Development and evaluation of an ovine antibody-based platform for treatment of Clostridium difficile infection

Abstract

Treatment of Clostridium difficile is a major problem as a hospital-associated infection which can cause severe, recurrent diarrhea. The currently available antibiotics are not effective in all cases and alternative treatments are required. In the present study, an ovine antibody-based platform for passive immunotherapy of C. difficile infection is described. Antibodies with high toxin-neutralizing titers were generated against C. difficile toxins A and B and were shown to neutralize three sequence variants of these toxins (toxinotypes) which are prevalent in human C. difficile infection. Passive immunization of hamsters with a mixture of toxin A and B antibodies protected them from a challenge with C. difficile spores in a dose-dependent manner. Antibodies to both toxins A and B were required for protection. The administration of toxin A and B antibodies up to 24 h postchallenge was found to reduce significantly the onset of C. difficile infection compared to nonimmunized controls. Protection from infection was also demonstrated with key disease isolates (ribotypes 027 and 078), which are members of the hypervirulent C. difficile clade. The ribotype 027 and 078 strains also have the capacity to produce an active binary toxin and these data suggest that neutralization of this toxin is unnecessary for the management of infection induced by these strains. In summary, the data suggest that ovine toxin A and B antibodies may be effective in the treatment of C. difficile infection; their potential use for the management of severe, fulminant cases is discussed.

Fig 1

Schematic of passive immunization study. Ovine antibody (Ab) was administered on the days indicated.

Fig 2

Protection from CDI by passive immunization with ovine antitoxin A/B. Graph shows survival postchallenge with C. difficile VPI 10463 spores in the hamster model. Animals were administered with IgG preparations purified from antisera raised using toxoid, and the specific neutralizing titers for toxins A and B were 800 and 100 U/mg, respectively. Three doses of antibody were administered as indicated, and each consisted of the following: ♦, 25 mg of antitoxin A/B IgG (1 × 10⁴ U for toxin A, 1.3 × 10³ U for toxin B); ▴, 2.5 mg of antitoxin A/B IgG (1 × 10³ U for toxin A, 1.3 × 10² U for toxin B); and •, 12.5 mg of nonspecific ovine IgG. Comparison of survival curves by log-rank analysis showed that for “♦” and “▴” values P < 0.001 compared to the control group (•).

Fig 3

Requirement of toxin A and B antibodies for protection from CDI. The graph shows survival postchallenge with C. difficile VPI 10463 spores in the hamster model. Animals were administered with IgG preparations purified from antisera raised using toxoid, and the specific neutralizing titers for toxins A and B were 800 and 400 U/mg, respectively. Animal groups were administered with three doses of antibody as indicated, and each consisted of the following: ▾, 25 mg of antitoxin A/B IgG (1 × 10⁴ U for toxin A, 5 × 10³ U for toxin B); ▵, 25 mg of anti-toxin A IgG (2 × 10⁴ U); ○, 25 mg of anti-toxin B (1 × 10⁴ U); and ☐, 12.5 mg of nonspecific ovine IgG. Log-rank analysis showed that for “▾” and “▵” values P < 0.001 compared to the controls (☐) and for “○” values P = 0.56.

Fig 4

Protection from challenge with 027 and 078 ribotype C. difficile strains. Graphs show the survival postchallenge with C. difficile 027 spores (A) and 078 spores (B) in the hamster model. IgG preparations were as described in Fig. 2. Animal groups were administered with 3 doses of antibody as indicated, and each consisted of the following: ▾, 25 mg of antitoxin A/B IgG (1 × 10⁴ U for toxin A, 1.3 × 10³ U for toxin B), or • and ■, 12.5 mg of nonspecific ovine IgG. A comparison of antibody-treated and control survival curves by log-rank analysis showed that P = 0.002 (A) and P < 0.001 (B).

Fig 5

Protective effects of toxin A and B antibodies administered after challenge. Graph shows survival postchallenge with C. difficile VPI 10463 spores in the hamster model. IgG preparations were as described in Fig. 3. Antibody treatment (two doses of 25 mg of antitoxin A/B IgG, administered 72 h apart, each containing 1 × 10⁴ U for toxin A and 5 × 10³ U for toxin B) was initiated: ▾, 24 h prechallenge; ☐, 5 h postchallenge; or ▵, 24 h postchallenge. A control group (•) received no antibody treatment. A comparison of survival curves by log-rank analysis showed that for “☐” values P = 0.001, for “▵” values P = 0.016, and for “▾” values P = 0.061 compared to the control group.