Molecular basis of antimalarial drug resistance in Plasmodium vivax

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Project Summary Malaria caused by infection with Plasmodium vivax is an enormous public health burden throughout the world, and the cause of significant morbidity. The antimalarial drug chloroquine is the first line of drug treatment for P. vivax in most countries, and has proven highly efficacious. However, chloroquine resistant (CQR) P. vivax infections have been widely reported, seriously hampering case management, malaria control efforts, and elimination programs. Most work on resistance mechanisms has focused on Plasmodium falciparum, while P. vivax remains poorly studied; nevertheless, P. vivax poses a major impediment to eradication. The molecular determinants of CQR and resistance to other antimalarials in P. vivax remain unclear, largely due to the lack of in vitro culture, precluding reverse genetics and robust drug assays. Several candidate drug transporter genes have been identified in P. vivax with polymorphism in sequence and expression level that could be associated with CQR and/or resistance to other antimalarials. These genes include pvmdr1, pvcrt-o and pvmrp1, orthologs of the Plasmodium falciparum pfmdr1, pfcrt and pfmrp1 genes, respectively, that are key determinants of antimalarial susceptibility in P. falciparum. Ex vivo P. vivax susceptibility to chloroquine is associated with specific P. vivax drug transporter polymorphisms in some but not all studies. It is not clear whether these discrepancies represent regional differences in parasite diversity and history of exposure to drugs; or result from technical differences, as no genetic validation has hitherto been possible. Here, we propose a comprehensive analysis of drug-resistance polymorphisms of P. vivax, utilizing two novel approaches leveraging the zoonotic macaque parasite Plasmodium knowlesi, which is much closer to P. vivax phylogenetically than is P. falciparum; possesses superior in vitro genetics with higher transfection efficiencies; and importantly, permits robust determinations of antimalarial drug susceptibility. We aim to utilize both cutting-edge evolutionary genomic and experimental forward genetic screening to identify putative determinants of P. vivax drug-resistance. We will subsequently use in vitro reverse genetic methods in P. knowlesi to functionally assess the importance of prioritized P. vivax genetic polymorphisms in mediating antimalarial drug-resistance. We hypothesize that these polymorphisms, either singly or in combination, have been selected in specific regions of the world by antimalarial use against P. vivax. The identification of specific polymorphisms that mediate different levels of susceptibility to chloroquine, and other antimalarial compounds in current clinical use, including artemisinin and its partner drugs, will be key to their use in surveillance, molecular epidemiological studies, and the design of strategies to prolong the usefulness of these drugs.