Virulence Reduction in<em> Yersinia pestis </em>by Combining Delayed Attenuation with Plasmid Curing

Background/Objectives: Yersinia pestis caused the three plague pandemics that claimed more than two hundred million human lives. There is still no vaccine that meets all WHO requirements, and many researchers continue to develop plague vaccines using various technological platforms. For example, researchers led by Roy Curtiss 3rd have developed a new approach to achieve controlled, delayed attenuation of bacterial pathogens. Mutants generated using this method were superior in protecting Y. pestis-infected mice immunized with strains generated using traditional gene knockout. However, further studies are needed to determine the safety and efficacy of these delayed-attenuated strains in other mammalian species in order to extrapolate on humans the data obtained in accordance with the FDA Animal Rule. In this study, we compared the role of delayed shutting down of the crp gene alone versus delayed silencing of the crp gene in combination with loss of the pPst plasmid or single crp knockout in the safety of mutants in mice and guinea pigs, as well as their ability to induce protective immunity in these two animal species. Methods: Three Y. pestis strains, a Δcrp mutant, a mutant with arabinose-dependent regulated crp expression (araC PBAD crp) or araC PBAD crp mutant cured of plasmid pPst, were derived from virulent wild-type strain 231. To evaluate the safety, outbred mice or guinea pigs were immunized subcutaneously with serial tenfold dilutions of mutated strains. For vaccine studies, immunized animals were subcutaneously challenged with 200 LD100 of the wild-type Y. pestis strain. Results: The challenge caused the death of 100% of naïve animals in controls. The Y. pestis strain 231Δcrp was nonlethal in mice at a dose of 107 CFU. The LD50 of 231Δcrp strain in guinea pigs increased at least by 107-fold compared to that of the wild-type strain. The LD50s of the 231PBAD-crp mutant in mice and guinea pigs were approximately 104-fold and 107-fold higher than those of Y. pestis 231, respectively. The 231PBAD-crp(pPst¯) strain did not cause death in mice (LD50&gt;107 CFU) and guinea pigs (LD50&gt;109 CFU) when administered subcutaneously and was capable of inducing intense protective immunity in both species of laboratory animals. Conclusions: Our research has shown once again the necessity of balance between safety and effectiveness demonstrating the feasibility of further investigation of crp mutants as promising candidate plague vaccines.