Construction of a multi-epitope vaccine against Helicobacter pylori fused with the CTLA-4 extracellular domain and expressed in BCG.

Owing to the high prevalence of Helicobacter pylori infections and the increasing rates of antibiotic resistance, the development of a potent vaccine against H. pylori infection has become an urgent need. In this study, we screened three linear B-cell dominant epitopes, 16 helper T-cell dominant epitopes, 20 cytotoxic T-cell (CTL) dominant epitopes, and conformational epitopes from the UreB, CagA, and VacA proteins using immunoinformatics approaches. To enhance vaccine affinity, dominant epitopes were linked to the extracellular domain of CTLA-4 using appropriate linkers, resulting in the formation of a multi-epitope vaccine. Subsequently, the rationality of the vaccine protein sequence was validated, and absence of allergenic properties was confirmed. Molecular dynamics and docking simulations demonstrated strong stability in the interactions between the vaccine proteins with B7, Toll like receptor (TLR)-2, and TLR-4. Additionally, immune simulations indicated that the constructed vaccine effectively induced both humoral and cellular immune responses. Ultimately, the refined DNA sequence of the vaccine was incorporated into the pMV261 vector of the Bacillus Calmette-Guerin (BCG) shuttle expression plasmid to enhance efficacy via BCG delivery. Preliminary experimental validation using western blotting and enzyme linked immunosorbent assay confirmed that the vaccine's structural integrity and its ability to elicit a strong immune response in the host, providing valuable insights for the advancement of H. pylori vaccine development.