Qβ-CSP <i>vs</i>. CSP in Montanide.

<p>Groups of 6 mice were vaccinated thrice with 2.5, 1 and 0.1 μg CSP or Qβ-CSP in Montanide. A, B show the individual data points and mean±SEM titers against full-length protein (A) and NANP repeat peptide (B) 2 weeks post 3^rd vaccination (2WP3). **** (p<0.0001 for ANOVA followed by Tukey’s multiple comparisons test); red data points correspond to mice protected 14 days post challenge and numbers (blue) were protected out of 6. C, Immunofluorescence image of methanol fixed sporozoites stained with 1:2500 dilution of anti-CSP pool (left) or Qβ-CSP serum pool (right) for the 1 μg dose groups. D, E show IgG1, IgG2b and IgG2c levels measured by Luminex and expressed as median fluorescence intensity (MFI) at 1:1000 serum dilution against the NANP peptide (D) or the C-term protein (E).</p

Combined ELISA titer and protection data of CSP <i>vs</i>. Qβ-CSP groups.

<p>Full-length (left) and NANP (right) titers were plotted for individual animals in Montanide, Alum and SE adjuvanted CSP and Qβ-CSP groups. Combined protection data is indicated (blue). Lines are mean±SEM and the P values are for unpaired T test performed on log transformed titers.</p

CD11c⁺NK1.1⁻ DC are constitutively present in the spleens of naïve mice but do not substantially increase following immunization.

<p>(A) CD11c⁺NK1.1⁻DC in <i>Pb</i>γ−spz-immune splenic MNC were identified according to the procedure described for HMNC in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0005075#pone-0005075-g001" target="_blank">Fig 1</a>. After exclusion of T cells, CD11c⁺ cells were segregated into CD11c⁺NK1.1⁺ and CD11c⁺NK1.1⁻ populations. Splenic mononuclear cells were isolated from individual mice before and after prime and boost immunizations with <i>Pb</i>γ−spz or uninfected mosquito debris (sham). Cells were stained with a cocktail of mAbs and NK1.1⁻ DC and CD8⁺ T_EM cells were identified by flow cytometry. (B) Panels show representative contour plots of DC in the spleens of naïve mice and in spleens of mice 6 days after either the 1°, 2° and 3° immunizations and in sham-immunized mice 6 days after the 3° immunization. The percentages of the CD11c⁺NK1.1⁻ DC in relation to the total SMNC/spleen for each representative mouse are indicated in each panel. (C) The results show the mean percentage ±SD of CD11c⁺NK1.1⁻ DC in total spleens of naïve mice, <i>Pb</i> γ−spz-immunized mice at day 6 after each immunization and in sham-immunized mice at day 6 after the 3° immunization. (D) Panels show representative contour plots of CD8⁺ T_CM cells and CD8⁺ T_EM cells in the spleens of naïve mice as well as of <i>Pb</i> γ−spz-immunized mice and sham-immunized mice at the same time-points described in (B). The numbers indicate the percentages of the T_CM and T_EM cells in the gated splenic CD3⁺CD8⁺ T cell population. (E) The results show the mean percentage ±SD of CD8⁺T_EM in the gated splenic CD3⁺CD8⁺ T cell population of naïve and immunized mice at day 6 after each immunization. Contour plots and bar graphs are representative of three individual mice per group in three independent experiments.</p

Hepatic cCD8α⁺ DC from <i>Pb</i>γ-spz-immunized and challenged mice mediate <i>in vitro</i> activation of naïve CD8⁺ T cells.

<p>HMNC were pooled from livers of <i>Pb</i>γ-spz-fully immunized and challenged mice (n = 18) and were incubated with a cocktail of biotinylated microbeads to deplete T cells, B cells, NK cells, granulocytes and macrophages as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0005075#s4" target="_blank">Materials and Methods</a>. DC subpopulations were further isolated from the enriched CD11c⁺NK1.1⁻ population by positive magnetic selection for cCD8α⁺DC and pDC and by negative selection for CD8α⁻DC, as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0005075#s4" target="_blank">Materials and Methods</a>. (A) Dot plots show the relative % of T cells and NK1.1 cells within the purified cCD8α⁺DC population. (B) CD8⁺ T cells were isolated from the spleens of naïve mice using magnetic beads and labeled with 2 µM CFSE. Dot plots show the gating scheme for the analysis of CFSE-labeled CD3⁺CD8⁺T cells for expression of the CD45RB^loCD44^hi phenotype. (C) cCD8α⁺ DC, cCD8α⁻ DC and pDC were purified from CD11c⁺NK1.1⁻ DC isolated from pooled livers 3 days after the challenge of <i>Pb</i>γ-spz-immunized mice. Liver DC subpopulations and CFSE-labeled splenic CD8⁺ T cells were co-cultured at a ratio of 1 DC : 2 CD8⁺T cells for 4 days. Cells were harvested, stained with a cocktail of mAbs and the % of CD3⁺CD8⁺CD45RB^loCD44^hi cells (CD8⁺ T_EM) was analyzed by flow cytometry. Results show contour plots of CD8⁺T cells co-cultured with cDC and pDC subpopulations. (D) Bar graphs show the percentage of CD8⁺ T_EM in the gated CD3⁺CD8⁺ T cell population after co-culture with cCD8α⁺ DC, cCD8α⁻ DC and pDC each isolated from <i>Pb</i>γ-spz-immunized-challenged mice.</p

Qβ-CSP <i>vs</i>. CSP in SE, GLA/SE and Alum.

<p>Groups of 10 mice received 3 doses, 3 weeks apart of 2.5 μg CSP+SE, Qβ-CSP+SE, CSP+GLA/SE, Qβ-CSP+GLA/SE or Qβ-CSP+Alum. A, B show the mean±SEM of the full-length and NANP-specific ELISA titers at 2 weeks post third vaccination. ** (p<0.01); * (p<0.05); red symbols represent protected mice and number (blue) protected out of 10. C, D, E and F are data from an independent 2-dose study. 15 mice received 2 doses of 2.5 μg CSP+GLA/SE and Qβ-CSP+GLA/SE, three weeks apart. C, D show the mean±SEM of full-length and NANP-specific ELISA titer from the 10 challenged mice at 2 weeks after the second vaccination. Red symbols were protected mice and number represent protected out of 10 (blue). E, F, IgG1, IgG2b and IgG2c responses of 15 mice measured by Luminex and expressed as MFI at 1:2000 dilution against NANP peptide (E) or C-term protein (F).</p

Qβ-CSP <i>vs</i>. CSP in Alum.

<p>Groups of 10 mice received 2 doses, 3 weeks apart of 2.5 μg CSP+Alum, Qβ-CSP+Alum or Qβ-CSP without an adjuvant. A, B show the mean±SEM of the full-length and NANP-specific ELISA titers at 2 weeks after the second vaccination. **** (p<0.0001); *** (p<0.001); * (p<0.05); red symbols represent protected mice and number (blue) protected out of 10. C, D, show the IgG1, IgG2b and IgG2c responses measured by Luminex and expressed as MFI at 1:1000 dilution against NANP peptide (C) or C-term protein (D).</p

CD8β mRNA is absent in purified liver cCD8α⁺DC.

<p>HMNC were pooled from livers of <i>Pb</i>γ-spz-fully immunized mice 6 days after the 3° immunization and were incubated with a cocktail of biotinylated microbeads to deplete T cells, B cells, NK cells, granulocytes and macrophages as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0005075#s4" target="_blank">Materials and Methods</a>. cCD8α⁺DC were further isolated from the enriched CD11c⁺NK1.1⁻ population by positive magnetic selection as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0005075#s4" target="_blank">Materials and Methods</a>. Staining with anti-CD8α, CD3, CD8b and CD11c was performed on permeabilized cells to reveal both the surface and the intracellular presence of these markers. (A) Dot plot show the relative % of T cells (red) and cCD8α⁺DC (blue) within the purified cCD8α⁺DC population. (B) Two-step quantitative real-time PCR was performed on RNA isolated from magnetic-bead purified liver cCD8α⁺DC (described in A). Ratio of CD8β/CD8α gene expression was calculated using standard curves for each gene. Measurements were done in duplicates in wells containing 1000, 1, 0.1 cells/well, or non-template control (NTC). Representative results of one out of two experiments are shown. (C) Histogram plots show expression of DEC 205, I-A^b and costimulatory molecules on the cCD8α⁺DC population (black lines). Grey lines represent staining of the isotype controls.</p

Numbers of splenic CD11c⁺NK1.1⁻ DC and cCD8α⁺DC in naïve and <i>Pb</i>γ-spz-immunized mice^a

a<p>SMNC were isolated from individual C57BL/6 mice before and after prime and boost immunizations with <i>Pb</i> γ−spz. Cells were stained with a cocktail of mAbs for identification of CD11c⁺NK1.1⁻ DC and cCD8α⁺DC as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0005075#s4" target="_blank">Materials and Methods</a> and analyzed by flow cytometry. Data represent the mean±SD of the number of cells of three mice per group and are representative of three independent experiments.</p

Characterization of Qβ-CSP vaccine.

<p>A, identity of the product bands was confirmed using reducing coomassie blue staining and western blot with polyclonal mouse anti-Qβ and anti-CSP antibodies. Lanes 1, 2, Qβ-CSP; lane 3, unconjugated Qβ; lane 4, unconjugated CSP. B, particle size distribution of Qβ-CSP by dynamic light scatter analysis. C, electron micrograph of the negatively stained Qβ-CSP. D, immunological reactivity of the soluble CSP and Qβ-CSP against CSP-specific mAbs targeting the NANP repeats, the C-term region or epitopes present only on full-length (FL) CSP. Mabs labelled as “Hu” were produced in humanized mice and “Mo” were produced in wild-type mice.</p

Production of the Qβ-CSP vaccine.

<p>A, Outline of the conjugation process. Numbers in parentheses correspond to the respective lane number in Fig 1B. B, CSP and Qβ proteins analyzed by reducing SDS-PAGE followed by coomassie blue staining (left) or western blot using anti-CSP polyclonal mouse antibodies (right). Lane 1, soluble CSP protein; 2, CSP diluted in urea; 3, SATA treated and desalted CSP; 4, Qβ protein; 5, SMPH treated Qβ; 6, desalted Qβ-Malemide; 7, deacetylated and desalted CSP; 8, CSP-Qβ conjugate; 9, desalted CSP-Qβ conjugate (final vaccine); M, molecular weight marker.</p