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1.
PLoS Pathog ; 20(5): e1011749, 2024 May.
Artículo en Inglés | MEDLINE | ID: mdl-38739648

RESUMEN

Hepatitis delta virus (HDV) infection represents the most severe form of human viral hepatitis; however, the mechanisms underlying its pathology remain incompletely understood. We recently developed an HDV mouse model by injecting adeno-associated viral vectors (AAV) containing replication-competent HBV and HDV genomes. This model replicates many features of human infection, including liver injury. Notably, the extent of liver damage can be diminished with anti-TNF-α treatment. Here, we found that TNF-α is mainly produced by macrophages. Downstream of the TNF-α receptor (TNFR), the receptor-interacting serine/threonine-protein kinase 1 (RIPK1) serves as a cell fate regulator, playing roles in both cell survival and death pathways. In this study, we explored the function of RIPK1 and other host factors in HDV-induced cell death. We determined that the scaffolding function of RIPK1, and not its kinase activity, offers partial protection against HDV-induced apoptosis. A reduction in RIPK1 expression in hepatocytes through CRISPR-Cas9-mediated gene editing significantly intensifies HDV-induced damage. Contrary to our expectations, the protective effect of RIPK1 was not linked to TNF-α or macrophage activation, as their absence did not alter the extent of damage. Intriguingly, in the absence of RIPK1, macrophages confer a protective role. However, in animals unresponsive to type-I IFNs, RIPK1 downregulation did not exacerbate the damage, suggesting RIPK1's role in shielding hepatocytes from type-I IFN-induced cell death. Interestingly, while the damage extent is similar between IFNα/ßR KO and wild type mice in terms of transaminase elevation, their cell death mechanisms differ. In conclusion, our findings reveal that HDV-induced type-I IFN production is central to inducing hepatocyte death, and RIPK1's scaffolding function offers protective benefits. Thus, type-I IFN together with TNF-α, contribute to HDV-induced liver damage. These insights may guide the development of novel therapeutic strategies to mitigate HDV-induced liver damage and halt disease progression.


Asunto(s)
Citocinas , Virus de la Hepatitis Delta , Hepatocitos , Proteína Serina-Treonina Quinasas de Interacción con Receptores , Animales , Ratones , Hepatocitos/metabolismo , Proteína Serina-Treonina Quinasas de Interacción con Receptores/metabolismo , Proteína Serina-Treonina Quinasas de Interacción con Receptores/genética , Citocinas/metabolismo , Virus de la Hepatitis Delta/fisiología , Hepatitis D/metabolismo , Muerte Celular , Ratones Endogámicos C57BL , Apoptosis , Ratones Noqueados , Humanos , Factor de Necrosis Tumoral alfa/metabolismo , Modelos Animales de Enfermedad
2.
Mol Ther ; 30(12): 3515-3541, 2022 12 07.
Artículo en Inglés | MEDLINE | ID: mdl-36203359

RESUMEN

Defective genes account for ∼80% of the total of more than 7,000 diseases known to date. Gene therapy brings the promise of a one-time treatment option that will fix the errors in patient genetic coding. Recombinant viruses are highly efficient vehicles for in vivo gene delivery. Adeno-associated virus (AAV) vectors offer unique advantages, such as tissue tropism, specificity in transduction, eliciting of a relatively low immune responses, no incorporation into the host chromosome, and long-lasting delivered gene expression, making them the most popular viral gene delivery system in clinical trials, with three AAV-based gene therapy drugs already approved by the US Food and Drug Administration (FDA) or European Medicines Agency (EMA). Despite the success of AAV vectors, their usage in particular scenarios is still limited due to remaining challenges, such as poor transduction efficiency in certain tissues, low organ specificity, pre-existing humoral immunity to AAV capsids, and vector dose-dependent toxicity in patients. In the present review, we address the different approaches to improve AAV vectors for gene therapy with a focus on AAV capsid selection and engineering, strategies to overcome anti-AAV immune response, and vector genome design, ending with a glimpse at vector production methods and the current state of recombinant AAV (rAAV) at the clinical level.


Asunto(s)
Terapia Genética , Estados Unidos , Humanos , United States Food and Drug Administration
3.
Proc Natl Acad Sci U S A ; 115(39): E9172-E9181, 2018 09 25.
Artículo en Inglés | MEDLINE | ID: mdl-30206152

RESUMEN

Genome-wide investigations of host-pathogen interactions are often limited by analyses of mixed populations of infected and uninfected cells, which lower sensitivity and accuracy. To overcome these obstacles and identify key mechanisms by which Zika virus (ZIKV) manipulates host responses, we developed a system that enables simultaneous characterization of genome-wide transcriptional and epigenetic changes in ZIKV-infected and neighboring uninfected primary human macrophages. We demonstrate that transcriptional responses in ZIKV-infected macrophages differed radically from those in uninfected neighbors and that studying the cell population as a whole produces misleading results. Notably, the uninfected population of macrophages exhibits the most rapid and extensive changes in gene expression, related to type I IFN signaling. In contrast, infected macrophages exhibit a delayed and attenuated transcriptional response distinguished by preferential expression of IFNB1 at late time points. Biochemical and genomic studies of infected macrophages indicate that ZIKV infection causes both a targeted defect in the type I IFN response due to degradation of STAT2 and reduces RNA polymerase II protein levels and DNA occupancy, particularly at genes required for macrophage identity. Simultaneous evaluation of transcriptomic and epigenetic features of infected and uninfected macrophages thereby reveals the coincident evolution of dominant proviral or antiviral mechanisms, respectively, that determine the outcome of ZIKV exposure.


Asunto(s)
Inmunidad Innata , Macrófagos/inmunología , Infección por el Virus Zika/inmunología , Virus Zika/inmunología , Efecto Espectador , Femenino , Humanos , Interferón beta/genética , Interferón beta/inmunología , Macrófagos/patología , Masculino , Proteolisis , ARN Polimerasa II/genética , ARN Polimerasa II/inmunología , Factor de Transcripción STAT2/genética , Factor de Transcripción STAT2/inmunología , Infección por el Virus Zika/patología
4.
J Hepatol ; 67(4): 669-679, 2017 10.
Artículo en Inglés | MEDLINE | ID: mdl-28527664

RESUMEN

BACKGROUND & AIMS: Studying hepatitis delta virus (HDV) and developing new treatments is hampered by the limited availability of small animal models. Herein, a description of a robust mouse model of HDV infection that mimics several important characteristics of the human disease is presented. METHODS: HDV and hepatitis B virus (HBV) replication competent genomes were delivered to the mouse liver using adeno-associated viruses (AAV; AAV-HDV and AAV-HBV). Viral load, antigen expression and genomes were quantified at different time points after AAV injection. Furthermore, liver pathology, genome editing, and the activation of the innate immune response were evaluated. RESULTS: AAV-HDV infection initiated HDV replication in mouse hepatocytes. Genome editing was confirmed by the presence of small and large HDV antigens and sequencing. Viral replication was detected for 45days, even after the AAV-HDV vector had almost disappeared. In the presence of HBV, HDV infectious particles were detected in serum. Furthermore, as observed in patients, co-infection was associated with the reduction of HBV antigen expression and the onset of liver damage that included the alteration of genes involved in the development of liver pathologies. HDV replication induced a sustained type I interferon response, which was significantly reduced in immunodeficient mice and almost absent in mitochondrial antiviral signaling protein (MAVS)-deficient mice. CONCLUSION: The animal model described here reproduces important characteristics of human HDV infection and provides a valuable tool for characterizing the viral infection and for developing new treatments. Furthermore, MAVS was identified as a main player in HDV detection and adaptive immunity was found to be involved in the amplification of the innate immune response. Lay summary: Co-infection with hepatitis B and D virus (HBV and HDV, respectively) often causes a more severe disease condition than HBV alone. Gaining more insight into HDV and developing new treatments is hampered by limited availability of adequate immune competent small animal models and new ones are needed. Here, a mouse model of HDV infection is described, which mimics several important characteristics of the human disease, such as the initiation and maintenance of replication in murine hepatocytes, genome editing and, in the presence of HBV, generation of infectious particles. Lastly, the involvement of an adaptive immunity and the intracellular signaling molecule MAVS in mounting a strong and lasting innate response was shown. Thus, our model serves as a useful tool for the investigation of HDV biology and new treatments.


Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/inmunología , Hepatitis D/inmunología , Interferón beta/biosíntesis , Inmunidad Adaptativa , Proteínas Adaptadoras Transductoras de Señales/deficiencia , Proteínas Adaptadoras Transductoras de Señales/genética , Animales , Línea Celular , Coinfección/inmunología , Coinfección/patología , Coinfección/virología , Dependovirus/genética , Modelos Animales de Enfermedad , Genoma Viral , Hepatitis B/complicaciones , Hepatitis B/inmunología , Hepatitis B/virología , Antígenos de la Hepatitis B/metabolismo , Virus de la Hepatitis B/genética , Virus de la Hepatitis B/inmunología , Hepatitis D/complicaciones , Hepatitis D/virología , Virus de la Hepatitis Delta/genética , Virus de la Hepatitis Delta/inmunología , Virus de la Hepatitis Delta/fisiología , Antígenos de Hepatitis delta/metabolismo , Humanos , Inmunidad Innata , Hígado/patología , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Transgénicos , Modelos Inmunológicos , Transducción de Señal/inmunología , Replicación Viral
6.
JHEP Rep ; 2(3): 100098, 2020 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-32382723

RESUMEN

BACKGROUND & AIMS: HDV infection induces the most severe form of human viral hepatitis. However, the specific reasons for the severity of the disease remain unknown. Recently, we developed an HDV replication mouse model in which, for the first time, liver damage was detected. METHODS: HDV and HBV replication-competent genomes and HDV antigens were delivered to mouse hepatocytes using adeno-associated vectors (AAVs). Aminotransferase elevation, liver histopathology, and hepatocyte death were evaluated and the immune infiltrate was characterized. Liver transcriptomic analysis was performed. Mice deficient for different cellular and molecular components of the immune system, as well as depletion and inhibition studies, were employed to elucidate the causes of HDV-mediated liver damage. RESULTS: AAV-mediated HBV/HDV coinfection caused hepatocyte necrosis and apoptosis. Activated T lymphocytes, natural killer cells, and proinflammatory macrophages accounted for the majority of the inflammatory infiltrate. However, depletion studies and the use of different knockout mice indicated that neither T cells, natural killer cells nor macrophages were necessary for HDV-induced liver damage. Transcriptomic analysis revealed a strong activation of type I and II interferon (IFN) and tumor necrosis factor (TNF)-α pathways in HBV/HDV-coinfected mice. While the absence of IFN signaling had no effect, the use of a TNF-α antagonist resulted in a significant reduction of HDV-associated liver injury. Furthermore, hepatic expression of HDAg resulted in the induction of severe liver damage, which was T cell- and TNF-α-independent. CONCLUSIONS: Both host (TNF-α) and viral (HDV antigens) factors play a relevant role in HDV-induced liver damage. Importantly, pharmacological inhibition of TNF-α may offer an attractive strategy to aid control of HDV-induced acute liver damage. LAY SUMMARY: Chronic hepatitis delta constitutes the most severe form of viral hepatitis. There is limited data on the mechanism involved in hepatitis delta virus (HDV)-induced liver pathology. Our data indicate that a cytokine (TNF-α) and HDV antigens play a relevant role in HDV-induced liver damage.

7.
Pathogens ; 4(1): 46-65, 2015 Feb 12.
Artículo en Inglés | MEDLINE | ID: mdl-25686091

RESUMEN

Hepatitis delta virus (HDV) is a defective RNA virus that has an absolute requirement for a virus belonging to the hepadnaviridae family like hepatitis B virus (HBV) for its replication and formation of new virions. HDV infection is usually associated with a worsening of HBV-induced liver pathogenesis, which leads to more frequent cirrhosis, increased risk of hepatocellular carcinoma (HCC), and fulminant hepatitis. Importantly, no selective therapies are available for HDV infection. The mainstay of treatment for HDV infection is pegylated interferon alpha; however, response rates to this therapy are poor. A better knowledge of HDV-host cell interaction will help with the identification of novel therapeutic targets, which are urgently needed. Animal models like hepadnavirus-infected chimpanzees or the eastern woodchuck have been of great value for the characterization of HDV chronic infection. Recently, more practical animal models in which to perform a deeper study of host virus interactions and to evaluate new therapeutic strategies have been developed. Therefore, the main focus of this review is to discuss the current knowledge about HDV host interactions obtained from cell culture and animal models.

8.
Comp Immunol Microbiol Infect Dis ; 34(3): 259-65, 2011 May.
Artículo en Inglés | MEDLINE | ID: mdl-21190734

RESUMEN

Recombinant adenoviral vectors have emerged as an attractive system for veterinary vaccines development. However, for poultry vaccination a very important criterion for an ideal vaccine is its low cost. The objective of this study was to test the ability of chicken CD154 to enhance the immunogenicity of an adenoviral vector-based vaccine against avian influenza virus in order to reduce the amount of antigen required to induce an effective immune response in avian. Chickens were vaccinated with three different doses of adenoviral vectors encoding either HA (AdHA), or HA fused to extracellular domain chicken's CD154 (AdHACD). Hemagglutination inhibition (HI) assay and relative quantification of IFN-γ showed that the adenoviral vector encoding for the chimeric antigen is able to elicit an improved humoral and cellular immune response, which demonstrated that CD154 can be used as a molecular adjuvant allowing to reduce in about 50-fold the amount of adenoviral vector vaccine required to induce an effective immune response.


Asunto(s)
Ligando de CD40/inmunología , Inmunidad Celular/inmunología , Inmunidad Humoral/inmunología , Proteínas Recombinantes de Fusión/inmunología , Vacunas Virales/inmunología , Adenoviridae/genética , Animales , Anticuerpos Antivirales/sangre , Ligando de CD40/genética , Línea Celular , Pollos , Orden Génico , Vectores Genéticos/genética , Interferón gamma/inmunología , Leucocitos Mononucleares/inmunología , Ratones , Proteínas Recombinantes de Fusión/genética , Vacunas Virales/genética
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