Comparison of the immune responses induced by oral immunization of mice with Lactobacillus casei-expressing porcine parvovirus VP2 and VP2 fused to Escherichia coli heat-labile enterotoxin B subunit protein

Abstract

The major structural protein VP2 of porcine parvovirus (PPV) was used as the model parvovirus antigen, which has been expressed in Lactobacillus casei fusing with Escherichia coli heat-labile enterotoxin B subunit (LTB) as mucosal adjuvant. The VP2-LTB DNA fragment was cloned into vector pPG611 or pPG612 to generated inducible surface-displayed and secretion expression systems based on xylose promoter, designated as rLc:pPG611-VP2-LTB (recombinant L. casei) and rLc:pPG612-VP2-LTB, respectively. Expression of the fusion protein was verified by SDS-PAGE, Western blot immunofluorescence and electron microscopy. It was observed that the level of IgG or sIgA from mice orally immunized with VP2-LTB was higher than that from mice received VP2 and negative control, which demonstrated significantly statistically different. Especially, the titer of IgG or sIgA in mice immunized with rLc:pPG612-VP2-LTB is the highest in this study. In summary, LTB as mucosal adjuvant was able to effectively facilitate induction of mucosal and systemic immunity by L. casei-expressing VP2 fusion protein.

Fig. 1

The scheme of recombinant vectors expressing VP2-LTB protein. The LTB fragment was cleaved with BamHI and XhoI restriction endonuclease and inserted into the corresponding sites of pPG611-VP2 and pPG612-VP2, respectively, giving rise to pPG611-VP2-LTB and pPG612-VP2-LTB.

Fig. 2

Secretion expression of VP2-LTB protein in rLc:pPG612-VP2-LTB. (a) Coomassie blue gel staining shows the expression of a 75,000 kDa fusion protein in lysates of rLc:pPG612-VP2-LTB (lane 2), but not in that of rLc:pPG612 (lane 3); lane 1, molecular mass marker. (b) Western blot analysis of VP2-LTB expression in recombinant strain. An immunoreactive band was observed (lane 1) in a similar position as observed in the SDS-PAGE shown in the panel (a), but not in that of rLc:pPG612 (lane 2). (c) 75,000 kDa fusion protein in supernatant of rLc:pPG612-VP2-LTB (lane 2), but not in that of rLc:pPG612 (lane 3); lane 1, molecular mass marker. (d) An immunoreactive band was observed (lane 2) in a similar position as observed in the SDS-PAGE shown in the panel (c), but not in that of rLc:pPG612 (lane 1).

Fig. 3

Expression of VP2-LTB protein in rLc:pPG611-VP2-LTB. (a) SDS-PAGE shows the expression of a 80,000 kDa fusion protein in lysates of rLc:pPG611-VP2-LTB (lane 3), but not in that of rLc:pPG611 (lane 2); lane 1, molecular mass marker. (b) Western blot analysis showed that an immunoreactive band in a similar position as observed in the SDS-PAGE in the panel (a).

Fig. 4

The immunofluorescence analysis of VP2-LTB surface-displayed expression by Lactobacillus casei. The rLc:pPG611-VP2-LTB and rLc:pPG611 was induced in basal MRS supplemented with xylose, the bacteria pellets were incubated with mouse anti-VP2 serum, FITC conjugated goat anti-mouse IgG. (a) There was green fluorescence on the surface of rLc:pPG611-VP2-LTB. (b) There was no immunofluorescence reaction on the cell surface rLc:pPG611.

Fig. 5

Identification of the fusion protein on the cell surface of recombinant L. casei labeled with McAb-colloid gold conjugates via immunoelectron microscopy (4000×). rLc:pPG611-VP2-LTB or rLc:pPG611 was grown in basal MRS supplemented with xylose followed by incubation with McAb-colloid gold conjugates. (a) The gold particles adhered to the cell surface of rLc:pPG611-VP2-LTB; (b) nothing happened to that of rLc:pPG611.

Fig. 6

Titers of mucosal and serum antibody. Feces, vaginal fluids, ophthalmic fluids and sera of mice received recombinant strains expressing VP2-LTB or VP2 and equivalent doses of L. casei harboring empty plasmid pPG611 were analyzed by ELISA, using the PPV as the coating antigen. Values are means (±S.D.) of three replicates per treatment in one experiment, repeated twice. (a) Feces were collected on 0, 7, 14, 21, 28, 35, 42, 49, 56 days and analyzed for the presence of PPV-VP2 specific IgA. (b) Vaginal fluids were obtained on 0, 7, 21, 35, 49 days for the analysis of specific IgA. (c) Ophthalmic fluids were performed as (b). (d) Sera were analyzed for the presence of PPV-VP2 specific IgG.

Fig. 7

IgG neutralization activity against PPV in vitro. Sera were prepared from mice administered with rLc:pPG612-VP2-LTB, rLc:pPG611-VP2-LTB, rLc:pPG612-VP2, rLc:pPG611-VP2 and rLc:pPG611. Results are mean values and standard errors of triplicates.