Immunogenic evaluation of nasal administration of conjugated IpaD with Co1 and STxB ligands against E.coli O157: H7 in mice

The most common cause of diarrhea is Shigella and no vaccine has been found so far. IpaD and B subunit of Shiga toxin proteins (STxB) play an important role in invasion, infection and pathogenesis caused by Shigella. On the other hand, Co1 ligand has introduced as one of the antigen delivery systems to M-cells in mucosal layer. In this study, nasal administration of antigen in mice, used to evaluation and comparison of immunogenicity of IpaD, IpaD-STxB and IpaD-Co1 proteins. The coding sequences of proteins cloned in pET28a vector and transformed to bacterial strain of E.coli BL21(DE3). Recombinant protein expression confirmed by using of Western bloting technique. The each one of the recombinant protein purified by affinity chromatography column and used for nasal administration in four times consecutively on mice. After the second nasal administration (first booster), a week after each administration, blood samples were collected and ELISA performed on serum. The ELISA results of serum titration showed that the highest amount of IgG has been produced against IpaD-STxB protein; and IpaD-Co1 was in next step. The immunogenicity was evaluated using active toxin E. coli O157:H7. The challenge results showed that immunized mice could endure 5 times the LD50 Shiga toxin E. coli O157: H7; but after injection of 10 times the LD50, the immunized mice by IpaD, IpaD-Co1 and IpaD-STxB were death after 24, 24 and 72 hours, respectively. According to these results recombinant protein IpaD-STxB founded to be more immunogenic than IpaD-Co1 against shiga toxin in mice.