Modeling hepatitis C virus kinetics during liver transplantation reveals the role of the liver in virus clearance.

While the liver, specifically hepatocytes, are widely accepted as the main source of hepatitis C virus (HCV) production, the role of the liver/hepatocytes in clearance of circulating HCV remains unknown. Frequent HCV kinetic data were recorded and mathematically modeled from five liver transplant patients throughout the anhepatic (absence of liver) phase and for 4 hr post-reperfusion. During the anhepatic phase, HCV remained at pre-anhepatic levels (n = 3) or declined (n = 2) with t1/2~1 hr. Immediately post-reperfusion, virus declined in a biphasic manner in four patients consisting of a rapid decline (t1/2 = 5 min) followed by a slower decline (t1/2 = 67 min). Consistent with the majority of patients in the anhepatic phase, when we monitored HCV clearance at 37°C from culture medium in the absence/presence of chronically infected hepatoma cells that were inhibited from secreting HCV, the HCV t1/2 in cell culture was longer in the absence of chronically HCV-infected cells. The results suggest that the liver plays a major role in the clearance of circulating HCV and that hepatocytes may be involved.

Intracellular Hepatitis C Virus Modeling Predicts Infection Dynamics and Viral Protein Mechanisms.

Hepatitis C virus (HCV) infection is a global health problem, with nearly 2 million new infections occurring every year and up to 85% of these infections becoming chronic infections that pose serious long-term health risks. To effectively reduce the prevalence of HCV infection and associated diseases, it is important to understand the intracellular dynamics of the viral life cycle. Here, we present a detailed mathematical model that represents the full hepatitis C virus life cycle. It is the first full HCV model to be fit to acute intracellular infection data and the first to explore the functions of distinct viral proteins, probing multiple hypotheses of cis- and trans-acting mechanisms to provide insights for drug targeting. Model parameters were derived from the literature, experiments, and fitting to experimental intracellular viral RNA, extracellular viral titer, and HCV core and NS3 protein kinetic data from viral inoculation to steady state. Our model predicts higher rates for protein translation and polyprotein cleavage than previous replicon models and demonstrates that the processes of translation and synthesis of viral RNA have the most influence on the levels of the species we tracked in experiments. Overall, our experimental data and the resulting mathematical infection model reveal information about the regulation of core protein during infection, produce specific insights into the roles of the viral core, NS5A, and NS5B proteins, and demonstrate the sensitivities of viral proteins and RNA to distinct reactions within the life cycle.IMPORTANCE We have designed a model for the full life cycle of hepatitis C virus. Past efforts have largely focused on modeling hepatitis C virus replicon systems, in which transfected subgenomic HCV RNA maintains autonomous replication in the absence of virion production or spread. We started with the general structure of these previous replicon models and expanded it to create a model that incorporates the full virus life cycle as well as additional intracellular mechanistic detail. We compared several different hypotheses that have been proposed for different parts of the life cycle and applied the corresponding model variations to infection data to determine which hypotheses are most consistent with the empirical kinetic data. Because the infection data we have collected for this study are a more physiologically relevant representation of a viral life cycle than data obtained from a replicon system, our model can make more accurate predictions about clinical hepatitis C virus infections.

Oral prenylation inhibition with lonafarnib in chronic hepatitis D infection: a proof-of-concept randomised, double-blind, placebo-controlled phase 2A trial.

BACKGROUND: Therapies for chronic hepatitis delta virus (HDV) infection are unsatisfactory. Prenylation is essential for HDV and inhibition abrogates HDV production in experimental models. In a proof-of-concept study, we aimed to assess the effect on HDV RNA levels, safety, and tolerability of the prenylation inhibitor lonafarnib in patients with chronic delta hepatitis. METHODS: In this phase 2A double-blind, randomised, placebo-controlled study, patients aged 18 years or older with chronic HDV infection were randomly assigned (3:1 in group 1 and 2:1 in group 2) to receive lonafarnib 100 mg (group 1) or lonafarnib 200 mg (group 2) twice daily for 28 days with 6 months' follow-up. Participants were randomised by random-number tables blocked in groups of four without stratification. Both groups enrolled six treatment participants and two placebo participants. Group 1 placebo patients received open-label lonafarnib as group 2 participants. The primary therapeutic endpoint was a decrease in HDV RNA viral titre in serum and the primary safety endpoint was the ability to tolerate the drug at the prescribed dose for the full 4-week duration, defined as drug discontinuation due to intolerance or grade 3/4 adverse events. This trial is registered with ClinicalTrials.gov, number NCT01495585. FINDINGS: Between Jan 19, 2012, and April 28, 2014, 14 patients were enrolled, of whom eight were assigned to group 1 and six were assigned to group 2. At day 28, compared with placebo, mean log HDV RNA declines from baseline were -0·73 log IU/mL in group 1 (95% CI 0·17-1·31; p=0·03) and -1·54 log IU/mL in group 2 (1·21-1·93; p<0·0001). Lonafarnib serum concentrations correlated with HDV RNA change (r(2)=0·78, p<0·0001). Model fits show that hepatitis B surface antigen (HBsAg) remained stable after a short pharmacological delay (0·75 days [SE 0·24]), lonafarnib effectiveness in blocking HDV production was greater in group 2 than in group 1 (0·952 [SE 0·06] vs 0·739 [0·05], p<0·001), and the HDV half-life was 1·62 days (0·07). There was no evidence of virological resistance. Adverse events were mainly mild to moderate with group 1 patients experiencing diarrhoea in three patients (50%) and nausea in two patients (33%) and in group 2 with all patients (100%) experiencing nausea, diarrhoea, abdominal bloating, and weight loss greater than 2 kg (mean of 4 kg). No treatment discontinuations occurred in any treatment groups. INTERPRETATION: Treatment of chronic HDV with lonafarnib significantly reduces virus levels. The decline in virus levels significantly correlated with serum drug levels, providing further evidence for the efficacy of prenylation inhibition in chronic HDV. FUNDING: National Institute of Diabetes and Digestive and Kidney Diseases and National Cancer Institute, National Institutes of Health, and Eiger Biopharmaceuticals Inc.

High-throughput screening (HTS) and hit validation to identify small molecule inhibitors with activity against NS3/4A proteases from multiple hepatitis C virus genotypes.

Development of drug-resistant mutations has been a major problem with all currently developed Hepatitis C Virus (HCV) NS3/4A inhibitors, including the two FDA approved drugs, significantly reducing the efficacy of these inhibitors. The high incidence of drug-resistance mutations and the limited utility of these inhibitors against only genotype 1 highlight the need for novel, broad-spectrum HCV therapies. Here we used high-throughput screening (HTS) to identify low molecular weight inhibitors against NS3/4A from multiple genotypes. A total of 40,967 compounds from four structurally diverse molecular libraries were screened by HTS using fluorescence-based enzymatic assays, followed by an orthogonal binding analysis using surface plasmon resonance (SPR) to eliminate false positives. A novel small molecule compound was identified with an IC50 value of 2.2 µM against the NS3/4A from genotype 1b. Mode of inhibition analysis subsequently confirmed this compound to be a competitive inhibitor with respect to the substrate, indicating direct binding to the protease active site, rather than to the allosteric binding pocket that was discovered to be the binding site of a few recently discovered small molecule inhibitors. This newly discovered inhibitor also showed promising inhibitory activity against the NS3/4As from three other HCV genotypes, as well as five common drug-resistant mutants of genotype 1b NS3/4A. The inhibitor was selective for NS3 from multiple HCV genotypes over two human serine proteases, and a whole cell lysate assay confirmed inhibitory activity in the cellular environment. This compound provides a lead for further development of potentially broader spectrum inhibitors.

Antioxidant and Anti-Hepatitis C Viral Activities of Commercial Milk Thistle Food Supplements.

Milk thistle dietary supplements that contain silymarin are widely marketed and used in the USA and other countries for liver enhancement and recovery. More recently, silymarin has also been identified as a possible antiviral for the treatment of hepatitis C virus (HCV) infection. To assess different brands of commercially sold silymarin, 45 products were collected from local stores and analyzed for their silymarin content, antioxidant activities, and antiviral activity against HCV. Antioxidant activity was measured as radical scavenging activity using DPPH and by estimating their antioxidant capacity as trolox equivalent. Anti-HCV activity was measured in an HCV genotype 1b replication inhibition assay. Samples were found to vary widely in their silymarin content, with some samples having none or very low concentrations while silymarin represented higher than 80% of other samples. Both antioxidant and anti-HCV activity correlated with the overall level of silymarin.

A transmembrane serine protease is linked to the severe acute respiratory syndrome coronavirus receptor and activates virus entry.

Spike (S) proteins, the defining projections of the enveloped coronaviruses (CoVs), mediate cell entry by connecting viruses to plasma membrane receptors and by catalyzing subsequent virus-cell membrane fusions. The latter membrane fusion requires an S protein conformational flexibility that is facilitated by proteolytic cleavages. We hypothesized that the most relevant cellular proteases in this process are those closely linked to host cell receptors. The primary receptor for the human severe acute respiratory syndrome CoV (SARS) CoV is angiotensin-converting enzyme 2 (ACE2). ACE2 immunoprecipitation captured transmembrane protease/serine subfamily member 2 (TMPRSS2), a known human airway and alveolar protease. ACE2 and TMPRSS2 colocalized on cell surfaces and enhanced the cell entry of both SARS S-pseudotyped HIV and authentic SARS-CoV. Enhanced entry correlated with TMPRSS2-mediated proteolysis of both S and ACE2. These findings indicate that a cell surface complex comprising a primary receptor and a separate endoprotease operates as a portal for activation of SARS-CoV cell entry.