Francisella tularensis is a Gram-negative facultative intracellular bacterium that can infect a variety of species and is the cause of the disease known as tularemia or rabbit fever (Sjostedt, 2007). Infection can result by exposure to the bacterium by contact with the skin, by ingestion, or by inhalation of aerosolized organisms (Ellis et al., 2002; Sjostedt, 2007). The precise course and kinetics of the disease varies with the Francisella strain and route of inoculation (Chen et al., 2003; Conlan et al., 2003). However, all routes of exposure can ultimately result in sepsis and widespread dissemination of the bacteria in the host (Conlan et al., 2003; Elkins et al., 2003). Inoculation with microorganisms in aerosolized form for some strains of F. tularensis has a remarkably low infectious dose (10 organisms or less) with a significant fatality rate if left untreated (Conlan et al., 2003; Twine et al., 2006). Tularemia can be treated with antibiotics if detected early (Hepburn and Simpson, 2008; Kman and Nelson, 2008). However, the possibility that the organism could be made antibiotic resistant, either through classic microbiologic means or by using recombinant DNA technology, is a considerable concern if the modified organism were then intentionally spread. The extreme virulence of certain strains, the ability to aerosolize the organisms and the ability of the organism to persist in the environment make it a potent potential bioweapon (Altman, 2002; Ellis et al., 2002). Indeed, both the former Soviet Union, as well as the United States, reportedly had a bioweapons program employing F. tularensis (Dennis et al., 2001; Fong and Alibek, 2005). Unfortunately, in comparison to other pathogenic microorganisms, the host response to F. tularensis is not yet well understood.
The immune response to Francisella tularensis appears complex. As might be anticipated for an intracellular organism, classical cellular immune responses appear to be critical. Studies using lymphocyte-deficient (CD4−, β2m−, TCR-γ−, TCR-β−, scid, nude) or lymphocyte-depleted (by using specific antibodies) mice have illustrated an important role for both CD4 and CD8 T cells (Conlan et al., 1994; Elkins et al., 1993; Elkins et al., 1996; Rhinehart-Jones et al., 1994; Yee et al., 1996). Interestingly, there is also an unusual Thy1+ αβ TCR+ CD4− CD8− NK1.1− T cell subset that has been shown to contribute to protection against F. tularensis challenge (Cowley and Elkins, 2003; Cowley et al., 2005). Recent studies have also suggested that IgA antibodies as well as CD4+ T cells may also play a role in the context of intranasal immunization with an inactivated strain of F. tularensis, in conjunction with IL-12 as an adjuvant (Baron et al., 2007). Thus, while the immune response against F. tularensis is clearly multifactorial, it seems that a cellular response, including CD4+ and CD8+ T cells, plays a critical role in protection.
There is no FDA approved vaccine for F. tularensis. During the 1940s an attenuated subsp. holarctica Live Vaccine Strain (LVS) was developed (Eigelsbach and Downs, 1961) as a vaccine candidate. This strain has proven invaluable for examining aspects of the F. tularensis-host interaction (Elkins et al., 2007). While LVS has greatly aided our understanding of F. tularensis biology and microbial host interactions, there are significant side effects to the use of LVS as a vaccine and the protection it affords is incomplete (Griffin et al., 2007; Saslaw et al., 1961a; Saslaw et al., 1961b). The drawbacks of the current LVS vaccine, and the possibility that F. tularensis might be used in a bioterror weapon, have added impetus to the identification of antigens recognized by the immune system. Currently, the nature of the protective antigens, indeed the molecular definition of any antigens in the cellular immune response, is limited. There have been only a few reports of immunostimulatory molecules for T cells in mice or humans (Golovliov et al., 1995; Lee et al., 2006; McMurry et al., 2007; Sjostedt et al., 1992; Sjostedt et al., 1991). Perhaps the best-characterized response is to the lipoprotein Tul4. Tul4 can be a target of the cellular and humoral immune response in both mice and humans (Golovliov et al., 1995; Sjostedt et al., 1992; Sjostedt et al., 1991). Mice are a natural host for F. tularensis infection and exhibit many of the same aspects of the infection in humans (Fortier et al., 1991). Interestingly, mice immunized with Salmonella typhimurium expressing Tul4 appeared to give partial protection as assessed by a decreased bacterial burden in spleen and liver (Sjostedt et al., 1992). It would be extremely valuable to define epitopes at the molecular level in mice so that the immune response could be quantitatively and qualitatively assessed. This would be a great aid in understanding the host immune response in the context of infection as well as helping to develop and assess vaccine vectors and immunization strategies. In the current study, we have defined in Tul4 a potential immunodominant epitope in B6 mice using a novel strategy and shown that it is an important epitope in the context of a F. tularensis infection in both the acute and memory immune response.