Selection of T-cell subset epitopes against F. hepatica using next generation phage display technology

Parasitic disease is a major cause of welfare and health issues in farmed livestock. Furthermore, the losses they cause and the need to provide veterinary care or drug treatments lead to massive economic losses in the UK and globally, e.g. US$2000M/yr for F. hepatica. The control of these diseases is primary through the application of drugs, however prolonged exposure of parasites has led to a rapid loss of drug efficacy due to the emergence of drug-resistant parasites. This has occurred in multiple parasite species and in response to multiple drug classes. Furthermore the continued use of some classes of drugs is no longer allowed due to the risk of entry of drug residues into the food chain. Moving forward the need for intensification of the domestic and international food supply will place increased pressure on farmers to ensure maximal output from their livestock. Correct control of parasitic disease is one route by which sustainable intensification can take place without compromising the health and welfare of livestock and ensuring economic returns. Prophylactic vaccination is the most suitable method by which this can be achieved as there are no concerns regarding drug residues or parasite resistance within the context of vaccination.
Identification and selection of successful vaccine targets for helminths has proven difficult and so progress in bringing such vaccines to market is slow. Helminths are complex pathogens which have multiple modes of evading the host immune response and so develop long-lived infections. This is accompanied by a dampening of the host immune response to avoid host and disease pathology. The manipulation of the host immune response by helminths, such as F. hepatica, makes selection of vaccine candidates eliciting protective immunity difficult. Where moderate protection has been achieved in the case of F. hepatica this has involved a mixed immune response, involving antibody and cell-mediated protection. These opposite arms of the response are orchestrated by T-helper 1 (Th1) and T-helper 2 (Th2) cells. These cells differ in their recognition of parasite epitopes, and the time at which they appear during infection, with Th1 cells peaking during the first two weeks of infection and Th2 cells peaking 6-8 weeks after infection.
We propose to isolate these cells from experimentally infected cattle and identify the parasite proteins they recognise by use of a novel technology called next generation phage display (NGPD). Animals experimentally infected with F. hepatica will develop robust immune responses generating large quantities of Th1 and Th2 cells in circulation. These can be identified by use of fluorescently tagged antibodies which recognize specific cell surface molecules which are unique to each cell type. These cells will be used to pan a phage display peptide library binding those for which they are specific. This will be conducted in two iterative rounds of panning to enrich the most frequently recognised peptides. Enriched peptides will be ranked by statistical analysis prior to being sythesised for testing using in vitro functional assays to determine the optimal composition of a multi-epitope vaccine. Synthesised peptides will be used in a flourescent screen. This will confirm those most commonly recognised by Th1 and Th2 cells in a mutually exclusive fashion. They will also be used to drive responses from Th1 and Th2 cells measuring production of key marker molecules to quantify the magnitude of response they can drive.
The development of this tool and its related technologies will enable us to deliver a multi-target vaccine for protection against Fasciola hepatica. Furthermore, we will be in a position to apply this much needed technology to combat other production-limiting infections enabling progress in the development of vaccines for the livestock sector.

Parasite infection, as caused by helminth worms, remains a major burden on the livestock industry. Unsustainable use of anthelminthics has led to a rise in drug resistance and concerns over drug residues in the food chain. Vaccination is the most feasible way forward in terms of parasite control. A lack of progress in the development of anti-parasite vaccines stems from the complexity of the pathogen, an inability to identify protective epitopes/vaccine targets and the mixed immune responses needed to mediate protective immunity. In this proposal we aim to develop tools to overcome the last two hurdles and deliver a portfolio of peptide vaccine candidates against a major helminth parasite, Fasciola hepatica. Fasciola hepatica induces a Th1 biased response within the first two weeks before switching to a Th2 response for the following 6-8 weeks, thereafter follows a prolonged period of immunosuppression where regulatory T-cells (T-regs) and their cytokine mediators, IL-10 and TGF-B1 dominate. Where experimental vaccination has shown some limited success, the type of responses needed to provide protection has been a mixed Th1/Th2 response overall. This paradigm fits with evidence in other tissue-resident helminths, e.g. Schistosoma mansoni and Trichinella spiralis larvae. With next generation sequencing linked phage display technology we will probe the epitope repertoire of specific Th1/Th2 cell subsets that contribute to F. hepatica immunity. Th1 and Th2 cell subsets will be sorted, at distinct times, from experimentally infected animals using high speed cell sorting. Phage display peptides will be bound to T-cell subsets and subject to DNA sequencing, statistical analysis (Z-scores) will identify enriched peptides and these candidates will be synthesized. Epitopes will be validated for their specificity by use of peptide staining and IFNg/IL-4 induction in ELISpot assays. Upon completion we will have produced a portfolio of T-cell-specific epitopes for vaccination.

Endemic and chronic parasite infections are a major burden and constraint on the ongoing requirement for increasing food production outputs in a sustainable and environmentally sound fashion. Our project will provide tools to combat a limiting factor in the farming sector, F. hepatica. The tools generated here will have both short- and long-term impacts in a variety of sectors including direct benefits to the farming sector while producing indirect benefits to wider members of society.

Livestock producers are under mounting pressure from external sources to produce increasing amounts of foodstuffs with diminishing profits and resources. Controlling infectious diseases will help to ease the burden on livestock producers both at home in the UK and globally where often livestock are the sole source of income for some small household farmers in underdeveloped countries. Aside from implications for production and financial outputs, F. hepatica infection can cause considerable health and welfare problems in the common UK hosts - sheep and cattle. Treatment with drugs to eliminate infection is now often incompatible with entry into the food chain. Helminth infections in particular are becoming increasingly resistant to drug treatment and the patterns of infection, especially for F. hepatica, are changing dramatically to increase prevalence year-on-year. The benefits of a novel more sustainable control mechanism such as vaccination would help to alleviate pressure on farmers aiding them to increase production in a sustainable economically viable fashion.

As transparency in the food production sector increases consumers are becoming increasingly aware of how their produce is being sourced and how it is being produced. Consumers are becoming more concerned with the health and welfare of animals involved in food production. Furthermore, they are now acutely aware of the impact of food production on the environment with respect to drug treatment/residues and contributions to pollution. Consumers will benefit from more sustainable farmed livestock through the reduction in F. hepatica increasing the quality of production, reducing animal disease and its negative impacts on welfare, and the overall reduction in the inputs needed to acquire the same, or more, outputs in terms of actual produce. This will translate into economic and sociological benefits to the consumer. This will generate a positive feedback loop whereby farmers who produce livestock in a more sustainable fashion, i.e. using vaccination for disease control, will be encouraged to do so by increasing consumer demand for their produce.

Our project will positively impact on wider society in a more indirect fashion. Environmental benefits from a more sustainable production method for livestock will offset growing concerns relating to the carbon footprint of food production. Societal concerns over the welfare and ethics of animal production can be in part met by the knowledge that farmers are implementing sustainable strategies to control parasitic disease in their livestock.

Government policy is directed at increasing the food security of the UK while promoting UK farming globally as an economic model. Our project is focused on producing tools to address one of the major limiting factors in UK farming today - F. hepatica. The outcomes of our project will promote sustainable growth and income development. Likewise it will help to address wider issues such as welfare and transparency within the food production sector, supporters of which form large pressure/lobby groups within parliament.

Within our pathways to Impact statement we detail our mechanisms for engaging with and promoting our research to the stakeholders we identify above.