MicrobiologyBytes

A paper in this week’s PLoS Medicine compares the activity of 50 current and experimental antimalarials against liver, sexual blood, and mosquito stages of selected human and nonhuman parasite species, including Plasmodium falciparum, Plasmodium berghei and Plasmodium yoelii. These results provide a valuable guide to help researchers decide which drugs and compounds show most promise as potential future antimalarial drugs for blocking the transmission of malaria.

The Activities of Current Antimalarial Drugs on the Life Cycle Stages of Plasmodium Life Cycle: A Comparative Study with Human and Rodent Parasites. (2012) PLoS Med 9(2): e1001169. doi:10.1371/journal.pmed.1001169
Background: Malaria remains a disease of devastating global impact, killing more than 800,000 people every year—the vast majority being children under the age of 5. While effective therapies are available, if malaria is to be eradicated a broader range of small molecule therapeutics that are able to target the liver and the transmissible sexual stages are required. These new medicines are needed both to meet the challenge of malaria eradication and to circumvent resistance.
Methods and Findings: Little is known about the wider stage-specific activities of current antimalarials that were primarily designed to alleviate symptoms of malaria in the blood stage. To overcome this critical gap, we developed assays to measure activity of antimalarials against all life stages of malaria parasites, using a diverse set of human and nonhuman parasite species, including male gamete production (exflagellation) in Plasmodium falciparum, ookinete development in P. berghei, oocyst development in P. berghei and P. falciparum, and the liver stage of P. yoelii. We then compared 50 current and experimental antimalarials in these assays. We show that endoperoxides such as OZ439, a stable synthetic molecule currently in clinical phase IIa trials, are strong inhibitors of gametocyte maturation/gamete formation and impact sporogony; lumefantrine impairs development in the vector; and NPC-1161B, a new 8-aminoquinoline, inhibits sporogony.
Conclusions: These data enable objective comparisons of the strengths and weaknesses of each chemical class at targeting each stage of the lifecycle. Noting that the activities of many compounds lie within achievable blood concentrations, these results offer an invaluable guide to decisions regarding which drugs to combine in the next-generation of antimalarial drugs. This study might reveal the potential of life-cycle–wide analyses of drugs for other pathogens with complex life cycles.

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The asexual erythrocytic phase of the life cycle of Plasmodium falciparum produces the clinical symptoms, disease and pathology associated with malaria. During this phase, merozoites released from schizont-infected erythrocytes invade uninfected erythrocytes. Invasion depends on distinct molecular interactions between ligands on the merozoite, the invasive form of the parasite, and host receptors on the erythrocyte membrane. To avoid infection, humans have evolved to eliminate or modify erythrocyte surface proteins that serve as receptors for parasite invasion. Perhaps one of the best examples of this evolutionary process is the loss of the Duffy blood group in Africa. Plasmodium vivax depends on two ligands for erythrocyte invasion: the Duffy-binding protein (DBP) that binds the Duffy blood group antigen and the reticulocyte homology protein that binds to an unknown receptor on reticulocytes.

Unlike P. vivax, P. falciparum has highly redundant, alternate invasion pathways that use several different receptor families. P. vivax has only one gene, DBP, in the Duffybinding-like erythrocyte-binding protein (DBL-EBP) family, whereas P. falciparum has four DBL-EBP genes: erythrocytebinding antigen 175 (EBA-175), erythrocyte-binding antigen 140 (BAEBL/EBA-140), erythrocyte-binding antigen 181 (JESEBL/ EBA-181), and erythrocyte-binding ligand-1 (EBL-1). Consequently, no erythrocyte has been identified that is refractory to P. falciparum invasion. A recent paper provides evidence that the fourth DBL-EBP family member, EBL-1, binds to glycophorin B.

Theoretical studies indicate that a null allele of glycophorin B would need to afford only a modest level of protection against malaria in heterozygous to increase in frequency from a single mutant to an allele frequency of 0.59. Assuming a constant population of size 1,000–10,000 individuals, one need invoke a selective advantage of only 1% in homozygous-null genotypes to have a single copy of the null mutant allele increase to a frequency of 59% across an interval of 100,000 years (5,000 generations). A shorter time entails stronger selection, but even for 10,000 years (500 generations), a selective advantage of only 10% in homozygous-null genotypes is required. Both cases require partial dominance corresponding to 10–20% as much protection in heterozygous genotypes as in the homozygous-null.

Glycophorin B is the erythrocyte receptor of Plasmodium falciparum erythrocyte-binding ligand, EBL-1. PNAS USA March 11, 2009
In the war against Plasmodium, humans have evolved to eliminate or modify proteins on the erythrocyte surface that serve as receptors for parasite invasion, such as the Duffy blood group, a receptor for Plasmodium vivax, and the Gerbich-negative modification of glycophorin C for Plasmodium falciparum. In turn, the parasite counters with expansion and diversification of ligand families. The high degree of polymorphism in glycophorin B found in malaria-endemic regions suggests that it also may be a receptor for Plasmodium, but, to date, none has been identified. We provide evidence from erythrocyte-binding that glycophorin B is a receptor for the P. falciparum protein EBL-1, a member of the Duffy-binding-like erythrocyte-binding protein (DBL-EBP) receptor family. The erythrocyte-binding domain, region 2 of EBL-1, expressed on CHO-K1 cells, bound glycophorin B+ but not glycophorin B-null erythrocytes. In addition, glycophorin B+ but not glycophorin B-null erythrocytes adsorbed native EBL-1 from the P. falciparum culture supernatants. Interestingly, the Efe pygmies of the Ituri forest in the Democratic Republic of the Congo have the highest gene frequency of glycophorin B-null in the world, raising the possibility that the DBL-EBP family may have expanded in response to the high frequency of glycophorin B-null in the population.

Related:

• Malaria: now you see me, now you don’t
• New ways to beat malaria
• Researchers characterize potential protein targets for malaria vaccine

Malaria is a major global health problem responsible for more than one million deaths annually. Severity of malaria disease is associated with the inability of host immune cells to efficiently eliminate malaria parasites from the blood. Little is known about immune regulatory factors controlling the onset of severe and potentially fatal malaria. Regulatory T (Treg) cells are a small specialized subset of immune cells that suppress the activation and expansion of effector immune cells which partake in parasite elimination. Scientists have now investigated the relationship between Treg cells, parasite burden, and disease severity in adult malaria patients with either uncomplicated or severe malaria.

They were able to demonstrate that Treg cell frequency was elevated in malaria patients and associated with high parasite burden in severe malaria but not in uncomplicated malaria. This type of cell turns off the immune system and can allow the parasite to grow uncontrollably. Comparison of Treg cell characteristics allowed them to identify a new highly suppressive subset of Treg cells that was elevated in severe malaria patients. When comparing Treg cell characteristics, the team was able to identify elevated levels of a new highly suppressive subset of Treg cells in those patients with severe malaria. The regulatory (Treg) cell subset associated with severe disease in humans expresses a unique combination of surface markers, including TNFRII. Regulatory T (Treg) cells are a small specialized subset of immune cells that suppress the activation and expansion of effector immune cells, which partake in parasite elimination.

These results indicate that severe malaria is accompanied by the induction of highly suppressive Treg cells that can promote parasite growth and caution against the induction of these Treg cells when developing effective malaria vaccines. It is estimated that 500 million people live in areas where there is a risk of getting malaria. The severe form of the disease causes death in 1-3 million people each year. Until now it had been largely unknown what bodily factors enable some patients to fight and survive the disease, while other patients contract the severe form of the disease and sometimes die. Targeting this cell type may lead to new drugs and immunotherapeutics against malaria. Further studies are needed to determine if this new cell may also be promoting severe forms of other inflammatory diseases.

Parasite-Dependent Expansion of TNF Receptor II–Positive Regulatory T Cells with Enhanced Suppressive Activity in Adults with Severe Malaria. 2009 PLoS Pathog 5(4): e1000402
Severe Plasmodium falciparum malaria is a major cause of global mortality, yet the immunological factors underlying progression to severe disease remain unclear. CD4+CD25+ regulatory T cells (Treg cells) are associated with impaired T cell control of Plasmodium spp infection. We investigated the relationship between Treg cells, parasite biomass, and P. falciparum malaria disease severity in adults living in a malaria-endemic region of Indonesia. CD4+CD25+Foxp3+CD127lo Treg cells were significantly elevated in patients with uncomplicated and severe malaria relative to exposed asymptomatic controls. In patients with SM, Treg cell frequency correlated positively with parasitemia and total parasite biomass, both major determinants for the development of severe and fatal malaria, and Treg cells were significantly increased in hyperparasitemia. There was a further significant correlation between Treg cell frequency and plasma concentrations of soluble tumor necrosis factor receptor II (TNFRII) in SM. A subset of TNFRII+ Treg cells with high expression of Foxp3 was increased in severe relative to uncomplicated malaria. In vitro, P. falciparum–infected red blood cells dose dependently induced TNFRII+Foxp3hi Treg cells in PBMC from malaria-unexposed donors which showed greater suppressive activity than TNFRII2 Treg cells. The selective enrichment of the Treg cell compartment for a maximally suppressive TNFRII+Foxp3hi Treg subset in severe malaria provides a potential link between immune suppression, increased parasite biomass, and malaria disease severity. The findings caution against the induction of TNFRII+Foxp3hi Treg cells when developing effective malaria vaccines.

Related:

• Malaria: now you see me, now you don’t
• New ways to beat malaria
• Researchers characterize potential protein targets for malaria vaccine