Biological functions that are predicted to be activated by CAV2 vector in CD103⁺ and CD11b⁺ -type DCs.

*<p>the expression of the underlined molecules is down-modulated.</p

Validation of gene expression induced by CAV2 vector with qRT-PCR.

<p>A selection of genes was checked for CAV2-induced upregulation using qRT-PCR on cDNA from the CD103⁺ and CD11b⁺ -type DCs (4 different sheep were used in total, 3 per validated gene). Each gene detection was normalized with GAPDH expression and the relative gene expressions (log2(CTgene-CTGAPDH)) are reported. The gene names printed with regular font were selected as being up-regulated in the CD11b⁺ type DC microarrays (p < 0.05 ANOVA followed by Benjamini-Hochberg FDR and > 2 fold), the gene names printed in italic are not represented by any probe on the ovine microarray, the underlined gene names were close to be selected by the variance analysis in the CD11b⁺ -type DCs.</p

Top canonical pathways induced by CAV2 vector in CD103⁺ and CD11b⁺ -type DCs.

<p>Top canonical pathways induced by CAV2 vector in CD103⁺ and CD11b⁺ -type DCs.</p

Transcriptional response to CAV2 vector in the CD103⁺ and CD11b⁺ -type DC subsets.

<p>(A)The mean fold induction of the selected genes up-regulated by Cav-null R⁰ are represented in graded yellow to red color (from 1 to >3) for both CD103⁺ and CD11b⁺ -type DCs from 4 sheep. The represented genes were selected from the significantly induced genes in the CD11b⁺ -type DC subset (222 genes, p<0.05, fold > 2) and they were ranked based on their fold induction in the CD11b⁺ -type DC subset. The significantly activated genes in the CD103⁺ subset (21 genes, p<0.05, fold > 2) are indicated by a star. (B) The mean fold reduction levels of the selected down-modulated genes by Cav-null R⁰ are represented in graded yellow to blue color (from 1 to <0.2) for both CD103⁺ and CD11b⁺ -type DCs from 4 sheep. The represented genes were selected from the significantly reduced genes in the CD11b⁺ -type DC subset (29 genes, p<0.05) and they were ranked based on their fold induction in the CD11b⁺ -type DC subset. No gene expression was significantly reduced in the CD103⁺ -type DC subset.</p

Antimicrobial gene network induced by CAV2 vector in CD11b⁺ -type DCs.

<p>An antimicrobial gene network centered on IFN was generated by the Ingenuity Pathways Analysis on the selected genes dys-regulated by CAV2 vector in CD11b⁺ -type DCs (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0052513#pone.0052513.s003" target="_blank">Table S1</a>, p < 0.05 and fold > 2, or p < 0.05 and folds confirmed by qRT-PCR). Molecule types are represented by symbols: diamonds (enzymes), triangles (kinase), square (cytokine), double circle (complex), oval (transcription regulator), circle (others).</p

Expression of VP7 antigen.

<p>Following transduction of CHO-CAR cells with Cav-VP7 R⁰, expression of bluetongue antigen was sought and detected by immunostaining using a MAb directed against the VP7 protein (A). Following transduction of RK13 cells with SG33-VP7, VP7 antigen was labeled using an anti-VP7 hyperimmune rabbit serum (C). Non-transduced cells were used as negative control (B and D).</p

Serum antibodies induced by Cav-VP7 R⁰ or Cav-G R⁰ in sheep.

<p>Sheep received two doses of Cav-VP7 R⁰ or Cav-G R⁰ or PBS (control groups) on D0 and D28; the sheep were challenged with two wild type strains of BTV on day D56. Sera were collected at days 0, 28, 56 and every two days until day 64. Antibody responses elicited against the VP7 protein were assessed by ELISA performed on plates coated with recombinant VP7 for 1/20 dilutions of individual sera are shown, for the group challenged with BTV2 (A) and with BTV8 (B).</p

Inoculation of sheep with Cav-VP7 R⁰ and SG33-VP7 elicits T-cell responses.

<p>Sheep were immunized twice at a four-week interval with recombinant vectors and cellular immune responses were analyzed at D60. Prescapular lymph node cells were isolated and cultured in complete medium alone or with the VP7 recombinant protein. The specific proliferation was evaluated as [the % divided T cells cultured with VP7 protein] − [the % divided T cells cultured in medium alone]. (A) The percentage of CD8⁺ T cells that had divided in the absence or presence of VP7 is shown (* statistical difference between immunized groups <i>p</i> = 0.0085). (B) The percentage of CD4⁺ T cells that had divided in the absence or presence of VP7 is shown. (C) PBMCs were labeled with CFSE and cultured in complete medium alone -or with either SG33 or SG33-VP7. VP7-specific CD4⁺ T cell proliferation was evaluated as [the % divided CD4⁺ T cells cultured with recombinant vector] − [the % divided CD4⁺ T cells cultured in medium alone]. Horizontal bars represent the means of proliferation for each group.</p

Serum antibodies induced by Cav-VP7 R⁰ and SG33-VP7 in sheep.

<p>Sheep were immunized twice with recombinant vectors. Antibody responses elicited against the VP7 protein were assessed by ELISA at day 60 following the first inoculation of Cav-VP7 R⁰ (A) or SG33-VP7 (B). Data are presented as means (with standard deviation) of individual ODs obtained for sheep inoculated with recombinant vector (black bar) or for control animals (white bar).</p

Evaluation of protection following vaccination with Cav-VP7 R⁰ and virulent challenge.

<p>(A) Evolution of mean threshold cycle (Ct) values (with standard deviation) of BTV-2 specific real-time RT-PCR (a) or BTV-8 specific real-time RT-PCR (b). (B) Mean cumulative scores (with standard deviation) after BTV-2 challenge in vaccinated (black bar) and control (white bar) groups.</p