Search

Immunization of mice with recombinant IgA1 protease of Neisseria meningitidis or several structural derivatives thereof protects the animals infected with a variety of deadly pathogens, including N. meningitidis serogroups A, B, and C and 3 serotypes of Streptococcus pneumonia. In sera of rabbits immunized with inactivated pneumococcal cultures, antibodies binding IgA1-protease from N. meningitidis serogroup B were detected. Thus, the cross-reactive protection against meningococcal and pneumococcal infections has been demonstrated in vivo. Presumably it indicates the presence of common epitopes in the N. meningitidis IgA1 protease and S. pneumoniae surface proteins.

We studied immunogenicity of two recombinant proteins FR.9 and FR.11-3 created on the basis of fragments of the primary structure of N. meningitidis IgA1 protease with different molecular weights containing different sets of T and B epitopes. The proteins actively protect animals infected with live virulent culture of meningococci, serogroups A, B, and C. Analysis of CD4+, CD8+, and CD19+ lymphocyte populations in mouse blood showed predominant contribution of different cell populations to the formation of immune response to different proteins. Injection of FR.11-3 protein to animals did no affect the immunoregulatory index, hence, this protein can be used for creation of immunologically safe vaccine preparation.

Four recombinant proteins, MA28–P1004LEH6, ME135–H328LEH6, MW329–H622LEH6 and MH835–P1004LEH6, were prepared based on the genomic sequence of IgA1 protease from Neisseria meningitidis serogroup B strain H44/76. The immunogenic and protective properties of these proteins were studied in a mouse model. The predicted T- and B-epitopes located in the N-terminal part of amino acid sequence of this enzyme are very important for the formation of effective protection against meningococci of the three main epidemic serogroups A, B, and C. The small-sized recombinant protein having the sequence ME135–H328LEH6 (molecular weight 23367 Da) appears to be as protective against meningococci of the tested serogroups as the high molecular MA28–P1004LEH6 (molecular weight 109019 Da), the latter being a large-sized analog of full-length IgA1 protease. These proteins can be promising candidates for a polyvalent meningococcal vaccine.

The study of enzymatic and protective properties of recombinant IgA1 protease in active and mutant form showed that active form of IgA1 protease exhibited species - and type-specificity for mouse and human immunoglobulins. Mutant form, which did not exhibit enzymatic activity, had protective properties against meningococcal infection, induced by meningococcus serogroup A, B and C protecting the mice from lethal infection by living virulent culture of heterologous serogroups of meningococcus. Obtained results make it possible to consider IgA1 protease as a perspective preparation at the stages of development of polyvalent vaccine for protection the people from meningococcal infection of various etiology.NIssledovanie fermentativnykh i protektivnykh svoĭstv rekombinantnoĭ IgA1 proteazy v aktivnoĭ i mutantnoĭ formakh pokazalo, chto aktivnaia forma IgA1 proteazy kharakterizuetsia vidovoĭ i tipovoĭ spetsifichnost'iu v otnoshenii immunoglobulinov myshi i cheloveka. Mutantnaia forma, ne obladaia fermentativnoĭ aktivnost'iu, proiavliala protektivnye svoĭstva v otnoshenii meningokokkovoĭ infektsii, vyzyvaemoĭ meningokokkami serogrupp A, V i S, zashchishchaia mysheĭ ot letal'nogo zarazheniia zhivoĭ virulentnoĭ kul'turoĭ geterologichnykh serogrupp meningokokka. Poluchennye rezul'taty pozvoliaiut rassmatrivat' IgA1 proteazu v kachestve perspektivnogo preparata pri sozdanii polivalentnoĭ vaktsiny dlia zashchity cheloveka ot meningokokkovoĭ infektsii razlichnoĭ étiologii.

A method of the isolation and purification of IgA1 protease from a culture of Neisseria meningitidis serogroup A has been developed. Three inactivated intermediates of the production of the meningococcal vaccine, a culture liquid, as well as a supernatant and precipitate obtained by the precipitation of bacterial cells by cetavlon, served as a starting material. The purity of IgA1 protease was determined by SDS-PAGE. An immunoenzyme assay for determining the IgA1 protease activity has been developed. The yield of the enzyme with a specific activity of 0.5 to 4 million units/mg from 103 g of the cetavlon precipitate (40 l of culture liquid) was about 600 µg. It was shown that IgA1 protease isolated from serogroup A meningococcus is capable of protecting experimental animals (mice) infected with meningococcus of serogroup B.

Using the genome sequence of IgA1 protease of N. meningitidis of serogroup B, four recombinant proteins of different structure and molecular weight were constructed. These proteins were equal in inducing the formation of specific antibodies to IgA1 protease and had protective properties against meningococci. In the sera of immunized mice, anti-IgA1 protease antibodies were detected by whole-cell ELISA, which indicated the presence of IgA1 protease on the surface of these bacteria. We hypothesized that the protective properties of IgA1 protease-based antigens and IgA1 protease analogs could be realized not only via impairment of bacterium adhesion to the mucosa, but also via suppression of this pathogen in the organism. The presented findings seem promising for using these proteins as the basis for anti-meningococcus vaccine.

A new approach to estimation of IgA subclass levels and IgA1/IgA2 ratio using enzymatically active and inactive forms of Neisseria meningitidis IgA1 protease was developed.The approach was tested using the sera of healthy volunteers and patients with meningococcal meningitis. There was a significant increase in the IgA1 level in patients with meningitis (mean titer 1:1546 ± 352) compared to healthy volunteers (mean titer 1:546 ± 282), while the IgA2 content remained unchanged. The IgA1/IgA2 ratio was 6.3 for the healthy volunteers and 12.8 for patients with meningitis. IgA2 for the patients with meningitis and the healthy volunteers were almost unchanged, 1:86 ± 61 and 1:121 ± 46, respectively.The proposed method is economical and reliable and can be used for evaluation of IgA1 and IgA2 in clinical laboratories or for research purposes.

Recombinant proteins (M1K2–N963-LEH6, MA28–N963-LEH6 and ME135–H328-LEH6) have been created on the basis of the genome sequence of IgA1 protease of N. meningitidis serogroup B strain H44/76. It is revealed that, similarly to the native enzyme isolated earlier from N. meningitidis serogroup A strain A208, these proteins induce formation of animal protection against the infection with the virulent strain of meningococcus serogroup B. It is shown that these compounds are promising as a basis for a polyvalent anti-meningococcal vaccine.

α2,3-Sialylation of the lactosamine type N-glycans with trans-sialidase from Trypanosomacruzi is reported. Trans-sialidase (160 kDa, pI 5.35–5.65) and its catalytic fragment (70 kDa, pI 6.0–6.3) were isolated from T. сruzi cells and immobilized on ConA-Sepharose. The resulting preparation retained its activity for several months and was repeatedly used for obtaining mono-, di-, tri-, and tetrasialylated 7-amino-4-metylcoumarin-labeled oligosaccharides with various numbers of antennas and for α2,3-sialylation of glycans within glycoproteins and neoglycoconjugates.

A method of the isolation and purification of IgAl protease from a culture of Neisseria meningitidis serogroup A has been developed. Three inactivated intermediates of the production of the meningococcal vaccine, a culture liquid, as well as a supernatant and sediment obtained by the precipitation of bacterial cells by cetavlon, served as a starting material. The purity of IgA1 protease was determined by SDS-PAGE. An immunoenzyme assay for determining the IgA1 protease activity has been devised. The yield of the enzyme with a specific activity of 0.5 to 4 million units/mg from 103 g of the cetavlon precipitate (40 l of culture liquid) was about 600 mug. It was shown that IgAl protease isolated from serogroup A meningococcus is capable of protecting experimental animals (mice) infected with meningococcus of serogroup B.

Alpha2,3-sialylation of the lactosamine type N-glycans with trans-sialidase from Trypanosoma cruzi is reported. Trans-sialidase (160 kDa, pI 5.35-5.65) and its catalytic fragment (70 kDa, pI 6.0-6.3) were isolated from T. cruzi cells and immobilized on ConA-Sepharose. The resulting preparation retained activity for several months and was repeatedly used for obtaining mono-, di-, tri-, and tetrasialylated 7-amino-4-metylcoumarine-labeled oligosaccharides with various numbers of antennas and for alpha2,3-sialylation of glycans within glycoproteins and neoglycoconjugates.

One hundred and eighty four strains of methicillin resistant Staphylococcus aureus (MRSA) isolated in pyosurgical and burn departments of Moscow, Minsk, Omsk, Tbilisi, Vologda, Smolensk and Dushanbe were differentiated with using phages of the International Set (BIS), two collections of experimental phages and two-probe fingerprinting. More than 50 per cent of the isolates could not be typed by the BIS phages. The Experimental Collection of the N.F. Gamaleya Research Institute of Epidemiology and Microbiology (Moscow) was more useful in the differentiation in comparison to the Experimental Collection of the London Health Centre. A new approach applied by us to the establishment of our collection i.e. screening of cultures with a definite specificity of the system of the restriction-modification (by a modified phage 85) followed by their differentiation with respect to the specificity of the prophages (by the induced phages with the respective modification specificity) provided reliable and reproducible results. Our collection made it possible to classify the phage type 85 strains which as was confirmed by the fingerprinting data belonged to 3 different genotype variants. The fingerprinting had no advantages over the typing by the phages of the more extended phage set and was recommended for differentiation of the strains (14 per cent) not sensitive to the phages used. A step-by-step scheme for typing MRSA easy for use in clinical and epidemiological laboratories is described.

A new collection of phages for typing methicillin-resistant S. aureus (MRSA) is proposed. The collection includes phage 85 (modified by the MRSA strain), capable of selecting strains with the similar specificity of the restriction-modification system, and 9 MRSA-induced phages. The latter differentiate MRSA strains according to the specificity of prophages present in bacterial cells. The use of this phage collection has permitted the typing of MRSA strains insensitive to the phages of the international collection. Among these cultures an epidemic strain has been detected and the source of its spread in the burn center has been established.

By inducing with mitomycin C the following phages were isolated from all the tested 32 methicillin resistant strains of S. aureus: the serogroup B phage was isolated from 2 strains, the serogroup B and F phages were isolated from 5 strains and the serogroup F phage was isolated from 25 strains. The phages were divided into 5 groups by the antiphage immunity. In group 1 of the phages 4 additional phages were specified. By the specificity of the prophages in the cultures all the strains were divided into 5 groups. Group 1 of the cultures was divided into 5 subgroups (A, B, C, D and E).

The functional activity of phagocytic cells in 52 salmonellosis patients was studied with regard to the following characteristics: percent share of phagocytosis, phagocytic index, nitro blue tetrazolium test results, digestive activity. In patients with the unfavorable course of salmonellosis (the formation of carrier state) disturbances in the bactericidal activity of neutrophils and monocytes were established. For 32 patients leukinferon was included in the complex of etiotropic and pathogenetic treatment. The preparation was introduced in 3 intramuscular injections of 10,000 I. U. at intervals of 48 hours (the course of treatment); 10 days after the last injection this course was repeated. The use of leukinferon restored the normal functioning of phagocytes and the number of T-lymphocytes.

Clinical trials of human leukocytic alpha-interferon for injections, leukinferon were performed in 51 patients with different forms of surgical purulent infections. It was shown that leukinferon lowered the terms of normalization of body's temperature, leukocytosis, respiratory neutrophilic outbreak and levels of active T-lymphocytes. The same was observed when leukinferon was used prophylactically in cardiosurgical patients. The effect of leukinferon depended on the level of radical operations on primary purulent foci and severity of the patient's state. Leukinferon had immunomodulatory properties and mainly influenced the system of neutrophilic phagocytes. The action was lymphocyte-mediated. The rapid effect of leukinferon makes it necessary to recommend it for treatment of patients with purulent infections as an agent of urgent immunomodulation.

It was experimentally demonstrated that cholinesterase activity increases in the gray matter of the cerebral hemispheres in tetanus and dysentery intoxications. However, there is no change in its activity in pneumococcal mococcal intoxication. The activity of the enzyme does not change in the cerebellum in tetanus and dysentery intoxications. The increase in activity of cholinesterase in all the above cases is proved statiscally.

The authors study the effect of compound B 58, a synthetic interferon inducer, on activity of natural killer cells (NKC) and cytostatic effectors in the mouse spleen. NKC activity in the spleen was determined in the 4-hour microtoxicity test against VAC-1 target cells labeled with /sup 51/Cr. /sup 3/H-thymidine was added to the effectors and targets. An increase in activity of the cellular mechanisms of natural antitumor resistance arising under the influence of compound B 58 is shown.

The average percentage of spontaneous plasmid-negative variants in an initial resistant population of staphylococci developingin vitro was 0.9±0.2 in broth and 1.2±0.3 in exudate; developingin vivo, in a subcutaneous suppurative inflammatory focus it was 6.3±2.0 and in the kidneys 9.5±2.6. The average percentage of spontaneous plasmid-negative variants in purulent exudatein vivo was significantly higher than in purulent exudatein vitro. The frequency of appearance of these variantsin vivo depends on the properties of the host.

The degree of the inhibiting concentration of isoniazid, kanamycin ethionamide, ethambutol and D,L-cycloserine with regard toM. tuberculosis var. avium increases with a decrease in the oxygen content of the medium. The inhibiting concentration of D-cycloserine does not depend on the oxygen content of the medium. The bactericidal effect of isoniazid on tuberculosis mycobacteria grows with an increase in the aeration of the medium and slows down with its decline. An analogous relationship in a somewhat lesser degree was revealed in experiments with D,L- and D-cycloserine and was manifest slightly, if at all, in experiments with ethionamide, ethambutol and kanamycin.

Five new 2,6-disubstituted derivatives of benzothiazole have been synthesized. The relation between the chemical structure and the biological activity of these compounds has been studied. It was found that the antimicrobial activity increases when sulfur in the number 2 position is oxidized to the sulfoxide and when the nitro group is introduced into the number 6 position in the corresponding sulfides. The preparations show no chemotherapeutic action in the animal body.

The frequency of the transduction of plasmids rms5, rms7, pT127, pC194, pS194 and pUB101 by phages belonging to serological group B (80, 52, 52A, 53, 85, phi 11, S2) in two systems was compared. In system 1 phages for transduction were obtained from plasmid-containing lysogenic donors in the process of induction with mitomycin C; in system 2 phages for transduction were obtained by their multiplication in plasmid-containing nonlysogenic donors. In system 1 the transduction of plasmids rms5, rms7, pT127, pS194 by phage 52A was found to occur with a greater (by 3-5 orders) frequency than in system 2 (the frequency of transduction was 10(-2) to 10(-4), and 10(-6) to 10(-8) respectively). A similar situation was observed with plasmids rms5 and rms7 and phage 52; plasmid pT127 and phage 53; but not observed with plasmids rms5 and rms7 and phages 80, phi 11 and S2; plasmids pC194 and pS194 and phage 53; plasmid pUB101 and phages 52A, 80 and phi 11; plasmids pC194, pS194 and pT127 and phage 85.

It has been established that the administration of homologous interferon to mice activates the production by splenocytes of a factor increasing the bactericidal properties of macrophages in the peritoneal exudate. The activation effect was dose-dependent. The maximum production was noted on day 3 after the injection of interferon, type I, or its inducers. Cell-producers of the factor are mainly lymphocytes.

The study of the staphylococcal population heterogeneity with respect to methicillin resistance by 2 methods revealed different numbers of the resistant cells in the population. Thus, when the microbial suspension was plated on an agarized medium with methicillin (50 gamma/ml), only 0.0007--0.0005 per cent of the resistant cells were found. When the colonies were replicated from a medium without methicillin to a medium containing methicillin (50 gamma/ml), 84.3--97.3 per cent of the resistant microbial cells were found in the population of the same strains. The main mechanism in the heterogeneity of the staphylococcal population with respect to methicillin resistance was impairement of the phenotype manifestation of the antibiotic resistance under definite conditions.

The dynamics of staphylococci sensitivity to antibiotics in purulent foci of 242 in patients was studied. Two criteria were used for estimation of the above process, i.e. (I) sensitivity to the antibiotics of the whole staphylococcal population isolated and (2) sensitivity to the antibiotics of separate strains isolated from the population. In 10 or more days the number of the in-patients with staphylococci resistant to streptomycin, monomycin or oxacillin in the purulent foci increased. The number of the in-patients with penicillin, neomycin or chloramphenicol resistant staphylocci was statistically doubtful. The average number of the antibiotic resistant microbial cells in the staphylococcal populations of the foci in the patients during their stay in the hospital increased 2-4 times.

The methicillin resistant strains were isolated from 62.2 per cent of the cases with acute purulent-inflammatory processes in the lungs. High resistance levels to all widely used antibiotics, stability to the effect of heavy metal salts, as well as virulence were characteristics of methicillin resistant staphylococci. Successful elimination of the determinants of methicillin resistance from some strains and their stability in the others indicate to the dual nature of methicillin resistance in clinical staphylococcal strains.

The experiments showed that among 110 Salmonella strains resistant to 1 or more antibiotics 54.5 per cent of the cultures transfered their resistance to E. coli when grown in vitro. The frequency of the transmissive drug resistance in Salmonella was directly proportional to the number of r-determinants in R-factor. The cultures resistant to ampicillin and kanamycin proved to be the most competent donors.

A method of site-specific cleavage of some tRNAs and rRNAs at the 7-methylguanine residue is described. After reduction of 7-methylguanine by sodium borohydride treatment in the presence of the exogenous carrier RNA methylated statistically by dimethylsulfate the polynucleotide chain is cut at the modified residue by aniline. It was shown that the previously used procedure which did not involve a methylated carrier RNA is not applicable for splitting rRNAs or low concentrations of tRNAs. The method described was successfully used for site-specific cleavage of 32P-terminally labelled yeast tRNAPhe and unlabelled E. coli 16S rRNA at 7-methylguanine position.

The concept on the epidemiological mechanism of formation of drug-resistant bacterial populations was substantiated. Investigations showed the epidemiological mechanism of formation of the drug-resistant populations of staphylococci in the purulent-inflammatory foci to be represented in 81.4 per cent of the patients by superinfection with resistant microbes. The latter group of the patients could be divided into two subgroups: in one of the subgroups (34.9 per cent) a reduction of sensitivity in the initial staphylococci was seen in the presence of donor bacteria which later either remained in the focus or were eliminated. In this case it could be supposed that there occurred a preliminary infection with the resistant staphylococci or with the transducing bacteriophages.

The antitoxic activity of leucocytic injection interferon I and immune interferon was shown in experimental erythrocyte hemolysis in the presence of staphylococcus alpha-toxin. The antitoxic effect was directly proportional to the interferon concentration in the medium and inversely proportional to the toxin concentration. Neutralization of the antiviral activity of leucocytic interferon did not lower its antitoxic effect. The highly purified and concentrated preparation of leucocytic injection interferon I and recombinant interferon had no antitoxic effect.

A method for analysis of antibiotic sensitivity restoration in staphylococcal populations in humans is described. The mechanism of streptomycin sensitivity restoration in staphylococcal populations on the nasal mucosa of 59 patients 7 months after discharging from surgical stationary was studied. Quantitative estimates for loss of superinfecting streptomycin-resistant staphylococci, initial streptomycin-resistant staphylococci and resistance determinants by the initial streptomycin-resistant staphylococci are presented.

Administration of leukocytic interferon to patients with sepsis and purulent resorptive fever resulted in a reliable increase in the indices of the host protective factors with respect to the infections. Addition of interferon to the surgical and antibacterial treatment promoted a decrease in the periods providing the clinical effect to 19.8 days against 43.8 days in the treatment without interferon.

The dynamics of sensitivity to penicillin of staphylococcal populations in purulent inflammatory foci of patients treated and not treated with antibiotics was estimated according to 4 indices. No reliable differences in the dynamics of sensitivity to penicillin were found in 2 groups of the patients, when estimation was performed with respect to the frequency of the penicillin resistant or penicillin sensitive staphylococci and detection of the penicillin resistant staphylococci by direct inoculation of the focal excretion to the medium with penicillin. A reliable increase in the percentage of the penicillin resistant staphylococci in the microbial population was observed only in the patients treated with penicillin.

The literature and experimental data on stability of resistance ot benzylpenicillin, streptomycin, tetracycline, chloramphenicol, erythromycin, kanamycin and methicillin in staphylococci stored for prolonged periods of time on artificial nutrient media were systematized. When the staphylococcal cultures were not in contact iwth the antibiotics for 5 to 18 months, sensitive cells to separate drugs were detected in 51.0--67.4 per cent of the strains. The number of such cells in the populations of some strains ranged from 1 to 100.0 per cent. The effect of the resistance multiplicity on the stability of resistance to benzylpenicillin, tetracycline, chloramphenicol and erythromycin was not observed.

Transmissivity of drug resistance in wild strains of E. coli was shown to be determined not only by the presence of the transmissive factor and r-determinants in the microbial cell, but also by the functional activity of the R-factor. The capacity of the R-factor for transmission depended to a certain extent on the number of the r-determinants in the R-factor and the presence of various r-determinants in the R-factor.

The effect of the in vivo treatment with synthetic interferon inducer B-58 on natural killer (NK) and cytostatic cell activity was studied in CBA and A/Sn mice. A marked increase in NK cell activity against target cells YAC-1 was observed in the spleen of CBA mice within 4 days after treatment. On the other hand, NK activity in A/Sn mice was not affected by B-58. However, B-58 was shown to enhance cytostatic cell activity both in CBA and A/Sn mice, when tested against target cells P 815.

Antistaphylococcal bacteriostatic and bactericidal activity was detected in the preparations of human leucocytic interferon of the native type. Such an activity could not be neutralized by the interferon antibodies. However, it was usually lost during the process of interferon purification. It is concluded that this antistaphylococcal activity is not related to interferon and is due to other factors which can be isolated with induced leucocytes.