Towards Development of an Edible Vaccine against Bovine Pneumonic Pasteurellosis Using Transgenic White Clover Expressing aMannheimia haemolyticaA1 Leukotoxin 50 Fusion Protein

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Abstract
Development of vaccines against bovine pneumonia pasteurellosis, or shipping fever, has focused mainly onMannheimia haemolyticaA1 leukotoxin (Lkt). In this study, the feasibility of expressing Lkt in a forage plant for use as an edible vaccine was investigated. Derivatives of theM. haemolyticaLkt in which the hydrophobic transmembrane domains were removed were made. Lkt66 retained its immunogenicity and was capable of eliciting an antibody response in rabbits that recognized and neutralized authentic Lkt. Genes encoding a shorter Lkt derivative, Lkt50, fused to a modified green fluorescent protein (mGFP5), were constructed for plant transformation. Constructs were screened by Western immunoblot analysis for their ability to express the fusion protein after agroinfiltration in tobacco. The fusion construct pBlkt50-mgfp5, which employs the cauliflower mosaic virus 35S promoter for transcription, was selected and introduced into white clover byAgrobacterium tumefaciens-mediated transformation. Transgenic lines of white clover were recovered, 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 temperature for 4 days. An extract containing Lkt50-GFP from white clover was able to induce an immune response in rabbits (via injection), and rabbit antisera recognized and neutralized authentic Lkt. This is the first demonstration of the expression of anM. haemolyticaantigen in plants and paves the way for the development of transgenic plants expressingM. haemolyticaantigens as an edible vaccine against bovine pneumonic pasteurellosis.