Fig 1.
Flowchart depicting the stages required in the in silico design of a multi-epitope vaccine against MPXV.
The process comprises numerous key phases, including target protein sequence retrieval, epitope prediction, vaccine construction, physicochemical characterization, 3D model prediction, molecular docking to immune receptor, MD simulation, and in silico cloning.
Fig 2.
A schematic representation of the multi-epitope vaccine construct against MPXV.
It includes 376 amino acids, 124 of which are adjuvant (grey). CTL, HTL, and LBL epitopes were represented by blue, green, and pink, respectively.
Table 1.
A list of the epitopes used in the proposed multi-epitope vaccine.
Fig 3.
Population coverage of selected CTL and HTL epitopes and their corresponding MHC alleles separately and in combination.
Table 2.
The physicochemical properties of a multi-epitope vaccine.
Fig 4.
The initial 3D structure of the multi-epitope vaccine is illustrated in green, and the refined 3D structure is presented in violet.
To compare the initial and refined models, the structures were superimposed.
Fig 5.
Comparing the quality of the initial and refined models of the vaccine construct’s three-dimensional structure using the Zlab server and the ProSA-web server.
(A) The Ramachandran plot analysis shows that in the initial model, 83.188%, 11.304%, and 5.507% of the residues are found in the highly preferred (green crosses), preferred (brown triangles), and questionable (red circles) regions, respectively; (B) whereas in the refined model, 94.783%, 4.638%, and 0.580% of the residues are found in the highly preferred, preferred, and questionable regions, respectively. (C) The initial model has a Z-score of -3.48, (D) while the refined model has a Z-score of -3.67.
Table 3.
The quality parameter scores of the models generated by the GalaxyRefine server.
Fig 6.
The discontinuous B-cell epitopes in the multi-epitope vaccine 3D model (A-H). The vaccine construct is depicted by gray sticks, while the discontinuous B-cell epitopes are shown by cyan spheres. (A) 8 residues with a score of 0.948; (B) 11 residues with a score of 0.833; (C) 6 residues with a score of 0.744; (D) 73 residues with a score of 0.732; (E) 16 residues with a score of 0.646; (F) 79 residues with a score of 0.646; (G) 6 residues with a score of 0.562; (H) 5 residues with a score of 0.515.
Table 4.
A list of the discontinuous B-cell epitopes predicted by the ElliPro server.
Fig 7.
The disulfide engineering of the multi-epitope vaccine’s 3D structure.
(A) The wild type; (B) The mutant type (the three introduced disulfide bonds are shown by red sticks).
Table 5.
List of residue pairs capable of forming disulfide bonds in the vaccine construct.
Fig 8.
The docked complex of the vaccine and TLR4.
The vaccine construct is shown in surface form, while TLR4 is shown in cartoon form.
Fig 9.
The interaction map of the vaccine construct with chains A and C from TLR4.
Hydrogen bonds are shown by dashed (green) lines with the length of the bond printed in the middle.
Table 6.
List of residues involved in the formation of hydrogen bonds between TLR4 (chains A) and the vaccine.
Table 7.
List of residues involved in the formation of hydrogen bonds between TLR4 (chain C) and the vaccine.
Fig 10.
The MD simulation of the vaccine-TLR4 docked complex in a period of 40 ns.
(A) The RMSD plot of TLR4 exhibits a consistent trend during the simulation period, while the vaccine’s RMSD plot begins with an infinitesimal variation and becomes stable at about 33 ns. (B) The RMSF plot shows that both TLR4 chains A and B show very mild fluctuations, while the vaccine construct shows high fluctuations.
Table 8.
Binding free energies of TLR4, vaccine, and TLR4-vaccine complex.
All values are given in kcal/mol.
Fig 11.
The sequence of the multi-epitope vaccine (shown in red) surrounded between HindIII (173) and BamHI (1307) into the pET-28a (+) expression vector (shown in black).