Production and characterization of an orally immunogenic Plasmodium antigen in plants using a virus-based expression system

Increasing numbers of plant-made vaccines and pharmaceuticals are entering the late stage of product development and commercialization. Despite the theoretical benefits of such production, expression of parasite antigens in plants, particularly those from Plasmodium, the causative parasites for malaria, have achieved only limited success. We have previously shown that stable transformation of tobacco plants with a plant-codon optimized form of the Plasmodium yoelii merozoite surface protein 4/5 (PyMSP4/5) gene resulted in PyMSP4/5 expression of up to ~0.25% of total soluble protein. In this report, we describe the rapid expression of PyMSP4/5 in Nicotiana benthamiana leaves using the deconstructed tobacco mosaic virus-based magnICON® expression system. PyMSP4/5 yields of up to 10% TSP or 1-2 mg/g of fresh weight were consistently achieved. Characterization of the recombinant plant-made PyMSP4/5 indicates that it is structurally similar to PyMSP4/5 expressed by Escherichia coli. It is notable that the plant-made PyMSP4/5 protein retained its immunogenicity following long-term storage at ambient temperature within freeze-dried leaves. With assistance from a mucosal adjuvant the PyMSP4/5-containing leaves induced PyMSP4/5-specific antibodies when delivered orally to naïve mice or mice primed by a DNA vaccine. This study provides evidence that immunogenic Plasmodium antigens can be produced in large quantities in plants using the magnICON® viral vector system.