Using single-cell RNAseq to investigate human malaria parasite transmission dynamics

Malaria is caused by a parasite that is transmitted exclusively by mosquitoes. The greatest malaria reduction and eradication success stories have been achieved by interrupting transmission. Historically, this has been through mosquito control. Targeting the small population of specialised parasites residing in the human host that are transmitted through mosquitoes would provide a similarly powerful malaria control method but we know too little about this population. Until now, the genes expressed by parasites have been analyzed by combining millions of parasites together. This approach confounds differences between individual parasites that could underlie success in getting into another host or in resisting the drugs we use to kill the parasites. We have recently developed a method to analyse single parasites one at a time. This technological leap has allowed us to understand parasites in the laboratory with more precision than ever before and importantly to understand how one parasite may differ from another during the whole life of the parasite both in the host and in the mosquito. Although the laboratory setting and lab strains of parasites are powerful tools for understanding parasite biology, in the lab we cannot understand the full diversity of parasites that exist in the wild causing devastating consequences for infected individuals. In this project we propose to characterise wild parasites at an individual level in partnership with Malian scientists. Our exploration will allow us to characterise the three main species of malaria parasite in sub-saharan Africa on a single parasite level for the first time. We will integrate the data into an interactive website called the Malaria Cell Atlas. This will become a key resource for the research community. We will then explore the changes from one patient to the other of the deadliest malaria species in both patients that are suffering from malaria symptoms and also from infected carriers who are not suffering from malaria, both of which contribute to the overall reservoir of parasites. Altogether, we will look at more than 300,000 individual parasites and get a very deep understanding of how individual parasites are both similar and different from each other. Understanding this infectious reservoir is pivotal to identifying how parasites efficiently get from one person to the next. Altogether our findings using cutting-edge tools to explore wild parasites will be key to understanding malaria and how to best control it.

Here we propose to explore natural parasite populations and within-host diversity by harnessing the power of single cell transcriptomics. We will create a Malaria Cell Atlas for three species of human malaria parasites, covering the asexual stages that cause disease and the sexual stages responsible for transmission by Anopheles vector mosquitoes. After we complete these atlases, we will dive deeply into transmission dynamics for P. falciparum, the most prevalent species in Africa and the species responsible for the majority of deaths. We will use single cell RNA-seq to interrogate parasites from both symptomatic and asymptomatic cases, exploring in more resolution than ever before possible the diversity of parasite populations: how many transcriptional states and how many genotypes exist in a single person; how does diversity compare between asexual and sexual parasites; do patterns change as the season progresses; do patterns differ between those silently carrying the parasite versus suffering disease. Finally, we will explore actual transmission dynamics of these deeply interrogated populations of parasites by investigating parasite genetic diversity in mosquitoes fed directly on these parasites. To do this, we have partnered with Malian scientists, and we will be bringing single cell RNAseq to West Africa for the first time. Our approach will derive deep and meaningful biological insights on within-host and within-vector diversity from samples that are directly sourced from a natural infection setting. In sum, will (i) generate high resolution transcriptional maps of the most prevalent human malaria species in the blood of infected patients, including pathogenic and transmission stages (ii) interrogate the inter-host variation in transcriptomic and genetic diversity in different reservoirs of the parasite contributing to further transmission (iii) understand the sexual reproduction and within-vector dynamics of the infectious reservoir of the disease.

The World Health Organization estimates that $5 billion is spent each year on malaria control. Research arising from this proposal will have long term benefits on spending that money more effectively because it will impact our understand on transmission. Beyond the impact on malaria control, our proposed research will also have a positive impact on global research by generating knowledge that will assist with the development of drug and vaccine targets for malaria control. Genes identified as highly variable or specifically expressed in wild strains of parasites at either the asexual and sexual stages are poor targets for drugs and vaccines due to their variability. The Malaria Cell Atlas will be a key resource to understand what genes are expressed more consistently across parasite genotypes and patients, and in what stages these genes are expressed. Our research could uncover new targets in the male and female gametocytes that may lead to the design of transmission blocking drugs and/or vaccines. Beyond drugs and vaccines, the knowledge we generate can be used to develop tools towards wide scale epidemiological characterization of malaria. From a wider perspective, the project may discover trends in vector-borne disease processes, which may be applicable to other infections such as the transmission of other parasites or arboviruses. Any arising IP from this research, where there might be clinical value, will be further evaluated for protection and development potential through discussions with the Translation Office.

Our research will also have impact on the specific country where we will carry out our research. Mali is on the DAC List of ODA Recipients as a Least Developed Country. The burden of Malaria in Mali remains unacceptably high. According to the latest World health organisation's Report of 2017, 2016 saw 90% of the 216 million cases of malaria occurring in sub-Saharan Africa and 91% of the 445,000 deaths also occurring in sub-Saharan Africa. In Mali, malaria is the leading cause of morbidity and mortality among pregnant women and children under five, accounting for 34% of the total number of outpatient visits for all age groups. In 2015 alone, 2.37 million cases of malaria were recorded in public health facilities, and it is estimated that 35% of children under 5 are infected. Malaria elimination requires thorough knowledge of parasite and mosquito, but the ability to do research on transmission biology is hampered by a lack of adequate insectaries around the country. While state of the art facilities do exist in Bamako, the capital city of Mali, malaria cases are less frequent and thus harder to access. Building an insectary next to an active medical clinic that serves both the 3,000 local inhabitants of Faladie and the 20,000 inhabitants of Ntjiba County benefits multiple facets of society from the researchers to the local people receiving healthcare and contributing to the science. As part of our research, an insectary will be built in the village of Faladie, where the burden of malaria is extremely high. Our studies will both benefit the local community as well as contribute to scientific knowledge in general. We estimate over the course of our activity there, thousands of children will receive treatment for malaria irrespective of whether they contribute to our study or not. The link between malaria and hampered socio-economic development has long been recognised. Decrease and eventual elimination of malaria will lead to healthier people, less poverty, more kids spending more time in classroom, more pregnant women delivering healthy babies and more socio-economic stability. The goal of malaria research is to decrease the burden of malaria, and ultimately to reach malaria elimination.