Research into the aetiological agent of the most widespread form of severe malaria, Plasmodium falciparum, has benefitted enormously from the ability to culture and genetically manipulate blood-stage forms of the parasite in vitro. However, most malaria outside Africa is caused by a distinct Plasmodium species, Plasmodium vivax, and it has become increasingly apparent that zoonotic infection by the closely related simian parasite Plasmodium knowlesi is a frequent cause of life-threatening malaria in regions of southeast Asia. Neither of these important malarial species can be cultured in human cells in vitro, requiring access to primates with the associated ethical and practical constraints. We report the successful adaptation of P. knowlesi to continuous culture in human erythrocytes. Humanadapted P. knowlesi clones maintain their capacity to replicate in monkey erythrocytes and can be genetically modified with unprecedented efficiency, providing an important and unique model for studying conserved aspects of malarial biology as well as speciesspecific features of an emerging pathogen.invasion | transfection T he development of a continuous culture system for asexual blood stages of the most deadly human malaria parasite, Plasmodium falciparum (1, 2), proved a milestone in malaria research, enabling genetic modification of the parasite (3), high-throughput drug screening (4), and other fundamental advances in parasite biology. Adaptation of other human malaria parasite species to in vitro culture has proved more challenging, and none of the additional four parasite species that cause human malaria can be continuously maintained in human RBC. This difficulty is a significant obstacle to studying these pathogens, which differ from P. falciparum in important aspects of biology and the pathology they cause. Furthermore, although considerable progress has been made in the development of transgenic technologies for Plasmodium, P. falciparum remains poorly amenable to genetic manipulation, with a typical transfection efficiency of only ∼10 −6 (5). Additional in vitro human malaria parasite models that are genetically tractable and that complement the P. falciparum system have tremendous potential.Much of the early work on the mechanics of RBC invasion by the malaria parasite used the simian parasite Plasmodium knowlesi. This species has a 24-h erythrocytic life cycle and large, long-lived invasive merozoites, facilitating the use of electron and video microscopy to dissect the dynamics of erythrocyte invasion (6-8). P. knowlesi can be cultured in vitro in rhesus monkey (Macaca mulata) RBC with rhesus or human serum (9, 10). Importantly, P. knowlesi is amenable to genetic manipulation, with reported transfection efficiencies similar to those achieved with the rodent malaria model Plasmodium berghei and far surpassing those attained in P. falciparum (10, 11). P. knowlesi is phylogenetically closely related to Plasmodium vivax, the most important cause of malaria outside of Africa (12), so its study can provide insights ...

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.

The dominant cause of malaria in Malaysia is now Plasmodium knowlesi, a zoonotic parasite of cynomolgus macaque monkeys found throughout South East Asia. Comparative genomic analysis of parasites adapted to in vitro growth in either cynomolgus or human RBCs identified a genomic deletion that includes the gene encoding normocyte-binding protein Xa (NBPXa) in parasites growing in cynomolgus RBCs but not in human RBCs. Experimental deletion of the NBPXa gene in parasites adapted to growth in human RBCs (which retain the ability to grow in cynomolgus RBCs) restricted them to cynomolgus RBCs, demonstrating that this gene is selectively required for parasite multiplication and growth in human RBCs. NBPXa-null parasites could bind to human RBCs, but invasion of these cells was severely impaired. Therefore, NBPXa is identified as a key mediator of P. knowlesi human infection and may be a target for vaccine development against this emerging pathogen.

In order to test the hypothesis that CD8 ؉ cytotoxic T lymphocytes mediate protection against acute superinfection, we depleted >99% of CD8 ؉ lymphocytes in live attenuated simian immunodeficiency virus macC8 (SIVmacC8) vaccinees from the onset of vaccination, maintained that depletion for 20 days, and then challenged with pathogenic, wild-type SIVmacJ5. Vaccinees received 5 mg per kg of humanized anti-CD8 monoclonal antibody (MAb) 1 h before inoculation, followed by the same dose again on days 3, 7, 10, 13, and 17. On day 13, peripheral CD8 ؉ T lymphocytes were >99% depleted in three out of four anti-CD8 MAb-treated vaccinees. At this time attenuated SIVmacC8 viral RNA loads in anti-CD8 MAb-treated vaccinees were significantly higher than control vaccinees treated contemporaneously with nonspecific human immunoglobulin. Lymphoid tissue CD8 ؉ T lymphocyte depletion was >99% in three out of four anti-CD8 MAb-treated vaccinees on the day of wild-type SIVmacJ5 challenge. All four control vaccinees and three out of four anti-CD8 MAb-treated vaccinees were protected against detectable superinfection with wild-type SIVmacJ5. Although superinfection with wild-type SIVmacJ5 was detected at postmortem in a single anti-CD8 MAb-treated vaccinee, this did not correlate with the degree of preceding CD8 ؉ T lymphocyte depletion. Clearance of attenuated SIVmacC8 viremia coincided with recovery of normal CD8 ؉ T lymphocyte counts between days 48 and 76. These results support the view that cytotoxic T lymphocytes are important for host-mediated control of SIV primary viremia but do not indicate a central role in protection against acute superinfection conferred by inoculation with live attenuated SIV.

South American Zika virus (ZIKV) recently emerged as a novel human pathogen, linked with neurological disorders. However, comparative ZIKV infectivity studies in New World primates are lacking. Two members of the Callitrichidae family, common marmosets (Callithrix jacchus) and red-bellied tamarins (Saguinus labiatus), were highly susceptible to sub-cutaneous challenge with the Puerto Rico-origin ZIKVPRVABC59 strain. Both exhibited rapid, high, acute viraemia with early neuroinvasion (3 days) in peripheral and central nervous tissue. ZIKV RNA levels in blood and tissues were significantly higher in New World hosts compared to Old World species (Macaca mulatta, Macaca fascicularis). Tamarins and rhesus macaques exhibited loss of zonal occludens-1 (ZO-1) staining, indicative of a compromised blood-brain barrier 3 days post-ZIKV exposure. Early, widespread dissemination across multiple anatomical sites distant to the inoculation site preceded extensive ZIKV persistence after 100 days in New and Old World lineages, especially lymphoid, neurological and reproductive sites. Prolonged persistence in brain tissue has implications for otherwise resolved human ZIKV infection. High susceptibility of distinct New World species underscores possible establishment of ZIKV sylvatic cycles in primates indigenous to ZIKV endemic regions. Tamarins and marmosets represent viable New World models for ZIKV pathogenesis and therapeutic intervention studies, including vaccines, with contemporary strains.