Volume 140, Issue 7, Pages 2064-2073 (June 2011) CD8+ T-Cell Response Promotes Evolution of Hepatitis C Virus Nonstructural Proteins  Marianne Ruhl, Torben Knuschke, Kevin Schewior, Lejla Glavinic, Christoph Neumann-Haefelin, Dae-In Chang, Marina Klein, Falko M. Heinemann, Hannelore Tenckhoff, Manfred Wiese, Peter A. Horn, Sergei Viazov, Ulrich Spengler, Michael Roggendorf, Norbert Scherbaum, Jacob Nattermann, Daniel Hoffmann, Jörg Timm  Gastroenterology  Volume 140, Issue 7, Pages 2064-2073 (June 2011) DOI: 10.1053/j.gastro.2011.02.060 Copyright © 2011 AGA Institute Terms and Conditions

Figure 1 (A) Maximum likelihood tree of hepatitis C virus (HCV) sequences (NS3–NS5B codon 2877 as aligned to NC_004102) from subjects of the East-German anti-D cohort and randomly chosen sequences from the HCV database.20 Sequences from the anti-D cohort fall into 3 distinct clades. The bootstrap values of the corresponding nodes are indicated. (B) Maximum likelihood tree of partial HCV NS3 sequences (NS3 codon 1027–1364) and clonal sequences from the infection source (immunoglobulin batches 8 and 12; colored in orange). Gastroenterology 2011 140, 2064-2073DOI: (10.1053/j.gastro.2011.02.060) Copyright © 2011 AGA Institute Terms and Conditions

Figure 2 Mutation rate inside and outside previously described CD8 epitopes. The frequency of (A) synonymous mutations and (B) nonsynonymous mutations per codon across the complete analyzed region (NS3–NS5B codon 2877 as aligned to NC_004102). (C–F) Frequency of mutations per codon in individual proteins (C) NS3/4A, (D) NS4B, (E) NS5A, and (F) NS5B. The corresponding P values for the comparison of mutation rates inside and outside previously described CD8 epitopes are shown. Gastroenterology 2011 140, 2064-2073DOI: (10.1053/j.gastro.2011.02.060) Copyright © 2011 AGA Institute Terms and Conditions

Figure 3 Identification of novel CD8 epitopes. (A) Residues potentially under selection pressure in the presence of a particular HLA class I allele (here HLA-B15) were identified. The graph indicates the P value for each amino acid position across the analyzed region. An arbitrarily chosen value of P < .01 was used as a cut-off for further analysis. A total of 10 position fulfilled these criteria (large dots), 4 of which were located in a short region located in NS5B (boxed area). (B) Detailed alignment of a short region in NS5B. The residues with a P < .01 are indicated with an arrow. Two predicted HLA-B15 motifs are present in this region (highlighted in gray). (C) The presence of CD8 epitopes was experimentally confirmed by antigen-specific expansion from peripheral blood mononuclear cells obtained from HLA-B15–positive patients and intracellular cytokine staining after 5 hours of restimulation with the same peptide. (D) Results of intracellular cytokine stainings for 4 additional novel CD8 epitopes. Gastroenterology 2011 140, 2064-2073DOI: (10.1053/j.gastro.2011.02.060) Copyright © 2011 AGA Institute Terms and Conditions

Figure 4 Frequency of previously described CD8 epitopes with statistical support for selection pressure. CD8 epitopes restricted by A alleles are compared to CD8 epitopes restricted by B alleles (Fisher's exact test). Novel CD8 epitopes identified in this study are indicated in gray. Gastroenterology 2011 140, 2064-2073DOI: (10.1053/j.gastro.2011.02.060) Copyright © 2011 AGA Institute Terms and Conditions

Supplementary Figure 1 Gastroenterology 2011 140, 2064-2073DOI: (10.1053/j.gastro.2011.02.060) Copyright © 2011 AGA Institute Terms and Conditions