Disulfide engineering in vaccine constructs.

<div> <i>Helicobacter pylori</i> is a gram-negative bacterium that persistently infects the human stomach, leading to peptic ulcers, gastritis, and an increased risk of gastric cancer. The extremophilic characteristics of this bacterium make it resistant to current drug treatments, and there are no licensed vaccines available against <i>H</i>. <i>pylori</i>. Computational approaches offer a viable alternative for designing antigenic, stable, and safe vaccines to control infections caused by this pathogen. In this study, we employed an immunoinformatic strategy to design a set of candidate multi-epitope subunit vaccines by combining the most potent B and T cell epitopes from three targeted antigenic proteins (BabA, CagA, and VacA). Out of the 12 hypothetical vaccines generated, two (HP_VaX_V1 and HP_VaX_V2) were found to be strongly immunogenic, non-allergenic, and structurally stable. The proposed vaccine candidates were evaluated based on population coverage, molecular docking, immune simulations, codon adaptation, secondary mRNA structure, and <i>in silico</i> cloning. The vaccine candidates exhibited antigenic scores of 1.19 and 1.01, with 93.5% and 90.4% of the most rama-favored regions, respectively. HP_VaX_V1 and HP_VaX_V2 exhibited the strongest binding affinity towards TLR-7 and TLR-8, as determined by molecular docking simulations (ΔG = −20.3 and −20.9, respectively). Afterward, multi-scale normal mode analysis simulation revealed the structural flexibility and stability of vaccine candidates. Additionally, immune simulations showed elevated levels of cell-mediated immunity, while repeated exposure simulations indicated rapid antigen clearance. Finally, <i>in silico</i> cloning was performed using the expression vector pET28a (+) with optimized restriction sites to develop a viable strategy for large-scale production of the chosen vaccine constructs. These analyses suggest that the proposed vaccines may elicit potent immune responses against <i>H</i>. <i>pylori</i>, but laboratory validation is needed to verify their safety and immunogenicity. </div>