age 184 Average 139 Average 123 Average 151 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. Listed are the 50 peptides with highest average ELISA units of the 402 peptides analyzed. ELISA units were calculated as 1,0006. 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. doi:10.1371/journal.pone.0064422.t002 membrane, outer membrane vesicles and outer membrane vesicles isolated from cultures grown in iron-depleted medium. Three candidates were found in the whole membrane and in outer membrane vesicles, and seven further candidates were detected in one of the three membrane preparations. than PD173074 antibody responses may contribute to protection against M. catarrhalis. Systemic human antibody responses against the selected antigens are not induced upon infection In order to evaluate the human immune response for the 8 selected recombinant antigens upon natural infection, additional serological studies were performed with ELISA and Luminex xMAPH technology, using a collection of 164 individual sera from children with otitis media collected during the acute and convalescent disease phase. Sera from healthy individuals were tested in parallel in order to compare antigen specific responses between healthy adults and children with otitis media. We detected antibodies against all eight antigens in the 20 paired acute/convalescent serum samples from children with otitis media, however IgG end titers were relatively low and no significant antigen specific seroconversion was detected in any of the donors. We also examined the median antibody titers between healthy donors and otitis media patients, however no statistically significant difference was seen. Moreover, we detected a decrease in median systemic IgG titers against the antigen MCR_1303 in convalescent sera compared to acute sera. These results are in agreement with the peptide ELISA data, as no increase in antibody titer was 22430212 detected for these antigens in sera from otitis media patients during an OM episode when the paired serum samples were collected. Three candidate vaccine antigens demonstrated protection in vivo Of the 23 candidates selected by the ANTIGENome technology, we evaluated 8 well conserved and readily recombinant expressed antigens that had shown some promise in a preliminary mouse study in more detail for their potential to elicit protective immune response in vivo. The rate of M. catarrhalis 23321512 clearance from mouse lungs in response to immunization with recombinant antigens was assessed using a mouse pulmonary clearance model. Mice were immunized intranasally 3 times at 3 week intervals and challenged intranasally with 40 mL of approximately 56106 live M. catarrhalis RH4 3 weeks after the last boost. Bacterial CFU were determined in lungs 6 hours post infection and systemic antibody titers after vaccination of mice were determined by ELISA. Groups of mice immunized with recombinant proteins MCR_1416, MCR_1303, MCR_0076-1, MCR_1010, MCR_0196, MCR_1003-1, MCR_0996 and MCR_0686 expressed in E. coli showed a greater or comparable clearance of bacteria from lungs compared to the positive c
