Production and assessment of ovine polyclonal antibodies to treat Clostridium difficile Infections
Cardiff Metropolitan University
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The clinical manifestations of Clostridium difficile infections (CDI) are due to the release of two powerful exotoxins, TcdA and TcdB. The aim of this study was to raise polyclonal antibodies (PcAb) in sheep that bind to and neutralise these toxins with a view to developing potent therapeutic agents for use in humans. Dose response studies were performed with recombinant fragments of each of the toxins, TxA4 and TxB4, in separate flocks of sheep. The PcAb produced showed high binding titres in an immunoassay and protected Vero cells against the two natural toxins in-vitro. TcdB was considerably more cytotoxic than TcdA with LC50 of 16 pg/mL and 1000 pg/mL respectively. The TxA4 immunogen stimulated a greater toxin neutralising immune response than TxB4. Thus, 1 mL of anti-TxA4 sera neutralised 1,800 μg of natural TcdA, whereas 1 mL of anti-TxB4 neutralised only 15 μg of TcdB. There was no correlation between binding titre and neutralising potency, suggesting that toxin inactivation was dependant on PcAb binding to specific regions on the natural toxins. Affinity purified PcAb, directed against distinct regions of TcdB, were fractionated and those directed to the central region possessed neutralising potencies of up to 250% greater than those directed elsewhere. These investigations identified the quantity and potency of specific PcAb directed to each of three distinct TcdB regions. A novel cell based assay, simulating the colonic epithelial barrier, was developed to investigate the translocation of TcdA and TcdB from simulated colonic lumen to the systemic circulation and to determine the effects of various toxin neutralising strategies. TcdA was shown to cause colonic epithelial damage allowing TcdB to translocate to the systemic circulation. Addition of neutralising PcAb either directly to simulated colonic lumen or systemic circulation provided protection to the intestinal epithelial layer and reduced or prevented toxin translocation.
PhD Thesis - School of Health Sciences
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