Variant antigen profiling: a novel genomic tool for diagnosis and surveillance of animal African trypanosomiasis.

Animal African trypanosomiasis (AAT) is a livestock disease with frequent and devastating effects on animal health and on the development of sub-Saharan nations. This neglected tropical disease is caused by the parasite Trypanosoma congolense, which is spread by biting flies and infects the blood causing chronic anaemia, wastage and ultimately death if untreated. Unlike Human African trypanosomiasis ('sleeping sickness'), which is caused by a related parasite T. brucei, and is declining after successful intervention and much research, AAT remains very common and poorly known. Yet the annual costs of disease and treatment are estimated at ~$4.5 billion across 37 countries. The FAO considers AAT to "lie at the heart of Africa's struggle against poverty", and central to its Millennium Goals. Most research on African trypanosomes is directed at the model organism T. brucei, but given the profound effects of AAT on animals and people, we need research dedicated to the veterinary parasites to determine how parasite variation affects the course and severity of disease.

All African trypanosomes use antigenic variation to avoid the immune response of their host. This involves the replacement of the protein coat covering the parasite surface each time the host produces antibodies to the protein. The protein is called the Variant Surface Glycoprotein (VSG) and is crucial to parasite survival and the severity of disease, determining the length of infection and resistance to innate host defences. The genomes of both T. brucei and T. congolense contain hundreds of VSG genes, of which just one is expressed at a time from a specific expression site. Together, this repertoire of alternative VSG genes is a ubiquitous, but highly variable, feature of trypanosome genomes that constantly changes in response to host immunity. We can use this repertoire to understand how parasites change through space and time, and what effect this has on disease. This project aims to measure variation in VSG repertoire across the T. congolense population, and then produce a novel tool for rapidly predicting the properties of a strain based on its VSG repertoire.

I will produce genome sequences for 40 isolates sampled from T. congolense infections of cows. Until now, analysis of VSG repertoires on a population scale has been avoided because their complexity makes it impossible to use standard tools. Previously, I discovered that T. congolense VSG are divided into distinct types, each defined by unique protein motifs. I will quantify the VSG repertoire in each T. congolense isolate by comparing its VSG to these protein motifs. The relative abundance of the VSG types will produce a 'variant antigen profile' (VAP) for each isolate. To give the VAP some predictive power, I will identify which VSG are actually used in antigenic variation, (and those that might perform some other role), by identifying the active VSG gene present in the specific expression site and expressed on the parasite cell surface. I will also identify significant correlations of VAPs with geography, time, disease severity and parasite relatedness. A tool to generate and interpret a VAP will be accessible online, making it possible to predict the properties of any T. congolense isolate from genome sequence data using variant antigen profiling.

AAT represents an enormous challenge to animal health and economic development in Africa and yet we have no detailed knowledge of how T. congolense varies and how this relates to the kind of disease we see. Such information would improve diagnosis, the efficiency of drug therapy and control measures, lead to earlier and more appropriate treatment for sick animals, and perhaps even identify proteins that could serve as vaccine targets. The time is right to develop a fast and simple approach to making sense of VSG diversity that can unlock the potential of trypanosome genomics for understanding AAT, estimating its risk and mitigating its effects.

Animal African trypanosomiasis (AAT) is a livestock disease caused by the parasite Trypanosoma congolense, which has a significant negative impact on animal health and productivity across sub-Saharan Africa. Variant Surface Glycoproteins (VSG) are expressed on the parasite surface where they protect against host immunity. The expressed VSG is periodically replaced to evade acquired immune responses through a process of antigenic variation. VSG genes are ubiquitous, highly variable and intimately associated with disease; thus, I aim to develop novel ways of quickly analysing VSG variation and so better understand antigenic variation, parasite transmission and disease phenotypes at the population scale. This project will quantify population variation in VSG repertoire through genome sequencing of 40 T. congolense isolates sampled from natural infections across Africa over a 50-year period. VSG will be extracted from genome sequences based on protein motifs identified previously and typed based on phylogenies revised using these new data. The distribution of total VSG among the various conserved phylotypes will produce a 'variant antigen profile' (VAP) for each strain. How the frequency of individual phylotypes changes through time and space will define specific VSG or VSG clades that are diagnostic of provenance (i.e. particular regions or outbreaks). To confirm which phylotypes encode functional variant antigens, I will characterize VSG expression sites by cloning T. congolense telomeres from ten strains into yeast using the telomere-assisted recombination (TAR) method. I will also identify expressed VSG sequences by mass-spectrometry to confirm that the active VSG is telomerically expressed. This work will result in a method for rapid analysis of VSG repertoire in trypanosome genome sequence data, which is currently lacking, a detailed understanding of VSG variation in T. congolense and of how this diversity relates to parasite populations and disease phenotypes.

>The UK science research community will benefit from new knowledge and tools relating to parasitology, veterinary science and genetics. These benefits will have an impact on our basic understanding, our ability to analyse highly abundant and variable gene families in diverse genomes on a population scale, and on future African collaborations. These are short term benefits likely to have impact by the end of the project.
>UK research capacity will benefit from the training of postgraduate and postdoctoral researchers and by establishing this laboratory for the high-throughput genomic analysis of parasite populations. This will have impact through the skills in genome analysis that individuals transfer to subsequent positions and through subsequent projects that apply this knowledge to other parasitic diseases in the UK and abroad. These are short term benefits likely to have impact by the end of the project.
>The UK public will benefit through learning about AAT and our efforts to control it. This will have impact by emphasising the generally unrecognized effects of veterinary disease on the prosperity of developing countries. These are short term benefits likely to have impact by the end of the project and ensured through the outreach activities of the IIGH.
>UK society will benefit through the contribution of this research towards the statutory commitment of the UK Government and its agencies towards international development. This will have impact by providing new knowledge that will facilitate African science, and by developing and entrenching collaborations with African research institutes. These are short term benefits likely to have impact by the end of the project.
>UK biotechnology companies could benefit from the commercialization of new tools and by providing diagnostic services to African veterinary sectors. AAT is endemic and the pressure for more livestock production in the coming decades is likely to make prevention of AAT more profitable. This is a long-term benefit, having impact once VAPs are proven and routinely applied.
>African farmers in AAT endemic regions will benefit through improved assessment of risk to their livestock, improved diagnosis of disease and prognosis of illness. This will allow individuals to prevent disease and to manage limited resources for therapy effectively, ultimately resulting in fewer deaths and greater productivity. These are medium term benefits likely to have impact once animal health agencies routinely apply VAPs in disease surveillance.
>African animal health practitioners will benefit through the routine application of VAPs during disease monitoring, providing more precise and data-rich diagnosis of AAT. This will have impact by allowing local researchers to take quicker action to prevent epidemic, to prioritise control measures depending on the nature of the parasite (and its VAP), and to build a population-wide understanding of disease phenotype for the first time. These are medium term benefits likely to have impact once VAPs are routinely using genomics in field surveillance.
>African veterinary policy makers will benefit from routine variant antigen profiling through the new information this provides on parasite population structure, transmission and the relationship between parasite diversity and disease phenotype. This will increase effectiveness of public services, allowing policy makers to assess risk on national scales and prioritize resource use to reduce disease burden. These are long term benefits likely to have impact after the utility of VAPs in disease surveillance is proven.
>African governments and societies will benefit from the precise and data-rich information provided by variant antigen profiling once routinely applied, through greater productivity and efficient land-use resulting from avoidance and prevention of AAT. These are long term benefits likely to have impact after VAPs are routinely used to assess risk and control AAT.