[PERSPECTIVES OF DEVELOPMENT OF LIVE RECOMBINANT ANTHRAX VACCINES BASED ON OPPORTUNISTIC AND APATHOGENIC MICROORGANISMS].

Live genetic engineering anthrax vaccines on the platform of avirulent and probiotic micro-organisms are a safe and adequate alternative to preparations based on attenuated Bacillus anthracis strains. Mucosal application results in a direct contact of the vaccine preparations with mucous membranes in those organs arid tissues of the macro-organisms, that are exposed to the pathogen in the first place, resulting in a development of local and systemic immune response. Live recombinant anthrax vaccines could be used both separately as well as in a prime-boost immunization scheme. The review focuses on immunogenic and protective properties of experimental live genetic engineering prearations, created based on members of geni of Salmonella, Lactobacillus and adenoviruses.

[CONTEMPORARY TENDENCIES IN CONSTRUCTING RECOMBINANT VACCINES FOR SPECIFIC PROPHYLAXIS OF PLAGUE].

An importance place in the system of prophylaxis measures against plague is allotted to vaccination of population contingents, that belong to risk groups for infection. The whole arsenal of accumulated knowledge on structure, properties, molecular nature, genetic determination, synthesis pathways, regulation and mechanisms of interaction with macroorganism of pathogenicity factors and immunogenicity of the infectious disease causative agent is used in the creation of new generation of vaccines. Contemporary technologies--genomics, proteomics, reverse vaccinology facilitate detection of protective antigens and help determine rational design of the vaccines. Main tendencies in development of recombinant live and chemical vaccines for specific prophylaxis of plague are presented in the review. Constructive approaches, that allow to produce highly effective and safe preparations are isolated.

[A METHOD FOR DIFFERENTIATION OF BACILLUS ANTHRACIS STRAINS AND PHYLOGENETICALLY RELATED SPECIES BASED ON DETERMINATION OF THE STRUCTURAL DIFFERENCESBETWEEN CHROMOSOMAL GENES FOR BIOSYNTHESIS OF FLAGELLIN AND METHIONINE].

Nucleotide sequence analysis of several genes responsible for the anthrax pathogen definitive properties--motility and penicillinase activity--determined a chromosomal locus promising for interspecies differentiation. We demonstrated that the gene fliC encoding flagellin synthesis contains extended region, distinguishing B. anthracis strains from the majority of non-pathogenic and opportunistic bacilli. A novel method for the anthrax pathogen indication and identification based on determination of the differences in the chromosomal genes fliC and hom2 structure was suggested. A total of 60 strains of different Bacillus spp. (B. anthracis, B. cereus, B. thuringiensis, B. mycoides, B. megaterium, B. subtilis, etc.) were tested using two chromosomal DNA targets. The algorithm developed in this work permits to detect the pathogenic microorganism and reliably differentiate it from other Bacillus spp. representatives. The introduction of primers complementary to specific sequences of pXO1 and pXQ2 plasmids into the multiplex PCR makes it possible to receive additional information on proposed virulence of the isolate.

[Immunogenicity and safety of a prototype chemical anthrax vaccine in laboratory animal models].

AIM: Evaluation of immune stimulating and toxic effects of a vaccine prototype protein components. MATERIALS AND METHODS: Linear mice, guinea pigs and rabbits were immunized subcutaneously once or twice by recombinant protective antigen (rPA), S-layer protein (EA1) or their complex. Innate immunity structure activation was registered by changes in Toll-like receptor (TLR) expression. Adaptive immune response parameters were determined by established methods. Toxicity of the preparations was determined using flow cytofluorometry and densitomorphometry. RESULTS: The ability of rPA and EA1 to activate structures of innate immunity - TLR 2 and 6 - was established. Features of anti-PA antibody titer dynamics for each of the animal species was determined, a comparison with antibody formation during immunization with Bacillus anthracis STI- 1 was carried out. 2 immunizations ofbiomodels with a complex preparation combined with an adjuvant provides protection from infection by a test-strain that is comparable with protectivity of a live vaccine. Evidences regarding damaging effect of rPA and EAI on cells and tissues of macro organism were not detected throughout the study. CONCLUSION: Aprototype of a chemical anthrax vaccine under development has high immunogenicity and its protein components are not toxic for laboratory animals based on the results of complex testing.

[Identification of the differences in the genes responsible for methionine biosynthesis in Bacillus anthracis strains and phylogeny-related species].

The comparative analysis of the gene sequences encoding the synthesis of enzymes responsible for the intermediary metabolism of methionine in Bacillus anthracis strains and in closely related bacterial species was carried out. Deletion of 42 nucleotides in the hom2 gene, which determines the homoserinedehydrogenase, is detected in all tested Bacillus anthracis strains. In the strains of other bacillar species hom2 gene mutation, which blocks up the tracts of methionine and threonine biosynthesis, was not identified. The single nucleotide polymorphism was determined in asd1, metX, and metH genes. It provides the identification of B. anthracis strains using sequencing technology.

[Immunogenicity of protective antigen extracted from asporogenic recombinant strain Bacillus anthracis].

AIM: To study the ability of recombinant protective antigen (PA) to stimulate adaptive immune response in laboratory animals. MATERIALS AND METHODS: Vaccine, recombinant, and reference strains of Bacillus anthracis were used in the study. Laboratory animals were immunized subcutaneously with two doses of antigenic preparation or one dose of B. anthracis strain. After inoculation with reference strain of B. anthracis, measurement of LD50 as well as indexes of immunity was performed by specified methods. RESULTS: It was revealed that asporogenic recombinant strain has stable biological characteristics during passages in vitro and is effective producer of PA. Using 2-stage chromatography, highly purified protein was obtained. Experiments on different biomodels--BALB/c mice, guinea pigs, and rabbits--demonstrated high protective activity of PA obtained from asporogenic producer. Increase of immunity index was noted when EA1 protein from S-layer was added to preparation for immunization. CONCLUSION: Immunity indexes determined in experiments on laboratory animals point to high protective efficacy of recombinant PA. Further studies of its interaction with macroorganism's innate and adaptive immunity systems are promising.

[Current views on pathogenicity and immunogenicity factors of Bacillus anthracis].

Bacillus anthracis is an etiological agent of extremely dangerous zooanthroponosis - anthrax. To date, significant volume of scientific data about structure, molecular nature, characteristics, genetic determination, regulation and action mechanisms of main pathogenicity factors of B. anthracis and its immunogenicity is accumulated. For study of integral picture ofpathogenesis and immunogenesis such global methodologies as complex analysis of structure and functions ofgenome, on the one hand, and studies of fine mechanisms of interactions of genes or certain parts of protein molecules, on the other, were used.

[Immunogenicity of the recombinant Bacillus strains with cloned gene of biosynthesis of protective antigen against Bacillus anthracis].

Microbe Russian Anti-Plague Research Institute, Saratov A hybrid plasmid pUB110PA-1 demonstrating stable functioning in the cells of Bacillus strains and containing the gene of biosynthesis of Bacillus anthracis protective antigen was constructed. The recombinant strains surpassing the anthrax vaccinal cultures in the secreted synthesis of the protective antigen were obtained and their immunological efficacy was assessed. A single inoculation of Guinea pigs with the dose of 5 x 107 spores of the recombinant strains imparted efficient protection against B. anthracis challenge. Immune responses were characterized by high indices of immunity and titers of antibodies to the protective antigen. In contrast to the anthrax vaccinal preparations, the gene-engineering strains imposed no residual virulence for BALB/n mice and Guinea pigs.

[Role of the components of the S-layer in immunogenicity of Bacillus anthracis].

The immunogenicity of proteins Sap and EA1, contained in B. anthracis S-layer, was evaluated in experiments on laboratory animals. These proteins were found to produce protective effect and could be regarded as additional immunogenic factors. The use of the newly constructed isogenic pair Sap+ and Sap- of B. anthracis strains made it possible to study the influence of Sap- mutation on the immunological properties of the causative agent of anthrax.

[study of the virulence of a recombinant strain of Bacillus anthracis, obtained as a result of transductive transfer of chromosomal genes from a strain of Bacillus cereus]. / Izuchenie virulentnosti rekombinantnogo shtamma Bacillus anthracis, poluchennogo v rezul'tate transduktsionnoi peredachi khromosomnykh genov iz shtamma Bacillus cereus.

Auxotrophic markers of B. anthracis strains differing them from other Bacillus representatives have been determined. Chromosome genes from prototrophic B. cereus strain were transduced into auxotrophic B. anthracis strain. The properties of transductants were studied in order to establish common transfer of chromosomal determinants responsible for realization of various signs. Transduction mating between species resulted in construction of prototroph B. anthracis strains (pX01- pX02+), whose derivatives are characterized by decreased virulence for laboratory animals.

[Contribution of determinants, located in Bacillus anthracis chromosomes, in realizing the pathogenic properties of the pathogen]. / Vyiasnenie uchastiia determinantov, lokalizovannykh v sostave khromosomy Bacillus anthracis, v realizatsii patogennykh svoistv vozbutitelia.

Comparative study of virulence of B. anthracis strains harbouring pXO1 and pXO2 plasmids in mice and guinea pigs showed that among six B. anthracis strains, three were 100-1000 times less virulent for guinea pigs. Genetic construction of B. anthracis strains using transduction and conjugation transfer of resident plasmids permitted us to rule out the effects of modified pXO1 and pXO2 replicons and to prove the existence of nonidentified chromosome locuses responsible for the development of an infectious process in anthrax, along with plasmid determinants of virulence.

[Correlation of the virulence of Bacillus anthracis with expression of signs, coded for by chromosomal genes]. / Korreliatsiia virulentnosti Bacillus anthracis s ékspressiei priznakov, kodiruemykh khromosomnymi genami.

Clonal analysis of Bacillus anthracis strains showed heterogeneity of the majority of populations by the proteolytic, hemolytic, and pigment-adsorbing activities. Phenotypes isolated within each population were subdivided into 4 subpopulations. The appearance of sub-populations of virulent and vaccine strains (B. anthracis Sterne, Zenkowskii) was due to the ability of clones adsorbing Congo red and possessing high activities of proteolytic and hemolytic enzymes to dissociate with the formation of phenotypes characterized by different expression of proteolysis, hemolysis, pigment adsorption, pigment production, and sporulation signs. A characteristic feature of vaccine strains of B. anthracis CTIl, Wright, and Pasteur is a relative homogeneity of their populations consisting mainly of cells with low activities of proteolytic, hemolytic, and pigment-adsorbing enzymes. Study of the heterogeneity of di-, mono-, and plasmid-free derivatives of B. anthracis showed that the process of phenotype formation did not depend on the plasmids proper. Evaluation of the virulence of clones isolated from highly virulent B. anthracis strain 81/1 showed that the processes associated with loss of sporulation capacity notably decrease the virulence for laboratory animals.