丙型肝炎病毒多表位融合蛋白的表达及免疫效果的评价
|
摘要
丙型肝炎是一种严重威胁人类生命健康和生活质量的传染性疾病,急性丙型肝炎患者的临床表现包括：乏力,食欲不振,肝脏肿大和叩击痛,部分病人可出现黄疸。这一疾病的致病原为丙型肝炎(丙肝)病毒(hepatitis C virus, HCV),属于黄病毒科丙型肝炎病毒属,它的基因组由一条单链正义RNA构成,全长约9600nt,只含有单一的开放读码框架,编码3011个氨基酸组成的聚合蛋白,该聚合蛋白在翻译的过程中或者翻译后被宿主和病毒蛋白酶切割成至少10种成熟的结构蛋白以及非结构蛋白,包括：核心蛋白C, El, E2, p7, NS2, NS3, NS4A, NS4B, NS5A和NS5B。
据WHO的统计资料表明,目前全世界约有1.7亿HCV感染者,并且每年新增感染者达300万～400万,所以,研制和开发HCV疫苗来预防HCV感染是当前全世界所共同面临的一个重大而且紧迫的难题。然而迄今为止尚无高效完善的HCV组织培养系统,致使传统减毒疫苗或者灭活疫苗的研制策略困难重重,因此关于HCV疫苗的研究主要集中在HCV重组亚单位蛋白的免疫特性上。此外,疫苗的免疫剂量,免疫方式和免疫程序都是值得深入探讨的问题。一个成功的HCV疫苗必须能够激发出较强的体液免疫反应和细胞免疫反应,体液免疫反应主要指诱导机体产生针对HCV包膜糖蛋白1和2(gpE1和gpE2)的病毒中和抗体,细胞免疫反应包括特异性的受MHC-Ⅱ类分子限制的CD4+辅助性T细胞以及受MHC-Ⅰ类分子限制的CD8+杀伤性T细胞的生成,上述两个类型的T细胞均能够分泌诸如干扰素γ(IFN-γ)之类的抗病毒细胞因子,其中CD8+杀伤性T细胞还具有清除被感染细胞的作用。
目前HCV疫苗研究领域中已经诞生了不少候选疫苗,这些研究一般都使用重组HCV包膜糖蛋白gpE1和gpE2作为疫苗中的抗原,但是无论如何在疫苗的前期研究工作中都需要候选分子的高效表达,以便在体外评价其免疫效果,针对HCV的高度变异性以及其他研究中表达的亚单位多肽诱发的体液免疫反应较弱等问题,我们选取了HCV基因组中相对保守并且也是T细胞表位非常集中的NS3区的一段780bp的DNA序列,另外为了提高对B细胞的激活能力,诱生出中和抗体,我们查阅文献后选取了三个保守的广谱中和抗原位点,将这些抗原表位融合表达,可望生成的融合蛋白具备较强的免疫原性并且能够对不同型,不同株的HCV提供保护作用,从而克服阻止HCV的感染,为丙型肝炎疫苗的研制作一些铺垫。本实验中通过微量中和试验以及CTL杀伤实验评价了HCV多表位融合蛋白的体液免疫原性和细胞免疫原性,为HCV疫苗的研制提供了一定的实验基础。另外,该融合蛋白也可作为临床诊断的候选抗原,在提升HCV诊断试剂的灵敏度和特异性方面具有一定的参考价值。
一、丙型肝炎病毒多表位融合蛋白在原核系统中的表达
根据文献报道：我们挑选出位于HCV包膜蛋白区的三个B细胞表位(aa192～aa202:YEVRNVSGVYH:aa313～aa327:ITGHRMAWDMMMNWS: aa702～aa710:PALSTGLIH)与NS3区一段T细胞表位非常集中的区域融合,利用原核表达系统,以pET-11d作为载体,在大肠埃希菌BL21(DE3)中经IPTG诱导表达获得融合蛋白,之后采用DEAE阴离子交换和镍离子柱亲和层析进行纯化后获得了HCV多表位融合蛋白。通过Western Blot验证融合蛋白的抗原活性。
二、丙型肝炎病毒感染模型的建立
长期以来,由于缺乏有效的HCV体外细胞培养体系,严重影响了对HCV生命周期的探讨及抗HCV药物的研发。本研究中,我们通过体外转录带有JFH1基因组全长的质粒从而获得了具有感染性的HCV JFH1毒株基因组全长RNA,之后进一步转染Huh7.5细胞建立了HCV的细胞培养体系,该体系能够产生有体外感染活性的HCVcc颗粒,我们使用反转录PCR以及间接免疫荧光两种技术检测到了转染后的细胞培养上清中HCVcc颗粒的存在,并且通过对Huh7.5细胞的再次感染证明了获得的HCVcc颗粒的感染性。
三、评价丙型肝炎病毒多表位融合蛋白的细胞免疫原性和体液免疫原性
我们通过流式细胞仪分析了免疫后的小鼠脾脏细胞以及外周血淋巴细胞经该多表位蛋白刺激后的增殖情况,显示出了比较好的细胞免疫效果,在小鼠体内产生了能够识别HCV相应抗原位点的典型特异CTL效应,说明免疫应答过程中的T淋巴细胞系统经过此抗原的刺激而得到了有效的激活,并在再次遇到相同的抗原肽时发挥其细胞毒性杀伤细胞效应。
另外,该蛋白在家兔体内所诱导的抗体水平迅速升高并且能够维持相当长的时间,当注射到家兔体内的抗原剂量为200ug/只时,HCV中和抗体的检测结果也为阳性,提示HCV多表位融合蛋白可能具有中和HCV野生毒株的能力,可以应用到HCV预防性疫苗的开发上。
Hepatitis C is a kind of infectious disease which seriously threatened public health and decreased the quality of everyone's life. The clinical symptoms of an acute infection with hepatitis C virus include fatigue, myalgia, nausea and vomiting. Other features are jaundice and the yellowish tone of the skin. Hepatitis C virus is the cause of this disease. It belongs to the Hepacivirus genus, Flaviviridae virus family and its genome consists of a single stranded positive sense RNA of approximately 9600 nucleotides, containing a single open reading frame. This open reading frame encodes a precursor poly-protein of 3011 amino acids followed by the cleavage performed by viral proteases or host cell peptidases in the subsequent co-and post-translationally procession to yield at least 10 matured structural and non-structural proteins including core protein C, envelope proteins E1 and E2, p7, NS2, NS3, NS4A, NS4B, NS5A and NS5B.
The hepatitis C virus is transmitted mainly through blood-to-blood. According to WHO's statistics, there are more than 170 million of the world population are chronic HCV carriers and 3～4 million HCV new cases were reported every year. It is estimated that there are approximate 40 million HCV carriers in Chinese population and the HCV prevalence is up to 3.2% in our country. Moreover, a large portion of those acute HCV infected individuals will become chronically and about 20% of the chronic infected will develop cirrhosis within a 10～30 year period, and these patients have an elevated risk of developing hepatocellular carcinoma. Histological analysis of liver biopsies is an important tool to determine and follow the chronic liver disease and the progression of fibrosis and cirrhosis. There are many reasons account for the viral immune escape and persistent infection. One of the main factor is that viral genome shows a considerable genetic heterogenecity because of the high mutation rate of 1.9×10-3 nucleotide conversion per gene site every year. Under the host immune pressure and the drug selective pressure, viral mutation occurs and the new kind of HCV quasispecies appears. That will lead to the host immune systems of some infected individuals can not spontaneously clear the viruses efficiently. Therefore, developing an ideal and effective HCV vaccine to prevent HCV infection is a very urgent problem all over the world. However, until very recently, HCV has not been propagated efficiently in cell culture. The major obstacle means that the use of inactivated or live, attenuated viral vaccines has not yet been available. Vaccine approaches have therefore included the use of adjuvanted recombinant polypeptide subunits of the virus in attempts to prime viral neutralizing antibodies to the envelope glycoproteins 1 and 2 (gpEl and gpE2), as well as priming MHC class-Ⅱ-restricted CD4+ T helper (Th) and MHC class-Ⅰ-restricted CD8+ cytotoxic lymphocyte (CTL) responses to these and other viral proteins. Both types of T cell can secrete antiviral cytokines such as interferon-γ(IFN-γ), and CD8+ CTLs have the potential to kill infected cells.
HCV vaccine will play a key role in the prevention of HCV infection. Recent studies have made a great progress in developing many HCV vaccine candidates. These studies involved the use of the recombinant HCV envelope glycoproteins gpEl and gpE2 as vaccine antigens. The high yield of the vaccine antigen is very important to the evaluation of immune response in vitro. However, the hypervariability of HCV genome and the weakness of the humoral immune response elicited by HCV subunit peptides have hindered the preparation of HCV vaccines. In order to overcome these obstacles, we selected a conserved HCV NS3 fragment of 780bp and it contains series of T-cell epitopes. Beside of this, our fusion protein also included three broad neutralized B-cell epitopes according to many publications of HCV study to improve the activities of HCV specific B cells and to elicit HCV neutralization antibodies. We expected it had strong immunogenicity and it can neutralize many kinds of HCV strains of different genotypes. Moreover, the micro neutralization test and CTL cytotoxicity assay used in our studies also provided an effective way to measure the humoral immune response and cellular immune response caused by HCV multi-epitope fusion protein. In addition, the fusion protein as a valuable candidate antigen can also be used in clinical diagnosis to improve sensitivity and specificity of present diagnosis reagent.
1. HCV multi-epitope fusion protein expressed in prokaryotic system
According to references, three conserved B-cell epitopes (aa192～aa202: YEVRNVSGVYH; aa313～aa327:ITGHRMAWDMMMNWS; aa702～aa710: PALSTGLIH) located within HCV envelop protein were chosen and fused with part of HCV NS3 protein containing series of T-cell epitopes. With the vector of pET-11d, fusion protein was expressed in Escherichia coli BL21(DE3) after induced by IPTG. The protein was then purified by DEAE negative ion exchange chromatography and Ni2+ affinity chromatography. Western Blot analysis was used to detect the antigenicity of the fusion protein.
2. Establish of HCV infectious model
Ever since a long time ago, researches about HCV life cycle and developments of anti-HCV drug have been hampered because the severe lack of effective cell culture system in vitro. In this study, we obtain HCV JFH-1 strain full length RNA transcribed from the plasmid template followed by transfection with Huh7.5 cells. RT-PCR and immunofluorescence assay were used in the detection of prepared HCVcc particles and the infectivity was measured by Huh7.5 cell infection.
3. Cellular immune response and humoral immune response induced by HCV multi-epitope fusion protein
The proliferation of PBMC and splenocytes of mice after antigen induction was analyzed by FACS. The results indicated that specific CD8+ cytotoxic lymphocytes could be detected in both PBMC and splenocytes and they could recognize specific antigen sites. We concluded that fusion protein was capable of elicit strong cellular immune response especially in the mice immunized with 100ug of fusion protein.
Furthermore, we investigated HCV specific neutralizing antibodies against the virus by micro-neutralization assay and we found that fusion protein was able to elicit neutralizing antibodies which was critical in viral clearance. Taken together the results of humoral immunity, viral neutralization and specific cellular responses suggest that HCV multi-epitope fusion protein is a potential candidate for designing a prophylactic and therapeutic vaccine against HCV.
据WHO的统计资料表明,目前全世界约有1.7亿HCV感染者,并且每年新增感染者达300万～400万,所以,研制和开发HCV疫苗来预防HCV感染是当前全世界所共同面临的一个重大而且紧迫的难题。然而迄今为止尚无高效完善的HCV组织培养系统,致使传统减毒疫苗或者灭活疫苗的研制策略困难重重,因此关于HCV疫苗的研究主要集中在HCV重组亚单位蛋白的免疫特性上。此外,疫苗的免疫剂量,免疫方式和免疫程序都是值得深入探讨的问题。一个成功的HCV疫苗必须能够激发出较强的体液免疫反应和细胞免疫反应,体液免疫反应主要指诱导机体产生针对HCV包膜糖蛋白1和2(gpE1和gpE2)的病毒中和抗体,细胞免疫反应包括特异性的受MHC-Ⅱ类分子限制的CD4+辅助性T细胞以及受MHC-Ⅰ类分子限制的CD8+杀伤性T细胞的生成,上述两个类型的T细胞均能够分泌诸如干扰素γ(IFN-γ)之类的抗病毒细胞因子,其中CD8+杀伤性T细胞还具有清除被感染细胞的作用。
目前HCV疫苗研究领域中已经诞生了不少候选疫苗,这些研究一般都使用重组HCV包膜糖蛋白gpE1和gpE2作为疫苗中的抗原,但是无论如何在疫苗的前期研究工作中都需要候选分子的高效表达,以便在体外评价其免疫效果,针对HCV的高度变异性以及其他研究中表达的亚单位多肽诱发的体液免疫反应较弱等问题,我们选取了HCV基因组中相对保守并且也是T细胞表位非常集中的NS3区的一段780bp的DNA序列,另外为了提高对B细胞的激活能力,诱生出中和抗体,我们查阅文献后选取了三个保守的广谱中和抗原位点,将这些抗原表位融合表达,可望生成的融合蛋白具备较强的免疫原性并且能够对不同型,不同株的HCV提供保护作用,从而克服阻止HCV的感染,为丙型肝炎疫苗的研制作一些铺垫。本实验中通过微量中和试验以及CTL杀伤实验评价了HCV多表位融合蛋白的体液免疫原性和细胞免疫原性,为HCV疫苗的研制提供了一定的实验基础。另外,该融合蛋白也可作为临床诊断的候选抗原,在提升HCV诊断试剂的灵敏度和特异性方面具有一定的参考价值。
一、丙型肝炎病毒多表位融合蛋白在原核系统中的表达
根据文献报道：我们挑选出位于HCV包膜蛋白区的三个B细胞表位(aa192～aa202:YEVRNVSGVYH:aa313～aa327:ITGHRMAWDMMMNWS: aa702～aa710:PALSTGLIH)与NS3区一段T细胞表位非常集中的区域融合,利用原核表达系统,以pET-11d作为载体,在大肠埃希菌BL21(DE3)中经IPTG诱导表达获得融合蛋白,之后采用DEAE阴离子交换和镍离子柱亲和层析进行纯化后获得了HCV多表位融合蛋白。通过Western Blot验证融合蛋白的抗原活性。
二、丙型肝炎病毒感染模型的建立
长期以来,由于缺乏有效的HCV体外细胞培养体系,严重影响了对HCV生命周期的探讨及抗HCV药物的研发。本研究中,我们通过体外转录带有JFH1基因组全长的质粒从而获得了具有感染性的HCV JFH1毒株基因组全长RNA,之后进一步转染Huh7.5细胞建立了HCV的细胞培养体系,该体系能够产生有体外感染活性的HCVcc颗粒,我们使用反转录PCR以及间接免疫荧光两种技术检测到了转染后的细胞培养上清中HCVcc颗粒的存在,并且通过对Huh7.5细胞的再次感染证明了获得的HCVcc颗粒的感染性。
三、评价丙型肝炎病毒多表位融合蛋白的细胞免疫原性和体液免疫原性
我们通过流式细胞仪分析了免疫后的小鼠脾脏细胞以及外周血淋巴细胞经该多表位蛋白刺激后的增殖情况,显示出了比较好的细胞免疫效果,在小鼠体内产生了能够识别HCV相应抗原位点的典型特异CTL效应,说明免疫应答过程中的T淋巴细胞系统经过此抗原的刺激而得到了有效的激活,并在再次遇到相同的抗原肽时发挥其细胞毒性杀伤细胞效应。
另外,该蛋白在家兔体内所诱导的抗体水平迅速升高并且能够维持相当长的时间,当注射到家兔体内的抗原剂量为200ug/只时,HCV中和抗体的检测结果也为阳性,提示HCV多表位融合蛋白可能具有中和HCV野生毒株的能力,可以应用到HCV预防性疫苗的开发上。
Hepatitis C is a kind of infectious disease which seriously threatened public health and decreased the quality of everyone's life. The clinical symptoms of an acute infection with hepatitis C virus include fatigue, myalgia, nausea and vomiting. Other features are jaundice and the yellowish tone of the skin. Hepatitis C virus is the cause of this disease. It belongs to the Hepacivirus genus, Flaviviridae virus family and its genome consists of a single stranded positive sense RNA of approximately 9600 nucleotides, containing a single open reading frame. This open reading frame encodes a precursor poly-protein of 3011 amino acids followed by the cleavage performed by viral proteases or host cell peptidases in the subsequent co-and post-translationally procession to yield at least 10 matured structural and non-structural proteins including core protein C, envelope proteins E1 and E2, p7, NS2, NS3, NS4A, NS4B, NS5A and NS5B.
The hepatitis C virus is transmitted mainly through blood-to-blood. According to WHO's statistics, there are more than 170 million of the world population are chronic HCV carriers and 3～4 million HCV new cases were reported every year. It is estimated that there are approximate 40 million HCV carriers in Chinese population and the HCV prevalence is up to 3.2% in our country. Moreover, a large portion of those acute HCV infected individuals will become chronically and about 20% of the chronic infected will develop cirrhosis within a 10～30 year period, and these patients have an elevated risk of developing hepatocellular carcinoma. Histological analysis of liver biopsies is an important tool to determine and follow the chronic liver disease and the progression of fibrosis and cirrhosis. There are many reasons account for the viral immune escape and persistent infection. One of the main factor is that viral genome shows a considerable genetic heterogenecity because of the high mutation rate of 1.9×10-3 nucleotide conversion per gene site every year. Under the host immune pressure and the drug selective pressure, viral mutation occurs and the new kind of HCV quasispecies appears. That will lead to the host immune systems of some infected individuals can not spontaneously clear the viruses efficiently. Therefore, developing an ideal and effective HCV vaccine to prevent HCV infection is a very urgent problem all over the world. However, until very recently, HCV has not been propagated efficiently in cell culture. The major obstacle means that the use of inactivated or live, attenuated viral vaccines has not yet been available. Vaccine approaches have therefore included the use of adjuvanted recombinant polypeptide subunits of the virus in attempts to prime viral neutralizing antibodies to the envelope glycoproteins 1 and 2 (gpEl and gpE2), as well as priming MHC class-Ⅱ-restricted CD4+ T helper (Th) and MHC class-Ⅰ-restricted CD8+ cytotoxic lymphocyte (CTL) responses to these and other viral proteins. Both types of T cell can secrete antiviral cytokines such as interferon-γ(IFN-γ), and CD8+ CTLs have the potential to kill infected cells.
HCV vaccine will play a key role in the prevention of HCV infection. Recent studies have made a great progress in developing many HCV vaccine candidates. These studies involved the use of the recombinant HCV envelope glycoproteins gpEl and gpE2 as vaccine antigens. The high yield of the vaccine antigen is very important to the evaluation of immune response in vitro. However, the hypervariability of HCV genome and the weakness of the humoral immune response elicited by HCV subunit peptides have hindered the preparation of HCV vaccines. In order to overcome these obstacles, we selected a conserved HCV NS3 fragment of 780bp and it contains series of T-cell epitopes. Beside of this, our fusion protein also included three broad neutralized B-cell epitopes according to many publications of HCV study to improve the activities of HCV specific B cells and to elicit HCV neutralization antibodies. We expected it had strong immunogenicity and it can neutralize many kinds of HCV strains of different genotypes. Moreover, the micro neutralization test and CTL cytotoxicity assay used in our studies also provided an effective way to measure the humoral immune response and cellular immune response caused by HCV multi-epitope fusion protein. In addition, the fusion protein as a valuable candidate antigen can also be used in clinical diagnosis to improve sensitivity and specificity of present diagnosis reagent.
1. HCV multi-epitope fusion protein expressed in prokaryotic system
According to references, three conserved B-cell epitopes (aa192～aa202: YEVRNVSGVYH; aa313～aa327:ITGHRMAWDMMMNWS; aa702～aa710: PALSTGLIH) located within HCV envelop protein were chosen and fused with part of HCV NS3 protein containing series of T-cell epitopes. With the vector of pET-11d, fusion protein was expressed in Escherichia coli BL21(DE3) after induced by IPTG. The protein was then purified by DEAE negative ion exchange chromatography and Ni2+ affinity chromatography. Western Blot analysis was used to detect the antigenicity of the fusion protein.
2. Establish of HCV infectious model
Ever since a long time ago, researches about HCV life cycle and developments of anti-HCV drug have been hampered because the severe lack of effective cell culture system in vitro. In this study, we obtain HCV JFH-1 strain full length RNA transcribed from the plasmid template followed by transfection with Huh7.5 cells. RT-PCR and immunofluorescence assay were used in the detection of prepared HCVcc particles and the infectivity was measured by Huh7.5 cell infection.
3. Cellular immune response and humoral immune response induced by HCV multi-epitope fusion protein
The proliferation of PBMC and splenocytes of mice after antigen induction was analyzed by FACS. The results indicated that specific CD8+ cytotoxic lymphocytes could be detected in both PBMC and splenocytes and they could recognize specific antigen sites. We concluded that fusion protein was capable of elicit strong cellular immune response especially in the mice immunized with 100ug of fusion protein.
Furthermore, we investigated HCV specific neutralizing antibodies against the virus by micro-neutralization assay and we found that fusion protein was able to elicit neutralizing antibodies which was critical in viral clearance. Taken together the results of humoral immunity, viral neutralization and specific cellular responses suggest that HCV multi-epitope fusion protein is a potential candidate for designing a prophylactic and therapeutic vaccine against HCV.
引文
1.Choo QL, Kuo G, Weiner AJ, et al. Isolation of cDNA clone derived from a blood-borne non-A, non-B viral hepatitis genome. Science 1989;244:359-62
2.Miller RH and Purcell RH. Hepatitis C virus shares amino acid sequence similarity with pestiviruses and flaviviruses as well as members of two plant virus supergroups. Proc Natl Acad Sci U S A 1990;87:2057-61
3.Matsuura Y, Suzuki T, Suzuki R, et al. Processing of El and E2 glycoprotein of hepatitis C virus expressed in mammalian and insect cells. Virology 1994;205:141-50
4.Choo QL, Kuo G, Ralston R, et al. Vaccination of chimpanzees against infection by the hepatitis C virus. Proc Natl Acad Sci U S A 1994;91:1294-8
5.Grakoui A, Wychowski C, Lin C, et al. Expression and identification of hepatitis C virus polyprotein cleavage products. J Virol 1993;67:1385-95
6.Davis GL. Hepatitis C virus genotypes and quasispecies. Am J Med 1999;107(6B):21-26
7.Williams I. Epidemiology of Hepatitis C in the United States. Am J Med 1999;107(6B):2-9
8.Hijikata M, Kato N, Ootsuyama Y, et al. Hypervariable regions in the putative glycoprotein of hepatitis C virus. Biochem Biophys Res Commun 1991;175:220-8
9.Santolini E, Migliaccio G and LaMonica N. Biosynthesis and biochemical properties of the hepatitis C virus core protein. J Virol 1994;68:3631-41
10.Zonaro A, Ravaggi A, Puoti M, et al. Differential pattern of sequence heterogeneity in the hepatitis C virus E1 and E2/NS1 proteins. J Hepatol 1994;21:858-65
11.Meunier JC, Fournillier A, Choukhi A, et al. Analysis of the glycosylation sites of hepatitis C virus (HCV) glycoprotein E1 and the influence of E1 glycans on the formation of the HCV glycoprotein complex. J Gen Virol 1999;80:887-96
12.Fournillier A, Depla E, Karayiannis P, et al. Expression of noncovalent hepatitis C virus envelope E1-E2 complexes is not required for the induction of antibodies with neutralizing properties following DNA immunization. J Virol 1999;73:7497-504
13. Emmanuel G, Cormier, Fay Tsamis, et al. CD81 is an entry coreceptor for hepatitis C virus. Proc Natl Acad Sci U S A 2004;101:7270-4
14.Shimizu YK, Hijikata M, Iwamoto A, et al. Neutralizing antibodies against hepatitis C virus and the emergence of neutralization escape mutant viruses. J Virol 1994;68:1494-500
15.Lechner S, Rispeter K, Meisel H, et al. Antibodies directed to envelope proteins of hepatitis C virus outside of hypervariable region 1. Virology 1998;243:313-21
16.Zibert A, Schreier E and Roggendorf M. Antibodies in human sera specific to hypervariable region 1 of hepatitis C virus can block viral attachment. Virology 1995;208:653-61
17.Bartenschlager R, Ahlborn-Laake L, Mous J, et al. Nonstructural protein 3 of the hepatitis C virus encodes a serine-type proteinase required for cleavage at the NS3/4 and NS4/5 junctions. J Virol 1993;67:3835-44
18.Tai CL, Chi WK, Chen DS, et al. The helicase activity associated with hepatitis C virus nonstructural protein 3(NS3). J Virol 1996;70:8477-84
19.Kim DW, Gwack Y, Han JH, et al. C-terminal domain of the hepatitis C virus NS3 protein contains an RNA helicase activity. Biochem Biophys Res Commun 1995;215:160-6
20.Ishido S, Fujita T and Hotta H. Complex formation of NS5B with NS3 and NS4A proteins of hepatitis C virus. Biochem Biophys Res Commun 1998;244:35-40
21.Satoh S, Tanji Y, Hijikata M, et al. The N-terminal region of hepatitis C virus nonstructural protein 3 (NS3) is essential for stable complex formation with NS4A. J Virol 1995;69:4255-60
22.Failla C, Tomei L and De Francesco R. Both NS3 and NS4A are required for proteolytic processing of hepatitis C virus nonstructural proteins. J Virol 1994;68:3753-60
23.Kim JL, Morgenstern KA, Lin C, et al. The crystal structure of hepatitis C virus NS3 protease domain complexed with a synthetic NS4A cofactor peptide. Cell 1996;87:343-55
24.Koch JO and Bartenschlager R. Modulation of hepatitis C virus NS5A hyperphosphorylation by nonstructural proteins NS3, NS4A and NS4B. J Virol 1999;73:7138-46
25.Neddermann P, Clementi A and De Francesco R. Hyperphosphorylation of the hepatitis C virus NS5A protein requires an active NS3 protease, NS4A, NS4B and NS5A encoded on the same polyprotein. J Virol 1999;73:9984-91
26.Ghosh AK, Steele R, Meyer K, et al. Hepatitis C virus NS5A protein modulates cell cycle regulatory genes and promotes cell growth. J Gen Virol 1999;80:1179-83
27.Ghosh AK, Majumder M, Steele R, et al. Hepatitis C virus NS5A protein protects against TNF-alpha mediated apoptotic cell death. Virus Res 2000;67:173-8
28.Enomoto N, Sakuma I, Asahina Y, et al. Comparison of full-length sequences of interferon-sensitive and resistant hepatitis C virus lb. Sensitivity to interferon is conferred by amino acid substitutions in the NS5A region. J Clin Invest 1995;96:224-30
29.Enomoto N, Sakuma I, Asahina Y, et al. Mutations in the nonstructural protein 5A gene and response to interferon in patients with chronic hepatitis C virus lb infection. N Engl J Med 1996;334:77-81
30.Ishii K, Tanaka Y, Yap CC, et al. Expression of hepatitis C virus NS5B protein: characterization of its RNA polymerase activity and RNA binding. Hepatology 1999;29:1227-35
31.Lohmann V, Korner F, Herian U, et al. Biochemical properties of hepatitis C virus NS5B RNA-dependant RNA polymerase and identification of amino acid sequence motifs essential for enzymatic activity. J Virol 1997;71:8416-28
32.Lohmann V, Roos A, Korner F, et al. Biochemical and kinetic analyses of NS5B RNA-dependent RNA polymerase of the hepatitis C virus. Virology 1998;249:108-18
33.Lin C, Lindenbach BD, Pragai BM, et al. Processing in the hepatitis C virus E2-NS2 region:identification of p7 and two distinct E2-specific products with different C termini. J Virol 1994;68:5063-73
34.Cooper S, Erickson AL, Adams EJ, et al. Analysis of a successful immune response against hepatitis C virus. Immunity 1999; 10:439-49
35.Cohen J. The scientific challenge of hepatitis C. Science 1999;285:26-30
36.Seeff LB. Natural history of Hepatitis C. Am J Med 1999;107(6B):10-15
37.Large MK, Kittlesen DJ and Hahn YS. Suppression of host immune response by the core protein of hepatitis C virus:possible implications for hepatitis C virus persistence. J Immunol 1999;162:931-8
38.Meunier JC, Russell RS, Goossens V, et al. Isolation and characterization of broadly neutralizing human monoclonal antibodies to the El glycoprotein of hepatitis C virus. J Virol 2008;82(2):966-973
39.Keck ZY, Sung VM, Perkins S, et al. Human monoclonal antibody to hepatitis C virus El glycoprotein that blocks virus attachment and viral infectivity. J Virol 2004;78(13):7257-7263
40.Perotti M, Mancini N, Diotti RA, et al. Identification of a broadly cross-reacting and neutralizing human monoclonal antibody directed against the hepatitis C virus E2 protein. J Virol 2008;82(2):1047-1052
41.Gordon EJ, Bhat R, Liu Q, et al. Immune responses to hepatitis C virus structural and nonstructural proteins induced by plasmid DNA immunization. J Inf Dis 2000; 181:42-50
42.Martin P, Perroche P, Chatel L, et al. Genetic immunization and comprehensive screening approaches in HLA-A2 transgenic mice lead to the identification of three novel epitopes in hepatitis C virus NS3 antigen. J Med Viral 2004;74(3):397-405
43.Guglietta S, Gerbuglia AR, Paociani V, et al. Positive selection of cytotoxic T lymphocyte escape variants during acute hepatitis C virus infection. Eur J Immunol 2005;35(9):2627-2637
44.Rosa D, Campagnoli S, Moretto C, et al. A quantitative test to estimate neutralizing antibodies to the hepatitis C virus:Cytofluorimetric assessment of envelope glycoprotein 2 binding to target cells. Proc Natl Acad Sci U S A 1996;93:1759-1763
45.Song MK, Lee SW, Suh YS, et al. Enhancement of immunoglobulin G2a and cytotoxic T-lymphocyte responses by a booster immunization with recombinant hepatitis C virus E2 protein in E2 DNA-primed mice. J Virol 2000;74:2920-2925
46.Okamoto H, Okada S, Sugiyama Y, et al. Nucleotide sequence of the genomic RNA of hepatitis C virus isolated from a human carrier:comparison with reported isolates for conserved and divergent regions. Virology 1991;72:2697-2704
47.Yanagi M, Purcell RH, Emerson SU, et al. Transcripts from a single full-length cDNA clone of hepatitis C virus are infectious when directly transfected into the liver of a chimpanzee. Proc Natl Acad Sci USA 1997;94:8738-8743
48.Blight KJ, McKeating JA and Rice CM. Highly permissive cell lines for subgenomic and genomic hepatitis C virus RNA replication. J Virol 2002;76:13001-13014
49.Tanaka J, Katayama K, Kumagai J, et al. Early dynamics of hepatitis C virus in the circulation of chimpanzees with experimental infection. Intervirology 2005;48:120-123
50.Cai Z, Zhang C, Chang KS, et al. Robust production of infectious hepatitis C virus from stably HCV cDNA-transfected human hepatoma cells. J Virol 2005;79:13963-13973
51.Yi M, Villanueva RA, Thomas DL, et al. Production of infectious genotype la hepatitis C virus (Hutchinson strain) in cultured human hepatoma cells. Proc Natl Acad Sci USA 2006; 103:2310-2315
52.Pietschmann T, Lohmann V, Kalll A, et al. Persistent and transient replication of full-length hepatitis C virus genomes in cell culture. J Virol 2002;76:4008-4021
53.Buonocore L, Blight KJ, Rice CM, et al. Characterization of vesicular stomatitis virus recombinants that express and incorporate high level of hepatitis C virus glycoproteins. J Virol 2002;76:6865-6872
54.Baumet TF, Ito S, Wong DT, et al. Hepatitis C virus structural proteins assemble into virus like particles in insect cells. J Virol 1998;72:3827-3836
55.Wakita T, Pietschmann T, Kato T, et al. Production of infectious hepatitis C virus in tissue culture from a cloned viral genome. Nat Med 2005;11:791-796
56.Zhong J, Gastaminza P, Cheng G, et al. Robust hepatitis C virus infection in vitro. Proc Natl Acad Sci USA 2005;102(26):9294-9299
57.Lindenbach BD, Evans MJ, Syder AJ, et al. Complete replication of hepatitits C virus in cell culture. Science 2005;309:623-626
58.Altman JD, Moss PA, Goulder PJ, et al. Phenotypic analysis of antigen-specific T lymphocytes. Science 1996;274:94-96
59.Benito JM, Lopez M, Lozano S, et al. Phenotype and functional characteristics of HIV-specific cytotoxic CD8+ T cells in chronically infected patients:dual effects of highly active antiretroviral therapy. J Acquir Immune Defic Syndr 2003;34:255-266
60.Radaelli A, Nacsa J, Tsai WP, et al. Prior DNA immunization enhances immune response to dominant and subdominant viral epitopes induced by a fowlpox-based SIVmac vaccine in long-term slow-progressor macaques infected with SIVmac251. Virology 2003;312:181-195
61.Hel Z, Nacsa J, Kelsall B, et al. Impairment of Gag-specific CD8(+) T-cell function in mucosal and systemic compartments of simian immunodeficiency virus ma. J Virol 2001;75:11483-11495
62.Falco DA, Nepomuceno RR, Krams SM, et al. Identification of Epstein-Barr virus-specific CD8+ T lymphocytes in the circulation of pediatric transplant recipients. Transplantation 2002;74:501-510
63.Chen G, Shankar P, Lange C, et al. CD8 T cells specific for human immunodeficiency virus, Epstein-Barr virus, and cytomegalovirus lack molecules for homing to lymphoid sites of infection. Blood 2001;98:156-164
64.Shankar P, Russo M, Harnisch B, et al. Impaired function of circulating HIV-specific CD8(+) T cells in chronic human immunodeficiency virus infection. Blood 2000;96:3094-3101
65.Wang B, Chen H, Jiang X, et al. Identification of an HLA-A*0201-restricted CD8+ T-cell epitope SSp-1 of SARSCoV spike protein. Blood 2004;
66.Vonderheide RH, Domchek SM, Schultze JL, et al. Vaccination of cancer patients against telomerase induces functional antitumor CD8+ T lymphocytes. Clin Cancer Res 2004;10:828-839
67.Zerbini A, Pilli M, Soliani P, et al. Ex vivo characterization of tumor-derived melanoma antigen encoding gene-specific CD8+cells in patients with hepatocellular carcinoma. J Hepatol 2004;40:102-109
68.Echchakir H, Dorothee G, Vergnon I, et al. Cytotoxic T lymphocytes directed against a tumor-specific mutated antigen display similar HLA tetramer binding but distinct functional avidity and tissue distribution. Proc Natl Acad Sci USA 2002;99:9358-9363
69.Wolfl M, Schalk S, Hellmich M, et al. Quantitation of MHC tetramer-positive cells from whole blood:evaluation of a single-platform, six-parameter flow cytometric method. Cytometry 2004;57A:120-130
70.Kosor E, Gagro A, Drazenovic V, et al. MHC tetramers:tracking specific immunity. Acta Med Croatica 2003;57:255-259
71.Lieberman SM, Evans AM, Han B, et al. Identification of the beta cell antigen targeted by a prevalent population of pathogenic CD8+ T cells in autoimmune diabetes. Proc Natl Acad Sci USA 2003; 100:8384-8388
72.Grakoui AN, Shoukry DJ, Woollard JH, et al. HCV persistence and immune evasion in the absence of memory T cell help. Science 2003;302:659-662
73.Shoukry NH, Grakoui M, Houghton DY, et al. Memory CD8+T cells are required for protection from persistent hepatitis C virus infection. J Exp Med 2003;197:1645-1655
74.Chang KM, Rehermann JG, McHutchison C, et al. Immunological significance of cytotoxic T lymphocyte epitope variants in patients chronically infected by the hepatitis C virus. J Clin Invest 1997;100:2376-2385
75.Altfeld MM, Addo R, Shankarappa PK, et al. Enhanced detection of human immunodeficiency virus type 1-specific T-cell responses to highly variable regions by using peptides based on autologous virus sequences. J Virol 2003;77:7330-7340
76.Day EB, Zeng PC, Doherty DC, et al. The context of epitope presentation can influence functional quality of recalled influenza A virus-specific memory CD8+T cells. J Immunol 2007; 179:2187-2194
77.Yerly DD, Heckerman TM, Allen JV, et al. Increased cytotoxic T-lymphocyte epitope variant cross-recognition and functional avidity are associated with hepatitis C virus clearance. J Virol 2008;82:3147-3153
78.Timm JG, Lauer DG, Kavanagh I, et al. CD8 epitope escape and reversion in acute HCV infection. J Exp Med 2004;200:1593-1604
79.Ward SG, Lauer R, Isba B, et al. Cellular immune responses against hepatitis C virus:the evidence base 2002. Clin Exp Immunol 2002; 128:195-203
80.Le Gall SP, Stamegna, and BD Walker. Portable flanking sequences modulate CTL epitope processing. J Clin Invest 2007;117:3563-3575
81.Sette AM, Newman B, Livingston D, et al. Optimizing vaccine design for cellular processing, MHC binding and TCR recognition. Tissue Antigens 2002;59:443-451
82.Frahm NK, Yusim TJ, Suscovich S, et al.2007. Extensive HLA class I allele promiscuity among viral CTL epitopes. Eur J Immunol 2007;37:2419-2433
83.Chen MM, Sallberg A, Sonnerborg O, et al. Limited humoral immunity in hepatitis C virus infection. Gastroenterology 1999;116:135-143
84.Farci PA, Shimoda A, Coiana G, et al. The outcome of acute hepatitis C predicted by the evolution of the viral quasispecies. Science 2000;288:339-344
85.Sheridan IO, Pybus EC, Holmes, et al. High-resolution phylogenetic analysis of hepatitis C virus adaptation and its relationship to disease progression. J Virol 2004;78:3447-3454
86.Seifert UH, Liermann V, Racanelli A, et al. Hepatitis C virus mutation affects proteasomal epitope processing. J Clin Invest 2004; 114:250-259
87.Lauer GM, Barnes M, Lucas J, et al. High resolution analysis of cellular immune responses in resolved and persistent hepatitis C virus infection. Gastroenterology 2004;127:924-936
[1]Simmonds P. Genetic diversity and evolution of hepatitis C virus—15 years on. J Gen Virol,2004,85:3173-3188.
[2]Lai ME, Mazzoleni AP, Argiolu F, et al. Hepatitis C virus in multiple episodes of acute hepatitis in polytransfused thalassaemic children. Lancet,1994,343:388-390.
[3]Wakita T, Pietschmann T, Kato T, et al. Production of infectious hepatitis C virus in tissue culture from a cloned viral genome. Nature Med,2005,11:791-796.
[4]Zhong J, Gastaminza P, Cheng G, et al. Robust hepatitis C virus infection in vitro. Proc Natl Acad Sci USA,2005,102:9294-9299.
[5]Lindenbach BD, Evans MJ, Syder AJ, et al. Complete replication of hepatitis C virus in cell culture. Science,2005,309:623-626.
[6]Polakos NK, Drane D, Cox J, et al. Characterization of hepatitis C virus core-specific immune responses primed in rhesus macaques by a nonclassical ISCOM vaccine. J Immunol,2001,166:3589-3598.
[7]Pearse MJ, Drane D. ISCOMATRIX adjuvant for antigen delivery. Adv Drug Deliv Rev,2005,57:465-474.
[8]Foy E, Li K, Sumpter RJ, et al. Control of antiviral defenses through hepatitis C virus disruption of retinoic acid-inducible gene-Ⅰ signaling. Proc Natl Acad Sci USA, 2005,102:2986-2991.
[9]Li K, Foy E, Ferreon JC, et al. Immune evasion by hepatitis C virus NS3/4A protease-mediated cleavage of the Toll-like receptor 3 adaptor protein TRIF. Proc Natl Acad Sci USA,2005,102:2992-2997.
[10]Crotta S, Stilla A, Wack A, et al. Inhibition of natural killer cells through engagement of CD81 by the major hepatitis C virus envelope protein. J Exp Med, 2002,195:35-41.
[11]Tseng CT, Klimpel GR. Binding of the hepatitis C virus envelope protein E2 to CD81 inhibits natural killer cell functions. J Exp Med,2002,195:43-49.
[12]Choo QL, Kuo G, Ralston R, et al. Vaccination of chimpanzees against infection by the hepatitis C virus. Proc Natl Acad Sci USA,1994,91:1294-1298.
[13]Pileri P, Uematsu Y, Campagnoli S, et al. Binding of hepatitis C virus to CD81. Science,1998,282:938-941.
[14]Rollier C, Depla E, Drexhage JA, et al. Control of heterologous hepatitis C virus infection in chimpanzees is associated with the quality of vaccine-induced peripheral T-helper immune response. J Virol,2004,78:187-196.
[15]Catalucci D, Sporeno E, Cirillo A, et al. An adenovirus type 5 (Ad5) amplicon-based packaging cell line for production of high-capacity helper-independent deltaEl-E2-E3-E4 Ad5 vectors. J Virol,2005,79:6400-6409.
[16]Brinster C, Chen M, Boucreux D, et al. Hepatitis C virus non-structural protein 3-specific cellular immune responses following single or combined immunization with DNA or recombinant Semliki Forest virus particles. J Gen Virol,2002,83: 369-381.
[17]Perri S, Greer CE, Thudium K, et al. An alphavirus replicon particle chimera derived from venezuelan equine encephalitis and sindbis viruses is a potent gene-based vaccine delivery vector. J Virol,2003,77:10394-10403.
[18]Abraham JD, Himoudi N, Kien F, et al. Comparative immunogenicity analysis of modified vaccinia Ankara vectors expressing native or modified forms of hepatitis C virus E1 and E2 glycoproteins. Vaccine,2004,22:3917-3928.
[19]Hagan DT, Singh M, Dong C, et al. Cationic microparticles are a potent delivery system for a HCV DNA vaccine. Vaccine,2004,23:672-680.
[20]Jeong SH, Qiao M, Hascimbeni M, et al. Immunization with hepatitis C virus-like particles induces humoral and cellular immune responses in nonhuman primates. J Virol,2004,78:6995-7003.
[21]Krahn MD, Baptiste A, Yi Q, et al. Potential cost-effectiveness of a preventive hepatitis C vaccine in high risk and average risk populations in Canada. Vaccine,2005, 23:1549-1558.
[22]Baumert TF, Wellnitz S, Aono S, et al. Antibodies against hepatitis C virus-like particles and viral clearance in acute and chronic hepatitis C. Hepatology,2000,32: 610-617.
[23]Nevens F, Roskams T, Vlierberghe H, et al. A pilot study of therapeutic vaccination with envelope protein E1 in 35 patients with chronic hepatitis C. Hepatology,2003,38:1289-1296.
[24]Abel K, Wang Y, Fritts L, et al. Deoxycytidyl-deoxyguanosine oligonucleotide classes A, B, and C induce distinct cytokine gene expression patterns in Rhesus monkey peripheral blood mononuclear cells and distinct alpha interferon responses in TLR9-expressing Rhesus monkey plasmacytoid dendritic cells. Clin Diagn Lab Immunol,2005,12:606-621.
[25]Oxman MN, Levin MJ, Johnson GR, et al. A vaccine to prevent herpes zoster and postherpetic neuralgia in older adults. N Engl J Med,2005,352:2271-2284.
2.Miller RH and Purcell RH. Hepatitis C virus shares amino acid sequence similarity with pestiviruses and flaviviruses as well as members of two plant virus supergroups. Proc Natl Acad Sci U S A 1990;87:2057-61
3.Matsuura Y, Suzuki T, Suzuki R, et al. Processing of El and E2 glycoprotein of hepatitis C virus expressed in mammalian and insect cells. Virology 1994;205:141-50
4.Choo QL, Kuo G, Ralston R, et al. Vaccination of chimpanzees against infection by the hepatitis C virus. Proc Natl Acad Sci U S A 1994;91:1294-8
5.Grakoui A, Wychowski C, Lin C, et al. Expression and identification of hepatitis C virus polyprotein cleavage products. J Virol 1993;67:1385-95
6.Davis GL. Hepatitis C virus genotypes and quasispecies. Am J Med 1999;107(6B):21-26
7.Williams I. Epidemiology of Hepatitis C in the United States. Am J Med 1999;107(6B):2-9
8.Hijikata M, Kato N, Ootsuyama Y, et al. Hypervariable regions in the putative glycoprotein of hepatitis C virus. Biochem Biophys Res Commun 1991;175:220-8
9.Santolini E, Migliaccio G and LaMonica N. Biosynthesis and biochemical properties of the hepatitis C virus core protein. J Virol 1994;68:3631-41
10.Zonaro A, Ravaggi A, Puoti M, et al. Differential pattern of sequence heterogeneity in the hepatitis C virus E1 and E2/NS1 proteins. J Hepatol 1994;21:858-65
11.Meunier JC, Fournillier A, Choukhi A, et al. Analysis of the glycosylation sites of hepatitis C virus (HCV) glycoprotein E1 and the influence of E1 glycans on the formation of the HCV glycoprotein complex. J Gen Virol 1999;80:887-96
12.Fournillier A, Depla E, Karayiannis P, et al. Expression of noncovalent hepatitis C virus envelope E1-E2 complexes is not required for the induction of antibodies with neutralizing properties following DNA immunization. J Virol 1999;73:7497-504
13. Emmanuel G, Cormier, Fay Tsamis, et al. CD81 is an entry coreceptor for hepatitis C virus. Proc Natl Acad Sci U S A 2004;101:7270-4
14.Shimizu YK, Hijikata M, Iwamoto A, et al. Neutralizing antibodies against hepatitis C virus and the emergence of neutralization escape mutant viruses. J Virol 1994;68:1494-500
15.Lechner S, Rispeter K, Meisel H, et al. Antibodies directed to envelope proteins of hepatitis C virus outside of hypervariable region 1. Virology 1998;243:313-21
16.Zibert A, Schreier E and Roggendorf M. Antibodies in human sera specific to hypervariable region 1 of hepatitis C virus can block viral attachment. Virology 1995;208:653-61
17.Bartenschlager R, Ahlborn-Laake L, Mous J, et al. Nonstructural protein 3 of the hepatitis C virus encodes a serine-type proteinase required for cleavage at the NS3/4 and NS4/5 junctions. J Virol 1993;67:3835-44
18.Tai CL, Chi WK, Chen DS, et al. The helicase activity associated with hepatitis C virus nonstructural protein 3(NS3). J Virol 1996;70:8477-84
19.Kim DW, Gwack Y, Han JH, et al. C-terminal domain of the hepatitis C virus NS3 protein contains an RNA helicase activity. Biochem Biophys Res Commun 1995;215:160-6
20.Ishido S, Fujita T and Hotta H. Complex formation of NS5B with NS3 and NS4A proteins of hepatitis C virus. Biochem Biophys Res Commun 1998;244:35-40
21.Satoh S, Tanji Y, Hijikata M, et al. The N-terminal region of hepatitis C virus nonstructural protein 3 (NS3) is essential for stable complex formation with NS4A. J Virol 1995;69:4255-60
22.Failla C, Tomei L and De Francesco R. Both NS3 and NS4A are required for proteolytic processing of hepatitis C virus nonstructural proteins. J Virol 1994;68:3753-60
23.Kim JL, Morgenstern KA, Lin C, et al. The crystal structure of hepatitis C virus NS3 protease domain complexed with a synthetic NS4A cofactor peptide. Cell 1996;87:343-55
24.Koch JO and Bartenschlager R. Modulation of hepatitis C virus NS5A hyperphosphorylation by nonstructural proteins NS3, NS4A and NS4B. J Virol 1999;73:7138-46
25.Neddermann P, Clementi A and De Francesco R. Hyperphosphorylation of the hepatitis C virus NS5A protein requires an active NS3 protease, NS4A, NS4B and NS5A encoded on the same polyprotein. J Virol 1999;73:9984-91
26.Ghosh AK, Steele R, Meyer K, et al. Hepatitis C virus NS5A protein modulates cell cycle regulatory genes and promotes cell growth. J Gen Virol 1999;80:1179-83
27.Ghosh AK, Majumder M, Steele R, et al. Hepatitis C virus NS5A protein protects against TNF-alpha mediated apoptotic cell death. Virus Res 2000;67:173-8
28.Enomoto N, Sakuma I, Asahina Y, et al. Comparison of full-length sequences of interferon-sensitive and resistant hepatitis C virus lb. Sensitivity to interferon is conferred by amino acid substitutions in the NS5A region. J Clin Invest 1995;96:224-30
29.Enomoto N, Sakuma I, Asahina Y, et al. Mutations in the nonstructural protein 5A gene and response to interferon in patients with chronic hepatitis C virus lb infection. N Engl J Med 1996;334:77-81
30.Ishii K, Tanaka Y, Yap CC, et al. Expression of hepatitis C virus NS5B protein: characterization of its RNA polymerase activity and RNA binding. Hepatology 1999;29:1227-35
31.Lohmann V, Korner F, Herian U, et al. Biochemical properties of hepatitis C virus NS5B RNA-dependant RNA polymerase and identification of amino acid sequence motifs essential for enzymatic activity. J Virol 1997;71:8416-28
32.Lohmann V, Roos A, Korner F, et al. Biochemical and kinetic analyses of NS5B RNA-dependent RNA polymerase of the hepatitis C virus. Virology 1998;249:108-18
33.Lin C, Lindenbach BD, Pragai BM, et al. Processing in the hepatitis C virus E2-NS2 region:identification of p7 and two distinct E2-specific products with different C termini. J Virol 1994;68:5063-73
34.Cooper S, Erickson AL, Adams EJ, et al. Analysis of a successful immune response against hepatitis C virus. Immunity 1999; 10:439-49
35.Cohen J. The scientific challenge of hepatitis C. Science 1999;285:26-30
36.Seeff LB. Natural history of Hepatitis C. Am J Med 1999;107(6B):10-15
37.Large MK, Kittlesen DJ and Hahn YS. Suppression of host immune response by the core protein of hepatitis C virus:possible implications for hepatitis C virus persistence. J Immunol 1999;162:931-8
38.Meunier JC, Russell RS, Goossens V, et al. Isolation and characterization of broadly neutralizing human monoclonal antibodies to the El glycoprotein of hepatitis C virus. J Virol 2008;82(2):966-973
39.Keck ZY, Sung VM, Perkins S, et al. Human monoclonal antibody to hepatitis C virus El glycoprotein that blocks virus attachment and viral infectivity. J Virol 2004;78(13):7257-7263
40.Perotti M, Mancini N, Diotti RA, et al. Identification of a broadly cross-reacting and neutralizing human monoclonal antibody directed against the hepatitis C virus E2 protein. J Virol 2008;82(2):1047-1052
41.Gordon EJ, Bhat R, Liu Q, et al. Immune responses to hepatitis C virus structural and nonstructural proteins induced by plasmid DNA immunization. J Inf Dis 2000; 181:42-50
42.Martin P, Perroche P, Chatel L, et al. Genetic immunization and comprehensive screening approaches in HLA-A2 transgenic mice lead to the identification of three novel epitopes in hepatitis C virus NS3 antigen. J Med Viral 2004;74(3):397-405
43.Guglietta S, Gerbuglia AR, Paociani V, et al. Positive selection of cytotoxic T lymphocyte escape variants during acute hepatitis C virus infection. Eur J Immunol 2005;35(9):2627-2637
44.Rosa D, Campagnoli S, Moretto C, et al. A quantitative test to estimate neutralizing antibodies to the hepatitis C virus:Cytofluorimetric assessment of envelope glycoprotein 2 binding to target cells. Proc Natl Acad Sci U S A 1996;93:1759-1763
45.Song MK, Lee SW, Suh YS, et al. Enhancement of immunoglobulin G2a and cytotoxic T-lymphocyte responses by a booster immunization with recombinant hepatitis C virus E2 protein in E2 DNA-primed mice. J Virol 2000;74:2920-2925
46.Okamoto H, Okada S, Sugiyama Y, et al. Nucleotide sequence of the genomic RNA of hepatitis C virus isolated from a human carrier:comparison with reported isolates for conserved and divergent regions. Virology 1991;72:2697-2704
47.Yanagi M, Purcell RH, Emerson SU, et al. Transcripts from a single full-length cDNA clone of hepatitis C virus are infectious when directly transfected into the liver of a chimpanzee. Proc Natl Acad Sci USA 1997;94:8738-8743
48.Blight KJ, McKeating JA and Rice CM. Highly permissive cell lines for subgenomic and genomic hepatitis C virus RNA replication. J Virol 2002;76:13001-13014
49.Tanaka J, Katayama K, Kumagai J, et al. Early dynamics of hepatitis C virus in the circulation of chimpanzees with experimental infection. Intervirology 2005;48:120-123
50.Cai Z, Zhang C, Chang KS, et al. Robust production of infectious hepatitis C virus from stably HCV cDNA-transfected human hepatoma cells. J Virol 2005;79:13963-13973
51.Yi M, Villanueva RA, Thomas DL, et al. Production of infectious genotype la hepatitis C virus (Hutchinson strain) in cultured human hepatoma cells. Proc Natl Acad Sci USA 2006; 103:2310-2315
52.Pietschmann T, Lohmann V, Kalll A, et al. Persistent and transient replication of full-length hepatitis C virus genomes in cell culture. J Virol 2002;76:4008-4021
53.Buonocore L, Blight KJ, Rice CM, et al. Characterization of vesicular stomatitis virus recombinants that express and incorporate high level of hepatitis C virus glycoproteins. J Virol 2002;76:6865-6872
54.Baumet TF, Ito S, Wong DT, et al. Hepatitis C virus structural proteins assemble into virus like particles in insect cells. J Virol 1998;72:3827-3836
55.Wakita T, Pietschmann T, Kato T, et al. Production of infectious hepatitis C virus in tissue culture from a cloned viral genome. Nat Med 2005;11:791-796
56.Zhong J, Gastaminza P, Cheng G, et al. Robust hepatitis C virus infection in vitro. Proc Natl Acad Sci USA 2005;102(26):9294-9299
57.Lindenbach BD, Evans MJ, Syder AJ, et al. Complete replication of hepatitits C virus in cell culture. Science 2005;309:623-626
58.Altman JD, Moss PA, Goulder PJ, et al. Phenotypic analysis of antigen-specific T lymphocytes. Science 1996;274:94-96
59.Benito JM, Lopez M, Lozano S, et al. Phenotype and functional characteristics of HIV-specific cytotoxic CD8+ T cells in chronically infected patients:dual effects of highly active antiretroviral therapy. J Acquir Immune Defic Syndr 2003;34:255-266
60.Radaelli A, Nacsa J, Tsai WP, et al. Prior DNA immunization enhances immune response to dominant and subdominant viral epitopes induced by a fowlpox-based SIVmac vaccine in long-term slow-progressor macaques infected with SIVmac251. Virology 2003;312:181-195
61.Hel Z, Nacsa J, Kelsall B, et al. Impairment of Gag-specific CD8(+) T-cell function in mucosal and systemic compartments of simian immunodeficiency virus ma. J Virol 2001;75:11483-11495
62.Falco DA, Nepomuceno RR, Krams SM, et al. Identification of Epstein-Barr virus-specific CD8+ T lymphocytes in the circulation of pediatric transplant recipients. Transplantation 2002;74:501-510
63.Chen G, Shankar P, Lange C, et al. CD8 T cells specific for human immunodeficiency virus, Epstein-Barr virus, and cytomegalovirus lack molecules for homing to lymphoid sites of infection. Blood 2001;98:156-164
64.Shankar P, Russo M, Harnisch B, et al. Impaired function of circulating HIV-specific CD8(+) T cells in chronic human immunodeficiency virus infection. Blood 2000;96:3094-3101
65.Wang B, Chen H, Jiang X, et al. Identification of an HLA-A*0201-restricted CD8+ T-cell epitope SSp-1 of SARSCoV spike protein. Blood 2004;
66.Vonderheide RH, Domchek SM, Schultze JL, et al. Vaccination of cancer patients against telomerase induces functional antitumor CD8+ T lymphocytes. Clin Cancer Res 2004;10:828-839
67.Zerbini A, Pilli M, Soliani P, et al. Ex vivo characterization of tumor-derived melanoma antigen encoding gene-specific CD8+cells in patients with hepatocellular carcinoma. J Hepatol 2004;40:102-109
68.Echchakir H, Dorothee G, Vergnon I, et al. Cytotoxic T lymphocytes directed against a tumor-specific mutated antigen display similar HLA tetramer binding but distinct functional avidity and tissue distribution. Proc Natl Acad Sci USA 2002;99:9358-9363
69.Wolfl M, Schalk S, Hellmich M, et al. Quantitation of MHC tetramer-positive cells from whole blood:evaluation of a single-platform, six-parameter flow cytometric method. Cytometry 2004;57A:120-130
70.Kosor E, Gagro A, Drazenovic V, et al. MHC tetramers:tracking specific immunity. Acta Med Croatica 2003;57:255-259
71.Lieberman SM, Evans AM, Han B, et al. Identification of the beta cell antigen targeted by a prevalent population of pathogenic CD8+ T cells in autoimmune diabetes. Proc Natl Acad Sci USA 2003; 100:8384-8388
72.Grakoui AN, Shoukry DJ, Woollard JH, et al. HCV persistence and immune evasion in the absence of memory T cell help. Science 2003;302:659-662
73.Shoukry NH, Grakoui M, Houghton DY, et al. Memory CD8+T cells are required for protection from persistent hepatitis C virus infection. J Exp Med 2003;197:1645-1655
74.Chang KM, Rehermann JG, McHutchison C, et al. Immunological significance of cytotoxic T lymphocyte epitope variants in patients chronically infected by the hepatitis C virus. J Clin Invest 1997;100:2376-2385
75.Altfeld MM, Addo R, Shankarappa PK, et al. Enhanced detection of human immunodeficiency virus type 1-specific T-cell responses to highly variable regions by using peptides based on autologous virus sequences. J Virol 2003;77:7330-7340
76.Day EB, Zeng PC, Doherty DC, et al. The context of epitope presentation can influence functional quality of recalled influenza A virus-specific memory CD8+T cells. J Immunol 2007; 179:2187-2194
77.Yerly DD, Heckerman TM, Allen JV, et al. Increased cytotoxic T-lymphocyte epitope variant cross-recognition and functional avidity are associated with hepatitis C virus clearance. J Virol 2008;82:3147-3153
78.Timm JG, Lauer DG, Kavanagh I, et al. CD8 epitope escape and reversion in acute HCV infection. J Exp Med 2004;200:1593-1604
79.Ward SG, Lauer R, Isba B, et al. Cellular immune responses against hepatitis C virus:the evidence base 2002. Clin Exp Immunol 2002; 128:195-203
80.Le Gall SP, Stamegna, and BD Walker. Portable flanking sequences modulate CTL epitope processing. J Clin Invest 2007;117:3563-3575
81.Sette AM, Newman B, Livingston D, et al. Optimizing vaccine design for cellular processing, MHC binding and TCR recognition. Tissue Antigens 2002;59:443-451
82.Frahm NK, Yusim TJ, Suscovich S, et al.2007. Extensive HLA class I allele promiscuity among viral CTL epitopes. Eur J Immunol 2007;37:2419-2433
83.Chen MM, Sallberg A, Sonnerborg O, et al. Limited humoral immunity in hepatitis C virus infection. Gastroenterology 1999;116:135-143
84.Farci PA, Shimoda A, Coiana G, et al. The outcome of acute hepatitis C predicted by the evolution of the viral quasispecies. Science 2000;288:339-344
85.Sheridan IO, Pybus EC, Holmes, et al. High-resolution phylogenetic analysis of hepatitis C virus adaptation and its relationship to disease progression. J Virol 2004;78:3447-3454
86.Seifert UH, Liermann V, Racanelli A, et al. Hepatitis C virus mutation affects proteasomal epitope processing. J Clin Invest 2004; 114:250-259
87.Lauer GM, Barnes M, Lucas J, et al. High resolution analysis of cellular immune responses in resolved and persistent hepatitis C virus infection. Gastroenterology 2004;127:924-936
[1]Simmonds P. Genetic diversity and evolution of hepatitis C virus—15 years on. J Gen Virol,2004,85:3173-3188.
[2]Lai ME, Mazzoleni AP, Argiolu F, et al. Hepatitis C virus in multiple episodes of acute hepatitis in polytransfused thalassaemic children. Lancet,1994,343:388-390.
[3]Wakita T, Pietschmann T, Kato T, et al. Production of infectious hepatitis C virus in tissue culture from a cloned viral genome. Nature Med,2005,11:791-796.
[4]Zhong J, Gastaminza P, Cheng G, et al. Robust hepatitis C virus infection in vitro. Proc Natl Acad Sci USA,2005,102:9294-9299.
[5]Lindenbach BD, Evans MJ, Syder AJ, et al. Complete replication of hepatitis C virus in cell culture. Science,2005,309:623-626.
[6]Polakos NK, Drane D, Cox J, et al. Characterization of hepatitis C virus core-specific immune responses primed in rhesus macaques by a nonclassical ISCOM vaccine. J Immunol,2001,166:3589-3598.
[7]Pearse MJ, Drane D. ISCOMATRIX adjuvant for antigen delivery. Adv Drug Deliv Rev,2005,57:465-474.
[8]Foy E, Li K, Sumpter RJ, et al. Control of antiviral defenses through hepatitis C virus disruption of retinoic acid-inducible gene-Ⅰ signaling. Proc Natl Acad Sci USA, 2005,102:2986-2991.
[9]Li K, Foy E, Ferreon JC, et al. Immune evasion by hepatitis C virus NS3/4A protease-mediated cleavage of the Toll-like receptor 3 adaptor protein TRIF. Proc Natl Acad Sci USA,2005,102:2992-2997.
[10]Crotta S, Stilla A, Wack A, et al. Inhibition of natural killer cells through engagement of CD81 by the major hepatitis C virus envelope protein. J Exp Med, 2002,195:35-41.
[11]Tseng CT, Klimpel GR. Binding of the hepatitis C virus envelope protein E2 to CD81 inhibits natural killer cell functions. J Exp Med,2002,195:43-49.
[12]Choo QL, Kuo G, Ralston R, et al. Vaccination of chimpanzees against infection by the hepatitis C virus. Proc Natl Acad Sci USA,1994,91:1294-1298.
[13]Pileri P, Uematsu Y, Campagnoli S, et al. Binding of hepatitis C virus to CD81. Science,1998,282:938-941.
[14]Rollier C, Depla E, Drexhage JA, et al. Control of heterologous hepatitis C virus infection in chimpanzees is associated with the quality of vaccine-induced peripheral T-helper immune response. J Virol,2004,78:187-196.
[15]Catalucci D, Sporeno E, Cirillo A, et al. An adenovirus type 5 (Ad5) amplicon-based packaging cell line for production of high-capacity helper-independent deltaEl-E2-E3-E4 Ad5 vectors. J Virol,2005,79:6400-6409.
[16]Brinster C, Chen M, Boucreux D, et al. Hepatitis C virus non-structural protein 3-specific cellular immune responses following single or combined immunization with DNA or recombinant Semliki Forest virus particles. J Gen Virol,2002,83: 369-381.
[17]Perri S, Greer CE, Thudium K, et al. An alphavirus replicon particle chimera derived from venezuelan equine encephalitis and sindbis viruses is a potent gene-based vaccine delivery vector. J Virol,2003,77:10394-10403.
[18]Abraham JD, Himoudi N, Kien F, et al. Comparative immunogenicity analysis of modified vaccinia Ankara vectors expressing native or modified forms of hepatitis C virus E1 and E2 glycoproteins. Vaccine,2004,22:3917-3928.
[19]Hagan DT, Singh M, Dong C, et al. Cationic microparticles are a potent delivery system for a HCV DNA vaccine. Vaccine,2004,23:672-680.
[20]Jeong SH, Qiao M, Hascimbeni M, et al. Immunization with hepatitis C virus-like particles induces humoral and cellular immune responses in nonhuman primates. J Virol,2004,78:6995-7003.
[21]Krahn MD, Baptiste A, Yi Q, et al. Potential cost-effectiveness of a preventive hepatitis C vaccine in high risk and average risk populations in Canada. Vaccine,2005, 23:1549-1558.
[22]Baumert TF, Wellnitz S, Aono S, et al. Antibodies against hepatitis C virus-like particles and viral clearance in acute and chronic hepatitis C. Hepatology,2000,32: 610-617.
[23]Nevens F, Roskams T, Vlierberghe H, et al. A pilot study of therapeutic vaccination with envelope protein E1 in 35 patients with chronic hepatitis C. Hepatology,2003,38:1289-1296.
[24]Abel K, Wang Y, Fritts L, et al. Deoxycytidyl-deoxyguanosine oligonucleotide classes A, B, and C induce distinct cytokine gene expression patterns in Rhesus monkey peripheral blood mononuclear cells and distinct alpha interferon responses in TLR9-expressing Rhesus monkey plasmacytoid dendritic cells. Clin Diagn Lab Immunol,2005,12:606-621.
[25]Oxman MN, Levin MJ, Johnson GR, et al. A vaccine to prevent herpes zoster and postherpetic neuralgia in older adults. N Engl J Med,2005,352:2271-2284.