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The emergence of SARS-CoV-2 and seasonal outbreaks of other respiratory viruses highlight the urgent need for broad-spectrum antivirals to treat respiratory tract infections. Stimulator of interferon genes (STING) is a key component of innate immune signaling and plays a critical role in protection of the host against viral infections. Previously the STING agonist diABZI-4, a diamidobenzimidazole-based compound, demonstrated protection against SARS-CoV-2 both in vitro and in vivo. However, its broad-spectrum antiviral activity against other respiratory viruses in human airway epithelial cells, which are the primary targets of these infections, is not well established. In this study, we demonstrated that diABZI-4 stimulated robust innate immune responses protecting lung cells against a wide range of respiratory viruses, including influenza A virus (IAV), common cold coronaviruses, SARS-CoV-2, human rhinovirus (HRV), and human parainfluenza virus. diABZI-4 was highly active in physiologically relevant human airway epithelial tissues grown at the air-liquid interface, blocking replication of IAV, SARS-CoV-2, and HRV in these tissues. Furthermore, treatment of macrophages with diABZI-4 resulted in the secretion of cytokines that protected the primary airway epithelial cells from IAV infection. Despite the promising in vitro pan-antiviral activity, intranasal administration of diABZI-4 in mice provided early, but not sustained, inhibition of IAV replication in the lungs. These data highlight the complexities of the relationship between timing of STING agonist-driven inflammatory responses and viral replication dynamics, emphasizing the development challenge posed by STING agonists as potential therapeutics against respiratory viruses.
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Inflammation in response to severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) infection drives severity of coronavirus disease 2019 (COVID-19) and is influenced by host genetics. To understand mechanisms of inflammation, animal models that reflect genetic diversity and clinical outcomes observed in humans are needed. We report a mouse panel comprising the genetically diverse Collaborative Cross (CC) founder strains crossed to human ACE2 transgenic mice (K18-hACE2) that confers susceptibility to SARS-CoV-2. Infection of CC x K18-hACE2 resulted in a spectrum of survival, viral replication kinetics, and immune profiles. Importantly, in contrast to the K18-hACE2 model, early type I interferon (IFN-I) and regulated proinflammatory responses were required for control of SARS-CoV-2 replication in PWK x K18-hACE2 mice that were highly resistant to disease. Thus, virus dynamics and inflammation observed in COVID-19 can be modeled in diverse mouse strains that provide a genetically tractable platform for understanding anti-coronavirus immunity.
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COVID-19 , Interferón Tipo I , Humanos , Ratones , Animales , Citocinas , SARS-CoV-2 , Ratones Transgénicos , Inflamación/genética , Modelos Animales de Enfermedad , PulmónRESUMEN
Inflammation in response to severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) infection drives severity of coronavirus disease 2019 (COVID-19) and is influenced by host genetics. To understand mechanisms of inflammation, animal models that reflect genetic diversity and clinical outcomes observed in humans are needed. We report a mouse panel comprising the genetically diverse Collaborative Cross (CC) founder strains crossed to human ACE2 transgenic mice (K18-hACE2) that confers susceptibility to SARS-CoV-2. Infection of CC x K18- hACE2 resulted in a spectrum of survival, viral replication kinetics, and immune profiles. Importantly, in contrast to the K18-hACE2 model, early type I interferon (IFN-I) and regulated proinflammatory responses were required for control of SARS-CoV-2 replication in PWK x K18-hACE2 mice that were highly resistant to disease. Thus, virus dynamics and inflammation observed in COVID-19 can be modeled in diverse mouse strains that provide a genetically tractable platform for understanding anti-coronavirus immunity.
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Kyasanur Forest disease virus (KFDV) and the closely related Alkhurma hemorrhagic disease virus (AHFV) are emerging flaviviruses that cause severe viral hemorrhagic fevers in humans. Increasing geographical expansion and case numbers, particularly of KFDV in southwest India, class these viruses as a public health threat. Viral pathogenesis is not well understood and additional vaccines and antivirals are needed to effectively counter the impact of these viruses. However, current animal models of KFDV pathogenesis do not accurately reproduce viral tissue tropism or clinical outcomes observed in humans. Here, we show that pigtailed macaques (Macaca nemestrina) infected with KFDV or AHFV develop viremia that peaks 2 to 4 days following inoculation. Over the course of infection, animals developed lymphocytopenia, thrombocytopenia, and elevated liver enzymes. Infected animals exhibited hallmark signs of human disease characterized by a flushed appearance, piloerection, dehydration, loss of appetite, weakness, and hemorrhagic signs including epistaxis. Virus was commonly present in the gastrointestinal tract, consistent with human disease caused by KFDV and AHFV where gastrointestinal symptoms (hemorrhage, vomiting, diarrhea) are common. Importantly, RNAseq of whole blood revealed that KFDV downregulated gene expression of key clotting factors that was not observed during AHFV infection, consistent with increased severity of KFDV disease observed in this model. This work characterizes a nonhuman primate model for KFDV and AHFV that closely resembles human disease for further utilization in understanding host immunity and development of antiviral countermeasures.
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Modelos Animales de Enfermedad , Virus de la Encefalitis Transmitidos por Garrapatas/patogenicidad , Encefalitis Transmitida por Garrapatas/virología , Fiebres Hemorrágicas Virales/virología , Macaca nemestrina , Animales , Chlorocebus aethiops , Citocinas/sangre , Virus de la Encefalitis Transmitidos por Garrapatas/genética , Virus de la Encefalitis Transmitidos por Garrapatas/inmunología , Encefalitis Transmitida por Garrapatas/inmunología , Encefalitis Transmitida por Garrapatas/patología , Femenino , Células HEK293 , Fiebres Hemorrágicas Virales/inmunología , Fiebres Hemorrágicas Virales/patología , Humanos , Ganglios Linfáticos/virología , Células Vero , ViremiaRESUMEN
An evolutionary arms race has been ongoing between retroviruses and their primate hosts for millions of years. Within the last century, a zoonotic transmission introduced the Human Immunodeficiency Virus (HIV-1), a retrovirus, to the human population that has claimed the lives of millions of individuals and is still infecting over a million people every year. To counteract retroviruses such as this, primates including humans have evolved an innate immune sensor for the retroviral capsid lattice known as TRIM5α. Although the molecular basis for its ability to restrict retroviruses is debated, it is currently accepted that TRIM5α forms higher-order assemblies around the incoming retroviral capsid that are not only disruptive for the virus lifecycle, but also trigger the activation of an antiviral state. More recently, it was discovered that TRIM5α restriction is broader than previously thought because it restricts not only the human retroelement LINE-1, but also the tick-borne flaviviruses, an emergent group of RNA viruses that have vastly different strategies for replication compared to retroviruses. This review focuses on the underlying mechanisms of TRIM5α-mediated restriction of retroelements and flaviviruses and how they differ from the more widely known ability of TRIM5α to restrict retroviruses.
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Cápside/inmunología , Inmunidad Innata , Virus ARN/inmunología , Virus ARN/metabolismo , Proteínas de Motivos Tripartitos/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo , Factores de Restricción Antivirales , Cápside/metabolismo , Proteínas Portadoras/genética , Flavivirus/inmunología , Flavivirus/metabolismo , Humanos , Virus ARN/clasificación , Virus ARN/genética , Retroviridae/inmunología , Retroviridae/metabolismo , Infecciones por Retroviridae/inmunología , Infecciones por Retroviridae/prevención & control , Proteínas de Motivos Tripartitos/genética , Proteínas de Motivos Tripartitos/inmunología , Ubiquitina-Proteína Ligasas/genética , Ubiquitina-Proteína Ligasas/inmunologíaRESUMEN
Although Powassan virus (POWV) is an emerging tick-transmitted flavivirus that causes severe or fatal neuroinvasive disease in humans, medical countermeasures have not yet been developed. Here, we developed a panel of neutralizing anti-POWV mAbs recognizing six distinct antigenic sites. The most potent of these mAbs bind sites within domain II or III of the envelope (E) protein and inhibit postattachment viral entry steps. A subset of these mAbs cross-react with other flaviviruses. Both POWV type-specific and cross-reactive neutralizing mAbs confer protection in mice against POWV infection when given as prophylaxis or postexposure therapy. Several cross-reactive mAbs mapping to either domain II or III also protect in vivo against heterologous tick-transmitted flaviviruses including Langat and tick-borne encephalitis virus. Our experiments define structural and functional correlates of antibody protection against POWV infection and identify epitopes targeted by broadly neutralizing antibodies with therapeutic potential against multiple tick-borne flaviviruses.
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Anticuerpos Monoclonales/inmunología , Anticuerpos Neutralizantes/inmunología , Virus de la Encefalitis Transmitidos por Garrapatas/inmunología , Encefalitis Transmitida por Garrapatas/inmunología , Vacunas Virales/inmunología , Animales , Anticuerpos Monoclonales/administración & dosificación , Anticuerpos Neutralizantes/administración & dosificación , Línea Celular , Chlorocebus aethiops , Reacciones Cruzadas/inmunología , Virus de la Encefalitis Transmitidos por Garrapatas/efectos de los fármacos , Virus de la Encefalitis Transmitidos por Garrapatas/genética , Virus de la Encefalitis Transmitidos por Garrapatas/fisiología , Encefalitis Transmitida por Garrapatas/prevención & control , Encefalitis Transmitida por Garrapatas/virología , Epítopos/inmunología , Células HEK293 , Humanos , Inmunoglobulina G/administración & dosificación , Inmunoglobulina G/inmunología , Ratones Endogámicos C57BL , Mutación , Células Vero , Proteínas del Envoltorio Viral/inmunología , Vacunas Virales/administración & dosificaciónRESUMEN
Mayaro virus (MAYV) is an alphavirus endemic to South and Central America associated with sporadic outbreaks in humans. MAYV infection causes severe joint and muscle pain that can persist for weeks to months. Currently, there are no approved vaccines or therapeutics to prevent MAYV infection or treat the debilitating musculoskeletal inflammatory disease. In the current study, a prophylactic MAYV vaccine expressing the complete viral structural polyprotein was developed based on a non-replicating human adenovirus V (AdV) platform. Vaccination with AdV-MAYV elicited potent neutralizing antibodies that protected WT mice against MAYV challenge by preventing viremia, reducing viral dissemination to tissues and mitigating viral disease. The vaccine also prevented viral-mediated demise in IFNâºR1-/- mice. Passive transfer of immune serum from vaccinated animals similarly prevented infection and disease in WT mice as well as virus-induced demise of IFNâºR1-/- mice, indicating that antiviral antibodies are protective. Immunization with AdV-MAYV also generated cross-neutralizing antibodies against two related arthritogenic alphaviruses-chikungunya and Una viruses. These cross-neutralizing antibodies were protective against lethal infection in IFNâºR1-/- mice following challenge with these heterotypic alphaviruses. These results indicate AdV-MAYV elicits protective immune responses with substantial cross-reactivity and protective efficacy against other arthritogenic alphaviruses. Our findings also highlight the potential for development of a multi-virus targeting vaccine against alphaviruses with endemic and epidemic potential in the Americas.
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Adenoviridae/genética , Alphavirus/inmunología , Fiebre Chikungunya/prevención & control , Virus Chikungunya/inmunología , Vacunas Virales/inmunología , Animales , Anticuerpos Neutralizantes/inmunología , Anticuerpos Antivirales/inmunología , Protección Cruzada/inmunología , Modelos Animales de Enfermedad , Femenino , Ingeniería Genética/métodos , Vectores Genéticos/genética , Inmunización , Masculino , Ratones , Ratones Endogámicos C57BL , Vacunas Virales/administración & dosificación , Vacunas Virales/genéticaRESUMEN
Repurposing FDA-approved drugs that treat respiratory infections caused by coronaviruses, such as SARS-CoV-2 and MERS-CoV, could quickly provide much needed antiviral therapies. In the current study, the potency and cellular toxicity of four fluoroquinolones (enoxacin, ciprofloxacin, levofloxacin, and moxifloxacin) were assessed in Vero cells and A549 cells engineered to overexpress ACE2, the SARS-CoV-2 entry receptor. All four fluoroquinolones suppressed SARS-CoV-2 replication at high micromolar concentrations in both cell types, with enoxacin demonstrating the lowest effective concentration 50 value (EC50) of 126.4 µM in Vero cells. Enoxacin also suppressed the replication of MERS-CoV-2 in Vero cells at high micromolar concentrations. Cellular toxicity of levofloxacin was not found in either cell type. In Vero cells, minimal toxicity was observed following treatment with ≥37.5 µM enoxacin and 600 µM ciprofloxacin. Toxicity in both cell types was detected after moxifloxacin treatment of ≥300 µM. In summary, these results suggest that the ability of fluoroquinolones to suppress SARS-CoV-2 and MERS-CoV replication in cultured cells is limited.
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Antibacterianos/farmacología , Antivirales/farmacología , Tratamiento Farmacológico de COVID-19 , Infecciones por Coronavirus/tratamiento farmacológico , Fluoroquinolonas/farmacología , Coronavirus del Síndrome Respiratorio de Oriente Medio/efectos de los fármacos , SARS-CoV-2/efectos de los fármacos , Células A549 , Enzima Convertidora de Angiotensina 2 , Animales , Línea Celular , Chlorocebus aethiops , Ciprofloxacina/farmacología , Enoxacino/farmacología , Humanos , Levofloxacino/farmacología , Moxifloxacino/farmacología , Células VeroRESUMEN
Chikungunya virus (CHIKV) infections can cause severe and debilitating joint and muscular pain that can be long lasting. Current CHIKV vaccines under development rely on the generation of neutralizing antibodies for protection; however, the role of T cells in controlling CHIKV infection and disease is still unclear. Using an overlapping peptide library, we identified the CHIKV-specific T cell receptor epitopes recognized in C57BL/6 infected mice at 7 and 14 days post-infection. A fusion protein containing peptides 451, 416, a small region of nsP4, peptide 47, and an HA tag (CHKVf5) was expressed using adenovirus and cytomegalovirus-vectored vaccines. Mice vaccinated with CHKVf5 elicited robust T cell responses to higher levels than normally observed following CHIKV infection, but the vaccine vectors did not elicit neutralizing antibodies. CHKVf5-vaccinated mice had significantly reduced infectious viral load when challenged by intramuscular CHIKV injection. Depletion of both CD4+ and CD8+ T cells in vaccinated mice rendered them fully susceptible to intramuscular CHIKV challenge. Depletion of CD8+ T cells alone reduced vaccine efficacy, albeit to a lesser extent, but depletion of only CD4+ T cells did not reverse the protective phenotype. These data demonstrated a protective role for CD8+ T cells in CHIKV infection. However, CHKVf5-vaccinated mice that were challenged by footpad inoculation demonstrated equal viral loads and increased footpad swelling at 3 dpi, which we attributed to the presence of CD4 T cell receptor epitopes present in the vaccine. Indeed, vaccination of mice with vectors expressing only CHIKV-specific CD8+ T cell epitopes followed by CHIKV challenge in the footpad prevented footpad swelling and reduced proinflammatory cytokine and chemokines associated with disease, indicating that CHIKV-specific CD8+ T cells prevent CHIKV disease. These results also indicate that a T cell-biased prophylactic vaccination approach is effective against CHIKV challenge and reduces CHIKV-induced disease in mice.
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Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD8-positivos/inmunología , Fiebre Chikungunya/prevención & control , Virus Chikungunya/inmunología , Vacunación , Vacunas Virales/inmunología , Animales , Fiebre Chikungunya/genética , Fiebre Chikungunya/inmunología , Virus Chikungunya/genética , Chlorocebus aethiops , Células HEK293 , Humanos , Ratones , Células 3T3 NIH , Células Vero , Vacunas Virales/genéticaRESUMEN
Tripartite motif-containing protein 5α (TRIM5α) is a cellular antiviral restriction factor that prevents early events in retrovirus replication. The activity of TRIM5α is thought to be limited to retroviruses as a result of highly specific interactions with capsid lattices. In contrast to this current understanding, we show that both human and rhesus macaque TRIM5α suppress replication of specific flaviviruses. Multiple viruses in the tick-borne encephalitis complex are sensitive to TRIM5α-dependent restriction, but mosquito-borne flaviviruses, including yellow fever, dengue, and Zika viruses, are resistant. TRIM5α suppresses replication by binding to the viral protease NS2B/3 to promote its K48-linked ubiquitination and proteasomal degradation. Importantly, TRIM5α contributes to the antiviral function of IFN-I against sensitive flaviviruses in human cells. Thus, TRIM5α possesses remarkable plasticity in the recognition of diverse virus families, with the potential to influence human susceptibility to emerging flaviviruses of global concern.
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Infecciones por Flavivirus/metabolismo , Péptido Hidrolasas/metabolismo , Complejo de la Endopetidasa Proteasomal/metabolismo , Proteínas de Motivos Tripartitos/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo , Proteínas Virales/metabolismo , Replicación Viral , Animales , Factores de Restricción Antivirales , Gatos , Chlorocebus aethiops , Células Dendríticas/metabolismo , Células Dendríticas/virología , Flavivirus/patogenicidad , Flavivirus/fisiología , Infecciones por Flavivirus/virología , Células HEK293 , Humanos , Unión Proteica , Proteolisis , Especificidad por Sustrato , Ubiquitinación , Células VeroRESUMEN
Alphaviruses are arthropod-transmitted RNA viruses that can cause arthralgia, myalgia, and encephalitis in humans. Since the role of cellular kinases in alphavirus replication is unknown, we profiled kinetic changes in host kinase abundance and phosphorylation following chikungunya virus (CHIKV) infection of fibroblasts. Based upon the results of this study, we treated CHIKV-infected cells with kinase inhibitors targeting the Src family kinase (SFK)-phosphatidylinositol 3-kinase (PI3K)-AKT-mTORC signaling pathways. Treatment of cells with SFK inhibitors blocked the replication of CHIKV as well as multiple other alphaviruses, including Mayaro virus, O'nyong-nyong virus, Ross River virus, and Venezuelan equine encephalitis virus. Dissecting the effect of SFK inhibition on alphavirus replication, we found that viral structural protein levels were significantly reduced, but synthesis of viral genomic and subgenomic RNAs was unaffected. By measuring the association of viral RNA with polyribosomes, we found that the SFK inhibitor dasatinib blocks alphavirus subgenomic RNA translation. Our results demonstrate a role for SFK signaling in alphavirus subgenomic RNA translation and replication. Targeting host factors involved in alphavirus replication represents an innovative, perhaps paradigm-shifting, strategy for exploring the replication of CHIKV and other alphaviruses while promoting antiviral therapeutic development.
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Infecciones por Alphavirus/tratamiento farmacológico , Alphavirus/efectos de los fármacos , Biosíntesis de Proteínas/efectos de los fármacos , Inhibidores de Proteínas Quinasas/farmacología , ARN Mensajero/genética , Familia-src Quinasas/genética , Alphavirus/genética , Infecciones por Alphavirus/virología , Animales , Antivirales/farmacología , Línea Celular , Chlorocebus aethiops , Genoma Viral/efectos de los fármacos , Genoma Viral/genética , Humanos , Ratones Noqueados , Fosfatidilinositol 3-Quinasas/genética , ARN Viral/genética , Transducción de Señal/efectos de los fármacos , Transducción de Señal/genética , Células Vero , Proteínas Virales/genética , Replicación Viral/efectos de los fármacos , Replicación Viral/genéticaRESUMEN
Zika virus (ZIKV) infection during pregnancy leads to an increased risk of fetal growth restriction and fetal central nervous system malformations, which are outcomes broadly referred to as the Congenital Zika Syndrome (CZS). Here we infect pregnant rhesus macaques and investigate the impact of persistent ZIKV infection on uteroplacental pathology, blood flow, and fetal growth and development. Despite seemingly normal fetal growth and persistent fetal-placenta-maternal infection, advanced non-invasive in vivo imaging studies reveal dramatic effects on placental oxygen reserve accompanied by significantly decreased oxygen permeability of the placental villi. The observation of abnormal oxygen transport within the placenta appears to be a consequence of uterine vasculitis and placental villous damage in ZIKV cases. In addition, we demonstrate a robust maternal-placental-fetal inflammatory response following ZIKV infection. This animal model reveals a potential relationship between ZIKV infection and uteroplacental pathology that appears to affect oxygen delivery to the fetus during development.
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Placenta/metabolismo , Circulación Placentaria , Complicaciones Infecciosas del Embarazo/inmunología , Infección por el Virus Zika/inmunología , Inmunidad Adaptativa , Animales , Encéfalo/embriología , Encéfalo/patología , Citocinas/sangre , Modelos Animales de Enfermedad , Femenino , Desarrollo Fetal , Feto/patología , Inmunidad Innata , Macaca mulatta , Imagen por Resonancia Magnética , Oxígeno/metabolismo , Permeabilidad , Placenta/inmunología , Placenta/patología , Placenta/virología , Embarazo , Complicaciones Infecciosas del Embarazo/metabolismo , Complicaciones Infecciosas del Embarazo/patología , Complicaciones Infecciosas del Embarazo/fisiopatología , Carga Viral , Infección por el Virus Zika/metabolismo , Infección por el Virus Zika/patología , Infección por el Virus Zika/fisiopatologíaRESUMEN
The type I interferon (IFN) system represents an essential innate immune response that renders cells resistant to virus growth via the molecular actions of IFN-induced effector proteins. IFN-mediated cellular states inhibit growth of numerous and diverse virus types, including those of known pathogenicity as well as potentially emerging agents. As such, targeted pharmacologic activation of the IFN response may represent a novel therapeutic strategy to prevent infection or spread of clinically impactful viruses. In light of this, we employed a high-throughput screen to identify small molecules capable of permeating the cell and of activating IFN-dependent signaling processes. Here we report the identification and characterization of N-(methylcarbamoyl)-2-{[5-(4-methylphenyl)-1,3,4-oxadiazol-2-yl]sulfanyl}-2-phenylacetamide (referred to as C11), a novel compound capable of inducing IFN secretion from human cells. Using reverse genetics-based loss-of-function assays, we show that C11 activates the type I IFN response in a manner that requires the adaptor protein STING but not the alternative adaptors MAVS and TRIF. Importantly, treatment of cells with C11 generated a cellular state that potently blocked replication of multiple emerging alphavirus types, including chikungunya, Ross River, Venezuelan equine encephalitis, Mayaro, and O'nyong-nyong viruses. The antiviral effects of C11 were subsequently abrogated in cells lacking STING or the type I IFN receptor, indicating that they are mediated, at least predominantly, by way of STING-mediated IFN secretion and subsequent autocrine/paracrine signaling. This work also allowed characterization of differential antiviral roles of innate immune signaling adaptors and IFN-mediated responses and identified MAVS as being crucial to cellular resistance to alphavirus infection.IMPORTANCE Due to the increase in emerging arthropod-borne viruses, such as chikungunya virus, that lack FDA-approved therapeutics and vaccines, it is important to better understand the signaling pathways that lead to clearance of virus. Here we show that C11 treatment makes human cells refractory to replication of a number of these viruses, which supports its value in increasing our understanding of the immune response and viral pathogenesis required to establish host infection. We also show that C11 depends on signaling through STING to produce antiviral type I interferon, which further supports its potential as a therapeutic drug or research tool.
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Alphavirus/metabolismo , Antivirales/farmacología , Fibroblastos/metabolismo , Proteínas de la Membrana/agonistas , Transducción de Señal/efectos de los fármacos , Proteínas Adaptadoras Transductoras de Señales/genética , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Proteínas Adaptadoras del Transporte Vesicular/genética , Proteínas Adaptadoras del Transporte Vesicular/metabolismo , Comunicación Autocrina/efectos de los fármacos , Comunicación Autocrina/genética , Fibroblastos/patología , Fibroblastos/virología , Humanos , Interferón Tipo I/genética , Interferón Tipo I/metabolismo , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Comunicación Paracrina/efectos de los fármacos , Comunicación Paracrina/genética , Receptor de Interferón alfa y beta/genética , Receptor de Interferón alfa y beta/metabolismo , Transducción de Señal/genéticaRESUMEN
Chikungunya virus (CHIKV) is a mosquito-borne virus that causes a febrile syndrome in humans associated with acute and chronic debilitating joint and muscle pain. Currently no licensed vaccines or therapeutics are available to prevent or treat CHIKV infections. We recently isolated a panel of potently neutralizing human monoclonal antibodies (mAbs), one (4N12) of which exhibited prophylactic and post-exposure therapeutic activity against CHIKV in immunocompromised mice. Here, we describe the development of an engineered CHIKV mAb, designated SVIR001, that has similar antigen binding and neutralization profiles to its parent, 4N12. Because therapeutic administration of SVIR001 in immunocompetent mice significantly reduced viral load in joint tissues, we evaluated its efficacy in a rhesus macaque model of CHIKV infection. Rhesus macaques that were treated after infection with SVIR001 showed rapid elimination of viremia and less severe joint infiltration and disease compared to animals treated with SVIR002, an isotype control mAb. SVIR001 reduced viral burden at the site of infection and at distant sites and also diminished the numbers of activated innate immune cells and levels of pro-inflammatory cytokines and chemokines. SVIR001 therapy; however, did not substantively reduce the induction of CHIKV-specific B or T cell responses. Collectively, these results show promising therapeutic activity of a human anti-CHIKV mAb in rhesus macaques and provide proof-of-principle for its possible use in humans to treat active CHIKV infections.
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Anticuerpos Monoclonales/administración & dosificación , Anticuerpos Antivirales/administración & dosificación , Fiebre Chikungunya/terapia , Factores Inmunológicos/administración & dosificación , Animales , Linfocitos B/inmunología , Fiebre Chikungunya/patología , Virus Chikungunya/inmunología , Modelos Animales de Enfermedad , Evaluación Preclínica de Medicamentos , Macaca mulatta , Linfocitos T/inmunología , Resultado del TratamientoRESUMEN
[This corrects the article DOI: 10.1371/journal.ppat.1006219.].
RESUMEN
Zika virus (ZIKV), an emerging flavivirus, has recently spread explosively through the Western hemisphere. In addition to symptoms including fever, rash, arthralgia, and conjunctivitis, ZIKV infection of pregnant women can cause microcephaly and other developmental abnormalities in the fetus. We report herein the results of ZIKV infection of adult rhesus macaques. Following subcutaneous infection, animals developed transient plasma viremia and viruria from 1-7 days post infection (dpi) that was accompanied by the development of a rash, fever and conjunctivitis. Animals produced a robust adaptive immune response to ZIKV, although systemic cytokine response was minimal. At 7 dpi, virus was detected in peripheral nervous tissue, multiple lymphoid tissues, joints, and the uterus of the necropsied animals. Notably, viral RNA persisted in neuronal, lymphoid and joint/muscle tissues and the male and female reproductive tissues through 28 to 35 dpi. The tropism and persistence of ZIKV in the peripheral nerves and reproductive tract may provide a mechanism of subsequent neuropathogenesis and sexual transmission.
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Infección por el Virus Zika/patología , Infección por el Virus Zika/virología , Animales , Separación Celular , Ensayo de Inmunoadsorción Enzimática , Femenino , Citometría de Flujo , Hibridación in Situ , Macaca mulatta , Masculino , Pruebas de Neutralización , Reacción en Cadena de la Polimerasa , Viremia/virología , Virus ZikaRESUMEN
Up to 80% of the cost of vaccination programmes is due to the cold chain problem (that is, keeping vaccines cold). Inexpensive, biocompatible additives to slow down the degradation of virus particles would address the problem. Here we propose and characterize additives that, already at very low concentrations, improve the storage time of adenovirus type 5. Anionic gold nanoparticles (10-8-10-6 M) or polyethylene glycol (PEG, molecular weight â¼8,000 Da, 10-7-10-4 M) increase the half-life of a green fluorescent protein expressing adenovirus from â¼48 h to 21 days at 37 °C (from 7 to >30 days at room temperature). They replicate the known stabilizing effect of sucrose, but at several orders of magnitude lower concentrations. PEG and sucrose maintained immunogenicity in vivo for viruses stored for 10 days at 37 °C. To achieve rational design of viral-vaccine stabilizers, our approach is aided by simplified quantitative models based on a single rate-limiting step.
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Vacunas contra el Adenovirus/farmacología , Estabilidad de Medicamentos , Excipientes/química , Inmunogenicidad Vacunal , Vacunas contra el Adenovirus/química , Vacunas contra el Adenovirus/inmunología , Animales , Frío , Almacenaje de Medicamentos/métodos , Estudios de Factibilidad , Oro/química , Semivida , Nanopartículas del Metal/química , Ratones , Modelos Animales , Modelos Biológicos , Polietilenglicoles/química , Sacarosa/química , Factores de TiempoRESUMEN
Chikungunya virus (CHIKV) is a reemerging alphavirus that causes acute febrile illness and severe joint pain in humans. Although acute symptoms often resolve within a few days, chronic joint and muscle pain can be long lasting. In the last decade, CHIKV has caused widespread outbreaks of unprecedented scale in the Americas, Asia, and the Indian Ocean island regions. Despite these outbreaks and the continued expansion of CHIKV into new areas, mechanisms of chikungunya pathogenesis and disease are not well understood. Experimental animal models are indispensable to the field of CHIKV research. The most commonly used experimental animal models of CHIKV infection are mice and nonhuman primates; each model has its advantages for studying different aspects of CHIKV disease. This review will provide an overview of animal models used to study CHIKV infection and disease and major advances in our understanding of chikungunya obtained from studies performed in these models.
Asunto(s)
Fiebre Chikungunya , Virus Chikungunya/patogenicidad , Modelos Animales de Enfermedad , Animales , Fiebre Chikungunya/virología , Virus Chikungunya/inmunología , Virus Chikungunya/fisiología , Ratones , PrimatesRESUMEN
Chikungunya virus (CHIKV) is a re-emerging global pathogen with pandemic potential, which causes fever, rash and debilitating arthralgia. Older adults over 65 years are particularly susceptible to severe and chronic CHIKV disease (CHIKVD), accounting for >90% of all CHIKV-related deaths. There are currently no approved vaccines or antiviral treatments available to limit chronic CHIKVD. Here we show that in old mice excessive, dysregulated TGFß production during acute infection leads to a reduced immune response and subsequent chronic disease. Humans suffering from CHIKV infection also exhibited high TGFß levels and a pronounced age-related defect in neutralizing anti-CHIKV antibody production. In vivo reduction of TGFß levels minimized acute joint swelling, restored neutralizing antibody production and diminished chronic joint pathology in old mice. This study identifies increased and dysregulated TGFß secretion as one key mechanism contributing to the age-related loss of protective anti-CHIKV-immunity leading to chronic CHIKVD.
Asunto(s)
Envejecimiento/inmunología , Fiebre Chikungunya/inmunología , Factor de Crecimiento Transformador beta/inmunología , Adulto , Anciano , Animales , Anticuerpos Neutralizantes/sangre , Anticuerpos Neutralizantes/inmunología , Anticuerpos Antivirales/sangre , Anticuerpos Antivirales/inmunología , Virus Chikungunya , Modelos Animales de Enfermedad , Susceptibilidad a Enfermedades , Ensayo de Inmunoadsorción Enzimática , Femenino , Citometría de Flujo , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , Reacción en Cadena de la Polimerasa , Factor de Crecimiento Transformador beta/biosíntesisRESUMEN
Pharmacologic stimulation of innate immune processes represents an attractive strategy to achieve multiple therapeutic outcomes including inhibition of virus replication, boosting antitumor immunity, and enhancing vaccine immunogenicity. In light of this we sought to identify small molecules capable of activating the type I interferon (IFN) response by way of the transcription factor IFN regulatory factor 3 (IRF3). A high throughput in vitro screen yielded 4-(2-chloro-6-fluorobenzyl)-N-(furan-2-ylmethyl)-3-oxo-3,4-dihydro-2H-benzo[b][1,4]thiazine-6-carboxamide (referred to herein as G10), which was found to trigger IRF3/IFN-associated transcription in human fibroblasts. Further examination of the cellular response to this molecule revealed expression of multiple IRF3-dependent antiviral effector genes as well as type I and III IFN subtypes. This led to the establishment of a cellular state that prevented replication of emerging Alphavirus species including Chikungunya virus, Venezuelan Equine Encephalitis virus, and Sindbis virus. To define cellular proteins essential to elicitation of the antiviral activity by the compound we employed a reverse genetics approach that utilized genome editing via CRISPR/Cas9 technology. This allowed the identification of IRF3, the IRF3-activating adaptor molecule STING, and the IFN-associated transcription factor STAT1 as required for observed gene induction and antiviral effects. Biochemical analysis indicates that G10 does not bind to STING directly, however. Thus the compound may represent the first synthetic small molecule characterized as an indirect activator of human STING-dependent phenotypes. In vivo stimulation of STING-dependent activity by an unrelated small molecule in a mouse model of Chikungunya virus infection blocked viremia demonstrating that pharmacologic activation of this signaling pathway may represent a feasible strategy for combating emerging Alphaviruses.