WO2021189571A1 - 一种高效诱导机体体液免疫应答的疫苗载体、其制备方法及用途 - Google Patents

一种高效诱导机体体液免疫应答的疫苗载体、其制备方法及用途 Download PDF

Info

Publication number
WO2021189571A1
WO2021189571A1 PCT/CN2020/085778 CN2020085778W WO2021189571A1 WO 2021189571 A1 WO2021189571 A1 WO 2021189571A1 CN 2020085778 W CN2020085778 W CN 2020085778W WO 2021189571 A1 WO2021189571 A1 WO 2021189571A1
Authority
WO
WIPO (PCT)
Prior art keywords
virus
cells
vaccine
vector
immune response
Prior art date
Application number
PCT/CN2020/085778
Other languages
English (en)
French (fr)
Inventor
徐建青
张晓燕
何欢
何香川
丁相卿
Original Assignee
上海市公共卫生临床中心
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by 上海市公共卫生临床中心 filed Critical 上海市公共卫生临床中心
Publication of WO2021189571A1 publication Critical patent/WO2021189571A1/zh

Links

Images

Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/46—Ingredients of undetermined constitution or reaction products thereof, e.g. skin, bone, milk, cotton fibre, eggshell, oxgall or plant extracts
    • 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
    • A61K39/39—Medicinal preparations containing antigens or antibodies characterised by the immunostimulating additives, e.g. chemical adjuvants
    • 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
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C12N15/86—Viral vectors
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
    • A61K2039/55505—Inorganic adjuvants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
    • A61K2039/55511—Organic adjuvants
    • A61K2039/55522—Cytokines; Lymphokines; Interferons
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
    • A61K2039/55511—Organic adjuvants
    • A61K2039/55522—Cytokines; Lymphokines; Interferons
    • A61K2039/55527—Interleukins
    • A61K2039/55533—IL-2
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
    • A61K2039/55511—Organic adjuvants
    • A61K2039/55566—Emulsions, e.g. Freund's adjuvant, MF59
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/57—Medicinal preparations containing antigens or antibodies characterised by the type of response, e.g. Th1, Th2
    • A61K2039/575—Medicinal preparations containing antigens or antibodies characterised by the type of response, e.g. Th1, Th2 humoral response
    • 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
    • C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
    • C12N2710/00011—Details
    • C12N2710/10011—Adenoviridae
    • C12N2710/10041—Use of virus, viral particle or viral elements as a vector
    • C12N2710/10043—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector
    • 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
    • C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
    • C12N2710/00011—Details
    • C12N2710/16011—Herpesviridae
    • C12N2710/16611—Simplexvirus, e.g. human herpesvirus 1, 2
    • C12N2710/16641—Use of virus, viral particle or viral elements as a vector
    • C12N2710/16643—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector
    • 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
    • C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
    • C12N2710/00011—Details
    • C12N2710/24011—Poxviridae
    • C12N2710/24041—Use of virus, viral particle or viral elements as a vector
    • C12N2710/24043—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector
    • 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
    • C12N2740/00—Reverse transcribing RNA viruses
    • C12N2740/00011—Details
    • C12N2740/10011—Retroviridae
    • C12N2740/15011—Lentivirus, not HIV, e.g. FIV, SIV
    • C12N2740/15041—Use of virus, viral particle or viral elements as a vector
    • C12N2740/15043—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector
    • 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
    • C12N2750/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssDNA viruses
    • C12N2750/00011—Details
    • C12N2750/14011—Parvoviridae
    • C12N2750/14111—Dependovirus, e.g. adenoassociated viruses
    • C12N2750/14141—Use of virus, viral particle or viral elements as a vector
    • C12N2750/14143—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • the present invention relates to the field of vaccines, in particular to a vaccine carrier capable of efficiently inducing a body fluid immune response, a preparation method and application thereof.
  • Vaccines are a very safe and effective way to fight or eliminate infectious diseases.
  • the development of a vaccine vector that can further effectively improve the level of antibody response is a very important goal in the field of vaccine research.
  • B cells are a group of lymphocytes that are crucial in humoral immune responses. Their main function in adaptive immune responses is to secrete antibodies, and they can also serve as antigen-presenting cells to present antigens and secrete cytokines. B cells can present antigens to T helper cells (Th). If activated T cells recognize the peptides presented by B cells, they will activate B by binding their own CD40 ligand to the CD40 receptor expressed by B cells. cell.
  • Th T helper cells
  • helper T lymphocyte subset 1 secretes IL-2
  • helper T lymphocyte subset 2 secretes IL-4
  • follicular helper T cells secretes IL-21
  • B cells can secrete large amounts of antibodies and act on antigen targets. Therefore, if a vaccine carrier carrying B cell activation-related molecules and target antigens can be developed, it is expected to efficiently activate B cells in the body and induce a strong humoral immune response.
  • the artificial antigen presenting cell (aAPC) system based on human tumor cells has been widely used to amplify tumor-specific cytotoxic T lymphocytes (CTL).
  • CTL cytotoxic T lymphocytes
  • the most commonly used aAPC is human red Leukemia K562 cell line. It lacks the endogenous expression of HLA-A, B and DR molecules, and is genetically engineered to express exogenous HLA-A, tumor antigens and other immune molecules. It can be used to amplify non-specific or specific antigens required for tumor immunotherapy. cell.
  • the invention makes full use of the characteristics of K562 cells, carries molecules related to B cell activation, and can maintain the survival and function of B cells in vitro; after carrying target antigens, it can be used as an effective vaccine carrier to induce the body to produce high-efficiency humoral immune responses.
  • the purpose of the present invention is to provide a vaccine vector capable of efficiently inducing the body's humoral immune response, and its preparation method and application.
  • the present invention provides a vaccine vector that efficiently induces the body's humoral immune response, characterized in that the vaccine vector is genetically engineered K562 cells.
  • the vaccine vector is K562 cells expressing molecules related to B cell activation through genetic engineering.
  • the molecules related to B cell activation include CD40L, IL-4, IL-21, and B cell activation.
  • Factor (BAFF) cholera toxin B subunit (CTB), CXC chemokine receptor type 5 (CXC chemokine receptor type 5, CXCR5) and a combination of the above different factors.
  • the vaccine vector K562 cells express CXCR5, CCR7 or CD62L through genetic engineering.
  • the vaccine carrier for efficiently inducing the body's humoral immune response is characterized in that the protein carrying the molecule related to B cell activation, wherein the protein carrying the molecule related to B cell activation includes a modified Fusion proteins formed by fusion expression of proteins, protein molecules or their partial sequences with other molecules, and protein-derived polypeptide fragments; fusion expression methods include fusion with antibody Fc sequence, fusion with different signal peptides, or fusion with cytokines.
  • the vaccine vector K562 cells are genetically engineered to express an immunogen, and the immunogen expression site includes intracellular, on cell membrane or secretory expression.
  • the source of the immunogen genetically engineered and expressed in the vaccine vector K562 cells includes viruses, bacteria, tumor antigens or human protein molecules.
  • the virus includes influenza virus, coronavirus, paramyxovirus, rubella virus, AIDS virus, hepatitis A virus, hepatitis B virus, hepatitis C virus, epidemic encephalitis virus, dengue virus, forest encephalitis Virus, West Nile virus, Zika virus, yellow fever virus, Hanta virus, Ebola virus, herpes virus, rabies virus, pox virus or Bunia virus.
  • influenza virus includes human influenza virus, avian influenza virus, and the coronavirus includes SARS, MERS, SARS-CoV-2, bat coronavirus (bat-CoV), and the paramyxovirus Including parainfluenza virus, measles virus, respiratory syncytial virus, mumps virus, Nipah virus, human metapneumovirus.
  • coronavirus includes SARS, MERS, SARS-CoV-2, bat coronavirus (bat-CoV)
  • paramyxovirus Including parainfluenza virus, measles virus, respiratory syncytial virus, mumps virus, Nipah virus, human metapneumovirus.
  • the viral immunogen is derived from viral envelope proteins, including hemagglutinin (HA), neuraminidase (NA), matrix protein (M), Envelope protein (Envelope), spike protein (Spike), membrane protein (Membrane, M), hemolysin (Haemolysin, HL), fusion protein (Fusion, F), adhesion protein (G).
  • viral envelope proteins including hemagglutinin (HA), neuraminidase (NA), matrix protein (M), Envelope protein (Envelope), spike protein (Spike), membrane protein (Membrane, M), hemolysin (Haemolysin, HL), fusion protein (Fusion, F), adhesion protein (G).
  • the tumor antigens include CD19, BCMA, CD20, CD22, CD30, CD33, CD38, CD47, CD52, CD70, CD117, CD123, CD133, CD138, CD147, CD171, RANKL, NKG2DL, HER2, MUC1, MUC16, CEA, EpCAM, IL-13R ⁇ 2, EGFR, EGFRvIII, GD2, DR5, EphA2, FR ⁇ , PSCA, PSMA, TARP, PIGF, cMet, VEGFR2, BCMA, CTLA-4, PD-L1, AFP, GPC3, AXL, ROR1, ROR2, FAP, Mesothelin, DLL3, CLD18, PDGF-R ⁇ , SLAMF7 and/or CCR4.
  • the human-derived protein molecules include protein molecules on human cell membranes.
  • the vaccine carrier carries the antigen
  • it is combined with different adjuvants to form a vaccine.
  • the adjuvants used include aluminum salt adjuvant, CpG adjuvant, manganese adjuvant, MF59 adjuvant, QS21 adjuvant, TLR ligand adjuvant, NOD pathway adjuvant, interferon adjuvant, cGAS/STING pathway adjuvant, cytokine adjuvant, PD-1 blocking antibody, CTLA-4 blocking antibody, TIGIT blocking antibody, ⁇ Galcer and its Derivative adjuvants
  • the cytokine adjuvants include IL-12, GM-CSF, IL-18, and IL-21.
  • the present invention provides a method for preparing a vaccine vector that efficiently induces a body fluid immune response, which is characterized in that it comprises the following steps:
  • CD40L, IL-4, IL-21, BAFF, cholera toxin B subunit, CXCR5, CCR7 or CD62L protein gene is expressed in a lentiviral vector, a plasmid vector, a poxvirus vector, an adenovirus vector, an adeno-associated virus vector , Herpes simplex virus vector or CMV vector.
  • CD40L, IL-4, IL-21, B cell activating factor, cholera toxin B subunit, CXCR5, CCR7 or CD62L protein genes are expressed in a lentiviral vector.
  • CD40L, IL-4, IL-21, BAFF, cholera toxin B subunit, CXCR5, CCR7 or CD62L protein gene can be expressed on K562 cells alone, or in combination of two or three, Four-four combinations, five-five combinations or six-six combinations, or all of them are expressed on K562 cells.
  • the present invention provides a method for using a vaccine vector that efficiently induces the body's humoral immune response, which is characterized in that live cell vector vaccines, inactivated cell vector vaccines, and exosomes secreted by cell vector vaccines are used as Vaccine for immunization; or load cell membrane expressing immunogen and B cell activation molecules on other nanoparticle materials to form nanoparticle vaccine for immunization.
  • X-rays, heat inactivation, fixatives, and lysing agents are used to inactivate the cell vaccine carrier.
  • the vaccine administration methods prepared by using the K562 vector include intravenous injection, subcutaneous injection, intradermal injection, intramuscular injection, mucosal administration, nose drops, eye drops, or aerosol immunization.
  • the K562 cell carrier is used for the survival and proliferation of B cells in vitro
  • the K562 cell carrier vaccine is used to carry antigens to immunize animals to obtain antibodies and use the antibodies for scientific research, clinical medicine, CAR-T recognition area, and BITE Recognition zone, and as a vaccine carrier to induce humoral immune response, it can be administered alone or in combination with other therapeutic techniques.
  • HIV-1 membrane protein subtypes used in vaccine vectors include HIV-1A, B, C, D, F, G, H, J, K subtypes and recombinant types formed between different types, including CRF01-AE , CRF07-BC, CRF08-BC, AG recombinant type.
  • sources of HIV-1 membrane proteins used in vaccine vectors include AE2F, RL42, and CN54.
  • the B cell activation-related molecular protein comprises a human amino acid sequence selected from the following:
  • amino acid sequence shown in SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5 or SEQ ID NO: 7 has at least 80%, at least 85%, at least 90%, at least 91%, at least A sequence of 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity.
  • the coding nucleotide sequence of the B cell activation-related molecular protein is shown in SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6 or SEQ ID NO: 8.
  • the B cell activation-related molecular protein may be the amino acid sequence shown in SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, or SEQ ID NO: 7 through one or more amino acid residue substitutions , Deletion or addition does not affect its molecular function.
  • the coding nucleotide sequence of the B cell activation-related molecular protein may be the nucleotide sequence shown in SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6 or SEQ ID NO: 8 through one or The substitution, deletion or addition of multiple nucleotides does not affect the function of the encoded protein.
  • the replicable and non-replicable forms of the cell vaccine vector and the secreted exosomes are used as vaccines for immunization.
  • vaccination methods include, but are not limited to, intravenous injection, intramuscular injection, intradermal injection, subcutaneous injection of the genital tract, rectum, and the like.
  • the HIV-1 membrane protein comprises an amino acid sequence selected from:
  • amino acid sequence shown in SEQ ID NO: 9 has at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96% , At least 97%, at least 98%, at least 99%, or 100% sequence identity.
  • the coding nucleotide sequence of the HIV-1 membrane protein is shown in SEQ ID NO: 10.
  • HIV-1 membrane protein may be formed by substituting, deleting or adding one or more amino acid residues to the amino acid sequence shown in SEQ ID NO: 9 without affecting its molecular function.
  • the coding nucleotide sequence of the HIV-1 membrane protein may be formed by replacing, deleting or adding one or more nucleotides to the nucleotide sequence shown in SEQ ID NO: 10 without affecting its coding protein Function.
  • the hemagglutinin (HA) form of influenza virus used in the vaccine vector includes but is not limited to simple membrane protein, modified membrane protein and fusion protein.
  • the HA protein comprises an amino acid sequence selected from:
  • amino acid sequence shown in SEQ ID NO: 11 has at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96% , At least 97%, at least 98%, at least 99%, or 100% sequence identity.
  • the coding nucleotide sequence of the HA protein is shown in SEQ ID NO: 12.
  • the HA protein may be formed by substituting, deleting or adding one or more amino acid residues to the amino acid sequence shown in SEQ ID NO: 11 without affecting its molecular function.
  • the coding nucleotide sequence of the HA protein may be formed by substituting, deleting or adding one or more nucleotides to the nucleotide sequence shown in SEQ ID NO: 12 without affecting its coding protein function.
  • the beneficial effect of the present invention is that the vaccine carrier can effectively activate B cells, enhance the survival of B cells, and promote the secretion of antibodies by B cells, and can also effectively stimulate the maturation and differentiation of B cells in vivo to induce effective antibody responses, and make them It has application prospects in preventing and reducing virus infections.
  • Figure 1 shows the construction of the lentiviral expression vector plasmid pHAGE-CTB-T2A-CD40L-puro and the verification of the expression of the piggyback protein.
  • the lentiviral expression vector plasmid ppHAGE-CTB-T2A-CD40L-puro expression map ( Figure 1a) including the lentiviral expression vector plasmid integrating human CD40L and CTB genes ( Figure 1a); the CD40L expression verification ( Figure 1a); 1b) Verification with CTB expression ( Figure 1c).
  • Figure 2 shows the construction of the lentiviral expression vector plasmid pHAGE-IL-4-T2A-IL-21-ZsGreen1 and the verification of the expression of the loaded cytokine.
  • Figure 3 shows the construction of the lentiviral expression vector plasmid pHAGE-BAFF-puro and the verification of the expression of the activation molecule.
  • Figure 4 shows the construction of the lentiviral expression vector plasmid pHAGE-IL-4-T2A-IL-21-IRES-CXCR5-ZsGreen1 and the expression verification of the activation molecule.
  • Including the lentiviral expression vector plasmid pHAGE-IL-4-T2A-IL-21-IRES-CXCR5-ZsGreen1 that integrates the human CXCR5 gene Figure 4a
  • the lentivirus packaged by the lentiviral expression vector after infection of K562 cells Verification of CXCR5 protein expression
  • Figure 5 shows the construction of the lentiviral expression vector plasmid pHAGE-AE2FSOSIP.664-ZsGreen1 and the verification of the expression of the antigen protein. Including the expression map of the lentiviral expression vector plasmid pHAGE-AE2FSOSIP.664-ZsGreen1 that integrates the AE2F SOSIP.664 gene ( Figure 5a); and the verification of the expression of AE2F SOSIP.664 after infection of K562 cells with the lentivirus packaged by the lentiviral expression vector ( Figure 5b).
  • Figure 6 shows the construction of the lentiviral expression vector plasmid (pHAGE-HA1-FLAG-CD8TM-ZsGreen) and the expression verification of the loaded antigen protein.
  • Figure 7 shows the effect of B cell activation-related factors on B cells in vitro.
  • Figure 7a is a flow cytometric analysis of K562 cells carrying the four B cell activation-related molecules of CD40L, IL-4, IL-21 and CTB and carrying BAFF, which can significantly improve the survival of B cells in vitro.
  • Figure 7b is an enzyme-linked immunosorbent assay (ELISA) analysis showing that K562 cells carrying the four B cell activation-related molecules of CD40L, IL-4, IL-21 and CTB and carrying BAFF can promote B cells in vitro Secret antibodies.
  • ELISA enzyme-linked immunosorbent assay
  • Figure 7c shows the flow cytometry analysis of K562 cells carrying the four B cell activation-related molecules of CD40L, IL-4, IL-21 and BAFF and carrying CXCR5, which can significantly improve the survival of B cells in vitro.
  • Figure 7d is an enzyme-linked immunosorbent assay (ELISA) analysis showing that K562 cells carrying CD40L, IL-4, IL-21 and BAFF four B cell activation-related molecules and carrying CXCR5 can promote B cells in vitro Secret antibodies.
  • ELISA enzyme-linked immunosorbent assay
  • Figure 8 shows the antibody induction effect of four-factor K562 cells with AE2F SOSIP.664 antigen in rhesus monkeys.
  • K562 cells loaded with four factors and carrying HIV-1 antigen AE2F SOSIP.664 can significantly increase the specific antibodies against HIV-1 after immunizing Chinese rhesus monkeys.
  • Figure 9 shows the antibody induction effect of four-factor K562 cells with HA antigen in rhesus monkeys. As shown in the figure, after immunizing Chinese rhesus monkeys with K562 cells loaded with four factors and carrying influenza virus antigen HA, the specific antibodies against HA protein can be significantly increased.
  • Example 1 Construction and expression verification of human CD40L and CTB gene lentiviral expression vector pHAGE-CTB-T2A-CD40L-puro
  • the synthesized fragment and vector plasmid by double enzyme digestion digest with Not I (Thermo Scientific Company, Catalog No. FD0596) and BamH I digest (Thermo Scientific Company, FD0054), and then cut the gel after gel electrophoresis, using Sanprep column format
  • the DNA gel recovery kit (Promega, catalog number A9282) recovers the digested fragments.
  • the gene recovery product was ligated with the digested linearized vector using T4 DNA ligase (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, the mutation site was corrected, and all sequences were verified to be correct, the human CD40L and CTB gene lentiviral expression plasmid (pHAGE-CTB-T2A-CD40L-puro) was successfully cloned, and the plasmid was constructed The spectrum is shown in Figure 1a.
  • Example 2 Construction and expression verification of human IL-4 and IL-21 gene lentiviral expression vector pHAGE-IL-4-T2A-IL-21-ZsGreen1
  • the DNA sequences of human-derived IL-4 and IL-21 are artificially synthesized, and T2A fragments are added between the two genes for dual expression.
  • the synthesized sequence is shown in SEQ ID NO: 4, with a NotI restriction site at the 5'end and a BamHI restriction site at the 3'end.
  • the synthesized fragment and vector plasmid were digested with Not I restriction enzyme (Thermo Scientific company, catalog number FD0596) and BamH I restriction enzyme digestion (Thermo Scientific company, FD0054), and the gel was cut and recovered, and the Sanprep column DNA gel recovery kit was used (Promega, Catalog No. A9282) The recovered fragments were digested with the vector.
  • the gene recovery product and the digested linearized vector were ligated with T4 DNA ligase (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 sequences were correct, the lentiviral expression plasmid of human IL-4 and IL-21 genes (pHAGE-IL-4-T2A- IL-21-ZsGreen1), the plasmid construction map is shown in Figure 2a.
  • Example 3 Construction and expression verification of human BAFF gene lentiviral expression vector pHAGE-BAFF-puro
  • the synthesized sequence is shown in SEQ ID NO: 6, with a NotI restriction site at the 5'end and a BamHI restriction site at the 3'end.
  • the synthesized fragment and vector plasmid were digested with Not I restriction enzyme (Thermo Scientific company, catalog number FD0596) and BamH I restriction enzyme digestion (Thermo Scientific company, FD0054), and the gel was cut and recovered, and the Sanprep column DNA gel recovery kit was used (Promega, Catalog No. A9282) The recovered fragments were digested with the vector.
  • the gene recovery product and the digested linearized vector were ligated with T4 DNA ligase (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, the mutation site was corrected, and all sequences were verified to be correct, the human BAFF gene lentiviral expression plasmid (pHAGE-BAFF-puro) was successfully cloned.
  • the plasmid construction map is shown in Figure 3a. .
  • K562 cells that survived the infection were taken, stained with anti-BAFF-APC antibody (BioLegend, model 366508), and finally analyzed by flow cytometry.
  • the positive cells were continuously enriched by flow cytometry, and finally K562 cells with high expression of BAFF were obtained.
  • the results show that after continuous enrichment, the target gene human BAFF can be highly expressed in K562 cells ( Figure 3b).
  • Example 4 Construction and expression verification of human CXCR5 gene lentiviral expression vector pHAGE-IL-4-T2A-IL-21-IRES-CXCR5
  • the synthesized sequence is shown in SEQ ID NO: 8, with a NotI restriction site at the 5'end and a SalI restriction site at the 3'end.
  • the synthesized fragment and vector plasmid were digested with Not I restriction enzyme (Thermo Scientific company, catalog number FD0596) and SalI restriction enzyme digestion (Thermo Scientific company, FD0644), and the gel was cut and recovered, and the Sanprep column DNA gel recovery kit was used (Promega, Catalog No. A9282) The recovered fragments were digested with the vector.
  • the gene recovery product and the digested linearized vector were ligated with T4 DNA ligase (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 plasmid of human CXCR5 gene (pHAGE-IL-4-T2A-IL-21-IRES-CXCR5) was successfully cloned. ), the plasmid construction map is shown in Figure 4a.
  • Example 5 Construction and expression verification of AE2F SOSIP.664 gene lentiviral expression vector pHAGE-AE2F SOSIP.664-ZsGreen1
  • the synthesized sequence is shown in SEQ ID NO: 10, with a NotI restriction site at the 5'end and a BamHI restriction site at the 3'end.
  • the synthesized fragment and vector plasmid were digested with Not I restriction enzyme (Thermo Scientific company, catalog number FD0596) and BamH I restriction enzyme digestion (Thermo Scientific company, FD0054), and the gel was cut and recovered, and the Sanprep column DNA gel recovery kit was used (Promega, Catalog No. A9282) The recovered fragments were digested with the vector.
  • the gene recovery product and the digested linearized vector were ligated with T4 DNA ligase (Thermo Scientific Company, Catalog No. 2011A).
  • the ligation product was transformed into E. coli Stable and grown overnight on a culture plate containing ampicillin.
  • a single colony was randomly selected for sequencing, the mutation site was corrected, and all sequences were verified to be correct, the human BAFF gene lentiviral expression plasmid (pHAGE-AE2F SOSIP.664-ZsGreen1) was successfully cloned.
  • the plasmid construction map is shown in the figure. Shown in 5a.
  • pHAGE-AE2F SOSIP.664- with AE2F SOSIP.664 ZsGreen1, lentiviral packaging plasmids psPAX and VSVG three plasmids were transfected into 293T cells at a mass ratio of 1:2:1.
  • Example 6 Construction and expression verification of the HA gene lentiviral expression vector pHAGE-HA1-FLAG-CD8TM-ZsGreen1
  • the synthesized sequence is shown in SEQ ID NO: 12, with a NotI restriction site at the 5'end and a BamHI restriction site at the 3'end.
  • the synthesized fragment and vector plasmid were digested with Not I restriction enzyme (Thermo Scientific company, catalog number FD0596) and BamH I restriction enzyme digestion (Thermo Scientific company, FD0054), and the gel was cut and recovered, and the Sanprep column DNA gel recovery kit was used (Promega, Catalog No. A9282) The recovered fragments were digested with the vector.
  • the gene recovery product and the digested linearized vector were ligated with T4 DNA ligase (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, the mutation site was corrected, and all sequences were verified to be correct, the human BAFF gene lentiviral expression plasmid (pHAGE-HA1-FLAG-CD8TM-ZsGreen1) was successfully cloned.
  • the plasmid construction map is as follows Shown in Figure 6a.
  • Example 7 The effect of factors related to B cell activation on B cells in vitro
  • the magnetic bead sorting method is used to sort B cells from human PBMC, and the human B cells (1E4) after magnetic bead sorting are placed in a 96-well plate with the same number of the following three kinds of cells: (1) K562 Control cells; (2) K562 cells loaded with CD40L, IL-4, IL-21 and CTB four B cell activating factors; (3) loaded with CD40L, IL-4, IL-21 and CTB four B cell activation Factor and K562 cells with BAFF. After a total of 14 days of incubation, flow cytometry was used to detect the survival and proportion of B cells in the wells; the human IgG detection kit (Thermo Scientific, article number 88-50550-22) was used to detect the IgG content.
  • K562 cells loaded with four factors including CD40L, IL-4, IL-21 and CTB four B cell activating factors
  • K562 cells with BAFF genes on the basis of four factors Figure 7a
  • IgG total antibody secretion level
  • the magnetic bead sorting method is used to sort B cells from human PBMC, and the human B cells (1E4) after magnetic bead sorting are placed in a 96-well plate with the same number of the following three kinds of cells: (1) K562 Control cells; (2) K562 cells loaded with CD40L, IL-4, IL-21 and BAFF four B cell activating factors; (3) loaded with CD40L, IL-4, IL-21 and BAFF four B cell activation Factor and K562 cells with CXCR5.
  • human B cells were labeled with CD19 molecules (Biolegend, catalog number HIB19), and flow cytometry was used to detect the number of B cells in the wells; and a human IgG detection kit (Thermo Scientific Company, catalog number 88-50550- 22) Detect the content of IgG.
  • K562 cells loaded with four factors can enhance B cell activity in vitro
  • the number of survivors can be increased by more than 3 times for the four factors, and nearly 4 times for the five factors.
  • the total antibody secretion level (IgG) of B cells can be increased by about 5 times in the four-factor group, and can be further increased to 11 times after carrying CXCR5 (Figure 7d).
  • Example 8 Evaluation of in vivo antibody induction of four-factor K562 cells with AE2F SOSIP.664 antigen
  • K562 cells loaded with four factors and carrying the HIV-1 antigen AE2F SOSIP.664 immunized Chinese rhesus monkeys which can significantly increase the specific antibodies against HIV-1, and the average titer can reach 1: 25600, indicating that the carrier of the present invention has strong immunogenicity after carrying HIV-1 antigen.
  • Example 9 Evaluation of in vivo antibody induction effect of four-factor K562 cells with HA antigen
  • the K562 cells loaded with four factors and influenza virus antigen HA can significantly increase the specific antibody against HA protein after immunization of Chinese rhesus monkeys, and the average titer can reach 1:25600; the second time After the booster immunization, the antibody titer can continue to increase to 1:51200, and the antibody concentration of the higher titer can be maintained until 21 weeks.
  • the carrier of the present invention not only has strong immunogenicity after carrying influenza antigen, but also can continuously induce a higher level of antibody response in the body.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Genetics & Genomics (AREA)
  • General Health & Medical Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Biotechnology (AREA)
  • Organic Chemistry (AREA)
  • Biomedical Technology (AREA)
  • General Engineering & Computer Science (AREA)
  • Veterinary Medicine (AREA)
  • Public Health (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Microbiology (AREA)
  • Wood Science & Technology (AREA)
  • Zoology (AREA)
  • Animal Behavior & Ethology (AREA)
  • Epidemiology (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Medicinal Chemistry (AREA)
  • Molecular Biology (AREA)
  • Botany (AREA)
  • Virology (AREA)
  • Biochemistry (AREA)
  • Physics & Mathematics (AREA)
  • Plant Pathology (AREA)
  • Biophysics (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Mycology (AREA)
  • Immunology (AREA)
  • General Chemical & Material Sciences (AREA)
  • Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)

Abstract

一种能够高效诱导机体体液免疫应答的疫苗载体、其制备方法及用途。所述疫苗载体是经由基因工程化表达B细胞活化相关的分子的K562细胞，所述B细胞活化相关的分子包括CD40L、IL-4、IL-21、BAFF、霍乱毒素B亚单位或CXCR5以及上述不同因子的组合。此外，所述疫苗载体能够有效活化B细胞，提升B细胞的生存，并促使B细胞分泌抗体。可很好的搭载抗原，在体内也可有效的刺激B细胞成熟分化从而诱导有效的抗体应答，使其在预防和降低病毒感染中有广泛的应用前景。

Description

一种高效诱导机体体液免疫应答的疫苗载体、其制备方法及用途 技术领域
本发明涉及疫苗领域，特别涉及一种能够高效诱导机体体液免疫应答的疫苗载体、其制备方法及用途。
背景技术
疫苗是一种非常安全有效的对抗或者清除传染性疾病的方式。开发出一种能够进一步有效的提高抗体应答水平的疫苗载体，是疫苗研究领域中非常重要的目标。
B细胞是体液免疫应答中至关重要的一群淋巴细胞，其在适应性免疫应答中的主要功能是分泌抗体，还能作为抗原递呈细胞发挥递呈抗原和分泌细胞因子的功能。B细胞可将抗原提呈给T辅助细胞(Th)，若活化的T细胞识别B细胞提呈的肽段，则通过其自身的CD40配体与B细胞表达的CD40受体相结合进而激活B细胞。与此同时，辅助性T淋巴细胞亚群1(Th1)分泌IL-2,辅助性T淋巴细胞亚群2(Th2)分泌IL-4，滤泡辅助性T细胞(Tfh)分泌IL-21,直接刺激B细胞，三者联合诱导B细胞增殖分化并促进抗体类型转换，最终分化成浆细胞和记忆B细胞。浆细胞可分泌大量的抗体并作用于抗原靶标。因此，如能开发一种搭载B细胞活化相关的分子与目标抗原的疫苗载体，有望高效活化体内B细胞，诱导出强劲的体液免疫应答。
基于人肿瘤细胞的人工抗原提呈细胞(artificial antigen presenting cell,aAPC)系统，已广泛用于扩增肿瘤特异性细胞毒性T淋巴细胞(cytotoxic T lymphocyte,CTL)，其中最常用的aAPC是人红白血病K562细胞株。它缺乏内源性表达HLA-A、B和DR分子，经基因工程表达外源性HLA-A、肿瘤抗原与其他免疫分子，可用于扩增肿瘤免疫治疗所需的抗原非特异性或特异性T细胞。
本发明充分利用K562细胞的特性，搭载B细胞活化相关的分子，可在体外维持B细胞的生存与功能；搭载目标抗原后可作为一种有效的疫苗载体，诱导机体产生高效体液免疫应答。
发明内容
本发明的目的是提供一种能够高效诱导机体体液免疫应答的疫苗载体、其制备方法及用途。
一方面，本发明提供一种高效诱导机体体液免疫应答的疫苗载体，其特征在于，所述疫苗载体是经由基因工程化的K562细胞。
在本发明的实施方式中，所述疫苗载体是经由基因工程化表达B细胞活化相关的分子的K562细胞，所述B细胞活化相关的分子包括CD40L、IL-4、IL-21、B细胞活化因子(BAFF)、霍乱毒素B亚单位(cholera toxin B subunit,CTB),C-X-C趋化因子受体5(C-X-C chemokine receptor type 5,CXCR5)以及上述不同因子的组合。
在本发明的实施方式中，所述疫苗载体K562细胞经由基因工程化表达CXCR5、CCR7或CD62L。
在本发明的实施方式中，所述高效诱导机体体液免疫应答的疫苗载体，其特征在于，搭载所述B细胞活化相关的分子的蛋白，其中搭载B细胞活化相关的分子的蛋白包括经过修饰的蛋白、蛋白分子或其部分序列与其他分子融合表达形成的融合蛋白、以及蛋白来源的多肽片段；融合表达的方式包括与抗体的Fc序列融合、与不同信号肽的融合或与细胞因子融合。
在本发明的实施方式中，所述疫苗载体K562细胞基因工程化表达免疫原，免疫原表达部位包括细胞内、细胞膜上或分泌性表达。
在本发明的实施方式中，疫苗载体K562细胞基因工程化表达的免疫原来源包括病毒、细菌、肿瘤抗原或人源蛋白分子。
在本发明的实施方式中，所述病毒包括流感病毒、冠状病毒、副粘病毒、风疹病毒、艾滋病病毒、甲肝病毒、乙肝病毒、丙肝病毒、流行性脑炎病毒、登革病毒、森林脑炎病毒、西尼罗病毒、寨卡病毒、黄热病毒、汉坦病毒、埃博拉病毒、疱疹病毒、狂犬病毒、痘病毒或布尼亚病毒。
在本发明的实施方式中，所述流感病毒包括人流感病毒、禽流感病毒，所述冠状病毒包括SARS、MERS、SARS-CoV-2、蝙蝠冠状病毒(bat-CoV),所述副粘病毒包括副流感病毒、麻疹病毒、呼吸道合胞病毒、腮腺炎病毒、尼帕病毒、人偏肺病毒。
在本发明的实施方式中，所述病毒免疫原来自于病毒囊膜蛋白，包括血凝素蛋白(Hemagglutinin,HA)、神经氨酸酶(Neuraminidase，NA)、基质蛋白(Matrix protein,M)、包膜蛋白(Envelope)、刺突蛋白(Spike)、膜蛋白(Membrane,M)、溶 血素(Haemolysin,HL)、融合蛋白(Fusion,F)、黏附蛋白(G)。
在本发明的实施方式中，所述肿瘤抗原，包括CD19、BCMA、CD20、CD22、CD30、CD33、CD38、CD47、CD52、CD70、CD117、CD123、CD133、CD138、CD147、CD171、RANKL、NKG2DL、HER2、MUC1、MUC16、CEA、EpCAM、IL-13Rα2、EGFR、EGFRvIII、GD2、DR5、EphA2、FRα、PSCA、PSMA、TARP、PIGF、cMet、VEGFR2、BCMA、CTLA-4、PD-L1、AFP、GPC3、AXL、ROR1、ROR2、FAP、Mesothelin、DLL3、CLD18、PDGF-Rα、SLAMF7和/或CCR4。
在本发明的实施方式中，所述人源蛋白分子，包括人的细胞膜上蛋白分子。
在本发的实施方式中，所述疫苗载体搭载抗原后，与不同的佐剂组合形成疫苗，所用佐剂包括铝盐佐剂、CpG佐剂、锰佐剂、MF59佐剂、QS21佐剂、TLR配体佐剂、NOD通路佐剂、干扰素佐剂、cGAS/STING通路佐剂、细胞因子佐剂、PD-1阻断抗体、CTLA-4阻断抗体、TIGIT阻断抗体、αGalcer及其衍生物佐剂，所述细胞因子佐剂包括IL-12、GM-CSF、IL-18、IL-21。
另一方面，本发明提供了一种高效诱导机体体液免疫应答的疫苗载体的制备方法，其特征在于，包括如下步骤：
1)分别构建含有CD40L、IL-4、IL-21、BAFF蛋白分子、霍乱毒素B亚单位、CXCR5、CCR7或CD62L的载体；
2)将含有CD40L、IL-4、IL-21、BAFF蛋白分子、霍乱毒素B亚单位、CXCR5、CCR7或CD62L的载体感染、转染或者电转处理K562细胞，进行优选单克隆整合有CD40L、IL-4、IL-21、BAFF、霍乱毒素B亚单位、CXCR5、CCR7或CD62L的细胞系，即得。
进一步地，将CD40L、IL-4、IL-21、BAFF、霍乱毒素B亚单位、CXCR5、CCR7或CD62L蛋白基因表达于慢病毒载体、质粒载体、痘病毒载体、腺病毒载体、腺相关病毒载体、单纯疱疹病毒载体或CMV载体。
进一步地，将CD40L、IL-4、IL-21、B细胞活化因子、霍乱毒素B亚单位、CXCR5、CCR7或CD62L蛋白基因表达于慢病毒载体。
进一步地，CD40L、IL-4、IL-21、BAFF、霍乱毒素B亚单位、CXCR5，CCR7或CD62L蛋白基因，可以是单独表达在K562细胞上，也可以是两两组合，或者三三组 合，四四组合，五五组合亦或是六六组合，也可以是全部都表达在K562细胞上。
再一方面，本发明提供了一种高效诱导机体体液免疫应答的疫苗载体的使用方法，其特征在于，用活的细胞载体疫苗、灭活的细胞载体疫苗以及细胞载体疫苗分泌的外泌体作为疫苗进行免疫接种；或将表达免疫原以及B细胞激活分子的细胞膜装载到其他纳米颗粒材料上形成纳米颗粒疫苗进行免疫接种。
进一步地，采用包括X-射线、热灭活、固定剂、裂解剂对细胞疫苗载体进行灭活。
进一步地，利用该K562载体所制备的疫苗给药方式，包括静脉注射、皮下注射、皮内注射、肌肉注射、黏膜给药、滴鼻、点眼、或气雾免疫接种。
进一步地，利用所述K562细胞载体进行体外B细胞的生存和增殖，利用所述K562细胞载体疫苗搭载抗原免疫动物获得抗体并将抗体用于科研、临床药物以及CAR-T的识别区、BITE的识别区，以及作为疫苗载体诱导体液免疫应答时可单独给药或与其他治疗技术联合应用。
进一步地，疫苗载体使用的HIV-1膜蛋白亚型包括HIV-1A、B、C、D、F、G、H、J、K亚型以及不同型别间形成的重组型，包括CRF01-AE、CRF07-BC、CRF08-BC、AG重组型。
进一步地，疫苗载体使用的HIV-1膜蛋白来源包括AE2F、RL42、CN54。
在本发明的实施方式中，优选地，所述B细胞活化相关分子蛋白包含选自以下的人源氨基酸序列：
(i)SEQ ID NO:1、SEQ ID NO:3、SEQ ID NO:5或SEQ ID NO:7所示的氨基酸序列；
(ii)与SEQ ID NO:1、SEQ ID NO:3、SEQ ID NO:5或SEQ ID NO:7所示的氨基酸序列相比具有一个或多个氨基酸的置换、缺失或添加(例如1个，2个，3个，4个或5个氨基酸的置换、缺失或添加)的序列；或
(iii)与SEQ ID NO:1、SEQ ID NO:3、SEQ ID NO:5或SEQ ID NO:7所示的氨基酸序列具有至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、至少99％或100％的序列同一性的序列。
优选地，所述B细胞活化相关分子蛋白的编码核苷酸序列如SEQ ID NO:2、SEQ ID NO:4、SEQ ID NO:6或SEQ ID NO:8所示。
另外，所述B细胞活化相关分子蛋白可以是将SEQ ID NO:1、SEQ ID NO:3、SEQ ID NO:5或SEQ ID NO:7所示氨基酸序列经过一个或多个氨基酸残基的取代，缺失或添加而形成的不影响其分子功能。
所述B细胞活化相关分子蛋白的编码核苷酸序列可以是将SEQ ID NO:2、SEQ ID NO:4、SEQ ID NO:6或SEQ ID NO:8所示的核苷酸序列经过一个或多个核苷酸的取代、缺失或添加而形成的不影响其编码蛋白质功能。
根据本发明所述的用途，其中包括该细胞疫苗载体的可复制与不可复制形式、及其分泌的外泌体作为疫苗进行免疫接种。
在本发明的一个具体实施方案中，在上述疫苗组合接种过程中，疫苗接种方式包括但不限于静脉注射、肌肉接种、皮内接种、皮下接种生殖道、直肠等。
在本发明的实施方式中，优选地，所述HIV-1膜蛋白包含选自以下的氨基酸序列：
(i)SEQ ID NO:9所示的氨基酸序列；
(ii)与SEQ ID NO:9所示的氨基酸序列相比具有一个或几个氨基酸的置换、缺失或添加(例如1个，2个，3个，4个或5个氨基酸的置换、缺失或添加)的序列；或
(iii)与SEQ ID NO:9所示的氨基酸序列具有至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、至少99％、或100％的序列同一性的序列。
优选地，所述HIV-1膜蛋白的编码核苷酸序列如SEQ ID NO:10所示。
另外，所述HIV-1膜蛋白可以是将SEQ ID NO:9所示氨基酸序列经过一个或多个氨基酸残基的取代、缺失或添加而形成的不影响其分子功能。
所述HIV-1膜蛋白的编码核苷酸序列可以是将SEQ ID NO:10所示的核苷酸序列经过一个或多个核苷酸的取代、缺失或添加而形成的不影响其编码蛋白质功能。
在本发明的一个具体实施方案中，疫苗载体使用的流感病毒的血凝素(HA)形式包括但不限于单纯膜蛋白、修饰后的膜蛋白与融合蛋白。
优选地，所述HA蛋白包含选自以下的氨基酸序列：
(i)SEQ ID NO:11所示的氨基酸序列；
(ii)与SEQ ID NO:11所示的氨基酸序列相比具有一个或几个氨基酸的置换、缺失或添加(例如1个，2个，3个，4个或5个氨基酸的置换、缺失或添加)的序列；或
(iii)与SEQ ID NO:11所示的氨基酸序列具有至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、至少99％、或100％的序列同一性的序列。
优选地，所述HA蛋白的编码核苷酸序列如SEQ ID NO:12所示。
另外，所述HA蛋白可以是将SEQ ID NO:11所示氨基酸序列经过一个或多个氨基酸残基的取代、缺失或添加而形成的不影响其分子功能。
所述HA蛋白的编码核苷酸序列可以是将SEQ ID NO:12所示的核苷酸序列经过一个或多个核苷酸的取代、缺失或添加而形成的不影响其编码蛋白质功能。
本发明的有益效果在于，所述疫苗载体能够有效活化B细胞，提升B细胞的生存，并促使B细胞分泌抗体，在体内也可有效的刺激B细胞成熟分化从而诱导有效的抗体应答，使其在预防和降低病毒感染中有应用前景。
附图说明
图1显示了慢病毒表达载体质粒pHAGE-CTB-T2A-CD40L-puro的构建与搭载蛋白的表达验证。包括整合人CD40L与CTB基因的慢病毒表达载体质粒ppHAGE-CTB-T2A-CD40L-puro表达图谱(图1a)；所述慢病毒表达载体包装的慢病毒感染K562细胞后，CD40L表达的验证(图1b)与CTB表达的验证(图1c)。
图2显示了慢病毒表达载体质粒pHAGE-IL-4-T2A-IL-21-ZsGreen1的构建与搭载细胞因子的表达验证。包括整合人IL-4和IL-21基因的慢病毒表达载体质粒pHAGE-IL-4-T2A-IL-21-ZsGreen1表达图谱(图2a)；所述慢病毒表达载体包装的慢病毒感染K562细胞后，IL4的表达验证(图2b)与IL-21表达的验证(图2c)。
图3显示了慢病毒表达载体质粒pHAGE-BAFF-puro的构建与活化分子的表达验证。包括整合人BAFF基因的慢病毒表达载体质粒pHAGE-BAFF-puro表达图谱(图 3a)；及所述慢病毒表达载体包装的慢病毒感染K562细胞后，BAFF蛋白表达的验证(图3b)。
图4显示了慢病毒表达载体质粒pHAGE-IL-4-T2A-IL-21-IRES-CXCR5-ZsGreen1的构建与活化分子的表达验证。包括整合人CXCR5基因的慢病毒表达载体质粒pHAGE-IL-4-T2A-IL-21-IRES-CXCR5-ZsGreen1表达图谱(图4a)；及所述慢病毒表达载体包装的慢病毒感染K562细胞后，CXCR5蛋白表达的验证(图4b)。
图5显示了慢病毒表达载体质粒pHAGE-AE2FSOSIP.664-ZsGreen1的构建与抗原蛋白表达验证。包括整合AE2F SOSIP.664基因的慢病毒表达载体质粒pHAGE-AE2FSOSIP.664-ZsGreen1表达图谱(图5a)；及所述慢病毒表达载体包装的慢病毒感染K562细胞后，AE2F SOSIP.664表达的验证(图5b)。
图6显示了慢病毒表达载体质粒(pHAGE-HA1-FLAG-CD8TM-ZsGreen)的构建与搭载抗原蛋白的表达验证。包括整合有HA的基因的慢病毒表达载体质粒pHAGE-HA1-FLAG-CD8TM-ZsGreen表达图谱(图6a)；及所述慢病毒表达载体包装的慢病毒感染K562细胞后，HA蛋白表达的验证(图6b)。
图7显示了含有B细胞活化相关因子体外对B细胞的作用。图7a为流式细胞技术分析转载有CD40L、IL-4、IL-21与CTB四种B细胞活化相关的分子并带有BAFF的K562细胞，在体外能明显提升B细胞的存活。图7b为酶联免疫吸附测定法(ELISA)分析显示，转载有CD40L、IL-4、IL-21与CTB四种B细胞活化相关的分子并带有BAFF的K562细胞，在体外能促进B细胞分泌抗体。图7c为流式细胞技术分析转载有CD40L、IL-4、IL-21与BAFF四种B细胞活化相关的分子并带有CXCR5的K562细胞，在体外能明显提升B细胞的存活。图7d为酶联免疫吸附测定法(ELISA)分析显示，转载有CD40L、IL-4、IL-21与BAFF四种B细胞活化相关的分子并带有CXCR5的K562细胞，在体外能促进B细胞分泌抗体。
图8所示为带有AE2F SOSIP.664抗原的四因子K562细胞在恒河猴体内抗体的诱导效果。如图所示，装载有四因子并带有HIV-1抗原AE2F SOSIP.664的K562细胞免疫中国恒河猴后，可显著提升针对HIV-1的特异性抗体。
图9所示为带有HA抗原的四因子K562细胞在恒河猴体内抗体的诱导效果。如图所示，装载有四因子并带有流感病毒抗原HA的K562细胞免疫中国恒河猴后，可显著提升针对HA蛋白的特异性抗体。
具体实施方案
下面将结合本发明实施例，对本发明实施例中的技术方案进行清楚、完整地描述，显然，所描述的实施例仅仅是本发明一部分实施例，而不是全部的实施例。基于本发明中的实施例，本领域普通技术人员在没有做出创造性劳动前提下所获得的所有其他实施例，都属于本发明保护的范围。阅读以下公开的实施方案的详细描述和所附权利要求后，本发明的这些和其它特征和优点将变得显而易见。
实施例一：人CD40L与CTB基因的慢病毒表达载体pHAGE-CTB-T2A-CD40L-puro的构建以及表达验证
1.1带人CD40L与CTB基因目的基因的pHAGE载体构建
人工合成人源CD40L与CTB的DNA序列：为了增强CTB的分泌，CTB的5’端加有CD8的信号肽，两个基因之间加T2A序列，进行基因的双表达。合成的序列如SEQ ID NO:2所示，序列5’端带有NotI酶切位点，3’端带有BamHI酶切位点。
双酶切合成的片段与载体质粒：用Not I酶切(Thermo Scientific公司，货号FD0596)与BamH I酶切(Thermo Scientific公司，FD0054)，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。
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，成功克隆出人CD40L与CTB基因的慢病毒表达质粒(pHAGE-CTB-T2A-CD40L-puro)，质粒构建图谱如图1a所示。
1.2含有人CD40L与CTB基因的载体pHAGE-CTB-T2A-CD40L-puro的慢病毒包装
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；将带有人CD40L与CTB的pHAGE-CTB-T2A-CD40L-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。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人CD40L与CTB慢病毒。
1.3含有人CD40L与CTB基因的K562细胞的表达验证
准备5×10 5的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步骤1.2中浓缩的病毒100μL，1000g，离心2小时。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1μg/mL的嘌呤霉素细胞培养基培养，最后能够存活的细胞便是整合有CD40L基因的细胞。取上述感染后存活的K562细胞，用anti-CD40L-PE的抗体(Biolegend公司，型号310805)进行染色，最后用流式细胞术(BD Pharminge公司，型号forttesa)分析。并将阳性的细胞不断的进行流式细胞分选富集，最终获得有高表达CD40L的K562细胞。结果显示，不断富集后的能够在K562细胞中高表达目的基因人CD40L(图1b)；所用的一抗分别为Anti-CTB的小鼠血清，二抗分别为HRP标记山羊抗小鼠(中杉金桥公司，货号zb-2305)。结果显示，利用蛋白质免疫印迹方法能检测到带有CD8信号肽的CTB蛋白的高表达，单独CTB有少量表达(图1c)。
实施例二：人IL-4与IL-21基因的慢病毒表达载体pHAGE-IL-4-T2A-IL-21-ZsGreen1的构建以及表达验证
2.1带人IL-4与IL-21目的基因的pHAGE载体构建
人工合成人来源IL-4与IL-21的DNA序列，两种基因之间加入T2A片段用于双表达。合成的序列如SEQ ID NO:4所示，序列5’端带有NotI酶切位点，3’端带有BamHI酶切位点。用Not I酶切(Thermo Scientific公司，货号FD0596)与BamH I酶切(Thermo Scientific公司，FD0054)双酶切合成的片段与载体质粒，并进行切胶回收，采用Sanprep柱式DNA胶回收试剂盒(Promega公司，货号A9282)回收片段与载体酶切片段。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，成功克隆出人来源IL-4与IL-21基因的慢病毒表达质粒(pHAGE-IL-4-T2A-IL-21-ZsGreen1)，质粒构建图谱如图2a所示。
2.2含有人IL-4与IL-21基因的载体pHAGE-IL-4-T2A-IL-21-ZsGreen1的慢病毒包装
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；将带有人IL-4与IL-21的pHAGE-IL-4-T2A-IL-21-ZsGreen1，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。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IL-4与IL-21慢病毒。
2.3含有人IL-4与IL-21基因的K562细胞的表达验证
准备5×10 5表达CD40L与CTB的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步骤1.2中浓缩的病毒100μL，1000g，离心2小时离心。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绿色荧光标签，将绿色荧光阳性的细胞不断的进行流式细胞分选富集，最终获得有高表达IL-4与IL-21的K562细胞。用anti-IL4-APC的抗体(Biolegend公司，型号500714)与anti-IL-21-BV421的抗体(BD Pharminge公司，型号564755)进行染色，最后用流式细胞术(BD Pharminge公司，型号forttesa)分析。结果显示，不断富集后能够在K562细胞中高表达目的基因人IL-4与IL-21(图2b，2c)。
实施例三：人BAFF基因的慢病毒表达载体pHAGE-BAFF-puro的构建以及表达验证
3.1带人BAFF目的基因的pHAGE载体构建
人工合成人BAFF基因的DNA序列。合成的序列如SEQ ID NO:6所示，序列5’端带有NotI酶切位点，3’端带有BamHI酶切位点。用Not I酶切(Thermo Scientific公司，货号FD0596)与BamH I酶切(Thermo Scientific公司，FD0054)双酶切合成的片段与载体质粒，并进行切胶回收，采用Sanprep柱式DNA胶回收试剂盒(Promega公司，货号A9282)回收片段与载体酶切片段。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，成功克隆出人BAFF基因的慢病毒表达质粒(pHAGE-BAFF-puro)，质粒构建图谱如图3a所示。
3.2含有人BAFF基因的载体pHAGE-BAFF-puro的慢病毒包装
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；将带有人BAFF的pHAGE-BAFF-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。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人BAFF慢病毒。
3.3含有人BAFF基因的K562细胞的表达验证
准备同时表达CD40L、CTB、IL-4与IL-21的5×10 5的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步骤1.2中浓缩的病毒100μL，1000g，离心2小时。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1μg/mL的嘌呤霉素细胞培养基培养，最后能够存活的细胞便是整合有CD40L基因的细胞。取上述感染后存活的K562细胞，用anti-BAFF-APC的抗体(BioLegend公司，型号366508)进行染色，最后用流式细胞术分析。并将阳性的细胞不断的进行流式细胞分选富集，最终获得有高表达BAFF的K562细胞。结果显示，不断富集后能够在K562细胞中高表达目的基因人BAFF(图3b)。
实施例四：人CXCR5基因的慢病毒表达载体pHAGE-IL-4-T2A-IL-21-IRES-CXCR5的构建以及表达验证
4.1带人CXCR5目的基因的pHAGE载体构建
人工合成人CXCR5基因的DNA序列。合成的序列如SEQ ID NO:8所示，序列5’端带有NotI酶切位点，3’端带有Sal I酶切位点。用Not I酶切(Thermo Scientific公司，货号FD0596)与Sal I酶切(Thermo Scientific公司，FD0644)双酶切合成的片段与载体质粒，并进行切胶回收，采用Sanprep柱式DNA胶回收试剂盒(Promega公司，货号A9282)回收片段与载体酶切片段。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，成功克隆出人CXCR5基因的慢病毒表达质粒(pHAGE-IL-4-T2A-IL-21-IRES-CXCR5)，质粒构建图谱如图4a所示。
4.2含有人CXCR5基因的载体pHAGE-IL-4-T2A-IL-21-IRES-CXCR5的慢病毒包装
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；将带有人CXCR5的pHAGE-IL-4-T2A-IL-21-IRES-CXCR5，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。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人BAFF慢病毒。
4.3含有人CXCR5基因的K562细胞的表达验证
准备5×10 5的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步骤1.2中浓缩的病毒100μL，1000g，离心2小时。离心感染结束后，继续在37℃，5％的孵箱培养12小时左右。取上述感染后存活的K562细胞，用anti-CXCR5-APC/Cy7的抗体(BioLegend公司，型号356926)进行染色，最后用流式细胞术分析。并将阳性的细胞不断的进行流式细胞分选富集，最终获得有高表达CXCR5的K562细胞。结果显示，不断富集后能够在K562细胞中高表达目的基因人CXCR5(图4b)。
实施例五：AE2F SOSIP.664基因的慢病毒表达载体pHAGE-AE2F SOSIP.664-ZsGreen1的构建以及表达验证
5.1带AE2F SOSIP.664目的基因的pHAGE载体构建
人工合成AE2F SOSIP.664基因的DNA序列。合成的序列如SEQ ID NO:10所示，序列5’端带有NotI酶切位点，3’端带有BamHI酶切位点。用Not I酶切(Thermo Scientific公司，货号FD0596)与BamH I酶切(Thermo Scientific公司，FD0054)双酶切合成的片段与载体质粒，并进行切胶回收，采用Sanprep柱式DNA胶回收试剂盒(Promega公司，货号A9282)回收片段与载体酶切片段。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，成功克隆出人BAFF基因的慢病毒表达质粒(pHAGE-AE2F SOSIP.664-ZsGreen1)，质粒构建图谱如图5a所示。
5.2含有AE2F SOSIP.664基因的载体pHAGE-AE2F SOSIP.664-ZsGreen1的慢病毒包装
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；将带有AE2F SOSIP.664的pHAGE-AE2F SOSIP.664-ZsGreen1，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。37℃，5％的孵箱培养48小时，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AE2F SOSIP.664慢病毒。
5.3含有AE2F SOSIP.664基因的K562细胞的表达验证
准备同时表达CD40L、CTB、IL-4与IL-21的5×10 5的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步骤1.2中浓缩的病毒100μL，1000g，离心2小时。离心感染结束后，用1μg/mL的嘌呤霉素细胞培养基，继续在37℃，5％的孵箱培养。取上述感染后的K562细胞，用anti-HIV-1的血清进行染色，最后用流式细胞术分析。并将阳性的细胞不断的进行流式细胞分选富集，最终获得有高表达AE2F SOSIP.664的K562细胞。结果显示，不断富集后能够在K562细胞中高表达目的基因AE2F SOSIP.664(图5b)。
实施例六：HA基因的慢病毒表达载体pHAGE-HA1-FLAG-CD8TM-ZsGreen1的构建以及表达验证
6.1带HA目的基因的pHAGE载体构建
人工合成HA基因的DNA序列。合成的序列如SEQ ID NO:12所示，序列5’端带有NotI酶切位点，3’端带有BamHI酶切位点。用Not I酶切(Thermo Scientific公司，货号FD0596)与BamH I酶切(Thermo Scientific公司，FD0054)双酶切合成的片段与载体质粒，并进行切胶回收，采用Sanprep柱式DNA胶回收试剂盒(Promega公司，货号A9282)回收片段与载体酶切片段。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，成功克隆出人BAFF基因的慢病毒表达质粒(pHAGE-HA1-FLAG-CD8TM-ZsGreen1)，质粒构建图谱如图6a所示。
6.2含有HA基因的载体pHAGE-HA1-FLAG-CD8TM-ZsGreen1的慢病毒包装
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；将带有HA的pHAGE-AE2F SOSIP.664-ZsGreen1，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。37℃，5％的孵箱培养48小时，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HA慢病毒。
6.3含有HA基因的K562细胞的表达验证
准备同时表达CD40L、CTB、IL-4与IL-21的5×10 5的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步骤1.2中浓缩的慢病毒100μL，1000g，离心2小时。离心感染结束后，用1μg/mL的嘌呤霉素细胞培养基，继续在37℃，5％的孵箱培养。取上述感染后的K562细胞，用anti-FLAG-PE的抗体进行染色(Biolegend公司，货号37310)，最后用流式细胞术分析。并将阳性的细胞不断的进行流式细胞分选富集，最终获得有高表达HA的K562细胞(图6b)。
实施例七：含有B细胞活化相关因子在体外对B细胞的作用
采用磁珠分选方法从人PBMC中分选B细胞，将磁珠分选后的人B细胞(1E4)、分别与同等数量的如下三种细胞放置于96孔板中共孵育：(1)K562对照细胞；(2)装载有CD40L、IL-4、IL-21与CTB四种B细胞活化因子的K562细胞；(3)装载有CD40L、IL-4、IL-21与CTB四种B细胞活化因子并带有BAFF的K562细胞。共孵育14天后，采用流式细胞术检测孔内的B细胞的存活情况及比例；用人IgG检测试剂盒(Thermo Scientific公司，货号88-50550-22)检测IgG的含量。
如图7所示，装载有四因子(包括CD40L、IL-4、IL-21与CTB四种B细胞活化因子)K562细胞、及在四因子基础上搭载BAFF基因K562细胞(图7a)，在体外能够提升B细胞的存活时间与增加细胞的数目。相比对照K562细胞，B细胞总抗体分泌水平(IgG)，在加持四因子组可提升约11倍，在此之上搭载BAFF之后可进一步提升至16倍(图7b)。
采用磁珠分选方法从人PBMC中分选B细胞，将磁珠分选后的人B细胞(1E4)、分别与同等数量的如下三种细胞放置于96孔板中共孵育：(1)K562对照 细胞；(2)装载有CD40L、IL-4、IL-21与BAFF四种B细胞活化因子的K562细胞；(3)装载有CD40L、IL-4、IL-21与BAFF四种B细胞活化因子并带有CXCR5的K562细胞。共孵育14天后，通过CD19分子标记人B细胞(Biolegend公司，货号HIB19)，采用流式细胞术检测孔内的B细胞的数目；并用人IgG检测试剂盒(Thermo Scientific公司，货号88-50550-22)检测IgG的含量。
装载有四因子(包括CD40L、IL-4、IL-21与BAFF四种B细胞活化因子)K562细胞、及在四因子基础上搭载CXCR5基因K562细胞(图7c)，在体外能够提升B细胞的存活数目，相较K562细胞组，四因子的能够提升超过3倍，而五因子的能近4倍。相比对照K562细胞，B细胞总抗体分泌水平(IgG)，在加持四因子组可提升约5倍，在此之上搭载CXCR5之后可进一步提升至11倍(图7d)。
实施例八：带有AE2F SOSIP.664抗原的四因子K562细胞的体内抗体的诱导评价
采用中国恒河猴(n＝3)进行免疫。免疫前2周采集本底血，第0周初免2.5×10 7的带有AE2F SOSIP.664抗原的四因子K562细胞，并在免疫后第1周以及第2周采集外周血，分离血浆，用Elisa的方法检测HIV-1特异性抗体的滴度。如图8所示，装载有四因子并带有HIV-1抗原AE2F SOSIP.664的K562细胞免疫中国恒河猴后，可显著提升针对HIV-1的特异性抗体，平均滴度可达到1：25600，说明本发明的载体搭载HIV-1抗原后，有较强的免疫原性。
实施例九：带有HA抗原的四因子K562细胞的体内抗体的诱导效果评价
采用中国恒河猴(n＝3)进行免疫。免疫前2周采集本底血，第0周初免1mg表达HA的DNA疫苗，免疫后2周采集外周血；在第4周第一次加强免疫1.5×10 7的带有HA抗原的四因子K562细胞，并在免疫后2周，4周分别采集外周血，分离血浆；在第11周第二次加强免疫2×10 7的带有HA抗原的四因子K562细胞，并在免疫后1周、3周、10周分别采集外周血，分离血浆，用Elisa的方法检测HA特异性抗体的滴度。
如图9结果显示，装载有四因子并带有流感病毒抗原HA的K562细胞免疫中国恒河猴后，可显著提升针对HA蛋白的特异性抗体，平均滴度可达到1：25600；第二次加强免疫后，能继续提升抗体滴度到1：51200，而且能够维持较高滴度的抗体浓度， 直至21周。该实施例说明，本发明载体搭载流感抗原后，不仅有较强的免疫原性，而且能够在体内持续诱导较高水平的抗体应答。

Claims (21)

  1. 一种高效诱导机体体液免疫应答的疫苗载体，其特征在于，所述疫苗载体是经由基因工程化的K562细胞。
  2. 根据权利要求1所述的高效诱导机体体液免疫应答的疫苗载体，其特征在于，所述疫苗载体是经由基因工程化表达B细胞活化相关的分子的K562细胞，所述B细胞活化相关的分子包括CD40L、IL-4、IL-21、B细胞活化因子、霍乱毒素B亚单位或C-X-C趋化因子受体5(CXCR5)以及上述不同因子的组合。
  3. 根据权利要求1所述的高效诱导机体体液免疫应答的疫苗载体，其特征在于，所述疫苗载体K562细胞经由基因工程化表达CXCR5、CCR7或CD62L。
  4. 根据权利要求2所述高效诱导机体体液免疫应答的疫苗载体，其特征在于，搭载所述B细胞活化相关的分子蛋白，其中搭载的B细胞活化相关的分子的蛋白包括经过修饰的蛋白、蛋白分子或其部分序列与其他分子融合表达形成的融合蛋白、以及蛋白来源的多肽片段；融合表达的方式包括与抗体的Fc序列融合、与不同信号肽的融合或与细胞因子融合。
  5. 根据权利要求1-4中任一项所述的高效诱导机体体液免疫应答的疫苗载体，其特征在于，所述疫苗载体K562细胞基因工程化表达免疫原，免疫原表达部位包括细胞内、细胞膜上或分泌性表达。
  6. 根据权利要求5所述的高效诱导机体体液免疫应答的疫苗载体，其特征在于，疫苗载体K562细胞基因工程化表达的免疫原来源包括病毒、细菌、肿瘤抗原或人源蛋白分子。
  7. 根据权利要求6所述的高效诱导机体体液免疫应答的疫苗载体，其特征在于，所述病毒包括流感病毒、冠状病毒、副粘病毒、风疹病毒、艾滋病病毒、甲肝病毒、乙肝病毒、丙肝病毒、流行性脑炎病毒、登革病毒、森林脑炎病毒、西尼罗病毒、寨卡病毒、黄热病毒、汉坦病毒、埃博拉病毒、疱疹病毒、狂犬病毒、痘病毒或布尼亚病毒。
  8. 根据权利要求7所述的高效诱导机体体液免疫应答的疫苗载体，其特征在于，所述流感病毒包括人流感病毒、禽流感病毒，所述冠状病毒包括SARS、MERS、SARS-CoV-2、蝙蝠冠状病毒(bat-CoV),所述副粘病毒包括副流感病毒、麻疹病毒、呼吸道合胞病毒、腮腺炎病毒、尼帕病毒、人偏肺病毒。
  9. 根据权利要求6所述的高效诱导机体体液免疫应答的疫苗载体，其特征在于，所述病毒免疫原来自于病毒囊膜蛋白，包括血凝素蛋白(Hemagglutinin,HA)、神经氨酸酶(Neuraminidase，NA)、基质蛋白(Matrix protein,M)、包膜蛋白(Envelope)、刺突蛋白(Spike)、膜蛋白(Membrane,M)、溶血素(Haemolysin,HL)、融合蛋白(Fusion,F)、黏附蛋白(G)。
  10. 根据权利要求6所述的高效诱导机体体液免疫应答的疫苗载体，其特征在于，所述肿瘤抗原，包括CD19、BCMA、CD20、CD22、CD30、CD33、CD38、CD47、CD52、CD70、CD117、CD123、CD133、CD138、CD147、CD171、RANKL、NKG2DL、HER2、MUC1、MUC16、CEA、EpCAM、IL-13Rα2、EGFR、EGFRvIII、GD2、DR5、EphA2、FRα、PSCA、PSMA、TARP、PIGF、cMet、VEGFR2、BCMA、CTLA-4、PD-L1、AFP、GPC3、AXL、ROR1、ROR2、FAP、Mesothelin、DLL3、CLD18、PDGF-Rα、SLAMF7和/或CCR4。
  11. 根据权利要求6所述的高效诱导机体体液免疫应答的疫苗载体，其特征在于，所述人源蛋白分子，包括人的细胞膜上蛋白分子。
  12. 根据权利要求6所述的高效诱导机体体液免疫应答的疫苗载体，其特征在于，所述疫苗载体搭载抗原后，与不同的佐剂组合形成疫苗，所用佐剂包括铝盐佐剂、CpG佐剂、锰佐剂、MF59佐剂、QS21佐剂、TLR配体佐剂、NOD通路佐剂、干扰素佐剂、cGAS/STING通路佐剂、细胞因子佐剂、PD-1阻断抗体、CTLA-4阻断抗体、TIGIT阻断抗体、αGalcer及其衍生物佐剂，所述细胞因子佐剂包括IL-12、GM-CSF、IL-18、IL-21。
  13. 根据权利要求1-3中任一项所述的高效诱导机体体液免疫应答的疫苗载体的制备方法，其特征在于，包括如下步骤：
    1)分别构建含有CD40L、IL-4、IL-21、BAFF蛋白分子、霍乱毒素B亚单位、CXCR5、CCR7或CD62L的载体；
    2)将含有CD40L、IL-4、IL-21、BAFF蛋白分子、霍乱毒素B亚单位、CXCR5、CCR7或CD62L的载体感染、转染或者电转处理K562细胞，进行优选单克隆整合有CD40L、IL-4、IL-21、BAFF、霍乱毒素B亚单位、CXCR5、CCR7或CD62L的细胞系，即得。
  14. 根据权利要求13所述的制备方法，其特征在于，将CD40L、IL-4、IL-21、B细胞活化因子、霍乱毒素B亚单位、CXCR5、CCR7或CD62L蛋白基因表达于慢病毒载体、质粒载体、痘病毒载体、腺病毒载体、腺相关病毒载体、单纯疱疹病毒载体或CMV载体。
  15. 根据权利要求13所述的制备方法，其特征在于，CD40L、IL-4、IL-21、BAFF、霍乱毒素B亚单位、CXCR5，CCR7或CD62L蛋白基因，可以是单独表达在K562细胞上，也可以是两两组合，或者三三组合，四四组合，五五组合亦或是六六组合，也可以是全部都表达在K562细胞上。
  16. 根据权利要求6所述的高效诱导机体体液免疫应答的疫苗载体的使用方法，其特征在于，用活的疫苗载体、灭活的疫苗载体以及疫苗载体分泌的外泌体作为疫苗进行免疫接种； 或将表达免疫原以及B细胞激活分子的疫苗载体细胞膜装载到其他纳米颗粒材料上形成纳米颗粒疫苗进行免疫接种。
  17. 根据权利要求6所述的高效诱导机体体液免疫应答的疫苗载体的使用方法，其特征在于，采用包括X-射线、热灭活、固定剂、裂解剂对疫苗载体进行灭活。
  18. 根据权利要求6所述的高效诱导机体体液免疫应答的疫苗载体的使用方法，其特征在于，利用所述K562疫苗载体制备的疫苗给药方式，包括静脉注射、皮下注射、皮内注射、肌肉注射、黏膜给药、滴鼻、点眼、或气雾免疫接种。
  19. 根据权利要求1所述的高效诱导机体体液免疫应答的疫苗载体的用途，其特征在于：利用所述K562细胞载体进行体外B细胞的生存和增殖，利用所述K562细胞载体搭载抗原制备成的疫苗免疫动物获得抗体并将抗体用于科研、临床药物以及CAR-T的识别区、BITE的识别区，以及作为疫苗载体诱导体液免疫应答时可单独给药或与其他治疗技术联合应用。
  20. 根据权利要求19所述的用途，所述疫苗载体搭载的HIV-1膜蛋白亚型包括HIV-1A、B、C、D、F、G、H、J、K亚型以及不同型别间形成的重组型，包括CRF01-AE、CRF07-BC、CRF08-BC、AG重组型。
  21. 根据权利要求20所述的用途，所述疫苗载体搭载的HIV-1膜蛋白来源包括AE2F、RL42、CN54。
PCT/CN2020/085778 2020-03-25 2020-04-21 一种高效诱导机体体液免疫应答的疫苗载体、其制备方法及用途 WO2021189571A1 (zh)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CN202010219186.5 2020-03-25
CN202010219186.5A CN111358953A (zh) 2020-03-25 2020-03-25 一种高效诱导机体体液免疫应答的疫苗载体、其制备方法及用途

Publications (1)

Publication Number Publication Date
WO2021189571A1 true WO2021189571A1 (zh) 2021-09-30

Family

ID=71199110

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2020/085778 WO2021189571A1 (zh) 2020-03-25 2020-04-21 一种高效诱导机体体液免疫应答的疫苗载体、其制备方法及用途

Country Status (2)

Country Link
CN (1) CN111358953A (zh)
WO (1) WO2021189571A1 (zh)

Families Citing this family (14)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
RU2733831C1 (ru) * 2020-07-08 2020-10-07 Федеральное государственное учреждение науки "Государственный научный центр вирусологии и биотехнологии "Вектор" Федеральной службы по надзору в сфере защиты прав потребителей и благополучия человека (ФБУН ГНЦ ВБ "Вектор" Роспотребнадзора) Искусственный ген, кодирующий бицистронную структуру, образованную последовательностями рецептор-связующего домена гликопротеина S коронавируса SARS-CoV-2, P2A-пептида и гликопротеина G VSV, рекомбинантная плазмида pStem-rVSV-Stbl_RBD_SC2, обеспечивающая экспрессию искусственного гена и рекомбинантный штамм вируса везикулярного стоматита rVSV-Stbl_RBD_SC2, используемого для создания вакцины против коронавируса SARS-CoV-2
RU2747467C1 (ru) * 2020-07-17 2021-05-05 Муса Тажудинович Абидов Способ защиты от коронавирусной инфекции
RU2733832C1 (ru) * 2020-07-28 2020-10-07 Федеральное бюджетное учреждение науки "Государственный научный центр вирусологии и биотехнологии "Вектор" Федеральной службы по надзору в сфере защиты прав потребителей и благополучия человека (ФБУН ГНЦ ВБ "Вектор" Роспотребнадзора) Искусственный ген Stbl_RBD_TrM_SC2, кодирующий бицистронную структуру, образованную последовательностями рецепторсвязывающего домена гликопротеина S коронавируса SARS-CoV-2, трансмембранного региона, P2A-пептида и гликопротеина G VSV, рекомбинантная плазмида pStem-rVSV-Stbl_RBD_TrM_SC2, обеспечивающая экспрессию искусственного гена, и рекомбинантный штамм вируса везикулярного стоматита rVSV-Stbl_RBD_TrM_SC2, используемый для создания вакцины против коронавируса SARS-CoV-2
RU2733834C1 (ru) * 2020-07-28 2020-10-07 Федеральное бюджетное учреждение науки Государственный научный центр вирусологии и биотехнологии "Вектор" Федеральной службы по надзору в сфере защиты прав потребителей и благополучия человека (ФБУН ГНЦ ВБ "Вектор" Роспотребнадзора) Искусственный ген EctoS_SC2, кодирующий эктодомен гликопротеина S коронавируса SARS-CoV-2 с C-концевым тримеризующим доменом, рекомбинантная плазмида pStem-rVSV-EctoS_SC2, обеспечивающая экспрессию искусственного гена, и рекомбинантный штамм вируса везикулярного стоматита rVSV-EctoS_SC2, используемый для создания вакцины против коронавируса SARS-CoV-2
CN111875680A (zh) * 2020-08-08 2020-11-03 武汉圣润生物科技有限公司 一种预防新型冠状病毒微颗粒的制备方法及应用
CN112386684B (zh) * 2020-11-12 2023-12-05 广东昭泰细胞生物科技有限公司 一种covid-19疫苗及其制备方法和应用
CN112439058A (zh) * 2020-11-25 2021-03-05 深圳市第二人民医院(深圳市转化医学研究院) 基于外泌体为载体的重组新型冠状病毒纳米疫苗方法
WO2022135563A1 (zh) * 2020-12-24 2022-06-30 上海市公共卫生临床中心 同时诱导抗多种病毒的免疫应答的方法
CN112679617B (zh) * 2021-01-15 2021-09-17 北京鼎成肽源生物技术有限公司 一种基于间皮素锚定的哺乳动物融合蛋白展示质粒、细胞系及应用
CN113249408B (zh) * 2021-06-23 2021-11-02 深圳湾实验室 一种靶向激活体液免疫和细胞免疫的核酸疫苗载体构建及应用
CN113666990A (zh) * 2021-08-24 2021-11-19 复旦大学 一种诱导广谱抗冠状病毒的t细胞疫苗免疫原及其应用
WO2023034991A1 (en) * 2021-09-02 2023-03-09 Kansas State University Research Foundation Mrna vaccine formulations and methods of using the same
CN114028539B (zh) * 2021-09-13 2024-04-26 北京大学 粘蛋白1在抑制冠状病毒中的应用
CN113797329A (zh) * 2021-10-19 2021-12-17 启锰生物科技(江苏)有限公司 一种二价锰佐剂和CpG佐剂的疫苗佐剂组合物及其制作方法

Citations (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101041816A (zh) * 2006-12-01 2007-09-26 扬州大学 人工抗原递呈细胞及其制备方法
KR20090062096A (ko) * 2007-12-12 2009-06-17 가톨릭대학교 산학협력단 Ｂ 세포의 활성화를 위한 세포성 ｃｄ40ｌ을 발현하는세포주
CN102586185A (zh) * 2012-03-12 2012-07-18 浙江中赢方舟生物工程股份有限公司 一种k562细胞扩增激活nk细胞的方法
CN102911918A (zh) * 2011-12-29 2013-02-06 上海交通大学医学院 一种基因工程细胞及其在nk细胞扩增中的应用
CN103333861A (zh) * 2013-06-03 2013-10-02 浙江省台州医院 特异性表达hla-g1抗原的细胞株k562
CN108300697A (zh) * 2017-01-13 2018-07-20 上海恒润达生生物科技有限公司 一种滋养细胞刺激nk细胞扩增的方法和用途
CN108607094A (zh) * 2016-12-12 2018-10-02 项雯华 由基因工程化的人造抗原提呈细胞的分泌小体所构建的t细胞疫苗及其制备方法和应用
CN109668819A (zh) * 2019-02-01 2019-04-23 上海市公共卫生临床中心 一种机体免疫功能评价的试剂盒及其使用方法和应用

Patent Citations (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101041816A (zh) * 2006-12-01 2007-09-26 扬州大学 人工抗原递呈细胞及其制备方法
KR20090062096A (ko) * 2007-12-12 2009-06-17 가톨릭대학교 산학협력단 Ｂ 세포의 활성화를 위한 세포성 ｃｄ40ｌ을 발현하는세포주
CN102911918A (zh) * 2011-12-29 2013-02-06 上海交通大学医学院 一种基因工程细胞及其在nk细胞扩增中的应用
CN102586185A (zh) * 2012-03-12 2012-07-18 浙江中赢方舟生物工程股份有限公司 一种k562细胞扩增激活nk细胞的方法
CN103333861A (zh) * 2013-06-03 2013-10-02 浙江省台州医院 特异性表达hla-g1抗原的细胞株k562
CN108607094A (zh) * 2016-12-12 2018-10-02 项雯华 由基因工程化的人造抗原提呈细胞的分泌小体所构建的t细胞疫苗及其制备方法和应用
CN108300697A (zh) * 2017-01-13 2018-07-20 上海恒润达生生物科技有限公司 一种滋养细胞刺激nk细胞扩增的方法和用途
CN109668819A (zh) * 2019-02-01 2019-04-23 上海市公共卫生临床中心 一种机体免疫功能评价的试剂盒及其使用方法和应用

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
LI, DONG ET AL.: "The Biological Function and Antitumour Effect of Interleukin-21", CHINESE BULLETIN OF LIFE SCIENCES, vol. 21, no. 4, 31 August 2009 (2009-08-31), pages 513 - 516, XP055853992 *
ZHU, YAN ET AL.: "Establishment of Leukemia Cell Lines Expressing IL-21 and Analysis of Anti-Tumor Function", BASIC & CLINICAL MEDICINE, vol. 34, no. 7, 31 July 2014 (2014-07-31), pages 961 - 967, XP055853984 *

Also Published As

Publication number Publication date
CN111358953A (zh) 2020-07-03

Similar Documents

Publication Publication Date Title
WO2021189571A1 (zh) 一种高效诱导机体体液免疫应答的疫苗载体、其制备方法及用途
US10570416B2 (en) TC-83-derived alphavirus vectors, particles and methods
Cheng et al. Cancer immunotherapy using Sindbis virus replicon particles encoding a VP22–antigen fusion
AU2017297610B2 (en) Compositions and methods for alphavirus vaccination
Salek-Ardakani et al. The TNFR family members OX40 and CD27 link viral virulence to protective T cell vaccines in mice
CN110464841B (zh) 免疫增强的药物组合物及其应用
CN112972668A (zh) 重组修饰的痘苗病毒安卡拉(mva)丝状病毒疫苗
Maamary et al. Newcastle disease virus expressing a dendritic cell-targeted HIV gag protein induces a potent gag-specific immune response in mice
JP2021138721A (ja) Ｈｉｖ予備免疫化および免疫療法
JP2006523224A (ja) 免疫原性組成物および方法
Gupta et al. Vaccination with a fusion protein that introduces HIV-1 gag antigen into a multitrimer CD40L construct results in enhanced CD8+ T cell responses and protection from viral challenge by vaccinia-gag
Cheng et al. Repeated DNA vaccinations elicited qualitatively different cytotoxic T lymphocytes and improved protective antitumor effects
US10052377B2 (en) Cellular vaccine and method of inducing an immune response in a subject
CN115819522B (zh) 一种带状疱疹病毒疫苗、表达蛋白及重组腺病毒制备与应用
Thompson et al. Nonmucosal alphavirus vaccination stimulates a mucosal inductive environment in the peripheral draining lymph node
CN115820742A (zh) 一种提高nk细胞慢病毒转导效率的方法
AU2017363967B2 (en) Viral vector constructs for expression of genetic adjuvants activating the CD40 and sting pathways
KR102678544B1 (ko) Cd40 및 sting 경로를 활성화하는 유전자 보조제의 발현을 위한 바이러스 벡터 작제물
WO2021217480A1 (zh) 一种病毒抗原-免疫共激活因子的双分子dna疫苗
KR102678519B1 (ko) Sting 경로를 활성화하는 유전자 보조제의 발현을 위한 바이러스 벡터 작제물
US11638752B2 (en) Viral vector constructs for expression of genetic adjuvants activating the STING pathway
KR101059721B1 (ko) 면역원성 조성물 및 의약
McKee et al. 559. Generating Powerful Immune Responses Against Lentiviral Antigens: Using rSV40 Vector-Delivered Proinflammatory Cytokines to Potentiate Immune Responses vs. rSV40-Delivered Lentiviral Antigens
CN118221830A (zh) T细胞抗原受体及其应用
CN111620955A (zh) 一种多靶位复合抗原及其应用

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 20926448

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 20926448

Country of ref document: EP

Kind code of ref document: A1