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Bacterial ghosts are intact walls of bacterial cells that are relieved of their contents through pores formed by mild biological or chemical methods. Methodology for generating bacterial ghosts increases the safety of killed vaccines while maintaining their antigenicity through milder preparation procedures. Moreover, bacterial ghosts can simultaneously carry several antigens or plasmid DNAs encoding protein epitopes. In recent years, there has been a growing interest in the development of prototype vaccines and systems for delivery of biologically active substances based on bacterial ghosts. This review discusses the progress in the development of this type of medications over the last years. Various methods of obtaining bacterial ghosts, their advantages and limitations are considered. The phage-mediated lysis of bacteria, molecular manipulations with lysis genes, difficulties encountered in scaling the biotechnological production of bacterial ghosts, and ways to overcome them are described in detail. The use of bacterial ghosts as alternative killed vaccines, adjuvants, recombinant antigenic platform, carrier of plasmid DNA by the models of pathogens of particularly dangerous infections of bacterial etiology is investigated.

Live vaccines induce both cellular and humoral immunity and are cheap and easy to use. The induction of immunity is provided through the reproduction of the vaccine strain in the host body without the development of the disease, since the bacterium to which it is necessary to induce the immunity is characterized by reduced virulence (attenuation). The first generation of attenuated strains was chosen from a variety of spontaneous or physically, chemically and biologically induced mutants after virulence assessment. The rapid development of molecular genetics makes it possible to significantly reduce the time of pathogen attenuation via obtaining knockout mutants with genes selected by a researcher or by inserting “avirulence genes” into the genome. But, given that the methodological aspects of the design of avirulent strains are basically clarified, the absence of officially established criteria for assessing the hazard in regulatory documents hinders the determination of the degree of attenuation. In this regard, there is a need for changes in the procedure for accounting and storage of bacterial cultures, as well as regulation of the process of transferring plague pathogen avirulent strains from the 1st into the 3rd pathogenicity group for subsequent use in the vaccine preparations development. Thereat, the requirements to methodological aspects of the safe generation of attenuated Yersinia pestis strains and the criteria for testing the virulence loss should be maintained at high levels

The causative agent of plague, Yersinia pestis, is classified as pathogenicity (hazard) group I agent, which means that the work with “wild type” strains should be carried out in BSL-3 facilities. Y. pestis EV NIIEG is a Δpgm strain, allowing experimental studies to be carried out in BSL-2 laboratories. However, the disease and its progression elicited by such strain do not entirely mirror the infection observed with fully virulent strains. Residual virulence of Y. pestis EV NIIEG strain for mice can be increased under in vivo iron supplementation. The aim of the study was to optimize methodological approaches to modeling experimental plague in laboratory animals following administration of attenuated Δpgm Y. pestis strains with iron dextran. Materials and methods. Simulation of plague infection in outbred mice was carried out through subcutaneous inoculation of Y. pestis EV NIIEG strain with iron dextran supplementation. The animal condition was assessed on a daily basis. In the course of the experiment, the pathological presentation and bacterial content in organs of mice were evaluated. Results and discussion. Mice inoculated subcutaneously with Y. pestis EV NIIEG strain in the presence of iron dextran developed a bubonic plague that resulted in lethal outcome with pathological changes of internal organs, characteristic of plague infection. In case of daily administration of iron, LD50 of Y. pestis EV NNIEG strain for the mice significantly exceeded the same one with a single injection. Differences in the survival rate among animals in the groups with a single and multiple administration of iron compared to the control group were statistically valid. Thus, attenuated Δpgm Y. pestis strains in the presence of iron dextran can be used to model experimental plague in mice with marked pathological changes and lethality in BSL-2 laboratories.

The causative agent of plague, Yersinia pestis, is a highly virulent bacterial pathogen and a potential bioweapon. Depending on the route of infection, two prevalent forms of the disease — bubonic and pneumonic, are known. The latter is featured by a high fatality rate. Mortality in untreated bubonic plague patients reaches up to 40—60%, whereas untreated pneumonic plague is always lethal. The development of the infectious process in susceptible host is accounted for by a whole set of pathogenicity factors in plague pathogen displaying various functional modalities being expressed depending on stage of infectious process, providing their coordinated expression. Knocking out any of such factors, in turn, may not either affect microbe virulence or lead to its attenuation. A search for new Yersinia pestis pathogenicity factors and subsequent development of highly effective subunit and live attenuated plague vaccines inducing development of pronounced cellular and humoral immune reactions, and/or assessment of their potential use as molecular targets for plague therapy still remain a pressing issue, as both currently licensed plague vaccines do not meet the WHO requirements, whereas strains of plague microbe isolated in Madagascar are resistant to all drugs recommended for plague antibacterial therapy. Here we summarize an impact of described and newly discovered pathogenicity factors into the virulence of Y. pestis strains and their protective anti-plague activity. An effect of loss of genes encoding regulatory proteins as well as mutations in the genes for various transport systems of Y. pestis on attenuation of virulent strains is described as well. Perspectives for introducing characterized antigens into prototype subunit vaccine as well as some other obtained mutants into prototypes of living attenuating vaccines were assessed. The use of antibiotics for plague treatment has been embraced by the World Health Organization Expert Committee on Plague as the “gold standard” treatment. However, concerns regarding development of antibioticresistant Y. pestis strains accounted for further exploring alternatives to plague therapy. Several research groups continue to seek for other alternative approaches, e. g. treatment with inhibitors of pathogenicity factors. Preliminary data attempting to treat plague patients with pathogenicity factor inhibitors are summarized. Antivirulence drugs targeting key microbial factors represent new promising therapeutic options in the fight against antibiotic-resistant bacteria.

To develop a modern plague vaccine, we used hypo-endotoxic Yersinia pestis bacterial ghosts (BGs) with combinations of genes encoding the bacteriophage ɸX174 lysis-mediating protein E and/or holin-endolysin systems from λ or L-413C phages. Expression of the protein E gene resulted in the BGs that retained the shape of the original bacterium. Co-expression of this gene with genes coding for holin-endolysin system of the phage L-413C caused formation of structures resembling collapsed sacs. Such structures, which have lost their rigidity, were also formed as a result of the expression of only the L-413C holin-endolysin genes. A similar holin-endolysin system from phage λ containing mutated holin gene S and intact genes R-Rz coding for the endolysins caused generation of mixtures of BGs that had (i) practically preserved and (ii) completely lost their original rigidity. The addition of protein E to the work of this system shifted the equilibrium in the mixture towards the collapsed sacs. The collapse of the structure of BGs can be explained by endolysis of peptidoglycan sacculi. Immunizations of laboratory animals with the variants of BGs followed by infection with a wild-type Y. pestis strain showed that bacterial envelopes protected only cavies. BGs with maximally hydrolyzed peptidoglycan had a greater protectivity compared to BGs with a preserved peptidoglycan skeleton.

Lipopolysaccharide (LPS), localized in the outer leaflet of the outer membrane, serves as the major surface component of the Gram-negative bacterial cell envelope responsible for the activation of the host’s innate immune system. Variations of the LPS structure utilized by Gram-negative bacteria promote survival by providing resistance to components of the innate immune system and preventing recognition by TLR4. This review summarizes studies of the biosynthesis of Yersinia pseudotuberculosis complex LPSs, and the roles of their structural components in molecular mechanisms of yersiniae pathogenesis and immunogenesis.

To develop a modern plague vaccine, we used hypo-endotoxic Yersinia pestis bacterial ghosts (BGs) with combinations of genes encoding the bacteriophage ɸX174 lysis-mediating protein E and/or holin-endolysin systems from λ or L-413C phages. Expression of the protein E gene resulted in the BGs that retained the shape of the original bacterium. Co-expression of this gene with genes coding for holin-endolysin system of the phage L-413C caused formation of structures resembling collapsed sacs. Such structures, which have lost their rigidity, were also formed as a result of the expression of only the L-413C holin-endolysin genes. A similar holin-endolysin system from phage λ containing mutated holin gene S and intact genes R-Rz coding for the endolysins caused generation of mixtures of BGs that had (i) practically preserved and (ii) completely lost their original rigidity. The addition of protein E to the work of this system shifted the equilibrium in the mixture towards the collapsed sacs. The collapse of the structure of BGs can be explained by endolysis of peptidoglycan sacculi. Immunizations of laboratory animals with the variants of BGs followed by infection with a wild-type Y. pestis strain showed that bacterial envelopes protected only cavies. BGs with maximally hydrolyzed peptidoglycan had a greater protectivity compared to BGs with a preserved peptidoglycan skeleton. [ABSTRACT FROM AUTHOR]