3  Results

3.1  T-cell epitopes prediction and VaxiJen scoring
Non-allergen proteins selected (Table 1) based on allergenicity scores (by deploying AllergenFP 1.0) New to NetMHCII PAN 3.2 and NETMHC 4.0 servers for determining 1-log50k value and affinity values for selecting the best possible pair of epitopes with their corresponding HLA alleles. In Table 2 and Table 3 , results for MHC Class I HLA and MHC Class II HLA alleles paired epitopes along with their VaxiJen scores were represented respectively, to obtain putative epitopes. The results are self-explanatory in Fig. 2 . Graphical representation of selected peptides for docking are based on their interaction with MHC Class I and Class II HLA alleles along with their antigenicity.
Table 2 Probable antigenic epitopes and MHC I Allele interaction based on NETMHC 4.0 server and VaxiJen 2.0 scores (≥1.0) for antigenicity prediction. (* - sign is used for no interaction any HLA Allele under consideration).
NCBI-GenBank ID MHC I Allele POS Core peptide 1-LOG50K Affinity(nM) Vaxijen score Antigenicity
QHD43417.1 HLA-A*68:01 7 FTIGTVTLK 0.853 4.9 2.0317 ANTIGEN
HLA-A*31:01 125 RLWLCWKCR 0.74 16.59 1.1604 ANTIGEN
QHD43418.1 – – – – – – NO INTERACTION
QHD43419.1 HLA-A*11:01 5 GTITVEELK 0.719 20.98 1.0976 ANTIGEN
QHD43421.1 HLA-A*11:01 109 ITLCFTLKR 0.71 22.97 2.0208 ANTIGEN
HLA-A*68:01 109 ITLCFTLKR 0.695 27.24 2.0208 ANTIGEN
HLA-A*23:01 107 VFITLCFTL 0.621 60.42 1.2490 ANTIGEN
QHD43423.2 – – – – – – NO INTERACTION
Table 3 Probable antigenic epitopes and MHC II Allele interaction based on NETMHC II PRED 3.2 server and VaxiJen 2.0 scores (≥1.0) for antigenicity prediction. (* - sign is used for no interaction any HLA Allele under consideration).
NCBI-GenBank ID MHC II Allele POS Core peptide 1- LOG50K Affinity(nM) Vaxijen score Antigenicity
QHD43417.1 – – – – – – NO INTERACTION
QHD43418.1 – – – – – – NO INTERACTION
QHD43419.1 HLA-DRB1*04:01 55 WLLWPVTA 0.282 2375.78 1.0631 ANTIGEN
QHD43421.1 HLA-DRB1*01:01 74 VYQLRARSV 0.484 267.05 1.3108 ANTIGEN
HLA-DRB1*07:01 74 VYQLRARSV 0.342 1235.08 1.3108 ANTIGEN
QHD43423.2 – – – – – – NO INTERACTION
Fig. 2 Graphical representation of selected peptides for docking based on their interaction with MHC Class I and Class II HLA alleles along with their Antigenicity.

3.2  Structural findings of epitope and MHC HLA-Alleles
Epitope structures were obtained by using the PEP-FOLD-3.5 web-server; the HLA allele's structures were retrieved from the RCSB-PDB database. In Table 4 the crystal structure/model structure details with reference PDB-Id is provided.
Table 4 Listing of MHC HLA-Alleles respective Crystal structures/Models with the PDB ID.
Allele Name Template structure (PDB-ID) Crystal structure/Model
HLA-A*11:01 2HN7 CRYSTAL STRUCTURE
HLA-A*23:01 3I6L CRYSTAL STRUCTURE
HLA-A*31:01 3RL1 CRYSTAL STRUCTURE
HLA-A*68:01 6PBH CRYSTAL STRUCTURE
HLA-DRB1*01:01 4AH2 CRYSTAL STRUCTURE
HLA-DRB1*04:01 5LAX CRYSTAL STRUCTURE
HLA-DRB1*07:01 6BIJ CRYSTAL STRUCTURE

3.3  Molecular docking analysis
It was found that FTIGTVTLK, ITLCFTLKR epitopes interacted with MHC class I HLA Alleles, and VYQLRARSV epitope interacted with MHC Class II HLA alleles with a perfect binding score and ACE values as shown in Table 5 . The ITLCFTLKR Epitope of the ORF-7A protein exhibits binding with 2 HLA alleles (HLA-A*11:01, HLA-A*68:01) of MHC Class I, while FTIGTVTLK Epitope of ORF-3a protein interact with 1 HLA Allele (HLA-A*68:01) of MHC Class I. The VYQLRARSV Epitope of ORF-7a protein interacts clearly with 2 HLA Alleles (HLA-DRB1*01:01, HLA-DRB1*07:01) of the MHC Class II domain.
Table 5 Binding Energies, Ace Values for Docked Complexes based on DINC Server and PatchDock Analysis for Putative Epitopes.
Epitope HLA Allele Binding score (kcal/mol) Patch dock score ACE Selection
FTIGTVTLK HLA-A*68:01 −8.80 8066 178.91 Selected
RLWLCWKCR HLA-A*31:01 −4.80 8916 117.53 Rejected
GTITVEELK HLA-A*11:01 −3.80 8040 78.78 Rejected
ITLCFTLKR HLA-A*11:01 −3.70 8206 −25.88 Selected
ITLCFTLKR HLA-A*68:01 −7.60 8136 184.55 Selected
VFITLCFTL HLA-A*23:01 −4.40 7706 −134.91 Rejected
WLLWPVTLA HLADRB1*04:01 −8.60 9432 −150.96 Rejected
VYQLRARSV HLADRB1*01:01 −6.20 6874 −168.74 Selected
VYQLRARSV HLADRB1*07:01 −6.20 6842 262.74 Selected
In Fig. 3, Fig. 4, Fig. 5 , interactions between a selected three T-Cell epitopes with respective MHC Class I and II HLA-Alleles via hydrogen bond formation and van der Waals interactions is depicted. After positive docking results, these epitopes were subjected to further Molecular dynamic simulation and biochemical parameters assessment. Fig. 6 represents a graphical plot of binding scores for epitopes interacting with HLA-Alleles.
Fig. 3 FTIGTVTLK Epitope interaction with an antigen-binding pocket of HLA-A*68:01, of MHC I-HLA Allele. Here, Threonine at 2nd,5th, and 7th position in the epitope generate preferably a hydrogen bond due to the presence of partially charged positive and negative atoms, and also 4th position Glutamic acid and lysine at 8th position side chains can form a salt bridge, while other amino acids result in van der Waals interactions.
Fig. 4 ITLCFTLKR Epitope interaction with an antigen-binding pocket of HLA-A*68:01, of MHC I-HLA Allele. Here, Threonine at 2nd and 6th position, as well as cysteine at the 4th position in epitope, generate preferably a hydrogen bond due to the presence of partially charged positive and negative atoms, while other amino acids result in van der Waals interactions.
Fig. 5 VYQLRARSV Epitope interaction with an antigen-binding pocket of HLA-DRB1*07:01, of MHCII-HLA Allele, Here, Tyrosine at 2nd, Glutamine at 3rd position and Serine at 8th position in epitope generate preferably a hydrogen bond due to the presence of partially charged positive and negative atoms, and 5th and 7tharginine residue side chains can form a salt bridge, while other amino acids result in van der Waals interactions.
Fig. 6 Binding energy graphical plot for selected Epitope and HLA-Allelic pair.

3.4  Molecular dynamics and simulation analysis
RMSD values and Atomic fluctuation per amino acid residue were obtained for Epitopes interacting with the HLA-Allele structure; this analysis allows a perfect pair selection and validation. Moreover, only two Epitope pairs, i.e., ITLCFTLKR and VYQLRARSV, were identified as probable T-cell epitopes and as putative vaccine specimens. Fig. 7 shows the RMSD Plot and Atomic fluctuation per residue for the ITLCFTLKR- HLA-A*68:01 complex, the RMSD Plot and Atomic fluctuation per residue for the VYQLRARSV- HLA-DRB1*07:01 complex. Both results were positive as best interactions, for protein-ligand docked complexes must possess RMSD values from 0 to 1.0 Å as a preferred range [13].
Fig. 7 A. RMSD Plot for ITLCFTLKR- HLA-A*68:01 complex, for each amino acid residue by Molecular dynamics analysis, B. B-Factor (atomic fluctuation) values per amino acid residue for Epitope ITLCFTLKR- HLA-A*68:01 docked complex, C. RMSD Plot for VYQLRARSV- HLA-DRB1*07:01, for each amino acid residue by Molecular dynamics analysis, D. B-Factor (atomic fluctuation) values per amino acid residue for Epitope VYQLRARSV- HLA-DRB1*07:01 docked complex.

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