Structure-Guided Design of a Synthetic Mimic of an Endothelial Protein C Receptor-Binding PfEMP1 Protein

Abstract

Structure-guided vaccine design provides a route to elicit a focused immune response against the most functionally important regions of a pathogen surface. This can be achieved by identifying epitopes for neutralizing antibodies through structural methods and recapitulating these epitopes by grafting their core structural features onto smaller scaffolds. In this study, we conducted a modified version of this protocol. We focused on the PfEMP1 protein family found on the surfaces of erythrocytes infected with Plasmodium falciparum. A subset of PfEMP1 proteins bind to endothelial protein C receptor (EPCR), and their expression correlates with development of the symptoms of severe malaria. Structural studies revealed that PfEMP1 molecules present a helix-kinked-helix motif that forms the core of the EPCR-binding site. Using Rosetta-based design, we successfully grafted this motif onto a three-helical bundle scaffold. We show that this synthetic binder interacts with EPCR with nanomolar affinity and adopts the expected structure. We also assessed its ability to bind to antibodies found in immunized animals and in humans from malaria-endemic regions. Finally, we tested the capacity of the synthetic binder to effectively elicit antibodies that prevent EPCR binding and analyzed the degree of cross-reactivity of these antibodies across a diverse repertoire of EPCR-binding PfEMP1 proteins. Despite our synthetic binder adopting the correct structure, we find that it is not as effective as the CIDRα domain on which it is based for inducing adhesion-inhibitory antibodies. This cautions against the rational design of focused immunogens that contain the core features of a ligand-binding site of a protein family, rather than those of a neutralizing antibody epitope. IMPORTANCE Vaccines train our immune systems to generate antibodies which recognize pathogens. Some of these antibodies are highly protective, preventing infection, while others are ineffective. Structure-guided rational approaches allow design of synthetic molecules which contain only the regions of a pathogen required to induce production of protective antibodies. On the surfaces of red blood cells infected by the malaria parasite Plasmodium falciparum are parasite molecules called PfEMP1 proteins. PfEMP1 proteins, which bind to human receptor EPCR, are linked to development of severe malaria. We have designed a synthetic protein on which we grafted the EPCR-binding surface of a PfEMP1 protein. We use this molecule to show which fraction of protective antibodies recognize the EPCR-binding surface and test its effectiveness as a vaccine immunogen.

Protein scaffolds: antibody alternatives for cancer diagnosis and therapy

“…The flexible motifs are well accepted by most scaffolds; in contrast, one should consider whether the scaffold's conformational folding path matches with rigid motifs. 184 Searching scaffolds from the PDB databases or de novo design library, and comparing similarity via PDBeFold or DALI is a good option; meanwhile, small protein inhibitors also require attention, such as typical kunitz, affimer, and knottin scaffolds. 185 It should be noted that some de novo scaffolds may be difficult to be expressed in E. coli due to their evolutionary preference inconsistence.…”

“…Sometimes, low stability may affect correct folding, and it is worth trying to introduce disulfide-bridges with appropriate distances to improve the overall stability of the scaffolds. 184 …”

“… 190 A helix-kinked-helix motif of PfEMP1 molecules which forms core binding site of endothelial protein C receptor was integrated to a three-helical bundle scaffold as a vaccine immunogen, the final binder could elicit antibodies, although not as effective as the whole CIDRalpha1 domain. 184 …”

“…Sometimes, low stability may affect correct folding, and it is worth trying to introduce disulfide-bridges with appropriate distances to improve the overall stability of the scaffolds. 184 Tetsuya's group grafted two CD25-binding residues GGGV-D of daclizumab and YGY-RS-Y of basiliximab onto a zinc-finger peptide scaffold to construct a small antibody mimetic library, three CD25-binding ligands with 30 nM affinity were successfully selected. 189 Cassie's group designed PD-1 agonist by inserting three binding motifs ''WDYKY'', ''ADYKR'', ''WDYKR'' into 34840 de novo-designed scaffolds, after Rosetta, fold-fromloopsm, flow cytometry sorting and affinity maturation processed, an agonist PD-MP1 were selected, monomeric PD-MP1 showed 6 mM affinity to mouse PD-1, and trimeric PD-MP1 strongly inhibited murine T cell activation in vitro.…”

“…190 A helixkinked-helix motif of PfEMP1 molecules which forms core binding site of endothelial protein C receptor was integrated to a three-helical bundle scaffold as a vaccine immunogen, the final binder could elicit antibodies, although not as effective as the whole CIDRalpha1 domain. 184 Another approach scaffold reshaping is commonly involved in scaffold selection, solvent-accessible surface area calculation, stability evaluation, library construction, and binder panning verification.…”