The nature of dengue virus specific long term memory T cell responses after natural infection

Ph.D. University of Hawaii at Manoa 2014.Includes bibliographical references.Background: All 4 serotypes of dengue virus can cause dengue fever and severe dengue (dengue hemorrhagic fever/dengue shock syndrome, DHF/DSS), mediated in part by cross-reactive T cells. Our understanding of how memory T-cells contribute to anti-DENV immunity is incomplete, and the duration and functional profile of dengue-specific T-cell-mediated immune responses is still not known. Due to a lack of an appropriate animal model and because cocirculation of all 4 dengue serotypes occurs in hyperendemic areas, research on memory T cells has been conducted in humans who have experienced multiple infections and are continuously being exposed to dengue antigens. Therefore, the purpose of this work was to assess the duration, memory phenotype, and effector function of dengue-specific T cell-mediated immune responses in volunteers, who experienced a single natural infection, 3, 9, or 60+ years previously. Methods: Volunteers who were infected with dengue virus in the 2001 Hawaii dengue outbreak were bled 3 years and 9 years after infection. Also volunteers who were infected with dengue in the 1943-44 dengue outbreak in the Pacific were enrolled and bled in 2005, six decades after infection. Negative control were volunteers who were dengue naïve. We tested plasma/serum collected from all subjects for the presence of anti-dengue neutralizing and total IgG antibodies. Proliferation, memory phenotype, cytokine profiles and crossreactive potential were examined using PBMCs from all subjects. Assays such as IFN-γ ELISpot, PKH-26 dye dilution proliferation, phenotyping of CD markers, dengue-specific MHC tetramer binding, and intracellular secretion of IFN-γ, MIP-1β and TNF-α were examined by flow cytometry. Results: The data in this work demonstrates that dengue-specific humoral and cell adaptive immune responses can persist for more than six decades in the absence of continual exposure to dengue antigens. Both CD4+ and CD8+ T-cells were involved in the proliferative responses, however there were marked differences between the two subsets with respect to longevity of responses. CD4+ responses declined gradually, whereas CD8+ T-cell memory declined more drastically with time, with substantially diminished responses apparent at 9 years. The functional phenotype analysis revealed that the multifunction cross-reactive response associated with anti-viral protection, that was observed at 3 years after infection, but greatly diminished with time, and was replaced by highly crossreacti ve monofunctional, TNF-α rich response. Significance: The dengue viruses cause more than 50 million symptomatic dengue infections annually, in more than 100 dengue endemic countries. Studies in endemic populations suggest that protective immunity is mediated by polyfunctional CD8+ T-cells, which secrete IFN-γ. We describe persistence of T-cell mediated immune responses for more than 60 years following a single dengue infection and show that in the absence of repeated exposures, CD8+ memory T-cell responses, including secretion of IFN-γ, decline markedly whereas CD4+ responses are maintained. This temporal decline in CD8+ T cell function, detectable as early as 6 years after infection, implies that T-cell mediated protection is progressively lost in the years following infection and suggests that boosting strategies may need to be incorporated into dengue vaccine regimens