Secreted complement regulatory protein clusterin interacts with dengue virus nonstructural protein 1

Vascular leakage and shock are the major causes of death in patients with dengue hemorrhagic fever (DHF) and dengue shock syndrome (DSS). It has been suggested that patients with an elevated level of the free soluble form of dengue virus (DV) nonstructural protein 1 (sNS1) are at risk of developing DHF. To understand the role of sNS1 in blood, we searched for the host molecule with which NS1 interacts in human plasma by affinity purification using a GST-fused NS1. Complement inhibitory factor clusterin (Clu), which naturally inhibits the formation of terminal complement complex (TCC), was identified by mass spectrometry. A recombinant sNS1 produced from 293T cells and sNS1 from DV-infected Vero cells interacted with human Clu. Since an activated complement system reportedly causes vascular leakage, the interaction between sNS1 and Clu may contribute to the progression of DHF.     

T cell responses to an HLA-B*07-restricted epitope on the dengue NS3 protein correlate with disease severity

Dengue hemorrhagic fever (DHF), the severe manifestation of dengue virus (DV) infection characterized by plasma leakage, is more common in secondary DV infections in previously infected individuals and is associated with high levels of immune activation. To determine the Ag specificity of this immune response, we studied the response to an HLA-B*07-restricted T cell epitope, residues 221-232 of the DV NS3 protein, in 10 HLA-B*07(+) Thai children who were studied during and after acute DV infections. Peptide-specific T cells were detected in 9 of 10 subjects. The frequency of peptide-specific T cells was higher in subjects who had experienced DHF than in those who had experienced DF. We also detected peptide-specific T cells in PBMC obtained at the time of the acute DV infection in 2 of 5 subjects. These data suggest that the NS3 (221-232) epitope is an important target of CD8(+) T cells in secondary DV infection and that the activation and expansion of DV-specific T cells is greater in subjects with DHF than in those with dengue fever. These findings support the hypothesis that activation of DV-specific CD8(+) T cells plays an important role in the pathogenesis of DHF.     

Proteomic analysis of endothelial cell autoantigens recognized by anti-dengue virus nonstructural protein 1 antibodies

We previously showed the occurrence of autoimmune responses in dengue virus (DV) infection, which has potential implications for the pathogenesis of dengue hemorrhagic syndrome. In the present study, we have used a proteomic analysis to identify several candidate proteins on HMEC-1 endothelial cells recognized by anti-DV nonstructural protein 1 (NS1) antibodies. The target proteins, including ATP synthase beta chain, protein disulfide isomerase, vimentin, and heat shock protein 60, co-localize with anti-NS1 binding sites on nonfixed HMEC-1 cells using immunohistochemical double staining and confocal microscopy. The cross-reactivity of anti-target protein antibodies with HMEC-1 cells was inhibited by NS1 protein pre-absorption. Furthermore, a cross-reactive epitope on NS1 amino acid residues 311-330 (P311-330) was predicted using homologous sequence alignment. The reactivity of dengue hemorrhagic patient sera with HMEC-1 cells was blocked by synthetic peptide P311-330 pre-absorption. Taken together, our results identify putative targets on endothelial cells recognized by anti-DV NS1 antibodies, where NS1 P311-330 possesses the shared epitope.     

Immunopathogenesis of dengue virus infection

Dengue virus infection causes dengue fever (DF), dengue hemorrhagic fever (DHF), and dengue shock syndrome (DSS), whose pathogeneses are not clearly understood. Current hypotheses of antibody-dependent enhancement, virus virulence, and IFN-gamma/TNFalpha-mediated immunopathogenesis are insufficient to explain clinical manifestations of DHF/DSS such as thrombocytopenia and hemoconcentration. Dengue virus infection induces transient immune aberrant activation of CD4/CD8 ratio inversion and cytokine overproduction, and infection of endothelial cells and hepatocytes causes apoptosis and dysfunction of these cells. The coagulation and fibrinolysis systems are also activated after dengue virus infection. We propose a new hypothesis for the immunopathogenesis for dengue virus infection. The aberrant immune responses not only impair the immune response to clear the virus, but also result in overproduction of cytokines that affect monocytes, endothelial cells, and hepatocytes. Platelets are destroyed by crossreactive anti-platelet autoantibodies. Dengue-virus-induced vasculopathy and coagulopathy must be involved in the pathogenesis of hemorrhage, and the unbalance between coagulation and fibrinolysis activation increases the likelihood of severe hemorrhage in DHF/DSS. Hemostasis is maintained unless the dysregulation of coagulation and fibrinolysis persists. The overproduced IL-6 might play a crucial role in the enhanced production of anti-platelet or anti-endothelial cell autoantibodies, elevated levels of tPA, as well as a deficiency in coagulation. Capillary leakage is triggered by the dengue virus itself or by antibodies to its antigens. This immunopathogenesis of DHF/DSS can account for specific characteristics of clinical, pathologic, and epidemiological observations in dengue virus infection.     

Vascular endothelium: the battlefield of dengue viruses

Increased vascular permeability without morphological damage to the capillary endothelium is the cardinal feature of dengue haemorrhagic fever (DHF)/dengue shock syndrome (DSS). Extensive plasma leakage in various tissue spaces and serous cavities of the body, including the pleural, pericardial and peritoneal cavities in patients with DHF, may result in profound shock. Among various mechanisms that have been considered include immune complex disease, T-cell-mediated, antibodies cross-reacting with vascular endothelium, enhancing antibodies, complement and its products, various soluble mediators including cytokines, selection of virulent strains and virus virulence, but the most favoured are enhancing antibodies and memory T cells in a secondary infection resulting in cytokine tsunami. Whatever the mechanism, it ultimately targets vascular endothelium (making it a battlefield) leading to severe dengue disease. Extensive recent work has been done in vitro on endothelial cell monolayer models to understand the pathophysiology of vascular endothelium during dengue virus (DV) infection that may be translated to help understand the pathogenesis of DHF/DSS. The present review provides a broad overview of the effects of DV infection and the associated host responses contributing towards alterations in vascular endothelial cell physiology and damage that may be responsible for the DHF/DSS.     

Re-evaluation of the pathogenic roles of nonstructural protein 1 and its antibodies during dengue virus infection

Dengue virus (DENV) infection can cause life-threatening dengue hemorrhagic fever (DHF) and dengue shock syndrome (DSS). Vascular leakage and abnormal hemorrhage are the two major pathogenic changes found in these patients. From previous studies, it is known that both antibodies and cytokines induced in response to DENV infection are involved in the immunopathogenesis of DHF/DSS. However, the role of viral factors during DENV infection remains unclear. Nonstructural protein 1 (NS1), which is secreted in the sera of patients, is a useful diagnostic marker for acute DENV infection. Nevertheless, the roles of NS1 and its antibodies in the pathogenesis of DHF/DSS are unclear. The focus of this review is to evaluate the possible contributions of NS1 and the antibodies it induces to vascular leakage and abnormal hemorrhage during DENV infection, which may provide clues to better understanding the pathogenesis of DHF/DSS.     

Role of cognitive parameters in dengue hemorrhagic fever and dengue shock syndrome

Dengue is becoming recognized as one of the most important vector-borne human diseases. It is predominant in tropical and subtropical zones but its geographical distribution is progressively expanding, making it an escalating global health problem of today. Dengue presents with spectrum of clinical manifestations, ranging from asymptomatic, undifferentiated mild fever, dengue fever (DF), to dengue hemorrhagic fever (DHF) with or without shock (DSS), a life-threatening illness characterized by plasma leakage due to increased vascular permeability. Currently, there are no antiviral modalities or vaccines available to treat and prevent dengue. Supportive care with close monitoring is the standard clinical practice. The mechanisms leading to DHF/DSS remains poorly understood. Multiple factors have been attributed to the pathological mechanism, but only a couple of these hypotheses are popular in scientific circles. The current discussion focuses on underappreciated factors, temperature, natural IgM, and endotoxin, which may be critical components playing roles in dengue pathogenesis.     

DENV NS1 and MMP-9 cooperate to induce vascular leakage by altering endothelial cell adhesion and tight junction

Dengue virus (DENV) is a mosquito-borne pathogen that causes a spectrum of diseases including life-threatening dengue hemorrhagic fever (DHF) and dengue shock syndrome (DSS). Vascular leakage is a common clinical crisis in DHF/DSS patients and highly associated with increased endothelial permeability. The presence of vascular leakage causes hypotension, circulatory failure, and disseminated intravascular coagulation as the disease progresses of DHF/DSS patients, which can lead to the death of patients. However, the mechanisms by which DENV infection caused the vascular leakage are not fully understood. This study reveals a distinct mechanism by which DENV induces endothelial permeability and vascular leakage in human endothelial cells and mice tissues. We initially show that DENV2 promotes the matrix metalloproteinase-9 (MMP-9) expression and secretion in DHF patients’ sera, peripheral blood mononuclear cells (PBMCs), and macrophages. This study further reveals that DENV non-structural protein 1 (NS1) induces MMP-9 expression through activating the nuclear factor κB (NF-κB) signaling pathway. Additionally, NS1 facilitates the MMP-9 enzymatic activity, which alters the adhesion and tight junction and vascular leakage in human endothelial cells and mouse tissues. Moreover, NS1 recruits MMP-9 to interact with β-catenin and Zona occludens protein-1/2 (ZO-1 and ZO-2) and to degrade the important adhesion and tight junction proteins, thereby inducing endothelial hyperpermeability and vascular leakage in human endothelial cells and mouse tissues. Thus, we reveal that DENV NS1 and MMP-9 cooperatively induce vascular leakage by impairing endothelial cell adhesion and tight junction, and suggest that MMP-9 may serve as a potential target for the treatment of hypovolemia in DSS/DHF patients.     

Identification of B cell epitopes of dengue virus 2 NS3 protein by monoclonal antibody

Dengue virus is a major international public health concern, and there is a lack of available effective vaccines. Virus-specific epitopes could help in developing epitope peptide vaccine. Previously, a neutralizing monoclonal antibody (mAb) 4F5 against nonstructural protein 3 (NS3) of dengue virus 2 (DV2) was developed in our lab. In this work, the B cell epitope recognized by mAb 4F5 was identified using the phage-displayed peptide library. The results of the binding assay and competitive inhibition assay indicated that the peptides, residues 460–469 (U460-469 RVGRNPKNEN) of DV2 NS3 protein, were the B cell epitopes recognized by mAb 4F5. Furthermore, the epitope peptides and a control peptide were synthesized and then immunized female BALB/c mice. ELISA analysis showed that immunization with synthesized epitope peptide elicited a high level of antibody in mice, and immunofluorescent staining showed that the antisera from fusion epitope-immunized mice also responded to DV2 NS3 protein, which further characterized the specific response of the present epitope peptide. Therefore, the present work revealed the specificity of the newly identified epitope (U460-469) of DV2 NS3 protein, which may shed light on dengue virus (DV) vaccine design, DV pathogenesis study, and even DV diagnostic reagent development.     

Roles of Small GTPase Rac1 in the Regulation of Actin Cytoskeleton during Dengue Virus Infection

Background

Increased vascular permeability is a hallmark feature in severe dengue virus (DV) infection, and dysfunction of endothelial cells has been speculated to contribute in the pathogenesis of dengue hemorrhagic fever/dengue shock syndrome (DHF/DSS). Rho-family GTPase Rac1 is a significant element of endothelial barrier function regulation and has been implicated in the regulation of actin remodeling and intercellular junction formation. Yet there is little evidence linking Rac1 GTPase to alteration in endothelial cell function induced by DV infection.

Methods and Findings

Here, we showed that actin is essential for DV serotype 2 (DV2) entry into and release from ECV304 cells, and Rac1 signaling is involved these processes. At early infection, actin cytoskeleton rearranged significantly during 1 hour post infection, and disrupting actin filament dynamics with jasplakinolide or cytochalasin D reduced DV2 entry. DV2 entry induced reduction of Rac1 activity within 1 hour post infection. The expression of dominant-negative forms of Rac1 established that DV2 entry is negatively regulated by Rac1. At late infection, actin drugs also inhibited the DV2 release and induced accumulation of viral proteins in the cytoplasm. Meanwhile, the activity of Rac1 increased significantly with the progression of DV2 infection and was up-regulated in transfected cells expressing E protein. Confocal microscopy showed that DV2 E protein was closely associated with either actin or Rac1 in DV2-infected cells. The interaction between E protein and actin was further confirmed by co-immunoprecipitation assay.

Conclusions

These results defined roles for actin integrity in DV2 entry and release, and indicated evidence for the participation of Rac1 signaling pathways in DV2-induced actin reorganizations and E-actin interaction. Our results may provide further insight into the pathogenesis of DHF/DSS.     

Analysis of Murine CD8+ T-Cell Clones Specific for the Dengue Virus NS3 Protein: Flavivirus Cross-Reactivity and Influence of Infecting Serotype

Serotype-cross-reactive dengue virus-specific cytotoxic T lymphocytes (CTL) induced during a primary dengue virus infection are thought to play a role in the immunopathogenesis of dengue hemorrhagic fever (DHF) during a secondary dengue virus infection. Although there is no animal model of DHF, we previously reported that murine dengue virus-specific CTL responses are qualitatively similar to human dengue virus-specific CTL responses. We used BALB/c mice to study the specificity of the CTL response to an immunodominant epitope on the dengue virus NS3 protein. We mapped the minimal H-2K^d-restricted CTL epitope to residues 298 to 306 of the dengue type 2 virus NS3 protein. In short-term T-cell lines and clones, the predominant CD8⁺ CTL to this epitope in mice immunized with dengue type 2 virus or vaccinia virus expressing the dengue type 4 virus NS3 protein were cross-reactive with dengue type 2 or type 4 virus, while broadly serotype-cross-reactive CTL were a minority population. In dengue type 3 virus-immunized mice, the predominant CTL response to this epitope was broadly serotype cross-reactive. All of the dengue virus-specific CTL clones studied also recognized the homologous NS3 sequences of one or more closely related flaviviruses, such as Kunjin virus. The critical contact residues for the CTL clones with different specificities were mapped with peptides having single amino acid substitutions. These data demonstrate that primary dengue virus infection induces a complex population of flavivirus-cross-reactive NS3-specific CTL clones in mice and suggest that CTL responses are influenced by the viral serotype. These findings suggest an additional mechanism by which the order of sequential flavivirus infections may influence disease manifestations.     

Human Apolipoprotein A-I Is Associated with Dengue Virus and Enhances Virus Infection through SR-BI

Diseases caused by dengue virus (DV) infection vary in severity, with symptoms ranging from mild fever to life threatening dengue hemorrhage fever (DHF) and dengue shock syndrome (DSS). Clinical studies have shown that significant decrease in the level of lipoproteins is correlated with severe illness in DHF/DSS patients. Available evidence also indicates that lipoproteins including high-density lipoprotein (HDL) and low-density lipoprotein (LDL) are able to facilitate cell entry of HCV or other flaviviruses via corresponding lipoprotein receptors. In this study, we found that pre-incubation of DV with human serum leads to an enhanced DV infectivity in various types of cells. Such enhancement could be due to interactions between serum components and DV particles. Through co-immunoprecipitation we revealed that apolipoprotein A-I (ApoA-I), the major protein component in HDL, is associated with DV particles and is able to promote DV infection. Based on that observation, we further found that siRNA knockdown of the scavenger receptor class B type I (SR-BI), the cell receptor of ApoA-I, abolished the activity of ApoA-I in enhancement of DV infection. This suggests that ApoA-I bridges DV particles and cell receptor SR-BI and facilitates entry of DV into cells. FACS analysis of cell surface dengue antigen after virus absorption further confirmed that ApoA-I enhances DV infection via promoting initial attachment of the virus to cells. These findings illustrate a novel entry route of DV into cells, which may provide insights into the functional importance of lipoproteins in dengue pathogenesis.     

Antibodies against dengue virus nonstructural protein-1 induce heme oxygenase-1 via a redox-dependent pathway in human endothelial cells

Heme oxygenase (HO)-1, the inducible isoform of the first and rate-limiting enzyme of heme degradation, affords anti-inflammatory protection via its cell-type-specific effects in endothelial cells (ECs). In dengue hemorrhagic fever (DHF), which is the life-threatening form of dengue virus (DV) infection, endothelial interactions of cross-reactive antibodies against the DV nonstructural glycoprotein-1 (NS1) are associated with endothelial dysfunction. In this study, we investigated whether anti-NS1 antibodies might regulate HO-1 gene expression in human ECs. Serum from DHF patients with high anti-NS1 titers and a monoclonal anti-NS1 antibody upregulated HO-1 gene expression in human umbilical vein ECs, which was blocked by purified NS1 antigen. Immunoprecipitation studies showed that anti-NS1 antibodies specifically bound to the oxidoreductase protein disulfide isomerase (PDI) on ECs. Moreover, anti-NS1-mediated HO-1 induction was reduced by inhibition of PDI enzyme activity. Reactive oxygen species, which were generated by NADPH oxidase and in turn activated the phosphatidylinositol 3-kinase (PI3K)/Akt cascade, were involved in this upregulation of HO-1 gene expression. Finally, apoptosis of ECs caused by anti-NS1 antibodies was increased by pharmacological inhibition of HO-1 enzyme activity. In conclusion, HO-1 gene expression is upregulated by anti-NS1 antibodies via activation of a redox-dependent PDI/PI3K/Akt-mediated pathway in human ECs.     

Dengue Virus Infection Mediates HMGB1 Release from Monocytes Involving PCAF Acetylase Complex and Induces Vascular Leakage in Endothelial Cells

High mobility group box 1 (HMGB1) protein is released from cells as a pro-inflammatory cytokine in response to an injury or infection. During dengue hemorrhagic fever (DHF)/dengue shock syndrome (DSS), a number of pro-inflammatory cytokines are released, contributing to disease pathogenesis. In this study, the release of HMGB1 from human myelogenous leukemia cell line K562 and primary peripheral blood monocytes (PBM) cells was examined during dengue virus (DV)-infection. HMGB1 was shown to translocate from cell nuclei to the cytoplasm in both K562- and PBM-infected cells. The translocation of HMGB1 from the nucleus to the cytoplasm was shown to be mediated by the host cell p300/CBP-associated factor (PCAF) acetylase complex in K562 cells. In addition, DV capsid protein was observed to be the putative viral protein in actuating HMGB1 migration from the nucleus to cytoplasm through the involvement of PCAF acetylase. HMGB1 was released from DV-infected K562 cells into the extracellular milieu in a multiplicity of infection (M.O.I.)-independent manner and its release can be inhibited by the addition of 1-5 mM of ethyl pyruvate (EP) in a dose-dependent manner. Application of DV-infected K562 cell culture supernatants to primary endothelial cells induced vascular permeability. In contrast, supernatants from DV-infected K562 cells treated with EP or HMGB1 neutralizing antibody were observed to maintain the structural integrity of the vascular barrier.     

The envelope glycoprotein domain III of dengue virus type 2 induced the expression of anticoagulant molecules in endothelial cells

Dengue virus (DV) causes a non-specific febrile illness known as Dengue fever (DF), and a severe life-threatening illness, Dengue hemorrhagic fever/Dengue shock syndrome (DHF/DSS). Hemostatic changes induced by this virus involve three main factors: thrombocytopenia, endothelial cell damage, and significant abnormalities of the coagulation and fibrinolysis systems. The pathogenesis of bleeding in DV infections remains unknown. In this article, we focused on the DV activating endothelial cells and altering the parameters of hemostasis system. The expression of hemostasis-related factors, Thrombomodulin, TF, TFPI, t-PA, and PAI-1, in DV-infected cells were determined by RT-PCR. Flow cytometry analysis and immunofluorescence staining confirmed that the expression levels of TM in the DV-infected HMEC-1 and THP-1 cells were increased. In addition, the purified recombinant domain III of the envelope glycoprotein of DV (EIII) could induce the expression of TM in the HMEC-1 cells and THP-1 cells. The TM expression induced by DV or EIII in the endothelial cells and monocytic cells suggests that the EIII of DV plays an important role in the pathogenesis of DHF/DSS.     

Protective and enhancing HLA alleles, HLA-DRB1*0901 and HLA-A*24, for severe forms of dengue virus infection, dengue hemorrhagic fever and dengue shock syndrome

Background

Dengue virus (DV) infection is one of the most important mosquito-borne diseases in the tropics. Recently, the severe forms, dengue hemorrhagic fever (DHF) and dengue shock syndrome (DSS), have become the leading cause of death among children in Southern Vietnam. Protective and/or pathogenic T cell immunity is supposed to be important in the pathogenesis of DHF and DSS.

Methodology/Principal Findings

To identify HLA alleles controlling T cell immunity against dengue virus (DV), we performed a hospital-based case control study at Children''s Hospital No.2, Ho Chi Minh City (HCMC), and Vinh Long Province Hospital (VL) in Southern Vietnam from 2002 to 2005. A total of 211 and 418 patients with DHF and DSS, respectively, diagnosed according to the World Health Organization (WHO) criteria, were analyzed for their characteristic HLA-A, -B and -DRB1 alleles. Four hundred fifty healthy children (250 from HCMC and 200 from VL) of the same Kinh ethnicity were also analyzed as population background. In HLA class I, frequency of the HLA-A*24 showed increased tendency in both DHF and DSS patients, which reproduced a previous study. The frequency of A*24 with histidine at codon 70 (A*2402/03/10), based on main anchor binding site specificity analysis in DSS and DHF patients, was significantly higher than that in the population background groups (HCMC 02-03 DSS: OR = 1.89, P = 0.008, DHF: OR = 1.75, P = 0.033; VL 02-03 DSS: OR = 1.70, P = 0.03, DHF: OR = 1.46, P = 0.38; VL 04-05 DSS: OR = 2.09, P = 0.0075, DHF: OR = 2.02, P = 0.038). In HLA class II, the HLA-DRB1*0901 frequency was significantly decreased in secondary infection of DSS in VL 04-05 (OR = 0.35, P = 0.0025, Pc = 0.03). Moreover, the frequency of HLA-DRB1*0901 in particular was significantly decreased in DSS when compared with DHF in DEN-2 infection (P = 0.02).

Conclusion

This study improves our understanding of the risk of HLA-class I for severe outcome of DV infection in the light of peptide anchor binding site and provides novel evidence that HLA-class II may control disease severity (DHF to DSS) in DV infection.     

Progress towards understanding the pathogenesis of dengue hemorrhagic fever

Dengue virus (DENV) is a mosquito-borne virus belonging to the Flaviviridae family. There are 4 serotypes of DENV that cause human disease through transmission by mosquito vectors. DENV infection results in a broad spectrum of clinical symptoms, ranging from mild fever to dengue hemorrhagic fever (DHF), the latter of which can progress to dengue shock syndrome (DSS) and death. Researchers have made unremitting efforts over the last half-century to understand DHF pathogenesis. DHF is probably caused by multiple factors, such as virus-specific antibodies, viral antigens and host immune responses. This review summarizes the current progress of studies on DHF pathogenesis, which may provide important information for achieving effective control of dengue in the future.

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A serotype-specific epitope of dengue virus 1 identified by phage displayed random peptide library

Abstract From a panel of monoclonal antibodies of dengue viruses, a serotype-specific epitope of dengue virus 1 was screened from a random peptide library displayed on phage. The epitope was the determinant reactive with monoclonal antibody 15F3-1 that was specific to dengue 1. The screening was monitored by a dot blotting procedure, and after three rounds of screening a consensus motif, HRYSWK, was found. This sequence matches the sequence HKYSWK, corresponding to the amino acid residues 885–890 of polyprotein or residues 111–116 of the non-structural protein 1 of dengue virus serotype 1. The linear epitope was confirmed by testing the antigenicity of chemically synthesized 8-branched peptide.