Plasmodium yoelii Sporozoites with Simultaneous Deletion of P52 and P36 Are Completely Attenuated and Confer Sterile Immunity against Infection

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 each shown to play an important albeit nonessential role in Plasmodium berghei sporozoite infectivity for the rodent host. Here, we generated p52/p36-deficient Plasmodium yoelii parasites by the simultaneous deletion of both genes using a single genetic manipulation. p52/p36-deficient 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-deficient sporozoites also showed normal motility and cell traversal activity. However, immunofluorescence analysis and electron microscopic observations revealed that p52/p36-deficient parasites did not form a PV within hepatocytes in vitro and in vivo. The p52/p36-deficient 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-deficient sporozoites were completely protected against infectious sporozoite challenge. Our results demonstrate for the first time the generation of two-locus gene deletion-attenuated parasites that infect the liver but do not progress to blood-stage infection. The study will critically guide the design of Plasmodium falciparum live attenuated malaria vaccines.

Preerythrocytic, live-attenuated Plasmodium falciparum vaccine candidates by design

“…Clearly, breakthrough blood-stage infections in human GAP vaccinations will not be acceptable. In the P. yoelii model, a p52 Ϫ /p36 Ϫ double-knockout GAP did not show any breakthrough infection when inoculated at high doses of 10 5 sporozoites (28). Furthermore, this model of a GAP vaccine induced sterile protection against high-dose i.v.…”

“…Previous experience with irradiation-attenuated rodent malaria parasites indicated that persistence of liver stages might be a prerequisite for protection (35). However, more recent data obtained for the p52 Ϫ /p36 Ϫ GAP rodent malaria line showed clearly that persistence of parasites is not necessary to induce and maintain protection against sporozoite challenge (28).…”

“…Deletion of the orthologous genes in the rodent malaria models had indicated that they are important for productive liver infection (11,12,28). Transmission to the mosquito vector and passage through the mosquito stages were not affected by deletion of either P52 or P36.…”

“…Furthermore, this model of a GAP vaccine induced sterile protection against high-dose i.v. challenge with 10 4 WT sporozoites and against challenge by infectious mosquito bite (28).…”

Knocking out malaria

“…In mouse models of malaria, their strategy produced attenuated P. yoelii and P. berghei strains that did not cause blood-stage infection but nevertheless protected against challenge by wild-type infectious sporozoites. 7,8 "We have recently engineered the first P. falciparum attenuated strains and will go into clinical trials in U.S. volunteers by spring of next year, " Kappe told SciBX.…”