Theoretical prediction of HER-2 epitopes.

*<p>potential calculated for every amino acid using DiscoTope software and characterizing immunogenic property of a given amino acid.</p

Mimotope sequences of affinity-selected phage.

a<p>Polyserum used for mimotope isolation.</p

Limited cross-reactivity between phage selected with different polysera.

<p>Selected phage were immobilized onto nitrocellulose membranes and reacted with three different sera overnight at 4°C (HER-2_FL – serum prepared from mice immunized with full-length HER-2; HER-2_ECD – serum prepared from mice immunized with soluble protein; HER-2_TUBO – serum from tumor bearing mice). Bound antibodies were detected with goat anti-mouse IgG HRP conjugated antibody and the signals were developed by ECL. <i>A.</i> phages selected with HER-2_FLserum, <i>B.</i> phages selected with HER-2_ECD-serum, <i>C.</i> phages selected with HER-2_TUBO -serum).</p

Three-dimensional modeling of putative epitopes.

<p>Three-dimensional model of rat HER-2 is presented in two orientations. <i>A.</i> clusters found for HER-2_FLserum, <i>B.</i> clusters found for HER-2_ECD-serum, <i>C.</i> clusters found for HER-2_TUBO –serum. Serum- specific clusters of amino acid pairs are numbered. Amino acid are coloured according to RasMol amino acid colour code: red = Glu and Asp, blue = Arg and Lys, brown = Pro, orange = Ser and Thr, Cyan = Asn and Gln, violet = Trp and Phe, purple = His.</p

Antigen structure influences the composition of the polyclonal response.

<p><i>A.</i> ELISA plate wells were coated with recombinant rat ECD and reacted with serial dilutions of mouse sera. <i>B.</i> Recombinant extracellular domain (ECD) of rat HER-2 was treated (<i>reduced protein</i>) or not (<i>native protein</i>) with β-mercaptoethanol and run in 10% SDS PAGE, transferred onto nitrocellulose membrane and probed with mouse sera diluted 1∶1000. <i>C.</i> 10⁶ Tubo cells over-expressing rat HER-2 were reacted with serial dilutions of mouse sera, probed with anti-mouse antibody conjugated with PE and fluorescence intensity was measured by flow cytometry. Each point is reflective of at least 10 000 events. <i>D.</i> Recombinant extracellular domain of rat HER-2 or human HER-2 were run in 10% SDS PAGE, transferred onto nitrocellulose membrane and probed with mouse sera diluted 1∶1000 or anti-HER-2 antibody. <i>E.</i> 500 TUBO cells were plated into 96 well plate in DMEM medium supplemented with 5% FBS and incubated 1–2 weeks in the presence of IgGs purified from mouse sera at a final concentration of 25 µg/ml.. The cell proliferation was measured according to manufacturer instruction by CellTiter 96® Aqueous Non-Radioactive Cell Proliferation Assay. The error bars reflect the mean+/−sem for 4 samples. These data represent the results of pooled serum from 5 vaccinated mice per group. Each experiment was replicated at least 3 times and representative results are shown.</p

Occurrence of statistically significant amino acids within mimotope collections.

a<p>Polyserum used for mimotope isolation.</p>b<p>The frequency was determined as: (# specific residues/total # of residues in the collection) ×100.</p

Occurrence of statistically significant amino acid pairs within mimotope collections.

a<p>Polyserum used for mimotope isolation.</p>b<p>Single letters amino acid codes (See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0005309#s3" target="_blank">Methods</a>).</p>c<p>Number of mimotopes carrying this generalized pair within the specific collection.</p>d<p>Unique pairs are presented in bold, pairs shared by two datasets are presented in italics.</p

Clusters predicted by computer algorithm.

<p>Clusters predicted by computer algorithm.</p

Age does not impact the frequency of functional virus-specific CD8+ T cells.

<p>A) Flow plots depict gating strategy for IFNγ+ CD8+ T cells. (B–D) Pooled WNV, CMV or EBV peptides were used to stimulated freshly thawed PBMCs isolated from WNV-naturally infected subjects 6–7 months post symptom onset. Scatter plots depict IFNγ+ CD8+ T cell responses only from reactive subjects (>0.05% and 3 fold above DMSO background); (B) WNV reactive subjects: 20/21 Young, 24/25 Mid-aged, 24/26 Aged; (C) CMV reactive subjects: 7/21 Young, 12/25 Mid-aged, 17/26 Aged; (D) EBV reactive subjects: 17/21 Young, 21/25 Mid-aged, 24/26 Aged. Means are displayed as horizontal lines. Statistical analysis performed by one-way ANOVA followed by Tukey's multiple comparison post-test.</p

Ageing results in increased frequencies of highly-differentiated CD8+ T cells and decreased frequencies of naïve CD8+ T cells.

<p>A) Flow plots depicting gating strategy for phenotypic analysis. B) Percentage of CD8+ CD28− T cells within the peripheral blood lymphocyte pool; C) Percentage of CD8+ CD28− CD57+ T cells (terminally differentiated cells) within the peripheral blood lymphocyte pool; D) Percentage of CD45RA+ CCR7+ (naïve) T cells within the peripheral blood lymphocyte pool. Data are group according to stratification described in the <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003076#s4" target="_blank">Materials and Methods</a>. Statistical analysis perfomed by one-way ANOVA with Tukey's mulitple comparison post-test. Box and whiskers plots are calculated at 95% confidenced interval.</p