Azithromycin disrupts apicoplast biogenesis in replicating and dormant liver stages of the relapsing malaria parasites Plasmodium vivax and Plasmodium cynomolgi.

The control and elimination of malaria caused by Plasmodium vivax is hampered by the threat of relapsed infection resulting from the activation of dormant hepatic hypnozoites. Currently, only the 8-aminoquinolines, primaquine and tafenoquine, have been approved for the elimination of hypnozoites, although their use is hampered by potential toxicity. Therefore, an alternative radical curative drug that safely eliminates hypnozoites is a pressing need. This study assessed the potential hypnozoiticidal activity of the antibiotic azithromycin, which is thought to exert antimalarial activity by inhibiting prokaryote-like ribosomal translation within the apicoplast, an indispensable organelle. The results show that azithromycin inhibited apicoplast development during liver-stage schizogony in P. vivax and Plasmodium cynomolgi, leading to impaired parasite maturation. More importantly, this study found that azithromycin is likely to impair the hypnozoite's apicoplast, resulting in the loss of this organelle. Subsequently, using a recently developed long-term hepatocyte culture system, this study found that this loss likely induces a delay in the hypnozoite activation rate, and that those parasites that do proceed to schizogony display liver-stage arrest prior to differentiating into hepatic merozoites, thus potentially preventing relapse. Overall, this work provides evidence for the potential use of azithromycin for the radical cure of relapsing malaria, and identifies apicoplast functions as potential drug targets in quiescent hypnozoites.

Novel thienopyrimidones targeting hepatic and erythrocytic stages of Plasmodium parasites with increased microsomal stability.

Based on the structure of a previously identified hit, Gamhepathiopine 1, which showed promising antiplasmodial activity, but poor microsomal stability, several strategies were investigated to improve the metabolic stability of the compounds. This included the introduction of fluorine or deuterium atoms, as well as carbocyclic groups. Among the new compounds, the 2-aminocyclobutyl derivative 5g demonstrated enhanced microsomal stability compared to compound 1, while retaining antiplasmodial activity against erythrocytic and hepatic stages of Plasmodium, without significant cytotoxicity against primary hepatocytes.

The claudin-like apicomplexan microneme protein is required for gliding motility and infectivity of Plasmodium sporozoites.

Invasion of host cells by apicomplexan parasites such as Toxoplasma and Plasmodium spp requires the sequential secretion of the parasite apical organelles, the micronemes and the rhoptries. The claudin-like apicomplexan microneme protein (CLAMP) is a conserved protein that plays an essential role during invasion by Toxoplasma gondii tachyzoites and in Plasmodium falciparum asexual blood stages. CLAMP is also expressed in Plasmodium sporozoites, the mosquito-transmitted forms of the malaria parasite, but its role in this stage is still unknown. CLAMP is essential for Plasmodium blood stage growth and is refractory to conventional gene deletion. To circumvent this obstacle and study the function of CLAMP in sporozoites, we used a conditional genome editing strategy based on the dimerisable Cre recombinase in the rodent malaria model parasite P. berghei. We successfully deleted clamp gene in P. berghei transmission stages and analyzed the functional consequences on sporozoite infectivity. In mosquitoes, sporozoite development and egress from oocysts was not affected in conditional mutants. However, invasion of the mosquito salivary glands was dramatically reduced upon deletion of clamp gene. In addition, CLAMP-deficient sporozoites were impaired in cell traversal and productive invasion of mammalian hepatocytes. This severe phenotype was associated with major defects in gliding motility and with reduced shedding of the sporozoite adhesin TRAP. Expansion microscopy revealed partial colocalization of CLAMP and TRAP in a subset of micronemes, and a distinct accumulation of CLAMP at the apical tip of sporozoites. Collectively, these results demonstrate that CLAMP is essential across invasive stages of the malaria parasite, and support a role of the protein upstream of host cell invasion, possibly by regulating the secretion or function of adhesins in Plasmodium sporozoites.

New antiplasmodial 4-amino-thieno[3,2-d]pyrimidines with improved intestinal permeability and microsomal stability.

The increasing number of Plasmodium falciparum strains resistant to current treatments justifies the urgent need to discover new compounds active on several stages of the parasite development. Based on the structure of Gamhepathiopine, a 2-tert-butylaminothieno[3,2-d]pyrimidin-4(3H)-one previously identified for its dual activity against the sexual and asexual stages of P. falciparum, 25 new 4-amino-substituted analogues were synthesized and evaluated on the erythrocytic and hepatic stages of Plasmodium. A promising compound, N2-(tert-butyl)-N [4]-(3-(dimethylamino)propyl)-6-(p-tolyl)thieno[3,2-d]pyrimidine-2,4-diamine, showed improved physicochemical properties, intestinal permeability (PAMPA model) and microsomal stability compared to Gamhepathiopine, while maintaining a good antiplasmodial activity on the erythrocytic stage of P. falciparum and on the hepatic stage of P. berghei.

Rapid and Specific Action of Methylene Blue against Plasmodium Transmission Stages.

Methylene blue (MB) is the oldest synthetic anti-infective. Its high potency against asexual and sexual stages of malaria parasites is well documented. This study aimed to investigate possible additional activities of MB in interfering with parasite transmission and determine target stages in Anopheles vectors and humans. MB's transmission-blocking activity was first evaluated by an ex vivo direct membrane feeding assay (DMFA) using Plasmodium falciparum field isolates. To investigate anti-mosquito stage activity, Plasmodium berghei-infected Anopheles stephensi mosquitoes were fed a second blood meal on mice that had been treated with methylene blue, 3, 6- and 15-days after the initial infectious blood meal. Anti-sporozoite and liver stage activities were evaluated in vitro and in vivo via sporozoite invasion and liver stage development assays, respectively. MB exhibited a robust inhibition of P. falciparum transmission in An. gambiae, even when added shortly before the DMFA but only a moderate effect against P. berghei oocyst development. Exposure of mature P. berghei and P. falciparum sporozoites to MB blocked hepatocyte invasion, yet P. berghei liver stage development was unaffected by MB. Our results indicate previously underappreciated rapid specific activities of methylene blue against Plasmodium transmission stages, preventing the establishment of both mosquito midgut and liver infections as the first essential steps in both hosts.

4-Substituted Thieno[3,2-d]pyrimidines as Dual-Stage Antiplasmodial Derivatives.

Malaria remains one of the major health problems worldwide. The increasing resistance of Plasmodium to approved antimalarial drugs requires the development of novel antiplasmodial agents that can effectively prevent and/or treat this disease. Based on the structure of Gamhepathiopine, a 2-tert-butylaminothieno[3,2-d]pyrimidin-4(3H)-one hit, active on the sexual and asexual stages of the parasite and thanked for the introduction of various substituents at position 4 of the thienopyrimidine core by nucleophilic aromatic substitution and pallado-catalyzed coupling reactions, a series of 4-substituted thieno[3,2-d]pyrimidines were identified as displaying in vitro activities against both the erythrocytic stage of P. falciparum and the hepatic stage of P. berghei. Among the 28 compounds evaluated, the chloro analogue of Gamhepathiopine showed good activity against the erythrocytic stage of P. falciparum, moderate toxicity on HepG2, and better activity against hepatic P. berghei parasites, compared to Gamhepathiopine.

Synthesis of antiplasmodial 2-aminothieno[3,2-d]pyrimidin-4(3H)-one analogues using the scaffold hopping strategy.

Gamhepathiopine (also known as M1), is a multi-stage acting antiplasmodial 2-tert-butylaminothieno[3,2-d]pyrimidin-4(3H)-one hydrochloride that was first described in 2015. The development of this compound is limited by poor microsomal stability, insufficient aqueous solubility and low intestinal permeability. In order to obtain new optimized derivatives, we conducted a scaffold hopping strategy from compound M1, resulting in the synthesis of 20 new compounds belonging to six chemical series. All the compounds were tested on the K1 multi-resistant strain of Plasmodium falciparum and the human HepG2 cell-line, to evaluate their antiplasmodial activity and their cytotoxicity. Analogues' biological results also highlighted the mandatory presence of a heteroatom at position 5 of the thieno[3,2-d]pyrimidin-4(3H)-one moeity for the antiplasmodial activity. However, modifications at position 7 were detrimental for the antiplasmodial activity. We identified furane bioisostere 3j as a promising candidate, showing good blood stage antiplasmodial activity, better water solubility and highly improved intestinal permeability in the PAMPA assay.

The Phosphodiesterase Inhibitor Tadalafil Promotes Splenic Retention of Plasmodium falciparum Gametocytes in Humanized Mice.

The persistence of erythrocytes infected with Plasmodium falciparum gametocytes in the bloodstream is closely related to the modulation of their mechanical properties. New drugs that increase the stiffness of infected erythrocytes may thus represent a novel approach to block malaria parasite transmission. The phosphodiesterase inhibitor tadalafil has been shown to impair the ability of infected erythrocytes to circulate in an in vitro model for splenic retention. Here, we used a humanized mouse model to address in vivo the effect of tadalafil on the circulation kinetics of mature gametocyte-infected erythrocytes. We show that stiff immature gametocyte-infected erythrocytes are retained in the spleen of humanized mice at rates comparable to that of the in vitro model. Accordingly, tadalafil-induced stiffening of mature gametocyte-infected erythrocytes impairs their circulation in the bloodstream and triggers their retention by the spleen. These in vivo results validate that tadalafil is a novel drug lead potentially capable of blocking malaria parasite transmission by targeting GIE mechanical properties.

Potent Antiplasmodial Derivatives of Dextromethorphan Reveal the Ent-Morphinan Pharmacophore of Tazopsine-Type Alkaloids.

The alkaloid tazopsine 1 was introduced in the late 2000s as a novel antiplasmodial hit compound active against Plasmodium falciparum hepatic stages, with the potential to develop prophylactic drugs based on this novel chemical scaffold. However, the structural determinants of tazopsine 1 bioactivity, together with the exact definition of the pharmacophore, remained elusive, impeding further development. We found that the antitussive drug dextromethorphan (DXM) 3, although lacking the complex pattern of stereospecific functionalization of the natural hit, was harboring significant antiplasmodial activity in vitro despite suboptimal prophylactic activity in a murine model of malaria, precluding its direct repurposing against the disease. The targeted N-alkylation of nor-DXM 15 produced a small library of analogues with greatly improved activity over DXM 3 against P. falciparum asexual stages. Amongst these, N-2'-pyrrolylmethyl-nor-DXM 16i showed a 2- to 36-fold superior inhibitory potency compared to tazopsine 1 and DXM 3 against P. falciparum liver and blood stages, with respectively 760 ± 130 nM and 2.1 ± 0.4 µM IC50 values, as well as liver/blood phase selectivity of 2.8. Furthermore, cpd. 16i showed a 5- to 8-fold increase in activity relative to DXM 3 against P. falciparum stages I-II and V gametocytes, with 18.5 µM and 13.2 µM IC50 values, respectively. Cpd. 16i can thus be considered a promising novel hit compound against malaria in the ent-morphinan series with putative pan cycle activity, paving the way for further therapeutic development (e.g., investigation of its prophylactic activity in vivo).

Artemisinin-independent inhibitory activity of Artemisia sp. infusions against different Plasmodium stages including relapse-causing hypnozoites.

Artemisinin-based combination therapies (ACT) are the frontline treatments against malaria worldwide. Recently the use of traditional infusions from Artemisia annua (from which artemisinin is obtained) or Artemisia afra (lacking artemisinin) has been controversially advocated. Such unregulated plant-based remedies are strongly discouraged as they might constitute sub-optimal therapies and promote drug resistance. Here, we conducted the first comparative study of the anti-malarial effects of both plant infusions in vitro against the asexual erythrocytic stages of Plasmodium falciparum and the pre-erythrocytic (i.e., liver) stages of various Plasmodium species. Low concentrations of either infusion accounted for significant inhibitory activities across every parasite species and stage studied. We show that these antiplasmodial effects were essentially artemisinin-independent and were additionally monitored by observations of the parasite apicoplast and mitochondrion. In particular, the infusions significantly incapacitated sporozoites, and for Plasmodium vivax and P. cynomolgi, disrupted the hypnozoites. This provides the first indication that compounds other than 8-aminoquinolines could be effective antimalarials against relapsing parasites. These observations advocate for further screening to uncover urgently needed novel antimalarial lead compounds.

A New Thienopyrimidinone Chemotype Shows Multistage Activity against Plasmodium falciparum, Including Artemisinin-Resistant Parasites.

Human malaria infection begins with a one-time asymptomatic liver stage followed by a cyclic symptomatic blood stage. For decades, the research for novel antimalarials focused on the high-throughput screening of molecules that only targeted the asexual blood stages. In a search for new effective compounds presenting a triple action against erythrocytic and liver stages in addition to the ability to block the transmission of the disease via the mosquito vector, 2-amino-thienopyrimidinone derivatives were synthesized and tested for their antimalarial activity. One molecule, named gamhepathiopine (denoted as "M1" herein), was active at submicromolar concentrations against both erythrocytic (50% effective concentration [EC50] = 0.045 µM) and liver (EC50 = 0.45 µM) forms of Plasmodium falciparum. Furthermore, gamhepathiopine efficiently blocked the development of the sporogonic cycle in the mosquito vector by inhibiting the exflagellation step. Moreover, M1 was active against artemisinin-resistant forms (EC50 = 0.227 µM), especially at the quiescent stage. Nevertheless, in mice, M1 showed modest activity due to its rapid metabolization by P450 cytochromes into inactive derivatives, calling for the development of new parent compounds with improved metabolic stability and longer half-lives. These results highlight the thienopyrimidinone scaffold as a novel antiplasmodial chemotype of great interest to search for new drug candidates displaying multistage activity and an original mechanism of action with the potential to be used in combination therapies for malaria elimination in the context of artemisinin resistance. IMPORTANCE This work reports a new chemical structure that (i) displays activity against the human malaria parasite Plasmodium falciparum at 3 stages of the parasitic cycle (blood stage, hepatic stage, and sexual stages), (ii) remains active against parasites that are resistant to the first-line treatment recommended by the World Health Organization (WHO) for the treatment of severe malaria (artemisinins), and (iii) reduces transmission of the parasite to the mosquito vector in a mouse model. This new molecule family could open the way to the conception of novel antimalarial drugs with an original multistage mechanism of action to fight against Plasmodium drug resistance and block interhuman transmission of malaria.

Preliminary studies on drug delivery of polymeric primaquine microparticles using the liver high uptake effect based on size of particles to improve malaria treatment.

Malaria is the most common parasitic disease around the world, especially in tropical and sub-tropical regions. This parasitic disease can have a rapid and severe evolution. It is transmitted by female anopheline mosquitoes. There is no reliable vaccine or diagnostic test against malaria; instead, Artesunate is used for the treatment of severe malaria and Artemisinin is used for uncomplicated falciparum malaria. However, these treatments are not efficient against severe malaria and improvements are needed. Primaquine (PQ) is one of the most widely used antimalarial drugs. It is the only available drug to date for combating the relapsing form of malaria. Nevertheless, it has severe side effects. Particle drug-delivery systems present the ability to enhance the therapeutic properties of drugs and decrease their side effects. Here, we report the development of Polymeric Primaquine Microparticles (PPM) labeled with 99mTc for therapeutic strategy against malaria infection. The amount of primaquine encapsulated into the PPM was 79.54%. PPM presented a mean size of 929.47 ± 37.72 nm, with a PDI of 0.228 ± 0.05 showing a homogeneous size for the microparticles and a monodispersive behavior. Furthermore, the biodistribution test showed that primaquine microparticles have a high liver accumulation. In vivo experiments using mice show that the PPM treatments resulted in partial efficacy and protection against the development of the parasite compared to free Primaquine. These results suggest that microparticles drug delivery systems of primaquine could be a possible approach for malaria prevention and treatment.

2-Phenoxy-3-Trichloromethylquinoxalines Are Antiplasmodial Derivatives with Activity against the Apicoplast of Plasmodium falciparum.

The malaria parasite harbors a relict plastid called the apicoplast. Although not photosynthetic, the apicoplast retains unusual, non-mammalian metabolic pathways that are essential to the parasite, opening up a new perspective for the development of novel antimalarials which display a new mechanism of action. Based on the previous antiplasmodial hit-molecules identified in the 2-trichloromethylquinoxaline series, we report herein a structure-activity relationship (SAR) study at position two of the quinoxaline ring by synthesizing 20 new compounds. The biological evaluation highlighted a hit compound (3i) with a potent PfK1 EC50 value of 0.2 µM and a HepG2 CC50 value of 32 µM (Selectivity index = 160). Nitro-containing (3i) was not genotoxic, both in the Ames test and in vitro comet assay. Activity cliffs were observed when the 2-CCl3 group was replaced, showing that it played a key role in the antiplasmodial activity. Investigation of the mechanism of action showed that 3i presents a drug response by targeting the apicoplast and a quick-killing mechanism acting on another target site.

The Host Protein Aquaporin-9 is Required for Efficient Plasmodium falciparum Sporozoite Entry into Human Hepatocytes.

Hepatocyte invasion by Plasmodium sporozoites represents a promising target for innovative antimalarial therapy, but the molecular events mediating this process are still largely uncharacterized. We previously showed that Plasmodium falciparum sporozoite entry into hepatocytes strictly requires CD81. However, CD81-overexpressing human hepatoma cells remain refractory to P. falciparum infection, suggesting the existence of additional host factors necessary for sporozoite entry. Here, through differential transcriptomic analysis of human hepatocytes and hepatoma HepG2-CD81 cells, the transmembrane protein Aquaporin-9 (AQP9) was found to be among the most downregulated genes in hepatoma cells. RNA silencing showed that sporozoite invasion of hepatocytes requires AQP9 expression. AQP9 overexpression in hepatocytes increased their permissiveness to P. falciparum. Moreover, chemical disruption with the AQP9 inhibitor phloretin markedly inhibited hepatocyte infection. Our findings identify AQP9 as a novel host factor required for P. falciparum sporozoite hepatocyte-entry and indicate that AQP9 could be a potential therapeutic target.

Chloroquine Potentiates Primaquine Activity against Active and Latent Hepatic Plasmodia Ex Vivo: Potentials and Pitfalls.

For a long while, 8-aminoquinoline compounds have been the only therapeutic agents against latent hepatic malaria parasites. These have poor activity against the blood-stage plasmodia causing acute malaria and must be used in conjunction with partner blood schizontocidal agents. We examined the impacts of one such agent, chloroquine, upon the activity of primaquine, an 8-aminoquinoline, against hepatic stages of Plasmodium cynomolgi, Plasmodium yoelii, Plasmodium berghei, and Plasmodium falciparum within several ex vivo systems-primary hepatocytes of Macaca fascicularis, primary human hepatocytes, and stably transformed human hepatocarcinoma cell line HepG2. Primaquine exposures to formed hepatic schizonts and hypnozoites of P. cynomolgi in primary simian hepatocytes exhibited similar 50% inhibitory concentration (IC50) values near 0.4 µM, whereas chloroquine in the same system exhibited no inhibitory activities. Combining chloroquine and primaquine in this system decreased the observed primaquine IC50 for all parasite forms in a chloroquine dose-dependent manner by an average of 18-fold. Chloroquine also decreased the primaquine IC50 against hepatic P. falciparum in primary human hepatocytes, P. berghei in simian primary hepatocytes, and P. yoelii in primary human hepatocytes. Chloroquine had no impact on primaquine IC50 against P. yoelii in HepG2 cells and, likewise, had no impact on the IC50 of atovaquone (hepatic schizontocide) against P. falciparum in human hepatocytes. We describe important sources of variability in the potentiation of primaquine activity by chloroquine in these systems. Chloroquine potentiated primaquine activity against hepatic forms of several plasmodia. We conclude that chloroquine specifically potentiated 8-aminoquinoline activities against active and dormant hepatic-stage plasmodia in normal primary hepatocytes but not in a hepatocarcinoma cell line.

Prediction of malaria transmission drivers in Anopheles mosquitoes using artificial intelligence coupled to MALDI-TOF mass spectrometry.

Vector control programmes are a strategic priority in the fight against malaria. However, vector control interventions require rigorous monitoring. Entomological tools for characterizing malaria transmission drivers are limited and are difficult to establish in the field. To predict Anopheles drivers of malaria transmission, such as mosquito age, blood feeding and Plasmodium infection, we evaluated artificial neural networks (ANNs) coupled to matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry (MS) and analysed the impact on the proteome of laboratory-reared Anopheles stephensi mosquitoes. ANNs were sensitive to Anopheles proteome changes and specifically recognized spectral patterns associated with mosquito age (0-10 days, 11-20 days and 21-28 days), blood feeding and P. berghei infection, with best prediction accuracies of 73%, 89% and 78%, respectively. This study illustrates that MALDI-TOF MS coupled to ANNs can be used to predict entomological drivers of malaria transmission, providing potential new tools for vector control. Future studies must assess the field validity of this new approach in wild-caught adult Anopheles. A similar approach could be envisaged for the identification of blood meal source and the detection of insecticide resistance in Anopheles and to other arthropods and pathogens.

Antigens reversibly conjugated to a polymeric glyco-adjuvant induce protective humoral and cellular immunity.

Fully effective vaccines for complex infections must elicit a diverse repertoire of antibodies (humoral immunity) and CD8+ T-cell responses (cellular immunity). Here, we present a synthetic glyco-adjuvant named p(Man-TLR7), which, when conjugated to antigens, elicits robust humoral and cellular immunity. p(Man-TLR7) is a random copolymer composed of monomers that either target dendritic cells (DCs) via mannose-binding receptors or activate DCs via Toll-like receptor 7 (TLR7). Protein antigens are conjugated to p(Man-TLR7) via a self-immolative linkage that releases chemically unmodified antigen after endocytosis, thus amplifying antigen presentation to T cells. Studies with ovalbumin (OVA)-p(Man-TLR7) conjugates demonstrate that OVA-p(Man-TLR7) generates greater humoral and cellular immunity than OVA conjugated to polymers lacking either mannose targeting or TLR7 ligand. We show significant enhancement of Plasmodium falciparum-derived circumsporozoite protein (CSP)-specific T-cell responses, expansion in the breadth of the αCSP IgG response and increased inhibition of sporozoite invasion into hepatocytes with CSP-p(Man-TLR7) when compared with CSP formulated with MPLA/QS-21-loaded liposomes-the adjuvant used in the most clinically advanced malaria vaccine. We conclude that our antigen-p(Man-TLR7) platform offers a strategy to enhance the immunogenicity of protein subunit vaccines.

Novel antimalarial chloroquine- and primaquine-quinoxaline 1,4-di-N-oxide hybrids: Design, synthesis, Plasmodium life cycle stage profile, and preliminary toxicity studies.

Emergence of drug resistance and targeting all stages of the parasite life cycle are currently the major challenges in antimalarial chemotherapy. Molecular hybridization combining two scaffolds in a single molecule is an innovative strategy for achieving these goals. In this work, a series of novel quinoxaline 1,4-di-N-oxide hybrids containing either chloroquine or primaquine pharmacophores was designed, synthesized and tested against both chloroquine sensitive and multidrug resistant strains of Plasmodium falciparum. Only chloroquine-based compounds exhibited potent blood stage activity with compounds 4b and 4e being the most active and selective hybrids at this parasite stage. Based on their intraerythrocytic activity and selectivity or their chemical nature, seven hybrids were then evaluated against the liver stage of Plasmodium yoelii, Plasmodium berghei and Plasmodium falciparum infections. Compound 4b was the only chloroquine-quinoxaline 1,4-di-N-oxide hybrid with a moderate liver activity, whereas compound 6a and 6b were identified as the most active primaquine-based hybrids against exoerythrocytic stages, displaying enhanced liver activity against P. yoelii and P. berghei, respectively, and better SI values than primaquine. Although both primaquine-quinoxaline 1,4-di-N-oxide hybrids slightly reduced the infection of mosquitoes, they inhibited sporogony of P. berghei and compound 6a showed 92% blocking of transmission. In vivo liver efficacy assays revealed that compound 6a showed causal prophylactic activity affording parasitaemia reduction of up to 95% on day 4. Absence of genotoxicity and in vivo acute toxicity were also determined. These results suggest the approach of primaquine-quinoxaline 1,4-di-N-oxide hybrids as new potential dual-acting antimalarials for further investigation.

Differential activity of methylene blue against erythrocytic and hepatic stages of Plasmodium.

BACKGROUND: In the context of malaria elimination/eradication, drugs that are effective against the different developmental stages of the parasite are highly desirable. The oldest synthetic anti-malarial drug, the thiazine dye methylene blue (MB), is known for its activity against Plasmodium blood stages, including gametocytes. The aim of the present study was to investigate a possible effect of MB against malaria parasite liver stages. METHODS: MB activity was investigated using both in vitro and in vivo models. In vitro assays consisted of testing MB activity on Plasmodium falciparum, Plasmodium cynomolgi and Plasmodium yoelii parasites in human, simian or murine primary hepatocytes, respectively. MB in vivo activity was evaluated using intravital imaging in BALB/c mice infected with a transgenic bioluminescent P. yoelii parasite line. The transmission-blocking activity of MB was also addressed using mosquitoes fed on MB-treated mice. RESULTS: MB shows no activity on Plasmodium liver stages, including hypnozoites, in vitro in primary hepatocytes. In BALB/c mice, MB has moderate effect on P. yoelii hepatic development but is highly effective against blood stage growth. MB is active against gametocytes and abrogates parasite transmission from mice to mosquitoes. CONCLUSION: While confirming activity of MB against both sexual and asexual blood stages, the results indicate that MB has only little activity on the development of the hepatic stages of malaria parasites.

A Specific PfEMP1 Is Expressed in P. falciparum Sporozoites and Plays a Role in Hepatocyte Infection.

Heterochromatin plays a central role in the process of immune evasion, pathogenesis, and transmission of the malaria parasite Plasmodium falciparum during blood stage infection. Here, we use ChIP sequencing to demonstrate that sporozoites from mosquito salivary glands expand heterochromatin at subtelomeric regions to silence blood-stage-specific genes. Our data also revealed that heterochromatin enrichment is predictive of the transcription status of clonally variant genes members that mediate cytoadhesion in blood stage parasites. A specific member (here called NF54varsporo) of the var gene family remains euchromatic, and the resultant PfEMP1 (NF54_SpzPfEMP1) is expressed at the sporozoite surface. NF54_SpzPfEMP1-specific antibodies efficiently block hepatocyte infection in a strain-specific manner. Furthermore, human volunteers immunized with infective sporozoites developed antibodies against NF54_SpzPfEMP1. Overall, we show that the epigenetic signature of var genes is reset in mosquito stages. Moreover, the identification of a strain-specific sporozoite PfEMP1 is highly relevant for vaccine design based on sporozoites.