WO2021254270A1 - 基于细胞膜展示冠状病毒免疫原以诱导中和抗体的方法 - Google Patents

基于细胞膜展示冠状病毒免疫原以诱导中和抗体的方法 Download PDF

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WO2021254270A1
WO2021254270A1 PCT/CN2021/099724 CN2021099724W WO2021254270A1 WO 2021254270 A1 WO2021254270 A1 WO 2021254270A1 CN 2021099724 W CN2021099724 W CN 2021099724W WO 2021254270 A1 WO2021254270 A1 WO 2021254270A1
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vaccine
protein
cell
cells
immunization
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French (fr)
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徐建青
张晓燕
何香川
丁龙飞
曹康丽
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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
    • 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
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues

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  • the present disclosure belongs to the field of biotechnology and vaccines. Specifically, the present disclosure relates to a method for displaying coronavirus immunogens based on cell membranes to induce neutralizing antibodies.
  • vaccines are the safest and most effective way for humans to actively prevent infectious diseases.
  • the working principle of vaccines is to induce specific immune responses against pathogens, including cellular immune responses and antibody responses, and form immunity through active immunity to inactivated pathogens or genetically engineered proteins or nucleic acid components with high immunogenicity. Memory, so that when there is a risk of infection again, the body’s immune system can react quickly and produce sufficient specific immune responses to block pathogens from invading target cells.
  • the main forms of existing vaccines are: inactivated virus vaccines, nucleic acid vaccines, protein subunit/virus-like particle vaccines, and bacterial/viral vector vaccines.
  • the virus-like particle vaccine has high immunogenicity, and at the same time, supplemented with adjuvant injection can produce a better immune protection effect.
  • exploring an immunogen display method that can express a form closer to the natural conformation will help improve the immunogenicity of the antigen and induce the production of neutralizing antibodies.
  • the epitope expressed on the membrane surface can be closer to the natural conformation of the viral envelope protein, and the glycosyl on its surface is similar to that of the infected virus, and the body needs to be truly reproduced.
  • the recognized antigen characteristics can be used as a preferred cell carrier for displaying enveloped virus antigens.
  • the K562 cell line is derived from human erythroleukemia and is characterized by the lack of endogenous expression of HLA-A, B, C (MHC-I), HLA-DR (MHC-II) and blood group antigen (A, B, O) molecules. It can avoid rejection reactions between the body, and is very sensitive to NK cell-mediated killing, and has no tumorigenicity in the body. Therefore, its application as an enveloped virus vaccine carrier has good safety and effectiveness.
  • Coronavirus is a type of positive-stranded single-stranded RNA virus with an envelope.
  • SARS-CoV-2 belongs to the ⁇ -coronavirus genus B subtype coronavirus, which is about 80% of the severe acute respiratory syndrome coronavirus (SARS-CoV). Homology, with extremely high transmission power and high pathogenicity in the population. Coronavirus mainly binds to host cell receptors through the spike protein Spike (S protein), which mediates virus invasion and determines the host tropism of the virus.
  • S protein spike protein
  • the receptor binding domain RBD located in the S protein S1 subunit can bind to the host cell surface receptor angiotensin converting enzyme 2 (ACE2), and then through the S protein S2 subunit to fuse the virus and the host cell membrane to promote its entry into susceptibility cell. Therefore, neutralizing antibodies targeting RBD, S1 and S protein epitopes can block viral RBD binding, interfere with S2-mediated membrane fusion and invasion, and inhibit viral replication, and can be used as candidate targets for coronavirus vaccine immunogens.
  • ACE2 angiotensin converting enzyme 2
  • This application provides a cell and related vaccine that can be effectively used to induce binding antibodies and neutralizing antibodies against the new coronavirus.
  • a cell displaying the new coronavirus SARS-CoV-2 spike protein S on its cell membrane surface is provided.
  • the spike protein S is selected from: (a) a polypeptide having the amino acid sequence shown in SEQ ID NO: 2; (b) a homologous polypeptide of the polypeptide described in (a), for example, it is the same as SEQ ID NO: : 2 has a homology 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%; ( c) A protein or polypeptide derived from (a) that has undergone substitution, deletion or addition of one or several amino acids in the amino acid sequence defined in (a) and is immunogenic.
  • the spike protein S is included in the fusion peptide, for example, the part fused with it is selected from: virus or host-derived protein, transferrin (Fn), human immunodeficiency virus (HIV) p24 protein, cyst The stem of a membrane virus, such as influenza virus HA2, HIV gp41, antibody Fc segment, GM-CSF, IL-21, CD40L or CD40 antibody.
  • virus or host-derived protein transferrin (Fn)
  • HAV human immunodeficiency virus
  • the cell contains a vector with a spike protein S coding sequence.
  • the spike protein S-encoding molecule is: (i) a molecule having a nucleotide sequence as shown in SEQ ID NO: 1; (ii) a molecule that hybridizes with (i) under stringent conditions; (iii) The sequence in (i) or (ii) has 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%, high Nucleotide molecules with homology of 99% or more; (iv) One or several nucleotides are substituted, deleted or added in the nucleotide sequence defined in (i) or (ii) and can express function Nucleotide molecule of a sex RBD immunogenic peptide.
  • the cell has been transferred into a vector with the spike protein S-encoding molecule described in any one of (i) to (iv).
  • the vector is selected from viral vectors, such as poxviruses (such as Tiantan strain, North American vaccine strain, Wyeth-derived strain, Lister strain, Ankara-derived strain, Copenhagen strain and New York strain poxvirus), 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.
  • poxviruses such as Tiantan strain, North American vaccine strain, Wyeth-derived strain, Lister strain, Ankara-derived strain, Copenhagen strain and New York strain poxvirus
  • adenovirus such as Ad5, Ad11, Ad26, Ad35, Ad68
  • lentiviral vector such as Ad5, Ad11, Ad26, Ad35, Ad68
  • lentiviral vector such as Ad5
  • adeno-associated virus such
  • the cell is a mammalian cell or an insect cell, such as K562, A549, HEK293, HeLa, CHO, NS0, SP2/0, PER.C6, Vero, RD, BHK, HT 1080, A549, Cos -7, ARPE-19, MRC-5 cells, High Five, Sf9, Se301, SeIZD2109, SeUCR1, Sf9, Sf900+, Sf21, BTI-TN-5B1-4, MG-1, Tn368, HzAm1, BM-N, Ha2302 , Hz2E5, Ao38.
  • an insect cell such as K562, A549, HEK293, HeLa, CHO, NS0, SP2/0, PER.C6, Vero, RD, BHK, HT 1080, A549, Cos -7, ARPE-19, MRC-5 cells, High Five, Sf9, Se301, SeIZD2109, SeUCR1, Sf9, Sf900+, Sf21
  • the cells are K562 cells, A549 cells, HEK293 cells.
  • the cell has a complete membrane structure displaying the spike protein S.
  • 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.
  • physical inactivation such as X-ray radiation, ultraviolet radiation
  • chemical inactivation such as ⁇ -propiolactone, formaldehyde, and paraformaldehyde fixation.
  • a vaccine or combination of vaccines against the new coronavirus SARS-CoV-2 is provided, which includes the cells of the present disclosure.
  • the form of the vaccine or vaccine combination is suitable for intramuscular vaccination, intradermal vaccination, subcutaneous vaccination, nasal drops, atomized inhalation, genital tract, rectal, oral administration or any combination thereof, preferably intramuscular injection.
  • the vaccine or vaccine combination includes 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, AS03 adjuvant, QS-21 adjuvant, CpG adjuvant, Poly I:C, E. coli Adhesin 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, AS03 adjuvant,
  • the vaccine combination further includes one or more other vaccines against the new coronavirus
  • the other vaccines include vaccines against coronavirus S, S1 or RBD, such as the S, S1 or RBD comes from including but not limited to SARS-CoV-2, SARS-CoV, MERS-CoV, HCoV-229E, HCoV-OC43, HCoV-NL63, HCoV-HKU1, bat-CoV.
  • the vaccine combination includes a combination of a nucleic acid vaccine (DNA or RNA vaccine) and a recombinant human-derived cell vector vaccine, and the components of the vaccine combination are vaccinated sequentially, preferably the DNA vaccine is vaccinated first.
  • a nucleic acid vaccine DNA or RNA vaccine
  • a recombinant human-derived cell vector vaccine a recombinant human-derived cell vector vaccine
  • the application of the cells of the present disclosure in preparing a vaccine for the prevention or treatment of the novel coronavirus SARS-CoV-2 is provided.
  • a cell of the present disclosure is provided, which is used to prevent or treat the novel coronavirus SARS-CoV-2.
  • a method for preventing or treating a novel coronavirus SARS-CoV-2 infection or related disorders comprising administering the cell or vaccine or vaccine composition of the present application to a subject in need.
  • a method for preparing a vaccine or a combination of vaccines against the novel coronavirus SARS-CoV-2 comprising:
  • the cells of the present disclosure display the coronavirus Spike protein immunogen on their cell membranes.
  • the cells of the present disclosure may be inactivated cells, and their inactivation methods include physical inactivation such as X-ray radiation and ultraviolet radiation; or chemical inactivation such as ⁇ -propiolactone and formaldehyde Formaldehyde fixation.
  • the inactivation of the cells of the present disclosure and other treatments do not destroy the integrity of their cell membranes.
  • the vaccine containing the cells of the present disclosure is used as a primary and/or booster vaccine. In some embodiments, the vaccine containing the cells of the present disclosure is used as a booster vaccine. In some embodiments, the vaccine containing the cells of the present disclosure is used as a booster vaccine after the initial immunization of the DNA vaccine.
  • Figure 1 Construction of lentiviral expression vector plasmid and cell membrane display Spike immunogen (S protein) expression verification.
  • FIG. 1 Identification of human leukocyte antigen (HLA) and human blood group antigen (A, B) expressed on K562 cell membrane.
  • HLA human leukocyte antigen
  • A, B human blood group antigen
  • FIG. 3 Comparison of the difference between the RBD specific binding antibody and neutralizing antibody induced by direct application of S protein epitope-related immunogens (RBD, S1) and K562 cells (K562-S protein) displaying S protein on the membrane to immunize mice .
  • RBD S protein epitope-related immunogens
  • K562-S protein K562 cells
  • mice were immunized with the immunization strategy in Table 1. Two weeks after the immunization, the ELISA method was used to detect the binding antibody titers induced by RBD, S1 protein immunization and K562-S protein immunization. The results showed that RBD protein was used for the second shot.
  • the RBD-specific binding antibody induced by immunization is weak, while S1 protein and K562-S protein immunization can produce higher antibody responses.
  • the K562 vaccine with S protein displayed on the membrane has a binding antibody titer of up to 40,000 ( Figure 3a) ); Consistent with the trend of induced binding antibodies, the neutralizing antibody titers against SARS-CoV-2 pseudovirus induced by K562-S protein 2 weeks after immunization were higher, with an average value of about 1200, and one mouse was able to Up to 4,500 (Figure 3b).
  • Figure 4 Using different vaccination routes to immunize the K562-S protein carrier vaccine, the difference in the induced RBD specific binding antibody and neutralizing antibody.
  • mice were immunized with the immunization strategy in Table 2, and the ELISA method was used to detect the binding antibody titers in the mouse serum at the first week, the second week, and the fourth week after immunization.
  • the immunization of K562 cells is divided into two methods: intraperitoneal and intramuscular injection. The results show that compared with the control group (K562), the experimental group (K562-S protein) can increase the titers of RBD-specific binding antibodies through intraperitoneal injection and intramuscular injection.
  • FIG. 5 Treatment of K562-S protein carrier vaccines with different inactivation methods can induce differences in RBD specific binding antibodies and neutralizing antibodies.
  • the K562-S protein cells were fixed by x-ray radiation and paraformaldehyde respectively, and the mice were immunized with the immunization strategy in Table 3, and the binding antibodies and neutralization in the mouse serum were detected in the first and second weeks after immunization.
  • Antibody titer The results of the ELISA method showed that the binding antibodies induced by the paraformaldehyde treatment group were higher in the first and second weeks after immunization, which was basically equal to or higher than that of the untreated group, while the binding antibodies induced by the x-ray treatment group were in the immune system.
  • FIG. 6 Sequential immunization with K562-S protein carrier vaccine can induce the production of RBD-specific binding antibodies and neutralizing antibodies.
  • the K562-S protein cells were fixed with paraformaldehyde, and the mice were randomly divided into 2 groups.
  • the mice were immunized with the immunization strategy in Table 4, and all the immunogens were inoculated by intramuscular injection. Detect the binding antibody and neutralizing antibody titers in the mouse serum at the first week and the second week after the second immunization.
  • the results of the ELISA method showed that the binding antibody titer induced by the K562-S protein immunization group in the first week after the second immunization could reach 6400 on average, and the binding antibody continued to maintain a high level in the second week (Figure 6a); at the same time, 2 Neutralizing antibodies against SARS-CoV-2 pseudovirus were not produced in the first week after the second immunization, and some mice were induced to produce neutralizing antibodies in the second week after immunization, with the highest titer reaching 324 ( Figure 6b).
  • the K562-S protein cell membrane was extracted, and the mice were immunized with the immunization strategy in Table 5. All immunogens were inoculated by intramuscular injection.
  • Figure 7 Different carrier vaccine immunizations based on S immunogen can induce the production of RBD-specific binding antibodies and neutralizing antibodies.
  • mice were randomly divided into 4 groups, and the mice were immunized with the immunization strategy in Table 6, and all the immunogens were inoculated by intramuscular injection.
  • the K562-S protein cells were fixed with paraformaldehyde, and then the inactivated K562-S vaccine and S trimer protein vaccine were combined with aluminum hydroxide adjuvant (Alum). Detect the binding antibody and neutralizing antibody titers in the mouse serum at the second week after the second immunization.
  • the results of the ELISA method showed that the binding antibody titer induced by the DNA-S group was weak after the second immunization, and the binding antibody GMT induced by the K562-S protein-Alum group was equivalent to that of the S-trimeric protein-Alum group, and could reach 100000 (Figure 7a); At the same time, the neutralizing antibody GMT of the anti-SARS-CoV-2 pseudovirus is basically the same as the binding antibody trend, and the GMT of the K562-S protein-Alum and S-trimeric protein-Alum groups are both higher than 1000 ( Figure 7b).
  • the S protein ELISA quantitative kit detects that the dose of S protein per 1e6 K562-S cells is approximately equal to 0.47 ⁇ g ( Figure 7c).
  • the antibody titer data of the S trimer protein group is converted into equal proportions to the K562-S vaccine immunization
  • the neutralizing antibody GMT of the protein vaccine group exceeded 100 after conversion
  • the neutralizing antibody GMT of the K562-S protein vaccine group exceeded 1000
  • the neutralizing GMT of the cell vaccine could reach about 13 times that of the protein vaccine ( Figure 7d).
  • n ⁇ indicates the multiple of phase difference
  • * indicates significant difference
  • mice were randomly divided into 7 groups, and the mice were immunized with the immunization strategy in Table 7.
  • the vaccination method and vaccine treatment were the same as above, and the compatible adjuvants were shown in Table 7.
  • Two weeks after the end of the immunization, the binding antibody titers and neutralizing antibody titers induced by the immunization of K562-S protein vaccines with different adjuvants were tested. The results showed that multiple adjuvants can increase the K562-S protein vaccine to varying degrees.
  • the AS03 milky adjuvant and the two combined adjuvants Alum+CpG and MnJ+CpG can induce the strongest immune response, with the highest neutralizing antibody titer, and GMT About 10,000; MnJ adjuvant alone and milky adjuvant MF59 are the second place; the traditional adjuvant Alum has the weakest lifting effect compared to other new or combined adjuvants, but it has been significantly improved compared to the control without adjuvant. And antibody GMT is about 1000; similarly, the trend of binding antibody is also consistent with neutralizing antibody ( Figure 8a, 8b).
  • K562-S protein carrier vaccine is compatible with superior adjuvants, which can induce the production of persistent RBD-specific binding antibodies and neutralizing antibodies.
  • ICR mice were randomly divided into 2 groups, and the mice were immunized with the immunization strategy in Table 8. All immunogens were inoculated by intramuscular injection. At different time points after the end of immunization, the binding antibody titers and neutralizing antibody titers induced by immunization with K562-S protein vaccine with different adjuvant compatibility were detected.
  • the present disclosure relates to the field of vaccines.
  • using the characteristics of biological cell membranes by displaying the coronavirus S protein on the surface of the cell vector vaccine membrane, the natural conformation of the enveloped virus protein is restored to the greatest extent; and, if the selected cell is, for example, K562 cells, it will not express the immunogen.
  • the cell membrane basically does not express molecules such as human leukocyte antigens (HLA) or blood group antigens (A, B, O) that can trigger rejection or hemolysis, making it safe as a vaccine carrier for membrane display immunogens
  • HLA human leukocyte antigens
  • A, B, O blood group antigens
  • the carrier vaccine displaying the S protein immunogen on the cell membrane is expected to be an effective coronavirus vaccine, inducing the body to produce specific neutralizing antibodies for the prevention of new coronavirus or multiple coronavirus-related diseases.
  • K562 cells In tumor treatment, genetically engineered K562 cells have been transformed into a whole-cell therapeutic vaccine that overexpresses GM-CSF cytokines.
  • the vaccine mainly secretes cytokines to play an adjuvant-like immune activation effect and stimulate antigenic growth. Ingest and induce the corresponding cellular immune response.
  • human-derived cells As a preventive vaccine carrier, especially in infectious diseases that are completely different from tumors in pathogenic mechanism, disease and development, and prevention and treatment methods.
  • the human-derived cell vector vaccine K562-S has excellent immunogenicity and immunoreactivity; it can effectively enhance its antibody response at a lower dose of immunogenic substances; through the use of compatible adjuvants, it can be further Improve its immune effect.
  • This application proves that the S protein vaccine based on human-derived cells is a form of vaccine that efficiently induces neutralizing antibodies against the new coronavirus, and its advantageous effects are unpredictable.
  • 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.
  • S protein As used herein, the terms "S protein”, “immunogenic peptide” and “immunogenic peptide of the present disclosure/application” are used interchangeably, and refer to the structure of the spike protein including the SARS-CoV-2 virus and It has a peptide that stimulates binding antibodies and neutralizes antibodies.
  • the S protein may be: (a) a polypeptide having the amino acid sequence shown in SEQ ID NO: 2; (b) a homologous polypeptide of the polypeptide described in (a), for example, it is the same as SEQ ID NO: 2 has a homology 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%; (c) A protein or polypeptide derived from (a) that has undergone substitution, deletion or addition of one or several amino acids in the amino acid sequence defined in (a) and is immunogenic.
  • the S protein may include modifications that facilitate the enhancement of its immunogenicity or reactivity or stability and/or be linked to other parts that facilitate the enhancement of its immunogenicity or reactivity or stability, for example, to enhance S Protein stability, increase neutralizing antibody response, formation of multimers, increase cellular response, etc.
  • the part that can be connected to the modified or unmodified S protein includes but is not limited to: virus or host-derived protein, transferrin, HIV p24, the stem of enveloped virus, such as influenza HA2, HIV gp41, antibody Fc segment , GM-CSF, IL-21, CD40L or CD40 antibody, etc.
  • S protein may also include its variant forms, 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) amino acid deletion, insertion and/or substitution, and the addition of one or several (usually within 20) at the C-terminus and/or N-terminus, more Preferably it is within 10, more preferably within 5) amino acid.
  • 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.
  • the S protein of the present application 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 or the like.
  • the cell membrane such as K562 cells
  • S protein coding molecule As used herein, the terms "S protein coding molecule", “S protein coding sequence” and the like are used interchangeably, and both refer to the nucleotide molecule encoding the immunogenic S protein described in the present disclosure.
  • the nucleic acid molecule can be selected from, for example: (i) a nucleotide molecule whose sequence is shown in SEQ ID NO: 1; (ii) a molecule that hybridizes with (i) under stringent conditions; (iii) and (i) Or (ii) the sequence has 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%.
  • Source-derived nucleotide molecules (iv) Substitution, deletion or addition of one or several nucleotides in the nucleotide sequence defined in (i) or (ii) and capable of expressing functional immunogenic S protein Nucleotide molecule.
  • 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 the S protein, 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.
  • an expression vector containing the S protein coding sequence and appropriate transcription/translation control signals can be constructed using conventional methods in the art. 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.
  • host cells that can display S protein on the cell membrane surface are preferred, such as K562 cells, A549 cells, HEK293 cells, and the like.
  • 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, NS0, SP2/0, PER.C6, Vero, RD, BHK, HT 1080, A549, Cos-7, ARPE-19 can be used.
  • 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 on the cell membrane. 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 containing the coronavirus Spike immunogen displayed on the cell membrane of the present disclosure.
  • the vaccine comprises a formulation of the S protein 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 A symptom.
  • 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 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.
  • the immunization dose of the recombinant plasmid vaccine used in this application is 10-200 ⁇ g/mouse in mice; the recombinant human-derived cell vector vaccine is 100,000-500,000 cells/mouse in mice; the protein immunization dose is 1- 20 ⁇ g/only.
  • the human immune dose of recombinant plasmid vaccine can be 0.1-100mg, such as 0.2-50mg, 0.5-10mg, 1-2mg/person; the human dose of recombinant human-derived cell vector vaccine can be 100,000-100,000,000 cells/person; protein
  • the immunized human dose can be 0.1-200 ⁇ g, 0.5-100 ⁇ g, 1-80 ⁇ g, 5-70 ⁇ g, 10-65 ⁇ g, 20-60 ⁇ g/person.
  • the cellular immunity of the present application can obtain a high immune effect with a very low S protein immunization dose.
  • the actual S protein immunization dose provided by each ⁇ 10 6 K562-S cells of the present application is about 0.3-0.6 ⁇ g, 0.4-0.5 ⁇ g, such as 0.47 ⁇ g.
  • the membrane-displayed S protein vaccine of the present application increases the level of neutralizing antibodies induced by the S trimeric protein vaccine by at least 10 times, such as 10-50 times, 10 ⁇ 30 times, 10 to 15 times. If supplemented with an appropriate adjuvant, the level can be increased by, for example, 2-9 times.
  • non-inactivated or inactivated cell vaccines can be used.
  • a paraformaldehyde fixative solution can be used for inactivation, such as paraformaldehyde with a concentration of 0.01% to 5% (g/ml; the solvent is PBS) Fixative.
  • 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) (the entire text is incorporated herein 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 adjuvant that can be used in the vaccine herein can be one or more selected from the group consisting of Alum, AS03, MF59, MnJ, CpG, or any combination thereof, such as Alum+CpG, MnJ+CpG Wait.
  • the level of neutralizing antibodies that the adjuvant-containing vaccine can induce is significantly increased, for example, 2-100 times, 5-90 times, 6-80 times, 8- 70 times, or any multiple range in between.
  • the adjuvant-containing vaccine has a duration of at least 5 months, such as half a year, 1 year, 1.5 years, or 2 years for inducing neutralizing antibodies.
  • 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 "Remington's Pharmaceutical Sciences” (Remington'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.
  • intramuscular injection, intraperitoneal injection, or a combination thereof is used.
  • 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, Rales, 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
  • cough such as cough, sputum, fever, Rales, wheezing, difficulty breathing, 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).
  • the present disclosure also provides a combination product, which includes the host cell and/or vaccine of the present disclosure, and may also contain one or more types that help prevent and/or treat the novel coronavirus infection or its The function of symptoms or other substances that 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.
  • 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 cell 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.
  • a recombinant DNA vaccine is used for systemic primary immunization to establish a systemic immune response, and then a cellular vaccine is used for one or more immune boosts.
  • 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 experimental animals, immunization methods, immunogens, pseudoviruses and detection methods involved in the examples are as follows:
  • mice 6-8 week old female C57/BL6 mice (Example 1-8); 6-8 week old female ICR mice (Example 9).
  • Immunization method intramuscular injection to the left and right hind limbs of the mouse, or intraperitoneal injection to the mouse.
  • Immunogen The S protein sequence is from Genebank: NC_045512.2, and its nucleotide sequence is shown in SEQ ID NO: 1, and its amino acid sequence is shown in SEQ ID NO: 2
  • Recombinant plasmid vaccine (DNA): pcDNA3.1 (empty), pcDNA3.1-S protein;
  • Protein subunit vaccine protein: RBD protein (Nanjing GenScript Biotechnology Co., Ltd., Z03483-1); S1 protein (Beijing Yiqiao Shenzhou Technology Co., Ltd., Z03501); S trimer protein (nearshore protein Technology Co., Ltd., DRA49);
  • Recombinant human cell vector vaccine K562, K562-S protein
  • Human-derived vector cell membrane fragments K562-cell membrane, K562-S protein-cell membrane;
  • the immunization dose and inactivation treatment dose of each immunogen in the combination are as follows:
  • Recombinant plasmid vaccine (DNA): plasmid (dissolved in sterile PBS), 100 ⁇ g/mouse, 100 ⁇ L;
  • Protein subunit vaccine protein: protein (dissolved in sterile PBS) and aluminum adjuvant (Aluminium, InvivoGen, Item No. 5200) were mixed in a volume ratio of 1:1 and then immunized, 10 ⁇ g/mouse, 100 ⁇ L;
  • Recombinant human cell vector vaccine 100 0000 cells/mouse, 100 ⁇ L, dissolved in sterile PBS;
  • K562 cell membrane fragment vaccine (K562-cell membrane): 50 ⁇ g/mouse, 100 ⁇ L, dissolved in sterile saline; (Thermo, Mem-PER TM Plus Membrane Protein Extraction Kit, 89842), about 50 ⁇ g cell membrane extracted from 1,000,000 cells , So the dose here is consistent with the recombinant human-derived cell vector vaccine;
  • K562-S vaccine compatible adjuvant use After paraformaldehyde inactivates the K562-S vaccine, mix the adjuvants AS03 and MF59 (InvivoGen) with the K562-S vaccine according to the adjuvant: vaccine volume ratio of 1:1 After immunization, the adjuvant MnJ (MnStarter Biotechnology Co., Ltd.) was mixed with K562-S vaccine at 100 ⁇ g per mouse and immunized, and the adjuvant CpG (InvivoGen) was mixed with K562-S vaccine at 30 ⁇ g per mouse. immunity;
  • the gene recovery product is ligated to the digested linearized vector with T4DNA ligase (Thermo Scientific, Catalog No. 2011A): The ligation product is 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 expression plasmid of the S protein gene (pcDNA3.1-S protein) and the lentiviral vector plasmid (pHAGE-S protein) were successfully cloned. -puro).
  • the gene recovery product is ligated to the digested linearized vector with T4DNA ligase (Thermo Scientific, Catalog No. 2011A): The ligation product is 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 plasmid (pHAGE-hACE2-puro) of the hACE2 gene was successfully cloned.
  • mice 4 weeks after the end of the last immunization, and before the mice were sacrificed, the peripheral whole blood of the mice was collected by removing the eyeballs, collected in a 1.5 mL EP tube, and allowed to stand at room temperature to allow the blood to coagulate naturally.
  • the mouse 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.
  • TMB 100 ⁇ l/well
  • Stop the reaction quickly add 100 ⁇ l/well of the stop solution to stop the reaction.
  • each well was added 50 ⁇ L SARS-CoV-2 virus dilutions false, false per well containing final virus 200TCID 50.
  • Example 1 Construction of lentiviral expression vector pHAGE-S protein-puro and verification of expression of coronavirus S protein displayed on K562 cell membrane
  • S protein as a biological cell membrane vector vaccine immunogen, we constructed a lentiviral expression vector of S protein, packaged the lentivirus to infect K562 cells, and screened positive clones expressing S protein on the membrane surface.
  • the experimental procedure is as follows: prepare 5 ⁇ 10 5 K562 cells, resuspend them with 500 ⁇ L of 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 ⁇ g/ml, and finally the cells that can survive are integrated with Spike gene 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 Zahongshan Jinqiao Company, catalog number ZB2304. The results showed that the high expression of S protein in K562-S protein cells could be detected by Western blotting, while the uninfected K562 cells did not express S protein ( Figure 1b).
  • 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.
  • ACE2-C-AVI-6his Shanghai Nearshore Technology Co., Ltd., model 0331753-4065
  • PE-streptavidin BD Pharminge company, product number 563259
  • flow cytometry BD Pharminge company, model Arial
  • Example 2 Expression and identification of human leukocyte antigen (HLA) and human blood group antigen (A, B) on K562 cell membrane.
  • HLA human leukocyte antigen
  • A, B human blood group antigen
  • HLA Human leukocyte antigen
  • MHC human histocompatibility complex
  • Classical HLA class I includes HLA-A, B, and C
  • HLA class II mainly includes HLA-DP, DQ and DR
  • HLA class I is almost distributed on the surface of all cells in the body
  • HLA class II is mainly located in macrophages and B lymph nodes Glycoproteins on the surface of cells. Therefore, the detection of HLA molecule expression on the surface of K562 cell membrane by flow cytometry staining can confirm the safety of its membrane components as an immunogen presentation carrier.
  • HLA-A, B, and C staining uses 293T cells as a positive control.
  • K562 cell membranes do not express HLA class I molecules (HLA-A, B, C) ( Figure 2a), and HLA-DR staining uses human B cells as Positive control, the results show that the K562 membrane components also do not express HLA class II molecules (HLA-DR) ( Figure 2b).
  • the above shows that the K562 cell membrane has a certain degree of safety.
  • the flow cytometry antibody used is APC anti-human HLA-A, B, C Antibody (purchased from biolegend, article number 311409), APC anti-human HLA-DR antibody (purchased from biolegend, article number 327022).
  • K562 cells are human erythroid cells, and the expression of their blood group antigens may also cause hemolysis in different blood groups. Western blotting is used to detect whether K562 cells have blood group antigen expression.
  • K562 cells do not express blood group antigens A and B ( Figure 2c), which can rule out the possibility of K562 cells causing hemolytic reactions in different organisms.
  • the antibody used is Blood Group AB antigen (Z5H-2/Z2A) FITC (purchased from Santa, catalog number) sc-52370). Based on this, it can be considered that K562 cell membrane components as immunogen presentation carriers will not stimulate adverse rejection reactions between different organisms.
  • Example 3 Comparison of direct application of S protein epitope-related immunogens (RBD, S1) and K562 cells (K562-S protein) displaying S protein on the membrane to immunize mice to induce specific RBD binding antibodies and neutralizing antibodies difference.
  • mice were randomly divided into 4 groups, and the mice were immunized with the immunization strategy in Table 1. All immunogens were inoculated by intramuscular injection. Two weeks after the end of immunization, the ELISA method was used to detect the binding antibody titers induced by RBD, S1 protein immunization and K562-S protein immunization. The results showed that the RBD specific binding antibody induced by the second injection of RBD protein immunization was weak. The S1 protein and K562-S protein immunization can produce a higher antibody response, and the K562 vaccine with S protein displayed on the membrane has a binding antibody titer of up to 400,000 or more ( Figure 3a).
  • Example 4 Applying different vaccination routes to immunize K562-S protein carrier vaccines to induce differences in RBD specific binding antibodies and neutralizing antibodies.
  • mice were randomly divided into 4 groups, and the mice were immunized with the immunization strategy in Table 2.
  • the ELISA method was used to detect the binding antibody titers in the mice's serum at the first, second, and fourth weeks after immunization.
  • the first injection of plasmid DNA immunization is all intramuscular injection
  • the second injection of K562 cell immunization is divided into two methods: intraperitoneal and intramuscular injection.
  • the results show that compared with the control group (K562), the experimental group (K562-S protein) can increase the titers of RBD-specific binding antibodies through intraperitoneal injection and intramuscular injection. Among them, the muscle immune effect is better ( Figure 4a).
  • mice boosted by K562-S protein with different immunization methods were boosted by K562-S protein with different immunization methods.
  • Example 5 Applying different inactivation methods to treat K562-S protein carrier vaccines, induced differences in RBD specific binding antibodies and neutralizing antibodies.
  • the K562-S protein cells were fixed by x-ray radiation and paraformaldehyde, and the mice were randomly divided into 4 groups.
  • the mice were immunized with the immunization strategy in Table 3, and all the immunogens were inoculated by intramuscular injection. Detection of binding antibody and neutralizing antibody titers in mouse serum at the first and second weeks after immunization.
  • the results of the ELISA method showed that the binding antibodies induced by the paraformaldehyde treatment group were higher in the first and second weeks after immunization, which was basically equal to or higher than that of the untreated group, while the binding antibodies induced by the x-ray treatment group were in the immune system. It was slightly higher than the untreated group at the first week after immunization, and weaker than the untreated and paraformaldehyde-fixed group at the second week after immunization (Figure 5a).
  • the K562-S protein cells were fixed with paraformaldehyde, and the mice were randomly divided into 2 groups.
  • the mice were immunized with the immunization strategy in Table 4, and all the immunogens were inoculated by intramuscular injection. Detect the binding antibody and neutralizing antibody titers in the mouse serum at the first week and the second week after the second immunization.
  • the results of the ELISA method showed that the binding antibody titer induced by the K562-S protein immunization group in the first week after the second immunization could reach 6400 on average, and the binding antibody continued to maintain a high level in the second week (Figure 6a).
  • DNA-pcDNA3.1-Spike primary immunization and K562-S protein boost immunization are the preferred immunization strategies.
  • Example 7 Comparison of direct application of multiple forms of S protein immunogen vaccine and K562 cells (K562-S protein) displaying S protein on the membrane to immunize mice to induce the difference between RBD specific binding antibodies and neutralizing antibodies.
  • mice C57/BL6 mice were randomly divided into 4 groups, and the mice were immunized with the immunization strategy in Table 6, and all immunogens were inoculated by intramuscular injection.
  • the ELISA method was used to detect the different forms of S protein vaccine DNA-S (ie, the aforementioned DNA-pcDNA3.1-spike), S trimer protein-Alum and K562-S protein-Alum immune induction
  • S protein vaccine DNA-S ie, the aforementioned DNA-pcDNA3.1-spike
  • S trimer protein-Alum S trimer protein-Alum
  • K562-S protein-Alum immune induction The resulting binding antibody titer and neutralizing antibody titer are shown in FIG. 7.
  • Example 8 Comparing the compatibility of different types of adjuvants with K562-S protein cell carrier vaccine, and the difference in the induced RBD specific binding antibody and neutralizing antibody.
  • mice C57/BL6 mice were randomly divided into 7 groups, and the mice were immunized with the immunization strategy in Table 7. All immunogens were inoculated by intramuscular injection. Two weeks after the end of the immunization, the binding antibody titers and neutralizing antibody titers induced by the K562-S protein vaccine immunization with different adjuvants were tested respectively, and the results are shown in Figure 8.
  • the tested multiple adjuvants can improve the immunogenicity of the K562-S protein vaccine to varying degrees.
  • the AS03 milky adjuvant and the two combined adjuvants Alum+CpG and MnJ+CpG can induce the strongest immune response.
  • the neutralizing antibody titer is the highest, with a GMT of about 10,000.
  • the MnJ adjuvant alone and the milky adjuvant MF59 come next; the traditional adjuvant Alum is compared to other new or combined adjuvants.
  • the lifting effect is the weakest, the neutralizing antibody GMT is about 1000, but compared with the control group without adjuvant, the traditional Alum adjuvant has significantly increased the amount of neutralizing antibody.
  • binding antibodies are also consistent with the trend of neutralizing antibodies.
  • Example 7 the conversion method in Example 7 was used to compare the immune effect of the membrane display K562-S vaccine containing different adjuvants with the protein vaccine of the same dose.
  • the membrane display K562-S vaccine was compared with the protein vaccine after using various adjuvants. Produce a significantly improved and better immune effect.
  • Example 9 The K562-S protein carrier vaccine is compatible with superior adjuvants, which can induce the production of persistent RBD-specific binding antibodies and neutralizing antibodies.
  • mice were randomly divided into 2 groups, and the mice were immunized with the immunization strategy in Table 8. All the immunogens were inoculated by intramuscular injection. At different time points after the end of the immunization, the binding antibody titers and neutralizing antibody titers induced by the immunization of K562-S protein vaccines with different adjuvant compatibility were detected, and the results are shown in Figure 9.

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Abstract

一种基于细胞膜展示冠状病毒免疫原以诱导中和抗体的方法。具体而言，提供了一种在其细胞膜表面展示新型冠状病毒SARS-CoV-2刺突蛋白S的细胞，包含所述细胞的针对新型冠状病毒SARS-CoV-2的疫苗或疫苗组合，所述细胞在制备用于预防或治疗新型冠状病毒SARS-CoV-2的疫苗中的应用及其制备方法。所述细胞和疫苗能够在体内高效活化B细胞，诱导中和抗体应答，在预防和降低新冠病毒感染中有广泛的应用前景。

Description

基于细胞膜展示冠状病毒免疫原以诱导中和抗体的方法 技术领域
本公开属于生物技术和疫苗领域。具体而言，本公开涉及一种基于细胞膜展示冠状病毒免疫原以诱导中和抗体的方法。
背景技术
迄今为止，疫苗是人类主动预防传染性疾病最为安全和有效的方式。疫苗的工作原理为通过主动免疫已灭活的病原体或基因工程化的具有较高免疫原性的蛋白质或核酸组分，诱发针对病原体的特异性免疫应答包括细胞免疫应答与抗体应答，同时形成免疫记忆，以便再次存在感染风险时，机体免疫系统能够迅速反应，产生充足的特异性免疫应答，阻断病原体入侵靶细胞。
现有疫苗的主要形式为：灭活病毒疫苗、核酸疫苗、蛋白亚单位/病毒样颗粒疫苗及细菌/病毒载体疫苗。其中病毒样颗粒疫苗的免疫原性较高，同时辅以佐剂注射能够产生较好的免疫保护效应。但对于一些无法自组装形成病毒天然结构的囊膜病毒，探寻能够表达更接近天然构象形式的免疫原展示方式，将有助于提高抗原的免疫原性并诱导产生中和抗体。
高等生物真核细胞特别是人源化细胞，其膜表面表达的抗原表位能够更接近病毒囊膜蛋白的天然构象形式，并保证其表面所含糖基与感染病毒类似，真实重现机体需要识别的抗原特性，可作为展示囊膜病毒抗原的优选细胞载体。K562细胞株来源于人红白血病，其特点是缺乏内源性表达HLA-A、B、C(MHC-I)、HLA-DR(MHC-II)和血型抗原(A、B、O)分子，能够免于机体间的排斥反应，同时对NK细胞介导的杀伤非常敏感，而且在机体内不具有成瘤性，因此应用其作为囊膜病毒疫苗载体具有较好的安全性和有效性。
冠状病毒是一类具有囊膜的正链单股RNA病毒，其中SARS-CoV-2属于β冠状病毒属B亚型冠状病毒，与严重急性呼吸综合征冠状病毒(SARS-CoV)约有80％同源性，在人群中具有极高的传播力和较高致病性。冠状病毒主要通过刺突蛋白Spike(S蛋白)与宿主细胞受体结合，介导病毒入侵并决定病毒的宿主嗜性。其中位于S蛋白S1亚基的受体结合域RBD，能够与宿主细胞表面受体血管紧张素转 化酶2(ACE2)结合，随后通过S蛋白S2亚基融合病毒和宿主细胞膜，促进其进入易感细胞。因此，靶向RBD、S1和S蛋白表位的中和抗体，能够阻断病毒RBD结合，干扰S2介导的膜融合、侵入，抑制病毒复制，可作为冠状病毒疫苗免疫原的候选靶点。
然而，如何针对这些免疫原制得可高效诱导结合抗体和中和抗体的疫苗仍然是本领域中急需攻克的难题。本领域中迫切需要开发出可有效产生冠状病毒中和抗体的新型疫苗。
发明内容
本申请中正是提供了一种可有效用于诱导针对新型冠状病毒的结合抗体和中和抗体的细胞及其相关疫苗。
在本公开的第一方面中，提供了一种在其细胞膜表面展示新型冠状病毒SARS-CoV-2刺突蛋白S的细胞。
在一些实施方式中，刺突蛋白S选自：(a)具有SEQ ID NO:2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。
在一些实施方式中，刺突蛋白S包含在融合肽中，例如与之融合的部分选自：病毒或宿主来源的蛋白，转铁蛋白(Fn)、人免疫缺陷病毒(HIV)p24蛋白、囊膜病毒的茎部，如流感病毒HA2、HIV的gp41、抗体Fc段、GM-CSF、IL-21、CD40L或CD40抗体。
在一些实施方式中，所述细胞包含具有刺突蛋白S编码序列的载体。
在一些实施方式中，所述刺突蛋白S编码分子为：(i)具有如SEQ ID NO：1所示核苷酸序列的分子；(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。
在一些实施方式中，所述细胞已转入了具有(i)～(iv)中任一项所述的刺突蛋白S编码分子的载体。
在一些实施方式中，所述载体选自病毒载体，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。
在一些实施方式中，所述细胞为哺乳动物细胞或昆虫细胞，如K562、A549、HEK293、HeLa、CHO、NS0、SP2/0、PER.C6、Vero、RD、BHK、HT 1080、A549、Cos-7、ARPE-19、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、Ao38。
在一些实施方式中，所述细胞为K562细胞、A549细胞、HEK293细胞。
在一些实施方式中，所述细胞具有展示所述刺突蛋白S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。
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的疫苗或疫苗组合，其包含本公开的细胞。
在一些实施方式中，所述疫苗或疫苗组合的形式适于肌肉接种、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，优选肌肉注射。
在一些实施方式中，所述疫苗或疫苗组合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、AS03佐剂、QS-21佐剂、CpG佐剂、Poly I:C、大肠杆菌粘附素及其他TLR配体、GM-CSF、IL-2、IL-3、IL-7、IL-11、IL-12、IL-18、IL-21等。
在一些实施方式中，所述疫苗组合还包含针对新型冠状病毒的一种或多种其他疫苗，例如所述其他疫苗包括针对冠状病毒S、S1或RBD的疫苗，例如所述的S、S1或RBD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。
在一些实施方式中，疫苗组合包含核酸疫苗(DNA或RNA疫苗)与重组人源细胞载体疫苗的组合，且所述疫苗组合中的组分前后序贯接种，优选先接种DNA疫苗。
在本公开的一个方面中，提供了本公开的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。
在本公开的一个方面中，提供了本公开的细胞，其用于预防或治疗新型冠状病毒SARS-CoV-2。
在本公开的一个方面中，提供了预防或治疗新型冠状病毒SARS-CoV-2感染或其相关病症的方法，所述方法包括给予有需要的对象本申请的细胞或疫苗或疫苗组合物。
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的疫苗或疫苗组合的方法，所述方法包括：
(a)提供本申请所述的细胞；
(b)将(a)中所提供的细胞与免疫学上或药学上可接受的载体组合。
在本公开的一些实施方式中，本公开的细胞在其细胞膜上展示冠状病毒Spike蛋白免疫原。
在本公开的一些实施方式中，本公开的细胞可为灭活的细胞，其灭活方式包括物理灭活如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。
在本公开的一些实施方式中，本公开细胞的灭活以及其他处理不破坏其细胞膜的完整性。
在本公开的一些实施方式中，包含本公开细胞的疫苗作为初免和/或加强免疫疫苗使用。在一些实施方式中，包含本公开细胞的疫苗作为加强免疫疫苗使用。在一些实施方式中，包含本公开细胞的疫苗在DNA疫苗初免后，作为加强免疫疫苗使用。
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。
附图说明
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。
图1：慢病毒表达载体质粒的构建与细胞膜展示Spike免疫原(S蛋白)的表达验证。
整合有S蛋白基因的慢病毒表达载体质粒pHAGE-S蛋白-puro表达图谱(图1a)；慢病毒表达载体pHAGE-S蛋白-puro包装的慢病毒感染K562细胞后，蛋白免疫印迹结果显示构建的K562-S蛋白细胞成功表达S蛋白(图1b)；流式结果显示S蛋白在K562细胞膜上成功表达，并通过多次染色后流式分选使K562-S蛋白细胞得以富集(图1c)。
图2：K562细胞膜上表达人白细胞抗原(HLA)和人血型抗原(A、B)的鉴定。
K562细胞膜上人白细胞抗原I类(HLA-A、B)和人白细胞抗原II类(HLA-DR)分子的表达鉴定，蛋白免疫印迹结果显示K562细胞膜上基本不表达两种分子，其膜成分进入机体将不会产生排斥反应(图2a和2b)；K562细胞膜上人血型抗原A、B分子表达鉴定，蛋白免疫印迹结果显示K562细胞基本不表达人血型抗原，其膜成分不会刺激不同血型机体产生溶血反应(图2c)；
图3：比较直接应用S蛋白表位相关免疫原(RBD、S1)及膜上展示S蛋白的K562细胞(K562-S蛋白)免疫小鼠，诱导产生的RBD特异性结合抗体和中和抗体差异。
以表1的免疫策略对小鼠进行免疫，免疫结束后2周，使用ELISA方法分别检测RBD、S1蛋白免疫和K562-S蛋白免疫诱导产生的结合抗体滴度，结果显示第二针使用RBD蛋白免疫诱导产生的RBD特异性结合抗体较微弱，而S1蛋白及K562-S蛋白免疫能够产生较高的抗体应答，其中膜上展示S蛋白的K562疫苗结合抗体滴度最高可达40 0000(图3a)；与诱导产生的结合抗体趋势一致，免疫2周后K562-S蛋白诱导产生的针对SARS-CoV-2假病毒的中和抗体滴度较高，均值在1200左右，其中有一只小鼠能够达到4,500(图3b)。
图4：采用不同接种途径免疫K562-S蛋白载体疫苗，诱导产生的RBD特异性结合抗体和中和抗体差异。
以表2的免疫策略对小鼠进行免疫，ELISA方法检测免疫后第1周、第2周及第4周小鼠血清中结合抗体滴度。K562细胞的免疫接种分为腹腔和肌肉注射两种方式。结果显示，实验组(K562-S蛋白)与对照组(K562)相比，通过腹腔注射和肌肉注射的两种免疫方式都能够提升RBD特异性结合抗体的滴度，其中肌肉免疫效果更佳(图4a)；同时检测肌肉免疫后第1周、第2周及第4周小鼠血清中中和抗体的滴度，结果显示，与对照组相比，免疫后1周实验组的中和抗体提升较弱，而免疫后2周和4周实验组中和抗体逐步提升，中和抗体滴度最高可达3500以上(图4b)；免疫后第4周，分别比较以腹腔和肌肉途径注射K562-S蛋白诱导的中和抗体，结果显示两组诱导的中和抗体均值都在1200左右，且肌肉免疫有一只小鼠能够达到3700以上(图4c)。
图5：不同灭活方式处理K562-S蛋白载体疫苗，诱导产生的RBD特异性结合抗体和中和抗体差异。
对K562-S蛋白细胞分别使用x射线辐射和多聚甲醛固定的方法，以表3的免疫策略对小鼠进行免疫，检测免疫后第1周、第2周小鼠血清中结合抗体和中和抗体滴度。应用ELISA方法检测结果显示，免疫后第1周、第2周多聚甲醛处理组诱导产生的结合抗体较高，与未处理组基本相当甚至更高，而x射线处理组诱导的结合抗体在免疫后第1周时略高于未处理组，在免疫后第2周弱于未处理和多聚甲醛固定组(图5a)；同时，免疫后第1周只有多聚甲醛处理组诱导产生了抗SARS-CoV-2假病毒的中和抗体，且免疫后第2周多聚甲醛处理组部分小鼠产生的中和抗体显著提升，最高可达800以上，而x射线处理组基本没有有效诱导出中和抗体，未处理组也仅能诱导出和免疫后第1周多聚甲醛处理组相当的中和抗体(图5b)；
图6：K562-S蛋白载体疫苗贯序免疫，能够诱导产生RBD特异性结合抗体和中和抗体。
对K562-S蛋白细胞使用多聚甲醛固定的方法，将小鼠随机分2组，以表4的免疫策略对小鼠进行免疫，免疫原全部以肌肉注射方式接种。检测2次免疫后第1周、第2周小鼠血清中结合抗体和中和抗体滴度。应用ELISA方法检测结果显示，2次免疫后第1周K562-S蛋白免疫组诱导产生的结合抗体滴度平均能够达到 6400，第2周结合抗体继续保持较高水平(图6a)；同时，2次免疫后第1周没有产生了抗SARS-CoV-2假病毒的中和抗体，而免疫后第2周部分小鼠能够被诱导产生中和抗体，最高滴度达到324(图6b)。提取K562-S蛋白细胞膜，以表5的免疫策略对小鼠进行免疫，免疫原全部以肌肉注射方式接种。检测2次免疫后第2周小鼠血清中结合抗体和中和抗体滴度。应用ELISA方法检测结果显示，2次免疫后第2周只有2只小鼠产生了滴度约为200的结合抗体(图6c)；同时，2次免疫后第2周只有1只小鼠产生了微弱的中和抗体，滴度约为20(图6d)。
图7：基于S免疫原的不同载体疫苗免疫，能够诱导产生RBD特异性结合抗体和中和抗体。
将小鼠随机分4组，以表6的免疫策略对小鼠进行免疫，免疫原全部以肌肉注射方式接种。对K562-S蛋白细胞使用多聚甲醛固定的方法，后对灭活的K562-S疫苗和S三聚体蛋白疫苗配伍氢氧化铝佐剂(Alum)使用。检测2次免疫后第2周小鼠血清中结合抗体和中和抗体滴度。应用ELISA方法检测结果显示，2次免疫后DNA-S组诱导的结合抗体滴度较弱，K562-S蛋白-Alum组诱导的结合抗体GMT与S三聚体蛋白-Alum组相当，平均能够达到100000(图7a)；同时，抗SARS-CoV-2假病毒的中和抗体GMT与结合抗体趋势基本一致，K562-S蛋白-Alum和S三聚体蛋白-Alum组GMT均高于1000(图7b)。S蛋白ELISA定量试剂盒检测每1e6个K562-S细胞数的S蛋白剂量约等于0.47μg(图7c)，将S三聚体蛋白组的抗体滴度数据等比例转换为与K562-S疫苗免疫剂量相同，转换后蛋白疫苗组中和抗体GMT超过100，K562-S蛋白疫苗组中和抗体GMT超过1000，细胞疫苗中和GMT可达蛋白疫苗的13倍左右(图7d)。
图中，n×表示相差倍数，*表示显著性差异。
图8：不同类型佐剂与K562-S蛋白载体疫苗配伍使用，诱导产生的RBD特异性结合抗体和中和抗体差异
将小鼠随机分7组，以表7的免疫策略对小鼠进行免疫，接种方式和疫苗处理同上，配伍佐剂见表7。免疫结束后2周，分别检测不同佐剂配伍的K562-S蛋白疫苗免疫诱导产生的结合抗体滴度和中和抗体滴度，结果显示多种佐剂均能不同程度的提高K562-S蛋白疫苗的免疫原性，在多种佐剂配伍形式中，AS03乳状 佐剂和两种联合型佐剂Alum+CpG和MnJ+CpG，能够诱导相对最强的免疫应答，中和抗体滴度最高，GMT约为10000；单独MnJ佐剂和乳状佐剂MF59次之；传统佐剂Alum相对于其他新型或联合佐剂的提升作用最弱，但相较于无佐剂对照而言已有明显提高，中和抗体GMT约为1000；同样的，结合抗体也与中和抗体的趋势一致(图8a，8b)。
图9：K562-S蛋白载体疫苗配伍优势佐剂使用，能够诱导产生持久性的RBD特异性结合抗体和中和抗体。
将ICR小鼠随机分2组，以表8的免疫策略对小鼠进行免疫，免疫原全部以肌肉注射方式接种。免疫结束后不同时间点，分别检测不同佐剂配伍的K562-S蛋白疫苗免疫诱导产生的结合抗体滴度和中和抗体滴度。
结果显示ICR小鼠在初免后6周(加强免疫后2周)结合抗体GMT分别达到102400(Alum)和557380(MnJ+CpG)，中和抗体GMT分别达到9982(Alum)和33649(MnJ+CpG)，且抗体应答随时间延长而逐渐减弱，MnJ+CpG联合佐剂诱导的抗体应答强度和持久性都要优于传统Alum佐剂，在初免后24周(免疫后5个月)时，抗体应答仍维持在较高水平，结合抗体GMT分别达到13825(Alum)和32254(MnJ+CpG)，中和抗体GMT分别达到535(Alum)和1045(MnJ+CpG)(图9a，9b)，该结果表明MnJ+CpG联合佐剂为优势佐剂，K562-S蛋白载体疫苗能够维持持久的免疫活性。
具体实施方式
本公开涉及疫苗领域。本申请中利用生物细胞膜的特性，通过在细胞载体疫苗膜表面展示冠状病毒S蛋白，最大程度还原囊膜病毒蛋白的天然构象；并且，若所选细胞为例如K562细胞时，其除表达免疫原外，细胞膜上基本不表达如人类白细胞抗原(HLA)或血型抗原(A、B、O)等会引发排斥或溶血反应的分子，使其作为膜展示免疫原的疫苗载体具有很好的安全性；据此，该种细胞膜上展示S蛋白免疫原的载体疫苗有望作为一种有效的冠状病毒疫苗，诱导机体产生特异性的中和抗体，用于预防新冠或多种冠状病毒相关的疾病。
在肿瘤治疗中，基因工程化的K562细胞曾被改造为一种过表达GM-CSF细胞因子的全细胞治疗性疫苗，该疫苗主要通过分泌细胞因子发挥类似佐剂的免疫激活作用，刺激抗原的摄取和诱发相应的细胞免疫反应。然而，目前仍无利用人 源细胞作为预防性疫苗载体的报道，特别是在与肿瘤在致病机理、病症及其发展、以及预防和治疗方法都截然不同的感染性疾病中未见报道。
在本申请中，基于自然感染过程中S蛋白在病毒膜上展示为天然构象的原理，我们应用人源细胞膜作为S蛋白展示的疫苗载体基质，研发基于人源细胞载体的新冠病毒疫苗。本设计使得S蛋白能够保持自然构象，也同时避免人体的排斥反应，减少疫苗的载体效应。
我们首次通过实验证明了人源细胞载体疫苗K562-S具有优异的免疫原性和免疫反应性；在较低免疫原性物质剂量下就能够有效提升其抗体应答；通过配伍佐剂使用，可进一步提高其免疫效果。本申请证明了基于人源细胞的S蛋白疫苗为一种高效诱导针对新冠病毒中和抗体的疫苗形式，其优势效应具有不可预知性。
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，可用于新冠病毒感染的预防和治疗。
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。
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％。
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。
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。
S蛋白及其编码分子
如本文所用，术语“S蛋白”、“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Spike蛋白结构且具有激发结合 抗体和中和抗体作用的肽。
在本公开的一些实施方式中，S蛋白可为：(a)具有SEQ ID NO：2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。
在一些情况下，S蛋白可包括有利于增强其免疫原性或反应性或稳定性的修饰和/或连接于有利于增强其免疫原性或反应性或稳定性的其他部分，以例如增强S蛋白稳定性、提高中和抗体应答、形成多聚体、增加细胞应答等。可与修饰或未修饰的S蛋白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、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。
S蛋白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、插入和/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。
本申请的S蛋白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。在本申请中，优选采用细胞膜(例如K562细胞)展示S蛋白，以最大程度还原囊膜病毒蛋白的天然构象，并提高安全性。
如本文所用，术语“S蛋白编码分子”、“S蛋白编码序列”等可互换使用，均是指编码本公开所述的免疫原性S蛋白的核苷酸分子。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 达功能性免疫原性S蛋白的核苷酸分子。
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。
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。
载体和宿主细胞
本公开还涉及包含S蛋白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。
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：
(1)用本公开的编码核苷酸分子，或用含有核苷酸分子的重组表达载体转化或转导合适的宿主细胞；
(2)在合适的培养基中培养的宿主细胞；
(3)从培养基或细胞中分离、纯化蛋白质或多肽。
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。
可采用本领域常规方法构建含S蛋白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等表达系统。
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。
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。本申请中优选可在细胞膜表面展示S蛋白的宿主细胞，例如K562细胞、A549细胞、HEK293细胞等。
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、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。
本公开的核苷酸分子在高等真核细胞中表达时，如果在载体中插入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。
在上面的方法中的重组多肽可在细胞膜上表达。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。
疫苗和免疫偶联物
本文中还提供了一种包含本公开的细胞膜上展示冠状病毒Spike免疫原的疫苗，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S蛋白的配制品，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。
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。
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。
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。
例如，本申请中所用重组质粒疫苗的免疫剂量在小鼠体内为10-200μg/只；重组人源细胞载体疫苗在小鼠体内为10 0000-500 0000个细胞/只；蛋白免疫剂量为1-20μg/只。重组质粒疫苗的人体免疫剂量可为0.1～100mg，例如0.2～50mg，0.5～10mg，1～2mg/人；重组人源细胞载体疫苗在人用剂量可为100 0000～100000000个细胞/人；蛋白免疫的人用剂量可为0.1～200μg，0.5～100μg，1～80μg，5～70μg，10～65μg，20-60μg/人。
采用本申请的细胞免疫能够以极低的S蛋白免疫剂量获得高免疫效果。例如，在一些实施方式中，每×10 6个本申请的K562-S细胞所能提供的实际S蛋白免疫剂量约为0.3～0.6μg，0.4～0.5μg，如0.47μg。以等量的S蛋白免疫剂量进行比较，本申请的膜展示S蛋白疫苗相较于S三聚体蛋白疫苗所诱导产生中和抗体的水平提高了至少10倍，例如10～50倍，10～30倍，10～15倍。若辅以适当的佐剂，则所述水平可再提高例如2～9倍。
在本申请中可采用未灭活或灭活的细胞疫苗，例如可采用多聚甲醛固定液进行灭活，如浓度可为0.01％～5％(g/ml；溶剂为PBS)的多聚甲醛固定液。
在疫苗制备和处理过程中，X射线辐照虽然是肿瘤相关治疗性细胞疫苗中的常用灭活方式，且不影响过表达的GM-CSF细胞因子的生物活性，但无法据此推 测X射线辐射对细胞疫苗表面展示蛋白的免疫原性的影响。辐照灭活可能对细胞疫苗的免疫活性造成一定不利影响，使其不易产生中和抗体，应慎用该灭活方式。同理，福尔马林溶液虽然也在全病毒疫苗灭活中使用，但人源细胞疫苗和全病毒疫苗的粒径、膜蛋白密度、膜成分等具有较多差异，在实际处理中也需调整，在保证其灭活完全的基础上，使其维持最大程度的免疫原性。
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。
在一些实施方式中，可用于本文疫苗的佐剂可为选自下组中的一种或多种：Alum、AS03、MF59、MnJ、CpG或它们的任意组合，例如Alum+CpG、MnJ+CpG等。优选地，相比不包含佐剂的相应疫苗，含佐剂的疫苗所能诱导产生的中和抗体水平显著提高，例如提高了2-100倍，5-90倍，6-80倍，8-70倍，或其间的任何倍数范围。优选地，相比不包含佐剂的相应疫苗，含佐剂的疫苗诱导产生中和抗体的持久性至少为5个月，例如半年、1年、1.5年、2年。
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》(Remington's Pharmaceutical Sciences)中。
本文疫苗组合物的形式可适于系统性或局部(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。在一些实施方式中，采用肌肉注射、腹腔注射或其组合。
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、啰音、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。
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)。
在本公开中还提供了一种组合产品，其包括本公开的宿主细胞和/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。
免疫方法
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：系统性免疫接种方式，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，初次免疫采用系统性接种或呼吸道内接种，优选系统性接种。
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。
在一些实施方式中，采用DNA疫苗进行初次免疫，并采用细胞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，可采用单一的全身系统免疫或呼吸道局部免疫方式，或采用两种免疫方式的组合。
在一些优选的实施方式中，采用重组DNA疫苗进行系统性初免，从而建立全身系统免疫应答，再用细胞疫苗进行一次或多种免疫加强。
采用本文的免疫方法可在呼吸道局部和全身系统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。
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。
实施例
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。
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。
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。
实施例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：
I.实验动物：6-8周龄雌性C57/BL6小鼠(实施例1-8)；6-8周龄雌性ICR小鼠(实施例9)。
II.免疫方式：对小鼠左右后肢分别进行肌肉注射，或对小鼠进行腹腔注射。
III.免疫原：S蛋白序列来自Genebank:NC_045512.2，其核苷酸序列如SEQ ID NO:1所示，其氨基酸序列如SEQ ID NO:2所示
1.重组质粒疫苗(DNA)：pcDNA3.1(空载)、pcDNA3.1-S蛋白；
2.蛋白亚单位疫苗(protein)：RBD蛋白(南京金斯瑞生物科技有限公司，Z03483-1)；S1蛋白(北京义翘神州科技有限公司，Z03501)；S三聚体蛋白(近岸蛋白质科技有限公司，DRA49)；
3.重组人源细胞载体疫苗：K562、K562-S蛋白；
4.人源载体细胞膜碎片：K562-细胞膜、K562-S蛋白-细胞膜；
IV.免疫原制备及免疫剂量：
所述组合中各个免疫原的免疫剂量及灭活处理剂量如下：
1.重组质粒疫苗(DNA)：质粒(溶于无菌PBS中)，100μg/只小鼠，100μL；
2.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，10μg/只小鼠，100μL；
3.重组人源细胞载体疫苗(K562)：100 0000个细胞/只小鼠，100μL，溶于无菌PBS中；
4.K562细胞膜碎片疫苗(K562-细胞膜)：50μg/只小鼠，100μL，溶于无菌生理盐水；(Thermo,Mem-PER TM Plus Membrane Protein Extraction Kit,89842),1000000个细胞约提取50μg细胞膜，因此此处剂量为与重组人源细胞载体疫苗一致；
5.X射线照射：50Gy；
6.多聚甲醛固定液浓度：4％，溶于无菌PBS中；
7.K562-S疫苗配伍佐剂使用：多聚甲醛灭活K562-S疫苗后，将佐剂AS03和MF59(InvivoGen)分别按照佐剂:疫苗的体积比为1:1与K562-S疫苗混合后进行免疫，佐剂MnJ(MnStarter生物技术有限公司)按照每只小鼠100μg与K562-S疫苗混合后进行免疫，佐剂CpG(InvivoGen)按照每只小鼠30μg与K562-S疫苗混合后进行免疫；
8.K562-S细胞的S蛋白含量：将一定数量的K562-S细胞裂解后，使用S
蛋白定量检测试剂盒-ELISA法测定S蛋白含量(Biodragon,BF03087)。
V.免疫间隔：
具体免疫间隔见下文表格。
VI.免疫原相关载体构建：
1.人工合成S蛋白DNA序列(SEQ ID NO:1)，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pcDNA3.1(购自优宝生物)/pHAGE-MCS-puro(购自上海鑫湾生物)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。
2.基因回收产物与酶切线性化载体用T4DNA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蛋白基因的表达质粒(pcDNA3.1-S蛋白)和慢病毒载体质粒(pHAGE-S蛋白-puro)。
VII.SARS-CoV-2包膜假病毒包装：
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；
2.吸取6μg SARS-CoV-2膜蛋白质粒pcDNA3.1-S蛋白和6μg pNL4-3Δenv(NIH AIDS Reagent Program,3418)骨架质粒，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；
3.用双无DMEM将24μL TurboFect稀释，终体积为500μL/样品，室温孵育5min；
4.将上述步骤2和3中的液体混匀，1000μL/样品终体积，室温孵育20min，孵育结束后加至预先铺好于10cm培养皿中的293T细胞。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；
5.培养结束后，收集10cm皿的细胞培养上清于15mL离心管里，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。
VIII.构建稳定表达hACE2受体的293T细胞：
1.人工合成人源ACE2(hACE2)序列(Genebank#NCBI_NP_001358344.1)，其核苷酸如SEQ ID NO:3所示，氨基酸序列如SEQ ID NO:4所示，其序列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。
2.基因回收产物与酶切线性化载体用T4DNA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)。
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。
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。
5.提前一天铺约5×10 5个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。
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)。
IV.检测方法：
采血：
小鼠：最后一次免疫结束后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。
ELISA方法检测结合抗体
1.提前一晚将RBD蛋白溶于包被液中，以1μg/ml的蛋白浓度包被于96孔平底板，包被体积为100μl/孔，4度冰箱中放置过夜。
2.洗板3次：300μl/孔加入PBST，停留1分钟后弃去孔内液体，最后一次在滤纸上扣干(后续洗板操作相同)。5％牛奶封闭2小时。
3.洗板1次后加样，100μl/孔加入倍比稀释的血清，室温放置3小时。
4.洗板5次后加检测抗体：100μl/孔加入稀释后的生物素化的抗小鼠抗体(购自北京中杉金桥，货号ZB-2305)。盖上封板膜，室温孵育1小时。
5.洗板5次后加入TMB：100μl/孔，室温避光孵育10-30分钟。
6.终止反应：迅速以100μl/孔加入终止溶液终止反应。
7.读板：加入终止溶液后10min内在λ＝450nm处读值(酶标仪，购自BioTek公司)。
293T-hACE2细胞检测中和抗体：
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。
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。
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％CO2孵育1h。
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。
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。
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。
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使细胞充分裂解；
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 PCTCN2021099724-appb-000001
96微孔板发光-检测仪检测荧光素酶活性。
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ID50。
Figure PCTCN2021099724-appb-000002
实施例1：慢病毒表达载体pHAGE-S蛋白-puro的构建及冠状病毒S蛋白在K562细胞膜上展示的表达验证
为了研究S蛋白作为生物细胞膜载体疫苗免疫原的功能，我们构建了S蛋白的慢病毒表达载体，并包装慢病毒感染K562细胞，筛选膜表面表达S蛋白的阳性克隆。
首先，我们合成了S蛋白基因，将其通过酶切连接至pHAGE-MCS-puro慢病毒表达载体上，构建形成了pHAGE-S蛋白-puro质粒(图1a)；将pHAGE-S蛋白-puro包装形成慢病毒后感染K562细胞，通过蛋白质免疫印迹方法鉴定K562-S蛋白细胞中S蛋白的表达(图1b)；同时进行流式细胞染色鉴定，发现S蛋白在细胞膜上有表达，而对照感染的细胞不能检测到相关蛋白的表达，并进一步对表达S蛋白的K562细胞进行富集(图1c)。
实验步骤如下：准备5×10 5个K562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Spike基因的细胞。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K562-S蛋白细胞S蛋白高表达，而未感染K562细胞不表达S蛋白(图1b)。
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蛋白细胞能够高表达目的基因S蛋白(图1c)。
实施例2.K562细胞膜上人白细胞抗原(HLA)和人血型抗原(A、B)的表达鉴定。
人白细胞抗原(HLA)系人组织相容性复合体(MHC)的表达产物，是构成移植排斥反应的重要抗原物质。经典的HLA I类包括HLA-A、B、C；HLA II类主要包括HLA-DP、DQ和DR；HLA I类几乎分布于身体全部细胞表面，HLA II类主 要定位于巨噬细胞和B淋巴细胞的表面的糖蛋白。因此，通过流式细胞染色检测K562细胞膜表面HLA分子的表达情况，能够确认其膜成分作为免疫原呈递载体的安全性。同时HLA-A、B、C染色以293T细胞为阳性对照，结果显示K562细胞膜上不表达HLA I类分子(HLA-A、B、C)(图2a)，HLA-DR染色以人B细胞为阳性对照，结果显示K562膜成分同样不表达HLA II类分子(HLA-DR)(图2b)，以上说明K562细胞膜具有一定的安全性，所用流式抗体为APC抗人HLA-A,B,C抗体(购自biolegend,货号311409)，APC抗人HLA-DR抗体(购自biolegend,货号327022)。此外，K562细胞作为人红系细胞，其血型抗原的表达也可能导致不同血型机体的溶血反应，通过蛋白免疫印迹检测K562细胞是否有血型抗原表达。
结果显示，K562细胞不表达血型抗原A、B(图2c)，可以排除K562细胞可能造成不同机体的溶血反应，所用抗体为Blood Group AB antigen(Z5H-2/Z2A)FITC(购自Santa，货号sc-52370)。据此，可以认为K562细胞膜成分作为免疫原呈递载体不会刺激不同机体间产生不良的排斥反应。
实施例3.比较直接应用S蛋白表位相关免疫原(RBD、S1)及膜上展示S蛋白的K562细胞(K562-S蛋白)免疫小鼠，诱导产生的RBD特异性结合抗体和中和抗体差异。
将小鼠随机分4组，以表1的免疫策略对小鼠进行免疫，免疫原全部以肌肉注射方式接种。免疫结束后2周，使用ELISA方法分别检测RBD、S1蛋白免疫和K562-S蛋白免疫诱导产生的结合抗体滴度，结果显示第二针使用RBD蛋白免疫诱导产生的RBD特异性结合抗体较微弱，而S1蛋白及K562-S蛋白免疫能够产生较高的抗体应答，其中膜上展示S蛋白的K562疫苗结合抗体滴度最高可达400000以上(图3a)。
与诱导产生的结合抗体趋势一致，免疫2周后K562-S蛋白诱导产生的针对SARS-CoV-2假病毒的中和抗体滴度较高，均值在1200左右，其中有一只小鼠能够达到4,500(图3b)。该实验证实，基于细胞膜上展示S蛋白的K562载体疫苗加强免疫，能够活化出与普通蛋白免疫相当甚至更高水平的中和抗体。
表1.分别以蛋白质和膜上展示免疫原加强免疫的小鼠实验分组
Figure PCTCN2021099724-appb-000003
Figure PCTCN2021099724-appb-000004
实施例4.应用不同接种途径免疫K562-S蛋白载体疫苗，诱导产生的RBD特异性结合抗体和中和抗体差异。
将小鼠随机分4组，以表2的免疫策略对小鼠进行免疫，ELISA方法检测免疫后第1周、第2周及第4周小鼠血清中结合抗体滴度。第一针质粒DNA免疫接种全部为肌肉注射，第二针K562细胞的免疫接种分为腹腔和肌肉注射两种方式。结果显示，实验组(K562-S蛋白)与对照组(K562)相比，通过腹腔注射和肌肉注射的两种免疫方式都能够提升RBD特异性结合抗体的滴度，其中肌肉免疫效果更佳(图4a)。
同时，检测肌肉免疫后第1周、第2周及第4周小鼠血清中中和抗体的滴度。结果显示，与对照组相比，免疫后1周实验组的中和抗体提升较弱，而免疫后2周和4周实验组中和抗体逐步提升，中和抗体滴度最高可达3500以上(图4b)；免疫后第4周，分别比较以腹腔和肌肉途径注射K562-S蛋白诱导的中和抗体，结果显示两组诱导的中和抗体均值都在1200左右，且肌肉免疫有一只小鼠能够达到3700以上(图4c)。
表2.不同免疫接种方式加强免疫K562-S蛋白的小鼠实验分组
Figure PCTCN2021099724-appb-000005
实施例5.应用不同灭活方式处理K562-S蛋白载体疫苗，诱导产生的RBD特异性结合抗体和中和抗体差异。
对K562-S蛋白细胞分别使用x射线辐射和多聚甲醛固定的方法，将小鼠随机分4组，以表3的免疫策略对小鼠进行免疫，免疫原全部以肌肉注射方式接种。检测免疫后第1周、第2周小鼠血清中结合抗体和中和抗体滴度。应用ELISA方法检测结果显示，免疫后第1周、第2周多聚甲醛处理组诱导产生的结合抗体较高，与未处理组基本相当甚至更高，而x射线处理组诱导的结合抗体在免疫后第1周时略高于未处理组，在免疫后第2周弱于未处理和多聚甲醛固定组(图5a)。
同时，免疫后第1周只有多聚甲醛处理组诱导产生了抗SARS-CoV-2假病毒的中和抗体，且免疫后第2周多聚甲醛处理组部分小鼠产生的中和抗体显著提升，最高可达800以上，而x射线处理组基本没有有效诱导出中和抗体，未处理组也仅能诱导出和免疫后第1周多聚甲醛处理组相当的中和抗体(图5b)。
表3.不同灭活方式加强免疫K562-S蛋白的小鼠实验分组
Figure PCTCN2021099724-appb-000006
实施例6.K562-S蛋白载体疫苗贯序免疫，能够诱导产生RBD特异性结合抗体和中和抗体。
对K562-S蛋白细胞使用多聚甲醛固定的方法，将小鼠随机分2组，以表4的免疫策略对小鼠进行免疫，免疫原全部以肌肉注射方式接种。检测2次免疫后第1周、第2周小鼠血清中结合抗体和中和抗体滴度。应用ELISA方法检测结果显示，2次免疫后第1周K562-S蛋白免疫组诱导产生的结合抗体滴度平均能够达到6400，第2周结合抗体继续保持较高水平(图6a)。
同时，2次免疫后第1周没有产生抗SARS-CoV-2假病毒的中和抗体，而免疫后第2周部分小鼠能够被诱导产生中和抗体，最高滴度达到324(图6b)；与以上结果比较发现2次K562-S蛋白贯序免疫与DNA-pcDNA3.1-RBD初免、K562-S蛋白加强免疫诱导产生的中和抗体水平相当，但2次K562-S蛋白贯序免疫没有DNA-pcDNA3.1-Spike初免、K562-S蛋白加强免疫诱导产生的中和抗体水平高。
该结果提示DNA-pcDNA3.1-Spike初免、K562-S蛋白加强免疫为优选的免疫策略。
表4.K562-S蛋白载体疫苗贯序免疫小鼠实验分组
Figure PCTCN2021099724-appb-000007
此外，还尝试了用从K562-S蛋白细胞提取的细胞膜以表5所示方案免疫小鼠。结果如图6c所示。结果显示采用K562提取的细胞膜作为免疫原免疫小鼠，其诱导结合抗体和中和抗体的效果明显弱于采用完整的K562细胞所能获得的效果。该结果表明S蛋白展示在结构完整的K562细胞膜上具有相对更优的免疫原性。
表5.K562-S蛋白-细胞膜贯序免疫小鼠实验分组
Figure PCTCN2021099724-appb-000008
实施例7.比较直接应用多形式的S蛋白免疫原疫苗及膜上展示S蛋白的K562细胞(K562-S蛋白)免疫小鼠，诱导产生的RBD特异性结合抗体和中和抗体差异。
将C57/BL6小鼠随机分4组，以表6的免疫策略对小鼠进行免疫，免疫原全部以肌肉注射方式接种。免疫结束后2周，使用ELISA方法分别检测不同形式S蛋白疫苗DNA-S(即前文所述的DNA-pcDNA3.1-spike)、S三聚体蛋白-Alum和K562-S蛋白-Alum免疫诱导产生的结合抗体滴度和中和抗体滴度，结果如图7所示。
结果显示：使用DNA-S免疫诱导产生的RBD特异性结合抗体和针对假病毒的中和抗体滴度相对较低，而S三聚体蛋白-Alum和K562-S蛋白-Alum免疫能够产生较高的抗体应答，其结合抗体几何平均滴度(GMT)均超过100000(图7a)，中和抗体GMT均超过1000(图7b)。
由于细胞疫苗(K562-S)和蛋白疫苗(S三聚体蛋白)的免疫剂量不同，通过S蛋 白ELISA定量试剂盒检测每只小鼠K562-S疫苗的实际S蛋白免疫剂量，结果显示免疫所用每1×10 6个K562-S细胞数的S蛋白剂量约等于0.47μg(图7c)。
通过将S三聚体蛋白组的抗体滴度数据等比例转换为与K562-S疫苗免疫剂量相同，比较其中和抗体滴度差异，结果显示蛋白疫苗组中和抗体GMT超过100，K562-S蛋白疫苗组中和抗体GMT超过1000，细胞疫苗中和GMT约是蛋白疫苗的13倍左右(图7d)。
该实验证实，基于细胞膜上展示S蛋白的K562载体疫苗能够有效活化出高水平的中和抗体，与相同剂量的蛋白疫苗相比，诱导的中和抗体滴度更高，说明细胞膜上展示的免疫原具有更高的免疫活性。
表6.分别以DNA、蛋白和膜上展示S免疫原疫苗免疫小鼠的实验分组
Figure PCTCN2021099724-appb-000009
实施例8.比较不同类型佐剂与K562-S蛋白细胞载体疫苗配伍使用，诱导产生的RBD特异性结合抗体和中和抗体差异。
将C57/BL6小鼠随机分7组，以表7的免疫策略对小鼠进行免疫，免疫原全部以肌肉注射方式接种。免疫结束后2周，分别检测不同佐剂配伍的K562-S蛋白疫苗免疫诱导产生的结合抗体滴度和中和抗体滴度，结果如图8所示。
结果显示：所测试的多种佐剂均能不同程度的提高K562-S蛋白疫苗的免疫原性，在多种佐剂配伍形式中，AS03乳状佐剂和两种联合型佐剂Alum+CpG和MnJ+CpG，能够诱导相对最强的免疫应答，中和抗体滴度最高，GMT约为10000；单独MnJ佐剂和乳状佐剂MF59次之；传统佐剂Alum相对于其他新型或联合佐剂的提升作用最弱，中和抗体GMT约为1000，但相较于无佐剂的对照组而言，传统Alum佐剂已明显提高了中和抗体量。同样的，结合抗体也与中和抗体的趋势一致。
该实验结果证明了K562-S与优势佐剂配伍能够显著提升细胞疫苗的免疫应 答强度。
此外，采用实施例7中换算方式将包含不同佐剂的膜展示K562-S疫苗与相同剂量的蛋白疫苗的免疫效果进行比较，采用各种佐剂后膜展示K562-S疫苗相对于蛋白疫苗能产生显著提高的更优异免疫效果。
表7.分别以不同佐剂配伍膜上展示S免疫原疫苗的小鼠实验分组
Figure PCTCN2021099724-appb-000010
实施例9.K562-S蛋白载体疫苗配伍优势佐剂使用，能够诱导产生持久性的RBD特异性结合抗体和中和抗体。
本组实验测试ICR品系小鼠对K562-S载体疫苗的免疫应答，选用传统佐剂Alum或新型联合佐剂MnJ+CpG配伍，并观察抗体应答的持久性。
将小鼠随机分2组，以表8的免疫策略对小鼠进行免疫，免疫原全部以肌肉注射方式接种。免疫结束后不同时间点，分别检测不同佐剂配伍的K562-S蛋白疫苗免疫诱导产生的结合抗体滴度和中和抗体滴度，结果如图9所示。
结果显示：ICR小鼠在初免后2周结合抗体GMT分别达到94810(Alum)和516064(MnJ+CpG)，中和抗体GMT分别达到9791(Alum)和29716(MnJ+CpG)，且抗体应答随时间延长而逐渐减弱，MnJ+CpG联合佐剂诱导的抗体应答强度和持久性都要优于传统Alum佐剂，在初免后24周(免疫后5个月)时，抗体应答仍维持在较高水平，结合抗体GMT分别达到29863(Alum)和129016(MnJ+CpG)，中和抗体GMT分别达到1849(Alum)和3249(MnJ+CpG)(图9a，9b)。
该结果表明K562-S蛋白载体疫苗在不同品系小鼠中均保持较优免疫原性，且MnJ+CpG联合佐剂为优势佐剂；疫苗诱导的抗体应答能够维持至少5个月，说明 配伍佐剂的K562-S疫苗的优越性和持久性。
表8.膜上展示S免疫原疫苗免疫ICR小鼠的实验分组
Figure PCTCN2021099724-appb-000011
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。

Claims (11)

  1. 一种在其细胞膜表面展示新型冠状病毒SARS-CoV-2刺突蛋白S的细胞。
  2. 如权利要求1所述的细胞，其中，所述刺突蛋白S的氨基酸序列如SEQ ID NO:2所示；或者，
    所述刺突蛋白S包含在融合肽中，例如与之融合的部分选自：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。
  3. 如权利要求1所述的细胞，其中，所述细胞包含具有刺突蛋白S编码序列的载体，例如，转入了具有SEQ ID NO:1所示核苷酸分子的载体。
  4. 如权利要求1所述的细胞，其中，所述细胞为哺乳动物细胞或昆虫细胞，如K562、A549、HEK293、HeLa、CHO、NS0、SP2/0、PER.C6、Vero、RD、BHK、HT 1080、A549、Cos-7、ARPE-19、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、Ao38，优选K562细胞、A549细胞、HEK293细胞，优选所述细胞具有展示所述刺突蛋白S的完整膜结构，优选每1×10 6个所述细胞提供约0.3～0.6微克(如0.47微克)的刺突蛋白S。
  5. 如权利要求1所述的细胞，其中，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。
  6. 一种针对新型冠状病毒SARS-CoV-2的疫苗或疫苗组合，其包含如权利要求1-5中任一项所述的细胞。
  7. 如权利要求6所述的疫苗或疫苗组合，其中，所述疫苗或疫苗组合的形式适于肌肉接种、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，优选肌肉注射。
  8. 如权利要求6所述的疫苗或疫苗组合，其中，所述疫苗组合还包含针对新型冠状病毒的一种或多种其他疫苗，例如所述其他疫苗包括针对冠状病毒S、S1或RBD的疫苗，例如所述的S、S1或RBD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；或者
    所述疫苗组合包含核酸疫苗(DNA或RNA疫苗)与重组人源细胞载体疫苗的 组合，且所述疫苗组合中的组分前后序贯接种，优选先接种DNA疫苗。
  9. 如权利要求1-5中任一项所述的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。
  10. 一种制备针对新型冠状病毒SARS-CoV-2的疫苗或疫苗组合的方法，所述方法包括：
    (a)提供如权利要求1-5中任一项所述的细胞；
    (b)将(a)中所提供的细胞与免疫学上或药学上可接受的载体或任选的佐剂组合。
  11. 如权利要求6-8中任一项所述的疫苗或疫苗组合、或如权利要求9所述的应用、或如权利要求10所述的方法，其中，所述疫苗或疫苗组合还包含佐剂，例如选自下组的佐剂：AS03、MF59、MnJ、CpG、氢氧化铝佐剂(如Alum)、完全弗氏佐剂、不完全弗氏佐剂、脂多糖(LPS)、RIBI佐剂或它们的任意组合，例如Alum和CpG、MnJ和CpG。
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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN115287271A (zh) * 2022-06-30 2022-11-04 中国食品药品检定研究院 一种检测SARS-CoV-2抗体中和活性的方法
CN115737798A (zh) * 2022-09-28 2023-03-07 四川农业大学 鸭il-7作为佐剂在鸭坦布苏病毒灭活疫苗中的应用
WO2023217206A1 (zh) * 2022-05-12 2023-11-16 中国科学院微生物研究所 新冠病毒嵌合核酸疫苗及其用途

Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101039955A (zh) * 2004-06-04 2007-09-19 巴斯德研究院 与sars冠状病毒刺突蛋白相关的核酸、多肽、表达方法和免疫原性组合物
WO2015143335A1 (en) * 2014-03-20 2015-09-24 The University Of North Carolina At Chapel Hill Methods and compositions for chimeric coronavirus spike proteins
CN111088283A (zh) * 2020-03-20 2020-05-01 苏州奥特铭医药科技有限公司 mVSV病毒载体及其病毒载体疫苗、一种基于mVSV介导的新冠肺炎疫苗
CN111217919A (zh) * 2020-03-04 2020-06-02 中山大学 一种基于火球菌铁蛋白的新型冠状病毒s蛋白双区域亚单位纳米疫苗
CN111228475A (zh) * 2020-02-21 2020-06-05 赛诺（深圳）生物医药研究有限公司 用于预防新型冠状病毒的生物制品
CN112409496A (zh) * 2020-11-26 2021-02-26 焦顺昌 一种跨膜表达新型冠状病毒抗原s2的融合蛋白、重组载体、重组树突状细胞及其应用

Patent Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101039955A (zh) * 2004-06-04 2007-09-19 巴斯德研究院 与sars冠状病毒刺突蛋白相关的核酸、多肽、表达方法和免疫原性组合物
WO2015143335A1 (en) * 2014-03-20 2015-09-24 The University Of North Carolina At Chapel Hill Methods and compositions for chimeric coronavirus spike proteins
CN111228475A (zh) * 2020-02-21 2020-06-05 赛诺（深圳）生物医药研究有限公司 用于预防新型冠状病毒的生物制品
CN111217919A (zh) * 2020-03-04 2020-06-02 中山大学 一种基于火球菌铁蛋白的新型冠状病毒s蛋白双区域亚单位纳米疫苗
CN111088283A (zh) * 2020-03-20 2020-05-01 苏州奥特铭医药科技有限公司 mVSV病毒载体及其病毒载体疫苗、一种基于mVSV介导的新冠肺炎疫苗
CN112409496A (zh) * 2020-11-26 2021-02-26 焦顺昌 一种跨膜表达新型冠状病毒抗原s2的融合蛋白、重组载体、重组树突状细胞及其应用

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
DATABASE Protein 18 July 2020 (2020-07-18), ANONYMOUS: "surface glycoprotein [Severe acute respiratory syndrome coronavirus 2]", XP055882285, retrieved from Genbank Database accession no. YP_009724390 *
WATANABE YASUNORI, ALLEN JOEL D., WRAPP DANIEL, MCLELLAN JASON S., CRISPIN MAX: "Site-specific glycan analysis of the SARS-CoV-2 spike.", SCIENCE, vol. 369, no. 6501, 4 May 2020 (2020-05-04), pages 330 - 333, XP055882284, ISSN: 0036-8075, DOI: 10.1126/science.abb9983 *

Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2023217206A1 (zh) * 2022-05-12 2023-11-16 中国科学院微生物研究所 新冠病毒嵌合核酸疫苗及其用途
CN115287271A (zh) * 2022-06-30 2022-11-04 中国食品药品检定研究院 一种检测SARS-CoV-2抗体中和活性的方法
CN115287271B (zh) * 2022-06-30 2024-04-09 中国食品药品检定研究院 一种检测SARS-CoV-2抗体中和活性的方法
CN115737798A (zh) * 2022-09-28 2023-03-07 四川农业大学 鸭il-7作为佐剂在鸭坦布苏病毒灭活疫苗中的应用
CN115737798B (zh) * 2022-09-28 2024-05-24 四川农业大学 鸭il-7作为佐剂在鸭坦布苏病毒灭活疫苗中的应用

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