[Show abstract][Hide abstract] ABSTRACT: Fighting viral infections is hampered by the scarcity of viral targets and their variability, resulting in devel-opment of resistance. Viruses depend on cellular molecules—which are attractive alternative tar-gets—for their life cycle, provided that they are dispensable for normal cell functions. Using the model organism Drosophila melanogaster, we iden-tify the ribosomal protein RACK1 as a cellular factor required for infection by internal ribosome entry site (IRES)-containing viruses. We further show that RACK1 is an essential determinant for hepatitis C virus translation and infection, indicating that its function is conserved for distantly related human and fly viruses. Inhibition of RACK1 does not affect Drosophila or human cell viability and proliferation, and RACK1-silenced adult flies are viable, indicating that this protein is not essential for general transla-tion. Our findings demonstrate a specific function for RACK1 in selective mRNA translation and un-cover a target for the development of broad antiviral intervention. INTRODUCTION
[Show abstract][Hide abstract] ABSTRACT: Hepatitis C virus (HCV)-induced, end-stage liver disease is a major indication for liver transplantation, but systematic graft reinfection accelerates liver disease recurrence. Transplantation recipients may be ineligible for direct-acting antivirals, owing to toxicity, resistance or advanced liver disease. Adoptive immunotherapy with liver graft-derived, ex vivo-activated lymphocytes was previously shown to prevent HCV-induced graft reinfections. Alternatively, the applicability and therapeutic efficacy of adoptive immunotherapy may be enhanced by 'ready for use' suicide gene-modified lymphocytes from healthy blood donors; moreover, conditional, prodrug-induced cell suicide may prevent potential side effects. Here, we demonstrate that allogeneic suicide gene-modified lymphocytes (SGMLs) could potently, dose- and time-dependently, inhibit viral replication. The effect occurs at effector:target cell ratios that exhibits no concomitant cytotoxicity toward virus-infected target cells. The effect, mediated mostly by CD56+ lymphocytes, is interleukin-2-dependent, IFN-γ-mediated and, importantly, resistant to calcineurin inhibitors. Thus, post-transplant immunosuppression may not interfere with this adoptive cell immunotherapy approach. Furthermore, these cells are indeed amenable to conditional cell suicide; in particular, the inducible caspase 9 suicide gene is superior to the herpes simplex virus thymidine kinase suicide gene. Our data provide in vitro proof-of-concept that allogeneic, third-party, SGMLs may prevent HCV-induced liver graft reinfection.Gene Therapy advance online publication, 13 November 2014; doi:10.1038/gt.2014.99.
[Show abstract][Hide abstract] ABSTRACT: Hepatitis C virus (HCV) is one of the major aetiologic agents that causes hepatocellular carcinoma (HCC) by generating an inflammatory, fibrogenic, and carcinogenic tissue microenvironment in the liver. HCV-induced HCC is a rational target for cancer preventive intervention because of the clear-cut high-risk condition, cirrhosis, associated with high cancer incidence (1% to 7% per year). Studies have elucidated direct and indirect carcinogenic effects of HCV, which have in turn led to the identification of candidate HCC chemoprevention targets. Selective molecular targeted agents may enable personalized strategies for HCC chemoprevention. In addition, multiple experimental and epidemiological studies suggest the potential value of generic drugs or dietary supplements targeting inflammation, oxidant stress, or metabolic derangements as possible HCC chemopreventive agents. While the successful use of highly effective direct-acting antiviral agents will make important inroads into reducing long-term HCC risk, there will remain an important role for HCC chemoprevention even after viral cure, given the persistence of HCC risk in persons with advanced HCV fibrosis, as shown in recent studies. The successful development of cancer preventive therapies will be more challenging compared to cancer therapeutics because of the requirement for larger and longer clinical trials and the need for a safer toxicity profile given its use as a preventive agent. Molecular biomarkers to selectively identify high-risk population could help mitigate these challenges. Genome-wide, unbiased molecular characterization, high-throughput drug/gene screening, experimental model-based functional analysis, and systems-level in silico modelling are expected to complement each other to facilitate discovery of new HCC chemoprevention targets and therapies.
[Show abstract][Hide abstract] ABSTRACT: Hepatitis C virus (HCV) infects an estimated more than 150 million people and is a leading cause of liver disease worldwide. The development of direct-acting antivirals (DAAs) will markedly improve the outcome of antiviral treatment with cure of the majority of treated patients. However, several hurdles remain before HCV infection can be considered a menace of the past: High treatment costs will most likely result in absent or limited access in middle and low resource countries and will lead to selective use even in wealthier countries. The limited efficacy of current HCV screening programs leads to a majority of cases being undiagnosed or diagnosed at a late stage and DAAs will not cure virus-induced end-stage liver disease such as hepatocellular carcinoma. Certain patient subgroups may not respond or not be eligible for DAA-based treatment strategies. Finally, reinfection remains possible, making control of HCV infection in people with ongoing infection risk difficult. The unmet medical needs justify continued efforts to develop an effective vaccine, protecting from chronic HCV infection as a mean to impact the epidemic on a global scale. Recent progress in the understanding of virus–host interactions provides new perspectives for vaccine development, but many critical questions remain unanswered. In this review, we focus on what is known about the immune correlates of HCV control, highlight key mechanisms of viral evasion that pose challenges for vaccine development and suggest areas of further investigation that could enable a rational approach to vaccine design. Within this context we also discuss insights from recent HCV vaccination studies and what they suggest about the best way to go forward.
[Show abstract][Hide abstract] ABSTRACT: Being the largest internal organ of the human body with the unique ability of self-regeneration, the liver is involved in a wide variety of vital functions that require highly orchestrated and controlled biochemical processes. Increasing evidence suggests that microRNA (miRNA) are essential for the regulation of liver development, regeneration and metabolic functions. Hence, alterations in intrahepatic miRNA networks have been associated with liver disease including hepatitis, steatosis, cirrhosis and hepatocellular carcinoma (HCC). miR-122 is the most frequent miRNA in the adult liver, and a central player in liver biology and disease. Furthermore, miR-122 has been shown to be an essential host factor for hepatitis C virus (HCV) infection and an antiviral target complementary to standard of care using direct-acting antivirals or interferon-based treatment. This review summarizes our current understanding of the key role of miR-122 in liver physiology and disease highlighting its role in HCC and viral hepatitis. We also discuss the perspectives of miRNA-based therapeutic approaches for viral hepatitis and liver disease.
[Show abstract][Hide abstract] ABSTRACT: The investigation of virus-induced liver disease and hepatocellular carcinoma needs small animal models modeling hepatitis C virus (HCV) infection and liver disease biology. A recent study published in Cell Research reports a novel mouse model which is permissive for chronic HCV infection and shows chronic liver injury including inflammation, steatosis and fibrosis.
[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: In this article, we address the design of innovative human serum albumin (HSA)-based nanoparticles loaded with silencing RNA and grafted with gadolinium complexes having average sizes ranging from ca. 50 to 150 nm according to the siRNA/HSA composition. The non-covalent siRNA/HSA assembly is formed on isobutyramide-modified mesoporous silica and the self-supported HSA-based nanoparticles are obtained following the silica template dissolution. These original protein particles provide simultaneous magnetic resonance imaging contrast enhancement and cellular in vitro gene silencing.
[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; · 54.42 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.