Live Plague Vaccine Research Articles

Live Plague Vaccine Development: Past, Present, and Future.

During the last 100 years, vaccine development has evolved from an empirical approach to one of the more rational vaccine designs where the careful selection of antigens and adjuvants is key to the desired efficacy for challenging pathogens and/or challenging populations. To improve immunogenicity while maintaining a favorable reactogenicity and safety profile, modern vaccine design must consider factors beyond the choice of target antigen alone. With new vaccine technologies currently emerging, it will be possible to custom-design vaccines for optimal efficacy in groups of people with different responses to vaccination. It should be noted that after a fairly long period of overwhelming dominance of papers devoted to subunit plague vaccines, materials devoted to the development of live plague vaccines have increasingly been published. In this review, we present our opinion on reasonable tactics for the development and application of live, safe, and protective human plague vaccines causing an enhanced duration of protection and breadth of action against various virulent strains in vaccination studies representing different ages, genders, and nucleotide polymorphisms of the genes responsible for immune response.

Study of allergic reaction to somatic antigen made from a live plague vaccine of the EV strain

Study of allergic reaction to somatic antigen made from a live plague vaccine of the EV strain

Possibility of Using Disposable Polymeric Containers in the Production Technology of Live Plague Vaccine

The aim of the work was to assess the possibility of using disposable polymeric containers in the production technology of the live plague vaccine.Materials and methods. We deployed the vaccine strain Yersinia pestis ЕV NIIEG for the work. A standard disposable 10 L Flexboy type polymeric container manufactured by Sartorius Stedim Biotech, equipped with Sartopore 2 filter capsules, was used for submerged cultivation of plague microbe inoculum. This method was compared to the regulated technology for obtaining seed cultures using glass bottles with a volume of 20 liters. The control of the produced seed cultures of the vaccine strain EV was performed in accordance with FS.3.3.1.0022.15. The cultivation of the seed culture in a disposable polymeric container was carried out in a liquid nutrient medium at a temperature of 26 to 28°C with continuous barbotage and mechanical agitation with a platform oscillation frequency of 80 to 90 per minute. For aeration, compressed air with a pressure of 0.3 to 0.4 kgf/cm2 was used. The volumetric flow rate of sterile air supplied for aeration ranged from 0.9 to 1.0 l/min.Results and discussion. The use of disposable polymeric containers made it possible to reduce the duration of the technological stage of obtaining a seed culture by 1.7 times and increase the yield of live microbes per unit volume of the nutrient medium by 2.8 times, as compared to the regulated production technology. Thus, the possibility and prospects of using disposable polymeric containers in the production technology of live plague vaccine at the stage of preparation of sowing cultures is evidenced.

Prospects for using flow cytometry in the quality control of live plague vaccines

Scientific relevance. The number of live bacteria is a quality parameter controlled at all stages of live plague vaccine production. Currently, live microbial cell counting uses a bacteriological method. However, flow cytometry has the potential to increase analytical accuracy and reduce testing time.Aim. This study aimed at testing the applicability of flow cytometry to assessing the quality of live plague vaccines.Materials and methods. The study quantified live microbial cells in 5 experimental batches of live plague vaccine as part of their quality control using the bacteriological method according to the State Pharmacopoeia of the Russian Federation (FS.3.3.1.0022.15). Cytofluorometry of the samples used the SynaptoGreen fluorescent dye.Results. The study quantified live microbial cells in live plague vaccine samples using the bacteriological method and flow cytometry. The results obtained by the bacteriological method ranged from 27.8±2.2 to 56.5±3.1% with an average of 39.8±5.4%. The results obtained by flow cytometry ranged from 29.2±1.2 to 59.1±2.1% with an average of 41.7±5.5%. The statistical analysis showed no significant difference between the results of vaccine quality control by both methods, as well as a high coefficient of determination.Conclusions. The results show that flow cytometry is an appropriate method for the quantification of live microbial cells as part of the quality control of plague vaccines. Being quick, easy, and highly informative, flow cytometry is preferable to traditional methods.

Increased expression of tyrosine protein phosphatase (CD45) on the surface of human blood granulocytes under the influence of the plague microbe vaccine strain <I>Yersinia pestis</I> EV NIIEG <I>ex vivo</I> and <I>in vivo</I>

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.

Evaluation of the applicability of immunochromatography to the identification of live plague vaccines and the tularaemia allergen (Tularin)

The regulatory standards require that the identification of live plague vaccines and the liquid tularaemia allergen (Tularin) should be performed by immunofluorescence. A major drawback of the recommended method is its labour intensive nature. However, immunochromatography represents an alternative method that offers a number of advantages, including rapid testing and easy result interpretation. The aim of the study was to assess the applicability of immunochromatography to the identification of live plague vaccines and the liquid tularaemia allergen (Tularin).Materials and methods. The authors performed identification tests using samples of the pharmacopoeia standard for live plague vaccines, three commercial batches of a live plague vaccine, and two batches of the liquid tularaemia allergen (Tularin). These samples were tested using immunochromatographic assay (ICA) reagent kits for rapid detection and identification of Yersinia pestis (ICA System for Y. pestis) and Francisella tularensis (ICA System for F. tularensis) manufactured by the State Scientific Center for Applied Microbiology and Biotechnology.Results. The findings show that immunochromatography is an effective, rapid, and species-specific method to confirm the presence of Y. pestis in a sample of a live plague vaccine or F. tularensis in a sample of the liquid tularaemia allergen (Tularin). To perform identification tests by immunochromatography, the authors recommend diluting live plague vaccine samples to a concentration of 109 bacterial cells/mL and using undiluted samples of the liquid tularaemia allergen (Tularin).Conclusions. The study results may support the inclusion of ICA into the regulatory standards for live plague vaccines and the liquid tularaemia allergen (Tularin) as an alternative identification method.

Direct Detection of Antibodies to <i>Yersinia pestis</i> Using Glass Microstructural Waveguides as an Express Method for Assessing Seroconversion in Individuals Vaccinated against Plague

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.

Activation of T-helper in vitro under the Influence of Yersinia pestis Antigens in People Vaccinated against the Plague

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.

Identifying correlates of protection from <i>Yersinia pestis</i> on a mouse model and assessing an opportunity for their use as markers of human vaccination efficiency

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.

Efficiency of human plague vaccination in tuvinian natural plague focus. Message 2: dynamics of immune status indicators after revaccination

Relevance. In Russia, the live plague vaccine (LPV) is used for specific prophylaxis of plague. Immunological monitoring of humans vaccinated by LPV in order to search for informative diagnostic markers, as well as to improve the tactics of epidemiological surveillance of plague enzootic territories is an urgent area of research.The aim is to assess the parameters of cellular and humoral immunity in humans revaccinated by LPV who permanently reside on territory of the Tuvinian natural plague focus.Materials and methods. The study involved 76 volunteers from the Republic of Tuva, revaccinated by LPV. Blood sampling was performed before vaccination and 1, 3, and 6 months after revaccination. The study included the determination of cytokine production (IFN-γ, IL-4, TNF-α), specific antibodies, immunoglobulins (IgM, IgG, IgA and IgE) and lymphocyte subpopulation composition (CD3, CD4, CD8, CD16, CD19, immunoregulatory index).Results. A decrease in IgG and IgM and an increase in IgA were found after vaccination with LPV and their increase after revaccination. Correlation relationships were revealed between immunoglobulins, B cells and IL-4. Revaccination leads to an increase seroconversion. The activation of humoral immunity in humans vaccinated against plague is also evidenced by dynamics of changes in the subpopulation composition: an increase in B-lymphocytes and natural killer cells, a decrease in T-helpers and immunoregulatory index, and cellular immunity stimulation is an increase in spontaneous and induced production of pro- and anti-inflammatory cytokines.Conclusion. It has been shown that LPV is capable of causing the body’s immune restructuring and activating the cellular and humoral mechanisms of immunological protection. For a complete understanding of the development and preservation of antiplague immunity, it is necessary to continue the annual immunological monitoring of the population living on the territory of the Tuvinian natural plague focus, using additional modern research methods.

Comparative Analysis of the Immunogenic Activity of the Plague Vaccine Depending on the Growing Medium

The aim was to carry out a comparative analysis of the immunogenic activity of the live plague vaccine obtained on various nutrient media.Materials and methods. The subject of the study was the blood of outbred white mice immunized with a series of live plague vaccine based on Yersinia pestis EV NIIEG strain, produced using experimental and regulated nutrient media. The immunogenic activity of vaccines was studied through flow cytometry. The intensity of antigen-reactivity of lymphocytes was determined in cell tests in vitro, analyzing the early activation marker CD25+ . For the specific activation of lymphocytes, a complex of water-soluble antigens of the plague microbe was used. To identify the interdependence between the presence of protective anti-plague immunity and the level of CD 25+ expression intensity, the ED50 of the series under study was determined by the standard method.Results and discussion. A comparative analysis of the immunogenic activity of the live plague vaccine obtained on the experimental nutrient medium with the vaccine produced on Hottinger’s agar has been performed. When animals were immunized with doses of 4·103 , 2·104 and 1·105 live microbial cells (regulated doses), the highest level of expression of CD25 marker by lymphocytes was on the day 14, with a subsequent decrease on the day 21 after vaccination. When determining immunogenicity using the conventional method, a high degree of direct correlation between the number of surviving animals and an increase in the level of lymphocytes expressing markers of early activation has been established. Comparison has revealed the general pattern: when the lowest immunizing dose (8·102 ) was administered, activation of early immunity markers was not observed. In case of immunization with higher doses on days 7, 14 and 21, a proportional increase in the number of CD25-positive lymphocytes after stimulation with a specific antigen under in vitro conditions is detected in the blood of biomodels.

Information support for monitoring post-vaccination immunity against plague

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.

Improvement of Microbial Cell Concentration Technology in the Production of Live Plague Vaccine in the Form of Orodispersible Tablets

The aim of the study was to improve the procedure for the Yersinia pestis EV strain cell concentration using the system for tangential flow microfiltration with the ASF-020 filter support unit.Materials and methods. The study used the vaccine Y. pestis EV strain derived from NIIEG cell line. Submerged cultivation of the native culture was performed using BIOR-0.25 reactor with automated control system. Microbial suspension concentrate was produced through microfiltration applying (Adaptive filtration system) AFS-009 and AFS-020 installations. The content of live microbial cells was determined by cytorefractometry. Assessment of the resistance of Y. pestis EV strain cells to technological factors was performed by photometric registration of changes in the optical density of bacterial suspension during the lytic response of cells to sodium dodecyl sulfate. Physical-chemical and immunobiological properties of the dry live plague vaccine were determined in accordance with the pharmacopoeial item.3.3.1.0021.15 of the State Pharmacopoeia of the Russian Federation, 14th edition.Results and discussion. The design features of the equipment introduced made it possible to carry out membrane filtration of microbial suspension, using BIOR-0.25 reactor as an intermediate storage unit, thereby excluding three technological stages. The total concentration of microbes in the suspension obtained by routine and improved methods was more than 150 billion microbial cells per ml. A comparative study of the effect of various hydrodynamic regimes in the working cavities of AFS-009 and AFS-020 filter units did not significantly affect the morphometric properties and resistance of microbial cultures to extreme (technological) factors. Based on the experimental data, the mass balance of the membrane filtration process has been determined. The optimized technology gave 0.13 liter yield of concentrate from 1 liter of native culture, and the process duration was reduced to 5 hours, the yield of the finished product in one production cycle was increased by 3 times. Thus, the process of concentrating Y. pestis EV strain cells during the production of the tablet form of live plague vaccine has been enhanced. A comparative study of the morphometric properties and resistance of plague microbe cultures to technological factors in the process of their concentration using optimized technology did not reveal any significant differences as compared to the routine one. Technological stage of concentrating has been reduced to 5 h term with a three-fold increase in the yield of finished product.

Effect of antiplague vaccination on phagocytic activity of human blood granulocytes

Anti-plague vaccination stimulates phagocytosis of Yersinia pestis cells by blood leukocytes of laboratory animals. However its effect on the phagocytic activity of human white blood cells towards plague microbes has not been studied. In this work, flow cytometry was used to study phagocytic activity of blood granulocytes towards Y. pestis, Escherichia coli and Staphylococcus aureus in the people vaccinated, or not vaccinated against plague. These persons inhabited the territories of natural plague foci in Russian Federation. The results of phagocytic activity were evaluated in microvolumes of whole blood. The induced IFNγ production was evaluated in blood by enzyme immunoassay technique, and the titers of specific antibodies to plague-specific F1 antigen were determined in blood serum samples. It was found, that the subjects who have never been vaccinated against plague had 3-fold lower granulocyte phagocytic activity towards Y. pestis than the persons repeatedly vaccinated with live plague vaccine, and 5-fold lower than phagocytosis of E. coli and S. aureus. The next annual revaccination caused additional stimulation of in vitro phagocytic capacity of blood leukocytes with respect to plague microbes. Individual values of phagocytic indices correlated with serum antibody titers (r = 0.65, p = 0.0004), and with levels of IFNγ production (r = 0.73, p = 0.0004). Thus, the obtained data confirmed dependence between phagocytic activity of human blood leukocytes and the pathogen type, thus allowing to demonstrate a connection between activated phagocytic response to Y. pestis cells in live plague-vaccinated individuals with other immune indexes of vaccine efficiency (specific antibodies to F1, induced IFNγ production). Further research in this direction will bring us closer to development of an informative test for assessing the intensity of antiplague immunity.

Yersinia Outer Membrane Vesicles as Potential Vaccine Candidates in Protecting against Plague.

Despite the relatively low incidence of plague, its etiological agent, Yersinia pestis, is an exceptional epidemic danger due to the high infectivity and mortality of this infectious disease. Reports on the isolation of drug-resistant Y. pestis strains indicate the advisability of using asymmetric responses, such as phage therapy and vaccine prophylaxis in the fight against this problem. The current relatively effective live plague vaccine is not approved for use in most countries because of its ability to cause heavy local and system reactions and even a generalized infectious process in people with a repressed immune status or metabolic disorders, as well as lethal infection in some species of nonhuman primates. Therefore, developing alternative vaccines is of high priority and importance. However, until now, work on the development of plague vaccines has mainly focused on screening for the potential immunogens. Several investigators have identified the protective potency of bacterial outer membrane vesicles (OMVs) as a promising basis for bacterial vaccine candidates. This review is aimed at presenting these candidates of plague vaccine and the results of their analysis in animal models.

Yersinia pestis Antigen F1 but Not LcrV Induced Humoral and Cellular Immune Responses in Humans Immunized with Live Plague Vaccine-Comparison of Immunoinformatic and Immunological Approaches.

The recent progress in immunoinformatics provided the basis for an accelerated development of target-specific peptide vaccines as an alternative to the traditional vaccine concept. However, there is still limited information on whether the in silico predicted immunoreactive epitopes correspond to those obtained from the actual experiments. Here, humoral and cellular immune responses to two major Yersinia pestis protective antigens, F1 and LcrV, were studied in human donors immunized with the live plague vaccine (LPV) based on the attenuated Y. pestis strain EV line NIIEG. The F1 antigen provided modest specific cellular (mixed T helper 1 (Th1)/Th2 type) and humoral immune responses in vaccinees irrespective of the amount of annual vaccinations and duration of the post-vaccination period. The probing of the F1 overlapping peptide library with the F1-positive sera revealed the presence of seven linear B cell epitopes, which were all also predicted by in silico assay. The immunoinformatics study evaluated their antigenicity, toxicity, and allergenic properties. The epitope TSQDGNNH was mostly recognized by the sera from recently vaccinated donors rather than antibodies from those immunized decades ago, suggesting the usefulness of this peptide for differentiation between recent and long-term vaccinations. The in silico analysis predicted nine linear LcrV-specific B-cell epitopes; however, weak antibody and cellular immune responses prevented their experimental evaluation, indicating that LcrV is a poor marker of successful vaccination. No specific Th17 immune response to either F1 or LcrV was detected, and there were no detectable serum levels of F1-specific immunoglobulin A (IgA) in vaccinees. Overall, the general approach validated in the LPV model could be valuable for the rational design of vaccines against other neglected and novel emerging infections with high pandemic potency.

Yersinia pestis Plasminogen Activator.

The Gram-negative bacterium Yersinia pestis causes plague, a fatal flea-borne anthropozoonosis, which can progress to aerosol-transmitted pneumonia. Y. pestis overcomes the innate immunity of its host thanks to many pathogenicity factors, including plasminogen activator, Pla. This factor is a broad-spectrum outer membrane protease also acting as adhesin and invasin. Y. pestis uses Pla adhesion and proteolytic capacity to manipulate the fibrinolytic cascade and immune system to produce bacteremia necessary for pathogen transmission via fleabite or aerosols. Because of microevolution, Y. pestis invasiveness has increased significantly after a single amino-acid substitution (I259T) in Pla of one of the oldest Y. pestis phylogenetic groups. This mutation caused a better ability to activate plasminogen. In paradox with its fibrinolytic activity, Pla cleaves and inactivates the tissue factor pathway inhibitor (TFPI), a key inhibitor of the coagulation cascade. This function in the plague remains enigmatic. Pla (or pla) had been used as a specific marker of Y. pestis, but its solitary detection is no longer valid as this gene is present in other species of Enterobacteriaceae. Though recovering hosts generate anti-Pla antibodies, Pla is not a good subunit vaccine. However, its deletion increases the safety of attenuated Y. pestis strains, providing a means to generate a safe live plague vaccine.

The Analysis of Factors Influencing Immunologic Reactivity in People Vaccinated with a Live Plague Vaccine

Introduction: An increase in epizootic activity has been registered in a number of plague foci in the Russian Federation over the past few years. As part of securing sanitary and epidemiologic wellbeing of the population living in the natural foci of the disease, a mass immunization with a live plague vaccine based of the Yersinia pestis EV line NIIEG vaccine strain was carried out. Objectives: The purpose of the study was to assess the influence of a complex of factors including age, gender, health status, the number of previous vaccinations against plague, blood groups, and HLA gene polymorphism on the state of the cellular and humoral immune response in people vaccinated with the live plague vaccine. Materials and methods: The analysis of venous blood of 347 volunteers included determination of the concentration of specific antibodies to the capsular antigen (F1) of plague microbe, spontaneous and induced production of marker cytokines (IFN-γ, TNF-α, and IL-4) by ELISA, and genes of the main histocompatibility complex (HLA) class II by real-time PCR. We also analyzed medical documentation (Form 025/u) and the results of a questionnaire-based survey of the vaccinated people. Results and discussion: We established the influence of various factors, including genetic ones, on marker indicators of the humoral and cellular immune response in persons vaccinated with the live plague vaccine. We also characterized the relationship between the level of specific antibodies to plague microbe F1 production and some cytokines and the age and the number of previous vaccinations in our volunteers. The most common gene variants of the main histocompatibility complex of class II (HLA-DQA1, HLA-DQB1 and HLA-DRB1) in the cohort were identified and possible relationships between the production of IFN-γ, TNF-α, IL-4 and allelic polymorphism of HLA class II genes were determined. Conclusions: Immunologic reactivity in people vaccinated with the live plague vaccine is mainly determined by age, the number of previous vaccinations against this infection, and individual characteristics of HLA gene polymorphism.

Monitoring of Stability of the Live Plague Vaccine Produced Using Condensed Corn Steep Extract Hydrolysate-Based Nutrient Medium

Objective of the study was to analyze the stability of the preparation of a live plague vaccine made using an experimental nutrient medium over its shelf life. Materials and methods . A series of live plague vaccine based on Yersinia pestis EV NIIEG strain prodused using experimental nutrient medium were utilized in this work. Most significant qualitative parameters of the preparation were studied: microbe cell content, viability, thermal stability, mass loss during the process of drying, and immunogenicity. Results and discussion . The stability of the vaccine produced using the nutrient medium based on hydrolysate of condensed corn steep extract was monitored over a specified shelf life (three years). A comparative analysis of the viability of all experimental vaccine series was carried out at certain time intervals - short-term storage (up to 3 months) and before the expiration date. During storage, there was an insignificant decrease in the percentage of living microbial cells, while in no series the viability decrease reached below the regulated levels of 25 %. Such a decrease is natural for a “live preparation” and is not critical within the obtained limits. The rest of the studied indicators practically did not change. Thus, the analysis of the data obtained indicates that in all the periods of observation the drug retained the stability of the main regulated indicators. The studies confirm the feasibility of using this referred nutrient medium based on hydrolyzed corn steep extract in industrial production of the plague live vaccine preparation.

Revelation of Immune Memory at the First Stage of Antigen-Specific Cell Response after Second Introduction of the Live Plague Vaccine

Background. The study of immune memory is necessary to evaluate the effectiveness of immunization against infection, including plague and to make a choice of vaccination scheme.Goals. The goal is to study the possible role of immune memory in the early stage of the antigen-specific response – the formation of cells with receptors for capsular (F1) and lipopolysaccharide (LPS) antigens of plague live EV vaccine.Methodology. Volunteers vaccinated with live plague vaccine EV for the first time (6 persons – group 1) and again (6 persons – group 2) were examined. In the mononuclear fraction of the blood of volunteers the cells binding antigens F1 and LPS Y. pestis (CBA) were determined.Results. In the volunteers group 2, the content of CBA at 2 days after vaccination was higher than in group 1. Between the 5th day and the end of the CBA detection, their content in group 2 decreased, and in group 1, it increased, but remained significantly less than in group 2 two days after immunization.Сonclusions. It is shown that the previous vaccination accelerates the first stage of the antigen-specific human response to second vaccination against plague. This reflects the role of immune memory in the formation of this stage of the immune response at vaccination against plague.