In vitro assembly of novel cholera toxin-like complexes

Cholera toxin (CT) is responsible for the major pathological features of cholera, but in addition to its cytotoxic properties, CT is a potent mucosal adjuvant when coadministered with antigens at mucosal sites. Discovery of CT adjuvanticity has prompted the generation of CT chimeras with reduced toxicity and improved efficiency for antigen presentation at mucosal sites. To date, chimeric forms of CT have been produced in bacterial strains by coexpressing the CT B subunit and a chimeric form of the CT A subunit consisting of a target protein antigen fused with the A2 polypeptide of CT. In this study, a chimeric protein consisting of green fluorescent protein (GFP) fused with polypeptide A2 was generated to investigate the feasibility of assembling CT holotoxin-like complexes in vitro. The assembly of such holotoxin-like complexes would expand the variety of antigenic compounds that could be incorporated into CT-based vaccines. In this study, GFP-A2/CTB complexes could be generated in vitro using a stepwise denaturation-renaturation process. These findings suggest that it is possible to generate novel mucosal vaccines consisting of macromolecules that are chemically coupled to polypeptide A2 and reconstituted into CT-like complexes in vitro.