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

Identification of B lymphocytes specific for HA from A and B influenza strains in <i>ex vivo</i> PBMCs samples.

<p>PBMCs from different anonymous blood donors were pre-incubated with vaccine mono-bulk subunits from the B/Brisbane/60/2008 (B/HA pretreatment), or the H3N2 A/Panama/2007/1999 strain (A/HA pretreatment) and then stained HSA, rH3 (from A/Brisbane/10/2007), rH1 (from A/California/07/2009), or B/HA (from B/Brisbane/60/2008), as indicated. <b>A.</b> Staining pattern observed on CD20⁺ cells in PBMCs stained with the different rHA bait. The rectangular gates identify brilliant HA+ B-cells; the dotted vertical lines mark the gates used to sort HA⁺ B-cells for the ELISPOT assays. <b>B.</b> Expression of the CD27 memory marker on HA⁺ and HA^neg B cells identified based on the sorting gates. <b>C.</b> H3⁺ (n = 15,234), H1⁺ (n = 6482) and B/HA⁺ (n = 26,803) B-cells identified in A were sorted, mixed with autologous CD20^neg cells (in the ratio of 1∶20, 1∶100 and 1∶33) and activated with CpG and IL-2 for 5 days <i>in vitro</i>. Unsorted PBMCs and CD20^neg cells mixed with HA^neg B cells were also cultured in the same manner, as controls. After 5 days cultured cells were harvested and assayed by ELISPOT for the number of cells secreting IgG and IgG specific for mono-bulk subunits from the vaccine strain homologous to the sorting bait. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0070620#s2" target="_blank">Results</a> are expressed as numbers of antibody secreting cells (ASC) normalized to 10⁶ cultured cells assayed by ELISPOT. Nd indicates undetectable ASC.</p

Molecular cloning of HA⁺ B lymphocytes.

<p>PBMCs from 4 anonymous blood bank donors were stained as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0070620#pone-0070620-g004" target="_blank">Figure 4B</a>. Single H1⁺, H3⁺, or H1^negH3^neg CD20⁺ B-cells were sorted to perform molecular cloning and analysis of their paired V_HV_L Ig regions as described in Material and Methods section. <b>A–E.</b> Distribution of V_H (A), D_H (B), J_H (C), V_k (D) and J_k (E) gene use across arrays of B-cells sorted from each donor (16 and 18 HA⁺ clones from donors #1 and #2; 35 HA⁺ and 20 HA^neg clones from donor 3; 16 HA⁺ and 16 HA^neg clones from donor #4. <b>F–I.</b> Number of mutations in H1⁺ and H1^neg CD20⁺ B-cells from donors #3 and #4, which cause dissimilar (F, H) or similar (G, I) amino acid substitutions in V_H (G,I) and V_L (F,H). NS and ** indicate not significant, or significant (p<0.036) difference between mean numbers of mutations by one-way Wilcoxon non-parametric test.</p

Vaccination induced changes in the pool of H1+ B-cells.

<p>PBMCs samples collected before (day 0) and at 3 and 6 weeks after vaccination from four seasonal influenza vaccinees, were pre-incubated with H3N2 subunit (from A/Panama/2007/1999) and then stained with rH1 A/Solomon Island/3/06) and mAbs anti-CD20, anti-CD27 and anti-human IgG. <b>A.</b> Dot plots gated on CD20⁺ B-cells showing the distribution of H1⁺ (middle panels) and H1^neg (bottom panels) B-cells from donor #a across: the mature memory (CD27⁺) and putatively naive (CD27^neg) CD20+ B-cell subsets (upper panels); un-switched mature memory (CD27⁺IgG^neg), IgG-switched mature (CD27⁺IgG⁺) and immature (CD27^negIgG⁺) memory B-cells. <b>B.</b> Numbers of circulating H1⁺ CD20⁺ B-cells in 4 vaccinees before and at 3 and 6 weeks after seasonal vaccination are overlaid with paired titers of antibodies inhibiting virus-induced hemmaglutination measured in their blood. The frequencies of H1⁺ B-cells are normalized according to the frequencies of CD20⁺ B-cells in 10⁶ PBMCs. <b>C.</b> Distribution of circulating H1⁺ and H1^neg B-cells across same subsets identified in <b>B</b> in all vaccinees.</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

Magnitude and quality of vaccine-specific CD4⁺ T-cell responses induced by 4C-Staph/T7-alum and 4C-Staph/alum.

<p>Splenocytes from single mice (n = 16) vaccinated with 4C-Staph/T7-alum, 4C-Staph-alum, T7-alum or alum by 12 days were stimulated or not with vaccine antigens <i>in vitro</i>, stained and analyzed by intracellular cytokine staining. CD4⁺CD44^high T cells producing IL-2, TNF, IL-4/IL-13, IFN-γ or IL-17A were identified (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0147767#pone.0147767.s001" target="_blank">S1 Fig</a> for gating strategy). The response of unstimulated cells was subtracted from that of stimulated cells. Data are the merge of four independent experiments. (<b>A</b>) Percentages of CD4⁺CD44^high T cells producing any combination of IL-2, TNF, IL-4/IL-13, IFN-γ or IL-17A in response to vaccine protein stimulation (CD4⁺CD44^high total CYT⁺ cells) were calculated applying Boolean gates. Bars represent mean ± SEM. *<i>p</i> < 0.05, **<i>p</i> < 0.01, ***<i>p</i> < 0.001 by one-way ANOVA and Sidak post-test. (<b>B</b>) Percentages of CD4⁺CD44^high T cells producing IL-2, TNF, IL-4/IL-13, IFN-γ and/or IL-17A in each of the possible combinations (CD4⁺CD44^high CYT⁺) in response to vaccine proteins stimulation calculated applying Boolean gates. No cells expressing more than 3 cytokines at once were detected. Bars represent mean ± SEM. *<i>p</i> < 0.05 by unpaired Student <i>t</i> test, two-tailed, and a partial permutation test.</p

One Dose of <i>Staphylococcus aureus</i> 4C-Staph Vaccine Formulated with a Novel TLR7-Dependent Adjuvant Rapidly Protects Mice through Antibodies, Effector CD4⁺ T Cells, and IL-17A

<div><p>A rapidly acting, single dose vaccine against <i>Staphylococcus aureus</i> would be highly beneficial for patients scheduled for major surgeries or in intensive care units. Here we show that one immunization with a multicomponent <i>S</i>. <i>aureus</i> candidate vaccine, 4C-Staph, formulated with a novel TLR7-dependent adjuvant, T7-alum, readily protected mice from death and from bacterial dissemination, both in kidney abscess and peritonitis models, outperforming alum-formulated vaccine. This increased efficacy was paralleled by higher vaccine-specific and α-hemolysin-neutralizing antibody titers and Th1/Th17 cell responses. Antibodies played a crucial protective role, as shown by the lack of protection of 4C-Staph/T7-alum vaccine in B-cell-deficient mice and by serum transfer experiments. Depletion of effector CD4⁺ T cells not only reduced survival but also increased <i>S</i>. <i>aureus</i> load in kidneys of mice immunized with 4C-Staph/T7-alum. The role of IL-17A in the control of bacterial dissemination in 4C-Staph/T7-alum vaccinated mice was indicated by <i>in vivo</i> neutralization experiments. We conclude that single dose 4C-Staph/T7-alum vaccine promptly and efficiently protected mice against <i>S</i>. <i>aureus</i> through the combined actions of antibodies, CD4⁺ effector T cells, and IL-17A. These data suggest that inclusion of an adjuvant that induces not only fast antibody responses but also IL-17-producing cell-mediated effector responses could efficaciously protect patients scheduled for major surgeries or in intensive care units.</p></div

One dose of 4C-Staph/T7-alum induces functional antibodies.

<p>BALB/c mice (n = 16) were immunized once with 4C-Staph/T7-alum or 4C-Staph/alum. Control mice were injected with T7-alum or alum alone. (<b>A</b>) Vaccine-specific serum IgG titers measured 12 (d12) and 32 (d32) days after vaccination. IgG concentrations in control sera (open symbols) are reported only for d32. Each symbol represents one mouse, and data are the merge of two independent experiments. Median with interquartile range of each group is also shown. *<i>p</i> < 0.05, ****<i>p</i> < 0.0001 by Kruskal-Wallis test and Dunn's multiple comparisons test. (<b>B</b>) Hla neutralizing activity of pooled sera from vaccinated mice (n = 16, same animals as in A) was assessed on rabbit RBCs and expressed as effective dilution that neutralized 50% of Hla lytic activity (ED₅₀). No hemolysis inhibition was detected (ED₅₀ < 6) in pre-immune sera or in sera from adjuvant-treated mice. Lack of overlap in the 95% confidence intervals between the ED₅₀ of sera from mice vaccinated with 4C-Staph/alum (30.3 to 51.4) vs. 4C-Staph/T7-alum (114.6 to 318.7) by 32 days indicates a difference significant with <i>p</i> < 0.05. Bars represent SEM. (<b>C</b>) Vaccine-specific IgG1 and IgG2a. Columns represent median MFI with interquartile range of pooled sera from vaccinated mice (n = 16, same pools as in B) bled at d32. **<i>p</i> < 0.01, ***<i>p</i> < 0.001 by unpaired Student <i>t</i> test, two-tailed. (<b>D</b>) Hla_H35L-specific IgM. Columns represent median MFI with interquartile range of sera from vaccinated mice (n = 12) bled at d12. IgM specific for EsxAB, FhuD2 and Csa1A were at the limit of detection (data not shown). Data shown are the merge of two independent experiments.</p

One dose of 4C-Staph/T7-alum induces protective antibodies.

<p>Sera from mice immunized with 4C-Staph/T7-alum (immune serum), or T7-alum as negative control (control serum), by 32 days were pooled and injected i.v. (150 μl/mouse) in naïve BALB/c mice (n = 16) 24 h before i.p. challenge with <i>S</i>. <i>aureus</i>. <b>(A</b>) Survival was monitored for 15 days post challenge. Data are the merge of two independent experiments. ***<i>p</i> < 0.001 by Log-rank test. <b>(B</b>) Fifteen days after <i>S</i>. <i>aureus</i> infection, survivors were euthanized, both kidneys were homogenized and CFU enumerated. Each symbol represents one mouse. <b>(C</b>) B cell/antibody-deficient J_H mice, or BALB/c (wt) as control, were immunized with 4C-Staph/T7-alum or T7-alum alone. Twelve days after vaccination, mice (n = 25 for 4C-Staph/T7-alum; n = 15 for T7-alum) were challenged i.p. with <i>S</i>. <i>aureus</i> and their survival was monitored for 15 days. Data are the merge of four independent experiments. ***<i>p</i> < 0.001 by Log-rank test.</p

One dose of 4C-Staph/T7-alum vaccine protects better than 4C-Staph/alum in kidney abscess and peritonitis models of <i>S</i>. <i>aureus</i> infection.

<p>BALB/c mice were immunized once i.m. with 4C-Staph/T7-alum or 4C-Staph/alum. Control mice were injected with T7-alum or alum alone. After 12 days, mice were challenged with <i>S</i>. <i>aureus</i> Newman strain. (<b>A</b>) Kidney abscess model. Mice (n = 28–32) were injected i.v. with 2 x 10⁷ CFU. Four days later, both kidneys of each mouse were homogenized in pool and CFU enumerated. Each symbol represents one mouse, and data are the merge of three independent experiments. Mean ± SEM of each group are shown. The dotted line indicates the lower limit of detection (LLD). *<i>p</i> < 0.05, **<i>p</i> < 0.01 by one-way ANOVA and Sidak's multiple comparisons test. Number of survivors with non-detectable CFU (CFU ND) in kidneys/total number of survivors and corresponding percentages are reported above the graph. (<b>B-C</b>) Peritonitis model. Mice (n = 32) were injected i.p. with 5 x 10⁸ CFU. Survival was monitored for 30 days after challenge. Data are the merge of three independent experiments. ***<i>p</i> < 0.001 by Log-rank test. (<b>C</b>) Thirty days after <i>S</i>. <i>aureus</i> infection, survivors were euthanized, both kidneys were homogenized and CFU enumerated. Each symbol represents one mouse. Mean ± SEM of each group is shown. **<i>p</i> < 0.01 by unpaired Student <i>t</i> test, one-tailed. Number of survivors with CFU ND in kidneys/total number of survivors and corresponding percentages are reported above the graph.</p