and the neutralizing antibody can be seen.
  • mice in each experimental group The histopathological results of the lungs of mice in each experimental group after being challenged with the new coronavirus prototype strain or Beta variant strain are shown in Figure 13G.
  • the lung pathological changes of the control group mice Sham
  • mice vaccinated with RBD-tr2, RBD-tr2-V2, or RBD-tr2-WTV2 exhibited only mild lung damage (Fig. 13G).
  • the present application provides a novel coronavirus multivalent antigen, its preparation method and application.
  • the novel coronavirus antigen can not only efficiently induce an immune response against the original virus strain, but also efficiently induce an immune response against a series of mutant virus strains; that is, the novel coronavirus multivalent antigen of the present application can activate broad-spectrum protective Antibodies can have a good preventive effect on the original strain of the new coronavirus and the current epidemic strain.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Virology (AREA)
  • General Health & Medical Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Organic Chemistry (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Molecular Biology (AREA)
  • Veterinary Medicine (AREA)
  • Public Health (AREA)
  • Animal Behavior & Ethology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Biophysics (AREA)
  • General Chemical & Material Sciences (AREA)
  • Oncology (AREA)
  • Communicable Diseases (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Biochemistry (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Genetics & Genomics (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Immunology (AREA)
  • Microbiology (AREA)
  • Mycology (AREA)
  • Epidemiology (AREA)
  • Peptides Or Proteins (AREA)
  • Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)

Abstract

La présente invention concerne un nouvel antigène multivalent de coronavirus, son procédé de préparation et son utilisation. Une séquence d'acides aminés du nouvel antigène de coronavirus comprend : une séquence d'acides aminés disposée dans un motif (A-B)-(A-B') ou une séquence d'acides aminés disposée dans un motif (A-B)-C-(A-B'), où A-B représente une séquence d'acides aminés partielle ou une séquence d'acides aminés plein longueur d'un domaine de liaison au récepteur (RBD) d'une protéine de spicule de surface du nouveau coronavirus, C représente une séquence d'acides aminés de liaison, et A-B' représente une séquence d'acides aminés obtenue par substitution de la séquence d'acides aminés de A-B avec un ou plusieurs acides aminés dans K417N, E484K ou N501Y. Par comparaison avec un dimère de répétition en tandem de deux protéines RBD de souche originale, le nouvel antigène multivalent de coronavirus selon la présente invention peut activer un anticorps de protection à large spectre, et a un bon effet préventif sur la souche originale et la souche épidémique actuelle.
PCT/CN2022/078426 2021-03-01 2022-02-28 Nouvel antigène multivalent de coronavirus, son procédé de préparation et son utilisation WO2022184027A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CN202110225584.2 2021-03-01
CN202110225584 2021-03-01

Publications (1)

Publication Number Publication Date
WO2022184027A1 true WO2022184027A1 (fr) 2022-09-09

Family

ID=81146201

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2022/078426 WO2022184027A1 (fr) 2021-03-01 2022-02-28 Nouvel antigène multivalent de coronavirus, son procédé de préparation et son utilisation

Country Status (2)

Country Link
CN (1) CN114369172A (fr)
WO (1) WO2022184027A1 (fr)

Families Citing this family (10)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN113150085B (zh) * 2021-04-27 2023-04-18 成都威斯克生物医药有限公司 抗SARS-CoV-2感染的组合物
CN114573667B (zh) * 2022-05-06 2022-08-02 艾棣维欣(苏州)生物制药有限公司 SARS-CoV-2病毒B.1.1.529突变株DNA疫苗及应用
CN115124626A (zh) * 2022-05-11 2022-09-30 中国科学院微生物研究所 串联式杂合三聚体新冠疫苗
CN117051019A (zh) * 2022-05-11 2023-11-14 江苏瑞科生物技术股份有限公司 新型冠状病毒疫苗及其制备方法和应用
CN114989308B (zh) * 2022-05-12 2023-04-04 中国科学院微生物研究所 新冠病毒嵌合核酸疫苗及其用途
CN115678906B (zh) * 2022-05-12 2023-09-19 中国科学院微生物研究所 经优化的新冠病毒嵌合核酸疫苗及其用途
CN114751992B (zh) * 2022-05-25 2024-05-31 中国科学院微生物研究所 一种冠状病毒的通用疫苗及其应用
CN115043915B (zh) * 2022-05-25 2023-10-24 中山大学 一种增强新冠病毒变异株免疫原性的方法及其应用
CN117327174A (zh) * 2022-06-24 2024-01-02 广东菲鹏制药股份有限公司 抗新型冠状病毒人源化多价结合蛋白及其应用
CN115197331A (zh) * 2022-06-28 2022-10-18 中国科学院微生物研究所 新冠病毒三聚体嵌合疫苗及其用途

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN111592602A (zh) * 2020-02-10 2020-08-28 中国科学院微生物研究所 一种β冠状病毒抗原、其制备方法和应用
WO2020198337A1 (fr) * 2019-03-25 2020-10-01 Ohio State Innovation Foundation Séquences d'arnm modifiées et leurs utilisations
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
CN112300253A (zh) * 2020-06-29 2021-02-02 斯克里普斯研究院 稳定的冠状病毒刺突(s)蛋白抗原和相关疫苗

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2020198337A1 (fr) * 2019-03-25 2020-10-01 Ohio State Innovation Foundation Séquences d'arnm modifiées et leurs utilisations
CN111592602A (zh) * 2020-02-10 2020-08-28 中国科学院微生物研究所 一种β冠状病毒抗原、其制备方法和应用
CN112300253A (zh) * 2020-06-29 2021-02-02 斯克里普斯研究院 稳定的冠状病毒刺突(s)蛋白抗原和相关疫苗
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

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
WANG ZIJUN, SCHMIDT FABIAN, WEISBLUM YISKA, MUECKSCH FRAUKE, BARNES CHRISTOPHER O., FINKIN SHLOMO, SCHAEFER-BABAJEW DENNIS, CIPOLL: "mRNA vaccine-elicited antibodies to SARS-CoV-2 and circulating variants", BIORXIV, 30 January 2021 (2021-01-30), XP055915931, DOI: 10.1101/2021.01.15.426911 *

Also Published As

Publication number Publication date
CN114369172A (zh) 2022-04-19

Similar Documents

Publication Publication Date Title
WO2022184027A1 (fr) Nouvel antigène multivalent de coronavirus, son procédé de préparation et son utilisation
CN113230395B (zh) 一种β冠状病毒抗原、β冠状病毒二联疫苗及其制备方法和应用
CN111592602B (zh) 一种β冠状病毒抗原、其制备方法和应用
WO2023186170A1 (fr) Antigène chimérique de variants delta et omicron de sars-cov-2, son procédé de préparation et son utilisation
WO2022089471A1 (fr) ANTIGÈNE MULTIMÈRE DE β-CORONAVIRUS, SON PROCÉDÉ DE PRÉPARATION ET SON UTILISATION
KR20160002938A (ko) 안정화된 가용성 예비융합 ｒｓｖ ｆ 폴리펩타이드
WO2017150683A1 (fr) Particule de type virale du virus zika
US10098943B2 (en) Flavivirus virus like particle
CN114106164B (zh) 抗新型冠状病毒s蛋白的单克隆抗体及其应用
CN113150084B (zh) 一种纳米化的冠状病毒抗原及其应用
EP2238160B1 (fr) Protéines multimères capables d'induire une réponse d'anticorps à l'encontre du bêta-amyloïde et leur utilisation
AU2009216696A1 (en) Protein nanocarriers, process for obtaining them and applications
EP4313138A1 (fr) Vaccin sous-unitaire contre le sars-cov-2
US9701720B2 (en) Epitope-scaffold immunogens against respiratory syncytial virus (RSV)
CN114702556A (zh) 一种冠状病毒rbd变异体及其应用
CN115724999B (zh) 串联式杂合三聚体新冠疫苗
US20030198947A1 (en) Hepatitis virus sentinel virus I (SVI)
WO2023226988A1 (fr) Procédé permettant d'améliorer l'immunogénicité d'un variant du sars-cov-2 et utilisation associée
KR102039059B1 (ko) 일본 뇌염 바이러스 피막 단백질의 돌출 도메인을 포함하는 단백질, 이의 동형이량체, 및 이의 용도
CN106749672B (zh) 一种丙型肝炎病毒融合抗原蛋白及其应用
RU2509805C2 (ru) СПОСОБ ПОЛУЧЕНИЯ РЕКОМБИНАНТНОГО АНТИГЕНА G2 ХАНТАВИРУСА ДОБРАВА В КЛЕТКАХ E.coli
EP2833901A1 (fr) Polypeptides et leur utilisation dans le traitement de l'infection à metaneumovirus (mpv)
CN117050193A (zh) 一种β冠状病毒重组嵌合抗原、其制备方法及应用
CN117917429A (zh) 新型冠状病毒抗原组合物、其制备方法和应用

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: 22762492

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 22762492

Country of ref document: EP

Kind code of ref document: A1