Achala Kamaladasa

Suppression of virus specific immune responses by IL-10 in acute dengue infection.

Background Elevated IL-10 has been shown to be associated with severe dengue infection (DI). We proceeded to investigate the role of IL-10 in the pathogenesis of acute DI. Materials and methods Ex vivo and cultured IFNγ ELISpot assays for dengue virus (DENV) NS3 protein and non dengue viral proteins were carried out in 26 patients with acute DI (16 with dengue haemorrhagic fever) and 12 healthy dengue seropositive individuals from Sri Lanka. DENV serotype specific (SS) responses were determined by using a panel of SS peptides. Results Serum IL-10 level were significantly higher (p = 0.02) in those who did not have in vitro responses to DENV-SS peptides (mean 144.2 pg/ml) when compared to those who responded (mean 75.7 pg/ml). DENV-NS3 specific ex vivo IFNγ ELISpot responses were also significantly lower (p = 0.0001) in those who did not respond to DENV-SS peptides (mean 42 SFU/million PBMCs) when compared to those who responded to DENV-SS peptides (mean 1024 SFU/million PBMCs). Serum IL-10 levels correlated significantly (p = 0.03) and inversely (Spearmans R = −0.45) with ex vivo DENV-NS3 specific responses but not with ex vivo non DENV specific responses (Spearmans R = −014, p = 0.52). Blockage of IL-10 in vitro significantly increased (p = 0.04) the ex vivo IFNγ ELISpot DENV-NS3 specific responses but had no effect on responses to non DENV proteins. Conclusion IL-10 appears to contribute to the pathogenesis of acute dengue infections by inhibiting DENV-specific T cell responses, which can be restored by blocking IL-10.

Dengue NS1 antigen as a marker of severe clinical disease

BackgroundEarly detection of complications significantly reduces dengue associated mortality and morbidity. We set out to determine if the NS1 rapid antigen detection test could be used as a point of care test to predict severe disease.Methods186 adult patients with confirmed dengue were enrolled during day 3-8 of illness. Clinical and laboratory parameters were recorded during the course of the illness and NS1 antigen levels were determined using both the Panbio dengue early ELISA (Panbio, Australia) and a NS1 rapid antigen detection kit (SD Bioline, South Korea).Results59.1% of patients presented to hospital on day 5-6 of illness when NS1 antigen positivity was significantly (p = 0.008) associated with severe dengue (odds ratio 3.0, 95% CI 1.39 to 6.47) and the NS1 antigen levels were significantly higher (p = 0.03) in those who went on to develop shock. Serum NS1 antigen levels significantly (p < 0.0001) and inversely correlated with the total white cell counts and lymphocyte counts. The bedside NS1 test showed comparable sensitivity (97.4%) and specificity (93.7%) to the laboratory NS1 test in our setting and cohort.ConclusionNS1 antigen positivity is associated with a higher risk of developing severe dengue especially when positive beyond day 5 of illness in our cohort, and while further validation studies are required, the test can therefore potentially be used as a bedside point of care test as a warning sign of severe dengue.

Functionality of Dengue Virus Specific Memory T Cell Responses in Individuals Who Were Hospitalized or Who Had Mild or Subclinical Dengue Infection

Background Although 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 findings Using 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/significance The 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.

Dengue NS1 antigen contributes to disease severity by inducing interleukin (IL)-10 by monocytes

Both dengue NS1 antigen and serum interleukin (IL)‐10 levels have been shown to associate with severe clinical disease in acute dengue infection, and IL‐10 has also been shown to suppress dengue‐specific T cell responses. Therefore, we proceeded to investigate the mechanisms by which dengue NS1 contributes to disease pathogenesis and if it is associated with altered IL‐10 production. Serum IL‐10 and dengue NS1 antigen levels were assessed serially in 36 adult Sri Lankan individuals with acute dengue infection. We found that the serum IL‐10 levels correlated positively with dengue NS1 antigen levels (Spearmans r = 0·47, P < 0·0001), and NS1 also correlated with annexin V expression by T cells in acute dengue (Spearmans r = 0·63, P = 0·001). However, NS1 levels did not associate with the functionality of T cell responses or with expression of co‐stimulatory molecules. Therefore, we further assessed the effect of dengue NS1 on monocytes and T cells by co‐culturing primary monocytes and peripheral blood mononuclear cells (PBMC), with varying concentrations of NS1 for up to 96 h. Monocytes co‐cultured with NS1 produced high levels of IL‐10, with the highest levels seen at 24 h, and then declined gradually. Therefore, our data show that dengue NS1 appears to contribute to pathogenesis of dengue infection by inducing IL‐10 production by monocytes.

Dengue NS1 antigen contributes to disease severity by inducing IL‐10 by monocytes

Both dengue NS1 antigen and serum interleukin (IL)‐10 levels have been shown to associate with severe clinical disease in acute dengue infection, and IL‐10 has also been shown to suppress dengue‐specific T cell responses. Therefore, we proceeded to investigate the mechanisms by which dengue NS1 contributes to disease pathogenesis and if it is associated with altered IL‐10 production. Serum IL‐10 and dengue NS1 antigen levels were assessed serially in 36 adult Sri Lankan individuals with acute dengue infection. We found that the serum IL‐10 levels correlated positively with dengue NS1 antigen levels (Spearmans r = 0·47, P < 0·0001), and NS1 also correlated with annexin V expression by T cells in acute dengue (Spearmans r = 0·63, P = 0·001). However, NS1 levels did not associate with the functionality of T cell responses or with expression of co‐stimulatory molecules. Therefore, we further assessed the effect of dengue NS1 on monocytes and T cells by co‐culturing primary monocytes and peripheral blood mononuclear cells (PBMC), with varying concentrations of NS1 for up to 96 h. Monocytes co‐cultured with NS1 produced high levels of IL‐10, with the highest levels seen at 24 h, and then declined gradually. Therefore, our data show that dengue NS1 appears to contribute to pathogenesis of dengue infection by inducing IL‐10 production by monocytes.

Expansion of highly activated iNKT cells with altered phenotype in acute dengue infection

Invariant natural killer T (iNKT) cells are capable of rapid activation and production of cytokines upon recognition of antigenic lipids presented by CD1d molecules. They have been shown to play a significant role in many viral infections and were observed to be highly activated in patients with acute dengue infection. In order to characterize further their role in dengue infection, we investigated the proportion of iNKT cells and their phenotype in adult patients with acute dengue infection. The functionality of iNKT cells in patients was investigated by both interferon (IFN)‐γ and interleukin (IL)−4 ex‐vivo enzyme‐linked immunospot (ELISPOT) assays following stimulation with alpha‐galactosyl‐ceramide (αGalCer). We found that circulating iNKT cell proportions were significantly higher (P = 0·03) in patients with acute dengue when compared to healthy individuals and were predominantly of the CD4+ subset. iNKT cells of patients with acute dengue had reduced proportions expressing CD8α and CD161 when compared to healthy individuals. The iNKT cells of patients were highly activated and iNKT activation correlated significantly with dengue virus‐specific immunoglobulin (Ig)G antibody levels. iNKT cells expressing Bcl‐6 (P = 0·0003) and both Bcl‐6 and inducible T cell co‐stimulator (ICOS) (P = 0·006) were increased significantly in patients when compared to healthy individuals. Therefore, our data suggest that in acute dengue infection there is an expansion of highly activated CD4+ iNKT cells, with reduced expression of CD161 markers.

Expansion of highly activated invariant natural killer T cells with altered phenotype in acute dengue infection

Invariant natural killer T (iNKT) cells are capable of rapid activation and production of cytokines upon recognition of antigenic lipids presented by CD1d molecules. They have been shown to play a significant role in many viral infections and were observed to be highly activated in patients with acute dengue infection. In order to characterize further their role in dengue infection, we investigated the proportion of iNKT cells and their phenotype in adult patients with acute dengue infection. The functionality of iNKT cells in patients was investigated by both interferon (IFN)‐γ and interleukin (IL)−4 ex‐vivo enzyme‐linked immunospot (ELISPOT) assays following stimulation with alpha‐galactosyl‐ceramide (αGalCer). We found that circulating iNKT cell proportions were significantly higher (P = 0·03) in patients with acute dengue when compared to healthy individuals and were predominantly of the CD4+ subset. iNKT cells of patients with acute dengue had reduced proportions expressing CD8α and CD161 when compared to healthy individuals. The iNKT cells of patients were highly activated and iNKT activation correlated significantly with dengue virus‐specific immunoglobulin (Ig)G antibody levels. iNKT cells expressing Bcl‐6 (P = 0·0003) and both Bcl‐6 and inducible T cell co‐stimulator (ICOS) (P = 0·006) were increased significantly in patients when compared to healthy individuals. Therefore, our data suggest that in acute dengue infection there is an expansion of highly activated CD4+ iNKT cells, with reduced expression of CD161 markers.

Regulatory T-cells in acute dengue viral infection.

Although regulatory T‐cells (Tregs) have been shown to be expanded in acute dengue, their role in pathogenesis and their relationship to clinical disease severity and extent of viraemia have not been fully evaluated. The frequency of Tregs was assessed in 56 adult patients with acute dengue by determining the proportion of forkhead box protein 3 (FoxP3) expressing CD4+ CD25+T‐cells (FoxP3+ cells). Dengue virus (DENV) viral loads were measured by quantitative real‐time polymerase chain reaction (PCR) and DENV‐specific T‐cell responses were measured by ex‐vivo interferon (IFN)‐γ enzyme‐linked immunospot (ELISPOT) assays to overlapping peptide pools of DENV‐NS3, NS1 and NS5. CD45RA and CCR4 were used to phenotype different subsets of T‐cells and their suppressive potential was assessed by their expression of cytotoxic T lymphocyte‐antigen 4 (CTLA‐4) and Fas. While the frequency of FoxP3+ cells in patients was significantly higher (P < 0·0001) when compared to healthy individuals, they did not show any relationship with clinical disease severity or the degree of viraemia. The frequency of FoxP3+ cells did not correlate with either ex‐vivo IFN‐γ DENV‐NS3‐, NS5‐ or NS1‐specific T‐cell responses. FoxP3+ cells of patients with acute dengue were predominantly CD45RA+ FoxP3low, followed by CD45RA‐FoxP3low, with only a small proportion of FoxP3+ cells being of the highly suppressive effector Treg subtype. Expression of CCR4 was also low in the majority of T‐cells, with only CCR4 only being expressed at high levels in the effector Treg population. Therefore, although FoxP3+ cells are expanded in acute dengue, they predominantly consist of naive Tregs, with poor suppressive capacity.

Ex Vivo ELISpot Assay to Investigate iNKT Cell Responses in Acute Dengue Infection

T cell receptors of the invariant Natural Killer T (iNKT) cells are downregulated with antigen stimulation. Therefore, identification of this cell population with flow cytometry in functionality studies is challenging. iNKT cells are known to produce both Th1 and Th2 cytokines immediately upon antigen stimulation. Therefore, we have used an ELISpot assay to determine the production of IFN-ɣ and IL-4 after stimulation with KRN7000, which has shown to bind to CD1d molecules and activate iNKTs in a similar fashion as α-GalCer. In this study, we observed that peripheral iNKT cells in patients with acute dengue show distinct production of IFN-ɣ, but not IL-4 with KRN7000 stimulation.