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

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

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

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

H1N1-specific CD4⁺IL-21⁺ICOS⁺CXCR5⁺ T_FH cells predict functional antibody responses.

<p>Correlations between the number of H1N1-specific CD4⁺IL-21⁺ICOS⁺CXCR5⁺ T_FH cells and H1N1-specific DHI responses measured at (A) day 28 and (B) day 168 after immunizzation. Dashed lines represent the least squares regressions fit to the data. R: Pearson product-moment correlation coefficient. <i>p</i>: correlation-associated p value.</p

H1N1-specific CD4⁺IL-21⁺ICOS⁺ T_H cells subsets expressing or not CXCR5 expand after influenza vaccination.

<p>Number of CD4⁺IL-21⁺ICOS⁺ T_H cells, showing a CXCR5⁺ (black) or CXCR5^- (gray) phenotype, in vaccinated participants after overnight stimulation with A/California/7/2009 (H1N1) antigen or SEB. Data show saline placebo (n=7), and merged TIIV (n=18) and ATIIV (n=17) cohorts at baseline (D0), day 7 (D7) and day 28 (D28) after a single dose of influenza vaccine. Data are expressed as number of cells in 10⁶ live CD4⁺ T cells; mean ± SEM is shown. Non-parametric Wilcoxon’s signed rank test was used for statistical analyses: *<i>p</i> < 0.05, **<i>p</i> < 0.01, and ***<i>p</i> < 0.001 compared to day 0.</p

B-cell and functional antibody responses after seasonal influenza vaccination.

<p>(<b>A</b>) Absolute number of plasmablasts (CD19⁺CD20^- CD38⁺) in 10⁶ live PBMCs acquired. (<b>B</b>) HI Geometric mean titers (GMT) for A H1N1 and H3N2, and B influenza strains at baseline (D0), 7 days (D7), 28 days (D28), and 168 days (D168) after a single dose of influenza vaccine. Data show three cohorts: saline placebo (n=7), TIIV (n=18), and ATIIV (n=17). (<b>C</b>) Geometric mean ratio (GMR) for all vaccine strains. Non-parametric Wilcoxon’s signed rank test was used for statistical analyses: *<i>p</i> < 0.05, **<i>p</i> < 0.01, and ***<i>p</i> < 0.001 compared to day 0; ^§<i>p</i> < 0.05, ^§§<i>p</i> < 0.01 and ^§§§<i>p</i> < 0.001 compared to saline placebo.</p

CONSORT Flow diagram.

<p>CONSORT Flow diagram.</p

T_FH1 ICOS⁺ cells predict functional antibody responses.

<p>Correlations between the number of total circulating CD4⁺ T_FH1 ICOS⁺ cells and the maximum DHI responses observed across the three influenza strains represented in the vaccine, measured at (A) day 28 and (B) day 168 after immunizzation. Dashed lines represent the least squares regressions fit to the data. R: Pearson product-moment correlation coefficient. <i>p</i>: correlation-associated p value.</p