Evaluating multiple loci modulating susceptibility of African malaria parasites to artemisinin

Combination drugs for treatment of malaria deployed globally since the start of the 21st century have provided a substantial public health benefit. Malaria deaths across Africa in particular have plummeted since a peak in the 1990s, when failure of the drugs chloroquine and sulphadoxine-pyrimethamine precipitated a rise in infant and child deaths. A key component in all the currently effective drug combinations is the plant-derived chemical called artemisinin. The parasite-killing ability of artemisinin has recently been threatened by the emergence of reduced susceptibility in the Mekong region: this is manifest as a slowing of artemisinin clearance of P. falciparum from the blood of treated malaria patients. Although under combination treatments these patients should eventually experience a full cure in most cases, in this region the combination partner drugs are also failing in some patients. Careful monitoring and vigilance of malaria drug resistance is therefore needed. A parasite gene has been identified in Cambodia and the surrounding region which contributes to the loss of effectiveness of the artemisinin drug - this gene, which encodes a kelch-domain protein and is called K13, has accumulated a variety of mutations. These are strongly associated with the loss of effectiveness of artemisinin against these parasites. However, this same phenomenon has not yet been observed in African malaria parasites - in the small number of cases where drug treatment does not work, K13 mutations are not implicated as the cause.

In the past 2 years, gene-editing studies by ourselves and others have successfully demonstrated a direct effect of 3 genes in reducing susceptibility to artemisinin : pfap2mu, pfubp1 and pfcoronin. Variants of these genes elicit increased parasite survival in vitro in the ring-stage artemisinin survival assay (RSA). In the case of trafficking adaptor potein subunit AP-2mu, we have shown that the role of this protein in P. falciparum is unlike in other organisms as it does not interact with clathrin, but is associated with an as yet unknown compartment in the cell that appears to also contain K13, although we found no evidence of direct interaction between AP-2mu and the K13 protein. There is, however, good evidence of direct interaction of AP-2mu with a different kelch protein , K10. Polymorphisms in the gene encoding the K10 protein have been previously identified as a genetic component of parasites from the Mekong region in which the K13-dependent reduced artemisinin susceptibility originally arose. This work was carried out by PhD student Ryan Henrici, who completed his doctoral studies in 2018 and has moved on.

We now request support to extend this work, using gene editing to test the effect of new mutations. In preliminary studies of both field samples and UK isolates, we have already identified a number of such new mutations in our current five genes of interest - pfk13, pfap2mu, pfubp1, pfcoronin and pfk10 (see Case for Support). For each new isolate (either from the UK or from our collaborator Dr Bismarck Dinko in Ho, Ghana) that is adapted to culture, we will fully test drug susceptibility, and genetically characterise them using genome sequencing. In gene-editing experiments we will then directly measure the impact of each candidate gene variant in both established laboratory lines and in our new cultures from UK and Ghanaian patients; any variant proven to generate a drug susceptibility phenotype may therefore be contributing to changing patterns of drug susceptibility, and could be developed as a surveillance marker for use in malaria endemic areas. Our work will thus assist our understanding of resistance to our current combination drugs, and help us to devise strategies for deploying these drugs carefully in both the UK and Africa, to maximise their useful life in curing malaria patients.

We have identified five genes (pfk13, pfap2mu, pfubp1, pfcoronin and pfk10), through testing in our laboratory or from the scientific literature, which are important for the effectiveness of artemisinin - the most important drug for treating malaria. We will establish an expanded collection of parasite isolates (100-200) from UK travellers with malaria, and also in a "cohort" of recent P. falciparum isolates from malaria patients in Ho, Ghana (50-100), being collected by our collaborator Dr Bismarck Dinko. All parasites that successfully grow in our laboratory will then be tested for drug susceptibility, focussing on artemisinin. Full genome sequence data for all parasites will be generated by co-Investigator Dr Susana Campino.
The application of CRISPR-Cas9 genome to Plasmodium falciparum provides an opportunity to directly test candidate drug resistance genes in transgenic parasites growing in the culture lab. We will use CRISPR editing to test gene variants of interest in our five top candidate genes. We will also have the capacity to test any genes identified in the course of our project, or in scientific journals. Pilot work by PhD student Ryan Henrici (who completed in 2018) sucessfully generated variants of two of these genes - pfap2mu and pfubp1 - in the standard laboratory line 3D7 and found that in both cases the transgenic clones were less susceptible to artemisinin, resembling resistant Cambodian parasites in phenotype. We will extend our genome editing work to natural parasite isolates from Africa, either collected from travellers presenting with malaria in the UK, or in the parasite cohort from Ghana. Thus we will have an opportunity to look at the impact of a variety of African "background" genotypes on changes in susceptibility caused by variants of interest in our top five candidates, and in any new candidates identified in the near future.

We have listed above potential academic beneficiaries of our project outputs.

We also have expectations of beneficiaries in wider society including:
- UK malaria patients
- Public Health England
- the National Malaria Control Program in Ghana
- WHO Technical Expert Group (TEG) on antimalarial drug efficacy
- WHO AFRO Region policy makers

Benefits for researchers will accrue from the proposed development, characterisation and provision as open access resources of new clonal lines of P. falciparum, corresponding full genome sequence databases and a collection of transgenic parasites with estabished drug susceptibility profiles. Drug and diagnostic development in the private sector could benefit from our work, as it will greatly expand the available panel of diverse parasite lines against which products can be tested in vitro. The Medicines for Malaria Venture (MMV), which successfully manages an impressive drug development pathway, has already utilised our panel of patient-derived P. falciparum isolates for testing the resistance profile of investigational compounds of interest. Further cloning out of these parasite lines, and the continuing collection of new isolates, can provide better defined and genetically characterised parasites for these investigations.

The UK NHS is a likely beneficiary, as this project will provide important new information about the performance of the current front-line chemotherapy for imported malaria in the UK. Our research team works closely with the Public Health England Malaria Reference Laboratory (MRL) and contributes to the establishment of drug resistance genotyping among returned travellers in the UK. Our data are transferred to the public domain each year by the PHE, and will inform the treatment guidelines published regularly by the Advisory Committee on Malaria Prevention in Travellers. Any new markers we identify will be translated into this process, so in close to real time we will have estimates of marker prevalences among Plasmodium falciparum-infected British travellers returning to the UK from a variety of African localities. This information can also be used to inform management of individual patients; in his role as a HCPC-registered Clinical Scientist in the through the MRL, and strong links to the Hospital for Tropical Diseases, Sutherland regularly assists UK clinicians with investigations into specific cases of apparent treatment failure. We currently have more than 13 suspected treatment failure cases at various stages of investigation

The WHO TEG will benefit from new in vitro artemisinin susceptibility data for African parasite isolates, whih is currently greatly lacking, and will also be in a good position to advocate wide-spread use of any genetic marker that we find is closely linked to susceptibility phenotypes of concern in the field. For example, this important expert group were instrumental in promoting the global testing of P. falciparum for K13 mutations directly implicated in falling drug efficacy in the Greater Mekong.

Given our focussed study of parasite isolates from Ho, our data will be directly communicated to the Ghanaian health authorities, and particularly to the NMCP; this may assist in decisions concerning the deployment of alternative or second-line treatment in any areas where evidence of AL treatment failure is found.