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Figure 4: Development of wild-type and Δp52 parasites in primary human hepatocytes.(A) Parasites at 20 hours. Extracellular parasites are visualised by staining with anti-PfCSP antibodies (secondary conjugated with ALEXA594, i.e. red fluorescence) before permeablisation (α-PfCS*) and all parasites are visualised by staining with anti-PfCSP antibodies (secondary conjugated with ALEXA488 i.e. green fluorescence) after permeablisation (α-PfCS**). The nuclei of the host cells are stained with DAPI (blue). (B) Parasites at day 3 or day 5. Nuclei of both the host cell and the merozoites inside the developing schizont are visible by DAPI staining (blue). Parasites are identified by anti-HSP70 staining (α-HSP70; secondary antibody conjugated with ALEXA488; green). Parasite lacking P52 expression fail to develop into schizonts and the only visible forms of the parasite are small ‘rounded’, possibly degenerate and/or extracelluar, forms. Scale bars in the DAPI panels represent a size of 10 µM.

Image Text (High Precision): DAPI p52

Other Images from "Gene Disruption of Plasmodium falciparum p52 Results in Attenuation of Malaria Liver Stage Development in Cultured Primary Human Hepatocytes":


Figure 1 Generation of P. falciparum parasites la...

Figure 2 Gliding Motility and Traversal Capacity ...

Figure 3 Invasion capacity of Wt and Δp52 sporozo...

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Abstract

Difficulties with inducing sterile and long lasting protective immunity against malaria with subunit vaccines has renewed interest in vaccinations with attenuated Plasmodium parasites. Immunizations with sporozoites that are attenuated by radiation (RAS) can induce strong protective immunity both in humans and rodent models of malaria. Recently, in rodent parasites it has been shown that through the deletion of a single gene, sporozoites can also become attenuated in liver stage development and, importantly, immunization with these sporozoites results in immune responses identical to RAS. The promise of vaccination using these genetically attenuated sporozoites (GAS) depends on translating the results in rodent malaria models to human malaria. In this study, we perform the first essential step in this transition by disrupting, p52, in P. falciparum an ortholog of the rodent parasite gene, p36p, which we had previously shown can confer long lasting protective immunity in mice. These P. falciparum P52 deficient sporozoites demonstrate gliding motility, cell traversal and an invasion rate into primary human hepatocytes in vitro that is comparable to wild type sporozoites. However, inside the host hepatocyte development is arrested very soon after invasion. This study reveals, for the first time, that disrupting the equivalent gene in both P. falciparum and rodent malaria Plasmodium species generates parasites that become similarly arrested during liver stage development and these results pave the way for further development of GAS for human use.


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