Immunogenicity of a neutralizing epitope from porcine epidemic diarrhea virus: M cell targeting ligand fusion protein expressed in transgenic rice calli

Abstract

To increase immune responses of plant-based vaccines in intestine mucosal immune systems, a synthetic neutralizing epitope (sCOE) gene of porcine epidemic diarrhea virus (PEDV) was fused with M cell-targeting ligand (Co1) and introduced into a plant expression vector under the control of rice amylase 3D promoter. The sCOE-Co1 fusion gene was introduced into rice calli via the particle bombardment-mediated transformation method. The stable integration and transcriptional expression of the sCOE-Co1 fusion gene was confirmed by genomic DNA PCR amplification and Northern blot analysis, respectively. The expression of the COE-Co1 fusion protein was confirmed by immunoblot analysis. The highest expression level of the COE-Co1 fusion protein reached 0.083 % of the total soluble protein according to quantitative densitometry of Western blot analysis. Mice immunized with transgenic rice calli protein extracts induced significant serum IgG and fecal IgA antibody levels against purified bacterial COE. The systemic and mucosal immune responses were confirmed by measuring COE-specific IgG and IgA antibody-secreting cells in the lymphocytes extracted from the spleen and COE-specific IgA antibody-secreting cells in the Peyer's patches from immunized mice. These results indicated that oral immunization of plant-produced COE-Co1 fusion protein could elicit efficient systemic and mucosal immune responses against the COE antigen. Key message Neutralizing epitope from porcine epidemic diarrhea virus-M cell targeting ligand fusion protein was produced in transgenic rice calli and elicited systemic and mucosal immune responses by oral administration in mice.

Fig. 1

Rice expression vector pMYV733 and genomic DNA PCR analysis. The synthetic COE gene (sCOE) was fused with the M cell-target peptide ligand, Co1, introduced into a plant expression vector under the control of a signal peptide (3Dsp), promoter (RAmy3D-p), and untranslated region (3′-UTR) of a rice amylase 3D gene, and transformed into rice calli. The hygromycin phosphotransferase (HPT) gene was used as a selection marker under the control of Cauliflower mosaic virus 35S promoter (35S-p) and terminator (35S-t). LB and RB are left and right border of T-DNA, respectively (a). Genomic DNA PCR amplification was conducted to amplify the sCOE–Co1 fusion gene in genomic DNA of wild-type and putative transgenic rice calli (b). Lane M is a 100 base-pair DNA ladder (ELPIS Biotech, Seoul, Korea). Lane PC is PCR products amplified from plasmid pMYV733 used as a positive control. Lane NC is a wild type used as a negative control. Lanes 1–14 are independent putative transgenic lines

Fig. 2

Northern blot analysis to determine the expression of the sCOE–Co1 fusion gene. Total RNA (30 μg) prepared from wild type and transformed rice calli were hybridized with ³²P-labeled sCOE–Co1 fusion gene probe. Lane NC is total RNA from wild-type rice calli. Lanes 1–12 are total RNA from transgenic rice calli

Fig. 3

Detection and N-glycosylation analysis of COE–Co1 fusion protein. Western blot analysis of COE–Co1 fusion protein in transgenic rice calli line #9 (a) and line #11 (b) with anti-COE antibody as the primary antibody. Lane M is prestained with SDS-PAGE standards (ELPIS Biotech). Lanes 10, 20 and 40 contain predetermined amounts (10, 20 and 40 ng) of COE protein purified in E. coli. Lane NC is protein extracts from wild-type rice calli. Lanes 1–5 are protein extracts from transgenic calli on 1–5 days after expression induction. Analysis of N-glycosylation of the COE–Co1 fusion protein (c). Lane N is protein extracts untreated with enzymes. Lanes F, F1 and H are protein extracts treated with enzymes PNGase F, Endo F1 and Endo H, respectively

Fig. 4

Measurement of protein expression level using quantitative densitometry. The expression levels of COE–Co1 fusion protein in transgenic rice calli line #9 (a) and line #11 (b) were evaluated under a sugar starvation condition on a time course and are presented as  %TSP

Fig. 5

Mouse feeding strategy and detection of COE-specific antibody. Protein extracts of transgenic rice calli expressing COE–Co1 fusion protein and wild type were concentrated by precipitating with ammonium sulfate for mouse oral immunization. Each mouse in the experimental group (n = 5) was gavaged with 2 ml of PBS buffer containing 40 μg of COE–Co1 fusion protein in total of 10 time feeds with an interval of 1 week (a). Blood and fecal samples were collected 10 days after 4, 6, 8, and 10-week feedings. The mouse sera and fecal samples were diluted 100-fold and 4-fold, respectively, and used to measure serum IgG (b) and fecal IgA (c) antibody levels against bacterial COE. The representative results show the average OD results from five immunized mice. Error bars represent standard deviation

Fig. 6

Measurement of COE-specific IgG and IgA antibody-secreting cells (ASCs) in immunized mice. The COE-specific ASCs per 10⁵ SPLs (a) or per 10⁶ PPLs (b) were determined using ELISPOT. SPL and PPL represent lymphocytes from the spleen and Peyer’s patches, respectively. Error bars represent standard deviations. *p < 0.05; **p < 0.01; ***p < 0.001 indicate significant differences between the values compared