Eric Soulier

Scavenger receptor class B type I is a key host factor for hepatitis C virus infection required for an entry step closely linked to CD81.

Hepatitis C virus (HCV) is a major cause of chronic hepatitis worldwide. Scavenger receptor class B type I (SR‐BI) has been shown to bind HCV envelope glycoprotein E2, participate in entry of HCV pseudotype particles, and modulate HCV infection. However, the functional role of SR‐BI for productive HCV infection remains unclear. In this study, we investigated the role of SR‐BI as an entry factor for infection of human hepatoma cells using cell culture–derived HCV (HCVcc). Anti–SR‐BI antibodies directed against epitopes of the human SR‐BI extracellular loop specifically inhibited HCVcc infection in a dose‐dependent manner. Down‐regulation of SR‐BI expression by SR‐BI–specific short interfering RNAs (siRNAs) markedly reduced the susceptibility of human hepatoma cells to HCVcc infection. Kinetic studies demonstrated that SR‐BI acts predominately after binding of HCV at an entry step occurring at a similar time point as CD81–HCV interaction. Although the addition of high‐density lipoprotein (HDL) enhanced the efficiency of HCVcc infection, anti–SR‐BI antibodies and SR‐BI–specific siRNA efficiently inhibited HCV infection independent of lipoprotein. Conclusion: Our data suggest that SR‐BI (i) represents a key host factor for HCV entry, (ii) is implicated in the same HCV entry pathway as CD81, and (iii) targets an entry step closely linked to HCV–CD81 interaction. (HEPATOLOGY 2007.)

Inhibition of hepatitis C virus infection by anti-claudin-1 antibodies is mediated by neutralization of E2–CD81–Claudin-1 associations†

The tight junction protein claudin‐1 (CLDN1) has been shown to be essential for hepatitis C virus (HCV) entry—the first step of viral infection. Due to the lack of neutralizing anti‐CLDN1 antibodies, the role of CLDN1 in the viral entry process is poorly understood. In this study, we produced antibodies directed against the human CLDN1 extracellular loops by genetic immunization and used these antibodies to investigate the mechanistic role of CLDN1 for HCV entry in an infectious HCV cell culture system and human hepatocytes. Antibodies specific for cell surface–expressed CLDN1 specifically inhibit HCV infection in a dose‐dependent manner. Antibodies specific for CLDN1, scavenger receptor B1, and CD81 show an additive neutralizing capacity compared with either agent used alone. Kinetic studies with anti‐CLDN1 and anti‐CD81 antibodies demonstrate that HCV interactions with both entry factors occur at a similar time in the internalization process. Anti‐CLDN1 antibodies inhibit the binding of envelope glycoprotein E2 to HCV permissive cell lines in the absence of detectable CLDN1‐E2 interaction. Using fluorescent‐labeled entry factors and fluorescence resonance energy transfer methodology, we demonstrate that anti‐CLDN1 antibodies inhibit CD81‐CLDN1 association. In contrast, CLDN1‐CLDN1 and CD81‐CD81 associations were not modulated. Taken together, our results demonstrate that antibodies targeting CLDN1 neutralize HCV infectivity by reducing E2 association with the cell surface and disrupting CD81‐CLDN1 interactions. Conclusion: These results further define the function of CLDN1 in the HCV entry process and highlight new antiviral strategies targeting E2‐CD81‐CLDN1 interactions. (HEPATOLOGY 2010.)

Monoclonal Anti-Claudin 1 Antibodies Prevent Hepatitis C Virus Infection of Primary Human Hepatocytes

BACKGROUND & AIMS Hepatitis C virus (HCV) infection is a challenge to prevent and treat because of the rapid development of drug resistance and escape. Viral entry is required for initiation, spread, and maintenance of infection, making it an attractive target for antiviral strategies. The tight junction protein claudin-1 (CLDN1) has been shown to be required for entry of HCV into the cell. METHODS Using genetic immunization, we produced 6 monoclonal antibodies against the host entry factor CLDN1. The effects of antibodies on HCV infection were analyzed in human cell lines and primary human hepatocytes. RESULTS Competition and binding studies demonstrated that antibodies interacted with conformational epitopes of the first extracellular loop of CLDN1; binding of these antibodies required the motif W(30)-GLW(51)-C(54)-C(64) and residues in the N-terminal third of CLDN1. The monoclonal antibodies against CLDN1 efficiently inhibited infection by HCV of all major genotypes as well as highly variable HCV quasispecies isolated from individual patients. Furthermore, antibodies efficiently blocked cell entry of highly infectious escape variants of HCV that were resistant to neutralizing antibodies. CONCLUSIONS Monoclonal antibodies against the HCV entry factor CLDN1 might be used to prevent HCV infection, such as after liver transplantation, and might also restrain virus spread in chronically infected patients.

Viral entry and escape from antibody-mediated neutralization influence hepatitis C virus reinfection in liver transplantation

End-stage liver disease caused by chronic hepatitis C virus (HCV) infection is a leading cause for liver transplantation (LT). Due to viral evasion from host immune responses and the absence of preventive antiviral strategies, reinfection of the graft is universal. The mechanisms by which the virus evades host immunity to reinfect the liver graft are unknown. In a longitudinal analysis of six HCV-infected patients undergoing LT, we demonstrate that HCV variants reinfecting the liver graft were characterized by efficient entry and poor neutralization by antibodies present in pretransplant serum compared with variants not detected after transplantation. Monoclonal antibodies directed against HCV envelope glycoproteins or a cellular entry factor efficiently cross-neutralized infection of human hepatocytes by patient-derived viral isolates that were resistant to autologous host-neutralizing responses. These findings provide significant insights into the molecular mechanisms of viral evasion during HCV reinfection and suggest that viral entry is a viable target for prevention of HCV reinfection of the liver graft.

Small molecule scavenger receptor BI antagonists are potent HCV entry inhibitors

BACKGROUND AND AIMS ITX 5061 is a clinical stage small molecule compound that promotes high-density lipoprotein (HDL) levels in animals and patients by targeting the scavenger receptor BI protein pathway. Since SR-BI is a known co-receptor for HCV infection, we evaluated these compounds for their effects on HCV entry. METHODS We obtained ITX 5061 and related compounds to characterize their interaction with SR-BI and effects on HCV entry and infection. RESULTS We confirmed that a tritium-labeled compound analog (ITX 7650) binds cells expressing SR-BI, and both ITX 5061 and ITX 7650 compete for HDL-mediated lipid transfer in an SR-BI dependent manner. Both molecules inhibit HCVcc and HCVpp infection of primary human hepatocytes and/or human hepatoma cell lines and have minimal effects on HCV RNA replication. Kinetic studies suggest that the compounds act at an early post-binding step. CONCLUSIONS These results suggest that the ITX compounds inhibit HCV infection with a mechanism of action distinct from other HCV therapies under development. Since ITX 5061 has already been evaluated in over 280 patients with good pharmacokinetic and safety profiles, it warrants proof-of-concept clinical studies in HCV infected patients.

Hepatitis C Virus Cell-Cell Transmission and Resistance to Direct-Acting Antiviral Agents

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.

Early Evolution of Hepatitis C Virus (HCV) Quasispecies after Liver Transplant for HCV-Related Disease

BACKGROUND End-stage liver disease as a result of chronic hepatitis C virus (HCV) infection is the main indication for liver transplant (LT), but allografts are systematically infected with HCV soon after transplant. Viral quasispecies are poorly described during the early posttransplant period. METHODS For 17 patients who received an LT for HCV disease, plasma viral quasispecies evolution was determined by sequence analysis of hypervariable region 1 of the E2 envelope gene before transplant (BT), after 7 days (D7), and after 1 month (M1). T helper (Th)1/Th2 cytokine levels were determined concomitantly. RESULTS HCV quasispecies showed a significant decrease in amino acid diversity at D7 and M1, compared with BT (P<.05). A correlation was observed between low plasma tumor necrosis factor-alpha levels at D7 and decreased quasispecies amino acid complexity at the same date. Nucleic acid diversity was lower for genotype 1 than for genotype 3 infection (P<.05). The complexity and diversity of amino acids were lower in patients with hepatocellular carcinoma (HCC) BT than in those without HCC (P<.05). Conserved amino acid residues within quasispecies were shared by the whole cohort before and after LT. CONCLUSION Viral structural and/or host immunological features could favor the emergence of fitter HCV strains after LT.

Frequent Compartmentalization of Hepatitis C Virus with Leukocyte-Related Amino Acids in the Setting of Liver Transplantation

BACKGROUND Nonrandom distribution of hepatitis C virus (HCV) quasispecies (compartmentalization between blood plasma and leukocytes) suggests the presence of HCV leukotropic variants. HCV compartmentalization in the setting of liver transplantation (LT) is poorly understood. To study HCV leukotropic variants, we investigated the evolution of HCV compartmentalization after immunosuppression in liver transplant recipients. METHODS Plasma and peripheral blood mononuclear cell (PBMC) samples were collected from 5 liver transplant recipients before and after LT. We used clone sequencing to analyze the hypervariable region 1 (HVR1)-E2(384-419) region, which plays a key role in HCV entry and the induction of neutralizing responses, and assessed compartmentalization through phylogenetic analyses and Mantels test. RESULTS Compartmentalization was frequent in the LT setting. HCV quasispecies were more homogeneous after LT in both the plasma and PBMC compartments, with a significant decrease in quasispecies complexity (P = .003) and genetic distances (P = .004) after transplantation. Our analysis identified 8 PBMC-related amino acid residues in HVR1. CONCLUSIONS HCV compartmentalization between plasma and PBMCs and the emergence of leukotropic variants could be potentiated by immunosuppression in liver transplant recipients. The identification of defined leukotropic variants may contribute to the understanding of virus-host interactions after transplantation.

Antiviral and immunoregulatory effects of indoleamine-2,3-dioxygenase in hepatitis C virus infection

In patients with hepatitis C virus (HCV) infection, enhanced activity of indoleamine-2,3-dioxygenase 1 (IDO) has been reported. IDO - a tryptophan-catabolizing enzyme - has been considered as both an innate defence mechanism and an important regulator of the immune response. The molecular mechanism of IDO induction in HCV infection and its role in the antiviral immune response remain unknown. Using primary human hepatocytes, we show that HCV infection stimulates IDO expression. IDO gene induction was transient and coincided with the expression of types I and III interferons (IFNs) and IFN-stimulated genes in HCV-infected hepatocytes. Overexpression of hepatic IDO prior to HCV infection markedly impaired HCV replication in hepatocytes, suggesting that IDO limits the spread of HCV within the liver. siRNA-mediated IDO knock-down revealed that IDO functions as an IFN-mediated anti-HCV effector. Hepatic IDO was most potently induced by IFN-γ, and ongoing HCV replication could significantly upregulate IDO expression. IRF1 (IFN-regulatory factor 1) and STAT1 (signal transducer and activator of transcription 1) regulated hepatic IDO expression. Hepatic IDO expression also had a significant inhibitory effect on CD4+ T-cell proliferation. Our data suggest that hepatic IDO plays a dual role during HCV infection by slowing down viral replication and also regulating host immune responses.

45 years after the discovery of human polyomaviruses BK and JC: Time to speed up the understanding of associated diseases and treatment approaches

Abstract Nearly 45 years after the discovery of the first two human polyomaviruses BK and JC, their life-long persistence and mechanisms of pathogenesis remain poorly understood and efficient antiviral treatments are severely lacking. In this review, we sought to provide an update on recent advances in understanding the life cycle of these two viruses, particularly focusing on their interaction with the host immune system and pathogenesis. We have also discussed novel treatment approaches and highlighted areas of future research.