The JI Acurri Cytometers
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     
 

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Data Supplement
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Sidney, J.
Right arrow Articles by Sette, A.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Sidney, J.
Right arrow Articles by Sette, A.
The Journal of Immunology, 2003, 171: 5964-5974.
Copyright © 2003 by The American Association of Immunologists

Simultaneous Prediction of Binding Capacity for Multiple Molecules of the HLA B44 Supertype 1

John Sidney*, Scott Southwood{dagger}, Valerie Pasquetto* and Alessandro Sette2,*

* Division of Translational Immunology and Biodefense, La Jolla Institute for Allergy and Immunology, San Diego, CA 92121; and {dagger} Epimmune Inc., San Diego, CA 92121


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
We selected for study a set of B44-supertype molecules collectively represented in >40% of the individuals in all major ethnicities (B*1801, B*4001, B*4002, B*4402, B*4403, and B*4501). The peptide-binding specificity of each molecule was characterized using single amino acid substitution analogues and nonredundant peptide libraries. In all cases, only peptide ligands with glutamic acid in position 2 were preferred. At the C terminus, each allele was associated with a unique but broad pattern of preferences, but all molecules tolerated hydrophobic/aliphatic (leucine, isoleucine, valine, methionine), aromatic (tyrosine, phenylalanine, tryptophan), and small (alanine, glycine, threonine) residues. Secondary anchor motifs were also defined for all molecules. Together, these features were used to define a B44 supermotif and a novel algorithm for calculating degeneracy scores that can be used to predict B44-supertype degenerate binders. Approximately 90% of the peptides with a B44 supermotif degeneracy score of >10 bound at least three of the six B44-supertype molecules studied with high affinity. Finally, a number of peptides derived from hepatitis B and C viruses, HIV, and Plasmodium falciparum have been identified that have degenerate B44 supertype-binding capacity. Taken together, these findings have important implications for epitope-based approaches to vaccination, immunotherapy, and the monitoring of immune responses.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Class I MHC molecules are extremely polymorphic, and in humans >700 HLA-A and -B allelic variants have already been described (1). At the functional level, it has been suggested that the majority of HLA class I variants can be classified into ~10 different major groups, or supertypes (2), characterized by overlapping peptide-binding motifs and repertoires. For example, previous studies have demonstrated that a number of HLA molecules, collectively designated as the A2 supertype, have similar peptide-binding motifs. The most common allelic molecules of the A2 supertype are A*0201, A*0202, A*0203, A*0206, and A*6802 when Caucasian, Black, and Oriental ethnicities are considered (2, 3, 4, 5, 6). Peptide epitopes (7, 8, 9, 10, 11, 12) derived from important pathogens, such as HIV, hepatitis B virus (HBV), 3 hepatitis C virus (HCV), and Plasmodium falciparum or cancer Ags, have been identified, which bind most or all of these allelic variants (degenerate binders). These supertopes are frequently recognized by exposed, infected, or immune individuals, demonstrating that they are indeed generated during the course of natural infection or cancer and that a repertoire of T cells capable of recognizing the corresponding epitope/HLA complex exists in humans (7, 8, 9, 10, 11, 12, 13, 14). The data obtained in the case of the A2 supertype have been extended to demonstrate that relevant supertopes can also be identified for the A3 and B7 supertypes (2, 4, 5, 7, 8, 15, 16, 17, 18, 19, 20). Finally, recent data suggest that HLA supertypic specificities extend to other primate species, such as chimpanzees, gorillas, and macaques (21, 22, 23, 24).

Previously, we predicted the existence of an HLA B44 supertype that encompasses molecules corresponding to the HLA B18, B40 (B60 and B61), B44, and B45 serological specificities (2, 4, 5). This prediction was based on reported peptide-binding motifs derived from sequencing pooled natural self ligands and sequences of individual self ligands and T cell epitopes. The structure of the B and F pockets of various MHC alleles was also considered in formulating this prediction. The predicted specificity of B44 supertype molecules is for acidic residues (glutamic acid and aspartic acid) in position 2, and hydrophobic or aromatic residues (leucine, isoleucine, valine, methionine, phenylalanine, tryptophan, tyrosine, and alanine) at the C terminus. More recently, similar motif specificities have been reported for the chimpanzee class I allele Patr A*16 (25) and the macaque class I molecule Mamu A*11 (21).

Until now, however, direct demonstration has been lacking that the apparent overlap in peptide-binding motifs of the B44-supertype molecules directly translates into overlapping peptide binding repertoires. This has been due in large part to the lack of quantitative binding assays to measure the binding capacity of potential peptide ligands to the various B44-supertype molecules. Furthermore, despite the availability of molecular binding assays for A2, A3, and B7 supertypes, bioinformatic methods allowing accurate predictions of peptides with supertype degenerate binding capacity has not been available.

In this study, we set out to establish quantitative assays for the most common molecules of the B44 supertype and to examine their associated peptide-binding motifs. We also wanted to determine whether it would be possible to identify peptide ligands capable of broad cross-reactivity with several B44-supertype molecules (supertopes), and if so, we were interested in defining the molecular basis for broad cross-reactivity.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Peptides

Peptides used were synthesized at Epimmune (San Diego, CA) as described elsewhere (26) or purchased as crude material from Mimotopes (Minneapolis, MN and Clayton, Victoria, Australia) or Pepscan (Lelystad, Netherlands). Peptides synthesized at Epimmune were typically purified to >95% homogeneity by reverse phase HPLC (26). Lyophilized peptides were resuspended at 4–20 mg/ml in 100% DMSO and then diluted to the required concentrations in 0.05% PBS (v/v), Nonidet P-40 (Fluka Biochemika, Buchs, Switzerland).

MHC purification

The EBV-transformed cell lines DUCAF (A*3002, B*1801), 2F7 (A*6801, B*4001), SWEIG (A*2902, B*4002), WT47 (A*3201, B*4402), PITOUT (A*2902, B*4403), and OMW (A*0201, B*4501) were used as the primary sources of MHC molecules. In some cases, to demonstrate assay specificity, MHC molecules were purified from 721.221 lines transfected with B*1801, B*4001, B*4002, A*0201, A*2902, A*3002, or A*3201 (Pure Protein LLD, Oklahoma City, OK) or an A*6801-transfected C1R line. Cells were maintained in vitro, and HLA molecules were purified from cell lysates as described elsewhere (27). Briefly, class I molecules were captured by repeated passage over protein A-Sepharose beads conjugated with the anti-HLA (A, B, C) Ab W6/32 (27). In some cases, HLA-A molecules were further purified from HLA-B and -C molecules by passage over a B1.23.2 (anti-HLA-B, -C, and some -A) (28, 29, 30, 31) column.

MHC-peptide-binding assays

Quantitative assays to measure the binding of peptides to soluble class I molecules are based on the inhibition of binding of a radiolabeled standard peptide, and were performed as previously described (27). Briefly, 1–10 nM concentrations of radiolabeled peptide was coincubated at room temperature with 1 nM to 1 µM of purified MHC in the presence of 1–3 µM human {beta}2-microglobulin (Scripps Laboratories, San Diego, CA) and a mixture of protease inhibitors. After a 2-day incubation, the percent of MHC-bound radioactivity was determined by size exclusion gel filtration chromatography using a TSK 2000 column. Alternatively, the percent of MHC-bound radioactivity was determined by capturing MHC/peptide complexes on W6/32 and/or B123.2 Ab-coated Optiplates (Packard Instrument, Meriden, CT), and measuring bound cpm using the TopCount (Packard Instrument) microscintillation counter.

Peptide 1420.12 (sequence SEIDLILGY), a sequence reported as a self peptide ligand of B44 molecules (32), was used as the radiolabeled probe and standard control for inhibition for the B*1801, B*4402, and B*4403 assays. The average IC50 values of this peptide in the B*1801, B*4402, and B*4403 assays were 3.1, 9.2, and 6.8 nM, respectively. For the B*4001 and B*4002 assays, peptide 1461.04 (sequence YEFLQPILL), a W1>Y analog of a peptide of unknown origin previously reported as a self peptide ligand of B40 (33), was used as the radiolabeled ligand and control for inhibition. The IC50 values of 1461.04 in the respective assays were 1.6 and 1.7 nM. The B*4501 radiolabeled ligand and control for inhibition was 1420.36, an artificial sequence (AEFKYIAAV) representing a consensus from analysis of pools of peptides eluted from B*4006 molecules (34); 1420.36 had an average IC50 of 4.9 nM in B*4501 assays. In the case of competitive assays, the concentration of peptide yielding 50% inhibition of the binding of the radiolabeled peptide was calculated. Peptides were tested at six different concentrations over a 100,000-fold range in three to five independent experiments.

Biochemical validation of assay specificity

The EBV-transformed cell lines used as the primary sources of purified MHC molecules express comparable amounts of A and B class I molecules. To validate the allelic specificity of the peptide-binding assays developed and to verify that the signals obtained were not due to the presence of contaminating amounts of HLA A molecules, MHC molecules purified from HLA A and/or B single allele-transfected cell lines were used in assay development studies. In these studies, the signal obtained in direct binding assays by a putative B loci ligand was compared with binding obtained using ligands for the coexpressed A molecule on either, or both (if available), the A or B allele (data not shown). Cross-inhibition studies were also performed, as well as inhibition of binding analyses using panels of peptides of known HLA-A or -B specificity.

More specifically, B*4001 molecules purified from the EBV-transformed homozygous clone 2F7 (A*6801, B*4001) bound peptide 1461.04 (sequence YEFLQPILL), a W1>Y analog of a peptide of unknown origin previously reported as an endogenously bound B40 ligand (33). The signal obtained could be readily inhibited by an excess amount of an unlabeled peptide. This binding was B*4001 specific because peptide 1461.04 did not bind MHC molecules purified from an A*6801 C1R line. Furthermore, the previously reported A*6801 high affinity binder 941.12 (17) was a poor inhibitor (IC50 > 10,000 nM) of 1461.04 binding (Table I). In a subsequent assay, two peptides reported as naturally processed ligands of B*40 molecules (33) were tested for their capacity to inhibit 1461.04 binding. Also tested were known ligands for other HLA-A and -B specificities (6, 17, 18, 32, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44). As shown in Table I, both peptides described as B*40 ligands inhibited 1461.04 binding with an IC50 of <25 nM. Conversely, none of the nine peptides with non-B44-supertype motifs inhibited 1461.04 binding with an IC50 of <10,000 nM.


View this table:
[in this window]
[in a new window]
  		Table I. HLA-B44 supertype binding capacity of various peptides of known HLA-A or -B specificity

 
In the case of B*4002, MHC molecules purified from the EBV-transformed homozygous cell line SWEIG (A*2902, B*4002) also bound peptide 1461.04. The 1461.04 signal obtained could be readily inhibited by an excess of unlabeled peptide. The B*4002 specificity of 1461.04 binding was demonstrated using A*2902 molecules purified from single class I allele C1R transfected cell lines. As shown in Table I, all three peptides previously reported as either natural B*40 ligands or as a B*4002 restricted CTL epitope inhibited 1461.04 binding with IC50 < 150 nM. Conversely, none of the nine peptides with non-B44-supertype motifs inhibited 1461.04 binding with high affinity (IC50 < 500 nM).

HLA B*4403 molecules purified from the EBV-transformed homozygous cell line PITOUT (A*2902, B*4403) bound peptide 1420.12 (sequence SEIDLILGY), an endogenous ligand of B44 molecules (32), and the HLA-B44-restricted epitope HIV gp1201–9 (sequence AENLWVTVY) (38). These peptides could readily inhibit the binding of each other but only poorly, if at all, the binding of a control A*2902 binding peptide (human J chain 101–109). Conversely, the human J chain 101–109 peptide did not inhibit the binding of either radiolabeled 1420.12 or gp1201–9. Specificity of the binding of human J chain 101–109 to A*2902 binding was further verified using A*2902 molecules purified from single MHC allele-transfected cells. As shown in Table I, six of eight peptides previously reported as endogenously bound to or restricted by HLA B*44 molecules bound with IC50 < 50 nM. Conversely, none of the nine ligands of other class I molecules bound with IC50 better than 500 nM.

HLA B*4402 molecules were purified from the EBV-transformed homozygous cell line WT47 (A*3201, B*4402) and, as in the case of B*4403, good binding was obtained using either the HLA-B44 epitope HIV gp1201–9 or peptide 1420.12, and the signal obtained could be inhibited by excess amounts of the unlabeled version of either peptide. Conversely, peptides previously identified as A*3201 ligands or epitopes could not inhibit binding (Table I) of either HIV gp1201–9 or peptide 1420.12, and neither HIV gp1201–9 or peptide 1420.12 could bind A*3201 molecules purified from single MHC allele-transfected cells.

A B*1801-specific binding assay was developed using class I molecules purified from the EBV-transformed homozygous cell line DUCAF (A*3002, B*1801) and peptide 1420.12. Specificity for B*1801 was confirmed using MHC purified from B*1801 and A*3002 single transfectants. Of 11 peptides, 6 reported as ligands for B*40 or B*44 molecules inhibited 1420.12 binding with IC50 of 10 nM or less (Table I). Conversely, of the nine peptides known to bind other HLA-A or B molecules, only the B7-supertype binder B35CON2 (16, 18) also bound B*1801. Additionally, five peptides previously reported as A*3002 ligands or epitopes were tested for their B*1801-binding capacity, and none of them bound with an IC50 of <30,000 nM (data not shown).

Finally, an HLA B*4501-specific binding assay was developed using MHC molecules purified from the EBV-transformed homozygous cell line OMW (A*0201, B*4501). Optimal binding to MHC purified with the B123.2 Ab was obtained using peptide 1420.36, an artificial sequence (AEFKYIAAV) representing a consensus from analysis of pools of peptides eluted from B*4006 molecules (34). No binding was detected in the same MHC preparations when HBV core 18–27 F6>Y, previously identified as a high affinity A*0201-binding peptide (3, 6, 26), was used as the radiolabeled ligand. Additional analyses, summarized in Table I, showed that 5 of 11 peptides previously identified as ligands or epitopes for B*40 or B*44 molecules could inhibit 1420.36 binding with IC50 < 500 nM. Conversely, none of the nine peptides with other motifs or known specificities bound B*4501 with an IC50 of <25,000 nM.

Bioinformatic analysis

In all assays, a relative binding value was calculated for each peptide by dividing the IC50 of the positive control for inhibition by the IC50 measured for each specific peptide tested. Standardized relative binding values also allow the calculation of a geometric mean, or average relative binding value (ARB), for all peptides with a particular characteristic (6, 17, 18, 26, 40, 45, 46, 47). Maps of secondary interactions influencing peptide binding to HLA B44-supertype molecules based on ARB were derived as previously described (6, 17, 18, 26, 40, 45, 46, 47). Essentially, all peptides of a given size (9 or 10 amino acids) and with at least 2 tolerated main anchor residues were selected for analysis. The binding capacity of peptides in each size group was analyzed by determining the ARB values for peptides that contain specific amino acid residues in specific positions. For determination of the specificity at main anchor positions, ARB values were standardized relative to the ARB of peptides carrying the residue associated with the best binding. For secondary anchor determinations, ARB values for peptides with a particular residue in a specific position were standardized relative to the ARB of the whole peptide set considered. Because of the rare occurrence of certain amino acids, for some analyses residues were grouped according to individual chemical similarities as previously described (6, 17, 18, 26, 40, 45, 46, 47).

This report describes for the first time an algorithm to predict degenerate binders of the B44 supertype. To predict degenerate binders, individual peptide sequences are scored using a coefficient matrix (see Results). As with other polynomial methods, the score for each peptide is calculated as the product of the coefficients corresponding to each peptide residue. On the basis of the product of the corresponding coefficients, peptides may be segregated into 4 categories, designated as positive, intermediate, low, and negative. These categories correspond to scores >=10, 1–10, 0.1–1, and <0.1, respectively.

The performance of predictive algorithms is compared using measures of sensitivity (SENS) and positive predictive value (PPV). SENS is the fraction of total possible positives that are identified using the specific algorithm or algorithm cut-off score, and PPV measures the fraction of peptides that are predicted to be positive that are actually positive. Accuracy, which is the fraction of correct predictions (true positives and true negatives), is also calculated, although this measure can be somewhat misleading if the data set used is skewed, or if the methodologies being compared have not been tested on an identical set of peptides. A further measure that is used for comparison purposes is EFF75, which is the efficiency of prediction (PPV) associated with selection of 75% of the total number of positive peptides.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Definition of a target set of common putative HLA B44-supertype molecules.

Previous studies (2, 4, 5) defined a putative B44 supertype encompassing various allelic variants of the B18, B37, B40, B41, B44, B45, B47, B49, and B50 Ags. The B44 supertype is defined by a shared peptide-binding specificity for peptides with the acidic residue glutamic acid in position 2 and a hydrophobic residue at the C terminus. However, whether these motif similarities correspond to significant overlaps in peptide binding repertoire was not addressed experimentally.

First, we selected a set of B44-supertype molecules representative of the alleles most frequent in major ethnic groups. Population-typing data collated from published sources (48) are summarized in Table II. On the basis of this data, the most frequent allele over all is B*4001. B*4001 is present in 24.6% of the Asian ethnicity and in >10%, on average, across the four major ethnicities studied. Two other alleles, B*4402 and B*4403, also have average frequencies of ~10%. B*4402 is notably frequent in the Black population (14.8%), whereas B*4403 is frequent in Caucasians (>16%). Three other alleles, B*1801, B*4501, and B*4002, representative of the B18, B45, and B61 Ags, respectively, are less frequent but still present in at least 7% of one or more major ethnic groups and >3% of the general population. All other alleles are found in <5% of all populations reported and <3% on average. Based on this analysis, a panel of six molecules (B*1801, B*4001, B*4002, B*4402, B*4403, and B*4501) predicted to be members of the HLA B44 supertype were selected for further studies defining their peptide-binding specificity and cross-reactivity.


View this table:
[in this window]
[in a new window]
  		Table II. Population frequencies of class I molecules potentially associated with an HLA-B44-supertype peptide-binding specificity

 
Determination of the peptide-binding motifs of common B44 supertype molecules using a panel of single substitution analogs

Initial binding assays (Table I) identified the HIV gp1201–9 peptide (sequence AENLWVTVY) as having the capacity to bind with high or intermediate affinity all of the molecules selected for study (B*4001, B*4002, B*4402, B*4403, B*1801, and B*4501). To define the primary motif associated with each molecule, a panel of analog peptides representing single amino acid substitutions of HIV gp1201–9 were synthesized and tested for their binding capacity relative to the binding capacity of the unsubstituted epitope (Fig. 1). As in previous studies (6, 18), in each position preferred residues were defined as those with relative binding (RB) of 0.1 or better. Residues with RB between 0.01 and 0.1 were defined as tolerated, and those with RB <0.01 were considered as nontolerated.



View larger version (34K):
[in this window]
[in a new window]
  		FIGURE 1. Binding capacity of single substitution analogs of HIV gp1201–9 to various molecules. Single amino acid substitutions of HIV gp1201–9 (sequence AENLWVTVY) were tested for their capacity to bind various HLA class I molecules predicted to be members of the B44 supertype. Binding data are expressed relative to the residue at each position with the highest binding affinity for the respective class I molecule. A dash indicates RB capacity <0.01. RB capacity values >=0.1 are highlighted in bold and indicate residues defined as preferred.

 
B*4001 The acidic residue glutamic acid was the only residue allowed in position 2. All other substitutions examined in this position were associated with a >100-fold reduction in binding capacity, demonstrating that B*4001 uses position 2 as a main anchor and has a very stringent B44 supermotif-like specificity for the acidic residue glutamic acid. At the C terminus, a preference for the aliphatic hydrophobic residues isoleucine, leucine, and methionine was noted. The small residues cysteine and valine were also preferred, but with somewhat lower binding capacity. Other small residues (alanine, threonine, and glycine), as well as aromatic (phenylalanine and tyrosine), polar (glutamine), and basic (histidine, arginine, and lysine) residues were tolerated, with RB in the 0.01–0.1 range. Significant influences on binding capacity were only rarely, if at all, noted at other positions.

B*4002 Again, at position 2 only glutamic acid was preferred or tolerated. All other substitutions examined were associated with a >100-fold reduction in binding capacity. At the C terminus, a preference for small (alanine and threonine) and aliphatic (leucine, isoleucine, valine, methionine) residues was noted. The aromatic residue phenylalanine, as well as the small residue glycine, was also preferred. Other noncharged substitutions (tyrosine, histidine, glutamine, tryptophan, serine, asparagine, and proline) were tolerated. At position 1, >100-fold reductions in binding capacity were noted in the context of the asparagine, aspartic acid, and proline substitutions. This pattern is similar to that observed in position 1 for other HLA molecules, including a majority of those in the B7 supertype (18), and is consistent with the designation of position 1 as an important B*4002 secondary anchor.

B*4403 For B*4403 it was also found that at position 2 only glutamic acid was preferred, and that all other substitutions were associated with a greater than 100-fold reduction in binding capacity. The C terminus was shown to have a preference for the large aliphatic residues methionine and isoleucine, and the aromatic residues tyrosine and phenylalanine. Other large residues (tryptophan, histidine, glutamine, and leucine), and the small residues alanine, glycine, and threonine were also preferred, but with RB in the 0.1–0.2 range. These data suggest that B*4403 is associated with a previously unappreciated broad specificity at the C terminus. Additional significant influences on binding capacity were only noted in 2 instances at position 1.

B*4402 At position 2, only glutamic acid was preferred or tolerated. At the C terminus a preference for the large aliphatic hydrophobic residues methionine, isoleucine, and leucine and the aromatic residues tyrosine, phenylalanine, and tryptophan was noted. Small (alanine, glycine, serine, threonine, cysteine, and valine), polar (glutamine, asparagine) and basic (histidine, arginine, and lysine) residues were tolerated, with RB between 0.01 and 0.07. Additional effects could be noted at positions 1, 3, and 4, where about one-half of the substitutions were associated with reductions in binding capacity in the 10- to 50-fold range, indicating their potential role as secondary anchors. Overall, the B*4402 motif appears distinct but remarkably similar to the peptide-binding motif defined above for B*4403 molecules.

B*1801 At position 2, only glutamic acid was preferred, but the polar residues serine, glutamine, and tyrosine, the acidic residue aspartic acid, and the aliphatic residue methionine were tolerated. All other substitutions tested were associated with >100-fold reduction in binding capacity. At the C terminus, only analogs carrying the aromatic residues tyrosine and phenylalanine (but not tryptophan) or methionine were preferred. All other substitutions were associated with a 100-fold or greater decrease in binding capacity.

B*4501 As with the other molecules examined, only glutamic acid was preferred at position 2. The aliphatic hydrophobic residue methionine was tolerated, but all other substitutions tested at positions 2 were associated with >100-fold reduction in binding capacity. Similar to the case with B*4403, influences associated with >100-fold reductions in peptide-binding capacity could not be noted at the C terminus. However, the small residues alanine and threonine were most preferred, and the next most preferred residues (glycine, serine, and valine) were also small. Interestingly, B*4501 binding capacity was found to also be dependent on the residue in position 1. Of nine substitutions tested, only the alanine residue was preferred.

Taken together, these data demonstrate that B*4001, B*4002, B*4402, B*4403, B*1801 and B*4501 all share a very stringent requirement for glutamic acid in position 2 of their peptide ligands. With the exception of B*1801, other residues are only rarely tolerated, and even then with relatively poor affinity. The presence of an additional primary anchor position was more difficult to identify, but the C terminus in each case appears to play an important role in determining peptide-binding capacity. Interestingly, and in contrast to position 2, in each case the specificity at the C terminus appears to be quite broad, with many residues being either tolerated or preferred. With the exception of position 1, only rarely were significant influences noted at other positions.

Library analysis of B44-supertype binding capacity

Next, a library of 549 peptides derived from various infectious disease and tumor Ags was assembled and tested in parallel for binding to B*1801, B*4001, B*4002, B*4402, B*4403, and B*4501. All of the peptides were between 8 and 13 residues long and had glutamic acid in position 2. At the C terminus, all peptides had phenylalanine, leucine, isoleucine, methionine, valine, alanine, tryptophan, tyrosine, glycine, or threonine, which are the residues noted in the single substitution analysis to be preferred in the context of two or more molecules.

Overall, between 22.8 and 46.6%, with an average of 34.6%, of the peptides in the library bound any one molecule. Peptides between 8 and 13 residues long bound with similar frequencies, although those 9–13 residues long were preferred. Examination of cross-reactivity patterns revealed that 37.0% of the peptides were degenerate, having the capacity to bind three or more molecules with an affinity of 500 nM or better. By contrast, in an additional library of 177 peptides with aspartic acid in position 2 and the same range of residues at the C terminus only between 0.6 and 9.0%, with an average of 3.6%, of peptides bound any one molecule. Furthermore, only 1.1% of the D2 peptides were degenerate (data not shown).

Next, the binding capacity associated with each C-terminal anchor residue was examined. The ARBs associated with peptides that bear a specific residue at the C terminus were calculated (Table III). These data are largely in agreement with patterns ascertained in the single substitution analyses, and indicate that each molecule is associated with a unique pattern of preferences. For example, B*1801 prefers the aromatic residues phenylalanine and tyrosine, B*4001 had a preference for the aliphatic residue leucine, and B*4501 had a preference for the small residues alanine and threonine. When considering the geometric mean of ARB across all six molecules, the aromatic residue phenylalanine and the aliphatic hydrophobic residues isoleucine and leucine had the highest ARB, although no residue was associated with an ARB of <0.24.


View this table:
[in this window]
[in a new window]
  		Table III. Differences and similarities in specificity of B44-supertype molecules for the C-terminal residue of their peptide ligandsa

 
To identify secondary anchor effects, the binding capacity of the library was further analyzed by determining the ARB values associated with the presence of specific residues in specific peptide positions (relative to the N terminus), as described in Materials and Methods. The resulting ARB values for each corresponding residue/position pair for 9- and 10-mer sequences are provided in Supplemental Data Tables S1–S64 for B*1801, B*4001, B*4002, B*4402, B*4403, and B*4501, respectively. Analysis of the data revealed that ARB <=0.33, or >=3, are roughly equivalent to 1 SD from the mean. Thus, for the present analyses, these thresholds have been used to indicate significant (positive or deleterious) influences on peptide-binding capacity. Summary maps, showing residue/position pairs with significant influences on binding capacity were also derived (Fig. 2) for 9- and 10-mer ligands.



View larger version (76K):
[in this window]
[in a new window]
  		FIGURE 2. Maps of the peptide-binding motifs of various B44-supertype molecules. Summary maps are of the peptide-binding motifs for 9-mer (a) and 10-mer (b) peptides for the B44-supertype molecules B*1801, B*4001, B*4002, B*4402, B*4403, and B*4501. At secondary anchor positions, residues shown as preferred (or deleterious) are associated with ARB values at least 3-fold greater than (or 3-fold less than) peptides of the same size carrying other residues at the same position. At the primary anchor positions, preferred residues are those associated with an average binding capacity within 3-fold of the optimal residue at the same position, and tolerated residues are those with an average binding capacity in the 0.01–0.33 range, as described for Table V; other residues are not allowed at the primary anchor positions.

 
Each molecule (and peptide size) was associated with a unique pattern of preferences, and in every position some secondary influence could be detected. However, common patterns were also readily discernable. Specifically, in the case of both 9- and 10-mer peptides, the majority (82.4%) of the negative influences involved the presence of acidic (aspartic acid and glutamic acid), basic (arginine, histidine, and lysine), polar (serine, threonine, glutamine, and asparagine) or proline residues. By contrast, in most instances (67.4%) preferred residues were aromatic (phenylalanine, tryptophan, or tyrosine) or hydrophobic (leucine, isoleucine, valine, or methionine). A preference for aromatic residues in position 3 was shared between 9- and 10-mers. Interestingly, many of the features identified are common to those noted in the context of A2-supertype molecules (6, 26).

Identification of HLA-B44 supertype cross-reactive epitopes (supertopes) and derivation of a detailed B44 supermotif

In the process of developing the peptide library used for the present study, conserved regions of the HBV, HCV, HIV, and P. falciparum genomes were screened to identify peptides with the B44 supermotif. A total of 177 peptides derived from these targets were synthesized and tested for binding. As a result, a number of B44 supertopes derived from important infectious disease Ags, and potentially useful for vaccine development, have been identified (Table IV). The data presented above (and Tables S1–S6 and Fig. 2) demonstrated that there are common patterns among the B44-supertype molecules with respect to secondary influences on peptide-binding capacity. On the basis of these observations, wesought to determine whether these commonalities could be used to predict B44-supertype degeneracy.


View this table:
[in this window]
[in a new window]
  		Table IV. Examples of peptides derived from various infectious agents associated with degenerate B44 supertype binding capacity

 
For this purpose we generated 9- and 10-mer specific coefficient matrices (Table V) representing the geometric mean of the ARB for each residue/position pair across the panel of B44-supertype molecules studied. For the C terminus, the coefficients represent the geometric mean values calculated when the entire library was analyzed. The resulting matrices were then used to provide coefficients to generate for each peptide in the library a Supermotif ARB (SARB) score by calculating the product of the coefficients corresponding to each peptide residue. Thus, for example, the peptide AEFKYIAAV was assigned a SARB of 41.6, representing the product of the A1(1.71), E2 (1.0), F3(4.36), K4(0.524), Y5(4.19), I6(2.94), A7(2.03), A8(1.71), and V9(0.249) coefficients. Fig. 3 provides a summary map highlighting B44-supertype ARB values with significant influences on binding capacity.


View this table:
[in this window]
[in a new window]
  		Table V. B44-supertype polynomial coefficients (SARB)

 


View larger version (20K):
[in this window]
[in a new window]
  		FIGURE 3. The B44 supermotif. Consensus summary of secondary and primary anchor influences on B44-supertype binding capacity of 9-mer (a) and 10-mer (b) peptides. At secondary anchor positions, residues shown as preferred (or deleterious) are associated with a geometric ARB value (across the set of 6 B44-supertype molecules tested) at least 3-fold greater than (or 3-fold less than) peptides of the same size carrying other residues at the same position. At the primary anchor positions, preferred residues are those associated with an average binding capacity within 3-fold of the optimal residue at the same position, as described in Table V.

 
Next, we analyzed whether the B44 SARB scores predicted the B44-supertype degeneracy of peptides in the analysis set. On the basis of SARB score, peptides were segregated into four categories, designated as positive, intermediate, low, and negative, corresponding to SARB scores >=10, 1–10, 0.1–1, and <0.1, respectively. As shown in Table VI, we found that these categories correlated with B44-supertype degeneracy. Specifically, 86.8% of the peptides with SARB >=10 were degenerate, compared with 59.8% of the intermediate, 19.8% of the low, and 3.4% of the negative peptides.


View this table:
[in this window]
[in a new window]
  		Table VI. SARB method efficiently identifies degenerate B44 supertype binders

 
Performance of the SARB method on a blind prediction

Test of the SARB method with an independent (blind) set of peptides that had not been used in any of the library analyses was also performed (Table VI). The blind set of peptides were derived from various human tumor-associated Ags, including carcinoembryonic Ag, MAGE 2, MAGE 3, p53, and Her2/neu. As with the original analysis, it was found that peptides could be distributed along a SARB scoring gradient that was largely reflective of frequency of supertype degeneracy. Specifically, 92.9% of the positive peptides (SARB >=10) and 82.1% of the intermediate peptides (SARB between 1 and 10) were degenerate, compared with only 27.3% of the negative peptides (SARB <0.1).

The performance of a predictive algorithm can be assessed with several measures. These include PPV, which is the fraction of peptides predicted to be positive that are actually positive, and SENS, which is the fraction of total positives identified using the selected cutoff score. Additional measures that can be used include accuracy, which is the fraction of correct predictions (a measure that can be severely inaccurate if the data set is skewed), and EFF75, which is the efficiency associated with selection of 75% of the total number of positive peptides. These performance measures, when using a SARB score of >=1 as the cutoff score, for both the analysis and blind sets are summarized in Table VI. As shown, the B44-SARB method performs well on all measures. Overall 86.1% of the predictions for the analysis set and 68.8% for the blind set were accurate. In the analysis set, 72% of the peptides scoring positive were indeed positive (PPV), and 80.6% of all positive peptides were identified (SENS). In the blind set, 85.7% of the positive predictions were true positives (PPV), although the SENS measure was somewhat lower (60%). Finally, when the cutoff score for the analysis library was set to allow identification of 75% of all positives (EFF75), an efficiency of 74.6% was noted. These values are comparable with those observed for algorithms that we and others have developed for predicting single MHC allele-peptide binding (47, 49, 50, 51) and underscore the effectiveness of the SARB method.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
We have examined the peptide-binding specificity of six different HLA-B molecules hypothesized to belong to the HLA-B44 supertype (2, 4). These molecules were selected on the basis of their frequency in major ethnic groups, thus affording coverage relevant for vaccine and diagnostic purposes. Molecular binding assays were established and validated using natural ligands or epitopes associated with various HLA-A and -B molecules. The peptide-binding specificity of these common B44-supertype molecules was further investigated using single substitution analog peptides and peptide libraries. Approximately 37% of the peptides associated with the B44 supermotif (glutamic acid in position 2 and phenylalanine, tryptophan, tyrosine, leucine, isoleucine, valine, methionine, alanine, glycine, or threonine at the C terminus) bound three or more supertype molecules with high affinity. This level of cross-reactivity is comparable with levels of cross-reactivity observed for the A2 and A3 supertypes (6, 17).

Comparison of the individual patterns of specificity revealed that for each molecule the dominant primary anchor was the residue in position 2 of peptide ligands, where invariably the only preferred residue was glutamic acid. The only other position at which a minority of the residues tested were preferred, indicative of a dominant influence on specificity, was the C terminus. However, in most cases, specificity at the C terminus was very broad, and only vague patterns of chemical similarity could be detected. In general, aliphatic hydrophobic (isoleucine, leucine, valine, and methionine), aromatic (phenylalanine, tryptophan, and tyrosine), and small (alanine, glycine, and threonine) residues were preferred or tolerated. The breadth of residues allowed at the C terminus suggests that it is the avoidance of certain residues, especially those with positive or negative charges, that confers binding capacity, rather than the dependence on the strength of a specific chemical interaction. This observation is supported by the remarkable frequency with which the presence of glycine at the C terminus was tolerated, or even preferred. In this context, it is reasonable to speculate that the narrow specificity of position 2 is balanced by the very broad specificity of the C terminus. This type of strategy might result in an optimal size of the repertoire of bound peptides and has also been noted for HLA B7-supertype molecules (16, 18), as well as Mamu A*01 (52, 53) and Mamu B*17 (54).

For positions 3 through C-1, most substitutions tested were preferred or tolerated, indicating that these positions in general have only relatively minor secondary influences on peptide-binding capacity. A common pattern of secondary influence was, however, suggested in position 1, where the acidic residue aspartic acid and the small residues valine and proline were not tolerated in most contexts. The negative influence of the presence of aspartic acid and proline on peptide-binding capacity in position 1 has been noted in the case of many other HLA-A and -B molecules (17, 18, 26, 45).

The peptide-binding motifs for B*4402 and B*4403 have been known since 1994 (37), and the motifs for B*4001 (B60) and B*4006 (B61) were reported in 1995 (34). Although noting the similarities in primary anchors, the authors of those studies did not hypothesize that these molecules could have peptide binding repertoires that would overlap with each other. Three other articles from the same period do note the fact that B37, B40 and B44 have similar primary anchor motifs, sharing a preference for peptides with negatively charged residues in position 2 (32, 55, 56). However, these studies did not report ligands that were bound by multiple alleles. More importantly, none of these authors speculated as to whether the peptide binding repertoires of these molecules overlap or whether they would comprise an HLA-supertype, as pointed out by Sidney, Sette and coworkers (2, 4, 5). This underlines the important distinction between demonstrating that molecules have similar primary anchor motifs and demonstrating that they have overlapping peptide-binding repertoires. For example, we have shown that the repertoires of HLA B7-supertype molecules apparently do not overlap with those of HLA C molecules sharing similar primary anchor motifs (16).

Our data fit well with previously described motifs obtained on the basis of elution and sequencing of natural ligands (32, 34, 37, 56). One notable exception is the B18 motif described by Bourgault-Villada et al. (57), which was based on the presence of acidic residues in position 3. We have found that B*1801 preferentially binds its ligands via the presence of glutamic acid in position 2. This motif is congruent with the sequence of a previously reported B18-restricted epitope (58).

This report is the first description of a B*4501 motif. Inclusion of B*4501 into the B44 supertype was originally suggested on the basis of the structure of the B and F pockets of the B*4501 molecule, hypothesized to engage the residue in position 2 and at the C terminus, respectively, of peptide ligands. B*4501 shares the narrow B44 supermotif specificity for glutamic acid in position 2 of its ligands. Also, like other B44-supertype molecules, B*4501 tolerates a range of hydrophobic, aromatic, and small residues at the C terminus, although a distinct preference for the small residues alanine and threonine was noted. B*4501 is prevalent in >10% of the North American Black population (Table I) and appears to be prevalent with similar frequency in several African Black populations (59) (D. Mann, unpublished observations). Thus, the development of a peptide-binding assay and the characterization of the specificity of this molecule is expected to be a valuable aid in the design and development of various vaccines targeting important diseases that are prevalent in sub-Saharan Africa, such as AIDS and malaria.

The detailed motifs we have derived provide coefficients for use in developing polynomial algorithms (8, 47) for predicting high affinity binders for 6 common B44-supertype molecules. Single allele algorithms often have some efficacy in also identifying peptides with supertype-binding capacity, reflecting the phenomenon that ligands with the highest binding affinity are often also the most degenerate binders (6, 7, 8, 15, 16, 17, 18). Until now, however, no algorithm has been described that specifically addresses prediction of degenerate binding. In fact, when development of specific algorithms to predict A2 supertype binders was attempted, it was met with limited success (A. Sette and J. Sidney, unpublished observations). This may reflect the fact that in the past, in most cases, only peptides binding with high affinity to the most common "prototype" allele (e.g., A*0201 in the case of the A2 supertype) were tested for cross-reactivity on other supertype molecules. Ultimately, this testing strategy results in the generation of data sets that are biased in terms of structural features, and might therefore be suboptimal for purposes of deriving supertype-specific algorithms. The recent availability of high throughput MHC peptide-binding assays has made it feasible in the present context to test all peptides for binding to all supertype alleles, rather than just selected subsets. The efficacy of the SARB method presented herein in the case of the B44 supertype suggests that the resulting availability of more balanced and unbiased data sets, which includes both positive and negative binders, is an important element for establishing algorithms predicting degenerate binding.

In addition to allowing accurate epitope prediction (17, 18, 26, 40, 45, 47, 60, 61), detailed peptide-binding motifs defining both primary and secondary anchor positions allow for the rational design of optimized ligands. For example, suboptimal main anchor residues may be replaced with more preferred residues, generating epitope analogues with increased binding affinity (18, 62, 63) and immunogenicity (62, 63, 64, 65, 66, 67). Evaluation of the efficacy of the detailed B44 supermotif to aid in the design of analogues with increased degeneracy will be the subject of future experiments, although the effectiveness of supermotifs in the design of degenerate binders has been demonstrated in the case of, for example, the B7 supermotif (18).

In conclusion, the data described herein formally demonstrates that B44-supertype molecules recognize similar features at primary and secondary anchor positions of their peptide ligands and also share largely overlapping peptide-binding repertoires. These results have significant implications for the design of epitope-based vaccine constructs and for monitoring immune responses. Indeed, the immunological relevance of supertype cross-reactivity at the peptide binding level has been demonstrated in a number of studies in both infectious disease (7, 8, 9, 12, 13, 15, 19, 68) and cancer (10, 11, 14, 20, 69, 70, 71) settings.


   Acknowledgments
 
We thank Carlos Betancourt, Rose Correa, Jamie Ahner, Pat Stuart, and Stephani Stewart for their expert technical assistance and Howard Grey and Bianca Mothe for their insightful comments.


   Footnotes
 
1 This work was supported in part by National Institutes of Health Contract NO1-AI-95362. Back

2 Address correspondence and reprint requests to Dr. Alessandro Sette, Division of Translational Immunology and Biodefense, La Jolla Institute for Allergy and Immunology, 10355 Science Center Drive, San Diego, CA 92121. E-mail address: alex{at}liai.org Back

3 Abbreviations used in this paper: HBV, hepatitis B virus; HCV, hepatitis C virus; RB, relative binding; ARB, average relative binding; SARB, supertype average relative binding; PPV, positive predictive value; SENS, sensitivity; EFF75, efficiency at 75% sensitivity. Back

4 The on-line version of this article contains supplemental material. Back

Received for publication June 23, 2003. Accepted for publication September 17, 2003.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Marsh, S. G., E. D. Albert, W. F. Bodmer, R. E. Bontrop, B. Dupont, H. A. Erlich, D. E. Geraghty, J. A. Hansen, B. Mach, W. R. Mayr, et al 2002. Nomenclature for factors of the HLA system, 2002. Hum. Immunol. 63:1213.[Medline]
  2. Sette, A., J. Sidney. 1999. Nine major HLA class I supertypes account for the vast preponderance of HLA-A and -B polymorphism. Immunogenetics 50:201.[Medline]
  3. del Guercio, M. F., J. Sidney, G. Hermanson, C. Perez, H. M. Grey, R. T. Kubo, A. Sette. 1995. Binding of a peptide antigen to multiple HLA alleles allows definition of an A2-like supertype. J. Immunol. 154:685.[Abstract]
  4. Sidney, J., H. M. Grey, R. T. Kubo, A. Sette. 1996. Practical, biochemical and evolutionary implications of the discovery of HLA class I supermotifs. Immunol. Today 17:261.[Medline]
  5. Sette, A., J. Sidney. 1998. HLA supertypes and supermotifs: a functional perspective on HLA polymorphism. Curr. Opin. Immunol. 10:478.[Medline]
  6. Sidney, J., S. Southwood, D. L. Mann, M. A. Fernandez-Vina, M. J. Newman, A. Sette. 2001. Majority of peptides binding HLA-A*0201 with high affinity crossreact with other A2-supertype molecules. Hum. Immunol. 62:1200.[Medline]
  7. Bertoni, R., J. Sidney, P. Fowler, R. W. Chesnut, F. V. Chisari, A. Sette. 1997. Human histocompatibility leukocyte antigen-binding supermotifs predict broadly cross-reactive cytotoxic T lymphocyte responses in patients with acute hepatitis. J. Clin. Invest. 100:503.[Medline]
  8. Doolan, D. L., S. L. Hoffman, S. Southwood, P. A. Wentworth, J. Sidney, R. W. Chesnut, E. Keogh, E. Appella, T. B. Nutman, A. A. Lal, et al 1997. Degenerate cytotoxic T cell epitopes from P. falciparum restricted by multiple HLA-A and HLA-B supertype alleles. Immunity 7:97.[Medline]
  9. Altfeld, M. A., B. Livingston, N. Reshamwala, P. T. Nguyen, M. M. Addo, A. Shea, M. Newman, J. Fikes, J. Sidney, P. Wentworth, et al 2001. Identification of novel HLA-A2-restricted human immunodeficiency virus type 1-specific cytotoxic T-lymphocyte epitopes predicted by the HLA-A2 supertype peptide-binding motif. J. Virol. 75:1301.[Abstract/Free Full Text]
  10. Kawashima, I., S. J. Hudson, V. Tsai, S. Southwood, K. Takesako, E. Appella, A. Sette, E. Celis. 1998. The multi-epitope approach for immunotherapy for cancer: identification of several CTL epitopes from various tumor-associated antigens expressed on solid epithelial tumors. Hum. Immunol. 59:1.[Medline]
  11. Rivoltini, L., D. J. Loftus, K. Barracchini, F. Arienti, A. Mazzocchi, W. E. Biddison, M. L. Salgaller, E. Appella, G. Parmiani, F. M. Marincola. 1996. Binding and presentation of peptides derived from melanoma antigens MART-1 and glycoprotein-100 by HLA-A2 subtypes. Implications for peptide-based immunotherapy. J. Immunol. 156:3882.[Abstract]
  12. Livingston, B. D., C. Crimi, J. Fikes, R. W. Chesnut, J. Sidney, A. Sette. 1999. Immunization with the HBV core 18–27 epitope elicits CTL responses in humans expressing different HLA-A2 supertype molecules. Hum. Immunol. 60:1013.[Medline]
  13. Khanna, R., S. R. Burrows, J. Nicholls, L. M. Poulsen. 1998. Identification of cytotoxic T cell epitopes within Epstein-Barr virus (EBV) oncogene latent membrane protein 1 (LMP1): evidence for HLA A2 supertype-restricted immune recognition of EBV-infected cells by LMP1-specific cytotoxic T lymphocytes. Eur. J. Immunol. 28:451.[Medline]
  14. Fleischhauer, K., S. Tanzarella, H. J. Wallny, C. Bordignon, C. Traversari. 1996. Multiple HLA-A alleles can present an immunodominant peptide of the human melanoma antigen Melan-A/MART-1 to a peptide-specific HLA-A*0201+ cytotoxic T cell line. J. Immunol. 157:787.[Abstract]
  15. Chang, K. M., N. H. Gruener, S. Southwood, J. Sidney, G. R. Pape, F. V. Chisari, A. Sette. 1999. Identification of HLA-A3 and -B7-restricted CTL response to hepatitis C virus in patients with acute and chronic hepatitis C. J. Immunol. 162:1156.[Abstract/Free Full Text]
  16. Sidney, J., M. F. del Guercio, S. Southwood, V. H. Engelhard, E. Appella, H. G. Rammensee, K. Falk, O. Rotzschke, M. Takiguchi, R. T. Kubo, et al 1995. Several HLA alleles share overlapping peptide specificities. J. Immunol. 154:247.[Abstract]
  17. Sidney, J., H. M. Grey, S. Southwood, E. Celis, P. A. Wentworth, M. F. del Guercio, R. T. Kubo, R. W. Chesnut, A. Sette. 1996. Definition of an HLA-A3-like supermotif demonstrates the overlapping peptide-binding repertoires of common HLA molecules. Hum. Immunol. 45:79.[Medline]
  18. Sidney, J., S. Southwood, M. F. del Guercio, H. M. Grey, R. W. Chesnut, R. T. Kubo, A. Sette. 1996. Specificity and degeneracy in peptide binding to HLA-B7-like class I molecules. J. Immunol. 157:3480.[Abstract]
  19. Threlkeld, S. C., P. A. Wentworth, S. A. Kalams, B. M. Wilkes, D. J. Ruhl, E. Keogh, J. Sidney, S. Southwood, B. D. Walker, A. Sette. 1997. Degenerate and promiscuous recognition by CTL of peptides presented by the MHC class I A3-like superfamily: implications for vaccine development. J. Immunol. 159:1648.[Abstract]
  20. Wang, R. F., S. L. Johnston, S. Southwood, A. Sette, S. A. Rosenberg. 1998. Recognition of an antigenic peptide derived from tyrosinase-related protein-2 by CTL in the context of HLA-A31 and -A33. J. Immunol. 160:890.[Abstract/Free Full Text]
  21. Dzuris, J. L., J. Sidney, E. Appella, R. W. Chesnut, D. I. Watkins, A. Sette. 2000. Conserved MHC class I peptide binding motif between humans and rhesus macaques. J. Immunol. 164:283.[Abstract/Free Full Text]
  22. Urvater, J. A., H. Hickman, J. L. Dzuris, K. Prilliman, T. M. Allen, K. J. Schwartz, D. Lorentzen, C. Shufflebotham, E. J. Collins, D. L. Neiffer, et al 2001. Gorillas with spondyloarthropathies express an MHC class I molecule with only limited sequence similarity to HLA-B27 that binds peptides with arginine at P2. J. Immunol. 166:3334.[Abstract/Free Full Text]
  23. McKinney, D. M., A. L. Erickson, C. M. Walker, R. Thimme, F. V. Chisari, J. Sidney, A. Sette. 2000. Identification of five different Patr class I molecules that bind HLA supertype peptides and definition of their peptide binding motifs. J. Immunol. 165:4414.[Abstract/Free Full Text]
  24. Bertoni, R., A. Sette, J. Sidney, L. G. Guidotti, M. Shapiro, R. Purcell, F. V. Chisari. 1998. Human class I supertypes and CTL repertoires extend to chimpanzees. J. Immunol. 161:4447.[Abstract/Free Full Text]
  25. Selby, M., A. Erickson, C. Dong, S. Cooper, P. Parham, M. Houghton, C. M. Walker. 1999. Hepatitis C virus envelope glycoprotein E1 originates in the endoplasmic reticulum and requires cytoplasmic processing for presentation by class I MHC molecules. J. Immunol. 162:669.[Abstract/Free Full Text]
  26. Ruppert, J., J. Sidney, E. Celis, R. T. Kubo, H. M. Grey, A. Sette. 1993. Prominent role of secondary anchor residues in peptide binding to HLA-A2.1 molecules. Cell 74:929.[Medline]
  27. Sidney, J., S. Southwood, C. Oseroff, M. F. Del Guercio, A. Sette, H. Grey. 1998. Measurement of MHC/peptide interactions by gel filtration. Current Protocols in Immunology 18.3.1. John Wiley & Sons, New York.
  28. Zinszner, H., M. Masset, J. F. Bourge, J. Colombani, D. Cohen, L. Degos, P. Paul. 1990. Nucleotide sequence of the HLA-A26 class I gene: identification of specific residues and molecular mapping of public HLA class I epitopes. Hum. Immunol. 27:155.[Medline]
  29. Drouet, M., L. Aussel, B. Drenou, R. Fauchet. 1995. Quantification and molecular analysis of plasmatic HLA-BCw molecules with a locus B and Cw HLA specific mAb (B1.23.2 mAb). Eur. J. Immunogenet. 22:363.[Medline]
  30. Sterkers, G., Y. Henin, J. Kalil, M. Bagot, J. P. Levy. 1983. Influence of HLA class I- and class II-specific monoclonal antibodies on DR-restricted lymphoproliferative responses. I. Unseparated populations of effector cells. J. Immunol. 131:2735.[Abstract]
  31. Rebai, N., B. Malissen. 1983. Structural and genetic analyses of HLA class I molecules using monoclonal xenoantibodies. Tissue Antigens 22:107.[Medline]
  32. DiBrino, M., K. C. Parker, D. H. Margulies, J. Shiloach, R. V. Turner, W. E. Biddison, J. E. Coligan. 1995. Identification of the peptide binding motif for HLA-B44, one of the most common HLA-B alleles in the Caucasian population. Biochemistry 34:10130.[Medline]
  33. Harris, P. E., A. Colovai, Z. Liu, R. Dalla Favera, N. Suciu-Foca. 1993. Naturally processed HLA class I bound peptides from c-myc-transfected cells reveal allele-specific motifs. J. Immunol. 151:5966.[Abstract]
  34. Falk, K., O. Rotzschke, M. Takiguchi, V. Gnau, S. Stevanovic, G. Jung, H. G. Rammensee. 1995. Peptide motifs of HLA-B58, B60, B61, and B62 molecules. Immunogenetics 41:165.[Medline]
  35. Scotet, E., J. David-Ameline, M. A. Peyrat, A. Moreau-Aubry, D. Pinczon, A. Lim, J. Even, G. Semana, J. M. Berthelot, R. Breathnach, M. Bonneville, E. Houssaint. 1996. T cell response to Epstein-Barr virus transactivators in chronic rheumatoid arthritis. J. Exp. Med. 184:1791.[Abstract/Free Full Text]
  36. Brichard, V. G., J. Herman, A. Van Pel, C. Wildmann, B. Gaugler, T. Wolfel, T. Boon, B. Lethe. 1996. A tyrosinase nonapeptide presented by HLA-B44 is recognized on a human melanoma by autologous cytolytic T lymphocytes. Eur. J. Immunol. 26:224.[Medline]
  37. Fleischhauer, K., D. Avila, F. Vilbois, C. Traversari, C. Bordignon, H. J. Wallny. 1994. Characterization of natural peptide ligands for HLA-B*4402 and -B*4403: implications for peptide involvement in allorecognition of a single amino acid change in the HLA-B44 heavy chain. Tissue Antigens 44:311.[Medline]
  38. Borrow, P., H. Lewicki, X. Wei, M. S. Horwitz, N. Peffer, H. Meyers, J. A. Nelson, J. E. Gairin, B. H. Hahn, M. B. Oldstone, G. M. Shaw. 1997. Antiviral pressure exerted by HIV-1-specific cytotoxic T lymphocytes (CTLs) during primary infection demonstrated by rapid selection of CTL escape virus. Nat. Med. 3:205.[Medline]
  39. Rickinson, A. B., D. J. Moss. 1997. Human cytotoxic T lymphocyte responses to Epstein-Barr virus infection. Annu. Rev. Immunol. 15:405.[Medline]
  40. Kondo, A., J. Sidney, S. Southwood, M. F. del Guercio, E. Appella, H. Sakamoto, H. M. Grey, E. Celis, R. W. Chesnut, R. T. Kubo, A. Sette. 1997. Two distinct HLA-A*0101-specific submotifs illustrate alternative peptide binding modes. Immunogenetics 45:249.[Medline]
  41. Kubo, R. T., A. Sette, H. M. Grey, E. Appella, K. Sakaguchi, N. Z. Zhu, D. Arnott, N. Sherman, J. Shabanowitz, H. Michel, et al 1994. Definition of specific peptide motifs for four major HLA-A alleles. J. Immunol. 152:3913.[Abstract]
  42. Krausa, P., C. Munz, W. Keilholz, S. Stevanovic, E. Y. Jones, M. Browning, M. Bunce, H. G. Rammensee, A. McMichael. 2000. Definition of peptide binding motifs amongst the HLA-A*30 allelic group. Tissue Antigens 56:10.[Medline]
  43. Harrer, E., T. Harrer, P. Barbosa, M. Feinberg, R. P. Johnson, S. Buchbinder, B. D. Walker. 1996. Recognition of the highly conserved YMDD region in the human immunodeficiency virus type 1 reverse transcriptase by HLA-A2-restricted cytotoxic T lymphocytes from an asymptomatic long-term nonprogressor. J. Infect. Dis. 173:476.[Medline]
  44. Jardetzky, T. S., W. S. Lane, R. A. Robinson, D. R. Madden, D. C. Wiley. 1991. Identification of self peptides bound to purified HLA-B27. Nature 353:326.[Medline]
  45. Kondo, A., J. Sidney, S. Southwood, M. F. del Guercio, E. Appella, H. Sakamoto, E. Celis, H. M. Grey, R. W. Chesnut, R. T. Kubo, et al 1995. Prominent roles of secondary anchor residues in peptide binding to HLA-A24 human class I molecules. J. Immunol. 155:4307.[Abstract]
  46. Southwood, S., J. Sidney, A. Kondo, M. F. del Guercio, E. Appella, S. Hoffman, R. T. Kubo, R. W. Chesnut, H. M. Grey, A. Sette. 1998. Several common HLA-DR types share largely overlapping peptide binding repertoires. J. Immunol. 160:3363.[Abstract/Free Full Text]
  47. Gulukota, K., J. Sidney, A. Sette, C. DeLisi. 1997. Two complementary methods for predicting peptides binding major histocompatibility complex molecules. J. Mol. Biol. 267:1258.[Medline]
  48. Gjertson, D. W., S.-H. Lee. 1998. HLA-A/B and -DRB1/DQB1 allele-level haplotype frequencies. D. W. Gjertson, and P. I. Terasaki, eds. HLA 1998 365. American Society for Histocompatibility and Immunogenetics, Lenexa.
  49. Udaka, K., H. Mamitsuka, Y. Nakaseko, N. Abe. 2002. Empirical evaluation of a dynamic experiment design method for prediction of MHC class I-binding peptides. J. Immunol. 169:5744.[Abstract/Free Full Text]
  50. Udaka, K., K. H. Wiesmuller, S. Kienle, G. Jung, H. Tamamura, H. Yamagishi, K. Okumura, P. Walden, T. Suto, T. Kawasaki. 2000. An automated prediction of MHC class I-binding peptides based on positional scanning with peptide libraries. Immunogenetics 51:816.[Medline]
  51. Peters, B., W. Tong, J. Sidney, A. Sette, Z. Weng. 2003. Examining the independent binding assumption for binding of peptide epitopes to MHC-I molecules. Bioinformatics 19:1765.[Abstract/Free Full Text]
  52. Sidney, J., J. L. Dzuris, M. J. Newman, R. P. Johnson, A. Kaur, K. Amitinder, C. M. Walker, E. Appella, B. Mothe, D. I. Watkins, A. Sette. 2000. Definition of the Mamu A*01 peptide binding specificity: application to the identification of wild-type and optimized ligands from simian immunodeficiency virus regulatory proteins. J. Immunol. 165:6387.[Abstract/Free Full Text]
  53. Allen, T. M., J. Sidney, M. F. del Guercio, R. L. Glickman, G. L. Lensmeyer, D. A. Wiebe, R. DeMars, C. D. Pauza, R. P. Johnson, A. Sette, D. I. Watkins. 1998. Characterization of the peptide binding motif of a rhesus MHC class I molecule (Mamu-A*01) that binds an immunodominant CTL epitope from simian immunodeficiency virus. J. Immunol. 160:6062.[Abstract/Free Full Text]
  54. Mothe, B. R., J. Sidney, J. L. Dzuris, M. E. Liebl, S. Fuenger, D. I. Watkins, A. Sette. 2002. Characterization of the peptide-binding specificity of Mamu-B*17 and identification of Mamu-B*17-restricted epitopes derived from simian immunodeficiency virus proteins. J. Immunol. 169:210.[Abstract/Free Full Text]
  55. Chelvanayagam, G.. 1996. A roadmap for HLA-A, HLA-B, and HLA-C peptide binding specificities. Immunogenetics 45:15.[Medline]
  56. Thorpe, C. J., P. J. Travers. 1994. Prediction of an HLA-B44 binding motif by the alignment of known epitopes and molecular modeling of the antigen binding cleft. Immunogenetics 40:303.[Medline]
  57. Bourgault Villada, I., N. Beneton, C. Bony, F. Connan, J. Monsonego, A. Bianchi, P. Saiag, J. P. Levy, J. G. Guillet, J. Choppin. 2000. Identification in humans of HPV-16 E6 and E7 protein epitopes recognized by cytolytic T lymphocytes in association with HLA-B18 and determination of the HLA-B18-specific binding motif. Eur. J. Immunol. 30:2281.[Medline]
  58. Steven, N. M., N. E. Annels, A. Kumar, A. M. Leese, M. G. Kurilla, A. B. Rickinson. 1997. Immediate early and early lytic cycle proteins are frequent targets of the Epstein-Barr virus-induced cytotoxic T cell response. J. Exp. Med. 185:1605.[Abstract/Free Full Text]
  59. Novitsky, V., P. O. Flores-Villanueva, P. Chigwedere, S. Gaolekwe, H. Bussman, G. Sebetso, R. Marlink, E. J. Yunis, M. Essex. 2001. Identification of most frequent HLA class I antigen specificities in Botswana: relevance for HIV vaccine design. Hum. Immunol. 62:146.[Medline]
  60. Parker, K. C., M. A. Bednarek, L. K. Hull, U. Utz, B. Cunningham, H. J. Zweerink, W. E. Biddison, J. E. Coligan. 1992. Sequence motifs important for peptide binding to the human MHC class I molecule, HLA-A2. J. Immunol. 149:3580.[Abstract]
  61. Milik, M., D. Sauer, A. P. Brunmark, L. Yuan, A. Vitiello, M. R. Jackson, P. A. Peterson, J. Skolnick, C. A. Glass. 1998. Application of an artificial neural network to predict specific class I MHC binding peptide sequences. Nat. Biotechnol. 16:753.[Medline]
  62. Pogue, R. R., J. Eron, J. A. Frelinger, M. Matsui. 1995. Amino-terminal alteration of the HLA-A*0201-restricted human immunodeficiency virus pol peptide increases complex stability and in vitro immunogenicity. Proc. Natl. Acad. Sci. USA 92:8166.[Abstract/Free Full Text]
  63. Bakker, A. B., S. H. van der Burg, R. J. Huijbens, J. W. Drijfhout, C. J. Melief, G. J. Adema, C. G. Figdor. 1997. Analogues of CTL epitopes with improved MHC class-I binding capacity elicit anti-melanoma CTL recognizing the wild-type epitope. Int. J. Cancer 70:302.[Medline]
  64. Parkhurst, M. R., M. L. Salgaller, S. Southwood, P. F. Robbins, A. Sette, S. A. Rosenberg, Y. Kawakami. 1996. Improved induction of melanoma-reactive CTL with peptides from the melanoma antigen gp100 modified at HLA-A*0201-binding residues. J. Immunol. 157:2539.[Abstract]
  65. Rosenberg, S. A., J. C. Yang, D. J. Schwartzentruber, P. Hwu, F. M. Marincola, S. L. Topalian, N. P. Restifo, M. E. Dudley, S. L. Schwarz, P. J. Spiess, J. R. , et al 1998. Immunologic and therapeutic evaluation of a synthetic peptide vaccine for the treatment of patients with metastatic melanoma. Nat. Med. 4:321.[Medline]
  66. Sarobe, P., C. D. Pendleton, T. Akatsuka, D. Lau, V. H. Engelhard, S. M. Feinstone, J. A. Berzofsky. 1998. Enhanced in vitro potency and in vivo immunogenicity of a CTL epitope from hepatitis C virus core protein following amino acid replacement at secondary HLA-A2.1 binding positions. J. Clin. Invest. 102:1239.[Medline]
  67. Ahlers, J. D., T. Takeshita, C. D. Pendleton, J. A. Berzofsky. 1997. Enhanced immunogenicity of HIV-1 vaccine construct by modification of the native peptide sequence. Proc. Natl. Acad. Sci. USA 94:10856.[Abstract/Free Full Text]
  68. Missale, G., A. Redeker, J. Person, P. Fowler, S. Guilhot, H. J. Schlicht, C. Ferrari, F. V. Chisari. 1993. HLA-A31- and HLA-Aw68-restricted cytotoxic T cell responses to a single hepatitis B virus nucleocapsid epitope during acute viral hepatitis. J. Exp. Med. 177:751.[Abstract/Free Full Text]
  69. Ressing, M. E., J. H. de Jong, R. M. Brandt, J. W. Drijfhout, W. E. Benckhuijsen, G. M. Schreuder, R. Offringa, W. M. Kast, C. J. Melief. 1999. Differential binding of viral peptides to HLA-A2 alleles. Implications for human papillomavirus type 16 E7 peptide-based vaccination against cervical carcinoma. Eur. J. Immunol. 29:1292.[Medline]
  70. Kawashima, I., V. Tsai, S. Southwood, K. Takesako, E. Celis, A. Sette. 1998. Identification of gp100-derived, melanoma-specific cytotoxic T-lymphocyte epitopes restricted by HLA-A3 super