PROJECT SUMMARY While the advent of antiretroviral therapy (ART) has dramatically reduced the morbidity and mortality associated with HIV infection, a cure is not achieved due to the persistence of latently-infected cells during treatment. Accumulating data suggest that HIV-infected individuals often experience persistent immune dysregulation, chronic inflammation, and accelerated aging even in the setting of ART-mediated viral suppression. These realities have created a pronounced interest in developing strategies to cure HIV infection. Identifying the host molecular determinants of HIV persistence and rebound kinetics following cessation of antiretroviral therapy (ART) will be critical in developing effective strategies to cure HIV infection. We recently applied a comprehensive systems profiling approach to plasma samples from HIV-infected individuals who underwent analytical treatment interruption (ATI), to identify circulating host factors that enable prediction of HIV rebound kinetics following ART interruption, and serve as potential pharmacological targets to promote durable virologic remission in the absence of ART. The host factor exhibiting the strongest statistical association with HIV rebound timing following ART cessation was circulating cell-free DNA (cfDNA) in plasma. Specifically, increased cfDNA abundance in plasma was associated with delayed time-to-rebound. cfDNA, released into circulation during programmed cell death, has been exploited extensively in the realm of clinical oncology (often termed “liquid biopsy”). However, cfDNA remains largely unexplored in the setting of HIV infection. In this R01 proposal, we rigorously explore cfDNA as a biomarker of HIV disease states, and we investigate the molecular and immunologic mechanisms linking cfDNA to viral expression and rebound. Our central hypotheses are that 1) cfDNA reflects the death rate of HIV-infected cells in vivo; and 2) cfDNA promotes an antiviral state in the host (e.g. induction of type I interferon response) which prevents viral rebound. Our project features an investigative team with basic, translational, and clinical expertise, and leverages large collections of clinical samples from well-characterized HIV-infected individuals. In Aim 1, we will apply next-generation sequencing (NGS) approaches to plasma samples from HIV-infected individuals to extend and enhance the prognostic significance of cfDNA as a biomarker of natural HIV control in vivo. In Aim 2, we will evaluate how the uptake and sensing of cfDNA by HIV target cells impacts cell fate and the reactivation and replication of HIV. In Aim 3, we will determine how HIV-associated mitochondrial dysfunction leads to cfDNA extrusion and ultimately induces type I interferon responses that prevent HIV rebound. Our project will advance the development and evaluation of HIV cure strategies.