Fig 1.
Workflow chart.
Fig 2.
Overlapping regions amongst the linear B cell epitopes predicted by the BepiPred method and other seven different B cell epitopes prediction methods.
B cell epitopes predicted by the BepiPred method and other different protein sequence based (Chou.., Emini.., Karplus.., Kolaskar.., and Parker..) and protein structure based (DiscoTope and ElliPro) prediction methods were found to have significant consensus. Consensus overlapping regions of BepiPred epitopes are underlined by the different color, corresponding to respective prediction method.
Fig 3.
Overlapping CTL, HTL and B cell epitopes.
Multiple sequence alignment performed by Clustal Omega at EBI to identify the consensus overlapping regions of CTL (red), HTL (blue) and B cell epitopes (green) amongst shortlisted epitopes. Epitopes with overlapping region amongst all the three types of epitopes (CTL, HTL and B Cell epitopes), epitopes with full sequence overlap and epitopes with the highest number of HLA allele binders were selected for further studies (encircled).
Fig 4.
Molecular docking analysis of CTL epitopes within the TAP transporter cavity.
Molecular interaction of the seven selected CTL epitopes (cyan sticks) with the TAP cavity (gray ribbon/sticks) is shown in detail. Panel (A) shows the binding of epitope at two different sites within TAP cavity, panel (B) and (C) show detailed molecular interaction between epitopes and TAP cavity; (a, b) show chain A and B of TAP transporter. H-bonds are highlighted as yellow dots. (*) Indicates binding energy, shown in kcal/mol.
Fig 5.
Design of Multi-Epitope Vaccine (MEVs).
(A) CTL and (B) HTL epitopes were linked by the short peptide linker ‘GGGGS’. Human Beta-Defensin 2 and Beta-Defensin 3 were used as an adjuvant at the N and C terminals respectively. The short peptide EAAAK was used to link the Beta-Defensin 2 and Beta-Defensin 3. Epitopes from different proteins were highlighted with different colors and the C terminal 6xHis is designated as His tag. *Indicates the epitopes common to Phosphoprotein, V Protein and W protein, dues to protein sequence similarity.
Fig 6.
Tertiary structure modelling of CTL and HTL Multi-Epitope Vaccines.
(A) & (F): Tertiary structural models of CTL and HTL MEVs showing epitopes in different colors corresponding to the epitopes color as shown in Fig 5. (B) & (G): Show the different domains of CTL and HTL MEVs. (C) & (H): The overlapping linear B cell epitope region present in CTL and HTL MEVs, shown by spheres. (D) & (I): From the CTL and HTL MEVs, the INF-γ inducing epitopes are shown in cyan, discontinuous B Cell epitopes are shown in magenta and the region common amongst INF-γ and discontinuous B Cell epitopes are shown in wheat color. (E) & (J): RAMPAGE analysis of the refined CTL and HTL MEV models.
Fig 7.
Molecular docking and dynamics simulation study of CTL and HTL MEVs with TLR3.
(A) CTL and (E) HTL MEVs (VIBGYOR) docked complex with TLR3 (gray). Both the complexes are forming several hydrogen bonds in the MEV and TLR3 interface, as shown by green dots. B-Factor of the docked MEVs is shown by a rainbow (VIBGYOR) presentation. The regions in blue being indicated stable and the region in red indicate unstable. In the above complexes, most of the region of docked MEVs is in blue and with the very small region is green, yellow or orange, hence the complexes are predicted to be very stable. (B) and (F), RMSD as generated by the molecular Dynamics simulation study of CTL, HTL MEVs and TLR3 complexes. (C) & (G) Rg (radius of gyration) across the time window of 100 nanosecond. (D) & (H), RMS fluctuation for all the atoms of the CTL, HTL MEVs and TLR3 complexes.