Two MHC Class I Molecules Associated with Elite Control of Immunodeficiency Virus Replication, Mamu-B*08 and HLA-B*2705, Bind Peptides with Sequence Similarity

MHC class I typing of macaques

Indian rhesus macaques (Macaca mulatta) were genotyped for Mamu-B*08 (6) along with the following MHC class I alleles, Mamu-A*01, -A*02, -A*08, -A*11, -B*01, -B*03, -B*04, -B*17, and -B*29 using PCR amplification with sequence-specific primers (PCR-SSP) as described previously (25).

Animals and virus infections

Macaques were infected with the pathogenic molecular clone SIVmac239 (26) (www.ncbi.nlm.nih.gov/GenBank/index.html; GenBank accession no. M33262) with the exception of macaque r99006, which was infected with an SIVmac239 recombinant virus bearing escape mutations in three CD8⁺ T cell epitopes (27). SIV viremia was monitored in the infected macaques by quantitative PCR as described previously (6, 28).

Seven macaques at varying disease courses in the chronic phase of SIV infection (>35 wk post-SIV infection) were utilized in this study. Four of the macaques were considered ECs with viral loads <1,000 vRNA copies/ml during this study (r00032, r02019, r98016, and r99006). The controller animal, r01027, maintained viral loads <20,000 vRNA copies/ml or a log less than the viral set point of typical progressor macaques (6), while macaque r91003 displayed a typical progressor disease course with viral loads ∼0.5 × 10⁶ vRNA copies/ml. Animal r00078 was previously an EC that had steadily increasing viral loads since 1 year after depletion of its CD8⁺ cells (28). At the time of the initial ELISPOT assays, the plasma virus concentrations in r00078 was ∼30,000 vRNA copies/ml.

All SIV-infected animals were maintained at the National Primate Research Center (University of Wisconsin Madison) and cared for according to the regulations and guidelines of the University of Wisconsin Institutional Animal Care and Use Committee.

Creation of stable MHC class I transfectants

A soluble Mamu-B*08 transfectant was produced in the MHC class I-deficient EBV-transformed B lymphoblastoid cell line 721.221. Briefly, a pcDNA 3.1⁻ vector (Invitrogen) encoding soluble Mamu-B*08 was created by removing the cytoplasmic and membrane domains with PCR mutagenesis. This construct was then transfected into the 721.221 cell line (29). A previously created membrane-bound Mamu-B*08 transfectant was also used in this study (7).

Mamu-B*08 peptide-binding motif from endogenous ligands

Transfected 721.221 cells producing soluble Mamu-B*08 were cultured in a hollow fiber bioreactor (Toray) in a CP2500 unit (Biovest International), and supernatant containing soluble Mamu-B*08 was collected. Approximately 25 mg of soluble Mamu-B*08 was affinity purified from supernatant with W6/32 Ab (30) coupled to Sepharose 4B matrix (GE Healthcare Systems). Bound peptide was eluted from Mamu-B*08 H and L chains with an acid boil and passed through an ultrafiltration stirred cell with a 3 kDa membrane (Millipore). Ten percent of the eluted peptide pool underwent 14 cycles of N-terminal Edman degradation sequencing on an ABI Procise 492 protein sequencer (Applied Biosystems). The relative dominance of amino acids at each position in the peptide pool was determined by calculating the fold increase in picomoles of an amino acid over the previous cycle. Amino acids exhibiting a fold increase of 2.0–2.49, 2.5–3.49, and ≥3.5, over the previous round are considered weak, strong, and dominant amino acids, respectively, at a given position in the peptide pool. The remaining peptide pool was fractionated by reversed-phase HPLC and sprayed via nanospray on an ESI-quadrupole time-of-flight Q-Star Elite mass spectrometer (Applied Biosystems). Approximately 40 peptide-containing fractions were generated. Individual peptide sequences were determined by MS/MS analysis. A peptide motif was compiled using resultant Edman sequencing data and individual peptide sequences (31).

Positional scanning combinatorial library and peptide synthesis

The PSCL was synthesized as described previously (32). Each pool in the library contains 9-mer peptides with one fixed residue at a single position. With each of the 20 naturally occurring residues represented at each position along the 9-mer backbone, the entire library consisted of 180 peptide mixtures.

Peptides used in screening studies were purchased as crude or purified material from Mimotopes, Pepscan Systems, A and A Labs, Genescript, or the Biotechnology Center at the University of Wisconsin Madison. Peptides synthesized for use as radiolabeled ligands were synthesized by A and A Labs and purified to >95% homogeneity by reversed-phase HPLC. Purity of these peptides was determined using analytical reversed-phase HPLC and amino acid analysis, sequencing, and/or mass spectrometry. Peptides were radiolabeled with the chloramine T method (33). Lyophilized peptides were resuspended at 4–20 mg/ml in 100% DMSO, then diluted to required concentrations in PBS + 0.05% (v/v) Nonidet P-40 (Fluka Biochemika).

SIV peptide sequences were derived from the SIVmac239 sequence, (www.ncbi.nlm.nih.gov/GenBank/index.html) GenBank accession no. M33262 (26).

MHC purification and peptide binding assays

MHC class I purification was performed using affinity chromatography as described previously (33, 34). The Mamu-B*08 and Mamu-B*03 molecules were purified from cell lysates of stable membrane-bound MHC class I transfectants using the anti-HLA class I (A, B, and C) Ab W6/32. HLA-B*2705 molecules were purified from the EBV-transformed homozygous cell line LG2 using the HLA-B and -C Ab B123.2 (35, 36, 37). Protein purity, concentration, and depletion efficiency steps were monitored by SDS-PAGE.

Quantitative assays for peptide binding to detergent-solubilized MHC class I molecules were based on the competitive inhibition of binding of a high-affinity radiolabeled standard probe peptide and performed as detailed in prior studies (33, 34, 37, 38). Peptides were tested at six different concentrations covering a 100,000-fold dose range in three or more independent assays. The radiolabeled peptides used for the Mamu-B*08, Mamu-B*03, and HLA-B*2705 assays were, respectively, peptide 3130.0006 (sequence RRDYRRGL, an N₁₇₅ to Y analog of the SIV Vif_172–179RL8 epitope), the artificial ligand 3130.0012 (sequence RRAARAEYL), and the human 60s rL28 38–46 peptide (sequence FRYNGLIHR). For each peptide, the concentration of peptide yielding 50% inhibition of the binding of the radiolabeled probe peptide (IC₅₀) was calculated. Under the conditions used, where [radiolabeled probe] < [MHC] and IC₅₀ ≥ [MHC], the measured IC₅₀s are reasonable approximations of the true K_d values (39).

Control for MHC specificity is implicit by the fact that in each binding assay, only one species of purified MHC class I molecule is utilized. Control wells to measure nonspecific (background) binding were also included. Specificity is further implied because not all peptides bound all three molecules, several peptides bound none of the three molecules, and each molecule tested bound a unique set of peptides. In each experiment, a titration of the unlabeled version of the radiolabeled probe was also tested as a positive control for inhibition.

Bioinformatic analysis

Analysis of the PSCL data was performed as described previously (40). Briefly, IC₅₀ nM values for each mixture were standardized as a ratio to the geometric mean IC₅₀ nM value of the entire set of 180 mixtures and then normalized at each position so that the value associated with optimal binding at each position corresponds to 1. For each position, an average (geometric) relative binding affinity (ARB) was calculated, and then the ratio of the ARB for the entire library to the ARB for each position was derived. We have denominated this ratio, which describes the factor by which the normalized geometric average binding affinity associated with all 20 residues at a specified position differs from that of the average affinity of the entire library, as the specificity factor (SF). As calculated, positions with the highest specificity will have the highest SF value. Primary anchor positions were then defined as those with an SF ≥ 2.4. This criterion identifies positions where the majority of residues are associated with significant decreases in binding capacity.

Secondary anchor designations were based on the SD of residue specific values at each position. Dominant secondary anchor positions were defined as those where the SD was >3 and the SF < 2.4, as well as positions associated with an SD > 2 if the SF is between 1.5 and 2.4. Weak secondary anchors have been defined as positions associated with a SD in the 2.5–3 range with an SF < 1.5, or an SF in the 1.5–2.4 range with an SD < 2.

To identify predicted binders, all possible 9-mer peptides in SIV sequences were scored using the matrix values derived from the PSCL analysis as described previously (40). The final score for each peptide represents the product of the corresponding matrix values for each peptide residue-position pair. Peptides of 8, 10, or 11 residues in length were also selected using the combinatorial library as described previously (41). Briefly, 8-mers were selected by scoring residues 1–7 with the corresponding residues in the 9-mer matrix, and the C terminus was scored using the corresponding 9-mer C-terminal value. Similarly, for 10- and 11-mer peptides, residues 1–8 were scored using the corresponding residues of the 9-mer matrix, and the C terminus with the 9-mer C-terminal residues. Peptides scoring among the top 2.5% (n = 82) for each size were selected. An additional set of 50 peptides representative of scores in each 10% range of the lower 97.5% was selected to evaluate the efficacy of the prediction approach.

IFN-γ ELISPOT assay

We performed ELISPOT assays as described previously (42). Briefly, PBMC were isolated from EDTA-anticoagulated blood using Ficoll-Paque PLUS (GE Healthcare Systems) and density centrifugation. 1 × 10⁵ PBMC were used per well in precoated ELISpot^PLUS kits (Mabtech) according to the manufacturer’s instructions for the detection of IFN-γ-secreting cells. All tests were performed in duplicate or triplicate using individual peptides at 10 μM. The positive control, Con A (Sigma-Aldrich), was used at a final concentration of 5 μg/ml. The negative control wells were devoid of any stimulation. The 96-well plates were incubated for 12–18 h at 37°C in 5% CO₂.

Wells were imaged and counted with an AID EliSpot reader version 4.0 (AID) and analyzed as described previously (38, 42). A response was considered positive if the mean number of spot-forming cells (SFC) from the duplicate (or triplicate) sample wells exceeded background (mean of wells without peptide stimulation) plus two SD. Background levels were subtracted from each well, and assay results are shown as SFC per 1 × 10⁶ PBMC. Responses of <50 SFC per 1 × 10⁶ PBMC were not considered positive.

Two of the 210 peptides binding Mamu-B*08 ≤ 500 nM were unable to be tested in ELISPOT assays (supplemental Table I).⁵ However, neither the Rev nor Nef peptide would be considered a novel response as both overlapped previously described Mamu-B*08-restricted epitopes by eight of nine amino acids.

Defining CD8⁺ T cell epitopes using peptide-specific CD8⁺ T cell lines

Previous methods used to define Mamu-B*08-restricted SIV epitopes were also utilized in this investigation (7). First, we generated peptide-specific CD8⁺ T cell lines. After several weeks of in vitro culture to increase specificity, we examined these cell lines in IFN-γ/TNF-α intracellular cytokine staining (ICS) assays with Mamu-B*08 transfectants to verify that immunogenic peptides were restricted by Mamu-B*08. These peptide-specific CD8⁺ T cell lines were used in similar ICS assays to confirm the minimal optimal epitope for each viral region. CD8⁺ T cell lines were cultured with media containing 100 U of IL-2/ml (National Institutes of Health AIDS Research and Reference Reagent Program).

MHC class I tetramer and surface staining

MHC class I tetramers were constructed for the six novel SIV epitopes with minor modifications as previously described (38, 43). We performed MHC class I tetramer stains on PBMC and CD8⁺ T cell lines to verify minimal optimal epitopes as previously described (38, 42).

Sequencing of plasma viral RNA (vRNA)

SIVmac239 sequencing data was obtained from other studies involving SIV-infected Mamu-B*08-positive macaques (7, 21). Viral sequencing was performed based on methods described previously (7, 21, 28, 44).

Sequencing of endogenously bound Mamu-B*08 ligands indicates peptide binding similarity to HLA-B27

With the emergence of SIV-infected Mamu-B*08-positive rhesus macaques as a model to study elite control of HIV replication, we decided to characterize a detailed peptide-binding motif of Mamu-B*08. We first defined a preliminary motif for Mamu-B*08 using an endogenous Mamu-B*08 peptide pool purified from Mamu-B*08 transfectants. By subjecting ten percent of the eluted Mamu-B*08 peptide pool to 14 cycles of N-terminal Edman degradation, we determined the dominance of particular amino acids at each position in the peptide pool. The pool sequencing analysis identified strong signals for R in position 2 and L at the C terminus (data not shown). We then fractioned the remaining peptide pool by reverse-phase HPLC into ∼40 peptide-containing fractions. From the various fractions spanning the HPLC chromatogram, 10 Mamu-B*08 ligands were selected and sequenced by MS/MS fragmentation (Table I⇓). In agreement with the binding motif determined by Edman sequencing, all ten peptides contained an R at position 2, and 8 of the 10 possessed L at the C terminus. The majority (8 of 10) of the peptides sequenced were nonamers, while the remaining two ligands were decamers.

We also tested the panel of sequenced ligands for their capacity to bind to Mamu-B*08. The ten endogenous peptides bound Mamu-B*08 with IC₅₀ values ≤165 nM, including eight that bound with much higher affinities of 17 nM or better (Table I⇑). All ten of these natural ligands also bound HLA-B*2705 with affinities ≤500 nM, indicating that the binding similarities of Mamu-B*08 and HLA-B*2705 extend beyond epitope sequence similarity.

Determination of Mamu-B*08 peptide-binding motif using a positional scanning combinatorial peptide library

To complement the pool sequencing analysis and derive a more detailed quantitative motif for Mamu-B*08, we next tested the capacity of a PSCL to bind purified Mamu-B*08 molecules. The Mamu-B*08 binding capacity (IC₅₀ nM) for each mixture was determined, and then normalized, as described in the Materials and Methods, to generate the resulting Mamu-B*08 matrix shown in Table II⇓. Analysis of the matrix using previous established criteria (40) confirmed position 2 and the C terminus as the main anchor positions for Mamu-B*08 binding. The second position was especially critical for binding with a SF of 28.4.

In agreement with the pool sequencing derived motif, at position 2 only R was preferred (Table II⇑). All other residues were associated with normalized average relative binding (ARB) values <0.05. At the C terminus, a broader range of residues was permitted. The PSCL analysis identified the aromatic residue F as the most preferred followed by the hydrophobic aliphatic residues L and I with ARB values of 0.673 and 0.375, respectively. The basic residue K and the hydrophobic residue M were tolerated with ARB values in the 0.1–0.12 range. All other residues were associated with affinities >10-fold lower than the optimal residue, F.

Influences on binding at other positions tended to be minor, reflected by relatively low SF and SD. In fact, none of the positions met the criterion we have utilized to define dominant secondary anchors (see Ref. 40 and Materials and Methods), and only position 1, associated with a SD of 2.7, appears to be a weak secondary anchor. In position 1, the basic residues R, H, and K, as well as M and S, were the most preferred, while the acidic residues D and E were two of the most deleterious.

In summary, we used the PSCL to define a detailed motif for Mamu-B*08. This motif was characterized by primary anchor specificity for the basic residue R in position 2 and a preference for hydrophobic (F, L, I, and M) and basic (K) residues at the C terminus (Fig. 2⇓).

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FIGURE 2.

Similarity of primary and secondary anchor positions among Mamu-B*08, Mamu-B*03, and HLA-B*2705. Primary and secondary anchor positions were defined using the PSCL matrix data (Table II and supplemental Table II),⁵ as described in Materials and Methods. Primary anchor positions are identified by bold font and blue shading. Dominant secondary positions are indicated by bold font and green shading, whereas weak secondary positions are highlighted by orange shading. Also shown are the most preferred residues at the corresponding position. In the case of the primary anchors, residues associated with an ARB > 0.1 are shown. At secondary positions, residues with an ARB > 0.3 are shown.

Identification of SIV-derived Mamu-B*08 binding peptides

PSCL-based matrices offer an efficient means to identify MHC class I binding peptides and the ability to identify nonmotif binders (40, 41, 45, 46, 47, 48, 49). To identify SIV-derived Mamu-B*08 binders, we used the Mamu-B*08 PSCL matrix to score all 9-mer peptides in the SIVmac239 proteome. After synthesizing the 9-mer peptides scoring in the upper 2.5% range (n = 82), we tested them for binding. An additional set of 50 lower scoring peptides, randomly selected to provide five peptides to represent each successive 10% scoring range, were also synthesized and tested. All of the SIV peptides and their accompanying binding data can be found in supplemental Table I.⁵ We discovered that 49 (60%) of the 82 peptides selected bound Mamu-B*08 with an affinity of ≤500 nM, including 16 peptides that bound with affinities of 10 nM or better. By contrast, none of the 50 peptides with scores beyond the upper 2.5% bound Mamu-B*08 with affinities ≤ 500 nM.

Both the endogenous ligands (Table I⇑) as well as the previously described Mamu-B*08-restricted SIV epitopes (7) demonstrated that Mamu-B*08 is not restricted to binding only nonamers. Thus, we applied the PSCL binding matrix, as described in Materials and Methods, to predict SIV-derived 8-, 10-, and 11-mer binders. For each size (8-, 10-, and 11-mer), the upper 2.5% scoring peptides (n = 82 for each size), plus an additional set of 50 peptides from poorer scoring ranges, were synthesized and tested for binding. In total 35 (42.7%), 58 (70.7%), and 37 (45.1%) of the 8-, 10-, and 11-mer peptide sets, respectively, bound with affinities of ≤500 nM. As with the 9-mer peptide set, none of the peptides from the lower scoring ranges bound with affinities ≤500 nM. Overall, considering all four size sets together, 179 of the 328 (54.6%) peptides scoring in the top 2.5% range were Mamu-B*08 binders. In the course of subsequent analyses probing lower scoring ranges, we found several additional Mamu-B*08 binders. In total, we identified 210 SIV-derived Mamu-B*08 binders (IC₅₀ ≤ 500 nM) from the 862 SIV-derived peptide candidates (supplemental Table I).⁵

Remarkable overlap in the peptide binding repertoires of Mamu-B*08 in comparison with HLA-B*2705

Given the similarities between epitopes restricted by Mamu-B*08, Mamu-B*03, and HLA-B*2705, we next sought to compare their respective PSCL-derived motifs, as well as investigate the extent, if any, that their repertoires overlapped. For this comparison, we created a PSCL matrix describing Mamu-B*03 binding (supplemental Table II-A).⁵ We also used the previously reported PSCL matrix-based motif for HLA-B*2705 (40) (supplemental Table II-B).⁵

The PSCL binding matrices indicated that Mamu-B*08, Mamu-B*03, and HLA-B*2705 share very similar primary anchor specificities. Position 2 was identified as the dominant anchor position for all three MHC class I molecules with an overwhelming preference for the positive residue R (Fig. 2⇑). Unlike Mamu-B*08, a second main anchor position was not identified for either Mamu-B*03 or HLA-B*2705. Instead, the C terminus of Mamu-B*03 was defined as a dominant secondary anchor. In this position, the most preferred residues were L, F, and I, representing a specificity pattern very similar to that of Mamu-B*08. Position 1 was also identified as a dominant secondary anchor and comparable to the Mamu-B*08 specificity, a preference for basic residues was noted. For HLA-B*2705, positions 1 and 3 as well as the C terminus were identified as weak secondary anchor positions. At the C terminus, the most preferred residues for HLA-B*2705 were F, R, K, L, I, V, and M, representing an overall pattern that resembles Mamu-B*08 (and Mamu-B*03) specificity, as well as previously published motifs for HLA-B*2705 (8, 9, 10, 11). Comparison of the specificity at the N terminus (P1) also revealed similarity to the preferences observed for Mamu-B*08 and -B*03 (Fig. 2⇑).

To determine whether these similar motifs would translate into overlapping binding repertoires, we assessed the binding patterns using 899 peptides. The 899 peptides tested included the SIV-derived peptides (supplemental Table I),⁵ the Mamu-B*08 endogenous ligands (Table I⇑), and a number of known HLA-B27-restricted T cell epitopes and endogenous ligands described in the literature (15, 50, 51, 52, 53, 54) (Table III⇓). We evaluated each peptide for its capacity to bind Mamu-B*08, Mamu-B*03, and HLA-B*2705 (Table IV⇓). Of the 899 peptides, 363 (40.4%) bound at least one of the three MHC class I molecules with an IC₅₀ ≤ 500 nM. The degree of cross-reactivity between binders (IC₅₀ ≤ 500 nM) was exceptionally high, as 241 (66.4%) bound more than one of the MHC class I molecules tested, including 167 (46%) that bound all three molecules. We also discovered the cross-reactivity pattern to be fairly uniform across the three MHC class I molecules. Specifically, between 63 and 85% of the repertoire of any allele overlapped with the repertoire of another allele. These levels of overlap are remarkable, and even higher than those typically seen among alleles within the same HLA supertypes (34, 55, 56, 57, 58). Correspondingly, monospecificity was low with only 7% of the Mamu-B*08 binders, 14% of the Mamu-B*03 binders, and 25% of the HLA-B*2705 binders failing to bind to another of the three MHC class I molecules.

Of the previously reported HLA-B27 epitopes, we found that 19 of the 24 peptides that bound HLA-B*2705 with an affinity ≤ 500 nM also bound Mamu-B*08 (Table III⇑). Fourteen of these epitopes bound both Mamu-B*03 and -B*08, again demonstrating the similar peptide binding specificity between Mamu-B*08, Mamu-B*03, and HLA-B*2705. Overall, this shared specificity is reflected in an extraordinary degree of repertoire overlap between the three MHC class I molecules.

Defining six novel SIV epitopes restricted by Mamu-B*08

The 500 nM threshold has been used to define candidate epitopes in previous SIV epitope screens (38, 59, 60, 61, 62) and has also been shown to be associated with in vivo T cell recognition in human, macaque, and murine systems (39, 63, 64, 65). On this basis, we examined whether the SIV-derived Mamu-B*08-binding peptides identified in the binding studies above were recognized in vivo using fresh PBMC from SIV-infected Mamu-B*08-positive macaques. Using IFN-γ ELISPOT assays, we tested seven macaques at varying disease courses in the chronic phase of SIV infection (>35 wk post-SIV infection). No Mamu-B*03-positive macaques were available for SIV immunogenicity testing because of the low frequency (<1%) of this allele in Indian rhesus macaque colonies (25).

We evaluated the antigenicity of 208 SIV-derived peptides in duplicate wells of two independent experiments. Seventy-five of these peptides (36%) demonstrated functional reactivity in at least one of the seven SIV-infected macaques (supplemental Table I).⁵ We also assessed two uninfected Mamu-B*08-positive macaques in IFN-γ ELISPOT assays. None of the peptides demonstrated reactivity in either of these control animals (data not shown).

We found a number of responses against previously characterized Mamu-B*08 epitopes in several macaques. Responses against Vif_123–131RL9, Vif_172–179RL8, Rev_12–20KL9, and Nef_137–146RL10 were the most prevalent and detected in at least five of the seven SIV-infected macaques (Fig. 3⇓). The Nef_137–146RL10-specific CD8⁺ T cell response was typically one of the strongest responses and exceeded 800 SFC/10⁶ PBMC in three macaques in agreement with previous data (7). In comparison, Rev_44–51RL8, Nef_8–16RL9, and Nef_246–254RL9 appeared subdominant during the chronic phase of SIV infection. We detected these responses in less than half of the macaques and at much lower levels, typically <200 SFC/10⁶ PBMC.

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FIGURE 3.

Ex vivo whole PBMC IFN-γ ELISPOT results of Mamu-B*08-restricted minimal optimal CD8⁺ T cell epitopes. Freshly isolated PBMC from seven Mamu-B*08-positive SIV-infected macaques were tested with Mamu-B*08 binding peptides in IFN-γ ELISPOT assays. Animals were tested during the chronic phase of SIV infection (>35 wk post-SIV infection). Results shown represent the 13 SIVmac239 epitopes known to be restricted by Mamu-B*08. Mean values and SDs from triplicate wells were calculated for each assay. Background (the mean of wells without peptide) levels were subtracted from each well. Mean responses < 50 SFC per 1 × 10⁶ PBMC were not considered positive. Assay results are shown as SFC per 1 × 10⁶ PBMC. Nef RL9* refers to the epitope at positions 8–16 in Nef, whereas Nef RL9 refers to the Nef epitope at positions 246–254. The SDs of Nef RL10 in r02019 (1522 SFC/10⁶ PBMC) and Nef RL10 in r99006 (1753 SFC/10⁶ PBMC) were 197 and 140, respectively.

Several responses were also directed against viral regions that did not contain epitopes known to be restricted by Mamu-B*08, warranting further investigation. After excluding the peptides that had considerable overlap (at least 6 aa) with the seven previously defined Mamu-B*08-restricted epitopes, 14 immunogenic viral regions, located in Gag, Pol, Vpx, Vpr, Env, and Nef, remained. We generated CD8⁺ T cell lines against 13 of the 14 novel regions. A peptide-specific CD8⁺ T cell line could not be generated against Nef_16–24LR9, likely because of its low frequency in a small number of macaques (data not shown).

Using the peptide-specific CD8⁺ T cell lines in conjunction with Mamu-B*08 transfectants in IFN-γ/TNF-α ICS assays, we established that 6 of the 14 regions with reactivity in ELISPOT assays were CD8⁺ T cell responses restricted by Mamu-B*08. Given that Indian rhesus macaques express upward of 12 or more MHC class I alleles (22, 66), it was not surprising that several of the novel responses detected in ELISPOT assays were restricted by other MHC class I molecules. We then used the Mamu-B*08-restricted, peptide-specific CD8⁺ T cell lines to identify the minimal optimal epitopes for cases where more than one peptide was reactive for that region. Minimal confirmation occurred by testing the functional avidity on peptide-specific CD8⁺ T cell lines and constructing MHC class I tetramers (data not shown). As with the previous seven epitopes, all six novel responses contained an R at position 2. Of the six novel epitopes, one was located in Gag (Gag_263–271YL9), one in Vpr (Vpr_62–70IF9), and four in Env (Env_524–532KF9, Env_573–581KL9, Env_717–725LF9, and Env_868–876RL9). Like the Nef_137–146RL10 epitope, the Env_573–581KL9 epitope was previously identified in Mamu-B*03-positive SIV-infected macaques (23, 67).

Similar to the previously identified SIV epitopes, the newly identified epitopes bound with extremely high affinity to Mamu-B*08. Overall, 10 of the 13 Mamu-B*08-restricted epitopes bound Mamu-B*08 with affinities ≤ 50 nM (Table V⇓). Interestingly, all 13 Mamu B*08 epitopes identified to date were among the top 0.58% scoring peptides corresponding to their size in the PSCL matrix-based predictions described above. For instance, all 10 of the SIV 9-mer peptides restricted by Mamu-B*08 would have been found by generating just 20 of the ∼3330 possible 9-mers. Of the 13 Mamu-B*08-restricted epitopes, 9 and 10 peptides, respectively, were found to bind HLA-B*2705 and Mamu-B*03 with affinities ≤ 50 nM. Overall, only one peptide failed to bind all three MHC class I molecules with an IC₅₀ ≤ 500 nM.

In comparison to responses directed against Vif_123–131RL9, Vif_172–179RL8, Rev_12–20KL9, and Nef_137–146RL10, the six novel responses appeared to be subdominant during the chronic phase of SIV infection (Fig. 3⇑). We detected these responses in up to three animals and typically at lower magnitudes. Interestingly, the SIV epitope in Gag p27 (Gag_263–271YL9) overlaps with an immunodominant CD8⁺ T cell epitope found in HLA-B*27-positive humans infected with HIV-1 (Gag_263–272KK10) and HIV-2 (Gag_263–272RK10) (68, 69, 70). However, unlike the immunodominance of this Gag epitope observed in HIV-infected individuals (13, 15, 18, 19, 20), we found Gag_263–271YL9-specific CD8⁺ T cells in only two of the seven SIV-infected animals (r99006 and r00078) at frequencies of 80 and 305 SFC/10⁶ PBMC, respectively (Fig. 3⇑).

Impact of viral variation in Mamu-B*08-restricted CD8⁺ T cell epitopes on Mamu-B*08 binding capacity

Previously, it has been shown that Mamu-B*08-specific CD8⁺ T cells selected for viral variation in several SIV epitopes (7, 21). We therefore tested the Mamu-B*08 binding capacity of a number of in vivo viral variants to determine whether they reduced the MHC class I peptide binding to Mamu-B*08 (Table VI⇓).

In general, the variant peptides studied were associated with substantial decreases in Mamu-B*08 binding capacity, with only a few having affinities within 3-fold of the WT epitope. Many of these peptides did not exhibit variation at position 2 and the C terminus, the primary anchor positions for Mamu-B*08 binding, and bound with a biologically relevant affinity of ≤500 nM (Table VI⇑). When variation was identified at the primary anchor positions, these peptides typically bound weakly, if at all, with the majority having affinities > 500 nM. We tested a number of peptides with variation in the primary anchor positions in IFN-γ ELISPOT assays. In comparison to the WT peptides, we found the variants to be greatly reduced in magnitude, and in some instances, no reactivity was observed (data not shown). However, ELISPOT is limited in its ability to test CD8⁺ T cell recognition of viral variants (71, 72). In the future, more extensive testing will be necessary to thoroughly examine the impact of these SIV variants on cellular recognition.

Although variation was found in the Nef_137–146RL10 epitope, these mutations did not greatly affect the binding capacity. In this case, as previously reported (7), an alanine to proline substitution at the residue immediately preceding the N terminus of the Nef_137–146RL10 epitope appears to affect peptide processing. A similar alanine to proline substitution immediately preceding the N-terminal residue of the epitope has also been shown to inhibit processing of an HLA-B57-restricted peptide in Gag of HIV (73).

The majority of the previously defined epitopes exhibited a wide range of viral variation. However, at the time of this study, we detected viral variation in only one of the newly discovered epitopes, Env_524–532KF9 (Table VI⇑). In this case, an F to Y substitution at the C terminus of Env_524–532KF9 reduced binding > 40-fold from 7.2 to 321 nM. The lack of viral variation was particularly surprising for the Env_573–581KL9 epitope. Viral escape from Env_573–581KL9-specific CD8⁺ T lymphocytes was previously found in Mamu-B*03-positive SIV-infected macaques during the chronic phase of SIV infection (23). However, we observed no such variation in our SIV-infected Mamu-B*08-positive infected macaques, despite detecting Env_573–581KL9-specific CD8⁺ T cells in three of the seven macaques tested during the chronic phase of infection. Two of these responses were >600 SFC/10⁶ PBMC (Fig. 3⇑).