A pangenome-based strategy for designing a multi-epitope vaccine against non-toxin antigens of necrotic enteritis-associated Clostridium perfringens.

Avian necrotic enteritis (NE), caused by Clostridium perfringens, poses a significant threat to the global poultry industry, exacerbated by rising antibiotic resistance. In this study, we applied a pangenome-guided reverse vaccinology approach to design a multi-epitope chimeric vaccine candidate. From the analysis of 45 genomes, our filtering pipeline identified three conserved and functionally synergistic target proteins: the type IV pilus assembly protein PilO and the prepilin peptidase-dependent protein A, both necessary for bacterial adhesion, and the regulatory protein MsrR, which is crucial for cell-wall integrity. High-affinity B-cell epitopes derived from these three proteins were assembled into an optimized chimeric construct. Structural modeling and molecular docking with Gallus gallus Toll-like receptors (TLRs) indicated strong binding affinity, while physicochemical analyses predicted high stability and antigenicity. We also propose an experimental validation plan encompassing recombinant production, formulation timeline, and in vivo endpoints in chickens, aligned with protein-based MEVs that have demonstrated efficacy in bacterial models. This work presents a rationally designed vaccine candidate that simultaneously targets the pathogen's offensive (adhesion) and defensive (cell integrity) mechanisms, offering a robust computational framework to accelerate the development of antibiotic alternatives in poultry production.