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

• Sponsoring Institution: National Institute of Food and Agriculture
• Project Status: ACTIVE
• Funding Source: AFRI COMPETITIVE GRANT
• Reporting Frequency: Annual
• Accession No.: 1027837
• Grant No.: 2022-67015-36337
• Cumulative Award Amt.: $625,000.00
• Proposal No.: 2021-07002
• Project Start Date: Jan 1, 2022
• Project End Date: Dec 31, 2025
• Grant Year: 2022
• Program Code: [A1221]- Animal Health and Production and Animal Products: Animal Health and Disease

Recipient Organization
IOWA STATE UNIVERSITY
S. AND 16TH ELWOOD
AMES,IA 50011

Performing Department
Vet Microbio & Preventive Med

Non Technical Summary
Bovine respiratory disease complex (BRDC) is a leading cause of morbidity and mortality in the U.S. Cattle industries. Economic costs to the worldwide cattle industry due to BRDC have been estimated as high as $3 billion annually. Despite widespread use of vaccines and antibiotics, the incidence of BRDC has remained unchanged for more than two decades. There is a significant need for effective, non-antibiotic treatments to prevent BRDC in the beef and dairy industries. The innate immune system is the body's first line of defense against infection. Itisbroadly specific and provides collateral protection against multiple types of invading pathogens. In a healthy animal, the innate immune system prevents most respiratory infections. However, stress or infection can impair the ability of the innate immune system to protect from disease. The idea of counteracting the known effects of stress 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. While vaccines target the adaptive immune system, immunomodulatory treatments instead target the innate immune system. Biodegradable polyanhydride nanoparticles (NPs) are known to provide sustained delivery of biological molecules and can be delivered intranasally, directly to the respiratory tract. Polyanhydride NPs can be used to deliver immunomodulatory payloads, therefore stimulating the innate immune system in the lungs. This project will optimize the use of intranasal, immunostimulatory NPs for induction of a potent innate immune response in the bovine respiratory tract. We will generate and test a panel of immunostimulatory NP formulations in primary bovine cell cultures (Objective 1), in mouse models of disease (Objective 2) and ultimately in cattle models of BRDC. At the conclusion of this project, we expect to have developed and characterized a novel, non-antibiotic, immunostimulatory treatment that will have the potential to improve the outcome of BRDC in beef and dairy cattle.

Animal Health Component

30%

Research Effort Categories

Basic

70%

Applied

30%

Developmental

0%

Classification

Knowledge Area (KA)	Subject of Investigation (SOI)	Field of Science (FOS)	Percent
311	3310	1090	50%
311	3410	1090	50%

Knowledge Area
311 - Animal Diseases;

Subject Of Investigation
3310 - Beef cattle, live animal; 3410 - Dairy cattle, live animal;

Field Of Science
1090 - Immunology;

Keywords

bovine respiratory disease

immunomodulator

innate immunity

intranasal

nanoparticle

Goals / Objectives
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.

Project Methods
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.

Progress 01/01/24 to 12/31/24

Outputs
Target Audience: Scientific results were shared with graduate students and scientists in the department during routine seminars and lab meetings. Scientific results were also shared with the larger University community at ISU Research Day. The data were also shared at the national Conference for Research Workers in Animal Diseases in January 2024, the American Association of Immunologists meeting in May 2024and at NanoVax 2024, a regional meeting held in Ames, IA in May 2024. Changes/Problems: We are advancing with our Objectives and have encountered no difficulties with executing our experiments as planned. We are moving into catttle work in the upcoming reporting period and will conduct the immunogenicity and efficacy experiments as planned, using the top 4 formulations that we have identified in our previous studies. What opportunities for training and professional development has the project provided? This project has provided training and professional development opportunities to a postdoctoral fellow, a graduate student and an undergraduate research assistant. The postdoctoral fellow had previous experience working with mice and rodent models. The current project has provided training in working with cell lines and rodents, lambsand in working with the nanoparticle formulations. The postdoc has provided training to the undergraduate student, teaching cell culture, qPCR and viability assays, as well as data analyses and statistics. This has benefited both the student, and provided mentoring experience for the postdoc. The graduate student is a chemical and biological engineer. She has gained training and professional development through generation and characterization of the various formulations for the project. Working with multiple chemistries, and different types of payloads, has taught her how to troubleshoot and optimize nanoparticle preparations, skills which will be necessary as she advances in her career. How have the results been disseminated to communities of interest? We have shared preliminary results with the department through standard work-in-progress meetings, and shared with the greater University community during Research Day, an internal celebration of research that includes lightning talks and poster presentations. We have also presented our data nationally at the Conference for Research Workers in Animal Diseases (Chicago, IL), American Association of Immunologists Meeting (Chicago, IL) and Nanovax 2024 (Ames, IA). What do you plan to do during the next reporting period to accomplish the goals? In the upcoming reporting period, we will be conducting the calf immunogenicity and efficacy studies. We have selected our top 4 nanoparticle formulations to be tested in the calf challenge model, and will also be collecting samples to understand immune responses and mechanisms of action of our various nanoparticle treatments.During the upcoming reporting period, we will also be writing and submitting a manuscript describing out work in the lamb model, and working towards a manuscript describing immune mechanisms.

Impacts
What was accomplished under these goals? Stressful management can lead to the development of Bovine respiratory disease (BRD) in calves, a multi-factorial disease where different viral and/or bacterial pathogens infect or colonize the respiratory tract. Despite the use of antimicrobials and vaccines, BRD prevalence remains high, leading to billions in economic losses to the cattle industry every year. With concerns mounting over antimicrobial resistance, efforts to develop immunomodulatory strategies generating broad-spectrum protection from pathogens causing BRD in cattle are required. Since polyanhydride (PA) nanoparticles have shown immunostimulatory properties when used as vaccine adjuvants in cattle, we have developed innate-immunostimulatory PA nanoparticles encapsulating pattern recognition receptors (PRRs) to use them as mucosal immunostimulants in cattle. In the prior reporting periods, we performed screening of a panel of 21 nanoparticle formulations using bovine and human cell lines, and primary bovine immune cells (turbinates and alveolar macrophages). This screening allowed us to move to 9 of the most promising nanoparticle formulations, and to move into in vivo mouse models. Through work with viral and bacterial modesl in mice, we were able to identify 6 leading formulations to advance to the next stage of screening. During this reporting period, we wrote and a submitted a manuscript summarizing our screening data and efficacy data in the mouse model of RSV infection. That manuscript is currently under review. In this reporting period, we also secured matching funds from an internal ISU grant and funds from the State of Iowa that enabled us to screen our leading candidates in a lamb model of BRSV infection. This allowed us to do additional testing in an important ruminant model of disease, and to become more confident in our selection of the best candidates to move into the calf model of disease. This work demonstrated that 4 formulations (CL413 20:80 CPH:SA, CL413 50:50 CPTEG:SA, CL413 20:80 CPTEG:CPH and PAM3CSK4 20:80 CPTEG:CPH) affored protection from clinical disease caused by BRSV infection in lambs, and the CL413 20:80 CPTEG:CPH formulation reduced lung viral loads compared to untreated lambs. In addition to the ruminant work, we are further refining our understanding of the mechanism of action of our leading nanoparticle formulations by examining immune responses that are generated in our mouse model. It seems that early immune cell recruitment may be one important component to protection, but other regulatory factors seem to also play a key role, such as induction of IL-10 and upregulation of proresolving mediators. We are continueing to explore these mechanisms, as this is key to selecting the optimum nanoparticle formulations for reducing disease in cattle. In the upcoming reporting period, we will be conducting the calf immunogenicity and efficacy studies. We have selected our top 4 nanoparticle formulations to be tested in the calf challenge model, and will also be collecting samples to understand immune responses and mechanisms of action of our various nanoparticle treatments. During the upcoming reporting period, we will also be writing and submitting a manuscript describing out work in the lamb model, and working towards a manuscript describing immune mechanisms.

Publications

• Type: Conference Papers and Presentations Status: Accepted Year Published: 2024 Citation: Diaz, FE, Grego, E, Narasimhan, B, JL McGill. 2024. Polyanhydride nanoparticles trigger effective antiviral responses against bovine and human RSV in vitro and in a murine RSV model. Conference for Research Workers in Animal Diseases. Chicago, IL.
• Type: Conference Papers and Presentations Status: Accepted Year Published: 2024 Citation: Diaz, FE, Grego, E, Narasimhan, B, JL McGill. 2024. Pattern recognition receptor agonist-loaded nanoparticles trigger effective antiviral responses against bovine and human Respiratory Syncytial Virus in vitro and in a murine RSV model. American Association of Immunologists Annual Meeting, Immunology 2024. Chicago, IL.
• Type: Conference Papers and Presentations Status: Accepted Year Published: 2024 Citation: Diaz, FE, Uslu, A, Grego, E, Narasimhan, B, JL McGill. 2024. Evaluation of Polyanhydride Nanoparticles as Innate Immunostimulants to Prevent Bovine Respiratory Disease in Cattle. Nanovax 2024. Ames, IA

Progress 01/01/23 to 12/31/23

Outputs
Target Audience:Scientific results were shared with graduate students and scientists in the department during routine seminars and lab meetings. Scientific results were also shared with the larger University community at ISU Research Day. The data were also shared at the national Conference for Research Workers in Animal Diseases and internationally at the 6th Annual Meeting ASOCHIN (Asociacion Chilena de Inmunologia) in Santa Cruz, Chile. Changes/Problems:We are advancing with our Objectives and have encountered no difficulties with executing our experiments as planned. Due to the high cost of cattle and the current market, we have made one minor change to our screening process. Rather than jumping directly from mice to cattle, we have chosen to use sheep as an in between step for screening the nanoparticle formulations. Sheep are a small ruminant and are susceptible to many of the same pathogens as cattle. This intermediate step will enable us to be confident in our selection of the best formulations, doses and timing, before moving into the cattle model. We do not anticipate this will slow or change our timeline for the project, and we believe this choice of an intermediate animal model is a conservative use of our time to ensure utlimate project success. What opportunities for training and professional development has the project provided?This project has provided training and professional development opportunities to a postdoctoral fellow, a graduate student and an undergraduate research assistant. The postdoctoral fellow had previous experience working with mice and rodent models. The current project has provided training in working with cell lines androdents and in working with the nanoparticle formulations. The postdoc has provided training to the undergraduate student, teaching cell culture, qPCR and viability assays, as well as data analyses and statistics. This has benefited both the student, and provided mentoring experience for the postdoc. The graduate student is a chemical and biological engineer. She has gained training and professional development through generation and characterization of the various formulations for the project. Working with multiple chemistries, and different types of payloads, has taught her how to troubleshoot and optimize nanoparticle preparations, skills which will be necessary as she advances in her career. How have the results been disseminated to communities of interest?We have shared preliminary results with the department through standard work-in-progress meetings, and shared with the greater University community during Research Day, an internal celebration of research that includes lightning talks and poster presentations. We have also presented our data national and internationally at the Conference for Research Workers in Animal Diseases (Chicago, IL), American Dairy Science Association Meeting (Ottawa Canada) and ASOCIAN (Association of Chilean Immunologists National Meeting, Santa Cruz, Chile). What do you plan to do during the next reporting period to accomplish the goals?We intend to complete our Objective 2 studies by screening of the nanoparticles against a bacterial pneumonia challenge in the mouse model, which will enablefurther downselection on the most promising nanoparticle formulations for the large animal studies. Before moving into cattle, which are currently extremely expensive, we have also chosen to use lambs as an intermediate step for screening nanoparticle efficacy and immunogenicity. In the first half of the Year, we will evaluate 6 of our most promising nanoparticle candidates in sheep.These studies will provide us with confidence to choose the best candidate formulations to move into the cattle projects In the second half of the reporting period.During the upcoming reporting period, we will also be writing and submitting a manuscript describing our work in the cell lines (Objective 1)and mouse models (Objective 2).

Impacts
What was accomplished under these goals? Stressful management can lead to the development of Bovine respiratory disease (BRD) in calves, a multi-factorial disease where different viral and/or bacterial pathogens infect or colonize the respiratory tract. Despite the use of antimicrobials and vaccines, BRD prevalence remains high, leading to billions in economic losses to the cattle industry every year. With concerns mounting over antimicrobial resistance, efforts to develop immunomodulatory strategies generating broad-spectrum protection from pathogens causing BRD in cattle are required. Since polyanhydride (PA) nanoparticles have shown immunostimulatory properties when used as vaccine adjuvants in cattle, we have developed innate-immunostimulatory PA nanoparticles encapsulating pattern recognition receptors (PRRs) to use them as mucosal immunostimulants in cattle. In the prior reporting period, we screened a panel of 18 NP formulations in a series ofin vitroassays in 3 different cell lines.Treatment of bovine turbinate cells (nasal epithelial cells) and alveolar macrophages with several different PA nanoparticles led to differential transcription of several effector molecules. Noteworthy, nanoparticles that contained CL413 (TL2/7 agonist), Pam3CSK4 (TLR2/1 agonist) and MPLA (TLR4 agonist) were also able to reduce bRSV infectious titersin vitro. We selected 9 of the most promising NP formulations to advance to in vivo studies. In the current reporting period, we completed a series of in vivo experiments using the mouse model. To date, we have primarily used RSV infection to screen PA nanoparticles for their ability to reduce disease.In most mouse studies, six- to eight-week-old B6 mice received nanoparticles (40 or 100ug/mice) intranasally (i.n), then lung samples were collected 3 or 7 days later for lung cytokine quantification. In infection studies, mice were treated with selected nanoparticles, then infected i.n. with RSV strain 1997, either 3, 7, or 14 days after treatment. Lung viral loads, weight change and respiratory function were analyzed to determine the efficacy of the nanoparticles in preventing RSV disease. Untreated cells (UT) and empty nanoparticles were used as controls.PA nanoparticles made of 1,8-bis-(p-carboxyphenoxy)- 3,6-dioxaoctane and 1,6-bis-(p-carboxyphenoxy)-hexane in a 20:80 ratio loaded with the CL413 agonist (CPTEG:CPH 20:80 CL413) given 3 or 14 days before infection were able to prevent weight loss (p = 0.008 and 0.04, respectively) and reduce lung viral loads (p = 0.0022 and 0.001, respectively) in RSV infected mice. Additional nanoparticles made from the same or different PA polymers and loaded with PAM3CSK and MPLA also showed protective effects in the RSV model. We are progressing with our Objectives in a manner that is consistent with our timeline. We intend to complete screening of the nanoparticles against a bacterial pneumonia challenge in the mouse model, to further downselect on the most promising nanoparticle formulations for the large animal studies.We have also chosen to use lambs as an intermediate step for screening nanoparticle efficacy and immunogenicity.In the first half of the Year, we will evaluate 6 of our most promising nanoparticle candidates in sheep.These studies will provide us with sufficient confidence to move into the cattle projects In the second half of the reporting period.During the upcoming reporting period, we will also be writing and submitting a manuscript describing out work in the cell lines and mouse models. Key Outcomes: The results of our in vitro and in vivo screening experiments have provided 9 optimum nanoparticle candidates that improve the outcome of experimental RSV infection in rodent models. These formulations have significant potential to show efficacy against a broad array of respiratory pathogens in cattle. This new knowledge will result in identification of promising, non-antimicrobial alternatives for treatment and prevention of bovine respiratory disease.

Publications

• Type: Conference Papers and Presentations Status: Accepted Year Published: 2023 Citation: Diaz, FE; Grego, EA; Uslu, A; Narasimhan, B; McGill, JL. Polyanhydride nanoparticles trigger effective antiviral responses against bovine and human RSV in vitro and in a murine RSV model. Conference for Research Workers in Animal Diseases 2024. Chicago, IL
• Type: Conference Papers and Presentations Status: Other Year Published: 2023 Citation: McGill, JL and Diaz, FE. Immunomodulation strategies to control respiratory disease in preweaned calves. American Dairy Science Association 2023. Ottawa, Canada.
• Type: Conference Papers and Presentations Status: Accepted Year Published: 2023 Citation: Diaz, FE and McGill, JL. Nonantibiotic strategies to control respiratory infections in calves and humans. ASOCHIN, Asociacion Chilena de Inmunologia. Santa Cruz, Chile.

Progress 01/01/22 to 12/31/22

Outputs
Target Audience:Scientific results were shared with graduate students and scientists in the department during routine seminars and lab meetings. Scientific results were also shared with thelarger University community at ISU Research Day. The data will be shared at the upcoming national Conference for Research Workers in Animal Diseases. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?This project has provided training and professional development opportunities to a postdoctoral fellow, a graduate student and an undergraduate research assistant. The postdoctoral fellow had previous experience working with mice and rodent models. The current project has provided training in working with cell lines and in working with the nanoparticle formulations. The postdoc has provided training to the undergraduate student, teaching cell culture, qPCR and viability assays, as well as data analyses and statistics. This has benefited both the student, and provided mentoring experience for the postdoc. The graduate student is a chemical and biological engineer. She has gained training and professional development through generation and characterization of the various formulations for the project. Working with multiple chemistries, and different types of payloads, has taught her how to troubleshoot and optimize nanoparticle preparations, skills which will be necessary as she advances in her career. How have the results been disseminated to communities of interest?We have shared preliminary results with the department through standard work-in-progress meetings, and shared with the greater University community during Research Day, an internal celebration of research that includes lightning talks and poster presentations. We have an accepted abstract and will be sharing a poster and presentation on our data at the upcoming national meeting, Conference for Research Workers in Animal Diseases. What do you plan to do during the next reporting period to accomplish the goals?We are planning and preparing to initiate the Objective 2 studies in mice. We have synthesized sufficient amounts of the 9 leading formulations, and have planned the first few rounds of in vivo mouse screening experiments. We intend to complete and analyze these experiments in Year 02 of this project. Following completion of the Objective 2 studies, we will have identified 4 leading formulations that can advance to trials in cattle.

Impacts
What was accomplished under these goals? In a healthy animal, the innate immune system prevents most respiratory infections. However, stress or infection can impair the ability of the innate immune system to protect from disease. Increasing attention has turned to the development of immunomodulators as a promising 'alternative' to antibiotic usage. While vaccines target the adaptive immune system, immunomodulatory treatments instead target the innate immune system. Biodegradable polyanhydride nanoparticles (NPs) are known to provide sustained delivery of biological molecules and can be delivered intranasally, directly to the respiratory tract. Polyanhydride NPs can be used to deliver immunomodulatory payloads, therefore stimulating the innate immune system in the lungs. This project is aimed at optimizingintranasal, immunostimulatory NPs for induction of a potent innate immune response in the bovine respiratory tract. In the current reporting period, we have made significant progress towards Objective 1 of our project. We have generated a panel of 18 NP formulations using combinations of innate immune agonists and various polyanhydride formulations. We have screened the activity of these 18 nanoparticles in bovine turbinate cells (an epithelial cell line from the respiratory tract), bovine alveolar macrophages, and bovine PBMC. We have generated qPCR results investigating gene expression of critical pro- and anti-inflammatory pathways andantimicrobial molecules. Candidate genes have included the type I IFNs, cathelicidins and beta-defensin, IL-10 as an anti-inflammatory marker; IL-6, TNF and IL-8 as proinflammatory markers, and others.We have also generated accompanying viability data to ensure good cell health, dose response curves and some preliminary data investigating the kinetics of the response induced by various NP formulations. As a result of this work, we have identified a panel of 9 promising NP formulations that will advance to the next Objective. Amongst our leading formulations are 20:80 CPTEG:CPH particles encapsulation PAM3CSK4, CpG and CL413, agonists of TLR2, TLR9 and TLR2/7, respectively.In the coming reporting period, we will be conducting screening and immunology assays with these 9 formulations in the mouse model. We have addressed any technical difficulties in the course of this year and are progressing with our Objectives in a manner that is consistent with our timeline. The experiments in Objective 2 will yield crucial, in vivo evidence to enable selection of the most promising formulations for use in cattle against BRD. Key Outcomes: The results of our initial screening experiments have provided 9 optimum nanoparticle candidates with the potential to serve as preventive immunostimulants that can prevent, or improve the outcome, of bovine respiratory disease in cattle. This new knowledge will result in identification of promising, non-antimicrobial alternatives for treatment and prevention of bovine respiratory disease.

Publications

• Type: Conference Papers and Presentations Status: Accepted Year Published: 2023 Citation: Diaz, FE; E Grego, Z Olsem, B Narasimhan, JL McGill. Polyanhydride nanoparticles induce innate activation of bovine epithelial cells and alveolar macrophages in vitro. Conference for Research Workers in Animal Diseases 2023. Chicago, IL.