Dengue virus NS1 triggers endothelial permeability and vascular leak, and vaccination with NS1 induces a protective antibody response.

Dengue is the most prevalent arboviral disease in humans and a major public health problem worldwide. Systemic plasma leakage, leading to hypovolemic shock and potentially fatal complications, is a critical determinant of dengue severity. Recently, we and others described a novel pathogenic effect of secreted dengue virus (DENV) non-structural protein 1 (NS1) in triggering hyperpermeability of human endothelial cells in vitro and systemic vascular leakage in vivo. NS1 was shown to activate toll-like receptor 4 signaling in primary human myeloid cells, leading to secretion of pro-inflammatory cytokines and vascular leakage. However, distinct endothelial cell-intrinsic mechanisms of NS1-induced hyperpermeability remained to be defined. The endothelial glycocalyx layer (EGL) is a network of membrane-bound proteoglycans and glycoproteins lining the vascular endothelium that plays a key role in regulating endothelial barrier function. Here, we demonstrate that DENV NS1 disrupts the EGL on human pulmonary microvascular endothelial cells, inducing degradation of sialic acid and shedding of heparan sulfate proteoglycans. This effect is mediated by NS1-induced expression of sialidases and heparanase, respectively. NS1 also activates cathepsin L, a lysosomal cysteine proteinase, in endothelial cells, which activates heparanase via enzymatic cleavage. Specific inhibitors of sialidases, heparanase, and cathepsin L prevent DENV NS1-induced EGL disruption and endothelial hyperpermeability. All of these effects are specific to NS1 from DENV1-4 and are not induced by NS1 from West Nile virus, a related flavivirus. Together, our data suggest an important role for EGL disruption in DENV NS1-mediated endothelial dysfunction during severe dengue disease. Dengue is the most prevalent mosquito-borne disease in humans and represents a major public health problem worldwide. Leakage of fluids and molecules from the bloodstream into tissues can lead to shock and potentially death and is a critical determinant of dengue disease severity. Recently, we showed that a secreted protein from dengue virus (DENV)-infected cells, non-structural protein 1 (NS1), can trigger increased leakage both in human cell culture and mouse models. It has been shown that NS1 can activate toll-like receptor 4 on peripheral blood mononuclear cells, leading to secretion of pro-inflammatory cytokines that can result in vascular leak. However, the mechanism by which NS1 triggers hyperpermeability directly in human endothelial cells remained undefined. The endothelial glycocalyx layer (EGL) is a network of membrane-bound molecules that lines endothelial cells on the inside of blood vessels, helping to regulate proper vascular function. Here, we show that DENV NS1 can disrupt the integrity of the EGL, inducing breakdown and shedding of key components. This is mediated by NS1 induction of cellular enzymes (e.g., sialidases, heparanase, and cathepsin L) that contribute to EGL alterations. Inhibitors that block these enzymes prevent both EGL disruption and endothelial permeability. These effects were all demonstrated to be specific to NS1 from DENV serotypes 1–4, as NS1 from the related West Nile Virus did not produce EGL alterations or increased leakage. Our study suggests a novel role for DENV NS1 in inducing EGL disruption to increase fluid leakage during severe dengue disease.

Flaviviruses cause systemic or neurotropic-encephalitic pathology in humans. The flavivirus nonstructural protein 1 (NS1) is a secreted glycoprotein involved in viral replication, immune evasion, and vascular leakage during dengue virus infection. However, the contribution of secreted NS1 from related flaviviruses to viral pathogenesis remains unknown. Here, we demonstrate that NS1 from dengue, Zika, West Nile, Japanese encephalitis, and yellow fever viruses selectively binds to and alters permeability of human endothelial cells from lung, dermis, umbilical vein, brain, and liver in vitro and causes tissue-specific vascular leakage in mice, reflecting the pathophysiology of each flavivirus. Mechanistically, each flavivirus NS1 leads to differential disruption of endothelial glycocalyx components, resulting in endothelial hyperpermeability. Our findings reveal the capacity of a secreted viral protein to modulate endothelial barrier function in a tissue-specific manner both in vitro and in vivo, potentially influencing virus dissemination and pathogenesis and providing targets for antiviral therapies and vaccine development.

Dengue virus (DENV) is the most prevalent medically important mosquito-borne virus in the world. Upon DENV infection of a host cell, DENV nonstructural protein 1 (NS1) can be found intracellularly as a monomer, associated with the cell surface as a dimer, and secreted as a hexamer into the bloodstream. NS1 plays a variety of roles in the viral life cycle, particularly in RNA replication and immune evasion of the complement pathway. Over the past several years, key roles for NS1 in the pathogenesis of severe dengue disease have emerged, including direct action of the protein on the vascular endothelium and triggering release of vasoactive cytokines from immune cells, both of which result in endothelial hyperpermeability and vascular leak. Importantly, the adaptive immune response generates a robust response against NS1, and its potential contribution to dengue vaccines is also discussed.

Dengue virus (DENV) is the most prevalent, medically important mosquito-borne virus. Disease ranges from uncomplicated dengue to life-threatening disease, characterized by endothelial dysfunction and vascular leakage. Previously, we demonstrated that DENV nonstructural protein 1 (NS1) induces endothelial hyperpermeability in a systemic mouse model and human pulmonary endothelial cells, where NS1 disrupts the endothelial glycocalyx-like layer. NS1 also triggers release of inflammatory cytokines from PBMCs via TLR4. Here, we examined the relative contributions of inflammatory mediators and endothelial cell-intrinsic pathways. In vivo, we demonstrated that DENV NS1 but not the closely-related West Nile virus NS1 triggers localized vascular leak in the dorsal dermis of wild-type C57BL/6 mice. In vitro, we showed that human dermal endothelial cells exposed to DENV NS1 do not produce inflammatory cytokines (TNF-α, IL-6, IL-8) and that blocking these cytokines does not affect DENV NS1-induced endothelial hyperpermeability. Further, we demonstrated that DENV NS1 induces vascular leak in TLR4- or TNF-α receptor-deficient mice at similar levels to wild-type animals. Finally, we blocked DENV NS1-induced vascular leak in vivo using inhibitors targeting molecules involved in glycocalyx disruption. Taken together, these data indicate that DENV NS1-induced endothelial cell-intrinsic vascular leak is independent of inflammatory cytokines but dependent on endothelial glycocalyx components. Dengue is the most prevalent mosquito-transmitted disease in humans and a significant public health issue worldwide. Severe dengue disease is characterized by vascular leak, which can lead to shock and potentially death. We previously demonstrated that nonstructural protein 1 (NS1), the only protein secreted from dengue virus (DENV)-infected cells, can both trigger vascular leak in mice when given systemically and increase permeability in human pulmonary endothelial cells via disruption of the endothelial glycocalyx-like layer, the molecular barrier that lines blood vessels. NS1 also triggers release of inflammatory cytokines from immune cells through activation of Toll-like receptor 4 (TLR4). Here, we explored the relative contributions of inflammatory molecules and the endothelial glycocalyx-like layer to NS1-mediated pathogenesis. Using cultured human dermal endothelial cells and mice genetically deficient for TLR4 or TNF-α receptor, we showed inflammatory signaling is not required for direct DENV NS1-mediated vascular leak. In contrast, inhibition of molecules involved in glycocalyx disruption blocked DENV NS1-induced vascular leak both in mice and in vitro. Altogether, our results indicate that disruption of endothelial glycocalyx components but not production of inflammatory cytokines is required for the direct action of DENV NS1 on endothelial cells and suggest potential molecular targets for treatment of severe dengue disease.

Two antibodies against flaviviruses: Flaviviruses are a group of RNA viruses that include the human pathogens dengue virus, Zika virus, and West Nile virus. The envelope protein (E) on the virus surface has been the target of vaccine development, but problems have arisen with antibodies against E, leading to enhanced infection. Now, Modhiran et al. and Biering et al. describe two different antibodies that bind to the flavivirus NS1 protein and prevent it from disrupting epithelial cells, which is associated with severe disease. Both antibodies cross-react with multiple flavivirus NS1 proteins. The antibodies reduce viremia and increase survival in mouse models of flavivirus disease. Both papers include structures of NS1 bound to an antibody, which give insight into the protective mechanism. Science , this issue p. 190 , p. 194