Original Article Investigation of Viral Escape Mutations Within HCV P7 During Treatment with Amantadine in Patients with Chronic Hepatitis C

Antiviral Therapy 2013; 18:803–811 (doi: 10.3851/IMP2663)

Original article Investigation of viral escape mutations within HCV p7 during treatment with amantadine in patients with chronic hepatitis C

Annika Vermehren1, Christoph Welsch1,2, Ulrike Elsler3,4, Johannes Vermehren1, Eva Herrmann5, Christoph Sarrazin1, Michael von Wagner1, Simone Susser1, Wolf Peter Hofmann6, Bernd Kronenberger1, Stefan Zeuzem1, Ulrike Mihm1*

1Medizinische Klinik 1, Universitätsklinikum Frankfurt, Frankfurt am Main, Germany 2Max-Planck-Institut für Informatik, Abteilung Bioinformatik und Angewandte Algorithmik, Saarbrücken, Germany 3Klinik für Innere Medizin II, Universitätsklinikum des Saarlandes, Homburg/Saar, Germany 4Present address: Gesundheitswesen Audi AG, Ingolstadt, Germany 5Institut für Biostatistik und mathematische Modellierung, Fachbereich Medizin, Goethe-Universität, Frankfurt am Main, Germany 6Polikum Gesundheitszentren, Berlin, Germany

*Corresponding author e-mail: [email protected]

Background: Combination of several direct-acting anti- patients, clonal sequencing was performed at baseline viral agents will be necessary to overcome viral resist- and after 2 weeks of amantadine/placebo monotherapy. ance in interferon-free treatment regimens for chronic Results: Changes of the relative frequency of amino acid HCV infection. HCV p7 inhibitors may be part of such substitutions by ≥20% between pretreatment and week combination regimens. Understanding why amantadine, 2 of monotherapy were considered potential resistance despite showing inhibition of HCV p7 in vitro, appears mutations if they were only found in patients receiving ineffective in clinical trials, may help in the design of amantadine but not in patients receiving placebo. Seven novel HCV p7 inhibitors. So far it is unknown whether substitutions fulfilling these criteria were identified in viral escape mutations within HCV p7 explain the inef- the subset of patients with clonal sequencing. However, fectiveness of amantadine in vivo. none of these substitutions were associated with treat- Methods: Pretreatment HCV p7 was directly sequenced in ment outcome in the complete cohort of patients receiv- 157 consecutive patients with chronic HCV genotype 1b ing triple therapy with amantadine. infection who had been treated with amantadine/placebo Conclusions: Potential viral escape mutations within HCV plus pegylated interferon (PEG-IFN)-α2a/ribavirin within p7 do not seem to play a major role for treatment response a multicentre clinical trial. Triple therapy was preceded to antiviral therapy with amantadine and PEG-IFN-α2a/rib- by 2 weeks of amantadine/placebo monotherapy. In nine avirin in patients with chronic HCV genotype 1b infection.

Introduction

Chronic HCV infection is a major cause for the direct-acting antiviral agents should target different development of liver cirrhosis and hepatocellular steps in the HCV lifecycle. HCV p7 inhibitors may carcinoma [1–3]. For the treatment of patients with be part of such future combination regimens [13,14]. chronic HCV genotype 1 infection the combination of HCV p7 has been shown to be essential for HCV infec- protease inhibitors with pegylated interferon (PEG- tivity in chimpanzees and for viral assembly and secretion IFN)-a and ribavirin has been licensed in Europe and in cell culture [15–21]. In vitro studies have shown that the US, and interferon-free treatment regimens seem HCV p7 homo-oligomerizes, like the functionally related feasible in the near future [4]. However, in interferon- influenza A virus M2, into a circular hexamer to form an free treatment regimens, combination of several direct- ion channel [15,22–26]. HCV p7 and influenza M2 ion acting antiviral agents will be necessary to overcome channel activity is abrogated by a number of compounds the problem of viral resistance [5–12]. Ideally, these including amantadine in vitro [13,14,22,24–27].

Although in vitro data provide a plausible mechanism obtained from each patient, and the study had been for an anti-HCV activity of amantadine, triple therapy approved by the local ethics committees of the par- combining amantadine with PEG-IFN-a and ribavirin ticipating study centres in accordance with the Decla- does not lead to higher rates of sustained virological ration of Helsinki. In that study, previously untreated response (SVR) than dual combination therapy without patients received amantadine sulfate 400 mg/day orally amantadine in treatment-naive patients [28–30]. or placebo in combination with PEG-IFN-a2a 180 mg Results of viral kinetic studies with a short initial once per week subcutaneously plus ribavirin 1,000 to drop and then rebound of HCV RNA early during 1,200 mg/day orally according to body weight. Before amantadine therapy suggest a rapid selection of viral the onset of combination therapy with PEG-IFN-a2a escape mutations [31,32]. This hypothesis is supported and ribavirin, amantadine/placebo was dose escalated by the knowledge of amantadine-resistant mutations in within 2 weeks in 100 mg steps weekly starting at 200 the functionally related influenza M2 protein [33,34]. mg/day. Patients without early virological response (<2

Furthermore, varying sensitivities towards amantadine log10 drop of HCV RNA at week 12 of combination depending on the HCV genotype and even subtype therapy) were considered as having a non-response have been reported suggesting that already minor vari- (NR) and treatment was discontinued. Patients with ations in the HCV amino acid (aa) sequence may alter early virological response continued antiviral combi- the sensitivity towards amantadine [13]. Recently, Fos- nation therapy for a full course of 48 weeks. SVR was ter et al. [35] and StGelais et al. [36] described aa sub- defined as negative HCV RNA at the end of treatment stitutions in HCV p7 conferring a lower sensitivity to and 24 weeks thereafter. A negative HCV RNA at the amantadine in vitro: L(50–55)A and L20F. In a clinical end of treatment that becomes detectable thereafter was study, L20F seemed to be associated with non-response classified as a relapse (REL). (NR) towards combination therapy with amantadine in Overall, 78 out of the 157 patients had been treated patients with HCV genotype 1b infection, but the effect within the amantadine group (NR n=9, SVR n=50 and was not statistically significant [37]. REL n=19), whereas 79 had received placebo (NR n=7, So far it is unknown whether pre-existing or emerg- SVR n=52 and REL n=20). In the present study, only ing viral escape mutations within HCV p7 are respon- patients infected with HCV subtype 1b were included sible for the ineffectiveness of amantadine in clinical because in a previous study no association of p7 muta- trials. Understanding the mechanisms of amantadine tions with response to amantadine therapy was observed resistance could help in the design of new and more in patients infected with HCV subtype 1a [37]. potent HCV p7 inhibitors. In the present study, pretreatment HCV p7 was Amplification of HCV p7 by RT-PCR and sequencing sequenced in 157 patients with chronic HCV genotype In all 157 patients, HCV p7 was sequenced in baseline 1b infection. Samples were derived from a clinical trial (pretreatment) samples. In addition, in nine patients comparing treatment with PEG-IFN-a2a/ribavirin plus (amantadine group: SVR n=3, REL n=2 and NR n=1; amantadine 400 mg/day with PEG-IFN-a2a/ribavirin placebo group: SVR n=2 and REL n=1) of the cohort, plus placebo. Before the onset of combination therapy cloning and sequencing of HCV p7 was performed at with PEG-IFN-a2a/ribavirin, amantadine/placebo was baseline and after the initial 2 weeks of monotherapy dose escalated within 2 weeks to reach the final 400 mg with amantadine/placebo. daily dose [38]. After extraction of HCV RNA from 100 ml of serum, In order to identify potential viral escape mutations, complementary DNA was generated by using appropri- cloning and sequencing of HCV p7 was performed ate specific antisense primers. before treatment and after the initial 2 weeks of aman- HCV p7 (nucleotide position 2568 to 2756 accord- tadine/placebo monotherapy in a subset of patients. To ing to HCV-J [39]) pretreatment sequences were ampli- explore the role of these identified mutations for viral fied by nested-PCR as follows: one cycle of initial escape to amantadine, their association with treatment denaturation (2 min), 45 cycles of denaturation (30 s)/ failure was investigated in all patients of the present annealing (60 s)/elongation (2 min) and one cycle of study receiving triple therapy with amantadine. final elongation (10 min). Denaturation was performed at 95°C, annealing at 53°C (first round PCR) or 59°C Methods (second round PCR) and elongation at 72°C. The first round of PCR was performed using external sense Patients (5′-GTGGACGTGCAATACCTGTACGG-3′; within In this study, HCV p7 was sequenced in 157 consecu- nucleotide positions 2418–2440 according to HCV-J tive patients with chronic HCV genotype 1b infec- [39]) and antisense primers (5′-ATTGTAACCACCAT- tion. Patients had been treated within the multicentre ATGAGCCTAG-3′; within nucleotide positions 2871– PRAMA study [38]. Written informed consent had been 2848 according to HCV-J [39]). In the second round of

the nested PCR, the internal sense (5′-TGCCTGCTT- Changes of the relative aa frequency by ≥20% between GTGGATGATGCTGC-3′; within nucleotide positions pretreatment and week 2 were considered potential 2525–2547 according to HCV-J [39]) and antisense resistance mutations if they were found only in patients primers (5′-AACCGCGCCTCCGCACGATGC-3′; receiving amantadine and were not present in patients within nucleotide positions 2795–2775 according to receiving placebo. HCV-J [39]) were used. For direct sequencing of HCV p7, 40 ml of the second-round PCR product was purified Statistical analyses with QIAquick PCR Purification Kit (Qiagen, Hilden, Data were analysed by Mann–Whitney U, Kruskal– Germany). PCR products were labelled for sequencing Wallis, Fisher–Freeman–Halton’s exact test and (BigDyeTM Terminator Cycle Sequencing Ready Reac- Wilcoxon–Rank tests, as appropriate. All tests were tion Kit; Applied Biosystems, Darmstadt, Germany) and two-tailed and P-values of <0.05 were considered sig- purified (DyEx 2.0 Spin Kit; Qiagen) according to the nificant. The numbers of mutations are given as mean manufacturer’s instructions. Sequencing of the positive- and standard error of mean. UCSF Chimera v1.5 was strand and negative-strand was performed on an ABI used for 3D structure analysis and visualization. PRISM® 3100 Genetic Analyzer (Applied Biosystems). In nine patients, cloning and sequencing of HCV p7 Results was performed at baseline and after the initial 2 weeks of monotherapy with amantadine/placebo according to the The pretreatment HCV p7 aa sequence was determined following protocol: for cloning, PCR products were gel- in 78 patients (SVR n=50, REL n=19 and NR n=9) who purified using the QIA-Quick Gel Extraction Kit (Qia- had been treated within the amantadine group (Fig- gen), ligated into the pCR-XL-TOPO vector (TOPO TA ure 1) and in 79 patients of the placebo group (data Cloning® Kit for Sequencing; Invitrogen, Carlsbad, CA, not shown). USA), and transformed into Escherichia coli competent In a subset of patients (amantadine group n=6 and cells. A mean number of 17 colonies (range 6–28) from placebo group n=3) cloning and sequencing with a patient samples, grown overnight at 37°C were picked. mean number of 17 clones (range 6–28) per patient was After purification with the High Pure Plasmid Isolation performed in pretreatment samples and after 2 weeks Kit (Roche Diagnostics, Mannheim Germany) molecular of amantadine or placebo monotherapy (Figure 2 and clones were subjected to sequence PCR according to the Additional file 1). Cloning and sequencing resulted in manufacturer’s instructions using the M13 forward and 96 sequences before treatment and in 101 sequences reverse primers (Big Dye Deoxy Terminators; Applied after 2 weeks of amantadine in the six patients (three Biosystems). Sequencing was performed using the 3130 patients with treatment failure, that is, one patient with xl Genetic Analyzer (Applied Biosystems). NR plus two patients with REL, and three patients with SVR) receiving amantadine monotherapy prior to anti- Sequence alignment and analysis viral combination therapy. In the three patients receiv- Phylogenetic analyses of the aa sequences were con- ing placebo before antiviral combination therapy, 66 ducted using Treecon software 1.3b [40]. Multiple sequences before treatment and 50 sequences after 2 alignments of the aa sequences and calculation of the weeks of placebo were generated. consensus sequences from all pretreatment sequences Shannon entropy and Hamming distance did not dif- were performed as previously described [41]. The fer between pretreatment samples and samples taken deduced pretreatment aa sequences of the HCV p7 after 2 weeks of amantadine monotherapy (median region were compared with the consensus sequences [range] Shannon entropy baseline versus week 2, 0.2978 of all pretreatment HCV-1b (con1b) isolates for assess- [0.0218–0.7570] versus 0.2000 [0.0638–0.5122]; ment of aa variations. P=0.438; median [range] Hamming distance baseline In nine patients, the quasispecies heterogeneity of versus week 2, 0.0071 [0.0020–0.0453] versus 0.0057 baseline samples was compared to the quasispecies [0.0019–0.0241]; P=0.438). heterogeneity after 2 weeks of amantadine/placebo For the detection of potential resistance mutations the monotherapy. For determination of genetic complexity relative frequencies of aa changes (towards the consensus and genetic diversity within HCV p7 the normalized sequence) in all 63 positions of HCV p7 were compared Shannon entropy and the Hamming distance were cal- between pretreatment and week 2 sequences for each of culated, respectively, as previously described [42]. For the nine patients with clonal sequencing, separately. There the detection of potential resistance mutations the rela- were aa substitutions with a change of their relative fre- tive frequencies of aa mutations (towards the consensus quency by >20% found in amantadine-treated patients sequence) in all 63 positions of HCV p7 were compared at the following positions of HCV p7: 16, 28, 34, 35, 40 between pretreatment and week 2 aa sequences for each and 42. In detail, the frequency of A16S increased from of the nine patients with clonal sequencing, separately. 0% to 28.57% during monotherapy with amantadine in

Antiviral Therapy 18.6 805 A Vermehren et al.

Figure 1. Sequence alignment of the amino acids 1–63 of HCV p7 in pretreatment isolates of patients treated with amantadine plus pegylated interferon-a2a/ribavirin

Amino acid (aa) residues are indicated by standard single-letter codes. Dashes indicate residues identical to the consensus sequence (con1b) calculated from all pretreatment isolates investigated. The aa positions harbouring potential viral escape mutations are highlighted by boxes shaded in light grey (aa positions 16, 28, 34, 35, 40 and 42) and dark grey (aa positions 20 and 50–55, which were shown to confer reduced amantadine sensitivity in vitro). NR, virological non-response; REL, virological relapse; SVR, sustained virological response.

Figure 2. Detailed description of amino acid changes between baseline and week 2 of amantadine monotherapy in three patients

B NN-term- helix TM1 helix TM2 helix Turn Basic loop C Membrane interface

Membrane TM2 hydrophobic helix core

Y42 A40 Membrane interface Basic R35 loop

(A) Sequence alignment of amino acids (aa) 1–63 of HCV p7. The aa residues are indicated by standard single-letter codes. Dashes indicate residues identical to the consensus sequence (Cons) calculated from all investigated isolates. A ‘0’ at the end of the sequence name denotes a pretreatment sequence and ‘w2’ indicates a sequence after 2 weeks of amantadine monotherapy. Exemplary sequences with the respective aa substitutions (bold) are shown. (Additional file 1 displays aa sequence alignments of all patients with clonal sequencing before and after 2 weeks of amantadine/placebo monotherapy). (B) Schematic representation of helical, turn and loop regions [43]. (C) Side view of the HCV p7 basic loop and transmembrane (TM)2 helix from the Protein Databank RCSB PDB structure 2K8J [48]. The 3D structure is shown as a ribbon model with aa positions that harbour potential escape mutations given as ball-and-stick models. Residue labelling is according to the p7 sequence alignment above.

patient 148bSVR3. In patient 140bSVR3, the frequency is, A40; Y42) after monotherapy with amantadine. In of A16V increased from 47.83% to 82.61% whereas the patient 144bREL3 the frequency of R35K increased from frequency of A40V and Y42H decreased from 43.48% 10.53% to 33.33%, whereas the frequency of A28K and and 30.43%, respectively, to 0% during monotherapy G34S decreased from 47.37% and 21.05%, respectively, with amantadine in the same patient. In both positions, to 0% during monotherapy with amantadine in that the aa substitutions in the baseline HCV p7 sequences patient. In both positions, the mutations in the baseline were replaced by the respective wild-type aa (as deter- sequences were replaced by the respective wild-type aa mined in the consensus sequence for all patients, that (A28; G34) after monotherapy with amantadine.

Antiviral Therapy 18.6 807 A Vermehren et al.

Interestingly, the naturally occurring variant A16T essential for p7 function [16,19,20,25]. A40V and was significantly more often observed in both pretreat- Y42H were also completely replaced by the wild-type ment and week 2 sequences of amantadine‑treated aa during amantadine monotherapy. While A40V is patients with treatment failure compared to patients a conservative aa substitution, histidine is a positive with SVR (pretreatment REL and NR versus SVR, charged residue containing an imidazole side chain 43/43 versus 0/53; week 2 REL and NR versus SVR, that is replaced by the aliphatic tyrosine containing a 48/48 versus 1/53; both P-values <0.001). phenol side chain. The location of the above-described residue The in vitro characterized viral escape mutations changes within the protein structure of p7 is depicted L(50–55)A were not present in any of the samples of the in Figure 2. The aa position 16 lies in the flexible present study. The L20F mutant was observed in only turn (15–18) linking the rigid N-terminal helix and two of our patients in whom clonal sequencing was per- the transmembrane helix 1 (TM1). While A16V rep- formed and the relative frequency increased only slightly resents a conservative aa change with both aa being and by <20% during amantadine monotherapy (from small, aliphatic and hydrophobic, A16S and A16T 0% [0/14] and 96% [22/23] before treatment to 7% are both non-conservative aa changes. In both sub- [1/14] and 100% [23/23] after 2 weeks of treatment). stitutions, the small, aliphatic, hydrophobic alanine is In order to further characterize the potential role of replaced by a polar uncharged residue containing a the above-described mutations for viral escape to an hydroxyl group in its side chain. The aa position 28 antiviral combination therapy with amantadine, their is situated within TM1 and has been suggested to be association with treatment response was investigated in one of the putative pore lining residues [43]. The pre- the complete cohort of patients receiving antiviral com- treatment substitution A28K was eliminated during bination therapy with amantadine (n=78). None of the monotherapy with amantadine and replaced by the pretreatment substitutions that were eliminated during wild-type aa. The large residue lysine with a positive amantadine monotherapy were associated with SVR. + charged side chain and e-amino group (NH3 ) was Neither was the wild-type in those cases associated with thereby replaced by the small, aliphatic and hydro- treatment failure. Moreover, none of the substitutions phobic alanine. The pretreatment substitution G34S with increased frequency during amantadine monother- was also eliminated during monotherapy with aman- apy was associated with treatment failure (Table 1). tadine and replaced by the wild-type aa. Thereby, the Finally, alignment of the aa sequences and calcula- larger polar uncharged serine containing a hydroxyl tion of the consensus sequence from all pretreatment group in its side chain was substituted by the smallest sequences was performed (Figure 1). The deduced aa glycine with hydrogen substituents as side chain. pretreatment aa sequences of the HCV p7 region G34S is located in the cytosolic loop, as is the con- were compared with the consensus sequences of all servative substitution R35K in position 35, which is pretreatment HCV-1b (con1b) isolates for assessment part of the dibasic motif that has been shown to be of aa variations (Figure 1). The total numbers of aa

Table 1. Association of viral escape mutations within HCV p7 with treatment response

P-value for SVR Substitution SVR (n=50) REL (n=19) NR (n=9) REL and NR (n=28) versus REL and NR

A16S 6.00 (3/50) 0.00 (0/19) 0.00 (0/9) 0.00 (0/28) 0.549 A16V 2.00 (1/50) 5.26 (1/19) 11.11 (1/9) 7.14 (2/28) 0.291 A16T 26.00 (13/50) 31.58 (6/19) 22.22 (2/9) 28.57 (8/28) 0.797 non-A16 34.00 (17/50) 36.84 (7/19) 30.00 (3/9) 35.71 (10/28) 1.000 A28K 0.00 (0/50) 0.00 (0/19) 0.00 (0/9) 0.00 (0/28) NA A28 98.00 (49/50) 100.0 (19/19) 100.00 (9/9) 100.0 (28/28) 1.000 G34S 0.00 (0/50) 0.00 (0/19) 0.00 (0/9) 0.00 (0/28) NA G34 100.0 (50/50) 100.0 (19/19) 100.00 (9/9) 100.0 (28/28) NA R35K 18.00 (9/50) 21.05 (4/19) 0.00 (0/9) 14.29 (4/28) 0.761 A40V 10.00 (5/50) 15.79 (3/19) 0.00 (0/9) 10.71 (3/28) 1.000 A40 90.00 (45/50) 84.21 (16/19) 100 (9/9) 89.29 (25/28) 1.000 Y42H 0.00 (0/50) 0.00 (0/19) 0.00 (0/9) 0.00 (0/28) NA Y42 100.0 (50/50) 100.0 (19/19) 100.00 (9/9) 100.0 (28/28) NA L20F 10.00 (5/50) 10.53 (2/19) 11.11 (1/9) 10.71 (3/28) 1.000 L(50–55)A 0.00 (0/50) 0.00 (0/19) 0.00 (0/9) 0.00 (0/28) NA

Data are percentage (n/total n). NA, not applicable; NR, virological non-response; REL, virological relapse; SVR, sustained virological response.

substitutions within HCV p7 did not differ between with variable sensitivities towards amantadine [13]. patients with different treatment outcomes to triple Moreover, the Y42H mutant was not detected in a sin- therapy (mean ±sem NR 1.89 ±0.31, REL 2.68 ±0.38 gle directly sequenced pretreatment sample from our and SVR 2.54 ±0.19; P=0.346). total cohort of 78 patients treated with amantadine. Therefore, replacement of the Y42H mutant by wild- Discussion type during amantadine monotherapy in one patient with clonal sequencing may seem to reflect elimina- In the present study Shannon entropy and Hamming tion of a less fit viral variant rather than replacement distance before treatment and after 2 weeks of mono- of a variant with ‘hypersensitivity’ to amantadine by therapy with amantadine did not differ between the two a potentially less sensitive wild-type variant. The same time points. The 2-week treatment duration may be too logic may also apply for wild-type replacements of short to reliably observe a selective pressure of amanta- A28K and G34S mutants during amantadine mono- dine on HCV p7 quasispecies. Thus, it is a limitation of therapy in another patient in whom clonal sequencing the present study that HCV RNA sampling was not per- was performed because here again these substitutions formed during the 2 weeks of amantadine monotherapy were not observed in any of the 78 patients treated with and hence no information with regard to viral kinetics amantadine and A28 and G34 also show a high con- during this time was available. However, Chan et al. [31] servation across different HCV genotypes with variable reported a transient viral decline by day 3 of amantadine sensitivities towards amantadine [13]. monotherapy with a viral rebound by as early as day In our study, the A16S substitution seems to be the 7, suggesting an early occurrence of resistance. Further- most interesting mutation that increased in frequency more, the lack of selective pressure on HCV genotype 1b during amantadine monotherapy. Position 16 lies quasispecies after 2 weeks of amantadine monotherapy within the flexible turn that links the rigid N-terminal in the present investigation is in-line with a previous pub- and TM1 helices [43]. It has been suggested that this lication by Castelain et al. [44], who did not observe a flexible turn may be involved in some rigid body move- reduction of the genetic diversity in patients with HCV ments of a-helices occurring for example during open- subtype 1b infection after 12 weeks of triple therapy with ing or closing of the HCV p7 ion channel pore [43]. It is amantadine and standard interferon-a2a/ribavirin. These well-conceivable that the substitution of the small, ali- observations speak against a strong selective pressure of phatic and hydrophobic alanine in this position to the amantadine in HCV genotype 1b infection. Despite this, polar, uncharged serine containing a hydroxyl group in we did observe aa substitutions with a change of their its side chain has a functional impact. However, none relative frequency by >20% between baseline and week of the above-described mutations that either increased 2 of amantadine monotherapy at several positions of or decreased during amantadine monotherapy were HCV p7, including 16, 28, 34, 35, 40 and 42. It cannot associated with treatment response or non-response. In be excluded that with a higher number of clones per sam- fact, they were quite evenly distributed among patients ple, additional potential viral escape mutations that occur with SVR and patients with treatment failure. Differ- only at low frequencies would have been detected [45]. ent explanations for this observation are conceivable: However, given the ineffectiveness of amantadine in large first, the described aa variations within HCV p7 may clinical trials, the clinical impact of such low-frequency not confer resistance to amantadine in vivo and may mutations is debatable. therefore not be linked to treatment failure. Therefore, Because non-conservative aa substitutions are more it could be assumed that HCV p7 sensitivity (or the likely to be of functional relevance than conservative absence thereof) to compounds such as amantadine are aa changes, it is interesting to know that the increase not sequence-specific, neither on the genotype/subtype of substitution A16S and the replacement of substitu- level nor due to variations within the HCV quasispe- tions A28K, G34S and Y42H by wild-type aa during cies. Second, based on our study we cannot exclude a amantadine monotherapy represent non-conservative relatively weak amantadine activity against HCV due to aa changes. In addition, A16T, which was present other mechanisms than escape mutations within HCV significantly more often in pretreatment and week 2 p7. Thus, patients may still respond to the combination sequences of patients with treatment failure compared of all three drugs although amantadine resistance may to patients with SVR, is also a non-conservative aa sub- have arisen or PEG-IFN-a treatment failure may still stitution. However, A16T is also present in the HCV occur in the absence of amantadine resistance. How- genotype 1a strain ‘H77’ and the HCV genotype 3a ever, both interpretations would result in the same con- strain ‘425’, which have both been proven to be fully clusion, namely, that potential viral escape mutations sensitive to amantadine in vitro [13]. Y42 has been within HCV p7 are not likely to play a major role for shown to be involved in the functioning of p7 [20] and treatment response in amantadine-based combination is highly conserved across different HCV genotypes therapies with PEG-IFN-a and ribavirin. Importantly,

Antiviral Therapy 18.6 809 A Vermehren et al.

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Kieffer TL, Sarrazin C, Miller JS, et al. Telaprevir and nar lipid bilayers versus liposome dye release) or HCV pegylated interferon-alpha-2a inhibit wild-type and resistant genotype 1 hepatitis C virus replication in patients. infectious culture systems that have shown conflicting Hepatology 2007; 46:631–639. results in the past [13,35,36,46,47]. While whole life 9. Reesink HW, Zeuzem S, Weegink C, et al. Rapid decline cycle systems are likely to represent the best approach of viral RNA in hepatitis C patients treated with VX-950: a Phase Ib, placebo-controlled, randomized study. to study what is going on in vivo, they do not allow Gastroenterology 2006; 131:997–1002. for the screening of potential HCV p7 inhibitors at high 10. Rodriguez-Torres M, Lawitz EJ, McHutchison J. Final throughput levels [47]. results of patients treated with peg-interferon-alfa-2A (PEG- In conclusion, our data do not support the hypoth- IFN) and ribavirin (RBV) follow-on therapy after 28-day treatment with the hepatitis C protease inhibitor telaprevir esis that pre-existing aa variations in HCV p7 or strong (VX-950), PEG-IFN and RBV. Hepatology 2007; 46 Suppl selective pressure on HCV p7 quasispecies with emer- 1:314A–315A. gence of viral escape mutations account for the observed 11. Sarrazin C, Kieffer TL, Bartels D, et al. Dynamic hepatitis C virus genotypic and phenotypic changes in patients treated lack of amantadine efficacy when given in combination with the protease inhibitor telaprevir. Gastroenterology with PEG-IFN-a2a/ribavirin in patients infected with 2007; 132:1767–1777. HCV genotype 1b. However, amantadine may still act 12. Sarrazin C, Rouzier R, Wagner F, et al. SCH 503034, a novel hepatitis C virus protease inhibitor, plus pegylated as a valuable compound to study the role of p7 function interferon alpha-2b for genotype 1 nonresponders. within the HCV life cycle. Gastroenterology 2007; 132:1270–1278. 13. Griffin S, Stgelais C, Owsianka AM, Patel AH, Rowlands D, Harris M. Genotype-dependent sensitivity of hepatitis C Acknowledgements virus to inhibitors of the p7 ion channel. Hepatology 2008; 48:1779–1790. The authors wish to thank Ursula Karey for skilled 14. Luscombe CA, Huang Z, Murray MG, Miller M, technical assistance. Wilkinson J, Ewart GD. A novel hepatitis C virus p7 ion channel inhibitor, BIT225, inhibits bovine viral diarrhea This study was supported by a Deutsche Forschungsge- virus in vitro and shows synergism with recombinant meinschaft (DFG) grant to UM, EH, CW, WPH, BK, SZ interferon-alpha-2b and nucleoside analogues. Antiviral Res 2010; 86:144–153. and CS (Klinische Forschergruppe, KFO 129, TP1, TP2, 15. 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Accepted 4 April 2013; published online 3 July 2013

Antiviral Therapy 18.6 811