NANOPARTICLE FORMULATIONS TO ACTIVATE INNATE IMMUNITY AND ENHANCE RESISTANCE TO BOVINE RESPIRATORY DISEASE

<b>Project Methods</b><br> Objective 1: Identify leading nanostimulant formulations for activation of innate immunity using bovine cell culture systemsCopolymers based on CPTEG:SA, CPTEG:CPH, and CPH:SA will be synthesizedusing melt polycondensation and characterized by1H NMR and gel permeation chromatography (GPC). Each PRR agonist will be loaded at a 5% w/w in the nanoparticles; thus, a dose of 500 μg will contain 25 μg of the PRR agonist.In vitro screening in bovine immune and epithelial cells:The first round of screening for innate immunostimulating activity will be conducted using bovine cells. Two types of cell culture will be used: PBEC, which will be representative of the cells lining the upper respiratory tract; and primary bovine alveolar macrophages (AMF). Our assays will focus on the ability of nanostimulant treatments to inducedirectantiviral and antibacterial activity in AMF and PBECs (i.e. reduction in viral loads, bacterial cell killing) andindirectactivity through inflammatory responses (i.e. chemokine production, cytokine production).PBEC will be prepared from 5 healthy lungs obtained from the local abattoir using published protocols. PBEC will be seeded in 12 well tissue culture plates. Wells be treated with 10 μg of innate agonist in 200 μg of NPs. Bovine AMF will be isolated from 5 healthy lungs obtained from the local abattoir. Cells will be seeded in triplicates in 12-well plates and stimulated with 10 μg of innate agonist in 200 μg of NPs. Both AMF and PBEC cultures will be harvested 18 hours after treatment for qPCR.Antibacterial assays in PBEC and AMF will be performed using published protocols. To assay for antibacterial activity, PBEC and AMF will be seeded in 12 well plates as above. Cell cultures will be incubated with nanostimulants for 24 hours (10 μg of innate agonist in 200 μg of NPs). Cultures treated with LPS will serve as a positive control. Additional controls will include cells treated with the PRR agonists (not NP encapsulated) or treated with empty NP (one of each type of chemistry). Cell cultures will then be treated with 1 MOIM. haemolyticastrain D153 or 1 MOIP. multocidafor 1, 3 and 5 h. At each timepoint, cell culture supernatants will be harvested for enumeration of extracellular bacteria.Objective 2: Identify leading nanostimulant formulations for activation of innate immunity in the respiratory tract using a mouse modelGroups of C57BL/6 mice (n=5 per group) will be treated intranasally with escalating doses of nanostimulant (1, 5 or 25 μg PRR agonist in 20, 100 or 200 μg NPs, respectively). Control mice will remain untreated or will receive empty NP (one group for each of the 3 formulations). Groups of mice will be sacrificed at intervals following treatment to evaluate the duration of innate immune activation: day 3, 5, 7, 10, 14, 1 month and 2 months. The lungs will be examined for signs of gross pneumonic lesions.In follow up experiments, groups of mice will be treated with 25 μg of the innate agonist encapsulated into 500 μg NP and sacrificed on day 5, 14 and 2 months after treatment (timing and dose to be informed by previous studies). Total RNA will be isolated from the lung and innate immune activation will be assessed by commercial Innate Immunity Gene Expression Panel (NanoString). We will also perform functional assays to determine the leading nanostimulants for induction of efficacious antiviral and antibacterial immunity in the respiratory tract. Groups of mice (n=5/group) will be treated with 25 μg of the innate agonist encapsulated into 500 μg NP. On day 5, 14 and 2 months after nanostimulant treatment, mice will be infected intranasally with 107TCID50BRSV strain 375 or 109colony forming units ofM. haemolyticastrain D153 . Mice infected with BRSV will be sacrificed on day 4 after infection and BRSV loads will be quantified by virus titration and qRT-PCR for the BRSV NS2 gene. Mice infected withM. haemolyticawill be sacrificed 24 h after infection and lung bacterial load will be determined by serial plating on blood agar.Objective 3: Determine the efficacy of leading nanostimulant formulations for activation of innate immunity in the bovine respiratory tract and for enhancing resistance to bovine respiratory disease complexIn Objective 3.1, we will determine the magnitude and duration of innate immune activation in the bovine respiratory tract. In Objective 3.2, we will determine the impact of nanostimulant treatment on resistance to an experimental viral/bacterial coinfection in weaned dairy calves.Objective 3.1: Evaluate magnitude and duration innate immune activation in the calf modelA total of 40, 2-3 month old Holstein steer calves will be divided into 5 groups of n=8 animals each. Each calf will receive 50 μg of the innate immune agonist encapsulated in 1 mg polyanhydride NP. The immunostimulant will be suspended in 5 mL of PBS and administered with a mucosal atomization device. Nasopharyngeal swabs and nasal fluid will be collected on days 1 (24 hours), 3, 5, 7, 10 and 14 after immunostimulant treatment. BAL samples will be collected on day 7 and 14 after treatment. Serum and PBMC will be collected on day 1, 7 and 14 after treatment. Cytospins and flow cytometry will be used to evaluate the numbers and relative frequency of lung-infiltrating innate immune populations collected from the BAL fluid, including neutrophils, macrophages, Natural Killer cells and gamma delta T cells. To evaluate innate immune function, cytokine production, phagocytosis and oxidative burst capacity will be evaluated in monocytes and neutrophils on days 0, 1, 7 and 14. AMF will be stimulatedin vitrowith LPS and Pam3CSK4 for 48 hours. Cell culture supernatants will be harvested and stored at -80º C for analysis by ELISA for secretion of IL-6, IL-1β and TNFα. Phagocytosis (measured by uptake of antibody-coated fluorescent beads) and oxidative burst (cleavage of DHR; Neutrophil/Monocyte Respiratory burst assay) will be evaluated using flow cytometry as we have used previously[110]. Serum will be analyzed for concentrations of IL-6 and haptoglobin.Objective 3.2: Evaluate impact of intranasal nanostimulant treatment on the outcome of experimental respiratory disease in the calfA total of 60, 2-3 month old Holstein steers will be enrolled in the trial. Calves will be blocked by age and divided into 5 groups of 12 animals per treatment. One group will serve as a negative control and will receive no nanostimulant treatments. The other four groups will receive one of the 4 leading nanostimulant formulations. Each calf will receive 50 μg of the innate immune agonist encapsulated in 1 mg polyanhydride NP as in Objective 3.1. Five days after treatment, calves will be challenged via aerosol inoculation with 104TCID50BRSV strain 375. On day 5 after viral infection, calves will be challenged via intratracheal inoculation with 5x108CFU of MH strain D153, (serotype A1). All animals will be monitored and scored daily for clinical signs including fever, respiratory rate, appetite and nasal discharge. Animals will be humanely euthanized on day 10 after infection. At necropsy, a veterinary pathologist (blinded to treatment) will assess and score the lungs and upper respiratory tract for gross and microscopic pathology. The BAL, lung tissue and nasopharyngeal swabs will be analyzed for viral burden using qRT-PCR for the BRSV NS2 geneand for bacterial burden by quantitative culture.