Project summary The long term goal of this application is to study HIV-1 reservoirs that persist in CNS and to understand the role of illicit drugs in establishing and reactivating the latent reservoirs. Persistence of latently infected cells in CNS poses a major barrier for HIV-1 eradication. Current strategies to eliminate the latent reservoirs include a “shock and kill” therapy and is aimed at peripheral blood, which constitutes only 1% of the total reservoirs. Whether it is possible to envision similar strategies for the eradication of HIV-infected CNS cells is currently unknown. In addition to the lack of knowledge about latency in CNS HIV+ cells and the effects of latency reversing agents, illicit drugs common within the HIV-infected populations constitute a further complexity. Many illicit drugs are known to stimulate HIV-1 replication. Since the current method to measure the peripheral blood HIV reservoir is not applicable to solid tissues or to cells that replicate poorly such as macrophages, microglia and astrocytes found in the brain. We have developed a novel, Single cell-single molecule, Multiplex, Immunofluorescence (IF) and RNA FISH-based Assay (SMIRA) to detect cells in which HIV-1 is actively replicating. Using automation, a large number of cells can be scanned. In this proposal, we will employ this novel method to first quantitate and characterize latency in cell line models, then the latent reservoirs in brain derived microglia and astrocytes and delineate the effect of three drugs of abuse (methamphetamine, cocaine and cannabis) on the efficiency of formation of latent cells, the rate of reactivation of latent cells, and establishment of latency across blood-brain-barrier (BBB). Three Aims are proposed. In Aim 1, we will study the role of illicit drugs on the reactivation of latently infected CNS-derived cells by establishing in vitro and ex vivo models of latency. First, we will employ immortalized human monocyte and microglial cell lines to study the kinetics of reactivation and the effect of METH, Cocaine and cannabis on the reactivation using SMIRA. This will be followed by similar studies using a primary CNS cells. In Aim 2, we will study the role of illicit drugs on the establishment of HIV reservoirs in CNS cells across BBB. Using SMIRA to detect HIV-1+ cells, we will determine the relative effect of drugs of abuse, antiretrovirals and the addition of LRAs to establish latency across BBB. In Aim 3, we will study the effect of illicit drugs on CNS HIV reservoirs in HIV-infected individuals. Employing brain autopsy samples from HIV+ individuals with or without illicit drug use history (from the Manhattan Brain Bank (NNTC), we will characterize the relative frequency of microglial and astrocyte latent cell r...