D. Lorne Tyrrell

HCV induces oxidative and ER stress, and sensitizes infected cells to apoptosis in SCID/Alb-uPA mice.

Hepatitis C virus (HCV) is a blood-borne pathogen and a major cause of liver disease worldwide. Gene expression profiling was used to characterize the transcriptional response to HCV H77c infection. Evidence is presented for activation of innate antiviral signaling pathways as well as induction of lipid metabolism genes, which may contribute to oxidative stress. We also found that infection of chimeric SCID/Alb-uPA mice by HCV led to signs of hepatocyte damage and apoptosis, which in patients plays a role in activation of stellate cells, recruitment of macrophages, and the subsequent development of fibrosis. Infection of chimeric mice with HCV H77c also led an inflammatory response characterized by infiltration of monocytes and macrophages. There was increased apoptosis in HCV-infected human hepatocytes in H77c-infected mice but not in mice inoculated with a replication incompetent H77c mutant. Moreover, TUNEL reactivity was restricted to HCV-infected hepatocytes, but an increase in FAS expression was not. To gain insight into the factors contributing specific apoptosis of HCV infected cells, immunohistological and confocal microscopy using antibodies for key apoptotic mediators was done. We found that the ER chaperone BiP/GRP78 was increased in HCV-infected cells as was activated BAX, but the activator of ER stress–mediated apoptosis CHOP was not. We found that overall levels of NF-κB and BCL-xL were increased by infection; however, within an infected liver, comparison of infected cells to uninfected cells indicated both NF-κB and BCL-xL were decreased in HCV-infected cells. We conclude that HCV contributes to hepatocyte damage and apoptosis by inducing stress and pro-apoptotic BAX while preventing the induction of anti-apoptotic NF-κB and BCL-xL, thus sensitizing hepatocytes to apoptosis.

HCV796: A selective nonstructural protein 5B polymerase inhibitor with potent anti‐hepatitis C virus activity In Vitro, in mice with chimeric human livers, and in humans infected with hepatitis C virus

Anti‐hepatitis C virus (HCV) drug development has been challenged by a lack of experience with inhibitors inclusive of in vitro, animal model, and clinical study. This manuscript outlines activity and correlation across such a spectrum of models and into clinical trials with a novel selective nonstructural protein 5B (NS5B) polymerase inhibitor, HCV796. Enzyme assays yielded median inhibitory concentration (IC50) values of 0.01 to 0.14 μM for genotype 1, with half maximal effective concentration (EC50s) of 5 nM and 9 nM against genotype 1a and 1b replicons. In the chimeric mouse model, a 2.02 ± 0.55 log reduction in HCV titer was seen with monotherapy, whereas a suboptimal dose of 30 mg/kg three times per day in combination with interferon demonstrated a 2.44 log reduction (P = 0.001 versus interferon alone) Clinical outcomes in combination with pegylated interferon and ribavirin have revealed additive efficacy in treatment naïve patients. Abnormal liver function test results were observed in 8% of HCV‐796 patients treated for over 8 weeks, resulting in suspension of further trial activity. Conclusion: The RNA‐dependent RNA polymerase inhibitor HCV796 demonstrated potent anti‐HCV activity consistently through enzyme inhibition assays, subgenomic replicon, and chimeric mouse studies. Strong correlations of outcomes in the mouse model were seen with subsequent clinical trials, including a plateau in dose‐related antiviral activity and additive impact from combination therapy with interferon. These outcomes demonstrate the utility of the range of in vitro and in vivo models now available for anti‐HCV drug development and support the potential utility of polymerase inhibitors in future combination therapies for HCV treatment. (HEPATOLOGY 2009.)

Anti-HCV therapies in chimeric scid-Alb/uPA mice parallel outcomes in human clinical application†

Compounds with in vitro anti‐hepatitis C virus (HCV) activity are often advanced directly into clinical trials with limited or no in vivo efficacy data. This limits prediction of clinical efficacy of compounds in the HCV drug pipeline, and may expose human subjects to unnecessary treatment effects. The scid‐Alb‐uPA mouse supports proliferation of transplanted human hepatocytes and subsequent HCV infection. Cohorts of genotype 1a HCV‐infected mice were treated with interferon α‐2b(IFN‐α), BILN‐2061 (anti‐NS3 protease), or HCV371 (anti‐NS5B polymerase). Mice treated with 1350IU/g/day IFN‐α intramuscularly for 10 to 28 days demonstrated reduced viral titers compared with controls in all five experiments (P < .05, t test); viral titers rebounded after treatment withdrawal. A more pronounced antiviral effect with IFN‐α was seen in genotype 3a–infected mice. Pilot studies with BILN2061 confirmed exposure to 10X replicon EC50 at trough and reduced viral titer over 2 log at 4 days. In a second 7‐day study, mean HCV RNA titers dropped 1.1 log in BILN2061‐treated animals, 0.6 log in IFN‐treated mice, and rose 0.2 log in controls (P = .013, ANOVA). Pre‐existing mutants with partial resistance to BILN2061 were identified by sequencing both the human inoculum and sera from treated mice. The polymerase inhibitor HCV371 yielded a decline in HCV titers of 0.3 log relative to vehicle‐treated controls (P = NS). Performance of all three antiviral regimens in the chimeric mouse model paralleled responses in humans. In conclusion, this system may help selection of lead compounds for advancement into human trials with an increased likelihood of clinical success while broadening the tools available for study of the biology of HCV infection. (HEPATOLOGY 2006;43:1346–1353.)

IFITM1 Is a Tight Junction Protein That Inhibits Hepatitis C Virus Entry

Type 1 interferon (IFN) continues to be the foundation for the current standard of care combination therapy for chronic hepatitis C virus (HCV) infection, yet the component interferon‐stimulated genes (ISGs) that mediate the antiviral actions of IFN are not fully defined. Interferon‐induced transmembrane protein 1 (IFITM1) is an ISG product that suppresses early stage infection by a number of viruses through an unknown mechanism of action. Moreover, the actions of IFITM1 on HCV infection are not fully elucidated. Here we identify IFITM1 as a hepatocyte tight junction protein and a potent anti‐HCV effector molecule. IFITM1 expression is induced early during IFN treatment of hepatocytes and accumulates at hepatic tight junctions in HCV‐infected human patient liver during IFN therapy. Additionally, we found that IFITM1 interacts with HCV coreceptors, including CD81 and occludin, to disrupt the process of viral entry. Thus, IFITM1 is an anti‐HCV ISG whose actions impart control of HCV infection through interruption of viral coreceptor function. Conclusion: This study defines IFITM1 as an ISG effector with action against HCV entry. Design of therapy regimens to enhance IFITM1 expression should improve the virologic response among HCV patients undergoing treatment with type I IFN. (HEPATOLOGY 2013)

A Hepatitis C Virus (HCV) Vaccine Comprising Envelope Glycoproteins gpE1/gpE2 Derived from a Single Isolate Elicits Broad Cross-Genotype Neutralizing Antibodies in Humans

Although a cure for HCV is on the near horizon, emerging drug cocktails will be expensive, associated with side-effects and resistance making a global vaccine an urgent priority given the estimated high incidence of infection around the world. Due to the highly heterogeneous nature of HCV, an effective HCV vaccine which could elicit broadly cross-neutralizing antibodies has represented a major challenge. In this study, we tested for the presence of cross-neutralizing antibodies in human volunteers who were immunized with recombinant glycoproteins gpE1/gpE2 derived from a single HCV strain (HCV1 of genotype 1a). Cross neutralization was tested in Huh-7.5 human hepatoma cell cultures using infectious recombinant HCV (HCVcc) expressing structural proteins of heterologous HCV strains from all known major genotypes, 1–7. Vaccination induced significant neutralizing antibodies against heterologous HCV genotype 1a virus which represents the most common genotype in North America. Of the 16 vaccinees tested, 3 were selected on the basis of strong 1a virus neutralization for testing of broad cross-neutralizing responses. At least 1 vaccinee was shown to elicit broad cross-neutralization against all HCV genotypes. Although observed in only a minority of vaccinees, our results prove the key concept that a vaccine derived from a single strain of HCV can elicit broad cross-neutralizing antibodies against all known major genotypes of HCV and provide considerable encouragement for the further development of a human vaccine against this common, global pathogen.

Rigid amphipathic fusion inhibitors, small molecule antiviral compounds against enveloped viruses

Antiviral drugs targeting viral proteins often result in prompt selection for resistance. Moreover, the number of viral targets is limited. Novel antiviral targets are therefore needed. The unique characteristics of fusion between virion envelopes and cell membranes may provide such targets. Like all fusing bilayers, viral envelopes locally adopt hourglass-shaped stalks during the initial stages of fusion, a process that requires local negative membrane curvature. Unlike cellular vesicles, however, viral envelopes do not redistribute lipids between leaflets, can only use the energy released by virion proteins, and fuse to the extracellular leaflets of cell membranes. Enrichment in phospholipids with hydrophilic heads larger than their hydrophobic tails in the convex outer leaflet of vesicles favors positive curvature, therefore increasing the activation energy barrier for fusion. Such phospholipids can increase the activation barrier beyond the energy provided by virion proteins, thereby inhibiting viral fusion. However, phospholipids are not pharmacologically useful. We show here that a family of synthetic rigid amphiphiles of shape similar to such phospholipids, RAFIs (rigid amphipathic fusion inhibitors), inhibit the infectivity of several otherwise unrelated enveloped viruses, including hepatitis C and HSV-1 and -2 (lowest apparent IC50 48 nM), with no cytotoxic or cytostatic effects (selectivity index > 3,000) by inhibiting the increased negative curvature required for the initial stages of fusion.

Half-Life of the Duck Hepatitis B Virus Covalently Closed Circular DNA Pool In Vivo following Inhibition of Viral Replication

ABSTRACT Covalently closed circular DNA (cccDNA) is a crucial intermediate in the replication of hepadnaviruses. We inhibited the replication of duck hepatitis B virus in congenitally infected ducks with a combination of lamivudine and a dideoxyguanosine prodrug. Inhibition of viral replication should prevent renewal of the cccDNA pool, and its decay was measured in liver biopsy samples collected over a 5-month period. In three ducks, the cccDNA pools declined exponentially, with half-lives ranging from 35 to 57 days. In two others, the pools declined exponentially for about 70 days but then stabilized at about 6 copies/diploid genome. The selection of drug-resistant virus mutants is an unlikely explanation for this unexpected stabilization of cccDNA levels. Liver sections stained for the cell division marker PCNA showed that animals in which cccDNA loss was continuous had significantly greater numbers of PCNA-positive nuclei than did those animals in which cccDNA levels had plateaued.

Zinc Finger Proteins Designed To Specifically Target Duck Hepatitis B Virus Covalently Closed Circular DNA Inhibit Viral Transcription in Tissue Culture

ABSTRACT Duck hepatitis B virus (DHBV) is a model virus for human hepatitis B virus (HBV), which infects approximately 360 million individuals worldwide. Nucleoside analogs can decrease virus production by inhibiting the viral polymerase; however, complete clearance by these drugs is not common because of the persistence of the HBV episome. HBV DNA is present in the nucleus as a covalently closed circular (cccDNA) form, where it drives viral transcription and progeny virus production. cccDNA is not the direct target of antiviral nucleoside analogs and is the source of HBV reemergence when antiviral therapy is stopped. To target cccDNA, six different zinc finger proteins (ZFP) were designed to bind DNA sequences in the DHBV enhancer region. After the binding kinetics were assessed by using electrophoretic mobility shift assays and surface plasmon resonance, two candidates with dissociation constants of 12.3 and 40.2 nM were focused on for further study. The ZFPs were cloned into a eukaryotic expression vector and cotransfected into longhorn male hepatoma cells with the plasmid pDHBV1.3, which replicates the DHBV life cycle. In the presence of each ZFP, viral RNA was significantly reduced, and protein levels were dramatically decreased. As a result, intracellular viral particle production was also significantly decreased. In summary, designed ZFPs are able to bind to the DHBV enhancer and interfere with viral transcription, resulting in decreased production of viral products and progeny virus genomes.

Host-Specific Response to HCV Infection in the Chimeric SCID-beige/Alb-uPA Mouse Model: Role of the Innate Antiviral Immune Response

The severe combined immunodeficiency disorder (SCID)-beige/albumin (Alb)-urokinase plasminogen activator (uPA) mouse containing a human-mouse chimeric liver is currently the only small animal model capable of supporting hepatitis C virus (HCV) infection. This model was utilized to characterize the host transcriptional response to HCV infection. The purpose of these studies was to investigate the genetic component of the host response to HCV infection and also to distinguish virus-induced gene expression changes from adaptive HCV-specific immune-mediated effects. Gene expression profiles from HCV-infected mice were also compared to those from HCV-infected patients. Analyses of the gene expression data demonstrate that host factors regulate the response to HCV infection, including the nature of the innate antiviral immune response. They also indicate that HCV mediates gene expression changes, including regulation of lipid metabolism genes, which have the potential to be directly cytopathic, indicating that liver pathology may not be exclusively mediated by HCV-specific adaptive immune responses. This effect appears to be inversely related to the activation of the innate antiviral immune response. In summary, the nature of the initial interferon response to HCV infection may determine the extent of viral-mediated effects on host gene expression.

Hepatitis C virus induced up‐regulation of microRNA‐27: A novel mechanism for hepatic steatosis

MicroRNAs (miRNAs) are small RNAs that posttranscriptionally regulate gene expression. Their aberrant expression is commonly linked with diseased states, including hepatitis C virus (HCV) infection. Herein, we demonstrate that HCV replication induces the expression of miR‐27 in cell culture and in vivo HCV infectious models. Overexpression of the HCV proteins core and NS4B independently activates miR‐27 expression. Furthermore, we establish that miR‐27 overexpression in hepatocytes results in larger and more abundant lipid droplets, as observed by coherent anti‐Stokes Raman scattering (CARS) microscopy. This hepatic lipid droplet accumulation coincides with miR‐27bs repression of peroxisome proliferator‐activated receptor (PPAR)‐α and angiopoietin‐like protein 3 (ANGPTL3), known regulators of triglyceride homeostasis. We further demonstrate that treatment with a PPAR‐α agonist, bezafibrate, is able to reverse the miR‐27b‐induced lipid accumulation in Huh7 cells. This miR‐27b‐mediated repression of PPAR‐α signaling represents a novel mechanism of HCV‐induced hepatic steatosis. This link was further demonstrated in vivo through the correlation between miR‐27b expression levels and hepatic lipid accumulation in HCV‐infected SCID‐beige/Alb‐uPa mice. Conclusion: Collectively, our results highlight HCVs up‐regulation of miR‐27 expression as a novel mechanism contributing to the development of hepatic steatosis. (Hepatology 2014;58:98–108)