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

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

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

A: Expression and purification of DENV-2 Ewt and Equad from Drosophila S2 culture supernatants; supernatant before induction (b.ind.), after 7 days of expression culture (7 d expr.), concentrated via tangential flow (Conc.) and the two step purification with immobilized imidazole affinity (IMAC) and size exclusion chromatography (SEC) were separated on a 10% SDS-PAGE gel under reducing conditions. B: 6 μg of purified DENV1-4 (D1-D4) Equad and DENV-2 Ewt proteins were analyzed with SDS-PAGE. Proteins were stained with Coomassie blue. Size of molecular weight markers in kilo Daltons is indicated on the left.

<p>A: Expression and purification of DENV-2 Ewt and Equad from Drosophila S2 culture supernatants; supernatant before induction (b.ind.), after 7 days of expression culture (7 d expr.), concentrated via tangential flow (Conc.) and the two step purification with immobilized imidazole affinity (IMAC) and size exclusion chromatography (SEC) were separated on a 10% SDS-PAGE gel under reducing conditions. B: 6 μg of purified DENV1-4 (D1-D4) Equad and DENV-2 Ewt proteins were analyzed with SDS-PAGE. Proteins were stained with Coomassie blue. Size of molecular weight markers in kilo Daltons is indicated on the left.</p

Antigen titration using the indicated amounts of DENV-1 (A), -2 (B), -3 (C) and -4 (D) Equad and DENV-2 Ewt (E) proteins with three different DENV, WNV and negative (NEG) human sera.

<p>Measurements were performed in duplicates in at least two independent experiments.</p

Alignment of the amino acid sequences containing the fusion loop domain from E proteins of DENV 1–4, WNV, JEV, YFV, TBEV and the four point mutations of the QUAD proteins.

<p>Amino acids numbering: 1 is start of the E protein.</p

300 ng per well of DENV-2 Ewt (A) and Equad (B) and 160 ng per well of a DENV 1–4 Equad mixture (C) were tested with DENV- WNV- and TBEV- infected and YFV-vaccinated sera compared to negative (NEG) samples in an IgG-ELISA (n = number of individuals).

<p>Bottom and top of the boxes are the first and third quartiles. The median signal is depicted as a line inside the box. Whiskers represent the 9^th and the 91^st percentile. Outliers in B and C are marked with numbers (1–5). Measurements were performed in duplicates in at least two independent experiments.</p

Detection of virus neutralizing antibodies.

<p>Five mice were vaccinated IM or topically with WNV-DermaVir nanoparticles and boosted either with WNV-DermaVir nanoparticles (IM) or E-protein combined with Matrix-M1 adjuvant (s.c.). Virus neutralization titers were determined by a focus reduction neutralization assay. Abbreviations: bE: boost E-protein, bDNA: boost WNV-DermaVir nanoparticles.</p

Induction of WNV-E specific IL-4 response by vaccination with WNV-DermaVir nanoparticles followed by a WNV-E protein boost.

<p>WNV-E specific IL-4 responses were determined by IL-4 ELISPOT. Splenocytes obtained two weeks after the boost were stimulated with WNV-E protein and the numbers of cells producing IL-4 per 3×10⁵ cells were determined in triplicate. Mice were primed ID (4 mice), IM (5 mice), topically (top, 5 mice) or not (−/, 5 mice) with WNV-DermaVir nanoparticles and boosted s.c. with E-protein combined with Matrix-M1.</p