Nadezhda A. Lipatnikova

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. Three Y. pestis strains, a Δcrp mutant, a mutant with arabinose-dependent regulated crp expression (araC P_BAD crp) or an araC P_BAD 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 (lethal dose in all exposed subjects) of the wild-type Y. pestis strain. 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 CFs. The LD₅₀ of the 231Δcrp strain in guinea pigs increased by at least 10⁷-fold compared to that of the wild-type strain. The LD₅₀s of the 231P_BAD-crp mutant in mice and guinea pigs were approximately 10⁴-fold and 10⁷-fold higher than those of Y. pestis 231, respectively. The 231P_BAD-crp(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 crp mutants as promising candidate plague vaccines.

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 P_BAD crp) or araC P_BAD 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 231P_BAD-crp mutant in mice and guinea pigs were approximately 104-fold and 107-fold higher than those of Y. pestis 231, respectively. The 231P_BAD-crp(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. 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.

Animal-model studies used to predict vaccine efficacy in humans are challenging because direct extrapolation of experimental results to humans is not possible for a number of reasons. Interspecies differences in pathogen-recognition receptors may explain differences between humans and rodents in the response to microbial vaccines. There are also species-specific B-cell and T-cell repertoires, as well as HLA presentation of dominant epitopes to CD4 and CD8 T cells. Because of these limitations, studies of candidate vaccines are conducted in several species of small and large animals, which is expected to yield data more transferable to humans. The aim of this study was a comparative assessment in mice and guinea pigs of the protective effect of the differing in antigenic composition 231ΔnlpD and EVΔlpxM plague pathogen strains upon a single immunization with them separately or in a mixture. Materials and methods. The protectivity of the studied strains was characterized by the immunity index in subcutaneous infection of mice and guinea pigs. Results. As in previous experiments [1], the introduction of monopreparations based on attenuated 231ΔnlpD or EVΔlpxM strains selectively induced protection only in mice or guinea pigs, respectively. Immunization with a mixture of attenuated strains provided an immunity index of more than 108 for both types of laboratory animals. Conclusion. Single-stage subcutaneous immunization of mice and guinea pigs with attenuated Y. pestis strains 231ΔnlpD and EVΔlpxM enhances immunity to plague following subcutaneous challenge with wild-type strain 231.

The goal of this study is to find out the effect of deletion of some genes encoding biogenesis of components of the outer membrane protein complex on the virulence of Yersinia pseudotuberculosis mutants. Materials and methods. Y. pseudotuberculosis surA, degP, bamB, and ompH mutants were generated by RedGam mutagenesis using suicide vectors. The constructed mutants were characterized by growth rate, sensitivity to antibiotics, bile salts, and sodium dodecyl sulfate, as well as to the bactericidal action of normal human serum. Total composition of the outer membrane proteins of the knockout mutants was determined by SDS-PAGE. The virulence of DsurA, DdegP, DbamB and DompH was studied after subcutaneous, intraperitoneal, or oral infection of mice. Results. It was found that BamB, OmpH and SurA proteins were necessary for maintaining the integrity of the Y. pseudotuberculosis cell membrane, SurA and BamB proteins are important for resistance to the bactericidal action of serum complement and a high degree of attenuation of Y. pseudotuberculosis strains with deleted surA, degP, bamB genes for outbred mice was revealed. Conclusion. The surA, degP, and bamB genes can be used as potential molecular targets for construction of Y. pseudotuberculosis vaccine strains.

Animal model studies used to predict vaccine efficacy in humans present a considerable challenge, as direct extrapolation of experimental results to humans

The goal of this study is to identify the effect of deletion of some genes encoding biogenesis of outer membrane protein complex components on the virulenc

Background/Objectives: Yersinia pestis is an important zoonotic pathogen responsible for the rare but deadly disease of people with bubonic, septic, or pneumonic forms of plague. The emergence of multidrug-resistant Y. pestis strains has attracted more and more researchers’ attention to the search for molecular targets for antivirulence therapy, including anti-nutritional-virulence therapy. The glnALG operon plays a crucial role in regulating the nitrogen content within a bacterial cell. This operon codes for three genes: the structural gene glnA and the two regulatory genes glnL and glnG. In this study, we tested the effect of the deletion of glnA and glnALG on the pathogenic properties of Y. pestis. Methods: To assess the contribution of nitrogen metabolism to Y. pestis virulence, knockout mutants ΔglnA and ΔglnALG were constructed. The former was unable to synthesize glutamine, while the latter was not only defective in glutamine synthesis but also lacked the two-component sensor–transcriptional activator pair GlnL and GlnG, which could partially compensate for the decrease in intracellular glutamine concentrations by transporting it from the host or by catabolic reactions. For vaccine studies, immunized mice and guinea pigs were injected s.c. with 200 LD₁₀₀ of the wild-type Y. pestis strain. Results: A single knockout mutation in the glnA gene did not affect the virulence of Y. pestis in mice and guinea pigs. Knockout of the entire glnALG gene cluster was required for attenuation in these animals. The ΔglnALG strain of Y. pestis did not cause death in mice (LD₅₀ > 10⁵ CFU) and guinea pigs (LD₅₀ > 10⁷ CFU) when administered subcutaneously and provided 100% protection of animals when subsequently infected with 200 LD₁₀₀ of the Y. pestis virulent wild-type strain 231. Conclusions: Y. pestis, defective in both the glutamine synthetase GlnA and the two-component sensor–transcriptional activator pair GlnL-GlnG, completely lost virulence and provided potent protective immunity to mice and guinea pigs subsequently challenged with a wild-type Y. pestis strain, demonstrating the potential use of the glnALG operon as a new molecular target for developing a safe and efficient live plague vaccine.

The aim of this work was to determine the role of periplasmic chaperone SurA in the pathogenesis of bubonic and pneumonic plague. Yersinia pestis surA knoc

Goal. Determination of the role of periplasmic chaperone SurA in the pathogenesis of bubonic and pneumonic plague. MATERIALS AND METHODS Yersinia pestis surA knockout mutants were generated by RedGam mutagenesis with the use of a suicide vector. The generated strains were characterized by their growth rate, sensitivity to antibiotics, bile acid salts and sodium dodecyl sulfate, as well as to the bactericidal action of normal human serum. Intracellular localization of the SurA protein was determined by DSN-PAAG and immunoblot methods. The virulence of the ΔsurA mutant was studied in subcutaneously or intranasally infected mice or rats. RESULTS The SurA-negative Y. pestis strain was characterized by an increase in the permeability of the outer membrane and sensitivity to antibiotics, as well as a decrease in virulence in a model of bubonic and pneumonic plague in two laboratory animal species. CONCLUSION The SurA protein can be considered as a molecular target for plague therapy. Reducing the virulence of a strain and its resistance to antibiotics by disrupting the biogenesis of outer membrane proteins may become a new strategy for solving the problem of combating multidrug resistance, and the widespread distribution of the protein among Gram-negative pathogens makes its use promising for the development of virulence inhibitors with a broad spectrum of action.

Over the past 200 years since the moment of E. Jenner’s discovery, vaccination continues to be the leading strategy for protection against infectious diseases, but commercially available live attenuated and inactivated vaccines have a number of serious drawbacks. Bacterial strains should be completely attenuated in live vaccines, while maintaining a high degree of immunogenicity. However, the majority of attenuation methods currently used makes potential vaccine strains more susceptible to the action of various host defenses, reducing the ability to persist in the body of the vaccinated individual in quantities and for periods sufficient for formation of long-term and intense immunity. Inactivation of microorganisms underlying the production of killed vaccines, applying various reagents and /or physical factors, can disrupt the native conformation of antigenic epitopes located on bacterial cell surface, which leads to a decrease in immunogenicity. This review examines a promising biotechnological platform for the development of vaccines based on the methodology of regulated delayed gene expression and repression of genes, which was developed to resolve the above-mentioned contradictions.