Neutralization of Clostridium difficile toxin A using antibody combinations

Abstract

The pathogenicity of Clostridium difficile (C. difficile) is mediated by the release of two toxins, A and B. Both toxins contain large clusters of repeats known as cell wall binding (CWB) domains responsible for binding epithelial cell surfaces. Several murine monoclonal antibodies were generated against the CWB domain of toxin A and screened for their ability to neutralize the toxin individually and in combination. Three antibodies capable of neutralizing toxin A all recognized multiple sites on toxin A, suggesting that the extent of surface coverage may contribute to neutralization. Combination of two noncompeting antibodies, denoted 3358 and 3359, enhanced toxin A neutralization over saturating levels of single antibodies. Antibody 3358 increased the level of detectable CWB domain on the surface of cells, while 3359 inhibited CWB domain cell surface association. These results suggest that antibody combinations that cover a broader epitope space on the CWB repeat domains of toxin A (and potentially toxin B) and utilize multiple mechanisms to reduce toxin internalization may provide enhanced protection against C. difficile-associated diarrhea.

Figure 1

Analysis of the neutralization capability of 3359 and 3358 combinations following a co-incubation with 80 ng (0.8 µg/mL) toxin A on CHO cell surfaces. (A) Titration of 3359 in the presence of 0, 0.5, 1 and 2 µg 3358. (B) Titration of 3358 in the presence of 0, 0.5, 1 and 2 µg 3359. The combination of the two antibodies (2 µg 3358 + 2 µg 3359) was significantly more neutralizing than 4 mg of 3358 or 4 mg of 3359 (p = 0.03 and p = 0.01, respectively).

Figure 2

Images from culture plates 48 h after seeding with CHO-K1 cells in the absence (A) and presence of toxin A (B). As a positive neutralization control, an anti-C. difficile toxin polyclonal antibody mixture (TE CHLAB^®) was added to the medium with toxin A (F). mAbs 3358 (C) and 3359 (D) were added separately and together (E) with toxin A to the culture media. Addition of 20 ng (0.2 mg/mL) toxin A to the CHO cell media led to >90% cell rounding following 2 days of incubation.

Figure 3

Antibody competition for ToxA:40R binding studied by surface plasmon resonance. Solutions containing 15 nM ToxA:40R were injected over surfaces containing immobilized rPCG-4 (circles), 3359 (triangles) or 3358 (squares). ToxA:40R was titrated with (A) rPCG-4 Fab, (B) rPCG-4 antibody, (C) 3359 or (D) 3358.

Figure 4

CHO cell surface binding of ToxA:11R in the presence and absence of anti-toxin A antibodies as determined by flow cytometry. Fluorescence of the CHO cell population (A) in the absence of ToxA:11R; (B) in the presence of 50 µg/mL ToxA:11R alone; and in the presence of (C) 250 µg/mL 3358, (D) 250 µg/mL 3359, (E) 250 µg/mL rPCG-4, (F) 250 µg/mL 3358 and 250 µg/mL 3359, (G) 250 µg/mL 3359 and 250 µg/mL rPCG-4 or (H) 250 µg/mL 3358 and 250 µg/mL rPCG-4.