Oral immunogenicity of the inactivated Vibrio cholerae whole-cell vaccine encapsulated in biodegradable microparticles.

Vibrio cholerae (VC)-loaded microparticles as an oral vaccine delivery system were prepared with 6% w/v poly(DL-lactide-co-glycolide)(PLG) in the oil phase as well as 10% w/v PVP and 5% w/v NaCl in the aqueous phase, by an water-in-oil-in-water emulsion/solvent extraction technique. VC was successfully entrapped in the microparticles with trapping efficiencies up to 97.8% and a loading level of 55.4+/-6.9 microg/mg. The microparticle delivery system with a particle size of 3.8 microm had different distribution of VC content in the core region (25.7+/-1.9 microg/mg) and surface (6.2+/-0.9 microg/mg). The immunogenic potential of VC-loaded microparticles in comparison with PLG microparticles or VC solution was evaluated in adult mice by oral immunization, in which mice received one dose of 20 mg VC-loaded microparticles or 20 mg VC-loaded microparticles physical mixed with amphotericin B. The control group received 20 mg PLG microparticle or VC solution. Serum samples were collected from all tested mice on the day of immunization and at 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10 weeks postimmunization. Sera were examined for vibriocidal antibodies by microtitration and Vibrio-specific serum IgG and IgM antibodies were assessed by the ELISA method. IgG and IgM antibodies to intact VC were detected in sera from all animals immunized with VC. The response was specific and of high magnitude. Significantly higher antibody responses were obtained when sera from both VC-loaded microparticles and VC-loaded microparticles physical mixed with amphotericin B immunized mice were titrated against VC. The immunogenicity of VC-loaded microparticles mixed with amphotericin B in evoking serum IgG and IgM responses was higher than that of VC-loaded microparticles only. These results demonstrate that VC-loaded microparticles physical mixed with amphotericin B and VC-loaded microparticles orally administered evoke Vibrio-specific serum IgG and IgM responses as well as vibriocidal antibody activity in mice. The VC incorporation, physicochemical characterization data, and the animal results obtained in this study may be relevant in optimizing the vaccine incorporation and delivery properties of these potential vaccine targeting carriers.