SUMMARY ABSTRACT There is substantial evidence in virologically-suppressed people with HIV (vsPWH) that HIV persists in monocytes and macrophages from blood and tissues. However, despite the likelihood that monocytes and macrophages contribute to the size of the HIV reservoir, and may derail cure-based efforts, limited studies exist investigating whether HIV within monocytes and macrophages can be reactivated to produce functional virus in vsPWH. Additionally, despite known sex-based differences in the CD4 reservoir and immunological response to HIV, there are no reported studies that have assessed sex-based differences in the myeloid (monocyte/macrophage) reservoir in vsPWH. The long-term goal of this proposal is to determine if the myeloid reservoir is a significant concern for cure-based efforts. The overall objectives are to (1) determine if there are sex-based differences in the myeloid reservoir, (2) determine the stability of the myeloid reservoir in the blood (monocyte derived macrophage, MDM) and brain (CNS) of vsPWH and SIV-ART macaques and (3) further develop an easily deployable method to assess the myeloid reservoir in vsPWH. The central hypothesis of this work is that there will be significant differences in the male and female MDM and CNS reactivatable reservoirs in both HIV and SIV, and that these myeloid reservoirs will be stable throughout ART suppression. The rationale for the proposed study is that delineating the size and stability of the MDM and CNS myeloid reservoirs, and potential sex-based differences, will allow for the development of cure-based strategies that appropriately target the myeloid reservoir in men and women with HIV. The central hypothesis will be tested by pursing two specific aims: 1. Define sex-based differences in the MDM HIV reservoir in vsPWH, and 2. Define sex-based differences in the MDM and CNS reservoirs in SIV-ART macaques. These aims will utilize novel HIV or SIV specific techniques, the monocyte derived macrophage quantitative viral outgrowth assay (MDM-QVOA), the CNS macrophage quantitative viral outgrowth assay (CNS-QVOA) and a myeloid adapted intact proviral DNA assay (mIPDA), to measure the replication-competent and intact viral reservoirs in MDM (vsPWH and SIV-ART model) and the CNS (SIV-ART macaques). This research proposal is innovative because it focuses on replication-competence rather than DNA, will elucidate potential sex-based differences and address the stability of myeloid reservoirs in vsPWH and SIV-ART macaques. Finally, this proposal is significant because it will provide essential insights into the monocyte and macrophage reservoir and help to determine if HIV establishes true latency in cells of myeloid origin. Ultimately, such knowledge has the potential to alter cure-based efforts and therapeutics in men and women with HIV as the majority of current efforts are entirely T cell focused.