Expression of vaccine antigens to edema disease in tobacco seeds and evaluation of immunogenicity on mouse model

Plant-derived vaccines present many potential advantages related to the management in intensive livestock. They could be administered without restraint of the animals, with low stress and without labour costs related to multiple injections of traditional vaccines. The aim of this study was the construction and subsequent evaluation in mouse model of transgenic tobacco seeds as edible vaccines for swine Edema disease. We focalized our attention on Verocitotoxic Escherichia coli strains (O138, O139, O141), responsible of Edema disease, that occurs in pigs approximately one week after weaning and is characterized by edema in various sites and by damages to vascular endothelium. The adhesion of bacterial strains is related to different fimbriae and Shiga-like toxins (VT2e), that play an important role in the pathogenesis. Structural parts of F18 fimbriae and B-subunit of VT2e genes were inserted in expression vectors, under control of GLOB promoter to obtain specific seed accumulation of heterologous proteins, and transformed in tobacco by agroinfection. We obtained two stable lines of transformed tobacco expressing the proteins in the seed: one included F18 gene (F18+) and another one included B-subunit of Vt2e gene (VT2e-B+). Tobacco lines were characterized by molecular and immunoenzymatic techniques for the expression of F18 and VT2e-b proteins. The amount of transgenic proteins was estimated at around 10ug/g of seeds. 14 Balb-c mice were divided randomly in two groups Control (CG) and Treatment (TG), with 7 mice each. Treatment diet, prepared as pellet to avoid different feed intakes in animals, contained 10% of tobacco seeds from F18+ and 10% of tobacco seeds from VT2e-b+. CG received a diet containing 20% of not-transgenic tobacco seeds. Treatments were administered on days 0,5,8,14,19,23. TG revealed an increment of fecal IgA at day 26, while CG at the same period decreased. The histometric data of the small intestine showed that TG crypts of the duodenum were significantly deeper than those of the CG. Immunostaining of the intestine showed that administration of transgenic tobacco seeds promotes a significant increase in the IgA-positive plasma cells production of the tonaca propria if compared to control group. In conclusion our findings suggest that tobacco seeds might be a potential source of oral vaccines.