A comprehensive survey of protein-protein interactions between Plasmodium falciparum merozoites and human receptors

Lead Research Organisation: The Wellcome Trust Sanger Institute
Department Name: Wellcome Trust Genome Campus

Abstract

Malaria is one of the most common infectious diseases in the world, with more than 300 million cases of malaria each year, leading to more than 1 million deaths, primarily in children under the age of five in Africa. Malaria is caused by single-celled Plasmodium parasites and one species is particularly deadly - Plasmodium falciparum - which is responsible for almost all malaria deaths. Plasmodium parasites have a complex life cycle, but all the symptoms of malaria are caused when they invade human red blood cells, also known as erythrocytes. The parasite uses the erythrocyte as a source of food, multiplies inside it, and, after 48 hours, breaks open the erythrocyte to release multiple new parasites. This cycle of invasion, multiplication and re-invasion results in high numbers of parasites in the blood stream.

Because Plasmodium parasites spend the majority of their life cycle inside human cells, they are difficult to target with vaccines, and there is currently no licensed vaccine for malaria. However, the process of erythrocyte invasion is one of the few stages of the parasite's life cycle when they are exposed to the host immune system, and is therefore a potential target for vaccine-induced invasion-blocking antibodies or drug development. For vaccines or drugs to be developed, it is critical that we understand which proteins on the parasite surface are binding directly to erythrocytes and the identity of the erythrocyte proteins that they are binding to. By first identifying these parasite-erythrocyte interactions, it may be possible to develop novel therapeutics that prevent invasion and hence cure or prevent malaria. Research over the last two decades has led to the identification of numerous P. falciparum proteins that may be involved in erythrocyte invasion, but only in very cases do we know which erythrocyte proteins they bind to. This is in large part because of the technical challenges involved: protein-protein interactions between the surface of cells are often very short-lived (lasting only a few seconds) and so are very difficult to detect using standard experimental approaches.

At the Wellcome Trust Sanger Institute we have recently developed a new approach to detect short-lived protein-protein interactions. Called AVEXIS (Avidity based Extracellular Interaction Screen), it uses libraries of proteins, expressed and purified in the lab, to detect novel interactions. Over the last two years we have started to apply this to the process of P. falciparum erythrocyte invasion and have created small pilot libraries of erythrocyte and P. falciparum proteins to screen for interactions. This preliminary work has identified two new parasite-erythrocyte interactions, one of which appears to play a very important role in erythrocyte invasion. However, this initial screen was limited in scope and we still do not have binding partners for the majority of P. falciparum proteins. In this research we will expand our libraries of parasite and erythrocyte proteins, and also include other components of the host blood which P. falciparum parasites may be interacting with, such as sugar structures that are present on cell surfaces. After screening the parasite and blood component libraries for interactions in an all vs. all manner, we will test whether any newly discovered interactions have roles in erythrocyte invasion using P. falciparum parasites that can be cultured in the lab. This research will build up the first systematic map of interactions between P. falciparum proteins and human blood cell components, and will identify protein-protein interactions that are of particular interest for vaccine or drug development. We will also deposit the DNA constructs that are used to create our recombinant protein libraries in not-for-profit reagent resource collections, where they can be freely accessed by other researchers and thereby empower malaria research in many labs across the globe.

Technical Summary

Plasmodium falciparum parasites cause more than a million deaths from malaria each year. All the symptoms and pathology of malaria are caused by the blood stages of P. falciparum development, which are initiated when merozoites invade human erythrocytes. Erythrocyte invasion is potentially an attractive vaccine target because it is essential to parasite survival and the extracellular ligands that catalyse it are exposed on the parasite surface. Although many candidate P. falciparum ligands have been identified, in very few cases are their host receptors known, in part because cell surface protein-protein interactions are often of very low affinity, making them difficult to detect using standard approaches.

We will address this important gap in our understanding using AVEXIS, a protein-protein interaction technology developed at the Sanger Institute specifically to detect low affinity interactions. A pilot screen has already identified two novel P. falciparum-erythrocyte interactions, including one that plays a critical role in invasion. To expand these studies, we will:
1) Construct a comprehensive library of recombinant P. falciparum merozoite proteins
2) Screen the library against human erythrocytes to identify erythrocyte binding proteins
3) Identify carbohydrate receptors for merozoite ligands by screening glycan arrays
4) Identify protein receptors by using AVEXIS to screen libraries of human erythrocyte receptors or other soluble blood components
5) Validate the function of identified interactions using in vitro cultured P. falciparum parasites.
This approach will generate the first biochemically validated map of interactions between P. falciparum merozoite proteins and human blood cell components, and will identify protein-protein interactions that are of particular interest for vaccine or drug development. All expression constructs will be deposited in not-for-profit reagent resource collections to empower research in other labs.

Planned Impact

Malaria is responsible for around 1 million deaths annually and the emergence and spread of drug-resistant strains is now a major global health concern. The economic cost of this disease is significant, estimated to represent up to 1.3% of GDP in countries with high transmission rates. The overarching, long-term goal of this research is to make a contribution towards an effective and cost-effective preventative treatment for malaria. The development of an effective vaccine for this disease would have a huge impact on global health and is a top priority for many public and privately led funding initiatives.

Scientific researchers working in the immediate subject area will be clear beneficiaries of this work, as outlined in the 'Academic Beneficiaries' section. In the context of translational impact, this research will be of particular benefit to researchers working within the global malaria vaccine initiatives both in the public and private sector. Merozoite proteins are regarded as excellent vaccine candidates. Should a single or multi-component blood-stage vaccine be an effective treatment for malaria, it is highly likely that one or more of the proteins within our recombinant merozoite protein resource will be a component of such a vaccine. Given our success in high level expression of functional proteins, the experience of expressing antigens in this project will be of clear utility to other vaccine projects focusing on the same targets. Furthermore, we have already shown that the combination of biochemical and in vitro approaches can make important functional insights into the role of merozoite proteins. The results of this work will therefore also help to rationalise antigen targets on the basis of function, and thereby provide a rational focus for vaccine development.

The ultimate aim of this research will be to identify candidate antigens that would lead towards a highly effective blood-stage vaccine, whether driven by ourselves or others. When this is achieved, the primary beneficiaries of this research would be the many billions of people living in malaria-endemic regions. A successful vaccine against malaria would therefore have a huge global health impact. This proposal is aimed at making a contribution towards this goal by expressing and determining the function of P. falciparum merozoite proteins. The timescale for any identified target/s to be used in a licensed vaccine is likely to be 10 to 20 years. The economic impact of an effective anti-malarial vaccine would be enormous, relieving a huge healthcare, economic and social burden from some of the poorest countries in the world, and also enabling non-native people travelling to and working in these countries to be effectively protected.

More broadly, the approach of producing recombinant protein libraries representing the surface repertoire of differentiated cell types to identify the receptors for pathogens is one that could be applied to other stages of the Plasmodium lifecycle, which also present important intervention targets. One important example is transmission-blocking vaccines, which have been identified as a high priority as part of the move towards malaria elimination. The same approaches could also be applied to other pathogens of global public health importance, including other parasites as well as viruses and bacteria.

Researchers employed on this grant would gain important skills necessary for producing and elucidating the function of P. falciparum merozoite proteins, which could play an important role in their own career development. These would include the expression systems used to produce the proteins and also the skills to culture and perform functional assays on P. falciparum parasites. The wider dissemination of these skills may lead to important insights in the function of merozoite proteins in other research environments.

Publications


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Osier FH (2014) New antigens for a multicomponent blood-stage malaria vaccine. in Science translational medicine

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Wanaguru M (2013) RH5-Basigin interaction plays a major role in the host tropism of Plasmodium falciparum. in Proceedings of the National Academy of Sciences of the United States of America




 
Title Parasite 
Description 'Parasite' has been created by artist Deborah Robinson working in close collaboration with Wellcome Trust Sanger Institute malaria researchers Julian Rayner and Oliver Billker. During a residency at the Sanger Institute, Deborah explored how genomics is being used to understand the biology of the parasite that causes malaria and how this may be used to establish new ways of preventing or treating the disease. For this exhibition Deborah has used sections of archival films which she found within the Wellcome Collection and Imperial War Museum Library and which depict the 'mass eradication' campaigns which characterised attempts to control the disease in the last century. She deploys software, developed with digital and sound artist David Strang, to corrode sections of this archival documentary footage and reminding us of the cyclical and recurrent nature of this deadly disease. This project was funded by Arts Council England and was displayed at the Ruskin Gallery in the Cambridge School of Art at Anglia Ruskin University until 18 July 2013. 
Type Of Art Film/Video/Animation 
Year Produced 2013 
Impact Talks about Parasite have taken place at: Inspace (Bio informatics gallery), Edinburgh University; Tent Gallery, Edinburgh University; St John's College, Cambridge; Exeter University. In 2013 Parasite was selected for inclusion in the Shanghai International Science and Art exhibition where it won an award for excellence in Science and Art. Solo exhibitions featuring Parasite have taken place at the Ruskin Gallery, Cambridge, July 2013, ICIA, University of Bath, March 2014. Heidelberg Castle, Germany, May 2014 (sponsored by The European Virtual Institute of Malaria Research and EVIMalaR) and at an international exhibition in Shanghai. It is now installed at the Wellcome Genome Campus Conference Centre. 
URL http://www.sanger.ac.uk/about/engagement/art.html
 
Description Institutional Links Collaboration - Colombia
Amount £230,000 (GBP)
Funding ID 172729106 
Organisation British Council 
Sector Charity/Non Profit
Country United Kingdom of Great Britain & Northern Ireland (UK)
Start 04/2015 
End 03/2017
 
Description MULTIVIVAX: Development of Effective Vaccines against Multiple Lifecycle Stages of Plasmodium vivax malaria
Amount € 351,298 (EUR)
Organisation European Union 
Sector Public
Country European Union (EU)
Start 01/2017 
End 12/2022
 
Description NIH R01
Amount $286,605 (USD)
Funding ID R01-AI-091595-01A Primary Award; 557892 Sub-award 
Organisation National Institutes of Health (NIH) 
Sector Public
Country United States of America
Start 12/2014 
End 08/2019
 
Title Plasmodium falciparum merozoite protein expression constructs I 
Description Eukaryotic expression constructs for 52 full-length Plasmodium falciparum merozoite secreted proteins and 10 processed fragments of P. falciparum merozoite secreted proteins have been generated in the first two years of the award. Constructs have been deposited into not-for-profit plasmid repository, Addgene. 
Type Of Material Technology assay or reagent 
Year Produced 2013 
Provided To Others? Yes  
Impact Screens for erythrocyte binding and erythrocyte receptors ongoing 
URL http://www.addgene.org/Gavin_Wright/
 
Title Plasmodium vivax merozoite protein expression constructs 
Description A library of 30 plasmids for expression of Plasmodium vivax merozoite proteins; made freely available through Addgene. 
Type Of Material Technology assay or reagent 
Year Produced 2015 
Provided To Others? Yes  
Impact We have applied these tools to large-scale immunoepidemiology studies; others are using them to do the same. 
URL https://www.addgene.org/Julian_Rayner/
 
Description Expression of Plasmodium knowlesi invasion ligands 
Organisation University of St Andrews
Department School of Medicine St Andrews
Country United Kingdom of Great Britain & Northern Ireland (UK) 
Sector Academic/University 
PI Contribution Plans underway to express fragments of Plasmodium knowlesi invasion ligands on the HEK293E expression system being used for Plasmodium falciparum expression
Collaborator Contribution Providing P. knowlesi invasion ligand sequences
Impact Some expression trials carried out; followup possible in the future but not a priority for either collaborating lab at this time.
Start Year 2012
 
Description Expression of Plasmodium vivax merozoite proteins 
Organisation National Institutes of Health (NIH)
Department National Institute of Allergy and Infectious Diseases (NIAID)
Country United States of America 
Sector Public 
PI Contribution Expression of a sub-library of Plasmodium vivax merozoite proteins for binding and immunoepidemiology studies
Collaborator Contribution Provided P. vivax infected patient serum from Cambodia, Papua New Guinea and Solomon Islands for immunoepidemiology studies
Impact Library expression now published. Initial immunoepidemiology screen under review in PLoS NTD.
Start Year 2012
 
Description Expression of Plasmodium vivax merozoite proteins 
Organisation Walter and Eliza Hall Institute (WEHI)
Country Australia, Commonwealth of 
Sector Academic/University 
PI Contribution Expression of a sub-library of Plasmodium vivax merozoite proteins for binding and immunoepidemiology studies
Collaborator Contribution Provided P. vivax infected patient serum from Cambodia, Papua New Guinea and Solomon Islands for immunoepidemiology studies
Impact Library expression now published. Initial immunoepidemiology screen under review in PLoS NTD.
Start Year 2012
 
Description Gathoni Kamuyu - PhD Student 
Organisation The Wellcome Trust Ltd
Department KEMRI-Wellcome Trust Research Programme
Country Kenya, Republic of 
Sector Multiple 
PI Contribution Gathoni is a graduate student, who is pursuing a PhD under Faith Osier's supervision. I am a co-supervisor on her PhD, and she is visiting my lab for a year, working on proteomic approaches to identify new vaccine targets. The expression system in HEK293E cells is at the core of the validation approaches.
Collaborator Contribution Gathoni's work is co-supervised by Faith Osier at KEMRI-Kilifi. This link enables access to invaluable longitudinal cohort data and samples.
Impact Future research directions developed. Publications expected in 2016/17.
Start Year 2013
 
Description Screening for targets of immunity in Colombia 
Organisation Universidad Nacional de Colombia
Country Colombia, Republic of 
Sector Academic/University 
PI Contribution Using P. falciparum and P. vivax antigen production in HEK293E cells to screen for targets of immunity, and detect asymptomatic infection, in communities on the Pacific Coast of Colombia
Collaborator Contribution Collection of samples, establishing community links and links to local health agencies
Impact Successful grant application, reported under relevant section
Start Year 2015
 
Description Testing Plasmodium protein binding to reticulocytes 
Organisation NHS Blood and Transplant (NHSBT)
Country United Kingdom of Great Britain & Northern Ireland (UK) 
Sector Public 
PI Contribution Testing binding of Plasmodium proteins to reticulocytes differentiated from HSCs at NHSBT. Initial data equivocal; collaboration may be restarted at a later date.
Collaborator Contribution Differentiating reticulocytes from HSCs and shipping to Sanger for testing
Impact Initial testing underway
Start Year 2012
 
Description Testing Plasmodium vivax vaccine candidates in ex vivo invasion assays 
Organisation Harvard School of Public Health
Country United States of America 
Sector Academic/University 
PI Contribution There is no in vitro culture system for Plasmodium vivax, so any vaccine testing will have to take place ex vivo. We have begun to collaborate with Prof. Manoj Duraisingh to carry out such assays at a field site in Goa, India, using antibodies raised against HEK239E expressed vaccine candidates.
Collaborator Contribution Short term ex vivo culture of patient P. vivax isolates.
Impact First joint publication planned for 2016; first invasion assays for late 2016; joint funding application planned for June 2016.
Start Year 2015
 
Description Trialing plant-based expression systems for Plasmodium proteins 
Organisation Planet Biotechnology
Country United States of America 
Sector Private 
PI Contribution Providing vectors for expression of Plasmodium proteins in tobacco systems. Trialing expressed proteins in in vitro Plasmodium invasion assays
Collaborator Contribution Expression trials
Impact Initial expression trials underway
Start Year 2012
 
Description Using recombinant proteins to identify new vaccine candidates 
Organisation The Wellcome Trust Ltd
Department KEMRI-Wellcome Trust Research Programme
Country Kenya, Republic of 
Sector Multiple 
PI Contribution Provided Plasmodium falciparum recombinant proteins produced from this funding and other sources for use in immunoepidemiology screens, and to generate antibodies for use in inhibition testing.
Collaborator Contribution Used the proteins to screen sera samples from longitudinal epidemiological cohorts
Impact Co-authored paper led by Kevin Marsh and Faith Osier; reported elsewhere. Other publications in development.
Start Year 2013
 
Description Video microscopy of Plasmodium erythrocyte invasion 
Organisation Burnet Institute
Department Centre for Infection
Country Australia, Commonwealth of 
Sector Charity/Non Profit 
PI Contribution Sharing protein and antibody reagents to test their ability to block invasion in video microscopy based phenotyping assays. Some initial exploratory work carried out at Burnet Institute, but now detailed followup being carried out by co-supervised PhD students at the Cavendish lab, to dissect the molecular steps that protein-ligand interactions catalyse during invasion.
Collaborator Contribution Video microscopy based phenotyping of invasion
Impact Papers planned for submission 2016/17
Start Year 2012
 
Description Video microscopy of Plasmodium erythrocyte invasion 
Organisation Department of Physics
Department Cavendish Laboratory
Country United Kingdom of Great Britain & Northern Ireland (UK) 
Sector Academic/University 
PI Contribution Sharing protein and antibody reagents to test their ability to block invasion in video microscopy based phenotyping assays. Some initial exploratory work carried out at Burnet Institute, but now detailed followup being carried out by co-supervised PhD students at the Cavendish lab, to dissect the molecular steps that protein-ligand interactions catalyse during invasion.
Collaborator Contribution Video microscopy based phenotyping of invasion
Impact Papers planned for submission 2016/17
Start Year 2012
 
Title Rh5 based blood stage vaccine for Plasmodium falciparum 
Description Currently seeking funding for Phase I/IIa clinical trial 
Type Therapeutic Intervention - Vaccines
Current Stage Of Development Refinement. Non-clinical
Year Development Stage Completed 2012
Development Status Actively seeking support
Impact Based on our previous work using the AVEXIS system, extension of which has now been supported by the MRC, PfRH5 has been identified as a high priority blood stage vaccine target. We are carrying out pre-clinical optimisation, and seeking funding for a Phase I/IIa trial. 
 
Description "Parasite" - visiting artist Deborah Robinson 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Type Of Presentation Keynote/Invited Speaker
Geographic Reach International
Primary Audience Public/other audiences
Results and Impact "Parasite", joint project with Oliver Billker and Deborah Robinson, visiting artist. Exhibited in Cambridge, Heidelberg, Shanghai, and now installed at the Wellcome Genome Campus Conference Centre. Presents a new perspective on malaria elimination and the cyclical nature of the scientific challenges that malaria causes.

First exhibit Anglia Ruskin University 06/2013. Plan for future exhibits in 2014
Year(s) Of Engagement Activity 2013,2015,2016
URL http://www.anglia.ac.uk/ruskin/en/home/microsites/ruskin_gallery/archive_201213/parasite.html
 
Description Blog on Malaria Challenge, an interactive website that explains malaria biology 
Form Of Engagement Activity A magazine, newsletter or online publication
Part Of Official Scheme? No
Type Of Presentation Paper Presentation
Geographic Reach International
Primary Audience Public/other audiences
Results and Impact I was recently involved in helping to create "Malaria Challenge", an online multimedia tool (www.yourgenome.org) to explain malaria to high school students. In September 2012, I wrote a blog about the website, which was posted on BioMedCentral's Open Access in the Developing World website.

Retweets and Facebook recommends
Year(s) Of Engagement Activity 2012
 
Description Grant Panel Member, Wellcome Trust Engagement Fellowships 
Form Of Engagement Activity A formal working group, expert panel or dialogue
Part Of Official Scheme? Yes
Type Of Presentation Keynote/Invited Speaker
Geographic Reach National
Primary Audience Public/other audiences
Results and Impact Member of the WT panel awarding Public Engagement Fellowships

Four fellowships awarded over the past two years
Year(s) Of Engagement Activity 2012,2013,2014
 
Description Royal Society Summer Exhibit 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Type Of Presentation Workshop Facilitator
Geographic Reach National
Primary Audience Public/other audiences
Results and Impact Scientific steering group for the Sanger Institute exhibit at the 2013 Royal Society Summer Exhibit. Involved in planning and delivery; also volunteered for 2 days at the exhibit.

Dessimination of research to the lay public
Year(s) Of Engagement Activity 2013
URL http://sse.royalsociety.org/2013/
 
Description Talk to visiting schools 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Type Of Presentation Keynote/Invited Speaker
Geographic Reach Local
Primary Audience Schools
Results and Impact Over the past year I have given four talks to visiting school groups (30+ pupils) and school teachers (further development courses) about the impact of genome sequencing on malaria research, which stimulated much discussion about malaria in general and vaccine development in particular,

Feedback from teachers and students reported high level of interest in malaria after the talk
Year(s) Of Engagement Activity 2012
 
Description Talks to undergraduate groups (Cambridge) 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Undergraduate students
Results and Impact Two talks, each reaching 20-30 biology/medical undergraduate students.
Year(s) Of Engagement Activity 2015,2016
 
Description Wellcome Trust Drama Workshop 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Type Of Presentation Keynote/Invited Speaker
Geographic Reach National
Primary Audience Public/other audiences
Results and Impact Participant, Wellcome Trust Drama Workshop, a forum for playwrights, producers and scientists to meet and discuss possible future collaborations

Established collaborative links
Year(s) Of Engagement Activity 2012
 
Description Winner of: I'm A Scientist, Get Me Out Of Here 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Schools
Results and Impact I'm a Scientist, Get me out of here! was not just about attracting students to study and launch careers in science, but also to equip all involved with ways to think scientifically and help them become responsible citizens.

This was a rewarding experience for me as a scientist. We are totally enthused by the format as a tool for science communication, and we are now trying to spread the word about this fantastic science communication tool to as many schools as we can, by talking to teachers and spreading the word on the web. To help with this, we used the money that I won on I'm a Scientist, Get me out of here! to get loads of CDs burned with a web tool to help explain the malaria life-cycle (Malaria Challenge). We have mailed copies out to all the schools that participated in this and distribute the rest for free as widely as possible to schools all over the country.
Year(s) Of Engagement Activity 2011
URL http://blog.wellcome.ac.uk/2011/06/16/any-questions-inside-i%E2%80%99m-a-scientist%E2%80%A6/