WO2021253172A1 - Method for inducing anti-novel coronavirus neutralizing antibody using receptor recognition domain - Google Patents

Method for inducing anti-novel coronavirus neutralizing antibody using receptor recognition domain Download PDF

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WO2021253172A1
WO2021253172A1 PCT/CN2020/096148 CN2020096148W WO2021253172A1 WO 2021253172 A1 WO2021253172 A1 WO 2021253172A1 CN 2020096148 W CN2020096148 W CN 2020096148W WO 2021253172 A1 WO2021253172 A1 WO 2021253172A1
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srbd
protein
rbd
cov
vaccine
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PCT/CN2020/096148
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French (fr)
Chinese (zh)
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徐建青
张晓燕
曹康丽
丁龙飞
何香川
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上海市公共卫生临床中心
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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
    • 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
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/62—DNA sequences coding for fusion proteins

Definitions

  • the present disclosure belongs to the field of biotechnology and vaccines. Specifically, the present disclosure relates to methods for inducing neutralizing antibodies against the new coronavirus (SARS-CoV-2) by using receptor recognition domains.
  • SARS-CoV-2 new coronavirus
  • MERS-CoV Middle East respiratory syndrome coronavirus
  • SARS-CoV severe acute respiratory syndrome coronavirus
  • SARS-CoV-2 The 2019 new type of coronavirus
  • Coronavirus contains four structural proteins, including spike protein (S protein), envelope protein, membrane protein and nucleocapsid protein. Among them, S protein plays the most important role in the process of virus attachment, fusion and entry, and is also the main target of antibodies, entry inhibitors and vaccines.
  • S protein mediates the virus into the host cell, firstly binds to the host receptor through the receptor binding domain (RBD) of the S1 subunit, and then fuses the virus and the host cell membrane through the S2 subunit.
  • RBD receptor binding domain
  • the non-neutralizing antibodies or low antibody levels produced by vaccine immunization may cause antibody-dependent enhancement (ADE) and enhance the pathogenicity of the virus.
  • ADE antibody-dependent enhancement
  • neutralizing antibodies block the virus from binding to cell surface receptors and neutralize the ability of the virus to infect; non-neutralizing antibodies bind to the virus, but in some cases, the Fc segment of the antibody binds to the cell surface Fc receptor, making it possible to infect Cells that express Fc receptors. Therefore, in order to reduce the side effects of ADE, the RBD region in the S protein of the new coronavirus will be the most effective target for vaccine development.
  • RBD The natural conformation of RBD in the S protein is a trimer. If RBD is purified alone, there will be less support from the S2 subunit trimer, and its conformation is likely to be changed. Therefore, how to maintain the RBD conformation is a problem that needs to be solved.
  • the specific RBD region of the new coronavirus is used as an immunogen, and a series of modifications are made to the RBD region at the same time, such as adding disulfide bonds, fusion proteins, and fusion cytokines, so that it can effectively induce anti-SARS-CoV-2. And antibodies.
  • an immunogenic peptide against the novel coronavirus SARS-CoV-2 which includes the RBD region of the spike protein S of the SARS-CoV-2 virus, and the RBD region is further processed by half Cystine modification forms the sRBD region.
  • the cysteine modification is to add a pair of cysteines at the root of the RBD domain to enable the formation of disulfide bonds.
  • the immunogenic peptide is selected from: (a) a polypeptide having the amino acid sequence shown in SEQ ID NO: 2, 4, 8 or 10; (b) a homology of the polypeptide described in (a) Polypeptide, for example, with SEQ ID NO: 2, 4, 8 or 10, it is higher than or equal to 90%, higher than or equal to 95%, higher than or equal to 96%, higher than or equal to 97%, higher than or equal to 98 %, higher than or equal to 99% homology; (c) The immunogenic protein or protein derived from (a) that has been substituted, deleted or added one or several amino acids in the amino acid sequence defined in (a) Peptides.
  • the amino acid sequence of the RBD region is shown in SEQ ID NO: 2
  • the amino acid sequence of the cysteine-modified sRBD region is shown in SEQ ID NO: 4.
  • the RBD region or the cysteine-modified sRBD region is included in the fusion peptide, for example, the part fused with it includes: virus or host-derived protein, transferrin (Fn), HIV p24 , The stem of enveloped virus, such as influenza HA2, HIV gp41, antibody Fc segment, GM-CSF, IL-21, CD40L or CD40 antibody.
  • virus or host-derived protein transferrin (Fn)
  • HIV p24 The stem of enveloped virus, such as influenza HA2, HIV gp41, antibody Fc segment, GM-CSF, IL-21, CD40L or CD40 antibody.
  • the fusion peptide may further include: signal peptide, connecting peptide, tag and other elements.
  • the signal peptide can be selected from: protein itself, CD33, CD8, CD16, mouse IgG1 antibody, influenza HA.
  • the connecting peptide can be selected from: (G4S) 3 , (G4S) n , GSAGSAAGSGEF, (Gly)6, EFPKPSTPPGSSGGAP, KESGSVSSEQLAQFRSLD, (Gly)8, EGKSSGSGSESKST.
  • Tags can be selected from: His-tag, AviTag, Calmodulin tag, polyglutamate tag, E-tag, FLAG tag, HA-tag, Myc-tag, S-tag, SBP-tag, Sof-tag 1, Sof-tag3, Strep -tag, TC tag, V5 tag, T7 tag, VSV tag, Xpress tag, 3X FLAG tag, Isopep tag, Spytag, Snoop tag and PNE tag.
  • the immunogenic peptide is sRBD-hFn, sRBD-HA2 and the like.
  • sequence of the fusion peptide is as shown in SEQ ID NO: 8 or SEQ ID NO: 10.
  • nucleotide molecule that encodes the immunogenic peptide of the present disclosure.
  • the nucleotide molecule is selected from: (i) nucleotide molecules whose sequence is as shown in SEQ ID NO: 1, 3, 7 or 9; (ii) and (i) under stringent conditions Hybrid molecules; (iii) and (i) or (ii) have a sequence higher than or equal to 90%, higher than or equal to 95%, higher than or equal to 96%, higher than or equal to 97%, higher than or equal to 98%, higher than or equal to 99% homology of nucleotide molecules; (iv) One or several nucleotides have been substituted, deleted or added in the nucleotide sequence defined in (i) or (ii) And can express the nucleotide molecule of functional RBD immunogenic peptide.
  • the coding sequence of the RBD region is shown in SEQ ID NO: 1
  • the coding sequence of the cysteine modified RBD region is shown in SEQ ID NO: 3, or the sequence of the nucleotide molecule Such as SEQ ID NO: 7 or SEQ ID NO: 9.
  • the nucleotide molecule further includes a sequence encoding a portion fused to an RBD region or a cysteine-modified RBD region.
  • a vector comprising the nucleotide molecule of the present disclosure.
  • the vector is: a viral vector, such as a poxvirus (such as a Tiantan strain, a North American vaccine strain, a Wyeth-derived strain, a Listeria strain, an Ankara-derived strain, a Copenhagen strain and a New York strain poxvirus), an adenovirus (such as Ad5, Ad11, Ad26, Ad35, Ad68), lentiviral vector, adeno-associated virus, herpes simplex virus, measles virus, reo virus, rhabdovirus, forest encephalitis virus, influenza virus, respiratory syncytial virus, spinal cord Poliovirus vector.
  • a viral vector such as a poxvirus (such as a Tiantan strain, a North American vaccine strain, a Wyeth-derived strain, a Listeria strain, an Ankara-derived strain, a Copenhagen strain and a New York strain poxvirus), an adenovirus (such as Ad5, Ad11, Ad26, Ad35, Ad68), lentiviral vector,
  • a host cell which contains the vector of the present disclosure and/or can express the immunogenic peptide of the present disclosure.
  • the host cell is a mammalian cell or an insect cell, such as HEK293, HeLa, K562, CHO, NS0, SP2/0, PER.C6, Vero, RD, BHK, HT 1080, A549, Cos- 7. ARPE-19 and MRC-5 cells; High Five, Sf9, Se301, SeIZD2109, SeUCR1, Sf9, Sf900+, Sf21, BTI-TN-5B1-4, MG-1, Tn368, HzAm1, BM-N, Ha2302 Hz2E5 and Ao38.
  • the host cell is K562.
  • the K562 cells comprise sRBD-HA2.
  • the present disclosure also provides a cell displaying the immunogenic peptide of the novel coronavirus SARS-CoV-2 described herein on its cell membrane surface.
  • the cell comprises a vector having the immunogenic peptide coding sequence described herein.
  • the cells are transferred into a vector with an immunogenic peptide coding sequence.
  • the cell has a complete membrane structure displaying the immunogenic peptide.
  • the cells are inactivated cells, for example, physical inactivation such as X-ray radiation, ultraviolet radiation; or chemical inactivation such as ⁇ -propiolactone, formaldehyde, and paraformaldehyde fixation.
  • a vaccine against the novel coronavirus SARS-CoV-2 which comprises the immunogenic peptides, nucleotide molecules, vectors and/or host cells of the present disclosure.
  • the use of the immunogenic peptides, nucleotide molecules, vectors and/or host cells of the present disclosure in the preparation of vaccines for the prevention or treatment of the novel coronavirus SARS-CoV-2 is also provided .
  • the immunogenic peptides, nucleotide molecules, vectors and/or host cells of the present disclosure are also provided, which are used to prevent or treat the new coronavirus SARS-CoV-2.
  • a method for preventing or treating the novel coronavirus SARS-CoV-2 comprising administering to a subject in need the immunogenic peptides, nucleotide molecules, and vectors of the present disclosure , Host cells and/or vaccines.
  • the vaccine is a nucleic acid vaccine (DNA or RNA vaccine), a recombinant protein subunit vaccine, a recombinant viral vector vaccine, a recombinant bacterial vector vaccine, a virus-like particle vaccine, a nanoparticle vaccine, and a cell vector vaccine.
  • the vaccine contains an adjuvant or is used in combination with an adjuvant, including but not limited to: aluminum adjuvant, cholera toxin and its subunits, oligodeoxynucleotide, manganese ion adjuvant, Colloidal manganese adjuvant, Freund's adjuvant, SAS adjuvant, MF59 adjuvant, QS-21 adjuvant, Poly I: C and other TLR ligands, GM-CSF, IL-2, IL-3, IL-7, IL-11, IL-12, IL-18, IL-21, etc.
  • an adjuvant including but not limited to: aluminum adjuvant, cholera toxin and its subunits, oligodeoxynucleotide, manganese ion adjuvant, Colloidal manganese adjuvant, Freund's adjuvant, SAS adjuvant, MF59 adjuvant, QS-21 adjuvant, Poly I: C and other TLR ligands, GM-CSF, IL
  • the form of the vaccine is suitable for the following vaccination methods: intramuscular vaccination, intradermal vaccination, subcutaneous vaccination, nose drops, nebulized inhalation, genital tract, rectum, oral administration or any combination thereof.
  • one or more of the vaccines are used for vaccination, such as combined vaccination or sequential vaccination.
  • one or more of the vaccines and other vaccines against new coronaviruses are used for vaccination, for example, the other vaccines include vaccines against coronavirus S or S1, for example, the S or S1 is derived from Including but not limited to SARS-CoV-2, SARS-CoV, MERS-CoV, HCoV-229E, HCoV-OC43, HCoV-NL63, HCoV-HKU1, bat-CoV, etc.
  • the vaccine comprises a combination of a nucleic acid vaccine (DNA or RNA vaccine) and a recombinant human-derived cell vector vaccine.
  • the vaccine comprises a combination of pcDNA3.1-sRBD-hFn and K562-HA2-sRBD.
  • the components of the vaccine combination are vaccinated sequentially, preferably the DNA vaccine is vaccinated first.
  • a method for preparing a vaccine against the novel coronavirus SARS-CoV-2 comprising:
  • FIG. 1 S protein and sRBD-hFn (disulfide bond modification) eukaryotic expression vector construction and protein expression:
  • Figure 1A Construction map of pcDNA3.1-sRBD-hFn and pcDNA3.1-S eukaryotic expression vector plasmids
  • FIG. 1B pcDNA3.1-sRBD-hFn and pcDNA3.1-S proteins were successfully expressed in 293T cells.
  • Figure 2 Construction of lentiviral expression vectors pHAGE-S-puro and pHAGE-sRBD-HA2-puro and verification of expression of S protein and sRBD-HA2 displayed on K562 cell membrane:
  • FIG. 2A Construction map of pHAGE-S-puro and pHAGE-sRBD-HA2-puro lentiviral expression vector plasmids
  • sRBD can bind to the ACE2 receptor.
  • FIG. 3 The induction effect of RBD protein on binding antibodies and neutralizing antibodies in mice:
  • mice used in the experiment were 6-8 weeks old C57/BL6, and the immunogen was the new coronavirus RBD protein and pcDNA3.1-S plasmid, and the proteins were all enhanced with aluminum adjuvant.
  • FIG. 3A ELISA method to detect the titers of bound antibodies in the serum of mice in the second week after the end of immunization.
  • the abscissa is the immunization group, and the ordinate is the titer of the bound antibody;
  • FIG. 3B 293T-ACE2 cells detect the titers of neutralizing antibodies in mouse serum at the 4th week after the end of immunization.
  • the abscissa is the immunization group, and the ordinate is the titer of neutralizing antibody (ID 50 ). * Indicates p ⁇ 0.05.
  • mice used in the experiment were 6-8 weeks old C57/BL6, and the immunogen was the new coronavirus S plasmid pcDNA3.1-S and the modified RBD plasmid pcDNA3.1-sRBD-hFn.
  • FIG. 4A ELISA method to detect the titers of bound antibodies in the serum of mice in the second week after the end of immunization.
  • the abscissa is the immunization group, and the ordinate is the titer of the bound antibody;
  • FIG. 4B 293T-ACE2 cells detect the titers of neutralizing antibodies in the serum of mice in the second week after the end of immunization.
  • the abscissa is the immunization group, and the ordinate is the titer of neutralizing antibody (ID 50 ).
  • FIG. 5 The induction effect of K562-HA2-sRBD and K562-S cells on mouse binding antibodies and neutralizing antibodies:
  • mice used in the experiment were 6-8 weeks old C57/BL6, and the immunogens were K562-HA2-sRBD and K562-S.
  • FIG. 5A ELISA method to detect the titers of bound antibodies in the serum of mice in the second week after the end of immunization.
  • the abscissa is the immunization group, and the ordinate is the titer of the bound antibody;
  • FIG. 5B 293T-ACE2 cells detect the titers of neutralizing antibodies in mouse serum at the second week after the end of immunization.
  • the abscissa is the immunization group, and the ordinate is the titer of neutralizing antibody (ID 50 ).
  • mice used in the experiment were 6-8 weeks old C57/BL6, and the immunogen was sRBD-hFn protein.
  • FIG. 6A ELISA method to detect the titers of bound antibodies in the serum of mice in the first week after immunization.
  • the abscissa is the immunization group, and the ordinate is the titer of bound antibody;
  • FIG. 6B 293T-ACE2 cells detect the titers of neutralizing antibodies in the serum of mice in the first week after the end of immunization.
  • the abscissa is the immunization group, and the ordinate is the titer of neutralizing antibody (ID 50 ).
  • the present disclosure relates to the field of vaccines, and in particular to a method for inducing neutralizing antibodies against the new coronavirus (SARS-Cov-2) by using the RBD region.
  • SARS-Cov-2 new coronavirus
  • Using the RBD region of the virus, the modified RBD region, and the RBD region fused with other proteins or cytokines as immunogens can reduce the antibody-dependent enhancement effect (ADE) caused by non-neutralizing antibodies or lower antibody levels, thereby Prevent new coronavirus infection.
  • ADE antibody-dependent enhancement effect
  • Animal experiment results confirm that the vaccine of the present disclosure is safe, can continuously produce high-titer neutralizing antibodies, and can be used for the prevention and treatment of the new coronavirus.
  • 0.1-2.5 mg/day includes 0.1 mg/day, 0.2 mg/day, 0.3 mg/day, etc. up to 2.5 mg/day.
  • RBD immunogenic peptide As used herein, the terms “RBD immunogenic peptide”, “immunogenic peptide against the novel coronavirus SARS-CoV-2” and “immunogenic peptide of the present disclosure/application” are used interchangeably and refer to It includes the RBD region of the spike protein S of the SARS-CoV-2 virus or the RBD region modified with cysteine (also referred to as the sRBD region herein), and has peptides that stimulate binding antibodies and neutralize antibodies.
  • the immunogenic peptide may be: (a) a polypeptide having the amino acid sequence shown in SEQ ID NO: 2, 4, 8, or 10; (b) the same polypeptide as described in (a) Source polypeptide, for example, with SEQ ID NO: 2, 4, 8 or 10, it is higher than or equal to 90%, higher than or equal to 95%, higher than or equal to 96%, higher than or equal to 97%, higher than or equal to 98%, higher than or equal to 99% homology; (c) A protein derived from (a) with immunogenicity after substitution, deletion or addition of one or several amino acids in the amino acid sequence defined in (a) Or polypeptide.
  • the immunogenic peptide may include other parts connected to the RBD region or the RBD region modified with cysteine, for example, to enhance the stability of the RBD region, improve the neutralizing antibody response, form multimers, and increase Cell response, etc.
  • Modifiable or unmodified RBD regions include, but are not limited to: virus or host-derived proteins, transferrin (Fn), HIV p24, the stem of enveloped viruses, such as influenza HA2, HIV gp41, antibodies Fc segment, GM-CSF, IL-21, CD40L or CD40 antibody, etc.
  • the sequence of the fusion peptide is shown in SEQ ID NO: 8 or SEQ ID NO: 10.
  • the fusion peptide may also include signal peptides, linkers, molecular tags and other elements.
  • the signal peptide element may refer to an amino acid sequence that has the function of guiding the secretion, localization and/or delivery of the fusion protein, and its length is usually 5-30 amino acids.
  • the signal peptide element may be selected from the group consisting of: protein signal peptide, CD33 protein signal peptide, CD8 protein signal peptide, CD16 protein signal peptide, mouse IgG1 antibody signal peptide, influenza HA protein signal peptide.
  • the linking peptide sequence may refer to a short peptide that serves to link different elements in the fusion protein herein, and its length is usually 1-50 (such as 5-50, 5-40, 10-40). Amino acids.
  • the connecting peptide does not affect or severely affect the formation of the correct folding and spatial conformation of the fusion protein of the present invention.
  • the connecting peptide component selected from: (G 4 S) 3 linker, (G4S) n, GSAGSAAGSGEF, (Gly) 6, EFPKPSTPPGSSGGAP, KESGSVSSEQLAQFRSLD, (Gly) 8, EGKSSGSGSESKST.
  • the immunogenic peptide may also include its variant form, such as one or more (usually 1-50, preferably 1-30, more preferably 1-20, most preferably 1-10, such as 1 , 2, 3, 4, 5, 6, 7, 8, 9 or 10) deletion, insertion and/or substitution of amino acids, and the addition of one or several (usually within 20) at the C-terminus and/or N-terminus , Preferably within 10, more preferably within 5) amino acids.
  • the function of the protein or polypeptide is usually not changed.
  • adding one or several amino acids to the C-terminus and/or N-terminus usually does not change the function of the protein or polypeptide.
  • the immunogenic peptide can be produced by recombinant expression under appropriate environment and conditions, for example, produced by the encoding nucleotide molecules, vectors, and host cells of the present disclosure; it can also be obtained by chemical synthesis, as long as it has the required amino acid sequence And immunogenicity and reactivity.
  • nucleic acid molecule can be selected from, for example: (i) a nucleotide molecule whose sequence is as shown in SEQ ID NO: 1, 3, 7 or 9; (ii) a molecule that hybridizes with (i) under stringent conditions; iii) The sequence in (i) or (ii) is higher than or equal to 90%, higher than or equal to 95%, higher than or equal to 96%, higher than or equal to 97%, higher than or equal to 98%, higher than Or a nucleotide molecule with 99% homology; (iv) One or several nucleotides are substituted, deleted or added in the nucleotide sequence defined in (i) or (ii) and can express functionality Nucleotide molecule of RBD immunogenic
  • stringent conditions refers to: (1) hybridization and elution at lower ionic strength and higher temperature, such as 0.2 ⁇ SSC, 0.1% SDS, 60°C; or (2) adding during hybridization There are denaturants, such as 50% (v/v) formamide, 0.1% calf serum/0.1% Ficoll, 42°C, etc.; or (3) only the identity between the two sequences is at least 50%, preferably 55 % Or more, 60% or more, 65% or more, 70% or more, 75% or more, 80% or more, 85% or more or 90% or more, more preferably 95% or more.
  • the full-length nucleotide sequence of the present disclosure or fragments thereof can usually be obtained by PCR amplification method, recombination method or artificial synthesis method.
  • primers can be designed according to the relevant nucleotide sequences disclosed in the present disclosure, and a commercially available cDNA library or a cDNA library prepared according to a conventional method known to those skilled in the art can be used as a template for amplification. Get the sequence. When the sequence is long, it is often necessary to perform two or more PCR amplifications, and then splice the amplified fragments together in the correct order.
  • the present disclosure also relates to a vector containing a nucleotide molecule encoding RBD, and a host cell produced by genetic engineering using the vector.
  • vector and “recombinant expression vector” are used interchangeably and refer to bacterial plasmids, bacteriophages, yeast plasmids, animal cell viruses, mammalian cell viruses or other vectors that are well known in the art.
  • An important feature of an expression vector is that it usually contains an origin of replication, a promoter, a marker gene, and translation control elements.
  • expression vectors containing RBD immunogenic peptide coding sequences and appropriate transcription/translation control signals can be used to construct expression vectors containing RBD immunogenic peptide coding sequences and appropriate transcription/translation control signals. These methods include in vitro recombinant DNA technology, DNA synthesis technology, and in vivo recombination technology.
  • the DNA sequence can be effectively linked to an appropriate promoter in the expression vector to guide mRNA synthesis.
  • the expression vector also includes a ribosome binding site for translation initiation and a transcription terminator. Expression systems such as pcDNA3.1 vector, pIRES2-EGFP vector, AdMaxTM, etc. can be used in the present disclosure.
  • the expression vector may contain one or more selectable marker genes to provide phenotypic traits for selecting transformed host cells, such as dihydrofolate reductase for eukaryotic cell culture, neomycin resistance, and green fluorescence Protein (GFP), or tetracycline or ampicillin resistance for E. coli.
  • selectable marker genes such as dihydrofolate reductase for eukaryotic cell culture, neomycin resistance, and green fluorescence Protein (GFP), or tetracycline or ampicillin resistance for E. coli.
  • a vector containing the above-mentioned appropriate DNA sequence and an appropriate promoter or control sequence can be used to transform an appropriate host cell so that it can express a protein or polypeptide.
  • the host cell can be a prokaryotic cell, such as a bacterial cell; or a lower eukaryotic cell, such as a yeast cell; or a higher eukaryotic cell, such as an animal cell.
  • Representative examples are: Escherichia coli, Streptomyces, Agrobacterium; fungal cells such as yeast; animal cells, etc.
  • host cells selected from the group consisting of HEK293, HeLa, CHO, K562, NS0, SP2/0, PER.C6, Vero, RD, BHK, HT 1080, A549, Cos-7, ARPE can be used -19 and MRC-5 cells; High Five, Sf9, Se301, SeIZD2109, SeUCR1, Sf9, Sf900+, Sf21, BTI-TN-5B1-4, MG-1, Tn368, HzAm1, BM-N, Ha2302, Hz2E5 and Ao38 .
  • Enhancers are cis-acting factors of DNA, usually about 10 to 300 base pairs, acting on promoters to enhance gene transcription. Those of ordinary skill in the art know how to select appropriate vectors, promoters, enhancers and host cells.
  • the recombinant polypeptide in the above method can be expressed in the cell or on the cell membrane or secreted out of the cell. If necessary, the physical, chemical, and other characteristics can be used to separate and purify the recombinant protein through various separation methods. These methods are well known to those skilled in the art. Examples of these methods include, but are not limited to: conventional renaturation treatment, treatment with protein precipitation agent (salting out method), centrifugation, osmotic sterilization, ultra-treatment, ultra-centrifugation, molecular sieve chromatography (gel filtration), adsorption layer Analysis, ion exchange chromatography, high performance liquid chromatography (HPLC) and various other liquid chromatography techniques and combinations of these methods.
  • a vaccine, or immune composition comprising the immunogenic peptide, nucleotide molecule, vector and/or host cell of the present disclosure.
  • the vaccine comprises a formulation of the immunogenic peptide and/or nucleic acid molecule of the present disclosure in a form that can be administered to a vertebrate (preferably a mammal), and it induces a protective immune response that improves immunity to prevent and/or alleviate the novel Coronavirus and/or at least one symptom thereof.
  • protective immune response refers to an immune response mediated by an immunogen against an infectious agent or disease, displayed by vertebrates (such as humans), preventing or reducing infection or reducing at least one disease symptom.
  • chordate subphylum refers to any member of the chordate subphylum, including but not limited to: humans and other primates, including non-human primates such as chimpanzees and other apes and monkeys Species; domestic animals such as cattle, sheep, pigs, goats, and horses; domestic mammals such as dogs and cats; laboratory animals, including rodents such as mice, rats, and guinea pigs; birds include domesticated, wild, and game birds such as chickens, fire Chickens and other quail chickens, ducks, and geese.
  • mammals include domesticated, wild, and game birds such as chickens, fire Chickens and other quail chickens, ducks, and geese.
  • mamammal and “animal” are included in this definition and are intended to cover adult, juvenile and newborn individuals.
  • the vaccine herein can be a recombinant protein vaccine, a recombinant DNA vaccine, a recombinant viral vector vaccine (e.g., adenovirus vector, poxvirus vector, adeno-associated virus vector, herpes simplex virus vector, cytomegalovirus vector), recombinant bacterial vector vaccine, recombinant yeast Vector vaccine or recombinant virus-like particle vaccine.
  • the vaccine herein is selected from a recombinant DNA vaccine, a recombinant adenovirus vector, a recombinant poxvirus vector, or a combination of two or three of them.
  • one or more vaccines selected from the following group or a combination thereof can be used: recombinant plasmid vaccine (DNA), such as a cysteine-modified RBD region coded with fusion with human recombinant Ferritin or HA2 Sequence DNA vaccine (such as pcDNA3.1-sRBD-hFn); recombinant protein subunit vaccine (protein), such as RBD protein (without disulfide bond modification), cysteine modified by fusion with human recombinant Ferritin or HA2 The RBD protein (sRBD-hFn protein, sRBD-HA2 protein); recombinant human cell vector vaccine, such as K562-HA2-sRBD.
  • DNA such as a cysteine-modified RBD region coded with fusion with human recombinant Ferritin or HA2 Sequence DNA vaccine (such as pcDNA3.1-sRBD-hFn); recombinant protein subunit vaccine (protein), such as RBD protein (without disulf
  • the vaccine composition herein includes an effective amount of the immunogen herein.
  • the vaccine composition of the present disclosure includes an immunogen in an amount sufficient to achieve the desired biological effect.
  • effective amount generally refers to the amount of an immunogen that can induce a protective immune response sufficient to induce immunity to prevent and/or reduce infection or disease and/or to reduce at least one symptom of infection or disease.
  • Adjuvants may also be included in the vaccines herein.
  • Adjuvants known to those of ordinary skill in the art can be used, for example, the adjuvants described in Vogel et al., "A Compendium of Vaccine Adjuvants and Excipients” (2nd edition) (incorporated in its entirety by reference).
  • known adjuvants include, but are not limited to: complete Freund's adjuvant, incomplete Freund's adjuvant, aluminum hydroxide adjuvant, lipopolysaccharide (LPS), RIBI adjuvant, MF-59, and the like.
  • the vaccine composition herein may also include pharmaceutically acceptable carriers, diluents, preservatives, solubilizers, emulsifiers and other auxiliary materials.
  • pharmaceutically acceptable carriers include, but are not limited to, water for injection, saline solution, buffered saline, dextrose, water, glycerol, sterile isotonic water buffer, and combinations thereof.
  • Pharmaceutically acceptable carriers, diluents and other excipients can be found in "Remngton's Pharmaceutical Sciences", for example.
  • the form of the vaccine composition herein may be suitable for systemic or topical (especially intra-respiratory) administration.
  • Methods of administering the vaccine composition include, but are not limited to: intramuscular inoculation, intradermal inoculation, subcutaneous inoculation, nasal drops, nebulized inhalation, genital tract, rectum, oral administration, or any combination thereof.
  • the vaccine herein prevents, eliminates or alleviates novel coronavirus infection or at least one symptom thereof in a subject, such as respiratory symptoms (such as nasal congestion, sore throat, hoarseness), headache, cough, sputum, fever, Rattle, wheezing, difficulty breathing, pneumonia caused by infection, severe acute respiratory syndrome, renal failure, etc.
  • respiratory symptoms such as nasal congestion, sore throat, hoarseness
  • headache such as nasal congestion, sore throat, hoarseness
  • headache such as nasal congestion, sore throat, hoarseness
  • headache such as nasal congestion, sore throat, hoarseness
  • headache such as nasal congestion, sore throat, hoarseness
  • cough such as cough, sputum, fever, Rattle, wheezing
  • difficulty breathing such as pneumonia caused by infection, severe acute respiratory syndrome, renal failure, etc.
  • This article also relates to an immunoconjugate (also called an immunoconjugate), which contains the immunogen herein and other substances conjugated with it.
  • the other substances can be targeted substances (such as a part that specifically recognizes a specific target), therapeutic substances (such as drugs, toxins, cytotoxic agents), and labeled substances (such as fluorescent markers, radioisotope markers).
  • a combination product which includes the immunogenic peptides, nucleotide molecules, vectors, host cells and/or vaccines of the present disclosure, and may also include one or more to help better Other substances that function to prevent and/or treat the new coronavirus infection or its symptoms or enhance the stability of the aforementioned substances.
  • other substances may include other vaccines against coronavirus S or S1, such as those from other vaccines including but not limited to SARS-CoV-2, SARS-CoV, MERS-CoV, HCoV-229E, HCoV-OC43, HCoV-NL63, HCoV -S or S1 vaccines of HKU1, bat-CoV; other active substances for diseases or disorders that benefit from T cell activation and/or memory immune response with T cells.
  • other vaccines against coronavirus S or S1 such as those from other vaccines including but not limited to SARS-CoV-2, SARS-CoV, MERS-CoV, HCoV-229E, HCoV-OC43, HCoV-NL63, HCoV -S or S1 vaccines of HKU1, bat-CoV; other active substances for diseases or disorders that benefit from T cell activation and/or memory immune response with T cells.
  • This article also provides a method for preventing and/or treating novel coronavirus infection and/or its symptoms, which comprises: administering at least one preventive and/or therapeutically effective amount of one or more vaccines of the present disclosure.
  • Available vaccination methods include, but are not limited to: systemic immunization methods, such as intramuscular injection, subcutaneous injection, and intradermal injection, etc.; and intra-respiratory tract immunization methods, such as atomization, nose drops, etc.
  • systemic vaccination or intra-respiratory vaccination is used for the initial immunization, preferably systemic vaccination.
  • the interval between every two vaccination is at least 1 week, for example, 2 weeks, 4 weeks, 2 months, 3 months, 6 months or longer intervals.
  • a DNA vaccine is used for the primary immunization, and a recombinant virus vaccine is used for one or more booster immunizations.
  • the immunization method of the present disclosure can adopt a "primary immunization-enhancement” or “primary immunization-enhancement-re-enhancement” approach, a single systemic immunization method or a local respiratory tract immunization method, or a combination of two immunization methods.
  • recombinant DNA vaccines are used for systemic primary immunization to establish a systemic immune response, and then other vaccines (such as recombinant adenovirus vaccines or recombinant poxvirus vaccines) are used for once.
  • other vaccines such as recombinant adenovirus vaccines or recombinant poxvirus vaccines
  • multiple immune boosts which may include at least one respiratory boost (for example, using an adenovirus vaccine).
  • the combination product herein can be provided in the form of a pharmaceutical pack or kit.
  • one or more vaccine compositions herein or one or more components thereof can be packaged in one or more containers, for example, packaged in the specified combination
  • the amount of the substance in a sealed container such as an ampoule or a sachet.
  • the vaccine composition can be provided in the form of liquid, sterile lyophilized powder or anhydrous concentrate, etc., and can be diluted, reconstituted and/or formulated with an appropriate liquid (such as water, saline, etc.) before use for administration To the appropriate concentration and form of the subject.
  • the combination product in the present disclosure can be used to locally induce high-level antigen-specific CD8+ T cell responses in the respiratory tract, so that it can be used in the prevention of respiratory pathogen infections, reducing the pathogenicity of respiratory pathogens, and the prevention and treatment of respiratory tumors prospect.
  • S and RBD sequences are from Genebank: NC_045512.2, Severe acute respiratory syndrome coronavirus 2 isolate Wuhan-Hu-1, complete genome; hFn sequence comes from Genebank: M97164.1, HA2 sequence comes from Genebank: AGI60292.1, specific sequence See sequence list.
  • Recombinant plasmid vaccine (DNA): pcDNA3.1-sRBD-hFn, pcDNA3.1-S, pcDNA3.1 (empty);
  • Recombinant protein subunit vaccine protein: RBD protein (without disulfide bond modification), sRBD-hFn protein;
  • Recombinant human cell vector vaccine K562, K562-S, K562-HA2-sRBD.
  • the immunogen doses used in the examples are as follows:
  • Recombinant plasmid vaccine 100 ⁇ g/mouse, 100 ⁇ L, dissolved in sterile saline;
  • Recombinant protein subunit vaccine protein: protein (dissolved in sterile PBS) and aluminum adjuvant (Aluminium, InvivoGen, Item No. 5200) are immunized at a volume ratio of 1:1, RBD protein, 10 ⁇ g/small Mouse, 100 ⁇ L; sRBD-hFn protein, 20 ⁇ g/mouse, 100 ⁇ L;
  • Recombinant human cell vector vaccine K562, K562-S, K562-HA2-sRBD: 1E6 cells/mouse, 100 ⁇ L, dissolved in sterile PBS.
  • the gene recovery product is ligated with the restriction digestion linearized vector with T4 DNA ligase (Thermo Scientific Company, Catalog No. 2011A): The ligation product is transformed into E.coli Stable, and placed on a culture plate containing ampicillin overnight Grow. On the second day, a single colony was randomly selected for sequencing, and the mutation site was corrected. After verifying that all the sequences were correct, the lentiviral expression plasmid (pHAGE-hACE2-puro) of the hACE2 gene was successfully cloned.
  • the peripheral whole blood of the mouse was collected by removing the eyeballs, collected in a 1.5mL EP tube, and allowed to stand at room temperature for natural coagulation.
  • the coagulated mouse The serum was centrifuged at 7000g for 15min. Transfer mouse serum to a new 1.5mL EP tube. Before the experiment, the sample needs to be inactivated at 56°C for 30 minutes to destroy the complement activity in the serum. Centrifuge briefly before inactivation to avoid residual samples on the tube wall and bottle cap. The liquid level of the water bath must be below the liquid level of the sample, but not more than the bottle cap.
  • each well was added 50 ⁇ L SARS-CoV-2 virus dilutions false, false per well containing final virus 200TCID 50.
  • Example 1 S and sRBD-hFn (disulfide bond modification) eukaryotic expression vector construction and protein expression
  • the sRBD-hFn gene adds a cysteine to the N and C ends of the RBD gene to form a disulfide bond at the root of the RBD to stabilize the structure of the RBD.
  • sRBD is expressed at the N-terminus of hFn, and the two are connected by a linker.
  • the gene recovery product was ligated with the digested linearized vector with T4 DNA ligase (Thermo Scientific, Catalog No. 2011A): the ligation product was transformed into E. coli Stable and placed on a culture plate containing ampicillin overnight Grow. On the second day, a single colony was randomly selected for sequencing, and the mutation site was corrected. After verifying that all the sequences were correct, the eukaryotic expression vectors pcDNA3.1-S and pcDNA3.1-sRBD-hFn of S and sRBD-hFn were successfully cloned.
  • the plasmid construction map is shown in Figure 1A.
  • WB Western blot
  • sRBD-hFn The structure of the expressed sRBD-hFn is: sRBD is expressed at the N-terminus of hFn, and the two are connected by (G4S) 3 .
  • Example 2 Construction of lentiviral expression vectors pHAGE-S-puro and pHAGE-sRBD-HA2-puro and verification of expression of S protein and sRBD-HA2 displayed on K562 cell membrane
  • the gene recovery product was ligated with the digested linearized vector using T4 DNA ligase method (Thermo Scientific Company, Catalog No. 2011A): The ligation product was transformed into E. coli Stable, and grown overnight on a culture plate containing ampicillin. On the second day, a single colony was randomly selected for sequencing, and the mutation site was corrected. After verifying that all the sequences were correct, the lentiviral expression vectors pHAGE-S-puro and pHAGE-sRBD-HA2-puro of S and sRBD-HA2 were successfully cloned. Figure 2A).
  • the pHAGE-S-puro and pHAGE-sRBD-HA2-puro were transfected into 293T cells, and the protein expression in K562-S cells was identified by Western blotting ( Figure 2B).
  • flow cytometric staining was performed to identify S protein and The sRBD-HA2 protein is expressed on the cell membrane and can bind to the receptor ACE2, while the expression of related proteins cannot be detected in the control infected cells, indicating that the conformation of the disulfide-modified RBD itself remains relatively intact and does not affect it.
  • Main functions Figure 2C).
  • the experimental procedure is as follows: Take a 6-well plate and inoculate about 6 ⁇ 10 5 293T cells in each well to ensure that the cell density reaches 90% during the second day of transfection; use pHAGE-S-puro and pHAGE-sRBD -HA2-puro were transfected into 293T cells (transfection reagent TurboFect). Incubate in a 5% incubator at 37°C for about 48 hours. The specific time depends on the cell conditions. The cells are collected and subjected to Western blot (WB) identification.
  • WB Western blot
  • the cells are cultured in RPMI (10% FBS) with a puromycin concentration of 4 ⁇ g/ml. Finally, the cells that can survive are integrated. S gene and sRBD-HA2 cells. Take the above-infected cells and use Western blotting to detect the expression of S protein.
  • the primary antibody used is ACE2-C-AVI-6his (Shanghai Nearshore Technology Co., Ltd., model 0331753-4065), and the secondary antibody is HRP-labeled goat antibody. Human antibody (Zhongshan Jinqiao Company, catalog number ZB2304). The results show that the high expression of the target protein can be detected by Western blotting (Figure 2B).
  • Flow cytometric staining method uses ACE2-C-AVI-6his (Shanghai Nearshore Technology Co., Ltd., model 0331753-4065)/PE-streptavidin (BD Pharminge company, product number 563259) for indirect staining, and finally uses flow cytometry ( BD Pharminge company, model Arial) enrichment.
  • the results showed that after continuous enrichment, more than 80% of K562-S cells and K562-HA2-sRBD cells could bind to ACE2, indicating that the conformation of the disulfide bond-modified RBD itself did not affect its main function (Figure 2C).
  • C57/BL6 mice were immunized with RBD protein (purchased from Nanjing GenScript Biotechnology), and the adjuvant was aluminum adjuvant. Two weeks after the completion of immunization, the immunization combination was evaluated to induce binding antibody titers against RBD protein and S1 protein. At the same time, the neutralizing antibody titers against SARS-CoV-2 pseudovirus were evaluated 4 weeks after the end of immunization.
  • RBD protein purchased from Nanjing GenScript Biotechnology
  • mice are randomly divided into 2 groups, and named as the control group and the RBD group according to the immunogen. Specific immunization combinations are shown in Table 1.
  • the RBD group produced binding antibody titers against RBD protein and S1 protein as shown in Figure 3A: The binding antibody titers against different proteins (S1 protein and RBD protein) were basically the same, with an average value of about 8000, and some mice did not have binding antibodies. The possible reason for being induced is the individual differences of mice.
  • the RBD group produced neutralizing antibody titers against the SARS-Cov-2 pseudovirus as shown in Figure 3B: except for one titer above 1000, the others were around 200, and some mice neutralized Antibodies are not induced, the possible reason is the individual differences of mice.
  • Example 4 Induction effect of plasmid pcDNA3.1-sRBD-hFn on mouse binding antibody and neutralizing antibody
  • the RBD plasmid (pcDNA3.1-sRBD-hFn) modified with disulfide bonds was used to immunize C57/BL6 mice, and 2 weeks after the completion of the immunization , To evaluate the immunization combination induced binding antibody titer against RBD protein and neutralizing antibody titer against SARS-CoV-2 pseudovirus.
  • mice were randomly divided into 2 groups, and named as the control group and the RBD-hFn group according to the immunogen of the second injection.
  • Specific immunization combinations are shown in Table 2.
  • the titers of antibodies against RBD protein produced by different immunization combinations are shown in Figure 4A: most of them are around 2000.
  • the neutralizing antibody titers against the SARS-CoV-2 pseudovirus produced by different immunization combinations are shown in Figure 4B: except for one neutralizing antibody with a titer greater than 80, the others are all at 20. about.
  • Example 5 Induction effect of K562-HA2-sRBD cells and K562-S cells on mouse binding antibodies and neutralizing antibodies
  • mice in the sRBD-hFn group were divided into two groups, and they were immunized again with K562-S and K562-HA2-sRBD. Two weeks after the immunization was completed, the immune combination was evaluated to induce RBD Protein binding antibody titer and neutralizing antibody titer against SARS-CoV-2 pseudovirus.
  • K562-HA2-sRBD cells can induce relatively higher binding antibodies and neutralizing antibodies in mice than K562-S cells.
  • Example 6 Induction effect of sRBD-hFn protein on mouse binding antibody and neutralizing antibody
  • the purified sRBD-hFn protein was used to immunize C57/BL6 mice.
  • the immunization combination induced the binding antibody titers against the RBD protein and the neutralizing antibody titers against the SARS-CoV-2 pseudovirus were evaluated.
  • mice were randomly divided into 2 groups, and named as the control group and the sRBD-hFn group according to the immunogen. Specific immunization combinations are shown in Table 4.
  • the antibody titers against the RBD protein produced are shown in Figure 6A: three mice in the sRBD-hFn group have a binding antibody of 52,000, except for one at 3200, the others are greater than 10,000.
  • the titers of neutralizing antibodies against the SARS-CoV-2 pseudovirus are shown in Figure 6B: there are three neutralizing antibodies, and the titers are all over a dozen.

Abstract

A method for inducing an anti-novel coronavirus neutralizing antibody using a receptor recognition domain is provided. Specifically, an immunogenic peptide for novel coronavirus SARS-CoV-2 is provided, comprising an RBD region of SARS-CoV-2 virus spike protein S or a cysteine-modified RBD region, and encoding a nucleotide molecule, a vector containing the nucleotide molecule, and a host cell. A vaccine for novel coronavirus SARS-CoV-2, preparation and application thereof are also provided. The vaccine is safe, can continuously produce high-potency neutralizing antibodies, and can be used in the prevention and/or treatment of novel coronavirus infections and symptoms thereof.

Description

利用受体识别域诱导抗新冠病毒中和抗体的方法Method for using receptor recognition domain to induce neutralizing antibodies against new coronavirus 技术领域Technical field
本公开属于生物技术和疫苗领域。具体而言，本公开涉及利用受体识别域诱导抗新冠病毒(SARS-CoV-2)中和抗体的方法。The present disclosure belongs to the field of biotechnology and vaccines. Specifically, the present disclosure relates to methods for inducing neutralizing antibodies against the new coronavirus (SARS-CoV-2) by using receptor recognition domains.
背景技术Background technique
迄今为止，已经确定了三种高致病性人类冠状病毒(CoVs)，包括中东呼吸综合征冠状病毒(MERS-CoV)、严重急性呼吸综合征(SARS)冠状病毒(SARS-CoV)和一种2019年新型冠状病毒(SARS-CoV-2，简称新冠病毒)。So far, three highly pathogenic human coronaviruses (CoVs) have been identified, including Middle East respiratory syndrome coronavirus (MERS-CoV), severe acute respiratory syndrome (SARS) coronavirus (SARS-CoV) and one The 2019 new type of coronavirus (SARS-CoV-2, referred to as the new coronavirus).
[根据细则9.2改正03.02.2021]　
目前，在全球至少85个国家和/或地区共报告了超750万例确诊的新冠病毒病例，包括超42万例死亡，WHO在3月11号宣布新冠疫情已发生全球大流行。目前，新冠病毒的中间宿主仍然未知，且没有有效的预防或治疗手段。因此，开发预防和治疗新冠病毒的疫苗和药物是亟待解决的关键问题。[Corrected in accordance with Rule 9.2 03.02.2021]
At present, a total of more than 7.5 million confirmed cases of the new crown virus have been reported in at least 85 countries and/or regions around the world, including more than 420,000 deaths. On March 11, the WHO announced that the new crown epidemic had a global pandemic. At present, the intermediate host of the new coronavirus is still unknown, and there is no effective prevention or treatment method. Therefore, the development of vaccines and drugs to prevent and treat the new coronavirus is a key issue that needs to be solved urgently.
众所周知，在感染性疾病的预防中，疫苗自问世以来就是最为有效和经济的措施，而目前常用的预防疫苗大部分都是以活化中和抗体为目标。因此，为了能够开发出预防新冠病毒的疫苗，我们需要寻找一种能够有效活化中和抗体的免疫原。As we all know, in the prevention of infectious diseases, vaccines have been the most effective and economical measures since they came out, and most of the current commonly used preventive vaccines are aimed at activating neutralizing antibodies. Therefore, in order to be able to develop a vaccine against the new coronavirus, we need to find an immunogen that can effectively activate neutralizing antibodies.
冠状病毒包含四种结构蛋白，包括突刺蛋白(S蛋白)、包膜蛋白、膜蛋白和核衣壳蛋白。其中，S蛋白在病毒的附着、融合和进入过程中起着最重要的作用，也是抗体、进入抑制剂和疫苗的主要靶点。S蛋白介导病毒进入宿主细胞，首先通过S1亚基的受体结合域(RBD)与宿主受体结合，然后通过S2亚基融合病毒和宿主细胞膜。Coronavirus contains four structural proteins, including spike protein (S protein), envelope protein, membrane protein and nucleocapsid protein. Among them, S protein plays the most important role in the process of virus attachment, fusion and entry, and is also the main target of antibodies, entry inhibitors and vaccines. The S protein mediates the virus into the host cell, firstly binds to the host receptor through the receptor binding domain (RBD) of the S1 subunit, and then fuses the virus and the host cell membrane through the S2 subunit.
WHO的《COVID-19全球研究路线图》里指出：冠状病毒疫苗免疫后的动物，再次暴露于活病毒时，可能发生更严重的症状。疫苗免疫产生的非中和抗体或较低的抗体水平可能会引起抗体依赖性增强效应(antibody-dependent enhancement，ADE)，增强病毒致病性。概括来说，中和抗体阻断病毒与细胞表面受体结合，中和病毒感染能力；非中和抗体结合病毒但在一些情况下，抗体Fc段与细胞表面Fc受体结合，使其可以感染表达Fc受体的细胞。因此，为了减少ADE副作用， 新冠病毒S蛋白中的RBD区将是最为有效的疫苗研发靶点。The WHO's "Global Research Roadmap for COVID-19" pointed out that animals immunized with coronavirus vaccines may experience more severe symptoms when they are exposed to live viruses again. The non-neutralizing antibodies or low antibody levels produced by vaccine immunization may cause antibody-dependent enhancement (ADE) and enhance the pathogenicity of the virus. In summary, neutralizing antibodies block the virus from binding to cell surface receptors and neutralize the ability of the virus to infect; non-neutralizing antibodies bind to the virus, but in some cases, the Fc segment of the antibody binds to the cell surface Fc receptor, making it possible to infect Cells that express Fc receptors. Therefore, in order to reduce the side effects of ADE, the RBD region in the S protein of the new coronavirus will be the most effective target for vaccine development.
RBD在S蛋白中的天然构象为三聚体，若将RBD单独纯化，那么少了S2亚基三聚体的支撑，其构象很有可能会被改变。因此如何维持RBD的构象是需要解决的问题。The natural conformation of RBD in the S protein is a trimer. If RBD is purified alone, there will be less support from the S2 subunit trimer, and its conformation is likely to be changed. Therefore, how to maintain the RBD conformation is a problem that needs to be solved.
本领域中迫切需要开发出可有效产生新冠病毒中和抗体的新型疫苗。There is an urgent need in this field to develop new vaccines that can effectively produce new coronavirus neutralizing antibodies.
发明内容Summary of the invention
本公开中采用新冠病毒的RBD特定区域作为免疫原，同时对RBD区进行一系列修饰，例如加入二硫键、融合蛋白、融合细胞因子，进而使其可有效诱导抗SARS-CoV-2的中和抗体。In the present disclosure, the specific RBD region of the new coronavirus is used as an immunogen, and a series of modifications are made to the RBD region at the same time, such as adding disulfide bonds, fusion proteins, and fusion cytokines, so that it can effectively induce anti-SARS-CoV-2. And antibodies.
在本公开的一个方面中，提供了一种针对新型冠状病毒SARS-CoV-2的免疫原性肽，其包括SARS-CoV-2病毒刺突蛋白S的RBD区，所述RBD区进一步经半胱氨酸修饰形成sRBD区。In one aspect of the present disclosure, there is provided an immunogenic peptide against the novel coronavirus SARS-CoV-2, which includes the RBD region of the spike protein S of the SARS-CoV-2 virus, and the RBD region is further processed by half Cystine modification forms the sRBD region.
在一些实施方式中，所述经半胱氨酸修饰为在RBD结构域的根部增加一对半胱氨酸，使之能够形成二硫键。In some embodiments, the cysteine modification is to add a pair of cysteines at the root of the RBD domain to enable the formation of disulfide bonds.
在一些实施方式中，所述免疫原性肽选自：(a)具有SEQ ID NO：2、4、8或10所示氨基酸序列的多肽；(b)(a)中所述多肽的同源多肽，例如其与SEQ ID NO：2、4、8或10具有高于或等于90％，高于或等于95％，高于或等于96％，高于或等于97％，高于或等于98％，高于或等于99％的同源性；(c)在(a)限定的氨基酸序列中经过取代、缺失或添加一个或几个氨基酸且具有免疫原性的由(a)衍生的蛋白质或多肽。In some embodiments, the immunogenic peptide is selected from: (a) a polypeptide having the amino acid sequence shown in SEQ ID NO: 2, 4, 8 or 10; (b) a homology of the polypeptide described in (a) Polypeptide, for example, with SEQ ID NO: 2, 4, 8 or 10, it is higher than or equal to 90%, higher than or equal to 95%, higher than or equal to 96%, higher than or equal to 97%, higher than or equal to 98 %, higher than or equal to 99% homology; (c) The immunogenic protein or protein derived from (a) that has been substituted, deleted or added one or several amino acids in the amino acid sequence defined in (a) Peptides.
在一些实施方式中，所述RBD区的氨基酸序列如SEQ ID NO：2所示，所述经半胱氨酸修饰的sRBD区的氨基酸序列如SEQ ID NO：4所示。In some embodiments, the amino acid sequence of the RBD region is shown in SEQ ID NO: 2, and the amino acid sequence of the cysteine-modified sRBD region is shown in SEQ ID NO: 4.
在一些实施方式中，所述RBD区或经半胱氨酸修饰的sRBD区包含在融合肽中，例如与之融合的部分包括：病毒或宿主来源的蛋白，转铁蛋白(Fn)、HIV p24、囊膜病毒的茎部，如流感HA2、艾滋病毒的gp41、抗体Fc段、GM-CSF、IL-21、CD40L或CD40抗体。In some embodiments, the RBD region or the cysteine-modified sRBD region is included in the fusion peptide, for example, the part fused with it includes: virus or host-derived protein, transferrin (Fn), HIV p24 , The stem of enveloped virus, such as influenza HA2, HIV gp41, antibody Fc segment, GM-CSF, IL-21, CD40L or CD40 antibody.
在一些实施方式中，所述融合肽可进一步包括：信号肽、连接肽、标签等元件。例如，信号肽可选自：蛋白自身、CD33、CD8、CD16、小鼠IgG1抗体、流 感HA。连接肽可选自：(G4S) 3、(G4S) n、GSAGSAAGSGEF、(Gly)6、EFPKPSTPPGSSGGAP、KESGSVSSEQLAQFRSLD、(Gly)8、EGKSSGSGSESKST。标签可选自：His-tag、AviTag、Calmodulin tag、polyglutamate tag、E-tag、FLAG tag、HA-tag、Myc-tag、S-tag、SBP-tag、Sof-tag 1、Sof-tag3、Strep-tag、TC tag、V5 tag、T7 tag、VSV tag、Xpress tag、3X FLAG tag、Isopep tag、Spytag、Snoop tag和PNE tag。 In some embodiments, the fusion peptide may further include: signal peptide, connecting peptide, tag and other elements. For example, the signal peptide can be selected from: protein itself, CD33, CD8, CD16, mouse IgG1 antibody, influenza HA. The connecting peptide can be selected from: (G4S) 3 , (G4S) n , GSAGSAAGSGEF, (Gly)6, EFPKPSTPPGSSGGAP, KESGSVSSEQLAQFRSLD, (Gly)8, EGKSSGSGSESKST. Tags can be selected from: His-tag, AviTag, Calmodulin tag, polyglutamate tag, E-tag, FLAG tag, HA-tag, Myc-tag, S-tag, SBP-tag, Sof-tag 1, Sof-tag3, Strep -tag, TC tag, V5 tag, T7 tag, VSV tag, Xpress tag, 3X FLAG tag, Isopep tag, Spytag, Snoop tag and PNE tag.
在一些实施方式中，所述免疫原性肽为sRBD-hFn、sRBD-HA2等。In some embodiments, the immunogenic peptide is sRBD-hFn, sRBD-HA2 and the like.
在一些实施方式中，所述融合肽的序列如SEQ ID NO：8或SEQ ID NO：10所示。In some embodiments, the sequence of the fusion peptide is as shown in SEQ ID NO: 8 or SEQ ID NO: 10.
在本公开的一个方面中，提供了一种核苷酸分子，其编码本公开的免疫原性肽。In one aspect of the present disclosure, there is provided a nucleotide molecule that encodes the immunogenic peptide of the present disclosure.
在一些实施方式中，所述核苷酸分子选自：(i)序列如SEQ ID NO：1、3、7或9所示的核苷酸分子；(ii)在严格条件下与(i)杂交的分子；(iii)与(i)或(ii)中序列具有高于或等于90％，高于或等于95％，高于或等于96％，高于或等于97％，高于或等于98％，高于或等于99％的同源性的核苷酸分子；(iv)在(i)或(ii)限定的核苷酸序列中经过取代、缺失或添加一个或几个核苷酸且能够表达功能性RBD免疫原性肽的核苷酸分子。In some embodiments, the nucleotide molecule is selected from: (i) nucleotide molecules whose sequence is as shown in SEQ ID NO: 1, 3, 7 or 9; (ii) and (i) under stringent conditions Hybrid molecules; (iii) and (i) or (ii) have a sequence higher than or equal to 90%, higher than or equal to 95%, higher than or equal to 96%, higher than or equal to 97%, higher than or equal to 98%, higher than or equal to 99% homology of nucleotide molecules; (iv) One or several nucleotides have been substituted, deleted or added in the nucleotide sequence defined in (i) or (ii) And can express the nucleotide molecule of functional RBD immunogenic peptide.
在一些实施方式中，RBD区的编码序列如SEQ ID NO：1所示，经半胱氨酸修饰的RBD区的编码序列如SEQ ID NO：3所示，或者所述核苷酸分子的序列如SEQ ID NO：7或SEQ ID NO：9所示。In some embodiments, the coding sequence of the RBD region is shown in SEQ ID NO: 1, and the coding sequence of the cysteine modified RBD region is shown in SEQ ID NO: 3, or the sequence of the nucleotide molecule Such as SEQ ID NO: 7 or SEQ ID NO: 9.
在一些实施方式中，所述核苷酸分子还包括编码与RBD区或与经半胱氨酸修饰的RBD区融合的部分的序列。In some embodiments, the nucleotide molecule further includes a sequence encoding a portion fused to an RBD region or a cysteine-modified RBD region.
在本公开的一个方面中，提供了一种载体，其包含本公开的核苷酸分子。In one aspect of the present disclosure, there is provided a vector comprising the nucleotide molecule of the present disclosure.
在一些实施方式中，所述载体为：病毒载体，如痘病毒(如天坛株、北美疫苗株、惠氏衍生株、李斯特株、安卡拉衍生株、哥本哈根株和纽约株痘病毒)、腺病毒(如Ad5、Ad11、Ad26、Ad35、Ad68)、慢病毒载体、腺相关病毒、单纯疱疹病毒、麻疹病毒、呼肠弧病毒、弹状病毒、森林脑炎病毒、流感病毒、呼吸道合胞病毒、脊髓灰质炎病毒载体。In some embodiments, the vector is: a viral vector, such as a poxvirus (such as a Tiantan strain, a North American vaccine strain, a Wyeth-derived strain, a Listeria strain, an Ankara-derived strain, a Copenhagen strain and a New York strain poxvirus), an adenovirus ( Such as Ad5, Ad11, Ad26, Ad35, Ad68), lentiviral vector, adeno-associated virus, herpes simplex virus, measles virus, reo virus, rhabdovirus, forest encephalitis virus, influenza virus, respiratory syncytial virus, spinal cord Poliovirus vector.
在本公开的一个方面中，提供了一种宿主细胞，其包含本公开的载体和/或能 表达本公开的免疫原性肽。In one aspect of the present disclosure, a host cell is provided, which contains the vector of the present disclosure and/or can express the immunogenic peptide of the present disclosure.
在一些实施方式中，所述宿主细胞为哺乳动物细胞或昆虫细胞，如HEK293、HeLa、K562、CHO、NS0、SP2/0、PER.C6、Vero、RD、BHK、HT 1080、A549、Cos-7、ARPE-19和MRC-5细胞；High Five、Sf9、Se301、SeIZD2109、SeUCR1、Sf9、Sf900+、Sf21、BTI-TN-5B1-4、MG-1、Tn368、HzAm1、BM-N、Ha2302、Hz2E5以及Ao38。In some embodiments, the host cell is a mammalian cell or an insect cell, such as HEK293, HeLa, K562, CHO, NS0, SP2/0, PER.C6, Vero, RD, BHK, HT 1080, A549, Cos- 7. ARPE-19 and MRC-5 cells; High Five, Sf9, Se301, SeIZD2109, SeUCR1, Sf9, Sf900+, Sf21, BTI-TN-5B1-4, MG-1, Tn368, HzAm1, BM-N, Ha2302 Hz2E5 and Ao38.
在一些实施方式中，所述宿主细胞为K562。在一些实施方式中，所述K562细胞包含sRBD-HA2。In some embodiments, the host cell is K562. In some embodiments, the K562 cells comprise sRBD-HA2.
由此，本公开中还提供了一种在其细胞膜表面展示本文所述新型冠状病毒SARS-CoV-2免疫原性肽的细胞。在一些实施方式中，所述细胞包含具有本文所述免疫原性肽编码序列的载体。在一些实施方式中，所述细胞转入了具有免疫原性肽编码序列的载体。在一些实施方式中，所述细胞具有展示所述免疫原性肽的完整膜结构。在一些实施方式中，所述细胞是已灭活的细胞，例如采用物理灭活如X-射线辐射、紫外辐射；或化学灭活如β-丙内酯、甲醛、多聚甲醛固定。Therefore, the present disclosure also provides a cell displaying the immunogenic peptide of the novel coronavirus SARS-CoV-2 described herein on its cell membrane surface. In some embodiments, the cell comprises a vector having the immunogenic peptide coding sequence described herein. In some embodiments, the cells are transferred into a vector with an immunogenic peptide coding sequence. In some embodiments, the cell has a complete membrane structure displaying the immunogenic peptide. In some embodiments, the cells are inactivated cells, for example, physical inactivation such as X-ray radiation, ultraviolet radiation; or chemical inactivation such as β-propiolactone, formaldehyde, and paraformaldehyde fixation.
在本公开的一个方面中，提供了一种针对新型冠状病毒SARS-CoV-2的疫苗，其包含本公开的免疫原性肽、核苷酸分子、载体和/或宿主细胞。In one aspect of the present disclosure, a vaccine against the novel coronavirus SARS-CoV-2 is provided, which comprises the immunogenic peptides, nucleotide molecules, vectors and/or host cells of the present disclosure.
在本公开的一个方面中，还提供了本公开的免疫原性肽、核苷酸分子、载体和/或宿主细胞在制备用于预防或治疗新型冠状病毒SARS-CoV-2的疫苗中的应用。In one aspect of the present disclosure, the use of the immunogenic peptides, nucleotide molecules, vectors and/or host cells of the present disclosure in the preparation of vaccines for the prevention or treatment of the novel coronavirus SARS-CoV-2 is also provided .
在本公开的一个方面中，还提供了本公开的免疫原性肽、核苷酸分子、载体和/或宿主细胞，其用于预防或治疗新型冠状病毒SARS-CoV-2。In one aspect of the present disclosure, the immunogenic peptides, nucleotide molecules, vectors and/or host cells of the present disclosure are also provided, which are used to prevent or treat the new coronavirus SARS-CoV-2.
在本公开的一个方面中，还提供了一种预防或治疗新型冠状病毒SARS-CoV-2的方法，所述方法包括给予有需要的对象本公开的免疫原性肽、核苷酸分子、载体、宿主细胞和/或疫苗。In one aspect of the present disclosure, there is also provided a method for preventing or treating the novel coronavirus SARS-CoV-2, the method comprising administering to a subject in need the immunogenic peptides, nucleotide molecules, and vectors of the present disclosure , Host cells and/or vaccines.
在一些实施方式中，所述疫苗为核酸疫苗(DNA或RNA疫苗)、重组蛋白亚单位疫苗、重组病毒载体疫苗、重组细菌载体疫苗、病毒样颗粒疫苗、纳米颗粒疫苗、细胞载体疫苗。In some embodiments, the vaccine is a nucleic acid vaccine (DNA or RNA vaccine), a recombinant protein subunit vaccine, a recombinant viral vector vaccine, a recombinant bacterial vector vaccine, a virus-like particle vaccine, a nanoparticle vaccine, and a cell vector vaccine.
在一些实施方式中，所述疫苗包含佐剂或与佐剂联用，所述佐剂包括但不限于：铝佐剂、霍乱毒素及其亚单位、寡脱氧核苷酸、锰离子佐剂、胶体锰佐剂、弗氏佐剂、SAS佐剂、MF59佐剂、QS-21佐剂、Poly I：C及其他TLR配体、GM-CSF、 IL-2、IL-3、IL-7、IL-11、IL-12、IL-18、IL-21等。In some embodiments, the vaccine contains an adjuvant or is used in combination with an adjuvant, including but not limited to: aluminum adjuvant, cholera toxin and its subunits, oligodeoxynucleotide, manganese ion adjuvant, Colloidal manganese adjuvant, Freund's adjuvant, SAS adjuvant, MF59 adjuvant, QS-21 adjuvant, Poly I: C and other TLR ligands, GM-CSF, IL-2, IL-3, IL-7, IL-11, IL-12, IL-18, IL-21, etc.
在一些实施方式中，所述疫苗的形式适于如下接种方式：肌肉接种、皮内接种、皮下接种、滴鼻、雾化吸入、生殖道、直肠、口服或其任意组合。In some embodiments, the form of the vaccine is suitable for the following vaccination methods: intramuscular vaccination, intradermal vaccination, subcutaneous vaccination, nose drops, nebulized inhalation, genital tract, rectum, oral administration or any combination thereof.
在一些实施方式中，采用一种或多种所述疫苗进行接种，例如联合接种或前后序贯接种。In some embodiments, one or more of the vaccines are used for vaccination, such as combined vaccination or sequential vaccination.
在一些实施方式中，采用一种或多种所述疫苗与其他针对新型冠状病毒的疫苗进行接种，例如所述其他疫苗包括针对冠状病毒S或S1的疫苗，例如所述的S或S1来自于包括但不限于SARS-CoV-2、SARS-CoV、MERS-CoV、HCoV-229E、HCoV-OC43、HCoV-NL63、HCoV-HKU1、bat-CoV等。In some embodiments, one or more of the vaccines and other vaccines against new coronaviruses are used for vaccination, for example, the other vaccines include vaccines against coronavirus S or S1, for example, the S or S1 is derived from Including but not limited to SARS-CoV-2, SARS-CoV, MERS-CoV, HCoV-229E, HCoV-OC43, HCoV-NL63, HCoV-HKU1, bat-CoV, etc.
在一些实施方式中，所述疫苗包含核酸疫苗(DNA或RNA疫苗)与重组人源细胞载体疫苗的组合。In some embodiments, the vaccine comprises a combination of a nucleic acid vaccine (DNA or RNA vaccine) and a recombinant human-derived cell vector vaccine.
在一些实施方式中，所述疫苗包含pcDNA3.1-sRBD-hFn与K562-HA2-sRBD的组合。在一些实施方式中，所述疫苗组合中的组分前后序贯接种，优选先接种DNA疫苗。In some embodiments, the vaccine comprises a combination of pcDNA3.1-sRBD-hFn and K562-HA2-sRBD. In some embodiments, the components of the vaccine combination are vaccinated sequentially, preferably the DNA vaccine is vaccinated first.
在本公开的一个方面中，提供了一种制备针对新型冠状病毒SARS-CoV-2的疫苗的方法，所述方法包括：In one aspect of the present disclosure, there is provided a method for preparing a vaccine against the novel coronavirus SARS-CoV-2, the method comprising:
(a)提供本公开的免疫原性肽、核苷酸分子、载体和/或宿主细胞；(a) Provide immunogenic peptides, nucleotide molecules, vectors and/or host cells of the present disclosure;
(b)将(a)中所提供的活性物质与免疫学上或药学上可接受的载体组合。(b) Combine the active substance provided in (a) with an immunologically or pharmaceutically acceptable carrier.
本领域的技术人员可对前述的技术方案和技术特征进行任意组合而不脱离本公开的发明构思和保护范围。本公开的其它方面由于本文的公开内容，对本领域的技术人员而言是显而易见的。Those skilled in the art can arbitrarily combine the aforementioned technical solutions and technical features without departing from the inventive concept and protection scope of the present disclosure. Other aspects of the present disclosure are obvious to those skilled in the art due to the content disclosed herein.
附图说明Description of the drawings
下面结合附图对本公开作进一步说明，其中这些显示仅为了图示说明本公开的实施方案，而不是为了局限本公开的范围。The present disclosure will be further described below in conjunction with the accompanying drawings, in which these displays are only for illustrating the embodiments of the present disclosure, and are not intended to limit the scope of the present disclosure.
图1：S蛋白及sRBD-hFn(二硫键改造)真核表达载体的构建及蛋白的表达：Figure 1: S protein and sRBD-hFn (disulfide bond modification) eukaryotic expression vector construction and protein expression:
图1A.pcDNA3.1-sRBD-hFn及pcDNA3.1-S真核表达载体质粒构建图谱；Figure 1A. Construction map of pcDNA3.1-sRBD-hFn and pcDNA3.1-S eukaryotic expression vector plasmids;
图1B.pcDNA3.1-sRBD-hFn及pcDNA3.1-S蛋白在293T细胞中成功表达。Figure 1B. pcDNA3.1-sRBD-hFn and pcDNA3.1-S proteins were successfully expressed in 293T cells.
图2：慢病毒表达载体pHAGE-S-puro及pHAGE-sRBD-HA2-puro的构建及S 蛋白和sRBD-HA2在K562细胞膜上展示的表达验证：Figure 2: Construction of lentiviral expression vectors pHAGE-S-puro and pHAGE-sRBD-HA2-puro and verification of expression of S protein and sRBD-HA2 displayed on K562 cell membrane:
图2A.pHAGE-S-puro及pHAGE-sRBD-HA2-puro慢病毒表达载体质粒构建图谱；Figure 2A. Construction map of pHAGE-S-puro and pHAGE-sRBD-HA2-puro lentiviral expression vector plasmids;
图2B.pHAGE-S-puro及pHAGE-sRBD-HA2-puro蛋白成功表达；Figure 2B. Successful expression of pHAGE-S-puro and pHAGE-sRBD-HA2-puro proteins;
图2C.sRBD能够与ACE2受体结合。Figure 2C. sRBD can bind to the ACE2 receptor.
图3：RBD蛋白对小鼠结合抗体和中和抗体的诱导效果：Figure 3: The induction effect of RBD protein on binding antibodies and neutralizing antibodies in mice:
实验所用小鼠为6-8周龄C57/BL6，免疫原为新冠病毒RBD蛋白及pcDNA3.1-S质粒，所述蛋白质均采用铝佐剂进行加强。The mice used in the experiment were 6-8 weeks old C57/BL6, and the immunogen was the new coronavirus RBD protein and pcDNA3.1-S plasmid, and the proteins were all enhanced with aluminum adjuvant.
图3A.ELISA方法检测免疫结束后第2周小鼠血清中结合抗体的滴度。横坐标为免疫组别，纵坐标为结合抗体的滴度；Figure 3A. ELISA method to detect the titers of bound antibodies in the serum of mice in the second week after the end of immunization. The abscissa is the immunization group, and the ordinate is the titer of the bound antibody;
图3B.293T-ACE2细胞检测免疫结束后第4周小鼠血清中中和抗体的滴度。横坐标为免疫组别，纵坐标为中和抗体的滴度(ID 50)。*表示p＜0.05。 Figure 3B. 293T-ACE2 cells detect the titers of neutralizing antibodies in mouse serum at the 4th week after the end of immunization. The abscissa is the immunization group, and the ordinate is the titer of neutralizing antibody (ID 50 ). * Indicates p<0.05.
图4：质粒pcDNA3.1-sRBD-hFn对小鼠结合抗体和中和抗体的诱导效果：Figure 4: Plasmid pcDNA3.1-sRBD-hFn induces binding antibodies and neutralizing antibodies in mice:
实验所用小鼠为6-8周龄C57/BL6，免疫原为新冠病毒S质粒pcDNA3.1-S及修饰后的RBD质粒pcDNA3.1-sRBD-hFn。The mice used in the experiment were 6-8 weeks old C57/BL6, and the immunogen was the new coronavirus S plasmid pcDNA3.1-S and the modified RBD plasmid pcDNA3.1-sRBD-hFn.
图4A.ELISA方法检测免疫结束后第2周小鼠血清中结合抗体的滴度。横坐标为免疫组别，纵坐标为结合抗体的滴度；Figure 4A. ELISA method to detect the titers of bound antibodies in the serum of mice in the second week after the end of immunization. The abscissa is the immunization group, and the ordinate is the titer of the bound antibody;
图4B.293T-ACE2细胞检测免疫结束后第2周小鼠血清中中和抗体的滴度。横坐标为免疫组别，纵坐标为中和抗体的滴度(ID 50)。 Figure 4B. 293T-ACE2 cells detect the titers of neutralizing antibodies in the serum of mice in the second week after the end of immunization. The abscissa is the immunization group, and the ordinate is the titer of neutralizing antibody (ID 50 ).
图5：K562-HA2-sRBD及K562-S细胞对小鼠结合抗体和中和抗体的诱导效果：Figure 5: The induction effect of K562-HA2-sRBD and K562-S cells on mouse binding antibodies and neutralizing antibodies:
实验所用小鼠为6-8周龄C57/BL6，免疫原为K562-HA2-sRBD及K562-S。The mice used in the experiment were 6-8 weeks old C57/BL6, and the immunogens were K562-HA2-sRBD and K562-S.
图5A.ELISA方法检测免疫结束后第2周小鼠血清中结合抗体的滴度。横坐标为免疫组别，纵坐标为结合抗体的滴度；Figure 5A. ELISA method to detect the titers of bound antibodies in the serum of mice in the second week after the end of immunization. The abscissa is the immunization group, and the ordinate is the titer of the bound antibody;
图5B.293T-ACE2细胞检测免疫结束后第2周小鼠血清中中和抗体的滴度。横坐标为免疫组别，纵坐标为中和抗体的滴度(ID 50)。 Figure 5B. 293T-ACE2 cells detect the titers of neutralizing antibodies in mouse serum at the second week after the end of immunization. The abscissa is the immunization group, and the ordinate is the titer of neutralizing antibody (ID 50 ).
图6：sRBD-hFn蛋白对小鼠结合抗体和中和抗体的诱导效果：Figure 6: Induction effect of sRBD-hFn protein on mouse binding antibody and neutralizing antibody:
实验所用小鼠为6-8周龄C57/BL6，免疫原为sRBD-hFn蛋白。The mice used in the experiment were 6-8 weeks old C57/BL6, and the immunogen was sRBD-hFn protein.
图6A.ELISA方法检测免疫后第1周小鼠血清中结合抗体的滴度。横坐标为 免疫组别，纵坐标为结合抗体的滴度；Figure 6A. ELISA method to detect the titers of bound antibodies in the serum of mice in the first week after immunization. The abscissa is the immunization group, and the ordinate is the titer of bound antibody;
图6B.293T-ACE2细胞检测免疫结束后第1周小鼠血清中中和抗体的滴度。横坐标为免疫组别，纵坐标为中和抗体的滴度(ID 50)。 Figure 6B. 293T-ACE2 cells detect the titers of neutralizing antibodies in the serum of mice in the first week after the end of immunization. The abscissa is the immunization group, and the ordinate is the titer of neutralizing antibody (ID 50 ).
具体实施方式detailed description
本公开涉及疫苗领域，特别涉及一种利用RBD区诱导抗新冠病毒(SARS-Cov-2)中和抗体的方法。采用该病毒的RBD区、改造的RBD区、融合其他蛋白或细胞因子的RBD区作为免疫原，可减少由非中和抗体或较低的抗体水平引起的抗体依赖性增强效果(ADE)，从而预防新冠病毒感染。动物实验结果证实，本公开的疫苗安全，可持续产生高效价中和抗体，可用于新冠病毒的预防和治疗。The present disclosure relates to the field of vaccines, and in particular to a method for inducing neutralizing antibodies against the new coronavirus (SARS-Cov-2) by using the RBD region. Using the RBD region of the virus, the modified RBD region, and the RBD region fused with other proteins or cytokines as immunogens can reduce the antibody-dependent enhancement effect (ADE) caused by non-neutralizing antibodies or lower antibody levels, thereby Prevent new coronavirus infection. Animal experiment results confirm that the vaccine of the present disclosure is safe, can continuously produce high-titer neutralizing antibodies, and can be used for the prevention and treatment of the new coronavirus.
本文中提供的所有数值范围旨在清楚地包括落在范围端点之间的所有数值及它们之间的数值范围。可对本公开提到的特征或实施例提到的特征进行组合。本说明书所揭示的所有特征可与任何组合物形式并用，说明书中所揭示的各个特征，可以任何可提供相同、均等或相似目的的替代性特征取代。因此除有特别说明，所揭示的特征仅为均等或相似特征的一般性例子。All numerical ranges provided herein are intended to clearly include all numerical values that fall between the end points of the range and numerical ranges between them. The features mentioned in the present disclosure or the features mentioned in the embodiments can be combined. All the features disclosed in this specification can be used in combination with any composition form, and each feature disclosed in the specification can be replaced by any alternative feature that can provide the same, equal or similar purpose. Therefore, unless otherwise specified, the disclosed features are only general examples of equal or similar features.
如本文所用，在数值或范围上下文中的“约”表示所引用或要求保护的数值或范围的±10％。As used herein, "about" in the context of a value or range means ±10% of the value or range cited or claimed.
应理解，当提供参数范围时，本发明同样提供了在该范围内的所有整数及其十分位小数。例如，“0.1-2.5毫克/天”包括0.1毫克/天、0.2毫克/天、0.3毫克/天等直至2.5毫克/天。It should be understood that when a parameter range is provided, the present invention also provides all integers and deciles within the range. For example, "0.1-2.5 mg/day" includes 0.1 mg/day, 0.2 mg/day, 0.3 mg/day, etc. up to 2.5 mg/day.
如本文所用，“含有”、“具有”或“包括”包括了“包含”、“主要由......构成”、“基本上由......构成”、和“由......构成”；“主要由......构成”、“基本上由......构成”和“由......构成”属于“含有”、“具有”或“包括”的下位概念。As used herein, "comprising", "having" or "including" includes "comprising", "consisting essentially of", "consisting essentially of", and "consisting of. ..... constituted"; "mainly constituted by", "basically constituted by ..." and "constituted by ..." belong to "contains" and "has "Or "include" subordinate concept.
RBD免疫原性肽及其编码分子RBD immunogenic peptide and its coding molecule
如本文所用，术语“RBD免疫原性肽”、“针对新型冠状病毒SARS-CoV-2的免疫原性肽”和“本公开/本申请的免疫原性肽”等可互换使用，是指包括SARS-CoV-2病毒刺突蛋白S的RBD区或经半胱氨酸修饰的RBD区(在本文中亦称sRBD区)、且具有激发结合抗体和中和抗体作用的肽。As used herein, the terms "RBD immunogenic peptide", "immunogenic peptide against the novel coronavirus SARS-CoV-2" and "immunogenic peptide of the present disclosure/application" are used interchangeably and refer to It includes the RBD region of the spike protein S of the SARS-CoV-2 virus or the RBD region modified with cysteine (also referred to as the sRBD region herein), and has peptides that stimulate binding antibodies and neutralize antibodies.
在本公开的一些实施方式中，免疫原性肽可为：(a)具有SEQ ID NO：2、4、8或10所示氨基酸序列的多肽；(b)(a)中所述多肽的同源多肽，例如其与SEQ ID NO：2、4、8或10具有高于或等于90％，高于或等于95％，高于或等于96％，高于或等于97％，高于或等于98％，高于或等于99％的同源性；(c)在(a)限定的氨基酸序列中经过取代、缺失或添加一个或几个氨基酸且具有免疫原性的由(a)衍生的蛋白质或多肽。In some embodiments of the present disclosure, the immunogenic peptide may be: (a) a polypeptide having the amino acid sequence shown in SEQ ID NO: 2, 4, 8, or 10; (b) the same polypeptide as described in (a) Source polypeptide, for example, with SEQ ID NO: 2, 4, 8 or 10, it is higher than or equal to 90%, higher than or equal to 95%, higher than or equal to 96%, higher than or equal to 97%, higher than or equal to 98%, higher than or equal to 99% homology; (c) A protein derived from (a) with immunogenicity after substitution, deletion or addition of one or several amino acids in the amino acid sequence defined in (a) Or polypeptide.
在一些情况下，免疫原性肽可包括与RBD区或经半胱氨酸修饰的RBD区连接的其他部分，以例如增强RBD区的稳定性、提高中和抗体应答、形成多聚体、增加细胞应答等。可修饰或未修饰的RBD区连接的部分包括但不限于：病毒或宿主来源的蛋白，转铁蛋白(Fn)、HIV p24、囊膜病毒的茎部，如流感HA2、艾滋病毒的gp41、抗体Fc段、GM-CSF、IL-21、CD40L或CD40抗体等。例如，所述融合肽的序列如SEQ ID NO：8或SEQ ID NO：10所示。In some cases, the immunogenic peptide may include other parts connected to the RBD region or the RBD region modified with cysteine, for example, to enhance the stability of the RBD region, improve the neutralizing antibody response, form multimers, and increase Cell response, etc. Modifiable or unmodified RBD regions include, but are not limited to: virus or host-derived proteins, transferrin (Fn), HIV p24, the stem of enveloped viruses, such as influenza HA2, HIV gp41, antibodies Fc segment, GM-CSF, IL-21, CD40L or CD40 antibody, etc. For example, the sequence of the fusion peptide is shown in SEQ ID NO: 8 or SEQ ID NO: 10.
在一些情况下，融合肽中还可包括信号肽、接头、分子标签等元件。例如，信号肽元件可指具有引导融合蛋白分泌、定位和/或输送功能的氨基酸序列，其长度通常为5-30个氨基酸。在一些实施方式中，信号肽元件可选自：蛋白自身信号肽、CD33蛋白信号肽、CD8蛋白信号肽、CD16蛋白信号肽、小鼠IgG1抗体信号肽、流感HA蛋白信号肽。例如，连接肽序列(或称接头)可指在本文的融合蛋白中起到连接不同元件作用的短肽，其长度通常为1～50(如5～50、5～40、10～40)个氨基酸。通常，连接肽不影响或严重影响本发明的融合蛋白形成正确的折叠和空间构象。在一些实施方式中，连接肽元件可选自：(G 4S) 3接头、(G4S) n、GSAGSAAGSGEF、(Gly)6、EFPKPSTPPGSSGGAP、KESGSVSSEQLAQFRSLD、(Gly)8、EGKSSGSGSESKST。 In some cases, the fusion peptide may also include signal peptides, linkers, molecular tags and other elements. For example, the signal peptide element may refer to an amino acid sequence that has the function of guiding the secretion, localization and/or delivery of the fusion protein, and its length is usually 5-30 amino acids. In some embodiments, the signal peptide element may be selected from the group consisting of: protein signal peptide, CD33 protein signal peptide, CD8 protein signal peptide, CD16 protein signal peptide, mouse IgG1 antibody signal peptide, influenza HA protein signal peptide. For example, the linking peptide sequence (or linker) may refer to a short peptide that serves to link different elements in the fusion protein herein, and its length is usually 1-50 (such as 5-50, 5-40, 10-40). Amino acids. Generally, the connecting peptide does not affect or severely affect the formation of the correct folding and spatial conformation of the fusion protein of the present invention. In some embodiments, the connecting peptide component selected from: (G 4 S) 3 linker, (G4S) n, GSAGSAAGSGEF, (Gly) 6, EFPKPSTPPGSSGGAP, KESGSVSSEQLAQFRSLD, (Gly) 8, EGKSSGSGSESKST.
免疫原性肽也可包括其变异形式，例如一个或多个(通常为1-50个，较佳地1-30个，更佳地1-20个，最佳地1-10个，例如1、2、3、4、5、6、7、8、9或10个)氨基酸的缺失、***和/或取代，以及在C末端和/或N末端添加一个或数个(通常为20个以内，较佳地为10个以内，更佳地为5个以内)氨基酸。例如，在本领域中，用性能相近或相似的氨基酸进行取代时，通常不会改变蛋白质或多肽的功能。又比如，在C末端和/或N末端添加一个或数个氨基酸通常也不会改变蛋白质或多肽的功能。The immunogenic peptide may also include its variant form, such as one or more (usually 1-50, preferably 1-30, more preferably 1-20, most preferably 1-10, such as 1 , 2, 3, 4, 5, 6, 7, 8, 9 or 10) deletion, insertion and/or substitution of amino acids, and the addition of one or several (usually within 20) at the C-terminus and/or N-terminus , Preferably within 10, more preferably within 5) amino acids. For example, in the art, when amino acids with similar or similar properties are substituted, the function of the protein or polypeptide is usually not changed. For another example, adding one or several amino acids to the C-terminus and/or N-terminus usually does not change the function of the protein or polypeptide.
免疫原性肽可在适当的环境和条件下通过重组表达产生，例如由本公开的编码核苷酸分子、载体、宿主细胞产生；也可通过化学合成等方式获得，只要其具有所需的氨基酸序列和免疫原性和反应性。The immunogenic peptide can be produced by recombinant expression under appropriate environment and conditions, for example, produced by the encoding nucleotide molecules, vectors, and host cells of the present disclosure; it can also be obtained by chemical synthesis, as long as it has the required amino acid sequence And immunogenicity and reactivity.
如本文所用，术语“免疫原性肽编码分子”、“RBD编码序列”等可互换使用，均是指编码本公开所述的免疫原性肽的核苷酸分子。所述核酸分子可选自，例如：(i)序列如SEQ ID NO：1、3、7或9所示的核苷酸分子；(ii)在严格条件下与(i)杂交的分子；(iii)与(i)或(ii)中序列具有高于或等于90％，高于或等于95％，高于或等于96％，高于或等于97％，高于或等于98％，高于或等于99％的同源性的核苷酸分子；(iv)在(i)或(ii)限定的核苷酸序列中经过取代、缺失或添加一个或几个核苷酸且能够表达功能性RBD免疫原性肽的核苷酸分子。As used herein, the terms "immunogenic peptide coding molecule", "RBD coding sequence" and the like are used interchangeably, and both refer to nucleotide molecules encoding the immunogenic peptides described in the present disclosure. The nucleic acid molecule can be selected from, for example: (i) a nucleotide molecule whose sequence is as shown in SEQ ID NO: 1, 3, 7 or 9; (ii) a molecule that hybridizes with (i) under stringent conditions; iii) The sequence in (i) or (ii) is higher than or equal to 90%, higher than or equal to 95%, higher than or equal to 96%, higher than or equal to 97%, higher than or equal to 98%, higher than Or a nucleotide molecule with 99% homology; (iv) One or several nucleotides are substituted, deleted or added in the nucleotide sequence defined in (i) or (ii) and can express functionality Nucleotide molecule of RBD immunogenic peptide.
如本文所用，术语“严格条件”是指：(1)在较低离子强度和较高温度下的杂交和洗脱，如0.2×SSC，0.1％SDS，60℃；或(2)杂交时加有变性剂，如50％(v/v)甲酰胺，0.1％小牛血清/0.1％Ficoll，42℃等；或(3)仅在两条序列之间的相同性至少在50％，优选55％以上、60％以上、65％以上、70％以上、75％以上、80％以上、85％以上或90％以上，更优选是95％以上时才发生杂交。As used herein, the term "stringent conditions" refers to: (1) hybridization and elution at lower ionic strength and higher temperature, such as 0.2×SSC, 0.1% SDS, 60°C; or (2) adding during hybridization There are denaturants, such as 50% (v/v) formamide, 0.1% calf serum/0.1% Ficoll, 42°C, etc.; or (3) only the identity between the two sequences is at least 50%, preferably 55 % Or more, 60% or more, 65% or more, 70% or more, 75% or more, 80% or more, 85% or more or 90% or more, more preferably 95% or more.
本公开的核苷酸全长序列或其片段通常可以用PCR扩增法、重组法或人工合成的方法获得。对于PCR扩增法，可根据本公开所公开的有关核苷酸序列来设计引物，并用市售的cDNA库或按本领域技术人员已知的常规方法所制备的cDNA库作为模板，扩增而得有关序列。当序列较长时，常常需要进行两次或多次PCR扩增，然后再将各次扩增出的片段按正确次序拼接在一起。The full-length nucleotide sequence of the present disclosure or fragments thereof can usually be obtained by PCR amplification method, recombination method or artificial synthesis method. For the PCR amplification method, primers can be designed according to the relevant nucleotide sequences disclosed in the present disclosure, and a commercially available cDNA library or a cDNA library prepared according to a conventional method known to those skilled in the art can be used as a template for amplification. Get the sequence. When the sequence is long, it is often necessary to perform two or more PCR amplifications, and then splice the amplified fragments together in the correct order.
载体和宿主细胞Vectors and host cells
本公开还涉及包含RBD编码核苷酸分子的载体，以及用该载体经基因工程产生的宿主细胞。The present disclosure also relates to a vector containing a nucleotide molecule encoding RBD, and a host cell produced by genetic engineering using the vector.
通过常规的重组DNA技术(Science，1984；224：1431)，可利用本公开的编码序列可用来表达或生产重组的免疫原性肽。一般来说有以下步骤：Through conventional recombinant DNA technology (Science, 1984; 224:1431), the coding sequence of the present disclosure can be used to express or produce recombinant immunogenic peptides. Generally speaking, there are the following steps:
(1)将本公开的编码核苷酸分子，或将含有核苷酸分子的重组表达载体转入合适的宿主细胞；(1) Transform the encoding nucleotide molecules of the present disclosure or the recombinant expression vector containing the nucleotide molecules into a suitable host cell;
(2)在合适的培养基中培养的宿主细胞；(2) Host cells cultured in a suitable medium;
(3)从培养基或细胞中分离、纯化蛋白质或多肽。(3) Separating and purifying proteins or polypeptides from culture media or cells.
本公开中，术语“载体”与“重组表达载体”可互换使用，指本领域熟知的细菌质粒、噬菌体、酵母质粒、动物细胞病毒、哺乳动物细胞病毒或其它载体。表达载体的一个重要特征是通常含有复制起点、启动子、标记基因和翻译控制元件。In the present disclosure, the terms "vector" and "recombinant expression vector" are used interchangeably and refer to bacterial plasmids, bacteriophages, yeast plasmids, animal cell viruses, mammalian cell viruses or other vectors that are well known in the art. An important feature of an expression vector is that it usually contains an origin of replication, a promoter, a marker gene, and translation control elements.
可采用本领域常规方法构建含RBD免疫原性肽编码序列和合适的转录/翻译控制信号的表达载体。这些方法包括体外重组DNA技术、DNA合成技术、体内重组技术等。所述的DNA序列可有效连接到表达载体中的适当启动子上，以指导mRNA合成。表达载体还包括翻译起始用的核糖体结合位点和转录终止子。本公开中可采用诸如pcDNA3.1载体、pIRES2-EGFP载体、AdMaxTM等表达***。Conventional methods in the art can be used to construct expression vectors containing RBD immunogenic peptide coding sequences and appropriate transcription/translation control signals. These methods include in vitro recombinant DNA technology, DNA synthesis technology, and in vivo recombination technology. The DNA sequence can be effectively linked to an appropriate promoter in the expression vector to guide mRNA synthesis. The expression vector also includes a ribosome binding site for translation initiation and a transcription terminator. Expression systems such as pcDNA3.1 vector, pIRES2-EGFP vector, AdMaxTM, etc. can be used in the present disclosure.
此外，表达载体可包含一个或多个选择性标记基因，以提供用于选择转化的宿主细胞的表型性状，如真核细胞培养用的二氢叶酸还原酶、新霉素抗性以及绿色荧光蛋白(GFP)，或用于大肠杆菌的四环素或氨苄青霉素抗性。In addition, the expression vector may contain one or more selectable marker genes to provide phenotypic traits for selecting transformed host cells, such as dihydrofolate reductase for eukaryotic cell culture, neomycin resistance, and green fluorescence Protein (GFP), or tetracycline or ampicillin resistance for E. coli.
包含上述的适当DNA序列以及适当启动子或者控制序列的载体，可以用于转化适当的宿主细胞，以使其能够表达蛋白质或多肽。宿主细胞可以是原核细胞，如细菌细胞；或是低等真核细胞，如酵母细胞；或是高等真核细胞，如动物细胞。代表性例子有：大肠杆菌，链霉菌属、农杆菌；真菌细胞如酵母；动物细胞等。在本公开中，可采用例如选自下组的宿主细胞：HEK293、HeLa、CHO、K562、NS0、SP2/0、PER.C6、Vero、RD、BHK、HT 1080、A549、Cos-7、ARPE-19和MRC-5细胞；High Five、Sf9、Se301、SeIZD2109、SeUCR1、Sf9、Sf900+、Sf21、BTI-TN-5B1-4、MG-1、Tn368、HzAm1、BM-N、Ha2302、Hz2E5以及Ao38。A vector containing the above-mentioned appropriate DNA sequence and an appropriate promoter or control sequence can be used to transform an appropriate host cell so that it can express a protein or polypeptide. The host cell can be a prokaryotic cell, such as a bacterial cell; or a lower eukaryotic cell, such as a yeast cell; or a higher eukaryotic cell, such as an animal cell. Representative examples are: Escherichia coli, Streptomyces, Agrobacterium; fungal cells such as yeast; animal cells, etc. In the present disclosure, for example, host cells selected from the group consisting of HEK293, HeLa, CHO, K562, NS0, SP2/0, PER.C6, Vero, RD, BHK, HT 1080, A549, Cos-7, ARPE can be used -19 and MRC-5 cells; High Five, Sf9, Se301, SeIZD2109, SeUCR1, Sf9, Sf900+, Sf21, BTI-TN-5B1-4, MG-1, Tn368, HzAm1, BM-N, Ha2302, Hz2E5 and Ao38 .
本公开的核苷酸分子在高等真核细胞中表达时，如果在载体中***增强子序列时将会使转录得到增强。增强子是DNA的顺式作用因子，通常大约有10到300个碱基对，作用于启动子以增强基因的转录。本领域一般技术人员都清楚如何选择适当的载体、启动子、增强子和宿主细胞。When the nucleotide molecules of the present disclosure are expressed in higher eukaryotic cells, if an enhancer sequence is inserted into the vector, the transcription will be enhanced. Enhancers are cis-acting factors of DNA, usually about 10 to 300 base pairs, acting on promoters to enhance gene transcription. Those of ordinary skill in the art know how to select appropriate vectors, promoters, enhancers and host cells.
在上面的方法中的重组多肽可在细胞内或在细胞膜上表达或分泌到细胞外。如果需要，可利用其物理的、化学的和其它特性通过各种分离方法分离和纯化重组的蛋白。这些方法是本领域技术人员所熟知的。这些方法的例子包括但并不限于：常规的复性处理、用蛋白沉淀剂处理(盐析方法)、离心、渗透破菌、超处理、超离心、分子筛层析(凝胶过滤)、吸附层析、离子交换层析、高效液相层析(HPLC) 和其它各种液相层析技术及这些方法的结合。The recombinant polypeptide in the above method can be expressed in the cell or on the cell membrane or secreted out of the cell. If necessary, the physical, chemical, and other characteristics can be used to separate and purify the recombinant protein through various separation methods. These methods are well known to those skilled in the art. Examples of these methods include, but are not limited to: conventional renaturation treatment, treatment with protein precipitation agent (salting out method), centrifugation, osmotic sterilization, ultra-treatment, ultra-centrifugation, molecular sieve chromatography (gel filtration), adsorption layer Analysis, ion exchange chromatography, high performance liquid chromatography (HPLC) and various other liquid chromatography techniques and combinations of these methods.
疫苗和免疫偶联物Vaccines and immunoconjugates
本文中还提供了一种包含本公开的免疫原性肽、核苷酸分子、载体和/或宿主细胞的疫苗，或称免疫组合物。该疫苗包含其形式能够被给予脊椎动物(优选哺乳动物)的本公开的免疫原性肽和/或核酸分子的配制品，并且其诱导提高免疫力的保护性免疫应答以预防和/或减轻新型冠状病毒和/或其至少一种症状。Also provided herein is a vaccine, or immune composition, comprising the immunogenic peptide, nucleotide molecule, vector and/or host cell of the present disclosure. The vaccine comprises a formulation of the immunogenic peptide and/or nucleic acid molecule of the present disclosure in a form that can be administered to a vertebrate (preferably a mammal), and it induces a protective immune response that improves immunity to prevent and/or alleviate the novel Coronavirus and/or at least one symptom thereof.
术语“保护性免疫应答”或“保护性应答”是指通过免疫原介导的针对传染原或疾病的免疫应答，通过脊椎动物(例如人)展现，预防或减轻感染或减少其至少一种疾病症状。The term "protective immune response" or "protective response" refers to an immune response mediated by an immunogen against an infectious agent or disease, displayed by vertebrates (such as humans), preventing or reducing infection or reducing at least one disease symptom.
术语“脊椎动物”或“对象”或“患者”是指脊索动物亚门的任何成员，包括但不限于：人和其他灵长类动物，包括非人灵长类动物诸如黑猩猩和其他猿和猴物种；家畜诸如牛、绵羊、猪、山羊和马；家养哺乳动物诸如狗和猫；实验室动物，包括啮齿动物诸如小鼠、大鼠和豚鼠；鸟包括驯养、野生和猎鸟诸如鸡、火鸡和其他鹑鸡类鸟、鸭、鹅。术语“哺乳动物”和“动物”被包括在这个定义中，旨在涵盖成年、幼年以及新生个体。The term "vertebrate" or "subject" or "patient" refers to any member of the chordate subphylum, including but not limited to: humans and other primates, including non-human primates such as chimpanzees and other apes and monkeys Species; domestic animals such as cattle, sheep, pigs, goats, and horses; domestic mammals such as dogs and cats; laboratory animals, including rodents such as mice, rats, and guinea pigs; birds include domesticated, wild, and game birds such as chickens, fire Chickens and other quail chickens, ducks, and geese. The terms "mammal" and "animal" are included in this definition and are intended to cover adult, juvenile and newborn individuals.
本文的疫苗可为重组蛋白疫苗、重组DNA疫苗、重组病毒载体疫苗(例如腺病毒载体、痘病毒载体、腺相关病毒载体、单纯疱疹病毒载体、巨细胞病毒载体)、重组细菌载体疫苗、重组酵母载体疫苗或重组病毒样颗粒疫苗。在一些实施方式中，本文的疫苗选自重组DNA疫苗、重组腺病毒载体、重组痘病毒载体或其中一种或两种或三种的组合。The vaccine herein can be a recombinant protein vaccine, a recombinant DNA vaccine, a recombinant viral vector vaccine (e.g., adenovirus vector, poxvirus vector, adeno-associated virus vector, herpes simplex virus vector, cytomegalovirus vector), recombinant bacterial vector vaccine, recombinant yeast Vector vaccine or recombinant virus-like particle vaccine. In some embodiments, the vaccine herein is selected from a recombinant DNA vaccine, a recombinant adenovirus vector, a recombinant poxvirus vector, or a combination of two or three of them.
在一些实施方式中，可采用选自下组的一种或多种疫苗或其组合：重组质粒疫苗(DNA)，如包含与人重组Ferritin或HA2融合的经半胱氨酸修饰的RBD区编码序列的DNA疫苗(例如pcDNA3.1-sRBD-hFn)；重组蛋白亚单位疫苗(protein)，如RBD蛋白(不包含二硫键修饰)、与人重组Ferritin或HA2融合的经半胱氨酸修饰的RBD蛋白(sRBD-hFn蛋白、sRBD-HA2蛋白)；重组人源细胞载体疫苗，如K562-HA2-sRBD。In some embodiments, one or more vaccines selected from the following group or a combination thereof can be used: recombinant plasmid vaccine (DNA), such as a cysteine-modified RBD region coded with fusion with human recombinant Ferritin or HA2 Sequence DNA vaccine (such as pcDNA3.1-sRBD-hFn); recombinant protein subunit vaccine (protein), such as RBD protein (without disulfide bond modification), cysteine modified by fusion with human recombinant Ferritin or HA2 The RBD protein (sRBD-hFn protein, sRBD-HA2 protein); recombinant human cell vector vaccine, such as K562-HA2-sRBD.
本文的疫苗组合物中包含有效量的本文免疫原。本公开的疫苗组合物中包含足以实现希望的生物效应的量的免疫原。术语“有效量”通常是指可以诱导足以诱 导免疫力的保护性免疫应答以预防和/或减轻感染或疾病和/或以减少感染或疾病的至少一种症状的免疫原的量。The vaccine composition herein includes an effective amount of the immunogen herein. The vaccine composition of the present disclosure includes an immunogen in an amount sufficient to achieve the desired biological effect. The term "effective amount" generally refers to the amount of an immunogen that can induce a protective immune response sufficient to induce immunity to prevent and/or reduce infection or disease and/or to reduce at least one symptom of infection or disease.
本文的疫苗中还可包含佐剂。可采用本领域普通技术人员已知的佐剂，例如Vogel等人，“A Compendium of Vaccine Adjuvants and Excipients”(第2版)中所记载的佐剂(通过引用以其全文结合在此)。已知佐剂的例子包括但不限于：完全弗氏佐剂、不完全弗氏佐剂、氢氧化铝佐剂、脂多糖(LPS)、RIBI佐剂、MF-59等。Adjuvants may also be included in the vaccines herein. Adjuvants known to those of ordinary skill in the art can be used, for example, the adjuvants described in Vogel et al., "A Compendium of Vaccine Adjuvants and Excipients" (2nd edition) (incorporated in its entirety by reference). Examples of known adjuvants include, but are not limited to: complete Freund's adjuvant, incomplete Freund's adjuvant, aluminum hydroxide adjuvant, lipopolysaccharide (LPS), RIBI adjuvant, MF-59, and the like.
本文的疫苗组合物还可包括药学上可接受的载体、稀释剂、防腐剂、增溶剂、乳化剂等辅料。例如，药学上可接受的载体是已知的，并且包括但不限于注射用水、盐溶液、缓冲盐水、右旋糖、水、甘油、无菌等渗水缓冲液及其组合。药学上可接受的载体、稀释剂和其他赋形剂可例如参见《雷明顿药物科学》(Remngton′s Pharmaceutcal Sciences)中。The vaccine composition herein may also include pharmaceutically acceptable carriers, diluents, preservatives, solubilizers, emulsifiers and other auxiliary materials. For example, pharmaceutically acceptable carriers are known, and include, but are not limited to, water for injection, saline solution, buffered saline, dextrose, water, glycerol, sterile isotonic water buffer, and combinations thereof. Pharmaceutically acceptable carriers, diluents and other excipients can be found in "Remngton's Pharmaceutical Sciences", for example.
本文疫苗组合物的形式可适于***性或局部(尤其是呼吸道内)给予。给予疫苗组合物的方法包括但不限于：肌肉接种、皮内接种、皮下接种、滴鼻、雾化吸入、生殖道、直肠、口服或其任意组合。The form of the vaccine composition herein may be suitable for systemic or topical (especially intra-respiratory) administration. Methods of administering the vaccine composition include, but are not limited to: intramuscular inoculation, intradermal inoculation, subcutaneous inoculation, nasal drops, nebulized inhalation, genital tract, rectum, oral administration, or any combination thereof.
在一些实施方式中，本文的疫苗预防、消除或减轻对象中的新型冠状病毒感染或其至少一种症状，例如呼吸道症状(如鼻塞、咽喉痛、声嘶)、头痛、咳嗽、痰、发热、哕音、喘息、呼吸困难、因感染引起的肺炎、严重急性呼吸综合症、肾衰竭等。In some embodiments, the vaccine herein prevents, eliminates or alleviates novel coronavirus infection or at least one symptom thereof in a subject, such as respiratory symptoms (such as nasal congestion, sore throat, hoarseness), headache, cough, sputum, fever, Rattle, wheezing, difficulty breathing, pneumonia caused by infection, severe acute respiratory syndrome, renal failure, etc.
本文中还涉及了一种免疫偶联物(也可称免疫缀合物)，其包含本文的免疫原以及与其偶联的其他物质。所述的其他物质可为靶向性物质(如特异性识别特定靶标的部分)、治疗性物质(如药物、毒素、细胞毒剂)、标记性物质(如荧光标记物、放射性同位素标记物)。This article also relates to an immunoconjugate (also called an immunoconjugate), which contains the immunogen herein and other substances conjugated with it. The other substances can be targeted substances (such as a part that specifically recognizes a specific target), therapeutic substances (such as drugs, toxins, cytotoxic agents), and labeled substances (such as fluorescent markers, radioisotope markers).
在本公开中还提供了一种组合产品，其包括本公开的免疫原性肽、核苷酸分子、载体、宿主细胞和/或疫苗，且还可包含一种或多种有助于更好发挥预防和/或治疗新型冠状病毒感染或其症状的功能或增强前述物质稳定性的其他物质。例如，其他物质可包括针对冠状病毒S或S1的其他疫苗，如来自于包括但不限于SARS-CoV-2、SARS-CoV、MERS-CoV、HCoV-229E、HCoV-OC43、HCoV-NL63、HCoV-HKU1、bat-CoV的S或S1疫苗；受益于T细胞活化和/或与T细胞的记忆性免疫反应的疾病或病症的其他活性物质。In the present disclosure, a combination product is also provided, which includes the immunogenic peptides, nucleotide molecules, vectors, host cells and/or vaccines of the present disclosure, and may also include one or more to help better Other substances that function to prevent and/or treat the new coronavirus infection or its symptoms or enhance the stability of the aforementioned substances. For example, other substances may include other vaccines against coronavirus S or S1, such as those from other vaccines including but not limited to SARS-CoV-2, SARS-CoV, MERS-CoV, HCoV-229E, HCoV-OC43, HCoV-NL63, HCoV -S or S1 vaccines of HKU1, bat-CoV; other active substances for diseases or disorders that benefit from T cell activation and/or memory immune response with T cells.
免疫方法Immunization method
本文还提供了一种用于预防和/或治疗新型冠状病毒感染和/或其症状的方法，其包括：至少一次给予预防和/或治疗有效量的本公开的一种或多种疫苗。可采用的接种方式包括但不限于：***性免疫接种方式，如肌肉注射、皮下注射和皮内注射等；呼吸道内免疫接种方式，如雾化、滴鼻等。在一些实施方式中，初次免疫采用***性接种或呼吸道内接种，优选***性接种。This article also provides a method for preventing and/or treating novel coronavirus infection and/or its symptoms, which comprises: administering at least one preventive and/or therapeutically effective amount of one or more vaccines of the present disclosure. Available vaccination methods include, but are not limited to: systemic immunization methods, such as intramuscular injection, subcutaneous injection, and intradermal injection, etc.; and intra-respiratory tract immunization methods, such as atomization, nose drops, etc. In some embodiments, systemic vaccination or intra-respiratory vaccination is used for the initial immunization, preferably systemic vaccination.
在本公开的一些实施方式中，每两次接种之间的间隔至少为1周，例如2周、4周、2个月、3个月、6个月或更长间隔。In some embodiments of the present disclosure, the interval between every two vaccination is at least 1 week, for example, 2 weeks, 4 weeks, 2 months, 3 months, 6 months or longer intervals.
在一些实施方式中，采用DNA疫苗进行初次免疫，并采用重组病毒疫苗进行一次或多次加强免疫。本公开的免疫方法可采用“初免-加强”或“初免-加强-再加强”的方式，可采用单一的全身***免疫或呼吸道局部免疫方式，或采用两种免疫方式的组合。In some embodiments, a DNA vaccine is used for the primary immunization, and a recombinant virus vaccine is used for one or more booster immunizations. The immunization method of the present disclosure can adopt a "primary immunization-enhancement" or "primary immunization-enhancement-re-enhancement" approach, a single systemic immunization method or a local respiratory tract immunization method, or a combination of two immunization methods.
根据不同载体疫苗的特点，在一些优选的实施方式中，采用重组DNA疫苗进行***性初免，从而建立全身***免疫应答，再用其他疫苗(例如重组腺病毒疫苗或重组痘病毒疫苗)进行一次或多种免疫加强，所述免疫加强可包括至少一次呼吸道内免疫加强(例如，采用腺病毒疫苗)。According to the characteristics of different vector vaccines, in some preferred embodiments, recombinant DNA vaccines are used for systemic primary immunization to establish a systemic immune response, and then other vaccines (such as recombinant adenovirus vaccines or recombinant poxvirus vaccines) are used for once. Or multiple immune boosts, which may include at least one respiratory boost (for example, using an adenovirus vaccine).
采用本文的免疫方法可在呼吸道局部和全身***有效建立的疫苗特异性免疫应答，有助于增强疫苗保护的有效性。Using the immunization method of this paper can effectively establish a vaccine-specific immune response in the local respiratory tract and the systemic system, which helps to enhance the effectiveness of vaccine protection.
以药物包或试剂盒的形式提供本文的组合产品可，例如可将本文的一种或多种疫苗组合物或其一种或多种成分包装在一个或多个容器中，例如包装在指明组合物的量的密封容器诸如安瓿或小药囊中。可以液体、无菌冻干粉或无水浓缩物等形式提供疫苗组合物，可在临用前用适当液体(例如水、盐水等)对其进行稀释、复原和/或配制以获得用于给予至对象的适当浓度和形式。The combination product herein can be provided in the form of a pharmaceutical pack or kit. For example, one or more vaccine compositions herein or one or more components thereof can be packaged in one or more containers, for example, packaged in the specified combination The amount of the substance in a sealed container such as an ampoule or a sachet. The vaccine composition can be provided in the form of liquid, sterile lyophilized powder or anhydrous concentrate, etc., and can be diluted, reconstituted and/or formulated with an appropriate liquid (such as water, saline, etc.) before use for administration To the appropriate concentration and form of the subject.
本公开中的组合产品可用于在呼吸道内局部诱导高水平抗原特异性CD8+T细胞应答的特征，使得其在预防呼吸道病原体感染、降低呼吸道病原体致病力和呼吸道肿瘤的预防治疗中均有应用前景。The combination product in the present disclosure can be used to locally induce high-level antigen-specific CD8+ T cell responses in the respiratory tract, so that it can be used in the prevention of respiratory pathogen infections, reducing the pathogenicity of respiratory pathogens, and the prevention and treatment of respiratory tumors prospect.
实施例Example
下面结合具体实施例，进一步阐述本公开。应理解，这些实施例仅用于说明本公开而不用于限制本公开的范围。本领域技术人员可对本公开做出适当的修改、变动，这些修改和变动都在本公开的范围之内。The present disclosure will be further explained below in conjunction with specific embodiments. It should be understood that these embodiments are only used to illustrate the present disclosure and not to limit the scope of the present disclosure. Those skilled in the art can make appropriate modifications and changes to the present disclosure, and these modifications and changes are all within the scope of the present disclosure.
下列实施例中未注明具体条件的实验方法，可采用本领域中的常规方法，例如参考《分子克隆实验指南》(第三版，纽约，冷泉港实验室出版社，New York：Cold Spring Harbor Laboratory Press，1989)或按照供应商所建议的条件。DNA的测序方法为本领域常规的方法，也可由商业公司提供测试。For the experimental methods that do not specify specific conditions in the following examples, conventional methods in the field can be used, for example, refer to "Molecular Cloning Experimental Guide" (third edition, New York, Cold Spring Harbor Laboratory Press, New York: Cold Spring Harbor Laboratory Press, 1989) or in accordance with the conditions recommended by the supplier. DNA sequencing methods are conventional methods in the field, and commercial companies can also provide tests.
除非另外说明，否则百分比和份数按重量计算。除非另行定义，文中所使用的所有专业与科学用语与本领域熟练人员所熟悉的意义相同。此外，任何与所记载内容相似或均等的方法及材料皆可应用于本公开方法中。文中所述的较佳实施方法与材料仅作示范之用。Unless otherwise stated, percentages and parts are calculated by weight. Unless otherwise defined, all professional and scientific terms used in the text have the same meaning as those familiar to those skilled in the art. In addition, any methods and materials that are similar or equivalent to the recorded content can be applied to the methods of the present disclosure. The preferred implementation methods and materials described in this article are for demonstration purposes only.
下述实验中涉及到的实验动物、免疫方式、免疫原、假病毒及检测方法如下：The experimental animals, immunization methods, immunogens, pseudoviruses and detection methods involved in the following experiments are as follows:
I.实验动物：6-8周龄雌性C57/BL6小鼠I. Experimental animals: 6-8 weeks old female C57/BL6 mice
II.免疫方式：对小鼠左、右后肢分别进行肌肉注射II. Immunization method: intramuscular injection into the left and right hind limbs of mice
III.免疫原的选择：III. Selection of immunogen:
S及RBD序列均来自Genebank：NC_045512.2，Severe acute respiratory syndrome coronavirus 2 isolate Wuhan-Hu-1，complete genome；hFn序列来自于Genebank：M97164.1，HA2序列来自于Genebank：AGI60292.1，具体序列见序列表。S and RBD sequences are from Genebank: NC_045512.2, Severe acute respiratory syndrome coronavirus 2 isolate Wuhan-Hu-1, complete genome; hFn sequence comes from Genebank: M97164.1, HA2 sequence comes from Genebank: AGI60292.1, specific sequence See sequence list.
1.重组质粒疫苗(DNA)：pcDNA3.1-sRBD-hFn、pcDNA3.1-S、pcDNA3.1(空载)；1. Recombinant plasmid vaccine (DNA): pcDNA3.1-sRBD-hFn, pcDNA3.1-S, pcDNA3.1 (empty);
2.重组蛋白亚单位疫苗(protein)：RBD蛋白(不包含二硫键修饰)、sRBD-hFn蛋白；2. Recombinant protein subunit vaccine (protein): RBD protein (without disulfide bond modification), sRBD-hFn protein;
3.重组人源细胞载体疫苗：K562、K562-S、K562-HA2-sRBD。3. Recombinant human cell vector vaccine: K562, K562-S, K562-HA2-sRBD.
IV.免疫原制备及免疫剂量：IV. Preparation of immunogen and immunization dose:
免疫原的制备参见实施例1See Example 1 for the preparation of immunogen
在实施例中所采用的免疫原免疫剂量如下：The immunogen doses used in the examples are as follows:
1.重组质粒疫苗(DNA)：100μg/只小鼠，100μL，溶于无菌生理盐水中；1. Recombinant plasmid vaccine (DNA): 100μg/mouse, 100μL, dissolved in sterile saline;
2.重组蛋白亚单位疫苗(protein)：蛋白质(溶于无菌PBS中)与铝佐剂(Aluminium，InvivoGen，货号5200)按照体积比1∶1混合后进行免疫，RBD蛋白，10μg/只小鼠，100μL；sRBD-hFn蛋白，20μg/只小鼠，100μL；2. Recombinant protein subunit vaccine (protein): protein (dissolved in sterile PBS) and aluminum adjuvant (Aluminium, InvivoGen, Item No. 5200) are immunized at a volume ratio of 1:1, RBD protein, 10μg/small Mouse, 100μL; sRBD-hFn protein, 20μg/mouse, 100μL;
3.重组人源细胞载体疫苗(K562、K562-S、K562-HA2-sRBD)：1E6个细胞/只小鼠，100μL，溶于无菌PBS中。3. Recombinant human cell vector vaccine (K562, K562-S, K562-HA2-sRBD): 1E6 cells/mouse, 100μL, dissolved in sterile PBS.
V.免疫间隔：V. Immunization interval:
具体免疫间隔见下文表格See the table below for specific immunization intervals
VI.SARS-CoV2包膜假病毒(Pseudovirus)包装：VI. SARS-CoV2 envelope pseudovirus (Pseudovirus) packaging:
1.转染前一天准备293T细胞，用于包装质粒的转染与表达。用DMEM完全培养基将细胞稀释至5×10 6个/mL细胞，取1mL稀释好的细胞，铺在10cm的皿中，37℃，5％CO 2，培养过夜； 1. Prepare 293T cells one day before transfection for transfection and expression of packaging plasmids. Dilute the cells to 5×10 6 cells/mL with DMEM complete medium, take 1 mL of the diluted cells, and spread them in a 10 cm dish at 37°C, 5% CO 2 , and culture overnight;
2.吸取SARS-CoV2膜蛋白质粒pcDNA3.1-S 4μg和pNL4-3Δenv骨架质粒8μg(NIH AIDS Reagent Program，3418)加入500μL双无(无血清、无双抗，双抗为青链霉素混合液)的DMEM中，室温孵育5min；2. Pipette SARS-CoV2 membrane protein particle pcDNA3.1-S 4μg and pNL4-3Δenv skeleton plasmid 8μg (NIH AIDS Reagent Program, 3418) and add 500μL double no (serum free, no double antibody, double antibody is penicillin streptomycin mixture) ) In DMEM, incubate at room temperature for 5 min;
3.用双无DMEM将24μL TurboFect(Thermo Fisher Scientific)稀释，终体积为500μL/样品，室温孵育5min；3. Dilute 24μL TurboFect (Thermo Fisher Scientific) with double-free DMEM to a final volume of 500μL/sample, and incubate at room temperature for 5 minutes;
4.将2与3两者混匀，1000μL/样品终体积，室温孵育20min，孵育结束后加到10cm培养皿的293T细胞中。6h后更换新鲜的15mL完全培养基，继续在细胞培养箱中培养48h；4. Mix 2 and 3 evenly, 1000μL/final volume of the sample, incubate at room temperature for 20min, and add to 293T cells in a 10cm petri dish after the incubation. After 6h, replace with fresh 15mL complete medium and continue to culture in the cell culture incubator for 48h;
5.培养结束后，收集10cm皿的细胞培养上清，于15mL离心管里，然后4000g，4℃，离心10min，用0.45μm的滤器过滤到新的15mL离心管中，冻存于-80℃保存，滴定后备用。5. After the completion of the culture, collect the cell culture supernatant from the 10cm dish, place it in a 15mL centrifuge tube, then centrifuge at 4000g at 4℃ for 10min, filter it with a 0.45μm filter into a new 15mL centrifuge tube, and store it at -80℃ Save it for later use after titration.
VII.构建稳定表达hACE2受体的293T细胞：VII. Construction of 293T cells stably expressing hACE2 receptor:
1.人工合成人源ACE2(hACE2)序列(Genebank#NCBI_NP_001358344.1)，如SEQ ID NO：13所示，序列5’端带有Age1酶切位点，3’端带有Xba1酶切位点，合成片段与载体质粒pHAGE-MCS-puro使用Age1酶切(Thermo Scientific公司，货号FD1464)与Xba1酶切(Thermo Scientific公司，FD0685)，并通过凝胶电泳后切胶回收，采用Sanprep柱式DNA胶回收试剂盒(Promega公司，货号A9282)回收酶切片段。1. Artificially synthesized human ACE2 (hACE2) sequence (Genebank#NCBI_NP_001358344.1), as shown in SEQ ID NO: 13, with Age1 restriction site at the 5'end and Xba1 restriction site at the 3'end , The synthetic fragment and the vector plasmid pHAGE-MCS-puro were digested with Age1 (Thermo Scientific, Catalog No. FD1464) and Xba1 (Thermo Scientific, FD0685), and recovered by gel electrophoresis, using Sanprep column DNA The gel recovery kit (Promega, catalog number A9282) recovers the digested fragments.
2.基因回收产物与酶切线性化载体用T4 DNA连接酶的方法连接(Thermo Scientific公司，货号2011A)：将连接产物转化至大肠杆菌E.coli Stable，在含氨苄霉素的培养板上过夜生长。第2天，随机挑取单菌落进行测序，突变位点校正，验证全部序列正确后，成功克隆出hACE2基因的慢病毒表达质粒(pHAGE-hACE2-puro)。2. The gene recovery product is ligated with the restriction digestion linearized vector with T4 DNA ligase (Thermo Scientific Company, Catalog No. 2011A): The ligation product is transformed into E.coli Stable, and placed on a culture plate containing ampicillin overnight Grow. On the second day, a single colony was randomly selected for sequencing, and the mutation site was corrected. After verifying that all the sequences were correct, the lentiviral expression plasmid (pHAGE-hACE2-puro) of the hACE2 gene was successfully cloned.
3.取10cm皿，在每个皿中接种约5×10 6个293T细胞，保证第二天转染时使细胞密度达90％为宜；将pHAGE-hACE2-puro，慢病毒包装质粒psPAX以及VSVG三种质粒，按照质量比1∶2∶1的比例转染293T细胞。 3. Take a 10cm dish and inoculate about 5×10 6 293T cells in each dish to ensure that the cell density reaches 90% during the next day of transfection; pHAGE-hACE2-puro, lentivirus packaging plasmid psPAX and The three plasmids of VSVG were transfected into 293T cells at a mass ratio of 1:2:1.
4. 37℃，5％的孵箱培养48小时左右，具体时间根据细胞情况而定，收集细胞上清。将收集的细胞上清用0.45μm的滤器进行过滤，再用PEG 8000进行浓缩，即可得到较为纯化的hACE2慢病毒。4. Incubate in a 5% incubator at 37°C for about 48 hours. The specific time depends on the cell condition. Collect the cell supernatant. Filter the collected cell supernatant with a 0.45μm filter, and then concentrate it with PEG 8000 to obtain a more purified hACE2 lentivirus.
5.提前一天铺5×10 5的293T细胞于12孔板的一个孔内，次日向铺好的细胞中加入步骤2中浓缩的病毒500μL，1000g，离心2小时。 5. Pour 5×10 5 293T cells in one well of a 12-well plate one day in advance, and add 500 μL of the virus concentrated in step 2 at 1000 g to the paved cells the next day, and centrifuge for 2 hours.
6.离心感染结束后，继续在37℃，5％的孵箱培养12小时左右，将培养基换成添加1μg/mL嘌呤霉素(puro)的细胞培养基培养，最后能够存活的细胞便是整合有hACE2基因的293T细胞，并通过流式分选筛选出稳定表达hACE2的293T细胞(能与S蛋白结合)。6. After the infection by centrifugation, continue to incubate at 37℃, 5% incubator for about 12 hours, change the medium to cell culture medium supplemented with 1μg/mL puromycin (puro), and finally the cells that can survive are 293T cells with hACE2 gene integrated, and 293T cells stably expressing hACE2 (which can bind to S protein) were screened by flow sorting.
VIII.检测方法：VIII. Detection method:
采血：Blood collection:
最后一次免疫结束后第4周，将小鼠脱颈处死前，通过摘眼球的方法采集小鼠外周全血，收集于1.5mL EP管中，室温静置使其自然凝血，凝固后的小鼠血清于7000g，离心15min。将小鼠血清转移至新的1.5mL EP管中。实验前需要将样品在56℃灭活30min，来破坏血清内的补体活性。灭活前短暂离心，避免管壁和瓶盖上的样品残存。水浴液面要没过样品液面，但不能超过瓶盖。On the 4th week after the last immunization, before the mouse was sacrificed, the peripheral whole blood of the mouse was collected by removing the eyeballs, collected in a 1.5mL EP tube, and allowed to stand at room temperature for natural coagulation. The coagulated mouse The serum was centrifuged at 7000g for 15min. Transfer mouse serum to a new 1.5mL EP tube. Before the experiment, the sample needs to be inactivated at 56°C for 30 minutes to destroy the complement activity in the serum. Centrifuge briefly before inactivation to avoid residual samples on the tube wall and bottle cap. The liquid level of the water bath must be below the liquid level of the sample, but not more than the bottle cap.
ELSIA检测结合抗体：ELSIA detects binding antibodies:
1.用4℃预冷的ELISA包被液稀释检测的抗原蛋白(S1或者RBD)，至终浓度为1μg/mL。在ELISA板的每孔加入100μL包被抗原溶液，4℃过夜；1. Dilute the detected antigen protein (S1 or RBD) with 4°C pre-cooled ELISA coating solution to a final concentration of 1μg/mL. Add 100μL of coating antigen solution to each well of the ELISA plate, overnight at 4°C;
2.第二天，取出ELISA板，弃掉包被液，用0.05％的PBST缓冲液洗板3次，每次220μL；2. The next day, take out the ELISA plate, discard the coating solution, and wash the plate 3 times with 0.05% PBST buffer, 220 μL each time;
3.洗涤完毕后，在吸水纸上拍干，每孔用200μL ELISA封闭液(0.5％脱脂奶粉，PBST溶解)进行封闭，室温封闭2h；3. After washing, pat dry on absorbent paper, and block each well with 200μL ELISA blocking solution (0.5% skimmed milk powder, dissolved in PBST), and block at room temperature for 2 hours;
4.封闭结束后，用0.05％的PBST洗板3次，每次220μL；4. After blocking, wash the plate 3 times with 0.05% PBST, 220 μL each time;
5.对于血清或者血浆，用ELISA样品稀释液(0.5％脱脂奶粉，PBST溶解)稀释，从1∶100起始，进行2倍比稀释。用未免疫的小鼠血清设置为阴性对照。设置Blank孔，只加样品稀释液，每个样品需做2个复孔，每孔终体积为100μL，室温孵育3h；5. For serum or plasma, dilute with ELISA sample diluent (0.5% skimmed milk powder, dissolved in PBST), starting from 1:100, and diluting at a 2-fold ratio. Serum from unimmunized mice was used as a negative control. Set up Blank wells, add only sample diluent, make 2 duplicate wells for each sample, each well will have a final volume of 100μL, and incubate at room temperature for 3h;
6.样品孵育结束后，继续用PBST洗板5次，每次220μL；6. After the sample incubation, continue to wash the plate 5 times with PBST, 220 μL each time;
7.用ELISA封闭液(0.5％脱脂奶粉，PBST溶解)稀释相对应比例的二抗(山羊抗鼠)，每孔加入100μL，室温孵育1-1.5h；7. Dilute the corresponding proportion of the secondary antibody (goat anti-mouse) with ELISA blocking solution (0.5% skimmed milk powder, dissolved in PBST), add 100μL to each well, and incubate for 1-1.5h at room temperature;
8.二抗孵育结束后，用0.05％的PBST洗板5次，每次220μL；8. After the secondary antibody incubation, wash the plate with 0.05% PBST 5 times, 220 μL each time;
9.取一对金银片OPD底物，溶解于20mL去离子水中，随后每孔加入100μL，避光反应5min；9. Take a pair of OPD substrates of gold and silver tablets, dissolve them in 20mL of deionized water, then add 100μL to each well, and react for 5min in the dark;
10.显色结束后，用50μL 2nM H 2SO 4进行终止，在酶标仪上读取OD 492-OD 630值； 10. After the color development is over, stop with 50μL 2nM H 2 SO 4 and read the OD 492 -OD 630 value on the microplate reader;
11.以最后一个稀释度OD492大于2倍的(negative mean+SD)值对应的血清稀释比的倒数作为抗体滴度。11. Take the reciprocal of the serum dilution ratio corresponding to the last dilution OD492 greater than 2 times (negative mean+SD) as the antibody titer.
293T-ACE2细胞检测中和抗体：293T-ACE2 cell detection neutralizing antibody:
1.取96孔透明底黑板进行中和实验，第一列设置细胞对照(CC)(150μL)，第二列设置病毒对照(VC)(100μL)，其他均为样品孔，对血清样品进行倍比稀释，最终孔中体积为100μL。1. Take 96-well transparent bottom blackboard for neutralization experiment, set cell control (CC) (150μL) in the first column, set virus control (VC) (100μL) in the second column, the others are sample wells, double the serum sample Dilute than, the final volume in the well is 100 μL.
2.除细胞对照组外，每孔加50μL SARS-CoV-2假病毒稀释液，使每孔最终含假病毒为200TCID 50。 2. In addition to the cell control group, each well was added 50μL SARS-CoV-2 virus dilutions false, false per well containing final virus 200TCID 50.
3.轻轻震荡混匀，将上述96孔底黑板置于细胞培养箱中，37℃，5％CO 2孵育1h。 3. Gently shake and mix, put the above 96-well bottom blackboard in a cell incubator, and incubate at 37°C and 5% CO 2 for 1 hour.
4.当孵育时间至20min时，开始准备293T-hACE2靶细胞，并用完全培养基将细胞稀释至10 5个细胞/mL。 4. When the incubation time to time 20min, 293T-hACE2 start preparing target cells, and the cells were diluted with complete medium to 105 cells / mL.
5.当孵育时间至1h，向96孔透明底黑板中每孔加100μL靶细胞，使每孔细胞为10 4个。 5. When the incubation time to IH, 96-well clear-bottom board in each well 100μL target cells per well of 10 4 cells.
6.前后左右轻轻晃动96孔透明底黑板，使孔中的细胞均匀分散，再将板子放入细胞培养箱中，37℃，5％CO 2培养48h。 6. Gently shake the 96-well transparent bottom blackboard back and forth to make the cells in the wells evenly dispersed, and then put the plate into the cell incubator, incubate at 37°C and 5% CO 2 for 48 hours.
7.培养48h后，从细胞培养箱中取出96孔透明底黑板，吸掉孔中上清，每孔加入100μL PBS清洗一遍，吸去PBS，每孔加入50μL 1×的裂解缓冲液(购自Promega公司Cat#E153A)，室温在水平摇床上孵育30min使细胞充***解；7. After culturing for 48 hours, remove the 96-well transparent bottom blackboard from the cell incubator, aspirate the supernatant from the wells, add 100μL PBS to each well to wash, aspirate the PBS, and add 50μL 1× lysis buffer (purchased from Promega Cat#E153A), incubate on a horizontal shaker for 30 minutes at room temperature to fully lyse the cells;
8.加30μL荧光素酶的底物(购自Promega公司，Cat#E1501)于96孔黑板中，用仪器
Figure PCTCN2020096148-appb-000001
96微孔板发光-检测仪检测荧光素酶活性。 8. Add 30μL of luciferase substrate (purchased from Promega, Cat#E1501) to the 96-well blackboard, and use the instrument
Figure PCTCN2020096148-appb-000001
The 96-well plate luminescence-detector detects luciferase activity.
9.导出荧光素读值，计算中和抑制率，结合中和抑制率结果，利用Graphpad Prism 5.0软件计算ID 50。 9. Export the fluorescein readings, calculate the neutralization inhibition rate, and use the Graphpad Prism 5.0 software to calculate the ID 50 in combination with the neutralization inhibition rate results.
Figure PCTCN2020096148-appb-000002
Figure PCTCN2020096148-appb-000002
实施例1：S及sRBD-hFn(二硫键改造)真核表达载体的构建及蛋白表达Example 1: S and sRBD-hFn (disulfide bond modification) eukaryotic expression vector construction and protein expression
为了研究RBD的功能，我们构建了S及sRBD-hFn的真核表达载体，并在体外细胞系中真核表达出成熟的蛋白质。In order to study the functions of RBD, we constructed eukaryotic expression vectors of S and sRBD-hFn, and eukaryotic expression of mature proteins in in vitro cell lines.
首先，我们人工合成了S基因(序列如SEQ ID NO：11所示)、sRBD-hFn基因(具体序列如SEQ ID NO：7所示，其中sRBD部分基于如SEQ ID NO：3所示的序列，hFn部分基于如SEQ ID NO：5所示的序列)，合成片段与载体质粒pcDNA3.1(购自优宝生物)使用BamHI酶切(Thermo Scientific公司，FD0054)与Not1酶切(Thermo Scientific公司，FD0596)，并通过凝胶电泳后切胶回收，采用Sanprep柱式DNA胶回收试剂盒(Promega公司，货号A9282)回收酶切片段。sRBD-hFn基因在RBD基因部分的N和C端各加了一个半胱氨酸，使之在RBD的根部形成二硫键，稳定RBD的结构。sRBD表达在hFn的N端，两者之间采用接头连接。First, we artificially synthesized the S gene (sequence shown in SEQ ID NO: 11), sRBD-hFn gene (specific sequence shown in SEQ ID NO: 7, and the sRBD part is based on the sequence shown in SEQ ID NO: 3 , HFn is partially based on the sequence shown in SEQ ID NO: 5), the synthetic fragment and vector plasmid pcDNA3.1 (purchased from Ubao Bio) were digested with BamHI (Thermo Scientific, FD0054) and Not1 (Thermo Scientific) , FD0596), and cut the gel after gel electrophoresis, and use the Sanprep column DNA gel recovery kit (Promega, Catalog No. A9282) to recover the digested fragments. The sRBD-hFn gene adds a cysteine to the N and C ends of the RBD gene to form a disulfide bond at the root of the RBD to stabilize the structure of the RBD. sRBD is expressed at the N-terminus of hFn, and the two are connected by a linker.
随后，基因回收产物与酶切线性化载体用T4 DNA连接酶的方法连接(Thermo Scientific公司，货号2011A)：将连接产物转化至大肠杆菌E.coli Stable，在含氨苄霉素的培养板上过夜生长。第2天，随机挑取单菌落进行测序，突变位点校正，验证全部序列正确后，成功克隆出S及sRBD-hFn的真核表达载体pcDNA3.1-S及pcDNA3.1-sRBD-hFn，质粒构建图谱如图1A。Subsequently, the gene recovery product was ligated with the digested linearized vector with T4 DNA ligase (Thermo Scientific, Catalog No. 2011A): the ligation product was transformed into E. coli Stable and placed on a culture plate containing ampicillin overnight Grow. On the second day, a single colony was randomly selected for sequencing, and the mutation site was corrected. After verifying that all the sequences were correct, the eukaryotic expression vectors pcDNA3.1-S and pcDNA3.1-sRBD-hFn of S and sRBD-hFn were successfully cloned. The plasmid construction map is shown in Figure 1A.
我们进一步检测了S及sRBD-hFn能否在真核细胞系293T中表达。We further tested whether S and sRBD-hFn can be expressed in the eukaryotic cell line 293T.
首先，取6孔板，在每个孔中接种约6×10 5个293T细胞，保证第二天转染时使细胞密度达90％为宜；用pcDNA3.1、pcDNA3.1-S及pcDNA3.1-sRBD-hFn分别转染293T细胞(转染试剂TurboFect)。37℃，5％的孵箱培养48小时左右，具体时间根据细胞情况而定，收集细胞，进行蛋白质免疫印迹(WB)鉴定，发现S在细胞中表达，大小为200KD左右；sRBD-hFn在细胞中表达，大小为55KD左右，而pcDNA3.1对照转染的细胞不能检测到sRBD-hFn蛋白的表达(图1B)。 First, take a 6-well plate and inoculate about 6×10 5 293T cells in each well to ensure that the cell density reaches 90% during the second day of transfection; use pcDNA3.1, pcDNA3.1-S and pcDNA3 .1-sRBD-hFn were transfected into 293T cells (transfection reagent TurboFect). Incubate in a 5% incubator at 37°C for about 48 hours. The specific time depends on the cell conditions. The cells are collected and subjected to Western blot (WB) identification. It is found that S is expressed in the cells and the size is about 200KD; sRBD-hFn is in the cells The expression of sRBD-hFn protein could not be detected in the cells transfected with pcDNA3.1 control (Figure 1B).
所表达的sRBD-hFn的结构为：sRBD表达在hFn的N端，两者之间采用(G4S) 3连接。 The structure of the expressed sRBD-hFn is: sRBD is expressed at the N-terminus of hFn, and the two are connected by (G4S) 3 .
实施例2：慢病毒表达载体pHAGE-S-puro及pHAGE-sRBD-HA2-puro的构建及S蛋白和sRBD-HA2在K562细胞膜上展示的表达验证Example 2: Construction of lentiviral expression vectors pHAGE-S-puro and pHAGE-sRBD-HA2-puro and verification of expression of S protein and sRBD-HA2 displayed on K562 cell membrane
为了验证二硫键修饰后的RBD(sRBD)的构象是否正确，我们构建了慢病毒表达载体pHAGE-S-puro及pHAGE-sRBD-HA2-puro，并将蛋白表达在K562细胞膜上，验证了蛋白的表达及构象。In order to verify whether the conformation of the disulfide bond modified RBD (sRBD) is correct, we constructed the lentiviral expression vectors pHAGE-S-puro and pHAGE-sRBD-HA2-puro, and expressed the protein on the K562 cell membrane to verify the protein Expression and conformation.
首先，我们人工合成了S基因(序列如SEQ ID NO：11所示)，sRBD-HA2基因(序列如SEQ ID NO：9所示)，合成片段序列5’端带有Not1酶切位点，3’端带有Xba1酶切位点，合成片段与载体质粒pHAGE-MCS-puro(购自上海鑫湾生物技术有限公司)使用Not1酶切(Thermo Scientific公司，FD0596)与Xba1酶切(Thermo Scientific公司，FD0685)，并通过凝胶电泳后切胶回收，采用Sanprep柱式DNA胶回收试剂盒(Promega公司，货号A9282)回收酶切片段。First, we artificially synthesized the S gene (sequence shown in SEQ ID NO: 11), sRBD-HA2 gene (sequence shown in SEQ ID NO: 9), and the synthesized fragment sequence has a Not1 restriction site at the 5'end, The 3'end has an Xba1 restriction site. The synthetic fragment and the vector plasmid pHAGE-MCS-puro (purchased from Shanghai Xinwan Biotechnology Co., Ltd.) were digested with Not1 (Thermo Scientific, FD0596) and Xba1 (Thermo Scientific). Company, FD0685), and cut the gel after gel electrophoresis, and use the Sanprep column DNA gel recovery kit (Promega Company, Catalog No. A9282) to recover the digested fragments.
基因回收产物与酶切线性化载体用T4 DNA连接酶的方法连接(Thermo Scientific公司，货号2011A)：将连接产物转化至大肠杆菌E.coli Stable，在含氨苄霉素的培养板上过夜生长。第2天，随机挑取单菌落进行测序，突变位点校正，验证全部序列正确后，成功克隆出S及sRBD-HA2的慢病毒表达载体pHAGE-S-puro及pHAGE-sRBD-HA2-puro(图2A)。The gene recovery product was ligated with the digested linearized vector using T4 DNA ligase method (Thermo Scientific Company, Catalog No. 2011A): The ligation product was transformed into E. coli Stable, and grown overnight on a culture plate containing ampicillin. On the second day, a single colony was randomly selected for sequencing, and the mutation site was corrected. After verifying that all the sequences were correct, the lentiviral expression vectors pHAGE-S-puro and pHAGE-sRBD-HA2-puro of S and sRBD-HA2 were successfully cloned. Figure 2A).
将pHAGE-S-puro及pHAGE-sRBD-HA2-puro转染293T细胞，通过蛋白质免疫印迹方法鉴定K562-S细胞中蛋白的表达(图2B)；同时进行流式细胞染色鉴定，发现S蛋白与sRBD-HA2蛋白在细胞膜上有表达，及都能够与受体ACE2结合，而对照感染的细胞不能检测到相关蛋白的表达，表明二硫键修饰的RBD本身的构 象保持的较为完整，不影响其主要功能(图2C)。The pHAGE-S-puro and pHAGE-sRBD-HA2-puro were transfected into 293T cells, and the protein expression in K562-S cells was identified by Western blotting (Figure 2B). At the same time, flow cytometric staining was performed to identify S protein and The sRBD-HA2 protein is expressed on the cell membrane and can bind to the receptor ACE2, while the expression of related proteins cannot be detected in the control infected cells, indicating that the conformation of the disulfide-modified RBD itself remains relatively intact and does not affect it. Main functions (Figure 2C).
实验步骤如下：取6孔板，在每个孔中接种约6×10 5个293T细胞，保证第二天转染时使细胞密度达90％为宜；用pHAGE-S-puro及pHAGE-sRBD-HA2-puro分别转染293T细胞(转染试剂TurboFect)。37℃，5％的孵箱培养48小时左右，具体时间根据细胞情况而定，收集细胞，进行蛋白质免疫印迹(WB)鉴定，发现S在细胞中表达，大小为200KD左右；sRBD-HA2在细胞中表达，大小为80KD左右，而pCDNA3.1对照转染的细胞不能检测到sRBD-hFn蛋白的表达(图2B)。准备5×10 5K562细胞，用500μL完全细胞培养基重悬，放置于12孔板的一个孔内。再向铺好的细胞中加入浓缩的慢病毒，1000g，离心2小时。离心感染结束后，继续在37℃，5％的孵箱培养48小时左右。取上述感染后的K562细胞，由于表达载体质粒带有嘌呤霉素抗性，将细胞在嘌呤霉素浓度为4μg/ml的RPMI(10％FBS)中培养，最后能够存活的细胞便是整合有S基因和sRBD-HA2的细胞。取上述感染后的细胞，使用蛋白质免疫印迹方法检测S蛋白表达，所用的一抗为ACE2-C-AVI-6his(上海近岸科技有限公司，型号0331753-4065)，二抗为HRP标记山羊抗人抗体(中杉金桥公司，货号ZB2304)。结果显示，利用蛋白质免疫印迹方法能检测到目的蛋白高表达(图2B)。 The experimental procedure is as follows: Take a 6-well plate and inoculate about 6×10 5 293T cells in each well to ensure that the cell density reaches 90% during the second day of transfection; use pHAGE-S-puro and pHAGE-sRBD -HA2-puro were transfected into 293T cells (transfection reagent TurboFect). Incubate in a 5% incubator at 37°C for about 48 hours. The specific time depends on the cell conditions. The cells are collected and subjected to Western blot (WB) identification. It is found that S is expressed in the cells and the size is about 200KD; sRBD-HA2 is in the cells The expression of sRBD-hFn protein could not be detected in the cells transfected with pCDNA3.1 control (Figure 2B). Prepare 5×10 5 K562 cells, resuspend them with 500 μL complete cell culture medium, and place them in one well of a 12-well plate. Then add concentrated lentivirus to the plated cells, 1000g, and centrifuge for 2 hours. After centrifugal infection, continue to incubate at 37°C, 5% incubator for about 48 hours. Take the above-infected K562 cells. Since the expression vector plasmid is resistant to puromycin, the cells are cultured in RPMI (10% FBS) with a puromycin concentration of 4 μg/ml. Finally, the cells that can survive are integrated. S gene and sRBD-HA2 cells. Take the above-infected cells and use Western blotting to detect the expression of S protein. The primary antibody used is ACE2-C-AVI-6his (Shanghai Nearshore Technology Co., Ltd., model 0331753-4065), and the secondary antibody is HRP-labeled goat antibody. Human antibody (Zhongshan Jinqiao Company, catalog number ZB2304). The results show that the high expression of the target protein can be detected by Western blotting (Figure 2B).
流式染色方法使用ACE2-C-AVI-6his(上海近岸科技有限公司，型号0331753-4065)/PE-streptavidin(BD Pharminge公司，货号563259)进行间接染色后，最后使用流式细胞分选(BD Pharminge公司，型号Arial)富集。结果显示，不断富集后，80％以上的K562-S细胞和K562-HA2-sRBD细胞能够与ACE2结合，说明二硫键修饰的RBD本身的构象并不影响其主要功能(图2C)。Flow cytometric staining method uses ACE2-C-AVI-6his (Shanghai Nearshore Technology Co., Ltd., model 0331753-4065)/PE-streptavidin (BD Pharminge company, product number 563259) for indirect staining, and finally uses flow cytometry ( BD Pharminge company, model Arial) enrichment. The results showed that after continuous enrichment, more than 80% of K562-S cells and K562-HA2-sRBD cells could bind to ACE2, indicating that the conformation of the disulfide bond-modified RBD itself did not affect its main function (Figure 2C).
实施例3：RBD蛋白对小鼠结合抗体和中和抗体的诱导效果Example 3: Induction effect of RBD protein on binding antibodies and neutralizing antibodies in mice
采用RBD蛋白(购买自南京金斯瑞生物科技)免疫C57/BL6小鼠，佐剂为铝佐剂，在完成免疫2周后，评价免疫组合诱导针对RBD蛋白和S1蛋白的结合抗体滴度。同时评价免疫结束4周后，针对SARS-CoV-2假病毒的中和抗体滴度。C57/BL6 mice were immunized with RBD protein (purchased from Nanjing GenScript Biotechnology), and the adjuvant was aluminum adjuvant. Two weeks after the completion of immunization, the immunization combination was evaluated to induce binding antibody titers against RBD protein and S1 protein. At the same time, the neutralizing antibody titers against SARS-CoV-2 pseudovirus were evaluated 4 weeks after the end of immunization.
实验步骤如下：将小鼠随机分为2组，根据免疫原分别命名为对照组、RBD组。具体免疫组合如表1所示。RBD组产生针对RBD蛋白和S1蛋白的结合抗体滴度如图3A所示：针对不同蛋白(S1蛋白和RBD蛋白)的结合抗体滴度基本一致， 均值都在8000左右，部分小鼠结合抗体没有被诱导起来，其可能的原因在于小鼠的个体差异性。免疫结束后4周，RBD组产生针对SARS-Cov-2假病毒的中和抗体滴度如图3B所示：除了一只滴度在1000以上，其他的都在200左右，部分小鼠中和抗体未被诱导起来，其可能的原因在于小鼠的个体差异性。The experimental procedure is as follows: the mice are randomly divided into 2 groups, and named as the control group and the RBD group according to the immunogen. Specific immunization combinations are shown in Table 1. The RBD group produced binding antibody titers against RBD protein and S1 protein as shown in Figure 3A: The binding antibody titers against different proteins (S1 protein and RBD protein) were basically the same, with an average value of about 8000, and some mice did not have binding antibodies. The possible reason for being induced is the individual differences of mice. 4 weeks after the end of immunization, the RBD group produced neutralizing antibody titers against the SARS-Cov-2 pseudovirus as shown in Figure 3B: except for one titer above 1000, the others were around 200, and some mice neutralized Antibodies are not induced, the possible reason is the individual differences of mice.
该实验证实，RBD蛋白可诱导结合抗体的产生(其效果与S蛋白诱导相似)，且单纯的RBD蛋白能够活化出较高水平的中和抗体。This experiment confirmed that RBD protein can induce the production of binding antibodies (its effect is similar to that of S protein induction), and that RBD protein alone can activate a higher level of neutralizing antibodies.
表1.RBD蛋白诱导结合抗体及中和抗体实验Table 1. RBD protein induced binding antibody and neutralizing antibody experiment
Figure PCTCN2020096148-appb-000003
Figure PCTCN2020096148-appb-000003
实施例4：质粒pcDNA3.1-sRBD-hFn对小鼠结合抗体及中和抗体的诱导效果Example 4: Induction effect of plasmid pcDNA3.1-sRBD-hFn on mouse binding antibody and neutralizing antibody
根据实施例1得出的结论(质粒能够正确表达出所需蛋白)，采用二硫键修饰后的RBD质粒(pcDNA3.1-sRBD-hFn)免疫C57/BL6小鼠，在完成免疫2周后，评价免疫组合诱导针对RBD蛋白的结合抗体滴度和针对SARS-CoV-2假病毒的中和抗体滴度。According to the conclusion drawn in Example 1 (the plasmid can correctly express the desired protein), the RBD plasmid (pcDNA3.1-sRBD-hFn) modified with disulfide bonds was used to immunize C57/BL6 mice, and 2 weeks after the completion of the immunization , To evaluate the immunization combination induced binding antibody titer against RBD protein and neutralizing antibody titer against SARS-CoV-2 pseudovirus.
实验步骤如下：将小鼠随机分为2组，根据第二针的免疫原分别命名为对照组和RBD-hFn组。具体免疫组合如表2所示。不同免疫组合产生针对RBD蛋白的结合抗体滴度如图4A所示：大部分都在2000左右。第二针免疫结束后2周，不同的免疫组合产生针对SARS-CoV-2假病毒的中和抗体滴度如图4B所示：除一只中和抗体滴度大于80，其他的都在20左右。The experimental procedure is as follows: the mice were randomly divided into 2 groups, and named as the control group and the RBD-hFn group according to the immunogen of the second injection. Specific immunization combinations are shown in Table 2. The titers of antibodies against RBD protein produced by different immunization combinations are shown in Figure 4A: most of them are around 2000. Two weeks after the second immunization, the neutralizing antibody titers against the SARS-CoV-2 pseudovirus produced by different immunization combinations are shown in Figure 4B: except for one neutralizing antibody with a titer greater than 80, the others are all at 20. about.
该实验证实，修饰后的RBD重组质粒疫苗能够在小鼠体内诱导出中和抗体。This experiment confirmed that the modified RBD recombinant plasmid vaccine can induce neutralizing antibodies in mice.
表2.修饰后的RBD-hFn质粒诱导结合抗体及中和抗体实验Table 2. The modified RBD-hFn plasmid induces binding antibody and neutralizing antibody experiment
Figure PCTCN2020096148-appb-000004
Figure PCTCN2020096148-appb-000004
实施例5：K562-HA2-sRBD细胞及K562-S细胞对小鼠结合抗体及中和抗体的诱导效果Example 5: Induction effect of K562-HA2-sRBD cells and K562-S cells on mouse binding antibodies and neutralizing antibodies
根据实施例4中得出的结果，将sRBD-hFn组的10只小鼠分为两组，再次免疫K562-S和K562-HA2-sRBD，在完成免疫2周后，评价免疫组合诱导针对RBD蛋白的结合抗体滴度和针对SARS-CoV-2假病毒的中和抗体滴度。According to the results obtained in Example 4, 10 mice in the sRBD-hFn group were divided into two groups, and they were immunized again with K562-S and K562-HA2-sRBD. Two weeks after the immunization was completed, the immune combination was evaluated to induce RBD Protein binding antibody titer and neutralizing antibody titer against SARS-CoV-2 pseudovirus.
具体免疫组合如表3所示。免疫结束后2周，不同免疫组合产生针对RBD蛋白的结合抗体滴度如图5A所示：K562-S组结合抗体滴度大部分在50000左右，K562-HA2-sRBD组结合抗体滴度大部分在80000左右。免疫结束后2周，不同的免疫组合产生针对SARS-CoV-2假病毒的中和抗体滴度如图5B所示：K562-S组中和抗体滴度大部分在350左右，K562-HA2-sRBD组中和抗体滴度有一只达到2000，其他在400左右。Specific immunization combinations are shown in Table 3. Two weeks after the end of immunization, the titers of binding antibodies against RBD protein produced by different immunization combinations are shown in Figure 5A: most of the titers of binding antibodies in the K562-S group are around 50,000, and most of the titers in the K562-HA2-sRBD group are binding antibodies. Around 80,000. Two weeks after the end of immunization, different immunization combinations produced neutralizing antibody titers against SARS-CoV-2 pseudovirus as shown in Figure 5B: The neutralizing antibody titers of K562-S group were mostly around 350, and K562-HA2- In the sRBD group, one of the neutralizing antibody titer reached 2000, and the other was around 400.
该实验证实，K562-HA2-sRBD细胞相比于K562-S细胞在小鼠体内能够诱导出相对更高的结合抗体及中和抗体。This experiment confirmed that K562-HA2-sRBD cells can induce relatively higher binding antibodies and neutralizing antibodies in mice than K562-S cells.
表3.K562-S及K562-HA2-sRBD诱导结合抗体及中和抗体实验Table 3. K562-S and K562-HA2-sRBD induced binding antibody and neutralizing antibody experiment
Figure PCTCN2020096148-appb-000005
Figure PCTCN2020096148-appb-000005
实施例6：sRBD-hFn蛋白对小鼠结合抗体及中和抗体的诱导效果Example 6: Induction effect of sRBD-hFn protein on mouse binding antibody and neutralizing antibody
采用纯化的sRBD-hFn蛋白免疫C57/BL6小鼠，在完成免疫1周后，评价免疫组合诱导针对RBD蛋白的结合抗体滴度和针对SARS-CoV-2假病毒的中和抗体滴度。The purified sRBD-hFn protein was used to immunize C57/BL6 mice. One week after the immunization, the immunization combination induced the binding antibody titers against the RBD protein and the neutralizing antibody titers against the SARS-CoV-2 pseudovirus were evaluated.
实验步骤如下：将小鼠随机分为2组，根据免疫原分别命名为对照组和sRBD-hFn组。具体免疫组合如表4所示。免疫结束后一周产生针对RBD蛋白的结合抗体滴度如图6A所示：sRBD-hFn组有三只小鼠的结合抗体达到52000，除一只在3200之外，其他的都大于一万。免疫结束后1周，产生针对SARS-CoV-2假病毒的中和抗体滴度如图6B所示：有三只有中和抗体，滴度都在十几。The experimental procedure is as follows: the mice were randomly divided into 2 groups, and named as the control group and the sRBD-hFn group according to the immunogen. Specific immunization combinations are shown in Table 4. One week after the end of the immunization, the antibody titers against the RBD protein produced are shown in Figure 6A: three mice in the sRBD-hFn group have a binding antibody of 52,000, except for one at 3200, the others are greater than 10,000. One week after the end of the immunization, the titers of neutralizing antibodies against the SARS-CoV-2 pseudovirus are shown in Figure 6B: there are three neutralizing antibodies, and the titers are all over a dozen.
表4.sRBD-hFn蛋白诱导结合抗体及中和抗体实验Table 4. sRBD-hFn protein induced binding antibody and neutralizing antibody experiment
Figure PCTCN2020096148-appb-000006
Figure PCTCN2020096148-appb-000006
在本公开提及的所有文献都在本申请中引用作为参考，就如同每一篇文献被单独引用作为参考那样。此外应理解，在阅读了本公开的上述讲授内容之后，本领域技术人员可以对本公开作各种改动或修改，这些等价形式同样落于本申请所附权利要求书所限定的范围。All documents mentioned in this disclosure are cited as references in this application, as if each document was individually cited as a reference. In addition, it should be understood that after reading the above teaching content of the present disclosure, those skilled in the art can make various changes or modifications to the present disclosure, and these equivalent forms also fall within the scope defined by the appended claims of the present application.
附表1.序列表对应信息Attached Table 1. Corresponding information of sequence table
Figure PCTCN2020096148-appb-000007
Figure PCTCN2020096148-appb-000007

Claims (10)

  1. 一种针对新型冠状病毒SARS-CoV-2的免疫原性肽，其包括SARS-CoV-2病毒刺突蛋白S的RBD区，其中，所述RBD区进一步经半胱氨酸修饰形成sRBD区。An immunogenic peptide for a novel coronavirus SARS-CoV-2, which includes the RBD region of the spike protein S of the SARS-CoV-2 virus, wherein the RBD region is further modified by cysteine to form an sRBD region.
  2. 如权利要求1所述的免疫原性肽，其中，所述RBD区的氨基酸序列如SEQ ID NO：2所示，所述经半胱氨酸修饰的sRBD区的氨基酸序列如SEQ ID NO：4所示。The immunogenic peptide of claim 1, wherein the amino acid sequence of the RBD region is shown in SEQ ID NO: 2, and the amino acid sequence of the cysteine-modified sRBD region is shown in SEQ ID NO: 4 Shown.
  3. 如权利要求1所述的免疫原性肽，其中，所述RBD区或经半胱氨酸修饰的sRBD区包含在融合肽中，例如与之融合的部分包括：病毒或宿主来源的蛋白，转铁蛋白(Fn)、HIV p24、囊膜病毒的茎部，如流感HA2、艾滋病毒的gp41、抗体Fc段、GM-CSF、IL-21、CD40L或CD40抗体，如所述融合肽的序列如SEQ ID NO：8或SEQ ID NO：10所示。The immunogenic peptide of claim 1, wherein the RBD region or the cysteine-modified sRBD region is contained in a fusion peptide, for example, the part fused with it includes a virus or host-derived protein, Ferritin (Fn), HIV p24, the stem of enveloped virus, such as influenza HA2, HIV gp41, antibody Fc segment, GM-CSF, IL-21, CD40L or CD40 antibody, such as the sequence of the fusion peptide SEQ ID NO: 8 or SEQ ID NO: 10 is shown.
  4. 一种核苷酸分子，其编码如权利要求1-3中任一项所述的免疫原性肽。A nucleotide molecule encoding the immunogenic peptide of any one of claims 1-3.
  5. 如权利要求4所述的核苷酸分子，其中，所述RBD区的编码序列如SEQ ID NO：1所示，所述经半胱氨酸修饰的RBD区的编码序列如SEQ ID NO：3所示，或者所述核苷酸分子的序列如SEQ ID NO：7或SEQ ID NO：9所示。The nucleotide molecule of claim 4, wherein the coding sequence of the RBD region is shown in SEQ ID NO: 1, and the coding sequence of the cysteine modified RBD region is shown in SEQ ID NO: 3 As shown, or the sequence of the nucleotide molecule is as shown in SEQ ID NO: 7 or SEQ ID NO: 9.
  6. 一种载体，其包含如权利要求4-5中任一项所述的核苷酸分子。A vector comprising the nucleotide molecule according to any one of claims 4-5.
  7. 一种宿主细胞，其包含如权利要求6所述的载体，且能表达如权利要求1-3中任一项所述的免疫原性肽。A host cell comprising the vector according to claim 6 and capable of expressing the immunogenic peptide according to any one of claims 1-3.
  8. 一种针对新型冠状病毒SARS-CoV-2的疫苗，其包含如权利要求1-3中任一项所述的免疫原性肽、如权利要求4-5中任一项所述的核苷酸分子、如权利要求6所述的载体和/或如权利要求7所述的宿主细胞。A vaccine against the novel coronavirus SARS-CoV-2, which comprises the immunogenic peptide according to any one of claims 1-3 and the nucleotide according to any one of claims 4-5 A molecule, a vector according to claim 6 and/or a host cell according to claim 7.
  9. 如权利要求1-3中任一项所述的免疫原性肽、如权利要求4-5中任一项所述的核苷酸分子、如权利要求6所述的载体和/或如权利要求7所述的宿主细胞在制备用于预防或治疗新型冠状病毒SARS-CoV-2的疫苗中的应用。The immunogenic peptide according to any one of claims 1 to 3, the nucleotide molecule according to any one of claims 4-5, the vector according to claim 6 and/or as claimed Application of the host cell described in 7 in the preparation of a vaccine for the prevention or treatment of the novel coronavirus SARS-CoV-2.
  10. 一种制备针对新型冠状病毒SARS-CoV-2的疫苗的方法，所述方法包括：A method for preparing a vaccine against the novel coronavirus SARS-CoV-2, the method comprising:
    (a)提供包含如权利要求1-3中任一项所述的免疫原性肽、如权利要求4-5中任一项所述的核苷酸分子、如权利要求6所述的载体和/或如权利要求7所述的宿主细胞；(a) Provide comprising the immunogenic peptide according to any one of claims 1-3, the nucleotide molecule according to any one of claims 4-5, the vector according to claim 6, and /Or the host cell of claim 7;
    (b)将(a)中所提供的活性物质与免疫学上或药学上可接受的载体组合。(b) Combine the active substance provided in (a) with an immunologically or pharmaceutically acceptable carrier.
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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US11547673B1 (en) 2020-04-22 2023-01-10 BioNTech SE Coronavirus vaccine
US11878055B1 (en) 2022-06-26 2024-01-23 BioNTech SE Coronavirus vaccine

Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20060188519A1 (en) * 2004-06-14 2006-08-24 To Cheung Peptides, antibodies, and methods for the diagnosis of SARS
WO2007059190A2 (en) * 2005-11-14 2007-05-24 Psma Development Company, Llc. Compositions of and methods of using stabilized psma dimers
CN101848719A (en) * 2007-06-25 2010-09-29 图兰恩教育基金管理人 Influenza inhibiting compositions and method
CN103842374A (en) * 2011-05-13 2014-06-04 诺华股份有限公司 Pre-fusion rsv f antigens
CN106928326A (en) * 2015-12-31 2017-07-07 中国科学院动物研究所 A kind of coronavirus vaccine of the receptor binding domain subunit based on dimerization
CN111217919A (en) * 2020-03-04 2020-06-02 中山大学 Novel coronavirus S protein double-region subunit nano vaccine based on pyrococcus ferritin
CN111217917A (en) * 2020-02-26 2020-06-02 康希诺生物股份公司 Novel coronavirus SARS-CoV-2 vaccine and preparation method thereof

Patent Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20060188519A1 (en) * 2004-06-14 2006-08-24 To Cheung Peptides, antibodies, and methods for the diagnosis of SARS
WO2007059190A2 (en) * 2005-11-14 2007-05-24 Psma Development Company, Llc. Compositions of and methods of using stabilized psma dimers
CN101848719A (en) * 2007-06-25 2010-09-29 图兰恩教育基金管理人 Influenza inhibiting compositions and method
CN103842374A (en) * 2011-05-13 2014-06-04 诺华股份有限公司 Pre-fusion rsv f antigens
CN106928326A (en) * 2015-12-31 2017-07-07 中国科学院动物研究所 A kind of coronavirus vaccine of the receptor binding domain subunit based on dimerization
CN111217917A (en) * 2020-02-26 2020-06-02 康希诺生物股份公司 Novel coronavirus SARS-CoV-2 vaccine and preparation method thereof
CN111217919A (en) * 2020-03-04 2020-06-02 中山大学 Novel coronavirus S protein double-region subunit nano vaccine based on pyrococcus ferritin

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
DATABASE Protein 23 April 2020 (2020-04-23), ANONYMOUS: "surface glycoprotein, partial [Severe acute respiratory syndrome coronavirus 2]", XP055881940, retrieved from Genbank Database accession no. QJD25214 *
DE TAEYE STEVEN W., GO EDEN P., SLIEPEN KWINTEN, DE LA PEÑA ALBA TORRENTS, BADAL KIMBERLY, MEDINA-RAMÍREZ MAX, LEE WEN-HSIN, DESAI: "Stabilization of the V2 loop improves the presentation of V2 loop–associated broadly neutralizing antibody epitopes on HIV-1 envelope trimers", JOURNAL OF BIOLOGICAL CHEMISTRY, vol. 294, no. 14, 1 April 2019 (2019-04-01), pages 5616 - 5631, XP055881943, ISSN: 0021-9258, DOI: 10.1074/jbc.RA118.005396 *

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US11547673B1 (en) 2020-04-22 2023-01-10 BioNTech SE Coronavirus vaccine
US11925694B2 (en) 2020-04-22 2024-03-12 BioNTech SE Coronavirus vaccine
US11878055B1 (en) 2022-06-26 2024-01-23 BioNTech SE Coronavirus vaccine

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