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.

The dry season is a major challenge for Plasmodium falciparum parasites in many malaria endemic regions, where water availability limits mosquitoes to only part of the year. How P. falciparum bridges two transmission seasons months apart, without being cleared by the host or compromising host survival is poorly understood. Here we show that low levels of P. falciparum parasites persist in the blood of asymptomatic Malian individuals during the 5-to 6-month dry season, rarely causing symptoms and minimally affecting the host immune response. Parasites isolated during the dry season are transcriptionally distinct from those of subjects with febrile malaria in the transmission season, reflecting longer circulation within each replicative cycle, of parasitized erythrocytes without adhering to the vascular endothelium. Low parasite levels during the dry season are not due to impaired replication, but rather increased splenic clearance of longer-circulating infected erythrocytes. We propose that P. falciparum virulence in areas of seasonal malaria transmission is regulated so that the parasite decreases its endothelial binding capacity, allowing increased splenic clearance and enabling several months of subclinical parasite persistence.

Erythrocyte invasion by malaria parasites is essential for blood-stage development and an important determinant of host range. In Plasmodium vivax, the interaction between the Duffy binding protein (DBP) and its cognate receptor, the Duffy antigen receptor for chemokines (DARC), on human erythrocytes is central to blood-stage infection. Contrary to this established pathway of invasion, there is growing evidence of P. vivax infections occurring in Duffy blood group-negative individuals, suggesting that the parasite might have gained an alternative pathway to infect this group of individuals. Supporting this concept, a second distinct erythrocyte binding protein (EBP2), representing a new member of the DBP family, was discovered in P. vivax and may be the ligand in an alternate invasion pathway. Our study characterizes this novel ligand and determines its potential role in reticulocyte invasion by P. vivax merozoites. EBP2 binds preferentially to young (CD71high) Duffy-positive (Fy+) reticulocytes and has minimal binding capacity for Duffy-negative reticulocytes. Importantly, EBP2 is antigenically distinct from DBP and cannot be functionally inhibited by anti-DBP antibodies. Consequently, our results do not support EBP2 as a ligand for invasion of Duffy-negative blood cells, but instead, EBP2 may represent a novel ligand for an alternate invasion pathway of Duffy-positive reticulocytes.