LAUSR

&NA; Ruminants are the major reservoirs of Escherichia coli O157:H7 and its fecal shedding mainly act as a source of entry of this pathogen into the human food chain. In humans, E. coli O157:H7 infection causes diarrhea, hemorrhagic colitis and hemolytic uremic syndrome. Intimate adherence of E. coli O157:H7 is mediated by Translocated intimin receptor (Tir) to which intimin binds in the host cell. Since E. coli O157:H7 colonizes intestinal epithelium, the mucosal vaccine has a potential to prevent its colonization. Zonula occludens toxin (Zot) of Vibrio cholerae transiently, reversibly alters epithelial tight junction structure to increase mucosal permeability of macromolecules via paracellular route. The C‐terminal region of Zot (&Dgr;G) responsible for this function could be used for mucosal antigen delivery. Therefore, we employed individual (Tir), cocktail (&Dgr;G + Tir), fusion protein (&Dgr;G‐Tir) and assessed the efficacy of its intranasal immunization on immunogenicity and fecal shedding of E. coli O157:H7 in streptomycin treated mouse model. Compared to control, &Dgr;G + Tir, &Dgr;G‐Tir immunized mice elicited significant antigen specific antibody titers in serum (IgG, IgA) and feces (IgA), whereas Tir immunized mice induced only serum IgG titer. Cytokine analysis revealed mixed Th1/Th2 type immune response in case of &Dgr;G + Tir, &Dgr;G‐Tir group while that of Tir group was solely Th2 type. Tir, &Dgr;G + Tir and &Dgr;G‐Tir immunized mice showed reduction in shedding of E. coli O157:H7 compared to control group. However, &Dgr;G‐Tir immunized group performed better than &Dgr;G + Tir, Tir group in reducing fecal shedding. Overall, our results demonstrate that intranasal immunization of &Dgr;G‐Tir induces effective systemic, mucosal, cellular immune responses and represents a promising mucosal subunit vaccine to prevent E. coli O157:H7 colonization. HighlightsCocktail/fusion protein &Dgr;G+Tir, &Dgr;G‐Tir with active fragment of Zot has been designed as an E. coli O157:H7 mucosal vaccine.&Dgr;G+Tir, &Dgr;G‐Tir induces Tir specific systemic, mucosal antibody response whereas Tir elicits only systemic response in mice.&Dgr;G+Tir, &Dgr;G‐Tir elicits mixed Th1/Th2 type cell mediated response while that of Tir group is solely Th2 type.&Dgr;G+Tir, &Dgr;G‐Tir group mice perform better in preventing E. coli O157:H7 colonization compared to Tir.Intranasal immunization of &Dgr;G‐Tir can serve as a mucosal candidate vaccine against E. coli O157:H7 colonization in mice.