Virulence Reduction in Yersinia pestis by Combining Delayed Attenuation with Plasmid Curing

<i>Yersinia pestis</i> 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 <i>Y. pestis</i>-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. Three <i>Y. pestis</i> strains, a Δ<i>crp</i> mutant, a mutant with arabinose-dependent regulated <i>crp</i> expression (<i>araC</i> P_BAD<i>crp</i>) or an <i>araC</i> P_BAD<i>crp</i> 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 (lethal dose in all exposed subjects) of the wild-type <i>Y. pestis</i> strain. The challenge caused the death of 100% of naïve animals in controls. The <i>Y. pestis</i> strain 231Δ<i>crp</i> was nonlethal in mice at a dose of 10⁷ CFs. The LD₅₀ of the 231Δ<i>crp</i> strain in guinea pigs increased by at least 10⁷-fold compared to that of the wild-type strain. The LD₅₀s of the 231P_BAD-<i>crp</i> mutant in mice and guinea pigs were approximately 10⁴-fold and 10⁷-fold higher than those of <i>Y. pestis</i> 231, respectively. The 231P_BAD-<i>crp</i>(pPst¯) strain did not cause death in mice (LD₅₀ > 10⁷ CFU) and guinea pigs (LD₅₀ > 10⁹ CFU) when administered subcutaneously and was capable of inducing intense protective immunity in both species of laboratory animals. Our research has shown once again the necessity of balance between safety and effectiveness demonstrating the feasibility of further investigation of <i>crp</i> mutants as promising candidate plague vaccines.