[PROFILES OF CYTOKINES IN MICE DURING IMMUNIZATION WITH ADTP- VACCINE WITH ACELLULAR PERTUSSIS COMPONENT].

AIM: Study cytokine status in mice immunized with vaccines containing acellular pertussis component. MATERIALS AND METHODS: Vaccines developed in Mechnikov RIVS - acellular pertussis vaccine (aPV) and adsorbed pertussis-diphtheria-tetanus vaccine (aDTaP), containing a complex of protective antigens of pertussis microbe - were used in the study. F1 (CBAxC57B16) line mice weighing 12 - 14 g were immunized intraperitoneally 3 times at an interval of 7 days with aPV and aDTaP at human immunization dose (0.5 ml), containing 25 µg of pertussis component. Intact mice were used as a control group. Levels of IFN-,γ, IL-2, IL-4, IL-5, IL-12 cytokines were de- termined after each immunization in enzyme immunoassay using commercial test-systems from Cusabio (China). RESULTS: An increase of levels of IFN-γ, IL-2, IL-5, IL-12 and lack of stimulation of production of IL-4 was established in dynamics of immune response after administration of aPV and aDTaP vaccines. CONCLUSION: The data obtained indicate that immunization of mice with aPV and aDTaP vaccines resulted in activation of production of cytokines characteristic for im- mune response during pertussis infection and immunization with whole-cellular aDTP-vaccines.

[Immunogenicity and safety of vaccine preparations based on circulating Bordetella pertussis strains].

AIM: Study specific activity and safety ofvaccine preparations based on circulating B. pertussis strains with currently predominating allele variants of pertussis toxin (ptxA1) and pertactin (prn2) genes. MATERIALS AND METHODS: B. pertussis strains isolated from pertussis patients in Moscow in 2001-2010 were grown in dense and liquid media. The content of separate antigens in B. pertussis strains was determined by EIA. Immunogenicity and safety of the preparations was determined in F1(CBAxC57B16) line mice. RESULTS: All the studied circulating B. pertussis strains expressed pertussis toxin (PT), filamentous hemagglutinin (FHA) and agglutinogens corresponding to the serovar. Whole-cell and acellular pertussis vaccines were prepared based on the circulating strains, and a highly productive recently isolated toxigenic B.pertussis strain that could be used for production ofpertussis vaccines was selected as a result of studies ofimmunogenic, toxic and sensibilizing properties. CONCLUSION: Vaccine preparations based on a B. pertussis strain adapted to growth in liquid media with pertussis toxin and pertactin ptxAl1 - prn2 gene allele variation characteristic for contemporary population are specifically active and safe.

[Immunobiological properties of Bordetella pertussis lipopolysaccharide in the acellular pertussis vaccine].

AIM: Study of Bordetella pertussis lipopolysaccharide (LPS) immunobiological properties in the acellular pertussis vaccine. MATERIALS AND METHODS: Experimental series of acellular pertussis vaccines (APV), lyophilized LPS were used. Antibody titers against LPS in mice sera were evaluated by using EIA with peroxidase conjugate of anti-species antibodies against mice IgG. LPS activity in B. pertussis antigen complex preparations was determined in quantitative chromogenic LAL-test by end point. APV protective activity was determined in mice test during intracerebral infection by B. pertussis strain No. 18323 virulent culture. APV safety was determined in the mice body weight change test. RESULTS: The presence of LPS in APV was shown in immune electrophoresis with purified B. pertussis LPS preparation as a control. Formalin treatment changes immunochemical properties of APV LPS that lead to the shift of precipitation bands with pertussis agglutinating sera from the start zone into cathode. The quantity of LPS in pertussis culture supernatants was on average 49050 +/- 6774 endotoxin units per ml (EU/ml). In APV preparations the quantity of LPS was on average 906 +/- 90 EU/ml, i.e. decreased by more than 50 times. An increase of antibody titers against B. pertussis LPS in mice sera after the APV immunization was shown in EIA, which gives evidence of its presence in immunogenic form in the complex preparations. The preclinical studies carried out show protective activity and specific safety of the experimental APV series. CONCLUSION: Formalin-neutralized APV preparation is a complex of protein antigens in association with LPS. Formalin treatment results in modification of LPS molecule that retains antigenic properties but is significantly less toxic.

[Epidemic process and vaccine prophylaxis of pertussis].

Analysis of data on epidemic process of pertussis infection in conditions of wide coverage of population by prophylactic vaccination is presented. Post-vaccination immunity against pertussis was shown to reduce with age; therefore a large part of vaccinated children becomes susceptible to this infection already in primary school age. Epidemic process of pertussis continues both as manifest form and as subclinical and atypical forms. The main reservoir of pertussis infection is older age population groups. The necessity to implement programs of revaccination against pertussis in older children, adolescents and adults is justified, Characteristics of vaccines for immune prophylaxis of pertussis infection are given.

[Preclinical studies of an adsorbed diphtheria-tetanus-pertussis vaccine (ADTP-vaccine) with acellular pertussis component].

AIM: Evaluate standardness of antigenic composition of pertussis component, completeness of sorption of pertussis, diphtheria and tetanus components, specific activity and safety of experimental series ofADTP-vaccine with acellular pertussis component (ADTaP-vaccine). MATERIALS AND METHODS: The content of separate antigens (pertussis toxin, filamentous hemagglutinin and agglutinogens 1, 2, 3) in samples of acellular pertussis component of ADTaP-vaccine and completeness of sorption of pertussis component of ADTaP-vaccine were evaluated by using enzyme immunoassay. Completeness of sorption of diphtheria and tetanus components were determined in flocculation reaction and antitoxin-binding reactions, respectively. Protective activity ofADTaP-vaccine was studied in model ofmeningoencephalitis development in mice infected with Bordetella pertussis (strain 18323) neurotropic virulent culture, protective activity oftetanus component - by survival of mice after administration of tetanus toxin, protective activity of diphtheria component - by survival of guinea pigs after administration of diphtheria toxin. Safety of preparations was evaluated in tests of acute and chronic toxicity with carrying out pathomorphologic studies including immature animals. RESULTS: All the studied experimental series ofADTaP-vaccine were standard by content of separate antigens of pertussis microbe. All the ADTaP-vaccine components were completely sorbed on aluminium hydroxide gel. By protective activity ADTaP preparations satisfied the WHO requirements. The preparations were non-toxic in acute and chronic toxicity and did not induce pathomorphologic changes including immature animals. CONCLUSION: Experimental samples of ADTaP-vaccine by specific activity and safety satisfied WHO requirements.

[Production of pertussis toxin by Bordetella pertussis strains isolated from patients with whooping cough].

AIM: To assess level of pertussin toxin (PT) production by vaccine strains of Bordetella pertussis and strains isolated from patients with whooping cough. MATERIALS AND METHODS: Concentration of PT in supernatants of microbial cultures of 3 vaccine strains and 25 strains of B. pertussis isolated from patients with pertussis in 2001 - 2005 was measured with enzyme immunoassay using gamma-globulin fractions of rabbit antiserum to PT as immunosorbent or included in peroxidase conjugates. RESULTS: Level of PT production by strains isolated from infected persons varied from 3 +/- 0.5 to 64.8 +/- 12.2 ng/MFU/ml: in 9 strains--from 3 +/- 0.5 to 9.4 +/- 2.1 ng/MFU/ml, in 7--10.5 +/- 1.8 to 18.4 +/- 2.6 ng/MFU/ml, and in 9--23.6 +/- 4.5 to 64.8 +/- 12.2 ng/MFU/ml. CONCLUSION: B. pertussis strains isolated from patients were heterogeneous on level of PT production. Difference in expression of PT between strains were as high as 20-fold. Conditionally low, moderate and high levels of PT production had 9 (36%), 7 (28%), and 9 (36%) of 25 studied strains. Three vaccine strains had levels of toxin production similar to recently isolated strains with moderate level of its production.

[Serological activity of vaccine and circulating Bordetella pertussis strains].

AIM: To study activity of vaccine and circulating strains of Bordetella pertussis in serological reactions with serum samples from healthy vaccinated children and children with pertussis infection. MATERIALS AND METHODS: One hundred forty-six serum samples from children with pertussis infection as well as 158 samples from healthy vaccinated children aged 3 - 5 years old were studied. Serologic activity of 3 vaccine strains and 7 strains of B. pertussis isolated from patients with pertussis in 2001 - 2005 against sera of children with pertussis infection or vaccinated children was assessed with hemagglutination assay (HA), radial gel immunodiffusion (RGI), and immunoelectrophoresis (IEP). RESULTS: In HA both serum samples of infected and vaccinated children were equally active in agglutination of microbial preparations prepared from vaccine or recently isolated strains of B. pertussis. RGI assay showed that 81 - 84% of serum samples from infected children and 17 -19% of samples from healthy vaccinated children reacted with vaccine strains, and 81 - 85% of samples from infected children and 16 - 20% of samples from healthy vaccinated children reacted with circulating strains: Sera from patients with pertussis formed identical lines of precipitation with vaccine and circulating strains in RGI assay and three types of precipitation arches profile in IEP. Sera from healthy vaccinated children formed identical precipitation arches with vaccine and circulating strains in RGI assay and one type of precipitation arches profile in IEP. CONCLUSION: Antibodies of patients with pertussis were equally active against vaccine and circulating strains of B. pertussis. Antibodies of vaccinated children were also equally active against vaccine and circulating strains although revealed more narrow spectrum of antigens compared to children with pertussis infection.

[Isotypes of anti-pertussis antibodies in pertussis patients at different stages of the disease].

AIM: Evaluation of anti-pertussis antibodies in pertussis patients at different stages after the onset of the disease. MATERIALS AND METHODS: Levels of IgG, IgG1, IgG2, IgG3, IgG4, IgA and IgM antibodies against the antigen complex of pertussis were evaluated by enzyme immunoassay (EIA). Sera samples were analyzed from 208 pertussis patients examined from week 1 to 10 after the onset of the disease. RESULTS: 51%, 82% and 86% pertussis patients, and 67%, 72% and 78% patients examined from week 1 to 3 after the onset of the disease had increased levels of IgM, IgA and IgG antibodies respectively. 85%, 70%, 74% and 68% pertussis patients, and 76%, 57%, 87%, 57% patients examined from week 1 to 3 after the onset of the disease had increased IgG1, IgG2, IgG3 and IgG4 levels respectively. 92% of all examined pertussis patients and 83% of patients examined from week 1 to 3 after the onset of the disease had an overall increase of anti-pertussis antibody levels. Increased level of IgM antibodies was detected predominately from week 1 to 5 after the onset of the disease. Most of the patients examined from week 3 to 10 after the onset of the disease had increased levels of IgA, IgG, IgG1, IgG2 and IgG4 antibodies, and IgG3 antibody level was increased predominately in patients examined from week 2 to 6 after the onset of the disease. CONCLUSION: Serological indicators of pertussis measured by EIA were observed in 83% of the patients examined at the early stages after the onset of the disease. Simultaneous measurement of IgA, IgG and IgM antibody levels is the most effective approach for serological diagnostics of pertussis due anti-pertussis antibodies isotype composition heterogeneity at different stages after the onset of the disease. Increased levels of IgM and IgG3 antibodies are serologic indicators of the acute phase of pertussis infection.

[Humoral immunity against pertussis and its prevalence in population].

Levels of IgG and IgA to complex of Bordetella pertussis antigens were assessed in 503 healthy children aged 1 - 14 years, 75 adolescents aged 15 - 17 years, and in 504 adults aged 18 - 54 years. The highest level of IgG was observed in children aged < 5 years. In older age groups progressive decrease of IgG level was noted, which more most prominent in 9 - 11 year-olds with subsequent stabilization of the level in adolescents and adults. Significant heterogeneity of IgG level was noted in all age groups. Rate of detection of increased IgA level correlated with age-related decrease of IgG level and increased from 2 - 5% in children aged 1 - 5 years to 12 - 16% in older children and adults. Obtained data point to low levels of immunity against pertussis in older children, adolescents and adults and high undetected incidence of pertussis in studied population.

[Distribution of specific IgG subclasses in patients with pertussis and in healthy persons].

AIM: To assess level of IgG1, IgG2, IgG3, and IgG4 to complex of antigens of Bordetella pertussis in patients with whooping cough and healthy children and adults. MATERIALS AND METHODS: Levels of anti-pertussis IgG subclasses in sera of patients with pertussis and healthy children and adults were measured with solid phase immunoenzyme assay using peroxidase-conjugated monoclonal antibodies to human IgG1, IgG2, IgG3, and IgG4. RESULTS: In patients with pertussis, IgG1-IgG3-IgG2-IgG4 type of distribution of subclasses with predominance of IgG1 and IgG3 was revealed. In healthy children and adults the character type of subclasses distribution was IgG1-IgG2-IgG4 with absent or low level of IgG3. CONCLUSION: Detection of specific IgG3 mainly in patients with pertussis allows to consider them as a reliable serological sign of pertussis infection.

[Improvement of pertussis diagnostics in adults with prolonged cough].

AIM: To study clinical and laboratory data and levels of IgG, IgG1, IgG2, IgG3, IgG4, IgA and IgM to Bordetella pertussis complex of antigens in adults with prolonged cough. MATERIALS AND METHODS: Antibody levels to Bordetella pertussis complex of antigens were measured by solid phase immunoenzyme assay. Clinical and laboratory methods included CBC, chest X-ray, measurement of respiratory function, allergologic tests. RESULTS: In 16 out of 75 studied patients (21%) serological signs that are characteristic for current pertussis infection (increased levels of specific IgG and IgA as well as IgG1 - IgG3 - IgG2 - IgG4 type of distribution of specific IgG subclasses) were observed. Clinical and laboratory parameters--course of disease, characteristics of cough, results of CBC--corresponded to diagnosis of pertussis. In other studied patients levels of specific antibodies did not differ from levels observed in healthy persons and observed clinical signs corresponded to other respiratory diseases. CONCLUSION: Obtained results prove the high incidence of pertussis in adults, its essential importance as etiologic factor of prolonged cough and high informative value of serologic tests.

[Antigenic composition and serologic characteristics of domestic acellular pertussis vaccine].

AIM: To assess antigenic composition consistency and serological characteristics of domestic acellular pertussis vaccine. MATERIALS AND METHODS: Amount of pertussis toxin, filamentous hemagglutinin, agglutinogens types 1, 2, and 3 in experimental batches of vaccine was measured by enzyme immunoassay. Levels of antibodies to aforementioned antigens as well as to lipopolysaccbaride in serum samples obtained from patients with pertussis and healthy vaccinated children were measured by the same method. The amount of lypopolysaccharide was determined by LAL test. RESULTS: Studied batches of vaccine were standard on amount of all protein antigens as well as lipopolysaccharide. Spectrum of antibodies to vaccine components in serum samples from patients with pertussis and healthy vaccinated children included antibodies to individual antigens: pertussis toxin, filamentous hemagglutinin, lipopolysaccharide, agglutinogens types 1, 2, and 3. CONCLUSION: Developed technology for manufacturing acellular pertussis vaccine allows to consistently produce preparations with standard amount of all components. Vaccine components interact with antibodies to wide spectrum of B. pertussis antigens.

[Diagnostic value of IgG, IgA, and IgM to Bordetella pertussis antigens in patients with pertussis].

Comparative study of IgG, IgA, and IgM levels to complex of antigens (CA) of vaccine strain No. 475 and separate antigens of Bordetella pertussis: pertussis toxin (PT), filamentous hemagglutinin (FHA), lypopolysaccharide (LPS), agglutinogens 1 (Aggl.1) and 2 (Aggl.2) was performed by ELISA in 80 patients with pertussis and 80 healthy vaccinated children. Antibodies to mentioned antigens were detected both in ill and healthy children but their levels were remarkably higher in patients. The most reliable serologic marker of pertussis was IgA, which were detected in the majority of patients. Detection rates of this class of antibodies to CA, PT, FHA, LPS, Aggl.1, and Aggl.2 were 91%, 77.5%, 69%, 59%, 80%, and 12%, respectively. Elevated levels of specific IgA were registered in 5% of healthy children. Obtained results showed high information value of detection of the IgA and IgG antibodies to CA, PT, FHA, and Aggl.1 using ELISA for pertussis diagnostics. Simplicity and economy of the CA obtainment allow to recommend CA-based ELISA for serologic diagnostics of pertussis.

[Acellular pertussis vaccine].

Protective, immunogenic, toxic, and sensitizing properties of acellular pertussis vaccine (aPV) developed according to original technology were studied, aPV had marked protective activity which lasted more than 2 years. Sera of mice immunized by aPV also possess protective properties, and they were more prominent than in sera of mice immunized by pertussis bacteria suspension (PS). Immune sera to aPV neutralized cytopathogenic effect of pertussis toxin (PT) on ovarian Chinese hamster cells in 1:250 dilution, whereas neutralizing activity of sera to PS was very low. Level of antibodies to PT was higher in rabbits immunized, according to schedules and dosage recommended for children, by aPV than by PS. High immunogenicity of aPV was proved also by levels of IgG to PT in sera of mice immunized three times by aPV in human dosage. During experiments on mice and guinea pigs aPV had mild toxicity, did not induce autoimmune process, did not have anaphylactogenic properties compared with bacterial suspension characterized by high anaphylactogenic activity. Histamine-sensitizing abilityof aPVwas 40 times lower than that of PS. Assessment of pyrogenic properties of aPV and PS performed on rabbits showed that aPV was 1,000 times less pyrogenic than PS. Obtained results demonstrate high protective and immunogenic properties of domestic acellular pertussis vaccine and its low toxic and sensitizing characteristics.

[Contents of antibodies to Bordetella pertussis antigens in patients with rheumatic diseases].

Study of presence of antibodies against pertussis in 72 rheumatic patients (with uvenile rheumatoid arthritis, systemic lupus erythematosus, etc.) aged 1-18 year old without history of pertussis was performed. Mean age of the patients was 10.6 +/- 0.48 year old, duration of illness--51.2 +/- 4.42 months. Immunosupressive therapy at the time of the study was conducted in 68 (94.4%) children. Using ELISA method, IgG to pertussis toxin (PT) and to antigens of acellular pertussis vaccine (aPV) were detected in 98.6% and 100% of children. High titers of antibodies were detected more frequently in 7-18 year old age group, which can indicate recent pertussis disease or infection. Vaccination history was studied in 131 children with rheumatic diseases. Incidence of pertussis in 43 unvaccinated children was 116.3 per 1000, and in 16 children with incomplete vaccination--62.5 per 1000. Out of 75 patients, who received vaccination series and revaccination, clinically distinct pertussis was not diagnosed.

[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.

[Modern strains of Bordetella pertussis: immunobiological properties and vaccine improvement].

Strains of B. pertussis isolated from patients in Moscow in 2001-2005 as well as strains included in locally produced diphtheria-tetanus-whole cell pertussis (DTP) vaccine were studied. Nucleotide sequences in genes of pertactin and S1-subunit of pertussis toxin of isolated strains, their immunobiological properties and opportunity to use for producing of the acellular pertussis vaccine were determined. Genes of pertactin and S1-subunit of pertussis toxin in the isolated wild strains differed from the same genes in strains included in the local DTP vaccine. Majority of the isolated strains belonged to serotype 1.0.3 and were markedly virulent.

[Humoral imunity to pertussis in children with diabetes of type 1].

To determine the state of humoral immunity to pertussis in children with insulin-dependent diabetes, IgG antibodies to pertussis toxin (PT) were determined in blood serum samples by means of EIA. In a group of children aged up to 6 years the highest percentage (100%) received the complete course of vaccination against pertussis with Russian adsorbed DPT vaccine, containing whole-cell pertussis monovaccine, while in a group over 6 years the complete vaccination course (3 vaccinations and 1 revaccination) had 53.4% of children. Pertussis morbidity was considerably higher in nonvaccinated subjects than in children with 4-fold vaccination (p < 0.001). The coefficient of association (Q) was 0.84. Children of all age groups were found to have low and average titers of antibodies to PT. The regressive analysis showed a decrease in antibodies in persons completely immunized against pertussis by the age of 6 years old. The presence of antibodies in nonimmunized persons showed that cases of pertussis or carrier state took place among the population. High titers of antibodies, indicative of recent cases of pertussis, were registered in all age groups, but high titers of antibodies were registered mostly in the group of children over 13 years old (p < 0.05), which confirmed an increase in pertussis morbidity in adolescents. Thus, vaccination against pertussis effectively protected children with diabetes of type 1, aged up to 6 years. For more prolonged protection the vaccination and revaccination of children aged over 4 years old is necessary.

[Protective activity of adsorbed D(a)PT vaccine with the acellular pertussis component and polyoxydonium].

The introduction of the immunomodulator polyoxidonium in an amount of 0.5 Mg/ml into adsorbed D(a)PT vaccine with the acellular pertussis component leads to the preservation of the protective activity of the pertusis component, diphtheria and tetanus toxoids, as well to the 4-time decrease of the content of adsorbent (aluminium hydroxide) from 2 to 0.5 mg/ml.

[Construction of plasmids coding for diphtheria toxin fragments]. / Konstruirovanie plazmid, kodiruiushchikh fragmenty difteriinogo toksina.

Plasmids coding different nontoxic derivatives (toxoids) of the diphtheria toxin were constructed. A secretion of toxoids that carry a signal sequence was found in the periplasmic space of E. coli and Erwinia carotovora. Toxoids without a signal sequence appear in the cytoplasm. We believe that the toxoids secreted in E. coli and E. carotovora cells undergo a limited proteolysis. According to the molecular weights of the fragments there are three targets for proteolysis. One of them being just between A- and B-fragments of the diphtheria toxin. The others are localised in the B-fragment. The role of E. coli signal peptidase in the specific cutting is discussed.