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Final Report Summary - EVIMALAR (Towards the establishment of a permanent European Virtual Institute dedicated to Malaria Research (EVIMalaR).)

Executive Summary:
The European Virtual Institute of MALAria Research (Evimalar) was a Network of Excellence (NoE) funded under FP7. It was an alliance of 54 full members drawn from European Union’s leading malaria research groups based in 36 institutions together with 6 leading African Institutions, the ICGEB from India and with representation from OzEMalaR, a network of Australian malaria researchers. A further 31 European groups were affiliated to Evimalar. The focus of the network was upon basic biological investigations of the malaria parasite and its interactions with both host and vector.
Membership of and affiliation to the network committed the participants to:
▪ Build upon their successful history of collaborative research – measurable output: academic publications and further grants held jointly by NoE members
▪ Combine their own expertise to produce a NoE that exploits natural synergies and complementarities in individual research perspectives resulting in a more integrated investigational and enhanced approach to the different research questions - measurable output: improved quality and diversity of publication output, publications and new research grants from new collaborations forged within the NoE
▪ Provide a management infrastructure for the network that will allow for the co-ordinated pursuit of the research, training, management and other activities detailed in this document
▪ Enhance and harmonise the experimental approaches to the subject areas in part through the measurable outputs of: the various use of shared technological platforms, exchange visits, shared PhD students recruited by the Evimalar PhD School established by the NoE as well as the creation of shared resources such as databases, reagent banks and agreed protocols for data collection. These resources and technological platforms were made available outwith the network through dedicated pages on the network website
▪ Created joint research programmes that will cover all thematic aspects stretching from basic research on the causative pathogen and its modes of transmission, to clinical investigation of pathogenesis - measurable output: Deliverables and Milestones that were defined in a Technical Annexe.
▪ Introduced and exploited new scientific concepts and approaches to maintain and define the state of the art in malaria research - measurable output: e.g. the development of systems biology approaches to the study of Plasmodium and host/vector interactions.
▪ Established an integrated work plan with the Australian Malaria Research Network – measurable output: itemised exchange visits, harmonisation of research, joint publications from novel collaborations
▪ Initiated further fostering of the next generation of malaria researchers through the establishment of an Evimalar fellowship programme
▪ Developed outreach programmes and tools for the education of the general public, dissemination of research and network activity information to peer groups around the world, established further network agreements to create and foster research exchange programmes.
▪ Took the initial steps towards the measurable output of the formation of a European Virtual Institute for Malaria Research beginning with the formation of a representative Legal Entity that it is hoped will assume certain management and administrational functions on behalf of the network going forward. It is also empowered by the network members to raise revenues on their behalf to fund long-term programmes of integrated research and training.

Project Context and Objectives:
BACKGROUND: Malaria is a public health problem today in more than 90 countries inhabited by some 2,400 million people (40 percent of the world's population). Malaria is estimated to cause up to 500 million clinical cases and nearly million deaths each year. Malaria is also spreading in to new areas, such as Central Asia, and Eastern Europe. More people are now dying of malaria than thirty years ago. Among the factors contributing to the raise of the mortality, they are the spreading of drug resistant parasite and insecticide resistant mosquitoes.
The recent successes in characterising genomes of Plasmodium sp. malaria parasites and their hosts provide a unique opportunity for developing novel strategies to control malaria. The size and complexity of this task require a concerted effort that no one laboratory, or even institution, has the resources or expertise to accomplish alone.
The objective of this proposal therefore was the creation of an interwoven network that integrates leading malaria research teams across Europe and African countries and provides central management, information and communication structures for strengthening European research in the field of malaria. Evimalar network will aim to promote the understanding in fundamental biology of the malaria parasite and its hosts including mechanisms involved in pathogenesis and transmission of the parasite.

General Objectives
Excellence of the Evimalar Network
The essence of Evimalar was:
• to integrate leading malaria research centres through a coherent, multidisciplinary programme of research.
• to bring together a critical mass of researchers (more than 150 scientists) from leading institutions (7 different European and six African malaria endemic countries),
• to implement jointly executed research, where Europe has proven excellence at the international level.
• to strengthen the excellence by tackling the fragmentation of European malaria research.
• to enhance the qualitative scope of existing studies in basic malaria and strengthen significantly Europe’s position as leader in the network’s field.
Evimalar built upon excellent networks that have been supported under the 5th and 6th framework programmes.

Impact of the Evimalar Network
An implemented European Network dedicated to high profile basic research on malaria will:
• exploit the wealth of information about the characterisation of the genomes of the malaria parasite and Anopheles vector in order to make a decisive impact in malaria research and control.
• feed the pipeline of new candidate molecules via existing or future integrated malaria research programmes on vaccines and drugs
• require concerted efforts to capitalise on recent technological breakthroughs
• mobilise the existing, but dispersed critical mass of expertise
• promote a new generation of well-trained young malaria scientists through the establishment of Evimalar PhD scholarship

Another key for the Network success was the spreading of excellence :
• with the creation of a Evimalar PhD programme to provide well-trained young scientists,
• with the improvement of the flow of information between the European scientific community, partners in developing countries and relevant organisations/institutions in order to facilitate co-operation.
• with fora to enhance general and political awareness of the importance of controlling malaria

Structuring ERA by establishment of a virtual European Institute for Malaria Research
In the long run, the objective of Evimalar was to create a European Institute where genuine joint strategies will be developed.
In order to have cohesion into the network, several integration activities will be implemented by Evimalar :
• Integration of new European participants with complementary of expertise into the network to strengthen the joint research activities, in parallel one of the scopes of Evimalar is to have a genuine involvement of research partners in developing countries.
• Sharing resources between the partners (knowledge and material pools, joint infrastructure)
• Implementation of interactive programmes for the training and exchange of personnel. This will be done through visits and planned short/long term exchange programmes in personnel within the Network for each joint research activity.
• Organisation of an annual meeting to present all Evimalar activities to the network participants
• Mobilisation of additional funding for malaria research

Evimalar’s Network was well structured and governed by different committees. A network management with different structures must be implemented to supervise the budget, to evaluate and monitor the scientific excellence and the integrating activities and to manage all legal issues.
The integrative networking of the major European Institutions and endemic country-based Institutions will provide thus the structure on which to build future spreading of the research efforts and agendas beyond the existing core group with the following objectives:
- To supply education and training of researchers, PhD students and technical personnel to excel in biology and pathology of the malaria parasite
- To disseminate the knowledge in relevant and accessible formats
- To provide access to core facilities, tools and platforms to Evimalar members
- To provide funding to develop innovative structures and technologies to Evimalar members including the participation of SMEs
- To provide a coherent and flexible management framework
- To integrate Evimalar NoE in the international activities
The final goal of the network is to build a virtual Institute that brings together leading European malaria teams and establish stable inter-institutional and structural scientific links via joint scientific and training programmes and shared infrastructures.
Evimalar NoE has to be perceived as an effective tool for health development, in term of output of appropriate research findings and model in researcher/user interactions and coordination among research institutions.

ORGANISATION OF THE PROJECT
The project was organised into 4 Work Packages (WP) covering 1. Research; 2. Coordination; 3. Spreading of Excellence and 4. Management.

WP 1 Research: was the major activity and organised into four work clusters that cover the diverse research interests of the group and each cluster has more focussed sub-activities
Cluster 1. Immunobiology and Pathophysiology:
We investigated the complex interactions between parasite and host that contribute to immunity and disease. The strength of the cluster lay in the extensive interactions and research plans already shared by the participants, and in the wide range of approaches, expertise and techniques available to dissect mechanisms of immunity and immunopathology. These included the use of field studies in areas of differing malaria endemicity, experimental infection of humans, animal models including humanised mice, transgenic parasites and in vitro systems for cellular and molecular immunology, and organ and parasite culture. To facilitate this, collaborative research, harmonisation of assays, use of shared platforms, exchange of students and postdocs and organisation of appropriate workshops within the network was undertaken within the following work packages (WP):
▪ WP1.1.1 An Analysis of the Host Parasite Interface;
▪ WP1.1.2 Malaria Pathogenesis;
▪ WP1.1.3 Towards a Greater Understanding of Mechanisms of Immunity.
Cluster 2. Parasite Molecular and Cell Biology:
Malaria parasites have developed sophisticated strategies that allow phenotypic variation without affecting the genotype. This allows the parasite to cope with a changing host environment such as the adaptive immune response, and to increase the duration of infection in the human host. It is generally achieved by the expression of clonally variant molecules either at the erythrocyte or merozoite surface. Switching of expression to another variant molecule avoids immune clearance and prolongs the period of infection. Very little is known about how one gene is brought to expression to the exclusion of all or most other members of the family. Available evidence suggests that there is active repression of the silent members at the transcriptional level involving epigenetic mechanisms. We propose to investigate whether active remodelling of chromatin via a number of distinct post-translational modifications and particular perinuclear sub-location of genes contributes to the molecular events that control gene expression in Plasmodium. The activities proposed will involve a general characterisation of the nuclear remodelling machinery, in particular the histone “writing” and “reading” machinery and the histone “code” in this pathogen.
Invasive stages of the malaria parasite have evolved a variety of strategies to establish infection in their host while evading the immune system and adapting to an intracellular life style. The parasite undergoes a lytic cycle whereby a single zoite produced by the previous cycle has to encounter a host cell, invade it, multiply to differentiate into a new zoite generation and escape to resume a new cycle. At the invasion and egress steps, the parasite cytoskeleton and the gliding motility apparatus play a crucial role. An increasing number of actors belonging to adhesins, motors, cytoskeletal components and proteases are involved, and critical signalling mechanisms are also implicated.
Despite its complex, multi-stage life cycle, the malaria pathogen is an obligate intracellular parasite, able to undergo replication only inside a host cell. As soon as it enters the cell, the parasite needs to initiate a dedicated programme of modification of the cell. After first constructing and modifying its PV, it has to produce de novo and export beyond its own plasma membrane boundaries a secretory membrane infrastructure (know as the tubovesicular network and Maurer’s clefts in erythrocytic stages), which is assembled in the host cell cytoplasm. It needs to synthesise and incorporate into the PV and host cell membranes new permeability pathways (transporters) for nutrient acquisition. It also has to export an entire range of distinct proteins involved in antigenic variation, immune evasion, and cytoadhesion. Particularly in the asexual and sexual erythrocytic stages, many of these parasite proteins are major determinants of virulence, sequestration and pathology. During its liver stage phase, the parasite has to overcome organ-specific problems, such as how to prevent apoptosis and immune recognition of the infected hepatocyte, how to modify the cell for optimal merozoite release and - in the case of P. vivax - how to differentiate into dormant hypnozoite forms. We investigated all of these different components in an integrated complementary fashion playing on the technical strengths of the collaborating groups in the following three WPs:
▪ WP1.2.1 Investigations of the Dynamics of Gene Expression and Protein Interactions;
▪ WP1.2.2 Understanding Mechanisms of Motility, Host Recognition, Invasion and Egress;
▪ WP1.2.3 Investigations of the Physiology and Biology of the Parasite and Parasitised Cell.
Cluster 3. Vector-Parasite Interactions:
Mosquitoes of the Anopheles genus represent a major threat for human health, as they are the exclusive vectors of malaria. A series of biological features, collectively known as vectorial capacity, renders Anopheles species very efficient vectors for the transmission of Plasmodium parasites, the causative agents of malaria. These include a genetically determined preference for blood meals on a human host (host-seeking behaviour) for egg development, a high reproductive rate and a long life span, combined with the innate ability to support parasite development. Therefore modulation of vector-parasite interactions, host-seeking behaviour, reproductive biology, and longevity offer key opportunities for interfering with malaria transmission. So far the most successful interventions that have led to a significant reduction in malaria transmission, chiefly exploited vector control strategies through the use of insecticides. However, recent insurgence of resistance in mosquitoes and the lack of novel insecticidal compounds constitute major hurdles in the fight against malaria. The combination of higher transmission rates due to the failure of traditional vector control measures and the rapid dissemination of drug resistance in Plasmodium parasites are escalating the number of malaria cases every year in the majority of disease-endemic countries, and lead to the re-introduction of malaria in some European countries. To efficiently and in a timely fashion identify and address global health issues, including emerging epidemics, novel alternative strategies are urgently needed to monitor risks and to roll back the disease.
The specific activities of Cluster 3 will integrate knowledge, research capacity and infrastructures of leading groups in the field of vector biology in Europe and Africa with the ultimate aim to reduce the mosquito vectorial capacity. Molecular interactions between malaria parasites and their hosts and vectors have been studied intensively in convenient but unnatural model systems, such as the triad Plasmodium berghei, Anopheles gambiae and the mouse. There are pragmatic reasons for this valuable model system approach that has been and will continue to be very fruitful. To complement this heuristic approach, a strong focus on coordinated studies on all three organisms that are actually implicated in natural cases of P. falciparum malaria will be developed. To this end, the impact of genetic variation in all three organisms implicated in malaria will be considered. The broad aims of Cluster 3 are to understand and describe at the molecular and nucleotide level the functional consequences of genetic variation in these organisms and their interactions. We have focused this project on A. gambiae, the most efficient vector of human malaria in the world. To help tackling the questions poised, a series of protocols and molecular tools will be developed which will be shared by the Network partners and made available to the Scientific Community. Overall, the joint scientific activities of this Cluster, articulated in 3 work packages described below, are anticipated to considerably expand our knowledge of the factors and pathways regulating the success of malaria transmission crucial for the development of transmission-blocking interventions.
▪ WP1.3.1 Illuminating Vector/Host/Parasite Interactions;
▪ WP1.3.2 Population genetics of Vector/Parasite/Host interactions
▪ WP1.3.3 Vector/Host Bioinformatics
Cluster 4. Modelling and Systems Biology:
Malaria research has entered the post-genomic era, the complete genome sequence of several Plasmodium species, strains and field isolates has been determined. However the complexity of this deadly parasite resides not simply in the composition of its structures, but more importantly in the wide-ranging networks of regulatory interactions within the parasite, and between the parasite and its hosts. This concept, termed ‘Systems Biology’, opens up new ways of thinking and working, integrating data from various high-throughput (HTP) disciplines to understand structural and regulatory networks of the cell. In an integrated approach to understand how parasite proliferation and differentiation is controlled we combined current, and newly generated knowledge from Plasmodium genomes, transcriptomes, proteomes, and metabolomes into systems databases that can be mined, interpreted by quantitative mathematical models, and experimentally tested. Whilst HTP technologies and advanced bioinformatics are now being applied to the study of malarial parasites in many participating European laboratories we recognised a clear and urgent need to develop new and effective interfaces between malaria researchers and systems biologists in Europe. This cluster sought to develop these interactions through consultation, and to encourage the training of young researchers in this innovative and important new science. We exploited two exemplar projects to capitalise on these beginnings, and develop new metabolomic studies on two specific aspects of parasite metabolism where we anticipated novel targets and pathways for intervention could be discovered. The data obtained from the ‘omics’ approaches were integrated and incorporated into a database that will be available to the wider malaria research community. We also recognised the necessity to extend the capabilities for parasite transgenesis to facilitate high-throughput reverse genetic analysis of protein function and thus analyse effectively the functions of the novel networks and molecules that systems approaches discover,
▪ WP1.4.1 The Structural and Functional Enhancement of Genome Annotation;
▪ WP1.4.2 Introduction of Systems Analysis to Plasmodium Expression Data;
▪ WP1.4.3 Exemplar Project 1: The regulation of Plasmodium Sexual Development;
▪ WP1.4.4 Exemplar project 2: Plasmodium metabolism and metabolomics;
▪ WP1.4.5 Further Development of Experimental Genetic Technologies.

WP 2 – Coordination: Consisted of the following major activities:
▪ NoE Executive committee Meetings (every 6 months)
▪ Strategic studies to address issues of scientific topicality
▪ Establishment of the Institutional Board
▪ Integrate new partners (Full and Affiliate members from Africa & Europe)
▪ Collaborate with other national/geographic networks of malaria researchers e.g. Australia
▪ Create platforms to share resources and technologies
▪ Communication and fund-raising
▪ Address and respond to development in ethical issues
▪ Form a Legal Entity capable of representing the network

WP3 - Spreading of Excellence
▪ Formed an Evimalar PhD School in conjunction with EMBL and produced 21 graduates
▪ Awarded a total of 4 post-doctoral fellowships
▪ Organised a total of >10 advanced training courses
▪ Organised an Annual NoE meeting in May 2010 in Heidelberg plus annual cluster research meetings
▪ Produced a web-site describing the network and its activities (www.evimalar.org)
▪ Communicated with other malaria stakeholders
▪ Supported communication with public and scientific community
▪ Supported communication with industry and SMEs

WP4 - Management
▪ Put in place general management structures: Director, Executive Committee, Research Cluster coordinators,
▪ Established a Management Office responsible for disbursement of funds
▪ Established the Institutional Board and an External Scientific Advisory Board (ESAB)
▪ Established structures for the evaluation of performance of all aspects of the network
▪ Established structures to monitor and promote gender equality
▪ Established structures to monitor ethical issues and compliance

Project Results:
Progress over the five years

OVERVIEW
EVIMALAR has been a great success at every level and has exceeded its ambition and stated aims as measured by overachievement of deliverables and milestones.
The outcomes are perhaps best summarised by a synopsis of the achievements of the many different activities of the Network and these are broken down on an annual basis so that granularity is evident and progress can be mapped across the lifetime of the activity.
Highlights include:

Year 1.
▪ A vibrant research programme has been established and is under active pursuit that has already resulted in joint publications.
▪ A kick-off meeting was successfully convened in Glasgow.
▪ All management structures are in place or are under development and according to schedule. The management office in Glasgow is fully staffed and operational. This office will shortly expand and will also serve as a focal point for investment from the host Institution.
▪ The Evimalar PhD School Office has been established and staffed. The first programme of projects (joint with PIs from two different countries jointly supervising each project) has been finalised. The first intake of students (11 from an initial applicant pool of >500) has been recruited, assigned to the project of their choice and started in post October 1 funded through the EMBL offices. The acceptance rate by the students of an offer to participate in the school was >90% (10/11). A rigorous evaluation scheme for applicants to the School has been established which includes an independent assessment of application and a two day interview process
▪ Each of these two initiatives has their own fully functional web site (www.evimalar.org, http://www.klinikum.uni-heidelberg.de/EVIMalaR-PhD-Programme.115126.0.html?&L=de).
▪ Two Evimalar post-doctoral fellowships have been awarded (to Dr. Kerstin Stegmann in Cluster Two and Dr. Valentina Mangano in Cluster Three) and a resource grant (to Prof Chris Newbold and Dr Matt Berriman, Oxford and Wellcome Trust Sanger Institute) created to allow the network to contribute directly to genome (parasite, vector) curation and analysis.
▪ The annual meeting was successfully held at EMBL, Heidelberg. Cluster meetings have been (or will shortly be) completed and have been very rewarding for the participants.
▪ A timetable for the Training Courses is in place and being implemented. These include training course to be held in Africa, training in advanced experimental genetics in malaria, advanced imaging technologies and metabolomics
▪ Ozemalar, the mechanism for liaison with the Australian Malaria Research Community has been established in Australia and funded by the NHMRC (http://www.ozemalar.org/). Additional funding for the Evimalar partnership has been obtained from the IRSES programme of the EC. The first calls and awards for proposed exchanges in both directions have been made and evaluated. The successful applicants are currently undertaking their exchanges.
▪ Support for a pictorial database of Plasmodium has been granted to Prof Hagai Ginsburg, University of Jerusalem (http://sites.huji.ac.il/malaria/).
▪ The ESAB has been appointed and performed its first advisory role at the annual meeting in May in Heidelberg. The membership of ESAB was (Prof Manuel Llinas, Princeton/Penn State University; Prof Dyann Wirth, Harvard; Prof Christine Clayton, Heidelberg Prof Laurent Renia, A-Star, Singapore; Prof Fidel Zavala, Johns Hopkins University and Prof. Ayoade Oduola WHO/Ibadan (Chair)
▪ Four new full members (Hernando Del Portillo (CRESIB, Barcelona, Spain); Richard Pleass (University of Nottingham, UK); Achidi Eric Akum (University of Buea, Cameron); Lyn-Marie Birkenholtz (University of Pretoria, South Africa) have been welcomed into the network following competitive calls announced on the Evimalar website and evaluation by the ESAB
▪ The Executive Committee has been constituted and all the network internal posts assigned. The Executive has met twice and has also made recommendations concerning fellowship awards, appointment of new Affiliates and engagement with scientists from other malaria-endemic regions.
▪ Formation of the Institutional Board is ongoing.
▪ Formation of a Legal Entity to represent the interests of the Network is under legal consultation.
▪ An advanced version (5th revision) of the Consortium Agreement has been produced and will shortly be circulated for final approval and signatures.
▪ All four Clusters have held their first independent annual meetings in London (Cluster 4), Stockholm (Cluster 3), Lisbon (Cluster 2) and Oxford (Cluster 3) respectively. Presentations were made by invited senior scientists including representatives of the partner Ozemalar network. The majority of the talks have been given by early stage researchers active in the network
▪ Evimalar performed outreach activities to peer groups. At the International Congress of Parasitology Associations (ICOPA) meeting held every four years and this year in Melbourne, Evimalar organised a symposium that described the network, showcased some of the research activities in all four clusters and the Ozemalar-Evimalar exchange programmes. Similar activities are planned for next year.

Research highlights from the individual clusters
Cluster 1: Immunobiology and Pathophysiology:
▪ We have identified members of the multigene family, cir, that are transcribed in during a blood-stage infection some of which are expressed on the surface of infected RBC and could be involved in sequestration.
▪ P. chabaudi can now be reliably tranfected with reporter constructs containing mCherry, Luciferase and GFP for binding studies and whole mouse imaging.
▪ P.berghei parasites in which a gene encoding a pexel-containing protein of 19kDa (SMAC) has been disrupted shows a strongly reduced capacity to sequester.
▪ We have generated new information on parasite effects on erythropoeisis demonstrating an association of haemozoin and 4-hydroxynonenal with malaria anaemia through inhibition of erythropoiesis in the bone marrow.
▪ We described P. vivax clinical malaria in Duffy-negative individuals from Madagascar, indicating that P. vivax has broken on its dependence on the Duffy antigen to invade red cells.
▪ IL-27 has been shown to play an essential role in protecting the host from the immunopathology associated with excessive Th-1-mediated inflammation in murine malaria.
▪ Antibodies to Palo Alto varO are associated with protection against clinical malaria in Benin.
▪ Despite substantial inter-clonal variation, VAR2CSA contains a number of functionally important and inter-clonally conserved antibody epitopes.
▪ B cell memory to malaria can persist for many years after last malaria infection in people exposed to only very infrequent reinfection.
▪ A C57Bl/6-P.berghei model has been established that shows lasting protection can be induced by sporozoite exposure under antibiotic cover.

Cluster 2. Parasite Molecular and Cell Biology:
▪ Release of UIS4 from expression regulation abolishes life cycle progression, highlighting the requirement for a fast switch mode from latency to intrahepatic transformation.
▪ The genetic elements that contribute to the 3D nuclear architecture of chromosomes in P. falciparum have been investigated in unprecedented details.
▪ Pfcrk-3, a transcriptional CDK-related kinase was shown to display protein kinase and histone deacetylase activities and to fulfil a crucial role in the erythrocytic asexual cycle.
▪ The subtilisin-like protease PfSUB1 is critical for egress and cGMP-dependent protein kinase PKG plays a key role in regulating discharge of PfSUB1 into the parasitophorous vacuole just prior to egress.
▪ Proteolytic shedding of AMA1 by PfSUB2 is also essential for parasite viability and may play an important role in evasion of antibody response.
▪ The rhomboid-like protease ROM4 is implicated into a novel function that triggers parasite replication following invasion in Toxoplasma.
▪ A major step forward has been achieved by the in-depth analyses of the step-wise processes of sporozoite in vitro gliding locomotion
▪ Host protein kinase-mediated signaling pathway involving RBC PAK1 and MEK1 is strongly stimulated in infected (versus uninfected) erythrocytes are essential to parasite survival in RBC.
▪ We produced the first GFP-expressing P. cynomolgi parasites, as a tool to study liver stage biology.
▪ Epitope tagged autophagy-related proteins such as ATG8 and ATG12 revealed active autophagous structures in P falciparum.

Cluster 3: Vector-parasite interactions
▪ Signal-independent activation of the Rel1 by depletion of Cactus repressed Vg expression after a bloodmeal and rendered mosquitoes sterile providing for the first time a molecular basis of the trade off between reproduction and immunity in the mosquitoes.

Cluster 4: Systems Biology and Technology Development.
▪ The genome sequence of P. falciparum (clone 3D7) is now closed with the exception of one gap. This now represents probably the most accurate genome of any eukaryotic organism.
▪ The genome of P. chabaudi is now virtually complete, and that of P. berghei is greatly improved.
▪ All three genomes have been re-annotated and available on public databases (deliverable predicted for M18).
▪ Importantly web services have been developed to facilitate the interoperability of GeneDB and PlasmoDB (WTSI2, UOXF2).
▪ A publically accessible database of genetically modified mutant rodent parasites (LUMC), has been linked both to PlasmoDB and GeneDB.
▪ Novel methods for the purification of stage 1 gametocytes of P. falciparum have permitted the identification of ca. 1400 proteins, this data is now submitted to PlasmoDB.
▪ Methodologies for the analysis of malarial ‘energy’ metabolomes have been established.
▪ Methodology for analysis of water soluble metabolites of phospholipid metabolism has been
▪ An inducible control system for malaria using the transactivator domains of the AP-2 transcription regulator has been developed and their response to Tetracycline demonstrated.
▪ 42 kinases have been shown to be essential in asexual bloodstage development, and 23 in the sexual and transmission stages.
▪ New PCR- and ligation-independent methods for the HTP production of genetic mutants are under development (WTSI2) , the Plasmodium artificial chromosome (L-PAC) has now been published

Progress in Year 2
Headlines
▪ A vibrant joint research programme has been established and has resulted in 177 peer-reviewed publications: all of these publications acknowledge the support of the EC in the form of the Evimalar Network of Excellence
▪ All management structures are in place or are under development and according to schedule. The management office in Glasgow is fully staffed and operational. This office has expanded and will also serve as a focal point for investment from the host Institution.
▪ The Evimalar PhD School continues to run well. The second programme of projects (joint with PIs from two different countries jointly supervising each project) has been finalised and the intake of students (12 from an initial applicant pool of >500) has been recruited, assigned to the project of their choice and started in post October 1, 2011, funded through the EMBL offices. The acceptance rate by the students of an offer to participate in the school was 100%. The rigorous evaluation scheme for applicants to the School which includes an independent assessment of application and a two day interview process once again proved its worth.
▪ Each of these two initiatives has their own fully functional web site (www.evimalar.org; http://www.klinikum.uni-heidelberg.de/EVIMalaR-PhD-Programme.115126.0.html?&L=de).
▪ Three Evimalar post-doctoral fellowships have been awarded to Carolina Nersesian (Bernhardt Nocht Institute for Tropical Medicine), Liliana Mancio Silva (Instituto de Medicina Molecular) and Paco Pino (University of Geneva). Applications were received in response to calls placed on the Evimalar website and the ESAB evaluated the applications
▪ The second annual meeting was successfully held at EMBL, Heidelberg. Prof Fotis Kafatos received the 2011 Evimalar award for lifetime achievement and contribution to malaria research. A bioinformatics course for students designed by personnel from WTSI and PlasmoDB was run in conjunction with this meeting
▪ A timetable for the Training Courses is in place and being implemented. Those already held include a training course in Uganda, training in advanced experimental genetics in malaria (at the Sanger Centre), those planned include repeats of the experimental genetics course as well as advanced imaging technologies and metabolomics.
▪ Ozemalar, the mechanism for liaison with the Australian Malaria Research Community has been established in Australia and funded by the NHMRC (http://www.ozemalar.org/) continues. Additional funding for the Evimalar partnership has been obtained from the IRSES programme of the EC and is being secured. The first exchanges have taken place and their activity reports received. Calls for applications and their evaluation are continuing.
▪ Support for a pictorial database of Plasmodium run by Prof Hagai Ginsburg, University of Jerusalem (http://sites.huji.ac.il/malaria/) continues. Discussions and plans for its long-term future are in train.
▪ The ESAB continued its advisory role at the 2nd annual meeting in May in Heidelberg. The first rotation of members of the committee is planned.
▪ Eight new full affiliates (James Brewer, University of Glasgow; Carlos Penha Goncalves, Instituto Gulbenkian de Ciencia; Alfred Cortes, Institute for Research in Biomedicine (IRB) Barcelona; Ken Vernick, Institut Pasteur; Sarah Butcher, Imperial College London; Sarah Reece, University of Edinburgh; Mario Recker, University of Oxford; Julian Rayner, Wellcome Trust Sanger Institute) have been welcomed into the network following competitive calls announced on the Evimalar website and evaluation by the ESAB
▪ The Executive Committee continues to fulfil its specified function. Prof Fotis Kafatos has demitted and discussions concerning a replacement will be held at the next Committee meeting in November 2011 (Rome). The Executive has met twice in the last 12 months (Heidelberg & Paris) and has also made recommendations concerning fellowship awards, appointment of new Affiliates and engagement with scientists from other malaria-endemic regions.
▪ Formation of the Institutional Board is complete and the initial Board meeting held (June, 6, 2011 in Leiden, NL).
▪ Formation of a Legal Entity to represent the interests of the Network is under legal consultation.
▪ The Consortium Agreement has been finalised, and signed and will shortly be submitted to the Scientific Officer of the EC.
▪ All four Clusters have held or will shortly hold independent annual meetings in Heidelberg (Cluster 4 with one planned for Rijswijk), Barcelona (Clusters 1 & 3) and Rome (Cluster 2) respectively. Presentations were made by invited senior scientists including representatives of the partner Ozemalar network. The majority of the talks have been given by early stage researchers active in the network
▪ Evimalar performed outreach activities to peer groups. Documentation is currently being exchanged with a National Science Funding agency in Brazil (CAPES).

Research highlights from the individual clusters
Cluster 1: Immunobiology and Pathophysiology:
• Chimeric models of rodent malaria parasites for the study of human parasite sequestration are nearing maturity and the individual members of the protein families involved identified. Various modes of parasite-infected red blood cell (iRBC) cytoadherence have been further clarified identifying specific sugars (e.g. ABO blood group tri-saccharides), cell types (eg platelets) and that the density of parasite-encoded receptors on iRBC is dependent on parasite maturity.
• The role of Stevor proteins in P. falciparum gametocyte sequestration has been established. There is a greater appreciation of the imbalance of pro-inflammatory cytokines in severe anaemia during P. falciparum infection which might suggest anti-anaemic therapies.
• A new blood stage vaccine candidate has been identified (PfRh5) and is undergoing initial trials.
• P. falciparum and P. vivax vaccine formulations and trials continue to be developed.
• Placental associated malaria is centred on PfEMP1 VAR2CSA expression which is bound by IgM which impairs host recognition however important epitopes are still available for host immune development.
• Human monoclonal antibodies that recognised different members of the parasite PfEMP1 repertoire on iRBC have been produced and have therapeutic potential.
• Importantly CD4+ T-cell responses to a small semi-conserved area of the DBL# domain of PfEMP-1, the DBL#-tag. CD4+IL4+ T cells specific for the DBL#-tag region of PfEMP1 are associated with a reduction in clinical malaria episodes

Cluster 2: Cell Biology.
• The quiescent hypnozoite form has been purified to 100% homogeneity and are undergoing biochemical characterisation.
• Host hepatocyte autophagy is important for parasite growth yet parasite derived protease inhibitors are essential at this point in the life cycle: host-derived lipoic acid is also essential at this stage.
• Cell cycle control checkpoints have been revealed as a novel target for anti-blood stage parasite therapies and the parasite remodels erythrocyte actin to deliver proteins to the infected cell surface a process that is impaired in sickle erythrocytes.
• The zoonotic human malaria pathogen Plasmodium knowlesi has been successfully adapted to long-term stable growth in vitro in human erythrocytes which will facilitate erythrocyte invasion studies.
• Kinase control of translational repression is essential for ookinete development.
• The further roles of different parasite proteases have been investigated in egress from hepatocytes and erythrocytes and their regulation by kinases has been demonstrated.
• Novel inducible gene disruption technologies have been developed and used to show that parasite gliding motility is not required for host cell invasion but absolutely required for egress.
• Assembly of the gliding apparatus is dependent upon numerous post-translational modifications of the actors in this highly regulated process.
• Greater insights into the regulation of parasite infrastructure have been obtained implicating an actin-myosin motor in nuclear architecture and specific proteins in invasion associated organelle positioning.

Cluster 3: Vector-Parasite Interactions.
• Significant progress has been made towards further identification of additional signaling pathways associated with mosquito immune responses to Plasmodium that involve variously GPCRs in combination with gut microbiota, transglutaminases and TEP1 independent pathways.
• 22 novel immune regulators in hemocytes were identified and included proteins putatively involved in many aspects of immunity.
• The development of robust functional cell-based assays paves the way for genome-wide functional screens to study the mosquito immune response to infections with human pathogens.
• Field studies have revealed that the genetic complexity of parasites is a potentially crucial component of mosquito–parasite interactions.
• Surveys of African immune responses have revealed a significant ethnic component that influences disease outcome

Cluster 4: Systems Biology and Technology Development.
• A suite of software tools forming the Post Assembly Genome Improvement Toolkit (PAGIT) has been published in Nature Protocols.
• An automated method of genome QC called REAPR has been developed and used to assert that 95% of the P. falciparum is now base-perfect.
• Numerous other Plasmodium genomes continue to be curated and improved.
• Ontological tools have been improved for Plasmodium and included in Malariaseek a new data sharing database that has been adopted and adapted from SYSMO and is already extensively used including newly developed phosphoproteomic and RNA-seq datasets.
• Hybrid optimisation method to allow stationary flux and kinetic parameters to be estimated for a metabolic network have been developed and applied to novel metabolomic datasets.
• Significant advances have been made for the genetic manipulation of P berghei including GIMO, recombineering and centromeric plasmid based approaches making this the most sophisticated species of Plasmodium for genetic manipulation.
Far from last and least an Evimalar Affiliate, Prof. Jules Hoffman was awarded a share of the Nobel Prize for Medicine in 2011.


Progress in Year 3
Headlines
▪ A vibrant joint research programme has been established and has resulted in 516 peer-reviewed publications, 214 of which were reported in Period 3: all of these publications acknowledge the support of the EC in the form of the EVIMalaR Network of Excellence
▪ All management structures are in place or are under development and according to schedule. The management office in Glasgow is fully staffed and operational. This office has expanded yet further and will also serve as a focal point for investment from the host Institution. The office has taken on additional EC–funded project management tasks
▪ The Evimalar PhD School continues to run well. The second programme of projects (joint with PIs from two different countries jointly supervising each project) has been finalised and the intake of students (12 from an initial applicant pool of >500) has been recruited, assigned to the project of their choice and started in post October 1, 2011, funded through the EMBL offices. The acceptance rate by the students of an offer to participate in the school was 100%. The rigorous evaluation scheme for applicants to the School which includes an independent assessment of application and a two day interview process once again proved its worth. Both cohorts of students are now recruited, registered with Universities who will award their degree and engaged in their projects – their collective mid-term review is planned for November, 2012 and will take place in Heidelberg.
▪ The third annual meeting of the Evimalar consortium (aka the eighth Biomalpar Meeting) was successfully held at EMBL, Heidelberg. Prof. Richard Carter (late of the University of Edinburgh) received the 2012 Evimalar award for lifetime achievement and contribution to malaria research. A bioinformatics course for students designed by personnel from WTSI and PlasmoDB was run in conjunction with this meeting.
▪ A timetable for the Training Courses is being implemented and further populated. Those already held in the reporting period include training in advanced experimental genetics in malaria (at the Sanger Centre), a 2-week parasitology workshop in Bamako, Mali, a metabolomics practical and theoretical workshop (in Glasgow). Those planned include repeats of the experimental genetics course as well as advanced imaging technologies.
▪ Ozemalar, the mechanism for liaison with the Australian Malaria Research Community has been established in Australia and funded by the NHMRC (http://www.ozemalar.org/) continues. Additional funding for the Evimalar partnership has been obtained from the IRSES programme of the EC. The first exchanges have taken place and their activity reports received. Calls for applications and their evaluation are continuing.
▪ Support for a pictorial database of Plasmodium run by Prof Hagai Ginsburg, University of Jerusalem (http://sites.huji.ac.il/malaria/) continues. Discussions and plans for its long-term future are in train.
▪ The ESAB continued its advisory role at the 3rd annual meeting in May 2012 in Heidelberg. The first rotation of members of the committee has occurred.
▪ Four new full affiliates (Hedda Wardemann, Max Planck, Berlin; Francis Ndungu, KEMRI Wellcome, Kenya; Margaret MacKinnon, KEMRI Wellcome, Kenya; Climent Casals-Pascual, Oxford, UK) have been welcomed into the network following competitive calls announced on the Evimalar website and evaluation by the ESAB
▪ The Executive Committee continues to fulfil its specified function. The Nobel Laureate Prof. Jules Hoffman has kindly agreed to succeed Prof Fotis Kafatos as an honorary member of the Executive. The Executive has met twice in the last 12 months (Heidelberg & Rome) and has also made recommendations concerning appointment of new Affiliates and engagement with scientists from other malaria-endemic regions.
▪ Formation of the Institutional Board is complete and the initial Board meeting held (June, 6, 2011 in Leiden, NL).
▪ Formation of a Legal Entity to represent the interests of the Network is under the final stages of preparation and will begin lobbying for continued support in 2012/13.
▪ The Consortium Agreement has been finalised, signed, submitted to the Scientific Officer of the EC and accepted.
▪ All four Clusters have held (Heidelberg, Rome & Barcelona) or will shortly hold independent annual meetings in Heidelberg (Cluster 4 with one planned for London/Hinxton), Berlin (Clusters 2 & 3) and Rome (Cluster 3) respectively. Presentations were made by invited senior scientists including representatives of the partner Ozemalar network. As always the majority of the talks have been given by early stage researchers active in the network
▪ Evimalar performed outreach activities to peer groups. Documentation is currently being exchanged with a National Science Funding agency in Brazil (CAPES).

Research highlights from the individual Clusters.
Cluster 1: Immunobiology and Pathophysiology:
▪ The role of hepcidin in the prevention of superinfection of children with malaria has been uncovered.
▪ RESA is inserted into parasitised erythrocytes by P falciparum leading to increased rigidity of the erythrocyte thereby aiding its retention in the spleen.
▪ The GT and AT heterozygous genotypes for the LT and IL-22 genes conferred 42% and 33% protection respectively against severe malaria.
▪ The first evidence for antibody-driven allelic diversification of the family in the field has been provided.

Cluster 2: Cell Biology.
▪ The importance of post-transcriptional regulation in sporozoite development has been demonstrated.
▪ Genome-wide epigenetic maps for P. falciparum blood stages have been produced. A global P. falciparum schizont phosphoproteome has been produced.
▪ The critical participation of the var gene intron in perinuclear localisation via an actin regulated mechanism has been demonstrated.
▪ The first application of an inducible transactivator system for controlled expression of genes in P. berghei has confirmed the essential nature of N-myristolyltransferase.
▪ Novel components of the glideosome in T. gondii have been identified and functionally characterised.
▪ The role of calcineurin and calcium signalling in microneme discharge has been demonstrated.
▪ Significant insights into sporozoite motility and adhesion have been generated.
▪ Osmiophilic bodies are undergoing in depth characterisation.
▪ The essential kinomes of P. berghei and P falciparum have been defined.
▪ The 5th human malaria P. knowlesi has been adapted to growth in vitro in human erythrocytes.
▪ The parasite-specific carbon dioxide-fixing enzyme PEPC is essential to P falciparum growth in vitro and therefore a validated drug target.
▪ Host 14-3-3 protein is involved in the assembly of functional cytoadherence complexes on the surface of the infected erythrocyte.
▪ Parasite autophagy is under investigation as is its ability to direct host-cell autophagy in concert with intracellular development.
Cluster 3: Vector-Parasite Interactions.
▪ The first key milestone towards development of genome-wide RNAi screens in An. gambiae cells has been completed identifying key genes that direct immune responses in hemocytes.
▪ Not all P. falciparum strains are susceptible to immune attach by TEP-1. Data suggest that coadaptation between vectors and parasites may act to minimize the infection impact on mosquito fitness, by selectively suppressing specific functional classes of genes.
▪ A trade-off between reproduction and immunity in Anopheline mosquitoes has been established.
▪ Preliminary results indicate that genes conferring resistance to severe malaria also play a role in protection against infection.
Cluster 4: Systems Biology and Technology Development.
▪ The genome sequence of P. falciparum (clone 3D7) is now closed with the exception of one gap. This now represents probably the most accurate genome of any eukaryotic organism.
▪ The genomes of two rodent malaria parasites, P. berghei and P chabaudi are significantly improved and nearly complete.
▪ Metabolome studies of P falciparum blood stages are very advanced.
▪ New methodologies have been developed for conditional knock out of genes in P. falciparum and P berghei.
▪ Global genome and post-genome datasets and databases for public dissemination and use have been produced.

Progress in Year 4
Headlines
▪ A vibrant joint research programme has been established and has resulted in 709 peer-reviewed publications, 193 of which were reported in Period 4: all of these publications acknowledge the support of the EC in the form of the EVIMalaR Network of Excellence
▪ All management structures are in place or are under development and according to schedule. The management office in Glasgow is fully staffed and operational. This office has expanded yet further and will also serve as a focal point for investment from the host Institution. The office has taken on additional EC–funded project management tasks
▪ The EVIMalaR PhD School continues to run well. Both cohorts of students have undergone their collective mid-term review in November 2012 Heidelberg. Progress was largely excellent. See (www.evimalar.org; http://www.klinikum.uni-heidelberg.de/EVIMalaR-PhD-Programme.115126.0.html?&L=de).
▪ The fourth annual meeting of the EVIMalaR consortium (aka the ninth BioMalPar Meeting) was successfully held at EMBL, Heidelberg. The annual bioinformatics course for students designed by personnel from WTSI and PlasmoDB was run in conjunction with this meeting. Cluster meetings were also planned around this meeting.
▪ The timetable for the Training Courses has been implemented and further populated. Those already held in the reporting period include a second training in advanced experimental genetics in malaria (at the Sanger Centre), an imaging course in Heidelberg, a discussion forum at MIM, Durban debating how to plan a scientific career in Africa and a second breakfast debating the position of women in science (in Africa). Those planned include repeats of the experimental genetics course as well as a full meeting on “Women in Science” as part of our equality agenda
▪ OZEMalaR and Ozmalnet, the mechanisms for liaison with the Australian Malaria Research Community has been established in Australia and funded by the NHMRC (http://www.ozemalar.org/) continues. Calls for applications and their evaluation are continuing with productive exchanges resulting.
▪ Support for a pictorial database of Plasmodium run by Prof Hagai Ginsburg, University of Jerusalem (http://sites.huji.ac.il/malaria/) continues. Discussions and plans for its long-term future are in train.
▪ The ESAB continued its advisory role at the 4th annual meeting in May 2013 in Heidelberg.
▪ Four new full affiliates (Carlota Dobano, CRESIB; Olivier Silvie, INSERM; David Conway, LSHTM, Taane Clark, LSHTM) have been welcomed into the network following competitive calls announced on the EVIMalaR website and evaluation by the ESAB.
▪ The Executive Committee continues to fulfil its specified function. The Executive has met twice in the last 12 months (Heidelberg & Berlin) and has also made recommendations concerning appointment of new Affiliates and engagement with scientists from other malaria-endemic regions.
▪ Formation of a Legal Entity to represent the interests of the Network is almost finalised and the lobbying process for continued support in 2012/13 has begun see example.
▪ All four Clusters have held (Heidelberg, Montpellier & Heraklion) or will shortly hold independent annual meetings in Heidelberg (Cluster 4 with one planned for London/Hinxton), Berlin (Clusters 2 & 3) and Rome (Cluster 3) respectively. As always the majority of the talks have been given by early stage researchers active in the network
▪ EVIMalaR performed outreach activities to peer groups, notably in Durban at the 2013 MIM meeting.

Research highlights from the individual clusters
Cluster 1: Immunobiology and Pathophysiology:
• Provided direct demonstration that changes in ring-stage RBC geometry contribute to their entrapment in the human spleen.
• CD34+ hematopoietic stem cells from bone marrow and peripheral blood produce (Hi CD71+) reticulocytes that can be invaded by P. vivax
• Constructed and validated a spleen on a chip
• Demonstrated the essential role of the spleen in inducing protective immune responses after immunisations with infected reticulocyte-derived exosomes.
• implemented the first optical projection tomography (OPT) imaging of the spleen in malaria
• IL-22 produced by T cells is important in controlling severity of blood-stage P. chabaudi malaria, probably by maintaining the integrity of epithelia.
• IL-27 receptor signalling restricts the formation of pathogenic, terminally differentiated Th1 cells during mouse malaria infection reducing the cytokine storm and associated pathology of the infection, but also limiting the extent of control of both primary and secondary malaria infections.
• hyper-susceptible children are numerous and major contributors to the disease burden and may necessitate specific prevention and control measures.
• Hemozoin decreases the migratory capacity of the neutrophils.
• Protein C/TM/ EPCR system mechanism for endothelial pathology in cerebral malaria has been developed and may yield novel anti-disease therapies based
• extensive parasite sequestration is not required for severe Plasmodium falciparum malaria,
• IL-21 from follicular helper T cells is necessary for B cell differentiation into antibody-secreting plasma cells, and for protection against blood stage mouse malaria.
• Parasite-specific CD4 T-cell degranulation together with 10 other cellular immune markers provides a combined immune signature of protection against malaria in the human Chloroquine Prophylaxis Sporozoite (CPS) challenge model.
• An efficient technique to reset epigenetic memory of var gene transcription in investigations of the relationship between PfEMP1 expression and IE adhesion phenotypes has been employed.
• a novel blood-stage malaria vaccine antigen target, PfRH5, that is capable of out-performing known candidate antigens and which induces broadly-neutralising, rather than strain-specific, antibody responses has been identified.

Cluster 2: Parasite Molecular and Cell Biology

• Sub-nanomolar oligoamines that interfere with epigenetic regulation and inhibit parasite growth have been developed
• A greater depth of histone variant deposition and epigenetic profiles has been established.
• A link between epigenetic regulation and parasite virulence has been established
• Specific AP2 proteins regulate non-var subtelomeric multigene family expression.
• Novel DiCre technologies have been developed for regulatable gene KO
• Rab 5A and C are essential for the biogenesis of parasite specific invasion related organelles
• Role of Ca++ and its multiple effector proteins in merozoites egress and erythrocyte invasion have been demonstrated.
• The role of vaccine candidate AMA-1 in parasite binding to RBCs has been defined.
• Molecular mechanisms of invasive organelle positioning within the parasite have been defined
• rhoptry protein secretion during erythrocyte invasion is being redefined
• The spectral nature of the gliding motility apparatus has been identified and is under further characterisation
• Protease mediated egress of parasites from infected RBC is regulated by cGMP-dependent protein kinase PKG
• Novel methods to investigate mutant parasites with altered mobility phenotypes have been produced
• P knowlesi adapted to long term culture in human erythrocytes
• haemoglobinopathies S and C, which protect carriers from severe malaria, impact on parasite actin dynamics and Maurer’s clefts morphology.
• P falciparum possesses a novel transport system for N-myristoylated proteins
• Parasite released protease inhibitors are essential for hepatocyte invasion and important for motility.
• phospoenolpyruvate carboxylase (PEPC) is essential in P. falciparum and a possible drug target.
• P cynomolgi cultured in hepatocytes forms atovaquone resistant hypnozoites that can reactivate
• Assays for the investigation of gametocyte cytoadherence in bone marrow have been developed.
• Drugs preventing P. falciparum gametocyte activation are being screened in a newly developed automated assay amenable to high throughput screens.
• An unexpected role of autophagy components in organelle biogenesis has been discovered.

Cluster 3: Vector-Parasite Interactions
• New components of the REL2 pathway are identified and generation of transgenic lines with tissue-specific activation of the REL2 pathway.
• Identification of the pathogen-associated prenyl pyrophosphate, (E)-4 Hydroxy-3-methyl-but-2-enyl pyrophosphate (HMBPP) is an important elicitor in common for both Plasmodium and gut bacteria in the mosquito.
• Discovery of a C3 convertase-like module in the A. gambiae hemolymph that is required for the accumulation of TEP1 on the surface of bacteria and Plasmodium parasites.
• Multiple signaling pathways are required for efficient Plasmodium killing in A. gambiae.
• Association of kdr mutation responsible for resistance to pyrethroids with the competence of the major malaria vector A. gambiae to transmit P. falciparum.
• Malaria parasites modulate expression of salivary proteins to favor their transmission.
• Demonstration of variability of African P. falciparum strains in their sensitivity towards the A. gambiae TEP1-mediated immunity, possibly determined by Pfs47 polymorphism.
• Metabolism pathways in the mosquito identified and experimentally shown to contribute to both parasite inhibition and egg development.
• Identification of genes that may be associated with the levels of A. gambiae infections with P. falciparum.
• Found protective association between the heterozygous AG genotype of the TNF -308 polymorphism.
• The population biology database is unique and is expected to be a major driver of meta-analysis of mosquito population data in the future.

Cluster 4: Systems Biology and Technology Development.
• Genomes of several rodent malaria parasite lines and strains have been characterised, also P reichenowi.
• Database of 1000’s of var genes produced.
• Strand specific RNAseq protocol developed and applied to P. knowlesi & P. reichenowi.
• Malar-a SEEK continues to improve accessibility and utility
• Phospholipid biosynthesis in P. falciparum has been extensively modelled integrating metabolomic data and general applicability of quantitative metabolomics is being explored.
• An unexpected compartmentalization of choline and ethanolamine metabolism has been identified in P. falciparum.
• Quantitative phenotyping of dozens of P. berghei mutants in a single infection is now possible.
• 1800 barcoded recombineering vectors are now available for .P berghei.

Progress in Year 5
Headlines
In general the progress has been excellent and to plan. Highlights include:
▪ A vibrant joint research programme has been established and has resulted in 1149 peer-reviewed publications, 228 of which were reported in Period 3: all of these publications acknowledge the support of the EC in the form of the EVIMalaR Network of Excellence
▪ All management structures are in place and the management office in Glasgow has expanded yet further and prompted the formation of an EU office in the host institution. The office continues to work on additional EC–funded project management tasks
▪ The initial graduates from the EVIMalaR PhD School have emerged and all graduates are on course to complete according to schedule. Progress has largely been excellent. See (www.evimalar.org; http://www.klinikum.uni-heidelberg.de/EVIMalaR-PhD-Programme.115126.0.html?&L=de).
▪ The fifth annual meeting of the EVIMalaR consortium (aka the tenth BioMalPar Meeting) was successfully held at EMBL, Heidelberg. The annual bioinformatics course for students designed by personnel from WTSI and PlasmoDB was run in conjunction with this meeting. Cluster meetings were also planned around this meeting.
▪ The timetable for the Training Courses has been implemented. Those already held in the reporting period include a third training in advanced experimental genetics in malaria (at the Sanger Centre), a further imaging course in Heidelberg, a discussion forum at Biomalpar X on Careers in Science. A full meeting on “Women in Science” was held in Rome as part of our gender equality agenda.
▪ OZEMalaR and Ozmalnet, the mechanisms for liaison with the Australian Malaria Research Community has been established in Australia and funded by the NHMRC (http://www.ozemalar.org/) continues. Calls for applications and their evaluation are continuing with productive exchanges resulting. This activity will continue after the completion of Evimalar NoE funding.
▪ Support for a pictorial database of Plasmodium run by Prof Hagai Ginsburg, University of Jerusalem (http://sites.huji.ac.il/malaria/) continues. The site is stored at Sanger and discussions area in progress with PlasmDB to represent and link to the knowledge it contains.
▪ The ESAB continued its advisory role at the 5th annual meeting in May 2013 in Heidelberg and are largely satisfied with our excellent progress.
▪ The Executive Committee continues to fulfil its specified function. The Executive has met twice in the last 12 months (Heidelberg & Copenhagen) and has also made recommendations concerning award of the Lifetime Achievement awards and the evaluation of small internal funding opportunities
▪ Formation of a Legal Entity to represent the interests of the Network with 13 members is almost finalised and the lobbying process for continued support continues.
▪ All four Clusters have held (Heidelberg, Copenhagen & Geneva) independent annual meetings in 2014. As always the majority of the talks have been given by early stage researchers active in the network
▪ EVIMalaR performed outreach activities to peer groups, notably in Heidelberg at the Biomalpar X meeting in May 2014.

Research highlights from the individual clusters
Cluster 1: Immunobiology and Pathophysiology:
• We identified Plasmodium RNA as a pathogen-associated molecular pattern capable of inducing type I IFN, which requires the cytosolic pattern recognition receptor MDA-5
• We show for the first time that the liver has sensor mechanisms for Plasmodium and that these mechanisms mediate an anti-parasite response.
• Identified the architecture of PfEMP1 using cryo-electron microscopy (C-shaped with mobile headstructure).
• The architecture of human IgM alone and in complex with PfEMP1 has been identified.
• The relationship between the specific PfEMP1 protein expressed at the surface of P. falciparum-infected erythrocytes and the density of knobs on the surface of such cells has been documented.
• P. berghei proteins located at the red blood cell membrane have been identified and are available to be used to generate chimeric P. berghei/P. falciparum proteins to examine properties of P. falciparum IE sequestration, in vivo.
• Provided further evidence of the pathological role of Haemozoinin in parasitized RBCs that generates 4-hydroxynonenal (4-HNE), a lipoperoxidation product that forms covalent conjugates with membrane proteins.
• Demonstratae that hypoargininemia during P. falciparum malaria may impair the arginine-NO production pathway and contribute to impaired RBCs in malaria patients with febrile temperatures.
• Mutant P. berghei lacking enzymes responsible for Hb proteolysis produced little or no HZ, were resistant to chloroquine but sensitive to artesunate. These findings have implications for drug development against P. vivax and P. ovale, which develop inside reticulocytes.
• We have shown show major abnormalities in the function of uninfected red blood cells in children with severe anaemia.

Cluster 2: Parasite Molecular and Cell Biology
• A second-generation of substituted oligoamines compounds with optimised pharmacokinetics, have been produced resulting in sub-nanomolar inhibition values.
• The polyamine analogue have pluripharmacology against proliferative and non-proliferative forms of the parasite.
• A bespoke transgenic P falciparum reporter line has been generated and is currently used to screen for anti-gametocyte compounds from the MMV Malaria Box.
• The palmitoyl transferase functions in P. berghei transmission stages have been systematically characterised.
• N-myristoyltransferase (NMT) has been validated as a new candidate drug target in malaria.
• Nucleosome variant positioning and epigenetic marking have been characterised in greater detail for P falciparum blood stage asexual parasites
• The DiCre-system has been optimised allowing medium throughput in combination with cas9/CRISPR system.
• inhibitors of CDPK1 with low nanomolar potency against the enzyme and their mode of action against the parasite in vitro have been identified..
• Glideosome heterogeneity is evident and functionally overlapping. Glideosome component function continues to be investigated
• Plasmodium Actin is unusual and makes shorter filaments than in most species and interacts with coronin which can also bind membranes and translocate during parasite invasion of host cells.
• The first crystal structure of PfSUB1 revealed both the detailed structure of the active site of the enzyme, as well as the presence of a unique regulatory ‘redox switch’ which likely governs protease activation in the parasite.
• Rises in both cAMP, which activates PfPKA, and Ca2+, which activates PfCDPK1, are required for microneme release
• Phosphoinositide Metabolism is linked to cGMP-Dependent Protein Kinase G and Essential Ca2+ Signals at Key Decision Points in the Life Cycle of Malaria Parasites.
• 2 Phospholipases are involved in Egress and are conserved among different Plasmodium species, suggesting a central function in Plasmodium biology.
• Deletion of PfEMP1 trafficking protein 1 (PfPTP1) leads to severe alterations in the architecture of Maurer's clefts. Furthermore, 2 major surface antigen families, PfEMP1 and STEVOR, are no longer displayed on the host cell surface leading to ablation of cytoadherence to host receptors.
• A genome scale vector resource enables high throughput reverse genetic screening in a malaria parasite.
• phosphoenolpyruvate carboxylase as a key enzyme in erythrocytic Plasmodium falciparum carbon metabolism and a potential drug target
• FACS purified in vitro cultured, 6-days old hypnozoites and developing liver stages can now be isolated and are being characterised in depth.
• A major cause of pathogenesis, VAR2CSA-mediated cytoadhesion of parasitized erythrocytes to the placenta, is dependent upon the density of its ligand chondroitin sulphate A.
• PfATG8 may be unique in having such a second role in apicoplast formation and maintenance in addition to the formation of autophagosomes required for classical autophagy,

Cluster 3: Vector-Parasite Interactions
• A new gustatory receptor GR9 expressed in the mosquito gut is shown to be involved in immune and homeostatic responses to the gut microbiota that significantly affect mosquito development of the malaria parasite.
• A novel bacterial receptor of the Nimrod superfamily is identified that protects mosquitoes against bacteria and malaria parasites.
• Characterisation of a new type of mosquito immune response against malaria parasites, termed anticipatory.
• Identification of a novel negative regulator of the TEP1 accumulation on the parasites.
• Characterisation of the P. berghei transcriptomes that control parasite development within the vector.
• Establishment of A. gambiae system for validation of malaria-blocking interventions.
• Demonstration that genetic background of both vector and parasite shape the outcome of malaria infections.
• Discovery that Plasmodium virulence in the liver stages is determined by the mosquito nutritional status.
• Characterization of microbial communities of emerging adult mosquitoes and their effect on Plasmodium development.
• Demonstration that individual blood meal experience affects host choice in An. coluzzi.
• Discovery that the infection cost of P. falciparum on An. coluzzii is stress dependent.
• Demonstration that malaria-infected mosquitoes produce progeny with reduced disease resistance.
• The presence of P. falciparum in salivary glands modulates the expression of salivary proteins.
• Polarization of human Th2-oriented immune responses and induction of tolerance are mediated by the A. gambiae salivary protein gSG6.
• Demonstration that antibody responses to the A. gambiae salivary protein gSG6 could be used as a measure of malaria exposure.

Cluster 4: Systems Biology and Technology Development.
• new reference genomes for Plasmodium berghei, P. chabaudi and P. yoelii have been published and the data made available via www.genedb.org and later www.PlasmoDB.org.
• draft whole genome sequence from further members of the Laverania subgenus including more P. reichenowi, P. gaboni, P. GorA, P. GorB and P. praefalciparum have been generated.
• Database of 1000’s of var genes produced.
• the mathematical modeling of metabolic phosphlipid pathways operating at the blood stage of P.knowlesi has been finalised.
• An unexpected compartmentalization of choline and ethanolamine metabolism has been identified in P. falciparum.
• A genetic screen of 1000 metabolism associated P. berghei genes using barcode sequencing of PlasmoGEM mutants has begun to reveal likely redundant and essential metabolic pathways in vivo.
• >2750 barcoded recombineering vectors are now available for P. berghei.
• The DiCre methods established for P. falciparum and T. gondii have been adapted to P. berghei and a new conditional protein degradation technology has been developed that allows access to all parasite life cycle stages

Perspective.
The Evimalar Network of Excellence has now terminated and the office in Glasgow will close shortly as reporting is completed. The graduation of 22 PhD students from the dedicated Evimalar PhD School based in Heidelberg is one of our most noteworthy achievements and it is hoped that they will go forward to form a new generation of malaria researchers. The research activity has been intense yielding 1149 peer-reviewed publications the vast majority of which were for collaborations that stemmed from within the network or its interactions with affiliated organisation such as Ozemalar. This interaction with the federated Australian malaria research community was also indicative of the global influence of the network.
Evimalar has been enshrined in an European Economic Interest Group (EEIG) that as such is capable of continuing as an independent entity and able to participate in bids for further funding from for example, the Commission. However despite being widely regarded as a success by every possible measure it is damning that continuity is not possible. The tropical regions of the world where malaria is rife are not silos of disease and poverty but in strict economic terms could be (cynically) viewed as under-resourced trading partners. Halving malaria would have an enormous effect on the global and therefore European economies and represent one of the best values for development investment (Copenhagen Consensus 2015 http://www.copenhagenconsensus.com/post-2015-consensus/), a consistently proffered opinion over the last decade. Therefore, the disappearance of malaria and other diseases of poverty that are not present and immediate threats for the member nations from the research portfolio of the European Commission is not only shameful from a humanitarian aspect but economically myopic. Of course global warming (should one believe in it) means the mosquito vectors of malaria will spread north accompanied by the wave of (illegal) immigration also marching north from the Mediterranean borders who themselves may be infected. Malaria has only been purged from Europe for 70 years and maybe returning. Europe is equipped with some of the finest malaria research laboratories in the world that Evimalar strengthened through the fostering of collaboration. National funding agencies do not fund international collaboration; representative Governments rightly expect that their contributions to the European project facilitate such endeavours. We urge the Commission to consider continuity.

Potential Impact:
The European Virtual Institute of MALAria Research (Evimalar) was a Network of Excellence (NoE) funded under FP7. It was an alliance of 54 full members drawn from European Union’s leading malaria research groups based in 36 institutions together with 6 leading African Institutions, the ICGEB from India and with representation from OzEMalaR, a network of Australian malaria researchers. A further 31 European groups were affiliated to Evimalar. The focus of the network was upon basic biological investigations of the malaria parasite and its interactions with both host and vector.

The Impact of Evimalar has been considerable. It has developed tools and mechanisms to both influence the future European research area but also to generate similar impact on a global level with a focus chiefly on Africa and Australia. Databases and technologies developed by the membership of Evimalar have a global influence and are used by the entire research community and are recoded in the main report and summarised here. The impact of Evimalar will be long lasting due to these advances, resources and the training that has been imparted during the course of the project.

Please refer to the attached Impact Report for further detail,

List of Websites:
www.evimalar.org

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UNIVERSITY OF GLASGOW
United Kingdom
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