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Multidrug-resistant Plasmodium falciparum parasites have emerged in Cambodia and neighboring countries in Southeast Asia, compromising the efficacy of first-line antimalarial combinations. Dihydroartemisinin + piperaquine (PPQ) treatment failure rates have risen to as high as 50% in some areas in this region. For PPQ, resistance is driven primarily by a series of mutant alleles of the P. falciparum chloroquine resistance transporter (PfCRT). PPQ resistance was reported in China three decades earlier, but the molecular driver remained unknown. Herein, we identify a PPQ-resistant pfcrt allele (China C) from Yunnan Province, China, whose genotypic lineage is distinct from the PPQ-resistant pfcrt alleles currently observed in Cambodia. Combining gene editing and competitive growth assays, we report that PfCRT China C confers moderate PPQ resistance while re-sensitizing parasites to chloroquine (CQ) and incurring a fitness cost that manifests as a reduced rate of parasite growth. PPQ transport assays using purified PfCRT isoforms, combined with molecular dynamics simulations, highlight differences in drug transport kinetics and in this transporter’s central cavity conformation between China C and the current Southeast Asian PPQ-resistant isoforms. We also report a novel computational model that incorporates empirically determined fitness landscapes at varying drug concentrations, combined with antimalarial susceptibility profiles, mutation rates, and drug pharmacokinetics. Our simulations with PPQ-resistant or -sensitive parasite lines predict that a three-day regimen of PPQ combined with CQ can effectively clear infections and prevent the evolution of PfCRT variants. This work suggests that including CQ in combination therapies could be effective in suppressing the evolution of PfCRT-mediated multidrug resistance in regions where PPQ has lost efficacy.

PfSPZ-CVac combines ‘PfSPZ Challenge’, which consists of infectious Plasmodium falciparum sporozoites (PfSPZ), with concurrent antimalarial chemoprophylaxis. In a previously-published PfSPZ-CVac study, three doses of 5.12x104 PfSPZ-CVac given 28 days apart had 100% vaccine efficacy (VE) against controlled human malaria infection (CHMI) 10 weeks after the last immunization, while the same dose given as three injections five days apart had 63% VE. Here, we conducted a dose escalation trial of similarly condensed schedules. Of the groups proceeding to CHMI, the first study group received three direct venous inoculations (DVIs) of a dose of 5.12x104 PfSPZ-CVac seven days apart and the next full dose group received three DVIs of a higher dose of 1.024x105 PfSPZ-CVac five days apart. CHMI (3.2x103 PfSPZ Challenge) was performed by DVI 10 weeks after the last vaccination. In both CHMI groups, transient parasitemia occurred starting seven days after each vaccination. For the seven-day interval group, the second and third vaccinations were therefore administered coincident with parasitemia from the prior vaccination. Parasitemia was associated with systemic symptoms which were severe in 25% of subjects. VE in the seven-day group was 0% (7/7 infected) and in the higher-dose, five-day group was 75% (2/8 infected). Thus, the same dose of PfSPZ-CVac previously associated with 63% VE when given on a five-day schedule in the prior study had zero VE here when given on a seven-day schedule, while a double dose given on a five-day schedule here achieved 75% VE. The relative contributions of the five-day schedule and/or the higher dose to improved VE warrant further investigation. It is notable that administration of PfSPZ-CVac on a schedule where vaccine administration coincided with blood-stage parasitemia was associated with an absence of sterile protective immunity. Clinical trials registration: NCT02773979., Author summary The world needs a protective malaria vaccine. One approach is to repeatedly administer whole sporozoites, the parasite form that is transmitted from mosquitos to humans. Without treatment, sporozoites enter the liver, grow for a week, and then infect red blood cells, causing clinical disease. Here, we gave a vaccine consisting of sporozoites with a drug that prevents red blood cell infections to eliminate clinical illness. This approach was protective in other studies so we initially evaluated a faster schedule where the vaccine was given weekly. Surprisingly, there was no protection observed. We determined that weekly intervals led the second and third vaccine doses to be administered just as the previous dose of sporozoites was transitioning from the liver to the blood stage. Even though blood stage infection was stopped in this study by the co-administered drug (chloroquine), we hypothesized that it was problematic to administer a vaccine during blood stage infection. Therefore, we gave the vaccinations every five days so that upon the second and third doses, there were no blood-stage parasites present. With five-day spacing and a higher dose, the vaccine protected 75% of participants. These findings suggest that blood-stage infections may hinder formation of protective responses to malaria.

Immunity against malaria depends on germinal center (GC)-derived antibody responses that are orchestrated by T follicular helper (TFH) cells. Emerging data show that the regulatory cytokine IL-10 plays an essential role in promoting GC B cell responses during both experimental malaria and virus infections. Here we investigated the cellular source and temporal role of IL-10, and whether IL-10 additionally signals to CD4 T-cells to support anti-Plasmodium humoral immunity. Distinct from reports of virus infection, we found that IL-10 was expressed by conventional, Foxp3-negative effector CD4 T cells and functioned in a B cell-intrinsic manner only during the first 96 hours of Plasmodium infection to support humoral immunity. The critical functions of IL-10 manifested only before the orchestration of GC responses and were primarily localized outside of B cell follicles. Mechanistically, our studies showed that the rapid and transient provision of IL-10 promoted B cell expression of anti-apoptotic factors, MHC class II, CD83, and cell-cell adhesion proteins that are essential for B cell survival and interaction with CD4 T cells. Together, our data reveal temporal features and mechanisms by which IL-10 critically supports humoral immunity during blood-stage Plasmodium infection, information that may be useful for developing new strategies designed to lessen the burden of malaria., Author summary Malaria is associated with the expression of both inflammatory and anti-inflammatory cytokines that together regulate disease severity and the function of parasite-specific immune cells subsets. Emerging data show that the anti-inflammatory cytokine Interleukin (IL)-10 can function to sustain an already established humoral immune response during acute and chronic virus infections. Unexpectedly, our results show that IL-10 functions only within the first 72–96 hours of Plasmodium blood-stage infection to promote initial helper T cell and B cell interactions, as well as B cell differentiation and survival. Our studies of the temporal features, spatial relationships, critical cellular sources and mechanisms by which IL-10 promotes humoral immunity during malaria highlight the distinct mechanisms governing anti-malarial immunity. This new information may lead to the identification of novel pathways and approaches for eliciting durable protection against malaria.

Plasmodium parasites are the causative agents of malaria, a disease with wide public health repercussions. Increasing drug resistance and the absence of a vaccine make finding new chemotherapeutic strategies imperative. Components of the ubiquitin and ubiquitin-like pathways have garnered increased attention as novel targets given their necessity to parasite survival. Understanding how these pathways are regulated in Plasmodium and identifying differences to the host is paramount to selectively interfering with parasites. Here, we focus on Nedd8 modification in Plasmodium falciparum, given its central role to cell division and DNA repair, processes critical to Plasmodium parasites given their unusual cell cycle and requirement for refined repair mechanisms. By applying a functional chemical approach, we show that deNeddylation is controlled by a different set of enzymes in the parasite versus the human host. We elucidate the molecular determinants of the unusual dual ubiquitin/Nedd8 recognition by the essential PfUCH37 enzyme and, through parasite transgenics and drug assays, determine that only its ubiquitin activity is critical to parasite survival. Our experiments reveal interesting evolutionary differences in how neddylation is controlled in higher versus lower eukaryotes, and highlight the Nedd8 pathway as worthy of further exploration for therapeutic targeting in antimalarial drug design., Author summary Ubiquitin and ubiquitin-like post-translational modifications are evolutionarily conserved and involved in fundamental cellular processes essential to all eukaryotes. As such, enzymatic components of these pathways present attractive targets for therapeutic intervention for both chronic and communicable diseases. Nedd8 modification of cullin ubiquitin E3 ligases is critical to the viability of eukaryotic organisms and mediates cell cycle progression and DNA damage repair. Given the complex lifecycle and unusual replication mechanisms of the malaria parasite, one would expect neddylation to be of central importance to its survival, yet little is known about this pathway in Plasmodium. Here we present our findings on how Nedd8 removal is controlled in Plasmodium falciparum and how this pathway differs to that of its human host.

Ozonide antimalarials, OZ277 (arterolane) and OZ439 (artefenomel), are synthetic peroxide-based antimalarials with potent activity against the deadliest malaria parasite, Plasmodium falciparum. Here we used a “multi-omics” workflow, in combination with activity-based protein profiling (ABPP), to demonstrate that peroxide antimalarials initially target the haemoglobin (Hb) digestion pathway to kill malaria parasites. Time-dependent metabolomic profiling of ozonide-treated P. falciparum infected red blood cells revealed a rapid depletion of short Hb-derived peptides followed by subsequent alterations in lipid and nucleotide metabolism, while untargeted peptidomics showed accumulation of longer Hb-derived peptides. Quantitative proteomics and ABPP assays demonstrated that Hb-digesting proteases were increased in abundance and activity following treatment, respectively. Ozonide-induced depletion of short Hb-derived peptides was less extensive in a drug-treated K13-mutant artemisinin resistant parasite line (Cam3.IIR539T) than in the drug-treated isogenic sensitive strain (Cam3.IIrev), further confirming the association between ozonide activity and Hb catabolism. To demonstrate that compromised Hb catabolism may be a primary mechanism involved in ozonide antimalarial activity, we showed that parasites forced to rely solely on Hb digestion for amino acids became hypersensitive to short ozonide exposures. Quantitative proteomics analysis also revealed parasite proteins involved in translation and the ubiquitin-proteasome system were enriched following drug treatment, suggestive of the parasite engaging a stress response to mitigate ozonide-induced damage. Taken together, these data point to a mechanism of action involving initial impairment of Hb catabolism, and indicate that the parasite regulates protein turnover to manage ozonide-induced damage., Author summary The ozonides are a novel class of fully synthetic antimalarial drugs with potent activity against all parasite species that cause malaria, including the deadliest, Plasmodium falciparum. With the emergence of resistance to current frontline artemisinin-based antimalarials, new drugs are urgently needed and a clear understanding of their mechanism of action is essential so that they can be optimally deployed in the field. Here, we studied the biochemical effects of two ozonides, OZ277 (marketed in India in combination with piperaquine) and OZ439 (in Phase IIb clinical trials) in P. falciparum parasites using an untargeted multi-omics approach consisting of proteomics, peptidomics and time-dependent metabolomics, along with activity-based protease profiling. We found that the ozonides initially disrupt haemoglobin metabolism and that they likely engage the parasite proteostatic stress response. Furthermore, when the duration of ozonide exposure was extended beyond 3 hours to reflect clinically-relevant exposure periods, additional parasite biochemical pathways were perturbed. This comprehensive analysis provides new insight into the antimalarial mode of action of ozonides and provides new opportunities for interventions to enhance their antimalarial efficacy.

The emergence of artemisinin (ART) resistance in Plasmodium falciparum intra-erythrocytic parasites has led to increasing treatment failure rates with first-line ART-based combination therapies in Southeast Asia. Decreased parasite susceptibility is caused by K13 mutations, which are associated clinically with delayed parasite clearance in patients and in vitro with an enhanced ability of ring-stage parasites to survive brief exposure to the active ART metabolite dihydroartemisinin. Herein, we describe a panel of K13-specific monoclonal antibodies and gene-edited parasite lines co-expressing epitope-tagged versions of K13 in trans. By applying an analytical quantitative imaging pipeline, we localize K13 to the parasite endoplasmic reticulum, Rab-positive vesicles, and sites adjacent to cytostomes. These latter structures form at the parasite plasma membrane and traffic hemoglobin to the digestive vacuole wherein artemisinin-activating heme moieties are released. We also provide evidence of K13 partially localizing near the parasite mitochondria upon treatment with dihydroartemisinin. Immunoprecipitation data generated with K13-specific monoclonal antibodies identify multiple putative K13-associated proteins, including endoplasmic reticulum-resident molecules, mitochondrial proteins, and Rab GTPases, in both K13 mutant and wild-type isogenic lines. We also find that mutant K13-mediated resistance is reversed upon co-expression of wild-type or mutant K13. These data help define the biological properties of K13 and its role in mediating P. falciparum resistance to ART treatment., Author summary The development of drug resistance in Plasmodium falciparum parasites presents a significant impediment to the global fight against malaria. Partial resistance to artemisinin (ART), the core component of current first-line drugs, has swept across Southeast Asia. In P. falciparum-infected patients, ART-resistant parasites show slow rates of clearance following treatment with an ART derivative or ART-based combination therapy. Resistance to partner drugs has also emerged in Southeast Asia, leading to frequent treatment failures. Single amino acid mutations in the P. falciparum K13 protein constitute the primary genetic cause of ART resistance and predict an increased risk of treatment failure. By generating monoclonal antibodies, we have investigated the subcellular localization of K13 in dihydroartemisinin-treated or untreated parasites. Analytical microscopy data localize K13 to or near the endoplasmic reticulum and vesicles that mediate intracellular trafficking, including plasma membrane-associated cytostomes that import host hemoglobin into the parasite. Co-immunoprecipitation experiments with K13-specific monoclonal antibodies identified multiple proteins associated with the endoplasmic reticulum, vesicular trafficking, the cytosol, or the mitochondria, with no apparent differences between K13 mutant and wild-type parasites. We also observed that overexpression of mutant or wild-type K13 in K13 mutant parasites could restore susceptibility, supporting the hypothesis that K13 mutations cause loss of function.

The malaria-causing blood stage of Plasmodium falciparum requires extracellular pantothenate for proliferation. The parasite converts pantothenate into coenzyme A (CoA) via five enzymes, the first being a pantothenate kinase (PfPanK). Multiple antiplasmodial pantothenate analogues, including pantothenol and CJ-15,801, kill the parasite by targeting CoA biosynthesis/utilisation. Their mechanism of action, however, remains unknown. Here, we show that parasites pressured with pantothenol or CJ-15,801 become resistant to these analogues. Whole-genome sequencing revealed mutations in one of two putative PanK genes (Pfpank1) in each resistant line. These mutations significantly alter PfPanK activity, with two conferring a fitness cost, consistent with Pfpank1 coding for a functional PanK that is essential for normal growth. The mutants exhibit a different sensitivity profile to recently-described, potent, antiplasmodial pantothenate analogues, with one line being hypersensitive. We provide evidence consistent with different pantothenate analogue classes having different mechanisms of action: some inhibit CoA biosynthesis while others inhibit CoA-utilising enzymes., Author summary The coenzyme A (CoA) biosynthetic pathway is under investigation as a target for the development of drugs aimed at several infectious agents, including malaria parasites. To synthesise CoA, the parasite scavenges the essential precursor pantothenate (vitamin B5). Several pantothenate analogues possess potent (nM) activity against the parasite, but their exact mechanism of action is unknown. We have generated mutant parasites that are resistant or hypersensitive to various pantothenate analogues. These parasites harbour mutations in a gene we now show codes for the first enzyme in the CoA biosynthesis pathway. This enzyme is not the target of the analogues, but instead converts them into antimetabolites that, depending on the analogue, either inhibit a CoA biosynthesis enzyme or downstream CoA-utilising enzymes.

Due to their remarkable parasitocidal activity, artemisinins represent the key components of first-line therapies against Plasmodium falciparum malaria. However, the decline in efficacy of artemisinin-based drugs jeopardizes global efforts to control and ultimately eradicate the disease. To better understand the resistance phenotype, artemisinin-resistant parasite lines were derived from two clones of the 3D7 strain of P. falciparum using a selection regimen that mimics how parasites interact with the drug within patients. This long term in vitro selection induced profound stage-specific resistance to artemisinin and its relative compounds. Chemosensitivity and transcriptional profiling of artemisinin-resistant parasites indicate that enhanced adaptive responses against oxidative stress and protein damage are associated with decreased artemisinin susceptibility. This corroborates our previous findings implicating these cellular functions in artemisinin resistance in natural infections. Genomic characterization of the two derived parasite lines revealed a spectrum of sequence and copy number polymorphisms that could play a role in regulating artemisinin response, but did not include mutations in pfk13, the main marker of artemisinin resistance in Southeast Asia. Taken together, here we present a functional in vitro model of artemisinin resistance that is underlined by a new set of genetic polymorphisms as potential genetic markers., Author summary The emergence of artemisinin resistance within and beyond Southeast Asia is a looming threat that needs to be promptly addressed. With this in mind, we derived several artemisinin-resistant parasite lines in vitro in order to fully characterize the resistance phenotype at the cellular and molecular levels. In addition to reinforcing the role of stress responses in mediating artemisinin resistance, we also identified novel genetic alterations that could also be responsible for causing artemisinin resistance. Collectively, this work provides additional insight in relation to, and beyond the paradigm of pfk13-driven artemisinin resistance and artemisinin response in P. falciparum. Understanding the processes that govern the acquisition of artemisinin resistance could aid in the development of strategies to prevent and contain it.

Resistance against all available antimalarial drugs calls for novel compounds that hit unexploited targets in the parasite. Here, we show that the recently discovered Plasmodium falciparum lactate/proton symporter, PfFNT, is a valid druggable target, and describe a new class of fluoroalkyl vinylogous acids that potently block PfFNT and kill cultured parasites. The original compound, MMV007839, is derived from the malaria box collection of potent antimalarials with unknown targets and contains a unique internal prodrug principle that reversibly switches between a lipophilic transport form and a polar, substrate-analogous active form. Resistance selection of cultured P. falciparum parasites with sub-lethal concentrations of MMV007839 produced a single nucleotide exchange in the PfFNT gene; this, and functional characterization of the resulting PfFNT G107S validated PfFNT as a novel antimalarial target. From quantitative structure function relations we established the compound binding mode and the pharmacophore. The pharmacophore largely circumvents the resistance mutation and provides the basis for a medicinal chemistry program that targets lactate and proton transport as a new mode of antimalarial action., Author summary The fight against malaria, i.e. one of the three major infectious diseases and transmitted by mosquitos, is conducted at three levels: i. transmission control (by attacking the mosquito vector or biological processes of vector infection), ii. vaccination (by stimulating the immune system to produce antibodies against molecular parasite surface structures), and iii. antimalarial drugs (by developing and applying small molecules that interfere with vital biochemical pathways). Despite strong efforts on levels i. and ii., small molecule drugs remain an indispensible antimalarial means; however, emergence and spreading of resistant malaria parasites against all currently used drugs pose a growing threat to treatment success. Therefore, novel drug targets need to be identified and exploited. Here, we show that a recently discovered lactic acid transporter, PfFNT, is a novel valid drug target and we provide first compounds that potently block transport and kill malaria parasites. Lactic acid is the metabolic end product of the parasites’ energy generation metabolism and interfering with this biochemical pathway represents a new mode of action against malaria parasites.

Therapeutics with novel modes of action and a low risk of generating resistance are urgently needed to combat drug-resistant Plasmodium falciparum malaria. Here, we report that the peptide vinyl sulfones WLL-vs (WLL) and WLW-vs (WLW), highly selective covalent inhibitors of the P. falciparum proteasome, potently eliminate genetically diverse parasites, including K13-mutant, artemisinin-resistant lines, and are particularly active against ring-stage parasites. Selection studies reveal that parasites do not readily acquire resistance to WLL or WLW and that mutations in the β2, β5 or β6 subunits of the 20S proteasome core particle or in components of the 19S proteasome regulatory particle yield only hundred-fold decreases in susceptibility. We observed no cross-resistance between WLL and WLW. Moreover, most mutations that conferred a modest loss of parasite susceptibility to one inhibitor significantly increased sensitivity to the other. These inhibitors potently synergized multiple chemically diverse classes of antimalarial agents, implicating a shared disruption of proteostasis in their modes of action. These results underscore the potential of targeting the Plasmodium proteasome with covalent small molecule inhibitors as a means of combating multidrug-resistant malaria., Author summary The spread of artemisinin-resistant Plasmodium falciparum malaria across Southeast Asia creates an imperative to develop new treatment options with compounds that are not susceptible to existing mechanisms of antimalarial drug resistance. Recent work has identified the P. falciparum proteasome as a promising drug target. Here, we report potent antimalarial activity of highly selective vinyl sulfone-conjugated peptide proteasome inhibitors, including against artemisinin-resistant P. falciparum early ring-stage parasites that are traditionally difficult to treat. Unlike many advanced antimalarial candidates, these covalent proteasome inhibitors do not readily select for resistance. Selection studies with cultured parasites reveal infrequent and minor decreases in susceptibility resulting from point mutations in components of the 26S proteasome, which we model using cryo-electron microscopy-based structural data. No parasites were observed to be cross-resistant to both compounds; in fact, partial resistance to one compound often created hypersensitivity to the other. We also document potent synergy between these covalent proteasome inhibitors and multiple classes of antimalarial agents, including dihydroartemisinin, the clinical candidate OZ439, and the parasite transmission-blocking agent methylene blue. Proteasome inhibitors have significant promise as components of novel combination therapies to treat multidrug-resistant malaria.

A major cause of the paucity of new starting points for drug discovery is the lack of interaction between academia and industry. Much of the global resource in biology is present in universities, whereas the focus of medicinal chemistry is still largely within industry. Open source drug discovery, with sharing of information, is clearly a first step towards overcoming this gap. But the interface could especially be bridged through a scale-up of open sharing of physical compounds, which would accelerate the finding of new starting points for drug discovery. The Medicines for Malaria Venture Malaria Box is a collection of over 400 compounds representing families of structures identified in phenotypic screens of pharmaceutical and academic libraries against the Plasmodium falciparum malaria parasite. The set has now been distributed to almost 200 research groups globally in the last two years, with the only stipulation that information from the screens is deposited in the public domain. This paper reports for the first time on 236 screens that have been carried out against the Malaria Box and compares these results with 55 assays that were previously published, in a format that allows a meta-analysis of the combined dataset. The combined biochemical and cellular assays presented here suggest mechanisms of action for 135 (34%) of the compounds active in killing multiple life-cycle stages of the malaria parasite, including asexual blood, liver, gametocyte, gametes and insect ookinete stages. In addition, many compounds demonstrated activity against other pathogens, showing hits in assays with 16 protozoa, 7 helminths, 9 bacterial and mycobacterial species, the dengue fever mosquito vector, and the NCI60 human cancer cell line panel of 60 human tumor cell lines. Toxicological, pharmacokinetic and metabolic properties were collected on all the compounds, assisting in the selection of the most promising candidates for murine proof-of-concept experiments and medicinal chemistry programs. The data for all of these assays are presented and analyzed to show how outstanding leads for many indications can be selected. These results reveal the immense potential for translating the dispersed expertise in biological assays involving human pathogens into drug discovery starting points, by providing open access to new families of molecules, and emphasize how a small additional investment made to help acquire and distribute compounds, and sharing the data, can catalyze drug discovery for dozens of different indications. Another lesson is that when multiple screens from different groups are run on the same library, results can be integrated quickly to select the most valuable starting points for subsequent medicinal chemistry efforts., Author Summary Malaria leads to the loss of over 440,000 lives annually; accelerating research to discover new candidate drugs is a priority. Medicines for Malaria Venture (MMV) has distilled over 25,000 compounds that kill malaria parasites in vitro into a group of 400 representative compounds, called the "Malaria Box". These Malaria Box sets were distributed free-of-charge to research laboratories in 30 different countries that work on a wide variety of pathogens. Fifty-five groups compiled >290 assay results for this paper describing the many activities of the Malaria Box compounds. The collective results suggest a potential mechanism of action for over 130 compounds against malaria and illuminate the most promising compounds for further malaria drug development research. Excitingly some of these compounds also showed outstanding activity against other disease agents including fungi, bacteria, other single-cellular parasites, worms, and even human cancer cells. The results have ignited over 30 drug development programs for a variety of diseases. This open access effort was so successful that MMV has begun to distribute another set of compounds with initial activity against a wider range of infectious agents that are of public health concern, called the Pathogen Box, available now to scientific labs all over the world (www.PathogenBox.org).

Among the several new antimalarials discovered over the past decade are at least three clinical candidate drugs, each with a distinct chemical structure, that disrupt Na+ homeostasis resulting in a rapid increase in intracellular Na+ concentration ([Na+]i) within the erythrocytic stages of Plasmodium falciparum. At present, events triggered by Na+ influx that result in parasite demise are not well-understood. Here we report effects of two such drugs, a pyrazoleamide and a spiroindolone, on intraerythrocytic P. falciparum. Within minutes following the exposure to these drugs, the trophozoite stage parasite, which normally contains little cholesterol, was made permeant by cholesterol-dependent detergents, suggesting it acquired a substantial amount of the lipid. Consistently, the merozoite surface protein 1 and 2 (MSP1 and MSP2), glycosylphosphotidylinositol (GPI)-anchored proteins normally uniformly distributed in the parasite plasma membrane, coalesced into clusters. These alterations were not observed following drug treatment of P. falciparum parasites adapted to grow in a low [Na+] growth medium. Both cholesterol acquisition and MSP1 coalescence were reversible upon the removal of the drugs, implicating an active process of cholesterol exclusion from trophozoites that we hypothesize is inhibited by high [Na+]i. Electron microscopy of drug-treated trophozoites revealed substantial morphological changes normally seen at the later schizont stage including the appearance of partial inner membrane complexes, dense organelles that resemble “rhoptries” and apparent nuclear division. Together these results suggest that [Na+]i disruptor drugs by altering levels of cholesterol in the parasite, dysregulate trophozoite to schizont development and cause parasite demise., Author Summary Malaria remains a major public health challenge in the world, especially with the realization that parasites causing the disease are becoming resistant to currently used antimalarial drugs. There are new antimalarial drugs under development, and among these are 3 clinical candidate drugs that have the propensity to cause sodium leakage into parasites growing inside human red blood cells. We have investigated events that lead to parasite death when sodium concentration inside the parasite increases. Our findings suggest that the drug-treated parasite rapidly acquires cholesterol and clusters containing lipid-embedded proteins MSP1 and MSP2 form within the plasma membrane. Because these changes are reversible when the drugs are removed, we suggest that there is an active process that keeps cholesterol out of the parasite. We also observed massive morphological changes resembling premature steps of parasite division following drug treatment. The changes we describe appear to be a direct consequence of increased sodium level in the parasites. We hypothesize that sodium influx constitutes a normal signaling process in malaria parasites and that the new antimalarial drugs initiate this process prematurely, which results in the death of the parasites.

In this study the ‘Malaria Box’ chemical library comprising 400 compounds with antiplasmodial activity was screened for compounds that perturb the internal pH of the malaria parasite, Plasmodium falciparum. Fifteen compounds induced an acidification of the parasite cytosol. Two of these did so by inhibiting the parasite’s formate nitrite transporter (PfFNT), which mediates the H+-coupled efflux from the parasite of lactate generated by glycolysis. Both compounds were shown to inhibit lactate transport across the parasite plasma membrane, and the transport of lactate by PfFNT expressed in Xenopus laevis oocytes. PfFNT inhibition caused accumulation of lactate in parasitised erythrocytes, and swelling of both the parasite and parasitised erythrocyte. Long-term exposure of parasites to one of the inhibitors gave rise to resistant parasites with a mutant form of PfFNT that showed reduced inhibitor sensitivity. This study provides the first evidence that PfFNT is a druggable antimalarial target., Author Summary The emergence and spread of Plasmodium falciparum strains resistant to leading antimalarial drugs has intensified the need to discover and develop drugs that kill the parasite via new mechanisms. Here we screened compounds that are known to inhibit P. falciparum growth for their effects on the pH inside the parasite. We identified fifteen compounds that decrease the pH inside the parasite, and determined the mechanism by which two of these, MMV007839 and MMV000972, disrupt pH and kill the parasite. The two compounds were found to inhibit the P. falciparum formate nitrite transporter (PfFNT), a transport protein that is located on the parasite surface and that serves to remove the waste product lactic acid from the parasite. The compounds inhibited both the H+-coupled transport of lactate across the parasite plasma membrane and the transport of lactate by PfFNT expressed in Xenopus oocytes. In addition to disrupting pH, PfFNT inhibition led to a build-up of lactate in the parasite-infected red blood cell and the swelling of both the parasite and the infected red blood cell. Exposing parasites to MMV007839 over a prolonged time period gave rise to resistant parasites with a mutant form of PfFNT that was less sensitive to the compound. This study validates PfFNT as a novel antimalarial drug target.

The evolution of drug resistance, a key challenge for our ability to treat and control infections, depends on two processes: de-novo resistance mutations, and the selection for and spread of resistant mutants within a population. Understanding the factors influencing the rates of these two processes is essential for maximizing the useful lifespan of drugs and, therefore, effective disease control. For malaria parasites, artemisinin-based drugs are the frontline weapons in the fight against disease, but reports from the field of slower parasite clearance rates during drug treatment are generating concern that the useful lifespan of these drugs may be limited. Whether slower clearance rates represent true resistance, and how this provides a selective advantage for parasites is uncertain. Here, we show that Plasmodium chabaudi malaria parasites selected for resistance to artesunate (an artemisinin derivative) through a step-wise increase in drug dose evolved slower clearance rates extremely rapidly. In single infections, these slower clearance rates, similar to those seen in the field, provided fitness advantages to the parasite through increased overall density, recrudescence after treatment and increased transmission potential. In mixed infections, removal of susceptible parasites by drug treatment led to substantial increases in the densities and transmission potential of resistant parasites (competitive release). Our results demonstrate the double-edged sword for resistance management: in our initial selection experiments, no parasites survived aggressive chemotherapy, but after selection, the fitness advantage for resistant parasites was greatest at high drug doses. Aggressive treatment of mixed infections resulted in resistant parasites dominating the pool of gametocytes, without providing additional health benefits to hosts. Slower clearance rates can evolve rapidly and can provide a strong fitness advantage during drug treatment in both single and mixed strain infections., Author Summary The evolution of drug resistance is a major challenge facing medicine in the 21st century. In the case of malaria parasites, this is particularly apparent, as the introduction of each drug has been followed by the rapid development and spread of resistant parasites. Without a constant supply of new drugs to replace those that are no longer effective, it is important to understand the processes that lead to the selection and spread of resistance though a parasite population, so that the useful lifespan of current drugs can be maximized. Here, we use a rodent malaria model system to try to select for reduced susceptibility to the current frontline malaria drug, artemisinin. We then examine the growth and transmission potential of resistant parasites in single infections and in competition with susceptible parasites (mixed infections) in drug-treated hosts. We show that parasites selected for reduced susceptibility to drugs have increased fitness in both situations. Our results also indicate that the consequences of different treatment regimes on the rate of spread of resistance should be evaluated and taken into account during regime choice.

Southeast Asia is an epicenter of multidrug-resistant Plasmodium falciparum strains. Selective pressures on the subcontinent have recurrently produced several allelic variants of parasite drug resistance genes, including the P. falciparum chloroquine resistance transporter (pfcrt). Despite significant reductions in the deployment of the 4-aminoquinoline drug chloroquine (CQ), which selected for the mutant pfcrt alleles that halted CQ efficacy decades ago, the parasite pfcrt locus is continuously evolving. This is highlighted by the presence of a highly mutated allele, Cam734 pfcrt, which has acquired the singular ability to confer parasite CQ resistance without an associated fitness cost. Here, we used pfcrt-specific zinc-finger nucleases to genetically dissect this allele in the pathogenic setting of asexual blood-stage infection. Comparative analysis of drug resistance and growth profiles of recombinant parasites that express Cam734 or variants thereof, Dd2 (the most common Southeast Asian variant), or wild-type pfcrt, revealed previously unknown roles for PfCRT mutations in modulating parasite susceptibility to multiple antimalarial agents. These results were generated in the GC03 strain, used in multiple earlier pfcrt studies, and might differ in natural isolates harboring this allele. Results presented herein show that Cam734-mediated CQ resistance is dependent on the rare A144F mutation that has not been observed beyond Southeast Asia, and reveal distinct impacts of this and other Cam734-specific mutations on CQ resistance and parasite growth rates. Biochemical assays revealed a broad impact of mutant PfCRT isoforms on parasite metabolism, including nucleoside triphosphate levels, hemoglobin catabolism and disposition of heme, as well as digestive vacuole volume and pH. Results from our study provide new insights into the complex molecular basis and physiological impact of PfCRT-mediated antimalarial drug resistance, and inform ongoing efforts to characterize novel pfcrt alleles that can undermine the efficacy of first-line antimalarial drug regimens., Author Summary Point mutations in the Plasmodium falciparum chloroquine resistance transporter (PfCRT) earlier thwarted the clinical efficacy of chloroquine, the former gold standard, and constitute a major determinant of parasite susceptibility to antimalarial drugs. Recently, we reported that the highly mutated Cambodian PfCRT isoform Cam734 is fitness-neutral in terms of parasite growth, unlike other less fit isoforms such as Dd2 that are outcompeted by wild-type parasites in the absence of CQ pressure. Using pfcrt-specific zinc-finger nucleases to genetically dissect the Cam734 allele, we report that its unique constituent mutations directly contribute to CQ resistance and collectively offset fitness costs associated with intermediate mutational steps. We also report that these mutations can contribute to resistance or increased sensitivity to multiple first-line partner drugs. Using isogenic parasite lines, we provide evidence of changes in parasite metabolism associated with the Cam734 allele compared to Dd2. We also observe a close correlation between CQ inhibition of hemozoin formation and parasite growth, and provide evidence that Cam734 PfCRT can modulate drug potency depending on its membrane electrochemical gradient. Our data highlight the capacity of PfCRT to evolve new states of antimalarial drug resistance and to offset associated fitness costs through its impact on parasite physiology and hemoglobin catabolism.

Neurological impairments are frequently detected in children surviving cerebral malaria (CM), the most severe neurological complication of infection with Plasmodium falciparum. The pathophysiology and therapy of long lasting cognitive deficits in malaria patients after treatment of the parasitic disease is a critical area of investigation. In the present study we used several models of experimental malaria with differential features to investigate persistent cognitive damage after rescue treatment. Infection of C57BL/6 and Swiss (SW) mice with Plasmodium berghei ANKA (PbA) or a lethal strain of Plasmodium yoelii XL (PyXL), respectively, resulted in documented CM and sustained persistent cognitive damage detected by a battery of behavioral tests after cure of the acute parasitic disease with chloroquine therapy. Strikingly, cognitive impairment was still present 30 days after the initial infection. In contrast, BALB/c mice infected with PbA, C57BL6 infected with Plasmodium chabaudi chabaudi and SW infected with non lethal Plasmodium yoelii NXL (PyNXL) did not develop signs of CM, were cured of the acute parasitic infection by chloroquine, and showed no persistent cognitive impairment. Reactive oxygen species have been reported to mediate neurological injury in CM. Increased production of malondialdehyde (MDA) and conjugated dienes was detected in the brains of PbA-infected C57BL/6 mice with CM, indicating high oxidative stress. Treatment of PbA-infected C57BL/6 mice with additive antioxidants together with chloroquine at the first signs of CM prevented the development of persistent cognitive damage. These studies provide new insights into the natural history of cognitive dysfunction after rescue therapy for CM that may have clinical relevance, and may also be relevant to cerebral sequelae of sepsis and other disorders., Author Summary Cerebral malaria (CM) is a deadly consequence of Plasmodium falciparum infection. Severe neurologic deficits are frequent during CM. Although most resolve within 6 months, several retrospective studies have described high frequencies of long-lasting cognitive impairment after an episode of CM. We developed behavioral tests to identify cognitive impairment due to experimental CM. During infection with Plasmodium berghei ANKA (PbA), mice susceptible to CM (C57BL/6) developed long-lasting cognitive impairment in contextual and aversive memory. The same profile was seen in Swiss Webster mice infected with Plasmodium yoelii XL, a lethal strain that also induces neurological dysfunctions in susceptible mice strains, confirming that the cognitive dysfunction is closely associated to the development of CM. Reactive oxygen species are described as mediators of neurological and cognitive impairment associated to sepsis and Alzheimer's disease. Here we found enhanced production of malondialdeyde and conjugated dienes in brains of PbA-infected C57BL/6 mice, indicating oxidative stress. Antioxidant therapy with N-acetylcisteine and desferroxamine, as an additive to chloroquine, prevented the cognitive impairment, confirming the importance of oxidative stress in CM-associated cognitive sequellae. Administration of additive antioxidants may be a successful therapeutic strategy to control long-lasting consequences of CM and in other severe systemic inflammatory syndromes with neurological involvement.

Mutant forms of the Plasmodium falciparum transporter PfCRT constitute the key determinant of parasite resistance to chloroquine (CQ), the former first-line antimalarial, and are ubiquitous to infections that fail CQ treatment. However, treatment can often be successful in individuals harboring mutant pfcrt alleles, raising questions about the role of host immunity or pharmacokinetics vs. the parasite genetic background in contributing to treatment outcomes. To examine whether the parasite genetic background dictates the degree of mutant pfcrt-mediated CQ resistance, we replaced the wild type pfcrt allele in three CQ-sensitive strains with mutant pfcrt of the 7G8 allelic type prevalent in South America, the Oceanic region and India. Recombinant clones exhibited strain-dependent CQ responses that ranged from high-level resistance to an incremental shift that did not meet CQ resistance criteria. Nonetheless, even in the most susceptible clones, 7G8 mutant pfcrt enabled parasites to tolerate CQ pressure and recrudesce in vitro after treatment with high concentrations of CQ. 7G8 mutant pfcrt was found to significantly impact parasite responses to other antimalarials used in artemisinin-based combination therapies, in a strain-dependent manner. We also report clinical isolates from French Guiana that harbor mutant pfcrt, identical or related to the 7G8 haplotype, and manifest a CQ tolerance phenotype. One isolate, H209, harbored a novel PfCRT C350R mutation and demonstrated reduced quinine and artemisinin susceptibility. Our data: 1) suggest that high-level CQR is a complex biological process dependent on the presence of mutant pfcrt; 2) implicate a role for variant pfcrt alleles in modulating parasite susceptibility to other clinically important antimalarials; and 3) uncover the existence of a phenotype of CQ tolerance in some strains harboring mutant pfcrt., Author Summary Plasmodium falciparum resistance to the antimalarial drug chloroquine has been found to result primarily from point mutations in PfCRT, which provide a highly sensitive marker of in vivo treatment failure and in vitro resistance. Debate has nonetheless continued about the singular role of mutant PfCRT and the contribution of the parasite genetic background. To address this, we have generated recombinant P. falciparum lines expressing a mutant pfcrt allele, or the reference wild type allele, in three distinct chloroquine-sensitive strains. Their analysis reveals a spectrum of responses ranging from high-level resistance to a previously unrecognized tolerance phenotype. The latter is characterized by virtually unchanged chloroquine IC50 values, significantly elevated IC90 values, and the ability to recrudesce after exposure to drug concentrations that are lethal to chloroquine-sensitive parasites. This tolerance phenotype was also observed in an isolate from French Guiana, confirming its presence in malaria-endemic regions. Mutant PfCRT significantly affected parasite responses to other antimalarials, including ones used in artemisinin-based combination therapies, in a strain-dependent manner. Our data suggest that successful CQ treatment of drug-resistant parasites is dependent on both host immunity and the strain-dependent extent to which mutant pfcrt imparts resistance.

The Thailand-Cambodia border is the epicenter for drug-resistant falciparum malaria. Previous studies have shown that chloroquine (CQ) and pyrimethamine resistance originated in this region and eventually spread to other Asian countries and Africa. However, there is a dearth in understanding the origin and evolution of dhps alleles associated with sulfadoxine resistance. The present study was designed to reveal the origin(s) of sulfadoxine resistance in Cambodia and its evolutionary relationship to African and South American dhps alleles. We sequenced 234 Cambodian Plasmodium falciparum isolates for the dhps codons S436A/F, A437G, K540E, A581G and A613S/T implicated in sulfadoxine resistance. We also genotyped 10 microsatellite loci around dhps to determine the genetic backgrounds of various alleles and compared them with the backgrounds of alleles prevalent in Africa and South America. In addition to previously known highly-resistant triple mutant dhps alleles SGEGA and AGEAA (codons 436, 437, 540, 581, 613 are sequentially indicated), a large proportion of the isolates (19.3%) contained a 540N mutation in association with 437G/581G yielding a previously unreported triple mutant allele, SGNGA. Microsatellite data strongly suggest the strength of selection was greater on triple mutant dhps alleles followed by the double and single mutants. We provide evidence for at least three independent origins for the double mutants, one each for the SGKGA, AGKAA and SGEAA alleles. Our data suggest that the triple mutant allele SGEGA and the novel allele SGNGA have common origin on the SGKGA background, whereas the AGEAA triple mutant was derived from AGKAA on multiple, albeit limited, genetic backgrounds. The SGEAA did not share haplotypes with any of the triple mutants. Comparative analysis of the microsatellite haplotypes flanking dhps alleles from Cambodia, Kenya, Cameroon and Venezuela revealed an independent origin of sulfadoxine resistant alleles in each of these regions., Author Summary Widespread resistance to chloroquine (CQ) and sulfadoxine-pyrimethamine (SP), the two least expensive and widely available antimalarial drugs, has become a major global public health challenge. It is known that point mutations in Plasmodium falciparum crt, dhfr and dhps genes contribute to resistance to CQ, pyrimethamine and sulfadoxine, respectively. CQ and pyrimethamine resistance spread to Africa and Asia from a few founding mutant lineages originating from the Thailand-Cambodia border. Here, we define the origins of dhps alleles in Cambodia and their relationships to African and South American counterparts. Three different triple mutant alleles including a novel allele comprised of 437G, 540N, and 581G mutations (S436 G437N540G581A613) were found in Cambodia as opposed to a single triple mutant allele in South America and two common double mutant alleles in Africa. Microsatellite data suggest strong selection operating on triple mutant alleles as compared to double and single mutants in Cambodia. We report three major independent origins for the double mutants and at least two independent origins for the highly resistant triple mutant dhps alleles in Cambodia. We also show that the resistant dhps alleles in Africa and South America have distinct origins from Cambodia. These results suggest that the evolution and spread of sulfadoxine resistance is different from CQ and pyrimethamine resistance.

When malaria parasites infect host red blood cells (RBC) and proteolyze hemoglobin, a unique, albeit poorly understood parasite-specific mechanism, detoxifies released heme into hemozoin (Hz). Here, we report the identification and characterization of a novel Plasmodium Heme Detoxification Protein (HDP) that is extremely potent in converting heme into Hz. HDP is functionally conserved across Plasmodium genus and its gene locus could not be disrupted. Once expressed, the parasite utilizes a circuitous “Outbound–Inbound” trafficking route by initially secreting HDP into the cytosol of infected RBC. A subsequent endocytosis of host cytosol (and hemoglobin) delivers HDP to the food vacuole (FV), the site of Hz formation. As Hz formation is critical for survival, involvement of HDP in this process suggests that it could be a malaria drug target., Author Summary Each year, more than one million people, most of them children under the age of 5, succumb to malaria, a devastating disease caused by Plasmodium parasites. The parasite resides inside the red blood cells of the host, where, during its development, it proteolyzes vast amounts of host hemoglobin. This degradation also releases heme, which is extremely toxic to the parasite. To protect itself (from the toxic effects of heme), the parasite converts free heme into hemozoin. This parasite-specific mechanism is widely accepted as the weakest link in its lifecycle and is targeted by several of the currently available antimalarial drugs, which prevent hemozoin formation by binding to heme. However, due to an incomplete understanding of the parasite processes that lead to hemozoin formation, a drug that specifically targets the parasite factors responsible for hemozoin production has never been developed. Here we identify and characterize Heme Detoxification Protein, a unique Plasmodium protein, which we show as the potent producer of hemozoin. We demonstrate that this protein is highly conserved across the Plasmodium genus, is extremely efficient in producing hemozoin, and is delivered to the food vacuole, the site of hemozoin formation, via a unique trafficking route. We also demonstrate the critical nature of this protein and suggest that it could be targeted to develop novel antimalarial drugs.

Cerebral malaria (CM) is associated with a high mortality rate, and long-term neurocognitive impairment in approximately one third of survivors. Adjunctive therapies that modify the pathophysiological processes involved in CM may improve outcome over anti-malarial therapy alone. PPARγ agonists have been reported to have immunomodulatory effects in a variety of disease models. Here we report that adjunctive therapy with PPARγ agonists improved survival and long-term neurocognitive outcomes in the Plasmodium berghei ANKA experimental model of CM. Compared to anti-malarial therapy alone, PPARγ adjunctive therapy administered to mice at the onset of CM signs, was associated with reduced endothelial activation, and enhanced expression of the anti-oxidant enzymes SOD-1 and catalase and the neurotrophic factors brain derived neurotrophic factor (BDNF) and nerve growth factor (NGF) in the brains of infected mice. Two months following infection, mice that were treated with anti-malarials alone demonstrated cognitive dysfunction, while mice that received PPARγ adjunctive therapy were completely protected from neurocognitive impairment and from PbA-infection induced brain atrophy. In humans with P. falciparum malaria, PPARγ therapy was associated with reduced endothelial activation and with induction of neuroprotective pathways, such as BDNF. These findings provide insight into mechanisms conferring improved survival and preventing neurocognitive injury in CM, and support the evaluation of PPARγ agonists in human CM., Author Summary Cerebral malaria (CM) is a severe complication of Plasmodium falciparum infection that is associated with long-term neurocognitive impairment in about a third of survivors even when optimal anti-malarial therapy is used. Since both the parasite and the host immune response to infection play a role in the development of CM, adjunctive therapies that modulate the host response, given in conjunction with anti-parasitic therapy, may improve survival and prevent neurocognitive injury. Here we examine the effects of PPARγ agonists on neurocongitive injury using a mouse model of CM. We demonstrate that PPARγ agonists, when administered with anti-malarials, protected mice from developing brain atrophy and neurocognitive impairment. This was associated with induction of anti-oxidant and neuroprotective pathways in the brains of infected mice. We also observed the same neuroprotective pathways induced in patients with falciparum malaria that received PPARγ adjunctive therapy. Our findings suggest that PPARγ agonists may be valuable in the treatment and prevention of CM-induced neurocognitive injury, and support the testing of PPARγ agonists in patients with CM.

Genome sequences of Plasmodium falciparum allow for global analysis of drug responses to antimalarial agents. It was of interest to learn how DNA microarrays may be used to study drug action in malaria parasites. In one large, tightly controlled study involving 123 microarray hybridizations between cDNA from isogenic drug-sensitive and drug-resistant parasites, a lethal antifolate (WR99210) failed to over-produce RNA for the genetically proven principal target, dihydrofolate reductase-thymidylate synthase (DHFR-TS). This transcriptional rigidity carried over to metabolically related RNA encoding folate and pyrimidine biosynthesis, as well as to the rest of the parasite genome. No genes were reproducibly up-regulated by more than 2-fold until 24 h after initial drug exposure, even though clonal viability decreased by 50% within 6 h. We predicted and showed that while the parasites do not mount protective transcriptional responses to antifolates in real time, P. falciparum cells transfected with human DHFR gene, and adapted to long-term WR99210 exposure, adjusted the hard-wired transcriptome itself to thrive in the presence of the drug. A system-wide incapacity for changing RNA levels in response to specific metabolic perturbations may contribute to selective vulnerabilities of Plasmodium falciparum to lethal antimetabolites. In addition, such regulation affects how DNA microarrays are used to understand the mode of action of antimetabolites., Author Summary Traditional knowledge of gene regulation, learned largely from non-pathogenic model organisms such as E. coli, yeast, and mice, suggests that RNA for metabolic pathways are regulated in large part by DNA-binding transcriptional factors that are responsive to cellular metabolic needs. We demonstrate that the malaria-causing Plasmodium falciparum parasites, under lethal drug pressure from an antifolate with a known mechanism of action, are incapable of large reproducible changes in RNA levels for the target pathways, or for any other gene throughout the genome. Small RNA changes, possibly informative of perturbed pathways, can be detected in dying parasites. In addition, significant RNA changes are seen when the hard-wired program, governing RNA levels, itself is altered. Our data formally proves that RNA levels for intermediary metabolism in malaria parasites are largely predetermined. We propose that as a parasite with a complex life cycle travels from one largely predictable intracellular biochemical environment to another, such hard-wiring may be sufficient to manage transcript levels for intermediary metabolism without employing sensory functions. Such a system-wide host–parasite difference in gene regulation may create unexpected pharmacological opportunities when important target pathways are rigid in the parasite but dynamically regulated in host cells.

The success of passive immunization suggests that antibody-based therapies will be effective at controlling malaria. We describe the development of fully human antibodies specific for Plasmodium falciparum by antibody repertoire cloning from phage display libraries generated from immune Gambian adults. Although these novel reagents bind with strong affinity to malaria parasites, it remains unclear if in vitro assays are predictive of functional immunity in humans, due to the lack of suitable animal models permissive for P. falciparum. A potentially useful solution described herein allows the antimalarial efficacy of human antibodies to be determined using rodent malaria parasites transgenic for P. falciparum antigens in mice also transgenic for human Fc-receptors. These human IgG1s cured animals of an otherwise lethal malaria infection, and protection was crucially dependent on human FcγRI. This important finding documents the capacity of FcγRI to mediate potent antimalaria immunity and supports the development of FcγRI-directed therapy for human malaria., Author Summary Malaria rivals HIV and tuberculosis as the world's most deadly infection killing a child every 30 seconds. Antibodies and their receptors (Fc-receptors) have been shown to be vital for the development of protective immunity, and as such they act as correlates of protection in studies aimed at defining the best antigens to incorporate into current vaccines. Understanding antibody types and Fc-receptors that optimally induce immunity is therefore vital to developing the best vaccines. Surrogate markers of antibody efficacy currently rely on in vitro assays that are laborious and difficult to reproduce. It remains unclear if such in vitro assays are predictive of functional immunity in humans due to the lack of suitable animal models permissive for Plasmodium falciparum. Here, we create a transgenic in vivo mouse model that has significant advantage over the use of new world primates, the only other model for human malaria. We demonstrate that this model defines an Fc-dependent mechanism of parasite destruction that cannot be assessed in current in vitro assays. The model provides both a test for therapeutic antibody efficacy prior to clinical trials in humans and an important tool in malaria vaccine development.