• We are sorry, but NCBI web applications do not support your browser and may not function properly. More information
Logo of immunresBiomed Central Web Sitesearchmanuscript Submissionregistrationthis articleJournal Front Page
Immunome Res. 2007; 3: 7.
Published online Aug 20, 2007. doi:  10.1186/1745-7580-3-7
PMCID: PMC2040137

In silico characterization of immunogenic epitopes presented by HLA-Cw*0401

Abstract

Background

HLA-C locus products are poorly understood in part due to their low expression at the cell surface. Recent data indicate that these molecules serve as major restriction elements for human immunodeficiency virus type 1 (HIV-1) cytotoxic T lymphocyte (CTL) epitopes. We report here a structure-based technique for the prediction of peptides binding to Cw*0401. The models were rigorously trained, tested and validated using experimentally verified Cw*0401 binding and non-binding peptides obtained from biochemical studies. A new scoring scheme facilitates the identification of immunological hot spots within antigens, based on the sum of predicted binding energies of the top four binders within a window of 30 amino acids.

Results

High predictivity is achieved when tested on the training (r2 = 0.88, s = 3.56 kJ/mol, q2 = 0.84, spress = 5.18 kJ/mol) and test (AROC = 0.93) datasets. Characterization of the predicted Cw*0401 binding sequences indicate that amino acids at key anchor positions share common physico-chemical properties which correlate well with existing experimental studies.

Conclusion

The analysis of predicted Cw*0401-binding peptides showed that anchor residues may not be restrictive and the Cw*0401 binding pockets may possibly accommodate a wide variety of peptides with common physico-chemical properties. The potential Cw*0401-specific T-cell epitope repertoires for HIV-1 p24gag and gp160gag glycoproteins are well distributed throughout both glycoproteins, with thirteen and nine immunological hot spots for HIV-1 p24gag and gp160gag glycoproteins respectively. These findings provide new insights into HLA-C peptide selectivity, indicating that pre-selection of candidate HLA-C peptides may occur at the TAP level, prior to peptide loading in the endoplasmic reticulum.

Background

Major histocompatibility complex (MHC) class I molecules, HLA-A, -B, and -C, are cell surface glycoproteins consisting of a polymorphic heavy α chain non-covalently linked to a light chain, β2-microglobulin (β2m). HLA-A and -B molecules play critical roles in cell mediated immune responses by binding short antigenic peptide fragments and presenting them on the surface of antigen-presenting cells for recognition by the CD8+ cytotoxic T lymphocyte (CTL). Although several HLA-C specificities with CTL epitopes have been reported [1,2], much remains unknown with regards to their role in the immune response against viral antigens in part due to their poor expression at the cell surface [3,4]. Recent research shows that this group of molecules plays a major role in the control of human immunodeficiency virus type 1 (HIV-1) infection [5]. Improved understanding of peptide binding to this group of molecules is important in the study of HIV-1 disease progression, as well as the design of effective HIV peptide vaccines.

The HLA-C allele, Cw*0401, is of particular interest in the study of HIV-1 disease progression because it is the restriction element for HIV-1 proteins [5]. Two HIV-1 proteins (p24gag and gp160gag) are currently known to be restricted by Cw*0401 [5]. Cw*0401 is present in approximately 10% of the general population [6]. The allele is expressed intracellularly in amounts comparable with HLA-A and -B molecules, but is poorly expressed at the cell surface [7,8]. Improved understanding of peptide binding to this molecule is important for elucidating its role in HIV-1 disease progression.

Computational strategies for prediction of peptide binding to HLA-A and -B molecules are relatively advanced [9], while sequence-based predictive models for HLA-C molecules have encountered limited success due to the lack of experimental training data [10]. Two matrix-based prediction algorithms for Cw*0401 were reported [11,12], but a sequence independent approach is still lacking. To overcome these limitations, we have developed a structure-based predictive technique that integrates the strength of Monte Carlo simulations and homology modeling [13-15]. This method utilizes a probe or "base fragment" to sample different regions of the receptor binding site, followed by loop closure and refinement of the entire class I peptide. The technique has been successfully applied to analyze peptides binding to a variety of MHC class II alleles [14,15]. In this work, we now extend our analysis to peptides presented by the class I HLA-C molecule. We investigated the HIV-1 p24gag and gp160gag peptide binding repertoire of Cw*0401 and illustrate that areas with high concentration of T-cell epitopes or "immunological hot spots" are potentially well distributed throughout both HIV-1 p24gag and gp160gag. We also show that Cw*0401 can possibly bind antigenic peptides in amounts comparable to both HLA-A and -B molecules. Characterization of predicted Cw*0401 binding sequences reveal that Cw*0401 may bind a large variety of amino acids at anchor positions with common physico-chemical properties which correlate well with existing experimental studies [11].

Results and discussion

Cw*0401 predictive model

High predictivity (r2 = 0.88, s = 3.56 kJ/mol, q2 = 0.84, spress = 5.18 kJ/mol) is achieved when tested on the training dataset of 6 Cw*0401 peptide sequences. The Cw*0401 predictive model outperforms the predictive models done by Rognan et al. [16] on training datasets of 5 A*0204 (r2 = 0.85, spress = 2.40 kJ/mol) and 37 2Kk (r2 = 0.78, spress = 3.16 kJ/mol) peptide sequences and is comparable with our previous DRB1*0402 (r2 = 0.90, s = 1.20 kJ/mol, q2 = 0.82, spress = 1.61 kJ/mol) and DQB1*0503 (r2 = 0.95, s = 1.20 kJ/mol, q2 = 0.75, spress = 2.15 kJ/mol) prediction models on a training set of 8 peptides [17]. The cross-validation coefficient q2 and the standard error of prediction spress are stable, with q2 = 0.84 and spress = 5.18 kJ/mol. This iterative regression procedure validates the internal consistency of the scoring function in the current model, rendering it suitable for predictions on the test dataset obtained from biochemical studies. The predictive performance of our model is further validated using the test dataset of 58 peptides. The external validation results indicate that our Cw*0401 predictive model is suitable for discriminating binding ligands from the background with high accuracy (AROC = 0.93) with sensitivity of 76% (SP = 0.95).

Characterization of Cw*0401 binding peptides

An in-depth analysis was performed to investigate the characteristics of Cw*0401 binding peptides. A panel of 2279 sequences was generated using an overlapping sliding window of size 9 across the entire p24gag [5] and gp160gag [18] glycoproteins and modeled into the binding groove of Cw*0401. From these sequences, a total of 877 binding sequences (predicted binders; SE = 76%, SP = 80%) were selected and a systematic analysis was performed to analyze the number of occurrence of individual amino acid residues and physico-chemical properties [19] at each position of Cw*0401 binding peptides.

Peptide position p2 is characterized by alanine (10%), glycine (13%), leucine (9%), serine (9%). 60% of the predicted residues at this position are hydrophobic in nature, while 93% are neutral. These properties correlate with the physico-chemical properties of existing binding motif (Tyr/Phe) at p2 [11] as well as with the observed conservation in the test data (Table (Table1:1: Phe – 58% and Tyr – 26%). The p3 position shows a strong preference for glycine (11%) and threonine (8%). Existing anchor residues at this position are aspartic acid and histidine, which accounts for 9% of the total position-specific composition in the dataset. The p4 position shares similar characteristics as p3 (glycine: 9%; threonine: 8%), with additional preference for leucine (8%). Similar results were obtained at the p5 position (alanine: 8%; glycine: 9%; leucine: 8%). At p6, characteristic residues include glycine (9%) and leucine (8%). This position is favored by neutral (81%) acyclic (89%), medium/large (77%), and hydrophobic (51%) residues. The physico-chemical properties of these residues are in agreement with Val/Ile/Leu as reported in earlier studies [11] and is comparable with the conservation in the test dataset reported in Table Table11 (Val – 29%, Ile – 12%, Leu and Pro – 10%). Finally, the p9 position was defined by six amino acids, including alanine (8%), glycine (10%), isoleucine (8%), leucine (8%), threonine (9%), and valine (8%). This position is primarily dominated by neutral (90%) and hydrophobic (58%) residues, and agrees with profiles of Leu/Phe as previously reported [11] and is consistent with the conservation in the test dataset reported in Table Table11 (Leu and Phe – 39%,). Collectively, our data indicates that individual binding pockets may not be highly specific as previously reported [11] but can rather accommodate a wide-range of anchor residues with common physico-chemical properties. We attribute the discrepancies to two possibilities: i) the lack of extensive research on Cw*0401 peptides; and ii) natural peptides carrying these residues may be present in small amount and were thus not detected by experimental studies.

Table 1
HLA-Cw4 dataset used in this study.

Prediction of p24gag and gp160gag immunological hot spots

The potential T-cell epitope repertoire (data not shown) and immunological hot spots (Figure (Figure1)1) for HIV-1 p24gag and gp160gag are well distributed throughout both glycoproteins. The known p24gag epitope (p24 168–175) and gp160gag epitope (gp160 375–383) were successfully predicted by our model at the threshold of -200 (SE = 76%, SP = 80%) [5,20]. At this threshold, the number of predicted hot spots for p24gag are fourteen (p24 20–75, 89–148, 143–262, 311–381, 447–506, 526–590, 664–727, 708–791, 873–930, 996–1053, 1054–1157, 1157–1316, 1308–1368, and 1378–1427), with an estimated FN = 3, and FP = 3 for SE = 0.76 and SP = 0.95 (Figure (Figure11 and Table Table2).2). For gp160gag we predict nine hot spots (gp160 44–89, 102–171, 174–289, 341–504, 489–559, 581–639, 667–721, 708–766, and 793–852), with an estimated FN = 2, FP = 2 for SE = 0.76 and SP = 0.95 (Figure (Figure11 and Table Table3).3). The results presented here indicate that Cw*0401 can bind antigenic peptides with specificities comparable to HLA-A and -B molecules, and any variability in antigen expression may be directly related to the loss or reduced cell surface expression of the molecule by mechanisms as yet unknown [8,21].

Table 2
Predicted Cw*0401-specific immunological hotspots for p24gag.
Table 3
Predicted Cw*0401-specific immunological hotspots for gp160gag.
Figure 1
Predicted start positions of Cw*0401-specific hotspots (sliding window size = 30) along (A) p24gag and (B) gp160gag glycoproteins. Scores are computed based on the sum of predicted binding energies of top four binders within the 30 amino acid sliding ...

Conclusion

Due to the low expression of HLA-C molecules at the cell surface, their role in cell mediated immune responses remain poorly understood. Collectively, the outcome of this analysis provides insights into the binding specificities of Cw*0401. Our data strongly indicate that Cw*0401 can bind antigenic peptides in amounts comparable to both HLA-A and -B molecules, and show the existence of a potentially large number of Cw*0401-specific T-cell epitopes that are evenly distributed throughout both HIV-1 p24gag and gp160gag glycoproteins. It remains to be determined what proportion of these peptides may be expressed at the cell surface and capable of eliciting functional responses. Probably, pre-selection of candidate HLA-C peptides may occur at the TAP level, prior to peptide loading in the ER [8]. Consequently, a higher concentration of peptides is necessary for complexation with HLA-C molecules, resulting in their release from TAP. This provides a possible explanation for the reduced cell surface expression of HLA-C molecules [8].

Methods

Data

Crystallographic data

The coordinates of Cw*0401 were obtained from the Protein Databank (PDB) with PDB code 1QQD [22]. The structure was relaxed by conjugate gradient minimization, using the Internal Coordinate Mechanics (ICM) software [23].

Experimental binding data

The dataset comprises a total of 64 (57 binders and 7 non-binders) 9-mer peptides (Table (Table1).1). The available dataset is divided into training and testing datasets. Peptides with experimental IC50 values were selected as training data for optimizing the empirical free energy function (refer Empirical Free Energy Function). Due to the lack of experimental data, only 9 peptides with experimental IC50 values were identified, six (three binders and three non-binders) of which were used for training, while the remainder (four non-binders) were included in the test dataset as true negatives. Therefore, the training dataset contained six peptides with experimentally determined IC50 values (2 high-affinity binders, 1 medium-affinity binder, and 3 non-binders) derived from biochemical studies [24], while the testing dataset comprised the remainder 58 peptides (54 binders and 4 non-binders) [10,18,21,24,25]. Experimental IC50 values were classified as follows – high-affinity binders: IC50 ≤ 500 nM, medium-affinity binders: 500 nM < IC50 ≤ 1500 nM, low-affinity binders: 1500 < IC50 ≤ 5000 nM and non-binders: 5000 < IC50.

HIV-1 sequence data

The sequences of HIV-1 p24gag and gp160gag glycoproteins were obtained from UniProt [26]. The accession numbers for p24gag and gp160gag glycoproteins used in this study are P04585 and P03377 respectively.

Model

Peptide docking

Docking was performed according to the procedure utilized in previous similar works [13-15]: (i) pseudo-Brownian rigid body docking of peptide fragments to the ends of the binding groove, (ii) central loop closure by satisfaction of spatial constraints, and (iii) refinement of the backbone and side-chain atoms of the ligand and receptor contact regions.

Empirical free energy function

The scoring function presented herein is based on the free energy potential in ICM [23]. Computation of the binding free energy was performed according to previous similar work based on the difference between the energy of the solvated complex and the sum of the energy of the solvated receptor and that of the peptide ligand, followed by optimization using experimental IC50 values [15]. This step was followed by 6-fold cross-validation for assessment of quality of the scoring function [15]. In k-fold cross-validation, k random, (approximately) equal-sized, disjoint partitions of the sample data were constructed, and all given models were trained on (k-1) partitions and tested on the excluded partition. The results were averaged after k such experiments, and thus the observed error rate may be taken as an estimate of the error rate expected upon generalization to new data. The predictive power of the models was assessed by the cross-validation coefficient q2 and the standard error of prediction spress.

Immunological hot spot prediction

In this study, 'immunological hot spots' are defined as antigenic regions of up to 30 amino acids and modeled according to previous similar work based on the sum of predicted binding energies of the top four binders within a window of 30 amino acids [27]. Where available, these predicted hotspots were validated with available experimentally determined sites.

Training, testing and validation

The free energy scoring function was calibrated using 6 peptides with experimental IC50 values and tested on a dataset 58 peptides (54 binders and 4 non-binders) obtained from biochemical studies (Table (Table1).1). The predictive performance of our model was assessed using sensitivity (SE), specificity (SP) and receiver operating characteristic (ROC) analysis [28]. SE = TP/(TP+FN) and SP = TN/(TN+FP), indicate percentages of correctly predicted binders and non-binders, respectively. TP (true positives) represents correctly predicted experimental binders and TN (true negatives) for experimental non-binders incorrectly predicted as binders. FN (false negatives) denotes experimental binders predicted as non-binders and FP (false positives) stands for experimental non-binders predicted as binders. The accuracy of our predictions was assessed by the ROC analysis where the ROC curve is generated by plotting SE as a function of (1-SP) for a complete range of classification thresholds. The area under the ROC curve (AROC) provides a measure of overall prediction accuracy, AROC < 70% for poor, AROC > 80% for good and AROC > 90% for excellent predictions [15].

Abbreviations

CTL, cytotoxic T lymphocyte; HLA, human leukocyte antigen; MHC, major histocompatibility complex; ER, endoplasmic reticulum; HIV, human immunodeficiency virus; ROC, receiver operating characteristic; SE, sensitivity; SP, specificity; TN, true negative; TP, true positive; FN, false negative; FP, false positive

Authors' contributions

JCT carried out the computational modeling studies, participated in data analysis and drafted the manuscript. ZHZ helped in data collection and computational modeling studies. JTA, TWT, VB participated in experimental design. SR conceived the study, and participated in its design and coordination and finalized the manuscript. All authors read and approved the final manuscript.

Acknowledgements

This work was partly funded by the National Institute of Allergy and Infectious Diseases, National Institute of Health, USA (Grant #5 U19 AI56541 & Contract #HHSN266200400085C).

References

  • Dill O, Kievits F, Koch S, Ivanyi P, Hämmerling GJ. Immunological function of HLA-C antigens in HLA-Cw3 transgenic mice. Proc Natl Acad Sci USA. 1988;85:5664–5668. doi: 10.1073/pnas.85.15.5664. [PMC free article] [PubMed] [Cross Ref]
  • Littaua RA, Oldstone MB, Takeda A, Debouck C, Wong JT, et al. An HLA-C-restricted CD8+ cytotoxic T-lymphocyte clone recognizes a highly conserved epitope on human immunodeficiency virus type 1 gag. J Virol. 1991;65:4051–4056. [PMC free article] [PubMed]
  • Snary D, Barnstable CJ, Bodmer WF, Crumpton MJ. Molecular structure of human histocompatibility antigens: the HLA-C series. Eur J Immunol. 1977;8:580–585. doi: 10.1002/eji.1830070816. [PubMed] [Cross Ref]
  • McCutcheon JA, Gumperz J, Smith KD, Lutz CT, Parham P. Low HLA-C expression at cell surfaces correlates with increased turnover of heavy chain mRNA. J Exp Med. 1995;181:2085–2095. doi: 10.1084/jem.181.6.2085. [PMC free article] [PubMed] [Cross Ref]
  • Korber BTM, Brander C, Haynes BF, Koup R, Moore JP, Walker BD, Watkind DI. HIV Molecular Immunology 2005. Los Alamos National Laboratory, Theoretical Biology and Biophysics, Los Alamos, New Mexico; 2005.
  • Bugawan TL, Klitz W, Blair A, Erlich HA. High-resolution HLA class I typing in the CEPH families: analysis of linkage disequilibrium among HLA loci. Tiss Antigens. 2000;56:392–404. doi: 10.1034/j.1399-0039.2000.560502.x. [PubMed] [Cross Ref]
  • Neefjes JJ, Ploegh HL. Allele and locus-specific differences in cell surface expression and the association of HLA class I heavy chain with β2-microglobulin: differential effects of inhibition of glycosylation on class I subunit association. Eur J Immunol. 1988;18:801–810. doi: 10.1002/eji.1830180522. [PubMed] [Cross Ref]
  • Neisig A, Melief CJ, Neefjes J. Reduced cell surface expression of HLA-C molecules correlates with restricted peptide binding and stable TAP interaction. J Immunol. 1998;160:171–179. [PubMed]
  • Brusic V, Bajic VB, Petrovsky N. Computational methods for prediction of T-cell epitopes – a framework for modelling, testing, and applications. Methods. 2004;34:436–443. doi: 10.1016/j.ymeth.2004.06.006. [PubMed] [Cross Ref]
  • Buchsbaum S, Barnea E, Dassau L, Beer I, Milner E, Admon A. Large-scale analysis of HLA peptides presented by HLA-Cw4. Immunogenetics. 2003;55:172–176. doi: 10.1007/s00251-003-0570-0. [PubMed] [Cross Ref]
  • Rammensee H, Bachmann J, Emmerich NP, Bachor OA, Stevanovic S. SYFPEITHI: database for MHC ligands and peptide motifs. Immunogenetics. 1999;50:213–219. doi: 10.1007/s002510050595. [PubMed] [Cross Ref]
  • DeLuca DS, Khattab B, Blasczyk R. A modular concept of HLA for comprehensive peptide binding prediction. Immunogenetics. 2007;59:25–35. doi: 10.1007/s00251-006-0176-4. [PubMed] [Cross Ref]
  • Tong JC, Tan TW, Ranganathan S. Modeling the structure of bound peptide ligands to major histocompatibility complex. Protein Sci. 2004;13:2523–2532. doi: 10.1110/ps.04631204. [PMC free article] [PubMed] [Cross Ref]
  • Tong JC, Bramson J, Kanduc D, Chow S, Sinha AA, Ranganathan S. Modeling the bound conformation of pemphigus vulgaris-associated peptides to MHC class II DR and DQ alleles. Immunome Res. 2006;2:1. doi: 10.1186/1745-7580-2-1. [PMC free article] [PubMed] [Cross Ref]
  • Tong JC, Zhang GL, Tan TW, August JT, Brusic V, Ranganathan S. Prediction of HLA-DQ3.2β ligands: Evidence of multiple registers in class II binding peptides. Bioinformatics. 2006;22:1232–1238. doi: 10.1093/bioinformatics/btl071. [PubMed] [Cross Ref]
  • Rognan D, Laumøller SL, Holm A, Buus S, Tschinke V. Predicting binding affinities of protein ligands from three-dimensional models: Application to Peptide Binding to Class I Major Histocompatibility Proteins. J Med Chem. 1999;42:4650–4658. doi: 10.1021/jm9910775. [PubMed] [Cross Ref]
  • Tong JC, Bramson J, Kanduc D, Sinha AA, Ranganathan S. Prediction of desmoglein-3 peptides reveals multiple shared T-cell epitopes in HLA DR4- and DR6-associated pemphigus vulgaris. BMC Bioinformatics. 2006;7:S7. doi: 10.1186/1471-2105-7-S5-S7. [PMC free article] [PubMed] [Cross Ref]
  • Johnson RP, Trocha A, Buchanan TM, Walker BD. Recognition of a highly conserved region of human immunodeficiency virus type 1 gp120 by an HLA-Cw4-restricted cytotoxic T-lymphocyte clone. J Virol. 1993;67:438–445. [PMC free article] [PubMed]
  • Sayle RA, Milner-White EJ. RASMOL: biomolecular graphics for all. Trends Biochem Sci. 1995;20:374. doi: 10.1016/S0968-0004(00)89080-5. [PubMed] [Cross Ref]
  • Brander C, Goulder PJR. Recent advances in HIV-1 CTL epitope characterization. In HIV molecular immunology database. In: Korber BTM, Brander C, Haynes BF, Koup R, Moore JP, Walker BD, Watkind DI, editor. HIV Molecular Database. Los Alamos National Laboratory. Los Alamos, New Mexico; 1999. pp. i1–i19.
  • Buseyne F, Stevanovic S, Rammensee HG, Riviere Y. Characterization of an HIV-1 p24gag epitope recognized by a CD8+ cytotoxic T-cell clone. Immunol Letters. 1997;55:145–149. doi: 10.1016/S0165-2478(97)02696-5. [PubMed] [Cross Ref]
  • Fan QR, Wiley DC. Structure of Hla-Cw4, a Ligand for the Kir2D Natural Killer Cell Inhibitory Receptor. J Exp Med. 1999;190:113–124. doi: 10.1084/jem.190.1.113. [PMC free article] [PubMed] [Cross Ref]
  • Abagyan RA, Totrov M. Ab initio folding of peptides by the optimal-bias Monte Carlo minimization procedure. J Comput Phys. 1999;151:402–421. doi: 10.1006/jcph.1999.6233. [Cross Ref]
  • Sidney J, del Guercio MF, Southwood S, Engelhard VH, Appella E, Rammensee HG, Falk K, Rötzschke O, Takiguchi M, Kubo RT, Grey HM, Sette A. Several HLA alleles share overlapping peptide specificities. J Immunol. 1995;154:247–259. [PubMed]
  • Rajagopalan S, Long EO. The direct binding of a p58 killer cell inhibitory receptor to human histocompatibility leukocyte antigen (HLA)-Cw4 exhibits peptide selectivity. J Exp Med. 1997;185:1523–1528. doi: 10.1084/jem.185.8.1523. [PMC free article] [PubMed] [Cross Ref]
  • The UniProt Consortium The Universal Protein Resource (UniProt) Nucleic Acids Res. 2007;35:D193–D197. doi: 10.1093/nar/gkl929. [PMC free article] [PubMed] [Cross Ref]
  • Srinivasan KN, Zhang GL, Khan AM, August JT, Brusic V. Prediction of class I T-cell epitopes: evidence of presence of immunological hot spots inside antigens. Bioinformatics. 2004;20:i297–i302. doi: 10.1093/bioinformatics/bth943. [PubMed] [Cross Ref]
  • Bradley AP. The use of the area under the ROC curve in the evaluation of machine learning algorithms. Pattern Recognition. 1997;30:1145–1159. doi: 10.1016/S0031-3203(96)00142-2. [Cross Ref]

Articles from Immunome Research are provided here courtesy of BioMed Central
PubReader format: click here to try

Formats:

Related citations in PubMed

See reviews...See all...

Cited by other articles in PMC

See all...

Links

Recent Activity

Your browsing activity is empty.

Activity recording is turned off.

Turn recording back on

See more...