Structural and functional characterisation of Trypanosoma cruzi antigens for drug design and vaccine applications

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T.cruzi parasites concentrate in the faeces of the triatomine insects and gain access to the blood stream upon deposition near the insect bite. When the parasites enter the bloodstream, they differentiate into a replicative form, which secretes the 80kDa enzyme prolyl oligopeptidase (Tc80) into the extracellular blood, which allows the invasion of mammalian cells by degrading their extracellular matrix components. Recombinant Tc80 has recently been shown to elicit a strong humoral response in immunised mice, which were also protected from a lethal dose of T. cruzi1 The absolute requirement for Tc80 in the invasion process, together with its conservation across different strains of T. cruzi, make this protein an exciting candidate for the development of a Chagas blocking vaccine.PhD objectives:Objective 1: Determine the effects of anti-Tc80 on Tc80 activity and parasite invasion The student will use biophysical techniques to quantify individual binding affinities and kineticproperties of anti-Tc80 mAbs in vitro, whilst in parallel cell-based assays will be performed using a T. cruzi fluorescent strain, which will allow to select mAbs, which have inhibitory effects on parasite invasion. This part of the project is in collaboration with parasitologist Prof. John Kelly at the London School of Hygiene and Tropical Medicine (LSHTM) in London.Objective 2: Determine the crystal structures of Tc80 and Tc80 in complex with specific mAbs The student will pursue the determination of the crystal structures of Tc80 and in complex with the best mAbs screened in objective 1, which will reveal the the molecular details of antibody-epitope recognition. This information will be exploited to guide future immunogen design (Campeotto et al., patent pending). Campeotto's group has routine access to Diamond Light Source synchrotron facilities (DLS, Oxford, UK) and the student will have the possibility of receiving further training at by the PI and by his collaborator at DLS, Dr Juan Sanchez-Weatherby. Objective 3: Design novel immunogenic molecules for vaccine applications The student will design in silico immunogens of Tc80 based on the structural information gained from objective 2 and based on bioinformatics and modelling analysis. These immunogens will be cloned, expressed, purified and conjugated to Virus-Like-Particles to boost the production of broadly neutralizing mAbs in mice2. Mice will be subsequently challenged with a lethal dose of the parasite.References to learn more:1. Bivona, A. E. et al. (2018). "Trypanosoma cruzi 80 kDa prolyl oligopeptidase (Tc80) as a novel immunogen for Chagas disease vaccine". PLOS Neglected Tropical Diseases, 12(3),e0006384-23. doi: http://doi.org/10.1371/journal.pntd.00063842. Brune D. K. and Howarth M. (2018). "New Routes and Opportunities for Modular Construction of Particulate Vaccines: Stick, Click, and Glue". Front.Immunol,https://doi.org/10.3389/fimmu.2018.01432