Escape from HLA-B*08-restricted CD8 T cells by hepatitis C virus is associated with fitness costs

Abstract

The inherent sequence diversity of the hepatitis C virus (HCV) represents a major hurdle for the adaptive immune system to control viral replication. Mutational escape within targeted CD8 epitopes during acute HCV infection has been well documented and is one possible mechanism for T-cell failure. HLA-B*08 was recently identified as one HLA class I allele associated with spontaneous clearance of HCV replication. Selection of escape mutations in the immunodominant HLA-B*08-restricted epitope HSKKKCDEL(1395-1403) was observed during acute infection. However, little is known about the impact of escape mutations in this epitope on viral replication capacity. Their previously reported reversion back toward the consensus residue in patients who do not possess the B*08 allele suggests that the consensus sequence in this epitope is advantageous for viral replication in the absence of immune pressure. The aim of this study was to determine the impact of mutational escape from this immunodominant epitope on viral replication. We analyzed it with a patient cohort with chronic HCV genotype 1b infection and in a single-source outbreak (genotype 1b). Sequence changes in this highly conserved region are rare and selected almost exclusively in the presence of the HLA-B*08 allele. When tested in the subgenomic replicon (Con1), the observed mutations reduce viral replication compared with the prototype sequence. The results provide direct evidence that escape mutations in this epitope are associated with fitness costs and that the antiviral effect of HLA-B*08-restricted T cells is sufficiently strong to force the virus to adopt a relatively unfavorable sequence.

FIG. 1.

Alignment of HCV sequences (amino acids 1390 to 1413 of the polyprotein) from subjects with chronic HCV genotype 1b infection. The HLA-B*08-restricted CD8 epitope HSKKKCDEL_1395-1403 is highlighted in gray. Viral loads of the samples are indicated (in IU/ml). ND, no data. Dots indicate that the residue corresponds to the consensus residue.

FIG. 2.

Relative replication efficiency of different variants compared to the Con1/ET replicon. (A) Luciferase activities after 48 h (top) and 72 h (bottom) are shown relative to the Con1/ET prototype (set to 100%). (B) A second series of experiments with independently constructed replicons was made. Relative luciferase activities after 48 h (top) and 72 h (bottom) are shown. Error bars represent SDs of three independent experiments.

FIG. 3.

Peptide-specific IFN-γ secretion of T cells stimulated for 5 h with different dilutions of synthetic peptide HSKKKCDEL (•), HSRKKCDEL (▾), HSKRKCDEL (▴), or HSKKKCDEF (⧫). Numbers of IFN-γ-positive cells as percentages of all CD8-positive T cells are shown as the result of intracellular cytokine stainings. Before restimulation, specific T cells were expanded in vitro for 10 days in the presence of the prototype peptide HSKKKCDEL.

FIG. 4.

Cross-recognition of T cells expanded for 10 days from the PBMC of R66 (top) or 06-75 (bottom) in the presence of the prototype or variant peptides, as indicated in the row labels by bold type and underlining. After in vitro expansion, cells were restimulated with the prototype or variant peptides (10 μg/ml), as indicated in the column labels, before intracellular cytokine staining for IFN-γ. The number of IFN-γ-positive and CD8-positive T cells is indicated in the upper right quadrant of each dot plot (relative to all CD8-postive T cells [percentage]).