Chicken intestinal organoids: a novel in vitro system to study mucosal vaccine targeting

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The project seeks to fully characterise an avian minigut model and to exploit the model's multifunctional potential. The ultimate goal is to use these chicken miniguts to investigate new strategies to target mucosal vaccines to M cells and enhance their uptake across the gut epithelium. Prior to this application, the expertise to prepare and maintain these intestinal organoids from the chicken intestine has been established in the applicants' labs at The Roslin Institute. Objective 1: Characterise the optimal growth conditions and cellular composition of avian gut organoids Preliminary data show chicken organoids, similar to mouse organoids, can be prepared and maintained from chicken small intestine using mammalian growth factors. Recently, we cloned chicken epithelial growth factor, R-spondin 1 and Noggin and the proteins were produced. The optimal concentrations required for growth and differentiation into crypt and villus domains will be established. Detailed cellular and molecular characterisation of the organoids is of great importance to show that chicken organoids are excellent in vitro models for the chicken gut. The composition of organoids prepared from different parts of the intestine will be analysed using state-of-the art technology present at the Roslin Institute. Bioimaging (FACS, live imaging, confocal and multiphoton microscopy), transcriptomics (RNAsequencing) and bioinformatics will be used to compare the cellular composition and gene expression profiles of these organoids to that in the gut in vivo. Objective 2: Study antigen uptake by M cells in the gut epithelium Little is known of the immunobiology of chicken M cells. Therefore the cellular and molecular mechanisms which influence antigen uptake in the gut epithelium will be studied. We have cloned and produced chicken RANKL, and in homology to the mammalian protein, its capacity to induce chicken M cells will be tested. Antigen uptake in the presence or absence of M cells will be studied using fluorescent pathogens, beads coated with PAMPs and surface modified nanoparticles such as PLGA. The expertise of MSD on adjuvants and vaccines will ensure that the student investigates the state-of-the-art mucosal delivery systems and how they influence the uptake of different vaccine preparations across the gut epithelium. The aim is to identify biomarkers that correlate with induction of beneficial innate responses and maintenance of gut integrity. Objective 3: to study host-pathogen interactions at the gut epithelium The organoids will also be used to study the cellular and molecular responses of the gut epithelium following exposure to important avian pathogens such as Eimeria and Salmonella. Organoids will be exposed to Eimeria sporozoites and attachment, invasion and proliferation within the epithelial cells monitored by bioimaging and transcriptomics as above. Our data show Salmonella in mammalian gut takes advantage of the M cells' capacity to transcytose antigen to infect the host. In parallel the student with also study how S. Typhimurium interacts with the avian gut epithelium.