Synthetic peptides derived from the G-H loop of the Foot-and Mouth Disease Virus (FMDV) capsid protein VP1 are relatively poor at recapitulating the native conformation present in the virus, and thus are often poor immunogens. We hypothesized that a candidate mucosal vaccine against FMDV could be developed using the non-toxic Pseudomonas aeruginosa exotoxin A (ntPE) and B subunit of E. coli heat labile enterotoxin (LTB) to deliver the G-H loop in its native conformation. PE, secreted by Pseudomonas aeruginosa is a mucosal adjuvant that binds to α2-macroglobulin receptor/Low density Lipoprotein receptor-related Protein (LRP1) /CD91 present on epithelial cells and APC plasma membranes to traverse the mucosal barrier. The LTB subunit, a 55KDa pentameric molecule is also a potent mucosal adjuvant and is responsible for binding to various eukaryotic cell receptors including GM1, glycosphingolipids, glycoprotein receptors, polyglycosilceramides and paraglobosides.
The chimeric protein ntPE-GH was generated by inserting the coding sequence of the G-H loop into an expression plasmid encoding ntPE, in place of the native Ib loop. Chimeric proteins LTA2B-GH and ntPE-LTA2B-GH were generated by inserting the coding sequence of the G-H loop at the C-terminus of LT where the toxic LTA1 domain is either removed or replaced with the protein of interest (ntPE) that allows the formation of holotoxin-like chimeras. These recombinant fusion proteins containing His6 tag were each expressed in E. coli , purified over a nickel resin and tested for their effectiveness in inducing anti-FMDV systemic and mucosal immune responses. In conclusion, this study demonstrates that the chimeric proteins ntPE-GH, LTA2B-GH and ntPE-LTA2B-GH could be used as a mucosal carriers/adjuvants to induce immune response against FMDV infection.
