Analysis of the diverse antigenic landscape of the malaria protein RH5 identifies a potent vaccine-induced human public antibody clonotype.

The highly conserved and essential Plasmodium falciparum reticulocyte-binding protein homolog 5 (PfRH5) has emerged as the leading target for vaccines against the disease-causing blood stage of malaria. However, the features of the human vaccine-induced antibody response that confer highly potent inhibition of malaria parasite invasion into red blood cells are not well defined. Here, we characterize 236 human IgG monoclonal antibodies, derived from 15 donors, induced by the most advanced PfRH5 vaccine. We define the antigenic landscape of this molecule and establish that epitope specificity, antibody association rate, and intra-PfRH5 antibody interactions are key determinants of functional anti-parasitic potency. In addition, we identify a germline IgG gene combination that results in an exceptionally potent class of antibody and demonstrate its prophylactic potential to protect against P. falciparum parasite challenge in vivo. This comprehensive dataset provides a framework to guide rational design of next-generation vaccines and prophylactic antibodies to protect against blood-stage malaria.

Human Antibodies that Slow Erythrocyte Invasion Potentiate Malaria-Neutralizing Antibodies.

The Plasmodium falciparum reticulocyte-binding protein homolog 5 (PfRH5) is the leading target for next-generation vaccines against the disease-causing blood-stage of malaria. However, little is known about how human antibodies confer functional immunity against this antigen. We isolated a panel of human monoclonal antibodies (mAbs) against PfRH5 from peripheral blood B cells from vaccinees in the first clinical trial of a PfRH5-based vaccine. We identified a subset of mAbs with neutralizing activity that bind to three distinct sites and another subset of mAbs that are non-functional, or even antagonistic to neutralizing antibodies. We also identify the epitope of a novel group of non-neutralizing antibodies that significantly reduce the speed of red blood cell invasion by the merozoite, thereby potentiating the effect of all neutralizing PfRH5 antibodies as well as synergizing with antibodies targeting other malaria invasion proteins. Our results provide a roadmap for structure-guided vaccine development to maximize antibody efficacy against blood-stage malaria.

Genome-Scale Identification of Essential Metabolic Processes for Targeting the Plasmodium Liver Stage.

Plasmodium gene functions in mosquito and liver stages remain poorly characterized due to limitations in the throughput of phenotyping at these stages. To fill this gap, we followed more than 1,300 barcoded P. berghei mutants through the life cycle. We discover 461 genes required for efficient parasite transmission to mosquitoes through the liver stage and back into the bloodstream of mice. We analyze the screen in the context of genomic, transcriptomic, and metabolomic data by building a thermodynamic model of P. berghei liver-stage metabolism, which shows a major reprogramming of parasite metabolism to achieve rapid growth in the liver. We identify seven metabolic subsystems that become essential at the liver stages compared with asexual blood stages: type II fatty acid synthesis and elongation (FAE), tricarboxylic acid, amino sugar, heme, lipoate, and shikimate metabolism. Selected predictions from the model are individually validated in single mutants to provide future targets for drug development.

γδ T cells suppress Plasmodium falciparum blood-stage infection by direct killing and phagocytosis.

Activated Vγ9Vδ2 (γδ2) T lymphocytes that sense parasite-produced phosphoantigens are expanded in Plasmodium falciparum-infected patients. Although previous studies suggested that γδ2 T cells help control erythrocytic malaria, whether γδ2 T cells recognize infected red blood cells (iRBCs) was uncertain. Here we show that iRBCs stained for the phosphoantigen sensor butyrophilin 3A1 (BTN3A1). γδ2 T cells formed immune synapses and lysed iRBCs in a contact, phosphoantigen, BTN3A1 and degranulation-dependent manner, killing intracellular parasites. Granulysin released into the synapse lysed iRBCs and delivered death-inducing granzymes to the parasite. All intra-erythrocytic parasites were susceptible, but schizonts were most sensitive. A second protective γδ2 T cell mechanism was identified. In the presence of patient serum, γδ2 T cells phagocytosed and degraded opsonized iRBCs in a CD16-dependent manner, decreasing parasite multiplication. Thus, γδ2 T cells have two ways to control blood-stage malaria-γδ T cell antigen receptor (TCR)-mediated degranulation and phagocytosis of antibody-coated iRBCs.

Breadth of Fc-mediated effector function correlates with clinical immunity following human malaria challenge.

Malaria is a life-threatening disease of global health importance, particularly in sub-Saharan Africa. The growth inhibition assay (GIA) is routinely used to evaluate, prioritize, and quantify the efficacy of malaria blood-stage vaccine candidates but does not reliably predict either naturally acquired or vaccine-induced protection. Controlled human malaria challenge studies in semi-immune volunteers provide an unparalleled opportunity to robustly identify mechanistic correlates of protection. We leveraged this platform to undertake a head-to-head comparison of seven functional antibody assays that are relevant to immunity against the erythrocytic merozoite stage of Plasmodium falciparum. Fc-mediated effector functions were strongly associated with protection from clinical symptoms of malaria and exponential parasite multiplication, while the gold standard GIA was not. The breadth of Fc-mediated effector function discriminated clinical immunity following the challenge. These findings present a shift in the understanding of the mechanisms that underpin immunity to malaria and have important implications for vaccine development.

Malaria: Biology and Disease.

Malaria has been a major global health problem of humans through history and is a leading cause of death and disease across many tropical and subtropical countries. Over the last fifteen years renewed efforts at control have reduced the prevalence of malaria by over half, raising the prospect that elimination and perhaps eradication may be a long-term possibility. Achievement of this goal requires the development of new tools including novel antimalarial drugs and more efficacious vaccines as well as an increased understanding of the disease and biology of the parasite. This has catalyzed a major effort resulting in development and regulatory approval of the first vaccine against malaria (RTS,S/AS01) as well as identification of novel drug targets and antimalarial compounds, some of which are in human clinical trials.

Extracellular Vesicles from Trypanosoma brucei Mediate Virulence Factor Transfer and Cause Host Anemia.

Intercellular communication between parasites and with host cells provides mechanisms for parasite development, immune evasion, and disease pathology. Bloodstream African trypanosomes produce membranous nanotubes that originate from the flagellar membrane and disassociate into free extracellular vesicles (EVs). Trypanosome EVs contain several flagellar proteins that contribute to virulence, and Trypanosoma brucei rhodesiense EVs contain the serum resistance-associated protein (SRA) necessary for human infectivity. T. b. rhodesiense EVs transfer SRA to non-human infectious trypanosomes, allowing evasion of human innate immunity. Trypanosome EVs can also fuse with mammalian erythrocytes, resulting in rapid erythrocyte clearance and anemia. These data indicate that trypanosome EVs are organelles mediating non-hereditary virulence factor transfer and causing host erythrocyte remodeling, inducing anemia.

Protein export into malaria parasite-infected erythrocytes: mechanisms and functional consequences.

Phylum Apicomplexa comprises a large group of obligate intracellular parasites of high medical and veterinary importance. These organisms succeed intracellularly by effecting remarkable changes in a broad range of diverse host cells. The transformation of the host erythrocyte is particularly striking in the case of the malaria parasite Plasmodium falciparum. P. falciparum exports hundreds of proteins that mediate a complex cellular renovation marked by changes in the permeability, rigidity, and cytoadherence properties of the host erythrocyte. The past decade has seen enormous progress in understanding the identity and function of these exported effectors, as well as the mechanisms by which they are trafficked into the host cell. Here we review these advances, place them in the context of host manipulation by related apicomplexans, and propose key directions for future research.

The PfRCR complex bridges malaria parasite and erythrocyte during invasion.

The symptoms of malaria occur during the blood stage of infection, when parasites invade and replicate within human erythrocytes. The PfPCRCR complex1, containing PfRH5 (refs. 2,3), PfCyRPA, PfRIPR, PfCSS and PfPTRAMP, is essential for erythrocyte invasion by the deadliest human malaria parasite, Plasmodium falciparum. Invasion can be prevented by antibodies3-6 or nanobodies1 against each of these conserved proteins, making them the leading blood-stage malaria vaccine candidates. However, little is known about how PfPCRCR functions during invasion. Here we present the structure of the PfRCR complex7,8, containing PfRH5, PfCyRPA and PfRIPR, determined by cryogenic-electron microscopy. We test the hypothesis that PfRH5 opens to insert into the membrane9, instead showing that a rigid, disulfide-locked PfRH5 can mediate efficient erythrocyte invasion. We show, through modelling and an erythrocyte-binding assay, that PfCyRPA-binding antibodies5 neutralize invasion through a steric mechanism. We determine the structure of PfRIPR, showing that it consists of an ordered, multidomain core flexibly linked to an elongated tail. We also show that the elongated tail of PfRIPR, which is the target of growth-neutralizing antibodies6, binds to the PfCSS-PfPTRAMP complex on the parasite membrane. A modular PfRIPR is therefore linked to the merozoite membrane through an elongated tail, and its structured core presents PfCyRPA and PfRH5 to interact with erythrocyte receptors. This provides fresh insight into the molecular mechanism of erythrocyte invasion and opens the way to new approaches in rational vaccine design.

Immune Sensing of Synthetic, Bacterial, and Protozoan RNA by Toll-like Receptor 8 Requires Coordinated Processing by RNase T2 and RNase 2.

Human toll-like receptor 8 (TLR8) activation induces a potent T helper-1 (Th1) cell response critical for defense against intracellular pathogens, including protozoa. The receptor harbors two distinct binding sites, uridine and di- and/or trinucleotides, but the RNases upstream of TLR8 remain poorly characterized. We identified two endolysosomal endoribonucleases, RNase T2 and RNase 2, that act synergistically to release uridine from oligoribonucleotides. RNase T2 cleaves preferentially before, and RNase 2 after, uridines. Live bacteria, P. falciparum-infected red blood cells, purified pathogen RNA, and synthetic oligoribonucleotides all required RNase 2 and T2 processing to activate TLR8. Uridine supplementation restored RNA recognition in RNASE2-/- or RNASET2-/- but not RNASE2-/-RNASET2-/- cells. Primary immune cells from RNase T2-hypomorphic patients lacked a response to bacterial RNA but responded robustly to small-molecule TLR8 ligands. Our data identify an essential function of RNase T2 and RNase 2 upstream of TLR8 and provide insight into TLR8 activation.

Talking to each other to initiate sexual differentiation.

During the blood stage of their life cycle, malaria parasites invade and replicate within host erythrocytes. Some parasites differentiate to form male and female gametocytes, enabling transmission to the insect vector. Two studies by Regev-Rudzki et al. and Mantel et al. reveal an unexpected role for cell-cell communication using extracellular vesicles in triggering the commitment of malaria parasites toward sexual differentiation.

Cell-cell communication between malaria-infected red blood cells via exosome-like vesicles.

Cell-cell communication is an important mechanism for information exchange promoting cell survival for the control of features such as population density and differentiation. We determined that Plasmodium falciparum-infected red blood cells directly communicate between parasites within a population using exosome-like vesicles that are capable of delivering genes. Importantly, communication via exosome-like vesicles promotes differentiation to sexual forms at a rate that suggests that signaling is involved. Furthermore, we have identified a P. falciparum protein, PfPTP2, that plays a key role in efficient communication. This study reveals a previously unidentified pathway of P. falciparum biology critical for survival in the host and transmission to mosquitoes. This identifies a pathway for the development of agents to block parasite transmission from the human host to the mosquito.

In vitro production of infectious Plasmodium falciparum sporozoites.

An effective vaccine is needed for the prevention and elimination of malaria. The only immunogens that have been shown to have a protective efficacy of more than 90% against human malaria are Plasmodium falciparum (Pf) sporozoites (PfSPZ) manufactured in mosquitoes (mPfSPZ)1-7. The ability to produce PfSPZ in vitro (iPfSPZ) without mosquitoes would substantially enhance the production of PfSPZ vaccines and mosquito-stage malaria research, but this ability is lacking. Here we report the production of hundreds of millions of iPfSPZ. iPfSPZ invaded human hepatocytes in culture and developed to mature liver-stage schizonts expressing P. falciparum merozoite surface protein 1 (PfMSP1) in numbers comparable to mPfSPZ. When injected into FRGhuHep mice containing humanized livers, iPfSPZ invaded the human hepatocytes and developed to PfMSP1-expressing late liver stage parasites at 45% the quantity of cryopreserved mPfSPZ. Human blood from FRGhuHep mice infected with iPfSPZ produced asexual and sexual erythrocytic-stage parasites in culture, and gametocytes developed to PfSPZ when fed to mosquitoes, completing the P. falciparum life cycle from infectious gametocyte to infectious gametocyte without mosquitoes or primates.

Plasmodium Egress Across the Parasite Life Cycle.

Human malaria, caused by infection with Plasmodium parasites, remains one of the most important global public health problems, with the World Health Organization reporting more than 240 million cases and 600,000 deaths annually as of 2020 (World malaria report 2021). Our understanding of the biology of these parasites is critical for development of effective therapeutics and prophylactics, including both antimalarials and vaccines. Plasmodium is a protozoan organism that is intracellular for most of its life cycle. However, to complete its complex life cycle and to allow for both amplification and transmission, the parasite must egress out of the host cell in a highly regulated manner. This review discusses the major pathways and proteins involved in the egress events during the Plasmodium life cycle-merozoite and gametocyte egress out of red blood cells, sporozoite egress out of the oocyst, and merozoite egress out of the hepatocyte. The similarities, as well as the differences, between the various egress pathways of the parasite highlight both novel cell biology and potential therapeutic targets to arrest its life cycle.

Plasmodium RON11 triggers biogenesis of the merozoite rhoptry pair and is essential for erythrocyte invasion.

Malaria is a global and deadly human disease caused by the apicomplexan parasites of the genus Plasmodium. Parasite proliferation within human red blood cells (RBCs) is associated with the clinical manifestations of the disease. This asexual expansion within human RBCs begins with the invasion of RBCs by P. falciparum, which is mediated by the secretion of effectors from 2 specialized club-shaped secretory organelles in merozoite-stage parasites known as rhoptries. We investigated the function of the Rhoptry Neck Protein 11 (RON11), which contains 7 transmembrane domains and calcium-binding EF-hand domains. We generated conditional mutants of the P. falciparum RON11. Knockdown of RON11 inhibits parasite growth by preventing merozoite invasion. The loss of RON11 did not lead to any defects in processing of rhoptry proteins but instead led to a decrease in the amount of rhoptry proteins. We utilized ultrastructure expansion microscopy (U-ExM) to determine the effect of RON11 knockdown on rhoptry biogenesis. Surprisingly, in the absence of RON11, fully developed merozoites had only 1 rhoptry each. The single rhoptry in RON11-deficient merozoites were morphologically typical with a bulb and a neck oriented into the apical polar ring. Moreover, rhoptry proteins are trafficked accurately to the single rhoptry in RON11-deficient parasites. These data show that in the absence of RON11, the first rhoptry is generated during schizogony but upon the start of cytokinesis, the second rhoptry never forms. Interestingly, these single-rhoptry merozoites were able to attach to host RBCs but are unable to invade RBCs. Instead, RON11-deficient merozoites continue to engage with RBC for prolonged periods eventually resulting in echinocytosis, a result of secreting the contents from the single rhoptry into the RBC. Together, our data show that RON11 triggers the de novo biogenesis of the second rhoptry and functions in RBC invasion.

Role of Rabenosyn-5 and Rab5b in host cell cytosol uptake reveals conservation of endosomal transport in malaria parasites.

Vesicular trafficking, including secretion and endocytosis, plays fundamental roles in the unique biology of Plasmodium falciparum blood-stage parasites. Endocytosis of host cell cytosol (HCC) provides nutrients and room for parasite growth and is critical for the action of antimalarial drugs and parasite drug resistance. Previous work showed that PfVPS45 functions in endosomal transport of HCC to the parasite's food vacuole, raising the possibility that malaria parasites possess a canonical endolysosomal system. However, the seeming absence of VPS45-typical functional interactors such as rabenosyn 5 (Rbsn5) and the repurposing of Rab5 isoforms and other endolysosomal proteins for secretion in apicomplexans question this idea. Here, we identified a parasite Rbsn5-like protein and show that it functions with VPS45 in the endosomal transport of HCC. We also show that PfRab5b but not PfRab5a is involved in the same process. Inactivation of PfRbsn5L resulted in PI3P and PfRab5b decorated HCC-filled vesicles, typical for endosomal compartments. Overall, this indicates that despite the low sequence conservation of PfRbsn5L and the unusual N-terminal modification of PfRab5b, principles of endosomal transport in malaria parasite are similar to that of model organisms. Using a conditional double protein inactivation system, we further provide evidence that the PfKelch13 compartment, an unusual apicomplexa-specific endocytosis structure at the parasite plasma membrane, is connected upstream of the Rbsn5L/VPS45/Rab5b-dependent endosomal route. Altogether, this work indicates that HCC uptake consists of a highly parasite-specific part that feeds endocytosed material into an endosomal system containing more canonical elements, leading to the delivery of HCC to the food vacuole.

Endoplasmic reticulum PI(3)P lipid binding targets malaria proteins to the host cell.

Hundreds of effector proteins of the human malaria parasite Plasmodium falciparum constitute a "secretome" carrying a host-targeting (HT) signal, which predicts their export from the intracellular pathogen into the surrounding erythrocyte. Cleavage of the HT signal by a parasite endoplasmic reticulum (ER) protease, plasmepsin V, is the proposed export mechanism. Here, we show that the HT signal facilitates export by recognition of the lipid phosphatidylinositol-3-phosphate (PI(3)P) in the ER, prior to and independent of protease action. Secretome HT signals, including those of major virulence determinants, bind PI(3)P with nanomolar affinity and amino acid specificities displayed by HT-mediated export. PI(3)P-enriched regions are detected within the parasite's ER and colocalize with endogenous HT signal on ER precursors, which also display high-affinity binding to PI(3)P. A related pathogenic oomycete's HT signal export is dependent on PI(3)P binding, without cleavage by plasmepsin V. Thus, PI(3)P in the ER functions in mechanisms of secretion and pathogenesis.

Identification of a divalent metal transporter required for cellular iron metabolism in malaria parasites.

Plasmodium falciparum malaria parasites invade and multiply inside red blood cells (RBCs), the most iron-rich compartment in humans. Like all cells, P. falciparum requires nutritional iron to support essential metabolic pathways, but the critical mechanisms of iron acquisition and trafficking during RBC infection have remained obscure. Parasites internalize and liberate massive amounts of heme during large-scale digestion of RBC hemoglobin within an acidic food vacuole (FV) but lack a heme oxygenase to release porphyrin-bound iron. Although most FV heme is sequestered into inert hemozoin crystals, prior studies indicate that trace heme escapes biomineralization and is susceptible to nonenzymatic degradation within the oxidizing FV environment to release labile iron. Parasites retain a homolog of divalent metal transporter 1 (DMT1), a known mammalian iron transporter, but its role in P. falciparum iron acquisition has not been tested. Our phylogenetic studies indicate that P. falciparum DMT1 (PfDMT1) retains conserved molecular features critical for metal transport. We localized this protein to the FV membrane and defined its orientation in an export-competent topology. Conditional knockdown of PfDMT1 expression is lethal to parasites, which display broad cellular defects in iron-dependent functions, including impaired apicoplast biogenesis and mitochondrial polarization. Parasites are selectively rescued from partial PfDMT1 knockdown by supplementation with exogenous iron, but not other metals. These results support a cellular paradigm whereby PfDMT1 is the molecular gatekeeper to essential iron acquisition by blood-stage malaria parasites and suggest that therapeutic targeting of PfDMT1 may be a potent antimalarial strategy.

Sequence elements within the PEXEL motif and its downstream region modulate PTEX-dependent protein export in Plasmodium falciparum.

The parasite Plasmodium falciparum causes the most severe form of malaria and to invade and replicate in red blood cells (RBCs), it exports hundreds of proteins across the encasing parasitophorous vacuole membrane (PVM) into this host cell. The exported proteins help modify the RBC to support rapid parasite growth and avoidance of the human immune system. Most exported proteins possess a conserved Plasmodium export element (PEXEL) motif with the consensus RxLxE/D/Q amino acid sequence, which acts as a proteolytic cleavage recognition site within the parasite's endoplasmic reticulum (ER). Cleavage occurs after the P1 L residue and is thought to help release the protein from the ER so it can be putatively escorted by the HSP101 chaperone to the parasitophorous vacuole space surrounding the intraerythrocytic parasite. HSP101 and its cargo are then thought to assemble with the rest of a Plasmodium translocon for exported proteins (PTEX) complex, that then recognises the xE/D/Q capped N-terminus of the exported protein and translocates it across the vacuole membrane into the RBC compartment. Here, we present evidence that supports a dual role for the PEXEL's conserved P2 ' position E/Q/D residue, first, for plasmepsin V cleavage in the ER, and second, for efficient PTEX mediated export across the PVM into the RBC. We also present evidence that the downstream 'spacer' region separating the PEXEL motif from the folded functional region of the exported protein controls cargo interaction with PTEX as well. The spacer must be of a sufficient length and permissive amino acid composition to engage the HSP101 unfoldase component of PTEX to be efficiently translocated into the RBC compartment.

Lipid droplet dynamics are essential for the development of the malaria parasite Plasmodium falciparum.

Lipid droplets (LDs) are organelles that are central to lipid and energy homeostasis across all eukaryotes. In the malaria-causing parasite Plasmodium falciparum the roles of LDs in lipid acquisition from its host cells and their metabolism are poorly understood, despite the high demand for lipids in parasite membrane synthesis. We systematically characterised LD size, composition and dynamics across the disease-causing blood infection. Applying split fluorescence emission analysis and three-dimensional (3D) focused ion beam-scanning electron microscopy (FIB-SEM), we observed a decrease in LD size in late schizont stages. LD contraction likely signifies a switch from lipid accumulation to lipid utilisation in preparation for parasite egress from host red blood cells. We demonstrate connections between LDs and several parasite organelles, pointing to potential functional interactions. Chemical inhibition of triacylglyerol (TAG) synthesis or breakdown revealed essential LD functions for schizogony and in counteracting lipid toxicity. The dynamics of lipid synthesis, storage and utilisation in P. falciparum LDs might provide a target for new anti-malarial intervention strategies.