Rational design of Francisella tularensis attenuated vaccine strains

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Francisella tularensis (Ft), one of the most infectious bacteria known, is a Category A Select Agent that poses a serious military and civilian bioterrorism threat. The only current effective vaccine against Ft is an attenuated Live Vaccine Strain (LVS) strain, which has been used successfully as a protective vaccine in humans in Russia and select groups who work with Ft in the US. However, LVS has not been licensed for use in the U.S., in part, because the mechanism of attenuation is unknown, the protective response in humans is not well-characterized, and it retains lethality in mice by some routes of infection. This proposal aims to systematically identify the LVS attenuating mutations to help further it's potential for licensure and to enable the rational design of new vaccine strains that protect against tularemia and perhaps other diseases as well. We have sequenced the genome of six virulent Ft strains for comparison to LVS, and a bias-reducing approach incorporating all sequenced Ft genomes has been implemented to generate a list of candidate attenuation mutations. This multi-genome approach is reducing the number of candidates that need to be subsequently constructed and tested in a murine infection model, thereby dramatically reducing the cost, time, and the number of animals needed to complete the initial aim of the project. Mutant strains of Ft will be constructed using newly-developed allelic exchange strategies for Francisella, to test the effect of each candidate attenuation mutation on Francisella pathogenicity in a murine infection model and determine which mutations are attenuating. This data will complement ongoing studies of the human response to LVS vaccination and will hopefully facilitate LVS licensure in the US. The data accumulated thus far suggests LVS is attenuated through novel mechanisms, which may provide insights into the rational design of vaccines for other pathogens. In subsequent years, we plan to leverage this data, along with detailed immunological profiling in the best animal models available, for use in the rational design of second generation Ft attenuated vaccines.