3.5  Toxicity analysis, Ramachandran Plot analysis, and population coverage results
ToxinPred 4.0 server results (in Table 6 .) represent Finalized T-cell Epitopes that were nontoxic from the biochemical perspective.
Table 6 Results of ToxinPred on probable antigens.
Peptide/Probable antigen SVM score Hydrophilicity Molecular weight Toxicity
FTIGTVTLK −1.36 −1.23 979.32 NON-TOXIN
GTITVEELK −0.98 0.34 989.27 NON-TOXIN
ITLCFTLKR −1.32 −0.41 1094.51 NON-TOXIN
VFITLCFTL −1.21 −1.52 1056.46 NON-TOXIN
WLLWPVTLA −1.18 −1.62 1098.49 NON-TOXIN
VYQLRARSV −1.07 −0.12 1091.40 NON-TOXIN
Ramachandran plot analysis, Fig. 8 A and B suggest that most of the residues are allowed in a favored region; this gives more confidence in the structural conformation for targeted T-Cell Epitopes.
Fig. 8 A. 99.8% residues of the ITLCFTLKR Epitope were in the allowed and favored region under Ramachandran Plot analysis. B. 99.8% residues of the VYQLRARSV Epitope were in the allowed and favored region under Ramachandran Plot analysis.
MHCPred results (Table 7 ) indicate quantitative estimation of IC50 values for both MHC I and MHC II alleles for respective Epitopes shows elicitation of an immune response when this data is deployed in a population coverage analysis.
Table 7 MHCPred results depict IC50 Values for HLA Alleles and confidence of the prediction.
HLA Alleles Amino acid groups Predicted -logIC50 (M) Predicted IC50 Value (nM) Confidence of prediction (Max = 1)
HLA-A*68:01 FTIGTVTLK 7.116 76.56 1.00
HLA-A*11:01 ITLCFTLKR 7.028 93.76 1.00
HLA-A*68:01 ITLCFTLKR 6.282 522.40 0.78
HLA-DRB1*01:01 VYQLRARSV 7.624 23.77 0.89
HLA-DRB1*07:01 VYQLRARSV 6.734 184.50 0.89
IEDB population coverage analysis suggests that ITLCFTLKR and VYQLRARSV epitopes exhibit a suitable population coverage, as depicted in the graphical representation of Fig. 9 A and B. This allows only two probable Epitopes for the final selection of vaccine crafting.
Fig. 9 A. Graphical representation of population conservancy analysis of ITLCFTLKR Epitope. B. Graphical representation of population conservancy analysis of VYQLRARSV Epitope.
In Table 8 , ProtParam analysis further reveals the stability of the considered epitopes and final revelation of one epitope ITLCFTLKR is screened out. This particular Epitope exhibits an instability index of 35.68, with a grand average of hydropathicity (GRAVY) calculated was 0.844, and the estimated half-life for this peptide was determined to be 20 h for mammalian reticulocytes.
Table 8 ProtParam analysis for selected epitopes.
Selected Epitope GRAVY Score Instability Index (Indication) Estimated Half-Life(Mammalian reticulocytes) Theoretical pI Aliphatic Index
ITLCFTLKR 0.844 35.68(Stable) 20 Hours 9.51 130.00
VYQLRARSV −0.067 70.73(Unstable) 100 Hours 10.83 118.89