丙型肝炎病毒新型DNA疫苗的免疫原性优化策略研究
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摘要
丙型肝炎病毒(HCV)是导致输血相关性和社区获得性肝炎的主要病原,感染者中大约70%发展为慢性肝炎,其中20%-30%进展到肝硬化,所有感染者罹患肝癌的风险增加。目前,唯一可用疗法是干扰素(IFN-α)单独或与利巴韦林联合使用,治疗费用昂贵,治愈率较低,可能产生严重的副作用。因此,发展一种抗丙型肝炎病毒的疫苗是目前控制HCV感染的有效策略。
研究表明,HCV感染的有效的预防与清除,需要强有力的和持久的T细胞介导的特异性免疫应答。在临床前研究中,DNA免疫已经成为一种能够诱导保护性T细胞免疫反应的有效和安全的疫苗研发策略。然而,在非人灵长类动物实验和人体试验中,DNA疫苗的免疫原性远不及其在小鼠试验中所表现出的强度。近来有一些提高DNA疫苗免疫原性的策略被应用到动物实验中,包括采用电转的方式提高DNA的摄入量和靶蛋白表达,融合表达抗原和功能蛋白以改善抗原特异性递呈,联合应用免疫刺激剂和分子佐剂以提高免疫应答水平等。
HCV实验性DNA疫苗的研究已有大量报导,并有几种疫苗进入临床试验。但其人群应用的障碍主要为免疫原性不理想。为研发新型HCV DNA疫苗并探讨优化其免疫原性的策略,本研究选取文献报道中已证实的在HCV感染清除中具有重要作用的靶抗原,包括结构蛋白(核壳蛋白C,包膜蛋白E1与E2)与相对保守的非结构蛋白3(NS3),应用复制型(pSCK)或非复制型(pVRC)质粒DNA疫苗载体,分别构建了几种能够表达NS3与Core的融合蛋白、E1E2蛋白、NS3及NS3与DEC205融合蛋白的DNA疫苗,瞬时转染鉴定其表达,采用直接注射或注射结合电转的方式免疫Balb/C小鼠,对其体液与细胞免疫效果进行研究,从而为研发新型HCV DNA疫苗并优化其免疫原性提供依据。主要研究结果如下：
1.为比较蛋白疫苗与DNA疫苗在小鼠中的免疫应答特点,同时优化免疫程序及评价电转对DNA疫苗免疫原性的影响,我们构建了NS3与Core融合蛋白的HCV DNA疫苗表达质粒,瞬时转染293T细胞后通过Western blot鉴定其表达,将该DNA疫苗质粒采用肌肉注射和皮内注射两种方式免疫小鼠,分别结合不同电转方式(2针阵列式电极电转或卡钳式电极电转)来提高免疫原性；以C44P蛋白疫苗肌肉注射免疫组为对照；通过ELISA和ELISPOT检验不同HCV疫苗的体液免疫和细胞免疫应答。结果表明：DNA疫苗免疫各组诱发的细胞免疫反应均强于蛋白疫苗组,DNA疫苗各组中肌肉注射组结合2针阵列式电极电转组要强于单独肌肉注射组,皮内注射结合卡钳式电极组要强于皮内注射结合2阵阵列式电极组,各质粒注射产生的抗体亚型以lgG1型为主。
2.为比较分析基于复制型载体pSCK与常规应用的非复制型载体pVRC的HCV DNA疫苗的免疫原性,分别采用表达包膜蛋白的pVRC-JFH1-E1E2、pVRC-HeBei-E1E2、pSCK-HeBei-E1E2质粒免疫小鼠,应用基于HeBei株包膜蛋白序列的特异性肽段或肽库进行的ELISPOT检测结果表明：基于复制型载体构建的DNA疫苗诱导产生的特异性IFN-γ高于非复制型载体构建的DNA疫苗,且其中皮内注射结合卡钳电极组产生的反应最强。以保守的肽段作为检测抗原进行ELISA分析特异性抗体应答,上述各组诱发产生的IgG滴度均低于1：50。
3.为进一步评价新型树突状细胞靶向的分子佐剂(DEC205单链抗体分子)对HCV DNA疫苗免疫原性的增强作用,我们构建了融合表达DEC205单链抗体分子与HCV NS3的DNA疫苗质粒pVRC-DEC-NS3,通过瞬时转染293细胞鉴定其表达,然后采用皮内注射结合卡钳电极电转的方式二针快速免疫小鼠,另设一组以同样方式注射质粒pVRC-DEC和pVRC-NS3的混合物,结果表明：对于pVRC-NS3单独免疫或者同pVRC-DEC共同免疫,无论是体液免疫方面还是细胞免疫方面应答水平均较低,但是采用NS3和DEC205的融合蛋白表达质粒pVRC-DEC-NS3免疫则免疫原性显著增强。综上所述,以HCV结构蛋白Core、E1、E2为靶抗原或者非结构蛋白NS3为靶抗原构建的新型DNA疫苗免疫小鼠,均可诱发特异性细胞免疫应答；应用电转技术有助于提高HCV DNA疫苗的免疫原性,使用皮内注射结合卡钳电极的注射方案诱导产生的细胞免疫反应最强；基于复制型载体构建的DNA疫苗诱导产生的特异性IFN-γ高于非复制型载体构建的DNA疫苗,并可降低疫苗应用剂量；HCV非结构蛋白NS3与DEC205单链抗体分子融合表达构成的DNA疫苗(pVRC-DEC-NS3)免疫后诱导的靶抗原特异的体液与细胞免疫应答明显强于非融合的HCV NS3 DNA疫苗(pVRC-NS3)。上述研究结果为新型HCV DNA疫苗(也包括其他病毒性疾病DNA疫苗)的研发及优化应用提供了重要的科学依据。
Hepatitis C virus (HCV) is the major etiological agent of transfusion-associated and community-acquired hepatitis worldwide. Approximately 70% of patients develop chronic hepatitis, of which 20 to 30% progress onto liver cirrhosis, and all cases of infection carry an increased risk of hepatocellular carcinoma. Presently, the only available therapies are alpha interferon (IFN-a) alone or in combination with ribavirin. Such treatments are expensive, show low-response rates, and carry the risk of significant side effects. Consequently, the development of a effective vaccine against hepatitis C infection remains a high priority goal.
Efficient vaccination against hepatitis C infection requires potent and sustained T cell-mediated immune responses. DNA immunization is one of the most evaluated approaches to obtain an effective vaccine against HCV infection. Several vaccine candidates have been evaluated in pre-clinic, but only a few have reached the clinical evaluation. Two DNA vaccine candidates against HCV have already reached clinical evaluation, and are well tolerated and immunogenic in HCV-chronically infected individuals.
DNA vaccination has emerged as an effective and safe strategy for inducing protective T cell immunity in preclinical models. However, experiments in nonhuman primates and human trials suggested that DNA vaccines are not nearly as immunogenic in these species as they are in rodents. In the last few years a group of technical refinements in DNA vaccines has allowed to increase their immunogenicity,include:1) involving gene delivery systems as electroporation to enhance DNA uptake and therefore expression; 2) targeting Dentritic cells(DCs) or other APCs via encoding fusion antigen through genetically fusing the Ag to molecules binding receptors at the cell surface of APCs;3)enhancing the immune respose by codelivery DNA vaccine with immunostimulatory or molecular adjuvants.
To develop novel HCV DNA vaccine candidates and explore the strategy enhancing the immune response during DNA vaccination,In this study, several plasmids were constructed to express coding antigen of HCV E1/E2 protein derived from JFH1/HeBei isolates, NS3, NS3 and Core fusion protein, NS3 fused with an antibody to DEC205(an endocytic receptor and expressed at high levels on DCs), and replicating(pSCK) or non-replicating(pVRC) backbone of DNA vaccine vectors was applied.The expression in vitro of novel DNA vaccine candidates were identified with Western Blotting (WB) after transient transfection. These plasmids immunized Balb/C mice via direct injection intramuscularly (i.m.) or intrademally(i.d.), or injection combined with electroporation. The humoral and cellular immune response after vaccination was measured by ELISA or Elispot assay,so as to provide a basis to optimize their immunogenicity.
The major findings are as follows:
1.To character immune responces between the protein and DNA vaccine in mouse and to optimize the immune protocol and evaluate the immunogenicy of DNA vaccine with electroporation. Construct a novel DNA vaccine expressing fusion protein coding partial NS3 and Core and then confirmed its expression in vitro by WB. The DNA vaccine was immunized mice twice by injection (i.m./i.d.),,and the technique of electroporation was used to facilitate DNA delivery in vivo. And the C44P protein was immunized as control. The results demonstrated that DNA vaccines induced stronger cellular responces in mice than protein vaccination. And Elispot assay showed that IFN-γinduced by injection(i.m.) combined with 2 needle array electrode were stronger than that of without electrical pulses. Injection (i.d.) combined with caliper electrodes induced more IFN-γsecreted than that combined with 2 needle array electrode. Subclass IgG1 was the predominant antibody detected in each DNA vaccination group.
2. To compare the immunogenicity of DNA vaccines based on replicative backbone vector (pSCK) to which based on common non-replicative backbone vector (pVRC),several DNA vaccine candidates were constructed expressing E1E2 derived from Hebei(1b) or JFH1(2a)isolate, named as pVRC-JFH1-E1E2, pVRC-HeBei-E1E2, pSCK-HeBei-E1E2. These DNA vaccines were used to immunize mice, and detect the specific cellular immune response by IFN-γELISPOT assay with spleen cells stimulated by synthetic peptides derived from amino acid sequence of HeBei isolate. The results showed that pSCK-HeBei-E1E2(derived from a replicating backbone vector) induce a significant increase in secreting anti-E1E2 specific IFN-γthan that of pVRC-HeBei-E1E2(derived from a non-replicating backbone vector), and pVRC-HeBei-E1E2 induced stronger cellular immune responses than pVRC-JFH1-E1E2. The results also indicated that immunization by intradermal injection with electroporation using caliper electrodes was mostly effective to improve the immunogenicity of DNA vaccines. And the total specific IgG titers induced were lower than 1:50 by ELISA with converse peptides as antigen in this study.
3. To evaluvate the effectiveness of a noval adjuvant to DNA vaccines by targeting the encoded protein to dendritic cells, we constructed a novel HCV DNA vaccine coding fusion protein, in which engineered NS3 protein into the heavy chain of anti-DEC-205 and nemed as pVRC-DEC-NS3, then transfect it into 293 cells to confirmed the expression in vitro. Immunize Balb/C mice by twice intradermal injection with electroporation using caliper electrodes, compared with the codelivery of pVRC-DEC or/and pVRC-NS3 only with the same immune protocol. The results indicated that the humoral immune responces as well as cellular immune responces induced by DNA vaccine pVRC-DEC-NS3 was significant increased compared to DNA vaccines expressing NS3 with or without the codelivery of the plasmid encoded anti-DEC-205 molecule.
In summary, all the novel HCV DNA vaccines constructed in this study,with either structural proteins (Core, E1/E2) or non-structural protein NS3 as the target antigen, could induce specific cellular immune response.Vaccination applying with electroporation can improve the immunogenicity of DNA vaccines. And plasmids immunized by intradermal injection with electroporation using caliper electrodes could induce most significant increased in CMI, compared with other immune protocol. DNA vaccine based on replicative backbone vector could induce more IFN-Y secreting than which based on a non-replicative vector. And DNA vaccine coding fusion protein, in which engineered target protein into the heavy chain of anti-DEC-205, would improve the immunogenicity of DNA vaccinaton. These data pave a way for the development and application of novel DNA vaccines against HCV infection and other persistant infection.,
研究表明,HCV感染的有效的预防与清除,需要强有力的和持久的T细胞介导的特异性免疫应答。在临床前研究中,DNA免疫已经成为一种能够诱导保护性T细胞免疫反应的有效和安全的疫苗研发策略。然而,在非人灵长类动物实验和人体试验中,DNA疫苗的免疫原性远不及其在小鼠试验中所表现出的强度。近来有一些提高DNA疫苗免疫原性的策略被应用到动物实验中,包括采用电转的方式提高DNA的摄入量和靶蛋白表达,融合表达抗原和功能蛋白以改善抗原特异性递呈,联合应用免疫刺激剂和分子佐剂以提高免疫应答水平等。
HCV实验性DNA疫苗的研究已有大量报导,并有几种疫苗进入临床试验。但其人群应用的障碍主要为免疫原性不理想。为研发新型HCV DNA疫苗并探讨优化其免疫原性的策略,本研究选取文献报道中已证实的在HCV感染清除中具有重要作用的靶抗原,包括结构蛋白(核壳蛋白C,包膜蛋白E1与E2)与相对保守的非结构蛋白3(NS3),应用复制型(pSCK)或非复制型(pVRC)质粒DNA疫苗载体,分别构建了几种能够表达NS3与Core的融合蛋白、E1E2蛋白、NS3及NS3与DEC205融合蛋白的DNA疫苗,瞬时转染鉴定其表达,采用直接注射或注射结合电转的方式免疫Balb/C小鼠,对其体液与细胞免疫效果进行研究,从而为研发新型HCV DNA疫苗并优化其免疫原性提供依据。主要研究结果如下：
1.为比较蛋白疫苗与DNA疫苗在小鼠中的免疫应答特点,同时优化免疫程序及评价电转对DNA疫苗免疫原性的影响,我们构建了NS3与Core融合蛋白的HCV DNA疫苗表达质粒,瞬时转染293T细胞后通过Western blot鉴定其表达,将该DNA疫苗质粒采用肌肉注射和皮内注射两种方式免疫小鼠,分别结合不同电转方式(2针阵列式电极电转或卡钳式电极电转)来提高免疫原性；以C44P蛋白疫苗肌肉注射免疫组为对照；通过ELISA和ELISPOT检验不同HCV疫苗的体液免疫和细胞免疫应答。结果表明：DNA疫苗免疫各组诱发的细胞免疫反应均强于蛋白疫苗组,DNA疫苗各组中肌肉注射组结合2针阵列式电极电转组要强于单独肌肉注射组,皮内注射结合卡钳式电极组要强于皮内注射结合2阵阵列式电极组,各质粒注射产生的抗体亚型以lgG1型为主。
2.为比较分析基于复制型载体pSCK与常规应用的非复制型载体pVRC的HCV DNA疫苗的免疫原性,分别采用表达包膜蛋白的pVRC-JFH1-E1E2、pVRC-HeBei-E1E2、pSCK-HeBei-E1E2质粒免疫小鼠,应用基于HeBei株包膜蛋白序列的特异性肽段或肽库进行的ELISPOT检测结果表明：基于复制型载体构建的DNA疫苗诱导产生的特异性IFN-γ高于非复制型载体构建的DNA疫苗,且其中皮内注射结合卡钳电极组产生的反应最强。以保守的肽段作为检测抗原进行ELISA分析特异性抗体应答,上述各组诱发产生的IgG滴度均低于1：50。
3.为进一步评价新型树突状细胞靶向的分子佐剂(DEC205单链抗体分子)对HCV DNA疫苗免疫原性的增强作用,我们构建了融合表达DEC205单链抗体分子与HCV NS3的DNA疫苗质粒pVRC-DEC-NS3,通过瞬时转染293细胞鉴定其表达,然后采用皮内注射结合卡钳电极电转的方式二针快速免疫小鼠,另设一组以同样方式注射质粒pVRC-DEC和pVRC-NS3的混合物,结果表明：对于pVRC-NS3单独免疫或者同pVRC-DEC共同免疫,无论是体液免疫方面还是细胞免疫方面应答水平均较低,但是采用NS3和DEC205的融合蛋白表达质粒pVRC-DEC-NS3免疫则免疫原性显著增强。综上所述,以HCV结构蛋白Core、E1、E2为靶抗原或者非结构蛋白NS3为靶抗原构建的新型DNA疫苗免疫小鼠,均可诱发特异性细胞免疫应答；应用电转技术有助于提高HCV DNA疫苗的免疫原性,使用皮内注射结合卡钳电极的注射方案诱导产生的细胞免疫反应最强；基于复制型载体构建的DNA疫苗诱导产生的特异性IFN-γ高于非复制型载体构建的DNA疫苗,并可降低疫苗应用剂量；HCV非结构蛋白NS3与DEC205单链抗体分子融合表达构成的DNA疫苗(pVRC-DEC-NS3)免疫后诱导的靶抗原特异的体液与细胞免疫应答明显强于非融合的HCV NS3 DNA疫苗(pVRC-NS3)。上述研究结果为新型HCV DNA疫苗(也包括其他病毒性疾病DNA疫苗)的研发及优化应用提供了重要的科学依据。
Hepatitis C virus (HCV) is the major etiological agent of transfusion-associated and community-acquired hepatitis worldwide. Approximately 70% of patients develop chronic hepatitis, of which 20 to 30% progress onto liver cirrhosis, and all cases of infection carry an increased risk of hepatocellular carcinoma. Presently, the only available therapies are alpha interferon (IFN-a) alone or in combination with ribavirin. Such treatments are expensive, show low-response rates, and carry the risk of significant side effects. Consequently, the development of a effective vaccine against hepatitis C infection remains a high priority goal.
Efficient vaccination against hepatitis C infection requires potent and sustained T cell-mediated immune responses. DNA immunization is one of the most evaluated approaches to obtain an effective vaccine against HCV infection. Several vaccine candidates have been evaluated in pre-clinic, but only a few have reached the clinical evaluation. Two DNA vaccine candidates against HCV have already reached clinical evaluation, and are well tolerated and immunogenic in HCV-chronically infected individuals.
DNA vaccination has emerged as an effective and safe strategy for inducing protective T cell immunity in preclinical models. However, experiments in nonhuman primates and human trials suggested that DNA vaccines are not nearly as immunogenic in these species as they are in rodents. In the last few years a group of technical refinements in DNA vaccines has allowed to increase their immunogenicity,include:1) involving gene delivery systems as electroporation to enhance DNA uptake and therefore expression; 2) targeting Dentritic cells(DCs) or other APCs via encoding fusion antigen through genetically fusing the Ag to molecules binding receptors at the cell surface of APCs;3)enhancing the immune respose by codelivery DNA vaccine with immunostimulatory or molecular adjuvants.
To develop novel HCV DNA vaccine candidates and explore the strategy enhancing the immune response during DNA vaccination,In this study, several plasmids were constructed to express coding antigen of HCV E1/E2 protein derived from JFH1/HeBei isolates, NS3, NS3 and Core fusion protein, NS3 fused with an antibody to DEC205(an endocytic receptor and expressed at high levels on DCs), and replicating(pSCK) or non-replicating(pVRC) backbone of DNA vaccine vectors was applied.The expression in vitro of novel DNA vaccine candidates were identified with Western Blotting (WB) after transient transfection. These plasmids immunized Balb/C mice via direct injection intramuscularly (i.m.) or intrademally(i.d.), or injection combined with electroporation. The humoral and cellular immune response after vaccination was measured by ELISA or Elispot assay,so as to provide a basis to optimize their immunogenicity.
The major findings are as follows:
1.To character immune responces between the protein and DNA vaccine in mouse and to optimize the immune protocol and evaluate the immunogenicy of DNA vaccine with electroporation. Construct a novel DNA vaccine expressing fusion protein coding partial NS3 and Core and then confirmed its expression in vitro by WB. The DNA vaccine was immunized mice twice by injection (i.m./i.d.),,and the technique of electroporation was used to facilitate DNA delivery in vivo. And the C44P protein was immunized as control. The results demonstrated that DNA vaccines induced stronger cellular responces in mice than protein vaccination. And Elispot assay showed that IFN-γinduced by injection(i.m.) combined with 2 needle array electrode were stronger than that of without electrical pulses. Injection (i.d.) combined with caliper electrodes induced more IFN-γsecreted than that combined with 2 needle array electrode. Subclass IgG1 was the predominant antibody detected in each DNA vaccination group.
2. To compare the immunogenicity of DNA vaccines based on replicative backbone vector (pSCK) to which based on common non-replicative backbone vector (pVRC),several DNA vaccine candidates were constructed expressing E1E2 derived from Hebei(1b) or JFH1(2a)isolate, named as pVRC-JFH1-E1E2, pVRC-HeBei-E1E2, pSCK-HeBei-E1E2. These DNA vaccines were used to immunize mice, and detect the specific cellular immune response by IFN-γELISPOT assay with spleen cells stimulated by synthetic peptides derived from amino acid sequence of HeBei isolate. The results showed that pSCK-HeBei-E1E2(derived from a replicating backbone vector) induce a significant increase in secreting anti-E1E2 specific IFN-γthan that of pVRC-HeBei-E1E2(derived from a non-replicating backbone vector), and pVRC-HeBei-E1E2 induced stronger cellular immune responses than pVRC-JFH1-E1E2. The results also indicated that immunization by intradermal injection with electroporation using caliper electrodes was mostly effective to improve the immunogenicity of DNA vaccines. And the total specific IgG titers induced were lower than 1:50 by ELISA with converse peptides as antigen in this study.
3. To evaluvate the effectiveness of a noval adjuvant to DNA vaccines by targeting the encoded protein to dendritic cells, we constructed a novel HCV DNA vaccine coding fusion protein, in which engineered NS3 protein into the heavy chain of anti-DEC-205 and nemed as pVRC-DEC-NS3, then transfect it into 293 cells to confirmed the expression in vitro. Immunize Balb/C mice by twice intradermal injection with electroporation using caliper electrodes, compared with the codelivery of pVRC-DEC or/and pVRC-NS3 only with the same immune protocol. The results indicated that the humoral immune responces as well as cellular immune responces induced by DNA vaccine pVRC-DEC-NS3 was significant increased compared to DNA vaccines expressing NS3 with or without the codelivery of the plasmid encoded anti-DEC-205 molecule.
In summary, all the novel HCV DNA vaccines constructed in this study,with either structural proteins (Core, E1/E2) or non-structural protein NS3 as the target antigen, could induce specific cellular immune response.Vaccination applying with electroporation can improve the immunogenicity of DNA vaccines. And plasmids immunized by intradermal injection with electroporation using caliper electrodes could induce most significant increased in CMI, compared with other immune protocol. DNA vaccine based on replicative backbone vector could induce more IFN-Y secreting than which based on a non-replicative vector. And DNA vaccine coding fusion protein, in which engineered target protein into the heavy chain of anti-DEC-205, would improve the immunogenicity of DNA vaccinaton. These data pave a way for the development and application of novel DNA vaccines against HCV infection and other persistant infection.,
引文
1. Rehermann, B. and M. Nascimbeni, Immunology of hepatitis B virus and hepatitis C virus infection. Nat Rev Immunol,2005.5(3):p.215-29.
2. Cohen, J.,1999, The scientific challenge of hepatitis C, Science,285:26-30
3. 中华医学会肝炎病学分会,中华医学会传染病分会.丙型肝炎防治指南[刀.中华医学杂志,2004,84(9)：775—780.
4. Seeff, L.B.,1999, Natural history of Hepatitis C, The American Journal of Medicine, Vol107(6B):10-15
5. McHutchison JG, Gordon SC, Schiff ER et al,1998, Inteferon alfa-2b alone or in combination with ribavirin as initial treatment for chronic hepatitis C, The New England journal of Medicine,339:1485-92.
6. Nelson, D. R., C. Marousis, G. L. Davis, C. M. Rice, J. Wong, M. Houghton,and J. Y. N. Lau. 1997. The role of hepatitis C virus-specific cytotoxic T lymphocytes in chronic hepatitis C. J. Immunol.158:1473-1481.
7. Scognamiglio, P., D. Accapezzato, M. A. Casciaro, A. Cacciani, M. Artini, G.Bruno, M. L. Chircu, J. Sidney, S. Southwood, S. Abrignani, A. Sette, and V.Barnaba.1999. Presence of effector CD81 T cells in hepatitis C virusexposed healthy seronegative donors. J. Immunol. 11:6681-6689.
8. Donnelly, J., J. Ulmer, J. Shiver, and M. Liu.1997. DNA Vaccines. Annu. Rev. Immunol. 15:617.
9. Folgori, A., et al., A T-cell HCV vaccine eliciting effective immunity against heterologous virus challenge in chimpanzees. Nat Med,2006.12(2):p.190-7.
10. Calarota, S., G. Bratt, S. Nordlund, J. Hinkula, A. C. Leandersson, E. Sandstrom,and B. Wahren.1998. Cellular cytotoxic response induced by DNA vaccination in HIV-1-infected patients. Lancet 351:1320.
11. Wang R., D. L. Doolan, T. P. Le, R. C. Hedstrom, K. M. Coonan, Y. Charoenvit,T. R. Jones, P. Hobart, M.Margalith, J. Ng, et al.1998. Induction of antigenspecificcytotoxic T lymphocytes in humans by a malaria DNA vaccine. Science 282:476.
12. MARGARET A. LIU,BRITTA WAHREN, GUNILLA B. KARLSSON HEDESTAM,2006. DNA Vaccines:Recent Developments and Future Possibilities. HUMAN GENE THERAPY 17:1051-1061.
13. Pierre Lemieux.2002.Technological advances to increase immunogenicity of DNA vaccines. Expert Rev. Vaccinesl(1),85-93
14. Mathiesen I. Electropermeabilization of skeletal muscle enhances gene transfer in vivo. Gene Therapy 6(4):508-14 Apr 1999.
15.Rizzuto G., Cappelletti M., Maione D., Savino R., Lazzaro D., Costa P., Mathiesen I., Cortese R., Ciliberto G., Laufer R., La Monica N., Fattori E. Efficient and regulated erythropoietin production by naked DNA injection and muscle electroporation. Proc Natl Acad Sci U S A; 96(11):6417-22. May 25 1999.
16. Shortman, K. and Caux, C.1997. Dendritic cell development:multiple pathways to nature's adjuvants. Stem Cells.15:409.
17. Nchinda G, Kuroiwa J, Oks M, Trumpfheller C, Park CG, Huang Y, Hannaman D, Schlesinger SJ, Mizenina O, Nussenzweig MC, Uberla K, Steinman RM. The efficacy of DNA vaccination is enhanced in mice by targeting the encoded protein to dendritic cells. J Clin Invest.2008 Apr;118(4):1427-36.
18. Bozzacco L, Trumpfheller C, Siegal FP, Mehandru S, Markowitz M, Carrington M, Nussenzweig MC, Piperno AG, Steinman RM. DEC-205 receptor on dendritic cells mediates presentation of HIV gag protein to CD8+ T cells in a spectrum of human MHC I haplotypes. Proc Natl Acad Sci U S A.2007 Jan 23; 104(4):1289-94. Epub 2007 Jan 17.
19. Bartenschlager R, Ahlborn-Laake L, Mous J, Jacobsen H,1993, 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.,67:3835-44
20. Bartenschlager R, Ahlborn-Laake L, Mous J, Jacobsen H,1994, Kinetic and structural analyses of hepatitis C virus polyprotein processing, J Virol.,68: 5045-55
21. Missale G, Bertoni R, Lamonaca V, Valli A, Massari M, Mori C, Rumi MG, Houghton M, Fiaccadori F, Ferrari C. Different clinical behaviors of acute hepatitis C virus infection are associated with different vigor of the anti-viral cell-mediated immune response. J Clin Invest.1996 Aug 1;98(3):706-14.
22. Diepolder HM, Gerlach JT, Zachoval R, Hoffmann RM, Jung MC, Wierenga EA, Scholz S, Santantonio T, Houghton M, Southwood S, Sette A, Pape GR. Immunodominant CD4+ T-cell epitope within nonstructural protein 3 in acute hepatitis C virus infection. J Virol.1997 Aug;71(8):6011-9.
23. Bassett SE, Guerra B, Brasky K, Miskovsky E, Houghton M, Klimpel GR, Lanford RE. Protective immune response to hepatitis C virus in chimpanzees rechallenged following clearance of primary infection. Hepatology.2001 Jun;33(6):1479-87.
24. Santolini E, Migliaccio G, LaMonica N,1994, Biosynthesis and biochemical properties of the hepatitis C virus core protein, J Virol.,68:3631-41
25. Yi M, Nakamoto Y, Kaneko S, Yamashita, T, Murakami S,1997, Delineation of regions important for heteromeric association of hepatitis C virus El and E2, Virology,231:119-29
26. Hijikata M, Kato N, Ootsuyama Y, Nakagawa M, Shimotohno K,1991a, Gene mapping of the putative structural region of the hepatitis C virus genome by in vitro processing analysis, PNAS USA.,88:5547-51.
27. Hijikata M, Kato N, Ootsuyama Y, Nakagawa M, Ohkoshi S, Shimotohno K, 1991b, Hypervariable regions in the putative glycoprotein of hepatitis C virus, Biochem Biophys Res Commun,175:220-8
28. Zonaro A, Ravaggi A, Puoti M, Kremsdorf D, Albertini A, Cariani E,1994, Differential pattern of sequence heterogeneity in the hepatitis C virus El and E2/NS1 proteins, J Hepatol.,21:858-65.
29. Shimizu YK, Hijikata M, Iwamoto A, Alter HJ, Purcell RH, Yoshikura H,1994, Neutralizing antibodies against hepatitis C virus and the emergence of neutralization escape mutant viruses, J Virol.,68:1494-500.
30. Lechner S, Rispeter K, Meisel H, Kraas W, Jung G, Roggendorf M, Zibert A, 1998, Antibodies directed to envelope proteins of hepatitis C virus outside of hypervariable region 1, Virology,243:313-21.
31. Cooper, S., A. L. Erickson, E. J. Adams, J. Kansopon, A. J. Weiner, D. Y. Chien, M. Houghton, P. Parham, and C. M. Walker.1999. Analysis of a successful immune response against hepatitis C virus. Immunity 4:439-449.
32. Sallusto F, Lanzavecchia A (2002). The instructive role of dendritic cells on T-cell responses. Arthritis Res.4 Suppl 3:S127-32.
33.Banchereau, J and Steinman RM. Dendritic cells and the control of immunity. Nature 1998; 392:245-52.
34. Choo, Q. L., G. Kuo, R. Ralston, A. Weiner, D. Chien, G. Van Nest, J. Han, K. Berger, K. Thudium, C. Kuo, J. Kansopon, J. McFarland, A. Tabrizi, K. Ching, B. Moss, L. B. Cummins, M. Houghton, and E. Muchmore.1994. Vaccination of chimpanzees against infection by the hepatitis C virus. Proc.Natl. Acad. Sci. USA 91:1294-1298.
35.Farci, P., A. Shimoda, D. Wong, T. Cabezon, D. De Gioannis, A. Strazzera,Y. Shimizu, M. Shapiro, H. J. Alter, and R. Purcell.1996. Prevention of hepatitis C virus infection in chimpanzees by hyperimmune serum against the hypervariable region1 of the envelope 2 protein. Proc. Natl. Acad. Sci. USA 96:15394-15399.
36. Ray RB, Lagging LM, Meyer K, Ray R,1996, Hepatitis C virus core protein cooperates with ras and transforms primary rat embryo fibroblasts to tumorigenic phenotype, J Virol.,70:4438-43
37. Ray RB, Steele R, Meyer K, Ray R,1997, Transcriptional repression of p53 promoter by hepatitis C virus core protein, J Biol Chem.,272:10983-6.
38. Ray RB, Meyer K, Steele R, Shrivastava A, Aggarwal BB, Ray R,1998b, Inhibition of tumor necrosis factor (TNF-a) mediated apoptosis by hepatitis C virus core protein, J Biol Chem.,273:2256-2259
39. Satoh S, Tanji Y, Hijikata M, Kimura K, Shimotohno K,1995, The N-terminal region of hepatitis C virus nonstructural protein 3 (NS3) is essential for stable complex formation with NS4A, J. Virol.,69:4255-60
40. ErtlHCJ,Xiang ZQ,Novel vaccine approaches.J immunol,1996;156(7):3579-3582
41. Wolff JA,Dowty ME,Jiao S,et al.Expression of naked plasmids by cultured myotubes and entry of plasmids into T tubules and caveolae of mammalian skeletal muscle.J Cell Sci,1992;103:1249-1259
42. Raz E,Carson PA,Parker SE,et al.Intradermal gene immunization:The possible role of DNA up-take in the induction of cellular immunity to viruses.Proe Natl Acad Sci USA,1994;91(12):9519-9523
43.Molnar MJ, Gilbert R, Lu Y, Liu AB, Guo A, Larochelle N, Orlopp K, Lochmuller H, Petrof BJ, Nalbantoglu J, Karpati G. Factors influencing the efficacy, longevity, and safety of electroporation-assisted plasmid-based gene transfer into mouse muscles. Mol Ther.2004 Sep;10(3):447-55.
44. Zampaglione I, Arcuri M, Cappelletti M, Ciliberto G, Perretta G, Nicosia A, La Monica N, Fattori E. In vivo DNA gene electro-transfer:a systematic analysis of different electrical parameters. J Gene Med.2005 Nov;7(11):1475-81.
45. Lee JS, Hadjipanayis AG, Welkos SL. Venezuelan equine encephalitis virus-vectored vaccines protect mice against anthrax spore challenge. Infect Immun.2003 Mar;71(3):1491-6.
46. Leitner WW, Hwang LN, Bergmann-Leitner ES, Finkelstein SE, Frank S, Restifo NP. Apoptosis is essential for the increased efficacy of alphaviral replicase-based DNA vaccines. Vaccine.2004 Mar 29;22(11-12):1537-44.
47. Frelin L, Alheim M, Chen A, Soderholm J, Rozell B, Barnfield C, Liljestrom P, Sallberg M. Low dose and gene gun immunization with a hepatitis C virus nonstructural (NS) 3 DNA-based vaccine containing NS4A inhibit NS3/4A-expressing tumors in vivo. Gene Ther.2003 Apr;10(8):686-99.
48. Hirao LA, Wu L, Khan AS, Satishchandran A, Draghia-Akli R, Weiner DB. Intradermal/subcutaneous immunization by electroporation improves plasmid vaccine delivery and potency in pigs and rhesus macaques.Vaccine.2008 Jan 17;26(3):440-8. Epub 2007 Nov 8.
1. Rehermann, B. and M. Nascimbeni, Immunology of hepatitis B virus and hepatitis C virus infection. Nat Rev Immunol,2005.5(3):p.215-29.
2. Cohen,J.,1999, The scientific challenge of hepatitis C, Science,285:26-30
3. Seeff, L.B.,1999, Natural history of Hepatitis C, The American Journal of Medicine, Vol107(6B):10-15
4. McHutchison JG, Gordon SC, Schiff ER et al,1998, Inteferon alfa-2b alone or in combination with ribavirin as initial treatment for chronic hepatitis C, The New England journal of Medicine,339:1485-92.
5. Nelson, D. R., C. Marousis, G. L. Davis, C. M. Rice, J. Wong, M. Houghton,and J. Y. N. Lau.1997. The role of hepatitis C virus-specific cytotoxic T lymphocytes in chronic hepatitis C. J. Immunol.158:1473-1481.
6. Scognamiglio, P., D. Accapezzato, M. A. Casciaro, A. Cacciani, M. Artini, G.Bruno, M. L. Chircu, J. Sidney, S. Southwood, S. Abrignani, A. Sette, and V.Barnaba.1999. Presence of effector CD81 T cells in hepatitis C virusexposed healthy seronegative donors. J. Immunol.11:6681-6689.
7. Donnelly, J., J. Ulmer, J. Shiver, and M. Liu.1997. DNA Vaccines. Annu. Rev. Immunol.15:617.
8. Folgori, A., et al., A T-cell HCV vaccine eliciting effective immunity against heterologous virus challenge in chimpanzees. Nat Med,2006.12(2):p.190-7.
9. MARGARET A. LIU,BRITTA WAHREN, GUNILLA B. KARLSSON HEDESTAM,2006. DNA Vaccines:Recent Developments and Future Possibilities. HUMAN GENE THERAPY 17:1051-1061.
10. Pierre Lemieux.2002.Technological advances to increase immunogenicity of DNA vaccines. Expert Rev. Vaccines1(1),85-93
11. Mathiesen I. Electropermeabilization of skeletal muscle enhances gene transfer in vivo. Gene Therapy 6(4):508-14 Apr 1999.
12. Rizzuto G., Cappelletti M., Maione D., Savino R., Lazzaro D., Costa P., Mathiesen I., Cortese R., Ciliberto G., Laufer R., La Monica N., Fattori E. Efficient and regulated erythropoietin production by naked DNA injection and muscle electroporation.Proc Natl Acad Sci U S A; 96(11):6417-22. May 25 1999.
13. Ray RB, Steele R, Meyer K, Ray R,1997, Transcriptional repression of p53 promoter by hepatitis C virus core protein, J Biol Chem.,272:10983-6.
14. Ray RB, Steele R, Meyer K, Ray R,1998a, Hepatitis C virus core protein represses p21 WAF1/Cipl/Sidl promoter activity, Gene,208:331-6
15. Ray RB, Meyer K, Steele R, Shrivastava A, Aggarwal BB, Ray R,1998b, Inhibition of tumor necrosis factor (TNF-a) mediated apoptosis by hepatitis C virus core protein, J Biol Chem.,273:2256-2259
16. Tokushige K, Wakita T, Pachuk C, Moradpour D, Weiner DB, Zurawski VR Jr, Wands JR. Expression and immune response to hepatitis C virus core DNA-based vaccine constructs. Hepatology.1996 Jul;24(1):14-20.
17. Ray RB, Lagging LM, Meyer K, Ray R,1996, Hepatitis C virus core protein cooperates with ras and transforms primary rat embryo fibroblasts to tumorigenic phenotype, J Virol.,70:4438-43
18. Moriya K, Fujie H, Shintani Y, Yotsuyanagi H, Tsutsumi T, Ishibashi K, Matsuura Y, Kimura S, Miyamura T, Koike K,1998, The core protein of hepatitis C virus induces hepatocellular carcinoma in transgenic mice, Nat Med.,4:1065-7
19. Large MK, Kittlesen DJ, Hahn YS,1999, Suppression of host immune response by the core protein of hepatitis C virus:possible implications for hepatitis C virus persistence, J. Immunol.,162:931-8.
20. Hijikata M, Kato N, Ootsuyama Y, Nakagawa M, Ohkoshi S, Shimotohno K, 1991b, Hypervariable regions in the putative glycoprotein of hepatitis C virus, Biochem Biophys Res Commun,175:220-8.
21. Weiner AJ, Brauer MJ, Rosenblatt J, Richman KH, Tung J, Crawford K, Bonino F, Saracco G, Choo QL, Houghton M, Han JH,1991, Variable and hypervariable domains are found in the regions of HCV corresponding to the flavivirus envelop and NS1 proteins and the pestivirus envelop glycoproteins, Virology,180:842-8
22. Shimizu YK, Hijikata M, Iwamoto A, Alter HJ, Purcell RH, Yoshikura H,1994, Neutralizing antibodies against hepatitis C virus and the emergence of neutralization escape mutant viruses, J Virol.,68:1494-500.
23. Tedeschi V, Akatsuka T, Shih JW, Battegay M, Feinstone SM. A specific antibody response to HCV E2 elicited in mice by intramuscular inoculation of plasmid DNA containing coding sequences for E2. Hepatology.1997 Feb;25(2):459-62.
24. Forns X, Emerson SU, Tobin GJ, Mushahwar IK, Purcell RH, Bukh J. DNA immunization of mice and macaques with plasmids encoding hepatitis C virus envelope E2 protein expressed intracellularly and on the cell surface. Hepatology. 2000 Sep;32(3):618-25.
25. Lee SW, Cho JH, Lee KJ, Sung YC. Hepatitis C virus envelope DNA-based immunization elicits humoral and cellular immune responses. Mol Cells.1998 Aug 31;8(4):444-51.
26. Kim DW, Gwack Y, Han JH, Choe J,1995, C-terminal domain of the hepatitis C virus NS3 protein contains an RNA helicase activity, Biochem Biophys Res Commun.,215:160-6
27. Tai CL, Chi WK, Chen DS, Hwang LH,1996, The helicase activity associated with hepatitis C virus nonstructural protein 3(NS3), J Virol.,70:8477-84
28. Sakamuro D, Furukawa T, Takegami T,1995, hepatitis C virus nonstructural protein NS3 transforms NIH 3T3 cells, J Virol.,69:3893-6
29. Failla C, Tomei L, De Francesco R,1994, Both NS3 and NS4A are required for proteolytic processing of hepatitis C virus nonstructural proteins, J Virol.,68: 3753-60.
30. Kim JL, Morgenstern KA, Lin C, Fox T, Dwyer MD, Landro JA, Chambers SP, Markland W, Lepre CA, O'Malley ET, Harbeson SL, Rice CM, Murcko MA, Caron PR, Thomson JA. Crystal structure of the hepatitis C virus NS3 protease domain complexed with a synthetic NS4A cofactor peptide. Cell.1996 Oct 18;87(2):343-55. Erratum in:Cell 1997 Apr 4;89(1):159.
31. Isaguliants MG, Petrakova NV, Mokhonov VV, Pokrovskaya K, Suzdaltzeva YG, Krivonos AV, Zaberezhny AD, Garaev MM, Smirnov VD, Nordenfelt E. DNA immunization efficiently targets conserved functional domains of protease and ATPase/helicase of nonstructural 3 protein (NS3) of human hepatitis C virus. Immunol Lett.2003 Jul 3;88(1):1-13.
32. Lang KA, Yan J, Draghia-Akli R, Khan A, Weiner DB. Strong HCV NS3-and NS4A-specific cellular immune responses induced in mice and Rhesus macaques by a novel HCV genotype 1a/1b consensus DNA vaccine. Vaccine.2008 Nov 18;26(49):6225-31. Epub 2008 Aug 8.
33. Tu H, Gao L, Shi ST, Taylor DR, Yang T, Mircheff AK, Wen Y, Gorbalenya AE, Hwang SB, Lai MM,1999, Hepatitis C virus RNA polymerase and NS5A complex with a SNARE-like protein, Virology,263:30-41.
34. Enomoto N, Sakuma I, Asahina Y, Kurosaki M, Murakami T, Yamamoto C, Ogura Y, Izumi N, Marumo F, Sato C,1996, Mutations in the nonstructural protein 5A gene and response to interferon in patients with chronic hepatitis C virus lb infection, N Engl J Med.,334:77-81
35. Masalova OV, Lesnova EI, Pichugin AV, Melnikova TM, Grabovetsky VV, Petrakova NV, Smirnova OA, Ivanov AV, Zaberezhny AD, Ataullakhanov RI, Isaguliants MG, Kushch AA. The successful immune response against hepatitis C nonstructural protein 5A (NS5A) requires heterologous DNA/protein immunization. Vaccine.2010 Feb 23;28(8):1987-96.
36. Jens Encke, Jasper zu Putlitz, Michael Geissler and Jack R. Wands. Proteins of the Hepatitis C Virus in a Responses Against the Nonstructural Cellular and Humoral Immune Genetic Immunization Generates. J. Immunol.1998; 161;4917-4923
37. Frelin L, Alheim M, Chen A, Soderholm J, Rozell B, Barnfield C, Liljestrom P, Sallberg M. Low dose and gene gun immunization with a hepatitis C virus nonstructural (NS) 3 DNA-based vaccine containing NS4A inhibit NS3/4A-expressing tumors in vivo. Gene Ther.2003 Apr;10(8):686-99.
38. Hariharan MJ, Driver DA, Townsend K, Brumm D, Polo JM, Belli BA, Catton DJ, Hsu D, Mittelstaedt D, McCormack JE, Karavodin L, Dubensky TW Jr, Chang SM, Banks TA. DNA immunization against herpes simplex virus:enhanced efficacy using a Sindbis virus-based vector. J Virol.1998 Feb;72(2):950-8.
39. Frelin L, Alheim M, Chen A, Soderholm J, Rozell B, Barnfield C, Liljestrom P, Sallberg M. Low dose and gene gun immunization with a hepatitis C virus nonstructural (NS) 3 DNA-based vaccine containing NS4A inhibit NS3/4A-expressing tumors in vivo. Gene Ther.2003 Apr;10(8):686-99.
40. Ma X, Forns X, Gutierrez R, Mushahwar IK, Wu T, Payette PJ, Bukh J, Purcell RH, Davis HL. DNA-based vaccination against hepatitis C virus (HCV):effect of expressing different forms of HCV E2 protein and use of CpG-optimized vectors in mice. Vaccine.2002 Sep 10;20(27-28):3263-71.
41. Hong Yu, Lorne A. Babiuk and Sylvia van Drunen Littel-van den Hurk. Priming with CpG-enriched plasmid and boosting with protein formulated with CpG oligodeoxynucleotides and Quil A induces strong cellular and humoral immune responses to hepatitis C virus NS3. Journal of General Virology (2004),85, 1533-1543.
42. Geissler M, Gesien A, Tokushige K, Wands JR. Enhancement of cellular and humoral immune responses to hepatitis C virus core protein using DNA-based vaccines augmented with cytokine-expressing plasmids. J Immunol.1997 Feb 1;158(3):1231-7.
43. Liao GY, Bi SL, Yang JY, Li WD, Chen JY, Zhang XW, Jiang SD.Construction of bicistronic vector and its application to combined DNA vaccine. Zhonghua Shi Yan He Lin Chuang Bing Du Xue Za Zhi.2006 Jun;20(2):75-7.
44. Ke JS, Zhao P, Cao J, Yu JP, Qi ZT. Enhancement of cellular immune response to DNA vaccine encoding hepatitis C virus core and envelope 2 fusion antigen by murine Fms-like tyrosine kinase 3 ligand. Sheng Wu Gong Cheng Xue Bao.2003 Mar; 19(2):158-62.
45. Jens Encke, Jomo Bernardin, Jasmin Geib, Gocha Barbakadze, Raymond Bujdoso, Wolfgang Stremmel. Genetic vaccination with Flt3-L and GM-CSF as adjuvants: Enhancement of cellular and humoral immune responses that results in protective immunity in a murine model of hepatitis C virus infection. World J Gastroenterol 2006 November 28; 12(44):7118-7125
46. Sallberg M, Frelin L, Weiland O. DNA vaccine therapy for chronic hepatitis C virus (HCV) infection:immune control of a moving target. Expert Opin Biol Ther. 2009 Jul;9(7):805-15. Review.
47. Ahlen G, Soderholm J, Tjelle T, et al. In Vivo Electroporation Enhances the Immunogenicity of Hepatitis C Virus Nonstructural 3/4A DNA by Increased Local DNA Uptake, Protein Expression, Inflammation, and Infiltration of CD3+ T Cells[J]. J. Immunol.,2007,179(7):4741-4753.
48. Capone S, Zampaglione I, Vitelli A, et al. Modulation of the immune response induced by gene electrotransfer of a hepatitis C virus DNA vaccine in nonhuman primates[J]. J. Immunol.,2006,177(10):7462-71.
49. Zucchelli S, Capone S, Fattori E, Folgori A, Di Marco A, Casimiro D, Simon AJ, Laufer R, La Monica N, Cortese R, Nicosia A..Enhancing B-and T-cell immune response to a hepatitis C virus E2 DNA vaccine by intramuscular electrical gene transfer.J Virol.2000 Dec;74(24):11598-607.
50. Schwendener RA, Ludewig B, Cerny A, Engler O. Liposome-based vaccines. Methods Mol Biol.2010;605:163-75.
51. Gao M, Wang HP, Wang YN, Zhou Y, Wang QL.HCV-NS3 Thl minigene vaccine based on invariant chain CLIP genetic substitution enhances CD4(+) Thl cell responses in vivo.Vaccine.2006 Jun 29;24(26):5491-7. Epub 2006 Apr 24.
52. Lemieux P. Technological advances to increase immunogenicity of DNA vaccines[J]. Expert Rev. Vaccines,2002,1(1):85-93.
53. Desjardins D, Huret C, Dalba C, Kreppel F, Kochanek S, Cosset FL, Tangy F, Klatzmann D, Bellier B. Recombinant retrovirus-like particle forming DNA vaccines in prime-boost immunization and their use for hepatitis C virus vaccine development. J Gene Med.2009 Apr;11(4):313-25.
54. Capone S, Meola A, Ercole BB, Vitelli A, Pezzanera M, Ruggeri L, Davies ME, Tafi R, Santini C, Luzzago A, Fu TM, Bett A, Colloca S, Cortese R, Nicosia A, Folgori A. A Novel Adenovirus Type 6 (Ad6)-Based Hepatitis C Virus Vector That Overcomes Preexisting Anti-Ad5 Immunity and Induces Potent and Broad Cellular Immune Responses in Rhesus Macaque. J Virol.2006 Feb;80(4):1688-99.
2. Cohen, J.,1999, The scientific challenge of hepatitis C, Science,285:26-30
3. 中华医学会肝炎病学分会,中华医学会传染病分会.丙型肝炎防治指南[刀.中华医学杂志,2004,84(9)：775—780.
4. Seeff, L.B.,1999, Natural history of Hepatitis C, The American Journal of Medicine, Vol107(6B):10-15
5. McHutchison JG, Gordon SC, Schiff ER et al,1998, Inteferon alfa-2b alone or in combination with ribavirin as initial treatment for chronic hepatitis C, The New England journal of Medicine,339:1485-92.
6. Nelson, D. R., C. Marousis, G. L. Davis, C. M. Rice, J. Wong, M. Houghton,and J. Y. N. Lau. 1997. The role of hepatitis C virus-specific cytotoxic T lymphocytes in chronic hepatitis C. J. Immunol.158:1473-1481.
7. Scognamiglio, P., D. Accapezzato, M. A. Casciaro, A. Cacciani, M. Artini, G.Bruno, M. L. Chircu, J. Sidney, S. Southwood, S. Abrignani, A. Sette, and V.Barnaba.1999. Presence of effector CD81 T cells in hepatitis C virusexposed healthy seronegative donors. J. Immunol. 11:6681-6689.
8. Donnelly, J., J. Ulmer, J. Shiver, and M. Liu.1997. DNA Vaccines. Annu. Rev. Immunol. 15:617.
9. Folgori, A., et al., A T-cell HCV vaccine eliciting effective immunity against heterologous virus challenge in chimpanzees. Nat Med,2006.12(2):p.190-7.
10. Calarota, S., G. Bratt, S. Nordlund, J. Hinkula, A. C. Leandersson, E. Sandstrom,and B. Wahren.1998. Cellular cytotoxic response induced by DNA vaccination in HIV-1-infected patients. Lancet 351:1320.
11. Wang R., D. L. Doolan, T. P. Le, R. C. Hedstrom, K. M. Coonan, Y. Charoenvit,T. R. Jones, P. Hobart, M.Margalith, J. Ng, et al.1998. Induction of antigenspecificcytotoxic T lymphocytes in humans by a malaria DNA vaccine. Science 282:476.
12. MARGARET A. LIU,BRITTA WAHREN, GUNILLA B. KARLSSON HEDESTAM,2006. DNA Vaccines:Recent Developments and Future Possibilities. HUMAN GENE THERAPY 17:1051-1061.
13. Pierre Lemieux.2002.Technological advances to increase immunogenicity of DNA vaccines. Expert Rev. Vaccinesl(1),85-93
14. Mathiesen I. Electropermeabilization of skeletal muscle enhances gene transfer in vivo. Gene Therapy 6(4):508-14 Apr 1999.
15.Rizzuto G., Cappelletti M., Maione D., Savino R., Lazzaro D., Costa P., Mathiesen I., Cortese R., Ciliberto G., Laufer R., La Monica N., Fattori E. Efficient and regulated erythropoietin production by naked DNA injection and muscle electroporation. Proc Natl Acad Sci U S A; 96(11):6417-22. May 25 1999.
16. Shortman, K. and Caux, C.1997. Dendritic cell development:multiple pathways to nature's adjuvants. Stem Cells.15:409.
17. Nchinda G, Kuroiwa J, Oks M, Trumpfheller C, Park CG, Huang Y, Hannaman D, Schlesinger SJ, Mizenina O, Nussenzweig MC, Uberla K, Steinman RM. The efficacy of DNA vaccination is enhanced in mice by targeting the encoded protein to dendritic cells. J Clin Invest.2008 Apr;118(4):1427-36.
18. Bozzacco L, Trumpfheller C, Siegal FP, Mehandru S, Markowitz M, Carrington M, Nussenzweig MC, Piperno AG, Steinman RM. DEC-205 receptor on dendritic cells mediates presentation of HIV gag protein to CD8+ T cells in a spectrum of human MHC I haplotypes. Proc Natl Acad Sci U S A.2007 Jan 23; 104(4):1289-94. Epub 2007 Jan 17.
19. Bartenschlager R, Ahlborn-Laake L, Mous J, Jacobsen H,1993, 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.,67:3835-44
20. Bartenschlager R, Ahlborn-Laake L, Mous J, Jacobsen H,1994, Kinetic and structural analyses of hepatitis C virus polyprotein processing, J Virol.,68: 5045-55
21. Missale G, Bertoni R, Lamonaca V, Valli A, Massari M, Mori C, Rumi MG, Houghton M, Fiaccadori F, Ferrari C. Different clinical behaviors of acute hepatitis C virus infection are associated with different vigor of the anti-viral cell-mediated immune response. J Clin Invest.1996 Aug 1;98(3):706-14.
22. Diepolder HM, Gerlach JT, Zachoval R, Hoffmann RM, Jung MC, Wierenga EA, Scholz S, Santantonio T, Houghton M, Southwood S, Sette A, Pape GR. Immunodominant CD4+ T-cell epitope within nonstructural protein 3 in acute hepatitis C virus infection. J Virol.1997 Aug;71(8):6011-9.
23. Bassett SE, Guerra B, Brasky K, Miskovsky E, Houghton M, Klimpel GR, Lanford RE. Protective immune response to hepatitis C virus in chimpanzees rechallenged following clearance of primary infection. Hepatology.2001 Jun;33(6):1479-87.
24. Santolini E, Migliaccio G, LaMonica N,1994, Biosynthesis and biochemical properties of the hepatitis C virus core protein, J Virol.,68:3631-41
25. Yi M, Nakamoto Y, Kaneko S, Yamashita, T, Murakami S,1997, Delineation of regions important for heteromeric association of hepatitis C virus El and E2, Virology,231:119-29
26. Hijikata M, Kato N, Ootsuyama Y, Nakagawa M, Shimotohno K,1991a, Gene mapping of the putative structural region of the hepatitis C virus genome by in vitro processing analysis, PNAS USA.,88:5547-51.
27. Hijikata M, Kato N, Ootsuyama Y, Nakagawa M, Ohkoshi S, Shimotohno K, 1991b, Hypervariable regions in the putative glycoprotein of hepatitis C virus, Biochem Biophys Res Commun,175:220-8
28. Zonaro A, Ravaggi A, Puoti M, Kremsdorf D, Albertini A, Cariani E,1994, Differential pattern of sequence heterogeneity in the hepatitis C virus El and E2/NS1 proteins, J Hepatol.,21:858-65.
29. Shimizu YK, Hijikata M, Iwamoto A, Alter HJ, Purcell RH, Yoshikura H,1994, Neutralizing antibodies against hepatitis C virus and the emergence of neutralization escape mutant viruses, J Virol.,68:1494-500.
30. Lechner S, Rispeter K, Meisel H, Kraas W, Jung G, Roggendorf M, Zibert A, 1998, Antibodies directed to envelope proteins of hepatitis C virus outside of hypervariable region 1, Virology,243:313-21.
31. Cooper, S., A. L. Erickson, E. J. Adams, J. Kansopon, A. J. Weiner, D. Y. Chien, M. Houghton, P. Parham, and C. M. Walker.1999. Analysis of a successful immune response against hepatitis C virus. Immunity 4:439-449.
32. Sallusto F, Lanzavecchia A (2002). The instructive role of dendritic cells on T-cell responses. Arthritis Res.4 Suppl 3:S127-32.
33.Banchereau, J and Steinman RM. Dendritic cells and the control of immunity. Nature 1998; 392:245-52.
34. Choo, Q. L., G. Kuo, R. Ralston, A. Weiner, D. Chien, G. Van Nest, J. Han, K. Berger, K. Thudium, C. Kuo, J. Kansopon, J. McFarland, A. Tabrizi, K. Ching, B. Moss, L. B. Cummins, M. Houghton, and E. Muchmore.1994. Vaccination of chimpanzees against infection by the hepatitis C virus. Proc.Natl. Acad. Sci. USA 91:1294-1298.
35.Farci, P., A. Shimoda, D. Wong, T. Cabezon, D. De Gioannis, A. Strazzera,Y. Shimizu, M. Shapiro, H. J. Alter, and R. Purcell.1996. Prevention of hepatitis C virus infection in chimpanzees by hyperimmune serum against the hypervariable region1 of the envelope 2 protein. Proc. Natl. Acad. Sci. USA 96:15394-15399.
36. Ray RB, Lagging LM, Meyer K, Ray R,1996, Hepatitis C virus core protein cooperates with ras and transforms primary rat embryo fibroblasts to tumorigenic phenotype, J Virol.,70:4438-43
37. Ray RB, Steele R, Meyer K, Ray R,1997, Transcriptional repression of p53 promoter by hepatitis C virus core protein, J Biol Chem.,272:10983-6.
38. Ray RB, Meyer K, Steele R, Shrivastava A, Aggarwal BB, Ray R,1998b, Inhibition of tumor necrosis factor (TNF-a) mediated apoptosis by hepatitis C virus core protein, J Biol Chem.,273:2256-2259
39. Satoh S, Tanji Y, Hijikata M, Kimura K, Shimotohno K,1995, The N-terminal region of hepatitis C virus nonstructural protein 3 (NS3) is essential for stable complex formation with NS4A, J. Virol.,69:4255-60
40. ErtlHCJ,Xiang ZQ,Novel vaccine approaches.J immunol,1996;156(7):3579-3582
41. Wolff JA,Dowty ME,Jiao S,et al.Expression of naked plasmids by cultured myotubes and entry of plasmids into T tubules and caveolae of mammalian skeletal muscle.J Cell Sci,1992;103:1249-1259
42. Raz E,Carson PA,Parker SE,et al.Intradermal gene immunization:The possible role of DNA up-take in the induction of cellular immunity to viruses.Proe Natl Acad Sci USA,1994;91(12):9519-9523
43.Molnar MJ, Gilbert R, Lu Y, Liu AB, Guo A, Larochelle N, Orlopp K, Lochmuller H, Petrof BJ, Nalbantoglu J, Karpati G. Factors influencing the efficacy, longevity, and safety of electroporation-assisted plasmid-based gene transfer into mouse muscles. Mol Ther.2004 Sep;10(3):447-55.
44. Zampaglione I, Arcuri M, Cappelletti M, Ciliberto G, Perretta G, Nicosia A, La Monica N, Fattori E. In vivo DNA gene electro-transfer:a systematic analysis of different electrical parameters. J Gene Med.2005 Nov;7(11):1475-81.
45. Lee JS, Hadjipanayis AG, Welkos SL. Venezuelan equine encephalitis virus-vectored vaccines protect mice against anthrax spore challenge. Infect Immun.2003 Mar;71(3):1491-6.
46. Leitner WW, Hwang LN, Bergmann-Leitner ES, Finkelstein SE, Frank S, Restifo NP. Apoptosis is essential for the increased efficacy of alphaviral replicase-based DNA vaccines. Vaccine.2004 Mar 29;22(11-12):1537-44.
47. Frelin L, Alheim M, Chen A, Soderholm J, Rozell B, Barnfield C, Liljestrom P, Sallberg M. Low dose and gene gun immunization with a hepatitis C virus nonstructural (NS) 3 DNA-based vaccine containing NS4A inhibit NS3/4A-expressing tumors in vivo. Gene Ther.2003 Apr;10(8):686-99.
48. Hirao LA, Wu L, Khan AS, Satishchandran A, Draghia-Akli R, Weiner DB. Intradermal/subcutaneous immunization by electroporation improves plasmid vaccine delivery and potency in pigs and rhesus macaques.Vaccine.2008 Jan 17;26(3):440-8. Epub 2007 Nov 8.
1. Rehermann, B. and M. Nascimbeni, Immunology of hepatitis B virus and hepatitis C virus infection. Nat Rev Immunol,2005.5(3):p.215-29.
2. Cohen,J.,1999, The scientific challenge of hepatitis C, Science,285:26-30
3. Seeff, L.B.,1999, Natural history of Hepatitis C, The American Journal of Medicine, Vol107(6B):10-15
4. McHutchison JG, Gordon SC, Schiff ER et al,1998, Inteferon alfa-2b alone or in combination with ribavirin as initial treatment for chronic hepatitis C, The New England journal of Medicine,339:1485-92.
5. Nelson, D. R., C. Marousis, G. L. Davis, C. M. Rice, J. Wong, M. Houghton,and J. Y. N. Lau.1997. The role of hepatitis C virus-specific cytotoxic T lymphocytes in chronic hepatitis C. J. Immunol.158:1473-1481.
6. Scognamiglio, P., D. Accapezzato, M. A. Casciaro, A. Cacciani, M. Artini, G.Bruno, M. L. Chircu, J. Sidney, S. Southwood, S. Abrignani, A. Sette, and V.Barnaba.1999. Presence of effector CD81 T cells in hepatitis C virusexposed healthy seronegative donors. J. Immunol.11:6681-6689.
7. Donnelly, J., J. Ulmer, J. Shiver, and M. Liu.1997. DNA Vaccines. Annu. Rev. Immunol.15:617.
8. Folgori, A., et al., A T-cell HCV vaccine eliciting effective immunity against heterologous virus challenge in chimpanzees. Nat Med,2006.12(2):p.190-7.
9. MARGARET A. LIU,BRITTA WAHREN, GUNILLA B. KARLSSON HEDESTAM,2006. DNA Vaccines:Recent Developments and Future Possibilities. HUMAN GENE THERAPY 17:1051-1061.
10. Pierre Lemieux.2002.Technological advances to increase immunogenicity of DNA vaccines. Expert Rev. Vaccines1(1),85-93
11. Mathiesen I. Electropermeabilization of skeletal muscle enhances gene transfer in vivo. Gene Therapy 6(4):508-14 Apr 1999.
12. Rizzuto G., Cappelletti M., Maione D., Savino R., Lazzaro D., Costa P., Mathiesen I., Cortese R., Ciliberto G., Laufer R., La Monica N., Fattori E. Efficient and regulated erythropoietin production by naked DNA injection and muscle electroporation.Proc Natl Acad Sci U S A; 96(11):6417-22. May 25 1999.
13. Ray RB, Steele R, Meyer K, Ray R,1997, Transcriptional repression of p53 promoter by hepatitis C virus core protein, J Biol Chem.,272:10983-6.
14. Ray RB, Steele R, Meyer K, Ray R,1998a, Hepatitis C virus core protein represses p21 WAF1/Cipl/Sidl promoter activity, Gene,208:331-6
15. Ray RB, Meyer K, Steele R, Shrivastava A, Aggarwal BB, Ray R,1998b, Inhibition of tumor necrosis factor (TNF-a) mediated apoptosis by hepatitis C virus core protein, J Biol Chem.,273:2256-2259
16. Tokushige K, Wakita T, Pachuk C, Moradpour D, Weiner DB, Zurawski VR Jr, Wands JR. Expression and immune response to hepatitis C virus core DNA-based vaccine constructs. Hepatology.1996 Jul;24(1):14-20.
17. Ray RB, Lagging LM, Meyer K, Ray R,1996, Hepatitis C virus core protein cooperates with ras and transforms primary rat embryo fibroblasts to tumorigenic phenotype, J Virol.,70:4438-43
18. Moriya K, Fujie H, Shintani Y, Yotsuyanagi H, Tsutsumi T, Ishibashi K, Matsuura Y, Kimura S, Miyamura T, Koike K,1998, The core protein of hepatitis C virus induces hepatocellular carcinoma in transgenic mice, Nat Med.,4:1065-7
19. Large MK, Kittlesen DJ, Hahn YS,1999, Suppression of host immune response by the core protein of hepatitis C virus:possible implications for hepatitis C virus persistence, J. Immunol.,162:931-8.
20. Hijikata M, Kato N, Ootsuyama Y, Nakagawa M, Ohkoshi S, Shimotohno K, 1991b, Hypervariable regions in the putative glycoprotein of hepatitis C virus, Biochem Biophys Res Commun,175:220-8.
21. Weiner AJ, Brauer MJ, Rosenblatt J, Richman KH, Tung J, Crawford K, Bonino F, Saracco G, Choo QL, Houghton M, Han JH,1991, Variable and hypervariable domains are found in the regions of HCV corresponding to the flavivirus envelop and NS1 proteins and the pestivirus envelop glycoproteins, Virology,180:842-8
22. Shimizu YK, Hijikata M, Iwamoto A, Alter HJ, Purcell RH, Yoshikura H,1994, Neutralizing antibodies against hepatitis C virus and the emergence of neutralization escape mutant viruses, J Virol.,68:1494-500.
23. Tedeschi V, Akatsuka T, Shih JW, Battegay M, Feinstone SM. A specific antibody response to HCV E2 elicited in mice by intramuscular inoculation of plasmid DNA containing coding sequences for E2. Hepatology.1997 Feb;25(2):459-62.
24. Forns X, Emerson SU, Tobin GJ, Mushahwar IK, Purcell RH, Bukh J. DNA immunization of mice and macaques with plasmids encoding hepatitis C virus envelope E2 protein expressed intracellularly and on the cell surface. Hepatology. 2000 Sep;32(3):618-25.
25. Lee SW, Cho JH, Lee KJ, Sung YC. Hepatitis C virus envelope DNA-based immunization elicits humoral and cellular immune responses. Mol Cells.1998 Aug 31;8(4):444-51.
26. Kim DW, Gwack Y, Han JH, Choe J,1995, C-terminal domain of the hepatitis C virus NS3 protein contains an RNA helicase activity, Biochem Biophys Res Commun.,215:160-6
27. Tai CL, Chi WK, Chen DS, Hwang LH,1996, The helicase activity associated with hepatitis C virus nonstructural protein 3(NS3), J Virol.,70:8477-84
28. Sakamuro D, Furukawa T, Takegami T,1995, hepatitis C virus nonstructural protein NS3 transforms NIH 3T3 cells, J Virol.,69:3893-6
29. Failla C, Tomei L, De Francesco R,1994, Both NS3 and NS4A are required for proteolytic processing of hepatitis C virus nonstructural proteins, J Virol.,68: 3753-60.
30. Kim JL, Morgenstern KA, Lin C, Fox T, Dwyer MD, Landro JA, Chambers SP, Markland W, Lepre CA, O'Malley ET, Harbeson SL, Rice CM, Murcko MA, Caron PR, Thomson JA. Crystal structure of the hepatitis C virus NS3 protease domain complexed with a synthetic NS4A cofactor peptide. Cell.1996 Oct 18;87(2):343-55. Erratum in:Cell 1997 Apr 4;89(1):159.
31. Isaguliants MG, Petrakova NV, Mokhonov VV, Pokrovskaya K, Suzdaltzeva YG, Krivonos AV, Zaberezhny AD, Garaev MM, Smirnov VD, Nordenfelt E. DNA immunization efficiently targets conserved functional domains of protease and ATPase/helicase of nonstructural 3 protein (NS3) of human hepatitis C virus. Immunol Lett.2003 Jul 3;88(1):1-13.
32. Lang KA, Yan J, Draghia-Akli R, Khan A, Weiner DB. Strong HCV NS3-and NS4A-specific cellular immune responses induced in mice and Rhesus macaques by a novel HCV genotype 1a/1b consensus DNA vaccine. Vaccine.2008 Nov 18;26(49):6225-31. Epub 2008 Aug 8.
33. Tu H, Gao L, Shi ST, Taylor DR, Yang T, Mircheff AK, Wen Y, Gorbalenya AE, Hwang SB, Lai MM,1999, Hepatitis C virus RNA polymerase and NS5A complex with a SNARE-like protein, Virology,263:30-41.
34. Enomoto N, Sakuma I, Asahina Y, Kurosaki M, Murakami T, Yamamoto C, Ogura Y, Izumi N, Marumo F, Sato C,1996, Mutations in the nonstructural protein 5A gene and response to interferon in patients with chronic hepatitis C virus lb infection, N Engl J Med.,334:77-81
35. Masalova OV, Lesnova EI, Pichugin AV, Melnikova TM, Grabovetsky VV, Petrakova NV, Smirnova OA, Ivanov AV, Zaberezhny AD, Ataullakhanov RI, Isaguliants MG, Kushch AA. The successful immune response against hepatitis C nonstructural protein 5A (NS5A) requires heterologous DNA/protein immunization. Vaccine.2010 Feb 23;28(8):1987-96.
36. Jens Encke, Jasper zu Putlitz, Michael Geissler and Jack R. Wands. Proteins of the Hepatitis C Virus in a Responses Against the Nonstructural Cellular and Humoral Immune Genetic Immunization Generates. J. Immunol.1998; 161;4917-4923
37. Frelin L, Alheim M, Chen A, Soderholm J, Rozell B, Barnfield C, Liljestrom P, Sallberg M. Low dose and gene gun immunization with a hepatitis C virus nonstructural (NS) 3 DNA-based vaccine containing NS4A inhibit NS3/4A-expressing tumors in vivo. Gene Ther.2003 Apr;10(8):686-99.
38. Hariharan MJ, Driver DA, Townsend K, Brumm D, Polo JM, Belli BA, Catton DJ, Hsu D, Mittelstaedt D, McCormack JE, Karavodin L, Dubensky TW Jr, Chang SM, Banks TA. DNA immunization against herpes simplex virus:enhanced efficacy using a Sindbis virus-based vector. J Virol.1998 Feb;72(2):950-8.
39. Frelin L, Alheim M, Chen A, Soderholm J, Rozell B, Barnfield C, Liljestrom P, Sallberg M. Low dose and gene gun immunization with a hepatitis C virus nonstructural (NS) 3 DNA-based vaccine containing NS4A inhibit NS3/4A-expressing tumors in vivo. Gene Ther.2003 Apr;10(8):686-99.
40. Ma X, Forns X, Gutierrez R, Mushahwar IK, Wu T, Payette PJ, Bukh J, Purcell RH, Davis HL. DNA-based vaccination against hepatitis C virus (HCV):effect of expressing different forms of HCV E2 protein and use of CpG-optimized vectors in mice. Vaccine.2002 Sep 10;20(27-28):3263-71.
41. Hong Yu, Lorne A. Babiuk and Sylvia van Drunen Littel-van den Hurk. Priming with CpG-enriched plasmid and boosting with protein formulated with CpG oligodeoxynucleotides and Quil A induces strong cellular and humoral immune responses to hepatitis C virus NS3. Journal of General Virology (2004),85, 1533-1543.
42. Geissler M, Gesien A, Tokushige K, Wands JR. Enhancement of cellular and humoral immune responses to hepatitis C virus core protein using DNA-based vaccines augmented with cytokine-expressing plasmids. J Immunol.1997 Feb 1;158(3):1231-7.
43. Liao GY, Bi SL, Yang JY, Li WD, Chen JY, Zhang XW, Jiang SD.Construction of bicistronic vector and its application to combined DNA vaccine. Zhonghua Shi Yan He Lin Chuang Bing Du Xue Za Zhi.2006 Jun;20(2):75-7.
44. Ke JS, Zhao P, Cao J, Yu JP, Qi ZT. Enhancement of cellular immune response to DNA vaccine encoding hepatitis C virus core and envelope 2 fusion antigen by murine Fms-like tyrosine kinase 3 ligand. Sheng Wu Gong Cheng Xue Bao.2003 Mar; 19(2):158-62.
45. Jens Encke, Jomo Bernardin, Jasmin Geib, Gocha Barbakadze, Raymond Bujdoso, Wolfgang Stremmel. Genetic vaccination with Flt3-L and GM-CSF as adjuvants: Enhancement of cellular and humoral immune responses that results in protective immunity in a murine model of hepatitis C virus infection. World J Gastroenterol 2006 November 28; 12(44):7118-7125
46. Sallberg M, Frelin L, Weiland O. DNA vaccine therapy for chronic hepatitis C virus (HCV) infection:immune control of a moving target. Expert Opin Biol Ther. 2009 Jul;9(7):805-15. Review.
47. Ahlen G, Soderholm J, Tjelle T, et al. In Vivo Electroporation Enhances the Immunogenicity of Hepatitis C Virus Nonstructural 3/4A DNA by Increased Local DNA Uptake, Protein Expression, Inflammation, and Infiltration of CD3+ T Cells[J]. J. Immunol.,2007,179(7):4741-4753.
48. Capone S, Zampaglione I, Vitelli A, et al. Modulation of the immune response induced by gene electrotransfer of a hepatitis C virus DNA vaccine in nonhuman primates[J]. J. Immunol.,2006,177(10):7462-71.
49. Zucchelli S, Capone S, Fattori E, Folgori A, Di Marco A, Casimiro D, Simon AJ, Laufer R, La Monica N, Cortese R, Nicosia A..Enhancing B-and T-cell immune response to a hepatitis C virus E2 DNA vaccine by intramuscular electrical gene transfer.J Virol.2000 Dec;74(24):11598-607.
50. Schwendener RA, Ludewig B, Cerny A, Engler O. Liposome-based vaccines. Methods Mol Biol.2010;605:163-75.
51. Gao M, Wang HP, Wang YN, Zhou Y, Wang QL.HCV-NS3 Thl minigene vaccine based on invariant chain CLIP genetic substitution enhances CD4(+) Thl cell responses in vivo.Vaccine.2006 Jun 29;24(26):5491-7. Epub 2006 Apr 24.
52. Lemieux P. Technological advances to increase immunogenicity of DNA vaccines[J]. Expert Rev. Vaccines,2002,1(1):85-93.
53. Desjardins D, Huret C, Dalba C, Kreppel F, Kochanek S, Cosset FL, Tangy F, Klatzmann D, Bellier B. Recombinant retrovirus-like particle forming DNA vaccines in prime-boost immunization and their use for hepatitis C virus vaccine development. J Gene Med.2009 Apr;11(4):313-25.
54. Capone S, Meola A, Ercole BB, Vitelli A, Pezzanera M, Ruggeri L, Davies ME, Tafi R, Santini C, Luzzago A, Fu TM, Bett A, Colloca S, Cortese R, Nicosia A, Folgori A. A Novel Adenovirus Type 6 (Ad6)-Based Hepatitis C Virus Vector That Overcomes Preexisting Anti-Ad5 Immunity and Induces Potent and Broad Cellular Immune Responses in Rhesus Macaque. J Virol.2006 Feb;80(4):1688-99.