Aminopeptidase substrate preference affects HIV epitope presentation and predicts immune escape patterns in HIV-infected individuals

Abstract

Viruses evade immune detection partly through immune-associated mutations. Analyses of HIV sequences derived from infected individuals have identified numerous examples of HLA-associated mutations within or adjacent to T cell epitopes, but the potential impact of most mutations on epitope production and presentation remains unclear. The multistep breakdown of proteins into epitopes includes trimming of N-extended peptides into epitopes by aminopeptidases before loading onto MHC class I molecules. Definition of sequence signatures that modulate epitope production would lead to a better understanding of factors driving viral evolution and immune escape at the population level. In this study, we identified cytosolic aminopeptidases cleavage preferences in primary cells and its impact on HIV Ag degradation into epitopes in primary human cell extracts by mass spectrometry and on epitope presentation to CTL. We observed a hierarchy of preferred amino acid cleavage by cytosolic aminopeptidases. We demonstrated that flanking mutations producing more or less cleavable motifs can increase or decrease epitope production and presentation by up to 14-fold. We found that the efficiency of epitope production correlates with cleavability of flanking residues. These in vitro findings were supported by in vivo population-level analyses of clinically derived viral sequences from 1134 antiretroviral-naive HIV-infected individuals: HLA-associated mutations immune pressures drove the selection of residues that are less cleavable by aminopeptidases predominantly at N-flanking sites, leading to reduced epitope production and immune recognition. These results underscore an important and widespread role of Ag processing mutations in HIV immune escape and identify molecular mechanisms underlying impaired epitope presentation.

Figure 1. Hierarchy of amino acid cleavage preference in human primary cells

A. Differential cleavage of Leu-amc and Tyr-amc. Leu-amc (black triangles) or Phe-amc (black circles) were added to PBMC cytosol preincubated with (plain black line) or without bestatin (dotted grey lines) and incubated for 1h at 37°C during which fluorescence emission was monitored every 5 minutes. B. PBMC extracts were preincubated with various inhibitors (bestatin, 1–10 phenantroline, puromycin, MG132, butabindide, E64, leupeptin) before addition of Leu-amc or Tyr-amc. The maximum slope of fluorescence emission over 1h was calculated for each condition. 100% represents the maximum slope of fluorescence emission for each substrate in the absence of inhibitor (5591 for Leu-amc and 1804 for Tyr-amc). Average and SD of 3 experiments using cytosol from 3 different donors. C. Maximum slope of fluorescence emission by each substrate in a 1-hour incubation with PBMC cytosol. 100% corresponds to the slope of Leu-amc hydrolysis (fluorescence values after subtraction of those in the presence of inhibitor) (4214 average fluorescence emission). Average of 5 healthy donors. D. Amino acid cleavage efficiency by cytosol from PBMC, or CD4 T cells and monocytes sorted from PBMC. Average of 4 healthy donors. 100% represents the maximum slope of Leu-amc cleavage by each subset (3231 for PBMC, 964 for CD4 T cells and 5211 for monocytes).

Figure 2. Flanking residues drive the amount and antigenicity of HIV peptides produced in PBMC cytosol

A. 3-extended KF11 peptides (KAFSPEVIPMF; aa 30–40 in p24) with double mutations flanking KF11 (WT VEE or mutants LL, MM, FF, KK, II, SS, HH, DD, VV) were degraded for 1h in PBMC cytosol. Cleavable motifs are bolded and poor substrates are italicized. Peptides KF11 (black bars), FF9 (grey bars) and SPEVIP (white bars) were identified by mass spectrometry. The intensity of the peak of the 3 peptides was compared between WT and mutants. Representative of 3 experiments. B. Correlation between the fold increase production of KF11 (open circles) and FF9 (black triangles) and the relative PBMC aminopeptidase-specific hydrolytic activity of each amino acid. C. Antigenicity of trimmed KF11 peptides. Degradation products of each extended KF11 peptides preceded with WT or cleavable motifs (left panel: WT EE black circles, FF: grey squares, KK grey circles, LL grey diamonds, MM grey triangles), or poorly cleavable motifs (right panel: WT EE black circles, SS: x, HH: open triangles, VV open diamonds, DD open squares, II: open circles) were purified at different time points, pulsed onto HLA-B57+ B cells used as targets in cytolysis assay with KF11-specific CTL. Average of 3 experiments.

Figure 3. The efficiency of endogenous processing and presentation of an aminopeptidase-dependent HIV epitope to CD8 T cells is driven by epitope-flanking motifs

A. Enhanced presentation of KF11 flanked by cleavable motifs. RNA encoding p24 with WT KF11 flanking motifs or mutated residues (LL, II) were transfected in HLA-B57+ B cells used as target in a cytolysis assay with KF11-specific CTL (black bars) or neighboring B57-restricted epitopes TW10 (TSTLQEQIGW; aa 108–117 in p24; grey bars). Cleavable motifs are bolded and poor substrates are italicized. Average of 3 transfection experiments. B. Ratio of KF11/TW10 epitope presentation by cells transfected with WT or mutated (LL, FF, FF, II, VV, DD, HH, SS) p24. For each transfection we calculated a ratio of an equivalent amount of exogenously pulsed KF11 or TW10 peptide needed to reach the same lysis percentage as those of transfected cells. Average of 3 transfection experiments. C. Correlation between the fold increase in KF11 presentation from various mutated p24 to CTL and the relative aminopeptidase activity against each residue. D. Correlation between the fold increase in KF11 presentation from various mutated p24 to CTL and the in vitro production of KF11 measured by mass spectrometry. Correlations with Spearman test.

Figure 4. An HLA-restricted epitope-flanking mutation toward a poorly cleavable aminopeptidase motif impairs epitope production and presentation

A. Peptide corresponding to HLA-B57-ISW9 with a 3-residue extension with WT (HQA-ISW9, black circles) or mutated HQP-ISW9 (grey triangles) were degraded in PBMC cytosol. The relative amount of 3-, 2-, 1-extended and epitope ISW9 (left to right) was identified by mass spectrometry over 60 minutes. The relative amount of each peptide in the total mix of degradation products is quantified at each time point. Average of 2 degradation experiments run twice on a mass spectrometer. 2 additional degradation experiments with less cytosol gave similar results albeit with slower degradation rates (not shown). B. Degradation products from HQA-ISW9 (black circles) or HQP-ISW9 (grey triangles) of each time point were pulsed onto HLA-B57 cells used as targets in a chromium assay with ISW9-specific CTL. Average of 3 killing assays run in triplicates. C. HLA-B57 cells were transfected with RNA encoding WT p24 (black bars), p24 with A146P mutation flanking ISW9 (grey bars) or GFP (white bars) and used as targets in a chromium-based killing assay with ISW9-, KF11, or TW10-specific CTL (3 epitopes located in p24 and restricted by HLA-B57). Epitope flanking sequences indicated above bars. Average of 3 transfections; killing assays run in triplicates.

Figure 5. HLA-restricted flanking mutations toward less cleavable motifs or intraepitopic mutations toward more cleavable aminopeptidase motifs reduce epitope production

A. Peptides including an HIV epitope with a 3-residue N-terminal extension corresponding to WT sequences (black bars) or with a HLA-restricted mutation at positions −3, −2, −1 (grey bars) were degraded in PBMC cytosol for 60 minutes. All degradation peptides were identified by mass spectrometry and the relative amount of the 3-extended peptide and epitopes were calculated. Names, sequences and HLA-restriction of each epitope are indicated below each panel. N-extended sequences, mutations and their locations are indicated above each panel. B. Same as A with HLA-restricted mutations located at positions 1 or 2 within epitopes. Average result of 3 mass spectrometry injections of one representative cytosolic degradation.

Figure 6. HIV evolution at the population level toward poorly cleavable flanking residues and more cleavable residues within epitopes

A. HLA-associated escape pathways located at positions −3 (diamond), −2 (inverted triangles), −1 (squares) flanking epitopes or at position 1 (circles), 2 (triangles), or 3 (stars) were identified in all optimally defined CTL epitopes across the HIV-1 proteome (except gp120) in a cohort of 1134 chronically HIV-infected, antiretroviral-naïve persons. A total of 88 HLA-restricted escape pathways (after exclusion of 14 mutations affecting binding to MHC-I) were identified. For each given HLA-restricted escape pathway we calculated the difference (delta; Δ) between the aminopeptidase cleavage capacity of the adapted (escape mutated) residue and that of non-adapted (usually consensus B) residue, based on the ranking established in figure 1C. A negative delta is indicative of a mutation toward a worse aminopeptidase substrate whereas a positive value shows a mutation toward a better aminopeptidase substrate. Wilcoxon Signed-rank test for deviation from a theoretical median delta score of 0 at position −1 (p=0.018). B. Same as in A except mutations are compared at positions −1, 1 and 2 only, for HLA alleles associated with protection (HLA B27, B57, B58) (left; grey symbols) or neutral or associated with progression (right; open symbols). C. Comparison of amino acids hydrolysis rates by PBMC cytosolic aminopeptidase established in figure 1 and ER aminopeptidases established by Schatz et al, J. Immunology 2008. Spearman correlation. D. Similar to A with except that the difference (delta; Δ) between the aminopeptidase cleavage capacity of the adapted (escape mutated) residue and that of non-adapted residue is based on the ranking of ER aminopeptidases established by Schatz et al. Wilcoxon Signed-rank test for deviation from a theoretical median delta score of 0 at position −1 p=0.018.