Ex Vivo ELISpot Assay to Investigate Dengue Virus Specific T-Cell Responses

Malavige, Gathsaurie Neelika

doi:10.1007/978-1-4939-8567-8_15

Cited by 5 publications

(4 citation statements)

References 12 publications

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“…DENV‐NS3 peptide array is comprised of 105, 14‐ to 17‐mer peptides while NS5 proteins were consisted of 156 peptides. The peptides were reconstituted as previously reported . DENV‐NS3 and ‐NS5 peptides were pooled separately to represent the DENV‐NS3 and ‐NS5 proteins.…”

Section: Methodsmentioning

confidence: 99%

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Association of dengue virus‐specific polyfunctional T‐cell responses with clinical disease severity in acute dengue infection

Wijeratne

Fernando

Gomes

et al. 2019

Immunity Inflam & Disease

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Introduction Although the role of dengue virus (DENV)‐specific T cells in the pathogenesis of acute dengue infection is emerging, the functionality of virus‐specific T cells associated with milder clinical disease has not been well studied. We sought to investigate how the functionality of DENV–NS3 and DENV–NS5 protein‐specific T cells differ in patients with dengue fever (DF) and dengue hemorrhagic fever (DHF). Methods Using intracellular cytokine assays, we assessed the production of interferon γ (IFNγ), tumor necrosis factor‐α (TNF‐α), macrophage inflammatory protein‐1β (MIP‐1β), and CD107a expression in adult patients with acute DF (n = 21) and DHF (n = 22). Results Quadruple cytokine‐producing, polyfunctional DENV–NS3‐ and DENV–NS5‐specific T cells were more frequent in those with DF when compared to those with DHF. While DENV–NS3‐ and DENV–NS5‐specific T cells in patients with DF expressed IFNγ > TNF‐α > MIP‐β > CD107a, T cells of those with DHF predominantly expressed CD107a > MIP‐1β > IFNγ > TNF‐α. Overall production of IFNγ or TNF‐α by DENV–NS3‐ and DENV–NS5‐specific T cells was significantly higher in patients with DF. The majority of NS3‐specific T cells in patients with DF (78.6%) and DHF (68.9%) were single‐cytokine producers; 76.6% of DENV–NS5‐specific T cells in those with DF and 77.1% of those with DHF, produced only a single cytokine. However, no significant association was found with polyfunctional T‐cell responses and the degree of viraemia. Conclusions Our results suggest that the functional phenotype of DENV‐specific T cells are likely to associate with clinical disease severity.

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Section: Methodsmentioning

confidence: 99%

“…The peptides were reconstituted as previously reported. 24 DENV-NS3 and -NS5 peptides were pooled separately to represent the DENV-NS3 and -NS5 proteins. Final concentration of dimethyl sulfoxide is less than 0.5%.…”

Section: Peptidesmentioning

confidence: 99%

Association of dengue virus‐specific polyfunctional T‐cell responses with clinical disease severity in acute dengue infection

Wijeratne

Fernando

Gomes

et al. 2019

Immunity Inflam & Disease

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“…Ex vivo ELISpot assays were performed as previously described [see detailed description under cultured ELISpot assays (14,31,32)]. In ex vivo ELISpot assays PBMCs 1 × 10 5 were added to each well and JEV-specific, conserved peptides were added at a final concentration of 10 µM as previously described and tested in duplicate (32) The spots were enumerated using an automated ELISpot reader (AID, Germany).…”

Section: Ex Vivo Elispot Assaysmentioning

confidence: 99%

Identification of Immune Responses to Japanese Encephalitis Virus Specific T Cell Epitopes

Pushpakumara

Jeewandara

Wijesinghe

et al. 2020

Front. Public Health

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Background: Due to the similarity between the dengue (DENV) and the Japanese encephalitis virus (JEV) there is potential for immune cross-reaction. We sought to identify T cell epitopes that are specific to JEV and do not cross react with DENV.Methodology: 20mer peptides were synthesized from regions which showed >90% conservation. Using IFNγ cultured ELISpot assays, we investigated JEV-specific T cell responses in DENV − and JEV − non-immune individuals (DENV − JEV − = 21), JEV seronegative and had not received the JE vaccine, but who were DENV seropositive (DENV + JEV − = 22), JEV + (seropositive for JEV and had received the JE vaccine), but seronegative for DENV (DENV − JEV + = 23). We further assessed the responses to these peptides by undertaking ex vivo IFNγ assays and flow cytometry.Results: None of DENV − JEV − individuals responded to any of the 20 JEV-specific peptides. High frequency of responses was seen to 6/20 peptides by individuals who were JEV + but DENV − , where over 75% of the individuals responded to at least one peptide. P34 was the most immunogenic peptide, recognized by 20/23 (86.9%) individuals who were DENV − JEV + , followed by peptide 3 and peptide 7 recognized by 19/23 (82.6%). Peptide 34 from the NS2a region, showed <25% homology with any flaviviruses, and <20% homology with any DENV serotype. Peptide 20 and 32, which were also from the non-structural protein regions, showed <25% homology with DENV. Ex vivo responses to these peptides were less frequent, with only 40% of individuals responding to peptide 34 and 16-28% to other peptides, probably as 5/6 peptides were recognized by CD4+ T cells.Discussion: We identified six highly conserved, T cell epitopes which are highly specific for JEV, in the Sri Lankan population. Since both JEV and DENV co-circulate in the same regions and since both JE and dengue vaccines are likely to be co-administered in the same geographical regions in future, these JEV-specific T cell epitopes would be useful to study JEV-specific T cell responses, in order to further understand how DENV and JEV-specific cellular immune responses influence each other.

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“…Another study showed that ILC produce higher IL-4/IL-13 or GATA3+ in dengue infection with warning signs compared to dengue infection without warning signs 32 . Additionally, dengue patients who had lower DENV-NS3 specific IFN-γ production by T cells since the onset of illness, before development of ‘critical phase’, were significantly likely to subsequently develop DHF 33 , 34 . Also, lung ILC2 from mice challenged with H1N1 virus subtype, containing a functional PB1-F2 virulence factor, induced a dominant IL-13 + CD8 T cell response, regardless of host IFN-γ expression 35 .…”

Section: Introductionmentioning

confidence: 99%

Dengue virus co-opts innate type 2 pathways to escape early control of viral replication

et al. 2022

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Mast cell products and high levels of type 2 cytokines are associated with severe dengue disease. Group 2 innate lymphoid cells (ILC2) are type-2 cytokine-producing cells that are activated by epithelial cytokines and mast cell-derived lipid mediators. Through ex vivo RNAseq analysis, we observed that ILC2 are activated during acute dengue viral infection, and show an impaired type I-IFN signature in severe disease. We observed that circulating ILC2 are permissive for dengue virus infection in vivo and in vitro, particularly when activated through prostaglandin D2 (PGD2). ILC2 underwent productive dengue virus infection, which was inhibited through CRTH2 antagonism. Furthermore, exogenous IFN-β induced expression of type I-IFN responsive anti-viral genes by ILC2. PGD2 downregulated type I-IFN responsive gene and protein expression; and urinary prostaglandin D2 metabolite levels were elevated in severe dengue. Moreover, supernatants from activated ILC2 enhanced monocyte infection in a GM-CSF and mannan-dependent manner. Our results indicate that dengue virus co-opts an innate type 2 environment to escape early type I-IFN control and facilitate viral dissemination. PGD2 downregulates type I-IFN induced anti-viral responses in ILC2. CRTH2 antagonism may be a therapeutic strategy for dengue-associated disease.

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Ex Vivo ELISpot Assay to Investigate Dengue Virus Specific T-Cell Responses

Cited by 5 publications

References 12 publications

Association of dengue virus‐specific polyfunctional T‐cell responses with clinical disease severity in acute dengue infection

Association of dengue virus‐specific polyfunctional T‐cell responses with clinical disease severity in acute dengue infection

Identification of Immune Responses to Japanese Encephalitis Virus Specific T Cell Epitopes

Dengue virus co-opts innate type 2 pathways to escape early control of viral replication

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