Fig 1.
Schematic representation of the workflow for the design of a multi-epitope vaccine against Helicobacter pylori.
Table 1.
Predicted LBL, CTL, and HTL epitopes from BabA, CagA, and VacA proteins for the development of multi-epitope vaccine.
Table 2.
Binding of CTL and HTL epitopes to MHC class I and II alleles.
Fig 2.
Country-specific population coverage of predicted CTL and HTL epitopes.
Population coverage for the epitopes is depicted by bar graphs, presented both collectively and separately for the selected CTL (red bars) and HTL (green bars) epitopes, along with their combined coverage (blue bars). Data indicates that the combined epitopes provide nearly 100% global population coverage, with only minimal variation among regions.
Fig 3.
3D structural models and epitope mapping of HP_VaX_V1 and HP_VaX_V2.
(a) & (b) 3D structures of both selected vaccines, HP_VaX_V1 and HP_VaX_V2. (c) & (d) Epitope sequences and linker/adjuvant arrangement for HP_VaX_V1 and HP_VaX_V2.
Fig 4.
Analysis of the Ramachandran plot of HP_VaX_V1 and HP_VaX_V2.
(a) HP_VaX_V1 exhibits a 93.5% presence inside the permissible zone, while 5.4% and 0.3% are found in the supplementary and highly permissive regions, respectively. Only 0.8% exhibited presence in the prohibited area. (b) HP_VaX_V2 exhibits a 90.4% presence inside the permissible range, with 6.8% and 1.6% presence in the supplementary and generously permissible regions, respectively. Only 1.2% of the data fell within the prohibited zone.
Fig 5.
ProSA validation and Z-score analysis of vaccine candidates HP_VaX_V1 and HP_VaX_V2.
(a) The Z-score for the ProSA validation of the HP_VaX_V1 vaccine model is –5.26, (b) The Z-score for the ProSA validation of the HP_VaX_V2 vaccine model is –3.64.
Table 3.
Physicochemical characteristics of vaccine candidates.
Fig 6.
B-cell epitope predictions for HP_VaX_V1.
The purple regions illustrate the epitopes as identified by the ElliPro webserver, along with their respective length and score listed below.
Fig 7.
B-cell epitope predictions for HP_VaX_V2.
The yellow regions illustrate the epitopes as identified by the ElliPro webserver, along with their respective length and score listed below.
Fig 8.
Molecular docking analysis of HP_VaX_V1 and HP_VaX_V2 with Toll-Like Receptors (TLRs).
Binding conformations of HP_VaX_V1 (blue) and HP_VaX_V2 (red) with eight Toll-Like Receptors (TLRs), including TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, and TLR9. The ΔG values (in kcal/mol) below each structure represent the binding affinities, with more negative values indicating stronger and more stable interactions.
Fig 9.
Comparative interaction network analysis of HP_VaX_V1 and HP_VaX_V2 with TLR7 and TLR8.
Detailed interaction networks between HP_VaX_V1 and TLR7 (left panel), and HP_VaX_V2 and TLR8 (right panel). The interactions include salt bridges, disulfide bonds, hydrogen bonds, and non-bonded contacts, visualized as connections between amino acid residues from the vaccine constructs and Toll-Like Receptors. Each interaction type is represented by a specific color-coded line, as indicated in the key below.
Fig 10.
Molecular dynamic simulation of the HP_VaX_V1-TLR7 and HP_VaX_V2-TLR8 constructs.
(a) Deformability of the protein structures, illustrating the flexibility at each residue. (b) B-factor (or temperature factor) analysis, showing the degree of atomic displacement. (c) Eigenvalues from the Normal Mode Analysis (NMA), indicating the stiffness of the modes. (d) Variance map, representing the contribution of each mode to the overall motion. (e) Co-variance map, highlighting correlated motions between residues. (f) Elastic network model, depicting the connections and interactions within the protein structure.
Fig 11.
Structural stability and residue flexibility analyses of the HP_VaX_V1-TLR7 and HP_VaX_V2-TLR8 protein complexes over a 100 ns MD simulation.
(A) Root Mean Square Deviation (RMSD) plot illustrating the structural stability of HP_VaX_V1-TLR7 (black) and HP_VaX_V2-TLR8 (red) complexes. (B) Root Mean Square Fluctuation (RMSF) plot showing the residue-wise flexibility of HP_VaX_V1-TLR7 (black) and HP_VaX_V2-TLR8 (red).
Fig 12.
Compactness and conformational stability analysis of vaccine constructs.
(A) Radius of Gyration (Rg) plot depicting the compactness of HP_VaX_V1 (black) and HP_VaX_V2 (red) over a 100 ns molecular dynamics simulation. (B) Principal Component Analysis (PCA) scatter plot illustrating the conformational stability of HP_VaX_V1 (black) and HP_VaX_V2 (red) by projecting their molecular dynamics trajectories onto principal eigenvectors.
Fig 13.
Free Energy Landscape (FEL) analysis of vaccine constructs.
(A) FEL map of HP_VaX_V1 constructed using the first two principal components (PC1 and PC2). The map reveals two prominent and deep energy basins, representing highly stable and energetically favorable conformational states. (B) FEL map of HP_VaX_V2 constructed using the same parameters. The map exhibits a well-defined single deep energy basin, suggesting a stable conformational state with limited structural deviations.
Fig 14.
Immune simulation results for HP_VaX_V1 and HP_VaX_V2 vaccine constructs.
(a) The black line represents antigen levels, signifying the production of immunoglobulin after immunization. (b) The B-cell population dynamics after multiple encounters with the antigen. (c) The dendritic cell population at various stages over a one-year period post-initial injection. (d) The Simpson index quantifies the interferon-gamma response within one year post-immunization. (e) The data for the population of Helper T-cells. (f) The concentration of Cytotoxic T-cells assessed over a one-year period after the initial antigen encounter.
Fig 15.
Computational predictions of secondary structures for HP_VaX_V1 and HP_VaX_V2 mRNAs.
Predicted minimum free energy (MFE) secondary structures of (A) HP_VaX_V1 mRNA (-760.60 kcal/mol) and (B) HP_VaX_V2 mRNA (-411.70 kcal/mol). Base pairs are represented by lines, with color gradients indicating base pairing probability.
Fig 16.
Schematic representation of recombinant expression plasmids for HP_VaX_V1 and HP_VaX_V2.
Circular maps of the pET-28a(+) expression vectors harboring the (a) HP_VaX_V1 and (b) HP_VaX_V2 gene inserts. Key features include the multiple cloning site (MCS), antibiotic resistance gene, origin of replication (ori), and T7 promoter. Restriction enzyme sites are indicated.