WO2023226988A1 - Procédé permettant d'améliorer l'immunogénicité d'un variant du sars-cov-2 et utilisation associée - Google Patents

Procédé permettant d'améliorer l'immunogénicité d'un variant du sars-cov-2 et utilisation associée Download PDF

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WO2023226988A1
WO2023226988A1 PCT/CN2023/095829 CN2023095829W WO2023226988A1 WO 2023226988 A1 WO2023226988 A1 WO 2023226988A1 CN 2023095829 W CN2023095829 W CN 2023095829W WO 2023226988 A1 WO2023226988 A1 WO 2023226988A1
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rbd
mutant
new coronavirus
protein
spike protein
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张辉
张贤涛
吴博霖
张译文
冯金珠
吴诗剑
何欣
陈然
邓洁仪
刘炳峰
张旭
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中山大学
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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
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/39—Medicinal preparations containing antigens or antibodies characterised by the immunostimulating additives, e.g. chemical adjuvants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
    • A61K2039/55505—Inorganic adjuvants
    • 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
    • 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 invention belongs to the technical field of biomedicine. More specifically, it relates to a method for enhancing the immunogenicity of new coronavirus variants and its application.
  • Omicron has rapidly evolved multiple new lineages based on the B.1.1.529 variant, including BA.1, BA.2, BA.3, BA.4 and BA.5.
  • Three subtypes of the Omicron mutant strains all have more than 30 mutations at more than 30 sites, and the number is much higher than that of previously discovered new coronavirus mutant strains, especially in the RBD region that directly binds to the receptor ACE2 to infect cells. mutation.
  • the extraordinary mutation of Omicron not only strengthens its ability to invade cells, but also significantly improves its ability to break through existing immunity.
  • the mutation in its RBD region caused significant immune evasion without significantly reducing its affinity with ACE2. Phenomenon.
  • the new coronavirus vaccine mainly uses the Spike protein or the RBD region on the Spike protein to design vaccine immunogens. Due to the high-frequency mutations of "Omicron" in the RBD region and even the complete S protein, its binding to ACE2 has undergone potential conformational changes during the process of invading cells. This change has a negative impact on the neutralizing ability of existing antibodies. The obvious escape effects, which threaten current strategies based on therapeutic antibodies, also have a serious impact on the immunity of existing vaccines, and it is necessary to further strengthen the mutation monitoring of the new coronavirus epidemic.
  • the technical problem to be solved by the present invention is to overcome the defects and deficiencies of poor immunogenicity of the existing new coronavirus mutant strains, provide a method and its application for enhancing the immunogenicity of the spike protein of the new coronavirus mutant strains, and overcome the shortcomings caused by the new coronavirus mutation. Violent mutations of strains cause immune escape and cause vaccine design problems with reduced immunogenicity.
  • the first purpose of the present invention is to provide a method for enhancing the immunogenicity of the spike protein of new coronavirus mutant strains.
  • the second purpose of the present invention is to provide a spike protein mutant of the new coronavirus variant strain with enhanced immunogenicity.
  • the third object of the present invention is to provide a product expressing the spike protein mutant.
  • the fourth object of the present invention is to provide the application of the spike protein mutant in the preparation of novel coronavirus antigens or anti-coronavirus drugs.
  • the fifth object of the present invention is to provide a new coronavirus antigen with enhanced immunogenicity.
  • the sixth object of the present invention is to provide a new coronavirus vaccine with enhanced immunogenicity.
  • the effectiveness of existing vaccines or immunity generated by past infections in preventing new coronavirus mutant strains has decreased.
  • the Omicron mutant strain of the new coronavirus on the one hand, causes severe evasion of existing vaccine immunity or immunity generated by previous infection; on the other hand, vaccines designed based on Omicron's own Spike (spike protein) or RBD (receptor binding domain) sequence
  • Omicron's own Spike spike protein
  • RBD receptor binding domain
  • the present invention has used the RBD of SARS-CoV-2 Omicron BA.2 strain Spike protein as a vaccine immunogen to design a nanoparticle vaccine based on Helicobacter pylori ferritin (the amino acid sequence is shown in SEQ ID NO.1) , compared with the corresponding nanoparticle vaccine of SARS-CoV-2 Delta, the titer of antigen-specific IgG antibodies produced by inducing immunity in mice is extremely low, suggesting that its violent mutation has produced a large reduction in immunogen, making it difficult to directly use the original viral protein. Sequence for vaccine application.
  • the present invention provides a method to enhance the immunogenicity of the new coronavirus mutant strain, that is, by targeting the receptor of the spike protein of the new coronavirus mutant strain with low immunogenicity.
  • the amino acid mutation in the binding domain can enhance the immunogenicity of the corresponding new coronavirus mutant strain by improving the immunogenicity of the spike protein of the new coronavirus mutant strain.
  • the method of enhancing the immunogenicity of the spike protein of the new coronavirus variant strain according to the present invention is: counting from the N-terminus of the spike protein, add the 371st, 373rd, 375th, 376th, 376th, and 376th of the spike protein of the new coronavirus variant strain.
  • Any 1 to 5 amino acids at positions 405, 408, 486, 493 or 505 are mutated into hydrophilic amino acids.
  • the hydrophilic amino acids include, but are not limited to, serine (S), glycine (G), tyrosine (Y), asparagine (N), glutamine (Q), threonine (T) or cysteine Acid (C).
  • the amino acid site of the present invention is located in the receptor binding domain (RBD) of the spike protein, and the new coronavirus mutant strain is an Omicron mutant strain.
  • RBD receptor binding domain
  • the Omicron mutant strains include but are not limited to BA.1, BA.2, BA.2.12.1, BA.4/5, BF.7, BA5.2, BF.7, XBB.1 ,XBB.1.15,XBB.1.16,XBB.1.9,BQ.1.
  • the present invention also provides a spike protein mutant of the new coronavirus mutant strain with enhanced immunogenicity.
  • the mutant is obtained by mutating the amino acids of the corresponding amino acid sites using the method of the present invention.
  • amino acid sequence of the receptor binding domain of the spike protein mutant of the present invention is such as SEQ ID NO.7, SEQ ID NO.9 ⁇ 14, SEQ ID NO.24 or SEQ ID NO.25 shown.
  • mutants for which no specific amino acid sequence is given since the original amino acid sequence is known, the mutation site and the amino acids before and after the mutation are also known, the mutated amino acid sequence can be known through substitution.
  • Nucleotide sequences encoding and expressing the spike protein mutants of the present invention can express the spike protein mutations of the present invention.
  • Recombinant vectors, expression cassettes, transgenic cell lines or recombinant bacteria should also be within the protection scope of the present invention. That is, the present invention applies to protect a product expressing the spike protein mutant.
  • the product is a recombinant vector or expression cassette containing a nucleotide sequence encoding the spike protein mutant.
  • the product is a transgenic cell line or recombinant bacteria expressing the spike protein mutant.
  • the spike protein mutant of the present invention can also be used directly as an antigen. Therefore, the present invention also applies for protection of the method for enhancing the immunogenicity of the spike protein of the new coronavirus mutant strain, the spike protein mutant of the new coronavirus mutant strain with enhanced immunogenicity, or the spike protein mutant encoding the new coronavirus mutant strain.
  • the recombinant vector or expression cassette of the nucleotide sequence is used in the preparation of novel coronavirus antigens or anti-coronavirus drugs.
  • the anti-COVID-19 drug may be a COVID-19 vaccine.
  • the present invention also provides a new coronavirus antigen with enhanced immunogenicity, which is a fusion protein obtained by fusion and expression of the spike protein mutant and the protein shown in SEQ ID NO.4 and SEQ ID NO.5 ;
  • the protein shown in SEQ ID NO.4 is used to assist RBD expression in eukaryotic cells and secreted into the culture supernatant to facilitate subsequent purification.
  • the protein shown in SEQ ID NO.5 can be combined with Ferritin ferritin nanoparticle structure SdCatcher for chemical connection.
  • the present invention also provides a new coronavirus vaccine with enhanced immunogenicity, which is prepared by using a protein containing the spike protein mutant of the present invention as an antigen or an antigen containing the new coronavirus antigen with enhanced immunogenicity. get.
  • the vaccine is prepared using the spike protein mutant of the present invention as an antigen or the new coronavirus antigen with enhanced immunogenicity.
  • the new coronavirus vaccine with enhanced immunogenicity of the present invention includes a multivalent new coronavirus prepared by using the spike protein mutant of the present invention as an antigen or the new coronavirus antigen with enhanced immunogenicity combined with other new coronavirus antigens. vaccine.
  • the new coronavirus vaccine of the present invention is a new coronavirus nanoparticle vaccine, that is, the above-mentioned RBD-HP_Ferritin nanoparticles are used as immunogens, emulsified with Alum adjuvant to make a vaccine, which induces an immune response and produces binding Antibodies to SARS-CoV-2 RBD.
  • the present invention uses the receptor binding domain (RBD) of the spike protein mutant as a single antigen fragment, realizes antigen multimerization based on the Helicobacter pylori multimeric protein, and constructs an RBD antigen multimer complex, while adding a signal peptide.
  • RBD-HP_Ferritin nanoparticles are prepared by completing the assembly of the connecting element peptide segments.
  • the immunogen RBD is displayed on the surface of the nanoparticles and emulsified with the Alum adjuvant.
  • the vaccine produced can induce an immune response and produce antibodies that bind RBD.
  • the present invention enhances the immunogenicity of the new coronavirus mutant strain by mutating the amino acid site of the spike protein of the new coronavirus mutant strain, and overcomes the defect of poor immunogenicity of the existing new coronavirus mutant strain.
  • the present invention also provides a new coronavirus vaccine with enhanced immunogenicity. Compared with a vaccine prepared using a virus strain without amino acid mutation as an immunogen, the vaccine can effectively improve the specificity after immunization.
  • the titer of neutralizing antibodies prevents the virus from invading the body.
  • Figure 1 is a schematic diagram of the plasmid structure expressing SARS-CoV-2 Omicron BA.2 RBD.
  • Figure 2 shows the expression staining results of SARS-CoV-2 Omicron BA.2 RBD and various mutant RBD proteins of the present invention in eukaryotic cells.
  • Figure 3 is a schematic diagram of the self-assembly construction of RBD-HP_Ferritin nanoparticle vaccine.
  • Figure 4 shows the gel electrophoresis detection results of SARS-CoV-2 Omicron BA.2 RBD mutation after being connected to Ferritin.
  • Figure 5 shows the purified molecular sieve diagram of SARS-CoV-2 Omicron BA.2 RBD nanoparticle antigen; among them, the left picture shows the molecular sieve diagram of BA.2_RBD-HP_Ferritin, the middle picture shows the molecular sieve diagram of BA.2-S375_RBD-HP_Ferritin, and the right picture is the molecular sieve diagram of Delta_RBD-HP_Ferritin.
  • Figure 6 shows the detection results of BA.2 RBD-specific IgG antibody titer levels in mouse serum detected by ELISA; * in the figure represents P ⁇ 0.05, ** represents P ⁇ 0.01, and **** represents P ⁇ 0.0001.
  • Figure 7 shows the detection results of the titer level of anti-virus-infected cells in the immune serum of mice 19 days after immunization through pseudovirus neutralization experiments; * in the figure indicates P ⁇ 0.05.
  • Figure 8 shows the detection results of BA.2 RBD-specific IgG antibody titer levels in the serum of immunized mice verified by ELISA method after expanding the number of mice; * in the figure indicates P ⁇ 0.05.
  • Figure 9 shows pseudoviruses after immunizing mice with RBD-specific nanoparticle vaccines prepared based on F375S mutant proteins of different Omicron family mutant strains (BA.1; BA.2; BA.2.12.1; BA.4/5). Neutralization experiment results; * in the figure indicates P ⁇ 0.05, ** indicates P ⁇ 0.01, and **** indicates P ⁇ 0.0001.
  • Figure 10 shows the pseudovirus neutralization experimental results after immunizing mice with the RBD-specific nanoparticle vaccine prepared by introducing the F375S site mutation into the full-length Omicron BA.2 Spike protein; * in the figure indicates P ⁇ 0.05.
  • Figure 11 shows the results of the pseudovirus neutralization experiment after immunizing mice with the RBD-specific nanoparticle vaccine of BA.5 (S375R493); * in the figure indicates P ⁇ 0.05, ** indicates P ⁇ 0.01, and *** indicates P ⁇ 0.001 , **** means P ⁇ 0.0001, ns means the difference is not significant.
  • Figure 12 is a schematic diagram of the BA.5(SR)RBD/Delta RBD bivalent vaccine and neutralization test, as well as the pseudovirus neutralization test results after immunizing mice; * in the figure represents P ⁇ 0.05, ** represents P ⁇ 0.01, *** means P ⁇ 0.001, ns means the difference is not significant.
  • Figure 13 shows the vaccines made using Ferritin, BA.2 RBD, BA.5(SR)RBD and the BA.5(SR)RBD/Delta RBD bivalent vaccine to immunize mice respectively with Delta, BA.2, BA.5 After wild-type virus infection, virus copy number analysis results in mouse lungs and tracheal tissues; **** indicates P ⁇ 0.0001.
  • Figure 14 shows mice immunized with vaccines made of Ferritin, BA.2 RBD, BA.5(SR)RBD and BA.5(SR)RBD/Delta RBD bivalent vaccine respectively and infected with Delta and BA.2 wild-type viruses. Finally, the results of H&E staining and immunohistochemical analysis of SARS-CoV-2 N protein in mouse lungs.
  • Figure 15 shows the results of the sequential immunization study of the BA.5(SR)RBD/Delta RBD bivalent vaccine on rhesus monkeys;
  • a in the picture is a schematic diagram of the sequential immunization study;
  • B in the picture is the sequential immunization BA of rhesus monkeys .5(SR)/Delta RBD bivalent vaccine 4 weeks later, the results of serum neutralization of live virus (FRNT50);
  • C in the figure is the sequential immunization of rhesus monkeys with BA.5(SR)/Delta RBD bivalent vaccine 4 Weeks later, the results of serum neutralization of various pseudoviruses (pVNT50);
  • * in the figure represents P ⁇ 0.05, *** represents P ⁇ 0.001.
  • Omicron BA.2 RBD and Gv are constructed according to the plasmid diagram in Figure 1 Gv-SARS-CoV-2 Omicron BA.2 RBD fusion protein (its amino acid sequence is shown in SEQ ID NO. .6), and at the same time, the code for the secreted peptide SP (its amino acid sequence is such as SEQ ID NO.4) is added to the 5' end of the nucleotide sequence encoding the Gv-SARS-CoV-2 Omicron BA.2 RBD fusion protein. Sequence, Gv and Omicron BA.2 RBD are separated by Linker GSG, and 6 ⁇ His and translation stop codon are added to the 3' end.
  • the constructed SP-Gv-SARS-CoV-2 Omicron BA.2 RBD-His was cloned into the Xho I and Xba I restriction sites of the expression vector (pcDNA3.1-Intron-WPRE) added with Intron and WPRE for enhanced expression. Between the points, the recombinant expression vector pcDNA3.1-Intron-SP-Gv-SARS-CoV-2 Omicron BA.2 RBD-His-IRES-GFP-WPRE was constructed (the schematic diagram of the plasmid structure is shown in Figure 1).
  • the specific mutations are as follows: D339G/F371S/F373S/F375S/A376T/N405D/S408R/N417K/K440N/N477S/K478S/A484E/R493Q/R498Q/Y501N/H505Y.
  • the above 16 Gv-SARS-CoV- 2 Omicron BA.2 RBD mutation plasmid (the amino acid sequences of the mutated RBD are shown in SEQ ID NO. 7 ⁇ 22); among them, taking D339G as an example, this means that the 339th amino acid is mutated from D to G.
  • the present invention also carried out F375Q and F375T mutations, whose amino acid sequences are shown in SEQ ID NO.24 and SEQ ID NO.25.
  • the present invention also constructed the Gv-SARS-CoV-2 Delta RBD recombinant plasmid (the amino acid sequence of Gv-SARS-CoV-2 Delta RBD is shown in SEQ ID NO. 23) and the Gv-Omicron BA.2 RBD recombinant plasmid, Used subsequently as a control.
  • the recombinant plasmid and recombinant mutant plasmid prepared in step 1 were transformed into DH5 ⁇ competent cells respectively, cultured at 37°C overnight, and positive clones were identified by PCR; the endotoxin-free plasmid was extracted and transfected into HEK293F using lipofectamine transfection protocol. Cells were harvested by centrifugation 4 days after transfection, and the target protein was purified.
  • the coupled Gv-SARS-CoV-2 Omicron BA.2 RBD-SdCatcher-HPF multimer antigen was purified by molecular sieve chromatography using Siperose6 Increase10/300 GL column (GE) to obtain twenty-tetramer RBD.
  • -HPF protein namely RBD-HP_Ferritin nanoparticles, its self-assembly construction schematic is shown in Figure 3.
  • the gel electrophoresis detection results of Gv-SARS-CoV-2 Omicron BA.2 RBD and Gv-SARS-CoV-2 Omicron BA.2 RBD mutation coupled with Ferritin are shown in Figure 4.
  • the purified molecular sieve diagram of SARS-CoV-2 Omicron BA.2 RBD nanoparticle vaccine is shown in Figure 5; the left picture in the picture is the molecular sieve diagram of BA.2_RBD-HP_Ferritin, and the middle is the molecular sieve diagram of BA.2-S375_RBD-HP_Ferritin. The picture on the right is the molecular sieve diagram of Delta_RBD-HP_Ferritin. It can be seen from Figure 4 and Figure 5 that Gv-SARS-CoV-2 Omicron BA.2 RBD and its mutant protein can be successfully displayed on the surface of nanoparticles to form macromolecular nanoparticle antigens.
  • mice On the 8th and 14th days of immunization, blood was collected from the orbits of the mice; after the mouse serum had been allowed to stand for a period of time for the serum to precipitate, it was centrifuged at 4°C and 2800rpm for 15 minutes, and the Anti-RBD IgG ELISA test was immediately performed. , detecting the BA.2 RBD specific IgG antibody titer level in mouse serum.
  • Omicron BA.2 RBD mutations at 371, 373, 376, 405, 408 and 505 also produced higher antibody titers than the natural Omicron BA.2 RBD.
  • the above results also show that this section The sequence is directly related to the low immunogenicity of Omicron BA.2.
  • the Spike protein of SARS-CoV-2 BA2 was synthesized and inserted into the pcDNA3.1 expression vector.
  • the SARS-CoV-2 BA2 Spike protein expression vector was co-transfected with pHIV-luciferase and psPAX2 plasmids into 293T cells. 5 hours after transfection, the cells were washed twice with PBS and replaced with serum-free DMEM medium to continue culturing; 48 hours later Collect the supernatant, centrifuge to remove cell debris, and dissolve it with a small volume of serum-free DMEM to obtain HIV-luc/SARS-CoV-2-BA2 S pseudovirus.
  • This pseudovirus can effectively simulate the process of wild-type SARS-CoV-2 invading cells; when it infects production cells or target cells, the expression of the luciferase reporter gene carried by the SARS-CoV-2 pseudovirus can accurately reflect viral infection. As a result, the results of the experimental system can be read accurately and quickly, and it can be used as an excellent antibody neutralization titer monitoring system.
  • TCID 50 was calculated by Reed-Muech method.
  • mice take the immune serum after immunization of mice, dilute the purified antibody 2 times, mix it with TCID 50 final concentration pseudovirus, and incubate it at 37°C for 1 hour; mix The solution was added to a 96-well plate in hACE 2-HEK293T cells with a density of about 70%; after 48 hours, discard the culture solution, wash the cells twice with PBS, add cell lysate, and detect the luciferase activity value. To detect the titer level of anti-viral infected cells in mice after immunization.
  • the present invention expanded the number of mice, that is, repeated the experiment three times, and detected the titer level of BA.2 RBD-specific IgG antibodies in the serum of immune mice through ELISA.
  • the results are shown in Figure 8.
  • the results shown show that there is a significant difference between the BA2-RBD(S375)-Ferritin and BA2-RBD-Ferritin groups (* represents p ⁇ 0.05). This result shows that the mutation at position 375 causes a higher level of neutralizing virus antibodies.
  • the present invention uses the method described in Example 1 to carry out F375S mutation on the spike proteins of different Omicron family mutant strains (BA.1; BA.2; BA.2.12.1; BA.4/5). After expression, the After using the corresponding mutant strain proteins, the method described in Example 2 was used to prepare RBD-specific nanoparticles for each mutant strain of the Omicron family (BA.1; BA.2; BA.2.12.1; BA.4/5). The granular vaccine was used to immunize BALB/c mice (10 ⁇ g/mouse) respectively, and the serum at the 4th week was collected, and a neutralization test was conducted according to the method described in Example 4.
  • the neutralizing antibody titer results of the RBD-specific nanoparticle vaccines prepared from the F375S mutation of different Omicron family mutant strains (BA.1; BA.2; BA.2.12.1; BA.4/5) against their respective pseudoviruses are as follows: As shown in Figure 9. As can be seen from Figure 9, compared with the prototype RBD nanoparticle vaccine, different Omicron family mutant strains have better immune response effects after S375F site mutation, and have higher neutralizing antibody titers (pVNT50).
  • the present invention also introduces the F375S site mutation into the full-length Omicron BA.2 Spike protein, makes the corresponding RBD-specific nanoparticle vaccine, immunizes BALB/c mice (10 ⁇ g/mouse), and detects it at the 4th week Neutralizing antibody titers against Omicron-specific pseudoviruses, the results are shown in Figure 10.
  • Figure 10 compared with the prototype Spike protein vaccine, the immune response after F375S site mutation is better and has a higher neutralizing antibody titer (pVNT50).
  • the present invention added new V486F and Q493R mutations based on the BA.5 (S375) RBD design.
  • the present invention uses BA.5 RBD as a template, carries out combined mutations of F375S, V486F and Q493R, abbreviated as BA.5 (S375R493) or BA.5 (SR), and makes the corresponding RBD-specific nanoparticle vaccine. .
  • the present invention also uses BA.5(SR)RBD and Delta RBD proteins to prepare a BA.5(SR)RBD/Delta RBD bivalent vaccine.
  • the bivalent vaccine Neutralization tests were conducted after immunizing k18-ACE2 mice (5 ⁇ g of each vaccine in the components, totaling 10 ⁇ g).
  • the schematic diagram of the BA.5(SR)RBD/Delta RBD bivalent vaccine and neutralization test, as well as the pseudovirus neutralization test results after immunizing mice, are shown in Figure 12.
  • Figure 12 compared with the monovalent vaccine, the BA.5(SR)RBD/Delta RBD bivalent vaccine can produce higher neutralizing protective antibody titers against the wild-type virus after 6 weeks.
  • the present invention also utilizes vaccines made of Ferritin, BA.2 RBD, BA.5(SR)RBD and BA.5(SR)RBD/Delta RBD bivalent vaccine to immunize (10 ⁇ g/mouse) k18-ACE2 mice respectively.
  • Prime/boost strategy after 6 weeks, mice were infected with Delta, BA.2, and BA.5 wild-type viruses. After 3 days, mouse lungs and tracheal tissues were taken, and RNA was extracted for virus copy number analysis. The results are as follows: As shown in Figure 13, it can be seen from Figure 13 that BA.5(SR)RBD and bivalent vaccine can protect mice from viral infection, and viral RNA cannot be detected.
  • the present invention also conducted sequential immunization studies on rhesus monkeys with the BA.5(SR)RBD/Delta RBD bivalent vaccine.
  • the process is as follows: immunize with 50 ⁇ g D614G RBD nanoparticle vaccine on days 0 and 28 (for preparation methods, please refer to patents CN111217919A and CN111217918A). On day 282, immunize with D614G RBD/Beta RBD (25 ⁇ g+25 ⁇ g) bivalent nanoparticle vaccine on day 747. , immune BA.5(SR)RBD/Delta RBD (25 ⁇ g+25 ⁇ g) bivalent nanoparticle vaccine.
  • the BA.5(SR)RBD/Delta RBD bivalent vaccine of the present invention can better induce the body to produce broad-spectrum neutralizing protective antibodies against a variety of mutant strains, and is effective against true viruses. and activity.

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Abstract

L'invention concerne un procédé permettant d'améliorer l'immunogénicité d'un variant du SARS-CoV-2 et une utilisation associée. La présente invention permet d'améliorer l'immunogénicité du variant du SARS-CoV-2 au moyen d'une mutation ponctuelle sur certaines positions d'acides aminés de la protéine de spicule du variant du SARS-CoV-2, et de surmonter le défaut de faible immunogénicité dans les mutants existants du SARS-CoV-2. Sur cette base, la présente invention concerne en outre un antigène présentant une immunogénicité améliorée et un vaccin contre le SARS-CoV-2. Par comparaison avec un vaccin préparé à partir d'un virus sans mutations d'acides aminés en tant qu'immunogène, le vaccin peut améliorer de manière efficace le titre d'anticorps spécifiques après vaccination, neutraliser le virus, et empêcher le virus d'envahir le corps humain.
PCT/CN2023/095829 2022-05-25 2023-05-23 Procédé permettant d'améliorer l'immunogénicité d'un variant du sars-cov-2 et utilisation associée WO2023226988A1 (fr)

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WO2021160346A1 (fr) * 2020-02-13 2021-08-19 Institut Pasteur Vaccin à base d'acide nucléique contre le coronavirus sars-cov-2
WO2021233885A1 (fr) * 2020-05-18 2021-11-25 Synthetic Vaccines Ltd Peptides mimotopes de la protéine spike du virus sars-cov-2
WO2022038501A1 (fr) * 2020-08-17 2022-02-24 Grifols Diagnostic Solutions Inc. Protéines de fusion comprenant le domaine de liaison de récepteur du sars-cov-2
CN114369172A (zh) * 2021-03-01 2022-04-19 中国科学院微生物研究所 一种新型冠状病毒多价抗原、其制备方法和应用

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CN114315989A (zh) * 2022-01-21 2022-04-12 国药中生生物技术研究院有限公司 一种重组新型冠状病毒蛋白疫苗、其制备方法和应用

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WO2021160346A1 (fr) * 2020-02-13 2021-08-19 Institut Pasteur Vaccin à base d'acide nucléique contre le coronavirus sars-cov-2
WO2021233885A1 (fr) * 2020-05-18 2021-11-25 Synthetic Vaccines Ltd Peptides mimotopes de la protéine spike du virus sars-cov-2
WO2022038501A1 (fr) * 2020-08-17 2022-02-24 Grifols Diagnostic Solutions Inc. Protéines de fusion comprenant le domaine de liaison de récepteur du sars-cov-2
CN112076315A (zh) * 2020-08-25 2020-12-15 中国农业科学院生物技术研究所 新冠病毒s蛋白和铁蛋白亚基融合的纳米抗原颗粒、新冠疫苗及其制备方法和应用
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