Antibody Against TcdB, but Not TcdA, Prevents Development of Gastrointestinal and Systemic Clostridium difficile Disease

Clostridium difficile, an anaerobic, spore-forming, gram-positive bacillus, is the leading cause of antibiotic-associated diarrhea in developing countries. A hypervirulent, epidemic strain, designated NAP1/027/BI, has caused an increase in C. difficile infection (CDI) case and fatality rates since the early 2000s [1, 2]. The symptoms of CDI, ranging from mild diarrhea to severe disease including pseudomembranous colitis, toxic megacolon, sepsis, shock, and even death, are thought to be caused mainly by 2 toxins—toxin A (TcdA) and toxin B (TcdB). Understanding the relative roles of these 2 toxins in disease pathogenesis is of great importance. Although the toxins have been investigated using numerous methods over the past several decades, the results have been conflicting. In earlier studies using culture supernatants or purified toxins in rodent or rabbit models, TcdA and/or TcdB were administered into the gastrointestinal (GI) tract directly via gavage or intestinal loop. Results indicated that TcdA was the essential virulence factor, with TcdB unable to induce lesions when administered in the absence of TcdA [3, 4]. When highly purified toxins are administered systemically to rodents, either together or separately, both are able to induce disease and death [5]. The finding of cardiotoxicity associated with TcdB in the zebrafish model more recently highlighted the potential for systemic actions of the toxins and the importance of TcdB, in particular [6], although this has yet to apply to mammalian species. Active and passive immunity have also been used to investigate the roles of the 2 toxins. Using toxoid for immunization in hamsters, 1 study found that immunization with both TcdA and TcdB toxoids was necessary for complete protection [7], but another study found that immunization with only TcdA toxoid was necessary for protection [8]. Investigation of naturally occurring serum antitoxin antibodies in human patients placed an emphasis on the importance of anti-TcdA in preventing recurrence of CDI [9]. Human monoclonal antibodies (HuMab) have been generated against both toxins, and studies in hamsters revealed that HuMab against both toxins prevented CDI in hamsters [10]. Likewise, studies in human patients also highlighted the importance of antibodies against both toxins, not just TcdA, for protection from recurrence [11]. In the past several years, methods for genetic manipulation of C. difficile have allowed for creation of isogenic mutant strains that produce only TcdA or TcdB. One such study showed TcdB to be the essential virulence factor in the hamster model [12], but another study found that both TcdA and TcdB are important for virulence [13]. The results of these numerous studies indicate that neither toxin can be ignored in terms of the importance in disease pathogenesis or for development of novel treatments and therapeutics. However, we are still left with many unanswered questions. In this study we used HuMabs [10] and alpaca polyclonal antibodies against TcdA and/or TcdB in the piglet model of CDI [14] to further investigate the roles of the 2 toxins in pathogenesis and to provide more information on the use of antitoxin antibodies for prevention and treatment of CDI in human patients.