1. CENTER FOR BIOLOGICAL SEQUENCE ANALYSISTECHNICAL UNIVERSITY OF DENMARK DTU B cell epitopes and B cell epitope predictions Morten Nielsen, CBS, BioCentrum, DTU ( mostly copied from Vsevolod Katritch’s, Siga presentation 2002 )

2. CENTER FOR BIOLOGICAL SEQUENCE ANALYSISTECHNICAL UNIVERSITY OF DENMARK DTU Algorithms for epitope prediction and selection B-cell epitopes Most epitope are structural epitopes sequence based methods are limited requires structure-based approach Antibody Fab fragment

3. CENTER FOR BIOLOGICAL SEQUENCE ANALYSISTECHNICAL UNIVERSITY OF DENMARK DTU B-cell epitope classification linear epitopes “Discontinuous epitope (with linear determinant) Discontinuous epitope B-cell epitope – structural feature of a molecule or pathogen, accessible and recognizable by B-cells

4. CENTER FOR BIOLOGICAL SEQUENCE ANALYSISTECHNICAL UNIVERSITY OF DENMARK DTU Sequence-based methods Protein hydrophobicity – hydrophilicity algorithms 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 Protein “antigenicity” prediction : Hopp and Woods, Parker, Protrusion Index (Thornton), Welling TSQDLSVFPLASCCKDNIASTSVTLGCLVTGYLPMSTTVT WDTGSLNKNVTTFPTTFHETYGLHSIVSQVTASGKWAKQ RFTCSVAHAESTAINKTFSACALNFIPPTVKLFHSSCNPVGD THTTIQLLCLISGYVPGDMEVIWLVDGQKATNIFPYTAPG TKEGNVTSTHSELNITQGEWVSQKTYTCQVTYQGFTFKD EARKCSESDPRGVTSYLSPPSPL  Same as protein surface accessibility  Predict “linear” epitopes only

5. CENTER FOR BIOLOGICAL SEQUENCE ANALYSISTECHNICAL UNIVERSITY OF DENMARK DTU Linear Epitopes (flexible loops) Con  only ~5% of epitopes can be classified as “linear”  weakly immunogenic in most cases  most epitope peptides does not provide antigen- neutralizing immunity  in many cases represent hypervariable regions (HIV, HCV etc.) 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

6. CENTER FOR BIOLOGICAL SEQUENCE ANALYSISTECHNICAL UNIVERSITY OF DENMARK DTU Prediction of linear epitopes

7. CENTER FOR BIOLOGICAL SEQUENCE ANALYSISTECHNICAL 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 probe Antibody Fab fragment Protrusion index

8. CENTER FOR BIOLOGICAL SEQUENCE ANALYSISTECHNICAL UNIVERSITY OF DENMARK DTU Rational B-cell epitope design Protein target choice Structural analysis of antigen X-ray 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 Model

9. CENTER FOR BIOLOGICAL SEQUENCE ANALYSISTECHNICAL UNIVERSITY OF DENMARK DTU Rational B-cell epitope design Surface accessibility Protrusion index Conserved sequence Glycosylation status Protein target choice Structural annotation Epitope prediction and ranking

10. CENTER FOR BIOLOGICAL SEQUENCE ANALYSISTECHNICAL 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

11. CENTER FOR BIOLOGICAL SEQUENCE ANALYSISTECHNICAL UNIVERSITY OF DENMARK DTU HIV gp120-CD4 epitope Binding of CD4 receptor Conformational changes in gp120 Opens chemokine-receptor binding site New highly concerved epitope Kwong et al.(1998) Nature 393, 648-658

12. CENTER FOR BIOLOGICAL SEQUENCE ANALYSISTECHNICAL UNIVERSITY OF DENMARK DTU HIV gp120-CD4 epitope Elicit broadly cross- reactive 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. First efforts to design single-chain analogue

13. CENTER FOR BIOLOGICAL SEQUENCE ANALYSISTECHNICAL UNIVERSITY OF DENMARK DTU HIV gp120-CD4 epitope reduce to minimal stable fold (iterative) optimize linker length 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 Further optimization of the epitope:

14. CENTER FOR BIOLOGICAL SEQUENCE ANALYSISTECHNICAL UNIVERSITY OF DENMARK DTU Homology modeling Alignment BLAB._ 0 : EKLKDNLYVYTTYNTFNGTKY-AANAVYLVTDKGVVVIDCPWGEDKFKSFTDEIYKKHGKKVIMNIATHS 1A8T.A 13 : TQLSDKVYTYVSLAEIEGWGMVPSNGMIVINNHQAALLDTPINDAQTEMLVNWVTDSLHAKVTTFIPNHW BLAB._ 69 : HDDRAGGLEYFGKIGAKTYSTKMTDSILAKENKPRAQYTFDNNKSFKVGKSEFQVYYPGKGHTADNVVVW 1A8T.A 83 : HGDCIGGLGYLQRKGVQSYANQMTIDLAKEKGLPVPEHGFTDSLTVSLDGMPLQCYYLGGGHATDNIVVW BLAB._ 139 : FPKEKVLVGGCIIKSADSKDLGYIGEAYVNDWTQSVHNIQQKFSGAQYVVAGHDDWKDQRSIQHTLDLIN 1A8T.A 153 : LPTENILFGGCMLKDNQTTSIGNISDADVTAWPKTLDKVKAKFPSARYVVPGHGNYGGTELIEHTKQIVN BLAB._ 209 : EYQQKQK 1A8T.A 223 : QYIESTS Sequence identity 27%

15. CENTER FOR BIOLOGICAL SEQUENCE ANALYSISTECHNICAL UNIVERSITY OF DENMARK DTU Homology Modeling Blue: Template Red: Model

16. CENTER FOR BIOLOGICAL SEQUENCE ANALYSISTECHNICAL UNIVERSITY OF DENMARK DTU Homology Modeling Protein sequence Known 3D structure Known 3D template(s) Model by homology Threading (seq-str. align.) Side chain prediction Loop building Local reliability prediction Model

17. CENTER FOR BIOLOGICAL SEQUENCE ANALYSISTECHNICAL UNIVERSITY OF DENMARK DTU Protein Model Health Evaluation Known 3D structure Maiorov, Abagyan, Proteins 1998 Cardozo, Abagyan, 2000 Model High energy strain Lower energy strain  Local alignment strength  Local Energy strain

18. CENTER FOR BIOLOGICAL SEQUENCE ANALYSISTECHNICAL UNIVERSITY OF DENMARK DTU Annotation of protein surface Contour-buildup algorithm (J.Str.Biol, 116, 138, 1996). Requires 3D structure Surface prediction using propensity scales (linear effects) Surface prediction using Neural networks (higher order effects)

19. CENTER FOR BIOLOGICAL SEQUENCE ANALYSISTECHNICAL UNIVERSITY OF DENMARK DTU Multichain protein design 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 B-cell epitope T-cell epitope

20. CENTER FOR BIOLOGICAL SEQUENCE ANALYSISTECHNICAL UNIVERSITY OF DENMARK DTU Conclusions Rational vaccines can be designed to induce strong and epitope-specific B-cell response Selection of protective B-cell epitope involves structural, functional and immunogenic analysis of the pathogenic proteins Structural modeling tools are critical in design of epitope mimics and optimal epitope presentation