Cholera toxin B (CTB) is functional as an adjuvant for cytoplasmatic proteins if directed to the endoplasmatic reticulum (ER), but not to the cytoplasm of plants

The mucosa-binding subunit B (CTB) of cholera toxin is frequently used to improve immunization. Up to now, the expression of CTB∷target antigen fusion proteins in plants has only been performed via the secretory pathway, or in chloroplasts. Thus, it has never been determined whether CTB can also enhance the immunogenicity of naturally cytosolically expressed proteins, such as rabbit haemorrhagic disease virus (RHDV) VP60; furthermore, the optimal compartment for protein partners with different needs has not been characterized. In order to enhance the immunogenicity of plant-derived VP60, we analyzed CTB and CTB∷VP60 accumulation via cytosolic versus secretory expression pathways. A synthetic CTB open reading frame (ORF), optimized with tobacco codons, was transferred to Nicotiana tabacum and Solanum tuberosum both with and without the codons for the CTB signal peptide. The genes were transcriptionally regulated by the CaMV 35S promoter. Biologically active, pentameric CTB complexes accumulated after expression of CTB with the signal peptide and ER-retention; whereas neither monomeric nor pentameric CTB molecules were detectable in transgenic plants expressing the signal peptide-deleted CTB. A similar result was obtained using tobacco plants expressing fusion proteins of the respective CTB variants and RHDV VP60. ER-directed and glycosylated CTB∷VP60 proteins induced anti-VP60 specific antibodies in rabbits using a low immunization dosage of approximately 0.4 ng antigenic VP60.