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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immunological monitoring of persons vaccinated with the live plague vaccine according to epidemic indications have been archived.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.

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.

As the reality of pandemic threats challenges humanity, exemplified during the ongoing SARS-CoV-2 infections, the development of vaccines targeting these etiological agents of disease has become increasingly critical. Of paramount concern are novel and reemerging pathogens that could trigger such events, including the plague bacterium Yersinia pestis. Y. pestis is responsible for more human deaths than any other known pathogen and exists globally in endemic regions of the world, including the four corners region and Northern California in the USA. Recent cases have been scattered throughout the world, including China and the USA, with serious outbreaks in Madagascar during 2008, 2013–2014, and, most recently, 2017–2018. This review will focus on recent advances in plague vaccine development, a seemingly necessary endeavor, as there is no Food and Drug Administration–licensed vaccine available for human distribution in western nations, and that antibiotic-resistant strains are recovered clinically or intentionally developed. Progress and recent development involving subunit, live-attenuated, and nucleic acid–based plague vaccine candidates will be discussed in this review. Key points • Plague vaccine development remains elusive yet critical. • DNA, animal, and live-attenuated vaccine candidates gain traction.

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.

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.

The variable response of wild mice to Yersinia pestis infection, the causative agent of plague, has generated much speculation concerning their role in the ecology of this potentially lethal disease. Researchers have questioned the means by which Y. pestis is maintained in nature and also sought methods for managing the disease. Here we assessed the efficacy of a new tool, the sylvatic plague vaccine (SPV), in wild-caught northern grasshopper mice (Onychomys leucogaster) and commercially acquired Sonoran deer mice (Peromyscus maniculatus sonoriensis). More than 40% of the animals survived a subcutaneous Y. pestis challenge of 175,000 colony forming units (over 30,000 times the white mouse 50% lethal dose) in both vaccine-treated and control groups. Our results indicate that SPV distribution is unlikely to protect adult mice from plague infection in field settings and corroborate the heterogeneous response to Y. pestis infection in mice reported by others.

Significance

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.

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.

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.

Yersinia pestis, the cause of plague, could be weaponized. Unfortunately, development of new vaccines is limited by lack of correlates of protection. We used pre- and post-vaccination sera and peripheral blood mononuclear cells from a flagellin adjuvanted F1/V vaccine trial to evaluate for protective markers. Here, we report for the first time in humans that inverse caspase-3 levels, which are measures of protective antibody, significantly increased by 29% and 75% on days 14 and 28 post-second vaccination, respectively. In addition, there were significant increases in T-cell responses on day 28 post-second vaccination. The strongest positive and negative correlations between protective antibody levels and gene expression signatures were identified for IFNG and ENSG00000225107 genes, respectively. Flagellin/F1/V subunit vaccine induced macrophage-protective antibody and significant CD4+ T-cell responses. Several genes associated with these responses were identified that could serve as potential correlates of protection.

Mice immunized with a combination of an adenovirus vector (Ad5-YFV) and live-attenuated (LMA)-based vaccines were evaluated for protective efficacy against pneumonic plague. While the Ad5-YFV vaccine harbors a fusion cassette of three genes encoding YscF, F1, and LcrV, LMA represents a mutant of parental Yersinia pestis CO92 deleted for genes encoding Lpp, MsbB, and Ail. Ad5-YFV and LMA were either administered simultaneously (1-dose regimen) or 21 days apart in various orders and route of administration combinations (2-dose regimen). The 2-dose regimen induced robust immune responses to provide full protection to animals against parental CO92 and its isogenic F1 deletion mutant (CAF−) challenges during both short- and long-term studies. Mice intranasally (i.n.) immunized with Ad5-YFV first followed by LMA (i.n. or intramuscularly [i.m.]) had higher T- and B-cell proliferative responses and LcrV antibody titers than those in mice vaccinated with LMA (i.n. or i.m.) first ahead of Ad5-YFV (i.n.) during the long-term study. Specifically, the needle- and adjuvant-free vaccine combination (i.n.) is ideal for use in plague regions of endemicity. Conversely, with a 1-dose regimen, mice vaccinated with Ad5-YFV i.n. and LMA by the i.m. route provided complete protection to animals against CO92 and its CAF− mutant challenges and elicited Th1/Th2, as well as Th17 responses, making it suitable for emergency vaccination during a plague outbreak or bioterrorist attack. This is a first study in which a viral vector-based and live-attenuated vaccines were effectively used in combination, representing adjuvant- and/or needle-free immunization, with each vaccine triggering a distinct cellular immune response. IMPORTANCE Yersinia pestis, the causative agent of plague, is a Tier-1 select agent and a reemerging human pathogen. A 2017 outbreak in Madagascar with >75% of cases being pneumonic and 8.6% causalities emphasized the importance of the disease. The World Health Organization has indicated an urgent need to develop new-generation subunit and live-attenuated plague vaccines. We have developed a subunit vaccine, including three components (YscF, F1, and LcrV) using an adenovirus platform (Ad5-YFV). In addition, we have deleted virulence genes of Y. pestis (e.g., lpp, msbB, and ail) to develop a live-attenuated vaccine (LMA). Both of these vaccines generated robust humoral and cellular immunity and were highly efficacious in several animal models. We hypothesized the use of a heterologous prime-boost strategy or administrating both vaccines simultaneously could provide an adjuvant- and/or a needle-free vaccine(s) that has attributes of both vaccines for use in regions of endemicity and during an emergency situation.

Pneumonic plague, caused by

Relatively recent advances in plague vaccinology have produced the recombinant fusion protein F1-V plague vaccine. This vaccine has been shown to readily protect mice from both bubonic and pneumonic plague. The protection afforded by this vaccine is solely based upon the immune response elicited by the F1 or V epitopes expressed on the F1-V fusion protein. Accordingly, questions remain surrounding its efficacy against infection with non-encapsulated (F1-negative) strains. In an attempt to further optimize the F1-V elicited immune response and address efficacy concerns, we examined the inclusion of multiple toll-like receptor agonists into vaccine regimens. We examined the resulting immune responses and also any protection afforded to mice that were exposed to aerosolizedYersinia pestis. Our data demonstrate that it is possible to further augment the F1-V vaccine strategy in order to optimize and augment vaccine efficacy.

Relatively recent advances in plague vaccinology have produced the recombinant fusion protein F1-V plague vaccine. This vaccine has been shown to readily protect mice from both bubonic and pneumonic plague. The protection afforded by this vaccine is solely based upon the immune response elicited by the F1 or V epitopes expressed on the F1-V fusion protein. Accordingly, questions remain surrounding its efficacy against infection with non-encapsulated (F1-negative) strains. In an attempt to further optimize the F1-V elicited immune response and address efficacy concerns, we examined the inclusion of multiple toll-like receptor agonists into vaccine regimens. We examined the resulting immune responses and also any protection afforded to mice that were exposed to aerosolized Yersinia pestis. Our data demonstrate that it is possible to further augment the F1-V vaccine strategy in order to optimize and augment vaccine efficacy.

We studied immunotropic properties of synthetic selenium-organic preparation 2,6-dipyridinium-9-selenabicyclo[3.3.1]nonyl dibromide (974zh). The experimental preparation reduced the cAMP/cGMP ratio, which indicated an increase in proliferative activity of cells of immunocompetent organs (thymus and spleen) in experimental animals. It was shown that 974zh intensified the immune response to Yersinia pestis EV thereby increasing the resistance to the plague agent.

Yersinia pestis is the causative agent of plague and is a re-emerging pathogen that also has the potential as a biological weapon, necessitating the development of a preventive vaccine. Despite intense efforts for the last several decades, there is currently not a vaccine approved by the FDA. The rF1-V vaccine adjuvanted with Alhydrogel is a lead candidate subunit vaccine for plague and generates a strong Th2-mediate humoral response with a modest Th1 cellular response. As immune protection against Y. pestis requires both humoral and Th1 cellular responses, modifying the rF1-V subunit vaccine formulation to include a robust inducer of Th1 responses may improve efficacy. Thus, we reformulated the subunit vaccine to include SA-4-1BBL, an agonist of the CD137 costimulatory pathway and a potent inducer of Th1 response, and assessed its protective efficacy against pneumonic plague. We herein show for the first time a sex bias in the prophylactic efficacy of the Alhydrogel adjuvanted rF1-V vaccine, with female mice showing better protection against pneumonic plague than male. The sex bias for protection was irrespective of the generation of comparable levels of rF1-V-specific antibody titers and Th1 cellular responses in both sexes. The subunit vaccine reformulated with SA-4-1BBL generated robust Th1 cellular and humoral responses. A prime-boost vaccination scheme involving prime with rF1-V + Alhydrogel and boost with the rF1-V + SA-4-1BBL provided protection in male mice against pneumonic plague. In marked contrast, prime and boost with rF1-V reformulated with both adjuvants resulted in the loss of protection against pneumonic plague, despite generating high levels of humoral and Th1 cellular responses. While unexpected, these findings demonstrate the complexity of immune mechanisms required for protection. Elucidating mechanisms responsible for these differences in protection will help to guide the development of better prophylactic subunit vaccines effective against pneumonic plague.

Electrophoretic, homogeneous, highly purified preparations of various isoforms of the immunodominant antigen V (LcrV protein) of Yersinia pestis have been obtained via chromatographic purification. A significant similarity in the chromatographic profiles of the LcrV antigen isoforms of different strains with tryptophan (W113) and glutamic acid (E113) residues at position 113, which differed from the isoform profile containing glycine residue LcrV (G113), has been recorded. The latter is characterized by the highest antigenic and protective activity as compared with other protein isoforms. This isoform almost completely protected the immunized group of animals and had the greatest ability to induce the production of antibodies, the titers of which reached 1 : 250 000.

To date, the technology of live plague vaccine production uses a combined method of concentrating microbial cells which consists of three operations with a total duration of 18 hours. The procedure of obtaining concentrate, which is used in the current vaccine production technology, has a number of disadvantages, namely: multiple operations, high energy consumption, long duration, and, as a consequence, low yield of concentrated suspension (0.04 l from 1 l of native culture). The aim of the study was to optimise the procedure of Yersinia pestis ЕV microbial cell concentration using the system for tangential flow microfiltration with the ASF-020 filter support unit. Materials and methods: the vaccine strain used in the study was Yersinia pestis ЕV derived from NIIEG cell line (the strain of the Research Institute of Epidemiology and Hygiene). Submerged cultivation of the native culture was performed using the BIOR-0.25 reactor. The content of live microbial cells was determined by cytorefractometry. Oxidative metabolism was assessed using the chronoamperometric method. Physico-chemical and immunobiological properties of the dry live plague vaccine were assessed according to the monograph FS.3.3.1.0022.15 of the State Pharmacopoeia of the Russian Federation, 14 edition. Results: the equipment’s design features made it possible to carry out membrane filtration of the microbial suspension using the BIOR-0.25 reactor as an intermediate storage unit, thereby excluding three technological stages. The total concentration of microbes in the suspension obtained by the routine and the optimised methods was not less than 120 billion microbial cells/ml. A comparative study of the effect of various hydrodynamic regimes in the working cavities of ASF-009 and ASF020 filter units did not significantly affect the morphometric and physiological properties of microbial cultures. Experimental data helped to determine the process mass balance of membrane filtration. The optimised technology gave 0.17 l yield of the concentrate from 1 l of native culture, and the process duration was reduced to 4 hours. Conclusions: the process of concentrating Y. pestis EV microbial cells during production of plague vaccines was optimised. A comparative study of morphometric and physiological properties of plague microbe cultures that was carried out during their concentration using the optimised technology did not reveal any significant differences as compared to the routine one.

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-1 β , IL-2, IL-4, IL-6, IL-8, IL-10, IL-18, IFN γ , TNF α (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 (TNF α , IL-1 β , 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 IFN γ , TNF α 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.

Three great plague pandemics, resulting in nearly 200 million deaths in human history and usage as a biowarfare agent, have made Yersinia pestis as one of the most virulent human pathogens. In late 2017, a large plague outbreak raged in Madagascar attracted extensive attention and caused regional panics. The evolution of local outbreaks into a pandemic is a concern of the Centers for Disease Control and Prevention (CDC) in plague endemic regions. Until now, no licensed plague vaccine is available. Prophylactic vaccination counteracting this disease is certainly a primary choice for its long-term prevention. In this review, we summarize the latest advances in research and development of plague vaccines.

The optimization of the drying schedule has been carried out to improve the quality indicators of the live plague vaccine. Based on the data obtained on the eutectic point of the vaccine suspension, the freezing temperature and freezing time were set to -50 °С and 6-7 h, respectively. A pressure of 40 mTorr over the surface of the drying suspension and 20 mTorr during the desorption were shown to be the best conditions for sublimation. The drying tests with different options for the shelf heating rate, vacuum depth and duration of intermediate temperature indicators were carried out to develop the improved freeze-drying mode providing the selection of the most adapted bacteria. A vaccine lyophilized under the developed conditions has low residual moisture (up to 2%) and high viability index that persists over the whole shelf life. lyophilization, sublimation, eutectic, live plague vaccine, residual moisture, viability

International audience; Immunization with the live-attenuated Yersinia pseudotuberculosis VTnF1 strain producing a Yersinia pestis F1 pseudocapsule efficiently protects mice against bubonic and pneumonic plague. In clinical trials, demonstration of a plague vaccine's efficacy in humans will not be feasible, and correlates of protection will be needed to bridge the immune response of protected animals to that of vaccinated humans. Using serum transfer and vaccination of antibody-deficient µMT mice, we established that both humoral and cellular responses elicited by VTnF1 independently conferred protection against bubonic plague. Thus, correlates were searched for in both responses, using blood only. Mice were vaccinated with increasing doses of VTnF1 to provide a range of immune responses and survival outcomes. The cellular response was evaluated using an in vitro IFNγ release assay, and IFNγ levels were significantly associated with protection, although some survivors were negative for IFNγ, so that IFNγ release is not a fully satisfactory correlate. Abundant serum IgG against the F1 capsule, Yop injectable toxins, and also non-F1 Y.pestis antigens were found, but none against the LcrV antigen. All readouts correlated to survival and to each other, confirming that vaccination triggered multiple protective mechanisms developing in parallel. Anti-F1 IgG was the most stringent correlate of protection, in both inbred BALB/c mice and outbred OF1 mice. This indicates that antibodies (Ab) to F1 play a dominant role for protection even in the presence of Ab to many other targets. Easy to measure, the anti-F1 IgG titer will be useful to evaluate the immune response in other animal species and in clinical trials.

Background . Plague is a dangerous natural-focal bacterial disease that can cause emergencies of interstate significance. In 2014–2016, three bubonic plague cases were registered among local residents in the Republic Altai. This circumstance was the reason for implementation of complex preventive measures. Comprehensive studies of the immune status of humans vaccinated (revaccinated) with live plague vaccine are presented in this paper. These people constantly reside and work at the territory of Gorno-Altai high-mountain natural plague focus. Methodology . The study of the clinical material (blood) from 60 volunteers included the determination of the functional activity of phagocytic cells, the production of IFN- γ , IL-4 and TNF- α cytokines, the titre of specific IgG anti-bodies to the capsular antigen of the plague microbe and the concentrations of the main classes of immunoglobulins in blood serum by enzyme immunoassay, as well as immunophenotyping of blood lymphocytes through flow cytometry and the de-termination of the alleles of HLA class II genes by a polymerase chain reaction. Results . The materials of these studies made it possible to determine a number of important parameters indicative of the cellular and humoral immunity activation in humans immunized against plague. Frequently occurring alleles of the HLA-DRB1, HLA-DQB1 and HLA-DQA1 genes were defined. Possible associations of these alleles with the levels of TNF- α and IL-4 production, as well as with the relative content of T-helpers and CD3-CD16+ cells were revealed. Conclusions . The obtained results indicate the immune reconstruction of the humans immunized against the plague. The immunological efficiency and safety of live plague vaccine were confirmed during the exacerbation of the epidemiological situation in active natural plague focus. Nevertheless, further immunological monitoring is necessary to fully characterize the immunological reactivity of the humans vaccinated (revaccinated) with live plague vaccine, and to improve the strategy for specific plague prevention in natural foci.

In accordance with the State Pharmacopoeia (SPh) requirements for live plague vaccine, a reference standard has to be used when testing the specific activity and thermal stability of plague vaccine commercial batches in order to assess the consistency and acceptability of the test results. Since there is no international reference standard for plague vaccine, the certification of a new batch of the industry reference standard (IRS) of live plague vaccine in terms of the above-mentioned quality parameters is an urgent challenge. Therefore, a certification programme for the industry reference standard was developed that establishes the design and scope of testing required to obtain statistically significant results. A candidate IRS was represented by a commercial batch of the product meeting the specification requirements for live plague vaccine. The certification parameters were: «Specific activity: microbial cell concentration», «Specific activity: live microbial cell percentage» and «Thermal stability». The article presents the results of the certification of a new batch of the live plague vaccine IRS, detailed evaluation of the candidate IRS in terms of: «Average weight and uniformity of weight», «Loss on drying», and statistical interpretation of the test results. It also summarises the results of the product testing in terms of «Specific activity: immunogenicity». The results of application of the previous batch of the live plague vaccine IRS (OSO 42-28-392-2013) and the results of monitoring the stability of its certification parameters demonstrated that the IRS shelf life could be extended by 6 months relative to the established period (from 2 to 2.5 years). All the certified and additional characteristics are reflected in the official documents for the scientific/technological product — live plague vaccine IRS, OSO 42-28-392-2017: passport, labelling and patient information leaflet.

Objective of the study was to analyze the basic quality parameters of the produced live plague vaccine and stability of the preparation during the storage process. Materials and methods . Specifications for batch series of the live plague vaccine on the basis of Yersinia pestis EV NIIEG, manufactured between 2012 and 2017, in compliance with the regulations of the normative documentation were assessed. The parameters under investigation were: viability, thermal resistance, absence of extraneous substances and fungi, weight loss in the process of drying, immunogenicity. Results and conclusions . Comparative analysis of the vaccine viability (quantity of live microbe cells) in the batches manufactured over the past 6 years was carried out. All the studied vaccine series have the appropriate indicators stated in the Specification to the Pharmacopeia Monograph and master formula which testifies to the stability of manufacturing line and basic live plague vaccine quality parameters throughout the whole term of storage. It is outlined that the production of the vaccine, corresponding to the requirements of the normative regulations, depends upon the strict adherence to the rules by the personnel, usage of high-grade stock and materials, validation of equipment and technological processes of manufacturing.

Melioidosis is a particularly dangerous infection caused by Burkholderia pseudomallei , against which a vaccine has not yet been created. In this regard, the development of effective treatment regimens and emergency prevention of melioidosis is very relevant. To improve the effectiveness of emergency prophylaxis of melioidosis, synthetic peptides (bestim, imunofan) and thiopoietin preparation glutoxim were used when combined with the antibiotic doxycycline. In addition, in experiments on white mice, the ability of heterologous vaccines (plague and tularemia), used in the emergency prevention mode, to increase the resistance of animals to melioidosis infection was assessed. It was shown that the most effective was imunofan, which, when combined with doxycycline, increased by 20% the survival rate of 5LD50 Burkholderia pseudomallei infection and significantly increased the average life span of mice infected with 5-12 LD50 (p < 0.05). The efficiency of use for stimulation of non-specific resistance to melioidosis of a heterologous plague vaccine EV, once administered 1 day prior to infection, protected 90% of mice from 6 LD50 Burkholderia pseudomallei and 60% - with an increase in the infectious dose of the pathogen of melioidosis up to 15 LD50. The same level of protection from melioidosis was provided by a 3 day course of antibiotic therapy with doxycycline. It was concluded that the tularemia vaccine is not suitable for immunostimulation in melioidosis due to its high residual virulence and reactogenicity.

Plague is a non-native disease in North America that reduces survival of many mammals. Previous studies have focused on epizootic plague which causes acute mortality events and dramatic declines in local abundance. We know much less about enzootic plague which causes less punctuated reductions in survival and abundance of infected populations. As a result, enzootic plague is much more difficult to detect because changes in population attributes are more subtle and Yersinia pestis prevalence is likely lower relative to epizootic plague outbreaks. The northern Idaho ground squirrel (Urocitellus brunneus) is a threatened species which coexists with Columbian ground squirrels (Urocitellus columbianus) and yellow-pine chipmunks (Neotamias amoenus) throughout their restricted distribution in central Idaho. Columbian ground squirrels and yellow-pine chipmunks are more abundant and widespread than northern Idaho ground squirrels and both are known hosts for plague. Hence, enzootic plague may be one cause of rarity for northern Idaho ground squirrels but its effect on this threatened species has not been evaluated. We conducted three controlled and randomized field experiments to examine the effects of plague in northern Idaho ground squirrels and the two coexisting species: 1) a plague vaccine experiment, 2) a paired flea-reduction experiment, and 3) a non-paired flea-reduction experiment. For Experiment 1, we hypothesized that if enzootic plague is present, vaccinated animals would have higher survival. Furthermore, Experiments 2 and 3 tested the prediction that untreated, control animals should have lower survival than those in areas where fleas are experimentally removed or reduced because fleas are the main vector for plague. In the plague vaccine experiment, vaccinated chipmunks had 4.65% higher apparent survival compared to chipmunks that received a placebo for intervals when the vaccine is believed to be effective. Apparent annual survival increased for all three species on experimental flea-reduction plots compared to non-treated plots for the paired experiment but results were mixed for the non-paired experiment. Taken together, our results suggest that enzootic plague is present and negatively impacting survival of northern Idaho ground squirrels and two coexisting species.

We report the whole-genome sequence of Yersinia pestis subsp. pestis bv. Antiqua strain 231 belonging to the 0.ANT3 phylogroup, the reference strain for testing plague vaccine protection in Russia. Genome sequencing was completed using the Oxford Nanopore MinION and Illumina platforms.

A plague vaccine with a fusion cassette of YscF, F1, and LcrV encoding genes in an adenovirus-5 vector (rAd5-YFV) is evaluated for efficacy and immune responses in mice. Two doses of the vaccine provides 100% protection when administered intranasally against challenge with Yersinia pestis CO92 or its isogenic F1 mutant in short- or long- term immunization in pneumonic/bubonic plague models. The corresponding protection rates drop in rAd5-LcrV monovalent vaccinated mice in plague models. The rAd5-YFV vaccine induces superior humoral, mucosal and cell-mediated immunity, with clearance of the pathogen. Immunization of mice with rAd5-YFV followed by CO92 infection dampens proinflammatory cytokines and neutrophil chemoattractant production, while increasing Th1- and Th2-cytokine responses as well as macrophage/monocyte chemo-attractants when compared to the challenge control animals. This is a first study showing complete protection of mice from pneumonic/bubonic plague with a viral vector-based vaccine without the use of needles and the adjuvant.

Bubonic plague results when Yersinia pestis is deposited in the skin via the bite of an infected flea. Bacteria then traffic to the draining lymph node (dLN) where they replicate to large numbers. Without treatment, this infection can result in highly fatal septicemia. Several plague vaccine candidates are currently at various stages of development, but no licensed vaccine is available in the United States. Though polyclonal and monoclonal antibodies (Ab) can provide complete protection against bubonic plague in animal models, the mechanisms responsible for this antibody-mediated immunity (AMI) to Y. pestis remain poorly understood. Here, we examine the effects of Ab opsonization on Y. pestis interactions with phagocytes in vitro and in vivo. Opsonization of Y. pestis with polyclonal antiserum modestly increased phagocytosis/killing by an oxidative burst of murine neutrophils in vitro. Intravital microscopy (IVM) showed increased association of Ab-opsonized Y. pestis with neutrophils in the dermis in a mouse model of bubonic plague. IVM of popliteal LNs after intradermal (i.d.) injection of bacteria in the footpad revealed increased Y. pestis-neutrophil interactions and increased neutrophil crawling and extravasation in response to Ab-opsonized bacteria. Thus, despite only having a modest effect in in vitro assays, opsonizing Ab had a dramatic effect in vivo on Y. pestis-neutrophil interactions in the dermis and dLN very early after infection. These data shed new light on the importance of neutrophils in AMI to Y. pestis and may provide a new correlate of protection for evaluation of plague vaccine candidates.