Host-based Prophylaxis Successfully Targets Liver Stage Malaria Parasites

Abstract

Eliminating malaria parasites during the asymptomatic but obligate liver stages (LSs) of infection would stop disease and subsequent transmission. Unfortunately, only a single licensed drug that targets all LSs, Primaquine, is available. Targeting host proteins might significantly expand the repertoire of prophylactic drugs against malaria. Here, we demonstrate that both Bcl-2 inhibitors and P53 agonists dramatically reduce LS burden in a mouse malaria model in vitro and in vivo by altering the activity of key hepatocyte factors on which the parasite relies. Bcl-2 inhibitors act primarily by inducing apoptosis in infected hepatocytes, whereas P53 agonists eliminate parasites in an apoptosis-independent fashion. In combination, Bcl-2 inhibitors and P53 agonists act synergistically to delay, and in some cases completely prevent, the onset of blood stage disease. Both families of drugs are highly effective at doses that do not cause substantial hepatocyte cell death in vitro or liver damage in vivo. P53 agonists and Bcl-2 inhibitors were also effective when administered to humanized mice infected with Plasmodium falciparum. Our data demonstrate that host-based prophylaxis could be developed into an effective intervention strategy that eliminates LS parasites before the onset of clinical disease and thus opens a new avenue to prevent malaria.

Figure 1

Bcl-2 family inhibition and P53 increase clears LS parasites by independent mechanisms. (a,b) Hepa 1–6 cells were infected with P. yoelii and treated with Nutlin-3 (20 µmol/l), ABT-737 (100 nmol/l), Obatoclax (100 nmol/l) with or without the pan-caspase inhibitor qVD-OPh (20 µmol/l). After 24 hours (a) or 48 hours (b), cultures were fixed and LS parasites were identified by UIS4 and HSP70 expression and quantified microscopically. (c,d) Hepa 1–6 cells were treated with Nutlin-3 (20 µmol/l), ABT-737 (100 nmol/l), or Obatoclax (100 nmol/l) for 24 hours (c), or 48 hours (d). Protein levels of P53 were determined by immunoblot. (e) A model for elimination of LSs by Obatoclax (orange) and Nutlin-3 (purple). NS (not significant), *P ≤ 0.05, **P ≤ 0.01.

Figure 2

Nutlin-3 and Obatoclax dramatically delays, or in some cases completely prevents, the onset of disease in vivo. BALB/cJ mice were treated with vehicle, Nutlin-3 (200 mg/kg/day), Obatoclax (5 mg/kg/day), or a combination of Nutlin-3 and Obatoclax beginning 24 hours prior to infection and then once daily for 3 days in total. Mice were infected intravenously with 1,000 (a) or 100 (b) P. yoelii sporozoites. Blood stage patency was monitored by Giemsa-stained thin blood smear beginning 3 days after infection and continuing through 2 weeks postinfection.

Figure 3

Serdemetan treatment decreases P. yoelii LS burden in Hepa 1–6 cells in a dose-dependent manner. (a) Hepa 1–6 cells were infected with P. yoelii sporozoites in chamber slides and treated with the indicated concentration of Serdemetan for 48 hours. Parasites were quantified microscopically by staining for UIS4 and HSP70. (b) Hepa 1–6 cells were treated with Serdemetan (10 µmol/l) for 48 hours and analyzed for P53 expression by immunoblot. (c) Hepa 1–6 cells were infected with P. yoelii and treated with Serdemetan (10 µmol/l) with or without the total caspase inhibitor qVD-OPh (20 µmol/l) for 24 hours. LS were quantified as in part (a). (d) Hepa 1–6 cells were infected with P. yoelii parasites and LS burden was quantified at 24 hours postinfection by microscopy as described in part a. Parasites were either treated with Serdemetan 24 hours before infection (-24 hours), 24 hours after infection (+24 hours) or both (-24 hours to +24 hours). NS (not significant), *P ≤ 0.05, **P ≤ 0.01 when compared with nontreated controls.

Figure 4

Combination treatment of Serdemetan and Obatoclax completely eliminates LS burden. (a) Hepa 1–6 cells were infected with P. yoelii sporozoites in chamber slides and treated with Serdemetan (10 µmol/l), Obatoclax (100 nmol/l), Serdemetan and Obatoclax, or media only (NT) for 48 hours. LS parasites were identified by UIS4 and HSP70 expression using fluorescence microscopy and quantified. NS (not significant), *P ≤ 0.05, **P ≤ 0.01 when compared with nontreated controls. (b) BALB/cJ mice were treated 24 hours prior to infection, the day of infection, and for 3 days following infection with vehicle only, Serdemetan (20 mg/kg/day) only, Serdemetan (20 mg/kg/day) and Obatoclax (5 mg/kg/day), or double Serdemetan (40 mg/kg/day) and Obatoclax (5 mg/kg/day). There was an additional condition where mice were treated for 24 hours prior to infection, the day of infection, and for 8 days following infection with Serdemetan (20 mg/kg/day) and Obatoclax (5 mg/kg/day). Patency was monitored for all conditions by thin blood smear and Giemsa stain.

Figure 5

Plasmodium falciparum is cleared from the liver of humanized mice treated with Serdemetan and Obatoclax. (a,b) FRG-HuHep mice were treated with either vehicle control or both 5 mg/kg of Obatoclax and 20 mg/kg Serdemetan by oral gavage once daily for 8 days. On the second day of treatment, mice were injected intravenously with 10⁶ P. falciparum GFP-luc transgenic parasites. Parasite load was assessed by IVIS. Mice with undetected parasite burdens are depicted in red.