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

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

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

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

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

CSP-specific IL-2⁺ T_E/EM and T_CM CD4⁺ T cells correlate with anti-CSP R region Ab titers.

<p>The figures display the log base 10 of the IgG level in micrograms per milliliter versus log of the percent of T cells expressing IL-2 as assayed by flow cytometry. Individuals who were protected from malaria by vaccination are noted with filled circles, while those not protected by open circles. The figure also shows the Spearman's correlation between the log IgG and log percent cells for the combined protected and non-protected individuals as well as a trend line.</p

Heterologous Prime-Boost Regimens with a Recombinant Chimpanzee Adenoviral Vector and Adjuvanted F4 Protein Elicit Polyfunctional HIV-1-Specific T-Cell Responses in Macaques

<div><p>HIV-1-specific CD4⁺ and CD8⁺ T lymphocytes are important for HIV-1 replication control. F4/AS01 consists of F4 recombinant fusion protein (containing clade B Gag/p24, Pol/RT, Nef and Gag/p17) formulated in AS01 Adjuvant System, and was shown to induce F4-specific polyfunctional CD4⁺ T-cell responses in humans. While replication-incompetent recombinant HIV-1/SIV antigen-expressing human adenoviral vectors can elicit high-frequency antigen-specific CD8⁺ T-cell responses, their use is hampered by widespread pre-existing immunity to human serotypes. Non-human adenovirus serotypes associated with lower prevalence may offer an alternative strategy. We evaluated the immunogenicity of AdC7-GRN (‘A’), a recombinant chimpanzee adenovirus type 7 vector expressing clade B Gag, RT and Nef, and F4/AS01 (‘P’), when delivered intramuscularly in homologous (PP or AA) and heterologous (AAPP or PPAA) prime-boost regimens, in macaques and mice. Vaccine-induced HIV-1-antigen-specific T cells in peripheral blood (macaques), liver, spleen, and intestinal and genital mucosa (mice) were characterized by intracellular cytokine staining. Vaccine-specific IgG antibodies (macaques) were detected using ELISA. In macaques, only the heterologous prime-boost regimens induced polyfunctional, persistent and balanced CD4⁺ and CD8⁺ T-cell responses specific to each HIV-1 vaccine antigen. AdC7-GRN priming increased the polyfunctionality of F4/AS01-induced CD4⁺ T cells. Approximately 50% of AdC7-GRN-induced memory CD8⁺ T cells exhibited an effector-memory phenotype. HIV-1-specific antibodies were detected with each regimen. In mice, antigen-specific CD4⁺ and CD8⁺ T-cell responses were detected in the mucosal and systemic anatomical compartments assessed. When administered in heterologous prime-boost regimens, AdC7-GRN and F4/AS01 candidate vaccines acted complementarily in inducing potent and persistent peripheral blood HIV-1-specific CD4⁺ and CD8⁺ T-cell responses and antibodies in macaques. Besides, adenoviral vector priming modulated the cytokine-expression profile of the protein-induced CD4⁺ T cells. Each regimen induced HIV-1-specific T-cell responses in systemic/local tissues in mice. This suggests that prime-boost regimens combining adjuvanted protein and low-seroprevalent chimpanzee adenoviral vectors represent an attractive vaccination strategy for clinical evaluation.</p></div

HIV-1-specific CD8+ T-cell responses in NHP.

<p>Rhesus macaques were immunized intramuscularly with F4/AS01 (‘P’) and/or AdC7-GRN (‘A’), according to the following regimens: weeks 0 and 4 (PP group), weeks 0 and 12 (AA group), weeks 0, 4, 16 and 28 (PPAA group) and weeks 0, 12, 24 and 28 (AAPP group). <b>A.</b> PBMCs were stimulated <i>in vitro</i> overnight with a pool of peptides covering the F4 antigen sequence at several time-points, and production of IL-2, IFN- and TNF- was measured by ICS. Frequencies of F4-specific CD8⁺ T cells were expressed as percentages of CD3⁺ CD8⁺ T cells expressing IFN-γ and/or TNF-α and/or IL2 over total CD3⁺ CD8⁺ T cells. Data are represented as group geometric mean frequencies. *: PPAA group: N = 8 at Weeks 0, 2 and 6, and N = 7 at all time-points thereafter. The dashed line indicates the assay cut-off value for CD8⁺ T cells (i.e., 0.14%). <b>B.</b> The cytokine co-expression profile of F4-specific CD8⁺ T cells at 2 weeks post last immunization is represented for each prime-boost regimen. Data are reported as median frequencies of responding CD8⁺ T cells expressing any combination of IL-2, IFN- and TNF-, with first and third quartiles measured. Pie charts show the group mean proportions of responding CD8⁺ T cells expressing (after <i>in vitro</i> stimulation) one, two or three cytokines (represented in light grey, dark grey and black, respectively) among IL-2, IFN- and TNF-. These group mean proportions were compared using Fisher’s exact test and a significance level of p<0.05. The number in the center of each pie represents the geometric mean of the total percentage of cytokine-expressing CD8⁺ T cells at 2 weeks post last immunization.</p

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

<p>PBMC were obtained at the Pre, Po2, DOC and PoCh timepoints from RTS,S-immunized subjects and were incubated for 18 h with co-stimulants (medium control) or co-stimulants plus a pool of 2 long contiguous CSP peptides as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0020775#s2" target="_blank">Methods</a>. Cultures also contained PE-conjugated-CCR7-specific Ab to detect CCR7⁺ cells. Cells were harvested and surface-labeled for CD3, CD4, CD8 and CD45RO and then stained for intracellular detection of IL-2, TNF-α and IFN-γ (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0020775#s2" target="_blank">Methods</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0020775#pone-0020775-g001" target="_blank">Fig. 1</a>). Cells were analyzed on an LSR-II flow cytometer to identify IL-2-producing CCR7⁺CD45RO⁺CD4⁺ T_CM cells and CCR7⁻CD45RO⁺CD4⁺ T_E/EM cells. The frequency of IL-2-producing CD4⁺ T cell subsets in the medium control cultures were subtracted from the corresponding values for the CD4⁺ T cell subsets in the cultures containing Ag. The resulting adjusted values are plotted as [A] the % IL-2 producing CD4⁺ T_CM cells and [B] the % IL-2 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

Phenotype of memory HIV-1-specific T cells in NHP at 6 months post last immunization.

<p>Frequencies and cytokine expression profile of F4-specific central memory (‘CM’; CD28⁺ CD95⁺) and effector memory (‘EM’; CD28^-CD95⁺) CD4⁺ and CD8⁺ T cells were determined at 6 months post last immunization by ICS after overnight <i>in vitro</i> stimulation of PBMCs with a pool of peptides covering the F4 sequence. Data are represented as group mean percentages +/- standard error of the mean (SEM).</p