Identification of a novel HLA-A24-restricted cytotoxic T-lymphocyte epitope within HIV-1 Nef

Cytotoxic T-lymphocyte (CTL) responses play a central role in controlling viral replication and in determining disease progression in HIV infection [1]. Identification of CTL epitopes is important in understanding the nature of the CTL response, and may be critical in vaccine design. In Caucasian populations, approximately 48% carry human leukocyte antigen (HLA)-A2, 31% HLA-A3, and 30% HLA-A1 [2]. The next most prevalent HLAA allele is HLA-A24, which is present in approximately 19% of Caucasians. However, in several large ethnic groups, HLA-A24 is the most common HLA-A allele, present in approximately 35% of Indian populations and found in 70% of the Japanese [2]. An HIV-specific HLA-A24-restricted epitope has now been described, which lies within a conserved region of HIV-1 Nef. At the time of this study, donor 106 (HLA class 1 tissue type: HLA-A1, A24, B27, B35, CW*0201, CW4) was asymptomatic and had been infected for approximately 6 years. His CD4 count was low (110 × 106 cells/l), but his viral load had also remained relatively low, in the range of 2000–10 000 RNA copies/ml plasma while off treatment (pre December 1996). Whilst on treatment with zidovudine and didanosine (started December 1996) his viral load was undetectable (< 400 copies/ml). Bulk-cultured lymphocytes were generated as previously described [3] by adding autologous phytohaemagglutinin-stimulated lymphoblasts to peripheral blood mononuclear cells and culturing for 14–21 days prior to assaying. Autologous Epstein–Barr virus-transformed B cells were pulsed with pools of overlapping 15–20mer peptides spanning the HIV-1 Gag and Nef proteins, and assayed against bulk-cultured CTL from donor 106. The predominant Nef response was to the HLA-B*2705-restricted epitope RRQDILDLWI (HIV-1LAI residues 105–114) [4]. Of the other responses identified, one was detected to the 20mer peptide in Nef HIV-1LAI (amino-acid sequence GPGVRYPLTFGWCYKLVPVE). The truncated peptides within this 20mer that were best recognised were the octamer RYPLTFGW and the nonamer VRYPLTFGW (Fig. 1). The octamer is therefore the likely cognate epitope, since truncation of the valine residue from the N-terminal end of the peptide did not reduce CTL recognition.Fig. 1: . (a) Recognition of truncated peptides within the 20mer Nef 14 by peptide-specific line from donor 106 [effector-to-target (E:T) ratio, 5:1]. (b) Recognition of peptides GVRYPLTFGW and RYPLTFGW presented by human leukocyte antigen (HLA)-A24-matched targets (□ and ▪) and HLA-Cw*0201-matched targets (○ and •). Effectors were the peptide-specific line from donor 106. (c) Peptide titres of truncated peptides within the 20mer Nef 14. Effectors were the peptide-specific line from donor 106 (E:T ratio, 20:1).The peptide-binding motif for HLA-A24, revealed from pool sequencing of peptides eluted from HLA-A24 molecules, was Tyr at position 2 in the peptide (P2), Ile, Leu or Phe at the C-terminal position (PC), with secondary anchors at position P5 (Ile or Val) and position P6 (Phe) [5]. This method was also performed by Kubo et al. [6], who also identified Tyr as the position P2 anchor, and Phe or Leu as the PC anchor. By binding polyalanine analogues to HLA-A24 molecules, Trp was identified as an equally strong-binding PC anchor. The epitope defined here as RYPLTFGW was therefore consistent with these data in having Tyr at position P2 and Trp at position PC. At position P6, the secondary anchor Phe was also present. The only other HLA-A24 CTL epitope defined to date has the sequence RYLKDQQLL [7], which is within HIV-1 gp41. Six pockets (A–F) within the peptide-binding groove of the HLA molecule have been described [8], into which particular residues of the peptide may be accommodated. The A pocket accommodates the residue at position P1; like HLA-A*0201, which has a similar electrostatic charge within the A pocket, hydrophobic or positively charged residues might be predicted at position P1 in the peptide [9], as seen in these two examples of HLA-A24-restricted epitopes. The presence of Tyr at position P2 was unusual. Large residues may be seen as a P2 anchor in HLA-CW-restricted epitopes [10]. HLA-A24 and -A23 (previously classified together as HLA-A9), in common with HLA-A19 subtypes (i.e., HLA-A29, A30, A31, A33), had a small residue at position 9 (Ser or Thr in place of Tyr or Phe). This would doubtless allow the HLA-A23 or -A24 B pocket more space in which to accommoate a large P2 anchor such as Tyr. Elution of peptides from HLA-A*3001 and A*3003 similarly provided a motif incorporating Tyr or Phe at position P2 in the peptide (P. Krausa, personal communication, 1997). Acknowledgement Overlapping peptides were supplied by the Medical Research Council AIDS Directed Programme (Dr H. Holmes).