Induction of antimalaria immunity by pyrimethamine prophylaxis during exposure to sporozoites is curtailed by parasite resistance

Abstract

Each year, infections with the protozoan parasite Plasmodium falciparum kill 1 million people, mostly children in Africa. Intermittent preventive treatment (IPT) with sulfadoxine-pyrimethamine (SP) reduces the incidence of malaria and aims to prevent mortality in infants, children, and pregnant women. There is contradictory evidence as to whether this strategy may generate additional protection against reinfection beyond the limited duration of the intervention. Previous work established that ablation of either liver-stage maturation or subsequent life cycle conversion by causal prophylactic drugs elicits protective immune responses against reinfections when drugs are no longer present. Here we show in the rodent malaria model that pyrimethamine, a component of SP, inhibits liver-stage development in vitro and in vivo, confirming the causal prophylactic activity of pyrimethamine. Repeated exposure to high doses of Plasmodium berghei sporozoites during pyrimethamine prophylaxis induced complete protection in C57BL/6 mice against challenge with high doses of sporozoites delivered intravenously 35 to 199 days later. Immunizations by infectious mosquito bites induced limited, inoculation-dependent protection against subsequent challenge by infected mosquito bites but provided partial protection against experimental cerebral malaria. Short-term pyrimethamine prophylaxis during intravenous transmission of sporozoites from a pyrimethamine-resistant strain delayed, but did not prevent, blood-stage infection. Our data provide a rationale for the notion of sustained protective efficacy of IPT based on the capacity of arrested, drug-sensitive liver-stage and/or suppressed blood-stage parasites to mount lasting protection.

Fig. 1.

Pyrimethamine treatment of preerythrocytic malaria parasites prevents blood-stage infection and inhibits the growth of susceptible and pyrimethamine-resistant parasites in vitro and in vivo. (A) Kaplan-Meier curve shows the time to patent blood-stage infection after oral treatment with pyrimethamine for 42 h. (B) Quantification by real-time PCR of parasite loads in infected livers at 42 h after sporozoite inoculation under oral treatment with pyrimethamine. WT parasites were used for infection of C57BL/6 mice. Relative expression levels of the Pb18S gene were normalized to the levels of the mouse GAPDH gene. (C) In vitro development of treated (1 μM pyrimethamine) and untreated (control) pyrimethamine-susceptible exoerythrocytic forms (green). Insets show the same images at a higher magnification. Bars, 10 μm.

Fig. 2.

Immunization of mice with mosquito bites (mosq.) under pyrimethamine (Pyr.) cover leads to a delay in the onset of blood-stage infection or to protection against reinfection relative to outcomes for nonimmunized mice. (A) Immunizations were carried out by bites of infectious Anopheles mosquitoes in two independent experiments. In the first experiment (Exp.), three consecutive immunizations were carried out by exposure to 10 mosquitoes/mouse, whereas in the second experiment, three immunizations with higher mosquito numbers (11 to 15 bites/mouse) were performed. (B) Times to patency for immunized and control animals after challenge by bite. (C) Percentages of immunized and control animals developing cerebral malaria.

Fig. 3.

Mice infected with pyrimethamine-resistant sporozoites show a delay in the onset of blood-stage infection after oral treatment with pyrimethamine. (A) Kaplan-Meier curve showing the time to patency of pyrimethamine-resistant (Pyr.-res.) and pyrimethamine-susceptible sporozoites. Animals were treated orally with pyrimethamine for 42 h. Each experimental group consisted of five C57BL/6 mice. (B) In vitro development of pyrimethamine-resistant exoerythrocytic forms (EEF) with different drug concentrations. Bars, 10 μm. (C) The effect of pyrimethamine on the growth of pyrimethamine-resistant and -susceptible parasites is illustrated by comparison of the EEF size to the size of the host cell nucleus. Fifty EEF were counted for each drug concentration. nd, none determined. (D) Quantification by real-time PCR of parasite loads in infected livers at 42 h after inoculation of pyrimethamine-resistant parasites with or without oral pyrimethamine prophylaxis. Relative expression levels of the Pb18S gene were normalized to those of the mouse GAPDH gene.

Fig. 4.

Breakthrough blood-stage infections curtail preerythrocytic immunity. (A) Animals (n = 18) were infected with pyrimethamine (pyr.)-resistant (res.) (n = 13) or pyrimethamine-sensitive (WT) (n = 5) sporozoites (spz.) under suboptimal pyrimethamine cover. All animals were monitored for parasitemia and were cured with 1.6 mg/day chloroquine (CQ) for 7 days, starting at day 14. Fourteen animals developed parasitemia between days 3 and 10 after infection, and 4 stayed malaria free. All treated animals and untreated control animals (−) (n = 4) were immunized with 10,000 irradiated (irr.) sporozoites at day 24. Finally, all mice were challenged with 10,000 WT sporozoites at day 43. Mice were sacrificed 42 h after the WT challenge, and livers were removed for RNA extraction and cDNA synthesis. (B) Quantitative RT-PCR data for control mice (n = 4) (black circles), blood-stage-negative mice given spz. and pyr. (n = 4) (green circles), and blood-stage-positive mice given spz. and pyr. (n = 14) (red circles). Relative expression levels of the Pb18S gene were normalized to those of the mouse GAPDH gene. *, P < 0.05; ***, P < 0.001.