Characterization of Schu S4 <i>aro</i> mutants as live attenuated tularemia vaccine candidates

There is a need for development of an effective vaccine against <i>Francisella tularensis</i>, as this potential bioweapon has a high mortality rate and low infectious dose when delivered via the aerosol route. Moreover, this Tier 1 agent has a history of weaponization. We engineered targeted mutations in the Type A strain <i>F. tularensis</i> subspecies <i>tularensis</i> Schu S4 in <i>aro</i> genes encoding critical enzymes in aromatic amino acid biosynthesis. <i>F. tularensis</i> Schu S4<i>ΔaroC</i>, Schu S4Δ<i>aroD</i>, and Schu S4Δ<i>aroC</i>Δ<i>aroD</i> mutant strains were attenuated for intracellular growth <i>in vitro</i> and for virulence <i>in vivo</i> and, conferred protection against pulmonary wild-type (WT) <i>F. tularensis</i> Schu S4 challenge in the C57BL/6 mouse model. <i>F. tularensis</i> Schu S4Δ<i>aroD</i> was identified as the most promising vaccine candidate, demonstrating protection against high-dose intranasal challenge; it protected against 1,000 CFU Schu S4, the highest level of protection tested to date. It also provided complete protection against challenge with 92 CFU of a <i>F. tularensis</i> subspecies <i>holarctica</i> strain (Type B). Mice responded to vaccination with Schu S4Δ<i>aroD</i> with systemic IgM and IgG2c, as well as the production of a functional T cell response as measured in the splenocyte-macrophage co-culture assay. This vaccine was further characterized for dissemination, histopathology, and cytokine/chemokine gene induction at defined time points following intranasal vaccination which confirmed its attenuation compared to WT Schu S4. Cytokine, chemokine, and antibody induction patterns compared to wild-type Schu S4 distinguish protective <i>vs</i>. pathogenic responses to <i>F. tularensis</i> and elucidate correlates of protection associated with vaccination against this agent.