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
We have determined the 1.8 Å X-ray crystal structure of nonlipidated (i.e., Nterminally truncated) nontypeable Haemophilus influenzae (NTHi; H. influenzae) protein D. Protein D exists on outer membranes of H. influenzae strains and acts as a virulence factor that helps invade human cells. Protein D is a proven successful antigen in animal models to treat obstructive pulmonary disease (COPD) and otitis media (OM), and when conjugated to polysaccharides also has been used as a carrier molecule for human vaccines, for example in GlaxoSmithKline Synflorix™. NTHi protein D shares high sequence and structural identify to the Escherichia coli (E. coli) glpQ gene product (GlpQ). E. coli GlpQ is a glycerophosphodiester phosphodiesterase (GDPD) with a known dimeric structure in the Protein Structural Database, albeit without an associated publication. We show here that both structures exhibit similar homodimer organization despite slightly different crystal lattices. Additionally, we have observed both the presence of weak dimerization and the lack of dimerization in solution during size exclusion chromatography (SEC) experiments yet have distinctly observed dimerization in native mass spectrometry analyses. Comparison of NTHi protein D and E. coli GlpQ with other homologous homodimers and monomers shows that the E. coli and NTHi homodimer interfaces are distinct. Despite this distinction, NTHi protein D and E. coli GlpQ possess a triose-phosphate isomerase (TIM) barrel domain seen in many of the other homologs. The active site of NTHi protein D is located near the center of this TIM barrel. A putative glycerol moiety was modeled in two different conformations (occupancies) in the active site of our NTHi protein D structure and we compared this to ligands modeled in homologous structures. Our structural analysis should aid in future efforts to determine structures of protein D bound to substrates, analog intermediates, and products, to fully appreciate this reaction scheme and aiding in future inhibitor design.
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.
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