A novel immuno-diagnostic strategy for invasive fungal infections in solid organ transplant patients.

Lead Research Organisation: Imperial College London
Department Name: Dept of Medicine

Abstract

The drugs used in patients who have organ transplants stop the immune system rejecting the transplant. However, they also impair the immune system‘s response to infection. A major problem for these patients is they become susceptible to life-threatening invasive fungal infections (IFIs).
My pilot studies in renal transplant patients indicate these infections may be due to deficiency of an immune-system activating chemical called interferon- . Furthermore I found that treating IFIs in transplant patients with interferon- injections may dramatically improve their chance of survival.
I now propose to define the mechanisms by which transplant drugs lead to susceptibility to IFIs in a mouse mouse model of invasive fungal infection where the mouse are treated with the same transplant drugs. This will enable me to understand how these immune deficits occur, and develop new diagnostic assays to be tested in parallel clinical studies in renal transplant patients with IFIs.
These studies will lead to the delivery of novel diagnostic assays to identify patients at risk of these infections and identify those patients with IFIs who can benefit from life saving interferon- therapy. It is further anticipated these diagnostic assays could have wider application to many infectious diseases in the future.

Technical Summary

Key aims and objectives of the project
1) Define the effects of transplant immuno-suppressant drugs on host immunity in the standard hydrocortisone-suppressed (HC) murine model of invasive aspergillosis (IA).
2) Identify the immune deficits that lead to susceptibility to IA in this model.
3) Develop novel diagnostic assays of susceptibility to invasive fungal infections in this model.
4) Validate these diagnostic assays through synergistic studies in renal transplant patients.
Design and Methodology
1) Define the additive effects of the transplant immuno-suppressants tacrolimus (FK506) and mycophenolate (MMF) on mortality in the HC-based model of invasive aspergillosis. Doses of FK506 and MMF will be increased until mortality increases from 50% to >80%. The model will be defined through histopathologiocal, pharmocokinetic, fungal burden and peripheral blood profiling.
2) Identify the key defects in immunity that increase susceptibility to IA in this model. Macrophage, T lymphocyte, NKT cell and dendritic cell populations, function and fungal responses in lung, liver, spleen and peripheral blood will be characterized by conidial phagocytosis assays, RT-PCR and Luminex cytokine assay, FACS analysis, NKT cell xenogenic target assays, and dendritic cell na?ve T-cell co-culture.
3) Develop peripheral blood mononuclear cell (PBMC) microarray and anti-Aspergillus T-cell Elispot IFN- assays that identify increased susceptibility to IA in FK506/MMF-immuno-suppressed mice. PBMCs from infected mice will be stimulated with Aspergillus antigens or control mitogens. Cytokine responses will be assayed by Agilent whole genome microarrays and correlated with immuno-suppressive drug levels and transcriptional markers of drug toxicity. Parallel anti-Aspergillus T-cell IFN- Elispot assays will be performed on peripheral blood. The Elispot assay will provide a highly sensitive means of quantifying the effects of FK506/MMF on T-cell mediated antifungal immunity.
4) Define the utility of the PBMC RT-PCR and Elispot assays described in Component 3 through a controlled clinical study of 40 renal transplant patients with invasive fungal infections. For the PBMC assays, select markers of susceptibility to invasive fungal infection will be translated into a rapid RT-PCR assay that can also identify patients who will benefit from IFN- therapy.
Scientific and Medical Opportunities
1) Deliver a model in which to define the immuno-pathogenesis of transplant infectious diseases.
2) Identify of the key immunological defects that predispose solid organ transplant patients to IFIs.
3) Deliver peripheral blood assays to identify susceptibility to IFI and guide immuno-therapy.
4) Provide a rational basis for future studies of biopharmaceutical modulators for IFIs.

Publications

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Armstrong-James D (2012) Renal allograft recipients fail to increase interferon-? during invasive fungal diseases. in American journal of transplantation : official journal of the American Society of Transplantation and the American Society of Transplant Surgeons

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Armstrong-James D (2012) Immunotherapy for fungal infections. in Current opinion in microbiology

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Armstrong-James D (2014) A neglected epidemic: fungal infections in HIV/AIDS. in Trends in microbiology

 
Description ISHAM Working Party on Fungal Respiratory Infections in Cystic Fibrosis
Geographic Reach Asia 
Policy Influence Type Influenced training of practitioners or researchers
URL http://www.isham.org/WorkingGroups/CysticFibrosis/
 
Description ISHAM Working Party on HIV Mycoses
Geographic Reach Multiple continents/international 
Policy Influence Type Membership of a guidance committee
 
Description Increasing recognition of the risk of fungal disease associated with clinically licensed inhibitors of Bruton's tyrosine kinase
Geographic Reach Multiple continents/international 
Policy Influence Type Citation in clinical reviews
 
Description Member, European Fungal Infection Study Group
Geographic Reach Asia 
Policy Influence Type Membership of a guidance committee
URL https://www.escmid.org/research_projects/study_groups/fungal_infection/
 
Description British Society of Antimicrobial Chemotherapy PhD Studentship
Amount £105,000 (GBP)
Organisation British Society for Antimicrobial Chemotherapy 
Sector Charity/Non Profit
Country United Kingdom
Start 04/2011 
End 04/2015
 
Description Imperial College Academic Health Science Centre Biomedical Research Centre
Amount £100,000 (GBP)
Organisation Imperial College London 
Department Imperial College Academic Health Science Centre
Sector Academic/University
Country United Kingdom
Start 04/2014 
End 03/2016
 
Description MRC Centenary Award
Amount £100,000 (GBP)
Organisation Medical Research Council (MRC) 
Sector Academic/University
Country United Kingdom
Start 01/2012 
End 01/2013
 
Description MRC Clinical Research Fellow
Amount £240,000 (GBP)
Organisation Medical Research Council (MRC) 
Sector Academic/University
Country United Kingdom
Start 10/2012 
End 09/2015
 
Description Research Grant
Amount £800,000 (GBP)
Funding ID NE/P001165/1 
Organisation Natural Environment Research Council 
Sector Public
Country United Kingdom
Start 11/2016 
End 10/2019
 
Description Role of airway macrophage Btk signalling in pulmonary aspergillosis
Amount £28,539 (GBP)
Funding ID MR/R014590/1 
Organisation Medical Research Council (MRC) 
Sector Academic/University
Country United Kingdom
Start 02/2018 
End 10/2018
 
Description Seed Award in Science
Amount £100,000 (GBP)
Funding ID 204566/Z/16/Z 
Organisation Wellcome Trust 
Sector Charity/Non Profit
Country United Kingdom
Start 11/2016 
End 05/2018
 
Description Wellcome Trust Strategic Award in Medical Mycology and Fungal Immunology
Amount £215,000 (GBP)
Organisation Wellcome Trust 
Department Wellcome Trust Strategic Award
Sector Charity/Non Profit
Country United Kingdom
Start 01/2015 
End 01/2018
 
Title Aspergillus high-throughput genome sequencing and phylogenetic analysis 
Description Next generation sequencing of Aspergillus fumigates and phylogenetic analysis of global population structure as detailed in "genomic context of amole-resistant Aspergillus fumigatus" publication 
Type Of Material Technology assay or reagent 
Year Produced 2015 
Provided To Others? Yes  
Impact we made the isolate collection globally available and several groups are now working on it with the benefit of the genome sequences 
 
Title Single cell imaging of fungal induce macrophage cell death 
Description We developed novel real-time confocal imaging approaches to show that macrophages transfer Aspergillus fumigatus between them during macrophage cell death and have set up the assays in transgenic zebrafish lines 
Type Of Material Technology assay or reagent 
Year Produced 2016 
Provided To Others? Yes  
Impact We have described a entirely novel form of macrophage cell death, which is cell to cell host mediated transfer of a pathogen i.e. Aspergillus fumigatus, and devloped the techniques to measure this. The work was publisghed in 2016 in the American Journal of Respiratory and critical care Medicine, the leading respiratory journal. 
URL http://www.atsjournals.org/doi/abs/10.1164/rccm.201601-0070OC?url_ver=Z39.88-2003&rfr_id=ori:rid:cro...
 
Title Transplant immunosuppression model for invasive aspergillosis 
Description We have performed extensive pharmakokinetic studies to determine the ideal dosing regimens for FK506 and mycophenolate to render mice susceptible to invasive aspergillosis 
Type Of Material Model of mechanisms or symptoms - mammalian in vivo 
Year Produced 2011 
Provided To Others? Yes  
Impact Greater understanding of the immunological factors that predispose to invasive aspergillosis in transplantation 
 
Title Zebrafish model for transplant aspergillosis 
Description A new model of zebrafish hind brain invasive aspergillosis in which shows that calcineurin inhibitors impair neutrophil recruitment to the site of infection leading to increased mortality 
Type Of Material Model of mechanisms or symptoms - non-mammalian in vivo 
Provided To Others? No  
Impact We are publishing a paper hopefully soon on this 
 
Title 24 Aspergillus fumigates genomes 
Description deposited in european nucleotide database.isolates deposited in NIAID Aspergillus repository 
Type Of Material Database/Collection of data 
Year Produced 2015 
Provided To Others? Yes  
Impact other groups are working on these isolates in the context of various virulence traits 
 
Description Fungal diagnostics in high-risk patients 
Organisation National Institute for Health Research
Department NIHR Imperial Biomedical Research Centre
Country United Kingdom 
Sector Academic/University 
PI Contribution Facilitation of a randomised study of fungal diagnostics in high risk patients
Collaborator Contribution Finincial support from the Imperial BRC and research infrastructure support from the Centre for Haematology
Impact This has enabled improved diagnostics for patients in West London
Start Year 2014
 
Description Genomic sequencing of azole-resistant Aspergillus fumigates 
Organisation Gilead Sciences, Inc.
Department Gilead
Country United Kingdom 
Sector Private 
PI Contribution Genome sequencing
Collaborator Contribution financial contribution
Impact mBio paper "genomic context of azalea resistance in Aspergillus fumigates" as detailed in publications
Start Year 2013
 
Description Innate antifungal responses in lung transplant recipients 
Organisation Royal Brompton & Harefield NHS Foundation Trust
Department Heart and Lung Transplantation Unit
Country United Kingdom 
Sector Hospitals 
PI Contribution We are analysing innate immune responses in alveolar macrophages from lung transplant recipients attending the Harefield Hospital
Collaborator Contribution I receive BAL samples from the Harefield, who also consent patients and have helped with relevant ethical permissions.
Impact Paper in review, Disease Models and Mechanisms
Start Year 2011
 
Description NK cell immunotherapy for aspergillosis 
Organisation University of Texas
Country United States 
Sector Academic/University 
PI Contribution I have two shared PhD students with Katy Rezvani, head of stem cell transplant research, at MD Anderson, the worlds largest stem cell transplant hospital. Our students are working on the role of NK cells in Aspergillosis and thier utility in cord blood transplantation and antifungal immunotherapy. I hope the collaboration will lead to major stdies of NK therapy for infectious diseases in immunocompromised hosts.
Collaborator Contribution Dr Rezvani funds one of the students, Tak Sekine, who is 50:50 between MD Anderson and Imperial, but registered through Imperial College London.
Impact Establishment of NK cell research within my laboratory.
Start Year 2011
 
Description Wellcome Trust Strategic Award in Medical Mycology and Fungal Immunology Clinical Research Fellowship 
Organisation University of Aberdeen
Department School of Medical Sciences Aberdeen
Country United Kingdom 
Sector Academic/University 
PI Contribution Award of a clinical PhD fellowship to Amelia Bercussson to study innate immunity to fungi in cystic fibrosis
Collaborator Contribution Collaboration with Adilia Warris at Aberdeen who will collaborate on innate immunity in cystic fibrosis with clinical samples and intellectual input/co-supervision
Impact PhD Clinical Fellowship as described.
Start Year 2014
 
Title Development of diagnostic algorithms for fungal infection in lung transplant recipients 
Description A new Aspergillus point of care assay (Chris Thornton, University of exeter) has been validated in lung transplant recipients at risk of invasive aspergillosis 
Type Diagnostic Tool - Non-Imaging
Current Stage Of Development Early clinical assessment
Year Development Stage Completed 2014
Development Status Under active development/distribution
Impact Improved case definition leading to better research outcomes 
URL http://www.iscadiagnostics.com/products/aspergillosis-flow-device/
 
Description Engagement for research with local Renal transplant patients group 
Form Of Engagement Activity A magazine, newsletter or online publication
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Participants in your research and patient groups
Results and Impact I am awaiting feedback from the Wets London Renal transplant Patients group and will give them a talk

DISSEMINATION OF INFORMATION ON PATIENT RESEARCH TO INVOLVED PATIENTS
Year(s) Of Engagement Activity 2014
 
Description Royal Society Summer Exhibition "Killer Fungus" 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Public/other audiences
Results and Impact A public outreach stall in the RSSE organised by the UK Mycology community (lead Aberdeen, Neil Gow).
Year(s) Of Engagement Activity 2016
URL https://royalsociety.org/science-events-and-lectures/summer-science-exhibition/exhibits/killer-fungu...