Attenuated parasites persist and induce cross-species protective immunity. We initially treated P. chabaudi parasitized rbcs (prbcs), 98% of which were at the early “ring” stage, with the seco-cyclopropyl pyrrolo indole analogs AS-I-145 (centanamycin), TH-III-149 (tafuramycin A), and AS-VIII-104 for 40 minutes in vitro. The prbcs were washed 3 times to remove excess drug, and then 106 treated cells were administered i.v. to immunodeficient SCID mice to ascertain attenuation. Mice were followed for 6 weeks for the appearance of any “breakthrough,” parasites but none were detected microscopically. We also analyzed the blood from SCID and normal mice receiving centanamycin-attenuated and WT parasites using a quantitative PCR (qPCR) assay based on the 18S rRNA gene of Plasmodium (Figure 1A). Based on this assay, we showed that most attenuated (and WT) parasites were removed from the blood within 5 minutes of administration. For up to 24 hours after administration of either attenuated or WT parasites to normal mice, DNA levels were similar. After that time, DNA levels then increased rapidly in mice given WT parasites as the infection progressed. However, DNA from the attenuated parasites persisted at low but fluctuating levels in the blood of SCID and normal mice for over 110 days. In a repeat experiment, we used 15 mice and divided them into 3 groups of 5 so that more blood could be taken from each mouse at each time point up to day 51 (collecting 50 μl instead of 20 μl of blood to increase the sensitivity of the assay) (Supplemental Figure 1; supplemental material available online with this article; doi: 10.1172/JCI66634DS1). As reported in Figure 1, DNA was detectable at low but fluctuating levels and all mice survived. It was not clear whether these low but fluctuating levels of DNA represented viable parasites or simply DNA from nonviable parasites. To explore further whether or not viable parasites were present in the blood of vaccinated mice, xenodiagnosis was undertaken. Blood from vaccinated mice was transferred to naive BALB/c mice (100 μl per mouse). None of the 20 mice that received the blood developed a patent parasitemia, suggesting that the parasites were not viable in the donors at the time of transfer.

Figure 1 Immunization with attenuated parasites. (A) Immunodeficient SCID mice (left panel) or immunocompetent A/J mice (right panel) were administered 106 (left panel) or 107 (right panel) P. chabaudi prbcs attenuated with 2 μM centanamycin or 107 WT parasites (right panel). qPCR was performed to estimate parasite density in the blood at various time points. y axes show the estimated parasite density. (B) To assess immunity, A/J mice (5 per group) were immunized with a single dose of 106P. chabaudi prbcs attenuated with centanamycin, TH-III-149, or AS-VIII-104, as indicated, or left untreated. All were challenged 5 weeks later with 105P. chabaudi prbcs i.v. (C) To assess immunity in a different mouse strain, C57BL/6 mice were either immunized with a single dose of 106P. chabaudi prbcs attenuated with centanamycin or given a saline injection (control) and challenged 9 weeks later with 105P. chabaudi prbcs i.v. (D) TH-III-149 (for P. vinckei challenge) or centanamycin (for P. yoelii challenge) were used to attenuate P. chabaudi, and mice were immunized with a single dose of 106 prbcs and challenged 4 weeks later with either 104P. vinckei or 104P. yoelii prbcs i.v. Plus signs indicate that mice succumbed to the infection.

Parasites attenuated with each of the seco-cyclopropyl pyrrolo indole analogs were next administered i.v. to immunocompetent A/J mice. The infection is lethal in this mouse strain. Five weeks after a single immunizing dose of 106 attenuated P. chabaudi parasites, the mice were challenged with 105 homologous WT parasites and were strongly protected (Figure 1B). Clinical scores were recorded for all mice and none of the vaccinated mice demonstrated any adverse clinical effects as a result of the vaccination or the challenge infection. Although P. chabaudi is not lethal in C57BL/6 mice, we observed that the centanamycin-attenuated vaccine could also induce strong antiparasitic immunity in this mouse strain (Figure 1C). In various experiments, control A/J mice immunized with uninfected rbcs treated with centanamycin, injected with saline, or receiving no treatment all developed a rapid infection following challenge and succumbed (data not shown). In other experiments, we observed protection following immunization with 104 attenuated parasites (data not shown) and also observed that 3 immunizations did not lead to enhanced protection over a single immunization (Supplemental Figure 2).

We next asked whether immunity was long lived and observed that A/J mice challenged 6 months after a single immunization with attenuated prbcs from a different parasite, P. yoelii, were strongly protected against homologous challenge (Supplemental Figure 3). To ask whether immunity resulted in cross-species protection, we vaccinated mice with centanamycin-attenuated parasites prepared from P. chabaudi strain AS, challenged them with the heterologous species, Plasmodium vinckei and P. yoelii, and also observed strong protection (Figure 1D). Mice were also vaccinated with centanamycin-attenuated parasites prepared from P. chabaudi strain AS and challenged initially with the homologous strain, then 3 months later with the heterologous strain, P. chabaudi AJ. We observed strong protection against the original homologous parasite and the subsequent heterologous challenge (Supplemental Figure 4).

Attenuated parasites induce a cellular immune response. To examine the nature of immunity, we initially determined the fate of attenuated P. chabaudi prbcs in vivo. We confirmed the rapid diminution of attenuated parasite numbers in the blood shown by qPCR (Figure 1) using an assay in which we tracked parasitized and normal rbcs by labeling the rbc membranes with DiI (22) and parasite DNA with Hoechst (HO) stain 34580. We demonstrated that chemically attenuated prbcs were removed from the circulation far more rapidly than normal rbcs (nrbcs) (Figure 2A). Attenuated parasites accumulated in the spleen and liver as determined by flow cytometry (Figure 2B) and were shown to be inside F4/80+ cells (macrophages) and CD11c+ dendritic cells (Figure 2, C and D). Confocal imaging confirmed that HO+DiI+ cells could be seen within F4/80+ cells in the spleen (Supplemental Figure 5B). These data suggested that attenuated parasites might rapidly activate the immune system.

Figure 2 Tracking of attenuated parasites stained with DiI and HO. rbcs from a P. chabaudi–infected mouse (parasitemia, 39.8%; Supplemental Figure 5A) were treated with centanamycin, stained with DiI and HO, and then injected i.v. into naive AJ mice (3 mice, 5 × 107 cells per mouse). rbcs from an uninfected mouse were stained with DiI alone and injected into other mice (3 mice, 5 × 107 cells per mouse). DiI+HO– cells (nrbcs) or DiI+HO+ cells (prbcs) in the peripheral blood of recipient mice were analyzed over time by flow cytometry and the percentage of these cells of the total injected nrbcs or prbcs calculated based on an assumption of the recipient mice each containing a total of 5 × 109 rbcs (A). Spleen and liver cells of recipient mice were prepared by mechanical disruption. rbcs were gated and DiI+HO+ cells identified. The percentages of DiI+HO+ cells of the total rbcs in the spleen and liver were then calculated (B). Free rbcs were then lysed using NH 4 Cl-Tris buffer to allow estimation of rbc uptake by white cells. Percentages of DiI+HO+F4/80+ or DiI+HO+CD11c+ cells in total F4/80+ (C) or CD11c+ cells (D) in the spleen and liver were then calculated. Two independent experiments were performed (total n = 4–6 mice per time point), and representative graphs are shown. Means ± SEM are shown.

To measure early activation of the immune system, we monitored CD11a and CD49d expression on T cells (23) and observed that within 5 days of administration of centanamycin-attenuated P. chabaudi prbcs, 1.2% of peripheral blood CD4+ T cells showed significant activation above the level of activation in mice that received a saline injection (P < 0.0001) and that the mean number of activated cells rose to 5% by 21 days (Figure 3A and Supplemental Figure 6). There was less activation of CD8+ cells. When tested 2 months after vaccination, spleen cells showed a strong proliferative response to homologous prbcs as well as to prbcs of the heterologous species P. yoelii, but not to uninfected rbcs (Figure 3B). Spleen cells from a naive mouse did not respond to the parasites.

Figure 3 Immune responses induced by chemically attenuated parasites. (A) C57BL/6 mice (12 per group) were vaccinated with 106P. chabaudi prbcs attenuated with centanamycin or injected with saline. Phenotypes of blood CD4+ and CD8+ cells were assessed on days 5 and 21 after vaccination. Values for means of the saline group were subtracted from values for each vaccinated mouse. Each circle represents 1 mouse. Horizontal bars represent means. One sample t test was performed comparing values to the saline mean of zero. (B) Three A/J mice were immunized with 3 doses of vaccine (each, 106 centanamycin-attenuated P. chabaudi prbcs), and spleen cells from these and a naive mouse were collected 8 weeks after the last immunization and cultured with indicated antigens; uptake of 3H thymidine was determined after 72 hours as described in Methods. (C) C57BL/6 mice (5 per group) were vaccinated with 106P. chabaudi prbcs or equivalent nrbcs attenuated with centanamycin and sacrificed on day 5. Spleen cells were stimulated in vitro for 4 hours with PMA and ionomycin in the presence of BFA and stained for IFN-γ. Percentage that was IFN-γ positive was determined by subtracting isotype control and values from mice injected with saline for CD4 and CD8 populations. (D) Three A/J mice were immunized with 3 doses of vaccine (each, 106 centanamycin-attenuated P. chabaudi prbcs), and spleen cells from these mice were collected 50 days after the last immunization, cultured with indicated antigens, and after 72 hours culture, supernatants collected and use for cytokine bead analysis as described in Methods. (E) C57BL/6 mice (CD45.2, 5 per group) were adoptively transferred with OT-II T cells (CD45.1), and 1 group received ovalbumin peptide 323–339 mixed with LPS i.p. 24 hours later, mice were immunized i.v. with 106P. chabaudi prbcs or equivalent nrbcs attenuated with centanamycin. Mice were bled on day 6 after immunization and the phenotype of donor and recipient cells determined by flow cytometry. Percentages were calculated by subtracting values from mice receiving OT-II cells alone.

To determine whether the T cells were functional, splenic CD4+ and CD8+ T cells were examined for intracellular IFN-γ 5 days after vaccination with centanamycin-attenuated parasites, centanamycin-treated nrbcs, or saline. Cells were activated in vitro for 4 hours with PMA/ionomycin prior to staining (as described in ref. 23). There were significantly more cytokine-positive cells in the spleens of vaccinated mice compared with the other groups of mice (Figure 3C). Furthermore, spleen cells from vaccinated mice produced larger amounts of IFN-γ and MCP-1 ex vivo in response to prbcs compared with nrbcs or medium alone (Figure 3D).

To confirm that vaccination induced expansion and activation of antigen-specific CD4+ T cells, rather than bystander activation, we transferred congenic (CD45.1) OVA-TCR transgenic OTII cells into CD45.2 recipients, which we vaccinated with attenuated parasites or OVA peptide/LPS, and observed activation of the OTII cells following OVA peptide administration, but not following vaccination with attenuated parasites. Conversely, we observed activation of CD45.2 CD4+ T cells following vaccination with centanamycin-treated prbcs, but not following administration of centanamycin-treated nrbcs or OVA. These data demonstrate that the vaccine induced expansion and activation of antigen-specific CD4+ T cells, without activation of bystander T cells (Figure 3E).

To determine whether the vaccine-induced, antigen-specific CD4+ T cells were protective, immunized mice were given antibodies to clear more than 98% of their CD4+ T cells or more than 89% of CD8+ T cells (Supplemental Figure 7) and then challenged. Control mice that received normal rat immunoglobulin showed complete protection, but CD4+ T cell–depleted mice all succumbed, albeit more slowly than naive mice (Supplemental Figure 8). When CD8+ T cells were depleted from immune mice, we observed no change in their level of immunity. Thus, CD8+ T cells alone are not able to provide complete protection, but they or other cell types may contribute a minor role.

Although CD4+ T cells were critical for protection, we were not able to transfer protection with purified CD4+ T cells. Thus, 107 CD45.2 splenic CD4+ T cells from C57BL/6 mice immunized 3 times with centanamycin-attenuated P. chabaudi prbcs or equivalent numbers of naive CD45.2 T cells were transferred into CD45.1 congenic naive mice that had been pretreated 3 days earlier with 1 dose of anti-CD4 mAb to deplete most endogenous T cells. CD4+ T cell engraftment was successful, as shown by flow cytometry with between 5% and 12% of peripheral blood T cells being of donor origin. Mice were challenged 11 days after transfer, but no protection was observed, despite control animals vaccinated with centanamycin-treated prbcs being protected.

We next asked whether mice immunized with either 104 or 106 chemically attenuated parasites developed parasite-specific antibodies. Using an ELISA to whole parasite antigens, we observed that while sera taken from mice that experienced multiple infections each followed by drug cure contained antibodies, sera from mice immunized with attenuated parasites did not (Supplemental Figure 9A). In a repeat experiment to confirm protection in the absence of antibodies, we observed that not only were antibodies not detectable after vaccination, but that following challenge (during which parasites were not detectable by microscopy in any of the vaccinated recipients), antibodies were also not detected (Supplemental Figure 9B). In a further experiment to ascertain any role for antibodies, sera from 10 vaccinated A/J mice and from 10 control mice were transferred into naive recipients (0.5 ml on each of days –1, 0, and 1) that were challenged on day 0. Mice that received serum from immunized mice as well as mice that received serum from naive controls all succumbed without any evidence of protection, but the serum donor mice were protected (Supplemental Figure 9C).

The above data suggest that antigen-presenting cells initiate an antiparasite CD4+ T cell response following uptake of rbcs containing attenuated parasites. To further understand the interactions between attenuated parasites and the cells of the immune system, we asked whether a preparation of lysed attenuated prbcs would also induce immunity. We immunized mice with intact or lysed attenuated prbcs and observed that when the rbc membranes were disrupted, immunity was not induced (Figure 4A). Therefore, the interaction between the attenuated parasites and the cells of the immune system requires the rbc membranes to remain intact. As the spleen is assumed to be a site where antiparasite immunity is induced (ref. 24 and Figure 2), we examined the importance of the spleen in the induction of protective immunity. We observed that, while eusplenic control mice could control parasite burden more effectively than splenectomized control mice, both groups of vaccinated mice could control parasite burden very effectively and to a similar level at 5 weeks after vaccination (Figure 4B). This demonstrated that short-term immunity could be induced and mediated in tissues other than the spleen, with one likely site being the liver (25, 26). However, in an experiment in which mice were challenged 12 weeks after vaccination, we observed that splenectomized mice were not protected, whereas sham-splenectomized mice were still protected. This may be due to the presence of spleen-resident memory cells mediating protection at later time points with effector cells, which may have a different tissue localization, mediating protection at earlier time points.

Figure 4 Vaccine protection requires an intact rbc membrane. The role of the spleen early (5 weeks) after vaccination. (A) To determine whether the rbc membrane must remain intact for vaccine efficacy, cohorts of 5 A/J mice were vaccinated with a single dose of 106P. chabaudi prbcs attenuated with centanamycin or a vaccine lysed with distilled water, then returned to isotonicity with 2× PBS. Naive mice served as a control. Five weeks later, mice were challenged with 105P. chabaudi prbcs i.v. and parasitemia monitored. Plus signs indicate that mice succumbed to the infection. (B) To determine the importance of the spleen to immunity early after vaccination, A/J mice were splenectomized and others had a sham splenectomy. Four weeks later, half were immunized i.v. with 3 doses of 106P. chabaudi prbcs attenuated by treatment with centanamycin, each 2 weeks apart. The other half received no treatment. Five weeks after the final dose of vaccine, all mice were challenged with 105 prbcs i.v. and parasitemia monitored. Plus signs indicate that mice succumbed to the infection.

We next determined whether centanamycin treatment would result in attenuation of the major human parasite P. falciparum. It was previously observed that centanamycin could kill P. falciparum in vitro (27). To analyze this further, we measured RNA transcription and DNA replication after centanamycin treatment of a synchronized P. falciparum culture containing initially only rings (Supplemental Figure 10). We showed that, over a 64-hour time period, very few centanamycin-treated cells progressed through the cell cycle and that there was near complete arrest of transcription. This level of attenuation is consistent with our observations in vivo (above).