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1.
Bull Exp Biol Med ; 157(5): 612-5, 2014 Sep.
Article in English | MEDLINE | ID: mdl-25257424

ABSTRACT

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.


Subject(s)
Glycoconjugates/pharmacology , Polysaccharides/chemistry , Streptococcus pneumoniae/chemistry , Animals , Carbohydrate Sequence , Mice , Mice, Inbred BALB C , Molecular Sequence Data
2.
Article in Russian | MEDLINE | ID: mdl-25816515

ABSTRACT

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.


Subject(s)
Adjuvants, Immunologic/administration & dosage , Aluminum Hydroxide/administration & dosage , Antibodies, Bacterial/biosynthesis , Antigens, Bacterial/immunology , Pneumococcal Vaccines/immunology , Pneumonia, Pneumococcal/prevention & control , Streptococcus pneumoniae/immunology , Adaptive Immunity/drug effects , Aluminum Hydroxide/immunology , Animals , Antigens, Bacterial/administration & dosage , Antigens, Bacterial/chemistry , Antigens, Bacterial/isolation & purification , Antigens, CD/genetics , Antigens, CD/immunology , Biomarkers/metabolism , CD8-Positive T-Lymphocytes/cytology , CD8-Positive T-Lymphocytes/drug effects , CD8-Positive T-Lymphocytes/immunology , Cytokines/genetics , Cytokines/immunology , Dendritic Cells/cytology , Dendritic Cells/drug effects , Dendritic Cells/immunology , Immunity, Innate/drug effects , Immunization , Immunoglobulin G/biosynthesis , Injections, Intraperitoneal , Male , Mice , Mice, Inbred BALB C , Mice, Inbred CBA , Pneumococcal Vaccines/administration & dosage , Pneumococcal Vaccines/chemistry , Pneumonia, Pneumococcal/immunology , Pneumonia, Pneumococcal/microbiology , Streptococcus pneumoniae/chemistry , Th1-Th2 Balance
3.
Article in Russian | MEDLINE | ID: mdl-23805647

ABSTRACT

AIM: Study protective activity of S. pneumoniae protein-containing antigen complex obtained from T3No.3 strain against infection by homologous pneumococcus strain. MATERIALS AND METHODS: S. pneumoniae T3No.3 (serotype 3) strain obtained from collection of pneumococcus strains of Mechnikov Research Institute of Vaccines and Sera was used in the study. S. pneumoniae protein-containing antigen complex was isolated by precipitation by 2 volumes of acetone of supernatant fraction of cultural medium used for pneumococcus cultivation. Molecular mass of proteins contained in S. pneumoniae antigen complex was determined by SDS electrophoresis in polyacrylamide gel. Protective activity of S. pneumoniae protein-containing antigen complex was studied in BALB/c line mice active protection experiments. Activity of mice immune sera obtained against whole-cell pneumococcus culture (T3No.3 strain) was determined in vitro by solid phase indirect EIA. RESULTS: The data obtained give evidence that the isolated protein-containing antigen complex from S. pneumoniae T3No.3 strain effectively protects mice from consequent infection by a homologous S. pneumoniae strain. S. pneumoniae protein-containing antigen complex sorbed on solid phase at 5 microg dose was established by using EIA to interact with homologous mice immune sera. CONCLUSION: The results of the carried out studies allow to move to studies of cross-activity of S. pneumoniae protein-containing antigen complex isolated from T3No.3 strain.


Subject(s)
Antigens, Bacterial/immunology , Bacterial Proteins/immunology , Pneumococcal Infections/immunology , Pneumococcal Vaccines/immunology , Streptococcus pneumoniae/immunology , Animals , Antigens, Bacterial/chemistry , Antigens, Bacterial/isolation & purification , Antigens, Bacterial/pharmacology , Bacterial Proteins/chemistry , Bacterial Proteins/isolation & purification , Bacterial Proteins/pharmacology , Female , Male , Mice , Mice, Inbred BALB C , Pneumococcal Infections/prevention & control , Pneumococcal Vaccines/chemistry , Pneumococcal Vaccines/isolation & purification , Pneumococcal Vaccines/pharmacology
4.
Article in Russian | MEDLINE | ID: mdl-23805648

ABSTRACT

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.


Subject(s)
Antigens, Bacterial , Bacterial Proteins , Streptococcus pneumoniae , Animals , Antigens, Bacterial/chemistry , Antigens, Bacterial/immunology , Antigens, Bacterial/isolation & purification , Bacterial Proteins/chemistry , Bacterial Proteins/immunology , Bacterial Proteins/isolation & purification , Culture Media/chemistry , Rabbits , Solubility , Streptococcus pneumoniae/chemistry , Streptococcus pneumoniae/growth & development , Streptococcus pneumoniae/immunology , Water/chemistry
5.
Article in Russian | MEDLINE | ID: mdl-24605656

ABSTRACT

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.


Subject(s)
Antibodies, Bacterial/immunology , Antigens, Bacterial/immunology , Streptococcus pneumoniae/immunology , Animals , Antigens, Bacterial/pharmacology , Cattle , Cross Reactions , Mice , Mice, Inbred BALB C , Species Specificity , Streptococcal Vaccines/immunology , Streptococcal Vaccines/pharmacology
6.
Article in Russian | MEDLINE | ID: mdl-24605676

ABSTRACT

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.


Subject(s)
Bacterial Capsules , Oligosaccharides , Polysaccharides, Bacterial , Streptococcus pneumoniae , Animals , Antibodies, Bacterial/immunology , Bacterial Capsules/chemistry , Bacterial Capsules/immunology , Cattle , Mice , Mice, Inbred BALB C , Oligosaccharides/chemical synthesis , Oligosaccharides/chemistry , Oligosaccharides/immunology , Oligosaccharides/pharmacology , Polysaccharides, Bacterial/chemical synthesis , Polysaccharides, Bacterial/chemistry , Polysaccharides, Bacterial/immunology , Polysaccharides, Bacterial/pharmacology , Serum Albumin, Bovine/chemistry , Serum Albumin, Bovine/immunology , Serum Albumin, Bovine/pharmacology , Streptococcus pneumoniae/chemistry , Streptococcus pneumoniae/immunology
7.
Article in Russian | MEDLINE | ID: mdl-23163034

ABSTRACT

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.


Subject(s)
Antibodies, Bacterial/biosynthesis , Antigens, Bacterial/immunology , Cross Protection , Pneumococcal Infections/prevention & control , Pneumococcal Vaccines/immunology , Streptococcus pneumoniae/immunology , Animals , Antibodies, Bacterial/immunology , Antigens, Bacterial/administration & dosage , Antigens, Bacterial/isolation & purification , Bacterial Capsules/chemistry , Bacterial Capsules/immunology , Humans , Immunization , Immunodiffusion , Mice , Mice, Inbred BALB C , Pneumococcal Infections/immunology , Pneumococcal Infections/microbiology , Pneumococcal Infections/mortality , Pneumococcal Vaccines/administration & dosage , Pneumococcal Vaccines/chemistry , Polysaccharides, Bacterial/administration & dosage , Polysaccharides, Bacterial/immunology , Polysaccharides, Bacterial/isolation & purification , Rabbits , Survival Rate
8.
Article in Russian | MEDLINE | ID: mdl-22308742

ABSTRACT

The problem of pneumococcal infections is pressing for the whole world. Existing vaccines based only on pneumococci polysaccharide antigens or polysaccharide antigens and diphtherial anatoxin are not capable of protecting from all serotypes of the microorganism. Reasonability of creation of pneumococcal vaccine based on surface proteins of Streptococcus pneumoniae is discussed in the literature. One of such key pneumococcal proteins is pneumococcal surface protein A (PSPA), because it is detected in all the S. pneumoniae strains, has cross activity and switches B-cell immune response to T-cell. Currently the development of conjugated vaccine based on surface proteins and capsule polysaccharides of pneumococcus seems promising.


Subject(s)
Bacterial Proteins/immunology , Pneumococcal Infections/prevention & control , Pneumococcal Vaccines/administration & dosage , Recombinant Proteins/immunology , Streptococcus pneumoniae/immunology , T-Lymphocytes/immunology , Vaccines, Conjugate/administration & dosage , B-Lymphocytes/immunology , Bacterial Proteins/chemistry , Bacterial Proteins/genetics , Drug Design , Humans , Pneumococcal Infections/immunology , Pneumococcal Vaccines/chemistry , Polysaccharides, Bacterial/chemistry , Polysaccharides, Bacterial/immunology , Protein Structure, Tertiary , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Streptococcus pneumoniae/drug effects , Vaccines, Conjugate/chemistry
9.
Article in Russian | MEDLINE | ID: mdl-21384595

ABSTRACT

Streptococcus pneumoniae cell wall and cytoplasmic proteins contribute directly to pathogenesis of pneumococcal infection. Protective effect of pneumococcal proteins such as pneumolysin (Ply), muramylamidase (LytA) and pneumococcal surface protein A (PspA). There is discussion in the literature about development of conjugared pneumococcal vaccines, which should include polysaccharides of invasive serotypes of pneumococci as well as protein antigens of this pathogen, for prevention of infections caused by S. pneumoniae. Researches suggest that such hybrid vaccines will be effective, first of all, for children < 2 years of age and elderly > 65 years old because immune response to polysaccharide vaccines either do not form at all or insufficient for prevention of pneumococcal infection.


Subject(s)
Pneumococcal Infections/prevention & control , Pneumococcal Vaccines/immunology , Streptococcus pneumoniae/immunology , Animals , Bacterial Proteins/chemistry , Bacterial Proteins/immunology , Cell Wall/immunology , Cytoplasm/immunology , Humans , N-Acetylmuramoyl-L-alanine Amidase/chemistry , N-Acetylmuramoyl-L-alanine Amidase/immunology , Polysaccharides/immunology , Streptolysins/chemistry , Streptolysins/immunology , Vaccines, Conjugate/immunology , Virulence Factors/chemistry , Virulence Factors/immunology
10.
Article in Russian | MEDLINE | ID: mdl-19621817

ABSTRACT

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.


Subject(s)
Bacterial Vaccines/immunology , HSP70 Heat-Shock Proteins/immunology , Lipopolysaccharides/immunology , Salmonella Infections/prevention & control , Salmonella typhimurium/immunology , Animals , Bacterial Vaccines/administration & dosage , Cell Line , Dose-Response Relationship, Immunologic , HSP70 Heat-Shock Proteins/administration & dosage , Humans , Injections, Intraperitoneal , Lipopolysaccharides/administration & dosage , Mice , Recombinant Proteins/administration & dosage , Recombinant Proteins/immunology , Salmonella Infections/blood , Toll-Like Receptor 2/metabolism , Toll-Like Receptor 4/metabolism , Vaccines, Synthetic/administration & dosage , Vaccines, Synthetic/immunology
11.
Article in Russian | MEDLINE | ID: mdl-18283735

ABSTRACT

Protective effect of immunization with heat shock protein (HSP) against bacterial and viral infections in mice was studied. Recombinant HSP 70 kDa of Mycobacterium tuberculosis contaminated with lypopolysaccharide (0.185 mcg/ml) was used for experiments. One intraperitoneal injection of 100 or 400 mcg of HSP induced rapid protection against intraperitoneal challen e with 125 LD50 of Salmonella typhimurium (on 3rd-6th day) and against intranasal challenge with 10 LD50 of avirulent for humans avian influenza virus H5N2 (A/ mallard/Pennsylvania/10218/84) (on 5th-8th day). Three daily injections with 10 mcg of HSP induced rapid, significant and long-term protection against S. typhimurium. Immunization with HSP protected 100% of mice during 3 days after the challenge, 50% of immunized animals survived during 21 days (duration of the study). All nonimmunized mice died on 6th day.


Subject(s)
Bacterial Proteins/administration & dosage , Heat-Shock Proteins/administration & dosage , Influenza A Virus, H5N2 Subtype , Lipopolysaccharides/administration & dosage , Orthomyxoviridae Infections/prevention & control , Salmonella Infections, Animal/prevention & control , Salmonella typhimurium , Vaccination , Animals , Bacterial Proteins/chemistry , Heat-Shock Proteins/chemistry , Immunization Schedule , Injections, Intraperitoneal , Mice , Molecular Weight , Mycobacterium tuberculosis/chemistry , Recombinant Proteins/administration & dosage , Recombinant Proteins/chemistry , Recombinant Proteins/metabolism , Vaccines, Combined/administration & dosage , Vaccines, Combined/chemistry , Vaccines, Synthetic/administration & dosage , Vaccines, Synthetic/chemistry
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