Running Title: Outer membrane protein-based vaccines for F. tularensis

ABSTRACT 2 Francisella tularensis is a Gram-negative intracellular bacterium and the causative agent of the 3 zoonotic disease tularemia. F. tularensis is a Category A select agent and thus a potential agent 4 of bioterrorism. Whereas a live, attenuated vaccine strain of F. tularensis (LVS) is the basis of an 5 investigational vaccine, it is not licensed for human use because of efficacy and safety concerns. 6 In the present study, we immunized mice with isolated native outer membrane proteins (OMPs), 7 ethanol-inactivated LVS (iLVS), or purified LVS lipopolysaccharide (LPS) and assessed the 8 ability of each vaccine preparation to protect mice against pulmonary challenge with the virulent 9 Type A F. tularensis strain SchuS4. Antibody isotyping indicated that both Th1 and Th2 10 antibody responses were generated in mice after immunization with OMPs or iLVS, whereas 11 LPS immunization resulted in IgA production only. In survival studies, OMP immunization 12 afforded the greatest level of protection (50% survival at 20 days post-infection with SchuS4), 13 with associated 3-log reductions in spleen and liver bacterial burdens (compared to non-14 vaccinated mice). Cytokine quantitation from the sera of SchuS4-challenged mice indicated that 15 OMP and iLVS immunizations induced high amounts of TNF-α and IL-2 production, whereas 16 only OMP immunization induced high levels of IL-10. By comparison, high levels of pro-17 inflammatory cytokines, including RANTES, G-CSF, IL-6, IL-1α, IL-12p40, and KC in non-18 vaccinated mice indicated that these cytokines may facilitate disease progression. Taken 19 together, this study demonstrates the potential utility of an OMP subunit (acellular) vaccine in 20 protecting mammals against Type A