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.