Corrigendum: Immune evasion of Plasmodium falciparum by RIFIN via inhibitory receptors.

This corrects the article DOI: 10.1038/nature24994.

Immune evasion of Plasmodium falciparum by RIFIN via inhibitory receptors.

Malaria is among the most serious infectious diseases affecting humans, accounting for approximately half a million deaths each year. Plasmodium falciparum causes most life-threatening cases of malaria. Acquired immunity to malaria is inefficient, even after repeated exposure to P. falciparum, but the immune regulatory mechanisms used by P. falciparum remain largely unknown. Here we show that P. falciparum uses immune inhibitory receptors to achieve immune evasion. RIFIN proteins are products of a polymorphic multigene family comprising approximately 150-200 genes per parasite genome that are expressed on the surface of infected erythrocytes. We found that a subset of RIFINs binds to either leucocyte immunoglobulin-like receptor B1 (LILRB1) or leucocyte-associated immunoglobulin-like receptor 1 (LAIR1). LILRB1-binding RIFINs inhibit activation of LILRB1-expressing B cells and natural killer (NK) cells. Furthermore, P. falciparum-infected erythrocytes isolated from patients with severe malaria were more likely to interact with LILRB1 than erythrocytes from patients with non-severe malaria, although an extended study with larger sample sizes is required to confirm this finding. Our results suggest that P. falciparum has acquired multiple RIFINs to evade the host immune system by targeting immune inhibitory receptors.

Plasmodium falciparum RIFIN is a novel ligand for inhibitory immune receptor LILRB2.

Plasmodium falciparum causes the most severe form of malaria. Acquired immunity against P. falciparum provides insufficient protection even after repeated infections. Therefore, P. falciparum parasites might exploit inhibitory receptors for immune evasion. P. falciparum RIFINs are products of a multigene family consisting of 150-200 genes. Previously, we demonstrated that some RIFINs downregulate the immune response through the leukocyte immunoglobulin-like receptor (LILR) family inhibitory receptor, LILRB1, and leukocyte-associated immunoglobulin-like receptor 1, LAIR1. In this study, we further analyzed the expression of inhibitory receptor ligands on P. falciparum-infected erythrocytes and found that P. falciparum-infected erythrocytes expressed ligands for another LILR family inhibitory receptor, LILRB2, that recognizes HLA class I molecules as a host ligand. Furthermore, we identified that a specific RIFIN was a ligand for LILRB2 by using a newly developed RIFIN expression library. In addition, the domain 3 of LILRB2 was involved in RIFIN binding, whereas the domains 1 and 2 of LILRB2 were involved in the binding to HLA class I molecules. These results suggest that inhibitory receptor LILRB2 is also targeted by RIFIN for immune evasion of P. falciparum similar to LILRB1 and LAIR1.

Structure-Activity Relationship of Anti-malarial Allylpyrocatechol Isolated from Piper betle.

Malaria disease remains a serious worldwide health problem. In South-East Asia, one of the malaria infection "hot-spots," medicinal plants such as Piper betle have traditionally been used for the treatment of malaria, and allylpyrocatechol (1), a constituent of P. betle, has been shown to exhibit anti-malarial activities. In this study, we verified that 1 showed in vivo anti-malarial activity through not only intraperitoneal (i.p.) but also peroral (p.o.) administration. Additionally, some analogs of 1 were synthesized and the structure-activity relationship was analyzed to disclose the crucial sub-structures for the potent activity.

An automated haematology analyzer XN-30 distinguishes developmental stages of falciparum malaria parasite cultured in vitro.

BACKGROUND: The automated haematology analyzer XN-30 (Sysmex, Kobe, Japan) easily and rapidly detects malarial parasites in clinical blood samples using flow cytometry. The XN-30 analyzer is able to distinguish each developmental stage by measuring DNA content and cell size. Thus, it was expected to be capable of quantifying the developmental stages of cultured falciparum parasite. To achieve this requirement, a modified algorithm was tested for its validity and reliability using in vitro cultured falciparum parasite. RESULTS: The XN-30 analyzer automatically measured each developmental stage as well as total parasitaemia. Comparison of the parasitaemia obtained using the XN-30 analyzer equipped with the modified algorithm with that obtained using microscopy examination of Giemsa-stained smears revealed the greater sensitivity and reproducibility of the former. The XN-30 analyzer also detected free merozoites and purified gametocytes. CONCLUSIONS: The XN-30 analyzer allows the precise recognition and enumeration of total and each developmental stages of cultured falciparum parasites, and permits the sensitive and reproducible calculation of parasitaemia. The results indicate the potential of the XN-30 analyzer for basic research on malarial biology, anti-malarial drug discovery, and evaluation of drug efficacy.

Detection of histidine-rich protein 2- and/or 3-deleted Plasmodium falciparum using the automated hematology analyzer XN-31: A proof-of-concept study.

Rapid diagnostic tests (RDTs) based on immunochromatographic detection of Plasmodium falciparum histidine-rich protein 2 (HRP2) have been frequently used for malaria diagnosis. The HRP2-based RDTs are highly sensitive and easy to use; however, their sensitivity may be low in detecting P. falciparum strains carrying deletion of the pfhrp2 and pfhrp3 genes encoding HRP2 and HRP3, respectively. The automated hematology analyzer XN-31, developed by Sysmex (Kobe, Japan) to aid in malaria diagnosis, has higher sensitivity than RDTs owing to a unique automated nucleic acid staining technology that has shown great potential in clinical settings. In this study, we compared the performance of the XN-31 analyzer and two RDTs to detect pfhrp2- and/or pfhrp3-deleted parasites cultured in vitro. The analyses showed that the analyzer was not only as sensitive to pfhrp2- and/or pfhrp3-deleted strains as it was to the wild-type strain but also had higher sensitivity than the RDTs. These results suggested that the XN-31 analyzer is useful for rapid and reliable detection of pfhrp2- and/or pfhrp3-deleted parasites in clinical settings.

Toll-like receptor 9 mediates innate immune activation by the malaria pigment hemozoin.

Malaria parasites within red blood cells digest host hemoglobin into a hydrophobic heme polymer, known as hemozoin (HZ), which is subsequently released into the blood stream and then captured by and concentrated in the reticulo-endothelial system. Accumulating evidence suggests that HZ is immunologically active, but the molecular mechanism(s) through which HZ modulates the innate immune system has not been elucidated. This work demonstrates that HZ purified from Plasmodium falciparum is a novel non-DNA ligand for Toll-like receptor (TLR)9. HZ activated innate immune responses in vivo and in vitro, resulting in the production of cytokines, chemokines, and up-regulation of costimulatory molecules. Such responses were severely impaired in TLR9-/- and myeloid differentiation factor 88 (MyD88)-/-, but not in TLR2, TLR4, TLR7, or Toll/interleukin 1 receptor domain-containing adaptor-inducing interferon beta-/- mice. Synthetic HZ, which is free of the other contaminants, also activated innate immune responses in vivo in a TLR9-dependent manner. Chloroquine (CQ), an antimalarial drug, abrogated HZ-induced cytokine production. These data suggest that TLR9-mediated, MyD88-dependent, and CQ-sensitive innate immune activation by HZ may play an important role in malaria parasite-host interactions.

New anti-malarial phenylpropanoid conjugated iridoids from Morinda morindoides.

A new phenylpropanoid conjugated iridoid together with four known congeners was isolated from Morinda morindoides, used for the therapy of malaria traditionally in some African countries, as anti-malarial principles through bioassay-guided separation. Furthermore, their absolute stereostructures were unambiguously established by a combination of modified Mosher's method and chemical correlation.

Implementation of a red blood cell-optical (RBO) channel for detection of latent iron deficiency anaemia by automated measurement of autofluorescence-emitting red blood cells.

Iron deficiency is the most common and widespread nutritional disorder worldwide. The automated haematology analyser XN-30 (Sysmex, Kobe, Japan) was developed to detect malaria-infected red blood cells (RBCs) in human blood samples using flow cytometry. The optical system of the analyser detects autofluorescence (AF)-emitting RBCs containing iron-deficient haem groups and would aid in the diagnosis of anaemia resulting from iron deficiency. Here, an RBC-optical (RBO) channel was devised and implemented on the analyser. In vitro analyses showed that the analyser detected AF-emitting RBCs treated with 5-aminolevulinic acid. Furthermore, the analyser detected AF-emitting RBCs in mice fed a low iron diet and infected with a rodent malaria parasite; it could also be effectively used in humans. This study demonstrates that the analyser can quantitatively and reproducibly detect AF-emitting RBCs and measure other haematological parameters, suggesting its usefulness for the initial evaluation of latent iron deficiency anaemia in conjunction with the diagnosis of malaria.

Adaptation of the Plasmodium falciparum FCB strain for in vitro and in vivo analysis in squirrel monkeys (Saimiri sciureus).

The asexual blood stages of the Plasmodium falciparum parasite are responsible for inducing the clinical symptoms and the most severe presentations of malaria infection that causes frequent mortality and morbidity in tropical and subtropical areas of the world, making the blood stages of infection a key target of new malaria treatment and prevention strategies. Progress towards the development of more effective treatment and prevention strategies has been hindered by the limited availability of infection models that permit the sequential analysis of blood stage parasites in vitro followed by in vivo analysis to confirm therapeutic benefits. To advance a model for in vitro and in vivo analysis of blood stage parasites, we examined nine laboratory strains of P. falciparum to determine which strains could adapt to growth in vivo in splenectomized squirrel monkeys (Saimiri sciureus). Only one of the nine laboratory strains tested, the FCB strain, adapted to in vivo growth. Morphological analysis show that the adapted ring-stage parasites have a different morphology from original parasites cultured in vitro, and more often they were found to localize at the edge of the infected red blood cell. No remarkable differences were observed for both trophozoites and schizonts. The adapted strain can be cultured back in vitro similar to the original parasite, indicating that the adapted parasite can develop both in vitro and in vivo. This squirrel monkey-adapted P. falciparum parasite is expected to be suitable and is advantageous for the research and development of vaccines and antimalarial drugs.

Plasmodium products persist in the bone marrow and promote chronic bone loss.

Although malaria is a life-threatening disease with severe complications, most people develop partial immunity and suffer from mild symptoms. However, incomplete recovery from infection causes chronic illness, and little is known of the potential outcomes of this chronicity. We found that malaria causes bone loss and growth retardation as a result of chronic bone inflammation induced by Plasmodium products. Acute malaria infection severely suppresses bone homeostasis, but sustained accumulation of Plasmodium products in the bone marrow niche induces MyD88-dependent inflammatory responses in osteoclast and osteoblast precursors, leading to increased RANKL expression and overstimulation of osteoclastogenesis, favoring bone resorption. Infection with a mutant parasite with impaired hemoglobin digestion that produces little hemozoin, a major Plasmodium by-product, did not cause bone loss. Supplementation of alfacalcidol, a vitamin D3 analog, could prevent the bone loss. These results highlight the risk of bone loss in malaria-infected patients and the potential benefits of coupling bone therapy with antimalarial treatment.

Antibody titres and boosting after natural malaria infection in BK-SE36 vaccine responders during a follow-up study in Uganda.

The malaria vaccine BK-SE36 is a recombinant protein (SE36) based on the Honduras 1 serine repeat antigen-5 of Plasmodium falciparum, adsorbed to aluminium hydroxide gel. The phase Ib trial in Uganda demonstrated the safety and immunogenicity of BK-SE36. Ancillary analysis in the follow-up study of 6-20 year-old volunteers suggest significant differences in time to first episodes of clinical malaria in vaccinees compared to placebo/control group. Here, we aimed to get further insights into the association of anti-SE36 antibody titres and natural P. falciparum infection. Children who received BK-SE36 and whose antibody titres against SE36 increased by ≥1.92-fold after vaccination were categorised as responders. Most responders did not have or only had a single episode of natural P. falciparum infection. Notably, responders who did not experience infection had relatively high anti-SE36 antibody titres post-second vaccination compared to those who were infected. The anti-SE36 antibody titres of the responders who experienced malaria were boosted after infection and they had lower risk of reinfection. These findings show that anti-SE36 antibody titres induced by BK-SE36 vaccination offered protection against malaria. The vaccine is now being evaluated in a phase Ib trial in children less than 5 years old.

Phase 1b randomized trial and follow-up study in Uganda of the blood-stage malaria vaccine candidate BK-SE36.

BACKGROUND: Up to now a malaria vaccine remains elusive. The Plasmodium falciparum serine repeat antigen-5 formulated with aluminum hydroxyl gel (BK-SE36) is a blood-stage malaria vaccine candidate that has undergone phase 1a trial in malaria-naive Japanese adults. We have now assessed the safety and immunogenicity of BK-SE36 in a malaria endemic area in Northern Uganda. METHODS: We performed a two-stage, randomized, single-blinded, placebo-controlled phase 1b trial (Current Controlled trials ISRCTN71619711). A computer-generated sequence randomized healthy subjects for 2 subcutaneous injections at 21-day intervals in Stage1 (21-40 year-olds) to 1-mL BK-SE36 (BKSE1.0) (n = 36) or saline (n = 20) and in Stage2 (6-20 year-olds) to BKSE1.0 (n = 33), 0.5-mL BK-SE36 (BKSE0.5) (n = 33), or saline (n = 18). Subjects and laboratory personnel were blinded. Safety and antibody responses 21-days post-second vaccination (Day42) were assessed. Post-trial, to compare the risk of malaria episodes 130-365 days post-second vaccination, Stage2 subjects were age-matched to 50 control individuals. RESULTS: Nearly all subjects who received BK-SE36 had induration (Stage1, n = 33, 92%; Stage2, n = 63, 96%) as a local adverse event. No serious adverse event related to BK-SE36 was reported. Pre-existing anti-SE36 antibody titers negatively correlated with vaccination-induced antibody response. At Day42, change in antibody titers was significant for seronegative adults (1.95-fold higher than baseline [95% CI, 1.56-2.43], p = 0.004) and 6-10 year-olds (5.71-fold [95% CI, 2.38-13.72], p = 0.002) vaccinated with BKSE1.0. Immunogenicity response to BKSE0.5 was low and not significant (1.55-fold [95% CI, 1.24-1.94], p = 0.75). In the ancillary analysis, cumulative incidence of first malaria episodes with ≥5000 parasites/µL was 7 cases/33 subjects in BKSE1.0 and 10 cases/33 subjects in BKSE0.5 vs. 29 cases/66 subjects in the control group. Risk ratio for BKSE1.0 was 0.48 (95% CI, 0.24-0.98; p = 0.04). CONCLUSION: BK-SE36 is safe and immunogenic. The promising potential of BK-SE36, observed in the follow-up study, warrants a double-blind phase 1/2b trial in children under 5 years. TRIAL REGISTRATION: Controlled-Trials.com ISRCTN71619711.

Lipocalin 2 bolsters innate and adaptive immune responses to blood-stage malaria infection by reinforcing host iron metabolism.

Plasmodium parasites multiply within host erythrocytes, which contain high levels of iron, and parasite egress from these cells results in iron release and host anemia. Although Plasmodium requires host iron for replication, how host iron homeostasis and responses to these fluxes affect Plasmodium infection are incompletely understood. We determined that Lipocalin 2 (Lcn2), a host protein that sequesters iron, is abundantly secreted during human (P. vivax) and mouse (P. yoeliiNL) blood-stage malaria infections and is essential to control P. yoeliiNL parasitemia, anemia, and host survival. During infection, Lcn2 bolsters both host macrophage function and granulocyte recruitment and limits reticulocytosis, or the expansion of immature erythrocytes, which are the preferred target cell of P. yoeliiNL. Additionally, a chronic iron imbalance due to Lcn2 deficiency results in impaired adaptive immune responses against Plasmodium parasites. Thus, Lcn2 exerts antiparasitic effects by maintaining iron homeostasis and promoting innate and adaptive immune responses.

Structure-activity relationship of anti-malarial spongean peroxides having a 3-methoxy-1,2-dioxane structure.

In order to study the structure-activity relationship of anti-malarial spongean peroxides, several analogues concerning with the 6-methoxyacetyl moiety and the 3-pentyl residue in methyl 2-(3-methoxy-3-pentyl-1,2-dioxan-6-yl)acetate were synthesized and evaluated for anti-malarial activity. The tert-butyl ester analogue 14 showed stability in mouse serum and a high selectivity index against the malaria parasite, Plasmodium falciparum, and the citronellyl analogue 31 exhibited the strongest in vitro anti-malarial activity among them, and the imidazole analogue 25 showed desirable in vivo anti-malarial activity against P. berghei infected mice.

Synthesis of a bioprobe for elucidation of target molecule of spongean anti-malarial peroxides.

The reactants of an anti-malarial peroxide having a 6-carbomethoxymethyl-3-methoxy-1,2-dioxane moiety treated with FeSO4 were analyzed. For mechanistic study of the anti-malarial peroxide, two biotinylated probes to elucidate the target molecules were designed and synthesized. The two synthesized probes showed potent anti-malarial activity, and one of them was proved to form an irreversible binding with protein in a model experiment.

New readily accessible peroxides with high anti-malarial potency.

In an explorative study for new anti-malarial substances using the methyl esters (1 and 2) of peroxyplakoric acids A(3) and B(3) as scaffolds, 6-carbomethoxymethyl-3-methoxy-3-pentyl-1,2-dioxane, which has been readily synthesized from 6-keto-alpha,beta-unsaturated ester, was found to exhibit potent anti-malarial activity with high selective toxicity.

New anti-malarial peroxides with in vivo potency derived from spongean metabolites.

The structure-activity relationship of the anti-malarial substance 3 having a 6-carbomethoxymethyl-3-methoxy-1,2-dioxane structure was studied. The ester portion of the peroxide 3, showing little in vivo efficacy in malaria-infected mice in spite of the potent in vitro activity, was hydrolyzed in serum to afford an inactive free acid 4. The amide analogues (8 and 9) robust to mouse serum were disclosed to exhibit in vivo anti-malarial potency.

Serine repeat antigen (SERA5) is predominantly expressed among the SERA multigene family of Plasmodium falciparum, and the acquired antibody titers correlate with serum inhibition of the parasite growth.

The Plasmodium falciparum serine repeat antigen (SERA) is one of the blood stage malaria vaccine candidates. The malaria genome project has revealed that SERA is a member of the SERA multigene family consisting of eight SERA homologues clustered on chromosome 2 and one SERA homologue on chromosome 9. Northern blotting and real time quantitative reverse transcription-PCR with five independent parasite strains, including three allelic representative forms of the SERA gene, have shown that all of the SERA homologues are transcribed most actively at trophozoite and schizont stages and that SERA5 (SERA/SERP) is transcribed predominantly among the family. Polyclonal antibodies were raised against recombinant proteins representing the N-terminal portions of four significantly transcribed SERA homologues (SERA3 to -6) in the center of the cluster on chromosome 2. Using these antibodies, indirect immunofluorescence microscopy detected the expression of SERA3 to -6, with similar localization, in all trophozoite- and schizont-infected erythrocytes. We have examined 40 sera from Ugandan adults for their antibody reactivity and found that enzyme-linked immunosorbent assay titer against SERA5 N-terminal domain, but not against other SERA proteins, is positively correlated with the inhibition of in vitro parasite growth by individual sera. Our data confirm the usefulness of the N-terminal domain of SERA5 as a promising malaria candidate vaccine.