Identification of Immunodominant HIV-1 Epitopes Presented by HLA-C*12:02, a Protective Allele, Using an Immunopeptidomics Approach

Identification of HLA-C*12:02-restricted HIV-1 peptides by LC-MS/MS.

721.221 cells expressing CD4 and HLA-C*12:02 (.221-C1202) were infected with the laboratory-adapted NL4-3 HIV-1 strain. HLA-peptide complexes were purified with the pan-anti-HLA class I antibody W6/32 (as described in Materials and Methods). Peptides were eluted, fractionated by high-performance liquid chromatography (HPLC), and then analyzed by LC-MS/MS. Two independent replicate experiments were performed. Background and nonspecifically coeluted peptides (56) were first removed from the two data sets, which yielded 7,598 and 7,039 C*12:02-restricted 8- to 12-mers (respectively). A total of 10,799 unique peptide sequences were identified in the two experiments (Fig. 1a). Examination of the length distribution of the 8- to 12-mer peptides identified indicated a typical HLA class I-restricted profile (Fig. 1b) (57). Analysis of the peptide motifs of 8- to 11-mers, which are the length of most of the reported CTL epitopes, revealed a preference for an alanine residue at the 2nd position (with other residues, including serine, glutamic acid, leucine, isoleucine, and valine, also being favored here) and a preference for residues including phenylalanine, tyrosine, leucine, or lysine at the C terminus in HLA-C*12:02-bound peptides (Fig. 1c).

Fifteen unique HIV-1 NL4-3-derived peptide sequences were detected across the two experiments (Table 1). Of these, Pol-IY11 and the corresponding consensus sequence of Nef-VY9 (Nef-MY9) were previously reported as immunodominant HLA-C*12:02-restricted epitopes (52, 53). The C-terminally extended Nef-VF10 peptide (VARELHPEYF) was also among the HIV-1 peptides identified. We previously demonstrated that the 9-mer version Nef-MY9 (MARELHPEY) was optimally recognized by T cells from subjects in an HIV-1-infected Japanese cohort (52).

Binding of the eluted peptides to HLA-C*12:02.

During immunoprecipitation (IP) of HLA-I–peptide complexes from single HLA allele-transfected 721.221 cells, a low level of “background” peptides is nonspecifically coeluted during the IP step (56). In addition, as the parental 721.221 cell line exhibits residual expression of HLA-C*01:02 (56), peptides restricted by this allele could have been copurified with HLA-C*12:02-bound peptides. Thus, we next sought to confirm the HLA restriction of the HIV-1 peptides identified from .221-C1202 cells. The corresponding consensus sequences of the relevant NL4-3 peptides in worldwide HIV-1 clade B viruses (Table 1) were synthesized and evaluated for binding to HLA-C*12:02. As Tat-MY9 is highly polymorphic at the 1st and 2nd residues in circulating HIV-1 subtype B strains (LANL database [www.hiv.lanl.gov]), this peptide was synthesized based only on the NL4-3 sequence. Env-VM9 was not evaluated for HLA-C*12:02 binding as it was located in a highly variable region of Env, which would confound subsequent analysis of epitope recognition by T cell responses in HIV-infected HLA-C*12:02 subjects (and limit the potential utility of this epitope for targeting by vaccines). Nef-MY10 was also excluded because it included the Nef-MY9 epitope.

Hence, 13 peptides were synthesized and tested for binding and stabilization of HLA-C*12:02 in an HLA stabilization assay using the Transporter 2 (TAP2)-defective RMA-S cell line expressing HLA-C*12:02 (RMA-S-C1202). It was found that 9/13 peptides (including the 2 previously reported epitopes Pol-IY11 and Nef-MY9) exhibited affinity for HLA-C*12:02 molecules, evidenced by the enhanced stabilization of surface HLA expression in a dose-dependent fashion (Fig. 2a). However, 4 peptides (Gag-MM11, Gag-LH8, Gag-FH9, and Nef-KF9) did not exhibit detectable binding to HLA-C*12:02 molecules in this assay, even when tested at a higher peptide concentration (300 μM) (Fig. 2b). To determine whether these peptides were capable of binding to HLA-C*12:02 but were of too low an affinity to enable detection of binding in the HLA stabilization assay in RMA-S cells, we measured the ability of these 4 peptides to stabilize HLA-C*12:02 using a peptide/HLA refolding assay. The refolding of the HLA monomer, β₂-microglobulin (β₂m), and peptide was performed under low-temperature conditions (see Materials and Methods), and the products generated were analyzed by fast protein liquid chromatography (FPLC). The refolded HLA-C*12:02 molecules formed a small peak that could be detected on the FPLC trace (data for the Nef-MY9 epitope are shown in Fig. 2c as an example). All four of the peptides tested and Pol-IY11 were shown to refold with HLA-C*12:02 heavy chain and β₂m molecules (Fig. 2d), confirming HLA-C*12:02 restriction.

T cell responses to HIV-1 peptides eluted from HLA-C*12:02.

To explore whether these HLA-C*12:02-restricted HIV-1 peptides were epitopes to which T cell responses were elicited during natural HIV-1 infection, we screened for CD8⁺ T cell responses to these 13 peptides in 20 HIV-1-infected HLA-C*12:02⁺ individuals using ex vivo gamma interferon (IFN-γ) enzyme-linked immunosorbent spot (ELISpot) assays. T cell responses to 4/13 peptides were detected in one or more individuals (Fig. 3a). These included the previously described Pol-IY11 and Nef-MY9 epitopes as well as two additional C*12:02-restricted peptides (Env-RL9 and Vif-DY9). Of the 20 individuals tested, 13 exhibited T cell responses to the Env-RL9 peptide, and 1 individual showed T cell reactivity to Vif-DY9. T cell responses to Pol-IY11 and Nef-MY9 were also detected in 5/20 individuals, consistent with results obtained in previous studies in Japanese cohorts, where responses to these epitopes were observed in a similar proportion of infected individuals (52, 53).

To confirm the HLA restriction of the T cell responses to Env-RL9 and Vif-DY9, we expanded T cells specific for Env-RL9 by stimulating PBMCs from the responder KI-1407 (A*24:02/24:02, B*52:01/52:01, and C*12:02/12:02) with the Env peptide and T cells specific for Vif-DY9 by stimulating PBMCs from the responder KI-1394 (A*02:01/24:02, B*35:01/52:01, and C*03:03/12:02) with the Vif peptide. The cultured T cells were tested for their ability to recognize the peptides of interest presented on 721.221 cells expressing each of the HLA alleles possessed by subject KI-1407 or KI-1394, reading out responses by intracellular cytokine staining (ICS). The T cells recognized .221-C1202 cells prepulsed with Env-RL9 but not prepulsed .221-A2402 or .221-B5201 cells (Fig. 3b), indicating that the T cell response to Env-RL9 was restricted by HLA-C*12:02. In contrast, Vif-DY9-reactive T cells expanded from subject KI-1394 showed a higher-magnitude response to Vif-DY9-pulsed HLA-B*35:01-expressing cells than to Vif-DY9-pulsed HLA-C*12:02-expressing cells, suggesting that the dominant Vif-DY9 response in this individual was HLA-B*35:01 restricted (Fig. 3c). Indeed, Vif-DY9 was reported to be an HLA-B*35:01-restricted epitope in a previous study (58). The Vif-DY9 peptide can bind to both HLA-C*12:02 and HLA-B*35:01, as these two alleles have similar peptide-binding motifs (59). The results presented here, together with multiple other reports indicating that a given peptide sequence may be presented by more than one HLA allele (LANL database [www.hiv.lanl.gov]), demonstrate the importance of confirming that responses observed in patients to peptides eluted from one HLA molecule are indeed mediated by T cells restricted by the allele of interest.

We next investigated whether Env-RL9-specific T cells could recognize NL4-3-infected .221-C1202 cells. Env-RL9-specific T cells recognized NL4-3-infected .221-C1202 but not uninfected .221-C1202 cells (Fig. 3d), confirming that Env-RL9 was a novel HLA-C*12:02-restricted epitope effectively presented on HIV-1-infected cells. The results from ex vivo ELISpot assays showed that the average magnitude of the T cell responses to Env-RL9 in individuals recognizing this epitope (575 spot-forming cells [SFC]/10⁶ PBMCs) was significantly higher (P = 0.014 and 0.01, respectively) than that of the responses to Pol-IY11 or Nef-MY9 in individuals recognizing these epitopes (130 and 167 SFC/10⁶ PBMCs, respectively) (Fig. 3a). Taken together, these results indicate that Env-RL9 is an immunodominant HLA-C*12:02-restricted T cell epitope.

Analysis of the sequence of Env-RL9 in the 20 HLA-C*12:02⁺ individuals screened for T cell responses revealed that more than 80% of these individuals had M at position 9 of the epitope (Fig. 4a). We therefore analyzed T cell responses to Env-RL9-9M and Env-RL9 using T cells expanded with Env-RL9 from epitope-reactive donors. The results showed that these CD8⁺ T cells recognized both epitopes (Fig. 4b). The HLA stabilization assay also showed no remarkable difference in binding affinity between Env-RL9 and Env-RL9-9M peptides (Fig. 4c). Thus, Env-RL9-specific T cells could effectively cross-recognize Env-RL9 and Env-RL9-9M. To enable evaluation of the impact of Env-RL9-specific T cells on clinical outcome in HIV-1-infected individuals, we screened an additional 20 HIV-1-infected HLA-C*12:02⁺ individuals for the presence of responses to this epitope. Altogether, 20/40 individuals showed responses to the RL9 peptide. However, there was no difference in plasma viral loads or CD4 T cell counts between responders and nonresponders (Fig. 4d), suggesting that the presence of Env-RL9-specific responses was not a dominant determinant of viral control and disease progression in this Japanese HIV-1-infected cohort.

HLA-C*12:02 binding and T cell recognition of HLA-C*12:02 peptide variants found in circulating HIV-1.

Having found no evidence of HLA-C*12:02-restricted T cell recognition of 10 of the 13 peptides shown to be presented with HLA-C*12:02 in cells infected with HIV-1 NL4-3, we sought to determine the impact that differences within these epitope sequences between the NL4-3/worldwide clade B viruses and those present in the subjects screened for T cell reactivity may have had on the detection of responses to these peptides. We first determined the amino acid sequences of the 10 peptides (9 nonrecognized peptides plus Vif-DY9) in the 20 HLA-C*12:02⁺ individuals in whom peptide-specific responses had been evaluated (Fig. 5a). The 20-subject consensus sequences of 6 of the peptides matched the worldwide subtype B consensus sequence (Fig. 5b). In contrast, for Gag-MM11, Gag-VM8, Pol-FF9, and Tat-MY9, we found that their 20-subject consensus sequences (Gag-MM11-7P8T, Gag-VM8-4P5T, Pol-FF9-8E, and Tat-MY9-1I2K4G) were different from those of the previously evaluated Japanese population subtype B consensus sequence peptides (Fig. 5b). After further analysis of amino acid substitutions in the patient autologous viruses used to form the whole-group consensus sequence, we selected 8 additional peptides with substitutions observed in more than 5 individuals (Fig. 6a). We synthesized these 4 patient-consensus and 8 additional variant peptides and tested their binding to the HLA-C*12:02 molecule at a concentration of 100 μM by an HLA stabilization assay (Fig. 6a). These peptides all showed measurable affinities for the HLA-C*12:02 molecule with the exception of Gag-MM11-8T and Tat-MY9-1T2K4G. Therefore, we went on to investigate T cell responses to these peptides in the 20 HIV-1-infected HLA-C*12:02⁺ individuals in whom responses to the NL4-3/worldwide subtype B consensus sequence peptides had previously been tested by an IFN-γ ELISpot assay. Since all 20 of these HLA-C*12:02⁺ individuals were also HLA-B*52:01⁺, we also tested for responses to two previously reported HLA-B*52:01-restricted epitopes (Gag-RI8 and Gag-WV8) (53) as well as the HLA-C*12:02-restricted Env-RL9 peptide in parallel (as positive controls). T cell responses to the positive-control peptides Env-RL9, Gag-RI8, and/or Gag-WV8 were observed in 16/20 of the subjects tested, but no T cell responses to the 12 test HLA-C*12:02 peptides were detected in these individuals (Fig. 6b). These results suggested that these peptides were not T cell epitopes, although we cannot exclude the possibility that they were subdominant epitopes, T cell responses to which were present in a minority (<5%) of HIV-1-infected HLA-C*12:02⁺ individuals.

Ethics statement.

All treatment-naive Japanese adult individuals chronically infected with HIV-1 subtype B were recruited from the National Center for Global Health and Medicine. The study was approved by the ethics committees of Kumamoto University (RINRI-1340 and GENOME-342) and the National Center for Global Health and Medicine (NCGM-A-000172-01). Written informed consent was obtained from all individuals for the collection of blood and their subsequent analysis according to the Declaration of Helsinki.

HLA genotyping.

HLA genotypes of the HLA-A, -B, and -C alleles were identified by the Luminex microbead method at the HLA laboratory (Japan).

Cell lines.

The HLA class Ia-deficient 721.221 cell line expressing CD4 and transfected with HLA-C*12:02 (.221-C1202) was generated in a previous study (54). The TAP2-deficient mouse RMA-S cell line expressing HLA-C*12:02 (RMA-S-C1202) was generated as previously described (71). Both cell lines were cultured in RPMI 1640 medium (Thermo Fisher) containing 10% fetal calf serum (FCS) and 0.15 mg/ml hygromycin B (Calbiochem).

HIV-1 NL4-3 infection.

.221-C1202 cells were infected with HIV-1 NL4-3 in a low volume of RPMI 1640 medium (Thermo Fisher) containing 10% fetal bovine serum (FBS), 2 mM l-glutamine, 100 U/ml penicillin, 100 μg/ml streptomycin, and 10 mM HEPES (R10). Fifty microliters of NL4-3 stock virus (reverse transcriptase [RT] value = 4 × 10² ng/ml) was added to 8 × 10⁶ .221-C1202 cells, and the cells were then incubated for 1.5 h at 37°C, after which 20 ml of R10 was added and the cells were cultured overnight. At day 1 postinfection, a further 20 ml R10 was added, and cells were split (1:2) on day 2. On day 3 postinfection, the proportion of cells infected with HIV-1 was determined by intracellular p24 staining, as previously described (72). Infected flasks were then harvested for immunoprecipitation of HLA-peptide complexes.

HLA class I immunoprecipitation.

A total of 1.5 × 10⁸ to 2 × 10⁸ .221-C1202 cells infected with NL4-3 were harvested, washed in phosphate-buffered saline (PBS), and lysed in 5 ml of lysis buffer (1% IGEPAL 630, 300 mM NaCl, and 100 mM Tris [pH 8.0] plus protease inhibitors) at 4°C for 45 min. Two centrifugation steps (2,000 × g for 10 min followed by 20,000 × g for 30 min at 4°C) were next employed to clear the lysates of infected cells prior to overnight capture of HLA-peptide complexes on W6/32-coated protein A‐Sepharose beads. W6/32-bound HLA-peptide complexes were sequentially washed with 10 to 20 ml of wash buffer 1 (0.005% IGEPAL, 50 mM Tris [pH 8.0], 150 mM NaCl, 5 mM EDTA), wash buffer 2 (50 mM Tris [pH 8.0], 150 mM NaCl), wash buffer 3 (50 mM Tris [pH 8.0], 400 mM NaCl), and, finally, wash buffer 4 (50 mM Tris [pH 8.0]) under gravity flow in Econo-Column glass chromatography columns (Bio-Rad). Peptide-HLA complexes were eluted from the beads with 5 ml of 10% acetic acid, and following drying under a vacuum, they were loaded onto a 4.6- by 50-mm ProSwift RP-1S column (Thermo Fisher Scientific) and eluted from an Ultimate 3000 HPLC system (Thermo Scientific) using a 500-μl/min flow rate over 10 min from 2 to 34% buffer B (0.1% trifluoroacetic acid [TFA] in acetonitrile) in buffer A (0.1% TFA in water). Alternate 1-ml fractions (odd and even) were divided into two separate pools and dried under vacuum prior to resuspension in loading buffer for LC-MS/MS analysis.

Liquid chromatography-tandem mass spectrometry.

Each HPLC-eluted sample was resuspended in 20 μl loading buffer, and 9 μl of it was injected onto a 3-μm-particle-size, 0.075-mm by 150-mm Acclaim PepMap rapid separation liquid chromatography (RSLC) C₁₈ column and further loaded onto a 2-μm-particle-size, 75-μm by 50-cm Acclaim PepMap RSLC C₁₈ column using an Ultimate 3000 nanoflow ultrahigh-performance liquid chromatography (nUPLC) system (Thermo Scientific). A linear gradient of 3 to 25% buffer B (0.1% formic acid and 5% dimethyl sulfoxide [DMSO] in acetonitrile) in buffer A (0.1% formic acid and 5% DMSO in water) was applied over 1 h to elute peptides from the column (flow rate of 250 μl/min). Peptides were introduced, using an Easy-Spray source at 2,000 V at 40°C, to a Fusion Lumos mass spectrometer (Thermo Scientific). The ion transfer tube temperature was set to 305°C. Full MS spectra were recorded from m/z 300 to 1,500 in an Orbitrap instrument at a resolution of 120,000 with an automatic gain control (AGC) target of 400,000. Precursors were selected in top-speed mode within a 2-s cycle time (accumulation time of 120 ms) and an isolation width of 1.2 atomic mass units (amu) for fragmentation. Higher-energy collisional dissociation (HCD) with a collision energy setting of 28 was performed on the peptides with a charge state of 2 to 4, while a higher collision energy of 32 was applied to singly charged precursor ions that were selected with lower priority. MS resolution was set at 120,000, and MS2 resolution was set at 30,000. All fragmented precursor ions were actively excluded from repeated selection for 30 s.

Analysis of LC-MS/MS data sets.

The analysis of all LC-MS/MS data sets (.raw files) was performed using PEAKS v8.0 software (Bioinformatic Solutions). No enzyme was specified during the peptide spectral matching, and mass tolerance settings of 5 ppm (for precursor ions) and 0.03 Da (for fragment ions) were used. Spectral sequence annotation was performed against the annotated Homo sapiens Swiss-Prot database appended with a 6-frame translation of the HIV-1 NL4-3 genome. A false discovery rate of 5% was set using a parallel decoy database search. Shannon sequence logos for all unique database-matched (Homo sapiens and HIV) 8-mer to 12-mer peptides were produced using the online tool Seq2logo v2.0 (73).

HLA stabilization assay.

The affinity of peptide binding to HLA-C*12:02 was examined using RMA-S-C1202 cells as previously described (67). Briefly, RMA-S-C1202 cells were cultured at 26°C for 16 h and then pulsed with peptides at 26°C for 1 h and subsequently incubated at 37°C for 3 h. Staining of cell surface HLA-C molecules was performed with DT-9, an HLA-C-specific monoclonal antibody (mAb) (a gift from Mary Carrington, NIH), and fluorescein isothiocyanate (FITC)-conjugated sheep anti-mouse IgG (Jackson ImmunoResearch). Staining data were acquired on a FACSCanto II instrument (BD Biosciences) and analyzed using FlowJo 10.5.3 software. Relative HLA expression was calculated as the ratio of the mean fluorescence intensity (MFI) of peptide-pulsed RMA-S-C1202 cells to that of control (non-peptide-pulsed) cells kept at 26°C.

Refolding assay.

Refolding of peptides with the HLA heavy chain and β₂m was performed as previously described (74). Briefly, HLA-C*12:02 heavy chain and β₂m were overexpressed in Escherichia coli as inclusion bodies and solubilized with 8 M urea. Before refolding, only β₂m was refolded and purified with Superdex 75 pg 26/600 (GE Healthcare) chromatography, and the solubilized HLA heavy chain was treated with 6 M guanidine hydrochloride at 4°C overnight. The dissolved HLA heavy chain, purified monomer β₂m, and peptide (final concentrations of 20 μM, 2 μM, and 85 μM, respectively) were diluted in 10 ml of refolding buffer (100 mM Tris-HCl [pH 8.0], 400 mM l-arginine, 2 mM EDTA-Na, 5 mM reduced l-glutathione, and 0.5 mM oxidized l-glutathione). After 48 h of slow stirring at 4°C, the peptide–HLA-C complex was then concentrated and purified by Superdex 75 pg 26/600 (GE Healthcare) chromatography.

ELISpot assay.

Peripheral blood mononuclear cells (PBMCs) were separated from whole blood and stored at −80°C. ELISpot assays were performed as previously described (52). Briefly, 1 × 10⁵ PBMCs from HIV-1-infected individuals and 100 nM each C*12:02-restricted HIV-1 peptide were added to 96-well polyvinylidene plates (Millipore) that had been coated overnight with 5 μg/ml anti-IFN-γ mAb 1-D1K (Mabtech). The plates were then incubated for 16 h at 37°C and subsequently washed with PBS before the addition of biotinylated anti-IFN-γ mAb (Mabtech) at 1 μg/ml. After the plates had been incubated at room temperature for 90 min, they were washed with PBS and then incubated with streptavidin-conjugated alkaline phosphatase (Mabtech) for 60 min at room temperature. Following washes with PBS, individual cytokine-producing cells were visualized as dark spots after a 20-min reaction with 5-bromo-4-chloro-3-indolyl phosphate and nitroblue tetrazolium in the presence of an alkaline phosphatase-conjugated substrate (Bio-Rad). The spots were counted with an Eliphoto-Counter (Minerva Teck). The number of spots was calculated per 10⁶ PBMCs. One hundred spots per 10⁶ PBMCs were defined as a positive response, as described previously (52).

Intracellular cytokine staining assay.

PBMCs from HIV-1-infected individuals were expanded by culture with each HIV-1 peptide for 2 weeks. .221-C1202 cells prepulsed with each HIV-1 peptide or .221-C1202 cells infected with HIV-1 NL4-3 were added to a 96-well plate together with bulk-cultured T cells, and the cells were incubated for 4 h at 37°C with brefeldin A (10 μg/ml). The cells were then stained with peridinin chlorophyll protein (PerCP)-labeled anti-CD3 mAb (BioLegend), phycoerythrin (PE)-labeled anti-CD4 mAb (BioLegend), and allophycocyanin (APC)-labeled anti-CD8 mAb (Dako) and subsequently fixed with 4% paraformaldehyde and incubated in permeabilization buffer (0.1% saponin–10% FBS–PBS). Thereafter, the cells were stained with FITC-labeled anti-IFN-γ mAb (BioLegend). Staining data were acquired on a FACSCanto II instrument (BD Biosciences) and analyzed using FlowJo 10.5.3 software.

Bulk HIV-1 sequencing.

Plasma was separated from whole blood and stored at −80°C. Bulk sequencing of plasma viral RNA from 20 HIV-1-infected patients was performed as described previously (75). Sequence logos were generated by using WebLogo, version 2.8.2 (76).

Statistical analyses.

Unless otherwise stated, the statistical significance of differences between groups was calculated by using the Mann-Whitney U test in GraphPad Prism v7.0. Differences were considered statistically significant at a P value of <0.05.