Rapid and iterative genome editing in the zoonotic malaria parasitePlasmodium knowlesi: New tools forP. vivaxresearch

Abstract

19Tackling relapsing Plasmodium vivax and zoonotic Plasmodium knowlesi infections is critical to 20 reducing malaria incidence and mortality worldwide. Understanding the biology of these important and 21 related parasites was previously constrained by the lack of robust molecular and genetic approaches. 22Here, we establish CRISPR-Cas9 genome editing in a culture-adapted P. knowlesi strain and define 23 parameters for optimal homology-driven repair. We establish a scalable protocol for the production of 24 repair templates by PCR and demonstrate the flexibility of the system by tagging proteins with distinct 25 cellular localisations. Using iterative rounds of genome-editing we generate a transgenic line expressing 26 P. vivax Duffy binding protein (PvDBP), a lead vaccine candidate. We demonstrate that PvDBP plays 27 no role in reticulocyte restriction but can alter the macaque/human host cell tropism of P. knowlesi. 28Critically, antibodies raised against the P. vivax antigen potently inhibit proliferation of this strain, 29providing an invaluable tool to support vaccine development. 30 31 32 33 Main Text: 34 Introduction: 35Malaria remains a serious health burden globally, with over 216 million cases annually (1). Plasmodium 36 falciparum is responsible for 99 % of estimated malaria cases in sub-Saharan Africa. Outside Africa, P. 37 vivax is the predominant parasite and causes ~7.4 million clinical cases annually (1). Despite extensive 38 efforts, in 2016 the number of malaria cases were on the rise again for the first time in several years. 39Achieving global malaria eradication requires new tools and approaches for addressing emerging drug 40 resistance, relapsing P. vivax infections, and emerging zoonotic P. knowlesi infections, which represent 41 significant causes of severe disease and death (2). 42Although P. vivax displays some distinctive features to P. knowlesi, including the formation of latent 43 hypnozoites stages in the liver and restriction to reticulocytes in the blood (3), the two parasites are 44 closely related, occupying a separate simian parasite clade to P. falciparum (4) Host cell invasion by P. 45 vivax and P. knowlesi relies on the Duffy binding proteins (DBP) PvDBP and PkDBPα, respectively, 46 both ligands for human red blood cell (RBC) Duffy antigen/receptor for chemokines (DARC) (5-8). 47The critical binding motif of the ligands is the cysteine-rich region 2 (DBP-RII), with ~70 % identity 48 between PkDBPα and PvDBP (9). Despite their similarity, PvDBP has also been implicated in both P. 49 vivax reticulocyte restriction (10) and as a host tropism factor preventing P. vivax from infecting 50 macaques (11). PvDBP-RII is also the leading blood stage vaccine candidate for P. vivax (12-14), with 51 antibodies targeting PvDBP-RII blocking parasite invasion in ex vivo P. vivax assays (15). P. knowlesi 52 additionally contains two PkDBPα paralogues, namely DBPβ and DBPγ which share high levels of 53 amino acid identity (68-88 %) to PkDBPα but bind to distinct receptors via N-glycolylneuramin...