Figure 1. Overview of experimental procedures.
To quantify transient blood-stage exposure during chemoprophylaxis and sporozoite (CPS) immunization female C57BL/6 were immunized three times at 2-week intervals with P. chabaudi AS-infected mosquito bites (typically 9.15 [median, range 6.9–13.6] [Spence et al., 2012]). Following each immunization mice received 100 mg per kg chloroquine (CQ) per os daily for 10 days, starting from the day of infection. A small blood sample was taken 48 hr after each mosquito transmission (before merozoite egress from the liver, P. chabaudi develops in the liver for 52 hr [Stephens et al., 2012]; erythrocytic cycle 0), and then every 24 hr until erythrocytic replication cycle 4. Blood parasitemia was analyzed by sensitive quantitative RealTime (qRT) PCR (Figure 2). Pre-erythrocytic immunity was evaluated in mice immunized three times with either P. chabaudi AS-infected mosquito bites (Figure 3A) or by intravenous (iv) injection of defined numbers of P. chabaudi CB sporozoites (Figure 3B). P. chabaudi CB was used since mosquitoes infected with this parasite harbor an increased number of sporozoites in their salivary glands (Spence et al., 2012), which made injections of high numbers of sporozoites technically feasible. Mice were challenged 100 days after the last immunization by mosquitoes infected with the respective homologous strain. Liver parasitemia was examined 42 hr after challenge by qRT PCR. Blood-stage immunity was assessed in mice immunized with P. chabaudi AS-infected mosquito bites by qRT PCR and thin blood film following homologous challenge with either infected mosquito bites (Figures 4A, 5C/D) or intraperitoneal (ip) injection of parasitized erythrocytes, which were either derived from a donor mouse infected by mosquito bite (recently mosquito transmitted, Figure 4B) or after 26–32 serial blood-passages (Figure 4C). Heterologous protection was assessed using P. chabaudi CB-infected mosquito bites (Figure 5A/B). To evaluate cross-stage protection mice received a first infection with P. chabaudi AS either by mosquito bite or by ip injection of recently mosquito transmitted parasitized erythrocytes. The resulting blood-stage infection was eventually self-cured without intervention. Mice were re-challenged 100 days after their first infection with P. chabaudi AS-infected mosquito bites and liver parasitemia was evaluated by qRT PCR (Figure 6).