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WO2011069408A1 - 一种多肽免疫原及其制备方法和应用 - Google Patents

一种多肽免疫原及其制备方法和应用 Download PDF

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WO2011069408A1
WO2011069408A1 PCT/CN2010/079040 CN2010079040W WO2011069408A1 WO 2011069408 A1 WO2011069408 A1 WO 2011069408A1 CN 2010079040 W CN2010079040 W CN 2010079040W WO 2011069408 A1 WO2011069408 A1 WO 2011069408A1
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amino acid
acid sequence
sequence
polypeptide
polypeptide immunogen
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French (fr)
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张嘎
张贲
韩斌
冯长访
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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
    • 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
    • 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
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A61P1/16—Drugs for disorders of the alimentary tract or the digestive system for liver or gallbladder disorders, e.g. hepatoprotective agents, cholagogues, litholytics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/20—Antivirals for DNA viruses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/40—Fusion polypeptide containing a tag for immunodetection, or an epitope for immunisation
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2730/00—Reverse transcribing DNA viruses
    • C12N2730/00011—Details
    • C12N2730/10011—Hepadnaviridae
    • C12N2730/10111—Orthohepadnavirus, e.g. hepatitis B virus
    • C12N2730/10134—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein

Definitions

  • the invention relates to a polypeptide immunogen and a preparation method thereof, and the application of the polypeptide immunogen in preparing a vaccine or a medicament for treating a chronic infection status of HBV and related diseases such as secondary liver cirrhosis and liver cancer, belonging to a vaccine or a medicament Medical technology field.
  • Hepatitis B is one of the infectious diseases that endanger the health of all human beings. Global hepatitis B virus infection accounts for about 30% of the population, and chronic hepatitis patients about 350 million. China is a highly endemic area. Hepatitis B virus carriers account for about 10% of the population (130 million), and chronic hepatitis patients are about 30 million. If they are unhealed, they can develop into cirrhosis and liver cancer.
  • the main method for controlling the prevalence of hepatitis B is injection of preventive hepatitis B vaccine, but it is not effective for people infected with hepatitis B virus, and the vaccination rate is not very high for various reasons. Therefore, for a long period of time, hepatitis B will still be It is one of the important diseases that endanger human health.
  • HBV as a hepadnavirus does not directly cause liver cell damage, and the pathological and clinical consequences of its infection depend on the immune mechanism.
  • HBV-specific cytotoxic T lymphocytes cytotoxic T The lymphocyte, CTL
  • CTL cytotoxic T The lymphocyte
  • Another object of the present invention is to provide use of the polypeptide immunogen for the preparation of a vaccine or medicament for the treatment of a chronic infection of HBV and its associated secondary cirrhosis, liver cancer and the like.
  • the polypeptide immunogen of the present invention comprises a polypeptide sequence comprising amino acid sequence 1, amino acid sequence 2, amino acid sequence 3 and amino acid sequence 4, wherein a linker peptide consisting of several amino acid residues between each amino acid sequence Segmental linkage or direct covalent linkage;
  • the amino acid sequence 1 is a mixed sequence of a Th cell epitope sequence or a Th cell epitope sequence covalently linked to a basic region sequence of an HIV-1 source-derived TAT protein;
  • the amino acid sequence 2 is the basic region sequence of the HIV-1 source-derived TAT protein;
  • the amino acid sequence 3 is a hepatitis C virus-derived CTL epitope sequence or a hepatitis B virus-derived CTL epitope sequence and A mixed sequence consisting of a covalently linked sequence of a basic region of an HIV-1 derived TAT protein;
  • the amino acid sequence 4 is a B cell epitope sequence derived from hepatitis B virus or a B cell derived from hepatitis
  • the order of arrangement of the polypeptide sequences of the present invention from the N-terminus to the C-terminus may be any combination of amino acid sequence 1, amino acid sequence 2, amino acid sequence 3, and amino acid sequence 4.
  • the preferred arrangement order is:
  • Amino acid sequence 1-amino acid sequence 3-amino acid sequence 4-amino acid sequence 2
  • Amino acid sequence 2-amino acid sequence 1-amino acid sequence 3-amino acid sequence 4,
  • amino acid sequence 2 when amino acid sequence 2 is present, amino acid sequences 1, 3, 4 cannot be a mixed sequence;
  • amino acid sequence 1 amino acid sequence 3-amino acid sequence 4
  • amino acid sequence 1, amino acid sequence 3, and amino acid sequence 4 contain at least one mixed sequence.
  • the amino acid sequence 1 of the present invention is the 830-843 amino acid sequence QYIKANSKFIGITE or the universal Th cell epitope PADRE or the mixed sequence RKKRRQRRRQYIKANSKFIGITE or the mixed sequence RKKRRQRRRPADRE on the Th cell epitope derived from tetanus toxoid;
  • the amino acid sequence 2 is HIV-1 The 48th-60th amino acid sequence GRKKRRQRRRPPQ or the 49th-57 amino acid sequence RKKRRQRRR or the 48th-57th amino acid sequence GRKKRRQRRR in the TAT protein;
  • the amino acid sequence 3 is the 18th to 27th amino acid sequence FLPSDFFPSV or the mixed sequence RKKRRQRRRFLPSDFFPSV on the HBV core antigen
  • the amino acid sequence 4 is HBV Pre The 14th amino acid sequence DPRVRGLYFPAGG or the 14-30 amino acid sequence DPRVRGLYFPAGGVSFG or the mixed sequence RKKRRQRRRDPRVR
  • the linking peptide between each amino acid sequence of the present invention is AAA, GGG or SSS; the polypeptide immunogen has a N-terminal amino acid sequence linked to a modified linking peptide KSS; the modification The palmitoyl group is covalently attached to the ⁇ or ⁇ amino group of the N-terminal lysine residue of the peptide KSS, respectively or simultaneously.
  • polypeptide immunogen of the present invention may have a plurality of polypeptide sequences, wherein the preferred polypeptide sequence is: CH3(CH2)14COKSSPADRESSSGRKKRRQRRRPPQAAAFLPSDFFPSVGGGDPRVRGLYFPAGGVSFG (polypeptide 1),
  • a method for preparing a polypeptide immunogen comprising the steps of:
  • the lysate is subjected to preliminary separation and purification by size exclusion chromatography, and then purified by reverse phase chromatography to obtain a polypeptide immunogen.
  • polypeptide immunogen for the preparation of a vaccine or medicament for the treatment of a persistent state of HBV chronic infection and its associated secondary cirrhosis, liver cancer disease.
  • the chronic infection status of HBV is chronic hepatitis B or hepatitis B virus carriers.
  • the vaccine or medicament also contains pharmaceutically acceptable excipients, adjuvants and carriers.
  • the vaccine or drug is administered by injection.
  • the invention adopts solid phase chemical synthesis method to prepare the immunogen, and determines the optimal temperature of the reaction system, the feed ratio, the peptide resin cracking method and time, the purification conditions and the like.
  • the reaction temperature is 30-40 ° C
  • the HOBT/DCC coupling strategy is adopted, and the amino acid sequence is continuously synthesized, and the optimum molar ratio of the raw material and the resin is 3:1, and the average molar ratio of each amino acid or component is The coupling efficiency is over 99.5%; the peptide resin is cleaved by trifluoroacetic acid method, and the optimal lysis time is 90 minutes.
  • Purification is carried out in two steps: (1) preliminary purification by size exclusion chromatography, dimethyl sulfoxide (DMSO) as mobile phase, optimal column temperature 20-40 ° C; (2) reversed phase chromatography Highly purified, the best loading conditions, the best chromatographic packing, the best column temperature and other factors were studied.
  • the optimal chromatography material is POROS 50 R1
  • the optimum column temperature is 22-34 °C.
  • the liposome dosage form was superior to the ethanol solution dosage form in inducing Th1 polarization and inducing CTL activity, and the pain of subcutaneous injection was significantly reduced; Liposomal lyophilized formulations are significantly superior in stability to liposomal suspension formulations.
  • the present invention sets the optimal formulation of the liposome injection of the polypeptide immunogen.
  • the above polypeptide immunogen soybean phospholipid, palmitic acid, vitamin E and cholesterol are contained.
  • the liposome lyophilized dosage form of the present invention contains human albumin, mannitol and phosphate in addition to the above-mentioned polypeptide immunogen, phospholipid, cholesterol, palmitic acid and vitamin E.
  • the polypeptide immunogen of the present invention and its dosage form may be any known immunological means such as subcutaneous injection, intradermal injection, intraperitoneal injection, intravenous injection or the like.
  • the immunization dose may be from 0.01 nmol to 20 nmol, and no additional adjuvant may be used.
  • Immunization of HBV transgenic mice stimulates lymphocyte activation and proliferation, Th1 polarization and CTL responses, and inhibits HBV replication, HBV in blood, in a dose-dependent manner The DNA copy number decreased, and the HbsAg and HbeAg titers decreased or disappeared. This potency is not achievable with single-epitope polypeptides or multi-epitope polypeptides alone.
  • the present invention determines a polypeptide immunogen having a specific skeleton structure based on a hepatitis B epitope map.
  • the polypeptide immunogen is effective in inducing Th1 polarization, HBV-specific CTL response, and inhibition of HBV replication in HBV transgenic mice.
  • the experimental results indicate that the above-mentioned polypeptide immunogen can be developed into a novel therapeutic vaccine or drug for hepatitis B and its secondary cirrhosis and liver cancer.
  • the Fmoc solid phase chemical synthesis method was employed. The carboxy terminus is fixed, and the peptide chain is extended from the C-terminus to the N-terminus, and is carried out on a synthesizer. Fmoc amino acid was used as raw material, the sample loading was 0.75mmol, side chain protected amino acids Ser(tBU), Thr(tBU), Tyr(tBU), Gln(Trt), Asp(OtBU), Glu(OtBU), Arg(pbf) Asn (Trt), Lys (Boc), Wang-resin is selected as the solid phase carrier, the loading amount is 0.25mmol, the molar ratio of raw material to resin is 3:1, and the first amino acid coupled to the resin is DIC/HOBT. Method coupling, activation of the remaining amino acids and palmitic acid and their coupling using the HOBT/DCC method.
  • TFA lysate (0.75 g phenol, 0.25 ml ethanedithiol, 0.5 ml thioanisole, 0.5 ml deionized water, 10.0 ml TFA) can minimize the occurrence of side reactions.
  • the reaction concentration was 40 mg/ml (40 mg refers to the mass of the polypeptide immunogen-resin synthesized in Example 1, and ml refers to the volume of the lysate), the reaction temperature was 25 ° C, and the reaction time was 1.5 hours.
  • the polypeptide molecule is cleaved from the resin, precipitated with diethyl ether, and rotary evaporated to give the crude peptide product.
  • the crude peptide product was subjected to SEC loading of 55.0 ml to carry out preliminary purification of the polypeptide molecule.
  • Chromatographic conditions chromatography system: P-6000 pump and AKTAeplorer 100; chromatography column: diameter 25 mm, column length 850 mm; filler Sephadex LH20; mobile phase: DMSO; flow rate: 2.0 ml/min. Good purification results were obtained.
  • HBV-DNA transgenic mice (ayw-type HBV whole gene (1.3 Kb) were used to infect Kunming mice). Animals were randomized into groups of 10, and polypeptide 5 was injected subcutaneously under the bilateral ribs and hind paws, and administered at 3 doses of 10, 100, and 1000 U/mouse, and boosted once a week for three times. IFN- ⁇ 2b (15000U/mouse) was used as a positive control drug, and physiological saline was used as a negative control drug. 10 days, 20 days, and 30 days after the end of the administration, the spleen of the mice was taken, and the spleen lymphocytes were isolated and stimulated with the test article 10 ng/ml for 3 days.
  • the supernatant was taken and detected by ELISA.
  • the secretion of cytokines such as IFN- ⁇ and IL-4 in the Qing dynasty was analyzed, and the effect of the test drug on the Th1/Th2 type polarization of T cells was analyzed. As a result, strong IFN- ⁇ secretion was detected, and no significant dose-effect relationship was observed in the detection of IL-4.
  • HBV-DNA transgenic mice (ayw-type HBV whole gene (1.3 Kb) were used to infect Kunming mice). Animals were randomized into groups of 10, and polypeptide 5 was injected subcutaneously under the bilateral ribs and hind paws, and administered at 3 doses of 10, 100, and 1000 U/mouse, and boosted once a week for three times. IFN- ⁇ 2b (15000U/mouse) was used as a positive control drug, and physiological saline was used as a negative control drug. The spleen of the mice was taken before, and 10, 20, and 30 days after the end of the last administration.
  • the spleen lymphocytes were isolated and stimulated with the test article 10 ng/ml for 3 days, and the IFN- ⁇ secretion was detected by ELI-SPOT method.
  • the frequency of expression of cells in peripheral blood lymphocytes The results showed that on the 30th day after the end of immunization, the frequency of expression of IFN- ⁇ secreting cells in peripheral blood lymphocytes increased with the increase of immunization dose, and the immune dose of 100 and 1000 U/mouse induced peripheral blood lymphocytes in vivo.
  • the frequency of expression of IFN- ⁇ secreting cells in cells increased significantly, and the highest detectable 3600 ⁇ IFN- ⁇ secreting cells/106 PBMC.
  • HbsAg viral surface antigen
  • HBV-DNA transgenic mice (ayw-type HBV whole gene (1.3 Kb) were used to infect Kunming mice). Animals were randomized into groups of 10, and polypeptide 5 was injected subcutaneously under the bilateral ribs and hind paws, and administered at 3 doses of 10, 100, and 1000 U/mouse, and boosted once a week for three times. IFN- ⁇ 2b (15000U/mouse) was used as a positive control drug, and physiological saline was used as a negative control drug. Blood was taken 10 days, 20 days, and 30 days after the end of the administration and the last administration, and the serum was separated, and the serum HbsAg was measured by ELISA. The results showed that serum HbsAg was significantly reduced in a dose- and time-dependent manner.
  • HBV-DNA transgenic mice (ayw-type HBV whole gene (1.3 Kb) were used to infect Kunming mice). Animals were randomly divided into groups of 10, and peptide 5 was injected subcutaneously under the bilateral ribs and hind paws, and administered at 3 doses of 10, 100, 1000 U/mouse, and boosted once a week for three times. IFN- ⁇ 2b (15000U/mouse) was used as a positive control drug, and physiological saline was used as a negative control drug. Blood was taken 10 days, 20 days, and 30 days after the end of the administration and the last administration. The serum was separated and the serum HBV was detected by quantitative PCR. DNA copy number. The results showed that serum HBV DNA copy number was significantly reduced in a dose- and time-dependent manner.

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Abstract

本发明公开了一种多肽免疫原及其制备方法。该多肽免疫原含有一个多肽序列，该多肽序列含有氨基酸序列1、氨基酸序列2、氨基酸序列3和氨基酸序列4。其中，各氨基酸序列之间由若干个氨基酸残基组成的连接肽段连接或直接共价连接。本发明还提供了该多肽免疫原在制备用于治疗HBV慢性感染持续状态及其相关的继发性肝硬化、肝癌等疾病的疫苗或药物中的应用。

Description

一种多肽免疫原及其制备方法和应用
技术领域
本发明涉及一种多肽免疫原及其制备方法，以及该多肽免疫原在制备用于治疗HBV慢性感染持续状态及其相关的继发性肝硬化、肝癌等疾病的疫苗或药物中的应用，属于医药技术领域。
背景技术
乙型病毒性肝炎是危害全人类健康的传染性疾病之一。全球乙型肝炎病毒感染者约占人群的30%，慢性肝炎患者约3.5亿。我国属高流行区，乙型肝炎病毒的携带者约占人口的10%（1.3亿），慢性肝炎患者约0.3亿，如其迁延不愈则可发展为肝硬化、肝癌。
目前控制乙型肝炎流行的主要方法是注射预防性乙肝疫苗，但其对乙肝病毒感染者无效，且因种种原因接种率也不是很高，因此，在今后相当长时期内，乙型肝炎仍将是危害人类健康的重要疾病之一。
目前已经证实：HBV作为一种嗜肝病毒，并不能直接引起肝细胞损伤，其感染的病理和临床后果取决于免疫机制。
作为细胞内感染，慢性持续性感染状态主要与机体细胞免疫应答太弱有关。其中，HBV特异性细胞毒性T淋巴细胞（cytotoxic T lymphocyte,CTL）反应决定了HBV感染的最终结果。感染HBV后CTL活性高者体内病毒被清除而痊愈；CTL活性低或测不到的感染者则发展为慢性持续感染状态，并进一步发展为肝硬化或肝癌。
因此，打破HBV持续感染患者的免疫耐受状态，在体内启动HBV特异性CTL反应，可治疗HBV慢性感染持续状态及防止其相关的继发性肝硬化、肝癌等疾病。
美国的一家研究机构在自己研究的基础上分别申请了4项美国专利。这些专利的主要内容是关于在HBV抗原上所确定的CTL表位。这些表位分别来源于核心抗原、表面抗原、多聚酶和X抗原。这些部位或结构，是HBV所诱导的CTL所识别的部位。我国科研人员闻玉梅发现抗原-抗体免疫原性复合物可以有效地在部分鸭中清除病毒及疾病抗原，由此认为发展成为供人体应用的治疗性疫苗是有可能的。
当前，对HBV慢性持续感染状态（包括病毒携带者、慢性迁延性肝炎和慢性活动性肝炎）尚无特效治疗方法。干扰素和拉米呋定虽有特定的治疗效果，但并不能清除病毒。因此，目前急需寻求有效治疗HBV慢性持续感染状态的新方法。
发明内容
本发明的目的在于提供一种多肽免疫原及其制备方法。
本发明的另一目的在于提供了该多肽免疫原在制备用于治疗HBV慢性感染持续状态及其相关的继发性肝硬化、肝癌等疾病的疫苗或药物中的应用。
本发明所述多肽免疫原，含有一个多肽序列，该多肽序列含有氨基酸序列1、氨基酸序列2、氨基酸序列3和氨基酸序列4，其中，各氨基酸序列之间由若干个氨基酸残基组成的连接肽段连接或直接共价连接；所述的氨基酸序列1是Th细胞表位序列或Th细胞表位序列与艾滋病毒HIV-1来源的TAT蛋白的碱性区域序列共价相连构成的混合序列；所述的氨基酸序列2是艾滋病毒HIV-1来源的TAT蛋白的碱性区域序列；所述的氨基酸序列3是乙型肝炎病毒来源的CTL表位序列或乙型肝炎病毒来源的CTL表位序列与艾滋病毒HIV-1来源的TAT蛋白的碱性区域序列共价相连构成的混合序列；所述的氨基酸序列4是乙型肝炎病毒来源的B细胞表位序列或乙型肝炎病毒来源的B细胞表位序列与艾滋病毒HIV-1来源的TAT蛋白的碱性区域序列共价相连构成的混合序列。
本发明所述的多肽序列从N端到C端的排列次序可以是氨基酸序列1、氨基酸序列2、氨基酸序列3、氨基酸序列4的任意组合，其中，优选的排列次序为：
氨基酸序列1-氨基酸序列2-氨基酸序列3-氨基酸序列4、
氨基酸序列1-氨基酸序列3-氨基酸序列4-氨基酸序列2、
氨基酸序列1-氨基酸序列4-氨基酸序列2-氨基酸序列3、
氨基酸序列2-氨基酸序列1-氨基酸序列3-氨基酸序列4、
和氨基酸序列1-氨基酸序列3-氨基酸序列4，
其中：当氨基酸序列2存在时，氨基酸序列1、3、4不能为混合序列；
当排列次序为氨基酸序列1-氨基酸序列3-氨基酸序列4时，氨基酸序列1、氨基酸序列3、氨基酸序列4中至少含有一混合序列。
本发明所述的氨基酸序列1是破伤风类毒素来源的Th细胞表位上的第830-843氨基酸序列QYIKANSKFIGITE或通用Th细胞表位PADRE或混合序列RKKRRQRRRQYIKANSKFIGITE或混合序列RKKRRQRRRPADRE；所述氨基酸序列2是HIV-1 TAT蛋白中第48-60氨基酸序列GRKKRRQRRRPPQ或第49-57氨基酸序列RKKRRQRRR或第48-57氨基酸序列GRKKRRQRRR;所述的氨基酸序列3是HBV核心抗原上的第18-27氨基酸序列FLPSDFFPSV或混合序列RKKRRQRRRFLPSDFFPSV；所述氨基酸序列4是HBV Pre S2来源的B细胞表位上的第14-26氨基酸序列DPRVRGLYFPAGG或第14-30氨基酸序列DPRVRGLYFPAGGVSFG或混合序列RKKRRQRRRDPRVRGLYFPAGGVSFG。
本发明所述的各氨基酸序列之间的连接肽段为AAA、GGG或SSS；所述的多肽免疫原其N端氨基酸序列的N端上连接着修饰性连接肽段KSS；所述的修饰性连接肽段KSS的N端的赖氨酸残基的α或ε氨基上分别或同时共价连接着棕榈酰基。
本发明所述的多肽免疫原，可以有多种多肽序列，其中优选的多肽序列为：CH3(CH2)14COKSSPADRESSSGRKKRRQRRRPPQAAAFLPSDFFPSVGGGDPRVRGLYFPAGGVSFG（多肽1）、
CH3(CH2)14COK(CH3(CH2)14CO)SSPADRESSSGRKKRRQRRRPPQAAAFLPSDFFPSVGGGDPRVRGLYFPAGGVSFG（多肽2）、
CH3(CH2)14COKSSQYIKANSKFIGITEAAAGRKKRRQRRRPPQAAAFLPSDFFPSVGGGDPRVRGLYFPAGGVSFG（多肽3）、
CH3(CH2)14COK(CH3(CH2)14CO)SSQYIKANSKFIGITEAAAGRKKRRQRRRPPQAAAFLPSDFFPSVGGGDPRVRGLYFPAGGVSFG（多肽4）、
CH3(CH2)14COKSSPADREAAAFLPSDFFPSVGGGDPRVRGLYFPAGGGRKKRRQRRRPPQ（多肽5）、
CH3(CH2)14COK(CH3(CH2)14CO)SSPADREAAAFLPSDFFPSVGGGDPRVRGLYFPAGGGRKKRRQRRRPPQ（多肽6）、
CH3(CH2)14COKSSQYIKANSKFIGITEAAAFLPSDFFPSVGGGDPRVRGLYFPAGGGRKKRRQRRRPPQ（多肽7）、
CH3(CH2)14COK(CH3(CH2)14CO)SSQYIKANSKFIGITEAAAFLPSDFFPSVGGGDPRVRGLYFPAGGGRKKRRQRRRPPQ（多肽8）、
CH3(CH2)14COKSSGRKKRRQRRRPPQSSSPADREAAAFLPSDFFPSVGGGDPRVRGLYFPAGGVSFG（多肽9）、
CH3(CH2)14COK(CH3(CH2)14CO)SSGRKKRRQRRRPPQSSSPADREAAAFLPSDFFPSVGGGDPRVRGLYFPAGGVSFG（多肽10）、
CH3(CH2)14COKSSGRKKRRQRRRPPQSSSQYIKANSKFIGITEAAAFLPSDFFPSVGGGDPRVRGLYFPAGGVSFG（多肽11）、
CH3(CH2)14COK(CH3(CH2)14CO)SSGRKKRRQRRRPPQSSSQYIKANSKFIGITEAAAFLPSDFFPSVGGGDPRVRGLYFPAGGVSFG（多肽12）、
CH3(CH2)14COKSSPADREAAAFLPSDFFPSVGGGRKKRRQRRRDPRVRGLYFPAGGVSFG（多肽13）
CH3(CH2)14COK(CH3(CH2)14CO)SSPADREAAAFLPSDFFPSVGGGRKKRRQRRRDPRVRGLYFPAG
GVSFG（多肽14）、
CH3(CH2)14COKSSPADREGGGDPRVRGLYFPAGGGRKKRRQRRRFLPSDFFPSV（多肽15）或
CH3(CH2)14COK(CH3(CH2)14CO)SSPADREGGGDPRVRGLYFPAGGGRKKRRQRRRFLPSDFFPSV（多肽16）。
一种多肽免疫原的制备方法，包括以下步骤：
a.多肽固相合成所述多肽免疫原-树脂，所述多肽免疫原-树脂表示与树脂结合的多肽免疫原;
b.对多肽免疫原-树脂进行裂解，得到裂解液；
c.将裂解液采用体积排阻层析进行初步分离纯化，然后通过反向层析纯化得到多肽免疫原。
多肽免疫原在制备用于治疗HBV慢性感染持续状态及其相关的继发性肝硬化、肝癌疾病的疫苗或药物中的应用。
所述的HBV慢性感染持续状态为慢性乙型肝炎或乙型肝炎病毒携带者。
所述的疫苗或药物还含有药学上可接受的辅料、佐剂和载体。
所述的疫苗或药物的给药方式为注射给药。
本发明常用氨基酸英文缩写含义为：
A Ala 丙氨酸
D Asp 天冬氨酸
E Glu 谷氨酸
F Phe 苯丙氨酸
G Gly 甘氨酸
I Ile 异亮氨酸
K Lys 赖氨酸
L Leu 亮氨酸
N Asn 天冬酰胺酸
P Pro 脯氨酸
Q Gln 谷氨酰胺酸
R Arg 精氨酸
S Ser 丝氨酸
T Thr 苏氨酸
V Val 缬氨酸
Y Tyr 酪氨酸
本发明采用固相化学合成方法制备该种免疫原，确定了反应体系的最佳温度、投料比、肽树脂裂解方法和时间、纯化条件等因素。
在本发明方法中，当反应温度在30-40℃时，采用HOBT/DCC偶联策略，氨基酸序列连续合成，原料和树脂的最佳摩尔投料比为3：1，每个氨基酸或组分平均偶联效率达99.5%以上；肽树脂裂解采用三氟乙酸法，最佳裂解时间为90分钟。
纯化采用两步法：（1）采用体积排阻层析法初步纯化，二甲基亚砜（DMSO）为流动相，最佳柱温为20-40℃；（2）采用反相层析法高度纯化，研究了其最佳上样条件、最佳层析填料、最佳柱温等因素。在本发明方法中，最佳层析填料是POROS 50 R1，最佳柱温为22-34℃。
经乙醇溶液剂型、脂质体混悬液体剂型、脂质体冻干剂型的比较，证明脂质体剂型在诱导Th1极化和诱导CTL活性方面优于乙醇溶液剂型，皮下注射疼痛感明显减轻；脂质体冻干剂型在稳定性方面明显优于脂质体混悬液剂型。
本发明设定所述多肽免疫原的脂质体注射液最佳处方。其中，含有上述多肽免疫原、大豆磷脂、棕榈酸、维生素E和胆固醇。
本发明所述脂质体冻干剂型除含上述多肽免疫原、磷脂、胆固醇、棕榈酸、维生素E外，还含有人白蛋白、甘露醇和磷酸盐。
本发明所述多肽免疫原及其剂型，可以使用任何一种公知的免疫方式，例如：皮下注射、皮内注射、腹腔注射、静脉注射等。免疫剂量可以是0.01nmol至20nmol，可以不使用另外的佐剂。用于免疫HBV转基因小鼠可以剂量依赖性方式刺激淋巴细胞活化和增殖，Th1极化和CTL应答，并抑制HBV复制，血中HBV DNA拷贝数下降，HbsAg和HbeAg滴度下降或消失。该效力为单独使用单表位多肽或多表位多肽所不能达到的。
本发明以乙型肝炎抗原表位图谱为基础，确定了具有特定骨架结构的多肽免疫原。该多肽免疫原在HBV转基因小鼠体内能有效激发Th1极化反应、HBV特异性CTL反应和抑制HBV的复制。实验结果表明上述多肽免疫原可开发成为一种新型乙型肝炎及其继发性肝硬化、肝癌的治疗用疫苗或药物。
具体实施方式
以下实施例仅用于解释本发明，但不能因此限制本发明。
实施例1
多肽免疫原合成通法
采用Fmoc固相化学合成方法。固定羧基端，由C端向N端延伸肽链，在合成仪上进行。Fmoc氨基酸为原料，上样量为0.75mmol,侧链保护氨基酸Ser(tBU)、Thr(tBU)、Tyr(tBU)、Gln(Trt)、Asp(OtBU)、Glu(OtBU)、Arg(pbf)、Asn(Trt)、Lys(Boc)，选用Wang-树脂为固相载体，上样量0.25mmol，原料和树脂摩尔比为3：1，偶联到树脂上的第一个氨基酸采用DIC/HOBT法偶联，其余氨基酸和棕榈酸的活化及其偶联采用HOBT/DCC法。
实施例2
多肽免疫原裂解通法
选用TFA裂解液（0.75g苯酚、0.25ml乙二硫醇、0.5ml苯甲硫醚、0.5ml去离子水、10.0mlTFA）,可最大限度的抑制副反应的发生。反应浓度40mg/ml（40mg指的是实施例1合成得到的多肽免疫原-树脂的质量，ml指的是裂解液的体积），反应温度为25℃，反应时间为1.5小时。将多肽分子从树脂上切割下来，乙醚沉淀，旋转蒸发得粗肽产品。
实施例3
多肽免疫原初步纯化通法
将粗肽产品采用SEC加样55.0ml，进行多肽分子的初步纯化。层析条件：层析系统：P-6000泵及AKTAeplorer 100；层析柱：直径25mm，柱长850mm；填料Sephadex LH20；流动相：DMSO；流速：2.0ml/min。得到较好的纯化结果。
实施例4
多肽免疫原纯化通法
采用层析柱APS 50/275 POROS 50 R1进行批量制备。肽树脂经过裂解和初步纯化后，收集样品分成7次进行高度纯化。层析条件：层析系统：AKTAexplorer 100；样品浓度：10.52mg/ml；加样量：11.0ml；层析柱：直径50mm，柱长275mm；填料POROS 50 R1；柱温34℃；流动相A：30%乙醇-10mmol/L磷酸；流动相B：90%乙醇-10mmol/L磷酸；梯度：0-50%B，5 CV；50-100%B，0.5CV；100-100%B，0.5CV；流速：100.0ml/min。将收集的洗脱峰合并液混匀后，存放于-20℃备用。本品的纯度为98.5%，收率为85%。
实施例5
在HBV转基因小鼠诱导Th1极化
采用HBV-DNA转基因小鼠（ayw型HBV全基因（1.3Kb）传染昆明种小鼠）。将动物随机分组，每组10只，于双侧肋下和后脚掌皮下注射多肽5，按10、100和1000U/鼠的3个剂量给药，每周加强免疫一次，共三次。设IFN-α 2b（15000U/鼠）为阳性对照药，设生理盐水为阴性对照药。给药前和最后一次给药结束后10天、20天、30天，取小鼠脾脏，分离脾脏淋巴细胞，用试品10ng/ml体外刺激3天，取上清，用ELISA法检测培养上清中IFN-γ、IL-4等细胞因子的分泌情况，分析受试药物体内诱导T细胞Th1/Th2型极化的功效。结果可检测到较强的IFN-γ分泌，IL-4的检测未见明显的剂量-效应关系。
实施例6
在HBV转基因小鼠诱导CTL活性
采用HBV-DNA转基因小鼠（ayw型HBV全基因（1.3Kb）传染昆明种小鼠）。将动物随机分组，每组10只，于双侧肋下和后脚掌皮下注射多肽5，按10、100和1000U/鼠的3个剂量给药，每周加强免疫一次，共三次。设IFN-α 2b（15000U/鼠）为阳性对照药，设生理盐水为阴性对照药。给药前和最后一次给药结束后10天、20天、30天，取小鼠脾脏，分离脾脏淋巴细胞，用试品10ng/ml体外刺激3天，用ELI-SPOT法检测IFN-γ分泌细胞在外周血淋巴细胞中的表达频率。结果表明，在结束免疫后第30天，随着免疫剂量的升高，外周血淋巴细胞中IFN-γ分泌细胞的表达频率升高，其中100和1000U/鼠的免疫剂量，体内诱导外周血淋巴细胞中IFN-γ分泌细胞的表达频率升高明显，最高可检测到3600±IFN-γ分泌细胞/106 PBMC.
实施例7
在HBV转基因小鼠抑制病毒表面抗原（HbsAg）
采用HBV-DNA转基因小鼠（ayw型HBV全基因（1.3Kb）传染昆明种小鼠）。将动物随机分组，每组10只，于双侧肋下和后脚掌皮下注射多肽5，按10、100和1000U/鼠的3个剂量给药，每周加强免疫一次，共三次。设IFN-α 2b（15000U/鼠）为阳性对照药，设生理盐水为阴性对照药。给药前和最后一次给药结束后10天、20天、30天，取血，分离血清，分别用ELISA法测定血清中HbsAg。结果表明血清HbsAg明显降低，且呈剂量依赖性和时间依赖性。
实施例8
在HBV转基因小鼠抑制病毒复制
采用HBV-DNA转基因小鼠（ayw型HBV全基因（1.3Kb）传染昆明种小鼠）。将动物随机分组，每组10只，于双侧肋下和后脚掌皮下注射多肽5，按10、100、1000U/鼠的3个剂量给药，每周加强免疫一次，共三次。设IFN-α 2b（15000U/鼠）为阳性对照药，设生理盐水为阴性对照药。给药前和最后一次给药结束后10天、20天、30天，取血，分离血清，分别用定量PCR法检测血清中HBV DNA拷贝数。结果表明血清HBV DNA拷贝数明显降低，且呈剂量依赖性和时间依赖性。

Claims (10)

  1. 一种多肽免疫原，其特征在于，所述多肽免疫原含有一个多肽序列，所述的多肽序列含有氨基酸序列1、氨基酸序列2、氨基酸序列3 和氨基酸序列4，其中，各氨基酸序列之间由若干个氨基酸残基组成的连接肽段连接或直接共价连接；所述的氨基酸序列1是Th细胞表位序列或Th细胞表位序列与艾滋病毒HIV-1来源的TAT蛋白的碱性区域序列共价相连构成的混合序列；所述的氨基酸序列2是艾滋病毒HIV-1来源的TAT蛋白的碱性区域序列；所述的氨基酸序列3是乙型肝炎病毒来源的CTL表位序列或乙型肝炎病毒来源的CTL表位序列与艾滋病毒HIV-1来源的TAT蛋白的碱性区域序列共价相连构成的混合序列；所述的氨基酸序列4是乙型肝炎病毒来源的B细胞表位序列或乙型肝炎病毒来源的B细胞表位序列与艾滋病毒HIV-1来源的TAT蛋白的碱性区域序列共价相连构成的混合序列。
  2. 根据权利要求1所述的多肽免疫原，其特征在于，所述的多肽序列从N端到C端的排列次序为：
    氨基酸序列1-氨基酸序列2-氨基酸序列3-氨基酸序列4、
    氨基酸序列1-氨基酸序列3-氨基酸序列4-氨基酸序列2、
    氨基酸序列1-氨基酸序列4-氨基酸序列2-氨基酸序列3、
    氨基酸序列2-氨基酸序列1-氨基酸序列3-氨基酸序列4、
    和氨基酸序列1-氨基酸序列3-氨基酸序列4，
    其中：当氨基酸序列2存在时，氨基酸序列1、3、4不能为混合序列；
    当排列次序为氨基酸序列1-氨基酸序列3-氨基酸序列4时，氨基酸序列1、氨基酸序列3、氨基酸序列4中至少含有一混合序列。
  3. 根据权利要求1或2所述的多肽免疫原，其特征在于，所述的氨基酸序列1是破伤风类毒素来源的Th细胞表位上的第830-843氨基酸序列QYIKANSKFIGITE或通用Th细胞表位PADRE或混合序列RKKRRQRRRQYIKANSKFIGITE或混合序列RKKRRQRRRPADRE；所述氨基酸序列2是HIV-1 TAT蛋白中第48-60氨基酸序列GRKKRRQRRRPPQ或第49-57氨基酸序列RKKRRQRRR或第48-57氨基酸序列GRKKRRQRRR；所述的氨基酸序列3是HBV核心抗原上的第18-27氨基酸序列FLPSDFFPSV或混合序列RKKRRQRRRFLPSDFFPSV；所述氨基酸序列4是HBV Pre S2来源的B细胞表位上的第14-26氨基酸序列DPRVRGLYFPAGG或第14-30氨基酸序列DPRVRGLYFPAGGVSFG或混合序列RKKRRQRRRDPRVRGLYFPAGGVSFG。
  4. 根据权利要求1或2所述的多肽免疫原，其特征在于，所述的各氨基酸序列之间的连接肽段为AAA、GGG或SSS；所述的多肽免疫原N端氨基酸序列的N端上连接着修饰性连接肽段KSS；所述的修饰性连接肽段KSS的N端的赖氨酸残基的α或ε氨基上分别或同时共价连接着棕榈酰基。
  5. 根据权利要求1所述的多肽免疫原，其特征在于，所述多肽序列为：CH3(CH2)14COKSSPADRESSSGRKKRRQRRRPPQAAAFLPSDFFPSVGGGDPRVRGLYFPAGGVSFG、
    CH3(CH2)14COK(CH3(CH2)14CO)SSPADRESSSGRKKRRQRRRPPQAAAFLPSDFFPSVGGGDPRVRGLYFPAGGVSFG、
    CH3(CH2)14COKSSQYIKANSKFIGITEAAAGRKKRRQRRRPPQAAAFLPSDFFPSVGGGDPRVRGLYFPAGGVSFG、
    CH3(CH2)14COK(CH3(CH2)14CO)SSQYIKANSKFIGITEAAAGRKKRRQRRRPPQAAAFLPSDFFPSVGGGDPRVRGLYFPAGGVSFG、
    CH3(CH2)14COKSSPADREAAAFLPSDFFPSVGGGDPRVRGLYFPAGGGRKKRRQRRRPPQ、
    CH3(CH2)14COK(CH3(CH2)14CO)SSPADREAAAFLPSDFFPSVGGGDPRVRGLYFPAGGGRKKRRQRRRPPQ、
    CH3(CH2)14COKSSQYIKANSKFIGITEAAAFLPSDFFPSVGGGDPRVRGLYFPAGGGRKKRRQRRRPPQ、
    CH3(CH2)14COK(CH3(CH2)14CO)SSQYIKANSKFIGITEAAAFLPSDFFPSVGGGDPRVRGLYFPAGGGRKKRRQRRRPPQ、
    CH3(CH2)14COKSSGRKKRRQRRRPPQSSSPADREAAAFLPSDFFPSVGGGDPRVRGLYFPAGGVSFG、
    CH3(CH2)14COK(CH3(CH2)14CO)SSGRKKRRQRRRPPQSSSPADREAAAFLPSDFFPSVGGGDPRVRGLYFPAGGVSFG、
    CH3(CH2)14COKSSGRKKRRQRRRPPQSSSQYIKANSKFIGITEAAAFLPSDFFPSVGGGDPRVRGLYFPAGGVSFG、
    CH3(CH2)14COK(CH3(CH2)14CO)SSGRKKRRQRRRPPQSSSQYIKANSKFIGITEAAAFLPSDFFPSVGGGDPRVRGLYFPAGGVSFG、
    CH3(CH2)14COKSSPADREAAAFLPSDFFPSVGGGRKKRRQRRRDPRVRGLYFPAGGVSFG、
    CH3(CH2)14COK(CH3(CH2)14CO)SSPADREAAAFLPSDFFPSVGGGRKKRRQRRRDPRVRGLYFPAGGVSFG、
    CH3(CH2)14COKSSPADREGGGDPRVRGLYFPAGGGRKKRRQRRRFLPSDFFPSV或
    CH3(CH2)14COK(CH3(CH2)14CO)SSPADREGGGDPRVRGLYFPAGGGRKKRRQRRRFLPSDFFPSV。
  6. 一种如权利要求1、2或5所述的多肽免疫原的制备方法，其特征在于包括以下步骤：
    a.多肽固相化学合成多肽免疫原-树脂，
    b.采用三氟乙酸法对多肽免疫原-树脂进行裂解，得到裂解液，
    c.将裂解液采用体积排阻层析进行分离纯化，然后通过反向层析纯化得到多肽免疫原。
  7. 如权利要求1、2或5所述的多肽免疫原在制备用于治疗HBV慢性持续感染状态及其相关的继发性肝硬化、肝癌疾病的疫苗或药物中的应用。
  8. 如权利要求7所述的多肽免疫原在制备用于治疗HBV慢性持续感染状态及其相关的继发性肝硬化、肝癌疾病的疫苗或药物中的应用，其特征在于所述HBV慢性感染持续状态为慢性乙型肝炎或乙型肝炎病毒携带者。
  9. 如权利要求7所述的多肽免疫原在制备用于治疗HBV慢性持续感染状态及其相关的继发性肝硬化、肝癌疾病的疫苗或药物中的应用，其特征在于，所述的疫苗或药物还含有药学上可接受的辅料、佐剂和载体。
  10. 如权利要求7所述的多肽免疫原在制备用于治疗HBV慢性持续感染状态及其相关的继发性肝硬化、肝癌疾病的疫苗或药物中的应用，其特征在于所述的疫苗或药物的给药方式为注射给药。
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