Abstract Evaluating curative interventions in older people on antiretroviral therapy (ART) is challenging due to the complex interplay between HIV infection, aging, and age-related comorbidities. We have recently shown in 14 people with HIV (PWH) receiving long-term ART that there are three patterns of intact proviral DNA decay: 1) a biphasic decline with markedly slower second phase decline; 2) an initial decline followed by a zero-slope plateau; and 3) an initial decline followed by later increases in intact proviral DNA. In this study, the two participants who had an increase in intact proviral DNA were females >60 years old and had been on ART for up to 20 years. This important result raises the question whether age is associated with a slowing decay in intact proviral DNA among PWH on long-term ART. Accordingly, we will extend these preliminary observations to a large longitudinal cohort (ACTG A5321) of men and women with a median duration of ART of 14 years (IQR 12 – 17) (range 5-24 years) and archived blood samples every six months. In addition, cellular senescence is also a factor that could affect HIV persistence as people age. Senescent cells are in a state of cell-cycle arrest and acquire the senescence associated secretory phenotype (SASP) resulting in an inflammatory state. If this inflammatory environment created by SASP leads to proliferation of HIV-infected cells, then senescent cells may play a role in slowing or reversing the decay of intact proviruses. The central hypothesis of this proposal is that markers of HIV persistence are more likely to slow their decline, or increase, with age on ART, in association with increasing markers of immune senescence. To address this hypothesis, we will 1) assess the longitudinal relationship between age/sex and the levels and trajectory of HIV-1 reservoirs on ART, and 2) determine the longitudinal relationships between the levels and trajectory of HIV persistence markers and SASP markers. The duration of ART in this ACTG A5321 cohort is much longer than in many other studies as participants have been in continuous follow-up since initiation of ART and have well-documented virologic suppression for approximately 20 years with detailed clinical, laboratory, reservoir data and additional blood samples from immediately pre-ART through the present day. We will evaluate 100 A5321 participants over multiple timepoints for levels of intact proviral DNA, total DNA, residual HIV-1 RNA using the single copy assay and assess the relationships between levels of these persistence markers and age/sex. In a subset of 25 participants, we will perform bulk integration site analysis to determine the clonality of cells carrying proviruses as a function of age. We will also measure a panel of SASP markers using a multiplex platform using samples from the 100 participants in Aim 1 to assess whether the measures of HIV persistence in Aim 1 are associated with the markers of senescence.