[CONSTRUCTION AND PROPERTIES OF THE FRANCISELLA TULARENSIS VACCINE STRAIN WITHOUT ONE COPY OF THE IGLC GENE AND WITHOUT RECA GENE].

The live vaccine based on the Francisella tularensis subsp. holarctica vaccine strain 15 NIIEG line is used in Russia against tularemia. This vaccine is highly effective, but fairly unstable. Therefore, development of stable live tularemia vaccine with minimal side effect is rather urgent. The method of allel removal in the F. tularensis vaccine strain was used to remove one copy of the iglC gene, which is required to provide intracellular production of the vaccine strain, as well as removal of the recA gene. The latter is crucial for homological recombination. pGM5 suicide vector based on pHV33 bireplicon plasmid was constructed to provide replacement of intact F. tularensis chromosome segments by modified segments. Modified chromosome segments contain F. Tularensis DNA fragment without iglC structural gene segment 545 p. b. (in pGMΔiglC plasmid), as well as DNA fragment containing no recA structural gene segment 1060 p.b. (pGMΔrecA plasmid). The constructed 15/23-1ΔrecA mutant, in contrast to the vaccine strain 15, was capable of reproducing in the macrophage-like cells J774A.1 line, whereas the efficiency of the reproduction was 8-10 times less. BALB/c mouse responded to immunization by the 15/23-1ΔrecA strain by smaller weight decrease (-2%) as compared to the strain 15 (-14%). Bacteria of the 15/23-1ΔrecA strain were virtually incapable of germinating from the BALB/c murine spleen 14 days after invasion, whereas bacteria of the strain 15 were found in the murine organs even after 21 days. The F. tularensis 15/23-1ΔrecA strain having smaller reaction ability can be used as a basis for construction of stable live safe tularemia vaccine.

Biological properties and structure of the lipopolysaccharide of a vaccine strain of Francisella tularensis generated by inactivation of a quorum sensing system gene qseC.

A knockout mutant with a deletion in a quorum sensing system gene qseC was generated from the vaccine strain Francisella tularensis 15 by site-directed mutagenesis. The variant with the inactivated gene qseC differed from the parental strain in growth rate on solid nutrient medium but had the same growth dynamics in liquid nutrient medium. The mutation abolished almost completely the resistance of the vaccine strain to normal rabbit serum and its ability to survive in macrophages; in addition, the strain lost the residual virulence. A significant phenotypic alteration was observed in the lipopolysaccharide of F. tularensis. Particularly, the mutant strain synthesized no noticeable amount of the lipopolysaccharide with the high-molecular-mass O-polysaccharide, presumably as a result of impairing biosynthesis of the repeating unit, namely, a loss of the ability to incorporate a formyl group, an N-acyl substituent of 4-amino-4,6-dideoxy-D-glucose.

[Anthrax: early steps of the intracellular stage of infection development].

It was shown that spore germination of different Bacillus anthracis strains in macrophage-like cells J774A.1 depended on the genotype of the strains. The virulent B. anthracis strains contain plasmids pXO1 and pX02 responsible for the synthesis of a toxin and a capsule, respectively. The loss of one of the plasmids results in the reduction of strain virulence. It was shown that effective survival of germinating spores in macrophages occurred in the presence of plasmid pXO1 only. The spores of the B. anthracis strains ?Ames and STI-Rif deprived of plasmid pXO1 were least adapted to passing through the intracellular stage. The B. anthracis strains 81/1 and 71/12 (carrying plasmids pXO1 and pXO2 and synthesizing the toxin and capsule) less effectively survived in the cytoplasm of macrophages than the strain STI-1 which has only the plasmid pXO1. It was found that the rate of synthesis of the capsule consisting of polymer gamma-D-glutamic acid depended on the ability of bacterial cells to escape from macrophages. In the B. anthracis strains carrying plasmid pXO2, capsule synthesis by vegetative cells was activated within macrophages that promoted a rapid escape of the vegetative cells from the macrophages. On the contrary, most of capsule-free cells of the vaccine strain STI-1 remained inside macrophages during the whole period of observation. Thus, integrated regulation of two processes, namely synthesis of the toxin components participating in the transition of the germinating cell from phagosome into cytoplasm, and synthesis of the capsule whose presence promotes rapid escape of bacterial cells from macrophages by presently unknown mechanism play the key role in anthrax development at early stages.

[Primary structure of the E. coli DNA-dependent RNA-polymerase beta'-subunit. Hydrolysis with trypsin]. / Pervichnaia struktura beta'-sub''edinitsy DNK-zavisomoi RNK-polimerazy E. coli. Gidroliz tripsinom.

The carboxymethylated beta'-subunit of DNA-dependent RNA polymerase was hydrolyzed with trypsin. The hydrolysate was separated on Bio-Gel P-4, followed by ion exchange chromatography, and was further purified by paper chromatography and electrophoresis. A mixture of large peptides was digested with Staphylococcus aureus protease, the fragments obtained were separated by an HPLC procedure. As a result, 172 peptides were isolated, the complete amino acid sequence for 162 and partial sequence for 10 of them being determined. In total, these peptides contain 862 amino acid residues.

[Detection of the functionally active domains in the molecule of the lethal factor of the anthrax exotoxin]. / Vyiavlenie funktsional'no aktivnykh domenov v molekule letal'nogo faktora sibireiazvennogo ékzotoksina.

Three functional domains were revealed in the molecule of the lethal factor of B. anthracis. They are located in the linear structure of the molecula as follows: the associative domain occupies the area from Lys39 to Met242, the stabilizing domain from Leu517 to Lys614, and the effector domain still further to the COOH-terminal Lys mino acid.

[Detection of the functionally active domains in the molecule of protective antigen of the anthrax exotoxin]. / Vyiavlenie funktsional'no aktivnykh domenov v molekule protektivnogo antigena sibireiazvennogo ékzotoksina.

Using the limited proteolysis method, we established that the protective antigen (PA) molecule consists of four functionally active domains. The shielding domain occupies an area in the linear structure of the molecule PA with NH4-terminal up to Lys166 and plays an important role in the proteolytic activation of PA. The associative domain situated in the Arg167-Met266 region is responsible for interactions with either lethal or edematous factors in self-assembly of the toxic complexes of the lethal or edematous toxin. The stabilizing domain occupies the Gly351 to Met434 area. On the one hand, this area promotes the formation of conformationally stable toxic complexes with the lethal factor, on the other, directly participates in the formation of the hydrophobic canal, through which the molecule of the lethal or edematous factor and, evidently, a fragment of PA molecule as well (from Arg167 to Gly314), including the associative gene, gets inside the target cell. The receptor domain representing a COOH-terminal region, starting from Leu663 amino acid, interacts with the specific receptors on macrophages and thus delivers the toxic complex to the target cell.

[Monoclonal antibodies to B.anthracis protective antigen are capable to neutralize and to enhance the anthrax lethal toxin action in vitro]. / Monoklonal'nye antitela k protektivnomu antigenu B.anthracis sposobny kak neitralizovat', tak i potentsirovat' deistvie sibireiazvennogo letal'nogo toksina in vitro.

Anthrax belongs to highly dangerous infections of man and animals. No effective treatment methods for pulmonary types of the disease have been yet developed. The existing anthrax vaccines were designed decades ago and need improvement to fit the large-scale vaccination of population. At the same time, the immunological properties of the anthrax vaccine main component, i.e. of the protective agent, have been poorly studied. We obtained, within the present case study, a panel of mouse monoclonal antibodies to the protective agent and investigated the properties of the highest-affine panel representatives. An unusual phenomenon was detected, which is related with enhancement of the anthrax toxin action on the mouse macrophage-like cell-line in presence of the 1F2 monoclonal antibody. The remaining analyzed antibodies, i.e. 6G8 and 6G7, were found to neutralize effectively the toxin action. The enhancing and neutralizing antibodies were proven to be specific to different domains of the protective antigen and to recognize epitopes in its composition. The antibody-mediated enhancement of the anthrax lethal action is a convincing argument for further development of a new-generation anthrax vaccine. Definition of the linear antigen determinants for neutralizing antibodies in the protective antigens is an important step in the development of the next-generation anthrax vaccine.

[Elucidation of functionally active domains in molecules of protective antigen bacillus anthracis's toxin]. / Vyiavlenie funktsional'no-aktivnykh domentov v molekulakh protektivnogo antigena i letal'nogo faktora sibireiazvennogo ékzotoksina.

Limited proteolysis has established that the protective antigen (PA) molecule consists of four functional-active domains. So, the shielding domain borrows an area in the linear structure of the PA molecule with NH2 of the end up to Lys 166 and plays a conducting role in the proteolytic activation of PA. The associative domain, engaging in the area Arg 167-Met266, plays a key role in the interaction with LF or EF at self-assembly toxic complexes LT or ET. The stabilizing domain borrows in the linear structure of the PA molecule are with Gly351 up to Met434. On the one hand, this area promotes formation with LF conformationally steady toxic complex's, and, on the other, takes a direct participation in the formation of a hydrophobic channel by which the molecule LF or EF enters the target cell. The receptor domain, representing a COOH-terminal area, starting from Leu663 amino acid, begins to interact with specific receptors on the macrophages and thus delivers the toxic complex to the target cell. It has been found that in the molecule of lethal factor there are 3 functionally active domains located in the linear structure of the molecule as follows: the associative domain borrows an area from Lys39 up to Met242, stabilizing and effector domains occupy areas from Leu517 to Lys614 and from that point to Lys COOH-terminal amino acid.

[Molecular mechanisms underlying bacillus anthracis infection at early stages and search for novel vaccines]. / Molekuliarnye mekhanizmy nachal'nykh étapov sibireiazvennoí infektsii i poisk novykh vaktsin.

The developmental mechanisms of anthrax immunity were studied. Immunization was found to generally generate specific antibodies and lysozyme. Collectively, all the factors are responsible for suppressing the development of spores in the body. This proves the fact that the immunity is directed not only towards the exotoxin of B. anthracis, but it affects mainly the formation of vegetative cells. On entering the immuned body, vegetative cells may cause B. anthracis infection because antitoxic antibodies have no effect on encapsulated cells. The findings indicate that any anti-anthrax vaccine strain must show a complete immunological response in the body, as well as constitute immunity to all pathogenetic factors of B anthracis.

Cloning and expression of protective antigens of Mycobacterium tuberculosis Ag85B and ESAT-6 in Francisella tularensis 15/10.

The possibility of expression of genes encoding mycobacterial antigens in Francisella tularensis 15/10 vaccine strain cells has been shown for the first time. To obtain stable and effective expression of mycobacterial antigens in the F. tularensis cells, the plasmid vector pPMC1 and hybrid genes consisting of the leader part FL of the F. tularensis membrane protein FopA and structural moieties of the mature protein Ag85B or the fused protein Ag85B-ESAT-6 were constructed. Recombinant strains F. tularensis RVp17 and RVp18 expressing protective mycobacterial antigens in the fused proteins FL-Ag85B and FL-Ag85B-ESAT-6, respectively, were obtained. Expression of the protective mycobacterial antigens in F. tularensis was analyzed using specific antisera to the recombinant proteins Ag85-(His)6 and ESAT-6-(His)6 isolated from Escherichia coli producer strains created on the basis of the pET23b(+) and pET24b(+) vectors. The expression of heterologous protective antigens in F. tularensis 15/10 is promising for creation of live recombinant anti-tuberculosis vaccines on the basis of the tularemia vaccine strain.