Preparation a Recombinant Form of Pneumolysin Protein from Streptococcus pneumoniae.

A recombinant form of pneumolysin from Streptococcus pneumoniae was obtained. By using Vector NTI Advance 11.0 bioinformatic analysis software, specific primers were designed in order to amplify the genome fragment of strain No. 3358 S. pneumoniae serotype 19F containing the nucleotide sequence encoding the full-length pneumolysin protein. A PCR product with a molecular weight corresponding to the nucleotide sequence of the S. pneumoniae genome fragment encoding the full-length pneumolysin was obtained. An expression system for recombinant pneumolysin in E. coli was constructed. Sequencing confirmed the identity of the inserted nucleotide sequence encoding the full-length recombinant pneumolysin synthesized in E. coli M15 strain. Purification of the recombinant protein was performed by affinity chromatography using Ni-Sepharose in 8 M urea buffer solution. Confirmation of the recombinant protein was performed by immunoblotting with monoclonal antibodies to pneumolysin.

[Immunogenic properties of experimental production series of Staphylovac-2 vaccine].

AIM: Study experimental production series of Staphylovac-2 by accumulation of specific IgG and safety. MATERIALS AND METHODS: Experimental production samples of staphylococci vaccines were studied by the accumulation of specific IgG in sera of immunized BALB/c line mice in EIA. Safety was evaluated in tests of acute and chronic toxicity including pathomorphologic and histologic, hematologic and biochemical studies, studies of the effect on central nervous system. RESULTS: A statistically significant (2.6 - 3.0 times) increase of IgG levels in sera of immunized mice compared with control was noted. In the experiments studying acute and chronic toxicity the increase in body mass and mass of internal organs differed from data obtained from control animals at no observation periods. None of the studied methods of safety evaluation showed differences of the studied vaccine series from the control. CONCLUSION: The recommended dose for subcutaneous administration into human of 200 µg is experimentally justified and could be the basis for carrying out clinical studies of staphylococci vaccines in humans.

[Study of protective activivty of "Staphylovac-2" vaccine].

AIM: Study the protective properties of "Staphylovac-2" vaccinie. MATERIALS AND METHODS: Samples of the vaccine manufactured by SPA "Microgen" based on the developed technology were studied in balb/c mice during 3- and 6-fold immunization schemes. Protective activity of the preparation was determined in experiments with active and passive protection during intraperitoneal infection, seeding of the causative agent from spleen and kidneys during intravenous infection, of animals. RESULTS: In experiments with active protection of mice for both 3- and 6-fold immunization schemes, a significant protective activity of the studied series was determined, compared with the control group of mice. Sera obtained after animal immunization (rabbits, mice) by staphylococcus vaccine had protective properties. A reduction of spleen and kidneys seeding by Staphylococcus aureus in immunized mice compared with the control group was detected in the model of generalized staphylococci infection. CONCLUSION: The preclinical studies carried out with the "Staphylovac-2" vaccine, developed baed on the complex of protective staplylococci antigens, have confirmed the high protective activity of the preparation.

[Genetic characteristics of Staphylococcus aureus No 6. strain--producer of secreted protein-containing compounds possessing protective properties].

AIM: Genotype characteristics of Staphylococcus aureus No 6: strain that is a producer of a protective protein complex. MATERIALS AND METHODS: Features of structure of 9 genes, that code synthesis of pathogenicity factors, of S. aureus--spa, coa, sea, seB, sec, pvl, tst-h, mecA and scc-mecA, that are responsible for synthesis of protein A, coagulase, enterotoxins A, B and C, Panton-Valentine toxin (PVL), heat shock syndrome protein, resistance to methicillin and staphylococci chromosomal cassette, respectively, were studied by amplification in PCR of the respective gene fragments with subsequent conduction of direct sequencing. RESULTS: The S. aureus No 6 strain under study possesses pvl gene fragments, as well as Spa and coagenes, detected in all the studied strains, that belong to t12507 and EMRSA-16 types, respectively. Sea, seb, sec genes responsible for.the synthesis of enterotoxins A, B and C were not detected in it, tst-h, mecA and scc-mecA gene fragments were not present. CONCLUSION: The detection of pvl gene fragment in the strain under study, on the one hand, and protective properties of the secreted protein-containing compound, on the other hand, give evidence in favor of the necessity of further analysis of extracellular proteome of S. aureus No 6.

[Analysis of extracellular proteome of Staphylococcus aureus 6 at the end of exponential growth phase].

AIM: Determine protein specter that Staphylococcus aureus synthesizes and secretes at early growth phase--the exponential phase. MATERIALS AND METHODS: Proteins secreted by S. aureus strain 6 into cultivation medium at the end of exponential growth phase (4.5 hours) were studied. 11 proteins were identified by liquid chromatography--mass-spectrometry method. RESULTS: Only in 3 of these proteins the presence of signal peptides was predicted, which indicates their extracellular localization; the rest of the proteins were localized predominantly in bacterial cytoplasm. 5 of 11 proteins function as enzymes of carbohydrate metabolism. Other extracellular proteins that could indicate its contamination with proteins from disrupted bacterial cells were not detected in S. aureus cultural liquid filtrate. It has been suggested that enzymes of carbohydrate metabolism can provide bacterial cells with energy necessary for passage from lag-phase into exponential growth phase. Superoxide dismutase enzyme probably provides the course of oxidation-reduction processes. Synthesis of other proteolytic enzymes and toxins is carried out at later stages of development of bacterial population. Immunization of mice with a mixture of 11 identified proteins showed their protective properties after infection by S. aureus 6 strain. CONCLUSION: Based on the above-mentioned, the complex of isolated proteins may be perspective in development of a new strategy of prophylaxis and therapy of staphylococcus infections.

[Novel type of vaccine with a combination of Toll like receptor agonists--TLR 1/2, 4, 5/6, 9].

AIM: Proof of therapeutic efficacy of a novel type of vaccine with a combination of natural Toll like receptor agonists (TLR) 1/2, 4, 5/6, 9 in infectious and noninfectious human pathology. MATERIALS AND METHODS: Immunovac-VP-4 vaccine, containing antigens of opportunistic microorganisms that are TLR 1/2, 4, 5/6, 9 ligands, was used for experiments and clinical trials. RESULTS: Immunovac-VP-4 activates innate immunity by inducing maturation of dendritic cells with expression of costimulating molecules on their membrane (CD40+, CD80+, CD86+), terminal differentiation molecules--CD83+ and antigen-presenting molecules (MHC class I and II); activates proinflammatory (TNFalpha, IL-6) and regulatory (IL-12, INFgamma) cytokine synthesis that programs T-lymphocyte differentiation by Th1 pathway; increases cytotoxic activity of natural killer cells, inhibits melanoma B16 growth and Lewis lung carcinoma metastasis. Therapeutic effect of Immunovac-VP-4 was evident regardless of pathology by a significant decrease of quantity and severity of recidives, improvement of all clinical parameters, reduction of quantity of administered pharmaceutical preparations including antibiotics and glucocorticosteroids. The rate of intercurrent acute respiratory viral illnesses and their bacterial complications decreased. Immunovac-VP-4 therapy modified course of illness from severe into milder forms. Positive therapeutic effect was 69.2 - 100%. CONCLUSION: High therapeutic effect of vaccine therapy is based on innate immunity activation by combining TLR agonists. Immunovac-VP-4 contains an optimal combination of natural TLR agonists that ensure high therapeutic effect in various nosological forms of infectious and noninfectious human pathology.

Growth-dependent release of carbohydrate metabolism-related and antioxidant enzymes from Staphylococcus aureus strain 6 as determined by proteomic analysis.

Proteins released into the culture medium by Staphylococcus aureus (S. aureus) strain 6 were determined at the end of the exponential growth phase (4.5 h). Eleven proteins were identified by liquid chromatography coupled with mass spectrometry. Three proteins were predicted to have signal peptides indicating their extracellular localization. The other proteins were presumably located in the cytoplasm of the bacteria. Five out of the 11 proteins were involved in carbohydrate metabolism. Other intracellular proteins of S. aureus were not detected in the culture medium. This indicates that the release of these 11 proteins was specific and that unspecific protein release due to damaged or dying bacteria did not play a role. It is suggested that enzymes associated with carbohydrate metabolism may provide the energy necessary for the transition of bacteria from a resting to a proliferative state. Another enzyme released by S. aureus, superoxide dismutase, may catalyze redox reactions in this context. The production of other proteolytic enzymes and toxins may take place at later stages of bacterial growth. A cocktail of these 11 proteins was used for the immunization of mice. Indeed, vaccination with these proteins prolonged the survival times of mice upon infection with S. aureus strain 6. Therefore, these proteins may have implications for the development of novel strategies for the prevention and therapy of S. aureus infections.

[Prevention and immunotherapy of staphylococcal infections with bacterial vaccines].

Pathogenesis of staphylococcal infection both local and systemic is associated with many pathogenicity factors, which in foreign literature are called virulence factors of Staphylococcus aureus, which were studied as potential candidates for vaccine development. Much difficulties are related to use of known experimental models, which virtually do not allow to determine direct appropriate effect by survival of animals, as well as to data about absence of correlation between increase of antibody titers in animals and protective effect of studied preparations. Despite the importance of the problem of prevalence and severity of staphylococcal infection and intensive research in order to determine protective components able to protect from infection caused by S. aureus, there are no licensed prophylactic preparations with proven efficacy.

[Characteristics of antigenic complexes of Staphylococcus aureus vaccine strains obtained in different cultivation conditions].

AIM: Assessment of characteristics of antigenic complexes of Staphylococcus aureus vaccine strains in different cultivation conditions. MATERIALS AND METHODS: S. aureus vaccine strains (No. 5, 9, 1986, 1991) were grown in liquid nutrient media--full value and semi-synthetic--as well as on solid medium. Reactor cultivation was performed in the fermenter ANKUM-2M. Complex of antigens were obtained by water extraction method applied to staphylococcal biomass inactivated with acetone and assessed by common methods on protein and carbohydrate content; specific activity was assessed by minimal inhibitory dose in passive hemagglutination inhibition assay. Study of acute toxicity was performed on outbred mice. RESULTS: Using strain no. 1991, model of reactor cultivation in full value medium with separation of biomass by microfiltration was validated on the basis of biomass and semiproduct of antigenic complex (acetone powder) yield as well as productivity of biomass cumulation. Study of antigenic complexes obtained from biomass of 4 strains during reactor cultivation compared with complexes extracted from cultures grown on solid medium revealed increased protein and decreased carbohydrate content but similar specific activity. It was demonstrated that complex of antigens obtained from cultures grown either by reactor cultivation or on solid medium were non-toxic. CONCLUSION: New technology for manufacturing staphylococcal complex of antigens with reactor cultivation of vaccine strains in full value medium with subsequent purification of antigenic complex from the biomass by microfiltration was developed. Results of the study demonstrated the usefulness of the developed technology for both further studies on a cellular staphylococcal vaccine and manufacture of staphylococcal component of "Immunovac" vaccine.

[Immunovac-VP-4 for prevention of acute respiratory diseases in children's communities].

AIM: Assessment of prophylactic effect of Immunovac-VP-4 against acute respiratory diseases (ARDs) in collectives of children. MATERIALS AND METHODS: Immunovac-VP-4 was used for prophylaxis of ARDs in communities of children (daycare centers, boarding school). During 3 consecutive controlled studies performed during peak incidence of epidemic influenza and ARD (1st study) and during pre-epidemic period (2nd and 3rd studies) the incidence of ARDs was studied in groups of vaccinated and non-vaccinated children. Criteria for assessment of ARDs incidence and their severity were as follows: number of ARD episodes per child, number of children with recurrent episodes of ARD during period of follow-up, duration of ARDs, and number of children with bacterial complications (bronchitis, otitis, etc.). Effect of intervention on immunologic parameters was assessed on levels of sIgA, IgG, IgA, and antibody titers in saliva. Schedule of vaccine administration was 3 daily intranasal and 6 - 9 oral administration of the vaccine with 3 - 4 days interval. RESULTS: Results of all three studies were virtually the same: decrease of number of ARD episodes and their duration; 3.1-fold reduction of number of children with recurrent infections during 14 months of follow-up, and 10.9--12-fold reduction during first 7 months of follow-up; decrease of bacterial complications rate (1.9% in children in intervention group, 11.9%--in control group). Increase of total immunoglobulin level and antibody titers to antigenic components of the vaccine from low baseline level was observed in saliva of vaccinated children. CONCLUSION: Immunovac-VP-4 results in enhanced and prolonged prophylactic effect on ARDs incidence and recommended for building of optimal system of prevention of polyetiologic complex of ARDs.

[Effect of acellular staphylococcal vaccine "Staphylovac" on several indicators of immune system in mice].

AIM: To study molecularand cellular mechanisms of action of "Staphylovac" vaccine on effectors of innate immunity in mice. MATERIALS AND METHODS: Dynamics of changes of mice spleen mononuclear leukocytes immunophenotype under the influence of the vaccine at determined time intervals was assessed in the study. RESULTS: In response to antigenic stimulation mice immune system reacted by increasing number of cells expressing molecules of T-cell subpopulations--CD3, CD4, CD8; B-cell subpopulation--CD19 and natural killers--NK. Activation of immune system of mice was noted which expressed as increased levels of molecules for antigenic presentation (MHCI, MHCII), regulatory molecules (CD4/CD25) as well as enhanced cytotoxicity potential and phagocytic activity. CONCLUSION: Staphylococcal vaccine as well as studied antigens of Staphylococcus aureus promote activation of immune system. Cytotoxic potential obtained by NK cells under their influence as well as enhancement of phagocytic activity of macrophages point to activation of innate immune mechanisms.

[Correction of cytostatic-induced immunosupression with staphylococcal vaccine in mice].

Studies aimed on evaluation of possibility for correction of cyclophosphan-induced immunosuppression 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 CD25 (assessment after 4 hours). Administration of SV along side with cytostatic does not influenced significantly on characteristics of CP-induced immunosuppression 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 along side with CP increased expression of MHC II on 38- and 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 3.3- and 2.3-fold (CD3), 12- and 4-fold (MHC I), and in 2.8- and 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.

[Protective activity of secreted proteins of Streptococcus pneumoniae and Klebsiella pneumoniae].

Protective efficacy of secreted proteins of Streptococcus pneumoniae and Klebsiella pneumoniae cultivated on cardiocerebral broth and semisynthetic growth medium respectively was studied in vivo. Fraction with molecular weight 30 - 50 kDa obtained by the method of membrane fractionation had high protective efficacy. Two-dose immunization of mice with this fraction provided 80 - 100% protection from infection by homologous strains of S. pneumoniae and K. pneumoniae. Cross-protective activity of the fraction was revealed when infecting immunized mice by different K-types of K. pneumoniae. Blood sera of mice immunized with 30 - 50 kDa fraction possessed preventive features protecting from infection 90% of animals while 100% of death in the control group. It was determined that protective efficacy of the mentioned fraction was determined by protein-containing antigens because proteolytic disruption of the protein component resulted in loss of protective properties of the preparation.

[Growth medium for Neisseria meningitidis isolation].

Growth medium for isolation of N. meningitidis, which do not require addition of serum and based on previously developed medium for cultivation of bacteria from Haemophilus genus (without growth factors V and X) was constructed. Selective properties of the medium in relation to meningococci were determined by addition of vancomycin and colistin--antibacterial supplement inhibiting growth of nonpathogenic Neisseria and outside microflora. Developed medium was successfully approved during examination of children for nasopharyngeal carriage of meningococci.

[Experimental, clinical and immunologic assessment of acellular staphylococcal vaccine "Staphylovac"].

Results of experimental, clinical and immunological effects of acellular dry staphylococcal vaccine "Staphylovac" developed in Mechnikov Research Institute of Vaccines and Sera are presented. Original mildly virulent strains of Staphylococcus aureus having high immunogenicity, and intra- and interspecies protective activity against different representatives of opportunistic microflora were used for construction of the preparation. Low-toxicity and weak anapylactogenicity of the vaccine were established. In experiments on mice, guinea pigs and rabbits significant protective, antigenic and immunomodulate activity of the preparation was revealed with low sensitization of animals. Clinical trials performed in different centers showed that inclusion of vaccinotherapy in complex treatment of chronic staphylococcal infections (chronic pyodermia, lung abscess etc.) resulted in prolonged pathologic locus, decrease of number and severity of exacerbations, prolongation of remission, and complete recovery in significant number of patients. Activation of innate and adaptive immunity was revealed in the same patients. It was shown on the large group of athletes that administration of the vaccine by aerosol route prevents disruption of immunologic adaptation occurring due to excess physical activity and stress situations during competitions.

[Protective effect of the aerosol immunization of mice with the polycomponent vaccine Immunovac VP-4 after the challenge of the animals with Klebsiella pneumoniae virulent strain].

Used four schemes of the administration of the preparation with different time of the exposition of the animals in an aerosol chamber were tested with their subsequent intraperitoneal challenge with K. pneumoniae virulent strain K16. Irrespective of the number of immunization courses, the administration of the preparation made at intervals of 1 day, or daily, did not ensure any protective effect, but only led to an insignificant increase in their survival time in comparison with nonimmunized animals. After intervals between immunizations were increased to 3 days the protective effect of aerosol immumization was obtained (the survival rate was 65-80 % and considerably differed from that of the controls). The protective effect of aerosol immunization thus obtained was comparable with the effectiveness immunization made in a single subcutaneous injection. Aerosol immunization resulted in low antibody titers to the antigens contained in the vaccine, while after a single subcutaneous injection high antibody titers to Klebsiella and Proteus antigens were detected. The antigen-stimulated blast transformation of spleen lymphocytes in mice subjected to aerosol immunizations in 5 exposures was high. After subcutaneous immunization significant changes in such characteristics were detected on day 15. The data thus obtained were indicative of good prospects in the development Immunovac VP-4 as the medicinal form intended for use in aerosols.

[Immunological characteristics in the therapy of atopic dermatitis in children with polycomponent vaccine Immunovac BN-4].

IgE-mediated reactions linked with the polarization of the immune process towards, mainly, the activation of Th2 lymphocytes which synthesized interleukins, responsible for switching over B lymphocytes to the production of IgE, were found to be the most important mechanism of the pathogenesis of atopic dermatitis (AD). The use of immunomodulating preparations, capable of changing unbalance in the system of Th1/Th2 cells, is one of promising approaches to the complex therapy of AD. Poly-component vaccine Immunovac BN-4 was included into the complex therapy of AD in children, The dynamics of immunological characteristics was studied for the period of 6 months after the end of the course of therapy. A considerable increase in the absolute and relative amount of lymphocytes with markers CD3, CD4, CD16, CD21, CD25, a rise in the levels of IgA, IgG and a decrease in the level of total IgE in the blood serum were established. The inclusion of the polycomponent vaccine into the complex therapy of AD may be recommended.

[Associated synthesis of some secreted pathogenicity factors of Klebsiella pneumoniae].

The study was carried out to evaluate the substrate specificity and activity of proteases secreted by strains of Klebsiella pneumoniae with various degree of virulence. The process included cultivation of the strains in semi-synthetic medium, after which the biomass was inactivated and the supernatant was separated from bacterial cells through centrifugation. Elastase-, trypsin-, and chymotrypsin-like proteolytic activity was measured in the supernatant and in all fractions obtained through gel-filtration, followed by DEAE-sepharose purification. Regardless of the degree of virulence, all the studied strains of K. pneumoniae secreted only one proteolitic enzyme, which was elastase with molecular weight of about 21 kDa. Addition of glycoprotein--the main structural component of eucaryotic cells--into the culture medium in the beginning of incubation, increased protein, polysaccharide, and lipopolysaccharide synthesis; proteolythic activity in the supernatant fluid increased from 7,476 to 15,731 mU/ml. The increase was associated with an elevation of polysaccharide synthesis from 173 to 349 mg dry weight. However, proteolythic activity per 1 gr of polysaccharide did not increase; it was 43.3 and 45.1 units, respectively. Thus, proteolytic activity increased in direct propotion to the increase of polysaccharide synthesis into the culture medium.

[Optimization of the therapy of atopic dermatitis by means of immunotherapy].

Taking into account disturbances in the functioning of the immune system in atopic dermatitis (AD) and the potentiating role of staphylococcal and other infections, the possibility of the optimization of the therapy of AD with the use of preparations having immunomodulating action and immunogenic activity is proposed. In the complex therapy of AD in children we used polycomponent vaccine Immunovac B-4, introduced intranasally and orally. Under the influence of immunotherapy the clinical characteristics of the patients had pronounced positive dynamics. A considerable decrease in the spread of the process, the degree of its severity and subjective symptoms was noted shortly after the course of vaccine treatment. Simultaneously the SCORAD index dropped from 64.5 to 39.4. During the later period of observation further decrease in the severity of the course of AD in children occurred, and the minimal characteristics were observed in 6 months of observation. At that time the SCORAD index fell to 19.9 +/- 1.34. The volume of pharmacotherapy and the number of acute respiratory infections considerably decreased, the positive dynamics of the quantitative and qualitative composition of the intestinal microflora was noted. The prolonged immunotherapeutic effect of the polycomponent vaccine made it possible to recommend the vaccine for the optimization of the therapy of AD.

[Protease activity of Klebsiella pneumoniae of different virulence]. / Proteaznaia aktivnost' Klebsiella pneumoniae razlichnoi virulentnosti.

The study of substrate specificity and activity of proteolytic enzymes secreted by K. pneumoniae strains with different virulence was carried out. The strains were cultivated in a liquid semi-synthetic medium. The biomass was inactivated, and the supernatant fluid was separated from microbial cells by centrifuging. In the supernatant thus obtained and in the fractions isolated by gel filtration with the subsequent purification on DEAE Sepharose elastase-like, trypsin-like and chemotrypsin-like proteolytic activity was determined. In K. pneumoniae strains with different virulence only a single proteolytic enzyme--elastase with a mol. wt. of 21 kD--was detected. The protease activity of the supernatant culture fluid did not depend on the virulence of the strain and was equal to 5,416-7,476 I.U./ml. The activity of the purified enzyme was 100% of the elastase-like activity of the supernatant culture fluid. The most virulent K. pneumoniae strain K2, whose LD50 for white mice was less than 10 microbial cells, was characterized by lower elastase-like activity. The absence of correlation between protease activity and K. pneumoniae virulence may be explained by the fact that surface glycoproteins of eukaryotic cells are glycosilated and thus slightly accessible for proteases.