Examination of putative outer membrane proteins in Moraxella catarrhalis: A genome mining approach for the identification of potential vaccine antigens
Ruckdeschel, Elizabeth Anne
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Chronic obstructive pulmonary disease (COPD) is the fourth leading cause of death in the United States and affects 24 million Americans. Moraxella catarrhalis is an important cause of respiratory infections in COPD. This organism is also a common cause of otitis media in children. Developing an effective vaccine to M. catarrhalis would reduce the morbidity, mortality, and financial costs associated with COPD and otitis media. Surface proteins from M. catarrhalis are attractive vaccine antigens. An ideal vaccine candidate has several characteristics including surface exposure, conservation among strains, expression during infection, and generation of a protective immune response. This thesis examined the overall hypothesis that conserved outer membrane proteins (OMPs) from Moraxella catarrhalis could act as vaccine antigens. Specifically the following hypotheses were examined (1) selected genes that encode surface proteins are differentially regulated during growth in a human simulated environment; (2) these antigens induce an antibody response in humans during infection; and (3) these OMPs generate a potentially protective immune response with antibodies directed at surface exposed epitopes. An unfinished genome sequence of a strain of M. catarrhalis available in GenBank was analyzed and open reading frames predicted to encode potential vaccine candidates were identified. Three genes, encoding proteins of approximately 22kDa, 75kDa, and 78kDa (named M oraxella s urface p roteins (Msp) msp22 , msp75 , and msp78 , respectively), were identified to be conserved by competitive hybridization using a microarray, polymerase chain reaction (PCR), and sequencing the genes in clinical isolates of M. catarrhalis. The genes were transcribed, translated, and expressed when M. catarrhalis was grown in vitro . To assess antibody responses in humans, these genes were first amplified by PCR and cloned into E. coli expression vectors. Recombinant proteins were generated and then studied in enzyme-linked immunosorbent assays (ELISAs) with pre-acquisition and post-clearance serum and sputum samples from 31 adults who had a known M. catarrhalis infection. New antibody responses to the three proteins were generated by a small proportion of patients with COPD indicating that these proteins were expressed during human infection. To examine if antibodies are directed at surface epitopes, immunized animals were tested in a mouse pulmonary clearance model using two routes of administration: subcutaneous and intranasal. Quantitative enzyme-linked immunosorbent assays were performed on sera and bronchoalveolar lavage (BAL) fluids from the immunized mice to characterize systemic and mucosal antibody responses. The antibodies, induced by immunization with recombinant proteins, were analyzed by flow cytometry. This method detected antibodies that bound to the bacterial surface of multiple M. catarrhalis strains. To evaluate the ability of these proteins to generate a protective effect after immunization, mice were challenged using a pulmonary clearance model. Mice immunized with recombinant Msp22 and Msp75 showed enhanced clearance of M. catarrhalis as compared to control mice. This observation suggests that immunization with either of these two proteins generates an immune response that mediates bacterial clearance from the lungs. Collectively the conservation among strains, immunogenicity, localization to the outer membrane, and the ability to enhance clearance in vivo indicate that two of the three proteins examined, Msp22 and Msp75, may make good vaccine antigens. These proteins should be studied further to determine if they are capable of inducing a protective response following immunization in humans.