Maria Teresa Catanese

A genetically humanized mouse model for hepatitis C virus infection

Hepatitis C virus (HCV) remains a major medical problem. Antiviral treatment is only partially effective and a vaccine does not exist. Development of more effective therapies has been hampered by the lack of a suitable small animal model. Although xenotransplantation of immunodeficient mice with human hepatocytes has shown promise, these models are subject to important challenges. Building on the previous observation that CD81 and occludin comprise the minimal human factors required to render mouse cells permissive to HCV entry in vitro, we attempted murine humanization via a genetic approach. Here we show that expression of two human genes is sufficient to allow HCV infection of fully immunocompetent inbred mice. We establish a precedent for applying mouse genetics to dissect viral entry and validate the role of scavenger receptor type B class I for HCV uptake. We demonstrate that HCV can be blocked by passive immunization, as well as showing that a recombinant vaccinia virus vector induces humoral immunity and confers partial protection against heterologous challenge. This system recapitulates a portion of the HCV life cycle in an immunocompetent rodent for the first time, opening opportunities for studying viral pathogenesis and immunity and comprising an effective platform for testing HCV entry inhibitors in vivo.

Real-time imaging of hepatitis C virus infection using a fluorescent cell-based reporter system.

Hepatitis C virus (HCV), which infects 2–3% of the world population, is a causative agent of chronic hepatitis and the leading indication for liver transplantation. The ability to propagate HCV in cell culture (HCVcc) is a relatively recent breakthrough and a key tool in the quest for specific antiviral therapeutics. Monitoring HCV infection in culture generally involves bulk population assays, use of genetically modified viruses and/or terminal processing of potentially precious samples. Here we develop a cell-based fluorescent reporter system that allows sensitive distinction of individual HCV-infected cells in live or fixed samples. We demonstrate use of this technology for several previously intractable applications, including live-cell imaging of viral propagation and host response, as well as visualizing infection of primary hepatocyte cultures. Integration of this reporter with modern image-based analysis methods could open new doors for HCV research.

High-Avidity Monoclonal Antibodies against the Human Scavenger Class B Type I Receptor Efficiently Block Hepatitis C Virus Infection in the Presence of High-Density Lipoprotein

ABSTRACT The human scavenger class B type 1 receptor (SR-B1/Cla1) was identified as a putative receptor for hepatitis C virus (HCV) because it binds to soluble recombinant HCV envelope glycoprotein E2 (sE2). High-density lipoprotein (HDL), a natural SR-B1 ligand, was shown to increase the in vitro infectivity of retroviral pseudoparticles bearing HCV envelope glycoproteins and of cell culture-derived HCV (HCVcc), suggesting that SR-B1 promotes viral entry in an HDL-dependent manner. To determine whether SR-B1 participates directly in HCV infection or facilitates HCV entry through lipoprotein uptake, we generated a panel of monoclonal antibodies (MAbs) against native human SR-B1. Two of them, 3D5 and C167, bound to conformation-dependent SR-B1 determinants and inhibited the interaction of sE2 with SR-B1. These antibodies efficiently blocked HCVcc infection of Huh-7.5 hepatoma cells in a dose-dependent manner. To examine the role of HDL in SR-B1-mediated HCVcc infection, we set up conditions for HCVcc production and infection in serum-free medium. HCVcc efficiently infected Huh-7.5 cells in the absence of serum lipoproteins, and addition of HDL led to a twofold increase in infectivity. However, the HDL-induced enhancement of infection had no impact on the neutralization potency of MAb C167, despite its ability to inhibit both HDL binding to cells and SR-B1-mediated lipid transfer. Of note, MAb C167 also potently blocked Huh-7.5 infection by an HCV strain recovered from HCVcc-infected chimpanzees. These results demonstrate that SR-B1 is essential for infection with HCV produced in vitro and in vivo and suggest the possible use of anti-SR-B1 antibodies as therapeutic agents.

Ultrastructural analysis of hepatitis C virus particles

Hepatitis C virus (HCV) is a major cause of chronic liver disease, with an estimated 170 million people infected worldwide. Low yields, poor stability, and inefficient binding to conventional EM grids have posed significant challenges to the purification and structural analysis of HCV. In this report, we generated an infectious HCV genome with an affinity tag fused to the E2 envelope glycoprotein. Using affinity grids, previously described to isolate proteins and macromolecular complexes for single-particle EM, we were able to purify enveloped particles directly from cell culture media. This approach allowed for rapid in situ purification of virions and increased particle density that were instrumental for cryo-EM and cryoelectron tomography (cryo-ET). Moreover, it enabled ultrastructural analysis of virions produced by primary human hepatocytes. HCV appears to be the most structurally irregular member of the Flaviviridae family. Particles are spherical, with spike-like projections, and heterogeneous in size ranging from 40 to 100 nm in diameter. Exosomes, although isolated from unfractionated culture media, were absent in highly infectious, purified virus preparations. Cryo-ET studies provided low-resolution 3D structural information of highly infectious virions. In addition to apolipoprotein (apo)E, HCV particles also incorporate apoB and apoA-I. In general, host apolipoproteins were more readily accessible to antibody labeling than HCV glycoproteins, suggesting either lower abundance or masking by host proteins.

Role of Scavenger Receptor Class B Type I in Hepatitis C Virus Entry: Kinetics and Molecular Determinants

ABSTRACT Scavenger receptor class B type I (SR-BI) is an essential receptor for hepatitis C virus (HCV) and a cell surface high-density-lipoprotein (HDL) receptor. The mechanism of SR-BI-mediated HCV entry, however, is not clearly understood, and the specific protein determinants required for the recognition of the virus envelope are not known. HCV infection is strictly linked to lipoprotein metabolism, and HCV virions may initially interact with SR-BI through associated lipoproteins before subsequent direct interactions of the viral glycoproteins with SR-BI occur. The kinetics of inhibition of cell culture-derived HCV (HCVcc) infection with an anti-SR-BI monoclonal antibody imply that the recognition of SR-BI by HCV is an early event of the infection process. Swapping and single-substitution mutants between mouse and human SR-BI sequences showed reduced binding to the recombinant soluble E2 (sE2) envelope glycoprotein, thus suggesting that the SR-BI interaction with the HCV envelope is likely to involve species-specific protein elements. Most importantly, SR-BI mutants defective for sE2 binding, although retaining wild-type activity for receptor oligomerization and binding to the physiological ligand HDL, were impaired in their ability to fully restore HCVcc infectivity when transduced into an SR-BI-knocked-down Huh-7.5 cell line. These findings suggest a specific and direct role for the identified residues in binding HCV and mediating virus entry. Moreover, the observation that different regions of SR-BI are involved in HCV and HDL binding supports the hypothesis that new therapeutic strategies aimed at interfering with virus/SR-BI recognition are feasible.

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)

Role of SR-BI in HCV entry: kinetics and molecular determinants

ABSTRACT Scavenger receptor class B type I (SR-BI) is an essential receptor for hepatitis C virus (HCV) and a cell surface high-density-lipoprotein (HDL) receptor. The mechanism of SR-BI-mediated HCV entry, however, is not clearly understood, and the specific protein determinants required for the recognition of the virus envelope are not known. HCV infection is strictly linked to lipoprotein metabolism, and HCV virions may initially interact with SR-BI through associated lipoproteins before subsequent direct interactions of the viral glycoproteins with SR-BI occur. The kinetics of inhibition of cell culture-derived HCV (HCVcc) infection with an anti-SR-BI monoclonal antibody imply that the recognition of SR-BI by HCV is an early event of the infection process. Swapping and single-substitution mutants between mouse and human SR-BI sequences showed reduced binding to the recombinant soluble E2 (sE2) envelope glycoprotein, thus suggesting that the SR-BI interaction with the HCV envelope is likely to involve species-specific protein elements. Most importantly, SR-BI mutants defective for sE2 binding, although retaining wild-type activity for receptor oligomerization and binding to the physiological ligand HDL, were impaired in their ability to fully restore HCVcc infectivity when transduced into an SR-BI-knocked-down Huh-7.5 cell line. These findings suggest a specific and direct role for the identified residues in binding HCV and mediating virus entry. Moreover, the observation that different regions of SR-BI are involved in HCV and HDL binding supports the hypothesis that new therapeutic strategies aimed at interfering with virus/SR-BI recognition are feasible.

Expression of Paramyxovirus V Proteins Promotes Replication and Spread of Hepatitis C Virus in Cultures of Primary Human Fetal Liver Cells

Here we demonstrate that primary cultures of human fetal liver cells (HFLC) reliably support infection with laboratory strains of hepatitis C virus (HCV), although levels of virus replication vary significantly between different donor cell preparations and frequently decline in a manner suggestive of active viral clearance. To investigate possible contributions of the interferon (IFN) system to control HCV infection in HFLC, we exploited the well‐characterized ability of paramyxovirus (PMV) V proteins to counteract both IFN induction and antiviral signaling. The V proteins of measles virus (MV) and parainfluenza virus 5 (PIV5) were introduced into HFLC using lentiviral vectors encoding a fluorescent reporter for visualization of HCV‐infected cells. V protein‐transduced HFLC supported enhanced (10 to 100‐fold) levels of HCV infection relative to untransduced or control vector‐transduced HFLC. Infection was assessed by measurement of virus‐driven luciferase, by assays for infectious HCV and viral RNA, and by direct visualization of HCV‐infected hepatocytes. Live cell imaging between 48 and 119 hours postinfection demonstrated little or no spread of infection in the absence of PMV V protein expression. In contrast, V protein‐transduced HFLC showed numerous HCV infection events. V protein expression efficiently antagonized the HCV‐inhibitory effects of added IFNs in HFLC. In addition, induction of the type III IFN, IL29, following acute HCV infection was inhibited in V protein‐transduced cultures. Conclusion: These studies suggest that the cellular IFN response plays a significant role in limiting the spread of HCV infection in primary hepatocyte cultures. Strategies aimed at dampening this response may be key to further development of robust HCV culture systems, enabling studies of virus pathogenicity and the mechanisms by which HCV spreads in its natural host cell population. (HEPATOLOGY 2011;)

Different Requirements for Scavenger Receptor Class B Type I in Hepatitis C Virus Cell-Free versus Cell-to-Cell Transmission

ABSTRACT Hepatitis C virus (HCV) is believed to initially infect the liver through the basolateral side of hepatocytes, where it engages attachment factors and the coreceptors CD81 and scavenger receptor class B type I (SR-BI). Active transport toward the apical side brings the virus in close proximity of additional entry factors, the tight junction molecules claudin-1 and occludin. HCV is also thought to propagate via cell-to-cell spread, which allows highly efficient virion delivery to neighboring cells. In this study, we compared an adapted HCV genome, clone 2, characterized by superior cell-to cell spread, to its parental genome, J6/JFH-1, with the goal of elucidating the molecular mechanisms of HCV cell-to-cell transmission. We show that CD81 levels on the donor cells influence the efficiency of cell-to-cell spread and CD81 transfer between neighboring cells correlates with the capacity of target cells to become infected. Spread of J6/JFH-1 was blocked by anti-SR-BI antibody or in cells knocked down for SR-BI, suggesting a direct role for this receptor in HCV cell-to-cell transmission. In contrast, clone 2 displayed a significantly reduced dependence on SR-BI for lateral spread. Mutations in E1 and E2 responsible for the enhanced cell-to-cell spread phenotype of clone 2 rendered cell-free virus more susceptible to antibody-mediated neutralization. Our results indicate that although HCV can lose SR-BI dependence for cell-to-cell spread, vulnerability to neutralizing antibodies may limit this evolutionary option in vivo. Combination therapies targeting both the HCV glycoproteins and SR-BI may therefore hold promise for effective control of HCV dissemination.

Successful anti‐scavenger receptor class B type I (SR‐BI) monoclonal antibody therapy in humanized mice after challenge with HCV variants with in vitro resistance to SR‐BI‐targeting agents

Hepatitis C virus (HCV)‐induced endstage liver disease is currently a major indication for liver transplantation. After transplantation the donor liver inevitably becomes infected with the circulating virus. Monoclonal antibodies (mAbs) against the HCV coreceptor scavenger receptor class B type I (SR‐BI) inhibit HCV infection of different genotypes, both in cell culture and in humanized mice. Anti‐SR‐BI mAb therapy is successful even when initiated several days after HCV exposure, supporting its potential applicability to prevent HCV reinfection of liver allografts. However, HCV variants with reduced SR‐BI dependency have been described in the literature, which could potentially limit the use of SR‐BI targeting therapy. In this study we show, both in a preventative and postexposure setting, that humanized mice infected with HCV variants exhibiting increased in vitro resistance to SR‐BI‐targeting molecules remain responsive to anti‐SR‐BI mAb therapy in vivo. A 2‐week antibody therapy readily cleared HCV RNA from the circulation of infected humanized mice. We found no evidence supporting increased SR‐BI‐receptor dependency of viral particles isolated from humanized mice compared to cell culture‐produced virus. However, we observed that, unlike wild‐type virus, the in vitro infectivity of the resistant variants was inhibited by both human high density lipoprotein (HDL) and very low density lipoprotein (VLDL). The combination of mAb1671 with these lipoproteins further increased the antiviral effect. Conclusion: HCV variants that are less dependent on SR‐BI in vitro can still be efficiently blocked by an anti‐SR‐BI mAb in humanized mice. Since these variants are also more susceptible to neutralization by anti‐HCV envelope antibodies, their chance of emerging during anti‐SR‐BI therapy is severely reduced. Our data indicate that anti‐SR‐BI receptor therapy could be an effective way to prevent HCV infection in a liver transplant setting. (Hepatology 2014;60:1508–1518)