Vaccine-specific PHPC T cell responses at late time points post-immunization.

<p><b>A)</b> PBMCs were isolated from blood samples taken between W40 and W47 PI for all animals (W28 to 35 PI for BX73). A portion of cells from each animal was used for ELISPOT assay to detect IFN-γ producing cells in response to medium, Gag and TRN pools of peptides (day 1). The other portion was cultured for 11 days in the presence or absence of relevant peptide pools, in medium supplemented on day 3 with recombinant simian IL-2 only and on day 7 with recombinant simian IL-2, IL-15 and IL-7 cytokines. On day 11, cells were collected, counted and used for ELISPOT assay using the same conditions as day 1 ELISPOT, to detect IFN-γ producing cells in response to Gag and TRN peptides (day 12). This assay is referred as PHPC assay for memory precursors with high proliferative capacity. Numbers of IFN-γ producing cells obtained per million of PBMCs against Gag + TRN at day 1 and day 12 are indicated in the <i>y</i>-axis. The number of median responses obtained among all 6 animals are represented (n = 457 spots/10⁶ at day 1 and n = 11628 spots/10⁶ at day 12). <b>B)</b> Memory phenotyping of Gag and TRN-specific T cells for animal BX72 at W40 PI. Day 1 and day 11 cultured PBMCs, as described in section A, were restimulated for 16 h in the presence or absence of Gag or TRN pool of peptides in medium containing Brefeldin A. Cells were then surface-stained with EMA, CD3, CD8, CD4, CD28, CD95 mAbs, permeabilized and stained with IFN-γ, IL-2 and TNF-α mAbs. Cells were gated on live lymphocytes (low FSC/SSC, EMA-, CD3⁺), bright CD8⁺ T cell populations (orange color) and CD4⁺ T cell populations (blue color). Antigen-specific T cells were identified by their capacity to secrete one or more cytokines (black dots). For simplicity, the percentage of total IFN-γ+ or TNF-α+ or IL-2+ antigen specific responses (black dots) obtained are indicated after the subtraction of background obtained with cells cultured with medium only. All black dots were superimposed to the total CD8⁺ or CD4⁺ T cell population and plotted against EM and CM memory markers (CD28 and CD95). <b>C)</b> The same procedure as described in B was performed on day 11-cultured PBMCs that have been isolated at W47 PI (W35 PI for BX73) from all six animals. The histograms represent the percentage of total IFN-γ+ (green) or TNF-α+ (red) or IL-2+ (blue) antigen-specific T cell responses obtained against Gag and TRN (indicated by G and M respectively in the histogram) within CD8⁺ (left histogram) and CD4⁺ (right histogram) T cells. Individual dot plot analysis for cytokine and memory phenotype shown in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0110883#pone-0110883-g004" target="_blank">Figure 4C</a>, are displayed in supplemental <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0110883#pone.0110883.s003" target="_blank">Figure S3</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0110883#pone.0110883.s004" target="_blank">S4</a>.</p

SHIV-specific CD4⁺ T cell recall responses.

<p>PBMCs were processed and labeled using the procedure described in the legend of <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0110883#pone-0110883-g002" target="_blank">Figure 2</a>. <b>A)</b> Flow cytometry data analyses were performed by gating on low FSC/SSC, EMA-, CD3⁺ and CD4⁺ T cell populations (colored in blue). Representative results obtained for macaques BX73, 80 and 83 at the pre-immunization time point and at the time of maximal immune response (IR) for Env antigen are displayed. Frequencies for Env-specific responses are reported as the percent of proliferating CD4⁺ T cells after the subtraction of backgrounds obtained with cells cultured for 5 days with medium alone and restimulated for 6 h with relevant peptides pools. <b>B)</b> Summary of the frequency of proliferating (CFSE low) CD4<i>⁺</i> T cells detected against each indicated antigen (Gag (blue), Env (red), TRN (green), Pol (purple)) in each immunized animal (BX72, 73, 78, 80, 83, 84) following weeks post-immunization. Of note, results obtained between W3 and W8 for BX83 were excluded due to non-specific T cell hyperactivation (indicated by an interrupted x-axis).</p

Polyfunctional SHIV-specific CD8⁺ T cell recall responses.

<p>At various indicated time points pre- and post-immunization PBMCs from vaccinated animals were labeled with CFSE and cultured in presence of specific pools of SHIV peptides Gag, Env, TRN, Pol or medium without peptide for 5 days. On day 5, cells were harvested and restimulated overnight with the same cocktail of peptides (designated as Gag(5d) Gag(16 h)) in the presence of costimulatory Abs and Brefeldin A. Cells were then surface-stained with EMA, CD3, CD8, CD4 mAbs, permeabilized and stained with IFN-γ, IL-2 and Granzyme B mAbs. For flow cytometry analysis, we initially gated on live lymphocytes (low FSC/SSC, EMA-, CD3⁺), bright CD8⁺ T cell populations (colored in orange). Antigen-specific T cells were identified by their capacity to proliferate, secrete cytokines and contain lytic molecules (black dots). <b>A)</b> Results for two representative animals (BX72 and BX78) were displayed. The proportion of cells producing IFN-γ (contour plot; upper number) and proliferating (CFSE dilution, contour plot, lower number) in response to specific antigens is indicated in each plot. Frequencies for antigen-specific responses are reported as the percent of cytokine-secreting and proliferating CD8⁺ T cells after the subtraction of backgrounds obtained with cells cultured for 5 days with medium only and restimulated for 6 h with relevant peptide pools. <b>B)</b> Summary of the frequencies of proliferating (CFSE low) CD8<i>⁺</i> T cells detected against each indicated antigen (Gag (blue), Env (red), TRN (green), Pol (purple)) in each immunized animal (BX72, 73, 78, 80, 83, 84) at various weeks post-immunization. Of note, results obtained between W3 and W8 for BX83 were excluded due to non-specific T cell hyperactivation (indicated by an interrupted x-axis).</p

Evaluation and characterization of immediate effector T cell responses induced by a single immunization with the CAL-SHIV-IN⁻ DNA vaccine.

<p><b>A)</b> PBMCs isolated from blood samples taken at the indicated weeks post-immunization (PI) were used for ELISPOT assay to detect IFN-γ producing cells in response to the pools of SIV Gag (indicated in blue), HIV Env (indicated in red), HIV Tat+Rev+ SIV Nef (TRN, indicated in green) and SIV Pol (indicated in purple) peptides. Numbers of IFN-γ producing cells obtained per million of PBMCs against all Ags tested are indicated in the <i>y</i>-axis. <b>B)</b> Mean and range of IFN-γ ELISPOT responses to Gag and TRN pools across 22 (for BX72, 78, 80, 83, 84) and 14 (for BX73) time-points collected. <b>C)</b> Memory phenotyping of Gag-specific T cells at W8 PI. PBMCs were restimulated for 16 h in presence or absence of Gag pool of peptides in medium containing Brefeldin A. Cells were then surface stained with EMA, CD3, CD8, CD4, CD28, CD95 mAbs, permeabilized and stained with IFN-γ, IL-2 and TNF-α mAbs. For analysis, cells were initially gated on live lymphocytes (low FSC/SSC, EMA-, CD3⁺), CD8 bright T cells (orange color) and CD4⁺ T cell populations (blue color). Antigen-specific T cells were identified by their capacity to secrete one or more cytokines (black dots). For each indicated animal (BX72, 78, 80, 84), we displayed the maximal response obtained at this time point within CD8⁺ or CD4⁺ T cell populations. For simplicity, the percentage of total responses (total black dots) obtained through all quadrants (IFN-γ+, IFN-γ+ &TNF-α+, IFN-γ+&IL-2+ and IL-2+) are indicated after the subtraction of background obtained with cells cultured with medium only. For memory T cell subset determinations, all black dots were superimposed to the total CD8⁺ or CD4⁺ T cell populations and plotted against memory markers (CD28 and CD95). The naïve population was defined as CD28+ CD95−, effector memory (EM) as CD28− CD95+ and central memory (CM) as CD28+ CD95+. D) Sequential evaluation of SIV Gag p55 specific antibody responses in immunized macaques (“homemade kit”). E) Sequential evaluation of HIV Env gp160 specific antibody responses in immunized macaques (Biorad, France).</p