ABSTRACT - OVERALL Congenital cytomegalovirus (cCMV) is a prevalent in utero infection, affecting approximately 1 in 200 newborns and leading to severe neurological impairment in 1 in 5 infected infants. Maternal CMV-specific adaptive immunity is partially protective against cCMV infection, with reduced transmission rates observed in chronically-infected women upon reinfection. However, challenges in CMV vaccine development include limited understanding of viral and immunological factors involved in cCMV transmission, strategies to counteract immune evasion mechanisms, and critical vaccine antigen targets. To address these challenges, our team developed and refined a nonhuman primate (NHP) model of cCMV infection in rhesus monkeys, which we demonstrated to closely mimic human transmission and fetal disease rates in our initial cycle of this P01 Program. Surprisingly, immune responses following maternal infection did not predict transmission risk, suggesting the need for pre-existing immunity different from natural infection. The NHP model also revealed T cell trafficking to the maternal-fetal interface following maternal CMV infection, the limited role of the viral pentameric glycoprotein complex in cCMV transmission, the role of Fc-mediated antibody responses in CMV containment, and viral immune evasion mechanisms as important factors in reducing CMV dissemination. Traditional vaccine approaches focused on neutralizing antibodies may be insufficient, prompting the evaluation of novel CMV vaccine candidates in the NHP model. We hypothesize that disarming the virus by eliciting immunity against key viral immune evasions and inducing cellular immunity at the maternal-fetal interface will prevent cCMV transmission following primary infection during pregnancy. The studies proposed in the renewal of this P01 Program will assess the role of CD8+ T cell responses at the maternal-fetal interface in preventing cCMV transmission (Project 1), the impact of vaccine-induced immunity against viral immune evasions, such as UL146 chemokine homologs (Project 2) and viral Fc receptors (Project 3), and test three novel vaccines targeting each of these novel immune mechanisms. The Program's ultimate goal is to develop an effective CMV vaccine, considering the limitations of previous approaches and targeting immune evasion proteins while eliciting local immunity at the maternal-fetal interface. The research will employ NHP models, placental organoid models, viral engineering, sequence analysis, and bioinformatics to de-risk these vaccine concepts before advancing to human efficacy trials. The successful development of a CMV vaccine is crucial for eliminating the most common infectious cause of birth defects and brain damage worldwide.