Binding of post-H5N1 vaccination polyclonal human serum to properly folded HA1 protein.

<p>Steady-state equilibrium analysis of the total binding antibodies in the polyclonal human vaccine sera to properly folded functional H5N1-A/Vietnam/1203/2004 HA1-His₆ was measured by SPR. Ten-fold diluted individual post-boost H5N1 vaccination sera from the three vaccine groups were injected simultaneously onto HA1 immobilized on a sensor chip through the free amine group and onto a blank flow cell, free of peptide. Maximum resonance unit (Max RU) values for HA1 binding by serum antibodies obtained from multiple individuals from either MF59-adjuvanted H5N3-primed (red symbols; average-1703 RU) or unadjuvanted H5N3-primed (green symbols; average-1408 RU) on day 21 following single booster vaccination or unprimed controls after 2 doses of MF-59-adjuvanted H5N1 vaccine (blue symbols; average-982 RU). The binding antibodies in the H5N3-primed vaccine groups were significantly higher compared to the unprimed vaccine group.</p

Geometric Mean and Standard deviations for the end-point microneutralization titers of three vaccine groups against diverse H5N1 virus strains.

a<p>MN Data are shown for post-H5N1 vaccination sera collected on day 21 following single H5N1 booster vaccination from the MF59-adjuvanted primed and nonadjuvanted primed or after two vaccinations in the unprimed control vaccine group.</p>b<p>Percentage of responders (fraction of subjects) demonstrating MN titers of ≥1∶40 are shown in the parenthesis.</p

Serum antibody off-rates to H5-Viet-rHA1 (but not rHA2) following heterologous prime-boost strongly correlate with the <i>in-vitro</i> neutralizing capacity against the homologous H5 vaccine viruses.

<p>Antibody off-rate constants were determined directly from the plasma sample interaction with H5N1 rHA1 or rHA2 protein using SPR in the dissociation phase. SPR analysis of post-boost vaccination human sera from all vaccine groups included in the vaccine trial was performed with rHA1 (A and B) or rHA2 (C and D) of the H5N1- A/Vietnam/1203/2004 strain. Each symbol represents one individual. Serum samples on day 21 following single H5N1 booster vaccination from the MF59-H5N3 adjuvanted primed (red symbols) or unadjuvanted H5N3 primed (green symbols) or after two MF59-H5N1 vaccinations in the unprimed control (blue symbols) vaccine group is shown. Antibody affinity of post-H5N1 vaccinated human sera against HA1 (but not HA2) of H5N1- A/Vietnam/1203/2004 correlated with the homologous MN titers against the A/Vietnam/1203/2004 (H5N1) booster vaccine virus (A) as well as A/duck/Singapore/1997 (H5N3) priming vaccine virus (B).</p

hIRA B cells are present in human tonsils.

<p>A) Gating strategy for the identification of hIRA B cells in tonsils. Tonsil-derived cells were obtained from tonsils of individuals undergoing routine tonsillectomy. After processing in the presence of DNAse and collagenase, samples were stained, fixed and analyzed by flow cytometry. Live cells were discriminated using Aqua Live/Dead staining kit, and singlet lymphocytes were selected on the basis of forward (FSC) and side (SSC) scatters. B cells were further identified as CD19⁺CD20⁺ and analyzed for the expression of GM-CSF. B) Quantification of hIRA B cells in tonsils. The number of CD19⁺CD20⁺GM-CSF⁺ cells was normalized on the number of total CD19⁺CD20⁺ lymphocytes after isotype background subtraction. The bar represents the geometric mean.</p

Effect of different stimuli on the frequencies of CD19⁺CD20⁺GM-CSF⁺ cells.

<p><i>Ex-vivo</i> cells from tonsils (5 x 10⁵ cells/well) were stimulated for 2 days at 37°C in the presence of IL-2 with or without polyclonal stimuli (anti-IgM 10 μg/ml or SAC 1:20.000 final) or TLR9 ligand (CpG at 10 μg/ml). Brefeldin A was added 3 h before fixation and staining.</p

Phenotypic properties of hIRA B cells from tonsils.

<p>Conventional CD19⁺CD20⁺GM-CSF^-(GM-CSF^neg) and hIRA CD19⁺CD20⁺GM-CSF⁺(GM-CSF^pos) B cells in cell suspensions from tonsils were analyzed by flow cytometry. A) Expression of CD5, CD43, CD11c and IgD in GM-CSF^neg versus GM-CSF^pos cells. The graphs show the geometric mean of fluorescence intensity (GeoMFI) for each marker after isotype background subtraction. Means and standard deviations are shown. P value was calculated using the non-parametric Mann–Whitney test (n = 4). B) Ig subclasses were analyzed within the hIRA B cell population. The plots are representative of n = 4.</p

hIRA B cells have phagocytic capacity.

<p>A) Tonsil-derived cells were incubated for 2 h at 37°C with heat-inactivated <i>S</i>. <i>aureus</i> at 1:10 (cells:bacteria) MOI. After staining with anti-CD19 (Blue), anti-GM-CSF (Orange), and anti-MPO (Red), different optical fields were analyzed by confocal microscopy. The enlargement (bright field merged with the GM-CSF channel, right panel) shows the presence of <i>S</i>. <i>aureus</i> internalized by a CD19⁺GM-CSF⁺MPO⁺ cell (white arrows) and two CD19⁺GM-CSF^-MPO^- cells with non-internalized bacteria (yellow arrows). B) <i>Left panels</i>: similar experimental approach described for panel A) with the addition of PhRodo labelled <i>S</i>. <i>aureus</i> bioparticles (green). The white arrows indicate not-internalized bioparticles (low fluorescence intensity), while bright fluorescent bioparticles localize within a CD19⁺GM-CSF⁺MPO⁺ cell (white square) but not within a conventional CD19⁺ cell (yellow square); <i>right panels</i>: control experiment where cells were incubated with PhRodo labelled <i>S</i>.<i>aureus</i> bioparticles (dark green) for 2 h at 4°C. Data are representative of 3 independent experiments. Scale bar = 10 μm.</p

Human IRA B cells reside within the follicles.

<p>A) Cell suspensions from tonsils were seeded onto poly-L-lysine coverslips and fixed with formaldehyde. Cells were incubated for 1 h with anti-CD19 (Green) and anti-GM-CSF (Red). The merge panel shows the co-localization of GM-CSF and CD19. The picture is representative of three different subjects. Scale bar = 10 μm. B) 8-μm tonsil tissue sections were fixed with formaldehyde and stained using anti-CD3 (T cell area, Blue), anti-CD19 (B cell area, Green) and anti-GM-CSF (Red). The enlargement shows the presence of GM-CSF⁺ cells within B cell follicles (white arrows). The panel is representative of 3 independent experiments using different tonsils where many sequential sections were screened (n>5). Scale bar = 50 μm. C) 8-μm tonsil tissue sections were fixed with formaldehyde and stained using anti-CD3 (T cell area, Blue), anti-IgD (mantle zone, Green) and anti-GM-CSF (Red). The panel is representative of 3 independent experiments using different tonsils where many sequential sections were screened (n>5). Scale bar = 50 μm.</p

Simultaneous identification of B lymphocytes specific for HA from different influenza strains in <i>ex vivo</i> PBMCs samples.

<p><b>A.</b> PBMCs from an anonymous blood donor were pre-incubated with subunits from either B/Brisbane/60/2008 or A/Panama/2007/1999 (H3N2), and then stained with anti-CD20 mAb, HSA conjugated with A488 and A647, with A647-rH3 (from A/Brisbane/10/2007) and A488-rH1 (from A/California/07/09), or with A647-rB/HA (from B/Brisbane/60/2008) and A488-rH1 (from A/California/07/09). The staining patterns observed in the CD20⁺ B-cell gate are shown. <b>B.</b> PBMCs from 16 anonymous blood donors were pre-saturated with B/Brisbane/60/2008 and then stained with anti-CD20 mAb, A647-rH3 (from A/Brisbane/10/2007) and A488-rH1 (from A/California/07/09). The scatter plot depicts paired values of H1⁺ (y-axis) and H3⁺ (x-axis) B-cells. The insert box plot depicts the distribution of H1⁺, H3⁺ and H1⁺H3⁺ B-cells in the same 16 donors. Mean values are indicated by dotted lines.</p

H1⁺ IgG⁺ MBCs frequencies measured by flow-cytometry and by ELISPOT correlated linearly.

<p><b>A–B.</b> Specificity of the staining with the rH1 bait from A/Solomon Island/3/06. PBMCs (1.6×10⁸) from anonymous blood donors were stained with Live/Dead, incubated with an H3N2 mono-bulk vaccine subunit (from A/Panama/2007/1999), and then stained with Alexa647-conjugated HSA (6×10⁷), or Alexa647-conjugated rH1 (1×10⁸), and with an antiCD20 mAb. <b>A.</b> Binding pattern of HSA (A647-HSA; left panel) and of rH1 (A647-rH1; right panel) in the CD20⁺ B-cell gates. <b>B.</b> H1⁺ B-cells identified in A were sorted (n = 8215), mixed with autologous CD20^neg cells in the ratio of 1∶50 and activated with CpG and IL-2 for 5 days <i>in vitro</i>. Unsorted PBMC and CD20^neg cells were also cultured in the same manner as controls. After 5 days, equal numbers of cultured cells were harvested and assayed by ELISPOT for numbers of cells secreting IgG and IgG specific for H1N1 (from A/Solomon Island/3/06). <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0070620#s2" target="_blank">Results</a> are expressed as number of antibody secreting cells (ASC) normalized to 10⁶ cultured cells assayed by ELISPOT. Nd indicates undetectable ASC. <b>C.</b> Distribution of IgG⁺ B-cells among H1^neg and H1⁺ B cells expressing or not the CD27 B cell memory marker; shown is one representative subject. <b>D.</b> Replicates of frozen PBMCs from 4 anonymous blood donors were assayed by conventional ELISPOT, or incubated with an H3N2 mono-bulk vaccine subunit and stained with rH1, and anti-CD20 plus anti-human IgG antibodies. The scatter plot depicts paired values of H1⁺ IgG⁺ B-cell frequencies measured by flow-cytometry (y-axis) and by ELISPOT (x-axis) across three different experimental sessions. Shown are: the regression line with the related 95% confidence interval (gray areas), slope, intercepts, R² and p-value. <b>E.</b> Variability plot showing mean standard deviations of the measurements done by ELISPOT and flow-cytometry. The three dotted lines mark the grand mean and the upper and lower control limits.</p