WO2018214699A1 - 免疫激动剂和组合物及其应用以及组合物的制备方法 - Google Patents

免疫激动剂和组合物及其应用以及组合物的制备方法 Download PDF

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WO2018214699A1
WO2018214699A1 PCT/CN2018/084771 CN2018084771W WO2018214699A1 WO 2018214699 A1 WO2018214699 A1 WO 2018214699A1 CN 2018084771 W CN2018084771 W CN 2018084771W WO 2018214699 A1 WO2018214699 A1 WO 2018214699A1
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immunoagonist
hepatitis
hbsag
composition
vaccine
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PCT/CN2018/084771
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French (fr)
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靳广毅
王竹林
胡云龙
唐黎
刘冰
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深圳大学
深圳市康居正医药科技有限公司
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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
    • A61K39/39—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
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
    • A61K31/519—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim ortho- or peri-condensed with heterocyclic rings
    • A61K31/52—Purines, e.g. adenine
    • 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
    • A61K39/385—Haptens or antigens, bound to carriers
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D473/00—Heterocyclic compounds containing purine ring systems
    • C07D473/02—Heterocyclic compounds containing purine ring systems with oxygen, sulphur, or nitrogen atoms directly attached in positions 2 and 6
    • C07D473/18—Heterocyclic compounds containing purine ring systems with oxygen, sulphur, or nitrogen atoms directly attached in positions 2 and 6 one oxygen and one nitrogen atom, e.g. guanine
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
    • A61K2039/55505—Inorganic adjuvants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/57—Medicinal preparations containing antigens or antibodies characterised by the type of response, e.g. Th1, Th2
    • A61K2039/575—Medicinal preparations containing antigens or antibodies characterised by the type of response, e.g. Th1, Th2 humoral response
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/60—Medicinal preparations containing antigens or antibodies characteristics by the carrier linked to the antigen

Definitions

  • the present invention relates to the technical field of the prevention and treatment of hepatitis B vaccine, and more particularly to immuno agonists and compositions, and uses thereof, and methods of preparing the compositions.
  • Hepatitis B vaccine is a drug used to prevent hepatitis B. After vaccination, it can stimulate the immune system to produce protective antibodies, so that the human body has the immunity to prevent hepatitis B and achieve the purpose of preventing hepatitis B infection.
  • HBV hepatitis B virus
  • preventive hepatitis B vaccine is basically ineffective. Therefore, there is a great need for a "therapeutic hepatitis B vaccine" that both prevents and eliminates chronic hepatitis B virus infection.
  • the object of the present invention is to provide an immunoagonist and a composition, and an application thereof, and a preparation method of the composition, which solve the problem that the hepatitis B vaccine in the prior art cannot simultaneously prevent and eliminate chronic infection of hepatitis B virus.
  • an immunoagonist having the following formula:
  • R is OH or SH.
  • the immunoagonist is a compound selected from the following structural formula I, structural formula II, structural formula III or structural formula IV:
  • the present invention also provides a composition for preparing a prophylactic and therapeutic hepatitis B vaccine comprising the above-mentioned immunoagonist, recombinant hepatitis B surface antigen and aluminum adjuvant, the composition of the recombinant hepatitis B surface antigen, aluminum adjuvant and immunoagonist
  • the ratio is X:Y:Z, where X, Y, and Z are numbers between 1 and 120, respectively, and represent the number of moles, volume, or mass.
  • the recombinant hepatitis B surface antigen is HBsAg
  • the amino acid sequence of HBsAg includes 200-400 amino acids
  • the molecular weight of HBsAg is 20,000 Da-44000 Da.
  • the amino acid sequence of HBsAg includes 200-250 amino acids; and the molecular weight of HBsAg is 20,000 Da-25000 Da.
  • the aluminum adjuvant is an aluminum hydroxide gel, aluminum phosphate, aluminum sulfate, ammonium alum or potassium alum.
  • the present invention also provides a method for preparing the above composition, which comprises: mixing a mother liquor of HBsAg in a solvent of DMSO or physiological saline with a solution of 9 volumes of an aluminum adjuvant to obtain a mixed solution, and then mixing the mixture with The immunoagonist was mixed in a 9:1 volume ratio to obtain a composition for preparing a therapeutic hepatitis B vaccine.
  • composition of the present invention is useful for improving the antibody titer and effector T cells against hepatitis B surface antigen HBsAg, and for preparing vaccines and medicaments for preventing and treating hepatitis B.
  • the present invention provides an immunoagonist for a chronic hepatitis B infected person and a composition for preparing a prophylactic and therapeutic hepatitis B vaccine It can inactivate and eliminate cells infected with hepatitis B virus in animals and humans on the basis of hepatitis B virus-specific antibodies that maintain the original prophylactic vaccine. It has dual functions of prevention and treatment; it has more than the original HBsAg antigen. Stronger immune activation effect, enhanced by immune cytokine induction, higher antibody production, especially high levels of ⁇ -interferon and IgG2a antibodies, Th1 type immune production, significant innovation for the removal of hepatitis B virus And practicality.
  • FIG. 1 is a synthetic process diagram of the immunoagonist SZU-103 represented by Structural Formula I;
  • Figure 2 is an ESI-MS diagram of the immunoagonist SZU-103 represented by Structural Formula I;
  • Figure 3 is a diagram showing the synthesis process of the immunoagonist SZU-117 represented by Structural Formula II;
  • Figure 4 is an ESI-MS diagram of the immunoagonist SZU-117 represented by Structural Formula II;
  • Figure 5 is a diagram showing the synthesis process of the immunoagonist SZU-114 represented by Structural Formula III;
  • Figure 6 is an ESI-MS diagram of the immunoagonist SZU-114 represented by Structural Formula III;
  • Figure 7 is a diagram showing the synthesis process of the immunoagonist SZU-122 represented by Structural Formula IV;
  • Figure 8 is an ESI-MS diagram of the immunoagonist SZU-122 represented by Structural Formula IV;
  • Figure 9A is a graph showing the effect of an immunoagonist represented by Formula I on the cytokine IL-6;
  • Figure 9B is a graph showing the effect of an immunoagonist represented by Formula I on the cytokine IFN ⁇ ;
  • Figure 9C is a graph showing the stimulatory effect of an immunoagonist represented by Formula I on the immune cytokine IL-12;
  • Figure 10A is a comparison diagram of an immune response of an immunoagonist represented by Formula I and a control group, respectively, which induces an HBsAg antigen-specific IgG1 antibody;
  • Figure 10B is a comparison diagram of an immune response of an immunogenic agonist represented by Formula I and a control group, respectively, which induces an HBsAg antigen-specific IgG2a antibody;
  • Figure 11A is a photograph showing an antigen-specific T cell response induced by an immunoagonist represented by Formula I as a hepatitis B vaccine adjuvant;
  • Figure 11B is a comparison diagram of an immunoagonist represented by Formula I as an adjuvant for hepatitis B vaccine and a control group, respectively, for inducing antigen-specific T cell responses;
  • Figure 12A is a fluorescent diagram of the effect of an immunoagonist represented by Formula I as a hepatitis B vaccine adjuvant to induce an antigen-specific CD4 + T cell response;
  • Figure 12B is a comparative diagram showing the effect of an immunoagonist represented by Formula I as a hepatitis B vaccine adjuvant and a control group in inducing antigen-specific CD4 + T cell responses, respectively;
  • Figure 12C is a fluorescent diagram of the effect of an immunoagonist represented by Formula I as a hepatitis B vaccine adjuvant to induce an antigen-specific CD8 + T cell response;
  • Fig. 12D is a comparative diagram showing the effect of an immunoagonist represented by Formula I as a hepatitis B vaccine adjuvant and a control group, respectively, in inducing an antigen-specific CD8 + T cell response.
  • the advantage of using small molecule immunoagonists as vaccine adjuvants is that they are structurally stable and easy to synthesize, purify and manufacture.
  • the shortcomings of small molecule immunoadjuvants are that the body absorbs quickly and metabolism is fast, so it cannot be well coordinated with antigenic proteins. metabolism.
  • the small molecule immunoagonist used in the present invention has a structural formula with an ⁇ , ⁇ -unsaturated carbonyl group, and has covalent adsorption to an antigenic protein (especially a protein containing a thiol residue); for aluminum atoms and aluminum ions
  • the empty atomic orbitals have electron adsorption.
  • the immunoagonist has the general formula:
  • SM360320 (1, 2) in Formula I is a TLR7 agonist (Proc Natl Acad Sci U S A. 2006 Feb 7; 103(6): 1828-33; Blood. 2011 May 26; 117(21): 5683-91 ).
  • the hydroxyl group (-OH) in the formula I may also be a sulfhydryl group (-SH) of a homologue.
  • SM360320 of Formula I has the tautomer of the same compound as Formula 1 and Formula 2:
  • the immunoagonist is a compound selected from the following structural formula I, structural formula II, structural formula III or structural formula IV:
  • the above structural formula is characterized by comprising a Toll-like receptor 7 (TLR7) agonist SM360320 and a compound system having an ⁇ , ⁇ -unsaturated carbonyl group.
  • TLR7 Toll-like receptor 7
  • the compound of Structural Formula I, Structural Formula II, Structural Formula III or Structural Formula IV may also be a hydroxyl group at the 8-position and a carbonyl tautomer.
  • the present invention also provides a composition for preparing a prophylactic and therapeutic hepatitis B vaccine comprising the above-mentioned immunoagonist and recombinant hepatitis B surface antigen and aluminum adjuvant, and the composition ratio of the recombinant hepatitis B surface antigen, the aluminum adjuvant and the immunoagonist is X: Y: Z, wherein X, Y, and Z are numbers between 1 and 120, respectively, and represent the number of moles, volume, or mass.
  • the recombinant hepatitis B surface antigen is HBsAg
  • the amino acid sequence of HBsAg includes 200-400 amino acids
  • the molecular weight of HBsAg is 20000Da-44000Da.
  • the amino acid sequence of HBsAg comprises 200-250 amino acids; the molecular weight of HBsAg is 20000Da-25000Da.
  • the typical amino acid sequence of HBsAg is as follows (containing 226 amino acids) (Chinese Journal of Bioengineering 1995, 15(2): 43-45):
  • the above aluminum adjuvant may be aluminum hydroxide gel, aluminum phosphate, aluminum sulfate, ammonium alum or potassium alum.
  • the present invention provides an immunoagonist and a composition for preparing a prophylactic and therapeutic hepatitis B vaccine for a chronic hepatitis B infection, which is capable of infecting an animal and a human body based on a hepatitis B virus-specific antibody which maintains the original prophylactic vaccine.
  • the cells of hepatitis B virus are inactivated and eliminated, which have the dual effects of prevention and treatment; they have stronger immune activation effect than the original HBsAg antigen, which is manifested by enhanced immune cytokine induction and higher antibody production effect, especially
  • the high level of gamma-interferon and IgG2a antibodies marks Th1 type immune production and is significantly innovative and practical for the removal of hepatitis B virus.
  • the above immuno agonists can be used for the preparation of a novel hepatitis B vaccine for prophylactic and therapeutic vaccines for hepatitis B.
  • Example 5 Preparation method for preparing a composition for preventing and treating hepatitis B vaccine
  • HBsAg HBsAg mother liquor (supplied by Shenzhen Kangtai Biological Products Co., Ltd.) and 9 volumes of aluminum adjuvant solution (containing Al(OH) 3 concentration of 1.44 mg/mL) were combined to obtain a concentration of HBsAg containing 22 ⁇ g/ A mixture of mL was mixed with a 10 mM immunoagonist in a 9:1 volume (9 volumes of vaccine solution: 1 volume of immuno agonist solution) to obtain a hepatitis B vaccine of the final composition.
  • M is the immunoagonist SZU-103.
  • the solvent for the 10 mM immunoagonist mother liquor was DMSO or physiological saline; the other aluminum adjuvant was replaced by the effective content of aluminum (the initial aluminum adjuvant solution contained 0.5 mg/mL of aluminum).
  • the form of HBsAg may be a monomer or a multimer, and the mother liquor has a concentration ranging from 220 ⁇ g/mL to 250 ⁇ g/mL in terms of HBsAg.
  • the biological activity test is specifically described below.
  • Example 6 In vitro cytokine stimulation
  • Spleen lymphocytes derived from Balb/c mice were used as a test system. 1 ⁇ 106/mL spleen lymphocytes were co-cultured with SZU-101, SZU-103, SZU-114, SZU-117 and SZU-122 at a concentration range of 0.03-40 ⁇ M for 24 hours. The supernatant was collected and detected by Elisa method. The concentration of IL6 and IFN ⁇ in the supernatant, R848 as a positive control, no addition as a negative control. Results As shown in Figures 9A, 9B and 9C, the stimulatory effects of immunoagonists on the immunocytokines IL-6, IL-12 and IFN- ⁇ were significant.
  • Example 7 Effect of an immunoagonist represented by Formula I as an adjuvant for hepatitis B vaccine to induce antigen-specific humoral immune response
  • mice (5 per group) were immunized on days 0, 14 and 28 (2 ⁇ g of HBsAg antigen mixed with 100 nmol of TLR7 agonist and 1.12 mg/mL of Al(OH)3 as another added adjuvant).
  • Mouse sera were isolated on day 35 and HBsAg-specific IgG1 and IgG2a antibody titers were measured by Elisa assay.
  • R848, commercial hepatitis B vaccine and PBS injection were used as a control group. The results are shown in Figures 10A and 10B, and the effect of the immunoagonist as an adjuvant for hepatitis B vaccine to induce an antigen-specific humoral immune response was apparent.
  • Example 8 Effect of an immunoagonist represented by Formula I as an hepatitis B vaccine adjuvant to induce an antigen-specific T cell response
  • mice (5 per group) were immunized on days 0, 14 and 28 (2 ⁇ g of HBsAg antigen mixed with 100 nmol of TLR7 agonist and 1.12 mg/mL of Al(OH)3 as another added adjuvant).
  • Mouse spleen lymphocytes were isolated on day 35, and HBsAg-specific T cell levels in spleen lymphocytes were detected by Elispot method.
  • R848, commercial hepatitis B vaccine and PBS injection were used as a control group. Referring to Fig. 11A and Fig. 11B, the results show that the immunoagonist represented by the general formula I has an effect of inducing an antigen-specific T cell response as a hepatitis B vaccine adjuvant.
  • Example 9 Effect of an immunoagonist represented by Formula I as a hepatitis B vaccine adjuvant to induce antigen-specific CD4+ and CD8+ T cell responses
  • mice (5 per group) were immunized on days 0, 14 and 28 (2 ⁇ g of HBsAg antigen mixed with 100 nmol of TLR7 agonist and 1.12 mg/mL of Al(OH)3 as another added adjuvant).
  • Mouse spleen lymphocytes were isolated on day 35, and the ratio of HBsAg-specific CD4+ and CD8+ T cells was identified by intracellular IFN ⁇ staining and flow cytometry.
  • R848 and commercial hepatitis B vaccine were used as a control group.
  • the results show that the immunoagonist represented by Formula I has a significant effect on the induction of antigen-specific CD4 + and CD8 + T cell responses as a hepatitis B vaccine adjuvant.

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Abstract

一种免疫激动剂及其组合物，以及它们在制备乙肝疫苗中的应用以及组合物的制备方法。所述组合物比原有HBsAg抗原具有更强的免疫激活效应，能够对感染乙肝病毒的细胞进行灭活和清除，具有预防和治疗的双重作用。

Description

免疫激动剂和组合物及其应用以及组合物的制备方法 技术领域
本发明涉及乙肝疫苗预防和治疗的技术领域，更具体地说，涉及免疫激动剂和组合物及其应用以及组合物的制备方法。
背景技术
乙肝疫苗是用于预防乙肝的药物。疫苗接种后，可刺激免疫系统产生保护性抗体，从而使人体具有了预防乙肝的免疫力，达到预防乙肝感染的目的。但是我国感染乙肝病毒(HBV)的人群有9000万人，其中慢性乙肝患者有2000万人。对于慢性乙肝感染者和患者，预防性乙肝疫苗基本无效。因此非常需要一种既能预防又能清除乙肝病毒慢性感染的“治疗性乙肝疫苗”。
发明内容
本发明的目的在于提供免疫激动剂和组合物及其应用以及组合物的制备方法，解决了现有技术中的乙肝疫苗不能同时预防和清除乙肝病毒慢性感染的问题。
本发明解决技术问题所采用的技术方案是：一种免疫激动剂，其通式如下：
Figure PCTCN2018084771-appb-000001
其中，R为OH或SH。
在本发明的免疫激动剂中，所述免疫激动剂是选自下述结构式I、结构式II、结构式III或结构式IV的化合物：
Figure PCTCN2018084771-appb-000002
本发明的上述的免疫激动剂在制备治疗性乙肝疫苗中的应用、以及在制备人和动物免疫调节药物中的应用。
本发明还提供一种用于制备预防和治疗乙肝疫苗的组合物，包含上述的免疫激动剂、重组乙肝表面抗原以及铝佐剂，所述重组乙肝表面抗原、铝佐剂和免疫激动剂的组成比例为X：Y：Z，其中，X、Y、 Z分别是1-120之间的数字且代表摩尔数、体积数或者质量数。
在本发明的组合物中，所述重组乙肝表面抗原、铝佐剂和免疫激动剂的组成比例为X：Y：Z＝1：20.36：20～31.8。
在本发明的组合物中，所述重组乙肝表面抗原为HBsAg，HBsAg的氨基酸序列包括200-400个氨基酸，HBsAg的分子量为20000Da-44000Da。
在本发明的组合物中，HBsAg的氨基酸序列包括200-250个氨基酸；HBsAg的分子量为20000Da-25000Da。
在本发明的组合物中，所述铝佐剂为氢氧化铝凝胶、磷酸铝、硫酸铝、铵明矾或钾明矾。
本发明还提供了上述的组合物的制备方法，其特征在于，包括：将以DMSO或生理盐水为溶剂的HBsAg母液与9倍体积的铝佐剂溶液混合得到混合液，再将该混合液与免疫激动剂按9：1体积比混合得到用于制备治疗性乙肝疫苗的组合物。
本发明的上述的组合物在用于提高针对乙肝表面抗原HBsAg的抗体效价和效应T细胞中应用、以及在制备预防和治疗乙肝的疫苗和药物中的应用。
实施本发明的免疫激动剂和组合物及其应用以及组合物的制备方法，具有以下有益效果：本发明针对慢性乙肝感染者提供一种免疫激动剂和用于制备预防和治疗乙肝疫苗的组合物，其在动物和人体内在保持原有预防性疫苗的乙肝病毒特异性抗体基础上，能够对感染乙肝病毒的细胞进行灭活和清除，具有预防和治疗的双重作用；其比原 有HBsAg抗原具有更强的免疫激活效应，表现在加强的免疫细胞因子诱导、更高的抗体产生效果，特别是γ-干扰素和IgG2a抗体的高水平标志Th1型免疫生成，对于清除乙肝病毒具有显著的创新性和实用性。
附图说明
图1为结构式I代表的免疫激动剂SZU-103的合成工艺图；
图2为结构式I代表的免疫激动剂SZU-103的ESI-MS图；
图3为结构式II代表的免疫激动剂SZU-117的合成工艺图；
图4为结构式II代表的免疫激动剂SZU-117的ESI-MS图；
图5为结构式III代表的免疫激动剂SZU-114的合成工艺图；
图6为结构式III代表的免疫激动剂SZU-114的ESI-MS图；
图7为结构式IV代表的免疫激动剂SZU-122的合成工艺图；
图8为结构式IV代表的免疫激动剂SZU-122的ESI-MS图；
图9A为通式I为代表的免疫激动剂对免疫细胞因子IL-6的激发效应图；
图9B为通式I为代表的免疫激动剂对免疫细胞因子IFNγ的激发效应图；
图9C为通式I为代表的免疫激动剂对免疫细胞因子IL-12的激发效应图；
图10A为通式I为代表的免疫激动剂和对照组分别诱导HBsAg抗原特异性IgG1抗体免疫反应对比图；
图10B为通式I为代表的免疫激动剂和对照组分别诱导HBsAg抗原特异性IgG2a抗体免疫反应对比图；
图11A为通式I为代表的免疫激动剂作为乙肝疫苗佐剂诱导抗原特异性T细胞反应的照片；
图11B为通式I为代表的免疫激动剂作为乙肝疫苗佐剂和对照组分别诱导抗原特异性T细胞反应的对比图；
图12A为通式I为代表的免疫激动剂作为乙肝疫苗佐剂诱导抗原特异性CD4 +T细胞反应的作用的荧光图；
图12B为通式I为代表的免疫激动剂作为乙肝疫苗佐剂和对照组分别诱导抗原特异性CD4 +T细胞反应的作用的对比图；
图12C为通式I为代表的免疫激动剂作为乙肝疫苗佐剂诱导抗原特异性CD8 +T细胞反应的作用的荧光图；
图12D为通式I为代表的免疫激动剂作为乙肝疫苗佐剂和对照组分别诱导抗原特异性CD8 +T细胞反应的作用的对比图。
具体实施方式
下面结合附图和实施例，对本发明的免疫激动剂和组合物及其应用以及组合物的制备方法作进一步说明：
使用小分子免疫激动剂作为疫苗佐剂的优点是其结构稳定，易于人工合成纯化和制造，但是小分子免疫佐剂的缺点是机体吸收快、代谢快，因此不能和抗原蛋白很好的协同分布代谢。本发明中应用的小分子免疫激动剂的结构式带有α,β-不饱和羰基基团，对抗原蛋白(特 别是含有巯基残基的蛋白)具有共价吸附作用；对铝原子和铝离子的空原子轨道具有电子吸附作用。本发明的免疫激动剂的这些优点大大提高了其和抗原的免疫协同作用，因此从机制上也具有显著的创新性。
该免疫激动剂，其通式如下：
Figure PCTCN2018084771-appb-000003
通式I中SM360320(1、2)是TLR7激动剂(Proc Natl Acad Sci U S A.2006 Feb 7；103(6):1828-33；Blood.2011 May 26；117(21):5683-91)。另外，通式I中的羟基基团(-OH)也可以是同系物的硫氢基(-SH)。
其中，通式I中的SM360320具有如式1和式2的同一化合物的互变异构体：
Figure PCTCN2018084771-appb-000004
具体地，免疫激动剂是选自下述结构式I、结构式II、结构式III或结构式IV的化合物：
Figure PCTCN2018084771-appb-000005
上述结构式特征为包含Toll样受体7(TLR7)激动剂SM360320与具有α,β-不饱和羰基基团的化合物体系。需要说明的是，结构式I、结构式II、结构式III或结构式IV的化合物也可以是8位羟基和羰基互变异构体。
本发明还提供一种用于制备预防和治疗乙肝疫苗的组合物，包含上述的免疫激动剂以及重组乙肝表面抗原和铝佐剂，重组乙肝表面抗原、铝佐剂和免疫激动剂的组成比例为X：Y：Z，其中，X、Y、Z分别是1-120之间的数字且代表摩尔数、体积数或者质量数。优选地，重组乙肝表面抗原、铝佐剂和免疫激动剂的质量比为X：Y：Z＝1：20.36：20～31.8。
其中，重组乙肝表面抗原为HBsAg，HBsAg的氨基酸序列包括200-400个氨基酸，HBsAg的分子量为20000Da-44000Da。优选地，HBsAg的氨基酸序列包括200-250个氨基酸；HBsAg的分子量为20000Da-25000Da。HBsAg的氨基酸典型代表序列如下(含226个氨 基酸)(《中国生物工程杂志》1995,15(2):43-45)：
Figure PCTCN2018084771-appb-000006
上述铝佐剂可以为氢氧化铝凝胶、磷酸铝、硫酸铝、铵明矾或钾明矾。
本发明针对慢性乙肝感染者提供一种免疫激动剂和用于制备预防和治疗乙肝疫苗的组合物，其在动物和人体内在保持原有预防性疫苗的乙肝病毒特异性抗体基础上，能够对感染乙肝病毒的细胞进行灭活和清除，具有预防和治疗的双重作用；其比原有HBsAg抗原具有更强的免疫激活效应，表现在加强的免疫细胞因子诱导、更高的抗体产生效果，特别是γ-干扰素和IgG2a抗体的高水平标志Th1型免疫生成，对于清除乙肝病毒具有显著的创新性和实用性。
上述免疫激动剂可用于制备新型乙肝疫苗，用于乙肝的预防性疫苗和治疗性疫苗。
下面通过具体实施例进行详细说明。
实施例1：结构式I代表的免疫激动剂(SZU-103)的合成
如图1所示，称取344mg(1mmol)SZU-008T和341mg(1.125mmol)利尿酸溶于8mL DMF中，加入HBTU(427mg，1.125mmol)、三乙胺(416μL，3mmol)和催化量的DMAP，室温下搅拌反应，过夜。将反应液倒入100mL水中，抽滤，滤渣经水洗、干燥得粗品。再经柱层析分离(DCM:MeOH＝20:1)，得白色固体475mg，产率75.5％。如图2所示，ESI-MS:m/z＝629.1[M+H] +。
实施例2：结构式II代表的免疫激动剂(SZU-117)的合成
如图3所示，在SZU-101(1g,0.002mol)的无水DMF溶液中，加入EDCI(0.0025mol)、HOBT(0.0025mol)后室温搅拌半小时。0℃下滴加DIEA(0.004mol)后加入N-Boc-哌嗪(0.0025mol)，室温反应过夜。TLC监测反应至结束，将反应液倒入水中，抽滤，水洗干燥后加入1:4TFA/DCM混合溶剂搅拌过夜。反应完成后，减压去除溶剂，用少量乙醇重新溶解，加入2N盐酸成盐。旋干后，加入乙醚研磨，过滤，得SZU-130，产率56％。
SZU-130(100mg)、丙烯酰氯和三乙胺在0℃下混合后，转入室温反应2h，将反应液倒入水中，抽滤，滤渣经水洗、干燥得粗品。再经柱层析分离，得白色固体80mg，产率72％。如图4所示，ESI-MS:m/z＝567.2[M+H] +。
实施例3：结构式III代表的免疫激动剂(SZU-114)的合成
如图5所示，将SZU-008T(350mg)、EDCI(172mg)、HOBT(172mg)溶于无水DMF中，0℃下滴加DIEA(0.35mL)。滴毕后，转入室 温继续反应30min后，加入117mg反式-4-二甲基胺基巴豆酸盐酸盐，室温反应过夜。LCMS监测，反应完毕后，将反应液倒入水中，抽滤，滤渣经水洗、干燥得粗品。再经制备液相分离，得白色固体130mg，产率33％。如图6所示，ESI-MS:m/z＝456.2[M+H] +。
实施例4：结构式IV代表的免疫激动剂(SZU-122)的合成如图7所示，SZU-008T(300mg)、丙烯酰氯和三乙胺在0℃下混合后，转入室温反应2h，将反应液倒入水中，抽滤，滤渣经水洗、干燥得粗品。再经柱层析分离，得白色固体280mg，产率80％。如图8所示，ESI-MS:m/z＝399.1[M+H] +。
实施例5：用于制备预防和治疗乙肝疫苗的组合物制备方法
将浓度为220μg/mL的HBsAg母液(深圳康泰生物制品股份有限公司提供)与9倍体积的铝佐剂溶液(含Al(OH) 3浓度为1.44mg/mL)混合得到含HBsAg浓度为22μg/mL的混合液，将该混合液与10mM的免疫激动剂按9:1体积混合(疫苗溶液9体积：免疫激动剂溶液1体积)得到最终组合物的乙肝疫苗。重量比(HBsAg)：(Al)：(M)＝X：Y：Z＝0.22mg：4.48mg：7mg＝1:20.36:31.8。其中M为免疫激动剂SZU-103。
以SZU-114替换SZU-103的制备方法同上，配制重量比(HBsAg)：(Al)：(M)＝X：Y：Z＝0.22mg：4.48mg：5mg＝1:20.36:22.7。
以SZU-117替换SZU-103的制备方法同上，配制重量比(HBsAg)：(Al)：(M)＝X：Y：Z＝0.22mg：4.48mg：6.3mg＝1:20.36:28.6。
以SZU-122替换SZU-103的制备方法同上，配制重量比(HBsAg)： (Al)：(M)＝X：Y：Z＝0.22mg：4.48mg：4.4mg＝1:20.36:20。
其中10mM免疫激动剂母液配置溶剂为DMSO或生理盐水；其它铝佐剂替换以铝的有效含量为准(初始铝佐剂溶液含铝0.5mg/mL)。HBsAg的形式可为单体或者多聚合体，其母液以HBsAg计算浓度范围为220μg/mL-250μg/mL之间。
下面具体说明生物活性测试。
实施例6：体外细胞因子激发作用
采用Balb/c小鼠来源的脾脏淋巴细胞作为测试体系。1×106/mL的脾脏淋巴细胞与浓度范围为0.03-40μM的SZU-101、SZU-103、SZU-114、SZU-117和SZU-122共同培养24小时，收集上清液后用Elisa方法检测上清液中IL6和IFNγ浓度，R848作为阳性对照，无添加作为阴性对照。结果如图9A、9B和9C，免疫激动剂对免疫细胞因子IL-6、IL-12和IFN-γ的激发效应显著。
实施例7：通式I为代表的免疫激动剂作为乙肝疫苗佐剂诱导抗原特异性体液免疫反应的作用
Balb/c小鼠(每组5只)在0、14和28天进行免疫(2μg的HBsAg抗原混合100nmol的TLR7激动剂，1.12mg/mL Al(OH)3作为另一添加佐剂)。在第35天分离获得小鼠血清，运用Elisa法检测血清中HBsAg特异性IgG1和IgG2a抗体滴度。R848、商用乙肝疫苗和PBS注射作为对照组。结果见图10A和10B，免疫激动剂作为乙肝疫苗佐剂诱导抗原特异性体液免疫反应的作用明显。
实施例8：通式I为代表的免疫激动剂作为乙肝疫苗佐剂诱导抗 原特异性T细胞反应的作用
Balb/c小鼠(每组5只)在0、14和28天进行免疫(2μg的HBsAg抗原混合100nmol的TLR7激动剂，1.12mg/mL Al(OH)3作为另一添加佐剂)。在第35天分离获得小鼠脾脏淋巴细胞，运用Elispot法检测脾脏淋巴细胞中HBsAg特异性T细胞水平。R848、商用乙肝疫苗和PBS注射作为对照组。见图11A和图11B，结果显示通式I为代表的免疫激动剂作为乙肝疫苗佐剂诱导抗原特异性T细胞反应的作用明显。
实施例9：通式I为代表的免疫激动剂作为乙肝疫苗佐剂诱导抗原特异性CD4+和CD8+T细胞反应的作用
Balb/c小鼠(每组5只)在0、14和28天进行免疫(2μg的HBsAg抗原混合100nmol的TLR7激动剂，1.12mg/mL Al(OH)3作为另一添加佐剂)。在第35天分离获得小鼠脾脏淋巴细胞，运用胞内IFNγ染色和流式细胞技术鉴定HBsAg特异性CD4+和CD8+T细胞比例。R848和商用乙肝疫苗作为对照组。见图12A、12B、12C和12D，结果显示通式I为代表的免疫激动剂作为乙肝疫苗佐剂诱导抗原特异性CD4 +和CD8 +T细胞反应的作用明显。
应当理解的是，对本领域普通技术人员来说，可以根据上述说明加以改进或变换，所有这些改进或变换都应属于本发明所附权利要求的保护范围之内。

Claims (10)

  1. 一种免疫激动剂，其通式如下：
    Figure PCTCN2018084771-appb-100001
    其中，R为OH或SH。
  2. 根据权利要求1所述的免疫激动剂，其特征在于，所述免疫激动剂是选自下述结构式I、结构式II、结构式III或结构式IV的化合物：
    Figure PCTCN2018084771-appb-100002
  3. 权利要求1或2所述的免疫激动剂在制备治疗性乙肝疫苗中的应用、以及在制备人和动物免疫调节药物中的应用。
  4. 一种用于制备预防和治疗乙肝疫苗的组合物，其特征在于，包含权利要求1或2所述的免疫激动剂、重组乙肝表面抗原以及铝佐剂，所述重组乙肝表面抗原、铝佐剂和免疫激动剂的组成比例为X：Y：Z，其中，X、Y、Z分别是1-120之间的数字且代表摩尔数、体积数或者质量数。
  5. 根据权利要求4所述的组合物，其特征在于，所述重组乙肝表面抗原、铝佐剂和免疫激动剂的组成比例为X：Y：Z＝1：20.36：20～31.8。
  6. 根据权利要求4所述的组合物，其特征在于，所述重组乙肝表面抗原为HBsAg，HBsAg的氨基酸序列包括200-400个氨基酸，HBsAg的分子量为20000Da-44000Da。
  7. 根据权利要求6所述的组合物，其特征在于，HBsAg的氨基酸序列包括200-250个氨基酸；HBsAg的分子量为20000Da-25000Da。
  8. 根据权利要求4所述的组合物，其特征在于，所述铝佐剂为氢氧化铝凝胶、磷酸铝、硫酸铝、铵明矾或钾明矾。
  9. 权利要求4-8中任一所述的组合物的制备方法，其特征在于，包括：将以DMSO或生理盐水为溶剂的HBsAg母液与9倍体积的铝佐剂溶液混合得到混合液，再将该混合液与免疫激动剂按9：1体积比混合得到用于制备治疗性乙肝疫苗的组合物。
  10. 权利要求4-8中任一所述的组合物在用于提高针对乙肝表面抗原HBsAg的抗体效价和效应T细胞中应用、以及在制备预防和治疗乙肝的疫苗和药物中的应用。
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