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Infection and Immunity, October 2005, p. 6608-6619, Vol. 73, No. 10
0019-9567/05/$08.00+0     doi:10.1128/IAI.73.10.6608-6619.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Protection against Hemorrhagic Colitis in an Animal Model by Oral Immunization with Isogeneic Rabbit Enteropathogenic Escherichia coli Attenuated by Truncating Intimin

Tonia S. Agin ,{dagger},{ddagger} Chengru Zhu,{ddagger} Laura A. Johnson, Timothy E. Thate, Zhuolu Yang, and Edgar C. Boedeker*

Center for Vaccine Development, University of Maryland School of Medicine, Baltimore, Maryland 21201

Received 6 December 2004/ Returned for modification 22 February 2005/ Accepted 12 July 2005

Strains of Shiga toxin (Stx)-producing Escherichia coli, also called enterohemorrhagic E. coli (EHEC), are important food-borne pathogens for humans. Most EHEC strains intimately adhere to the intestinal mucosa in a characteristic attaching and effacing (A/E) pattern, which is mediated by the bacterial adhesin intimin. Subsequent release of Stx1 and/or Stx2 leads to the frequent development of hemorrhagic colitis and, less commonly, to hemolytic-uremic syndrome. The aim of the present study was to develop an attenuated A/E E. coli strain for use as a vaccine against EHEC infection encoding a truncated intimin lacking adhesive capacity, but which would still express somatic antigens, other products of the locus of enterocyte effacement pathogenicity island, and an immunogenic remnant of the intimin molecule. A single-nucleotide deletion was generated in the eae gene in the prototype rabbit A/E E. coli strain RDEC-1 (O15:H–), which resulted in truncation of intimin by 81 C-terminal residues (860 to 939 amino acids) containing a disulfide loop. Inoculation of rabbits with large doses of the truncated intimin mutant (RDEC-1{Delta}eae860-939) was well tolerated, as observed by the absence of clinical signs of disease or evidence of intestinal A/E lesions. The efficacy of RDEC-1{Delta}eae860-939 as a vaccine was evaluated by orogastric inoculation of rabbits with RDEC-1{Delta}eae860-939 followed by challenge with the virulent strain RDEC-H19A, an Stx1-producing derivative of wild-type RDEC-1 capable of inducing hemorrhagic colitis in rabbits. Following RDEC-H19A challenge, nonimmunized control rabbits exhibited characteristic weight loss with watery to bloody diarrhea and demonstrated intimate bacterial attachment, effacement of microvilli, submucosal edema, mucosal heterophile infiltrates, and Shiga toxin-induced vascular lesions. In contrast, the RDEC-1{Delta}eae860-939-immunized rabbits showed no clinical signs of disease, maintained normal weight gain, had reduced fecal shedding of challenge organisms, and showed an absence of gross or microscopic lesions in the intestinal mucosa. Serum antibodies specific to intimin were detected among rabbits immunized with RDEC-1{Delta}eae860-939, indicating that truncation of the intimin functional domain not only attenuated bacterial virulence, but also retained at least some of the immunogenicity of native intimin. Although it is not possible to gauge the exact contribution of residual intimin immunity to protection, this attenuation strategy for A/E E. coli strains shows promise for the development of effective vaccines to prevent EHEC infection in humans and animals.

* Corresponding author. Mailing address: Center for Vaccine Development, University of Maryland School of Medicine, 685 West Baltimore Street, Baltimore, MD 21201. Phone: (410) 706-0330. Fax: (410) 706-6205. E-mail: eboedeke{at}medicine.umaryland.edu.

Editor: V. J. DiRita

{dagger} Present address: Pfizer Animal Health, Pfizer Inc., Kalamazoo, MI 49001.

{ddagger} T.S.A and C.Z. contributed equally to this paper.

Infection and Immunity, October 2005, p. 6608-6619, Vol. 73, No. 10
0019-9567/05/$08.00+0     doi:10.1128/IAI.73.10.6608-6619.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.



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