A systems biology approach to understand immunity and pathogenesis of malaria in children

Lead Research Organisation: University of Oxford
Department Name: Tropical Medicine

Abstract

Malaria is a major health problem, with approximately half of the world's population at risk. Most malaria cases and deaths occur in sub-Saharan Africa and are caused by the species "Plasmodium falciparum", with an annual mortality rate of up to 700,000, mostly among children.. Despite many years of work and investment, we still do not have a highly effective vaccine. One reason for this is that we have an incomplete understanding of the interaction between malaria parasites and immunity. Immune responses to Plasmodium are complex, and protective immunity in malaria endemic areas develops only after several years of exposure. We know from our long-term studies of longitudinal cohorts that the outcome of malaria infection in children is extremely variable such that some children have two or three clinical episodes while others have very frequent clinical episodes or severe episodes without apparently becoming immune. Malaria can have a profound suppressive impact on immune responses. Our hypothesis is that these children with frequent and symptomatic malaria infections are caught in a causal "loop" (or "vicious cycle") whereby malaria episodes lead to impaired immunity to malaria, which in turn leads to further episodes of malaria.

In our first phase of analysis, we will investigate immune responses in a cohort of children developing immunity to P.falciparum malaria, for whom we have detailed life histories of malaria exposure. We will compare those immune responses of children with a history of repeated and clinical malaria episodes with those of children with a normal number of clinical malaria episodes (matching age, location and malaria exposure). We will also examine responses in a third group of children who live nearby, but are not exposed to malaria. This first phase of analysis will establish the analytical methodology, determine patterns of responses, and generate models and hypotheses that can be tested in the second phase (see below). We will combine transcriptomic, flow-cytometric, cytokine analyses, and malaria-specific B cell/antibody responses to compare as comprehensively as possible the features of the host response in the two groups.

A single "snap shot" of immune responses may represent only the endpoint of immune processes and may not reflect those that are causally related to differences in malaria outcome. Therefore, in a second phase of testing, we will collect and store samples from the whole cohort prior to surveillance for clinical malaria episodes. Samples will be stored, and then tested and analysed when parallel groups to those describe for the first phase above can be identified (i.e. multiple episodes vs normal episodes). These samples will be analysed by the laboratory and analytical teams blinded to epidemiological data, and the association between the signatures identified in the first phase and malaria episodes in prospect will be examined. We will undertake a further cross-sectional assessment of the children in this cohort in the second year, in order to assess the stability of these markers over time.

Our studies will identify immune response patterns that can be used as predictive markers of the child's ability to develop immunity to clinical malaria, and will be of great value in informing rational vaccine design, and monitoring vaccine efficacy and therapeutics. We will establish a set of robust analytical and modeling tools, which can be used in further studies, which will combine genetic variation in parasite and host, and which will inform mechanistic studies in animal models. Our unique dataset will be available for other researchers, and the expertise acquired will help establish a systems immunology expertise in the UK.

Technical Summary

We will examine immune response profiles of children developing immunity to Plasmodium falciparum malaria, in order to define a response pattern that distinguishes children who have suffered very frequent clinical malaria episodes from those who have an average number of clinical. We will then sample a cohort prior to surveillance, store the samples, and then test once clinical data are available to examine whether these profiles can prospectively predict multiple clinical episodes and failure to acquire immunity.
Using RNA sequencing we will determine the transcriptome profile of the immune response. This will be combined with flow cytometric analyses on leukocytes in whole blood, and measurements of cytokines, chemokines and P. falciparum specific B cell/antibody responses.
At each analytical step we will identify potentially important 'predictors', which could be individual up- or down-regulated genes, gene clusters, regulatory pathways etc. These will be collated and iteratively used to build up our predictive model. We will concentrate on the most significant pathways for more in depth analysis by developing mathematical descriptions for pathways and then predict the effects of perturbations in the study population. Detailed cytometric analyses (CyTOF and Flow) and functional B-cell and T-cell assays will be performed, and will inform mechanistic animal model studies. During this study we will develop novel analytical tools and train scientists in systems immunology, build up large databases of childrens' immune responses, and define biomarkers that will predict frequent malaria episodes in children.

Planned Impact

This project seeks to use a systems approach to define the characteristics of protective immunity to the malaria parasite, Plasmodium falciparum, and to understand how the host response contributes to severe malaria in children. We believe our studies will be the first comprehensive dataset obtained from children in an endemic area of P. falciparum transmission that will link epidemiological and clinical data on malaria to a large range of immune parameters such as the gene expression of immune-related genes, cytokine and chemokine production, the breadth of B-cell/antibody responses and functional capacity of immune cells.
Our data set will be of great value, and will be available to other Malaria immunologists, epidemiologists, and vaccine developers, who will be able to interrogate the data for their particular areas of immunological or other interest. The data set can also be used by any interested parties to define pathways, cells and molecules of interest that can be then investigated in mechanistic studies in experimental models. This would be an excellent way to bridge the gap between experimental models and human infection and disease by focussing on those responses that are directly relevant to human malaria.
The plasma remaining from this study will be available to colleagues, who are already in the process of developing various P. falciparum proteomes for screening for vaccine candidate antigens and developing protein micro-arrays targeting blood-stage merozoite and variable surface antigens, respectively, and will be able to link the resultant data sets with the results of this study.
We will collect samples such as P. falciparum DNA and RNA as well as human DNA in addition to the human RNA, peripheral blood cells and plasma to be used in our immunological studies. These samples will then be integrated into existing and planned large-scale studies funded by other sources such as Malariagen (www.malariagen.net), a network that integrates malaria epidemiology with genome studies on both the host and the malaria. This will enable us to expand our systems approach to bring together genetics with gene expression, functional immune responses and parasite variation.
This has huge potential for identifying markers for protective immunity and for identifying risk factors for severe disease. The data from this study will identify potential immune modulatory pathways that can be interrogated by interested immunologists from anywhere in the world. Furthermore the studies will provide important novel information on the relationships between genetic variability, gene expression and functional responses. Within the time scale of this project we will train young postgraduate and postdoctoral scientists in bioinformatics, predictive modeling and systems approaches to study human immunology and thereby establish a cohort of scientists who will further and expand this type of research in the UK and in Africa.
Identification of patterns of immune responses that can be used as predictive markers of the child's ability or not to develop immunity will be of great value in informing rational vaccine design, monitoring vaccine efficacy and determining indicators for children at risk of severe disease. Our findings will aid in designing vaccines to target the most protective responses can be used by those organisations such as WHO, MVI, Gates Foundation, MRC, Wellcome Trust and pharmaceutical companies involved in furthering development of vaccines and therapeutic interventions.
Information that would accelerate malaria vaccine development against an infection to which half of the world's population are exposed would have a significant impact on the educational and economic development of those affected countries, and therefore in the longer term also on the global economy and British economy.
 
Description Sofja Kovalevskaja Award
Amount € 1,600,000 (EUR)
Funding ID 3.2 - 1184811 - KEN - SKP 
Organisation Alexander von Humboldt Foundation 
Sector Public
Country Germany, Federal Republic of
Start 11/2015 
End 10/2021
 
Description Strategic Award
Amount £4,500,000 (GBP)
Organisation The Wellcome Trust Ltd 
Department Wellcome Trust Strategic Award
Sector Charity/Non Profit
Country United Kingdom of Great Britain & Northern Ireland (UK)
Start 11/2015 
End 11/2020
 
Title CyTOF panels 
Description deveoplement of panels of antibodies for Mass cytometry, including isotyope labelling of certin purtified antibodies 
Type Of Material Technology assay or reagent 
Provided To Others? No  
Impact This panel will enable a very detailed analysis of PBMC responses to Plasmodium falciparum-infected red blood cells from exposed children in Kilifi, Kenya. The panel will be used to determine the phenotype of responding cells from selected children in the next longitudinal blled 
 
Title CytoF Panels 
Description developed panels for antibodies for mass cytometry, including isotope labelling of selected Abs 
Type Of Material Technology assay or reagent 
Provided To Others? No  
Impact this panel will enable a detailed investigation of PBMC responses to Plasmodium falciparum infected red blood cells of exposed children in Kilifi , Kenya using Mass Cytometry, 
 
Title FLow cytometry deconvolution panel for human whole blood 
Description flow cytometry panel to detemine the major cellular components of human peripheral blood for later deconvoultion studies 
Type Of Material Technology assay or reagent 
Year Produced 2015 
Provided To Others? Yes  
Impact this enables us to detemine cell specific signatures from whole blood RNA seq data 
 
Title FLow cytometry deconvoution panel for whole blood 
Description wehave developed a flow cytometry panels to deterine the cellaur components of peripheral blood, whic can be used to detemine cell specific signatures from RNA sequencing. 
Type Of Material Technology assay or reagent 
Year Produced 2015 
Provided To Others? Yes  
Impact we will now be able to deconvolute our RNA seq data and assign cell specific signatures. 
 
Title RNA extraction from 1ml Blood 
Description Protocol developed and optimised for extraction from 1ml of blood in Tempus tubes. (Manufacturers protocol requires 3 ml) 
Type Of Material Biological samples 
Provided To Others? No  
Impact This is a critical step in enabling transcriptome analysis from blood samples of infants 
 
Title bar coding of PBMC samples for Cytof 
Description bar coding allows multiplexing samples for Cytof analysis 
Type Of Material Technology assay or reagent 
Provided To Others? No  
Impact this allows us to save money of reagents and analysis time as multiple samples can be analysed in a single tube 
 
Title RNA-Seq analysis of first cross-sectional bleed 
Description This reports provides an overview of the RNA-Seq data analysis pipeline from the first cross-sectional bleed (X1) using open-source software R 
Type Of Material Data analysis technique 
Provided To Others? No  
Impact The report, and the the pipeline described therein, constitutes a major part of the general, data analytic methodology of this project. It is being described a general as possible to facilitate similar analysis on unrelated data sets in the future (both for this project and others). 
 
Title SIMS LabKey 
Description Broad-based collaborations are becoming increasingly common among disease researchers. This is true for the SIMS project, where several partners are generating data ranging from clinical, RNA-Seq, proteomics and immunological data sets. To enable the researchers to organize and integrate these heterogeneous data early in the discovery process, standardize processes are needed to manage and integrate the data to gain new insights into pooled data and collaborate securely. We are implementing the open source LabKey server (www.labkey.org), a web-based data integration platform, to meet the needs of the project to manage the multi-dimensional data being generated in the SIMS project. The SIMS LabKey instance is hosted and managed by KWTRP. 
Type Of Material Data handling & control 
Provided To Others? No  
Impact Is providing a shared platform for all participants in the project to gain access to all the data being generated including SOPs and other relevant documents. Also server as a communication tool for the project collaborators. 
URL http://labkey.kemri-wellcome.org:8080/labkey
 
Title analysis of protein microarray data 
Description Generic pipeline to analyse antibody profiles based on protein microarray data and develop predictive models of clinical infections using random forests machine learning algorithm 
Type Of Material Data analysis technique 
Provided To Others? No  
Impact Powerful technique to (i) identify important variables from high dimensional data sets, and (ii) develop predictive models of infection outcomes based on individual antibody profiles 
 
Title cluster analysis of gene expression data in R 
Description Pipeline to perform rudimentary cluster analysis of gene expression data 
Type Of Material Computer model/algorithm 
Provided To Others? No  
Impact Speedup of performaing cluster analysis using generic R script 
 
Title differentail gene expression analysis in R 
Description R-based pipeline to perform differential gene expression analysis; currently based on microarray data but can be used as generic tool 
Type Of Material Computer model/algorithm 
Provided To Others? No  
Impact This pipeline should speed-up process of performing rudimentary gene expression analysis using opensource statistical software package R 
 
Description CyTOf analysis 
Organisation King's College London (KCL)
Country United Kingdom of Great Britain & Northern Ireland (UK) 
Sector Academic/University 
PI Contribution a member of the Langhporne lab will work on the Cytof
Collaborator Contribution DR Susanne Heck at Knigs will provide advice and train gin on the cytof
Impact noe yet it only started this year
Start Year 2014
 
Description Cytof analysis of human PBMC responding to malaria 
Organisation Guy's and St Thomas' NHS Foundation Trust
Country United Kingdom of Great Britain & Northern Ireland (UK) 
Sector Public 
PI Contribution multiparameter mass cytometry to investigate leukocyte responses to Plasmodium falciparum. design of study and optimisation of of cell preparation, completed testing of panel of reagents and analysed subpopulations of PBMC in malaria naive donors
Collaborator Contribution advised on design and testing a panel of markers to delineate different populations of PBMC in P. falciparum- infected children. Training in the use of the Cytof and Cytobank.
Impact a panel of tested reagents suitable to investigate PBMC responses in malaria
Start Year 2015
 
Description Multi-centre cohort study - new partners 
Organisation Kintampo Health Research Centre
Country Ghana, Republic of 
Sector Hospitals 
PI Contribution Collaborations over shared samples and data to be generated to address research questions. Agreements to support capacity building of African researchers from these institutions
Collaborator Contribution Contribution of samples and epidemiological expertise on malaria from the respective countries
Impact Supported scientists from 2 african countries to attend the Federation of African Immunological Societies (FAIS) conference in Nairobi this year (2014) Two scientists, one from Institut Pasteur Dakar, Senegal and one from Kintampo Health Research Centre visiting the KEMRI/Wellcome Trust Research Programme for research training visits for one and six months respectively in 2016
Start Year 2014
 
Description Multi-centre cohort study - new partners 
Organisation London School of Hygiene and Tropical Medicine (LSHTM)
Country United Kingdom of Great Britain & Northern Ireland (UK) 
Sector Academic/University 
PI Contribution Collaborations over shared samples and data to be generated to address research questions. Agreements to support capacity building of African researchers from these institutions
Collaborator Contribution Contribution of samples and epidemiological expertise on malaria from the respective countries
Impact Supported scientists from 2 african countries to attend the Federation of African Immunological Societies (FAIS) conference in Nairobi this year (2014) Two scientists, one from Institut Pasteur Dakar, Senegal and one from Kintampo Health Research Centre visiting the KEMRI/Wellcome Trust Research Programme for research training visits for one and six months respectively in 2016
Start Year 2014
 
Description Multi-centre cohort study - new partners 
Organisation Pasteur Institute Dakar
Country Senegal, Republic of 
Sector Charity/Non Profit 
PI Contribution Collaborations over shared samples and data to be generated to address research questions. Agreements to support capacity building of African researchers from these institutions
Collaborator Contribution Contribution of samples and epidemiological expertise on malaria from the respective countries
Impact Supported scientists from 2 african countries to attend the Federation of African Immunological Societies (FAIS) conference in Nairobi this year (2014) Two scientists, one from Institut Pasteur Dakar, Senegal and one from Kintampo Health Research Centre visiting the KEMRI/Wellcome Trust Research Programme for research training visits for one and six months respectively in 2016
Start Year 2014
 
Description Multi-centre cohort study - new partners 
Organisation University of Bamako
Department Malaria Research and Training Centre (MRTC) Bamako
Country Mali, Republic of 
Sector Academic/University 
PI Contribution Collaborations over shared samples and data to be generated to address research questions. Agreements to support capacity building of African researchers from these institutions
Collaborator Contribution Contribution of samples and epidemiological expertise on malaria from the respective countries
Impact Supported scientists from 2 african countries to attend the Federation of African Immunological Societies (FAIS) conference in Nairobi this year (2014) Two scientists, one from Institut Pasteur Dakar, Senegal and one from Kintampo Health Research Centre visiting the KEMRI/Wellcome Trust Research Programme for research training visits for one and six months respectively in 2016
Start Year 2014
 
Description Towards a highly effective multi-component vaccine against Plasmodium falciparum malaria 
Organisation Heidelberg University Hospital
Country Germany, Federal Republic of 
Sector Hospitals 
PI Contribution Acquired new funding from the Alexander Humboldt Foundation to build on the existing work
Collaborator Contribution Provided transgenic parasites to test potential vaccine candidates discovered in the "antigen discovery" part of the ongoing project.
Impact Work ongoing
Start Year 2016
 
Description F, Osier attended Women in Science event, University of Warwick 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Postgraduate students
Results and Impact We had a lively debate where we discussed the challenges and successes of women in science, engineering and mathematics. The audience were engaged and participated actively. they were mostly young women in social sciences and basic laboratory sciences who were all interested in developing research careers. It id too early to tell the impact of the meeting but the initial feedback from attendees was encouraging and the discussions showed that these were issues they were thinking about as they went about their studies
Year(s) Of Engagement Activity 2017
URL https://www2.warwick.ac.uk/research/priorities/internationaldevelopment/research/annualthemes/gender...
 
Description World malaria day at Mill Hill 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Other audiences
Results and Impact we had an exhibition at the Mill Hill laboratory for world malaria day together with the other malaria groups. It was very well received within the institute and in 2017 it is planned to extend this to Midland rd site and for a Crick Late for the general public.
Year(s) Of Engagement Activity 2016
 
Description community sensitization (systems immunology study) 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? Yes
Geographic Reach Local
Primary Audience Professional Practitioners
Results and Impact They medical officers in charge of health provision for the study population that we will recruit from are now aware of the systems immunology for malaria susceptibility that is just about to start. This meeting opened up the process for community engagement and will be followed by several meetings of sensitizing the potential study participants of the new study.

The Kilifi County health provision team are happy to be involved in the new study, and happy to support our community engagement activities.
Year(s) Of Engagement Activity 2014,2015