A live attenuated strain of Yersinia pestis KIM as a vaccine against plague

Yersinia pestis, the causative agent of plague, is a potential weapon of bioterrorism. Y. pestis evades the innate immune system by synthesizing tetra-acylated lipid A with poor Toll-like receptor 4 (TLR4)-stimulating activity at 37°C, whereas hexa-acylated lipid A, a potent TLR4 agonist, is made at lower temperatures. Synthesis of Escherichia coli LpxL, which transfers the secondary laurate chain to the 2'-position of lipid A, in Y. pestis results in production of hexa-acylated lipid A at 37°C, leading to significant attenuation of virulence. Previously, we described a Y. pestis vaccine strain in which crp expression is under the control of the arabinose-regulated araC P_BAD promoter, resulting in a 4-5 log reduction in virulence. To reduce the virulence of the crp promoter mutant further, we introduced E. coli lpxL into the Y. pestis chromosome. The χ10030(pCD1Ap) (ΔlpxP32::P_lpxL lpxL ΔP_crp21::TT araC P_BAD crp) construct likewise produced hexa-acylated lipid A at 37°C and was significantly more attenuated than strains harboring each individual mutation. The LD₅₀ of the mutant in mice, when administered subcutaneously or intranasally was >10⁷-times and >10⁴-times greater than wild type, respectively. Mice immunized subcutaneously with a single dose of the mutant were completely protected against a subcutaneous challenge of 3.6×10⁷ wild-type Y. pestis and significantly protected (80% survival) against a pulmonary challenge of 1.2×10⁴ live cells. Intranasal immunization also provided significant protection against challenges by both routes. This mutant is an immunogenic, highly attenuated live Y. pestis construct that merits further development as a vaccine candidate.