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 degrees 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 degrees 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 chi 10030( pCD1Ap) (Delta lpxP32::P-lpxL lpxL Delta P-crp21::TT araC P-BAD crp) construct likewise produced hexa-acylated lipid A at 37 degrees 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 > 10(7)-times and > 10(4)-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 x 10(7) wild-type Y. pestis and significantly protected (80% survival) against a pulmonary challenge of 1.2 x 10(4) 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. (C) 2011 Elsevier Ltd. All rights reserved.