Vol 86, No 1 (2009)

Polymorphism of tox and dtxR genes responsible for diphtheria toxin synthesis was revealed. Seven point mutations in tox gene were detected; study of their combinations allowed to determine 10 allelic variants of the tox gene in C.diphtheriae strains. Majority of mutations did not lead to changes in substitutions in amino acid sequence of diphtheria toxin. In tox gene from 2 strains of mitis biovar, ribotype «Otchakov» isolated in Saint-Petersburg, mutatio n in position 1252 (G → C) , which corre- sponds to change of glycine on arginine in amino acid sequence of diphtheria toxin (G393R), was identified. Mutation localizes in R domain of fragment B of diphtheria toxin. In the dtxR gene 16 point mutations were registered; study of their combinations allowed to determine 10 allelic variants of the dtxR gene. Two mutations led to amino acid substitutions in regulatory protein DtxR: in position 640 (C→A), which corresponds to change of leucine on isoleucine (L214I), and in position 440 (C→T), which corresponds to change of alanine on valine (A147V). Mutation A147V is characteristic for all strains of epidemic clonal group (strains of biovar gravis , ribotype «Sankt-Peterburg/Rossija», enzymetypes of complex 8), dominated in Russia during diphtheria epidemic at 1990s. Strains of this group were characterized by high level of diphtheria toxin production.

Phase I clinical trial of inactivated virosomal split influenza vaccine «Grifor®» was conducted in the Mechnikov Research Institute of Vaccines and Sera as accredited base for such trials. Forty healthy volunteers (males and females) aged 18—50 years consented to participate in the trial. Reactogenicity, safety, and immunogenicity of new Russian influenza vaccine were assessed. Analysis of obtained results showed that there was evidence of safety and low reactogenicity of the vaccine as well as of its high immunogenic characteristics, which satisfied both the EMEA’s Committee for Proprietal Medicinal Products criteria and requirements of Federal Service for Surveillance for Protection of Consumers Rights and Human Welfare (MU 3.3.2.1758-03) for inactivated influenza vaccines.

Immunogenic properties of recombinant protein ScaAB mixed with polypeptide P6 as well as conjugate of the former with capsular polysaccharide type III (PSIII) were assessed. Protein-polysaccharide conjugate was synthesized by reductive amination method. Immunogenicity was studied on the mouse model using aluminium hydroxide as adjuvant. Antibody titers in antisera were measured by immunoenzyme assay. Functional activity of antibodies was evaluated by opsonophagocytic test. Immunization with ScaAB-PSIII conjugate resulted in increased immune response to both ScaAB and polysaccharide. Administration of ScaAB and P6 proteins mixture compared to their separated administration led to increased of antibody titer and prolonged circulation of specific antibodies. Injection of studied vaccines increased opsonizing activity of antisera compared to immunization with uncombined components.

Studies aimed on evaluation of possibility for correction of cyclophosphan-induced immunosupression in BALB/c mice by using acellular staphylococcal vaccine «Staphylovac» (SV). Cyclophosphan (CP) administered to mice four times with 24 hours intervals decreased levels of T-, B-, T-regulatory (T-reg CD4/CD25/Foxp3) lymphocytes, increased quantity of cells expressing early activation marker D25 (assessment after 4 hours). Administration of SV alongside with cytostatic does not influenced significantly on characteristics of CP-induced immunosupression at the moment of its assessment. Twenty four hours after administration of CP or SV with CP level of cells expressing CD3 and MHC I continued to decrease as compared with control. Compared with administration of CP only or with control group, SV administered alongside with CP increased expression of MHC II on 38and 1.8-fold respectively. Levels of CD4, CD25, CD8, and CD19 cells in these groups were already closer to control values that points to the beginning of restoration of some disturbances in mechanisms of immunoregulation. Five days after administration of CP or CP+SV levels of CD3, MHC I, and CD8 lymphocytes significantly increased, although were lower than in the control group in 3.3and 2.3-fold (CD3), 12and 4-fold (MHC I), and in 2.8and 1.8-fold (CD8) respectively. Levels of NK, NKT were higher as compared to control. CP continued to decrease levels of CD4 and CD19 cells and simultaneously increased level of T-regulatory cells, which play key role in suppression of immune response. Administration of SV during CP course corrected levels of cells expressing these markers. It was established that under the influence of SV, cytotoxic potential of NK cells and proliferative activity of lymphocytes were restored.

Levels of IgG and IgA to complex of Bordetella pertussis antigens were assessed in 503 healthy children aged 1 — 14 years, 75 adolescents aged 15 — 17 years, and in 504 adults aged 18 — 54 years. The highest level of IgG was observed in children aged <5 years. In older age groups progressive decrease of IgG level was noted, which more most prominent in 9 — 11 year-olds with subsequent stabilization of the level in adolescents and adults. Significant heterogeneity of IgG level was noted in all age groups. Rate of detection of increased IgA level correlated with age-related decrease of IgG level and increased from 2 — 5% in children aged 1 — 5 years to 12 — 16% in older children and adults. Obtained data point to low levels of immunity against pertussis in older children, adolescents and adults and high undetected incidence of pertussis in studied population.

During spring-autumn period of 2006 Borrelia were isolated for the first time in Russia from blood of 79 patients treated in Perm City Clinical Hospital for Infectious Diseases No. 1 with diagnosis «tickborne borreliosis, manifestive form with migrating erythema, localized stage». Ten primary isolates (12.7% of total seeded samples) were obtained by seeding plasma samples on the BSK medium. Their subsequent identification by polymerase chain reaction-restriction fragments length polymorphism revealed presence of Borrelia garinii NT29 in all patients. Length of sequenced fragment of rrfA-rrlB region was 253 b.p. Seven isolates had 100% and 3 — 99.6% similarity with typical strain NT29 (L30130). Nucleotide sequences of 4 obtained isolates were deposited in GenBank database (No. AM932199 — AM932202). It was proposed that B.garinii NT29 more frequently than other Borrelia species can be an etiologic agent of tick-borne borreliosis not only in Perm region but also in whole Russia.

Results of study of microecological disorders in oral cavity of patients with non-specific ulcerative colitis (NSUC) and Crohn’s disease (CD) and control subjects (patients with hypertension). Condition of mucosa was assessed on the basis of morphological data and electrophoretic mobility of cell nuclei, whereas structure of microbiocenosis and metabolic activity of microflora - on the basis of saliva bacterial culture and contents and profile of volatile fat acids in it. Detection rate of negative charge of the cell nuclei (decrease of functional activity of epithelium) was significantly higher in patients with NSUC and CD (66.6%) compared with controls (10%). This fact was directly related with hypercolonization of oral cavity by Gram-negative microflora. Lesions of mucosa which are characteristic of NSUC and CD and determined by pathologic immune mechanisms correlated with quantity of pathogenic microflora ( Staphylococcus aureus and Candida ). Marked differences of chromatograms’ patterns were observed in patients with NSUC and CD indicating the suppression of anaerobic microflora in patients with CD and hypercolonization of oral cavity by anaerobic microflora in majority of patients with NSUC.

Testing of immunocompromised patients for markers of β-herpesviruses — human herpesvirus type 6 (HHV-6) and cytomegalovirus (CMV), as well as γ-herpesvirus — Epstein-Barr virus (EBV), revealed that all mentioned infections are frequently detected, mainly as mixed infections. Chronic HHV6 infection was diagnosed in more than half of the patients, whereas markers of acute phase of CMV and EBV infections were detected in 25% and 15% of patients respectively.

Attempt to consider an infection as a model of associative symbiosis was made. Assessing symbiosis as a biologic basis of infectious process, author pays attention to change of paradigm in symbiology and emergence of a new concept - associative symbiosis. Main structural and functional elements of associative symbiosis are reviewed and their analogy to vectors of infectious process was established. Special attention is paid to associative symbionts (pathogenic and opportunistic microorganisms) as well as to their universal way of interaction with host and dominating normal flora. It was pointed out that the basis of interaction between symbionts is determined by change of persistence potential of both infectious agent and commensal microorganisms including indigenous microflora during interactions with each other. Data characterizing the role of symbionts intercellular interactions on the level pro-prokaryotes, pro-eukaryotes during infection and unveiling pathogenetic basis of infectious processes are provided.