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.

Preparation and evaluation of chitosan nanoparticles containing CtxB antigen against Vibrio cholera.

Vibrio cholera is a Gram-negative pathogen that causes diarrheal disease. The B subunit of Chlora toxin (CtxB) is one of the most important antigens of Vibrio cholera in which mediates the attachment of the bacteria to target cells. The aim of this study was to prepare chitosan nanoparticles containing CtxB and evaluate the effect of the antigen entrapment on the immunogenicity of this antigen. For this, the pET28a vector was induced using IPTG. Recombinant CtxB protein was expressed and purified using Ni-NTA column and finally was confirmed by western blotting. Following the confirmation of the protein entrapment onto the chitosan nanoparticles, the formulation was prescribed to BALB/c mice in three groups, including oral, oral-injection and injection groups. Serum and fecal IgA and IgG were evaluated by ELISA test. Finally, challenge of immunized mice was performed using Ctx toxin and rabbit ileal loop test. Using SDS-PAGE and western blotting, the 17.5 kDa recombinant CtxB was confirmed. Size electrical charge and of nanoparticles were determined and approved by Zetasizer. Nanoparticles prescription showed 1/102400 IgG endpoint titers for injection groups and 1/1600, 1/6400 for oral, oral-injection groups respectively and Serum and fecal IgA endpoint titers showed above 1/160 in all groups. Furthermore, immunized mice were able to neutralize Ctx toxin by ileal loop test. The CtxB is a suitable immunogen of V. cholera to be incorporated in both protective and preventive vaccines. Chitosan nanoparticles improve the immune responses and it may be used as a carrier for vaccine delivery.