The role and diagnosis of complement dysregulation in disease

Lead Research Organisation: Cardiff University
Department Name: School of Medicine

Abstract

The proposed research addresses basic questions of how a central part of the immune system is controlled. It is relevant because understanding of these mechanisms and their disturbance in disease will better equip us to identify those at risk of disease to target prevention and treatment.
Complement is part of our immune defence against infection, a group of proteins in blood plasma that recognise, attack and destroy bacteria. This role of complement is important for health and people with complement defects are susceptible to infections. However, this defence comes at a price. Because of its powerful cell-killing properties, complement must be tightly controlled to prevent damage to our own cells resulting in disease. Recently, it has become apparent that small inherited changes in complement proteins, when present in specific combinations, affect this control and greatly increase the risk that complement will attack our own cells and damage organs, particularly kidney and eye. These include a common form of irreversible kidney failure in children (aHUS) and the commonest cause of blindness in the elderly (AMD). We plan to study the ways in which these small inherited changes (either common and called polymorphisms or rare and called mutations) in complement lead to loss of control. To do this we will first gain an understanding of how complement is normally activated and controlled using tools to study the ways that proteins interact. We will use these tools to test how the polymorphisms and mutations weaken control. This knowledge will be used to benefit health in two ways. First, we will develop simple and inexpensive blood tests to identify people carrying risky polymorphisms; those at risk can then be monitored closely to reduce the risk of getting disease. Second, better understanding of the problems in control will enable the design of appropriate treatments to restore control in the complement system to prevent and treat disease.
The work will be performed by a team with proven expertise in complement biology and genetics that has already made contributions to understanding how complement drives disease. The work involves analyses of proteins, either obtained from patients or healthy volunteers or manufactured in the laboratory, to discover how small changes in these proteins predispose to common diseases such as AMD and lethal disorders such as aHUS.

Technical Summary

The proposed work will address the hypothesis that subtle effects on activation and regulation caused by common polymorphisms and rare mutations in components and regulators of the complement system synergise to cause dysregulation of complement that predisposes to disease. The hypothesis is built upon accumulating clinical evidence from patients with specific renal and ocular diseases but may have much broader relevance across many pathologies linked by chronic, low-grade inflammation and activation of the alternative pathway of complement. The research objectives are: first, to address how common polymorphisms (individual and in combination) in complement components and regulators affect the assembly, activity and regulation of the convertase enzymes; second, to determine how rare mutations in components and regulators identified in patients affect convertase assembly, activity and regulation; third, to develop tools for the rapid and efficient identification of disease-predisposing combinations of complement polymorphisms in plasma samples from individuals. The work will utilise state-of-the-art methods for analysing the effects of these polymorphisms and mutations on alternative pathway convertase function and regulation. These will include measures of intermolecular interactions using surface plasmon resonance, conformational changes using dual polarisation interferometry, and convertase function using assays of C3 cleavage, convertase decay and haemolytic capacity. These studies will utilise complement proteins and regulators either purified from plasma or recombinantly expressed as appropriate. The results will explain how individual mutations predispose to pathology and identify those combinations of complement polymorphisms that confer high risk of dysregulation and disease. To facilitate identification of individuals with high risk combinations of polymorphisms we will use proven methods to develop novel monoclonal antibodies that distinguish in plasma the relevant polymorphic variants. These will be used to develop simple immunoassays optimised for routine use to enable screening of relevant populations for disease risk. These assays will not only identify the complement polymorphic status of an individual but also quantify the various isoforms of components and regulators in plasma, information that further aids identification of those at risk of disease. The findings will be disseminated through publication and presentation at research and clinical meetings. Commercially exploitable findings will be patented prior to publication. Commercial partners will be involved at an early stage of assay development.

Publications


10 25 50
Alcorlo M (2011) Unique structure of iC3b resolved at a resolution of 24 Å by 3D-electron microscopy. in Proceedings of the National Academy of Sciences of the United States of America
Brown JH (2012) Postpartum aHUS secondary to a genetic abnormality in factor H acquired through liver transplantation. in American journal of transplantation : official journal of the American Society of Transplantation and the American Society of Transplant Surgeons
Goicoechea De Jorge E (2013) Dimerization of complement factor H-related proteins modulates complement activation in vivo. in Proceedings of the National Academy of Sciences of the United States of America
Harris CL (2012) The complotype: dictating risk for inflammation and infection. in Trends in immunology
Heurich M (2011) Common polymorphisms in C3, factor B, and factor H collaborate to determine systemic complement activity and disease risk. in Proceedings of the National Academy of Sciences of the United States of America
 
Description Complement standardistaion through ICS/ECN
Geographic Reach Multiple continents/international 
Policy Influence Type Membership of a guidance committee
 
Description Project grant
Amount $49,986 (USD)
Organisation Kidneeds  
Sector Charity/Non Profit
Country United States of America
Start 11/2012 
End 10/2013
 
Title Complotype assay 
Description An in vitro assay using purified complement proteins that can illustrate the effect on overall complement activity of combining different polymorphic variants of complement activators and regulators. 
Type Of Material Model of mechanisms or symptoms - in vitro 
Year Produced 2009 
Provided To Others? Yes  
Impact Five publications since 2009 use this methodology. Visitors have come to Cardiff from external collaborating labs to train in these techniques and take them back to their Institutes. 
 
Title Monoclonal antibodies 
Description Antibodies to disease-associated proteins to enable better analysis of their role in pathology. 
Type Of Material Antibody 
Year Produced 2011 
Provided To Others? Yes  
Impact Collaborative venture to generate antibodies to proeins involved in retinal diseases; anibodies (hybridomas) have been generated in Cardiff and will be sent to Edinburgh (collaboration 2). 
 
Title Monoclonal antibodies 
Description Hybridomas secreting antibodies specific for complement proteins and polymorphic variants. 
Type Of Material Antibody 
Provided To Others? No  
Impact Will contribute to future publications and collaborations. Our aim is to generate multiplexed assay panels for screening patient samples. 
 
Title Separation of Factor H allele products by affinity chromatography 
Description Antibodies have been developed on previous funding which can identify variants of factor H differing by just one amino acid. We have now developed this further by using these allele-specific antibodies to separate factor H variants in plasma of heterozygous individuals. This has proved valuable in patients with factor H-related pathologies as we can separate the variants and use the assays described in 7.1 (complotype assay) to dissect the mechanism underlying their disease. 
Type Of Material Technology assay or reagent 
Provided To Others? No  
Impact Published in Blood, 2011 Francis NJ et al. Oral presentation at national BSI meeting, 2010. Oral presentation at international ECN meeting, Leiden 2011. 
 
Description Edinburgh 1 
Organisation University of Edinburgh
Department School of Chemistry Edinburgh
Country United Kingdom of Great Britain & Northern Ireland (UK) 
Sector Academic/University 
PI Contribution Disease-causing mutations have been described in complement factor H by others. The Edingburgh group generated recombinant forms of these proteins and the Cardiff group characterised their functional deficit.
Collaborator Contribution Sharing of reagents and expertise.
Impact Manuscript published in JBC Oral presentation at International Complement Workshop, new york, August 2010 Multi-disciplinary: Edinburgh group -protein expression and structural biology; Cardiff group -functional analyses.
Start Year 2009
 
Description Edingburgh 2 collaboration 
Organisation Medical Research Council (MRC)
Department MRC Human Genetics Unit
Country United Kingdom of Great Britain & Northern Ireland (UK) 
Sector Public 
PI Contribution Functional analysis of a mutant protein thought to interfere with a complement protein. We are currently making monoclonal antibodies against this protein.
Collaborator Contribution A manuscript hs been prepared.
Impact Manuscript submitted. Monoclonal antibodies have been generated and will be shared across groups.
Start Year 2010
 
Description Imperial collaboration 
Organisation Faculty of Medicine
Department Department of Medicine
Country United Kingdom of Great Britain & Northern Ireland (UK) 
Sector Academic/University 
PI Contribution Functional analysis of a mutant complement protein which causes aHUS. Advice on protein purification etc. Have hosted the RA from Imperial in the Cardiff lab to teach new techniques. Generated data for publication.
Collaborator Contribution Clinical analyses, protein purification, functional analyses.
Impact Collaborator on a fellowship to further this work. Work was presented orally at International Complement meeting (Leiden) in 2011. Some of the work incorporated into 2013 paper with Lea, Oxford (PNAS).
Start Year 2010
 
Description Madrid Collaboration 
Organisation Spanish Ministry of Economy and Competitiveness
Department Spanish National Research Council (CSIC)
Country Spain, Kingdom of 
Sector Public 
PI Contribution Experimental data, sharing of reagents and research tools, frequent meetings (at each institute) or by conference call.
Collaborator Contribution Collaborative research project, sharing experimental expertise, knowledge, data and reagents. Involves travel between the two labs for discussions (mini-conferences) and experimental work.
Impact Many publications in section 2 are collaborative with the Madrid lab. High impact -PNAS, JCI, HMG... Three further manuscripts are in preparation. Multi-disciplinary: cardiff group -protein biochemistry, functional assays; Madrid group -genetics, links to clinicians.
 
Description Newcastle collaboration 
Organisation Newcastle University
Department Institute of Cellular Medicine
Country United Kingdom of Great Britain & Northern Ireland (UK) 
Sector Academic/University 
PI Contribution First and joint senior author manuscript published in Blood. Newcastle identified disease-causing mutation in complement proteins using genetic techniques; Cardiff purified the mutant protein from plasma and functionally characterised the defect. Further manuscripts published.
Collaborator Contribution Sharing of reagents, expertise and data
Impact Manuscript published in Blood. Oral presentation at International complement Workshop, new york August 2010 Oral presentation at BSI meeting, liverpool, December 2010. Multi-disciplinary. Newcastle group: clinicians and geneticists; Cardiff group: protein biochemists and experts in functional analyses.
Start Year 2009
 
Description Oxford 
Organisation University of Oxford
Department Sir William Dunn School of Pathology
Country United Kingdom of Great Britain & Northern Ireland (UK) 
Sector Academic/University 
PI Contribution Purified plasma proteins using novel antibodies to send to oxford for structural analyses. Oxford have sent recombinant reagents to cardiff for functional analyses.
Collaborator Contribution Shared reagents and research tools. Experimental data. Published.
Impact Shared reagents and expertise. Published PNAS. Multi-disciplinary: structural biology (Oxford), functional analyses (Cardiff).
Start Year 2011
 
Description USA autoantibody collaboration 
Organisation Wexner Medical Center
Department Department of Internal Medicine
Country United States of America 
Sector Academic/University 
PI Contribution Identification of autoantibodies against complement proteins in a cohort of SLE patients.
Collaborator Contribution Provision of samples. Some analyses of complement proteins and antibodies.
Impact Collaboration instigated in August 2010; predict to publish in 2012/2013
Start Year 2010
 
Description Beacon Researchers in Schools 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? Yes
Geographic Reach Local
Primary Audience Schools
Results and Impact Postdoctoral scientist employed on grant talked to 6th form students about immunological research and described the project. In 2010 the students attended the laboratory to see how a research lab works.

Students were very enthusiastic and keen to visit the lab.
Year(s) Of Engagement Activity 2009