[SPECIFIC IMMUNE RESPONSE TO CERTAIN CAPSULE POLYSACCHARIDES OF STREPTOCOCCUS PNEUMONIAE IN HEALTHY BLOOD DONORS AND INDIVIDUALS IMMUNIZED WITH PNEUMOCOCCAL VACCINES].

AIM: Evaluate humoral immune IgG response to 13 specific capsule polysaccharides in healthy blood donors and individuals immunized with pneumococcal vaccines. MATERIALS AND METHODS: A solid-phase EIA method was used with sorption on polystyrol of pneumococcal CPS isolated by us (1, 3, 4, 6A, 6B, 7F, 9N, 9V, 14, 18C, 19A, 19F, 23F). Blood sera of 140 healthy donors, 42 conscripts (young males) vaccinated with Pneumo-23 and 5 adults that are frequently ill with various bronchial-lung diseases for a long time and vaccinated with Prevenar-13 preparation were analyzed. IgG level was expressed in conventional units. RESULTS: The mean level of antibodies against pneumococcal CPS in donors was at the level of 70. c.u.; in conscripts during the prevaccination period--50 c.u.; in frequently ill for a long time--90 c.u. In 14 of 140 donors increased antibody levels against one or several CPS simultanously were observed. During post-vaccinal period in vaccinated with Pneumo-23 an increase of antibody levels against 12 - 13 CPS varying by specificity was observed only in 9 of 42 individuals; 1 individual did no have an increase of antibody levels against any of the 13 CPS; in 32 of 42--the antibody levels against 1 or several CPS did not increase. In 5 individuals vaccinated with Prevenar-13 a rise of antibody levels against 1 - 9 CPS, that are included in the vaccine, was not detected. CONCLUSION: Thus, clinical trials during registration of newly developed vaccines should, in our opinion, include evaluation of the initial levels of specific antibodies against each polysaccharide component of the vaccine in blood sera of all groups of volunteers. This would allow to evaluate the effectiveness of the vaccination.

[Experience of application of pneumococcus vaccines for studies of humoral immunity].

AIM: Evaluate possible use of Pneumo-23 pneumococcus polysaccharide vaccine and Prevenar-7 conjugated vaccine in EIA as antigens for determination of IgG levels against capsule polysaccharides. MATERIALS AND METHODS: Solid phase EIA method with sorption on polystyrol of commercial vaccines Penumo-23 and Prevenar-7 was used in the study. Blood sera of 41 children before immunization and sera of 8 children before and after vaccination with Pneumo-23 and Prevenar-7 were analyzed. IgG level was determined in standard units (u.). RESULTS: Mean level of antibodies in groups of unimmunized children against antigens of both vaccines were in the range of 52.3-69.1 u. (p > 0.05). The number of children with diagnostically significant level of antibodies (114 - 120 u.) was 2.4% in the control group (1/41) when Pneumo-23 antigens were used and 7.3% (3/41) when Prevenar-7 antigens were used. After vaccination with Pneumo-23 the fraction of diagnostically significant level of antibodies against Pneumo-23 antigens was on average higher by 1.8 times than in pre-vaccination period in 62.5% of children, and against Prevenar-7 antigens - by 1.6 times higher in 75% of children. After immunization with Prevenar-7 vaccine the level of antibodies increased by 3-4 times against antigens of both vaccines and reached diagnostically significant in 100% of cases. CONCLUSION: Pneumo-23 and Prevenar-7 pneumococcal vaccines may be used as antigens for determination of antibodies against capsule polysaccharides of Streptococcus pneumoniae in EIA. Higher sensitivity of EIA based on Prevenar-7 allows to recommend this test for studies of postvaccination immunity in immunized with both conjugated and non-conjugated polysaccharide pneumococcal vaccines.

[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.

[Assessment of protective effect of heat shock protein-lypopolysaccharide of Salmonella typhimurium recombinant construction in mice].

AIM: To study protective activity of recombinant construction of heat-shock protein with lypopolysaccharide (rcHSP-LPS) as well as its variants (with destroyed protein or bounded LPS) against Salmonella typhimurium. It was also planned to study the ability of rcHSP-LPS to interact with toll-like receptors (TLRs) expressed on continuous cell lines. MATERIALS AND METHODS: One of the following preparations was administered to outbred mice: rcHSP-LPS; rcHSP-LPS treated by polymyxin B (PMB) for bounding of LPS - rc(HSP-LPS)PMB; rcHSP-LPS in which protein was treated by boiling during 30 min--rc (HSP-LPS)B; LPS (E. coli K-235); polymyxin B (PMB). Twenty-four hours after single or last administration of rcHSP-LPS, each mice was intraperitoneally inoculated with 63 LD50 of S. typhimurium 415 contained in 0.5 ml of physiologic solution. Antibody titer to LPS of Salmonella typhimurium was measured by immunoenzyme assay. RESULTS: It was demonstrated that rcHSP-LPS administered 24 hours before inoculation induced resistance to S. typhimurium infection. Protection formed after 3 injections of rcHSP-LPS with 10 mcg in each or single injection with 100 mcg/mouse. Forty to eighty percent of immunized mice survived after challenge while 90% of control animals died. Destroy of the HSP by boiling of the construction led to loss of protective effect. Bounding of LPS by PMB did not lead to loss of protective properties of the construction but they expressed only after its multiple administration with 10 mcg per mouse. LPS of E. coli in dose 0.0266 mcg per mouse as well as PMB did not influence the course of S. typhimurium infection in mice. CONCLUSION: It was shown that rcHSP-LPS effectively protects mice from S. typhimurium infection by activating innate immunity; one of the possible mechanisms for such protection determined by interaction with TLRs 2 and 4 was considered. Other studies are needed in order to elucidate other mechanisms of innate immunity, which can be activated by rcHSP-LPS.

[Immunological effect of vaccination against pneumococcal infection in HIV-infected children].

AIM: To measure level of antibodies to pneumococcal antigens in HIV-infected children vaccinated in the age > 2 y.o. in order to assess clinical effect of vaccination. MATERIALS AND METHODS: Levels of IgG and IgM were measured by ELISA in 16 HIV-infected children > 24 months of age vaccinated against pneumococcal infection with Pneumo 23 vaccine on IIA-B stage of the disease. When the study was conducted, children did not receive antiretroviral therapy. Control group was represented by 47 children of the same age born from HIV-negative women. RESULTS: It was determined that HIV-infected children had high baseline levels of IgG and IgM to antigens of Streptococcus pneumoniae to 2 years of age, which is indirect evidence of previous pneumococcal infection. Increase of antibody levels after vaccination to polysaccharides (PS) of S. pneumoniae serotypes 3, 6B, 9N, 23F as well as to mix of PS included in the vaccine was not observed compared to the control group. Despite the absence in dynamics of IgG and IgM levels, decrease of acute respiratory, infections incidence on 34.6 - 36.4% was noted in HIV-infected participants during 1-year follow-up, which can be associated with immunocorrecting effect of PS contained in the Pneumo 23 vaccine. It was assumed that significant clinical and immunological effect of vaccination could be obtained by administration of pneumococcal conjugate vaccine in younger age, before realization of HIV-infection. CONCLUSION: Vaccination against pneumococcal infection is indicated for HIV-infected children; it promotes decrease of rate of intercurrent infections on the background of the main disease.

[Autoantibodies after vaccination against pneumococcal and influenza infections in children and adolescents with type I diabetes mellitus].

AIM: To study the influence of vaccination against pneumococcal and influenza infections on the levels of autoantibodies to DNA, pancreatic and adrenal tissues as well as on populations of lymphocytes and levels of immunoglobulins in children and adolescents with type I diabetes mellitus (DM1). MATERIALS AND METHODS: One hundred and thirty children and adolescents 2 - 18 years of age were followed during 12 months. One hundred participants were vaccinated against pneumococcal infection with Pneumo 23 vaccine. Twenty-eight of them (28%) were also vaccinated against influenza with subunit vaccine Grippol. Vaccination was performed during intensified insulin treatment. Unvaccinated group consisted from 30 children of the same age. During vaccination 59 (59%) children were in phase of diabetes compensation and 41 (41%)--in phase of subcompensation. RESULTS: In 5 out of 10 participants with high level of antibodies (Abs) to native DNA (n-DNA) before vaccination with Pneumo 23, normalization of this level 1 year after vaccination was noted. Normalization of level of Abs to denaturated DNA (d-DNA) was observed in 2 out of 7 patients with high levels to d-DNA before vaccination. Normalization of level of Abs to n-DNA was observed in 2 out of 3 patients after combined vaccination. It is important to note that vaccination of children in subcompensation phase of DM1 did not result in changes of levels of autoantibodies. One year after vaccination decrease of IgG, IgA, and IgM levels to normal range was noted. Absence of increase of HLA-DR and CD16 levels in postimmunization period in children with DM1 vaccinated even in subcompensation phase is also an indirect evidence of absence of autoimmune process activation. CONCLUSION: Use of Pneumo 23 vaccine or its combination with Grippol vaccine in patients with DM1 did not result in increase of levels of autoantibodies to n-DNA, d-DNA and pancreatic tissue, was not able to initiate or lead to disease progression as well as positively influenced on the immune response with tendency to normalization of the several arms of the immune system and, at the same time, did not result in activation of autoimmune process.

[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.

[Effect of recombinant heat-shock protein (rHSP70) of Mycobacterium tuberculosis on immunogenicity of Haemophilus influenzae type B capsular polysaccharide].

AIM: To study effect of recombinant heat-shock protein (rHSP70) of Mycobacterium tuberculosis on immunogenicity of Haemophilus influenzae type b capsular polysaccharide (CPSHib). MATERIALS AND METHODS: Capsular polysaccharide was obtained by precipitation with cetavlon, antibody titers and Toll-like receptors (TLRs) were detected by enzyme immunoassay and flow cytometry respectively. RESULTS: rHSP70 modified immune response to chemically conjugated and unconjugated CPSHib. rHSP70 enhanced expression of TLRs 2, 4, 9 on mice splenocytes; increased levels of CD3+, CD8+, NK, CD3/NK (NKT) lymphocytes. Levels of CD4+, CD25+ (markers of early activation of T-helpers) as well as MHC class II molecules were increased that could be appraised as a shift from T-independent to T-dependent immune response. Difference in antibody titers after 2- or 3-dose immunization of mice with 5 mcg/dose of CPSHib in mixture or conjugated with rHSP70 was not revealed. Level of antibodies to rHSP70 in serum samples of mice immunized with CPSHib conjugated with rHSP70 was 6.55 - 8.4 times higher compared to unimmunized animals. Antibodies, which have common antigenic epitopes to human organs and tissues, were not detected. CONCLUSION: rHSP70 modifies immune response to CPSHib.

[Pertussis toxin and cross-reactive antigens in dynamics of Bordetella pertussis cultivation].

Cultures of Bordetella pertussis from phases of exponential growth, retarded growth and from stationary phase were obtained during periodic dynamic cultivation. Preparations for intravenous immunization of rabbits were made from these cultures. Levels of IgG to pertussis toxin, cell walls preparations from 12 bacterial species, 4 organo-specific antigens, and 7 organospecific human antigens were measured in obtained sera. It was shown that higher levels of IgG to pertussis toxin were found in sera of rabbits immunized with cultures from exponential growth phase whereas decrease of this level in 8 times was observed in sera of rabbits immunized with cultures from retarded growth phase or end of stationary phase. After immunization with culture from exponential growth phase increase of IgG levels to cross-reactive antigens was not observed compared to levels of these antibodies in control sera obtained before immunization. After immunization with cultures from retarded growth phase or end of stationary phase increase of IgG levels to preparations of cell walls of Staphylococcus aureus, S. epidermidis, Pseudomonas aeruginosa, Klebsiella pneumoniae, to denaturated DNA, elastin, and renal and liver microsomal fractions was detected compared to control sera. Described data can substantiate usefulness of obtaining the most specific diagnostic sera and test-systems using cultures of B. pertussis from the phase of exponential growth.

[Immunity to antigens of nontypeable Haemophilus influenzae strains].

Nonencapsulated (nontypeable) Haemophilus influenzae (NTHi) is a Gram-negative coccobacillus colonizing upper respiratory tract of most healthy people and causing such diseases as otitis media, sinusitis, exacerbations of chronic obstructive pulmonary disease, and bronchitis. NTHi may cause systemic infection. As a result, over the past decade the bacterium has been the subject of intense research. However immune response to NTHi has not been well characterized. Data on research of immune response to NTHi are presented.

[Klebsiella pneumoniae secreted protein-containing antigens in the system of adaptive immunity].

The study was aimed at the evaluation of the antigenic properties of K. pneumoniae secreted protein-containing antigens with a molecular weightt of 21 and 34-35 kD, obtained from supernatant culture fluid. As confirmed by the method of flow cytofluorimetry, the protein-containing fractions belonged to the secreted components of the microbial cell. The fraction with a molecular weight of 34-35 kD possessed high antigenic activity and contributed to the formation of specific antibodies after the immunization of mice. At the same time none of the protein fractions lead to an increase in the level of autoantibodies in mouse blood sera to organ-unspecific and organ-specific antigens. As revealed by the method of solid-phase, in 6 (27.3%) from 22 patients of patients with rhizomelic spondylitis had an increased level of IgG to K. pneumoniae cell-wall antigens with a molecular weight of 34-35 kD. An increase in the level of IgG to the secreted protein-containing fraction with a molecular weight of 34-35 kD was detected only in one patient (4.5%) (p<0.05).

[Immunologic autoagression in patients with diabetic retinopathy under type I diabetes mellitus].

Presence of IgG autoantibodies to broad spectrum of tissues' antigens in patients with diabetic retinopathy under type I diabetes mellitus was studied. Increased levels of antibodies to different antigens were observed in 56% of patients. Increased levels of antibodies to hypophysis, denaturated DNA, and myelin basic protein were detected most frequently. Correlation in presence of autoantibodies of different specificity with diabetes length as well as with frequency of respiratory infections was established. There was relation between level of antibodies to DNA and increased permeability of microvascular network of retina as well as between presence of autoantibodies to myelin basic protein and encephalopathy in patients with type I diabetes mellitus.

[Level of IgG antibodies to bacteria of 12 species in the blood sera of rabbits, immunized with preparations obtained from Neisseria meningitidis, grown under the stress conditions].

The blood sera of rabbits, immunized with preparations obtained from N. meningitidis of serogroups A, B or C, cultivated under the stress conditions, were studied. These sera were found to contain IgG antibodies not only to N. meningitidis antigens, but also to the bacterial antigens of 12 species. The sera of rabbits, immunized with meningococcal preparation of serogroup A, were found to have the elevated levels of IgG antibodies, in comparison with the control, to the antigens of 3 other bacterial species; the blood sera of rabbits, immunized with meningococcal preparation of serogroup B, were found the elevated levels of IgG antibodies to the antigens of 11 other bacterial species; and the blood sera of rabbits, immunized with meningococcal preparation of serogroup C, to the antigens of 9 other bacterial species. The study of serogroup B meningococci, used as an example, revealed the influence of the growth phase of the culture on the content of cross-reacting antigens. Their greatest amount was determined at the stationary phase when the stressor effect on the culture reached its maximum and their least amount, at the exponential phase when the stressor effect on the culture was minimal. It was, therefore, found to be expedient to obtain immunodiagnostic and test systems from N. meningitidis cultures, grown to middle of the exponential phase of growth.

[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.

[Influence of the growth phase of Neisseria meningitidis on the accumulation of antigens cross-reacting with human antigens in the culture].

The content of N. meningitidis antigens with epitopes cross-reacting with human antigens was studied in the dynamics of the meningococci cultivation. The absence of these antigens in the culture in the middle of the exponential phase of growth and their presence in the culture at the end of the stationary growth phase of growth were shown. Thus the expediency of using the culture in the middle of the exponential phase of growth for obtaining meningococcal (serogroups A, B and C) vaccines was demonstrated. The trend, found to be promising for the development of immunologically safe vaccines which contained no antigens having common epitopes with human antigens, was determined.

[Specific features of humoral immunity in cryptogenic fibrosing alveolitis patients]. / Osobennosti gumoral'nogo immuniteta u bol'nykh kriptogennym fibroziruiushchim al'veolitom.

Cryptogenic fibrosing alveolitis (CFA) is a severe autoimmune disease of unclear etiology and prognostically unfavorable. The complexity of the diagnostics of this disease makes it necessary to search for new methods; for this reason immunity in CFA patients must be studied. The study of humoral organ-specific, organ-unspecific and antibacterial immunity of CFA patients revealed that the latter differed from the members of the groups used for comparison by a higher frequency of positive reactions in EIA determinations of IgG antibodies to cytokeratin-8 and Moraxella catarrhalis antigens. In addition, only in CFA patients a high degree of correlation (r=0.88) between these results was established. This made it possible to propose to use these reactions for confirming the diagnosis of CFA and suggested the probable role of M. catarrhalis in triggering autoimmune reactions characteristic of this disease.

[Cross-reactions between the antigens of healthy pulmonary tissue and Moraxella catarrhalis]. / Perekrestnye vzaimodeistviia mezhdu antigenami zdorovoi legochnoi tkani i Moraxella catarrhalis.

The study of cross-reactions between healthy pulmonary tissue antigens and Moraxella catarrhalis with the use of SDS-electrophoresis and immunoblotting revealed that in the component of healthy pulmonary tissue with a mol. wt. of 40 kD epitopes existed to which antibodies were produced, capable of cross reaction with the components of M. catarrhalis with a mol. wt. of 35 kD and 70 kD. In addition, the presence of cross-reactions between cytokeratin-8, protein contained in healthy pulmonary tissue, and M. catarrhalis antigens was established.