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Mobile laboratories for implementing anti-epidemic measures in the territories of natural plague foci perform an important function of ensuring sanitary-epidemiological well-being of the country. The aim of the study was to determine the range of areas and tactics for deploying mobile laboratories to ensure the current tasks of immunological monitoring over the health status of the population at risk in the territory of a natural plague focus. Retrospective analysis of the results of long-term immunological monitoring of the risk contingent vaccinated against plague was carried out. The possibilities and prospects for using a mobile laboratory for immunological monitoring within the framework of ensuring the tasks and functions of the Rospotrebnadzor have been determined. Mobile laboratories for immunological monitoring expand the capacities to solve operational issues related to the provision of specific prophylaxis of particularly dangerous infectious diseases, and their deployment as part of other mobile units of the Sanitary-Epidemiological Service will contribute to a more accurate delimitation of the territory, volume, scope and priority of any given preventive measure.

The aim of the work was to study the effect of the azoximer bromide immunoadjuvant (polyoxidonium, PO) on certain molecular-genetic and proteomic properties of Yersinia pestis EV NIIEG strain, when added to the culture medium. Materials and methods. Y. pestis EV NIIEG was grown at 28 °C for 48 hours on LB agar pH 7.2 (Miller), with and without PO (EV+PO). Whole-genome sequencing of EV and EV+PO strains was performed on the Ion S5 XG generation II platform. Whole-genome SNP analysis and search for marker SNPs were conducted in the Wombac 2.0 program. Mass-spectra of Y. pestis EV extracts and EV+PO cells were recorded using a Microflex LT mass spectrometer. Protective properties of the test cultures were evaluated by the integral ImD50 index in BALB/c mice when infected with Y. pestis 231(708). Results and discussion. Comparative analysis has not revealed deletions, insertions and single nucleotide polymorphisms in the structure of Y. pestis EV+PO strain genome leading to a violation of the production of pathogenicity, immunogenicity and ensuring the vital activity factors of the plague pathogen. The MALDI-TOF mass spectrometry has shown that Y. pestis EV+PO strains changed the intensity of 22 % of the total number of peaks in the range of 2000–20000 Da. Most of the altered peaks in the UniProtKB protein bank belong to uncharacterized proteins and metabolic proteins. At the same time, the ImD50 was 2–3.3 times lower in cultures grown with the addition of PO than in Y. pestis EV. Thus, the addition of PO to Y. pestis EV NIIEG culture medium does not cause changes in the genes of pathogenicity and vital activity support factors of plague pathogen, but modulates its protein profile, which is accompanied by an increase in the protective potential of the EV vaccine strain.

Ensuring the preventive anti-plague vaccination and the rational use of immunoprophylaxis means entail an adequate and timely assessment of their effectiveness. In the absence of mass cases of plague, it is impossible to objectively assess the actual (epidemiological) effectiveness of anti-plague vaccination means and methods, therefore, the analysis of efficacy shifts towards studying individual indicators of the immune response to the introduction of a vaccine, and the immunological efficacy of vaccination becomes a key factor that ultimately ensures its epidemiological effect. The aim of the work – as part of a comprehensive evaluation – was to determine the nature of individual immune response and characterize the group immune responsiveness of persons vaccinated against plague in order to build up a methodology for individual and group adjustment of the anti-plague vaccination regimen. Materials and methods. We used an array of information data obtained during monitoring of post-vaccination immunity in individuals immunized with live plague vaccine (LPV), presented in the form of a database. To analyze diseases in the post-vaccination period among the vaccinated population, a five-year retrospective cohort study was conducted to determine the incidence of acute and exacerbation of chronic diseases in people vaccinated and not vaccinated against plague. Results and discussion. Persons vaccinated against plague were ranked according to the level of immune responsiveness. The nature of individual immune response was revealed and the group immunoreactivity of vaccinated individuals was characterized. The possibility of predicting the intensity of the immune response to vaccination has been determined. The frequency of adverse events following immunization (AEFI) in individuals annually vaccinated against plague has been assessed. As a result of the distribution of vaccinated individuals according to the level of immune responsiveness and taking into account AEFI, the possibility of forecasting the individual and group immunoreactivity of vaccinated individuals has been determined to justify timely correction of the LPV administration regimen.

An important area of research concerns monitoring of immune response features in patients with SARS-CoV-2 infection as well as their analysis, as compared with characteristics of vaccine-mediated protection, in order to specify the determinants of cellular immune response. The aim of our work was to compare the state of cellular immune response in patients who underwent COVID-19, and in persons vaccinated with a peptide vaccine preparation. The study involved volunteers who suffered with COVID-19 of varying severity (n = 30), as well as persons who completed the full course of vaccination with the peptide vaccine (n = 27). For comparison, we took blood specimens from the volunteers before vaccination. Immunophenotyping of leukocytes was performed by the Lyse/No-Wash procedure (BD Bioscience, USA), and Cyto-Stat monoclonal antibodies (CD45-FITC, CD4-PE, CD8-ECD, CD3-PC5), CD45RA-PC7, CD45RO-PE (Beckman Coulter, USA), and analyzed with a DakoCytomation flow cytometer (Denmark). Determination of intracellular IFNγ (CD4+IFNγ+) was performed with the standard technique. Cytokine production was determined using reagent kits for detection of IFNγ, TNFα, IL-4, IL-8, IL-10 (Vector-Best JSC, Russia) with automatic enzyme immunoassay analyzer LAZURIT (Dynex Technologies, USA). As based on the results obtained, we have shown that cellular immunity was developed after vaccination and infection with COVID-19. However, the most pronounced immune response was recorded in the COVID-19 reconvalescents, i.e., more than 60% of these patients showed an increased number of CD4+T-memory helper cells (8.7 (0.5-12.1) % versus 0.3 (0.1-0.5) % in the comparison group, p < 0.05) as well as proportion of CD4+IFNγ+T lymphocytes (4.2 (1.8-4.3) % versus 0.4 (0-0.8) % in the comparison group, p < 0.05). Moreover, we revealed an increased functional reserve of cells in terms of TNFα, IL-8, IL-10 production. One month after vaccination of volunteers with the peptide-based preparation, the total pool of memory T lymphocytes was apparently dominated by CD8+T memory cells (CD45+CD8+CD45RA-CD45RO+). A significant increase was found in the average levels of CD4+IFNγ+ activated cells (8.2-fold), as well as in values of ConA-induced IL-4 production (3.3 (1.1-4.5) pg/mL, and 2.8 (1.7-3.9) pg/mL, respectively versus 1.3 (0.1-2.4) pg/mL in the control group, p < 0.05). The data obtained are in accordance with information available in the literature concerning development of cellular immune responses to SARS-CoV-2, which results from a past illness, or measures for the specific prevention of COVID-19. Further search for cellular correlates of protection against a new coronavirus infection will allow us to revise the current vaccination strategy and develop an optimal approach to COVID-19 prevention.

Tyrosine protein phosphatase (common leukocyte antigen CD45) regulates FcᵧR-mediated cell signaling and secretory function of neutrophilic granulocytes (NG) when interacting with antigen-antibody immune complexes. The aim of the work is to study changes in the expression of CD45 on the surface of human granulocytes in ex vivo modeling of bacteremia by live cells of the plague microbe vaccine strain Yersinia pestis EV NIIEG and to evaluate the priming effect of the live plague vaccine (LPV) in vivo in terms of this parameter. The expression density of CD45 on NG was determined by flow cytometry in conventional units of fluorescence intensity (MFI) after staining the cells with the CD45-FITC labeled mouse antibody reagent (Backman Coulter, USA) during immunophenotyping of blood leukocytes according to the Lyse/No Wash protocol. In donors not previously vaccinated against plague (group 1), the value of the indicator was assessed before and 30 min, 2 h, 6 h after the addition of Y pestis EV cells to whole blood at a dose of 108 mc/ml, as well as 1 month and 6 months after the primary anti-plague vaccination. In individuals who had previously been repeatedly vaccinated with LPV in the territory of the natural plague focus (group 2), CD45 expression on blood granulocytes was determined one year after the last annual vaccination, and then 1 month and 6 months after revaccination. Getting into human blood, living cells of the vaccine strain Y pestis EV of the plague microbe induced a change in the NC phenotype already after 30 minutes, associated with a 3.5-fold increase in the surface expression of CD45, which remained at an elevated level for 6 hours. The studied indicator depended ex vivo on the degree of resistance of plague microbes to phagocytosis and killing of NG. Plague vaccination had a similar stimulating effect on human peripheral blood NG in vivo. Under the influence of HPV, CD45 expression increased on blood NG in groups 1 and 2 one month after vaccination, and the changes persisted in volunteers for 6 months. The experimental data obtained in the work may reflect the result of NG priming with lipopolysaccharide and other Y. pestis antigens. The registered functional activation of NG by expression of tyrosine protein phosphatase probably indicates the formation of “immune memory” at the level of innate immunity cells under the influence of LPV, the functioning of which explains the development of a faster and more intense antigen-specific immune response to repeated introduction of the plague vaccine into the body.

Relevance. As part of ensuring sanitary and epidemiological well­-being, in the territory of natural plague foci of the Russian Federation, according to epidemiological indications, the population is vaccinated against this infection. The lack of a unified scheme for evaluating the effectiveness of vaccination dictates the need to develop universal express methods that allow screening studies of anti­-plague immunity, including in the field.Aims. To evaluate the effectiveness of the express method for the direct detection of antibodies to Yersinia pestis in biological material using microstructural glass waveguides with a hollow core (MGW HC) as immunosensors in studying the dynamics of seroconversion in people vaccinated with the plague live people.Materials and methods. In the study, blood serum samples were taken from 30 individuals vaccinated according to epidemic indications with the live plague vaccine (PLV) and 30 volunteers who were not vaccinated and did not have a history of contact with the plague agent.Results. An assessment was made of the possibility of using the method of direct detection of antibodies using MGW HC at different time intervals according to the receipt of blood sera in the framework of monitoring vaccinated individuals from among the inhabitants of the Caspian sandy natural plague focus. The presence of specific antibodies to the F1 protein in the blood serum of vaccinated volunteers was revealed both with the help of ELISA and with the use of MGW HC. The speed of the method of direct detection of antibodies (maximum 2 minutes per test), the absence of the need to use species­specific secondary antibodies, enzymes and substrates and additional costs for laborious sample preparation were noted.Conclusions. The method of direct detection of antibodies using glass microstructural waveguides is promising for introduction into the list of express methods for assessing the immunological effectiveness of anti­plague vaccination.

Relevance. The search for informative markers for assessing the immunological efficacy of a live plague vaccine remains an urgent scientific task. The goal of the studyAims was to make a comparative assessment of the level helper T-cells activation by CD69 and HLA-DR markers, in people, vaccinated against plague, in an in vitro test, using the disintegrated by ultrasonic Y. pestis cells, grown at temperature 28 °C as a specific stimulator.Materials & Methods. A cytofluorimetric analysis of blood samples of 45 individuals vaccinated against the plague was carried out. The dependence of the result of cytological analysis of the two studied cell markers on the immunoregulatory index (IRI) of the vaccinated donor at the time of vaccination was revealed.Results. It was found that for newly vaccinated individuals, the T-helpers were more intensive and prolonged by the early activation marker, while for those revaccinated with the immunoregulatory index, at the time of vaccination more than 1.5, the more intense cellular response was by the late activation marker.Conclusions. Thus, the possibility of quantitative evaluation of the immunological efficacy of vaccination against plague, based on the identification of lymphocyte activation markers when stimulated with a specific antigen, is confirmed.

Complexity and multifactorial nature of potential pathogenic consequences of SARS-CoV-2 infection in human body, discovery of new virus-induced mechanisms triggering a cascade of pathological responses in the cells of host organism leading to development of multiple organ failure elicited increasing interest in morpho-functional state of blood cells in reconvalescent persons after COVID-19 infection. The aim of the present work is to characterize morphofunctional pattern of blood cells at different periods of recovery, depending on the severity of COVID-19. We examined 55 convalescents after bearing COVID-19 infection: Group I included the convalescents 30 days after the disease (n = 39); Group II consisted of the persons 60 days after recovery (n = 16); Group III included clinically healthy volunteers with no history of clinical SARS-CoV-2 infection (n = 11). The cells were examined by means of Olympus CX41 microscope (Olympus, Japan), and VZ-C31S digital videocamera (VideoZavr, Russia) using the VideoZavr software (version 1.5). Assessment of neutrophil populations in the whole blood samples was performed with BD Accuri C6 Plus flow cytometer (USA) with automatic differentiation of cells between lymphocytes and monocytes, according to the degree of granularity. Cytokine production was determined using commercial kits for detection of IFNγ, TNFα, IL-4, IL-8, IL-10 (JSC Vector-Best, Russia), IL-17A (eBioscience, Austria) was assayed with automatic enzyme immunoassay analyzer “LAZURIT” (Dynex Technologies, USA). Among the convalescents who suffered the moderate-degree COVID-19 (45.5% and 50% of cases, respectively) on days +30 and +60 after clinical recovery, a significantly increased ratio of morphologically altered forms of erythrocytes (echinocytes, ovalocytes, dacryocytes, codocytes) was noted as compared with group III (p = 0.00001 and p = 0.001, respectively). Regardless of clinical severity of the disease; a mean of 40.6% convalescents from groups I and II had moderate disturbances in the neutrophil morphology (cytoplasmic vacuolization, chromatin decondensation at the pre-netosis stage, transformation of cells by the netosis type), and, in 27.4% of cases, the areas of neutrophilplatelet aggregation were seen. In blood supernates from recovered patients, we have revealed a significantly decreased content of IFNγ (P = 0.02), TNFα (p = 0.03), IL-10 (p = 0.04) and IL-17A (p = 0.02). The revealed morphological and functional changes in blood cells in the persons who underwent COVID-19 infection suggest long-term maintenance of toxic damage to erythrocytes, neutrophils and lymphocytes over the recovery period. The effects of the detected morphological and functional disorders of blood cells following COVID-19 recovery leading to increase in blood viscosity and microcirculation, formation of neutrophil-platelet aggregates, may cause higher risks of thrombotic complications at the long-range period as well as decreased levels of regulatory cytokines, thus confirming slow recovery of the lymphocyte populations (Th1, Th2, Th17) of the immune system.

In case no assessment of changes in incidence rate can be used as an indicator for effectiveness of applied live plague vaccine, it is really necessary to search for other, particularly immunological correlates for vaccine-based protection. The aim of this study was to reveal immunological correlates for plague protection in mice immunized with Yersinia pestis EV NIIEG, and assess dynamics changes in select markers of plague vaccinated subjects. BALB/c mice were immunized with Y. pestis EV at dose of 2 102, 1 103, 5 103, 2.5 104 CFU, and on day 21 they were challenged with Y. pestis 231 at a dose of 400 LD50. Immunogenicity was calculated by the Kerber method and ImD50 was determined. Volunteers 20 subjects who were first vaccinated with live plague vaccine and 20 subjects who were not vaccinated against the plague. Blood cytokine production was measured on the LAZURIT analyzer (Dynex Technologies, USA) in mouse groups before Y. pestis 231 infection on day 14 as well as 21 days after vaccination, in humans before vaccination, 1, 6 and 12 months after vaccination. The immunized mice showed a significant increase (by 2.2 times) in the induced production of IFN and a moderate increase in the concentration of TNF, IL-10 and IL-17A on day 14 of disease. A high correlation was found between the survival rate of animals and the level of antigen-/mitogen-induced IFN production (r = 0.94, p = 0.039), both on day 14 and 21, as well as a noticeable relationship with the level of produced IL-10 and IL-17A on day 14. One month after vaccination volunteers had significantly increase by month 6 (p 0.05) levels of IFN, TNF, IL-10, IL-17A, although only for IFN and IL-17A, the persistence of induced production was noted at a fairly high level for up to a year. Thus, IFN and IL-17A can be considered as possible informative correlates of mouse protection against Y. pestis on days 14 and 21, considering the increase in the induced production of these cytokines as adequate markers of the protective efficacy of immunization, and assessing dynamics in these parameters of volunteers vaccinated with the plague live vaccine, the increase in the levels of IFN and IL-17A can be considered as a favorable prognostic marker of the immunological efficacy of the vaccine in the period from the month 6 to 12 of observation.

Introduction. Disease severity in hospitalized COVID-19 patients correlates with the relative content in the blood of a specific low-density neutrophilic granulocyte (NG) population, whose cells are characterized by reduced granularity, high heterogeneity in the expression of FcᵧRIIIb (CD16) and a tendency to spontaneous autolysis (netosis).The aim of the study was to compare the intensity of NG degranulation and the FcᵧRIIIb expression by these cells in blood of patients with COVID-19 and convalescents.Materials and methods. The blood leukocytes of 40 patients diagnosed with COVID-19, 33 convalescents and 20 healthy donors (control) were examined using flow cytometry. To identify NG (CD16+-granulocytes) and to assess the surface expression of the netosis molecular trigger (FcᵧRIIIb), a single-color reagent of labeled monoclonal antibodies CD16-FITC was used. Immunophenotyping of lymphocytes was performed using two four-color reagents. In addition, cell debris content was determined in each Lyse/No-Wash-stained whole blood sample. The presence of neutrophils at the stage of netosis was confirmed by microscopy. Cytokine production was determined on an automatic enzyme immunoassay analyzer.Results. Against the background of characteristic changes in the lymphocyte subpopulation composition and cytokine production, in blood of COVID-19 patients with acute pneumonia, hypoxia and tachycardia a more intense degranulation of NG (2.6 times), higher degree of CD16 expression heterogeneity (1.8 times) and an increased leukocytolysis intensity (1.6 times) were observed compared to convalescents who have undergone severe and moderate forms of the disease. In convalescents, the degree of differences of these indicators compared to control values varied in concordance with the disease severity.Conclusion. The nature of changes in the parameters studied in COVID-19 patients in the acute phase of the disease and during the period of convalescence confirms the possible mechanism of the development of complications due to an imbalance between the activity of elastase released from NG granules and its plasma inhibitor α1 -antitrypsin.

Background. The live-attenuated vaccine based on the Yersinia pestis strain EV line NIIEG is still used in Russia, providing protective efficacy against plague. Nevertheless, there is an urgent need for developing new ways to increase the immunogenicity of the Y. pestis EV NIIEG vaccine strain. In this study, the ability of direct action of immunoadjuvant azoximer bromide (polyoxidonium, PO) on the immunobiological properties of vaccine strain Y. pestis EV NIIEG during cultivation on a dense nutrient medium was evaluated. Materials & Methods. Y.pestis EV NIIEG, cultivated at 28 °С for 48 h on LB agar, Miller pH 7.2 ± 0.1 (Sigma-Aldrich, USA) with the addition of PO and without. MALDI-TOF mass-spectrometry was deployed for the obtainment of mass-spectra of ribosomal proteins from Y. pestis EV NIIEG cells on the MicroflexTM LT mass spectrometer (Bruker Daltonics, Germany). Protective efficacy was evaluated under subcutaneously challenge guinea pigs and mice BALB's with 400 LD50 doses of the Y. pestis 231, Y. pestis P-13268 Vietnam (MLD=5 CFU). Antibody titers to F1 in serum were determined using an ELISA. Results. The addition of the therapeutic concentration of PO in the cultivation medium induced a significant increase in the immunogenicity of Y. pestis EV NIIEG that resulted in enhancement of serum antibody levels against Y. pestis F1 antigen and several times the growth of protective efficacy in the bubonic plague model on two types of experimental animals. ImD50 of the vaccine strain Y. pestis EV NIIEG, cultivated with PO, was significantly (p < 0,05) lower in comparison to ImD50 for Y. pestis EV NIIEG in standard cultivation conditions. One year of storage at a temperature of 4 °С did not alter the protective properties of the vaccine strain Y. pestis EV NIIEG, cultivated with PO. Conclusions. Morphological studies confirmed the absence of influence PO introduction into the cultivation environment on the safety of the vaccine strain. MALDI-TOF MS profile of the Y. pestis EV NIIEG, cultivated with PO, had peaks characteristic features. The mass peak at m/z 3,061 was significantly down-regulated and new mass peaks at m/z 2,759, m/z 3,533 were determined. These changes are accompanied by the increase of Y. pestis EV NIIEG immunogenicity.

Community-acquired pneumonia is a polyetiological infectious disease of bacterial or viral nature. In most cases, it is caused by pathogens such as Streptococcus pneumoniae, Mycoplasma pneumoniae, Haemophilus influenzae, Chlamydophila pneumoniae, Staphylococcus aureus, Legionella spp. However, despite that a wide range of modern diagnostic methods have been introduced into laboratory practice, the etiology of the disease can be determined only in 40–60% of cases. Here, we analyzed the causative agents of community-acquired pneumonia and relevant markers samples of clinical material collected from patients undergoing treatment in the medical facilities of the city of Saratov and the Saratov Region during the period from July to August, 2020, coupled to increase number of cases of new coronavirus infection COVID-19. Clinical material (oropharyngeal swabs, saliva, sputum, blood and blood serum) was obtained from 129 patients and investigated by using microbiological, immunoserological and molecular genetic methods. For this, there were used transport and nutrient media, diagnostic drugs, reagents, discs with antibacterial domestic and foreign drugs registered in the Russian Federation. As a result, 82 (63.6%) patients were found to have markers of the pathogen COVID-19 (viral RNA or specific antibodies of IgM and/or IgG classes), whereas 42% of cases had overlapped RT-PCR and ELISA data. There was a predominance of severe clinical forms of the disease in the group of patients with pneumonia caused by SARS-CoV-2 virus (17.1%), compared with pneumonia of another etiology (12.8%). No marked differences in specific pattern of the accompanying microflora and its sensitivity to antibacterial agents were observed in persons with/without markers of COVID-19. While being examined by bacteriological methods, S. aureus, K. pneumoniae, S. pneumoniae, M. pneumoniae, Acinetobacter spp., both in monoculture and in associations, were isolated from samples of clinical material collected from the majority of patients. In 49.6% of cases, pathogenic microflora, including pathogens causing community-acquired pneumonia, has not been identified, which may be due to the use of antibacterial drugs before the collection of clinical material and treatment applied due to comorbid chronic diseases.

The COVID-19 pandemic continues, causing colossal damage to the population and the global economy. As COVID-19 is studied, new data are emerging regarding the risk of severe coronavirus infection in patients with α1-antitrypsin deficiency. α1 -Antitrypsin is the main inhibitor and key endogenous regulator of the serine leukocyte proteinase activitry released from the granules of activated neutrophils to the cell surface and into the extracellular space. It has been established that the number of cases of severe course and death of COVID-19 in the territories of 68 countries of the world correlates with the frequency of the spread of mutations in the proteinase inhibitor gene among the population of these countries, at which the concentration of α1-antitrypsin in the human blood plasma is 10 times lower than normal. All this contributes to the revision of a number of provisions of the pathogenesis and therapy of a new coronavirus infection.The review presents an analysis of the literature on the role of an inhibitor of serine leukocyte proteinases in protecting the body from COVID-19. The participation of α1-antitrypsin in the inhibition of SARS-CoV-2 penetration into the respiratory tract epithelial cells, in the protection of the vascular endothelium, blood plasma proteins and elastin of the lung tissue from the damaging effect of leukocyte elastase released during neutrophil degranulation and the formation of neutrophil extracellular traps (NETs) is considered. The role of a1-antitrypsin in suppressing inflammation by limiting the secretion of proinflammatory cytokines and neutrophil extracellular traps into the blood has been shown. The individual links in the pathogenesis of the new coronavirus infection have been detailed, which will allow revising the strategy for reducing the risks of severe course of COVID-19.

Aim. To characterize the influence of azoximer bromide (polyoxidonium, PO) in cultivation conditions on the morpho- and nanomechanical cell surface properties of Y. pestis EV NIIEG vaccine strain and its derivatives Y. pestis КМ218 (pYT–, pYV–, pYP–), Y. pestis КМ216 (pYT–, pYV–, pYP+), Y. pseudotuberculosis, Y. enterocolitica by atomic force microsopy (AFM), as well as on the adhesion of cells Y. pestis EV NIIEG to human collagen type IV.Materials and methods. The measurements were carried out using the Solver P47-PRO probe microscope (NT-MDT, Russia), standard methods of semi-contact AFM and AFM imaging analysis program. The adhesion of Y. pestis EV NIIEG to type IV collagen was determined by the number of cells binding to glass slides covered with human collagen type IV.Results. The introduction of PO in the cultivation environment caused changes in the morphometric parameters of the cells of Y. pestis EV NIIEG vaccine strain and its isogenic derivatives (increase in volume, flatten ingested (S/H), index I (W/H). These changes were accompanied by the transformation of nanomechanical properties of the cell surface (reducing the root mean square, adhesion force), which countenance was associated with the plasmid profile. The lesser decrease of adhesion force in the absence of changes of the index I was observed in cells Y. pseudotuberculosis and Y. enterocolitica with plasmid pYV. In the strain Y. enterocolitica KM383 (pYV–) PO did not induce significant changes in the indicators studied. The introduction of the PO into the cultivation environment decreased the ability of Y. pestis EV cells to bind to human collagen type IV. Modification by PO the adhesive properties of the vaccine strain Y. pestis EV NIIEG was accompanied by an increase in its immunogenicity.

Relevance. The widespread use of information technologies, both when creating a database based on the results of immunological monitoring of persons vaccinated with the live plague vaccine, and for predicting the effectiveness of measures for specific prevention of plague in a specific territory, is aimed at ensuring epidemiological surveillance of plague in the territories of natural foci of infection. The aim of the work was to analyze the application of the created replenished database, which allows accumulating, archiving and systematizing the results of immunological monitoring of persons vaccinated against plague for informational support of sanitary and epidemiological surveillance of plague in the territories of natural foci of infection.Materials and methods. The database included the results of a prospective cohort observational study. The database was created on the Microsoft Access 8.0 platform, an interactive software development environment for Visual Basic 6.0 with VB (Visual Basic) and / or SQL (structured query language) queries. To store the data, TXT files connected to the database were used.Results. Permanent (region of residence, age, sex, blood group, genovariants of the human leukocyte antigen HLA) and variable (indicators of cellular and humoral immunity) parameters obtained as a result of long-term immunological monitoring of persons vaccinated with the live plague vaccine according to epidemic indications have been archived. Using the information support of the Database, an assessment of the state of anti-plague immunity was carried out and screening indicators of the immune status of the contingent at risk were determined, characterizing the individual response to vaccination.Conclusions. The introduction of information technologies, which are the basis for multifactorial risk management of vaccine prevention, based on the use of modern databases for collecting, storing and analyzing data, makes it possible to make management decisions to optimize the volume and timing of measures for specific prevention of plague in the territories of natural foci of infection and in institutions that ensure the implementation of work with pathogens of especially dangerous infections.

Aim. To characterize the intracellular expression of cytokines by spleen T-helpers and the spontaneous production of cytokines in the blood of BALB/c mice immunized with Yersinia pestis EV NIIEG against the background of immunomodulation.Materials and methods. Intracellular expression of CD4+IFN-γ+, CD4+IL-4+, CD4+IL-17+ was determined in mice spleen cell suspensions by flow cytometry, IFN-γ and IL-10 were measured in ELISA in blood supernatants on day 3 and day 21 after the immunization with Y. pestis EV against the background of immunomodulation. On day 21 after the immunization animals were infected by Y. pestis 231 at a dose of 400 LD50.Results. Differences in cytokine response to studied drugs, correlated with CD4+IFN-γ+ levels in animals, were identified. On day 3, a significant decrease in CD4+IFN-γ+ was observed in response to Y. pestis EV and to recombinant gamma interferon (Ingaron). A significant increase in CD4+IFN-γ+ was detected in response to vaccine strain administered with azoximer bromide (Polyoxidonium). Intracellular expression of IFN-γ, IL-4 and IL-17 increased on day 21by an average of 2,3 times when immunomodulators were used in the immunization schedule. In addition, on day 21 a significant (p ˂ 0.05) increase in the proportion of T-helpers expressing IFN-γ, as well as in level of spontaneous IFN-γ production in blood supernatants was observed only in animals immunized by schedules that included immunomodulators. After the challenge with Y. pestis 231 of animals previously immunized by schedules that included Polyoxidonium, the correlation analysis confirmed the association (r = 0,94; p = 0,0004) of mice survival with intensity of CD4+IFN-γ+ expression.Conclusion. The data obtained confirm the effectiveness of Polyoxidonium application in experimental animal Y. pestis EV immunization schedule and the usefulness of intracellular cytokine expression measurement for assessment of the level of protection following the immunization.

The global community is experiencing one of the largest infectious disease outbreaks in the 21st century. In the Saratov Region, the first case of new coronavirus infection was confirmed on March 19, 2020.The maximum increase in cases was noted between May 15 and June 30, during that time the total number of infected people in the region increased from 1526 to 6444. Since July 2020, a stable incidence level of new coronavirus infection has been observed in the Saratov Region, without a steady decline.The aim of the study was to assess the status of population immunity to the SARS-CoV-2 virus among residents of Saratov and the Saratov Region under the COVID-19 epidemic.Materials and methods. In the period from June 23 to July 26, 2020, a serological study of blood samples from 3372 volunteers of different age groups was conducted. The content of antibodies to SARS-CoV-2 was determined by ELISA using a set of reagents “ELISA anti-SARS-CoV-2 IgG” produced by the State Scientific Center of Applied Microbiology and Biotechnology of the Rospotrebnadzor (Russia).Results and discussion. In general, the incidence of COVID-19 in the Saratov Region is taking place against the background of moderate seroprevalence to the SARS-CoV-2 virus, accompanied by a high incidence of non-apparent (asymptomatic) forms of the infectious process. The absence of clinical symptoms of the disease, in the context of the limited use of methods for determining the RNA of the SARS-CoV-2 virus in PCR (11 % of the region’s population) makes it difficult to assess the real spread of the virus in the population and to establish the timing of the formation of persistent herd immunity. A low rate of antibody response among individuals with a positive result of PCR analysis, as well as among volunteers who had an infection in May, June 2020, indicates a weak formation of the immune response, or the prevalence of individuals reacting mainly by activating the cellular link of the immune system in the population. The obtained results, although they need to be explained in a number of respects, can be applied to the organization of preventive measures, including vaccination, in the region.

The purpose of the study was to determine the effect of Yersinia pestis EV NIIEG on the process of neutrophil extracellular traps formation in vivo when modeling plague infection and assess their contribution to antiplague protection.Materials and methods. BALB/c mice, which were immunized subcutaneously with the Y. pestis EV NIIEG vaccine strain, were used in the study. Animals were infected with a virulent strain Y. pestis 231 at a dose of 20 LD50 (103 CFU). To evaluate the contribution of neutrophil extracellular traps (NETs) to antibacterial protection, an experimental model was used based on fermenting NETs in the abdominal cavity of mice with nuclease. To calculate the number of NETs in peritoneal exudate (PE) fluorescent microscopy was applied. Phagocytic activity of PE cells was determined by flow cytometry. Bactericidal effect of NETs was recorded using bacteriological method.Results and discussion. In pre-immunized mice, the process of NETs formation in response to the reintroduction of plague microbe living cells was 5 times more intense than in intact animals and was accompanied by a significant increase in the killing of Y. pestis cells in PE. The use of micrococcus nuclease in the experiment for fermentation of the NETs, produced in the body of immunized animals, provided evidence of NET participation in conferring anti-infective protection against plague infection. Thus, the established fact of the NET formation in case of Y. pestis infection of mice immunized with Y. pestis EV NIIEG vaccine strain and the influence of this process on the effectiveness of protection against plague is the basis for further clarifying the immunopathogenetic role of neutrophil granulocytes in plague.

Abstract. Objective of the work was to conduct a comparative assessment of the immune-modulating effect of the combined use of Yersinia pestis EV NIIEG vaccine strain with Polyoxidonium and Ingaron preparations on a BALB/c mouse model.Materials and methods. Mice of the BALB/c line were immunized subcutaneously with Yersinia pestis EV NIIEG culture at a dose of 2.5 104 m.c. (1st group), in combination with Ingaron at a dose of 150 IU (2nd group) or with Polyoxidonium at a dose of 4 мg (3rd group), the 4th group is intact mice. On days 3, 7, 21 and 90 after immunization, the subpopulation composition of lymphocytes, the production of mediators of the cellular response (INF-ɣ and IL-10), the titers of specific antibodies to the capsular antigen of plague microbe (F1), the nuclear apparatus of lymphocytes, and the nature of histological changes in the organs of mice were determined. Characterization of immunogenic (protective) activity of the combined use of Y. pestis EV NIIEG with immune-modulators against Y. pestis 231 in experiments on BALB/c mice was performed on the 21st day after immunization through determining the number of dead animals and their average life expectancy.Results and discussion. The combined administration of Y. pestis EV NIIEG vaccine strain with Polyoxidonium or Ingaron to experimental animals allowed us to establish differences in the response of the immune system of biomodels, due to the mechanism of action of a specific immune-modulator. It has been established that both Polyoxidonium and Ingaron combined with Y. pestis EV NIIEG enhance the response of immune-competent cells in experimental animals, contribute to the activation of the humoral response and the production of mediators of the cellular response, do not have a damaging effect on the tissue of the macroorganism. At the same time, the efficacy of using combined vaccination of Y. pestis EV NIIEG with immune-modulators in the inoculation test is confirmed for Polyoxidonium only.

Aim. To compare by flow cytometry the cellular immune status indicators in persons, living on the territory of the Caspian sandy natural plague focus for epidemic indications. Materials and methods. Whole blood leukocytes of 120 people were examined by flow cytometry with using the two four-color labeled monoclonal antibody reagents (Cyto-Stat CD45-FITC, CD4-PE, CD8-ECD, CD3-PC5 и Cyto-Stat CD45-FITC, CD56-PE, CD19-ECD, CD3-PC5) and by means of CD95-FITC reagent. The results were taken into account before and in a month and 6 months after vaccination. We were determined in the blood the relative content of lymphocytes, monocytes, granulocytes, leukocytes carrying an early apoptosis marker (CD95), T and B- lymphocytes, NK cells as well T – lymphocyte helpers (Th) and cytotoxic T-lymphocytes (Tcyt) for immunoregulatory index (IRI) calculation. Results. Before anti-plague vaccination in 14 people (11,7%) were found abnormally low immunoregulatory index values characterized the immunodeficiency state (IRI

In theRussian Federation, Y. pestis NIIEG strain-based live attenuated vaccine is used for immunization against plaque on epidemiological indications, displaying high efficiency. However, individual fluctuations in adaptive immunity after vaccination necessitate conducting a search for genes underlying variability of developing immune response. Of note, HLA (Human Leukocyte Antigen) gene polymorphism plays an important role in this process. In our study, we identified HLA class II haplotypes for HLA-DRB1, HLA-DQA1, and HLA-DQB1 in 120 residents of theterritoryofPre-Caspiannatural plague focus, who were immunized against plague. In addition, level of TNFα production correlated with detecting allelic groups HLA-DRB1*04 (р = 0.05) and DRB1*12 (р = 0.01). The data obtained show that HLA class II gene polymorphism can affect the level of cytokine secretion in response to plague immunization. Examining the genes regulating immune factor production will allow to get better insight into the mechanisms underlying immune response variations after vaccination as well as contribute to predicting immunogenicity and efficiency of developing vaccine preparations.

At present, the search for biomarkers, which indicate the presence of tense antiplague immunity, is continuing. The aim of the study was to determine the parameters of cellular and humoral immunity in persons vaccinated with the plague vaccine according to epidemiological indications and to characterize the correlation interrelations between them in the dynamics of the antiplague response formation. Blood samples from 114 people living in the territory of the Pre-Caspian sandy natural plague foci were investigated. Using commercial kits, the production of cytokines IL-1β, IL-2, IL-4, IL-6, IL-8, IL-10, IL-18, IFNγ, TNFα (Vector-Best, Russia) IL-17A (eBioscience, Austria), immunoglobulins IgG, IgM, IgA (Vector-Best, Russia) was determined on the enzyme immunoassay analyzer LAZURIT (Dynex Technologies, США). Blood leukocyte immunophenotyping was carried out using f luorochrome-labeled monoclonal antibodies Cyto-Stat CD45-FITC, CD4-PE, CD8-ECD, CD3-PC5 (Beckman Coulter, США) on the CyAn ADP DakoCytomation f low cytometer (Denmark). Obtained data analysis revealed a number of differences in the production of detectable cytokines (TNFα, IL-1β, IL-2, IL-4, IL-6, IL-17) between groups of vaccinated and first-vaccinated people. However, human blood cells, regardless of the multiplicity of the graft, retained their functional reactivity and were able to actively secrete the majority of the analyzed cytokines (8 of 10) in response to stimulation with concanavalin A. Diagnostically significant changes in the concentration of IFNγ, TNFα and IL-4 were detected for the characteristics of a specific antiplague response. For these marker cytokines, the greatest number of correlations have been revealed, both with each other and with changes in the level of other parameters: cytokines, immunoglobulins, T cells. The revealed multiple correlation links between the cytokine and immune status indices indicate the activation of both the cellular and humoral components of the antiplague immune response. The informative value of the complex analysis application of correlation interrelations between the humoral and cellular immunity parameters is shown to assess the level of immunological efficacy (actual vaccination) of vaccination in persons vaccinated against plague.

Current areal of anthrax among live stock and wild animals covers almost all the continents. The cause of human infection is conventionally considered to be the contact with diseased animals in the process of carrying for an animal, forced slaughter or further trimming of carcasses, as well as contact with infected raw materials of animal origin. Licensed vaccines has made an invaluable contribution to the improvement of epidemiological situation on anthrax, however, development of the vaccines complying with current scientific progress remains relevant. We have constructed a vaccine prototype containing recombinant protective antigen and S-layer EA1 protein and added state-of-the-art CpG 2006 adjuvant to the formulation. We have demonstrated the advantage of the lyophilized form of the preparation. Objective — obtainment of anthrax vaccine prototype in lyophilized state and assessment of the prototype compliance with the requirements to vaccine preparations. Materials and methods. Isolation of rPA and EA1 protein was carried out using producer-strain B. anthracis 55ΔTПА-1Spo– on an integrated end-to-end manufacturing line, including concentration, diafiltration and two-phase chromatography. CpG 2006 adjuvant was synthesized according to known sequences. Components were mixed and lyophilized in sublimation unit. Combination of 1% sucrose and 3% glycine was used as cryoprotector. Effectiveness and safety of the preparation were evaluated on the model of BALB/c mice and guinea pigs applying immunological, morphometric, and histological assays. Antibody titers in sera of immunized animals were evaluated using standard ELISA procedures. Protective properties were investigated through LD50 values for the test-strain used on immunized and control animals and immunity index. Results. We have performed complex investigation of the anthrax vaccine prototype, containing B. anthracis 55ΔTПА-1Spo– proteins as main and supplementary antigens, as well as CpG 2006 adjuvant, stabilizers and preservative agent. The prototype meets the requirements to immunobiological medicinal drugs by all physical-chemical properties. Our preparation does not have a toxic effect on the organism of laboratory animals in case of subcutaneous and intraperitoneal administration of single human dose. Pathomorphological study of guinea pigs’ organs immunized with double dose of the vaccine prototype has not revealed any evidence of damaging effect on the cells and tissues of macroorganism. The prototype protects BALB/c mice from the infection with B. anthracis 71/12 test-strain.

The article covers the issues related to the scientific substantiation and methodological support of immunological monitoring of persons vaccinated against plague upon epidemic indications. The problematic issues of the methodology for the assessment of immunological efficiency (efficacy) of plague live vaccine (PLV) are noted. The current tasks and possible prospects for the introduction of immunological monitoring of persons vaccinated against plague upon epidemic indications have been defined. The algorithm of efficacy estimation of plague live vaccine in vaccinated (revaccinated) persons has been tested under real conditions. Analysis of the results of efficacy evaluation of plague live vaccine among vaccinated (revaccinated) people against plague living in the territories of natural foci of this infection has been performed. Demonstrated is the possibility of using immunological monitoring results in creating an objective basis for improving the strategy of specific plague prevention in natural foci of this infection. The priority areas for further optimization of the specific plague prevention in the territories of natural foci of the infection, including those related to the formation of individual regimen revaccination tactics, taking into account the possibilities of creating modern and effective vaccines, are outlined.

Relevance. In 2005 International Health Regulations, the plague, is on the list of dangerous infectious diseases that can cause emergency situations of interstate importance. Even single cases of human plague are considered as the basis for carrying out preventive measures. The paper presents the results of immunological monitoring conducted on the territory of the Republic of Kalmykia in order to assess the immunological efficacy and safety of the plague live vaccine The paper presents the results of immunological monitoring in the territory of the Kalmyk Republic over the individuals vaccinated against plague due to epidemiological reasons. Materials and methods. Studies of immunological efficacy of live plague vaccine were conducted alongside stepwise assessment of cellular and humoral components of innate and adaptive immunity in persons revaccinated against plague, using a complex of advanced informative tests. Results and conclusions. It is established that before the second revaccination all the surveyed persons retained expressed immune response by the mixed or cellular type, characterized by high level of spontaneous and induced production of Th1-associated cytokines. Activation of Th1 immune reaction was registered one month after the scheduled revaccination; immune response change-over from Th1 to Th2 type – after 6 months of observation, and retention of adaptive immunity by mixed type at the moderate level – in a year. Specific humoral immunity developed in 85% of the surveyed persons, but throughout the whole investigation the dynamics of antibody titers to plague microbe F1 individualized and did not coincide with cellular immunity indicators. Performed complex study has confirmed the relative safety of the live plague vaccine.

In view of increasing epidemic danger potential of some natural plague foci and performing measures to provide specific prevention of this infection, it becomes necessary to carefully monitor the state of immunity in vaccinated (revaccinated) individuals. The aim of the study was to assess humoral and cellular immunity levels in the persons after repeated anti-plague revaccination performed for appropriate epidemiological indications. Evaluation of immune responses to anti-plague revaccination was carried out according to studies of blood samples from 20 people aged 24 to 53 years living in the Caspian sandy natural plague focus located at the territory in Kalmykia Republic. Specific antibody titers to the F1 antigen of plague microbe were assessed in blood serum by immunoassay testing using an “ELISA-AT-F1 YERSINIA PESTIS” test system (Russian Research Anti-Plague Institute “Microbe”, Russia), along with spontaneous and concanavalin A-induced production of different cytokines, i.e., IFNγ, TNFα, IL-4, IL-10 (Vector-Best, Russia), IL-17A (Bender Medsystems, Austria). The studies were performed before vaccination, and 1, 6, 12 month after the booster vaccination.The results of the study indicate that immunological reconstitution after revaccination with live plague vaccine occurs occurred mostly according to mixed Th1/Th2 type. Detection of antibodies to the F1 specific plague microbe antigen before and 1 month after revaccination (65 and 85%, respectively) was indicative of development of humoral response. The most informative cytokine markers (IFNγ and TNFα) for evaluation of an anti-plague cellular immune response have been identified. The threefold increase in IFNγ induced production before revaccination indicates to initial immunological competence of all the examined individuals. A high correlation between TNFα and other cytokine levels was determined. The fact of high correlation for the studied cytokines (TNFα, IFNγ, IL-10) indicated to a synchronism in the immunocompetent T helper cell reaction. By measuring the levels of spontaneous and induced cytokine production levels, one may indirectly judge on degree of anti-plague cell immunity in humans.

The causes of emergence and development of pneumonic plague outbreak in 2017 on Madagascar were analyzed. Summarized were the literature data, characterizing the spatial and biocoenotic structure of natural plague focus on Madagascar, assessed were the risk factors of plague infection among rural and urban population of the island, as well as the properties of Yersinia pestis strains isolated there in the XX century. It is substantiated that the distinctive type of plague outbreaks on Madagascar is a rural one. It is demonstrated that one of the main causes of wide spread of plague in 2017 is importation of the infection from central regions of the Republic of Madagascar into big cities with further formation of multiple epidemic foci. Confirmed was the circulation of Y. pestis strains that belong to the third branch of irradiation of oriental biovar (1.ORI3). Discussed are the issues of diagnostics and treatment of plague in epidemic foci in the territory of the Republic of Madagascar. The evidence is provided to the fact that the plague outbreak in 2017 on Madagascar is the consequence of a lower surveillance level, first and foremost undervaluation of specific and non-specific prophylaxis capacities regarding this infection.

Objective of the study is to evaluate the effect of immunomodulators on the intensity of the post-apoptotic lysis of sensitized organism leukocytes in the presence of specific antigens of tularemia microbe in vitro.Materials and methods. Flow cytometry method was used to determine the relative content of apoptotic and proliferating splenocytes obtained from mice, immunized against tularemia against the background of immunomodulation.Results and conclusions. Obtained is the evidence that is consistent with modern data on the massive leukocyte apoptosis and post-apoptotic leukocyte autolysis (secondary necrosis) in case of tularemia infection. Given the important role of secondary necrosis in the systemic inflammatory response development, the use of immunomodulators suppressing macrophage apoptosis and dead leukocyte lysis, emerging in the course of interaction with Francisella tularensis antigens, may be promising in order to reduce the live tularemia vaccine reactogenicity.

Objective of the study was to determine allelic variants of HLA II class haplotypes in persons living in the Republic of Kalmykia in the territory of Pre-Caspian sandy natural focus of the plague, immunized for epidemic reasons with live plague vaccine and search for associations of HLA class II haplotypes with peculiarities of post-vaccinal immunity development.Materials and methods. 20 individuals took part in the study. HLA typing was performed applying multiplex PCR. Production of immune-regulatory cytokines and antibody titers to fraction 1 of the plague microbe was determined using enzyme immunoassay. Statistical processing of the results was carried out using standard programs.Results and conclusions. Allelic variants of haplotypes HLA-DQA1, HLA-DQB1 and HLA-DRB1 class II of the main histocompatibility complex of 20 persons residing in Lagansky and Chernozemelsky districts of the Republic of Kalmykia have been identified. Determined have been the differences in the ratio of allelic variants of HLA-DQA1 and cytokine production INF-γ, TNF-α, IL-4 and IL-10 by the areas of residence. Association of the HLA-DRB1*01 allele with a high level of spontaneous and induced IL-10 cytokine production has been revealed at various times after booster vaccination. Further study of genes that regulate the development of immunity, along with immunological methods will make it possible to personalize the use of the existing vaccine against plague, and predict the immunogenicity and effectiveness of preventive drugs under development.

Objective of the study is to specify the character and dynamics of morpho-functional changes in the organs of peripheral immune system of BALB/c mice in case of immunization with vaccine Yersinia pestis EV NIIEG strain against the background of immune modulation. Materials and methods. Applying a combination of histological, histo-chemical, immune histo-chemical methods, and morphometric analysis, defined has been the targeting of immunological processes in peripheral lymphoid organs of BALB/c mice. Results and conclusions. Revealed has been stimulating effect of polyoxidonium in concomitant use of Yersinia pestis EV NIIEG, on the processes of cell proliferation in T-zones of lymphoid organs, on activation of T- and B-lymphocyte sub-population in them by reference to the increment in the amount of specifically stained with CD25 marker cells and nuclei of lymphocytes with high argyrophil granule content, indirectly characterizing the functional state of nuclei apparatus, as well as on response intensification of neuroendocrine environment of lymphoid structures. Based on the results of complex morphological analysis, specified have been the peculiarities of morphofunctional changes in immune competent organs of biomodels, being indicative of polyoxidonium potential to increase the effectiveness of vaccination against plague.

Objective of the study is to sum-up the results of prophylactic (anti-epidemic) measures associated with elimination of epidemic focus in Kosh-Agach Region of the Republic of Altai in 2016. Materials and methods. Utilized were the data of reporting and source documentation from Altai Plague Control Station, Rospotrebnadzor Administration in the Republic of Altai, RusRAPI “Microbe”, and Irkutsk RAPI. Results and conclusions. It is pointed out that carried out in 2016 complex of organizational, anti-epidemic, and sanitary-prophylactic activities against plague is the importnant stage of rehabilitation of Gorno-Altai high-mountain natural plague focus, the final goal of which is maximally possible decrement of risks of primary human infection with plague, and in case of occurrence of such – performing of prompt response measures on localization and elimination of epidemic focus. It has been substantiated that for epidemic risk minimization in 2017 it is necessary to continue prophylactic vaccination, desinsection and deratization in the areas of predictive epizootic situation aggravation, which is contained in “Complex Action Plan of the Rospotrebnadzor Institutions for Sanitation and Rehabilitation of Gorno-Altai high-mountain natural plague focus in Kosh-Agach Region of the Altai Republic in 2017”. It is also necessary to provide for implementation of the Program on lowering risks of importation from cross-border Sailyugemsky natural focus and spread of plague in the territory of the Russian Federation, within the frames of RF Government Order No 1864-p, dated 05.09.2016.

Aim. Evaluate the effect of serotonin on morphometric and topographic characteristics of cells of vaccine strain of F. tularensis 15 NIIEG (Cap+) and its non-capsule variant F. tularensis KM-9 (Cap ). Materials and methods. Analysis was carried out by methods of densitomorphometry and atomic-force microscopy. Results. Cultivation of F. tularensis 15 NIIEG (Cap+) in FT agar has shown that in the presence of serotonin the thickness of capsule-like substance, surrounding bacterial cells, has increased on average 3 times and was 95.1±0.56 nm against 31.7±0.18 nm in control (without serotonin). During similar cultivation of F. tularensis KM-9 (Cap") such phenomenon was noted detected. Capsule-like substance of tularemia microbe was established to play an important role in biofilm formation. Conclusion. The data obtained prove an importance of phenotype of tularemia microbe and allow to assume a special role of serotonin in the processes of formation of biofilm community, in which the bacteria are protected from damaging factors of the environment.

Investigated is the effect of azoximer bromide (polioxidonium - PO) and dalargin (DA) on the immune system cell reactivity in the anti-tularemia vaccine prosess modeling in Balb/c mice. The animals were immunized subcutaneously with a dose of 104 live microbial cells of vaccine strain Francisella tularensis 15 NIIEG. PO or DA subcutaneously administered 60 min before vaccination, respectively, in doses of 4 mg and 2 mg. On 3rd and 21st days of immunogenesis the peripheral organs of the immune system and the contents of the abdominal cavity were investigated. Cell response to vaccination and investigated immunomodulators evaluated by flow cytometry (CyAn ADP) for two light scatter parameters and for changing the proliferating and apoptotic cell number. Additionally, for the characterization of changes in the functional status of lymphocytes nuclei the light microscopy was used and titers of specific antibodies were determined. Installed is the different response of intact mice lymphocytes and phagocytes to PO and DA introduction. As distinct from PO, DA stimulated the lymphocete proliferation and macrophage apoptosis by acting on their cytoplasmic granules. PO activated the migration of phagocytes with high cytoplasmic granules from mice abdominal cavity in the spleen. Only when using the PO as an immunomodulator in anti-tularemia vaccine process modeling recorded a significant increase in specific antibody titers against the background of a long-term functional splenocyte activation and macrophages reduced macrophage damage intensity in the spleen and abdomen.

Identified are the complete nucleotide sequences of two cryptic plasmids - pCKF from the Central-Caucasian high-mountain plague focus and pTP33 - from Tuva mountain plague focus in Russia. It is established that the size of the pCKF is 5.4 kbp, and its G-C pair composition accounts for 38.4 %. The plasmid contains 8 open reading frames, encoding transport and secretion functions, in particular, secretion systems of the VI type. The size of another cryptic plasmid, pTP33, is 33.8 kbp, and G-C pair composition accounts for 50.3 %. It contains 52 open reading frames, the majority of which have been classified as phage proteins. Hence, it is inferred that pTP33 is a ring genome of phage. It also has two genes of two-component protein system, toxin-antitoxin, YoeB/YefM, that affects replication apparatus of bacteria.

Studied has been the state of the nucleolar apparatus in lymphocytes of periphery lymphoid organs in laboratory animals vaccinated against plague and tularemia in order to evaluate predictive capability of the parameter in the context of pre-clinical evaluation of the vaccines under development, against particularly dangerous infections. For the detection of silver adjoining proteins, contained in the nucleolar organizer region (AgNOR), W.M.Howell and D.A.Black staining of semi-thin paraffin sections of lymphoid organs of the immunized BALB/c mice has been performed. For this purpose, ready-assembled panel, manufactured at BioVirtum, has been deployed. Under assessment of the functional state of immunogenesis organs in vaccinated against plague and tularemia animals revealed has been the increase in numbers of cells with three and more AgNOR-positive nucleoli in the nucleus of the lymphocytes of periphery organs of the biomodels’ immune system. It indirectly manifests proliferative capacity of immune system cellular component. Informativity of the quantitative evaluation of the cell proliferative activity by the reference to the status changes of AgNOR in the nuclei of immunogenesis organs’ lymphocytes makes it possible to entertain the possibility of introducing this method into the existing morphological vaccine quality assessment scheme at the stage of the pre-clinical trials.

one. The data obtained has substantiated the lack of an expressed damaging effect of the recombinant protective antigen on the cells and tissues of the macro­organism, as well as possibility of its application as one of the core components of the chemical anthrax vaccine.

Objective of this review is to analyze diachronically paradigm shift as regards problems of specific plague prophylaxis and appreciate contribution of the present-day scientific discoveries in the sphere of plague agent investigations and peculiarities of its interaction with host organism to the solution of topical issues of vaccine development that will be safe and tangibly effective against this particularly dangerous disease. Outlined is the historical background of the conceptual evolution concerning specific plague prophylaxis and events that are landmarked with eminent scientific discoveries by A.Yersin, French researcher and microbiologist. Given are the data on the current state of plague immune-prophylaxis both in Russia and around the world. Through the prism of the latest researches that assume application of advanced technological resource of medical sciences (molecular biology, biotechnology, bioinformatics, molecular immunology) put forward is the prospective of search and construction of safe and effective anti-plague next generation vaccines.

Designed is an experimental preparation of a prototype chemical tularemia vaccine (PCTV). It is composed of protective antigenic complex (PAC) of tularemia microbe and S-layer protein (Slp) of plague microbe. Determined is optimum ratio of these components in the preparation and schedule of its administration. Displayed are the results of its testing as regards physical-chemical properties, reactogenicity, specific activity and impact on the immune system of laboratory animals. It is found out that preparation of the prototype is non-toxic for white mice and Guinea pigs and has no damaging effect on their immune systems. Single-stage subcutaneous immunization with PCTV induces elaboration of high-level adaptive immunity in laboratory animals within 14–21 days: specific antibody generation and stimulation of immune system cell component. PCTV protective index for white mice in case of experimental tularemia, caused by Francisella tularensis subsp. holarctica, is 87,5 % on average; in case of infecting with F. tularensis subsp. nearctica – 50 %; and high-level immunity in both cases. High potency of the experimental preparation against tularemia caused by subsp. holarctica (protective index is 75 %) and high-grade immunity persistence is verified on the model of Guinea pigs too.

Research objective consisted in the development of a unified approach to the evaluation of severity of experimental tularemia disease based on the morphometric analysis. Experimental tularemia infection was morbidized in Guinea pigs, both female and male, using subcutaneous inoculation with virulent tularemia microbe cultures. Subsequent to the results of morphometric analysis, selected was a range of indicators characterizing intensiveness of dystrophy, necrobiotic and necrotic processes, infiltrate changes within cells and tissues of the infected animals to fullest extent, and reflecting a life-support system functioning state adequately. Thus, application of quantitative counting to distinguish the changes in experimental animals, while modeling specific infectious process, allowed for standardization and verification of severity evaluation approach and facilitated increase in quality of experimental investigations.

Carried out was immunobiological evaluation of cholera toxin B subunit preparations obtained using different experimental and production methods. It was demonstrated that B subunit preparations were non-toxic for biomodels and did not cause significant pathological alterations in their organs and tissues. They also did not alter the condition of immunocompetent cells at different stages of their cycle, and promote anti-toxic antibodies production. Thus, B subunit preparations under study can be considered as promising components of cholera vaccine.

Worked out is the algorithm of cholera vaccines quality based on application of the methods of quantitative registration of formalized parameters which characterize pathologic and adaptive processes in the intestine of the immunized laboratory animals. The following parameters were selected as formalized ones: quantity of interepithelial lymphocytes, morpho-functional condition of apudocytes and scyphiform cells. Morphometric parameters are presented as associations of indicators in the form of indices and coefficients in order to carry out interrelated assessment of separate parts of homeostasis. It was determined that in case the assessed parameters were in the limits identified in corresponding controls, adaptation condition of the organism and alterations described in histological assay were considered as benign and characterized safety or efficiency of tested cholera vaccines.

Carried out was morphometric analysis of cells' reaction of APUD system (neuroendocrinal cells) of guinea pigs immunized with live plague vaccine by aerogenic method. The results of investigations were compared with characteristics of the state of biomodel's internal organs. Elucidated was that the changes of morphofunctional state of neuroendocrinal cells in lungs and organs of immunity were of phase character suggesting the involvement of APUD system elements in the inhalant method of antigenic material intake. Assessment of APUD-system cell's reaction enables to estimate indirectly the nature of adaptive and compensatory processes in biomodel in the course of anti-plague vaccination.

Comparison of clinical and morphological analysis data and the results of morphometric assessment of changes in suckling rabbits infected intra-intestinally with cholera vibrios with different set of virulence determinants proved the presence of ctxA gene in Vibrio cholerae genome to be the necessary condition for the development of choleragen reaction. Experimental infection in this case is followed by the excess of the considered morphometric parameters in kidneys and liver in comparison with those of the intact animals. Changes in suckling rabbits infected with ctxA- strains are mainly of the adaptive and compensatory character and are determined by ZOT and ACE toxins of Vibrio cholerae to some extent. The reaction of the suckling rabbits` intestinal apudocytes depends on the genotype of V. cholerae strains used for challenge and correlates with the intensity of morphological changes in the intestine and internal organs of experimental animals.

Evaluated were alterations in thymus, regional and remote lymphatic glands, and spleen of biomodels as well as neuroendocrinal cells reaction in these organs in simulating anti-tularemia vaccinal process. Received are quantitative characteristics of neuroendocrinal environment state of biomodels' lymphoid organs on the 5 day after administration of anti-tularemia vaccine in different doses. Elucidated were certain principles in cells' reaction of APUD-system of biomodel's lymphoid organs and lungs, associated with nature of their alterations. Identified were the prospects of application of the APUD-system cells quantitative characteristics in view of improving and optimizing the evaluation methods as regards safety and efficiency of live vaccines.

Data on adaptive-compensatory process formation in detoxication and adaptation functional systems of test animals during the plague infection modeling were obtained by means of morphometric analysis. The latter included characterization of apudocytes condition in the number of organs. Changes of apudocytes activity and quantity in immunocompetent organs and pulmonary tissue of biomodels were determined. Morphometric indices selected for registration were shown to allow characterizing the severity of experimental infectious process.

The constructed avirulent biplasmid Vibrio cholerae strain KM182 producing cholera toxin B subunit and Escherichia coli colonization factor CFA/1 (providing for antitoxic and anti-colonization immunity formation, correspondently) was demonstrated to protect immunized model laboratory animals from experimental cholera caused by the virulent Vibrio cholerae O139 strain. The optimal doses for immunization and challenging were determined.