WO2022002180A1 - 融合蛋白疫苗平台的构建与应用 - Google Patents

融合蛋白疫苗平台的构建与应用 Download PDF

Info

Publication number
WO2022002180A1
WO2022002180A1 PCT/CN2021/103931 CN2021103931W WO2022002180A1 WO 2022002180 A1 WO2022002180 A1 WO 2022002180A1 CN 2021103931 W CN2021103931 W CN 2021103931W WO 2022002180 A1 WO2022002180 A1 WO 2022002180A1
Authority
WO
WIPO (PCT)
Prior art keywords
vaccine
antigen
seq
sars
cov
Prior art date
Application number
PCT/CN2021/103931
Other languages
English (en)
French (fr)
Inventor
傅阳心
彭华
孙士玉
Original Assignee
中国科学院生物物理研究所
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
Priority claimed from CN202010623708.8A external-priority patent/CN113876938B/zh
Application filed by 中国科学院生物物理研究所 filed Critical 中国科学院生物物理研究所
Priority to JP2022580808A priority Critical patent/JP2023531291A/ja
Priority to AU2021301953A priority patent/AU2021301953A1/en
Priority to EP21833302.9A priority patent/EP4176893A4/en
Priority to US18/003,872 priority patent/US20230293679A1/en
Priority to CA3184383A priority patent/CA3184383A1/en
Priority to CN202111680865.3A priority patent/CN115137812A/zh
Publication of WO2022002180A1 publication Critical patent/WO2022002180A1/zh

Links

Images

Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/04—Antibacterial agents
    • 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
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • 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
    • A61K39/0005—Vertebrate antigens
    • A61K39/0011—Cancer antigens
    • A61K39/001102—Receptors, cell surface antigens or cell surface determinants
    • A61K39/001103—Receptors for growth factors
    • A61K39/001106—Her-2/neu/ErbB2, Her-3/ErbB3 or Her 4/ErbB4
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/12—Viral antigens
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • 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
    • 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
    • A61K39/46—Cellular immunotherapy
    • A61K39/464—Cellular immunotherapy characterised by the antigen targeted or presented
    • A61K39/4643—Vertebrate antigens
    • A61K39/4644—Cancer antigens
    • A61K39/464402—Receptors, cell surface antigens or cell surface determinants
    • A61K39/464403—Receptors for growth factors
    • A61K39/464404—Epidermal growth factor receptors [EGFR]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/0012—Galenical forms characterised by the site of application
    • A61K9/0019—Injectable compositions; Intramuscular, intravenous, arterial, subcutaneous administration; Compositions to be administered through the skin in an invasive manner
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/20—Antivirals for DNA viruses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/52—Cytokines; Lymphokines; Interferons
    • C07K14/555—Interferons [IFN]
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/62—DNA sequences coding for fusion proteins
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/54—Medicinal preparations containing antigens or antibodies characterised by the route of administration
    • A61K2039/541—Mucosal route
    • A61K2039/543—Mucosal route intranasal
    • 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
    • A61K2039/57—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
    • 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
    • A61K2039/60—Medicinal preparations containing antigens or antibodies characteristics by the carrier linked to the antigen
    • A61K2039/6031—Proteins
    • 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
    • A61K2039/60—Medicinal preparations containing antigens or antibodies characteristics by the carrier linked to the antigen
    • A61K2039/6031—Proteins
    • A61K2039/605—MHC molecules or ligands thereof
    • 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
    • A61K2039/60—Medicinal preparations containing antigens or antibodies characteristics by the carrier linked to the antigen
    • A61K2039/6031—Proteins
    • A61K2039/6056—Antibodies
    • 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
    • A61K2039/60—Medicinal preparations containing antigens or antibodies characteristics by the carrier linked to the antigen
    • A61K2039/6031—Proteins
    • A61K2039/6075—Viral proteins
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/52—Constant or Fc region; Isotype
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/30—Non-immunoglobulin-derived peptide or protein having an immunoglobulin constant or Fc region, or a fragment thereof, attached thereto
    • 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
    • C12N2710/16211—Lymphocryptovirus, e.g. human herpesvirus 4, Epstein-Barr Virus
    • C12N2710/16234—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein
    • 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
    • C12N2710/16634—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein
    • 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
    • C12N2710/16711—Varicellovirus, e.g. human herpesvirus 3, Varicella Zoster, pseudorabies
    • C12N2710/16734—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein
    • 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
    • C12N2730/00—Reverse transcribing DNA viruses
    • C12N2730/00011—Details
    • C12N2730/10011—Hepadnaviridae
    • C12N2730/10111—Orthohepadnavirus, e.g. hepatitis B virus
    • C12N2730/10134—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein
    • 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
    • C12N2760/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses negative-sense
    • C12N2760/00011—Details
    • C12N2760/16011—Orthomyxoviridae
    • C12N2760/16111—Influenzavirus A, i.e. influenza A virus
    • C12N2760/16134—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein
    • 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
    • C12N2770/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses positive-sense
    • C12N2770/00011—Details
    • C12N2770/20011—Coronaviridae
    • C12N2770/20034—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein

Definitions

  • the invention belongs to the technical field of genetic engineering and biomedicine, and in particular relates to a vaccine, such as a vaccine comprising a fusion protein containing interferon-target antigen-immunoglobulin Fc region (antibody) as the main backbone.
  • a vaccine such as a vaccine comprising a fusion protein containing interferon-target antigen-immunoglobulin Fc region (antibody) as the main backbone.
  • the vaccine of the present invention can be used as a vaccine platform for preventing hepatitis B virus (HBV) infection, HPV, EBV, HIV, SARA-COV2, influenza virus infection and the occurrence of HPV, EBV-related tumors and treating chronic hepatitis B (chronic hepatitis B). , CHB) infection, treatment of HBV, HP, EBV-related tumors.
  • HBV hepatitis B virus
  • HBV hepatocellular carcinoma
  • Chronic HBV infection is one of the main diseases that threaten human health. It is urgent to explore an effective immunotherapy strategy for chronic hepatitis B. The development of a therapeutic vaccine for chronic hepatitis B is of great social and economic significance.
  • Vaccines are the best way to prevent and control influenza. Vaccines can reduce the incidence of disease and reduce the severity of infection, especially in young children and the elderly who are at risk of complications from influenza. Even if currently approved influenza vaccines confer good protection in healthy young adults, there are still some issues that need to be addressed. For example, some vaccines rely on chicken embryos, such as fire-fighting influenza vaccines and attenuated influenza vaccines. One disadvantage of these vaccines is that if the circulating strains are of avian origin, the epidemic will lead to vaccines and chicken embryos. Demand increases, which can lead to problems with the supply of chicken embryos [6] .
  • influenza vaccines take a lot of time to produce.
  • the elderly are more likely to develop severe syndrome, and the standard vaccine is generally not effective for the elderly, and the immune system of the elderly will gradually weaken with age [7] .
  • an influenza vaccine with strong immunogenicity that can be rapidly produced without relying on chicken embryos is urgently needed for the epidemic of influenza viruses.
  • Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the pathogen causing the 2019 coronavirus disease (COVID) pandemic.
  • the clinical symptoms caused by SARS-CoV-2 mainly include asymptomatic infection, mild influenza-like symptoms, Pneumonia and severe acute respiratory distress syndrome, which in severe cases can cause death in infected patients.
  • the vaccine is the basic countermeasure to control and terminate the new crown pandemic [9] .
  • 2019-nCoV mutants poses new challenges to the existing vaccine candidates and the control of the epidemic [10] . Therefore, a vaccine that is potent and can also work on 2019-nCoV mutants is not suitable for the current epidemic situation. is urgently needed.
  • the half-life of the antigen will be significantly prolonged, and the Fc region of the immunoglobulin can bind to the Fc receptor on the surface of the antigen-presenting cell to promote the antigen-presenting cell to the antigen. Processing and commission [11-13] .
  • type I interferon As an antiviral cytokine, type I interferon has many biological activities, one of which is to stimulate immune cells [14] . IFN ⁇ can strongly induce the differentiation and activation of human DC cells [15] .
  • type I interferon acts on immature DCs, it can promote the expression of MHC molecules and costimulatory molecules on the surface of DCs, such as: MHC class I, CD80 and CD86, thereby enhancing the ability of DCs to activate T cells [16-18] . It has been reported that after vaccinia virus and lymphocytic chorioencephalitis virus (LCMV) infection, type I interferon can promote the antigen-presenting ability of DCs [19-21] . In addition, after acting on DCs, type I interferon can promote the migration of DCs to lymph nodes by up-regulating the expression of chemokine receptors, thereby promoting the activation of T cells [22,23] .
  • LCMV lymphocytic chorioencephalitis virus
  • type I interferon can be used as an immune adjuvant.
  • the study of Le Bon et al. showed that when mice were immunized with weak immunogens, type I interferon exhibited a strong immune adjuvant effect in mice, inducing the production of long-lasting antibodies and immune memory [24] .
  • the main cell group in which type I interferons were found to play a role were DC cells.
  • the use of antibodies to target the delivery of the vaccine to DC stimulates DC activation and cross-presentation, which will further enhance the activity and titer of the vaccine.
  • Vaccines are an effective way to prevent and control major outbreaks of infectious diseases.
  • There are various types of vaccines one of which is protein subunit vaccines.
  • the immunogenicity of pure protein subunit vaccines is generally poor, which often limits the use of protein subunit vaccines. Therefore, a universal protein subunit vaccine platform is urgently needed.
  • the inventors propose an interferon alpha-virus antigen, bacteria or tumor-immunoglobulin Fc region fusion protein vaccine platform to enhance the body's response to viruses, bacteria or tumors. antigenic response.
  • the present invention provides a type I interferon-protein antigen-immunoglobulin Fc vaccine platform, wherein type I interferon can act on antigen presenting cells to make them mature and migrate so as to better exert antigen presentation and activate T cells
  • type I interferon can act on antigen presenting cells to make them mature and migrate so as to better exert antigen presentation and activate T cells
  • the Fc part of the vaccine platform can combine with Fc receptors on the surface of antigen-presenting cells to enhance the uptake of antigens by antigen-presenting cells to further help antigen-presenting cells to function.
  • the inventors propose that fusion of Th cell helper epitopes can further enhance the immune response effect of type I interferon-protein antigen-immunoglobulin Fc vaccine, and is an important component of the vaccine.
  • the inventors propose that antibodies such as anti-PD-L1 can be used to replace Fc, and the vaccine can be delivered to DCs in a targeted manner to stimulate DC activation and cross-presentation, which will further enhance the activity and titer of the vaccine.
  • the present invention can be used as a preventive and therapeutic vaccine for viral infection, bacterial infection or tumor and other diseases.
  • the present invention provides a vaccine comprising an interferon-target antigen-immunoglobulin Fc region (or antibody) fusion protein (attached Th epitope).
  • the present invention also provides fusion proteins (attached Th epitopes) comprising interferon-target antigen-immunoglobulin Fc region (or antibody) for use in the preparation of prophylactic or therapeutic compositions or kits (eg, pharmaceutical or vaccine compositions or kits).
  • the vaccine of the present invention can be produced by eukaryotic cell expression system, and vaccinated by immunization route such as subcutaneous/muscular or nasal cavity.
  • the antibody as a structural unit is not particularly limited, and can include, for example, an intact antibody or a fragment of an antibody, such as antibody heavy and light chains, or a single-chain antibody, which can be targeted for DC activation.
  • Antibodies including anti-PD-L1, anti-DEC205, anti-CD80/86 and other antibodies.
  • the target antigen described herein is not particularly limited and can be any suitable antigen.
  • the target antigens described herein can be, for example, tumor antigens and/or pathogen antigens (eg, viral or bacterial antigens).
  • the target antigen described herein can be, for example, a tumor antigen, such as a protein molecule that is highly expressed by tumor cells, for example, human epidermal growth factor receptor 2 (HER2/neu), epidermal growth factor (Epidermal growth factor receptor, EGFR).
  • the target antigen used in the vaccines provided herein can be, for example, a mutated target antigen that is different from the wild type.
  • the target antigens described herein can be, for example, mutants of tumor antigens and/or pathogen antigens (eg, viral or bacterial antigens).
  • the target antigen can be, for example, the full length or S1 region of the SARS-COV-2 virus S protein, for example, the target antigen can be the antigen shown in SEQ ID NO.76 or SEQ ID NO.77.
  • the wild-type target antigen refers to the immunogenic protein expressed by the virus or other infectious agent or tumor encoded by the wild-type gene (the wild-type gene refers to the most alleles in nature, which are used in biological experiments. It is often used as a standard control gene), such as the Spike protein (S protein) derived from the original wild-type strain of SARS-CoV-2.
  • S protein Spike protein
  • the mutated target antigen refers to the mutant viral protein expressed by the mutant virus strain encoded by the mutant gene mutated from the wild-type gene, such as the point mutation of the S protein of different mutant new coronaviruses that have been found Including: NTD region 69-70 deletion, Y144 deletion, 242-244 deletion, L18F, D80A, D215, R246I mutation, RBD region K417, E484, N501Y and other mutations, L452R mutation, T478K mutation, D614G, H655Y mutation.
  • these point mutations exist in different combinations in the B.1.1.7 (Alpha) mutant from the United Kingdom, the South African B.1.351 (Beta) mutant, the Brazilian P1 (Gamma) mutant, the Indian B.1.617, B. 1.617.1 (Kappa), B.1.617.2 (Delta), B.1.617.3 mutant strain, California B.1.429 mutant strain and other mutant new coronaviruses.
  • the mutated target antigen may include, for example, natural point mutation/deletion mutation/gain mutation/truncation, artificial point mutation/deletion mutation/gain mutation/truncation, any combination of natural or artificial mutations, mutations Subtypes are generated later, wherein the target antigen can be a tumor antigen, a pathogen antigen, such as a virus (eg, SARS-COV-2) or a bacterial antigen.
  • a pathogen antigen such as a virus (eg, SARS-COV-2) or a bacterial antigen.
  • the target antigen used in the vaccine provided by the present invention is a mutated viral antigen
  • the mutated viral antigen can be a mutant of SARS-COV-2, including, for example, SARS-COV-2 protein (eg One or more of S protein, N protein, M protein, E protein) natural point mutation/deletion mutation/increase mutation/truncation, artificial point mutation/deletion mutation/increase mutation/truncation, natural or artificial Any combination of mutations, the resulting subtype after mutation, for example, the mutant viral antigen can be a mutant of the full length of S protein, S1 region, and RBD region, for example, the mutant viral antigen can include SARS-COV-2 One or more of the following mutations of the S protein: NTD region 69-70 deletion, Y144 deletion, 242-244 deletion, L18F, D80A, D215, R246I mutation, RBD region K417, E484, N501Y mutation, L452R mutation, T478
  • references to target antigens herein generally include wild-type target antigens and mutant target antigens, unless expressly stated otherwise or clearly limited by context.
  • the purpose of the present invention is to provide a vaccine platform composed of interferon (IFN) and tumor, bacterial or viral antigens (pres1 antigen of hepatitis B virus, SARS-COV2 RBD antigen, influenza HA antigen, human papilloma virus HPV E7 antigen, hepatitis B virus surface antigen (HBsAg) antigen or peptide, herpes zoster virus (VZV) gE antigen, Ebstein-Barr virus (EBV) EBNA1/LMP2/gp350, herpes simplex virus 2 (HSV-2) gD antigen, HIV (HIV) gp120 antigen-immunoglobulin Fc region (or antibody) composition (attached Th epitope).
  • IFN interferon
  • bacterial or viral antigens pres1 antigen of hepatitis B virus, SARS-COV2 RBD antigen, influenza HA antigen, human papilloma virus HPV E7
  • the fusion protein can be a homologous or heterodimeric protein.
  • the fusion protein is In the case of a dimer, interferon, target antigen, immunoglobulin Fc region (or antibody Ab) as structural units can exist in the first polypeptide chain and/or the second polypeptide chain, and the existence of each structural unit is not With particular limitation, for example, it can exist in one chain at the same time, or any one or more structural units can exist in one chain, and another one or more structural units can exist in the other chain.
  • the interferon of the present invention can be selected from interferon I, type II interferon and type III interferon, such as IFN- ⁇ , IFN- ⁇ , IFN- ⁇ , IFN- ⁇ 1 (IL-29), IFN- ⁇ 2 (IL-2) -28a), IFN- ⁇ (IL-28b) and IFN- ⁇ ;
  • the IFN can be derived from human or murine; preferably type I interferon IFN- ⁇ (SEQ ID NO. 1, SEQ ID NO. 21, SEQ ID NO. 21, SEQ ID NO. 22).
  • the immunoglobulin Fc region of the present invention can be selected from the constant region amino acid sequences of IgG1, IgG2, IgG3 and IgG4/or IgM, preferably IgG1 (SEQ ID NO.2, SEQ ID NO.23, SEQ ID NO.24).
  • the fusion polypeptides of the present invention may also optionally comprise one or more Th cell helper epitopes and/or linking fragments (linkers).
  • the fusion protein when the fusion protein is a dimer, optionally the fusion protein may also be in any one or both chains of the homodimer or heterodimer (ie, the first polypeptide chain and/or the heterodimer). or the second polypeptide chain) contains one or more Th cell helper epitopes and/or linking fragments.
  • the various building blocks of the fusion protein can be linked by appropriate linking fragments (linkers).
  • the linker fragment that can be used in the vaccine of the present invention is not particularly limited, and can be any suitable peptide fragment known in the art.
  • the connecting fragment of each structural unit described in the present invention can be a flexible polypeptide sequence, and can be connecting fragment 1 and 2, for example, as shown in the amino acid sequences of SEQ ID NO.4 and SEQ ID NO.25.
  • the N-terminus of the polypeptide sequence composed of each structural unit of the present invention contains a corresponding signal peptide capable of promoting protein secretion, for example, as shown in the amino acid sequence of SEQ ID NO.5.
  • Preferred antigens described in the present invention include hepatitis B Pres1 antigen, including ad subtype (SEQ ID NO. 6) ay subtype (SEQ ID NO. 26), HBV HBsAg antigen (each subtype and peptide segment), including adr subtype (SEQ ID NO.7), adw subtype (SEQ ID NO.27), ayw subtype (SEQ ID NO.28), SARA-COV2 RBD antigen (SEQ ID NO.8), influenza virus HA antigen (SEQ ID NO.8) NO.9), HPV E7 antigen (SEQ ID NO.10).
  • Herpes virus VZV-gE antigen SEQ ID NO. 91
  • EBV-gp350 antigen SEQ ID NO. 92
  • HSV-2-gD antigen SEQ ID NO. 93
  • the homodimeric protein described in the present invention comprises a first polypeptide and a second polypeptide, and the first polypeptide and the second polypeptide are completely identical.
  • the first polypeptide and the second polypeptide are IFN-tumor or virus antigens from N segment to C segment (pres1 antigen of hepatitis B, SARS-COV2 RBD antigen, influenza HA antigen, HPV E7 antigen, HBsAg antigen, VZV-gE antigen, EBV EBNA1/LMP2/gp350, HSV-2-gD antigen, HIV gp120 antigen)-immunoglobulin Fc region; or a polypeptide containing a Pan epitope.
  • SEQ ID NO. 11, 12, 13, 14, 29, 30, 31, 32, 38, 39, 40, 47, 48, 49, 50, 51, 56, 57, 59, 58, 65, 66, 67 the amino acid sequence shown in 68.
  • the heterodimer of the present invention comprises a first polypeptide and a second pair of peptides, the first polypeptide and the second polypeptide are not the same polypeptide, and the first polypeptide is from the C segment to the N-terminus They are IFN-immunoglobulin Fc regions, respectively, including nucleotide sequences shown in SEQ ID NO.
  • the second polypeptide from segment C to segment N is tumor or virus, respectively Antigens (Hepatitis B Pres1 antigen, SARS-COV2 RBD antigen, influenza HA antigen, HPV E7 antigen, VZV-gE antigen, EBV EBNA1/LMP2/gp350, HSV-2-gD antigen, HIV gp120 antigen)-immunoglobulin Fc region; Comprising SEQ ID NO. 16, 17, 18, 19, 34, 35, 36, 37, 43, 44, 45, 46, 52, 53, 54, 55, 61, 62, 63, 64, 70, 71, 72 , the amino acid sequence shown in 73.
  • Antigens Hepatitis B Pres1 antigen, SARS-COV2 RBD antigen, influenza HA antigen, HPV E7 antigen, VZV-gE antigen, EBV EBNA1/LMP2/gp350, HSV-2-gD antigen, HIV gp120 antigen
  • the present invention also provides coding for the above-mentioned IFN-tumor or virus antigen hepatitis B Pres1 antigen, HBsAg antigen or peptide segment, SARS-COV2 RBD antigen, influenza HA antigen, HPV E7 antigen, VZV-gE antigen, EBV EBNA1/LMP2/gp350, HSV- 2-gD antigen, amino acid sequence of HIV gp120 antigen-immunoglobulin Fc vaccine platform.
  • the present invention also relates to nucleotide fragments encoding the vaccine platform and fusion protein.
  • the present invention also relates to the preparation method of the fusion protein or vaccine platform, for example, the preparation method comprises the following steps:
  • an expression vector comprising the encoding gene encoding the fusion protein or the vaccine platform, preferably, the expression vector is a pEE12.4 expression vector;
  • the host cell comprises the expression vector by transiently transfecting the host cell, preferably, the host cell is a 293F cell;
  • the present invention further includes the application of the vaccine platform, the vaccine platform can be used as a hepatitis B preventive vaccine, the vaccine platform can be used as a hepatitis B therapeutic vaccine, the vaccine platform can be used as an influenza preventive vaccine, and the vaccine platform can be used as SARA- The use of preventive vaccines against COV2, influenza, HPV, VZV, EBV, HSV-2, and HIV, and the use of the vaccine platform as preventive vaccines against HPV and EBV-related tumors.
  • the present invention includes adjuvants used in the vaccine platform, wherein the adjuvants include aluminum adjuvant (Alum), Toll-like receptor 4 activator ligand MPLA, Toll-like receptor 9 ligand, M59, oligodeoxy Nucleotides (CpG-ODN) and Freund's adjuvant.
  • the adjuvants include aluminum adjuvant (Alum), Toll-like receptor 4 activator ligand MPLA, Toll-like receptor 9 ligand, M59, oligodeoxy Nucleotides (CpG-ODN) and Freund's adjuvant.
  • the present invention includes the clinical use of the vaccine platform as HBV therapeutic vaccine combined with hepatitis B virus envelope protein HBsAg vaccine in the treatment of chronic hepatitis B virus infection.
  • the present invention includes the clinical use of the vaccine platform as an HBV therapeutic vaccine combined with nucleoside or nucleotide analogs in the treatment of chronic hepatitis B virus infection.
  • the present invention includes the combined application of the vaccine platform as HBV, influenza, SARA-COV2, HPV, VZV, EBV, HSV-2, HIV preventive or therapeutic vaccine, etc. with antiviral drugs and other therapeutic methods; HBV, HPV, Combination of prophylactic or therapeutic vaccines for EBV-related tumors with antiviral antitumor drugs and therapies.
  • the vaccine platform as HBV, influenza, SARA-COV2, HPV, VZV, EBV, HSV-2, HIV preventive or therapeutic vaccine, etc.
  • the present invention includes the vaccine platform as a multivalent combined vaccine composed of a component of the vaccine and other virus or pathogen or tumor vaccines.
  • the present invention includes any fusion protein vaccine of the vaccine platform and adenovirus vaccine or mRNA vaccine or inactivated vaccine or DNA vaccine of the same virus, pathogen or tumor for immunization in a sequential or simultaneous immunization program.
  • the present invention includes the full-length sequence and any truncated sequence of the vaccine platform antigen, such as SEQ ID NO.76, SEQ ID NO.77, SEQ ID NO.78.
  • the present invention contains any possible mutants of the fusion protein vaccine antigen, including natural point mutations/deletion mutations/truncations, any combination of natural point mutations, subtypes produced after mutation, and the inventors' artificial enhancement
  • the artificial point mutation/deletion mutation/truncated body and other mutant sequences constructed by the vaccine effect such as SEQ ID NO.79, SEQ ID NO.80, SEQ ID NO.81, SEQ ID NO.82, SEQ ID NO.83 , SEQ ID NO.84.
  • any one of the vaccines of the present invention as a component of the vaccine is a multivalent combined vaccine composed of another vaccine of the present invention or other vaccines different from the vaccine of the present invention, such as other viruses or pathogens or tumor vaccines, for example, SARS of the present invention -
  • a multivalent vaccine combining COV-2 fusion protein vaccine and influenza vaccine or other vaccines for example, any vaccine of the present invention and adenovirus vaccine or mRNA vaccine or inactivated vaccine or DNA vaccine of the same virus, pathogen, tumor in sequence Immunization by sequential or simultaneous immunization programs, such as SARS-COV-2 fusion protein vaccine and SARS-COV-2 adenovirus vaccine or mRNA vaccine or inactivated vaccine or DNA vaccine Immunization in sequential or simultaneous immunization programs, such as sequential immunization
  • the sequence can be: 1) first immunize the SARS-COV-2 fusion protein vaccine of the present invention, and then immunize the adenovirus vaccine or mRNA vaccine or inactivated vaccine or DNA vaccine of
  • the present invention includes but is not limited to the following beneficial effects:
  • the antigens of the IFN-tumor or viral antigen-immunoglobulin Fc (or antibody) vaccine platform provided by the present invention can be varied in various components, and can be either tumor-associated antigens or virus-specific antigens, which enhances the The flexibility of using the vaccine platform is enhanced, and the scope of use of this vaccine platform is also enhanced.
  • the IFN-tumor or viral antigen-immunoglobulin Fc (or antibody) vaccine platform provided by the present invention wherein the interferon (IFN) can enhance the migration and maturation of antigen-presenting cells, and increase the co-stimulatory fraction of its expression. , so that it is more conducive to the presentation of antigen to T cells, and the Fc region (or antibody) in the vaccine platform, on the one hand, enhances the molecular weight of the antigen to increase its half-life, and on the other hand, the Fc region (or antibody) can interact with the antigen.
  • the combination of Fc receptors on the surface of presenting cells promotes the processing and uptake of antigens by antigen-presenting cells, which is more conducive to the generation of immune responses.
  • the IFN-tumor or viral antigen-immunoglobulin Fc (or antibody) vaccine platform provided by the present invention is expressed by eukaryotic HEK293 cell expression system.
  • the biological functions are closer to those of natural protein molecules.
  • the IFN-tumor or viral antigen-immunoglobulin Fc (or antibody) vaccine platform provided by the present invention has two structures of homologous or heterodimer, and has better selection for different antigens.
  • the IFN-tumor or viral antigen-immunoglobulin Fc vaccine platform provided by the present invention can stimulate immune responses by fusing Th cell auxiliary epitopes, such as Pan epitopes, using DC targeting antibodies such as anti-PD-L1, etc.
  • Th cell auxiliary epitopes such as Pan epitopes
  • DC targeting antibodies such as anti-PD-L1, etc.
  • Various types of adjuvants can activate DCs to enhance DC cross-presentation and generate strong B-cell and T-cell immune responses.
  • the IFN-tumor or viral antigen-immunoglobulin Fc (or antibody) vaccine platform provided by the present invention can be widely used not only as a preventive vaccine, but also as a therapeutic vaccine.
  • the IFN-tumor or viral antigen-immunoglobulin Fc (or antibody) vaccine platform provided by the present invention can not only be used alone, but also can be combined with existing HBsAg commercial vaccines, combined with nucleoside/nucleotide analogs together as a therapeutic vaccine.
  • the vaccine platform provided by the present invention can be used as a multivalent combined vaccine composed of other viruses or pathogens or tumor vaccines.
  • Any fusion protein vaccine in the vaccine platform provided by the present invention can be immunized with adenovirus vaccine or mRNA vaccine or inactivated vaccine or DNA vaccine of the same virus, pathogen or tumor in a sequential or simultaneous immunization program.
  • Any mutants that may appear of the vaccine platform antigen provided by the present invention including natural point mutation/deletion mutation/increase mutation/truncation, any combination of natural point mutations, subtypes generated after mutation, and the present invention.
  • SEQ ID NO.1 Mouse mIFN ⁇ 4 amino acid sequence (mIFN ⁇ )
  • SEQ ID NO.21 Human IFN ⁇ 2 amino acid sequence (hIFN ⁇ )
  • SEQ ID. NO. 22 Human mutant IFN ⁇ 2 (Q124R) amino acid sequence (hmIFN ⁇ )
  • SEQ ID NO.2 Human IgG1-Fc amino acid sequence
  • SEQ ID NO.3 Th auxiliary epitope Pan HLA DR-binding epitope (PADER) amino acid sequence
  • SEQ ID NO.4 Linker 1 amino acid sequence:
  • SEQ ID NO.5 Signal peptide amino acid sequence:
  • SEQ ID NO.6 Amino acid sequence of HBV Pres1 (subtype ad)
  • SEQ ID NO. 26 Amino acid sequence of HBV Pres1 (subtype ay)
  • SEQ ID NO.7 Amino acid sequence of HBV HBsAg (adr subtype)
  • SEQ ID NO.27 Amino acid sequence of HBV HBsAg (adw subtype)
  • SEQ ID NO.28 Amino acid sequence of HBV HBsAg (ayw subtype)
  • SEQ ID NO. 8 Amino acid sequence of SARS-CoV-2 RBD
  • SEQ ID NO.9 HA amino acid sequence of influenza virus
  • SEQ ID NO. 10 Amino acid sequence of HPV-E7 antigen
  • Murine IFN vaccine mIFN ⁇ -antigen-Fc sequence
  • SEQ ID NO.12 mIFN ⁇ -RBD(SARS-CoV-2)-Fc amino acid sequence in homodimer
  • SEQ ID NO.15 The first chain mIFN ⁇ -Fc-hole amino acid sequence in the heterodimer
  • SEQ ID NO. 16 The second chain Pres1-Fc-knob amino acid sequence in the heterodimeric mIFN ⁇ -Pres1-Fc
  • SEQ ID NO.17 The second chain RBD(SARS-CoV-2)-Fc-knob amino acid sequence in the heterodimeric mIFN ⁇ -RBD(SARA-CoV-2)-Fc
  • SEQ ID NO. 18 Second chain HA-Fc-knob amino acid sequence in heterodimeric mIFN ⁇ -HA-Fc
  • SEQ ID NO. 19 Second chain E7-Fc-knob amino acid sequence in heterodimeric mIFN ⁇ -E7(HPV)-Fc
  • SEQ ID NO.33 The first chain mIFN ⁇ -Fc-hole amino acid sequence in the heterodimer
  • SEQ ID NO. 34 The second chain Pan-Pres1-Fc-knob amino acid sequence in the heterodimeric mIFN-Pan-Pres1-Fc
  • SEQ ID NO.35 The second chain Pan-RBD(SARS-CoV-2)-Fc-knob amino acid sequence in the heterodimeric mIFN ⁇ -Pan-RBD(SARA-CoV-2)-Fc
  • SEQ ID NO. 36 The second chain Pan-HA-Fc-knob amino acid sequence in the heterodimeric mIFN ⁇ -Pan-HA-Fc
  • SEQ ID NO. 37 Second chain Pan-E7-Fc-knob amino acid sequence in heterodimeric mIFN ⁇ -Pan-E7(HPV)-Fc
  • SEQ ID NO.38 amino acid sequence of hIFN ⁇ -Pres1-Fc in homodimer
  • SEQ ID NO.40 amino acid sequence of hIFN ⁇ -HA-Fc in homodimer
  • SEQ ID NO.42 The first chain hIFN-Fc-hole amino acid sequence in the heterodimer
  • SEQ ID NO. 43 The second chain Pres1-Fc-knob amino acid sequence in the heterodimeric hIFN ⁇ -Pres1-Fc
  • SEQ ID NO.44 The second chain RBD(SARS-CoV-2)-Fc-knob amino acid sequence in the heterodimeric hIFN ⁇ -RBD(SARA-CoV-2)-Fc
  • SEQ ID NO.45 Second chain HA-Fc-knob amino acid sequence in heterodimeric hIFN ⁇ -HA-Fc
  • SEQ ID NO. 46 Second chain E7(HPV)-Fc-knob amino acid sequence in heterodimeric hIFN ⁇ -E7(HPV)-Fc
  • SEQ ID NO.47 amino acid sequence of hIFN ⁇ -Pan-Pres1-Fc in homodimer
  • SEQ ID NO.49 amino acid sequence of hIFN ⁇ -Pan-HA-Fc in homodimer
  • SEQ ID NO.51 The first chain hIFN ⁇ -Fc-hole amino acid sequence in the heterodimer
  • SEQ ID NO. 52 The second chain Pan-Pres1-Fc-knob amino acid sequence in the heterodimeric hIFN ⁇ -Pan-Pres1-Fc
  • SEQ ID NO.53 The second chain Pan-RBD(SARS-CoV-2)-Fc-knob amino acid sequence in the heterodimeric hIFN ⁇ -Pan-RBD(SARA-CoV-2)-Fc
  • SEQ ID NO. 54 Second chain Pan-HA-Fc-knob amino acid sequence in heterodimeric hIFN ⁇ -Pan-HA-Fc
  • SEQ ID NO. 55 Second chain Pan-HA-Fc-knob amino acid sequence in heterodimeric hIFN ⁇ -Pan-E7(HPV)-Fc
  • SEQ ID NO.60 The first chain hmIFN-Fc-hole amino acid sequence in the heterodimer
  • SEQ ID NO.61 The second chain Pres1-Fc-knob amino acid sequence in the heterodimeric hmIFN ⁇ -Pres1-Fc
  • SEQ ID NO.62 The second chain RBD(SARS-CoV-2)-Fc-knob amino acid sequence in the heterodimeric hmIFN ⁇ -RBD(SARA-CoV-2)-Fc
  • SEQ ID NO. 63 The second chain HA-Fc-knob amino acid sequence in the heterodimeric hmIFN ⁇ -HA-Fc
  • SEQ ID NO. 64 Second chain HA-Fc-knob amino acid sequence in heterodimeric hmIFN ⁇ -E7(HPV)-Fc
  • SEQ ID NO. 68 hmIFN ⁇ -Pan-E7(HPV)-Fc amino acid sequence in homodimer
  • SEQ ID NO.69 The first chain hmIFN ⁇ 4-Fc-hole amino acid sequence in the heterodimer
  • SEQ ID NO. 70 The second chain Pan-Pres1-Fc-knob amino acid sequence in the heterodimeric hmIFN ⁇ -Pan-Pres1-Fc
  • SEQ ID NO.71 The second chain Pan-RBD(SARS-CoV-2)-Fc-knob amino acid sequence in the heterodimeric hmIFN ⁇ -Pan-RBD(SARA-CoV-2)-Fc
  • SEQ ID NO. 72 The second chain Pan-HA-Fc-knob amino acid sequence in the heterodimeric hmIFN ⁇ -Pan-HA-Fc
  • SEQ ID NO. 73 The second chain Pan-HA-Fc-knob amino acid sequence in the heterodimeric hmIFN ⁇ -Pan-E7(HPV)-Fc
  • SEQ ID NO.20 ScFv (PD-L1) amino acid sequence
  • SEQ ID NO.74 Anti-PD-L1 VH amino acid sequence
  • SEQ ID NO.75 Anti-PD-L1 VL amino acid sequence
  • SEQ ID NO.76 Amino acid sequence of SARS-CoV-2 Spike protein
  • SEQ ID NO.80 Amino acid sequence of SARS-CoV-2 South African mutant strain (B.1.351, Beta) RBD protein
  • SEQ ID NO.81 Amino acid sequence of SARS-CoV-2 Brazilian mutant strain (P.1) RBD protein
  • VZV Envelope glycoprotein E (aa 31-538)
  • FIG. Schematic diagram of the arrangement and combination of vaccine platforms in the form of homodimers in the order of interferon-linked fragment 1-target antigen-immunoglobulin Fc (or antibody);
  • Figure 2 A schematic diagram of the combination of interferon-linked fragment 1-IgG1-hole and target antigen-IgG1-knob (or antibody) in the form of a heterodimer for the vaccine platform;
  • FIG. 1 The schematic diagram of the vaccine platform in the form of heterodimers, respectively, according to the combination of interferon-linked fragment 1-IgG1-knob and target protein-IgG1-hole (or antibody);
  • FIG. Schematic diagram of the orderly arrangement and combination of the vaccine platform in the form of homodimers, interferon-linking fragment 1-Th cell helper epitope-linking fragment 2-target antigen-immunoglobulin Fc (or antibody);
  • Figure 5 Schematic diagram of the combination of interferon-linker fragment 1-IgG1-hole and Th cell helper epitope-linker fragment 2-target antigen-IgG1-knob (or antibody) for the vaccine platform in the form of heterodimers, respectively ;
  • Figure 6 Schematic diagram of the combination of interferon-linker fragment 1-IgG1-knob and Th cell helper epitope-linker fragment 2-target antigen-IgG1-hole (or antibody) for the vaccine platform in the form of heterodimers, respectively .
  • FIG. 8 Compared with free preS1, the fusion proteins preS1-Fc and IFN-preS1-Fc can significantly enhance the immunity of antigen molecules and can cause the production of broad-spectrum neutralizing antibodies.
  • (b) Three HBV genotypes stably carrying mice (n 4) were injected intravenously with serum from mice immunized with IFN ⁇ -Pres1-Fc protein, and the changes of Pres1 antigen in serum were detected 12 hours later.
  • IFN ⁇ -Pres1-Fc can be used as a hepatitis B preventive vaccine.
  • C57/BL6 mice were subcutaneously immunized with free hepatitis B Pres1, Pres1-Fc, IFN ⁇ -Pres1-Fc proteins, and 28 days after inoculation, 1x10 11 ⁇ g of AAV-HBV1.3 virus was infected by tail vein injection.
  • Pres1 levels in serum were detected at the indicated time points.
  • Serum HBsAg levels were detected by Elisa method at 1, 2, 3, and 4 weeks.
  • IFN ⁇ -Pres1-Fc as a therapeutic vaccine for chronic B infection.
  • Pres1 by subcutaneous immunization
  • IFN ⁇ -Pres1-Fc protein immunized once every 2 weeks of isolation for a total of three immunizations.
  • Th cell helper epitopes enhance antibody responses to IFN ⁇ -Pres1-Fc vaccines
  • IFN-Pan-preS1-Fc can significantly enhance the immunogenicity of antigen molecules.
  • IFN ⁇ -Pan-Pres1-Fc as a therapeutic vaccine for chronic B infection.
  • IFN ⁇ -Pres1-Fc combined with HBsAg commercial vaccine breaks immune tolerance against HBsAg and induces HBsAg-HBsAb seroconversion.
  • HBV Carrier mice were subcutaneously immunized with IFN ⁇ -Pres1-Fc and HBsAg commercial vaccine, once every two weeks for a total of three times.
  • IFN ⁇ -RBD(SARS-CoV2)-Fc elicits stronger antibody responses than free SARS-Cov2 RBD protein.
  • FIG. 16 Detection of antiserum RBD-specific antibodies produced by immunization with IFN ⁇ -Pan-RBD (original strain)-Fc and IFN ⁇ -Pan-RBD (SARS-CoV-2 South African mutant)-Fc.
  • IFN ⁇ -Pan-RBD original strain of SARS-CoV-2
  • IFN ⁇ -Pan-RBD SARS-CoV-2 South African mutant
  • FIG. (a) Mouse IFN ⁇ -RBD-Fc, Mouse IFN ⁇ -Pan-RBD-Fc protein SDS-PAGE electrophoresis identification map. (b) Human IFN ⁇ -RBD-Fc, Human IFN ⁇ -Pan-RBD-Fc protein SDS-PAGE identification map.
  • Pan (Pan DR-binding epitope) CD4 T cell helper epitope can further enhance the immunogenicity of IFN ⁇ -RBD-Fc.
  • Mice were inoculated with 10 ⁇ g Mouse IFN ⁇ -RBD-Fc, Mouse IFN ⁇ -Pan-RBD-Fc or 10 ⁇ g Human IFN ⁇ -RBD-Fc, Human IFN ⁇ -Pan-RBD-Fc protein by intramuscular immunization, 14 days after inoculation Give a booster immunization.
  • the mouse serum was collected on the 7th, 14th, and 28th days after immunization, and the levels of RBD-specific antibodies in the mouse serum were detected by ELISA. *,p ⁇ 0.05;****,p ⁇ 0.0001.
  • Aluminum adjuvant can enhance the specific humoral immune response induced by Human IFN ⁇ -RBD-Fc, Human IFN ⁇ -Pan-RBD-Fc protein.
  • C57BL/6 mice were vaccinated with 10 ⁇ g of Human IFN ⁇ -RBD-Fc or Human IFN ⁇ -Pan-RBD-Fc protein on days 0 and 14 with (AL+) or without the addition of aluminum adjuvant (AL-) adjuvant , the mouse serum was collected on the 7th, 14th, and 28th days after inoculation, and the SARS-CoV-2 RBD-specific antibody level in the mouse serum was detected by ELISA.
  • FIG. 20 Intranasal immunization with IFN-Pan-RBD-Fc induces the production of high titers of RBD-specific IgG, IgA neutralizing antibodies.
  • 6-8 weeks C57BL/6 mice were divided into 5 groups, 10 mice in each group, immunized by intranasal immunization with 10 ⁇ g of IFN ⁇ -pan-RBD-Fc or the same moles of RBD, RBD-Fc, IFN ⁇ -RBD -Fc protein, the volume of nasal drops is 10uL/mouse. Mice were immunized on days 0 and 14 using two immunization schedules.
  • mice serum was collected on the 7th, 14th, 21st, 28th, 35th, and 42nd day after immunization, respectively, and the levels of IgG(a) and IgA(b) of the new crown RBD-specific antibodies in the serum of each group were detected by ELISA; Serum for in vitro neutralization experiments of SARS-CoV-2 pseudovirus (c).
  • Statistical method one-way ANOVA, *p ⁇ 0.05 with significant difference, **p ⁇ 0.01, ***p ⁇ 0.001, ****p ⁇ 0.0001.
  • FIG. 21 IFN-Pan-RBD-Fc intranasal immunization of the unadjuvanted new crown vaccine induces the secretion of high titers of RBD-specific IgG and IgA neutralizing antibodies in nasopharyngeal and lung tissues. 6-8 weeks C57BL/6 mice were killed after 28 days of immunization, and the nasal mucosa of the mice was collected and disrupted with a tissue homogenizer. The homogenized liquid was centrifuged at high speed at 13,000 rpm for 10 minutes, and the supernatant was taken as nasal mucosa supernatant (NMDS).
  • NMDS nasal mucosa supernatant
  • IgG (a) and IgA (b) of new coronavirus RBD-specific antibodies in the nasal mucosal supernatant (NMDS) of each group were detected by ELISA; the 28-day serum was taken for in vitro neutralization experiments of SARS-CoV-2 pseudovirus (c ).
  • Statistical method one-way ANOVA, *p ⁇ 0.05 with significant difference, **p ⁇ 0.01, ***p ⁇ 0.001, ****p ⁇ 0.0001.
  • FIG 22 Intranasal immunization with IFN-Pan-RBD-Fc of the unadjuvanted new crown vaccine induces high titers of RBD-specific IgG and IgA antibodies in lung tissue. 6-8 week C57BL/6 mice were sacrificed 28 days after immunization (Figure 19). In the lungs of mice, use a 1ml syringe to suck about 0.8ml of HBSS+100uMEDTA, inject it into the tracheal intubation, gently blow and suck three times, suck out the liquid, and collect it into a centrifuge tube. Obtain 2 ml of lung lavage fluid.
  • mice lung lavage fluid was centrifuged at 500 g for 5 minutes, and the supernatant was the mouse lung lavage fluid (BALF).
  • BALF mouse lung lavage fluid
  • FIG. 23 Her2 vaccine protein expression and purification.
  • the related proteins were expressed and purified in 293F cells, and the size and purity of the proteins were detected by SDS-PAGE and Coomassie brilliant blue staining.
  • FIG. 24 Analysis of the antitumor activity of Her2 vaccines IFN ⁇ -3-Fc and IFN ⁇ -pan-3-Fc.
  • the TUBO breast cancer model mice were constructed, and the tumor size was 50-80 mm3.
  • the mice were injected with the relevant fusion protein intratumorally, once a week, for a total of 3 treatments.
  • the dose of IFN ⁇ -3-Fc is 10ug//dose/mouse, other fusion proteins are administered in equimolar, and CpG is used as an adjuvant. Tumor size was measured and tumor growth curves were drawn.
  • Figure 26 Identification pattern of IFN-HA1-Fc fusion protein by SDS-PAGE electrophoresis.
  • FIG. 27 Inoculation of mice with 10ug IFN by intramuscular immunization
  • Mice were inoculated with tumors or the same molar amount of HA1 protein, and a booster immunization was performed 14 days after the primary vaccination.
  • the mouse serum was collected on the 28th day after immunization, and the level of HA1-specific antibody in the mouse serum was detected by ELISA.
  • Mice were infected with 1000 PFU of A/PR8 influenza virus by nasal infection at 42 days after immunization. From the third day after virus infection, mice were observed and their body weight changes were recorded.
  • (a) The mouse serum was collected 28 days after the primary immunization, and the level of HA1-specific antibody in the mouse serum was detected by ELISA.
  • the vaccine platform of interferon-target antigen-immunoglobulin Fc (or antibody) structural unit due to three structural units, the first structural unit is the interferon part, and the second structural unit is the immunoglobulin Fc region (or antibody) , the third unit is the target antigen.
  • the three structural units can be arranged and combined in any form and the target antigen can be linked to the Th cell helper epitope through the linker sequence 2. Its representation is as follows:
  • Fig. 1 is a schematic diagram showing that the vaccine platform is arranged and combined in the form of homodimer according to the sequence of interferon-linked fragment 1-target antigen-immunoglobulin Fc.
  • FIG. 2 is a schematic diagram of the combination of the interferon-linked fragment 1-IgG1-hole and the target antigen-IgG1-knob, respectively, of the vaccine platform in the form of a heterodimer.
  • Figure 3 Schematic diagram of the combination of the interferon-linked fragment 1-IgG1-knob and the target protein-IgG1-hole in the form of a heterodimer of the vaccine platform.
  • Fig. 4 is a schematic diagram showing that the vaccine platform is arranged and combined in the order of interferon-linker fragment 1-Th cell helper epitope-linker fragment 2-target antigen-immunoglobulin Fc in the form of homodimer.
  • FIG. 5 is a schematic diagram of the combination of interferon-linker fragment 1-IgG1-hole and Th cell helper epitope-linker fragment 2-target antigen-IgG1-knob, respectively, of the vaccine platform in the form of a heterodimer.
  • FIG. 6 is a schematic diagram of the combination of interferon-linker fragment 1-IgG1-knob and Th cell helper epitope-linker fragment 2-target antigen-IgG1-hole, respectively, of the vaccine platform in the form of a heterodimer.
  • the cell suspension to be transfected was counted, and the cell density was expected to be 2.5-3.5 ⁇ 10 6 cells/ml. Then, the cell suspension was centrifuged at 1,000 rpm for 5 min, and the supernatant was discarded.
  • the plasmid/PEI mixture was added to the cell suspension, placed in an incubator at 37° C., 8% CO 2 , and 85 rpm for cultivation, while supplemented with growth factor 50ug/L LONG TM R3IGF-1.
  • Sample preparation transfer the suspended cell culture solution to a 500ml centrifuge bucket, centrifuge at 8,000rpm for 20min, discard the precipitate, filter the supernatant through a 0.45 ⁇ M filter to remove impurities, and then add a final concentration of 0.05% NaN3 to prevent bacteria during purification Pollution.
  • the identification chart of the protein by SDS-PAGE electrophoresis is shown in FIG. 7 .
  • mice C57BL/6 male mice (5-8 weeks old) were purchased from Beijing Weitong Lihua Laboratory Animal Technology Co., Ltd.; goat anti-mouse IgG labeled with horseradish peroxidase (HRP) was purchased from Beijing Kangwei Biotechnology Co., Ltd. Technology Co., Ltd.; 96-well ELISA assay plate was purchased from Corning Costar Company; ELISA color developing solution was purchased from eBioscience Company; the microplate reader SPECTRA max PLUS 384 was purchased from Molecular Company of the United States. The aluminum adjuvant used was purchased from SIGMA Company.
  • Pres1 fusion protein immunized mice 80 pmol IFN-Pres1-Fc or 80 pmol Pres1-Fc, Pres1 protein was mixed with aluminum adjuvant, and then the mice were subcutaneously immunized. Sera from mice were collected by orbital bleeding at the indicated time points for antibody detection.
  • mice with continuous and stable expression of HBV antigens were screened for the test.
  • the selected mice (4 mice/group) were injected intravenously with 200ul/mice of serum from mice immunized with IFN ⁇ -Pres1-Fc. After 12 hours, the mouse serum was collected, and the changes of Pres1 antigen in the mice before and after the injection of antiserum were detected by Elisa.
  • Pres1 (2ug/ml) coating solution was added to Elisa plate (Corning 9018) in a system of 50ul per well, and coated overnight at 4°C. Wash once with PBS, 260ul per well. Block with 5% blocking solution (5% FBS) at 37°C for two hours. Serum samples were diluted with PBS (1:10, 1:100, 1:1000, 1:10000), and 50ul per well was added to a blocked Elisa plate and incubated at 37°C for 1 hour.
  • PBST was washed 5 times, 260ul each time, 50ul enzyme-conjugated secondary antibody (enzyme-conjugated anti-mouse IgG-HRP 1:5000 diluted by PBS) was added to each well, and incubated at 37°C for 1 hour. Washed with PBST five times, per 260ul, OPD TMB 100ul / hole, incubated in the dark at room temperature, waiting chromogenic substrate; 50ul per well of stop solution (2N H 2 SO 4) Color development was stopped microplate reading plate, OD450-630.
  • enzyme-conjugated secondary antibody enzyme-conjugated secondary antibody
  • IFN ⁇ -Pres1-Fc can be used as a hepatitis B preventive vaccine
  • mice C57BL/6 (6-8) weeks male mice were purchased from Beijing Weitong Lihua Biotechnology Co., Ltd., and HBsAg detection kit was purchased from Shanghai Kehua Bioengineering Co., Ltd.
  • AAV-HBV 1.3 virus was purchased from Guangzhou Paizhen Biotechnology Co., Ltd.
  • Other experimental materials are the same as in Example 3.
  • mice were immunized subcutaneously with 80 pmol of different forms of Pres1 vaccine, including pure Pres1, Pes1-Fc, and IFN ⁇ -Pres1-Fc protein.
  • mouse serum was collected and mice were infected with 1x10 11 vg AAV-HBV 1.3 virus, and then collect mouse serum every week to detect anti-Pres1 antibody, HBsAg, and Pres1 antigen in serum for four consecutive weeks.
  • Peripheral HBV-DNA levels in mice were detected at the third week.
  • Pres1 antibody XY007 (4ug/ml) coating solution was added to Elisa plate (Corning 9018) in a system of 50ul per well, and coated overnight at 4°C. Wash once with PBS, 260ul per well. Block with 5% blocking solution (5% FBS) at 37°C for two hours. Serum samples were diluted with PBS (1:10, 1:100), and 50ul per well was added to a sealed Elisa plate. Two duplicate wells were set for each dilution, and incubated at 37°C for 1 hour.
  • mice in the IFN ⁇ -Pres1-Fc immunized group could produce high levels of Pres1 antibodies before virus inoculation, and the antibodies continued to maintain a high level during virus infection as shown in Figure 9(a).
  • IFN-Pres1-Fc vaccine immunization can significantly prevent HBV infection, and the anti-preS1 antibody produced after immunization can quickly and completely clear the preS1 antigen in serum Figure 9(b), and in IFN
  • the majority of mice in the -Pres1-Fc immunized group that received virus infection were negative for peripheral HBsAg Figure 9 (c, d).
  • the above experimental results show that IFN-Pres1-Fc, as a vaccine, can effectively prevent HBV infection, as shown in Figure 9.
  • Example 5 IFN ⁇ -Pres1-Fc as a therapeutic vaccine for chronic B infection
  • C57BL/6 male mice (4 weeks) were purchased from Beijing Weitong Lihua Biotechnology Co., Ltd.
  • AAV-HBV 1.3 was purchased from Guangzhou Paizhen Biotechnology Co., Ltd.
  • the HBsAg detection kit was purchased from Shanghai Kehua Biotechnology Co., Ltd., and other experimental materials were the same as those in Example 4.
  • HBV Carrier mice 4-week-old HBV C57BL/6 mice were injected with 1x10 11 vg AAV-HBV 1.3 virus through the tail vein, and the HBV antigen HBsAg was detected at 1-6 weeks to screen out the stable expression of HBsAg. mice, as HBV Carrier mice for experiments.
  • mice were injected subcutaneously with 80 pmol of different forms of Pres1 protein, once every two weeks for a total of three immunizations.
  • the mouse serum was collected on the 14th day of immunization, and the mouse serum was collected once a week thereafter, and the levels of anti-Pres1 antibody, HBsAG, and Pres1 antigen in the mouse serum were detected by ELISA.
  • the content of HBV-DNA in the peripheral blood of mice was detected after the last blood draw.
  • T cell helper epitopes enhance antibody responses to IFN ⁇ -Pres1-Fc vaccines
  • mice with Pres1 fusion protein subcutaneously immunize mice with 80 pmol of IFN-Pan-Pres1-Fc containing Pan epitope or 80 pmol of IFN-Pan-Pres1-Fc, Pres1-Fc, and Pres1 proteins. Sera from mice were collected by orbital bleeding at the indicated time points for antibody detection.
  • IFN-Pan-preS1-Fc could significantly enhance the immunogenicity of antigen molecules, and could induce the production of broad-spectrum neutralizing antibodies.
  • Example 7 IFN ⁇ -Pan-Pres1-Fc as a therapeutic vaccine for chronic B infection
  • mice C57BL/6 male mice (4 weeks) were purchased from Beijing Weitong Lihua Biotechnology Co., Ltd. AAV-HBV
  • HBsAg detection kit was purchased from Shanghai Kehua Biotechnology Co., Ltd., and other experimental materials were the same as those in Example 4.
  • HBV Carrier mice 4-week-old HBV C57BL/6 mice were injected with 1x10 11 vg AAV-HBV 1.3 virus through the tail vein, and the HBV antigen HBsAg was detected at 1-6 weeks to screen out the stable expression of HBsAg. mice, as HBV Carrier mice for experiments.
  • mice were injected subcutaneously with 80 pmol of different forms of Pres1 protein, once every two weeks for a total of three immunizations.
  • the mouse serum was collected on the 14th day of immunization, and the mouse serum was collected once a week thereafter, and the levels of anti-Pres1 antibody, HBsAG, and Pres1 antigen in the mouse serum were detected by ELISA.
  • the content of HBV-DNA in the peripheral blood of mice was detected after the last blood draw.
  • mice We detected the serum preS1 antigen, as well as the changes of Pres1 antibody and HBsAg in the serum of mice immunized with IFN-Pan-Pres1-Fc vaccine for hours. The results showed that after immunization with the IFN-Pan-Pres1-Fc vaccine, the mice produced high levels of anti-Pres1 antibodies as shown in Figure 12(a). And the preS1 antigen in the serum can be completely cleared as shown in Figure 12(b), and the HBsAg in the serum also decreased to a certain extent 12(c), while the untreated control group and the Pres1 vaccine immunization group had no therapeutic effect. . And HBV DNA was also significantly decreased in the IFN ⁇ -Pan-Pres1-Fc immunized group, as shown in Figure 12(d).
  • Example 8 IFN ⁇ -Pan-Pres1-Fc combined with HBsAg commercial vaccine breaks immune tolerance against HBsAg and induces HBsAg-HBsAb seroconversion.
  • C57BL/6 male mice (4 weeks) were purchased from Beijing Weitong Lihua Biotechnology Co., Ltd.
  • AAV-HBV 1.3 was purchased from Guangzhou Paizhen Biotechnology Co., Ltd.
  • the HBsAg detection kit was purchased from Shanghai Kehua Biotechnology Co., Ltd.
  • the Anti-HBsAg kit was purchased from Beijing Wantai Biopharmaceutical Co., Ltd.
  • the commercial HBsAg vaccine was purchased from Amy Hanxin Vaccines (Dalian) Co., Ltd.
  • Other experimental materials are the same as in Example 7.
  • HBV Carrier mice 4-week-old HBV C57BL/6 mice were injected with 1x10 11 vg AAV-HBV 1.3 virus through the tail vein, and the HBV antigen HBsAg was detected at 1-6 weeks to screen out the stable expression of HBsAg. mice, as HBV Carrier mice for experiments.
  • mice The screened HBV Carrier mice were immunized with 80 pmol IFN ⁇ -pan-Pres1-Fc, and 2 ⁇ g of commercial HBsAg vaccine was immunized twice in a row with an interval of 14 days.
  • Mouse serum was collected 14 days after the first immunization, and then every week, and the changes of anti-Pres1, Pres1, anti-HBsAg, and HBsAg in serum were detected. And in the last mouse serum collection, the level of HBV-DNA in serum was detected.
  • mice Balb/c male and female mice (6-8 weeks) were purchased from Beijing Weitong Lihua Laboratory Animal Technology Co., Ltd., and the SARS-CoV-2 RBD protein used was purchased from Beijing Keyue Zhongkai Biotechnology Co., Ltd. 293-hACE2 cells were provided by Professor Zhang Zheng (Shenzhen Third People's Hospital). The Luciferase Reporter Assay Kit was purchased from promega.
  • IFN ⁇ -RBD SARS-Cov-2
  • RBD-Fc 10ug RBD protein
  • 10ug RBD protein 10ug RBD protein was mixed with aluminum adjuvant and then immunized mice subcutaneously.
  • the serum of the mice was collected by orbital blood sampling for the detection of new crown-specific antibodies.
  • RBD Antigen coating solution was added to Elisa plate (Corning 9018) in a system of 100ul per well, and coated overnight at 4°C. Wash once with PBS, 260ul per well. Block with 100 ul of 5% blocking solution (5% FBS) at 37°C for two hours. Serum samples were diluted with PBS (1:10, 1:100, 1:1000, 1:10000, 1:100000%), and 100ul per well was added to a blocked Elisa plate and incubated at 37°C for 1 hour.
  • PBST was washed 5 times, 260ul each time, 100ul enzyme-conjugated secondary antibody (enzyme-conjugated anti-mouse IgG-HRP 1:5000 diluted by PBS) was added to each well, and incubated at 37°C for 1 hour. Wash five times with PBST, 260ul each time, add substrate TMB 100ul/well, incubate at room temperature for 15 minutes in the dark, and wait for the substrate to develop color. Add 50ul of stop solution (2N H 2 SO 4 ) to each well to stop the color development, read the plate with a microplate reader, OD450-630.
  • enzyme-conjugated secondary antibody enzyme-conjugated secondary antibody
  • the titer calculation method is to select the maximum dilution ratio with a positive result, multiply the (0.1) dilution ratio by the OD value/Cutoff value corresponding to the dilution ratio, and the obtained value is the antibody titer corresponding to the serum.
  • Example 10 Detection of antiserum RBD-specific antibodies produced by FN ⁇ -Pan-RBD (original strain)-Fc and IFN ⁇ -RBD (SARS-CoV-2 South African mutant)-Fc immunization.
  • mice Balb/c male and female mice (6-8 weeks) were purchased from Beijing Weitong Lihua Laboratory Animal Technology Co., Ltd., and the RBD protein of the original strain of SARS-CoV-2 was purchased from Beijing Keyue Zhongkai Biotechnology Co., Ltd.
  • the RBD protein of the South African mutant strain of SARS-CoV-2 used was purchased from Beijing Yiqiao Shenzhou Technology Co., Ltd.
  • IFN ⁇ -Pan-RBD original strain
  • IFN ⁇ -RBD SARS-CoV-2 South African mutant
  • IFN ⁇ -Pan-RBD original strain
  • IFN ⁇ -Pan-RBD SARS-CoV-2 South African mutant
  • IFN ⁇ -Pan-RBD SARS-CoV-2 South African mutant
  • the ELISA results for the South African mutant RBD also showed that IFN ⁇ -Panan-RBD (original strain)-Fc immunized mice, the same as IFN ⁇ -Pan-RBD (SARS-CoV-2 South African mutant)-Fc immunized mice, induced The generated antibodies could all bind to the South African mutant RBD, and their binding abilities were comparable (Fig. 16d).
  • mice C57BL/6 female mice (6-8 weeks) were purchased from Beijing Weitong Lihua Biotechnology Co., Ltd.
  • SARS-CoV-2 RBD protein used in ELISA was purchased from Beijing Keyue Zhongkai Biotechnology Co., Ltd.
  • Mouse IFN ⁇ -RBD-Fc, Mouse IFN ⁇ -Pan-RBD-Fc, Human IFN ⁇ -RBD-Fc, Human IFN ⁇ - Pan-RBD-Fc proteins were all produced in our laboratory, and other experimental materials were the same as those in Example 3.
  • mice (2) immunize mice with vaccine protein.
  • 10 ⁇ g Mouse IFN ⁇ -RBD-Fc, Mouse IFN ⁇ -Pan-RBD-Fc or 10 ⁇ g Human IFN ⁇ -RBD-Fc, Human IFN ⁇ -Pan-RBD-Fc vaccine proteins were mixed with 20 ⁇ g aluminum adjuvant overnight, respectively, and then immunized by muscle.
  • Mice were vaccinated and given a booster 14 days after the primary vaccination.
  • Mouse serum was collected on days 7, 14, and 28 after immunization, and the levels of RBD-specific antibodies in mouse serum were detected by ELISA.
  • the titer calculation method selects the maximum dilution ratio that the result is positive, and multiplies the (X) dilution ratio with the corresponding OD value/Cutoff value (0.1) of the dilution ratio, and the obtained value is the antibody titer corresponding to this serum.
  • Pan (Pan DR-binding epitope) CD4 T cell helper epitope can enhance the immunity of Mouse IFN ⁇ -RBD-Fc and Human IFN ⁇ -RBD-Fc.
  • the experimental results showed that Mouse IFN ⁇ -Pan-RBD-Fc was comparable to Mouse IFN ⁇ -RBD-Fc, Human IFN ⁇ -Pan-RBD-Fc on day 7, day 14, and day 28 after vaccine protein immunization.
  • pan epitope can lead to higher production of RBD-specific antigens.
  • mice C57BL/6 female mice (6-8 weeks) were purchased from Beijing Weitong Lihua Biotechnology Co., Ltd.; SARS-CoV-2 RBD protein used in ELISA was purchased from Beijing Keyue Zhongkai Biotechnology Co., Ltd. Human IFN ⁇ -RBD-Fc and Human IFN ⁇ -Pan-RBD-Fc proteins for immunization were produced in our laboratory. Other experimental materials are the same as in Example 3.
  • mice were inoculated with 10 ⁇ g Human IFN ⁇ -RBD-Fc and Human IFN ⁇ -Pan-RBD-Fc protein by intramuscular immunization, 14 days after inoculation A booster immunization.
  • the mouse serum was collected on the 7th, 14th, and 28th day after immunization, and the RBD-specific antibody level in the mouse serum was detected by ELISA.
  • RBD Antigen coating solution was added to Elisa plate (Corning 9018) in a system of 100ul per well, and coated overnight at 4°C. Wash once with PBS, 260ul per well. Block with 100 ul of 5% blocking solution (5% FBS) at 37°C for two hours. Serum samples were diluted with PBS (1:10, 1:100, 1:1000, 1:10000, 1:100000%), and 100ul per well was added to a blocked Elisa plate and incubated at 37°C for 1 hour.
  • the titer calculation method is to select the maximum dilution ratio with a positive result, multiply the (0.1) dilution ratio by the OD value/Cutoff value corresponding to the dilution ratio, and the obtained value is the antibody titer corresponding to the serum.
  • the application of aluminum adjuvant can enhance the immunogenicity of Human IFN ⁇ -RBD-Fc and Human IFN ⁇ -Pan-RBD-Fc proteins.
  • unadjuvanted Human IFN ⁇ -RBD-Fc, Human IFN ⁇ -Pan-RBD-Fc vaccines can generate high titer antibody responses, aluminum adjuvant-assisted Human IFN ⁇ -RBD-Fc and Human IFN ⁇ -Pan-RBD-Fc proteins Compared with the adjuvant-free adjuvant group, the RBD-specific antibody response level could be further improved on the 7th, 14th, and 28th days after inoculation.
  • the experimental animals were purchased from Beijing Weitong Lihua Laboratory Animal Co., Ltd., and the animals used were 6-8 week old C57BL/6 mice; animal certificate number: No.110011200106828974; RBD protein for immunization was purchased from Beijing Keyue Zhongkai Biotechnology Co., Ltd.; RBD-Fc, IFN ⁇ -RBD-Fc and IFN-pan-RBD-Fc proteins were produced by our laboratory; all adjuvants were purchased from SERVA, Germany; horseradish peroxidase (HRP) labeled goat anti-mouse IgG was purchased from Beijing Kangwei Biotechnology Co., Ltd.; 96-well ELISA assay plate was purchased from Corning Costar Company; ELISA color developing solution was purchased from eBioscience Company; the microplate reader SPECTRA max PLUS 384 was purchased from American Molecular Company; Purchased from Beijing Haonuosi Technology Co., Ltd.
  • RBD protein for immunization was purchased from Beijing Keyue Zhongka
  • mice 6-8 weeks old mice were divided into 5 groups, 10 mice in each group, immunized by intranasal immunization with 10ug of IFN ⁇ -pan-RBD-Fc or the same moles of RBD, RBD-Fc, IFN ⁇ -RBD-Fc protein. , the intranasal dose is 10uL per mouse.
  • Mice were immunized on days 0 and 14 using two immunization schedules.
  • the mouse serum was collected on the 7th, 14th, 21st, 28th, 35th, and 42nd day after immunization, and the SARS-CoV-2 RBD-specific antibody content in the serum of each group was detected by ELISA; -2 Pseudovirus neutralization experiments in vitro.
  • RBD, RBD-Fc protein twice intranasal immunization can cause a certain degree of antibody response
  • serum IgG and IgA levels induced by IFN ⁇ -pan-RBD-Fc at the same time point after intranasal immunization twice All were significantly higher than the RBD, RBD-Fc and IFN-RBD-Fc groups.
  • the pseudovirus neutralization test results showed that compared with the RBD and RBD-Fc immunization groups, IFN-RBD-Fc could induce higher levels of neutralizing antibodies. produce.
  • mice 6-8 weeks old mice were divided into 4 groups, 5 mice in each group, immunized by intranasal immunization with 10ug of IFN ⁇ -pan-RBD-Fc or the same moles of RBD, RBD-Fc, IFN ⁇ -RBD-Fc protein. , the dose is 10uL per mouse.
  • Mice were immunized on days 0 and 14 using two immunization schedules. On the 28th day after immunization, the mouse nasal mucosa supernatant and lung lavage fluid were collected, and the serum levels of SARS-CoV-2 RBD-specific antibodies in each group were detected by ELISA. SARS-CoV-2 neutralization experiment in serum and nasal mucosa supernatant.
  • mice Obtaining the Nasal Mucosa Supernatant and Bronchoalveolar Lavage Fluid of Mice: After the mice were dormantly sacrificed, the nasal mucosa of the mice was taken and crushed with a tissue homogenizer. The homogenized liquid was centrifuged at high speed at 13,000 rpm for 10 minutes, and the supernatant was taken as nasal mucosa supernatant (NMDS). In the lungs of mice, use a 1ml syringe to suck about 0.8ml of HBSS+100uMEDTA, inject it into the tracheal intubation, gently blow and suck three times, suck out the liquid, and collect it into a centrifuge tube.
  • NMDS nasal mucosa supernatant
  • the mouse lung lavage fluid was centrifuged at 500 g for 5 minutes, and the supernatant was the mouse lung lavage fluid (BALF), which was precipitated as lymphocytes in the mouse lung for further analysis.
  • BALF mouse lung lavage fluid
  • IFN ⁇ -pan-RBD-Fc protein can induce strong local IgG antibody response and IgA mucosal immunity in nasal mucosa after two intranasal immunization.
  • the reaction intensity of IFN ⁇ -pan-RBD-Fc protein was stronger than that of RBD and RBD-Fc groups.
  • the results of pseudovirus neutralization experiments showed that the IFN ⁇ -pan-RBD-Fc protein immunization group could induce higher titers of neutralizing antibodies at the nasal mucosa.
  • IFN ⁇ -pan-RBD-Fc fusion protein twice intranasally immunized C57BL/6 mice also caused strong IgG antibody and IgA antibody secretion in local lung tissue.
  • Her2 belongs to the HER family of type I transmembrane growth factor receptors and consists of an extracellular ligand-binding domain, a transmembrane domain, and an intracellular tyrosine kinase domain. Once the ligand binds to the extracellular domain, the HER protein will dimerize and trans-phosphorylate its intracellular domain. The phosphorylated tyrosine residues can bind to a variety of intracellular signaling molecules and activate downstream signaling pathways. Regulate gene transcription. The regulated genes are mostly related to the processes of cell proliferation, survival, differentiation, angiogenesis, invasion and metastasis.
  • the extracellular segment of Her2 protein is large, with more than 600 amino acids, and can be divided into four domains, namely domains I, II, III, and IV.
  • the currently approved Trastuzumab mainly binds to domain IV
  • Pertuzumab mainly binds to domain II
  • the polypeptide vaccine E75 that is undergoing clinical trials targets domain III. It shows that there are some important sites in different domains, which may mediate anti-tumor effects.
  • the patent research selects tumor antigen Her2 as the target, constructs IFN-Her2-Fc and IFN-Pan-Her2-Fc to construct fusion protein vaccines, and analyzes in vivo antitumor activity and vaccine immunity. active.
  • BALB/c female mice (6-8 weeks) were purchased from Beijing Weitong Lihua Biotechnology Co., Ltd.; TUBO cells were derived from TCGA; other materials were the same as in Example 3.
  • expression plasmids were constructed for the domains III and IV of the extracellular segment of mouse Her2, (respectively denoted as: IFN ⁇ -3-Fc, IFN ⁇ -pan-3-Fc, IFN ⁇ -pan-4-Fc and IFN ⁇ -4-Fc ), expressed and purified related proteins in human 293F cell line. Protein size and purity were identified by SDS-PAGE and Coomassie brilliant blue staining.
  • TUBO is a breast cancer cell line derived from BALB-NeuT mice used to study the growth and treatment of Her2-positive breast cancers. Antitumor activity of IFN ⁇ in protein was detected using TUBO tumor.
  • TUBO breast cancer model mice 5*10 5 TUBO cells were subcutaneously inoculated into BALB/C mice, and the tumor size was 50-80 mm 3 , once a week, for a total of 3 treatments.
  • the dose of IFN ⁇ -3-Fc was 10ug/mouse, other drugs were administered in equimolar, and CpG was used as an adjuvant. Tumor size was measured and tumor growth curves were drawn.
  • mice 6-8 week old BALB/C female mice were subcutaneously vaccinated with unadjuvanted HER2 domain V fusion protein vaccines 4-Fc, IFN ⁇ -4-Fc and IFN ⁇ -pan-4-Fc once a week for a total of 3 times.
  • the immunization dose was IFN ⁇ -4-Fc 10ug/mouse, and other proteins were inoculated in equimolar.
  • Venous blood was collected 14 days and 21 days after immunization, and the antibody level of Her2-specific IgG was detected by ELISA.
  • the size of the Her2 fusion protein basically meets the expectations, and the purity meets the experimental requirements.
  • IFN ⁇ -3-Fc (62.6kDa), IFN ⁇ -pan-3-Fc (63.9kDa), IFN ⁇ -pan-4-Fc (74.9kDa) and IFN ⁇ -4-Fc (73.6kDa).
  • the protein Under non-deformed conditions, the protein is in a dimerized state, which is consistent with the automatic dimerization of Fc fragments.
  • IFN ⁇ and pan helps to increase the immunogenicity of 4-Fc and induce a stronger antigen-specific antibody response, so IFN-Pan-HER2-Fc and IFN-Pan-HER2-Fc are potential antibodies against Effective tumor vaccine for Her2-positive tumors.
  • mice BALB/c female mice (6-8 weeks) were purchased from Beijing Weitong Lihua Biotechnology Co., Ltd.; HA1 (A/PR8) protein used in ELISA was purchased from Beijing Yiqiao Shenzhou Biotechnology Co., Ltd.; HA1 used for immunization Protein protein (A/PR8) was purchased from Beijing Yiqiao Shenzhou Biotechnology Co., Ltd., IFN ⁇ -HA1-Fc was produced in our laboratory; H1N1 (A/PR8) influenza virus used to infect mice was produced in our laboratory; other experiments The materials are the same as those in Example 3. method:
  • mice were immunized with HA1 and IFN ⁇ -HA1-Fc protein. After mixing 10ug IFN ⁇ -HA1-Fc or the same molar amount of HA1 protein with 20 ⁇ g aluminum adjuvant overnight, mice were inoculated by intramuscular immunization, and a booster immunization was performed 14 days after the primary inoculation. The mouse serum was collected on the 28th day after immunization, and the level of HA1-specific antibody in the mouse serum was detected by ELISA.
  • HA1 (2 ⁇ g/ml) coating solution was added to Elisa plate (Corning 9018) in a system of 100ul per well, and coated overnight at 4°C. Wash once with PBS, 260ul per well. Block with 100 ul of 5% blocking solution (5% FBS) at 37°C for two hours. Serum samples were diluted with PBS (1:10, 1:100, 1:1000, 1:10000, 1:100000%), and 100ul per well was added to a blocked Elisa plate and incubated at 37°C for 1 hour.
  • the titer calculation method is to select the maximum dilution ratio with a positive result, multiply the (0.1) dilution ratio by the OD value/Cutoff value corresponding to the dilution ratio, and the obtained value is the antibody titer corresponding to the serum.
  • IFNa-Pan-VZV-gE-Fc The design, plasmid construction and protein purification of IFNa-Pan-VZV-gE-Fc, IFNa-Pan-EBV-gp350-Fc, and IFNa-Pan-HSV-2-gD-Fc proteins are as shown in Examples 1 and 2.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Animal Behavior & Ethology (AREA)
  • Veterinary Medicine (AREA)
  • Public Health (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Organic Chemistry (AREA)
  • Virology (AREA)
  • Epidemiology (AREA)
  • Microbiology (AREA)
  • Oncology (AREA)
  • General Chemical & Material Sciences (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Immunology (AREA)
  • Mycology (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Engineering & Computer Science (AREA)
  • Genetics & Genomics (AREA)
  • Communicable Diseases (AREA)
  • Molecular Biology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Biotechnology (AREA)
  • Zoology (AREA)
  • Biomedical Technology (AREA)
  • Dermatology (AREA)
  • Cell Biology (AREA)
  • Biophysics (AREA)
  • Biochemistry (AREA)
  • General Engineering & Computer Science (AREA)
  • Wood Science & Technology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Physics & Mathematics (AREA)
  • Plant Pathology (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Toxicology (AREA)
  • Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
  • Peptides Or Proteins (AREA)

Abstract

本发明涉及融合蛋白疫苗平台的构建与应用。本发明提供一种疫苗，其包含含有干扰素-靶抗原-免疫球蛋白Fc区（或抗体）和Th细胞辅助表位的融合蛋白。本发明还涉及含有干扰素-靶抗原-免疫球蛋白Fc（或抗体）区和Th细胞辅助表位的融合蛋白用于制备预防性或治疗性组合物的用途。本发明的疫苗可以通过真核细胞表达系统产生，制备野生型和各种突变抗原的疫苗，通过皮下/肌肉或者鼻腔等免疫途径进行接种，能够引起机体强烈的免疫应答。本发明的疫苗可以作为预防性或治疗性疫苗来使用。

Description

融合蛋白疫苗平台的构建与应用 技术领域
本发明属于基因工程和生物医学技术领域，具体涉及一种疫苗，例如包含含有干扰素-靶抗原-免疫球蛋白Fc区(抗体)为主要骨架的融合蛋白的疫苗。本发明的疫苗可以作为疫苗平台，用于预防乙肝病毒(HBV)感染、HPV、EBV、HIV、SARA-COV2、流感病毒感染和HPV、EBV相关肿瘤的发生以及治疗慢性乙型肝炎(chronic hepatitis B，CHB)感染、治疗HBV、HP、EBV相关肿瘤的用途。
背景技术
全球约有2.57亿慢性病毒感染者，每年约有8.87万人死于HBV所引起的终末期肝病，包括肝功能衰竭、肝硬化、肝细胞肝癌 [1-3]。肝硬化病人中大约有30％由HBV所引起，大约40％的肝细胞肝癌(HCC)由HBV所引起 [4]。乙型肝炎病毒感染依旧是全球重大公共健康问题。但是目前仍旧没有有效治疗慢性乙型肝炎的策略，现有的HBV治疗方式主要包括抗病毒药物(核苷/核苷酸类似物)和干扰素，它们虽然有一定的治疗效果疗效，但是通常不能诱导有效的免疫应答，从而无法彻底清除HBV感染；而且长期用药导致的副作用较大，抗病毒药物也会产生耐药性。慢性HBV感染是威胁人类身体健康的主要疾病之一，探索慢性乙型肝炎有效的免疫治疗策略迫在眉睫，研制慢性乙型肝炎的治疗性疫苗具有十分重要的社会意义和经济意义。
季节性流感每年会导致1-4百万人产生严重的疾病并且导致20-50万人死亡 [5]。疫苗途径是预防和控制流感的最佳方式，疫苗能够减少疾病的发生并且能够降低感染的严重性，特别是在幼儿和老年人这种有流感并发症的风险群体。即使当前批准的流感疫苗能够在健康的年轻成人体内产生良好的保护效果，但是依旧存在一些需要解决的问题。比如有些疫苗的产生途径依赖于鸡胚，如灭火的流感疫苗和减毒的流感疫苗，这些疫苗的一个缺点是如果流行的毒株是禽类起源的，同时疾病的流行会导致疫苗和鸡胚的需求增加，这会导致鸡胚的供应出现问题 [6]。另一个不利因素是这些疫苗的产生需要大量的时间。对于流感病毒来说老年人更容易产生严重的综合征，同时标准的疫苗对于老年人来说一般效果十分不理想，老年人对于免疫系统会随着年龄的增长而逐渐衰弱 [7]。鉴于当前流感疫苗所遇见的问题，针对于流感病毒的流行当前迫切需要一种有较强免疫原性，不依赖于鸡胚，可以快速产生的流感疫苗。
严重急性呼吸系统综合冠状病毒2(SARS-CoV-2)是引起2019冠状病毒病(COVID)大流行的病原体，SARS-CoV-2引起的临床症状主要包括无症状感染、轻度流感样症状、肺炎和严重的急性呼吸窘迫综合征，在严重的情况下会引起感染患者的死亡。现在对于新型冠状病毒还没有特效药出现，疫苗是控制与终止新冠大流行的基本对策 [9]。另外，新冠病毒突变体的出现对现有的候选疫苗和疫情的控制都提出了新的挑战 [10]，因此一种强效并且可以对新冠病毒突变体同样发挥作用的疫苗在当前疫情状况下是迫切需要的。
当把抗原与免疫球蛋白Fc区相连接时，抗原的半衰期会得到明显的延长，并且免疫球蛋白Fc区可以与抗原提呈细胞表面的Fc受体相结合从而促进抗原提呈细胞对抗原的加工与提成 [11-13]。I型干扰素作为一种抗病毒细胞因子有许多生物活性，其中一个就包含对免疫细胞的刺激作用 [14]。IFNα可以强有力的诱导人的DC细胞的分化与活化 [15]。I型干扰素作用于未成熟的DCs后，能够促进DCs细胞表面的MHC分子和共刺激分子的表达，如：MHC class I、CD80和CD86，进而增强DCs活化T细胞的能力 [16-18]。有报道指出，在牛痘病毒和淋巴细胞性脉络丛脑炎病毒(LCMV)感染后，I型干扰素能够促进DCs的抗原递呈能力 [19-21]。此外，I型干扰素作用于DCs后能够通过上调趋化因子受体的表达，促进DCs迁移到淋巴结，进而促进T细胞的激活 [22,23]。最近越来越多的研究表明I型干扰素可作为免疫佐剂来使用。Le Bon等人的研究表明当以弱的免疫原免疫小鼠时，I型干扰素在小鼠体内展现出了强劲的免疫佐剂作用诱导产生了长效抗体和免疫记忆 [24]，作者也发现I型干扰素发挥作用的主要细胞类群为DC细胞。同时利用抗体，将疫苗靶向递送给DC，刺激DC活化和交叉递呈功能，将进一步增强疫苗的活性和效价。
本发明存在提供增强机体对病毒、细菌或肿瘤抗原的反应的疫苗平台的需要。
发明内容
疫苗是预防和控制重大突发传染病的有效方式，疫苗的形式有各种类型，其中重要的一类为蛋白亚单位疫苗。一般来说单纯的蛋白亚单位疫苗，其免疫原性一般较差，这往往限制了蛋白亚单位疫苗的使用。因此一种通用型蛋白亚单位疫苗平台是十分迫切需要的。依据免疫球蛋白Fc区域和I型干扰素对免疫系统的影响，发明人特提出干扰素α-病毒抗原、细菌或肿瘤-免疫球蛋白Fc区融合蛋白疫苗平台来增强机体对病毒、细菌或肿瘤抗原的反应。本发明提供了一种I型干扰素-蛋白抗原-免疫球蛋白Fc疫苗平台，其中I型干扰素可以作用于抗原提呈细胞使其成熟与迁移从而更好的发挥抗原 提呈与激活T细胞的作用，另一方面该疫苗平台的Fc部分可以与抗原提成细胞表面的Fc受体相结合从未增强抗原提成细胞对抗原的摄取从而进一步帮助抗原提呈细胞发挥作用。发明人提出融合Th细胞辅助表位，可以进一步提升I型干扰素-蛋白抗原-免疫球蛋白Fc疫苗的免疫应答效果，是该疫苗的重要组成成分。发明人提出可以利用anti-PD-L1等抗体替换Fc，将疫苗靶向递送给DC，刺激DC活化和交叉递呈功能，将进一步增强疫苗的活性和效价。本发明作为一种新型疫苗平台可以作为病毒感染、细菌感染或肿瘤等疾病的预防性和治疗性疫苗来使用。
在一些实施方案中，本发明提供一种疫苗，其包含含有干扰素-靶抗原-免疫球蛋白Fc区(或抗体)的融合蛋白(附加Th表位)。在一些实施方案中，本发明还提供含有干扰素-靶抗原-免疫球蛋白Fc区(或抗体)的融合蛋白(附加Th表位)用于制备预防性或治疗性组合物或试剂盒(例如药物或疫苗组合物或试剂盒)的用途。本发明的疫苗可以通过真核细胞表达系统产生，通过皮下/肌肉或者鼻腔等免疫途径进行接种。对于本发明的融合多肽，其中作为结构单元的抗体(简称Ab)没有特别限制，可以包括例如完整抗体或抗体的片段，例如抗体重链和轻链，或单链抗体，可以为DC靶向活化抗体，包括anti-PD-L1，anti-DEC205,anti-CD80/86等抗体。
在一些实施方案中，本文所述靶抗原没有特别限制，可以为任何适当抗原。在一些实施方案中，本文所述靶抗原可以为例如肿瘤抗原和/或病原体抗原(如病毒或细菌抗原)。在一些实施方案中，本文所述靶抗原可以为例如肿瘤抗原，例如肿瘤细胞高表达蛋白分子，例如，人表皮生长因子受体2(human epidermal growth factor receptor 2，HER2/neu)、表皮生长因子(Epidermal growth factor receptor，EGFR)。
在一些实施方案中，本发明提供的疫苗中使用的靶抗原可以为例如与野生型不同的突变的靶抗原。在一些实施方案中，本文所述靶抗原可以为例如肿瘤抗原和/或病原体抗原(如病毒或细菌抗原)的突变体。在一些实施方案中，所述靶抗原可以为例如SARS-COV-2病毒S蛋白全长或S1区，例如所述靶抗原可以为SEQ ID NO.76或SEQ ID NO.77所示的抗原。在本文中，野生型靶抗原是指由野生型基因编码的病毒或其他感染原或肿瘤所表达的具有免疫原性的蛋白(野生型基因指自然界中占多数的等位基因，在生物学实验中常作为标准对照基因)，如来源于SARS-CoV-2最初野生型毒株的Spike蛋白(S蛋白)。在本文中，突变的靶抗原(突变体)是指由野生型基因突变而来的突变型基因编码的突变病毒株所表达的突变病毒蛋白，例如已发现的不同突变新冠病毒S蛋白的点突变包含：NTD区域69-70缺失、Y144缺失、242-244缺失、L18F、D80A、D215、R246I突变，RBD区域K417、E484、N501Y等突变、L452R突变、T478K 突变，D614G，H655Y突变。例如这些点突变以不同的组合方式存在于来源于英国B.1.1.7(Alpha)突变株、南非B.1.351(Beta)突变株、巴西P1(Gamma)突变株、印度B.1.617、B.1.617.1(Kappa)、B.1.617.2(Delta)、B.1.617.3突变株、加州B.1.429突变株等突变的新冠病毒。在一些实施方案中，突变的靶抗原可以包括例如天然点突变/缺失突变/增多突变/截短体、人工点突变/缺失突变/增多突变/截短体、天然或人工突变的任何组合、突变后所产生的亚型，其中所述靶抗原可以是肿瘤抗原，病原体抗原，如病毒(例如SARS-COV-2)或细菌抗原。在一些实施方案中，本发明提供的疫苗中使用的靶抗原是突变的病毒抗原，例如所述突变的病毒抗原可以为SARS-COV-2的突变体，包括例如SARS-COV-2蛋白(例如S蛋白、N蛋白、M蛋白、E蛋白中的一个或多个)的天然点突变/缺失突变/增多突变/截短体、人工点突变/缺失突变/增多突变/截短体、天然或人工突变的任何组合、突变后所产生的亚型，例如所述突变的病毒抗原可以为S蛋白全长、S1区、RBD区的突变体，例如所述突变的病毒抗原可以包含SARS-COV-2的S蛋白的下述一个或多个突变：NTD区域69-70缺失、Y144缺失、242-244缺失、L18F、D80A、D215、R246I突变，RBD区域K417、E484、N501Y突变、L452R突变、T478K突变，D614G，H655Y突变，例如例如所述突变的病毒抗原可以包含存在于来源于英国B.1.1.7(Alpha)突变株、南非B.1.351(Beta)突变株和巴西P1(Gamma)突变株、印度B.1.617、B.1.617.1(Kappa)、B.1.617.2(Delta)、B.1.617.3突变株、加州B.1.429突变株中的突变，例如所述突变的病毒抗原可以包含SEQ ID NO.79、SEQ ID NO.80、SEQ ID NO.81中任一个所示突变的突变体，例如所述突变的病毒抗原可以为包含SEQ ID NO.79、SEQ ID NO.80、SEQ ID NO.81、SEQ ID NO.82、SEQ ID NO.83、SEQ ID NO.84中任一个所示序列的突变体。在本文中，除非另有明确说明或上下文明确限制，本文提及靶抗原一般包括野生型靶抗原和突变体靶抗原。
本发明的目的是提供了一种疫苗平台，所述疫苗平台由干扰素(IFN)与肿瘤、细菌或者病毒抗原(乙肝病毒Pres1抗原，SARS-COV2 RBD抗原，流感HA抗原，人乳头状瘤病毒HPV E7抗原，乙肝病毒表面抗原(HBsAg)抗原或肽段，带状疱疹病毒(VZV)gE抗原，埃博斯坦-巴尔病毒(EBV)EBNA1/LMP2/gp350，单纯疱疹病毒2(HSV-2)gD抗原，艾滋病毒(HIV)gp120抗原-免疫球蛋白Fc区域(或抗体)组成(附加Th表位)。所述融合蛋白可以为同源或者异源二聚体蛋白。当所述融合蛋白为二聚体时，作为结构单元的干扰素、靶抗原、免疫球蛋白Fc区(或抗体Ab)可以存在于第一多肽链和/或第二多肽链中，各结构单元的存在方式没有特别限制，例如可以同时存在于一条链中，也可以在一条链中存在任意一个或多个结构单元，而在另 一条链中存在另外的一个或多个结构单元。
本发明所述干扰素可以选自I干扰素、II型干扰素和III型干扰素，例如IFN-α、IFN-β、IFN-γ、IFN-λ1(IL-29)、IFN-λ2(IL-28a)、IFN-λ(IL-28b)和IFN-ω；所述IFN可来自人源或鼠源；优选I型干扰素IFN-α(SEQ ID NO.1、SEQ ID NO.21、SEQ ID NO.22)。
本发明所述免疫球蛋白Fc区可选自IgG1、IgG2、IgG3和IgG4/或IgM的恒定区氨基酸序列，优选IgG1(SEQ ID NO.2、SEQ ID NO.23、SEQ ID NO.24)。
本发明的融合多肽还可以任选地包含一个或多个Th细胞辅助表位和/或连接片段(接头)。例如，当所述融合蛋白为二聚体时，任选地所述融合蛋白还可以在同源二聚体或者异源二聚体的任意一条或两条链(即第一多肽链和/或第二多肽链)中包含一个或多个Th细胞辅助表位和/或连接片段。如本领域技术人员已知的，在融合蛋白的各个结构单元之间可以通过适当的连接片段(接头)连接。可以在本发明的疫苗中使用的连接片段没有特别限制，可以是本领域已知的任何适当肽片段。本发明所述各个结构单元的连接片段可以为柔性多肽序列，可以为连接片段1和2，例如SEQ ID NO.4、SEQ ID NO.25氨基酸序列所示。
本发明所述各个结构单元组成的多肽序列N端均含有能够促进蛋白分泌的相应信号肽，例如SEQ ID NO.5氨基酸序列所示。
本发明所描述的优选抗原包括乙肝Pres1抗原，包含ad亚型(SEQ ID NO.6)ay亚型(SEQ ID NO.26)，HBV HBsAg抗原(各个亚型和肽段)，包含adr亚型(SEQ ID NO.7)，adw亚型(SEQ ID NO.27)，ayw亚型(SEQ ID NO.28)，SARA-COV2 RBD抗原(SEQ ID NO.8)，流感病毒HA抗原(SEQ ID NO.9)，HPV E7抗原(SEQ ID NO.10)。疱疹病毒VZV-gE抗原(SEQ ID NO.91)，EBV-gp350抗原(SEQ ID NO.92)，HSV-2-gD抗原(SEQ ID NO.93)。
本发明所描述的同源二聚体蛋白包含第一条多肽与第二条多肽，第一条多肽与第二条多肽完全相同。第一条多肽与第二条多肽从N段到C段顺序为IFN-肿瘤或者病毒抗原(乙肝Pres1抗原，SARS-COV2 RBD抗原，流感HA抗原，HPV E7抗原，HBsAg抗原，VZV-gE抗原，EBV EBNA1/LMP2/gp350，HSV-2-gD抗原，HIV gp120抗原)-免疫球蛋白Fc区域；或含有Pan表位的多肽。包含SEQ ID NO.11、12、13、14、29、30、31、32、38、39、40、47、48、49、50、51、56、57、59、58、65、66、67、68所示氨基酸序列。
本发明所述异源二聚体包含第一条多肽和第二条对肽，所述第一条多肽与第二条多肽不为同一条多肽，所述第一条多肽从C段到N端分别为IFN-免疫球蛋白Fc区域， 包含SEQ ID NO.15、33、42、51、60、69所示核苷酸序列；所述第二条多肽从C段到N段分别为肿瘤或者病毒抗原(乙肝Pres1抗原，SARS-COV2 RBD抗原，流感HA抗原，HPV E7抗原，VZV-gE抗原，EBV EBNA1/LMP2/gp350，HSV-2-gD抗原，HIV gp120抗原)-免疫球蛋白Fc区域；包含SEQ ID NO.16、17、18、19、34、35、36、37、43、44、45、46、52、53、54、55、61、62、63、64、70、71、72、73所示氨基酸序列。
本发明还提供编码上述IFN-肿瘤或者病毒抗原乙肝Pres1抗原，HBsAg抗原或肽段，SARS-COV2 RBD抗原，流感HA抗原，HPV E7抗原，VZV-gE抗原，EBV EBNA1/LMP2/gp350，HSV-2-gD抗原，HIV gp120抗原-免疫球蛋白Fc疫苗平台的氨基酸序列。
本发明还涉及编码所述疫苗平台、融合蛋白的核苷酸片段。
本发明还涉及所述的融合蛋白或疫苗平台的制备方法，例如所述的制备方法包括如下步骤：
(1)构建包含所述编码所述融合蛋白或或疫苗平台的编码基因的表达载体，优选的，所述的表达载体是pEE12.4表达载体；
(2)通过瞬时转染宿主细胞的方法构建包含所述表达载体的宿主细胞，优选的，所述的宿主细胞是293F细胞；
(3)培养所述宿主细胞并收集细胞上清；
(4)通过ProteinA/G的亲和层析柱纯化蛋白纯化所述融合蛋白或疫苗平台。
本发明另外包含该疫苗平台的应用，该疫苗平台可作为乙肝预防性疫苗的使用，该疫苗平台作为乙肝治疗性疫苗的使用，该疫苗平台作为流感预防性疫苗的使用，该疫苗平台作为SARA-COV2、流感、HPV、VZV、EBV、HSV-2、HIV预防性疫苗的使用，述疫苗平台作为HPV、EBV相关肿瘤预防性疫苗的使用。
本发明包含所述疫苗平台所使用的佐剂，其中所述佐剂包括铝佐剂(Alum)、Toll样受体4激活剂配体MPLA、Toll样受体9配体，M59，寡聚脱氧核苷酸(CpG-ODN)和弗氏佐剂。
本发明包含所述疫苗平台作为HBV治疗性疫苗联合乙型肝炎病毒包膜蛋白HBsAg疫苗进行慢性乙型肝炎病毒感染治疗过程中的临床使用。
本发明包含所述疫苗平台作为HBV治疗性疫苗联合核苷或者核苷酸类似物进行慢性乙型肝炎病毒感染治疗过程中的临床使用。
本发明包含所述疫苗平台作为HBV、流感、SARA-COV2、HPV、VZV、EBV、 HSV-2、HIV预防性或治疗性疫苗等与抗病毒药物和其他治疗方法的联合应用；HBV、HPV、EBV相关肿瘤预防性或治疗性疫苗与抗病毒抗肿瘤药物和疗法的联合应用。
本发明包含所述疫苗平台作为疫苗的一个成分与其他病毒或病原体或肿瘤疫苗组成的多价联用疫苗。
本发明包含所述疫苗平台的任何一种融合蛋白疫苗与同一病毒、病原体或肿瘤的腺病毒疫苗或mRNA疫苗或灭活疫苗或DNA疫苗按照先后顺序或同时免疫程序进行免疫。
本发明包含所述疫苗平台抗原的全长序列和任何截短体序列，例如SEQ ID NO.76、SEQ ID NO.77、SEQ ID NO.78。
本发明含所述融合蛋白疫苗抗原的可能出现的任何突变体，包括天然点突变/缺失突变/截短体、天然点突变的任何组合、突变后所产生的亚型、以及本专利发明人为增强疫苗效果所构建的人工的点突变/缺失突变/截短体等突变序列，例如SEQ ID NO.79、SEQ ID NO.80、SEQ ID NO.81、SEQ ID NO.82、SEQ ID NO.83、SEQ ID NO.84。
本发明任何一种疫苗作为疫苗的一个成分与本发明的另一种疫苗或不同于本发明疫苗的其他疫苗如其他病毒或病原体或肿瘤疫苗组成的多价联用疫苗，例如，本发明的SARS-COV-2融合蛋白疫苗与流感疫苗或其他疫苗联用的多价疫苗，例如本发明任何一种疫苗与同一病毒、病原体、肿瘤的腺病毒疫苗或mRNA疫苗或灭活疫苗或DNA疫苗按照先后顺序或同时免疫程序进行免疫，例如SARS-COV-2融合蛋白疫苗与SARS-COV-2的腺病毒疫苗或mRNA疫苗或灭活疫苗或DNA疫苗按照先后顺序或同时免疫程序进行免疫，例如先后免疫顺序可以为：1)先免疫接种本发明的SARS-COV-2融合蛋白疫苗，后免疫SARS-COV-2的腺病毒疫苗或mRNA疫苗或灭活疫苗或DNA疫苗；2)先免疫SARS-COV-2的腺病毒疫苗或mRNA疫苗或灭活疫苗或DNA疫苗，后免疫ARS-COV-2融合蛋白疫苗；3)SARS-COV-2融合蛋白疫苗与SARS-COV-2的腺病毒疫苗或mRNA疫苗或灭活疫苗或DNA疫苗同时免疫。如本领域已知的，在联合使用时，联合使用的疫苗可以制备为方便使用的试剂盒。
相比与现有技术本发明包括但不限于下列有益效果：
1.本发明提供的IFN-肿瘤或者病毒抗原-免疫球蛋白Fc(或抗体)疫苗平台其抗原可以是多种组分变化的，既可以为肿瘤相关抗原也可以为病毒特异型抗原，这增强了疫苗平台使用的灵活性，也增强了此疫苗平台的使用范围。
2.本发明提供的IFN-肿瘤或者病毒抗原-免疫球蛋白Fc(或抗体)疫苗平台，其中的干扰素(IFN)可以增强抗原提呈细胞的迁移与成熟，使其表达的共刺激分增加， 从而使其更有利于向T细胞呈递抗原，同时疫苗平台中的Fc区域(或抗体)，一方面增强了抗原的分子量从而增加了其半衰期，另一方面Fc区(或抗体)可以与抗原提呈细胞表面的Fc受体相结合从而促进抗原提呈细胞对抗原的加工与提成，从而更有利于免疫反应的产生。
3.本发明提供的IFN-肿瘤或者病毒抗原-免疫球蛋白Fc(或抗体)疫苗平台，有真核HEK293细胞表达系统表达，HEK293细胞表达的蛋白不管在分子结构还是理化特征和蛋白修饰及蛋白的生物学功能都与天然蛋白分子更加接近。
4.本发明提供的IFN-肿瘤或者病毒抗原-免疫球蛋白Fc(或抗体)疫苗平台，具有同源或者异源二聚体的两种结构，针对不同的抗原有更优秀的选择。
5.本发明提供的IFN-肿瘤或者病毒抗原-免疫球蛋白Fc疫苗平台，可以通过融合Th细胞辅助表位，例如Pan表位、利用anti-PD-L1等DC靶向抗体、添加刺激免疫应答的各类佐剂，从而激活DC增强DC交叉递呈，产生强大的B细胞、T细胞免疫应答。
6.本发明提供的IFN-肿瘤或者病毒抗原-免疫球蛋白Fc(或抗体)疫苗平台，使用范围广泛不仅可以作为预防性疫苗使用，同时也可以作为治疗性疫苗使用。
7.本发明提供的IFN-肿瘤或者病毒抗原-免疫球蛋白Fc(或抗体)疫苗平台，不仅可以单独使用，同时也可以联合现有的HBsAg商业化疫苗、联合核苷/核苷酸类似物一起作为治疗性疫苗来使用。
8.本发明提供的疫苗平台作为疫苗的一个成分可以与其他病毒或病原体或肿瘤疫苗组成的多价联用疫苗。
9.本发明提供的疫苗平台中任何一种融合蛋白疫苗可以与同一病毒、病原体或肿瘤的腺病毒疫苗或mRNA疫苗或灭活疫苗或DNA疫苗按照先后顺序或同时免疫程序进行免疫。
10.本发明所提供的疫苗平台抗原的全长序列和任何截短体序列。
11.本发明所提供的疫苗平台抗原的可能出现的任何突变体，包括天然点突变/缺失突变/增多突变/截短体、天然点突变的任何组合、突变后所产生的亚型、以及本专利发明人为增强疫苗效果所构建的人工的点突变/缺失突变/增多突变/截短体等突变序列。
本文涉及的序列信息：
1.单元构件序列：
SEQ ID NO.1：小鼠mIFNα4氨基酸序列(mIFNα)
Figure PCTCN2021103931-appb-000001
SEQ ID NO.21：Human IFNα2氨基酸序列(hIFNα)
Figure PCTCN2021103931-appb-000002
SEQ ID.NO.22：Human mutant IFNα2(Q124R)氨基酸序列(hmIFNα)
Figure PCTCN2021103931-appb-000003
SEQ ID NO.2：人IgG1-Fc氨基酸序列
Figure PCTCN2021103931-appb-000004
SEQ ID No.23：异源二聚体Fc-hole
Figure PCTCN2021103931-appb-000005
SEQ ID No.24：异源二聚体Fc-knob
Figure PCTCN2021103931-appb-000006
Figure PCTCN2021103931-appb-000007
SEQ ID NO.3：Th辅助表位Pan HLA DR-binding epitope(PADER)氨基酸序列
Figure PCTCN2021103931-appb-000008
SEQ ID NO.4：Linker 1氨基酸序列：
Figure PCTCN2021103931-appb-000009
SEQ ID NO.25：Linker 2氨基酸序列：
Figure PCTCN2021103931-appb-000010
SEQ ID NO.5：信号肽氨基酸序列：
Figure PCTCN2021103931-appb-000011
SEQ ID NO.6：HBV Pres1(ad亚型)的氨基酸序列
Figure PCTCN2021103931-appb-000012
SEQ ID NO.26：HBV Pres1(ay亚型)的氨基酸序列
Figure PCTCN2021103931-appb-000013
SEQ ID NO.7：HBV HBsAg(adr亚型)的氨基酸序列
Figure PCTCN2021103931-appb-000014
SEQ ID NO.27：HBV HBsAg(adw亚型)的氨基酸序列
Figure PCTCN2021103931-appb-000015
SEQ ID NO.28：HBV HBsAg(ayw亚型)的氨基酸序列
Figure PCTCN2021103931-appb-000016
SEQ ID NO.8：SARS-CoV-2 RBD的氨基酸序列
Figure PCTCN2021103931-appb-000017
SEQ ID NO.9：流感病毒的HA氨基酸序列
Figure PCTCN2021103931-appb-000018
SEQ ID NO.10：HPV-E7抗原的氨基酸序列
Figure PCTCN2021103931-appb-000019
2.鼠源IFN疫苗mIFNα-抗原-Fc序列：
SEQ ID NO.11：同源二聚体中mIFNα-Pres1-Fc氨基酸序列
Figure PCTCN2021103931-appb-000020
Figure PCTCN2021103931-appb-000021
SEQ ID NO.12：同源二聚体中mIFNα-RBD(SARS-CoV-2)-Fc氨基酸序列
Figure PCTCN2021103931-appb-000022
SEQ ID NO.13：同源二聚体中mIFNα-HA-Fc氨基酸序列
Figure PCTCN2021103931-appb-000023
Figure PCTCN2021103931-appb-000024
SEQ ID NO.14：同源二聚体中mIFNα-E7(HPV)-Fc氨基酸序列
Figure PCTCN2021103931-appb-000025
SEQ ID NO.15：异源二聚体中第一条链mIFNα-Fc-hole氨基酸序列
Figure PCTCN2021103931-appb-000026
SEQ ID NO.16：异源二聚体mIFNα-Pres1-Fc中第二条链Pres1-Fc-knob氨基酸序列
Figure PCTCN2021103931-appb-000027
SEQ ID NO.17：异源二聚体mIFNα-RBD(SARA-CoV-2)-Fc中第二条链RBD(SARS-CoV-2)-Fc-knob氨基酸序列
Figure PCTCN2021103931-appb-000028
SEQ ID NO.18：异源二聚体mIFNα-HA-Fc中第二条链HA-Fc-knob氨基酸序列
Figure PCTCN2021103931-appb-000029
SEQ ID NO.19：异源二聚体mIFNα-E7(HPV)-Fc中第二条链E7-Fc-knob氨基酸序列
Figure PCTCN2021103931-appb-000030
3.鼠源IFN含Pan表位的疫苗IFNα-Pan-抗原-Fc序列：
SEQ ID NO.29：同源二聚体中mIFNα-Pan-Pres1-Fc氨基酸序列
Figure PCTCN2021103931-appb-000031
SEQ ID NO.30：同源二聚体中mIFNα-Pan–RBD(SARS-CoV-2)-Fc氨基酸序列
Figure PCTCN2021103931-appb-000032
SEQ ID NO.31：同源二聚体中mIFNα-Pan-HA-Fc氨基酸序列
Figure PCTCN2021103931-appb-000033
Figure PCTCN2021103931-appb-000034
SEQ ID NO.32：同源二聚体中mIFNα-Pan-E7(HPV)-Fc氨基酸序列
Figure PCTCN2021103931-appb-000035
SEQ ID NO.33：异源二聚体中第一条链mIFNα-Fc-hole氨基酸序列
Figure PCTCN2021103931-appb-000036
SEQ ID NO.34：异源二聚体mIFN-Pan-Pres1-Fc中第二条链Pan-Pres1-Fc-knob氨基酸 序列
Figure PCTCN2021103931-appb-000037
SEQ ID NO.35：异源二聚体mIFNα-Pan-RBD(SARA-CoV-2)-Fc中第二条链Pan-RBD(SARS-CoV-2)-Fc-knob氨基酸序列
Figure PCTCN2021103931-appb-000038
SEQ ID NO.36：异源二聚体mIFNα-Pan-HA-Fc中第二条链Pan-HA-Fc-knob氨基酸序列
Figure PCTCN2021103931-appb-000039
Figure PCTCN2021103931-appb-000040
SEQ ID NO.37：异源二聚体mIFNα-Pan-E7(HPV)-Fc中第二条链Pan-E7-Fc-knob氨基酸序列
Figure PCTCN2021103931-appb-000041
4.人源IFN疫苗hIFNα-抗原-Fc序列：
SEQ ID NO.38：同源二聚体中hIFNα-Pres1-Fc氨基酸序列
Figure PCTCN2021103931-appb-000042
SEQ ID NO.39：同源二聚体中hIFNα-RBD(SARS-CoV-2)-Fc氨基酸序列
Figure PCTCN2021103931-appb-000043
Figure PCTCN2021103931-appb-000044
SEQ ID NO.40：同源二聚体中hIFNα-HA-Fc氨基酸序列
Figure PCTCN2021103931-appb-000045
SEQ ID NO.41：同源二聚体中hIFNα-E7(HPV)-Fc氨基酸序列、
Figure PCTCN2021103931-appb-000046
Figure PCTCN2021103931-appb-000047
SEQ ID NO.42：异源二聚体中第一条链hIFN-Fc-hole氨基酸序列
Figure PCTCN2021103931-appb-000048
SEQ ID NO.43：异源二聚体hIFNα-Pres1-Fc中第二条链Pres1-Fc-knob氨基酸序列
Figure PCTCN2021103931-appb-000049
SEQ ID NO.44：异源二聚体hIFNα-RBD(SARA-CoV-2)-Fc中第二条链RBD(SARS-CoV-2)-Fc-knob氨基酸序列
Figure PCTCN2021103931-appb-000050
SEQ ID NO.45：异源二聚体hIFNα-HA-Fc中第二条链HA-Fc-knob氨基酸序列
Figure PCTCN2021103931-appb-000051
Figure PCTCN2021103931-appb-000052
SEQ ID NO.46：异源二聚体hIFNα-E7(HPV)-Fc中第二条链E7(HPV)-Fc-knob氨基酸序列
Figure PCTCN2021103931-appb-000053
5.人源IFN含Pan表位的疫苗IFNα-Pan-抗原-Fc序列：
SEQ ID NO.47：同源二聚体中hIFNα-Pan-Pres1-Fc氨基酸序列
Figure PCTCN2021103931-appb-000054
SEQ ID NO.48：同源二聚体中hIFNα-Pan-RBD(SARS-CoV-2)-Fc氨基酸序列
Figure PCTCN2021103931-appb-000055
SEQ ID NO.49：同源二聚体中hIFNα-Pan-HA-Fc氨基酸序列
Figure PCTCN2021103931-appb-000056
SEQ ID NO.50：同源二聚体中hIFNα-Pan-E7(HPV)-Fc氨基酸
Figure PCTCN2021103931-appb-000057
Figure PCTCN2021103931-appb-000058
SEQ ID NO.51：异源二聚体中第一条链hIFNα-Fc-hole氨基酸序列
Figure PCTCN2021103931-appb-000059
SEQ ID NO.52：异源二聚体hIFNα-Pan-Pres1-Fc中第二条链Pan-Pres1-Fc-knob氨基酸序列
Figure PCTCN2021103931-appb-000060
SEQ ID NO.53：异源二聚体hIFNα-Pan-RBD(SARA-CoV-2)-Fc中第二条链Pan-RBD(SARS-CoV-2)-Fc-knob氨基酸序列
Figure PCTCN2021103931-appb-000061
Figure PCTCN2021103931-appb-000062
SEQ ID NO.54：异源二聚体hIFNα-Pan-HA-Fc中第二条链Pan-HA-Fc-knob氨基酸序列
Figure PCTCN2021103931-appb-000063
SEQ ID NO.55：异源二聚体hIFNα-Pan-E7(HPV)-Fc中第二条链Pan-HA-Fc-knob氨基酸序列
Figure PCTCN2021103931-appb-000064
6.人源突变IFN疫苗hmIFNα-Pan-抗原-Fc序列：
SEQ ID NO.56：同源二聚体中hmIFNα-Pres1-Fc氨基酸序列
Figure PCTCN2021103931-appb-000065
SEQ ID NO.57：同源二聚体中hmIFNα-RBD(SARS-CoV-2)-Fc氨基酸序列
Figure PCTCN2021103931-appb-000066
SEQ ID NO.58：同源二聚体中hmIFNα-HA-Fc氨基酸序列
Figure PCTCN2021103931-appb-000067
Figure PCTCN2021103931-appb-000068
SEQ ID NO.59：同源二聚体中hmIFNα-E7(HPV)-Fc氨基酸序列
Figure PCTCN2021103931-appb-000069
SEQ ID NO.60：异源二聚体中第一条链hmIFN-Fc-hole氨基酸序列
Figure PCTCN2021103931-appb-000070
SEQ ID NO.61：异源二聚体hmIFNα-Pres1-Fc中第二条链Pres1-Fc-knob氨基酸序列
Figure PCTCN2021103931-appb-000071
Figure PCTCN2021103931-appb-000072
SEQ ID NO.62：异源二聚体hmIFNα-RBD(SARA-CoV-2)-Fc中第二条链RBD(SARS-CoV-2)-Fc-knob氨基酸序列
Figure PCTCN2021103931-appb-000073
SEQ ID NO.63：异源二聚体hmIFNα-HA-Fc中第二条链HA-Fc-knob氨基酸序列
Figure PCTCN2021103931-appb-000074
SEQ ID NO.64：异源二聚体hmIFNα-E7(HPV)-Fc中第二条链HA-Fc-knob氨基酸序列
Figure PCTCN2021103931-appb-000075
Figure PCTCN2021103931-appb-000076
7.人源突变IFN含Pan表位疫苗hmIFNα-Pan表位-抗原-Fc序列
SEQ ID NO.65：同源二聚体中hmIFNα-Pan-Pres1-Fc氨基酸序列
Figure PCTCN2021103931-appb-000077
SEQ ID NO.66：同源二聚体中hmIFNα-Pan–RBD(SARS-CoV-2)-Fc氨基酸序列
Figure PCTCN2021103931-appb-000078
SEQ ID NO.67：同源二聚体中hmIFNα-Pan-HA-Fc氨基酸序列
Figure PCTCN2021103931-appb-000079
SEQ ID NO.68：同源二聚体中hmIFNα-Pan-E7(HPV)-Fc氨基酸序列
Figure PCTCN2021103931-appb-000080
SEQ ID NO.69：异源二聚体中第一条链hmIFNα4-Fc-hole氨基酸序列
Figure PCTCN2021103931-appb-000081
Figure PCTCN2021103931-appb-000082
SEQ ID NO.70：异源二聚体hmIFNα-Pan-Pres1-Fc中第二条链Pan-Pres1-Fc-knob氨基酸序列
Figure PCTCN2021103931-appb-000083
SEQ ID NO.71：异源二聚体hmIFNα-Pan-RBD(SARA-CoV-2)-Fc中第二条链Pan-RBD(SARS-CoV-2)-Fc-knob氨基酸序列
Figure PCTCN2021103931-appb-000084
SEQ ID NO.72：异源二聚体hmIFNα-Pan-HA-Fc中第二条链Pan-HA-Fc-knob氨基酸序列
Figure PCTCN2021103931-appb-000085
Figure PCTCN2021103931-appb-000086
SEQ ID NO.73：异源二聚体hmIFNα-Pan-E7(HPV)-Fc中第二条链Pan-HA-Fc-knob氨基酸序列
Figure PCTCN2021103931-appb-000087
8.替代Fc的抗体序列
SEQ ID NO.20：ScFv(PD-L1)氨基酸序列
Figure PCTCN2021103931-appb-000088
SEQ ID NO.74：Anti-PD-L1 VH氨基酸序列
Figure PCTCN2021103931-appb-000089
SEQ ID NO.75：Anti-PD-L1 VL氨基酸序列
Figure PCTCN2021103931-appb-000090
Figure PCTCN2021103931-appb-000091
9.其它病毒抗原序列
SEQ ID NO.76 SARS-CoV-2 Spike蛋白的氨基酸序列
Figure PCTCN2021103931-appb-000092
SEQ ID NO.77 SARS-CoV-2 S1蛋白的氨基酸序列
Figure PCTCN2021103931-appb-000093
Figure PCTCN2021103931-appb-000094
SEQ ID NO.78 SARS-CoV-2原始毒株RBD蛋白的氨基酸序列
Figure PCTCN2021103931-appb-000095
SEQ ID NO.79 SARS-CoV-2英国突变株(B.1.1.7,Alpha)RBD蛋白的氨基酸序列
Figure PCTCN2021103931-appb-000096
SEQ ID NO.80 SARS-CoV-2南非突变株(B.1.351,Beta)RBD蛋白的氨基酸序列
Figure PCTCN2021103931-appb-000097
SEQ ID NO.81 SARS-CoV-2巴西突变株(P.1)RBD蛋白的氨基酸序列
Figure PCTCN2021103931-appb-000098
SEQ ID NO.82 SRAS-CoV-2加州突变株(B.1.429)RBD蛋白的氨基酸序列
Figure PCTCN2021103931-appb-000099
Figure PCTCN2021103931-appb-000100
SEQ ID NO.83 SRAS-CoV-2印度B.1.617、、B.1.617.1(Kappa)、B.1.617.3突变株RBD蛋白的氨基酸序列
Figure PCTCN2021103931-appb-000101
SEQ ID NO.84 SRAS-CoV-2印度二代B.1.617.2(Delta)突变株RBD蛋白的氨基酸序列
Figure PCTCN2021103931-appb-000102
10.其它肿瘤抗原序列：
实施例中涉及的鼠源Her2胞外段结构域II、III和IV的氨基酸序列：
SEQ ID NO.85 Mouse Her2-extracellular domain 2：
Figure PCTCN2021103931-appb-000103
SEQ ID NO.86 Mouse Her2-extracellular domain 3：
Figure PCTCN2021103931-appb-000104
SEQ ID NO.87 Mouse Her2-extracellular domain 4：
Figure PCTCN2021103931-appb-000105
实施例中涉及的人源Her2胞外段结构域II、III和IV的氨基酸序列：
SEQ ID NO.88 Human Her2-extracellular domain 2：
Figure PCTCN2021103931-appb-000106
Figure PCTCN2021103931-appb-000107
SEQ ID NO.89 Human Her2-extracellular domain 3：
Figure PCTCN2021103931-appb-000108
SEQ ID NO.90 Human Her2-extracellular domain 4：
Figure PCTCN2021103931-appb-000109
11.实施例中涉及的疱疹病毒抗原序列：
SEQ ID NO.91 VZV Envelope glycoprotein E(aa 31-538)
Figure PCTCN2021103931-appb-000110
SEQ ID NO.92 EBV Envelope glycoprotein GP350(aa 1-425)
Figure PCTCN2021103931-appb-000111
Figure PCTCN2021103931-appb-000112
SEQ ID NO.93 HSV-2 Envelope glycoprotein gD(aa 26-339)
Figure PCTCN2021103931-appb-000113
附图说明
图1.疫苗平台以同源二聚体的形式，按照干扰素-连接片段1-靶抗原-免疫球蛋白Fc(或抗体)顺序排列组合的示意图；
图2.疫苗平台以异源二聚体的形式，按照干扰素-连接片段1-IgG1-hole、靶抗原-IgG1-knob(或抗体)组合的示意图；
图3.疫苗平台以异源二聚体的形式，分别按照干扰素-连接片段1-IgG1-knob组合和靶蛋白-IgG1-hole(或抗体)的示意图；
图4.疫苗平台以同源二聚体的形式，按照干扰素-连接片段1-Th细胞辅助表位-连接片段2-靶抗原-免疫球蛋白Fc(或抗体)顺序排列组合的示意图；
图5.疫苗平台以异源二聚体的形式，分别按照干扰素-连接片段1-IgG1-hole和Th细胞辅助表位-连接片段2-靶抗原-IgG1-knob(或抗体)组合的示意图；
图6.疫苗平台以异源二聚体的形式，分别按照干扰素-连接片段1-IgG1-knob和Th细胞辅助表位-连接片段2-靶抗原-IgG1-hole(或抗体)组合的示意图。
图7.Pres1-Fc、IFN-Pres1-Fc非变性蛋白SDS-PAGE电泳鉴定图
图8.相比于游离preS1，融合蛋白preS1-Fc和IFN-preS1-Fc，能显著增强抗原分子的免疫性，并能引起广谱的中和性抗体的产生。(a)对C57/BL6(n＝8/组)小鼠采用皮下免疫的方式接种游离的乙肝Pres1、Pres1-Fc、IFNα-Pres1-Fc蛋白，在指定的时间采用Elisa的方法检测血清中Pres1特异性抗体的水平。(b)三种HBV基因型稳定携带小鼠(n＝4)通过静脉注射的方式注射来自IFNα-Pres1-Fc蛋白免疫小鼠的血清，12小时后检测血清中Pres1抗原的变化。
图9.IFNα-Pres1-Fc可应用为乙型肝炎预防性疫苗。C57/BL6小鼠皮下免疫接种游 离的乙肝Pres1、Pres1-Fc、IFNα-Pres1-Fc蛋白，接种28天时，采用尾静脉注射的方式感染1x10 11μg的AAV-HBV1.3病毒。(a)病毒接种前及病毒接种后第1、2、3、4周血清Anti-Pres1水平。(b)在指定时间点检测血清中Pres1的水平。(c)通过Elisa的方式检测第1、2、3、4周血清HBsAg的水平。(d)AAV-HBV1.3病毒接种后，HBsAg阳性小鼠的比例。
图10.IFNα-Pres1-Fc作为慢性乙型感染的治疗性疫苗。C57/BL6小鼠通过尾静脉注射的方式感染1x10 11μg的AAV-HBV1.3病毒，感染6周后筛选出稳定感染的小鼠(n＝8/组)，通过皮下免疫的方式接种重组Pres1、IFNα-Pres1-Fc蛋白，每隔离2周免疫一次一共免疫三次。(a)血清中Anti-Pres1抗原的检测；(b)血清中Pres1抗原的检测；(c)小鼠血清中HBV相关抗原HBsAg的水平检测
图11.Th细胞辅助表位增强IFNα-Pres1-Fc疫苗的抗体应答
相比于IFN-preS1-Fc，IFN-Pan-preS1-Fc能显著增强抗原分子的免疫原性。对C57/BL6(n＝8/组)小鼠采用皮下免疫的方式接种不含铝佐剂的乙肝Pres1、Pres1-Fc、IFNα-Pres1-Fc蛋白，在指定的时间采用Elisa的方法检测血清中Pres1特异性抗体的水平。
图12.IFNα-Pan-Pres1-Fc作为慢性乙型感染的治疗性疫苗。C57/BL6小鼠通过尾静脉注射的方式感染1x10 11μg的AAV-HBV1.3病毒，感染6周后筛选出稳定感染的小鼠(n＝8/组)，通过皮下免疫的方式接种重组Pres1、IFNα-Pres1-Fc蛋白，每隔离2周免疫一次一共免疫三次。(a)血清中Anti-Pres1抗原的检测；(b)血清中Pres1抗原的检测；(c)小鼠血清中HBV相关抗原HBsAg的水平检测；(d)通过QPCR的方式检测小鼠血清中HBV-DNA的水平。
图13.IFNα-Pres1-Fc联合HBsAg商业化疫苗，打破针对于HBsAg引起的免疫耐受，诱导HBsAg-HBsAb血清学转换。HBV Carrier小鼠皮下免疫IFNα-Pres1-Fc与HBsAg商业化疫苗，每两周免疫一次一共免疫三次。(a)HBV Carrier鼠中血清中Pres1的水平，(b)HBsAg的水平，(c)血清中Anti-Pres1的水平(d)，血清中Anti-HBsAg的水平(e)血清中HBV-DNA的水平。***,p<0.001
图14.IFNα-RBD(SARS-CoV2)-Fc相比游离的SARS-Cov2 RBD蛋白，可以引起更强的抗体反应。对Balb/c(n＝8/组)小鼠采用皮下免疫的方式接种游离的SARS-Cov-2RBD、RBD-Fc、IFNα-RBD-Fc蛋白，在指定的时间采用Elisa的方法检测血清中SARS-Cov2 S蛋白特异性抗体的水平。****，p<0.0001。
图15.IFNα-RBD(SARS-CoV2)-Fc免疫小鼠后产生高滴度抗病毒血清，在体外 细胞实验中，可以完全阻断SARS-CoV2假病毒感染。
图16.IFNα-Pan-RBD(原始毒株)-Fc与IFNα-Pan-RBD(SARS-CoV-2南非突变株)-Fc免疫所产生的抗血清RBD特异性抗体检测。(a)IFNα-Pan-RBD(SARS-CoV-2原始毒株)-Fc SDS-PAGE电泳鉴定图。(b)IFNα-Pan-RBD(SARS-CoV-2南非突变株)-Fc SDS-PAGE电泳鉴定图。(c)IFNα-Pan-RBD(原始毒株)-Fc与IFNα-Pan-RBD(SARS-CoV-2南非突变株)-Fc免疫后14天血清中RBD特异性抗体与原始毒株RBD的结合情况。(d)IFNα-Pan-RBD(原始毒株)-Fc与IFNα-Pan-RBD(SARS-CoV-2南非突变株)-Fc免疫后14天血清中RBD特异性抗体与南非突变株RBD的结合情况。
图17.(a)Mouse IFNα-RBD-Fc、Mouse IFNα-Pan-RBD-Fc蛋白SDS-PAGE电泳鉴定图谱。(b)Human IFNα-RBD-Fc、Human IFNα-Pan-RBD-Fc蛋白SDS-PAGE电泳鉴定图谱。
图18.Pan(Pan DR-binding epitope)CD4T细胞辅助表位可进一步增强IFNα-RBD-Fc的免疫原性。通过肌肉免疫的方式对小鼠接种10μg Mouse IFNα-RBD-Fc、Mouse IFNα-Pan-RBD-Fc或10μg Human IFNα-RBD-Fc、Human IFNα-Pan-RBD-Fc蛋白，在接种后的14天进行一次加强免疫。分别在免疫后的第7、14、28天收集小鼠血清，并通过ELISA的方法对小鼠血清中的RBD特异性抗体进行水平进行检测。*,p<0.05；****,p<0.0001.
图19.铝佐剂可以增强Human IFNα-RBD-Fc、Human IFNα-Pan-RBD-Fc蛋白诱导的特异型体液免疫应答。C57BL/6小鼠在第0天和第14天在添加(AL+)或者不添加铝佐剂(AL-)辅助的情况下接种10μg Human IFNα-RBD-Fc或Human IFNα-Pan-RBD-Fc蛋白，在接种后的第7、14、28天收集小鼠血清，通过ELISA的方法检测小鼠血清中的SARS-CoV-2 RBD特异性抗体水平。*,p<0.05；****,p<0.0001.
图20.IFN-Pan-RBD-Fc滴鼻免疫可诱导产生高滴度RBD特异的IgG、IgA中和抗体抗体。6-8周C57BL/6小鼠分为5组，每组10只，分别通过滴鼻免疫的方式免疫10μg的IFNα-pan-RBD-Fc或者相同摩尔数的RBD、RBD-Fc、IFNα-RBD-Fc蛋白，滴鼻剂体积为10uL/小鼠。采用两次免疫程序在第0天与14天免疫小鼠。分别在免疫后第7、14、21、28、35、42天收集小鼠血清，采用ELISA方法检测各组血清中新冠RBD特异性抗体IgG(a)和IgA(b)水平；取42天的血清，进行SARS-CoV-2假病毒体外中和实验(c)。统计方法：one-way ANOVA，*p<0.05有显著性差异，**p<0.01，***p<0.001，****p<0.0001。
图21.无佐剂新冠疫苗IFN-Pan-RBD-Fc滴鼻免疫诱导鼻咽部和肺组织内分泌高滴度RBD特异性IgG、IgA中和抗体。6-8周C57BL/6小鼠免疫28天后处死，取小鼠鼻黏膜，用组织匀浆破碎仪破碎。将匀浆后的液体13000rpm高速离心10分钟，取其上清即为鼻黏膜上清(NMDS)。采用ELISA方法检测各组鼻黏膜上清(NMDS)中新冠RBD特异性抗体IgG(a)和IgA(b)水平；取28天的血清，进行SARS-CoV-2假病毒体外中和实验(c)。统计方法：one-way ANOVA，*p<0.05有显著性差异，**p<0.01，***p<0.001，****p<0.0001。
图22.无佐剂新冠疫苗IFN-Pan-RBD-Fc滴鼻免疫诱导肺组织内分泌高滴度RBD特异性IgG和IgA抗体。6-8周C57BL/6小鼠免疫28天后(如图19)处死。小鼠肺部，用1ml注射器吸取约0.8ml的HBSS+100uMEDTA，注入气管插管，轻柔地反复吹吸三遍后，将液体吸出，收集到离心管里，重复该步骤三遍，最终大约能获得2ml的肺灌洗液。将小鼠肺灌洗液500g离心5分钟，上清即为小鼠肺灌洗液(BALF)，采用ELISA方法检测各组小鼠肺灌洗液(BALF)中新冠RBD特异性抗体IgG(a)和IgA(b)含量。统计方法：one-way ANOVA，*p<0.05有显著性差异，**p<0.01，***p<0.001，****p<0.0001。
图23.Her2疫苗蛋白表达和纯化。293F细胞中表达并纯化相关蛋白，SDS-PAGE和考马斯亮蓝染色检测蛋白的大小和纯度。a.IFNα-3-Fc(62.6kDa)；b.IFNα-pan-3-Fc(63.9kDa)；c.IFNα-pan-4-Fc(74.9kDa)和IFNα-4-Fc(73.6kDa)。
图24.分析Her2疫苗IFNα-3-Fc和IFNα-pan-3-Fc的抗肿瘤活性。构建TUBO乳腺癌模型小鼠，肿瘤大小在50-80mm3给药治疗，给小鼠瘤内注射相关融合蛋白，每周一次，共治疗3次。给药剂量为IFNα-3-Fc是10ug//剂/小鼠，其他融合蛋白等摩尔给药，CpG作为佐剂。测量肿瘤大小，绘制肿瘤生长曲线。
图25.分析IFNα和Pan提高Her2抗原免疫原性功能。6-8周的BALB/C小鼠(n＝5)，皮下接种小鼠Her2融合蛋白疫苗4-Fc、IFNα-4-Fc和IFNα-pan-4-Fc进行免疫，无添加佐剂。免疫剂量为IFNα-4-Fc 10ug/剂//小鼠，其他融合蛋白等摩尔接种。在免疫接种后14天、21天采静脉血，用ELISA检测Her2血清中特异性IgG的抗体水平。
图26.IFN-HA1-Fc融合蛋白白SDS-PAGE电泳鉴定图谱。
图27.通过肌肉免疫的方式对小鼠接种10ug IFN方式对小鼠接种瘤或相同摩尔量的HA1蛋白，并在初次接种后的14天进行一次加强免疫。在免疫后的第28天收集小鼠血清，并通过ELISA的方法对小鼠血清中的HA1特异性抗体进行水平进行检测。小鼠在免疫后的42通过鼻腔感染的方式感染1000PFU的A/PR8流感病毒，在病毒感 染后的第三天起，对小鼠进行观察并对其体重变化进行记录。(a)初次免疫28天后收集小鼠血清，通过ELISA的方法检测小鼠血清中HA1特异性抗体水平。(b)在病毒感染后小鼠体重变化。
图28
IFNa-Pan-VZV-gE-Fc、IFNa-Pan-EBV-gp350-Fc、IFNa-Pan-HSV-2-gD-Fc蛋白SDS-PAGE电泳鉴定图。
具体实施方式
为使本发明目的、技术方案和优势更加清楚明了，下面将参考实施例和附图详细描述本发明。所介绍实施例仅是举例说明本发明，而不是意图限定本发明的范围，所述实例仅是本发明的一部分，而不是全部的实施例。本发明的范围由后附的权利具体要求限定。
实施例1.疫苗平台的设计
干扰素-靶抗原-免疫球蛋白Fc(或抗体)结构单元的疫苗平台，由于三个结构单元构成，第一结构单元为干扰素部分，第二结构单元为免疫球蛋白Fc区(或抗体)，第三单元为靶抗原。现实构建中，三个结构单元可以以任意形式排列组合并且靶抗原可以通过连接序列2与Th细胞辅助表位相连接。其代表形式如下：
图1.为所述疫苗平台以同源二聚体的形式，按照干扰素-连接片段1-靶抗原-免疫球蛋白Fc顺序排列组合的示意图。
图2.为所述疫苗平台以异源二聚体的形式，分别按照干扰素-连接片段1-IgG1-hole和靶抗原-IgG1-knob组合的示意图。
图3.为所述疫苗平台以异源二聚体的形式，按照干扰素-连接片段1-IgG1-knob和靶蛋白-IgG1-hole组合的示意图。
接下来我们通过连接片段2，将靶抗原与细胞辅助表位相连接，然后与其他两个疫苗平台组件相结合其代表形式如下：
图4.为所述疫苗平台以同源二聚体的形式，按照干扰素-连接片段1-Th细胞辅助表位-连接片段2-靶抗原-免疫球蛋白Fc顺序排列组合的示意图。
图5.为所述疫苗平台以异源二聚体的形式，分别按照干扰素-连接片段1-IgG1-hole和Th细胞辅助表位-连接片段2-靶抗原-IgG1-knob组合的示意图。
图6.为所述疫苗平台以异源二聚体的形式，分别按照干扰素-连接片段1-IgG1-knob 和Th细胞辅助表位-连接片段2-靶抗原-IgG1-hole组合的示意图。
实施例2.疫苗平台的构建纯化和生产
我们以乙型肝炎病毒Pres1、冠状病毒SARS-CoV-2 RBD蛋白同源二聚体形式为例对该疫苗平台的表达生产进行描述。
1、载体的构建、宿主细胞的转染及诱导表达
1.1、以PEE12.4为载体通过分子克隆的方式将疫苗结构单元构建到该载体上，从而得到可以表达融合蛋白的质粒，然后瞬时转染293F细胞的,收集培养上清，最后通过Protein A亲和层析柱纯化目标蛋白。
载体构建(以含有HBV preS1抗原为例)
(1)PEE12.4-HindIII-信号肽1-干扰素素-BsiwI-Pres1-BstbI-hIgG1-EcoRI
(2)PEE12.4-HindIII-信号肽1-干扰素-BsiwI-RBD(SARS-CoV-2)-BstbI-hIgG1-EcoRI
(3)PEE12.4-HindIII-信号肽1-干扰素-Bsiwi-PADER-Pres1-hIgG1-EcoRI
(4)PEE12.4-HindIII-信号肽1-干扰素-Bsiwi-PADER-RBD(SARS-CoV-2)-hIgG1-EcoRI
各个融合蛋白片段之间的连接序列为
(1)干扰素与Pres1之间为连接片段1
(2)干扰素与RBD(SARS-CoV-2)之间为连接片段1
(3)干扰素与PADER之间连接序列为连接片段1，PADER与Pres1之间连接片段为连接片段2
(4)干扰素与PADER之间连接序列为连接片段1，PADER与RBD(SARS-CoV-2)之间连接片段为连接片段2
1.2、瞬时转染快速表达目的蛋白：
(1)细胞复苏：Freestyle 293F细胞以3×10 7cells/ml的浓度于CD OptiCHOTM media(含10％DMSO)中冻存。从液氮中取出后在37℃水浴锅中快速溶化，加入到含有10ml OptiCHOTM media的15ml离心管中离心，1,000rpm，5min。弃上清，将细胞沉淀悬浮培养于30ml OptiCHOTM media中，37℃，8％CO 2，135rpm。4天后将细胞进行扩大培养，扩大培养时浓度不要超过3×106cells/ml。
(2)转染前两天，准备悬浮培养的293F细胞用于瞬时转染(200ml)，接种密度为0.6-0.8×10 6cells/ml。
(3)两天后对待转染细胞悬液进行计数，预计细胞密度2.5-3.5×10 6cells/ml，随后取细胞悬液1,000rpm离心5min，弃上清。
(4)用50ml的新鲜的Freestyle 293media重新悬浮细胞，再次1,000rpm离心5min，弃上清液。
(5)用200ml Freestyle 293media重新悬浮293F细胞。
(6)用5ml Freestyle 293media培养基稀释600μg质粒，并利用0.22μM滤器过滤除菌。
(7)用5ml Freestyle 293media培养基稀释1.8mg PEI并利用0.22μM滤器过滤除菌。随后立即将5ml质粒和5ml的PEI混匀，室温静置5分钟。
(8)将质粒/PEI混合物加入细胞悬液中，放置在37℃，8％CO 2，85rpm培养箱中培养，同时补加生长因子50ug/L LONG TM R3IGF-1。
(9)4小时后补加200ml EX-CELLTM 293media培养基和2mM Glutamine，将转速调至135rpm继续培养。
(10)24小时后加入细胞增殖抑制剂3.8mM VPA，72小时后加入40ml medium D继续培养，转然后6-8天(细胞存活率低于70％)收集上清液进行下一步纯化。
1.3、融合蛋白的收集、纯化和电泳验证
2.利用Protein A进行目的蛋白纯化：
(1)样品准备：将悬浮细胞培养液转至500ml离心桶中离心，8,000rpm，20min弃沉淀，将上清经0.45μM滤器过滤除去杂质，然后加入终浓度为0.05％NaN3防止纯化过程中细菌污染。
(2)组装层析柱：取适量Protein A Agarose(每1ml Protein A纯化20mg human Fc融合蛋白计算)混匀后加入层析柱，室温静置约10min，待Protein A与20％乙醇溶液分层后把底部的出口打开，让乙醇溶液通过重力作用缓慢流出。
(3)分别用10倍柱体积的蒸馏水和Binding buffer(20mM sodium phosphate+0.15M NaCl，pH 7.0)冲洗和平衡层析柱。
(4)利用恒流泵进行上样，流速为10倍柱体积/小时，收集流穿液，重复上样2次。
(5)用10倍柱体积以上的Binding buffer冲洗柱子，洗去杂蛋白，冲洗至流出液无蛋白检出。
(6)使用Elution Buffer(0.1M Glycine,pH 2.7)进行洗脱，洗脱液分管收集，每1ml收集1管，并采用蛋白指示液(Bio-Rad protein assay)观察洗脱峰。将洗脱峰 的收集管混合后加入适量的1M Tris，pH 9.0中和(调节pH值至6-8，应于所纯化蛋白等电点相差0.5以上)。
(7)利用Zeba脱盐离心柱或浓缩离心柱将目的蛋白溶液置换到所需要的缓冲液中(注意调节缓冲液pH，避开蛋白的等电点)。利用BSA为标准品，通过SDS-PAGE电泳和NanoDrop2000确定蛋白浓度。
(8)洗脱结束后，依次使用20倍柱体积的蒸馏水冲洗柱子，再用10倍柱体积的20％乙醇冲洗柱子，最后乙醇溶液要浸没凝胶介质，4℃保存。
3.所述蛋白SDS-PAGE电泳鉴定图如图7所示。
实施例3.IFNα-Pres1-Fc，Pres1-Fc相较于单纯的Pres1抗原在小鼠体内可以引起更强的免疫应答。
材料：C57BL/6雄性小鼠(5-8)周购买于北京维通利华实验动物技术有限公司；辣根过氧化物氧化酶(HRP)标记的山羊抗小鼠IgG买自北京康为生物技术有限公司；96孔ELISA测定板购自Corning Costar公司；ELISA显色液购自eBioscience公司；所用酶标仪SPECTRA max PLUS 384购自美国Molecular公司。所用铝佐剂购自SIGMA公司。
方法：
(1)Pres1融合蛋白免疫小鼠，将80pmol IFN-Pres1-Fc或者80pmol的Pres1-Fc、Pres1蛋白与铝佐剂混合后皮下免疫小鼠。在指定的时间点通过眼眶取血的方式收集小鼠的血清，进行抗体检测。
(2)IFNα-Pres1-Fc所产生的抗体对不同基因型的HBV病毒有广泛的中和作用。5周年龄雄性C57BL/6通过尾静脉的方式感染AAV-HBV 1.3(其中HBV基因型为B、C、D型)1x10 11vg病毒，6周后筛选HBV抗原持续稳定表达的小鼠进行试验。将筛选出的小鼠(4只/组)通过静脉注射的方式注射来自IFNα-Pres1-Fc免疫小鼠的血清200ul/只。12小时后，收集小鼠血清，通过Elisa的方式检测注射抗血清前后小鼠体内Pres1抗原的变化。
(3)ELISA检测血清中anti-Pres1特异性抗体。将Pres1(2ug/ml)包被液以每孔50ul的体系加入到Elisa板(Corning 9018)中，4℃包被过夜。用PBS洗一遍，260ul每孔。5％的封闭液(5％FBS)37℃封闭两小时。用PBS稀释血清样品(1:10,1:100，1:1000,1:10000),每孔加50ul到封闭好的Elisa板中37℃孵育1小时。PBST洗5次，每次260ul，每孔加入50ul酶标二抗(enzyme-conjugated  anti-mouse IgG-HRP 1:5000 diluted by PBS)，37℃孵育1小时。用PBST洗五遍，每次260ul，加底物TMB 100ul/孔，室温避光孵育，等待底物显色；每孔加50ul终止液(2N H 2SO 4)终止显色，酶标仪读板，OD450-630。
结果：游离的Pres1免疫原性较弱，当在Pres1的基础上加上IFNα、Fc部分构成IFNα-Pres1-Fc融合蛋白时其免疫原性得到了极大的提升，如图8(a)所示。并且由IFNα-Pres1-Fc所引起的抗体，能对不同的HBV基因型病毒产生广泛的中和作用，如图8(b)所示。
实施例4.IFNα-Pres1-Fc可作为乙肝预防性疫苗来使用
材料：C57BL/6(6-8)周雄性小鼠购自北京维通利华生物技术有限公司，HBsAg检测试剂盒购自上海科华生物工程股份有限公司。AAV-HBV 1.3病毒购买自广州派真生物技术有限公司。其他实验材料同实施例3。
方法：
(1)小鼠通过皮下免疫的方式接种80pmol不同形式的Pres1疫苗包括单纯的Pres1、Pes1-Fc、IFNα-Pres1-Fc蛋白，在免疫28天时，收集小鼠血清并对小鼠感染1x10 11vg AAV-HBV 1.3病毒，之后每周收集小鼠血清检测血清中的anti-Pres1抗体、HBsAg、Pres1抗原，连续检测四周。在第三周时检测小鼠外周HBV-DNA水平。
(2)ELISA检测血清中Pres1特异性抗原。抗原包被：将Pres1抗体XY007(4ug/ml)包被液以每孔50ul的体系加入到Elisa板(Corning 9018)中，4℃包被过夜。用PBS洗一遍，260ul每孔。用5％的封闭液(5％FBS)37℃封闭两小时。用PBS稀释血清样品(1:10,1:100),每孔加50ul到封闭好的Elisa板中，每个稀释度设置两个复孔，37℃孵育1小时。PBST洗5次，每次260ul，每孔加入50ul酶结合物(来自科华HBsAg检测Kit)，37℃孵育1小时。用PBST洗五遍，每次260ul，加底物TMB 100ul/孔，室温避光孵育，等待底物显色；每孔加50ul终止液(2N H2SO4)终止显色，酶标仪读板，OD450-630。
结果：IFNα-Pres1-Fc免疫组的小鼠在接种病毒前即能产生高水平的Pres1抗体并且抗体在病毒感染过程中持续维持在较高水平如图9(a)。相较于未进行蛋白免疫组而言，IFN-Pres1-Fc疫苗免疫能够显著预防HBV感染，免疫后产生的抗-preS1抗体能够很快完全清除血清中preS1抗原图9(b),并且在IFN-Pres1-Fc免疫组接受病毒感染的小鼠大 部分表现为外周HBsAg阴性图9(c、d)。以上实验结果表明，IFN-Pres1-Fc作为疫苗，能够有效预防HBV感染，如图9所示。
实施例5.IFNα-Pres1-Fc作为慢性乙型感染的治疗性疫苗
材料：C57BL/6雄性小鼠(4周)购自北京维通利华生物技术有限公司。AAV-HBV 1.3购自广州派真生物技术有限公司。HBsAg检测试剂盒购买自上海科华生物技术有限公司，其他实验材料同实施例4。
方法：
(1)HBV Carrier小鼠的筛选，4周龄HBV C57BL/6小鼠通过尾静脉的方式注射1x10 11vg AAV-HBV 1.3病毒，1-6周通过检测HBV抗原HBsAg，筛选出HBsAg稳定表达的小鼠，作为HBV Carrier小鼠进行实验。
(2)筛选出的小鼠通过皮下注射的方式注射80pmol不同形式的Pres1蛋白，每两周进行一次一共进行三次免疫。在免疫14天时收集小鼠血清，之后每周采集一次小鼠血清，通过ELISA检测小鼠血清中抗-Pres1抗体、HBsAG、Pres1抗原的水平。在最后一次取血后检测小鼠外周血中的HBV-DNA含量。
结果：我们检测IFN-Pres1-Fc疫苗免疫Carrier小鼠后其血清中preS1抗原，以及血清中Pres1抗体和HBsAg的变化情况。结果显示，在IFNα-Pres1-Fc疫苗免疫之后，小鼠高水平的anti-Pres1抗体的产生如图10(a)所示，并且伴有血清中preS1抗原能够被完全清除,如图10(b)所示，。同时血清中的HBsAg也有一定程度的下降如图10(c)，而未处理对照组以及单独Pres1疫苗免疫组都没有治疗效果图10所示。
实施例6.T细胞辅助表位增强IFNα-Pres1-Fc疫苗的抗体应答
材料：同实施例3
方法：
(1)Pres1融合蛋白免疫小鼠，将80pmol含有Pan表位的IFN-Pan-Pres1-Fc或者80pmol的IFN-Pan-Pres1-Fc、Pres1-Fc、Pres1蛋白皮下免疫小鼠。在指定的时间点通过眼眶取血的方式收集小鼠的血清，进行抗体检测。
(2)ELISA检测血清中anti-Pres1特异性抗体，同实施例3。
结果：相比于IFN-preS1-Fc等融合蛋白疫苗，IFN-Pan-preS1-Fc能显著增强抗原分子的免疫原性，并能引起广谱的中和性抗体的产生。对C57/BL6(n＝8/组)小鼠采用皮下免疫的方式接种不含铝佐剂的乙肝Pres1、Pres1-Fc、IFNα-Pres1-Fc蛋白，在指定的 时间采用Elisa的方法检测血清中Pres1特异性抗体的水平。
实施例7.IFNα-Pan-Pres1-Fc作为慢性乙型感染的治疗性疫苗
材料：C57BL/6雄性小鼠(4周)购自北京维通利华生物技术有限公司。AAV-HBV
1.3购自广州派真生物技术有限公司。HBsAg检测试剂盒购买自上海科华生物技术有限公司，其他实验材料同实施例4。
方法：
(1)HBV Carrier小鼠的筛选，4周龄HBV C57BL/6小鼠通过尾静脉的方式注射1x10 11vg AAV-HBV 1.3病毒，1-6周通过检测HBV抗原HBsAg，筛选出HBsAg稳定表达的小鼠，作为HBV Carrier小鼠进行实验。
(2)筛选出的小鼠通过皮下注射的方式注射80pmol不同形式的Pres1蛋白，每两周进行一次一共进行三次免疫。在免疫14天时收集小鼠血清，之后每周采集一次小鼠血清，通过ELISA检测小鼠血清中抗-Pres1抗体、HBsAG、Pres1抗原的水平。在最后一次取血后检测小鼠外周血中的HBV-DNA含量。
结果：我们检测IFN-Pan-Pres1-Fc疫苗免疫Carrier小时小鼠后其血清中preS1抗原，以及血清中Pres1抗体和HBsAg的变化情况。结果显示，在IFN-Pan-Pres1-Fc疫苗免疫之后，小鼠产生高水平的anti-Pres1抗体的产生如图12(a)所示。并且伴有血清中preS1抗原能够被完全清除如图12(b)所示，同时血清中的HBsAg也有一定程度的下降12(c)，而未处理对照组以及单独Pres1疫苗免疫组都没有治疗效果。并且HBV DNA在IFNα-Pan-Pres1-Fc免疫组中也有明显的下降如图12(d)所示。
实施例8.IFNα-Pan-Pres1-Fc联合HBsAg商业化疫苗，打破针对于HBsAg引起的免疫耐受，诱导HBsAg-HBsAb血清学转换。
材料：C57BL/6雄性小鼠(4周)购自北京维通利华生物技术有限公司。AAV-HBV 1.3购自广州派真生物技术有限公司。HBsAg检测试剂盒购买自上海科华生物技术有限公司，Anti-HBsAg试剂盒购买自北京万泰生物制药有限公司。商业化HBsAg疫苗购买自艾美汉信疫苗(大连)有限公司。其他实验材料同实施例7。
方法：
(1)HBV Carrier小鼠的筛选，4周龄HBV C57BL/6小鼠通过尾静脉的方式注射1x10 11vg AAV-HBV 1.3病毒，1-6周通过检测HBV抗原HBsAg，筛选出HBsAg稳定表达的小鼠，作为HBV Carrier小鼠进行实验。
(2)筛选得出的HBV Carrier小鼠，免疫80pmol IFNα-pan-Pres1-Fc，同时免疫商业化HBsAg疫苗2μg连续免疫两次，每次间隔14天。在第一次免疫后14天收集小鼠血清，之后每周采集小鼠血清，并检测血清中anti-Pres1、Pres1、anti-HBsAg、HBsAg的变化。并在最后一次小鼠血清采集时，检测血清中HBV-DNA的水平。
结果：我们发现，以IFNα-Pan-Pres1-Fc联合商业化HBsAg作为治疗慢性乙型肝炎的策略，最终能够打破HBsAg耐受。HBV耐受小鼠中产生的免疫反应能够完全清除血清中preS1抗原能够被图13(a)，而且血清中存在高浓度的Pres1抗体13(c)。令人兴奋的是，IFN-Pan-Pres1-Fc疫苗同时有效清除血清中HBsAg，同时诱导了部分的血清学HBsAb的转化图13(b)和4(d)，这在临床上被认为是HBV治愈的关键指标。另外我们分别通过荧光定量PCR(real-time PCR)的方法检测了外周血中HBV相关的DNA的表达水平，结果显示，相对于对照组而言，以IFNα-Pan-Pres1-Fc联合商业化HBsAg的免疫方式能够最终降低外周HBV DNA的水平图13(e)，通过以上结果，我们发明了联合以IFNα-Pan-Pres1-Fc和商业化HBsAg疫苗治疗慢性乙型肝炎的疫苗策略
实施例9.IFNα-RBD(SARS-CoV2)-Fc相比游离的SARS-Cov2 RBD蛋白，可以引起更强的抗体反应
材料：Balb/c雌雄小鼠(6-8周)购买自北京维通利华实验动物技术有限公司，所用SARS-CoV-2 RBD蛋白购买自北京科跃中楷生物技术有限公司。293-hACE2细胞来自张政教授(深圳第三人民医院)提供。 Luciferase Reporter检测试剂盒购买自promega公司。
其他实验材料同实施例3。
方法：
(1)IFNα-RBD(SARS-Cov-2)-Fc融合蛋白免疫小鼠，将10ug IFNα-RBD-Fc、RBD-Fc或者10ug RBD蛋白与铝佐剂混合后皮下免疫小鼠。在免疫28天时通过眼眶取血的方式收集小鼠的血清，进行新冠特异性抗体的检测。
(2)检测血清SARS-cov2 RBD抗体。抗原包被：将RBD(1.5ug/ml)包被液以每孔100ul的体系加入到Elisa板(Corning 9018)中，4℃包被过夜。用PBS洗一遍，260ul每孔。用5％的封闭液(5％FBS)100ul，37℃封闭两小时。用PBS稀释血清样品(1:10,1:100，1:1000,1:10000,1:100000…),每孔加100ul到封闭好的Elisa板中37℃孵育1小时。PBST洗5次，每次260ul，每孔加入100ul酶标二抗(enzyme-conjugated anti-mouse IgG-HRP  1:5000 diluted by PBS)，37℃孵育1小时。用PBST洗五遍，每次260ul，加底物TMB 100ul/孔，室温避光孵育15分钟，等待底物显色。每孔加50ul终止液(2N H 2SO 4)终止显色，酶标仪读板，OD450-630。滴度计算方法，选取结果为阳性的最大稀释倍数，以该稀释倍数对应的OD值/Cutoff值(0.1)乘(X)稀释倍数,所得的值为该血清对应的抗体滴度。
(3)SARS-CoV-2 S蛋白假病毒体外中和实验。抗血清按1：3稀释加入到96孔板中，50ul带有luciferase spike蛋白的假病毒颗粒加入到孔中，病毒抗体混合物37℃放置1小时，每孔10^4的293-hACE2细胞加入到96孔板中。将96孔板放置到37℃细胞培养箱，48小时后检测luciferase的活性。
结果：游离的新冠RBD免疫原性较弱，当在新冠RBD多肽蛋白区域的基础添加IFNα、Fc部分构成IFNα-RBD-Fc融合蛋白时，其免疫原性得到了极大的提升，如图14所示。并且由IFNα-RBD-Fc所引起的抗体，能够在体外阻断SARS-CoV-2 S蛋白的假病毒对细胞的感染如图15所示。
实施例10.FNα-Pan-RBD(原始毒株)-Fc与IFNα-RBD(SARS-CoV-2南非突变株)-Fc免疫所产生的抗血清RBD特异性抗体检测。
材料：Balb/c雌雄小鼠(6-8周)购买自北京维通利华实验动物技术有限公司，所用SARS-CoV-2原始毒株RBD蛋白购买自北京科跃中楷生物技术有限公司。所用SARS-CoV-2南非突变毒株RBD蛋白购买自北京义翘神州科技有限公司。
其他实验材料同实施例3。
方法:
(1)IFNα-Pan-RBD(原始毒株)-Fc与IFNα-RBD(SARS-CoV-2南非突变株)-Fc蛋白构建表达方法同实施例2。
(2)IFNα-Pan-RBD(原始毒株)-Fc与IFNα-Pan-RBD(SARS-CoV-2南非突变株)-Fc融合蛋白免疫小鼠，将10ug IFNα-Pan-RBD(原始毒株)-Fc或者IFNα-Pan-RBD(SARS-CoV-2南非突变株)-Fc蛋白与铝佐剂混合后皮下免疫小鼠。在免疫14天时通过眼眶取血的方式收集小鼠的血清，进行新冠特异性抗体的检测。
(3)ELISA方法分析抗体应答同实施例9。
结果：SDS-PAGE结果显示出IFNα-Pan-RBD(SARS-CoV-2原始毒株)-Fc正确的条带大小，说明成功构建、表达、纯化突变的新冠病毒IFNα-RBD(SARS-CoV-2原始毒株)-Fc 疫苗蛋白(图16a)，SDS-PAGE结果显示出IFNα-Pan-RBD(SARS-CoV-2南非突变株)-Fc正确的条带大小，说明成功构建、表达、纯化突变的新冠病毒IFNα-RBD(SARS-CoV-2南非突变株)-Fc疫苗蛋白(图16b)。ELISA结果显示IFNα-Panan-RBD(原始毒株)-Fc免疫小鼠，同IFNα-Pan-RBD(SARS-CoV-2南非突变株)-Fc免疫小鼠，诱导产生的抗体，都可以与原始毒株新冠病毒RBD蛋白结合，并且两者诱导产生的抗体与原始毒株RBD的结合能力并无显著性差异(图16c)。同时针对南非突变株RBD的ELISA结果也显示IFNα-Panan-RBD(原始毒株)-Fc免疫小鼠，同IFNα-Pan-RBD(SARS-CoV-2南非突变株)-Fc免疫小鼠，诱导产生的抗体，均可与南非突变株RBD结合，并且其结合能力相当(图16d)。
实施例11.
材料：C57BL/6雌性小鼠(6-8周)购自北京维通利华生物技术有限公司。ELISA所用SARS-CoV-2 RBD蛋白购买自北京科跃中楷生物技术有限公司；免疫用的Mouse IFNα-RBD-Fc、Mouse IFNα-Pan-RBD-Fc、Human IFNα-RBD-Fc、Human IFNα-Pan-RBD-Fc蛋白均产自本实验室，其他实验材料同实施例3。
方法：
(1)融合蛋白设计、质粒构建和蛋白纯化方法同实施例1和2所示。
(2)对小鼠进行疫苗蛋白的免疫。10μg Mouse IFNα-RBD-Fc、Mouse IFNα-Pan-RBD-Fc或10μg Human IFNα-RBD-Fc、Human IFNα-Pan-RBD-Fc疫苗蛋白分别与20μg铝佐剂混合过夜后，通过肌肉免疫的方式对小鼠接种，并在初次接种后的14天进行一次加强免疫。在免疫后的第7、14、28天收集小鼠血清，并通过ELISA的方法对小鼠血清中的RBD特异性抗体进行水平进行检测。
(3)检测血清SARS-cov2 RBD抗体。抗原包被：将RBD(1.5ug/ml)包被液以每孔100ul的体系加入到Elisa板(Corning 9018)中，4℃包被过夜。用PBS洗一遍，260ul每孔。用5％的封闭液(5％FBS)100ul，37℃封闭两小时。用PBS稀释血清样品(1:10,1:100，1:1000,1:10000,1:100000…),每孔加100ul到封闭好的Elisa板中37℃孵育1小时。PBST洗5次，每次260ul，每孔加入100ul酶标二抗(enzyme-conjugated anti-mouse IgG-HRP 1:5000 diluted by PBS)，37℃孵育1小时。用PBST洗五遍，每次260ul，加底物TMB 100ul/孔，室温避光孵育15分钟，等待底物显色。每孔加50ul终止液(2N H2SO4)终止显色，酶标仪读板，OD450-630。滴度计算方法，选取结果为 阳性的最大稀释倍数，以该稀释倍数对应的OD值/Cutoff值(0.1)乘(X)稀释倍数,所得的值为该血清对应的抗体滴度。
结果：
如图17所示，蛋白经过表达纯化后，SDS-PAGE结果表明蛋白大小符合预期并且在目标位置处展现出单一条带。
如图18所示，Pan(Pan DR-binding epitope)CD4T细胞辅助表位的加入可以增强Mouse IFNα-RBD-Fc与Human IFNα-RBD-Fc的免疫性。实验结果显示不管是在疫苗蛋白免疫后的第7天还是第14天、28天，Mouse IFNα-Pan-RBD-Fc相较于Mouse IFNα-RBD-Fc，、Human IFNα-Pan-RBD-Fc相比于Human IFNα-RBD-Fc，pan表位的加入均能引起更高的RBD特异性抗原的产生。
实施例.12
材料：C57BL/6雌性小鼠(6-8周)购自北京维通利华生物技术有限公司；ELISA所用SARS-CoV-2 RBD蛋白购买自北京科跃中楷生物技术有限公司。免疫用的Human IFNα-RBD-Fc、Human IFNα-Pan-RBD-Fc蛋白均产自本实验室。其他实验材料同实施例3。
方法：
(1)Human IFNα-RBD-Fc、Human IFNα-Pan-RBD-Fc蛋白免疫小鼠。10ug Human IFNα-RBD-Fc或Human IFNα-Pan-RBD-Fc蛋白与铝佐剂混合过夜，作为含有铝佐剂的疫苗样品，另一组10ug Human IFNα-RBD-Fc或Human IFNα-Pan-RBD-Fc蛋白经PBS稀释，作为不含佐剂的疫苗样品。在有铝佐剂存在或者没有铝佐剂存在的情况下，通过肌肉免疫的方式对小鼠接种10μg Human IFNα-RBD-Fc、Human IFNα-Pan-RBD-Fc蛋白，在接种后的14天进行一次加强免疫。分别在免疫后的第7、14、28天收集小鼠血清，并通过ELISA的方法对小鼠血清中的RBD特异性抗体水平进行检测。
(2)检测血清SARS-cov2 RBD抗体。抗原包被：将RBD(1.5ug/ml)包被液以每孔100ul的体系加入到Elisa板(Corning 9018)中，4℃包被过夜。用PBS洗一遍，260ul每孔。用5％的封闭液(5％FBS)100ul，37℃封闭两小时。用PBS稀释血清样品(1:10,1:100，1:1000,1:10000,1:100000…),每孔加100ul到封闭好的Elisa板中37℃孵育1小时。PBST洗5次，每次260ul，每孔加入100ul酶标二抗(enzyme-conjugated anti-mouse IgG-HRP 1:5000 diluted by PBS)，37℃孵育1小时。用PBST洗五遍，每次260ul，加底物TMB 100ul/孔，室温避光孵育15分钟，等待底物显色。每孔加50ul 终止液(2N H2SO4)终止显色，酶标仪读板，OD450-630。滴度计算方法，选取结果为阳性的最大稀释倍数，以该稀释倍数对应的OD值/Cutoff值(0.1)乘(X)稀释倍数,所得的值为该血清对应的抗体滴度。
结果：
如图19所示，铝佐剂的应用可以增强Human IFNα-RBD-Fc、Human IFNα-Pan-RBD-Fc蛋白的免疫原性。虽然无佐剂Human IFNα-RBD-Fc、Human IFNα-Pan-RBD-Fc疫苗可以产生高滴度抗体应答，但是铝佐剂辅助的Human IFNα-RBD-Fc和Human IFNα-Pan-RBD-Fc蛋白在接种后的第7天、14天、28天相比于无佐剂辅助组均可进一步提高RBD特异性抗体应答水平。
实施例13.
材料：
实验动物购自北京维通利华实验动物有限公司，所用动物为6-8周C57BL/6小鼠；动物合格证编号：No.110011200106828974；免疫用的RBD蛋白购自北京科跃中楷生物技术有限公司；RBD-Fc，IFNα-RBD-Fc及IFN-pan-RBD-Fc蛋白为本实验室生产；所有佐剂购自德国SERVA公司；辣根过氧化为酶(HRP)标记山羊抗小鼠IgG购自北京康为生物技术有限公司；96孔ELISA测定板购自Corning Costar公司；ELISA显色液购自eBioscience公司；所用酶标仪SPECTRA max PLUS 384购自美国Molecular公司；组织匀浆破碎仪购自北京昊诺斯科技有限公司。
方法：
6-8周小鼠分为5组，每组10只，分别通过滴鼻免疫的方式免疫10ug的IFNα-pan-RBD-Fc或者相同摩尔数的RBD、RBD-Fc、IFNα-RBD-Fc蛋白，滴鼻剂量为10uL每只小鼠。采用两次免疫程序在第0天与14天免疫小鼠。分别在免疫后第7、14、21、28、35、42天收集小鼠血清，采用ELISA方法检测各组血清中SARS-CoV-2RBD特异性抗体含量；取28天的血清，进行SARS-CoV-2假病毒体外中和实验。
结果：
如图20所示，RBD,RBD-Fc蛋白两次滴鼻免疫可以引起一定程度的抗体反应，IFNα-pan-RBD-Fc两次滴鼻免疫后同时间点所诱导产生的血清IgG和IgA水平均都明显高于RBD，RBD-Fc与IFN-RBD-Fc组，假病毒中和实验结果显示相比于RBD和RBD-Fc免疫组，IFN-RBD-Fc可以诱导更高水平的中和抗体产生。
实施例14.
材料：
同实施例10
方法：
6-8周小鼠分为4组，每组5只，分别通过滴鼻免疫的方式免疫10ug的IFNα-pan-RBD-Fc或者相同摩尔数的RBD、RBD-Fc、IFNα-RBD-Fc蛋白，剂量为10uL每只小鼠。采用两次免疫程序在第0天与14天免疫小鼠。在免疫后第28天收集小鼠鼻黏膜上清以及肺灌洗液，采用ELISA方法检测各组血清中SARS-CoV-2 RBD特异性抗体含量,采用SARS-CoV-2假病毒中和实验测血清和鼻粘膜上清的新冠假病毒中和实验。
免疫实验动物小鼠鼻黏膜上清和肺泡灌洗液获取：小鼠休眠处死后，取小鼠鼻黏膜，用组织匀浆破碎仪破碎。将匀浆后的液体13000rpm高速离心10分钟，取其上清即为鼻黏膜上清(NMDS)。小鼠肺部，用1ml注射器吸取约0.8ml的HBSS+100uMEDTA，注入气管插管，轻柔地反复吹吸三遍后，将液体吸出，收集到离心管里，重复该步骤三遍，最终大约能获得2ml的肺灌洗液。将小鼠肺灌洗液500g离心5分钟，上清即为小鼠肺灌洗液(BALF)，沉淀为小鼠肺中有的淋巴细胞，可进行进一步分析。
结果：
如图21所示，IFNα-pan-RBD-Fc蛋白相比于RBD和RBD-Fc蛋白，两次滴鼻免疫的可以引起强烈的鼻粘膜局部IgG抗体反应和IgA黏膜免疫。IFNα-pan-RBD-Fc蛋白反应强度均强于RBD、RBD-Fc组。假病毒中和实验结果显示，IFNα-pan-RBD-Fc蛋白免疫组在鼻粘膜处可以诱导更高滴度的中和抗体产生。
如图22所示，IFNα-pan-RBD-Fc融合蛋白两次滴鼻免疫C57BL/6小鼠在局部肺组织同样引起强烈的IgG抗体和IgA抗体分泌。假病毒中和实验结果显示IFNα-pan-RBD-Fc相比于RBD、RBD-Fc其诱导产生了更高滴度的中和抗体。
实施例15.
Her2属于I型跨膜生长因子受体HER家族，由胞外配体结合结构域、跨膜结构域和胞内酪氨酸激酶结构域组成。一旦配体结合到胞外结构域，HER蛋白将二聚化并反式磷酸化其胞内结构域，磷酸化的酪氨酸残基可与多种胞内信号分子结合，激活下游信号通路，调控基因转录。被调控的基因多与细胞增殖、存活、分化、血管生成、侵袭和转移等过程有关。Her2蛋白的胞外段较大，有600多个氨基酸，可分为四个结构域，分别为结构域I、II、III、IV。目前批准的Trastuzumab主要结合在结构域IV， Pertuzumab主要结合在结构域II，正在开展临床试验的多肽类疫苗E75则靶向结构域III。说明不同的结构域中都具有一些重要的位点，可能介导抗肿瘤作用。为研究该疫苗平台用于肿瘤预防与治疗，该专利研究选取肿瘤抗原Her2作为靶点，构建IFN-Her2-Fc和IFN-Pan-Her2-Fc构建融合蛋白疫苗，分析体内抗肿瘤活性和疫苗免疫活性。
材料和方法：
材料：
BALB/c雌性小鼠(6-8周)购自北京维通利华生物技术有限公司；TUBO细胞来源于TCGA；其他材料同实施例3。
方法：
(1)融合蛋白设计、质粒构建构建和蛋白纯化方法如实施例1和2所示。
首先针对小鼠Her2胞外段的结构域III和IV构建表达质粒，(分别记作：IFNα-3-Fc、IFNα-pan-3-Fc、IFNα-pan-4-Fc和IFNα-4-Fc)，在人293F细胞系中表达并纯化相关蛋白。利用SDS-PAGE和考马斯亮蓝染色对蛋白的大小和纯度进行鉴定。
(2)IFNα-3-Fc和IFNα-pan-3-Fc直接抗肿瘤活性分析
TUBO是来源于BALB-NeuT小鼠的乳腺癌细胞系，用于研究Her2阳性乳腺癌的生长和治疗。利用TUBO肿瘤检测蛋白中IFNα的抗肿瘤活性。构建TUBO乳腺癌模型小鼠，5*10 5TUBO细胞皮下接种于BALB/C小鼠，肿瘤大小在50-80mm 3给药治疗，每周一次，共治疗3次。给药剂量为IFNα-3-Fc是10ug/小鼠，其他药物等摩尔给药，CpG作为佐剂。测量肿瘤大小，绘制肿瘤生长曲线。
(3)分析IFNα和Pan对Her2疫苗免疫原性的提升
6-8周的BALB/C雌性小鼠皮下接种不含佐剂的HER2结构域V融合蛋白疫苗4-Fc、IFNα-4-Fc和IFNα-pan-4-Fc，每周1次，共接种3次。免疫剂量为IFNα-4-Fc 10ug/小鼠，其他蛋白等摩尔接种。在免疫接种后14天、21取静脉血，用ELISA方法检测Her2特异性IgG的抗体水平。
结果：
(1)如图23所示，Her2融合蛋白大小基本符合预期，纯度达到实验要求。IFNα-3-Fc(62.6kDa)、IFNα-pan-3-Fc(63.9kDa)、IFNα-pan-4-Fc(74.9kDa)和IFNα-4-Fc(73.6kDa)。非变形条件下，蛋白处于二聚体状态，符合Fc片段自动二聚化的特性。
(2)如图24所示，与对照组相比，肿瘤内注射Her2融合蛋白IFNα-pan-3-Fc和 IFNα-3-Fc显著抑制TUBO肿瘤的生长，且控制效果与IFNα-Fc组相当。说明蛋白疫苗中IFNα的活性良好，并没有出现空间位阻等因素影响其IFNα活性，可用于进一步探究其在抗肿瘤免疫中的功效和机制。
(3)如图25所示，与对照组相比，Her2融合蛋白疫苗免疫后14天和21天，4-Fc、IFNα-4-Fc和IFNα-pan-4-Fc均能诱导明显的Her2特异性IgG抗体反应；相比于4-Fc，IFNα-4-Fc和IFNα-pan-4-Fc诱导的抗体滴度有增加的趋势。而且，免疫后21天时，IFNα-pan-4-Fc诱导的抗体滴度显著高于4-Fc组。说明IFNα和pan的添加有助于增加4-Fc的免疫原性，诱导出更加强烈的抗原特异性抗体反应，因此IFN-Pan-HER2-Fc和IFN-Pan-HER2-Fc是有潜力的抗Her2阳性肿瘤的有效肿瘤疫苗。
实施例16.
材料：BALB/c雌性小鼠(6-8周)购自北京维通利华生物技术有限公司；ELISA所用HA1(A/PR8)蛋白购买自北京一翘神州生物技术有限公司；免疫用的HA1蛋白蛋白(A/PR8)购买自北京一翘神州生物技术有限公司，IFNα-HA1-Fc产自本实验室；感染小鼠所用的H1N1(A/PR8)流感病毒由本实验室所生产；其他实验材料同实施例三。方法：
(1)IFNα-HA1-Fc蛋白的设计、质粒构建和蛋白纯化同实施例1和2所示。
(2))HA1、IFNα-HA1-Fc蛋白免疫小鼠。10ug IFNα-HA1-Fc或相同摩尔量的HA1蛋白分别与20μg铝佐剂混合过夜后，通过肌肉免疫的方式对小鼠接种，并在初次接种后的14天进行一次加强免疫。在免疫后的第28天收集小鼠血清，并通过ELISA的方法对小鼠血清中的HA1特异性抗体进行水平进行检测。
(3)检测血清HA1抗体。抗原包被：将HA1(2μg/ml)包被液以每孔100ul的体系加入到Elisa板(Corning 9018)中，4℃包被过夜。用PBS洗一遍，260ul每孔。用5％的封闭液(5％FBS)100ul，37℃封闭两小时。用PBS稀释血清样品(1:10,1:100，1:1000,1:10000,1:100000…),每孔加100ul到封闭好的Elisa板中37℃孵育1小时。PBST洗5次，每次260ul，每孔加入100ul酶标二抗(enzyme-conjugated anti-mouse IgG-HRP 1:5000 diluted by PBS)，37℃孵育1小时。用PBST洗五遍，每次260ul，加底物TMB 100ul/孔，室温避光孵育15分钟，等待底物显色。每孔加50ul终止液(2N H2SO4)终止显色，酶标仪读板，OD450-630。滴度计算方法，选取结果为阳性的最大稀释倍数，以该稀释倍数对应的OD值/Cutoff值(0.1)乘(X)稀释倍数,所得的值为该血清对应的抗体滴度。
(4)小鼠免疫42天后将小鼠麻醉通过滴鼻的方式对小鼠感染1000PFU A/PR8流感病毒，感染后的第三天起，每两天对小鼠进行一次观察并称量小鼠体重。
结果：
如图26所示，蛋白经表达纯化后，经SDS-PAGE对蛋白的大小和纯度进行检测结果显示在目的条带大小位置展现出了单一的条带。如图27所示，IFNα-HA1-Fc相比与HA1蛋白可以诱导产生更高滴度的HA1特异性抗体(图27a)，表明该疫苗平台可以提升HA1蛋白的免疫原性。小鼠经过攻毒后其体重会发生明显的变化，但是IFNα-HA1-Fc免疫组小鼠体重相比与PBS组和HA1蛋白免疫组，其体重会得到快速的回升，表明了IFNα-HA1-Fc疫苗免疫对流感病的的感染表现出了良好的保护性(图27b)。
实施例17
材料和方法：
IFNa-Pan-VZV-gE-Fc、IFNa-Pan-EBV-gp350-Fc、IFNa-Pan-HSV-2-gD-Fc蛋白的设计、质粒构建和蛋白纯化同实施例1和2所示。
结果：
如图28所示，IFNa-Pan-VZV-gE-Fc、IFNa-Pan-EBV-gp350-Fc、IFNa-Pan-HSV-2-gD-Fc融合蛋白经表达纯化后，经SDS-PAGE对蛋白的大小和纯度进行检测结果显示在目的条带位置正确。
参考文献：
[1]WHO W.Global hepatitis report 2017[J].Geneva:World Health Organization,2017,
[2]RAZAVI-SHEARER D,GAMKRELIDZE I,NGUYEN M H,et al.Global prevalence,treatment,and prevention of hepatitis B virus infection in 2016:a modelling study[J].The lancet Gastroenterology&hepatology,2018,3(6):383-403.
[3]SCHWEITZER A,HORN J,MIKOLAJCZYK R T,et al.Estimations of worldwide prevalence of chronic hepatitis B virus infection:a systematic review of data published between 1965and 2013[J].The Lancet,2015,386(10003):1546-55.
[4]VOS T,ABAJOBIR A A,ABATE K H,et al.Global,regional,and national incidence,prevalence,and years lived with disability for 328 diseases and injuries for 195 countries,1990–2016:a systematic analysis for the Global Burden of Disease Study 2016[J].The  Lancet,2017,390(10100):1211-59.
[5]KRAMMER F,SMITH G J D,FOUCHIER R A M,et al.Influenza[J].Nature reviews Disease primers,2018,4(1):3.
[6]HANNOUN C.The evolving history of influenza viruses and influenza vaccines[J].Expert review of vaccines,2013,12(9):1085-94.
[7]HOUSER K,SUBBARAO K.Influenza vaccines:challenges and solutions[J].Cellhost&microbe,2015,17(3):295-300.
[8]HU B,GUO H,ZHOU P,et al.Characteristics of SARS-CoV-2 and COVID-19[J].Nature Reviews Microbiology,2020,1-14.
[9]LURIE N,SAVILLE M,HATCHETT R,et al.Developing Covid-19 vaccines at pandemic speed[J].New England Journal of Medicine,2020,382(21):1969-73.
[10]LIQ,WU J,NIE J,et al.The impact of mutations in SARS-CoV-2 spike on viral infectivity and antigenicity[J].Cell,2020,182(5):1284-94.e9.
[11]KONTERMANN R E.Strategies for extended serum half-life of protein therapeutics[J].Current opinion in biotechnology,2011,22(6):868-76.
[12]MEKHAIEL D N,CZAJKOWSKY D M,ANDERSEN J T,et al.Polymeric human Fc-fusion proteins with modified effector functions[J].Scientific reports,2011,1(1):1-11.
[13]ROOPENIAN D C,AKILESH S.FcRn:the neonatal Fc receptor comes of age[J].Nature reviews immunology,2007,7(9):715-25.
[14]VILCEK J.Fifty years of interferon research:aiming at a moving target[J].Immunity,2006,25(3):343-8.
[15]BRACCI L,LA SORSA V,BELARDELLI F,et al.Type I interferons as vaccine adjuvants against infectious diseases and cancer[J].Expert review of vaccines,2008,7(3):373-81.
[16]HAHM B,TRIFILO M J,ZUNIGA E I,et al.Viruses evade the immune system through type I interferon-mediated STAT2-dependent,but STAT1-independent,signaling[J].Immunity,2005,22(2):247-57.
[17]ITO T,AMAKAWA R,INABA M,et al.Differential regulation of human blood dendritic cell subsets by IFNs[J].The Journal of Immunology,2001,166(5):2961-9.
[18]MONTOYA M,SCHIAVONI G,MATTEI F,et al.Type I interferons produced by dendritic cells promote their phenotypic and functional activation[J].Blood,2002,99(9): 3263-71.
[19]LE BON A,ETCHART N,ROSSMANN C,et al.Cross-priming of CD8+ T cells stimulated by virus-induced type I interferon[J].Nature immunology,2003,4(10):1009-15.
[20]LE BON A,DURAND V,KAMPHUIS E,et al.Direct stimulation of T cells by type I IFN enhances the CD8+ T cell response during cross-priming[J].The Journal of Immunology,2006,176(8):4682-9.
[21]SPADARO F,LAPENTA C,DONATI S,et al.IFN-α enhances cross-presentation in human dendritic cells by modulating antigen survival,endocytic routing,and processing[J].Blood,The Journal of the American Society of Hematology,2012,119(6):1407-17.
[22]PARLATO S,SANTINI S M,LAPENTA C,et al.Expression of CCR-7,MIP-3β,and Th-1 chemokines in type I IFN-induced monocyte-derived dendritic cells:importance for the rapid acquisition of potent migratory and functional activities[J].Blood,The Journal of the American Society of Hematology,2001,98(10):3022-9.
[23]ROUZAUT A,GARASA S,TEIJEIRA
Figure PCTCN2021103931-appb-000114
et al.Dendritic cells adhere to and transmigrate across lymphatic endothelium in response to IFN‐α[J].European journal of immunology,2010,40(11):3054-63.
[24]LE BON A,SCHIAVONI G,D'AGOSTINO G,et al.Type I interferons potently enhance humoral immunity and can promote isotype switching by stimulating dendritic cells in vivo[J].Immunity,2001,14(4)：461-70.

Claims (10)

  1. 一种疫苗，其包含含有干扰素-靶抗原-免疫球蛋白Fc区(或抗体Ab)为结构单元的融合蛋白，
    其中所述干扰素为第一结构单元，可以是I型干扰素、II型干扰素和/或III型干扰素例如IFN-α、IFN-β、IFN-γ、IFN-λ1(IL-29)、IFN-λ2(IL-28a)、IFN-λ(IL-28b)和IFN-ω，所述干扰素可来自人源或鼠源，优选所述干扰素为I型干扰素，例如IFN-α，例如小鼠IFN-α4、人IFN-α2、人IFN-α2的突变体(结合人和小鼠的IFN受体)，例如SEQ ID NO.1、SEQ ID NO.21、SEQ ID NO.22氨基酸序列所示，
    其中所述靶抗原为第三结构单元，其所述靶抗原可以是例如肿瘤抗原，病原体抗原，如病毒或细菌抗原，其中所述靶抗原可以是例如与野生型不同的突变的靶抗原，包括例如野生型抗原的天然点突变/缺失突变/增多突变/截短体、人工点突变/缺失突变/增多突变/截短体、天然或人工突变的任何组合、突变后所产生的亚型，其中所述病毒可以是例如SARS-COV-2，或者其中所述靶抗原可以是例如SARS-COV-2病毒S蛋白全长或S1区，例如所述靶抗原可以为SEQ ID NO.76或SEQ ID NO.77所示的抗原，
    其中所述免疫球蛋白Fc区(或抗体)为第二结构单元，可以为IgG1、IgG2、IgG3、IgG4和/或IgM的恒定区氨基酸序列，例如IgG1的Fc区，以及用于形成异源二聚体的IgG1-Fc-hole和IgG1-Fc-knob的氨基酸序列SEQ ID NO.2、SEQ ID NO.23、SEQ ID NO.24所示的Fc区，其中作为第二结构单元的所述抗体(包括例如抗体重链和轻链，或单链抗体，简称Ab)可以为DC靶向活化抗体，包括anti-PD-L1，anti-DEC205,anti-CD80/86等抗体，
    任选地所述疫苗可以为靶向性疫苗，任选地所述融合蛋白还可以包含一个或多个Th细胞辅助表位和/或连接片段。
  2. 权利要求1所述的疫苗，其中所述靶抗原是病毒抗原，所述病毒可以为例如HBV，HPV，VZV，EBV，HSV-2，HIV,流感病毒，冠状病毒，如SARS-COV，SARS-COV-2，MERS-CoV，例如，所述抗原可以是HBV抗原，例如HBV Pres1抗原，HBsAg抗原或肽段，例如ad亚型或ay亚型HBV Pres1抗原，例如SEQ ID NO.6氨基酸序列所示的ad亚型HBV Pres1抗原，例如SEQ ID NO.26氨基酸序列所示的ay亚型HBV Pres1抗原；例如HBV HBsAg抗原(包含各个亚型和肽段)，例如SEQ ID NO.7氨基酸序列所示的adr亚型HBV HBsAg抗原，例如SEQ ID NO.27氨基酸序列所示的 adw亚型HBV HBsAg抗原，例如SEQ ID NO.28氨基酸序列所示的ayw亚型HBV HBsAg抗原；例如，所述抗原可以是例如SARS-COV-2抗原，例如SARS-COV2 RBD抗原，例如SEQ ID NO.8氨基酸序列所示的SARS-COV2 RBD抗原；例如流感病毒抗原，例如流感病毒HA抗原，例如SEQ ID NO.9氨基酸序列所示流感病毒HA抗原；例如HPV抗原，例如SEQ ID NO.10氨基酸序列所示的HPV E7抗原；例如gE抗原，例如SEQ ID NO.91氨基酸序列所示的带状疱疹病毒(VZV)gE抗原；例如EBV-gp350，例如SEQ ID NO.92氨基酸序列所示的埃博斯坦-巴尔病毒(EBV)gp350蛋白；例如gD抗原，例如SEQ ID NO.93氨基酸序列所示的单纯疱疹病毒2(HSV-2)gD抗原；所述抗原可以是例如EBV EBNA1/LMP2，VZV-IE62，HSV-2 ICP0，HIV gp120抗原；
    其中所述靶抗原可以是突变的病毒抗原，例如前述任一病毒抗原的突变体，例如SARS-COV-2的突变体，包括例如SARS-COV-2蛋白(例如S蛋白、N蛋白、M蛋白、E蛋白中的一个或多个)的天然点突变/缺失突变/增多突变/截短体、人工点突变/缺失突变/增多突变/截短体、天然或人工突变的任何组合、突变后所产生的亚型，例如所述突变的病毒抗原可以为野生型SARS-COV-2的S蛋白全长(SEQ ID NO.76)、S1区(SEQ ID NO.77)、RBD区(SEQ ID NO.78)的突变体，例如所述突变的病毒抗原可以包含SARS-COV-2的S蛋白的下述一个或多个突变：NTD区域69-70缺失、Y144缺失、242-244缺失、L18F、D80A、D215、R246I突变，RBD区域K417、E484、N501Y，L452R突变，D614G，H655Y突变，例如所述突变的病毒抗原可以包含存在于来源于英国B.1.1.7(501Y.1)突变株、南非B.1.351(501Y.2)突变株和巴西P1(501Y.3)突变株、加州B.1.429突变株中的突变，例如所述突变的病毒抗原可以包含SEQ ID NO.79、SEQ ID NO.80、SEQ ID NO.81、SEQ ID NO.82中任一个所示突变的突变体，例如所述突变的病毒抗原可以为包含SEQ ID NO.79、SEQ ID NO.80、SEQ ID NO.81、SEQ ID NO.82中任一个所示序列的突变体，
    所述病毒抗原可以融合表达增强B细胞和T细胞应答的辅助多肽表位，可以位于抗原表位的N端或C端，例如以Pan HLA DR-binding表位(PADER)，例如SEQ ID NO.3其氨基酸序列所示；
    所述各个结构单元的连接片段为柔性多肽序列，可以为连接片段1和2，例如SEQ ID NO.4、SEQ ID NO.25氨基酸序列所示，
    所述结构单元组成的每一条多肽序列N端可以均含有能够促进蛋白分泌的相应信号肽，例如SEQ ID NO.5氨基酸序列所示，
    所述疫苗可以通过真核表达系统产生，例如通过真核表达系统293F、CHO细胞产 生。
  3. 根据权利要求1或2所述的疫苗，其中所述靶抗原是肿瘤抗原，例如肿瘤细胞高表达蛋白分子，例如，所述抗原可以是人表皮生长因子受体2(human epidermal growth factor receptor 2，HER2/neu)和表皮生长因子(Epidermal growth factor receptor，EGFR)；例如，肿瘤细胞高表达蛋白分子Her2及其各功能区和截短体，例如SEQ ID NO.85、86、97、88、89、90所示的抗原及其突变体。
  4. 权利要求1-3任一项所述的疫苗，其中所述融合蛋白为同源二聚体或者异源二聚体融合蛋白，任选地所述融合蛋白还可以在同源二聚体或者异源二聚体的任意一条或两条链(即第一多肽链和/或第二多肽链)中包含一个或多个Th细胞辅助表位和/或连接片段，
    任选地所述同源二聚体融合蛋白包含第一多肽链和第二多肽链，所述第一多肽链和第二多肽链完全相同，例如所述第一多肽链和第二多肽链自N端到C端依次包含IFN、靶抗原和免疫球蛋白Fc区(或Ab)，或三个结构单元任意组合次序的多肽，并生成同源二聚体；优选N端到C端依次包含IFN、靶抗原和免疫球蛋白Fc区(或Ab)；其还可以包含Th细胞辅助表位的融合蛋白；
    任选地所述异源二聚体融合蛋白包含第一多肽链和第二多肽链，所述第一多肽链和第二多肽链不同，例如所述第一多肽链可以从N端到C端依次包括IFN和免疫球蛋白Fc区(或Ab)，或从N端到C端依次包括免疫球蛋白Fc区(或Ab)和IFN，所述第二条肽链可以包括靶抗原和免疫球蛋白Fc区(或Ab)，其中靶抗原可以位于N端，免疫球蛋白区(或Ab)可以位于C端，或免疫球蛋白区(或Ab)可以位于N端，靶抗原可以位于C端；或三个结构单元任意组合次序的多肽，并生成异源二聚体；优选IFN和靶抗原位分别位于两个多肽的N端，免疫球蛋白Fc区(或Ab)位于两个多肽C端；其还可以包含Th细胞辅助表位的融合蛋白。
  5. 权利要求4所述的疫苗，其中
    1)所述同源二聚体的第一多肽和第二多肽可以包含SEQ ID NO.11、12、13、14、29、30、31、32、38、39、40、47、48、49、50、51、56、57、59、58、65、66、67、68所示氨基酸序列，
    2)所述异源二聚体第一多肽可以包含SEQ ID NO.15、33、42、51、60、69所示核苷酸序列，第二多肽包含SEQ ID NO.16、17、18、19、34、35、36、37、43、44、45、46、52、53、54、55、61、62、63、64、70、71、72、73所示氨基酸序列，
    3)所述抗体可以包括DC靶向抗体，免疫检查点阻断抗体，免疫激活抗体等，例 如含有anti-PD-L1抗体(SEQ ID NO.20)，anti-DEC205抗体,anti-CD80/86等抗体氨基酸序列的疫苗。
  6. 编码权利要求1-5任一项所述疫苗中的融合蛋白的核酸分子，包含所述核酸分子的表达载体，或者包含所述核酸分子或表达载体的宿主细胞，例如真核细胞。
  7. 权利要求1-5任一项所述的疫苗中的融合蛋白在制备组合物或试剂盒，例如药物或免疫原性组合物或试剂盒，重组微生物或者细胞系中的应用。
  8. 权利要求7所述的应用，其中所述组合物或试剂盒用于肿瘤或病原体的预防或治疗，例如病毒或细菌的预防或治疗，所述病毒可以为HBV，HPV，EBV，流感病毒，HIV，冠状病毒，如SARS-COV，SARS-COV-2，MERS-CoV，例如，所述组合物或试剂盒用作乙肝预防性或治疗性疫苗，HBV预防性或治疗性疫苗，流感预防性或治疗性疫苗，SARS-COV2预防性或治疗性疫苗，HPV预防性或治疗性疫苗，HPV相关肿瘤预防性或治疗性疫苗，EBV预防性或治疗性疫苗，EBV相关肿瘤预防性或治疗性疫苗，HIV预防性或治疗性疫苗。
  9. 根据权利要求1-5任一项所述的疫苗或根据权利要求7或8所述的应用，其中所述疫苗、所述组合物或试剂盒可以肌内、静脉内、经皮、皮下或者鼻腔等免疫途径进行接种，其中所述疫苗、所述组合物或试剂盒还可以包括佐剂，所述佐剂可以包括铝佐剂(Alum)、Toll样受体4激活剂配体MPLA、Toll样受体9配体，寡聚脱氧核苷酸(CpG-ODN)，M59和弗氏佐剂。
  10. 根据权利要求1-5任一项所述的疫苗或根据权利要求7或8所述的应用，其中所述疫苗可以与另外的预防性或治疗性疗法联合使用，例如所述疫苗可以为HBV治疗性疫苗，所述HBV治疗性疫苗可以与另外的预防性或治疗性HBV疗法联合使用，例如所述HBV治疗性疫苗可以与乙型肝炎病毒包膜蛋白HBsAg疫苗联合使用，例如用于慢性乙型肝炎病毒感染治疗，例如所述HBV治疗性疫苗可以联合核苷或者核苷酸类似物，例如用于慢性乙型肝炎病毒感染治疗，例如用于流感、SARS-COV2、HPV、EBV、HIV预防性或治疗性疫苗等与抗病毒药物和其他治疗方法的联合应用；HPV、EBV相关肿瘤预防性或治疗性疫苗与抗病毒抗肿瘤药物和疗法的联合应用，例如，任何一种根据权利要求1-5任一项所述的疫苗作为疫苗的一个成分与其他病毒或病原体或肿瘤疫苗组成的多价联用疫苗，例如，任何一种根据权利要求1-5任一项所述的SARS-COV-2疫苗与流感疫苗或其他疫苗联用的多价疫苗，例如任何一种根据权利要求1-5任一项所述的疫苗与同一病毒、病原体、肿瘤的腺病毒疫苗或mRNA疫苗或灭活疫苗或DNA疫苗按照先后顺序或同时免疫程序进行免疫，例如SARS-COV-2融合 蛋白疫苗与SARS-COV-2的腺病毒疫苗或mRNA疫苗或灭活疫苗或DNA疫苗按照先后顺序或同时免疫程序进行免疫，例如先后免疫顺序可以为：1)先免疫接种本发明的SARS-COV-2融合蛋白疫苗，后免疫；2)先免疫SARS-COV-2的腺病毒疫苗或mRNA疫苗或灭活疫苗或DNA疫苗，后免疫ARS-COV-2融合蛋白疫苗；3)SARS-COV-2融合蛋白疫苗与SARS-COV-2的腺病毒疫苗或mRNA疫苗或灭活疫苗或DNA疫苗同时免疫。
PCT/CN2021/103931 2020-07-01 2021-07-01 融合蛋白疫苗平台的构建与应用 WO2022002180A1 (zh)

Priority Applications (6)

Application Number Priority Date Filing Date Title
JP2022580808A JP2023531291A (ja) 2020-07-01 2021-07-01 融合タンパク質ワクチンプラットフォームの構築と応用
AU2021301953A AU2021301953A1 (en) 2020-07-01 2021-07-01 Construction and application of fusion protein vaccine platform
EP21833302.9A EP4176893A4 (en) 2020-07-01 2021-07-01 CONSTRUCTION AND APPLICATION OF A FUSION PROTEIN-BASED VACCINE PLATFORM
US18/003,872 US20230293679A1 (en) 2020-07-01 2021-07-01 Construction and application of fusion protein vaccine platform
CA3184383A CA3184383A1 (en) 2020-07-01 2021-07-01 Construction and application of fusion protein vaccine platform
CN202111680865.3A CN115137812A (zh) 2021-03-31 2021-12-31 融合蛋白疫苗平台的构建与应用

Applications Claiming Priority (4)

Application Number Priority Date Filing Date Title
CN202010623708.8 2020-07-01
CN202010623708.8A CN113876938B (zh) 2020-07-01 2020-07-01 融合蛋白疫苗平台的构建与应用
CN202110353488.6 2021-03-31
CN202110353488 2021-03-31

Publications (1)

Publication Number Publication Date
WO2022002180A1 true WO2022002180A1 (zh) 2022-01-06

Family

ID=79315093

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2021/103931 WO2022002180A1 (zh) 2020-07-01 2021-07-01 融合蛋白疫苗平台的构建与应用

Country Status (6)

Country Link
US (1) US20230293679A1 (zh)
EP (1) EP4176893A4 (zh)
JP (1) JP2023531291A (zh)
AU (1) AU2021301953A1 (zh)
CA (1) CA3184383A1 (zh)
WO (1) WO2022002180A1 (zh)

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN114621357A (zh) * 2022-05-17 2022-06-14 康希诺生物股份公司 一种带状疱疹亚单位疫苗及其制备方法
WO2022242649A1 (zh) * 2021-05-18 2022-11-24 珠海市丽珠单抗生物技术有限公司 一种新型冠状病毒SARS-CoV-2突变体疫苗与应用
CN116284454A (zh) * 2023-03-15 2023-06-23 中国科学院微生物研究所 用于预防猪繁殖与呼吸综合征的融合蛋白组合物及其相关生物材料与应用
WO2023236726A1 (zh) * 2022-06-07 2023-12-14 中国人民解放军军事科学院军事医学研究院 基于金属硫蛋白家族的免疫佐剂及其应用

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20100261248A1 (en) * 2003-11-13 2010-10-14 Hanmi Pham Co., Ltd. Pharmaceutical Composition Comprising An Immunoglobulin FC Region as a Carrier
CN104403004A (zh) * 2014-11-24 2015-03-11 苏州丁孚靶点生物技术有限公司 抗体-干扰素异二聚体的制备和用途
US20190225673A1 (en) * 2016-09-30 2019-07-25 Baylor College Of Medicine Antibody based gene therapy with tissue-directed expression

Family Cites Families (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
BR9915548A (pt) * 1998-10-16 2001-08-14 Biogen Inc Proteìnas de fusão de interferon-beta e usos
SK782002A3 (en) * 1999-07-21 2003-08-05 Lexigen Pharm Corp FC fusion proteins for enhancing the immunogenicity of protein and peptide antigens
MX2008013993A (es) * 2006-05-03 2009-05-11 Univ Colorado Combinación adyuvante sinergistica de anticuerpo agonista de cd40/interferona tipo 1, conjugados que la contienen y uso de los mismos como un terapeutico para aumentar la inmunidad celular.
CN102180971B (zh) * 2011-03-04 2013-10-30 中国人民解放军军事医学科学院生物工程研究所 重组β淀粉样肽B细胞表位多肽嵌合抗原、其制备方法和应用

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20100261248A1 (en) * 2003-11-13 2010-10-14 Hanmi Pham Co., Ltd. Pharmaceutical Composition Comprising An Immunoglobulin FC Region as a Carrier
CN104403004A (zh) * 2014-11-24 2015-03-11 苏州丁孚靶点生物技术有限公司 抗体-干扰素异二聚体的制备和用途
US20190225673A1 (en) * 2016-09-30 2019-07-25 Baylor College Of Medicine Antibody based gene therapy with tissue-directed expression

Non-Patent Citations (25)

* Cited by examiner, † Cited by third party
Title
BRACCI LLA SORSA VBELARDELLI F ET AL.: "Type I interferons as vaccine adjuvants against infectious diseases and cancer [J", EXPERT REVIEW OF VACCINES, vol. 7, no. 3, 2008, pages 373 - 81
CHEN QIN-YUN, WAN YAN-MIN;ZHANG XIAO-YAN: "Advances in Research on Ig G-Fc Fusion Proteins", CHINESE JOURNAL OF BIOLOGICALS, vol. 28, no. 11, 23 November 2015 (2015-11-23), China , pages 1223 - 1227, XP055885500, ISSN: 1004-5503, DOI: 10.13200/j.cnki.cjb.001153 *
HAHM BTRIFILO M JZUNIGA E I ET AL.: "Viruses evade the immune system through type I interferon-mediated STAT2-dependent, but STAT1-independent, signaling [J", IMMUNITY, vol. 22, no. 2, 2005, pages 247 - 57
HANNOUN C: "The evolving history of influenza viruses and influenza vaccines [J", EXPERT REVIEW OF VACCINES, vol. 12, no. 9, 2013, pages 1085 - 94
HOUSER KSUBBARAO K: "Influenza vaccines: challenges and solutions [J", CELL HOST & MICROBE, vol. 17, no. 3, 2015, pages 295 - 300, XP055572607, DOI: 10.1016/j.chom.2015.02.012
HU B, GUO H, ZHOU P: "Characteristics of SARS-CoV-2 and COVID-19 [J]", NATURE REVIEWS MICROBIOLOGY, 2020, pages 1 - 14
ITO TAMAKAWA RINABA M ET AL.: "Differential regulation of human blood dendritic cell subsets by IFNs [J", THE JOURNAL OF IMMUNOLOGY, vol. 166, no. 5, 2001, pages 2961 - 9, XP002971753
KONTERMANN R E.: "Strategies for extended serum half-life of protein therapeutics [J", CURRENT OPINION IN BIOTECHNOLOGY, vol. 22, no. 6, 2011, pages 868 - 76, XP028397475, DOI: 10.1016/j.copbio.2011.06.012
KRAMMER FSMITH G J DFOUCHIER R A M ET AL.: "Influenza [J", NATURE REVIEWS DISEASE PRIMERS, vol. 4, no. 1, 2018, pages 3
LE BON ADURAND VKAMPHUIS E ET AL.: "Direct stimulation of T cells by type I IFN enhances the CD8+ T cell response during cross-priming [J", THE JOURNAL OF IMMUNOLOGY, vol. 176, no. 8, 2006, pages 4682 - 9, XP002516827
LE BON AETCHART NROSSMANN C ET AL.: "Cross-priming of CD8+ T cells stimulated by virus-induced type I interferon [J", NATURE IMMUNOLOGY, vol. 4, no. 10, 2003, pages 1009 - 15, XP037924575, DOI: 10.1038/ni978
LE BON ASCHIAVONI GD'AGOSTINO G ET AL.: "Type I interferons potently enhance humoral immunity and can promote isotype switching by stimulating dendritic cells in vivo [J", IMMUNITY, vol. 14, no. 4, 2001, pages 461 - 70, XP001008314, DOI: 10.1016/S1074-7613(01)00126-1
LI QWU JNIE J ET AL.: "The impact of mutations in SARS-CoV-2 spike on viral infectivity and antigenicity [J", CELL, vol. 182, no. 5, 2020, pages 1284 - 94
LURIE NSAVILLE MHATCHETT R ET AL.: "Developing Covid-19 vaccines at pandemic speed [J", NEW ENGLAND JOURNAL OF MEDICINE, vol. 382, no. 21, 2020, pages 1969 - 73
MEKHAIEL D NCZAJKOWSKY D MANDERSEN J T ET AL.: "Polymeric human Fc-fusion proteins with modified effector functions [J", SCIENTIFIC REPORTS, vol. 1, no. 1, 2011, pages 1 - 11, XP002708372, DOI: 10.1038/srep00124
MONTOYA MSCHIAVONI GMATTEI F ET AL.: "Type I interferons produced by dendritic cells promote their phenotypic and functional activation [J", BLOOD, vol. 99, no. 9, 2002, pages 3263 - 71
PARLATO SSANTINI S MLAPENTA C ET AL.: "Expression of CCR-7, MIP-30, and Th-1 chemokines in type I IFN-induced monocyte-derived dendritic cells: importance for the rapid acquisition of potent migratory and functional activities [J", BLOOD, THE JOURNAL OF THE AMERICAN SOCIETY OF HEMATOLOGY, vol. 98, no. 10, 2001, pages 3022 - 9
QING-TONG WANG; YU-KUN MA; BEI HUANG; DAN-DAN LIU; WEI WEI;: "Effect of rhTACI-Ig fusion protein on antigen-specific T cell responses from keyhole limpet haemocyanin challenged mice", MOLECULAR IMMUNOLOGY, vol. 49, no. 1, 12 September 2011 (2011-09-12), GB , pages 380 - 386, XP028328180, ISSN: 0161-5890, DOI: 10.1016/j.molimm.2011.09.007 *
RAZAVI-SHEARER DGAMKRELILLZE INGUYEN M H ET AL.: "Global prevalence, treatment, and prevention of hepatitis B virus infection in 2016: a modelling study [J", THE LANCET GASTROENTEROLOGY & HEPATOLOGY, vol. 3, no. 6, 2018, pages 383 - 403
ROOPENIAN D CAKILESH S: "FcRn: the neonatal Fc receptor comes of age [J", NATURE REVIEWS IMMUNOLOGY, vol. 7, no. 9, 2007, pages 715 - 25
ROUZAUT AGARASA STEIJEIRA A ET AL.: "Dendritic cells adhere to and transmigrate across lymphatic endothelium in response to IFN - a [J", EUROPEAN JOURNAL OF IMMUNOLOGY, vol. 40, no. 11, 2010, pages 3054 - 63, XP071224635, DOI: 10.1002/eji.201040523
SCHWEITZER AHORN JMIKOLAJCZYK R T ET AL.: "Estimations of worldwide prevalence of chronic hepatitis B virus infection: a systematic review of data published between 1965 and 2013 [J", THE LANCET, vol. 386, no. 10003, 2015, pages 1546 - 55
SPADARO FLAPENTA CDONATI S ET AL.: "IFN-a enhances cross-presentation in human dendritic cells by modulating antigen survival, endocytic routing, and processing [J", BLOOD, THE JOURNAL OF THE AMERICAN SOCIETY OF HEMATOLOGY, vol. 119, no. 6, 2012, pages 1407 - 17, XP086694233, DOI: 10.1182/blood-2011-06-363564
VILCEK J: "Fifty years of interferon research: aiming at a moving target [J", IMMUNITY, vol. 25, no. 3, 2006, pages 343 - 8
VOS TABAJOBIR A AABATE K H ET AL.: "Global, regional, and national incidence, prevalence, and years lived with disability for 328 diseases and injuries for 195 countries, 1990-2016: a systematic analysis for the Global Burden of Disease Study 2016 [J", THE LANCET, vol. 390, no. 10100, 2017, pages 1211 - 59

Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2022242649A1 (zh) * 2021-05-18 2022-11-24 珠海市丽珠单抗生物技术有限公司 一种新型冠状病毒SARS-CoV-2突变体疫苗与应用
CN114621357A (zh) * 2022-05-17 2022-06-14 康希诺生物股份公司 一种带状疱疹亚单位疫苗及其制备方法
WO2023236726A1 (zh) * 2022-06-07 2023-12-14 中国人民解放军军事科学院军事医学研究院 基于金属硫蛋白家族的免疫佐剂及其应用
CN116284454A (zh) * 2023-03-15 2023-06-23 中国科学院微生物研究所 用于预防猪繁殖与呼吸综合征的融合蛋白组合物及其相关生物材料与应用
CN116284454B (zh) * 2023-03-15 2023-11-21 中国科学院微生物研究所 用于预防猪繁殖与呼吸综合征的融合蛋白组合物及其相关生物材料与应用

Also Published As

Publication number Publication date
CA3184383A1 (en) 2022-01-06
AU2021301953A1 (en) 2023-02-09
EP4176893A1 (en) 2023-05-10
US20230293679A1 (en) 2023-09-21
EP4176893A4 (en) 2024-10-23
JP2023531291A (ja) 2023-07-21

Similar Documents

Publication Publication Date Title
WO2022002180A1 (zh) 融合蛋白疫苗平台的构建与应用
JP6449384B2 (ja) 抗原特異的ｔ細胞反応を誘導するための免疫原エンハンサーとしての融合タンパク質
CN113321739B (zh) 一种covid-19亚单位疫苗及其制备方法与应用
CN113876938B (zh) 融合蛋白疫苗平台的构建与应用
WO2021103434A1 (zh) 一种重组水痘带状疱疹病毒疫苗
TW201712119A (zh) 新穎桿狀病毒載體及使用方法
CA2900318C (en) Induction of cross-reactive cellular response against rhinovirus antigens
CN115137812A (zh) 融合蛋白疫苗平台的构建与应用
JP2015514132A (ja) ヒト呼吸器合胞体ウイルスコンセンサス抗原、核酸構築物、およびそれらから作製されるワクチン、ならびにその使用方法
CN111556896A (zh) 交叉免疫抗原疫苗及其制备方法
WO2019076218A1 (zh) H3n2亚型流感病毒血凝素蛋白的突变体及其应用
WO2022148374A1 (zh) 抗冠状病毒的全人广谱中和抗体76e1及其应用
Kawai et al. Intranasal immunization with an RBD-hemagglutinin fusion protein harnesses preexisting immunity to enhance antigen-specific responses
US20210322544A1 (en) Vaccine composition comprising hepatitis b virus-like particle as adjuvant
WO2002012535A1 (en) Use of anti-ceacam antibodies for stimulating b cells in the production of monoclonal antibodies or in immunotherapy
WO2020143799A1 (zh) 广谱性中和流感病毒的全人抗体
WO2017120280A1 (en) Antigen targeting to porcine langerin
WO2024067785A1 (zh) 基于干扰素协同免疫检查点阻断抗体治疗慢性乙型肝炎
WO2022242649A1 (zh) 一种新型冠状病毒SARS-CoV-2突变体疫苗与应用
WO2023025257A1 (zh) 一种β冠状病毒异源多聚体抗原、其制备方法和应用
Zhang et al. IFN-α armed gE elicits superior immunogenicity compared to unmodified antigens and flagellin armed gE in mice
WO2023020637A1 (es) Antigenos quimericos para el control de coronavirus y composiciones que los comprenden
JP2007509081A (ja) 治療用の医薬組成物
JPWO2005090576A1 (ja) 百日咳感染症予防用ｄｎａ構築物
BR122024015865A2 (pt) Nanopartícula, seu uso, proteína de fusão, molécula de ácido nucleico, vacina contra o vírus do epstein-barr e método para a sua produção

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: 21833302

Country of ref document: EP

Kind code of ref document: A1

ENP Entry into the national phase

Ref document number: 2022580808

Country of ref document: JP

Kind code of ref document: A

ENP Entry into the national phase

Ref document number: 3184383

Country of ref document: CA

NENP Non-entry into the national phase

Ref country code: DE

ENP Entry into the national phase

Ref document number: 2021833302

Country of ref document: EP

Effective date: 20230201

ENP Entry into the national phase

Ref document number: 2021301953

Country of ref document: AU

Date of ref document: 20210701

Kind code of ref document: A