[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.

[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.

[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.

[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.

[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.

[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.

[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.