Exploring the structural basis to develop efficient multi-epitope vaccines displaying interaction with HLA and TAP and TLR3 molecules to prevent NIPAH infection, a global threat to human health

Abstract

Nipah virus (NiV) is an emerging zoonotic virus that caused several serious outbreaks in the south asian region with high mortality rates ranging from 40 to 90% since 2001. NiV infection causes lethal encephalitis and respiratory disease with the symptom of endothelial cell-cell fusion. No specific and effective vaccine has yet been reported against NiV. To address the urgent need for a specific and effective vaccine against NiV infection, in the present study, we have designed two Multi-Epitope Vaccines (MEVs) composed of 33 Cytotoxic T lymphocyte (CTL) epitopes and 38 Helper T lymphocyte (HTL) epitopes. Out of those CTL and HTL combined 71 epitopes, 61 novel epitopes targeting nine different NiV proteins were not used before for vaccine design. Codon optimization for the cDNA of both the designed MEVs might ensure high expression potential in the human cell line as stable proteins. Both MEVs carry potential B cell linear epitope overlapping regions, B cell discontinuous epitopes as well as IFN-γ inducing epitopes. Additional criteria such as sequence consensus amongst CTL, HTL and B Cell epitopes was implemented for the design of final constructs constituting MEVs. Hence, the designed MEVs carry the potential to elicit cell-mediated as well as humoral immune response. Selected overlapping CTL and HTL epitopes were validated for their stable molecular interactions with HLA class I and II alleles and in case of CTL epitopes with human Transporter Associated with antigen Processing (TAP) cavity. The structure based epitope cross validation for interaction with TAP cavity was used as another criteria choosing final epitopes for NiV MEVs. Finally, human Beta-defensin 2 and Beta-defensin 3 were used as adjuvants to enhance the immune response of both the MEVs. Molecular dynamics simulation studies of MEVs-TLR3 ectodomain (Human Toll-Like Receptor 3) complex indicated the stable molecular interaction. We conclude that the MEVs designed and in silico validated here could be highly potential vaccine candidates to combat NiV infections, with great effectiveness, high specificity and large human population coverage worldwide.

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.