Plasmepsin 4-Deficient Plasmodium berghei Are Virulence Attenuated and Induce Protective Immunity against Experimental Malaria

Plasmodium parasites lacking plasmepsin 4 (PM4), an aspartic protease that functions in the lysosomal compartment and contributes to hemoglobin digestion, have only a modest decrease in the asexual blood-stage growth rate; however, PM4 deficiency in the rodent malaria parasite Plasmodium berghei results in significantly less virulence than that for the parental parasite. P. berghei Δpm4 parasites failed to induce experimental cerebral malaria (ECM) in ECM-susceptible mice, and ECM-resistant mice were able to clear infections. Furthermore, after a single infection, all convalescent mice were protected against subsequent parasite challenge for at least 1 year. Real-time in vivo parasite imaging and splenectomy experiments demonstrated that protective immunity acted through antibody-mediated parasite clearance in the spleen. This work demonstrates, for the first time, that a single Plasmodium gene disruption can generate virulence-attenuated parasites that do not induce cerebral complications and, moreover, are able to stimulate strong protective immunity against subsequent challenge with wild-type parasites. Parasite blood-stage attenuation should help identify protective immune responses against malaria, unravel parasite-derived factors involved in malarial pathologies, such as cerebral malaria, and potentially pave the way for blood-stage whole organism vaccines.The digested vacuole (DV) of malaria parasites performs hemoglobin degradation, which is a crucial process for parasite growth and survival within the host erythrocyte. In Plasmodium falciparum, the most important human malaria parasite, this is achieved with the contribution of several digestive vacuole proteases including three aspartic proteinases, the plasmepsins (PM) PfPM1, PfPM2, and PfPM4 and one histo-aspartic protease, PfHAP.^1–5 The plasmepsins have long been studied as potential drug targets and subjected to functional and biochemical studies with the hope that inhibiting them would halt hemoglobin digestion and result in parasite death. Surprisingly, the systematic disruption of either individual or different combinations of the plasmepsin genes did not result in any striking growth defect. Presumably, this is due to redundant enzyme systems for digesting hemoglobin, which involve cysteine proteases, metalloproteases, and aminopeptidases, and to the presence of multiple pathways for the uptake of extracellular amino acids.^6–8 The P. falciparum and Plasmodium reichnowi clades differ from other Plasmodium species in that they have four genes encoding DV plasmepsins. In P. falciparum only the disruption of all four plasmepsin genes, which eliminates all aspartic protease activity from the DV, resulted in delayed in vitro schizont maturation accompanied by reduced formation of hemozoin (an insoluble crystal produced during hemoglobin degradation) and less efficient processing of endosomal vesicles in the DV.⁴We here investigated the impact of the loss of the various functions of the DV plasmepsins on parasite virulence by disrupting the single gene encoding the DV plasmepsin 4 (pm4) in the rodent malaria parasite Plasmodium berghei. This parasite is a well established and tractable model to study the function of Plasmodium genes in vivo and replicates several key features of human cerebral malaria.^9,10 The phenotypic analysis of loss-of-function mutants has been used to gain an insight into a variety of host-parasite interactions.¹¹ In this study, we confirm that the disruption of PM4, which results in loss of all aspartic proteinase activity targeted to its lysosomal compartments, has only a modest effect on the intraerythrocytic development of P. berghei parasites, but we observed dramatic differences in the virulence of these parasites compared with that of wild-type parasites. Specifically, we report the growth and multiplication characteristics of Δpm4 parasites in different mouse strains and demonstrate that these parasites neither induce experimental cerebral malaria (ECM) in ECM-susceptible mice nor kill the host by hemolytic anemia in ECM-resistant mice. In these latter mice, Δpm4 parasites induce a self-resolving infection, which generates spleen-dependent protective immune responses. This is the first report of a mutant P. berghei parasite that does not induce cerebral complications as the result of a single gene mutation.