Variant antigens at the infected red cell surface in Plasmodium falciparum malaria

Abstract: Disease severity in Plasmodium falciparum infections is, to a large extent, a consequence of the parasite's efficient escape from die human host defences. Sequestration and antigenic variation are two evasion mechanisms featured by P.falciparum. Sequestering infected erythrocytes (PRBC) that bind to receptors on the vascular lining (endothelial cytoadherence) arid to uninfected erythrocytes (rosetting) are accumulated in the brain, placenta and other organs, avoiding in this way the circulation and their destruction upon passage through the spleen. The investigations included in this thesis were undertaken to study the molecular background underlying the adhesive interaction(s) between PRBC and other vascular cells. In particular, emphasis was made in the identification of parasite-derived molecules expressed de novo on the infected erythrocyte surface that could mediate adhesive functions and display antigenic variation. Monomorphic and isogenic populations of parasites expressing distinct adhesive profiles were generated by phenotypic selection, cloning, and sub-cloning. A unique P.falciparum was characterized expressing a pan-adhesive phenotype linked to erythrocyte rosetting, a previously identified correlate of cerebral malaria. The PRBC of this parasite not only bound uninfected erythrocytes but also formed agglutinates of infected cells, adhered to endothelial cells, and bound to CD36, to immunoglobulins, and to the terminal blood group A saccharide. A novel adhesive phenotype was identified, in which PRBC spontaneously autoagglutinated with each other. The linkage of rosetting, autoagglutination, and cytoadherence involved the co-expression on a single PRBC of ligands with multiple specificities and the binding to two or more receptors on erythrocytes and to at least two other cell adhesion molecules, including a new endothelial cell receptor for P.falciparuminfected erythrocytes. The receptor was found to be the plateletlendothelial cell adhesion molecule-1 (PECAMA-1/CD31). The binding of PRBC to this receptor was characterized and mapped to the N-terminal Ig-like homology domains in the molecule. Specificity for PECAM-1 was common among clinically isolated parasites, suggesting that this receptor is of importance in the sequestration occurring during natural infections. Analysis of the PRBC surface of parasite lines, clones, and sub-clones, led to the identification of a variable high molecular weight antigen, the expression of which changed concomitantly with switches in binding phenotype. Using singlecell reverse transcription (RT)-PCR and cDNA cloning, the sequence of the gene encoding a rosetting ligand expressed on the PRBC surface was determined. The rosetting ligand belongs to the var/Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1) family of clonally variant antigens. Heparan sulphate (HS) or HS-like glycosaminoglycans were identified as the erythrocytic receptors specifically recognized by the inserting ligand. The transcription of var genes at different developmental intraerythrocytic stages was studied in bulk cultures and in single parasites. The repertoire of var genes was estimated to be around 50 per haploid genome. Many of these genes were found to be transcriptionally active very early in the erythrocytic cycle, and unique patterns (type and number) of var transcripts were detected in each individual PRBC as early as 2-4 hours post-invasion. Chromosomal mapping of transcriptionally active var indicated that their distribution reflects that of var loci in the genome, including a pronounced clustering in chromosome 4. This relaxed transcription was curtailed to a monogenic expression of a single detectable var transcript encoding the expressed PfEMP1 as the parasite progressed from the young ring to the mature trophozoite stage, between 8 and 16 hours after RBC invasion. The transcriptionally controlled expression of var is believed to lie beneath the allelic exclusion mechanism governing the display of only one (or a major) PfEMP1 variant per individual PRBC. A second large multicopy gene family, the rif/RIFIN comprising 200-250 genes, was found to encode polypeptides targeted to the PRBC surface. These clonally variant antigens with yet unknown function were found to be prominently expressed by fresh isolates of P.falciparum and recognized in a specific fashion by immune sera that agglutinated the infected cells, suggesting the presence of epitopes in RIFIN targeted by agglutinating and potentially protective antibodies. When analyzed by two-dimensional electrophoresis, the RIFIN resolved into several isoforms in a slightly acidic pI range, indicating molecular microheterogeneity, a possible novel source of antigenic diversity in P.falciparum. Taken together, these data illustrate the vast capacity and plasticity of the recognition/binding mechanisms used by P.falciparum intraerythrocytic stages to ensure sequestration. The data also show how the parasite controls the exposure of its large, but not infinite, adhesive and antigenic repertoire. The finding of a new large family of clonally variant surface proteins underscores antigenic polymorphism as a probably absolute requirement in molecules expressed by the parasite outside its host cell. The results suggest that any strategy aiming at the development of prophylactics targeting these molecules must take into account extensive molecular variation.