Bovine respiratory disease complex (BRDC) is a leading cause of morbidity and mortality in the beef and dairy industries.The innate immune system is the body's first line of defense against infection. Itisbroadly specific and therefore provides collateral protection against an array of invading bacteria and viruses. In a healthy animal, the innate immune system prevents a majority of respiratory infections. The idea of counteracting the known effects of stress or viral infection to enhance an animal's innate state of disease resistance is appealing. Therefore, increasing attention has turned to the development of immunomodulators as a promising 'alternative' to antibiotic usage. Immunomodulators engage or prime the host's own innate immune system to defend against infectious agents. While vaccines are effective at activating the adaptive immune system, immunomodulatory agents instead engage the innate arm of the immune system. A handful of immunomodulators have recently reached the commercial market with label claims of reducing or preventing BRDC. However, there is clear room for improvement, as field trials have shown only moderate to no effects on disease incidence or severity.Biodegradable polymer-based nanoparticles (NPs) are notable for their ability to provide sustained delivery of biological molecules. Among these polymers, polyanhydrides have the potential to be delivered needle free via the mucosa and are stable even at room temperature, eliminating the need for cold chain storage.A novel amphiphilic polyanhydride NP-based delivery system has been developed by Co-I Dr. Narasimhan at the Iowa State University Nanovaccine Institute. Polyanhydride NPs have intrinsic pathogen-mimmicking and immunostimulating propertiesincluding the ability to induce macrophages, monocytes and dendritic cells to secrete proinflammatory cytokines and upregulate costimulatory and MHC molecules. As a delivery vehicle, the immunomodulatory properties of the polyanhydride NP can be further exploited by incorporation of additional stimulatory payloads such as pattern recognition receptor (PRR) agonists.The respiratory tract is the point of entry for respiratory pathogens. While systemic immunity can provide a degree of protection, the mucosal immune system is the critical first line of defense. Induction of a potent innate immune response in the nasopharynx and respiratory tracts can suppress pathogen invasion at the initial site of infection. Given that BRDC often accompanies a 'stress' event that negatively impacts systemic immune function, induction of local, innate immune mechanisms may be less vulnerable to the inhibitory processes of stress.To date, there have been few attempts to develop an immunomodulator that specifically targets the mucosal innate immune system.Therefore, in the current proposal,we will develop a slow-eroding, intranasal, polyanhydride NP-based immunostimulant (nanostimulant) to promote effective activation of innate immune responses in the respiratory tract that will reduce the incidence and severity of BRDC. Wehypothesizethat the intranasal administration of nanostimulants with tunable erosion properties will have the capacity to: 1) activate the bovine innate immune system in the nasopharynx and lungs; 2) prolong innate immune priming in the respiratory tract; and 3) enhance resistance to BRDC in the calf by enhancing innate immunity in the nasopharynx and lungs. We will test this hypothesis in the following three objectives:Objective 1: Identify leading nanostimulant formulation(s) for activation of innate immunity using bovine cell culture systems;Objective 2: Identify leading nanostimulant formulation(s) for activation of innate immunity in the respiratory tract using a mouse model; andObjective 3: Determine the efficacy of lead nanostimulant formulation(s) for activation of innate immunity in thebovine respiratory tract and for enhancing resistance to BRDC.In Objective 1, we will narrow down the candidate nanostimulants by 50% based on their capacity to activate broad innate responses in bovine bronchial epithelial cells and in bovine PBMC. In Objective 2, we will capture the complexity of thein vivoinnate immune response using a mouse model. Mice will be treated with the remaining 9 nanostimulant formulations and assessed for safety andin vivoactivity by targeted gene expression arrays and functional responses to a model viral and bacterial pathogen. Following the completion of the muring studies, four nanostimulants will progress for further testing. In Objective 3, we will assess the final four lead nanostimulants in the calf. We will determine the magnitude and duration of the innate activation in the nasopharynx and lungs and the efficacy of nanostimulant treatment for protecting against a viral/bacterial coinfection model of BRDC. At all stages in the process, we will take iterative approaches to improve upon the nanostimulant formulations, adjusting doses, chemistries and PRR agonists as needed to ensure selection of ideal candidates at all stages of the project.