The development of sensitive methods to quantify specfic antibodies directed against Clostridium difficile toxins and their use to provide improved immunotherapeutic products (Presentation)

Student: Christopher Phillips
Company: MicroPharm Ltd
Academic Supervisor: Professor Rose Cooper & Professor Ken Jones

Immunotherapy for Severe C. Difficile Infection

Over the past decade the ‘superbug’, Clostridium difficile, has become the most common hospital acquired infection in the developed world. Epidemic strains have emerged which exhibit enhanced virulence factors including multidrug resistance, increased capacity to form highly resistant spores and produce significantly more toxins A and B than conventional strains. Leakage of toxin into the blood ultimately leads to multi organ failure. These factors help explain why C. difficile infection (CDI) is becoming more frequent, severe and difficult to treat.

Currently, there are approximately 20,000 CDI cases reported in the UK per annum with up to 25% of patients experiencing a relapse of symptoms after withdrawal of antibiotic treatment. Of those patients who suffer a relapse, up to 65% suffer from multiple recurrences and in severe or complicated cases, surgical intervention is often necessary. Presently, the ‘in hospital’ mortality rate for patients infected with the more virulent strains of C. difficile is up to 37%. The cost to the UK healthcare system is reported to be approximately £7,000 per CDI case with an estimated cost worldwide of over $10 billion per annum. Clearly, alternative treatments are urgently required.

MicroPharm, a small bio-pharmaceutical company based in West Wales, specialises in producing antibodies to treat acute toxic events, such as snake bites and drug overdose. Recently, recombinant fragments based on C. difficile toxins A and B have been used to raise antibodies in sheep which were shown to have high toxin neutralising activity against their respective native toxins in a cell based assay.

In the present study, we assessed a range of doses of either toxin A or B by immunising two flocks of 15 sheep and monitoring the optimum dose required to elicit high titres of anti-toxin. Both flocks were sub-divided into five groups, each receiving a different dose (from 50μg to 2mg), with serum samples taken and re-immunisation given at four week intervals. Binding titre was assessed by enzyme immunoassay and potency was measured by a cell based toxin neutralisation assay.

Toxin B was found to be nearly 100 fold more toxic than A with the effective concentration required to cause cytopathic effect in 50% of the cells (LC50) measured at 1000 pg/mL and 16 pg/mL for each respectively. In the dose ranging study, toxin A produced a better response with resulting antitoxin shown to neutralise 1,800 μg of toxin per mL of antisera (neutralising potency) compared to 15 μg/mL for B.

Binding titre was found to be higher than neutralising potency in all dose groups in the first 14 weeks of immunisations with no dose dependant response evident. However, at week 22 the neutralising potency of the higher dose groups began to trend upwards with no corresponding increase in binding titre.

These results demonstrate that C. difficile anti-toxin can be raised in sheep and that higher immunising doses result in an increased response, in terms of toxin neutralising potency. This is an important step towards the development of an immunotherapeutic for the treatment of severe cases of CDI.