The importance of the F4 receptor in post-weaned pigs In eliciting F4 specific immune responses in the intestine
In this Master’s dissertation, various doses of solubulized crude F4 fimbrial protein in conjunction with the adjuvants CpG ODN and porcine â-defensin 1 (pBD-1) were used to enhance the F4-specific intestinal immune response against Enterotoxigenic Escherichia coli (ETEC) F4 in post-weaned pigs. Using the mechanically shearing method we isolated the F4 fimbrial protein of ETEC with a molecular weight of 26 kDa. We verified this using a Western blot probed with a rabbit anti-F4 fimbrial antibody. Binding of the F4 fimbrial protein to the F4 receptor (F4R), present on the brush border of the villi in the small intestine of pigs, was demonstrated using an in vitro villus adhesion assay (IVVA). To demonstrate specificity rabbit polyclonal and mouse monoclonal anti-F4 antibodies, or the F4 protein were used to inhibit the adhesion of ETEC F4ac to F4R positive (F4Rpos) villi. To examine immunogenicity of the 500 micrograms (ìg) of the F4 were administered into surgically created jejunal gut-loops in pigs. Three weeks later Peyer’s patches (PP) from immunized and control loops as well as gut-wall tissue were analyzed for their F4-specific antibody secreting cells (ASCs) by a modified enzyme linked immunosorbent spot (ELISPOT) assay. The F4-specific immune response in the serum was analyzed by an enzyme linked immunosorbant assay (ELISA). High numbers of F4-specific ASCs were isolated from the loops of pigs that contained high levels of the F4R. Conversely nominal or low numbers of F4-specific ASCs were found in loops of pigs expressing low levels of the F4R or no F4R (F4Rneg). The IVVA was used to categorize the pigs into either F4Rpos or F4Rneg animals. Next three different concentrations of the crude F4 protein 50, 250, and 500 µg in the loops of individual pigs were used to analyze if dose affected the F4-specific immune response. Interestingly dose had no effect on the magnitude of the response. Therefore we hypothesized that the F4-specific immune response in the loops could be enhanced through the use of the adjuvants CpG ODN 2007 and pBD-1. The F4 protein was co-administered with either CpG ODN 2007 or pBD-1 and immune responses were assessed after 3 weeks. However neither CpG ODN 2007 nor PBD-1 at the doses used made an improvement in the immune response. Thus, these results demonstrated that the expression level of the F4R was the most important parameter for eliciting of the local immune response against the F4 protein. Furthermore our studies revealed that both F4Rneg and F4Rpos pigs responded to F4 immunization, however the former respond only nominally to F4-immunization in the loops. Moreover, an inverse relationship existed between the level of the F4-specific IgG in the serum and the F4-specific immune response seen in the loops. Thus our findings have important implications for oral vaccination using fimbrial based antigens (Ags) that utilize a receptor for their immunogenicity. Our results indicate that only animals with high levels of enterocyte F4R will have the ability to elicit high levels of protective F4-specific anti-fimbrial antibodies in their intestine after oral immunization. Therefore unless an effective adjuvant is available, animals with low to moderate levels of the fimbrial receptor in their small intestine will mount only weak immune responses making herd immunity after vaccination currently unattainable.
Gut-loop model, crude F4
Master of Science (M.Sc.)