Summary 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.

Accuracy of aminoacylation is dependent on maintaining fidelity during attachment of amino acids to cognate tRNAs. Cis- and trans-editing protein factors impose quality control during protein translation, and 8 of 36 Plasmodium falciparum aminoacyl-tRNA synthetase (aaRS) assemblies contain canonical putative editing modules. Based on expression and localization profiles of these 8 aaRSs, we propose an asymmetric distribution between the parasite cytoplasm and its apicoplast of putative editing-domain containing aaRSs. We also show that the single copy alanyl- and threonyl-tRNA synthetases are dually targeted to parasite cytoplasm and apicoplast. This bipolar presence of two unique synthetases presents opportunity for inhibitor targeting their aminoacylation and editing activities in twin parasite compartments. We used this approach to identify specific inhibitors against the alanyl- and threonyl-tRNA synthetases. Further development of such inhibitors may lead to anti-parasitics which simultaneously block protein translation in two key parasite organelles, a strategy of wider applicability for pathogen control.

Understanding mechanisms of antibody synergy is important for vaccine design and antibody cocktail development. Examples of synergy between antibodies are well-documented, but the mechanisms underlying these relationships often remain poorly understood. The leading blood-stage malaria vaccine candidate, CyRPA, is essential for invasion of Plasmodium falciparum into human erythrocytes. Here we present a panel of anti-CyRPA monoclonal antibodies that strongly inhibit parasite growth in in vitro assays. Structural studies show that growth-inhibitory antibodies bind epitopes on a single face of CyRPA. We also show that pairs of non-competing inhibitory antibodies have strongly synergistic growth-inhibitory activity. These antibodies bind to neighbouring epitopes on CyRPA and form lateral, heterotypic interactions which slow antibody dissociation. We predict that such heterotypic interactions will be a feature of many immune responses. Immunogens which elicit such synergistic antibody mixtures could increase the potency of vaccine-elicited responses to provide robust and long-lived immunity against challenging disease targets.

The IL-33-ST2 pathway is an important initiator of type 2 immune responses. We previously characterised the HpARI protein secreted by the model intestinal nematode Heligmosomoides polygyrus, which binds and blocks IL-33. Here, we identify H. polygyrus Binds Alarmin Receptor and Inhibits (HpBARI) and HpBARI_Hom2, both of which consist of complement control protein (CCP) domains, similarly to the immunomodulatory HpARI and Hp-TGM proteins. HpBARI binds murine ST2, inhibiting cell surface detection of ST2, preventing IL-33-ST2 interactions, and inhibiting IL-33 responses in vitro and in an in vivo mouse model of asthma. In H. polygyrus infection, ST2 detection is abrogated in the peritoneal cavity and lung, consistent with systemic effects of HpBARI. HpBARI_Hom2 also binds human ST2 with high affinity, and effectively blocks human PBMC responses to IL-33. Thus, we show that H. polygyrus blocks the IL-33 pathway via both HpARI which blocks the cytokine, and also HpBARI which blocks the receptor.

Basigin is an essential host receptor for invasion of Plasmodium falciparum into human erythrocytes, interacting with parasite surface protein PfRH5. PfRH5 is a leading blood-stage malaria vaccine candidate and a target of growth-inhibitory antibodies. However, basigin is not alone on the erythrocyte surface. Instead, we show that it is exclusively found in one of two macromolecular complexes, bound predominantly to either plasma membrane Ca2+-ATPase 1/4, PMCA1/4, or monocarboxylate transporter 1, MCT1. PfRH5 binds to either of these complexes with a higher affinity than to isolated basigin ectodomain, making it likely that these are the physiological targets of PfRH5. PMCA-mediated Ca2+ export is not affected by PfRH5, ruling this out as the mechanism underlying changes in calcium flux at the interface between an erythrocyte and the invading parasite. However, our studies rationalise the function of the most effective growth inhibitory antibodies targeting PfRH5. While these antibodies do not reduce the binding of PfRH5 to monomeric basigin, they do reduce its binding to basigin-PMCA and basigin-MCT complexes. This indicates that the most effective PfRH5-targeting antibodies inhibit growth by sterically blocking the essential interaction of PfRH5 with basigin in its physiological context.

Malaria parasite erythrocytic stages comprise of repeated bursts of parasites via cyclical invasion of host erythrocytes using dedicated receptor-ligand interactions. A family of erythrocyte-binding proteins from Plasmodium knowlesi (Pk) and Plasmodium vivax (Pv) attach to human Duffy antigen receptor for chemokines (DARC) via their Duffy binding-like domains (DBLs) for invasion. Here we provide a novel, testable and overarching interaction model that rationalizes even contradictory pieces of evidence that have so far existed in the literature on Pk/Pv-DBL/DARC binding determinants. We further address the conundrum of how parasite-encoded Pk/Pv-DBLs recognize human DARC and collate evidence for two distinct DARC integration sites on Pk/Pv-DBLs.

The symptoms of malaria occur during the blood stage of infection, when parasites invade and replicate within human erythrocytes. The five-component PfPCRCR complex, containing PfRH5, PfCyRPA, PfRIPR, PfCSS and PfPTRAMP, is essential for erythrocyte invasion by the deadliest human malaria parasite, Plasmodium falciparum. Invasion can be prevented by antibodies or nanobodies against each of these five conserved proteins, making them the leading blood stage malaria vaccine candidates. However, little is known about the molecular mechanism by which PfPCRCR functions during invasion. Here we present the structure of the PfRCR complex, containing PfRH5, PfCyRPA and PfRIPR, determined by cryogenic-electron microscopy. This reveals that PfRIPR consists of an ordered multi-domain core flexibly linked to an elongated tail. We test the hypothesis that PfRH5 opens to insert into the membrane, but instead show that a rigid, disulphide-locked PfRH5 can mediate efficient erythrocyte invasion. Finally, we show that the elongated tail of PfRIPR, which is the target of growth-neutralising antibodies, binds to the PfCSS-PfPTRAMP complex on the parasite membrane. Therefore, a modular PfRIPR is linked to the merozoite membrane through an elongated tail, while its structured core presents PfCyRPA and PfRH5 to interact with erythrocyte receptors. This provides novel insight into the mechanism of erythrocyte invasion and opens the way to new approaches in rational vaccine design.

Trypanosoma brucei (T. brucei) is responsible for the fatal human disease called African trypanosomiasis, or sleeping sickness. The causative parasite, Trypanosoma, encodes soluble versions of inorganic pyrophosphatases (PPase), also called vacuolar soluble proteins (VSPs), which are localized to its acidocalcisomes. The latter are acidic membrane-enclosed organelles rich in polyphosphate chains and divalent cations whose significance in these parasites remains unclear. We here report the crystal structure of T. brucei brucei acidocalcisomal PPases in a ternary complex with Mg 2؉ and imidodiphosphate. The crystal structure reveals a novel structural architecture distinct from known class I PPases in its tetrameric oligomeric state in which a fused EF hand domain arranges around the catalytic PPase domain. This unprecedented assembly evident from Tb b VSP1 crystal structure is further confirmed by SAXS and TEM data. SAXS data suggest structural flexibility in EF hand domains indicative of conformational plasticity within Tb b VSP1.African trypanosomiasis, commonly known as sleeping sickness, affects ϳ50,000 inhabitants of sub-Saharan Africa yearly (1) with 60 million people at risk of infection (2). Sleeping sickness is caused by two subspecies of T. brucei: T. brucei gambiense and T. brucei rhodesiense. The former alone accounts for ϳ98% of the cases in humans and livestock (1). T. brucei brucei is another subspecies of T. brucei that is used as an experimental model in laboratory to study sleeping sickness, and this parasite infects animals causing a disease called nagana (3). Left untreated, sleeping sickness is fatal, and there are currently two drugs used to treat the initial phase of the disease: suramin and pentamidine. These are employed in treatment of trypanosomiasis caused by T. brucei rhodesiense and T. brucei gambiense infections (4). For the treatment of second or neurological phase, an arsenic-based drug called melarsopol is used. However, this drug causes severe side effects and can sometimes be lethal (5). A newer and much more expensive drug, eflornithine, is effective only against T. brucei gambiense. (6). A combination of another drug, nifurtimox, with eflornithine has been used for treatment, but unfortunately it is not effective against T. brucei gambiense (6). Therefore, there is a pressing case to find new, safe, inexpensive, and broad spectrum drugs for treating sleeping sickness in humans and livestock.Soluble inorganic pyrophosphatase (PPase, EC 3.6.1.1) 3 is a ubiquitous and essential enzyme that hydrolyzes the PP i generated during cellular processes such as DNA replication and protein translation (7-9). Soluble PPases comprise two families that differ in both sequence and structure (10, 11). Family/class I PPases occur in all types of cells from bacteria to humans, whereas class II occurs exclusively in bacteria (11-12). Both classes of enzymes require divalent metal cations for catalysis (13-15). PPases from wide sources have been characterized, but those from Saccharomyces cerevisi...