PMC

Survival curves of BALB/c mice that were injected i.p. with 250 μl of pooled F. tularensis LVS-immune serum, FopA-immune serum or normal serum. Serum injections were performed 24 hrs before and 24 hrs after i.d. challenge with 2LD₅₀ of F. tularensis LVS. Each group consisted of 5 mice.

Mice were immunized with liposomal rFopA or empty liposomes in the presence of IL-12 as adjuvant and mean CFU were measured in the indicated tissues on days 1, 5, and 7 post i.n challenge. The error bars represent standard deviation from groups of 5-6 mice.

A. Survival of unvaccinated C57BL/6 mice or mice immunized with a sublethal dose of F.tularensis LVS, or with liposomal rFopA after i.d. challenge with a lethal dose of F. tularensis LVS. Challenge was performed 35 days after the final immunization. The FopA-vaccinated and unvaccinated groups consisted of 5 and 6 mice, respectively, while the LVS-immune group consisted of 3 mice. B. Survival of C57BL/6 mice immunized with either 10⁸ CFU of UV-fixed F. tularensis LVS, liposomal FopA, or empty liposomes, after i.n challenge with F. tularensis. Challenge was performed 21 days after the final immunization. Each group consisted of 8 mice. C. Survival of unvaccinated C57BL/6 mice or mice immunized with liposomal rFopA after i.d. challenge with a lethal dose of F. tularensis SchuS4. The FopA-vaccinated and unvaccinated groups consisted of 4 and 5 mice, respectively.

A. Coomassie-stained polyacrylamide gel of rFopA-expressing E. coli BL21(DE3)omp8 preparations. The fraction tested is indicated above the gel. Arrows indicate the FopA/DsbA fusion protein. B. Coomassie-stained polyacrylamide gel of the Triton X-114 and Sarkosyl-soluble fractions bound to Ni-NTA beads, before and after thrombin treatment, and the purified FopA protein obtained after thrombin cleavage. “Boiled” samples were heated in SDS loading buffer for 10 min before being loaded onto the polyacrylamide gel, while “RT” samples were left at room temp. “Induced” samples were derived from bacteria incubated in the presence of 0.3 mM IPTG, while “Uninduced” samples were derived from bacteria that were incubated in the absence of IPTG.

A. ELISA results showing the reactivity of either FopA immune sera, or sera collected from mice receiving sham immunizations with empty liposomes, with wells coated with rFopA. Each line represents the binding curve of an individual mouse. B. ELISA results showing the reactivity of sera from liposomal rFopA-immunized mice with wells coated with wildtype F. tularensis LVS (top) or F. tularensis LVS fopA:Tn5 mutant (bottom). Each line represents the binding curve of an individual mouse. C. Western blot (left) and coomassie-stained polyacrylamide gel (right) analysis of reactivity of pooled anti-rFopA serum against wildtype and fopA:Tn5 LVS cell lysates. 3μg of total protein from each lysate were loaded into each well. The PAGE analysis of the samples demonstrates equal loading of protein in each lane.