Plasmepsin 4-deficient Plasmodium berghei are virulence attenuated and induce protective immunity against experimental malaria

Abstract

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 Deltapm4 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.

Figure 1

The course of infection of Δpm4 parasites in different mouse lines/parasite strains combinations. Cumulative death and parasitemia levels are shown in the left and right panels, respectively. A: BALB/c mice infected with 10⁷ wild-type (wt) (n = 30) or Δpm4 parasites of lines Δpm4cl1 (n = 50) and Δpm4cl6 (n = 50). All wild-type infected mice died within the 3rd week after infection with high parasitemia, whereas all mice infected with Δpm4 parasites survived infection. B: NIH Swiss mice infected with 10⁷ wild-type (cl15cy1) (n = 10) or Δpm4 parasites of line 688cl2 (n = 10). All NIH Swiss mice infected with wild-type (cl15cy1) parasites developed high parasitemia, and experiments were terminated on day 15 after infection at a parasitemia 80%. All mice infected with Δpm4 parasites after the peak of parasitemia were able to control the infection and completely cleared the parasites. C: C57BL/6 mice infected with 10⁵ wild-type (n = 15) or Δpm4cl1 (n = 15) and Δpm4cl6 (n = 50) parasites Ninety percent of wild-type-infected C57BL/6 mice developed ECM at day 6 to 8 after infection. None of the mice infected with Δpm4 parasites showed signs of cerebral complications. Most Δpm4-infected mice (80%) developed high parasitemia and died in the 3rd week after infection. The surviving mice cleared the parasites. D: Swiss-OF1 mice infected with either 10⁵ wild-type (cl15cy1) (n = 10) or Δpm4 parasites of lines 688cl2 (n = 13) or line 688cl3 (n = 6). All Swiss-OF1 mice developed ECM at day 6 to 8 after infection at a parasitemia ranging from 15 to 20%, whereas the mice infected with Δpm4 parasites did not show cerebral complications. These mice developed high parasitemia, and experiments were terminated between day 14 and 16 after infection at a parasitemia >30%. ***P < 0.0001.

Figure 2

Distribution of luciferase expressing Δpm4⁺ and wt⁺ parasites in live mice as visualized by real time in vivo imaging through measurement of luciferase activity. A: A time course of an atypical distribution pattern of schizonts in mice with synchronous infections of Δpm4⁺ and wt⁺ parasites at different time points (hours) after the injection of purified merozoites. Schizont sequestration at 19 and 21 hours in wt⁺-infected mice (belly fat tissue, lungs, and spleen) and reinvasion of ring forms at 23 and 25 hours, resulting in bioluminescence in the whole body. In Δpm4⁺ parasites schizont sequestration and invasion start later (21 and 23 hours), but the distribution remains similar. B: Bioluminescence in organs of mice infected either with wt⁺ or Δpm4⁺ and isolated at 21 and 23 hours after infection, respectively. C: Quantification of luciferase signals in organs of mice infected with wt⁺ and Δpm4⁺ (six mice per group) showed no significant differences in parasite distribution. D: Parasite load and distribution in mice treated with IgG obtained from Δpm4 convalescent mice and infected with wt⁺⁺ parasites. IgG purified from the serum of animals infected with Δpm4cl6 parasites at day 18 or 40 after injection were given to BALB/c mice at days −1, 0, and + 1 relative to the time of i.v. inoculation with wt⁺⁺ parasites. As a control, mice were treated with IgG from wild-type infected mice obtained at day 18 postinfection. Nontreated: BALB/c mice infected with the wt⁺⁺ parasite line only. Parasites were visualized at day 2 after infection of the mice with the wt⁺⁺ parasites. E: Quantification of luciferase signals in organs of mice (six mice per group) treated with the different IgG samples shows a significant increase in parasite accumulation in the spleen of mice treated with IgG from convalescent animals 40 days postinfection. F: The course of the parasitemia in mice treated with the different IgG samples shows a significant reduction of the parasite load in mice treated with IgG that was collected from convalescent animals (day 40 after infection).*P < 0.05. RLU, relative light unit.

Figure 3

Analysis of cerebral complications in mice infected with wt⁺ and Δpm4⁺ parasites. A: Histological analysis of the brains of C57BL/6 mice with asynchronous infections at day 7 after injection of wt⁺ or Δpm4⁺ parasites. Longitudinal sections of the brain, stained with H&E, show extensive hemorrhagic areas in wt⁺-infected animals (black arrows), which are absent in Δpm4⁺-infected mice. In brains of wt⁺-infected mice, infected erythrocytes, recognized by pigment granules, were found as infiltrates in brain tissue (red arrow). B: Representative digital images of Evans Blue dye extrusion analysis of brains of wt⁺ or Δpm4⁺-infected C57BL/6 mice at day 7 postinfection. The blue staining of the brains shows vascular leakage in wt⁺-infected animals, which is absent in the brains of Δpm4⁺-infected mice. C: Bioluminescent images of brain isolated from C57BL/6 mice at day 7 after infection with 10⁵ wt⁺ or 10⁵ to 10⁶ Δpm4⁺ parasites showing a significant higher parasite load (see D) in brains of wt⁺ than in brains of Δpm4⁺-infected mice as a result of differences in accumulation of infected erythrocytes in brain tissue. Percentages shown represent the parasitemias at the time of collection of the brains. D: Differences in luminescence signal between the brains of mice infected with either wt⁺ or Δpm4⁺ parasites (n = 6) showing the differences in parasite load. *P < 0.05.

Figure 4

The role of the spleen in parasite clearance and protective immunity. A: Macroscopic observation of spleens from BALB/c mice infected with either 10⁷ wild-type or Δpm4cl6 parasites. This analysis revealed that at 21 days postinfection the spleens of Δpm4-infected mice (lower panel) were much bigger than those from wild-type infected animals (upper panel). This observation was confirmed by comparing the spleen weight in either wild-type or Δpm4cl6-infected mice at different time points (7, 14, 21, and 40 days) after infection. Numbers represent the mean values ± SD of spleen weight of eight animals. B: Cumulative death (left panel) and levels of parasitemia (right panel) of either intact (gray) or splenectomized (black) BALB/c mice infected with 10⁷ Δpm4cl6 parasites. ***P < 0.0001. C: Cumulative death (left panel) of splenectomized Δpm4 convalescent BALB/c mice (convalescent splenectomized [c.s.]) after a challenge with wild-type parasites. As a control Δpm4 convalescent intact BALB/c mice (convalescent not splenectomized [c.n.s.]) and naïve intact BALB/c mice (naïve not splenectomized [n.n.s]) were challenged with the same dose of wild-type parasites. Right panel: Time course of parasitemia in eight individual convalescent splenectomized BALB/c mice (M1–M8) after a challenge with wild-type parasites. None of the Δpm4 convalescent immune mice was able to control the parasitemia when challenged after splenectomy. All mice showed oscillating levels of parasitemia that increased sharply in a few days, leading to the death of the animals. ***P < 0.0001.