Characterize replication competent myeloid reservoirs in the central nervous system

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SUMMARY The eradication of HIV-1(HIV) infection must address all tissues where infection persists. Clearance of infection in the central nervous system (CNS) is not achieved by antiretroviral therapy (ART) alone, just as in the periphery. Latent HIV reservoirs in the CNS may allow viral rebound upon discontinuation of ART, as HIV can egress from the brain into the peripheral blood. However, studies are needed to elucidate the contributions of subsets of latently infected CNS cells to ongoing HIV persistence in the CNS. Circulating T cells have been well characterized as the major HIV reservoirs in the peripheral blood, and may circulate into the CNS, contributing to HIV persistence in the brain. Nevertheless, it is still not known whether T cells are the only major viral reservoir in the CNS. Myeloid cells are a major cellular compartment of the immune system infected by HIV in the brain. In vivo imaging and immunostaining studies have revealed that brain myeloid cells (BMCs) harbor HIV DNA and produce HIV RNA and proteins. However, it is not clear whether BMCs, and especially long-lived microglia, are latently infected, and if they are true HIV reservoirs encoding replication-competent HIV despite durable, successful ART. Our team at the UNC HIV Cure Center has recently established a protocol to isolate highly pure myeloid cells from the brain of SIV-infected macaques, in whom ART was interrupted. These SIV containing BMCs can be cultured for many generations ex vivo. Further application of this technology to other fully ART-suppressed animals found that BMCs contain proviral SIV DNA, and that SIV RNA can be effectively induced by latency reversal agents. This platform to study myeloid cells ex vivo allows rigorous characterization of brain myeloid cells to address their role as true HIV reservoirs that can produce replication- competent viruses. Our preliminary data highlights that epigenetic regulation, such as histone deacetylation and histone methylation, may play an essential role in the modulation of HIV latency in the CNS. We hypothesize that BMCs are latently infected by HIV and harbor replication competent HIV. In this proposal, we will isolate highly pure brain myeloid cells from different regions of the brain in both people living with HIV (PLWH) enrolled in the “Last Gift” cohort and fully ART-suppressed SIV-infected rhesus macaques. We will examine whether BMCs harbor replication-competent HIV or SIV using the intact proviral DNA assay (IPDA) and quantitative viral outgrowth assay (QVOA) (Aim1). We will examine whether latent HIV in myeloid cells responds to the currently available latency reversal agents related to epigenetic regulation of HIV latency (Aim 2), which may provide clues to the mechanisms that allow HIV latency in the brain. Our study will address critical gaps in knowledge to better understand whether BMCs serve as true HIV reservoirs and how HIV persists in these CNS cells.