Campylobacter phase variation and its impact on immunity and vaccine development.

Campylobacter is the commonest cause of bacterial food poisoning in the UK. One of the most frequent sources of infection for humans comes from eating undercooked poultry or cross contamination by uncooked meat during food preparation. The main problem is that Campylobacter can readily colonise poultry to very high levels without causing disease while ingestion of small amounts of bacteria by humans leads to severe gastroenteritis. These infections can result in complications as the bacterium interacts with the human immune system to cause irritable bowl syndrome and Guillain-Barré syndrome, which is an effect on the nerves of patients causing paralysis. Control of Campylobacter in poultry is predicted to significantly reduce human cases of infection. One route to achieve this is by vaccination. Vaccines induce antibodies to specific surface components of bacteria. These antibodies bind to the bacteria cells and interfere with the ability of these cells to colonise the bacterial hosts. Vaccines for Campylobacter are available but have a variable efficacy in preventing the bacterium from colonising chickens. One reason this approach is a problem is because Campylobacter naturally induces the production of antibodies against its surface components but these antibodies do not prevent colonisation or clear the bacteria from birds. Given that a range of antibody responses are generated, it is surprising that these adaptive immune responses do not clear the bacteria. This suggests that the bacteria must have a way of avoiding antibody-mediated control. We believe that Campylobacter can avoid antibodies by switching 'off' the expression of key surface molecules so that they are not recognised by the chicken's antibodies. These switches can occur at high frequencies in a process known as phase variation, which is widespread in bacterial species and is usually associated with surface components. In Campylobacter, phase variation is mediated by repeat sequences in the DNA of the bacteria which undergo a higher than normal frequency of mutation during replication of the bacteria and result in genes being switched 'on' or 'off'. If an antibody acts against the product of one of these phase variable genes, this generates selection for a change in the component by phase variation facilitating persistence of the Campylobacter in the birds. This process is reversible allowing the bacteria to recover the function of the gene when the antibodies dissipate. There is only a limited amount of data on how this process works when Campylobacter infects chickens. This project will identify the frequency of these switches and how the production of antibody impacts on selection of phase variants by the immune system. Specifically, we will investigate whether phase variation is involved in avoidance of antibodies during natural infections and following vaccination. We will assess some novel vaccine candidates for which we have evidence that phase variation may interfere with the effectiveness of induced antibodies and will modify some genes so they cannot switch 'off'. This data will not only be important in understanding how this pathogen survives in poultry but also in humans where the surface components play a major role in disease. The data generated in this project will aid in the development of effective vaccines to protect both animals and man against infections by this bacterium.

Phase variation (PV) of surface determinants influences both commensal and pathogenic behaviour in many bacterial species. PV in C. jejuni is mediated by mutations in polyC/G tracts. Each genome of C. jejuni contains 16 or more phase variable genes whose products modify capsule, lipo-oligosaccharide and flagella. These surface structures are major targets for host adaptive immunity. We have shown that some phase-variable genes are involved in persistence of C. jejuni in the avian GI-tract suggesting that understanding PV could improve the efficacy of current attempts to limit colonisation. .Reduction of C. jejuni in the poultry food chain would reduce the very high incidence of C. jejuni-associated gasteroenteritis. Colonisation of chickens by C. jejuni induces adaptive immune responses however there is limited evidence for this response promoting clearance. Our published and unpublished data indicates that PV of surface components, including glycosylation of flagella, facilitates colonisation and spread in flocks. There is therefore a strong rationale for investigating the role of PV in immune avoidance and host colonisation. We have already tested the methods required to study PV in vivo and in vitro and shown by analysis of input and output populations from chickens that we can accurately monitor PV in vivo. We will assess PV of 29 genes and the immune responses against C. jejuni following inoculation of chickens with strain 11168. Inoculation of naïve and immunised chickens will permit a thorough assessment of the impact of immune responses on colonisation and selection of phase variants. In silico models will be used to determine whether changes in proportions of phase variants are due to drift, bottlenecks or selection. Outcomes will include an improved understanding of the impact of PV on the course of infections and the variability of vaccine responses. Long-term benefits will comprise identification of novel vaccine strategies.

Campylobacter jejuni is largest cases of bacterial food-borne gastroenteritis and therefore leads to a significant number of rare but severe neurological complications. Plans for engagement of specific potential beneficiaries are highlighted below. Outreach to other beneficiaries will be pursued through publication of research in peer-reviewed journals, presentations at international and national meetings, involvement in local developmental networks and web-accessible resources. Development of vaccines to reduce colonisation of chickens by C. jejuni is an important aim of both policy-holders and commercial companies with interests in the farming sector. Pfizer is a company with a significant interest in C. jejuni and public health. This proposal could impact on development of a vaccine by identifying whether bacterial surface structures are subject to immune selection, by defining specific vaccine targets and by generating attenuated strains, with lower levels of colonisation, for use as live vaccines. An efficacious Campylobacter vaccine could have an immense effect on the burden of food-borne disease, and hence on public health, but would also be highly lucrative for the company involved in production of the vaccine. This research will identify potential vaccine targets/strategies which would then require 3-6 years for further testing and development. Jones, Bayliss and Barrow have experience of working with companies interested in vaccine development (Sanofi Pasteur, Intervet, Pfizer and Lohmann Animal Health) and so will be able to arrange for commercial exploitation of any relevant findings. Intervention strategies to prevent cases of Campylobacteriosis in humans are being explored by governmental agencies. The identification of the risk factors associated with different strains and isolates of C. jejuni would inform development of these strategies. Research in this proposal will define whether phase variation contributes to the poor efficacy observed in many vaccine studies and allow rational modification no vaccine strategies to improve their efficacy. Full-implementation of a targeted strategy of this type could reduce drive forward effective vaccine based control for both animal and human use. The research in this proposal will provide a significant resource for informing lay people about the contributions of genetic mechanisms to the ability of bacteria to cause disease. The results of this research will provide basic material for lectures and tutorial discussions within the higher education establishments in which all the applicants are based. In addition there is the potential for communicating simpler findings to a more general lay audience. This latter aim will be achieved in collaboration with GENIE (www.le.ac.uk/genetics/genie/), which is a Centre for Excellence in Teaching of Genetics based in the same department as Bayliss. This proposal will generate appropriate visual aids and interactive computer models to communicate concepts such as how rapid generation of genetic variants enables bacterial pathogens to adapt to changes in their environment which can be communicated to the wider educational community. All the applicants have significant teaching experience and Jones is involved in widening participation day-course delivery and has recently completed an MA in Higher education and has carried out projects using computer based delivery of interactive teaching videos and reviewed for the Wikivet project. The University of Nottingham is pursuing an open access policy for teaching materials. Bayliss has delivered presentations for the Royal Society Schools weeks.