Defining the molecular basis of H7 flagellin and as an adhesin and mucosal adjuvant for vaccine development

Enterohaemorrhagic Escherichia coli (EHEC) O157:H7 are bacteria that cause serious gastrointestinal disease in humans. Toxins released by the bacteria cause life-threatening damage to the kidneys and brain. Cattle are the main reservoir host for the bacteria and humans become infected initially by ingesting food or drink that has been contaminated, directly or indirectly, from animal faeces. One way to guard against human infection with EHEC O157 is to block or limit shedding of the organism from cattle. This can be done via vaccination of cattle. Our previous research has demonstrated that the whip-like 'flagella' of EHEC O157 that are usually used for bacterial movement, can bind to the intestinal lining of cattle to promote colonisation. If we vaccinate cattle with purified flagella, specific antibodies are generated in the gut that block this binding and limit bacterial colonisation. However, a subset of these antibodies have a negative impact in that they bind to a region of the flagella that interferes with how the host recognizes and responds to the real infection when it occurs. There are two main aims of the research. The first is to define the specific region within the flagellin molecule required for flagella binding and to use this in a vaccine preparation to study how it limits colonisation of cattle by EHEC O157. It is anticipated that by using only the region of flagellin required for binding in the gastrointestinal tract of cattle or by altering recognition of the region that stimulates inflammatory responses in mammals, we should be able to produce an effective vaccine containing flagellin that significantly reduces EHEC O157 shedding from cattle. The second part of the research is to understand how the vaccination of cattle with whole flagellin stimulates such a good antibody response on the surface of the animals gastrointestinal tract and then exploit this understanding to generate responses to other molecules linked to flagellin. Finding ways to stimulate this 'mucosal' immunity is important for the development of vaccines to many infectious diseases.

Enterohaemorrhagic Escherichia coli (EHEC) O157:H7 is an important food-borne pathogen that causes serious human infections ranging in symptoms from self-limiting diarrhoea or haemorrhagic colitis to life-threatening haemolytic-uremic syndrome. Cattle are the primary asymptomatic reservoir for EHEC O157 and human infections can occur by consumption of contaminated food or drink, direct contact with animals or person-to-person spread. A key objective is to reduce the carriage or prevalence of EHEC O157:H7 in cattle to limit human exposure. Development of effective interventions in cattle will rely on understanding the bacterial factors involved in colonization of the gastrointestinal tract. We have demonstrated that EHEC O157 predominately colonises the terminal rectum of cattle and that H7 flagella functions as an adhesin for EHEC O157 when binding to epithelial cells cultured from this rectal site. Comparative binding studies with other flagella indicate that distinct motifs in the central (D2 and D3) domains are likely to confer the binding specificity. Systemic immunization of cattle with purified H7 flagellin reduces the proportion of animals that become colonised. However, in calves that do become colonised, bacterial shedding levels are at least as high as non-vaccinated controls. Both IgG and IgA are induced following H7 flagellin immunization and inhibit EHEC O157:H7 adherence in vitro. However, IgG blocks TLR5 signalling and this may impact on shedding levels following immunization. The aims are: (1) to define the region of FliC(H7) required for binding specificity and to generate an H7 flagellin fragment or H7 variant that can be used to induce antibodies that block binding but that do not inhibit TLR5 signalling by wild type H7 flagellin; (2) to investigate how systemic vaccination with H7 flagellin results in specific IgA responses and to use this information to generate a mucosal response to a heterologous antigen.

The translational potential of this area of work is already evident through patent submission (PCT/GB2008/003515) by our grouping. This project, as shown by the investment form Novartis Animal Health (NAH), has two areas that could be translated for commercial benefit. The first is use of H7 or modified H7 flagella in a cattle vaccine to prevent or limit EHEC O157 shedding which therefore could have a direct impact on food safety, human health and the sustainability of UK farming. In addition to NAH, we are in discussion with Bioniche which already has a provisional licence for an EHEC O157 cattle vaccine in Canada and wishes to pursue development and use in Europe potentially through our grouping. They may consider licensing an H7 derivative to add to their current vaccine as we have data that this improves the efficacy of the vaccine. This proposal also aims to develop a mucosal adjuvant technology based on flagellin hybrids that could be applied to other antigens, initially targeting ruminant diseases. We consider that the research will add to the knowledge economy, hopefully providing opportunities to develop other vaccines. The research fits directly under the BBSRC priorities of 'Animal Health', 'Food Security' and 'Global Security' in carrying out research on host-pathogen interactions that can be applied to reduce the threat from infectious diseases to the UK, thereby improving the sustainability of agriculture and reducing our dependence on imported food. Also, functional understanding of this TLR5 agonist should allow its exploitation for therapeutic purposes, in line with the BBSRC drive to 'synthetic biology' in which key molecules are used directly or modified for therapeutic or diagnostic purposes. Another pathway to impact under discussion is to spin out the technology as a small company in the UK working in partnership with NAH to develop animal vaccine adjuvants and provide a 'consultancy' link to the animal research community based at Roslin/SAC and MRI in the Edinburgh area along with the University of Glasgow. This would continue to support a multi-institute grouping that was established as a result of DEFRA Fellowship funding in 1999 and provides a world-leading centre for expertise on VTEC/EHEC and is also currently expanding into other significant zoonotic diseases. The grouping has introduced many veterinary scientists into research that have gone on to contribute in companies, further research and to agencies, such as EFSA. This type of research support underpins the capacity to train undergraduate and post-graduate researchers in this important, inter-disciplinary area. Students that train in the laboratory experience a wide-range of skills and build their value for future contribution to scientific advancement and wealth creation. The wider grouping, including SAC, has not only disseminated information in journals and at conferences, but has provided reports for government agencies including DEFRA, FSA and HPS that has impacted on advice and policy (e.g. information on EHEC O157 rectal carriage; Godstone petting farm enquiry in Southern England). Collaborators at both SAC and Moredun have extensive connections into the agricultural industry and prepare articles and hold events for knowledge exchange with farmers and producers. Significant efforts have been made by the grouping in Scotland to raise awareness of O157 carriage by cattle and sheep and to provide information for risk management by rural communities and countryside users. Stakeholders include a broad range of groups not only connected with farming and food production but those associated with recreational use of land/water. An additional impact at the educational level comes from articles for the press and the radio. Under this grant, we would aim to continue with these different modes of information transfer but our primary focus will be with vaccine development and liaison with industry to push translation of the work into interventions.