[Influence of siRNA complexes on the reproduction of influenza A virus (Orthomyxoviridae: Alphainfluenzavirus) in vivo].

INTRODUCTION: Influenza is one of the most pressing global health problems. Despite the wide range of available anti-influenza drugs, the viral drug resistance is an increasing concern and requires the search for new approaches to overcome it. A promising solution is the development of drugs with action that is based on the inhibition of the activity of cellular genes through RNA interference. AIM: Evaluation in vivo of the preventive potential of miRNAs directed to the cellular genes FLT4, Nup98 and Nup205 against influenza infection. MATERIALS AND METHODS: The A/California/7/09 strain of influenza virus (H1N1) and BALB/c mice were used in the study. The administration of siRNA and experimental infection of animals were performed intranasally. The results of the experiment were analyzed using molecular genetic and virological methods. RESULTS: The use of siRNA complexes Nup98.1 and Nup205.1 led to a significant decrease in viral reproduction and concentration of viral RNA on the 3rd day after infection. When two siRNA complexes (Nup98.1 and Nup205.1) were administered simultaneously, a significant decrease in viral titer and concentration of viral RNA was also noted compared with the control groups. CONCLUSIONS: The use of siRNAs in vivo can lead to an antiviral effect when the activity of single or several cellular genes is suppressed. The results indicate that the use of siRNAs targeting the cellular genes whose expression products are involved in viral reproduction is one of the promising methods for the prevention and treatment of not only influenza, but also other respiratory infections.

Preparation a Recombinant Form of Pneumolysin Protein from Streptococcus pneumoniae.

A recombinant form of pneumolysin from Streptococcus pneumoniae was obtained. By using Vector NTI Advance 11.0 bioinformatic analysis software, specific primers were designed in order to amplify the genome fragment of strain No. 3358 S. pneumoniae serotype 19F containing the nucleotide sequence encoding the full-length pneumolysin protein. A PCR product with a molecular weight corresponding to the nucleotide sequence of the S. pneumoniae genome fragment encoding the full-length pneumolysin was obtained. An expression system for recombinant pneumolysin in E. coli was constructed. Sequencing confirmed the identity of the inserted nucleotide sequence encoding the full-length recombinant pneumolysin synthesized in E. coli M15 strain. Purification of the recombinant protein was performed by affinity chromatography using Ni-Sepharose in 8 M urea buffer solution. Confirmation of the recombinant protein was performed by immunoblotting with monoclonal antibodies to pneumolysin.

Recombinant Pneumolysin of Pneumococci Induces TLR4 Expression and Maturation of Dendritic Cells In Vitro.

Pneumolysin (Ply) is a target for the development of serotype-independent pneumococcal vaccines, an important condition for the efficacy of which is their ability to activate innate immunity with the subsequent formation of adaptive immunity. In this study, the ability of recombinant full-length Ply (rPly) of pneumococci to induce TLR expression and maturation of dendritic cells generated from mouse bone marrow was evaluated. It was shown that rPly in vitro increased the number of dendritic cells expressing Toll-like receptor 4 (TLR4) on the membrane. rPly caused maturation of dendritic cells generated from mouse bone marrow, which manifested in a decrease in the number of progenitor cells (CD34), an increase in the number of cells expressing the adhesion molecule CD38, costimulatory molecules CD80 and CD86, molecules of terminal differentiation of dendritic cells CD83, as well as molecules of antigenic presentation of the major histocompatibility complex class II.

[Hydrogen effect on the mechanisms of mucosal immunity in patients with COVID-19].

AIM: To study the inhalation of an active form of hydrogen effect to mucosal and system immunity in a rehabilitation program for health workers. MATERIALS AND METHODS: The study involved patients that survived COVID-19 after therapy with inhaled hydrogen for 90 minutes (n=30), and a control group of patients treated according to standard protocol for managing patients that survived COVID-19 during the rehabilitation period (n=30). Biomaterial was carried out in 2 stages: on the first day of the study, before the accepted therapy and on the 10th day of the study. The indicators of humoral and cellular immunity were studied. The levels of secretory immunoglobulin A (sIgA) and IgG were investigated using the method of enzyme-linked immunosorbent assay. Phagocytosis was assessed on a Beckman Coulter FC-500 flow cytometer. Statistical data processing was carried out in the GraphPad Prism 7.00 software using nonparametric methods. RESULTS: It was shown that the phagocytic index (PI) of monocytes in nasal scrapings after inhaled hydrogen treatment did not significantly change relative to the first day of treatment and control, while the PI of granulocytes in nasal scrapings significantly increased relative to the first day by 2.5 times (p=0.000189), as well as relative to the control by 1.1 times (p=0.047410). PI of monocytes in pharyngeal scrapings showed a significant increase relative to the first day of treatment by 2.8 times (p=0.041103), however, did not differ relative to the control. PI of granulocytes of pharyngeal scraping did not differ significantly relative to the first day and control. PI of granulocytes and blood monocytes of the studied group did not change significantly. PI of granulocytes and monocytes of peripheral blood relative to control during therapy did not change. The sIgA level in nasal scrapings significantly increased by 2.9 times, while in pharyngeal scrapings the level of sIgA significantly decreased by 2 times. Сonclusion. We have shown an increase in granulocytes PI in the nasal cavity and oral monocytes, as well as in the level of sIgA in the nasal cavity during therapy with active hydrogen. The data obtained indicate the effectiveness of therapy, which can be used both in the treatment of COVID-19, and in post-COVID syndrome as an additional therapy. The absence of changes in blood parameters, as well as individual links in nasal and pharyngeal scrapings, requires further study to develop ways to overcome treatment tolerance.

[The prospects for the use of drugs based on the phenomenon of RNA interference against HIV infection].

The human immunodeficiency virus (HIV) is currently one of the most pressing global health problems. Since its discovery in 1978, HIV has claimed the lives of more than 35 million people, and the number of people infected today reaches 37 million. In the absence of highly active antiretroviral therapy (HAART), HIV infection is characterized by a steady decrease in the number of CD4+ T-lymphocytes, but its manifestations can affect the central nervous, cardiovascular, digestive, endocrine and genitourinary systems. At the same time, complications induced by representatives of pathogenic and opportunistic microflora, which can lead to the development of bacterial, fungal and viral concomitant infections, are of particular danger. It should be borne in mind that an important problem is the emergence of viruses resistant to standard therapy, as well as the toxicity of the drugs themselves for the body. In the context of this review, of particular interest is the assessment of the prospects for the creation and clinical use of drugs based on small interfering RNAs aimed at suppressing the reproduction of HIV, taking into account the experience of similar studies conducted earlier. RNA interference is a cascade of regulatory reactions in eukaryotic cells, which results in the degradation of foreign messenger RNA. The development of drugs based on the mechanism of RNA interference will overcome the problem of viral resistance. Along with this, this technology makes it possible to quickly respond to outbreaks of new viral diseases.

Quantitative Assay of B. pertussis Lipopolysaccharide.

We studied activity of Bordetella pertussis LPS in the LAL test. The mean activity of various series of LPS preparations obtained from B. pertussis cells ranged from 1,950,000 to 2,940,000 endotoxin units/µg (EU/µg). Activity of the LPS preparation obtained from the culture medium supernatant was significantly higher (4,640,000 EU/µg). Activity of the control standard E. coli 055:B5 LPS was 19,500±500 EU/µg. These data indicate that activity of the obtained preparations of B. pertussis LPS in the LAL test is 100-200 times higher than activity of E. coli LPS used as a reference control. It was concluded that the results of the LAL test when assessing the permissible content of B. pertussis endotoxins require correction, probably by introducing a correction factor.

[Vaccination with virus-like particles containing hemagglutinin protects the lungs of mice with postifluenza bacterial pneumonia: virological, microbiological and clinical data].

INTRODUCTION: Influenza is a severe viral disease, a frequent complication of which is a secondary bacterial pneumonia. Influenza vaccines prevent secondary bacterial complications. Virus-like particles are one of the promising areas for the development of new vaccines. The aim of this work is to study the correlation of the pathomorphological characteristics of the lungs with clinical, virological, and microbiological markers of the disease at vaccination with virus-like particles (VLPs), containing hemagglutinin (HA) of influenza virus (HA-Gag-VLPs) in a murine model of secondary bacterial pneumonia induced by S. pneumoniae after influenza infection. MATERIAL AND METHODS: BALB/c mice were vaccinated with VLPs containing influenza HA. After 21 days, mice were infected with two strains of influenza viruses, homologous and non-homologous, and 5 days after viral infection, were infected with S. pneumoniae. The vaccination effect was evaluated by morphological, virological (titer of the virus in the lungs) and microbiological (titer of bacteria in the lungs) data, and was confirmed by clinical data (survival, change in body weight). RESULTS: Immunization with HA-Gag-VLPs, followed by infection with a homologous influenza virus and S. pneumoniae, reduced the area of foci of inflammation, inhibited the replication of the virus and bacteria in the lungs, and also protected animals from death and reduced their weight loss. Immunization with HA-Gag-VLPs upon infection with a heterologous strain and S. pneumoniae did not affect these criteria. CONCLUSION: The immunization with HA-Gag-VLPs prevented the viral replication, providing a reduction of S. pneumoniae titer and the degree of lung damage, protecting animals from the disease in a murine model of secondary bacterial pneumonia, induced by S. pneumoniae, after influenza infection with homologous strain of the virus.

Application of bacterial therapeutic vaccine Immunovac-VP4 in the treatment of pollinosis.

AIM: The aim of the study was to study the effectiveness of the complex use of bacterial therapeutic vaccine Immunovac-VP4 and allergen-specific immune therapy (ASIT) in pollinosis in children and adults. MATERIALS AND METHODS: Bacterial therapeutic vaccine Immunovac-VP4 was used annually, nasal and oral administration in patients before the course of ASIT standardized aqueous-salt solutions of allergens. RESULTS: The therapeutic application of bacterial vaccines, Immunoac-ВП4 before the course ASIT has helped to reduce the frequency of acute respiratory infections in 8,5 times in comparison with the control group. Clinical efficacy of complex treatment according to the results of the survey of patients in 7 years after the start of therapy was 90%. There was a significant decrease In IgG4 to causally significant allergens, General immnunoglobulin E (IgE) and a tendency to decrease IgE. CONCLUSION: The use of bacterial therapeutic vaccine Immunovac-VP4, which is a natural ligand of toll-like receptors in combination with ASIT, seems to be an effective and promising direction in the treatment of allergic diseases.

[MICROBIOCENOSIS, IMMUNE SYSTEM AND HEREDITY].

The formation of pro-/eukaryotic systems is the general biological mechanism of formation and variability of the phenotype of plants, animals, human beings under the influence of external wednesday, i.e. formation of adaptive potency conditions to external wednesday that increases the <> prokaryotic structures in sustaining body health. Prominent role in the formation of the phenotype of micro media, immunological tolerance (immunological programming), as a basis for the formation of individual pro-/eukaryotic interactions in perinatal age, the dominant role of maternal influence in this process on the one hand, micro-variability due to external stress impact on the other, makes it possible to consider pro-/eukaryotic interaction as a possible mechanism of perinatal programming and epigenetics inheritance and therefore, as one possible approach for correction of chronic and congenital pathology This points to the need to improve monitoring of the formation microbiocenosis of children, improve the methods of assessment and correction.

[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.

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

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

[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.

[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.

[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.

[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.

[Dynamics of autoantibodies in elderly persons in the process of vaccination with Grippol and multicomponent vaccine VP-4]. / Dinamika autoantitel u pozhilykh liudei v protsesse vaktsinatsii grippom i polikomponentnoi vaktsinoi VP-4.

The dynamics of antibodies to organ specific and organ nonspecific antigens in the process of combined immunization with Grippol, an influenza polymer subunit vaccine and polycomponent vaccine VP-4 used for prophylaxis of acute respiratory infections, was under study. Persons aged 65 years and older were vaccinated. Grippol alone was introduced in a single subcutaneous injection into 92 persons and Grippol in combination with vaccine VP-4--to 103 persons. B[symbol: see text]-4 Vaccine was introduced intranasally and orally (6-8 doses). The administration of vaccine VP-4 was started simultaneously with vaccination with Grippol. Prior to immunization and 1 and 5 months later autoantibodies to the following antigens were detected: DNA (native and denaturated), collagen, elastin, myelin basic protein, microsomal fractions of kidneys, lungs, heart, liver, intestine, pituitary body, thyroid gland, pancreas, adrenal glands, ovaries, mucous and muscular layers of stomach. The number of persons with the level of antibodies at least to one of the antigens under study exceeding the normal values prior to vaccination varied from 19.4 +/- 8.6% to 41.5 +/- 7.7%, the average values of positive sera being 0.26 +/- 0.05 to 0.32 +/- 0.08 delta OD. One and 5 months after vaccination both values varied within the same limits in both groups. Immunization with Grippol as well as with its combination with vaccine VP-4 was found to increased spectrum of antibodies to tissue antigens and their increased content. The data give evidence that Grippol and vaccine Vp-4, introduced according to the immunization schedule used in these experiments, do not induce development of autoimmune processes.

[Specific anti-Yersinia G- and M-antibodies in healthy blood donors and in patients with alimentary intoxication]. / Spetsificheskie protivoiersinioznye antitela klassov G i M u zdorovykh donorov krovi i bol'nykh pishchevoi toksikoinfektsiei.

The work deals with the results of determination of specific antibodies in blood donors of Moscow and Tula and in patients with alimentary toxicoinfection, made with the use of enzyme immunoassay on the basis of Yersinia enterocolitica lipopolysaccharides (LPS), serovars O3 and O9. The sera of patients with alimentary toxicoinfection were found to yield positive reactions with Y. enterocolitica LPS in 35.9% of cases (the number of such reactions obtained with blood donor sera was 3 times less). The presence of cross reactions between Y. enterocolitica LPS and the microsomal antigens of the thyroid gland was established. A high detection rate of antibodies to the microsomal antigens of the thyroid gland among blood donors of Tula was registered.