Efficient processing of the immunodominant, HLA-A*0201-restricted human immunodeficiency virus type 1 cytotoxic T-lymphocyte epitope despite multiple variations in the epitope flanking sequences

Abstract

Immune escape from cytotoxic T-lymphocyte (CTL) responses has been shown to occur not only by changes within the targeted epitope but also by changes in the flanking sequences which interfere with the processing of the immunogenic peptide. However, the frequency of such an escape mechanism has not been determined. To investigate whether naturally occurring variations in the flanking sequences of an immunodominant human immunodeficiency virus type 1 (HIV-1) Gag CTL epitope prevent antigen processing, cells infected with HIV-1 or vaccinia virus constructs encoding different patient-derived Gag sequences were tested for recognition by HLA-A*0201-restricted, p17-specific CTL. We found that the immunodominant p17 epitope (SL9) and its variants were efficiently processed from minigene expressing vectors and from six HIV-1 Gag variants expressed by recombinant vaccinia virus constructs. Furthermore, SL9-specific CTL clones derived from multiple donors efficiently inhibited virus replication when added to HLA-A*0201-bearing cells infected with primary or laboratory-adapted strains of virus, despite the variability in the SL9 flanking sequences. These data suggest that escape from this immunodominant CTL response is not frequently accomplished by changes in the epitope flanking sequences.

FIG. 1

Recognition of the SL9 epitope expressed by different vaccinia virus constructs. HLA-A*0201-positive B-LCL cells were infected overnight with vaccinia virus constructs or pulsed with optimal peptides (SL9 for clones 115.D4 and 13010.B17) or, in this experiment, with the HLA-B52 restricted peptide 122E (Gag p24, RMYSPTSI, amino acids 275 to 282) recognized by clone 19-203 for 90 min. The effector/target ratio was 5:1. This experiment was repeated three times with different CTL clones and target cell lines from three different donors, yielding the same pattern of recognition.

FIG. 2

Processing of SL9 from patient LWF-derived Gag sequences expressed as vaccinia virus minigene constructs and episomal vectors. (A) Vaccinia virus construct expression system. HLA-A*0201-positive B-LCL cells were infected overnight with vaccinia virus constructs expressing patient LWF-derived p17 sequences or the SL9 epitope only. (B) Episomal vector expression system. Alternatively, HMY-A2 cells were stably transfected with episomal vectors expressing SL9 with or without three additional N-terminal amino acid residues. B-LCL and HMY-A2 target cells were ⁵¹Cr labeled for 90 min and incubated with a polyclonal SL9-specific CTL line at the indicated effector/target ratios for 4 h in a standard cytotoxicity assay.

FIG. 3

Inhibition of viral replication by SL9-specific CTL clones and peptide titration of SL9 and the NL4-3 encoded variant V821/T84V. (a and b) HLA-A*0201-expressing CD4 cells were infected with viral isolates from subjects VI-06 or 053i and cultured in the presence (◊) or absence (⧫) of the SL9-specific CTL cone 115D4. (c and d) HLA-A*0201-positive T1 cells (c) and HLA-A*0201-negative H9 cells (d) were infected with HIV-1 IIIB and cultured in the presence of SL9-specific CTL clone 115D4 (◊) or clone 161jXA/14 (▵) or without CTL (⧫). (e) T1 cells were infected with HIV-1 NL4-3 isolate and cultured with the same clones as for panels c and d. The production of p24 was measured after 3 to 15 days. (f) For peptide titrations, clones 115.D4 and 161j.XA/14 were both tested on the SL9 sequences expressed by HIV-1 HXB2 (HIV-1 IIIB, SL9 consensus sequence) and by HIV-1 NL4-3 (SL9 variant V82I/T84V). Peptide titration was carried out with HLA-A*0201-positive EBV-transformed B-LCL cells as target cells in a standard ⁵¹Cr release assay.