International Clostridium difficile symposium edition:4 location:Bled, Slovenia date:20-22 September 2012
Introduction - Clostridium difficile associated disease (CDAD) is increasing in incidence and severity with significant morbidity and mortality. Antibiotic treatment is not invariably successful with a relapse rate around 30%. Since only toxin A and/or B (TcdA, TcdB) producing strains are proven to be pathogenic, we aim to develop a novel treatment modality based on targeted mucosal delivery of anti- Clostridium difficile toxin-antigen binding fragments.
Methods - SJL/J mice were immunized with Clostridium difficile toxoid by a standard protocol for hybridoma generation. Monoclonal antibody (MA) production was evaluated on coated TcdA and TcdB. Selected MAs were evaluated for their in vitro neutralization capacities using CCL-186 fibroblasts and TcdA or TcdB at 570 pM or 56 pM, respectively. Affinities were determined on a Biacore 3000. Hemagglutination experiments were performed on rabbit red blood cells (1%, 16 nM TcdA) and relative epitope-mapping was based on pair-wise combination of MAs in sandwich-type ELISAs. In vivo neutralization capacities of the MAs were evaluated by mouse ileal loops in C57BL/6 mice (65 nM TcdA, 4 hours incubation, 2 cm loops either with or without 72 – 113 uM MA). Gene expression was analyzed by standard RNA extraction and RealTime-PCR.
Results - Out of 99 monoclonal antibodies that were reactive against TcdA and/or TcdB, 4 neutralized TcdA in vitro (average EC50: 15 nM), 6 neutralized TcdB in vitro (average EC50: 3,02 nM) and 1 antibody neutralized both toxins (EC50-TcdA: 13 nM, EC50-TcdB: 140 nM) in vitro. Hemagglutination against TcdA showed that 4 anti-TcdA MAs were targeting the receptor binding domain and 1 was not. Biacore affinity studies on 4 MAs against TcdA demonstrated high affinity against TcdA and moreover, very slow dissociation rates. Relative epitope mapping resulted in the selection of 6 neutralizing MAs representing 3 distinct epitopes at each toxin. In vivo neutralization activitity was confirmed for three selected MAs by a decrease of the expression at mRNA level of IL6, CXCL1 and TNFα after injection in the ileal loop of TcdA and the MA, compared to injection with TcdA alone.
Discussion - This study identifies several monoclonal antibodies with high affinity and bioactivity in vitro as well as in vivo. We will further seek in vivo confirmation in the well-established Syrian gold hamster model of CDAD.