Profiling of host and parasite factors associated with organ dysfunction in severe malaria

Severe Plasmodium falciparum malaria is a complex disease with a wide spectrum of clinical manifestations, which accounts for the majority of malaria-associated deaths. In low transmission settings like Asia, it predominantly affects adults, who often develop brain, lungs, liver or kidney dysfunction, either alone or in combination. The mechanisms leading to these complications have never been systematically investigated, but the binding of Plasmodium falciparum-parasitised red blood cells to the vessels walls of these organs, as well as the immune response of the host are likely to be involved.

Recent results suggest that a specific type of the parasite surface protein PfEMP1 that binds to two receptors called ICAM-1 and EPCR on the vessel walls are associated with the development of cerebral malaria in African children, the form of severe disease that affects the brain. We therefore postulated that distinct combinations of parasite PfEMP1 types and receptors on endothelial cells lining the vasculature are associated with specific organ dysfunctions during severe malaria.

To test our hypothesis and investigate the parasite and human host factors leading to complications in severe malaria, we intend leverage the enrolment and in-depth clinical classification of Plasmodium falciparum-infected patients as part of our ongoing International Center of Excellence for Malaria Research (ICEMR) at Ispat General Hospital in Rourkela, India. Fatal cases of severe malaria with clinically-defined brain, lung, liver or kidney dysfunction will be recruited, and samples from these tissues will be collected using minimally invasive autopsy techniques. We will use a digestion protocol that dissociates all the cells contained in a tissue to generate single-cell suspensions for each organ sample. Cells will then be sorted to obtain purified populations of parasitised red blood cells, endothelial cells, and immune cells. We will profile PfEMP1 variants, endothelial receptors, and immune cells type and quantity in all affected organs, as well as signatures of dysfunction in the plasma of these patients. In addition, we will perform an extensive analysis of the structural damage of the brain, lungs, liver and kidneys in fatal severe malaria.

By combining these complementary approaches, our project will provide an exhaustive profiling of the factors leading to specific organ dysfunction in severe malaria. This has never been undertaken before, in part due to the reluctance of the population to authorise full autopsies. Our project circumvents this issue by implementing a fast and mark-free needle-based tissue sampling technique. The information generated by our project will have the potential to help clinicians and scientists assessing new therapies to increase patient survival, refine tools to identify malaria patients at risk of developing organ failures and improve the design of future malaria vaccines.

In low transmission settings, patients with severe falciparum malaria develop brain, lungs, liver and kidney dysfunction, either alone or in combination. The mechanisms leading to these complications have never been systematically investigated. We propose to implement minimally invasive tissue sampling techniques in well-characterised patients admitted at Ispat General Hospital in India to analyse for the first time multi-omics host and parasite factors leading to specific organ dysfunction in severe malaria. We will collect samples of brain, lung, liver, kidney and blood tissue from fatal cases with or without cerebral malaria, acute respiratory distress syndrome, jaundice and acute kidney injury. Half of the solid tissues will be used for the first extensive immunohistopathology analysis of its kind, and the rest digested to isolate parasitized red blood cells, endothelial cells, and immune cell populations. We will determine the subset of circulating parasite variants that sequestered in different organs of fatal malaria cases and their association with specific clinical complications by cloning, sequencing and comparing DBLa tags between tissues. In parallel, we will perform deep RNA sequencing to characterise global transcription profiles of endothelial cells from the same organs and identify potential PfEMP1/endothelial receptor combinations associated with specific organ dysfunctions. We will also determine the type, amount and transcriptome of immune cell populations present in each organ through single-cell transcriptomics. Lastly, we will carry out global metabolomic and complex lipidomic profiling of the plasma from fatal cases, non-fatal matched cases and uncomplicated malaria controls to identify signatures associated with specific organ failures and with fatality. The primary outcome of this project will be a better understanding of the factors influencing the development of organ dysfunction in severe malaria, with a view to developing new therapies.

In the long term, the main beneficiaries of this research project will be populations living in malaria-endemic areas. While a significant reduction in transmission has been achieved over the past decade, the burden of severe malaria remains high and still leads to approximately 400 thousand deaths annually worldwide. Because artemisinin resistance in on the rise in Southeast Asia, and the leading pre-erythrocytic vaccine candidate RTS,S is only moderately effective to prevent severe infections, a better understanding of the pathophysiology of this complex disease is pivotal to develop new therapeutic approaches and inform the design of improved vaccines.

We propose to carry out an extensive characterisation of the different PfEMP1 variants sequestered within organs affected during severe malaria, including the brain, lungs, liver, and kidneys. In parallel, we will also profile the endothelial cell receptors in the microvasculature of these organs. This will constitute an important step towards the identification of novel vaccine antigens and will inform strategies targeting epitopes shared between clinically relevant PfEMP1 variants. The international malaria community, as well as partners in the pharmaceutical industry, will benefit directly and indirectly from the data generated by our project.

In addition, the histopathology analysis we intend to carry out will be combined with the screening and quantification of immune cell populations within brain, lung, liver and kidney tissues to identify potential immune mechanisms leading to specific organ dysfunction in severe malaria. The generated datasets will be available to immunologists nationally and internationally, for comparison with other immune-driven pathologies and assessment of potential adjunct therapies.

Lastly, the data generated by our metabolomic and lipidomic phenotyping will allow the identification of biomarkers associated with malaria infection severity, specific organ dysfunction, and clinical outcome. Such candidates will be available for further commercial development either by our team or by industrial partners.

Our team of investigators has strong collaborative links with Ispat General Hospital in Rourkela, India, a partner Institution in this application, but also the Swiss Tropical and Public Health Institute and its sites in Mali and in the Democratic Republic of Congo. Our existing collaborations with these academic groups overseas and in malaria-endemic areas places us in an ideal position to use the data generated through this project for translational research, ultimately leading to improved diagnosis and outcome for severe malaria patients.