Evaluation of the long-term memory T cell in mice after immunization with a live tularemia vaccine

The vaccine strain F. tularensis 15 NIIEG induces long-lived cell-mediated immunity but exhibits a certain reactogenicity and genetic instability. Progress in development of a vaccine against tularemia has been limited by a lack of information regarding the mechanisms required to protect against this disease. The BALB/c mouse is the most commonly used animal to study tularemia due to its relatively low cost, well-characterized genetics, available immunological tools and mouse infection with virulent F. tularensis recapitulates human disease.CD4+ and CD8+T cells are known to be critical for the formation of protective immunity but the relative roles of memory T cell subpopulations in long lived protection against virulent strains of F. tularensis are not well established. We hypothesized that this immunity depends on central (TCM) and effector memory (TEM) T cells and their functional activity. In this study we have dissected the T cell immune response in BALB/c mice 30, 60 and 90 days after subcutaneous vaccination with 15 NIIEG.Multiparametric flow cytometry were used to characterize in vitro recall responses of splenocytes to F. tularensis antigen. TEM cells were identified as CD3+CD4+CD44+CD62L- and CD3+CD8+CD44+CD62L-, TCM cells as CD3+CD4+CD44+CD62L+ and CD3+CD8+CD44+CD62L+, respectively. The functional activity of memory T cells was assessed by the following parameters: the level of expression of the activation marker CD69 and cytokine-producing activity by staining with the intracellular cytokines IFNg and TNFa.Thus, development of a long-lived vaccine directed against F. tularensis is dependent on identifying not only the correlates of immunity present early after vaccination, but also those that persist in the host after the effector phase has ended. The maintenance of long-term protective immunity initiated by vaccination with F. tularensis strain 15 NIIEG has been shown to require the presence of antigen-specific CD4+ and CD8+ memory T cells producing IFNg and TNFa and expressing the activation marker CD69. A decrease in count and functional activity of CD8+TCM and CD8+TEM was detected in the long term after vaccination. The detected parameters of functional activity of memory T cells can be used as criteria for evaluation of protective immunity against virulent strains of F. tularensis

Study of the UV Irradiation and Nalidicsic Acid Effect on the RecA-protein Induction in <i>Francisella tularensis</i> 15/10 Cells

Studied is the UV irradiation and nalidicsic acid effect on the RecA-protein synthesis in Francisella tularensis 15/10 cells. Obtained is the specific murine serum to the recombinant RecA-protein. The results of immunoblotting with this specific serum demonstrate that the amount of RecA-protein in Francisella tularensis 15/10 cells is not increased after the exposure to these damaging factors

Immunobiological Properties of <i>Francisella tularensis</i> 15/10 Strain with Deleted recA Gene

Deletion of recA gene in Francisella tularensis 15/10 genome leads to the increase in its sensitivity to ultraviolet irradiation, reduction of the homologous recombination capacity, and a slight decline of virulence for mice. Efficacy of Francisella tularensis 15/10ΔrecA reproduction within microphage-like cells, its resistance to normal rabbit serum, and protective properties on the mouse model of tularemia are the same as in original Francisella tularensis 15/10

Construction and Investigation of the Vaccine Strain Francisella tularensis without iglC genes. Communication 1

Using site-specific mutagenesis constructed were the variants of the vaccine strain F. tularensis subsp. holarctica 15 with the deleted iglC genes. Identified was the fact that genome of the strain 15 contained two copies of iglC gene. Deletion of one of them as well as both had little effect on the cultural-morphological and growth properties of the microbe. At the same time F. tularensis 15 lacking one copy of the iglC gene propagated in mice macrophages several times slower, than the original strain. Inactivation of both of the copies in the chromosome leaded to the emergence of a variant incapable of intracellular reproduction. This capacity in F. tularensis 15/23-2 with two inactivated iglC gene copies was partially recovered after integration of a complementing plasmid. Therewith the data mentioned above testifies to the significance of iglC gene for the process of reproduction in macrophages

Construction and Investigation of the Variants of the Vaccine Strain <I>Francisella tularensis</I> Lacking <I>iglC </I>Genes. Communication 2

for BALB/c mice by an order while retaining protective properties. Strain Francisella tularensis 15 lacking two copies of the iglC gene cannot replicate in mice organism, induces weak humoral response, is fully avirulent, and has decreased protective activity. Treatment of mice with sera obtained from the animals immunized on day 49 post immunization both with the stock F. tularensis 15 strain and its variants with one or two inactivated iglC genes has provided for 100 % protection from challenge with 200 DCL of F. tularensis 15 strain. However in case of BALB/c mice exposure to virulent F. tularensis 503 and F. tularensis Schu strains (50 DCL and 25 DSL, respectively), treated with immune sera 24 hours before, registered has been only mean lifetime increase

Infectious Sensitivity of BALB/c Mice to Infestation with <I>Photorhabdus asymbiotica</I> and <I>Photorhabdus temperata</I>

) KOE). Identified are the patho-morphological changes in the organs of animals, inoculated with Photorhabdus spp. strains

Dynamics of Antibody Response to <i>Yersinia pestis</i> Proteins in Plague Affected Guinea Pigs

Designing of new means for the specific prevention of plague, especially protein subunit vaccines, is impossible without studying the role of individual antigens in the manifestation of the pathogenic and immunogenic properties of Yersinia pestis. The aim of the present study was to determine the antibody levels to Y. pestis antigens in guinea pigs that survived infection with sub-lethal doses of virulent plague agent strains using enzyme immunoassay (ELISA). Materials and methods. Guinea pigs were inoculated subcutaneously with 30 CFU of the wild type Y. pestis subsp. Pestis strain 231 or non-capsular Y. pestis subsp. pestis Caf1-negative strain 358/12. Blood samples from sick or recovered guinea pigs were collected on day 15, 30, 60, and 90 after infection. The antibody response was assessed by 18 recombinant Y. pestis proteins in ELISA. Results and discussion. Heterogeneity of the antibody responses to the majority of the antigens with variation of IgG titers from animal to animal has been revealed. We observed increase in antibody titers by day 90 for the most analyzed antigens in the sera of the guinea pigs injected with wild type Y. pestis 231. On the contrary we found reduction in antibody titers by day 90 in case of inoculation with Y. pestis 358/12. The preservation of antibodies to Y. pestis proteins of different localization in the organism of the guinea pigs, as well functional activity, and the degree of representation on the surface of bacterial cell for a prolonged period of time indicates the multiplex nature of the plague immunity formation. Our findings are significant for the future design and development of effective vaccines against plague and the search for new targets for diagnostics of this disease

Определение противококлюшных антител у школьников с длительным кашлем

Objective: to assess anti-pertussis immunity in schoolchildren aged 7–17 who complained of a prolonged cough during the 11-year follow-up period.  Materials and methods. The study included 1046 patients aged 7 to 17 years who applied to the Consultative and Diagnostic Center of the G.N. Gabrichevsky Research Institute of Epidemiology and Microbiology with complaints of prolonged cough in the period from 2010 to 2020. Blood serums were examined in ELISA with the determination of IgM, IgG, IgA antibodies using RIDASCREEN test system (Germany).  Results. An active infection with the detection of IgM and/ or IgA, IgG antibodies above threshold levels was detected in 51,3% of children with prolonged cough, while annually in a fairly high percentage throughout the follow-up period. Active pertussis infection, established based on the detection of IgM, IgG, IgA antibodies above thresholds in blood serum samples, prevailed in children 12–15 years old, accounting for more than 60% in children with prolonged cough. Antipertussis immunity as a result of childhood vaccination or previous disease was detected in 16.1-20.2% of people in the period 2010–2014 and in 12,8-20,9% in 2015–2020.  Conclusion. The results obtained by us on the study of anti-pertussis immunity in schoolchildren confirm the presence of active latent circulation of the pathogen whooping cough among children of this age cohort and, therefore, the presence of unaccounted for cases of the disease. This confirms the importance of timely diagnosis of pertussis, isolation of children for the period of active infection and justifies the need for the widespread introduction of a second revaccination against pertussis. Цель: оценка противококлюшного иммунитета у школьников 7–17 лет, обратившихся с жалобами на длительный кашель, в течение 11-летнего периода наблюдения.  Материалы и методы. В исследование включено 1046 пациентов в возрасте от 7 до 17 лет, обратившихся в консультативно-диагностический центр Московского научно-исследовательского института эпидемиологии и микробиологии им. Г.Н. Габричевского с жалобами на длительный кашель в период с 2010 по 2020 г. Сыворотки крови исследовали в ИФА с определением IgM, IgG, IgA антител с помощью тест-системы «RIDASCRЕЕN» (Германия). Результаты. Активная инфекция с выявлением антител классов IgM и (или) IgA, IgG выше пороговых уровней выявлена у 51,3% детей с длительным кашлем, при этом ежегодно в достаточно высоком проценте на протяжении всего периода наблюдения. Активная коклюшная инфекция, установленная на основании выявления в образцах сывороток крови антител IgM, IgG, IgA выше пороговых значений, преобладала у детей 12–15 лет, составляя выше 60% у детей с длительным кашлем. Противококлюшный иммунитет в результате проведенной в детстве вакцинации или перенесенного заболевания выявили у 16,1–20,2% лиц в период 2010–2014 гг. и у 12,8–20,9% – в 2015–2020 гг.  Заключение. Полученные нами результаты по изучению противококлюшного иммунитета у школьников подтверждают наличие активной скрытой циркуляции возбудителя коклюша среди школьников и, следовательно, наличие недоучтенных случаев заболевания. Это подтверждает важность своевременной диагностики коклюша, изоляции детей на период активной инфекции и обосновывает необходимость повсеместного введения второй ревакцинации против коклюша.

Диагностическая ценность метода ИФА при коклюше у детей

Because of low effectiveness of laboratory methods for diagnosing pertussis it is important to look for new ways of verification of this infection. The article presents the analysis of the diagnostic value of ELISA method, which involves the identification of antibodies of different isotypes (IgM, IgG, IgA) to pertussis toxoid (PT) and filamentous haemagglutinin (FHA). The study included 279 children: 114 were under 1 year of age, 165 — older than 1 year. The pertussis was confirmed in 74.3 ± 2.6% of patients by using ELISA method. A significant proportion of seronegative patients (46.1 ± 6.2 per cent) was revealed in the group of patients under 1 year. The pattern of production of antibodies in unvaccinated children was different. It depended on the age of the children and timing of illness. A low proportion of diagnostically significant indicators of IgM-antibodies at 2—3 weeks of illness was typical for patients under 1 year of age (e.g. 6.7 ± 6.5% as compared to 20.0 ± 7.9% and 50.0 ± 15.3 — 1—3 and 4—6 years of age). The diagnosis of pertussis in children under 1 year of age was confirmed mainly by the detection of IgG, starting from the 4th week of the disease. In the examination of vaccinated children diagnostically significant levels of IgA and IgG were identified (even in the late stages of the disease). Thus, the results of the analysis show special significance of using ELISA method for the diagnosis of pertussis in vaccinated children.Недостаточная эффективность используемых методов лабораторной диагностики коклюша в практическом здравоохранении диктует необходимость расширения спектра средств верификации этой инфекции. В статье представлен анализ диагностической ценности метода ИФА, который предусматривает выявление антител различных изотипов (IgM, IgG, IgA) к коклюшному токсину и филаментозному гемагглютинину. Всего обследовано 279 детей, среди которых 114 были в возрасте до 1 года, 165 — старше 1 года. Использование метода ИФА в целом позволило подтвердить диагноз коклюша у 74,3 ± 2,6% больных. При проведении сравнительного анализа его эффективности у детей различного возраста установлено, что в группе больных в возрасте до 1 года выявлен значительный удельный вес серонегативных пациентов, составивших 46,1 ± 6,2%. Анализ частоты выявления антител различных классов у непривитых детей разного возраста выявил отличия в характере динамики продукции антител, превышающих пороговый уровень, в зависимости от сроков болезни и возрастных групп. Для пациентов раннего возраста был характерен низкий удельный вес больных с диагностически значимыми показателями IgM-антител на 2—3-й неделях болезни (например, на 2-й неделе у 6,7 ± 6,5% против 20,0 ± 7,9% и 50,0 ± 15,3 — 1—3 и 4—6 лет соответственно). Диагноз коклюша у детей до 1 года был подтвержден преимущественно выявлением антител IgG класса, начиная с 4 недели болезни. Сравнительный анализ частоты обнаружения антител различных классов у привитых детей показал значительный удельный вес больных с диагностически значимыми уровнями IgA наряду с высоким уровнем продукции IgG, причем и на поздних сроках болезни, что позволяет принимать это во внимание как важный серологический критерий диагностики коклюша у привитых детей