Evaluation of an M Cell–Targeted FedA Fusion Protein as an Oral Vaccine Candidate for Post-Weaning Diarrhea

Post-weaning diarrhea (PWD), primarily caused by enterotoxigenic Escherichia coli (ETEC), remains a major constraint on swine production worldwide due to its impact on piglet health, growth performance, and farm economics. Fimbrial adhesins F4 and F18 play central roles in ETEC pathogenesis by mediating intestinal colonization. However, effective vaccines against F18⁺ ETEC are still lacking, largely because of the weak immunogenicity and structural instability of F18 fimbriae. The major fimbrial subunit FedA has emerged as a promising antigen candidate, but successful oral vaccination requires efficient antigen delivery to mucosal immune sites. In this study, we aimed to enhance FedA immunogenicity by fusing it to PEP, an M cell–targeting peptide designed to improve antigen uptake in the intestinal mucosa. The fedA gene was cloned into the pET22b-pep vector to generate the pET22b-pep-fedA construct and expressed in E. coli BL21(DE3). The recombinant PEP–FedA fusion protein was purified to greater than 90% homogeneity using immobilized metal affinity chromatography. Oral immunization in mice elicited robust mucosal and systemic immune responses, as evidenced by significantly increased fecal secretory IgA and serum IgG levels compared with FedA alone. Moreover, fecal IgA specifically recognized F18⁺ ETEC without cross-reactivity to F4⁺ ETEC. These findings demonstrate that PEP-mediated M cell targeting substantially enhances FedA immunogenicity and highlight the potential of this strategy for developing effective oral vaccines against PWD.