Integrative 16S rRNA characterization, pan-genome, and immunoinformatics approaches for the design of a multi-epitope vaccine against Bacillus cereus, a foodborne pathogen.

Bacillus cereus is a spore-forming foodborne pathogen responsible for diarrheal and emetic syndromes, as well as severe opportunistic infections. Its persistence in diverse environments and intrinsic resistance mechanisms highlight the urgent need for effective preventive strategies, particularly in the absence of licensed vaccines. In this study, an integrated computational and immunoinformatics approach was employed to identify conserved antigenic targets and design a multi-epitope vaccine candidate against Bacillus cereus. Pan-genome analysis enabled the identification of conserved antigenic proteins, followed by epitope prediction and construction of a multi-epitope vaccine. The designed construct exhibited strong antigenicity, stability, and solubility, with 97.3% of residues located in the favored regions of the Ramachandran plot and a ProSA-web Z-score within the range of native proteins. Molecular docking analysis demonstrated a favorable binding affinity for Toll-like receptor 2 (TLR2), while molecular dynamics simulations confirmed the structural stability of the complex. Codon optimization enhanced expression potential by reducing the GC content from 70.10% to 56.80%. Immune simulations predicted robust IgG responses, Th1-skewed cytokine production, and the development of immunological memory. Overall, the designed multi-epitope vaccine demonstrates strong in silico immunogenic potential, broad global population coverage (96.98%), and favorable expression feasibility. These findings support the proposed construct as a promising vaccine candidate; however, experimental validation through in vitro and in vivo studies is required to confirm its efficacy.