Additional file 5: Figure S4. of Optimization and evaluation of Luminex performance with supernatants of antigen-stimulated peripheral blood mononuclear cells

Non-linear relationship between %CV and mean cytokine concentration. %CVs concentration (mean CVs for four different donors and four conditions [NS, PPD, SEB and ESAT-6 stimulations] calculated using culture triplicates for each of the 11 cytokines analyzed) plotted against corresponding mean concentrations for Ozyme, Millipore and Bio-Rad kits. (PDF 98 kb

Association between baseline 25OH Vitamin D level and antibody response to hepatitis B vaccination in the ANRS HB03 VIHVAC B trial (proportional logistic regression model, multivariable analysis, n = 333 with non missing covariates).

<p>Association between baseline 25OH Vitamin D level and antibody response to hepatitis B vaccination in the ANRS HB03 VIHVAC B trial (proportional logistic regression model, multivariable analysis, n = 333 with non missing covariates).</p

Modeling of IFN-γ producing cytotoxic CD8+ T cells (CD8+ IFN-γ+ T cells) as a function of IL-2 producing CD4+ helper T cells (CD4+ IL-2+ T cells) and time (model 1 and model 2) in the rAd5-rAd5 group, HVTN 068 trial.

<p>Modeling of IFN-γ producing cytotoxic CD8+ T cells (CD8+ IFN-γ+ T cells) as a function of IL-2 producing CD4+ helper T cells (CD4+ IL-2+ T cells) and time (model 1 and model 2) in the rAd5-rAd5 group, HVTN 068 trial.</p

Correlations between baseline 25OHD level and change in cellular immune responses between W0 and W4 in the group having received the 7-valent conjugate vaccine prime containing the diphtheria-derived carrier protein CRM₁₉₇ at W0, ANRS-114 PNEUMOVAC sub-study (n = 11).

<p>Correlations between baseline 25OHD level and change in cellular immune responses between W0 and W4 in the group having received the 7-valent conjugate vaccine prime containing the diphtheria-derived carrier protein CRM₁₉₇ at W0, ANRS-114 PNEUMOVAC sub-study (n = 11).</p

Kinetics of IL-2 producing CD4+ helper T cells (CD4+ IL-2+ T cells) and IFN-γ producing cytotoxic CD8+ T (CD8+ IFN-γ+ T cells) responses over time after Env ex-vivo stimulation of PBMC in the rAd5-rAd5 and DNA-rAd5 groups respectively, HVTN 068 trial.

<p>(A) CD4+ IL-2+ T cells responses in the rAd5-rAd5 group. (B) CD8+ IFN-γ+ T cells responses in the rAd5-rAd5 group. (C) CD4+ IL-2+ T cells responses in the DNA-rAd5 group. (D) CD8+ IFN-γ+ T cells responses in the DNA-rAd5 group.</p

Baseline characteristics of the study populations in the ANRS HB03 VIHVAC B trial and the ANRS 114-PNEUMOVAC sub-study.

<p>Baseline characteristics of the study populations in the ANRS HB03 VIHVAC B trial and the ANRS 114-PNEUMOVAC sub-study.</p

HIV-specific responses are significantly upregulated after the vaccination with LIPO-5-DC vaccine.

<p>Each patient is designated with its individual color/symbol code (refer to <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004752#ppat.1004752.s008" target="_blank">S1 Table</a>). Symbols are attributed based on maximum viral load rebound after treatment interruption as indicated in the figure. (A) Representative plots for a single patient prior (wk -4) and after (wk 16) the vaccination. Cells were stimulated with gag p24 pool, LIPO-5 vaccine or CMV lysate as a control, stained and analyzed by flow cytometry 44 hours later. Plots show viable CD4⁺ T cells. (B) Graphs show the percentages of antigen-specific cells (CD134⁺CD25⁺) among CD4⁺ T cells after the stimulation with the indicated antigens (n = 14). (C) IFN-γ, TNF-α and IL-2 production among LIPO-5- specific cells (CD134⁺CD25⁺) (n = 14). (D) Graph shows the correlation between the relative increase in LIPO-5-specific response (response after the vaccination-response before vaccination) and maximal viral load rebound after HAART interruption (n = 14). Data were analyzed by Wilcoxon matched-pairs signed rank test. *p < 0.05; **p < 0.01; ***p < 0.001. Spearman coefficient is indicated (r) as well as p value.</p

HVTN 068 trial design with repeated immunogenicity measurements–cellular and antibody responses—during the 52 weeks of follow-up.

<p>HVTN 068 trial design with repeated immunogenicity measurements–cellular and antibody responses—during the 52 weeks of follow-up.</p

Tregs can suppress HIV-specific responses in vitro.

<p>(A) Percentages of gag p24-specific Tregs (CD134⁺CD25⁺CD39⁺FoxP3⁺) or IFN-γ-producing cells after stimulation in whole or Tregs-depleted fractions. (B) Representative plots of suppression assays in which depleted Tregs from (A) (lower panel) or CD25^lo fraction (upper panel) were cocultured in 1:2 ratio with CFSE-labeled PBMCs in overnight culture in the presence of 2μg/mL of gag p24 peptide pool, 1μg/mL of αCD28 and αCD49d and 10μg/mL of Brefeldin A. (C) Graphs showing percent of both CD4⁺ and CD8⁺ IFN- γ-secretion suppression (n = 9). Data were analyzed by Wilcoxon matched-pairs signed rank test. *p < 0.05; **p < 0.01; ***p < 0.001.</p

Ex-vivo phenotype.

<p>Percentages of CD4⁺, CD8⁺ T cells, CD4⁺/CD8⁺ T-cells ratios as well as the percentages of CD4⁺CD25^hiCD127^loFoxP3⁺ Tregs (among CD4⁺ T cells) at week -4 (prior vaccination) and week 16 (post vaccination). Medians with IQR values are indicated. P values are calculated based on Wilcoxon matched-pairs signed rank test. All cell staining and flow cytometry analysis was performed on freshly thawed samples from n = 14 HIV-1 infected individuals included in this study.</p><p>Ex-vivo phenotype.</p