Anti-E1E2 antibodies status prior therapy favors direct-acting antiviral treatment efficacy.

INTRODUCTION: Presence of anti-E1E2 antibodies was previously associated with spontaneous cure of hepatitis C virus (HCV) and predictive before treatment of a sustained virological response (SVR) to bi- or tri-therapy in naïve or experienced patients, regardless of HCV genotype. We investigated the impact of anti-E1E2 seroprevalence at baseline on treatment response in patients receiving direct-acting antiviral (DAA) therapy. MATERIAL AND METHODS: We screened anti-E1E2 antibodies by ELISA in serum samples collected at treatment initiation for two groups of patients: 59 with SVR at the end of DAA treatment and 44 relapsers after DAA treatment. Nineteen patients received a combination of ribavirin (RBV) or PEG-interferon/ribavirin with sofosbuvir or daclatasvir and others received interferon-free treatment with DAA±RBV. HCV viral load was measured at different time points during treatment in a subgroup of patients. RESULTS: A significant association was observed between presence of anti-E1E2 and HCV viral load<6log10 prior treatment. Among patients with anti-E1E2 at baseline, 70% achieved SVR whereas among patients without anti-E1E2, only 45% achieved SVR. Conversely, 66% of patients experiencing DAA-failure were anti-E1E2 negative at baseline. In the multivariate analysis, presence of anti-E1E2 was significantly associated with SVR after adjustment on potential cofounders such as age, sex, fibrosis stage, prior HCV treatment and alanine aminotransferase (ALT) level. CONCLUSIONS: The presence of anti-E1E2 at treatment initiation is a predictive factor of SVR among patients treated with DAA and more likely among patients with low initial HCV viral load (<6log10). Absence of anti-E1E2 at baseline could predict DAA-treatment failure.

New insights into HCV replication in original cells from Aedes mosquitoes.

BACKGROUND: The existing literature about HCV association with, and replication in mosquitoes is extremely poor. To fill this gap, we performed cellular investigations aimed at exploring (i) the capacity of HCV E1E2 glycoproteins to bind on Aedes mosquito cells and (ii) the ability of HCV serum particles (HCVsp) to replicate in these cell lines. METHODS: First, we used purified E1E2 expressing baculovirus-derived HCV pseudo particles (bacHCVpp) so we could investigate their association with mosquito cell lines from Aedes aegypti (Aag-2) and Aedes albopictus (C6/36). We initiated a series of infections of both mosquito cells (Ae aegypti and Ae albopictus) with the HCVsp (Lat strain - genotype 3) and we observed the evolution dynamics of viral populations within cells over the course of infection via next-generation sequencing (NGS) experiments. RESULTS: Our binding assays revealed bacHCVpp an association with the mosquito cells, at comparable levels obtained with human hepatocytes (HepaRG cells) used as a control. In our infection experiments, the HCV RNA (+) were detectable by RT-PCR in the cells between 21 and 28 days post-infection (p.i.). In human hepatocytes HepaRG and Ae aegypti insect cells, NGS experiments revealed an increase of global viral diversity with a selection for a quasi-species, suggesting a structuration of the population with elimination of deleterious mutations. The evolutionary pattern in Ae albopictus insect cells is different (stability of viral diversity and polymorphism). CONCLUSIONS: These results demonstrate for the first time that natural HCV could really replicate within Aedes mosquitoes, a discovery which may have major consequences for public health as well as in vaccine development.

Anti-E1E2 antibodies do predict response to triple therapy in treatment-experienced Hepatitis C Virus-cirrhosis cases.

BACKGROUND AND AIMS: We previously showed that pre-treatment serum anti-E1E2 predicted hepatitis C virus (HCV) RNA viral kinetics (VKs) and treatment outcome in patients with chronic hepatitis C receiving pegylated interferon/ribavirin (Peg-IFN/RBV) double therapy. Here, we determined whether baseline anti-E1E2 was correlated with the on-treatment VK and could predict virological outcome in treatment-experienced HCV-infected cirrhotic patients receiving protease inhibitor-based triple therapy. METHODS: Sera from 19 patients with HCV genotype 1 infection and compensated cirrhosis who failed to respond to a prior course of Peg-IFN/RBV were selected at time 0 before starting triple therapy with boceprevir or telaprevir. We assessed patients with sustained viral response 12 weeks after the end of triple therapy (SVR12) by analyzing VKs at weeks 4, 12, 24, 36, 48 (end of treatment) and 60. RESULTS: Patients baseline characteristics were similar to the well-defined CUPIC cohort (age, HCV subtype, baseline viremia, and treatment history). Among the 19 patients, 11 achieved an SVR12. Fifteen patients were positive for pre-treatment anti-E1E2 and all of them achieved SVR12. Moreover, anti-E1E2 and SVR12 correlated with prior response to IFN/RBV therapy (relapse, partial or null response). CONCLUSIONS: Baseline anti-E1E2 could be considered as a new biomarker to predict SVR12 after triple therapy in this most difficult-to-treat population. These results warrant further validation on larger cohorts including patients receiving highly effective direct-acting antivirals to explore whether this test could help in better defining treatment duration for these very costly molecules.

Predictors of the therapeutic response in hepatitis C. A 2013 update.

Chronic hepatitis C is a major cause of cirrhosis and hepatocellular carcinoma. Current therapy based on pegylated-interferon-α (PEG-IFN) and ribavirin (RBV) combination has limited efficacy and is poorly tolerated. Disease progression is highly variable and pre-therapeutic prediction of response to treatment remains difficult. Although viral kinetics proved most useful to monitor duration of therapy, other predictors would be helpful to identify patients with the best chance of subsequent treatment response prior initiation of antiviral therapy (double or triple therapy). The predictive power of IL28B polymorphism is well-recognized and has become the reference biomarker for clinicians in patients treated with double therapy. The combination of serum IP-10 and IL28B SNPs increases predictive value of treatment response. Recently, anti-E1E2 antibodies appear to closely correlate with therapeutic outcome and predict the complete elimination of HCV. They may represent a new relevant prognostic biomarker of double therapy response. Since the introduction of triple therapy including protease inhibitors (telaprevir/boceprevir), the major priority is to help patients who failed on double therapy, and there is now an urgent need for robust pre-therapeutic predictors of response to better select the patients to treat. Indeed, the relevance of IL28B polymorphism and IP-10 serum concentration are limited in triple therapy. Many new drugs are currently under investigation and there is hope that effective and well-tolerated IFN-free regimens may become a part of future therapy. In this context, this will help to identify the most powerful predictive marker and/or to assess the benefit of anti-E1E2 in decision to treat.

Pretreatment predictive factors for hepatitis C therapy outcome: relevance of anti-E1E2 antibodies compared to IP-10 and IL28B genotypes.

BACKGROUND: Unique serum anti-E1E2 antibodies were shown to be associated with spontaneous recovery or predictive of sustained virological response (SVR) in patients with chronic hepatitis C receiving pegylated interferon/ribavirin (PEG-IFN/RBV) therapy. The objectives were to establish the relationship between pretreatment anti-E1E2 titres and HCV RNA kinetics during PEG-IFN/RBV therapy, and to examine whether the combined determination of interleukin (IL)28B rs12979860 and rs8099917, pretreatment inducible protein (IP)-10 levels and/or anti-E1E2 improved the prediction of SVR. METHODS: Sera from 26 treatment-naive consecutive HCV patients treated with PEG-IFN/RBV for 48 weeks were analysed. Serum anti-E1E2 titres and pretreatment IP-10 levels were measured by enzyme-linked immunosorbent assays. The IL28B variants were determined using genotyping real-time polymerase chain reaction method. Viral decline was measured at weeks (W) 4 and 12 and SVR assessed 6 months after the end of therapy. RESULTS: Baseline anti-E1E2 titres were correlated with HCV RNA decline at W4 and W12 and were highly predictive of SVR with 100% of patients negative for anti-E1E2 failing to achieve SVR. Receiver operating characteristic curve analyses indicate that the best prediction of SVR (AUC 0.990) was obtained with the combination of anti-E1E2 and IP-10 levels. Predictive values were better than those obtained with IP-10 alone or in combination with IL28B variants. CONCLUSIONS: Pretreatment serum anti-E1E2 response predicts HCV RNA clearance kinetics and treatment outcome. The combination of anti-E1E2 and IP-10 significantly improved the prediction of treatment response. This warrants further investigation and validation on larger cohorts of patients in the context of new therapeutic strategies.

Long-term propagation of serum hepatitis C virus (HCV) with production of enveloped HCV particles in human HepaRG hepatocytes.

UNLABELLED: HepaRG human liver progenitor cells exhibit morphology and functionality of adult hepatocytes. We investigated the susceptibility of HepaRG hepatocytes to in vitro infection with serum-derived hepatitis C virus (HCV) particles (HCVsp) and the potential neutralizing activity of the E1E2-specific monoclonal antibody (mAb) D32.10. The infection was performed using HCVsp when the cells actively divided at day 3 postplating. HCV RNA, E1E2, and core antigens were quantified in HCV particles recovered from culture supernatants of differentiated cells for up to 66 days. The density distributions of particles were analyzed on iodixanol or sucrose gradients. Electron microscopy (EM) and immune-EM studies were performed for ultrastructural analysis of cells and localization of HCV E1E2 proteins in thin sections. HCV infection of HepaRG cells was documented by increasing production of E1E2-core-RNA(+) HCV particles from day 21 to day 63. Infectious particles sedimented between 1.06 and 1.12 g/mL in iodixanol gradients. E1E2 and core antigens were expressed in 50% of HCV-infected cells at day 31. The D32.10 mAb strongly inhibited HCV RNA production in HepaRG culture supernatants. Infected HepaRG cells frozen at day 56 were reseeded at low density. After only 1-3 subcultures and induction of a cell differentiation process the HepaRG cells produced high titer HCV RNA and thus showed to be sustainably infected. Apolipoprotein B-associated empty E1E2 and complete HCV particles were secreted. Characteristic virus-induced intracellular membrane changes and E1E2 protein-association to vesicles were observed. CONCLUSION: HepaRG progenitor cells permit HCVsp infection. Differentiated HepaRG cells support long-term production of infectious lipoprotein-associated enveloped HCV particles. The E1E2-specific D32.10 mAb neutralizes the infection and this cellular model could be used as a surrogate infection system for the screening of entry inhibitors.

Expression of E1E2 on hepatitis C RNA-containing particles released from primary cultured human hepatocytes derived from infected cirrhotic livers.

OBJECTIVE: To determine whether liver-derived hepatitis C RNA-containing particles express the E1E2 discontinuous antigenic determinant defined by unique monoclonal antibody (mAb) D32.10 which recognizes three highly conserved segments in E1 (aa297-306) and E2 (aa480-494 and aa613-621) envelope glycoproteins. METHODS: Human hepatocytes were isolated from HCV-infected cirrhotic explanted livers. The liver-derived hepatitis C virus (HCV) particles released from three distinct cultures (genotypes 1b and 2b) were characterized. HCV RNA+ was quantified by real-time RT-PCR. The E1E2 antigenic activity was assessed by indirect ELISA and immunoblotting using D32.10. The density distributions of HCV RNA and E1E2 antigen were determined by isopycnic sucrose density gradients. HCV E1E2, E2 and core antigens were detected in the cells by immunochemical staining. RESULTS: Liver-derived HCV particles contained HCV RNA (106-107 copies/mg of protein) and core proteins and expressed the E1E2/D32.10 epitope. HCV RNA and E1E2 cosedimented between 1.15 and 1.25 g/ml in sucrose gradients. Moreover, the mAb D32.10 detected E1E2 by immunostaining in HCV-infected hepatocytes in parallel with E2 and core antigens. CONCLUSION: Our results provide evidence that the mAb D32.10 recognizes E1E2 envelope complexes expressed in the cell cytoplasm and on the surface of HCV RNA-containing particles released from short-term cultures of in vivo infected hepatocytes.

Association of anti-E1E2 antibodies with spontaneous recovery or sustained viral response to therapy in patients infected with hepatitis C virus.

UNLABELLED: The monoclonal antibody (mAb) D32.10 recognizes a discontinuous epitope encompassing three regions E1 (amino acids 297-306), E2A (amino acids 480-494), and E2B (amino acids 613-621) juxtaposed on the surface of serum-derived hepatitis C virus (HCV) particles (HCVsp). The mAb D32.10 inhibits efficiently and specifically the binding of HCVsp to human hepatocytes. Therefore, we investigated the clinical relevance of anti-E1E2A,B response in the serum of patients infected with HCV. To this end, an enzyme-linked immunosorbent assay (ELISA) using synthetic E1-, E2A-, and E2B-derived peptides was used. The ELISA was validated in terms of sensitivity, specificity, and test efficiency. The detection of the anti-E1E2 D32.10 epitope-binding antibodies during natural HCV infection in more than 300 HCV-positive sera demonstrated significantly (P < 0.001) higher prevalence of these antibodies: (1) in patients who spontaneously cured HCV infection (46 of 52, 88.5%) showing high titers (70% ≥ 1/1000) compared to never-treated patients with chronic hepatitis C (7 of 50, 14%) who actively replicated the virus, and (2) in complete responders (20 of 52, 38.5%) who cleared virus following treatment and achieved a sustained viral response compared to nonresponders (4 of 40, 10%). Serum anti-E1E2 antibodies were monitored before, during, and after the current standard-of-care therapy (pegylated interferon plus ribavirin) in responder and nonresponder patients. Optimal cutoff values were assessed by receiver operating characteristic curve analysis. One month prior to therapy initiation, the threshold of 1131 (optical density × 1000) gave 100% and 86% positive and negative predictive values, respectively, for achieving or not achieving a sustained viral response. CONCLUSION: The anti-E1E2 D32.10 epitope-binding antibodies are associated with control of HCV infection and may represent a new relevant prognostic marker in serum. This unique D32.10 mAb may also have immunotherapeutic potential.

Inhibition of the binding of HCV serum particles to human hepatocytes by E1E2-specific D32.10 monoclonal antibody.

The aim of this study was to determine the inhibition of binding activity of the monoclonal antibody (mAb) D32.10 which recognizes a highly conserved discontinuous antigenic determinant (E1:297-306, E2:480-494, and E2:613-621) expressed on the surface of serum-derived HCV particles (HCVsp) of genotypes 1a, 1b, 2a, and 3a. To this end, an in vitro direct cell-binding assay based on the attachment of radiolabeled HCVsp was developed, and Scatchard plots were used to analyze ligand-receptor binding data. HCV adsorption was also assessed by quantitating cell-associated viral RNA by a real-time RT-PCR method. Saturable concentration-dependent specific binding of HCVsp to Huh-7 or HepaRG cells was demonstrated. The Scatchard transformed data showed two-site interaction for Huh-7 and proliferative HepaRG cells: the high-affinity binding sites (K(d1) = 0.1-0.5 microg/ml) and the low-affinity binding sites (K(d1) = 5-10 microg/ml), and one-site high-affinity binding model between E1E2/D32.10-positive HCVsp and hepatocyte-like differentiated HepaRG cells. The E1E2-specific mAb D32.10 inhibited efficiently (>60%) and selectively the binding with an IC(50)

Efficient hepatitis C antigen immunohistological staining in sections of normal, cirrhotic and tumoral liver using a new monoclonal antibody directed against serum-derived HCV E2 glycoproteins.

Detection and localization of Hepatitis C Virus (HCV) in liver tissue is useful for diagnostic purposes as well as to elucidate the mechanisms by which the virus participates in hepatocarcinogenesis. However, so far, a sensitive method for HCV detection at the cellular level is lacking. We describe here the application of a novel antibody, D4.12.9, developed against serum-derived HCV RNA-positive particles, for the detection of E2 proteins by immunohistochemistry in fixed, archived specimens including liver biopsies of HCV-infected patients and surgical specimens of hepatocellular carcinoma. We demonstrate that D4.12.9 is a powerful tool for sensitive and specific detection of HCV, independently of viral genotype. This approach has applications to diagnosis as well as exploratory pathological studies.

Enveloped particles in the serum of chronic hepatitis C patients.

HCV particles were isolated from the plasma of chronically infected patients. The virus was analysed by sucrose density gradient centrifugation. The fractions were tested for viral RNA, core antigen and envelope proteins by using a monoclonal antibody directed against the natural E1E2 complex (D32.10). Two populations of particles containing RNA plus core antigen were separated: the first with a density of 1.06-1.08 g/ml did not contain the envelope proteins; the second with a density between 1.17 and 1.21 g/ml expressed both E1 and E2 glycoproteins. Electron microscopy of the enveloped population after immunoprecipitation with D32.10 showed spherical particles with a rather featureless surface and with a diameter around 40 nm. Immuno-gold staining gave evidence that the E1E2 complex was indeed positioned at the surface of these particles.

Origin and characterization of a human bipotent liver progenitor cell line.

BACKGROUND & AIMS: Liver progenitor cells may be important in carcinogenesis resulting from human chronic liver diseases. The HepaRG cell line has been established from a liver tumor associated with chronic hepatitis C. We observed that these cells showed an evident morphological heterogeneity, displaying both hepatocyte-like and biliary-like epithelial phenotypes. Our goal was to determine whether they could share some features with liver progenitor cells. METHODS: Phenotypic studies using immunofluorescence, immunoblotting, and flow cytometry were performed at different culture stages. RESULTS: HepaRG cells progressively exhibited polarized and functional hepatocytes and bile duct-like cell features under defined conditions. Cytokeratin 18 and 19 coexpression was, however, observed all along the maturation process together with oval cell-specific markers (M2-PK, OV-1, OV-6, and CD34); kinetics and expression profiles were dependent on the cell population. In addition, a strong commitment toward the hepatocytic lineage could be observed in the presence of epidermal growth factor. Immunohistochemistry on the fibrotic liver showing the atypical ductular reaction from which HepaRG cells originated displayed a comparable immunophenotype specifically restricted to bile neoductules. CONCLUSIONS: HepaRG cells constitute the first described human hepatic bipotent progenitor cell line regarding phenotype and histological origin.

Latent hepatitis B virus (HBV) infection and HBV DNA integration is associated with further transformation of hepatoma cells in vitro.

Hepatitis B virus (HBV) is the major cause of chronic liver disease and hepatocellular carcinoma in the world, with more than 400 million people infected worldwide. To date, there is no reliable model for the study of the many aspects of HBV infection, despite the use of the chimpanzee. Although several alternative methods have been previously developed for the in vitro study of HBV infection, there is still an urgent need for new in vitro infection models, including for the ability of HBV to integrate into the host cell genome. Here we describe a process to improve infection of the human hepatoma cell lines HepG2 and HuH-7 in vitro with HBV originating from human blood. As shown previously for infection of hepatocytes with hepatitis C virus (HCV), the removal of the cell-bound lipoproteins prior to the addition of the viral inoculum to the cells could also be critical for the uptake of HBV via lipoprotein (LDL)-related receptors. Induction by insulin and dexamethasone led to an increase of HBsAg expression at the cell surface in association with the integration of the viral DNA into the host genome and HBx RNA detection. This integration process was also shown to be associated with cytopathic changes and further phenotypic transformations of the cells.

Mapping of a conformational epitope shared between E1 and E2 on the serum-derived human hepatitis C virus envelope.

Monoclonal antibody D32.10 produced by immunizing mice with a hepatitis C virus (HCV)-enriched pellet obtained from plasmapheresis of a chronically HCV1b-infected patient binds HCV particles derived from serum of different HCV1a- and HCV1b-infected patients. Moreover, this monoclonal has been shown to recognize both HCV envelope proteins E1 and E2. In an attempt to provide novel insight into the membrane topology of HCV envelope glycoproteins E1 and E2, we localized the epitope recognized by D32.10 on the E1 and/or E2 sequence using Ph.D.-12 phage display peptide library technology. Mimotopes selected from the phage display dodecapeptide library by D32.10 shared partial similarities with 297RHWTTQGCNC306 of the HCV E1 glycoprotein and with both 613YRLWHYPCT621 and 480PDQRPYCWHYPPKPC494 of the HCV E2 glycoprotein. Immunoreactivity of D32.10 with overlapping peptides corresponding to these three HCV regions confirmed these localizations and suggested that the three regions identified are likely closely juxtaposed on the surface of serum-derived particles as predicted by the secondary model structure of HCV E2 derived from the tick-borne encephalitis virus E protein. This assertion was supported by the detection of specific antibodies directed against these three E1E2 regions in sera from HCV-infected patients.

EWI-2 is a new component of the tetraspanin web in hepatocytes and lymphoid cells.

Several tetraspanins bind directly to a few molecular partners to form primary complexes, which might assemble through tetraspanin-tetraspanin interactions to form a network of molecular interactions, the tetraspanin web. We have produced a monoclonal antibody directed to a 63 kDa molecule (determined under non-reducing conditions) associated with CD9. This molecule was first identified by MS as a molecule with four Ig domains, EWI-2. Like the related molecule CD9P-1, EWI-2 was found to be a partner not only for CD9, but also for CD81, a tetraspanin required for hepatic infection by the parasite responsible for malaria, and also a putative hepatitis C virus receptor. Using chimaeric CD9/CD82 molecules, two separate regions of CD9 of 40 and 47 amino acids were demonstrated to confer the ability to interact with EWI-2. Both EWI-2 and CD9P-1 were detected in the human liver at the surface of hepatocytes and were found to associate with CD81 on freshly isolated hepatocytes. EWI-2 also co-localized with CD81 in the liver. CD9P-1 was not detected on most peripheral blood cells, whereas EWI-2 was expressed on the majority of B-, T- and natural killer cells and was not detected on monocytes, polynuclear cells or platelets. This distribution is identical to that of CD81. Finally, EWI-2 associated with all tetraspanins studied after lysis under conditions preserving tetraspanin-tetraspanin interactions, showing that EWI-2 is a new component of the tetraspanin web.