Novel vaccines are urgently needed to reduce the burden of severe malaria. Using a differential whole-proteome screening method, we identified Plasmodium falciparum schizont egress antigen-1 ...(PfSEA-1), a 244-kilodalton parasite antigen expressed in schizont-infected red blood cells (RBCs). Antibodies to PfSEA-1 decreased parasite replication by arresting schizont rupture, and conditional disruption of PfSEA-1 resulted in a profound parasite replication defect. Vaccination of mice with recombinant Plasmodium berghei PbSEA-1 significantly reduced parasitemia and delayed mortality after lethal challenge with the Plasmodium berghei strain ANKA. Tanzanian children with antibodies to recombinant PfSEA-1A (rPfSEA-1A) did not experience severe malaria, and Kenyan adolescents and adults with antibodies to rPfSEA-1A had significantly lower parasite densities than individuals without these antibodies. By blocking schizont egress, PfSEA-1 may synergize with other vaccines targeting hepatocyte and RBC invasion.
Malaria caused by Plasmodium falciparum remains the leading single-agent cause of mortality in children
, yet the promise of an effective vaccine has not been fulfilled. Here, using our previously ...described differential screening method to analyse the proteome of blood-stage P. falciparum parasites
, we identify P. falciparum glutamic-acid-rich protein (PfGARP) as a parasite antigen that is recognized by antibodies in the plasma of children who are relatively resistant-but not those who are susceptible-to malaria caused by P. falciparum. PfGARP is a parasite antigen of 80 kDa that is expressed on the exofacial surface of erythrocytes infected by early-to-late-trophozoite-stage parasites. We demonstrate that antibodies against PfGARP kill trophozoite-infected erythrocytes in culture by inducing programmed cell death in the parasites, and that vaccinating non-human primates with PfGARP partially protects against a challenge with P. falciparum. Furthermore, our longitudinal cohort studies showed that, compared to individuals who had naturally occurring anti-PfGARP antibodies, Tanzanian children without anti-PfGARP antibodies had a 2.5-fold-higher risk of severe malaria and Kenyan adolescents and adults without these antibodies had a twofold-higher parasite density. By killing trophozoite-infected erythrocytes, PfGARP could synergize with other vaccines that target parasite invasion of hepatocytes or the invasion of and egress from erythrocytes.
Abstract
Spreading antimalarial resistance threatens effective treatment of malaria, an infectious disease caused by Plasmodium parasites. We identified a compound, BCH070, that inhibits asexual ...growth of multiple antimalarial-resistant strains of Plasmodium falciparum (half maximal inhibitory concentration IC50 = 1–2 µM), suggesting that BCH070 acts via a novel mechanism of action. BCH070 preferentially kills early ring-form trophozoites, and, importantly, equally inhibits ring-stage survival of wild-type and artemisinin-resistant parasites harboring the PfKelch13:C580Y mutation. Metabolomic analysis demonstrates that BCH070 likely targets multiple pathways in the parasite. BCH070 is a promising lead compound for development of new antimalarial combination therapy that retains activity against artemisinin-resistant parasites.
BCH070 inhibits growth of antimalarial-resistant strains of Plasmodium falciparum, including artemisinin-resistant parasites. BCH070 is active during a unique life-cycle stage, likely targeting multiple parasite pathways. Thus, BCH070 is a strong lead candidate in the development of novel antimalarial combination therapy.
Abstract
Background
In holoendemic areas, children suffer the most from Plasmodium falciparum malaria, yet newborns and young infants express a relative resistance to both infection and severe ...malarial disease (SM). This relative resistance has been ascribed to maternally-derived anti-parasite immunoglobulin G; however, the targets of these protective antibodies remain elusive.
Methods
We enrolled 647 newborns at birth from a malaria-holoendemic region of Tanzania. We collected cord blood, measured antibodies to Plasmodium falciparum Schizont Egress Antigen-1 (PfSEA-1), and related these antibodies to the risk of severe malaria in the first year of life. In addition, we vaccinated female mice with PbSEA-1, mated them, and challenged their pups with P. berghei ANKA parasites to assess the impact of maternal PbSEA-1 vaccination on newborns’ resistance to malaria.
Results
Children with high cord-blood anti–PfSEA-1 antibody levels had 51.4% fewer cases of SM compared to individuals with lower anti–PfSEA-1 levels over 12 months of follow-up (P = .03). In 3 trials, pups born to PbSEA-1–vaccinated dams had significantly lower parasitemia and longer survival following a P. berghei challenge compared to pups born to control dams.
Conclusions
We demonstrate that maternally-derived, cord-blood anti–PfSEA-1 antibodies predict decreased risk of SM in infants and vaccination of mice with PbSEA-1 prior to pregnancy protects their offspring from lethal P. berghei challenge. These results identify, for the first time, a parasite-specific target of maternal antibodies that protect infants from SM and suggest that vaccination of pregnant women with PfSEA-1 may afford a survival advantage to their offspring.
Maternally-derived, cord-blood anti–Plasmodium falciparum Schizont Egress Antigen-1 antibodies significantly decrease infants’ risks of severe Plasmodium falciparum malaria. The vaccination of mice with P. berghei Schizont Egress Antigen-1 prior to pregnancy protects offspring from lethal P. berghei parasite challenges.
We previously identified a Plasmodium falciparum (Pf) protein of unknown function encoded by a single-copy gene, PF3D7_1134300, as a target of antibodies in plasma of Tanzanian children in a ...whole-proteome differential screen. Here we characterize this protein as a blood-stage antigen that localizes to the surface membranes of both parasitized erythrocytes and merozoites, hence its designation as Pf erythrocyte membrane and merozoite antigen 1 (PfEMMA1). Mouse anti-PfEMMA1 antisera and affinity-purified human anti-PfEMMA1 antibodies inhibited growth of P. falciparum strains by up to 68% in growth inhibition assays. Following challenge with uniformly fatal Plasmodium berghei (Pb) ANKA, up to 40% of mice immunized with recombinant PbEMMA1 self-cured, and median survival of lethally infected mice was up to 2.6-fold longer than controls (21 vs. 8 d, P = 0.005). Furthermore, high levels of naturally acquired human anti-PfEMMA1 antibodies were associated with a 46% decrease in parasitemia over 2.5 yr of follow-up of Tanzanian children. Together, these findings suggest that antibodies to PfEMMA1 mediate protection against malaria.
eNOS (endothelial nitric oxide synthase) contains a MAPK (mitogen-activated protein kinase)-binding site associated with a major eNOS control element. Purified ERK (extracellular-signal-regulated ...kinase) phosphorylates eNOS with a stoichiometry of 2-3 phosphates per eNOS monomer. Phosphorylation decreases NO synthesis and cytochrome c reductase activity. Three sites of phosphorylation were detected by MS. All sites matched the SP and TP MAPK (mitogen-activated protein kinase) phosphorylation motif. Ser602 lies at the N-terminal edge of the 42-residue eNOS AI (autoinhibitory) element. The pentabasic MAPK-binding site lies at the opposite end of the AI, and other critical regulatory features are between them. Thr46 and Ser58 are located in a flexible region associated with the N terminus of the oxygenase domain. In contrast with PKC (protein kinase C), phosphorylation by ERK did not significantly interfere with CaM (calmodulin) binding as analysed by optical biosensing. Instead, ERK phosphorylation favours a state in which FMN and FAD are in close association and prevents conformational changes that expose reduced FMN to acceptors. The close associations between control sites in a few regions of the molecule suggest that control of signal generation is modulated by multiple inputs interacting directly on the surface of eNOS via overlapping binding domains and tightly grouped targets.
A single step novel multiplex polymerase chain reaction (PCR) has been developed for simultaneous detection of human filarial parasites,
Brugia malayi and
Wuchereria bancrofti, from blood samples and ...mosquitoes. The primers used were novel and have been tested with the parasite DNA amplifying 188
bp (
BM) and 129
bp (
WB) DNA fragments, specific to
B. malayi and
W. bancrofti, respectively, in a single reaction. The specificity of the PCR product was confirmed by DNA sequencing and slot blot hybridization assay. The test was found highly sensitive for both
B. malayi and
W. bancrofti by detecting the parasitaemia up to the level of one microfilaria per reaction. The assay was further evaluated on 98 blood samples and 144 mosquito samples collected from filarial endemic areas. The PCR was found to be more efficient in comparison to microscopy by detecting 8% and 5% more filarial parasites in field-collected blood and mosquito samples, respectively. This novel PCR that offers scope for simultaneous detection of both the parasites may be used as a diagnostic tool for the detection of filariasis in population and can be adopted for rapid surveillance and monitoring of mosquitoes for use in the effective control of filariasis.
Abstract only
Nitric Oxide synthases produce NO as a signal and as a cytotoxin in immune response. In the tethered shuttle model for NOS reductase kinetics, the FMN domain moves between NADPH ...dehydrogenase and oxygenase catalytic sites. FMN fluorescence is sensitive to NOS conformation; eNOS and nNOS holoenzymes have states with lifetimes of ~4.3ns, 1 ns, and 90 ps. Two domain oxyFMN constructs (truncated before the FAD binding domain) and holoenzyme share open conformations with lifetimes of ~4.3 ns. The holoenzyme majority state has a lifetime of ~90 ps due to FAD/FMN proximity as in crystal structures (Wang et al, Garcin et al). In oxyFMN ~25% of FMN has a lifetime of 0.9 ns, which we attribute to quenching by heme. Calmodulin activation of NOS releases the FMN binding domain from the ‘dehydrogenase’ complex, increasing the populations of the long lifetime states. Calmodulin binding shortens the average lifetime of oxyFMN constructs, because CaM promotes association of the FMN binding domain with the oxygenase domain, and favors the 1 ns state over the 4.3 ns state. Similar effects help explain modulation of NOS activity by covalent modification and protein‐protein interactions, e.g., in the HSP90‐eNOS complex.
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