Chicken intestinal organoids: a novel in vitro system to study mucosal vaccine targeting
Lead Research Organisation:
University of Edinburgh
Department Name: The Roslin Institute
Abstract
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.
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.
Publications
Nash TJ
(2021)
Inside-out chicken enteroids with leukocyte component as a model to study host-pathogen interactions.
in Communications biology
Studentship Projects
| Project Reference | Relationship | Related To | Start | End | Student Name |
|---|---|---|---|---|---|
| BB/M014819/1 | 01/10/2015 | 30/11/2020 | |||
| 1658198 | Studentship | BB/M014819/1 | 01/10/2015 | 30/09/2019 |
| Description | We have developed an in vitro system to grow chicken intestinal organoids, which have the potential to be implemented in both research and industry intestinal-based activities. The organoids grow inside-out so they have an accessible apical epithelial surface in contract with the classical mammalian intestinal organoid with an internal lumen. The organoids also have a unique lamina propria component including immune cells so we can study intestinal epithelial-immune cell interactions. We have already modelled infections and immune responses with salmonella typhimurium, eimeria tenella and Influenza A virus. Since the organoids can be successfully cryopreserved, this intestinal model will reduce the use of animals in research. |
| Exploitation Route | This in vitro model system can be utilised in research and industry for use in chicken intestinal applications. |
| Sectors | Agriculture, Food and Drink,Environment,Pharmaceuticals and Medical Biotechnology |
| Description | A patent application regarding this work has been filed and a licence agreement with an industrial partner is being negotiated. |
| First Year Of Impact | 2019 |
| Sector | Agriculture, Food and Drink,Education |
| Impact Types | Economic |
| Description | Chicken enteroids: exploring their potential as mucosal immunological model |
| Amount | £13,275 (GBP) |
| Funding ID | FTMA Panel- Email-10/12/19 |
| Organisation | Biotechnology and Biological Sciences Research Council (BBSRC) |
| Sector | Public |
| Country | United Kingdom |
| Start | 01/2020 |
| End | 03/2020 |
| Description | General Scheme - Lonneke Vervelde - Chicken Intestinal Organoids: A Novel In Vitro System To Study Mucosal Vaccine Targeting |
| Amount | £24,000 (GBP) |
| Funding ID | WT 5906748 |
| Organisation | Intervet International B.V. |
| Sector | Private |
| Country | Netherlands |
| Start | 09/2017 |
| End | 08/2021 |
| Description | Research Grant - Lonneke Vervelde - Development of in vitro chicken enterocyte and organoid cultures |
| Amount | £136,852 (GBP) |
| Funding ID | WT 6571120 |
| Organisation | Novozymes |
| Sector | Public |
| Country | Denmark |
| Start | 12/2018 |
| End | 11/2019 |
| Title | avian intestinal organoids |
| Description | We have developed complex multilobulated 3D chicken enteroids from intestinal embryonic villi and adult crypts. These avian enteroids develop optimally in suspension without the structural support required to produce mammalian enteroids, resulting in an inside-out enteroid conformation with media-facing apical brush borders. Histological and transcriptional analyses show these enteroids comprise of differentiated intestinal epithelial cells bound by cell-cell junctions, and notably, include intraepithelial leukocytes and an inner core of lamina propria leukocytes. The advantageous polarisation of these enteroids has enabled infection of the epithelial apical surface with Salmonella Typhimurium, influenza A virus and Eimeria tenella without the need for micro-injection. We have created a comprehensive model of the chicken intestine which has the potential to explore epithelial and leukocyte interactions and responses in host-pathogen, food science and pharmaceutical research. |
| Type Of Material | Technology assay or reagent |
| Year Produced | 2021 |
| Provided To Others? | Yes |
| Impact | The development of this tools has led to follow up funding: BB/V019899/1 in which the technology is employed to study determinants of virulence or emerging virulent non-notifiable avian influenza viruses. The technology may be developed as diagnostic tool to rapidly asses virulence of viruses and thereby may reduce the number of animals used for IVPI scores. BB/V011286/1 in which the technology is employed to study the effect of innate immune pressure and host jumps on evolution of newly emerging avian influenza viruses. |
| Title | Avian Enteroids |
| Description | mRNA expression datasets for chicken enteroids at 0, 3 and 7 days of culture have been deposited in the European Nucleotide Archive (ENA) at EMBL-EBI under accession number PRJEB37491 |
| Type Of Material | Database/Collection of data |
| Year Produced | 2020 |
| Provided To Others? | Yes |
| Impact | Content in publication 'Inside-Out Chicken Enteroids with Leukocyte Component as a Model to Study Host-Pathogen Interactions' |
| URL | https://www.ebi.ac.uk/ena/data/view/PRJEB37491 |
| Description | MSD Animal Health |
| Organisation | MSD Animal Health |
| Country | United Kingdom |
| Sector | Private |
| PI Contribution | Our research team has provided adequate facilities, materials, equipment, supervision and training for the my work on the Project. |
| Collaborator Contribution | MSD provided me with a placement at its premises for 10 days in 2019. During the placement MSD provided adequate facilities, equipment, supervision and training for my work on the Project, as well as paying the travel and accommodation costs incurred during the placement. |
| Impact | Development of a technique to isolate sporozoites from Eimeria. |
| Start Year | 2017 |
| Title | Avian Enteroids |
| Description | There is provided an in vitro three dimensional cell construct for use as a model of the avian intestine derived from avian intestinal tissue comprising avian cells organised into intestinal villi and crypts. Suitably the construct comprises an exterior surface that mimics the apical surface of a chicken intestine. Also provided are methods of making the cell construct and use of the construct as an in vitro intestinal model system to examine an agent including, but not limited to a microbe, a vaccine, a pharmaceutical, a feed additive, a toxin, a pre-biotic, postbiotic, pre pro post biotic, therapeutic, a cell, gene construct, protein, immune-modulator, an intestinal effector agent, a candidate intestinal effector agent, cell signalling inhibitor, or cell signalling activator. |
| IP Reference | PCT/GB2020/051607 |
| Protection | Patent application published |
| Year Protection Granted | 2020 |
| Licensed | No |
| Impact | A patent application regarding this work has been filed and a licence agreement with an industrial partner is being negociated. |
| Description | Invited lecture at MSD-Animal Health |
| Form Of Engagement Activity | A talk or presentation |
| Part Of Official Scheme? | No |
| Geographic Reach | International |
| Primary Audience | Industry/Business |
| Results and Impact | Invited lecture at MSD-Animal Health in Boxmeer the Netherlands, with WebEx connection to all other location o MSD -AH around the world. |
| Year(s) Of Engagement Activity | 2019 |
| Description | One Health workshop |
| Form Of Engagement Activity | Participation in an activity, workshop or similar |
| Part Of Official Scheme? | No |
| Geographic Reach | Local |
| Primary Audience | Undergraduate students |
| Results and Impact | 30 veterinary, medical and dentistry students from The University of Edinburgh attended an online 'One Health; Organoids as Model Systems' workshop designed to inspire students to consider careers in research. This INSPIRE workshop was funded by the Wellcome Trust and coordinated by the Academy of Medical Sciences and the Edinburgh One Health Society. There were questions and discussions on the subjects of poultry and organoid research. afterwards and very positive feedback from the students who attended the course. |
| Year(s) Of Engagement Activity | 2020 |
| Description | Organoid Symposium Poster |
| Form Of Engagement Activity | Participation in an activity, workshop or similar |
| Part Of Official Scheme? | No |
| Geographic Reach | International |
| Primary Audience | Other audiences |
| Results and Impact | Presenter a poster on 'Inside-Out Chicken Enteroids with Leukocyte Component as a Model to Study Host-Pathogen Interactions' presented at the virtual EMBO EMBL Symposium: Organoids: Modelling Organ Development and Disease in 3D Culture. Sparked questions and discussions. Approached by industry afterwards to offer help on the enteroids. |
| Year(s) Of Engagement Activity | 2020 |
| Description | Poster presentation at British Society of Immunology Conference |
| Form Of Engagement Activity | Participation in an activity, workshop or similar |
| Part Of Official Scheme? | No |
| Geographic Reach | International |
| Primary Audience | Other audiences |
| Results and Impact | Poster presentation at the virtual British Society of Immunology - connecting immunology in the time of COVID conference. The poster sparked question from attendees. |
| Year(s) Of Engagement Activity | 2020 |
| Description | Talk and poster at Edinburgh Infectious Diseases Symposium |
| Form Of Engagement Activity | Participation in an activity, workshop or similar |
| Part Of Official Scheme? | No |
| Geographic Reach | International |
| Primary Audience | Other audiences |
| Results and Impact | Talk and poster presentation at the Edinburgh Infectious Disease Symposium with over 300 attendees globally. Questions and discussions were sparked. |
| Year(s) Of Engagement Activity | 2020 |
| Description | Talk at Avian Research Symposium |
| Form Of Engagement Activity | A talk or presentation |
| Part Of Official Scheme? | No |
| Geographic Reach | Local |
| Primary Audience | Postgraduate students |
| Results and Impact | Presentation given to Edinburgh University scientists, veterinarians and students at the Avian Research Symposium. Sparked questions and discussions afterwards. |
| Year(s) Of Engagement Activity | 2021 |
| Description | Talk given at RDSVS |
| Form Of Engagement Activity | A talk or presentation |
| Part Of Official Scheme? | No |
| Geographic Reach | International |
| Primary Audience | Professional Practitioners |
| Results and Impact | Talk on project given to qualified veterinary surgeons as part of their 10 year reunion, demonstrating the different career paths available outside of general practice. This sparked questions and discussions on the project field as well as research careers in general. |
| Year(s) Of Engagement Activity | 2018 |
| Description | Talk given to MSD DIscovery & Technology Dept |
| Form Of Engagement Activity | A talk or presentation |
| Part Of Official Scheme? | No |
| Geographic Reach | International |
| Primary Audience | Industry/Business |
| Results and Impact | Talk summarising the findings of the project so far to ~15 members of MSD's Discovery and Technology department. MSD used the content of the talk to suggest ideas for further research in this field, and invited further presentations on this topic to other poultry-based industries/businesses. |
| Year(s) Of Engagement Activity | 2019 |
| Description | Talk to Easter Bush Campus |
| Form Of Engagement Activity | A talk or presentation |
| Part Of Official Scheme? | No |
| Geographic Reach | Local |
| Primary Audience | Postgraduate students |
| Results and Impact | Online presentation on the development, characterisation and utilisation of the chicken enteroids as a host-pathogen model. Attended by scientists and veterinary professionals from the Roslin Institute, Moredun Institute and RDSVS. Sparked questions and discussion afterwards including discussions about the utilisation of the enteroids in nutritional research. |
| Year(s) Of Engagement Activity | 2021 |