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

Abstract

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.

Fig. 1

PEDV S protein immunodominant region S1 expressed in PG1-S1. Western blot analysis showed an immunoreactive band of 75-kDa fusion protein in lysates of PG1-S1 (lane 1), but not in that of PG1 (lane 2)

Fig. 2

The immunofluorescence analysis of S1 surface-displayed expression by L. casei. a There was green fluorescence on the surface of PG1-S1, b there was no immunofluorescence reaction on the PG1 cell surface

Fig. 3

Secretory expression of PEDV nucleocapsid (N) in PG2-N. An immunoreactive band of 55-kDa fusion protein in lysates and supernatant (lanes 1 and 3), but not in that of PG2, was shown by Western blot analysis

Fig. 4

IgA and IgG levels of mucosal and serum samples (specific for S1). Samples of nasal (a), feces (b), ophthalmic (c), vaginal lavage fluids (d), intestinal mucus (e), and serum (f) from mice immunized with PG1-S1+ PG1-N, PG2-S1+ PG2-N, PG1-S1, PG2-S1, PG1, and PG2. Values are means (±standard deviation) of three replicates per treatment in one experiment, which was repeated twice. *P < 0.001

Fig. 5

IgA and IgG levels of mucosal and serum samples (specific for N). Samples of nasal (a), feces (b), ophthalmic (c), vaginal lavage fluids (d), intestinal mucus (e), and serum (f) from mice immunized with PG1-S1+ PG1-N, PG2-S1+ PG2-N, PG1-S1, PG2-S1, PG1, and PG2. Values are means (±standard deviation) of three replicates per treatment in one experiment, which was repeated twice. *P < 0.001

Fig. 6

Antibody neutralizing activity against PEDV in vitro. a Serum and b intestinal mucus prepared from mice immunized with PG1-S1+ PG1-N, PG2-S1+ PG2-N, PG1, and PG2. Results are mean values and standard errors of triplicates

Fig. 7

Cytokine responses of spleen cells from mice immunized with PG1-S1+ PG1-N, PG2-S1+ PG2-N, PG1, and PG2. Bars represent the means (±standard deviation) of three replicates per treatment in one experiment, which was repeated twice. *P < 0.001