The design of an AIDS vaccine is complicated by the fact that any HIV/SIV-specific immune responses aimed at preventing transmission or disease progression inevitably result in the generation of activated CD4+ T cells that may paradoxically facilitate transmission and/or disease progression. Indeed, increased HIV acquisition in the ineffective Step/Phambili trials and the lack of efficacy in HVTN-505 indicate that vaccine-elicited CD4+ T-cell responses can mitigate protection and, in some cases, increase acquisition. The overarching goal of this Program Project grant is to test the hypothesis that durable and balanced HIV-specific humoral immune responses are needed to provide effective protection from mucosal challenge, and avoid paradoxical effects that may enhance virus acquisition. This project focuses on the question of whether vaccine-induced changes in the mucosal micro-environment are factors influencing, and often undermining, the protective efficacy of humoral immunity in rhesus macaques (RMs) and by extension, humans. Three main aspects of the mucosal environment will be examined: (1) the role of activated CD4+ T cells as potential targets for the virus; (2) the role of the innate immune response, and in particular type I interferon (IFN-I) and interferon-stimulated gene (ISG)- mediated pathways, as antiviral but also pro-inflammatory factors; and (3) the potential role of abortive infection and induction of pyroptotic cell death in quiescent mucosal CD4+ T cells as a source of inflammatory/activating signals facilitating spread of the transmitted founder virus populations. The studies proposed in in this project leverage animals and samples from Project 1 (which will elucidate the humoral determinants for persistent anti- gp120 responses) and Project 2 (in which the protection conferred by passive administration of an anti-gp120 antibody will be tested in the setting of concomitant exposure to a vector-based vaccine known to induce strong mucosal cellular immune responses to HIV/SIV). There are three specific aims. Aim 1: To examine how the in vivo interventions in Projects 1 and 2 affect the number, phenotype, activation state, histological location, and gene expression of mucosal CD4+ T cells. Aim 2: To examine how the in vivo interventions in Projects 1 and 2 affect the innate immune environment of mucosal tissues, with specific focus on IFN-I and ISGs. Aim 3: To determine whether abortive infection and inflammatory pyroptosis occur in mucosal CD4+ T cells of vaccinated macaques and promote expansion and dissemination of SHIV infection. This work will provide a comprehensive picture of the mucosal micro-environment in the macaque vaccine model, within the context of settings where protective anti-gp120 antibodies are present. This information will validate hypotheses regarding the causal relationships between balanced mucosal immune profiles and protective efficacy via humoral immunity. Such information will have significant translational impact on the development of future HIV vaccine strategies.