Development of Live Attenuated Bacterial Vaccines to Prevent Carriage of Shiga Toxin-Producing E. Coli in Cattle

NON-TECHNICAL SUMMARY: EHEC (enterohemorrhagic E. coli) are important foodborne pathogens. The major reservoirs of EHEC are cattle. Asymptomatic cattle are colonized by and shed these organisms leading to contamination of livestock-derived food products and water, and subsequent human EHEC infections. The current proposal, based on extensive studies on the pathogenesis of EHEC and mechanism of protection using a rabbit model, represents a novel and practical strategy, which should induce effective local mucosal immunity thereby preventing EHEC colonization in the gut. An effective vaccination protocol for STEC in cattle would provide safer meat and a cleaner environment.
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APPROACH: PCR-based techniques will be used by utilizing primers derived from the to the target genes to create defined mutations (stxAB, eae, ler). The defined mutation will be introduced to the wild-type EHEC O157, O26,or O111 by site-directed mutagenesis using suicide vector or lambda Red recombinase systems. The phenotypes of the mutants will be examined for in vitro Stx cytotoxicity, production of relevant proteins and adherence on cultured cells and for in vivo immunogenicity by oral or rectal immmunization of mice. The immune responses to immunization protocols will be determined by ELISA for specific IgG and IgA titers to the whole bacteria or intimin, the major adhesion of EEHEC.

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PROGRESS: 2004/12 TO 2006/12 <BR>
The long-term goal of this proposal 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: Objective One: To delete, by site-directed mutagenesis, the genes encoding Shiga toxin from STEC strains of serogroup O157, O26, and O111; 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; 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; Objective Four: to determine the immunogenicity of attenuated mutants by oral and intrarectal immunization in mice. Studies and results: STEC strains were obtained from the STEC Center(NFSTC) at Michigan State University (MSU). The defined deletion mutations were made in the target genes (eae, stxAB, and ler) by single overlap extension PCR, without introducing any antibiotic marker. The double mutations, (eae and stx2AB) have been made in O157:H7 strain 86-24 and triple mutations, (eae, stx1A and stx2AB) were made in another O157:H7 strain EDL932. The strain 86-24(d-eae/d-stx2AB) was further characterized for in vitro and in vivo virulence. We demonstrated that this mutant expressed truncated intimin and is deficient by HeLa cell adherence assay and Shiga-toxin production compared to the parent strain. Immunogenicity of 86-24(d-eae/d-stx2AB) has been tested in mice. Three groups of mice (oral immunization, rectal immunization, and naive) were evaluated. They were vaccinated twice at 2 weeks intervals, and serum were collected at day 0, 14, and 28, and intestinal secretion samples were collected at day 28. ELISA was performed to analyse antibody titers against O157 LPS. We observed different patterns following initial and boost vaccination indicating possible mucosal immunity following initial vaccination. Oral vaccination of mice with this attenuated mutant strain induced measurable serum IgG and fecal IgA specific to O157 lipopolysacharide antigen. Rectal immunization induced no significant increase of antibody titers. The construction of 86-24(d-ler/d-stx2AB, the regulatory mutant is problematic. Conjugation of 86-24(d-stx2AB) with delivery suicide plasmid was successful. However, integration of deletion-mutated ler into 86-24 chromosome has not been achieved in several attempts. We obtained 86-24(d-ler/d-stx2AB by alternative approach. The defined deletion mutations in O26 eae, ler and stx1AB, and O111 eae and stx1AB have been made and the suicide delivery vector systems have been constructed. Attempts are being made to generate mutations in the wild-type background. The P.D. has accepted an offer an University of New Mexico School of Medicine, Albuquerque, NM. The transfer of the current grant is in progress.
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IMPACT: 2004/12 TO 2006/12<BR>
EHEC (enterohemorrhagic E. coli) are important foodborne pathogens. Asymptomatic cattle are colonized by and shed these organisms leading to the contamination of livestock-derived food products and water, and subsequent human EHEC infections. Previous studies in our laboratory demonstrated that a defined truncated intimin mutant of rabbit attaching and effacing E. coli is immunogenic after orogastric administration and protective against challenge with the WT parent strain and related shiga toxin producing strains. We have constructed desired double mutant of O157:H7 (eae/Stx2AB) and shown that they are deficient in Shiga-toxin production and adherence to cultured epithelial cells. We demonstrated measurable serum IgG and fecal IgA specific to O157 lipopolysacharide antigen following oral or rectal immunization of mice with this attenuated mutant strain. Thus, we have demonstrated that this mutant is attenuated and immunogenic in a mouse model. Immunization of animals with this double mutant represents a novel and practical vaccination strategy to induce effective local mucosal immunity, thereby preventing carriage of EHEC in cattle.