Tularemia is a severe zoonotic disease in humans with fatality rates exceeding 30% in untreated subjects. Francisella tularensis, the causative organism, is a gram-negative coccobacillus and a facultative intracellular pathogen. Infection can occur by direct contact with infected animals or tissues as well as by inhalation; inhalation causes the most severe form of the disease. There is concern that F. tularensis could be used as a biological weapon. A vaccine is needed to protect against the threat posed by the intentional release of this organism, particularly against infection by inhalation. An attenuated strain dubbect the Live Vaccine Strain (LVS) was developed in the 1950s and gives good protection, however this protection breaks down at higher challenge doses and there are concerns about reversion. We have previously shown that aerosol prime-boost vacination with a live attenuated vaccine, S4aroD, provides good protection in an outbred rabbit model against aerosol challenge with virulent F. tularensis SCHU S4. Antibody in plasma corresponded with the level of protection, which was determined by the route of vaccination, the vaccine used, and the number of doses. In this proposal we will evaluate whether restricting deposition of S4aroD to the upper or lower respiratory tract impacts the subsequent immune response and protection against challenge with SCHU S4. Our hypothesis is that deposition of LVS in the lower respiratory tract will provide good immunity against aerosol challenge with SCHU S4 while deposition of S4aroD in the upper respiratory tract may not generate effective immunity. The first aim will develop the technical procedures necessary to generate large particle aerosols and to evaluate deposition of S4aroD in these particles in the respiratory tract of rabbits. In the second aim, rabbits will be vaccinated with S4arpD by large- or small-particle aerosol; we will then compare the immunological response and subsequent protection against aerosol challenge with SCHU S4. The information gained under this proposal will explore whether regional deposition in the respiratory tract impacts subsequent protection, information important for generation of immunity in the respiratory tract and protection against pathogens entering the respiratory tract.