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

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.

Structural basis for inhibition of Plasmodium vivax invasion by a broadly neutralizing vaccine-induced human antibody.

The most widespread form of malaria is caused by Plasmodium vivax. To replicate, this parasite must invade immature red blood cells through a process requiring interaction of the P. vivax Duffy binding protein (PvDBP) with its human receptor, the Duffy antigen receptor for chemokines. Naturally acquired antibodies that inhibit this interaction associate with clinical immunity, suggesting PvDBP as a leading candidate for inclusion in a vaccine to prevent malaria due to P. vivax. Here, we isolated a panel of monoclonal antibodies from human volunteers immunized in a clinical vaccine trial of PvDBP. We screened their ability to prevent PvDBP from binding to the Duffy antigen receptor for chemokines, and their capacity to block red blood cell invasion by a transgenic Plasmodium knowlesi parasite genetically modified to express PvDBP and to prevent reticulocyte invasion by multiple clinical isolates of P. vivax. This identified a broadly neutralizing human monoclonal antibody that inhibited invasion of all tested strains of P. vivax. Finally, we determined the structure of a complex of this antibody bound to PvDBP, indicating the molecular basis for inhibition. These findings will guide future vaccine design strategies and open up possibilities for testing the prophylactic use of such an antibody.

Structural basis for therapeutic inhibition of complement C5.

Activation of complement C5 generates the potent anaphylatoxin C5a and leads to pathogen lysis, inflammation and cell damage. The therapeutic potential of C5 inhibition has been demonstrated by eculizumab, one of the world's most expensive drugs. However, the mechanism of C5 activation by C5 convertases remains elusive, thus limiting development of therapeutics. Here we identify and characterize a new protein family of tick-derived C5 inhibitors. Structures of C5 in complex with the new inhibitors, the phase I and phase II inhibitor OmCI, or an eculizumab Fab reveal three distinct binding sites on C5 that all prevent activation of C5. The positions of the inhibitor-binding sites and the ability of all three C5-inhibitor complexes to competitively inhibit the C5 convertase conflict with earlier steric-inhibition models, thus suggesting that a priming event is needed for activation.