Recent studies on Hodgkin’s lymphoma (HL) have indicated that patients with active disease display functional impairment of Ag-specific CD8+ T cells due to expansion of regulatory T cells at sites of disease and in the peripheral blood. Adoptive cellular immunotherapy based on EBV-specific CD8+ T cells has been explored with limited success to date. It has been proposed that improved targeting of these CD8+ T cells toward viral Ags that are expressed in HL may enhance future therapeutic vaccine strategies. In this study, we have developed a novel replication-deficient adenoviral Ag presentation system that is designed to encode glycine alanine repeat-deleted EBV nuclear Ag 1 covalently linked to multiple CD8+ T cell epitopes from latent membrane proteins 1 and 2. A single stimulation of CD8+ T cells from healthy virus carriers, and patients with HL with this adenoviral construct in combination with IL-2, was sufficient to reverse the functional T cell impairment and restored both IFN-γ production and cytolytic function. More importantly, these activated CD8+ T cells responded to tumor cells expressing membrane proteins and recognized novel EBNA1 epitopes. Flow cytometric analysis revealed that a large proportion of T cells expanded from patients with HL were CD62L high and CD27 high, and CCR7 low, consistent with early to mid effector T cells. These findings provide an important platform for translation of Ag-specific adoptive immunotherapy for the treatment of EBV-associated malignancies such as HL and nasopharyngeal carcinoma.