Identification of potential inhibitors of casein kinase 2 alpha of Plasmodium falciparum with potent in vitro activity.

Drug resistance to commercially available antimalarials is a major obstacle in malaria control and elimination, creating the need to find new antiparasitic compounds with novel mechanisms of action. The success of kinase inhibitors for oncological treatments has paved the way for the exploitation of protein kinases as drug targets in various diseases, including malaria. Casein kinases are ubiquitous serine/threonine kinases involved in a wide range of cellular processes such as mitotic checkpoint signaling, DNA damage response, and circadian rhythm. In Plasmodium, it is suggested that these protein kinases are essential for both asexual and sexual blood-stage parasites, reinforcing their potential as targets for multi-stage antimalarials. To identify new putative PfCK2α inhibitors, we utilized an in silico chemogenomic strategy involving virtual screening with docking simulations and quantitative structure-activity relationship predictions. Our investigation resulted in the discovery of a new quinazoline molecule (542), which exhibited potent activity against asexual blood stages and a high selectivity index (>100). Subsequently, we conducted chemical-genetic interaction analysis on yeasts with mutations in casein kinases. Our chemical-genetic interaction results are consistent with the hypothesis that 542 inhibits yeast Cka1, which has a hinge region with high similarity to PfCK2α. This finding is in agreement with our in silico results suggesting that 542 inhibits PfCK2α via hinge region interaction.

Gas6 drives Zika virus-induced neurological complications in humans and congenital syndrome in immunocompetent mice.

Zika virus (ZIKV) has the ability to cross placental and brain barriers, causing congenital malformations in neonates and neurological disorders in adults. However, the pathogenic mechanisms of ZIKV-induced neurological complications in adults and congenital malformations are still not fully understood. Gas6 is a soluble TAM receptor ligand able to promote flavivirus internalization and downregulation of immune responses. Here we demonstrate that there is a correlation between ZIKV neurological complications with higher Gas6 levels and the downregulation of genes associated with anti-viral response, as type I IFN due to Socs1 upregulation. Also, Gas6 gamma-carboxylation is essential for ZIKV invasion and replication in monocytes, the main source of this protein, which was inhibited by warfarin. Conversely, Gas6 facilitates ZIKV replication in adult immunocompetent mice and enabled susceptibility to transplacental infection. Our data indicate that ZIKV promotes the upregulation of its ligand Gas6, which contributes to viral infectivity and drives the development of severe adverse outcomes during ZIKV infection.

Rosettes integrity protects Plasmodium vivax of being phagocytized.

Plasmodium vivax is the most prevalent cause of malaria outside of Africa. P. vivax biology and pathogenesis are still poorly understood. The role of one highly occurring phenotype in particular where infected reticulocytes cytoadhere to noninfected normocytes, forming rosettes, remains unknown. Here, using a range of ex vivo approaches, we showed that P. vivax rosetting rates were enhanced by plasma of infected patients and that total immunoglobulin M levels correlated with rosetting frequency. Moreover, rosetting rates were also correlated with parasitemia, IL-6 and IL-10 levels in infected patients. Transcriptomic analysis of peripheral leukocytes from P. vivax-infected patients with low or moderated rosetting rates identified differentially expressed genes related to human host phagocytosis pathway. In addition, phagocytosis assay showed that rosetting parasites were less phagocyted. Collectively, these results showed that rosette formation plays a role in host immune response by hampering leukocyte phagocytosis. Thus, these findings suggest that rosetting could be an effective P. vivax immune evasion strategy.

Plasmodium vivax in Hematopoietic Niches: Hidden and Dangerous.

Estimation of Plasmodium vivax biomass based on circulating biomarkers indicates the existence of a predominant biomass outside of the circulation that is not captured by peripheral parasitemia, in particular in patients with complicated outcomes. A series of recent studies have suggested that the hematopoietic niche of the bone marrow (BM) is a major reservoir for parasite replication and the development of transmission stages. However, significant knowledge gaps remain in our understanding of host-parasite interactions, pathophysiology, and the implications for treatment and diagnosis of such a reservoir. Here, we discuss the current status of this emerging research field in the context of P. vivax.

Inflammasome activation and IL-1 signaling during placental malaria induce poor pregnancy outcomes.

Placental malaria (PM) is associated with severe inflammation leading to abortion, preterm delivery, and intrauterine growth restriction. Innate immunity responses play critical roles, but the mechanisms underlying placental immunopathology are still unclear. Here, we investigated the role of inflammasome activation in PM by scrutinizing human placenta samples from an endemic area and ablating inflammasome components in a PM mouse model. The reduction in birth weight in babies from infected mothers is paralleled by increased placental expression of AIM2 and NLRP3 inflammasomes. Using genetic dissection, we reveal that inflammasome activation pathways are involved in the production and detrimental action of interleukin-1ß (IL-1ß) in the infected placenta. The IL-1R pharmacological antagonist Anakinra improved pregnancy outcomes by restoring fetal growth and reducing resorption in an experimental model. These findings unveil that IL-1ß-mediated signaling is a determinant of PM pathogenesis, suggesting that IL-1R antagonists can improve clinical outcomes of malaria infection in pregnancy.

Chemical Genomic Profiling Unveils the in Vitro and in Vivo Antiplasmodial Mechanism of Açaí (Euterpe oleracea Mart.) Polyphenols.

Malaria remains a major detrimental parasitic disease in the developing world, with more than 200 million cases annually. Widespread drug-resistant parasite strains push for the development of novel antimalarial drugs. Plant-derived natural products are key sources of antimalarial molecules. Euterpe oleracea Martius ("açaí") originates from Brazil and has anti-inflammatory and antineoplasic properties. Here, we evaluated the antimalarial efficacy of three phenolic fractions of açaí; total phenolics (1), nonanthocyanin phenolics (2), and total anthocyanins (3). In vitro, fraction 2 moderately inhibited parasite growth in chloroquine-sensitive (HB3) and multiresistant (Dd2) Plasmodium falciparum strains, while none of the fractions was toxic to noncancer cells. Despite the limited activity in vitro, the oral treatment with 20 mg/kg of fraction 1 reduced parasitemia by 89.4% in Plasmodium chabaudi-infected mice and prolonged survival. Contrasting in vitro and in vivo activities of 1 suggest key antiplasmodial roles for polyphenol metabolites rather than the fraction itself. Finally, we performed haploinsufficiency chemical genomic profiling (HIP) utilizing heterozygous Saccharomyces cerevisiae deletion mutants to identify molecular mechanisms of açaí fractions. HIP results indicate proteostasis as the main cellular pathway affected by fraction 2. These results open avenues to develop açaí polyphenols as potential new antimalarial candidates.

Antiplasmodial and trypanocidal activity of violacein and deoxyviolacein produced from synthetic operons.

BACKGROUND: Violacein is a deep violet compound that is produced by a number of bacterial species. It is synthesized from tryptophan by a pathway that involves the sequential action of 5 different enzymes (encoded by genes vioA to vioE). Violacein has antibacterial, antiparasitic, and antiviral activities, and also has the potential of inducing apoptosis in certain cancer cells. RESULTS: Here, we describe the construction of a series of plasmids harboring the complete or partial violacein biosynthesis operon and their use to enable production of violacein and deoxyviolacein in E.coli. We performed in vitro assays to determine the biological activity of these compounds against Plasmodium, Trypanosoma, and mammalian cells. We found that, while deoxyviolacein has a lower activity against parasites than violacein, its toxicity to mammalian cells is insignificant compared to that of violacein. CONCLUSIONS: We constructed E. coli strains capable of producing biologically active violacein and related compounds, and propose that deoxyviolacein might be a useful starting compound for the development of antiparasite drugs.

Immunization with the MAEBL M2 Domain Protects against Lethal Plasmodium yoelii Infection.

Malaria remains a world-threatening disease largely because of the lack of a long-lasting and fully effective vaccine. MAEBL is a type 1 transmembrane molecule with a chimeric cysteine-rich ectodomain homologous to regions of the Duffy binding-like erythrocyte binding protein and apical membrane antigen 1 (AMA1) antigens. Although MAEBL does not appear to be essential for the survival of blood-stage forms, ectodomains M1 and M2, homologous to AMA1, seem to be involved in parasite attachment to erythrocytes, especially M2. MAEBL is necessary for sporozoite infection of mosquito salivary glands and is expressed in liver stages. Here, the Plasmodium yoelii MAEBL-M2 domain was expressed in a prokaryotic vector. C57BL/6J mice were immunized with doses of P. yoelii recombinant protein rPyM2-MAEBL. High levels of antibodies, with balanced IgG1 and IgG2c subclasses, were achieved. rPyM2-MAEBL antisera were capable of recognizing the native antigen. Anti-MAEBL antibodies recognized different MAEBL fragments expressed in CHO cells, showing stronger IgM and IgG responses to the M2 domain and repeat region, respectively. After a challenge with P. yoelii YM (lethal strain)-infected erythrocytes (IE), up to 90% of the immunized animals survived and a reduction of parasitemia was observed. Moreover, splenocytes harvested from immunized animals proliferated in a dose-dependent manner in the presence of rPyM2-MAEBL. Protection was highly dependent on CD4(+), but not CD8(+), T cells toward Th1. rPyM2-MAEBL antisera were also able to significantly inhibit parasite development, as observed in ex vivo P. yoelii erythrocyte invasion assays. Collectively, these findings support the use of MAEBL as a vaccine candidate and open perspectives to understand the mechanisms involved in protection.

Glycophorin C (CD236R) mediates vivax malaria parasite rosetting to normocytes.

Rosetting phenomenon has been linked to malaria pathogenesis. Although rosetting occurs in all causes of human malaria, most data on this subject has been derived from Plasmodium falciparum. Here, we investigate the function and factors affecting rosette formation in Plasmodium vivax. To achieve this, we used a range of novel ex vivo protocols to study fresh and cryopreserved P vivax (n = 135) and P falciparum (n = 77) isolates from Thailand. Rosetting is more common in vivax than falciparum malaria, both in terms of incidence in patient samples and percentage of infected erythrocytes forming rosettes. Rosetting to P vivax asexual and sexual stages was evident 20 hours postreticulocyte invasion, reaching a plateau after 30 hours. Host ABO blood group, reticulocyte count, and parasitemia were not correlated with P vivax rosetting. Importantly, mature erythrocytes (normocytes), rather than reticulocytes, preferentially form rosetting complexes, indicating that this process is unlikely to directly facilitate merozoite invasion. Although antibodies against host erythrocyte receptors CD235a and CD35 had no effect, Ag-binding fragment against the BRIC 4 region of CD236R significantly inhibited rosette formation. Rosetting assays using CD236R knockdown normocytes derived from hematopoietic stem cells further supports the role of glycophorin C as a receptor in P vivax rosette formation.

Fucosylated chondroitin sulfate inhibits Plasmodium falciparum cytoadhesion and merozoite invasion.

Sequestration of Plasmodium falciparum-infected erythrocytes (Pf-iEs) in the microvasculature of vital organs plays a key role in the pathogenesis of life-threatening malaria complications, such as cerebral malaria and malaria in pregnancy. This phenomenon is marked by the cytoadhesion of Pf-iEs to host receptors on the surfaces of endothelial cells, on noninfected erythrocytes, and in the placental trophoblast; therefore, these sites are potential targets for antiadhesion therapies. In this context, glycosaminoglycans (GAGs), including heparin, have shown the ability to inhibit Pf-iE cytoadherence and growth. Nevertheless, the use of heparin was discontinued due to serious side effects, such as bleeding. Other GAG-based therapies were hampered due to the potential risk of contamination with prions and viruses, as some GAGs are isolated from mammals. In this context, we investigated the effects and mechanism of action of fucosylated chondroitin sulfate (FucCS), a unique and highly sulfated GAG isolated from the sea cucumber, with respect to P. falciparum cytoadhesion and development. FucCS was effective in inhibiting the cytoadherence of Pf-iEs to human lung endothelial cells and placenta cryosections under static and flow conditions. Removal of the sulfated fucose branches of the FucCS structure virtually abolished the inhibitory effects of FucCS. Importantly, FucCS rapidly disrupted rosettes at high levels, and it was also able to block parasite development by interfering with merozoite invasion. Collectively, these findings highlight the potential of FucCS as a candidate for adjunct therapy against severe malaria.

Adherence to human lung microvascular endothelial cells (HMVEC-L) of Plasmodium vivax isolates from Colombia.

BACKGROUND: For years Plasmodium vivax has been considered the cause of benign malaria. Nevertheless, it has been observed that this parasite can produce a severe disease comparable to Plasmodium falciparum. It has been suggested that some physiopathogenic processes might be shared by these two species, such as cytoadherence. Recently, it has been demonstrated that P. vivax-infected erythrocytes (Pv-iEs) have the capacity to adhere to endothelial cells, in which intercellular adhesion molecule-1 (ICAM-1) seems to be involved in this process. METHODS: Adherence capacity of 21 Colombian isolates, from patients with P. vivax mono-infection to a microvascular line of human lung endothelium (HMVEC-L) was assessed in static conditions and binding was evaluated at basal levels or in tumor necrosis factor (TNF) stimulated cells. The adherence specificity for the ICAM-1 receptor was determined through inhibition with an anti-CD54 monoclonal antibody. RESULTS: The majority of P. vivax isolates, 13 out of 21 (61.9%), adhered to the HMVEC-L cells, but P. vivax adherence was at least seven times lower when compared to the four P. falciparum isolates. Moreover, HMVEC-L stimulation with TNF led to an increase of 1.6-fold in P. vivax cytoadhesion, similar to P. falciparum isolates (1.8-fold) at comparable conditions. Also, blockage of ICAM-1 receptor with specific antibodies showed a significant 50% adherence reduction. CONCLUSIONS: Plasmodium vivax isolates found in Colombia are also capable of adhering specifically in vitro to lung endothelial cells, via ICAM-1 cell receptor, both at basal state and after cell stimulation with TNF. Collectively, these findings reinforce the concept of cytoadherence for P. vivax, but here, to a different endothelial cell line and using geographical distinct isolates, thus contributing to understanding P. vivax biology.

In vitro and in vivo assessment of the anti-malarial activity of Caesalpinia pluviosa.

BACKGROUND: To overcome the problem of increasing drug resistance, traditional medicines are an important source for potential new anti-malarials. Caesalpinia pluviosa, commonly named "sibipiruna", originates from Brazil and possess multiple therapeutic properties, including anti-malarial activity. METHODS: Crude extract (CE) was obtained from stem bark by purification using different solvents, resulting in seven fractions. An MTT assay was performed to evaluate cytotoxicity in MCF-7 cells. The CE and its fractions were tested in vitro against chloroquine-sensitive (3D7) and -resistant (S20) strains of Plasmodium falciparum and in vivo in Plasmodium chabaudi-infected mice. In vitro interaction with artesunate and the active C. pluviosa fractions was assessed, and mass spectrometry analyses were conducted. RESULTS: At non-toxic concentrations, the 100% ethanolic (F4) and 50% methanolic (F5) fractions possessed significant anti-malarial activity against both 3D7 and S20 strains. Drug interaction assays with artesunate showed a synergistic interaction with the F4. Four days of treatment with this fraction significantly inhibited parasitaemia in mice in a dose-dependent manner. Mass spectrometry analyses revealed the presence of an ion corresponding to m/z 303.0450, suggesting the presence of quercetin. However, a second set of analyses, with a quercetin standard, showed distinct ions of m/z 137 and 153. CONCLUSIONS: The findings show that the F4 fraction of C. pluviosa exhibits anti-malarial activity in vitro at non-toxic concentrations, which was potentiated in the presence of artesunate. Moreover, this anti-malarial activity was also sustained in vivo after treatment of infected mice. Finally, mass spectrometry analyses suggest that a new compound, most likely an isomer of quercetin, is responsible for the anti-malarial activity of the F4.

Immunogenic properties of a recombinant fusion protein containing the C-terminal 19 kDa of Plasmodium falciparum merozoite surface protein-1 and the innate immunity agonist FliC flagellin of Salmonella typhimurium.

In a recent study, we demonstrated the immunogenic properties of a new malaria vaccine polypeptide based on a 19 kDa C-terminal fragment of the merozoite surface protein-1 (MSP1(19)) from Plasmodium vivax and an innate immunity agonist, the Salmonella enterica serovar Typhimurium flagellin (FliC). Herein, we tested whether the same strategy, based on the MSP1(19) component of the deadly malaria parasite Plasmodium falciparum, could also generate a fusion polypeptide with enhanced immunogenicity. The His(6)FliC-MSP1(19) fusion protein was expressed from a recombinant Escherichia coli and showed preserved in vitro TLR5-binding activity. In contrast to animals injected with His(6)MSP1(19), mice subcutaneously immunised with the recombinant His(6)FliC-MSP1(19) developed strong MSP1(19)-specific systemic antibody responses with a prevailing IgG1 subclass. Incorporation of other adjuvants, such as CpG ODN 1826, complete and incomplete Freund's adjuvants or Quil-A, improved the IgG responses after the second, but not the third, immunising dose. It also resulted in a more balanced IgG subclass response, as evaluated by the IgG1/IgG2c ratio, and higher cell-mediated immune response, as determined by the detection of antigen-specific interferon-gamma secretion by immune spleen cells. MSP1(19)-specific antibodies recognised not only the recombinant protein, but also the native protein expressed on the surface of P. falciparum parasites. Finally, sera from rabbits immunised with the fusion protein alone inhibited the in vitro growth of three different P. falciparum strains. In summary, these results extend our previous observations and further demonstrate that fusion of the innate immunity agonist FliC to Plasmodium antigens is a promising alternative to improve their immunogenicity.

Ciliary neurotrophic factor fused to a protein transduction domain retains full neuroprotective activity in the absence of cytokine-like side effects.

Ciliary neurotrophic factor (CNTF) is a multifunctional cytokine that can regulate the survival and differentiation of many types of developing and adult neurons. CNTF prevents the degeneration of motor neurons after axotomy and in mouse mutant progressive motor neuronopathy, which has encouraged trials of CNTF for human motor neuron disease. Given systemically, however, CNTF causes severe side effects, including cachexia and a marked immune response, which has limited its clinical application. The present work describes a novel approach for administering recombinant human CNTF (rhCNTF) while conserving neurotrophic activity and avoiding deleterious side effects. rhCNTF was fused to a protein transduction domain derived from the human immunodeficiency virus-1 TAT (transactivator) protein. The resulting fusion protein (TAT-CNTF) crosses the plasma membrane within minutes and displays a nuclear localization. TAT-CNTF was equipotent to rhCNTF in supporting the survival of cultured chicken embryo dorsal root ganglion neurons. Local or subcutaneous administration of TAT-CNTF, like rhCNTF rescued motor neurons from death in neonatal rats subjected to sciatic nerve transection. In contrast to subcutaneous rhCNTF, which caused a 20-30% decrease in body weight in neonatal rats between postnatal days 2 and 7 together with a considerable fat mobilization in brown adipose tissue, TAT-CNTF lacked such side effects. Together, these results indicate that rhCNTF fused with the protein transduction domain/TAT retains neurotrophic activity in the absence of CNTFs cytokine-like side effects and may be a promising candidate for the treatment of motor neuron and other neurodegenerative diseases.

A biotechnological product and its potential as a new immunomodulator for treatment of animal phlebovirus infection: Punta Toro virus.

Intracellular pathogens with widespread drug-resistance contribute substantially to the increasing rates in morbidity and mortality due to emerging and reemerging diseases. Thus, the development of new drugs, including those that can enhance the immune response, is urgently needed. The immunomodulator, P-MAPA, a proteinaceous aggregate of ammonium and magnesium phospholinoleate-palmitoleate anhydride derived from Aspergillus oryzae, have been shown to induce antitumor activities. The ability of this compound to elicit protective immunity against viral infections has not been fully explored. Here, we report findings on the use of P-MAPA as an antiviral agent in a mouse model of acute phleboviral (Punta Toro virus) disease. A dose administered i.p. 24h post-infectious challenge (100mg/kg dose of P-MAPA) was remarkably effective at preventing death due to Punta Toro virus infection. This dose also reduced systemic viral burden and liver discoloration assayed on day 3 of infection. Taken together, our findings indicate that non-specific immunotherapy with P-MAPA appears to be an effective treatment for blocking Punta Toro virus-induced disease and suggest that further exploration with other viral disease models is warranted.

Violacein extracted from Chromobacterium violaceum inhibits Plasmodium growth in vitro and in vivo.

Violacein is a violet pigment extracted from the gram-negative bacterium Chromobacterium violaceum. It presents bactericidal, tumoricidal, trypanocidal, and antileishmanial activities. We show that micromolar concentrations efficiently killed chloroquine-sensitive and -resistant Plasmodium falciparum strains in vitro; inhibited parasitemia in vivo, even after parasite establishment; and protected Plasmodium chabaudi chabaudi-infected mice from a lethal challenge.

Cytotoxic activity of violacein in human colon cancer cells.

Several studies have shown that violacein, a purple pigment extracted from Chromobacterium violaceum, is capable to induce apoptosis in a variety of cancer cells, including those leukemia cell lines. Herein, we examined the effects of violacein on reactive oxygen species (ROS) production during the apoptotic colon cancer cell death. We demonstrate that violacein mediates ROS production followed by activation of Caspase-3, release of cytochrome c, and calcium release to citosol in Caco-2 cells. Moreover, presence of ROS scavengers such as N-acetyl-cysteine (NAC) diminishes ROS cytotoxicity induced by violacein in Caco-2 cells, indicating that violacein mediates cellular critical mechanisms in the triggering of apoptotic tumor cell death. These data also imply that violacein-induced ROS are collectively key mediators of mitochondrial membrane collapse, leading to cytochrome c release, and culminating in tumor apoptosis. Unlike in Caco-2 cells, violacein was incapable of increasing ROS levels in HT29 cells, suggesting the existence of violacein cell-type specific mechanisms. Those findings bring light to the violacein cytotoxic mechanism studies, indicating that oxidative stress play a role in the violacein-induced cytotoxicity.

Placenta cryosections for study of the adhesion of Plasmodium falciparum-infected erythrocytes to chondroitin sulfate A in flow conditions.

The adhesion of Plasmodium falciparum-infected erythrocytes (IEs) to chondroitin-4-sulfate (CSA) via the PfEMP1-CSA parasite ligand domain is correlated with placental malaria in primigravidae. The recent identification of parasite genes encoding CSA adhesion molecules and the development of pan-reactive monoclonal antibodies against the Pf(CSA) ligand have opened up new avenues for the development of anti-IE sequestration therapies for the prevention of placental malaria. A model closely mimicking placental sequestration of IEs during pregnancy is needed for the preclinical and clinical evaluation of candidate molecules for the induction of antibodies that could protect pregnant women from placental malaria. We found that normal placenta cryosections were a specific and highly consistent support for the binding of IEs to CSA in flow conditions under physiological conditions. This model makes possible the quantitative and qualitative analysis of IE adhesion. We identified distinct CSA-binding phenotypes within the FCR3(CSA)-selected parasites in flow analyses, but not in static analyses. We also analyzed inhibitors of placental parasite binding such as soluble CSA and antibodies directed against the Pf(CSA) ligand. Our data demonstrate that placenta cryosections could be used to standardize assays between laboratories, potentially advancing the development of therapies against placental malaria.

Immunization with recombinant duffy binding-like-gamma3 induces pan-reactive and adhesion-blocking antibodies against placental chondroitin sulfate A-binding Plasmodium falciparum parasites.

Maternal malaria is associated with the sequestration, in the placenta, of Plasmodium falciparum-infected erythrocytes onto chondroitin sulfate A (CSA), via the duffy binding-like (DBL)-gamma3 domain of the P. falciparum erythrocyte membrane protein 1 (PfEMP1(CSA)) (DBL-gamma3(CSA)). The production of antibodies against CSA-binding infected erythrocytes (IEs(CSA)) is correlated with resistance to maternal malaria in multiparous women. We produced recombinant DBL-gamma3(CSA) (rDBL-gamma3(CSA)) in insect cells, corresponding to 2 variant DBL-gamma3(CSA) subtypes that mediate binding to CSA in laboratory lines and placental isolates. Both recombinant cysteine-rich DBL-gamma3(CSA) domains blocked IEs(CSA) binding to CSA. Immunization of mice, with the rDBL-gamma3(CSA)-FCR3 and rDBL-gamma3(CSA)-3D7 domains, resulted in the generation of antibodies recognizing homologous and heterologous rDBL-gamma3(CSA), a finding indicating conserved epitopes inducing a pan-reactive immune response. Mouse monoclonal antibodies (MAbs) against both recombinant proteins were pan-reactive with various IEs(CSA). One MAb efficiently inhibited and reversed IE(CSA) cytoadhesion to endothelial cells in vitro. Thus, DBL-gamma3(CSA) is the target of inhibitory and pan-reactive antibodies. Saimiri sciureus monkeys immunized with FCR3-rDBL-gamma3(CSA) developed pan-reactive and inhibitory antibodies, a finding suggesting that the development of a vaccine to prevent maternal malaria is feasible.