Abstract Antiretroviral treatment (ART) has dramatically improved outcomes for children living with HIV (CHIV) but poses challenges including poor adherence, side-effects, stigma, and drug resistance. An HIV cure would address these challenges but has been elusive due to the latent HIV reservoir that persists despite ART. To advance pediatric HIV cure, it is important to identify determinants and consequences of the pediatric HIV reservoir. Recent adult studies have shown differences in the decline of total versus intact HIV reservoir and have demonstrated antigen-related clonal proliferation of the reservoir, with enriched HIV provirus in memory T- cells responding to CMV, EBV, HIV, influenza, or tetanus antigens. Acute CMV frequently occurs concurrent with HIV infection in infancy and elicits a substantial immune response; this may result in increased HIV-proviral infection of CMV-responsive immune cells. Indeed, we recently found that earlier and cumulative CMV viremia were associated with increased HIV total reservoir size in CHIV. There are few epidemiologic studies to estimate population-level impact of antigen-specific proliferation; and scant data from pediatric cohorts on intact HIV reservoir. In addition to impeding eradication of HIV, the HIV reservoir may also influence neurocognitive outcomes due to the trafficking between systemic and CNS compartments potentially via HIV reservoir- containing immune cells. We have had 4 cycles of a renewed NIH R01 (NICHD R01-023412). Cycle 3 of the R01 involved a cohort of early-treated Kenyan infants (OPH cohort) which we have continued to follow-up to examine HIV reservoir. Our team has optimized a novel intact proviral DNA assay (IPDA) for Kenyan subtypes which provides quantitative estimates of intact and defective virus. Building on this work, we propose a competitive renewal of our R01 to examine the influence of cumulative and episodic CMV/EBV activation on the intact HIV reservoir and the relationship between HIV reservoir and long-term neurocognitive outcomes. Aim 1. Compare reservoir clonal dynamics in children who initiated ART during concurrent acute HIV/ acute CMV infection (early-ART) to children who initiated ART during chronic HIV/CMV (late-ART) and to mothers who initiated ART during chronic HIV/CMV. Aim 2. Quantify the influence of CMV/EBV antigenic stimulation on HIV intact/defective reservoir size in longitudinal molecular epidemiologic studies over >15-year follow-up. Aim 3. Determine influence of total and intact HIV reservoir on long-term neurocognitive outcomes. These aims will provide novel data on the pediatric HIV reservoir that will inform strategies for HIV cure and to optimize neurocognitive outcomes in CHIV.