Search

Relevance. The development of effective and safe vaccines for pertussis prevention remains an urgent public health challenge.Aim. To study the protective activity and safety of acellular pertussis vaccine (AcPV) containing a complex of protective antigens from freshly isolated and vaccine strains of Bordetella pertussis.Materials and methods. Freshly isolated (No. 287, and No. 317) and vaccine (No. 305 and No. 475) B. pertussis strains with «non-vaccine» and «vaccine» allelic variants of the pertussis toxin (PT) subunit A gene, the PT promoter gene, the pertactin gene, the fimbria 2 gene, and the fimbria 3 gene strains were used for the production of AcPV.Results. All the studied variants of AcPV were harmless in the test of changes in the body weight of mice and sensitivity to histamine. The protective activity of AcPV3 (strains No. 287, No. 317 and No. 305) and AcPV1 (strains No. 287, No. 305 and No. 475) was higher than that of AcPV2 (strains No. 317, No. 305, and No. 475). IgG antibody titers to PT were also higher in mice immunized with AcPV1 and AcPV3.Conclusion. The higher protective activity of AcPV3 and AcPV1 may be associated with the genotype of strain No. 287, which has a ptxP3 PT promoter and is characterized by an increased level of PT production and high virulence. The most promising for further preclinical and clinical studies is AcPV3, which contains 2/3 of the antigens of the dominant «non-vaccine» genotype and 1/3 of the «vaccine» genotype, corresponding to the genes of PT, pertactin and fimbria to the currently circulating B. pertussis strains.

Study cytokine status in mice immunized with vaccines containing acellular pertussis component.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).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.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.

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.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.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.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.

Study of Bordetella pertussis lipopolysaccharide (LPS) immunobiological properties in the acellular pertussis vaccine.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.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.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.

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).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.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.Experimental samples of ADTaP-vaccine by specific activity and safety satisfied WHO requirements.

There are presented the results of genotoxicologic, immunologic and allergologic examinations which were conducted within the framework of integrated medical and biological assessment of genetically modified second generation soybean event MON 89788. Analysis of damages of DNA and structural chromosome aberrations, assessment of the allergenic potential and immunoreactive properties has not confirmed any genotoxic, allergenic and immunotoxic effect of soybean event MON 89788.

To study pathogenic characteristics of B. pertussis strains isolated from patients during different periods of pertussis infection epidemic process.Strains of B. pertussis isolated in Moscow during 1967 - 1971, 1980 - 1985, and 2001 - 2005 were studied. Nutrient media: Bordet-Gengou blood agar, casein-charcoal agar.mice - F1 hybrids (CBA x C57BL6). Pathogenic characteristics of strains were studied by assessment of virulence (LD50), leukocytosis-stimulating (LS units) and histamine-sensitizing (HSD50) activities of cultures. Genotyping was performed using standard equipment and reagents for DNA isolation, amplification, sequencing and detection of results.On the sample of 164 strains, pathogenic and genotypic characteristics of B. pertussis populations circulated during 1967 - 1971, 1980 - 1985, and 2001 - 2005. Majority of B. pertussis strains isolated in 1967 - 1971 and strains circulated during current phase of epidemic process were virulent (80.75% and 81.8% respectively) and had significant leukocytosis-stimulating and histamine-sensitizing activity, whereas strains isolated from patients with pertussis in 1980 - 1985 characterized by lower virulence and toxicity. Genotyping showed strains carrying "non-vaccine" allele ptxA1, which emerged in the middle of 1970s, totally displaced strains with "vaccine" alleles ptxA2 and ptxA4.Adaptive changes of B. pertussis driven by increased vaccination coverage involve both ptxA gene and pathogenic characteristics of infectious agent in the range of genotypically homogenous population with domination of strains, which have high levels of virulence and toxicity.

To assess antigenic composition consistency and serological characteristics of domestic acellular pertussis vaccine.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.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.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.

There are presented the results of genotoxicologic, immunologic and allergologic examinations which were conducted within the framework of integrated medical and biological assessment of genetically modified rootworm Diabrotica spp.-protected maize event MIR604. Analysis of damages of DNA and structural chromosome aberrations, assessment of the allergenic potential and immunoreactive properties has not confirmed any genotoxic, allergenic and immunotoxic effect of maize event MIR604.

There are presented the results of genotoxicologic, immunologic and allergologic examinations which were conducted within the framework of integrated medical and biological assessment of genetically modified rootworm Diabrotica spp.--protected and glyphosate tolerant maize event MON 88017. Analysis of damages of DNA and structural chromosome aberrations, assessment of the allergenic potential and immunoreactive properties has not confirmed any genotoxic, allergenic and immunotoxic effect of maize event MON 88017.

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.

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.

As shown in this work, the synthetic immunomodulator glucosaminylmuramyldipeptide (GMDP) can be included into acellular pertussis vaccine (APV). The optimal doses of GMDP, ranging from 0.001 to 0.0001 microg, have been found. These doses enhance the protective activity of APV, especially its low-active doses. GMDP decrease the manifestations of toxic, anaphylactogenic and pyrogenic properties of APV, which may lead to the decrease of the antigenic load of APV on the body of the vaccines and thus to lessening the side-effects of vaccination. GMDP has been shown to considerably increase, in comparison with common pertussis vaccine and APV, the percentage of phagocytizing leukocytes by day 14. The immunization of mice with APV with and without GMDP in doses of 0.01 and 0.001 microg leads to a change in T-lymphocyte/B-lymphocyte ratio in the population of spleen lymphocytes.

The immunomodulating activity of acellular pertussis vaccine (APV) and adsorbed DPT vaccine with acellular pertussis component (DPTA vaccine) was studied. The study revealed that only large doses of APV, 10 immunizing doses (ID), suppressed humoral and cell-mediated response to sheep red blood cells (SRBC). 1 ID produced no influence on the formation of antibody producing cells, but increased the development of delayed hypersensitivity (DH) to SRBC. The modulation of cell-mediated immune response, induced by APV, returned to normal after the injection of purified staphylococcal toxoid, used as immunomodulator, in doses of 0.15 BU per mouse and 1.5 BU per mouse. DPTA vaccine containing 1 ID, as well as 10 ID, produced no immunomodulating effect. This was established by the evaluation of humoral response to SRBC in CBA mice and the study of the formation of DH to SRBC in BALB/c mice. As indicated by the total of the presented data, the inclusion of APV into DPTA vaccine enhanced the immunological safety of its pertussis component.

Toxic properties of acellular pertussis vaccine (APV) and morphological changes in white mice in response to intramuscular injection of APV (without or with immunomodulator glucosaminylmuramyl dipeptide-GMDP) were under study. APV used in these experiments was developed at the Mechnikov Research Institute for Vaccines and Sera (the Russian Acad. Med. Sci.) on the basis of Bordetella pertussis cultures in synthetic fluid culture media. In experiments on acute and chronic toxicity of APV (without GMDP) increased tissue immunity reactions in spleen, thymus, liver, lungs and intestinal wall was detected. There was no difference in immunomorphological reactions in mice receiving APV with different doses of GMDP, but some difference was observed in time dynamics of tissue immunity reactions. A small dose of GMDP should be preferred (0.0001 microgram) which results in gradual growth of tissue immunity reactions less pronounced toxic reactions caused be the APV injection.

The humoral response of mice and rabbits to the injection of whole-cell pertussis vaccine (PV) and acellular pertussis vaccine (APV), developed at the Mechnikov Research Institute for Vaccines and Sera (Russian Acad. Med. Sci.) in Moscow, was studied. In the sera of immunized animals antibodies to the antigenic complex were determined in the direct hemagglutination (DHA) test, specific antibodies to filamentous hemagglutinin (FHA) and pertussis toxin (PT)--in the enzyme immunoassay (EIA) and antibodies neutralizing PT in a cytopathogenic dose (CPD)--in neutralization test on Chinese hamster ovary (CHO) cells. In mice and rabbits immunized with APV the antibody titers determined in the DHA test were higher than those in the animals immunized with PV. Specific antibodies titers to FHA and PT in the sera of rabbits immunized with APV were also higher than those in the sera of rabbits immunized with PV. High dilutions of sera taken from the animals immunized with APC neutralized 4-16 doses of PT in the neutralization test on CHO cells. The most important result of this study was the detection of a more pronounced immune response in the animals immunized with APV in comparison with that induced by PV according to the results obtained in EIA and in the test of PT CPD neutralization on CHO cells.

The influence of whole-cell and acellular pertussis vaccines, introduced both alone and in combination with N-acetylglucosaminylmuramyl-2-alanine-D-isoglutamine (GMDP) on the activity of two enzymes of peritoneal exudate macrophages (5'-nucleotidase and Na+K(+)-adenosine triphosphatase) was studied. The study revealed that both pertussis vaccines exhibited immunomodulating properties, these properties being most pronounced in whole-cell pertussis vaccine. The use of GMDP in combination with pertussis vaccines led to changes in the enzymatic activity of peritoneal exudate macrophages, which was indicative of a decrease in the immunomodulating action of pertussis preparations.

The immunogenic and protective properties of acellular pertussis vaccines, prepared on the basis of B. pertussis multicomponent protective complex and the preparation containing only pertussis toxin, were studied. The study revealed that multicomponent preparations containing pertussis toxin (PT), filamentous hemagglutinin, agglutinogens, pertactin and adenylate cyclase possessed more pronounced immunobiological and protective properties in comparison with the monovalent preparation of PT, which was indicative of the expediency of developing acellular pertussis vaccines on the basis of the polyvalent protective complex as minor protective antigens seemed to enhance the protective action of pertussis toxoid, the main protective antigen.

A highly effective technology for obtaining a protective antigenic complex, including the main Bordetella pertussis protective antigens (pertussis toxin, filamentous hemagglutinin, agglutinogens, pertactin and adenylate cyclase) and isolated from the supernatant of B. pertussis cultivation medium, has been developed. A new method for the detoxication of antigenic complex which more available to preserve the protective property was suggested. Experimental batches of monovaccine and adsorbed DPT vaccine with the acellular pertussis component, possessing high protective activity and low histamine-sensitizing properties, have been obtained. The stability of protective properties both in liquid and lyophilized acellular pertussis preparations has been noted.

The possibility of obtaining native pertussis preparations with high immunobiological activity has been shown. To obtain such preparations, the use of B. pertussis selected strain, its growth under the conditions of static cultivation in a synthetic culture medium and the use of supernatants with the maximum content of protective antigens with the aim of obtaining protective complexes are necessary.

Changes in the histamine-sensitizing activity of whole-cell pertussis vaccine (PV) under the action of immunomodulators (IM) of bacterial (peptide and peptidoglycans), synthetic (peptidoglycan) and vegetable origin have been studied. The study has revealed that these IM, introduced orally and parenterally, exhibit histamine-sensitizing activity, depending on the nature of IM and the optimum selection of the doses of IM and PV.

As revealed in animal experiments, glucosaminylmuramyl dipeptide (GMDP), the synthetic analog of muramyl dipeptide, when introduced intraperitoneally in a single injection or orally, exhibits adjuvant activity with respect to Citrobacter 0-antigens, Shigella flexneri and enhances the protective properties of dysentery and pertussis vaccines. The stimulating properties of GMDP depend on its dose, the route of its administration, the time elapsed after its administration, its ratio to the concomitant doses of bacterial antigens and to the dose of the virulent culture used for challenge.

The action of peptidoglycans (PG) of different origin has been experimentally studied in vivo. In these experiments PG of bacterial origin, such as blastolysin (BL), and synthetic PG, viz. muramyldipeptide (MDP) and its analog glucosaminylmuramyldipeptide (GMDP) have been used. Their toxicity, allergenic action, their effect on the phagocytic activity of peritoneal exudate macrophages (PEM), the accumulation of antibody-producing cells in the spleen, antibody titer in the blood serum and delayed hypersensitivity to nonbacterial antigens have been determined. As revealed in this study, BL does not differ from MDP in its toxicity and allergenic action. The phagocytic activity of PEM under the influence of BL only insignificantly differs from their activity under the influence of MDP, but is lower than under the influence of GMDP. The adjuvant action of BL is somewhat higher than that of synthetic PG.

The influence of pertussis preparations, introduced by oral and parenteral routes, on the detoxifying function of the liver and the state of the nervous system of the animals was studied by methods used in pharmacology and toxicology. The use of these methods made it possible to find out side effects produced by corpuscular pertussis vaccine, introduced parenterally, on the detoxifying function of the liver and the state of the nervous system of the animals. The negative influence on the nervous system was more pronounced after the injection of the commercial adsorbed diphtheria-pertussis-tetanus vaccine used in this investigation than after the injection of pertussis monovaccine. The oral administration of corpuscular pertussis vaccine exerted no negative influence on the above-mentioned body functions of the animals.

The influence of bacterial and synthetic peptidoglycans on the toxicity of acellular pertussis preparations (APP) has been studied in the mouse weight gain test and in the endotoxic shock development test on mice treated with Actinomycin D. The data obtained in these tests indicate that the immunomodulators (IM) under study are capable of changing (increasing or decreasing, depending on the dose) the toxic properties of APP. The antitoxic action of IM, established in this study, depends on the combination of the doses of IM and APP and the time elapsed from the time of immunization. The possibility of using these IM in a dose of 0.0001 microgram for reducing the LD50 of APP has been established. The use of IM belonging to the group of bacterial synthetic peptidoglycans for the development of acellular pertussis vaccines with reduced reactogenicity has been shown to hold much promise.

The authors present the results of study of humoral immunity in rabbits after oral and subcutaneous immunization with pertussis vaccine. There was an increase in agglutinin titre and of the protective properties of the serum both after the oral administration of the vaccine according to two different schemes and after the subcutaneous immunization. However, rabbits immunized orally displayed a more prolonged (150-day) persistence of agglutinin titres, as well as of preventive and protective activity of the sera than rabbits immunized subcutaneoulsy.

The influence of extracts from oak bark, St. John's-wort leaves and pine buds on natural immunity characteristics of mice has been studied. The injection of these extracts into mice has been found to enhance their resistance to infection with Staphylococcus aureus and Bordetella pertussis virulent cultures, to decrease the enzymatic activity of 5'-nucleotidase in the peritoneal exudate macrophages of mice and to increase the level of lysozyme in their blood. The action of these extracts has proved to depend on their dosage and the time of observation.

The influence of blastolysin, the cell-wall glycopeptide of Lactobacillus bulgaricus, on the main immunobiological properties of pertussis vaccine, its immunogenicity and toxicity, has been studied. The use of blastolysin, introduced as a stimulating agent in definite doses and following definite administration schedules, has been shown to permit achieving not only an increase in the immunogenicity of the corpuscular pertussis vaccine, but also a considerable decrease in its toxicity and histamine-sensitizing activity.