Most pathogens invade hosts via mucosal surfaces. Therefore, the induction of a mucosal immune response is necessary to prevent the invasion. Also, mucosal immunization can overcome the problems of parenteral immunization such as no induction of mucosal immune response, needle born infections, poor compliance, injection site pain, and local side effects from injections. Of the mucosal immunization methods, oral vaccinations have received attention because of their easy and acceptable properties. However, the antigens for oral vaccinations must be able to resist the harsh environment of the gastrointestinal tract, including various gastric acids, hydrolytic enzymes, and degradation of the ingested antigens. Therefore, development of an oral vaccine delivery system is needed to overcome these problems. Several delivery systems have been developed and among them, polymers exhibiting mucoadhesive or pH-sensitive properties have been reported to deliver the vaccines successfully through oral administration. Thiolated eudragit microspheres have both mucoadhesive and pH-sensitive properties. We tried to develop an oral vaccine delivery system against enterotoxigenic Escherichia coli (ETEC) using thiolated eudragit microspheres. ETEC is one of main causes of diarrhea or edema disease in piglets. The first infection of ETEC occurs by binding fimbriae of ETEC with porcine enterocytes, so F4 or F18 fimbriae, which is well-known virulence factor of ETEC, was selected as antigen protein of the vaccine.
We selected ETEC strains with multiplex PCR for F4 and F18 and purified F4 or F18 proteins by mechanical shearing and heat shock method. After that, the fimbriae proteins were loaded into thiolated eudragit microspheres (TEMS) and the characteristic feature of the vaccine was evaluated. The average particle sizes of TEMS, F4-loaded TEMS, and F18-loaded TEMS were measured as 4.2 ± 0.75 ㎛, 4.7 ± 0.50 ㎛, and 4.5 ± 0.37 ㎛, respectively. F4 is more efficiently encapsulated than F18 in the loading with TEMS. In the release test, F4 and F18 fimbriae were protected in acidic circumstances, whereas most were released at pH 7.4 of intestine circumstances.
For in vitro test, Raw 264.7 cells and mouse splenocytes were stimulated with unloaded TEMS, F4, F18, and F4 or F18 loaded TEMS and production levels of various cytokines were evaluated. Production of TNF-α and NO from Raw 264.7 cells was increased in a time-dependent manner after exposure to all groups, whereas only F4- or F18-loaded TEMS stimulated IL-6 secretion. The levels of IFN-γ from mouse splenocytes after exposure to F4 or F18 were increased while IL-4 was not detectable. With these results, in vitro immune stimulating activity of the vaccine was investigated.
For in vivo assay, we orally vaccinated mice with TEMS, F4, F18, F4-loaded TEMS, and F18-loaded TEMS. Mice that were orally administered with F4 or F18 loaded TEMS showed higher antigen-specific IgG antibody responses in serum and antigen-specific IgA in saliva and feces than mice that were immunized with antigens only. In addition, oral vaccination of F4 or F18 loaded TEMS resulted in higher numbers of IgG and IgA antigen-specific antibody secreting cells in the spleen, lamina propria, and Peyer’s patches of immunized mice than other groups. Moreover, TEMS administration loaded with F4 or F18 induced mixed Th1 and Th2 type responses based on similarly increased levels of IgG1 and IgG2a.
These results suggest that F4 or F18 loaded TEMS may be a promising candidate for an oral vaccine delivery system to elicit systemic and mucosal immunity against ETEC.