Specificity of activation of C7 clone and protection<b>.</b>

<p>Mice were first injected (iv) or not with 10³ live wild-type (<i>wt</i>) ANKA <i>Pb</i>SPZ 10 h before they received or not 7×10⁵ naïve-BALB/c hepatocytes and C7 clone as indicated above. Infected hepatocytes were isolated from BALB/c mice injected with 10⁶ ANKA <i>wt Pb</i>SPZ 2 h earlier; and naïve BALB/c hepatocytes were isolated from naïve BALB/c mice. Mice were considered protected if they remained parasite negative 2 weeks after infection.</p

BALB/c mice injected with iSPZ-loaded hepatocytes are protected against SPZ challenge.

<p>BALB/c mice were injected (IS transfer) with 7×10⁵<i>Pb</i>iSPZ-infected or naïve BALB/c hepatocytes. Infected hepatocytes were obtained from BALB/c mice immunized with 10⁶ ANKA <i>wt</i> iSPZ 2 h earlier. Recipient mice were then challenged with two different doses (2×10³ and 5×10³, respectively, A and B) of live <i>Pb</i>SPZ one week later. Mice were considered protected if they remained parasite negative 2 weeks after challenge.</p

Infected hepatocytes present a <i>Pb</i>CSP-specific epitope to primed CD8+ T-cells and protect mice against SPZ challenge.

<p>Each recipient <i>TAP−/−</i> mouse (H-2K^b) received 7×10⁵<i>Pb</i>SPZ-infected BALB/c (H-2k^d) hepatocytes by IS transfer as described in Materials and Methods. Before IS injection, infected hepatocytes were isolated from BALB/c mice that were injected (iv) with 10⁶ANKA <i>wt Pb</i>SPZ 2 h earlier. C7 and S14 (20 million cells per mouse, iv) were injected into the corresponding group 4 h after IS transfer. Mice were protected if they remained parasite negative 2 weeks after infected hepatocyte transfer.</p

Frequency of <i>Pb</i>CSP-specific CD8+ T-cells in different organs of BALB/c mice immunized with <i>wt</i> or <i>spect (</i>−<i>)</i> iSPZ.

<p>BALB/c mice (4 mice per group) were immunized (iv) with <i>wt</i> or <i>spect (</i>−<i>)</i> ANKA <i>Pb</i>iSPZ (one dose of 1×10⁵ iSPZ). Seven (7) days later, PBL, liver and spleen cells of mice were isolated to determine frequency of <i>Pb</i>CSP-specific CD8+ T-cells by flow cytometry (FACScan). The CD8+ T-cells were double stained with PE-conjugated <i>Pb</i>CSP epitope tetramer and FITC-conjugated anti-mouse CD8b antibody. The naive group received neither <i>wt</i> nor <i>spect (</i>−<i>)</i> iSPZ. p-value compares statistically significant mean of CD8+ T-cell frequency between <i>wt</i> and <i>spect (</i>−<i>)</i> iSPZ-immunized groups.</p

Relative comparative <i>wt</i> and <i>spect (</i>−<i>)</i> parasite DNA load in spleen and liver of immunized mice.

<p>To compare relative load of <i>wt</i> and <i>spect (</i>−<i>)</i> parasite DNA in liver and spleen, BALB/c (3 mice per group) were immunized iv on tail with 1×10⁵ iSPZ (<i>wt</i> or <i>spect (</i>−<i>)</i>) in 500 µl of RPMI. Two hours later, a real-time PCR was performed following extraction of respective parasite RNA. Every sample was done in duplicate. To avoid any contamination by eventual parasite from blood, the liver and spleen were perfused with PBS before performing the RT-PCR. Figures <b>a</b> and <b>b</b> represent <i>wt</i> and <i>spect (</i>−<i>)</i> parasite DNA load, respectively, in the liver and spleen 2 h after iSPZ injection (iv). Naïve mice (receiving only 500 µl iv of DMEM) were used as a control.</p

Both <i>wt</i> and <i>spect (</i>−<i>) Pb</i>iSPZ immunization protect mice against SPZ challenge.

<p>One week after 1×10⁵<i>Pb</i>iSPZ (<i>wt</i> or <i>spect (</i>−<i>)</i>) immunization, BALB/c mice were challenged with 1×10⁴ live ANKA <i>wt Pb</i>SPZ. Parasitemia was checked at 1 week and 2 weeks post challenge and mice were considered protected when they remained parasite negative 2 weeks after challenge.</p

Protection from infection in cyt c- and PBS- treated mice.

<p>Protection of mice after cyt c or PBS treatment from live SPZ challenge of two independent experiments (5 mice per group in each experiment). Mice were either pre-treated iv for 3 days with 5 mg/mouse of horse cyt c per day in 100 µl of PBS or with 100 µl of PBS alone. On the last day of treatment, mice were immunized iv with 1×10⁵<i>Pb</i>iSPZ in 100 µl of RPMI. One week later, mice were challenged with 5×10³ live <i>Pb</i>SPZ. Parasitemia was checked at 1 and 2 weeks after challenge. Mice were considered protected if they remained parasite negative 2 weeks after challenge.</p

Level of <i>Pb</i>CSP-specific CD8+ T-cells in different organs of BALB/C mice after treatment with either cyt c or PBS and immunization with <i>Pb</i>iSPZ.

<p>Two groups of BALB/c mouse were pre-treated (iv through the tail vein) with 5 mg/mouse of cyt c per day for 3 days in 100 µl of PBS or with 100 µl of PBS alone. Immediately after the last treatment, mice were immunized with 1×10⁵<i>Pb</i>iSPZ in 100 µl of RPMI. One-week later, <i>Pb</i>CSP epitope-specific CD8+ T-cell frequency (2 mice per group of treatment) was evaluated in peripheral blood lymphocytes (PBL), liver, lymph nodes (LN) and spleen. Naïve (receiving neither cyt c, PBS nor iSPZ) mice were used as negative control. Inserted panel shows the fold change in frequency and the percentage of inhibition of <i>Pb</i>CSP-specific CD8+ T-cells in cyt c-treated compared to PBS alone-treated groups.</p