New approaches to a livestock trypanosomiasis vaccine: targeting the bite-site by immunization with novel metacyclic-stage parasite antigens

We will express recombinant forms of newly-discovered, parasite proteins that are specific to the surface of infective-stage cells, and examine their ability to protect vaccinated mice against infection by African trypanosomes.

African animal trypanosomiasis (AAT) is a livestock disease caused by vector-borne, blood parasites (Trypanosoma congolense and T. vivax), and endemic in 37 sub-Saharan countries. AAT causes chronic anaemia and muscle wastage, resulting in death if untreated. The UN FAO considers AAT to "lie at the heart of Africa's struggle against poverty" with 50 million cattle at risk and billions of dollars lost in agricultural productivity annually. Resolving AAT is an enormous challenge because trypanocidal drugs frequently solicit parasite resistance, vector control is typically unsustainable, while vaccines are unavailable because the trypanosome surface is enveloped by a variable surface glycoprotein (VSG) coat. Serial replacement of the active VSG through antigenic variation allows the parasite population to evade host immunity indefinitely. Antigenic variation is the common thread linking the physiology and epidemiology of AAT, and key to understanding and preventing disease.

Nevertheless, a vaccine remains the most sustainable solution to AAT, and thus, discovering invariant parasite antigens that elicit protective immunity is a priority. A decade of trypanosome genomics and gene expression analysis has revolutionized our understanding of the antigenic landscape. Our work has identified parasite-specific proteins expressed uniquely during the metacyclic life stage in the fly mouthparts. We have shown that metacyclic protein architecture is distinct, and unlike the VSG-dominated surface of bloodstream form trypanosomes. These stage-specific proteins are typically invariant and produce strong immune responses. Our hypothesis is that immunizing with these metacyclic antigens can provide the host immune system with a 'standing-start' in fighting infection, producing antibodies that neutralize metacyclics before they migrate into the bloodstream and, critically, before they become antigenically variant. We aim to test this by evaluating a panel of novel antigens for their protective properties and correlates of protection.

Our motivation is to translate this knowledge into an effective vaccination strategy. In this project, our approach has several innovations. First, we examine host-parasite interactions at the bite site, where the immunological response initiates. Second, we focus on the metacyclic stage of the trypanosome life cycle prior to the onset of antigenic variation. Third, we use a natural challenge model that produces an authentic initiation to infection. We have four specific objectives. 1) Recombinant protein expression in a range of combinations. 2) Vaccinate mice using different antigen formulations. 3) Infect mice via infected tsetse bite and compare the infection in vaccinated and control animals. 4) Profile gene expression around the bite site using transcriptomics of mouse leucocytes and parasites to identify correlates of protection, while providing the first global analysis of host-parasite interactions during the first days of infection.

This project will identify antigens for an experimental multivalent vaccine against metacyclic T. congolense and T. vivax, while establishing the immunological correlates of protection. Besides the immediate application of this new knowledge to efficacy trials of an AAT vaccine, it will also transform our understanding of host-parasite interactions at the bite site. The potential commercial application of AAT vaccines is enormous, and the likely positive effect on animal health and livestock productivity across the world would be profound. As one of the world's foremost veterinary diseases, a sustainable solution to AAT would be a seminal breakthrough leading to improved health and wealth in the world's poorest countries.

We will produce recombinant parasite antigens found on the surface of metacyclic-stage trypanosomes using a eukaryotic expression system, and examine their protective properties in a murine model under natural challenge.

Vaccines against livestock trypanosomes (T. congolense and T. vivax) have been neglected because long-lasting protective immunity is prevented by antigenic variation of the Variant Surface Glycoprotein (VSG) coating these parasites. However, this dogma is based on one model species (T. brucei), and the assumption that a vaccine must work against bloodstream-form parasites, fully enveloped by VSG. We have discovered novel, parasite-specific proteins on the surface of metacyclic-stage T. congolense and T. vivax that are not antigenically variant but are naturally immunogenic.

Our hypothesis is that, in nature, antibodies to invariant antigens are diluted by the immuno-dominance of VSG, once the parasite migrates to the blood and begins VSG switching. We propose that immunizing against metacyclic-stage antigens would give host immunity a 'standing start', able to neutralize metacyclics at the bite site before onset of antigenic variation.

Our aim is to formulate a vaccine based on metacyclic-stage antigens and establish immune correlates. We have four objectives: 1) To express recombinant T. congolense and T. vivax metacyclic-stage proteins using a eukaryotic (mammalian) expression system that will ensure native folding and decoration; 2) To vaccinate mice with diverse antigens; 3) To compare patent infections in vaccinated and control mice using natural (tsetse) challenge; and 4) To identify correlates of protection from host leucocyte transcriptomes.

Our novel approach focuses on the bite site as essential to the outcome of infection, and the metacyclic cell-surface as immunological target, and using tsetse to model authentic infections. This project will establish the basis for a future clinical trials.

Parties who will benefit from the project:

1.Academic scientists: molecular parasitologists and parasite immunologists in the UK and elsewhere working on trypanosomiasis and other vector-borne diseases will benefit from our new knowledge and the resources for future functional experiments we make freely available.
2.Parasitology discipline: our field will benefit from fresh approaches to vaccination against trypanosomes. Challenging the dogma will encourage renewed discussion of the best strategy for control of parasitic disease, extending to funding agencies and other charitable stakeholders.
3.Animal health NGOs: organizations like GALV-med and Gates Foundation, which specialize in novel translational solutions, will benefit by developing the new knowledge we create.
4.Animals and their owners: livestock across sub-Saharan Africa and elsewhere are routinely exposed to AAT and will experience reduced infection, mortality and morbidity due to a vaccine developed through our work. Such relief will benefit small-scale pastoralists or farmers reliant on animals for labour, by increasing their productivity and thereby their wealth and nutrition.
5.Livestock producers: reduced losses to commercial producers due to AAT will increase productivity and agricultural output.
6.Overseas economies: many countries in which AAT is endemic have agrarian economies and will benefit from decreased AAT due to both increased productivity and reduced disease control costs. This will remove one barrier to greater socio-economic development.
7.UK economy: commercial exploitation of our protective antigens will benefit the economy.
8.UK public

Impacts of the project that will bring benefit:

1.Academic scientists (knowledge): we will create new knowledge about how novel parasite antigens elicit immune responses in hosts, about the contribution of fly bite to immunological response, and about the molecular host-parasite interaction during early infection.
2.Academic scientists (resources): plasmids containing our novel antigen genes, recombinant proteins and select anti-sera, will be freely available to the community for use in functional experiments. This grant will sustain facilities of unique usefulness to the trypanosome research community (i.e. tsetse insectary, high-throughput protein expression).
3.Parasitology discipline: by challenging the consensus that vaccination against trypanosomes is impossible, we will encourage facilitate progress towards this important aim.
4.Animal health NGOs: protective antigens that we identify will facilitate the work of NGOs (e.g. GALVmed, BMGF), seeking to develop novel interventions from new basic knowledge.
5.Reducing AAT (animals): protective antigens would provide a vaccine that would reduce animal mortality and morbidity. These antigens may be cross-reactive with homologs in human trypanosome species, and so protect against both human and animal trypanosomiasis.
6.Reducing AAT (people): reduction in AAT will increase the wealth and prosperity of farmers in the developing world, from small-scale farms using animals for draught to large-scale livestock production systems.
7.Reducing AAT (societies): reduction in AAT will reduce economic loss and government costs in disease control, leading to greater socio-economic development and food security. This will contribute to UK Government commitments towards international development.
8.UK economy: our protective antigens may be patented for commercial application, and exploitation of a successful vaccine may contribute to the wider UK economy.
9.Public: we will inform public perception of infection biology through dissemination of our work through national and international media outlets and social media such as Twitter; participation in events such as SciBar (organised by the British Science Association), Pint of Science, and Ignite Liverpool; and by working with local schools to foster scientific curiosity in neglected diseases.