Human sera selected for antigen identification by peptide library screening.

<p>Pool P39 consisted of individual sera collected from OM patients during the convalescent disease phase. Pool IC20 contained sera from healthy individuals. The additional 2 pools were used as controls for otitis media-unrelated antigen reactivity: Pool P37 (patients with asthma, age: 5–17 years). Pool P36 (patients with respiratory allergies, 8 months– 18 years of age).</p

Msp22 shows heme-dependent peroxidase activity.

<p>The specificity of the heme stain for Msp22 is demonstrated by staining of lysates from the wild type, and gene deletion mutant strains as well as the BBH18 strain transformed with pEMCJH04-KAN-Msp22. Hemoglobin (positive control), BSA (negative control). wt, wild type <i>M. catarrhalis</i> BBH18; wt c*, wild type <i>M. catarrhalis</i> BBH18 transformed with pEMCJH04-KAN-Msp22; msp22Δ, msp22 gene deletion mutant; msp22Δ c* msp22 gene deletion mutant transformed with pEMCJH04-KAN-Msp22. The position of Msp22 is marked with an arrow.</p

Structural features of 8 potential <i>M. catarrhalis</i> vaccine candidates.

<p>MCR_0076, TonB-dependent receptor; MCR_0196, MltB; lytic murein transglycosylase; MCR_0686, peptide methionine sulfoxide reductase MsrA/MsrB; MCR_0996, hypothetical protein; MCR_1003, LysM domain protein; MCR_1010, D-alanyl-D-alanine carboxypeptidase; MCR_1303, oligopeptide ABC transport system substrate binding protein; MCR_1416, cytochrome c class II, Msp22. SP, signal peptide; LP, signal peptide for lipidation; Plug, an independent folding subunit blocking the pore until the channel is bound by a ligand; PGBD1, peptidoglycan binding-like; MsrA, methionine sulfoxide reductase A; SelR, seleno protein R; LysM, lysine motif; SBP bac 5, bacterial extracellular solute-binding protein family 5. Light grey bars represent the recombinant protein (fragments). Thin black bars delineate epitope containing regions covered by clones selected by the ANTIGENome technology with human IgGs.</p

ELISA data for the 50 most reactive <i>M. catarrhalis</i> peptides – ELISA titers for individual sera.

<p>For legend see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0064422#pone-0064422-t002" target="_blank">Table 2</a>.</p

Detection of recombinant MCR_1416 (Msp22) expressed and purified from <i>M. catarrhalis</i>.

<p>Equal volumes of eluates obtained from IMAC columns from extracts of <i>M. catarrhalis</i> complemented with His-tagged MCR_1416 (eluate A) or wild type strain (not complemented, negative control) were separated by SDS-PAGE and immunoblotted using immune serum against recombinant Msp22 (left panel) and antibody against the His-tag (right panel).</p

ELISA data for the 50 most reactive <i>M. catarrhalis</i> peptides – Average ELISA titers for groups of sera.

<p>The peptides are named by the ORF followed by a number indicating the individual peptide for the respective ORF. Individual sera were obtained from asthma patients and healthy individuals and convalescent sera patients with otitis media (OM). Listed are the 50 peptides with highest average ELISA units of the 402 peptides analyzed. ELISA units were calculated as 1,000×[(A₄₀₅ wells with serum) – (A₄₀₅ wells with secondary antibody alone)]. The serum ELISA units were additionally corrected for the background reactivity of sera with streptavidin, by subtracting the values obtained with streptavidin coated wells in the absence of peptide from the values obtained in the wells containing bound peptides.</p

Pulmonary clearance of <i>M. catarrhalis</i> RH4 after intranasal challenge following intranasal immunization with 8 selected antigens.

<p>Pulmonary clearance 6 hours after intranasal challenge with ∼5×10⁶ CFU <i>M. catarrhalis</i>, in mice immunized with purified, IC31^® adjuvanted recombinant proteins, IC31^® adjuvant without proteins in PBS, or PBS without adjuvant. The mean values of the combined, normalized results from 2 to 6 independent experiments are shown. Error bars represent the standard error of the mean. (A) Bacterial CFU recovered from all experiments; (B) bacterial CFU recovered from experiments after exclusion of sterile lung cultures. Black bars: negative and positive controls (data from 6 experiments), grey bars: data from 2 to 3 independent experiments in which different antigens were tested. (C) ELISA measuring IgG levels to the respective recombinant proteins in serum from mice immunized intranasally with purified recombinant proteins as noted below the x-axis. For the controls (IC31^® alone or PBS), IgG levels were determined using a mix of all recombinant proteins. Endpoint titers were expressed as the last dilution that gave an absorbance of at least 0.1 at 405 nm. Median values with the interquartile range from 2 to 6 independent experiments using 10 sera (10 mice per group) per experiment are shown. **, statistically highly significant (P<0.01), *, statistically significant (P<0.05).</p