Volume 132, Issue 2, Pages 667-678 (February 2007) Hepatitis C Virus Continuously Escapes From Neutralizing Antibody and T-Cell Responses During Chronic Infection In Vivo  Thomas von Hahn, Joo Chun Yoon, Harvey Alter, Charles M. Rice, Barbara Rehermann, Peter Balfe, Jane A. McKeating  Gastroenterology  Volume 132, Issue 2, Pages 667-678 (February 2007) DOI: 10.1053/j.gastro.2006.12.008 Copyright © 2007 AGA Institute Terms and Conditions

Figure 1 Evolving anti-HCV E1E2 response in patient H over time. (A) Lines show serum ALT (- - -) and viral load (◊ and grey line) over 26 years of chronic infection. Bars represent the serum dilution capable of neutralizing HCVpp-H77 (□) or HCVpp -J6 (■) infectivity by 90% (id90). Arrows indicate samples from which E1E2 sequences were amplified and included in the phylogenetic analysis. (B) The reactivity of sequential serum samples with antigens representing HCV E1E2 sequences by EIA. Antigens tested were as follows: ◊, H77 HVR peptide; ⧫, H77 E1E2; Δ, J6 E1E2; ▲, H77 sE2. (C) HCVpp H77 infection of Hep3B cells in the presence of neutralizing E2 antibodies 9/27 and 11/20 (at 1 μg/mL) or H serum (week 9 at 1:300; week 244 at 1:6000, and week 759 at 1:10,000 dilution) with or without H77 sE2 (5 μmol/L) or HVR peptide (5 μmol/L; mean of n = 3 ± SD). Mock, mock infected cells; no env, cells infected with an envelope-deficient pseudoparticle; rlu, relative light units. Gastroenterology 2007 132, 667-678DOI: (10.1053/j.gastro.2006.12.008) Copyright © 2007 AGA Institute Terms and Conditions

Figure 2 Generation of HCVpp-bearing glycoprotein clones from H serum samples. (A) The phylogenetic tree displays the relationships between E1E2 sequences amplified from H serum samples. The 2 digits after H define the year of the serum sample from which the clone was obtained. *Clones that gave rise to infectious HCVpp. (B) Infectivity of functional HCVpp generated using sequences amplified from H serum samples. This constitutes the set of HCVpp that was used for subsequent analyses (mean of 3 experiments ± SD). Gastroenterology 2007 132, 667-678DOI: (10.1053/j.gastro.2006.12.008) Copyright © 2007 AGA Institute Terms and Conditions

Figure 3 Loss of antigen recognition by monoclonal antibodies raised against the H77 sequence over time. Lysates from 293T-cells expressing the H-derived E1E2 clones were immobilized on GNA-coated EIA plates and the ability of 4 neutralizing anti-E2 MAbs raised against the H77 sequence (3/11, 7/16, 9/27, 11/20) to bind the respective glycoproteins was tested. To control for variation in antigen expression level, optical density readings were normalized relative to the signal obtained with a nonneutralizing antibody, 9/75, that recognizes an epitope that was fully conserved among all H isolates. The value obtained with H77 E1E2 as the capture antigen was normalized to 1. Gastroenterology 2007 132, 667-678DOI: (10.1053/j.gastro.2006.12.008) Copyright © 2007 AGA Institute Terms and Conditions

Figure 4 Identification of T-cell epitopes in the E1E2. (A) Patient H’s PBMCs were stimulated with 15-mer peptides corresponding to the H77 E1E2 sequence. The number of peptide-specific interferon-γ–producing cells was determined by enzyme-linked immunospot analysis. (B) The 3 most vigorously recognized peptides indicated in A were retested on purified CD4 and CD8 cells isolated from patient H. Gastroenterology 2007 132, 667-678DOI: (10.1053/j.gastro.2006.12.008) Copyright © 2007 AGA Institute Terms and Conditions

Figure 5 Escape from CD4 and CD8 T-cell recognition owing to mutations in the H 2003 E1E2 consensus sequence. CD4 or CD8 T cells obtained in 2003 were stimulated with peptides representing the E1 226-240, E1 296-310, and E2 436-450 epitopes in their original 1977 (Wt) form or with mutations that were present in the 2003 consensus (Mut). Interferon-γ responses were assessed by enzyme-linked immunospot assay. Gastroenterology 2007 132, 667-678DOI: (10.1053/j.gastro.2006.12.008) Copyright © 2007 AGA Institute Terms and Conditions