Malaria liver stages represent an ideal therapeutic target with a bottleneck in parasite load and reduced clinical symptoms; however, current in vitro pre-erythrocytic (PE) models for Plasmodium vivax and P. falciparum lack the efficiency necessary for rapid identification and effective evaluation of new vaccines and drugs, especially targeting late liver-stage development and hypnozoites. Herein we report the development of a 384-well plate culture system using commercially available materials, including cryopreserved primary human hepatocytes. Hepatocyte physiology is maintained for at least 30 days and supports development of P. vivax hypnozoites and complete maturation of P. vivax and P. falciparum schizonts. Our multimodal analysis in antimalarial therapeutic research identifies important PE inhibition mechanisms: immune antibodies against sporozoite surface proteins functionally inhibit liver stage development and ion homeostasis is essential for schizont and hypnozoite viability. This model can be implemented in laboratories in disease-endemic areas to accelerate vaccine and drug discovery research.

Malaria parasites transmitted by mosquito bite are remarkably efficient in establishing human infections. The infection process requires roughly 30 minutes and is highly complex as quiescent sporozoites injected with mosquito saliva must be rapidly activated in the skin, migrate through the body, and infect the liver. This process is poorly understood for Plasmodium vivax due to low infectivity in the in vitro models. To study this skin-to-liver-stage of malaria, we used quantitative bioassays coupled with transcriptomics to evaluate parasite changes linked with mammalian microenvironmental factors. Our in vitro phenotyping and RNA-seq analyses revealed key microenvironmental relationships with distinct biological functions. Most notable, preservation of sporozoite quiescence by exposure to insect-like factors coupled with strategic activation limits untimely activation of invasion-associated genes to dramatically increase hepatocyte invasion rates. We also report the first transcriptomic analysis of the P. vivax sporozoite interaction in salivary glands identifying 118 infection-related differentially-regulated Anopheles dirus genes. These results provide important new insights in malaria parasite biology and identify priority targets for antimalarial therapeutic interventions to block P. vivax infection.

BackgroundA stenogamous colony of Anopheles cracens (A. dirus B) established 20 years ago in a Thai insectary proved susceptible to Plasmodium vivax. However, routine sporozoite production by feeding on field-collected blood samples has not been described. The setting-up of an A. cracens colony in an insectary on the Thai-Myanmar border and the process of using P. vivax field samples for the production of infectious sporozoites are described.MethodsThe colony was started in 2012 from egg batches that were sent from the Department of Parasitology, Faculty of Medicine, University of Chiang Mai, to the Shoklo Malaria Research Unit (SMRU), on wet filter paper in sealed Petri dishes. From May 2013 to December 2014, P. vivax-infected blood samples collected from patients seeking care at SMRU clinics were used for membrane feeding assays and sporozoite production.ResultsMosquitoes were fed on blood samples from 55 patients, and for 38 (69 %) this led to the production sporozoites. The average number of sporozoites obtained per mosquito was 26,112 (range 328–79,310). Gametocytaemia was not correlated with mosquito infectiousness (p = 0.82), or with the number of the sporozoites produced (Spearman’s ρ = −0.016, p = 0.905). Infectiousness did not vary with the date of collection or the age of the patient. Mosquito survival was not correlated with sporozoite load (Spearman’s ρ = 0.179, p = 0.282).ConclusionConsistent and routine P. vivax sporozoites production confirms that A. cracens is highly susceptible to P. vivax infection. Laboratory-bred colonies of this vector are suitable for experimental transmission protocols and thus constitute a valuable resource.Electronic supplementary materialThe online version of this article (doi:10.1186/s12936-015-0830-0) contains supplementary material, which is available to authorized users.

The pre-erythrocytic stages of Plasmodium vivax and Plasmodium falciparum remain challenging for experimental research in part due to limited access to sporozoites. An important factor limiting availability is the laboratory support required for producing infected mosquitoes and the ephemeral nature of isolated extracellular sporozoites. This study was undertaken to investigate methods to improve the availability of this limited resource by extending the longevity of the extracellular sporozoites after mosquito dissection. Our goal in this study was to determine whether buffer conditions more closely mimicking the insect microenvironment could prolong longevity of ex vivo P. vivax and P. falciparum sporozoites. The study compared the current standard dissection buffer RPMI1640 to Hank's Balanced Salt Solution with 1g/L glucose (HBSS-1) or 2g/L glucose (HBSS-2) and Grace's Insect Medium for ability to extend longevity of ex vivo P. vivax and P. falciparum sporozoites. The effect of each buffer on sporozoite viability was evaluated by measuring sporozoite gliding motility at 0, 4, 8, and 24h post-dissection from mosquito salivary glands. Comparisons of mean gliding percentages of ex vivo sporozoites in the different buffers and time points found that RPMI and Grace's both showed strong gliding at 0h. In contrast, by 4h post-dissection sporozoites in RPMI consistently had the lowest gliding activity, whereas sporozoites in Grace's had significantly more gliding compared to all other buffers at almost all time points. Our results indicate that P. vivax and P. falciparum sporozoites maintained in insect media rather than the standard dissection buffer RPMI and HBSS retain viability better over time.

Immunoglobulin (Ig)A antibodies play a critical role in protection against mucosal pathogens. However, the role of serum IgA in immunity to nonmucosal pathogens, such as Plasmodium falciparum, is poorly characterized, despite being the second most abundant isotype in blood after IgG. Here, we investigated the circulating IgA response in humans to P. falciparum sporozoites that are injected into the skin by mosquitoes and migrate to the liver via the bloodstream to initiate malaria infection. We found that circulating IgA was induced in three independent sporozoite-exposed cohorts: individuals living in an endemic region in Mali, malaria-naïve individuals immunized intravenously with three large doses of irradiated sporozoites, and malaria-naïve individuals exposed to a single controlled mosquito bite infection. Mechanistically, we found evidence in an animal model that IgA responses were induced by sporozoites at dermal inoculation sites. From malaria-resistant individuals, we isolated several IgA monoclonal antibodies that reduced liver parasite burden in mice. One antibody, MAD2-6, bound to a conserved epitope in the amino terminus of the P. falciparum circumsporozoite protein, the dominant protein on the sporozoite surface. Crystal structures of this antibody revealed a unique mode of binding whereby two Fabs simultaneously bound either side of the target peptide. This study reveals a role for circulating IgA in malaria and identifies the amino terminus of the circumsporozoite protein as a target of functional antibodies.

An intrahepatic Plasmodium vivax liver stage schizont and hypnozoite develop in a microfeature-based, 384-well culture system for primary human hepatocytes.

The Antarctic sponge Dendrilla antarctica is rich in defensive terpenoids with promising antimicrobial potential. Investigation of this demosponge has resulted in the generation of a small chemical library containing diterpenoid secondary metabolites with bioactivity in an infectious disease screening campaign focused on Leishmania donovani, Plasmodium falciparum, and methicillin-resistant Staphylococcus aureus (MRSA) biofilm. In total, eleven natural products were isolated, including three new compounds designated dendrillins B–D (10–12). Chemical modification of abundant natural products led to three semisynthetic derivatives (13–15), which were also screened. Several compounds showed potency against the leishmaniasis parasite, with the natural products tetrahydroaplysulphurin-1 (4) and dendrillin B (10), as well as the semisynthetic triol 15, displaying single-digit micromolar activity and low mammalian cytotoxicity. Triol 15 displayed the best profile against the liver-stage malaria parasites, while membranolide (5) and dendrillin C (11) were strong hits against MRSA biofilm cultures.

Mass drug administration of ivermectin has been proposed as a possible malaria elimination tool. Ivermectin exhibits a mosquito‐lethal effect well beyond its biological half‐life, suggesting the presence of active slowly eliminated metabolites. Human liver microsomes, primary human hepatocytes, and whole blood from healthy volunteers given oral ivermectin were used to identify ivermectin metabolites by ultra‐high performance liquid chromatography coupled with high‐resolution mass spectrometry. The molecular structures of metabolites were determined by mass spectrometry and verified by nuclear magnetic resonance. Pure cytochrome P450 enzyme isoforms were used to elucidate the metabolic pathways. Thirteen different metabolites (M1‐M13) were identified after incubation of ivermectin with human liver microsomes. Three (M1, M3, and M6) were the major metabolites found in microsomes, hepatocytes, and blood from volunteers after oral ivermectin administration. The chemical structure, defined by LC‐MS/MS and NMR, indicated that M1 is 3″‐O‐demethyl ivermectin, M3 is 4‐hydroxymethyl ivermectin, and M6 is 3″‐O‐demethyl, 4‐hydroxymethyl ivermectin. Metabolic pathway evaluations with characterized cytochrome P450 enzymes showed that M1, M3, and M6 were produced primarily by CYP3A4, and that M1 was also produced to a small extent by CYP3A5. Demethylated (M1) and hydroxylated (M3) ivermectin were the main human in vivo metabolites. Further studies are needed to characterize the pharmacokinetic properties and mosquito‐lethal activity of these metabolites.