Understanding the basis of strain restricted immunity to Theileria parva

Over 20% of people in sub-Saharan Africa are classified as living below the poverty line and many of these people reside in rural communities that are heavily dependent on livestock for their livelihoods. Infectious diseases have a major impact on livestock production in these regions. One of the most important diseases of cattle is East Coast fever (ECF) caused by a tick-borne parasite Theileria parva. This disease is present in 16 countries in eastern and southern Africa and it is the most economically important cattle disease in 11 of these countries. Estimates of economic losses due to ECF exceed 300 million US dollars annually. Imported breeds of cattle, which are increasingly being used to satisfy demands for milk production, are particularly susceptible. Although treatment of infected animals with anti-parasitic drugs and application of chemicals to prevent tick infestation can be used to control ECF, these methods are difficult to apply and generally are unaffordable by poor livestock keepers. Vaccination, therefore offers a more sustainable means of controlling the disease. Cattle can be immunised against the disease by infection with live parasites and simultaneous drug treatment, but the immunity induced by one parasite strain is not effective against all other strains. Use of a mixture of 3 parasite strains for vaccination has been found to give broad protection. This vaccine has been used locally with some success, but widespread application has been hindered by difficulties in production and distribution. The antigenic composition of the vaccine is poorly defined and there remain questions as to whether the content of parasite strains is optimal for obtaining robust immunity. More recently, antigens recognised by the protective cellular immune responses have been identified, with the aim of investigating their potential for developing a subunit vaccine. Preliminary evidence indicates that several of these antigens vary between parasite strains. Hence, understanding antigenic variability in T. parva is a key issue both for improving the current live vaccine and for the design of a sub-unit vaccine. The overall aim of the current project is to understand the antigenic basis of strain restricted immunity to T. parva, in order to develop improved vaccines against ECF. The key elements of the proposed research are: - An analysis of T. parva isolates from naturally infected cattle in different regions of Kenya, to determine the nature and extent of antigenic diversity in the parasite population. - An analysis of antigenic diversity in the 3 parasite isolates that make up the current live vaccine. - A detailed study of how variation in the antigens affects the ability of the cellular immune response to recognise different parasite strains. - An analysis of the specificity of cellular immune responses in cattle immunised sequentially and simultaneous with different parasite strains, to understand how these immunisation procedures affect the fine specificity of the immune response. - Analysis of immune Friesian cattle of different MHC genotypes for their ability to recognise currently defined T. parva antigens, in order to identify priorities for further antigen screening. It is anticipated that the results will provide methods for improving quality control of the current live vaccine, identify parasite strains that could be incorporated into an improved second generation live vaccine and indicate which and how many antigens will need to be incorporated into a subunit vaccine. Realisation of the project objectives will ultimately result in improved vaccines for ECF leading to better control of the disease. This will help to secure the assets and increase the incomes of smallholder and pastoral livestock keepers in affected regions. Improved disease control will result in increased production of meat and milk and reduce the levels of poverty in these vulnerable communities.

The specific objectives are to: 1. Utilise known CD8 T cell antigens as markers to determine the extent of antigenic variability in field populations of T. parva: Parasites will be isolated from cattle in different regions of Kenya, cloned and subjected to genotyping with satellite DNA markers and multi-locus sequencing of antigen-encoding genes. 2. Evaluate antigenic variability in the live vaccine in relation to that of field populations: Cultures of the live 'infection and treatment' vaccine parasites will be cloned in vitro by limiting dilution and large sets of clones subjected to genotyping and multi-locus antigen typing. 3. Determine how amino acid changes in the antigens affect CD8 T cell recognition and whether there is evidence of immune-imposed selection: Antigen-presenting cells pulsed with peptides representing allelic variants of defined epitopes in the T. parva antigens will be screened for recognition of reference sets of epitope-specific CD8 T cell clones Peptides containing single substitutions will also be tested to identify the residues involved in MHC binding and T cell receptor recognition. 4. Determine the influence of immunisation with heterologous parasite strains on the composition and strain specificity of the CD8 T cell response: Cattle will be immunised simultaneously or sequentially with different cloned parasites and the parasite strain specificity of CD8 T cell responses will be analysed at the whole population and clonal levels. Cattle of MHC genotypes known to respond to the Tp1 and Tp2 antigens will be used in these experiments. 5. Determine whether Friesian cattle carrying common class I MHC haplotypes recognise currently defined antigens: CD8 T cell lines derived from MHC-typed Friesian cattle of different MHC genotypes will be tested for recognition of the defined antigens using sets of overlapping peptides for each antigen. Based on the results, T cell lines will be selected for further antigen screening.

Potential beneficiaries and how they will benefit: Livestock producers, particularly poor smallholder and pastoralist farmers: By undertaking research that will improve the control of ECF through vaccination, the project aims to help secure the assets and increase the income of smallholder and pastoral livestock keepers. Socio-economic analyses predict that widespread introduction of an effective vaccine would result in annual on-farm yield gains of 243.8 million litres of milk and 34,000 tonnes of meat. In addition, the reduced acaricide dipping of cattle would result in savings of >$100 million per year and reduced detrimental impact on the environment. The above benefits translate into increased incomes and improved nutrition and welfare for a large proportion of the estimated 280 million people who live in ECF-endemic regions, many of whom are classified as living in poverty. Vaccine producers: The outputs of the research will provide tools to improve the quality control and efficacy of the live vaccine and information that could be exploited to develop alternative vaccines. They will therefore be of direct value to companies and organisations currently involved in production and distribution of the live vaccine, as well as larger Animal Health Companies who may be interested in commercialisation of sub-unit vaccines. Animal health advisory and regulatory authorities: Access to the results and conclusions of the study will benefit those organisations involved in formulating national disease control policy, including Departments of Veterinary Services, by allowing more informed decisions on where and how vaccination is to be used and deciding on actions in the event of vaccine breakdowns. National Agriculture Research Institutes: Access to the data and the biological reagents generated in the project will enhance the ability of National Institutes to conduct disease surveillance and to undertake their own independent research activities into East Coast fever. The theileria research community: The project will not only provide information that will influence the direction of other researchers in the field and foster collaborative opportunities but also generate a reference set of parasite isolates and reagents that will be made available to the community. The wider scientific community: The research findings will be of significant comparative interest for scientists working on other parasites that exhibit strain restricted immunity, eg. Plasmodium and Eimeria species. Engagement with the beneficiaries: - The project will exploit ILRI's and Edinburgh's close links with vaccine producers and, via interactions with GALVmed, identify others organisations with an interest in vaccine production, to ensure that improvements in the live vaccine are rapidly translated into commercial products. - ILRI's network of links with National organisations throughout Africa, and their in-house expertise on socio-economics and policy, will be utilised to ensure effective and appropriate dissemination of the project findings to those involved in formulating and implementing disease control strategies. This will be achieved by Institute reports, newsletters, via it's website and at meetings of National representatives. In addition, the project will organise a workshop to publicise the findings. - Communication with end-users (farmers) will be organised through intermediary organisations that have regular contact with the communities and can deliver the information in an appropriate format. - A publicly accessible database containing Information on the parasite isolates and derivative reagents, as well as result of the analyses, will be generated and reagents made available to other researchers upon request. - Findings of the project will be publishing in scientific journals and in other literature directed to policy-makers and end-users, and will be presented at national and international scientific meetings.