ABSTRACTImmunization strategies that generate either CD4 or CD8 T cell responses are relatively well described, but less is known with regard to optimizing regimens to induce both CD4 and CD8 memory T cells. Considering the importance of both CD4 and CD8 T cells in the control of intracellular pathogens such asLeishmania donovani, we wanted to identify vaccines that could raise both CD4 and CD8 T cell responses and determine how to configure immunization strategies to generate the best combined protective T cell response. We examined responses generated against theLeishmaniavaccine antigen F3 following its administration in either recombinant form with the Toll-like receptor 4 (TLR4) agonist-containing adjuvant formulation GLA-SE (F3+GLA-SE) or as a gene product delivered in an adenoviral vector (Ad5-F3). Homologous immunization strategies using only F3+GLA-SE or Ad5-F3 preferentially generated either CD4 or CD8 T cells, respectively. In contrast, heterologous strategies generated both antigen-specific CD4 and CD8 T cells. Administration of F3+GLA-SE before Ad5-F3 generated the greatest combined CD4 and CD8 responses. Cytotoxic CD8 T cell responses were highest when Th1 cells were generated prior to their induction by Ad5-F3. Finally, a single immunization with a combination of F3+GLA-SE mixed with Ad5-F3 was found to be sufficient to provide protection against experimentalL. donovaniinfection. Taken together, our data delineate immunization regimens that induce antigen-specific CD4 and CD8 T cell memory responses, and identify a single immunization strategy that could be used to rapidly provide protection against intracellular pathogens in regions where access to health care is limited or sporadic.