Current methodologies to quantify latent HIV-1 infection demand high cell numbers, require repeated leukapheresis of subjects and highly expert molecular biology techniques. For HIV cure regimens to reach the clinic, efficient and high-throughput diagnostic assays will be needed to confirm disruption of latent HIV-1 infection, preferably while patients continue antiretroviral therapy (ART). This proposal will examine whether HIV-1 specific CD8 T cells, which are highly sensitive biomarkers of HIV-1 viremia, can be used as a diagnostic measure of HIV-1 reactivation, even in the presence of ART. To examine whether the HIV-1 specific CD8 T cell response will be upregulated by HIV-1 protein expression that follows the disruption of HIV latency, studies will use flow cytometry to firstly measure variability of HIV-1 specific CD8 T cell frequency and phenotype over time in durably ART-treated, HIV-1 infected subjects. Statistical testing will then be performed to calculate baseline variability and define assay criteria to detect significant differences in response frequencies and specificities. Sensitivity of T cell assays in patient samples will be examined firstly by in vitro studies, treating resting CD4 T cells isolated from ART treated aviremic subjects with/or without HDAC inhibitors to assess whether treatment and in vitro reactivation induces autologous HIV-1 specific CD8 T cell proliferation. Those T cell assays/parameters that are most sensitive for detection of reactivation will then be tested using samples from subjects before and after receipt of vorinostat. A key challenge to the development of a CD8 reactivation assay is sensitivity because latently infected cells number only a few in a million. In parallel with studies of patient samples, in vitro HIV-1 infection and latency models will be established. HIV-1 CD8 T cell clones will be examined for their ability to detect low multiplicity of HIV-1 infection with and without anti-retroviral drugs that block different HIV-1 replication pathways. Models of HIV- 1 latency in CD4 T cells will also be examined for their ability induce CD8 T cell responses. From this work, we will develop statistical and mathematical models to predict in vivo T cell sensitivity to low frequency reactivation of latently infected cells.