WO2021115410A1 - 免疫刺激组合物及其用途 - Google Patents

免疫刺激组合物及其用途 Download PDF

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WO2021115410A1
WO2021115410A1 PCT/CN2020/135572 CN2020135572W WO2021115410A1 WO 2021115410 A1 WO2021115410 A1 WO 2021115410A1 CN 2020135572 W CN2020135572 W CN 2020135572W WO 2021115410 A1 WO2021115410 A1 WO 2021115410A1
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saponins
composition according
cpg
vaccine
immunostimulatory composition
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PCT/CN2020/135572
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English (en)
French (fr)
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葛君
李建强
孙娇娇
周童
任苏林
顾月
黄红颖
王世伟
黄精俸
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南京远大赛威信生物医药有限公司
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Priority to EP20897830.4A priority Critical patent/EP4074335A1/en
Priority to JP2022535201A priority patent/JP2023506440A/ja
Priority to AU2020403296A priority patent/AU2020403296A1/en
Priority to CA3161628A priority patent/CA3161628A1/en
Priority to KR1020227023837A priority patent/KR20220114031A/ko
Priority to US17/784,089 priority patent/US20230040021A1/en
Priority to BR112022011445A priority patent/BR112022011445A2/pt
Publication of WO2021115410A1 publication Critical patent/WO2021115410A1/zh

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/12—Viral antigens
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • 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
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A61K31/711—Natural deoxyribonucleic acids, i.e. containing only 2'-deoxyriboses attached to adenine, guanine, cytosine or thymine and having 3'-5' phosphodiester links
    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/12—Viral antigens
    • A61K39/245—Herpetoviridae, e.g. herpes simplex virus
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/12—Viral antigens
    • A61K39/29—Hepatitis virus
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/12—Viral antigens
    • A61K39/29—Hepatitis virus
    • A61K39/292—Serum hepatitis virus, hepatitis B virus, e.g. Australia antigen
    • 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
    • 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
    • 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
    • A61P31/20—Antivirals for DNA 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
    • A61P31/22—Antivirals for DNA viruses for herpes viruses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P33/00—Antiparasitic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A61P37/04—Immunostimulants
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
    • A61K2039/55511—Organic adjuvants
    • A61K2039/55561—CpG containing adjuvants; Oligonucleotide containing adjuvants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
    • A61K2039/55511—Organic adjuvants
    • A61K2039/55577—Saponins; Quil A; QS21; ISCOMS
    • 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
    • A61K2039/572—Medicinal preparations containing antigens or antibodies characterised by the type of response, e.g. Th1, Th2 cytotoxic 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
    • A61K2039/57—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
    • A61K2039/70—Multivalent vaccine
    • 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
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • the invention belongs to the field of biopharmaceuticals. Specifically, the present invention relates to an immunostimulatory composition comprising saponins and CpG oligodeoxynucleotides, or the immunostimulatory composition is composed of an adjuvant comprising saponin and CpG oligodeoxynucleosides. Acid composition. Wherein, the CpG oligodeoxynucleotide sequence has two or more copies of 5'-TTCGTT-3' motif or 5'-TCGTCGTCG-3' motif.
  • the invention also relates to the pharmaceutical use of the immunostimulatory composition.
  • CpG oligodeoxynucleotide is a new type of immunostimulant discovered in recent years. Its chemical essence is a deoxy oligonucleotide containing cytosine guanine dinucleotide, which has immunity similar to that of natural CpG pattern recognition receptors. The reaction can bind to the Toll-like receptors on the cell membrane and effectively trigger the mammalian immune response through the TLR9 signaling pathway.
  • the immune response caused by CpG is mainly Th1 type, which can induce the conversion of Th2 type immune response to Th1, thereby stimulating cellular immunity.
  • T cells, B cells, NK cells and other immunologically active cells By activating T cells, B cells, NK cells and other immunologically active cells, a large number of cytokines are produced, thereby enhancing the body's specific and non-specific immune effects. It is an important link connecting natural immunity and acquired immunity.
  • Saponins are a class of glycosides in which aglycones are triterpenoids or spirostane compounds, and belong to plant-derived adjuvants. Among them, Quercus saponins (QS) are saponins extracted from Quillaja saponaria. In the QS series, QS-21 is currently the most widely reported type of adjuvant, but QS-21 can induce cell hemolysis and has certain systemic side effects. And local side effects. Alving et al. (ALVING CR, MATYAS G, BECK Z, et al.
  • Ng et al. (NG H, FERNANDO G J P, DEPELSENAIRE A C I, et al. Scientific Reports, 2016, 6(1): 228-230.) use a subcutaneous transfer technology-nanopatch, which is composed of QS-21 Adjuvant complex.
  • nanopatch can significantly reduce the dosage of antigen and QS-21 and induce higher IgG titer (Han Ziyi, Zeng Zhongliang, Modern Agricultural Science and Technology, 2019(14):220- 221.).
  • the inventor unexpectedly discovered an immunostimulatory composition with stronger immune effect.
  • the saponins and CpG oligodeoxynucleotides It exhibits highly effective synergy and can mediate a stronger immune response.
  • Application of the immunostimulatory composition to different antigens or antigen compositions has significant advantages.
  • an object of the present invention is to provide an immunostimulatory composition, which can be used to prepare a variety of drugs to obtain high-efficiency immunostimulatory properties.
  • Another object of the present invention is to provide a vaccine adjuvant that can strongly elicit a mammalian immune response.
  • the present invention provides an immunostimulatory composition comprising saponin and CpG oligodeoxynucleotide, or consisting of an adjuvant comprising saponin and CpG oligodeoxynucleotide, wherein The CpG oligodeoxynucleotide sequence has two or more copies of 5'-TTCGTT-3' motif or 5'-TCGTCGTCG-3' motif.
  • the immunostimulatory composition according to the present invention wherein the sequence of the CpG oligodeoxynucleotide is selected from any one of the following: CpG T1: TCG TTC GTT CGT TCG TTC GTT (SEQ ID NO: 6) , CpG T2: TCG TTC GTT CGT TCG TTC GTT CGT T (SEQ ID NO: 7) and CpG T3: TCG TCG TCG TCG TCG TCG TCG TCG (SEQ ID NO: 8).
  • the sequence of the CpG oligodeoxynucleotide is CpG T1: TCG TTC GTT CGT TCG TTC GTT (SEQ ID NO: 6).
  • the saponins are selected from the group consisting of saponins of Quercus saponins, ginsenosides, platycodon saponins, astragalus saponins, notoginseng saponins, glycyrrhiza saponins, albizia bark saponins, Ophiopogon japonicus saponins, saikosaponins or One or more of Panax Japonicus saponins; preferably, the saponins are Quillaja saponins, ginsenosides, platycodin, or astragaloside IV; more preferably, the saponins are QS-7, QS-17, QS-18 or QS-21; more preferably, the Quercus saponin is QS-21; the ginsenoside may be ginsenoside Rg1, ginsenoside Rg3, ginsenoside Rb1 or ginsen
  • immunostimulatory composition wherein the adjuvant containing saponin is an immunostimulatory complex adjuvant (Iscom adjuvant).
  • immunostimulatory complex adjuvant Iscom adjuvant
  • the immunostimulatory composition according to the present invention wherein the CpG oligodeoxynucleotide comprises a phosphorothioate linkage.
  • the CpG oligodeoxynucleotide is a thio-oligodeoxynucleotide, preferably a full-thio-oligodeoxynucleotide.
  • the immunostimulatory composition according to the present invention wherein the weight ratio of the CpG oligodeoxynucleotide to the saponin is 1-40:0.1-2, preferably 2-40:0.1-2, more preferably It is 2:1.
  • the present invention also provides a pharmaceutical composition, which comprises the immunostimulatory composition, and an antigen or an antigen composition.
  • the pharmaceutical composition according to the present invention wherein the antigen or antigen composition is selected from any one of the following: human immunodeficiency virus, human herpes virus, varicella-zoster virus, human cell hypertrophy virus, A, Hepatitis B, C or E virus, respiratory syncytial virus, human papilloma virus, influenza virus, Mycobacterium tuberculosis, Salmonella, Neisseria, such as Neisseria meningitidis or Neisseria gonorrhoeae, Borrelia genus such as return Treponema or Dotonella, Chlamydia such as Chlamydia trachomatis, Bordetella such as Bordetella pertussis, Plasmodium such as Plasmodium falciparum, Plasmodium malariae, egg Plasmodium ovale, Plasmodium vivax or Plasmodium knowlesi or Toxoplasma genus such as Toxoplasma.
  • composition according to the present invention wherein the human herpes virus is HSV1 or HSV2.
  • composition according to the present invention wherein the antigen is a tumor antigen.
  • the present invention provides a vaccine comprising the immunostimulatory composition.
  • the vaccine according to the present invention wherein the vaccine is a vaccine for preventing viral, bacterial and/or parasitic infections, or the vaccine is a vaccine for treating viral, bacterial and/or parasitic infections by immunotherapy.
  • the present invention provides the use of the immunostimulatory composition in the preparation of a medicament for causing a cytolytic T cell response.
  • the present invention provides the use of the immunostimulatory composition in the preparation of a medicament for inducing an interferon gamma response in a mammal.
  • the present invention provides the use of the immunostimulatory composition in the preparation of a vaccine for preventing viral, bacterial and/or parasitic infections.
  • the present invention provides the use of the immunostimulatory composition in the preparation of a vaccine for the treatment of viral, bacterial and/or parasitic infections by immunotherapy.
  • the present invention provides the use of the immunostimulatory composition in the preparation of a vaccine for the treatment of tumors by immunotherapy.
  • the present invention also provides a method for inducing a cytolytic T cell response, the method comprising administering to a subject in need an effective amount of a pharmaceutical composition comprising the immunostimulatory composition of the present invention.
  • the present invention also provides a method for inducing an interferon gamma response in a mammal, the method comprising administering an effective amount of a pharmaceutical composition comprising the immunostimulatory composition of the present invention to a subject in need.
  • the present invention also provides a method for preventing infection by viruses, bacteria and/or parasites, the method comprising administering to a subject in need a preventively effective amount of a vaccine comprising the immunostimulatory composition of the present invention.
  • the present invention also provides a method for treating viral, bacterial and/or parasitic infections with immunotherapy, the method comprising administering to a subject in need an effective amount of a vaccine comprising the immunostimulatory composition of the present invention.
  • the present invention also provides a method for treating tumors, the method comprising administering to a subject in need a therapeutically effective amount of a pharmaceutical composition comprising the immunostimulatory composition of the present invention.
  • the immunostimulatory composition provided by the present invention achieves unexpected technical effects and can mediate a stronger immune response.
  • the immunostimulatory effect of CpG T1-T3 alone is weaker than that of CpG1018, CpG7909, CpG1826, etc., but after combined application with QS-21, the immunostimulatory composition exhibits unexpected synergistic effects, and the immune effect is significantly enhanced.
  • the hepatitis B therapeutic vaccine containing the immunostimulatory composition can break through the immune tolerance of the transgenic mice and produce high-titer anti-HBsAg antibodies, anti-HBcAg antibodies, and neutralizing antibodies.
  • Various test results show that the vaccine can significantly eliminate hepatitis B virus in transgenic mice through multiple immunizations. After the immunization process, the HBsAb level is close to saturation, which can maintain a stable and long-lasting immune effect. The average HBsAg decline rate is maintained at Around 92%.
  • the hepatitis B vaccine containing the immunostimulatory composition can induce strong HBsAg and HBcAg specific IFN- ⁇ levels, and the immune effect is significantly better than that of the adjuvant alone, and also significantly better than the existing CPG adjuvant and QS-21 The combination.
  • the shingles vaccine containing the immune stimulating composition also proves that the immune stimulating composition has superior immune stimulating effects.
  • Cellular immunity experiments have proved that the vaccine can induce a strong level of herpes gE protein-specific IFN- ⁇ , and the protein immune effect is significantly better than that of a single adjuvant.
  • Humoral immunity experiments also proved that the vaccine can produce higher levels of herpes gE protein-specific IgG/IgG1/IgG2a antibodies, and the effect is better than a single adjuvant, and it is also significantly better than the existing combination of CPG adjuvant and QS-21.
  • the immunostimulatory composition provided by the present invention has a superior immunostimulatory effect.
  • the immunostimulatory composition of the present invention has CpG T1-T3 and QS- 21 shows highly effective synergy and can mediate a stronger immune response. It has significant advantages when applied to different antigens or antigen compositions. Therefore, as a new adjuvant, the immunostimulatory composition of the present invention has high clinical application value and broad market prospects.
  • Figure 1 shows the effect of different CPG oligodeoxynucleotides on the secretion level of HBsAg antigen-specific IFN- ⁇ ;
  • Figure 2 shows the effect of different CPG oligodeoxynucleotides on the secretion level of HBcAg antigen-specific IFN- ⁇ ;
  • Figure 3 shows the effect of different immunostimulatory compositions according to the present invention on the secretion level of HBsAg antigen-specific IFN- ⁇ ;
  • Figure 4 shows the effect of different immunostimulatory compositions according to the present invention on the secretion level of HBcAg antigen-specific IFN- ⁇ ;
  • Figure 5 shows the effect of different doses of immunostimulatory composition according to the present invention on the secretion level of HBsAg antigen-specific IFN- ⁇ ;
  • Figure 6 shows the effect of different doses of immunostimulatory composition according to the present invention on the secretion level of HBcAg antigen-specific IFN- ⁇ ;
  • Figure 7 shows the effect of hepatitis B vaccine containing the immunostimulatory composition of the present invention on the level of HBsAg in serum;
  • Figure 8 shows the effect of hepatitis B vaccine containing the immunostimulatory composition of the present invention on the level of HBsAb in serum;
  • Figure 9 shows the effect of hepatitis B vaccine containing the immunostimulatory composition of the present invention on the secretion level of HBsAg antigen-specific IFN- ⁇ ;
  • Figure 10 shows the effect of hepatitis B vaccine containing the immunostimulatory composition of the present invention on the secretion level of HBcAg antigen-specific IFN- ⁇ ;
  • Figure 11 shows the effect of hepatitis B vaccine containing the immunostimulatory composition of the present invention on the levels of HBsAg antigen-specific IgG antibodies and their subtypes in mouse serum;
  • Panel A HBsAb IgG levels in the serum of each group of mice
  • Panel B HBsAb IgG1 levels in the serum of each group
  • Panel C HBsAb IgG2a levels in the serum of each group of mice
  • Panel D Level of the serum of each group of mice The ratio of HBsAb IgG2a to IgG1;
  • Figure 12 shows the effect of hepatitis B vaccine containing the immunostimulatory composition of the present invention on the levels of HBcAg antigen-specific IgG antibodies and their subtypes in mouse serum;
  • panel A HBcAb IgG levels in the serum of each group of mice
  • panel B HBcAb IgG1 levels in the serum of each group of mice
  • panel C HBcAb IgG2a levels in the serum of each group of mice
  • panel D levels of the serum of each group of mice The ratio of HBcAb IgG2a to IgG1;
  • Figure 13 shows the effect of the herpes zoster vaccine containing the immunostimulatory composition of the present invention on the secretion level of herpes gE antigen-specific IFN- ⁇ ;
  • Figure 14 shows the effect of the herpes zoster vaccine containing the immunostimulatory composition of the present invention on the levels of antigen-specific IgG antibodies and their subtypes in the serum of mice
  • panel A IgG levels in the serum of mice in each group
  • panel B IgG1 levels in the serum of mice in each group
  • panel C IgG2a levels in the serum of mice in each group
  • panel D IgG2a and IgG1 in the serum of mice in each group Ratio.
  • Figure 15 shows the effect of immunostimulatory compositions containing different saponins according to the present invention on the secretion level of herpes gE antigen-specific IFN- ⁇ .
  • the terms “pharmaceutical composition”, “combination drug” and “drug combination” are used interchangeably, which means at least one drug and optional pharmaceutically acceptable excipients combined together to achieve a specific purpose A combination of agents or excipients.
  • the pharmaceutical composition includes combinations separated in time and/or space, as long as they can work together to achieve the purpose of the present invention.
  • the ingredients eg, gE protein, QS-21, CpG oligodeoxynucleotide
  • the ingredients contained in the pharmaceutical composition may be administered to the subject as a whole, or separately administered to the subject.
  • the ingredients contained in the pharmaceutical composition are separately administered to the subject, the ingredients may be administered to the subject simultaneously or sequentially.
  • CpG oligodeoxynucleotide or “CpG-ODN” as used herein refers to a short single-stranded synthetic DNA molecule containing one or more "CpG" units, where C represents cytosine and G represents bird Purine, p represents a phosphodiester bond.
  • C represents cytosine and G represents bird Purine
  • p represents a phosphodiester bond.
  • the CpG oligodeoxynucleotide is unmethylated.
  • the CpG-ODN comprises a phosphorothioate linkage or phosphorothioate backbone.
  • the CpG-ODN is phosphorothioate oligodeoxynucleotide (i.e., thiooligodeoxynucleotide).
  • all internucleotide linkages in the CpG-ODN are phosphorothioate linkages, that is, the CpG-ODN is a full-thio-oligodeoxynucleotide.
  • the CpG-ODN contains two or more copies of the 5'-TTCGTT-3' motif or 5'-TCGTCGTCG-3' motif.
  • the CpG-ODN has a sequence selected from the following: TCG TTC GTT CGT TCG TTC GTT (SEQ ID NO: 6), TCG TTC GTT CGT TCG TTC GTT CGT T (SEQ ID NO: 7) or TCG TCG TCG TCG TCG TCG (SEQ ID NO: 8), preferably TCG TTC GTT CGT TCG TTC GTT (SEQ ID NO: 6).
  • Ginsenosides platycodon saponins, astragalus saponins, notoginseng saponins, glycyrrhiza saponins, albizia bark saponins, Ophiopogon saponins, saikosaponins or panax japonicus saponins
  • Ginsenoside is a kind of sterol compound, which mainly exists in medicinal materials of the genus Ginseng, and is the active ingredient in ginseng.
  • the ginsenoside is preferably monomers such as ginsenoside Rg1, ginsenoside Rg3, ginsenoside Rb1, ginsenoside Re, etc., or a mixture of two or more saponins monomers;
  • platycodin is preferably platycodin D.
  • Astragalus saponins are preferably monomers such as Astragaloside IV (Astragaloside IV), Astragaloside I, Astragaloside II, or a mixture of two or more of them; 3.
  • Heptasaponins are preferably notoginsenoside R1, etc.; Ophiopogon japonicus saponins are preferably Ophiopogon japonicus saponins D, etc.; Saikosaponins are preferably saikosaponin a, saikosaponin d or a mixture of both; Albizia saponins are preferably Albizia bark total Saponins and the like; glycyrrhizin are preferably total glycyrrhiza saponins and the like; and ginseng saponins are preferably total saponins of ginseng and the like.
  • Iscom adjuvant used herein is an immunostimulatory complex adjuvant, specifically Iscom matrix (ISCOM MATRIX) that does not contain an antigen, and is an adjuvant with a cage structure composed of phospholipids, saponins, and cholesterol.
  • terapéuticaally and/or prophylactically effective amount refers to a dose sufficient to show its benefit to the subject to which it is administered.
  • the actual amount administered, as well as the rate and time course of administration, will depend on the condition and severity of the individual being treated.
  • the prescription of treatment (such as the decision on dosage, etc.) is ultimately the responsibility of the general practitioner and other doctors and rely on them to make decisions, usually taking into account the disease to be treated, the individual patient’s condition, the delivery site, the method of application, and what is already known to the doctor. Know other factors.
  • mammal refers to humans, but can also be other animals, such as wild animals (such as herons, storks, cranes, etc.), domestic animals (such as ducks, geese, etc.) or laboratory animals (such as orangutans, monkeys, etc.) Rats, mice, rabbits, guinea pigs, groundhogs, ground squirrels, etc.).
  • wild animals such as herons, storks, cranes, etc.
  • domestic animals such as ducks, geese, etc.
  • laboratory animals such as orangutans, monkeys, etc.
  • Rats mice, rabbits, guinea pigs, groundhogs, ground squirrels, etc.
  • composition of the present invention may also contain additional additives, such as pharmaceutically acceptable carriers or additives, especially when it is in the form of a pharmaceutical preparation.
  • Preferred pharmaceutical carriers are especially water, buffered aqueous solutions, preferably isotonic saline solutions such as PBS (phosphate buffered saline), glucose, mannitol, dextrose, lactose, starch, magnesium stearate, cellulose, magnesium carbonate, 0.3 % Glycerin, hyaluronic acid, ethanol or polyalkylene glycols such as polypropylene glycol, triglycerides, etc.
  • the type of pharmaceutical carrier used depends in particular on whether the composition according to the invention is formulated for oral, nasal, intradermal, subcutaneous, intramuscular or intravenous administration.
  • the composition according to the invention may contain wetting agents, emulsifiers or buffer substances as additives.
  • compositions, vaccine or pharmaceutical preparation according to the present invention can be administered by any suitable route, such as oral, nasal, intradermal, subcutaneous, intramuscular or intravenous administration.
  • HBsAg stock solution the amino acid sequence of HBsAg protein is shown in SEQ ID NO:1.
  • the HBsAg protein is prepared from HBsAg gene recombinant yeast cells.
  • the types of yeast cells include Hansenula, Saccharomyces cerevisiae and Pichia pastoris, preferably Hansenula.
  • HBsAg gene recombinant Hansenula cells are fermented and cultured, and the bacteria are harvested. It is purified by sterilization treatment, silica gel adsorption, column chromatography and TFF.
  • HBcAg stock solution the amino acid sequence of HBcAg protein is shown in SEQ ID NO: 2.
  • the HBcAg protein is prepared from HBcAg gene recombinant yeast cells, and the types of yeast cells include Hansenula, Saccharomyces cerevisiae and Pichia pastoris, preferably Hansenula.
  • yeast cells include Hansenula, Saccharomyces cerevisiae and Pichia pastoris, preferably Hansenula.
  • HBcAg gene recombinant Hansenula cells are fermented and cultured, and the cells are harvested. After bacteriostasis, ammonium sulfate, column chromatography and TFF purification, the HBcAg stock solution is prepared.
  • the oligodeoxynucleotide is a synthetically prepared oligodeoxynucleotide sequence fragment, which contains one or more CpG motifs.
  • the oligodeoxynucleotide sequence used in this example is shown in Table 1:
  • the phosphoramidite tetrazole active intermediate Enter the synthesis column to form the phosphoramidite tetrazole active intermediate, which undergoes a condensation reaction with the deprotected nucleotides on CpG; 3) Connection: the phosphoramidite tetrazole active intermediate encounters the deprotection on CpG When the nucleotide of the protective group, it will have an affinity reaction with its 5'hydroxyl group, condense and remove the tetrazole. At this time, the oligonucleotide chain will extend forward by one base; 4) Oxidation: the nucleotide is single in the condensation reaction.
  • the body is connected to the oligonucleotide connected to CpG through a phosphorous ester bond, and the phosphorous ester bond is unstable and easy to be hydrolyzed by acid or alkali.
  • a thio reagent is used to oxidize the phosphoramidite to a phosphorous double bond.
  • This terminal hydroxyl after the above five steps, a deoxynucleotide is connected to the nucleotide of CpG; repeat the above deprotection, activation, ligation, oxidation, and sealing process to obtain a crude DNA fragment; finally It can be subjected to post-synthesis treatments such as cutting, deprotection, purification, and quantification.
  • mice C57BL/6(N) mice, male, 4 weeks old, 135 animals, Shanghai Lingchang Experimental Animal Technology Co., Ltd.
  • each injection volume is 100 ⁇ L/mouse.
  • group A is a negative control with 100 ⁇ L of PBS solution per mouse.
  • the stimulator HBsAg-specific peptide library PS4 and HBcAg-specific peptide library PCP were used to stimulate splenocytes; according to the kit instructions, use ELISPOT kit (BD company) to detect the secretion level of HBsAg and HBcAg antigen-specific IFN- ⁇ ; use ImmunoSPOT Series 3
  • the enzyme-linked spot analyzer reads the number of spots measured by the ELISPOT kit (for specific operation steps, refer to Example 7 of Chinese Patent CN104043120B).
  • the HBsAg-specific peptide library sequence refers to Example 7 of Chinese Patent CN104043120B; the HBcAg-specific peptide library sequence is shown in SEQ ID NO: 16-30.
  • mice C57BL/6(N) mice, male, 4 weeks old, 81 animals, Shanghai Lingchang Experimental Animal Technology Co., Ltd.,
  • each injection volume is 100 ⁇ L/mouse.
  • group A is a negative control with 100 ⁇ L of PBS solution per mouse.
  • Example 4 The influence of different content adjuvants on the immune effect of the pharmaceutical composition
  • mice C57BL/6(N) mice, male, 4 weeks old, 60 animals, Shanghai Lingchang Experimental Animal Technology Co., Ltd.
  • HBsAg protein, HBcAg protein and CpG T1 are all prepared in Example 1;
  • C57BL/6(N) mice male, 4 weeks old, 81 mice, Shanghai Lingchang Laboratory Animal Technology Co., Ltd., rAAV8-HBV adenovirus, purchased from Beijing Wujiahe Institute of Molecular Medicine Co., Ltd.
  • RAAV8-HBV adenovirus was injected into the upper tail vein of C57BL/6(N) mice to establish a mouse model of continuous rAAV8-HBV infection with C57BL/6(N).
  • mice see Table 5.
  • the injection volume per mouse is 100 ⁇ L/mouse, and group A is the negative control, and 100 ⁇ L/mouse of PBS solution is injected.
  • mice All mice were given intramuscular injection once every 2 weeks, and the site of inoculation was the right back thigh. A total of 6 doses were administered, respectively, at the 4th, 6th, and 6th weeks after the tail vein injection of the rAAV8-HBV virus. 8 weeks, 10th week, 12th week, 14th week administration. Blood is collected every 2 weeks after the start of the administration, and the blood collection times are the 4th week, the 6th week, the 8th week, the 10th week, the 12th week, the 14th week, the 16th week, the 18th week, and the 20th week. Week, 22nd week. All mice were sacrificed on the 22nd week.
  • Example 6 The effect of hepatitis B vaccine on the level of HBsAg in serum
  • Serum HBsAg detection steps entrust Nanjing Gulou Hospital to test.
  • a two-step immunoassay method first detect the binding between the sample and the paramagnetic microparticles coated with hepatitis B surface antibody. After washing, add the hepatitis B surface antibody conjugate labeled with acridinium ester, and then after washing, add the pre-excitation solution and The challenge solution is added to the reaction mixture, and the relative luminescence value (RLU) of the test sample is measured.
  • RLU relative luminescence value
  • the HBsAg content in the sample is positively correlated with RLU.
  • the HBsAg concentration in the mouse serum sample is determined by the ARCHTITECT HBsAg standard curve generated, and the final mouse The HBsAg concentration in the serum sample is 50 to 200 times the measured value.
  • the HBsAg level of this group decreased by more than 30% after the second immunization (the 6th week), and after the third immunization (the 8th week)
  • the HBsAg level dropped by more than 70%, and the average drop rate remained around 92% after the 14th week of immunization.
  • H still maintained a stable immune effect after the 14th week of immunization, and the immune level was significantly better than that of group I.
  • Serum HBsAb testing steps entrust Nanjing Gulou Hospital to test.
  • a two-step immunoassay method is used, that is, the test sample is mixed with recombinant HBsAg (rHBsAg) coated paramagnetic microparticles, washed, and the acridinium ester-labeled rHBsAg conjugate is added, and then washed, and the pre-excitation solution and excitation solution are added to the reaction In the mixture, determine the relative luminescence value (RLU) of the test sample.
  • the HBsAb content in the sample is positively correlated with RLU.
  • the HBsAb concentration in the mouse serum sample is determined by the generated ARCHTITECT HBsAb calibration curve, and the final HBsAb in the mouse serum sample The concentration is 50 to 200 times the measured value.
  • Evaluation index If the number of spots in the control wells ⁇ 5SCF, and the number of spots in the sample wells ⁇ 10SCF, it is judged as positive; if 5SFC ⁇ the number of spots in the control well ⁇ 10SCF, and the number of spots in the sample wells/the number of control wells ⁇ 2, it is judged as positive; If the number of spots in the control hole>10SFC, and the number of spots in the sample hole/the number of spots in the control hole ⁇ 3, it is judged as positive.
  • the results of ELISPOT spots are shown in Figures 9 and 10.
  • the analysis results show that the HBsAg-specific IFN- ⁇ positive conversion rate of the F group-I group is 100%, and the HBcAg-specific IFN- ⁇ positive conversion rate is both 100%.
  • the H group vaccine containing the immune stimulant can induce higher HBsAg and HBcAg specific IFN- ⁇ levels, which are greater than 2350SFC/10 6 spleen cells and greater than 1250SFC/10 6 spleen cells, respectively, compared with the CpG group alone (F Compared with QS-21 group (G group), there is a significant difference.
  • Detection steps Coat 96-well microtiter plate with purified HBsAg and HBcAg to form a solid phase antigen. After blocking, the serum to be tested is diluted with a certain initial dilution as multiples, and multiple dilutions are set , Add the diluted serum sample to the 96-well microtiter plate, and then combine with the HRP-labeled anti-IgG/IgG1/IgG2a antibody to form an antigen-antibody (serum)-enzyme-labeled antibody complex, and finally add the substrate TMB Develop the color and measure the absorbance (OD value) at 450nm wavelength with a microplate reader.
  • the depth of the color is positively correlated with the level of HBsAg and HBcAg specific antibodies IgG/IgG1/IgG2a in the test sample.
  • the "OD value of absorbance” The relationship curve of serum sample dilution factor (Log)" to determine the antibody titer.
  • the levels of HBsAb IgG antibodies and subtypes in the serum of mice were detected by ELISA at different times in each group as shown in Figure 11.
  • the H group vaccine containing the immune stimulant produced higher titers of anti-HBsAg-specific IgG/IgG1/IgG2a antibodies, and with the increase in the number of immunizations, the antibody level continued to increase.
  • the specific antibody titer can reach 5.4 logarithmic values or more; no specific antibody is detected in the A group-D group, although the E group-G group has a certain level of HBsAg-specific IgG/IgG1/IgG2a antibody , But the antibody level was significantly lower than that of the H group, and the anti-HBsAg-specific IgG and IgG2a antibody levels produced by the dual adjuvant control (group I) were significantly lower than that of the H group.
  • the levels of HBcAb IgG antibodies and subtypes in the serum of mice detected by ELISA at different times in each group are shown in Figure 12.
  • the H group vaccine containing the immune stimulant produced higher titers of anti-HBcAg-specific IgG/IgG1/IgG2a antibodies, and with the increase in the number of immunizations, the antibody level continued to increase.
  • the specific antibody titer can reach more than 4.8 logarithms; no specific antibody is detected in the A group-D group, although the E group-G group has a certain level of HBcAg-specific IgG/IgG1/IgG2a antibody , But the antibody level was significantly lower than that of the H group.
  • group H is more inclined to the Th1 pathway.
  • Figure D shows that the specific antibody IgG2a shows a significant upward trend, reflecting that the vaccine of group H can promote the conversion of anti-HBcAg antibody subtypes, and the conversion efficiency is significantly higher than that of the dual adjuvant control (group I).
  • mice C57BL/6(N) mice, female, 5 weeks old, 48 mice, purchased from Shanghai Slack Laboratory Animal Co., Ltd.
  • Herpes gE protein The amino acid sequence is shown in SEQ ID NO: 31.
  • the fermentation broth was dialyzed into a solution containing 50 mM Tris-HCl, 500 mM NaCl, and 20 mM imidazole in a 4°C chromatography cabinet at a dialysis ratio of 1:100.
  • the dialysis was performed every 4 hours for a total of 3 times; the sample was collected through a nickel column.
  • Purify collect the target protein peak samples for SDS-PAGE detection, combine the purer purification solution, and dialyze the solution containing 20mM phosphate and 150mM NaCl in a 4°C chromatography cabinet for 24h, the dialysis ratio is 1:100, and change every 8h Liquid once; pass the sample through a 0.22um sterile filter membrane and store in a refrigerator at 4°C for later use.
  • the prepared herpes gE protein stock solution requires that its purity be greater than 95%, the protein content is not less than 200 ⁇ g/ml, and the endotoxin level is not more than 0.1 Eu/ug.
  • mice see Table 7.
  • the injection volume per mouse is 100 ⁇ L/mouse, and group A is the negative control, and 100 ⁇ L/mouse of PBS solution is injected.
  • mice All groups were given intramuscular injection once every two weeks, and the site of inoculation was the right back thigh. Two consecutive administrations, namely injections in the 0th week and the 2nd week, respectively, and all groups were injected in the 4th week. The mice were sacrificed.
  • the detection steps and evaluation indicators are the same as in Example 2; the sequence of the gE-specific peptide library is shown in SEQ ID NO. 32-46.
  • Detection procedure Collect blood and separate serum on the 28th day after immunization (place the whole blood in a constant temperature incubator at 37°C for 40 minutes, centrifuge at 12000 rpm and 4°C for 10 minutes; aspirate the supernatant, freeze it at -20°C for later use), follow the reagents Box instructions, use an ELISA kit (Shanghai Kehua) to detect the positive conversion rate of the herpes gE protein-specific antibody.
  • a blank control, a negative control, and a sample to be tested are set up in the test, each with two parallel wells, the negative control is negative mouse serum; in addition to the blank control, each well is added with a negative control or a sample to be tested, and then an enzyme conjugate After mixing and sealing the plate, incubate at 37°C for 30 minutes; wash each well with washing solution, add developer A and developer B to each well, mix and seal the plate and incubate at 37°C for 15 minutes; add stop solution to each well to mix Evenly; use a microplate reader to read the OD value of each well at 450nm wavelength.
  • Example 13 The effect of different saponins on the efficacy of recombinant shingles vaccine composition
  • mice C57BL/6(N) mice, female, 5 weeks old, 48 mice, purchased from Shanghai Slack Laboratory Animal Co., Ltd.
  • QS-21 (CAS.NO.A010-023, purchased from BRENNTAG); Ginsenoside Rg1 (CAS: 22427-39-0, purchased from Nanjing Chunqiu Bioengineering Co., Ltd.), Astragaloside IV (CAS: 84687- 43-4, purchased from Nanjing Chunqiu Biological Engineering Co., Ltd.), Platycodin D (CAS: 58479-68-8, purchased from Hubei Yunmei Technology Co., Ltd.), Iscom adjuvant (purchased from Shanghai Xiyuan Biological Technology Co., Ltd.)
  • each injection volume is 100 ⁇ L/mouse.
  • the control group was injected with 100 ⁇ L of PBS solution per mouse.
  • the immune composition provided by the present invention has a superior immune stimulating effect. Compared with the combination of a single adjuvant, other CPG adjuvants and QS21, CpG T1 ⁇ T3 and QS-21 show a highly effective synergistic effect. Mediates a stronger immune response. It has significant advantages when applied to different antigens or antigen compositions. Therefore, as a new type of adjuvant, the immune composition has high clinical application value and broad market prospects.

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Abstract

提供了一种免疫刺激组合物，其包含皂苷和CpG寡聚脱氧核苷酸或者其由包含皂苷的佐剂和CpG寡聚脱氧核苷酸组成，其中所述CpG寡聚脱氧核苷酸序列中具有两个或两个以上拷贝的5'-TTCGTT-3'基序或5'-TCGTCGTCG-3'基序；还提供了所述免疫刺激组合物在制备用于治疗疾病的药物中的用途。

Description

免疫刺激组合物及其用途 技术领域
本发明属于生物制药领域。具体地，本发明涉及一种免疫刺激组合物，所述免疫刺激组合物包含皂苷和CpG寡聚脱氧核苷酸，或者所述免疫刺激组合物由包含皂苷的佐剂和CpG寡聚脱氧核苷酸组成。其中，所述CpG寡聚脱氧核苷酸序列具有两个或两个以上拷贝的5’-TTCGTT-3’基序或5’-TCGTCGTCG-3’基序。本发明还涉及所述免疫刺激组合物的制药用途。
背景技术
CpG寡聚脱氧核苷酸是近年来发现的一类新型免疫刺激剂，其化学本质为含有胞嘧啶鸟嘌呤二核苷酸的脱氧寡核苷酸，具有与天然CpG模式识别受体相似的免疫反应，能与细胞膜上的Toll样受体结合，通过TLR9信号通路有效引发哺乳动物免疫反应。CpG引起的免疫反应以Th1型为主，可诱导Th2型免疫应答向Th1转换，从而激发细胞免疫。通过激活T细胞、B细胞、NK细胞等免疫活性细胞，大量地产生多种细胞因子，从而增强机体的特异性和非特异性免疫效应，是连接天然免疫和获得性免疫的重要纽带。
皂苷是苷元为三萜或螺旋甾烷类化合物的一类糖苷，属植物来源佐剂。其中，皂树皂苷(QS)是从皂树中提取得到的皂苷，在QS系列中QS-21是目前报道最广泛的一类佐剂，但QS-21可诱导细胞溶血，有一定系统毒副作用和局部的毒副作用。Alving等(ALVING CR,MATYAS G,BECK Z,et al.Revue Roumaine de Chimie,2016,61(8):631-635.)研究发现，以ALF脂质体结合MPLA和QS-21作为佐剂对抗HIVgp140蛋白可有效增加血清中抗体滴度。Ng等(NG H,FERNANDO G J P,DEPELSENAIRE A C I,et al.Scientific Reports,2016,6(1):228-230.)使用一种皮下传递技术-纳米贴片，与QS-21构成佐剂复合物。结果表明，纳米贴片与传统肌内注射相比，可显著减少抗原和QS-21的使用剂量以及诱导更高的IgG滴度(韩子怡，曾忠良，现代农业科技，2019(14):220-221.)。
现有技术(WO2001051083A3)曾报道关于包含皂苷与CpG寡聚脱氧核苷酸的免疫刺激组合物，其中CpG寡聚脱氧核苷酸涉及CpG1826和CpG7909。然而，由于CpG寡聚脱氧核苷酸具备结构多样性，不同序列的CpG佐剂间的效果差异巨大。
因此，当前对免疫效果更强的佐剂以及药物存在需求。
发明内容
针对现有技术的不足，发明人在进行了大量研究之后，出乎意料的发现了免疫效果更强的免疫刺激组合物，在所述组合物中，皂苷和CpG寡聚脱氧核苷酸之间表现出高效的协同作用，可以介导更强烈的免疫应答。将所述免疫刺激组合物应用于不同的抗原或抗原组合物中，均具有显著性优势。
因此，本发明的一个目的是提供一种免疫刺激组合物，所述组合物可用于制备多种药物，以获得高效的免疫刺激原性。
本发明的另一个目的是提供一种疫苗佐剂，所述疫苗佐剂能强烈地引发哺乳动物的免疫反应。
本发明的目的是通过以下技术方案实现的：
一方面，本发明提供了一种免疫刺激组合物，所述免疫刺激组合物包含皂苷和CpG寡聚脱氧核苷酸，或者由包含皂苷的佐剂和CpG寡聚脱氧核苷酸组成，其中所述CpG寡聚脱氧核苷酸序列具有两个或两个以上拷贝的5’-TTCGTT-3’基序或5’-TCGTCGTCG-3’基序。
根据本发明所述的免疫刺激组合物，其中，所述CpG寡聚脱氧核苷酸的序列选自以下中的任一种：CpG T1：TCG TTC GTT CGT TCG TTC GTT(SEQ ID NO:6)、CpG T2：TCG TTC GTT CGT TCG TTC GTT CGT T(SEQ ID NO:7)和CpG T3：TCG TCG TCG TCG TCG TCG TCG(SEQ ID NO:8)。
优选地，所述CpG寡聚脱氧核苷酸的序列为CpG T1：TCG TTC GTT CGT TCG TTC GTT(SEQ ID NO:6)。
根据本发明所述的免疫刺激组合物，其中，所述皂苷选自皂树皂苷、人参皂苷、桔梗皂苷、黄芪皂苷、三七皂苷、甘草皂苷、合欢皮皂苷、麦冬皂苷、柴胡皂苷或竹节参皂苷的一种或多种；优选地，所述皂苷为皂树皂苷、人参皂苷、桔梗皂苷或黄芪甲苷；更优选地，所述皂树皂苷为QS-7、QS-17、QS-18或QS-21；更优选地，所述皂树皂苷为QS-21；所述人参皂苷可以为人参皂苷Rg1、人参皂苷Rg3、人参皂苷Rb1或人参皂苷Re；所述桔梗皂苷为桔梗皂苷D、桔梗皂苷D2或其两者的混合物；所述黄芪皂苷可以为黄芪甲苷(黄芪皂苷IV)、黄芪皂苷I、黄芪皂苷II或其中两种或两种以上皂苷单体的混合物；所述三七皂苷可以为三七皂苷R1；所述麦冬皂苷可以为麦冬皂苷D；所述柴胡皂苷可以为柴胡皂苷a、柴胡皂苷d或其两者的混合物；所述合欢皮皂苷可以为合欢皮总皂苷；所述甘草皂苷可以为甘草总皂苷；所述竹节参皂苷可以为竹节参总皂苷。
根据本发明所述的免疫刺激组合物，其中，所述包含皂苷的佐剂为免疫刺激复合物佐剂(Iscom佐剂)。
根据本发明所述的免疫刺激组合物，其中，所述CpG寡聚脱氧核苷酸 包含硫代磷酸酯连接。特别地，所述CpG寡聚脱氧核苷酸为硫代寡聚脱氧核苷酸，优选为全硫代寡聚脱氧核苷酸。
根据本发明所述的免疫刺激组合物，其中，所述CpG寡聚脱氧核苷酸与所述皂苷的重量比为1～40∶0.1～2，优选为2～40∶0.1～2，进一步优选为2∶1。
另一方面，本发明还提供了一种药物组合物，所述药物组合物包含所述免疫刺激组合物，以及抗原或抗原组合物。
根据本发明所述的药物组合物，其中，所述抗原或抗原组合物选择下述中任一种：人体免疫缺陷病毒，人疱疹病毒，水痘-带状疱疹病毒，人体细胞肥大病毒，甲、乙、丙或戊型肝炎病毒，呼吸合胞体病毒，人体乳头状瘤病毒，流感病毒，结核分枝杆菌，沙门氏菌，奈瑟菌如脑膜炎奈瑟菌或淋病奈瑟菌，疏螺旋体属如回归热螺旋体或杜通氏螺旋体，衣原体属如沙眼衣原体，博德特氏杆菌属如百日咳博德特氏菌，疟原虫属如恶性疟原虫(Plasmodium falciparum)、三日疟原虫(Plasmodium malariae)、卵形疟原虫(Plasmodium ovale)、间日疟原虫(Plasmodium vivax)或诺氏疟原虫(Plasmodium knowlesi)或弓形虫属如弓形虫。
根据本发明所述的药物组合物，其中，所述人疱疹病毒是HSV1或HSV2。
根据本发明所述的药物组合物，其中，所述抗原为肿瘤抗原。
再一方面，本发明提供了一种疫苗，所述疫苗包含所述免疫刺激组合物。
根据本发明所述的疫苗，其中所述疫苗为用于预防病毒、细菌和/或寄生虫感染的疫苗，或所述疫苗为以免疫疗法治疗病毒、细菌和/或寄生虫感染的疫苗。
再一方面，本发明提供了所述免疫刺激组合物在制备用于引起细胞溶解的T细胞应答的药物中的用途。
在一些具体实施方案中，本发明提供了所述免疫刺激组合物在制备用于在哺乳动物中诱发干扰素γ应答的药物中的用途。
在一些具体实施方案中，本发明提供了所述免疫刺激组合物在制备用于预防病毒、细菌和/或寄生虫感染的疫苗中的用途。
在一些具体实施方案中，本发明提供了所述免疫刺激组合物在制备用于以免疫疗法治疗病毒、细菌和/或寄生虫感染的疫苗中的用途。
在一些具体实施方案中，本发明提供了所述免疫刺激组合物在制备用于以免疫疗法治疗肿瘤的疫苗中的用途。
本发明还提供了一种引起细胞溶解的T细胞应答的方法，所述方法包括给予有需要的受试者有效量的包含本发明所述的免疫刺激组合物的药物组 合物。
本发明还提供了一种在哺乳动物中诱发干扰素γ应答的方法，所述方法包括给予有需要的受试者有效量的包含本发明所述的免疫刺激组合物的药物组合物。
本发明还提供了一种预防病毒、细菌和/或寄生虫感染的方法，所述方法包括给予有需要的受试者预防有效量的包含本发明所述的免疫刺激组合物的疫苗。
本发明还提供了一种以免疫疗法治疗病毒、细菌和/或寄生虫感染的方法，所述方法包括给予有需要的受试者有效量的包含本发明所述的免疫刺激组合物的疫苗。
本发明还提供了一种治疗肿瘤的方法，所述方法包括给予有需要的受试者治疗有效量的包含本发明所述的免疫刺激组合物的药物组合物。
本发明提供的免疫刺激组合物达到了出乎意料的技术效果，可介导更强的免疫应答。单独使用CpG T1～T3的免疫刺激作用弱于CpG1018、CpG7909和CpG1826等，但与QS-21联合应用后，所述免疫刺激组合物表现出出人意料的协同作用，免疫效果显著增强。
本发明研究发现，含有所述免疫刺激组合物的乙肝治疗性疫苗可以突破转基因小鼠的免疫耐受，产生高滴度的抗HBsAg抗体、抗HBcAg抗体、中和抗体。各项检测结果均显示，该疫苗通过多次免疫可以显著清除转基因小鼠体内的乙肝病毒，在免疫过程结束后，HBsAb水平接近饱和，可以保持稳定长效的免疫效果，HBsAg平均下降率维持在92％左右。同时含有所述免疫刺激组合物的乙肝疫苗可诱导产生较强的HBsAg和HBcAg特异性IFN-γ水平，免疫效果显著优于单独佐剂，也显著优于现有的CPG佐剂和QS-21的组合。
含有该免疫刺激组合物的带状疱疹疫苗也证明该免疫刺激组合物具有优越的免疫刺激作用。细胞免疫实验证明该疫苗可诱导产生较强的疱疹gE蛋白特异性IFN-γ水平，蛋白免疫效果显著优于单一佐剂。体液免疫实验也证明该疫苗可产生较高的疱疹gE蛋白特异性IgG/IgG1/IgG2a抗体水平，且效果优于单一佐剂，也显著优于现有的CPG佐剂和QS-21的组合。
综上，本发明提供的免疫刺激组合物具有优越的免疫刺激作用，相比单一佐剂、现有的CPG佐剂和QS21的组合，本发明的免疫刺激组合物中CpG T1～T3与QS-21表现出高效的协同作用，可以介导更强烈的免疫应答。应用于不同的抗原或抗原组合物，均有显著性优势。因此，本发明的免疫刺激组合物作为一种新型佐剂，具有很高的临床应用价值和广阔的市场前景。
附图的简要说明
以下，结合附图来详细说明本发明的实施方案，其中：
图1显示了不同的CPG寡聚脱氧核苷酸对HBsAg抗原特异性IFN-γ分泌水平的影响；
图2显示了不同的CPG寡聚脱氧核苷酸对HBcAg抗原特异性IFN-γ分泌水平的影响；
图3显示了根据本发明的不同的免疫刺激组合物对HBsAg抗原特异性IFN-γ分泌水平的影响；
图4显示了根据本发明的不同的免疫刺激组合物对HBcAg抗原特异性IFN-γ分泌水平的影响；
图5显示了根据本发明的不同剂量的免疫刺激组合物对HBsAg抗原特异性IFN-γ分泌水平的影响；
图6显示了根据本发明的不同剂量的免疫刺激组合物对HBcAg抗原特异性IFN-γ分泌水平的影响；
图7显示了含有本发明所述的免疫刺激组合物的乙肝疫苗对血清中HBsAg水平的影响；
图8显示了含有本发明所述的免疫刺激组合物的乙肝疫苗对血清中HBsAb水平的影响；
图9显示了含有本发明所述的免疫刺激组合物的乙肝疫苗对HBsAg抗原特异性IFN-γ分泌水平的影响；
图10显示了含有本发明所述的免疫刺激组合物的乙肝疫苗对HBcAg抗原特异性IFN-γ分泌水平的影响；
图11显示了含有本发明所述的免疫刺激组合物的乙肝疫苗对小鼠血清中HBsAg抗原特异性IgG抗体及其亚型水平的影响；
其中，A图：各组小鼠血清的HBsAb IgG水平；B图：各组小鼠血清的HBsAb IgG1水平；C图：各组小鼠血清的HBsAb IgG2a水平；D图：各组小鼠血清的HBsAb IgG2a与IgG1的比值；
图12显示了含有本发明所述的免疫刺激组合物的乙肝疫苗对小鼠血清中HBcAg抗原特异性IgG抗体及其亚型水平的影响；
其中，A图：各组小鼠血清的HBcAb IgG水平；B图：各组小鼠血清的HBcAb IgG1水平；C图：各组小鼠血清的HBcAb IgG2a水平；D图：各组小鼠血清的HBcAb IgG2a与IgG1的比值；
图13显示了含有本发明所述的免疫刺激组合物的带状疱疹疫苗对疱疹gE抗原特异性IFN-γ分泌水平的影响；
图14显示了含有本发明所述的免疫刺激组合物的带状疱疹疫苗对小鼠 血清中抗原特异性IgG抗体及其亚型水平的影响
其中，A图：各组小鼠血清的IgG水平；B图：各组小鼠血清的IgG1水平；C图：各组小鼠血清的IgG2a水平；D图：各组小鼠血清的IgG2a与IgG1的比值。
图15显示了根据本发明的包含不同皂苷的免疫刺激组合物对疱疹gE抗原特异性IFN-γ分泌水平的影响。
定义
除非另有定义，本文使用的所有科技术语具有本领域普通技术人员所理解的相同含义。关于本领域的定义及术语，专业人员具体可参考Current Protocols in Molecular Biology(Ausubel)。氨基酸残基的缩写是本领域中所用的指代20个常用L-氨基酸之一的标准3字母和/或1字母代码。
尽管本发明以广义范围显示数字范围和参数近似值，但是具体实施例中所示的数值尽可能准确的进行记载。然而，任何数值本来就必然含有一定的误差，其是由它们各自的测量中存在的标准偏差所致。另外，本文公开的所有范围应理解为涵盖其中包含的任何和所有子范围，例如记载的“2至40”的范围应认为包含最小值2和最大值40之间(包含端点)的任何和所有子范围，也就是说，所有以最小值2或更大起始的子范围，例如2至6.1，以及以最大值40或更小终止的子范围，例如5.5至40。另外，任何称为“并入本文”的参考文献应理解为以其整体并入。
另外应注意，如本说明书中所使用的，单数形式包括其所指对象的复数形式，除非清楚且明确的限于一个所指对象。术语“或”可与术语“和/或”互换使用，除非上下文另有清楚指明。
本文使用的术语“药物组合物”、“组合药物”和“药物组合”可互换地使用，其表示组合在一起以实现某种特定目的的至少一种药物以及任选的可药用赋形剂或辅料的组合。在某些实施方案中，所述药物组合物包括在时间和/或空间上分开的组合，只要其能够共同作用以实现本发明的目的。例如，所述药物组合物中所含的成分(例如gE蛋白、QS-21、CpG寡聚脱氧核苷酸)可以以整体施用于对象，或者分开施用于对象。当所述药物组合物中所含的成分分开地施用于对象时，所述成分可以同时或依次施用于对象。
本文使用的术语“CpG寡聚脱氧核苷酸”或“CpG-ODN”是指短的单链合成DNA分子，其含有一个或更多个“CpG”单元，其中C表示胞嘧啶，G表示鸟嘌呤，p表示磷酸二酯键。特别地，所述CpG寡聚脱氧核苷酸是非甲基化的。在一些实施方案中，所述CpG-ODN包含硫代磷酸酯连接或硫代磷酸酯骨架。也就是说，在一些实施方案中，所述CpG-ODN为硫代磷酸酯 寡聚脱氧核苷酸(即硫代寡聚脱氧核苷酸)。优选地，所述CpG-ODN中所有核苷酸间连接均为硫代磷酸酯连接，即所述CpG-ODN为全硫代寡聚脱氧核苷酸。在另一些实施方案中，所述CpG-ODN包含两个或两个以上拷贝的5’-TTCGTT-3’基序或5’-TCGTCGTCG-3’基序。特别地，所述CpG-ODN具有选自下列的序列：TCG TTC GTT CGT TCG TTC GTT(SEQ ID NO:6)、TCG TTC GTT CGT TCG TTC GTT CGT T(SEQ ID NO:7)或TCG TCG TCG TCG TCG TCG TCG(SEQ ID NO:8)，优选地为TCG TTC GTT CGT TCG TTC GTT(SEQ ID NO:6)。
本文使用的“人参皂苷、桔梗皂苷、黄芪皂苷、三七皂苷、甘草皂苷、合欢皮皂苷、麦冬皂苷、柴胡皂苷或竹节参皂苷”是指是存在于对应植物中的活性成分，例如人参皂苷是一种固醇类化合物，主要存在于人参属药材中，是人参中的活性成分。在一些实施方案中，所述人参皂苷优选为人参皂苷Rg1、人参皂苷Rg3、人参皂苷Rb1、人参皂苷Re等单体或其中两个或两个以上皂苷单体的混合物；桔梗皂苷优选为桔梗皂苷D、桔梗皂苷D2或其两者的混合物；黄芪皂苷优选为黄芪甲苷(黄芪皂苷IV)、黄芪皂苷I、黄芪皂苷II等单体或其中两个或两个以上皂苷单体的混合物；三七皂苷优选为三七皂苷R1等；麦冬皂苷优选为麦冬皂苷D等；柴胡皂苷优选为柴胡皂苷a、柴胡皂苷d或其两者的混合物；合欢皮皂苷优选为合欢皮总皂苷等；甘草皂苷优选为甘草总皂苷等；竹节参皂苷优选为竹节参总皂苷等。
本文使用的“Iscom佐剂”为免疫刺激复合物佐剂，具体地为不包含抗原的Iscom基质(ISCOM MATRIX)，是由磷脂、皂苷、胆固醇组成的笼状结构的佐剂。
本文使用的“治疗和/或预防有效量”或“有效量”是指足以显示其对于所施用对象益处的剂量。施用的实际量，以及施用的速率和时间过程会取决于所治疗者的自身情况和严重程度。治疗的处方(例如对剂量的决定等)最终是全科医生及其它医生的责任并依赖其做决定，通常考虑所治疗的疾病、患者个体的情况、递送部位、施用方法以及对于医生来说已知的其它因素。
本文所使用的术语“哺乳动物”是指人类，也可以是其它动物，如野生动物(如苍鹭、鹳、鹤等)，家畜(如鸭、鹅等)或实验动物(如猩猩、猴子、大鼠、小鼠、兔子、豚鼠、土拨鼠、地松鼠等)。
在另一些实施方案中，本发明组合物还可包含另外的添加剂，如可药用载体或添加剂，尤其是当它以药物制剂形式存在时。
优选的药物载体尤其是水，缓冲水溶液，优选等渗盐溶液如PBS(磷酸盐缓冲液)、葡萄糖、甘露醇、右旋葡萄糖、乳糖、淀粉、硬脂酸镁、纤维素、碳酸镁、0.3％甘油、透明质酸、乙醇或聚亚烷基二醇如聚丙二醇、甘油 三酯等。所用药物载体的类型尤其依赖于根据本发明的组合物是否配制为用于口服、鼻、皮内、皮下、肌内或静脉施用。根据本发明的组合物可包含润湿剂、乳化剂或缓冲液物质作为添加剂。
根据本发明的药物组合物、疫苗或者药物制剂可通过任何适宜的途径施用，例如可口服、鼻、皮内、皮下、肌内或静脉内施用。
以下结合附图通过具体实施方式的描述对本发明作进一步说明，但这并非是对本发明的限制，本领域技术人员根据本发明的基本思想，可以作出各种修改或改进，但是只要不脱离本发明的基本思想，均在本发明的范围之内。
实施发明的最佳实施方式
以下参照具体的实施例来说明本发明。本领域技术人员能够理解，这些实施例仅用于说明本发明，其不以任何方式限制本发明的范围。
下述实施例中的实验方法，如无特殊说明，均为常规方法。下述实施例中所用的原料、试剂材料等，如无特殊说明，均为市售购买产品。
实施例1 制备本发明的免疫刺激组合物和乙肝疫苗
1、HBsAg原液：HBsAg蛋白的氨基酸序列如SEQ ID NO:1所示。
HBsAg蛋白由HBsAg基因重组酵母细胞制备，酵母细胞种类包括汉逊酵母、酿酒酵母和毕赤酵母，优选为汉逊酵母。具体制备步骤参考中国专利申请CN108330145A，HBsAg基因重组汉逊酵母细胞经发酵培养，收获菌体。经破菌处理和硅胶吸附、柱层析和TFF等步骤纯化。
2、HBcAg原液：HBcAg蛋白的氨基酸序列如SEQ ID NO:2所示。
HBcAg蛋白由HBcAg基因重组酵母细胞制备，酵母细胞种类包括汉逊酵母、酿酒酵母和毕赤酵母，优选汉逊酵母。具体制备步骤参考中国专利申请CN108047316A，HBcAg基因重组汉逊酵母细胞经发酵培养，收获菌体。经破菌处理和硫酸铵、柱层析和TFF等步骤纯化，制备HBcAg原液。
3、QS-21：购自BRENNTAG公司，CAS.NO.A010-023。
4、CPG寡聚脱氧核苷酸原料的制备方法：
寡聚脱氧核苷酸是合成制备的寡聚脱氧核苷酸序列片段，其含有一个或多个CpG基序，本实施例使用寡聚脱氧核苷酸序列见表1：
表1 CPG寡聚脱氧核苷酸的具体序列
Figure PCTCN2020135572-appb-000001
具体制备方法：使用常规固相亚磷酰胺三酯法化学合成方法制备，由3’端开始，1)脱保护基：先用三氯乙酸脱去连接在CpG上的核苷酸的保护基团DMT(二甲氧基三苯甲基)，获得游离的5’羟基，以供下一步缩合反应使用；2)活化：将亚磷酰胺保护的核苷酸单体与四氮唑活化剂混合并进入合成柱，形成亚磷酰胺四唑活性中间体，此中间体与CpG上已脱保护基的核苷酸发生缩合反应；3)连接：亚磷酰胺四唑活性中间体遇到CpG上已脱保护基的核苷酸时，将与其5’羟基发生亲和反应，缩合并脱去四唑，此时寡核苷酸链向前延长一个碱基；4)氧化：缩合反应时核苷酸单体是通过亚磷酯键与连在CpG上的寡核苷酸连接，而亚磷酯键不稳定，易被酸或碱水解，此时使用硫代试剂将亚磷酰胺氧化为硫磷双键的磷酸三酯，从而得到稳定的寡核苷酸；5)封闭：缩合反应后为了防止连在CpG上的未参与反应的5’羟基在随后的循环反应中被延伸，常通过乙酰化来封闭此端羟基；经过以上五个步骤后，一个脱氧核苷酸就连到CpG的核苷酸上；重复以上的脱保护基、活化、连接、氧化、封闭过程即可得到一个DNA片段粗品；最后对其 进行切割、脱保护基、纯化、定量等合成后处理即可。
5、使用PBS溶液(购自Hyclone公司)将HBsAg原液和HBcAg原液分别稀释至200μg/ml和100μg/ml；使用PBS溶液将各CPG原料分别溶解并稀释至100μg/ml，供下一步使用。
实施例2 CPG寡聚脱氧核苷酸的筛选实验
1、实验动物：C57BL/6(N)小鼠，雄性，4周龄，135只，上海灵畅实验动物技术有限公司。
2、实验分组：见表2，每次注射量为100μL/只。其中A组为阴性对照，PBS溶液100μL/只。
表2 实验动物分组
Figure PCTCN2020135572-appb-000002
3、实验步骤：小鼠免疫后第7天取脾，按常规方法制备脾淋巴细胞，具体如下：无菌操作取脾脏：用无菌镊子及剪刀剪取脾脏，放于70μm细胞滤网中，置于含有2ml预冷处理的2％FBS(购自GIBCO公司)-PBS的平皿中；用研磨棒研磨脾脏，脾脏细胞通过筛目进入平皿中，得到细胞悬液， 用巴氏吸管将悬液放入经40μm细胞滤网过滤(购自BD公司)的50ml无菌离心管；500×g，4℃离心5分钟；弃去上清，加入2ml 1×破红剂(购自BD公司)重悬细胞，4℃避光静置5分钟，以破碎红细胞；加入10ml 2％FBS-PBS终止破红反应；500×g，4℃离心5分钟；弃去上清，加入5ml 2％FBS-PBS重悬细胞备用。分别使用刺激物HBsAg特异性肽库PS4和HBcAg特异性肽库PCP刺激脾细胞；按照试剂盒说明书，使用ELISPOT试剂盒(BD公司)检测HBsAg和HBcAg抗原特异性IFN-γ分泌水平；使用ImmunoSPOT Series 3酶联斑点分析仪读取ELISPOT试剂盒测出的斑点数(具体操作步骤参考中国专利CN104043120B的实施例7)。
其中，HBsAg特异性肽库序列参考中国专利CN104043120B的实施例7；HBcAg特异性肽库序列见SEQ ID NO：16～30。
4、实验结果：ELISPOT斑点结果见图1和图2，结果显示，不同序列的B型CpG佐剂具有不同的免疫效果，其中，CpG T1～T3、CpG1018、CpG7909、CpG 1826和CpG 684整体优于A型CpG佐剂和C型CpG佐剂，而CpG1618和CPG D2的免疫效果较差，诱导产生HBsAg和HBcAg特异性IFN-γ水平均低于A型CpG佐剂和C型CpG佐剂。
实施例3 免疫刺激组合物的筛选实验
1、实验动物：C57BL/6(N)小鼠，雄性，4周龄，81只，上海灵畅实验动物技术有限公司，
2、实验分组：见表3，每次注射量为100μL/只。其中A组为阴性对照，PBS溶液100μL/只。
表3 实验动物分组
Figure PCTCN2020135572-appb-000003
3、实验步骤：同实施例2。
4、实验结果：ELISPOT斑点结果见图3和图4，结果显示，CpG T1～T3与QS21联合应用后产生了高效的协同作用，诱导产生HBsAg和HBcAg特异性IFN-γ水平显著高于CpG1018、CpG7909等其他几种CpG佐剂，具有出人意料的免疫效果。
实施例4 不同含量佐剂对药物组合物免疫效果的影响
1、实验动物：C57BL/6(N)小鼠，雄性，4周龄，60只，上海灵畅实验动物技术有限公司。
2、试剂材料：
1)HBsAg蛋白、HBcAg蛋白和CpG T1均由实施例1制得；
2)QS-21(CAS.NO.A010-023，购自BRENNTAG公司)
3)使用PBS溶液(购自Hyclone公司)将HBsAg原液、HBcAg原液分别稀释至200μg/ml、100μg/ml，使用PBS溶液将QS21分别稀释至5μg/ml、50μg/ml、100μg/ml，使用PBS溶液将CpG T1溶解并分别稀释至50μg/ml、100μg/ml、2mg/ml，使用PBS溶液将CPG7909溶解并稀释至100μg/ml，供下一步使用。
3、实验分组：见表4，每次注射量为100μL/只。其中A组为阴性对照，PBS溶液100μL/只。
4、实验步骤：同实施例2。
5、实验结果：ELISPOT斑点结果见图5和图6，结果显示，CpG T1与QS21的剂量变化对疫苗组合物的均有显著性影响，高于剂量5的免疫刺激组合物，诱导产生HBsAg和HBcAg特异性IFN-γ水平显著高于CPG7909组，但由于种属差异性，佐剂剂量进一步增加诱导效果并无显著性增加，推测原因为小鼠无法准确反映佐剂的免疫强度。
剂量1、2、4与CPG7909组的免疫刺激效果相当，但是使用的佐剂剂量低于同等CPG7909组，因此也具有一定优势。
表4 实验动物分组
Figure PCTCN2020135572-appb-000004
实施例5 乙肝疫苗的实验组设置及免疫流程
1、实验动物及模型建立：
C57BL/6(N)小鼠，雄性，4周龄，81只，上海灵畅实验动物技术有限公司，rAAV8-HBV腺病毒，购自北京五加和分子医学研究所有限公司。在C57BL/6(N)小鼠上尾静脉注射rAAV8-HBV腺病毒，建立rAAV8-HBV持续感染C57BL/6(N)的小鼠模型。
2、试剂材料：
1)HBsAg蛋白：实施例1制得。
2)HBcAg蛋白：实施例1制得。
3)使用PBS溶液(购自Hyclone公司)将HBsAg原液、HBcAg原液和QS-21分别稀释至200μg/ml、100μg/ml、50μg/ml，使用PBS溶液将CpG溶解并稀释至100μg/ml，供下一步使用。
3、实验分组：见表5，每只小鼠每次注射量为100μL/只，其中A组为阴性对照，注射PBS溶液100μL/只。
表5 实验动物分组及每组注射量
Figure PCTCN2020135572-appb-000005
4、动物免疫：所有组别每2周进行1次肌肉注射，接种部位为右侧后大腿，共给药6次，分别于尾静脉注射rAAV8-HBV病毒后第4周、第6周、第8周、第10周、第12周、第14周给药。给药开始后每2周采1次血，采血时间分别为第4周、第6周、第8周、第10周、第12周、第14周、第16周、第18周、第20周、第22周。第22周将所有小鼠处死。
实施例6 乙肝疫苗对血清中HBsAg水平的影响
1、血清HBsAg的检测步骤：委托南京鼓楼医院检测。
使用两步免疫测定法，首先检测样本和包被乙型肝炎表面抗体的顺磁微粒子结合，经过洗涤，加入吖啶酯标记的乙型肝炎表面抗体结合物，再经过洗涤，加入预激发液和激发液到反应混合物中，测定检测样本的相对发光值(RLU)，样本中的HBsAg含量与RLU之间成正相关性，通过产生的ARCHTITECT HBsAg标准曲线测定小鼠血清样本中HBsAg浓度，最终小鼠血清样本中HBsAg浓度为测定值的50～200倍。
2、结果分析(图7)：含有本发明所述免疫刺激物的H组疫苗，对应的HBsAg水平呈明显下降趋势，在免疫过程结束后(第14周起)，保持稳定长效的免疫效果，相比单独的CpG组(F组)和QS-21组(G组)，具有显著性优势。H组HBsAg水平从起始>6350IU/ml降低到50IU/ml左右，该组在第二次免疫(第6周)后HBsAg水平即下降30％以上，在第三次免疫(第8周)后HBsAg水平下降70％以上，且在第14周免疫结束后平均下降率维持在92％左右。具有优越的免疫效果。与双佐剂对照(I组)相比，H在第14周免疫结束后依旧维持稳定的免疫效果，免疫水平显著优于I组。
实施例7 乙肝疫苗的体液免疫效果评价
1、血清HBsAb的检测步骤：委托南京鼓楼医院检测。
使用两步免疫测定法，即首先检测样本和重组HBsAg(rHBsAg)包被顺磁微粒子混合，经过洗涤，加入吖啶酯标记的rHBsAg结合物，再经过洗涤，加入预激发液和激发液到反应混合物中，测定检测样本的相对发光值(RLU)，样本中的HBsAb含量与RLU之间成正相关性，通过产生的ARCHTITECT HBsAb校准曲线测定小鼠血清样本中HBsAb浓度，最终小鼠血清样本中HBsAb浓度为测定值的50～200倍。
2、结果分析(图8)：含有所述免疫刺激物的H组疫苗在第二次免疫(第6周)后开始产生HBsAb(>10mIU/ml)，且随着免疫次数的增加，HBsAb水平呈现不断增长的趋势，且增长趋势显著优于单独的CpG组(F组)和QS-21组(G组)。免疫结束后两周(第十六周)之后HBsAb水平接近饱和，可达4.0个对数值即10000mIU/ml左右的HBsAb水平，与双佐剂对照(I组)相比，产生的抗体水平也具有显著性优势。
实施例8 乙肝疫苗的细胞免疫效果评价
1、检测步骤：同实施例2。
2、评价指标：若对照孔斑点数≤5SCF，样品孔斑点数≥10SCF，判定为阳性；若5SFC＜对照孔斑点数≤10SCF，样品孔斑点数/对照孔斑点数≥2，判定为阳性；若对照孔斑点数＞10SFC，样品孔斑点数/对照孔斑点数≥3，判定为阳性。
3、实验结果：
表6 脾细胞分泌的HBsAg和HBcAg特异性IFN-γ的转阳率
Figure PCTCN2020135572-appb-000006
细胞免疫水平检测结果：ELISPOT斑点结果见图9和10，分析结果显示，F组-I组的HBsAg特异性IFN-γ阳转率均为100％，HBcAg特异性IFN-γ阳转率均为100％。含有所述免疫刺激物的H组疫苗可诱导产生较高的HBsAg和HBcAg特异性IFN-γ水平，分别大于2350SFC/10 6脾细胞和大于1250SFC/10 6脾细胞，与单独的CpG组(F组)和QS-21组(G组)相比，具有显著性差异。H组与双佐剂对照(I组)相比，后者诱导产生的HBsAg和HBcAg特异性IFN-γ水平约为1630SFC/10 6脾细胞和750SFC/10 6脾细胞左右，显著低于H组。
实施例9 药物组合物在血清中HBsAg和HBcAg特异性抗体检测
1、检测步骤：用纯化的HBsAg和HBcAg包被96孔酶标板，形成固相抗原，经过封闭处理后，将待测血清以一定的起始稀释度作倍比稀释，设多个稀释度，在96孔酶标板中加入倍比稀释后的血清样本，再与HRP标记的抗IgG/IgG1/IgG2a抗体结合，形成抗原-抗体(血清)-酶标抗体复合物，最后加入底物TMB显色，并用酶标仪测定450nm波长下的吸光度(OD值)，显色颜色的深浅与检测样本中的HBsAg和HBcAg特异性抗体IgG/IgG1/IgG2a水平呈正相关，通过拟合“吸光度OD值-血清样本稀释倍数(Log)”的关系曲线，进行抗体滴度的判定。
2、结果分析：
1)血清中HBsAb IgG抗体及亚型检测结果：
各组不同时间用ELISA方法检测小鼠血清中HBsAb IgG抗体及亚型水平如图11所示。含有所述免疫刺激物的H组疫苗产生较高滴度的抗HBsAg特异性IgG/IgG1/IgG2a抗体，且随着免疫次数的增加，抗体水平不断增长，在第六次免疫(第14周)时抗体水平接近饱和，特异性抗体滴度可达5.4个对数值以上；A组-D组未检测到特异性抗体，E组-G组虽然产生一定的HBsAg特异性IgG/IgG1/IgG2a抗体水平，但抗体水平显著低于H组，双佐剂对照(I组)产生的抗HBsAg特异性IgG抗体和IgG2a抗体水平显著低于H组。
2)血清中HBcAb IgG抗体及亚型检测结果：
各组不同时间用ELISA方法检测小鼠血清中HBcAb IgG抗体及亚型水平如图12所示。含有所述免疫刺激物的H组疫苗产生较高滴度的抗HBcAg特异性IgG/IgG1/IgG2a抗体，且随着免疫次数的增加，抗体水平不断增长，在第六次免疫(第14周)时抗体水平接近饱和，特异性抗体滴度可达4.8个对数值以上；A组-D组未检测到特异性抗体，E组-G组虽然产生一定的HBcAg特异性IgG/IgG1/IgG2a抗体水平，但抗体水平显著低于H组。且H 组更偏向于Th1通路，D图可见特异性抗体IgG2a呈显著上升趋势，反映出H组疫苗可以促进抗HBcAg抗体亚型转变，且转变效率显著高于双佐剂对照(I组)。
实施例10 带状疱疹疫苗的实验组设置
1、实验动物及模型建立：
C57BL/6(N)小鼠，雌性，5周龄，48只，购于上海斯莱克实验动物有限责任公司。
2、试剂材料：
1)疱疹gE蛋白：氨基酸序列如SEQ ID NO：31所示。
制备步骤参考文献Thomsson E,Persson L等在《Journal of Virological Methods》2011，第175卷，第1期，第53-59页中的报道，具体步骤如下：根据目的蛋白序列对其核酸序列进行优化，使其密码子符合哺乳动物表达体系，并对目的基因进行合成；将合成的目的基因通过酶切连接的方式与pcDNA3.1(+)质粒连接，转化Top 10感受态，挑取阳性单克隆，对阳性单克隆进行测序验证；大量扩增单克隆菌体，用无内毒素质粒抽提试剂盒进行大量抽提符合细胞转染的质粒；通过瞬时转染方式用质粒转染CHO悬浮细胞，CHO细胞活力低于70％或发酵时间大于7天时，在4℃下用5000rpm离心30min收集发酵液上清。将发酵液在4℃层析柜中透析至含50mMTris-HCl、500mM NaCl、20mM咪唑的溶液中，透析比例1:100，每4h透析一次，共透析3次；通过镍柱对收集样品经行纯化，对收集目的蛋白峰样品进行SDS-PAGE检测，合并较纯的纯化液，用含20mM磷酸盐、150mM NaCl的溶液在4℃层析柜中透析24h，透析比例1:100，每8h换液一次；将样品过0.22um无菌滤膜，4℃冰箱中保存备用。
制备的疱疹gE蛋白原液要求其纯度大于95％，蛋白含量不低于200μg/ml，内毒素水平不高于0.1Eu/ug。
2)使用PBS溶液(购自Hyclone公司)将疱疹gE原液分别稀释至50μg/ml和10μg/ml，使用PBS溶液将QS-21分别稀释至50μg/ml和10μg/ml，使用PBS溶液将CpG分别稀释至100μg/ml和20μg/ml，使用PBS溶液将CpG7909分别稀释至100μg/ml和20μg/ml。
3、实验分组：见表7，每只小鼠每次注射量为100μL/只，其中A组为阴性对照，注射PBS溶液100μL/只。
表7 实验动物分组及每组注射量
Figure PCTCN2020135572-appb-000007
4、动物免疫：所有组别每2周进行1次肌肉注射，接种部位为右侧后大腿，连续给药2次，即分别于第0周和第2周注射给药，第4周将所有小鼠处死。
实施例11 带状疱疹疫苗的细胞免疫药效验证
1、检测步骤和评价指标同实施例2；其中gE特异性肽库序列见SEQ ID NO.32～46。
2、实验结果：各组小鼠脾细胞分泌gE特异性IFN-γ的T淋巴细胞斑点数水平如图13所示，gE特异性IFN-γ阳转率结果如表8所示。结果表明：免疫高剂量的E、F组对应的脾细胞分泌gE特异性IFN-γ的T淋巴细胞斑点数水平(>4000SFC/10 6脾细胞)均显著高于低剂量的G、H组；其中，E组、G组(CpGT1+QS-21)对应的脾细胞分泌gE特异性IFN-γ的T淋巴细胞斑点数水平高于同剂量的F组、H组(CpG7909+QS-21)，E-H组IFN-γ的转阳率均为100％。
表8 脾细胞分泌的SgE特异性IFN-γ的转阳率
组别 A B C D E F G H
转阳数量/只 1/6 0/6 4/6 5/6 6/6 6/6 6/6 6/6
转阳率/％ 16.7 0 66.7 83.3 100 100 100 100
实施例12 带状疱疹疫苗的体液免疫药效验证
1、检测步骤：免疫后第28天进行采血，分离血清(全血置于37℃恒温培养箱放置40min，12000rpm、4℃离心10min；吸取上清，冻存于-20℃备 用)，按照试剂盒说明书，使用ELISA试剂盒(上海科华)检测产生的疱疹gE蛋白特异性抗体阳转率。检测设空白对照、阴性对照和待测样品，每种两个平行孔，其中阴性对照为阴性小鼠血清；除空白对照外，各孔分别加入阴性对照或待测样品，再加入酶结合物，混匀封板后37℃孵育30分钟；使用洗涤液洗涤各孔，每孔加入显色剂A液和显色剂B液，混匀封板后37℃孵育15分钟；每孔加入终止液混匀；使用酶标仪读取450nm波长处的各孔OD值。
2、实验结果：ELISA检测小鼠血清中抗原特异性IgG抗体及亚型水平如图14所示，结果表明：含有本发明所述免疫刺激物的E组免疫效果显著优于单独的CpG组(C组)和QS-21组(D组)和双佐剂对照(F组)，且对应的IgG和IgG2a抗体水平与两组均存在显著差异，即在QS-21中加入CpG可提高相应的体液免疫水平。
实施例13 不同皂苷对重组带状疱疹疫苗组合物的药效影响
1、实验动物及模型建立：
C57BL/6(N)小鼠，雌性，5周龄，48只，购于上海斯莱克实验动物有限责任公司。
2、试剂材料：
1)疱疹gE蛋白由实施例10获得、CpG T1、CpG 7909均由实施例1制得；
2)QS-21(CAS.NO.A010-023，购自BRENNTAG公司)；人参皂苷Rg1(CAS:22427-39-0，购自南京春秋生物工程有限公司)、黄芪甲苷(CAS:84687-43-4，购自南京春秋生物工程有限公司)、桔梗皂苷D(CAS:58479-68-8，购自湖北云镁科技有限公司)，Iscom佐剂(购自上海熹垣生物科技有限公司)
3)使用PBS溶液(购自Hyclone公司)将疱疹gE原液稀释至50μg/mL，使用PBS溶液将各皂苷分别稀释至50μg/mL，使用PBS溶液将CpG T1和CpG 7909分别溶解并稀释至100μg/mL，供下一步使用。
3、实验分组：
见表9，每次注射量为100μL/只。其中对照组注射PBS溶液100μL/只。
4、实验步骤：同实施例2。
5、实验结果：
ELISPOT斑点结果见图15，结果显示，CpG T1与各皂苷联合应用后均产生了高效的协同作用，诱导产生gE特异性IFN-γ水平显著高于其他CpG和皂苷的组合物，其中，QS21的效果最佳。
表9 实验动物分组
Figure PCTCN2020135572-appb-000008
综上所述，本发明提供的免疫组合物具有优越的免疫刺激作用，相比单一佐剂、其他CPG佐剂和QS21的组合，CpG T1～T3与QS-21表现出高效的协同作用，可以介导更强烈的免疫应答。应用于不同的抗原或抗原组合物，均有显著性优势。因此，该免疫组合物作为一种新型佐剂，具有很高的临床应用价值和广阔的市场前景。
尽管以上已经对本发明作了详细描述，但是本领域技术人员理解，在不偏离本发明的精神和范围的前提下可以对本发明进行各种修改和改变。本发明的权利范围并不限于上文所作的详细描述，所述修改和改变应归属于权利要求书的范围。虽然以上仅描述了本发明的具体实施方式的范例，但本领域的技术人员应当理解，以上这些仅为举例说明，本发明的保护范围是由所附权利要求书限定的。本领域的技术人员在不背离本发明的原理和实质的前提下，可以对这些实施方式做出多种变更或修改，但这些变更或修改均应落入本发明的保护范围。

Claims (23)

  1. 一种免疫刺激组合物，其包含皂苷和CpG寡聚脱氧核苷酸，或者其由包含皂苷的佐剂和CpG寡聚脱氧核苷酸组成，其中所述CpG寡聚脱氧核苷酸序列具有两个或两个以上拷贝的5’-TTCGTT-3’基序或5’-TCGTCGTCG-3’基序。
  2. 根据权利要求1所述的免疫刺激组合物，其中，所述CpG寡聚脱氧核苷酸的序列选自以下中的任一种：CpG T1：TCG TTC GTT CGT TCG TTC GTT(SEQ ID NO:6)、CpG T2：TCG TTC GTT CGT TCG TTC GTT CGT T(SEQ ID NO:7)和CpG T3：TCG TCG TCG TCG TCG TCG TCG(SEQ ID NO:8)；
    优选地，所述CpG寡聚脱氧核苷酸的序列为CpG T1：TCG TTC GTT CGT TCG TTC GTT(SEQ ID NO:6)。
  3. 根据权利要求1或2所述的免疫刺激组合物，其中，所述皂苷选自皂树皂苷、人参皂苷、桔梗皂苷、黄芪皂苷、三七皂苷、甘草皂苷、合欢皮皂苷、麦冬皂苷、柴胡皂苷或竹节参皂苷中的一种或多种。
  4. 根据权利要求3所述的免疫刺激组合物，其中，所述皂树皂苷为QS-7、QS-17、QS-18或QS-21，优选地，所述皂树皂苷为QS-21；所述人参皂苷为人参皂苷Rg1、人参皂苷Rg3、人参皂苷Rb1或人参皂苷Re；所述桔梗皂苷为桔梗皂苷D、桔梗皂苷D2或其两者的混合物；所述黄芪皂苷为黄芪甲苷、黄芪皂苷I、黄芪皂苷II或其中两种或两种以上皂苷单体的混合物；所述三七皂苷为三七皂苷R1；所述麦冬皂苷为麦冬皂苷D；所述柴胡皂苷为柴胡皂苷a、柴胡皂苷d或其两者的混合物；所述合欢皮皂苷为合欢皮总皂苷；所述甘草皂苷为甘草总皂苷；所述竹节参皂苷为竹节参总皂苷。
  5. 根据权利要求1至4中任一项所述的免疫刺激组合物，其中所述包含皂苷的佐剂为Iscom佐剂。
  6. 根据权利要求1至5中任一项所述的免疫刺激组合物，其中所述CpG寡聚脱氧核苷酸包含硫代磷酸酯连接。
  7. 根据权利要求5所述的免疫刺激组合物，其中所述CpG寡聚脱氧核 苷酸是全硫代寡聚脱氧核苷酸。
  8. 根据权利要求1至7中任一项所述的免疫刺激组合物，所述CpG寡聚脱氧核苷酸与皂苷的重量比为1～40:0.1～2，优选为2～40:0.1～2；更优选地为2:1。
  9. 一种药物组合物，其包含权利要求1至8中任一项所述的免疫刺激组合物，以及抗原或抗原组合物。
  10. 根据权利要求9所述的药物组合物，其中，所述抗原或抗原组合物选自下述中的任一种：人体免疫缺陷病毒，人疱疹病毒，水痘-带状疱疹病毒，人体细胞肥大病毒，甲、乙、丙或戊型肝炎病毒，呼吸合胞体病毒，人体乳头状瘤病毒，流感病毒，结核分枝杆菌，沙门氏菌，奈瑟菌如脑膜炎奈瑟菌或淋病奈瑟菌，疏螺旋体属如回归热螺旋体或杜通氏螺旋体，衣原体属如沙眼衣原体，博德特氏杆菌属如百日咳博德特氏菌，疟原虫属如恶性疟原虫(Plasmodium falciparum)、三日疟原虫(Plasmodium malariae)、卵形疟原虫(Plasmodium ovale)、间日疟原虫(Plasmodium vivax)或诺氏疟原虫(Plasmodium knowlesi)或弓形虫属如弓形虫。
  11. 根据权利要求10所述的药物组合物，其中，所述人疱疹病毒是HSV1或HSV2。
  12. 根据权利要求9所述的药物组合物，其中，所述抗原为肿瘤抗原。
  13. 一种疫苗，其包含权利要求1至8中任一项所述的免疫刺激组合物；
    优选地，所述疫苗为用于预防病毒、细菌和/或寄生虫感染的疫苗，或所述疫苗为以免疫疗法治疗病毒、细菌和/或寄生虫感染的疫苗。
  14. 权利要求1至8中任一项所述的免疫刺激组合物在制备用于引起细胞溶解的T细胞应答的药物中的用途。
  15. 权利要求1至8中任一项所述的免疫刺激组合物在制备用于在哺乳动物中诱发干扰素γ应答的药物中的用途。
  16. 权利要求1至8中任一项所述的免疫刺激组合物在制备用于预防病 毒、细菌和/或寄生虫感染的疫苗中的用途。
  17. 权利要求1至8中任一项所述的免疫刺激组合物在制备用于以免疫疗法治疗病毒、细菌和/或寄生虫感染的疫苗中的用途。
  18. 权利要求1至8中任一项所述的所述免疫刺激组合物在制备用于以免疫疗法治疗肿瘤的疫苗中的用途。
  19. 一种引起细胞溶解的T细胞应答的方法，所述方法包括给予有需要的受试者有效量的如权利要求9至12中任一项所述的药物组合物。
  20. 一种在哺乳动物中诱发干扰素γ应答的方法，所述方法包括给予有需要的受试者有效量的如权利要求9至12中任一项所述的药物组合物。
  21. 一种预防病毒、细菌和/或寄生虫感染的方法，所述方法包括给予有需要的受试者预防有效量的如权利要求13所述的疫苗。
  22. 一种以免疫疗法治疗病毒、细菌和/或寄生虫感染的方法，所述方法包括给予有需要的受试者治疗有效量的如权利要求13所述的疫苗。
  23. 一种治疗肿瘤的方法，所述方法包括给予有需要的受试者治疗有效量的如权利要求9至12中任一项所述的药物组合物。
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