The Expression of Cytokines in CS-stimulated Cells Before and After Vaccination.

<p>Expression of IFN-γ, MIG, TGF-β1, FoxP3 and IL-10 was measured by real-time RT-PCR in total PBMCs following 12 hour culture with the vaccine antigen CS in subjects who received vaccination with RTS,S/AS02A and MVA-CS. Results are expressed as copy number relative to the housekeeping gene HPRT. Median values (9 subjects) are shown. Vac+7 =  seven days after the final vaccination, Vac+28 = 28 days after the final vaccination, each subject received two doses of RTS,S/AS02A and one dose of MVA-CS. A) IFN-g mRNA, B) MIG mRNA, C) TGF-β1 mRNA, D) FoxP3 mRNA, E) IL-10 mRNA and F) Correlation of MIG mRNA expression at day 7 with days to parasitemia following sporozoite challenge, n = 6–12.</p

The Correlation of IL-10 expression with FoxP3 and TGFβ1 mRNA Expression.

<p>For the A) baseline (r = 0.557 <i>P</i> = 0.030), b) 7 days following vaccination (r = 0.695 P = 0.019) and C) 28 days following vaccination time points (r = 0.554 <i>P</i> = 0.048) IL-10mRNA expression correlates with FoxP3 expression. At the 28 days time point D) IL-10 mRNA also correlates with TGF-β1 mRNA expression (r = 0.642 <i>P</i> = 0.023). The volunteers with sterile protection are indicated by open circles and triangles indicate volunteers who did not enter the challenge study. Correlations were performed using Spearman's one-sided test, n = 9–12.</p

Foldchange of Gene Expression at Each Timepoint in CS-Stimulated Cells Compared to Unstimulated Cells.

<p>Day 0 =  baseline prior to vaccination.</p><p>Vac+7 =  seven days after the final vaccination.</p><p>Vac+28 = 28 days after the final vaccination.</p

Inverse Correlation between Anti-inflammatory Cytokines and Antibody Response on the Day of Malaria Challenge.

<p>The anti-CS IgG antibody response on the day of malaria challenge induced by vaccination with RTS,S and MVA-CS inversely correlates with the anti-inflammatory cytokines TGF-β1 and IL-10 measured in unstimulated PBMC. A) TGF-β1 at baseline inversely correlates with anti-CS IgG measured on day of challenge r = −0.644 <i>P</i> = 0.022. B) TGF-β1 at day 7 inversely correlates with anti-CS IgG measured on day of challenge, r = −0.670 <i>P</i> = 0.009. C) IL-10 mRNA at baseline inversely correlates with anti-CS IgG measured on day of challenge r  = −0.554 <i>P</i> = 0.031. D) IL-10 mRNA at day 28 inversely correlates with anti-CS IgG measured on day of challenge r = −0.762 <i>P</i> = 0.005. The volunteers with sterile protection are indicated by open circles and triangles indicate volunteers who did not enter the challenge study. Correlations were performed using Spearman's two-tailed test, n = 9–12.</p

The Expression of Cytokines in Unstimulated Cells Before and After Vaccination.

<p>Expression of IFN-γ, MIG, TGF-β1, FoxP3 and IL-10 was measured by real-time RT-PCR in total PBMCs following 12 hour culture in media only in subjects who received vaccination with RTS,S/AS02A and MVA-CS. Results are expressed as copy number relative to the housekeeping gene HPRT. Median values (6–12 subjects) are shown. Vac+7 =  seven days after the final vaccination, Vac+28 = 28 days after the final vaccination, each subject received two doses of RTS,S/AS02A and one dose of MVA-CS. A) IFN-g mRNA, B) MIG mRNA, C) TGF-β1 mRNA, D) FoxP3 mRNA, E) IL-10 mRNA and F) Correlation of MIG mRNA expression at day 7 with days to parasitemia following sporozoite challenge.</p

Per cell expression levels of IL-1β and TNF by each monocyte subset before and after stimulation.

<div><p>Geometric mean fluorescence (GMF) intensities are shown by monocyte subset for IL-1β (left column) and TNF (right column) in the basal (top row) state, and after oxLDL (middle row) or LPS (bottom row) stimulation. In HIV-infected subjects, IL-1β was expressed at highest levels by the Mono1 (CD14++CD16-) and Mono2 (CD14++CD16+) populations. TNF expression was observed in all subsets after stimuli, with the brightest monocyte subsets being Mono2 (CD14++CD16+) and Mono3 (CD14+ CD16+). Statistical significance was adjusted for multiple comparisons (p = 0.05/4 comparisons per stimulation condition: p = 0.0125).</p> <p>**** p < 0.0001, *** p < 0.001, ** p < 0.01, * p < 0.0125.</p></div

Monocyte production of pro-inflammatory cytokines in the basal state and upon stimulation.

<div><p>HIV-infected subjects are shown in red circles and HIV-uninfected subjects are shown in blue triangles. In the no stimulation condition (basal state), HIV-infected subjects showed higher levels of IL-1β (upper left panel) and IL-8 (lower left panel). Upon stimulation with either oxLDL or LPS, HIV-infected subjects exhibited higher levels of IL-1β, IL-8 and IL-6 (upper right panel) compared to the HIV-uninfected subjects. While HIV-1 infected subjects did tend to have higher TNF (lower right panel) responses upon stimulation, these differences were not significant.</p> <p>**** p < 0.0001, *** p < 0.001, ** p < 0.01.</p></div

Gating strategy for identification of total monocytes, monocyte subsets and detection of cytokine expression.

<p>Gating Strategy. Panel A. Identification of total monocytes from peripheral blood mononuclear cells by exclusion of doublets, dead cells, CD3, CD56, CD19, CD20 and low HLA-DR expressing cells. Double-negative CD14-CD16- cells were also excluded. Forward scatter (FSC) vs. side scatter (SSC) plots are shown comparing total PBMC (doublet and dead cells excluded) and total monocytes. Panel B. Monocyte subsets were identified based on the expression of CD14 and CD16 (left column). The diagram at the bottom of the left column is a visual guide for the terminology of monocyte subsets employed in this report. Intracellular cytokines (IL-1β, IL-8/CXCL8, IL-6 and TNF) produced in total monocytes were detected in response to no stimulus, oxidized low density lipoprotein (oxLDL) or lipopolysaccharide (LPS). Fluorescence minus one control condition, in which the antibody conjugate in question is omitted to guide creation of the gate that defines positive expression of that target, is shown on the bottom row. The subject presented is HIV-infected and displays high but representative responses to stimuli.</p

HIV-1 infected subjects with well-controlled viremia have a greater proportion of classical (Mono1, CD14++CD16-) monocytes and a lower frequency of intermediate (Mono2, CD14++CD16+) monocytes, which correlates with production of IL-1β.

<p>HIV-infected subjects are shown in red circles, and HIV-uninfected subjects are shown in blue triangles. Panel A. HIV-infected subjects had a greater fraction of monocytes that fall into the Mono1 (CD14++CD16- classical) subset, and a lower fraction that fall into the Mono2 (CD14++CD16+ intermediate) subsets. Upper right diagram represents monocyte gating scheme (Mono 1-4). Panel B. A lower proportion of monocytes in the Mono2 subset was associated with higher basal IL-1β production. **** p < 0.0001, *** p < 0.001,.</p

Comparison of cross-sectional HIV incidence assay results from dried blood spots and plasma

<div><p>Background</p><p>Assays have been developed for cross-sectional HIV incidence estimation using plasma samples. Large scale surveillance programs are planned using dried blood spot (DBS) specimens for incidence assessment. However, limited information exists on the performance of HIV cross-sectional incidence assays using DBS.</p><p>Methods</p><p>The assays evaluated were: Maxim HIV-1 Limiting Antigen Avidity EIA (LAg-Avidity), Sedia HIV-1 BED-Capture EIA (BED-CEIA), and CDC modified BioRad HIV-1/2 Plus O Avidity-based Assay (CDC-BioRad Avidity) using pre-determined cutoff values. 100 matched HIV-1 positive plasma and DBS samples, with known duration of infection, from the Consortium for the Evaluation and Performance of HIV Incidence Assays repository were tested. All assays were run in duplicate. To examine the degree of variability within and between results for each sample type, both categorical and continuous results were analyzed. Associations were assessed with Bland Altman, R² values and Cohen’s kappa coefficient (ĸ).</p><p>Results</p><p>Intra-assay variability using the same sample type was similar for all assays (R² 0.96 to 1.00). The R² values comparing DBS and plasma results for LAg-Avidity, BED-CEIA, and CDC-BioRad Avidity were 0.96, 0.94, and 0.84, respectively. The concordance and ĸ values between DBS and plasma for all three assays were >87% and >0.64, respectively. The Bland-Altman analysis showed significant differences between plasma and DBS samples. For all three assays, a higher number of samples were classified as recent infections using DBS samples.</p><p>Conclusions</p><p>DBS and plasma sample results were highly correlated. However, when compared to plasma, each assay performed somewhat differently in DBS at the lower and higher ends of the dynamic range. DBS samples were more likely to be classified as recently infected by all three assays, which may lead to overestimation of incidence in surveys using performance criteria derived for plasma samples.</p></div