In silico design and experimental validation of a multi-epitope vaccine candidate against Helicobacter hepaticus associated chronic liver inflammation

Background Helicobacter hepaticus is an emerging enterohepatic pathogen associated with chronic gastrointestinal and hepatic inflammatory disorders. Despite its clinical relevance, effective vaccination strategies remain unavailable due to antigenic variability, immune evasion, and limited induction of durable cellular immunity. Methods An integrated reverse vaccinology and experimental validation approach was employed to design a multi-epitope vaccine against H. hepaticus . Two conserved outer membrane proteins, BamA and an organic solvent tolerance protein, were selected based on antigenicity, surface exposure, and safety profiling. Immunodominant cytotoxic and helper T-cell epitopes were predicted, screened for immunogenicity, allergenicity, and toxicity, and assembled into a rationally designed construct incorporating human β-defensin-3 as an adjuvant. Structural modeling, conformational B-cell epitope mapping, molecular docking, molecular dynamics simulations, and in silico immune simulations were performed. The construct was recombinantly expressed in Escherichia coli and evaluated through biophysical, antibacterial, and immunological assays. Results The vaccine candidate exhibited favorable physicochemical properties, structural stability, and strong binding affinity toward Toll-like receptor 2. In silico immune simulations predicted robust Th1-biased immune responses with effective memory formation. Recombinant expression yielded a soluble, monodisperse protein. Functional assays demonstrated significant inhibition of H. hepaticus growth and host cell adhesion. In vitro immunogenicity studies revealed strong peripheral blood mononuclear cell proliferation, enhanced CD4 + and CD8 + T-cell activation, elevated Th1 cytokine secretion, and antigen-specific antibody production. Conclusion The proposed multi-epitope vaccine candidate induces coordinated innate and adaptive immune responses and exhibits promising antibacterial activity against H. hepaticus . This study provides a comprehensive and translational framework for epitope-based vaccine development and supports further in vivo evaluation of the candidate vaccine.