Background and aims: During chronic hepatitis C virus (HCV) infection, CD8+ T-cells become functionally exhausted, undergoing progressive phenotypic changes, i.e., overexpression of "inhibitory" molecules such as PD-1 (programmed cell death protein 1) and/or Tim-3 (T-cell immunoglobulin and mucin domain-containing molecule-3). The extreme intrahost genetic diversity of HCV is a major mechanism of immune system evasion, facilitating epitope escape. The aim of the present study was to determine whether T-cell exhaustion phenotype in chronic HCV infection is related to the sequence repertoire of NS3 viral immunodominant epitopes. Methods: The study population was ninety prospective patients with chronic HCV genotype 1b infection. Populations of peripheral blood CD8+ T-cells expressing PD-1/Tim-3 were assessed by multiparametric flow cytometry, including HCV-specific T-cells after magnetic-based enrichment using MHC-pentamer. Autologous epitope sequences were inferred from next-generation sequencing. The correction of sequencing errors and genetic variants reconstruction was performed using Quasirecomb. Results: There was an interplay between the analyzed epitopes sequences and exhaustion phenotype of CD8+ T-cells. A predominance of NS31406 epitope sequence, representing neither prototype KLSGLGLNAV nor cross-reactive variants (KLSSLGLNAV, KLSGLGINAV or KLSALGLNAV), was associated with higher percentage of HCV-specific CD8+PD-1+Tim-3+ T-cells, P=0.0102. Variability (at least two variants) of NS31406 epitope sequence was associated with increased frequencies of global CD8+PD-1+Tim-3+ T-cells (P=0.0197) and lower frequencies of CD8+PD-1-Tim-3- T-cells (P=0.0079). In contrast, infection with NS31073 dominant variant epitope (other than prototype CVNGVCWTV) was associated with lower frequency of global CD8+PD-1+Tim-3+ T-cells (P=0.0054). Conclusions: Our results indicate that PD-1/Tim-3 receptor expression is largely determined by viral epitope sequence and is evident for both HCV-specific and global CD8+ T-cells, pointing to the importance of evaluating autologous viral epitope sequences in the investigation of CD8+ T-cell exhaustion in HCV infection.Keywords: PD-1; T-cell exhaustion; Tim-3; epitope sequence; hepatitis C virus.

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