A Combination of Three Fully Human Toxin A- and Toxin B-Specific Monoclonal Antibodies Protects against Challenge with Highly Virulent Epidemic Strains of Clostridium difficile in the Hamster Model

Abstract

Clostridium difficile infection (CDI) is the principal cause of nosocomial diarrhea and pseudomembranous colitis associated with antibiotic therapy. Recent increases in the number of outbreaks attributed to highly virulent antibiotic-resistant strains underscore the importance of identifying efficacious alternatives to antibiotics to control this infection. CDI is mediated by two large exotoxins, toxins A and B. Strong humoral toxin-specific immune responses are associated with recovery and a lack of disease recurrence, whereas insufficient humoral responses are associated with recurrent CDI. Multiple approaches targeting these toxins, including intravenous immunoglobulin, neutralizing polymers, active vaccines, and, most recently, monoclonal antibodies (MAbs), have been explored, with various degrees of success. In this study, we describe the characterization of the first MAbs isolated from healthy human donors using a high-throughput B-cell cloning strategy. The MAbs were selected based on their ability to inhibit the actions of toxins A and B in vitro and because of their in vivo efficacy in a hamster challenge model. A potent 2-MAb cocktail was identified and then further potentiated by the addition of a second anti-toxin B MAb. This 3-MAb combination protected animals against mortality and also reduced the severity and duration of diarrhea associated with challenge with highly virulent strains of C. difficile toxinotypes 0 and III. This highly efficacious cocktail consists of one MAb specific to the receptor binding domain of toxin A and two MAbs specific to nonoverlapping regions of the glucosyltransferase domain of toxin B. This MAb combination offers great potential as a nonantibiotic treatment for the prevention of recurrent CDI.

FIG 1

Binding epitopes of B1, B2, B4, and B6 anti-toxin B MAbs. (A) Immunoblot of cloned fragments using candidate MAbs. (B) Dot blot of cloned CTDs of various toxinotypes with MAb B6. MWM, molecular weight marker.

FIG 2

Survival (A) and protection against illness (B) in hamsters treated with 50-mg/kg/dose of A2+B2, A2+B1, A2+B4, or A2+B6 MAb combinations following challenge with clinical C. difficile strain 630. X, death. The numbers in parentheses are the doses in milligrams per kilogram of body weight.

FIG 3

Survival (A) and protection against illness (B) in hamsters treated with 6-mg/kg/dose of A2+B1, A2+B2, or A2+B4 MAb combinations following challenge with clinical C. difficile strain 630. X, death. The numbers in parentheses are the doses in milligrams per kilogram of body weight.

FIG 4

Survival in hamsters treated with the A2+B2 MAb combination postchallenge with C. difficile strain VPI 10463. The numbers in parentheses are the doses in milligrams per kilogram of body weight.

FIG 5

Survival in hamsters treated with the A2+B2 MAb combination postchallenge with C. difficile clinical isolate 13695#7. The numbers in parentheses are the doses in milligrams per kilogram of body weight.

FIG 6

Survival (A) and protection against illness (B) in three-MAb-cocktail-treated hamsters postchallenge with C. difficile strain 13695#7. The numbers in parentheses are the doses in milligrams per kilogram of body weight.

FIG 7

Survival of hamsters treated with total 50 mg/kg/dose of individual or combinations A2, B2, and B1 MAbs following challenge with C. difficile strain 630. The numbers in parentheses are the doses in milligrams per kilogram of body weight.