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Severe COVID-19 is associated with epithelial and endothelial barrier dysfunction within the lung as well as in distal organs. While it is appreciated that an exaggerated inflammatory response is associated with barrier dysfunction, the triggers of vascular leak are unclear. Here, we report that cell-intrinsic interactions between the Spike (S) glycoprotein of SARS-CoV-2 and epithelial/endothelial cells are sufficient to induce barrier dysfunction in vitro and vascular leak in vivo, independently of viral replication and the ACE2 receptor. We identify an S-triggered transcriptional response associated with extracellular matrix reorganization and TGF-ß signaling. Using genetic knockouts and specific inhibitors, we demonstrate that glycosaminoglycans, integrins, and the TGF-ß signaling axis are required for S-mediated barrier dysfunction. Notably, we show that SARS-CoV-2 infection caused leak in vivo, which was reduced by inhibiting integrins. Our findings offer mechanistic insight into SARS-CoV-2-triggered vascular leak, providing a starting point for development of therapies targeting COVID-19.
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COVID-19 , SARS-CoV-2 , Humanos , Enzima Convertidora de Angiotensina 2 , Glicoproteína de la Espiga del Coronavirus/genética , Células Endoteliales , Integrinas , Peptidil-Dipeptidasa A/genética , Factor de Crecimiento Transformador betaRESUMEN
The flavivirus nonstructural protein 1 (NS1) is secreted from infected cells and contributes to endothelial barrier dysfunction and vascular leak in a tissue-dependent manner. This phenomenon occurs in part via disruption of the endothelial glycocalyx layer (EGL) lining the endothelium. Additionally, we and others have shown that soluble DENV NS1 induces disassembly of intercellular junctions (IJCs), a group of cellular proteins critical for maintaining endothelial homeostasis and regulating vascular permeability; however, the specific mechanisms by which NS1 mediates IJC disruption remain unclear. Here, we investigated the relative contribution of five flavivirus NS1 proteins, from dengue (DENV), Zika (ZIKV), West Nile (WNV), Japanese encephalitis (JEV), and yellow fever (YFV) viruses, to the expression and localization of the intercellular junction proteins ß-catenin and VE-cadherin in endothelial cells from human umbilical vein and brain tissues. We found that flavivirus NS1 induced the mislocalization of ß-catenin and VE-cadherin in a tissue-dependent manner, reflecting flavivirus disease tropism. Mechanistically, we observed that NS1 treatment of cells triggered internalization of VE-cadherin, likely via clathrin-mediated endocytosis, and phosphorylation of ß-catenin, part of a canonical IJC remodeling pathway during breakdown of endothelial barriers that activates glycogen synthase kinase-3ß (GSK-3ß). Supporting this model, we found that a chemical inhibitor of GSK-3ß reduced both NS1-induced permeability of human umbilical vein and brain microvascular endothelial cell monolayers in vitro and vascular leakage in a mouse dorsal intradermal model. These findings provide insight into the molecular mechanisms regulating NS1-mediated endothelial dysfunction and identify GSK-3ß as a potential therapeutic target for treatment of vascular leakage during severe dengue disease.
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Associations between vaccine breakthrough cases and infection by different SARS coronavirus 2 (SARS-CoV-2) variants have remained largely unexplored. Here we analysed SARS-CoV-2 whole-genome sequences and viral loads from 1,373 persons with COVID-19 from the San Francisco Bay Area from 1 February to 30 June 2021, of which 125 (9.1%) were vaccine breakthrough infections. Vaccine breakthrough infections were more commonly associated with circulating antibody-resistant variants carrying ≥1 mutation associated with decreased antibody neutralization (L452R/Q, E484K/Q and/or F490S) than infections in unvaccinated individuals (78% versus 48%, P = 1.96 × 10-8). Differences in viral loads were non-significant between unvaccinated and fully vaccinated cases overall (P = 0.99) and according to lineage (P = 0.09-0.78). Symptomatic vaccine breakthrough infections had comparable viral loads (P = 0.64), whereas asymptomatic breakthrough infections had decreased viral loads (P = 0.023) compared with infections in unvaccinated individuals. In 5 cases with serial samples available for serologic analyses, vaccine breakthrough infections were found to be associated with low or undetectable neutralizing antibody levels attributable to an immunocompromised state or infection by an antibody-resistant lineage. Taken together, our results show that vaccine breakthrough infections are overrepresented by antibody-resistant SARS-CoV-2 variants, and that symptomatic breakthrough infections may be as efficient in spreading COVID-19 as unvaccinated infections, regardless of the infecting lineage.
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Anticuerpos Antivirales/sangre , Vacuna BNT162/inmunología , COVID-19/epidemiología , SARS-CoV-2/genética , SARS-CoV-2/inmunología , Adolescente , Adulto , Anciano , Anticuerpos Neutralizantes/sangre , Anticuerpos Neutralizantes/inmunología , Anticuerpos Antivirales/inmunología , Vacuna BNT162/administración & dosificación , COVID-19/inmunología , Vacunas contra la COVID-19/inmunología , Estudios de Cohortes , Femenino , Genoma Viral , Humanos , Masculino , Persona de Mediana Edad , Mutación , Filogenia , San Francisco/epidemiología , Vacunación , Carga Viral/estadística & datos numéricos , Secuenciación Completa del Genoma , Adulto JovenRESUMEN
Severe COVID-19 is associated with epithelial and endothelial barrier dysfunction within the lung as well as in distal organs. While it is appreciated that an exaggerated inflammatory response is associated with barrier dysfunction, the triggers of this pathology are unclear. Here, we report that cell-intrinsic interactions between the Spike (S) glycoprotein of SARS-CoV-2 and epithelial/endothelial cells are sufficient to trigger barrier dysfunction in vitro and vascular leak in vivo , independently of viral replication and the ACE2 receptor. We identify an S-triggered transcriptional response associated with extracellular matrix reorganization and TGF-ß signaling. Using genetic knockouts and specific inhibitors, we demonstrate that glycosaminoglycans, integrins, and the TGF-ß signaling axis are required for S-mediated barrier dysfunction. Our findings suggest that S interactions with barrier cells are a contributing factor to COVID-19 disease severity and offer mechanistic insight into SARS-CoV-2 triggered vascular leak, providing a starting point for development of therapies targeting COVID-19 pathogenesis.
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Arthropod-borne rickettsial pathogens cause mild and severe human disease worldwide. The tick-borne pathogen Rickettsia parkeri elicits skin lesions (eschars) and disseminated disease in humans; however, inbred mice are generally resistant to infection. We report that intradermal infection of mice lacking both interferon receptors (Ifnar1-/-;Ifngr1-/-) with as few as 10 R. parkeri elicits eschar formation and disseminated, lethal disease. Similar to human infection, eschars exhibited necrosis and inflammation, with bacteria primarily found in leukocytes. Using this model, we find that the actin-based motility factor Sca2 is required for dissemination from the skin to internal organs, and the outer membrane protein OmpB contributes to eschar formation. Immunizing Ifnar1-/-;Ifngr1-/- mice with sca2 and ompB mutant R. parkeri protects against rechallenge, revealing live-attenuated vaccine candidates. Thus, Ifnar1-/-;Ifngr1-/- mice are a tractable model to investigate rickettsiosis, virulence factors, and immunity. Our results further suggest that discrepancies between mouse and human susceptibility may be due to differences in interferon signaling.
Tick bites allow disease-causing microbes, including multiple species of Rickettsia bacteria, to pass from arthropods to humans. Being exposed to Rickettsia parkeri, for example, can cause a scab at the bite site, fever, headache and fatigue. To date, no vaccine is available against any of the severe diseases caused by Rickettsia species. Modelling human infections in animals could help to understand and combat these illnesses. R. parkeri is a good candidate for such studies, as it can give insight into more severe Rickettsia infections while being comparatively safer to handle. However, laboratory mice are resistant to this species of bacteria, limiting their use as models. To explore why this is the case, Burke et al. probed whether an immune mechanism known as interferon signalling protects laboratory rodents against R. parkeri. During infection, the immune system releases molecules called interferons that stick to 'receptors' at the surface of cells, triggering defense mechanisms that help to fight off an invader. Burke et al. injected R. parkeri into the skin of mice that had or lacked certain interferon receptors, showing that animals without two specific receptors developed scabs and saw the disease spread through their body. Further investigation showed that two R. parkeri proteins, known as OmpB or Sca2, were essential for the bacteria to cause skin lesions and damage internal organs. Burke et al. then used R. parkeri that lacked OmpB or Sca2 to test whether these modified, inoffensive microbes could act as 'vaccines'. And indeed, vulnerable laboratory mice which were first exposed to the mutant bacteria were then able to survive the 'normal' version of the microbe. Together, this work reveals that interferon signalling protects laboratory mice against R. parkeri infections. It also creates an animal model that can be used to study disease and vaccination.
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Estudios de Asociación Genética , Receptores de Interferón/deficiencia , Receptores de Interferón/genética , Infecciones por Rickettsia/inmunología , Animales , Médula Ósea , Femenino , Inmunidad Innata , Inflamación , Listeria monocytogenes , Macrófagos , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Receptor de Interferón alfa y beta/genética , Rickettsia , Infecciones por Rickettsia/patología , GarrapatasRESUMEN
We identified an emerging severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variant by viral whole-genome sequencing of 2,172 nasal/nasopharyngeal swab samples from 44 counties in California, a state in the western United States. Named B.1.427/B.1.429 to denote its two lineages, the variant emerged in May 2020 and increased from 0% to >50% of sequenced cases from September 2020 to January 2021, showing 18.6%-24% increased transmissibility relative to wild-type circulating strains. The variant carries three mutations in the spike protein, including an L452R substitution. We found 2-fold increased B.1.427/B.1.429 viral shedding in vivo and increased L452R pseudovirus infection of cell cultures and lung organoids, albeit decreased relative to pseudoviruses carrying the N501Y mutation common to variants B.1.1.7, B.1.351, and P.1. Antibody neutralization assays revealed 4.0- to 6.7-fold and 2.0-fold decreases in neutralizing titers from convalescent patients and vaccine recipients, respectively. The increased prevalence of a more transmissible variant in California exhibiting decreased antibody neutralization warrants further investigation.
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Anticuerpos Neutralizantes/inmunología , COVID-19/inmunología , COVID-19/transmisión , SARS-CoV-2/genética , Glicoproteína de la Espiga del Coronavirus/inmunología , Anticuerpos Monoclonales/inmunología , Anticuerpos Antivirales/inmunología , Humanos , Mutación/genética , Secuenciación Completa del Genoma/métodosRESUMEN
We identified a novel SARS-CoV-2 variant by viral whole-genome sequencing of 2,172 nasal/nasopharyngeal swab samples from 44 counties in California. Named B.1.427/B.1.429 to denote its 2 lineages, the variant emerged around May 2020 and increased from 0% to >50% of sequenced cases from September 1, 2020 to January 29, 2021, exhibiting an 18.6-24% increase in transmissibility relative to wild-type circulating strains. The variant carries 3 mutations in the spike protein, including an L452R substitution. Our analyses revealed 2-fold increased B.1.427/B.1.429 viral shedding in vivo and increased L452R pseudovirus infection of cell cultures and lung organoids, albeit decreased relative to pseudoviruses carrying the N501Y mutation found in the B.1.1.7, B.1.351, and P.1 variants. Antibody neutralization assays showed 4.0 to 6.7-fold and 2.0-fold decreases in neutralizing titers from convalescent patients and vaccine recipients, respectively. The increased prevalence of a more transmissible variant in California associated with decreased antibody neutralization warrants further investigation.
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Medically important flaviviruses cause diverse disease pathologies and collectively are responsible for a major global disease burden. A contributing factor to pathogenesis is secreted flavivirus nonstructural protein 1 (NS1). Despite demonstrated protection by NS1-specific antibodies against lethal flavivirus challenge, the structural and mechanistic basis remains unknown. Here, we present three crystal structures of full-length dengue virus NS1 complexed with a flavivirus-cross-reactive, NS1-specific monoclonal antibody, 2B7, at resolutions between 2.89 and 3.96 angstroms. These structures reveal a protective mechanism by which two domains of NS1 are antagonized simultaneously. The NS1 wing domain mediates cell binding, whereas the ß-ladder triggers downstream events, both of which are required for dengue, Zika, and West Nile virus NS1-mediated endothelial dysfunction. These observations provide a mechanistic explanation for 2B7 protection against NS1-induced pathology and demonstrate the potential of one antibody to treat infections by multiple flaviviruses.
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Anticuerpos Neutralizantes/química , Anticuerpos Antivirales/química , Virus del Dengue/inmunología , Proteínas no Estructurales Virales/inmunología , Virus del Nilo Occidental/inmunología , Virus Zika/inmunología , Animales , Anticuerpos Monoclonales/química , Anticuerpos Monoclonales/inmunología , Anticuerpos Neutralizantes/inmunología , Anticuerpos Antivirales/inmunología , Reacciones Cruzadas , Cristalografía por Rayos X , Dengue/prevención & control , Dengue/terapia , Endotelio/inmunología , Glicocálix/inmunología , Humanos , Ratones , Conformación Proteica en Lámina beta , Dominios Proteicos , Proteínas no Estructurales Virales/química , Fiebre del Nilo Occidental/prevención & control , Fiebre del Nilo Occidental/terapia , Infección por el Virus Zika/prevención & control , Infección por el Virus Zika/terapiaRESUMEN
Given the limited availability of serological testing to date, the seroprevalence of SARS-CoV-2-specific antibodies in different populations has remained unclear. Here, we report very low SARS-CoV-2 seroprevalence in two San Francisco Bay Area populations. Seroreactivity was 0.26% in 387 hospitalized patients admitted for non-respiratory indications and 0.1% in 1,000 blood donors in early April 2020. We additionally describe the longitudinal dynamics of immunoglobulin-G (IgG), immunoglobulin-M (IgM), and in vitro neutralizing antibody titers in COVID-19 patients. The median time to seroconversion ranged from 10.3-11.0 days for these 3 assays. Neutralizing antibodies rose in tandem with immunoglobulin titers following symptom onset, and positive percent agreement between detection of IgG and neutralizing titers was >93%. These findings emphasize the importance of using highly accurate tests for surveillance studies in low-prevalence populations, and provide evidence that seroreactivity using SARS-CoV-2 anti-nucleocapsid protein IgG and anti-spike IgM assays are generally predictive of in vitro neutralizing capacity.
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Anticuerpos Neutralizantes/sangre , Betacoronavirus/inmunología , Infecciones por Coronavirus/epidemiología , Neumonía Viral/epidemiología , Anticuerpos Antivirales/inmunología , COVID-19 , Prueba de COVID-19 , Técnicas de Laboratorio Clínico , Infecciones por Coronavirus/sangre , Infecciones por Coronavirus/diagnóstico , Infecciones por Coronavirus/inmunología , Humanos , Inmunoglobulina G/inmunología , Inmunoglobulina M/inmunología , Pandemias , Neumonía Viral/sangre , Neumonía Viral/inmunología , SARS-CoV-2 , San Francisco/epidemiología , Sensibilidad y Especificidad , Estudios Seroepidemiológicos , Pruebas Serológicas/métodosRESUMEN
We report very low SARS-CoV-2 seroprevalence in two San Francisco Bay Area populations. Seropositivity was 0.26% in 387 hospitalized patients admitted for non-respiratory indications and 0.1% in 1,000 blood donors. We additionally describe the longitudinal dynamics of immunoglobulin-G, immunoglobulin-M, and in vitro neutralizing antibody titers in COVID-19 patients. Neutralizing antibodies rise in tandem with immunoglobulin levels following symptom onset, exhibiting median time to seroconversion within one day of each other, and there is >93% positive percent agreement between detection of immunoglobulin-G and neutralizing titers.
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BACKGROUND: Dengue virus (DENV) can cause life-threatening disease characterized by endothelial dysfunction and vascular leakage. DENV nonstructural protein 1 (NS1) induces human endothelial hyperpermeability and vascular leak in mice, and NS1 vaccination confers antibody-mediated protective immunity. We evaluated the magnitude, cross-reactivity, and functionality of NS1-specific IgG antibody responses in sera from a phase 2 clinical trial of Takeda's live-attenuated tetravalent dengue vaccine candidate (TAK-003). METHODS: We developed an enzyme-linked immunosorbent assay to measure anti-DENV NS1 IgG in sera from DENV-naive or preimmune subjects pre- and postvaccination with TAK-003 and evaluated the functionality of this response using in vitro models of endothelial permeability. RESULTS: TAK-003 significantly increased DENV-2 NS1-specific IgG in naive individuals, which cross-reacted with DENV-1, -3, and -4 NS1 to varying extents. NS1-induced endothelial hyperpermeability was unaffected by prevaccination serum from naive subjects but was variably inhibited by serum from preimmune subjects. After TAK-003 vaccination, all samples from naive and preimmune vaccinees completely abrogated DENV-2 NS1-induced hyperpermeability and cross-inhibited hyperpermeability induced by DENV-1, -3, and -4 NS1. Inhibition of NS1-induced hyperpermeability correlated with NS1-specific IgG concentrations. Postvaccination sera also prevented NS1-induced degradation of endothelial glycocalyx components. CONCLUSION: We provide evidence for functional NS1-specific IgG responses elicited by a candidate dengue vaccine. CLINICAL TRIALS REGISTRATION: NCT01511250.
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Vacunas contra el Dengue/inmunología , Inmunoglobulina G/sangre , Inmunoglobulina G/metabolismo , Proteínas no Estructurales Virales/inmunología , Adolescente , Adulto , Línea Celular , Niño , Preescolar , Reacciones Cruzadas , Células Endoteliales , Humanos , Lactante , Persona de Mediana Edad , Vacunas Atenuadas , Adulto JovenRESUMEN
BACKGROUND: During pregnancy, the Zika flavivirus (ZIKV) infects human placentas, inducing defects in the developing fetus. The flavivirus nonstructural protein 1 (NS1) alters glycosaminoglycans on the endothelium, causing hyperpermeability in vitro and vascular leakage in vivo in a tissue-dependent manner. The contribution of ZIKV NS1 to placental dysfunction during ZIKV infection remains unknown. METHODS: We examined the effect of ZIKV NS1 on expression and release of heparan sulfate (HS), hyaluronic acid (HA), and sialic acid on human trophoblast cell lines and anchoring villous explants from first-trimester placentas infected with ZIKV ex vivo. We measured changes in permeability in trophoblasts and stromal cores using a dextran-based fluorescence assay and changes in HA receptor expression using immunofluorescent microscopy. RESULTS: ZIKV NS1 in the presence and absence of ZIKV increased the permeability of anchoring villous explants. ZIKV NS1 induced shedding of HA and HS and altered expression of CD44 and lymphatic endothelial cell HA receptor-1, HA receptors on stromal fibroblasts and Hofbauer macrophages in villous cores. Hyaluronidase was also stimulated in NS1-treated trophoblasts. CONCLUSIONS: These findings suggest that ZIKV NS1 contributes to placental dysfunction via modulation of glycosaminoglycans on trophoblasts and chorionic villi, resulting in increased permeability of human placentas.
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Placenta/metabolismo , Proteínas no Estructurales Virales/metabolismo , Infección por el Virus Zika/transmisión , Virus Zika/metabolismo , Femenino , Glicosaminoglicanos/metabolismo , Humanos , Transmisión Vertical de Enfermedad Infecciosa , Permeabilidad , Placenta/virología , Embarazo , Complicaciones Infecciosas del Embarazo/virología , Infección por el Virus Zika/virologíaRESUMEN
Arthropod-borne flaviviruses cause life-threatening diseases associated with endothelial hyperpermeability and vascular leak. We recently found that vascular leak can be triggered by dengue virus (DENV) non-structural protein 1 (NS1) via the disruption of the endothelial glycocalyx-like layer (EGL). However, the molecular determinants of NS1 required to trigger EGL disruption and the cellular pathway(s) involved remain unknown. Here we report that mutation of a single glycosylated residue of NS1 (N207Q) abolishes the ability of NS1 to trigger EGL disruption and induce endothelial hyperpermeability. Intriguingly, while this mutant bound to the surface of endothelial cells comparably to wild-type NS1, it was no longer internalized, suggesting that NS1 binding and internalization are distinct steps. Using endocytic pathway inhibitors and gene-specific siRNAs, we determined that NS1 was endocytosed into endothelial cells in a dynamin- and clathrin-dependent manner, which was required to trigger endothelial dysfunction in vitro and vascular leak in vivo. Finally, we found that the N207 glycosylation site is highly conserved among flaviviruses and is also essential for West Nile and Zika virus NS1 to trigger endothelial hyperpermeability via clathrin-mediated endocytosis. These data provide critical mechanistic insight into flavivirus NS1-induced pathogenesis, presenting novel therapeutic and vaccine targets for flaviviral diseases.
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Virus del Dengue/patogenicidad , Proteínas no Estructurales Virales/fisiología , Sustitución de Aminoácidos , Sitios de Unión/genética , Permeabilidad Capilar , Línea Celular , Virus del Dengue/genética , Virus del Dengue/fisiología , Endocitosis/fisiología , Células Endoteliales/fisiología , Células Endoteliales/virología , Glicocálix/fisiología , Glicosilación , Células HEK293 , Humanos , Modelos Biológicos , Modelos Moleculares , Mutagénesis Sitio-Dirigida , Mutación , Estructura Cuaternaria de Proteína , Proteínas no Estructurales Virales/química , Proteínas no Estructurales Virales/genéticaRESUMEN
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.
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Virus del Dengue/genética , Células Endoteliales/virología , Glicocálix/virología , Proteínas no Estructurales Virales/genética , Animales , Encéfalo/patología , Encéfalo/virología , Línea Celular , Permeabilidad de la Membrana Celular , Dengue/genética , Dengue/metabolismo , Dengue/patología , Virus del Dengue/metabolismo , Virus del Dengue/patogenicidad , Dermis/patología , Dermis/virología , Virus de la Encefalitis Japonesa (Especie)/genética , Virus de la Encefalitis Japonesa (Especie)/metabolismo , Virus de la Encefalitis Japonesa (Especie)/patogenicidad , Células Endoteliales/patología , Expresión Génica , Glicocálix/química , Humanos , Hígado/patología , Hígado/virología , Pulmón/patología , Pulmón/virología , Masculino , Ratones , Especificidad de Órganos , Cultivo Primario de Células , Venas Umbilicales/patología , Venas Umbilicales/virología , Proteínas no Estructurales Virales/química , Proteínas no Estructurales Virales/metabolismo , Replicación Viral , Virus del Nilo Occidental/genética , Virus del Nilo Occidental/metabolismo , Virus del Nilo Occidental/patogenicidad , Virus de la Fiebre Amarilla/genética , Virus de la Fiebre Amarilla/metabolismo , Virus de la Fiebre Amarilla/patogenicidad , Virus Zika/genética , Virus Zika/metabolismo , Virus Zika/patogenicidadRESUMEN
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.
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Virus del Dengue/inmunología , Virus del Dengue/fisiología , Dengue/inmunología , Dengue/virología , Interacciones Huésped-Patógeno , Proteínas no Estructurales Virales/inmunología , Proteínas no Estructurales Virales/metabolismo , Citocinas/metabolismo , Dengue/prevención & control , Vacunas contra el Dengue/inmunología , Vacunas contra el Dengue/aislamiento & purificación , Virus del Dengue/patogenicidad , Endotelio Vascular/fisiología , Endotelio Vascular/virología , Evasión Inmune , Permeabilidad , ARN Viral/metabolismo , Replicación ViralRESUMEN
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.
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Virus del Dengue/metabolismo , Dengue/metabolismo , Endotelio Vascular/metabolismo , Glicocálix/metabolismo , Leucocitos Mononucleares/metabolismo , Proteínas no Estructurales Virales/metabolismo , Animales , Citocinas/genética , Citocinas/metabolismo , Dengue/genética , Virus del Dengue/genética , Endotelio Vascular/patología , Endotelio Vascular/virología , Glicocálix/genética , Humanos , Leucocitos Mononucleares/patología , Leucocitos Mononucleares/virología , Ratones , Ratones Noqueados , Receptor Toll-Like 4/genética , Receptor Toll-Like 4/metabolismo , Proteínas no Estructurales Virales/genéticaRESUMEN
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.
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Permeabilidad Capilar/fisiología , Células Endoteliales/patología , Glicocálix/patología , Proteínas no Estructurales Virales/metabolismo , Western Blotting , Línea Celular , Virus del Dengue/metabolismo , Ensayo de Inmunoadsorción Enzimática , Glicocálix/virología , Humanos , Microscopía FluorescenteRESUMEN
Dengue remains the most prevalent arthropod-borne viral disease in humans. While probing for blood vessels, Aedes aegypti and Ae. albopictus mosquitoes transmit the four serotypes of dengue virus (DENV1-4) by injecting virus-containing saliva into the skin. Even though arthropod saliva is known to facilitate transmission and modulate host responses to other pathogens, the full impact of mosquito saliva on dengue pathogenesis is still not well understood. Inoculating mice lacking the interferon-α/ß receptor intradermally with DENV revealed that mosquito salivary gland extract (SGE) exacerbates dengue pathogenesis specifically in the presence of enhancing serotype-cross-reactive antibodies-when individuals already carry an increased risk for severe disease. We further establish that SGE increases viral titers in the skin, boosts antibody-enhanced DENV infection of dendritic cells and macrophages in the dermis, and amplifies dendritic cell migration to skin-draining lymph nodes. We demonstrate that SGE directly disrupts endothelial barrier function in vitro and induces endothelial permeability in vivo in the skin. Finally, we show that surgically removing the site of DENV transmission in the skin after 4 hours rescued mice from disease in the absence of SGE, but no longer prevented lethal antibody-enhanced disease when SGE was present. These results indicate that SGE accelerates the dynamics of dengue pathogenesis after virus transmission in the skin and induces severe antibody-enhanced disease systemically. Our study reveals novel aspects of dengue pathogenesis and suggests that animal models of dengue and pre-clinical testing of dengue vaccines should consider mosquito-derived factors as well as enhancing antibodies.
Asunto(s)
Acrecentamiento Dependiente de Anticuerpo/inmunología , Movimiento Celular , Culicidae/virología , Dengue/transmisión , Células Endoteliales/virología , Insectos Vectores/patogenicidad , Saliva/metabolismo , Animales , Permeabilidad Capilar , Quimiotaxis de Leucocito/inmunología , Culicidae/metabolismo , Dengue/inmunología , Virus del Dengue/inmunología , Virus del Dengue/patogenicidad , Modelos Animales de Enfermedad , Células Endoteliales/inmunología , Citometría de Flujo , Insectos Vectores/inmunología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Saliva/inmunología , Saliva/virología , Piel/irrigación sanguínea , Piel/inmunologíaRESUMEN
The four dengue virus serotypes (DENV1 to DENV4) are mosquito-borne flaviviruses that cause up to ~100 million cases of dengue annually worldwide. Severe disease is thought to result from immunopathogenic processes involving serotype cross-reactive antibodies and T cells that together induce vasoactive cytokines, causing vascular leakage that leads to shock. However, no viral proteins have been directly implicated in triggering endothelial permeability, which results in vascular leakage. DENV nonstructural protein 1 (NS1) is secreted and circulates in patients' blood during acute infection; high levels of NS1 are associated with severe disease. We show that inoculation of mice with DENV NS1 alone induces both vascular leakage and production of key inflammatory cytokines. Furthermore, simultaneous administration of NS1 with a sublethal dose of DENV2 results in a lethal vascular leak syndrome. We also demonstrate that NS1 from DENV1, DENV2, DENV3, and DENV4 triggers endothelial barrier dysfunction, causing increased permeability of human endothelial cell monolayers in vitro. These pathogenic effects of physiologically relevant amounts of NS1 in vivo and in vitro were blocked by NS1-immune polyclonal mouse serum or monoclonal antibodies to NS1, and immunization of mice with NS1 from DENV1 to DENV4 protected against lethal DENV2 challenge. These findings add an important and previously overlooked component to the causes of dengue vascular leak, identify a new potential target for dengue therapeutics, and support inclusion of NS1 in dengue vaccines.