Prediction of Epitope Based Vaccine Candidates against Macaca fascicularis PV Type 2 Virus Using In-silico Approaches

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Papillomavirus, epitope, antigenicity prediction, physicochemical characterization, population coverage analysis
Papillomaviruses are the causing agents of benign tumors in their hosts, i.e., mammals and birds, across the world.They have circular double stranded DNA genome.In order to combat the viral infection in Macaca fascicularis PV type 2, a computational pipeline was employed in this study for the prediction of viral protein targeting peptides for vaccine discovery.Epitope prediction enabled the identification of multi-peptides suitable for vaccine development.Further in-depth analysis for immunogenicity and toxicity prediction scrutinized the optimal candidate for target based designing of vaccines.Immunogenic and physicochemical properties of proteins E1, E2, E4, E6, E7, L1, and L2 of Macaca fascicularis PV type 2 revealed their instability index, molecular weight, and antigenic potential.The predicted epitopes may lead to promising targets for broad spectrum vaccine designing against the viral strain of Macaca fascicularis PV type 2.
In order to combat the infection caused by Macaca fascicularis PV type 2, there is an urgent need to develop effective therapeutic vaccine providing protection at inter-and intra-species level.Overcoming the limitation caused by time consuming, labor intensive, and expensive traditional methods of generating monoclonal antibodies, in silico epitope identification is considered as a potential route of the development of broad spectrum vaccine.Invading pathogenesis by identifying B-cell epitopes initiating humoral immune response by antigen-antibody interaction (Getzoff et al., 1988;Somvanshi and Seth, 2009) along with antigens binding to HLA class I (CD8+ T-cells) and HLA class II (CD4+ T-cells) alleles with specificity and sensitivity was used earlier against several viruses (Singh et al., 2009;Somvanshi et al., 2008a;Somvanshi et al., 2008b).Additionally, population coverage analysis was included in the study to identify the epitope(s) restricting the limitation of extreme polymorphism among maximal population setting.The consequential epitopes of the present study would be a germane initiator for potential vaccine development against Macaca fascicularis PV type 2.

Data-set Collection:
A complete set of bioinformatics tools and softwares were used in a sequential manner for the complete  ProtParam (Gasteiger et al., 2005), an online protein analysis tool on EXPASY server was used for the appraisal of various physicochemical properties including molecular weight, amino acid composition, extinction coefficient (Gill and Hippel, 1989), theoretical pI, and grand average of hydropathicity (Kyte and Doolittle, 1982), aliphatic index (Ikai, 1980), and instability index (Guruprasad et al., 1990).

Epitope Prediction:
Possible epitopes (B-cell and Tcell for MHC Class I & II) in protein primary sequences were screened out using ABCpred server (www.imtech.res.in/abcpred) and Immune Epitope Database tool (www.iedb.org).Both tools involve combinatorial machine learning algorithmic approach for epitope prediction.Peptide property calculator (https://www.genscript.com), a freely available tool to determine the best solvent for a peptide was used for peptide property calculation.

Population Coverage Analysis:
Afterwards, population coverage analysis was done by using Immune Epitope Database and Analysis Resource (IEDB) Population Coverage tool available at http://tools.immuneepitope.org/tools/population/iedb_input for the identification of all the possible binding alleles (MHC Class I and MHC Class II) with respect to the identified T-cell epitopes (Bui et al., 2006).

RESULTS AND DISCUSSION
Macaca fascicularis PV type 2 (MfPV2) genome, isolated from exophytic skin of hand and feet of cynomolgus monkey (M.fascicularis) is a double stranded DNA virus of size 7632 base pairs and includes seven proteins (E1, E2, E4, E6, E7, L1, and L2).The complete genomic sequence was retrieved from NCBI Genome database (NC_015691) in GenBank format.In the present study, sequence based analysis along with physicochemical characterization, epitope prediction and population coverage analysis was accomplished on the protein sequences of Macaca fascicularis PV type 2. A highly proficient computational pipeline involving bioinformatics tools was developed to retrieve a vast amount of data to identify potential vaccine candidates.In order to predict the biological activity of the proteins, physicochemical characterization was performed.
The physicochemical properties of the identified proteins (Somvanshi and Seth, 2009) were computed using ProtParam server (Table 1).The instability index is an estimate of the stability of a protein in a test tube, consequently seven proteins were found stable in nature ranged between (42.97-59.19).The sequential addition of hydropathy values of each amino acid residue divided by the number of amino acids is the indicator of protein hydrophobicity (GRAVY).It is calculated as a sum of hydropathy values of all the amino acids residues divided by the number of residues in the sequence.Increasing positive score shares directly proportional relationship with hydrophobicity.Theoretical isoelectric point deciphers pH dependent characteristics of a protein in a suitable medium ranging between 4.61-10.2.Immunogenicity prediction declares the potential of the proteins to acts as probable antigen.
Information about the epitopic regions or antigenic determining factor is necessary for designing active inhibitors in contrast to active viral proteins.T-cell and B-cell antibodies recognize a specific part of antigen to bind with specificity, termed as epitope or antigenic determinant.
Antibodies recognize specific regions (antigenic determinants or B-cell epitopes) and bind to the antigens with the specificity.Understanding the antigen-antibody interaction pattern determine the viral pathogenesis.B-cell epitopes were determined for seven proteins of Macaca fascicularis PV type 2 (Table 2).Paratope is a part of an antibody, assists in recognizing the antigenic determinant.Based on the foreignness characteristics, epitopes are typically non-self-protein sequences resulting from the host that can be recognized are also epitopes.Two classes of proteins antigens are known based on amino acid sequence composition (a) conformational epitopes and (b) linear epitopes (Huang and Hond, 2006)  Their immunogenic potential correlates with other viruses and laid the foundation of synthetic biology by developing synthetic peptides for vaccine development.In addition, toxicity prediction enabled behavioral study of peptides under different environmental conditions.
The population coverage analysis depicted potential application of the probable vaccine at a global level by providing maximal coverage ranging from 36.09-99.09%(Table 4 & Figure 1).The population setting at global level was analyzed showing maximum percentage at South East Asia and Central Africa.Gupta S, Kapoor P, Chaudhary K, Gautam A, Kumar R (2013) In silico approach for predicting toxicity of peptides and proteins.PLoS ONE 8:e73957.Guruprasad K, Reddy BV, Pandit MW (1990) Correlation between stability of a protein and its dipeptide composition: a novel approach for predicting in vivo stability of a protein from its primary sequence.

Fig. 1 :
Fig. 1: Representing average hit and PC90 analysis for MHC Class I and MHC Class II, respectively (blue = average hit for MHC Class I, red = PC90 for MHC Class I, green = average hit for MHC Class II, and purple = PC90 for MHC Class II).

Table 1 :
Immunogenic and physicochemical properties of proteins of Macaca fascicularis PV type 2.

Table 2 :
Predicted B-cell epitopes in Macaca fascicularis PV type 2.

Table 3 :
Predicted T-cell epitopes (MHC Class I and MHC Class II) in Macaca fascicularis PV type 2.

Table 4 :
Population coverage analysis (%) of MHC Class-I and MHC Class-II.