Abstract
Fine mapping of human cytotoxic T lymphocyte (CTL) responses against hepatitis C virus (HCV) is based on external loading of target cells with synthetic peptides which are either derived from prediction algorithms or from overlapping peptide libraries. These strategies do not address putative host and viral mechanisms which may alter processing as well as presentation of CTL epitopes. Therefore, the aim of this proof-of-concept study was to identify naturally processed HCV-derived major histocompatibility complex (MHC) class I ligands. To this end, continuous human cell lines were engineered to inducibly express HCV proteins and to constitutively express high levels of functional HLA-A2. These cell lines were recognized in an HLA-A2-restricted manner by HCV-specific CTLs. Ligands eluted from HLA-A2 molecules isolated from large-scale cultures of these cell lines were separated by high performance liquid chromatography and further analyzed by electrospray ionization quadrupole time of flight mass spectrometry (MS)/tandem MS. These analyses allowed the identification of two HLA-A2-restricted epitopes derived from HCV nonstructural proteins (NS) 3 and 5B (NS31406–1415 and NS5B2594–2602). In conclusion, we describe a general strategy that may be useful to investigate HCV pathogenesis and may contribute to the development of preventive and therapeutic vaccines in the future.
Highlights
With an estimated 120–180 million chronically infected individuals, hepatitis C virus (HCV) is a leading cause of chronic hepatitis, liver cirrhosis and hepatocellular carcinoma worldwide [1]
To identify naturally processed HCV major histocompatibility complex (MHC) class I ligands by mass spectrometry (MS), as described in this manuscript, U-2 OS human osteosarcoma-derived cell lines UNS3-4A-24 and UHCVcon-57.3 [8,9] were used
This is a prerequisite for the elution of HCV proteins from MHC class I molecules isolated from these cells and demonstrates that HCV protein expression does not interfere with HLA-A2 surface expression
Summary
With an estimated 120–180 million chronically infected individuals, HCV is a leading cause of chronic hepatitis, liver cirrhosis and hepatocellular carcinoma worldwide [1]. The development of preventive and therapeutic vaccines against hepatitis C is of major public health importance [3]. Innate and adaptive immune responses to HCV have been studied in great detail [4]. The majority of patients fail to eliminate HCV and develop chronic infection (reviewed in [5,6]). The high genetic variability of HCV significantly contributes to the escape from the immune system and complicates the development of an efficient vaccine [7]. More recent data indicate that there is protective immunity against HCV [4]
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