Molecular and structural characterization of broadly neutralizing anti-HCV antibodies

项目摘要 目前迫切需要一种丙型肝炎病毒疫苗。全球超过1.7亿人感染丙型肝炎病毒 每年在美国导致的死亡人数比艾滋病毒还多。而直接作用抗病毒(DAA)疗法 革命性的丙型肝炎病毒治疗，由于高危人群频繁再次感染，丙型肝炎大流行的控制仍然具有挑战性 个人和高比例的无症状携带者继续感染他人。约30%的 感染丙型肝炎病毒的人会自发清除感染，我们之前已经展示了 丙型肝炎病毒的自发清除与广谱中和抗体的早期发展有关 (抗病毒抗体)。在多种动物模型中，bNAbs对丙型肝炎病毒感染也有保护作用。 不幸的是，到目前为止，针对丙型肝炎病毒的疫苗还没有诱导出足够的保护性bNAb滴度。我们的无能 诱导有效的bNAbs的部分原因是我们对分子和结构相互作用的理解很差。 BNAbs与丙型肝炎病毒包膜蛋白(E1和E2)之间的相互作用。我们的初步工作表明信封 远离bNAb结合位点的序列多态对中和有强烈的、意想不到的影响 敏感度。这些数据和在HIV中迅速出现的工作表明，这些关键的bNAb-包膜相互作用 需要在三维(结构)背景下理解。我们假设分子和结构 对bNAb-E2相互作用的分析将使我们能够合理地设计稳定的丙型肝炎病毒包膜蛋白 非常适合结构和疫苗研究的bNab表位，以及具有增强中和功能的bNAbs 效力和广度，更好地定义了疫苗应该诱导的理想抗体。 我们已经鉴定了一组不同的独特的丙型肝炎病毒包膜蛋白，并分离出了一些最广泛的 迄今为止所描述的中和抗丙型肝炎病毒的单抗。在目标1中，我们将从功能和分子上 描述这组不同的、自然产生的丙型肝炎病毒包膜变异体与该组之间的相互作用 ，这将使我们能够确定E2中和敏感性的氨基酸决定因素以及 赋予bNAbs中和效力和广度的体细胞突变。在目标2中，我们将定义生化 以及影响丙型肝炎病毒包膜蛋白稳定性和天然折叠的分子因素。我们将克隆的不止是 100个不同的天然丙型肝炎病毒E2变异体，并确定与体外稳定表达E2相关的多态。 在目标3中，我们将确定广泛和有效的丙型肝炎病毒中和的结构相关性。我们将会明晰 丙型肝炎病毒E2与不同广度和效力的bNAbs形成复合体。我们将使用通过以下方式获得的数据 Three的目标是设计具有优化的bNAb表位的稳定的E2变体，这将是未来理想的试剂 结构分析和疫苗研究。此外，我们将设计具有更强中和能力的bNAbs。 和广度，这将定义理想的抗体，可以由疫苗诱导，也可能是有用的 治疗剂。通过这些研究，我们将促进丙型肝炎疫苗的合理设计。

Project Summary A vaccine against Hepatitis C virus (HCV) is urgently needed. HCV infects over 170 million people worldwide and kills more people in the United States annually than HIV. While direct-acting antiviral (DAA) therapy has revolutionized HCV care, control of the HCV pandemic remains challenging due frequent reinfection in high-risk individuals and a high proportion of asymptomatic carriers who continue to infect others. Approximately 30% of individuals who become infected with HCV spontaneously clear the infection, and we have previously shown that this spontaneous clearance of HCV is associated with early development of broadly neutralizing antibodies (bNAbs) against the virus. BNAbs are also protective against HCV infection in multiple animal models. Unfortunately, to date, vaccines against HCV have not induced adequate titers of protective bNAbs. Our inability to induce potent bNAbs is in part due to our poor understanding of the molecular and structural interactions between bNAbs and HCV envelope proteins (E1 and E2). Our preliminary work indicates that envelope sequence polymorphisms distant from bNAb binding sites have a strong, unexpected influence on neutralization sensitivity. These data and rapidly emerging work in HIV indicate that these crucial bNAb-envelope interactions need to be understood in a three dimensional (structural) context. We hypothesize that molecular and structural analysis of bNAb-E2 interactions will allow us to rationally design stable HCV envelope proteins with optimized bNAb epitopes that are ideal for structural and vaccine studies as well as bNAbs with enhanced neutralizing potency and breadth, better defining the ideal antibodies that should be induced by a vaccine. We have characterized a diverse panel of unique HCV envelope proteins and isolated some of the most broadly neutralizing anti-HCV monoclonal antibodies described to date. In Aim 1, we will functionally and molecularly characterize interactions between this panel of diverse, naturally occurring HCV envelope variants and the panel of bNAbs, which will allow us to identify amino acid determinants of neutralization sensitivity of E2 as well as somatic mutations conferring neutralizing potency and breadth to bNAbs. In Aim 2, we will define biochemical and molecular factors influencing stability and native folding of HCV envelope proteins. We will clone more than 100 distinct natural HCV E2 variants and identify polymorphisms associated with stable in vitro E2 expression. In Aim 3, we will determine structural correlates of broad and potent neutralization of HCV. We will crystallize HCV E2 in complex with bNAbs of varying breadth and potency. We will use the data acquired through these three aims to design stable E2 variants with optimized bNAb epitopes that will be ideal reagents for future structure analyses and vaccine studies. In addition, we will design bNAbs with enhanced neutralizing potency and breadth that will define the ideal antibodies that could be induced by a vaccine and may also be useful therapeutic agents. Through these investigations, we will advance rational design of an HCV vaccine.