Quantifying antiviral activity optimizes drug combinations against hepatitis C virus infection

Abstract

With the introduction of direct-acting antivirals (DAAs), treatment against hepatitis C virus (HCV) has significantly improved. To manage and control this worldwide infectious disease better, the "best" multidrug treatment is demanded based on scientific evidence. However, there is no method available that systematically quantifies and compares the antiviral efficacy and drug-resistance profiles of drug combinations. Based on experimental anti-HCV profiles in a cell culture system, we quantified the instantaneous inhibitory potential (IIP), which is the logarithm of the reduction in viral replication events, for both single drugs and multiple-drug combinations. From the calculated IIP of 15 anti-HCV drugs from different classes [telaprevir, danoprevir, asunaprevir, simeprevir, sofosbuvir (SOF), VX-222, dasabuvir, nesbuvir, tegobuvir, daclatasvir, ledipasvir, IFN-α, IFN-λ1, cyclosporin A, and SCY-635], we found that the nucleoside polymerase inhibitor SOF had one of the largest potentials to inhibit viral replication events. We also compared intrinsic antiviral activities of a panel of drug combinations. Our quantification analysis clearly indicated an advantage of triple-DAA treatments over double-DAA treatments, with triple-DAA treatments showing enhanced antiviral activity and a significantly lower probability for drug resistance to emerge at clinically relevant drug concentrations. Our framework provides quantitative information to consider in designing multidrug strategies before costly clinical trials.

Keywords: HCV; antiviral; instantaneous inhibitory potential; mathematical model; replicon.

Fig. 1.

Schematics of the anti-HCV drug targets and the experimental system. (A) HCV life cycle and drug targets. After entry into the host cell, HCV genomic RNA is translated into viral precursor polyprotein and processed into functional proteins (C, E1, E2, p7, NS2, NS3, NS4A, NS4B, NS5A, and NS5B). HCV RNA replicates inside the isolated membrane compartments derived from the endoplasmic reticulum (ER), and assembles into viral particles on lipid droplets, which traffic through the Golgi and are released outside of the cell. PIs (TPV, DPV, ASV, and SMV) inhibit the processing step, and drugs such as NI (SOF), NNIs (VX, DAS, NSV, and TGV), NS5AIs (DCV and LDV), and CIs (CsA and SCY) target HCV RNA replication. IFNs (IFN-α and IFN-λ1) supposedly inhibit at least the step(s) of translation and replication. (B) HCV replication activity was evaluated using an HCV subgenomic replicon (genotype 1b, strain NN) carrying a fusion of the firefly luciferase gene (Luc) with the neomycin phosphotransferase (Neo^r). The replicon autonomously and persistently replicates in Huh-7 cells. Cells treated with drugs were incubated for 72 h and then harvested for luciferase assay. Inhibition of HCV replication was measured by the luciferase activity in drug-treated cells, relative to activity in DMSO-treated cells.

Fig. 2.

Quantification of the IIP of single-HCV drugs. (A) Log–log plots of dose–response curves normalized by $I{C}_{50}$, determined from the replicon assay, of PIs (TPV, DPV, SMV, and ASV; red), the NI (SOF; blue), NNIs (VX, DAS, NSV, and TGV; orange), NS5AIs (DCV and LDV; green), IFNs (IFN-α and IFN-λ1; cyan), and CIs (CsA and CSY; purple). Each point represents the mean of three experiments. (B) IIP of classes or subclasses of antiviral drugs, normalized by $I{C}_{50}$, calculated from the experimentally measured $f_u$ by Eq. 1. (C) IIP values at drug concentration $D=100\times I{C}_{50}$ (IIP₁₀₀) determined by extrapolation.

Fig. 3.

Quantification of inhibitory potential of anti-HCV drug multicombinations. (A) IIP^com of antiviral drug double combinations was calculated from the measured $f_u^{\text{com}}$ by Eq. 1. Fifty-two double combinations of interclass (or subclass) antiviral drugs were analyzed using the HCV replicon assay. Each point represents the mean of three experiments. Drugs were concentrated at a constant ratio from their initial concentrations $D_{\text{initial}}=0.25\times I{C}_{50}$, where the $I{C}_{50}$ values were determined in separate single-drug experiments. (B) IIP^com of antiviral drug triple combinations was calculated from the measured $f_u^{\text{com}}$ by Eq. 1. Eight triple combinations of antiviral drugs were analyzed using the HCV replicon assay. Each point represents the mean of three experiments. Drugs were concentrated at a constant ratio from their initial concentrations $D_{\text{initial}}=0.25\times I{C}_{50}$, where the $I{C}_{50}$ values were determined in separate single-drug experiments.

Fig. 4.

Quantification of the risk of HCV drug resistance. The fraction of unaffected HCV replication events $f_u^{Bcom}$ of each double-drug (A) and triple-drug (B) combination at clinical concentrations is shown. The expected number of newly produced mutants with one-nucleotide (blue) and two-nucleotide (red) substitutions after the first day of double-drug (C) and triple-drug (D) combination treatment is shown. Each number is calculated by multiplying the number of newly produced mutants per day and the fraction of production events unaffected by a drug combination as follows: ${10}^{12}\times P_1\times f_u^{com}$ and ${10}^{12}\times P_2\times f_u^{com}$, where $P_1$ and $P_2$ are the probability of one and two mutations occurring in the HCV genome after one replication event. The y axis shows the number of all possible one-nucleotide and two-nucleotide mutants ($2.9\times {10}^4$ and $4.1\times {10}^8$, respectively). Thus, if the bar faces to the left for a drug combination, it means that the expected number of newly produced mutants is below the number of all possible mutants under the corresponding treatment, suggesting drug-resistant mutants are unlikely to occur.