Abstract
Francisella tularensis is an intracellular bacterium that causes tularemia. Progress in creating a safe and effective vaccine for the prevention of tularemia is challenging due to a lack of knowledge about immunological parameters indicative of protective adaptive immunity. Objective of the research was to assess the effect of modifications of the F. tularensis 15 NIIEG genome on the immunogenic and protective properties of F. tularensis 15/23-1ΔrecA and F. tularensis 15/23-1/sodBΔrecA strains. Materials and methods. Multi-parameter flow cytometry and the measurement of secreted cytokines were used to characterize the responses of mouse spleen lymphocytes in response to re-stimulation of F. tularensis with acid-insoluble complex (AIC) in vitro. Also, the titers of specific antibodies to F. tularensis lipopolysaccharide in blood serum were analyzed by enzyme-linked immunosorbent assay. Results and discussion. It has been shown that immunization with the studied strains led to a significant increase in CD4+ and/or CD8+ T cells capable of expressing functional markers: CD69, CD25 and/or CD28; an increase in the subpopulation of T-helpers synthesizing IFN-γ. In the body of immune mice, a pool of B-lymphocytes was formed, capable of secreting IFN-γ in response to their stimulation with AIC. Immunization with the strain 15/23-1/sodBΔrecA provided 70% protection in mice from intranasal infection with a virulent strain of F. tularensis SchuS4. More pronounced protective properties were associated with the activation of not only B-lymphocytes and T-helpers, but also with the simultaneous activation of cytotoxic T-lymphocytes.
Highlights
Objective of the research was to assess the effect of modifications of the F. tularensis 15 NIIEG genome on the immunogenic and protective properties of F. tularensis 15/23-1∆recA and F. tularensis 15/23-1/sodB∆recA strains
Multi-parameter flow cytometry and the measurement of secreted cytokines were used to characterize the responses of mouse spleen lymphocytes in response to re-stimulation of F. tularensis with acid-insoluble complex (AIC) in vitro
The titers of specific antibodies to F. tularensis lipopolysaccharide in blood serum were analyzed by enzyme-linked immunosorbent assay
Summary
Целью исследований являлась оценка влияния модификаций генома F. tularensis 15 НИИЭГ на иммуногенные и протективные свойства штаммов F. tularensis 15/23-1∆recA и F. tularensis 15/23-1/sodB∆recA. Многопараметрическая проточная цитометрия и измерение секретируемых цитокинов использованы для характеристики реакций лимфоцитов селезенки мышей на рестимуляцию кислотонерастворимым комплексом F. tularensis в системе in vitro, также проведен анализ титров специфических антител к липополисахариду F. tularensis в сыворотке крови методом иммуноферментного анализа. В работе показано, что иммунизация исследуемыми штаммами приводила к достоверному увеличению CD4+ и/или CD8+ Т‐клеток, способных экспрессировать функциональные маркеры CD69, CD25 и/или CD28, а также к увеличению субпопуляции Т‐хелперов, синтезирующих IFN-γ. В организме иммунных мышей формировался пул В‐лимфоцитов, способных секретировать IFN-γ в ответ на их стимуляцию кислотонерастворимым комплексом. Иммунизация штаммом 15/23-1/sodB∆recA обеспечивала 70 % защиту у мышей от интраназального заражения вирулентным штаммом F. tularensis SchuS4. Более выраженные протективные свойства связаны с активацией не только В-лимфоцитов и Т-хелперов, но также с одновременной активацией цитотоксических Т‐лимфоцитов. State Scientific Center for Applied Microbiology and Biotechnology, Obolensk, Russian Federation
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