Vertical transmission of cytomegalovirus (CMV) is the single most common congenital infection worldwide, often resulting in deafness and neurodevelopmental delay for afflicted children. CMV related neurologic complications are more frequent and severe following primary maternal infection during pregnancy, and therefore a vaccine to prevent maternal acquisition of CMV during pregnancy is the leading strategy to reduce the incidence of congenital CMV disease. The most successful maternal immunization regimen tested to date is a CMV glycoprotein B (gB) subunit vaccine combined with MF59 adjuvant, which achieved a promising 50% efficacy in multiple phase II clinical trials. However, efforts to improve this vaccine have been slowed by an incomplete understanding of the determinants of protection provided by this moderately-effective vaccine. Previous studies and recent work in our nonhuman primate model of congenital CMV transmission have indicated that maternal CMV-specific antibody responses modulate the risk of congenital CMV infection. Yet there remains major a gap in knowledge impeding further vaccine development on: (1) what antibody functions must be elicited by an efficacious vaccine to prevent congenital CMV and (2) whether antibody responses targeting the gB protein alone, and not other CMV glycoproteins, are sufficient for an effective vaccine. The development of novel, high-throughput methods within the vaccine immunology field has made it possible to investigate epitope targets/antibody functions protective of CMV acquisition as well as mechanisms of viral immune evasion. Interestingly, gB/MF59-elicited plasma antibodies were generally non-neutralizing, and therefore vaccine-induction of neutralizing antibodies does explain the 50% partial efficacy observed in human clinical trial. Thus, we hypothesize that the efficacy of gB/MF59 vaccination is dependent upon non- neutralizing antibody effector functions and viral immune evasion of the gB-targeted responses. We will first identify the fine specificities, properties, and effector functions of vaccine-elicited antibodies in gB/MF59 recipients using multiplex ELISA, linear peptide binding, Fc glycosylation/FcR binding, and assessment of non- neutralizing activities (ADCC and ADCP). Second, through deep sequencing of viral variants and subsequent viral sieve analysis, we will investigate sequence diversity at the loci of gB antigenic epitopes to determine if viral immune evasion of antibody responses is associated with vaccine failure. This work will address research priorities for CMV vaccine development, and will thus set the stage for rational design of a future glycoprotein- based vaccine for the elimination of congenital CMV infection.