Vol. 59, No. 3
INFECTION AND IMMUNITY, Mar. 1991, p. 1192-1195
0019-9567/91/031192-04$02.00/0 Copyright © 1991, American Society for Microbiology
Protection against Experimental Pseudomembranous Colitis in Gnotobiotic Mice by Use of Monoclonal Antibodies against Clostridium difficile Toxin A G. CORTHIER,1* M. C. MULLER,' T. D. WILKINS,2 D. LYERLY,2 AND R. L'HARIDON3 Laboratoire d'Ecologie et de Physiologie du Systeme Digestif' and Laboratoire de Virologie et d'Immunologie Moleculaire,3 Centre de Recherches de Jouy, 78352 Jouy-en-Josas, France, and Department ofAnaerobic Microbiology, Virginia Polytechnic Institute and State University, Blacksburg, Virginia 240612 Received 7 June 1990/Accepted 24 November 1990
The pathogenicity of Clostridium difficile is due to the production of two toxins (toxins A and B). We prepared monoclonal antibodies against toxin A and determined whether axenic mice passively immunized with the monoclonal antibodies were protected against C. difficile disease. The mice were kept in an isolator and were given ascites fluid intravenously prior to challenge with a toxinogenic strain of C. difficile. Control mice and mice receiving ascites fluid devoid of toxin antibody died within 2 days and had high levels of toxins A and B in their feces. Mice that received ascites fluid containing high amounts of toxin A monoclonal antibodies directed against the repeating units of the toxin survived. In protected mice, toxin B levels were similar to those in dying mice, but toxin A levels were greatly reduced. These data show that passive immunity induced by monoclonal antibodies against toxin A was effective against pseudomembranous cecitis.
precipitates toxin A and neutralizes the enterotoxic activity of the toxin. In the following study, we prepared a series of MAbs against toxin A and compared these MAbs with MAb PCG-4 for their reaction with toxin A. Studies were then done to determine whether the passive systemic immunity afforded by the MAbs was protective against C. difficile disease in axenic mice inoculated with a toxigenic strain of C. difficile. Our results show that the MAb developed in this study reacted with the repeating units located on the COOH terminus of toxin A and that these MAbs as well as MAb PCG-4 effectively protected the mice. C. difficile strain VPI 10463 (referred to as VPI) was obtained from the anaerobe collection of the Department of Anaerobic Microbiology, Virginia Polytechnic Institute and State University (Blacksburg, Va.). The strain was grown in brain-heart infusion (BHI) broth (Difco Laboratories, Detroit, Mich.). For plate counts, samples were homogenized, serially diluted, and plated on BHI agar. Colony counting was performed after incubation at 37°C for 2 days. The toxin A preparation was obtained by using C. difficile grown within a dialysis bag in flasks containing autoclaved BHI. Flasks were incubated for 4 days at 37°C in an anaerobic chamber. Toxin A was purified as described previously (27). The purified toxin gave a single band in polyacrylamide gel electrophoresis (PAGE) (16) and Western immunoblot analysis (28) and had a molecular mass of 400,000 Da. C3He/J axenic adult mice were reared in a Trexler-type isolator fitted with a rapid transfer system (La Calhene, Velizy, France) and fed a rodent diet (R03-40, UAR, Villemoisson, France) ad libitum. All materials used for the mice were sterilized by heat or gamma irradiation. Pseudomembranous cecitis was induced as follows. Axenic mice were inoculated through the orogastric route with 1 ml of a 24-h culture of C. difficile VPI (ca. 108 vegetative cells per ml). Under these conditions, mice developed a disease characterized by an intense cecal abrasion together with a severe inflammatory process. All the animals died within 2 days (3, 4). For passive protection studies, ascites fluids diluted 1:3 were injected intravenously (0.2 ml
Clostridium difficile, which causes pseudomembranous colitis and antibiotic-associated diarrhea (2, 10, 17, 20, 26), produces two toxins, designated toxins A and B (27). Toxin A is a tissue-damaging enterotoxin, whereas toxin B is a potent cytotoxin. It has been suggested that the tissue damage caused by toxin A represents the initial stage of the disease and that this action may allow toxin B to act (23). It appears that toxin A acts after it binds to its receptor via the series of repeating units located at the COOH terminus of the toxin molecule (7, 15). Several animal models (hamsters, hares, guinea pigs, prairie dogs, monkeys, and gnotobiotic mice) have been used to study the disease. The most commonly used model consists of conventional hamsters treated with clindamycin (26). Another model consists of axenic rodents colonized with C. difficile (4, 6). In both models, the disease develops within several days if highly toxigenic strains are used. The results of previous studies have shown that vaccination against both toxins is needed to protect conventional hamsters colonized with highly toxigenic strains (8, 11, 13, 14, 18). For example, Libby et al. (18) showed that active immunity against toxin A was not sufficient to protect hamsters against experimental infection. These observations have been substantiated by the works of other investigators (13, 14), who demonstrated that active immunity against both toxins was protective. Kim et al. (13) showed, however, that vaccination against toxin A but not against toxin B could be sufficient. The antibody-mediated protection can be transferred from the mother to litters through milk (12-14). The results of several studies have shown that hamsters also can be passively immunized against C. difficile with Clostridium sordellii antitoxin (1, 8). At present, little is known about the influence of passive systemic immunity against toxin A. Monoclonal antibodies (MAbs) have been prepared by several teams (22, 25). MAb PCG-4 (22) is an immunoglobulin G2a (IgG2a) isotype which *
Corresponding author. 1192
VOL. 59, 1991
NOTES
1193
TABLE 1. Screening of ascites fluid containing MAbs against toxin A ELISA titera
Neutralization Immunoblot Agarose . (no. of survivors/ .. .
MAb
Native toxin A
A9 141-2
C1l PCG-4 D19
Recombinant peptide
Istp Isotype
5 tested) 1v41.LUII 51
IgG ^f arte maw/ml UL a3ILt) content
4.2 4.2 5.2 5.3
3.4 4.2 4.3 4.3
+ + + +
+ + + +
5 4
IgG2a(K) IgGl(K)
3 0
IgGl(K) IgG2a(K)
0.8 11 0.1 8
INFECTION AND IMMUNITY, Mar. 1991, p. 1192-1195
0019-9567/91/031192-04$02.00/0 Copyright © 1991, American Society for Microbiology
Protection against Experimental Pseudomembranous Colitis in Gnotobiotic Mice by Use of Monoclonal Antibodies against Clostridium difficile Toxin A G. CORTHIER,1* M. C. MULLER,' T. D. WILKINS,2 D. LYERLY,2 AND R. L'HARIDON3 Laboratoire d'Ecologie et de Physiologie du Systeme Digestif' and Laboratoire de Virologie et d'Immunologie Moleculaire,3 Centre de Recherches de Jouy, 78352 Jouy-en-Josas, France, and Department ofAnaerobic Microbiology, Virginia Polytechnic Institute and State University, Blacksburg, Virginia 240612 Received 7 June 1990/Accepted 24 November 1990
The pathogenicity of Clostridium difficile is due to the production of two toxins (toxins A and B). We prepared monoclonal antibodies against toxin A and determined whether axenic mice passively immunized with the monoclonal antibodies were protected against C. difficile disease. The mice were kept in an isolator and were given ascites fluid intravenously prior to challenge with a toxinogenic strain of C. difficile. Control mice and mice receiving ascites fluid devoid of toxin antibody died within 2 days and had high levels of toxins A and B in their feces. Mice that received ascites fluid containing high amounts of toxin A monoclonal antibodies directed against the repeating units of the toxin survived. In protected mice, toxin B levels were similar to those in dying mice, but toxin A levels were greatly reduced. These data show that passive immunity induced by monoclonal antibodies against toxin A was effective against pseudomembranous cecitis.
precipitates toxin A and neutralizes the enterotoxic activity of the toxin. In the following study, we prepared a series of MAbs against toxin A and compared these MAbs with MAb PCG-4 for their reaction with toxin A. Studies were then done to determine whether the passive systemic immunity afforded by the MAbs was protective against C. difficile disease in axenic mice inoculated with a toxigenic strain of C. difficile. Our results show that the MAb developed in this study reacted with the repeating units located on the COOH terminus of toxin A and that these MAbs as well as MAb PCG-4 effectively protected the mice. C. difficile strain VPI 10463 (referred to as VPI) was obtained from the anaerobe collection of the Department of Anaerobic Microbiology, Virginia Polytechnic Institute and State University (Blacksburg, Va.). The strain was grown in brain-heart infusion (BHI) broth (Difco Laboratories, Detroit, Mich.). For plate counts, samples were homogenized, serially diluted, and plated on BHI agar. Colony counting was performed after incubation at 37°C for 2 days. The toxin A preparation was obtained by using C. difficile grown within a dialysis bag in flasks containing autoclaved BHI. Flasks were incubated for 4 days at 37°C in an anaerobic chamber. Toxin A was purified as described previously (27). The purified toxin gave a single band in polyacrylamide gel electrophoresis (PAGE) (16) and Western immunoblot analysis (28) and had a molecular mass of 400,000 Da. C3He/J axenic adult mice were reared in a Trexler-type isolator fitted with a rapid transfer system (La Calhene, Velizy, France) and fed a rodent diet (R03-40, UAR, Villemoisson, France) ad libitum. All materials used for the mice were sterilized by heat or gamma irradiation. Pseudomembranous cecitis was induced as follows. Axenic mice were inoculated through the orogastric route with 1 ml of a 24-h culture of C. difficile VPI (ca. 108 vegetative cells per ml). Under these conditions, mice developed a disease characterized by an intense cecal abrasion together with a severe inflammatory process. All the animals died within 2 days (3, 4). For passive protection studies, ascites fluids diluted 1:3 were injected intravenously (0.2 ml
Clostridium difficile, which causes pseudomembranous colitis and antibiotic-associated diarrhea (2, 10, 17, 20, 26), produces two toxins, designated toxins A and B (27). Toxin A is a tissue-damaging enterotoxin, whereas toxin B is a potent cytotoxin. It has been suggested that the tissue damage caused by toxin A represents the initial stage of the disease and that this action may allow toxin B to act (23). It appears that toxin A acts after it binds to its receptor via the series of repeating units located at the COOH terminus of the toxin molecule (7, 15). Several animal models (hamsters, hares, guinea pigs, prairie dogs, monkeys, and gnotobiotic mice) have been used to study the disease. The most commonly used model consists of conventional hamsters treated with clindamycin (26). Another model consists of axenic rodents colonized with C. difficile (4, 6). In both models, the disease develops within several days if highly toxigenic strains are used. The results of previous studies have shown that vaccination against both toxins is needed to protect conventional hamsters colonized with highly toxigenic strains (8, 11, 13, 14, 18). For example, Libby et al. (18) showed that active immunity against toxin A was not sufficient to protect hamsters against experimental infection. These observations have been substantiated by the works of other investigators (13, 14), who demonstrated that active immunity against both toxins was protective. Kim et al. (13) showed, however, that vaccination against toxin A but not against toxin B could be sufficient. The antibody-mediated protection can be transferred from the mother to litters through milk (12-14). The results of several studies have shown that hamsters also can be passively immunized against C. difficile with Clostridium sordellii antitoxin (1, 8). At present, little is known about the influence of passive systemic immunity against toxin A. Monoclonal antibodies (MAbs) have been prepared by several teams (22, 25). MAb PCG-4 (22) is an immunoglobulin G2a (IgG2a) isotype which *
Corresponding author. 1192
VOL. 59, 1991
NOTES
1193
TABLE 1. Screening of ascites fluid containing MAbs against toxin A ELISA titera
Neutralization Immunoblot Agarose . (no. of survivors/ .. .
MAb
Native toxin A
A9 141-2
C1l PCG-4 D19
Recombinant peptide
Istp Isotype
5 tested) 1v41.LUII 51
IgG ^f arte maw/ml UL a3ILt) content
4.2 4.2 5.2 5.3
3.4 4.2 4.3 4.3
+ + + +
+ + + +
5 4
IgG2a(K) IgGl(K)
3 0
IgGl(K) IgG2a(K)
0.8 11 0.1 8
