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WO2023025257A1 - 一种β冠状病毒异源多聚体抗原、其制备方法和应用 - Google Patents

一种β冠状病毒异源多聚体抗原、其制备方法和应用 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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PCT/CN2022/114892
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English (en)
French (fr)
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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
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    • 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
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    • 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
    • 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

涉及一种β冠状病毒异源多聚体抗原、其制备方法和应用，该β冠状病毒异源多聚体抗原的氨基酸序列包括：多个相互连接的、来自于β冠状病毒的单体，每个来自于β冠状病毒的单体为β冠状病毒刺突蛋白的受体结合区的部分氨基酸序列或全部氨基酸序列，单体的数目为≥3的整数，所述β冠状病毒异源多聚体抗原的多个单体中包括来自于2个异源β冠状病毒的单体或来自于3个以上异源β冠状病毒的单体。所述β冠状病毒RBD多聚体可以稳定的表达，免疫小鼠后可以诱导强烈的对多种β冠状病毒的免疫反应，仅一种抗原蛋白就可实现多价疫苗的免疫效果。

Description

一种β冠状病毒异源多聚体抗原、其制备方法和应用
交叉引用
本申请要求于2021年8月26日提交的、申请号为202110987719.9、发明名称为“一种β冠状病毒异源多聚体抗原、其制备方法和应用”的发明专利申请的优先权益，其全部内容通过引用并入本文。
技术领域
本申请涉及生物医药领域，具体涉及一种β冠状病毒异源多聚体抗原、其制备方法和应用。
背景技术
冠状病毒科含有4个冠状病毒属，分别是α、β、γ、δ。严重急性呼吸综合征冠状病毒(SARS-CoV)、中东呼吸综合征冠状病毒(MERS-CoV)以及新型冠状病毒(2019-nCoV，后又被命名为SARS-CoV-2)都是属于β冠状病毒属。它们都是正链RNA囊膜病毒，能够广泛感染人和动物，感染人后可以导致严重疾病甚至死亡。新冠病毒通过人血管紧张素转换酶2(human angiotensin-converting enzyme 2，hACE2)进入细胞，hACE2受体在动静脉内皮细胞，动脉平滑肌细胞，肠上皮细胞和肺泡，支气管等呼吸系统器官都有分布，病毒可以感染这些含有hACE2受体的细胞。此外，还有一些冠状病毒可以感染人类，包括β冠状病毒属的HCoV-OC43和HKU1，α冠状病毒属的HCoV-NL63和HCoV-229E，这些冠状病毒感染人后症状相对较轻。
MERS病例主要集中在中东和欧洲，中东以外国家的确诊病例发病前多有中东地区工作史或旅居史，死亡率高达约40％。除人传人的病例外，MERS-CoV主要由单峰驼传播给人，中东地区饲养着大量的单峰驼，有研究报道MERS-CoV已经在沙特的单峰驼中形成地方性流行，因此MERS-CoV具有较大的传播风险，目前尚没有疫苗和药物使用。当前，新冠病毒在全球范围内大流行，造成了严重危害。新发和再发传染病日益频繁，因此，开发一种冠状病毒疫苗抗原设计方法，实现一个抗原可以高效预防多种病毒，具有极大的应用价值。
β冠状病毒的包膜上主要有3种糖蛋白：S蛋白(刺突蛋白，Spike protein，S)、E蛋白(囊膜蛋白，Envelope protein，E)蛋白和M蛋白(膜蛋白，Membrane protein，M)。S蛋白和冠状病毒入侵细胞的过程密切相关，而且是疫苗研发中重要抗原，可以产生中和抗体。其中S蛋白的受体结合区(receptor binding domain，RBD)是机体诱导产生中和抗体的最主要的抗原靶区 域。
发明内容
发明目的
本申请的目的在于提供一种β冠状病毒异源多聚体抗原、其制备方法和应用。本申请基于β冠状病毒的RBD蛋白能激发机体产生中和抗体，且二聚体RBD蛋白比单体RBD蛋白能更有效的激发机体免疫反应的结论，通过将3个以上β冠状病毒异源单体串联起来，获得了一种β冠状病毒异源多聚体抗原，并使用该抗原获得了相应疫苗。该疫苗能够刺激小鼠产生强烈的对多种β冠状病毒的抗体反应，实现一种抗原高效预防多种病毒的效果。
一种β冠状病毒异源多聚体抗原，其氨基酸序列包括：多个相互连接的、来自于β冠状病毒的单体，每个来自于β冠状病毒的单体为β冠状病毒刺突蛋白的受体结合区的部分氨基酸序列或全部氨基酸序列，单体的数目为≥3的整数，所述β冠状病毒异源多聚体抗原的多个单体中包括来自于2个异源β冠状病毒的单体或来自于3个以上异源β冠状病毒的单体。
上述β冠状病毒异源多聚体抗原在一种可能的实现方式中，β冠状病毒为严重呼吸综合征冠状病毒、中东呼吸综合征冠状病毒或2019新型冠状病毒。
上述β冠状病毒异源多聚体抗原在一种可能的实现方式中，所述β冠状病毒异源多聚体抗原的多个单体中包括来自于中东呼吸综合征冠状病毒的单体和来自于2019新型冠状病毒的单体。
上述β冠状病毒异源多聚体抗原在一种可能的实现方式中，单体的数目为3个或4个。
上述β冠状病毒异源多聚体抗原在一种可能的实现方式中，当所述β冠状病毒异源多聚体抗原的多个单体中包括2个或3个以上来自于同源β冠状病毒的单体时，来自于同源β冠状病毒的单体先相互连接，再与来自于异源β冠状病毒的单体连接。
上述β冠状病毒异源多聚体抗原在一种可能的实现方式中，单体之间直接串联或通过连接氨基酸序列进行串联；可选地，不同位置的连接氨基酸序列相互独立地选自以下序列：(GGS)n连接序列，其中n表示GGS的个数，n为≥1的整数；进一步可选地，n为选自1-10的整数；更进一步可选地，n为选自1-5的整数。GGS三个字母分别表示氨基酸G、G、S。不同位置的连接氨基酸序列相互独立是指：连接从N端起第一个和第二个单体的连接氨基酸序列可以不同于连接从N端起第二个和第三个单体的连接氨基酸序列，依此类推。
上述β冠状病毒异源多聚体抗原在一种可能的实现方式中，当所述β冠状病毒异源多聚体抗原的多个单体中包括2个或3个以上来自于同源β冠状病毒的单体时，相互连接的、来自于同源β冠状病毒的单体的氨基酸序列为完全相同的序列。即同源串联的序列之间完全相同，为多 个重复序列。当然，来自于同源β冠状病毒的单体并不一定完全相同，如来自于同源β冠状病毒的单体可以一个较长、一个较短。
上述β冠状病毒异源多聚体抗原在一种可能的实现方式中，β冠状病毒的刺突蛋白的受体结合区的部分氨基酸序列为β冠状病毒的刺突蛋白的受体结合区的全部氨基酸序列的至少50％、60％、70％、80％、90％、95％、99％。
上述β冠状病毒异源多聚体抗原在一种可能的实现方式中，多个相互连接的、来自于异源β冠状病毒的单体为：
一个来自于中东呼吸综合征冠状病毒的单体与相互串联的两个来自于2019新型冠状病毒的单体相互连接。即：中东呼吸综合征冠状病毒的单体-2019新型冠状病毒的单体-2019新型冠状病毒的单体；
或，相互串联的两个来自于中东呼吸综合征冠状病毒的单体与相互串联的两个来自于2019新型冠状病毒的单体相互连接。即：中东呼吸综合征冠状病毒的单体-中东呼吸综合征冠状病毒的单体-2019新型冠状病毒的单体-2019新型冠状病毒的单体。
上述β冠状病毒异源多聚体抗原在一种可能的实现方式中，β冠状病毒异源抗原的氨基酸序列包括选自以下氨基酸序列的任意一种：
序列如SEQ ID NO:7所示，即：一个中东呼吸综合征冠状病毒RBD 367-602位氨基酸与两个2019新型冠状病毒RBD 319-537位氨基酸直接串联；
序列如SEQ ID NO:8所示，即：两个中东呼吸综合征冠状病毒RBD 367-602位氨基酸与两个2019新型冠状病毒RBD 319-537位氨基酸直接串联。
其中：中东呼吸综合征冠状病毒RBD的367-602区域来源于MERS-CoV的刺突蛋白序列(NCBI上的GenBank：AFS88936.1)的E367-N602区域；2019新型冠状病毒RBD的319-537区域来源于2019新型冠状病毒的WH01株的S刺突蛋白序列(NCBI上的GenBank：YP_009724390)的R319-K537区域。
本申请还提供了一种制备上述β冠状病毒异源多聚体抗原的方法，包括以下步骤：在编码上述β冠状病毒异源多聚体抗原的核苷酸序列的5’端加入编码信号肽的序列，3’端加上编码组氨酸标签的序列以及终止密码子，进行克隆表达，筛选正确的重组子，然后转染表达系统的细胞进行表达，表达后收集细胞上清，纯化得到β冠状病毒异源多聚体抗原。
上述方法在一种可能的实现方式中，所述表达系统的细胞包括为哺乳动物细胞、昆虫细胞、酵母细胞或细菌细胞，可选地；所述哺乳动物细胞包括HEK 293T细胞、HEK 293F细胞或CHO细胞，所述细菌细胞包括大肠杆菌细胞。
本申请还提供了一种编码上述β冠状病毒异源多聚体抗原的多核苷酸、一种包括上述多核 苷酸的重组载体、一种包括上述重组载体的表达系统细胞。
本申请还提供了一种上述β冠状病毒异源多聚体抗原、编码上述β冠状病毒异源多聚体抗原的多核苷酸、包括上述多核苷酸的重组载体或包括上述重组载体的表达系统细胞在制备β冠状病毒疫苗中的应用。
本申请还提供了一种β冠状病毒疫苗，包括上述β冠状病毒多聚体抗原和佐剂。
上述β冠状病毒疫苗在一种可能的实现方式中，所述佐剂选自铝佐剂、MF59佐剂、类MF59佐剂。
本申请还提供了一种β冠状病毒DNA疫苗，其包括有：包含编码上述β冠状病毒异源多聚体抗原的DNA序列的重组载体。
本申请还提供了一种β冠状病毒mRNA疫苗，其包括有：包含编码上述β冠状病毒异源多聚体抗原的mRNA序列的重组载体。
本申请还提供了一种β冠状病毒病毒载体疫苗，其包括有：包含编码上述β冠状病毒异源多聚体抗原的核苷酸序列的重组病毒载体；可选地，病毒载体选自以下的一种或几种：腺病毒载体、痘病毒载体、流感病毒载体、腺相关病毒载体、水疱性口炎病毒载体(Vesicular Stomatitis Virus，VSV)。
附图说明：
图1为本申请实施例1中的MERS-RBD和SARS-CoV-2-RBD串联二聚体(MC-RBD-tr2)抗原蛋白进行分子筛分析与凝胶电泳分析；
图2为本申请实施例4中的单链异源三聚体抗原蛋白进行分子筛分析与凝胶电泳分析；
图3为本申请实施例4中的单链异源四聚体抗原蛋白进行分子筛分析与凝胶电泳分析；
图4为本申请实施例5中各组免疫小鼠实验的免疫策略示意图；
图5为本申请实施例5中的小鼠免疫后19天的血清按照实施例6中的ELISA方法检测到的针对MERS-RBD的特异性IgG结合抗体水平的结果图；
图6为本申请实施例5中的小鼠免疫后19天的血清按照实施例6中的ELISA方法检测到的针对SARS-CoV-2-RBD的特异性IgG结合抗体水平的结果图；
图7为本申请实施例5中的小鼠免疫后35天的血清按照实施例6中的ELISA方法检测到的针对MERS-RBD的特异性IgG结合抗体水平的结果图；
图8为本申请实施例5中的小鼠免疫后35天的血清按照实施例6中的ELISA方法检测到的针对SARS2-RBD的特异性IgG结合抗体水平的结果图；
图9为本申请实施例7中获得的免疫后第35天的小鼠血清通过假病毒中和试验(实施例7)检测的抗SARS-CoV-2的中和抗体结果图，如实施例8中所检测的；
图10为本申请实施例10中，通过SARS-CoV-2活病毒攻毒保护试验和RT-qPCR实验，检测SARS-CoV-2活病毒攻毒后的肺组织病毒载量结果图；其中，纵坐标为每克肺组织中新冠病毒sgRNA的拷贝数，横坐标显示了免疫组类别。
具体实施方式
新冠病毒在全球范围内大流行，造成了严重危害。新发和再发传染病日益频繁，因此，开发一种冠状病毒疫苗抗原设计方法，实现一个抗原可以高效预防多种病毒，具有极大的应用价值。在该研究中，我们首先选择了新冠病毒和MERS-CoV。MERS具有较高的死亡率，可以人传人，也可以由骆驼传播给人，中东地区饲养着大量的单峰驼，有研究报道MERS-CoV已经在沙特的单峰驼中形成地方性流行，因此MERS-CoV具有较大的传播风险。我们以新冠病毒和MERS-CoV设计多价抗原作为模型，开发新型的疫苗设计方法。在前期研究中，我们发现了基于新冠病毒的单链二聚体RBD蛋白制备的亚单位疫苗比单体RBD蛋白疫苗具有更高的免疫原性，可以诱导产生更好的抗体水平(PMID:32645327)。为了进一步提高疫苗效果和实现同时预防多种冠状病毒，我们设计了来源于不同病毒的异源RBD多聚体抗原，检测得到的蛋白是否可以比二聚体RBD蛋白免疫动物后诱导更强的免疫反应，以及对多种病毒同时具有较好的效果，用于疫苗研发。
实施例1：MERS-CoV RBD和SARS-CoV-2 RBD嵌合二聚体(MC-RBD-tr2)蛋白的表达纯化
把MERS-RBD(367位-602位)氨基酸序列与SARS-CoV-2-RBD(319位-537位)氨基酸串联，命名为MC-RBD-tr2(其序列如SEQ ID NO:1所示)，在该序列的C端加上6个组氨酸，N端连接MERS-S蛋白自身信号肽(MIHSVFLLMFLLTPTES，SEQ ID NO:2)。将编码带有MERS-S蛋白自身信号肽和组氨酸的MC-RBD-tr2的核苷酸序列插入到pCAGGS载体的EcoRI和XhoI酶切位点(上述核苷酸序列如SEQ ID NO:3所示，该核苷酸序列包括了编码组氨酸和信号肽的序列)。启动子上游含有Kozak序列GCCGCCACC。通过分子克隆得到表达异源二聚体的质粒pCAGGS-MC-RBD-tr2。将该质粒转染HEK293F细胞，5天后收集上清，离心去除沉淀再通过0.22μm的滤膜过滤，进一步除去杂质。将所得细胞上清在4℃通过镍亲和柱(His Trap,GE Healthcare)吸附，用缓冲液A(20mM Tris,150mM NaCl,pH 8.0)洗涤，除去非特 异结合蛋白。然后用缓冲液B(20mM Tris,150mM NaCl,pH 8.0,300mM咪唑)将目的蛋白从His Trap上洗脱下来，并用30kD浓缩管将洗脱液浓缩换液30倍以上至分子筛层析缓冲液PBS(8mM Na 2HPO4,136mM NaCl,2mM KH 2PO4,2.6mM KCl,pH7.2)终体积小于1ml。再通过Superdex TM200 Increase 10/300GL柱子(GE Healthcare)进行分子筛层析进一步纯化目的蛋白。分子筛层析缓冲液为PBS缓冲液。经过分子筛层析，典型的分子筛层析图如图1所示：取洗脱体积14ml附近的洗脱峰进行SDS-PAGE分析，目的蛋白在非还原条件下(不加DTT)和还原条件下(加DTT)大小约为60Kd，证明该峰是二聚体(单体的大小为30Kd)。取洗脱体积16ml附近的洗脱峰进行SDS-PAGE分析，目的蛋白在非还原条件下(不加DTT)和还原条件下(加DTT)大小约为30kD，证明该峰主要是RBD单体。
实施例2：串联重复RBD二聚体蛋白的表达纯化
为了与串联重复RBD二聚体抗原作对比，分别构建表达单链重复二聚体MERS-RBD-tr2(两个重复的MERS-RBD 367位-602位氨基酸序列直接串联，其氨基酸序列如SEQ ID NO:4所示)、SARS2-RBD-tr2(两个重复的SARS-CoV-2-RBD 319位-537位氨基酸序列直接串联，其氨基酸序列如SEQ ID NO:5所示)的质粒。在以上序列的C端加上6个组氨酸，N端连接MERS-S蛋白自身信号肽(MIHSVFLLMFLLTPTES，SEQ ID NO:2)。将编码带有MERS-S蛋白自身信号肽和组氨酸的MERS-RBD-tr2或SARS-CoV-2-RBD-tr2的核苷酸序列插入到pCAGGS载体EcoRI和XhoI酶切位点。启动子上游含有Kozak序列GCCACC。通过分子克隆得到表达重复二聚体的质粒pCAGGS-MERS-RBD-tr2、pCAGGS-SARS-CoV-2-RBD-tr2(具体步骤详见Dai,L.,et al.,A Universal Design of Betacoronavirus Vaccines against COVID-19,MERS,and SARS.Cell,2020.182(3):p.722-733e11.)。
实施例3：MERS-RBD、SARS-CoV-2 RBD单链异源多聚体的设计及制备
MERS-RBD和SARS-CoV-2-RBD的N端和C端各有一段柔性序列，我们尝试将三个或四个异源RBD亚基之间串联起来获得RBD单链异源三聚体或RBD单链异源四聚体，以期诱导产生既针对MERS-CoV、又针对SARS-CoV-2两种冠状病毒的抗体反应。
我们设计了如下单链异源三聚体和单链异源四聚体的构建：
(1)把一个MERS-RBD部分序列367位-602位氨基酸与两个SARS-CoV-2-RBD部分序列 319位-537位氨基酸串联，命名为MCC-RBD-tr3(其氨基酸序列如SEQ ID NO:6所示)；
(2)把两个MERS-RBD部分序列367位-602位氨基酸与两个SARS-CoV-2-RBD部分序列319位-537位氨基酸串联，命名为MMCC-RBD-tr4(其氨基酸序列如SEQ ID NO:7所示)。
将编码带有N端连接信号肽(MERS-S蛋白自身信号肽MIHSVFLLMFLLTPTES，SEQ ID NO:2)和C端6个组氨酸的MCC-RBD-tr3或MMCC-RBD-tr4的DNA序列插入到pCAGGS载体的EcoRI和XhoI酶切位点之间(编码带有N端连接信号肽和组氨酸的MCC-RBD-tr3的核苷酸序列如SEQ ID NO:8所示、编码带有N端连接信号肽和组氨酸的MMCC-RBD-tr4的核苷酸序列如SEQ ID NO:9所示)。启动子上游含有Kozak序列GCCACC。通过分子克隆得到表达异源三聚体和异源四聚体的质粒pCAGGS-MCC-RBD-tr3、pCAGGS-MMCC-RBD-tr4。
实施例4：MERS-RBD和SARS-CoV-2 RBD单链异源三聚体和四聚体蛋白的表达纯化
使用HEK293F细胞表达MCC-RBD-tr3单链异源三聚体和MMCC-RBD-tr4单链异源四聚体。将质粒pCAGGS-MCC-RBD-tr3、pCAGGS-MMCC-RBD-tr4分别转染HEK293F细胞，5天后收集上清，离心去除沉淀再通过0.22μm的滤膜过滤，进一步除去杂质。将所得细胞上清在4℃通过镍亲和柱(His Trap,GE Healthcare)吸附，用缓冲液A(20mM Tris,150mM NaCl,pH 8.0)洗涤，除去非特异结合蛋白。然后用缓冲液B(20mM Tris,150mM NaCl,pH 8.0,300mM咪唑)将目的蛋白从His Trap上洗脱下来，并用30kD浓缩管将洗脱液浓缩换液30倍以上至分子筛层析缓冲液PBS(8mM Na 2HPO4,136mM NaCl,2mM KH 2PO4,2.6mM KCl,pH7.2)终体积小于1ml。再通过Superdex TM200 Increase 10/300GL柱子(GE Healthcare)进行分子筛层析进一步纯化目的蛋白，分子筛层析缓冲液为PBS缓冲液。经过分子筛层析，MCC-RBD-tr3(图2)、MMCC-RBD-tr4(图3)分别在14ml和13ml左右有一个洗脱峰，进行SDS-PAGE分析，显示非还原(不加DTT)和还原(加DTT)条件下MCC-RBD-tr3和MMCC-RBD-tr4蛋白大小分别约为90KD和120KD左右，为三聚体和四聚体。
由图2、图3可见，MCC-RBD-tr3和MMCC-RBD-tr4都能够正确的折叠和分泌出来，且通过纯化能够获得高纯度的抗原蛋白。
实施例5：MCC-RBD-tr3和MMCC-RBD-tr4蛋白免疫小鼠实验
我们将不同的抗原组分与类MF59佐剂——SWE佐剂(SEPPIC公司)进行混合后免疫小鼠。小鼠分组情况如表1。小鼠免疫实验设置的免疫组使用MCC-RBD-tr3和MMCC-RBD-tr4 作为免疫原；PBS作为阴性对照；阳性对照组分别是MERS-CoV和SARS-CoV-2的同源二聚体作为免疫原(MERS-RBD-tr2和SARS2-RBD-tr2)。
表1冠状病毒RBD单链异源三聚体和四聚体疫苗免疫小鼠分组及剂量
Figure PCTCN2022114892-appb-000001
本申请所使用的BALB/c小鼠从维通利华公司购买，均为雌性，6-8周龄。免疫策略如图4。将抗原蛋白用PBS稀释至200μg/ml，类MF59佐剂与免疫原按照体积比1:1的比例混合乳化制备成疫苗。混合后的疫苗对BALB/c小鼠进行免疫，采用肌肉注射的方式，所有小鼠分别在第0天、第21天接受2次疫苗免疫，每次100μl的接种体积(其中50μl抗原与50μl佐剂混合，200μg/ml*50μl＝10μg)。第19天和第35天对小鼠进行取血离心收集血清，于-80℃冰箱保存，之后用于滴定抗原特异性抗体滴度。
实施例6：酶联免疫吸附试验(ELISA)检测疫苗产生的抗原特异性抗体滴度
(1)将MERS-CoV或者SARS-CoV-2的RBD单体蛋白用ELISA包被液(索莱宝,C1050)稀释至3μg/ml，96孔ELISA板(Coring，3590)每孔加入100μl，4℃放置12小时。
(2)倒掉包被液，加入PBS，洗一遍。使用PBS配置的5％脱脂牛奶作为封闭液，加入96孔板中，每孔100μl，封闭，室温放置1小时。封闭完后用PBS溶液洗一遍。
(3)封闭期间稀释小鼠血清。血清样品也用封闭液稀释。血清样品从20倍起始按照4倍梯度依次稀释。第一个孔加入152μl封闭液和8μl的血清混匀，第二个稀释度为封闭液120μl和第一个孔的溶液40μl混匀，依次稀释。稀释完之后在ELISA板中每孔加入100μl，阴性对照为加入封闭液，37度孵育1.5小时，之后使用PBST洗4遍。
(4)加入使用封闭液1:2000稀释的偶联HRP的羊抗鼠二抗(Abcam，ab6789)，37℃孵育1.5小时，之后PBST洗5-6遍。加入60μl TMB显色液显色，反应适当时间后加入60μl 2M盐酸终止反应，在酶标仪上检测OD450读值。抗体滴度值被定义为反应值大于2.5倍阴性对照值的血清最高稀释倍数。当最低稀释倍数(检测限)的反应值仍小于2.5倍背景值时，该样品的滴度定义为最低稀释倍数的一半即1:10。
结果分析：
一次免疫后的血清针对MERS-RBD的ELISA结果如图5，MCC-RBD-tr3和MMCC-RBD-tr4均能诱导产生针对MERS-RBD的特异性IgG，滴度高达10 3以上，其中，MMCC-RBD-tr4组 与MERS-RBD-tr2组相比有显著差异(**p<0.01)，MCC-RBD-tr3组与MERS-RBD-tr2组相比有显著差异(*p<0.05)；但是MCC-RBD-tr3组与MMCC-RBD-tr4组没有显著差异。
一次免疫后的血清针对SARS-CoV-2-RBD的ELISA结果如图6，MCC-RBD-tr3和MMCC-RBD-tr4均能诱导产生针对SARS-CoV-2-RBD的特异性IgG，滴度高达10 3以上，与SARS2-RBD-tr2组相比都有显著差异(*p<0.05)。
两次免疫后的血清针对MERS-RBD的ELISA结果如图7，MCC-RBD-tr3和MMCC-RBD-tr4均能诱导产生了1:10 5以上的抗原特异性IgG滴度。MMCC-RBD-tr4组与MERS-RBD-tr2组相比有显著差异(*p<0.05)。
两次免疫后的血清针对SARS-CoV-2-RBD的ELISA结果如图8，MCC-RBD-tr3诱导产生了1:10 5以上的抗原特异性IgG滴度，MMCC-RBD-tr4诱导产生了1:10 6以上的抗原特异性IgG滴度；MCC-RBD-tr3组和MMCC-RBD-tr4组与PBS组相比都有显著差异(****P<0.0001)，然而与SARS2-RBD-tr2组相比没有统计学差异。
以上结果说明，MCC-RBD-tr3和MMCC-RBD-tr4能同时诱导小鼠产生针对MERS-CoV和SARS-CoV-2的高水平的RBD特异性IgG，与相同剂量的同源二聚体诱导产生的抗体水平相当(SARS-CoV-2)，甚至更好(MERS-CoV)。
实施例7：MC-RBD-tr2和MMCC-RBD-tr4蛋白免疫小鼠实验
为了进一步比较MMCC-RBD-tr4异源多聚体与MC-RBD-tr2异源二聚体的免疫原性，我们将不同的抗原组分与SWE佐剂进行混合后免疫小鼠。小鼠分组情况如表2。小鼠免疫实验设置的免疫组使用MC-RBD-tr2和MMCC-RBD-tr4作为免疫原；PBS作为阴性对照；阳性对照组是SARS-CoV-2的同源二聚体作为免疫原(SARS2-RBD-tr2)。
表2冠状病毒RBD单链异源三聚体和四聚体疫苗免疫小鼠分组及剂量
Figure PCTCN2022114892-appb-000002
本申请所使用的BALB/c小鼠从维通利华公司购买，均为雌性，6-8周龄。免疫策略同实施例5。第35天对小鼠进行取血离心收集血清，于-80℃冰箱保存，之后用于滴定中和抗体滴度。
实施例8：免疫血清的假病毒中和实验
我们将实施例7中获得的免疫后第35天(即两次免疫后14天)的血清倍比稀释(20倍起始，按2倍梯度进行稀释)，所得系列稀释液分别与100TCID 50的SARS-CoV-2假病毒(按照Jianhui Nie et al.Establishment and validation of a pseudovirus neutralization assay for SARS-CoV-2,Emerging Microbes&Infections,2020,VOL.9中描述的方法制得)混合，37℃共孵育1小时。将混合液加入到已铺满Huh7细胞的96孔板中。24小时弃掉培养液后加入细胞裂解液，检测荧光素酶活性值。
两次免疫后血清针对SARS-CoV-2的中和抗体水平如图9所示，图9结果显示：SARS-CoV-2RBD-tr2、MC-RBD-tr2和MMCC-RBD-tr4都能诱导小鼠产生1:10 3以上(90％中和效价，pVNT 90)的、针对SARS-CoV-2的中和抗体，且MMCC-RBD-tr4较MC-RBD-tr2组诱导产生的中和抗体水平显著提高(*p<0.05)。此外，与SARS2-RBD-tr2组相比，MC-RBD-tr2组诱导产生中和抗体水平较弱(*p<0.05)，但是多聚体的MMCC-RBD-tr4产生中和抗体的水平与同源二聚体相当(ns)，这一结果说明了MMCC-RBD-tr4较MC-RBD-tr2具有更好的免疫原性，刺激产生更高水平的中和抗体。
实施例9：免疫血清的SARS-CoV-2原始毒株真病毒中和
本实施例中，采用实施例7中两次免疫后的血清，进行SARS-CoV-2原始毒株真病毒的中和实验。
具体地，我们将实施例7中获得的免疫后第35天(即两次免疫后14天)的血清进行倍比稀释(20倍起始，按2倍梯度进行稀释)，所得系列稀释液分别与100TCID 50的SARS-CoV-2真病毒(hCoV-19/China/CAS-B001/2020,GISAID No.EPI_ISL_514256-7)混合，37℃共孵育1小时；将混合液加入到已铺满Vero-E6细胞的96孔板中；72小时后，显微镜下观察细胞病变(cytopathic effect,CPE)。该实验在中科院微生物所生物安全3级实验室(BSL3)完成。
结果显示：MC-RBD-tr2诱导小鼠产生1:1413以上滴度的中和抗体，相比之下，MMCC-RBD-tr4诱导产生的中和抗体滴度在1:2100以上，显著高于MC-RBD-tr2组；这些结果说明，通过SARS-CoV-2真病毒中和实验证实了，与MC-RBD-tr2相比，MMCC-RBD-tr4能够诱导小鼠产生更高的抗SARS-CoV-2的中和抗体。
实施例10：活病毒攻毒保护实验和RT-qPCR实验检测肺组织病毒载量
对实施例7中两次免疫后的小鼠，通过滴鼻感染表达hACE2的腺病毒，在肺部瞬时表达SARS-CoV-2的受体hACE2，从而使得小鼠对SARS-CoV-2易感；5天后，用5x 10 5TCID 50 的SARS-CoV-2原始毒株活病毒(hCoV-19/China/CAS-B001/2020,GISAID No.EPI_ISL_514256-7)攻毒；攻毒后3天，将小鼠解剖，取肺组织，研磨后取上清匀浆，提取病毒RNA。采用定量PCR实验，检测病毒载量(sgRNA)，具体地，取核酸5μl配制PCR反应体系，在Bio-Rad荧光定量PCR仪上进行实时荧光RT-PCR反应。引物序列如下：
正向引物序列是：CGATCTCTTGTAGATCTGTTCTC(SEQ ID NO:10)；
反向引物序列是：ATATTGCAGCAGTACGCACACA(SEQ ID NO:11)；
荧光探针序列是：FAM-ACACTAGCCATCCTTACTGCGCTTCG(SEQ ID NO:12)-TAMRA；
qRT-PCR实验使用天根生化科技公司的FastKing一步法反转录-荧光定量试剂盒(探针法，货号FP314)，按照试剂盒说明书方法进行实验操作；反应参数为：50℃ 30min、95℃ 3min一个循环；95℃ 15s、60℃ 30s，循环40次，延伸后采集荧光信号。
实验结果如图10所示，图10显示：新冠病毒活病毒攻毒3天后，MMCC-RBD-tr4组7只小鼠中有6只小鼠的肺组织病毒载量清零，即，病毒清零率为大约86％；而SARS2-RBD-tr2组和MC-RBD-tr2组分别仅有2只和1只小鼠的肺组织病毒载量清零，其病毒清零率分别为约29％和14％。这一结果表明，MMCC-RBD-tr4作为疫苗有着非常显著的、针对SARS-CoV-2活毒攻毒的保护效果，且保护效果明显优于MC-RBD-tr2和SARS2-RBD-tr2。
最后应说明的是：以上实施例仅用以说明本申请的技术方案，而非对其限制；尽管参照前述实施例对本申请进行了详细的说明，本领域的普通技术人员应当理解：其依然可以对前述各实施例所记载的技术方案进行修改，或者对其中部分技术特征进行等同替换；而这些修改或者替换，并不使相应技术方案的本质脱离本申请各实施例技术方案的精神和范围。
工业实用性
本申请提供了一种β冠状病毒异源多聚体抗原、其制备方法和应用。本申请的β冠状病毒异源多聚体抗原不仅可以稳定地表达，在其免疫小鼠后还可以诱导强烈的、针对多种β冠状病毒的免疫反应，仅一种抗原蛋白就可实现多价疫苗的免疫效果，因此，可用于β冠状病毒的疫苗制备。

Claims (21)

  1. 一种β冠状病毒异源多聚体抗原，其特征在于：氨基酸序列包括：多个相互连接的、来自于β冠状病毒的单体，每个来自于β冠状病毒的单体为β冠状病毒刺突蛋白的受体结合区的部分氨基酸序列或全部氨基酸序列，单体的数目为≥3的整数，所述β冠状病毒异源多聚体抗原的多个单体中包括来自于2个异源β冠状病毒的单体或来自于3个以上异源β冠状病毒的单体。
  2. 根据权利要求1所述的β冠状病毒异源多聚体抗原，其特征在于：β冠状病毒为严重呼吸综合征冠状病毒、中东呼吸综合征冠状病毒或2019新型冠状病毒。
  3. 根据权利要求1所述的β冠状病毒异源多聚体抗原，其特征在于：所述β冠状病毒异源多聚体抗原的多个单体中包括来自于中东呼吸综合征冠状病毒的单体和来自于2019新型冠状病毒的单体。
  4. 根据权利要求1所述的β冠状病毒异源多聚体抗原，其特征在于：单体的数目为3个或4个。
  5. 根据权利要求1所述的β冠状病毒异源多聚体抗原，其特征在于：当所述β冠状病毒异源多聚体抗原的多个单体中包括2个或3个以上来自于同源β冠状病毒的单体时，来自于同源β冠状病毒的单体先相互连接，再与来自于异源β冠状病毒的单体连接。
  6. 根据权利要求1所述的β冠状病毒异源多聚体抗原，其特征在于：单体之间直接串联或通过连接氨基酸序列进行串联；可选地，不同位置的连接氨基酸序列相互独立地选自以下序列：(GGS)n连接序列，其中n表示GGS的个数，n为≥1的整数；进一步可选地，n为选自1-10的整数；更进一步可选地，n为选自1-5的整数。
  7. 根据权利要求5所述的β冠状病毒异源多聚体抗原，其特征在于：当所述β冠状病毒异源多聚体抗原的多个单体中包括2个或3个以上来自于同源β冠状病毒的单体时，相互连接的、来自于同源β冠状病毒的单体的氨基酸序列为完全相同的序列。
  8. 根据权利要求1所述的β冠状病毒异源多聚体抗原，其特征在于：β冠状病毒的刺突蛋白的受体结合区的部分氨基酸序列为β冠状病毒的刺突蛋白的受体结合区的全部氨基酸序列的至少50％、60％、70％、80％、90％、95％、99％。
  9. 根据权利要求1所述的β冠状病毒异源多聚体抗原，其特征在于：多个相互连接的、来自于异源β冠状病毒的单体为：一个来自于中东呼吸综合征冠状病毒的单体与相互串联的两个来自于2019新型冠状病毒的单体相互连接；
    或，多个相互连接的、来自于异源β冠状病毒的单体为：相互串联的两个来自于中东呼吸综合征冠状病毒的单体与相互串联的两个来自于2019新型冠状病毒的单体相互连接。
  10. 根据权利要求1所述的β冠状病毒异源多聚体抗原，其特征在于：β冠状病毒异源抗原的氨基酸序列包括选自以下氨基酸序列的任意一种：
    序列如SEQ ID NO:7所示；
    序列如SEQ ID NO:8所示。
  11. 一种制备上述β冠状病毒异源多聚体抗原的方法，其特征在于：包括以下步骤：在编码上述β冠状病毒异源多聚体抗原的核苷酸序列的5’端加入编码信号肽的序列，3’端加上编码组氨酸标签的序列以及终止密码子，进行克隆表达，筛选正确的重组子，然后转染表达系统的细胞进行表达，表达后收集细胞上清，纯化得到β冠状病毒异源多聚体抗原。
  12. 根据权利要求11所述的方法，其特征在于：所述表达系统的细胞包括为哺乳动物细胞、昆虫细胞、酵母细胞或细菌细胞，可选地；所述哺乳动物细胞包括HEK 293T细胞、HEK 293F细胞或CHO细胞，所述细菌细胞包括大肠杆菌细胞。
  13. 一种编码权利要求1-10之一所述的β冠状病毒异源多聚体抗原的多核苷酸。
  14. 一种包括权利要求13所述的多核苷酸的重组载体。
  15. 一种包括权利要求14所述的重组载体的表达系统细胞。
  16. 一种权利要求1-10之一所述的β冠状病毒异源多聚体抗原、权利要求13所述的多核苷酸、权利要求14所述的重组载体或权利要求15所述的表达系统细胞在制备β冠状病毒疫苗中的应用。
  17. 一种β冠状病毒疫苗，其特征在于：包括权利要求1-10之一所述的β冠状病毒异源多聚体抗原和佐剂。
  18. 根据权利要求17所述的β冠状病毒疫苗，其特征在于：所述佐剂选自铝佐剂、MF59佐剂或类MF59佐剂。
  19. 一种β冠状病毒DNA疫苗，其特征在于：包括有：包含编码权利要求1-10之一所述的β冠状病毒异源多聚体抗原的DNA序列的重组载体。
  20. 一种β冠状病毒mRNA疫苗，其特征在于：包括有：包含编码权利要求1-10之一所述的β冠状病毒异源多聚体抗原的mRNA序列的重组载体。
  21. 一种β冠状病毒病毒载体疫苗，其特征在于：包括有：包含编码权利要求1-10之一所述的β冠状病毒异源多聚体抗原的核苷酸序列的重组病毒载体；可选地，病毒载体选自以下的一种或几种：腺病毒载体、痘病毒载体、流感病毒载体、腺相关病毒载体、水疱性口炎病毒载体(Vesicular Stomatitis Virus，VSV)。
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