Additional file 1: of The therapeutic HIV Env C5/gp41 vaccine candidate Vacc-C5 induces specific T cell regulation in a phase I/II clinical study

Supporting data. (XLSX 16 kb

Insignificant relations between T cell activation and R_AC induced by Env and Gag peptide panels.

<p><b>A.</b> Relations between regulation and activation to Env (left panels) and Gag (right panels) within the CD4⁺ and CD8⁺ T cell subsets respectively, correlation coefficients indicated. <b>B.</b> Relations between Env- and Gag-induced regulation within the CD4⁺ (right panel) and the CD8⁺ (right panel) T cell subsets, respectively, correlation coefficients indicated.</p

Relations between Env- and Gag-induced R_AC and activation and markers related to progression of chronic HIV infection.

<p>R_AC and corresponding activation (x-axis), for simplicity represented as overall CD3+ estimates (CD4⁺ plus CD8⁺ combined) induced by Env and Gag peptide panels, respectively, and in relations to various progression markers (y-axis). Spearman rank r and corresponding p-values indicated, values with p<0.10 bolded.</p

Relations between total CD3⁺ T cell activation and R_AC induced by Env and Gag peptide panels.

<p><b>A.</b> Relations between total CD3⁺ T cell regulation calculated after Env and Gag peptide panel stimulations, respectively. Low regulators (<b>+</b>), Gag regulators (▵) and Pan regulators (⧫) indicated as defined in the text. <b>B.</b> Relations between total CD3⁺ T cell activation to Env and Gag, respectively, in the same patient groups as in panel A.</p

Distributions between regulator subgroups.

<p>Box and whisker plots representing medians, interquartile ranges and overall ranges for cytokines in snap-frozen plasma (upper two panels), CD4 counts and CD4 loss rates (two middle panels) and CD8 counts as well as HIV RNA levels (two lower panels). “Low regulators” (Low) as defined in the text are represented as one group, whereas the “High regulator” patients are split into “Gag regulators” (Gag) and “Pan regulators” (Pan), respectively. Significant differences p<0.05 between groups (Mann-Whitney) indicated (*).</p

Characteristics of regulator groups.

<p>Comparisons between patient groups, p<0.05 bolded, p<0.10 italic.</p

Cohort characteristics.

<p>Cohort characteristics.</p

Comparison of CD25⁺HLA-DR⁺ expression and CFSE^dim as measures for antigen-induced activation and regulation of T cells.

<p>CFSE-labelled PBMC from 28 HIV-infected individuals were stimulated for 6 days with peptides encoding either HIV p24 consensus regions (HIV Ag) or a pool of commonly encountered non-HIV viral peptides (Non-HIV Ag). <b>A.</b> Co-expression of CD25 and HLA-DR on live CD8⁺CD3⁺ T cells on non-divided CFSE^high (left panel) and proliferated CFSE^dim T cells (right panel), showing excessive difference in fractions of CD25⁺HLA-DR⁺ in the activated subset to the right. <b>B.</b> Scatter plots of activation measured in the same culture by CFSE^dim or HLA-DR⁺CD25⁺, respectively, to non-HIV (<b>+</b>) and HIV antigen (□) within the CD8⁺ (left panel) and CD4⁺ (right panel) T cell subsets. Significant and high correlations obtained for both Non-HIV antigens (CD8⁺, r = 0.92, p<0.001: CD4⁺ r = 0.64, p<0.001) and HIV Gag p24 (CD8⁺, r = 0.90, p<0.001: CD4⁺ r = 0.71, p<0.001).</p

Schematic outline and examples of the T cell regulation assay.

<p><b>A.</b> Schematic outline of the assay measuring antigen-induced cytokine-mediated induced regulation (R_AC) of T cell activation by IL-10 and TGF-ß. Left panel shows a conventional T cell assay where the final activation measurements are regarded as net results of proinflammatory and regulatory signals. Right panel show a possible outcome when blocking T cell regulatory cytokines. T cell regulation (<b>Δ</b>) is here defined as the difference in activation responses to the same antigen between these panels. <b>B.</b> Detailed example calculating classical activation (upper panels) and regulation (calculated from activation in the presence blocking mAbs to IL-10 and TGF, lower panels), all gated for viable CD8⁺ CD3⁺ T cells. Activation in both upper and lower panels calculated as the difference (<b>Δ</b>) in CD25⁺HLA-DR⁺ fractions between control and antigen-stimulated (Ag⁺). Note a typical slight increase in background activation (lower left panel). Regulation (R_AC) calculated as the difference (<b>Δ</b>) between activation in cytokine-blocked culture and conventional activation culture. <b>C.</b> Scatter plots of regulation calculated in the same cultures by CFSE^dim or HLA-DR⁺CD25⁺, respectively, for the CD8⁺ (λ, r = 0.67, p<0.001) and CD4⁺ (Υ, r = 0.45, p = 0.026) T cell subsets.</p

Vacc-4x Ig antibody levels.

<p>Changes (Δ) from baseline to end of study in rectal IgA, nasal IgA and serum IgG antibody levels in the four dose groups. Adj  =  adjuvant, LD  =  low, MD  =  median and HD  =  high dose. IgA antibody levels are adjusted to total IgA in rectal and nasal samples (please note different scales). Data are given as medians, interquartile and overall ranges. Differences between dose groups and the adjuvant group with p-values less than 0.10 are indicated (Mann-Whitney U test). Kruskal-Wallis ANOVA analysis for all four groups yielded p = 0.044 (rectal IgA), p = 0.093 (nasal IgA) and p = 0.067 (serum IgG).</p