Transmissibility of clinically relevant atovaquone-resistant Plasmodium falciparum by anopheline mosquitoes

Rising numbers of malaria cases and deaths underscore the need for new interventions. Long-acting injectable medications, such as those now in use for HIV prophylaxis, offer the prospect of a malaria “chemical vaccine”, combining the efficacy of a drug (like atovaquone) with the durability of a biological vaccine. Of concern, however, is the possible selection and transmission of drug-resistant parasites. We addressed this question by generating clinically relevant, highly atovaquone-resistant, Plasmodium falciparum mutants competent to infect mosquitoes. Isogenic paired strains, that differ only by a single Y268S mutation in cytochrome b, were evaluated in parallel in southeast Asian ( Anopheles stephensi ) or African ( Anopheles gambiae ) mosquitoes, and thence in humanized mice. Fitness costs of the mutation were evident along the lifecycle, in asexual parasite growth in vitro and in a progressive loss of parasites in the mosquito. In numerous independent experiments, microscopic exam of salivary glands from hundreds of mosquitoes failed to detect even one Y268S sporozoite, a defect not rescued by coinfection with wild type parasites. Furthermore, despite uniformly successful transmission of wild type parasites from An. stephensi to FRG NOD huHep mice bearing human hepatocytes and erythrocytes, multiple attempts with Y268S-fed mosquitoes failed: there was no evidence of parasites in mouse tissues by microscopy, in vitro culture, or PCR. These studies confirm a severe-to-lethal fitness cost of clinically relevant atovaquone-resistant P. falciparum in the mosquito, and they significantly lessen the likelihood of their transmission in the field. Significance New tools are needed to protect individuals from malaria and to control malaria in the field. Atovaquone plus proguanil is a commonly used and well-tolerated medicine to prevent malaria. No drug resistance has been reported from its prophylactic use, but tablets must be taken daily. Giving atovaquone as a single injection may provide much longer-lasting protection, against both falciparum and vivax malaria, but there is concern this may create drug resistance. In this study we showed that clinically relevant atovaquone-resistant malaria parasites survive poorly, if at all, in mosquitoes, and that mosquitoes do not transmit drug-resistant parasites to humanized mice. These findings lessen the likelihood that an atovaquone “chemical vaccine” would lead to the spread of atovaquone resistance. ### Competing Interest Statement Andrew Owen: Director of Tandem Nano Ltd; personal fees from Gilead and Assembly Biosciences; co-investigator on funding received by the University of Liverpool from ViiV Healthcare and Gilead Sciences. David Sullivan: Founder and Board Member AliquantumRx (macrolides for malaria); expert legal consultant on malaria cases (Mabery Firm); stock ownership or options AliquantumRx; US patents 7,270,948, 9,568,471, 9,642,865; royalties from Binax Inc/D/B/A Inverness Medical for plasmids for HRP aldolase for malaria diagnostic test; NIH R21TR001737 Quantum model repurposing of cethromycin for liver stage malaria. Abhai Tripathi, Godfree Mlambo, Rahul Bakshi, Andrew Owen, Theresa Shapiro are co-inventors of atovaquone long-acting injectable formulation PCT/GB2017/051746.