乙肝病毒体内外感染模型的建立及其在乙肝新药药效学评价中的应用
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摘要
乙肝病毒(hepatitis B virus, HBV),是目前已知的感染人类最小的双链DNA病毒,属于嗜肝DNA病毒科(hepadnavividae)。乙肝病毒感染肝细胞后,能引起一系列的疾病,如急性肝炎、慢性肝炎、肝硬化、肝癌等,还往往伴有严重的并发症,然而在过去的三十年由于缺乏理想合适的经济型HBV感染模型,对HBV的感染机制和发病机制,特别是早期感染机制不清楚。
乙肝病毒有严格的宿主特异性,除了黑猩猩以外缺乏理想的易感动物模型。虽然黑猩猩能被乙肝病毒感染,而且跟人有亲源关系,具有类似人的乙肝病毒感染史,但是属于濒危动物,来源困难、价格昂贵,存在伦理等问题,所以应用受到限制,不能作为常规实验动物进行研究。因此需要来源广泛且经济适用的小型动物代替黑猩猩作为HBV感染模型。
虽然目前已经有一系列经济型的小鼠模型在使用,如HBV转基因小鼠、HBV转染小鼠模型、免疫缺陷嵌合肝小鼠模型,但都存在各自的缺点。转基因小鼠模型存在遗传不稳定、对HBV免疫耐受、病毒复制过程缺少cccDNA,不适合用于HBV体内复制机制和病毒清除机制的研究。HBV转染小鼠模型,因为分泌的HBV病毒不能感染健康的小鼠肝细胞,不适合作为HBV感染机制、发病机制的模型进行研究,作为抗病毒药物药效的评价模型也不是很理想,因为病毒很快就被清除。嵌合肝免疫缺陷小鼠模型,嵌合了人肝细胞,因此可以作为研究HBV感染机制、抗病毒药物药效学评价的模型,但是人肝细胞来源困难,虽然可以用树鼩肝细胞代替,但是这样还不如直接使用树鼩模型。
早在90年代瑞士和德国科学家就发现树鼩对HBV具有易感性,他们进行了体内外实验获得证实。他们在树鼩原代肝细胞体外感染HBV实验中,肝细胞内能检测到HBV DNA、HBV RNA,且细胞上清中有HBsAg和HBeAg的分泌,表明树鼩肝细胞能被HBV感染,而且病毒能在树鼩肝细胞进行繁殖,树鼩肝细胞对HBV的易感性后来也被大量的实验证明。树鼩在体内感染HBV实验中,他们发现树鼩肝组织中有HBV DNA的表达,并且血液中出现了HBsAg,接着产生了HBsAb、HBeAb、HBcAb,病毒很快就被清除,表明树鼩体内能感染HBV,并且属于一种急性自限性感染。
既然证实树鼩体内外均能被HBV有效感染,具有完整的病毒感染和复制过程,而且感染后能引起机体免疫系统应答并被被机体清除,甚至可能引起肝组织病理变化,这些表明树鼩不仅可用于研究HBV感染机制和机体免疫清除病毒机制,而且可作为抗HBV新药药效学评价的模型,大量的体外研究证实了树鼩肝细胞的这种应用价值。但是,一直以来没有科学家在树鼩体内进行抗HBV药物药效学的评价研究工作,一方面是由于树鼩没有稳定的品系,不像其它动物品系那样具有很好的重复性,另一方面,没有人进行树鼩感染HBV后详细的病程研究,对树鼩感染HBV后体内病毒繁殖情况以及是否引起树鼩病理变化不清楚。
不过这给我们一个提示,如果对树鼩感染HBV后的详细病程进行深入研究,以树鼩在体模型作为抗乙肝病毒新药药效学评价模型将更有说服力。基于此,我们做了大量的树鼩在体感染HBV实验,研究感染后的病理过程。首先摸索树鼩的有效HBV感染剂量,再确定采血时间点,通过对血液化验分析、肝组织免疫组化和病理分析,深入研究树鼩感染HBV后详细的病理过程,并建立病程图。本次实验不仅有助于深入研究树鼩感染HBV后的发病机制,而且能给新药药效学的评价体系提供参考数据,具有重要的医学研究意义。
目前临床对乙肝的治疗策略仅限于对HBV在病人体内的复制进行干预,主要通过干扰素和核苷类药物应用。虽然这些药能暂时能有效控制住病毒的复制,但是不能完全清除病毒,一旦停药,仍会复发。而且干扰素存在着副作用、稳定性差、半衰期短等特点,核苷类药物则容易引起乙肝病毒突变株和耐药株,虽然通过联合用药可以提高疗效,且降低乙肝病毒突变株和耐药株的产生几率,但是这种抑制病毒复制的治疗方案不能从根本上治愈乙肝。
近年来通过HepRG细胞系和树鼩原代肝细胞模型的研究发现乙肝病毒表面抗原preS1蛋白可能与HBV结合宿主肝细胞有关,肝细胞的HBV受体的结合位点可能就位于preS1上,而且大量体外的研究证实了preS1 N端的48AA,可能就是HBV结合肝细胞步骤中的关键位点,而且来源于preS1 N端的48AA的衍生肽经豆蔻酰修饰后具有很强的阻断病毒感染肝细胞的效果, IC50约为8nM。但是目前这种衍生肽的药效学评价仅限于体外研究,没有在动物体内进一步评价。这提示我们,如果在动物,如树鼩体内进一步评价这种衍生肽的药效,更能体现出阻断乙肝病毒感染的药效。
综上所述,我们决定建立树鼩体内外感染乙肝病毒模型并应用于乙肝病毒表面抗原preS1衍生肽L47的药效评价。我们首先在体外实验中证实L47阻断HBV感染药效,然后在树鼩体内进一步进行抗乙肝病毒药效学评价。
第一部分实验,我们建立树鼩体内外感染乙肝病毒模型,并进行鉴定。首先建立树鼩肝细胞体外感染乙肝病毒模型,通过不连续蔗糖梯度离心获得的乙肝病病毒用于体外感染实验,两步灌注法分离树鼩肝细胞,进行原代培养作为病毒感染对象,用不同感染复数的乙肝病毒进行孵育感染,发现乙肝病毒能有效感染树鼩肝细胞,并在肝细胞内的繁殖,病毒表达量与感染复数成良好的依赖关系,表明我们成功建立了HBV体外感染模型;然后我们建立树鼩体内感染乙肝病毒模型,通过摸索有效HBV感染剂量和采血时间点,并对采集到的血液标本进行病毒血清学定量检测、ALT生化定量测定、HBV DNA荧光定量PCR检测,我们不但证实了HBV感染树鼩后能在肝组织内大量繁殖,而且发现HBV引起树鼩急性肝炎。
第二部分实验,在建立好的体外感染模型上证实L47具有阻断HBV感染树鼩肝细胞的药效,发现L47,半抑制率IC50约为2.5nM(n=7),低于报道的类似衍生肽8nM的半抑制率,表明能更有效阻断病毒感染,具有更好的开发和应用前景;在树鼩体内感染乙肝病毒模型上进一步评价L47阻断HBV感染的药效作用,发现0.08mg/kg的L47剂量能非常显著的降低树鼩体内的病毒复制和预防病毒感染引起的肝功异常。
Hepatitis B virus (HBV) is a small circular DNA virus, belonging to the hepadnavirus family. The virus attacks liver cells and can cause lifelong infection, scarring of the liver, liver failure, and death. Despite considerable advances in the understanding of the natural history of HBV infection, most of them,especially, the early steps,remain unclear as the lack of practicable primate little animal model in the past three decades.
Chimpanzee was the first animal described for its susceptibility to HBV infection. However, chimpanzees are relatively rare and expensive, protected by law, no longer represent an appropriate model for HBV studies. The restriction of HBV infection to chimpanzee has increased demand for small animal models for studies HBV.
A series of mouse models represent more accessible for HBV studies and evaluation of anti-HBV drug, but none of they could replace the chimpanzees. Transgenic mouse and hydrodynamic HBV mouse model are not susceptible to HBV infection, only be used for evaluation of antiviral drug. In addition, uPA-mouse transplanted human hepatocytes or tupaia hepatocytes can be used for HBV entry mechanism studies, but human hepatocytes are scare and difficulte to work, furthermore, tupaias may be better choice for HBV studies.
Despite the narrow species specificity of hepadnaviruses, the susceptibility of tupaias for human HBV is approved. HBV infection in vitro, results in viral DNA and RNA synthesis in tupaia hepatocytes, and secretion hepatitis B surface antigen (HBsAg) and hepatitis B e antigen (HBeAg) into culture medium. Moreover, tupaias can be infected with HBV in vivo, resulting in viral DNA replication and gene expression in tupaia livers, similar to acute self-limited hepatitis B in human, HBsAg is rapidly cleared from serum, followed by seroconversion to HBeAb and HBsAb.
These reports strongly support tupaia as a valid model for experimental studies of HBV infection and evaluation of anti-HBV drug. However, evaluation of anti-HBV drug in vivo had been hampered by the lack of longitudinal analyses evaluating the clinical development and pathology of tupaias after HBV infection.
HBV is one of the major threaten to human health and it was estimated that about more than 400 million people worldwide are chronically infected with HBV. Since currently available therapy strategies are restricted to anti-HBV replication by interferon- alpha (IFN-a) and nucleos(t)ide analogues, although, combination therapies, such as lamivudine combined with hepatitis B immunoglobulin or adefovir have been widely adopted for improve efficacy and avoid drug-resistant virus strains, but none of them could effectively eliminate HBV in patient, the number of HBV-infected people and HBV-related deaths continues to increase worldwide.
Monoclonal antibodies against the S-domain were showed neutralized infectivity of HBV for primay human hepatocytes and primary tupaia hepatocytes cultures previously, and envelope protein-derived entry inhibitors resulted from the development of the HBV-susceptible cell line HepaRG. These inhibitors are acylated peptides derived from HBV preS1 domain could block virus attached to hepatocytes in vitro, especially, the preS1-derived peptides (HBVpreS/2-48myr) which can effectively block HBV infection at very low doses and bound strongly to hepatocytes but not to non hepatic cells. Thus, the N-terminal 47 amino acid residue of preS1 domain, are important in mediating attachment of HBV to hepatocytes.
These dates indicat that myristoylated preS1-derived peptides (HBVpreS/2-48myr) are promising candidates for specific antiviral therapy against hepatitis B infections, leading to open a new perspectives for the future therapy of chronic hepatitisB and the emergence of resistant strains after antiviral treatment. But these preS1-derived peptides have not be examined in tupaias for evaluation of block virus infection in vivo
To validate whether L47 synthetic peptides derived from preS1 can impede HBV infection in tupaias, we verified its efficacy against virus attaching to tupaia hepatocytes in vitro model at first, and found its IC50 less than 2.5nM, then we used HBV-infected tupaias model for evaluation of L47 after longitudinal analyses of HBV pathogensis during the course of infection within 6 weeks. In this case, HBV caused tupaias hepatitis during the acute phase of infection, wherase HBV infection was constrained in tupaias treated with L47 at 0.08 mg/kg dose.
乙肝病毒有严格的宿主特异性,除了黑猩猩以外缺乏理想的易感动物模型。虽然黑猩猩能被乙肝病毒感染,而且跟人有亲源关系,具有类似人的乙肝病毒感染史,但是属于濒危动物,来源困难、价格昂贵,存在伦理等问题,所以应用受到限制,不能作为常规实验动物进行研究。因此需要来源广泛且经济适用的小型动物代替黑猩猩作为HBV感染模型。
虽然目前已经有一系列经济型的小鼠模型在使用,如HBV转基因小鼠、HBV转染小鼠模型、免疫缺陷嵌合肝小鼠模型,但都存在各自的缺点。转基因小鼠模型存在遗传不稳定、对HBV免疫耐受、病毒复制过程缺少cccDNA,不适合用于HBV体内复制机制和病毒清除机制的研究。HBV转染小鼠模型,因为分泌的HBV病毒不能感染健康的小鼠肝细胞,不适合作为HBV感染机制、发病机制的模型进行研究,作为抗病毒药物药效的评价模型也不是很理想,因为病毒很快就被清除。嵌合肝免疫缺陷小鼠模型,嵌合了人肝细胞,因此可以作为研究HBV感染机制、抗病毒药物药效学评价的模型,但是人肝细胞来源困难,虽然可以用树鼩肝细胞代替,但是这样还不如直接使用树鼩模型。
早在90年代瑞士和德国科学家就发现树鼩对HBV具有易感性,他们进行了体内外实验获得证实。他们在树鼩原代肝细胞体外感染HBV实验中,肝细胞内能检测到HBV DNA、HBV RNA,且细胞上清中有HBsAg和HBeAg的分泌,表明树鼩肝细胞能被HBV感染,而且病毒能在树鼩肝细胞进行繁殖,树鼩肝细胞对HBV的易感性后来也被大量的实验证明。树鼩在体内感染HBV实验中,他们发现树鼩肝组织中有HBV DNA的表达,并且血液中出现了HBsAg,接着产生了HBsAb、HBeAb、HBcAb,病毒很快就被清除,表明树鼩体内能感染HBV,并且属于一种急性自限性感染。
既然证实树鼩体内外均能被HBV有效感染,具有完整的病毒感染和复制过程,而且感染后能引起机体免疫系统应答并被被机体清除,甚至可能引起肝组织病理变化,这些表明树鼩不仅可用于研究HBV感染机制和机体免疫清除病毒机制,而且可作为抗HBV新药药效学评价的模型,大量的体外研究证实了树鼩肝细胞的这种应用价值。但是,一直以来没有科学家在树鼩体内进行抗HBV药物药效学的评价研究工作,一方面是由于树鼩没有稳定的品系,不像其它动物品系那样具有很好的重复性,另一方面,没有人进行树鼩感染HBV后详细的病程研究,对树鼩感染HBV后体内病毒繁殖情况以及是否引起树鼩病理变化不清楚。
不过这给我们一个提示,如果对树鼩感染HBV后的详细病程进行深入研究,以树鼩在体模型作为抗乙肝病毒新药药效学评价模型将更有说服力。基于此,我们做了大量的树鼩在体感染HBV实验,研究感染后的病理过程。首先摸索树鼩的有效HBV感染剂量,再确定采血时间点,通过对血液化验分析、肝组织免疫组化和病理分析,深入研究树鼩感染HBV后详细的病理过程,并建立病程图。本次实验不仅有助于深入研究树鼩感染HBV后的发病机制,而且能给新药药效学的评价体系提供参考数据,具有重要的医学研究意义。
目前临床对乙肝的治疗策略仅限于对HBV在病人体内的复制进行干预,主要通过干扰素和核苷类药物应用。虽然这些药能暂时能有效控制住病毒的复制,但是不能完全清除病毒,一旦停药,仍会复发。而且干扰素存在着副作用、稳定性差、半衰期短等特点,核苷类药物则容易引起乙肝病毒突变株和耐药株,虽然通过联合用药可以提高疗效,且降低乙肝病毒突变株和耐药株的产生几率,但是这种抑制病毒复制的治疗方案不能从根本上治愈乙肝。
近年来通过HepRG细胞系和树鼩原代肝细胞模型的研究发现乙肝病毒表面抗原preS1蛋白可能与HBV结合宿主肝细胞有关,肝细胞的HBV受体的结合位点可能就位于preS1上,而且大量体外的研究证实了preS1 N端的48AA,可能就是HBV结合肝细胞步骤中的关键位点,而且来源于preS1 N端的48AA的衍生肽经豆蔻酰修饰后具有很强的阻断病毒感染肝细胞的效果, IC50约为8nM。但是目前这种衍生肽的药效学评价仅限于体外研究,没有在动物体内进一步评价。这提示我们,如果在动物,如树鼩体内进一步评价这种衍生肽的药效,更能体现出阻断乙肝病毒感染的药效。
综上所述,我们决定建立树鼩体内外感染乙肝病毒模型并应用于乙肝病毒表面抗原preS1衍生肽L47的药效评价。我们首先在体外实验中证实L47阻断HBV感染药效,然后在树鼩体内进一步进行抗乙肝病毒药效学评价。
第一部分实验,我们建立树鼩体内外感染乙肝病毒模型,并进行鉴定。首先建立树鼩肝细胞体外感染乙肝病毒模型,通过不连续蔗糖梯度离心获得的乙肝病病毒用于体外感染实验,两步灌注法分离树鼩肝细胞,进行原代培养作为病毒感染对象,用不同感染复数的乙肝病毒进行孵育感染,发现乙肝病毒能有效感染树鼩肝细胞,并在肝细胞内的繁殖,病毒表达量与感染复数成良好的依赖关系,表明我们成功建立了HBV体外感染模型;然后我们建立树鼩体内感染乙肝病毒模型,通过摸索有效HBV感染剂量和采血时间点,并对采集到的血液标本进行病毒血清学定量检测、ALT生化定量测定、HBV DNA荧光定量PCR检测,我们不但证实了HBV感染树鼩后能在肝组织内大量繁殖,而且发现HBV引起树鼩急性肝炎。
第二部分实验,在建立好的体外感染模型上证实L47具有阻断HBV感染树鼩肝细胞的药效,发现L47,半抑制率IC50约为2.5nM(n=7),低于报道的类似衍生肽8nM的半抑制率,表明能更有效阻断病毒感染,具有更好的开发和应用前景;在树鼩体内感染乙肝病毒模型上进一步评价L47阻断HBV感染的药效作用,发现0.08mg/kg的L47剂量能非常显著的降低树鼩体内的病毒复制和预防病毒感染引起的肝功异常。
Hepatitis B virus (HBV) is a small circular DNA virus, belonging to the hepadnavirus family. The virus attacks liver cells and can cause lifelong infection, scarring of the liver, liver failure, and death. Despite considerable advances in the understanding of the natural history of HBV infection, most of them,especially, the early steps,remain unclear as the lack of practicable primate little animal model in the past three decades.
Chimpanzee was the first animal described for its susceptibility to HBV infection. However, chimpanzees are relatively rare and expensive, protected by law, no longer represent an appropriate model for HBV studies. The restriction of HBV infection to chimpanzee has increased demand for small animal models for studies HBV.
A series of mouse models represent more accessible for HBV studies and evaluation of anti-HBV drug, but none of they could replace the chimpanzees. Transgenic mouse and hydrodynamic HBV mouse model are not susceptible to HBV infection, only be used for evaluation of antiviral drug. In addition, uPA-mouse transplanted human hepatocytes or tupaia hepatocytes can be used for HBV entry mechanism studies, but human hepatocytes are scare and difficulte to work, furthermore, tupaias may be better choice for HBV studies.
Despite the narrow species specificity of hepadnaviruses, the susceptibility of tupaias for human HBV is approved. HBV infection in vitro, results in viral DNA and RNA synthesis in tupaia hepatocytes, and secretion hepatitis B surface antigen (HBsAg) and hepatitis B e antigen (HBeAg) into culture medium. Moreover, tupaias can be infected with HBV in vivo, resulting in viral DNA replication and gene expression in tupaia livers, similar to acute self-limited hepatitis B in human, HBsAg is rapidly cleared from serum, followed by seroconversion to HBeAb and HBsAb.
These reports strongly support tupaia as a valid model for experimental studies of HBV infection and evaluation of anti-HBV drug. However, evaluation of anti-HBV drug in vivo had been hampered by the lack of longitudinal analyses evaluating the clinical development and pathology of tupaias after HBV infection.
HBV is one of the major threaten to human health and it was estimated that about more than 400 million people worldwide are chronically infected with HBV. Since currently available therapy strategies are restricted to anti-HBV replication by interferon- alpha (IFN-a) and nucleos(t)ide analogues, although, combination therapies, such as lamivudine combined with hepatitis B immunoglobulin or adefovir have been widely adopted for improve efficacy and avoid drug-resistant virus strains, but none of them could effectively eliminate HBV in patient, the number of HBV-infected people and HBV-related deaths continues to increase worldwide.
Monoclonal antibodies against the S-domain were showed neutralized infectivity of HBV for primay human hepatocytes and primary tupaia hepatocytes cultures previously, and envelope protein-derived entry inhibitors resulted from the development of the HBV-susceptible cell line HepaRG. These inhibitors are acylated peptides derived from HBV preS1 domain could block virus attached to hepatocytes in vitro, especially, the preS1-derived peptides (HBVpreS/2-48myr) which can effectively block HBV infection at very low doses and bound strongly to hepatocytes but not to non hepatic cells. Thus, the N-terminal 47 amino acid residue of preS1 domain, are important in mediating attachment of HBV to hepatocytes.
These dates indicat that myristoylated preS1-derived peptides (HBVpreS/2-48myr) are promising candidates for specific antiviral therapy against hepatitis B infections, leading to open a new perspectives for the future therapy of chronic hepatitisB and the emergence of resistant strains after antiviral treatment. But these preS1-derived peptides have not be examined in tupaias for evaluation of block virus infection in vivo
To validate whether L47 synthetic peptides derived from preS1 can impede HBV infection in tupaias, we verified its efficacy against virus attaching to tupaia hepatocytes in vitro model at first, and found its IC50 less than 2.5nM, then we used HBV-infected tupaias model for evaluation of L47 after longitudinal analyses of HBV pathogensis during the course of infection within 6 weeks. In this case, HBV caused tupaias hepatitis during the acute phase of infection, wherase HBV infection was constrained in tupaias treated with L47 at 0.08 mg/kg dose.
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