Structure guided mimicry of an essential P. falciparum receptor-ligand complex enhances cross neutralizing antibodies

Invasion of human red blood cells (RBCs) by Plasmodium falciparum (Pf) merozoites relies on the interaction between two parasite proteins, apical membrane antigen 1 (AMA1) and rhoptry neck protein 2 (RON2) ^1,2 . Antibodies to AMA1 confer limited protection against P. falciparum in non-human primate malaria models ^3,4 . However, clinical trials with recombinant AMA1 alone (apoAMA1) saw no protection, likely due to inadequate levels of functional antibodies ^5-8 . Notably, immunization with AMA1 in its ligand bound conformation using RON2L, a 49 amino acid peptide from RON2, confers superior protection against P. falciparum malaria by enhancing the proportion of neutralizing antibodies ^9,10 . A limitation of this approach, however, is that it requires the two vaccine components to form a complex in solution. To facilitate vaccine development, we engineered chimeric antigens by strategically replacing the AMA1 DII loop that is displaced upon ligand binding with RON2L. Structural characterization of the fusion chimera, Fusion-F _D12 to 1.55 Å resolution showed that it closely mimics the binary receptor-ligand complex. Immunization studies showed that Fusion-F _D12 immune sera neutralized parasites more efficiently than apoAMA1 immune sera despite having an overall lower anti-AMA1 titer, suggesting improvement in antibody quality. Furthermore, immunization with Fusion-F _D12 enhanced antibodies targeting conserved epitopes on AMA1 resulting in greater neutralization of non-vaccine type parasites. Identifying epitopes of such cross-neutralizing antibodies will help in the development of an effective, strain-transcending malaria vaccine. Our fusion protein design is a robust vaccine platform that can be enhanced by incorporating polymorphisms in AMA1 to effectively neutralize all P. falciparum parasites.