Institute for Bioscience and Biotechnology Research, University of Maryland, Rockville, MD 20850.
Department of Cell Biology and Molecular Genetics, University of Maryland, College Park, MD 20742.
Proc Natl Acad Sci U S A. 2021 Jan 19;118(3). doi: 10.1073/pnas.2015149118.
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）是全球范围内的主要健康负担，需要开发预防性疫苗来控制或消除这种病毒。该病毒的高度变异性是开发有效 HCV 疫苗的一个重大障碍，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 跨膜结构域使得难以生产出同质可溶性形式的复合物，这种形式能够反映其在病毒包膜上的状态。为了能够对 E1E2 疫苗进行合理设计以及进行结构特征描述，我们设计了一种可溶性、分泌型的 E1E2（sE1E2）。与其他病毒的可溶性糖蛋白设计一样，它包含一个支架，在没有跨膜结构域的情况下强制组装，同时还包含一个弗林切割位点，以允许类似天然的异二聚化。与全长 E1E2 相比，这种 sE1E2 组装成的形式更接近预期的大小。通过与一系列构象特异性单克隆抗体（包括两种针对天然 E1E2 及其主要受体 CD81 的中和抗体）的高亲和力结合，证实了其保留了天然结构元件。最后，sE1E2 在体内能够引发强大的中和抗体。这种设计的 sE1E2 不仅可以深入了解天然 E1E2 组装的决定因素，还可以作为生产未来结构和疫苗研究用 E1E2 的平台，从而能够对基于 E1E2 的抗原进行合理优化。
