High throughput approaches to strain-transcending malaria vaccine candidate selection

P. vivax - found across South America and southeast Asia - is the most widespread of the Plasmodium spp. infecting humans and is increasingly acknowledged to cause a significant burden of disease. A particular hurdle in P. vivax research (unlike P. falciparum) has been the inability to culture these parasites, due to their preference for invading reticulocytes ('young' red blood cells, RBCs). In culture, reticulocytes mature quickly into normocytes (fully formed RBCs), leaving an insufficient population for subsequent parasite generations to invade. A major step forward in P. vivax research came in 2013 with the adaptation of the closely related simian parasite, P. knowlesi, to grow in human RBCs by Moon et al. The clear benefit of this technique is in aiding P. knowlesi research, as the parasite is an emerging zoonotic health threat in Asia and is notoriously misdiagnosed by microscopy. Excitingly, these parasites were also shown to be efficiently genetically modified using the CRISPR/Cas9 system. P. vivax and P. knowlesi are closely related and utilise similar invasion ligands to undergo intraerythrocytic replication, including the use of two key protein superfamilies, the reticulocyte binding-like (RBL) ligands and the Duffy-binding proteins (DBPs), to select and invade host erythrocytes. Orthologue replacement of P. knowlesi DBPa with PvDBP - both of which recognise the same receptor on human erythrocytes, DARC - in this model has allowed in vitro assessment of monoclonal antibodies generated by volunteers immunised with PvDBP-RII-based vaccines, permitting rational vaccine design prior to necessitating CHMI. Recent work by a previous member of the lab additionally identified that, in P. knowlesi, the essential RBL and DBP proteins act sequentially during erythrocytic invasion, mediating host cell deformation and merozoite reorientation, respectively. This raises the question as to whether both protein families should be assessed as vaccine targets - potentially used in tandem to improve vaccine efficacy.

This project focuses on four key questions. Firstly, I will use both wild-type and PvDBP-expressing P. knowlesi lines to investigate how the DBP-DARC interaction mediates host cell tropism, with specific focus on the similarities and differences of how binding affinity of PvDBP and PkDBPa is affected by mutations in host cell DARC.

Secondly, I will explore whether P. vivax RBLs are responsible for mediating host cell restriction to reticulocytes; my particular focus will be on PvRBP2b and PvRBP1a, as these proteins have recently identified host cell receptors which are both significantly upregulated in reticulocytes compared to normocytes. I will be expressing these proteins in an inducible PkNBPXa complementation line, which will permit investigation of potential reticulocyte-restricting invasion genes over a limited number of cycles in vitro without compromising the ability of the line to be grown in normocytes long-term.
Thirdly, I will be using a combination of monoclonal antibodies blocking PvDBP-DARC recognition, alongside newly purified antibodies recognising PkNBPXa, to investigate whether there is synergy when blocking both RBL and DBP binding to host erythrocytes. If so, targeting both families of proteins in a single vaccine might provide more robust protection against P. vivax and P. knowlesi malaria.
Finally, time permitting, these investigations can be taken one step further to assess how variation in RBL and DBP genes in P. vivax and P. knowlesi may alter the efficacy of vaccines. As these parasite proteins are immunogenic, they are susceptible to selection and significant variability between parasite isolates; an effective vaccine must be able to target a wide range of variants to provide strain-transcending immunity. By expressing different variants of PvRBPs and PkNBPXa, the cross-reactivity of different antibodies can be assessed in a long-term culture model.