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

Background: African swine fever (ASF) is a deadly, highly contagious disease with major global pig industry impacts, highlighting the urgent need for an effective vaccine. Methods: In this study, six antigenic proteins (K78R, A104R, E120R, E183L, D117L, and H171R) of ASFV were fused with the LPxTG surface anchoring polypeptide i.e., LP3065 of Lactobacillus plantarum WCFS1, resulting in the generation of the recombinant Lactiplantibacillus plantarum NC8 (rNC8) strains. The surface expression of ASFV antigenic proteins was confirmed by immunofluorescence and Western blotting assays. The peptides (DCpep) targeting dendritic cells (DCs) were also fused with LP3065 to bind to mouse DCs. The recombinant strains were administered via intragastric (I.G.), intranasal (I.N.), and intravenous (I.V.) routes in mice at 109 CFU/mouse. Results: In the I.V. immunized mice, the higher IgG titers were induced by rNC8-K78R and rNC8-A104R, while rNC8-E120R, rNC8-E183L, rNC8-D117L, rNC8-H171R induced low IgG titers, and rNC8-E120R remained insignificant. In the I.G. immunized mice, rNC8-E120R, rNC8-E183, rNC8-K78R, and rNC8-A104R induced higher sIgA levels, while rNC8-H171R and rNC8-D117L remained not significant in the feces. In the bronchioalveolar lavage fluid (BALF) of the I.G. immunized mice, rNC8-D117L, rNC8-K78R, and rNC8-A104R induced higher levels of sIgA, while rNC8-E120R, rNC8-E183L, and rNC8-H171R were insignificant. In the feces of the I.N. immunized mice, rNC8-E120R, rNC8-K78R, and rNC8-A104R induced higher levels of sIgA, while rNC8-D117L remained insignificant. In the BALF of the I.N. immunized mice, rNC8-E120R, rNC8-K78R, and rNC8-A104R induced higher sIgA levels, while rNC8-H171R remained insignificant. Th1 (IFN-γ and IL-2) and Th2 (IL-4 and IL-10) responses were significant in the sera of the I.G. and I.N. immunized mice, whereas IL-10 levels remained insignificant in both groups. Flow cytometry showed a significant increase in CD3+CD4+ T cells in both the I.N. and I.G. groups, while CD3+CD8+ T cells were elevated only in the I.N. group compared with the control groups. Conclusion: This multiple antigen cocktail represents a probiotic-based ASF vaccine candidate that warrants further evaluation.