Journal of Viral Hepatitis
S. De Meyer, A. Ghys, G. R. Foster, M. Beumont, B. Van Baelen, T.-I. Lin, I. Dierynck, H. Ceulemans, G. Picchio
J Viral Hepat. 2013;20(6):395-403.
Abstract and Introduction
Abstract
Study C209 evaluated the activity of telaprevir in treatment-naïve patients with genotypes 2 or 3 (G2, G3) hepatitis C virus (HCV) infection. Telaprevir monotherapy showed potent activity against HCV G2, but limited activity against G3. This analysis was performed to characterize HCV viral variants emerging during telaprevir-based treatment of G2/G3 HCV-infected patients. Patients were randomized to receive 2 weeks of treatment with telaprevir (telaprevir monotherapy), telaprevir plus peginterferon alfa-2a and ribavirin (triple therapy), or placebo plus peginterferon alfa-2a and ribavirin (control), followed by 22–24 weeks of peginterferon/ribavirin alone. Viral breakthrough was defined as an increase >1 log10 in HCV RNA from nadir, or HCV RNA >100 IU/mL in patients previously reaching <25 IU/mL. Twenty-three patients (47%) had G2 and 26 (53%) had G3 HCV. Viral breakthrough occurred during the initial 2-week treatment phase in six G2 patients (66.7%; subtypes 2, 2a and 2b) and three G3 patients (37.5%; all subtype 3a), all in the telaprevir monotherapy arm. Four breakthrough patients (three G2, one G3) subsequently achieved sustained virologic response (SVR). In all patients with breakthrough and available sequence data, mutations associated with reduced susceptibility to telaprevir in genotype 1 (G1) HCV were observed. No novel G2/G3-specific mutations were associated with telaprevir resistance. The telaprevir resistance profile appeared consistent across HCV genotypes 1, 2 and 3. Although viral breakthrough with resistance occurred in patients receiving telaprevir monotherapy, half of these patients achieved an SVR upon addition of peginterferon/ribavirin highlighting the importance of combination therapy.
Introduction
Chronic hepatitis C virus (HCV) infection is a major global healthcare burden.[1] Six HCV genotypes and over 100 subtypes are documented,[2] each with a distinct geographical distribution. Genotype 1 (G1) HCV predominates in Europe, the United States and Japan, G2 predominates in Mediterranean countries and the Far East, and G3 is common in the Indian subcontinent.[3,4] An increase in the proportion of patients with G3 HCV infection was recently observed in Europe.[5]
Two direct-acting antiviral (DAA) agents that target the HCV NS3-4A serine protease, telaprevir[6] and boceprevir,[7] showed substantial efficacy against chronic HCV G1 infection[8–12] and were recently approved in the United States and Europe. Preliminary in vitro data suggested that telaprevir also has activity against G2/G3 HCV.[13] Therefore, the Phase IIa C209 study evaluated the activity of telaprevir, administered for 15 days with or without peginterferon/ribavirin, on G2/G3 HCV infection. When administered as monotherapy, telaprevir had significant and rapid antiviral activity against G2 HCV, but little or no activity against G3.[14]
G1 HCV variants associated with decreased susceptibility to telaprevir were previously identified in patients not achieving an SVR. The single amino acid changes V36A/M, T54A/S, R155K/T and A156S confer lower-level in vitro resistance to telaprevir (3- to 25-fold increase in replicon 50% inhibitory concentration [IC50]), whereas single change A156T/V and double change V36M+R155K confer higher-level resistance (>25-fold increase in replicon IC50).[15] Whether similar variants are associated with lack of response to telaprevir in G2/G3 HCV infection is unknown. Therefore, this subanalysis of the C209 study characterized HCV viral variants emerging with telaprevir-based therapy in G2/G3 HCV-infected patients.
Materials and Methods
The methodology for this Phase IIa, multicentre, partially blinded, randomized, multiple-dose trial in treatment-naïve patients with chronic G2 or G3 HCV infection, was described elsewhere (ClinicalTrials.gov NCT00561015).[14]
Study Design
After a 6-week screening period, patients were randomized 1:1:1 to receive 2 weeks of telaprevir 750 mg every 8 h (q8 h; telaprevir monotherapy), telaprevir 750 mg q8 h plus peginterferon alfa-2a 180 μg once-weekly and ribavirin 400 mg twice daily (bid; triple therapy), or placebo q8 h plus peginterferon alfa-2a 180 μg once-weekly and ribavirin 400 mg bid (control). This 2-week investigational treatment period was followed by a standard-treatment phase where patients received peginterferon alfa-2a plus ribavirin for an additional 24 weeks (telaprevir monotherapy arm) or 22 weeks (other arms). After treatment completion, patients were followed up for 24–48 weeks.
Primary and secondary endpoints are reported elsewhere.[14] Viral breakthrough (vBT) was defined as an increase >1 log10 in HCV RNA from nadir or HCV RNA >100 IU/mL in patients who previously reached HCV RNA <25 IU/mL, confirmed by two consecutive samples. Patients who had an unconfirmed vBT during telaprevir monotherapy were still considered to have vBT. SVR was defined as undetectable HCV RNA at end of treatment (EOT) and 24 weeks afterwards.
The study was approved by each centre's institutional review board and conducted in accordance with the Declaration of Helsinki and Good Clinical Practice guidelines. Written informed consent was obtained from all patients.
Virologic Assessments
Hepatitis C virus RNA levels were evaluated using the Roche COBAS TaqMan HCV test version 2.0 (Roche Molecular Systems Inc., Branchburg, NJ, USA), with a limit of quantification (LOQ) of 25 IU/mL. Samples containing HCV RNA below the LOQ were reported as '<25 IU/mL detected'; those with no HCV RNA as '<25 IU/mL, target not detected' (also described as undetectable HCV RNA).
Samples for viral sequencing were obtained at baseline; days 2, 3, 4, 8, 12 and 15; weeks 4, 6 and 14; at EOT; 4, 8, 12 and 24 weeks after EOT; and 24 weeks after relapse (if any). Population-based NS3 sequence analysis was conducted in all baseline samples and in postbaseline samples from telaprevir-treated patients with vBT and/or not achieving an SVR. Population-based sequencing of the HCV NS3 region used subtype-specific amplification and sequencing protocols (Virco BVBA, Beerse, Belgium). Clonal sequencing of the NS3 region was conducted in a subset of samples. For both population-based and clonal sequencing, total RNA was extracted from plasma. The RNA was reverse transcribed, and cDNA was amplified in a one-step polymerase chain reaction (PCR) using the SuperScriptTM III One-Step Reverse Transcriptase (RT)-PCR System (Life Technologies, Carlsbad, CA, USA). An amplicon encompassing the NS3 region was further amplified during a nested PCR (Expand high Fidelity PCR system, Roche Applied Science, Penzberg, Germany). For population-based sequencing, PCR products were purified and directly sequenced in both directions using Big Dye terminator sequencing chemistry (Life Technologies). In case of clonal sequencing, the PCR product was first cloned using a TOPO TA cloning kit (Life Technologies). DNA from 20 to 30 clones per sample was purified and sanger sequenced as described above. The genotype and subtype of each sample were determined based on the NS5B region. The lower limit of detection for the sequencing assay was ~1000 IU/mL HCV RNA.
For phenotypic analyses, cDNA encoding NS3 amino acids 1–181 was cloned and expressed using Escherichia coli Rosetta2 (DE3) (Novagen, Madison, WI, USA). Telaprevir susceptibility was tested using a fluorescence resonance energy transfer cleavage assay with the retS1 peptide substrate (Anaspec, San Jose, CA, USA). Briefly, NS3-4A was pre-incubated with telaprevir for 10 min. The retS1 substrate was added, and fluorescence continuously measured for 20 min (excitation 355 nm, emission 510 nm). The IC50 value was calculated from the inhibition values of a series of telaprevir concentrations. Fold change in IC50 values (FC), compared with wild-type (Con1b) HCV, was calculated.
Statistical Analyses
To identify mutations potentially associated with reduced susceptibility to telaprevir, the frequency of every amino acid at every position was compared between telaprevir-exposed and -unexposed samples. Statistical significance was defined as P < 0.05 in Fisher's exact test; Bonferroni correction was used when multiple amino acid values or positions were explored simultaneously. For G3 analyses, unexposed samples comprised 102 G3a HCV sequences from combined public Los Alamos and internal HCV sequence databases. No formal statistical analyses were performed for G2, due to the low number of available sequences from unexposed samples.
Results
Patient disposition and baseline characteristics were reported in detail elsewhere[14] and were generally well-balanced across treatment groups and genotypes. Twenty-three patients (47%) had G2 HCV and 26 (53%) had G3 HCV. The HCV G2 subtypes were as follows: 2 (n = 7), 2c (n = 6), 2b (n = 4), 2a (n = 3), 2i (n = 1) and 2k (n = 1); G3 subtypes were 3a (n = 22) and 3b (n = 3). No sequence information was available for one G2 and one G3 patient. Forty patients (82%) completed the study, including follow-up.
G2 HCV-infected Patients
Antiviral Activity. During the investigational phase, vBT was observed in 6/9 G2 patients (66.7%) in the telaprevir monotherapy arm, compared with no vBT in the triple therapy and control arms (). One patient experienced vBT during the standard-treatment phase (control arm). One patient in the telaprevir monotherapy arm relapsed. HCV RNA profiles during telaprevir monotherapy are shown in Fig. 1a.
Table 1. Telaprevir antiviral activity in patients infected with either G2 or G3 HCV. Data are shown as n (%)
| | Patients infected with G2 HCV | Patients infected with G3 HCV |
| T (n = 9) | T/PR (n = 5) | PR (n = 9) | T (n = 8) | T/PR (n = 9) | PR (n = 9) |
| Virologic response* |
| By end of telaprevir/placebo treatment | 0 | 2 (40.0) | 2 (22.2) | 0 | 2 (22.2) | 1 (11.1) |
| By end of treatment | 8 (88.9) | 5 (100) | 8 (88.9) | 6 (75.0) | 9 (100) | 9 (100) |
| Cumulative vBT |
| By end of telaprevir/placebo treatment | 6 (66.7) | 0 | 0 | 3 (37.5) | 0 | 0 |
| By end of treatment | 6 (66.7) | 0 | 1 (11.1) | 3 (37.5) | 0 | 0 |
| Relapse | 1 (12.5)† | 0 | 0 | 2 (33.3)§ | 3 (33.3)¶ | 2 (22.2)¶ |
| Missing follow-up data | 2 (22.2)‡ | 0 | 0 | 0 | 0 | 3 (33.3)** |
| SVR | 5 (55.6) | 5 (100) | 8 (88.9) | 4 (50.0) | 6 (66.7) | 4 (44.4) |
G, genotype; HCV, hepatitis C virus; T, telaprevir monotherapy, telaprevir 750 mg every 8 h (q8 h); T/PR, triple combination therapy, telaprevir 750 mg q8 h plus peginterferon alfa-2a 180 μg once-weekly and ribavirin 400 mg bid; PR, peginterferon/ribavirin, placebo q8 h plus peginterferon alfa-2a 180 μg once-weekly and ribavirin 400 mg bid; SVR, sustained virologic response; vBT, viral breakthrough. *Undetectable HCV RNA; †Eight patients had undetectable HCV RNA at end of treatment; ‡One patient discontinued due to noncompliance and one patient lost to follow-up; §Six patients with undetectable HCV RNA at end of treatment; ¶Nine patients with undetectable HCV RNA at end of treatment; **Three patients lost to follow-up.
Figure 1.
HCV RNA levels in individual patients over time. HCV RNA profiles during telaprevir monotherapy in patients infected with (a) G2 HCV and (b) G3 HCV are shown. LOQ was 25 IU/mL; HCV RNA values below LOQ were imputed with an arbitrary value of 17.5 for <25 IU/mL detectable and five for undetectable HCV RNA. LOQ, limit of quantification; HCV, hepatitis C virus Baseline Virologic Data. Baseline population-based sequences were available for 22/23 G2 HCV-infected patients; no patient had mutations associated with decreased telaprevir susceptibility in G1 HCV. Clonal sequence analysis was conducted in the six patients with vBT: 1/27 clones in one patient had the T54A or R155K mutation at baseline; 1/22 clones in another patient had the A156V mutation at baseline (Fig. 2a).
Figure 2.
Variants detected by clonal sequencing in patients with vBT. Clonal sequence data are shown for (a) G2 and (b) G3 HCV-infected patients with vBT in the telaprevir monotherapy arm (20–40 clones/timepoint). Mutations at positions 36, 54, 155 and 156 were considered for this analysis. G, genotype The mean G2 HCV telaprevir FC at baseline (vs wild type) was 2.4 (range, 0.2–6.6). For the seven telaprevir monotherapy patients with available phenotypic data, there was no clear relationship between baseline telaprevir FC and the change in HCV RNA from baseline to day 3 (Fig. 3a). However, the two patients with the lowest telaprevir FC at baseline had the greatest change in HCV RNA at day 3.
Figure 3.
HCV RNA reduction vs in vitro telaprevir susceptibility. Change in HCV RNA from baseline to day 3 is shown against baseline telaprevir FC in telaprevir monotherapy patients infected with (a) G2 HCV and (b) G3 HCV. G, genotype; HCV, hepatitis C virus; FC, fold change in IC50 Genotypic Characterization of Viral Variants. Figure 4 shows HCV RNA, genotypic and phenotypic profiles over time for the six G2 HCV-infected patients (two G2b, one G2a and three G2 unknown subtype) in the telaprevir monotherapy arm with vBT.
Figure 4.
HCV RNA, genotypic and phenotypic profiles for individual G2 HCV-infected patients. Profiles are shown for (a) G2a and G2b and (b) G2 unknown subtype HCV-infected patients in the telaprevir monotherapy arm with vBT, during both the investigational (panel i) and standard (panel ii) treatment phases. HCV; hepatitis C virus; TVR disc, discontinuation of telaprevir; PR disc, discontinuation of peginterferon/ribavirin; LOQ, limit of quantification; FC, fold change in IC50 In all five patients with vBT for whom sequences were available, emerging mutations associated with reduced telaprevir susceptibility in G1 HCV were observed using population sequencing. No sequence could be obtained for the day 15 sample of patient 5 due to low HCV RNA levels (101 IU/mL). At the time of vBT, T54A alone (n = 2), R155K alone (n = 1), A156S alone (n = 1) and the T54A+R155K combination (n = 1) were found. T54A was detected in one patient (patient 3) before vBT and A156S was detected in another patient (patient 1) after vBT. Clonal sequence analysis confirmed that in patients with vBT, mutations associated with reduced telaprevir susceptibility in G1 HCV emerged and became the dominant quasispecies (Fig. 2a).
In addition to the mutations associated with reduced susceptibility to telaprevir in G1 HCV, a number of other amino acid changes from baseline were identified in telaprevir monotherapy patients with vBT: Q32H, S146P, V163I, S166A and T179A (Fig. 4). However, no mutation appeared consistently across patients.
Of the six patients with vBT in the telaprevir monotherapy arm, five had undetectable HCV RNA at the end of peginterferon/ribavirin treatment, showing that the emerging variants remained sensitive to peginterferon/ribavirin. Three of these patients, including two with emerging mutations, achieved an SVR. For the patient who relapsed, no mutations associated with reduced susceptibility to telaprevir or other HCV protease inhibitors were detected at follow-up (week 24).[15]
Phenotypic Characterization of Viral Variants. Paired genotypic and phenotypic data were available for 3/6 G2 patients with vBT. At the time of vBT, two patients (patients 3 and 4, both G2b) had an emerging R155K mutation associated with a >9-fold increase from baseline in telaprevir FC. The same two patients had an emerging T54A mutation. In patient 3, T54A was not associated with an increase in telaprevir FC; while in patient 4, it was not present in the expression construct used for phenotyping. At the time of vBT, patient 6 (G2) had an emerging A156S mutation associated with a >48-fold increase from baseline in FC.
Introducing the R155K mutation into the baseline NS3 sequence of patients 3 and 4 by site-directed mutagenesis increased the telaprevir FC from 6.4 and 5.4 (without R155K) to >76.9 and >100, respectively. Introducing T54A into the baseline NS3 sequence of patient 4 increased the FC from 5.4 to 39.5. Introducing the A156S mutation into the baseline NS3 sequence of patients 1 (G2a) and 6 increased the FC from 1.2–1.6 to >58.8–>71.4.
G3 HCV-infected Patients
Antiviral Activity. During the investigational phase, vBT was observed in 3/8 G3 patients (37.5%) in the telaprevir monotherapy arm, compared with no vBT in the triple therapy and control arms (). As telaprevir monotherapy only produced slight decreases in HCV RNA levels (Fig. 1b), most patients did not meet the definition for vBT. Relapse was observed in two patients in the telaprevir monotherapy arm, three patients in the triple therapy arm and two control patients.
Table 1. Telaprevir antiviral activity in patients infected with either G2 or G3 HCV. Data are shown as n (%)
| | Patients infected with G2 HCV | Patients infected with G3 HCV |
| T (n = 9) | T/PR (n = 5) | PR (n = 9) | T (n = 8) | T/PR (n = 9) | PR (n = 9) |
| Virologic response* |
| By end of telaprevir/placebo treatment | 0 | 2 (40.0) | 2 (22.2) | 0 | 2 (22.2) | 1 (11.1) |
| By end of treatment | 8 (88.9) | 5 (100) | 8 (88.9) | 6 (75.0) | 9 (100) | 9 (100) |
| Cumulative vBT |
| By end of telaprevir/placebo treatment | 6 (66.7) | 0 | 0 | 3 (37.5) | 0 | 0 |
| By end of treatment | 6 (66.7) | 0 | 1 (11.1) | 3 (37.5) | 0 | 0 |
| Relapse | 1 (12.5)† | 0 | 0 | 2 (33.3)§ | 3 (33.3)¶ | 2 (22.2)¶ |
| Missing follow-up data | 2 (22.2)‡ | 0 | 0 | 0 | 0 | 3 (33.3)** |
| SVR | 5 (55.6) | 5 (100) | 8 (88.9) | 4 (50.0) | 6 (66.7) | 4 (44.4) |
G, genotype; HCV, hepatitis C virus; T, telaprevir monotherapy, telaprevir 750 mg every 8 h (q8 h); T/PR, triple combination therapy, telaprevir 750 mg q8 h plus peginterferon alfa-2a 180 μg once-weekly and ribavirin 400 mg bid; PR, peginterferon/ribavirin, placebo q8 h plus peginterferon alfa-2a 180 μg once-weekly and ribavirin 400 mg bid; SVR, sustained virologic response; vBT, viral breakthrough. *Undetectable HCV RNA; †Eight patients had undetectable HCV RNA at end of treatment; ‡One patient discontinued due to noncompliance and one patient lost to follow-up; §Six patients with undetectable HCV RNA at end of treatment; ¶Nine patients with undetectable HCV RNA at end of treatment; **Three patients lost to follow-up.
Baseline Virologic Data. Baseline population-based sequences were available for 24/26 G3 HCV-infected patients; no patient had mutations associated with decreased telaprevir susceptibility in G1 studies. No patient with vBT had known baseline mutations by clonal sequence analysis (Fig. 2b).
The mean telaprevir FC at baseline for G3 HCV (vs wild type) was 7.0 (range, 2.5–14.5). Unexpectedly, patients with the highest baseline telaprevir FC tended to show a greater change in HCV RNA between baseline and day 3 (Fig. 3b). However, patient numbers were small.
Genotypic Characterization of Viral Variants. All three telaprevir monotherapy patients with vBT had HCV subtype 3a (Fig. 5); R155K was present at vBT in two patients. In the third patient, no mutations associated with reduced telaprevir susceptibility in G1 studies were detected at vBT, but R155K was detected later. Clonal sequence analysis confirmed the results obtained by population sequencing (Fig. 2b). Two of the three patients with vBT had undetectable HCV RNA at the end of peginterferon/ribavirin treatment; one subsequently achieved an SVR, the other relapsed.
Figure 5.
HCV RNA, genotypic and phenotypic profiles for individual G3 HCV-infected patients. Profiles are shown for G3 HCV-infected patients in the telaprevir monotherapy arm with vBT, for both the investigational (panel i) and standard (panel ii) treatment phases. HCV; hepatitis C virus; TVR disc, discontinuation of telaprevir; PR disc, discontinuation of peginterferon/ribavirin, LOQ, limit of quantification; FC, fold change in IC50 For patients without vBT, sequences of samples collected during the investigational phase were available for 4/5 patients in the telaprevir monotherapy arm and 1/7 patients in the triple therapy arm. No mutations associated with decreased telaprevir susceptibility in G1 HCV were detected by population sequencing. For patients who relapsed, sequences of samples were available for 4/5 patients in the telaprevir monotherapy and triple therapy arms. In patient 9, the R155K mutation was detected at time of relapse.
In addition to mutations associated with reduced susceptibility to telaprevir in G1, several other amino acid changes from baseline (T47A/T, A67A/V, K92K/N, A98A/T, S101A/T and P146S) were identified in G3 telaprevir monotherapy patients during or at the end of the investigational phase or at time of relapse. None of the NS3 region amino acid changes associated with telaprevir exposure reached statistical significance by Fisher's exact test.
Phenotypic Characterization of Viral Variants. Paired genotypic and phenotypic data were available for the three G3 patients with vBT. The emergence of R155K was associated with a >5-fold increase from baseline in FC.
Introduction of the R155K mutation into the NS3 baseline sequence of patients 7 and 9 (both G3a) by site-directed mutagenesis led to an FC increase from 9.4-10 (without R155K) to >58.8–>90.9 (with R155K).
Discussion
In treatment-naïve patients, telaprevir monotherapy had significant antiviral activity against G2 HCV but little or no activity against G3 HCV.[14] All vBTs occurred during telaprevir monotherapy, and most NS3 mutations emerging during breakthrough occurred at amino acid positions previously reported to be associated with reduced telaprevir susceptibility in G1 HCV. The finding that telaprevir monotherapy is associated with a high rate of vBT in G2/G3 HCV is similar to previous observations in G1 HCV,[15,16] and highlights the importance of combining telaprevir with peginterferon/ribavirin to control the emergence of resistant variants that remain susceptible to these agents.
In the telaprevir monotherapy arms, vBT was reported in six of nine patients with G2 HCV and three of eight patients with G3 HCV. Seven of these nine patients with vBT had undetectable HCV RNA at the end of subsequent treatment with peginterferon/ribavirin, and four achieved an SVR. The fact that G2 and G3 HCV variants with reduced telaprevir susceptibility remained sensitive to, and can be eradicated by, peginterferon/ribavirin extends similar observations with G1 HCV.[15]
Three mutations associated with reduced telaprevir susceptibility in G1 HCV (R155K, T54A, A156S) were seen in G2 HCV patients with vBT; two (R155K and T54A) were seen in G3 HCV patients with vBT. As in G1a HCV,[16] all emergent mutations required only a single nucleotide change from baseline. Several additional mutations were identified by the present analyses, but these did not occur consistently across patients and were not previously reported for other HCV protease inhibitors.[2,15,17] Moreover, none of these additional mutations occurred with significantly higher frequency than in HCV samples from patients not exposed to telaprevir. However, small sample sizes and a restricted diversity of subtypes limited the robustness of our findings and, for G2, statistical analyses could not be performed. Furthermore, in samples with these additional mutations, resistance could be explained by known telaprevir-resistant mutations using site-directed mutagenesis, although we had not formally introduced the additional mutations into baseline samples to determine their impact on telaprevir susceptibility. In spite of this, our data suggest a lack of G2- or G3-specific mutations for telaprevir resistance. Therefore, the resistance profile appears consistent across G1, G2 and G3.
The NS3 region of HCV shows some differences between genotypes. For example, position 36 of the NS3 region, identified as a site for telaprevir-resistant G1 mutations, contains leucine (L) in patients with G2 and G3 HCV, but valine (V) in G1 HCV. The impact of this amino acid change, if any, on telaprevir susceptibility is not fully understood and may be genotype and subtype dependent. For example, some G2 NS3 proteins carrying an 'L' at position 36 exhibited telaprevir FC values similar to those observed with G1 proteins carrying a 'V' at position 36 (Janssen Infectious Diseases BVBA, data on file). G3 NS3 proteins carrying an 'L' at position 36 exhibited on average higher FC values, although changes at other amino acid positions could have caused this decrease in susceptibility. This type of finding may underlie the differences observed in this study between G2 and G3 in baseline telaprevir FC, as well as the differences in viral activity of telaprevir against the two genotypes.
G2-infected patients with vBT during telaprevir therapy had subtype 2b, 2a or an unknown subtype, but none had 2c. Although patient numbers were small, this suggests that vBT may be less common with subtype 2c. All G3-infected patients with vBT had subtype 3a. The lack of vBT among subtype 3b patients could relate to the lack of telaprevir activity. The R155K mutation only emerged in G2-infected patients with subtype 2b; in G2a viruses, the emergence of a 'K' at position 155 would require a double nucleotide change. The R155K mutation was dominant in G3a HCV patients with vBT.
For patients with G2 HCV in the telaprevir monotherapy arm, the greatest HCV RNA decline from baseline to day 3 was observed in those with the highest telaprevir susceptibility at baseline. Conversely, in G3 patients, the greatest HCV RNA decline from baseline to day 3 was in those with the lowest baseline telaprevir susceptibility. Patient numbers were small, and the phenotype data were obtained with a biochemical assay, which is less reliable than a cell-based assay. However, this inverse correlation might relate to the low efficacy of telaprevir monotherapy in G3 patients. As previously observed in G1 HCV, emergence of the R155K mutation at vBT was associated with a high FC increase, and site-directed mutagenesis confirmed that R155K is associated with reduced susceptibility to telaprevir in G2/G3 patients. In addition, the A156S mutation was associated with reduced telaprevir susceptibility in G2 HCV.
Clonal sequence analysis confirmed that in most instances of vBT in patients with G2/G3 HCV, mutations associated with reduced telaprevir susceptibility in G1 HCV emerged and became the dominant quasispecies. In two patients, a mutation associated with reduced telaprevir susceptibility was detected at baseline in one clone. However, a mutation found in one clone of 22–27 clones can be the result of a PCR amplification error and not a true mutation present in the clonal population.
In addition to the limitations already mentioned for this study, the sample size was small, and a proportion of patients were unfortunately lost to follow-up. Small samples, however, are a common characteristic of proof-of-principle studies exploring the intrinsic activity of an antiviral agent.
In summary, telaprevir showed activity against G2 HCV in treatment-naïve patients, but limited or no activity against G3 HCV. The resistance profile of telaprevir in G2 and G3 HCV appears to involve similar amino acid substitutions as previously observed in G1 HCV. All cases of vBT occurred in the telaprevir monotherapy arms, highlighting the importance of combining telaprevir with peginterferon/ribavirin to avoid the emergence of resistant variants. A high proportion of patients who experienced vBT with telaprevir monotherapy still achieved an SVR after peginterferon/ribavirin treatment, indicating that the emergence of variants with reduced susceptibility to telaprevir can be controlled with these agents.
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Abbreviations
HCV, hepatitis C virus; G, genotype; SVR, sustained virologic response; DAA, direct-acting antiviral; IC50, 50% inhibitory concentration; q8h, every 8 h; bid, twice daily; vBT, viral breakthrough; EOT, end of treatment; LOQ, limit of quantification; FC, fold change in IC50; L, leucine; V, valine; T, telaprevir monotherapy; T/PR, triple combination therapy; PR, peginterferon/ribavirin; TVR disc, discontinuation of telaprevir; PR disc, discontinuation of PR; PCR, polymerase chain reaction.
Acknowledgements
We express our gratitude to the patients who participated in the study, as well as the study centre staff and Janssen Infectious Diseases personnel. We acknowledge Tom Westgate and Joanne Williams (Medical Writers, Gardiner-Caldwell Communications, Macclesfield, UK) for assistance in drafting the manuscript and collating author contributions. The clinical trial was sponsored by Janssen Pharmaceuticals and Vertex Pharmaceuticals.
J Viral Hepat. 2013;20(6):395-403. © 2013 Blackwell Publishing
Source
weeks to non-cirrhotic patients who have failed previous PegIFN and RBV treatment.
