Mutation Rate of the Hepatitis C Virus NS5B in Patients Undergoing Treatment With Ribavirin Monotherapy

GASTROENTEROLOGY 2007;132:1757–1766

Mutation Rate of the Hepatitis C Virus NS5B in Patients Undergoing Treatment With Ribavirin Monotherapy GLEN LUTCHMAN,* SUSAN DANEHOWER,‡ BYUNG– CHEOL SONG,* T. JAKE LIANG,* JAY H. HOOFNAGLE,* MICHAEL THOMSON,‡ and MARC G. GHANY*

See editorial on page 2050. Background & Aims: Error catastrophe from an increase in mutation rate may be a possible mechanism of action of ribavirin in chronic hepatitis C (CHC). We sought to evaluate the mutagenic potential of ribavirin in vivo and to determine if conserved regions of hepatitis C virus (HCV) NS5B are mutated during ribavirin therapy. Methods: Thirty-one patients with CHC genotype 1 who participated in a randomized, placebo-controlled trial of ribavirin for 48 weeks were studied. After 48 weeks, patients on placebo were crossed-over to open-label ribavirin for 48 weeks. Viral RNA was extracted from paired, stored sera at day 0 and week 24 during the randomized phase and weeks 48, 52, and 72 during the cross-over phase. The entire NS5B region was sequenced and the mutation rates were calculated. Results: An increase in mutation rate was observed after 4 weeks (4.4 ⴛ 10ⴚ2 vs 2.1 ⴛ 10ⴚ3 per site/y, P ⴝ .02) but not after 24 weeks (4.0 ⴛ 10ⴚ3 vs. 5.5 ⴛ 10ⴚ3 per site/y, P ⴝ .1) in patients who crossed over to ribavirin. Similarly, during the randomized phase no increase in the number of mutations or the mutation rate was observed at week 24 between the ribavirin- and placebo-treated patients 6.6 vs 4.3 ⴛ 10ⴚ3 per site/y, respectively (P ⴝ .4). No mutations were observed in conserved regions of NS5B. Conclusions: Ribavirin therapy is associated with an early, transient increase in the mutation rate of HCV. Lethal mutagenesis and error catastrophe is unlikely to be the sole mechanism of action of ribavirin during therapy for CHC.

R

ibavirin is a synthetic guanosine nucleoside analogue that has a broad spectrum of antiviral activity.1 It has limited effectiveness when used as monotherapy for respiratory syncytial virus and Lassa fever.2,3 Interestingly, ribavirin has only minimal antiviral activity when used as monotherapy,4 – 6 but doubles the sustained viral clearance rates when combined with interferon/peginterferon for the treatment of chronic hepatitis C (CHC).7,8 This is

primarily through the prevention of virologic relapse after discontinuation of therapy, but ribavirin may act synergistically with interferon to achieve the initial clearance of virus.8 –10 The exact mechanism through which ribavirin produces its antiviral response in patients with CHC is not clear. Four possible mechanisms have been proposed based on in vitro data. First, ribavirin may have an immunomodulatory effect favoring an antiviral T-helper cell-1 (Th1) cytokine response with production of tumor necrosis factor-␣ and interferon ␥.11,12 Second, ribavirin monophosphate has been shown to be a competitive inhibitor of inosine monophosphate dehydrogenase, the rate-limiting step in the production of guanosine triphosphate, an important molecule for RNA synthesis. Depletion of intracellular guanosine triphosphate may inhibit viral replication indirectly.13 Third, the intracellular metabolite of ribavirin, ribavirin triphosphate, may directly inhibit the HCV-RNA– dependent RNA polymerase (RdRp) by acting as a substrate for the RdRp, leading to mis-incorporation or premature primer chain termination and inhibition of replication.14,15 Finally, ribavirin acts as a viral mutagen, causing error catastrophe and lethal mutagenesis.16,17 Many RNA viruses circulate as quasispecies with marked genetic variation as a result of the infidelity of the RNA polymerase. This high mutation rate provides a selection advantage to the virus for escaping the host immune response and allowing for adaptation to different host environments. However, a minor increase in the mutation rate above a certain threshold may drastically reduce the viability of viral progeny and lead to lethal mutagenesis and error catastrophe. This phenomenon has been shown in vitro for poliovirus, GB virus B, and in hepatitis C virus (HCV) using subgenomic and binary T7/HCV complementary DNA replicon systems.16,18 –22 Abbreviations used in this paper: CHC, chronic hepatitis C; PCR, polymerase chain reaction; RdRp, RNA-dependent RNA polymerase; Th1, T-helper cell-1. © 2007 by the AGA Institute 0016-5085/07/$32.00 doi:10.1053/j.gastro.2007.03.035

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*Liver Diseases Branch, National Institutes of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland; and the ‡Virology Department, GlaxoSmithKline, Research Triangle Park, North Carolina

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Ribavirin increased the mutation rate by causing misincorporation of nucleoside bases.14,21,23 A small in vivo study suggested that ribavirin use in CHC was associated with an increase in the mutation rate of HCV. In this study, the error rate determined by the evolution of the nucleotide sequence of the NS5B coding region of the viral genome was higher in 5 patients treated with ribavirin for 36 weeks compared with 2 patients who received placebo or 8 patients who received interferon.24 In addition, a mutation conferring resistance to ribavirin, F415Y, was observed in all ribavirintreated patients. However, this study was limited by the small number of patients and the fact that only a portion of NS5B was analyzed. More importantly, the observation of a ribavirin-resistant variant might negate the possibility of ribavirin-induced mutagenesis. Recently, another study of 34 patients with genotype 1b infection treated with 600 – 800 mg/day of ribavirin for 4 weeks reported an increase in the mutation rates of NS5A and NS5B. The number of mutations within NS5A correlated with subsequent sustained virologic response to interferon and ribavirin.25 However, mutations were observed in only a third of patients. Furthermore, an analysis of the NS3 region in 4 patients treated with ribavirin showed no overall increase in the viral mutation rate.26 Thus, the issue of whether ribavirin acts as a viral mutagen in vivo remains controversial. The specific aims of this study were to determine if ribavirin treatment is associated with an increase in the mutation rate of the NS5B coding region of the HCV genome and to examine whether any mutations emerge in conserved regions of NS5B in patients with CHC.

Materials and Methods Patients Fifty-eight patients with CHC participated in a randomized, double-blind, placebo-controlled trial of ribavirin at the Clinical Center of the National Institutes of Health from 1992 to 1994.4 Patients were randomized to receive oral ribavirin 1200 mg or matching placebo daily for 48 weeks and were monitored by monthly biochemical, hematologic, and virologic testing (Quantiplex HCV RNA branched DNA assay version 1.0; Chiron Corporation, Emeryville, CA), as well as collection of a research serum sample that was stored at ⫺70°C. At the end of the trial consenting patients in the placebo arm were offered open-label ribavirin in a cross-over trial. For the present study the cohort was confined to patients infected with genotype 1 to avoid genotypic-related sequence differences within NS5B during the analysis. Forty-three of the 58 patients were infected with HCV genotype 1 virus and therefore were eligible, but paired, stored serum samples from day 0 and week 24 of treatment were available for 38 patients (Figure 1A). In 3 patients for whom the day 0 sample was not accessible,

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Figure 1. (A) Derivation of the study cohort. Fifty-eight patients were randomized to ribavirin or placebo, 29 patients per arm. Forty-three were infected with genotype 1 virus and paired sera were available for 38 patients, 19 from each arm. Complete sequences were obtained from 18 patients randomized to ribavirin and 13 patients who received placebo. Complete paired sequences were obtained for 10 of 13 and 8 of 13 cross-over patients at weeks 72 and 52, respectively. (B) Study design and time points evaluated. Patients were randomized to ribavirin 1200 mg/day or matching placebo for 48 weeks. Consenting patients randomized to placebo then were crossed over to ribavirin for 48 weeks. Serum samples were evaluated at day 0 and week 24 during the randomized phase and at weeks 48, 52, and 72 during the cross-over phase. Bracketed time points indicate the periods of unpaired and paired comparisons.

the next available sample from the closest time point before randomization was used (study weeks -16, -29, and -53). Similarly, in 1 patient for whom the week 24 sample was not available, the sample from the next closest time point was used (study week 34). Patients who were crossed over to ribavirin monotherapy after 48 weeks of placebo were sequenced at weeks 48, 52, and 72 (ie, before starting and after 4 and 24 weeks of ribavirin treatment) (Figure 1B). All patients were participants in clinical research protocols that had been approved by the National Institutes of Diabetes and Digestive and Kidney Diseases Institutional Review Board and gave written informed consent for evaluation and treatment.

Sequence Analysis Viral RNA was extracted from 200 ␮L of stored serum using the QIAamp Viral RNA Mini Kit (Qiagen, Valencia, CA). HCV RNA was reverse transcribed using the SuperScript III reverse transcriptase (RT) system (In-

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Table 1. Primer Sequences for Reverse Transcription and PCR Reaction

Primers

Reverse transcription

Genotype 1a 5=-AAAAAAAAAAAACAGGAAATGGCCTATTG-3= Mixed in equal concentration with 5=-AAAAAAAAAAAACAGGAAATGGCTTAAG-3= Genotype 1b 5=-AGTGKTTAGCTCCCCGTTCA-3= Genotype 1a (all mixed in equal concentration): 5=-AAAAAAAAAAAACAGGAAATGGCCTATTG-3= 5=-AAAAAAAAAAAACAGGAAATGGCTTAAG-3= 5=-TCCTCAACTTCCGGCATTAC-3= Genotype 1b (all mixed in equal concentration): 5=-AGTGKTTAGCTCCCCGTTCA-3= 5=- TGTGGCRGCARGAGATGG3= 5=-AGTGKTTAGCTCCCCGTTCAY-3= Genotype 1a (mixed in equal concentration) 5=-TCCTCAACTTCCGGCATTAC-3= 5=-ATTGGCCTGGAGTGGTTAGCC-3= 5=-AAGAGGCCGGAGTGTTTACCC-3= Genotype 1b (mixed in equal concentration) 5=-CTCAGCGACGGGTCTTGGT-3= 5=-CTCAGCGACGGGTCTTGGTC-3= 5=-TAGCTCCCCGTTCAYCGGTTG-3=

PCR reaction

Second PCR reaction

vitrogen Corporation, Carlsbad, CA) with an equal mixture of 2 primers for genotype 1a and a single primer for genotype 1b (Table 1). A nested polymerase chain reaction (PCR) was performed using the transcribed DNA from the RT reaction using Platinum Taq DNA Polymerase High Fidelity (Invitrogen Corporation) to amplify a 1773– base pair fragment between nucleotides 7601 and 9374. A mixture of 3 primers in equal concentrations was used for genotype 1a and a mixture of 3 primers in equal concentrations was used for genotype 1b (Table 1). The following PCR reaction conditions were used: denaturing at 94°C for 1 minute, followed by denaturing at 94°C for 30 seconds, annealing at 55°C for 15 seconds, and extension at 68°C for 2 minutes for 30 cycles, followed by a final extension at 72°C for 7 minutes. The second PCR reaction was performed with 5 ␮L of DNA from the first PCR reaction using Platinum Taq DNA Polymerase High Fidelity and a mixture of 3 primers in equal concentration for genotype 1a, and a mixture of 3 primers in equal concentration for genotype 1b (Table 1). The same reaction conditions were used as for the firstround PCR. The PCR products were visualized on a 1% agarose gel stained with ethidium bromide to ensure the correct amplicon size and then purified using the QIA quick PCR purification kit (Qiagen). The purified DNA was sequenced bidirectionally with overlapping primers using

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the BigDye Terminator v1 Cycle sequencing kit (Applied Biosystems, Foster City, CA) and then resolved on an Applied Biosystems 3730xl DNA analyzer. The sequence data were aligned and analyzed using Sequencher version 4.0.5 (Gene Codes Corporation, Ann Arbor, MI).

Clonal Analysis Viral RNA was extracted and reverse transcribed as described previously. After nested PCR, the amplified DNA was TA-cloned using the Topo-TA cloning vector according to the manufacturer’s instructions (Invitrogen Corporation). The plasmid containing the cloned DNA was transformed into competent Escherichia coli bacteria and plated on Luria Broth-ampicillin plates. After 16 hours of incubation at 37°C, 10 clones were selected randomly from each patient sample and sequenced as previously described.

Statistical Analysis and Calculation of Mutation Rates The reference sequence for genotype 1a was H7727 and for 1b was J4L6S.28 Comparisons of paired sequences were made at day 0 and at week 24 during the randomized phase and weeks 48 and 52 and weeks 48 and 72 during the cross-over phase (Figure 1B). The number of nonsynonymous and synonymous mutations for each patient pair was determined. The error generation rate for each patient then was calculated as follows: total number of mutations observed after therapy (both synonymous and nonsynonymous)/number of base pairs sequenced in NS5B (1773)/interval between samples analyzed (years). The ratio of the number of nonsynonymous to synonymous mutations (dn/ds) and the Hamming distance were calculated for each paired sequence. These 2 parameters describe the sequence variation between a given pair of sequences, with the Hamming distance defined by the following equation: Hd ⫽ (1 ⫺ s) ⫻ 100, where s is the fraction of shared sites in the 2 aligned sequences. Similar calculations and comparisons were performed for the subset of patients who were crossed over to ribavirin from the placebo arm. For the clonal analysis, each clone from the week 48 sample was compared with each clone from the week 52 sample and the mean number of mutations was used to calculate the error rate. A similar analysis was performed to calculate the Hamming distance. Statistical analysis was performed using Graphpad v4.0 (Prism, San Diego, CA) software. All analyses were interpreted using a 2-sided ␣ value of 0.05. The standard t test, Mann–Whitney test, and Wilcoxon ranked sum test, as well as the Fisher exact test were used as indicated to assess the differences in the absolute and normalized number of errors and the error generation rate between ribavirin- and placebo-treated patients.

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Table 2. Baseline Demographics and Clinical Characteristics of the Patients in the Randomized and Cross-Over Phases of the Study

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N Age, y Number of men Race, white/black/other Body mass index, kg/m2 Duration of infection, y HCV genotype, 1a/1b ALT level, IU/mL Aspartate aminotransferase level, IU/mL Total bilirubin, mg/dL Albumin level, g/dL White blood cell level, 103/mm3 Hematocrit level, % Platelet level, 103/mm3 Branched DNA, 105 copies/mL Histologic activity index, 0–22 Fibrosis, 0–4 Interval between samples, wk

Placebo

Placebo crossed over to ribavirin

P value (placebo vs ribavirin)

18 42.5 (32.1–66.1) 15 16/1/1 28.1 (21.4–37.8) 5.5 (0.7–19.5) 10/8 190.6 (105–496) 108.8 (64–296)

13 44.0 (31.8–63.9) 5 12/1/0 26.1 (17.7–32.4) 7.6 (0.7–17.2) 8/5 160.2 (66–448) 105.4 (53–240)

10 47.4 (37.9–63.9) 4 9/1/0 27.7 (22.8–46.9) 10.0 (3.3–19.2) 6/4 122.3 (57–256) 94.0 (42–176)

NSa NSb NSa NSa NSb NSb NSb NSb NSb

0.8 (0.4–1.5) 4.3 (3.8–4.7) 5.7 (3.5–7.9) 46.0 (42.3–53.0) 202.2 (147–312) 4.2 (0.4–16.9) 11.3 (5–16) 1.9 (0–4) 27.9 (19–84.9)

0.6 (0.3–1.0) 4.1 (3.7–4.6) 5.8 (3.2–9.0) 41.6 (35.3–49.2) 205.6 (97–347) 4.1 (0.4–8.5) 11.3 (7–16) 1.8 (0–4) 28.8 (24.0–53.0)

0.8 (0.4–1.2) 4.0 (3.6–4.4) 6.3 (2.8–10.5) 41.4 (36.6–48.3) 190 (105–304) 4.1 (0.5–7.5) 11.0 (7–16) 1.3 (0–3) 26.6 (24–34)

NSb NSb NSb ⬍.001b NSb NSb NSb NSb NSb

All patients

Ribavirin

31 42.3 (31.8–66.1) 20 28/2/1 27.8 (17.7–46.9) 6.2 (0.7–19.5) 18/13 181.5 (66–496) 113.6 (53–332) 0.7 (0.3–1.5) 4.2 (3.7–4.7) 5.8 (3.2–9.0) 44.0 (35.3–53.0) 200.5 (97–347) 3.9 (0.4–16.9) 11.3 (5–16) 1.9 (0–4) 29.9 (19.0–84.9)

NOTE. Continuous data are presented as mean (range). a␹2 test. bStudent t test.

Results Of the 43 patients infected with genotype 1 virus who participated in the study, paired serum samples were available for 38 patients, 19 of whom received ribavirin. Complete, paired sequence data were obtained for 18 of 19 (10 genotype 1a) ribavirin-treated and 13 of 19 (8 genotype 1a) placebo-treated patients (Figure 1). Thirteen of the 19 placebo-treated patients consented to participate in the open-label cross-over trial to receive ribavirin monotherapy for 48 weeks. Paired stored samples were available for 11 patients and complete, paired sequence data were available for 10 of 11 (weeks 48 and 72) and 8 of 10 (weeks 48 and 52, Figure 1A).

Baseline Clinical Characteristics The baseline patient characteristics for the 2 groups during the randomized phase of the trial are shown in Table 2. The mean age of the cohort was 43.3 years (range, 32– 66 y), two thirds were male and the majority were Caucasian. The estimated mean duration of infection was 6 years, with a range of 0.7–20.0 years. All patients had alanine aminotransferase (ALT) levels greater than 1.5 times the upper limit of normal at entry into the trial (mean ALT level, 182 IU/L). All patients had compensated liver disease and none had received prior antiviral therapy. Eighteen patients were infected with HCV genotype 1a and 13 patients were infected with genotype 1b. The mean viral load was 3.9 ⫻ 105 copies/mL by the branched DNA assay. The mean necroinflammatory score was 9.4 and no patient had cirrhosis.

The ribavirin (n ⫽ 18) and placebo (n ⫽ 13) groups were well matched for baseline clinical characteristics with the exception that patients randomized to ribavirin had a significantly higher mean hematocrit level compared with the placebo group. This may have been because there was a greater proportion of men in the ribavirin group.

Comparison of Laboratory Data Between Week 24 and Baseline There was no measurable difference at week 24 vs baseline (day 0) for any parameter evaluated in the placebo group. In particular, serum ALT, hematocrit, and HCV RNA levels were unchanged. In contrast, the ribavirin group had significantly lower serum ALT levels, and not unexpectedly lower hematocrit levels and higher reticulocyte counts at week 24 compared with baseline.4 Notably, there was no difference in the HCV-RNA level. Comparing the week 24 results, the ribavirin-treated group had significantly lower ALT and higher total bilirubin levels and reticulocyte counts compared with the placebo group. There was no difference in the HCV-RNA levels consistent with other reports of a lack of antiviral response to ribavirin monotherapy.5,6,9

Mutation Frequency and Pattern in the Ribavirin- and Placebo-Treated Groups To determine if ribavirin increases the mutation rate of HCV we compared the number of synonymous, nonsynonymous, and the total number of mutations

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Table 3. Number of Nonsynonymous and Synonymous Mutations and Error Rate in the Patients During the Randomized Phase Number of synonymous mutations b/w wk 24 vs day 0

Number of nonsynonymous mutations b/w wk 24 vs day 0

Total number of mutations

Error rate (mutations/genome/y), mean (range)

49 90 NSa

6 13 NSa

55 103 NSa

4.3E-03 (0–15) 6.6E-03 (0–40) NSb

Placebo (n ⫽ 13) Ribavirin (n ⫽ 18) P value aMann–Whitney

test. t test.

within the HCV polymerase NS5B in patients after 24 weeks of ribavirin or placebo using the day 0 sample as the comparator sequence. There were numerically more synonymous (90 vs 49), nonsynonymous (13 vs 6), and total number of mutations (103 vs 55) in the ribavirintreated group compared with the placebo-treated group, respectively, but the differences were not statistically significant. Importantly, the error rate was no different between the ribavirin-treated (6.6 ⫻ 10⫺3 per site/y) and the placebo-treated patients (4.3 ⫻ 10⫺3 per site/y) (Table 3). In addition, the distribution of number of mutations per patient, in any of the ranges analyzed (0, ⱕ1, ⱕ2, ⱕ10, and ⱕ20), also was not significantly different between the ribavirin and placebo patients, supporting the previous results that ribavirin has little mutagenic potential (Table 4). We also examined the pattern of nucleotide substitutions between the 2 groups of patients. As a purine analogue, once incorporated into RNA, ribavirin may base pair with cytidine and uracil and could be mutagenic by promoting A-G and G-A transitions. G-A and C-T were the most commonly observed transitions in ribavirin-treated patients, but the frequency was not greater than A-G and T-C (Figure 2A). These 4 substitution patterns accounted for 90% of the mutations observed. The mean ratios of nonsynonymous to synonymous substitutions and mean Hamming distance were not significantly different between the ribavirin- and placebotreated groups (Figure 2B).

Mutation Frequency and Pattern in the Cross-Over Patients: Week 24 Comparison Paired samples were available for 11 of 13 and complete paired sequence data were available for 10 of 11 patients who crossed over to ribavirin (Figure 1A). A total of 42 mutations were observed in these 10 patients dur-

Table 4. Distribution of Number of Mutations Between the Ribavirin- and Placebo-Treated Patients Number of mutations per patient

Ribavirin (n ⫽ 18)

Placebo (n ⫽ 13)

P valuea

0 ⱕ1 ⱕ2 ⱕ10 ⱕ20

4 7 10 14 17

5 7 8 11 13

NS NS NS NS NS

aFisher

exact test.

Figure 2. (A) Proportion of nucleotide transition mutations at week 24 in ribavirin- and placebo-treated patients. The 4 transitions shown represent 90% of the observed mutations. (B) Hamming distance at week 24 between ribavirin- and placebo-treated patients.

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bUnpaired

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Table 5. Error Rates, Hamming Distance, and Number of Mutations While on Placebo and After 4 and 24 Weeks of Ribavirin in the Patients Who Were Crossed Over to Ribavirin Day 0 (wk 24 vs 0)

Week 4 (wk 52 vs 48)

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Patient

Error rate

HD

No. of mts

1 2 3 4 5 6 7 8 9 10 Mean 1-8 Mean 1-10

6.1 ⫻ 10⫺3 0 0 1.2 ⫻ 10⫺3 2.3 ⫻ 10⫺3 7.6 ⫻ 10⫺3 0 0 8.1 ⫻ 10⫺3 1.4 ⫻ 10⫺2 2.1 ⴛ 10ⴚ3 4.0 ⴛ 10ⴚ3

2.8 ⫻ 10⫺1 0 0 5.6 ⫻ 10⫺2 1.1 ⫻ 10⫺1 4 ⫻ 10⫺1 0 0 5.6 ⫻ 101 9.6 ⫻ 10⫺1 7.4 ⫻ 10⫺2 2.4 ⫻ 10⫺1

5 0 0 1 2 7 0 0 10 17 1.9 4.2

Error rate

HD

2.9 ⫻ 10⫺2 0 1.1 ⫻ 10⫺1 4.4 ⫻ 10⫺2 2.2 ⫻ 10⫺2 8 ⫻ 10⫺2 3.7 ⫻ 10⫺2 2.9 ⫻ 10⫺2

2.3 ⫻ 10⫺1 0 8.4 ⫻ 10⫺1 3.4 ⫻ 10⫺1 1.7 ⫻ 10⫺1 5.6 ⫻ 10⫺1 2.8 ⫻ 10⫺1 1.7 ⫻ 10⫺1

4.4 ⴛ 10ⴚ2

3.2 ⫻ 10⫺1

Week 24 (wk 72 vs 48) No. of mts 4 0 15 6 3 10 5 3

5.8

Error rate

HD

No. of mts

2.4 ⫻ 10⫺3 1.8 ⫻ 10⫺3 3.4 ⫻ 10⫺3 2.2 ⫻ 10⫺3 3.4 ⫻ 10⫺3 1.1 ⫻ 10⫺2 1.2 ⫻ 10⫺3 0 1.5 ⫻ 10⫺2 1.4 ⫻ 10⫺2 3.1 ⴛ 10ⴚ3 5.5 ⴛ 10ⴚ3

1.1 ⫻ 10⫺1 1.1 ⫻ 10⫺1 1.7 ⫻ 10⫺1 1.1 ⫻ 10⫺1 1.7 ⫻ 10⫺1 5.1 ⫻ 10⫺1 5.6 ⫻ 10⫺2 0 7.3 ⫻ 10⫺1 6.8 ⫻ 10⫺1 1.6 ⫻ 10⫺1 2.7 ⫻ 10⫺1

2 2 3 2 3 9 1 0 13 12 2.8 4.7

NOTE. Statistical comparisons of mean error rates in bold: 2.1 ⫻ 10⫺3 vs 4.4 ⫻ 10⫺2 (P ⫽ .02); 4.4 ⫻ 10⫺2 vs 3.1 ⫻ 10⫺3 (P ⫽ .01); 4.0 ⫻ 10⫺3 vs 5.5 ⫻ 10⫺3 (P ⫽ .1). HD, hamming distance; mts, mutations.

ing the initial 24 weeks (week 24 vs day 0) while receiving placebo in the randomized phase of the trial, of which 38 were synonymous and 4 were nonsynonymous (Table 5). When the patients were crossed over to ribavirin we observed a total of 47 mutations between weeks 72 and 48, of which 33 were synonymous and 14 were nonsynonymous. Of the 14 nonsynonymous mutations, 3 represented mutations back to wild-type virus (virus isolated at day 0). The number of nonsynonymous mutations between weeks 72 and 48 was significantly higher compared with between week 24 and day 0 (14 vs 4; P ⫽ .03). However, the calculated error rates between weeks 72 and 48 (5.5 ⫻ 10⫺3 per site/y) and week 24 vs day 0 (4.0 ⫻ 10⫺3 per site/y) were not significantly different (Table 5). In these 10 patients, A-G, C-T, G-A, and T-C mutations accounted for the majority (87%) of the nucleotide changes observed between weeks 72 and 48, and there was no significant difference in the proportion of A-G or G-A mutations (Figure 3A). Similarly, there was no significant difference in the mean dn/ds ratio or the Hamming distance when comparisons were made between weeks 72 vs 48 and week 24 vs day 0 (Figure 3B). Thus, this analysis in which each patient served as their own control reinforced the findings of the randomized phase that ribavirin has a nonsignificant effect on the mutation rate of NS5B after 24 weeks of administration.

Mutation Frequency in the Cross-Over Patients: Week 4 Comparison A similar analysis was performed comparing the week 48 to 52 samples in patients who crossed over to ribavirin to assess if ribavirin had a mutagenic effect at an earlier time point. Week 52 samples were available from 8 of 10 patients and complete sequence data were obtained for all patients. Forty-six mutations were observed,

Figure 3. (A) Proportion of nucleotide transition mutations and (B) Hamming distances comparing day 0 to week 24 (period on placebo) with weeks 48 to 72 (24 weeks of ribavirin) in the cross-over patients. The 4 transitions shown represent 87% of the observed mutations.

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34 synonymous and 12 nonsynonymous (Table 5). The week 4 calculated mean error rate was significantly higher compared with the periods when these patients were receiving placebo (4.4 ⫻ 10⫺2 vs 2.1 ⫻ 10⫺3 per site/y; P ⫽ .02) and 24 weeks of ribavirin (4.4 ⫻ 10⫺2 vs 3.1 ⫻ 10⫺3 per site/y; P ⫽ .01). However, the Hamming distance, the calculation of which is time independent, showed no difference at weeks 52 and 72 (3.2 ⫻ 10⫺1 vs 1.6 ⫻ 10⫺1; P ⫽ .1). To validate the results of direct sequencing, virus from 3 patients at weeks 48 and 52 were cloned and 10 clones from each time point were sequenced. The mean calculated error rate was 1.23 ⫻ 10⫺1 per site/y. However, the individual error rates in 2 of 3 patients were similar to the rate obtained with direct sequencing (5.8 ⫻ 10⫺2 vs 2.9 ⫻ 10⫺2 per site/y [patient 1] and 2.6 ⫻ 10⫺1 vs 1.1 ⫻ 10⫺1 site/y [patient 3]) (Table 5). The mean Hamming distance derived from the cloning and direct sequencing results was similar (data not shown).

Location of Nonsynonymous Mutations Within NS5B At treatment week 24 compared with day 0, 13 nonsynonymous mutations were observed in the ribavirin-treated vs 6 in the placebo-treated patients.29 None of the mutations affected the conserved regions of NS5B (Figure 4), but there appeared to be clustering of mutations at positions 405-431 and 455-533, which represented 9 of 13 mutations. The F415Y mutation previously reported to be associated with ribavirin resistance was noted in 1 subject with genotype 1a infection who received ribavirin. Interestingly, this patient had a marked improvement in serum ALT (468 to 13 IU/mL) and HCV RNA levels (18.7 to 3.6 ⫻ 105 copies/mL) at the end of 24 weeks of therapy.

Prevalence of the F415Y Mutation During the randomized phase, 1 of 10 HCV genotype 1a patients receiving ribavirin developed the

CLINICAL–LIVER, PANCREAS, AND BILIARY TRACT

Figure 4. HCV NS5B illustrating the major conserved domains and location of mutations observed during the initial 24 weeks of the randomized phase. Mutations in the ribavirin-treated patients are shown in red and the placebo-treated patients are shown in green. A clustering was observed between amino acids 405– 431 and 455–533.

F415Y mutation. In the cross-over trial, 4 of 6 HCV genotype 1a patients developed the F415Y mutation. The F415Y mutation was not observed in any patient with HCV genotype 1b treated with ribavirin and in no patient who received placebo, regardless of genotype. Thus, the F415Y mutation was observed in 5 of 16 HCV genotype 1a vs 0 of 13 genotype 1b patients treated with ribavirin and 0 of 8 genotype 1a patients treated with placebo. There was no significant difference in the serum ALT or HCV-RNA levels at week 24 between patients with and without the F415Y mutation who received ribavirin. Considering all patients who received ribavirin for 24 weeks, there was no difference in the mean ALT level (47.4 vs 78 IU/L; P ⫽ .1) and branched DNA level (8.1 vs 4.5 ⫻ 105 copies/mL; P ⫽ .7) between those with (n ⫽ 5) vs those without (n ⫽ 23) the F415Y mutation. The F415Y mutation persisted in follow-up samples sequenced 1 year after the discontinuation of ribavirin in all 5 patients (data not shown). Two of 5 patients subsequently were treated with interferon and ribavirin and both achieved a sustained virologic response.

Discussion There has been much speculation about the mechanism of action of ribavirin when combined with interferon for the therapy of chronic HCV infection. This study assessed the mutagenic potential of ribavirin in vivo from a well-characterized cohort of patients with chronic HCV infection. We analyzed the mutagenic potential of ribavirin in a number of ways: comparing the number of nonsynonymous and synonymous mutations, examining the pattern and distribution of mutations, calculating the error rate, and performing a clonal analysis between ribavirin- and placebo-treated patients. We observed a small, albeit significant, increase in the error rate and the number of mutations after 4 weeks of ribavirin, suggesting that ribavirin may be a viral mutagen in vivo. However, the effect appeared to be transient

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and abrogated with continued use because analysis after 24 weeks of ribavirin revealed neither a significant increase in the number of mutations nor the error rate within NS5B of HCV. This was observed both in patients treated during the randomized and cross-over phases (in which patients served as their own controls) of the trial. Furthermore, at treatment week 24, the frequency of nonsynonymous mutations was not significantly different between the 2 groups and no mutations were detected in the conserved regions of NS5B. The previously reported F415Y mutation24 was observed in almost a fifth of patients treated with ribavirin but its clinical significance is unclear because it did not seem to affect response to ribavirin or future response to combination therapy. The results of this study appear to be relevant to our understanding of the mechanism of action of ribavirin in CHC. The results support but do not confirm recent in vivo and mathematic models on the mechanism of action of ribavirin. Pawlotsky at al9 observed a transient antiviral effect at day 2 or 3 in 4 of 7 patients treated with weight-based ribavirin, but no further effect with continued use. An early but nonsustained mutagenic effect would explain the viral kinetics observed by Pawlotsky et al.9 Similarly, an early mutagenic effect also supports a recent mathematic model that postulated that ribavirin causes a fraction of newly produced virions to be noninfectious owing to lethal mutagenesis as opposed to an immunomodulatory effect.30 In this model it is predicted that ribavirin affects second-phase clearance of HCV RNA and the effect is most pronounced when interferon’s effectiveness is suboptimal.30 In our study and others, ribavirin was not mutagenic in every patient. These findings raise the issue of whether a sustained virologic response is achieved only in patients who experience viral mutagenesis. A recent clinical study suggested that mutations in NS5A correlated with eventual sustained virologic response.25 The estimated mutation rate of 6.6 ⫻ 10⫺3 per site/y after 24 weeks of ribavirin was lower than that reported by Young et al24 of 9.1 ⫻ 10⫺3 site/y after 36 weeks of ribavirin. This may be because of differences in the region of the viral genome sequenced, differences in virus isolates, differences in viral response to host immune pressure, or the method used to calculate the error rate.31,32 However, the mutation rate at week 4 of 4.4 ⫻ 10⫺2 per site/y was similar to the rate of 1.3 ⫻ 10⫺2 per site/y reported by Asahina et al25 for the NS5B region at a comparable time point. Ribavirin is metabolized to ribavirin triphosphate within hepatocytes and erythrocytes by intracellular adenosine kinase.33 This metabolite has the ability to pair both with cytosine and uracil and be incorporated in the place of guanine or adenosine. Therefore, its mis-incorporation during viral replication results in an increase in transition mutations (A-G and G-A).16 This mutagenic effect of ribavirin has been reported previously using various in vitro systems.20,21 We found no significant

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difference in the frequency of A-G and G-A transitions between the 2 groups. Furthermore, ribavirin did not appear to have a preference for a particular site within NS5B because there was no difference in the dn/ds ratio between the ribavirin and placebo groups. Similarly, the number of nonsynonymous mutations, which more accurately reflects external pressure rather than the infidelity of the RdRp, was no different between the 2 groups. Taken together, the results suggest that ribavirin is a weak mutagen at best and does not preferentially mutate sites within NS5B. A second aim of the study was to examine whether treatment with ribavirin resulted in any mutations in the conserved regions of NS5B. The NS5B is the virally encoded RdRp and similar to other viral polymerases contains a finger, palm, and thumb domain with 5 conserved motifs in the palm, A, B, C, D, E, a sixth in the finger domain, F, and a seventh in the thumb domain, a unique 12–amino acid ␤-hairpin (amino acids 443– 454) (Figure 4). We observed no mutations in any of the conserved regions of NS5B in the ribavirin-treated patients. Furthermore, none of the important or critical residues within NS5B responsible for its enzymatic activity, template and primer binding, NS5A binding, and for replication in the replicon system were affected by ribavirin therapy. A clustering of mutations was noted in amino acids (aa) 405– 431 (upstream of the thumb region) and (aa) 455– 533 (downstream of the ␤-hairpin), which theoretically could lead to conformational changes that may affect the ability of NS5B to use templates. We plan to characterize the effects of these mutations on the activity of the RdRp using the recently reported full-length infectious replicon.34 The previously reported F415Y mutation reputed to confer resistance to ribavirin was observed in 5 of 16 genotype 1a patients (1 of 10 in the randomized phase and 4 of 6 patients in the open-labeled phase). An analysis of ALT and HCV-RNA levels comparing levels before and after 24 weeks of ribavirin showed no significant difference between those with and without the mutation, suggesting a lack of clinical resistance as previously reported.24 Whether this mutation is associated with ribavirin resistance is unclear. Tyrosine (Y) is found at position 415 in all genotypes except genotypes 1a and 6a, which probably explains why the mutation was not found in genotype 1b patients.35 Moreover, in clinical studies there does not appear to be any difference in response to combination therapy between genotype 1a and 1b patients, which argues against this representing a true ribavirin-resistant mutant. In addition, a ribavirinresistant mutant might be predicted to show no increase in mutation rate, which is contrary to a previous report by Young et al.24 Our study had several limitations. HCV exists as a quasispecies and sequencing the dominant species may underestimate the true mutation rate because viral species with higher mutation rates may be less fit for repli-

cation. Thus, the mutation rate of the dominant sequence may not be representative of the mutation rate of the whole viral population. The results from the clonal analysis were quite similar to those obtained with direct sequencing and therefore we believe sequencing the dominant species should provide a good estimate of the error rate. We assumed that the development of mutations was linear over time in our error rate calculations and that some mutations may be advantageous and become fixed over time, leading to a decrease of the error rate, but this assumption also could lead to an overestimation of the error rate at earlier time points. Finally, given the high replication rate and short half-life of HCV, the ideal time to assess for mutations during ribavirin therapy might be early after achievement of a steady serum concentration of ribavirin. In conclusion, our data suggest that ribavirin is associated with a small, early increase in the mutation rate in vivo that is not sustained over time, perhaps owing to our inability to detect viruses with impaired replication as a result of the accumulation of mutations or as a result of the development of resistant virions.36 Lethal mutagenesis may contribute to but is unlikely to be the sole mechanism of action of ribavirin in the treatment of chronic HCV infection in clinical practice. Future research is needed in this area, which may help with the design of novel antiviral therapies. References 1. Sidwell RW, Huffman JH, Khare GP, Allen LB, Witkowski JT, Robins RK. Broad-spectrum antiviral activity of Virazole: 1-betaD-ribofuranosyl-1,2,4-triazole-3-carboxamide. Science 1972;177: 705–706. 2. Huggins JW. Prospects for treatment of viral hemorrhagic fevers with ribavirin, a broad-spectrum antiviral drug. Rev Infect Dis 1989;11(Suppl 4):S750 –S761. 3. Ventre K, Randolph A. Ribavirin for respiratory syncytial virus infection of the lower respiratory tract in infants and young children. Cochrane Database Syst Rev 2004:CD000181. 4. Di Bisceglie AM, Conjeevaram HS, Fried MW, Sallie R, Park Y, Yurdaydin C, Swain M, Kleiner DE, Mahaney K, Hoofnagle JH. Ribavirin as therapy for chronic hepatitis C. A randomized, doubleblind, placebo-controlled trial. Ann Intern Med 1995;123: 897–903. 5. Dusheiko G, Main J, Thomas H, Reichard O, Lee C, Dhillon A, Rassam S, Fryden A, Reesink H, Bassendine M, Norkrans G, Cuypers T, Lelie N, Telfer P, Watson J, Weegink C, Sillikens P, Weiland O. Ribavirin treatment for patients with chronic hepatitis C: results of a placebo-controlled study. J Hepatol 1996;25:591–598. 6. Bodenheimer HC Jr, Lindsay KL, Davis GL, Lewis JH, Thung SN, Seeff LB. Tolerance and efficacy of oral ribavirin treatment of chronic hepatitis C: a multicenter trial. Hepatology 1997;26: 473– 477. 7. Manns MP, McHutchison JG, Gordon SC, Rustgi VK, Shiffman M, Reindollar R, Goodman ZD, Koury K, Ling M, Albrecht JK. Peginterferon alfa-2b plus ribavirin compared with interferon alfa-2b plus ribavirin for initial treatment of chronic hepatitis C: a randomised trial. Lancet 2001;358:958 –965. 8. Fried MW, Shiffman ML, Reddy KR, Smith C, Marinos G, Goncales FL Jr, Haussinger D, Diago M, Carosi G, Dhumeaux D, Craxi A, Lin A, Hoffman J, Yu J. Peginterferon alfa-2a plus ribavirin for chronic hepatitis C virus infection. N Engl J Med 2002;347:975–982.

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Received December 21, 2005. Accepted February 15, 2007. Address requests for reprints to Marc G. Ghany, MD, Liver Diseases Branch, National Institutes of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Building 10, Room 9B-16, 10 Center Drive, MSC 1800, Bethesda, Maryland 20892-1800. e-mail: [email protected]. Supported by the Intramural Research Program of the National Institutes of Health, National Institutes of Diabetes and Digestive and Kidney Diseases.