A murine model of falciparum-malaria by in vivo selection of competent strains in non-myelodepleted mice engrafted with human erythrocytes

Abstract

To counter the global threat caused by Plasmodium falciparum malaria, new drugs and vaccines are urgently needed. However, there are no practical animal models because P. falciparum infects human erythrocytes almost exclusively. Here we describe a reliable falciparum murine model of malaria by generating strains of P. falciparum in vivo that can infect immunodeficient mice engrafted with human erythrocytes. We infected NOD(scid/beta2m-/-) mice engrafted with human erythrocytes with P. falciparum obtained from in vitro cultures. After apparent clearance, we obtained isolates of P. falciparum able to grow in peripheral blood of engrafted NOD(scid/beta2m-/-) mice. Of the isolates obtained, we expanded in vivo and established the isolate Pf3D7(0087/N9) as a reference strain for model development. Pf3D7(0087/N9) caused productive persistent infections in 100% of engrafted mice infected intravenously. The infection caused a relative anemia due to selective elimination of human erythrocytes by a mechanism dependent on parasite density in peripheral blood. Using this model, we implemented and validated a reproducible assay of antimalarial activity useful for drug discovery. Thus, our results demonstrate that P. falciparum contains clones able to grow reproducibly in mice engrafted with human erythrocytes without the use of myeloablative methods.

Figure 1. Selection of immunodeficient mice.

The percentage of hE (TER-119⁻ or human glycophorin A⁺ cells) in peripheral blood of NIH-III^{beige/xid/nude}, CB17^scid, CB17^scid/beige, NOD^scid and NOD^{scid/β2m−/−} murine strains upon daily intraperitoneal injection of hE is shown. The regression curves for fitting to an exponential association equation are shown for CB17^scid, CB17^scid/beige, NOD^scid and NOD^{scid/β2m−/−}. The strain NIH-III^{beige/xid/nude} was discarded from the study at the point indicated (★). Data are the mean±SE of n = 4 mice per data point.

Figure 2. Optimized selection and characterization of competent P. falciparum isolates.

(A) A cohort C1 of mice is conditioned for 7–10 days with daily i.p. injections of hE. Then, the mice are infected by i.p. or i.v. route with 25·10⁶ or 50·10⁶ parasitized erythrocytes from in vitro cultures. Parasitemia in peripheral blood is followed up to 1 month after infection or until about 1% of parasitemia is achieved (see point B). Mice having productive infections are used as donors to infect by i.v. route a cohort C2 of conditioned mice. After 1 week of infection, the mice with the highest parasitemia is selected as donor for next in vivo passage. After ten passages showing a stable kinetics of growth (see point C), a final expansion step is performed to establish a standard P. falciparum strain, which is frozen and used in experiments. (B). Course of parasitemia in peripheral blood from the mice that originated the competent isolates Pf3D7^0087/N9 (i.p. infection), PfV1/S^0176/N7 (i.p. infection) and PfV1/S^0176/N10 (i.v. infection). (C) Dot plot analysis of the kinetic stability of the isolate Pf3D7^0087/N9 growth during sequential i.v. infections with 20·10⁶ P. falciparum-infected erythrocytes. Stability in 10 sequential i.v. infections was used as the criterion for establishing the reference standard Pf3D7^0087/N9 strain. Each data point is the mean of three mice per in vivo passage for ten consecutive passages. (D) Microsatellite PfRRM of the P. falciparum 3D7 and Pf3D7^0087/N9 strains. (E) Growth of Pf3D7^0087/N9 in different strains of HM after i.v. infection with 20·10⁶ Pf3D7^0087/N9 parasites. Data are the mean±SE of four mice·group⁻¹.

Figure 3. Engraftment of hE in NOD^{scid/β2m−/−}.

(A) Long term kinetics of engraftment of NOD^{scid β2m−/−} mice with hE. Data are the mean percentage±SE of hE (TER-119⁻ or human glycophorin-A⁺ mouse) erythrocytes pooled from 142 mice. The inset plot shows the effect of human serum deprivation from daily-inoculated hE on engraftment of hE in peripheral blood of NOD^{scid β2m−/−} (n = 3 mice·group⁻¹). Data shown are from a representative experiment out of three. (B) Histological analysis of brain, kidney, liver and spleen of NOD^{scid/β2m−/−} control, conditioned with hE and conditioned mice infected with P. falciparum (day 8 after infection). Non-infected mice conditioned with hE showed a marked vascular congestion compared to control mice. Infected mice, showed decreased vascular congestion, increased numbers of myelomonocytic cells and enhanced phagocytic activity in the spleen (×600 magnification). (C) Concentration of erythrocytes, hE and mE in peripheral blood of NOD^{scid β2m−/−} during conditioning before i.v. infection with P. falciparum. Data are the mean±SE of n = 21 mice.

Figure 4. Infection dynamics in HM infected with Pf3D7^0087/N9.

Concentration of hE, ihE and mE in peripheral blood of infected mice. Data are the mean±SE of three mice per data point. The results are from a representative experiment out of three. (B) Abrogation of infection-induced elimination of hE by treatment with chloroquine (10 mg·Kg⁻¹) and exponential growth of Pf3D7^0087/N9 during recrudescence. Data are the mean concentration±SE of three mice per data point. The results are from a representative experiment out of two. (C) The effect of splenectomy on the percentage of hE and parasites in peripheral blood. The data are the means±SE of n = 6 mice·group⁻¹ pooled from two independent experiments.

Figure 5. Characterization of the P. falciparum-malaria model for antimalarial drug testing.

(A) Kinetics of parasitemia in peripheral blood of HM infected i.v. with 20·10⁶ Pf3D7^0087/N9 parasites. Data are the mean±SE from 88 mice. The inset plot shows the regression line to fit an exponential growth model of the means of parasitemia of the pooled data up to day 7 after infection. (B) Giemsa-stained smears from peripheral blood of HM infected i.v. with Pf3D7^0087/N9 showing the different stages of P. falciparum: ring (upper row on the left) to mature schizont (lower row on the right) (×1000 magnification). (C) Replication of Pf3D7^0087/N9 in hE. Flow cytometry panels depict erythrocyte populations in control (left panel), uninfected HM (middle panel) and infected HM (right panel). The percentage of mE TER-119⁺ cells is indicated. Pf3D7^0087/N9 (parasitemia 6%) are in TER-119⁻SYTO-16⁺ events. Pycnotic forms of P. falciparum were found in Giemsa stained-cytospin preparations of mE TER-119⁺ purified immunomagnetically. Viable parasites were found in purified hE TER-119⁻. Data are representative of three independent experiments. (D) Q–Q plot of normality for the variable log₁₀ (parasitemia at day 7) showing observed values vs expected normal values (n = 327 mice).

Figure 6. Validation of the P. falciparum 4-day test.

(A) Therapeutic efficacy of chloroquine, artesunate, and pyrimethamine using the standard P. falciparum 4-day test. Data are the mean parasitemia±SE of n = 3 mice·group⁻¹ from a single experiment using 45 mice. (B, C, D, E) Representative blood smears 48 h after starting treatment with saline, chloroquine (20 mg·Kg⁻¹), artesunate (25 mg·Kg⁻¹) or pyrimethamine (20 mg·Kg⁻¹), respectively. The remaining parasites in peripheral blood after treatment with chloroquine or artesunate were pycnotic cells and disrupted trophozoites. Pyrimethamine led to swollen late trophozoites with prominent granules of hemozoin (×1000 magnification). (F) Logarithmic growth of Pf3D7^0087/N9 during recrudescence after treatment with chloroquine (10 and 20 mg·Kg⁻¹, p.o., u.i.d.) in a standard 4-day test. Data are the mean parasitemia±SE of three mice/group. (G) Exposure-therapeutic efficacy relationships of chloroquine in the P. yoelii or the P. falciparum murine models of malaria. Data are the mean concentration of chloroquine in blood of n = 3 mice·group⁻¹.

Figure 7. Therapeutic efficacy of diamidine derivatives against P. berghei, P. vinckei and P. falciparum.

(A, B, C) Therapeutic efficacy of pentamidine (40 mg·Kg⁻¹, u.i.d., s.c.), DB75 (10 mg·Kg⁻¹, u.i.d., s.c.) or DB289 (100 mg·Kg^-1, u.i.d., p.o.) against P. berghei, P. vinckei and P. falciparum, respectively. We started treatment when parasitemias where comparable and administered compounds for 4 days. (D, E) Giemsa-stained blood smears from mice infected with P. berghei obtained 48 h after starting treatment with vehicle or DB75 at 10 mg·Kg⁻¹, respectively (×1000 magnification). Neither relevant cellular damage nor significant inhibition of parasitemia were observed at the time of sampling. (F, G) Giemsa-stained blood smears from mice infected with P. vinckei obtained 48 h after starting treatment with vehicle or DB75 at 10 mg·Kg⁻¹, respectively (×1000 magnification). Parasites from treated mice were mostly abnormal late trophozoites. (H, I) Giemsa-stained blood smears from mice infected with P. falciparum obtained 48 h after starting treatment with vehicle or DB75 at 10 mg·Kg⁻¹, respectively (×1000 magnification). Parasites from treated mice were mostly abnormal late trophozoites.