IFNγ response by liver and spleen CD8+CD44hi T cells.

<p>At day 62, liver and spleen cells collected from immune RAS or CPS mice were stimulated for 24 hours ex vivo with cryo-conserved <i>Pb</i>spz. IFNγ response was assessed by intracellular cytokine staining prior to flow-cytometry measurement (2 experiments). The percentage of IFNγ+ lymphocytes upon stimulation with PMA and ionomycin was similar between RAS (n = 15), CPS (n = 10) and naïve (n = 6) mice in the liver (6.9%, 5.2%, 5.1%) or spleen (1.33%, 1.65%, 1.1%). * = p<0.05, ** = p<0.001.</p

Long-term RAS and CPS^a protection following <i>P. berghei</i> sporozoite challenge^b.

a<p>CPS mice received 24-days chloroquine treatment. Three of the six naïve mice challenged at t = 3months receive the same chloroquine treatment.</p>b<p>Mice were challenged by i.v. injection of 10.000 WT sporozoites. Protection was defined as negative blood-smears at day 21 after challenge.</p

Sporozoite and blood-stage specific IgG.

<p>Plasma were collected from mice immunized by RAS or CPS before (C-1) and 6 to 21 days after (C+6; C+21) challenge. Levels of anti-sporozoite or anti-blood-stage IgG antibodies were determined by ELISA (n_RAS = 5; n_CPS = 5; n_naïve = 9). Error bars represent standard error of the mean (SEM). ** = p<0.005, *** = p<0.0001.</p

Phenotypic analyses CD44hi T-cells.

<p>(A) Gating strategy. After lymphocytes gating based on forward-scatter (FSC) and side-scatter (SSC) properties, CD4+ and CD8+ T cells were selected. Total memory T cells were gated based on high CD44 expression. T cells with effector memory (T_EM) and central memory (T_CM) phenotype were identified based on CD62L expression. (B) CD8+CD44hi and CD4+CD44hi T-cell pool at day 40 post-immunization with high or lose dose. Composition of the CD8+CD44hi and CD4+CD44hi T-cell pool was assessed in the liver (left panel), spleen (central panel) or PBMC (right panel) of mice immunized by high and low dose of RAS or CPS. Results are from 2 independent experiments (n_RAS = 10; n_CPS = 10; n_naïve = 13) and cells from individual mice assayed. Error bars represent standard error of the mean (SEM). * = p<0.05, ** = p<0.005, *** = p<0.0001.</p

Declined specific IFNγ response by hepatic CD8+ memory T cells over time.

<p>Sporozoite specific IFNγ response by hepatic CD8+CD44hi T cells was measured by intracellular staining at 3 months post-immunization (A) – individual values and median are plotted. Longevity of the specific (sporozoites) and non-specific (PMA/Ionomycin) IFNγ response was further assessed 6 and 9 months after immunization (B) – error bars represent SEM. * = p<0.05, ** = p<0.001.</p

Re-exposure by sporozoite challenge increases memory CD8+ T cell response.

<p>Levels of CD8+ T_EM were measured before (C-1) and after (C+21) challenge at 3, 6 or 9 months after RAS and CPS immunization (A) – individual values for each time point and median are plotted. IFNγ response by hepatic CD8+CD44hi T cells was measured by intracellular staining before (C-1) and after (C+21) challenge at 3, 6 or 9 months post-immunization (B) – error bars represent SEM. * = p<0.05, ** = p<0.005, *** = p<0.0001.</p

RAS and CPS^a protection upon <i>P. berghei</i> sporozoite challenge^b.

a<p>CPS mice received 24-days chloroquine treatment. All immunized mice were challenged 17 days after CQ treatment.</p>b<p>Cumulative data from three experiments. In two experiments, mice were challenged by i.v. injection of 10.000 WT sporozoites. In one experiment, mice were challenged by bites of 5–11 infected mosquitoes. Protection was defined as negative blood-smears at day 21 after challenge.</p

CD4+ and CD8+ T_EM cells in response to challenge.

<p>Changes in the effector memory CD8+ and CD4+ T-cell compartment from the liver, spleen and PBMC of mice immunized with low dose of RAS (n = 15) or CPS (n = 12) were evaluated at various time-points around challenge (C-1, C+6, C+21). Results are from 3 independent experiments (n_naïve = 18) and cells from individual mice assayed. Error bars represent standard error of the mean (SEM). * = p<0.05, *** = p<0.0001.</p

Attraction of malaria infected mosquitoes to human odor.

<p>(<b>A</b>) Total number of landings by uninfected (green bars) and <i>P. falciparum</i> infected (red bars) <i>An. gambiae s.s.</i> females in response to no odor (left bars) or human odor (right bars). Error bars represent the standard error of the mean. (<b>B</b>) Simplified overview of our hypothesis on the effect of <i>P. falciparum</i> infection of <i>An. gambiae s.s.</i> on human malaria risk via alterations in the olfactory system of the vector (OBPs: odorant-binding proteins, ORs/IRs: olfactory and ionotropic receptors; R0: the basic reproductive number).</p

Demographic information.

<p>Demographic information.</p