Structure, antigenicity, and function of HCMV fusogen gB

项目摘要/摘要 人巨细胞病毒(HCMV)是一种非常成功的人类病原体，可感染世界上大部分地区的 终生的人口。虽然通常没有症状，但巨细胞病毒感染可在 免疫功能受损的患者，如移植受者，以及新生儿发育异常。 因此，迫切需要预防措施来防止病毒传播，特别是从母亲到胎儿。 人巨细胞病毒糖蛋白B(GB)是一种关键的病毒穿透蛋白，也是宿主防御的优势抗原。 不出所料，GB是目前所有候选HCMV疫苗的组成部分。然而，疫苗的努力是 至少在一定程度上，是因为对gb特异性免疫反应的不完全理解。此外， GB对膜融合的作用机理需要进一步阐明。我们知识中的这些鸿沟 GB功能和抗原性在很大程度上源于对不活跃的融合后形式的研究 不能充分概括主动的、再灌流形式的GB。我们已经成功地设计了 再融合形式的可溶性人巨细胞病毒gB胞外区。在这里，我们建议描述它的结构， 抗原性，以及控制其活性的机制。在目标1中，我们将确定高分辨率 人巨细胞病毒gB及其与已知中和抗体的复合体的结构。我们还将 为下游应用(生化研究和免疫原开发)稳定输注GB。在……里面 目的2.从HCMV阳性血清中分离和鉴定预融合特异性中和抗体 捐赠者。在目标3中，我们将研究可能抑制GB预融合形式的调控机制。这个 这项提议之所以具有创新性，是因为据我们所知，这是第一次将人巨细胞病毒gb的可溶性预融合形式 被成功地设计出来。这项提议意义重大，因为它将产生新的机械知识和 提供可帮助开发最佳疫苗和被动免疫的新试剂 应对巨细胞病毒的策略。

PROJECT SUMMARY/ABSTRACT Human cytomegalovirus (HCMV) is a highly successful human pathogens that infects much of the world’s population for life. While often asymptomatic, HCMV infections can cause severe disseminated disease in immunocompromised patients, e.g., transplant recipients, and developmental abnormalities in neonates. Hence, there is an urgent need for prophylactics to prevent viral transmission, especially from mother to fetus. HCMV glycoprotein B (gB) is both a key viral penetration protein and a dominant antigen for host defense. Unsurprisingly, gB is a component of all current HCMV vaccine candidates. However, vaccine efforts are stymied, at least in part, by an incomplete understanding of gB-specific immune responses. Moreover, the mechanistic contributions of gB to membrane fusion require further clarification. These gaps in our knowledge of both gB function and antigenicity largely stem from the reliance of research on the inactive, postfusion form of gB that cannot adequately recapitulate the active, prefusion form. We have successfully engineered soluble HCMV gB ectodomain in a prefusion form. Here, we propose to characterize its structure, antigenicity, and the mechanisms that control its activity. In Aim 1, we will determine the high-resolution structures of the prefusion HCMV gB and its complexes with known neutralizing antibodies. We will also stabilize prefusion gB for downstream applications (biochemical research and immunogen development). In Aim 2, we will isolate and characterize prefusion-gB-specific neutralizing antibodies from HCMV-seropositive donors. In Aim 3, we will investigate the regulatory mechanism that may restrain gB in its prefusion form. The proposal is innovative because, to our knowledge, this is the first time the soluble prefusion form of HCMV gB was successfully engineered. The proposal is significant because it will yield new mechanistic knowledge and provide new reagents that could aid in the development of optimal vaccines and passive immunization strategies against HCMV.