ANTIGENIC STRUCTURE OF HEPATITIS C VIRUS

对丙型肝炎病毒的T细胞和B细胞免疫反应正在研究中。 在此之前，我们开发并鉴定了人类和小鼠的单克隆 抗丙型肝炎病毒结构基因的抗体；使用重组 杆状病毒表达抗原。我们已经证明了针对e1的抗体 E2能与天然病毒粒子结合，核心区抗体能与病毒粒子结合 被洗涤剂剥去了信封。T细胞研究仍在继续 识别细胞毒性T细胞和辅助T细胞表位的努力。 我们正在研究慢性粒细胞白血病患者的增殖和CTL反应 慢性丙型肝炎病毒对多肽抗原和抗原的感染 重组DNA系统。我们之前发现了一个CTL表位 老鼠和人类都能识别的核心区域。使用预测 从丙型肝炎病毒多蛋白中提取的人类白细胞抗原A2基序，我们鉴定出另外五个 核心蛋白3个，NS4B和NS5B各1个。我们发现 慢性丙型肝炎病毒感染的人类白细胞抗原A2患者对至少一种 CTL检测中的这些表位，但2名康复的患者 丙型肝炎病毒无反应。我们还开发了一种新的方法来识别 CTL表位不依赖于对已知基序的选择。我们 对纯化的丙型肝炎病毒NS3蛋白进行蛋白水解酶消化，分离出 用高效液相色谱(HPLC)分析得到的多肽。每一组分被用来驱动同源基因 靶细胞和在CTL试验中识别的细胞是 已排序。然后通过合成来绘制准确的表位图 覆盖在反应肽上的重叠的十肽。我们 在NS3蛋白中发现了一个新的表位，患者对其具有 慢性丙型肝炎病毒感染会有反应。这个表位也只有 表达人类白细胞抗原A2的患者，即使该多肽不包含 识别的A2结合基序。实验性丙型肝炎病毒基因疫苗 正在研究中。BALB/c小鼠被证明在 注射表达丙型肝炎病毒E2的质粒组。我们已经延长了这些 实验包括丙型肝炎病毒核心基因和E1基因以及E2基因。 接种的小鼠对特定的丙型肝炎病毒既有抗体又有CTL反应 抗原。体外免疫相关性正在开发中，它将 协助评估这些基于质粒基的疫苗。其他内容 现在正在评估包括细胞因子基因的结构 非结构基因和非结构基因的表达以及特异性CTL表位 非特定辅助表位。

Both T cell and B cell immune responses to HCV are being investigated. Previously we developed and characterized human and mouse monoclonal antibodies to the HCV structural genes; core, E1 and E2 using recombinant baculovirus expressed antigens. We have shown that the antibodies ot E1 and E2 can bind to native virion and antibody to core can bind to virion stripped of its envelope by detergent. T cell studies are continuing in an effort to identify both cytotoxic T cell and helper T cell epitopes. We are studying the proliferative and CTL responses of patients with chronic HCV infections to peptide antigens and antigens expessed by recombinant DNA systems. We previously identified a CTL epitope in the core region that is recognized by both mice and humans. Using predicted HLA A2 motifs from the HCV polyprotein, we identified five additional epitopes, 3 in the core protein, 1 in NS4B and 1 in NS5B. We found that HLA A2 patients with chronic HCV infections react to at least one of these epitopes in CTL assays but that 2 patients who had recovered from HCV did not react. We have also developed a new approach to identifying CTL epitopes that is not dependent on selecting for known motifs. We made proteolytic digests purified HCV NS3 protein and separated the resulting peptides by HPLC. Each fraction was used to pulse syngenetic target cells and those that were recognized in a CTL assay were sequenced. The precise epitope was then mapped by synthesizing overlapping decapeptides that covered the reactive peptide. We identified a new epitope in the NS3 protein to which patients with chronic HCV infections react. This epitope was also recognized only by patients who expressed HLA A2 even though the peptide did not contain a recognized A2 binding motif. Experimental genetic vaccines against HCV are being studied. BALB/c mice have been shown to develop antibody after injection with a plasmid that expresses HCV E2. We have extended these experiments to include the HCV core and E1 genes as well as E2. Inoculated mice exhibit both antibody and CTL responses to specific HCV antigens. In vitro correlates of immunity are being developed that will assist in evaluation of these plasmid based vaccines. Additional constructs are now being evaluated that include genes for cytokine expression as well as specific CTL epitopes from non-structural genes and non-specific helper epitopes.