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 PBAD 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::PlpxL lpxL ΔPcrp21::TT araC PBAD 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.

The ppGpp molecule is part of a highly conserved regulatory system for mediating the growth response to various environmental conditions. This mechanism may represent a common strategy whereby pathogens such as Yersinia pestis, the causative agent of plague, regulate the virulence gene programs required for invasion, survival and persistence within host cells to match the capacity for growth. The products of the relA and spoT genes carry out ppGpp synthesis. To investigate the role of ppGpp on growth, protein synthesis, gene expression and virulence, we constructed a ΔrelA ΔspoT Y. pestis mutant. The mutant was no longer able to synthesize ppGpp in response to amino acid or carbon starvation, as expected. We also found that it exhibited several novel phenotypes, including a reduced growth rate and autoaggregation at 26°C. In addition, there was a reduction in the level of secretion of key virulence proteins and the mutant was>1,000-fold less virulent than its wild-type parent strain. Mice vaccinated subcutaneously (s.c.) with 2.5×104 CFU of the ΔrelA ΔspoT mutant developed high anti-Y. pestis serum IgG titers, were completely protected against s.c. challenge with 1.5×105 CFU of virulent Y. pestis and partially protected (60% survival) against pulmonary challenge with 2.0×104 CFU of virulent Y. pestis. Our results indicate that ppGpp represents an important virulence determinant in Y. pestis and the ΔrelA ΔspoT mutant strain is a promising vaccine candidate to provide protection against plague.

Two mutant strains of Yersinia pestis KIM5؉, a ⌬crp mutant and a mutant with arabinose-dependent regulated delayed-shutoff crp expression (araC P BAD crp), were constructed, characterized in vitro, and evaluated for virulence, immunogenicity, and protective efficacy in mice. Both strains were highly attenuated by the subcutaneous (s.c.) route. The 50% lethal doses (LD 50 s) of the ⌬crp and araC P BAD crp mutants were approximately 1,000,000-fold and 10,000-fold higher than those of Y. pestis KIM5؉, respectively, indicating that both strains were highly attenuated. Mice vaccinated s.c. with 3.8 ؋ 10 7 CFU of the ⌬crp mutant developed high anti-Y. pestis and anti-LcrV serum IgG titers, both with a strong Th2 bias, and induced protective immunity against subcutaneous challenge with virulent Y. pestis (80% survival) but no protection against pulmonary challenge. Mice vaccinated with 3.0 ؋ 10 4 CFU of the araC P BAD crp mutant also developed high anti-Y. pestis and anti-LcrV serum IgG titers but with a more balanced Th1/Th2 response. This strain induced complete protection against s.c. challenge and partial protection (70% survival) against pulmonary challenge. Our results demonstrate that arabinose-dependent regulated crp expression is an effective strategy to attenuate Y. pestis while retaining strong immunogenicity, leading to protection against the pneumonic and bubonic forms of plague.Bubonic and pneumonic plague are zoonotic diseases endemic in many parts of the world, including the United States, and have resulted in over 200 million deaths over the course of human history (51). The etiological agent of plague is Yersinia pestis. Although the number of confirmed plague cases that occur worldwide has stabilized over the last 50 years at an average of about 1,700 per year, plague remains a serious public health threat in some regions of the world and outbreaks still occur (19). In addition to the potential for natural infections, Y. pestis is generally considered to be among the top five potential biological weapons (19). Recent efforts to create a safe and effective pneumonic plague vaccine have focused on the development of recombinant subunit vaccines that elicit antibodies against two well-characterized Y. pestis antigens, the F1 capsule and the virulence protein LcrV (2,8,40,53). A plague vaccine based on live attenuated Y. pestis provides the theoretical advantage of simultaneously priming against many antigens, thereby greatly enhancing the likelihood of broadbased protection. In the past, live attenuated strains were generated by selection, rather than precise genetic manipulation, thus raising concern about their genetic composition and stability. The live EV76 vaccine is an apparent pgm mutant that has been used in some countries (49). However, a concern is that the EV76 vaccine strain can cause disease in primates, raising questions about its suitability as a human vaccine (29). Nevertheless, as recently as 2002, USAMRIID researchers noted, "Despite their drawbacks, there is ample evidence that live-a...

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