Plasma vRNA levels after IV challenge with SIVmac239.

<p>(A) unimmunized macaques, (B) SHIV-immunized macaques and (C) progesterone-treated, SHIV-immunized macaques. In panel (D), cumulative vRNA levels over the 20 weeks of observation were transformed into areas under the curve (AUC) and the median AUC of the 3 groups of animals were compared. The Kruskal-Wallis test and a pair-wise comparison between the 2 groups of immunized animals and the unimmunized group using Dunn's multiple comparisons test were performed.</p

Changes in peripheral CD4⁺ T cell populations after IV SIVmac239 challenge.

<p>(A) Number of CD4⁺ T cells, and (B) the percentage of CD4⁺ CD95⁺ T cells in blood. (λ) Naïve control macaques (n = 7), (○) SHIV-immunized macaques (n = 5) and (5) Depo-Provera® SHIV-immunized macaques (n = 6). The number of CD4⁺ T cells (p = 0.0007) and the percentages of CD4⁺ CD95⁺ T cell (p<0.0001) were significantly higher (Friedman test) in the Depo-Provera®-treated, SHIV-immunized macaques than in the naïve control group.</p

Frequencies of regulatory CD4⁺ T cells after progesterone treatment and IV SIVmac239 challenge.

<p>A) Percentages of CD4⁺ CD152⁺ T cells in PBMC. B) Percent of CD4⁺ FoxP3⁺ HLA-DR⁺ T cells after challenge in PBMC. C) Percentages of CD4⁺ CD152⁺ T cells in axillary LN of the vaccinated animals at 12 weeks post-challenge. D) Percentages of CD4⁺ FoxP3⁺ HLA-DR⁺ T cells in axillary LN of the vaccinated animals at 12 weeks post-challenge. (λ) Naïve control macaques (n = 7), (○) SHIV-immunized macaques (n = 5) and (5) Depo-Provera®-treated, SHIV-immunized macaques (n = 6).</p

SIV-specific T cell responses after IV SIVmac239 challenge.

<p>(A) Mean number of SIV Gag p27 interferon (IFN)-γ secreting cells in PBMC before and after challenge. (B) Median area under the curve for the total IFN-γ secreting cells in each group from week 2 to 16 PC, the time-points for which samples were available. (C) Mean SIV-specific T cell proliferative responses in PBMC expressed as the stimulation index (SI) before and after SIV challenge. See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0009814#s2" target="_blank"><i>Methods</i></a> for details. (D) Median area under the curve for the cumulative SI in each group from week -4 to 20 PC. (λ) Naïve control macaques (n = 7), (○) SHIV-immunized macaques (n = 5) and (5) Depo-Provera® SHIV-immunized macaques (n = 6). P values of a T test are indicated.</p

Frequency and functional capacity of Gag CM9- and GY9-specific CD8⁺ T cells of SHIV-immunized animals after IV SIVmac239 challenge.

<p>SIV-specific CD8⁺ T cell responses in cryopreserved PBMC from a <i>Mamu</i>-A*01 positive animal and a <i>Mamu</i>-A*02 positive animal in each vaccinated group are shown as pie charts. The number of positive SIV-specific T cells normalized to 10⁵ CD3⁺ T cells is shown for each response as the white number in the center of the pie chart. Each portion of a pie chart indicates the percentage of SIV-specific T cells that responded with one, two, or three functions; and the colored arcs around the pie show the cytokine or combination of cytokines comprising each response. ND not done, as samples were not available.</p

Cytokine mRNA levels after IV SIVmac239 challenge.

<p>Changes in gene expression are shown as the fold-change relative to the levels on the day of challenge for (A) TNF-α and (B) TGF-β. (C) Spearman rank correlation of TGF-β at 4 weeks PC, for all animals, with plasma viral RNA AUC between 1 and 20 weeks PC. (λ) Naïve control macaques (n = 7), (○) SHIV-immunized macaques (n = 5) and (5) Depo-Provera®-treated, SHIV-immunized macaques (n = 6).</p

Frequencies of activated T cells after progesterone treatment and IV SIVmac239 challenge.

<p>A) Percent of CD3⁺ CD4⁺ T cells expressing CD38 but not HLA-DR in PBMC. B) Percent of CD3⁺ CD8⁺ T cells expressing CD38 but not HLA-DR in PBMC. D) Percent of CD3⁺ CD8⁺ T cells expressing Ki-67⁺ in PBMC. E) Percent of CD3⁺ CD8⁺ T cells expressing caspase-3⁺ in PBMC. (○) SHIV-immunized macaques (n = 5) and (5) Depo-Provera®-treated, SHIV-immunized (n = 6).</p

Effect of Ad5hr infection on the frequency of circulating CD4+T cells expressing activation markers.

<p>A) The percentage of CD95+/CD3+/CD4+ cells expressing CCR5. B) The percentage of CD3+/CD4+ cells expressing a high level of HLA-DR. C) The percentage of CD3+/CD4+ cells expressing Ki67. Arrows on the X axis indicate the time of Ad5hr inoculation. The mean values for all animals are indicated by blue bars. P values ≤0.05 from the ANOVA and Dunnetts's post-hoc tests are shown.</p

Ad5 hexon-specific T cell responses in PBMCs after Ad5hr infection as detected by multiparameter flow cytometry.

<p>Pie charts present the average CD4+ or CD8+T cell responses for all positive animals at weeks 2, 10 and 14 from the first Ad5hr inoculation, as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0106004#s4" target="_blank">Materials and Methods</a>. Pie slice colors denote the number of positive functions (expression of IL-2, IFN-γ, TNF and CD107 degranulation). Pie arc colors denote the 4 functions separately. Red numbers in the center of pie charts are the mean ± standard deviation of hexon-specific T cells normalized to 10⁵ CD3+ T cells. Fractional numbers below a pie chart refer to the number of animals in a group of 18 that responded.</p

Effect of Ad5hr infection on proinflammatory cytokine and chemokine gene expression in PBMC.

<p>A) The fold-change of IFN-gammamRNA in PBMCs; B) IFN-α; C) IL-1; D) TNF and E) CCL20. The fold-change in target gene mRNA levels in study animals post-Ad5hr infection relative to the same target gene mRNA level before Ad5hr infection was calculated as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0106004#s4" target="_blank">Materials and Methods</a>. Arrows on the X axis indicate the time of Ad5hr inoculation. The mean values for all animals are indicated by blue bars P values ≤0.05 from the ANOVA and Dunnetts's post-hoc tests are shown.</p