[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.

[Genomic polymorphism of the main subspecies of the plague agent strains].

Genomic fingerprinting analysis of plague agent strains of the main subspecies isolated in natural foci of various types in the Russian Federation and neighboring countries suggests their genetic polymorphism, while they are similar in phenotypic properties. The strains of the main subspecies, Y. pesis subsp. Pestis, fall into four genetic variants, each of them being associated with specific carrier species. The conserved genomic fingerprinting profile of each genovariant of Y. pesis subsp. Pestis strains ensures the suggested methodic approach to be promising for the intraspecies differentiation of plague agent strains (including atypical strains). Correlation of genovariants with carrier species permits their application for research into enzootic territories, where carrier change-over takes place.

[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.

[Analysis of genomic polymorphism of typical and atypical strains of the plague pathogen using polymerase chain reaction with universal primers]. / Analiz genomnogo polimorfizma tpovykh i atipichnykh shtammov bozbuditelia chumy s ispol'zovaniem polimeraznoi tsepnoi reaktsii s universal'nymi praimerami.

An analysis of genome polymorphism of the Y. pestis strains by using the method of polymerase chain reaction (PCR) for the tandem repeats of bacteriophage M13 DNA revealed a species similarity of both typical and atypical (according to diagnostic signs) plague-microbe strains. Strain Y. pestis A-1726 with the atypical differential-and-diagnostic properties, without the amplicon specified for Y. pestis and sized 1000 b.p., was identified among 27 analyzed Y. pestis strains. The amplicon profiles of the basic Y. pestis subtype were found to be different from such profiles of other Y. pestis subtypes.

[The range of pathogen variation due to discussion of different hypotheses of plaque]. / O diapazone izmenchivosti vozbuditelia v sviazi s obsuzhdeniem razlichnykh gipotez énzootii chumy.

The paper reviews the hypotheses that explain the mechanism of plague enzooty in natural foci, which are based on a concept of a wide range of plague microbial variability. A comparative analysis of the parameters of variability in the experimentally obtained plague microbial strains and "atypical" natural isolates of the causative agent has led to the conclusion that the mechanism of adaptive variability is due to a phenotypic change in ontogenesis that reflects the philogenetic pathway of the adaptability of a plague microbe to constantly changing living conditions in the ecological niche assimilated by the causative agent.

[Functional phenotypic variability in a plague pathogen and plague enzootics]. / Funktsional'naia fenotipicheskaia izmenchivost' vozbuditelia i problemy énzootii chumy.

In the bacterial population, phenotypic variability plays a part of structural and functional subsystem that occupies a special place in the relations of heterogenic populations by performing an important adaptive function in the common system of ecological connections of parasitocenosis. At the same time regularly varying microorganism phenotypes act as an independent system that are closely related with the conditions of the niches occupied by the causative agent. Each subsystem as part of parasitocenosis is provided by its intrinsic adaptive mechanisms, which in combination ensures the stability of biocenosis based on self-regulation of evolutionarily established ecosystems. With the total complexity of parasitocenosis, the causative agent of plague is essential in forming its focus. For objective analysis of an epizootic process, it is necessary to include the established mechanisms of phenotypic variability of the causative agent of plague as a key link of the structural and functional interaction ecosystem that determines the mechanism of plague entozootics.

[Genotyping Yersinia pestis strains from various natural foci]. / Genotipirovanie shtammov Yersinia pestis iz razlichnykh prirodnykh ochagov.

Seven genetic variants of Yersinia pestis were detected by finger-printing of 85 strains of this bacterium from natural foci by means of a BX probe. Variants of Y. pestis strains correlate with certain species of carriers.

[Comparative study of the structure of some genetic probes used for typing Yersinia pestis strains]. / Sravnitel'noe izuchenie struktury nekotorykh geneticheskikh zondov, ispol'zuemykh dlia tipirovaniia shtammov Yersinia pestis.

A nucleotide sequence common for genetic probes used for detection and investigation of Y. pestis strains MK, IS100, and HRSIII was identified on the basis of restriction, hybridization, and computer analysis. This region of chromosomal DNA is a part of low-molecular BX-probe (about 170 bp) we have developed. The results of genomic fingerprinting of Y. pestis strains by the BX-probe are promising as regards its future utilization for typing the strains isolated in various endemic areas.

[Molecular genetic analysis of DNA structure of pFra plasmids in plague pathogen of varying biovar]. / Molekuliarno-geneticheskii analiz struktury DNK plazmid pFra shtammov vozbuditelia chumy razlichnykh biovarov.

Data on comparative molecular genetic analysis of pFra plasmids from plague bacillus belonging to either two biovars (antiqua and orientalis) are presented. It was established that during evolution these replicons were rearranged, which resulted in the differences between pFra plasmids of plague bacillus of antiqua biovar (Yersinia pestis 231 and Y. pestis 358/12) and that of orientalis biovar (Y. pestis A1122 and Y. pestis EV). These included inversions of two plasmid DNA regions and size differences of 3 kb.

[Molecular genetic methods for detecting and identifying the plague microbe]. / Molekuliarno-geneticheskie sposoby vyiavleniia i identifikatsii chumnogo mikroba.

The review analyzes the data on the new designed drugs and ways of identification and detection of the plague causative agent. It gives the results obtained from molecular genetic studies in designing the diagnostic test system based on DNA [correction of DNK] probes and in developing a way of detecting the plague causative agent by the polymerase chain reaction.

[The determination of the content of the cholera toxin gene in the composition of the DNA from Vibrio cholerae strains by means of the nested polymerase chain reaction]. / Opredelenie soderzhaniia gena kholernogo toksina v sostave DNK iz shtammov Vibrio cholerae metodom "gnezdovoi" polimeraznoi tsepnoi reaktsii.

The possibility of detecting cholera toxin genes in V.cholerae enterotoxigenic strains by the method of "nested" polymerase chain reaction with the use of primers on the DNA area of operon ctx of AB genes. The possibility of the detection of several V.cholerae cells by this method was shown with the use of a series of bacterial lysate dilutions. The newly developed test system for the detection of cholera toxin gene on the basis of the analysis of bacterial lysates of V.cholerae nontoxigenic strains, as well as Escherichia coli toxigenic and nontoxigenic strains, was shown to be highly specific.

[Phenotypic expression of the Ca2+-dependence trait in recombinant cells of Yersinia pestis (Lehmann, Neumann)]. / Fenotipicheskaia ékspressiia priznaka Ca2+-zavisimosti v rekombinantnykh kletkakh Yersinia pestis (Lehmann, Neumann)]

A conceptual model of the Yersinia pestis Ca(2+)-dependence mechanism is proposed. The model is based on data from analyses of peculiarities of recombinant cells of this plague-causing agent carrying the cloned first Bg/II fragment of the Ca(2+)-dependence plasmid (pCaD) and a combination of this fragment and other plasmid pCaD fragments. The data obtained also allowed a revision of the role of the lcr GVH locus of pCaD in this phenomenon.

[A DNA analysis of Vibrio cholerae strains by the polymerase chain reaction]. / Analiz DNK shtammov Vibrio cholerae metodom polimeraznoi tsepnoi reaktsii.

The method for the analysis of cholera toxin gene in V. cholerae strains was developed on the basis of polymerase chain reaction (PCR). This specific and highly sensitive method using primers affecting the site of the DNA of the operon of cholera toxin gene made it possible to identify one copy of V. cholerae genome. For the first time the content of cholera toxin gene in 4 V. cholerae (eltor) strains, obtained from the clinical material of cholera patients in Tajikistan and Dagestan, was shown with the use of PCR.

[Genetic research on plague pathogen in plague control]. / Geneticheskie issledovaniia vozbuditelia chumy v praktike protivochymnoi raboty.

The results of molecular-genetic studies performed by Russian specialists in plague research are discussed. On their basis, new concepts concerning the factors determining virulence of the plague bacterium were formulated, and certain aspects of Yersinia taxonomy were clarified.