Plain PPS-1 booster abrogates the Pnc1-TT-induced GC reaction in mice primed as neonates.

<p>(A–C) Mean number of follicles and (D) ratio of GC/follicle per section in consecutive sections from spleen of mice that received booster by either s.c. (left columns) or i.n. (right columns) route with saline (open columns), PPS-1+LT-K63 (black columns), Pnc1-TT+LT-K63 (grey columns) and unvaccinated controls (stribe columns), as indicated. Spleens were removed 7 days after booster in mice primed as neonates with Pnc1-TT+LT-K63 by the same route as the booster. Half of the spleen was snap frozen, serial cryosection prepared, cutting into 7 µm sections at four levels, starting 700 µm into the tissue and the levels separated by 210 µm. Section from all 4 levels were stained with (A) anti-IgM, (B) anti-IgG, (C) PNA, and results (mean±SD) shown for each group. (D) Mean ratio of GC/follicle was calculated for every spleen at all 4 levels for individual mice and results (mean±SD) shown for each group. Statistical difference between vaccinated groups is stated in the graphs. Results in A–D are from one representative of two independent experiments (8 mice per group) showing comparable results.</p

Plain PPS-1 booster s.c. abrogates the Pnc1-TT-induced GC reaction in mice primed as neonates.

<p>Active germinal centers in spleen sections were enumerated with PNA staining (upper panels). Double fluorescent staining was performed with PNA and MOMA-1 (metallophilic marginal macrophages) to show the follicular structure (top panel). IgM⁺ and IgG⁺ follicles were identified with anti-IgM (middle panel) and anti-IgG (lower panel) staining, 7 days after booster with 5.0 µg PPS-1 and 5.0 µg LT-K63 s.c. (left) or i.n. (right). Spleen sections, 7 µm, were prepared from four different levels in the spleen, starting 700 µm into the tissue and each level separated by 210 µm. One representative section per group is shown. Results are from one representative of two independent experiments (8 mice per group) showing comparable results.</p

Repeated plain PPS-1 boosters i.n. reduce PPS-1-specific long-lived plasma cell pool in the bone marrow.

<p>PPS-1-specific IgG⁺ AbSCs, shown as number of spots (mean±SD) per 10⁶ cells, in spleen (A) and bone marrow (B) measured by ELISPOT, and PPS-1-IgG Abs (mean EU/ml±SD) in serum (C) measured by ELISA, at day 7, 23 and 39 after mice receiving i.n. booster with saline, PPS-1+LT-K63 or unvaccinated controls. Statistical difference between test groups and unvaccinated controls are indicated as; * P<0.05; ** P≤0.001, and the difference between vaccinated groups is stated in the graphs. The results are shown for one of two independent experiments (eight mice/group for each time point), showing comparable results.</p

PPS-1 booster depletes Pnc1-TT-induced memory cells in spleen preventing homing of PPS-1-specific AbSCs to BM.

<p>PPS-1-specific (A–B) and TT-specific (D–E) IgG⁺ AbSCs, shown as number of spots (mean±SD) per 10⁶ cells, in spleen (A and D) and bone marrow (B and E) measured by ELISPOT, and PPS-1- (C) and TT-specific (F) IgG Abs (mean EU/ml±SD) in serum measured by ELISA, at day 7, 23 and 39 after booster with saline (open squares), PPS-1+LT-K63 (filled squares) or unvaccinated controls (open circles). The results shown are from one of two independent experiments (eight mice/group for each time point) showing comparable results.</p

A single PPS-1 booster s.c. is sufficient to completely deplete PPS-1-specific-memory established by neonatal Pnc1-TT-priming.

<p>PPS-1-specific IgG⁺ AbSCs, shown as number of spots (mean±SD) per 10⁶ cells, in spleen (A) and bone marrow (B) measured by ELISPOT, and PPS-1-specific IgG Abs (mean EU/ml±SD) in serum (C) measured by ELISA, at day 7, 23 and 39 after mice receiving s.c. booster with saline, PPS-1+LT-K63 or unvaccinated controls. Statistical difference between test groups and unvaccinated controls are indicated as; * P<0.05; ** P≤0.001, and the difference between vaccinated groups is stated in the graphs. The results are shown for one of two independent experiments (eight mice/group for each time point), showing comparable results.</p

PPS-1 booster abrogation of Pnc1-TT-induced GCs reduces levels, avidity and protective efficacy of PPS-1-specific IgG.

<p>PPS-1-specific IgG levels (mean EU/ml±SD) in serum (A) and their avidity index (mean AI±SD) (B) measured by ELISA −2 days before and 7, 23 and 39 days after s.c. (left panels) or i.n. (right panels) booster with saline (open squares), PPS-1+LT-K63 (filled squares) in mice primed with Pnc1-TT as neonates or unvaccinated controls (open circles). Six weeks after the booster the mice were challenged with live <i>S. pneumoniae</i> serotype 1 to assess protection against bactermia and lung lung infection <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0072588#pone.0072588-Jakobsen1" target="_blank">[26]</a>. Pneumococcal colony forming units (CFU/ml) in blood (C, left graph) and lungs (C, right graph) 24 h after challenge shown for each mouse (N = 8/group), the median for each group is indicated by a line. Statistical difference in bacteremia or lung infection between vaccinated groups is indicated in the graphs. The results shown are from one of two independent experiments (eight mice/group for each time point) showing comparable results.</p

Subcutaneous administration of PPS-1 booster depletes Pnc1-TT-induced PPS-1-specific AbSC pool in the spleen.

<p>PPS-1 (upper panels) and TT (lower panels) specific IgG⁺ AbSC measured by ELISPOT in spleen (A and B) and bone marrow (C and D), shown as number of spots (mean±SD) per 10⁶ spleen cells and PPS-1- and TT-specific IgG levels (mean EU/ml±SD) in serum measured by ELISA (E and F). Results are shown for mice boosted by either i.n. or s.c. route with saline (open columns), PPS-1+LT-K63 (black columns), Pnc1-TT+LT-K63 (grey columns) and unvaccinated controls (stribe columns), as indicated. Statistical difference between vaccinated groups is indicated in the graphs. All vaccinated mice had significantly higher frequency of PPS-1 -specific IgG⁺ AbSCs in spleen and BM and also higher serum IgG anti-PPS-1 levels compared to unvaccinated controls (B–F), except mice that received PPS-1 booster s.c. which had comparable frequency of PPS-1-specific IgG⁺ AbSCs in spleen (A). The results shown in A–F are from one of two independent experiments showing comparable results (8 mice per group in each experiment).</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

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