Oral Delivery of Lactococcus lactis Expressing Full-Length S Protein via Alginate–Chitosan Capsules Induces Immune Protection Against PEDV Infection in Mice

<b>Background/Objectives</b>: Porcine epidemic diarrhea (PED) is a highly contagious enteric infectious disease that causes severe morbidity and mortality in piglets, posing significant economic losses to the swine industry worldwide. Oral vaccines based on <i>Lactococcus lactis</i> offer a promising approach due to their safety and genetic manipulability. This study aims to develop and evaluate an oral <i>L. lactis</i>-based vaccine expressing the full-length PEDV S protein. <b>Methods</b>: A recombinant <i>L. lactis</i> strain expressing the PEDV S protein was constructed and encapsulated in alginate-chitosan microcapsules. Vaccine stability was tested in simulated digestive fluids, and mice were orally immunized. Immune responses were evaluated by measuring specific antibodies, cytokines, and lymphocyte proliferation. <b>Results</b>: The recombinant <i>L. lactis</i> NZ3900/pNZ8149-S strain successfully expressed the full-length PEDV S protein and maintained stable plasmid inheritance. Oral immunization in mice induced detectable PEDV-specific immune responses. Both encapsulated and non-encapsulated vaccines stimulated the production of IgG and sIgA antibodies, as well as cytokines associated with Th1 and Th2 responses. Notably, encapsulation with alginate-chitosan significantly enhanced bacterial survival in digestive conditions and further amplified immune responses, including higher antibody titers, elevated levels of IFN-γ, IL-4, and IL-10, and greater lymphocyte proliferation, indicating improved immune memory. <b>Conclusions</b>: The oral <i>L. lactis</i> NZ3900/pNZ8149-S vaccine expressing the PEDV S protein effectively induced systemic and mucosal immunity in mice. Encapsulation with alginate-chitosan further enhanced its immunogenicity and stability in gastrointestinal conditions. These results suggest that both the engineered <i>L. lactis</i> strain and the encapsulation strategy contribute to the development of a promising oral vaccine platform for controlling PEDV in swine populations.