Structure, antigenicity, and function of HCMV fusogen gB

项目总结/摘要 人巨细胞病毒（HCMV）是一种非常成功的人类病原体，感染了世界上大部分地区， 人口终身。虽然通常无症状，但HCMV感染可导致严重的播散性疾病， 免疫功能低下的患者，例如，移植受体和新生儿发育异常。 因此，迫切需要预防病毒传播的药物，特别是从母亲到胎儿。 HCMV糖蛋白B（gB）是病毒的一种重要穿透蛋白，也是宿主防御的主要抗原。 不出所料，gB是目前所有HCMV候选疫苗的组分。然而，疫苗的努力是 至少部分是由于对gB特异性免疫反应的不完全理解而受阻。而且 gB对膜融合的机制贡献需要进一步澄清。我们知识上的这些空白 对gB功能和抗原性的研究主要源于对无活性的融合后形式的依赖 不能充分再现活性的融合前形式的gB。我们成功地设计了 融合前形式的可溶性HCMV gB胞外域。在这里，我们建议描述其结构， 抗原性和控制其活性的机制。在目标1中，我们将确定高分辨率 融合前HCMV gB及其与已知中和抗体的复合物的结构。我们还将 稳定融合前gB用于下游应用（生物化学研究和免疫原开发）。在 目的2：从HCMV感染者中分离和鉴定融合前gB特异性中和抗体， 捐助者。在目标3中，我们将研究可能抑制gB在其融合前形式的调节机制。的 该提案是创新的，因为据我们所知，这是第一次将可溶性融合前形式的HCMV gB 被成功地设计出来。这个建议意义重大，因为它将产生新的机械知识， 提供新的试剂，可以帮助开发最佳疫苗和被动免疫 针对HCMV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.