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

Abstract

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...

A New Stochastic Model for Subgenomic Hepatitis C Virus Replication Considers Drug Resistant Mutants

“…It was demonstrated previously that the resistance observed during in vitro replicon studies results from the outgrowth of preexisting NS5B mutations rather than from the generation of mutations after the onset of antiviral suppression [8]. Our modeling results are consistent with this evidence: in the whole pool of mutant RNAs the preexisting NS3 protease mutants prevail over mutants generated during the wild-type viral RNA replication in the presence of drug.…”

Antiviral response and resistance analysis of treatment‐naïve HCV‐infected patients receiving single and multiple doses of GS‐9190

“…Similar observations have been made with the class of non‐nucleoside analogue (NNI) inhibitors of NS5B . In contrast, resistance to nucleoside analogue inhibitors (NI) of NS5B is less frequent .…”

Development and application of a fast, reproducible assay to measure HCV NS3 protease activity using Escherichia coli lysate

“…NS3 mutants conferring reduced susceptibility to PIs have been shown to exist at relatively high frequencies within the treatment-naïve HCV quasispecies population. The NS3 PIs historically as a class have a lower barrier to resistance than nucleoside inhibitors, and resistance-associated mutations can be rapidly selected with monotherapy treatment in vivo and in vitro (Robinson et al, 2011). For PIs in development, characterization of activity against known PI mutants, genotype-dependent susceptibility, and phenotyping and cross-resistance analysis of patient sequences are important components of the development process.…”