Philip Meuleman

Morphological and biochemical characterization of a human liver in a uPA‐SCID mouse chimera

A small animal model harboring a functional human liver cell xenograft would be a useful tool to study human liver cell biology, drug metabolism, and infections with hepatotropic viruses. Here we describe the repopulation, organization, and function of human hepatocytes in a mouse recipient and the infections with hepatitis B virus (HBV) and hepatitis C virus (HCV) of the transplanted cells. Homozygous urokinase plasminogen activator (uPA)‐SCID mice underwent transplantation with primary human hepatocytes, and at different times animals were bled and sacrificed to analyze plasma and liver tissue, respectively. The plasma of mice that were successfully transplanted contained albumin and an additional 21 human proteins. Liver histology showed progressive and massive replacement of diseased mouse tissue by human hepatocytes. These cells were accumulating glycogen but appeared otherwise normal and showed no signs of damage or death. They formed functional bile canaliculi that connected to mouse canaliculi. Besides mature hepatocytes, human hepatic progenitor cells that were differentiating into mature hepatocytes could be identified within liver parenchyma. Infection of chimeric mice with HBV or HCV resulted in an active infection that did not alter the liver function and architecture. Electron microscopy showed the presence of viral and subviral structures in HBV infected hepatocytes. In conclusion, human hepatocytes repopulate the uPA+/+‐SCID mouse liver in a very organized fashion with preservation of normal cell function. The presence of human hepatic progenitor cells in these chimeric animals necessitates a critical review of the observations and conclusions made in experiments with isolated “mature” hepatocytes. Supplementary material for this article can be found on the HEPATOLOGY website (http://www.interscience.wiley.com/jpages/0270‐9139/suppmat/index.html). (HEPATOLOGY 2005;41:847–856.)

Anti‐CD81 antibodies can prevent a hepatitis C virus infection in vivo

The viral life cycle of the hepatitis C virus (HCV) has been studied mainly using different in vitro cell culture models. Studies using pseudoviral particles (HCVpp) and more recently cell culture–derived virus (HCVcc) suggest that at least three host cell molecules are important for HCV entry in vitro: the tetraspanin CD81, the scavenger receptor class B member I, and the tight junction protein Claudin‐1. Whether these receptors are equally important for an in vivo infection remains to be demonstrated. We show that CD81 is indispensable for an authentic in vivo HCV infection. Prophylactic treatment with anti‐CD81 antibodies completely protected human liver‐uPA‐SCID mice from a subsequent challenge with HCV consensus strains of different genotypes. Administration of anti‐CD81 antibodies after viral challenge had no effect. Conclusion: Our experiments provide evidence for the critical role of CD81 in a genuine HCV infection in vivo and open new perspectives for the prevention of allograft reinfection after orthotopic liver transplantation in chronically infected HCV patients. (HEPATOLOGY 2008;48:1761–1768.)

The green tea polyphenol, epigallocatechin‐3‐gallate, inhibits hepatitis C virus entry

Hepatitis C virus (HCV) is a major cause of liver cirrhosis and hepatocellular carcinoma. Current antiviral therapy fails to clear infection in a substantial proportion of cases. Drug development is focused on nonstructural proteins required for RNA replication. Individuals undergoing orthotopic liver transplantation face rapid, universal reinfection of the graft. Therefore, antiviral strategies targeting the early stages of infection are urgently needed for the prevention of HCV infection. In this study, we identified the polyphenol, epigallocatechin‐3‐gallate (EGCG), as an inhibitor of HCV entry. Green tea catechins, such as EGCG and its derivatives, epigallocatechin (EGC), epicatechin gallate (ECG), and epicatechin (EC), have been previously found to exert antiviral and antioncogenic properties. EGCG had no effect on HCV RNA replication, assembly, or release of progeny virions. However, it potently inhibited Cell‐culture–derived HCV (HCVcc) entry into hepatoma cell lines as well as primary human hepatocytes. The effect was independent of the HCV genotype, and both infection of cells by extracellular virions and cell‐to‐cell spread were blocked. Pretreatment of cells with EGCG before HCV inoculation did not reduce HCV infection, whereas the application of EGCG during inoculation strongly inhibited HCV infectivity. Moreover, treatment with EGCG directly during inoculation strongly inhibited HCV infectivity. Expression levels of all known HCV (co‐)receptors were unaltered by EGCG. Finally, we showed that EGCG inhibits viral attachment to the cell, thus disrupting the initial step of HCV cell entry. Conclusion: The green tea molecule, EGCG, potently inhibits HCV entry and could be part of an antiviral strategy aimed at the prevention of HCV reinfection after liver transplantation. (HEPATOLOGY 2011)

Cell Culture Adaptation of Hepatitis C Virus and In Vivo Viability of an Adapted Variant

ABSTRACT Production of infectious hepatitis C virus in cell culture has become possible because of the unique properties of the JFH1 isolate. However, virus titers are rather low, limiting the utility of this system. Here we describe the generation of cell culture-adapted JFH1 variants yielding higher titers of infectious particles and enhanced spread of infection in cultured cells. Sequence analysis of adapted genomes revealed a complex pattern of mutations that differed in two independent experiments. Adaptive mutations were observed both in the structural and in the nonstructural regions, with the latter having the highest impact on enhancement of virus titers. The major adaptive mutation was identified in NS5A, and it enhanced titers of three intergenotypic chimeras consisting of the structural region of a genotype 1a, 1b, or 3a isolate and the remainder of the JFH1 isolate. The mutation resides at the P3 position of the NS5A-B cleavage site and slows down processing, implying that subtle differences in replication complex formation appear to determine the efficiency of virus formation. Highly adapted JFH1 viruses carrying six mutations established a robust infection in uPA-transgenic SCID mice xenografted with human hepatocytes. However, the mutation in NS5A which enhanced virus titers in cell culture the most had reverted to wild type in nearly half of the viral genomes isolated from these animals at 15 weeks postinoculation. These results argue for some level of impaired fitness of this mutant in vivo.

Scavenger Receptor BI and BII Expression Levels Modulate Hepatitis C Virus Infectivity

ABSTRACT Hepatitis C virus (HCV) enters cells via a pH- and clathrin-dependent endocytic pathway. Scavenger receptor BI (SR-BI) and CD81 are important entry factors for HCV internalization into target cells. The SR-BI gene gives rise to at least two mRNA splice variants, SR-BI and SR-BII, which differ in their C termini. SR-BI internalization remains poorly understood, but SR-BII is reported to endocytose via a clathrin-dependent pathway, making it an attractive target for HCV internalization. We demonstrate that HCV soluble E2 can interact with human SR-BI and SR-BII. Increased expression of SR-BI and SR-BII in the Huh-7.5 hepatoma cell line enhanced HCV strain J6/JFH and JFH infectivity, suggesting that endogenous levels of these receptors limit infection. Elevated expression of SR-BI, but not SR-BII, increased the rate of J6/JFH infection, which may reflect altered intracellular trafficking of the splice variants. In human plasma, HCV particles have been reported to be complexed with lipoproteins, suggesting an indirect interaction of the virus with SR-BI and other lipoprotein receptors. Plasma from J6/JFH-infected uPA-SCID mice transplanted with human hepatocytes demonstrates an increased infectivity for SR-BI/II-overexpressing Huh-7.5 cells. Plasma-derived J6/JFH infectivity was inhibited by an anti-E2 monoclonal antibody, suggesting that plasma virus interaction with SR-BI was glycoprotein dependent. Finally, anti-SR-BI antibodies inhibited the infectivity of cell culture- and plasma-derived J6/JFH, suggesting a critical role for SR-BI/II in HCV infection.

Production of infectious genotype 1b virus particles in cell culture and impairment by replication enhancing mutations.

With the advent of subgenomic hepatitis C virus (HCV) replicons, studies of the intracellular steps of the viral replication cycle became possible. These RNAs are capable of self-amplification in cultured human hepatoma cells, but save for the genotype 2a isolate JFH-1, efficient replication of these HCV RNAs requires replication enhancing mutations (REMs), previously also called cell culture adaptive mutations. These mutations cluster primarily in the central region of non-structural protein 5A (NS5A), but may also reside in the NS3 helicase domain or at a distinct position in NS4B. Most efficient replication has been achieved by combining REMs residing in NS3 with distinct REMs located in NS4B or NS5A. However, in spite of efficient replication of HCV genomes containing such mutations, they do not support production of infectious virus particles. By using the genotype 1b isolate Con1, in this study we show that REMs interfere with HCV assembly. Strongest impairment of virus formation was found with REMs located in the NS3 helicase (E1202G and T1280I) as well as NS5A (S2204R), whereas a highly adaptive REM in NS4B still allowed virus production although relative levels of core release were also reduced. We also show that cells transfected with the Con1 wild type genome or the genome containing the REM in NS4B release HCV particles that are infectious both in cell culture and in vivo. Our data provide an explanation for the in vitro and in vivo attenuation of cell culture adapted HCV genomes and may open new avenues for the development of fully competent culture systems covering the therapeutically most relevant HCV genotypes.

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.

The human liver-uPA-SCID mouse: a model for the evaluation of antiviral compounds against HBV and HCV.

The study of the hepatitis B virus (HBV) and the hepatitis C virus (HCV) has long been hampered by the lack of a suitable small animal model. Both viruses could only be studied in humans or in chimpanzees. Recently, a new chimeric mouse model was developed that was permissive for HBV and HCV infection. In this model, uPA+/+-SCID mice, suffering from a transgene-induced liver disease, are transplanted early after birth with primary human hepatocytes. These human hepatocytes integrate in the parenchyma and progressively repopulate the diseased mouse liver without losing their normal metabolic functions. Successfully transplanted mice can then be infected with HBV and HCV. In this review, we describe the characteristics of this chimeric mouse model in more detail and give an overview of how this model has already contributed to the development of new antiviral compounds for the treatment of viral hepatitis.

A human monoclonal antibody targeting scavenger receptor class B type I precludes hepatitis C virus infection and viral spread in vitro and in vivo.

Endstage liver disease caused by chronic hepatitis C virus (HCV) infection is the leading indication for liver transplantation in the Western world. However, immediate reinfection of the grafted donor liver by circulating virus is inevitable and liver disease progresses much faster than the original disease. Standard antiviral therapy is not well tolerated and usually ineffective in liver transplant patients, whereas anti‐HCV immunotherapy is hampered by the extreme genetic diversity of the virus and its ability to spread by way of cell‐cell contacts. We generated a human monoclonal antibody against scavenger receptor class B type I (SR‐BI), monoclonal antibody (mAb)16‐71, which can efficiently prevent infection of Huh‐7.5 hepatoma cells and primary hepatocytes by cell‐culture‐derived HCV (HCVcc). Using an Huh7.5 coculture system we demonstrated that mAb16‐71 interferes with direct cell‐to‐cell transmission of HCV. Finally we evaluated the in vivo efficacy of mAb16‐71 in “human liver urokinase‐type plasminogen activator, severe combined immune deficiency (uPA‐SCID) mice” (chimeric mice). A 2‐week anti‐SR‐BI therapy that was initiated 1 day before viral inoculation completely protected all chimeric mice from infection with serum‐derived HCV of different genotypes. Moreover, a 9‐day postexposure therapy that was initiated 3 days after viral inoculation (when viremia was already observed in the animals) suppressed the rapid viral spread observed in untreated control animals. After cessation of anti‐SR‐BI‐specific antibody therapy, a rise of the viral load was observed. Conclusion: Using in vitro cell culture and human liver‐chimeric mouse models, we show that a human mAb targeting the HCV coreceptor SR‐BI completely prevents infection and intrahepatic spread of multiple HCV genotypes. This strategy may be an efficacious way to prevent infection of allografts following liver transplantation in chronic HCV patients, and may even hold promise for the prevention of virus rebound during or following antiviral therapy. (HEPATOLOGY 2012)

Polyclonal immunoglobulins from a chronic hepatitis C virus patient protect human liver–chimeric mice from infection with a homologous hepatitis C virus strain

The role of the humoral immune response in the natural course of hepatitis C virus (HCV) infection is widely debated. Most chronically infected patients have immunoglobulin G (IgG) antibodies capable of neutralizing HCV pseudoparticles (HCVpp) in vitro. It is, however, not clear whether these IgG can prevent a de novo HCV infection in vivo and contribute to the control of viremia in infected individuals. We addressed this question with homologous in vivo protection studies in human liver–urokinase‐type plasminogen activator (uPA)+/+ severe combined immune deficient (SCID) mice. Chimeric mice were loaded with chronic phase polyclonal IgG and challenged 3 days later with a 100% infectious dose of the acute phase H77C virus, both originating from patient H. Passive immunization induced sterilizing immunity in five of eight challenged animals. In the three nonprotected animals, the HCV infection was attenuated, as evidenced by altered viral kinetics in comparison with five control IgG‐treated animals. Plasma samples obtained from the mice at viral challenge neutralized H77C‐HCVpp at dilutions as high as 1/400. Infection was completely prevented when, before administration to naïve chimeric mice, the inoculum was pre‐incubated in vitro at an IgG concentration normally observed in humans. Conclusion: Polyclonal IgG from a patient with a long‐standing HCV infection not only displays neutralizing activity in vitro using the HCVpp system, but also conveys sterilizing immunity toward the ancestral HCV strain in vivo, using the human liver–chimeric mouse model. Both experimental systems will be useful tools to identify neutralizing antibodies for future clinical use. (HEPATOLOGY 2008.)