Received date: December 01, 2012; Accepted date: January 27, 2013; Published date: January 31, 2013
Citation: Gomase VS, Chitlange NR, Sherkhane AS, Changbhale SS, Kale KV (2013) Prediction of Wuchereria Bancrofti Troponin Antigenic Peptides: Application in Synthetic Vaccine Design to Counter Lymphatic Filariasis. J Vaccines Vaccin 4:169. doi: 10.4172/2157-7560.1000169
Copyright: © 2013 Gomase VS, et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
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Wuchereria bancrofti is a threadlike nematode one of the causative worm of lymphatic filariasis in which the
lymphatic and genital organs get disabled either temporarily or permanently; till date no effective drug or vaccine has been discovered to treat lymphatic filariasis. In this analysis we have predicted antigenic peptides from Wuchereria bancrofti troponin protein for synthetic peptide vaccine design against lymphatic filariasis because with a single protein subunit immune response can be generated in large population. Analysis shows predicted epitopes of Wuchereria bancrofti troponin protein are important determinant for protection against lymphatic filariasis. In this assay we have analysed the binding affinity of Wuchereria bancrofti troponin protein having 136 amino acids, which shows 128 nonamers. In this research work, we predicted CTL-epitopes by two different methods namely SVM (Support Vector Machine) and ANN (Artificial Neural Network), SVM based prediction shown sixteen valid epitopes having optimal score of 1.129 at cut off 0.36 whereas ANN based prediction shown thirty-one valid epitopes having optimal score of 1.000 at cut off 0.51. We also predicted cascade SVM based TAP binders and four potential antigenic epitopes as, 31-LRKLIRK-37, 49-DEFCALVYTVANT-61, 87-SRPTLKALLKE-97, 108-EAAVDE-113 (optimal propensity 1.223) predicted on the basis of highest local hydrophilicity; in addition to this we also have experimentally predicted tertiary structure of the two longest potential epitopes those can aid our understanding in
sequence-structure-function relationship of Wuchereria bancrofti troponin protein towards synthetic peptide vaccine design. Thus a small fragment of troponin protein can produce immune response against activity of Wuchereria bancrofti. This approach can be applied for designing subunit and synthetic peptide vaccines
Lymphatic filariasis; Parasitic disease; Antigenic peptides; MHC; SVM; ANN; CTL; Nonamers; Synthetic peptide vaccines
MHC: Major Histocompatibility Complex; CTL: Cytotoxic T lymphocytes; TAP: Transporter associated with Antigen Processing; SVM: Support Vector Machine; ANN: Artificial Neural Network
Lymphatic filariasis also known Elephantiasis is a parasitic infection caused by filarial roundworm, Wuchereria bancrofti. The infection is usually acquired in childhood, its indication of the existence occur later in life, causing temporary or permanent disability of an infected organ, causes severe damage and painful swelling, disfiguring swelling of the legs and genital organs is a classic symptom of late disease stage. In many tropical countries, lymphatic filariasis has a major social and economic impact, though World Health Organization is trying to eliminate it completely by the year 2020 yet there is no effective drug or vaccine has been invented to treat/prevent Elephantiasis (WHO, lymphatic Filariasis).
An Infected mosquito deposits larvae on the individual’s skin while biting and larvae enter Wound. The Larvae migrates into lymphatic system where they grow, Adult male worms are about 3-4 cm long while female worms are 8-10 cm long. When male and female worms mate they form nests; these nests cause blockages in the human lymphatic system resulting symptoms like swelling and fever. Female worm produces microscopic worms called microfilaria. When mosquito bites to an infected individual, ingests microfilaria with blood and infects to healthy individual, Microfilaria develops into adult worm over a week and the cycle continues [WHO, lymphatic Filariasis].
This approach is based on the phenomenon of cross-protection  hereby an individual infected with a mild strain of pathogen possess immunity against more severe strain of the same pathogen. Body proteins are necessary for its production in or on all food commodities. An exemption from the requirement of a tolerance is established for residues of the drugs or chemicals. The lymphatic system is an important component of the body’s immune system, it include several centres of initial infection so the immune response can be generated in the lymph organ even with single antigen subunit.
MHC class binding peptides
The new paradigm in vaccine design is emerging; following essential discoveries in immunology and development of new CTL binding peptides prediction tools . MHC molecules are cell surface glycoproteins, which take active part in host immune reactions. The involvement of MHC class-I in response to almost all antigens and considering its binding affinity with peptides of 9 amino acids, we have predicted the 9 amino acid long epitopes from extracted data MHCBN Comprehensive data base of MHC-I and MHC-II binding as well as non-binding epitopes. MHC molecules have been well characterized in terms of their role in immune reactions. They bind to several peptide fragments generated after proteolytic cleavage of antigen . This binding act like red flags for specific antigen and generates immune response against the parent antigen. So an antigen from Wuchereria bancrofti troponin subunit can induce immune response against Wuchereria bancrofti activity. CTL epitopes are most suitable for subunit vaccine development because with single epitope, the immune response can be generated in large population. MHC peptide complexes will be translocated on the surface of antigen presenting cells (APCs). This theme is implemented in developing subunit and synthetic peptide vaccines [4-7]. One of the important problems in subunit vaccine design is to search for antigenic regions in an antigen protein  that can stimulate T-cells called T-cell epitopes. In literature, fortunately, a large amount of data about such peptides is available. Previously and presently, a number of databases have been developed to provide comprehensive information related to T-cell epitopes [9-13].
Protein sequence analysis
Wuchereria bancrofti troponin protein is a diagnostic antigen against lymphatic filariasis. The antigenic protein sequence of W. bancrofti troponin antigen protein was analyzed to study the antigenicity solvent accessible regions and MHC class binding peptide, which allows potential drug targets to predict active sites against Lymphatic filariasis.
A Wuchereria bancrofti troponin (gi- 373938659) protein sequence is 136 amino acids long as-MFDRGKQGYIMATQIGQIMHAMEQDF DEKQLRKLIRKFDADGSGKLEFDEFCALVYTVANTVDKETLQKEL REAFRLFDKEGNGYISRPTLKALLKEIADDLSDEQLEAAVDEIDED GSGKIEFEEFWELMAGDAD
Antigenicity Prediction program results those segments from Wuchereria bancrofti troponin antigen protein that are likely to be antigenic by eliciting an antibody response. Antigenic epitopes are determined using the Gomase, (2007), Hopp and Woods, Welling, Parker, B-EpiPred Server and Kolaskar and Tongaonkar antigenicity methods [14-18].
Protein secondary structure prediction
The important concepts in secondary structure prediction are identified as: residue conformational propensities, sequence edge effects, hydrophobicity moments, insertions and Deletions positions in aligned homologous sequence, moments of conservation, autocorrelation, residue ratios, secondary structure feedback effects and filtering [19,20].
Finding the location in solvent accessible regions
Finding the location in solvent accessible regions in protein, type of plot determines the hydrophobic and hydrophilic scales and it is utilized for prediction. This may be useful in predicting membranespanning domains, potential antigenic sites and regions that are likely exposed on the protein surface [21-41].
CTL epitope prediction
The CTL Epitopes of Wuchereria bancrofti troponin are obtained from MHCBN comprehensive database of MHC binding and nonbinding peptides using two different methods firstly with Support Vector Machine based method (Cut off is 0.36) and then by Artificial Neural Network based method (Cut off is 0.51). In this work predicted MHC-Peptide binding is a log-transformed value related to the IC50 values in nM units.
The average accuracy of Support Vector Machine (SVM) based epitope prediction method is ~76% at cut off 0.36. SVM has been trained on the binary input of single amino acid sequence. In Case of Artificial Neural Network ANN based epitope prediction method the average accuracy is ~74% at the cut off score 0.51 .
TAP binding prediction
TAP (Transporter associated with Antigen Peptide) play an important role in transportation of MHC-Peptide complexes, which elicits the immune response for clearing various intracellular infections. The prediction of TAP binding peptides is crucial in identifying the MHC class-I restricted T cell epitopes. The Prediction is based on cascade SVM, using properties of amino acid sequence at correlation coefficient of 0.88 as per Jack-Knife validation test .
Peptide structure prediction
Peptide Structure prediction is a challenge, which would aid our understanding of sequence–structure–function relationships towards synthetic peptide vaccine design. Peptide Structure prediction depends on the complexity and the accuracy of the models used to represent them. We have used Hidden Markov Model derived structural alphabet (SA) based tool that discretizes peptide experimental conformation as series of overlapping fragments of four residues length. The predicted structure can be considered to have an average accuracy of ~1.1A rootmean- square deviation (RMSd) [43-46].
Antigenic peptides prediction
In this assay we predicted the antigenic determinants by finding the area of highest local hydrophilicity. The Hopp & Woods scale was designed to predict the locations of antigenic determinants in linear antigen protein sequence, assuming that the antigenic determinants would be exposed on the protein surface and thus would be located in hydrophilic regions (Figure 1). Its values are derived from the transferfree energies for amino acid side chains between ethanol and water. Welling antigenicity plot gives value as the log of the quotient between percentage in a sample of known antigenic regions and percentage in average proteins (Figure 2). We also studied B-EpiPred Server, Parker, Kolaskar and Tongaonkar antigenicity methods and the predicted antigenic fragments can bind to MHC molecule is the first bottlenecks in vaccine design (Figures 3-6).
The Robson and Garnier method applied for secondary structure prediction of the Wuchereria bancrofti troponin protein. Each residue have specific assigned value for alpha helix (Shown in Red), beta sheet (Shown in Blue) and coil (Shown in Pink) using a window of 7 residues (Figure 7). Using these information parameters, the likelihood of a given residue assuming each of the four possible conformations alpha, beta, reverse turn or coils calculated and the conformation with the largest likelihood is assigned to the residue.
Solvent accessible regions
Solvent accessible scales for delineating hydrophobic and hydrophilic characteristics of amino acids and scales are developed for predicting potential antigenic sites of globular proteins, which are likely to be rich in charged and polar residues. It was shown that Wuchereria bancrofti troponin protein is hydrophobic in nature and contains segments of low complexity and high-predicted flexibility (Figures 8-28).
Prediction of MHC binding peptides
MHC molecules are cell surface glycoproteins, which take active part in host immune reactions and involvement of MHC class-I and MHC II in response to almost all antigens so these MHC binding peptides are sufficient for eliciting the desired immune response. In analysis we determined the CTL binding regions, TAP binding regions and several potential antigenic epitopes. In this study we predicted the binding affinity of Wuchereria bancrofti troponin protein having 136 amino acids, which shows number of peptides. The CTL epitope prediction is based on an elegant machine learning technique Support Vector Machine (SVM) and Artificial Neural Network (ANN). SVM has been trained on the binary input of single amino acid sequence. In this assay we have predicted the binding affinity of Wuchereria bancrofti troponin protein sequence having 136 amino acids, which shows 128 nonamers.The SVM based CTL epitopes, 86-ISRPTLKAL, 46-LEFDEFCAL, 84-GYISRPTLK, 122-IEFEEFWEL, 47-EFDEFCALV, 29-KQLRKLIRK, 80-KEGNGYISR, 87-SRPTLKALL, 69-KELREAFRL, 50-EFCALVYTV, 72-REAFRLFDK, 2-FDRGKQGYI, 11-MATQIGQIM, 4-RGKQGYIMA, 90-TLKALLKEI, 123-EFEEFWELM (optimal score is 1.129); the ANN based CTL epitopes recorded are, 60-NTVDKETLQ, 12-ATQIGQIMH, 36-RKFDADGSG, 85-YISRPTLKA, 109-AAVDEIDED, 76-RLFDKEGNG, 20-HAMEQDFDE, 69-KELREAFRL, 94-LLKEIADDL, 32-RKLIRKF-DA, 50-EFCALVYTV, 67-LQKELREAF, 112-DEIDEDGSG, 54-LVYTVANTV, 98-IADDLSDEQ, 41-DGSGKLEFD, 89-PTLKALLKE, 79-DKEGNGYIS, 82-GNGYISRPT, 104-DEQLEAAVD, 100-DDLSDEQLE, 40-ADGSGKLEF, 62-VDKETLQKE, 27-DEKQLRKLI, 90-TLKALLKEI, 108-EAAVDEIDE, 42-GSGKLEFDE, 52-CALVYTVAN, 43-SGKLEFDEF, 61-TVDKETLQK, 53-ALVYTVANT, which represented predicted binders from Wuchereria bancrofti troponin protein (Tables 1 and 2). In addition to the CTL epitopes we also have predicted several high Affinity TAP binders as 102-LSDEQLEAA, 23-EQDFDEKQL, 26-FDEKQLRKL, 12-ATQIGQIMH, 92-KALLKEIAD, 73-EAFRLFDKE, 98-IADDLSDEQ, 57-TVANTVDKE, 82-GNGYISRPT,125-EEFWELMAG, 35-IRKFDADGS, 9-YIMATQIGQ, 21-AMEQDFDEK, 74-AFRLFDKEG, 95-LKEIADDLS, 44-GKLEFDEFC, 11-MATQIGQIM, 36-RKFDADGSG, 94-LLKEIADDL, 66-TLQKELREA, 51-FCALVYTVA, 48-FDEFCALVY, 124-FEEFWELMA, 33-KLIRKFDAD, 37-KFDADGSGK, 45-KLEFDEFCA, 22-MEQDFDEKQ, 99-ADDLSDEQL, 53-ALVYTVANT, 96-KEIADDLSD, 63-DKETLQKEL, 70-ELREAFRLF, 90-TLKALLKEI, 10-IMATQIGQI, 88-RPTLKALLK, 24-QDFDEKQLR, 123-EFEEFWELM, 52-CALVYTVAN, 120-GKIEFEEFW, 14-QIGQ-IMHAM, 116-EDGSGKIEF, these TAP binders are important in generating immune response (Table 3). The optimal propensity for the Wuchereria bancrofti troponin protein found is 1.223 (Figure 4). All residues having above 1.0 propensity are always potentially antigenic (Table 4). The predicted segments in Wuchereria bancrofti troponin protein are 31-LRKLIRK-37, 49-DEFCALVYTVANT-61, 87-SRPTLKALLKE- 97, 108-EAAVDE-113 Fragments identified through this approach tend to be high-efficiency binders, which is a larger percentage of their atoms are directly involved in binding as compared to larger molecules. These MHC binding peptides are sufficient for inducing the desired immune response. Predicted MHC binding regions in Wuch-ereria bancrofti troponin sequence and these are actively taking part in immune reactions.
|Peptide rank||Start position||Sequence||Score|
Table 1: SVM based Predicted CTL-Epitopes of Wuchereria bancrofti troponin protein.
|Peptide rank||Start position||Sequence||Score|
Table 2: ANN based Predicted CTL-Epitopes of Wuchereria bancrofti troponin protein.
|Peptide rank||Start position||Sequence||Score|
Table 3: Cascade SVM based High affinity TAP epitopes of Wuchereria bancrofti troponin protein.
|No.||Start position||End position||Peptide||Peptide length|
Table 4: Potential Antigenic epitopes of Wuchereria bancrofti troponin protein.
Tertiary structure prediction of the predicted epitopes
We have predicted the experimentally confirmed structures of the two potential epitopes 49-DEFCALVYTVANT-61, 87-SRPTLKALLKE-97, these are the longest epitopes predicted from the Wuchereria bancrofti troponin protein showing the highest pick. The peptide structures are validated by applying A Hidden Markov Model based approach which predicts the tertiary confirmation of the peptide sequence at accuracy ~1.1 RMSd. The validated structures are shown in Balls & Sticks model over solvent-accessible surface (VDW+1.4 angstrom) (Figures 29 and 30) [47,48].
Gomase method (2007), B-EpiPred Server, Hopp and Woods, Welling, Parker, Kolaskar and Tongaonkar antigenicity scales were designed to predict the locations of antigenic determinants in Wuchereria bancrofti troponin protein sequence. It shows beta sheets regions, which have higher antigenic response than helical region of this peptide and shows high antigenicity (Figures 1-5) [49-52]. We also found the Sweet hydrophobicity, Kyte & Doolittle hydrophobicity, Abraham & Leo, Bull & Breese hydrophobicity, Guy, Miyazawa hydrophobicity, Roseman hydrophobicity, Cowan HPLC pH 7.5 hydrophobicity, Rose hydrophobicity, Eisenberg hydrophobicity, Manavalan hydrophobicity, Black hydrophobicity, Fauchere hydrophobicity, Janin hydrophobicity, Rao & Argos hydrophobicity, Wolfenden hydrophobicity, Wilson HPLC hydrophobicity, Cowan HPLC pH 3.4, Tanford hydrophobicity, RF mobility hydrophobicity and Chothia hydrophobicity scales, Theses scales are essentially a hydrophilic index, with a polar residues assigned negative values (Figures 8-28). In this assay we predicted the binding affinity of Wuchereria bancrofti troponin protein having 136 amino acids, which shows 128 nonamers. We predicted SVM and ANN based CTL epitopes (Tables 1 and 2) [53,54].
We have determined sixteen CTL predicted epitopes (Optimal score is 1.129) at cut off 0.36 using SVM based method (Table 1) and thirty one CTL predicted epitopes using ANN based method at cut off 0.51 (Optimal score is 1.000) (Table 2) which represents predicted peptide binders from Wuchereria bancrofti troponin protein. We also have predicted Cascade SVM based forty one High affinity TAP binding epitopes (Optimal score is 8.641) (Table 3) in addition to four potentially antigenic peptides recognized by antibodies of the immune system for troponin protein in the region of maximum local hydrophilicity (Table 4) . Kolaskar and Tongaonkar antigenicity are the sites of molecules that are recognized by antibodies of the immune system for troponin protein, analysis shows epitopes present in the troponin protein eliciting desired immune response . The region of maximal hydrophilicity is likely to be an antigenic site, having hydrophobic characteristics, because C- terminal regions of Wuchereria bancrofti troponin protein is solvent accessible and unstructured, antibodies against those regions are also likely to recognize the native protein. For the prediction of antigenic determinant site of Wuchereria bancrofti troponin protein, we predicted four antibody recognized antigenic determinant sites in the Wuchereria bancrofti troponin sequence. The highest pick is recorded between sequence of amino acid in the region are 49- DEFCALVYTVANT-61 and 87-SRPTLKALLKE-97 (Table 4) . We also predicted the surface accessible tertiary structure for the peptide recorded with the highest pick; these are the longest peptides predicted in the antigenic sequence of the Wuchereria bancrofti troponin protein, the experimentally confirmed structures of the predicted peptides are considered to have an average accuracy of ~1.1A RMSd, the predicted peptide structures can be applied in synthetic peptide vaccine design approach to understand the sequence–structure–function relationship of the Wuchereria bancrofti troponin protein [57-59].
This method will be useful in cellular immunology, Vaccine design, immunodiagnostics, immunotherapeutics and molecular understanding of autoimmune susceptibility. Wuchereria bancrofti troponin protein sequence contains multiple antigenic components to direct and empower the immune system to protect an individual from lymphatic filariasis disease. MHC molecules are cell surface proteins, which actively participates in host immune reactions and involvement of MHC class in response to almost all antigens and it give effects on target sites. Predicted MHC binding regions acts like red flags for specific antigen and generate immune response against the whole antigen. So a small antigen fragment can generate immune response against entire antigen. The method integrates prediction of Peptide-MHC class binding; proteosomal C terminal cleavage and TAP transport efficiency. This approach is implemented in designing subunit and synthetic peptide vaccines.