Plasmodium yoelii sporozoites with simultaneous deletion of P52 and P36 are completely attenuated and confer sterile immunity against infection

Authors:
Labaied M, Harupa A, Dumpit RF, Coppens I, Mikolajczak SA, Kappe SH
In:
Source: Infect Immun
Publication Date: (2007)
Issue: 75(8): 3758-68
Research Area:
Parasitology
Cells used in publication:
Plasmodium yoelii
Species: unicellular
Tissue Origin: blood
Platform:
Nucleofector™ I/II/2b
Experiment
Transfection was performed using the Nucleofector® device (Amaxa GmbH). 1.0Ü?107 purified P. yoelii mature schizonts were mixed with 14μg of the replacement fragment excised by KpnI/SacII digestion per 10μl of Tris-EDTA (pH 8.0) and 100μl of Human T cell Nucleofector® solution (Amaxa GmbH). Parasites were transfected using the electroporation program U-033 available in the Nucleofector® device and injected intravenously into naܯve SW recipient mice.
Abstract
Malaria infection starts when sporozoites are transmitted to the mammalian host during a mosquito bite. Sporozoites enter the blood circulation, reach the liver and infect hepatocytes. The formation of a parasitophorous vacuole (PV), establishes their intracellular niche. Recently, two members of the 6-cys domain protein family, P52 and P36, were shown to each play an important albeit non-essential role in Plasmodium berghei sporozoite infectivity for the rodent host. Here, we generated p52(-)/p36(-) deficient Plasmodium yoelii parasites by simultaneous deletion of both genes using a single genetic manipulation. p52(-)/p36(-) parasites exhibited normal progression through the life cycle during blood stage infection, transmission to mosquitoes, mosquito stage development and sporozoite infection of the salivary glands. p52(-)/p36(-) sporozoites also showed normal motility and cell traversal activity. However, immunofluorescence analysis and electron microscopic observations revealed that p52(-)/p36(-) parasites did not form a PV within hepatocytes in vitro and in vivo. The p52(-)/p36(-) parasites localized as free entities in the host cell cytoplasm or the host cell nucleoplasm and did not develop as liver stages. Consequently they did not cause blood stage infections even at high sporozoite inoculation doses. Mice immunized with p52(-)/p36(-) sporozoites were completely protected against infectious sporozoite challenge. Our results demonstrate for the first time the generation of two-loci gene deletion-attenuated parasites that infect the liver but do not progress to blood stage infection. The study will critically guide the design of P. falciparum live attenuated malaria vaccines.