Escape in one of two cytotoxic T-lymphocyte epitopes bound by a high-frequency major histocompatibility complex class I molecule, Mamu-A*02: a paradigm for virus evolution and persistence?

Abstract

It is now accepted that an effective vaccine against AIDS must include effective cytotoxic-T-lymphocyte (CTL) responses. The simian immunodeficiency virus (SIV)-infected rhesus macaque is the best available animal model for AIDS, but analysis of macaque CTL responses has hitherto focused mainly on epitopes bound by a single major histocompatibility complex (MHC) class I molecule, Mamu-A*01. The availability of Mamu-A*01-positive macaques for vaccine studies is therefore severely limited. Furthermore, it is becoming clear that different CTL responses are able to control immunodeficiency virus replication with varying success, making it a priority to identify and analyze CTL responses restricted by common MHC class I molecules other than Mamu-A*01. Here we describe two novel epitopes derived from SIV, one from Gag (Gag(71-79) GY9), and one from the Nef protein (Nef(159-167) YY9). Both epitopes are bound by the common macaque MHC class I molecule, Mamu-A*02. The sequences of these two eptiopes are consistent with the molecule's peptide-binding motif, which we have defined by elution of natural ligands from Mamu-A*02. Strikingly, we found evidence for the selection of escape variant viruses by CTL specific for Nef(159-167) YY9 in 6 of 6 Mamu-A*02-positive animals. In contrast, viral sequences encoding the Gag(71-79) GY9 epitope remained intact in each animal. This situation is reminiscent of Mamu-A*01-restricted CTL that recognize Tat(28-35) SL8, which reproducibly selects for escape variants during acute infection, and Gag(181-189) CM9, which does not. Differential selection by CTL may therefore be a paradigm of immunodeficiency virus infection.

FIG. 1.

Vaccinated animal 87082 makes CD8-positive T-cell responses to regions of viral Gag and Nef proteins. At 1 week after the boost with recombinant MVA expressing SIV proteins, PBMC from animal 87082 were isolated and tested in ICS against pools of 15mer peptides (A and C), as described in Materials and Methods. (B and D) If a peptide pool sensitized cells to produce IFN-γ and CD69 (see Gag pool B and Nef pool D), its constituent peptides were then tested with PBMC from the second week after immunization. The 15mer peptides Gag 17 and 18 (B) and Nef 63 and 64 (D) were found to stimulate CD8-positive lymphocytes. The sequences of the regions where these peptides overlap are boxed in dark gray.

FIG. 2.

Definition of minimal optimal CD8-positive T-cell epitopes. T-cell lines specific for Gag 17 and Nef 64 were generated and tested in ICS against a range of concentrations of overlapping peptides in order to determine which peptide results in maximal stimulation of the T-cell line. The two 9mer peptides, Gag 17-4 (GSENLKSLY; Gag_71-79 GY9) and Nef 139 (YTSGPGIRY; Nef_159-167 YY9), optimally stimulated the Gag 17-specific (A) or the Nef 64-specific T cells (B).

FIG. 3.

Mamu-A*02 restricts SIV-derived CTL epitopes. Cells of the MHC class I-negative human B-lymphoblastoid cell line 721.221, transfected with Mamu-A*02 (Mamu-A*02.221) or Mamu-A*04 (Mamu-A*04.221), were pulsed with the appropriate peptide and used in the ICS assay, together with peptide-specific T-cell lines from animal 87082. Both T-cell lines specific for Gag_71-79 GY9 (A) and for Nef_159-167 YY9 (B) were stimulated by Mamu-A*02-transferents pulsed with the relevant peptide but not by Mamu-A*04 transferents.

FIG. 4.

Pooled motif and individual ligand sequences for Mamu-A*02. (A) Peptides eluted from Mamu-A*02 were pooled and subjected to Edman sequencing. The resultant data were analyzed as previously described (6) to generate a peptide motif based on the fold increase in picomoles over the prior round of sequencing. Dominant residues (in boldface) exhibited a ≥3.5-fold increase over the prior round; strong residues (in blue) exhibited a 2.5- to 3.5-fold increase; weak residues (in green) exhibited a 2.0- to 2.5-fold increase. (B) Individual ligand sequences were derived from MS/MS sequencing of selected ions. The two new Gag and Nef SIV epitopes and the previously described Env epitope (39) are listed below the naturally loaded ligands.

FIG. 5.

Amino acid variation accumulates in the Mamu-A*02-restricted Nef_159-167 YY9 epitope during acute infection with SIVmac239. Virus isolated from Mamu-A*02-positive monkeys 4 to 16 weeks after infection with SIVmac239 was analyzed by direct sequencing of viral amplicons spanning the SIV genes nef and gag. The Mamu-A*02-restricted Nef_159-167 YY9 epitope accumulated amino acid variation in all animals analyzed, whereas the second Mamu-A*02-restricted epitope Gag_71-79 GY9 did not. Sites of mixed-base heterogeneity are shown in black; codons containing these sites are boxed, with the encoded amino acid(s) indicated in black.

FIG. 6.

Sequencing of individually cloned viral cDNA amplicons. At least 10 individual nef amplicons derived from chronic-phase vRNA of each animal were cloned and sequenced as described in the text. No wild-type sequences were detected within the Nef_159-167 YY9 epitope, whereas there was relatively little variation outside the epitope sequence (see Table 2). The sequence of the clonal SIVmac239 inoculum is indicated at the top; the epitope is indicated by a yellow box.

FIG. 7.

Mutant peptides reduce recognition by Nef_159-167 YY9-specific CTL. T-cell lines specific for Nef_159-167 YY9 were generated from four Mamu-A*02-positive, SIV-infected animals by using thawed PBMC from weeks 4 (animal 87082), 6 (animal 95084), 8 (animal 96020), or 10 (animal 97086) postchallenge. These T-cell lines were then tested in ICS against a range of concentrations of the index peptide and variant peptides representing some of the mutant epitope sequences detected in these animals (see Fig. 6).