Project SummarySecreted extracellular vesicles (EVs) may play a role in biological processes and disease pathogenesis. Impactof these EVs on Human Immunodeficiency Virus type 1(HIV-1) infection has only recently started to beinvestigated. In fact, EVs such as exosomes have been shown to influence cells within the central nervoussystem (CNS) and modulate immune responses to pathogens. The HIV Negative factor (Nef) is released fromnef-transfected or HIV-infected immune cells in exosomes, extracellular nano-sized vesicles generally used forpara- or intercellular communication – delivery of antigen, modification of gene expression, and modulation ofimmune responses. Interestingly, microglia infected with HIV or transfected with a nef-gfp expression plasmidrelease Nef in exosomes. However, the role this extracellular exosomal Nef (exNef) may have in HIV replicationwithin the CNS and neuropathogenesis is unknown. It is known that HIV infects cells within the brain, persistswithin the CNS despite successful combination anti-retroviral therapy (cART), and causes neurocognitiveimpairments such as HIV-associated Neurological Disorder (HAND). Although cART significantly lowersperipheral viral load to undetectable levels(aviremia), HAND is still observed in among 40% of virally suppressedHIV+ individuals. Together these findings suggest that in the presence of cART a novel mechanism notassociated with the HIV is at play to induce neurocognitive impairment. Substance abuse could also play a keyrole in HIV disease progression and the onset of neurocognitive impairment. Opiates such as heroin, and itsactive metabolite morphine have been shown to increase the rate of HIV disease progression to NeuroAIDS andincrease both the risk and severity of HAND in people living with HIV/AIDS (PLWHAs). Given that almost one-third of PLWHAs report heroin abuse, it is important to understand how opiates and cART interplay in HIVdisease to cause neurocognitive impairment in order to improve the quality of life for these HIV+ individuals. Wehypothesize that opiate-induced modifications in Nef+ EV composition and release exacerbates exNefassociated neuronal damage and leads to greater neurocognitive impairment in the context of cART. Inthis proposal we will investigate in the context of cART and opiates, the impact of extracellular vesicles,specifically exNef released from HIV-infected (or nef- transfected) microglia on neurons in order to understandthe mechanism(s) that underlie HIV-induced neurocognitive impairment/HAND during aviremia. We will alsoperform a cross-sectional study comparing cerebral spinal fluid(CSF) exNef in PLWHAs on cART with ahistory/current opiate use and neurocognitive impairment/HAND. Findings from this proposal will allow us todemonstrate the role of EVs in the neuropathogenesis induced by the interplay of opiates and HIV in the CNS.