Additional file 7: of GOexpress: an R/Bioconductor package for the identification and visualisation of robust gene ontology signatures through supervised learning of gene expression data

Scoring table for GO terms produced by GOexpress using the ExpressionSet described in the paper (see Additional file 1). (XLSX 871 kb

Description of Circulating Clones.

*<p>Not detected.</p>**<p>Serotype switch.</p>***<p>Serotype switch.</p

Bar chart showing rank order and cumulative serotype distribution (%).

<p>Bar chart showing rank order and cumulative serotype distribution (%).</p

Genomic diversity of serotypes targeted by PCV13.

<p>Genomic diversity of serotypes targeted by PCV13.</p

Variation of IgG antigen targets between individuals and with age.

<p><b>(A-C)</b> Results for six Malawian subjects (labelled A to G). <b>(A)</b> whole cell ELISAs to 4 <i>S</i>. <i>pneumoniae</i> strains (serotype 4, 14, 9C and 1). <b>(B)</b> Immunoblots of IgG binding to the wild-type <i>S</i>. <i>pneumoniae</i> strain D39 (10 μg / lane, red boxes highlight bands that visibly vary in intensity between subjects). <b>(C)</b> MFI of IgG binding to selected protein antigens measured using a Luminex assay. Data points represent mean (SEMs) of two technical duplicates for IgG binding. For immunoblots and the Luminex assay, sera were diluted to 1/1000. <b>(D)</b> Levels of serum IgG binding to specific protein antigens aged (mean age 67.2 years, black symbols) and young subjects (mean age 31.2 years, empty symbols) measured by multiplex MSD (only results for antigens with stronger responses are shown as means and SEM). <b>(E-F)</b> Levels of serum IgG binding for each individual aged (black symbols) and young (empty symbols) adult subjects to the protein antigens PspC (E) and PcpA (F) measured by MSD. <i>P</i> values were calculated using unpaired T tests, with bars representing means for the group.</p

IVIG improves macrophage and neutrophil opsonophagocytosis of both encapsulated an unencapsulated <i>S</i>. <i>pneumoniae</i> TIGR4.

<p>(<b>A</b>) MFI and example of a histogram of the fluorescence of RAW macrophages incubated with fluorescent (FAM-SE labelled) encapsulated (clear columns) and unencapsulated (black columns) <i>S</i>. <i>pneumoniae</i> TIGR4 strains with and without addition of 10% IVIG. <i>P</i> values were calculated using unpaired 2-tailed Student t-test. <b>(B</b>) MFI of fluorescence intensity of human neutrophils following incubation with FAM-SE labelled <i>S</i>. <i>pneumoniae</i> TIGR4 encapsulated (clear columns) and unencapsulated (filled columns) strains pre-opsonised with increasing concentrations of IVIG. <i>P</i> values were calculated using one-way ANOVAs. (<b>C</b>) Relative survival of <i>S</i>. <i>pneumoniae</i> TIGR4 encapsulated (+cps) and unencapsulated (-cps) strains following incubation with human neutrophils in the presence of 10% IVIG. Percentage survival calculated relative to survival of identical strain incubated without IVIG, with the surviving CFU / ml shown for control versus 10% IVIG above each column. For all panels, data are presented as means and SDs of three to four technical replicates and are representative of experiments repeated at least twice. <i>P</i> values were calculated using unpaired 2-tailed Student t-test (<b>A</b> and <b>C</b>) or one way ANOVAs (<b>B</b>).</p

Passive vaccination with IVIG protects against IPD in murine models of <i>S</i>. <i>pneumoniae</i> infection.

<p>Mice were passively vaccinated by i.p. administration of 12.8 mg of IVIG (Intratect) or PBS 3 hours before challenge with <i>S</i>. <i>pneumoniae</i>. (<b>A</b>) and <b>(B)</b> Concentration of human IgG measured by ELISA after <b>(A)</b> in mouse sera 3 h post-IVIG (n = 4), and (<b>B</b>) in mouse BALF recovered from mice 3 h post-IVIG and immediately before and at 2.5 and 24 h after i.n. challenge with 10⁷ CFU of TIGR4 strain <i>S</i>. <i>pneumoniae</i> (n = 4 to 6). <b>(C)</b> Correlation between concentration of murine albumin (mg/ml) and human IgG (μg/ml) in BALF 24 hr following invasive i.n. challenge in IVIG-treated mice (n = 6); <i>P</i> and r² values were calculated using the F test. <b>(D)</b> Bacterial CFU (log₁₀) recovered from BALF 2.5 hr following IN challenge with 5x10⁵ CFU of TIGR4 of IVIG-treated or PBS-treated control mice (n = 6 or 7). <b>(E)</b> Bacterial CFU (log₁₀) recovered from BALF, lung tissue or blood 24 hr following IN challenge with 10⁷ CFU of TIGR4 of IVIG-treated or PBS-treated control mice (n = 6). <b>(F)</b> Bacterial CFU (log₁₀) recovered from blood 4 hr following i.v. challenge with 5x10⁵ CFU of TIGR4 of IVIG-treated or PBS-treated mice (n = 5). <b>(G)</b> Effect of administration of i.v. 100 μl liposomal clodronate (5mg/ml) to mice on the numbers of F4/80+ve splenocytes measured by flow cytometry (n = 6), with data presented as means, error bars represent SDs, and <i>P</i> values were calculated using unpaired 2-tail Student t-test. <b>(H)</b> Effect of clodronate or PBS administration on bacterial CFU (log₁₀) recovered from the blood of IVIG-treated mice 4 hr following i.v. challenge with 5x10⁵ CFU of TIGR4 (n = 5). (<b>I)</b> Bacterial CFU (log₁₀) recovered from the lungs of neutrophil depleted mice (by prior treatment with the antiLy6 antibody 1A8) given either IVIG or PBS 24 hr and then inoculated i.n. with 5x10⁶ CFU of TIGR4 (n = 11 or 12). For <b>(A, B, D, E, F, H, I)</b>, symbols represent data from individual mice, bars represent group means, and <i>P</i> values were calculated using unpaired 2-tail Student t-test. Dashed lines represent the limit of detection.</p

Identification of protein antigens recognised by different sources of pooled IgG.

<p>(<b>A</b>) Immunoblots of IgG (1/1000) binding to the wild-type <i>S</i>. <i>pneumoniae</i> strain D39 and isogenic mutant strains (10 μg) lacking specific surface proteins using IVIG. Boxes highlight missing bands corresponding to the molecular weight for the protein(s) absent in the mutant strains. (<b>B</b>) Immunoblots of IgG binding to selected recombinant <i>S</i>. <i>pneumoniae</i> proteins (0.5 μg / lane) probed with IVIG (1/500). (<b>C</b>) Immunoblots of IgG binding to wild-type <i>S</i>. <i>pneumoniae</i> D39 strain (10 μg / lane) using pooled sera from different geographical regions, commercial IVIG (1/3300) from Europe (Intratect) or USA (Vigam) and from Malawi. (<b>D</b>) Linear regression of the rank order of strength of IgG binding to different protein antigens between serum pooled from donors from Malawi and IVIG preparations obtained from the USA (Vigam) or Europe (Intratect). Data points represent the rank order of each protein antigen for the mean MFI of two technical duplicates for IgG binding measured using the Luminex assay. <i>P</i> and R² values were obtained using F tests.</p

Absorption of anti-capsular antibody does not prevent IVIG protection against murine models of IPD.

<p><b>(A)</b> Mean (SD) anti-PsaA titres measured by ELISA in IVIG following depletion of type 4 specific IgG by pre-absorption with type 4 capsule expressing <i>S</i>. <i>mitis</i>. (<b>B</b>) Mean (SD) anti-capsular serotype 4 titre IgG titre measured by ELISA in type 4 specific IgG-depleted IVIG. (<b>B</b>) Mean (SD) MFI of anti-human IgG binding to wild-type (clear columns) and unencapsulated (black columns) <i>S</i>. <i>pneumoniae</i> TIGR4 following incubation in 1% or 10% in type 4 specific IgG-depleted IVIG. (<b>C</b>) Bacterial surface IgG binding measured by flow cytometry to <i>S</i>. <i>pneumoniae</i> TIGR4 incubated in PBS (grey column), 1% or 10% mock absorbed IVIG with (filled columns) or type 4 specific IgG-depleted IVIG (open columns). For panels (A) to (C) data are presented as means and SDs of three to four technical replicates and representative of experiments repeated at least twice. (<b>D</b>) Log₁₀ CFU recovered from BALF, lungs, and blood 24 h after i.n. challenge with 1x10⁷ CFU <i>S</i>. <i>pneumoniae</i> TIGR4 for mice pre-treated with 12.8 mg of type 4 specific IgG-depleted IVIG or PBS. (<b>E</b>) Log₁₀ CFU recovered from blood 4 h after i.v. challenge with 5x10⁵ CFU <i>S</i>. <i>pneumoniae</i> TIGR4 for mice pre-treated with PBS (open circles), or 12.8 mg of mock absorbed IVIG (black circles) or type 4 specific IgG-depleted IVIG (grey circles). Data for panels <b>(D)</b> and <b>(E)</b> were obtained using IVIG depleted of capsular antibody on separate occasions; symbols represent data from individual mice, bars represent group means, and <i>P</i> values were calculated using unpaired 2-tail Student t-test <b>(D)</b> or one way ANOVA <b>(E).</b> Dashed lines represent the limit of detection.</p

eIVIG immune recognition of <i>S</i>. <i>pneumoniae</i>.

<p><b>(A-D)</b> Whole cell ELISAs of IgG binding to solid-phase <i>S</i>. <i>pneumoniae</i> of increasing concentrations of eIVIG made using TIGR4 <b>(A and B)</b> or D39 <b>(C</b> and <b>D)</b> or IVIG. Binding was assessed to the solid phase strain that was homologous (<b>A</b> and <b>D</b>) or heterologous (<b>B</b> and <b>C</b>) to the enrichment strain. (<b>E-F</b>) Bacterial surface IgG binding measured by flow cytometry to <i>S</i>. <i>pneumoniae</i> TIGR4 <b>(E)</b> or D39 <b>(F)</b> incubated in either PBS (shaded area) or TIGR4 eIVIG at 30 μg/ml (solid line). <i>P</i> values were calculated using linear regression for panels A, B, C, and D, and unpaired 2-tailed Student t-tests for panels E and F.</p