1. September 2K3Bioinformatics Centre, University of Pune, Pune. 1 Role of Bioinformatics in designing vaccines Urmila Kulkarni-Kale Information Scientist Bioinformatics Centre University of Pune, Pune 411 007 India urmila@bioinfo.ernet.in

2. September 2K3Bioinformatics Centre, University of Pune, Pune. 2 Biological Research Biology is study of life and is a descriptive science. Macro micro properties Research methods are In vivo In vitro In silico Diversity: forms and functions

3. September 2K3Bioinformatics Centre, University of Pune, Pune. 3 Bioinformatics is a “scientific discipline that encompasses all aspects of biological information acquisition, processing, storage, distribution, analysis and interpretation”. What is Bioinformatics?

4. September 2K3Bioinformatics Centre, University of Pune, Pune. 4 BIOLOGY PHYSIOMICS CELLOMICS BIOTECH EVOLUTION INFOTECH ONTOLOGY PROTEOMICS MOLECULARMODELING MATHEMATICS METABOLOMICS TRANSCRIPTOMICS GENOMICS STATISTICS Bioinformatics combines the tools of Biology, Chemistry, Mathematics, Statistics and Computer Science to understand Life & its processes. Bioinformatics bridges many disciplines

5. September 2K3Bioinformatics Centre, University of Pune, Pune. 5 BIOLOGY PHYSIOMICS CELLOMICS BIOTECH EVOLUTION INFOTECH ONTOLOGY PROTEOMICS MOLECULARMODELING MATHEMATICS METABOLOMICS TRANSCRIPTOMICS GENOMICS STATISTICS The “omics” Series Omics is Latin word for ‘Give us money’! Genomics Gene identification & characterization Transcriptomics Expression profiles of mRNA Proteomics functions & interactions of proteins Structural Genomics Large scale structure determination Cellinomics Metabolic Pathways Cell-cell interactions Pharmacogenomics Genome-based drug design

6. September 2K3Bioinformatics Centre, University of Pune, Pune. 6 Nature of Biological data Post-Genomic era: Genomes Proteomes Metabolomes

7. September 2K3Bioinformatics Centre, University of Pune, Pune. 7 Approaches for vaccine development  Recombinant DNA vaccines  Peptide Vaccines  Polytope Vaccines

8. September 2K3Bioinformatics Centre, University of Pune, Pune. 8 Vaccine development In Post-genomic era: Reverse Vaccinology Approach. Rappuoli R. (2000). Reverse vaccinology. Curr Opin Microbiol. 3:445-450.

9. September 2K3Bioinformatics Centre, University of Pune, Pune. 9 Genome Sequence Proteomics Technologies In silico analysis DNA microarrays High throughput Cloning and expression In vitro and in vivo assays for Vaccine candidate identification Global genomic approach to identify new vaccine candidates

10. September 2K3Bioinformatics Centre, University of Pune, Pune. 10 In Silico Analysis Gene/Protein Sequence Database Disease related protein DB Candidate Epitope DB VACCINOME Peptide Multiepitope vaccines Epitope prediction

11. September 2K3Bioinformatics Centre, University of Pune, Pune. 11

12. September 2K3Bioinformatics Centre, University of Pune, Pune. 12 Epitopes … B-cell epitopesT h -cell epitopes

13. September 2K3Bioinformatics Centre, University of Pune, Pune. 13 Methods to identify epitopes 1.Immunochemical methods ELISA : Enzyme linked immunosorbent assay Immunoflurorescence Radioimmunoassay 2.X-ray crystallography: Ag-Ab complex is crystallized and the structure is scanned for contact residues between Ag and Ab. The contact residues on the Ag are considered as the epitope. 3.Prediction methods: Based on the X-ray crystal data available for Ag-Ab complexes, the propensity of an amino acid to lie in an epitope is calculated.

14. September 2K3Bioinformatics Centre, University of Pune, Pune. 14 Epitope prediction methods B cell epitope prediction algorithms : Hopp and Woods –1981 Welling et al –1985 Parker & Hodges - 1986 Kolaskar & Tongaonkar – 1990 Kolaskar & Urmila Kulkarni - 1999 T cell epitope prediction algorithms : Margalit, Spouge et al - 1987 Rothbard & Taylor – 1988 Stille et al –1987 Tepitope -1999

15. September 2K3Bioinformatics Centre, University of Pune, Pune. 15 Resources Programs available: ANTIGEN: Kolaskar & Tongaonkar’s method. Available in EMBOSS program as antigenic. url: http://bioweb.pasteur.fr/seqanal/interfaces/antigeni c.html EPIPLOT: Compilation of T and B cell prediction algorithms. Stand-alone program for PC. Databases of interest: BIMAS SYFPEITHI

16. September 2K3Bioinformatics Centre, University of Pune, Pune. 16 Prediction of epitopes  Knowledge of antigenic structure  Delineation of sequential and conformational epitopes  Knowledge of the 3-D structure of antigen  A method to map conformational epitopes

17. September 2K3Bioinformatics Centre, University of Pune, Pune. 17 Conformational epitope prediction method

18. September 2K3Bioinformatics Centre, University of Pune, Pune. 18

19. September 2K3Bioinformatics Centre, University of Pune, Pune. 19

20. September 2K3Bioinformatics Centre, University of Pune, Pune. 20

21. September 2K3Bioinformatics Centre, University of Pune, Pune. 21 Methods & Materials

22. September 2K3Bioinformatics Centre, University of Pune, Pune. 22

23. September 2K3Bioinformatics Centre, University of Pune, Pune. 23 We Have Chosen JE Virus, Because  JE virus is endemic in South-east Asia including India.  JE virus causes encephalitis in children between 5-15 years of age with fatality rates between 21-44%.  Man is a "DEAD END" host.

24. September 2K3Bioinformatics Centre, University of Pune, Pune. 24 We Have Chosen JE Virus, Because Killed virus vaccine purified from mouse brain is used presently which requires storage at specific temperatures and hence not cost effective in tropical countries. Protective prophylactic immunity is induced only after administration of 2-3 doses. Cost of vaccination, storage and transportation is high.

25. September 2K3Bioinformatics Centre, University of Pune, Pune. 25 Why Synthetic Peptide Vaccines?  Chemically well defined, selective and safe.  Stable at ambient temperature.  No cold chain requirement hence cost effective in tropical countries.  Simple and standardised production facility.

26. September 2K3Bioinformatics Centre, University of Pune, Pune. 26 Egp of JEV as an Antigen  Is a major structural antigen.  Responsible for viral haemagglutination.  Elicits neutralising antibodies.  ~ 500 amino acids long.  Structure of extra-cellular domain (399) was predicted using knowledge-based homology modeling approach.

27. September 2K3Bioinformatics Centre, University of Pune, Pune. 27

28. September 2K3Bioinformatics Centre, University of Pune, Pune. 28

29. September 2K3Bioinformatics Centre, University of Pune, Pune. 29 Multiple alignment of Predicted T H -cell epitope in the JE_Egp with corresponding epitopes in Egps of other Flaviviruses 426457 JE DFGSIGGVFNSIGKAVHQVFGGAFRTLFGGMS MVE DFGSVGGVFNSIGKAVHQVFGGAFRTLFGGMS WNE DFGSVGGVFTSVGKAIHQVFGGAFRSLFGGMS KUN DFGSVGGVFTSVGKAVHQVFGGAFRSLFGGMS SLE DFGSIGGVFNSIGKAVHQVFGGAFRTLFGGMS DEN2 DFGSLGGVFTSIGKALHQVFGAIYGAAFSGVS YF DFSSAGGFFTSVGKGIHTVFGSAFQGLFGGLN TBE DFGSAGGFLSSIGKAVHTVLGGAFNSIFGGVG COMM DF S GG S GK H V G F G Multiple alignment of JE_Egp with Egps of other Flaviviruses in the YSAQVGASQ region. 151 183 JE SENHGNYSAQVGASQAAKFTITPNAPSITLKLG MVE STSHGNYSTQIGANQAVRFTISPNAPAITAKMG WNE VESHG ‑‑‑‑ KIGATQAGRFSITPSAPSYTLKLG KUN VESHGNYFTQTGAAQAGRFSITPAAPSYTLKLG SLE STSHGNYSEQIGKNQAARFTISPQAPSFTANMG DEN2 HAVGNDTG ‑‑‑‑‑ KHGKEIKITPQSSTTEAELT YF QENWN ‑‑‑‑‑‑‑‑ TDIKTLKFDALSGSQEVEFI TBE VAANETHS ‑‑‑‑ GRKTASFTIS ‑‑ SEKTILTMG

30. September 2K3Bioinformatics Centre, University of Pune, Pune. 30 STEPS in Homology Modeling Template structure (PDB entry: 1SVB). (Rey et al., 1995). Alignment of Egp of JEV and Egp of TBEV. Definition of SCRs and Loops. Assignment of Initial co-ordinates to Backbone & Side-chains. Rotamer search for the favored side-chain conformations.

31. September 2K3Bioinformatics Centre, University of Pune, Pune. 31 Model Refinement PARAMETERS USED force field:AMBER all atom Dielectric const:Distance dependent Optimisation:Steepest Descents & Conjugate Gradients. rms derivative 0.1 kcal/mol/A for SD rms derivative 0.001 kcal/mol/A for CG Biosym from InsightII, MSI and modules therein.m

32. September 2K3Bioinformatics Centre, University of Pune, Pune. 32 ORDER OF REFINEMNT of MODEL  Loops  MD at 300  K for 500ps and equilibration of 100ps.  SCRs adjacent to the loop: SCR n-1, loop n, SCR n+1  Domains: I, II, III  Full molecule

33. September 2K3Bioinformatics Centre, University of Pune, Pune. 33 Model For Solvated Protein  Egp of JEV molecule was soaked in the water layer of 10A .  4867 water molecules were added.  The system size was increased to 20,648 atoms from 6047.

34. September 2K3Bioinformatics Centre, University of Pune, Pune. 34 Model Evaluation I:Energy Profile

35. September 2K3Bioinformatics Centre, University of Pune, Pune. 35 Model Evaluation II: Ramachandran Plot

36. September 2K3Bioinformatics Centre, University of Pune, Pune. 36

37. September 2K3Bioinformatics Centre, University of Pune, Pune. 37

38. September 2K3Bioinformatics Centre, University of Pune, Pune. 38 Strain specific properties: JEVN & JEVS

39. September 2K3Bioinformatics Centre, University of Pune, Pune. 39 Peptide Modeling Initial random conformation Force field: Amber Distance dependent dielectric constant 4r ij Geometry optimization: Steepest descents & Conjugate gradients Molecular dynamics at 400 K for 1ns Peptides are: SENHGNYSAQVGASQ NHGNYSAQVGASQ YSAQVGASQ YSAQVGASQAAKFT NHGNYSAQVGASQAAKFT SENHGNYSAQVGASQAAKFT 149 168

40. September 2K3Bioinformatics Centre, University of Pune, Pune. 40

41. September 2K3Bioinformatics Centre, University of Pune, Pune. 41

42. September 2K3Bioinformatics Centre, University of Pune, Pune. 42

43. September 2K3Bioinformatics Centre, University of Pune, Pune. 43 Publication/Patent A.S. Kolaskar and Urmila Kulkarni-Kale, 1999 - Prediction of three-dimensional structure and mapping of conformational epitopes of envelope glycoprotein of japanese encephalitis virus,Virology, 261, 31-42. Chimeric T helper - B cell peptide as a vaccine for Flaviviruses Gore, MM; Dewasthaly, SS; Kolaskar, AS; Kulkarni-Kale, Urmila

44. September 2K3Bioinformatics Centre, University of Pune, Pune. 44 Epitope prediction: References Hopp, Woods, 1981, Prediction of protein antigenic determinants from amino acid sequences, PNAS U.S.A 78, 3824-3828 Parker, Hodges et al, 1986, New hydrophilicity scale derived from high performance liquid chromatography peptide retention data: Correlation of predicted surface residues with antigenicity and X- ray derived accessible sites, Biochemistry:25, 5425-32 Kolaskar, Tongaonkar, 1990, A semi empirical method for prediction of antigenic determinants on protein antigens, FEBS 276, 172-174 Men‚ndez-Arias, L. & Rodriguez, R. (1990), A BASIC microcomputer program forprediction of B and T cell epitopes in proteins, CABIOS, 6, 101-105 Peter S. Stern (1991), Predicting antigenic sites on proteins, TIBTECH, 9, 163-169 A.S. Kolaskar and Urmila Kulkarni-Kale, 1999 - Prediction of three- dimensional structure and mapping of conformational epitopes of envelope glycoprotein of Japanese encephalitis virus,Virology, 261, 31-42

45. September 2K3Bioinformatics Centre, University of Pune, Pune. 45 Acknowledgements Department of Biotechnology, Govt. of India. Immunology Div., NIV.