Multiple HLA A11-restricted cytotoxic T-lymphocyte epitopes of different immunogenicities in the Epstein-Barr virus-encoded nuclear antigen 4

Abstract

Epstein-Barr virus (EBV), a ubiquitous herpesvirus, induces potent HLA class I-restricted cytotoxic T-lymphocyte (CTL) responses. Analyses of target antigen choice have shown that the very strong CTL responses which are often observed through the HLA All allele map are due almost entirely to a single transformation-associated EBV protein, the nuclear antigen EBNA4. Here, we sought to determine the number and relative immunogenicities of HLA All-restricted epitopes within this 938-amino-acid protein. An initial screening with a series of recombinant vaccinia virus vectors encoding progressively truncated forms of EBNA4 was followed by peptide sensitization experiments using overlapping 14-or 15-mers from the entire sequence. These two approaches allowed the identification of five epitope regions located between residues 101 and 115, 416 and 429, 396 and 410, 481 and 495, and 551 and 564 of the EBNA4 molecule. CTL preparations from all seven HLA All-positive donors tested had demonstrable reactivities against the 416-to-429 peptide, whereas reactivities against the other epitopes either tended to be lost on serial passage or, for some of the donors, were never detected. The immunodominance of the 416-to-429 epitope was further supported by peptide dilution assays using polyclonal effectors and by CTL cloning experiments. Analysis of the 416-to-429 region identified the nanomer 416-424 (IVTDFSVIK) as the cognate peptide. This peptide was able to sensitize targets to lysis by All-restricted CTL clones at concentrations as low as 5 x 10-14 M.

Activation of epitope-specific memory cytotoxic T lymphocyte responses by synthetic peptides

“…Cell lines were maintained in RPMI 1640 supplemented with 2 mM glutamine, antibiotics, and 10% heatinactivated fetal calf serum (FCS). Phytohaemagglutinin (PHA)activated blasts were obtained by stimulation of peripheral blood lymphocytes (PBL) with 1 g/ml of purified PHA (Wellcome) for 3 days and expanded in medium supplemented with IL-2 as described [26].…”

“…The IVTDFSVIK (IVT) and AVFSRKSDAK (AVF) peptides, corresponding to amino acids 416-424 and 399-408 of the EBV nuclear antigen-4 (EBNA4) [25,26], were synthesized by solidphase method as previously described [31]. Crude deprotected peptides were purified by high performance liquid chromatography (HPLC); purity was >98%.…”

“…Cloning was done by limiting dilution 1 day after the third stimulation (day 15). Single cells were seeded in 96-well plates in 200 l medium containing 30% MLA144 culture supernatant, 10 U/ml human recombinant IL-2 and 10 5 irradiated (30 Gy) allogeneic PHA-activated PBL as feeder [26]. Growing cultures were transferred into 48-well plates and expanded in IL-2-conditioned medium.…”

“…For the peptide sensitization assays using PHA blasts, 4 2 10 3 cells were placed in triplicate wells of 96 V-shaped well plates. Peptides were added to each well, and the plates were incubated for 1 h at 378C before addition of the effectors [26]. T2/A11 cells were cultured at 268C overnight and then treated with different concentrations of peptide for 3 h at 378C.…”

“…We have previously shown that HLA-A11-restricted EBV-specific CTL responses are made up of reactivities to five different peptides from the EBNA4 antigen. The immunodominant epitope was identified within the nonamer peptide IVTDFSVIK (IVT), corresponding to amino acids 416-424, while a subdominant epitope was localized within residues 399-408, corresponding to the decamer peptide AVFSRKSDAK (AVF) [25,26].…”