Intranasal immunization of cocktail/fusion protein containing Tir along with ΔG active fragment of Zot as mucosal adjuvant confers enhanced immunogenicity and reduces E. coli O157:H7 shedding in mice.

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 (ΔG) responsible for this function could be used for mucosal antigen delivery. Therefore, we employed individual (Tir), cocktail (ΔG + Tir), fusion protein (ΔG-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, ΔG + Tir, ΔG-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 ΔG + Tir, ΔG-Tir group while that of Tir group was solely Th2 type. Tir, ΔG + Tir and ΔG-Tir immunized mice showed reduction in shedding of E. coli O157:H7 compared to control group. However, ΔG-Tir immunized group performed better than ΔG + Tir, Tir group in reducing fecal shedding. Overall, our results demonstrate that intranasal immunization of ΔG-Tir induces effective systemic, mucosal, cellular immune responses and represents a promising mucosal subunit vaccine to prevent E. coli O157:H7 colonization.