Our recent identification of epitopes recognised by CD8+ T cells, which are evoked by malaria liver stages, provides the basis to address fundamental parasitological and immunological questions: Are these proteins essential for the parasite life cycle? Do responses to these targets contribute to protection? How are these targets efficiently presented to CD8+ T cells? We will generate a series of Plasmodium berghei transgenic parasites as tools to answer these questions.
We propose a novel strategy that combines methods for positive/negative selection and flow cytometry-mediated sorting of fluorescent transgenic parasites to mutate candidate genes, along with integration of a green fluorescent protein and luciferase expression cassette in the parasite genome. While 'traditional' protocols require up to 30 mice for each cloning step, our strategy will allow the rapid generation of transgenic parasites using as few as three to five mice (Reduction). During the generation of the transgenic parasites, we will administer pyr and 5C (selection drugs) in the drinking water, not by injection (Refinement).
The various transgenic parasites will be assessed for their ability to complete the life cycle in both Anopheles stephensi mosquitoes and mice. Since the transgenic parasites express luciferase, their development in the liver can be evaluated by in vivo imaging (Refinement and Reduction). This method permits the non-invasive detection of parasites in intact animals and allows for the observation of the same animal as infection progresses. Gene-deficient (KO) parasites will be characterised based on their interactions with hepatocytes. Parasites with mutated epitopes (EM) will be used to immunise mice to determine the contribution of specific epitopes in protective immunity. Parasites expressing the ovalbumin epitope (OvaEpiSwaps) will be used to develop in vitro assays (Replacement) for antigen processing and presentation.