title: Identification of naturally processed hepatitis C virus-derived major histocompatibility complex class I ligands.
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.
authors.affiliation: Department of Medicine II, University of Freiburg, Freiburg, Germany.
authors.forename: Benno
authors.initials: B
authors.lastname: Wölk
authors.forename: Claudia
authors.initials: C
authors.lastname: Trautwein
authors.forename: Benjamin
authors.initials: B
authors.lastname: Büchele
authors.forename: Nadine
authors.initials: N
authors.lastname: Kersting
authors.forename: Hubert E
authors.initials: HE
authors.lastname: Blum
authors.forename: Hans-Georg
authors.initials: HG
authors.lastname: Rammensee
authors.forename: Andreas
authors.initials: A
authors.lastname: Cerny
authors.forename: Stefan
authors.initials: S
authors.lastname: Stevanovic
authors.forename: Darius
authors.initials: D
authors.lastname: Moradpour
authors.forename: Volker
authors.initials: V
authors.lastname: Brass
mesh_terms: D000595:Amino Acid Sequence; D045744:Cell Line, Tumor; D018604:Epitope Mapping; D005818:Genetic Engineering / Q000379:methods*; D015789:HLA-A2 Antigen / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D016174:Hepacivirus / Q000276:immunology / Q000378:metabolism*; D006801:Humans; D008024:Ligands; D013045:Species Specificity; D013602:T-Lymphocytes, Cytotoxic / Q000276:immunology / Q000821:virology; D017361:Viral Nonstructural Proteins / Q000737:chemistry / Q000276:immunology; D014764:Viral Proteins / Q000235:genetics* / Q000276:immunology / Q000378:metabolism*; D012697:Serine Endopeptidases; D043583:Nucleoside-Triphosphatase; D053487:DEAD-box RNA Helicases; D000085242:Viral Proteases; D012324:RNA-Dependent RNA Polymerase
chemical_list: D015789:HLA-A2 Antigen; D008024:Ligands; D017361:Viral Nonstructural Proteins; D014764:Viral Proteins; C084422:NS3 protein, hepatitis C virus; D012697:Serine Endopeptidases; D043583:Nucleoside-Triphosphatase; D053487:DEAD-box RNA Helicases; D000085242:Viral Proteases; C087036:NS-5 protein, hepatitis C virus; D012324:RNA-Dependent RNA Polymerase