WO2021115408A1 - 药物组合物及其用途 - Google Patents

药物组合物及其用途 Download PDF

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WO2021115408A1
WO2021115408A1 PCT/CN2020/135570 CN2020135570W WO2021115408A1 WO 2021115408 A1 WO2021115408 A1 WO 2021115408A1 CN 2020135570 W CN2020135570 W CN 2020135570W WO 2021115408 A1 WO2021115408 A1 WO 2021115408A1
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pharmaceutical composition
hepatitis
saponins
composition according
cpg
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PCT/CN2020/135570
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English (en)
French (fr)
Inventor
李建强
葛君
孙娇娇
周童
任苏林
谭昌耀
顾月
黄红颖
王世伟
陈晓晓
黄精俸
王晓东
陈悦
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南京远大赛威信生物医药有限公司
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Application filed by 南京远大赛威信生物医药有限公司 filed Critical 南京远大赛威信生物医药有限公司
Priority to US17/784,081 priority Critical patent/US20230073321A1/en
Priority to JP2022535199A priority patent/JP2023506439A/ja
Priority to EP20897828.8A priority patent/EP4074334A1/en
Publication of WO2021115408A1 publication Critical patent/WO2021115408A1/zh
Priority to ZA2022/07790A priority patent/ZA202207790B/en

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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
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    • 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
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    • 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.
  • the present invention relates to a pharmaceutical composition comprising hepatitis B surface antigen, hepatitis B core antigen and an immunostimulatory composition.
  • the immunostimulatory composition comprises saponins and CpG oligodeoxynucleotides, or the immunostimulatory composition is composed of an adjuvant comprising saponin and CpG oligodeoxynucleotides, wherein the CpG oligodeoxynucleotides There are two or more copies of 5'-TTCGTT-3' motif or 5'-TCGTCGTCG-3' motif in the nucleotide sequence.
  • the present invention also relates to the use of the pharmaceutical composition for the treatment of hepatitis B virus infection and/or hepatitis B virus-mediated diseases.
  • HBV infection is one of the serious public health problems worldwide. HBV infection is an important cause of chronic hepatitis B, liver cirrhosis and hepatocellular carcinoma (Fattovich G.J. Hepatol. 2008; 48: 335-352).
  • Commonly used drugs for clinical treatment of chronic HBV infection mainly include nucleoside analogs and interferons. Nucleoside analogs cannot completely eliminate cccDNA in hepatocytes, and long-term use can easily lead to the emergence of drug-resistant mutant strains and rebound after drug withdrawal (Kwon H, Lok AS. Nat Rev Gastroenterol Hepatol. 2011; 8: 275-284). Interferon is not suitable for asymptomatic HBV carriers. In chronic HBV patients, the incidence of HBeAg seroconversion after six months of use is only 33%, and the large side effects of interferon also limit its application (Tang SX, Yu GL. Lancet 1990; 335(8684): 302).
  • the currently widely used hepatitis B protein vaccine induces humoral immunity to produce protective neutralizing antibodies to achieve the purpose of prevention.
  • neutralizing antibodies can only eliminate extracellular virus particles, while intracellular viruses mainly rely on specific cellular immune responses, such as Th1 cytokines such as IFN- ⁇ produced by helper T cells and CD4+ T cells.
  • Th1 cytokines such as IFN- ⁇ produced by helper T cells and CD4+ T cells.
  • CTL virus-specific cytotoxic T lymphocytes
  • Chinese patent CN104043120B provides a therapeutic hepatitis B vaccine, which includes hepatitis B surface antigen (HBsAg), hepatitis B core antigen (HBcAg), oligodeoxynucleotide (CpG), which can break through the immune tolerance of hepatitis B and is used for Treatment of viral hepatitis B, especially chronic hepatitis B.
  • HBsAg hepatitis B surface antigen
  • HBcAg hepatitis B core antigen
  • CpG oligodeoxynucleotide
  • CpG Commonly used adjuvant oligodeoxynucleotide
  • CpG oligodeoxynucleotide
  • its chemical nature is an oligodeoxynucleotide containing cytosine guanine dinucleotide, which has similar characteristics to natural CpG pattern recognition receptors
  • the immune response can bind to the Toll-like receptors on the cell membrane and effectively trigger the mammalian immune response through the TLR9 signaling pathway.
  • Saponins adjuvants are glycosides in which aglycones are triterpenoids or spirostane compounds, and are plant-derived adjuvants.
  • Quercus saponins are saponins extracted from Quillaja saponins.
  • QS-21 is the most widely reported type of adjuvant, but QS-21 can induce cell hemolysis, with certain systemic and local conditions. Toxic 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 present invention contains a double adjuvant, and the saponins and CpG oligodeoxynucleotides exhibit high-efficiency synergistic effects, which can mediate a stronger immune response.
  • the present invention also provides the use of the pharmaceutical composition for the treatment of hepatitis B virus infection and/or hepatitis B virus-mediated diseases.
  • the present invention provides a pharmaceutical composition comprising:
  • Hepatitis B surface antigen active fragments of said antigen, variants of said antigen, or a mixture of at least two of them
  • Hepatitis B core antigen active fragments of said antigen, variants of said antigen, or a mixture of at least two of them
  • An immunostimulatory composition comprising saponin and CpG oligodeoxynucleotides, or the immunostimulatory composition consists of an adjuvant comprising saponin and CpG oligodeoxynucleotides; wherein The CpG oligodeoxynucleotide sequence has two or more copies of 5'-TTCGTT-3' motif or 5'-TCGTCGTCG-3' motif.
  • 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)
  • CpG T3 TCG TCG TCG (SEQ ID NO: 7)
  • CpG T3 TCG TCG TCG (SEQ ID NO: 6) ID NO: 8);
  • the sequence of the CpG oligodeoxynucleotide is CpG T1: TCG TTC GTT CGT TCG TTC GTT (SEQ ID NO: 6).
  • the pharmaceutical composition according to the present invention wherein the saponins may be selected from Quercus saponins, ginsenosides, platycodon saponins, astragalus saponins, notoginseng saponins, glycyrrhizin, albizia bark saponins, Ophiopogon japonicus saponins, saikosaponins or One or more of Panax Japonicus saponins.
  • the saponins are Quercus saponins, ginsenosides, platycodin or Astragaloside IV; more preferably, the saponins are QS-7, QS-17, QS-18 or QS-21; further preferably The saponins of Quercus saponins are QS-21; the ginsenosides may be ginsenoside Rg1, ginsenoside Rg3, ginsenoside Rb1 or ginsenoside Re; the platycodon saponins may be platycodin D, platycodin D2, or both The mixture; the astragaloside can be astragaloside IV (astragaloside IV), astragaloside I, astragaloside II and other monomers or a mixture of two or more saponin monomers; the notoginsenoside can be three Heptaponin R1; the Ophiopogon japonicus saponins can be Ophiopogon japonicus sapononins
  • composition according to the present invention wherein the adjuvant containing saponin is an immunostimulatory complex adjuvant (Iscom adjuvant).
  • immunostimulatory complex adjuvant Iscom adjuvant
  • composition according to the present invention wherein the CpG oligodeoxynucleotide may comprise phosphorothioate linkage; preferably, the CpG oligodeoxynucleotide is a thiooligodeoxynucleotide , More preferably a full thio-oligodeoxynucleotide.
  • 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.
  • the weight ratio of the CpG oligodeoxynucleotide to the saponin is 1:0.1, 1:1, 1:2, 2:0.1, 2:1, 40:0.1, 40:1 or 20:1, Preferably it is 1:1, 2:1, 40:0.1, 40:1 or 20:1, and more preferably 2:1.
  • composition according to the present invention wherein the hepatitis B surface antigen comprises or consists of the sequence shown in SEQ ID NO:1.
  • the hepatitis B core antigen comprises or consists of the sequence shown in SEQ ID NO: 2; preferably, the active fragment of the hepatitis B core antigen comprises SEQ ID NO: 2
  • the pharmaceutical composition according to the present invention wherein the weight ratio between components i), ii) and iii) of the pharmaceutical composition is 4:2:1.1-42, preferably 4:2:2.1- 42.
  • the weight ratio between the components i), ii) and iii) of the pharmaceutical composition is 4:2:1.1, 4:2:2, 4:2:3, 4:2:2.1, 4:2:40.1, 4:2:41 or 4:2:42, preferably 4:2:2, 4:2:3, 4:2:40.1, 4:2:41 or 4:2:42, More preferably, it is 4:2:3.
  • composition according to the present invention wherein the pharmaceutical composition further comprises:
  • the present invention provides a hepatitis B preventive vaccine or hepatitis B therapeutic vaccine, which comprises the pharmaceutical composition; preferably, the vaccine is a hepatitis B therapeutic vaccine.
  • the present invention provides the use of the pharmaceutical composition in the preparation of a medicament for the prevention and/or treatment of hepatitis B virus infection and/or hepatitis B virus-mediated diseases; preferably, the B Hepatitis B virus infection and/or hepatitis B virus-mediated diseases are selected from hepatitis B, liver cirrhosis and liver cancer.
  • the present invention provides the use of the pharmaceutical composition in the preparation of a medicament for generating humoral and/or cellular immune responses against hepatitis B virus in a subject.
  • the present invention provides the use of the pharmaceutical composition in the preparation of a medicament for subtype conversion of hepatitis B core antibody in a subject.
  • the present invention provides the use of the pharmaceutical composition in the preparation of a medicament for breaking the immune tolerance of hepatitis B virus in a subject.
  • the present invention provides a method for preventing and/or treating hepatitis B virus infection and/or hepatitis B virus-mediated diseases, the method comprising administering to a subject in need of prevention and/or treatment An effective amount of the pharmaceutical composition; preferably, the hepatitis B virus infection and/or hepatitis B virus-mediated disease is selected from hepatitis B, liver cirrhosis and liver cancer.
  • the present invention provides a method for generating a humoral and/or cellular immune response against hepatitis B virus in a subject, the method comprising administering an effective amount of a pharmaceutical composition to a subject in need.
  • the present invention provides a method for subtype conversion of hepatitis B core antibody in a subject, the method comprising administering an effective amount of a pharmaceutical composition to a subject in need.
  • the present invention provides a method for breaking through hepatitis B virus immune tolerance in a subject, the method comprising administering an effective amount of a pharmaceutical composition to a subject in need.
  • the pharmaceutical composition of the present invention contains two adjuvants, which has unexpected technical effects and can mediate a stronger immune response.
  • the immunostimulatory effects of CpG T1, CpG T2, and CpG T3 alone are weaker than CpG1018, CpG7909, CpG1826, etc., but after combined application with QS21, the adjuvant 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.
  • the vaccine can significantly eliminate hepatitis B virus in transgenic mice through multiple immunizations.
  • 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 immune stimulant can induce stronger HBsAg and HBcAg specific IFN- ⁇ levels, and the immune effect is significantly better than that of a single adjuvant, and is significantly better than the combination of other CPG adjuvants and QS21.
  • 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 immunostimulatory compositions containing different saponins on the secretion level of HBsAg antigen-specific IFN- ⁇ ;
  • Figure 14 shows the effect of immunostimulatory compositions containing different saponins on the secretion level of HBcAg 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 components eg, HBsAg, HBcAg, QS-21, CpG oligodeoxynucleotides
  • the ingredients contained in the pharmaceutical composition 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 guanine , P represents a phosphodiester bond.
  • the CpG oligodeoxynucleotide is unmethylated.
  • the CpG-ODN includes a phosphorothioate linkage or a phosphorothioate backbone.
  • the CpG-ODN is phosphorothioate oligodeoxynucleotide (ie, thiooligodeoxynucleotide).
  • all nucleosides in the CpG-ODN are all phosphorothioate linkages, that is, the CpG-ODN is a full thio-oligodeoxynucleotide.
  • the CpG-ODN contains two or more copies of 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 effective amount refers to a dose sufficient to show a 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.
  • QS-21 purchased from BRENNTAG company, CAS.NO.A010-023.
  • the oligodeoxynucleotide is a synthetically prepared oligodeoxynucleotide sequence fragment, which contains one or more CpG motifs.
  • the CPG sequence used in this example is shown in Table 1:
  • the oligonucleotide chain will extend forward by one base;
  • 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. At this time, a thio reagent is used to oxidize the phosphoramidite to a phosphorous double bond.
  • Blocking After the condensation reaction, in order to prevent the 5'hydroxyl group attached to the CpG from being extended in the subsequent cycle reaction, it is often blocked by acetylation.
  • 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 processing 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 Series3
  • 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 NOs: 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 transformation of anti-HBcAg antibody subtypes, and the conversion efficiency is significantly higher than that of the dual adjuvant control (group I).
  • Example 10 The influence of different saponins on the immune effect of pharmaceutical compositions
  • mice C57BL/6(N) mice, male, 4 weeks old, 40 animals, Shanghai Lingchang Experimental Animal Technology Co., Ltd.
  • HBsAg protein, HBcAg protein and CpG T1 are all prepared in Example 1;
  • 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.) .
  • the pharmaceutical composition of the present invention uses a dual adjuvant combination. Compared with the combination of a single adjuvant, other CPG adjuvants and QS21, the CpG T1-T3 of the present invention is combined with various adjuvants, especially QS- 21 shows highly effective synergy and can mediate a stronger immune response.
  • the pharmaceutical composition containing the immunostimulatory composition has significant advantages, exhibits high-efficiency immunotherapy effects, has high clinical application value and broad market prospects.

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Abstract

属于生物制药领域，具体涉及一种药物组合物。所述药物组合物包含乙肝表面抗原、乙肝核心抗原和免疫刺激组合物，其中，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。

Description

药物组合物及其用途 技术领域
本发明属于生物制药领域。具体地，本发明涉及一种药物组合物，其包含乙肝表面抗原、乙肝核心抗原和免疫刺激组合物。其中，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中具有两个或两个以上拷贝的5’-TTCGTT-3’基序或5’-TCGTCGTCG-3’基序。本发明还涉及所述药物组合物用于治疗乙型肝炎病毒感染和/或乙型肝炎病毒介导的疾病的用途。
背景技术
乙型肝炎病毒(HBV)感染是世界范围的严重公共卫生问题之一。HBV感染是导致慢性乙型肝炎、肝硬化和肝细胞癌的重要原因(Fatt ovich G.J.Hepatol.2008；48：335-352)。临床治疗慢性HBV感染的常用药物主要有核苷类似物和干扰素。核苷类似物无法完全清除肝细胞内的cccDNA，且长期使用易导致耐药突变株的出现以及停药后反弹(Kwon H，Lok AS.Nat Rev Gastroenterol Hepatol.2011；8：275-284)。干扰素不适合用于无症状的HBV携带者，在慢性HBV患者中，使用半年后HBeAg血清学转换发生率仅为33％，而且干扰素副作用较大也限制其应用(Tang SX，Yu GL.Lancet 1990；335(8684)：302)。
目前广泛应用的乙肝蛋白疫苗通过诱导体液免疫，产生保护性中和抗体，达到预防的目的。大量的研究发现中和抗体仅能够消除胞外病毒颗粒，而胞内感染的病毒则主要依赖特异性的细胞免疫反应，辅助性T细胞、CD4+T细胞产生的IFN-γ等Th1型细胞因子，尤其是病毒特异性的杀伤性T淋巴细胞(cytotoxic T lymphocyte，CTL)来清除(ChinR，Lacamini S.Rev Med Viorl.2003：13(4)：255-72)。细胞免疫反应的强弱直接决定着乙肝的预后。因此，理想的治疗性乙肝疫苗需要同时诱导特异性的体液免疫和细胞免疫，突破乙肝的免疫耐受。例如，中国专利CN104043120B提供了一种治疗性乙肝疫苗，其包括乙肝表面抗原(HBsAg)、乙肝核心抗原(HBcAg)、寡聚脱氧核苷酸(CpG)，可以突破乙肝的免疫耐受，用于病毒性乙型肝炎，尤其是慢性乙型肝炎的治疗。
发明人对现有技术进行了深入研究，发现佐剂对治疗性乙肝疫苗的治疗 作用起到重要作用。常用佐剂寡聚脱氧核苷酸(CpG)作为一种免疫刺激组合物，其化学本质为含有胞嘧啶鸟嘌呤二核苷酸的寡脱氧核苷酸，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。皂苷佐剂是一类苷元为三萜或螺旋甾烷类化合物的糖苷，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***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佐剂涉及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乙肝治疗剂存在需求。
发明内容
针对现有技术的不足，发明人在进行了大量研究后，出乎意料地发现了免疫效果更强的免疫刺激组合物，以及包含所述免疫刺激组合物的药物组合物。本发明提供的药物组合物包含双佐剂，并且其中皂苷和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。本发明还提供了所述药物组合物用于治疗乙型肝炎病毒感染和/或乙型肝炎病毒介导的疾病的用途。
本发明的目的是通过以下技术方案实现的：
一方面，本发明提供一种药物组合物，其包含：
i)乙肝表面抗原、所述抗原的活性片段、所述抗原的变体，或者其中至少两种的混合物，
ii)乙肝核心抗原、所述抗原的活性片段、所述抗原的变体，或者其中至少两种的混合物，
iii)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。
根据本发明所述的药物组合物，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)。
根据本发明所述的药物组合物，其中，所述皂苷可以选自皂树皂苷、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。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；进一步优选地，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；所述桔梗皂苷可以为桔梗皂苷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等单体或其中两个或两个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等；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等；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。
根据本发明所述的药物组合物，其中，所述包含皂苷的佐剂为免疫刺激复合物佐剂(Iscom佐剂)。
根据本发明所述的药物组合物，其中，所述CpG寡聚脱氧核苷酸可以包含硫代磷酸酯连接；优选地，所述CpG寡聚脱氧核苷酸为硫代寡聚脱氧核苷酸，更优选地为全硫代寡聚脱氧核苷酸。
根据本发明所述的药物组合物，其中，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。
优选地，所述CpG寡聚脱氧核苷酸与皂苷的重量比为1∶0.1、1∶1、1∶2、2∶0.1、2∶1、40∶0.1、40∶1或20∶1，优选为1∶1、2∶1、40∶0.1、40∶1或20∶1，进一步优选为2∶1。
根据本发明所述的药物组合物，其中，所述乙肝表面抗原包含如SEQ ID NO:1所示的序列或由其组成。
根据本发明所述的药物组合物，其中，所述乙肝核心抗原包含如SEQ ID NO:2所示的序列或由其组成；优选地，所述乙肝核心抗原的活性片段包含SEQ ID NO:2中的第1位～第X位的连续氨基酸或由其组成，其中，X可以为149至183之间的整数；进一步优选地，X为152至183之间的整数。
根据本发明所述的药物组合物，其中，所述药物组合物的组分i)、ii)和iii)之间的重量比为4∶2∶1.1～42，优选为4∶2∶2.1～42。
进一步优选地，所述药物组合物的组分i)、ii)和iii)之间的重量比为4∶2∶1.1、4∶2∶2、4∶2∶3、4∶2∶2.1、4∶2∶40.1、4∶2∶41或4∶2∶42，优选为4∶2∶2、4∶2∶3、4∶2∶40.1、4∶2∶41或4∶2∶42，进一步优选为4∶2∶3。
根据本发明所述的药物组合物，其中，所述药物组合物还包含：
iv)可药用载体。
另一方面，本发明提供了一种乙肝预防性疫苗或乙肝治疗性疫苗，其包含所述药物组合物；优选地，所述疫苗为乙肝治疗性疫苗。
再一方面，本发明提供了所述药物组合物在制备用于预防和/或治疗乙型肝炎病毒感染和/或乙型肝炎病毒介导的疾病的药物中的用途；优选地，所述乙型肝炎病毒感染和/或乙型肝炎病毒介导的疾病选自乙型肝炎、肝硬化和肝癌。
再一方面，本发明提供了所述药物组合物在制备用于在对象中产生针对乙型肝炎病毒的体液免疫和/或细胞免疫应答的药物中的用途。
再一方面，本发明提供了所述药物组合物在制备用于使对象中乙肝核心抗体发生亚型转变的药物中的用途。
再一方面，本发明提供了所述药物组合物在制备用于在对象中突破乙型肝炎病毒免疫耐受的药物中的用途。
再一方面，本发明提供了一种预防和/或治疗乙型肝炎病毒感染和/或乙型肝炎病毒介导的疾病的方法，所述方法包括给予有需要的受试者预防和/或治疗有效量的药物组合物；优选地，所述乙型肝炎病毒感染和/或乙型肝炎病毒介导的疾病选自乙型肝炎、肝硬化和肝癌。
另一方面，本发明提供了一种在对象中产生针对乙型肝炎病毒的体液免疫和/或细胞免疫应答的方法，所述方法包括给予有需要的受试者有效量的药物组合物。
另一方面，本发明提供了一种使对象中乙肝核心抗体发生亚型转变的方法，所述方法包括给予有需要的受试者有效量的药物组合物。
另一方面，本发明提供了一种在对象中突破乙型肝炎病毒免疫耐受的方 法，所述方法包括给予有需要的受试者有效量的药物组合物。
本发明的药物组合物中包含两种佐剂，具有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、CpG T2和CpG T3的免疫刺激作用弱于CpG1018、CpG7909和CpG1826等，但与QS21联合应用后，所述佐剂表现出出人意料的协同作用，免疫效果显著增强。含有该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，显著优于其他CPG佐剂和QS21的组合。
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显示了含有不同皂苷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14显示了含有不同皂苷的免疫刺激组合物对HBcAg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“1.1至42”的范围应认为包含最小值1.1和最大值42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1.1或更大起始的子范围，例如1.1至6.1，以及以最大值42或更小终止的子范围，例如5.5至42。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HBsAg、HBcAg、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免疫刺激组合物的筛选实验准备
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，本实施例使用的CPG序列见表1：
表1 CPG寡聚脱氧核苷酸的具体序列
Figure PCTCN2020135570-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 PCTCN2020135570-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 Series3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 PCTCN2020135570-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，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 PCTCN2020135570-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 PCTCN2020135570-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；实验分组见表6。
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。
表6脾细胞分泌的HBsAg和HBcAg特异性IFN-γ的转阳率
Figure PCTCN2020135570-appb-000006
Figure PCTCN2020135570-appb-000007
3、实验结果：
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不同皂苷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，40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)；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HBsAg原液、HBcAg原液分别稀释至200μg/ml、100μg/ml，将各皂苷分别稀释至50μg/ml，使用PBS溶液将CpG T1溶解并稀释至100μg/ml，供下一步使用。
3、实验分组：见表7，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13和图14，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HBsAg和HBcAg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7实验动物分组
Figure PCTCN2020135570-appb-000008
综上所述，本发明所述的药物组合物应用双佐剂组合，相比单一佐剂、其他CPG佐剂和QS21的组合，本发明的CpG T1～T3与各种佐剂尤其是QS-21表现出高效的协同作用，可以介导更强烈的免疫应答。含有该免疫刺激组合物的药物组合物具有显著性优势，表现出高效的免疫治疗作用，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 (27)

  1. 一种药物组合物，所述药物组合物包含：
    i)乙肝表面抗原、所述抗原的活性片段、所述抗原的变体，或者其中至少两种的混合物，
    ii)乙肝核心抗原、所述抗原的活性片段、所述抗原的变体，或者其中至少两种的混合物，
    iii)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所述的药物组合物，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所述的药物组合物，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所述的药物组合物，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中任一项所述的药物组合物，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中任一项所述的药物组合物，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1至6中任一项所述的药物组合物，其中，所述CpG寡聚脱氧核苷酸为硫代寡聚脱氧核苷酸。
  8. 根据权利要求1至7中任一项所述的药物组合物，其中，所述CpG寡聚脱氧核苷酸为全硫代寡聚脱氧核苷酸。
  9. 根据权利要求1至8中任一项所述的药物组合物，其中，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。
  10. 根据权利要求1至9中任一项所述的药物组合物，其中，所述CpG寡聚脱氧核苷酸与皂苷的重量比为2∶1。
  11. 根据权利要求1至10中任一项所述的药物组合物，其中，所述乙肝表面抗原包含如SEQ ID NO:1所示的序列或由其组成；所述乙肝核心抗原包含如SEQ ID NO:2所示的序列或由其组成。
  12. 根据权利要求1至11中任一项所述的药物组合物，其中，所述乙肝核心抗原的活性片段包含SEQ ID NO:2中的第1位～第X位连续氨基酸或由其组成，其中，X为149至183之间的整数。
  13. 根据权利要求12所述的药物组合物，其中，所述乙肝核心抗原的活性片段包含SEQ ID NO:2中的第1位～第X位连续氨基酸或由其组成，其中，X为152至183之间的整数。
  14. 根据权利要求1至13中任一项所述的药物组合物，其中，所述药物组合物的组分i)、ii)和iii)之间的重量比为4∶2∶1.1～42，优选为4∶2∶2.1～42。
  15. 根据权利要求1至14中任一项所述的药物组合物，其中，所述药物 组合物的组分i)、ii)和iii)之间的重量比为4∶2∶3。
  16. 根据权利要求1至15中任一项所述的药物组合物，其中，所述药物组合物还包含：
    iv)可药用载体。
  17. 一种乙肝预防性疫苗或乙肝治疗性疫苗，其包含如权利要求1-16中任一项所述的药物组合物。
  18. 如权利要求1-16中任一项所述的药物组合物在制备用于预防和/或治疗乙型肝炎病毒感染和/或乙型肝炎病毒介导的疾病的药物中的用途。
  19. 如权利要求18所述的用途，其中，所述乙型肝炎病毒感染和/或乙型肝炎病毒介导的疾病选自乙型肝炎、肝硬化和肝癌。
  20. 如权利要求1-16中任一项所述的药物组合物在制备用于在对象中产生针对乙型肝炎病毒的体液免疫和/或细胞免疫应答的药物中的用途。
  21. 如权利要求1-16中任一项所述的药物组合物在制备用于使对象中乙肝核心抗体发生亚型转变的药物中的用途。
  22. 如权利要求1-16中任一项所述的药物组合物在制备用于在对象中突破乙型肝炎病毒免疫耐受的药物中的用途。
  23. 一种预防和/或治疗乙型肝炎病毒感染和/或乙型肝炎病毒介导的疾病的方法，所述方法包括给予有需要的受试者预防和/或治疗有效量的如权利要求1-16中任一项所述的药物组合物。
  24. 根据权利要求23所述的方法，其中，所述乙型肝炎病毒感染和/或乙型肝炎病毒介导的疾病选自乙型肝炎、肝硬化和肝癌。
  25. 一种在对象中产生针对乙型肝炎病毒的体液免疫和/或细胞免疫应答的方法，所述方法包括给予有需要的受试者有效量的如权利要求1-16中任一项所述的药物组合物。
  26. 一种使对象中乙肝核心抗体发生亚型转变的方法，所述方法包括给予有需要的受试者有效量的如权利要求1-16中任一项所述的药物组合物。
  27. 一种在对象中突破乙型肝炎病毒免疫耐受的方法，所述方法包括给予有需要的受试者有效量的如权利要求1-16中任一项所述的药物组合物。
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