Local and systemic neutralizing antibody responses induced by intranasal immunization with the nontoxic binding domain of toxin a from Clostridium difficile

Fourteen of the 38 C-terminal repeats from Clostridium difficile toxin A (1 4CDTA) were cloned and expressed either with an N-terminal polyhistidine ta g (14CDTA-HIS) or fused to the nontoxic binding domain from tetanus toxin ( 14CDTA-TETC). The recombinant proteins were successfully purified by bovine thyroglobulin affinity chromatography. Both C. difficile toxin A fusion pr oteins bound to known toxin A ligands present on the surface of rabbit eryt hrocytes. Intranasal immunization of BALB/c mice with three separate 10-mu g doses of 14CDTA-HIS or -TETC generated significant levels of anti-toxin A serum antibodies compared to central animals. The coadministration of the mucosal adjuvant heat labile toxin (LT) from Escherichia coli (1 mu g) sign ificantly increased the anti-toxin B response in the serum and at the mucos al surface. Importantly, the local and systemic antibodies generated neutra lized toxin A cytotoxicity. Impressive systemic and mucosal anti-toxin A re sponses were also seen following coadministration of 14CDTA-TETC with LTR72 , an LT derivative with reduced toxicity which shows potential as a mucosal adjuvant for humans.