Gastrointestinal infections are among the leading causes of childhood mortality around the world and are responsible for approximately 2 million deaths every year (12). Enteropathogenic Escherichia coli (EPEC) is one of the main causes of childhood diarrhea in developing countries and has been considered a worldwide pathogen (7,18,31,59). EPEC has been classified into typical or atypical according to the expression or lack of expression, respectively, of the bundle-forming pilus, encoded by the EAF plasmid (1, 50). Epidemiological studies suggest that the prevalence of atypical EPEC has increased in acute or persistent diarrhea cases in both children and adults (23,29,52,62). EPEC colonizes the intestinal mucosa, causing the characteristic attaching and effacing (A/E) lesions. The formation of the A/E lesion involves a type III secretion system (T3SS), encoded by the LEE pathogenicity island, which delivers a range of effector proteins into host cells. Using this strategy, the pathogen triggers actin polymerization, producing a pedestal-shaped structure to which the bacteria strongly attach through the interaction of the adhesin intimin with the translocated receptor Tir (27,38). Intimin is a 94-kDa protein inserted into the bacterial outer membranes from typical and atypical EPEC, enterohemorrhagic E. coli (EHEC), and the mouse pathogen Citrobacter rodentium. This adhesin is encoded by the eae gene and is essential for virulence (28,48). The N-terminal region is conserved among intimins, whereas the C-terminal portion is highly variable and has been used to classify intimin into 29 types that can be related to tropism for different intestinal tissues (26,35,49). Among them, the ␤ subtype is one of the intimins most frequently expressed by EPEC isolates (6,56,60). EPEC and EHEC infections can be prevented by breastfeeding (17,57), and antibodies against intimin are usually observed in the milk of women living in areas of endemicity (58,70). In addition, these antibodies are able to inhibit the adhesion of EPEC to epithelial cells in vitro (15,19). Several vaccination strategies using intimin have reduced colonization or mortality in animal models of infection with rabbit enteropathogenic E. coli (REPEC), EHEC, or C. rodentium (4,30,36,39), suggesting that intimin is a good candidate for the formulation of vaccines against these infections. A minimal intimin fragment able to bind to the receptor Tir is composed of the last 190 amino acids, but high-affinity binding was observed only with a larger fragment comprising the last 280 amino acid residues (Int 280 ) (8). In fact, subcutaneous immunization of mice with the Int 280 fragment from ␣-intimin reduced intestinal colonization by a C. rodentium strain expressing an intimin from the same subtype (30). However, analysis of the immunodominant regions inside Int 280 have shown that antibodies against this fragment were directed to 2 regions in the N terminus of Int 280 , more precisely, a