Comprehensive antigen screening identifies Moraxella catarrhalis proteins that induce protection in a mouse pulmonary clearance model

Abstract

Moraxella catarrhalis is one of the three most common causative bacterial pathogens of otitis media, however no effective vaccine against M. catarrhalis has been developed so far. To identify M. catarrhalis vaccine candidate antigens, we used carefully selected sera from children with otitis media and healthy individuals to screen small-fragment genomic libraries that are expressed to display frame-selected peptides on a bacterial cell surface. This ANTIGENome technology led to the identification of 214 antigens, 23 of which were selected by in vitro or in vivo studies for additional characterization. Eight of the 23 candidates were tested in a Moraxella mouse pulmonary clearance model, and 3 of these antigens induced significantly faster bacterial clearance compared to adjuvant or to the previously characterized antigen OmpCD. The most significant protection data were obtained with the antigen MCR_1416 (Msp22), which was further investigated for its biological function by in vitro studies suggesting that Msp22 is a heme binding protein. This study comprises one of the most exhaustive studies to identify potential vaccine candidate antigens against the bacterial pathogen M. catarrhalis.

Figure 1. Structural features of 8 potential M. catarrhalis vaccine candidates.

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.

Figure 2. Pulmonary clearance of M. catarrhalis RH4 after intranasal challenge following intranasal immunization with 8 selected antigens.

Pulmonary clearance 6 hours after intranasal challenge with ∼5×10⁶ CFU M. catarrhalis, 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).

Figure 3. Detection of recombinant MCR_1416 (Msp22) expressed and purified from M. catarrhalis.

Equal volumes of eluates obtained from IMAC columns from extracts of M. catarrhalis 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).

Figure 4. Msp22 shows heme-dependent peroxidase activity.

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 M. catarrhalis BBH18; wt c*, wild type M. catarrhalis 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.