PROJECT SUMMARY Despite the relative success of pneumococcal conjugate vaccines (PCVs) against pneumococcal otitis media (OM), the incidence of all-cause OM remains unacceptably high worldwide and broad use of PCVs has also changed the microbiology of OM to one in which nontypeable Haemophilus influenzae (NTHI) predominates. Additionally, whereas the importance of biofilms in OM pathogenesis, chronicity and recurrence is recognized, and the recalcitrance of biofilms to antimicrobials is well accepted, we nonetheless still treat OM with oral antibiotics that do not reach levels in the middle ear required to kill planktonic bacteria, let alone those resident in a biofilm. The use of broad-spectrum antibiotics is also not without consequence. Rashes and diarrhea are common, and we now know that early life use of antibiotics disrupts development of the very gut microbiome that is essential for normal human immune system development. Due to global concerns about these issues, in a recent Nature commentary, thought leaders encouraged us to consider new ways to combine the protection offered by effective induced antibodies with the more appropriate use of antibiotics as our “last hope against multi-drug resistant bacteria and persistent disease”(55). With the recent recognition that bacteria which cause persistent diseases exist in not only planktonic or biofilm-resident states, but also a third distinct newly released (NRel) state that is the most sensitive to killing by antibiotics, we envision use of specifically targeted antibodies to release bacteria from their highly resistant biofilms so they can now be killed by both host immune effectors and, if necessary, traditional antibiotics but now used at a markedly reduced dose and for a limited time. We demonstrate via many new preliminary data that NRel NTHI are highly unique and further, that NRel NTHI populations are distinct from each other, dependent upon the specific effector that induced their release. Here, we strive to combine our expertise in immunology with both our advanced understanding of biofilm structural biology and our growing appreciation of the NRel NTHI state. Integration of these concepts provides us with the opportunity to develop a truly novel approach that we believe could offer a viable option with which to combat chronic and recurrent OM. We will utilize our understanding of both the NRel phenotype and the activity of specifically directed antibodies to leverage their combined power. In Aim 1, we will use established in vitro assays to determine how environmental conditions (singly and in combination) affect the kinetics, extent of duration and phenotype of NRel bacteria as a target for intervention to identify the control points and rate-limiting steps that confer greatest vulnerability. In Aim 2, we will further define the significant vulnerability of the NRel state in OM through changes in release kinetics, gene expression and clearance of otopathogenic biofilms by innate ...