Defining mechani1sms of CD8+ T-cell mediated immunity - using an integrated longitudinal model to achieve an elusive goal.

Vaccines have been amongst the most successful and cost-effective health intervention strategies implemented and have played a critical role in reducing the incidence of many human and animal diseases. However, for a number of diseases (e.g. HIV, malaria, African Swine Fever) for which CD8+ T-cell mediated immunity is thought to make a substantial contribution to immunity, there are currently no available vaccines that elicit protective CD8+ T-cell responses. Viral-vectored vaccines are the delivery system that has shown most promise, especially in model species. However, in target human or animal species these vaccines, although usually highly immunogenic (induce T-cell responses), have demonstrated only a limited ability to generate protective CD8+ T-cells. The discrepancy between immunogenicity and protection indicates that qualitative, rather than quantitative, parameters of the vaccine-induced T-cell responses are deficient. As yet our understanding of what these parameters are is limited and this 'knowledge-gap' prevents a fully rational approach to design of novel, efficacious vaccine delivery platforms.

Theileria parva, a tick-borne protozoan parasite of cattle, causes major economic losses in livestock farming in sub-Saharan Africa of ~$600M/yr. This loss is largely borne by small-holder farmers and can be devastating for some of the most vulnerable people in low/middle income countries. Natural immunity to T. parva is associated with CD8+ T-cell responses, which can be mimicked under experimental conditions using either an 'infection-and-treatment method' (ITM) or autologous T. parva-infected cell line (ACL) immunisation which have been used to study and characterise protective immunity. A major aim of T. parva research has been the development of a subunit vaccine that could be deployed as part of sustainable T. parva control programme. Trials using a variety of viral vectors (canarypox, Ad, MVA) to deliver known T. parva CD8+ T-cell antigens have demonstrated immunogenicity but only limited protection from in vivo challenge. Recent data has implicated defects in Ad/MVA-induced CD4+ T-cell responses as contributing to deficient CD8+ T-cell functionality.

Development and trialling of novel viral vectors requires substantial investments of time and resources and is limited by the availability of suitable candidates. Although disappointing, failure of current viral vectors to induce protective CD8+ T-cell responses can be exploited to understand how the induced T-cell responses differ from equivalent but protective T-cell responses.

In this project we propose to use a 'longitudinal' model of T. parva immunisation in which the T-cell responses of individuals are intensively analysed following sequential administration of a non-protective (Ad/MVA) and protective (ITM) immunisation to enable the parameters that influence the transition of phenotypes to be analysed and so define the immunological parameters that determine protective efficacy of CD8+ T-cells. By facilitating intra-animal comparisons this model has a number of advantages over conventional cohort studies. The objectives of the project are to define:

1. How does the function and transcriptome of non-protective CD8+ T-cells induced by Ad/MVA heterologous prime-boost differ from protective T. parva-specific CD8+ T-cells?
2. How does the function and transcriptome of CD4+ T-cells induced by Ad/MVA heterologous prime-boost differ from those associated with protection from T. parva infection?
3. How dependent on CD4+ T-cells is the functional competency of CD8+ T-cells?

This information will provide the data to understand the fundamental immunological mechanisms that dictate CD8+ T-cell vaccine success or failure and can be used to inform the design/engineering of improved novel vaccine vectors for T. parva and other human and veterinary pathogens.

Virally vectored vaccines have been shown to be highly immunogenic in eliciting CD8+ T-cell responses; however in humans and veterinary target species these responses confer only limited protection. Failure to understand the qualitative defects of vaccine-induced CD8+ T-cells that lead to functional deficiencies results in a substantial empiricism in the design of new candidate vaccine delivery systems, retarding the development of urgently required vaccines. Comparative analysis of how non-protective vaccine-induced and equivalent but protective T-cell responses differ offers the most direct route to delineate which parameters are critical in determining the protective efficacy of CD8+ T-cells.

Building on data from recent Theileria parva Ad/MVA vaccine trials, this proposal aims to exploit a novel 'longitudinal' T. parva model (in which animals serially receive an immunogenic but non-protective Ad/MVA heterologous prime-boost immunisation followed by a protective 'infection-and treatment method' immunisation) to conduct comprehensive functional and transcriptomic analysis of CD8+ and CD4+ T-cell responses as they transition from a non-protective to a protective phenotype. The longitudinal model offers a number of advantages over conventional 'cohort studies', including the application of single-cell RNA sequencing to track the transcriptional changes within individual clonotypes. By enabling intra-individual comparison of protective and non-protective T-cells, the study offers a direct route to defining correlates for protection using a model that is difficult to replicate in other relevant pathogen models. The data generated from this study will define fundamental immunological mechanisms that determine vaccine efficacy and will have relevance not only to T. parva but to other human and veterinary pathogens.

Theileria parva remains one of the most economically significant diseases of cattle in a large area of sub-Saharan Africa and novel control approaches remain a priority for improving livestock productivity in this region. By identifying the immunological parameters that dictate CD8+ T-cell protective efficacy this project aims to generate the data that can accelerate development of next generation vaccines. As well as the direct impact of this research on T. parva, the continued need for vaccines capable of inducing protective CD8+ T-cell responses for many of the major human and veterinary pathogens means the output of the project will have impact in the broader academic community.

Academic research communities: see Academic Beneficiaries for the direct impact of the project in fundamental academic research.

Animal Heath NGOs, funders and industry: the combination of the currently available vaccine delivery platforms' failure to induce protective CD8+ T-cells and the paucity of data to support the rational development of novel alternatives has become a major hurdle in advancing vaccine development against T. parva. The potential to progress our understanding of relevant 'correlates of protection' will be of interest to NGOs such as GALVmed and BMGF that seek to translate new knowledge acquired from such studies into novel interventions for tackling livestock diseases that have profound consequences on the livelihoods of small-holder farmers in sub-Saharan Africa. If successful these groups could be engaged to support further work applying the system (e.g. to other current vaccine delivery platforms to provide a detailed profile of the potential 'defects'; potentially with the aim of establishing if combination of multiple platforms can have synergistic benefits by reciprocal compensation of the defects of the individual platforms).

Livestock owners and agricultural production in LMICs: the impact on livestock owners and agricultural production in LMICs will realistically be a longer term effect (by definition this project doesn't aim to generate a novel vaccine but to generate the data that can accelerate future vaccine development). The benefits will come from the direct consequence of provision of an effective vaccine; reduced animal losses and costs of disease control resulting in improved food and financial security, improved animal health and welfare and reduced environmental impact from the use of acaricides. There will also be indirect benefits as East Coast Fever (ECF) has indirectly constrained the genetic improvement of local agricultural production by limiting the feasibility of introducing the more productive European (Bos taurus) cattle due to their high levels of susceptibility to ECF.

UK governmental policy and BBSRC's strategic priorities: by focusing on a pathogen that is prevalent in sub-Saharan Africa the proposal falls within the remit of the International Development Act (2002) which defines the UK government's statutory requirement to provide assistance leading to the reduction of poverty in LMICs and contribute towards the achievement of the Millennium Development Goals. The project is also aligned with the BBSRC's strategic priority for Agriculture and food security, contributing to a 'programme on veterinary vaccinology to accelerate research into next generation vaccines to combat major diseases of livestock', and by providing opportunities for the PDRA to receive training in a portfolio of immunological and bioinformatic skills will help to 'address skills shortages in areas of specialist research expertise' which includes veterinary vaccinology and scientists with cross-over expertise in immunology and bioinformatics.