Recombinant porcine rotavirus VP4 and VP4-LTB expressed in Lactobacillus casei induced mucosal and systemic antibody responses in mice

Abstract

Background: Porcine rotavirus infection is a significant cause of morbidity and mortality in the swine industry necessitating the development of effective vaccines for the prevention of infection. Immune responses associated with protection are primarily mucosal in nature and induction of mucosal immunity is important for preventing porcine rotavirus infection.

Results: Lactobacillus casei expressing the major protective antigen VP4 of porcine rotavirus (pPG612.1-VP4) or VP4-LTB (heat-labile toxin B subunit from Echerichia coli) (pPG612.1-VP4-LTB) fusion protein was used to immunize mice orally. The expression of recombinant pPG612.1-VP4 and pPG612.1-VP4-LTB was confirmed by SDS-PAGE and Western blot analysis and surface-displayed expression on L. casei was verified by immunofluorescence. Mice orally immunized with recombinant protein-expressing L. casei produced high levels of serum immunoglobulin G (IgG) and mucosal IgA. The IgA titers from mice immunized with pPG612.1-VP4-LTB were higher than titters from pPG612.1-VP4-immunized mice. The induced antibodies demonstrated neutralizing effects on RV infection.

Conclusion: These results demonstrated that VP4 administered in the context of an L. casei expression system is an effective method for stimulating mucosal immunity and that LTB served to further stimulate mucosal immunity suggesting that this strategy can be adapted for use in pigs.

Figure 1

Expression of VP4 and VP4-LTB in rLc393:pPG612.1-VP4 and pPG612.1-VP4-LTB. Total cell lysates were analysed by SDS-PAGE. Coomassie blue gel staining shows the expression of a 27 KD and 40 KD fusion protein in lysates of rLc393 induced by xylose (Fig. 1A, lane 3 and Fig. 1B, lane 3), but not in basal MRS with glucose (Fig. 1A, lane 2 and Fig. 1B, lane 2).

Figure 2

Western-blotting analysis of VP4 and vp4-LTB expression in recombinant strain. Immunoreactive bands were observed (Fig. 2A, lane 2 and Fig. 3B, lane 2) in the similar position as shown in the SDS-PAGE, however, there were no immunoblots in the same cell lysates induced by glucose (Fig. 2A, lane 3 and Fig. 3B, lane 1).

Figure 3

Immunofluorescence analysis. Wild-type L. casei 393 was induced by xylose, the result of immunofluorescence was negative, and the cells were dyed red by Evans blue (A). When pPG612.1-VP4 and pPG612.1-VP4-LTB were induced by xylose, there were green-yellow fluorescence reaction on the surface of the cells (B, C).

Figure 4

Specifis IgG antibodies in serum. Serum from groups of mice (10 mice every group) immunized orally with pPG612.1-VP4, pPG612.1-VP4-LTB and equivalent dose of pPG612.1 were analyzed for the presence of anti-VP4 specific IgG by ELISA. IgG titers of serum in mice given pPG612.1-VP4 or pPG612.1-VP4-LTB were similar but higher than that of mice given pPG612.1. ** P < 0.01 significant difference between IgG titers of serum in mice given pPG612.1-VP4 and pPG612.1 on day 7, 21 and 35. Results are the IgG titers ± standard errors of the means in each group.

Figure 5

Specific IgA levels in ophthalmic and vaginal wash were detected after oral immunization (10 mice every group administered with different recombinant strains). Specific IgA antibody titers were detectable in the mice immuned with pPG612.1-VP4 and pPG612.1-VP4-LTB after the first administration (Fig. 5A, B). Statistically significant difference (** P < 0.01) was observed in ophthalmic and vaginal wash of mice administered with recombinant strains after seven days. IgA levels elicited by pPG612.1-VP4-LTB were higher than those elicited following pPG612.1-VP4 immunization and the difference is significant statistically (** P < 0.01). Bars represent the IgA titers ± standard errors of the means in each group.

Figure 6

Specific IgA levels in fecal pellets after oral immunization. The mice (10 every group) received three consecutive immunization, three times at 2-week intervals. The control group of mice received the same dose of pPG612.1. Fecal pellets were collected 1, 2, and 7 days after every immunization. Both of the groups immuned with pPG612.1-VP4 or pPG612.1-VP4-LTB produced specific IgA. Statistically significant difference (** P < 0.01) was observed in fecal pellets of mice administered with recombinant strains after one day. The levels of IgA in fecal pellets induced by pPG612.1-VP4 appeared lower than those induced by pPG612.1-VP4-LTB (*P < 0.05,**P < 0.01). Results are the IgA titers ± standard errors of the means in each group.

Figure 7

Neutralization ability of the sera prepared from mice immunized with pPG612.1-VP4 and pPG612.1-VP4-LTB. The maximum reduction of CPE, expressed as a percentage of CPE obtained for the negative control samples, by using sera collected from mice fed with pPG612.1-VP4 or pPG612.1-VP4-LTB, was 50.28% ± 0.83% or 56.06% ± 0.77%, respectively. Statistically significant difference (** P < 0.01) was observed in sera of mice administered with recombinant strains. The neutralizing efficacy of anti-VP4 IgG from mice immunized with pPG612.1-VP4 was lower than antibodies obtained from mice immunized with pPG612.1-VP4-LTB and the difference was significant statistically (* P < 0.05,**P < 0.01). Results are mean values and standard errors (error bars) of triplicates.

Figure 8

Target amplification fragments of VP4 and VP4-LTB fusion gene. Lane 1,5: Blank controls; Lanes 2: Target amplification fragment of VP4 gene; Lanes 3: 2000 bp DNA marker; Lane 4:Target amplification fragment of VP4-LTB fusion gene.