ABSTRACT – PROJECT 2 Congenital cytomegalovirus (cCMV) infection is the leading infectious cause of congenital defects and the development of vaccines and treatments to prevent cCMV have long been recognized as top public health goals. Unfortunately, vaccines targeting human CMV (HCMV) glycoproteins or based on attenuated HCMV have not yet yielded a licensed vaccine, most likely since they were unable to provide immunity against infection that is better than the limited immunity provided by natural infection. The overall goal of this project is to explore a novel and innovative approach to prevent cCMV infection by targeting the function of viral chemokine-like proteins. This approach is based on the suprising finding that rhesus CMV (RhCMV) lacking the UL146 family of viral CXC chemokine-like proteins shows highly limited dissemination, reduced plasma viremia and delayed shedding during primary infection of rhesus macaques (RM). Since viral dissemination is essential for congenital infection, we hypothesize that UL146 family members are required for transmission to the fetus, and that interfering with their role in dissemination could prevent congenital infection. The goals of this project are therefore to identify and characterize the UL146 family proteins required for dissemination and congenital infection and to develop UL146 family targeting strategies to prevent congenital infection. In Aim 1 we will determine which of the six RhCMV UL146 family proteins are required for dissemination, and whether HCMV UL146 and/or UL147 can substitute for their RhCMV homologs. Since this family displays homology to host chemokines, we will also examine the chemokine structural requirements for dissemination. We will begin characterizing possible mechanisms of dissemination by studying UL146-mediated spreading in placental organoid cultures and by characterizing how the cellular microenvironment is affected by viral chemokines using immunohistochemistry and single cell transcriptomics and proteomics. In Aim 2 we will examine dissemination of UL146 family-deleted RhCMV to tissues upon intravenous inoculation during primary infection and transmission to the fetus in CD4+ T cell depleted dams. We will additionally examine whether the minimal set of UL146 family members required for dissemination can restore congenital infection. In Aim 3 we will evaluate whether UL146 family proteins are immunogenic during primary infection and whether immunization against the minimal set of UL146 family members required for dissemination will limit dissemination upon primary infection. Finally, we will determine whether IgG from these immunized animals can protect against congenital infection, either on their own or in combination with IgG treatments evaluated in Project 3. We expect that a limited number of UL146 family members support viral dissemination and congenital infection and that anti-UL146 immunity can limit dissemination and congenital infection. By providing a be...