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• W2022832216 abstract "Background: Enterotoxigenic Escherichia coli (ETEC), defined by their production of heat labile (LT) and/or heat stable (ST) toxins, are major causes of infantile diarrhea in developing countries and of travelers' diarrhea in visitors to the developing world. Despite our understanding of ETEC pathogenesis, no broadly protective ETEC vaccine is available. This is largely a result of the difficulty in inducing immunity to the small ST molecule. Our goal is to develop a broadly protective live attenuated vaccine against ETEC by taking advantage of the recent demonstration (Zhang et al.,2013,PLoSONe 8 (10):e77386) that genetically produced fusions of mutant ST with LT components can induce effective immunity against both toxins. Methods: To achieve this aim, we engineered an attenuated strain of E. coli, which we have developed as a vaccine vector for mucosal antigen delivery, to express immunogenic fusion constructs of LT and mutant ST. Our vaccine vector strain ZCR533, is a derivative of O157:H7 enterohemorrhagic E. coli (EHEC) strain 86-24 attenuated by deleting its genes for Shiga toxin production and truncating its major adhesin, intimin, such that it can no longer adhere intimately, but can still induce anti-intimin antibody. To express LT-ST fusions in immunogenic form in our vector strain, we used restriction-free cloning technology to incorporate them into the passenger domain of an autotransporter protein (EspP) expressed on a medium copy number plasmid. This system is designed to express the toxin antigens in either surface-bound or secreted forms, and to provide effective delivery to the mucosal immune system as we have done for other toxin antigens. Results: The incorporation of the LT-ST fusions into the passenger domain of the EspP autotransporter plasmids was confirmed by PCR and DNA sequencing. Protein expression by ZCR533 containing the autotransporter plasmids was confirmed by SDS-PAGE, and Western blot of whole cell lysates and culture supernates, using poly-clonal antisera to the LT component. Moreover, appropriate expression of the LT-ST fusions on the bacterial surface of ZCR533 was confirmed by immuno-fluorescent staining with the specific antisera. Conclusions: These studies show that immunogenic fusions of LT and mutant ST can be expressed on the surface, and in the culture supernates, of our attenuated E. coli vaccine vector ZCR533 via the EspP autotransporter. Since our recent studies using the similarly-sized Shiga toxin B subunit antigen have indicated the validity of this approach to mucosal immunization against toxin antigens, these studies could lead to the development of broadly protective vaccines directed against both LTand ST-producing ETEC, as well as against attaching/ effacing E. coli strains expressing intimin." @default.
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• W2022832216 date "2014-05-01" @default.
• W2022832216 modified "2023-09-27" @default.
• W2022832216 title "Mo1948 Toward a Broadly Protective Vaccine Against Enterotoxigenic E.coli (ETEC) Infections" @default.
• W2022832216 doi "https://doi.org/10.1016/s0016-5085(14)62538-5" @default.
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