Hepatitis C virus (HCV) is a major worldwide health burden, and a preventive vaccine is needed for global control or eradication of this virus. A substantial hurdle to an effective HCV vaccine is the high variability of the virus, leading to immune escape. The E1E2 glycoprotein complex contains conserved epitopes and elicits neutralizing antibody responses, making it a primary target for HCV vaccine development. However, the E1E2 transmembrane domains that are critical for native assembly make it challenging to produce this complex in a homogenous soluble form that is reflective of its state on the viral envelope. To enable rational design of an E1E2 vaccine, as well as structural characterization efforts, we have designed a soluble, secreted form of E1E2 (sE1E2). As with soluble glycoprotein designs for other viruses, it incorporates a scaffold to enforce assembly in the absence of the transmembrane domains, along with a furin cleavage site to permit native-like heterodimerization. This sE1E2 was found to assemble into a form closer to its expected size than full-length E1E2. Preservation of native structural elements was confirmed by high-affinity binding to a panel of conformationally specific monoclonal antibodies, including two neutralizing antibodies specific to native E1E2 and to its primary receptor, CD81. Finally, sE1E2 was found to elicit robust neutralizing antibodies in vivo. This designed sE1E2 can both provide insights into the determinants of native E1E2 assembly and serve as a platform for production of E1E2 for future structural and vaccine studies, enabling rational optimization of an E1E2-based antigen.

丙型肝炎病毒 (HCV) 是全球主要的健康负担，需要一种预防性疫苗来全球控制或根除该病毒。有效的 HCV 疫苗的一个重大障碍是病毒的高度变异性，导致免疫逃逸。E1E2 糖蛋白复合物包含保守表位并引发中和抗体反应，使其成为 HCV 疫苗开发的主要目标。然而，对于天然组装至关重要的 E1E2 跨膜结构域使得以反映其在病毒包膜上的状态的均质可溶形式生产这种复合物具有挑战性。为了能够合理设计 E1E2 疫苗以及进行结构表征工作，我们设计了一种可溶性分泌形式的 E1E2 (sE1E2)。与其他病毒的可溶性糖蛋白设计一样，它结合了一个支架以在没有跨膜结构域的情况下强制组装，以及一个弗林蛋白酶切割位点以允许类似天然的异二聚化。发现该 sE1E2 组装成比全长 E1E2 更接近其预期大小的形式。天然结构元素的保存通过与一组构象特异性单克隆抗体的高亲和力结合得到证实，包括两种特异性针对天然 E1E2 及其主要受体 CD81 的中和抗体。最后，发现 sE1E2 在体内引发强大的中和抗体。这种设计的 sE1E2 既可以深入了解天然 E1E2 组装的决定因素，又可以作为用于未来结构和疫苗研究的 E1E2 生产平台，从而能够合理优化基于 E1E2 的抗原。