The virulent intracellular pathogen Francisella tularensis subsp. tularensis (Ftt) causes an acute, lethal disease called tularemia. Surviving tularemia depends on the presence of an effective adaptive immune response. However, the nature of such a response has not been elucidated. Thus, we designed in vitro and in vivo models to characterize the protective immune responses with the goal of applying these features to novel vaccines. Utilizing unlicensed vaccine strains with varying efficacy, we found that vaccinated mice lacking CD4+ T cells succumbed rapidly to Ftt challenge, surviving only 1 day longer than naïve animals. These data indicated that vaccine-mediated protection requires a pool of CD4+ T cells capable of immediately controlling Ftt replication. We found effective vaccination correlated with the presence of poly-functional CD4+ T cells producing IFN-γ, TNF-α, and IL-2 and these purified pulmonary and splenic CD4+ T cells controlled Ftt replication in vitro. To follow the antigen-specific response, we generated vaccine and Ftt strains expressing the well-characterized epitope gp61 from LCMV. Immune animals had persistent numbers of gp61-specific CD4+ T cells in their lymph nodes and spleens, whereas vaccinated, but non-immune animals did not. Therefore, we hypothesized the expansion of high avidity, antigen specific CD4+ T cells would convert a poorly efficacious vaccine to one that engenders protection. As predicted, inclusion of the gp61 epitope in both the vaccinating strain and virulent Ftt challenge strain converted a vaccine that failed evoke adequate T cell responses to one that was 100% protective. Together our work has revealed successful vaccines directed against an aggressive, highly pathogenic organism requires a large pool of high avidity, poly-functional CD4+ T cells. Moreover, the elucidation of Francisella epitopes that elicit high-avidity CD4+ T cell responses, specifically in humans, will be required for successful vaccine development

Learning Objective 1: Learn about in vitro and in vivo immune response models to Francisella                                                 tularensis subsp. tularensis (Ftt) infection.

Learning Objective 2: Learn how effective vaccination correlates with the presence of poly-                                                       functional CD4+ T cells.