Induction of immune responses in mice after intragastric administration of Lactobacillus casei producing porcine parvovirus VP2 protein

Abstract

Lactobacillus casei ATCC 393 was selected as an antigen delivery vehicle for mucosal immunization against porcine parvovirus (PPV) infection. A 64-kDa fragment of PPV major protective antigen VP2 protein was used as the parvovirus antigen model. A recombinant Lactobacillus expressing VP2 protein was constructed with plasmid pPG611.1, where expression and localization of the VP2 protein from recombinant Lc393-rPPV-VP2 was detected via sodium dodecyl sulfate-polyacrylamide gel electrophoresis, Western blotting, and immunofluorescence. Both local mucosal and systemic immune responses against PPV were induced in BALB/c mice immunized orally with the recombinant Lactobacillus expressing VP2 protein. The induced antibodies demonstrated neutralizing effects on PPV infection. These data indicated that the use of recombinant lactobacilli could be a valuable strategy for future vaccine development of PPV.

FIG. 1.

Map of pPG611.1-VP2, including the Cm resistance determinant, repA and repC replication elements, signal peptide (SP) obtained from the lactococcal Usp45 protein containing the xylose operon promoter, VP2 gene obtained from PPV genome DNA by PCR, and the cell wall anchor motif obtained from Streptococcus pyogenes M6 protein.

FIG. 2.

The bile tolerance of L. casei 393, from using the plate method to enumerate the amounts of lactobacilli that survived in different concentrations of bile. Results were mean values of amounts of bacteria ± standard errors (error bars) of the means.

FIG. 3.

Expression of VP2 protein in rLc393-rPPV-VP2. Total cell lysates were analyzed by SDS-PAGE and Western blotting with specific antiserum. (A) Coomassie blue gel staining shows the expression of a 74-kDa fusion protein in lysates from rLc393-rPPV-VP2 induced by xylose (lanes 2 and 3) but not by glucose (lane 4), or wild-type L. casei 393 was grown in xylose (lane 5). Lane 1, molecular mass marker. (B) An immunoreactive band was detected (lane 1) in a position similar to that observed via SDS-PAGE, as shown in panel A. No immunoreactive bands were observed for cell lysates from recombinant cells induced by glucose (lane 2).

FIG. 4.

Localization analysis of VP2 expression from Lc393-rPPV-VP2 by Western blotting. Lane 1, total lysates of xylose-inducible cells expressing VP2 fusion; lane 2, supernatant of the same cells treated with lysozyme; lane 3, supernatant of cells treated with protease, followed by lysozyme; lane 4, supernatant of cells treated with only protease.

FIG. 5.

The immunofluorescence reaction of VP2 protein on the cell surface of recombinant Lc393-rPPV-VP2. (A) When rLc393-rPPV-VP2 was grown in MRS medium containing glucose, no fluorescence appeared on the cell surfaces, and the bacteria were dyed red by Evans blue. (B) When rLc393-rPPV-VP2 was induced by xylose, there was a green-yellow fluorescence reaction on the surface of the bacteria. (C) When wild-type L. casei 393 was induced by xylose, the result of immunofluorescence was negative, and the bacteria were dyed red by Evans blue.

FIG. 6.

Expression and identification of VP2 protein in rLc393-rPPV-VP2 subcultured for 70 generations, without Cm^r selection. Lane 1, molecular mass marker. Lane 3, Coomassie blue gel staining shows that a 74-kDa fusion protein was expressed from rLc393-rPPV-VP2 subcultured for 70 generations, without Cm selection, induced by xylose but not glucose, in a position similar to that observed via SDS-PAGE, as shown in Fig. 3A (lane 2). Lane 4, an immunoreactive band of protein expressed by rLc393-rPPV-VP2 subcultured for 70 generations was detected in the same position as that in lane 3. No immunoreactive bands were observed in the cell lysates from the recombinant cells induced by glucose (lane 5).

FIG. 7.

Anti-VP2-specific IgA levels in intestinal lavage fluid after intragastric immunization. The mice received three consecutive doses of 10⁹ Lc393-rPPV-VP2 bacilli, three times at 2-week intervals. Control mice received 10⁹ Lc393-pPG611.1 bacilli, while the negative control group received PBS. Intestinal lavage fluids collected on days 18, 32, and 46 after the first immunization were analyzed via ELISA by using PPV as the coating antigen. Bars represent the IgA titers ± standard errors of the means in each group.

FIG. 8.

Anti-PPV-VP2 serum IgG response induced after intragastric immunization with rLc393-rPPV-VP2. Sera from groups of mice immunized orally with 10⁹ rLc393-rPPV-VP2 and equivalent doses of Lc393-pPG611.1 and negative control sera from mice that received PBS were analyzed for the presence of anti-PPV-VP2-specific IgG by ELISA, using the PPV as the coating antigen. Bars represent the IgG titers ± standard errors of the means in each group.

FIG. 9.

Inhibition of viral plaque formation by (A) intestinal lavage fluids and (B) sera prepared from mice immunized with rLc393-rPPV-VP2. The maximum reduction in the number of plaques, expressed as a percentage of plaques obtained for the negative control samples, by using (A) intestinal lavage fluids or (B) sera collected from mice fed with Lc393-rPPV-VP2, was 31.2% ± 1.09% or 80.5% ± 1.13%, respectively. Results are mean values and standard errors (error bars) of triplicates.