LAUSR

Enterotoxigenic Escherichia coli (ETEC), defined by the production of heat labile (LT) and/or heat stable (ST) toxins, are major causes of diarrhea in animals, children in developing countries and to travelers. No broadly protective ETEC vaccine is available, largely because of the difficulty in inducing immunity to the small ST molecule. To take advantage of the demonstration (Liu et al., 2011; Zhang et al., 2013, 2010) that genetically produced fusions of mutant ST with LT subunits can induce effective immunity against both toxins, we engineered a live attenuated vaccine vector strain of E. coli (ZCR533), expressing the immunogenic LT-ST fusions. To present the LT-ST fusions to the mucosal immune system, we used restriction-free cloning to incorporate them into the passenger domain of the autotransporter protein (EspP) expressed on a medium copy number plasmid. This versatile system permits expression of incorporated antigens in either surface-bound or secreted forms by the ZCR533 vector, for delivery to the mucosal inductive sites. Incorporation of the fusions into EspP plasmids was confirmed by PCR and DNA sequencing. Protein expression was confirmed by Western blot of whole cell lysates and culture supernatents using polyclonal antisera to LT. Expression of the surface-targeted fusion on the surface of ZCR533 was confirmed by immuno-fluorescent staining. These studies show that antigenic LT-ST fusions can be surface-expressed or secreted, by our attenuated E. coli ZCR533 vaccine vector via the EspP autotransporter. These constructs could serve as broadly protective vaccine candidates to protect against both LT- and ST-producing ETEC.