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.
In vitro culture of Plasmodium vivax liver
stages underlies key understandings of the fundamental biology of
this parasite, particularly the latent, hyponozoite stage, toward
drug and vaccine development. Here, we report systematic production
of Plasmodium vivax sporozoites in colonized Anopheles darlingi mosquitoes in the Peruvian Amazon. Human
subject-derived P. vivax-infected blood was
fed to Anopheles darlingi females using standard
membrane feedings assays. Optimizing A. darlingi infection and sporozoite production included replacement of infected
patient donor serum with naïve donor serum, comparing anticoagulants
in processing blood samples, and addition of penicillin–streptomycin
and ATP to infectious blood meals. Replacement of donor serum by naïve
serum in the P. vivax donor blood increased
oocysts in the mosquito midgut, and heparin, as anticoagulant, was
associated with the highest sporozoite yields. Maintaining blood-fed
mosquitoes on penicillin–streptomycin in sugar significantly
extended mosquito survival which enabled greater sporozoite yield.
In this study, we have shown that a robust P. vivax sporozoite production is feasible in a malaria-endemic setting where
infected subjects and a stable A. darlingi colony
are brought together, with optimized laboratory conditions.
An intrahepatic Plasmodium vivax liver stage schizont and hypnozoite develop in a microfeature-based, 384-well culture system for primary human hepatocytes.
Drug resistance and a dire lack of transmission-blocking antimalarials hamper malaria elimination. Here, we present the pantothenamide MMV693183 as a first-in-class acetyl-CoA synthetase (AcAS) inhibitor to enter preclinical development. Our studies demonstrate attractive drug-like properties and in vivo efficacy in a humanized mouse model of Plasmodium falciparum infection. The compound shows single digit nanomolar in vitro activity against P. falciparum and P. vivax clinical isolates, and potently blocks P. falciparum transmission to Anopheles mosquitoes. Genetic and biochemical studies identify AcAS as the target of the MMV693183-derived antimetabolite, CoA-MMV693183. Pharmacokinetic-pharmacodynamic modelling predict that a single 30 mg oral dose is sufficient to cure a malaria infection in humans. Toxicology studies in rats indicate a > 30-fold safety margin in relation to the predicted human efficacious exposure. In conclusion, MMV693183 represents a promising candidate for further (pre)clinical development with a novel mode of action for treatment of malaria and blocking transmission.
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