Evaluation of Francisella tularensis Δ pdpC as a candidate live attenuated vaccine against respiratory challenge of Tularemia in C 57 BL / 6 J mouse model Running title : Evaluation of Δ pdpC in mice

Francisella tularensis, which causes tularemia, is an intracellular and Gram-negative bacterium. F. tularensis has received significant attention in recent decades because of their history as biological weapons. Thus, the development of novel vaccines directed against tularemia has been an important goal. The attenuated F. tularensis strain ΔpdpC, in which the pathogenicity determinant protein C gene (pdpC) was disrupted by TargeTron mutagenesis, was investigated as a potential vaccine candidate for tularemia in the present study. C57BL/6J mice immunized subcutaneously with 1 × 106 colony-forming units (CFUs) of ∆pdpC were challenged intranasally with 100 × the median lethal dose (LD50) of a virulent SCHU P9 strain at 21 days after immunization. Thirty eight % of C57BL/6J mice were protected against challenge with 100 LD50 of the SCHU P9 strain. Conversely, all unimmunized mice succumbed to death on 6 days after challenge. Compared with that in unimmunized mice, survival rates were significantly higher in vaccinated groups. In addition, ∆pdpC was passaged serially in mice to confirm its stable attenuation. Low bacterial loads persisted in mouse spleens during the 1st to 10th passages. Statistically significant changes in the number of CFUs were not observed during in vivo passage of ∆pdpC. The inserted intron sequences for disrupting pdpC were completely maintained even after the 10th passage in mice. Considering the stable attenuation and intron sequences, it was suggested that ΔpdpC is a promising tularemia vaccine candidate.