Development of a highly effective vaccine or antibodies for the prevention and ultimately elimination of malaria is urgently needed. Here we report the isolation of a number of human monoclonal ...antibodies directed against the Plasmodium falciparum (Pf) circumsporozoite protein (PfCSP) from several subjects immunized with an attenuated Pf whole-sporozoite (SPZ) vaccine (Sanaria PfSPZ Vaccine). Passive transfer of one of these antibodies, monoclonal antibody CIS43, conferred high-level, sterile protection in two different mouse models of malaria infection. The affinity and stoichiometry of CIS43 binding to PfCSP indicate that there are two sequential multivalent binding events encompassing the repeat domain. The first binding event is to a unique 'junctional' epitope positioned between the N terminus and the central repeat domain of PfCSP. Moreover, CIS43 prevented proteolytic cleavage of PfCSP on PfSPZ. Analysis of crystal structures of the CIS43 antigen-binding fragment in complex with the junctional epitope determined the molecular interactions of binding, revealed the epitope's conformational flexibility and defined Asn-Pro-Asn (NPN) as the structural repeat motif. The demonstration that CIS43 is highly effective for passive prevention of malaria has potential application for use in travelers, military personnel and elimination campaigns and identifies a new and conserved site of vulnerability on PfCSP for next-generation rational vaccine design.
Primaquine (PQ) is an essential antimalarial drug but despite being developed over 70 years ago, its mode of action is unclear. Here, we demonstrate that hydroxylated-PQ metabolites (OH-PQm) are ...responsible for efficacy against liver and sexual transmission stages of Plasmodium falciparum. The antimalarial activity of PQ against liver stages depends on host CYP2D6 status, whilst OH-PQm display direct, CYP2D6-independent, activity. PQ requires hepatic metabolism to exert activity against gametocyte stages. OH-PQm exert modest antimalarial efficacy against parasite gametocytes; however, potency is enhanced ca.1000 fold in the presence of cytochrome P450 NADPH:oxidoreductase (CPR) from the liver and bone marrow. Enhancement of OH-PQm efficacy is due to the direct reduction of quinoneimine metabolites by CPR with the concomitant and excessive generation of H
O
, leading to parasite killing. This detailed understanding of the mechanism paves the way to rationally re-designed 8-aminoquinolines with improved pharmacological profiles.
The global decline in malaria has stalled
, emphasizing the need for vaccines that induce durable sterilizing immunity. Here we optimized regimens for chemoprophylaxis vaccination (CVac), for which ...aseptic, purified, cryopreserved, infectious Plasmodium falciparum sporozoites (PfSPZ) were inoculated under prophylactic cover with pyrimethamine (PYR) (Sanaria PfSPZ-CVac(PYR)) or chloroquine (CQ) (PfSPZ-CVac(CQ))-which kill liver-stage and blood-stage parasites, respectively-and we assessed vaccine efficacy against homologous (that is, the same strain as the vaccine) and heterologous (a different strain) controlled human malaria infection (CHMI) three months after immunization ( https://clinicaltrials.gov/ , NCT02511054 and NCT03083847). We report that a fourfold increase in the dose of PfSPZ-CVac(PYR) from 5.12 × 10
to 2 × 10
PfSPZs transformed a minimal vaccine efficacy (low dose, two out of nine (22.2%) participants protected against homologous CHMI), to a high-level vaccine efficacy with seven out of eight (87.5%) individuals protected against homologous and seven out of nine (77.8%) protected against heterologous CHMI. Increased protection was associated with Vδ2 γδ T cell and antibody responses. At the higher dose, PfSPZ-CVac(CQ) protected six out of six (100%) participants against heterologous CHMI three months after immunization. All homologous (four out of four) and heterologous (eight out of eight) infectivity control participants showed parasitaemia. PfSPZ-CVac(CQ) and PfSPZ-CVac(PYR) induced a durable, sterile vaccine efficacy against a heterologous South American strain of P. falciparum, which has a genome and predicted CD8 T cell immunome that differs more strongly from the African vaccine strain than other analysed African P. falciparum strains.
The development of therapies and vaccines for human hepatropic pathogens requires robust model systems that enable the study of host-pathogen interactions. However, in vitro liver models of infection ...typically use either hepatoma cell lines that exhibit aberrant physiology or primary human hepatocytes in culture conditions in which they rapidly lose their hepatic phenotype. To achieve stable and robust in vitro primary human hepatocyte models, we developed micropatterned cocultures (MPCCs), which consist of primary human hepatocytes organized into 2D islands that are surrounded by supportive fibroblast cells. By using this system, which can be established over a period of days, and maintained over multiple weeks, we demonstrate how to recapitulate in vitro hepatic life cycles for the hepatitis B and C viruses and the Plasmodium pathogens P. falciparum and P. vivax. The MPCC platform can be used to uncover aspects of host-pathogen interactions, and it has the potential to be used for drug and vaccine development.
Liver sinusoidal endothelial cells (LSECs) differ, both structurally and functionally, from endothelial cells (ECs) lining blood vessels of other tissues. For example, in contrast to other ECs, LSECs ...possess fenestrations, have low detectable levels of platelet endothelial cell adhesion molecule 1 expression, and in rat tissue, they distinctively express a cell surface marker recognized by the SE‐1 antibody. These unique phenotypic characteristics seen in hepatic tissue are lost over time upon culture in vitro; therefore, this study sought to systematically examine the effects of microenvironmental stimuli—namely, extracellular matrix and neighboring cells, on the LSEC phenotype in vitro. In probing the role of the underlying extracellular matrix, we identified collagen I and collagen III as well as mixtures of collagen I/collagen IV/fibronectin as having a positive effect on LSEC survival. Furthermore, using a stable hepatocellular model (hepatocyte–fibroblast) we were able to prolong the expression of both SE‐1 and phenotypic functions of LSEC such as factor VIII activity and AcLOL uptake in cocultured LSECs through the production of short‐range paracrine signals. In the course of these experiments, we identified the antigen recognized by SE‐1 as CD32b. Conclusion: Collectively, this study has identified several microenvironmental regulators of liver sinusoidal endothelial cells that prolong their phenotypic functions for up to 2 weeks in culture, enabling the development of better in vitro models of liver physiology and disease. (HEPATOLOGY 2009.)
During invasion, Plasmodium, the causative agent of malaria, wraps itself in a parasitophorous vacuole membrane (PVM), which constitutes a critical interface between the parasite and its host cell. ...Within hepatocytes, each Plasmodium sporozoite generates thousands of new parasites, creating high demand for lipids to support this replication and enlarge the PVM. Here, a global analysis of the total lipid repertoire of Plasmodium-infected hepatocytes reveals an enrichment of neutral lipids and the major membrane phospholipid, phosphatidylcholine (PC). While infection is unaffected in mice deficient in key enzymes involved in neutral lipid synthesis and lipolysis, ablation of rate-limiting enzymes in hepatic PC biosynthetic pathways significantly decreases parasite numbers. Host PC is taken up by both P. berghei and P. falciparum and is necessary for correct localization of parasite proteins to the PVM, which is essential for parasite survival. Thus, Plasmodium relies on the abundance of these lipids within hepatocytes to support infection.
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•Lipid composition of P. berghei-infected hepatocytes is altered during infection•Plasmodium liver stage infection does not require de-novo-synthesized neutral lipids•Plasmodium takes up host phosphatidylcholine (PC), which associates with the PVM•Host cell de novo PC synthesis contributes to PVM integrity and parasite survival
Plasmodium replication in hepatocytes requires abundant lipid resources. By analyzing the lipidome of P. berghei-infected cells, Itoe et al. reveal enrichment of phosphatidylcholine, a major membrane phospholipid. Targeted silencing of host genes involved in de novo phosphatidylcholine synthesis show that these pathways are critical for Plasmodium liver-stage infection.
Malaria eradication is a major goal in public health but is challenged by relapsing malaria species, expanding drug resistance, and the influence of host genetics on antimalarial drug efficacy. To ...overcome these hurdles, it is imperative to establish in vitro assays of liver-stage malaria for drug testing. Induced pluripotent stem cells (iPSC) potentially allow the assessment of donor-specific drug responses, and iPSC-derived hepatocyte-like cells (iHLCs) can facilitate the study of host genetics on host-pathogen interactions and the discovery of novel targets for antimalarial drug development. We establish in vitro liver-stage malaria infections in iHLCs using P. berghei, P. yoelii, P. falciparum, and P. vivax and show that differentiating cells acquire permissiveness to malaria infection at the hepatoblast stage. We also characterize antimalarial drug metabolism capabilities of iHLCs using prototypical antimalarial drugs and demonstrate that chemical maturation of iHLCs can improve their potential for antimalarial drug testing applications.
•iPSC-derived hepatocyte-like cells (iHLCs) can host liver-stage malaria in vitro•iHLCs become permissive to Plasmodium infection at the hepatoblast stage•Plasmodium-infected iHLCs are sensitive to atovaquone but not primaquine•Small molecule-mediated maturation of iHLCs confers primaquine sensitivity
In this article, Bhatia and colleagues show that iPSC-derived hepatocyte-like cells (iHLCs) can be infected with Plasmodium parasites of the species that cause human malaria, starting from the hepatoblast stage. They also demonstrate that further maturation of iHLCs by small molecules confers drug sensitivity to a model antimalarial, primaquine, that requires hepatic bioactivation.
Homeostasis of mammalian cell function strictly depends on balancing oxygen exposure to maintain energy metabolism without producing excessive reactive oxygen species. In vivo, cells in different ...tissues are exposed to a wide range of oxygen concentrations, and yet in vitro models almost exclusively expose cultured cells to higher, atmospheric oxygen levels. Existing models of liver-stage malaria that utilize primary human hepatocytes typically exhibit low in vitro infection efficiencies, possibly due to missing microenvironmental support signals. One cue that could influence the infection capacity of cultured human hepatocytes is the dissolved oxygen concentration. We developed a microscale human liver platform comprised of precisely patterned primary human hepatocytes and nonparenchymal cells to model liver-stage malaria, but the oxygen concentrations are typically higher in the in vitro liver platform than anywhere along the hepatic sinusoid. Indeed, we observed that liver-stage Plasmodium parasite development in vivo correlates with hepatic sinusoidal oxygen gradients. Therefore, we hypothesized that in vitro liver-stage malaria infection efficiencies might improve under hypoxia. Using the infection of micropatterned co-cultures with Plasmodium berghei, Plasmodium yoelii or Plasmodium falciparum as a model, we observed that ambient hypoxia resulted in increased survival of exo-erythrocytic forms (EEFs) in hepatocytes and improved parasite development in a subset of surviving EEFs, based on EEF size. Further, the effective cell surface oxygen tensions (pO2) experienced by the hepatocytes, as predicted by a mathematical model, were systematically perturbed by varying culture parameters such as hepatocyte density and height of the medium, uncovering an optimal cell surface pO2 to maximize the number of mature EEFs. Initial mechanistic experiments revealed that treatment of primary human hepatocytes with the hypoxia mimetic, cobalt(II) chloride, as well as a HIF-1α activator, dimethyloxalylglycine, also enhance P. berghei infection, suggesting that the effect of hypoxia on infection is mediated in part by host-dependent HIF-1α mechanisms.
The activation of hepatic stellate cells (HSCs) is a critical event in hepatic fibrosis, because these cells are the main producers of extracellular matrix proteins in the liver and contribute to the ...modulation of inflammatory responses via the secretion of several cytokines and the expression of adhesion molecules. The goal of the present study was to characterize cell surface proteins that regulate HSC activation. To this end, a panel of monoclonal antibodies (mAbs) was generated. mAb 14.27 recognized a protein of 45 kd that was highly expressed on HSCs. Affinity purification of this protein followed by sequencing revealed that protein to be CD38. We subsequently demonstrated that CD38 was constitutively expressed by HSCs and that its expression increased after in vitro and in vivo activation. mAb 14.27 induced an increase in cytosolic Ca2+ levels in HSCs, showing that it functions as an agonistic antibody. Moreover, the effects mediated by the CD38 mAb included induction of the proinflammatory cytokine interleukin-6 and up-regulation of the adhesion molecules intercellular adhesion molecule-1, vascular cell adhesion molecule-1, and neural cell adhesion molecule. Collectively, our data suggest that CD38 can act as a regulator of HSC activation and effector functions.
The aim of this study was to characterize the reactivity of monoclonal antibodies (mAbs) that had been submitted to the HLDA8 Workshop. The lineage specificity of target molecules was tested by ...analyzing their expression patterns on blood cells, leukocytes, and lymphocyte subsets. The expression of target molecules during B cell development, ranging from early precursors to plasma cells, was analyzed using a large panel of B cell lines. Our results have permitted us to characterize the expression of 10 new CD molecules: CD316 (HM1.24, BST2), CD268 (BAFF-R, TNFRSF13C), CD269 (BCMA, TNFRF17), CD267 (TACI, TNFRSF13B), CD275 (ICOSL, B7H2), CD254 (TRANCE, TNFSF11), CD252 (OX40L TNFSF4), CD315 (CD9-P), CD316 (EWI-2, PGRL), and CD307 (IRTA-2 or FcRH5). Three of these new CDs, CD267, CD269, and CD307 presented a B cell-restricted expression pattern. MAbs against these novel cell-surface molecules may offer new tools for research, diagnosis, and therapy.
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