Protective immune response to foot-and-mouth disease virus with VP1 expressed in transgenic plants

Abstract

It has been reported recently that genes encoding antigens of bacterial and viral pathogens can be expressed in plants in a form in which they retain native immunogenic properties. The structural protein VP1 of foot-and-mouth disease virus (FMDV), which has frequently been shown to contain critical epitopes, has been expressed in different vectors and shown to induce virus-neutralizing antibodies and protection in experimental and natural hosts. Here we report the production of transformed plants (Arabidopsis thaliana) expressing VP1. Mice immunized with leaf plant extracts elicited specific antibody responses to synthetic peptides representing amino acid residues 135 to 160 of VP1, to VP1 itself, and to intact FMDV particles. Additionally, all of the immunized mice were protected against challenge with virulent FMDV. To our knowledge, this is the first study showing protection against a viral disease by immunization with an antigen expressed in a transgenic plant.

FIG. 1

Detection of the VP1 gene in transgenic plants by PCR. Plant DNA was isolated from cell extracts, and PCR was performed with a pair of primers designed to amplify the region between positions 369 and 490 of the VP1 gene. Lanes: A, nontemplate; B and E, DNA from pRok-transformed plants; C, D, and F, DNA from pRok.VP1-transformed plants; G, pRok.VP1 plasmid.

FIG. 2

Detection of VP1 in transgenic plants by ELISA. Ninety-six-well Immulon 2 ELISA plates (Dynatech) were coated with a rabbit anti-p135-160 antiserum in carbonate buffer (pH 9.6) for 12 h at 4°C. The plates were then washed three times with PBST and blocked with 3% horse serum in PBST for 1 h at 37°C (all subsequent steps were performed with this buffer). Then, a fourfold dilution of extracts from the plants to be tested were added and incubated for 1 h at 37°C. The plates were washed, and a pool of mouse anti-p135-160 antisera was added. The plates were then washed three times and incubated for 1 h at 37°C with peroxidase-labeled rabbit anti-mouse immunoglobulin antibodies (Dakkopats). After three washes, the reaction was developed by the addition of O-phenylenediamine–H₂O₂ in citrate buffer (pH 5) and read 10 min later at 490 nm in an MR 500 Microplate Reader (Dynatech). Results are presented as rough optical density (OD) readings. Positive control is an extract of a plant transformed with pRok with the addition of p135-160 (10μg/ml).

FIG. 3

ELISA detection of antibodies to p135-160 (A) and anti-FMDV particles (B) in mice immunized with transformed plants. Briefly, the assay was performed exactly as described previously for detection of VP1 in transgenic plants regarding the capture of antigen to the plate and the blocking, washing, developing, and reading steps (20). Antigen was added after the capture antibody (p135-160 at a concentration of 10 μg/ml and purified inactivated FMDV at a concentration of 1 μg/ml) and incubated for 1 h at 37°C. Sera were tested in a fourfold dilution series in blocking buffer. Serum titers are expressed as the log of the reciprocal of the highest serum dilution which presents optical density (OD) readings above the mean OD readings plus 5 standard deviations of sera from five animals immunized with plants transformed with pRok.

FIG. 4

Anti-VP1 antibodies detected by Western blotting. FMDV (produced, inactivated, and purified as described by Berinstein et al. [3]) was resuspended in sample buffer (50 mM Tris [pH 7.5], 1 mM phenylmethylsulfonyl fluoride, 4 M urea, 1% sodium dodecyl sulfate [SDS], 2 mM dithiothreitol, and 2% 2-β-mercaptoethanol), boiled 3 min, subjected to SDS–12.5% polyacrylamide gel electrophoresis, and blotted to an Immobilon P (Millipore) membrane. The membrane was blocked overnight with PBST containing 3% skim milk (all subsequent steps were performed with this buffer) and incubated with the corresponding mouse sera (diluted 1/50) for 2 h at 37°C. The membrane was washed and incubated with an alkaline phosphatase-labeled anti-mouse immunoglobulin rabbit antiserum (Dakkopats) for 1 h at 37°C and then washed three more times, and the reaction was developed by the addition of the substrate nitroblue tetrazolium–4-chloro-3-indolylphosphate. Sera from mice immunized with plant extracts were used for the reaction. Lanes A through C correspond to anti-p135-160 antiserum, a pool of sera from mice immunized with pRok-transformed plants, and a pool of sera from mice immunized with pRok.VP1-transformed plants, respectively.