Chitosan nano-structure loaded with recombinant E. coli O157:H7 antigens as a vaccine candidate can effectively increase immunization capacity.

Escherichia coli O157:H7 is considered as emerging foodborne pathogens that occur globally. Three major virulence protein factors; EspA(E), intimin(I), Tir(T) and Stx2 toxin have been found to be highly associated with bloody diarrhoea or, Haemolytic Uremic Syndrome. In this study, a trivalent recombinant EIT in combination with the binding domain of STX toxin were encapsulated with chitosan nanoparticles as a combination vaccine candidate. Mice were immunized either subcutaneously or orally with these antigens and challenged with E. coli O157:H7. Results of the binding inhibition assay with caco2 cell monolayer show a significant reduction in the adhesion percentage of pre-treated E. coli O157:H7 with immunized mice sera. Evaluation of neutralizing abilities of immune sera pre-incubated with CD50 dose of STX2 by Vero cells cytotoxicity neutralization assay shows less morphological reforms in comparison with the control groups. Results of mice mortality challenge with STX2 demonstrate around 66% of survived in immunized mice. In a challenge experiment with E. coli O157:H7, all the immunized mice showed a significant decrease in bacterial colonization and shedding. The results indicate that the use of multiple recombinant proteins in combination with natural nanostructure effectively evocated strong humoral and mucosal response, increasing the protection capacity of the synthetic antigen.

Prevention of EHEC infection by chitosan nano-structure coupled with synthetic recombinant antigen.

One of highly effective methods for prevention and control of Entrohemorragic Esherichia coli (EHEC) infections is to use vaccination against extremely immunogenic part of attachment factors. In this study rEIT (EspA, Intimin, Tir) was produced in bacteria and then encapsulated with chitosan nanoparticle as a candidate nanovaccine. A chimeric trivalent recombinant protein which was previously found to provide reasonable immunogenicity against E.coli O157:H7 was used as a base. Mice immunized orally with chitosan based nanoparticle containing rEIT antigen. The rEIT-specific immune responses (IgG and IgA) were measured by indirect ELISA. In challenging tests different groups of immunized mice were infected orally with E.coli O157:H7. The results showed that the recombinant nanovaccine candidate could induce the strong humoral and mucosal immune responses and protect the mice from live EHEC O157:H7 challenge. Higher titers of serum anti rEIT IgG were achieved after the last immunization in all of the groups. Comparison of the amount of IgA titers in serum and feces showed higher values for the latter. In vitro study of binding inhibition assay on Caco-2 cell monolayers by pre-incubated antisera with EHEC bacteria, showed that immunized mice antibody could reduce adhesion properties of E. coli O157:H7. In a challenging study with EHEC bacteria, reduction in number of colonies was observed in all of the immunized groups for over two weeks. Results from the present study prove that nanovaccine candidate with rEIT can reduce signs and symptoms of EHEC infections. This novel approach can be a new strategy for inducing immunity against E. coli O157:H7. This study suggests the use of oral -injection combined vaccination routes comparing to other methods available in order to achieve higher humoral and mucosal immunogenicity levels.