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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.

The aim of the study was to investigate the effect of experimental bases on the growth qualities of liquid nutrient media at the stage of obtaining the biomass of plague microbe vaccine strain using submerged cultivation.Materials and methods. Yersinia pestis EV NIIEG vaccine strain was used in the work. The cultivation was carried out in a 5 L bioreactor with automatic stirrer control. We used 28 variants of nutrient media obtained through combining five types of bases and six growth stimulants. Nutrient media without the addition of growth-stimulating additives were used as controls. The following parameters were assessed in the yield biomass: the total number of microbial cells, pH, the percentage of viable microbial cells.Results and discussion. On experimental nutrient media, biomasses of the plague microbe vaccine strain have been produced using submerged cultivation. After evaluating bacterial suspensions by the main indicators, the quality of the obtained suspensions has been compared depending on the nutrient medium used. The most promising bases identified are pancreatic hydrolyzate of casein with dry enzymatic peptone and acid hydrolyzate of corn syrup, especially in combination with such growth stimulants as sodium sulfite, ferrous ammonium sulfate or ammonium molybdate.

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.

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.

Research objective – studying of a possibility of application antigen – stimulated cellular in vitro tests and technology of the cytometric analysis for control of immunogene activity of batches of vaccine plague live.Materials and methods. As biomodels used white laboratory mice, immunized commercial medicine of vaccine of the plague NIIEG line, live from a strain of Yersinia pestis EV, in doses – 8 х 102, 4 х 103, 2 х 104 and 1 х 105 of living microbic cells. Blood for a research was taken from intact mice and on 7, 14 and 21 days after immunization. The intensity of an antigenreaktivnost of lymphocytes was defined in cellular in vitro tests, analyzing a marker of early activation (CD45+CD3+CD25+) of lymphocytes with use of the monoclonal antibodies conjugated from fluorokhroma. As specific antigen used a complex of water-soluble antigens of a plague microbe.Results. As a result of a research it is shown that at the animals vaccinated by doses 4 х 103 – 1 х 105 living microbic cells, the highest level of an expression activation marker lymphocytes at anti-gene stimulation of in vitro is registered on 14 days after immunization, at the same time the quantity of CD25 – positive lymphocytes are on average 6.8 times higher, than in control group. High degree of direct link (coefficient of correlation of r = 1,000) quantities of the survived animals with increase in level of lymphocytes, expressiruyushchy markers of early activation – CD25 is established.Conclusions. The offered technique can be used as the additional test when studying degree of immunogenicity of new (kandidatny) vaccines against plague.

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.

Objective of the study was to test the nutrient medium based on the enzymatic hydrolysate of corn extract condensed for a scaled production of live plague vaccine and to check the quality of the obtained batches against the specified parameters. Materials and methods .A dense nutrient medium based on corn extract was used to grow biomass in the process of live plague vaccine production. The quality parameters of the vaccine preparation obtained were studied by the regulated methods set forth in the regulatory documentation. Results and conclusions. The vaccine was monitored at all stages of its manufacture, including control of the finished dosage form, in strict accordance with the approved regulatory documentation. All the experimental production series complied with the specified indices. Approbation of the production cycle environment for live plague vaccine manufacturing showed efficiency of the conditions and the possibility of environment’s application in the industrial production of the preparation.

The formation of immunity against infectious diseases is accompanied by an immunoallergic alteration of the organism, while the intensity of the allergic reaction is associated with the presence of specific immunity. Skin allergotesting is often used to determine the intensity of sensitization of the body. When determining the immunity of vaccines against the plague, previously an allergen was suggested as a pestin PP — a polypeptide polysaccharide complex of a plague microbe. The authors optimized the technique for obtaining the preparation of pestin with preservation of its chemical composition, high specificity and allergenic activity. It is known that a lack of allergic test in vivo is a high risk of formation of adverse reactions. A method for estimating adaptive antiplague immunity with the allergen pestin in antigen-specific cellular tests in vitro is proposed. The preparation of the allergen by a modified procedure was carried out by hydrolysis of the biomass of the vaccine strain of the plague microbe Yersinia pestis EV of the NIIEG line, followed by filtration and lyophilization of the precipitate. In the preparation obtained, the pH and the protein concentration were determined. To check the specificity, the samples were subjected to spectrophotometric and chromatographic analysis. To assess specific activity, blood samples of 17 people immunized with the plague live vaccine from the Yersinia pestis EV strain of the NIIEG line were used for epidemic indications. As a comparative control, a sterile isotonic sodium chloride solution was used. The contingent was examined before vaccination on days 7, 21 and 3 months after immunization by evaluating the expression intensity of basophils CD63. Biochemical analysis of the obtained by the modified procedure of the pestin and derivatives allowed to judge the qualitative composition, to show the absence of impurities of the protein nature, as well as to determine the carbohydrate profile. The use of the drug as an allergen to assess the formation of antiplague immunity in the vaccinated contingent confirmed its specificity. The obtained data showed the possibility and prospect of using the Pestin PP as a test allergen for the establishment of the reaction of activation of basophils in vitro.

Objective of the study was to develop a solid medium on the basis of enzyme digest of corn-steep extract for manufacturing of live plague vaccine and storage of plague agent strains. Materials and methods. Vaccine strain and virulent strains of Yersinia pestis, nutrient media for accumulation and storage. Investigated parameters were assessed according to regulatory documentation. Results and conclusions. Developed has been nutrient medium based on enzyme digest of corn-steep extract with growth stimulation additives – Mohr’s salt and sodium sulphite. Studied have been its physical-chemical and biological properties. Approbation of the medium in manufacturing laboratory has revealed its high efficiency and possibility of usage in industrial production of live plague vaccine. Batches of preparation with optical concentration of 100 mlrd/ml and (68.2±0.9) % viability have been manufactured. Application of the stated medium allows for increase in biomass output and decrease in prime cost of final product. Confirmed has been the possibility to store the virulent plague agent strains on the medium at (4±2) °C for 18 months without reduction of the culture viability.

The possibility of post-vaccination anti-plague immunity evaluation was researched using antigen-stimulated cells tests in vitro and cytometry analysis. The object of study — the blood samples of 17 people immunised by the live plague vaccine (Yersinia pestis EV) epicutaneously. Blood taking was carried out before vaccination and after immunisation on 7 and on 21 days, in 3 and in 6 months. Intensity antigen reactivity of lymphocytes was detected by cell tests in vitro, analysing markers of early (CD45+CD3+CD25+) and late (CD45+CD3+HLA-DR+) lymphocyte activation using flow cytometry. The complex of water-soluble Y. pestis antigens and allergen — pestin PP was tested as antigen. The high stimulating potential was defined of the water-soluble antigens Y. pestis complex. It is shown that coefficient of stimulation of relative level T- lymphocytes which express receptors for IL-2 was positive for all observation times after immunisation. The coefficient of stimulation had maximum values at 21 days (56.37%) and at 3 (47.41%) months. In identifying HLADR-positive lymphocytes before vaccination, the negative coefficient of stimulation was indicated on 7 and 21 days and the positive coefficient of stimulation was indicated at 3 and at 6 months. Analysis of intensity expression of early and late lymphocyte activation markers dynamics showed the possibility and prospect of application of cellular in vitro tests for the laboratory evaluation of specific reactivity of cellular immunity in both the early (7 days) and late (6 months) periods after vaccination. The results can be the basis for developing a new algorithm for assessment of immunological effectiveness of vaccination people against plague. It is the algorithm based on the identification of lymphocyte activation markers by antigen stimulation in conditions in vitro.

Over the years, the production release of the plague vaccine is well developed its technology. The technological cycle of production of the preparation consists of regulated steps, however, despite their effectiveness it is necessary to modernize the manufactoring process, for example, solutions for some of the pressing needs of the customers, in particular, small groups of immunization. Our research has focused on obtaining experimental samples plague vaccine smaller compared to the commercial vaccine, the number of doses per vial prepared in a biomass production unit (ACM-Sh) surface by cultivation using all regulated processing steps, except step of combining content two swabs, and then an additional dilution of the cell suspension stabilizer. However, the time information and the subsequent preparation of such a vaccine is excluded us, since biomass is the second flush in quantitative terms is a ready raw material for the preparation of reduced dosage. The benefits of receiving the vaccine reduced the number of doses directly from the biomass of the second flush with the concentration of microbial cells Yersinia pestis EV 20–40 × 109 biotechnology greatly simplify the manufacture of such a preparation. The experimental vaccine series were tested by major regulated parameters: optical concentration, vitality, thermal stability, the loss on drying. In addition, the vaccine was prefabricated with high baseline viability to extreme temperatures (37±1)°C for 24 hours to exclude enough viable microbial cells for subsequent stabilization indicator of viability during storage. It should be noted that all the experimental samples preserved viability index not lower regulated (25%) during the experiment, in contrast to the commercial preparation. To determine the stability of the formulation during storage (over 3 years) was a comparative analysis of the viability of the experimental and commercial lots. To assess post vaccination immune analyzed the immune response to the introduction of a plague vaccine using FACSCalibur flow cytometer, considering that this technology has a high specificity, sensitivity and informativity. With regard to the immunogenic properties, the active component is recorded at a very high level as the white mice, and guinea pigs. Thus, the main biological indicators derived preparations (viability, thermal stability, storage stability) exceed those of commercial analog and provide effective immunological alterations and highly immunogenic in experimental animals.

Comparative analysis of specified indices (viability, thermostability) of plague live vaccine preparations, produced in accordance with different biotechnological production schemes, have been carried out. Shown is that the combination of optimal temperature for Yersinia pestis strain EV biomass propagation (21±1) °C as well as optimal concentration proportion of bacteria and stabilizer in suspension with decreased optical density and volume facilitates effectual moisture removal out of preparation, thus stabilizing microbial cells viability index in the process of storage.

The preference of the (21±1) °C temperature for cultivation of the vaccine strain Yersinia pestis EV in the live plague vaccine biomass production to (27±1) °C is experimentally confirmed. An original interpretation of the mechanism of small cells stability that are formed at (21±1) °C has been proposed. This interpretation stipulates a smaller damaging effect of steam molecules during sublimation in the process of lyophilization.