Towards development of an edible vaccine against bovine pneumonic pasteurellosis using transgenic white clover expressing a Mannheimia haemolytica A1leukotoxin 50 fusion protein

Development of vaccines against bovine pneumonia pasteurellosis, or shippin g fever, has focused mainly on Mannheimia haemolytica A1 leukotoxin (Lkt). In this study, the feasibility of expressing Lkt in a forage plant for use as an edible vaccine was investigated. Derivatives of the M. haemolytica Lk t in which the hydrophobic transmembrane domains were removed were made. Lk t66 retained its immunogenicity and was capable of eliciting an antibody re sponse in rabbits that recognized and neutralized authentic Lkt. Genes enco ding a shorter Lkt derivative, Lkt50, fused to a modified green fluorescent protein (mGFP5), were constructed for plant transformation. Constructs wer e screened by Western immunoblot analysis for their ability to express the fusion protein after agroinfiltration in tobacco. The fusion construct pBlk t50-mgfp5, which employs the cauliflower mosaic virus 35S promoter for tran scription, was selected and introduced into white clover by Agrobacterium t umefaciens-mediated transformation. Transgenic lines of white clover were r ecovered, and expression of Lkt50-GFP was monitored and confirmed by laser confocal microscopy and Western immunoblot analysis. Lkt50-GFP was found to be stable in clover tissue after drying of the plant material at room temp erature for 4 days. An extract containing Lkt50-GFP from white clover was a ble to induce an immune response in rabbits (via injection), and rabbit ant isera recognized and neutralized authentic Lkt. This is the first demonstra tion of the expression of an M. haemolytica antigen in plants and paves the way for the development of transgenic plants expressing M. haemolytica ant igens as an edible vaccine against bovine pneumonic pasteurellosis.