Dror I. Baruch

The pathogenic basis of malaria

Malaria is today a disease of poverty and underdeveloped countries. In Africa, mortality remains high because there is limited access to treatment in the villages. We should follow in Pasteurs footsteps by using basic research to develop better tools for the control and cure of malaria. Insight into the complexity of malaria pathogenesis is vital for understanding the disease and will provide a major step towards controlling it. Those of us who work on pathogenesis must widen our approach and think in terms of new tools such as vaccines to reduce disease. The inability of many countries to fund expensive campaigns and antimalarial treatment requires these tools to be highly effective and affordable.

Cloning the P. falciparum gene encoding PfEMP1, a malarial variant antigen and adherence receptor on the surface of parasitized human erythrocytes

Plasmodium falciparum-infected human erythrocytes evade host immunity by expression of a cell-surface variant antigen and receptors for adherence to endothelial cells. These properties have been ascribed to P. falciparum erythrocyte membrane protein 1 (PfEMP1), an antigenically diverse malarial protein of 200-350 kDa on the surface of parasitized erythrocytes (PEs). We describe the cloning of two related PfEMP1 genes from the Malayan Camp (MC) parasite strain. Antibodies generated against recombinant protein fragments of the genes were specific for MC strain PfEMP1 protein. These antibodies reacted only with the surface of MC strain PEs and blocked adherence of these cells to CD36 but without effect on adherence to thrombospondin. Multiple forms of the PfEMP1 gene are apparent in MC parasites. The molecular basis for antigenic variation in malaria and adherence of infected erythrocytes to host cells can now be pursued.

Genetic diversity and chloroquine selective sweeps in Plasmodium falciparum

Widespread use of antimalarial agents can profoundly influence the evolution of the human malaria parasite Plasmodium falciparum. Recent selective sweeps for drug-resistant genotypes may have restricted the genetic diversity of this parasite, resembling effects attributed in current debates to a historic population bottleneck. Chloroquine-resistant (CQR) parasites were initially reported about 45 years ago from two foci in southeast Asia and South America, but the number of CQR founder mutations and the impact of chlorquine on parasite genomes worldwide have been difficult to evaluate. Using 342 highly polymorphic microsatellite markers from a genetic map, here we show that the level of genetic diversity varies substantially among different regions of the parasite genome, revealing extensive linkage disequilibrium surrounding the key CQR gene pfcrt and at least four CQR founder events. This disequilibrium and its decay rate in the pfcrt-flanking region are consistent with strong directional selective sweeps occurring over only ∼20–80 sexual generations, especially a single resistant pfcrt haplotype spreading to very high frequencies throughout most of Asia and Africa. The presence of linkage disequilibrium provides a basis for mapping genes under drug selection in P. falciparum.

Classification of adhesive domains in the Plasmodium falciparum erythrocyte membrane protein 1 family.

The Plasmodium falciparum Erythrocyte Membrane Protein 1 (PfEMP1) family of cytoadherent proteins has a central role in disease from malaria infection. This highly diverse gene family is involved in binding interactions between infected erythrocytes and host cells and is expressed in a clonally variant pattern at the erythrocyte surface. We describe by sequence analysis the structure and domain organization of 20 PfEMP1 from the GenBank database. Four domains comprise the majority of PfEMP1 extracellular sequence: the N-terminal segment (NTS) located at the amino terminus of all PfEMP1, the C2, the Cysteine-rich Interdomain Region (CIDR) and the Duffy Binding-like (DBL) domains. Previous work has shown that CIDR and DBL domains can possess adhesive properties. CIDR domains grouped as three distinct sequence classes (alpha, beta, and gamma) and DBL domains as five sequence classes (alpha, beta, gamma, delta, and epsilon). Consensus motifs are described for the different DBL and CIDR types. Whereas the number of DBL and CIDR domains vary between PfEMP1, PfEMP1 domain architecture is not random in that certain tandem domain associations--such as DBLalphaCIDRalpha, DBLdeltaCIDRbeta, and DBLbetaC2--are preferentially observed. This conservation may have functional significance for PfEMP1 folding, transport, or binding activity. Parasite binding phenotype appears to be a determinant of infected erythrocyte tissue tropism that contributes to parasite survival, transmission, and disease outcome. The sequence classification of DBL and CIDR types may have predictive value for identifying PfEMP1 domains with a particular binding property. This information might be used to develop interventions targeting parasite binding variants that cause disease.

Abnormal display of PfEMP-1 on erythrocytes carrying haemoglobin C may protect against malaria.

Haemoglobin C, which carries a glutamate-to-lysine mutation in the β-globin chain, protects West African children against Plasmodium falciparum malaria. Mechanisms of protection are not established for the heterozygous (haemoglobin AC) or homozygous (haemoglobin CC) states. Here we report a marked effect of haemoglobin C on the cell-surface properties of P. falciparum-infected erythrocytes involved in pathogenesis. Relative to parasite-infected normal erythrocytes (haemoglobin AA), parasitized AC and CC erythrocytes show reduced adhesion to endothelial monolayers expressing CD36 and intercellular adhesion molecule-1 (ICAM-1). They also show impaired rosetting interactions with non-parasitized erythrocytes, and reduced agglutination in the presence of pooled sera from malaria-immune adults. Abnormal cell-surface display of the main variable cytoadherence ligand, PfEMP-1 (P. falciparum erythrocyte membrane protein-1), correlates with these findings. The abnormalities in PfEMP-1 display are associated with markers of erythrocyte senescence, and are greater in CC than in AC erythrocytes. Haemoglobin C might protect against malaria by reducing PfEMP-1-mediated adherence of parasitized erythrocytes, thereby mitigating the effects of their sequestration in the microvasculature.

Immunization of Aotus monkeys with a functional domain of the Plasmodium falciparum variant antigen induces protection against a lethal parasite line

Immunity to Plasmodium falciparum in African children has been correlated with antibodies to the P. falciparum erythrocyte membrane protein 1 (PfEMP1) variant gene family expressed on the surface of infected red cells. We immunized Aotus monkeys with a subregion of the Malayan Camp variant antigen (MCvar1) that mediates adhesion to the host receptor CD36 on the endothelial surface and present data that PfEMP1 is an important target for vaccine development. The immunization induced a high level of protection against the homologous strain. Protection correlated with the titer of agglutinating antibodies and occurred despite the expression of variant copies of the gene during recurrent waves of parasitemia. A second challenge with a different P. falciparum strain, to which there was no agglutinating activity, showed no protection but boosted the immune response to this region during the infection. The level of protection and the evidence of boosting during infection encourage further exploration of this concept for malaria vaccine development.

Molecular basis for the dichotomy in Plasmodium falciparum adhesion to CD36 and chondroitin sulfate A

Plasmodium falciparum-infected erythrocytes adhere dichotomously to the host receptors CD36 and chondroitin sulfate A (CSA). This dichotomy is associated with parasite sequestration to microvasculature beds (CD36) or placenta (CSA), leading to site-specific pathogenesis. Both properties are mediated by members of the variant P. falciparum erythrocyte membrane protein 1 (PfEMP-1) family and reside on nonoverlapping domains of the molecule. To identify the molecular basis for the apparent dichotomy, we expressed various domains of PfEMP-1 individually or in combination and tested their binding properties. We found that the CD36-binding mode of the cysteine-rich interdomain region-1 (CIDR1) ablates the ability of the Duffy binding-like γ domain to bind CSA. In contrast, neither a non-CD36-binding CIDR1 nor an intercellular adhesion molecule 1 binding domain had any affect on CSA binding. Our findings point out that interactions between different domains of PfEMP-1 can alter the adhesion phenotype of infected erythrocytes and provide a molecular basis for the apparent dichotomy in adhesion. We suggest that the basis for the dichotomy is structural and that mutually exclusive conformations of PfEMP-1 are involved in binding to CD36 or CSA. Furthermore, we propose a model explaining the requirement for structural dichotomy between placental and nonplacental isolates.

The surface variant antigens of Plasmodium falciparum contain cross-reactive epitopes

Plasmodium falciparum parasites evade the host immune system by clonal expression of the variant antigen, P. falciparum erythrocyte membrane protein 1 (PfEMP1). Antibodies to PfEMP1 correlate with development of clinical immunity but are predominantly variant-specific. To overcome this major limitation for vaccine development, we set out to identify cross-reactive epitopes on the surface of parasitized erythrocytes (PEs). We prepared mAbs to the cysteine-rich interdomain region 1 (CIDR1) of PfEMP1 that is functionally conserved for binding to CD36. Two mAbs, targeting different regions of CIDR1, reacted with multiple P. falciparum strains expressing variant PfEMP1s. One of these mAbs, mAb 6A2-B1, recognized nine of 10 strains tested, failing to react with only one strain that does not bind CD36. Flow cytometry with Chinese hamster ovary cells expressing variant CIDR1s demonstrated that both mAbs recognized the CIDR1 of various CD36-binding PfEMP1s and are truly cross-reactive. The demonstration of cross-reactive epitopes on the PE surface provides further credence for development of effective vaccines against the variant antigen on the surface of P. falciparum-infected erythrocytes.

A recombinant peptide based on Pf EMP-1 blocks and reverses adhesion of malaria-infected red blood cells to CD36 under flow

During falciparum malaria infection, severe complications ensue because parasitized red blood cells (PRBCs) adhere to endothelial cells and accumulate in the microvasculature. At the molecular level, adhesion is mediated by interaction of Plasmodium falciparum erythrocyte membrane protein 1 (Pf EMP‐1) on the PRBC surface with receptors on the surface of endothelial cells, including CD36. We have shown that a recombinant 179‐residue subfragment of Pf EMP‐1 (rC1‐2[1–179]), which encompasses the CD36‐binding region, inhibits and reverses adhesion of PRBCs to CD36 under physiologically relevant flow conditions. rC1‐2[1–179] inhibited adhesion in a concentration‐dependent manner over the range 100 pM to 2 μM, with up to 99% of adhesion blocked at the highest concentration tested. The antiadhesive activity of rC1‐2[1–179] was not strain specific and almost totally ablated adhesion of four different parasite lines. Furthermore, rC1‐2[1–179] showed remarkable ability to progressively reverse adhesion when flowed over adherent PRBCs for 2 h. The effect of rC1‐2[1–179] was, however, specific for CD36‐mediated adhesion and had no effect on adhesion mediated by CSA. Interference with binding of PRBCs to the vascular endothelium using rC1‐2[1–179] or smaller organic mimetics may be a useful therapeutic approach to ameliorate severe complications of falciparum malaria.

Identification of a 67-amino-acid region of the Plasmodium falciparum variant surface antigen that binds chondroitin sulphate A and elicits antibodies reactive with the surface of placental isolates.

The complications of malaria in pregnancy are caused by the massive sequestration of parasitized erythrocytes (PE) in the placenta. Placental isolates of Plasmodium falciparum are unusual in that they do not bind the primary microvasculature receptor CD36 but instead bind chondroitin sulphate A (CSA). Pregnant mothers develop antibodies that recognize placental variants worldwide, suggesting that a vaccine against malaria in pregnancy is possible. Some members of the Duffy binding‐like γ (DBL‐γ) domain of the large and diverse P. falciparum erythrocyte membrane protein‐1 (PfEMP‐1) family, when expressed on Chinese hamster ovary (CHO) cells, bind CSA. To characterize better the molecular requirements for DBL‐γ adhesion to CSA, we determined the binding of various DBL‐γ domains. Most DBL‐γ did not bind CSA, and no conserved region was identified that strictly differentiated binders from non‐binders. Structure–function analysis of the FCR3‐CSA DBL‐γ domain localized the minimal CSA binding region to a 67‐residue fragment. This region was partially conserved among some binding sequences. Serum from a rabbit immunized with the minimal domain reacted with CSA‐binding parasite lines, but not with non‐CSA‐adherent PE lines that adhered to CD36 and other receptors. The identification of a minimal binding region from a highly variable cytoadherent family may have application for a vaccine against malaria in pregnancy.