HCV C-Fc融合基因疫苗修饰的树突状细胞功能研究
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摘要
丙型肝炎是由丙型肝炎病毒(HCV)引起的慢性传染性疾病。丙型肝炎是严重威胁人类健康的常见疾病，全球约有1.7亿感染者。由于目前还没有确实有效的治疗方法，也还没有研制出疫苗，因此感染者中绝大部分可发展成慢性感染、进而部分可发展成肝硬化和肝细胞癌，因此危害极大。目前亟需发展一种有效的预防和治疗方法。
由于HCV疫苗研究的重要性，自1989年HCV基因获得克隆以来，人们一直在致力于疫苗的研究，但进展缓慢。原因是多方面的，归纳起来主要有三点：(1)HCV复制力差，体内滴度低，不易传代培养，不能大量制备。(2)HCV感染力弱，目前没有理想的细胞模型，除黑猩猩外，目前无其它易感染动物模型。(3)HCV基因具有高变异特性，以及HCV编码的不少蛋白质具有致癌性，因此，抗原的选择受到一定的限制。
当然，在HCV基因组已完全搞清、基因工程技术发展的今天，疫苗免疫原的制备应已不成问题，病毒基因表达质粒或重组病毒蛋白质均已可以大量制备。细胞和动物模型曾是影响HCV疫苗研究的又一因素，虽然目前还不能完全令人满意，但已发现不少人类细胞株，如肝细胞、T、B细胞株，可和HCV呈高亲和力结合及为HCV染感，并能为中和性抗体所中和，因此可以评
第四军医大学博士学位论文
价免疫机体中和性抗体的产生。丙型肝炎病毒(HCV)基因疫苗
诱导特异性免疫应答产生己被大量的实验室证实，但普遍存在
着抗体滴度不高，CTL杀伤率不强等问题。其原因可能与HCV
感染后，抗原提呈细胞(APC)，尤其是树突状细胞(dendri tic
cells，DC)数量减少、功能降低有关。如何绕过或提高DC免
疫功能低下，诱导出更强有力的、更具广泛性的免疫应答，应
当成为基因疫苗和免疫治疗鱼待解决的突出问题之一。
治疗性疫苗，是当今一个吸引人的、有着广阔应用前景的研
究领域。树突状细胞作为体内重要的专职抗原提呈细胞(APC)，
是机体T细胞特异免疫应答的直接启动和调控者。近年来，树突状
细胞的分化发育、抗原加工提呈机理及其在肿瘤、感染、自身免
疫性疾病和移植排斥中的作用己经成为免疫学的前沿领域，从树
突状细胞中寻找免疫新分子是目前免疫学领域的一大研究热点。
对DC的研究不仅有助于深刻了解机体免疫应答的调控机制，而且
可以了解DC对相关疾病发生的影响从而制定相应的防治措施。
25年前Steinman和Cohn在深入地研究了DC在造血器官
组织中的分布，识别，分化和功能特点。发现它们有激发抗原
特异免疫应答的能力，非常适合作为疫苗制剂。DC干细胞在体
外容易从血液和骨髓中分离和操作，使得临床应用具有可行性。
用DC作为肿瘤疫苗制剂的实验己经开始。用药途径和剂量的选
择，给药的时间，被提呈的抗原性质，理想的成熟期是以现有
的DC生物学和从体外研究和动物模型获得的治疗作用知识为基
础的。DC免疫治疗的继续进展和精密的治疗策略将不仅依赖于
实验室研究的结果，更以人体临床试验的结果为基础。业已证
实，DC是目前发现的功能最强的抗原提呈细胞，也是唯一能激
活初始T细胞的抗原呈递细胞。因此，应用抗原或抗原多肤体
外冲击致敏DC，然后将之回输或免疫接种荷瘤宿主或带病毒特
异性抗原的宿主，进行免疫治疗;利用DC与肿瘤细胞融合成为
第四军医大学博士学位论文
新型带有DC功能及肿瘤特异抗原的融合细胞瘤苗的报道;以及
利用病毒载体将带有肿瘤或病毒抗原的编码基因转染DC，使之
在DC内持续表达相应特异性抗原，诱导产生特异性抗肿瘤或抗
病毒免疫反应〔们等方法应运而生。这些方法存在着DC激活时间
短，治疗不彻底或利用病毒载体的安全性等问题。
病毒已经形成了许多用以逃脱宿主免疫反应的机制。这包括:
减少抗原表位的表达，MHC表位的基因突变，CTL克隆的耗竭，下
调 MHC一I类与MHC/肤复合物的表达，产生免疫细胞因子与受体，
以及下调某些决定性的细胞因子。所以，一个理想的抗原提呈策
略，应当是全方位、强有力的诱导CD扩CTL、CD扩Th及B细胞应
答，这样才能更有效的清除病原体及肿瘤细胞。近年来，以免疫
球蛋白(主要是工gGI)的Fc段为基础构建的新型免疫分子进展很
快，有望用于新型免疫复合制剂的研制。工gGI的Fab与FC是功能
相对独立的片段，Fab中的可变区负责与抗原结合，而Fc段能与
免疫效应细胞的F。Y受体(Fc YR)结合，发挥其调节免疫效应、
细胞炎症反应、细胞毒效以及激活吞噬细胞的作用。DC本身表面
带有Fc丫R，因此由FC YR介导的吞噬细胞内在化的过程可加强免
疫应答的抗原提呈。
本研究旨在构建出包含fJCVC一Fc融合基因的可分泌型真核
表达载体，转染经体外活化的DC，利用已建立的表达HCvC抗
原的靶细胞(SPZ/O一HCV一C)及HCV感染荷瘤鼠(BALB/c小鼠)模
型，观察HCVC一Fc融合基因疫苗修饰的DC，诱导的特异性CTL、
抗体强度，以及对T细胞的增殖作用。研究基因疫苗转染前后，
DC对抗原提呈作用的变化，探索HCVC一Fc融合基因疫苗修饰的
DC，用于预防和治疗HCV感染的可能性和可行性。
研究内容及实验结果如下:
1本文选用人工gGIFc段基因作为目的基?
Hepatitis C virus (HCV) is the major causative agent of non-A, non-B hepatitis throughout the world with more than 170 million people infected . The majority of infected patients are unable to clear the virus and many develop chronic liver disease, cirrhosis and hepatocellular carcinoma . It is not clearly known how an individual develops a chronic hepatitis virus carrier state; however, a defective immune response of the host is thought to play a critical role in the underlying pathogenetic mechanism.
Dendritic cells and Langerhans cells are specialized for the recognition of pathogens and have a pivotal role in the control of immunity.Inadequate Ag presentation by APCs contributes to the failure of the human immune system to mount effective immune responses against chronic infection and tumors. Accumulating evidence indicates that a vaccine or immunotherapy, which can induce combined CD4+ and CD8+ T cell and B cell immune responses, is likely the most effective one to prevent or control chronic infections such as hepatitis virus infection, or tumors.
Efficient Ag presentation is essential to induce effective cellular and humoral immune responses. Thus, one central goal of current immunotherapy and vaccine development is to enhance Ag presentation to induce potent and broad immune responses. Here, a novel Ag presentation strategy is developed by transducing dendritic cells (DCs) to produce an Ag for presentation as an exogenous Ag to efficiently induce both humoral and cellular immunity. The principle of this strategy is illustrated by genetically modifying DCs to secrete a model hepatitis C virus Ag fused with a cell-binding domain and to process the fusion Ag as an exogenous Ag after receptor-mediated internalization for MHC class I and II presentation. Vigorous Ag-specific CD4+ helper and CD8+ cytotoxic T cell, as well as B cell, responses were induced by the transduced DCs in mouse models. Thus, this novel strategy uses a receptor-mediated internalization process to efficiently induce all arms of the adaptive immunity and may provide a powerful means to develop potent vaccines and immunotherapies.
In this study, we design a novel Ag presentation strategy by transducing DCs to produce and secrete a fusion protein consisting of a hepatitis C virus (HCV) core protein HCV C Ag fused with a cell-binding domain such as the Fc fragment of IgG. The secreted fusion proteins, in addition to inducing Ab responses, are transported back to DCs via receptor-mediated internalization. It has been demonstrated that Ag presentation by receptor-mediated internalization of DCs can be enhanced up to 1000-fold, compared with fluid phase Ag pinocytosis. As a result, the fusion Ags are processed in the endosomal pathway and presented by DCs as exogenous for MHC-II presentation to induce CD4+ Th cells. The internalized Ags can also be presented to MHC-I (cross-priming) by
DCs to directly activate CTLs. Thus, this strategy uses a unifying mechanism to activate all arms of the adaptive immunity. In this study, we demonstrate that this receptor-mediated Ag presentation strategy is able to induce vigorous Th cell, CTL, and B cell responses against the model HCV nucleocapsid protein in mouse models. The results are as following:
1 Construction and expression of fusion proteins HCV C-Fc
To construct a recombinant cherimal plasmid of HCV-Fc which can express HCV core gene and IgG Fc gene ,which should be useful to transfect dendritic cells and changed into dendritic cell vaccince. The IgG Fc gene derived from the plasmid pCMVsFc by using polymerase chain reaction(PCR) was inserted into the backward position of cytomegalovirus(CMV) immediate early promotor element of HCV C plasmid(pcDNA3HCV C) then the recombinant plasmid pcDNA3.HCV-Fc was obtained. Resules showed that the insert DNA of pcDNA3 HCV-Fc was conformed HCV core and Fc gene by endonuclease, PCR and sequencing. HCV core gene and Fc gene was expressed transiently with Lipofectamine 2000 coated in human hepatoblastoma 7721 cells which conformed by immunofluorescence.Recombina
由于HCV疫苗研究的重要性，自1989年HCV基因获得克隆以来，人们一直在致力于疫苗的研究，但进展缓慢。原因是多方面的，归纳起来主要有三点：(1)HCV复制力差，体内滴度低，不易传代培养，不能大量制备。(2)HCV感染力弱，目前没有理想的细胞模型，除黑猩猩外，目前无其它易感染动物模型。(3)HCV基因具有高变异特性，以及HCV编码的不少蛋白质具有致癌性，因此，抗原的选择受到一定的限制。
当然，在HCV基因组已完全搞清、基因工程技术发展的今天，疫苗免疫原的制备应已不成问题，病毒基因表达质粒或重组病毒蛋白质均已可以大量制备。细胞和动物模型曾是影响HCV疫苗研究的又一因素，虽然目前还不能完全令人满意，但已发现不少人类细胞株，如肝细胞、T、B细胞株，可和HCV呈高亲和力结合及为HCV染感，并能为中和性抗体所中和，因此可以评
第四军医大学博士学位论文
价免疫机体中和性抗体的产生。丙型肝炎病毒(HCV)基因疫苗
诱导特异性免疫应答产生己被大量的实验室证实，但普遍存在
着抗体滴度不高，CTL杀伤率不强等问题。其原因可能与HCV
感染后，抗原提呈细胞(APC)，尤其是树突状细胞(dendri tic
cells，DC)数量减少、功能降低有关。如何绕过或提高DC免
疫功能低下，诱导出更强有力的、更具广泛性的免疫应答，应
当成为基因疫苗和免疫治疗鱼待解决的突出问题之一。
治疗性疫苗，是当今一个吸引人的、有着广阔应用前景的研
究领域。树突状细胞作为体内重要的专职抗原提呈细胞(APC)，
是机体T细胞特异免疫应答的直接启动和调控者。近年来，树突状
细胞的分化发育、抗原加工提呈机理及其在肿瘤、感染、自身免
疫性疾病和移植排斥中的作用己经成为免疫学的前沿领域，从树
突状细胞中寻找免疫新分子是目前免疫学领域的一大研究热点。
对DC的研究不仅有助于深刻了解机体免疫应答的调控机制，而且
可以了解DC对相关疾病发生的影响从而制定相应的防治措施。
25年前Steinman和Cohn在深入地研究了DC在造血器官
组织中的分布，识别，分化和功能特点。发现它们有激发抗原
特异免疫应答的能力，非常适合作为疫苗制剂。DC干细胞在体
外容易从血液和骨髓中分离和操作，使得临床应用具有可行性。
用DC作为肿瘤疫苗制剂的实验己经开始。用药途径和剂量的选
择，给药的时间，被提呈的抗原性质，理想的成熟期是以现有
的DC生物学和从体外研究和动物模型获得的治疗作用知识为基
础的。DC免疫治疗的继续进展和精密的治疗策略将不仅依赖于
实验室研究的结果，更以人体临床试验的结果为基础。业已证
实，DC是目前发现的功能最强的抗原提呈细胞，也是唯一能激
活初始T细胞的抗原呈递细胞。因此，应用抗原或抗原多肤体
外冲击致敏DC，然后将之回输或免疫接种荷瘤宿主或带病毒特
异性抗原的宿主，进行免疫治疗;利用DC与肿瘤细胞融合成为
第四军医大学博士学位论文
新型带有DC功能及肿瘤特异抗原的融合细胞瘤苗的报道;以及
利用病毒载体将带有肿瘤或病毒抗原的编码基因转染DC，使之
在DC内持续表达相应特异性抗原，诱导产生特异性抗肿瘤或抗
病毒免疫反应〔们等方法应运而生。这些方法存在着DC激活时间
短，治疗不彻底或利用病毒载体的安全性等问题。
病毒已经形成了许多用以逃脱宿主免疫反应的机制。这包括:
减少抗原表位的表达，MHC表位的基因突变，CTL克隆的耗竭，下
调 MHC一I类与MHC/肤复合物的表达，产生免疫细胞因子与受体，
以及下调某些决定性的细胞因子。所以，一个理想的抗原提呈策
略，应当是全方位、强有力的诱导CD扩CTL、CD扩Th及B细胞应
答，这样才能更有效的清除病原体及肿瘤细胞。近年来，以免疫
球蛋白(主要是工gGI)的Fc段为基础构建的新型免疫分子进展很
快，有望用于新型免疫复合制剂的研制。工gGI的Fab与FC是功能
相对独立的片段，Fab中的可变区负责与抗原结合，而Fc段能与
免疫效应细胞的F。Y受体(Fc YR)结合，发挥其调节免疫效应、
细胞炎症反应、细胞毒效以及激活吞噬细胞的作用。DC本身表面
带有Fc丫R，因此由FC YR介导的吞噬细胞内在化的过程可加强免
疫应答的抗原提呈。
本研究旨在构建出包含fJCVC一Fc融合基因的可分泌型真核
表达载体，转染经体外活化的DC，利用已建立的表达HCvC抗
原的靶细胞(SPZ/O一HCV一C)及HCV感染荷瘤鼠(BALB/c小鼠)模
型，观察HCVC一Fc融合基因疫苗修饰的DC，诱导的特异性CTL、
抗体强度，以及对T细胞的增殖作用。研究基因疫苗转染前后，
DC对抗原提呈作用的变化，探索HCVC一Fc融合基因疫苗修饰的
DC，用于预防和治疗HCV感染的可能性和可行性。
研究内容及实验结果如下:
1本文选用人工gGIFc段基因作为目的基?
Hepatitis C virus (HCV) is the major causative agent of non-A, non-B hepatitis throughout the world with more than 170 million people infected . The majority of infected patients are unable to clear the virus and many develop chronic liver disease, cirrhosis and hepatocellular carcinoma . It is not clearly known how an individual develops a chronic hepatitis virus carrier state; however, a defective immune response of the host is thought to play a critical role in the underlying pathogenetic mechanism.
Dendritic cells and Langerhans cells are specialized for the recognition of pathogens and have a pivotal role in the control of immunity.Inadequate Ag presentation by APCs contributes to the failure of the human immune system to mount effective immune responses against chronic infection and tumors. Accumulating evidence indicates that a vaccine or immunotherapy, which can induce combined CD4+ and CD8+ T cell and B cell immune responses, is likely the most effective one to prevent or control chronic infections such as hepatitis virus infection, or tumors.
Efficient Ag presentation is essential to induce effective cellular and humoral immune responses. Thus, one central goal of current immunotherapy and vaccine development is to enhance Ag presentation to induce potent and broad immune responses. Here, a novel Ag presentation strategy is developed by transducing dendritic cells (DCs) to produce an Ag for presentation as an exogenous Ag to efficiently induce both humoral and cellular immunity. The principle of this strategy is illustrated by genetically modifying DCs to secrete a model hepatitis C virus Ag fused with a cell-binding domain and to process the fusion Ag as an exogenous Ag after receptor-mediated internalization for MHC class I and II presentation. Vigorous Ag-specific CD4+ helper and CD8+ cytotoxic T cell, as well as B cell, responses were induced by the transduced DCs in mouse models. Thus, this novel strategy uses a receptor-mediated internalization process to efficiently induce all arms of the adaptive immunity and may provide a powerful means to develop potent vaccines and immunotherapies.
In this study, we design a novel Ag presentation strategy by transducing DCs to produce and secrete a fusion protein consisting of a hepatitis C virus (HCV) core protein HCV C Ag fused with a cell-binding domain such as the Fc fragment of IgG. The secreted fusion proteins, in addition to inducing Ab responses, are transported back to DCs via receptor-mediated internalization. It has been demonstrated that Ag presentation by receptor-mediated internalization of DCs can be enhanced up to 1000-fold, compared with fluid phase Ag pinocytosis. As a result, the fusion Ags are processed in the endosomal pathway and presented by DCs as exogenous for MHC-II presentation to induce CD4+ Th cells. The internalized Ags can also be presented to MHC-I (cross-priming) by
DCs to directly activate CTLs. Thus, this strategy uses a unifying mechanism to activate all arms of the adaptive immunity. In this study, we demonstrate that this receptor-mediated Ag presentation strategy is able to induce vigorous Th cell, CTL, and B cell responses against the model HCV nucleocapsid protein in mouse models. The results are as following:
1 Construction and expression of fusion proteins HCV C-Fc
To construct a recombinant cherimal plasmid of HCV-Fc which can express HCV core gene and IgG Fc gene ,which should be useful to transfect dendritic cells and changed into dendritic cell vaccince. The IgG Fc gene derived from the plasmid pCMVsFc by using polymerase chain reaction(PCR) was inserted into the backward position of cytomegalovirus(CMV) immediate early promotor element of HCV C plasmid(pcDNA3HCV C) then the recombinant plasmid pcDNA3.HCV-Fc was obtained. Resules showed that the insert DNA of pcDNA3 HCV-Fc was conformed HCV core and Fc gene by endonuclease, PCR and sequencing. HCV core gene and Fc gene was expressed transiently with Lipofectamine 2000 coated in human hepatoblastoma 7721 cells which conformed by immunofluorescence.Recombina
引文
1 Anderson WF. Gene therapy: The best of times, the worst of times. Science, 2000, 288(5466):627
2.Kanto T, Hayashi N, Takehara T, et al. Impaired allostimulatory capacity of peripheral blood dendritic cells recoveredfrom hepatitis C virus-infected individuals. J Immunol ,1999:162(9):5584
3.Saito T, Sherman GJ, Kurokhchi K, et al. Plasmid DNA-based immunization for hepatitis C virus structural protein: immune responses in mice. Gastroenterology, 1997,112(4):1321
4 冯志华，周永兴，贾战生，等。含有丙型肝炎病毒核心基因表达质粒的构建及其基因免疫．中国免疫学杂志 2000；16(4)：136-137
5 郝春秋，周永兴，冯志华，等。HCV结构基因腺病毒表达载体骨架质粒pAd．HCV-S的构建、鉴定及表达。医学研究生学报2001；(5)：121-123
6.Auffermann-Gretzinger S, Keeffe EB, Levy S. Impaired dendritic cell maturation in patients with chronic, but not resolved, hepatitis C virus infection[J]. Blood,2001:97(10):3171-3175
7 Bruna-Romero O, Lasarte JJ, Wilkinson G, et al. Induction of cytotoxic T-cell response against hepatitis C virus
structural antigens using a defective recombinant adenovirus. Hepatology, 1997;25:470-476
2.Kanto T, Hayashi N, Takehara T, et al. Impaired allostimulatory capacity of peripheral blood dendritic cells recoveredfrom hepatitis C virus-infected individuals. J Immunol ,1999:162(9):5584
3.Saito T, Sherman GJ, Kurokhchi K, et al. Plasmid DNA-based immunization for hepatitis C virus structural protein: immune responses in mice. Gastroenterology, 1997,112(4):1321
4 冯志华，周永兴，贾战生，等。含有丙型肝炎病毒核心基因表达质粒的构建及其基因免疫．中国免疫学杂志 2000；16(4)：136-137
5 郝春秋，周永兴，冯志华，等。HCV结构基因腺病毒表达载体骨架质粒pAd．HCV-S的构建、鉴定及表达。医学研究生学报2001；(5)：121-123
6.Auffermann-Gretzinger S, Keeffe EB, Levy S. Impaired dendritic cell maturation in patients with chronic, but not resolved, hepatitis C virus infection[J]. Blood,2001:97(10):3171-3175
7 Bruna-Romero O, Lasarte JJ, Wilkinson G, et al. Induction of cytotoxic T-cell response against hepatitis C virus
structural antigens using a defective recombinant adenovirus. Hepatology, 1997;25:470-476