Nasal immunogenicity induced by STxB and STxB-IpaD antigens in laboratory rats

  Introduction: Shigella and E. coli bacteria are the most common cause of Diarrhea, though as yet no effective vaccine against them has been produced. IpaD protein plays an important role in invasion and infection caused by Shigella. Another major virulence factor in Shigella dysenteriae type 1 and E. coli O157: H7 is Shigella enterotoxin or (STxB). IpaD protein in combination with STxB can produce a suitable candidate vaccine. In this study, the nasal STxB and STxB-IpaD fused recombinant proteins antibody titers and their immunogenicity were assessed and compared in rats .   Materials and Methods: (pET28- stxB) and (pET28-ipaD-stxB) vectors were prepared at Biology center of the Imam Hussein University (AS). Transformation of these plasmids into E. coli BL21 DE3 bacteria were confirmed by PCR and enzymatic digestion. Production of recombinant proteins were confirmed by SDS-PAGE and Western blotting. Expression of fused STxB-IpaD and STxB antigens were performed under induction of IPTG. After protein purifications, using affinity chromatography, antigens were prescribed nasally to five groups of rats in four consecutive sessions. Later the polyclonal antibodies produced in rat sera were measured .   Results : ELISA showed that antibody titers were increased by the combination of IpaD to STxB compared to that produced against single STxB antigen. The immunized Rats with StxB antigen were able to tolerate up to six fold of LD50, while rats that were immunized with STxB-IpaD combined antigens were able tolerate up to tenfold of LD50 for E. coli O157: H7 Shiga toxin .   Conclusion: The protein produced from the fusion of ipaD and stxB genes, can increase the effect of single STxB antigen immunogenicity. Recombinant proteins fused with STxB protein without chemicals adjuvants can be recommended in the form of nasal drops as possible candidates for vaccines against E. coli and Shigella types .