Rapid and iterative genome editing in the malaria parasite Plasmodium knowlesi provides new tools for P. vivax research

Abstract

Tackling relapsing Plasmodium vivax and zoonotic Plasmodium knowlesi infections is critical to reducing malaria incidence and mortality worldwide. Understanding the biology of these important and related parasites was previously constrained by the lack of robust molecular and genetic approaches. Here, we establish CRISPR-Cas9 genome editing in a culture-adapted P. knowlesi strain and define parameters for optimal homology-driven repair. We establish a scalable protocol for the production of repair templates by PCR and demonstrate the flexibility of the system by tagging proteins with distinct cellular localisations. Using iterative rounds of genome-editing we generate a transgenic line expressing P. vivax Duffy binding protein (PvDBP), a lead vaccine candidate. We demonstrate that PvDBP plays no role in reticulocyte restriction but can alter the macaque/human host cell tropism of P. knowlesi. Critically, antibodies raised against the P. vivax antigen potently inhibit proliferation of this strain, providing an invaluable tool to support vaccine development.

UsingPlasmodium knowlesias a model for screeningPlasmodium vivaxblood-stage malaria vaccine targets reveals new candidates

“…The low level of inhibition observed with anti-PvMSP7.1 and PvMSP3.10 could be due to the low degree of homology between Pv and Pk homologs, and the lack of inhibition observed with the anti-PvMSP3.10 was consistent with the absence of cross-reactivity with PkMSP3 homologues in immunofluorescence assays. Strong inhibition with anti-PvDBP, the only P. vivax blood stage vaccine target in the advanced stage of vaccine development, was confirmatory and comparable to other studies (46). Antibodies to two other targets, Pv12 and PvGAMA, had broadly similar IC 50 s to anti-PvDBP, while antibodies to Pv41, which interacts with Pv12 (32), also had strong inhibition.…”

“…We have explored whether P. knowlesi, which has a close phylogenetic relationship with P. vivax (39,40) and has been adapted to in vitro culture in human erythrocytes (41,50), could be used to screen for P. vivax blood-stage vaccine candidates, as it can for drug-resistance candidates (51). Such an approach has proven viable to explore the most advanced P. vivax blood-stage vaccine candidate, PvDBP (46). In this case we sought to apply the P. knowlesi model to systematically screen for new blood-stage antigens, using a panel of polyclonal antibodies generated against candidates from a previously published library of P. vivax schizont expressed proteins (32).…”

Use of a highly specific kinase inhibitor for rapid, simple and precise synchronization ofPlasmodium falciparumandPlasmodium knowlesiasexual stage parasites

“…Of the PKG inhibitors used for synchronization of parasite cultures, Compound 2 has been the most commonly used [14, 38, 42, 44, 45, 54, 55, 61]. However, the results shown here indicate that ML10 has advantages over Compound 2.…”

“…An additional application of the very tight synchronization is the preparation of parasites for schizont-stage transfection, which requires extremely synchronized parasites [6, 59]. Compound 2 has been used previously to aid synchronization of P. knowlesi for transfection [44]. Furthermore, ML10 has recently been used to block the proliferation of asexual stages in a P. falciparum culture induced to form gametocytes [60].…”

The enigmatic mechanisms by which Plasmodium vivax infects Duffy-negative individuals

“…Such an approach could also make use of the Saimiri and Aotus nonhuman primate model-as both species can be infected by P. vivax, while PvDBP can bind only to Aotus erythrocytes and not to Saimiri ones, indicating an alternative invasion pathway reminiscent of what is observed for Duffy-negative human erythrocytes [90]. Alternative models to decipher erythrocyte invasion mechanisms could also rely on the genetically tractable P. knowlesi that shares many biological similarities with P. vivax, including the requirement of the Duffy receptor for erythrocyte invasion [91] or even on P. cynomolgi, closely related to P. vivax and recently adapted to in vitro culture [92].…”