Dissecting and exploiting vector components of Leishmania transmission.

Leishmaniasis is a zoonotic vector-borne disease, caused by a parasitic protozoa transmitted by the bite of a bloodfeeding sandfly. Symptoms range from painful skin ulcers, through to infection of the liver and spleen, which is fatal if not treated. Wild rodents and domestic dogs are major reservoirs of infection, which can amplify human infection in endemic areas. There is an urgent need for new preventive measures to control leishmaniasis, in people and in the reservoir hosts, since we have a limited range of effective drugs and currently there is no vaccine. Immunological studies of leishmaniasis have traditionally used mice infected by needle injection of parasites into the skin. It is now very clear, from my own work and that of others, that this does not properly reflect the lesions that develop after natural infection initiated by sandfly bite. I have therefore developed, and evaluated, a protocol by which infection can be induced in the most naturalistic way possible, using infected sandfly bites. Sandflies carry Leishmania parasites in their gut. The parasite produces a gel - promastigote secretory gel (PSG) - that acts as a plug in the fly's gut, forcing it to expel the plug before it can feed on animals. I have shown that infected sand flies regurgitate both parasites and this PSG gel-plug during bloodfeeding and have found that co-injection of purified PSG gel with parasites greatly exacerbated Leishmania infections in mice. This revealed PSG to be a missing and important part of natural infection. I have shown that to promote Leishmania infection in the skin, the gel entice immune cells (macrophages) to the site of the sandfly bite. Macrophages normally kill invading parasites by ingesting and digesting them. However, within the first few days of infection, the gel modifies the function of the macrophages (via a process known as alternative activation) so that they engulf but do not kill the Leishmania; indeed these alternatively activated macrophages provide essential nutrients to the parasites so that they can grow faster. In this way Leishmania parasites are very cunning - they make PSG in order to inhibit the immune system so that they can establish a skin infection. Macrophages receive instructions from T lymphocytes to either kill or not kill the Leishmania that they ingest. How the PSG, macrophage and T cells interact in the skin following natural infection is poorly understood and characterisation of these interactions is a major focus of this project. I have gathered evidence showing that PSG operates through a newly described immune pathway involving the receptor ST2 and its corresponding cytokine, interleukin-33 (IL-33) which are involved in the body's response to allergy and wound healing (eg. a sandfly bite). Therefore, I propose to investigate the immune mechanism(s) which allow PSG to promote Leishmania infection in the skin via this novel signalling pathway, and how the wound response to the sandfly bite interacts with PSG to promote long term infection. From my studies, a striking feature of PSG in skin is its ability to facilitate wound closure, dependent on IL-33. Therefore, an intriguing possibility may arise as a spin-off from my proposed research - that PSG may be developed as a novel treatment for speeding up wound healing in humans and in animals. This may increase healing from surgery, or aid the healing of chronic wounds which are a persistent problem of the elderly, diabetic and obese in humans, and a recurrent problem in livestock. It is expected that the proposed work will not only reveal novel insights into the earliest immune events that govern natural Leishmania infection, but may also reveal potentially exploitable information regarding the response of skin to infection and wounding. I also hope that my studies will help us to design more effective Leishmania vaccines that will help to control the spread of the infection from its animal reservoir into humans

All Leishmania infections start in the skin following transmission by phlebotomine sandflies, however, we know very little about the dynamics of natural infection, or the immune response following infected sandfly bite. Leishmania secrete unique mucin-like proteophosphoglycans which condense within the sandfly midgut into a gel, termed promastigote secretory gel (PSG). I have shown that PSG is regurgitated by sandflies along with parasites during bloodfeeding and this significantly exacerbates parasite growth and chronic cutaneous lesion development in mice. Building on the discovery of this new component of the infectious inoculum, my recent work shows that PSG modulates macrophage alternative activation and specifically interacts with ST2 (a receptor with toll-like motifs) or its ligand, IL-33, to exacerbate cutaneous leishmaniasis. Using a competent and transmissible Leishmania-sandfly combinations I propose to model the dynamics of PSG, parasite and vector saliva delivery by individual sandflies, dissect the mechanism(s) of PSGs interactions with the ST2-IL-33 signalling pathway and determine how Leishmania, through secretion of PSG, subverts the wound healing process initiated by the sandfly bite. A range of gene-targeted mouse models will be used in combination with methods I have developed for reproducibly infecting mice with the bites of individual infected flies and my recently acquired skills in immunology to define the interaction between PSG and macrophages for the successful establishment of parasites in the skin. During these studies I expect to gain unique insight into the function of ST2/IL-33 in the skin and in wound healing. Following from my preliminary studies, I will also test the potential of PSG as a novel wound healing therapy. By understanding the early events in the pathogenesis of leishmaniasis initiated by sandfly bite I will provide the framework within which future vaccines against leishmaniasis will be developed and evaluated.

In addition to the direct biomedical and commercial applications of my work to develop novel wound healing drugs and modulators of the IL-33/ST2 signalling pathways, as described above under 'Exploitation', there are numerous other potential impacts of my work: 1. First UK facility for screening candidate drugs and vaccines for leishmaniasis transmitted by the natural route of infection: Challenge by sandfly bite is the 'gold standard' to which any drug or vaccine must protect against in the field. This will be a unique facility in the UK for academia and industry. 2. 3Rs: Refining the current Leishmania infection model will reduce the number of experimental animals required for anti-Leishmania drug and vaccine screening. This has a both economic as well as ethical impact, since it is estimated that global pharmaceutical companies annually waste $8 billion in research and development of unsuitable molecules. Finding candidate molecules which protect against infected fly bite will speed up the process of vaccine discovery, save money and reduce the number of animals required. 3. Teaching and training in entomology: re-establishing sandflies at the LSHTM will provide a significant teaching resource for student projects, demonstrations and practicals. Entomology skills have declined in the UK in recent years and if I am able to establish myself as an independent researcher and lecturer at the LSHTM I will aim to enthuse and train new generations of sandfly biologists. 4. Novel epidemiological tools for sandfly and Leishmania research: Antibodies to components of sandfly saliva can be used as an independent and direct measure of sandfly biting of both humans and animal reservoir hosts. This allows the distribution of the disease to be mapped; this is especially important for identifying animal reservoirs of infection prior to introduction of control programmes (e.g. novel insecticides, traps etc) and for monitoring the impact of these programmes. Establishing a sandfly colony as a source of sand fly saliva will enable me, and my collaborators, to develop and apply these methods more widely. This approach may also be adapted for the incrimination of new vector species from the field, and as a surveillance strategy to track the spread or introduction of Leishmania vectors into new areas such as northern Europe and, eventually, the UK. This has the potential to guide policy on vector control and evaluating risk of infection. Currently, I am developing such a tool for the visceral leishmaniasis-elimination programme in India (Clements et al 2009, in press). I will endeavour to optimise these impacts by: 1. Engaging with the pharmaceutical industry to develop collaborations for drug and vaccine screening. Professor Croft already has established collaborations with Pfizer and Glaxosmithkline; these companies will be the starting point for my discussions. 2. Engaging with the leishmaniasis control commmunity to maximise the use and contribution of the sandfly colony to the mapping of Leishmania transmission. I have already established links to trials in South America for novel odour-baited sandfly traps against canine leishmaniasis, the Kalanet programme in India (impregnated bednets) and Intervet (impregnated dog collars). 3. Contributing to the teaching and supervision of post graduate students at the LSHTM, lecturing on the MSc courses in Medical Parasitology and Biology and Control of Disease Vectors, supervising Masters and PhD student projects. 4. Actively communicating my research to the public: I will participate in the LSHTM high school 'access to science' programme. I am part of a team that will participate in the 350th anniversary of the Royal Society summer exhibition to highlight the importance of leishmaniasis lab and field research. I will use a wide range of media to disseminate my findings to a larger audience, and have done so recently via the BBC News Online.