Vaccination with live Plasmodium yoelii blood stage parasites under chloroquine cover induces cross-stage immunity against malaria liver stage

Abstract

Immunity to malaria has long been thought to be stage-specific. In this study we show that immunization of BALB/c mice with live erythrocytes infected with nonlethal strains of Plasmodium yoelii under curative chloroquine cover conferred protection not only against challenge by blood stage parasites but also against sporozoite challenge. This cross-stage protection was dose-dependent and long lasting. CD4(+) and CD8(+) T cells inhibited malaria liver but not blood stage. Their effect was mediated partially by IFN-gamma, and was completely dependent of NO. Abs against both pre-erythrocytic and blood parasites were elicited and were essential for protection against blood stage and liver stage parasites. Our results suggest that Ags shared by liver and blood stage parasites can be the foundation for a malaria vaccine that would provide effective protection against both pre-erythrocytic and erythrocytic asexual parasites found in the mammalian host.

FIGURE 1

Self-cured infection induced a protective immunity against blood stages but not against liver parasites. Groups of BALB/c mice were i.v. injected with 4000 P. yoelii 265BY sporozoite. All mice develop patent self-limiting blood parasitemia that cured by day 20. The mice were then rested for 15 days before challenge. A, Sterile protection in self-cured mice. Self-cured naive mice were challenged with 4000 homologous sporozoites. All naive mice (n = 5) developed patent blood stage parasitemia after sporozoite challenge. A mouse was considered protected if it did not develop patent blood stage parasitemia by day 10 after sporozoite challenge. Significant difference (p < 0.01) between self-cured naive mice was determined by Fisher’s exact test. B, Groups of naive BALB/c mice were infected allowed to self-cure as in A before challenge with 35,000 live sporozoites, so that the extent of hepatic parasite development could be assessed (29). Results were expressed as mean liver parasite load in log units ± SEM of n = 5 mice.

FIGURE 2

Immunization with iRBC under chloroquine cover induces protective immunity against a sporozoite challenge. A, Blood stage parasitemia in BALB/c mice challenged with 4000 homologous sporozoites 25 days after immunization with 10⁵ Py-iRBC or 10⁵ normal RBC under 10 days chloroquine cover. Parasitemia was monitored in each animal by microscopic examination of Giemsa-stained blood smears. Only points at which parasitemia was patent were plotted. A different symbol is assigned to each animal in the group. The number of mice with patent parasitemia relative to the total number of mice in the group is shown (top right corner). B C, Liver parasite loads in immunized mice challenged with sporozoites. Groups of BALB/c mice were either immunized with matching numbers of normal RBC (nRBC), with 10⁶ Py-iRBC (B) or 10⁶ P. berghei ANKA iRBC (Pb-iRBC) (C) on the day when a 10-day chloroquine treatment was initiated, or treated with chloroquine alone (CQ). Mice were then challenged with 35,000 P. yoelii 265BY sporozoites 25 days or more after immunization, liver parasite development was quantified. Results were expressed as mean liver parasite load log units ± SEM of n = 5 mice. Reduction of parasite load was more than 95% when the arithmetic values were used for calculation. *, p < 0.05, vs control mice using ANOVA followed by Tukey’s test.

FIGURE 3

Immunization with iRBC under chloroquine cover induce a protective T cell response against liver but not blood stage parasites. A, Groups of BALB/c mice were immunized with 10⁶ iRBC under chloroquine cover, then i.v. challenged with 35,000 sporozoites 15 days after the last immunizing dose so that the extent of hepatic parasite development could be assessed (29). On day 1 day 0 before the challenge, immunized mice were injected with control rat IgG, rat anti-CD8, or rat anti-CD4. Results were expressed as mean liver parasite load log units ± SEM of n = 5 mice. *, p < 0.05 vs control mice using ANOVA followed by Tukey’s test corresponding to a reduction in parasite load of >95% when the arithmetic values were used for calculation. B, Groups of BALB/c mice were immunized with 10⁶ Py-iRBC under chloroquine cover as in A. Control groups received chloroquine alone. Immunized mice were injected with the control IgG, anti-CD8 or anti-CD4 Abs as described. Mice were challenged with 4000 live sporozoites infection was determined on Giemsa-stained blood smears over a 10-day period. All control mice treated with chloroquine mice (n = 10) developed patent blood stage parasitemia after sporozoite challenge. This experiment was performed twice. C, Groups of BALB/c mice were immunized with 10⁶ iRBC under chloroquine cover as in A. Control groups received chloroquine alone. Immunized mice were injected with the control IgG, anti-CD8 or anti-CD4 Abs as described. Mce were challenged with 105 P. yoelii 265BY iRBC. These mice were completely protected against a challenge with blood stage parasites.

FIGURE 4

Protection induced by vaccination with Py-iRBC followed by chloroquine treatment is mediated by IFN-γ. A, Concentrations of IFN-γ in the sera collected 42 h after challenge with 2500 sporozoites, from control mice or mice immunized with 10⁶ Py-iRBC or normal RBC under 10-day chloroquine cover starting on the day of immunization. Sera were collected 42 h after challenge with 35,000 sporozoites. Results are expressed as mean ± SEM of n = 5 mice. *, p < 0.05 vs control mice using ANOVA followed by Tukey’s test. B, Groups of BALB/c mice were injected with 10⁶ Py-iRBC treated with chloroquine from day 0 to 10 starting the day of Py-iRBC inoculation. They were challenged i.v. with 4000 sporozoites at least 15 days after the last chloroquine injection. Immunized mice were injected with the rat Abs control rat IgG or anti-IFN-γ. Data are cumulative results of two experiments with n = 4–5 mice per experiment. All control mice treated with chloroquine alone (n = 10) developed patent blood stage parasitemia after sporozoite challenge. Protection was defined as the percentage of the mice that did not develop patent blood stage over 10 days postchallenge in the group that were challenged by sporozoites. **, p <0.05 between immunized mice treated with rat IgG immunized mice treated with anti-IFN—γ Abs, for values on day 10, as calculated by Fisher’s exact test. Number in brackets represents the percentage of protection.