Similar Prevalence of Low-Abundance Drug-Resistant Variants in Treatment-Naive Patients with Genotype 1a and 1b Hepatitis C Virus Infections as Determined by Ultradeep Pyrosequencing

<div><p>Background and Objectives</p><p>Hepatitis C virus (HCV) variants that confer resistance to direct-acting-antiviral agents (DAA) have been detected by standard sequencing technology in genotype (G) 1 viruses from DAA-naive patients. It has recently been shown that virological response rates are higher and breakthrough rates are lower in G1b infected patients than in G1a infected patients treated with certain classes of HCV DAAs. It is not known whether this corresponds to a difference in the composition of G1a and G1b HCV quasispecies in regards to the proportion of naturally occurring DAA-resistant variants before treatment.</p><p>Methods</p><p>We used ultradeep pyrosequencing to determine the prevalence of low-abundance (<25% of the sequence reads) DAA-resistant variants in 191 NS3 and 116 NS5B isolates from 208 DAA-naive G1-infected patients.</p><p>Results</p><p>A total of 3.5 million high-quality reads of ≥200 nucleotides were generated. The median coverage depth was 4150x and 4470x per NS3 and NS5B amplicon, respectively. Both G1a and G1b populations showed Shannon entropy distributions, with no difference between G1a and G1b in NS3 or NS5B region at the nucleotide level. A higher number of substitutions that confer resistance to protease inhibitors were observed in G1a isolates (mainly at amino acid 80 of the NS3 region). The prevalence of amino acid substitutions that confer resistance to NS5B non-nucleoside inhibitors was similar in G1a and G1b isolates. The NS5B S282T variant, which confers resistance to the polymerase inhibitors mericitabine and sofosbuvir, was not detected in any sample.</p><p>Conclusion</p><p>The quasispecies genetic diversity and prevalence of DAA-resistant variants was similar in G1a and G1b isolates and in both NS3 and NS5B regions, suggesting that this is not a determinant for the higher level of DAA resistance observed across G1a HCV infected patients upon treatment.</p></div

Low-abundance NS3 PI-resistant amino acid substitutions found in 20/136 G1a and in 5/55 G1b treatment-naïve HCV samples.

1<p>In parenthesis, abundance of the variant is indicated (in % and number of reads in which the variant is detected over the number of total reads).</p

List of known <i>in</i><i>vitro</i> and <i>in</i><i>vivo</i> amino acid substitutions resistant to NS5B polymerase inhibitors used in this study (NS5B residues 244–496).

<p>*<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0105569#pone.0105569-Lam1" target="_blank">[6]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0105569#pone.0105569-Tong1" target="_blank">[9]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0105569#pone.0105569-Thompson1" target="_blank">[11]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0105569#pone.0105569-Troke1" target="_blank">[15]</a>–<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0105569#pone.0105569-Lawitz1" target="_blank">[18]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0105569#pone.0105569-Lam2" target="_blank">[54]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0105569#pone.0105569-Shih1" target="_blank">[55]</a>.</p><p>**L320F when in combination with L159F confers low level resistance to MCB <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0105569#pone.0105569-Tong1" target="_blank">[9]</a>, L320I or V321I when in combination with C223H confers low level resistance to GS-938 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0105569#pone.0105569-Lam2" target="_blank">[54]</a>.</p

Contribution of each mutational category (transition and transversion) to the development of drug-resistant substitutions (variants) in the NS3 and NS5B proteins reported in this study.

<p>The genetic barrier score (GBS) was calculated according to the model described by Van de Vijver et al.(50) A score of 1 is assigned to transitions (A↔G and C↔T) and 2.5 to transversions (A↔C, A↔T, G↔C and G↔T). Natural polymorphisms are indicated in italic. Numbers of isolates are indicated in parenthesis.</p

Low-abundance NS5B drug-resistant variants found in 30/77 G1a and in 12/39 G1b treatment-naïve HCV samples.

1<p>In parenthesis, abundance of the variant is indicated (in % and number of reads in which the mutation is detected over the number of total reads).</p

Amino acid diversity in the NS3 protease region of 136 G1a and 55 G1b isolates from treatment-naïve HCV infected subjects.

<p>Superscript numbers represent the number of isolates with the particular noted residue. Amino acid representing the wild type is indicated in bold. Residues known for resistance substitutions are underlined.</p

List of known <i>in</i><i>vitro</i> and <i>in</i><i>vivo</i> amino acid substitutions resistant to NS3 protease inhibitors used in this study (NS3 residues 31 to 175).

<p>*For conciseness, 2 representatives from each PI class were chosen <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0105569#pone.0105569-DeMeyer1" target="_blank">[1]</a>–<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0105569#pone.0105569-Jacobson1" target="_blank">[3]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0105569#pone.0105569-Lenz1" target="_blank">[35]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0105569#pone.0105569-Lenz2" target="_blank">[37]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0105569#pone.0105569-Lin1" target="_blank">[50]</a>–<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0105569#pone.0105569-LePogam4" target="_blank">[53]</a>.</p

Amino acid diversity in the NS5B polymerase region of 77 G1a and 39 G1b isolates from treatment-naïve HCV infected subjects.

<p>Numbers in superscript represent the number of isolates with a particular noted residue. Amino acid representing the wild type is indicated in bold. Residues known for resistance substitutions are underlined. Note: For residue 426, the M426L has not been described as a mutation conferring resistance (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0105569#pone-0105569-t002" target="_blank">Table 2</a>).</p

Patient disposition in DYNAMO 1.

<p>BOC, boceprevir; MCB, mericitabine; P/R, peginterferon alfa-2a + ribavirin.</p

SVR12 rates by treatment arm in the overall populations and by HCV genotype and presence/absence of bridging fibrosis or cirrhosis in DYNAMO 1 (a) and DYNAMO 2 (b).

<p>BOC, boceprevir; MCB, mericitabine; P/R, peginterferon alfa-2a + ribavirin; TVR, telaprevir.</p