No abstract

erythrocyte membrane protein 1 (PfEMP1) mediates parasite sequestration to the cerebral microvasculature via binding of DBLβ domains to intercellular adhesion molecule 1 (ICAM1) and is associated with severe cerebral malaria. In a cohort of 187 young children from Papua New Guinea (PNG), we examined baseline levels of antibody to the ICAM1-binding PfEMP1 domain, DBLβ3, in comparison to four control antigens, including NTS-DBLα and CIDR1 domains from another group A variant and a group B/C variant. Antibody levels for the group A antigens were strongly associated with age and exposure. Antibody responses to DBLβ3 were associated with a 37% reduced risk of high-density clinical malaria in the follow-up period (adjusted incidence risk ratio [aIRR] = 0.63 [95% confidence interval {CI}, 0.45 to 0.88; = 0.007]) and a 25% reduction in risk of low-density clinical malaria (aIRR = 0.75 [95% CI, 0.55 to 1.01; = 0.06]), while there was no such association for other variants. Children who experienced severe malaria also had significantly lower levels of antibody to DBLβ3 and the other group A domains than those that experienced nonsevere malaria. Furthermore, a subset of PNG DBLβ sequences had ICAM1-binding motifs, formed a distinct phylogenetic cluster, and were similar to sequences from other areas of endemicity. PfEMP1 variants associated with these DBLβ domains were enriched for DC4 and DC13 head structures implicated in endothelial protein C receptor (EPCR) binding and severe malaria, suggesting conservation of dual binding specificities. These results provide further support for the development of specific classes of PfEMP1 as vaccine candidates and as biomarkers for protective immunity against clinical malaria.

This 2-step screening approach should be efficient for identification of antiadhesion drug candidates for falciparum malaria.

Tandem Oligonucleotide Repeat Cascade Amplification (TORCA) based on signal rather than target amplification under isothermal conditions was developed for nucleic acid assays. The initial signal was generated by hybridization of single stranded DNA targets to immobilized recognition probes followed by hybrid cleavage with specific restriction endonuclease (REase), and release of trigger oligonucleotides (Tr1). The signal amplification chamber contained two bead types carrying single-stranded amplification probes and two amplification REases. The probes consisted of multiple tandem repeats of either Tr1 or another trigger Tr2, with the tandem-Tr1 anchored to the beads through the antisense Tr2 linker and vice versa. Addition of the recognition reaction solution and Tr1 hybridization to the anti-Tr1 linkers started cleavage and release of additional Tr1 and Tr2, resulting in exponential signal amplification. The cleavage cascade also released horseradish peroxidase (HRP) pre-attached to the amplification probes, and the resultant signal was measured colorimetrically. A TORCA assay was developed for detection of Plasmodium falciparum parasites in blood. It had the detection limit in the attomolar concentration range, successfully detecting sub-microscopic P . falciparum infections at less than 0.75 infected erythrocytes per microliter. Further TORCA optimization will likely produce the quantitative isothermal alternative to PCR at a fraction of its cost.

Major complications and mortality from Plasmodium falciparum malaria are associated with cytoadhesion of parasite-infected erythrocytes (IE). The main parasite ligands for cytoadhesion are members of the P. falciparum erythrocyte membrane protein 1 (PfEMP1) family. Interactions of different host receptor-ligand pairs may lead to various pathological outcomes, like placental or cerebral malaria. It has been shown previously that IE can bind integrin αVβ3. Using bead-immobilized PfEMP1 constructs, we have identified that the PFL2665c DBLδ1_D4 domain binds to αVβ3 and αVβ6. A parasite line expressing PFL2665c binds to surface-immobilized αVβ3 and αVβ6; both are RGD motif-binding integrins. Interactions can be inhibited by cyloRGDFV peptide, an antagonist of RGD-binding integrins. This is a first, to the best of our knowledge, implication of a specific PfEMP1 domain for binding to integrins. These host receptors have important physiological functions in endothelial and immune cells; therefore, these results will contribute to future studies and a better understanding, at the molecular level, of the physiological outcome of interactions between IE and integrin receptors on the surface of host cells.