WO2024002129A1 - Nouveau vaccin chimérique trimère de coronavirus et son utilisation - Google Patents

Nouveau vaccin chimérique trimère de coronavirus et son utilisation Download PDF

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WO2024002129A1
WO2024002129A1 PCT/CN2023/103052 CN2023103052W WO2024002129A1 WO 2024002129 A1 WO2024002129 A1 WO 2024002129A1 CN 2023103052 W CN2023103052 W CN 2023103052W WO 2024002129 A1 WO2024002129 A1 WO 2024002129A1
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vaccine
amino acid
acid sequence
new coronavirus
seq
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Chinese (zh)
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高福
王奇慧
戴连攀
杜沛
齐建勋
徐坤
马雪慧
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中国科学院微生物研究所
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    • 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
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    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • 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
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    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
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    • 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
    • A61K2039/54—Medicinal preparations containing antigens or antibodies characterised by the route of administration
    • A61K2039/541—Mucosal route
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    • A61K2039/542—Mucosal route oral/gastrointestinal
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    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/54—Medicinal preparations containing antigens or antibodies characterised by the route of administration
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    • A61K2039/543—Mucosal route intranasal
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    • A61K39/00—Medicinal preparations containing antigens or antibodies
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    • A61K2039/55505—Inorganic adjuvants
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    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • 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
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    • C12N2760/00011—Details
    • C12N2760/20011—Rhabdoviridae
    • C12N2760/20211—Vesiculovirus, e.g. vesicular stomatitis Indiana virus
    • C12N2760/20241—Use of virus, viral particle or viral elements as a vector
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    • 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

  • This application relates to the field of biomedicine, specifically to a novel coronavirus heterotrimeric chimeric antigen peptide, its encoding nucleic acid and its application in the preparation of a novel coronavirus vaccine.
  • Novel coronavirus pneumonia is an acute respiratory infectious disease caused by infection with a new coronavirus (also known as SARS-CoV-2).
  • the new coronavirus belongs to the genus ⁇ -coronavirus of the family Coronaviridae. It has an envelope and is a single-stranded positive-strand RNA virus.
  • the spike protein (also known as S protein) on the surface of the new coronavirus is responsible for the binding and membrane fusion between the virus and the host cell membrane receptor.
  • RBD receptor binding domain
  • the COVID-19 epidemic is still severe around the world, and new coronavirus mutant strains continue to emerge and spread; in particular, there are many subtypes of the new coronavirus Omicron mutant strain (for example, BA.1, BA.2, BA.1.1 , BA.3 subtype, etc.), has a transmission speed exceeding Delta and has become the dominant strain in the world; among them, the BA.2 subtype strain has a transmission speed higher than other Omicron subtypes and now occupies the largest Proportion.
  • the S protein of the Omicron variant has more than 50 amino acid mutations, which is much more than the previous Delta variant.
  • the immune response stimulated by the existing vaccines designed and developed based on the new coronavirus prototype strain is ineffective in the face of mutant strains (such as Omicron mutations). strain), the efficacy is greatly reduced.
  • the phenomenon of this mutant strain breaking through vaccine and antibody protection is called immune escape.
  • the phenomenon of immune evasion is particularly obvious in the various subtypes of Omicron mutant strains.
  • this application provides a novel coronavirus heterotrimeric chimeric antigen peptide, a polynucleotide encoding the same, and nucleic acid products related to the polynucleotide, based on the aforementioned Vaccines or immunogenic compositions of antigenic peptides or polynucleotides, and the application of the aforementioned products in the preparation of new coronavirus vaccines.
  • Recombinant protein vaccines or various types of nucleic acid vaccines (especially the latter) based on the chimeric antigen peptide or its encoding nucleic acid can effectively stimulate strong immune responses against the original strain and multiple mutant strains of the new coronavirus, and have broad spectrum and relatively high immunity. Strong immunogenicity.
  • A-B represents the amino acid sequence of the RBD domain of the S protein of the new coronavirus Delta variant strain or a part thereof, or is at least 90%, 92%, 95%, 96%, 97%, 98% or 99% identical to and identical to it. or substantially the same immunogenic amino acid sequence;
  • A-B' represents the amino acid sequence of the RBD domain of the S protein of the new coronavirus Omicron variant BA.2 subtype or a part thereof, or has at least 90%, 92%, 95%, 96%, 97%, 98% or An amino acid sequence that is 99% identical and has the same or substantially the same immunogenicity;
  • A-B represents the amino acid sequence of the S protein RBD domain of the new coronavirus Omicron variant strain BA.4/BA.5 subtype or a part thereof, or has at least 90%, 92%, 95%, 96%, 97%, 98 % or 99% identity and having the same or substantially the same immunogenicity as an amino acid sequence;
  • a part of the RBD domain of the S protein of the new coronavirus Delta variant strain is at least 70%, 80%, 85%, 90%, 92%, 95% of its entire amino acid sequence. , 96%, 97%, 98% or 99%;
  • a part of the RBD domain of the S protein of the new coronavirus Omicron variant BA.2 subtype is at least 70%, 80%, 85%, 90%, 92%, 95%, 96 of its entire amino acid sequence %, 97%, 98% or 99%;
  • a part of the RBD domain of the S protein of the new coronavirus Omicron variant strain BA.4/BA.5 subtype is at least 70%, 80%, 85%, 90%, 92%, of its entire amino acid sequence. 95%, 96%, 97%, 98% or 99%;
  • n 0,1,2 or 3.
  • the amino acid sequence of the S protein RBD domain of the new coronavirus Delta variant strain or a part thereof is as shown in SEQ ID NO: 1, or the amino acid sequence as shown in SEQ ID NO: 1 is An amino acid sequence obtained by substituting, deleting or adding one or more amino acids and having the same or substantially the same immunogenicity;
  • amino acid sequence of the new coronavirus Omicron variant BA.2 subtype S protein RBD domain or part thereof is as shown in SEQ ID NO:2, or the amino acid sequence as shown in SEQ ID NO:2 is An amino acid sequence obtained by substituting, deleting or adding one or more amino acids and having the same or substantially the same immunogenicity;
  • amino acid sequence of the new coronavirus Omicron variant BA.4/BA.5 subtype S protein RBD domain or part thereof is as shown in SEQ ID NO:3, or as shown in SEQ ID NO:3
  • n 0, 1 or 2.
  • A-B represents the amino acid sequence as shown in SEQ ID NO:1
  • A-B' represents the amino acid sequence as shown in SEQ ID NO:2
  • A-B" represents the amino acid sequence as shown in SEQ ID NO:3 The amino acid sequence shown;
  • the recombinant chimeric antigen peptide has the amino acid sequence shown in SEQ ID NO:4.
  • the present application provides a polynucleotide encoding the recombinant chimeric antigen as described in the above first aspect.
  • the polynucleotide is a nucleotide sequence optimized by human codons, and can be DNA or mRNA;
  • the polynucleotide is a DNA molecule, preferably, the DNA molecule has the DNA sequence shown in SEQ ID NO:5.
  • the polynucleotide is an mRNA molecule, preferably, the mRNA molecule has an mRNA sequence as shown in SEQ ID NO: 6.
  • the present application provides a nucleic acid construct comprising the polynucleotide as described in the second 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 above third aspect.
  • the present application provides a host cell, which is transformed or transfected with the polynucleotide as described in the second aspect, the nucleic acid construct as described in the third aspect, or the polynucleotide as described in the fourth aspect.
  • Expression vector
  • the present application provides the recombinant chimeric antigen peptide as described in the first aspect, the polynucleotide as described in the second aspect, the nucleic acid construct as described in the third aspect, the fourth aspect as described above.
  • the new coronavirus is one or more selected from the following: original strain of SARS-CoV-2, mutant strain of SARS-CoV-2 Alpha (B.1.1.7), Beta (B.1.351 ), Gamma(P.1), Kappa(B.1.617.1), Delta (B.1.617.2), Omicron subtypes BA.1, BA.1.1, BA.2, BA.2.12.1, BA.3, BA.4, BA.5.
  • the vaccine is used for separate immunization or sequential immunization with other types of new coronavirus vaccines; further preferably, the other types of new coronavirus vaccines are inactivated vaccines.
  • the present application provides a vaccine or immunogenic composition, which includes the recombinant chimeric antigen peptide as described in the first aspect, the polynucleotide as described in the second aspect, the third aspect as described above.
  • the vaccine or immunogenic composition is a novel coronavirus recombinant protein vaccine, which includes the recombinant chimeric antigen peptide 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 DNA sequence constructed into the eukaryotic expression vector and encoding the recombinant chimeric antigen peptide as described in the first aspect above is preferably the DNA sequence shown in SEQ ID NO: 5;
  • 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, and the mRNA vaccine includes:
  • the vaccine or immunogenic composition is a novel coronavirus-viral vector vaccine, which includes:
  • the DNA sequence constructed into the viral backbone vector and encoding the recombinant chimeric antigen peptide as described in the first aspect above is preferably the DNA sequence shown in SEQ ID NO: 5;
  • 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 gums sachets, film-coated tablets, 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 present application provides a kit, which includes the vaccine or immunogenic composition as described in the seventh aspect above, and optionally other types of new coronavirus vaccines, the vaccine or immunogenic composition
  • the composition is packaged separately from other types of novel coronavirus vaccines as described;
  • novel coronavirus vaccines are novel coronavirus inactivated vaccines.
  • This application provides a recombinant chimeric antigen peptide of the receptor-binding domain of the new coronavirus Delta variant, Omicron variant BA.2 subtype, and Omicron variant BA.4/BA.5 subtype, and a polynuclear polypeptide encoding the same.
  • nucleotides and various types of vaccine products based on the chimeric antigen peptide or its polynucleotide; recombinant protein vaccines or various types of nucleic acid vaccines (especially the latter) based on the chimeric antigen peptide or its encoding nucleic acid can effectively stimulate the The strong immune response of the original strain and multiple mutant strains of the new coronavirus has broad spectrum and strong immunogenicity.
  • nucleic acid vaccines including DNA vaccines, mRNA vaccines, and viral vector vaccines
  • nucleic acid encoding the chimeric antigen peptide target multiple new coronavirus strains especially a series of subtypes BA of the currently popular Omicron variant strains.
  • BA.1.1, BA.2, BA.2.12.1, BA.3, BA.4, and BA.5 can all provide strong immune protection efficacy, and when combined with other types of vaccines (such as inactivated vaccines) Sequential immunization can induce a significantly higher level of immune response against various strains of the new coronavirus (i.e., broad spectrum). It is very suitable for the current complex epidemic prevention and control, and has potential clinical application value and prospects.
  • Figure 1 is the new coronavirus prototype strain RBD trimer mRNA vaccine (referred to as PPP mRNA vaccine, as a control vaccine) constructed in Example 1 of the present application, which is composed of the new coronavirus Delta variant RBD, Omicron variant BA.2 subtype RBD and Schematic structural diagram of the chimeric RBD trimer mRNA vaccine (referred to as DOO mRNA vaccine, representing the vaccine of this application) formed by serial connection of Omicron variant BA.4/BA.5 subtype RBDs; each segment of the mRNA vaccine is marked on the figure , where 5'UTR represents the 5' untranslated region, 3'UTR represents the 3' untranslated region, SP represents the signal peptide sequence, Poly(A) represents the poly(A) tail, and Protype RBD represents the RBD of the prototype strain.
  • PPP mRNA vaccine referred to as a control vaccine
  • Delta RBD represents the RBD sequence of the Delta variant
  • Omicron(BA.2)RBD represents the RBD sequence of the Omicron variant BA.2 subtype
  • Omicron(BA.4/5)RBD represents the Omicron variant BA.4/BA RBD sequence of .5 subtype.
  • Figure 3 shows the detection of the serum collected on the 14th day after immunizing mice with PPP or DOO mRNA vaccines by enzyme-linked immunosorbent assay (ELISA) against the new coronavirus prototype strain, Delta variant strain, and Omicron strain as described in Example 3 of the present application. Bar chart of binding antibody titer levels to RBD antigens of mutant subtypes BA.1, BA.1.1, BA.2, BA.2.12.1, and BA.4/5.
  • ELISA enzyme-linked immunosorbent assay
  • Figure 4 shows the detection of serum collected on the 28th day after immunizing mice with PPP or DOO mRNA vaccines by enzyme-linked immunosorbent assay (ELISA) against the new coronavirus prototype strain, Delta variant strain, and Omicron strain as described in Example 4 of the present application.
  • ELISA enzyme-linked immunosorbent assay
  • Figure 5 shows that the serum collected on the 14th day after immunizing mice with the mRNA vaccine neutralized the new coronavirus prototype strain, Delta variant strain, Omicron variant subtype BA.1, BA.1.1, and The NT 50 values of the pseudoviruses BA.2, BA.2.12.1, BA.3, and BA.4/5 are as recorded in Example 6; among them, the above figure is a bar graph, and the abscissa shows Vaccine types are PPP vaccine, DOO vaccine and negative control LNP respectively. The ordinate shows the log 10 value of pVNT 50 titer; the figure below is based on the GMT value of each experimental group in the bar chart (i.e., the geometric mean titer degree) radar chart.
  • Figure 6 shows that the serum collected on the 28th day after immunizing mice with the mRNA vaccine neutralized the new coronavirus prototype strain, Delta variant strain, Omicron variant strain subtypes BA.1, BA.1.1, and The NT 50 values of the pseudoviruses BA.2, BA.2.12.1, BA.2.75, BA.3, and BA.4/5 are as described in Example 7; where, the upper figure is a bar graph, and the horizontal The coordinates show the vaccine types, which are PPP vaccine, DOO vaccine and negative control LNP respectively. The ordinate shows the log 10 value of pVNT 50 titer; the figure below is based on the GMT value of each experimental group in the bar chart (i.e. , geometric mean titer) radar chart produced.
  • the bar chart i.e. , geometric mean titer
  • Figure 7 shows the detection of mouse spleen cells collected on the 21st day after mice were immunized with PPP or DOO mRNA vaccines as described in Example 8 of the present application by enzyme-linked immunospot assay (ELISPOT).
  • ELISPOT enzyme-linked immunospot assay
  • Figure 8 shows that the serum collected on day 56 after sequential immunization of mice with inactivated vaccine and mRNA vaccine neutralized the new coronavirus prototype strain, Delta variant strain, Omicron variant strain BA.1, and The NT 50 values of pseudoviruses of BA.1.1, BA.2, BA.2.12.1, BA.3, and BA.4/5 subtypes are as described in Example 10; where, the upper figure is a bar graph , the abscissa shows the types of mRNA vaccines used for sequential immunization with inactivated vaccines, which are PPP, DOO mRNA vaccines and negative control LNP. The ordinate shows the log 10 value of pVNT 50 titer; the figure below is the basic A radar plot was created of the GMT values (i.e., geometric mean titers) of each experimental group in the bar graph.
  • GMT values i.e., geometric mean titers
  • Figure 9 shows the enzyme-linked immunospot test (ELISPOT) described in Example 11 of the present application.
  • ELISPOT enzyme-linked immunospot test
  • Example 1 RBD trimer mRNA vaccine of the new coronavirus prototype strain (referred to as PPP mRNA vaccine, as a control) as well as the Delta variant strain, Omicron variant strain BA.2 subtype and Omicron variant strain BA.4/BA.5 subtype Construction, in vitro preparation and packaging of chimeric RBD trimer mRNA vaccine (referred to as DOO mRNA vaccine, representing the mRNA vaccine of this application)
  • the basic plasmid used for in vitro transcription of the mRNA vaccine is pUC57, provided by Nanjing GenScript Biotechnology Co., Ltd.
  • the DNA expression element of the mRNA vaccine was introduced through conventional molecular biology methods. Including: (1) T7 promoter, (2) DNA coding region of the mRNA vaccine (DOO)
  • the DNA coding sequence of the mRNA vaccine is as shown in SEQ ID NO: 5
  • the DNA coding sequence of the PPP mRNA vaccine as a control is as shown in SEQ ID NO: 7
  • (3) 5' UTR sequence upstream of the coding region (the 5' UTR sequences of the two mRNA vaccines are the same, both are sequences shown in SEQ ID NO: 8)
  • signal peptide coding The sequence (i.e., the signal peptide coding sequence of the SP, PPP vaccine and DOO vaccine is shown in SEQ ID NO:9 and SEQ ID NO:10 respectively), and (5) the downstream 3' end UTR sequence (the 3' end UTR sequence of the two mRNA vaccines)
  • the '-end UTR sequences are the same, as shown in SEQ ID NO: 11
  • the capping enzyme kit Cap1 capping enzyme kit (M082-01B, Suzhou Nearshore Protein Technology Co., Ltd.) was used to cap the 5' end of the purified in vitro transcribed mRNA with Cap1 to meet the requirements for eukaryotic expression. Conditions for translation in cells; thereafter, the mRNA was purified again using the same lithium chloride precipitation method as above to obtain purified 5'-end capped mRNA.
  • HEK293T cells in a 12-well plate so that the cell density reaches about 50% the next day; add 1 ⁇ g of DOO or PPP mRNA together with TransIT-mRNA reagent (2 ⁇ l) and enhancement reagent (2 ⁇ l) into 100 ⁇ l of serum-free Opti-MEM, respectively. Incubate for 3 minutes, then drop into a 12-well plate; 36 hours after transfection, collect the supernatant.
  • step (1) Mix the supernatant sample obtained in step (1) with the loading buffer containing dithiothreitol (DTT), and separate by 10% SDS-PAGE; after the separation is completed, transfer the membrane to transfer the protein to the PVDF membrane; then, Block the membrane with 5% skim milk dilute, incubate the PVDF membrane with SARS-CoV-2 Spike/RBD primary antibody (Sino Biological) and goat anti-rabbit IgG-HRP (EASYBio) secondary antibody for 1 hour each; finally, use Beyotime Beyo ECL Plus color developer.
  • DTT dithiothreitol
  • FIG. 2 shows the expression of DOO or PPP mRNA in cells.
  • the expression result of DOO mRNA in cells has only a single band, which is about the same size as the PPP mRNA vaccine.
  • the prepared 5'-end capped mRNA is mixed and packaged. After packaging is completed, use centrifugation or dialysis to replace the buffer solution with PBS. After completing the packaging, Thermo Fisher's Quan-iT Ribogreen RNA reagent kit was used to identify the mRNA packaging efficiency. The packaging efficiency met the standards of mRNA vaccines.
  • mice of the BALB/c strain purchased from Viton Lever
  • the experimental groups were divided into an mRNA vaccine immunization group and a negative control group, in which the mRNA vaccine immunization group
  • the groups include PPP mRNA vaccine immunization group and DOO mRNA vaccine immunization group, and the negative control group is the LNP immunization group.
  • All mice in the mRNA vaccine immunization group were immunized with the same designed mRNA vaccine (i.e., PPP or DOO mRNA vaccine) on days 0 and 14, and the mice in the negative control group were injected with the same amount at the same time. Empty LNP.
  • the vaccination method was intramuscular injection, and the vaccination dose was 10 ⁇ g of mRNA vaccine or empty LNP per mouse each time; mouse serum samples were collected on the 14th and 28th days respectively to test the binding antibody titer and pseudogene of the immune serum. Virus neutralizing antibody titers; mouse spleen samples were collected on day 21 to test T cell immunity.
  • enzyme-linked immunosorbent assay was used to detect the sera collected from the mice in each experimental group in the above Example 2 on the 14th day of the immunization program against the new coronavirus prototype strain, Delta variant strain, and Omicron virus.
  • ELISA enzyme-linked immunosorbent assay
  • the new coronavirus prototype strain (SEQ ID NO: 12), Delta variant strain (SEQ ID NO: 13), Omicron variant strain subtype BA.1 (SEQ ID NO: 14), BA.1.1 (SEQ ID NO: 14), and NO:15), BA.2 (SEQ ID NO:16), BA.2.12.1 (SEQ ID NO:17), BA.4/5 (SEQ ID NO:18) RBD antigen protein (0.2 ⁇ g/ml ) to coat the ELISA plate, and block the coated ELISA plate in 5% skim milk for 1 hour; then, the serum collected from the mice in each experimental group on the 14th day of the immunization program in Example 2 was incubated at 56°C Incubate for 30 minutes for inactivation; perform a three-fold gradient dilution of the inactivated serum sample starting from 1:200 or 1:1000, then add the dilution to each well, and then incubate the ELISA plate at 37°C for 1 hour.
  • the endpoint titer is defined as: the absorbance produced by the serum (as mentioned above, the absorbance at 450nm minus the absorbance at 630nm) is greater than the background value by 2.1 times.
  • the corresponding serum dilution factor is lower than the detection
  • the limiting antibody titer is defined as one-third of the detection limit.
  • the serum collected from the mice in the DOO mRNA vaccine immunization group of this application on the 14th day of the immunization program is specific to the new coronavirus Omicron variant subtypes BA.1, BA.1.1, BA.2, and BA.
  • the specific antibody titers produced by the RBD antigen proteins of 2.12.1 and BA.4/5 were significantly higher than those of the PPP mRNA vaccine, and the specific antibodies produced by the RBD antigen proteins of the new coronavirus prototype strain and Delta variant strain The titer level is comparable to the PPP mRNA vaccine, both at a higher level.
  • the DOO mRNA vaccine of the present application can induce a higher level of humoral immunity than the PPP mRNA vaccine.
  • it produces significantly higher humoral immunity than the PPP mRNA vaccine against each subtype of the new coronavirus Omicron variant strain. Immunity level.
  • Example 3 the same method as in Example 3 was used to further detect the sera collected from the mice in each immune group in Example 2 on the 28th day of the immunization program against the new coronavirus prototype strain, Delta variant strain, Specific binding antibody titer levels for RBD antigens of Omicron variant subtypes BA.1, BA.1.1, BA.2, BA.2.12.1, BA.2.75, BA.3, and BA.4/5; experimental procedures
  • the amino acid sequences of the RBD antigen proteins of the Omicron variant subtypes BA.2.75 and BA.3 used in the research are shown in SEQ ID NO: 27 and 28 respectively.
  • the serum collected from mice in each experimental group on the 28th day of the immunization program was against the new coronavirus prototype strain, Delta variant strain, and Omicron variant strain subtypes BA.1, BA.1.1, BA.2, BA.2.12.1, and BA.
  • the binding antibody titer levels of RBD antigens of .2.75, BA.3, and BA.4/5 are shown in Figure 4.
  • the specific antibody titer levels produced by the RBD antigen proteins of .1.1, BA.2, BA.2.12.1, BA.2.75, BA.3, and BA.4/5 were all significantly higher than those of the PPP mRNA vaccine; and, for The specific antibody titers produced by the RBD antigen proteins of the new coronavirus prototype strain and the Delta variant strain are also at high levels, indicating that they can also be used for the prevention and/or treatment of these virus strains.
  • the DOO mRNA vaccine of the present application can induce a higher level of humoral immunity than the PPP mRNA vaccine.
  • it produces a significantly higher level of humoral immunity than the PPP mRNA vaccine against each subtype of the new coronavirus Omicron variant strain.
  • the S encoding the new coronavirus prototype strain, Delta variant strain and Omicron variant strain subtypes BA.1, BA.1.1, BA.2, BA.2.12.1, BA.3, BA.4/5 and BA.2.75 respectively The nucleotides of the last 18 amino acids of the protein were removed, and the resulting nucleotides were named WT-S-del18, Delta-S-del18, BA.1-S-del18, BA.1.1-S-del18, and BA.2.
  • the culture medium was DMEM medium containing 10% FBS.
  • the immune mouse serum collected on the 14th day of the immunization program in the above Example 2 was tested for the new coronavirus prototype strain, Delta mutant strain, Omicron mutant strain subtypes BA.1, BA.1.1, 50% pseudovirus neutralization titer (pVNT 50 ) of pseudoviruses BA.2, BA.2.12.1, BA.3, and BA.4/5; the specific detection methods are as follows:
  • mice serum of each experimental group collected on the 14th day in Example 2 was incubated at 56°C for 30 minutes for inactivation; the inactivated serum samples were diluted, and a 2-fold gradient dilution was performed starting from 1:80. .
  • each pseudovirus was mixed with an equal volume of diluted serum and incubated at 37°C for 1 hour. Add 100 ⁇ l of the virus-serum mixture to the pre-plated Vero cells in a 96-well plate.
  • the CQ1 confocal image cytometer was used to detect the number of transduction units (TU) to calculate the response of the immune mouse serum to the above-mentioned new coronavirus prototype strain, Delta variant strain and Omicron variant strain subtypes BA.1 and BA. Neutralization ability of pseudoviruses 1.1, BA.2, BA.2.12.1, BA.3, and BA.4/5.
  • the serum neutralizing antibody titer levels induced by the DOO mRNA vaccine of this application against each subtype of the Omicron variant strain are much higher than those of the PPP mRNA vaccine; specifically, the serum neutralizing antibody titer levels induced by the BA.1 subtype
  • the level of neutralizing antibody titers induced by the DOO mRNA vaccine is 75 times higher than that of the PPP mRNA vaccine; for the neutralizing antibody titer level induced by the BA.1.1 subtype, the DOO mRNA vaccine is more than 72 times higher than that of the PPP mRNA vaccine; for BA.
  • the neutralizing antibody titer level induced by the .2 subtype, DOO mRNA vaccine is 66.7 times higher than that of PPP mRNA vaccine; for the neutralizing antibody titer level induced by BA.2.12.1 subtype, DOO mRNA vaccine is higher than PPP mRNA vaccine Up to 56.5 times; for the neutralizing antibody titer level induced by the BA.3 subtype, the DOO mRNA vaccine is 43.8 times higher than the PPP mRNA vaccine; for the neutralizing antibody titer level induced by the BA.4/5 subtype, the DOO The mRNA vaccine is 33.5 times higher than the PPP mRNA vaccine; and for the neutralizing antibody titer level induced by the Delta variant, the DOO mRNA vaccine is nearly 2 times higher than the PPP mRNA vaccine.
  • the DOO mRNA vaccine of this application can induce significantly higher neutralizing antibody titer levels against various strains of the new coronavirus (especially against the Delta variant strain and the Omicron variant strain subtypes), indicating that it is effective against All strains of the new coronavirus (especially against the Delta variant and Omicron variant subtypes) will have significantly higher immune protection efficacy. That is, the DOO mRNA vaccine of the present application has a broad spectrum and significantly enhanced immunogen. sex.
  • the immune mouse serum collected on the 28th day of the immunization program in the above Example 2 was tested for the new coronavirus prototype strain, Delta mutant strain, Omicron mutant strain subtypes BA.1, BA.1.1, 50% pseudovirus neutralization titers (pVNT 50 ) of pseudoviruses BA.2, BA.2.12.1, BA.2.75, BA.3, and BA.4/5; the specific detection method is the same as in Example 6.
  • the upper bar chart of Figure 6 shows the neutralization of the new coronavirus prototype strain, Delta variant strain and Omicron variant strain subtypes BA.1, BA.1.1, BA in the serum of each immune group .2, pVNT 50 (i.e., 50% pseudovirus neutralization titer) levels (expressed in log10) of pseudoviruses of BA.2.12.1, BA.2.75, BA.3, and BA.4/5, bars
  • pVNT 50 i.e., 50% pseudovirus neutralization titer
  • the number above each bar represents the geometric mean titer (GMT) of all samples in the experimental group; the radar chart below is based on the GMT value of each experimental group in the bar chart above.
  • the DOO mRNA vaccine of the present application can induce significantly higher neutralizing antibody titer levels against various strains of the new coronavirus (especially against the Delta variant strain and the Omicron variant strain subtypes). It shows that it will have significantly higher immune protection efficacy against various strains of the new coronavirus (especially against the Delta variant strain and Omicron variant strain subtypes). That is, the DOO mRNA vaccine of the present application has a broad spectrum and significantly enhanced immunity. of immunogenicity.
  • Example 8 Evaluation of the level of cellular immunity induced by mRNA vaccines
  • mice from each experimental group collected on the 21st day in Example 2 were used to detect the level of cellular immunity induced by the mRNA vaccine.
  • the specific methods are as follows:
  • a cell homogenizer to prepare mouse spleen cells into single-cell homogenate in 1 ml of serum-free DMEM, filter with a 40 ⁇ m cell filter, and use red blood cell lysis buffer (Beijing Solebao Technology Co., Ltd., R1010) to lyse red blood cells; then , cells were washed with washing solution (PBS+0.5% FBS), stained with 0.4% trypan blue solution (Gibco, 15250061), and counted using a CelldropFL automatic cell counter.
  • the positive control wells were stimulated with phytohemagglutinin (PMA) to produce non-specific cellular immune responses, and the negative control wells were not stimulated with the peptide library. Then, the cells were discarded and the 96-well plate was sequentially incubated with biotinylated IFN ⁇ antibody, streptavidin-HRP antibody, and chromogenic substrate. When spots appear on the bottom of the plate, rinse the sample thoroughly with deionized water to stop color development. Finally, ImmunoCapture6.5.0 was used to take pictures and count the number of spots.
  • PMA phytohemagglutinin
  • the experimental groups are as follows: three times inactivated vaccine immunization group (referred to as "IV” group), two times inactivated vaccine + PPP mRNA vaccine immunization group (i.e., PPP sequential immunization group, referred to as "PPP” group), two times inactivated vaccine +DOO mRNA vaccine immunization group (i.e., DOO sequential immunization group, referred to as "DOO” group) and twice inactivated vaccine adjuvant + LNP immunization group (i.e., LNP sequential immunization group, referred to as "LNP” group), as negative control group).
  • IV three times inactivated vaccine immunization group
  • PPP PPP sequential immunization group
  • DOO DOO sequential immunization group
  • LNP twice inactivated vaccine adjuvant + LNP immunization group
  • IV group All mice were vaccinated with one dose of inactivated vaccine on days 0, 21 and 42;
  • PPP sequential immunization group and DOO sequential immunization all mice were vaccinated with one dose of inactivated vaccine on day 0 and day 21, and then received one dose of each mRNA vaccine on day 42;
  • LNP sequential immunization group All mice were vaccinated with Al adjuvant, the adjuvant of the inactivated vaccine, on days 0 and 21, and then were vaccinated with empty LNP on day 42.
  • the vaccination method for each of the above vaccines is intramuscular injection.
  • the vaccination dose of the inactivated vaccine is 2.6U per mouse each time (0.4 doses of the human dose).
  • the vaccination dose of each mRNA vaccine or empty LNP is 2.6U per mouse. 10 ⁇ g each time for mice.
  • Mouse serum samples and mouse spleen samples were collected on day 56 to test the binding antibody titer and pseudovirus neutralizing antibody titer of the immune serum, respectively, as well as to test the level of T cell immunity.
  • Example 10 Evaluation of the inhibitory effect of sequentially immunized mouse serum on pseudoviruses of various strains of the new coronavirus
  • Example 5 the pseudovirus packaged in Example 5 was used, and the method described in Example 6 was used to detect the sensitivity of the mouse sera of each immune group collected on the 56th day in Example 9 against the new coronavirus prototype strain and Delta.
  • the DOO mRNA vaccine sequential immunization group targets Delta variants and Omicron variants BA.1, BA.1.1, BA.2, BA.2.12.1, and BA.3
  • the neutralizing antibody titer levels induced by pseudoviruses of the , BA.4/5 subtypes were significantly higher than those of the PPP mRNA vaccine; the neutralizing antibody titer levels induced by the DOO mRNA vaccine sequential immunization group against the new coronavirus prototype strain were significantly higher than those of the PPP mRNA vaccine.
  • the PPP mRNA vaccines were comparable, both at higher levels, indicating that they can also be used for prevention and/or treatment of the prototype strain.
  • the DOO mRNA vaccine sequential immunization group targets the new coronavirus prototype strain, Delta variant strain, and Omicron variant strain BA.1, BA.1.1, and BA. 2.
  • the neutralizing antibody titer levels induced by pseudoviruses of BA.2.12.1, BA.3, and BA.4/5 subtypes were significantly higher than those of inactivated vaccines.
  • Example 11 Evaluation of cellular immunity levels induced by sequential immunization
  • Example 8 the method described in Example 8 and the spleen samples of mice in each immunized group collected on day 56 in Example 9 were used to detect the level of cellular immunity induced by the sequential immunization of the mRNA vaccine.
  • the recombinant protein vaccine or various types of nucleic acid vaccines based on the novel coronavirus heterotrimeric chimeric antigen peptide or its encoding nucleic acid of the present application can effectively stimulate the original strain and multiple mutations of the novel coronavirus.
  • the strong immune response of strains (especially the subtypes of Omicron mutant strains) has broad spectrum and strong immunogenicity, and is expected to become a broad-spectrum vaccine to prevent the new coronavirus.

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Abstract

L'invention concerne un nouveau peptide antigénique chimérique trimérisé hétérologue de coronavirus, un polynucléotide codant pour celui-ci ou un produit d'acide nucléique associé au polynucléotide, un vaccin ou une composition immunogène basée sur le peptide antigénique ou le polynucléotide, et l'utilisation de ces produits dans un nouveau vaccin contre le coronavirus.
PCT/CN2023/103052 2022-06-28 2023-06-28 Nouveau vaccin chimérique trimère de coronavirus et son utilisation WO2024002129A1 (fr)

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WO2021239147A1 (fr) * 2020-05-29 2021-12-02 中国科学院微生物研究所 ANTIGÈNE DU β-CORONAVIRUS, VACCIN BIVALENT DU β-CORONAVIRUS, LEURS PROCÉDÉS DE PRÉPARATION ET LEURS APPLICATIONS
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