Immunogenic Responses Elicited by a Pool of Recombinant Lactiplantibacillus plantarum NC8 Strains Surface-Displaying Diverse African Swine Fever Antigens Administered via Different Immunization Routes in a Mouse Model

<b>Background:</b> African swine fever (ASF) is a highly contagious and often deadly disease that poses a major threat to swine production worldwide. The lack of a commercially available vaccine underscores the critical need for innovative immunization strategies to combat ASF. <b>Methods:</b> Six ASFV antigenic proteins (K78R, A104R, E120R, E183L, D117L, and H171R) were fused with the <i>Lactiplantibacillus plantarum</i> WCFS1 surface anchor LP3065 (LPxTG motif) to generate recombinant <i>Lactiplantibacillus plantarum</i> NC8 (rNC8) strains. The surface expression was confirmed using immunofluorescence and Western blotting assays. Additionally, the dendritic cell-targeting peptides (DCpep) were co-expressed with each antigen protein. Mice were immunized at a dosage of 10⁹ colony-forming units (CFU) per strain per mouse via intragastric (I.G.), intranasal (I.N.), and intravenous (I.V.) routes. The bacterial mixture was heat-inactivated by boiling for 15 min to destroy viable cells while preserving antigenic structures. I.V. administration caused no hypersensitivity, confirming the method's safety and effectiveness. <b>Results:</b> Following I.G. administration, rNC8-E120R, rNC8-E183L, rNC8-K78R, and rNC8-A104R induced significant levels of secretory immunoglobulin A (sIgA) in fecal samples, whereas rNC8-H171R and rNC8-D117L failed to induce a comparable response. Meanwhile, rNC8-D117L, rNC8-K78R, and rNC8-A104R also elicited significant levels of sIgA in bronchoalveolar lavage fluid (BALF). Following I.N. immunization, rNC8-E120R, rNC8-K78R, and rNC8-A104R significantly increased sIgA levels in both fecal and BALF immunization. In contrast, I.V. immunization with heat-inactivated rNC8-K78R and rNC8-A104R induced robust serum IgG titers, whereas the remaining antigens elicited minimal or insignificant responses. Flow cytometry analysis revealed expanded CD3⁺CD4⁺ T cells in mice immunized via the I.N. and I.G. and CD3⁺CD4⁺ T cells only in those immunized via the I.N. route. Th1 responses were also significant in the sera of mice immunized via the I.G. and I.N. routes. <b>Conclusions:</b> The rNC8 multiple-antigen cocktail elicited strong systemic and mucosal immune responses, providing a solid foundation for the development of a probiotic-based vaccine against ASF.