о.а.Волох, м.В.антонычева, н.Г.авдеева, е.м.кузнецова, к.и.холматов, Д.н.бибиков, а.к.никифоров жидКая Питательная Среда для глуБинного Культивирования туляреМийного МиКроБа ФКУЗ «Российский научно-исследовательский противочумный институт «Микроб», Саратов, Российская Федерация в работе представлена эффективная жидкая питательная среда, использование которой позволяет при глубинном культивировании вакцинного штамма туляремийного микроба получать высокие концентрации жизнеспособной биомассы с низкой степенью диссоциации, что актуально при производстве живых вакцин. в качестве питательной основы новой питательной среды использовали сухой ферментативный гидролизат фибрина, приготовленный из отхода производства антирабического иммуноглобулина.

Objective of the study was to optimize the conditions of submerged recurrent cultivation and the composition of the nutrient medium for obtaining the cell mass of Yersinia pseudotuberculosis strains, used as an adsorbent in the preparation of diagnostic plague immunoglobulins. Materials and methods. We utilized Y. pseudotuberculosis adsorbent strains 6; 31; 68; 69; and 70 belonging to V, I, III, IV, and V serotypes, respectively, received from the State Collection of Pathogenic Bacteria at the premises of the RusRAPI “Microbe”. The cultivation process was carried out on an incubator-shaker, laboratory and pilot fermenters with variation of the process parameters, different options for feeding and nutrient media. Results and discussion. In the course of work, the optimal parameters of recurring submerged cultivation have been established. It was found that the highest biomass yield is provided by a combination of a nutrient medium – a carbon substrate in the form of a broth, based on an enzymatic fibrin hydrolysate, with the addition of galactose as a substrate feeding. Thereat, the morphology and immunochemical properties of microbial cells obtained through modified preparation process do not differ from those produced in the control run. The optimization of the parameters for Y. pseudotuberculosis cell mass cultivation with subsequent upscaling has been performed.

ЭКСПЕРИМЕНТАЛЬНЫЙ ПРЕПАРАТ дЛя СПЕцИфИчЕСКоЙ ПРофИЛАКТИКИ ТУЛяРЕМИИ ФКУЗ «Российский научно-исследовательский противочумный институт «Микроб», Саратов, Российская ФедерацияСконструирован экспериментальный препарат прототипной химической туляремийной вакцины (ПХТВ). В состав ПХТВ входит протективный антигенный комплекс (ПАК) туляремийного микроба и белок S-слоя (Slp) чумного микроба. Определено оптимальное соотношение компонентов и схема введения препарата. Представлены результаты испытаний его физико-химических свойств, реактогенности, специфической активности и влияния на иммунную систему лабораторных животных. Установлено, что препарат прототипа химической туляремийной вакцины не токсичен для белых мышей и морских свинок и не оказывает повреждающего действия на их иммунную систему. Однократная подкожная иммунизация лабораторных животных препаратом ПХТВ вызывает формирование напряженного адаптивного иммунитета к 14-21-м суткам: выработку специфических антител и стимуляцию клеточного звена иммунитета. Индекс защиты препарата ПХТВ для белых мышей при экспериментальной туляремии, вызванной Francisella tularensis subsp. holarctica, составил в среднем 87,5 %, при инфицировании F. tularensis subsp. nearctica -50 %, с высокой напряженностью иммунитета в обоих случаях. На модели морских свинок была подтверждена высокая эффективность экспериментального препарата против туляремии голарктического подвида (индекс защиты 75 %) и длительность напряженного иммунитета. Ключевые слова: Francisella tularensis, антиген, химические вакцины.Designed is an experimental preparation of a prototype chemical tularemia vaccine (PCTV). It is composed of protective antigenic complex (PAC) of tularemia microbe and S-layer protein (Slp) of plague microbe. Determined is optimum ratio of these components in the preparation and schedule of its administration. Displayed are the results of its testing as regards physical-chemical properties, reactogenicity, specific activity and impact on the immune system of laboratory animals. It is found out that preparation of the prototype is non-toxic for white mice and Guinea pigs and has no damaging effect on their immune systems. Single-stage subcutaneous immunization with PCTV induces elaboration of high-level adaptive immunity in laboratory animals within 14-21 days: specific antibody generation and stimulation of immune system cell component. PCTV protective index for white mice in case of experimental tularemia, caused by Francisella tularensis subsp. holarctica, is 87,5 % on average; in case of infecting with F. tularensis subsp. nearctica -50 %; and high-level immunity in both cases. High potency of the experimental preparation against tularemia caused by subsp. holarctica (protective index is 75 %) and high-grade immunity persistence is verified on the model of Guinea pigs too.

Objective: to study the possibility of applying electro-optical analysis for the assessment of cell viability of tularemia microbe vaccine strain at different stages of experimental live tularemia vaccine production.Materials and methods. The research object was a cell culture of Francisella tularensis 15 NIIEG.  Investigations were carried out at all stages of experimental live tularemia vaccine (ELTV) manufacturing according to an advanced technology: cultivation, concentrating, diafiltration, mixing with drying media, stabilization, and storage (two-year period of observation). Electro-optical analysis by the parameter “polarizability anisotropy” of bacterial cell was conducted with the help of EloTrace (EloSystems, Germany). Total concentration of cells was evaluated using density metering at 590 nm and spectrometry – at 650 nm. Viability was assessed through inoculation of plates with FT-agar.Results and discussion. The experiment has demonstrated that the change in polarizability anisotropy of the cell at the frequencies of 900 kHz and 2100 kHz, reflecting the state of cytoplasm and cytoplasmic membrane, respectively, is the earliest response to changes in vital indicators of bacterial culture in the process of cultivation. Thereby, the decrease in viability of F. tularensis cells occurrs well before the decrease in cell concentration. We have shown the preservation of viability of F. tularensis 15 NIIEG cells at all stages of experimental live tularemia vaccine production. Electro-optical analysis allows for registering the changes in vital parameters of microorganism cells in real-time mode, while the assessment of viability applying bacteriological method takes up to 5 days. Different stages of tularemia vaccine manufacturing have impact on the vital indicators of F. tularensis cells, and electro-optical analysis is a prospect method of control of such parameter as “Specific activity (the number of live microbial cells)”.

Carried out has been comparative assessment between the performances of liquid nutrient pancreatic fibrin overcook-based and bakery yeast autolyzate-based media and conventionally used in manufacturing of cholera vaccine media for submerged cultivation of Vibrio cholerae 569B and V. cholerae M-41 strains. Results of investigation of media quality biological predictors (morphological and biochemical property stability, efficacy of biomass and protective antigen accumulation), which are fibrin hydrolyzate and bakery yeast autolyzate-based, suggest the possibility of using them for the production of cholera vaccine. Deployment of inedible raw material-based media in manufacturing of cholera vaccine is a prospective technology in view of reduction of medical-prophylactic preparation costs. Moreover it allows for solving the problem of protein waste-product disposal, which is generated in the process of anti-rabies immunoglobulin manufacturing, thus decreasing ecological impact on the environment.