We have sequenced the Pan troglodytes class I (Patr) molecules from three common chimpanzees and expressed them as single molecules in a class I-deficient cell line. These lines were utilized to obtain purified class I molecules to define the peptide binding motifs associated with five different Patr molecules. Based on these experiments, as well as analysis of the predicted structure of the B and F polymorphic MHC pockets, we classified five Patr molecules (Patr-A*0101, Patr-B*0901, Patr-B*0701, Patr-A*0602, and Patr-B*1301) within previously defined supertype specificities associated with HLA class I molecules (HLA-A3, -B7, -A1, and -A24 supertypes). The overlap in the binding repertoire between specific HLA and Patr class I molecules was in the range of 33 to 92%, depending on the particular Patr molecule as assessed by the binding of HIV-, hepatitis B virus-, and hepatitis C virus-derived epitopes. Finally, live cell binding assays of nine chimpanzee-derived B cell lines demonstrated that HLA supertype peptides bound to Patr class I molecules with frequencies in the 20–50% range.

Preclinical studies to evaluate the efficacy of vaccine candidates may include nonhuman primate models such as chimpanzees. These models are attractive for several reasons. Chimpanzees are closely related to humans from an evolutionary standpoint. As such, they can be experimentally infected with a number of human viral pathogens, such as hepatitis B and C viruses (HBV and HCV)3 (1, 2), HIV (3), and Rous sarcoma virus (4), as well as bacterial and protozoal infections, such as leprosy and tuberculosis (5). Some viral infections, such as HBV, follow a clinical course very similar to the human disease (1). Chimpanzees provide the only known animal model for HCV infection, and animals develop a chronic infection (2). Thus, chimpanzees may provide an excellent disease model system both for the disease progression, as well as for vaccine testing. In contrast to the attractive features of chimpanzee models of human disease, relatively little information is available regarding the frequency and specificity of chimpanzee class I and class II MHC molecules. Thus, to allow identification of potential ligands for immune response monitoring as well as for rational design of experimental vaccines, a need exists to define the peptide binding motifs of common Pan troglodytes class I (Patr) molecules.

The organization and structure of MHC genes are also conserved in humans and both chimpanzee species, P. troglodytes (the common chimpanzee) and Pan paniscus (the pygmy chimpanzee or bonobo) (6, 7). All three species have orthologous class I MHC loci designated A, B, and C that display a remarkable degree of allelic variation. Most of the nucleotide diversity is clustered in exons 2 and 3 encoding the α1 and α2 domains of class I MHC proteins, and facilitates binding of a constellation of self and non-self peptides important for CD8+ T cell control of tumors and intracellular parasites. Diversity is generated in a two-step process involving point mutation followed by recombination events that include allele and gene conversions (8). Of all nonhuman primate species, the extent of class I allelic diversity is most thoroughly characterized in populations of captive common chimpanzees. Remarkably, the majority of nucleotide polymorphisms that define the A, B, and C loci are conserved in humans and chimpanzees, indicating that they were transmitted from a common ancestor some 7–10 million years ago (9). Although these genes have no general species-defining characteristics, it is also true that identical class I alleles have not yet been found in humans and chimpanzees. Nevertheless, domains of the class I MHC peptide binding groove are highly conserved in the most closely related molecules found in human and chimpanzee species, suggesting the potential for presentation of an overlapping or identical set of peptides to CD8+ T cells. This is illustrated by a study of common and pygmy chimpanzees (10). Although these species diverged about 2.5 million years ago, their class I complexes, designated Patr (P. troglodytes) and Papa (P. paniscus), encode alleles that present a viral peptide across the species barrier. Thus, Patr-A*0401 and Papa-A*06 class I molecules differed by six amino acids, but both presented a peptide derived from HCV with equal efficiency to a CD8+ CTL line derived from an infected common chimpanzee (10).

This similarity was further underlined by recent studies that demonstrated that HLA supertypes extend to chimpanzees (11). Specifically, it was shown that peptides characterized by cross-reactive binding capacity for multiple common members of the human HLA-A2 or -B7 supertypes also frequently bound with appreciable affinity to chimpanzee-derived BCL lines. In the series of experiments described herein, we sought to confirm and expand these observations, utilizing a larger panel of radiolabeled peptide ligands. Our goals were to identify the specific Patr class I molecules capable of cross-binding human HLA supertype peptides and to establish their peptide binding motifs.

EBV-transformed B cell lines (BCL) had previously been established from PBMC of nine healthy young adult chimpanzees as described (11). BCL were grown in culture medium consisting of RPMI 1640 medium with HEPES (Life Technologies, Rockville, MD) supplemented with 10% FBS, 4 mM l-glutamine, 50 μM 2-ME, 0.5 mM sodium pyruvate, 100 μg/ml streptomycin, and 100 U/ml penicillin (Gemini BioProducts, Calabasas, CA).

Peptides were either synthesized at Epimmune (San Diego, CA) as previously described (12) or purchased as crude material from Mimotopes (San Diego, CA). Peptides synthesized at Epimmune were purified to >95% homogeneity by reverse phase HPLC. The purity of these synthetic peptides was assayed on an analytical reverse-phase column, and their composition was ascertained by mass spectrometry analysis.

Isolation of total RNA from BCL, first-strand cDNA synthesis, and Patr class I amplification was performed as described elsewhere (13). Patr class I genes amplified by PCR with primers specific for products of the A, B, or C loci were cloned into the expression plasmid PBJ1neo (14) and bidirectional DNA sequencing was performed by fluorescent dye termination using an ABI 377 automated sequencer (Perkin-Elmer Biosystems, Foster City, CA). A consensus sequence was derived by sequencing at least three clones per allele, and a clone identical with the consensus sequence was then selected for transfection into the class I-deficient human BCL 721.221. Detailed methods for transfection of these cells have been described in detail (15). Briefly, 10–20 μg of plasmid DNA was electroporated into 721.221 cells (Model 600 ECM electroporation system; BTX, San Diego, CA). Cells were then cultured for 2 days in RPMI containing 30% FCS. After that, cells were maintained in RPMI supplemented with Geneticin (1.5 mg/ml of active drug; Life Technologies). Cultures were then screened for actively growing transfectants and sorted by flow cytometry using the fluorescein-conjugated mAb W6/32 (16).

Transfected 721.221 cells expressing single Patr molecules were used as the source of class I molecules. Lysates were prepared from cell pellets, and class I molecules were purified as previously described (17). Briefly, cells were grown in roller bottles and lysed at a concentration of 108 cells/ml in 50 mM Tris-HCl, pH 8.5, containing 1% Nonidet P-40 (Fluka Biochemika, Buchs, Switzerland), 150 mM NaCl, 5 mM EDTA, and 2 mM PMSF. The lysates were passaged through 0.45-μM filters and cleared of nuclei and debris by centrifugation at 10,000 × g for 20 min. MHC molecules were then purified by affinity chromatography. Columns of inactivated Sepharose CL4B and protein A-Sepharose (Amersham Pharmacia Biotech, Piscataway, NJ) were used as precolumns. Subsequently, a column of the anti-HLA (A, B, and C) mAb W6/32 (18) was used to capture Patr molecules. Protein purity and concentration were monitored by SDS-PAGE.

Quantitative assays for the binding of peptides to soluble class I molecules on the basis of the inhibition of binding of a radiolabeled standard probe peptide to detergent-solubilized MHC molecules were performed as previously described (17). The various peptides used as radiolabeled probes are shown in Table I. Briefly, 1–10 nM radiolabeled probe peptide, iodinated (125I; Amersham, Piscataway, NJ) by the chloramine T method, was coincubated at room temperature with various amounts of MHC in the presence of 1 μM human β2-microglobulin (Scripps Laboratories, San Diego, CA) and a cocktail of protease inhibitors (containing a final concentration of 1.07 mg/ml EDTA, 62.5 μg/ml pepstatin A, 325 μg/ml phenanthroline, 250 μg/ml PMSF, and 60 μg/ml TLCK (Sigma, St. Louis, MO, and Calbiochem, La Jolla, CA)). At the end of a 2-day incubation period, the percent of MHC-bound radioactivity was determined by size exclusion gel filtration chromatography on a TSK 2000 column (TosoHaas, Montgomeryville, PA), or by capture on mAb W6/32-coated Flash plates (NEN, Boston, MA) counted on a TopCount instrument (Packard, Meriden, CT).

Table I.

Peptides used as radiolabeled probes for binding to chimpanzee class I molecules

PeptideSequencePrototype HLA SpecificityReference
A3Con1 KVFPYALINK A3 19 
Hu J chain 102–110 YTAVVPLVY A1 20 
Hu J chain 5–13 APRTLVYLL B7 21 
A24Con1 AYIDNYNKF A24 19 
PeptideSequencePrototype HLA SpecificityReference
A3Con1 KVFPYALINK A3 19 
Hu J chain 102–110 YTAVVPLVY A1 20 
Hu J chain 5–13 APRTLVYLL B7 21 
A24Con1 AYIDNYNKF A24 19 

In the case of competitive assays, the concentration of peptide yielding 50% inhibition of the binding of the radiolabeled probe peptide was calculated. Peptides were usually tested at one or two high doses, and the IC50 of peptides yielding positive inhibition were determined in subsequent experiments, in which two to six further dilutions were tested, as necessary. MHC concentrations yielding ∼15% binding of the radiolabeled probe peptide were used for all competitive inhibition assays. Because under these conditions [label] < [MHC] and IC50 ≥ [MHC], the measured IC50 values are reasonable approximations of the true Kd values. Each competitor peptide was tested in two to four completely independent experiments. Unlabeled probe peptide was also tested in each experiment, and its IC50 was measured as a positive control for inhibition.

This assay was performed as a modification of the assay described by Bertoni et al. (11). BCL were washed once in complete RPMI with 5% FBS and incubated overnight at room temperature to increase the class I expression on the cell surface. The next morning, the cells were washed twice in RPMI without FBS, and resuspended in the same medium with human β2-microglobulin at a final concentration of 3 μg/ml. Ninety-six-well tissue culture plates were prepared containing, in duplicate wells, 105 cpm radiolabeled peptide, 10-fold serially diluted competitor peptides (from 10 μg/ml down to 100 pg/ml final concentration), and a protease inhibitor mixture as described above. Finally, 2 × 106 BCL were added to each well, and the plates were incubated at 20°C for 4 h. At the end of the incubation period, the excess label was removed by washing the cells three times with RPMI, and free and cell-bound peptides were separated by centrifugation of the cells through a FBS gradient. The cells were then counted on a gamma counter.

As a prelude to the molecular definition of Patr molecules capable of binding prototype HLA supertype peptides, the specific class I MHC molecules expressed by BCL derived from three chimpanzees, Wilma (1558), Hoss (1530), and Barry (1564), were identified by PCR amplification with Patr A- and B-specific primers, as previously described (13). Remarkably, 10 of 11 class I A and B alleles identified in these animals (Table II) were described previously in other studies of chimpanzees housed at a variety of facilities in Europe and North America. The only unique allele was Patr-A*0602 (GenBank accession number AF302080), a subtype that differs from Patr-A*0601 by one nonsynonymous nucleotide substitution in exon 2.

Table II.

Patr alleles expressed by three chimpanzee-derived BCL lines

Chimpanzee BCLPatr Molecules
1558 A*0901 
 A*1401 
 B*0101 
 B*0301 
  
1530 A*0101 
 A*0701 
 B*0802 
 B*2401 
  
1564 A*0602 
 A*0701 
 B*0901 
 B*1301 
Chimpanzee BCLPatr Molecules
1558 A*0901 
 A*1401 
 B*0101 
 B*0301 
  
1530 A*0101 
 A*0701 
 B*0802 
 B*2401 
  
1564 A*0602 
 A*0701 
 B*0901 
 B*1301 

Class I Patr molecules were isolated by standard affinity chromatography methods from the single Patr transfectants described above. These purified class I molecules were then tested for their capacity to bind the prototype HLA supertype peptides listed in Table I. The peptides chosen represent four of the nine major HLA supertype binding specificities currently known (22). More specifically, the A3Con1 peptide (19) was utilized as prototype A3 supertype binder. In addition, the binding capacity of the Hu J chain 102–110 and A24Con1 peptides, chosen as representative ligands of the recently described A1 and A24 supertypes (22), respectively, were also investigated. Finally, the previously described B7-specific natural ligand, Hu J chain 5–13 (21), was chosen as the prototype B7 supertype ligand.

Specific signals were detected in the case of several Patr molecule/radiolabeled peptide combinations, with 10–15% of the labeled peptide bound by as little as 1–10 nM concentrations of purified class I molecules in most cases. More specifically, Patr-A*0101 bound the A3 Con1 peptide (Fig. 1,A). Patr-B*0901 bound Hu J chain 102–110 (Fig. 1,B), and Patr-B*1301 selectively bound the Hu J chain 5–13 ligand (Fig. 1,C). Weaker binding was also noted for the A24Con1 peptide to Patr-A*0701 (Fig. 1 D). In all of these instances, the binding was inhibitable by excess unlabeled peptide, with IC50 in the range of 0.4 - 165 nM (data not shown).

FIGURE 1.

Binding of radio-labeled HLA supertype peptides to representative Patr class I molecules. Radio-labeled A3Con1 (▴), Hu J chain 102–110 (♦), Hu J chain 5–13 (▵), and A24Con1 (○) were incubated with purified preparations of Patr-A*0101 (A), -B*0901 (B), -B*1301 (C), or -A*0701 (D), as described.

FIGURE 1.

Binding of radio-labeled HLA supertype peptides to representative Patr class I molecules. Radio-labeled A3Con1 (▴), Hu J chain 102–110 (♦), Hu J chain 5–13 (▵), and A24Con1 (○) were incubated with purified preparations of Patr-A*0101 (A), -B*0901 (B), -B*1301 (C), or -A*0701 (D), as described.

Close modal

Additional experiments showed that the prototype HLA-A*0101 Hu J chain 102–110 peptide also bound Patr-A*0602, and the A3Con1 peptide also weakly bound Patr-A*0901 and -A*0602 (data not shown). No binding was observed to Patr-A*1401, -B*2401, or -B*0101 molecules with the set of radiolabeled probes utilized. Thus, the ligand specificity of these molecules remains undetermined. In conclusion, these results illustrate the establishment of molecular Patr/peptide binding assays, which can be utilized to analyze in detail the functional binding specificity of several Patr class I molecules.

To define the peptide binding motifs of the various chimpanzee-derived class I molecules described above, panels of single amino acid substituted analogues of the various prototype peptide ligands were tested. For this analysis, a main anchor residue is arbitrarily defined as a residue in which >50% of the substitutions tested are associated with a greater than 10-fold change in binding capacity. Likewise, a secondary anchor is defined as a residue in which some (but 50% or less) of the substitutions tested are associated with a >10-fold change in binding capacity.

The specificity of the Patr-A*0101 class I molecule was analyzed first. Nonconservative amino acid substitutions (K or D) were introduced at every position of the A3Con1 peptide, and their binding capacity was quantified in the Patr-A*0101 binding assay (Fig. 2 A). Decreases in binding affinity of >10-fold were detected only at the C terminus.

FIGURE 2.

Peptide binding motifs of Patr-A*0101 and HLA-A*0301. Single amino acid substitutions of the A3Con1 peptide (KVFPYALINK) were used as competitive inhibitors of binding of radio-labeled A3Con1 to Patr-A*0101 (A) and HLA-A*0301 (B). Binding is expressed as relative to the binding of A3Con1 (0.7 nM). The amino acid substitution and position of the substitution (P1–P10) are indicated.

FIGURE 2.

Peptide binding motifs of Patr-A*0101 and HLA-A*0301. Single amino acid substitutions of the A3Con1 peptide (KVFPYALINK) were used as competitive inhibitors of binding of radio-labeled A3Con1 to Patr-A*0101 (A) and HLA-A*0301 (B). Binding is expressed as relative to the binding of A3Con1 (0.7 nM). The amino acid substitution and position of the substitution (P1–P10) are indicated.

Close modal

To better define anchor specificity, the effect of six additional substitutions was investigated at both position two and the C terminus. At position two, all substitutions tested, apart from aspartic acid (D), had little or no effect (all analogues bound within 10-fold of the wild-type peptide). By contrast, at position 10, a conservative replacement of the C-terminal K with an R led to increased binding capacity, whereas most other substitutions were associated with 10- to 1000-fold decreases in binding affinity. Interestingly, alanine was also tolerated at the C terminus. In conclusion, the positively charged C termini of the peptide represents the main Patr-A*0101 anchor residue, and position 2 is defined as a secondary anchor.

For the sake of comparison, the same set of substitutions was also tested for their capacity to bind the human class I molecule, HLA-A*0301, which is a prototype molecule of the HLA-A3 supertype (Fig. 2 B). In agreement with previous studies (19), significant effects were noted at position 2 and the C terminus.

In position two, whereas aromatic or hydrophobic substitutions had no effect or even increased binding capacity, the two charged residues K and D were associated with decreased binding, and modest decreases were also noted with the polar N substitution. At the C terminus, a binding pattern very similar to that observed with Patr-A*0101 was noted. The only significant difference was associated with the A substitution, which was associated with higher binding capacity in the case of Patr-A*0101, but had little effect on HLA-A*0301 binding. In addition, a significant decrease in binding was also noted for the D substitution at position 1, which is a secondary anchor residue for HLA-A*0301 binding (23). Taken together, these results illustrate how a similar, but clearly distinct, fine binding specificity is associated with the two class I molecules Patr-A*0101 and HLA-A*0301.

The binding motifs associated with the two alleles Patr-B*0901 and Patr-A*0602 were analyzed next, following the same strategy outlined above. These two alleles are of interest because they both bind Hu J chain 102–110, a naturally occurring high affinity ligand of HLA-A*0101, the prototype molecule of the A1-supertype (22). Nonconservative substitutions (K) were introduced at every position of the Hu J chain 102–110 peptide, and the binding capacity of the corresponding peptides was quantified in Patr-B*0901 and Patr-A*0602 binding assays (Fig. 3). Greater than 10-fold decreases in binding affinity were detected at positions 2 and the C terminus of both molecules, suggesting their potential role as main peptide-binding anchors. In addition, >10-fold effects were also detected in positions 3, 7, and 8 for Patr-B*0901 and 3, 6, and 7 for Patr-A*0602. We speculated that these positions might act as secondary anchor residues.

FIGURE 3.

Peptide binding motifs of Patr-B*0901 and -A*0602. Single amino acid substitutions of the Hu J chain 102–110 peptide (YTAVVPLVY) were used as competitive inhibitors of binding of radio-labeled Hu J chain 102–110 to Patr-B*0901 (A) and Patr-A*0602 (B). Binding is expressed as relative to the binding of Hu J chain 102–110 (6.4 nM for Patr-B*0901 and 0.4 nM for Patr-A*0602). The amino acid substitution and position of the substitution (P1–P9) are indicated.

FIGURE 3.

Peptide binding motifs of Patr-B*0901 and -A*0602. Single amino acid substitutions of the Hu J chain 102–110 peptide (YTAVVPLVY) were used as competitive inhibitors of binding of radio-labeled Hu J chain 102–110 to Patr-B*0901 (A) and Patr-A*0602 (B). Binding is expressed as relative to the binding of Hu J chain 102–110 (6.4 nM for Patr-B*0901 and 0.4 nM for Patr-A*0602). The amino acid substitution and position of the substitution (P1–P9) are indicated.

Close modal

To better define the anchor specificity, the effect of 5–10 additional substitutions at various positions was investigated. In the case of Patr-B*0901 (Fig. 3 A), small residues such as S or T were preferred in position 2. A substitution of A was tolerated but associated with about 10-fold lower binding capacity. All other substitutions at this position were associated with >100-fold decreases in binding capacity. At the C termini, aromatic (W, F, and Y) and hydrophobic (L) residues were associated with increased binding capacity, whereas all other substitutions tested (N, D, A, and K) were associated with decreases in binding capacity of 100-fold or greater.

Position 3 appeared to function as a secondary anchor, with predilection for negatively charged residues (D and E). Significant decreases were also noted for the F and K substitutions. An important role was also ascribed to positions 7 and 8, which appeared to prefer negatively charged or amide (position 7) or uncharged (position 8) residues, respectively. Overall, the Patr-B*0901 peptide binding specificity closely resembles the known binding specificity of the human HLA-A*0101 allele, which is associated with a preference for S or T in 2, D or E in 3, and aromatic residues at the C termini (19).

The specificity of Patr-A*0602 was examined next. For this allele, position 2 was less important, and only D, N, and K substitutions were not accepted. By contrast, position 9 was very selective, and all substitutions tested at this position led to 100- to 10,000-fold decreases in binding. Position 3 was also an important anchor for Patr-A*0602, with most substitutions negatively impacting binding. Furthermore, a very prominent role in determining binding capacity appeared to be played by positions 6 and 7, where most of the substitutions tested also impacted binding. At position 6, only P was tolerated, whereas, at position 7, only L was tolerated.

Based on these results, we conclude that Patr-B*0901 and Patr-A*0602 share overlapping but yet distinct peptide binding motifs. In particular, the motif recognized by the Patr-B*0901 molecule is most similar to the motif associated with the human class I molecule HLA-A*0101. The motif associated with Patr-A*0602 has also several unique features, including the apparent lack of strict dependence on position 2 as an anchor, and the reliance of positions 3, 6, and 7 as additional potentially main anchors.

Additional experiments were designed to define the peptide binding motifs recognized by Patr-B*1301 and Patr-A*0701. First, the specificity of Patr-B*1301 was defined following a strategy similar to the one described above for the other Patr class I molecules (Fig. 4 A). Position 2 and the C terminus were mapped as the main anchor residues, and position 7 and possibly 6 were identified as secondary anchors. Notably, only P was allowed at position 2, and all other substitutions were associated with >100-fold decreases in binding capacity. At the C terminus, a broad specificity for either aromatic or aliphatic residues was detected. It was noted that this main anchor specificity is essentially identical with the known binding specificity of the human class I molecule HLA-B*0702, in particular, and the B7-supertype in general (21). Additionally, the Patr-B*1301 motif described is in accordance with previously described HCV epitopes targeted by CD8+ T cells from an infected chimpanzee (16).

FIGURE 4.

Peptide binding motifs of Patr-B*1301 and -A*0701 molecules. A, Single amino acid substitutions of the Hu J chain 5–13 peptide (APRTLVYLL) were used as competitive inhibitors of binding of radiolabeled Hu J chain 5–13 to Patr-B*1301. Binding is expressed as relative to the binding of Hu J chain 5–13 (1.3 nM). The amino acid substitution and position of the substitution (P1–P9) are indicated. B, Single amino acid substitutions of the A24Con1 peptide (AYIDNYNKF) were used as competitive inhibitors of binding of radio-labeled A24Con1 to Patr-A*0701. Binding is expressed as relative to the binding of A24Con1 (164.4 nM). The amino acid substitution and position of the substitution (P1–P9) are indicated.

FIGURE 4.

Peptide binding motifs of Patr-B*1301 and -A*0701 molecules. A, Single amino acid substitutions of the Hu J chain 5–13 peptide (APRTLVYLL) were used as competitive inhibitors of binding of radiolabeled Hu J chain 5–13 to Patr-B*1301. Binding is expressed as relative to the binding of Hu J chain 5–13 (1.3 nM). The amino acid substitution and position of the substitution (P1–P9) are indicated. B, Single amino acid substitutions of the A24Con1 peptide (AYIDNYNKF) were used as competitive inhibitors of binding of radio-labeled A24Con1 to Patr-A*0701. Binding is expressed as relative to the binding of A24Con1 (164.4 nM). The amino acid substitution and position of the substitution (P1–P9) are indicated.

Close modal

As described above, the Patr-A*0701 molecule was found to bind the A24Con1 peptide (Fig. 1,D). A number of different single amino acid substitutions of the A24Con1 peptide were synthesized and tested for binding in the Patr-A*0701 binding assay (Fig. 4 B). Greater than 10-fold effects were noted for positions 2 and 6 and the C terminus. The most drastic effects were seen at position 2 and the C terminus, which were designated as primary anchor residues, and were associated with a specificity for aromatic/aliphatic residues in 2, and aliphatic or F at the C terminus. Position 6 appeared to function as a secondary anchor residue, with positive effects noted in the case of hydrophobic residues (F, L, and A), and detrimental effects associated with charged substitutions (D and K). This motif is similar to the previously described HLA-A*2402 motif (19, 24).

The data presented in the preceding sections define peptide motifs specific for several Patr class I molecules. These motifs, summarized in Table III, share several general features in common with specific HLA supertype motifs, summarized in Table IV. To determine whether these peptide binding specificities could be correlated with structure, the Patr residues predicted to form the B and F pockets (25) were tabulated, and compared with the B and F pockets of HLA class I molecules whose peptide binding specificity was also known (Table V). For this analysis, we examined the polymorphic residues known to form the B and F pockets of HLA-A*02, as originally defined by Saper et al. (26). A structural model of the HLA-A*0201 molecule can be accessed online in the Protein Data Bank at http://www.rcsb.org/pdb/(PDB ID, 3HLA).

Table III.

Summary of allele-specific motifs of Patr class I molecules

Patr AlleleResidueCorresponding HLA Supertype
12345678C terminus 9/10
A*0101  a       RKA A3 
B*0901  ST ∗    DN[EQ]b ∗ WFLY A1 
A*0602  ∗ ANS[QT]   L[IVM]  A1 
B*1301      ∗  FLMIV B7 
A*0701  YMPFL    ∗   LVAFMI A24 
Patr AlleleResidueCorresponding HLA Supertype
12345678C terminus 9/10
A*0101  a       RKA A3 
B*0901  ST ∗    DN[EQ]b ∗ WFLY A1 
A*0602  ∗ ANS[QT]   L[IVM]  A1 
B*1301      ∗  FLMIV B7 
A*0701  YMPFL    ∗   LVAFMI A24 
a

∗, Secondary anchor position.

b

Bracketed residues are hypothesized as acceptable anchors on the basis of chemical similarity.

Table IV.

HLA supertype motifs corresponding to the Patr motifs identified in Table III (reviewed in Ref. 22 )

HLA SupertypeResidue
12345678C terminus 9/10
A*03  AVILMST       RK 
A*01  STIVLM       YWF 
B*07        ALIMVFWY 
A*24  FWYLVIMT       FIYWLM 
HLA SupertypeResidue
12345678C terminus 9/10
A*03  AVILMST       RK 
A*01  STIVLM       YWF 
B*07        ALIMVFWY 
A*24  FWYLVIMT       FIYWLM 
Table V.

Comparison of binding pocket residues for HLA and Patr molecules

AlleleB Pocket PreferenceB Pocket ResiduesAlleleF Pocket PreferenceF Pocket Residues
94563666770778081116
Patr-B*0901 ST Patr-A*0101 RKA 
HLA-A*0101 TSM HLA-A*0301 KRY 
Patr-B*0101 Unknown F M E N M S Patr-A*0401-2 Unknown D T L D 
Patr-B*1301 Patr-A*0602 
HLA-B*0702 HLA-A*0101 
Patr-A*0701 YMPFL Patr-A*0601, -A*1101 Unknown N T L D 
HLA-A*2402 YFW Patr-B*0901 WFLY 
Patr-A*0401-2, Unknown S M E S V S HLA-A*2402 FLIW N I A Y 
A*0601, -A*0901        Patr-B*0101 Unknown N I A Y 
        Patr-B*1301 FMLIV 
        HLA-B*0702 LIVM 
        Patr-B*1601, -B*1701 Unknown S N L Y 
AlleleB Pocket PreferenceB Pocket ResiduesAlleleF Pocket PreferenceF Pocket Residues
94563666770778081116
Patr-B*0901 ST Patr-A*0101 RKA 
HLA-A*0101 TSM HLA-A*0301 KRY 
Patr-B*0101 Unknown F M E N M S Patr-A*0401-2 Unknown D T L D 
Patr-B*1301 Patr-A*0602 
HLA-B*0702 HLA-A*0101 
Patr-A*0701 YMPFL Patr-A*0601, -A*1101 Unknown N T L D 
HLA-A*2402 YFW Patr-B*0901 WFLY 
Patr-A*0401-2, Unknown S M E S V S HLA-A*2402 FLIW N I A Y 
A*0601, -A*0901        Patr-B*0101 Unknown N I A Y 
        Patr-B*1301 FMLIV 
        HLA-B*0702 LIVM 
        Patr-B*1601, -B*1701 Unknown S N L Y 

In the case of the B pocket (Table V), it was noted that class I molecules with a preference for S or T, such as Patr-B*0901 and HLA-A*0101, were associated with the consensus motif of M at residue 45, E at residue 63, N at residue 66, and M at residue 67. Interestingly, Patr-B*0101, whose peptide binding specificity in unknown, shares the same consensus residues in the B pocket, and thus would be predicted to share the same preference for binding small residues. Alleles with a preference for P (Patr-B*1301 and HLA-B*0702) were also associated with a common B pocket structural motif (Y 9, E 45, N 63, I 66, Y 67, and Q 70). No other Patr molecules have been identified with an identical pocket structure. Finally, Patr-A*0701 and HLA-A*2402 share a similar binding pocket, with identity of S 9, M 45, E 63, and V 67. The B pocket of Patr-A*0701 lacks the positively charged residues seen at positions 66 (K) and 70 (H) of HLA-A*2402, which may account for its ability to accommodate a larger repertoire of residues. A similar pattern of residues was also observed in Patr-A*0401, -A*0402, -A*0601, and -A*0901. These alleles would be predicted to share a preference for hydrophobic or aromatic residues with Patr-A*0701. In fact, three of four minimal optimal HCV epitopes presented to CD8+ CTL by either Patr-A*0601 or -A*0901 have a tryptophan at position 2 (C.M.W., unpublished data).

In the case of the F pocket (Table V), alleles with a preference for positively charged residues at the C terminus (Patr-A*0101 and HLA-A*0301) share an identical F pocket, lined with D at residue 77, T at residue 80, L at residue 81, and D at residue 116. Patr-A*0401 and -A*0402 also share an identical F pocket, and would be predicted have a preference for peptides with positively charged residues at the C terminus.

Patr-A*0602 and HLA-A*0101, which have a shared preference for Y at the C terminus, share an identical F pocket with N 77, T 80, L 81, and D 116. This exact pattern is also seen in Patr-A*0601 and -A*1101, which would thus be predicted also to be specific for peptides with Y at the C terminus. A different pattern of F pocket residues is seen in Patr-B*0901 and HLA-A*2402, which share N 77, I 80, and A 81, and have either D or Y at position 116. These alleles have similar specificity in terms of C-terminal residues of their peptide ligands. Patr-B*0101 shares an identical binding pocket with HLA-A*2402, and would be expected to show a preference for similar residues.

Finally, an identical pattern of F pocket residues is seen for Patr-B*1301 and HLA-B*0702, which bind peptides ending with hydrophobic nonpolar residues. The residues lining this pocket are S 77, N 80, L 81, and Y 116. Patr-B*1601 and -B*1701 also share this pocket structure, and would be predicted to bind peptides with hydrophobic residues at the C terminus. It should also be noted that Patr-B*1301 and HLA-B*0702 are identical in both the B and F pockets. In conclusion, the similarities between HLA and Patr molecules at the level of binding motifs and described in the preceding sections are also mirrored by structural similarities in the B and F bonding pockets of the same HLA and Patr molecules.

Based on the data presented above, it may be hypothesized that the repertoire of peptides bound by certain chimpanzee and human class I molecules would overlap significantly. To test this theory, we measured the capacity of known HLA supertype epitopes to bind appropriate Patr alleles. The Patr-B*1301 allele, whose peptide binding motif was shown above to be similar to the one recognized by the human HLA-B*0702 molecule, was investigated first. Specifically, nine HBV-derived peptides carrying the HLA-B7 supermotif, and capable of binding to multiple HLA-B7 supertype molecules (27), were tested for their capacity to bind Patr-B*1301. When the results (Table VI) were compared with previously published data regarding the capacity of the same peptides to bind purified HLA-B*0702, a large overlap became apparent. In fact, all of the peptides that bound HLA-B*0702 with IC50 of <500 nM, also bound Patr-B*1301 (Table VI).

Table VI.

Cross-reactivity between selected HLA and Patr class I molecules: binding of HBV-derived HLA B7 motif-bearing peptides to Patr-B*1301

ProteinPeptide SequenceIC50 (nM)
B*1301B*0702
Env 313 IPIPSSWAF 0.5 42 
X 58 LPVCAPSSA 3.6 115 
Pol 640 YPALMPLYA 8.4 306 
Pol 429 HPAAMPHLL 15.7 56 
Core 19 LPSDFFPSV 29.1 1774 
Pol 530 FPHCLAFSYM 40.9 56 
Pol 640 YPALMPLY 209 a 
Pol 640 YPALMPLYACI 361 1375 
Pol 541 FPHCLAFSY 545 — 
ProteinPeptide SequenceIC50 (nM)
B*1301B*0702
Env 313 IPIPSSWAF 0.5 42 
X 58 LPVCAPSSA 3.6 115 
Pol 640 YPALMPLYA 8.4 306 
Pol 429 HPAAMPHLL 15.7 56 
Core 19 LPSDFFPSV 29.1 1774 
Pol 530 FPHCLAFSYM 40.9 56 
Pol 640 YPALMPLY 209 a 
Pol 640 YPALMPLYACI 361 1375 
Pol 541 FPHCLAFSY 545 — 
a

—, IC50 > 10,000 nM.

Similar experiments were performed with larger sets of peptides derived from human disease targets for which chimpanzees have been utilized as possible animal models (HBV, HIV, and HCV). In all cases, the capacity of known HLA supertype binding epitopes to bind a Patr allele associated with a similar or overlapping peptide binding motif was measured. The results of this analysis are summarized in Table VII. A significant degree of overlap was noted in the case of the Patr-A*0101 and the HLA-A3 supertype, where in total 23 of 25 (92%) HLA-A*0301-binding peptides also bound Patr-A*0101. A high degree of overlap was also noted in the case of Patr-B*1301 and HLA-B*0702, where 12 of 13 (92%) HLA-B*0702-binding peptides also bound to Patr-B*1301. The overlap in peptide binding between Patr-B*0901 and HLA-A*0101 was also considerable, as 12 of 21 (57%) of HLA-A*0101 peptides also bound Patr-B*0901. An intermediate degree of overlap was noted in the case of the Patr-A*0701 and HLA-A*2402, where 7 of 14 (50%) HLA-A*2402 binders also bound Patr-A*0701. Finally, a lower degree of overlap was noted in the case of the Patr-A*0602 and the HLA-A*0101, where only 8 of 21 HLA-A*0101 binders (38%) also bound Patr-A*0602. This is not surprising, as the position 2 and 3 specificities of Patr-A*0602 and HLA-A*0101 are not as tightly correlative as noted for the other Patr/HLA overlaps described above.

Table VII.

Cross-reactivity between selected HLA and Patr class I molecules: number of epitopes binding to a given HLA molecule and cross-reactive on Patr molecules of similar specificity

HLA MoleculeA*0301B*0702A*0101A*0101A*2402
Patr MoleculeA*0101B*1301B*0901A*0602A*0701
Peptide origin      
HCV 7 /8 1 /2 5 /7 2 /7 1 /4 
HBV 8 /8 5 /5 5 /10 4 /10 1 /1 
HIV 8 /9 6 /6 2 /4 1 /4 5 /9 
      
Total 23/25 (92%) 12/13 (92%) 12/21 (57%) 7/21 (33%) 7/14 (50%) 
HLA MoleculeA*0301B*0702A*0101A*0101A*2402
Patr MoleculeA*0101B*1301B*0901A*0602A*0701
Peptide origin      
HCV 7 /8 1 /2 5 /7 2 /7 1 /4 
HBV 8 /8 5 /5 5 /10 4 /10 1 /1 
HIV 8 /9 6 /6 2 /4 1 /4 5 /9 
      
Total 23/25 (92%) 12/13 (92%) 12/21 (57%) 7/21 (33%) 7/14 (50%) 

A previous study (11) has demonstrated that peptides characterized by the capacity to bind multiple common members of the HLA-A2 or -B7 supertypes, also frequently bind, with appreciable affinity, to chimpanzee-derived BCL. Herein, we sought to expand these observations to the HLA-A3, -A1, and -A24 supertypes, utilizing a panel of chimpanzee-derived lines previously characterized for their capacity to bind HLA-A2 and -B7 supertype peptides by Bertoni and colleagues (11). Accordingly, radiolabeled HLA-A1, -A3, and -A24 prototype peptides (Table I) were tested for their capacity to bind live chimpanzee BCL.

The results obtained are summarized in Table VIII. Significant binding was noted for various peptide/BCL combinations with absolute bound cpm values ranging from 1032 to 8533 cpm. Binding of the HLA-A3 supertype binder was detected in 5 of 9 (56%) of the cell lines tested. Likewise, binding of the HLA-A1 and -A24 supertype binders was detected in 2 of 9 (22%) and 5 of 9 (56%) of the cell lines tested. The binding was specific, in that no label bound all lines. Rather, every label and cell line was associated with a unique binding pattern.

Table VIII.

Binding of 125I-labeled HLA prototype peptides to chimpanzee BCL

Chimp BCLSignal (IC50)
A3Con1Hu J Chain 102–110A24Con1
1558 a — — 
1530 1867 (31) — 3105 (33) 
1564b 5307 (385) 8131 (4.9) 2933 (53) 
1580b — — 4875 (25) 
1573b — 8533 (1.3) 2687 (33) 
1578 — — 2970 (41) 
5829 1032 (270) — — 
5835 1732 (182) — — 
5867 3099 (219) — — 
Chimp BCLSignal (IC50)
A3Con1Hu J Chain 102–110A24Con1
1558 a — — 
1530 1867 (31) — 3105 (33) 
1564b 5307 (385) 8131 (4.9) 2933 (53) 
1580b — — 4875 (25) 
1573b — 8533 (1.3) 2687 (33) 
1578 — — 2970 (41) 
5829 1032 (270) — — 
5835 1732 (182) — — 
5867 3099 (219) — — 
a

—, Signal <1000 cpm or IC50 >10000 nM. Values are calculated as averages of at least two independent experiments.

b

Chimps 1564 and 1580 are brothers, and chimp 1573 shares the sire of 1564 and 1580. All other chimps are apparently unrelated.

In all cases, the binding could be inhibited by excess unlabeled peptide. The IC50 values recorded in such inhibition experiments are also shown in Table VIII, in parentheses. These values ranged between 1.3 and 385 nM, indicating that a range of different affinities was detected. A general correlation was also noted, as expected, between the magnitude of the signal (in terms of bound cpm) and the affinity of interaction (estimated by IC50 values). In conclusion, the results presented herein demonstrate that HLA-A3, -A1, and -A24 prototype supertype peptides bind with appreciable frequencies to Patr molecules on chimpanzee cells isolated from chimpanzees in primate centers.

In the series of experiments described herein, the Patr A and B alleles expressed from three chimpanzee-derived EBV-immortalized lines were identified by DNA sequencing. These alleles were then singly transfected into class I-deficient cell lines that were subsequently utilized as a source of purified MHC molecules. Binding assays for five of the Patr class I molecules were established using HLA supertype peptides that bind to HLA-A3, -B7, -A1, and -A24 molecules.

These results allow, for the first time, a glimpse of the peptide motifs associated with specific Patr class I molecules. The general features of these motifs were similar to those associated with human HLA class I molecules. Position 2 and the C terminus played, in most cases, the role of main anchors, with residues 3, 6, 7, and 8 playing the role of additional, mostly secondary, anchors. Positions 4–5 appeared to be the most permissive in terms of class I binding, and probably correspond to positions protruding away from the class I molecule and accessible for TCR recognition (25, 26).

The definition of peptide-binding motifs for Patr class I molecules will allow a search for chimpanzee-restricted disease-specific peptides predicted to bind to the various Patr alleles, and potentially induce an immune response against the disease of interest. In turn, this will allow more exact monitoring of CTL response, and also provide the molecular basis for the design and testing of epitope-based vaccines in chimpanzees.

Our results confirm the results of Bertoni et al. (11) that HLA supertypes extend to chimpanzees. Furthermore, our study extends these observations to include the HLA-A3, -A1 and -A24 supertypes. The present study unequivocally identifies some of the Patr molecules capable of cross-reacting with human HLA molecules at the level of peptide binding specificity.

In terms of specific motifs, the A3Con1 peptide, which binds several HLA molecules of the HLA-A3 supertype (23), also binds to purified chimpanzee Patr-A*0101. The motif defined for Patr-A*0101 overlaps considerably with the motif defined previously for HLA-A*0301, and differs primarily at position 2, where the chimpanzee class I molecule is associated with a less-stringent specificity.

The HLA-A1 prototype peptide, Hu J Chain 102–110 (20), bound to two purified Patr molecules, Patr-B*0901 and Patr-A*0602. Like HLA-A*0101, Patr-B*0901 preferred serine or threonine at position 2. In the case of Patr-A*0602, position 2 played the role of secondary anchor, and an unusual pattern of main anchors at positions 3 (alanine), 6 (proline), and 7 (leucine) was detected instead. The apparent main anchor role of positions 6 and 7 may be related to conformational constraints related to the presence of a P residue in position 6, and might be a peculiarity of this particular peptide ligand. Indeed, additional high affinity A*06 ligands already identified do not carry P in position 6 (data not shown).

Patr-B*1301 binds the B7 supertype binding peptide Hu J Chain 5–13 L7→Y. The Patr B*1301 molecule was cloned from cells from chimpanzee 1564, which bound the radiolabeled B7 peptide in the previous study (11). In this study, the B7 peptide bound only this single Patr molecule, as would be predicted from the previous results. The motif defined for Patr-B*1301 is virtually identical with the motif previously defined for HLA-B*0702 (28, 29), and consists of P at position 2 and aromatic or hydrophobic residues at the C terminus of the peptide. Finally, the HLA-A*2402-binding peptide, A24Con1, bound Patr-A*0701. The motif for this Patr molecule was shown to be indeed similar to that of HLA-A*2402.

We have also analyzed the residues lining the B and F pockets of various Patr class I molecules and compared them with the residues present in the same positions of various human HLA class I molecules. In many cases, similarities in both F and B pockets were detected, and these provide a structural basis for the molecular cross-reactivities detected at the level of binding specificity. This type of analysis should allow rational prediction of motifs for Patr molecules for which motifs have not been yet determined. For example, Patr-B*0101, a molecule utilized in our study, did not bind any of the radiolabeled HLA-restricted peptides. Examination of the residues lining the B pocket revealed a very similar pattern with HLA-A*0101 and Patr-B*0901 (Table V), molecules for which a motif of S or T at position 2 is known. The residues lining the F pocket were identical with HLA-A*2402. Thus, the motif for Patr-B*0101 would be predicted to have S or T at position 2 and F, L, I, or W at the C terminus. This prediction is now being tested experimentally.

In future studies, it might be of interest to elute naturally processed peptides from the same Patr class I molecules. This represents an alternative and complementary method to define MHC-specific motifs. Indeed, in a previous study, the motifs for HLA-A1, -A3, -A11, and -A24 were determined both by acid elution of peptides as well as by the method described herein (19). Equivalent results were reached by both methods, but the method based on peptide-binding assays allows a more accurate quantification of the binding potential of disease-specific epitopes.

It is interesting to interpret the results presented in this study in the context of known epitopes recognized by CD8+ T cells from HCV-infected chimpanzees. For instance, based on similar B and F pocket structures, HLA-A*2402 and Patr-B*0101 should bind peptides with T in position 2 and I in position 9. Patr-B*0101 does indeed present an epitope from the HCV nonstructural 3 (NS3) protein (YTGDFDSVI) that fits this prediction (30). Another NS3 epitope (VPHPNIEEV) has the position 2 (P) and 9 (V) motif that facilitates peptide binding to Patr-B*1301 and HLA-B*0702 molecules (30). Finally, a positively charged amino acid at the COOH terminus of a peptide serves as an anchor for binding to HLA-A*0301, Patr-A*0101, and probably Patr-A*0401. Earlier studies demonstrated that Patr-A*0401-restricted T cells target an HCV E2 epitope containing either K or R at its COOH terminus (30), and two of three Patr-A*0101-restricted HCV epitopes also contain a K residue at this position (data not shown). Thus, not only do human supertype motifs facilitate peptide binding to chimpanzee class I molecules in vitro, they probably also shape the immune response against virus infection in this animal model.

Conservation of peptide binding pockets is probably rooted in the evolutionary relationship between HLA, Patr, and Papa class I molecules. Six families of HLA-A alleles can be defined based on serologic cross-reactivities and shared nucleotide polymorphisms (31, 32, 33). Three families represented by the HLA-A9, -A80, and -A1/A3/A11 molecules probably derived from an ancient A3 lineage, whereas the other three families represented by HLA-A2, -A10, and -A19 derived from an ancient A2 lineage. Perhaps unexpectedly, all Patr-A alleles characterized to date are related to only the HLA-A1/A3/A11 family (6, 7, 34, 35, 36). This supports the concept of transspecies evolution involving transmission of MHC alleles from one species to the next (9), and indicates that chimpanzees may have inherited only part of the human allelic repertoire from a common ancestor. Evolution of MHC molecules in both species occurs by reassortment of point mutations through intra- and interlocus recombination (8). At least for the A locus, this process has been relatively slow (35), and could provide a partial explanation for common peptide binding pockets found in contemporary molecules such as HLA-A*0301 and Patr-A*0101.

Human and chimpanzee class I B alleles evolved somewhat more rapidly because of a higher rate of recombination at this locus (8, 37). Although this has obscured lineage relationships between class I B alleles from Homo sapiens and P. troglodytes, some domains display sequence similarities. This is most evident for the α-1 domain of HLA-B*0702 and related molecules such as Patr-B*1301 in common chimpanzees and Papa-B*01 and -B*04 in bonobos (37). A high degree of sequence homology in the α-1 domain, which forms the B pocket of the peptide-binding groove, indicates that these molecules arose from a common ancestor and that structural or functional constraints limit mutations. The α-2 domain comprising the F pocket is considerably more divergent in class I B molecules of chimpanzees and humans, although amino acid residues governing peptide binding are similar in HLA-B*0702 and Patr-B*1301 molecules (37). Why molecules that diverged several million years ago have conserved peptide-binding motifs is not clear, but we speculate that they might mediate effective immune responses against pathogens common to both species.

Finally, we would like to underline the implications of the present study for the design and testing of vaccines in chimpanzees. The high degree of cross-reactivity of human epitopes for certain Patr molecules may allow the utilization of human epitopes to monitor CTL responses in chimpanzees, as well as for the design of human epitope-based vaccines that could be tested for efficacy in chimpanzees. Based on similarities detected, we have also determined the degree of cross-reactivity between specific Patr and HLA molecules, in terms of their capacity to bind various epitopes, as candidates for inclusion in multiepitope vaccines.

A significant degree of cross-reactivity was detected between several Patr and HLA class I molecules. These data, together with the live cell binding presented here and in the previous study of Bertoni (11), suggest that the degree of cross-reactivity between HLA supertypes and Patr class I molecules will be appreciable. This provides further data to support the concept that the design and testing of experimental vaccines destined for human use in chimpanzees will be a valuable approach.

We thank Dr. Robert Purcell for providing us with the chimpanzee cells. We also thank Rhonda Skvoretz and Rose Corea for skilled technical assistance in Patr MHC purification and binding assays. The expert secretarial assistance of Denise Porter is also gratefully acknowledged.

1

This study was supported by National Institutes of Health Grants SBIR AI38620-03 and RO1 AI47367, and with federal funds from National Institute for Allergy and Infectious Diseases, National Institutes of Health, under Contract N01-AI-95362.

3

Abbreviations used in this paper: HBV, hepatitis B virus; HCV, hepatitis C virus; Patr, Pan troglodytes class I; Papa, Pan paniscus class I; BCL, B cell line.

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