Modeling population heterogeneity in viral dynamics for chronic hepatitis C infection: Insights from Phase 3 telaprevir clinical studies.

Viral dynamic modelling has proven useful for designing clinical studies and predicting treatment outcomes for patients infected with the hepatitis C virus. Generally these models aim to capture and predict the on-treatment viral load dynamics from a small study of individual patients. Here, we explored extending these models (1) to clinical studies with numerous patients and (2) by incorporating additional data types, including sequence data and prior response to interferon. Data from Phase 3 clinical studies of the direct-acting antiviral telaprevir (T; total daily dose of 2250 mg) combined with pegylated-interferon alfa and ribavirin (PR) were used for the analysis. The following data in the treatment-naïve population were reserved to verify the model: (1) a T/PR regimen where T was dosed every 8 h for 8 weeks (T8(q8h)/PR) and (2) a T/PR regimen where T was dosed twice daily for 12 weeks (T12(b.i.d.)/PR). The resulting model accurately predicted (1) sustained virologic response rates for both of these dosing regimens and (2) viral breakthrough characteristics of the T8(q8h)/PR regimen. Since the observed viral variants depend on the T exposure, the second verification suggested that the model was correctly sensitive to the different T regimen even though the model was developed using data from another T regimen. Furthermore, the model predicted that b.i.d. T dosing was comparable to q8h T dosing in the PR-experienced population, a comparison that has not been made in a controlled clinical study. The methods developed in this work to estimate the variability occurring below the limit of detection for the viral load were critical for making accurate predictions.

Modeling viral evolutionary dynamics after telaprevir-based treatment.

For patients infected with hepatitis C virus (HCV), the combination of the direct-acting antiviral agent telaprevir, pegylated-interferon alfa (Peg-IFN), and ribavirin (RBV) significantly increases the chances of sustained virologic response (SVR) over treatment with Peg-IFN and RBV alone. If patients do not achieve SVR with telaprevir-based treatment, their viral population is often significantly enriched with telaprevir-resistant variants at the end of treatment. We sought to quantify the evolutionary dynamics of these post-treatment resistant variant populations. Previous estimates of these dynamics were limited by analyzing only population sequence data (20% sensitivity, qualitative resistance information) from 388 patients enrolled in Phase 3 clinical studies. Here we add clonal sequence analysis (5% sensitivity, quantitative) for a subset of these patients. We developed a computational model which integrates both the qualitative and quantitative sequence data, and which forms a framework for future analyses of drug resistance. The model was qualified by showing that deep-sequence data (1% sensitivity) from a subset of these patients are consistent with model predictions. When determining the median time for viral populations to revert to 20% resistance in these patients, the model predicts 8.3 (95% CI: 7.6, 8.4) months versus 10.7 (9.9, 12.8) months estimated using solely population sequence data for genotype 1a, and 1.0 (0.0, 1.4) months versus 0.9 (0.0, 2.7) months for genotype 1b. For each individual patient, the time to revert to 20% resistance predicted by the model was typically comparable to or faster than that estimated using solely population sequence data. Furthermore, the model predicts a median of 11.0 and 2.1 months after treatment failure for viral populations to revert to 99% wild-type in patients with HCV genotypes 1a or 1b, respectively. Our modeling approach provides a framework for projecting accurate, quantitative assessment of HCV resistance dynamics from a data set consisting of largely qualitative information.

Genotypic and phenotypic analyses of hepatitis C virus variants observed in clinical studies of VX-222, a nonnucleoside NS5B polymerase inhibitor.

VX-222, a thiophene-2-carboxylic acid derivative, is a selective nonnucleoside inhibitor of the hepatitis C virus (HCV) NS5B RNA-dependent RNA polymerase. In phase 1 and 2 clinical studies, VX-222 demonstrated effective antiviral efficacy, with substantial reductions in plasma HCV RNA in patients chronically infected with genotype 1 HCV. To characterize the potential for selection of VX-222-resistant variants in HCV-infected patients, the HCV NS5B gene was sequenced at baseline and during and after 3 days of VX-222 dosing (monotherapy) in a phase 1 study. Variants with the substitutions L419C/I/M/P/S/V, R422K, M423I/T/V, I482L/N/T, A486S/T/V, and V494A were selected during VX-222 dosing, and their levels declined over time after the end of dosing. Phenotypic analysis of these variants was conducted using HCV replicons carrying site-directed mutations. Of the 17 variants, 14 showed reduced susceptibility to VX-222 compared with the wild type, with the L419C/S and R422K variants having higher levels of resistance (>200-fold) than the rest of the variants (6.8- to 76-fold). The M423I and A486S variants remained susceptible to VX-222. The 50% effective concentration (EC50) for the L419P variant could not be obtained due to the poor replication of this replicon. The majority of the variants (15/17) were less fit than the wild type. A subset of the variants, predominately the L419S and R422K variants, were observed when the efficacy and safety of VX-222- and telaprevir-based regimens given for 12 weeks were investigated in genotype 1 HCV-infected patients in a phase 2 study. The NS3 and NS5B variants selected during the dual combination therapy showed reduced susceptibility to both telaprevir and VX-222 and had a lower replication capacity than the wild type. The phase 1b study has the ClinicalTrials.gov identifier NCT00911963, and the phase 2a study has ClinicalTrials.gov identifier NCT01080222.

Evolution of treatment-emergent resistant variants in telaprevir phase 3 clinical trials.

BACKGROUND: Telaprevir (TVR), a hepatitis C virus (HCV) NS3/4A protease inhibitor, has been approved to treat genotype 1 HCV. To understand the clinical impact of TVR-resistant variants, we analyzed samples from patients in phase 3 clinical trials to determine the frequency and retention of TVR-resistant variants in patients who did not achieve sustained virologic response (SVR). METHODS: A total of 1797 patients were treated with TVR. Resistant variants (V36A/G/I/L/M, T54A/S, I132V [subtype 1a only], R155G/K/T/M, A156F/N/S/T/V, and D168N) were identified after treatment failure and at visits thereafter, by direct (population) sequencing of the NS3/4A region. Kaplan-Meier analysis was used to determine median time to loss of these variants. RESULTS: Resistant variants were observed in 77% (299/388) of patients who did not achieve SVR. Resistance occurred more commonly in subtype 1a (86%; 232/269) than subtype 1b infections (56%; 67/119). After treatment failure, 355 patients had at least 1 follow-up visit (median follow-up period: 9.6 months). Of patients with resistance at time of failure and at least 1 follow-up visit, 60% (153/254) lost resistance. Kaplan-Meier analysis, including all patients with any sequence data after treatment failure, indicated that median time to wild type was 10.6 months (95% confidence interval [CI], 9.47-12.20) in subtype 1a and 0.9 months (95% CI, 0.00-2.07) in subtype 1b infections. CONCLUSIONS: After failure to achieve SVR with TVR-based treatment, resistant variants are observed in most patients. However, presumably due to the lower fitness of those variants, they tend to be replaced with wild-type virus over time.

Development of a sensitive RT-PCR method for amplifying and sequencing near full-length HCV genotype 1 RNA from patient samples.

BACKGROUND: Direct-acting antiviral (DAAs) agents for hepatitis C virus (HCV) span a variety of targets, including proteins encoded by the NS3/4A, NS4B, NS5A, and NS5B genes. Treatment with DAAs has been shown to select variants with sequence changes in the HCV genome encoding amino acids that may confer resistance to the treatment. In order to assess these effects in patients, a Reverse Transcription Polymerase Chain Reaction (RT-PCR) method was developed to sequence these regions of HCV from patient plasma. METHODS: A method was developed to amplify and sequence genotype 1 HCV RNA from patient plasma. Optimization of HCV RNA isolation, cDNA synthesis, and nested PCR steps were performed. The optimization of HCV RNA isolation, design of RT-PCR primers, optimization of RT-PCR amplification conditions and reagents, and the evaluation of the RT-PCR method performance is described. RESULTS: The optimized method is able to successfully, accurately, and reproducibly amplify near full-length genotype 1 HCV RNA containing a wide range of concentrations (103 to 108 IU/mL) with a success rate of 97%. The lower limit of detection was determined to be 1000 IU/mL HCV RNA. CONCLUSIONS: This assay allows viral sequencing of all regions targeted by the most common DAAs currently in development, as well as the possibility to determine linkage between variants conferring resistance to multiple DAAs used in combination therapy.

Futility rules for telaprevir combination treatment for patients with hepatitis C virus infection.

For patients treated with telaprevir, peginterferon, and ribavirin, futility rules have been developed to prevent needless drug exposure and minimize development of drug-resistant variants for patients who have little or no chance of achieving a sustained virologic response. We performed retrospective analyses of data from phase 3 trials and validated the current futility rule. All therapy should be stopped for treatment-naive and treatment-experienced patients if hepatitis C virus RNA levels are greater than 1000 IU/mL at weeks 4 or 12, or if hepatitis C virus RNA is detectable at week 24.

Hepatitis C virus variants with decreased sensitivity to direct-acting antivirals (DAAs) were rarely observed in DAA-naive patients prior to treatment.

The prevalence of naturally occurring hepatitis C virus (HCV) variants that are less sensitive to direct-acting antiviral (DAA) inhibitors has not been fully characterized. We used population sequence analysis to assess the frequency of such variants in plasma samples from 3,447 DAA-naive patients with genotype 1 HCV. In general, HCV variants with lower-level resistance (3- to 25-fold increased 50% inhibitor concentration [IC(50)]) to telaprevir were observed as the dominant species in 0 to 3% of patients, depending on the specific variant, whereas higher-level resistant variants (>25-fold-increased IC(50)) were not observed. Specific variants resistant to NS5A inhibitors were predominant in up to 6% of patients. Most variants resistant to nucleo(s/t)ide active-site NS5B polymerase inhibitors were not observed, whereas variants resistant to non-nucleoside allosteric inhibitors were observed in up to 18% of patients. The presence of DAA-resistant variants in NS5A, NS5B, or NS3 (including telaprevir-resistant variants), in baseline samples of treatment-naive patients receiving a telaprevir-based regimen in phase 3 studies did not affect the sustained viral response (SVR). Treatment-naive patients with viral populations containing the telaprevir-resistant variants NS3 V36M, T54S, or R155K at baseline achieved a 74% SVR rate, whereas patients with no resistant variants detected prior to treatment achieved a 76% SVR rate. The effect of specific resistant variant frequency on response to various DAA treatments in different patient populations, including interferon nonresponders, should be further studied.

Compensatory substitutions in the HCV NS3/4A protease cleavage sites are not observed in patients treated unsuccessfully with telaprevir combination treatment.

BACKGROUND: Development of compensatory mutations within the HIV p7/p1 and p1/p6 protease cleavage site region has been observed in HIV-infected patients treated with protease inhibitors. Mechanisms of fitness compensation may occur in HCV populations upon treatment of HCV protease inhibitors as well. FINDINGS: In this study, we investigated whether substitutions in protease cleavage site regions of HCV occur in response to a treatment regimen containing the NS3/4A protease inhibitor telaprevir (TVR). Evaluation of viral populations from 569 patients prior to treatment showed that the four NS3/4A cleavage sites were well conserved. Few changes in the cleavage site regions were observed in the 159 patients who failed TVR combination treatment, and no residues displayed evidence of directional selection after the acquisition of TVR-resistance. CONCLUSIONS: Cleavage site mutations did not occur after treatment with the HCV protease inhibitor telaprevir.

Hepatitis C viral evolution in genotype 1 treatment-naïve and treatment-experienced patients receiving telaprevir-based therapy in clinical trials.

BACKGROUND: In patients with genotype 1 chronic hepatitis C infection, telaprevir (TVR) in combination with peginterferon and ribavirin (PR) significantly increased sustained virologic response (SVR) rates compared with PR alone. However, genotypic changes could be observed in TVR-treated patients who did not achieve an SVR. METHODS: Population sequence analysis of the NS3•4A region was performed in patients who did not achieve SVR with TVR-based treatment. RESULTS: Resistant variants were observed after treatment with a telaprevir-based regimen in 12% of treatment-naïve patients (ADVANCE; T12PR arm), 6% of prior relapsers, 24% of prior partial responders, and 51% of prior null responder patients (REALIZE, T12PR48 arms). NS3 protease variants V36M, R155K, and V36M+R155K emerged frequently in patients with genotype 1a and V36A, T54A, and A156S/T in patients with genotype 1b. Lower-level resistance to telaprevir was conferred by V36A/M, T54A/S, R155K/T, and A156S variants; and higher-level resistance to telaprevir was conferred by A156T and V36M+R155K variants. Virologic failure during telaprevir treatment was more common in patients with genotype 1a and in prior PR nonresponder patients and was associated with higher-level telaprevir-resistant variants. Relapse was usually associated with wild-type or lower-level resistant variants. After treatment, viral populations were wild-type with a median time of 10 months for genotype 1a and 3 weeks for genotype 1b patients. CONCLUSIONS: A consistent, subtype-dependent resistance profile was observed in patients who did not achieve an SVR with telaprevir-based treatment. The primary role of TVR is to inhibit wild-type virus and variants with lower-levels of resistance to telaprevir. The complementary role of PR is to clear any remaining telaprevir-resistant variants, especially higher-level telaprevir-resistant variants. Resistant variants are detectable in most patients who fail to achieve SVR, but their levels decline over time after treatment.

Characterization of V36C, a novel amino acid substitution conferring hepatitis C virus (HCV) resistance to telaprevir, a potent peptidomimetic inhibitor of HCV protease.

We characterized a novel substitution conferring moderate resistance to telaprevir, a peptidomimetic inhibitor of hepatitis C virus protease. V36C conferred a 4.0-fold increase in the telaprevir 50% inhibitory concentration in an enzyme assay and a 9.5-fold increase in the replicon model. The replication capacity of a replicon harboring V36C was close to that of the wild-type protease. This case emphasizes the complexity of hepatitis C virus resistance to protease inhibitors.

Natural prevalence of hepatitis C virus variants with decreased sensitivity to NS3.4A protease inhibitors in treatment-naive subjects.

BACKGROUND: The prevalence and clinical implications of naturally occurring variants that are resistant to hepatitis C virus (HCV) protease inhibitors in treatment-naive patients has not been reported. We report here the prevalence of such variants and their effect on clinical response. METHODS: Population sequence analysis of the NS3.4A protease was conducted in 570 treatment-naive subjects. RESULTS: Most subjects (98%) had wild-type virus. The remaining subjects had the following variants present in significant proportions (100%): V36M, 0.9%; R155K, 0.7%; V170A, 0.2%; and R109K, 0.2%. The V36M, R109K, and V170A substitutions confer low-level resistance (<7-fold) to protease inhibitors in replicon cells. The R155K substitution confers low-level resistance to telaprevir (TVR) and boceprevir and confers high-level resistance (>70-fold) to BILN 2061 and ITMN-191. Five subjects with the V36M or R109K variant were treated with 8-24 weeks of TVR and peginterferon-alpha2a (P) with or without ribavirin (R). Four achieved a sustained viral response, and 1 was lost to follow-up. In subjects with the R155K variant, TVR/PR provided greater antiviral activity than PR alone; however, the antiviral response was lower than that observed in subjects with wild-type virus. CONCLUSION: High levels of naturally occurring protease inhibitor-resistant variants were uncommon (<1% each) in HCV treatment-naive patients. TVR/PR efficiently inhibited V36M and R109K variants and contributed partial antiviral activity against the R155K variant. As new HCV agents are evaluated in clinical trials, it will be important to monitor the effect of baseline variants on sensitivity.

Phenotypic characterization of resistant Val36 variants of hepatitis C virus NS3-4A serine protease.

In patients chronically infected with hepatitis C virus (HCV) strains of genotype 1, rapid and dramatic antiviral activity has been observed with telaprevir (VX-950), a highly selective and potent inhibitor of the HCV NS3-4A serine protease. HCV variants with substitutions in the NS3 protease domain were observed in some patients during telaprevir dosing. In this study, purified protease domain proteins and reconstituted HCV subgenomic replicons were used for phenotypic characterization of many of these substitutions. V36A/M or T54A substitutions conferred less than eightfold resistance to telaprevir. Variants with double substitutions at Val36 plus Thr54 had approximately 20-fold resistance to telaprevir, and variants with double substitutions at Val36 plus Arg155 or Ala156 had >40-fold resistance to telaprevir. An X-ray structure of the HCV strain H protease domain containing the V36M substitution in a cocomplex with an NS4A cofactor peptide was solved at a 2.4-A resolution. Except for the side chain of Met36, the V36M variant structure is identical to that of the wild-type apoenzyme. The in vitro replication capacity of most variants was significantly lower than that of the wild-type replicon in cells, which is consistent with the impaired in vivo fitness estimated from telaprevir-dosed patients. Finally, the sensitivity of these replicon variants to alpha interferon or ribavirin remained unchanged compared to that of the wild-type.

Phenotypic and structural analyses of hepatitis C virus NS3 protease Arg155 variants: sensitivity to telaprevir (VX-950) and interferon alpha.

Telaprevir (VX-950) is a highly selective, potent inhibitor of the hepatitis C virus (HCV) NS3.4A serine protease. It has demonstrated strong antiviral activity in patients chronically infected with genotype 1 HCV when dosed alone or in combination with peginterferon alfa-2a. Substitutions of Arg(155) of the HCV NS3 protease domain have been previously detected in HCV isolates from some patients during telaprevir dosing. In this study, Arg(155) was replaced with various residues in genotype 1a protease domain proteins and in genotype 1b HCV subgenomic replicons. Characterization of both the purified enzymes and reconstituted replicon cells demonstrated that substitutions of Arg(155) with these residues conferred low level resistance to telaprevir (<25-fold). An x-ray structure of genotype 1a HCV protease domain with the R155K mutation, in a complex with an NS4A co-factor peptide, was determined at a resolution of 2.5A. The crystal structure of the R155K protease is essentially identical to that of the wild-type apoenzyme (Protein Data Bank code 1A1R) except for the side chain of mutated residue 155. Telaprevir was docked into the x-ray structure of the R155K protease, and modeling analysis suggests that the P2 group of telaprevir loses several hydrophobic contacts with the Lys(155) side chain. It was demonstrated that replicon cells containing substitutions at NS3 protease residue 155 remain fully sensitive to interferon alpha or ribavirin. Finally, these variant replicons were shown to have reduced replication capacity compared with the wild-type HCV replicon in cells.