Dissecting the Red Blood Cell Invasion Pathways of the Malaria Parasite Plasmodium knowlesi

Lead Research Organisation: London Sch of Hygiene and Trop Medicine
Department Name: Infectious and Tropical Diseases

Abstract

Malaria is one of the most important infectious diseases of man with more than half of the world's population living at risk of the disease, and resulting in more than half a million deaths per year. The disease is caused by Plasmodium species, single-celled parasites that can be transferred to humans by the bite of an infected mosquito. Symptoms of the disease result from the parasite destroying red blood cells by first entering them, growing and replicating inside them, before bursting out and invading other red blood cells in a continuous cycle. The parasites produce a range of adhesive proteins enabling them to bind to specific proteins on the surface of red blood cells and establish the process of red blood cell invasion. Because of their crucial role in the invasion process these parasite proteins are important vaccine targets. They also determine how effectively parasites can replicate and so can affect disease severity as well as determining which hosts are susceptible to malaria.

In this project I will investigate the role of these proteins during the invasion process using a malaria parasite known as Plasmodium knowlesi. This parasite naturally infects macaque monkeys in South-East Asia, and was recently found to be a significant cause of severe and fatal human infections. In recent work I have developed methods to grow this parasite in culture with human red blood cells for the first time, and established highly efficient techniques to genetically modify the parasite. I will use these techniques to generate parasites in which I have deleted genes encoding the adhesive proteins. This will enable me to determine which are essential for invasion and which can be deleted without any effect on the invasion process. Using similar techniques I will also add fluorescent "tags" to each of the proteins coded by the target genes, so that I can determine where the adhesive proteins are in the cell and where they move during the invasion process. The "tags" will also allow me to identify parasite proteins that interact with the adhesive proteins as well as what they specifically bind to on the host red blood cell surface. I will analyse both the gene deletion and "tagged" parasite lines using cutting edge imaging technologies including electron tomography and super resolution microscopy, which have never before been used to visualise invasion of this parasite species. This will provide critical insight into the mechanism of invasion of all malaria parasites, as well as identifying precisely which parasite proteins and host proteins are required for P. knowlesi to invade human red blood cells. The latter is of particular importance as it may explain how a macaque malaria parasite is able to spread to infect humans and determine the potential for emergence of human-to-human transmission of the parasite.

Whilst there is currently no vaccine for malaria, there is great interest and several vaccine candidates under development for the most common and serious cause of malaria P. falciparum. However, vaccine development for the second most common cause of malaria P. vivax, is hampered by the fact that it cannot be grown in the laboratory. This means that testing new vaccines would involve infecting people or non-human primates with P. vivax. The primary vaccine candidate for P. vivax is one of the parasite's adhesive proteins. P. knowlesi is closely related to P. vivax and also uses a similar version of this adhesive protein. By genetically modifying P. knowlesi to replace the gene encoding the adhesive protein with the version from P. vivax, it will be possible to determine whether a vaccine can induce antibodies that kill parasites in culture, before it is necessary to test it in people. Thus I will use the unique biology of P. knowlesi along with the technical advantages of the model to not only study the process of invasion but also generate important tools to expedite the development of vital malaria vaccines.

Technical Summary

Clinical symptoms of malaria are associated with the cycles of parasite invasion of, and multiplication within, host red blood cells (RBCs). The ligands used by the parasite to target host cell receptors and invade RBCs are key determinants of virulence. Two parasite protein families are implicated in this process, the reticulocyte binding-like (RBL) and Duffy binding-like (DBL) proteins, but little is known about their role or their host cell receptors. I will define the role of these protein families in Plasmodium knowlesi, a parasite causing severe and fatal disease in South East Asia. Taking advantage of the high transfection efficiency and unique capability to grow this parasite in two different host cells, I will generate gene knock-out lines for each of the 5 P. knowlesi DBL/RBL genes - to define which proteins are essential for invasion of the two different host RBC. Transgenic parasite lines will be generated in which each of the RBL/DBL proteins has been modified by the addition of a fluorescent/epitope tag, which will be used to determine subcellular localisation, and through immunoprecipitation, identify host cell receptors and interacting parasite proteins. I will use electron tomography and super resolution microscopy for the phenotypic analysis of knock-out and tagged lines to determine the precise role of each protein in the invasion process. To identify proteins that are essential for invasion of both host RBC, I will adapt the Cas9 and DiCre conditional recombinase system for use in P. knowlesi. Finally, I will develop a transgenic P. knowlesi model to assess both naturally acquired and vaccine induced antibodies targeting P. vivax, an important malaria parasite that currently lacks an in vitro model. The unique advantages of P. knowlesi will provide an unprecedented insight into the mechanistic role of RBL/DBL proteins during invasion, and novel tools to support development of vaccines to target them.

Planned Impact

Malaria is one of the most important infectious diseases of man, with 3.2 billion living at risk of the disease and between 350 and 500 million clinical cases a year. My research is driven by my desire to produce an impact on affected communities. My research proposal will help to unravel the process of red blood cell (RBC) invasion by malaria parasites, an important target for novel drugs and vaccines. The P. knowlesi malaria parasite that I will use to study this is recognised as an important emergent cause of severe zoonotic infections in South East Asia. Malaysia is currently working toward the elimination of malaria and whilst control measures are resulting in a decline in other human malaria parasites, P. knowlesi cases have risen >10-fold in areas of Malaysian Borneo between 2004-2011. Dealing with a malaria parasite with an animal reservoir will require the development of novel intervention strategies. In addressing parasite host cell tropism, this project will identify parasite factors which may be causing this rise in human infections. Through my Winston Churchill Memorial Trust Fellowship to Malaysia, I have developed strong links with both Malaysian Government Institutes (IMR), and field work teams, which I will use to convey my findings, inform policy and target field work. It was clear from my time in Malaysia that the UK retains a reputation for excellence in tropical disease through its historic links and continued commitment to global health research. I have already provided the P. knowlesi parasite line that I have developed to two Malaysian Institutions and provided training to a Malaysian PhD student seconded to my lab for 3 months to help establish its use in laboratories in Malaysia. Through the CDA I will continue this high level of support and interaction with Malaysian research groups, in alignment with the MRC's strategic aim 2 "Going Global".
By defining the ligands essential for human red blood invasion by P. knowlesi, I will identify novel vaccine candidates which in the long term could be used in humans or even the macaque reservoir. In addition to the potential validation of novel vaccine targets for P. knowlesi, I will collaborate with Simon Draper to develop a transgenic P. knowlesi model to test existing vaccine candidates for P. vivax. This means that my research can have an immediate impact on the development of malaria vaccines, since it establishes an important translational path to facilitate the development of novel vaccine targets as they emerge from my basic research in P. knowlesi.
My work will impact directly on healthcare in the UK. I have already provided parasite DNA and preparations from cultured parasites to the UK Malaria Reference laboratory to aid diagnosis of P. knowlesi infections in returning travellers. I regularly attend the All-party Parliamentary Committee for Malaria and Neglected Tropical Diseases at Westminster (MRC strategic Aim Two: Research to people). This allows me the opportunity to discuss my research with policy makers, including politicians, officials from the UK Department for International Development and key non-governmental organisations. In addition to the training and career development opportunities afforded to me (see Career Intentions), the project will provide a postdoctoral researcher with training in a superb collection of research skills. The combination of exposure to cutting edge imaging skills and training in a new malaria model with wide potential will be highly prized by the malaria research community, and the novelty of the system will provide plentiful opportunities to develop independence.
Finally, a significant hurdle to the delivery of impact for neglected tropical diseases is the relative lack of economic incentives for investment from Industry. Public awareness has a major role to play in promoting the required investment and I will use my experience of public engagement to highlight this (See communications plan for details).

Publications

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Benavente ED (2018) A reference genome and methylome for the Plasmodium knowlesi A1-H.1 line. in International journal for parasitology

 
Description BBSRC London Interdiscplinary Doctoral Programme Studentship "Combining next-generation sequencing and CRISPR/Cas9 Genome editing to unravel genetic variation in host cell preference and invasion in malaria parasites"
Amount £84,000 (GBP)
Organisation Biotechnology and Biological Sciences Research Council (BBSRC) 
Sector Public
Country United Kingdom
Start 10/2018 
End 10/2021
 
Description Bloomsbury Colleges Studentship
Amount £62,600 (GBP)
Organisation Bloomsbury Colleges 
Sector Academic/University
Country United Kingdom
Start 09/2016 
End 08/2019
 
Description Building an international collaboration to explore invasion biology and vaccine development for the malaria parasites Plasmodium vivax and P. knowlesi
Amount £10,000 (GBP)
Funding ID MC_PC_18012 
Organisation Medical Research Council (MRC) 
Sector Public
Country United Kingdom
Start 05/2018 
End 05/2019
 
Description Malaria Mode of Action Studies (Collaboration with Dundee Drug Discovery Unit)
Amount £40,000 (GBP)
Organisation Bill and Melinda Gates Foundation 
Sector Charity/Non Profit
Country United States
Start 12/2018 
End 12/2020
 
Description Sir Henry Wellcome Postdoctoral Fellowship, 'Unravelling the intracellular interactions and signalling of Apical membrane antigen-1 (AMA1) in malaria parasites.'
Amount £250,000 (GBP)
Funding ID 210861/Z/18/Z 
Organisation Wellcome Trust 
Sector Charity/Non Profit
Country United Kingdom
Start 06/2018 
End 06/2022
 
Title CRISPR-Cas9 genome editing in Plasmodium knowlesi 
Description We have adapted CRISPR Cas9 genome editing to the malaria parasite P. knowlesi for the first time. Our approach enables the generation of transgenic parasites without integration of a selectable marker, which allows iterative genomic modifications. We have also demonstrated that this approach is compatible with PCR generated constructs and thus is highly scalable. 
Type Of Material Technology assay or reagent 
Year Produced 2019 
Provided To Others? Yes  
Impact We have shared plasmid constructs and methods with more than 6 labs globally, and have also had interest from industrial partners. The method has enabled the generation of P. knowlesi lines expressing critical P. vivax vaccine antigens, and thus is supporting the development of vaccines as well as work to understand basic biology of malaria parasites. 
 
Title Development of transgenic Plasmodium knowlesi models for vaccine development against Plasmodium vivax. 
Description Development and testing vaccines against Plasmodium vivax is hampered by the fact that this parasite cannot be maintained in continuous culture. Thus sera and antibodies derived from animal and human vaccination trials must be tested either in low throughput assays using ex vivo cultured clinical isolates or P. vivax maintained in non-human primate models. Using CRISPR-Cas9 we have genetically engineered P. knowlesi to express a lead P. vivax blood stage vaccine candidate, known as PvDBP. The P. knowlesi line uses this protein to bind to and invade red blood cells and using a scalable parasite growth assay can be used to accurately identify and characterise antibodies which are able to block invasion in P. vivax. 
Type Of Material Cell line 
Year Produced 2019 
Provided To Others? Yes  
Impact The tool has been used in conjunction with a Phase Ia vaccine trial to identify the first vaccine induced strain transcending human monoclonal antibody against P. vivax. It will now also be integral to an ongoing phase II trial of a vaccine against PvDBP. The line has been requested by 5 different laboratories across the world and MTA are currently being drafted to provide the line for academic uses. 
 
Description In vitro Drug Sensitivity of the Zoonotic Malaria Parasite Plasmodium knowlesi cultured in human erythrocytes 
Organisation Medicines for Malaria Venture (MMV)
Country Switzerland 
Sector Charity/Non Profit 
PI Contribution This collaboration was established in response to a joint application to the Medicines for Malaria Venture with Colin Sutherland (LSHTM), to provide funding for the development of an in vitro drug sensitivity assay for the malaria parasite Plasmodium knowlesi. Current drug testing regimes rely heavily on the use of P. falciparum for in vitro testing. This project will utilise the P. knowlesi line that our lab has previously adapted to culture in human red blood cells, to develop and validate a medium/high throughput assay for in vitro drug sensitivity testing. P. knowlesi is closely related to P. vivax (the second most common cause of malaria infection), which currently lacks an in vitro model, thus an in vitro drug assay for a second human malaria parasite will be invaluable to ensure novel antimalarials are effective against all species of human malaria. We have supported the work through providing technical assistance to the Sutherland lab in establishing the parasite culture and in vitro assays. We are also providing ongoing support contributing to conceptual design of project, experimental design and analysis of data. Work is now coming to an end and a manuscript summarising the work will be submitted within the next month. Our work identified key differences between drug sensitivity of P. knowlesi and P. falciparum, and provided a critical tool for early evaluation of new investigational antimalarial compounds.
Collaborator Contribution The MMV have provided financial support for this project (including reagent and staff costs for Sutherland lab). They have also provided access to their extensive compound library. Direction and focus of project both in terms of assay development and drug class focus are determined through regular interactions between the LSHTM research groups and MMV project manager.
Impact Three papers have been published, in the Journal of Antimicrobial Chemotherapy(DOI: 10.1093/jac/dkx279), International Journal of Parasitology Drugs and Drug Resistance (doi: 10.1016/j.ijpddr.2019.02.004) and Antimicrobial Agents and Chemotherapy (doi: 10.1128/AAC.02549-19). Two additional manuscripts are in preparation.
Start Year 2015
 
Description Lab Adaptation and Drug Susceptibility of Non-falciparum Parasites 
Organisation Medicines for Malaria Venture (MMV)
Country Switzerland 
Sector Charity/Non Profit 
PI Contribution This collaboration was established in response to a joint application to the Medicines for Malaria Venture with Donelly van Schalkwyk and Colin Sutherland (LSHTM), to provide funding to attempt culture adaptation of P. malariae and to undertake ex vivo drug screening against clinical isolates of non-falciparum malaria parasites. Only two species of malaria parasite (P. falciparum and P. knowlesi) can be cultured in vitro, but to ensure that drugs under development are effective against all species of human infecting malaria parasites it is essential that we develop ways of testing drugs against P. malariae, P. ovale and P. vivax. iWe have supported the work through providing technical assistance to Dr. van Schalkwyk in parasite adaptation procedures as well as providing ongoing support contributing to conceptual design of project, experimental design and analysis of data.
Collaborator Contribution The MMV have provided financial support for this project (including reagent and staff costs for Dr van Schalkwyk). They have also provided access to their extensive compound library. Direction and focus of project both in terms of assay development and drug class focus are determined through regular interactions between the LSHTM research groups and MMV project manager.
Impact A manuscript is in preparation to describe work ex vivo assay data for non-falciparum malaria parasites.
Start Year 2017
 
Description LSHTM Work Placement Scheme 
Form Of Engagement Activity Participation in an open day or visit at my research institution
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Schools
Results and Impact 2017 Student Work Placement Programme (Department for Education and The Challenge) for college level applied science students. Providing work placement (56 days over 5 months) for two 16-18 old students currently taking Applied Sciences BTEC. Students worked in the lab with team to learn a range of molecular biology techniques including western blotting, PCR and bacterial cloning.
Year(s) Of Engagement Activity 2018