Production of orally immunogenic bacterial protein in transgenic plants

Abstract

The binding subunit of Escherichia coli heat labile enterotoxin (LT-B) is a highly active oral immunogen. Stable genetic transformation of tobacco and potato was achieved using the gene encoding LT-B or a chimeric gene encoding an LT-B fusion protein with a C-terminal microsomal retention sequence (SEKDEL). The transgenic plants expressed the foreign peptides, which retained the property of binding ganglioside, the natural ligand for LT-B on intestinal epithelia. Immunization of mice with partially purified leaf extracts delivered by oral intubation elicited serum and secretary anti-LT-B immunoglobulins which neutralized the enterotoxin in in-vitro cell protection assays. Direct feeding of fresh transgenic potato tubers expressing the LT-B fusion protein also elicited anti-LT-B serum and secretary antibodies. In order to obtain higher levels of expression of LT-B in plants, a synthetic gene was designed with optimal plant-codon usage. The putative polyadenylation signals and MRNA instability signals were removed. Studies of expression of the designed gene encoding LT-B suggests an elevated level of expression compared to the native LT-B gene. These experiments demonstrate that a candidate oral immunogen can be produced in plants and, more importantly, serum and mucosal immune responses can be elicited when it is presented simply as a component of a food source; this is "proof of concept" that plants can be used to produce and deliver recombinant oral (edible) vaccines.