Kaplan Meier plot showing fraction of the RTS,S/AS01_E vaccinees experiencing an episode of clinical malaria, divided according to antibody and TNF+ only CD4+ cell anti-CSP responses.

<p>The 4 groups are as follows; 1) anti-CSP antibody titers below 40 EU/ml, TNF+ only CD4+ cells on CSP stimulation below median; 2) anti-CSP antibody titers above 40 EU/ml, TNF+ only CD4+ cells on CSP-peptide stimulation below median; 2) anti-CS antibody titers below 40 EU/ml, TNF+ CD4+ cells on CSP-peptide stimulation above median; 2) anti-CSP antibody titers above 40 EU/ml, TNF+ only CD4+ cells on CSP-peptide stimulation above median. The anti-CSP antibody titers are applied as a time-varying covariate.</p

Box plots for cytokine positive T cell frequencies on stimulation with CSP peptides pre-vaccination, 1 month after the last vaccination and 12 months after the last vaccination, shown by cytokine and by vaccination group.

<p>Box plots show medians and inter-quartile ranges, the whiskers show 5^th to 95^th centiles, and outliers are shown by circles. Vaccination group is indicated by “RTS” for RTS,S/AS01_E and “Ctl” for rabies control. T cell phenotype is indicated by + and − for the cytokines shown far left. Significance is indicated by a horizontal line at p<0.0003 (using a Bonferroni correction for multiple comparisons).</p

A representative example of the identification and quantitation of RTS,S/AS01_E induced CSP-peptide reactive CD4+ T cells producing various cytokines.

<p>A) CD4+/CD8+ T cells were gated off the side scatter (SCC) Vs CD3 gate, after gating on lymphocytes on the SSC vs forward scatter (FSC) density plot. B) Determination of the percentages of IL2+, TNF+ and IFNγ CD4+ T cells following <i>in vitro</i> stimulations with nothing (Media control), CSP and SEB. C) An example of a typical gating tree showing the identification and quantitation of CD4+ T cells producing various combinations of IL2, TNF and IFNγ, following stimulation with nothing (media control), CSP or SEB, is shown. The resultant data was expressed as percentages of cytokine positive CD4+ T cells.</p

Geometric means and 95% confidence intervals for cellular responses to CSP by vaccination group and t-test on log-transformed values for significance of difference.

<p>Pre-vac = prior to vaccination, Vac+1 = 1 month after final vaccination, Vac+12 = 12 months after final vaccination.</p

Hazard Ratios (HR) and 95% Confidence intervals from Cox regression models for the effect of CD4+ cellular responses to CSP on clinical malaria episodes.

<p>Hazard Ratios are adjusted by age (as a continuous variable), distance from the dispensary (continuous variable), bednet use, location of residence (in 4 groupings) and, when all vaccinees are included in an analysis, by vaccination group.</p><p>“At least TNF+” refers to all cells producing TNF, including polyfunctional cells (i.e. producing TNF with IL2, TNF with IFNγ, or TNF with IFNγ and IL2). The parallel definition applies to “at least IL2+”. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0052870#s2" target="_blank">Results</a> that are significant after a Bonferroni correction (i.e.<0.002) are shown in bold.</p

Frequency of CD4^<b>+</b> T_CM and CD4⁺T_E/EM cells producing IL-2 upon recall with CSP peptides.

<p>PBMC obtained at the indicated time points from RTS,S-immunized subjects were incubated as described for <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0020775#pone-0020775-g002" target="_blank">Fig. 2</a> with CSP peptides as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0020775#s2" target="_blank">Methods</a>. Cells were then analyzed as described in the legend to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0020775#pone-0020775-g001" target="_blank">Fig. 1</a>. The values are plotted as the frequency of IL-2-producing CD4⁺ T_CM cells (open bars) and IL-2-producing CD4⁺ T_E/EM cells (closed bars) for protected (right-hand panels) and non-protected (left-hand panels) subjects. Error bars indicate 95% confidence intervals. *p<0.05; **p<0.01; ***p<0.001.</p

Frequency of CD4⁺T_CM and T_E/EM cells producing TNF-α upon recall with CSP peptides.

<p>PBMC were obtained at the indicated time points from RTS,S-immunized subjects and were incubated as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0020775#pone-0020775-g003" target="_blank">Fig. 3</a> with CSP peptides as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0020775#s2" target="_blank">Methods</a>. Cells were then analyzed as described in the legend to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0020775#pone-0020775-g001" target="_blank">Fig. 1</a>. The values are plotted as [A] the % TNF-α-producing CD4⁺ T_CM cells and [B] the % TNF-α-producing CD4⁺ T_E/EM cells for protected (closed symbol) and non-protected (open symbol) subjects. Statistically significant differences between pre-immune and post-vaccination time points are indicated at the top of the graph. Statistically significant differences between values for protected and non-protected subjects are as indicated within the body of the graph. *p<0.05; **p<0.01; ***p<0.001.</p

Frequency of CD4^<b>+</b> T_CM and CD4^<b>+</b>T_EM cells producing IFN-γ upon recall with CSP peptides.

<p>PBMC were obtained at the indicated time points from RTS,S-immunized subjects and were incubated as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0020775#pone-0020775-g003" target="_blank">Fig. 3</a> with CSP peptides as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0020775#s2" target="_blank">Methods</a>. Cells were then analyzed as described in the legend to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0020775#pone-0020775-g001" target="_blank">Fig. 1</a>. The resulting values are plotted as the % of [A] IFN-γ producing CD4⁺ T_CM cells and [B] IFN-γ producing CD4⁺ T_E/EM cells for protected (closed symbol) and non-protected (open symbol) subjects. Statistically significant differences between pre-immune and post-vaccination time points are indicated at the top of the graph. Statistically significant differences between values for protected and non-protected subjects are as indicated within the body of the graph. *p<0.05; **p<0.01; ***p<0.001.</p

Number of cells per subset on the DOC.

a<p>per µl of peripheral blood.</p>b<p>mean ± SEM.</p>c<p>Mann-Whitney <i>U</i>-test.</p

Identification of RTS,S vaccine-induced CSP-specific CD4⁺ T cell memory subsets.

<p>PBMCs from an RTS,S-immunized volunteer were stimulated in vitro for 18 h with anti-CD28 and anti-CD49d plus a pool of 2 long contiguous CSP peptides in the presence of PE-conjugated anti-CCR7 Ab. Cells were harvested and surface-labeled for CD3, CD4, CD8, CD45RO and UV viability dye and then stained intra-cellularly for IL-2, TNF-α and IFN-γ. Cells were acquired on an LSR-II flow cytometer and analyzed using FlowJo. (A) Gating strategy used to identify CD4⁺CD45RO⁺CCR7⁺ T_CM and CD4⁺CD45RO⁺CCR7⁻ T_E/EM cells. Numbers indicate the percentage of cells in each gate. (B) Detection of IL-2, TNF-α and IFN-γ production by CD4⁺ T_CM and CD4⁺T_E/EM cells. Numbers indicate the percentage of cytokine⁺ cells for each memory cell type. (C) Boolean gating analysis was used to divide cytokine-producing cells into seven distinct populations based on their production of IL-2 (2), TNF-α (T) and IFN-γ (G) in any combination. Each population is shown as two dot plots: the upper is TNF-α vs IL-2 and the lower is IFN-γ vs. IL-2. Numbers indicate the percentage of cytokine⁺ cells for each memory subtype.</p