Characterization of Schu S4 delta aroD as a putative vaccine candidate against pulmonary tularemia

Abstract

Francisella tularensis is the etiological agent of the human disease tularemia and a Tier 1 Select Agent. There is a need for an efficacious vaccine against this pathogen due to its low infectious dose, high mortality rate, and ability to be spread by aerosol. To this end, we have engineered a defined live attenuated strain derived from the highly human virulent F. tularensis WT strain Schu S4, designated Schu S4ΔaroD; this strain lacks aroD, the 3rd enzyme in the chorismate biosynthesis pathway used for synthesis of aromatic amino acids. Schu S4ΔaroD is attenuated for growth in broth cultures and in both J774 and primary murine peritoneal macrophages, with a pulmonary LD50 > 105 CFU in C57BL/6 mice (compared to WT Schu S4 LD50 < 10 CFU). Intranasal immunization with Schu S4ΔaroD protects against high-dose WT pulmonary challenge in C57BL/6 mice. A single 50 CFU dose of Schu S4ΔaroD generated 80% protection against 100 CFU challenge, and addition of a 105 CFU booster to the initial 50 CFU vaccination is 80% and 40% protective against 500 or 1000 CFU challenge, respectively. Further studies revealed that a 103 or 105 CFU priming dose followed by a 108 CFU boost is 100% protective against 1000 CFU WT Schu S4 pulmonary challenge. This level of protection has not previously been demonstrated by any tularemia vaccine candidate. Bacterial burden following i.n. vaccination found that regardless of initial dose, immunized mice show identical bacterial loads (107 CFU/g tissue in the lung and 105 CFU/g tissue in the liver) at the peak of infection. However, time to peak burden was dose-dependent, as animals receiving a higher initial inoculum (at least 5600 CFU) reach this peak at day 3, as opposed to day 7 in lower initial inoculum groups. Peak bacterial burden correlates with maximal associated histopathology in the liver and peak pro-inflammatory cytokine and chemokine production in lungs, liver, and spleen (TNF-α, IL-1β, Cox-2, KC, iNOS, MCP-1). Our study shows Schu S4ΔaroD serves as an efficacious defined live attenuated vaccine against pulmonary tularemia. We have revealed a dose-dependent response to Schu S4ΔaroD, which may help determine the protective capacity of this vaccine and assist in development of vaccine-associated correlates of protection in the mouse model.