Characterisation of a host receptor for Plasmodium falciparum-infected erythrocyte rosette formation

Abstract

Plasmodium falciparum (P. falciparum) rosetting, the binding of two or more uninfected erythrocytes to an infected erythrocyte, is a key virulence factor associated with severe malaria. Rosette formation is mediated by P. falciparum erythrocyte membrane protein 1 (PfEMP1) expressed on the surface of infected erythrocytes. Several molecules have been proposed as host rosetting receptors (such as Blood Group A antigen and Complement Receptor 1), but none of these can account for rosetting interactions across all parasite lines, suggesting that major host receptors remain unidentified. The Wrightb blood group antigen, which is formed by a physical interaction between Band 3 and Glycophorin A, has been identified by the Rowe lab as a potential novel rosetting receptor. Antibody fragments targeting Wrightb disrupt rosettes in several P. falciparum lines, but the mechanism of action of this Fab is largely unknown. This thesis aimed to characterise this key receptor-ligand interaction implicated in rosetting and explore the therapeutic potential of anti-Wrightb reagents.

To determine whether Wrightᵇ or Band 3 is a rosetting receptor, I tested the ability of Band 3-transfected K562 cells and naturally occurring Glycophorin null erythrocytes to form rosettes with purified P. falciparum-infected erythrocytes. The results showed that Band 3- transfected cells (expressing the Wrightᵇ antigen) form rosettes with two parasite lines, whereas wild type K562 cells (expressing Glycophorin A alone) do not. Rosette frequency and rosette size did not vary across the glycophorin genotypes examined, showing that Glycophorin A and Wrightᵇ are not essential for rosetting with the parasite lines tested. Thus, these data suggest that Band 3, rather than Wrightᵇ, is a rosetting receptor.

Secondly, I aimed to identify the parasite protein that adheres to Band 3 to form rosettes. Experiments were performed to determine whether anti-Band 3 Fabs blocked recombinant PfEMP1 from binding to erythrocytes and whether they co-localised on the surface of uninfected erythrocytes. I found that the binding of recombinant PfEMP1 from rosetting lines was inhibited by Fabs targeting a variety of erythrocyte surface receptors and that PfEMP1 proteins co-occurred with several Fabs on the surface of normal erythrocytes. Recombinant PfEMP1 domains did not bind to Band 3 peptides, did not compete for binding on the surface of erythrocytes and did not disrupt rosetting in homologous or heterologous P. falciparum parasite lines. These data show that recombinant PfEMP1 domains from rosetting variants likely bind to several receptors on erythrocytes but direct adhesion between Band 3 and PfEMP1 DBLα was not confirmed.

Thirdly, the rosette-disrupting ability of the BRIC 14 Fab was further characterised across varying concentrations, multiple parasite lines and blood types, in addition to fresh clinical isolates from children with severe malaria. I found that whilst BRIC 14 was effective in lab-adapted lines, it was not able to disrupt rosettes in fresh or recently lab-adapted Ugandan clinical isolates. To test the rosette-busting potential of additional anti-Wrightb reagents, the anti-Wrightb scFv from Villa et al, (2018) was constructed. The scFv was unable to disrupt rosettes in historically lab-adapted and recently lab-adapted parasite lines, agreeing with previous results that showed that the Wrightb antigen is not essential for rosetting. As these data demonstrated a limited role for anti-Wrightᵇ reagents as anti-rosetting treatments, Band 3 peptides were tested for rosette-disrupting abilities. Although Band 3 was identified in Chapter 3 as a rosetting receptor, the Band 3 peptides were also unable to disrupt rosetting, demonstrating the difficulty in developing rosette-disrupting drugs.