WO2021239147A1 - 一种β冠状病毒抗原、β冠状病毒二联疫苗及其制备方法和应用 - Google Patents

一种β冠状病毒抗原、β冠状病毒二联疫苗及其制备方法和应用 Download PDF

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WO2021239147A1
WO2021239147A1 PCT/CN2021/097463 CN2021097463W WO2021239147A1 WO 2021239147 A1 WO2021239147 A1 WO 2021239147A1 CN 2021097463 W CN2021097463 W CN 2021097463W WO 2021239147 A1 WO2021239147 A1 WO 2021239147A1
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coronavirus
antigen
amino acid
acid sequence
vaccine
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高福
戴连攀
郑天依
韩雨旋
徐坤
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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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P11/00—Drugs for disorders of the respiratory system
    • 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
    • A61K2039/54—Medicinal preparations containing antigens or antibodies characterised by the route of administration
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/70—Multivalent vaccine
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2770/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses positive-sense
    • C12N2770/00011—Details
    • C12N2770/20011—Coronaviridae
    • C12N2770/20034—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein

Definitions

  • the invention relates to the field of biomedicine, in particular to a ⁇ -coronavirus antigen and ⁇ -coronavirus dual vaccine, and a preparation method and application thereof.
  • Coronaviruses belong to the family of Coronaviruses, which contains 4 genera of Coronaviruses. Severe Acute Respiratory Syndrome Coronavirus (SARS-CoV), Middle East Respiratory Syndrome Coronavirus (MERS-CoV) and New Coronavirus (2019-nCoV) (hereinafter referred to as nCoV) belong to the ⁇ -coronavirus genus, ⁇ -coronavirus The genus contains 4 subgroups (A, B, C, D), SARS-CoV and nCoV belong to the B subgroup, and MERS-CoV belongs to the C subgroup. They are all positive-strand RNA enveloped viruses, which can infect humans and animals widely. At present, there are seven types of coronaviruses that can infect humans.
  • SARS-CoV SARS-CoV
  • MERS-CoV MERS-CoV
  • nCoV which belong to the ⁇ -coronavirus genus
  • the surface spike protein (S protein) is the main neutralizing antigen of coronavirus.
  • the receptor binding domain (RBD) of the S protein of MERS-CoV, SARS-CoV, and 2019-nCoV is considered to be the most important antigen target area that induces the body to produce neutralizing antibodies.
  • RBD can focus the neutralizing antibodies produced by the body's stimulation on the receptor binding of the virus, which can improve the immunogenicity and immune efficiency of the vaccine.
  • MERS-CoV invades cells through the binding of RBD to the host cell receptor (CD26, also known as DPP4).
  • CD26 also known as DPP4
  • both SARS-CoV and 2019-nCoV are found to enter cells through their RBD binding to the host cell receptor ACE2.
  • the purpose of the present invention is to provide a ⁇ -coronavirus antigen and ⁇ -coronavirus dual vaccine, and a preparation method and application thereof.
  • the present invention is based on the conclusion that the RBD proteins of nCoV, MERS-CoV, and SARS-CoV can stimulate the body to produce neutralizing antibodies, and that the dimer RBD protein can more effectively stimulate the body’s immune response than the monomer RBD protein.
  • NCoV-RBD and SARS-RBD are connected in series to obtain a ⁇ -coronavirus antigen, and use the ⁇ -coronavirus antigen to obtain a ⁇ -coronavirus dual vaccine, which can stimulate mice to produce strong Compared with using a ⁇ -coronavirus RBD monomer alone as the antigen in the vaccine, it can stimulate mice to produce a more pronounced and strong antibody response.
  • an embodiment of the present invention provides a ⁇ -coronavirus antigen.
  • the amino acid sequence of the ⁇ -coronavirus antigen in the order from N-terminal to C-terminal includes: according to (AB)-(A'-B') Amino acid sequence arranged in a pattern or an amino acid sequence arranged in a (AB)-C-(A'-B') pattern, where: AB represents a partial or all amino acid sequence from the receptor binding region of the surface spike protein of ⁇ coronavirus Sequence, A'-B' represents a partial or all amino acid sequence of the receptor binding region of the surface spike protein from another ⁇ coronavirus, C represents the linking amino acid sequence, and the ⁇ coronavirus antigen is a single-stranded isoform Source dimer structure.
  • the "species” in “another species” is not the species in the "genus, genus, and species” in taxonomy, but it just means that AB and A'-B' come from the ⁇ -coronavirus genus with different nomenclature Coronavirus, this expression also echoes "heterogeneous”. For example, when A-B comes from SARS-CoV, A’-B’ cannot also come from SARS-CoV, but A’-B’ can come from nCoV and MERS-CoV of the ⁇ -coronavirus genus.
  • the sequence to the C-terminus can be either: a partial amino acid sequence or the entire amino acid sequence of the receptor binding region of the surface spike protein from nCoV-(C, if necessary)-the receptor binding region of the surface spike protein from SARS-CoV Part of the amino acid sequence or all of the amino acid sequence of, can also be: the partial amino acid sequence or the entire amino acid sequence of the receptor binding region of the surface spike protein from SARS-CoV-(C, if necessary)-the surface spike protein from nCoV Part of the amino acid sequence or the entire amino acid sequence of the receptor binding region. And so on.
  • the beta coronavirus is selected from: severe respiratory syndrome coronavirus, Middle East respiratory syndrome coronavirus or new coronavirus.
  • the A-B sequence is derived from Middle East respiratory syndrome coronavirus
  • the A’-B’ sequence is derived from severe respiratory syndrome coronavirus
  • the A-B sequence is from the Middle East Respiratory Syndrome coronavirus, and the A’-B’ sequence is from the new coronavirus;
  • the A-B sequence is from the new coronavirus
  • the A’-B’ sequence is from the severe respiratory syndrome coronavirus.
  • the partial amino acid sequence of the receptor binding region of the surface spike protein from the Middle East respiratory syndrome coronavirus is SEQ ID NO. 7 or SEQ ID NO. 13. These two sequences are derived from the MERS-CoV S protein (sequence such as GenBank: AFS88936.1) RBD (367-606) and (367-602) sequences. When selecting the RBD(367-602) sequence, the protein expression can be increased by preventing the mismatch of the disulfide bond of the cysteine at position 603.
  • the partial amino acid sequence of the receptor binding region of the surface spike protein from the novel coronavirus is SEQ ID NO. 8 or SEQ ID NO. 14. These two sequences are derived from the S protein sequence of the WH01 strain of 2019-nCoV (sequence such as GenBank: YP_009724390) RBD (319-541) and (319-537) sequences. When selecting the RBD(319-537) sequence, the protein expression can be increased by preventing the mismatch of the disulfide bond of the cysteine at position 538.
  • the partial amino acid sequence of the receptor binding region of the surface spike protein from the severe respiratory syndrome coronavirus is SEQ ID NO. 9 or SEQ ID NO. 15. These two sequences are derived from the SARS-CoV S protein (sequence such as GenBank: NP_828851) RBD (306-527) and (306-523) sequences. When selecting the RBD(306-523) sequence, the protein expression can be increased by preventing the mismatch of the disulfide bond of the cysteine at position 524.
  • the amino acid sequence of the beta coronavirus antigen is selected from: SEQ ID NO. 1, SEQ ID NO. 2 or SEQ ID NO. 3.
  • the embodiment of the present invention also provides a method for preparing the above-mentioned ⁇ -coronavirus antigen, which includes the following steps: adding a sequence encoding a signal peptide to the 5'end of the nucleotide sequence encoding the above-mentioned ⁇ -coronavirus antigen, and adding a coding sequence to the 3'end.
  • the histidine sequence is 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 the ⁇ -coronavirus antigen.
  • the cells of the expression system include mammalian cells, insect cells, yeast cells, or bacterial cells, optionally; the mammalian cells include 293T cells or CHO cells, and the bacterial cells Including E. coli cells.
  • the embodiment of the present invention also provides a polynucleotide encoding the above-mentioned ⁇ -coronavirus antigen.
  • the embodiment of the present invention also provides a recombinant vector including the above-mentioned polynucleotide.
  • the embodiment of the present invention also provides an expression system cell including the aforementioned recombinant vector; preferably, the expression system cell is a mammalian cell, and further preferably, the mammalian cell is a 293T cell or a CHO cell.
  • the embodiment of the present invention also provides the above-mentioned ⁇ -coronavirus antigen, the polynucleotide encoding the above-mentioned ⁇ -coronavirus antigen, the recombinant vector including the above-mentioned polynucleotide, and the expression system cells including the above-mentioned recombinant vector are used in the preparation of the ⁇ -coronavirus dual vaccine Applications.
  • the above-mentioned ⁇ -coronavirus dual vaccine includes: Middle East respiratory syndrome coronavirus-severe respiratory syndrome coronavirus dual vaccine, new coronavirus-severe respiratory syndrome coronavirus dual vaccine, Middle East Respiratory Syndrome Coronavirus-New Coronavirus Combination Vaccine.
  • the embodiment of the present invention also provides a ⁇ -coronavirus dual vaccine, including the above-mentioned ⁇ -coronavirus antigen and an adjuvant.
  • the above-mentioned ⁇ -coronavirus dual vaccine includes: Middle East respiratory syndrome coronavirus-severe respiratory syndrome coronavirus dual vaccine, new coronavirus-severe respiratory syndrome coronavirus dual vaccine, Middle East Respiratory Syndrome Coronavirus-New Coronavirus Combination Vaccine.
  • the adjuvant is selected from aluminum adjuvant, MF59 adjuvant and MF59-like adjuvant.
  • the volume ratio of the aforementioned beta coronavirus antigen and adjuvant is 1:1-2; alternatively, it is 1:1.
  • the present invention also provides a ⁇ -coronavirus dual DNA vaccine, which contains a DNA sequence encoding the above-mentioned ⁇ -coronavirus antigen.
  • the present invention also provides a ⁇ -coronavirus dual mRNA vaccine, which comprises an mRNA sequence encoding the above-mentioned ⁇ -coronavirus antigen.
  • the present invention also provides a ⁇ -coronavirus dual virus vector vaccine, which comprises a polynucleotide encoding the above-mentioned ⁇ -coronavirus antigen; optionally, the virus vector is selected from one or more of the following: adenovirus vector, pox Viral vectors, influenza virus vectors, adeno-associated virus vectors.
  • the ⁇ -coronavirus antigen in the embodiment of the present invention is used as the antigen in the ⁇ -coronavirus dual vaccine, which can produce two kinds of antibodies, which reduces the number of vaccine injections by the public and enhances compliance.
  • the ⁇ -coronavirus antigen in the examples of the present invention is used as the antigen in the ⁇ -coronavirus dual vaccine. Compared with using two ⁇ -coronavirus RBD monomers separately as the antigen in the vaccine, it can stimulate mice to produce more significant Strong antibody response. For example, when the ⁇ -coronavirus antigen contains both MERS RBD and nCoV RBD, it can produce higher levels of anti-MERS RBD and nCoV RBD than when using MERS RBD monomer and nCoV RBD monomer separately as vaccine antigens. nCoV RBD specific antibody level.
  • Figure 1 is a Western blot of the coronavirus RBD dual vaccines MERS-SARS, MERS-nCoV, and nCoV-SARS in Example 1 of the present invention.
  • Figure 2 shows the molecular sieve analysis and gel electrophoresis analysis of the MERS-nCoV dual vaccine antigen in Example 2 of the present invention.
  • Figure 3 shows the molecular sieve analysis and gel electrophoresis analysis of the nCoV-SARS dual vaccine antigen in Example 2 of the present invention.
  • Figure 4 shows the molecular sieve analysis and gel electrophoresis analysis of the MERS-SARS dual vaccine antigen in Example 2 of the present invention.
  • Figure 5 is a schematic diagram of the mouse immunization process in Example 3 of the present invention.
  • Fig. 6 shows the serum nCoV-RBD-specific antibody titers induced on the 19th day after the mice are immunized with the dual vaccine in Example 4 of the present invention.
  • Figure 7 shows the serum MERS-RBD-specific antibody titers induced on the 19th day after the mice are immunized with the dual vaccine in Example 4 of the present invention.
  • Fig. 8 shows the serum SARS-RBD-specific antibody titers induced on the 19th day after the mice are immunized with the dual vaccine in Example 4 of the present invention.
  • Figure 9 shows the serum nCoV-RBD specific antibody titers induced on the 33rd day after the mice are immunized with the dual vaccine in Example 4 of the present invention.
  • Fig. 10 shows the serum MERS-RBD-specific antibody titers induced on the 33rd day after the mice are immunized with the dual vaccine in Example 4 of the present invention.
  • Figure 11 shows the serum SARS-RBD-specific antibody titers induced on the 33rd day after the mice are immunized with the dual vaccine in Example 4 of the present invention.
  • Fig. 12 is the detection result of serum neutralizing antibody titers against the new coronavirus nCoV after the secondary immunization of mice with the dual vaccine in Example 5 of the present invention.
  • Fig. 13 is the detection result of serum neutralizing antibody titers against MERS coronavirus after the secondary immunization of mice with the dual vaccine in Example 6 of the present invention.
  • Figure 14 shows the qRT-PCR experiment in Example 8 of the present invention to detect the total viral load (including replication-competent live virus, residual dead virus, or dead virus) in the lungs of mice 5 days after virus challenge (5dpi) Viral genome fragment).
  • Fig. 15 shows the viral load of the replication-capable live virus in the lungs of mice 5 days (5dpi) after virus challenge by qRT-PCR experiment in Example 8 of the present invention.
  • Fig. 16 shows the detection result of serum neutralizing antibody titers against MERS coronavirus after the secondary immunization of rhesus monkeys with dual vaccine in Example 10 of the present invention.
  • Figure 17 is the detection result of serum neutralizing antibody titers against the new coronavirus nCoV after the secondary immunization of rhesus monkeys with dual vaccine in Example 10 of the present invention.
  • the MERS-RBD (367-602) and (367-606) sequences in the following examples are derived from the MERS-CoV S protein (sequence such as GenBank: AFS88936.1).
  • the SARS-RBD (306-523) and (306-527) sequences are derived from the SARS-CoV S protein (sequence such as GenBank: NP_828851).
  • the nCoV-RBD (319-537) and (319-541) sequences are derived from the S protein sequence of the WH01 strain of 2019-nCoV (such as GenBank: YP_009724390).
  • Example 1 Design and preparation of single-chain heterodimers of MERS, SARS, nCoV RBD
  • MERS-nCoV MERS-nCoV NO.1
  • SEQ ID NO.1 MERS-nCoV-RBD 319-537
  • SEQ ID NO.2 Concatenate the partial sequence of nCoV-RBD (319-537) with the partial sequence of SARS-RBD (306-523), named nCoV-SARS (SEQ ID NO.2)
  • MERS-RBD partial sequence 367-602 and SARS-RBD partial sequence (306-523) in series, named MERS-SARS (SEQ ID NO.3).
  • the upstream of the promoter contains the Kozak sequence gccacc.
  • the plasmids pCAGGS-MERS-nCoV, pCAGGS-nCoV-SARS and pCAGGS-MERS-SARS expressing three heterodimers were obtained by molecular cloning.
  • the above plasmids were transfected into 293T cells. After 48 hours, the supernatant was taken. The N-terminus of the target protein had a signal peptide.
  • the expression of the target protein was detected by Western Blot. The Western Blot results are shown in Figure 1.
  • the expression cells can all stably express MERS-nCoV, nCoV-SARS and MERS-SARS heterodimer RBD protein.
  • the monomer MERS-RBD (367-606) (SEQ ID NO. 7), nCoV-RBD (319-541) (SEQ ID NO. 8) and SARS-RBD ( 306-527) (SEQ ID NO. 9) plasmid.
  • the MERS-RBD (367-606), nCoV-RBD (319-541) and SARS-RBD (306-527) genes were added to the 5'end to encode the signal peptide sequence, and the 3'end was added to encode 6 histidines
  • the sequence (SEQ ID NO.10, SEQ ID NO.11, SEQ ID NO.12, this sequence does not include the sequence encoding histidine and signal peptide), cloned into the pCAGGS vector EcoRI and XhoI restriction sites, The upstream of the promoter contains the Kozak sequence gccacc.
  • the plasmids pCAGGS-MERS-RBD, pCAGGS-nCoV-RBD, and pCAGGS-SARS-RBD expressing three monomeric RBD protein antigens were obtained by molecular cloning.
  • Example 2 Expression and purification of single-chain heterodimer RBD protein of MERS, SARS, nCoV
  • HEK293T cells were used to express MERS-nCoV, nCoV-SARS and MERS-SARS single-chain heterodimer RBD proteins.
  • the plasmids pCAGGS-MERS-nCoV, pCAGGS-nCoV-SARS and pCAGGS-MERS-SARS were respectively transfected into HEK293T cells. After 72 hours, the supernatant was collected, centrifuged to remove the precipitate and filtered through a 0.22 ⁇ m filter membrane to further remove impurities. The cell supernatant was adsorbed through a nickel affinity column (Histrap, GE Healthcare) at 4°C.
  • the target protein was further purified by molecular sieve chromatography on Superdex TM 200Increase 10/300GL column (GE Healthcare).
  • the molecular sieve chromatography buffer is PBS buffer (8mM Na 2 HPO4, 136 mM NaCl, 2 mM KH 2 PO 4 , 2.6 mM KCl, pH 7.2).
  • MERS-nCoV Figure 2
  • nCoV-SARS Figure 3
  • MERS-SARS Figure 4
  • SDS-PAGE analysis shows that non-reducing ( Under the conditions of no DTT and reduction (with DTT), the protein is about 62KD, which is a dimer.
  • Example 3 Experiment of immunizing mice with MERS-nCoV and nCoV-SARS protein
  • mice with the purified single-chain heterodimer protein were purchased from Weitong Lihua Company, all females, 6-8 weeks old.
  • the grouping of mice (5 mice per group) and vaccine dose are shown in Table 1.
  • Mouse immunization experiment settings dual vaccine group, which uses MERS-nCoV and nCoV-SARS (obtained in Example 2) as immunogens to immunize mice; single vaccine control group, which uses MERS-CoV and SARS-CoV, respectively
  • the RBD monomer protein of the S protein of the three viruses and nCoV were used as immunogens to immunize mice; the negative control group used PBS to treat the mice.
  • the immunogen was diluted with PBS to 200 ⁇ g/ml, and the MF59-like adjuvant-AddaVax TM and the immunogen were mixed and emulsified according to a volume ratio of 1:1 to prepare a vaccine.
  • the mixed vaccine was used to immunize BALB/c mice with 5 mice in each group.
  • the experimental procedure of mice is shown in Figure 5.
  • blood was collected from mice by centrifugation and the serum was stored in a refrigerator at -80°C, and then used to titrate antigen-specific antibody titers and pseudovirus neutralization titers.
  • Example 4 Enzyme-linked immunosorbent assay (ELISA) to detect the titer of antigen-specific antibodies produced by vaccines
  • Figure 6 shows the ELISA results of immune serum against the new coronavirus nCoV-RBD.
  • the MERS-nCoV dual vaccine induced an antigen-specific IgG titer of more than 1:1000, which was significantly higher than the level of specific antibodies induced by the MERS-RBD monomer (***P ⁇ 0.001), and compared with the nCoV-RBD single
  • the level of specific antibodies induced by the body was significantly increased (****P ⁇ 0.0001), which was significantly higher than the level of specific antibodies induced by the PBS control immunization group (****P ⁇ 0.0001).
  • the level of specific antibodies against nCoV-RBD induced by the MERS-nCoV dual vaccine was also significantly higher than that of nCoV-SARS (***P ⁇ 0.001), as shown in Figure 6.
  • the ELISA results of the immune serum against the new coronavirus MERS-RBD are shown in Figure 7.
  • the MERS-nCoV dual vaccine induced an antigen-specific IgG titer of more than 1:5000.
  • the level of specific antibody induced by nCoV-RBD monomer was significantly increased (****P ⁇ 0.0001), compared with The level of specific antibodies induced by the PBS control immunization group was significantly increased (****P ⁇ 0.0001), as shown in Figure 7.
  • the ELISA results of the immune serum against the new coronavirus SARS-RBD are shown in Figure 8.
  • the nCoV-SARS dual vaccine induces an antigen-specific IgG titer of more than 1:500, which is significantly higher than the specific antibody levels induced by nCoV-RBD monomer, SARS-RBD and PBS (*P ⁇ 0.05; **P ⁇ 0.01).
  • mice were immunized for the second time on the 21st day, and the serum after the second immunization was collected on the 33rd day.
  • the antibody titers of the mouse serum on the 33rd day (ie, after the second immunization) of the ELISA experiment are shown in Figures 9, 10 and 11.
  • the results showed that RBD heterodimer induced higher levels of antibodies after immunization of mice, and there was a significant difference in the level of antibody response between RBD heterodimer and RBD monomer induced mice.
  • MERS-nCoV bivalent vaccine induced more than 5 1:10 antigen-specific IgG titers than MERS-RBD monomer induced specific antibody levels significantly increased (**** P ⁇ 0.0001), compared nCoV-RBD
  • the level of specific antibodies induced by monomer was significantly increased (****P ⁇ 0.0001), which was significantly higher than that of the PBS control immunization group (****P ⁇ 0.0001).
  • the level of specific antibodies against nCoV-RBD induced by the MERS-nCoV dual vaccine was also significantly higher than that of nCoV-SARS (****P ⁇ 0.0001).
  • MERS-nCoV bivalent vaccine antigen specific IgG titers induced nearly 6 1:10, more MERS-RBD monomer induced specific antibody levels significantly increased (** P ⁇ 0.01), induced more monomers nCoV
  • the level of specific antibodies was significantly increased (**P ⁇ 0.01), which was significantly higher than the level of specific antibodies induced by the PBS control immunization group (**P ⁇ 0.01).
  • the ELISA results of the second immune mouse serum against the new coronavirus SARS-RBD are shown in Figure 11.
  • the secondary immunization of the nCoV-SARS dual vaccine induced an antigen-specific IgG titer of more than 1:10 5 , which was significantly higher than the specific antibody levels induced by the SARS RBD monomer, nCoV-RBD monomer and PBS group. (***P ⁇ 0.001;****P ⁇ 0.0001).
  • Example 5 Pseudovirus neutralization experiment detects the neutralizing antibody titer against the new coronavirus produced by the vaccine
  • the serum obtained in Example 3 was diluted in multiples, and the obtained serial dilutions were mixed with 100 TCID 50 pseudovirus respectively, and incubated at 37° C. for 1 hour. Add the mixture to a 96-well plate that has been contaminated with Huh7 cells. After incubating at 37°C for 24 hours, the culture medium was discarded, the cells were washed twice with PBS, and the cell lysate was added to detect the luciferase activity value.
  • the immunogenicity test results of the serum after the second immunization are shown in Figure 12.
  • the results of Figure 12 show that the MERS-nCoV dual vaccine produced neutralizing antibodies after the second immunization.
  • the neutralizing effect of mouse serum is 90%.
  • the price (NT 90 ) can reach more than 1:1000.
  • only four of the eight mice immunized with nCoV RBD monomer produced weak neutralizing antibodies (two of them had NT 90 of 1:20 and the other two had NT 90 of 1:40).
  • Example 6 Pseudovirus neutralization experiment to detect the neutralizing antibody titer of the anti-MERS tubular virus produced by the vaccine
  • the serum obtained in Example 3 was diluted in multiples, and the obtained serial dilutions were mixed with 100 TCID 50 pseudovirus respectively, and incubated at 37° C. for 1 hour. Add the mixture to a 96-well plate that has been contaminated with Huh7 cells. After 48 hours of incubation at 37°C, the culture medium was discarded, the cells were washed twice with PBS, and the cell lysate was added to detect the luciferase activity value.
  • the immunogenicity test results of the serum after the second immunization are shown in Figure 13, and the results of Figure 13 show that the MERS-nCoV dual vaccine produced neutralizing antibodies after the second immunization, and NT 90 can reach more than 1:1000;
  • the level of neutralizing antibody induced by MERS RBD homodimer is equivalent (ns). This result shows that the MERS-nCoV dual vaccine can induce a high level of antibody response in mice through pseudovirus neutralization experiments and is a good candidate for MERS-CoV vaccine.
  • mice Thirty-two BALB/c mice were randomly divided into 4 groups with 8 mice in each group. All BALB/c mice were purchased from Weitong Lihua Company, all females, 6-8 weeks old.
  • Mouse immunization experiment settings the dual vaccine group, which uses the MERS-nCoV dual vaccine (obtained in Example 2) as the immunogen to immunize mice; the single vaccine control group, which uses the RBD single of the S protein of the new coronavirus Somatic protein and RBD homodimer protein were used as immunogens to immunize mice; negative control group, which used PBS to treat mice.
  • the vaccine dose for immunization is the same as that in Table 1 in Example 3.
  • mice 39 days after the second immunization (ie, 74 days after the first immunization), we infect mice with Ad5-ACE2-deficient adenovirus by intranasal drip to induce the expression of human ACE2 receptor protein in the lungs of mice, and then The mice were transferred to the BSL-3 laboratory of the Institute of Microbiology (A). On the 79th day after immunization, the mice were intranasally infected with the new coronavirus (hCoV-19/China/CAS-B001/2020 (GISAID databases EPI_ISL_514256-7) virus strain). The dose of virus infection is: virus stock solution 50 ⁇ L/mouse. The route of challenge is nasal infection.
  • the specific operation is as follows: the diluted tribromoethanol (concentration of 20 mg/ml) is injected into the intraperitoneal cavity (i.p.) of the mouse for anesthesia, and 250 ⁇ L is injected into the intraperitoneal cavity of each mouse. After the mice are completely anesthetized, use a 200 ⁇ L pipette to suck up 50 ⁇ L of virus liquid to perform nasal drip operation on the mice.
  • mice Five days after the virus challenge, the mice were euthanized. Then, the lungs were dissected out, placed in a 2mL tube (weighed in advance), weighed, and the net weight of the lungs was calculated. Grind mouse lungs with a grinder, and then separate the supernatant. After virus inactivation of the obtained supernatant, it was taken out of the (A) BSL-3 laboratory. Use the kit (QIAamp Viral RNA Mini Kit) to extract viral RNA from the above supernatant.
  • kit QIAamp Viral RNA Mini Kit
  • Example 8 Using qRT-PCR experiment to detect the viral load in the lungs of mice 5 days (5dpi) after the new coronavirus challenge
  • the qRT-PCR method is used to detect the viral load in the lungs of mice after virus challenge;
  • the probes and primers used can be combined with the N gene of the new coronavirus genome, and their sequences are as follows:
  • N-F GACCCCAAAATCAGCGAAAT (SEQ ID NO: 16);
  • N-R TCTGGTTACTGCCAGTTGAATCTG (SEQ ID NO: 17);
  • N-probe ACCCCGCATTACGTTTGGTGGACC (SEQ ID NO: 18);
  • sgRNA-F CGATCTCTTGTAGATCTGTTCTC (SEQ ID NO: 19);
  • sgRNA-R TGTGTGCGTACTGCTGCAATAT (SEQ ID NO: 20);
  • sgRNA-probe ACACTAGCCATCCTTACTGCGCTTCG (SEQ ID NO: 21);
  • the results of qRT-PCR detection are shown in Figure 15.
  • the results of Figure 15 show that: in the MERS-nCoV dual vaccine group, no replicating virus was detected at all, while in the PBS negative control group and nCoV RBD monomer control group, replicating virus
  • the results show that the MERS-nCoV dual vaccine has a good protective effect on mice against new coronavirus infections.
  • Example 9 Conducting a protection experiment against virus challenge in a rhesus monkey model
  • the neutralizing antibody titers against the new coronavirus nCoV and MERS coronavirus in the rhesus monkey serum obtained in Example 9 were detected.
  • the serum immunogenicity test results against MERS coronavirus and nCoV new coronavirus are shown in Figure 16 and Figure 17, respectively; the results of Figure 16 and Figure 17 show that the MERS-nCoV vaccine immunization group after the second immunization , Produced neutralizing antibodies against the new coronavirus and anti-MERS coronavirus.
  • the NT 90 against the MERS coronavirus reached about 1:10 or more ( Figure 16), and the NT 90 against the new coronavirus could reach about 1:100 or more ( Figure 17); the neutralizing antibodies of the PBS control group are all negative.
  • the MERS-nCoV dual vaccine can induce neutralizing antibody response in rhesus monkeys through pseudovirus neutralization experiments, and it is a good dual candidate vaccine against the new coronavirus and MERS coronavirus.

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Abstract

本发明涉及一种β冠状病毒抗原、β冠状病毒二联疫苗及其制备方法和应用。所述β冠状病毒抗原的氨基酸序列按照从N端到C端的顺序包括：按照(A-B)-(A'-B')样式排列的氨基酸序列或(A-B)-C-(A'-B')样式排列的氨基酸序列，其中：A-B表示来自β冠状病毒的表面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，A'-B'表示来自另一种β冠状病毒的表面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，C表示连接氨基酸序列，所述β冠状病毒抗原为单链异源二聚体结构。使用该β冠状病毒抗原获得了一种β冠状病毒二联疫苗，该二联疫苗能够刺激小鼠产生强烈的抗体反应。

Description

一种β冠状病毒抗原、β冠状病毒二联疫苗及其制备方法和应用
交叉引用
本申请要求于2020年5月29日提交的、申请号为202010479851.4、发明名称为“一种β冠状病毒抗原、β冠状病毒二联疫苗及其制备方法和应用”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
本发明涉及生物医药领域，具体涉及一种β冠状病毒抗原、β冠状病毒二联疫苗及其制备方法和应用。
背景技术
冠状病毒属于冠状病毒科，冠状病毒科又含有4个冠状病毒属。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)(以下简称为nCoV)都是属于β冠状病毒属，β冠状病毒属含有4个亚群(A,B,C,D)，SARS-CoV和nCoV属于B亚群，MERS-CoV属于C亚群。它们都是正链RNA囊膜病毒，能够广泛感染人和动物，目前已鉴定出能够感染人类的冠状病毒共有七种，其中，同属于β冠状病毒属的SARS-CoV、MERS-CoV以及nCoV具有高致命性，在人类历史上引发了三次严重的疾病流行。因此，开发相应疫苗有着重要意义。
表面刺突蛋白(S蛋白)是冠状病毒的主要中和抗原。MERS-CoV、SARS-CoV、2019-nCoV的S蛋白的受体结合区(Receptor Binding Domain,RBD)被认为是诱导机体产生中和抗体的最主要的抗原靶区域。RBD作为疫苗能够将机体刺激产生的中和抗体更加聚焦在针对病毒的受体结合，可以提高疫苗的免疫原性和免疫效率。MERS-CoV通过RBD与宿主细胞的受体(CD26，又名DPP4)结合而侵入细胞。此外，SARS-CoV和2019-nCoV都发现通过其RBD与宿主细胞受体ACE2结合而进入细胞。
公开于该背景技术部分的信息仅仅旨在增加对本发明的总体背景的理解，而不应当被视为承认或以任何形式暗示该信息构成已为本领域一般技术人员所公知的现有技术。
发明内容
发明目的
本发明的目的在于提供一种β冠状病毒抗原、β冠状病毒二联疫苗及其制备方法和应用。本发明基于nCoV、MERS-CoV、SARS-CoV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MERS-RBD、nCoV-RBD和SARS-RBD两两串联起来，获得了一种β冠状病毒抗原，并使用该β冠状病毒抗原获得了一种β冠状病毒二联疫苗，该二联疫苗能够刺激小鼠产生强烈的抗体反应，并且相对于单独使用一种β冠状病毒RBD单体作为疫苗中的抗原能够刺激小鼠产生更显著强烈的抗体反应。
解决方案
为实现本发明目的，本发明实施例提供了一种β冠状病毒抗原，所述β冠状病毒抗原的氨基酸序列按照从N端到C端的顺序包括：按照(A-B)-(A’-B’)样式排列的氨基酸序列或(A-B)-C-(A’-B’)样式排列的氨基酸序列，其中：A-B表示来自β冠状病毒的表面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，A’-B’表示来自另一种β冠状病毒的表面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，C表示连接氨基酸序列，所述β冠状病毒抗原为单链异源二聚体结构。
此处，“另一种”中的“种”并非生物分类学中“界门纲目科属种”中的种，只是表示A-B和A’-B’是来自β冠状病毒属中具有不同命名的冠状病毒，该种表述也与“异源”相互呼应。如：当A-B来自于SARS-CoV时，A’-B’不能也来自于SARS-CoV，但A’-B’可以来自于β冠状病毒属的nCoV、MERS-CoV。
此处，当A-B和A’-B’的来源选定时，(A-B)-(A’-B’)样式排列或(A-B)-C-(A’-B’)样式排列按照从N端到C端的顺序既可以是：来自nCoV的表面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-(C，如果必要)-来自SARS-CoV的表面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，也可以是：来自SARS-CoV的表面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-(C，如果必要)-来自nCoV的表面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。以此类推。
在一种可能的实现方式中，β冠状病毒选自：严重呼吸综合征冠状病毒、中东呼吸综合征冠状病毒或新型冠状病毒。
在一种可能的实现方式中，A-B序列来自于中东呼吸综合征冠状病毒，A’-B’序列来自于严重呼吸综合征冠状病毒；
或，A-B序列来自于中东呼吸综合征冠状病毒，A’-B’序列来自于新型冠状病毒；
或，A-B序列来自于新型冠状病毒，A’-B’序列来自于严重呼吸综合征冠状病毒。
在一种可能的实现方式中，来自中东呼吸综合征冠状病毒的表面刺突蛋白的受体结合区的部分氨基酸序列为SEQ ID NO.7或SEQ ID NO.13。这两条序列分别为来自于MERS-CoV S蛋白(序列如GenBank:AFS88936.1)RBD(367-606)、(367-602)序列。当选择RBD(367-602)序列时可以通过防止603位的半胱氨酸发生二硫键错配，增加蛋白表达量。
在一种可能的实现方式中，来自新型冠状病毒的表面刺突蛋白的受体结合区的部分氨基酸序列为SEQ ID NO.8或SEQ ID NO.14。这两条序列分别来自于2019-nCoV的WH01株的S蛋白序列(序列如GenBank:YP_009724390)RBD(319-541)、(319-537)序列。当选择RBD(319-537)序列时可以通过防止538位的半胱氨酸发生二硫键错配，增加蛋白表达量。
在一种可能的实现方式中，来自严重呼吸综合征冠状病毒的表面刺突蛋白的受体结合区的部分氨基酸序列为SEQ ID NO.9或SEQ ID NO.15。这两条序列分别来自于SARS-CoV S蛋白(序列如GenBank:NP_828851)RBD(306-527)、(306-523)序列。当选择RBD(306-523)序列时可以通过防止524位的半胱氨酸发生二硫键错配，增加蛋白表达量。
在一种可能的实现方式中，β冠状病毒抗原的氨基酸序列选自：SEQ ID NO.1、SEQ ID NO.2或SEQ ID NO.3。
本发明实施例还提供了制备上述β冠状病毒抗原的方法，包括以下步骤：在编码上述的β冠状病毒抗原的核苷酸序列的5’端加入编码信号肽的序列，3’端加上编码组氨酸的序列，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冠状病毒抗原。
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293T细胞或CHO细胞，所述细菌细胞包括大肠杆菌细胞。
本发明实施例还提供了一种编码上述的β冠状病毒抗原的多核苷酸。
本发明实施例还提供了一种包括上述的多核苷酸的重组载体。
本发明实施例还提供了一种包括上述的重组载体的表达系统细胞；优选地，所述表达系统细胞为哺乳动物细胞，进一步优选地，所述哺乳动物细胞为293T细胞或CHO细胞。
本发明实施例还提供了上述β冠状病毒抗原、编码上述β冠状病毒抗原的多核苷酸、包括上述多核苷酸的重组载体、包括上述重组载体的表达系统细胞在制备β冠状病毒二联疫苗中的应用。
在一种可能的实现方式中，上述β冠状病毒二联疫苗包括：中东呼吸综合征冠状病毒-严重呼吸综合征冠状病毒二联疫苗、新型冠状病毒-严重呼吸综合征冠状病毒二联疫苗、中东呼吸综合征冠状病毒-新型冠状病毒二联疫苗。
本发明实施例还提供了一种β冠状病毒二联疫苗，包括上述的β冠状病毒抗原和佐剂。
在一种可能的实现方式中，上述β冠状病毒二联疫苗包括：中东呼吸综合征冠状病毒-严重呼吸综合征冠状病毒二联疫苗、新型冠状病毒-严重呼吸综合征冠状病毒二联疫苗、中东呼吸综合征冠状病毒-新型冠状病毒二联疫苗。
在一种可能的实现方式中，所述佐剂选自铝佐剂、MF59佐剂和类MF59佐剂。
在一种可能的实现方式中，上述的β冠状病毒抗原和佐剂的体积比为1：1-2；可选地，为1：1。
本发明还提供了一种β冠状病毒二联DNA疫苗，其包含编码上述β冠状病毒抗原的DNA序列。
本发明还提供了一种β冠状病毒二联mRNA疫苗，其包含编码上述β冠状病毒抗原的mRNA序列。
本发明还提供了一种β冠状病毒二联病毒载体疫苗，其包含编码上述β冠状病毒抗原的多核苷酸；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。
有益效果
(1)本发明实施例中的β冠状病毒抗原作为β冠状病毒二联疫苗中的抗原使用，可以产生两种抗体，减少了公众注射疫苗的次数，增强了依从性。
(2)本发明实施例中的β冠状病毒抗原作为β冠状病毒二联疫苗中的抗原使用，相对于分别单独使用两种β冠状病毒RBD单体作为疫苗中的抗原能够刺激小鼠产生更显著强烈的抗体反应，如当β冠状病毒抗原同时包含MERS RBD和nCoV RBD时，其相较于分别单独使用MERS RBD单体和nCoV RBD单体作为疫苗抗原时，能产生更高的针对MERS RBD和nCoV RBD的特异性抗体水平。
附图说明
一个或多个实施例通过与之对应的附图中的图片进行示例性说明，这些示例性说明并不构成对实施例的限定。在这里专用的词“示例性”意为“用作例子、实施例或说明性”。这里作为“示例性”所说明的任何实施例不必解释为优于或好于其它实施例。
图1是本发明实施例1中冠状病毒RBD二联苗MERS-SARS,MERS-nCoV,nCoV-SARS的Western Blot。
图2是本发明实施例2中MERS-nCoV二联苗抗原的分子筛分析与凝胶电泳分析。
图3是本发明实施例2中nCoV-SARS二联苗抗原的分子筛分析与凝胶电泳分析。
图4是本发明实施例2中MERS-SARS二联苗抗原的分子筛分析与凝胶电泳分析。
图5是本发明实施例3中小鼠免疫的过程示意图。
图6是本发明实施例4中二联苗免疫小鼠后第19天诱导产生的血清nCoV-RBD特异性抗体滴度。
图7是本发明实施例4中二联苗免疫小鼠后第19天诱导产生的血清MERS-RBD特异性抗体滴度。
图8是本发明实施例4中二联苗免疫小鼠后第19天诱导产生的血清SARS-RBD特异性抗体滴度。
图9是本发明实施例4中二联苗免疫小鼠后第33天诱导产生的血清nCoV-RBD特异性抗体滴度。
图10是本发明实施例4中二联苗免疫小鼠后第33天诱导产生的血清MERS-RBD特异性抗体滴度。
图11是本发明实施例4中二联苗免疫小鼠后第33天诱导产生的血清SARS-RBD特异性抗体滴度。
图12是本发明实施例5中二联苗二次免疫小鼠后针对新冠病毒nCoV的血清中和抗体滴度检测结果。
图13是本发明实施例6中二联苗二次免疫小鼠后针对MERS冠状病毒的血清中和抗体滴度检测结果。
图14是本发明实施例8中，通过qRT-PCR实验，检测病毒攻毒后5天(5dpi)小鼠肺脏中总的病毒载量(包括有复制能力的活病毒，残留的死病毒，或者病毒基因组片段)。
图15是本发明实施例8中，通过qRT-PCR实验，检测病毒攻毒后5天(5dpi)小鼠肺脏中有复制能力的活病毒的病毒载量。
图16是本发明实施例10中，二联苗二次免疫恒河猴后，针对MERS冠状病毒的血清中和抗体滴度检测结果。
图17是本发明实施例10中，二联苗二次免疫恒河猴后，针对新冠病毒nCoV的血清中和抗体滴度检测结果。
具体实施方式
为使本发明实施例的目的、技术方案和优点更加清楚，下面将对本发明实施例中的技术方案进行清楚、完整地描述，显然，所描述的实施例是本发明一部分实施例，而不是全部的 实施例。基于本发明中的实施例，本领域普通技术人员在没有作出创造性劳动前提下所获得的所有其他实施例，都属于本发明保护的范围。
另外，为了更好的说明本发明，在下文的具体实施方式中给出了众多的具体细节。本领域技术人员应当理解，没有某些具体细节，本发明同样可以实施。在一些实施例中，对于本领域技术人员熟知的原料、元件、方法、手段等未作详细描述，以便于凸显本发明的主旨。
除非另有其它明确表示，否则在整个说明书和权利要求书中，术语“包括”或其变换如“包含”或“包括有”等等将被理解为包括所陈述的元件或组成部分，而并未排除其它元件或其它组成部分。
以下实施例中MERS-RBD(367-602)、(367-606)序列来自于MERS-CoV S蛋白(序列如GenBank:AFS88936.1)。SARS-RBD(306-523)、(306-527)序列来自于SARS-CoV S蛋白(序列如GenBank:NP_828851)。nCoV-RBD(319-537)、(319-541)序列来自于2019-nCoV的WH01株的S蛋白序列(序列如GenBank:YP_009724390)。
实施例1：MERS、SARS、nCoV RBD单链异源二聚体的设计及制备
我们设计通过将两个异源RBD亚基之间串联起来，获得单链RBD异源二聚体，以期诱导同时产生针对两种冠状病毒的中和抗体。
我们设计了三种单链异源二聚体的构建：(1)把MERS-RBD部分序列(367-602)与nCoV-RBD部分序列(319-537)串联，命名为MERS-nCoV(SEQ ID NO.1)；(2)把nCoV-RBD部分序列(319-537)与SARS-RBD部分序列(306-523)串联，命名为nCoV-SARS(SEQ ID NO.2)；(3)把MERS-RBD部分序列(367-602)与SARS-RBD部分序列(306-523)串联，命名为MERS-SARS(SEQ ID NO.3)。将编码以下氨基酸序列的DNA序列插入到pCAGGS载体EcoRI和XhoI酶切位点之间：以上3条氨基酸序列C端加上6个组氨酸、N端连接信号肽(MIHSVFLLMFLLTPTES)(SEQ ID NO.4、SEQ ID NO.5、SEQ ID NO.6，该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gccacc。通过分子克隆得到表达三种异源二聚体的质粒pCAGGS-MERS-nCoV、pCAGGS-nCoV-SARS和pCAGGS-MERS-SARS。
以上质粒转染293T细胞，48小时后，取上清，目的蛋白N端带有信号肽，通过蛋白印迹法(Western Blot)检测目的蛋白的表达。Western Blot结果如图1所示，表达细胞均能稳定的表达MERS-nCoV、nCoV-SARS和MERS-SARS异源二聚体RBD蛋白。
为了与单体RBD抗原做对比，分别构建表达单体MERS-RBD(367-606)(SEQ ID NO.7)、nCoV-RBD(319-541)(SEQ ID NO.8)和SARS-RBD(306-527)(SEQ ID NO.9)的质粒。将MERS-RBD(367-606)、nCoV-RBD(319-541)和SARS-RBD(306-527)基因分别在5’端加入编码信号肽的序列，3’端加入编码6个组氨酸的序列(SEQ ID NO.10,SEQ ID NO.11, SEQ ID NO.12，该序列未包括编码组氨酸和信号肽的序列)，克隆到pCAGGS载体EcoRI和XhoI酶切位点之间，启动子上游含有Kozak序列gccacc。通过分子克隆得到表达三种单体RBD蛋白抗原的质粒pCAGGS-MERS-RBD、pCAGGS-nCoV-RBD、pCAGGS-SARS-RBD。
实施例2：MERS、SARS、nCoV单链异源二聚体RBD蛋白的表达纯化
使用HEK293T细胞表达MERS-nCoV、nCoV-SARS和MERS-SARS单链异源二聚体RBD蛋白。将质粒pCAGGS-MERS-nCoV、pCAGGS-nCoV-SARS和pCAGGS-MERS-SARS分别转染HEK293T细胞，72小时后收集上清，离心去除沉淀再通过0.22μm的滤膜过滤，进一步除去杂质。将细胞上清在4℃通过镍亲和柱(Histrap,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Histrap上洗脱下来，并用10kD浓缩管将洗脱液浓缩换液30倍以上至缓冲液A,终体积小于1ml。再通过Superdex TM200Increase 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(8mM Na 2HPO4,136mM NaCl,2mM KH 2PO 4,2.6mM KCl,pH 7.2)。经过分子筛层析，MERS-nCoV(图2)、nCoV-SARS(图3)和MERS-SARS(图4)在14.5-15ml左右均有一个洗脱峰，进行SDS-PAGE分析，显示非还原(不加DTT)和还原(加DTT)条件下蛋白都是62KD左右，为二聚体。
由图2、图3、图4可见，MERS-nCoV、nCoV-SARS以及MERS-SARS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3：MERS-nCoV和nCoV-SARS蛋白免疫小鼠实验
为了进一步检测二联疫苗的免疫原性，我们将纯化的单链异源二聚体蛋白免疫BALB/c小鼠。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。小鼠分组(每组5只)及疫苗剂量见表1。小鼠免疫实验设置：二联疫苗组，其使用MERS-nCoV和nCoV-SARS(实施例2获得)作为免疫原对小鼠进行免疫；单疫苗对照组，其分别使用MERS-CoV、SARS-CoV和nCoV这三种病毒S蛋白的RBD单体蛋白作为免疫原对小鼠进行免疫；阴性对照组，其使用PBS处理小鼠。
表1冠状病毒RBD单链异源二聚体疫苗免疫小鼠分组及剂量
Figure PCTCN2021097463-appb-000001
Figure PCTCN2021097463-appb-000002
将免疫原用PBS稀释至200μg/ml，将类MF59佐剂——AddaVax TM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小鼠进行免疫每组5只。小鼠实验流程如图5所示，通过肌肉注射的方式，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+50μl佐剂混合，200μg/ml*500μl＝10μg)。第19天、第33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和假病毒中和滴度。
实施例4：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、SARS或者nCoV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2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：
一免后血清的免疫原性检测结果如图6、7和8所示。结果显示单链RBD异源二聚体在免疫后均产生了相应的抗体。
免疫血清针对新冠病毒nCoV-RBD的ELISA结果如图6。MERS-nCoV二联苗诱导了约1:1000以上的抗原特异性IgG滴度，较MERS-RBD单体所诱导的特异性抗体水平显著提高(***P<0.001)，较nCoV-RBD单体诱导的特异性抗体水平显著提高(****P<0.0001)，较PBS对照免疫组诱导的特异性抗体水平显著提高(****P<0.0001)。此外，MERS-nCoV二联苗诱导的针对nCoV-RBD的特异性抗体水平也较nCoV-SARS的产量显著提高(***P<0.001)， 如图6。
免疫血清针对新冠病毒MERS-RBD的ELISA结果如图7。MERS-nCoV二联苗诱导了约1:5000以上的抗原特异性IgG滴度。较MERS-RBD单体所诱导的特异性抗体水平显著提高(****P<0.0001)，较nCoV-RBD单体诱导的特异性抗体水平显著提高(****P<0.0001)，较PBS对照免疫组诱导的特异性抗体水平显著提高(****P<0.0001)，如图7。
免疫血清针对新冠病毒SARS-RBD的ELISA结果如图8。nCoV-SARS二联苗一次免疫诱导了约1:500以上的抗原特异性IgG滴度，较nCoV-RBD单体、SARS-RBD和PBS都诱导的特异性抗体水平都显著的提高(*P<0.05；**P<0.01)。
第21天小鼠进行第二次免疫，第33天收集二免后的血清，ELISA实验第33天(即二免后)的小鼠血清抗体滴度，如图9、10和11所示。结果显示RBD异源二聚体在免疫小鼠后诱导了更高水平的抗体，且RBD异源二聚体与RBD单体诱导小鼠的抗体反应水平有显著差异。
二免小鼠血清针对新冠病毒nCoV-RBD的ELISA结果如图9。MERS-nCoV二联苗诱导了1:10 5以上的抗原特异性IgG滴度，较MERS-RBD单体所诱导的特异性抗体水平显著提高(****P<0.0001)，较nCoV-RBD单体诱导的特异性抗体水平显著提高(****P<0.0001)，较PBS对照免疫组诱导的特异性抗体水平显著提高(****P<0.0001)。此外，MERS-nCoV二联苗诱导的针对nCoV-RBD的特异性抗体水平也较nCoV-SARS的产量显著提高(****P<0.0001)。
二免小鼠血清针对新冠病毒MERS-RBD的ELISA结果如图10。MERS-nCoV二联苗诱导了将近1:10 6的抗原特异性IgG滴度，较MERS-RBD单体所诱导的特异性抗体水平显著提高(**P<0.01)，较nCoV单体诱导的特异性抗体水平显著提高(**P<0.01)，较PBS对照免疫组诱导的特异性抗体水平显著提高(**P<0.01)。
二免小鼠血清针对新冠病毒SARS-RBD的ELISA结果如图11。nCoV-SARS二联苗二次免疫诱导了超过1:10 5以上的抗原特异性IgG滴度，较SARS RBD单体、nCoV-RBD单体和PBS组所诱导的特异性抗体水平都显著的提高(***P<0.001；****P<0.0001)。
实施例5假病毒中和实验检测疫苗产生的抗新冠病毒的中和抗体滴度
将实施例3中获得的血清进行倍比稀释，所得系列稀释液分别与100TCID 50假病毒混合，于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。于37℃孵育24小时后，弃掉培养液，用PBS洗涤细胞2次，加入细胞裂解液，检测荧光素酶活性值。
二次免疫后的血清的免疫原性检测结果如图12所示，图12结果显示：MERS-nCoV二联疫苗在二次免疫之后，产生了中和抗体，小鼠血清的90％中和效价(NT 90)可达1:1000以 上。而以nCoV RBD单体免疫的8只小鼠中仅有四只产生了微弱的中和抗体(其中2只的NT 90为1:20，另外2只的NT 90为1:40)。该结果说明，通过假病毒中和实验证实了MERS-nCoV二联疫苗能够诱导的中和抗体水平远比nCoV RBD单体的高(**p<0.01)，其是很好的新冠病毒候选疫苗。
实施例6假病毒中和实验检测疫苗产生的抗MERS管状病毒的中和抗体滴度
将实施例3中获得的血清进行倍比稀释，所得系列稀释液分别与100TCID 50假病毒混合，于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。于37℃孵育48小时后，弃掉培养液，用PBS洗涤细胞2次，加入细胞裂解液，检测荧光素酶活性值。
二次免疫后的血清的免疫原性检测结果如图13所示，图13结果显示：MERS-nCoV二联疫苗在二次免疫之后，产生了中和抗体，NT 90可达1:1000以上；与MERS RBD同源二聚体诱导的中和抗体水平相当(n.s.)。该结果说明，通过假病毒中和实验证实了MERS-nCoV二联疫苗能够诱导小鼠产生高水平的抗体反应，是很好的MERS-CoV候选疫苗。
实施例7小鼠模型中针对病毒攻毒的保护实验
将32只BALB/c小鼠随机分成4组，每组8只。所有BALB/c小鼠从维通利华公司购买，均为雌性，6-8周龄。小鼠免疫实验设置：二联疫苗组，其使用MERS-nCoV二联疫苗(实施例2获得)作为免疫原对小鼠进行免疫；单疫苗对照组，其分别使用新冠病毒的S蛋白的RBD单体蛋白和RBD同源二聚体蛋白作为免疫原对小鼠进行免疫；阴性对照组，其使用PBS处理小鼠。免疫的疫苗剂量同实施例3中的表1。第二次免疫后39天(即，首次免疫后第74天)，我们对小鼠进行滴鼻感染Ad5-ACE2缺陷型腺病毒，以诱导小鼠肺部表达人源ACE2受体蛋白，随后将小鼠转入微生物所(A)BSL-3实验室。在免疫后第79天，对小鼠进行滴鼻感染新冠病毒(hCoV-19/China/CAS-B001/2020(GISAID databases EPI_ISL_514256-7)病毒株)。病毒感染剂量为：病毒原液50μL/只小鼠。攻毒途径为滴鼻感染。具体操作如下：将稀释好的三溴乙醇(浓度为20mg/ml)，对小鼠腹腔(i.p.)注射，以进行麻醉，每只小鼠腹腔注射250μL。待小鼠完全麻醉后，用200μL移液器吸取50μL病毒液对小鼠进行滴鼻操作。
在病毒攻毒后5天，对小鼠实施安乐死。然后，解剖取出肺脏，将其置于2mL管(已预先称取其重量)中，称重，计算出肺脏的净重量。使用研磨仪研磨小鼠肺脏，之后分离上清。对所得上清进行病毒灭活后，将其带出(A)BSL-3实验室。使用试剂盒(QIAamp Viral RNA Mini Kit)，从上述上清中提取病毒RNA。
实施例8通过qRT-PCR实验，检测新冠病毒攻毒后5天(5dpi)小鼠肺脏的病毒载量
本实施例中，使用qRT-PCR方法检测病毒攻毒后小鼠肺脏的病毒载量；所用探针和引物可以结合在新冠病毒基因组N基因上，其序列分别如下：
N-F:GACCCCAAAATCAGCGAAAT(SEQ ID NO:16)；
N-R:TCTGGTTACTGCCAGTTGAATCTG(SEQ ID NO:17)；
N-probe:ACCCCGCATTACGTTTGGTGGACC(SEQ ID NO:18)；
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qRT-PCR实验操作；实验结果表明，MERS-nCoV二联疫苗组小鼠的病毒载量比PBS阴性对照组低了约1000倍(如图14所示)。由于在病毒攻毒中给小鼠滴入了较大量的病毒颗粒，靶向病毒基因组N基因的探针引物不能区分检测到的是有活性的病毒还是残留的死病毒或者是病毒基因组片段，因此，我们又使用了另外一套可与新冠病毒sgRNA结合的探针引物进行检测，由于sgRNA只在有活性的病毒细胞内复制过程中产生，因此sgRNA的量可代表有复制能力的活病毒的量。用于检测sgRNA的引物探针序列如下：
sgRNA-F:CGATCTCTTGTAGATCTGTTCTC(SEQ ID NO:19)；
sgRNA-R:TGTGTGCGTACTGCTGCAATAT(SEQ ID NO:20)；
sgRNA-probe:ACACTAGCCATCCTTACTGCGCTTCG(SEQ ID NO:21)；
qRT-PCR检测结果如图15所示，图15结果显示：在MERS-nCoV二联疫苗组，完全检测不到复制型病毒，而在PBS阴性对照组和nCoV RBD单体对照组，复制型病毒仍维持在较高水平，该结果说明，MERS-nCoV二联疫苗对小鼠抵抗新冠病毒感染具有很好的保护效果。
实施例9在恒河猴模型中进行针对病毒攻毒的保护实验
为了进一步检测MERS-nCoV二联疫苗的保护效力，我们在恒河猴模型中进行了针对病毒攻毒的保护实验。为此，我们用纯化的MERS-nCoV二联疫苗蛋白对恒河猴进行免疫。所使用的恒河猴来源：中国医学科学院医学实验动物研究所，4-6岁，级别SARS-CoV-2Free。恒河猴分组(每组3只)及疫苗剂量见表2。小鼠免疫实验设置：二联疫苗组，其使用MERS-nCoV作为免疫原对恒河猴进行免疫：阴性对照组，其使用PBS处理恒河猴。
表2：恒河猴的实验分组和给药剂量
Figure PCTCN2021097463-appb-000003
操作过程如下：将免疫原MERS-nCoV蛋白用PBS稀释至100μg/ml，将类MF59佐剂与上述免疫原稀释液按照体积比1:1的比例混合乳化，制备成疫苗；通过肌肉注射的方式，将 所有恒河猴分别在第0天、第28天进行2次疫苗免疫接种，每次500μl的接种体积(其中250μl免疫原稀释液+250μl佐剂混合，每次接种免疫原的实际用量为100μg/ml*250μl＝25μg)。第35天，对恒河猴进行取血，离心收集血清，于-80℃冰箱保存，之后用于假病毒中和滴度的滴定。
实施例10通过假病毒中和实验，检测恒河猴在疫苗免疫后产生的中和抗体滴度
按照实施例5、6所述的具体操作方法，检测实施例9中获得的恒河猴血清中针对新冠病毒nCoV和MERS冠状病毒的中和抗体滴度。二次免疫后，针对MERS冠状病毒和nCoV新冠病毒的血清免疫原性检测结果分别如图16、图17所示；图16和图17的结果显示，MERS-nCoV疫苗免疫组在二次免疫之后，产生了抗新冠病毒和抗MERS冠状病毒的中和抗体，其中，抗MERS冠状病毒的NT 90达约1:10以上(图16)，抗新冠病毒的NT 90可达到约1:100以上(图17)；PBS对照组的中和抗体均为阴性。该结果说明，通过假病毒中和实验证实了MERS-nCoV二联疫苗能够诱导恒河猴产生中和抗体反应，是很好的抗新冠病毒和MERS冠状病毒的二联候选疫苗。
最后应说明的是：以上实施例仅用以说明本发明的技术方案，而非对其限制；尽管参照前述实施例对本发明进行了详细的说明，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发明各实施例技术方案的精神和范围。

Claims (18)

  1. 一种β冠状病毒抗原，其特征在于：所述β冠状病毒抗原的氨基酸序列按照从N端到C端的顺序包括：按照(A-B)-(A’-B’)样式排列的氨基酸序列或(A-B)-C-(A’-B’)样式排列的氨基酸序列，其中：A-B表示来自β冠状病毒的表面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，A’-B’表示来自另一种β冠状病毒的表面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，C表示连接氨基酸序列，所述β冠状病毒抗原为单链异源二聚体结构。
  2. 根据权利要求1所述的β冠状病毒抗原，其特征在于：β冠状病毒选自：严重呼吸综合征冠状病毒、中东呼吸综合征冠状病毒或新型冠状病毒。
  3. 根据权利要求1所述的β冠状病毒抗原，其特征在于：A-B序列来自于中东呼吸综合征冠状病毒，A’-B’序列来自于严重呼吸综合征冠状病毒；
    或，A-B序列来自于中东呼吸综合征冠状病毒，A’-B’序列来自于新型冠状病毒；
    或，A-B序列来自于新型冠状病毒，A’-B’序列来自于严重呼吸综合征冠状病毒。
  4. 根据权利要求3所述的β冠状病毒抗原，其特征在于：来自中东呼吸综合征冠状病毒的表面刺突蛋白的受体结合区的部分氨基酸序列为SEQ ID NO.7和SEQ ID NO.13；
    和/或，来自新型冠状病毒的表面刺突蛋白的受体结合区的部分氨基酸序列为SEQ ID NO.8或SEQ ID NO.14；
    和/或，来自严重呼吸综合征冠状病毒的表面刺突蛋白的受体结合区的部分氨基酸序列为SEQ ID NO.9或SEQ ID NO.15。
  5. 根据权利要求1所述的β冠状病毒抗原，其特征在于：β冠状病毒抗原的氨基酸序列选自：SEQ ID NO.1、SEQ ID NO.2或SEQ ID NO.3。
  6. 一种制备权利要求1-5之一所述的β冠状病毒抗原的方法，其特征在于：包括以下步骤：在编码权利要求1-5之一所述的β冠状病毒抗原的核苷酸序列的5’端加入编码信号肽的序列，3’端加上编码组氨酸的序列，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冠状病毒抗原。
  7. 根据权利要求6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293T细胞或CHO细胞，所述细菌细胞包括大肠杆菌细胞。
  8. 一种编码权利要求1-5之一所述的β冠状病毒抗原的多核苷酸。
  9. 一种包括权利要求8所述的多核苷酸的重组载体。
  10. 一种包括权利要求9所述的重组载体的表达系统细胞；优选地，所述表达系统细胞 为哺乳动物细胞；进一步优选地，所述哺乳动物细胞为293T细胞或CHO细胞。
  11. 一种权利要求1-5之一所述的β冠状病毒抗原、权利要求8所述的多核苷酸、权利要求9所述的重组载体或权利要求10所述的表达系统细胞在制备β冠状病毒二联疫苗中的应用。
  12. 一种β冠状病毒二联疫苗，包括权利要求1-5之一所述的β冠状病毒抗原和佐剂。
  13. 根据权利要求12所述的β冠状病毒二联疫苗，其特征在于：所述β冠状病毒二联疫苗包括：中东呼吸综合征冠状病毒-严重呼吸综合征冠状病毒二联疫苗、新型冠状病毒-严重呼吸综合征冠状病毒二联疫苗、中东呼吸综合征冠状病毒-新型冠状病毒二联疫苗。
  14. 根据权利要求12或13所述的β冠状病毒二联疫苗，其特征在于：所述佐剂选自铝佐剂、MF59佐剂和类MF59佐剂。
  15. 根据权利要求12-14任一项所述的β冠状病毒二联疫苗，其特征在于：权利要求1-5之一所述的β冠状病毒抗原和佐剂的体积比为1：1-2；可选地，为1：1。
  16. 一种β冠状病毒二联DNA疫苗，其包含编码权利要求1-5之一所述的β冠状病毒抗原的DNA序列。
  17. 一种β冠状病毒二联mRNA疫苗，其包含编码权利要求1-5之一所述的β冠状病毒抗原的mRNA序列。
  18. 一种β冠状病毒二联病毒载体疫苗，其包含编码权利要求1-5之一所述的β冠状病毒抗原的多核苷酸；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。
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