COMPUTATIONAL APPROACH TO IDENTIFICATION

AND IMMUNOGENIC CHARACTERISATION OF CHAP’S

PROTEINPREMNATH.D *

EPITOPE BASED VACCINES

Advantages :

•Can be more potent

•Can be controlled better

•Can target multiple conserved epitopes in rapidly mutating pathogens like HIV and Hepatitis C virus (HCV)

•Can induce subdominant epitopes (e.g. against tumor antigens where there is tolerance against dominant epitopes)

•Can be designed to break tolerance

•Can overcome safety concerns associated with entire organisms or proteins

Targets for vaccine therapy•Disease Specific Antigens (DSA)•Disease Associated Antigens (DAA)

OBJECTIVES OF THE PRESENT STUDY

The governing peptide/MHC and TCR/pMHC interactions recognising and binding ,leading to successful epitope prediction

PATHWAY OF CLASS I MHC PROTEINS

EPITOPES • In protein antigens epitopes can be defined in terms of:

• Amino acid composition

• Protein location

• Length (5-15 amino acids)

• Immunodominant epitopes:

• Epitopes bound by a greater proportion

of antibodies than others in a normal

in vivo immune response.

• Also known as Major Antigenic Sites.

• Epitopes can be divided into 2 classes:

• Discontinuous epitopes

• Continuous (linear) epitopes

GENERAL WAYS OF PREDICTING EPITOPES

• By 3D structure of protein• Can be very accurate• Not always available + proteins change structure

• By properties that epitopes have• Hydrophilicity• Readily available, but not very accurate

Charprons protein against Fish Disease of pickkrettesia salmon

• Gram-negative bacterium, Piscirickettsia salmonis• In common with many systemic, chronic inflammatory diseases of

salmonids, the internal signs include a swollen and discoloured kidney and an enlarged spleen.• chronic inflammatory diseases of salmonids

COMPUTATIONAL STRATEGY Homology modeling.Peptide prediction.

PROT PARM svrMHC SVM MAST SIGNAL IP NET chop tool

CHARMmQ-site finder.Autodock 3.0.5.ZDOCKproRDOCKpro.

HOMOLOGY MODELED CHAPRONS PROTEIN

Protparam Results :

molecular weight of the chaps was 5735.6. The amino acid composition are Ala 12, Arg 3.9, Asn 4.2, Asp

6.1, Cys 0.4, Gln 2.8%, Glu 8.8%, Gly 9.5%, His 0.4%, Ile 7.2%, Leu .3%, Lys 7.0%, Met 4.0%, Phe

2.0%, Ser 5.1%, Thr 5.9, Trp 0.0%, Tyr 0.7%, Val 9.4%.The negatively charged residues are Asp+Glu is

81 and the positively charged residues are 59( Arg + Lys). The domain was predicted from the SMART

tool from the position 1-19 the protein was found to be disordered protein and the PFam was observed as

23-525 score.

>gi|30525581|gb|AAP32277.1| immunogenic protein ChaPs [Piscirickettsia salmonis]MSAKEVRFGTGSRQKMLDGVNLLANAVKVTLGPRGRNVILEKSFGAPTITKDGVSVAKEIELSDKFENMGAQMVKEVASKSNDDAGDGTTTATVLAQAII GVKSVAAGMNPMDLKRGIDKATIAAVAALKDLSTPCTDNKAIAQVGTISANSDEEIGSIIAKAMEKVPTDGVITVEEGSSLENELDVVEGMQFDRGYLSPYFVNKQEKMIAEIESPFILLVDKKISNIRELLPTLESVAKSGKPLFIIAEDVEGEALATLVVNNIRGIVKVCAVKAPGFGDRRKAMLEDIAILTGGTVISEEVGLDLEKATLEHLGTAKRIVVTKDNTTVIDGAGEQNAIEARVTQRAQVEETSSDYDREKLQERVAKLSGGVAVIKVGAATEIEMKEKKDRVDDALHATRAAVEEGVVPGGGVALVRAMAAVKALDFANDEQAQGANILLRAMSAPLRQIVENAGSEAAVILDKIVNGEGNFGYNAATNEFGDMIEMGILDPTKVTRSALQNAASIAGLMITTEAMVAELPKEDSAGGAGMPDMG

PEPTIDE IDENTIFICATION

SERVER used – svrMHC

SVM and MAST

The (SVM) support vector machine shows 0.474 identity. This result concludes that the prediction by SVM is non Allergen. The MAST and other blast algorithm of Allerg predicts also shown that the given protein is not a Allergen. Next to that the B-cell epitopes and T-cell epitopes are predicted in the chaps protein. From the results it was observed that 36 B-cell epitopes were identified from 181 total residues of chaps protein.

Signal Ip

NetPhos 2.0 Server - prediction results

The phosphorylation tools are used to predict the phosphorylation site in the chaps’ protein. It was found that Serine, Threonine and Tyrosine amino acids involved in the phosphorylation are 13,18,19,23 and 7 The MHC binding complexes are identified in the chaps protein using the Net phos tool. The Net phos tool predict that the 74 position of amino acid sequences are identified as the MHC binding peptide from 245 sequences

PEPTIDES – FROM DOCKING STUDIES

Tool used - AUTODOCK

SELECTED ANTIGENIC PEPTIDE

nanomer 344-352 [TSSAYGGGA]

MHC/PEPTIDE COMPLEX

AUTODOCK RESULT

From the autodock result the peptide strongly bind with the region of TYR59,LYS66 3HLA mhc protein

FROM PROTEIN TO IMMUNOGENS

From protein to immunogen 20%processing and 5%HLA binding 50%T-cell response has been functioning in peptide vaccine

PROTEIN-PROTEIN DOCKING

• computational determination of the structure of protein complexes from individual protein structures • Biological activity depends on the specific molecular

recognition of proteins• Virtually every cellular process is regulated by protein–protein

interactions–Signal transduction cascades e.g. cancer

• Understanding mechanism of action of therapeutically interesting proteins (protein therapeutics)

–In the area of rational approaches for drug development and treatment of disease

STRUCTURE OF pMHC/TCRs COMPLEX

MHC/peptide TCRs alpha chain

pMHC/TCRs

ZDOCKpro RESULT

BINDING SITE OF pMHC/TCRs COMPLEX

The pMHC/TCRs complex has to be bind to the specific binding site amino acid LYS41,SER39 from the qrn-d alpha chain protein and Leu230,Glu232 pMHC complex

OBSERVATION The epitope peptide TSSAYGGGA (344-352) has the highest MHC binder identified from auto dock result through peptide /MHC complex. The result obtain from the ZDOCKpro have shown that strong binding RMSD value of 7.96 by the amino acid LYS41 ,SER39 from the complex protein.

The project concludes the immunogenic analysis and model of chaps suggested that this protein is a good target for vaccine design for the fish disease pickkerittisea salmon.

THANK YOU