An Immunoinformatics Approach to Design Synthetic Peptide Vaccine from Dendroaspis polylepis polylepis Dendrotoxin-K(DTX-K)

Dendroaspis polylepis polylepis commonly known as black mamba is the aggressive and highly venomous land snake; Dendroaspis polylepis polylepis venom contains Dendrotoxin-K (DTX-K), which has ability to kill a mouse within 5 minutes after bite. The dendrotoxin is highly specific and virulently toxic protein of low molecular weight that can spread very rapidly within the bitten tissue, so black mamba venom is the most rapid-acting of all snake venoms. Dendrotoxin inhibits the exogenous process of muscle contraction by means of the sodium potassium pump. Dendrotoxin-K is a selective blocker of voltage-gated potassium channels [1,2].


Introduction
Dendroaspis polylepis polylepis commonly known as black mamba is the aggressive and highly venomous land snake; Dendroaspis polylepis polylepis venom contains Dendrotoxin-K (DTX-K), which has ability to kill a mouse within 5 minutes after bite. The dendrotoxin is highly specific and virulently toxic protein of low molecular weight that can spread very rapidly within the bitten tissue, so black mamba venom is the most rapid-acting of all snake venoms. Dendrotoxin inhibits the exogenous process of muscle contraction by means of the sodium potassium pump. Dendrotoxin-K is a selective blocker of voltage-gated potassium channels [1,2].

Strategy
The phenotype of the resistant transgenic plants includes fewer centers of initial virus infection, a delay in symptom development, and low bacterial accumulation. Protoplasts from disease resistant transgenic plants are also resistant, suggesting that the protection is largely operational at the cellular level. Transgenic plants expressing nucleocapsid protein are protected against infection by bacteria but are susceptible to bacterial DNA, indicating that the protection may primarily involve an inhibition of bacterial cell wall. This approach is based on the phenomenon of cross-protection [3], hereby a plant infected with a mild strain of bacteria is protected against a more severe strain of the same bacteria. Plant 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 biological plant pesticide.

MHC class binding peptides
The new paradigm in vaccine design is emerging, following essential discoveries in immunology and development of new MHC Class-I binding peptides prediction tools [4][5][6][7]. 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 the variable length of interacting peptides make the study of MHC Class I molecules very interesting. MHC molecules have been well characterized in terms of their role in immune reactions. They bind to some of the peptide fragments generated after proteolytic cleavage of antigen [8]. This binding acts like red flags for antigen specific and to generate immune response against the parent antigen. So a small fragment of antigen can induce immune response against whole antigen. Antigenic peptides 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 Abstract Dendroaspis polylepis polylepis is the most toxic snake commonly known as black mamba, the black mamba venom contains Dendrotoxin-K which is highly specific and virulently toxic protein. Antigenic peptides of Dendrotoxin toxic protein are most suitable for peptide vaccine development because with single epitope, the immune response can be generated in large population. Analysis shows MHC class II binding peptides of antigenic protein from Dendroaspis polylepis polylepis DTX-K are important determinant for protection against several venom toxins. In this assay we predicted the binding affinity of Dendroaspis polylepis polylepis DTX-K protein having 79 amino acids, which shows71nonamers. In this analysis, we found the High affinity TAP Transporter peptide regions as, 37-KRKIPSFYY (score-9.550), 45-YKWKAKQCL (Score-8.581) 36-CKRKIPSFY (Score-7.685), 24-AKYCKLPLR (Score-7.669), 42-SFYYKWKAK (Score-6.859), 31-LRIGPCKRK (Score-6.848) 65-NRFKTIEEC (Score-6.698), 25-KYCKLPLRI (Score-6.632), 49-AKQCLPFDY (Score-6.576), 66-RFKTIEECR (Score-6.464), 47-WKAKQCLPF (Score-6.197), 23-AAKYCKLPL (Score-6.166). We also found the SVM based MHCII-IAb peptide regions, 61-GGNANRFKT, 12-TLWAELTPV, 41-PSFYYKWKA, 25-KYCKLPLRI (optimal score is 0.946); MHCII-IAd peptide regions, 2-GHLLLLLGL, 57-SGCGGNAN, 3-HLLLLLGLL, 1-SGHLLLLLG (optimal score is 0.488); MHCII-IAg7 peptide regions 60-CGGNANRFK, 21-SGAAKYCKL, 61-GGNANRFKT, 20-VSGAAKYCK (optimal score is 1.468); and MHCII-RT1.B peptide regions 46-KWKAKQCLP, 24-AKYCKLPLR, 10-LLTLWAELT, 45-YKWKAKQCL (optimal score is 0.569) which represented predicted binders from dendrotoxin. The method integrates prediction of peptide MHC class I binding; proteasomal C terminal cleavage and TAP transport efficiency of the Dendroaspis polylepis polylepis DTX-K. Thus a small fragment of antigen can induce immune response against whole antigen. This theme is implemented in designing subunit and synthetic peptide vaccines.
translocated on the surface of antigen presenting cells (APCs). This theme is implemented in designing subunit and synthetic peptide vaccines [9]. One of the important problems in subunit vaccine design is to search antigenic regions in an antigen [10] that can stimulate T cells called T-cell epitopes. In literature, fortunately, a large amount of data about such peptides is available. Pastly and presently, a number of databases have been developed to provide comprehensive information related to T-cell epitopes [11][12][13][14].

Protein sequence analysis
The antigenic protein sequence of Dendroaspis polylepis polylepis DTX-K was analyzed to study the antigenicity [15], solvent accessible regions and MHC class peptide binding, which allows potential drug targets to identify active sites against plant diseases.

Prediction of protein secondary structure
The important concepts in secondary structure prediction are identified as: residue conformational propensities, sequence edge effects, moments of hydrophobicity, position of insertions and Deletions in aligned homologous sequence, moments of conservation, auto-correlation, residue ratios, secondary structure feedback effects, and filtering [21,22].

Prediction of MHC binding peptide
The MHC peptide binding is predicted using neural network strained on C terminals of known epitopes. In analysis predicted MHC/ peptide binding is a log-transformed value related to the IC50 values in nM units. MHC2Pred predicts peptide binders to MHCI and MHCII molecules from protein sequences or sequence alignments using Position Specific Scoring Matrices (PSSMs). Support Vector Machine (SVM) based method for prediction of promiscuous MHC class II binding peptides. The average accuracy of SVM based method for 42 alleles is ~80%. For development of MHC binder, an elegant machine learning technique SVM has been used. SVM has been trained on the binary input of single amino acid sequence. In addition, we predicts those MHCI ligands whose C-terminal end is likely to be the result of proteosomal cleavage [43][44][45].

Result and Interpretation
A antigenic sequence is 79 residues long as-GEDGYIADGDNCT YICTFNNYCHALCTDKKGDSGACDWWVPYGVVCWCEDLPTP VPIRGSGKCR

Prediction of antigenic peptides
In these methods we found the antigenic determinants by finding the area of greatest local hydrophilicity. The Hopp-Woods scale was designed to predict the locations of antigenic determinants in a protein, assuming that the antigenic determinants would be exposed on the surface of the protein 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 study 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 (Figure 3-6).

Secondary alignment
The Robson and Garnier method predicted the secondary structure of the Dendroaspis polylepis polylepis DTX-K. Each residue is assigned values for alpha helix, beta sheet, turns and coils 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 a Dendroaspis polylepis polylepis DTX-K is hydrophobic in nature and contains segments.

Prediction of MHC binding peptides
These MHC binding peptides are sufficient for eliciting the desired immune response. The prediction is based on cascade support vector machine, using sequence and properties of the amino acids. The correlation coefficient of 0.88 was obtained by using jack-knife validation test. In this test, we found the MHCI and MHCII binding regions (Tables 1 and 2). 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. In this assay we predicted the binding affinity of Dendroaspis polylepis polylepis DTX-K having 79 amino acids, which shows different nonamers (Tables 1 and 2). For development of MHC binder prediction method, an elegant machine learning technique support vector machine (SVM) has been used. SVM has been trained on the binary input of single amino acid sequence. In this assay we predicted the binding affinity of Dendroaspis polylepis polylepis DTX-K sequence (IsTX) having 79 amino acids, which shows 71nonamers. Small peptide regions found as High affinity TAP Transporter peptide regions as, 37   (Score-6.576), 66-RFKTIEECR (Score-6.464), 47-WKAKQCLPF (Score-6.197), 23-AAKYCKLPL (Score-6.166). We also found the SVM based MHCII-IAb peptide regions, 61-GGNANRFKT, 12-TLWAELTPV, 41-PSFYYKWKA, 25-KYCKLPLRI (optimal score is 0.946); MHCII-IAd peptide regions, 2-GHLLLLLGL, 57-SGCGGNAN, 3-HLLLLLGLL, 1-SGHLLLLLG (optimal score is 0.488); MHCII-IAg7 peptide regions 60-CGGNANRFK, 21-SGAAKYCKL, 61-GGNANRFKT, 20-VSGAAKYCK (optimal score is 1.468); and MHCII-RT1.B peptide regions 46-KWKAKQCLP, 24-AKYCKLPLR, 10-LLTLWAELT, 45-YKWKAKQCL (optimal score is 0.569) which represented predicted binders from Dendroaspis polylepis polylepis DTX-K. ( Table 2). The predicted binding affinity is normalized by the 1% fractil. The MHC peptide binding is predicted using neural networks trained on C terminals of known epitopes. In analysis predicted MHC/peptide binding is a log-transformed value related to the IC50 values in nM units. These MHC binding peptides are sufficient for eliciting the desired immune response. Predicted MHC binding regions in an antigen sequence and there are directly associated with immune reactions, in analysis we found the MHCI and MHCII binding region.

Future Perspectives
This method will be useful in cellular immunology, Vaccine design, immunodiagnostics, immunotherapeutics and molecular understanding of autoimmune susceptibility. Dendroaspis polylepis polylepis DTX-K sequence involved multiple antigenic components to direct and empower the immune system to protect the host from the dendrotoxin. MHC molecules are cell surface proteins, which take active part in host immune reactions and involvement of MHC class in response to almost all antigens and it give effects on specific sites. Predicted MHC binding regions acts like red flags for antigen specific and generate immune response against the parent antigen. So, a small fragment of antigen can induce immune response against whole antigen. The method integrates prediction of peptide MHC class binding; proteosomal C terminal cleavage and TAP transport efficiency. This theme is implemented in designing subunit and synthetic peptide vaccines.