Molecular and structural characterization of broadly neutralizing anti-HCV antibodies

项目摘要 丙型肝炎病毒（HCV）的疫苗是迫切需要的。HCV感染全球超过1.7亿人 在美国每年死于艾滋病的人比死于艾滋病的人还多。虽然直接作用的抗病毒药物（DAA）治疗 尽管丙型肝炎护理发生了革命性变化，但由于高危人群的频繁再感染，丙型肝炎大流行的控制仍然具有挑战性。 感染者中有很大一部分是无症状携带者，他们继续感染其他人。大约30%的 感染HCV的个体会自发清除感染，我们之前已经证明， HCV的自发清除与广泛中和抗体的早期发展有关， （bNAbs）对抗病毒。BNAb在多种动物模型中也具有抗HCV感染的保护作用。 不幸的是，迄今为止，针对HCV的疫苗尚未诱导足够滴度的保护性bNAb。我们无法 诱导有效的bNAb的部分原因是我们对分子和结构相互作用的理解不足， bNAb和HCV包膜蛋白（E1和E2）之间的差异。我们的初步工作表明 远离bNAb结合位点的序列多态性对中和作用具有强烈的、意想不到的影响 灵敏度这些数据和在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）环境中理解。我们假设分子和结构 bNAb-E2相互作用的分析将使我们能够合理地设计稳定的HCV包膜蛋白， 对于结构和疫苗研究理想的bNAb表位以及具有增强的中和性的bNAb 效力和广度，更好地定义疫苗应诱导的理想抗体。 我们已经鉴定了一组不同的独特HCV包膜蛋白，并分离出一些最广泛的 迄今为止描述的中和抗HCV单克隆抗体。在目标1中，我们将从功能上和分子上 表征这组不同的、天然存在的HCV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， 赋予bNAb中和效力和宽度的体细胞突变。在目标2中，我们将定义生物化学 以及影响HCV包膜蛋白的稳定性和天然折叠的分子因素。我们将克隆更多的 100种不同的天然HCV E2变体，并鉴定与体外E2稳定表达相关的多态性。 在目标3中，我们将确定广泛和有效中和HCV的结构相关性。我们将结晶 HCV E2与不同宽度和效力的bNAb复合。我们将使用通过这些获取的数据 三个目标是设计具有优化的bNAb表位的稳定的E2变体，这将是未来的理想试剂。 结构分析和疫苗研究。此外，我们将设计具有增强的中和效力的bNAb， 这将定义疫苗可以诱导的理想抗体， 治疗剂。通过这些研究，我们将促进HCV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.