Defining P. falciparum resistance to artemisinin-based combination therapies

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PROJECT SUMMARY The worldwide adoption of artemisinin (ART)-based combination therapies (ACTs) has been instrumental in halving the global burden of Plasmodium falciparum (Pf) malaria since the early 2000s; however, progress has stalled since 2015. Malaria’s impact remains vast, with an estimated 405,000 deaths in 2018. Now, Pf resistance to ART derivatives and their ACT partner drugs threatens to overwhelm control efforts. Parasites resistant to ART and piperaquine (PPQ), mediated primarily by mutations in the genes k13 and pfcrt respectively, have swept through the low-transmission setting of Southeast Asia. The development of ACT resistance in Africa, with 94% of the global malaria burden, would be calamitous. A major warning sign is the increasing prevalence of an ART resistance-conferring K13 mutant in Rwanda, and of other K13 mutations in nearby countries. Herein we confront the prospect of ACT resistance emerging in Africa. In Aim 1, we will test the hypothesis that African Pf strains rarely pose a biological obstacle to K13 mutations driving ART resistance. By gene editing geographically diverse African strains, we will identify the most resistant mutations and assess the impact of the parasite background, which can substantially influence the levels of resistance and fitness. Having recently completed two Pf genetic crosses between Cambodian ART-resistant K13 mutants and the drug-sensitive African parasite NF54, we will map and confirm secondary ART resistance modulators. We will also test whether resistance can be attributed to loss-of-function mutations that restrict hemoglobin endocytosis in early ring stages. In Aim 2, we will test the hypothesis that mutant K13 will exert a substantial fitness cost in African parasites, which could impede its spread in high-transmission settings. One approach will be to quantify growth rates of barcoded K13 mutant and wild-type isogenic lines. We also hypothesize that ART resistance in Asian parasites evolved via a gain of K13 mutations combined with compensatory fitness mutations, and will use our genetic cross progeny to map and confirm fitness modulators. In Aim 3, we will pursue determinants of Pf resistance to the ACT partner drugs PPQ, lumefantrine (LMF), pyronaridine (PND) and amodiaquine (ADQ) in African parasites. Gene editing will be used to test the hypothesis that PPQ resistance can arise through single point mutations in African PfCRT haplotypes. Efforts to identify determinants of resistance to LMF and PND will employ selections with hyper- mutable African lines. We will also leverage the discovery in one of our genetic crosses of a two-component basis of ADQ resistance that includes pfcrt and an unknown determinant on chromosome 12. The identification of these markers will provide a valuable tool to screen for ADQ resistance. This proposal, which aligns with the NIAID priority of supporting research on antimicrobial drug resistance, is designed to proactively prepare for the dire possibility of ART and ACT resistance emerging in Africa by identifying causal determinants and mutations and assessing whether fitness costs can impede the spread of resistance in African transmission settings.