ABSTRACT Recently, we described a heterologous prime-boost strategy using plasmid DNA followed by replication-defective human recombinant adenovirus type 5 as a powerful strategy to elicit long-lived CD8 ⁺ T-cell-mediated protective immunity against experimental systemic infection of mice with a human intracellular protozoan parasite, Trypanosoma cruzi . In the present study, we further characterized the protective long-lived CD8 ⁺ T cells. We compared several functional and phenotypic aspects of specific CD8 ⁺ T cells present 14 or 98 days after the last immunizing dose and found the following: (i) the numbers of specific cells were similar, as determined by multimer staining or by determining the number of gamma interferon (IFN-γ)-secreting cells by enzyme-linked immunospot (ELISPOT) assay; (ii) these cells were equally cytotoxic in vivo ; (iii) following in vitro stimulation, a slight decline in the frequency of multifunctional cells (CD107a ⁺ IFN-γ ⁺ or CD107a ⁺ IFN-γ ⁺ tumor necrosis factor alpha positive [TNF-α ⁺ ]) was paralleled by a significant increase of CD107a singly positive cells after 98 days; (iv) the expression of several surface markers was identical, except for the reexpression of CD127 after 98 days; (v) the use of genetically deficient mice revealed a role for interleukin-12 (IL-12)/IL-23, but not IFN-γ, in the maintenance of these memory cells; and (vi) subsequent immunizations with an unrelated virus or a plasmid vaccine or the depletion of CD4 ⁺ T cells did not significantly erode the number or function of these CD8 ⁺ T cells during the 15-week period. From these results, we concluded that heterologous plasmid DNA prime-adenovirus boost vaccination generated a stable pool of functional protective long-lived CD8 ⁺ T cells with an effector memory phenotype.