Acute otitis media (AOM) is a middle ear infection, which is the most common ailment in infants and young children. In the United States, three bacteria (commensal organisms in the nasopharynx) are the most common causes of AOM: Streptococcus pneumoniae, Moraxella catarrhalis, and nontypeable Haemophilus influenzae (NTHi). This study focuses on NTHi, which also causes chronic obstructive pulmonary disease (COPD), sinusitis, and other respiratory illnesses. OMP26 and Protein D are two leading candidates for a protein vaccine to prevent NTHi infections. Scientists have evaluated both of these NTHi proteins, with promising results. However, results from our preliminary study in mice showed that OMP26 and Protein D, when administered as a single vaccine formulation, leads to suppression of Protein D antibodies. We hypothesize that OMP26 physically interacts with Protein D, which results in the antibody suppression, but alternative mechanisms are also being explored. The aim of this work was to elucidate the inter‐protein interactions between OMP26 and Protein D, with the goal of understanding how and why Protein D antibody suppression occurs in mice. To study these interactions, we have begun to employ several biochemical and biophysical methods, including co‐immunoprecipitation experiments, size exclusion chromatography, and x‐ray crystallography.
Protein D is a leading vaccine candidate for nontypeable Haemophilus influenzae (NTHi), a Gram‐negative bacterium causing both lower and upper respiratory illnesses, such as acute otitis media (AOM), also known as an ear infection. We recently discovered that when Protein D is mixed with outer membrane protein 26 (OMP26), another leading vaccine candidate for NTHi, mice fail to produce antibodies to Protein D. Toward understanding the mechanism of antibody suppression, we performed co‐immunoprecipitation and protein‐protein interaction studies, as well as in vivo mouse experiments. Preliminary results suggest a direct interaction between Protein D and OMP26. We propose that OMP26 interacts with Protein D and either prevents its interaction with host immune cells or alters its conformation and/or epitopes. Further biochemical and biophysical structure studies are proposed to determine how and why Protein D antibody suppression occurs to inform the creation of a multivalent protein‐based vaccine (PBV) for NTHi.
Two leading protein vaccine candidates from Nontypeable Haemophilus Influenzae (NTHi) are outer membrane proteins Omp26 and Protein D. When administered individually, Omp26 and Protein D elicit an antibody response in mice; when combined, the Protein D antibody response is significantly suppressed. Here, we describe a biochemical approach toward elucidating the interactions between Omp26 and Protein D, which we propose contribute to the suppression of Protein D‐specific antibody production. Preliminary data from enzyme‐linked immunosorbent assays and other standard protein detection methods suggest that Protein D binds strongly to Omp26 in vitro and perhaps in vivo in mice.
Peptidoglycan associated lipoprotein (Pal) is an outer membrane protein from Gram‐negative bacteria, including Escherichia coli (E. coli). E. coli has two subpopulations of Pal: a periplasmic subpopulation bound to peptidoglycan and a surface exposed subpopulation. When the bacteria are cultured under normal conditions in rich media, only a small percentage of cells contain surface exposed Pal. Under more stressful culture conditions (ex. increased temperature, the presence of antibiotics), E. coli will release outer membrane vesicles (OMVs). Using ultracentrifugation and immunoblotting, we have shown that Pal is released from E. coli in these OMV's. We also used flow cytometry to measure the surface exposed Pal subpopulations in hyper‐vesiculating E. coli cells and their OMVs. Preliminary data suggest that the surface exposed Pal subpopulations are increased in vesiculating cells and OMVs.Support or Funding InformationReceived: Rochester Institute of Technology FEAD grant to Dr. Lea Vacca Michel Applying for: RIT Honors program $500 travel stipend to Nicole Pannullo Applying for: RIT National Technical Institute for the Deaf Student Travel Award to Nicole PannulloThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
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