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

Abstract

Fourteen of the 38 C-terminal repeats from Clostridium difficile toxin A (14CDTA) were cloned and expressed either with an N-terminal polyhistidine tag (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 proteins bound to known toxin A ligands present on the surface of rabbit erythrocytes. Intranasal immunization of BALB/c mice with three separate 10-microg doses of 14CDTA-HIS or -TETC generated significant levels of anti-toxin A serum antibodies compared to control animals. The coadministration of the mucosal adjuvant heat labile toxin (LT) from Escherichia coli (1 microg) significantly increased the anti-toxin A response in the serum and at the mucosal surface. Importantly, the local and systemic antibodies generated neutralized toxin A cytotoxicity. Impressive systemic and mucosal anti-toxin A responses 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.

FIG. 1

Thyroglobulin affinity-purified 14CDTA-HIS (A) and 14CDTA-TETC (B) in SDS–10% PAGE gels stained with Coomassie blue. Lanes 1, cell lysates before affinity column; lanes 2, cell lysates after affinity column; lanes 3, purified protein collected from column (approximately 3 μg); lanes M, prestained molecular weight markers (apparent molecular masses are shown in kilodaltons).

FIG. 2

Immunoblot of affinity-purified 14CDTA-HIS (lanes 1) and 14CDTA-TETC (lanes 2) against the toxin A-specific monoclonal antibody PCG-4 (A) or anti-TT polyclonal antiserum (B). Lanes M, molecular weight markers (apparent molecular masses are shown in kilodaltons).

FIG. 3

Mean anti-toxin A total Ig responses in the serum of i.n. immunized BALB/c mice. Antibody titers were measured by ELISA in serum taken after one dose (day 19), two doses (day 34), and three doses (day 47) of antigen. Mean titers are shown ± SDs from five mice. Individual preimmune titers have been subtracted from the calculated titer of each corresponding mouse.

FIG. 4

Mucosal toxin A-specific IgA responses generated by three i.n. doses of antigen in nasal (A) and pulmonary (B) lavage samples. Responses show the variation in the immune responses between individual mice within each group. Bars represent mean antibody titers.

FIG. 5

Mean anti-TT total Ig responses in the serum of i.n. immunized BALB/c mice. Antibody titers were measured by ELISA in serum taken after one dose (day 19), two doses (day 34), and three doses (day 47) of antigen. Mean titers are shown ± SDs from five mice. Individual preimmune titers have been subtracted from the calculated titer of each corresponding mouse.

FIG. 6

Mucosal TT-specific IgA responses generated by three i.n. doses of antigen in nasal (A) and pulmonary (B) lavage. Responses show the variation in the immune responses between individual mice in each group. Bars represent mean antibody titers.