Background/Aims: Identification of epitopes recognized by cytotoxic T lymphocytes (CTL) in hepatitis C virus (HCV) proteins is of importance because they can be used for vaccination, treatment of infection or monitoring of immune responses. Our purpose was to characterize new CTL epitopes in HCV structural proteins.
Methods: Peptides were synthesized and tested in HLA-A2 binding assays. Binder peptides were used to stimulate peripheral blood mononuclear cells from HCV+ patients and controls, and activity measured in chromium release and ELISPOT assays.
Results: Twenty binder peptides were found, and stimulation of HCV+ patient cells with nine peptides showing high binding ability led to the growth of CD8+ CTL recognizing peptide E2(614-622) in association with HLA-A2. Peptide E2(614-622) was recognized by 30% of HLA-A2+ patients with chronic HCV infection, but no responses were observed in control groups. Five peptides derived from region E2(614-622) from 26 different viral isolates bound to HLA-A2 molecules, and all of them but one, containing Phe at position 622, were recognized by E2(614-622) specific CTL.
Conclusions: These results show that peptide E2(614-622) belongs to a highly conserved region of HCV E2, and might be a good candidate to induce anti-HCV CTL responses in HLA-A2+ subjects.
Hepatitis C virus (HCV) is a single stranded RNA virus responsible for the majority of non-A non-B hepatitis [1]. Infection with HCV frequently evolves to chronicity, and ultimately to cirrhosis and hepatocellular carcinoma [2]. After infection, patients elicit a wide range of antibodies against viral proteins [3], which do not seem to be relevant to clear infection. CD4+ T-cell responses are also elicited [4], [5], [6], [7], [8], [9], [10], although they are very weak or undetectable in chronically infected patients. As in other viral infections, CD8+ cytotoxic T lymphocytes (CTL) have been detected in HCV patients [11], [12], [13], [14], [15], [16], [17], and recent reports show their importance in viral clearance in the acute phase of the disease [18] and in HCV-exposed seronegative donors [19], [20]. CTL usually eliminate infected cells after recognition of viral antigens presented as short peptides in the HLA molecules. These peptides are produced after processing of viral proteins and they are presented by HLA molecules on the cell surface [21]. The use of synthetic peptides has allowed the characterization of these epitopes recognized by CTL. These peptides usually contain certain amino acids at some positions called anchor positions that define sequence patterns or motifs [22]. By using these HLA binding motifs, several CTL epitopes from HCV antigens have been synthesized and tested [13], [14], [23], [24], [25]. These peptides have first been tested in binding assays and binder peptides used to stimulate PBMC from HCV patients. However, not all experimentally identified peptides as CTL epitopes fulfil these motifs [17], indicating that other peptides without the common binding motif may exist and have not yet been identified. Identification of CTL epitopes in the HCV proteins is of great importance, because they may be useful for vaccine development, treat chronic infection or monitor anti-HCV CTL immune responses.
In order to identify new CTL epitopes from HCV structural proteins we synthesized 9-mer peptides predicted as binders to HLA-A2 by an algorithm, and overlapping 14-15-mer peptides spanning almost the entire structural region. We show below that binding to HLA-A2, one of the more common HLA molecules, can be detected using both types of peptides. Moreover, in vitro stimulation of cells from HCV infected patients allowed us to identify CTL activity against peptide E2(614-622), not previously characterized as CTL epitope. These findings are discussed in more detail below.
Peptides were prepared manually as described [26]. Purity of the peptides was always above 80% as judged by HPLC. Nine-mer peptides were selected using an algorithm developed in our laboratory based on published data [22]. This algorithm searches for peptides containing the HLA-A2 binding motif.
Cell lines T2 (gift of Dr Jay A. Berzofsky (NIH, Bethesda, USA)), JY (HLA-A2.1, B7, Cw7), kindly gift of Dr Andreas Cerny (Bern, Switzerland), and BE (HLA-A24/26, B27/44), generated in our laboratory,
In order to identify HLA-A2 binding peptides from HCV structural proteins that could be potential CTL epitopes, we used an algorithm that predicts peptide binding and synthesized thirty two 9-mer peptides. Binding of these 9-mer peptides to HLA-A2 was measured using T2 cells. Four peptides from core, 2 from E1 and 3 from E2 showed some degree of binding to HLA-A2 (Table 1). To study if other peptides not predicted by algorithms could bind to HLA-A2, we measured binding using 117 overlapping
CTL are important effector cells in the control of many viral diseases [33], [34]. In the case of HCV infection, many individuals become chronically infected despite the presence of CTL. Although this may argue against the role of CTL in the control of HCV infection, recent reports show the importance of CTL in the final outcome of the infection [18], [35], in the protection of individuals in contact with HCV patients [19], [20] or in patients clearing HCV after α-IFN treatment [36]. Thus,
This work was supported by a grant from FIS (00/0536). The authors thank Dr J.A. Berzofsky and Dr A. Cerny for the kind gift of T2 and JY cell lines respectively, and the Department of Radiotherapy of the University Clinic of Navarra for the irradiation of the samples.
