Preclinical Evaluation of a Phage T4‐Based Multi‐Epitope Nanoparticle Vaccine Against Porcine Epidemic Diarrhea Virus

Porcine epidemic diarrhea virus (PEDV) continues to threaten the global swine industry, and currently available vaccines often provide incomplete protection against emerging virulent strains. In this study, we developed a phage T4-based multi-epitope nanoparticle vaccine targeting PEDV. B and T cell epitopes derived from the PEDV spike (S) protein were identified through immunoinformatic analyses and assembled into a tandem epitope construct (Pep) using flexible linkers. In silico analyses predicted favorable antigenic properties of Pep and a stable interaction with Toll-like receptor 3 (TLR3). The recombinant Pep protein was expressed in Escherichia coli and subsequently displayed on T4 phage to generate the T4-Pep vaccine. Characterization revealed that each phage carried approximately 800 copies of Pep. In mice, immunization with T4-Pep induced significantly higher PEDV-specific IgG levels and virus-neutralizing antibody titers than immunization with the Pep. Following PEDV challenge, T4-Pep-immunized mice showed improved protection and reduced intestinal damage, as confirmed by histopathological analysis. Together, these findings highlight the potential of phage T4 as an efficient and immunogenic platform for delivering viral epitopes, offering a promising strategy for the development of vaccines against PEDV and related pathogens.