© 2016 Wolters Kluwer Health, Inc. Background: The causes of HIV-vaccines failure are poorly understood. Therapeutic vaccination with modified vaccinia Ankara (MVA)-B in HIV-1-infected individuals did not control the virus upon analytical treatment interruption (ATI). We investigated whether the functional characteristics of HIV-specific CD8+ T-cell responses stimulated by this vaccine, and the level of exhaustion of these cells might explain these results. Methods: Twenty-one HIV-1 chronically infected patients on combination antiretroviral therapy, included in the therapeutic vaccine trial RISVAC03, were studied: 13 immunized and eight controls. Functional characteristics, cytotoxic potential and exhaustion of HIV-specific CD8+ T cells, were evaluated by polychromatic flow cytometry. Differences between groups were tested using nonparametric tests. Results: MVA-B vaccine induced an increase in HIV-specific CD8+ T-cell response, but also increased their levels of exhaustion. At week 18 (following three immunizations) the level of response increased with respect to baseline (P=0.02). A significant increase at weeks 18 and 24 (ATI) in granzyme B content was also observed. Interestingly, an increase in expression of exhaustion markers was found at weeks 18 (P=0.006) and 24 (P=0.01). However, there was no significant change in the functional profile of vaccine-induced CD8+ cells. At week 36, in parallel to the rebound of plasma viremia after 12 weeks ATI, a significant increase in the level of CD8+ response, in granzyme B content and in exhaustion markers expression, was observed in both groups. Conclusion: We show that therapeutic vaccination with MVA-B tilts the balance between activation and regulation of the response of HIV-specific CD8+ T cells towards regulation, which impacts on the viral rebound after ATI.