Plasmodium vivax spleen-dependent protein 1 and its role in extracellular vesicles-mediated intrasplenic infections.

Recent studies indicate that human spleen contains over 95% of the total parasite biomass during chronic asymptomatic infections caused by Plasmodium vivax. Previous studies have demonstrated that extracellular vesicles (EVs) secreted from infected reticulocytes facilitate binding to human spleen fibroblasts (hSFs) and identified parasite genes whose expression was dependent on an intact spleen. Here, we characterize the P. vivax spleen-dependent hypothetical gene (PVX_114580). Using CRISPR/Cas9, PVX_114580 was integrated into P. falciparum 3D7 genome and expressed during asexual stages. Immunofluorescence analysis demonstrated that the protein, which we named P. vivax Spleen-Dependent Protein 1 (PvSDP1), was located at the surface of infected red blood cells in the transgenic line and this localization was later confirmed in natural infections. Plasma-derived EVs from P. vivax-infected individuals (PvEVs) significantly increased cytoadherence of 3D7_PvSDP1 transgenic line to hSFs and this binding was inhibited by anti-PvSDP1 antibodies. Single-cell RNAseq of PvEVs-treated hSFs revealed increased expression of adhesion-related genes. These findings demonstrate the importance of parasite spleen-dependent genes and EVs from natural infections in the formation of intrasplenic niches in P. vivax, a major challenge for malaria elimination.

Morphological and Transcriptional Changes in Human Bone Marrow During Natural Plasmodium vivax Malaria Infections.

BACKGROUND: The presence of Plasmodium vivax malaria parasites in the human bone marrow (BM) is still controversial. However, recent data from a clinical case and experimental infections in splenectomized nonhuman primates unequivocally demonstrated the presence of parasites in this tissue. METHODS: In the current study, we analyzed BM aspirates of 7 patients during the acute attack and 42 days after drug treatment. RNA extracted from CD71+ cell suspensions was used for sequencing and transcriptomic analysis. RESULTS: We demonstrated the presence of parasites in all patients during acute infections. To provide further insights, we purified CD71+ BM cells and demonstrated dyserythropoiesis and inefficient erythropoiesis in all patients. In addition, RNA sequencing from 3 patients showed that genes related to erythroid maturation were down-regulated during acute infections, whereas immune response genes were up-regulated. CONCLUSIONS: This study thus shows that during P. vivax infections, parasites are always present in the BM and that such infections induced dyserythropoiesis and ineffective erythropoiesis. Moreover, infections induce transcriptional changes associated with such altered erythropoietic response, thus highlighting the importance of this hidden niche during natural infections.

Plasmodium vivax spleen-dependent genes encode antigens associated with cytoadhesion and clinical protection.

Plasmodium vivax, the most widely distributed human malaria parasite, causes severe clinical syndromes despite low peripheral blood parasitemia. This conundrum is further complicated as cytoadherence in the microvasculature is still a matter of investigations. Previous reports in Plasmodium knowlesi, another parasite species shown to infect humans, demonstrated that variant genes involved in cytoadherence were dependent on the spleen for their expression. Hence, using a global transcriptional analysis of parasites obtained from spleen-intact and splenectomized monkeys, we identified 67 P. vivax genes whose expression was spleen dependent. To determine their role in cytoadherence, two Plasmodium falciparum transgenic lines expressing two variant proteins pertaining to VIR and Pv-FAM-D multigene families were used. Cytoadherence assays demonstrated specific binding to human spleen but not lung fibroblasts of the transgenic line expressing the VIR14 protein. To gain more insights, we expressed five P. vivax spleen-dependent genes as recombinant proteins, including members of three different multigene families (VIR, Pv-FAM-A, Pv-FAM-D), one membrane transporter (SECY), and one hypothetical protein (HYP1), and determined their immunogenicity and association with clinical protection in a prospective study of 383 children in Papua New Guinea. Results demonstrated that spleen-dependent antigens are immunogenic in natural infections and that antibodies to HYP1 are associated with clinical protection. These results suggest that the spleen plays a major role in expression of parasite proteins involved in cytoadherence and can reveal antigens associated with clinical protection, thus prompting a paradigm shift in P. vivax biology toward deeper studies of the spleen during infections.