[Production of mycobacterial antigenes merged with cellulose binding protein domain in order to produce subunit vaccines against tuberculosis].

Protein genes Ag85A, Esat-6, and Cfp10 of Mycobacterium tuberculosis were sequenced using the database GenBank to implement selection and synthesis of primer pairs of given genes. PCR was used to obtain target amplicons of the genes. Chromosome DNA of M. tuberculosis H37Rv was used as the DNA amplification matrix. The PCR products were obtained using the plasmid pQE6, cloned, and amplified in the Escherichia coli M15 strain. Chimere products containing mycobacterial genes and cellulose binding protein domain (CBD), were obtained using the plasmid treated with restriction endonucleases. CBD fragment obtained using similar treatment of the ptt10 plasmid. The plasmids containing merged sequences of mycobacterial genes-antigenes and CBD were selected. The 3 mycobacterial genes were expressed in the E. coli M15 cells resulting in biosynthesis of corresponding recombinant proteins of expected molecular weight. Concentration of CBD, Cfp10-CBD, Ag85A-CBD, and ESAT6-CBD was 20%, 15%, and 15% total protein, respectively. The resulting chimere proteins provide high affinity for cellulose and high stability. Immobilization of CBD-containing recombinant proteins proceeds as one-stage process providing target protein purification and adsorption on cellulose. The vaccines produced using this technology are inexpensive because of low cost of cellulose sorbents as well as simultaneous use of cellulose for purification and immobilization of protein. Many cellulose preparations are not toxic, biocompatible, and widely used in medicine.

Outer membranes of a lipopolysaccharide-protein complex (LPS-17 kDa protein) as chemical tularemia vaccines.

Immunisation with outer membranes of Francisella tularensis induced an efficient protection in guinea pigs against challenge with the virulent strains 503 or 144/713 (type B biovar holarctica), both clinical isolates, and prevented the development of typical signs of infection in hamadryads (baboons), challenged with the virulent strain Schu (type A, biovar tularensis) of F. tularensis. Immunisation with a lipopolysaccharide protein complex isolated from the outer membranes afforded protection in CBA mice against challenge with strain 503. Another LPS-protein complex obtained by the simple mixture of LPS preparations from strain 503 and a 17-kDa membrane protein from the avirulent R-variant of the vaccine strain 15 also demonstrated protective properties against experimental tularemia in mice.

Structure of the O-antigen of Francisella tularensis strain 15.

The O-specific polysaccharide, obtained by mild acid degradation of the lipopolysaccharide of Francisella tularensis strain 15, contained 2-acetamido-2,6-dideoxy-D-glucose (D-QuiNAc), 4,6-dideoxy-4-formamido-D-glucose (D-Qui4NFm), and 2-acetamido-2-deoxy-D-galacturonamide (D-GalNAcAN) in the ratios 1:1:2. Tri- and tetra-saccharide fragments were obtained on treatment of the polysaccharide with anhydrous hydrogen fluoride and partial hydrolysis with 0.1 M hydrochloric acid, respectively. On the basis of 1H- and 13C-n.m.r. spectroscopy of the polysaccharide and the saccharides, it was concluded that the O-antigen had the structure: ----4)-alpha-D-GalpNAcAN-(1----4)-alpha-D-GalpNAcAN-(1----3) -beta-D-QuipNAc-(1----2)-beta-D-Quip4NFm-(1----. This O-antigen is related in structure to those of Pseudomonas aeruginosa O6, immunotype 1, and IID 1008, and Shigella dysenteriae type 7.

[Biochemical, antigenic and protective properties of the outer membrane of tularemia pathogens]. / Izuchenie biokhimicheskikh, antigennykh i protektivnykh svoistv vneshnei membrany vozbuditelia tuliaremii.

The outer membranes of Francisella tularensis were studied. The membranes were identified morphologically, immunologically and biochemically. They contained 12-20% of protein, 15-30% of carbohydrates, up to 40% of lipids. The main integral proteins of the outer membranes were the 47, 43, 17 and 12 kD proteins. The main protein 63 kD was not integral. The lipopolysaccharides isolated from the outer membranes and acetone-dried cells did not possess the protective properties in experimental tularemia. The preparations of outer membranes possessed the protective properties for mice infected with the virulent strain 503. Chitosan amplified the protective properties of outer membranes.

[Physicochemical properties of the Teschen disease virus]. / Fiziko-khimicheskie svoistva virusa bolezni Teshena.

The physical stability of Teschen disease virus (TDV) was tested. It was found that 8 M urea at 37 degrees C and 0.5% Tween-20 for 1-2 hours destroyed TDV with formation of morphologically distinct subunits. The morphology of TDV virions and its subunits formed under the effect of urea and Tween-20 was studied. Sedimentation constant (120 S) and the polypeptide composition of TDV were determined. In TDV, major polypeptides (VP1 less than or equal to 4) and minor polypeptides (VP1m and VP2m) were found their molecular weights being 82,000, 67,000, 35,000, 30,000, 77,000, and 46,000 daltons respectively.

[The effect of antigenic fractions of the outer membrane in Francisella tularensis on the functional activity of macrophages]. / Vliianie antigennykh fraktsii vneshnei membrany Francisella tularensis na funktsional'nuiu aktivnost' makrofagov.

The influence of different gel-chromatographic antigenic fractions (GAF) of the membrane of F. tularensis, strain A'Cole, on different forms of reactivity of mouse peritoneal macrophages, such as the adhesion, ingestion and presentation of antigen on the cell surface, has been immunologically evaluated. GAF isolated from F. tularensis have been shown to produce a pronounced modulating effect on all forms of macrophagal functional activity under study. Thus, GAF II with a molecular weight of 85-200 kD inhibits the adhesion, ingestion and presentation of antigens and, on the contrary, GAF IV with a molecular weight of 15-35 kD stimulates these functions.

[The effect of the antigenic fractions of the outer membrane of Francisella tularensis on the T-cell immunity indices]. / Vliianie antigennykh fraktsii vneshnei membrany Francisella tularensis na nekotorye pokazateli T-kletochnogo zvena immuniteta.

The immunological evaluation of the influence of individual gel-chromatographic antigenic fractions (GAF) of F. tularensis outer membrane on different forms of T-cell reactiveness, such as delayed hypersensitivity (DH), proliferation of lymphocytes in the reaction of blast transformation (RBT) and mixed lymphocyte culture (MLC), has been made. As revealed in this study, GAF isolated from F. tularensis produce a pronounced immunomodulating effect on the processes linked with polyclonal activation of T-lymphocytes. Thus, GAF II with a molecular weight of 85-200 kD inhibits the maturation and activity of T-effectors of DH, the proliferation of lymphocytes in RBT and MLC. On the contrary, GAF IV with a molecular weight of 15-35 kD produces a stimulating effect on T-cells in the immune system in all the parameters under study.

[The LPS-protein complex from the outer membrane of Francisella tularensis]. / Issledovanie LPS-belkovogo kompleksa iz vneshnei membrany Francisella tularensis.

LPS-protein complex containing proteins of 15 kD, 17 kD and 19 kD was isolated from F. tularensis outer membrane by solving with sodium deoxycholate with the subsequent gel filtration on Sephacryl S-200. Protein of 17 kD constituted the main protein component of the complex. The LPS-protein ratio of this complex was 1:1. Proteins contained in LPS-protein complex have mainly the alpha-spiral structure. In the absence of detergent these proteins and LPS formed micelles with molecular weight exceeding 10(7) D. LPS-protein complex was shown to have a protective effect in mice infected with F. tularensis virulent strain 503.

[The determination of the antigenic determinant of protective monoclonal antibodies specific to the Francisella tularensis lipopolysaccharide]. / Opredelenie antigennoi determinanty preventivnykh monoklonal'nykh antitel, spetsifichnykh k lipopolisakharidu Francisella tularensis.

F. tularensis lipopolysaccharide (LPS) was studied with the use of monoclonal antibodies (McAb) having protective properties. The binding site of these McAb (IgG2a) is localized on the O-chain of LPS. In contrast to LPS isolated from vaccine strain 15, LPS isolated from F. tularensis cells in the R-form has no O-chains and does not interact with McAb.

[The use of immunoenzyme analysis and immunoblotting for the serological characterization of mycobacterial antigens]. / Ispol'zovanie immunofermentnogo analiza i immunoblottinga dlia serologicheskoi kharakteristiki antigenov mikobakterii.

The enzyme immunoassay and immunoblotting were used for the study of the serological activity of different mycobacterial antigens and the spectrum of antibodies to them in patients with different forms of tuberculosis and healthy persons. Antibodies in patients' sera were shown to bind antigens with different molecular weight. The level and spectrum of antibodies to purified protein fraction I made it possible to differentiate between patients with various forms of tuberculosis and healthy persons.

[The opsonizing activity of the sera of hamadryas baboons immunized with a preparation of the outer membranes of Francisella tularensis (based on data from the luminol-dependent luminescence method)]. / Opsoniziruiushchaia aktivnost' syvorotok obez'ian gamadrilov, immunizirovannykh preparatom vneshnikh membran Francisella tularensis (po dannym metoda liuminolzavisimoi khemiliuminestsentsii).

The opsonizing properties of sera obtained from hamadryas baboons immunized with the preparation of F. tularensis outer membranes (OM) were studied with the use of luminol-dependent chemiluminescence (CL) of whole blood. The immunization of monkeys with the OM preparation was shown to lead to the formation of functionally active antibodies possessing opsonizing properties with respect to virulent F. tularensis. Immune sera obtained from the animals immunized with live vaccine and from those immunized with OM preparation had no essential differences in their opsonizing properties. The level of IgG antibodies in immune sera correlated with the CL parameters of whole blood in the presence of F. tularensis opsonized with these sera. Increased CL of phagocytes observed after addition of bacteria and immune sera under test to whole blood taken from a nonimmune donor made it possible to evaluate the functional activity of antibodies, thus permitting its use as a test for the evaluation of the effectiveness of new vaccine preparations.

[The preventive activity of monoclonal antibodies specific to the lipopolysaccharide of Francisella tularensis]. / Preventivnaia aktivnost' monoklonal'nykh antitel, spetsifichnykh k lipopolisakharidu Francisella tularensis.

The preventive activity of five monoclonal antibodies (McAb) in experimental tularemia was evaluated. McAb produced by hybridoma FB11-k (IgG2a), specific to F. tularensis lipopolysaccharide, prevented the death of mice and guinea pigs infected with F. tularensis virulent strain 503 of the holarctic subspecies.

[The immunological efficacy of Francisella tularensis outer membranes for hamadryas baboons]. / Immunologicheskaia éffektivnost' vneshnikh membran Francisella tularensis dlia obez'ian-gamadril.

The protective properties of the preparation of F. tularensis outer membranes (OM), obtained from F. tularensis vaccine strain 15, were studied in experiments on hamadryas baboons challenged subcutaneously with F. tularensis virulent strain Schu (nonarctic subspecies). The subcutaneous immunization with the OM preparation prevented the development of clinically pronounced infection in more than 70% of the monkeys challenged with F. tularensis strain Schu in a dose of 787 live microbial cells 30 days after immunization. Antibody titers determined in the immunized monkeys with the use of the agglutination test (AT) and the passive hemagglutination test (PHAT) were usual in minimal diagnostic limits (1:80 for AT and 1:320 for PHAT) and did not significantly rise by day 20 after immunization. In all intact animals infected with F. tularensis strain Schu the development of the infectious process was registered, which was accompanied by a rise in temperature exceeding 39.5 degrees C and a rise in the titer of specific antibodies.

[The protective properties of the outer membranes of Francisella tularensis in an experimental infection in guinea pigs]. / Zashchitnye svoistva vneshnykh membran Francisella tularensis pri éksperimental'noi infektsii morskikh svinok.

Subcutaneous immunization, made in a single injection, with outer membrane preparations obtained from F.tularensis vaccine strain 15 and virulent strain A'Cole results in intensive immunity to tularemia in guinea pigs, ensuring the protection of 60-100% of the animals within a month after challenge with F.tularensis virulent strain 503 in a dose of 1,000 DCL. The development of protective effect induced by F.tularensis outer membranes can be observed during the first 24 hours and reaches its maximum by days 15-21 after immunization.

Structural and functional similarity between Yersinia pestis capsular protein Caf1 and human interleukin-1 beta.

A comparative study of the structural and functional properties of recombinant Yersinia pestis Caf1 and human IL-1beta was performed. According to Fourier transform infrared spectroscopy (FTIR) and circular dichroism (CD) data, IL-1beta and Caf1 are typical beta-structural proteins. Neither protein interacts with the hydrophobic probe ANS (8-anilino-1-naphthalenesulfonate) under physiological conditions. Specific binding of Caf1 [K(d) = (5.4 +/- 0.1) x 10(-10) M] to interleukin-1 receptors (IL-1Rs) on the surface of finite mouse fibroblasts (line NIH 3T3) was observed. Caf1 is able to inhibit high-affinity binding of (125)I-labeled IL-1beta to NIH 3T3 cells, and in the presence of Caf1, the binding of [(125)I]IL-1beta is characterized by a K(d) of (2.0 +/- 0.3) x 10(-9) M. Caf1 binding to IL-1R could reflect adhesive properties of the capsular subunits responsible for the contact of bacteria with the host immunocompetent cells. In its turn, this may represent a signal for the initiation of the expression and secretion of the proteins of Y. pestis Yop virulon. Thus, these results help to explain the importance of Caf1 in the interaction of Y. pestis with the host immune system.