WO2023186170A1 - Antigène chimérique de variants delta et omicron de sars-cov-2, son procédé de préparation et son utilisation - Google Patents

Antigène chimérique de variants delta et omicron de sars-cov-2, son procédé de préparation et son utilisation Download PDF

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WO2023186170A1
WO2023186170A1 PCT/CN2023/085915 CN2023085915W WO2023186170A1 WO 2023186170 A1 WO2023186170 A1 WO 2023186170A1 CN 2023085915 W CN2023085915 W CN 2023085915W WO 2023186170 A1 WO2023186170 A1 WO 2023186170A1
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rbd
amino acid
omicron
acid sequence
delta
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高福
戴连攀
徐坤
郑天依
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中国科学院微生物研究所
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    • 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
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    • A61P31/14—Antivirals for RNA viruses
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    • 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
    • C12N15/70—Vectors or expression systems specially adapted for E. coli
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    • 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
    • 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
    • A61K2039/70—Multivalent vaccine
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
    • C07K2319/02—Fusion polypeptide containing a localisation/targetting motif containing a signal sequence
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/20—Fusion polypeptide containing a tag with affinity for a non-protein ligand
    • C07K2319/21—Fusion polypeptide containing a tag with affinity for a non-protein ligand containing a His-tag
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • 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
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    • 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
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • This application relates to the field of biomedicine, specifically to a new coronavirus Delta and Omicron variant chimeric antigen, its preparation method and application.
  • Novel coronavirus pneumonia is an acute respiratory infectious disease caused by infection with a new coronavirus (also known as new coronavirus, SARS-CoV-2).
  • the new coronavirus belongs to the beta-coronavirus genus of the family Coronaviridae. It has an envelope and is a positive-strand RNA virus.
  • the spike (S) protein on the surface of the new coronavirus is responsible for the receptor recognition and membrane fusion of the virus.
  • the receptor binding domain (RBD) on the S protein is an important vaccine target. It stimulates the production of neutralizing antibodies and has immunity. Focused advantages. In the early days of the COVID-19 epidemic, we urgently developed the recombinant subunit protein vaccine ZF2001 based on the COVID-19 RBD dimer, which showed good immunogenicity and protective effects in subsequent clinical trials.
  • the COVID-19 epidemic is still severe around the world.
  • New coronavirus mutant strains continue to emerge and spread, some of which can escape the immune response of existing vaccines and cause breakthrough infections.
  • the Delta and Omicron mutant strains have swept the world and become the dominant epidemic strains.
  • the Omicron mutant strain has as many as 32 S protein mutation sites. It has severe immune evasion against the humoral immune response activated by new coronavirus neutralizing antibody drugs and vaccines, posing severe challenges to the current epidemic prevention and control.
  • the purpose of this application is to provide a recombinant chimeric antigen of the new coronavirus Delta and Omicron mutant strains, its related products, and its preparation methods and applications.
  • the recombinant antigen according to the present application is (1) the specific amino acid sequence of the RBD protein from the new coronavirus Delta variant strain or is at least 90%, 92%, 95%, 96%, 97%, 98% or 99% identical to it specific amino acid sequence and (2) a specific amino acid sequence of the RBD protein from the new coronavirus Omicron variant strain or an amino acid with at least 90%, 92%, 95%, 96%, 97%, 98% or 99% identity thereto
  • the single-chain dimer formed by directly connecting the sequences or connecting them through appropriate connecting sequences can effectively activate broad-spectrum protective antibodies and can effectively prevent or prevent the original strain and various current mutant strains. treatment effect.
  • this application provides a recombinant chimeric antigen of the new coronavirus Delta and Omicron variants.
  • the amino acid sequence of the recombinant antigen includes: (A-B)-(A-B') or (A-B)-C -Amino acid sequences arranged in the (A-B') pattern, wherein:
  • A-B represents an amino acid sequence as set forth in SEQ ID NO:1 or an amino acid sequence having at least 90%, 92%, 95%, 96%, 97%, 98% or 99% identity thereto,
  • A-B’ represents an amino acid sequence as set forth in SEQ ID NO:2 or an amino acid sequence having at least 90%, 92%, 95%, 96%, 97%, 98% or 99% identity thereto, and
  • C represents a connection sequence shown as (GGS) n , where n represents the number of GGS, and n is an integer between 1 and 10, preferably 1-5 integers between.
  • the amino acid sequence of the recombinant antigen includes: an amino acid sequence arranged in the (A-B)-(A-B’) pattern, wherein:
  • A-B represents the amino acid sequence shown in SEQ ID NO: 1 or an amino acid sequence having at least 90%, 92%, 95%, 96%, 97%, 98% or 99% identity thereto, preferably as SEQ ID NO: The amino acid sequence shown in 1;
  • A-B' represents the amino acid sequence shown in SEQ ID NO:2 or an amino acid sequence having at least 90%, 92%, 95%, 96%, 97%, 98% or 99% identity with it, preferably as SEQ The amino acid sequence shown in ID NO:2.
  • amino acid sequence of the recombinant antigen is shown in SEQ ID NO: 3.
  • this application provides a method for preparing a recombinant antigen as described in the first aspect, which includes the following steps:
  • the cells of the expression system include mammalian cells, Insect cells, yeast cells, or bacterial cells;
  • the mammalian cells include HEK293T cells, HEK293F cells, Expi293F cells or CHO cells;
  • the bacterial cells comprise E. coli cells.
  • the present application provides a polynucleotide encoding the recombinant antigen as described in the first aspect.
  • the polynucleotide is a nucleotide sequence optimized by human codons, and can be DNA or mRNA;
  • the polynucleotide is the nucleotide sequence shown in SEQ ID NO:4.
  • the present application provides a nucleic acid construct comprising the polynucleotide as described in the third aspect above, and optionally, at least one expression control element operably linked to the polynucleotide.
  • the present application provides an expression vector comprising the nucleic acid construct described in the fourth aspect.
  • the present application provides a transformed cell, which includes the polynucleotide as described in the third aspect, the nucleic acid construct as described in the fourth aspect, or the expression vector as described in the fifth aspect. .
  • the present application provides the recombinant antigen as described in the first aspect, the polynucleotide as described in the third aspect, the nucleic acid construct as described in the fourth aspect, the fifth aspect as described above The application of the expression vector or the transformed cells as described in the sixth aspect above in the preparation of a new coronavirus vaccine.
  • the present application provides a vaccine or immunogenic composition, which includes the recombinant antigen as described in the first aspect, the polynucleotide as described in the third aspect, the fourth aspect as described above
  • the vaccine or immunogenic composition is a novel coronavirus recombinant protein vaccine, which includes the recombinant antigen and adjuvant as described in the first aspect above;
  • the adjuvant is one or more selected from the following adjuvants: aluminum adjuvant, MF59 adjuvant and MF59-like adjuvant.
  • the vaccine or immunogenic composition is a novel coronavirus DNA vaccine, which includes:
  • the eukaryotic expression vector is selected from pGX0001, pVAX1, pCAGGS and pCDNA series vectors.
  • the vaccine or immunogenic composition is a novel coronavirus mRNA vaccine, which includes an mRNA sequence encoding a recombinant antigen as described in the first aspect above and lipid nanoparticles.
  • the vaccine or immunogenic composition is a novel coronavirus-viral vector vaccine, which includes:
  • nucleic acid sequence encoding the recombinant antigen described in the first aspect is constructed into the viral backbone vector
  • the viral backbone vector is selected from one or more of the following viral vectors: adenovirus vector, poxvirus vector, influenza virus vector, and adeno-associated virus vector.
  • the vaccine or immunogenic composition is in the form of a nasal spray, oral preparation, suppository or parenteral preparation;
  • the nasal spray is selected from aerosols, sprays and powder sprays;
  • the oral preparation is selected from tablets, powders, pills, powders, granules, fine granules, soft/hard capsules, film-coated agents, pellets, sublingual tablets and ointments;
  • the parenteral preparation is a transdermal preparation, an ointment, a plaster, a topical liquid, an injectable or a pushable preparation.
  • the inventor of the present application has designed a recombinant chimeric antigen of the new coronavirus Delta and Omicron mutant strains.
  • the recombinant antigen consists of (1) the specific amino acid sequence of the RBD protein from the new coronavirus Delta mutant strain or has at least 90% similarity with it.
  • Figure 1 shows the new coronavirus prototype strain RBD dimer (referred to as prototype RBD-dimer), Delta variant strain RBD dimer (referred to as Delta RBD-dimer), and Omicron variant strain RBD dimer constructed and expressed in Example 1 of the present application.
  • Structural schematic diagram of the polymer referred to as Omicron RBD-dimer
  • the chimeric RBD dimer formed by connecting Delta RBD and Omicron RBD referred to as Delta-Omicron chimeric RBD-dimer, representing the recombinant antigen of the present application.
  • Figure 2 is the absorbance curve of the Delta-Omicron chimeric RBD-dimer protein purified using a nickel affinity column as described in Example 1 of the present application, as well as the SDS-PAGE identification results of the collected elution peaks.
  • the position indicated by the arrow is the target The elution peak where the protein is located.
  • Figure 3 is the absorbance curve of the eluate containing the Delta-Omicron chimeric RBD-dimer protein purified by the nickel affinity column and subjected to molecular sieve chromatography (to further purify the protein) as described in Example 1 of the present application, and SDS-PAGE identification results of the collected elution peaks (under non-reducing or reducing conditions), the position indicated by the arrow is the elution peak where the target protein is located.
  • Figure 4 is the absorbance curve of the eluate containing the prototype RBD-dimer protein purified by the nickel affinity column and subjected to molecular sieve chromatography (to further purify the protein) as described in Example 1 of the present application, as well as the collected elution SDS-PAGE identification results of peaks.
  • the position indicated by the arrow is the elution peak where the target protein is located.
  • Figure 5 is the absorbance curve of molecular sieve chromatography (to further purify the protein) of the eluate containing the Delta RBD-dimer protein purified by the nickel affinity column as described in Example 1 of the present application, and the collected elution SDS-PAGE identification results of peaks.
  • the position indicated by the arrow is the elution peak where the target protein is located.
  • Figure 6 is the absorbance curve of the eluate containing the Omicron RBD-dimer protein purified by the nickel affinity column and subjected to molecular sieve chromatography (to further purify the protein) as described in Example 1 of the present application, as well as the collected elution SDS-PAGE identification results of peaks.
  • the position indicated by the arrow is the elution peak where the target protein is located.
  • Figure 7 is a schematic diagram of the three-dimensional structure of the RBD protein of the new coronavirus prototype strain from different perspectives as described in Example 2 of the present application. In it, the mutated amino acid positions of the Delta and Omicron mutant strains in the RBD protein, the new coronavirus receptor are marked. Binding epitopes of human hACE2 and 5 representative antibodies (CB6, CV07-270, C110, S309 and CR3022).
  • Figure 8 shows the novel coronavirus receptor protein hACE2 and the representative antibodies CB6, CV07-270, C110, S309 and CR3022 of five different antibody epitopes and the antigens detected through surface plasmon resonance experiments as described in Example 2 of the present application. Protein binding affinity data for epitope identification of antigenic proteins.
  • Figure 9 is the absorbance curve of the complex of Delta-Omicron chimeric RBD-dimer protein and CB6 Fab purified by molecular sieve chromatography technology as described in Example 3 of the present application. Among them, one elution peak is Delta-Omicron chimeric RBD. -Dimer protein complex with CB6 Fab, another elution peak is excess CB6 Fab.
  • Figure 10 is a schematic diagram of the cryo-electron microscopy structure of the complex of Delta-Omicron chimeric RBD-dimer protein and CB6 Fab as described in Example 3 of the present application.
  • Figure 11 is a schematic diagram of the mutation sites of the S protein of the new coronavirus Alpha, Beta, Delta and Omicron mutant strains relative to the S protein of the new coronavirus prototype strain as described in Example 4 of the present application.
  • Figure 12 is the neutralization titer results of the serum collected from mice after the second immunization with the immunogen against the pseudovirus of the new coronavirus prototype strain and each new coronavirus mutant strain as described in Example 4 of the present application.
  • Figure 13 is the detection result of the viral load in the lung tissue of mice collected on the 3rd day after the Delta mutant strain was challenged with the mice in each immune group as described in Example 5 of the present application, where gRNA represents viral genomic RNA and sgRNA Represents viral subgenomic RNA.
  • Figure 14 is the neutralizing resistance of the serum of immunized mice to the Delta variant pseudovirus as described in Example 5 of the present application. Correlation analysis between the body titer and the viral gRNA load in the lung tissue of the immunized mice after challenge with the Delta mutant strain.
  • Figure 15 is the detection result of the viral load in the lung tissue of mice collected on the 3rd day after the mice in each immune group were challenged with Omicron mutant strains as described in Example 5 of the present application, where gRNA represents viral genomic RNA and sgRNA Represents viral subgenomic RNA.
  • Figure 16 is described in Example 5 of the present application.
  • Figure 17 is a representative HE staining pathological picture of the lung tissue of each group of mice after challenging each group of immunized mice with Delta or Omicron mutant strains as described in Example 5 of the present application.
  • Example 1 Construction, expression and purification of SARS-CoV-2 prototype strain RBD dimer, Delta variant RBD dimer, Omicron variant RBD dimer and Delta-Omicron chimeric RBD dimer protein
  • the RBD dimer of the new coronavirus prototype strain referred to as prototype RBD-dimer
  • the RBD dimer of the Delta variant strain referred to as Delta RBD-dimer
  • the Omicron variant strain were designed respectively.
  • the constructs of RBD dimer (referred to as Omicron RBD-dimer) and chimeric RBD dimer formed by connecting Delta RBD and Omicron RBD referred to as Delta-Omicron chimeric RBD-dimer
  • the amino acid sequences of the above four constructs are optimized using human codons, and the corresponding DNA coding sequences are as shown in SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12 and SEQ ID NO:14; in these The Kozak sequence gccacc is added to the upstream of the DNA coding sequence.
  • SEQ ID NO:8 SEQ ID NO:10, SEQ ID NO:12 and SEQ ID NO:14
  • the Kozak sequence gccacc is added to the upstream of the DNA coding sequence.
  • These four DNA sequences containing the Kozak sequence were synthesized by Suzhou Jinweizhi Biotechnology Co., Ltd.; the four synthesized DNA sequences were cloned into pCAGGS through the EcoRI and XhoI restriction sites.
  • Plasmids were obtained to express the prototype RBD-dimer, Delta RBD-dimer, Omicron RBD-dimer and Delta-Omicron chimeric RBD-dimer expression plasmids pCAGGS-prototype, pCAGGS-Delta, pCAGGS-Omicron and pCAGGS-D-O chimeric.
  • the plasmid expressing the Delta-Omicron chimeric RBD-dimer protein was transfected into Expi293F TM cells. After 5 days, the supernatant was collected, centrifuged to remove the precipitate, and then filtered through a 0.22 ⁇ m filter to further remove impurities.
  • the obtained cell supernatant was adsorbed through a nickel affinity column (His Trap, GE Healthcare) at 4°C, washed with buffer A (20mM Tris, 150mM NaCl, pH 8.0) to remove non-specific binding proteins, and then washed with buffer B ( 20mM Tris, 150mM NaCl, pH 8.0, 300mM imidazole) to elute the target protein from His Trap, collect the eluate at the elution peak, and use it for SDS-PAGE identification of the target protein and subsequent molecular sieve chromatography.
  • buffer A (20mM Tris, 150mM NaCl, pH 8.0
  • buffer B 20mM Tris, 150mM NaCl, pH 8.0, 300mM imidazole
  • the nickel affinity column chromatography curve of the Delta-Omicron chimeric RBD-dimer protein and the SDS-PAGE identification results of its elution peak are shown in Figure 2.
  • the position pointed by the arrow is the elution peak where the target protein is located. .
  • the molecular sieve chromatography curve of the Delta-Omicron chimeric RBD-dimer protein and the SDS-PAGE identification results of the elution peaks are shown in Figure 3.
  • the arrow indicates the elution peak corresponding to the target protein; in addition , the SDS-PAGE gel electrophoresis analysis of the elution peak showed that the size of the eluted protein was correct, proving that the Delta-Omicron chimeric RBD-dimer protein was purified, and it can be seen from the electrophoresis bands that the purified target protein was also With higher purity and yield.
  • the three proteins of prototype RBD-dimer, Delta RBD-dimer and Omicron RBD-dimer were expressed and purified using the same method. Briefly, their expression plasmids were transfected into Expi293F TM cells respectively. After 5 days, the supernatant was collected, centrifuged and Filter to remove impurities.
  • the prototype RBD-dimer protein was 16/600 200pg column (GE Healthcare) was used for molecular sieve chromatography, and the two proteins Delta RBD-dimer and Omicron RBD-dimer were used for molecular sieve chromatography on a Superdex TM 200 Increase 10/300GL column (GE Healthcare) to further purify the target protein; prototype RBD
  • the molecular sieve chromatography curves of -dimer, Delta RBD-dimer and Omicron RBD-dimer and the SDS-PAGE identification results of their elution peaks are shown in Figure 4, Figure 5 and Figure 6 respectively.
  • the inventor used surface plasmon resonance technology (Surface Plasmon Resonance, SPR) to identify the RBD binding motif (RBM) of the antigen protein and the exposure of the main neutralizing antibody epitope, and detected the antigen protein's response to the human receptor of the new coronavirus - human Affinity for angiotensin-converting enzyme (hACE2), as well as for the representative monoclonal antibody CB6 targeting 5 different epitopes in the SARS-CoV-2 RBD (see A human neutralizing antibody targets for specific information on this antibody the receptor-binding site of SARS-CoV-2.Nature, 2020, PMID: 32454512), CV07-270 (for specific information about this antibody, please see A Therapeutic Non-self-reactive SARS-CoV-2 Antibody Protects from Lung Pathology in a COVID-19 Hamster Model.
  • SPR Surface Plasmon Resonance
  • the monomeric RBD protein of the prototype strain the monomeric RBD protein of the Delta variant strain
  • the affinity of the monomeric RBD protein of the Omicron variant strain and the Delta-Omicron chimeric RBD-dimer protein to the hACE2 receptor and the above five monoclonal antibodies were comparatively analyzed.
  • the affinity test method is as follows: This test is performed using BIAcore8000 (GE Healthcare) instrument. Before testing, the target antigen protein was changed into PBS-T buffer (10mM Na 2 HPO 4 , 2mM KH 2 PO 4 , pH 7.4, 137mM NaCl, 2.7mM KCl, 0.005% Tween 20). First, the antigen protein is immobilized on the CM5 chip using the amino coupling method, with a target response value of about 1000RU; then, the antibody Fab protein is diluted twice, and the diluent is used as the mobile phase, flowing through the fixed chip in sequence at a speed of 30 ⁇ L/min. Antigen protein, different real-time binding response signals are obtained. 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 between the antigen protein and the antibody is obtained.
  • PBS-T buffer 10mM Na 2 HPO 4 , 2mM KH 2 PO 4 , pH 7.4,
  • the Delta-Omicron chimeric RBD-dimer protein can bind to all representative monoclonal antibodies tested, and the affinity value of the Delta-Omicron chimeric RBD-dimer protein for binding to each monoclonal antibody is consistent with Delta or Omicron
  • the two monomeric RBD proteins have similar affinity for binding to each monoclonal antibody, which shows that the Delta-Omicron chimeric RBD-dimer protein combines the epitope characteristics of Delta and Omicron and well exposes the receptor binding sites and shows the major neutralizing antibody epitope conformations.
  • Example 3 Electron microscope structure analysis of the complex of the Delta-Omicron chimeric RBD-dimer protein and CB6 Fab of the present application
  • Delta-Omicron chimeric RBD-dimer protein and CB6 Fab protein were mixed and incubated at 4°C for 12 hours.
  • Molecular sieve chromatography (pH8.0) was then carried out through Superdex TM 200 Increase 10/300GL column (GE Healthcare) to purify the complex of Delta-Omicron chimeric RBD-dimer protein and CB6 Fab protein.
  • the molecular sieve chromatography curve is as shown in the figure As shown in 9; in addition, the eluates at the two elution peaks were collected and subjected to SDS-PAGE identification.
  • Quantifoil grid (specification 1.2/1.3) for sample preparation in advance and perform glow discharge hydrophilization treatment. Then, the prepared complex of Delta-Omicron chimeric RBD-dimer protein and CB6 Fab was dropped onto the prepared network, and the automatic sample preparation machine Vitrobot Mark IV was used to quickly insert the network into liquid ethane to complete sample preparation. .
  • FIG. 10 The cryo-electron microscope image of the complex of the Delta-Omicron chimeric RBD-dimer protein and CB6 Fab of this application is shown in Figure 10. It can be seen from Figure 10: In the complex of the Delta-Omicron chimeric RBD-dimer protein and CB6 Fab, Delta RBD and Omicron RBD are symmetrically distributed and present a "double lung shape"; moreover, Delta RBD can bind CB6 Fab, and the main epitope of the Delta-Omicron chimeric RBD-dimer protein is fully exposed, which is beneficial to activating the immune response.
  • Example 4 Detection of humoral immune response induced by Delta-Omicron chimeric RBD-dimer protein
  • the specific immunization program is as follows:
  • the mutant strains include Alpha, Beta, and Delta. , Omicron mutant strains.
  • the mutation sites of the S protein of each mutant strain relative to the S protein of the prototype strain are shown in Figure 11.
  • the new coronavirus pseudovirus used in this example is a pseudovirus displaying the S protein of the new coronavirus prepared based on the vesicular stomatitis virus (VSV) skeleton.
  • VSV vesicular stomatitis virus
  • the preparation method please refer to the method section of the published paper of this research group (Effects of a Prolonged 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 the pseudovirus) is as follows: in a 96-well plate, The immune mouse serum was diluted with a 2-fold gradient, and then mixed with the pseudovirus respectively. The blank culture medium was also mixed with the pseudovirus as a control, and incubated at 37°C for 1 hour. Transfer the immune serum-pseudovirus mixture to a 96-well plate covered with Vero cells.
  • the geometric mean (GMT) of the neutralizing antibody titer of the immune mouse serum against the prototype strain pseudovirus is 3009, but the neutralizing effect against some mutant strains of the pseudovirus has decreased. , including Beta (GMT, 1112), Delta (GMT, 2059), Omicron (GMT, 374).
  • the neutralizing antibody titer GMT of the immunized mouse serum against Delta pseudovirus is 16722, but the neutralizing antibody titer GMT against Beta pseudovirus is 756, and the neutralizing antibody titer against Omicron pseudovirus is 756.
  • the titer GMT is 633, which is not effective against Beta and Omicron mutant strains.
  • the neutralizing antibody titers GMT of the immunized mouse serum against the pseudovirus were 4518 (prototype) and 5576 (Alpha), respectively. 2263 (Beta), 38387 (Delta) and 7194 (Omicron).
  • the neutralizing antibody GMT of the serum of mice immunized with the Delta-Omicron chimeric RBD-dimer protein vaccine was higher than that of the other three protein vaccines. , showing the advantages of strong immunogenicity and broad spectrum.
  • mice prepared as in Example 4 were tested respectively. Live virus challenge experiments of SARS-CoV-2 Delta and Omicron mutant strains.
  • mice in each experimental group were subjected to a live virus challenge experiment with the new coronavirus Delta variant strain, and the other five mice in each experimental group were subjected to a live virus challenge experiment with the new coronavirus Omicron variant strain. Since BALB/c mice are not susceptible to the Delta mutant strain, the following Delta mutant strain challenge experimental method was used: intranasally transduced 8 ⁇ 10 9 vp recombinant adenovirus type 5 (Ad5- hACE2), a model of transient expression of hACE2 was established. Five days after transducing Ad5-hACE2, 6 ⁇ 10 5 TCID 50 Delta variant strain (CCPM-BV-049-2105-8) was intranasally infected.
  • Ad5- hACE2 intranasally transduced 8 ⁇ 10 9 vp recombinant adenovirus type 5
  • TCID 50 Delta variant strain CCPM-BV-049-2105-8
  • mice are directly infected with 6 ⁇ 10 5 TCID 50 new coronavirus Omicron mutant strain (BA.1, CCPM-BV-049-2112-18) through intranasal drip.
  • BA.1, CCPM-BV-049-2112-18 new coronavirus Omicron mutant strain
  • mice On the third day after infection with the new coronavirus Delta or Omicron mutant strain, the mice were euthanized and dissected; the lungs of each mouse were removed and divided into two parts: one part was homogenized and ground, the viral nucleic acid was extracted, and qRT-PCR was used to sick Viral viral genome gRNA and subgenomic sgRNA were quantified.
  • gRNA represents the entire viral nucleic acid
  • sgRNA represents the viral nucleic acid in the process of replication, which is an indicator of the viral replication level
  • the other part was fixed with paraformaldehyde, and then subjected to hematoxylin and Eosin (H&E) staining was used to observe the tissue pathology.
  • H&E hematoxylin and Eosin
  • the method for detecting viral gRNA and sgRNA is as follows: After homogenizing mouse lung tissue, take the tissue homogenate supernatant and use the Direct-zol RNA MiniPrep kit (Zymo Research Company, Cat. No. R2052) to extract viral RNA. SARS-CoV-2-specific quantitative reverse transcription PCR (qRT) was performed on the CFX384 Touch real-time PCR detection system (Bio-Rad) using the TaqMan Fast Virus 1-Step Master Mix Kit (Thermo Fisher Scientific, Cat. No. 4444436) -PCR) detection. Two sets of primers and probes were used to detect SARS-CoV-2 Delta and Omicron virus genomic gRNA respectively, and a set of primers and probes were used to detect Delta and Omicron virus sgRNA.
  • qRT quantitative reverse transcription PCR
  • the primer and probe sequences for detecting SARS-CoV-2 Delta gRNA are as follows:
  • the primer sequence for detecting SARS-CoV-2 Omicron gRNA is the same as Delta, namely (SEQ ID NO: 15) and (SEQ ID NO: 16).
  • the probe sequence for detecting SARS-CoV-2 Omicron gRNA is as follows:
  • the primer and probe sequences for detecting SARS-CoV-2 Delta and Omicron sgRNA are as follows:
  • sgRNA-F,CGATCTCTTGTAGATCTGTTCTC (SEQ ID NO: 19);
  • sgRNA-R,ATATTGCAGCAGTACGCACACA (SEQ ID NO: 20);
  • sgRNA-probe FAM-ACACTAGCCATCCTTACTGCGCTTCG (SEQ ID NO: 21)-BHQ1.
  • the viral load detection results in the lung tissue of mice are shown in Figure 13. It can be seen from Figure 13 that for mice challenged with the Delta variant strain of the new coronavirus, the control group mice tested to high levels of gRNA (mean: 1.09 ⁇ 10 copies/g lung tissue) and sgRNA (mean: 1.70 ⁇ 10 copies/g lung tissue), compared to those detected in mice after vaccination Viral loads (including gRNA and sgRNA) were significantly reduced, with the average values of lung tissue gRNA in the prototype RBD-dimer and Delta-Omicron chimeric RBD-dimer immunization groups being 1.43 ⁇ 10 8 copies/g and 2.37 ⁇ 10 7 copies respectively. /g lung tissue.
  • the viral load detection results of the mouse lung tissue are shown in Figure 15. From Figure 15, it can be seen that for the mice challenged with the Omicron mutant strain of the new coronavirus, the control group mice tested to high levels of gRNA (mean: 1.04 ⁇ 10 9 copies/g lung tissue) and sgRNA (mean: 1.73 ⁇ 10 7 copies/g lung tissue). In contrast, the prototype RBD-dimer immune panel and Delta-Omicron embedded The average values of lung tissue gRNA in the RBD-dimer immunized group were 3.68 ⁇ 10 7 copies/g and 1.61 ⁇ 10 7 copies/g lung tissue, respectively.
  • mice in the Delta-Omicron chimeric RBD-dimer vaccine group had detectable lung tissue viral sgRNA, indicating that they completely inhibited viral replication, compared with 2 of 5 mice in the prototype RBD-dimer group.
  • the sgRNA was positive, and the mean titer of the prototype RBD-dimer group was 2.41 ⁇ 10 4 copies/g lung tissue, indicating that compared with the prototype RBD-dimer, the Delta-Omicron chimeric RBD-dimer has an inhibitory effect on the Omicron variant of the new coronavirus. Significantly better.
  • mice in each experimental group after being challenged with the new coronavirus Delta variant or Omicron variant are shown in Figure 17.
  • the lung pathological changes of mice in the control group showed moderate to severe lesions after being infected with the new coronavirus Delta or Omicron mutant strains.
  • mice vaccinated with prototype RBD-dimer and Delta-Omicron chimeric RBD-dimer vaccines only showed mild lung damage and significantly alleviated pneumonia ( Figure 17).
  • mice showed that the Delta-Omicron chimeric RBD-dimer had a better protective effect compared with the prototype RBD-dimer ( Figure 17).
  • These lung histopathological results were consistent with the above lung tissue viral gRNA. The measured trends are consistent, proving that the Delta-Omicron chimeric RBD-dimer protein vaccine can indeed provide a relatively balanced and efficient protection against different strains of the new coronavirus, especially the recently popular Delta and Omicron variants.
  • the RBD recombinant chimeric antigens of the new coronavirus Delta and Omicron variants provided by this application can be very Efficiently activating broad-spectrum protective antibodies can have a very good preventive or therapeutic effect on the original strain of the new coronavirus and various current mutant strains, which is of far-reaching significance for the prevention and control of the global new coronavirus epidemic.

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Abstract

La présente demande concerne un antigène chimérique de variants delta et omicron du SARS-CoV-2, son procédé de préparation et son utilisation. L'antigène recombinant selon la présente invention est formé par : (1) une séquence d'acides aminés spécifique à partir d'une protéine RBD de variant delta de SARS-CoV-2 ; et (2) une séquence d'acides aminés spécifique à partir d'une protéine RBD de variant omicron de SARS-CoV-2 au moyen d'une séquence de liaison appropriée ou d'une liaison directe en série. Par rapport à un homodimère RBD d'une souche de SARS-CoV-2 d'origine ou d'un variant de celle-ci, l'antigène recombinant selon la présente demande peut activer plus efficacement des anticorps protecteurs à large spectre, et peut également obtenir un bon effet de prévention ou de traitement sur la souche d'origine et divers variants existants.
PCT/CN2023/085915 2022-04-01 2023-04-03 Antigène chimérique de variants delta et omicron de sars-cov-2, son procédé de préparation et son utilisation WO2023186170A1 (fr)

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