An overview of used E-protein derived peptides and their characteristics.

<p>Amino acid sequences of the E-protein derived peptides used in this study, their expression level in <i>E. coli</i>, and the presence of predicted MHC class I or class II epitopes in the different domains of the E-protein compared to known human T-cell epitopes. The sequence of the peptides can be found in Chabierski et al. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0115343#pone.0115343-Chabierski1" target="_blank">[24]</a>.</p><p>Abbreviations. E: WNV envelope protein, M: WNV membrane protein, NS: WNV non-structural protein ++: very high expression, +: high expression, +/−: moderate expression and –: very low expression.</p><p>An overview of used E-protein derived peptides and their characteristics.</p

Expression and purification of recombinant GST tagged peptides.

<p>SDS-PAGE showing crude lysates of protein-expressing bacteria and purification steps of peptide E471 showing a moderate expression (a) and peptide E131 showing very low expression (b). Lane 1, crude lysate; lane 2, lysate supernatant after centrifugation; lane 3, size marker; lanes 4–5, respectively elution fraction 1 and 2 of recombinant peptide after glutathione affinity purification.</p

Location of the peptide sequences in the E protein that, based on our <i>in vivo</i> experiments, contain strong CD4+ (underlined) and CD8+ (bold) T cell epitopes.

<p>The shown amino acid sequence is that of the E protein of lineage 1 WNV strain Ita09. Sequences that are in bold and underlined contain strong CD4+ as well as CD8+ T cell epitopes.</p

Detection of cellular and humoral immune response following pDNA-based vaccination.

<p>IFN-γ production by (a) CD4-depleted and (c) CD8-depleted splenocytes after stimulation with purified recombinant GST tagged E-protein derived peptides. The WNV E-protein specific T-cell repertoire in BALB/c mice was expanded by two DNA vaccinations. Splenocytes obtained two weeks after the boost were stimulated with different recombinant GST tagged E-protein derived peptides and the numbers of cells producing IFN-γ were determined via ELISPOT. (b) Detection of serum IgG1 and IgG2a titers to the WNV E-protein two weeks after the boost via ELISA.</p

Purification and testing of recombinant proteins.

<p><b>A</b>: SDS-PAGE showing crude lysates of protein-expressing bacteria and purification steps. M, size marker; Lane 1, culture expressing GST (arrowhead); lane 2, culture expressing peptide C_41–70 (asterisk); lane 3, peptide C_41–70 after glutathione-affinity-purification; lane 4, peptide C_41–70 after gelfiltration. <b>B</b>: ELISA using serum I3 on GST purified only with glutathione sepharose (white column, 500 ng) or additionally with gel filtration chromatography (black column, 2000 ng). Values are the mean of two independent experiments (performed in duplicate), error bars represent the standard deviation. Statistical analysis to evaluate the difference between the two signals was performed by using an unpaired t-test, (asterisks, p<0,001).</p

Analysis of the binding property of selected recombinant peptides with human sera in an ELISA.

<p>30-mer peptides spanning the WNV-proteins capsid, prM/M and E, fused to GST, were incubated with human sera from outbreaks in Italy (I1-8), Greece (G1–G15) and USA (US1-2). Those peptides displaying signals with an OD >0,5 are shown (the total number of peptides is shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0066507#pone.0066507.s001" target="_blank">Figure S1</a>).Values represent the absorption over cut-off (mean of four negative sera plus two standard deviations) and are derived from at least two independent experiments (performed in duplicate). Error bars represent the standard deviation. The background (binding of the serum to GST) was subtracted.</p

Optimizing antibody binding to selected peptides.

<p>ELISA plates were coated with increasing amounts of the indicated peptides fused to GST and incubated with human sera: WNV positive sera I2, I4; G11, G12, G15 and negative serum N5. Values are the mean of two independent experiments (performed in duplicate), error bars represent the standard deviation.</p

Competition of antibody binding between selected peptides and recombinant E-ectodomain.

<p>The indicated sera were pre-incubated with recombinant peptides E_71–100, E_231–260, E_371–400 (all fused to GST) or GST alone, before they were bound to recombinant E-ectodomain of WNV. Values are derived from at least two independent experiments (performed in duplicate). Error bars represent the standard deviation. Statistical analysis to evaluate the difference between the signals of the competitor peptide and the control competitor (GST alone) were performed by using a Mann-Whitney Rank Sum Test (asterisks, p<0,05).</p

Amino acid sequences of the fragments used in this study (lineage 1 WNV strain Ita09).

<p>Amino acid sequences of the fragments used in this study (lineage 1 WNV strain Ita09).</p

Analysis of binding properties of human sera with recombinant E ectodomain in an ELISA.

<p>Sera used in this study were incubated with the recombinant E-ectodomain protein (lineage 1). Mean values of two independent experiments (performed in duplicate) are shown, and error bars represent the standard deviation.</p