Anastasia S. Vagaiskaya

The objective of the study was to develop an algorithm for constructing recombinant Yersinia pestis strains with regulated gene expression. Materials and m

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.

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.

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.

During the last 100 years, vaccine development has evolved from an empirical approach to one of the more rational vaccine designs where the careful selection of antigens and adjuvants is key to the desired efficacy for challenging pathogens and/or challenging populations. To improve immunogenicity while maintaining a favorable reactogenicity and safety profile, modern vaccine design must consider factors beyond the choice of target antigen alone. With new vaccine technologies currently emerging, it will be possible to custom-design vaccines for optimal efficacy in groups of people with different responses to vaccination. It should be noted that after a fairly long period of overwhelming dominance of papers devoted to subunit plague vaccines, materials devoted to the development of live plague vaccines have increasingly been published. In this review, we present our opinion on reasonable tactics for the development and application of live, safe, and protective human plague vaccines causing an enhanced duration of protection and breadth of action against various virulent strains in vaccination studies representing different ages, genders, and nucleotide polymorphisms of the genes responsible for immune response.

Background/Objectives Bacterial ghosts (BGs), non-living empty envelopes of bacteria, are produced either through genetic engineering or chemical treatment of bacteria, retaining the shape of their parent cells. BGs are considered vaccine candidates, promising delivery systems, and vaccine adjuvants. The practical use of BGs in vaccine development for humans is limited because of concerns about the preservation of viable bacteria in BGs. Methods: To increase the efficiency of Klebsiella pneumoniae BG formation and, accordingly, to ensure maximum killing of bacteria, we exploited previously designed plasmids with the lysis gene E from bacteriophage φX174 or with holin–endolysin systems of λ or L-413C phages. Previously, this kit made it possible to generate bacterial cells of Yersinia pestis with varying degrees of hydrolysis and variable protective activity. Results: In the current study, we showed that co-expression of the holin and endolysin genes from the L-413C phage elicited more rapid and efficient K. pneumoniae lysis than lysis mediated by only single gene E or the low functioning holin–endolysin system of λ phage. The introduction of alternative lysing factors into K. pneumoniae cells instead of the E protein leads to the loss of the murein skeleton. The resulting frameless cell envelops are more reminiscent of bacterial sacs or bacterial skins than BGs. Although such structures are less naive than classical bacterial ghosts, they provide effective protection against infection by a hypervirulent strain of K. pneumoniae and can be recommended as candidate vaccines. For our vaccine candidate generated using the O1:K2 hypervirulent K. pneumoniae strain, both safety and immunogenicity aspects were evaluated. Humoral and cellular immune responses were significantly increased in mice that were intraperitoneally immunized compared with subcutaneously vaccinated animals (p < 0.05). Conclusions: Therefore, this study presents novel perspectives for future research on K. pneumoniae ghost vaccines.

During the last 100 years, vaccine development has evolved from an empirical approach to one of more rational vaccine designs where careful selection of antigens and adjuvants is key to the desired efficacy for challenging pathogens and/or challenging populations. To improve immunogenicity while maintaining a favorable reactogenicity and safety profile, modern vaccine design must consider factors beyond the choice of target antigen alone. With new vaccine technologies currently emerging, it will be possible to custom-design vaccines for optimal efficacy in groups of people with different response to vaccination. It should be noted that after a rather long period of overwhelming dominance of the number of papers devoted to subunit plague vaccines, materials devoted to the development of live plague vaccines are increasingly being published. In this review, we present our opinion on reasonable tactics of development and application of live, safe and protective human plague vaccines causing sufficient duration of protection and breadth of action against various virulent strains in vaccination studies representing different age, gender and nucleotide polymorphisms of the genes responsible for immune response.

Brucellosis is a zoonotic disease caused by Brucella. In the Russian Federation, a live vaccine based on the attenuated strain B. abortus 19BA is used to p