Nontypeable Haemophilus influenzae (NTHi) has emerged as a dominant mucosal pathogen causing acute otitis media (AOM) in children, acute sinusitis in children and adults, and acute exacerbations of chronic bronchitis in adults. Consequently, there is an urgent need to develop a vaccine to protect against NTHi infection. A multi-component vaccine will be desirable to avoid emergence of strains expressing modified proteins allowing vaccine escape. Protein D (PD), outer membrane protein (OMP) 26, and Protein 6 (P6) are leading protein vaccine candidates against NTHi. In pre-clinical research using mouse models, we found that recombinantly expressed PD, OMP26, and P6 induce robust antibody responses after vaccination as individual vaccines, but when PD and OMP26 were combined into a single vaccine formulation, PD antibody levels were significantly lower. We postulated that PD and OMP26 physiochemically interacted to mask PD antigenic epitopes resulting in the observed effect on antibody response. However, column chromatography and mass spectrometry analysis did not support our hypothesis. We postulated that the effect might be in vivo through the mechanism of protein vaccine immunologic antigenic competition. We found when PD and OMP26 were injected into the same leg or separate legs of mice, so that antigens were immunologically processed at the same or different regional lymph nodes, respectively, antibody levels to PD were significantly lower with same leg vaccination. Different leg vaccination produced PD antibody levels quantitatively similar to vaccination with PD alone. We conclude that mixing PD and OMP26 into a single vaccine formulation requires further formulation studies.Nontypeable Haemophilus influenzae (NTHi) has become the most common cause of acute otitis media (AOM) in children in the US, persistent AOM, and recurrent AOM [1][2][3][4][5]. Treatment of AOM in children has an annual cost of over $6 billion in the US [6,7].NTHi also causes acute sinusitis and conjunctivitis in children and adults and acute exacerbations of chronic obstructive pulmonary disease (COPD) in adults [8][9][10][11]. COPD is the third leading cause of death in the US, affecting at least 24 million people, and US
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.
Sepsis is a condition in which the body’s inflammatory response to an infection is overwhelming and unregulated. Sepsis‐related inflammation can lead to tissue damage, organ failure, and even death. Once diagnosed, most sepsis patients are put on a regimen of antibiotics, especially if a bacterial infection is the suspected cause of sepsis. We proposed that at least some antibiotics may enhance the release of potentially toxic and inflammatory molecules from bacteria. Specifically, we considered several clinically relevant antibiotics and their effect on the release of outer membrane vesicles (OMVs) from Gram‐negative Escherichia coli. We employed ultracentrifugation to purify the OMVs and Western blotting to quantify the OMVs released from E. coli. Preliminary results suggest that, using our methods, some antibiotics enhance the production of OMVs from E. coli.
Outer membrane vesicles (OMVs) are 20‐250 nm particles released from Gram‐negative bacteria, including commensal and pathogenic Escherichia coli(E. coli). OMVs contain outer membrane and periplasmic proteins, toxins, nucleic acids, and pieces of peptidoglycan. OMV production is enhanced by environmental stressors, and OMVs are thought to facilitate bacterial cell growth and division, to contribute to bacterial communication (quorum sensing), and can also act as decoys during antibiotic or host immune system attacks. We aim to optimize E. coli OMV production and purification using ultracentrifugation and alternative techniques, to characterize OMVs using immunoblotting and nanoparticle tracking analysis (NTA), and to ultimately determine how OMVs can be used as molecular biomarkers for E. coli infections and Gram‐negative sepsis. Preliminary studies have shown that antibodies to peptidoglycan associated lipoprotein (Pal) can be employed to successfully identify E. coli OMVs.
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