Chimeric hepatitis C vaccine vector

描述(申请人提供)：超过1.7亿人感染丙型肝炎病毒(丙型肝炎病毒)，并面临肝功能衰竭和肝细胞癌的风险。丙型肝炎病毒是黄病毒科的一种囊膜RNA病毒，也包括黄病毒属和害虫病毒属。通过操纵这些RNA病毒的互补DNA基因组并产生可在细胞培养中分析的感染性病毒粒子的能力，促进了对后一类病毒的分子分析。黄热病病毒(YFV)17D疫苗株非致病背景下表达致病性黄病毒囊膜糖蛋白的嵌合黄病毒很容易产生，是安全有效的活疫苗。由于无法在细胞培养或小动物模型中培养病毒，阻碍了了解丙型肝炎病毒生命周期和开发抗病毒药物和疫苗的努力。尽管对丙型肝炎病毒体外传播的限制包括与病毒包膜糖蛋白与细胞受体相互作用有关的限制(S)，但额外的障碍可能包括在病毒粒子蛋白的细胞内运输和组装过程中出现的那些障碍。在这个小型的研究资助计划中，我们描述了利用17D YFV株载体的主干来表达丙型肝炎病毒包膜糖蛋白的初步研究，并开发了一个强大的体外系统来研究丙型肝炎病毒的结构、功能和免疫学。具体目标是：(1)获得携带丙型肝炎病毒囊膜糖蛋白基因的重组YFV基因组；(2)评估丙型肝炎病毒囊膜糖蛋白的结构完整性和嵌合病毒粒子的组装和分泌能力。我们将确定嵌合病毒粒子是否能够感染人肝细胞癌细胞。重组基因组可作为天然丙型肝炎病毒包膜糖蛋白复合体的来源，用于生化分析。如果嵌合病毒粒子被组装和分泌，那么丙型肝炎病毒包膜糖蛋白可能介导进入适当的靶细胞。活的嵌合病毒将有助于在细胞培养中研究丙型肝炎病毒的结合和进入，以及病毒粒子的组装和形态发生，并可能为开发减毒丙型肝炎疫苗提供一个起点。

DESCRIPTION (provided by applicant): Over 170 million persons are infected with Hepatitis C virus (HCV) and are at risk for liver failure and hepatocellular carcinoma. HCV is an enveloped RNA virus in the Flaviviridae family, which also includes the Flavivirus and Pestivirus genera. Molecular analysis of these latter genera has been facilitated by the ability to manipulate the complementary DNA genomes of these RNA viruses and to generate infectious virions that can be analyzed in cell culture. Viable chimeric Flaviviruses can be readily generated, and those expressing the envelope glycoproteins of pathogenic Flaviviruses within the nonpathogenic background of the 17D vaccine strain of the Yellow Fever Virus (YFV) have been shown to be safe and effective live vaccines. Efforts to understand the HCV life-cycle and to develop antiviral drugs and vaccines have been hindered by the inability to grow the virus in cell culture or in small animal models. Although restrictions on the in vitro propagation of HCV include those related to the interaction of the viral envelope glycoproteins with cellular receptor(s), additional barriers may include those that arise during the intracellular trafficking and assembly of the virion proteins. In this Small Research Grant proposal, we describe pilot studies to utilize the backbone of the strain 17D YFV vector to express the HCV envelope glycoproteins and develop a robust in vitro system to study HCV structure, function, and immunology. Specific Aims are: (1) to generate recombinant strain 17D YFV genomes bearing the HCV envelope glycoprotein genes, and (2) to assess the structural integrity of the HCV envelope glycoproteins and the ability of chimeric virion particles to be assembled and secreted. We will determine whether the chimeric virions are able to infect human hepatocellular carcinoma cells. Recombinant genomes may serve as a source of native HCV envelope glycoprotein complex for biochemical analysis. If chimeric virions are assembled and secreted, then the HCV envelope glycoproteins may mediate entry into appropriate target cells. Viable chimeric viruses will enable the study in cell culture of HCV binding and entry, as well as virion assembly and morphogenesis, and may provide a starting point towards the development of an attenuated HCV vaccine.