Surface-displayed porcine epidemic diarrhea viral (PEDV) antigens on lactic acid bacteria

Abstract

In this report, for surface display of viral antigen on lactobacilli, we have developed a surface antigen display system using the poly-gamma-glutamate synthetase A protein (pgsA) of Bacillus subtilis as an anchoring matrix. Recombinant fusion proteins comprised of pgsA and neucleocapsid protein of PEDV were stably expressed in Lactobacillus casei. Surface location of fusion protein was verified by ELISA, immunofluoresence microscopy. Oral and intranasal inoculations of recombinant L. casei into pregnant sow and mice resulted in high levels of serum immunoglobuline G (IgG) and mucosal IgA, as demonstrated by rnELISA(recombinant N protein ELISA) using recombinant N protein. Absorbance of IgG in pregnant sow sera highly increased duration of the experiment. More importantly, the level of IgA in colostrum were increased significantly higher than that of IgG. The IgG levels of the piglets were increased after suckling colostrum secreted from sows previously inoculated recombinant L. casei. These results indicate that mucosal immunization with recombinant L. casei expressing PEDV N protein (neucleoprotein of PEDV) on its surface elicited high levels of mucosal IgA and circulation IgG immune responses against the antigen N of PEDV.

Figure 1

Detection of pHCE1LB-pgsA-PEDN expressed on Lactobacillus casei by Western Blot with anti-PEDN (left) and anti-pgsA (right) polyclonal antibodies. Lane 1 shows whole-cell lysates of wild-type (parental vector); lane 2 shows the recombinant L. casei. Protein bands of ∼90 kDa, corresponding to the expected size of pgsA-PEDN were detected.

Figure 2

Confirmation of surface-displayed PEDN on L. casei by ELISA.

Figure 3

Representative immunofliuoresence images of control cells harboring pHCE1LB-pgsA and recombinant L. casei cells expressing pgsA-PEDN. Bright-field images are shown in the left.

Figure 4

Antigenicity of surface displayed PED Ag in lactic acid bacteria display. Anti-PEDN mouse serum IgG absorbances following Intranasal, oral immunization with recombinant L. casei. Statistical comparisons between groups were made by the Mann–Whitney U-test.

Figure 5

Anti-PEDVN mucosal IgA antibody responses. Intestinal and bronchoalveoar lavage fluids, harvested from mice sacrificed 56 or 70 days postimmunization, were analyzed by rnELISA. Absorbance 450 nm of samples from animals immunized orally (A) or intranasally (B) are shown. Fluids from control animals are shown as black bars. The error bars represent standard deviations.

Figure 6

Anti-PEDN pregnant sow serum IgG absorbances following oral immunization with recombinant L. casei. Coating Ag: 100 ng/well; serum: 1:100; conjugation: 1:3000.

Figure 7

Detection of PEDN IgA and IgG levels in colostrum secreted from sow previously Inoculated recombinant L. casei (A). Coating Ag: 100 ng/well for PEDN colostrums: 1:100; conjugation: 1:3000. Colostrum from control animals are shown as black bars. The error bars represent standard deviations. PEDN IgG antibody in piglets serum on day 10 after sucking colostrums secreted from gilts previously inoculated recombinant PEDN L. casei (B). Coating Ag: 100 ng/well; serum: 1:100; conjugation: 1:3000, lane 1, negative control; lanes 2–7. The piglet sera randomly selected from offerings of the five immunized pregnant sows.

Figure 8

inhibition of viral plaque formation by (A) serum IgG prepared from sow fed with recombinant L. casei and piglets serum on day 10 after sucking colostrums and (B) colostrum IgA. (A) The number of plaque formed using purified IgG treatment. Control: IgG from sow sera unimmunized with recombinant L. casei; sow sera: IgG from sow sera on day 70 after immunization; piglets serum: IgG from piglet sera on day 10 after sucking colostrums; P.C: positive control IgG. (B) The number of plaque formed using purified IgA treatment. Control: IgA from sows’ colostrum unimmunized with recombinant L. casei; colostrum: IgA from immunized sows’ colostrum immunized with recombinant L. casei; P.C: positive control IgA. The assays were performed in duplicate, and the graphs are representative of two independent experiments. The error bars represent standard deviations.