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

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. Six ASFV antigenic proteins (K78R, A104R, E120R, E183L, D117L, and H171R) were fused with the WCFS1 surface anchor LP3065 (LPxTG motif) to generate recombinant 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 10 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. 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 CD3CD4 T cells in mice immunized via the I.N. and I.G. and CD3CD4 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. 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.