Evgeny Michailovich Zaitsev

Introduction. Whooping cough remains an urgent health problem worldwide, including in countries with high vaccination rates, where, since the 1990s, there has been an increase in the incidence of whooping cough, an increase in the severity of the disease and mortality. In this situation, it is necessary to create a new generation of acellular pertussis vaccines (аPV) that can more effectively affect the colonization, persistence and transmission of Bordetella pertussis. One of the possible directions for improving the vaccine prophylaxis of pertussis infection is the creation of aPV based on protective antigens isolated from biofilm cultures of B. pertussis. The aim of the study was to research the level of IgG antibodies to the antigens of the pertussis pathogen: adhesins — filamentous hemagglutinin (FHA), pertactin (PRN) and toxins — pertussis toxin (PT), lipopolysaccharide (LPS) in mice immunized with experimental aPV based on antigenic complexes (AC) isolated from biofilm and planktonic cultures of B. pertussis. Materials and methods. Experimental aPV based on AC isolated from the culture medium of biofilm (aPV-B) and planktonic (aPV-P) cultures of B. pertussis strain No. 317 (serotype 1.2.3) were used in the experiments. IgG titers of antibodies to PT, FHA, PRN and LPS in blood sera of mice immunized with aPV-B and aPV-P was determined in ELISA. Results. The titers of IgG antibodies to adhesins (FHA and PRN) in the aPV-B group were 8 and 4 times higher, respectively, compared with aPV-P, in the absence of significant differences in the titers of IgG antibodies to PT and LPS. Conclusion. The higher ability of aPV-B to induce an immune response to B. pertussis adhesins compared to aPV-P, in the absence of significant differences between them in stimulating IgG antibodies to toxins, indicates the advantage of using antigenic complexes from biofilm cultures to create aPV of a new type.

Relevance. An increase in the incidence of whooping cough, a high proportion of severe forms of the disease, and a decrease in the sensitivity of circulating strains of B. pertussis to antibiotics require the development of more effective etiotropic therapies, including those capable of influencing biofilm forms of the whooping cough pathogen, which differ from planktonic cells by increased resistance to the host immune system and antibacterial drugs.Аim of the work is to study the effect of trypsin and lidase in combination with gentamycin on the growth of biofilms of Bordetella pertussis strains on an abiotic substrate.Materials and methods. In the experiments B. pertussis strains isolated in the Russian Federation from whooping cough patients in 2001‒2010 were used: No. 178 (serotype 1.2.0), No. 287 (serotype 1.0.3) and No. 317 (serotype 1.2.3), grown on a dense nutrient medium. The intensity of biofilm formation in a liquid nutrient medium in the presence of trypsin (10 mcg/ml), lidase (20 IU/ml), gentamycin (2.0 mg/ml, 0.4 mg/ml and 0.08 mg/ml) and their combinations in roundbottomed polystyrene 96­well plates was evaluated by staining with 0.1% gentian­violet solution.Results. Gentamycin partially suppressed the formation of biofilms and caused partial destruction of the formed biofilms in the absence of growth of microbial colonies when sowing supernatants from biofilm cultures on a dense nutrient medium. The minimum suppressive concentration of gentamycin (MSC) was 2 mg/ml. Trypsin completely suppressed the growth of biofilms and caused the complete destruction of the formed biofilms. Lidase also suppressed the growth of biofilms, but less effectively affected the formed biofilms. The growth of colonies typical of B. pertussis was noted when sowing supernatants from biofilm cultures in the presence of trypsin and lidasе on a dense nutrient medium. Trypsin in combination with all the studied concentrations of gentamycin completely suppressed the growth of biofilms (MSC 0.08 mg/ml), and in combination with gentamycin at a concentration of 2.0 mg/ml caused complete destruction of biofilms in the absence of microbial growth on a dense nutrient medium. Lidase in combination with all the studied concentrations of gentamycin also suppressed the formation of biofilms (MSC 0.08 mg/ml), and in combination with gentamycin at a concentration of 2.0 mg/ml caused partial destruction of the formed biofilms in the absence of microbial growth on a dense nutrient medium.Conclusion. The synergistic effect of the combination of trypsin and lidase with gentamycin on growing and formed biofilms of B. pertussis strains was revealed. The combined use of trypsin or lidase with gentamicin reduced its MSC for growing biofilms by 25 times. The most pronounced effect on the formed biofilms was the combination of trypsin with gentamycin at a concentration of 2 mg/ml, which caused their complete destruction and death of planktonic cells. The effect of the combination of lidase with gentamycin on the formed biofilms was less pronounced.

Aim. Study the effect of trypsin, lidase (hyaluronidase) and fluimucil (N-acetyl-L-cysteine) on the growth of biofilms of Bordetella pertussis strains on the abiotic substrate. Materials and methods. In the experiments, the strains of the main B. pertussis serotypes isolated in the Russian Federation from whooping cough patients in 20012010 were used: No. 178 (serotype 1.2.0), No. 287 (serotype 1.0.3) and No. 317 (serotype 1.2.3), grown on a dense nutrient medium. The intensity of biofilm formation in a liquid nutrient medium in the presence of trypsin, lidase and fluimucil in round-bottomed polystyrene 96-well plates was estimated by staining with 0.1% gentian-violet solution. Results. Trypsin suppressed the growth of biofilms and destroyed the formed biofilms. Lidase suppressed the growth of biofilms less actively, without affecting the formed biofilms. Fluimucil did not affect both the growth of biofilms and the formed biofilms. The growth of colonies typical for B. pertussis was noted when planting fluids from cultures in the presence of preparations, as well as from culture control wells on a dense nutrient medium. Conclusion. The different effect of the drugs studied by us may be related to the different quantitative content of targets for trypsin (proteins), lidase (mucopolysaccharides, containing uronic acids), fluimucil (acid mucopolysaccharides) in the biofilm matrix. The growth of the typical morphological properties of the colony of B. pertussis during the sowing of culture seedlings on a dense nutrient medium testifies to the destruction of the biofilm matrix by trypsin and lidase in the absence of influence on plankton cells.

Since the last century, European countries began to actively use soybean seeds as food additives or for food production with 100% milk and animal proteins replaced with soy. In Russia, soy is a relatively new agricultural crop, which recently began to be used in food production. Meanwhile, some proteins of soybeans have pronounced allergenic properties. The World Health Organization classified eight soy proteins (Gly m 1Gly m 8) as allergens capable of causing clinically significant immunoglobulin (Ig)E-mediated sensitization in patients with allergic diseases. Moreover, most of these allergens were included in the World Health Organization nomenclature over the past 15 years. Additionally, the presence of other soybean allergens that can bind to sIgE has scientific evidence. Moreover, some soy allergens have homology in amino acid sequence and three-dimensional similarity to proteins of other plants and animals. Therefore, questions of IgE-cross-reactivity are becoming relevant. Thus, this review aimed to analyze and summarize scientific data on the chemical structure, properties, and allergenic activity of soy and soybean proteins (soybean seeds) and their cross-reactions with proteins of animal and plant origin.

Relevance. Whooping cough remains a pressing public health problem worldwide, including in countries with high vaccination rates. One of the probable causes of the ongoing epidemic process of pertussis infection is B. pertussis biofilms, which differ from plankton cultures by an altered gene expression spectrum and are highly resistant to environmental conditions, antibiotics, and immune factors.Aims. Analysis of literature data on the genetic and molecular - cellular mechanisms of biofilm formation by bacteria of the genus Bordetella, as well as approaches to the search for means aimed at suppressing the growth of biofilms and the destruction of formed biofilms in the macroorganism.Conclusions. Biofilm formation by microbes of the genus Bordetella is a complex multistage process regulated by genetic signaling systems: the Bvg AS system and the 2-nucleotide (p) ppGrp system, as well as other regulatory proteins and the polysaccharide complex. The matrix of B. pertussis biofilms consists of extracellular DNA, proteins, and a polysaccharide polymer that play an important role in the formation of biofilms in the respiratory tract and on abiotic surfaces. The genetic and molecular-cellular processes of biofilm formation and maintenance, as well as the various components of the biofilm matrix, can serve as targets for new antimicrobial drugs and more effective pertussis vaccines that will better control the entire pertussis infection cycle, including colonization, persistence, and transmission of the causative agent. One of the approaches to the development of new-generation cell-free pertussis vaccines is the identification of new biofilm-associated antigens that can induce effective cellular and humoral responses. The search for drugs that can destroy biofilms, including substances that affect the matrix and facilitate the access of antibacterial drugs to microbial cells, is promising.

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.

Aim. Study of the effect of antibodies to agglutinogens 1 and 2, filamentous hemagglutinin (FHA) and pertussis toxin (PT) on the formation of biofilms by Bordetella pertussis strains on the abiotic substrate.Materials and methods. Vaccine-derived and freshly isolated strains of B. pertussis were used. Cultures of strains grown on a dense nutrient medium were used as an inoculum for obtaining biofilms. The intensity of biofilm formation in round-bottomed polystyrene 96-well plates in the presence of antisera to agglutinogens 1 and 2, antiserum to FHA, and monoclonal antibodies (MСА) to the S1, S2, and S3 subunits of PT was evaluated by staining with 0.1% gentian-violet solution.Results. Most of the studied strains were sensitive to antibodies, which was manifested in complete suppression of biofilm formation. All strains were sensitive to antiserum to agglutinogen 1, antiserum to FHA, and MCA to the S2 subunit of KT. Three out of 4 studied strains with this agglutinogen in their composition were sensitive to antiserum to agglutinogen 2: No. 475 (serotype 1.2.3), No. 317 (serotype 1.2.3) and No. 178 (serotype 1.2.0). Relative resistance to antiserum was detected only in serotype 1.2.0 strain No. 305, but with minimal dilution, the intensity of biofilm formation was 1.8 times lower than in the control group. Strains No. 703 (serotype 1.0.3) and No. 287 (serotype 1.0.3) that did not have agglutinogen 2 were resistant to antiserum. Four and 5 out of the 6 strains used were sensitive to the S1 and S3 subunits of PT, respectively. Strain No. 305 was resistant to MCA to the S1 and S3 subunits, and strain No. 287 to MCA to the S1 subunit. At the same time, the intensity of biofilm formation was 2 and 1.8 times lower than in the control at the minimum MCA dilution.Conclusion. These data indicate that the growth of biofilms of B. pertussis strains is suppressed by antibodies both to the surface structures of the microbial cell (agglutinogens 1 and 2, FHA) and to the S1, S2 and S3 subunits of PT.

Aim. To study the sensitivity of biofilms of vaccine and freshly isolated strains of Bordetella pertussis to antibiotics.Materials and methods. Vaccine and freshly isolated strains of B. pertussis were used. Cultures of strains grown on dense nutrient medium were used as inoculate for biofilms production. The intensity of biofilm formation in round-bottomed polystyrene 96-well plates was estimated by staining with 0.1% gentian-violet solution. The following antibiotics were used in experiments: penicillins (ampicillin), cephalosporins (ceftriaxone), aminoglycosides (gentamicin), macrolides (erythromycin).Results. The highest resistance to antibiotics was demonstrated by the vaccine strain No. 305 and freshly isolated strain No. 211, sensitive only to erythromycin. Vaccine strain No. 703 was sensitive to gentamicin and ampicillin and showed resistance to erythromycin and ceftriaxone. Vaccine strain No. 475 was sensitive to all tested antibiotics. The Tohama 1 strain was resistant to ampicillin and sensitive to other antibiotics. Freshly isolated strains No. 178 and No. 162 were resistant to ceftriaxone and sensitive to gentamicin, erythromycin and penicillin. Minimal inhibitory concentrations of tested antibiotics ranged from 0.2 μg/ml to 5.0 μg/ml.Conclusion. These data indicate the heterogeneity of vaccine and freshly isolated strains of B. pertussis in sensitivity to antibiotics. The greatest activity was shown by erythromycin, which suppressed the growth of biofilms of 6 out of 7 strains. The least effective was ceftriaxone, which suppressed the growth of biofilms of only 2 strains.

Aim. Study of the sensitivity of Bordetella pertussis biofilms to polyvalent pertussis serum.Materials and methods. The intensity of biofilm formation by strains of B. pertussis in round-bottomed polystyrene 96-well plates in the presence of polyvalent pertussis serum was estimated by staining with 0.1% gentian-violet solution. The serum titer was estimated as a highest dilution, which suppressed the growth of biofilm cultures.Results. Serum titers that completely suppressed the formation of biofilms by the studied strains ranged from : 1,000 to 1 : 20,000. Vaccine strain No. 475a (serotype 1.2.3) was characterized by the highest sensitivity to serum, the titer of which was 1 : 20,000. Vaccine strain No. 305 (serotype 1.2.0) and strains isolated from whooping cough patients in the period from 2001 to 2010: No. 287 (serotype 1.0.3), No. 178 (serotype 1.2.0), No. 317 (serotype 1.2.3) were sensitive to serum in the titers 1 : 1,000-1 : 2,000. Vaccine strain No. 703 (serotype 1.0.3) was resistant to all serum dilutions studied. When sowing supernatants from wells with biofilms on a dense nutrient medium, the growth of colonies typical for B. pertussis was observed. Similar results were obtained when seeding the supernatants of the cultures from the wells with no biofilm.Conclusion. These data indicate that B. pertussis strains are heterogeneous in sensitivity to pertussis serum. The growth of colonies typical for B. pertussis when culture supernatants are sown on a dense nutrient medium, in the absence of biofilms in the wells, indicates that the biofilm formation is suppressed by inhibiting the adhesion of microbial cells on the substrate, and not due to the bactericidal action of the serum.

Aim. Development of technology for the manufacture of cell-free pertussis vaccine (CPV) from a freshly isolated strain of B. pertussis No. 211 serotype 1.2.3 and the study of its protective activity and safety in comparison with the preparation of vaccine strains. Materials and methods. Following B. pertussis strains were used: freshly isolated strain No. 211, serotype 1.2.3; vaccine strains No. 305, serotype 1.2.0, and No. 475a, serotype 1.2.3. According to the original method, a CPV was obtained from the supernatant of the liquid culture medium of B. pertussis strain No. 211 and its protective and toxic properties were studied. Results. Studies have shown that the use of enriched nutrient media for the cultivation of the strain and the increase in the duration of the the detoxification period of the protective antigen complex isolated from the culture medium are needed to obtain a CPV vaccine consisting of antigens of a freshly isolated strain. CPV obtained from the freshly isolated strain had protectivity 1.7 times higher compared to those of CPV obtained from vaccine strains, was nontoxic and had a low sensitizing properties. The results indicate that the freshly isolated strain No. 211 is a promising candidate for use in the development of pertussis vaccines.