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Pernille Haste Andersen, Ph.d. student Immunological Bioinformatics CBS, DTU CENTER FOR BIOLOGICAL SEQUENCE ANALYSIS TECHNICAL UNIVERSITY OF DENMARK DTU B cell epitopes and predictions • What is a B-cell epitope? • How can you predict B-cell epitopes? • B-cell epitopes in rational vaccine design. – Case story: Using B-cell epitopes in rational vaccine design against HIV. CENTER FOR BIOLOGICAL SEQUENCE ANALYSIS TECHNICAL UNIVERSITY OF DENMARK DTU Outline B-cell epitopes Antibody Fab fragment Accessible structural feature of a pathogen molecule. Antibodies are developed to bind the epitope specifically using the complementary determining regions (CDRs). CENTER FOR BIOLOGICAL SEQUENCE ANALYSIS TECHNICAL UNIVERSITY OF DENMARK DTU What is a B-cell epitope? Binding strength Salt bridges Hydrogen bonds Hydrophobic interactions Van der Waals forces CENTER FOR BIOLOGICAL SEQUENCE ANALYSIS TECHNICAL UNIVERSITY OF DENMARK DTU The binding interactions Many of the charged groups and hydrogen bonding partners are present on highly flexible amino acid side chains. Most crystal structures of epitopes and antibodies in free and complexed forms have shown conformational rearrangements upon binding. “Induced fit” model of interactions. CENTER FOR BIOLOGICAL SEQUENCE ANALYSIS TECHNICAL UNIVERSITY OF DENMARK DTU B-cell epitopes are dynamic B-cell epitope – structural feature of a molecule or pathogen, accessible and recognizable by B-cells Linear epitopes One segment of the amino acid chain Discontinuous epitope (with linear determinant) Discontinuous epitope Several small segments brought into proximity by the protein fold CENTER FOR BIOLOGICAL SEQUENCE ANALYSIS TECHNICAL UNIVERSITY OF DENMARK DTU B-cell epitope classification Antibody FAB fragment complexed with Guinea Fowl Lysozyme (1FBI). Black: Light chain, Blue: Heavy chain, Yellow: Residues with atoms distanced < 5Å from FAB antibody fragments. Guinea Fowl Lysozyme KVFGRCELAAAMKRHGLDNYRGYSLGNWVCAAKFESNFNSQNRNTDGS DYGVLNSRWWCNDGRTPGSRNLCNIPCSALQSSDITATANCAKKIVSDG GMNAWVAWRKCKGTDVRVWIKGCRL CENTER FOR BIOLOGICAL SEQUENCE ANALYSIS TECHNICAL UNIVERSITY OF DENMARK DTU Binding of a discontinuous epitope • Linear epitopes: – Chop sequence into small pieces and measure binding to antibody • Discontinuous epitopes: – Measure binding of whole protein to antibody • The best annotation method : X-ray crystal structure of the antibody-epitope complex CENTER FOR BIOLOGICAL SEQUENCE ANALYSIS TECHNICAL UNIVERSITY OF DENMARK DTU B-cell epitope annotation • Databases: AntiJen, IEDB, BciPep, Los Alamos HIV database, Protein Data Bank • Large amount of data available for linear epitopes • Few data available for discontinuous CENTER FOR BIOLOGICAL SEQUENCE ANALYSIS TECHNICAL UNIVERSITY OF DENMARK DTU B-cell epitope data bases CENTER FOR BIOLOGICAL SEQUENCE ANALYSIS TECHNICAL UNIVERSITY OF DENMARK DTU B cell epitope prediction Protein hydrophobicity – hydrophilicity algorithms Parker, Fauchere, Janin, Kyte and Doolittle, Manavalan Sweet and Eisenberg, Goldman, Engelman and Steitz (GES), von Heijne Protein flexibility prediction algorithm Karplus and Schulz Protein secondary structure prediction algorithms GOR II method (Garnier and Robson), Chou and Fasman, Pellequer Protein “antigenicity” prediction : Hopp and Woods, Welling TSQDLSVFPLASCCKDNIASTSVTLGCLVTG YLPMSTTVTWDTGSLNKNVTTFPTTFHETY GLHSIVSQVTASGKWAKQRFTCSVAHAES TAINKTFSACALNFIPPTVKLFHSSCNPVGD THTTIQLLCLISGYVPGDMEVIWLVDGQKA TNIFPYTAPGTKEGNVTSTHSELNITQGEW VSQKTYTCQVTYQGFTFKDEARKCSESDPR GVTSYLSPPSPL CENTER FOR BIOLOGICAL SEQUENCE ANALYSIS TECHNICAL UNIVERSITY OF DENMARK DTU Sequence-based methods for prediction of linear epitopes • The Parker hydrophilicity scale • Derived from experimental data D E N S Q G K T R P H C A Y V M I F L W 2.46 1.86 1.64 1.50 1.37 1.28 1.26 1.15 0.87 0.30 0.30 0.11 0.03 -0.78 -1.27 -1.41 -2.45 -2.78 -2.87 -3.00 Hydrophilicity CENTER FOR BIOLOGICAL SEQUENCE ANALYSIS TECHNICAL UNIVERSITY OF DENMARK DTU Propensity scales: The principle ….LISTFVDEKRPGSDIVEDLILKDENKTTVI…. (-2.78 + -1.27 + 2.46 +1.86 + 1.26 + 0.87 + 0.3)/7 = 0.39 Prediction scores: 0.38 0.1 0.6 0.9 1.0 1.2 2.6 1.0 0.9 0.5 -0.5 Epitope CENTER FOR BIOLOGICAL SEQUENCE ANALYSIS TECHNICAL UNIVERSITY OF DENMARK DTU Propensity scales: The principle • A Receiver Operator Curve (ROC) is useful for finding a good threshold and rank methods CENTER FOR BIOLOGICAL SEQUENCE ANALYSIS TECHNICAL UNIVERSITY OF DENMARK DTU Evaluation of performance • Pellequer found that 50% of the epitopes in a data set of 11 proteins were located in turns •Turn propensity scales for each position in the turn were used for epitope prediction. Pellequer et al., Immunology letters, 1993 1 4 2 3 CENTER FOR BIOLOGICAL SEQUENCE ANALYSIS TECHNICAL UNIVERSITY OF DENMARK DTU Turn prediction and B-cell epitopes • Extensive evaluation of propensity scales for epitope prediction • Conclusion: – Basically all the classical scales perform close to random! – Other methods must be used for epitope prediction CENTER FOR BIOLOGICAL SEQUENCE ANALYSIS TECHNICAL UNIVERSITY OF DENMARK DTU Blythe and Flower 2005 • Parker hydrophilicity scale • Hidden Markow model • Markow model based on linear epitopes extracted from the AntiJen database • Combination of the Parker prediction scores and Markow model leads to prediction score • Tested on the Pellequer dataset and epitopes in the HIV Los Alamos database CENTER FOR BIOLOGICAL SEQUENCE ANALYSIS TECHNICAL UNIVERSITY OF DENMARK DTU BepiPred: CBS in-house tool A Pos 1 7.28 Pos 2 9.39 C D ………… G S T V W Pos 3 0.3 Pos 4 5.2 Pos 5 7.9 ….LISTFVDEKRPGSDIVEDLILKDENKTTVI…. 2.46+1.86+1.26+0.87+0.3 = 6.75 Prediction value CENTER FOR BIOLOGICAL SEQUENCE ANALYSIS TECHNICAL UNIVERSITY OF DENMARK DTU Hidden Markow Model Evaluation on HIV Los Alamos data set CENTER FOR BIOLOGICAL SEQUENCE ANALYSIS TECHNICAL UNIVERSITY OF DENMARK DTU ROC evaluation • Pellequer data set: – Levitt – Parker – BepiPred AROC = 0.66 AROC = 0.65 AROC = 0.68 – Levitt – Parker – BepiPred AROC = 0.57 AROC = 0.59 AROC = 0.60 • HIV Los Alamos data set CENTER FOR BIOLOGICAL SEQUENCE ANALYSIS TECHNICAL UNIVERSITY OF DENMARK DTU BepiPred performance • BepiPred conclusion: – On both of the evaluation data sets, Bepipred was shown to perform better – Still the AROC value is low compared to T-cell epitope prediction tools! – Bepipred is available as a webserver: www.cbs.dtu.dk/services/BepiPred CENTER FOR BIOLOGICAL SEQUENCE ANALYSIS TECHNICAL UNIVERSITY OF DENMARK DTU BepiPred • • • • • Pro easily predicted computationally easily identified experimentally immunodominant epitopes in many cases do not need 3D structural information easy to produce and check binding activity experimentally • • • • Con only ~10% of epitopes can be classified as “linear” weakly immunogenic in most cases most epitope peptides do not provide antigenneutralizing immunity in many cases represent hypervariable regions CENTER FOR BIOLOGICAL SEQUENCE ANALYSIS TECHNICAL UNIVERSITY OF DENMARK DTU Prediction of linear epitopes Linear methods for prediction of B cell epitopes have low performances The problem is analogous to the problems of representing the surface of the earth on a two-dimensional map Reduction of the dimensions leads to distortions of scales, directions, distances The world of B-cell epitopes is 3 dimensional and therefore more sophisticated methods must be developed Regenmortel 1996, Meth. of Enzym. 9. CENTER FOR BIOLOGICAL SEQUENCE ANALYSIS TECHNICAL UNIVERSITY OF DENMARK DTU Sequence based prediction methods • Use of the three dimensional structure of the pathogen protein • Analyze the structure to find surface exposed regions • Additional use of information about conformational changes, glycosylation and trans-membrane helices CENTER FOR BIOLOGICAL SEQUENCE ANALYSIS TECHNICAL UNIVERSITY OF DENMARK DTU So what is more sophisticated? Structural determination • X-ray crystallography • NMR spectroscopy Both methods are time consuming and not easily done in a larger scale Structure prediction • Homology modeling • Fold recognition Less time consuming, but there is a possibility of incorrect predictions, specially in loop regions CENTER FOR BIOLOGICAL SEQUENCE ANALYSIS TECHNICAL UNIVERSITY OF DENMARK DTU How can we get information about the three-dimensional structure? • Homology/comparative modeling >25% sequence identity (seq 2 seq alignment) • Fold-recognition/threading <25% sequence identity (Psi-blast search/ seq2str alignment) • Ab initio structure prediction 0% sequence identity CENTER FOR BIOLOGICAL SEQUENCE ANALYSIS TECHNICAL UNIVERSITY OF DENMARK DTU Protein structure prediction methods •Look at the surface of your protein •Analyse for turns and loops •Analyse for amino acid composition •Find exposed Bepipred epitopes CENTER FOR BIOLOGICAL SEQUENCE ANALYSIS TECHNICAL UNIVERSITY OF DENMARK DTU Annotation of protein surface probe Antibody Fab fragment Protrusion index CENTER FOR BIOLOGICAL SEQUENCE ANALYSIS TECHNICAL UNIVERSITY OF DENMARK DTU Q: What can antibodies recognize in a protein? A: Everything accessible to a 10 Å probe on a protein surface Novotny J. A static accessibility model of protein antigenicity. Int Rev Immunol 1987 Jul;2(4):379-89 • CBS server for prediction of discontinuous epitopes – http://www.cbs.dtu.dk/services/DiscoTope/ • Uses protein structure as input • Combines propensity scale values of amino acids in discontinuous epitopes with surface exposure CENTER FOR BIOLOGICAL SEQUENCE ANALYSIS TECHNICAL UNIVERSITY OF DENMARK DTU Use the DiscoTope server CENTER FOR BIOLOGICAL SEQUENCE ANALYSIS TECHNICAL UNIVERSITY OF DENMARK DTU CENTER FOR BIOLOGICAL SEQUENCE ANALYSIS TECHNICAL UNIVERSITY OF DENMARK DTU Green: Predicted epitopes Black: Experimental epitopes CENTER FOR BIOLOGICAL SEQUENCE ANALYSIS TECHNICAL UNIVERSITY OF DENMARK DTU Malaria protein AMA1. >PATHOGEN PROTEIN KVFGRCELAAAMKRHGLDNYRG YSLGNWVCAAKFESNF Rational Vaccine Design CENTER FOR BIOLOGICAL SEQUENCE ANALYSIS TECHNICAL UNIVERSITY OF DENMARK DTU Rational vaccine design • Protein target choice • Structural analysis of antigen Model CENTER FOR BIOLOGICAL SEQUENCE ANALYSIS TECHNICAL UNIVERSITY OF DENMARK DTU Rational B-cell epitope design Known structure or homology model Precise domain structure Physical annotation (flexibility, electrostatics, hydrophobicity) Functional annotation (sequence variations, active sites, binding sites, glycosylation sites, etc.) Known 3D structure • Protein target choice • Structural annotation • Epitope prediction and ranking Surface accessibility Protrusion index Conserved sequence Glycosylation status CENTER FOR BIOLOGICAL SEQUENCE ANALYSIS TECHNICAL UNIVERSITY OF DENMARK DTU Rational B-cell epitope design • • • • Protein target choice Structural annotation Epitope prediction and ranking Optimal Epitope presentation Fold minimization, or Design of structural mimics Choice of carrier (conjugates, DNA plasmids, virus like particles) Multiple chain protein engineering CENTER FOR BIOLOGICAL SEQUENCE ANALYSIS TECHNICAL UNIVERSITY OF DENMARK DTU Rational B-cell epitope design epitope T-cell epitope Rational optimization of epitope-VLP chimeric proteins: Design a library of possible linkers (<10 aa) Perform global energy optimization in VLP (virus-like particle) context Rank according to estimated energy strain CENTER FOR BIOLOGICAL SEQUENCE ANALYSIS TECHNICAL UNIVERSITY OF DENMARK DTU Multi-epitope protein design B-cell Using B-cell epitopes in the rational vaccine design against HIV The gp120-CD4 epitope CENTER FOR BIOLOGICAL SEQUENCE ANALYSIS TECHNICAL UNIVERSITY OF DENMARK DTU Case story Binding of CD4 receptor Conformational changes in gp120 Opens chemokinereceptor binding site New highly conserved epitopes Kwong et al.(1998) Nature 393, 648-658 CENTER FOR BIOLOGICAL SEQUENCE ANALYSIS TECHNICAL UNIVERSITY OF DENMARK DTU HIV gp120-CD4 epitope SIV gp120 no ligands Human gp120 complex with CD4 and 17b antibody Chen et al. Nature 2005 Kwong et al. Nature 1998 CENTER FOR BIOLOGICAL SEQUENCE ANALYSIS TECHNICAL UNIVERSITY OF DENMARK DTU Conformational changes in gp120 Efforts to design a epitope fusion protein Elicit broadly crossreactive neutralizing antibodies in rhesus macaques. This conjugate is too large(~400 aa) and still contains a number of irrelevant loops Fouts et al. (2000) Journal ofVirology, 74, 11427-11436 Fouts et al. (2002) Proc Natl Acad Sci U S A. 99, 11842-7. CENTER FOR BIOLOGICAL SEQUENCE ANALYSIS TECHNICAL UNIVERSITY OF DENMARK DTU HIV gp120-CD4 epitope Further optimization of the epitope: reduce to minimal stable fold (iterative) find alternative scaffold to present epitope (miniprotein mimic) Martin & Vita, Current Prot. An Pept. Science, 1: 403-430. Vita et al.(1999) PNAS 96:13091-6 CENTER FOR BIOLOGICAL SEQUENCE ANALYSIS TECHNICAL UNIVERSITY OF DENMARK DTU HIV gp120-CD4 epitope • Rational vaccines can be designed to induce strong and epitope-specific B-cell responses • Selection of protective B-cell epitopes involves structural, functional and immunogenic analysis of the pathogenic proteins • When you can: Use protein structure for prediction • Structural modeling tools are helpful in prediction of epitopes, design of epitope mimics and optimal epitope presentation CENTER FOR BIOLOGICAL SEQUENCE ANALYSIS TECHNICAL UNIVERSITY OF DENMARK DTU Conclusions