[Show abstract][Hide abstract] ABSTRACT: The type I interferon (IFN) response protects cells from viral infection by inducing hundreds of interferon-stimulated genes (ISGs), some of which encode direct antiviral effectors. Recent screening studies have begun to catalogue ISGs with antiviral activity against several RNA and DNA viruses. However, antiviral ISG specificity across multiple distinct classes of viruses remains largely unexplored. Here we used an ectopic expression assay to screen a library of more than 350 human ISGs for effects on 14 viruses representing 7 families and 11 genera. We show that 47 genes inhibit one or more viruses, and 25 genes enhance virus infectivity. Comparative analysis reveals that the screened ISGs target positive-sense single stranded RNA viruses more effectively than negative-sense single stranded RNA viruses. Gene clustering highlights the cytosolic DNA sensor cyclic GMP-AMP synthase (cGAS, also known as MB21D1) as a gene whose expression also broadly inhibits several RNA viruses. In vitro, lentiviral delivery of enzymatically active cGAS triggers a STING-dependent, IRF3-mediated antiviral program that functions independently of canonical IFN/STAT1 signalling. In vivo, genetic ablation of murine cGAS reveals its requirement in the antiviral response to two DNA viruses, and an unappreciated contribution to the innate control of an RNA virus. These studies uncover new paradigms for the preferential specificity of IFN-mediated antiviral pathways spanning several virus families. (Hepatology 2014;)
[Show abstract][Hide abstract] ABSTRACT: Hepatitis C virus (HCV) particles associate with lipoproteins and infect cells using at least four cell entry factors. These include the scavenger receptor class B type I (SR-BI), CD81, claudin 1 (CLDN1) and occludin (OCLN). Little is known about specific functions of individual host factors during HCV cell entry and viral domains that mediate interaction with these factors. The HVR1 within the viral envelope protein 2 (E2) is involved in usage of SR-BI and conceals the viral CD81 binding site. Moreover, deletion of this domain alters the density of virions. We compared lipoprotein interaction, surface attachment, receptor usage and cell entry between wild type HCV and a viral mutant lacking this domain. Deletion of HVR1 did not affect CD81, CLDN1 and OCLN usage. However, unlike wild type HCV, HVR1-deleted viruses were not neutralized by antibodies and small molecules targeting SR-BI. Nevertheless, modulation of SR-BI cell surface expression altered infection efficiency of both viruses to similar levels. Analysis of affinity purified virions revealed comparable levels of ApoE incorporation into viruses with or without HVR1. However, ApoE incorporated into these viruses was differentially recognized by ApoE-specific antibodies. Thus, SR-BI has at least two functions during cell entry. One of them can be neutralized by SR-BI-targeting molecules and it is critical only for wild type HCV. The other one is important for both viruses, but apparently is not inactivated by those SR-BI-binding antibodies and small molecules evaluated here. In addition, HVR1 modulates the conformation and/or epitope exposure of virus particle assocated ApoE.
[Show abstract][Hide abstract] ABSTRACT: Background
Hepatitis B surface antigen (HBsAg) clearance is the main indicator of viral cure in patients infected with the hepatitis B virus (HBV).AimsWe sought to identify the parameters associated with HBsAg loss in a well-characterized real-life clinical cohort of chronically HBV-infected patients.Methods
Patients with chronic HBV infection were prospectively included, classified according to the disease stage, and followed up to determine parameters associated with HBsAg clearance.ResultsIn total, 315 patients were followed up for a mean of almost 6 years. At study entry, 109 (34.6%) were inactive HBsAg carriers, 204 (64.8%) had chronic active hepatitis (CAH), and two (0.6%) were immune-tolerant carriers. During follow-up, 128 (62.7%) of the 204 patients with CAH received antiviral therapy. Sixty-nine had HBeAg-positive CAH: 55 (79.7%) were treated and 14 (20.3%) untreated. One hundred thirty-five had HBeAg-negative CAH: 73 (54.1%) were treated and 62 (45.9%) untreated. Inactive carriers showed an annual HBsAg clearance incidence rate of 23.4 cases per 1,000 persons-years, which was higher than that of CAH groups. The clearance incidence rates (in cases per 1,000 persons-years) of CAH groups were: treated HBeAg-positive (20.7), untreated HBeAg-positive (19.1), treated HBeAg-negative (10.1), and untreated HBeAg-negative (8.1). Older age (p=0.001) and inactive carrier status (p=0.019) were independent predictors of HBsAg clearance.Conclusions
In a well-characterized real-life clinical cohort of chronically HBV-infected patients in various disease phases, older age and inactive HBsAg carrier status were the only predictors of HBsAg clearance, whereas anti-HBV therapy only marginally increased annual incidence of HBsAg loss.This article is protected by copyright. All rights reserved.
Liver international: official journal of the International Association for the Study of the Liver 08/2014; · 3.87 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: In spite of the high variability of its sequence, Hepatitis C virus (HCV) envelope glycoprotein E2 contains several conserved regions. In this study, we explored the structural and functional features of the highly conserved E2 aa502-520 segment that had been proposed as a fusion peptide and that has been shown to strongly overlap with a potential conserved neutralizing epitope. For this purpose, we used reverse genetics to introduce point mutations within this region, and we characterized the phenotype of these mutants in the light of the recently published structure of E2. The functional analyses showed that their phenotype is in agreement with the position of the corresponding residues in E2 crystal structure. In contrast, our data ruled out the involvement of this region in membrane fusion and they indicate that alternative conformations would be necessary to expose the potential neutralizing epitope present in this segment. Of particular interest, we identified three specific mutations (Y507L, V514A and V515A) located within this neutralizing epitope, which only mildly reduced infectivity and showed no assembly defect. These mutations modulated HCV dependence on the viral receptor SRB1 and/or they also modulated virion sensitivity to neutralizing antibodies. Importantly, their characterization also showed that amino acids Y507, V514 and V515 contribute to E2 interaction with HCV receptor CD81. In conclusion, our data show that the highly conserved E2 aa502-520 segment plays a key role in cell entry by influencing the association of the viral particle with co-receptors and neutralizing antibodies.
[Show abstract][Hide abstract] ABSTRACT: Although direct-acting antiviral agents (DAAs) have markedly improved the outcome of treatment in chronic HCV infection, there continues to be an unmet medical need for improved therapies in difficult-to-treat patients as well as liver graft infection. Viral entry is a promising target for antiviral therapy.
[Show abstract][Hide abstract] ABSTRACT: Hepatitis C virus (HCV) is transmitted between hepatocytes via classical cell entry but also uses direct cell-cell transfer to infect neighboring hepatocytes. Viral cell-cell transmission has been shown to play an important role in viral persistence allowing evasion from neutralizing antibodies. In contrast, the role of HCV cell-cell transmission for antiviral resistance is unknown. Aiming to address this question we investigated the phenotype of HCV strains exhibiting resistance to direct-acting antivirals (DAAs) in state-of-the-art model systems for cell-cell transmission and spread. Using HCV genotype 2 as a model virus, we show that cell-cell transmission is the main route of viral spread of DAA-resistant HCV. Cell-cell transmission of DAA-resistant viruses results in viral persistence and thus hampers viral eradication. We also show that blocking cell-cell transmission using host-targeting entry inhibitors (HTEIs) was highly effective in inhibiting viral dissemination of resistant genotype 2 viruses. Combining HTEIs with DAAs prevented antiviral resistance and led to rapid elimination of the virus in cell culture model. In conclusion, our work provides evidence that cell-cell transmission plays an important role in dissemination and maintenance of resistant variants in cell culture models. Blocking virus cell-cell transmission prevents emergence of drug resistance in persistent viral infection including resistance to HCV DAAs.
[Show abstract][Hide abstract] ABSTRACT: The molecular mechanisms that link IFN-λ3 genotypes to differential induction of interferon (IFN)-stimulated genes (ISGs) in the liver of patients with chronic hepatitis C (CHC) are not known. We measured the expression of IFN-λ and of the specific IFN-λ receptor chain (IFN-λR1) in 122 liver biopsies of patients with CHC and 53 control samples. The IFN-λ3 genotype was not associated with differential expression of IFN-λ, but rather IFN-λR1. In a series of 30 primary human hepatocyte (PHH) samples, IFN-λR1 expression was low but could be induced with IFN-α. IFN-α-induced IFN-λR1 expression was significantly stronger in PHHs carrying the minor IFN-λ3 allele. The analysis of liver biopsies of patients with CHC revealed a strong association of high IFN-λR1 expression with elevated ISG expression, with IFN-λ3 minor alleles, and with nonresponse to pegylated IFN-α and ribavirin. The findings provide a missing link between the IFN-λ3 genotype and the associated phenotype of treatment nonresponse.
Journal of Experimental Medicine 04/2014; · 13.21 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Chronic hepatitis C is a major cause of liver cirrhosis and hepatocellular carcinoma worldwide. Some 130 million to 170 million people, or about 3% of the world's population, are chronically infected with the hepatitis C virus (HCV). In the United States, chronic hepatitis C, the most common cause of liver-related death and reason for liver transplantation, recently eclipsed human immunodeficiency virus (HIV) infection as a cause of death. The development of direct-acting antiviral agents (DAAs) has revolutionized HCV treatment by offering genuine prospects for the first comprehensive cure of a chronic viral infection in humans. This success can be traced . . .
New England Journal of Medicine 04/2014; · 51.66 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Laparoscopic liver resection (LLR) is growing in popularity, but the short- and long-term outcome of patients undergoing LLR for hepatocellular carcinoma (HCC) has not yet been established.
A literature search was performed using PubMed, Scopus, and Web of Science (WoS) from cited English and Chinese publications. Clinical and survival parameters were extracted. The search was last conducted in October 2013. After application of selective criteria, 24 remaining original studies with more than 15 patients were analyzed.
In the Western experience, mean operative time was between 150 to 300 minutes, and mean blood loss ranged from 55 to 452 mL. Transfusion was required in all series, ranging from 2.8% to 50%. The conversion rate ranged from 5% to 19.4%. Three cases of death were reported. General morbidity rate ranged from 1.5% to 25%. Specific complications were divided into hemorrhage (2.4% to 25%), ascites (3.7% to 15.3%), and biliary collection (0.6% to 5%). Liver insufficiency was reported in two cases. Mean hospital stay ranged from 5.4 to 15 days. In all case-matched studies, LLR was statistically associated with a shorter hospital stay. The 5-year overall survival rate ranged from 55% to 70%. No trocar-site recurrence was observed. The recurrence rate ranged from 21.4% to 50%. Comparative studies did not demonstrate any significant difference in terms of recurrence between LLR and open liver resection (OLR). In the Middle Eastern experience, mean operative time ranged from 147 to 325 minutes, and mean blood loss ranged from 88 to 808 mL. Transfusion was required, ranging from 1.8% to 19.2%. The conversion rate ranged from 1.8% to 18.6%, and four series reported no conversion. There was no mortality. The main specific complication was ascites (1.7% to 26.6%). A biliary collection was reported in only two series (10.7% and 13.3%), and only one case of postoperative liver insufficiency was reported. Mean hospital stay ranged from 4 to 11.5 days. Statistically, three comparative studies reported a shorter postoperative hospital stay following LLR versus OLR. The 5-year overall survival rate ranged from 50% to 76.6%. Comparative studies did not demonstrate any significant difference in terms of overall survival and recurrence rate between LLR and OLR. No trocar-site recurrence was reported. The recurrence rate ranged from 26.9% to 45.5%, and two series reported no recurrence.
Laparoscopic surgery should be considered an acceptable alternative for the treatment of HCC.
Hepatobiliary surgery and nutrition. 04/2014; 3(2):60-72.
[Show abstract][Hide abstract] ABSTRACT: The high variability and the limited knowledge of the structure of the hepatitis C virus (HCV) envelope glycoproteins (GP) are challenging hurdles for vaccine design. Recently, Kong et al. published a new model of HCV E2 GP structure in Science, revealing a globular structure, starkly contrasting from the extended model of class II fusion proteins from other Flaviviridae viruses.
[Show abstract][Hide abstract] ABSTRACT: Chronic hepatitis B and C virus infections are major causes of liver disease and hepatocellular carcinoma worldwide. Although both viruses infect hepatocytes, the molecular virology and cellular biology of their respective replication cycles differ. Viral entry is the first step of the life cycle and recent developments in functional genomic and proteomic methodologies have increased our understanding of the entry pathways for these two important human pathogens. In this review we provide a comparative analysis of the internalization routes for these viruses and highlight differences and how they impact the viral life cycle, immune responses and development of antivirals.
[Show abstract][Hide abstract] ABSTRACT: Tetraspanins are integral transmembrane proteins organized in microdomains displaying specific and direct interactions with other tetraspanins and molecular partners. Among them, CD81 has been implicated in a variety of physiological and pathological processes. CD81 also plays a crucial role in pathogen entry into host cells, including hepatitis C virus (HCV) entry into hepatocytes. HCV is a major cause of liver cirrhosis and hepatocellular carcinoma. HCV entry into hepatocytes is a complex process that requires the coordinated interaction of viral and host factors for the initiation of infection, including CD81, scavenger receptor BI, claudin-1, occludin, membrane-bound host cell kinases, Niemann-Pick C1 Like 1, Harvey rat sarcoma viral oncogene homolog (HRas), CD63 and transferrin receptor 1. Furthermore, recent data in HCV model systems have demonstrated that targeting critical components of tetraspanins and associated cell membrane proteins open new avenues to prevent and treat viral infection.
[Show abstract][Hide abstract] ABSTRACT: Hepatitis C virus (HCV) is a major cause of liver disease worldwide and HCV infection represents a major health problem. HCV associates with host lipoproteins forming host/viral hybrid complexes termed lipoviral particles. Apolipoprotein E (apoE) is a lipoprotein component that interacts with heparan sulfate proteoglycans (HSPG) to mediate hepatic lipoprotein uptake, and may likewise mediate HCV entry. We sought to define the functional regions of apoE with an aim to identify critical apoE binding partners involved in HCV infection. Using adenoviral vectors and siRNA to modulate apoE expression we show a direct correlation of apoE expression and HCV infectivity, whereas no correlation exists with viral protein expression. Mutating the HSPG binding domain (HSPG-BD) of apoE revealed key residues that are critical for mediating HCV infection. Furthermore, a novel synthetic peptide that mimics apoE's HSPG-BD directly and competitively inhibits HCV infection. Genetic knockdown of the HSPG proteins syndecan (SDC) 1 and 4 revealed that SDC4 principally mediates HCV entry. Our data demonstrate that HCV uses apoE-SDC4 interactions to enter hepatoma cells and establish infection. Targeting apoE-SDC interactions could be an alternative strategy for blocking HCV entry, a critical step in maintaining chronic HCV infection.
PLoS ONE 01/2014; 9(4):e95550. · 3.53 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Until recently, there were no effective drugs available blocking coronavirus (CoV) infection in humans and animals. We have shown before that CsA and FK506 inhibit coronavirus replication (Carbajo-Lozoya et al., 2012; Pfefferle, 2011). Here we demonstrate that CsD Alisporivir, NIM811 as well as novel non-immunosuppressive derivatives of CsA and FK506 strongly inhibit the growth of human coronavirus HCoV-NL63 at low micromolar, non-cytotoxic concentrations in cell culture. We show by qPCR analysis that virus replication is diminished up to four orders of magnitude to background levels. Knockdown of the cellular Cyclophilin A (CypA/PPIA) gene in Caco-2 cells prevents replication of HCoV-NL63, suggesting that CypA is required for virus replication. Collectively, our results uncover Cyclophilin A as a host target for CoV infection and provide new strategies for urgently needed therapeutic approaches.
[Show abstract][Hide abstract] ABSTRACT: Hepatitis C virus (HCV) is a major cause of liver cirrhosis and hepatocellular carcinoma. Furthermore, HCV-induced liver disease is the leading indication for liver transplantation. The recent introduction of direct-acting antivirals (DAAs) has revolutionized HCV treatment by making possible the cure of the majority of patients. However, their efficacy and safety in difficult-to-treat patients such as patients receiving immunosuppression, those with advanced liver disease, co-morbidity and HIV/HCV-co-infection remain to be determined. Furthermore, prevention of liver graft infection remains a pressing issue. HCV entry inhibitors target the very first step of the HCV life cycle and efficiently inhibit cell-cell transmission – a key prerequisite for viral spread. Because of their unique mechanism of action on cell-cell transmission they may provide a promising and simple perspective for prevention of liver graft infection. A high genetic barrier to resistance and complementary mechanism of action compared to DAAs makes entry inhibitors attractive as a new strategy for treatment of multi-resistant or difficult-to-treat patients. Clinical studies are needed to determine the future role of entry inhibitors in the arsenal of antivirals to combat HCV infection. This article forms part of a symposium in Antiviral Research on “Hepatitis C: next steps toward global eradication.”
[Show abstract][Hide abstract] ABSTRACT: Cell therapy based on alloreactivity has completed clinical proof of concept against hematological malignancies. However, the efficacy of alloreactivity as a therapeutic approach to treat solid tumors is unknown. Using cell culture and animal models, we aimed to investigate the efficacy and safety of allogeneic suicide gene-modified killer cells as a cell-based therapy for hepatocellular carcinoma (HCC), for which treatment options are limited. Allogeneic killer cells from healthy donors were isolated, expanded, and phenotypically characterized. Antitumor cytotoxic activity and safety were studied using a panel of human or murine HCC cell lines engrafted in immunodeficient or immunocompetent mouse models. Human allogeneic suicide gene-modified killer cells (aSGMKCs) exhibit a high, rapid, interleukin-2-dependent, and non-major histocompatibility complex class I-restricted in vitro cytotoxicity toward human hepatoma cells, mainly mediated by natural killer (NK) and NK-like T cells. In vivo evaluation of this cell therapy product demonstrates a marked, rapid, and sustained regression of HCC. Preferential liver homing of effector cells contributed to its marked efficacy. Calcineurin inhibitors allowed preventing rejection of allogeneic lymphocytes by the host immune system without impairing their antitumor activity. Our results demonstrate proof of concept for aSGMKCs as immunotherapy for HCC and open perspectives for the clinical development of this approach.Molecular Therapy (2014); doi:10.1038/mt.2013.277.