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

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

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

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