A Multi-Epitope FimH-CsgA-OmpA-Based Vaccine Induces Broad Anti-Adhesion Activity and Protects Chickens Against Avian Pathogenic Escherichia coli O2 Challenge

Avian pathogenic Escherichia coli (APEC) causes substantial economic losses in the poultry industry, and vaccines capable of providing protection across diverse APEC strains remain needed. In this study, a FimH-CsgA-OmpA multiepitope fusion antigen (MEFA) was constructed and evaluated for immunogenicity, in vitro anti-adhesion activity, and protective efficacy against APEC challenge. Conserved immunodominant B-cell epitopes (BCEs) from CsgA and OmpA were incorporated into surface-exposed regions of a FimH scaffold. Structural modeling predicted that the inserted epitopes remained surface accessible without major disruption of the overall FimH-like fold, and immunoblotting confirmed that the recombinant fusion protein retained immunoreactivity with antibodies against FimH, CsgA, and OmpA. In mice, immunization with the fusion protein, either alone or formulated with Freund’s adjuvant, elicited FimH-, CsgA-, and OmpA-specific IgG responses and enhanced TNF-α and IL-6 production after antigen restimulation of splenocytes. Immune sera significantly inhibited the adhesion of APEC serotypes O1, O2, O8, O78, and O141 to DF-1 cells. In chickens, immunization induced FimH-, CsgA-, and OmpA-specific IgY responses. Following challenge with APEC-XM (O2), vaccinated chickens showed improved survival, reduced gross lesions, and a lower frequency of challenge-strain recovery from systemic tissues than PBS-treated controls. These findings demonstrate broad in vitro anti-adhesion activity across five APEC serotypes and protection against the tested APEC O2 challenge. Heterologous in vivo protection remains to be established.