Structural and biophysical approaches to the understanding of immunity and host-pathogen interactions in human malaria

Malaria remains a global health challenge with significant morbidity and mortality. This thesis describes host-pathogen interactions in two malaria parasites with the greatest human impact, Plasmodium falciparum and Plasmodium vivax. I aimed to shed light on the fundamental biology of antibody responses against P. falciparum and the invasion of erythrocytes by P. vivax, offering potential insights into design and development of novel vaccines and antibody therapeutics. Previous efforts over many years in P. falciparum vaccine development led to the recent approval of the RTS,S/AS01 vaccine, which is a recombinant vaccine based on P. falciparum circumsporozoite protein (PfCSP). This vaccine, while a milestone, has certain limitations in terms of efficacy and durability of the antibodies. My thesis research has provided structural insights into the antibody responses against the CSP. Crystal structures of several diverse antibody Fabs targeting the central NANP repeat region of CSP reveal dominant usage of germline-encoded aromatic residues to bind to their repeat paratopes, which frequently comprise conserved secondary structural motifs formed by the NPNA sequence. The structures also reinforce the importance of homotypic Fab-Fab interactions among anti-NANP antibodies that lead to an increase in affinity due to an avidity effect. These findings support the hypothesis that PfCSP may function as an immune decoy, potentially limiting the maturation of B-cell responses and may explain non-durable antibody responses seen in the current vaccines. Additionally, this study identifies antibodies targeting the less-explored N- and C-terminal domains of PfCSP that inhibit parasite infection in vivo, suggesting a potential role of these two domains in protective humoral immunity against P. falciparum. The characterization of these antibodies also reveals a new, highly conserved C-terminal epitope, representing a potential strain-transcending target that could be incorporated into future vaccines. The second part of this thesis explores P. vivax Duffy-binding protein (PvDBP), an essential component in the invasion of human red blood cells, binds to its receptor, DARC. Sulfation of tyrosine residues on DARC affects its binding to PvDBP, but this interaction with sulfated tyrosine has not been visualized as previous studies only show a small, non-sulfated, helical peptide from DARC binding to region II of PvDBP (PvDBP-RII). Here, the structure of PvDBP-RII bound to sulfated DARC peptide shows a sulfate on Y41 binds to a positively charged pocket on PvDBP-RII. Affinity measurement shows that sulfated Y41 on DARC is crucial to high-affinity binding to PvDBP-RII, and the importance of tyrosine sulfation is also confirmed in growth-inhibition experiments in parasites. This thesis also introduces a promising vaccine candidate based on a conserved subdomain of PvDBP. This candidate, termed ‘Interface,’ demonstrates superior thermal stability and immunogenicity compared to the existing construct, holding promise for large-scale production and distribution in malaria-endemic regions. Overall, this thesis advances our understanding of the immune responses against these promising vaccine candidates and the biology of P. vivax invasion and paves the way for the development of next-generation malaria vaccines and antibody therapeutics.

Keywords: antibody responses
malaria
circumsporozoite protein
Duffy antigen/receptor for chemokine
Plasmodium falciparum
Duffy-binding protein
Plasmodium vivax
Advisor: Wilson, IA
Higgins, MK