Gathsaurie Neelika Malavige

Dengue viral infections

Dengue viral infections are one of the most important mosquito borne diseases in the world. They may be asymptomatic or may give rise to undifferentiated fever, dengue fever, dengue haemorrhagic fever (DHF), or dengue shock syndrome. Annually, 100 million cases of dengue fever and half a million cases of DHF occur worldwide. Ninety percent of DHF subjects are children less than 15 years of age. At present, dengue is endemic in 112 countries in the world. No vaccine is available for preventing this disease. Early recognition and prompt initiation of appropriate treatment are vital if disease related morbidity and mortality are to be limited. This review outlines aspects of the epidemiology of dengue infections, the dengue virus and its mosquito vector, clinical features and pathogenesis of dengue infections, and the management and control of these infections.

Varicella zoster virus glycoprotein E-specific CD4+ T cells show evidence of recent activation and effector differentiation, consistent with frequent exposure to replicative cycle antigens in healthy immune donors.

Varicella zoster viru (VZV)‐specific T cell responses are believed to be vital in recovery from primary VZV infection and also in the prevention of viral reactivation. While glycoprotein E (gE) is the most abundant and one of the most immunogenic proteins of the virus, there are no data addressing potential T cell epitopes within gE, nor the phenotype of specific T cells. Using interferon gamma enzyme‐linked immunospot assays and intracellular cytokine assays, we identified gE‐specific immune responses in 20 adult healthy immune donors which were found to be dominated by the CD4+ subset of T cells. We characterized three immune dominant epitopes within gE restricted through DRB1*1501, DRB1*07 and DRB4*01, and used DRB1*1501 class II tetrameric complexes to determine the ex vivo frequency and phenotype of specific T cells. In healthy immune donors, the cells were largely positive for CCR7, CD28 and CD27, but expressed variable CD62L and low levels of cutaneous lymphocyte associated antigen with evidence of recent activation. In summary, we show that circulating gE‐specific CD4+ T cells are detected at a relatively high frequency in healthy immune donors and show evidence of recent activation and mixed central and effector memory phenotype. These data would be compatible with frequent exposure to replicative cycle antigens in healthy donors and are consistent with a role for gE‐specific CD4+ T cells in the control of viral replication.

Cellular and Cytokine Correlates of Severe Dengue Infection

Background The occurrence of dengue haemorrhagic fever (DHF) is thought to result from a complex interplay between the virus, host genetics and host immune factors. Existing published data are not consistent, in part related to relatively small sample sizes. We set out to determine possible associations between dengue virus (DEN-V) NS3 specific T cells and cytokine and chemokine levels and the pathogenesis of severe disease in a large cohort of individuals with DHF. Methodology/Principal Findings By using ex vivo IFNγ ELISpot assays we determined DENV-NS3 specific responses in patients with varying severity of DHF. Other cytokines produced by DENV-NS3 specific T cells were determined by using multiple bead array analysis (MBAA). We also determined the serum cytokine levels using MBAA, lymphocyte subsets and Annexin V expression of lymphocytes in patients with varying severity of DHF. Of the 112 DHF patients studied, 29 developed shock. Serum IL-10 and IP-10 levels positively and significantly correlated with T cell apoptosis while IL-10 levels inversely correlated with T cell numbers. In contrast, TGFß showed a very significant (P<0.0001) and positive correlation (Spearman’s R = 0.65) with the platelet counts, consistent with platelet release. We found that whilst patients with severe dengue had lower total T cell numbers, the DV-NS3 specific T cells persisted and produced high levels of IFNγ but not TNFα, IL-3, IL-13, IL-2, IL-10 or IL-17. Conclusions/Significance Our data suggest that serum IL-10, TNFα and TGFβ differentially associate with dengue disease severity.

Serum IL-10 as a marker of severe dengue infection

BackgroundSeveral studies have shown that serum IL-10, IFNγ and MIF are elevated in patients in severe dengue (SD) and could be used as potential biomarkers. We proceeded to determine if these cytokines could be used as biomarkers in a large cohort of adult dengue patients with varying severity of dengue infection.MethodsSerum IL-10 levels were determined in 259 of whom 40 had severe dengue infection. Serum IFNγ and IFNα levels were done in 78 and MIF levels were done in 65 patients with acute dengue infection. Clinical features and laboratory investigations were undertaken during the febrile and critical phase.ResultsWe found that serum IL-10 levels were significantly higher (p = 0.001) in patients with SD, when compared to those with non SD. Serum IL-10 levels significantly and inversely correlated with white cell counts (R = −0.23, p = 0.0002) and lymphocyte counts (R = −0.29, p < 0.0001) but significantly and positively correlated with aspartate tranaminase levels (R = 0.16, p = 0.01) and alanine transaminase levels (R = 0.22, p = 0.007). However, IL-10 levels did not have a good predictive value in discriminating those who were likely to develop SD (AUC = 0.66). Serum IFNγ levels were also significantly higher (p = 0.04) in patients with SD when compared to non SD. There was no difference (p = 0.34) in serum IFNα levels and serum MIF levels (p = 0.15) in patients with SD and non SD.ConclusionAlthough serum IL-10 was significantly elevated in patients with SD it had a poor discriminatory value in identifying those with SD and non SD and therefore, is unsuitable to be used as a robust biomarker in this cohort.

Viral Load, Clinical Disease Severity and Cellular Immune Responses in Primary Varicella Zoster Virus Infection in Sri Lanka

Background In Sri Lanka, varicella zoster virus (VZV) is typically acquired during adulthood with significant associated disease morbidity and mortality. T cells are believed to be important in the control of VZV replication and in the prevention of reactivation. The relationship between viral load, disease severity and cellular immune responses in primary VZV infection has not been well studied. Methodology We used IFNγ ELISpot assays and MHC class II tetramers based on VZV gE and IE63 epitopes, together with quantitative real time PCR assays to compare the frequency and phenotype of specific T cells with virological and clinical outcomes in 34 adult Sri Lankan individuals with primary VZV infection. Principal Findings Viral loads were found to be significantly higher in patients with moderate to severe infection compared to those with mild infection (p<0.001) and were significantly higher in those over 25 years of age (P<0.01). A significant inverse correlation was seen between the viral loads and the ex vivo IFNγ ELISpot responses of patients (P<0.001, r = −0.85). VZV-specific CD4+ T cells expressed markers of intermediate differentiation and activation. Conclusions Overall, these data show that increased clinical severity in Sri Lankan adults with primary VZV infection associates with higher viral load and reduced viral specific T cell responses.

Patterns of disease in Sri Lankan dengue patients

Background: Dengue is the most important mosquito borne viral infection in the world. Nearly 90% of infections occur in children. At present, prospective information on clinical and laboratory findings in South Asian children with dengue is generally lacking. Aim: To describe patterns of clinical disease in a cohort of children hospitalised with dengue during a major dengue epidemic in Sri Lanka. Results: A total of 104 children were studied during a three month period. Eighteen had dengue fever (DF) and 86 had dengue haemorrhagic fever (DHF). Of those with DHF, 34, 23, 27, and 2 had DHF grade I, II, III, and IV respectively. Based on dengue serology testing, 13 of the DF patients had a primary infection and 5 had secondary dengue infections. In contrast, 68 of the children with DHF had secondary and 18 had primary dengue infections. Oral candidiasis was seen in 19 children. The odds ratio for children with secondary dengue infection to develop DHF was 9.8 (95% CI 3.1 to 31.2). Conclusion: Studies on patterns of paediatric dengue disease in different regions should help clinicians and health administrators make more informed and evidence based health planning decisions. It should also help towards mapping out dengue trends on a global scale. Oral candidiasis has not been previously documented in children suffering with acute dengue in Sri Lanka or elsewhere. Studying underlying reasons for this manifestation during future dengue epidemics may provide useful leads in understanding overall dengue pathogenesis.

Seroprevalence of varicella zoster virus infections in Colombo District, Sri Lanka

BACKGROUND Although Varicella Zoster virus (VZV) infections occur worldwide, the epidemiology is remarkably different in tropical and temperate climates. VZV infections result in significant morbidity and mortality among adults in Sri Lanka. AIMS For future VZV vaccination strategies, we set to determine the age-specific seroprevalence rate of VZV infections in Colombo, Sri Lanka. MATERIALS AND METHODS The study was carried out from 1999 to 2000. Multi-stage cluster sampling technique was used to collect 913 blood samples, which were tested for the presence of VZV-specific IgG antibodies. RESULTS VZV seroprevalence rates were markedly lower in all age groups when compared to temperate climates. The seroprevalence rates increased with age in both the rural and urban populations. Of those aged 60 years, only 50% in the rural population and 78.9% in the urban population were immune to VZV. Seroprevalence rates of VZV infections were significantly different between the urban and rural populations (P< 0.001), with VZV-specific IgG antibodies detected in 47.5% in the urban population and 27.9% in the rural population. It was found that 56.2% (131) of females of childbearing age were nonimmune to VZV. CONCLUSIONS These findings highlight the need for a VZV vaccination program, which is likely to have a huge impact on the incidence of chickenpox and its associated morbidity and mortality.

Persistent High Frequencies of Varicella-Zoster Virus ORF4 Protein-Specific CD4+ T Cells after Primary Infection

ABSTRACT Open reading frame 4 (ORF4) of varicella-zoster virus (VZV) encodes an immediate-early protein that is believed to be important for viral infectivity and establishing latency. Evidence suggests that VZV-specific T cells are crucial in the control of viral replication, but there are no data addressing the existence of potential ORF4 protein-specific CD4+ T cells. We tested the hypothesis that VZV ORF4 protein-specific CD4+ T cells could be identified and characterized within the peripheral blood of healthy immune donors following primary infection. Gamma interferon (IFN-γ) immunosorbent assays were used to screen peripheral blood mononuclear cells obtained from healthy seropositive donors for responses to overlapping ORF4 peptides, viral lysate, and live vaccine. High frequencies of ORF4 protein-specific T cells were detected ex vivo in individuals up to 52 years after primary infection. Several immunogenic regions of the ORF4 protein were identified, including a commonly recognized epitope which was restricted through HLA-DRB1*07. Total ORF4 protein-specific responses comprised 19.7% and 20.7% of the total lysate and vaccine responses, respectively, and were dominated by CD4+ T cells. Indeed, CD4+ T cells were found to dominate the overall virus-specific IFN-γ cellular immune response both ex vivo and after expansion in vitro. In summary, we have identified an ORF4 protein as a novel target antigen for persistent VZV-specific CD4+ T cells, with implications for disease pathogenesis and future vaccine development.

HLA Class I and Class II Associations in Dengue Viral Infections in a Sri Lankan Population

Background HLA class I and class II alleles have been shown to be associated with the development of dengue hemorrhagic fever (DHF)/dengue shock syndrome (DSS) in different populations. However, the majority of studies have been based on limited numbers of patients. In this study we aimed to investigate the HLA-class I and class II alleles that are positively and negatively associated with the development of DSS in a cohort of patients with DHF and also the alleles associated with development of DHF during primary dengue infections in a Sri Lankan population. Methodology/Principal Findings The allele frequencies of HLA class I and class II alleles were compared in 110 patients with DHF and 119 individuals from the population who had never reported a symptomatic dengue infection at the time of recruitment. We found that HLA-A*31 (corrected P = 0.01) and DRB1*08 (corrected P = 0.009) were associated with susceptibility to DSS when infected with the dengue virus, during secondary dengue infection. The frequency of DRB1*08 allele was 28.7 times higher than in the normal population in patients with DSS. HLA-A*31 allele was increased 16.6 fold in DHF who developed shock when compared to those who did not develop shock. A*24 (corrected P = 0.03) and DRB1*12 (corrected P = 0.041) were strongly associated with the development of DHF during primary dengue infection. Conclusions/Significance These data suggest that certain HLA alleles confer susceptibility/protection to severe dengue infections. As T cell epitope recognition depend on the HLA type of an individual, it would be now important to investigate how epitope specific T cells associate with primary and secondary dengue infections and in severe dengue infections.

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.