Transmissible gastroenteritis virus (TGEV) is a well-characterized model of mucosal immunity in swine. The S protein of TGEV forms a large trimeric structure protruding from the viral envelope and likely facilitates the attachment to the epithelial cells lining the intestine. It is also the major antigen responsible for the generation of neutralizing IgA antibodies, which are secreted from the epithelia, thereby inhibiting viral attachment. Antibodies can also be secreted in the milk of a sow which has been infected or inoculated, thereby providing passive immunity to suckling piglets. It is our objective to synthesize the S protein in plants and to generate a lactogenic immune response in the sow through feeding of the plant material. We have expressed the S protein and portions thereof in leaf tissue of alfalfa and tobacco using a variety of vector constructs and have tested these tissues in mice and piglets.

Expression of a Swine Viral Epitope as a Fusion Protein in Plants. Andrea Bailey*, W. Yu, T. Tuboly, E. Nagy and Larry Erickson. Plant Agriculture Department - Biotechnology Division, University of Guelph, Guelph, Ontario, Canada N1G 2W1

Swine transmissible gastroenteritis virus (TGEV) can infect pigs of all ages but is most severe on very young piglets, and the high mortality associated with such early infection can result in substantial losses to the pork industry. Current preventative therapy involves orally vaccinating sows with live virus, thereby triggering an immune response that generates neutralizing antibodies in milk and colostrum. These antibodies provide passive immunity to suckling piglets. There is an increasing interest in developing a plant-based TGEV vaccine delivery system. In this work, an epitope has been selected from the TGEV spike (S) protein for expression in transgenic tobacco and alfalfa. The S protein is the dominant protein recognized in the immune response to TGEV, and previous attempts to produce this protein in tobacco have resulted in degraded mRNA and virtually no protein. In response to this, a fusion protein approach was adopted here. Epitope D is a linear, neutralizing epitope from the S protein. It was fused to the 3' end of a glycoprotein (GPX) found only in the pollen of alfalfa, and this chimeric construct was expressed in the leaves of tobacco and alfalfa. Preliminary data indicate a relatively high level of expression, as detected by anti-GPX polyclonal serum. As well, the immunogenicity of this protein will be evaluated by intramuscular injection in mice.

The spike (S) glycoprotein in the envelope of transmissible gastroenteritis virus (TGEV) of swine is believed to be the major antigen to generate neutralizing IgA antibodies to this virus. Expression of the S protein in plants, therefore, would be an economical method to produce an edible vaccine against TGEV. The original S gene, however, failed to be expressed in a detectable amount in plants despite using various promoters or/and adding endoplasmic reticulum-retention tail, SEKDEL, to construct recombinant vectors. Based on the fact that the nucleotide fragments coding for all the epitopes of the antigen are located within the first 1800 bp from the N-terminus, we redesigned an 1850 bp truncated S gene using plant favored genetic codons, removing potential RNA-instablizing sequences, and replacing the original signal peptide with a tobacco pathogen-related signal peptide. The redesigned artificial S gene was synthesized using over-lapped PCR and multiple site-directed mutagensis techniques. The synthetic gene was then successfully expressed in both tobacco and alfalfa after gene transformation via Agrobacterium-mediated method. Intramuscular injection into pigs using the protein extracts from the transgenic plants resulted in the generation of neutralizing antibodies to TGEV. Data from animal feeding trials using the plant materials will be also presented.

Syndrome Oral Vaccine in Plants The Production  of a Porcine Reproductive and Respiratory J. Zhang1,4 ,  R. Rymeson3,4, D. Yoo2, J. Brandle3, L. Erickson1 1. Department of Plant Agriculture, University of Guelph;   2. Department of Pathobiology, University of Guelph, Guelph On. N1G 2W1;  3.Agriculture and Agri-food Canada, 1391 Sandford Street, London, Ontario, Canada N5V 4T3; 4. These authors contributed equally to this reseach.

Porcine Reproductive and Respiratory Syndrome (PRRS) is a major worldwide viral disease of swine. PRRS infections  result in chronic low levels of abortion , stillbirths or weak, slow-growing piglets. Among the identified PRRS open reading frames  in the viral genome, ORF-5 is a major structural protein and the most probable candidate antigen for developing a recombinant subunit vaccine. A native cDNA clone encoding ORF 5 was  ligated to  pBIN1-containing the “Super-promoter” (courtesy of S. Gelvin, Perdue University) and transferred to tobacco (PetH4) and alfalfa (N4) using Agrobacterium.  Immunochemical and molecular analysis of the transgenic plants has shown the presence of  ORF-5 protein in R0 and R1 transgenic tobacco plants and in R0 transgenic alfalfa plants, the Western data also suggest  the presence of ORF 5 protein as a dimer or trimer, as well as a monomer. This is the first demonstration of  a PRRS viral gene  expressed in  plants. Such transgenic plants could potentially provide an inexpensive recombinant oral vaccine for PRRS (Click here to view Posters).