Enrichment of phage clones predicted to display authentic NadA fragments on their surface after selection with a serum pool from volunteers immunized with the Bexero vaccine.

<p>Frequency values reported in the vertical axis in panels A–C refer to the occurrence, per single amino acid position, of sequences predicted to express authentic NadA fragments, relative to those predicted to express irrelevant or no polypeptides. The inset in figure A reports the same data with a higher y-axis magnification. The horizontal axis reports the amino acid positions of the translated NadA sequence. A, unselected library; B and C, library outputs after one and two rounds of selection, respectively. D, Cumulative enrichment factors for each amino acid position derived from NadA fragments obtained after one (blue line) and two (red line) rounds of selection; colored bars in the horizontal axis refer to NadA domains; the area between the dashed vertical lines correspond to the cell binding region of NadA <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0114159#pone.0114159-Tavano1" target="_blank">[18]</a>. E and F, enrichment factors of NadA fragments after one and two rounds of selection, respectively. Only the fragments laying in the upper quartile of enrichment factors values are shown.</p

Occurrence of cumulative amino acid positions in NadA fragments obtained after two rounds of selection, as determined by traditional random picking of 50 plaques followed by Sanger sequencing of individual clones.

<p>Occurrence of cumulative amino acid positions in NadA fragments obtained after two rounds of selection, as determined by traditional random picking of 50 plaques followed by Sanger sequencing of individual clones.</p

Effects of active immunoprotection with the 6pGST FbsA fragment in adult and neonatal mouse models of GBS sepsis.

<p><b>A</b>. Immunoprotection in adult mice. Five-week-old CD1 mice underwent three immunizations with the 6p FbsA fragment fused to GST (6pGST) or with GST alone. At 3 weeks after the last immunizations mice were challenged by the i.p. injection of GBS strain COH1 (5x10⁷ CFUs) and lethality was observed daily. *, p<0.05 relative to GST-immunized mice by Kaplan-Meier survival plots. Shown are the cumulative results of two independent experiments. <b>B</b>. Effect of maternal immunization on survival of experimentally infected pups. Female CD1 mice (5 wk old) were immunized three times with the 6p FbsA fragment fused to GST (6pGST) or with GST alone. Mice were then time-mated and two-day-old pups were infected s.c. with 250 CFUs of GBS strain 6313. *, p<0.05 relative to GST-immunized mice by Kaplan-Meier survival plots.</p

Properties of the antigen-specific phage library before and after selection with a pool of serum samples from volunteers immunized with the Bexero anti-MenB vaccine.

<p>A–C, abundance of “natural frame” <i>nadA</i> fragments in the library before (A) and after the first and second rounds of selection (B and C, respectively). Each point represents the number of unique fragments (vertical axis) displaying the number of copies indicated in the horizontal axis; D–F, <i>nadA</i> fragment length distribution before (D) and after the first and second rounds of selection (E and F, respectively).</p

Schematic outline of the epitope mapping approach.

<p>The gene encoding the antigen is fragmented by DNAse digestion and the gene fragments are inserted into lambda phage vectors. The phage library is mixed with immune serum and phage particles binding to immunoglobulins are separated using Protein-G coated magnetic beads. The inserts of the phage population obtained after selection are massively sequenced and compared with those of the original unselected library using an <i>ad hoc</i> developed software which identifies the region(s) of the antigen targeted by serum antibodies.</p

Immunoreactivity of selected phage clones corresponding to different fragments of the <i>N. meningitidis</i> NadA antigen.

<p>Immunoreactivity of selected phage clones corresponding to different fragments of the <i>N. meningitidis</i> NadA antigen.</p

Effects of deletion of the <i>fbsa</i> gene on GBS virulence in adult and neonatal mouse models of GBS sepsis.

<p>A. Eight-week-old CD1 mice of either sex were injected i.p. with 2x10⁸ CFUs of GBS strain 6313 or of its <i>fbsa</i>-deleted (Δ-<i>fbsa</i>) mutant. Shown are cumulative survival data from two independent experiments. *, p<0.05 relative to wild-type by Kaplan-Meier survival plots. <b>B</b>. Two-day-old CD1 pups were infected s.c. with 250 CFUs of GBS strain 6313 or of its <i>fbsA</i>-deleted (Δ-<i>fbsA</i>) mutant. *, p<0.05 relative to wild-type by Kaplan-Meier survival plots. Shown are cumulative survival data from two independent experiments.</p

Interaction of Fng with recombinant FbsA fragments.

<p><b>A</b>. Dose-dependent binding of Fng to recombinant FbsA fragments. 5pGST and 6pGST were coated onto microtiter plates (500 ng/well) and incubated with increasing amounts of Fng, followed by mouse anti-Fng IgG and HRP-conjugated rabbit anti-mouse IgG. Values represent the means of triplicate samples ± S.E. This experiment was performed three times with similar results. <b>B</b> and <b>C</b>. Surface Plasmon Resonance analysis of the interaction of 5pGST and 6pGST with Fng. 5pGST (panel B) and 6pGST (panel C) were captured on a BIAcore sensor chip coated with goat anti-GST IgG. Human Fng (2.92 nM to 750 nM) was then flowed over the chip surface. The data shown are representative of three individual experiments.</p

Inhibition of the candidacidal activity of sera from animals immunized with plasmids containing selected peptide sequences (pT.TK1, left panel and pT.TK4, right panel) by laminarin, a soluble β-1,3-glucan, pustulan, a soluble β-1,6-D-glucan, and keyhole limpet hemocyanin (KLH)-conjugated peptides.

<p>Inhibition of the candidacidal activity of sera from animals immunized with plasmids containing selected peptide sequences (pT.TK1, left panel and pT.TK4, right panel) by laminarin, a soluble β-1,3-glucan, pustulan, a soluble β-1,6-D-glucan, and keyhole limpet hemocyanin (KLH)-conjugated peptides.</p

Binding of Fng to phage particles in the presence and in the absence of inhibitors.

<p><b>A</b>. Binding to Fng of increasing numbers of 5p or 6p lambda phage (λ5p or λ6p) particles. Plates were coated with Fng, and phage particles were added at the indicated PFU numbers followed by anti-lambda phage rabbit IgG and alkaline phosphatase-labeled goat anti-rabbit IgG. Error bars represent means ± standard deviations from three independent experiments; *, p<0.05 by analysis of variance followed by the Student Newman Keuls test. <b>B</b> and <b>C</b>. Inhibition of binding of 5p or 6p lambda phage particles (λ5p or λ6p, 10⁸ PFU) to immobilized Fng in the presence of increasing concentrations of recombinant FbsA fragments (5pGST and 6pGST in panels B and C, respectively) used as inhibitors. Data are from one experiment, representative of three producing similar results.</p