Recombinant Pneumolysin of Pneumococci Induces TLR4 Expression and Maturation of Dendritic Cells In Vitro.

Pneumolysin (Ply) is a target for the development of serotype-independent pneumococcal vaccines, an important condition for the efficacy of which is their ability to activate innate immunity with the subsequent formation of adaptive immunity. In this study, the ability of recombinant full-length Ply (rPly) of pneumococci to induce TLR expression and maturation of dendritic cells generated from mouse bone marrow was evaluated. It was shown that rPly in vitro increased the number of dendritic cells expressing Toll-like receptor 4 (TLR4) on the membrane. rPly caused maturation of dendritic cells generated from mouse bone marrow, which manifested in a decrease in the number of progenitor cells (CD34), an increase in the number of cells expressing the adhesion molecule CD38, costimulatory molecules CD80 and CD86, molecules of terminal differentiation of dendritic cells CD83, as well as molecules of antigenic presentation of the major histocompatibility complex class II.

Synthetic Analogs of Streptococcus pneumoniae Capsular Polysaccharides and Immunogenic Activities of Glycoconjugates.

Streptococcus pneumoniae is a Gram-positive bacterium (pneumococcus) that causes severe diseases in adults and children. It was established that some capsular polysaccharides of the clinically significant serotypes of S. pneumoniae in the composition of commercial pneumococcal polysaccharide or conjugate vaccines exhibit low immunogenicity. The review considers production methods and structural features of the synthetic oligosaccharides from the problematic pneumococcal serotypes that are characterized with low immunogenicity due to destruction or detrimental modification occurring in the process of their preparation and purification. Bacterial serotypes that cause severe pneumococcal diseases as well as serotypes not included in the composition of the pneumococcal conjugate vaccines are also discussed. It is demonstrated that the synthetic oligosaccharides corresponding to protective glycotopes of the capsular polysaccharides of various pneumococcal serotypes are capable of inducing formation of the protective opsonizing antibodies and immunological memory. Optimal constructs of oligosaccharides from the epidemiologically significant pneumococcal serotypes are presented that can be used for designing synthetic pneumococcal vaccines, as well as test systems for diagnosis of S. pneumoniae infections and monitoring of vaccination efficiency .

Wavelet compression of off-axis digital holograms using real/imaginary and amplitude/phase parts.

Compression of digital holograms allows one to store, transmit, and reconstruct large sets of holographic data. There are many digital image compression methods, and usually wavelets are used for this task. However, many significant specialties exist for compression of digital holograms. As a result, it is preferential to use a set of methods that includes filtering, scalar and vector quantization, wavelet processing, etc. These methods in conjunction allow one to achieve an acceptable quality of reconstructed images and significant compression ratios. In this paper, wavelet compression of amplitude/phase and real/imaginary parts of the Fourier spectrum of filtered off-axis digital holograms is compared. The combination of frequency filtering, compression of the obtained spectral components, and extra compression of the wavelet decomposition coefficients by threshold processing and quantization is analyzed. Computer-generated and experimentally recorded digital holograms are compressed. The quality of the obtained reconstructed images is estimated. The results demonstrate the possibility of compression ratios of 380 using real/imaginary parts. Amplitude/phase compression allows ratios that are a factor of 2-4 lower for obtaining similar quality of reconstructed objects.

[BALANCE OF Thl/Th2/Th9/Thl7/Th22 CYTOKINES IN POST-OPERATION PERIOD IN PATIENTS WITH MALIGNANT TUMOR OF LIVER].

AIM: Evaluate cytokine status in patients with malignant liver cells after surgery. MATERIALS AND METHODS: 33 patients aged 35 to 76 years were included into the study. Blood was obtained before the operation and in the post-operation period: after 6 and 24 hours and at day 7 Cytokine profile (IL-Ib, IL-2, TNF-α, IFN-γ, IL-12p70, IL-4, IL-5,IL-6, IL-10, IL-13, IL-9, I-17a, IL-22) was evaluated using Multiplex- 13 system (Bender MedSystems, Austria). RESULTS: In patients levels of all the studied cytokines (Thl/Th2/Th9/Thl7/Th22) were already increased before the operations, that gives evidence of the presence of an inflammatory proc- ess connected with activation of immune system effectors. CONCLUSION: Disbalance of cytokine system helper cells resulting in functional and organic alterations through induction of the "cytokine storm" may aggravate the state of these patients. Further studies on the correction of cytokine system in these patients are thus needed.

[SYNTHETIC CONJUGATED ANALOGUES OF CAPSULE POLYSACCHARIDES OF PNEUMOCOCCUS - AN INSTRUMENT FOR DETECTION OF POST-VACCINATION ANTIBODIES].

AIM: Evaluation ofthe ability of capsule polysaccharides (CP) of Streptococcus pneumoniae serotype 3 and 14 and their synthetic structure analogues, conjugated with bovine serum albumin (BSA), to detect antibodies in post-vaccination sera of mice. Materials andmethods. Oligosaccharides correspond- ing to one, one and a half and two repeating links of serotype 3 and 14 S. pneumoniae CP were synthe- sized, their conjugates with BSA were produced by squarate method as well. Ligand content-per BSA molecule was controlled by MALDI-TOF spectrometry. Immune sera were obtained after 2 intraperi- toneal administrations to mice of glucoconjugates adsorbed on aluminum hydroxide or 13-valent pneumococcal conjugated vaccine. Determination of levels of post-vaccination class G antibodies and their sub-isotypes was carried out in EIA. RESULTS: Immunization of mice with neoglucoconjugates resulted in formation of predominantly IgGl recognizing serotype 3 and 14 S. pneumoniae C. IgG1 in mice immunized with a 13-valent conjugated vaccine recognized serotype 3 S. pneumoniae CP, but detected serotype 14 S. pneumoniae CP weakly. All the conjugated synthetic oligosaccharides were characterized by a high ability to bind antibodies in blood of mice immunized with the polysaccharide conjugated vaccine. BSA-tetrasaccharide of serotype 3 S. pneumoniae and BSA-tetrasaccharide of serotype 14 S.pneumoniae were characterized by the highest ability to detect IgG1 against C. CONCLUSION: Synthetic oligosaccharides, conjugated with BSA protein-carrier, may be used to develop diagnostic test-systems for determination of antibodies in post-vaccination sera.

[EPITOPIC SPECIFICITY OF A SYNTHETIC DISACCHARIDE, RECURRING LINK OF CAPSULE POLYSACCHARIDE CHAIN OF SEROTYPE 3 STREPTOCCUS PNEUMONIAE].

AIM: Study epitopic specificity of synthetic disaccharide, recurring link of serotype 3 S. pneumoniae, conjugated with bovine serum albumin (BSA). MATERIALS AND METHODS: Conjugate of the synthetic disaccharide with BSA was obtained by squarate method. Antigenic activity of the conjugate was studied in competitive EIA. Titers of IgG against capsule polysaccharide of serotype 3 S. pneumoniae were determined in EIA by using sera of mice immunized twice with disaccharide conjugate sorbed onto aluminum hydroxide. RESULTS: Disaccharide conjugate used as a well-covering antigen (4 µg/well) in EIA was characterized by a high degree of specificity and interacted only with IgG against serotype 3 S. pneumoniae in antimicrobial sera of animals without reacting with antibodies (ABs) against other pneumococcus serotypes (6B, 10A, 19A, 19F, 23F). Disaccharide conjugated with BSA was determined in competitive EIA to inhibit bonding of ABs to disaccharide by 78.8%, bacterial capsule polysaccharide by 56.9%, BSA did not inhibit the sera activity. The study of sera of mice immunized by serotype 3 S. pneumoniae disaccharide conjugate in EIA, where capsule polysaccharide was used as a plate-sorbed antigen, has established the presence of IgG against capsule polysaccharide at a titer of 1:1600. CONCLUSION: The disaccharide that is a single recurring link of serotype 3 S. pneumoniae contains a key epitope of capsule polysaccharide. The synthetic disaccharide could be used as a component of multivalent conjugated pneumococcal vaccines and for development of diagnostic test-systems.

Protective activity of a glycoconjugate based on a synthetic hexasaccharide related to a fragment of capsular Streptococcus pneumoniae serotype 14 polysaccharide chain.

We studied the effects of immunization with a conjugate of carrier protein and hexasaccharide ligand related to a fragment of capsular of Str. pneumoniae serotype 14 polysaccharide chain on activation of innate and adaptive immunity. It was found that two-fold immunization with the glycoconjugate adsorbed on aluminum hydroxide significantly increased the titer of IgG antibodies to capsular polysaccharide in the blood and protected 100% mice from infection with Str. pneumoniae serotype 14. Enhanced bactericidal activity of peripheral blood lymphocytes of mice was found 4 and 24 h after the first immunization with the immobilized glycoconjugate. Adsorption of the glycoconjugate on aluminum hydroxide resulted in modification of the immune processes at the stage of activation of innate immunity and subsequent strengthening of the adaptive immunity.

[Influence of various bacterial ligand application methods on cytokine expression].

AIM: Study the production of cytokines in mice during vaccination with polycomponent Immunovac-VP-4 vaccine containing TLR ligands with various administration methods. MATERIALS AND METHODS: Immunovac-VP-4 was administered to mice by subcutaneous, intranasal or per oral methods. The preparation was administered nasally at a single dose of 500 microg in the volume of 30 microl. Per oral single dose was 2000 microg in the volume of 0.5 ml. 200 microg of the preparation was administered subcutaneously. Cytokines in blood sera were determined by EIA 8 hours after the administration of the vaccine. RESULTS: In mice 8 hours afterthe single administration of Immunovac-VP-4 the levels of IL-1beta, IL-6, IL- 12, IL-5 increased significantly. However their concentration differed depending on the method of administration. The most active expression of cytokines was observed during subcutaneous administration. The indexes of cytokine expression were significantly higher (p < 0.05) than during non-parenteral administration methods. CONCLUSION: Mucosal application methods along with parenteral were established to be able to activate effector mechanisms of immune repose with its consequent polarization by Th1/Th2 pathways. These mechanisms lay the groundwork for development of antigen-specific immune responses against antigens/pathogens.

[Effect of aluminium hydroxide on innate immunity and immunogenicity of bacterial and synthetic antigens of Streptococcus pneumoniae].

AIM: Study the effect of aluminium hydroxide on molecular-cell mechanisms of innate immunity activation and its adjuvant effect on immunogenicity of natural bacterial and synthetic pneumococci antigens. MATERIALS AND METHODS: Surface markers of dendritic cells (DC), mononuclear leukocytes (ML) and cytokine levels were determined by flow cytometry; IgG titers--by EIA. Protective activity was evaluated in experiments with active protection of mice from infection with virulent pneumococci strains. RESULTS: Aluminium hydroxide increased the ML content of mice spleen expressing TLR2 and TLR4. Its addition into the culture of immature DC induced the appearance of a population of cells with mature DC markers--CD83, CD80, CD86, however, the level of undifferentiated cells (CD34) and cells with adhesion molecules (CD11c, CD38) did not change. DC produced IL-1ß, IL-5, IL-10, IFNγ into the cultivation medium. An increase of cytokine production took place 2 hours after the administration into mice and was retained for the observation period (24 hours). Th1 (IFNγ, TNFα) and Th2 (IL-5, IL-10, GM-CSF) cytokine production gave evidence on immune response polarization by Th1/Th2, type. After 2 administrations of aluminium hydroxide into mice the number of ML with CD19+, CD5+, NK1.1+, CD25+, MHCII+ markers increased during decrease of CD3+, CD4+ and CD8+ T-lymphocytes. Adaptive immunity activation was characterized by high IgG titers to pneumococci capsule polysaccharide and protection of 90 - 100% of the mice against infection with lethal doses of S. pneumoniae strains, was detected during 2-fold immunization of mice with conjugates of synthetic pneumococci oligosaccharides with BSA,sorbed onto aluminium hydroxide, whereas natural bacterial antigens provided 90 - 100% survival of animals during immunization without the adjuvant. CONCLUSION: Data are provided on the effect of aluminium hydroxide on key effectors of innate immunity: DC, ML, TLRs and cytokine production. A reasonable administration of this adjuvant was shown to be in association with conjugates of pneumococci synthetic oligosaccharides with a carrier protein.

[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.

[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.

Development of approaches to creation of experimental test system for evaluation of antigenic activity of synthetic oligosaccharide ligands related to fragments of the Streptococcus pneumoniae type 14 capsular polysaccharide.

A conjugate of a synthetic hexasaccharide fragment of the Streptococcus pneumoniae type 14 capsular polysaccharide with bovine serum albumin (BSA) has been prepared. The antigenic activity and specificity of this conjugate are comparable with those of natural antigens of S. pneumoniae type 14. The data suggest that the resulting synthetic conjugate can be used as a coating antigen in an experimental test system (based on enzyme immunoassay) for evaluating the antigenic activity and specificity of synthetic oligosaccharide ligands and for testing specimens of natural capsular polysaccharides and immune sera.

[Effect of strain-producer and cultivation medium on cross antigenic activity of Streptococcus pneumoniae water soluble antigens].

AIM: Production of water soluble protein-containing antigens from various strains of S. pneumoniae during cultivation in complete and semi-synthetic culture media as well as selection of strains with cross antigenic activity. MATERIALS AND METHODS: S. pneumoniae 3, 6A, 6B, 14, 10A, 18A, 19A, 19F, 23F serotype strains were cultivated in brain-heart broth and semi-synthetic medium with addition of aminopeptide for 24 hours at 37 degrees C for the production of water soluble antigens. The antigens were obtained by a method of triple water extraction from acetone dried microbial cells. Chemical composition of preparations, electrophoresis mobility of protein-containing components of preparations and cross antigenic activity in gel immune diffusion reaction by using rabbit hyperimmune sera were studied. RESULTS: In studies of 10 pneumococcus strains from various serotypes a method of microbial cell inactivation by acetone was selected that allows to produce preparations with high protein content (25.5 - 53.1%). Electrophoretic separation of the preparations revealed difference in the preparations obtained from various pneumococcus strains in the layout of major protein lines in the 8 - 95 kDa range. The most virulent and immunogenic S. pneumoniae strain that during cultivation in semi-synthetic medium was characterized by intraspecies cross antigenic activity and in gel immune diffusion reacted with all the studied sera against 3, 14, 18C, 23F serotype strains was selected. CONCLUSION: The study resulted in the selection of a technologically simple method of production of pneumococcus antigens with high protein content and showed that only 1 of the studied preparations produced from a virulent strain with poorly expressed S. pneumoniae capsule during cultivation in semi-synthetic medium has the highest cross antigenic activity.

[Strain differences of intra-species immunogenic activity of Streptococcus pneumoniae antigen components].

AIM: Study intra-species immunogenic activity of antigenic protein-polysaccharide components of S. pneumoniae. MATERIALS AND METHODS: Antigenic components of serotype 3, 6A, 6B, 14, 10A, 18A, 19A, 19F, 23F and unencapsulated S. pneumoniae strains were obtained by water extraction method. Synthetic hexasaccharide--corresponding to the structure of S. pneumoniae serotype 14 capsule polysaccharide repeated unit chain fragment was used as a reference preparation. Molecular mass of antigenic components was determined in SDS-electrophoresis. Antibody titers in blood sera of immunized mice were evaluated by solid-phase EIA method. Protective activity of preparations was studied in mice after 2 immunizations with consequent infection by virulent S. pneumoniae serotype 3 and 6B strains. RESULTS: Preparations from serotype 6A, 6B, 14, 19A, 19F, 23F strains in reaction with anti-microbial sera were characterized by cross serologic activity (IgG titers of 1200 - 12 800). The lowest serologic activity was detected in S. pneumoniae serotype 3 and unencapsulated strain preparations. Conjugate of synthetic hexasaccharide and bovine serum albumin interacted only with homologous antimicrobial sera up to titers of 600 +/- 89.4 and did not react with sera against serotypes 19A and 19E Cross serologic activity of preparations is probably determined by the presence of protein fractions that were detected in SDS-electrophoresis. This is confirmed by high intra-species cross protective activity of preparations from serotype 6B and 10 A strains that protect 90 - 100% of mice from infection by heterologous S. pneumoniae strains. CONCLUSION: Use of strains with cross antigenic and protective activity for production of immunogenic protein-containing fractions with the aim of enchanting and broadening specter of protective activity of vaccine preparations that are constructed based on capsule polysaccharides of S. pneumoniae is appropriate.

[Immunogenic activity of a conjugate of synthetic hexasaccharide related to a streptococcus pneumoniae serotype 14 capsule polysaccharide chain fragment].

AIM: Study antigenic and immunogenic activity of a conjugate of synthetic hexasaccharide related to a S. pneumoniae serotype 14 capsule polysaccharide chain fragment with bovine serum albumin (BSA). MATERIALS AND METHODS: Synthetic glucoconjugate based on BSA protein carrier and hexasaccharide ligand reflecting capsule polysaccharide chain fragment was obtained by using squarate method. Natural polysaccharide-protein complex from S. pneumoniae serotype 14 strain was obtained from cultural fluid supernatant by acetone precipitation. IgG titer against hexasaccharide/capsule polysaccharide was determined in antimicrobial sera and sera of mice immunized with glucoconjugate by EIA method. RESULTS: Immunogenic activity ofglucoconjugate based on BSA protein carriers and synthetic hexasaccharide reflecting S. pneumoniae serotype 14 capsule protein chain fragment was established. After 2 immunizations antibodies against hexasaccharide ligand and BSA were determined in blood sera of mice. Antibody titers against hexasaccharide exceeded the level in intact mice by 4.2 times. BSA in the conjugate did not have effect on production of antibodies against hexasaccharide. CONCLUSION: The developed experimental test-system based on synthetic glucoconjugate is useful for evaluation of level of antibodies against S. pneumoniae serotype 14 in infected and, probably, carriers of bacteria.

[Study of cross-activity of Streptococcus pneumoniae antigen preparations].

AIM: Study cross-activity of S. pneumoniae antigen preparations. MATERIALS AND METHODS: Antigen preparations were obtained by ultrasound disintegration (from bacteria in R-form), extraction with water (from serotype 3 bacteria), cetavlon and trichloroacetic acid (from serotype 6A bacteria). Chemical composition and immunochemic properties of preparations were studied by contemporary methods as well as in experiments with direct and cross-protection of mice from infection. RESULTS: 3 of 4 preparations (except ultrasound disintegrate) had approximately 30% of protein. In immunodiffusion reaction they interacted with hyper immune rabbit sera obtained against 12 various pneumococcus serotypes--1, 3, 4, 6A, 6B, 9V, 9N, 14, 18C, 19A, 19F and 23F. In animal experiments 30 - 70% of mice were protected from subsequent infection with knowingly high dose of homologous and 3 heterologous pneumococcus strains. In immunoblotting the highest number of components serologically active with heterologous sera was formed by cetavlon extract (12 - 23). Addition of capsule polysaccharides to the preparation increased its cross-protective activity. CONCLUSION: By data set and the highest yield, water extract is reasonable for isolation of cross-reactive proteins of pneumococcus. Development of another method of extraction from cultural fluid is necessary for obtaining extracellular protein antigens. Generation of vaccines containing cross-reactive proteins of pneumococcus and capsule polysaccharides is a promising direction.

[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.

[Protective activity of Immunovac-VP-4 vaccine against avian influenza virus H5N2 administered by different methods].

AIM: To experimentally assess protective effect of Immunovac-VP-4 vaccine against avian influenza virus H5N2. MATERIALS AND METHODS. Immunization of mice with polycomponent vaccine Immunovac-VP-4 was performed using oral or mucosal route of administration (intranasally, orally, and with combined nasal-oral method). Immunized mice were inoculated intranasally by influenza virus H5N2 adapted for mice. Survival of mice in experimental and control (intact) groups was assessed daily during 14 days. Survival and death rates of mice were determined. Levels of cytokines in sera of mice from both groups were measured by enzyme immunoassay. RESULTS: Half of experimental animals survived after triple subcutaneous administration of vaccine in dose 20 mcg and subsequent intranasal challenge with avian influenza virus H5N2. Single subcutaneous immunization with dose 400 mcg resulted in survival of 80 +/- 12.6% of mice after challenge. Triple intranasal and combined intranasal-oral immunization as well as after triple subcutaneous immunization resulted in survival of half of challenged mice. In control group challenge was lethal for 90 - 100% of mice. Same methods of immunization lead to increase of IL-6, IL-12, IL-15, and IFN-gamma levels. CONCLUSION: Data about significant protective effect after immunization with Immunovac-VP-4 against avian influenza virus H5N2 were obtained. Immunovac-VP-4 administered by mentioned routes activated nasal-associated lymphoid tissue providing first line defense at entry site of influenza infection, which demonstrates need to further study of this vaccine during development of strategy for non-specific prophylaxis of influenza infection.

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.

[Effect of methods of application of bacterial ligands on activation of lymphoid organs' effectors in mice].

AIM: Evaluation of differentiation dynamics and localization of various populations of lymphocytes as well as their expression of TLRs during different methods of administration (intranasal, oral, and subcutaneous) of bacterial ligands of opportunistic microorganisms (Immunovac-VP-4 vaccine) in experiment on mice. MATERIALS AND METHODS: Polycomponent vaccine Immunovac-VP-4 consisting of ligands of 4 opportunistic bacteria was administered to CBA line mice. Groups of mice were immunized orally, subcutaneously, or intranasally. Number of mononuclear leukocytes, as well as levels of cytokines, lymphocytic antigens, and cytotoxic activity of cells were measured. RESULTS: It was demonstrated that modification of immunophenotype of lymphocytes and cytotoxic activity of NK cells depends from route of administration of Immunovac-VP-4 because the most intensive activation of cells occurred in organs proximal to place of vaccine application. However, already 1 day after administration of vaccine there was intensive exchange between lymphoid cells not only in nasal associated lymphoid tissue, bronchial associated lymphoid tissue, and gut associated lymphoid tissue but also in spleen that points to integration of fine mechanisms of mucosal and systemic immune response regulation. CONCLUSION: Development of noninvasive methods of vaccination is an optimal way of safe and mass prevention of infectious diseases.