Novel biotherapeutics for the prevention and control of Campylobacter spp. in chickens

The most important food-poisoning bacterium both in terms of incidence and in the damage that it causes is Campylobacter. In the UK, out of an estimated one million cases of foodborne disease each year, Campylobacter is responsible for up to 750,000 cases, 22,000 hospitalisations and 110 deaths. Current estimates by the Food Standards Agency indicate that the cost of human campylobacteriosis in the UK is around £1B, out of a total of around £1.5B for all foodborne infections.

Campylobacter is found in the gastrointestinal tracts of animals destined for human consumption, with faecal contamination of meat during processing a recognised route of transmission to humans. Up to 80% of raw chicken sold in the UK is contaminated with Campylobacter, and studies suggest that the consumption of poultry is responsible for 50-70% of all endemic infections that are reported. The current level of contamination of raw poultry on sale in the UK presents an unacceptably high public health burden, and the number of human campylobacteriosis cases continues to increase. Alternative strategies are needed to reduce Campylobacter in chickens and the associated disease burden in humans.

Once a chicken is colonised with Campylobacter, antibodies against the bacterium are generated and a decrease in the number of Campylobacter colonising the intestinal tract has been observed. These data suggest that passive immunotherapy using anti-Campylobacter antibodies could be an approach for interfering with colonisation in chickens. The approach could also benefit humans and other animals. This is supported by the evidence that, in the general population, humoral immune response to a number of Campylobacter antigens is developed in most people upon infection, and epidemiological studies indicate that the immunity is crucial for the development of protection against Campylobacter disease.

Antibody fragments or similar small peptide molecules are under-used as therapeutics. We contend that they have potential as therapeutic and prophylactic treatments for chickens, humans and other animals against Campylobacter. We propose to use antibody library technologies, protein-directed panning and early engagement of functional screens to identify single-chain Fvs (scFvs) that block or impair the activity of Campylobacter proteins. We plan to deliver the molecules via a harmless probiotic bacterium, Bacillus subtilis, either in vegetative cell or spore form, that has been genetically altered to synthesise or release the antibody fragment in situ within the chicken gastrointestinal tract, thus providing a continuous source of scFv in vivo for therapeutic and prophylactic applications. This local in situ production of scFv has many advantages over delivery of the same reagent by conventional means. For example, removing downstream processing steps, reducing the cost of goods, and doing away with any requirement for complex dosing regimens or delivery vehicles. Such a therapeutic could be delivered in food or drink. Alternatively, the bacteria and/or bacterial spores could be used to produce the scFv(s) cheaply and easily for more traditional methods of drug delivery.

Campylobacter spp. are the most prevalent cause of bacterial diarrhoeal disease worldwide. There is an urgent need to develop novel therapeutic options. We contend that there is an opportunity to utilise antibody fragments (scFvs), to bind to, and inhibit the activity of, key surface-exposed proteins that are essential for Campylobacter jejuni and Campylobacter coli viability and/or colonisation of chickens, without the requirement to induce the effector-arm of the immune system or prime innate immunity.

Using data derived from our previously funded functional genomic, genomic and bioinformatics approaches, we have prioritised three antigens that are core C. jejuni and C. coli extracellular or outer membrane proteins that are either 'essential' (required for fitness) or 'conditionally essential' (required for colonisation of chickens. We plan to screen single-chain variable fragment (scFv) antibody libraries against the three recombinant proteins as well as whole C. jejuni and C. coli. By performing multiple rounds of affinity selection against the antigens and the whole bacteria, a population of scFvs that can access the antigenic target on the bacteria will be isolated. From the enriched population, unique scFvs will be screened via in vitro assays for their ability to bind to C. jejuni and C. coli in a strain-independent manner. Following affinity selection, scFvs will be screened for functional activity against C. jejuni and C. coli. Subsequently, for the target-independent screens, the target proteins for the functional scFvs will be determined. Following library screening, the DNA pool representing the selected antibody population will be recovered and full-length open reading frames will be cloned into a suitable vector and introduced into B. subtilis in order to generate spores or vegetative cells producing scFv against C. jejuni and/or C. coli antigens. The activity of these scFvs will be assessed in in vitro co-culture experiments.