WO2023025257A1 - Antigène hétéromultimère de coronavirus bêta, son procédé de préparation et son utilisation - Google Patents

Antigène hétéromultimère de coronavirus bêta, son procédé de préparation et son utilisation Download PDF

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WO2023025257A1
WO2023025257A1 PCT/CN2022/114892 CN2022114892W WO2023025257A1 WO 2023025257 A1 WO2023025257 A1 WO 2023025257A1 CN 2022114892 W CN2022114892 W CN 2022114892W WO 2023025257 A1 WO2023025257 A1 WO 2023025257A1
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coronavirus
antigen
monomers
heteromultimer
rbd
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Chinese (zh)
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高福
戴连攀
郑天依
徐坤
安亚玲
韩雨旋
胥森瑜
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中国科学院微生物研究所
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    • 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
    • A61K39/215—Coronaviridae, e.g. avian infectious bronchitis virus
    • 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
    • C07K14/08—RNA viruses
    • C07K14/165—Coronaviridae, e.g. avian infectious bronchitis virus
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K19/00—Hybrid peptides, i.e. peptides covalently bound to nucleic acids, or non-covalently bound protein-protein complexes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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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
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/62—DNA sequences coding for fusion proteins
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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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
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    • 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

  • the present application relates to the field of biomedicine, in particular to a betacoronavirus heteromultimeric antigen, its preparation method and application.
  • Coronaviridae contains 4 genera of coronaviruses, ⁇ , ⁇ , ⁇ , and ⁇ .
  • Severe acute respiratory syndrome coronavirus (SARS-CoV), Middle East respiratory syndrome coronavirus (MERS-CoV) and new coronavirus (2019-nCoV, later named SARS-CoV-2) are all betacoronaviruses belongs to. They are positive-strand RNA enveloped viruses that can widely infect humans and animals, and can cause severe disease and even death in humans.
  • the new coronavirus enters cells through human angiotensin-converting enzyme 2 (hACE2), and hACE2 receptors are distributed in arteriovenous endothelial cells, arterial smooth muscle cells, intestinal epithelial cells, alveoli, bronchi and other respiratory organs , the virus can infect these cells that contain the hACE2 receptor.
  • hACE2 receptors are distributed in arteriovenous endothelial cells, arterial smooth muscle cells, intestinal epithelial cells, alveoli, bronchi and other respiratory organs , the virus can infect these cells that contain the hACE2 receptor.
  • coronaviruses that can infect humans, including HCoV-OC43 and HKU1 of the betacoronavirus genus, HCoV-NL63 and HCoV-229E of the alphacoronavirus genus, and these coronaviruses have relatively mild symptoms after infecting humans.
  • MERS cases are mainly concentrated in the Middle East and Europe. Most of the confirmed cases in countries outside the Middle East have a history of working or living in the Middle East before onset, and the mortality rate is as high as about 40%.
  • MERS-CoV is mainly transmitted to humans by dromedary camels. There are a large number of dromedary camels in the Middle East. Studies have reported that MERS-CoV has become endemic in dromedary camels in Saudi Arabia. Therefore, MERS -CoV has a greater risk of transmission, and there is currently no vaccine and drug in use.
  • the COVID-19 pandemic is spreading around the world, causing serious harm. Emerging and re-emerging infectious diseases are becoming more and more frequent. Therefore, it is of great application value to develop a coronavirus vaccine antigen design method to realize that one antigen can effectively prevent multiple viruses.
  • S protein Session protein, S
  • E protein envelope protein, Envelope protein, E
  • M protein membrane protein, Membrane protein, M
  • S protein Spike protein, S
  • E protein envelope protein, Envelope protein, E
  • M protein membrane protein, Membrane protein, M
  • the S protein is closely related to the process of coronavirus invading cells, and is an important antigen in vaccine development, which can produce neutralizing antibodies.
  • the receptor binding domain (RBD) of the S protein is the most important antigen target region for the body to induce neutralizing antibodies.
  • the purpose of this application is to provide a betacoronavirus heteromultimeric antigen, its preparation method and application.
  • This application is based on the conclusion that the RBD protein of ⁇ -coronavirus can stimulate the body to produce neutralizing antibodies, and the dimeric RBD protein can stimulate the body's immune response more effectively than the monomeric RBD protein.
  • a ⁇ -coronavirus heterologous multimer antigen was obtained by connecting them in series, and a corresponding vaccine was obtained using this antigen.
  • the vaccine can stimulate mice to produce a strong antibody response to a variety of betacoronaviruses, achieving the effect of one antigen efficiently preventing multiple viruses.
  • a ⁇ -coronavirus heteromultimer antigen the amino acid sequence of which includes: a plurality of interconnected monomers from ⁇ -coronaviruses, each monomer from ⁇ -coronaviruses being the spike protein of ⁇ -coronaviruses
  • the partial amino acid sequence or the entire amino acid sequence of the receptor binding region, the number of monomers is an integer ⁇ 3
  • the multiple monomers of the ⁇ -coronavirus heteromultimer antigen include those from 2 heterologous ⁇ -coronaviruses monomers or monomers from more than 3 heterologous betacoronaviruses.
  • the ⁇ -coronavirus is severe respiratory syndrome coronavirus, Middle East respiratory syndrome coronavirus or 2019-nCoV.
  • the multiple monomers of the ⁇ -coronavirus heteromultimeric antigen include monomers from Middle East respiratory syndrome coronavirus and monomers from A monomer of the 2019 novel coronavirus.
  • the number of monomers is 3 or 4.
  • the multiple monomers of the ⁇ -coronavirus hetero-multimeric antigen include 2 or more monomers from homologous ⁇ -coronavirus
  • the monomers from the homologous ⁇ coronavirus are connected to each other first, and then connected to the monomers from the heterologous ⁇ coronavirus.
  • the monomers are connected in series directly or by linking amino acid sequences; optionally, the linking amino acid sequences at different positions are independently selected from the following sequences: : (GGS) n connection sequence, wherein n represents the number of GGS, and n is an integer ⁇ 1; further optionally, n is an integer selected from 1-10; further optionally, n is selected from 1- Integer of 5.
  • the three letters GGS represent the amino acids G, G, and S, respectively.
  • the linking amino acid sequences at different positions are mutually independent means that the linking amino acid sequence linking the first and second monomers from the N-terminus may be different from the linking amino acid sequence linking the second and third monomers from the N-terminus ,So on and so forth.
  • the multiple monomers of the ⁇ -coronavirus hetero-multimeric antigen include 2 or more monomers from homologous ⁇ -coronavirus
  • the amino acid sequences of monomers from homologous betacoronaviruses that are connected to each other are identical sequences. That is, the homologous tandem sequences are completely identical and are multiple repeated sequences.
  • the monomers from homologous ⁇ coronaviruses are not necessarily identical, for example, the monomers from homologous ⁇ coronaviruses can be longer and one shorter.
  • the partial amino acid sequence of the receptor-binding region of the spike protein of ⁇ -coronavirus is the entirety of the receptor-binding region of the spike protein of ⁇ -coronavirus At least 50%, 60%, 70%, 80%, 90%, 95%, 99% of the amino acid sequence.
  • multiple interconnected monomers derived from heterologous ⁇ -coronaviruses are:
  • MERS-CoV One monomer from MERS-CoV is connected to two monomers from 2019-nCoV in series. That is: monomer of Middle East respiratory syndrome coronavirus - monomer of 2019 new coronavirus - monomer of 2019 new coronavirus;
  • the two monomers from MERS-CoV connected in series are connected with the two monomers from 2019-nCoV in series. Namely: monomer of MERS-CoV-monomer of MERS-CoV-monomer of 2019-nCoV-monomer of 2019-nCoV.
  • the amino acid sequence of the ⁇ -coronavirus heterologous antigen includes any one selected from the following amino acid sequences:
  • SEQ ID NO: 7 The sequence is shown in SEQ ID NO: 7, that is: one Middle East respiratory syndrome coronavirus RBD 367-602 amino acids and two 2019-nCoV RBD 319-537 amino acids are directly connected in series;
  • SEQ ID NO: 8 The sequence is shown in SEQ ID NO: 8, namely: two RBD 367-602 amino acids of the Middle East respiratory syndrome coronavirus and two 2019-nCoV RBD 319-537 amino acids are directly connected in series.
  • the 367-602 region of the Middle East respiratory syndrome coronavirus RBD is derived from the E367-N602 region of the MERS-CoV spike protein sequence (GenBank on NCBI: AFS88936.1); the 319-537 region of the 2019 novel coronavirus RBD The R319-K537 region of the S spike protein sequence (GenBank on NCBI: YP_009724390) derived from the WH01 strain of the 2019 novel coronavirus.
  • the present application also provides a method for preparing the above-mentioned ⁇ -coronavirus heteromultimer antigen, comprising the following steps: adding a coding signal peptide to the 5' end of the nucleotide sequence encoding the above-mentioned ⁇ -coronavirus hetero-multimer antigen The sequence of encoding the histidine tag and the stop codon are added to the 3' end, cloned and expressed, the correct recombinant is screened, and then transfected into the cells of the expression system for expression. After expression, the cell supernatant is collected and purified to obtain ⁇ Coronavirus heteromultimeric antigen.
  • the cells of the expression system include mammalian cells, insect cells, yeast cells or bacterial cells, optionally; the mammalian cells include HEK 293T cells, HEK 293F cells or CHO cells, the bacterial cells include E. coli cells.
  • the present application also provides a polynucleotide encoding the above-mentioned ⁇ -coronavirus heteromultimer antigen, a recombinant vector comprising the above-mentioned polynucleotide, and an expression system cell comprising the above-mentioned recombinant vector.
  • the present application also provides the above-mentioned ⁇ -coronavirus heteromultimer antigen, polynucleotide encoding the above-mentioned ⁇ -coronavirus heteromultimer antigen, a recombinant vector comprising the above-mentioned polynucleotide, or an expression system comprising the above-mentioned recombinant vector Use of cells in the preparation of betacoronavirus vaccines.
  • the present application also provides a ⁇ -coronavirus vaccine, comprising the above-mentioned ⁇ -coronavirus multimer antigen and an adjuvant.
  • the adjuvant is selected from aluminum adjuvants, MF59 adjuvants, and MF59-like adjuvants.
  • the present application also provides a ⁇ -coronavirus DNA vaccine, which includes: a recombinant vector comprising a DNA sequence encoding the above-mentioned ⁇ -coronavirus heteromultimer antigen.
  • the present application also provides a ⁇ -coronavirus mRNA vaccine, which includes: a recombinant vector comprising the mRNA sequence encoding the above-mentioned ⁇ -coronavirus heteromultimer antigen.
  • the present application also provides a ⁇ -coronavirus virus vector vaccine, which includes: a recombinant virus vector comprising a nucleotide sequence encoding the above-mentioned ⁇ -coronavirus heteromultimer antigen; optionally, the virus vector is selected from the following One or more: adenovirus vector, poxvirus vector, influenza virus vector, adeno-associated virus vector, vesicular stomatitis virus vector (Vesicular Stomatitis Virus, VSV).
  • adenovirus vector comprising a nucleotide sequence encoding the above-mentioned ⁇ -coronavirus heteromultimer antigen
  • the virus vector is selected from the following One or more: adenovirus vector, poxvirus vector, influenza virus vector, adeno-associated virus vector, vesicular stomatitis virus vector (Vesicular Stomatitis Virus, VSV).
  • Figure 1 is the molecular sieve analysis and gel electrophoresis analysis of the MERS-RBD and SARS-CoV-2-RBD tandem dimer (MC-RBD-tr2) antigenic protein in Example 1 of the present application;
  • Figure 2 shows the molecular sieve analysis and gel electrophoresis analysis of the single-chain heterotrimeric antigen protein in Example 4 of the present application;
  • Figure 3 shows the molecular sieve analysis and gel electrophoresis analysis of the single-chain heterotetramer antigen protein in Example 4 of the present application;
  • Example 4 is a schematic diagram of the immunization strategy of each group of immunized mice experiments in Example 5 of the present application;
  • Figure 5 is a graph showing the results of the level of specific IgG-binding antibodies against MERS-RBD detected in the serum of mice 19 days after immunization in Example 5 according to the ELISA method in Example 6;
  • Figure 6 is a graph showing the results of specific IgG-binding antibody levels against SARS-CoV-2-RBD detected in the serum of mice 19 days after immunization in Example 5 according to the ELISA method in Example 6;
  • Figure 7 is a graph showing the results of the level of specific IgG-binding antibodies against MERS-RBD detected in the serum of mice 35 days after immunization in Example 5 of the present application according to the ELISA method in Example 6;
  • Figure 8 is a graph showing the results of the level of specific IgG-binding antibodies against SARS2-RBD detected in the serum of mice 35 days after immunization in Example 5 of the present application according to the ELISA method in Example 6;
  • Fig. 9 is the result figure of the neutralizing antibody against SARS-CoV-2 detected by the mouse serum on the 35th day after immunization obtained in Example 7 of the present application by the pseudovirus neutralization test (Example 7), as in Example 8 detected in
  • Figure 10 is a graph showing the results of lung tissue viral load after SARS-CoV-2 live virus challenge through SARS-CoV-2 live virus challenge protection test and RT-qPCR experiment in Example 10 of the present application; wherein, The ordinate is the copy number of the new coronavirus sgRNA per gram of lung tissue, and the abscissa shows the immune group category.
  • MERS has a high mortality rate and can be transmitted from person to person or from camels to people. There are a large number of dromedary camels in the Middle East. Studies have reported that MERS-CoV has become endemic in dromedary camels in Saudi Arabia. Therefore, MERS-CoV has a greater risk of transmission.
  • Example 1 Expression and purification of MERS-CoV RBD and SARS-CoV-2 RBD chimeric dimer (MC-RBD-tr2) protein
  • MERS-RBD The amino acid sequence of MERS-RBD (position 367-602) is concatenated with the amino acid sequence of SARS-CoV-2-RBD (position 319-537), named MC-RBD-tr2 (its sequence is shown in SEQ ID NO: 1) , 6 histidines are added to the C-terminus of the sequence, and the N-terminus is connected to the signal peptide of the MERS-S protein itself (MIHSVFLLMFLLTPTES, SEQ ID NO: 2).
  • nucleotide sequence encoding MC-RBD-tr2 with MERS-S protein self signal peptide and histidine into the EcoRI and XhoI restriction sites of pCAGGS vector (above-mentioned nucleotide sequence is as SEQ ID NO:3 As shown, the nucleotide sequence includes sequences encoding histidine and signal peptide).
  • the upstream of the promoter contains the Kozak sequence GCCGCCACC.
  • the plasmid pCAGGS-MC-RBD-tr2 expressing heterodimer was obtained by molecular cloning.
  • the plasmid was transfected into HEK293F cells, and the supernatant was collected 5 days later, centrifuged to remove the precipitate, and then filtered through a 0.22 ⁇ m filter membrane to further remove impurities.
  • the obtained cell supernatant was adsorbed by a nickel affinity column (His Trap, GE Healthcare) at 4°C, and washed with buffer A (20 mM Tris, 150 mM NaCl, pH 8.0) to remove non-specific binding proteins.
  • the target protein was further purified by molecular sieve chromatography on a Superdex TM 200 Increase 10/300GL column (GE Healthcare).
  • the molecular sieve chromatography buffer is PBS buffer.
  • a typical molecular sieve chromatogram is shown in Figure 1: the elution peak near the elution volume of 14ml was taken for SDS-PAGE analysis, and the target protein was analyzed under non-reducing conditions (without DTT) and reducing conditions ( Adding DTT) the size is about 60Kd, which proves that the peak is a dimer (the size of the monomer is 30Kd). Take the elution peak near the elution volume of 16ml for SDS-PAGE analysis. The size of the target protein is about 30kD under non-reducing conditions (without DTT) and reducing conditions (with DTT), which proves that the peak is mainly RBD monomer.
  • MERS-RBD-tr2 two repeated MERS-RBD 367-602 amino acid sequences are directly connected in series, and its amino acid sequence is as shown in SEQ ID NO: 4
  • SARS2-RBD-tr2 two repeated SARS-CoV-2-RBD 319-537 amino acid sequences are directly concatenated, and its amino acid sequence is shown in SEQ ID NO: 5) plasmids.
  • Embodiment 3 Design and preparation of MERS-RBD, SARS-CoV-2 RBD single-chain heteromultimer
  • MERS-RBD and SARS-CoV-2-RBD each have a flexible sequence.
  • MERS-RBD partial sequence The 367-602 amino acids of a MERS-RBD partial sequence are concatenated with the 319-537 amino acids of two SARS-CoV-2-RBD partial sequences, named MCC-RBD-tr3 (its amino acid sequence is as SEQ ID shown in NO:6);
  • MCC-RBD-tr3 or MMCC-RBD-tr4 with N-terminal linking signal peptide (MERS-S protein self-signal peptide MIHSVFLLMFLLTPTES, SEQ ID NO: 2) and C-terminal 6 histidines was inserted into Between the EcoRI and XhoI restriction sites of the pCAGGS vector (the nucleotide sequence of the MCC-RBD-tr3 with the N-terminal connection signal peptide and histidine is shown in SEQ ID NO: 8, and the encoding has the N-terminal The nucleotide sequence of MMCC-RBD-tr4 connecting signal peptide and histidine is shown in SEQ ID NO:9).
  • the upstream of the promoter contains the Kozak sequence GCCACC.
  • the plasmids pCAGGS-MCC-RBD-tr3 and pCAGGS-MMCC-RBD-tr4 expressing heterotrimer and heterotetramer were obtained by molecular cloning.
  • Example 4 Expression and purification of MERS-RBD and SARS-CoV-2 RBD single-chain heterotrimeric and tetrameric proteins
  • HEK293F cells were used to express MCC-RBD-tr3 single-chain heterotrimer and MMCC-RBD-tr4 single-chain heterotetramer. Plasmids pCAGGS-MCC-RBD-tr3 and pCAGGS-MMCC-RBD-tr4 were transfected into HEK293F cells respectively. After 5 days, the supernatant was collected, centrifuged to remove the precipitate, and then filtered through a 0.22 ⁇ m filter membrane to further remove impurities.
  • the obtained cell supernatant was adsorbed by a nickel affinity column (His Trap, GE Healthcare) at 4°C, and washed with buffer A (20 mM Tris, 150 mM NaCl, pH 8.0) to remove non-specific binding proteins. Then use buffer B (20mM Tris, 150mM NaCl, pH 8.0, 300mM imidazole) to elute the target protein from His Trap, and use a 30kD concentrator tube to concentrate the eluate and change the solution more than 30 times to molecular sieve chromatography buffer PBS (8mM Na2HPO4, 136mM NaCl, 2mM KH2PO4 , 2.6mM KCl, pH7.2) The final volume was less than 1ml.
  • the target protein was further purified by molecular sieve chromatography on a Superdex TM 200 Increase 10/300GL column (GE Healthcare), and the molecular sieve chromatography buffer was PBS buffer.
  • MCC-RBD-tr3 (Fig. 2) and MMCC-RBD-tr4 (Fig. 3) had an elution peak at about 14ml and 13ml respectively.
  • SDS-PAGE analysis showed non-reduction (without DTT)
  • the sizes of MCC-RBD-tr3 and MMCC-RBD-tr4 proteins are about 90KD and 120KD respectively under reducing (plus DTT) conditions, and they are trimers and tetramers.
  • mice We mixed different antigen components with MF59-like adjuvant——SWE adjuvant (SEPPIC Company) to immunize mice.
  • the grouping of the mice is shown in Table 1.
  • MCC-RBD-tr3 and MMCC-RBD-tr4 were used as immunogens in the immune group set up in the mouse immunization experiment; PBS was used as a negative control; the positive control group was the homodimer of MERS-CoV and SARS-CoV-2 respectively.
  • Immunogens (MERS-RBD-tr2 and SARS2-RBD-tr2).
  • the BALB/c mice used in this application were purchased from Weitong Lihua Company, all of them were female, aged 6-8 weeks.
  • the immunization strategy is shown in Figure 4.
  • the antigenic protein was diluted to 200 ⁇ g/ml with PBS, and the MF59-like adjuvant and the immunogen were mixed and emulsified at a volume ratio of 1:1 to prepare a vaccine.
  • On the 19th day and the 35th day blood was collected from the mice and the serum was collected by centrifugation, stored in a -80°C refrigerator, and then used to titrate the titer of the antigen-specific antibody
  • Embodiment 6 Enzyme-linked immunosorbent assay (ELISA) detects the antigen-specific antibody titer that vaccine produces
  • MCC-RBD-tr3 and MMCC-RBD-tr4 can induce the production of specific IgG against MERS-RBD with a titer as high as 10 3 or more.
  • MMCC - RBD-tr4 group is significantly different from MERS-RBD-tr2 group (**p ⁇ 0.01)
  • MCC-RBD-tr3 group is significantly different from MERS-RBD-tr2 group (*p ⁇ 0.05); But there was no significant difference between the MCC-RBD-tr3 group and the MMCC-RBD-tr4 group.
  • MCC-RBD-tr3 induced an antigen-specific IgG titer above 1: 105
  • MMCC-RBD-tr4 induced Antigen-specific IgG titer above 1 :106
  • MCC-RBD-tr3 group and MMCC-RBD-tr4 group had significant difference compared with PBS group (****P ⁇ 0.0001), however compared with SARS2-RBD -tr2 group compared with no statistical difference.
  • MCC-RBD-tr3 and MMCC-RBD-tr4 can simultaneously induce mice to produce high levels of RBD-specific IgG against MERS-CoV and SARS-CoV-2, which is similar to that induced by homodimers at the same dose.
  • Antibody levels produced were comparable (SARS-CoV-2), or even better (MERS-CoV).
  • Example 7 MC-RBD-tr2 and MMCC-RBD-tr4 protein immunization experiments on mice
  • mice In order to further compare the immunogenicity of MMCC-RBD-tr4 heterodimer and MC-RBD-tr2 heterodimer, we mixed different antigen components with SWE adjuvant and immunized mice. The grouping of the mice is shown in Table 2.
  • the immune group set up in the mouse immunization experiment used MC-RBD-tr2 and MMCC-RBD-tr4 as immunogens; PBS was used as a negative control; the positive control group was a homodimer of SARS-CoV-2 as an immunogen (SARS2- RBD-tr2).
  • mice used in this application were purchased from Weitong Lihua Company, all of them were female, aged 6-8 weeks.
  • the immunization strategy is the same as in Example 5. On the 35th day, blood was collected from the mice and centrifuged to collect serum, stored in a -80°C refrigerator, and then used for titration of neutralizing antibody titers.
  • Embodiment 8 the pseudovirus neutralization experiment of immune serum
  • the MC-RBD-tr2 group induced a weaker level of neutralizing antibodies (*p ⁇ 0.05), but the level of neutralizing antibodies produced by the multimeric MMCC-RBD-tr4 was comparable to that of the SARS2-RBD-tr2 group.
  • the homodimers were equivalent (ns), which indicated that MMCC-RBD-tr4 had better immunogenicity than MC-RBD-tr2 and stimulated higher levels of neutralizing antibodies.
  • Example 7 the serum after two immunizations in Example 7 was used to carry out the neutralization experiment of the true virus of the original strain of SARS-CoV-2.
  • mice induced mice to produce neutralizing antibody titers above 1:1413, in contrast, MMCC-RBD-tr4 induced neutralizing antibody titers above 1:2100, significantly higher than MC-RBD-tr2 group; these results indicate that, compared with MC-RBD-tr2, MMCC-RBD-tr4 can induce mice to produce higher anti-SARS- Neutralizing antibodies to CoV-2.
  • Example 10 Live virus challenge protection experiment and RT-qPCR experiment to detect viral load in lung tissue
  • mice immunized twice in Example 7 the adenovirus expressing hACE2 was infected by nasal drip, and the receptor hACE2 of SARS-CoV-2 was transiently expressed in the lungs, thereby making the mice susceptible to SARS-CoV-2 ;
  • challenge with 5x 10 5 TCID 50 of the original strain live virus of SARS-CoV-2 hCoV-19/China/CAS-B001/2020, GISAID No.EPI_ISL_514256-7
  • 3 days after the challenge The mice were dissected, the lung tissue was taken, and the supernatant was homogenized after grinding to extract viral RNA.
  • a quantitative PCR experiment was used to detect the viral load (sgRNA). Specifically, 5 ⁇ l of nucleic acid was taken to prepare a PCR reaction system, and a real-time fluorescent RT-PCR reaction was performed on a Bio-Rad fluorescent quantitative PCR instrument.
  • the primer sequences are as follows:
  • the forward primer sequence is: CGATCTCTTGTAGATCTGTTCTC (SEQ ID NO: 10);
  • the reverse primer sequence is: ATATTGCAGCAGTACGCACACA (SEQ ID NO: 11);
  • the fluorescent probe sequence is: FAM-ACACTAGCCATCCTTACTGCGCTTCG(SEQ ID NO:12)-TAMRA;
  • the qRT-PCR experiment used the FastKing one-step reverse transcription-fluorescence quantitative kit (probe method, product number FP314) of Tiangen Biochemical Technology Co., Ltd., and the experimental operation was carried out according to the kit instruction method; the reaction parameters were: 50°C 30min, 95°C A cycle of 3 minutes; 95°C for 15s, 60°C for 30s, cycle 40 times, and collect fluorescence signals after extension.
  • the kit instruction method the reaction parameters were: 50°C 30min, 95°C A cycle of 3 minutes; 95°C for 15s, 60°C for 30s, cycle 40 times, and collect fluorescence signals after extension.
  • Figure 10 shows that after 3 days of challenge with the live virus of the new coronavirus, the viral load in the lung tissue of 6 mice out of 7 mice in the MMCC-RBD-tr4 group was cleared, that is, the virus cleared The zero rate was about 86%; while only 2 and 1 mice in the SARS2-RBD-tr2 group and MC-RBD-tr2 group had their lung tissue viral loads cleared, respectively, and their virus cleared rates were about 29% and 14%.
  • This result shows that MMCC-RBD-tr4 has a very significant protective effect against SARS-CoV-2 live virus challenge as a vaccine, and the protective effect is significantly better than MC-RBD-tr2 and SARS2-RBD-tr2.
  • the application provides a betacoronavirus heteromultimer antigen, its preparation method and application.
  • the ⁇ -coronavirus heteromultimer antigen of the present application can not only be stably expressed, but also induce a strong immune response against a variety of ⁇ -coronaviruses after immunization of mice, and only one antigenic protein can achieve multivalent The immune effect of the vaccine, therefore, can be used in the preparation of a betacoronavirus vaccine.

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Abstract

La présente invention concerne un antigène hétéromultimère de coronavirus bêta, son procédé de préparation et son utilisation. L'antigène hétéromultimère de coronavirus bêta a une séquence d'acides aminés comprenant une pluralité de monomères provenant d'un coronavirus bêta qui sont liés l'un à l'autre, chaque monomère du coronavirus bêta étant une séquence d'acides aminés partielle ou une séquence d'acides aminés entière d'une région de liaison au récepteur d'une protéine de spicule de coronavirus bêta ; le nombre de monomères est un nombre entier supérieur ou égal à trois ; et la pluralité de monomères de l'antigène hétéromultimère de coronavirus bêta comprenant des monomères provenant de deux coronavirus bêta hétérologues ou de monomères à partir d'au moins trois coronavirus bêta hétérogènes. Le multimère RBD de coronavirus bêta peut être exprimé de manière stable, et peut induire une forte réponse immunitaire contre divers coronavirus bêta après immunisation de souris, de sorte qu'une seule protéine d'antigène puisse atteindre l'effet immunitaire d'un vaccin multivalent.
PCT/CN2022/114892 2021-08-26 2022-08-25 Antigène hétéromultimère de coronavirus bêta, son procédé de préparation et son utilisation WO2023025257A1 (fr)

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