Principles of Quantitative Morphometric Assessment of Cholera Vaccines Safety

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

Estimation of the Severity of Experimental Tularemia against a Number of Morphometric Characteristics

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

Morphofunctional characteristics of blood cells in reconvalescents after suffering COVID-19

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

Nucleolar Apparatus of Lymphocytes as an Indicator of Lymphoid Organs’ Functional Activity in the Context of Pre-Clinical Vaccine Evaluation

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

Development of Amplification Test-Systems for Tularemia Agent Detection

Developed is the method for tularemia microbe DNA detection using PCR with electrophoretic and hybridization-fluorescent registration of results. iglBC genes have been chosen as DNA-matrixes, being species-specific ones for tularemia agent. Based on the results obtained constructed have been preparations for tularemia microbe DNA detection in biological material and environmental samples applying PCR with electrophoretic registration of results and real-time PCR: “Gene Francisella tularensis - REP” and “Gene Francisella tularensis RHF”, respectively. Identified are the package contents to be included into the test-systems. Sensitivity and specificity of the designed panels are validated through investigations of tularemia agent bacterial emulsions and suspensions from small mammals’ organs, from ticks, fleas and mosquitoes, as well as through studies of soil and surface water samples, sputum and human blood probes, experimentally contaminated with tularemia agent. Test-systems demonstrate high sensitivity (1·103 microbe cells/ml) and specificity (100 %), irrespective of the type of test material

Increasing the immunogenic and protective activity of the vaccine strain Yersinia pestis EV line NIIEG using synthetic immunomodulators

Introduction. One of the main tasks of specific plague prevention remains the development of vaccines and their application schemes aimed at improving the effectiveness of vaccination through the use of adjuvants and immunomodulators. The purpose of the study were comparative evaluation of the effect of drugs from the group of synthetic immunomodulators on the immunogenic and protective properties of the Yersinia pestis EV line NIIEG vaccine strain in model animal experiments. Materials and methods. White mice and guinea pigs were immunized with the vaccine strain Y. pestis EV line NIIEG. The animals of the experimental groups were injected with oligopeptides O1 (Threonyl-glutamyl-lysyl-lysyl-arginyl-arginyl-glutamyl-threonyl-valyl-glutamyl-arginyl-glutamyl-lysyl-glutamate), O2 (Glutamyl-cysteinyl-glycine disodium) and O3 (Arginyl-alpha-aspartyl-lysyl-valyl-tyrosyl-arginine) one hour before vaccination or three times before infection. On days 3, 14 and 21, antibody and cytokine products were studied. Animals were infected with the test strain Y. pestis 231(708) at a dose of 400 LD50. Results. It was found that a single administration of immunomodulators 1 hour before vaccination did not change the susceptibility of animals to the plague microbe: ImD50 = 5860 (O1); 5860 (O2); 6454 (O3) and 6876 (control) CFU for white mice and 446 (O1), 551 (O2), 446 (O3) and 578 (control) CFU for guinea pigs. Three-time administration of drugs to vaccinated animals led to a decrease in the ImD50 Y. pestis EV line NIIEG indicators in the group with O1 by 2.2 times (mice) and 1.8 times (guinea pigs), with O2 and O3 by 1.2 times, regardless of the biomodel compared with the control. The stimulating effect of O1 and O3 on the production of antibodies to F1 of the plague microbe and cytokines interferon-gamma, interleukin-10 has been established. Conclusion. The stimulating potential of synthetic immunomodulators on the immune system of biomodels immunized by Y. pestis EV line NIIEG has been demonstrated, which determines the prospects of research to improve the schemes of prophylactic vaccination against plague

EFFECT OF IMMUNOMODULATORS AND ANTI-TULAREMIA VACCINATION ON APOPTOSIS AND LYSIS OF SPLENOCYTES INTERACTING WITH TULYARIN IN VITRO

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

Historical and Modern Views on the Problem of Specific Plague Prophylaxis

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

Setting-up of the Database on the Nucleotide Sequences of the Genomes of the Strains of Bacterial and Viral Infections Agents of the I–II Pathogenicity Groups

Set-up is database containing characteristics of variable tandem repeats, oligo-nucleotide primers and probes of the genomes of the strains bacterial and viral infections agents of the I-II pathogenicity groups. It allows for accumulation of information on genome regions. Using the incorporated VBA one can rapidly analyze the data and carry out substantiated calculation of gene-diagnostic preparations based on various PCR frameworks

Changes in Biomodels in Response to Recombinant Protective Anthrax Antigen Inoculation

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