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.
An analysis of trends in the development of situation on brucellosis in the world over past decade and the data on the main risk factors for the occurrence of epidemiological complications regarding this infection in various regions of the world are provided in the paper. An expert assessment of the current epizootiological and epidemiological situation on brucellosis, the coverage of population and animals with immunization in the Russian Federation is given. Over 9 months of 2021, 210 potentially hazardous as regards brucellosis in cattle areas and 24 sites – as regards brucellosis in small ruminants – were registered in Russia. Compared to the same period in 2020, there was a decrease in the number of newly identified hazardous sites for bovine brucellosis by 35.8 % (117 areas). However, long-term upward trend in epizootiological adversity for bovine brucellosis in Russia persists. The epidemiological situation on brucellosis in the country for the period of 2012–2021 is characterized as unfavorable. Decrease in the number of newly detected human brucellosis cases (by 25.1 % of long-term average values) is observed against the background of persistent unfavorable epizootic conditions for brucellosis among epidemiologically significant species of small ruminants and cattle in regions with developed animal husbandry. In 2021, clusters of human cases were registered in the Republic of Dagestan and Penza Region. In the Republic of Dagestan, against the background of aggravation of epizootiological and epidemiological situation on brucellosis, there was also an alarming trend towards prevalence of a relatively high incidence among minors. The proportion of cases of brucellosis among children under the age of 17 in the Republic amounted to 60.3 % of the total number of minors with newly diagnosed brucellosis in Russia over the past 10 years. Taking into account current epizootic, epidemic situations and the long-term dynamics of the development of situation on brucellosis in the Russian Federation, the incidence of brucellosis among the population is predicted to be 10–15 % lower than the average long-term values – 0.18–0.20 per 100000 of the population – in 2022. The number of human cases of brucellosis can range from 250 to 300.
Резюме. Бруцеллез остается одной из наиболее актуальных опасных инфекций в регионах с развитым живот-новодством. Исключительный полиморфизм симптомов, многообразие форм болезни, малая информатив-ность результатов рутинного лабораторного общеклинического обследования нередко приводят к диагно-стическим ошибкам на догоспитальном этапе. Усовершенствование комплекса лабораторной диагностики бруцеллезной инфекции требует разработки современных дополнительных методов верификации, осно-ванных на клеточных факторах иммунитета как ведущих в иммуногенезе и патогенезе бруцеллеза. Учиты-вая ведущую роль клеточного иммунитета в формировании защиты от большинства бактериальных особо опасных инфекций, изучение клеточной реакции в ответ на антигенную стимуляцию следует считать наи-более информативным (маркерным) и объективным при оценке иммунологической перестройки организма при болезни или вакцинации. Перспективными показателями специфической клеточной антигенреактив-ности могут выступать следующие маркеры (рецепторы) активации лимфоцитов: CD25 -высокоаффинный рецептор интерлейкина 2 (IL-2Ra), маркер ранней активации Т-лимфоцитов; HLA-DR -антиген главного комплекса гистосовместимости класса II, экспрессия маркера ассоциирована не только с поздней, но и с дли-тельной активацией лимфоцитов; CD95 (Fas, APO-1) -рецептор индукции апоптоза («клеточной смерти»), маркер «поздней» активации (представлен преимущественно на CD4 + лимфоцитах) и Fas L (CD178) -ре-цептор индукции апоптоза, экспрессируется в основном на CD8 + клетках. Цель работы -оценить возмож-ность и перспективность применения технологии проточной цитофлуориметрии и клеточных тестов in vitrо для диагностики острого бруцеллеза. В исследовании участвовало 35 человек с диагнозом «острый бруцеллез» и 12 человек -не больные, не переболевшие бруцеллезом, не вакцинированные против бруцеллеза (конт-рольная группа). Материалом исследования послужила венозная кровь. Определяли количество лимфоци-тов, экспрессирующих рецепторы CD25, HLA-DR, CD95, CD95L (CD178) при активации специфическим антигеном. Полученные результаты обрабатывали статистически с использованием приложений Microsoft Excel 2010. В ходе исследования установлено, что интенсивность антиген-стимулированной активации лим-фоцитов in vitro можно использовать в качестве маркера острой бруцеллезной инфекции у человека. Наибо-лее перспективными показателями активации лимфоцитов in vitro, можно считать рецепторы к IL-2 (CD25)
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.
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