[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.

[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.

[Influence of nutrient medium composition on the production of capsule polysaccharide by Streptococcus pneumonia 19A serotype].

AIM: Evaluate accumulation of capsule polysaccharide by Streptococcus pneumoniae 19A strain in semisynthetic nutrient medium including various amino acid sources. MATERIALS AND METHODS: Comparative evaluation of the production of capsule polysaccharide by the strain belonging to one of the most widespread S. pneumoniae serotype (19A) was performed by using rocket immunoelectrophoresis. The bacteria were cultivated in semisynthetic liquid nutrient media of varying composition. RESULTS: Among 4 sources of nitrogen (aminopeptide, acid and pancreatic hydrolysate of casein, soy peptone) added to salt nutrient medium supplemented with glucose and vitamins, casein and soy peptone were shown to promote the maximum synthesis of capsule polysaccharide independently of the cultivation time. Supplementation of the medium with sulfates of iron, zinc and manganese, as well as pH decrease to acid values significantly reduced the level of capsule polysaccharide in the culture liquid. The maximum growth of bacteria was observed at 11 hours after the start of cultivation in a 10 L volume fermenter in semisynthetic nutrient medium with soy peptone. Accumulation of capsule polysaccharide in the culture liquid continued to the end of the observation period (24 hours) and by the end of the process reached 193 mcg/ml. CONCLUSION: Further study of influence of vitamins, carbohydrates, CO2 concentration on the synthesis of high molecular capsule polysaccharide by bacteria belonging to various pneumococcus serotypes is reasonable.

[Subtyping of lipooligosaccharides of non-typeable haemophilus influenzae isolated from children with bronchopulmonary diseases].

AIM: Subtyping of lipooligosaccharides (LOS) of non-typeable strains of Haemophilus influenzae (NTHi) isolated from children with bronchopulmonary diseases. MATERIALS AND METHODS: Lipooligosaccharides obtained from 62 acapsular strains of H. influenzae were studied by vertical SDS-electrophoresis in PAAG. RESULTS: Majority of LOS formed electrophoretically mobile components in low molecular mass zone. Obtained results allowed to differentiate 23 subtypes of LOS. Lipooligosaccharides of majority of strains (67.7%) belonged to one of 10 main subtypes, 30.6% of strains belonged to mixed subtypes because they had signs of 2-3 subtypes. CONCLUSION: Strains possessing LOS of three subtypes--VI, VII, and X--were significantly more prevalent in pediatric patients (p < 0.05). More than one third (43.5%) of studied NTHi strains belonged to these subtypes.

[Immunobiologic characteristics of carbohydrate-containing preparations of Haemophilus influenzae].

AIM: Comparative assessment of immunobiological characteristics of 3 antigenic preparations containing capsular polysaccharide of Haemophilus influenzae type b (CPS Hib). MATERIALS AND METHODS: The following preparations were assessed: CPS Hib obtained by using cetavlon; hydroxylamine preparation of Hib (HAP Hib); mixture of CPS Hib and lipooligosaccharide of non-typeable H. influenzae (LOS NTHi) detoxified by hydroxylamine hydrochloride. Effects of these preparations on immunophenotype of mononuclear leukocytes of mice spleen as well as on spectrum and level of cytokines in serum were studied. RESULTS: It was shown that mixture of CPS Hib and detoxified LOS NTHi has low toxicity and most protective activity during Hib challenge leading to activation of innate immunity effectors and initiation of adaptive immune response. CONCLUSION: Obtained data provide perspective for development of preparation able to protect from infections caused by both capsular and acapsular strains of H. influenzae.

[Influence of the different detoxifying agents on toxic characteristics of Haemophilus influenzae lypooligosaccharides].

AIM: To study toxicity of lypooligosaccharides (LOS) of non-typable Haemophilus influenzae (NTHi) strain and products of their detoxication obtained using different reagents. MATERIALS AND METHODS: LPS was obtained from the NTHi strain grown on solid brain-heart infusion nutrient medium using previously described method of isolation and purification of LOS. Obtained LPS was treated in same conditions by one of the 3 detoxifying agents: anhydrous hydrazine (AH), alkali (NaOH), and hydrochloric hydroxylamine (HH). Toxicity of LOS and its detoxified derivatives was measured on outbred mice which were administered 0.5 ml of actinomycin D intraperitoneally 1 day before immunization. Death of animals was assessed on day 2 after immunization. Polyacrylamide gel electrophoresis was used for study the influence of detoxifying agents on physico-chemical properties of LOS. RESULTS: As a result of treatment of NTHi No.45 LOS by different detoxifying agents, 3 preparations of detoxified LOS (d-LOS) and 3 preparations from precipitates (nd-LOS) were obtained. Preparation d-LOSAH was the least toxic. Toxic properties of nd-LOSHH did not reliably change. PAAG electrophoresis showed that virtually all detoxified preparations were characterized by higher migration of lypooligosaccharide components compared to original LOS of NTHi No. 45, which indicates the lowering of LOS molecular weight after treatment by detoxifying agents, associated with elimination of lipid A higher fatty acids. CONCLUSION: Analysis of effects of detoxifying agents indicates the need to select individual conditions for treatment by each of them.

[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.

[Influence of nicotinamide adenine dinucleotide and hemin concentrations on the growth of Haemophilus influanzae type b and the synthesis of capsular polysaccharide].

In the process the cultivation of H. influenzae, type b, in semisynthetic nutrient medium with aminopeptide base the growth of the bacteria and the synthesis of capsular polysaccharide were shown to depend on the concentrations of aminopeptide, nicotinamide adenine nucleotide (NAD) and hemin. An increase in the concentrations of NAD and hemin stimulated the growth of H. influenzae and inhibited the synthesis of capsular polysaccharide. Similar effect was observed in the simultaneous increase of NAD and hemin concentrations. At elevated concentrations of NAD and hemin and the content of aminopeptide equal to 350 mI/l the maximum weight of biomass was achieved. The increase of hemin concentration had no influence on the growth of H. influenzae, type b, and the synthesis of capsular polysaccharide.

[Influence of the aminopeptide concentration on the growth of Haemophilus influenzae, type b, and the synthesis of its capsular polysaccharide].

The influence of the aminopeptide concentration on the growth of H. influenzae b culture and the synthesis of H. influenzae b capsular polysaccharide was determined. The maximum amount of capsular polysaccharide was accumulated at the concentration of aminopeptide in the culture fluid reaching 50 ml/l. An increase in the aminopeptide concentration led to a decreased amount of synthesized polysaccharide and an increased amount of biomass. The decrease of the aminopeptide concentration to 10 ml/l resulted in decreased amounts of both biomass and synthesized polysaccharide.

[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.

[Properties of the isolated Bacillus subtilis strains and their influence on the intestinal microflora of experimental mice]. / Svoistva vydelennykh shtammov Bacillus subtilis i ikh vliianie na intestinal'nuiu mikrofloru éksperimental'nykh myshei.

The cultural, physiologo-biochemical adhesive and antagonistic properties of B. subtilis strains with good prospects for use as biotherapeutic preparations were studied. For further studies B. subtilis strain No. 1719 was chosen. In experiments on non-inbred white mice the animals were treated by the preparation Cifran used for their selective decontamination from opportunistic microflora and for the creation of the state of dysbiosis. The influence of the spore-forming microbe on the parietal microflora of the large intestine of the animals was shown. Reliable data on the changes in the number of microorganisms (CFU/ml) per 1 cm of the surface of the large intestine were established. As markers making it possible to evaluate the action of biotherapeutic and other medicinal remedies, easily determinable ratios of lac+/lac- of bacteria and Staphylococcus aureus/Staphylococcus spp. was proposed.

[Effectiveness of the oral administration of tomicide in experimental infection]. / Otsenka éffectivnosti énteral'nogo primeneniia "tomitsida" pri éksperimental'noi infektsii.

Experimental data on the oral administration of Tomicid using different schemes for the protection of white mice from staphylococcal infection are presented. The use of Tomicid, administered in the maximum dose admissible for mice, ensured the protection of up to 2/3 of the total number of mice. A single oral administration of the preparation immediately after infection protected 1/3 of the survived mice from local staphylococcal infection. Good prospects of using Tomicid for the prevention of catarrhal diseases in guinea pigs were established.

[Cultivation of Haemophilus influenzae, serotype B, in amino peptide-based semisynthetic nutrient medium]. / Kul'tivirovanie Haemophilus influenzae serotipa B na polusinteticheskoi pitatel'noi srede na osnove aminopeptida.

The work shows the possibility of the cultivation of H. influenzae, serotype b, in semisynthetic nutrient medium with amino peptide as the only source of amino acids, glucose--as the main source of carbon and energy and containing, in addition, the necessary growth factors and vitamins.

[Specific characteristics of composition of synthetic nutrient media for cultivation of Hemophilus influenzae type b from the standpoint of bacterial metabolism]. / Osobennosti sostava sinteticheskikh pitatel'nykh sred dlia kul'tivirovaniia Hemophilus influenzae type b s pozitsii sovremennykh predstavlenii o metabolizme étikh bakterii.

In many countries vaccination against Haemophilus influenzae of type b (Hib) has permitted the liquidation of severe generalized forms of infections caused by these bacteria. The vaccine is obtained on the basis of Hib capsular polysaccharide. To obtain pure capsular polysaccharide, Hib should be cultivated on synthetic nutrient media. The present review deals with the data substantiating the advantages of using synthetic nutrient media for the cultivation of these bacteria with a view to obtaining pure capsular polysaccharide.

[The optimization of the technology for culturing vaccinal strains of Shigella flexneri]. / Optimizatsiia tekhnologii kul'tivirovaniia vaktsinal'nykh shtammov Shigella flexneri.

The drainage-filling cultivation process for three Shigella flexneri vaccine strains in small-capacity fermenters has been developed. The study has shown that after hydroxylamine treatment the yield of the antigenic component, calculated per 1000 million microbial cells, is tenfold higher in comparison with the traditional method.

[Effect of immunomodulators of various origin on the histamine-sensitizing activity of whole-cell pertussis vaccine]. / Vliianie immunomoduliatorov razlichnogo proiskhozhdeniia na gistaminsensibiliziruiushchuiu aktivnost' korpuskuliarnoi kokliushnoi vaktsiny.

Changes in the histamine-sensitizing activity of whole-cell pertussis vaccine (PV) under the action of immunomodulators (IM) of bacterial (peptide and peptidoglycans), synthetic (peptidoglycan) and vegetable origin have been studied. The study has revealed that these IM, introduced orally and parenterally, exhibit histamine-sensitizing activity, depending on the nature of IM and the optimum selection of the doses of IM and PV.

[An increase in the immunogenicity of bacterial antigens under the influence of one of the derivatives of muramyl dipeptide]. / Povyshenie immunogennosti bakterial'nykh antigenov pod vliianiem odnogo iz derivatov muramildipeptida.

As revealed in animal experiments, glucosaminylmuramyl dipeptide (GMDP), the synthetic analog of muramyl dipeptide, when introduced intraperitoneally in a single injection or orally, exhibits adjuvant activity with respect to Citrobacter 0-antigens, Shigella flexneri and enhances the protective properties of dysentery and pertussis vaccines. The stimulating properties of GMDP depend on its dose, the route of its administration, the time elapsed after its administration, its ratio to the concomitant doses of bacterial antigens and to the dose of the virulent culture used for challenge.