In silico design of a VacA-based multi-epitope Subunit Vaccine Candidate for immunoprotection against Helicobacter...

Helicobacter pylori, a globally prevalent gastric pathogen associated with gastric carcinoma, ulcers of the bowel, and persistent gastritis where the vacuolating cytotoxin A (vacA), a major virulence factor, that promotes cellular damage and immune evasion. Despite increasing antibiotic resistance, no licensed vaccine exists for effective prevention or treatment. In present study, we implemented an all-encompassing immunoinformatic strategy in developing a novel vacA- driven multi-epitope subunit vaccine (MESV). Utilising the IEDB database, cytotoxic as well as helper T-cell epitopes were determined based on the vacA sequence of proteins and examined for antigenic properties, non-allergenicity and nontoxic effects. The mapped epitopes were coupled via AAY and GPGPG linkers, with the 50S ribosomal protein L7/L12 incorporated as an N-terminal adjuvant through an EAAAK linker for enhancing immunogenicity. The designed vaccine construct comprised 257 amino acids, demonstrated an antigenicity score of 0.8287 along with non-allergenic property and exhibited a high solubility score of 0.9444. Structural modeling using trRosetta followed by refinement and validation through GalaxyRefine, ProSA, and PROCHECK confirmed a stable 3D conformation with desirable quality metrics reported (Z-score: -5.93; 94.4 % residues in favored regions). Molecular docking and molecular dynamics simulations using GROMACS demonstrated strong and stable binding with toll-like receptors (TLRs 2/4/5/9), especially TLR4 shows highest stability and binding affinity suggesting effective innate immune activation. Immune simulations predicted potent humoral and cellular responses characterized by elevated IgM, IgG, IFN-γ, and IL-2 levels, as well as long-term memory cell formation. Codon optimization (CAI: 0.98; GC: 49.4 %) and in silico cloning into the pET28a (+) vector indicated efficient potential expression in E. coli K12. Thus, the outcomes of our study propose the vacA-based MESV as a promising, stable, and immunogenic vaccine construct targeting H. pylori, warranting future in vitro and in vivo evaluation to substantiate its protective efficacy.