Molecular and structural characterization of broadly neutralizing anti-HCV antibodies

总结 全世界有7000多万人感染丙型肝炎病毒（HCV），疫苗的开发 对于根除疾病至关重要。虽然直接作用的抗病毒药物对治疗非常有效， 大多数接受调查的国家没有走上实现世卫组织消除丙型肝炎病毒作为公共卫生目标的轨道。 到2030年，大多数国家的HCV发病率将上升。幸运的是，有强有力的证据表明， 疫苗诱导高滴度的广泛中和抗体（bNAb;能够中和 不同的HCV变体）可以提供针对人HCV感染的保护。然而，我们无法 用疫苗刺激bNAbs抗HCV，因为我们还没有确定抗HCV的全谱 对中和宽度至关重要或表征HCV包膜糖蛋白（E1和E2）的抗体 有利于bNAb选择和成熟的遗传和结构特征。本提案的总体目标是 分离大量具有代表性的E1 E2特异性bNAb、bNAb未突变的祖先和bNAb 来自Elite中和剂（EN）的中间体，具有广泛中和血浆和自发性的个体 清除HCV感染。在目的1中，我们将从E1 E2特异性B细胞中分离单克隆抗体（mAb 分离自EN和慢性持续性感染（CP）的对照。我们将推断未突变的种系bNAb 并使用纵向E1 E2特异性B细胞的B细胞受体测序来鉴定bNA B遗传 中间体的我们将比较EN与CP的中和广度、效力、表位和遗传特征 单克隆抗体在目标2中，我们将使用X射线晶体学或冷冻-EM技术来比较EN bNAb或 与可溶性E2或E1 E2异二聚体复合的CP mAb。通过比较mAb-E1 E2与CP之间的相互作用， 单克隆抗体，bNAb未突变的祖先，bNAb中间体和成熟的bNAb，我们将定义结构和遗传 E1 E2的特性是开发中和宽度所必需的。总之，这些研究将确定一个 与HCV自发清除相关的大量代表性bNAb，定义了关键表位残基 和E1 E2中的结构特征，其可以被稳定化以优化疫苗抗原。这些研究将提供信息， 基于结构的设计努力，以改善基于E1 E2的疫苗候选物，这是一个紧迫的挑战， 全球公共卫生影响。

Summary More than 70 million people worldwide are infected with hepatitis C virus (HCV), and development of a vaccine for HCV is essential for disease eradication. Although direct-acting antivirals are highly effective for treatment, the majority of countries surveyed are not on track to reach the WHO goal of eliminating HCV as a public health problem by 2030, with most countries seeing rising incidence of HCV. Fortunately, there is strong evidence that vaccine-induction of high titers of broadly neutralizing antibodies (bNAbs; antibodies capable of neutralizing diverse HCV variants) could provide protection against human HCV infection. However, we are unable to stimulate bNAbs against HCV with a vaccine because we have not defined the full spectrum of anti-HCV antibodies that are critical for neutralizing breadth or characterized the HCV envelope glycoprotein (E1 and E2) genetic and structural features that favor bNAb selection and maturation. The overarching goal of this proposal is to isolate a large and representative set of E1E2-specific bNAbs, bNAb unmutated ancestors, and bNAb intermediates from Elite Neutralizers (EN), individuals with broadly neutralizing plasma and spontaneous clearance of HCV infection. In Aim 1, we will isolate monoclonal antibodies (mAbs) from E1E2-specific B cells isolated from EN and from controls with chronic, persistent infection (CP). We will infer unmutated germline bNAb ancestors and use B cell receptor-sequencing of longitudinal E1E2-specific B cells to identify bNAb genetic intermediates. We will compare neutralizing breadth, potency, epitopes, and genetic features for EN vs. CP mAbs. In Aim 2, we will use X-ray crystallography or cryo-EM techniques to compare structures of EN bNAbs or CP mAbs in complex with soluble E2 or E1E2 heterodimers. By comparing mAb-E1E2 interactions among CP mAbs, bNAb unmutated ancestors, bNAb intermediates, and mature bNAbs, we will define structural and genetic features of E1E2 necessary for the development of neutralizing breadth. Together, these studies will identify a large, representative set of bNAbs associated with spontaneous clearance of HCV, defining key epitope residues and structural features in E1E2 that could be stabilized to optimize vaccine antigens. These studies will inform structure-based design efforts to improve E1E2-based vaccine candidates, which is an urgent challenge with global public health implications.