[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.

[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.

[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.

[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.

[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 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.

[Cross infection of guinea pigs with different pneumococcal serotypes in the presence of the carrier state]. / Perekrestnoe zarazhenie morskikh svinok pnevmokokkom raznykh serotipov na fone nositel'stva.

The possibility of the pneumococcal cross infection of guinea pigs in experimental conditions, the time course of the distribution of pneumococci in their organs, and the duration, within the time limits of the experiment, of persistence of the given infective agent were studied. Normal animals placed in the same room with infected ones were shown to become the carriers of definite pneumococcal serotypes. As a result, these studies revealed that nasopharyngeal carriership and infection of different organs were not directly interrelated and the method of infection of guinea pigs did not influence the time course of distribution of pneumococci in their organs. The data on the duration of persistence of the infective agent, as well as on the importance of this phenomenon for determination of the relationship between pneumococcal carriership and disease, are presented.

[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.

[Interaction of a cAMP-deficient Yersinia pseudotuberculosis mutant with a macrophage culture in vitro]. / Vzaimodeistvie tsAMP-defitsitnogo mutanta Yersinia pseudotuberculosis s kul'turoi makrofagov in vitro.

The behavior of a virulent Y. pseudotuberculosis strain and its c-AMP-deficient mutant possessing lower virulence in their in vitro interaction with the culture of peritoneal macrophages of white mice, as well as a change in the level of c-AMP in macrophages in the process of the phagocytosis of the initial and mutant strains were studied. The c-AMP-deficient mutant was shown to retain its ability to infect macrophages and to multiply in them, while loosing its cytotoxic effect. Macrophages obtained from mice previously immunized with the mutant acquired the capacity of resisting the cytotoxic action of the initial virulent strain. The mutant proved to be unable to elevate the level of c-AMP in macrophages in the process of phagocytosis, while the initial strain caused a considerable increase in the level of c-AMP in macrophages.

[Behavior of cAMP-deficient mutants of Salmonella typhimurium in macrophage cultures]. / Povedenie tsAMF-defitsitnykh mutantov Salmonella typhimurium v kul'ture makrofagov.

The behavior of c-AMP-deficient S. typhimurium mutants in the culture of peritoneal macrophages of white mice and the antibacterial activity of immune macrophages obtained from mice previously immunized with c-AMP-deficient S. typhimurium mutants were studied; c-AMP-deficient mutants were shown to have a lesser cytotoxic effect on macrophages than the initial virulent strain, while retaining their capacity for intracellular proliferation. Immune macrophages acquired the ability to withstand the cytotoxic action of the virulent strain.

[Mechanism of action of N-acetylglucosaminyl-N-acetylmuramylalanyl-D-isoglutamine on the nonspecific anti-infection resistance of mice]. / K mekhanizmu deistviia N-atsetilgliukozaminil-N-atsetilmuramilalanil-D-izoglutamina na nespetsificheskuiu protivoinfektsionnuiu rezistentnost' myshei.

The stimulating influence of glucose-containing muramyldipeptide (GMDP) on the nonspecific resistance of mice was shown to depend on the features of the pathogenesis of the infection. Thus, the intraperitoneal injection of GMDP increased the survival rate of mice infected with Escherichia coli, but had no stimulating effect on the resistance of the animals to Salmonella typhimurium natural infection in whose pathogenesis macrophages played an essential role. Experiments demonstrated that GMDP was capable of enhancing the ingestive function of macrophages, but did not increase their bactericidal activity with respect to this infection.

[Effect of insulin and isoproterenol on macrophage phagocytic activity]. / Vliianie insilina i izoproterenola na fagotsitarnuiu aktivnost' makrofagov.

To reveal the influence of cyclic adenosine monophosphate (cAMP) on the completion of the phagocytosis of salmonellae, the influence of insulin and isoproterenol on the phagocytic activity of peritoneal macrophages obtained from mice infected with S. typhimurium strains differing in virulence was studied in vitro. The study showed that isoproterenol, while increasing the intracellular content of cAMP, suppressed the bactericidal properties of macrophages with respect to salmonellae, whereas insulin decreased the level of cAMP in the cells and thus facilitated more rapid and complete digestion of ingested bacteria irrespective of their virulence.

[STP (the substance produced by a strain of Streptococcus sp. Thom-1606) as a stimulant of the nonspecific resistance of the macroorganism]. / STP (substantsiia, produtsiruemaia shtammom Streptococcus sp. TOM-1606) kak stimuliator nespetsificheskoi rezistentnosti makroorganizma.

Preparation STP, a new immunostimulating agent, is a substance produced by Streptococcus strain sp. Thom-1606 and capable of enhancing the nonspecific activity of the body as shown in animal experiments. The optimal dose-time parameters of the administration of the immunostimulator have been established by the method of the mathematical planning of experiments. As a result, the survival of all animals used in the experiment has been achieved. Mouse peritoneal macrophages have been found to form the population of target cells whose phagocytic activity is enhanced under the effect of the immunostimulating agent STP.

[Mechanism of the nonspecific protective action of Shigella antigens]. / O mekhanizme nespetsificheskogo protektivnogo deistviia antigenov shigell.

The mechanism promoting the nonspecific action of antigens obtained from S. flexneri and S. sonnei by a sparing method has been studied. These antigens stimulate the T- and B- systems of immunity, that is followed by activation of myelopoiesis and the humoral protective factors of the body, which seems to underlie the formation of resistance to infection caused by nonspecific microorganisms.

[The protective properties of myelopeptides in the development of infectious processes caused by bacteria of the genus Salmonella]. / Protektivnye svoistva mielopeptidov pri razvitii infektsionnykh protsessov, vyzvannykh bakteriiami roda Salmonella.

The protective properties of myelopeptides in the development of bacterial infection in mice and young pigs, caused by S. typhimurium 415, S. cholerae-suis 1422 and 370, have been studied. Myelopeptides have been found to possess protective properties when injected into animals infected with S. typhimurium and S. cholerae-suis in lethal doses. The best protective effect (survival rate of 100%) has been achieved by the injection of myelopeptides 24 hours before challenge. Myelopeptides have also been found to promote the weight gain of young pigs infected with S. cholerae-suis.

[Field trials with an oral cell-free vaccine made from Shigella flexneri]. / Polevye opyty s oral'noi beskletochnoi vaktsinoi iz Shigella flexneri.

The data on the study of the reactogenicity, safety and prophylactic potency of a new acellular vaccine prepared from S. flexneri 2a antigenic complexes are presented. According to the results of two epidemic experiments, the vaccine, introduced by oral administration, showed low reactogenicity, safety and sufficient prophylactic potency. The vaccine decreased morbidity rate in dysentery caused by S. flexneri 2a and ensured the protection of 74% (72-80%) of the vaccinees. The complete course of immunization consisting of three administrations followed by the booster administration induces the formation of specific immunity whose duration is sufficient for ensuring the protection of immunized persons during the epidemiologically unfavorable period (for at least 3 months).