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The objective of this study is to evaluate the impact of therapeutic HIV vaccination on the HIV reservoir and assess the relationship of the viral reservoir with HIV-specific immune status and viral rebound kinetics.A retrospective analysis of ACTG A5197, a randomized, placebo-controlled trial of a therapeutic rAd5 HIV-1 gag vaccine.Participants received vaccine/placebo at weeks 0, 4 and 26 prior to a 16-week analytic treatment interruption (ATI) at week 38. Cell-associated HIV-1 RNA and DNA (CA-RNA and CA-DNA) and HIV-1 residual viremia were quantified at weeks 0, 8 and 38. HIV-specific CD4+/CD8+ activity was assessed by an intracellular cytokine staining assay.At study entry, CA-RNA and CA-DNA levels were correlated inversely with the numbers of HIV-specific CD4+ interferon-γ producing cells (CA-RNA: r = −0.23, P = 0.03 and CA-DNA: r = −0.28, P < 0.01, N = 93). Therapeutic HIV vaccination induced HIV-specific CD4+ activity, but did not significantly affect levels of CA-RNA or CA-DNA. Vaccine recipients with undetectable residual viremia at week 8 had higher frequencies of HIV-specific CD4+ and CD8+ interferon-γ producing cells (undetectable versus detectable residual viremia: 277 versus 161 CD4+ cells/106 lymphocytes, P = 0.03 and 1326 versus 669 CD8+ cells/106 lymphocytes, P = 0.04). Pre-ATI CA-RNA and CA-DNA were associated with post-ATI plasma HIV set point (CA-RNA: r = 0.51, P < 0.01 and CA-DNA: r = 0.47, P < 0.01).Vaccine-induced T-cell responses were associated with a modest transient effect on residual viremia, but more potent immune responses and/or combination treatment with latency-reversing agents are needed to reduce the HIV reservoir. HIV reservoir measures may act as biomarkers of post-ATI viral rebound kinetics.NCT00080106.