Selection, transmission, and reversion of an antigen-processing cytotoxic T-lymphocyte escape mutation in human immunodeficiency virus type 1 infection

Abstract

Numerous studies now support that human immunodeficiency virus type 1 (HIV-1) evolution is influenced by immune selection pressure, with population studies showing an association between specific HLA alleles and mutations within defined cytotoxic T-lymphocyte epitopes. Here we combine sequence data and functional studies of CD8 T-cell responses to demonstrate that allele-specific immune pressures also select for mutations flanking CD8 epitopes that impair antigen processing. In persons expressing HLA-A3, we demonstrate consistent selection for a mutation in a C-terminal flanking residue of the normally immunodominant Gag KK9 epitope that prevents its processing and presentation, resulting in a rapid decline in the CD8 T-cell response. This single amino acid substitution also lies within a second HLA-A3-restricted epitope, with the mutation directly impairing recognition by CD8 T cells. Transmission of the mutation to subjects expressing HLA-A3 was shown to prevent the induction of normally immunodominant acute-phase responses to both epitopes. However, subsequent in vivo reversion of the mutation was coincident with delayed induction of new CD8 T-cell responses to both epitopes. These data demonstrate that mutations within the flanking region of an HIV-1 epitope can impair recognition by an established CD8 T-cell response and that transmission of these mutations alters the acute-phase CD8(+) T-cell response. Moreover, reversion of these mutations in the absence of the original immune pressure reveals the potential plasticity of immunologically selected evolutionary changes.

FIG. 1.

Decline of HLA-A3-restricted RK9 and KK9 CD8 T-cell responses in subject AC-38. HIV-1 Gag-specific CD8⁺ T-cell responses were followed longitudinally in HLA-A3-positive subject AC-38 during untreated HIV-1 infection using the IFN-γ ELISPOT assay and described optimal epitope peptides. The magnitude of these responses is indicated in SFC per 10⁶ PBMC.

FIG. 2.

Impaired recognition of variant RK9 epitope in subject AC-38. (A) Peptide-specific responses exhibited by RK9-specific CD8⁺ T-cell lines were quantified by flow cytometry using intracellular IFN-γ staining. The wild-type peptide (RK9) was recognized by 28.1% of CD8⁺ T cells, while the RQ9 variant peptide was poorly recognized (0.8%). (B) Peptide-specific responses exhibited by autologous unstimulated PBMC from subject AC-38 were quantified by IFN-γ ELISPOT. Each of the variant RK9 peptides showed reduced reactivity compared to the autologous peptide (RLRPGGKKK).

FIG. 3.

KK9- and RK9-specific CD8 T-cell lines fail to recognize target cells infected with vaccinia virus expressing the K₂₈Q variant. Cell lines were tested for recognition of target cells infected with vaccinia virus expressing either wild-type sequences of RK9 and KK9 (Vac T135) or the K₂₈Q variant sequence (Vac T142). KK9- and RK9-specific cell lines failed to recognize target cells infected with vaccinia virus expressing the K₂₈Q variant, while target cells infected with the wild-type vaccinia virus induced strong IFN-γ production. A control CD8⁺ T-cell line specific for the HLA-B14-restricted p24 Gag epitope DRFYKTLRA (DA9) that was conserved in both vaccinia viruses (T135 and T142) recognized infected target cells equally well. Target cells infected with the control vaccinia virus Lac were used as negative controls.

FIG. 4.

Impaired recognition of variant RK9 epitope in subject AC-33. Peptide-specific responses exhibited by autologous unstimulated PBMC from subject AC-33 were quantified by IFN-γ ELISPOT. Each of the variant RK9 peptides showed reduced reactivity compared to the autologous peptide (RLRPGSKKK).