An effective preventive vaccine for hepatitis C virus (HCV) remains a major unmet need. Antigenic region 3 (AR3) on the E1E2 envelope glycoprotein complex overlaps with the CD81 receptor binding site and represents an important epitope for broadly neutralizing antibodies (bNAbs) and is therefore important for HCV vaccine design. Most AR3 bNAbs utilize the V_H1-69 gene and share structural features that define the AR3C-class of HCV bNAbs. In this work, we identify recombinant HCV glycoproteins based on a permuted E2E1 trimer design that bind to the inferred V_H1-69 germline precursors of AR3C-class bNAbs. When presented on nanoparticles, these recombinant E2E1 glycoproteins efficiently activate B cells expressing inferred germline AR3C-class bNAb precursors as B cell receptors. Furthermore, we identify critical signatures in three AR3C-class bNAbs that represent two subclasses of AR3C-class bNAbs that will allow refined protein design. These results provide a framework for germline-targeting vaccine design strategies against HCV.

有效的丙型肝炎病毒（HCV）预防疫苗仍然是一个未满足的主要需求。E1E2 包膜糖蛋白复合物上的抗原区 3 (AR3) 与 CD81 受体结合位点重叠，代表广泛中和抗体 (bNAb) 的重要表位，因此对于 HCV 疫苗设计非常重要。大多数 AR3 bNAb 利用V _H 1-69基因，并具有定义 AR3C 类 HCV bNAb 的结构特征。在这项工作中，我们基于排列的 E2E1 三聚体设计鉴定了重组 HCV 糖蛋白，该三聚体设计与AR3C 类 bNAb 的推断V _H 1-69 种系前体结合。当出现在纳米颗粒上时，这些重组 E2E1 糖蛋白有效激活 B 细胞，将推断的种系 AR3C 类 bNAb 前体表达为 B 细胞受体。此外，我们还确定了三种 AR3C 类 bNAb 中的关键特征，这些特征代表了 AR3C 类 bNAb 的两个子类，从而可以进行精细的蛋白质设计。这些结果为针对 HCV 的种系靶向疫苗设计策略提供了框架。