Toward development of an oral, plant-based vaccine against Escherichia coli O157:H7.

摘要

Enterohemorrhagic Escherichia coli (EHEC) O157:H7 is the most common infectious cause of bloody diarrhea in the United States, and a sequela of this infection, the hemolytic uremic syndrome (HUS), is the primary cause of acute renal failure in children in the U.S. The majority of U.S. cases of EHEC O157:H7 have occurred as a consequence of ingestion of undercooked, contaminated hamburger or produce and/or water contaminated with bovine manure. Intimin is the primary adhesin of EHEC O157:H7, and is required for colonization of neonatal calves. I hypothesized that an intimin-based vaccination strategy in calves might reduce colonization of cattle with EHEC O157:H7. To test this concept in a small animal model, I developed transgenic tobacco plant cells that expressed the full length or carboxy-terminal portion (Int261) of EHEC O157:H7 intimin and then immunized mice parenterally with intimin expressed from the plant cells, or fed mice the transgenic plant cells, or both. I was able to show that these mice not only generated an intimin-specific mucosal immune response when primed parenterally and boosted orally but also exhibited a reduced duration of EHEC O157:H7 fecal shedding after challenge. These results suggest that transgenic plants are attractive and feasible production and delivery systems for an intimin-based vaccine for cattle, and such a vaccine can reduce the duration of EHEC O157:H7 shedding in a small animal model. In addition, Shiga toxin type 2 (Stx2) is another important EHEC O157:H7 virulence factor that plays a critical role in the development of potentially fatal HUS in humans. I developed a toxoid of Stx2 by making site-directed changes to the nucleotide sequence of the Stx2 A subunit gene that abrogated cytotoxicity in vitro and in vivo. The Stx2 toxoid elicited toxin-neutralizing antibody when parenterally injected in mice. I also optimized both the Stx2 A toxoid subunit and the B subunit for expression in plants and, thus laid the groundwork for future expression of this Stx2 toxoid molecule in plants.