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

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

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

A Human Anti-M2 Antibody Mediates Antibody-Dependent Cell-Mediated Cytotoxicity (ADCC) and Cytokine Secretion by Resting and Cytokine-Preactivated Natural Killer (NK) Cells

<div><p>The highly conserved matrix protein 2 (M2) is a good candidate for the development of a broadly protective influenza vaccine that induces long-lasting immunity. In animal models, natural killer (NK) cells have been proposed to play an important role in the protection provided by M2-based vaccines through a mechanism of antibody-dependent cell-mediated cytotoxicity (ADCC). We investigated the ability of the human anti-M2 Ab1-10 monoclonal antibody (mAb) to activate human NK cells. They mediated ADCC against M2-expressing cells in the presence of Ab1-10 mAb. Furthermore, NK cell pro-inflammatory cytokine and chemokine secretion is also enhanced when Ab1-10 mAb is present. We also generated cytokine-preactivated NK cells and showed that they still displayed increased effector functions in the presence of Ab1-10 mAb. Thus, our study has demonstrated that human resting and cytokine-preactivated NK cells may have a very important role in the protection provided by anti-M2 Abs.</p></div

The anti-M2e Ab1-10 mAb induces cytokine-preactivated NK cell-mediated ADCC.

<p>293FT or M2-293FT cells were used as targets and control (cultured with low concentrations of IL-15) NK cells (A) and cytokine-preactivated (pretreated with IL-12, IL-15 and IL-18) NK cells (B) were used as effector cells in an ADCC assay. Effector and target cells were co-incubated at different ratios either in the presence or absence of Ab1-10 mAb (Ab). The percentage of specific lysis is shown. Error bars represent the SEM from four independent experiments with NK cells from four donors.</p

Cytokine release by freshly isolated NK cells in the presence of Ab1-10 mAb.

<p>Freshly isolated NK cells were either left untreated or co-cultured with both 293FT cells and M2-293FT cells in the presence and absence of Ab1-10 mAb (Ab). Culture supernatants were harvested and tested for the secretion of human cytokines and chemokines using flow-cytometric bead analysis. The values on the y-axis correspond to the concentrations of TNF-α, IFN-γ, MIP-1α, MIP-1β and RANTES in pg/ml. Graph bars represent the average ± SEM. Data shown are from five independent experiments with freshly isolated NK cells from five donors (* P<0.05, ** P<0.01).</p

Human Ab1-10 mAb recognizes M2e on animal cells.

<p>(A) 293FT cells (left) and M2-293FT cells (right) were cell surface stained with the human anti-M2 Ab1-10 mAb (upper panel) and the mouse anti-M2 14C2 mAb (lower panel) and analyzed by flow cytometry. Shaded histograms represent unstained cells. The binding of the isotype controls (dotted line) and anti-M2e specific mAbs (black line) is shown. (B) Uninfected A549 (left) and influenza infected (right) A549-H1N1 cells were cell surface stained with the human anti-M2 Ab1-10 mAb. The binding of secondary Ab alone (grey histogram) and the binding of Ab1-10 mAb (black line histogram) is shown.</p

The anti-M2e Ab1-10 mAb induces freshly isolated NK cell-mediated ADCC.

<p>(A) Effectors (freshly isolated NK cells) and targets (293FT and M2-293FT cells) were co-incubated at different ratios either in the presence or absence of Ab1-10 mAb (Ab). The percentage of specific lysis is shown. Error bars represent the standard error of the mean (SEM) from five independent experiments with freshly isolated NK cells from five donors (* P<0.05, ** P<0.01). (B) Influenza infected A549 cells were co-cultured with human NK cells from healthy donors in the absence or presence of Ab1-10 mAb (Ab). The percentage of lysis is shown. Error bars represent the SEM from four independent experiments with freshly isolated NK cells from four donors (* P<0.05).</p