[Show abstract][Hide abstract] ABSTRACT: Dengue virus (DENV) is the leading cause of mosquito-borne viral illness and death in humans. Like many viruses, DENV has evolved potent mechanisms that abolish the antiviral response within infected cells. Nevertheless, several in vivo studies have demonstrated a key role of the innate immune response in controlling DENV infection and disease progression. Here, we report that sensing of DENV infected cells by plasmacytoid dendritic cells (pDCs) triggers a robust TLR7-dependent production of IFNα, concomitant with additional antiviral responses, including inflammatory cytokine secretion and pDC maturation. We demonstrate that unlike the efficient cell-free transmission of viral infectivity, pDC activation depends on cell-to-cell contact, a feature observed for various cell types and primary cells infected by DENV, as well as West Nile virus, another member of the Flavivirus genus. We show that the sensing of DENV infected cells by pDCs requires viral envelope protein-dependent secretion and transmission of viral RNA. Consistently with the cell-to-cell sensing-dependent pDC activation, we found that DENV structural components are clustered at the interface between pDCs and infected cells. The actin cytoskeleton is pivotal for both this clustering at the contacts and pDC activation, suggesting that this structural network likely contributes to the transmission of viral components to the pDCs. Due to an evolutionarily conserved suboptimal cleavage of the precursor membrane protein (prM), DENV infected cells release uncleaved prM containing-immature particles, which are deficient for membrane fusion function. We demonstrate that cells releasing immature particles trigger pDC IFN response more potently than cells producing fusion-competent mature virus. Altogether, our results imply that immature particles, as a carrier to endolysosome-localized TLR7 sensor, may contribute to regulate the progression of dengue disease by eliciting a strong innate response.
[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 one of the most important emerging viral diseases globally. The majority of symptomatic infections result in a relatively benign disease course. However, a small proportion of patients develop severe clinical manifestations, including bleeding, organ impairment, and endothelial dysfunction with increased capillary permeability causing hypovolaemic shock that can lead to cardiovascular collapse. Evidence is increasing that dengue can also cause myocardial impairment, arrhythmias and, occasionally, fulminant myocarditis. No antiviral agents or vaccines are licensed for dengue, and treatment remains supportive with judicious fluid replacement for patients with severe disease. Defining the role of cardiac dysfunction in the haemodynamic compromise of severe dengue has potentially important management implications. In this Review, we will outline the current understanding of the cardiovascular manifestations of dengue, including myocardial and vascular involvement, and conclude with a discussion of the available therapeutic options and potential future research directions.
[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; · 5.03 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; · 5.03 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Humans express four MHC-like CD1 molecules CD1a, b, c and d that are capable of presenting a wide variety of self or foreign lipid antigens to T cells. Much progress has been made in elucidating the function of CD1d-restricted NKT cells in both innate and adaptive immune responses. However, knowledge of the other CD1 molecules is less well defined in terms of lipid presentation and immune regulation. We have previously shown that immunoglobulin-like transcript 4 (ILT4) binds to CD1d and inhibits its recognition by NKT cells. In this study, we show that CD1c can also interact specifically with ILT4 with a higher affinity than that of CD1d. Furthermore, changes in CD1c expression seem to modulate CD1d function; up-regulation of CD1c enhances NKT recognition of CD1d and down-regulation reduces CD1d recognition. We propose that CD1c can act as a sink for the inhibitory receptor ILT4: when CD1c is up-regulated, ILT4 is recruited to CD1c, thus reducing the inhibitory effect of ILT4 on CD1d recognition. Consequently, CD1c could be a potential target for modulating NKT activity.Keywords: NKT, CD1d, CD1c, ILT4, antigen presentation.
International Immunology 08/2012; 24(11):729-37. · 3.14 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: It has been widely demonstrated that a quantitative and/or qualitative impairment of regulatory T cells (T(regs)) play a fundamental role in the initiation and persistence of rheumatoid arthritis (RA) in animal models and in patients. In the present work it is demonstrated that partial myeloablation induces a relative expansion of T(regs) that is sufficient to mediate immunological tolerance.
(1) To test the ability of low-intensity myeloablation mediated T(reg) activation to prevent and/or to treat experimental arthritis using the collagen-induced arthritis (CIA) model and (2) to clarify the role of T(reg) in mediating the beneficial effect.
Low-dose irradiation was used before the induction of arthritis or at the onset of disease. The role of T(regs) (CD4CD25forkhead box P3 (FoxP3)(+) cells) and their suppressive activity was assessed by testing their functional activities ex vivo after the treatment and by their in vivo depletion before the treatment.
It was observed that irradiation ameliorated CIA before or after disease induction. T(regs) appear to play a fundamental role in the therapeutic efficacy of irradiation, because the depletion of CD25 or folate receptor (FR)4(+) cells with specific antibodies before the treatment abolished the beneficial effects. The therapeutic efficacy was associated with an increment in the proportion of T(regs) despite the overall reduction in lymphocyte counts. Furthermore, a decline in the percentage of CD4CD25FoxP3(+) T(regs) was associated with disease flare.
In vivo T(reg) expansion is a feasible and effective approach in the treatment of autoimmune diseases.
Annals of the rheumatic diseases 05/2010; 69(8):1519-26. · 8.11 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: The genome of the severe acute respiratory syndrome-associated coronavirus (SARS-CoV) contains eight open reading frames (ORFs) that encode novel proteins. These accessory proteins are dispensable for in vitro and in vivo replication and thus may be important for other aspects of virus-host interactions. We investigated the functions of the largest of the accessory proteins, the ORF 3a protein, using a 3a-deficient strain of SARS-CoV. Cell death of Vero cells after infection with SARS-CoV was reduced upon deletion of ORF 3a. Electron microscopy of infected cells revealed a role for ORF 3a in SARS-CoV induced vesicle formation, a prominent feature of cells from SARS patients. In addition, we report that ORF 3a is both necessary and sufficient for SARS-CoV-induced Golgi fragmentation and that the 3a protein accumulates and localizes to vesicles containing markers for late endosomes. Finally, overexpression of ADP-ribosylation factor 1 (Arf1), a small GTPase essential for the maintenance of the Golgi apparatus, restored Golgi morphology during infection. These results establish an important role for ORF 3a in SARS-CoV-induced cell death, Golgi fragmentation, and the accumulation of intracellular vesicles.
Journal of Virology 11/2009; 84(2):1097-109. · 5.08 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Abnormal glycosylation is one of the hallmarks of the cancer cell and is associated with tumor invasion and metastasis. The development of tumor-associated carbohydrate antigen (TACA) vaccines has been problematic due to poor immunogenicity. However, when appropriate targets can be identified, passive immunization with monoclonal antibodies (mAbs) directed against TACAs has been shown to have antitumor activity. Fas ligand (FasL) is a transmembrane protein that induces apoptosis in cells expressing its receptor, Fas. When grafted into mice, FasL-expressing tumor cells break immunologic tolerance to self-antigens and induce antibody-mediated tumor immunity. Here, five IgM mAbs were produced from mice vaccinated with FasL-expressing B16F10 mouse melanoma cells. They recognize various syngeneic and allogeneic murine tumor cell lines. One mAb, TM10, recognizes a range of human tumor cell lines, including melanoma, prostate, and ovarian cancer. It does not bind to untransformed cells. The epitopes recognized by all the mAbs were carbohydrates expressed on proteins. Using carbohydrate microarrays, the antigenic targets of TM10 were found to be high-mannose core structures of N-linked glycans. In normal cells, high-mannose clusters are hidden by extensive saccharide branching but they become exposed in cancer cells as a result of abnormal glycosylation pathways. Vaccination with FasL-expressing tumors therefore enables the immune system to break tolerance to self-antigens, allowing identification of novel TACAs that can form the basis of future humoral anticancer therapy.
Cancer Research 03/2009; 69(5):2018-25. · 9.28 Impact Factor