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Case Study #2 How Bioinformatics Aids in Vaccine Development / Peptide Vaccine Development Using Bionformatics Approaches Ashok Kolaskar University of Pune, Pune 411 007, India. [email protected] Emerging and re-emerging infectious diseases threats, 1980-2001 Viral - Bolivian hemorrhagic fever-1994,Latin America Bovine spongiform encephalopathy-1986,United Kingdom Creulzfeldt-Jackob disease(a new variant V-CID)/mad cow disease-1995-96, UK/France Dengue fever-1994-97,Africa/Asia/Latin America/USA Ebola virus-1994,Gabon;1995,Zaire;1996,United States(monkey) Hantavirus-1993,United States; 1997, Argentina HIV subtype O-1994,Africa Influenza A/Beijing/32/92, A/Wuhan/359/95, HS:N1-1993,United States; 1995,China; 1997, Hongkong Japanese Encephalitis-1995, Australia Lassa fever-1992,Nigeria Measles-1997, Brazil Monkey pox-1997,Congo Morbillivirus – 1994, Australia O’nyong-nyong fever-1996,Uganda Polio-1996,Albania Rift Valley fever-1993,Sudan Venezuelan equine encephalitis-1995-96,Venezuela/Colombia West Nile Virus-1996,Romania Yellow fever-1993,Kenya;1995,Peru Emerging and re-emerging infectious diseases threats contd., • Parasitic - African trypanosomiasis-1997,Sudan - Ancylcostoma caninum(eosinophilic enteritis)1990s,Australia - Cryptosporiadiasis-1993+,United States - Malaria-1995-97,Africa/Asia/Latin America/United states - Metorchis-1996,Canada - Microsporidiosis-Worldwide • Fungal - Coccidiodomycosis-1993,United States - Penicillium marneffi Emerging and re-emerging infectious diseases threats contd. • Bacterial – Anthrax-1993,Caribbean – Cat scratch disease/Bacillary angiomatosis(Bartonella henseiae)-1900s, USA – Chlamydia pneumoniae(Pneumonia/Coronary artery disease?)-1990s, USA(discovered 1983) – Cholera-1991,Latin America – Diphtheria-1993,Former Soviet Union – Ehrlichia chaffeensis,Human monocytic ahrlichiosis(HME)-United States – Ehrlichia phagocytophilia,Human Granulocytic ehrlichis(HGE)-United States – Escherichia coli O157-1982-1997,United States;1996,Japan – Gonorrhea(drug resistant)-1995,United States – Helicobacter pylori(ulcers/cancer_-worldwide(discovered 1983) – Leptospirosis-195,Nicaragun – Lyme disease(Borrelia burgdorferi)-1990s,United states – Meningococcal meningitis(serogroup A)-1995-1997,West Africa – Pertussis-1994,UK/Netherlands;1996,USA – Plague-1994,India – Salmonella typhimurium DT104(drug resistant)-1995,USA – Staphylococcus aureus(drug resistant)-1997,United States/Japan – Toxic strep-United States – Trench fever(Barnionella quintana)-1990s,United States – Tuberculosis(highly transmissible)-1995,United states – Vibrio cholerae 0139-1992,Southern Asia Types of Vaccines • • • • • • • Killed virus vaccines Live-attenuated vaccines Recombinant DNA vaccines Genetic vaccines Subunit vaccines Polytope/multi-epitope vaccines Synthetic peptide vaccines Molecular vaccines in phase III clinical trials and beyond Company Product/Type Status Aphton Indication Gastrimmune Phase III Blocks hormone Gastrin 17 Avax Technologies M-Vax Vaccine Phase III Inc. Autologous cellular vaccine AVI Biopharma Avicin (CTP-7) Phase II/III Inc/Immunotherapy Peptide conjugate Corp caccine GI Cancers Biomira Inc/Chiron Corp Phase III Metastatic breast cancer Phase III e1999 Phase III e2000 Prostate cancer Theratope vaccine Synthetic cancer vaccine Jenner Biotherapies OncoVax-P Vaccine Inc. Jenner Biotherapies OncoVax-CL Inc. Vaccine Melanoma Pancreatic Colorectal and lung cancer Contd../ Company Progenics Pharmaceuticals/B MS Ribi Immunochem Product/Type Status GMK Phase III Ganglioside conjugate vaccine Melacine Allogeneic BLA tumour specific filing e1999 vaccine Agouron Remune Inactivated Phase III Pharmaceuticals Inc HIV therapeutic vaccine Aviron FluMist Intranasal flu PLA submitted (but refused) mid-98 Bio-Technology Bio-Hep-B Phase III trials General Corp Generally engineered Completed vacine Chiron Corp Pertugen (acellular MAA filed pertussis) Subunit vaccine Indication Melanoma Melanoma HIV/AIDS Influenza in children/adults Hepatitis B Pertussis Contd../ Company Product/Type Status Indication Chiron Corp Influenza + MF-59 Phase III Subunit vaccine Hepatitis A Chiron Copr Hepatitis A Vaccine Phase III (Europe) Hepatitis A NABI StaphVAX Polysaccharide conjugate vaccine Phase III Nosocomial Staphylococc us aureus bacteremia OraVAX Inc Arilvax Live Phase III 1999 Yellow fever (Peptide attenuated vaccine Therapeutics Ltd) SB LYMErix FDA approval Lyme disease Recombinanat OspA 5/98 vaccine SB Recombinant subunit Phase III Genital vaccine herpes prophylaxis VaxGen Inc AIDSVax Phase III HIV/AIDS Recombinant gp 120 SB Recombinant subunit Phase III end Chronic hepatitis B Therapeutic vaccine 1998 Systems with potential use as T-cell vaccines CD4 + T-cell vaccines Killed microbe Live attenuated microbe Synthetic peptide coupled to protein Recombinant microbial protein bearing CD4+ T-cell epitope CD8+ T-cell vaccines Live attenuated microbe Synthetic peptide delivered in liposomes or ISCOMs - Chimeric virus expressing CD4+ T-cell epitope Chimeric virus expressing CD8+ T-cell epitope Chimeric Ig Self-molecule expressing CD8+ T-cell epitope Chimeric-peptide-MHC class II complex Chimeric peptide-MHC Class I complex Receptor-linked peptide Naked DNA expressing CD4+ T-cell epitope Naked DNA expressing CD8+ T-cell epitope Abbreviations: Ig, Immunoglobulin, ISCOM, immune-stimulating complex; MHC,Major histocompability complex. 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. Epitopes … B-cell epitopes Th-cell epitopes What Are Epitopes? Antigenic determinants or Epitopes are the portions of the antigen molecules which are responsible for specificity of the antigens in antigen-antibody (Ag-Ab) reactions and that combine with the antigen binding site of Ab, to which they are complementary. Epitopes could be contiguous (when Ab binds to a contiguous sequence of amino acids) non-contiguous (when Ab binds to non-contiguous residues, brought together by folding). Sequential epitopes are contiguous epitopes. Conformational epitopes are noncontiguous antigenic determinants. Properties of Amino Acids: predictors for Epitopes Sequential epitope prediction methods Theoretical methods are based on properties of amino acids and their propensity scales. Hopp & Woods, 1981. Parker et al., 1986 Kolaskar & Tongaonkar, 1990. The accuracy of prediction: 50-75%. Conformational epitope prediction method Kolaskar & Kulkarni-Kale, 1999. Antigenic determinants of Egp of JEV Kolaskar & Tongaonkar approach Peptide vaccines to be launched in near future • • • • • • Foot & Mouth Disease Virus (FMDV) Human Immuno Deficiency Virus (HIV) Metastatic Breast Cancer Pancreatic Cancer Melanoma Malaria Retro-Inverso peptides Potential Synthetic peptide Vaccines • A retro structure is obtained by synthesizing peptides in reverse order, in which the direction of peptide bond is reversed and side-chains are oriented in the manner similar to that in D-enantiomer, which explains why these two analogs cross-react immunochemically. • When both of these transformations are combined in the form of an all-D-retro or Retro-Inverso (RI) peptide, the side-chains are oriented as in original Lpeptide. • As a result, antibodies raised against the L- or the allD-retro form cross-react strongly with both structures. • Ex: RI peptide as vaccine candidate for FMDV (Muller, S., Brown, F, MHV Van Regenmortel) Various transformations on side-chain orientation in a model tetrapeptide Mirror symmetry between L- and Dforms of peptides Note that the retro modification does not take the peptide through an axis of symmetry. Schematic representation of the natural(L) IRGERA peptide of histone H3 1 Cys and 2Gly were added at N’term for conjugation with carrier. Vaccine development In Post-genomic era: Reverse Vaccinology Approach. Reverse Vaccinology • Advantages – – – – – Fast access to virtually every antigen Non-cultivable can be approached Non abundant antigens can be identified Antigens not expressed in vitro can be identified. Non-structural proteins can be used • Disadvantages – Non proteinous antigens like polysaccharides, glycolipids cannot be used. Genome Sequence Proteomics Technologies In silico analysis IVET, STM, 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 In Silico Analysis Peptide Multitope vaccines VACCINOME Candidate Epitope DB Epitope prediction Disease related protein DB Gene/Protein Sequence Database Genome sequencing & Identification of vaccine Candidates of Neisseria meningitidis • Complete Genome Sequence of Neisseria meningitidis Serogroup B Strain MC58. – Tettelin, et al., (2000). Science. 287:1089-1815. • Identification of Vaccine Candidates Against Serogroup B Meningococcus by Whole-Genome Sequencing. – Pizza, et al., (2000). Science. 287:1816-1820. Genome analysis of Neisseria meningitidis serogroup B strain MC58 • • • • Genome size: 2,272,351 bp Predicted coding regions: 2158 Function annotations: 1158 (53.7%) 3 major islands of horizontal transfer have been observed. – 2 contains genes encoding proteins involved in pathogenicity – 1 contains a hypothetical protein • It contains more genes that undergo phase variation than any pathogen studied to date, a mechanism that controls their expression and contributes to the evasion of the host immune system. Neisseria meningitidis (Meningococcus) • Is a gram negative Proteobacterium. • Is a cause of life threatening invasive bacterial infections especially in infants. • Causes Meningitis & Septicemia, which are significant public health problem. • The fatality rate ranges from 5-15% and up to 25% of survivors are left with neurological sequelae. • There are 5 pathogenic serogroups (A,B,C,Y and W135) based on chemical composition of distinctive capsular polysaccharide typing. • Although the vaccine exists, its impact has been limited. Comparative Genomics: key to identify the genes responsible for pathogenesis of meningitis • Complete genomes of 2 strains of Neisseria meningitidis are available. – Serogroup B strain MC58 – Serogroup A strain Z2491 • Complete genome of Haemophilus influenzae, another pathogen responsible for meningitis and the first organism to be sequenced completely is also available. • Comparative genomics of these three genomes provides an opportunity to define a common subset of genes that are responsible for pathogenesis of this disease. Comparison of ORFs of two strains of N.meningitidis Dendogram showing genetic relation ship among 107 N.meningitidis stains Synthetic Peptide Vaccine Case Study: Design and Development of Synthetic Peptide vaccine against Japanese encephalitis virus 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. MULTIPLE SEQUENCE ALIGNMENT OF FLAVIVIRUSES. PREDICTED ANTIGENIC DETERMINANTS USING KOLASKAR & TONGAONKAR, 1990 ARE MARKED. PART 1/5 JE FNCLGMGNRDFIEGASGATWVDLVLEGDSCLTIMANDKPTLDVRMINIEASQLAEVRSYC MVE FNCLGMSSRDFIEGASGATWVDLVLEGDSCITIMAADKPTLDIRMMNIEATNLALVRNYC WNE FNCLGMSNRDFLEGVSGATWVDLVLEGDSCVTIMSKDKPTIDVKMMNMEAANLADVRSYC KUN FNCLGMSNRDFLEGVSGATWVDLVLEGDSCVTIMSKDKPTIDVKMMNMEAANLAEVRSYC SLE FNCLGTSNRDFVEGASGATWIDLVLEGGSCVTVMAPEKPTLDFKVMKMEATELATVRKYC DEN2 MRCIGMSNRDFVEGVSGGSWVDIVLEHGSCVTTMAKNKPTLDFELIKTEAKQPATLRKYC YF AHCIGITDRDFIEGVHGGTWVSATLEQDKCVTVMAPDKPSLDISLETVAIDRPAEVRKVC TBE SRCTHLENRDFVTGTQGTTRVTLVLELGGCVTITAEGKPSMDVWLDAIYQENPAKTREYC * ***. * * . . ** *.* . **..* . * * * JE MVE WNE KUN SLE DEN2 YF TBE YHASVTDISTVARCPTTGEAHNEKRADSSYVCKQGFTDRGWGNGCGLFGKGSIDTCAKFS YAATVSDVSTVSNCPTTGESHNTKRADHNYLCKRGVTDRGWGNGCGLFGKGSIDTCAKFT YLASVSDLSTRAACPTMGEAHNEKRADPAFVCKQGVVDRGWGNGCGLFGKGSIDTCAKFA YLATVSELSTKAACPTMGEAHNDKRADPSFVCKQGVVDRGWGNGCGLFGKGSIDTCAKFA YEATLDTLSTVARCPTTGEAHNTKRSDPTFVCKRDVVDRGWGNGCGLFGKGSIDTCAKFT IEAKLTNTTTESRCPTQGEPSLNEEQDKRFVCKHSMVDRGWGNGCGLFGKGGIVTCAMFR YNAVLTHVKINDKCPSTGEAHLAEENEGDNACKRTYSDRGWGNGCGLFGKGSIVACAKFT LHAKLSDTKVAARCPTMGPATLAEEHQGGTVCKRDQSDRGWGNHCGLFGKGSIVACVKAA * . **. * **. ****** ******* * .* PART 2/5 JE CTS--KAIGRTIQPENIKYEVGIFVHGTTTSENHGNYSAQVGASQAAKFTITPNAPSITL MVE CSN--SAAGRLILPEDIKYEVGVFVHGSTDSTSHGNYSTQIGANQAVRFTISPNAPAITA WNE CTT--KATGWIIQKENIKYEVAIFVHGPTTVESHG----KIGATQAGRFSITPSAPSYTL KUN CST--KATGRTILKENIKYEVAIFVHGPTTVESHGNYFTQTGAAQAGRFSITPAAPSYTL SLE CKN--KATGKTILRENIKYEVAIFVHGSTDSTSHGNYSEQIGKNQAARFTISPQAPSFTA DEN2 CKK--NMEGKVVQPENLEYTIVITPHSGEEHAVGNDTG-----KHGKEIKITPQSSTTEA YF CAK--SMSLFEVDQTKIQYVIRAQLHVGAKQENWN--------TDIKTLKFDALSGSQEV TBE CEAKKKATGHVYDANKIVYTVKVEPHTGDYVAANETHS----GRKTASFTIS--SEKTIL . * . . * * JE MVE WNE KUN SLE DEN2 YF TBE KLGDYGEVTLDCEPRSGLNTEAFYVMTVGS------KSFLVHREWFHDLALPWTSPSSTKMGDYGEVTVECEPRSGLNTEAYYVMTIGT------KHFLVHREWFNDLLLPWTSPASTKLGEYGEVTVDCEPRSGIDTSAYYVMSVGE------KSFLVHREWFMDLNLPWSSAGSTKLGEYGEVTVDCEPRSGIDTSAYYVMTVGT------KTFLVHREWFMDLNLPWSSAESNNMGEYGTVTIDCEARSGINTEDYYVFTVKE------KSWLVNRDWFHDLNLPWTSPATTELTGYGTVTMECSPRTGLDFNEMVLLQMEN------KAWLVHRQWFLDLPLPWLPGADTQ EFIGYGKATLECQVQTAVDFGNSYIAEMET------ESWIVDRQWAQDLTLPWQSGSGGTMGEYGDVSLLCRVASGVDLAQTVILELDKTVEHLPTAWQVHRDWFNDLALPWKHEGAQ** .. * . . . . * * * ** *** PART 3/5 JE --AWRNRELLMEFEEAHATKQSVVALGSQEGGLHQALAGAIVVEYSSS----VKLTSGHL MVE --EWRNREILVEFEEPHATKQSVVALGSQEGALHQALAGAIPVEFSSST---LKLTSGHL WNE --TWRNRETLMEFEEPHATKQSVVALGSQEGALHQALAGAIPVEFSSNT---VKLTSGHL KUN --VWRNRETLMEFEEPHATKQSVIALGSQEGALHQALAGAIPVEFSSNT---VKLTSGHL SLE --DWRNRETLVEFEEPHATKQTVVALGSQEGRPATALAGAIPATVSSST---LTLQSGHL DEN2 GSNWIQKETLVTFKNPHAKKQDVVVLGSQEGAMHTALTGATEIQMSSG----NLLFTGHL YF --VWREMHHLVEFEPPHAATIRVLALGNQEGSLKTALTGAMRVTKDTNDNNLYKLHGGHV TBE --NWNNAERLVEFGAPHAVKMDVYNLGDQTGVLLKALAGVPVAHIEGTK---YHLKSGHV * *. * ** * ** * * **.* * **. JE MVE WNE KUN SLE DEN2 YF TBE KCRLKMDKLALKGTTYGMCTE-KFSFAKNPADTGHGTVVIELSYSGSDGPCKIPIVSVAS KCRVKMEKLKLKGTTYGMCTE-KFTFSKNPADTGHGTVVLELQYTGSDGPCKIPISSVAS KCRVKMEKLQLKGTTYGVCSK-AFKFARTPADTGHGTVVLELQYTGTDGPCKVPISSVAS KCRVKMEKLQLKGTTYGVCSK-AFRFLGTPADTGHGTVVLELQYTGTDGPCKIPISSVAS KCRAKLDKVKIKGTTYGMCDS-AFTFSKNPTDTGHGTVIVELQYTGSNGPCRVPISVTAN KCRLRMDKLQLKGMSYSMCTG-KFKVVKEIAETQHGTIVIRVQYEGDGSPCKIPFEIMDSCRVKLSALTLKGTSYKICTD-KMFFVKNPTDTGHGTVVMQVKVS-KGAPCRIPVIVADD TCEVGLEKLKMKGLTYTMCDKTKFTWKRAPTDSGHDTVVMEVTFS-GTKPCRIPVRAVAH * . . .** .* .* ... * *... . **..* PART 4/5 JE LNDMTPVGRLVTVNPFVATSSANSKVLVEMEPPFGDSYIVVGRGDKQINHHWHKAGSTLG MVE LNDMTPVGRMVTANPYVASSTANAKVLVEIEPPFGDSYIVVGRGDKQINHHWHKEGSSIG WNE LNDLTPVGRLVTVNPFVSVATANSKVLIELEPPFGDSYIVVGRGEQQINHHWHKSGSSIG KUN LNDLTPVGRLVTVNPFVSVSTANAKVLIELEPPFGDSYIVVGRGEQQINHHWHKSGSSIG SLE LMDLTPVGRLVTVNPFISTGGANNKVMIEVEPPFGDSYIVVGRGTTQINYHWHKEGSSIG DEN2 LEKRHVLGRLITVNPIVTE--KDSPVNIEAEPPFGDSYIIIGVEPGQLKLNWFKKGSSIG YF LTAAINKGILVTVNPIASTN--DDEVLIEVNPPFGDSYIIVGRGDSRLTYQWHKEGSSIG TBE GSPDVNVAMLITPNPTIEN---NGGGFIEMQLPPGDNIIYVG----ELSHQWFQKGSSIG ..* ** .* * ** * .* . * . **..* JE MVE WNE KUN SLE DEN2 YF TBE KAFSTTLKGAQRLAALGDTAWDFGSIGGVFNSIGKAVHQVFGGAFRTLFGGMSWITQGLM KAFSTTLKGAQRLAALGDTAWDFGSVGGVFNSIGKAVHQVFGGAFRTLFGGMSWISQGLL KAFTTTLRGAQRLAALGDTAWDFGSVGGVFTSVGKAIHQVFGGAFRSLFGGMSWITQGLL KAFTATLKGAQRLAALGDTAWDFGSVGGVFTSVGKAVHQVFGGAFRSLFGGMSWITQGLL KALATTWKGAQRLAVLGDTAWDFGSIGGVFNSIGKAVHQVFGGAFRTLFGGMSWITQGLL QMFETTMRGAKRMAILGDTAWDFGSLGGVFTSIGKALHQVFGAIYGAAFSGVSWTMKILI KLFTQTMKGVERLAVMGDTAWDFSSAGGFFTSVGKGIHTVFGSAFQGLFGGLNWITKVIM RVFQKTKKGIERLTVIGEHAWDFGSAGGFLSSIGKAVHTVLGGAFNSIFGGVGFLPKLLL . * .* *.. .*. **** * ** *.** .* * * . * *. . .. PART 5/5 JE GALLLWMGVNARDRSIALAFLATGGVLVFLATNVHA MVE GALLLWMGVNARDKSIALAFLATGGVLLFLATNVHA WNE GALLLWMGINARDRSIAMTFLAVGGVLLFLSVNVHA KUN GALLLWMGINARDRSIALTFLAVGGVLLFLSVNVHA SLE GALLLWMGLQARDRSISLTLLAVGGILIFLATSVQA DEN2 GVIITWIGMNSRSTSLSVTLVLVGIVTLYLGVMVQA YF GAVLIWVGINTRNMTMSMSMILVGVIMMFLSLGVGA TBE GVALAWLGLNMRNPTMSMSFLLAGGLVLAMTLGVGA * . *.*.. * ..... . * . . . * * Multiple alignment of Predicted TH-cell epitope in the JE_Egp with corresponding epitopes in Egps of other Flaviviruses 426 457 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 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. Model Refinement PARAMETERS USED • force field: • Dielectric const: • Optimisation: AMBER all atom Distance dependent 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 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. Model Evaluation I:Energy Profile TBE JEV Model Evaluation II: Ramachandran Plot An Algorithm to Identify Conformational Epitopes Calculate the percent accessible surface area (ASA) of the amino acid residues. If ASA 30%, then residue was termed as accessible residues. A contiguous stretch of more than three accessible residues was termed as the antigenic determinant. …Cont. A determinant is extended to N- and Cterminals, only if, accessible amino acid(s) are present after an inaccessible amino acid residue. A list of sequential antigenic determinants was prepared. Peptide Modeling Initial random conformation Force field: Amber Distance dependent dielectric constant 4rij Geometry optimization: Steepest descents & Conjugate gradients Molecular dynamics at 400 K for 1ns Peptides are: SENHGNYSAQVGASQ NHGNYSAQVGASQ YSAQVGASQ YSAQVGASQAAKFT NHGNYSAQVGASQAAKFT SENHGNYSAQVGASQAAKFT 149 168 Prediction of conformations of the antigenic peptides Lowest energy Allowed conformations were obtained using multiple MD simulations: – Initial conformation: random, allowed – Amber force field with distance dependent dielectric constant of 4*rij – Geometry optimization using Steepest descents & Conjugate gradient – 10 cycles of molecular dynamics at 400 K; each of 1ns duration, with an equilibration for 500 ps – Conformations captured at 10ps intervals, followed by energy minimization of each – Analysis of resulting conformations to identify the lowest energy, geometrically and stereochemically allowed conformations MD simulations of following peptides were carried out B Cell Epitopes: SENHGNYSAQVGASQ NHGNYSAQVGASQ YSAQVGASQ YSAQVGASQAAKFT NHGNYSAQVGASQAAKFT 149 T-helper Cell Epitope: 436 445 SIGKAVHQVF 168 SENHGNYSAQVGASQAAKFT Chimeric B+Th Cell Epitope With Spacer: SENHGNYSAQVGASQAAKFTSIGKAVHQVF Structural comparison of Egps of Nakayama and Sri Lanka strains of JEV. Single amino acid differences are highlighted.