Peculiarities of the Formation of Antimeningococcus Immunity in Mice Immunized with Fragments of N. meningitidis IgA1 Protease.

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.

Serological Analysis of Immunogenic Properties of Recombinant Meningococcus IgA1 Protease-Based Proteins.

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.

[Protective properties of recombinant IgA1 protease from meningococcus].

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.

[Isolation and determination of activity of IgA1 protease from Neisseria meningitidis].

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.

Mucoadjuvant properties of lipo- and glycoconjugated derivatives of oligochitosans.

Chitosan, (1-4)-2-amino-2-deoxy-beta-d-glucan, is a deacetylated form of chitin, an abundant biodegradable, positively charged natural polysaccharide. Chitosan is used for antigen delivery through mucosal barrier due to its ability to disrupt tight junctions. Here we produced new water-soluble low-molecular weight chitosan (LMW-Chi) lipid derivatives and compared their ability to stimulate humoral response with the effect of unmodified LMW-Chi or its oligosaccharide derivatives. LMW-Chi effectively penetrated into macrophage-like, lymphoid and epithelial cells. It also stimulated in mice IgG production to model proteins delivered either by subcutaneous or intranasal routes. Adjuvant effect of chitosan derivatives was comparable to or lower than that of unmodified LMW-Chi. Thus, it is possible that adjuvant effect is induced by unmodified glucosamine units of chitosan.

[Sialylation of N-carbohydrate chains of glycoproteins with immobilized trans-sialidase from Trypanosoma cruzi]. / Sialilirovanie N-uglevodnykh tsepei glikoproteinov s pomoshch'iu immobilizovannoi trans-sialidazy Trypanosoma cruzi.

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.

[A system of step-by-step differentiation of methicillin-resistant Staphylococcus aureus]. / Sistema poétapnogo differentsirovaniia metitsillinorezistentnykh shtammov Staphylococcus aureus]

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 Staphylococcus aureus]. / Novaia kollektsiia fagov dlia tipirovaniia metitsillino-rezistentnykh Staphylococcus aureus.

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.

[Conjugative plasmid transfer in Staphylococcus aureus?]. / Kon''iugativnyi perenos plazmid u Staphylococcus aureus?

The microbiological and electron-microscopic study of the transfer of conjugative (pG873) and nonconjugative (rms7 and pE994) plasmids in two systems, on nitrocellulose filters and in a fluid culture medium, was carried out. In both systems the low frequency of the transfer of plasmid pG873 or the absence of the transfer of plasmids rms7 and pE994 were observed when nonlysogenic recipients were used for crossing. The presence of prophage in recipient cells increased the rate of the detection of gentamicin-sensitive transconjugants 100-fold and provided to reveal the transfer rms7 and pE994 plasmids. The electron-microscopic study of specimens with lysogenic recipients revealed a picture which can be interpreted as the fusion of two cells. Such picture was not observed in crossings with a nonlysogenic recipient and in preparations obtained from separate donor and recipient cultures.

[Phage-mediated conjugative transfer of plasmids in Staphylococcus aureus]. / Oposredovannyi fagom kon''iugativnyi perenos plazmid u Staphylococcus aureus.

It was shown possible to transfer nonconjugative plasmids during joint cultivation of the donor and recipient cells by transduction and phage-mediated conjugation. In the latter case it was necessary that the phage in the medium was free and the prophage was present in the recipient cells. Differences in the regularities of the transfer of the nonconjugative plasmids mobilized by the conjugative plasmid or phage were observed.

[Differentiation of methicillin-resistant strains of Staphylococcus aureus by prophage specificity]. / Differentsiatsiia metitsillinorezistentnykh Staphylococcus aureus po spetsifichnosti profagov.

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).

[Effect of prophages on transfer frequency of conjugative plasmid G 873]. / Vliianie profagov na chastotu peredachi kon''iugativnoi plazmidy G 873.

Transfer of the conjugative plasmid G873 on filters and mixed cultivation of the donor and recipient cells in liquid media is described. In the both systems the use of the lysogenic recipient cells (phages of serogroups B and F) in the crossings increased mor than 100-fold the frequency of plasmid transfer. The conjugative transfer of the plasmid in the mixed cultivation system was proved. The conjugative transfer required the presence (while not obligatory) of calcium chloride and was restricted by the serum factors.

A new approach to establishing the set of phages for typing methicillin-resistant Staphylococcus aureus.

A new approach to using experimental phages for typing methicillin-resistant S. aureus (MRSA) non-sensitive to the phages of International Basic Set (IBS) is described. The collection Includes phage 85, modified on a culture of MRSA, and 5 phages induced from MRSA strains isolated in clinics of Moscow in 1975-76. Firstly, the modified phage selects cultures according to the specific character of its restriction-modification system, then the induced phages differentiate the selected strains into 5 groups (1, 2, 3, 4, 5) based on the specificity of the prophages they contain. Group 1 strains can further be differentiated into 5 subgroups (A, B, C, D, E) by additional phages. Forty-one MRSA strains isolated in 1987-90 in various hospitals of Moscow showing no sensitivity to IBS phages, were lysed by the modified phage, 15 of them belonging to Group 2 and isolated in the traumatological hospital, 26 belonging to Group 1 and were circulating in the burn center. Twenty-three strains of Group 1 appertain to subgroup 1B and were isolated over a 4-year period from the burned surface of patients and from the throat of a medical staff carrier.

Lysogeny of methicillin-resistant Staphylococcus aureus and the role of prophages in transfer of conjugative and non-conjugative plasmids.

The lysogenicity of 49 strains of methicillin-resistant S. aureus (MRSA) isolated in Moscow clinics in the 1970s and '80s was studied by the method of mitomycin C induction. It was found that one strain had phage of serogroup B, 33 strains had serogroup F phages and 15 strains had phages of both serogroups. In the course of genetic crossing on nitrocellulose filters it was demonstrated that serogroups B and F prophages contained in recipient cells 1) increase the frequency of transfer of conjugative plasmid pG873 and 2) mobilize transfer of non-conjugative plasmids pE994 and rms7.

[The effect of leukinferon on the activity of phagocytosing cells in human salmonellosis]. / Vliianie leikinferona na aktivnost' fagotsitiruiushchikh kletok pri sal'monellezakh u liudei.

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.

[The mechanism of the resistance of methicillin-resistant Staphylococcus aureus to phages from the International Collection]. / K mekhanizmu ustoichivosti metitsillinrezistentnykh Staphylococcus aureus k fagam Mezhdunarodnogo nabora.

The resistance of methicillin-resistant staphylococci to phage 85 is due to the presence of a certain system restriction modification in microbial cells. The loss of the capacity for restricting phage DNA by the cell as the consequence of the loss of the mec determinant is not accompanied by the loss of its capacity for modifying phage DNA.

[Clinical and laboratory effect of leukinferon in purulent infections]. / Kliniko-laboratornyi éffekt leikinferona pri gnoinoi infektsii.

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.

Association between phage DNA injection and generation of the protonmotive force in Staphylococcus aureus.

Suppression of protonmotive force generators in staphylococci lead to a loss of phage infection efficiency. KCN inhibited phage infection by 49.5-53.5%; DCCD by 51.0-61.4%; CCCP by 59.2-68.8%. Suppression tock place at the stage of phage DNA transport. Valinomycin in concentration of 0.5 microM evoked dissipation of the membrane potential, nigericin caused a reduction of the gradient pH in the staphylococci membrane at a concentration of 12.0 microM. Individually, these antibiotics did not have an essential influence on the efficiency of phage infection but when combined, a maximum inhibition of phage infection (64.5%) occurred at the stage of introducing phage DNA into the bacterial cell implicating the participation of the membrane potential and pH gradient in the transportation of phage nucleic acid to the staphylococcal cell.