An official website of the United States government.
The long term goal of this work is to develop live, attenuated STEC vaccine candidates for cattle based on the most common STEC serotypes and to establish their attenuation and immunogenicity in mice. There are four specific objectives of this proposal: <P>
Objective One: To delete, by site-directed mutagenesis, the genes encoding Shiga toxin from STEC strains of serogroup O157, O26, and O111; <P>Objective Two: To fully attenuate STEC virulence by modifying the bacterial adhesive protein intimin (encoded by the eae gene) or by inactivating the regulator Ler (ler), which up-regulates the expression of virulence genes encoded on the locus of the enterocytes effacement (LEE) pathogenicity island; <P>Objective Three: To characterize the isogeneic mutants by examination of protein profiles and in vitro assays for Stx production and adherence to cultured cell lines; <P>Objective Four: to determine the immunogenicity of attenuated mutants by oral and intrarectal immunization in mice. <P>
The constructed organisms deficient in Shiga toxin production and adhesion to the host will be fully attanuated and harmless to animals. However, vaccination of animals (such as cattle) with these live, attenuated organisms will induce measurable level of protection against infection with the virulent EHEC organisms.
NON-TECHNICAL SUMMARY: STEC (Shiga toxin-producing E. coli), or EHEC (enterohemorrhagic E. coli) are important foodborne pathogens. The major reservoirs of EHEC are cattle leading to contamination of livestock-derived food products and water and subsequent human STEC infections. We hypothesize that immunization of cattle with live attenuated vaccine(s) will induce protective immunity against STEC thus to prevent asymptomatic carriage in cattle. To address the above hypothesis, we propose to develop multivalent live attenuated STEC vaccines based on the most prevalent STEC strains of O157, O111, O26 serotypes. Double mutations targeted both on Stx and eae genes will fully attenuate STEC virulence. Thus they are safe for use in cattle. Taking advantage of live bacteria being most effective mucosal vaccine we anticipate that vaccination with desired multivalent, live attenuated STEC will induce protective immune responses in cattle. The vaccine candidates which we will develop can be tested for immunogenicity and protection efficacy in cattle through future studies.
<P>
APPROACH: The targeted genes encoding Shiga toxin and bacterial attachment to host intestine will be deleted by site-directed mutagenesis. The isogeneic mutants will be characterized by examination of protein profiles and in vitro assays for Stx production and adherence to cultured cell lines. Bactreial attenuation will be tested in vivo in mouse model. The protective efficacy will be examined by challenging mice with adhesive EHEC organisms deficient in Shiga toxin production.