[Show abstract][Hide abstract] ABSTRACT: Dengue viral infection is a global health threat without vaccine or specific treatment. The clinical outcome varies from asymptomatic, mild dengue fever (DF) to severe dengue hemorrhagic fever (DHF). While adaptive immune responses were found to be detrimental in the dengue pathogenesis, the roles of earlier innate events remain largely uninvestigated. Invariant natural killer T (iNKT) cells represent innate-like T cells that could dictate subsequent adaptive response but their role in human dengue virus infection is not known. We hypothesized that iNKT cells play a role in human dengue infection.
[Show abstract][Hide abstract] ABSTRACT: Dengue is a rapidly spreading vector-borne disease estimated to infect 400 million people worldwide. To date, there are no licensed treatments or vaccines. The last few years have seen significant developments in dengue control strategies. In this review, we will address four key areas: vaccines, vector control, antivirals and immunotherapeutics.
The first generation of dengue vaccines is able to induce good serological responses in test individuals. However, the recent Sanofi-Pasteur trial in Thailand found that a good serological response did not correlate with clinical protection. This trial did not demonstrate an increase in cases of severe disease following immunization, suggesting that concerns over vaccine-related immune enhancement may have been overcome. The bacterium Wolbachia appears to control dengue proliferation in Aedes mosquitoes, and field studies are underway. A large number of antivirals are in early-stage development and may prove useful in epidemics. Monoclonal antibodies have been postulated to have a clinical role. Whether their clinical application is feasible has yet to be seen.
Marked improvements in our knowledge of dengue have been made over the recent years. Sadly, clinical application remains some years away.
Current Opinion in Infectious Diseases 10/2013; · 4.87 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Here we present an approach that advances the throughput of a genetic analysis of a positive-sense RNA virus by simplifying virus construction. It enabled comprehensive dissection of a complex, multi-gene phenotype through rapid derivation of a large number of chimeric viruses and construction of a mutant library directly from a virus pool. The versatility of the approach described here expands the applicability of diverse genetic approaches to study these viruses.
[Show abstract][Hide abstract] ABSTRACT: The envelope (E) protein of dengue virus (DENV) is the major target of neutralizing antibodies (Abs) and vaccine development. Previous studies of human dengue-immune sera reported a significant proportion of anti-E Abs were cross-reactive to all four DENV serotypes and to one or more other flaviviruses, known as group-reactive (GR). Based on the studies of mouse anti-E monoclonal antibodies (mAbs), GR mAbs were non- or weakly neutralizing compared with type-specific mAbs; GR response was thus not regarded as important for vaccine strategy. We investigated the epitopes, binding avidity and neutralization potency of 32 human GR anti-E mAbs. In addition to fusion loop (FL) residues in E protein domain II, human GR mAbs recognized an epitope involving both FL and bc loop residues in domain II. The neutralization potency and binding avidity of GR mAbs derived from secondary DENV infection were stronger than those derived from primary infection. GR mAbs derived from primary DENV infection primarily block attachment, whereas those derived from secondary infection block DENV at post-attachment. Analysis of repertoire of anti-E mAbs dereived from patients with primary DENV infection revealed that the majority were GR, low avidity and weakly neutralizing, whereas those from secondary infection were primarily GR, high avidity and potent neutralizing. Our findings suggest the weakly neutralizing GR anti-E Abs generated from primary DENV infection become potent neutralizing against four serotypes after secondary infection. The observations that the dengue immune status of host affects the quality of cross-reactive Abs generated have implications for new strategies of DENV vaccine.
[Show abstract][Hide abstract] ABSTRACT: Dengue disease is a mosquito-borne infection caused by Dengue virus. Infection may be asymptomatic or variably manifest as mild Dengue fever (DF) to the most severe form, Dengue hemorrhagic fever (DHF). Mechanisms that influence disease severity are not understood. Complement, an integral component of the immune system, is activated during Dengue infection and the degree of activation increases with disease severity. Activation of the complement alternative pathway is influenced by polymorphisms within activation (factor B rs12614/rs641153, C3 rs2230199) and regulatory (complement factor H [CFH] rs800292) proteins, collectively termed a complotype. Here, we tested the hypothesis that the complotype influences disease severity during secondary Dengue infection. In addition to the complotype, we also assessed two other disease-associated CFH polymorphisms (rs1061170, rs3753394) and a structural polymorphism within the CFH protein family. We did not detect any significant association between the examined polymorphisms and Dengue infection severity in the Thai population. However, the minor allele frequencies of the factor B and C3 polymorphisms were less than 10% so our study was not sufficiently powered to detect an association at these loci. We were also unable to detect a direct interaction between CFH and Dengue NS1 using both recombinant NS1 and DV2-infected culture supernatants. We conclude that the complotype does not influence secondary Dengue infection severity in the Thai population.
[Show abstract][Hide abstract] ABSTRACT: PURPOSE OF REVIEW: Dengue is one of the most rapidly spreading vector-borne diseases in the world, with the incidence increasing 30-fold in the past 50 years. There are currently no licensed treatments or vaccines for dengue. This review covers the recent advances in our understanding of dengue pathogenesis, including host and viral determinants. RECENT FINDINGS: The pathogenesis of severe dengue is thought to be immune-mediated due to the timing of the clinical manifestations and higher incidence in secondary infections with a heterologous serotype. Recent evidence has provided further information of neutralizing versus enhancing monoclonal antibodies and their target epitopes on the dengue virion, which has major implications for vaccine design. The role of T-cell immunopathology has also been advanced with recent evidence of cross-reactive high pro-inflammatory cytokine producing T cells predominating in severe dengue. Recent large genome-wide association studies have identified specific susceptibility loci associated with severe disease. Epidemiological studies have served to define certain at-risk groups and specific viral virulence factors have recently been described. SUMMARY: The pathogenesis of dengue is likely to be a complex interplay of host immunity and genetic predisposition combined with certain viral virulence factors. Better understanding of the underlying mechanisms leading to severe dengue is crucial if we are to develop prognostic markers, novel diagnostics and therapeutics and ultimately a balanced and safe vaccine.
Current Opinion in Infectious Diseases 02/2013; · 4.87 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Dengue is a flavivirus which is endemic throughout tropical and sub-tropical regions across the globe (Figure 1) (Pinheiro and Corber, 1997). Case reports describe dengue-like illnesses dating back to the late 18th century (Rush, 1789). It now causes disease predominantly in children and adolescents who live in the tropics, and in travellers (Gubler, 1997; Wilder-Smith and Schwartz, 2005).
British journal of hospital medicine (London, England: 2005) 04/2012; 73(4):C60-4. · 0.25 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Dengue virus infections are still increasing at an alarming rate in tropical and subtropical countries, underlying the need for a dengue vaccine. Although it is relatively easy to generate Ab responses to dengue virus, low avidity or low concentrations of Ab may enhance infection of FcR-bearing cells with clinical impact, posing a challenge to vaccine production. In this article, we report the characterization of a mAb, 2H12, which is cross-reactive to all four serotypes in the dengue virus group. Crystal structures of 2H12-Fab in complex with domain III of the envelope protein from three dengue serotypes have been determined. 2H12 binds to the highly conserved AB loop of domain III of the envelope protein that is poorly accessible in the mature virion. 2H12 neutralization varied between dengue serotypes and strains; in particular, dengue serotype 2 was not neutralized. Because the 2H12-binding epitope was conserved, this variation in neutralization highlights differences between dengue serotypes and suggests that significant conformational changes in the virus must take place for Ab binding. Surprisingly, 2H12 facilitated little or no enhancement of infection. These data provide a structural basis for understanding Ab neutralization and enhancement of infection, which is crucial for the development of future dengue vaccines.
The Journal of Immunology 04/2012; 188(10):4971-9. · 5.52 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The human C-type lectin-like molecule CLEC5A is a critical macrophage receptor for dengue virus. The binding of dengue virus
to CLEC5A triggers signaling through the associated adapter molecule DAP12, stimulating proinflammatory cytokine release.
We have crystallized an informative ensemble of CLEC5A structural conformers at 1.9-Å resolution and demonstrate how an on-off
extension to a β-sheet acts as a binary switch regulating the flexibility of the molecule. This structural information together
with molecular dynamics simulations suggests a mechanism whereby extracellular events may be transmitted through the membrane
and influence DAP12 signaling. We demonstrate that CLEC5A is homodimeric at the cell surface and binds to dengue virus serotypes
1–4. We used blotting experiments, surface analyses, glycan microarray, and docking studies to investigate the ligand binding
potential of CLEC5A with particular respect to dengue virus. This study provides a rational foundation for understanding the
dengue virus-macrophage interaction and the role of CLEC5A in dengue virus-induced lethal disease.
Journal of Biological Chemistry 07/2011; 286(27):24208-24218. · 4.65 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Dengue virus receptors are relatively poorly characterized, but there has been recent interest in 2 C-type lectin molecules, dendritic cell-specific intercellular adhesion molecule 3 (ICAM-3)-grabbing nonintegrin (DC-SIGN) and its close homologue liver/lymph node-specific ICAM-3-grabbing integrin (L-SIGN), which can both bind dengue and promote infection. In this report we have studied the interaction of dengue viruses produced in insect cells, tumor cell lines, and primary human dendritic cells (DCs) with DC-SIGN and L-SIGN. Virus produced in primary DCs is unable to interact with DC-SIGN but remains infectious for L-SIGN-expressing cells. Skin-resident DCs may thus be a site of initial infection by insect-produced virus, but DCs will likely not participate in large-scale virus replication during dengue infection. These results reveal that differential glycosylation of dengue virus envelope protein is highly dependent on cell state and suggest that studies of virus tropism using virus prepared in insect cells or tumor cell lines should be interpreted with caution.
The Journal of Infectious Diseases 06/2011; 203(12):1775-83. · 5.85 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The human C-type lectin-like molecule CLEC5A is a critical macrophage receptor for dengue virus. The binding of dengue virus to CLEC5A triggers signaling through the associated adapter molecule DAP12, stimulating proinflammatory cytokine release. We have crystallized an informative ensemble of CLEC5A structural conformers at 1.9-Å resolution and demonstrate how an on-off extension to a β-sheet acts as a binary switch regulating the flexibility of the molecule. This structural information together with molecular dynamics simulations suggests a mechanism whereby extracellular events may be transmitted through the membrane and influence DAP12 signaling. We demonstrate that CLEC5A is homodimeric at the cell surface and binds to dengue virus serotypes 1-4. We used blotting experiments, surface analyses, glycan microarray, and docking studies to investigate the ligand binding potential of CLEC5A with particular respect to dengue virus. This study provides a rational foundation for understanding the dengue virus-macrophage interaction and the role of CLEC5A in dengue virus-induced lethal disease.
Journal of Biological Chemistry 05/2011; 286(27):24208-18. · 4.65 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The evolution of dengue viruses has resulted in four antigenically similar yet distinct serotypes. Infection with one serotype likely elicits lifelong immunity to that serotype, but generally not against the other three. Secondary or sequential infections are common, as multiple viral serotypes frequently cocirculate. Dengue infection, although frequently mild, can lead to dengue hemorrhagic fever (DHF) which can be life threatening. DHF is more common in secondary dengue infections, implying a role for the adaptive immune response in the disease. There is currently much effort toward the design and implementation of a dengue vaccine but these efforts are made more difficult by the challenge of inducing durable neutralizing immunity to all four viruses. Domain 3 of the dengue virus envelope protein (ED3) has been suggested as one such candidate because it contains neutralizing epitopes and it was originally thought that relatively few cross-reactive antibodies are directed to this domain. In this study, we performed a detailed analysis of the anti-ED3 response in a cohort of patients suffering either primary or secondary dengue infections. The results show dramatic evidence of original antigenic sin in secondary infections both in terms of binding and enhancement activity. This has important implications for dengue vaccine design because heterologous boosting is likely to maintain the immunological footprint of the first vaccination. On the basis of these findings, we propose a simple in vitro enzyme-linked immunosorbent assay (ELISA) to diagnose the original dengue infection in secondary dengue cases.
Journal of Virology 10/2010; 85(1):410-21. · 5.08 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Dengue infections are increasing at an alarming rate in many tropical and subtropical countries, where epidemics can put health care systems under extreme pressure. The more severe infections lead to dengue hemorrhagic fever (DHF), which can be life threatening. A variety of viral and host factors have been associated with the severity of dengue infections. Because secondary dengue infection is more commonly associated with DHF than primary infections, the acquired immune response to dengue, both B cells and T cells have been implicated. In this study, we set out to study T-cell responses across the entire dengue virus proteome and to see whether these were related to disease severity in a cohort of dengue-infected children from Thailand. Robust responses were observed in most infected individuals against most viral proteins. Responses to NS3 were the most frequent, and there was a very strong association between the magnitude of the response and disease severity. Furthermore, in DHF, cytokine-high CD107a-negative cells predominated.
Proceedings of the National Academy of Sciences 09/2010; 107(39):16922-7. · 9.81 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Dengue virus co-circulates as four serotypes, and sequential infections with more than one serotype are common. One hypothesis for the increased severity seen in secondary infections is antibody-dependent enhancement (ADE) leading to increased replication in Fc receptor-bearing cells. In this study, we have generated a panel of human monoclonal antibodies to dengue virus. Antibodies to the structural precursor-membrane protein (prM) form a major component of the response. These antibodies are highly cross-reactive among the dengue virus serotypes and, even at high concentrations, do not neutralize infection but potently promote ADE. We propose that the partial cleavage of prM from the viral surface reduces the density of antigen available for viral neutralization, leaving dengue viruses susceptible to ADE by antibody to prM, a finding that has implications for future vaccine design.
[Show abstract][Hide abstract] ABSTRACT: Severe dengue virus (DV) infections can cause the life-threatening condition dengue hemorrhagic fever, which is characterized by a severe plasma leak, thrombocytopenia, hemorrhage, and, in severe cases, circulatory collapse and death. There is now much evidence that pre-existing immunity to DV can enhance disease when an individual becomes infected on a second or sequential occasion. It has been shown that in contrast to infected dendritic cells (DC), noninfected bystander DC underwent maturation in dengue infection. In this study, we show that TNF-alpha and type I IFN contribute to the maturation of bystander DC, whereas the inhibition of DV-infected DC maturation can be overcome by activated T cells. Furthermore, IFN-gamma-inducible chemokines, CXCL9, 10, and 11 produced by infected DC are greatly amplified in the presence of DV-specific T cells. The chemokine secretion is also enhanced in coculture of HUVEC with either DV-infected DC or activated T cells. Finally, we found a close correlation between the serum level of these three chemokines and disease severity.
The Journal of Immunology 12/2008; 181(9):5865-74. · 5.52 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Effective vaccines should confer long-term protection against future outbreaks of severe acute respiratory syndrome (SARS) caused by a novel zoonotic coronavirus (SARS-CoV) with unknown animal reservoirs. We conducted a cohort study examining multiple parameters of immune responses to SARS-CoV infection, aiming to identify the immune correlates of protection. We used a matrix of overlapping peptides spanning whole SARS-CoV proteome to determine T cell responses from 128 SARS convalescent samples by ex vivo IFN-gamma ELISPOT assays. Approximately 50% of convalescent SARS patients were positive for T cell responses, and 90% possessed strongly neutralizing Abs. Fifty-five novel T cell epitopes were identified, with spike protein dominating total T cell responses. CD8(+) T cell responses were more frequent and of a greater magnitude than CD4(+) T cell responses (p < 0.001). Polychromatic cytometry analysis indicated that the virus-specific T cells from the severe group tended to be a central memory phenotype (CD27(+)/CD45RO(+)) with a significantly higher frequency of polyfunctional CD4(+) T cells producing IFN-gamma, TNF-alpha, and IL-2, and CD8(+) T cells producing IFN-gamma, TNF-alpha, and CD107a (degranulation), as compared with the mild-moderate group. Strong T cell responses correlated significantly (p < 0.05) with higher neutralizing Ab. The serum cytokine profile during acute infection indicated a significant elevation of innate immune responses. Increased Th2 cytokines were observed in patients with fatal infection. Our study provides a roadmap for the immunogenicity of SARS-CoV and types of immune responses that may be responsible for the virus clearance, and should serve as a benchmark for SARS-CoV vaccine design and evaluation.
The Journal of Immunology 10/2008; 181(8):5490-500. · 5.52 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: T-cell recognition of the antigenic peptides presented by MHC class I molecules normally triggers protective immune responses, but can result in immune enhancement of disease. Cross-reactive T-cell responses may underlie immunopathology in dengue haemorrhagic fever. To analyze these effects at the molecular level, the functional MHC class I molecule HLA-A*1101 was crystallized bound to six naturally occurring peptide variants from the dengue virus NS3 protein. The crystals contained high levels of solvent and required optimization of the cryoprotectant and dehydration protocols for each complex to yield well ordered diffraction, a process that was facilitated by the use of a free-mounting system.
Acta Crystallographica Section F Structural Biology and Crystallization Communications 06/2007; 63(Pt 5):386-92. · 0.55 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Vascular leakage and shock are the major causes of death in patients with dengue hemorrhagic fever (DHF) and dengue shock syndrome (DSS). Thirty years ago, complement activation was proposed to be a key underlying event, but the cause of complement activation has remained unknown.
The major nonstructural dengue virus (DV) protein NS1 was tested for its capacity to activate human complement in its membrane-associated and soluble forms. Plasma samples from 163 patients with DV infection and from 19 patients with other febrile illnesses were prospectively analyzed for viral load and for levels of NS1 and complement-activation products. Blood and pleural fluids from 9 patients with DSS were also analyzed.
Soluble NS1 activated complement to completion, and activation was enhanced by polyclonal and monoclonal antibodies against NS1. Complement was also activated by cell-associated NS1 in the presence of specific antibodies. Plasma levels of NS1 and terminal SC5b-9 complexes correlated with disease severity. Large amounts of NS1, complement anaphylatoxin C5a, and the terminal complement complex SC5b-9 were present in pleural fluids from patients with DSS.
Complement activation mediated by NS1 leads to local and systemic generation of anaphylatoxins and SC5b-9, which may contribute to the pathogenesis of the vascular leakage that occurs in patients with DHF/DSS.
The Journal of Infectious Diseases 05/2006; 193(8):1078-88. · 5.85 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Dengue virus infection poses a growing public health and economic burden in a number of tropical and subtropical countries. Dengue circulates as a number of quasispecies, which can be divided by serology into four groups or serotypes. An interesting feature of Dengue, recognized over five decades ago, is that most severe cases that show hemorrhagic fever are not suffering from a primary infection. Instead, they are reinfected with a virus of different serotype. This observation poses considerable problems in vaccine design, and it is therefore imperative to gain a full understanding of the mechanisms underlying this immunological enhancement of disease. In this study, we examined a T cell epitope restricted by HLA-A*24, a major MHC class I allele, in Southeast Asia in a cohort of children admitted to a hospital with acute Dengue infection. The cytokine profiles and the degranulation capacity of T cells generated to this epitope are defined and compared across different viral serotypes. Cross-reactive Dengue-specific T cells seem to show suboptimal degranulation but high cytokine production, which may contribute to the development of the vascular leak characteristic of Dengue hemorrhagic fever.
The Journal of Immunology 04/2006; 176(6):3821-9. · 5.52 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The enhancement of severe disease upon secondary infection makes dengue almost unique among infectious pathogens and presents a serious challenge to vaccine design. Several key observations have been made which shed light onto this phenomenon particularly that antibodies can enhance Fc receptor-dependent uptake of virus into macrophages thereby increasing virus replication. Furthermore there seems to be a relationship between the peak virus load and disease severity. However, a second key feature of dengue is that the life-threatening symptoms do not correlate with the period of high viraemia; instead they occur at a time when the virus load is in steep decline. The coincidence of severe disease manifestations with defervescence and virus control suggests that the symptoms may be a consequence of the immune response to the virus rather than virus induced cytopathology. One of the key elements in the immune response to viruses are T cells which can both secrete a host of inflammatory cytokines and also be directly cytotoxic to infected cells. There are a number of experimental models of T cell-induced immunopathology including in responses to viruses. Particularly interesting in this respect are models of RSV-induced immunopathology, which have direct relevance to vaccine design as a formalin-inactivated vaccine to RSV actually enhanced disease in children when they became naturally infected with RSV, an echo of the disease enhancement seen in dengue. We will present an analysis of CD8+ T cell responses to a number of novel T cell epitopes during dengue infection and also analyse the function and cytokine secretion of these cells. We suggest that an exaggerated and partially misdirected T cell response seen in secondary dengue infection may be part of the complex series of events leading to dengue haemorrhagic fever and shock.
Novartis Foundation symposium 02/2006; 277:164-71; discussion 171-6, 251-3.