Novel Hepatitis C Virus Reporter Replicon Cell Lines Enable Efficient Antiviral Screening against Genotype 1a

Robinson, Margaret; Yang, Huiling; Sun, Siu-Chi Chang; Peng, Betty; Yang, Tian; Pagratis, Nikos; Greenstein, Andrew E.; Delaney, William E.

doi:10.1128/aac.00289-10

Cited by 51 publications

(59 citation statements)

References 41 publications

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“…2B). To compare the EC 50 values obtained by this method with those generated in replicon cell lines encoding a more traditional reporter, we performed parallel antiviral assays using the luciferase-encoding genotype 1a replicon cell line 51C-RFP-1a (15). We observed a significant correlation (R 2 = 0.88, P = 0.0001) in EC 50 values generated with luciferase and fluorescent protein reporter replicons (Fig.…”

Section: Construction and Validation Of Fluorescent Protein-labeled Rmentioning

confidence: 90%

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Preexisting drug-resistance mutations reveal unique barriers to resistance for distinct antivirals

Robinson

Yang

Delaney

et al. 2011

Proc. Natl. Acad. Sci. U.S.A.

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Clinical trials of direct-acting antiviral agents in patients chronically infected with hepatitis C virus (HCV) have demonstrated that viral resistance is detected rapidly during monotherapy. In patients, HCV does not exist as a single, genetically homogenous virus but rather as a population of variants termed "quasispecies." Preexisting variants resistant to specific antiviral drugs, overlooked in traditional hit-to-lead discovery efforts, may be responsible for these poor clinical outcomes. To enable real-time studies of resistance emergence in live cells, we established fluorescent protein-labeled HCV replicon cell lines. We validated these cell lines by demonstrating that antiviral susceptibility and the selection of signature resistance mutations for various drug classes are similar to traditional replicon cell lines. By quantifying the kinetics and uniformity of replication within colonies of drug-resistant fluorescent replicon cells, we showed that resistance emerged from a single cell and preexisted in a treatment-naive replicon population. Within this population, we determined the relative frequency of preexisting replicons capable of establishing foci during treatment with distinct antivirals. By measuring relative frequency as a function of dose, we quantitatively ranked distinct antiviral molecules on the basis of their distinct barriers to resistance. These insights into RNA virus quasispecies structure provide guidance for selecting clinical drug concentrations and selecting antiviral drug combinations most likely to suppress resistance.evolution | virology R esistance to antiviral drugs is a significant worldwide health problem. Although we typically discuss drug resistance in language suggesting that resistance "emerges" after administration of an antiviral drug, classical microbial genetics suggests that drug-resistant mutants preexist in viral populations. The Luria and Delbruck experiment (1) provided the first evidence that drug-resistance mutations could preexist. In infected patients, hepatitis C virus (HCV), a positive-strand RNA virus, is composed of a swarm of genetically heterogeneous variants termed "quasispecies." A central question is whether this diversity is so pronounced that drug-resistance mutations preexist in a drugnaïve HCV population. Despite the importance of drug resistance in driving therapeutic outcomes, identification of rare variants within polymorphic virus populations has been fundamentally difficult in vitro and in vivo.Although many new classes of direct-acting anti-HCV drugs are in clinical development, viral resistance to these agents generally is detected within a few days of monotherapy (2, 3). Further supporting the model of preexisting resistance, ultradeep sequencing experiments identified mutations, before drug exposure, that confer resistance to NS3 protease inhibitors (4, 5). Although drugdiscovery campaigns traditionally have prioritized leads and lead classes on the basis of potency and toxicity, quantitative comparisons of the frequency of drug-resista...

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Section: Construction and Validation Of Fluorescent Protein-labeled Rmentioning

confidence: 90%

“…1A). Similarly, stable cell lines with the genotype 1a RFP-encoding replicon were established by transfecting a genotype 1a-permissive cell line, 51C (15), with 1a-RFP replicon RNA (Fig. 1A).…”

Section: Construction and Validation Of Fluorescent Protein-labeled Rmentioning

confidence: 99%

Preexisting drug-resistance mutations reveal unique barriers to resistance for distinct antivirals

Robinson

Yang

Delaney

et al. 2011

Proc. Natl. Acad. Sci. U.S.A.

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“…Resistance mutations were introduced into the GT1a (15) or GT1b replicon (16) by site-directed mutagenesis and tested in transient-transfection assays, as previously described (14). Briefly, NS5B mutations were introduced into a plasmid carrying the gene encoding the PI-hRluc replicon using a QuikChange II XL mutagenesis kit, according to the manufacturer's instructions (Stratagene, La Jolla, CA).…”

Section: Compoundsmentioning

confidence: 99%

Clinical and In Vitro Resistance to GS-9669, a Thumb Site II Nonnucleoside Inhibitor of the Hepatitis C Virus NS5B Polymerase

Dvory‐Sobol

Voitenleitner

Mabery

et al. 2014

Antimicrob Agents Chemother

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bTreatment with GS-9669, a novel nonnucleoside inhibitor (site II) of hepatitis C virus (HCV) nonstructural 5B (NS5B) polymerase, resulted in significant antiviral activity in HCV genotype (GT) 1 patients dosed at 50 and 500 mg once daily (QD) and at 50, 100, and 500 mg twice daily (BID) for 3 days. This report characterizes the virologic resistance to GS-9669 in vitro and in GT1 HCV-infected patients from a phase I clinical study. An in vitro resistance selection study with GS-9669 revealed substitutions at several NS5B residues that conferred resistance. The M423 variants were selected at low drug concentrations (5؋ the 50% effective concentration [EC 50 ]), and the L419, R422, and I482 variants were selected at higher drug concentrations (20؋ the EC 50 ). H epatitis C virus (HCV) infects an estimated 170 million people worldwide (1). HCV infection can lead to cirrhosis, hepatocellular carcinoma, or other complications. Until recently, the standard of care for the treatment of chronic HCV infection consisted of 24 to 48 weeks of pegylated interferon (PEG-IFN) and ribavirin (RBV) (2), which are associated with significant side effects, including fever, fatigue, anemia, leukopenia, thrombocytopenia, and depression (3, 4). A sustained virologic response (SVR) occurs in only 42% to 53% of patients with genotype (GT) 1 or GT4 HCV and up to 78% to 82% of patients infected with GT2 or GT3 HCV (5, 6). Novel direct-acting antiviral agents (DAAs) are being developed in combination with PEG-IFN-RBV and are also being pursued as components of IFN-free and IFN-and RBV-free regimens to improve efficacy and shorten treatment duration. Two protease inhibitors (PIs) approved for the treatment of HCV, telaprevir and boceprevir, have demonstrated significantly improved SVR rates when given in combination with PEG-IFN-RBV in GT1 patients (60 to 75% for combination compared with 38 to 46% for PEG-IFN-RBV only) (7,8). However, these new agents require thrice-daily dosing and are associated with more frequent occurrences of and severe anemia and rash (9, 10). Two HCV drugs received FDA approval at the end of 2013, simeprevir (Olysio), a nonstructural 3/4A (NS3/4A) protease inhibitor in combination with PEG-IFN-RBV, and sofosbuvir (Sovaldi), a nucleotide inhibitor, which is the first drug that has demonstrated safety and efficacy for treating non-genotype-1 HCV infection without the need to coadminister PEG-IFN.GS-9669 (Fig. 1) is a novel thumb site II nonnucleoside inhibitor (NNI) of the HCV NS5B RNA polymerase, with a binding affinity of 1.4 nM for the GT1b NS5B protein. It is a selective inhibitor of HCV RNA replication, with a mean 50% effective concentration (EC 50 ) of Յ11 nM in GT1 and GT5 replicon assays (11). Other NNIs currently in phase II clinical studies include BI-207127 and BMS-791325 (binding to thumb site I), filibuvir and lomibuvir (binding to thumb site II), setrobuvir, ABT-072, and ABT-333 (binding to palm site I), and tegobuvir (also binding in the palm) (12). In a phase Ib study of filibuvir, resistance-associat...

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“…Subgenomic replicons GT3a-PI-hRluc, GT4aED43SG, and GT6aRlucNeoSG were established recently (18,23,24). Replicons H77, HSG-51, HSG-57, 1a-HGluc-1, 1a-HGluc-2, 1a-Sf9Rluc-1, 1a-Sf9Rluc-2, Con-1, Huh-luc, and SL-3 have previously been described (25,26).…”

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confidence: 99%

In Vitro Antiviral Activity and Resistance Profile Characterization of the Hepatitis C Virus NS5A Inhibitor Ledipasvir

Cheng

Yang

Doehle

et al. 2016

Antimicrob Agents Chemother

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Ledipasvir (LDV; GS-5885), a component of Harvoni (a fixed-dose combination of LDV with sofosbuvir [SOF]), is approved to treat chronic hepatitis C virus (HCV) infection. Here, we report key preclinical antiviral properties of LDV, including in vitro potency, in vitro resistance profile, and activity in combination with other anti-HCV agents. LDV has picomolar antiviral activity against genotype 1a and genotype 1b replicons with 50% effective concentration (EC 50 ) values of 0.031 nM and 0.004 nM, respectively. LDV is also active against HCV genotypes 4a, 4d, 5a, and 6a with EC 50 values of 0.11 to 1.1 nM. LDV has relatively less in vitro antiviral activity against genotypes 2a, 2b, 3a, and 6e, with EC 50 values of 16 to 530 nM. In vitro resistance selection with LDV identified the single Y93H and Q30E resistance-associated variants (RAVs) in the NS5A gene; these RAVs were also observed in patients after a 3-day monotherapy treatment. In vitro antiviral combination studies indicate that LDV has additive to moderately synergistic antiviral activity when combined with other classes of HCV direct-acting antiviral (DAA) agents, including NS3/4A protease inhibitors and the nucleotide NS5B polymerase inhibitor SOF. Furthermore, LDV is active against known NS3 protease and NS5B polymerase inhibitor RAVs with EC 50 values equivalent to those for the wild type.

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Novel Hepatitis C Virus Reporter Replicon Cell Lines Enable Efficient Antiviral Screening against Genotype 1a

Cited by 51 publications

References 41 publications

Preexisting drug-resistance mutations reveal unique barriers to resistance for distinct antivirals

Preexisting drug-resistance mutations reveal unique barriers to resistance for distinct antivirals

Clinical and In Vitro Resistance to GS-9669, a Thumb Site II Nonnucleoside Inhibitor of the Hepatitis C Virus NS5B Polymerase

In Vitro Antiviral Activity and Resistance Profile Characterization of the Hepatitis C Virus NS5A Inhibitor Ledipasvir

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