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. This platform was recently used to generate transgenic P. knowlesi lines which express key P. vivax surface proteins, including PvDBP, which helps P. vivax attach to and invade RBCs. PvDBP is currently being researched as a promising P. vivax vaccine target, so this cell culture platform permits in vitro screening of P. vivax vaccine candidates which was not previously possible. Furthermore, this novel P. knowlesi culture can be used to investigate protein interactions and invasion biology of this zoonotic parasite. To select which PkNBPXa/PvRBP2b alleles to express, sequence analysis of published genomes and clinical isolates will be conducted to establish the most commonly expressed alleles. Mapping these sequences against location may also identify geographically distinct alleles. This would permit future clinical work to be targeted to relevant regions which may experience transmission of genetically distinct strains. For the second objective, a novel yeast display library technique developed in this laboratory will be used. Using nanobodies will allow easier manipulation and production in this system, and more targeted specificity. The ability to neutralise parasites (e.g. by inhibiting further growth by preventing merozoite attachment and/or invasion) can be assessed in multiple ways, for example, by tracking growth curves throughout the 24 hour P. knowlesi replication cycle, or by imaging parasites directly to visualise invasion phenotypes.The first year of the PhD will be centred around generating the required transgenic parasite lines and developing the barcoded yeast expression libraries for nanobodies. These high-throughput screens will hopefully identify variable regions of interest which can be examined in greater depth in subsequent years. In later stages of the project, work may be undertaken with groups working on controlled human malaria infections and clinical trials of vaccine candidates to identify protective antibody responses in vitro.

Project details

P. vivax
Vaccines (Immune Correlates)

Sep 2021 — Mar 2025

United Kingdom