BackgroundSri Lanka has been affected by epidemics of dengue infections for many decades and the incidence and severity of dengue infections have been rising each year. Therefore, we investigated the age stratified seroprevalence of dengue infections in order to facilitate future dengue vaccine strategies. In addition, since the symptomatic dengue infections have increased during the past few decades, we also investigated the possible association with Japanese Encephalitis Virus (JEV) antibody seropositivity with symptomatic dengue in a community cohort in Sri Lanka.Methods1689 healthy individuals who were attending a primary health care facility were recruited. Dengue and JEV antibody status was determined in all individuals and JEV vaccination status was recorded.Results1152/1689 (68.2%) individuals were seropositive for dengue and only 133/1152 (11.5%) of them had been hospitalized to due to dengue. A significant and positive correlation was observed for dengue antibody seropositivity and age in children (Spearmans R = 0.84, p = 0.002) and in adults (Spearmans R = 0.96, p = 0.004). We observed a significant rise in the age stratified seroprevalence rates in children over a period of 12 years. For instance, in year 2003 the annual seroconversion rate was 1.5% per annum, which had risen to 3.79% per annum by 2014. We also found that both adults (p<0.001) and in children (p = 0.03) who were hospitalized due to dengue were more likely to be seropositive for JEV antibodies. However, 244 (91.4%) of adults who were seropositive for JEV had not had the JEV vaccine.ConclusionsDengue seroprevalence rates have risen significantly over the last 12 years in Sri Lanka, possibly due to increased transmission. As individuals who were hospitalized due to dengue were more likely to be seropositive for JEV, the possibility of cross-reactive assays and/or of JEV infection on immunity to the DENV and clinical disease severity should be further investigated.

BackgroundVascular leak is the hallmark of severe dengue infections and leads to complications such as shock and multi-organ failure. Although many mediators have been implicated in the vascular leak in dengue, the role of sphingosine 1-phosphate (S1P) has not been investigated.Metholodology/Principal findingsAs S1P has been shown to be important in barrier integrity, we assessed the S1P levels in 28 patients with acute dengue and 12 healthy individuals. The S1P levels were significantly lower in patients with acute dengue (p = 0.002) and the levels in patients with grade IV dengue haemorrhagic fever (DHF) were significantly lower than those with dengue fever (p = 0.005). We then investigated the kinetics of S1P levels throughout the course of the illness in another 32 patients in serum samples obtained twice a day. We found that S1P levels were low throughout the course of illness and S1P levels were <0.5 µM in 12/23 patients with DHF when compared to 1/9 with DF.Conclusions/SignificanceAs S1P has shown to be important in the endothelial barrier integrity and increases transendothelial resistance, low levels of S1P in acute dengue infection are likely to contribute to increased vascular permeability.

BackgroundAlthough antibody responses to dengue virus (DENV) in naturally infected individuals have been extensively studied, the functionality of DENV specific memory T cell responses in relation to clinical disease severity is incompletely understood.Methodology/Principal findingsUsing ex vivo IFNγ ELISpot assays, and by determining cytokines produced in ELISpot supernatants, we investigated the functionality of DENV-specific memory T cell responses in a large cohort of individuals from Sri Lanka (n=338), who were naturally infected and were either hospitalized due to dengue or had mild or sub clinical dengue infection. We found that T cells of individuals with both past mild or sub clinical dengue infection and who were hospitalized produced multiple cytokines when stimulated with DENV-NS3 peptides. However, while DENV-NS3 specific T cells of those with mild/sub clinical dengue infection were more likely to produce only granzyme B (p=0.02), those who were hospitalized were more likely to produce both TNFα and IFNγ (p=0.03) or TNFα alone.We have also investigated the usefulness of a novel T cell based assay, which can be used to determine the past infecting DENV serotype. 92.4% of DENV seropositive individuals responded to at least one DENV serotype of this assay and none of the seronegatives responded. Individuals who were seronegative, but had received the Japanese encephalitis vaccine too made no responses, suggesting that the peptides used in this assay did not cross react with the Japanese encephalitis virus.Conclusions/significanceThe types of cytokines produced by DENV-specific memory T cells appear to influence the outcome of clinical disease severity. The novel T cell based assay, is likely to be useful in determining the past infecting DENV serotype in immune-epidemiological studies and also in dengue vaccine trials.

Background: Patients with severe dengue infection have shown to have impaired dengue virus (DV) specific T cell responses and higher viral loads. Our previous data have shown that DV-specific T cell responses are suppressed by IL-10. Therefore, we set out to investigate the possible mechanisms of inhibition of DV-specific T cell responses in patients with acute dengue infection.Methods & Materials: 18 adult patients with acute confirmed dengue infection were recruited following informed written consent. The first blood sample was collected during day 3-5 of illness and the second sample was collected 2 days later. Clinical disease severity was classified according to the WHO 2011 dengue guidelines. Flow-cytometry was done on freshly extracted peripheral blood mononucleocytes (PBMCs). Intracellular cytokine staining was done by stimulating PBMCs with DV-NS3 overlapping peptides. Expression of CTLA-4, TIM-3, PD-1, CD27 and CD28 were determined in T cells in general and DV-specific T cells. IL-10 quantitative ELISA was done in the patients and in 10 healthy DV seropositive individuals.Results: Expression of IL-10R was significantly higher in both CD4+ T cells (p = 0.007) and CD8+ T cells (p = 0.005) in patients with acute dengue when compared to healthy individuals. Expression of CTLA-4 was also significantly higher (p < 0.0001) among T cells in patients with acute dengue (mean 411.3, SD ± 100.2 MFI) when compared to healthy individuals (mean 256.4, SD ± 84.4 MFI). Expression of CTLA-4 was also significantly higher (p = 0.009) in DV-NS3 specific T cells (mean 419.5; SD ± 69.5) when compared to CD3+ T cells in general (mean 386.6; SD ± 86.81 MFI) in the same patient. Although not significant (p = 0.11) serum IL-10 levels correlated with CTLA-4 expression in T cells (spearman's r = 0.48). CTLA-4 expression also significantly (p = 0.04) correlated with serum alanine transaminase levels (spearman's r = 0.59). PD-1 expression was higher in T cells in patients with acute dengue (mean 106.8, SD ± 65.43 MFI) when compared to healthy individuals (mean 88.71, SD ± 22.77MFI), although not significant (p = 0.85).Conclusion: CTLA-4 expression appears to be higher in T cells in patients with acute dengue and significantly higher in DV-specific T cells, suggesting a role in inhibition of DV-specific immune responses.