Assembly of cholera toxin-antigen fusion proteins in transgenic potato

One of the most significant problems challenging the protective efficacy of plant delivered oral vaccines is the relatively low level of antigenic proteins generally synthesized in plant tissues. To circumvent this difficulty, we explored the feasibility of using cholera toxin as an enterocyte targeted carrier molecule for delivery of multiple antigens to the mucosal immune system. Potato plants were engineered to synthesize two cholera toxin fusion proteins. The cholera toxin B subunit (CTB) was linked to a rotavirus enterotoxin NSP4 epitope and the cholera toxin A2 subunit (CTA2) was fused to the enterotoxigenic E. coli (ETEC) colonization factor CFA/I. The fusion proteins were detected in transformed tuber tissues by immunoblot assay and quantitated by enzyme linked immunosorbent assay (ELISA). The CTB-NSP4 protein expression level was found to be approximately 0.01% of total soluble plant protein (TSP) and the CFA/I-CTA2 protein expression level was about 0.002% of TSP. Assembly of the CFA/I-CTA2 subunit into the CTB-NSP4 pentamer was detected by immunoblot staining methods. The CFA/I-CTA2-CTB-NSP4 pentamer fusion protein complex retained its binding affinity for G_M1 ganglioside enterocyte membrane receptors. Our experimental results demonstrate for the first time that plant synthesized cholera toxin A and B subunit fusion proteins synthesized in planta interact in a coordinated fashion to generate a multimeric carrier complex for targeted delivery of multiple antigens to the mucosal surface.