Hepatitis C Viral Entry Inhibitors Prolong Viral Suppression by Replication Inhibitors in Persistently-Infected Huh7 Cultures

Caroline O Bush,Rudolf K F Beran,William E Delaney,Andrew E Greenstein,Ravi Jhaveri

doi:10.1371/journal.pone.0065273

Abstract

Efforts to treat HCV patients are focused on developing antiviral combinations that lead to the eradication of infection. Thus, it is important to identify optimal combinations from the various viral inhibitor classes. Based on viral dynamic models, HCV entry inhibitors are predicted to reduce viral load in a monophasic manner reflecting the slow death rate of infected hepatocytes (t1/2 = 2–70 days) and the protection of naïve, un-infected cells from HCV infection. In contrast, replication inhibitors are predicted to reduce viral load in a biphasic manner. The initial rapid reduction phase is due to the inhibition of virus production and elimination of plasma virus (t1/2∼3 hours). The second, slower reduction phase results from the elimination of infected hepatocytes. Here we sought to compare the ability of HCV entry and replication inhibitors as well as combinations thereof to reduce HCV infection in persistently-infected Huh7 cells. Treatment with 5×EC50 of entry inhibitors anti-CD81 Ab or EI-1 resulted in modest (≤1 log10 RNA copies/ml), monophasic declines in viral levels during 3 weeks of treatment. In contrast, treatment with 5×EC50 of the replication inhibitors BILN-2016 or BMS-790052 reduced extracellular virus levels more potently (∼2 log10 RNA copies/ml) over time in a biphasic manner. However, this was followed by a slow rise to steady-state virus levels due to the emergence of resistance mutations. Combining an entry inhibitor with a replication inhibitor did not substantially enhance the rate of virus reduction. However, entry/replication inhibitor and replication/replication inhibitor combinations reduced viral levels further than monotherapies (up to 3 log10 RNA copies/ml) and prolonged this reduction relative to monotherapies. Our results demonstrated that HCV entry inhibitors combined with replication inhibitors can prolong antiviral suppression, likely due to the delay of viral resistance emergence.

Highlights

Researchers are actively working to develop inhibitors of several stages of the hepatitis C viral (HCV) lifecycle including entry, replication, and assembly [1,2,3,4,5]
We sought to determine whether HCV entry inhibitor monotherapy could significantly reduce extracellular viral levels over time in HCV persistently-infected cultures
We showed that HCV entry inhibitor monotherapy only slowly reduced extracellular viral levels in persistently-infected cell cultures where most of the cells are infected

Summary

Introduction

Researchers are actively working to develop inhibitors of several stages of the hepatitis C viral (HCV) lifecycle including entry, replication, and assembly [1,2,3,4,5]. A curative antiviral therapy for HCV-infected patients will likely be comprised of a combination of two or more distinct viral inhibitors. Significant progress has been made toward understanding HCV entry [6,7] and developing inhibitors of this process [2,7,8,9,10,11]. HCV entry is initiated by the attachment of viral envelope proteins (E1 and E2) to glycosaminoglycans [12] followed by a post-attachment stage which includes specific binding to cellular receptors and subsequent uptake into the cell. The tyrosine kinases epidermal growth factor receptor and ephrin receptor A2 are thought to act as HCV entry co-factors by modulating the interaction between CD81 and claudin 1 [18]

Methods

Results

Conclusion