PROJECT 1. Virology, Epigenetics, Integration (VENI) Research Project - ABSTRACT Person-to-person variability in the size, distribution and dynamics of the viral reservoir is substantial. There is also considerable heterogeneity within a person with HIV (PWH) across their tissues. Thus, a ‘one size fit all’ HIV cure strategy will likely fail, as each reservoir is governed by different viral and host factors in its HIV Obligate MicroEnvironment (HOME). In response to the Understanding HIV Reservoir Dynamics RFA (AI-21-013), our overall HOME project is designed to elucidate the viral and host mechanisms governing reservoir dynamics throughout the body of PWH on and off antiretroviral therapy (ART). Our “Virology, EpigeNetics, Integration” (VENI) Research Project (RP) will focus on the viral factors that govern the continuum of HIV reservoir dynamics in PWH including: HIV reservoir size and genetic composition, HIV integration landscape, tissue density of clonally expanded virus, epigenetic marks and proviral chromatin accessibility of intact provirus. (The complementary VIDI RP will evaluate the cellular, immune, drug, human epigenetics and architectural HOMEs, and the VICI RP will integrate and analyze all data.) For experimental planning, the HIV reservoir dynamic continuum is simplified as the following reservoir states on and off ART: · Leaves HOME when HIV (re)activates from tissues during ART (i.e., ‘ready to move once ART is interrupted’, like packing its bag and getting ready to leave) and causes rebound viremia during ART interruption. · Comes HOME when HIV (re)populates tissues during viremia off ART and through the spread of clonally expanded HIV-infected cells while on ART. (Clonal expansion is like adding family to the home.) · Stays HOME when HIV persists in tissue reservoirs during ART and viral suppression in plasma. The feasibility of our VENI RP is greatly enhanced by the knowledge gained from our previous P01 (AI131385) and its Last Gift Cohort, which collects pre- and post-mortem specimens from PWH who did (n=5) or did not stop ART (n=15) before death. The HOME program will allow the continuation of this unique cohort while greatly expanding the science to study HIV reservoirs across the human body at the single genome and cellular level. To achieve our objectives and optimize allocated resources, we developed an Adaptive Learning strategy that consists of Mapping and Confirmation phases. During the Mapping phase, the VENI RP will map all samples for HIV reservoir size, composition, and transcriptional activity, and the VIDI RP will map the ART and cellular milieus in these samples. In the Confirmation phase, viable cells from the samples-of-interest will be more deeply characterized by the VENI RP to determine viral and provial epigenetic factors at the single genome level associated with pre-defined reservoir states (i.e., leaving, coming, staying HOME). During analysis of these data, the VICI RP will make use of data from b...