High-level mucosal and systemic immune responses induced by oral administration with Lactobacillus-expressed porcine epidemic diarrhea virus (PEDV) S1 region combined with Lactobacillus-expressed N protein.

To develop effective mucosal vaccine formulation against porcine epidemic diarrhea virus (PEDV) infection, the DNA fragments encoding spike protein immunodominant region S1 and nucleocapsid N of PEDV were inserted into pPG1 (surface-displayed) or pPG2 (secretory) plasmids followed by electrotransformation into Lactobacillus casei (Lc) to yield four recombinant strains: PG1-S1, PG2-S1, PG1-N, and PG2-N. After intragastric administration, it was observed that live Lc-expressing S1 protein combined with Lc-expressing N protein could elicit much more potent mucosal and systemic immune responses than the former alone (P < 0.001), however slightly inferior to the latter alone (P > 0.05). Furthermore, the surface-displayed mixture (PG1-S1+ PG1-N) revealed stronger immunogenicity than the secretory mixture (PG2-S1+ PG2-N) as well as PEDV-neutralizing potency in vitro (P < 0.001). On 49th day after the last immunization, splenocytes were prepared from mice immunized with surface-displayed mixture, secretory mixture and negative control to be stimulated by purified N and S protein, respectively. The results of ELISA analysis showed that N protein was capable of inducing a higher level of IL-4 (P < 0.001) and IFN-γ (P < 0.001) than S1 protein in the immunized mice. Taken together, Lc-expressed N protein as molecular adjuvant or immunoenhancer was able to effectively facilitate the induction of mucosal and systemic immune responses by Lc-expressing S1 region.