Immunogenicity of enterotoxigenic Escherichia coli outer membrane vesicles encapsulated in chitosan nanoparticles

Objective(s): Enterotoxigenic Escherichia coli (ETEC) is an important cause of diarrheal disease in humans, particularly in children under ۵ years and travelers in developing countries. To our knowledge, no vaccine is licensed yet to protect against ETEC infection. Like many Gram-negative pathogens, ETEC can secrete outer membrane vesicles (OMVs). These structures contain various immunogenic virulence proteins such as LT and therefore can be used as vaccine candidates. In this study we attempted to isolate the OMVs of ETEC cultivated at different temperatures and evaluate their immunogenicity and protective efficacy in a murine model of infection. Materials and Methods: OMVs was purified from bacterial supernatant by ultracentrifugation. OMVs were encapsulated in chitosan nanoparticles prepared by ionic gelation method within a layer of Eudragit L۱۰۰ for oral delivery.  Female BALB/c mice of ۹ weeks’ old were immunized by parenteral injection and oral administration with free and encapsulated OMVs obtained from bacteria cultivated at ۳۷°C and ۴۲°C. The serum samples were collected and the antibody titers were measured by an enzyme-linked immunosorbent assay (ELISA). Results: The protein concentrations of OMVs were ۳.۴۷ mg/ml and ۲.۴۶ mg/ml for bacteria grown at ۳۷°C and ۴۲°C respectively. OMVs loaded into nanoparticles (NP-OMVs) were homogeneous and spherical in shape, with a size of ۵۳۲ nm. The encapsulation efficiency of NP was ۹۰%. Mice immunized with OMVs, inhibited the ETEC colonization in their small intestine and induced production of antibodies against LT toxin. Conclusion: The results obtained in this research place OMVs among promising candidates to be used for vaccination.

Keywords:

Enterotoxigenic Escherichia coli , virulence factors , Outer membrane vesicle Chitosan nanoparticle