Source: UNIV OF WISCONSIN submitted to
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
Funding Source
Reporting Frequency
Accession No.
Grant No.
Project No.
Proposal No.
Multistate No.
Program Code
Project Start Date
Mar 15, 2016
Project End Date
Mar 14, 2019
Grant Year
Project Director
Recipient Organization
21 N PARK ST STE 6401
MADISON,WI 53715-1218
Performing Department
Non Technical Summary
According to 2013 US agriculture statistics, the combined value of production and sales from broilers, eggs, turkeys, and chickens was $44.1 billion; of this, broiler sales comprised 70 percent of the industry value. The economic success of the poultry industry in the USA hinges on extensive use of vaccines to control bacterial and viral infections. Most of the current antibiotics are not used in food animals to curb the problem of spreading drug-resistant pathogens and anti-viral agents are expensive to use on poultry. Fortunately, vaccination programs are available to combat many of the important pathogens that impact broilers during their short-lived cycle of production (~8 weeks). However, these vaccines do not provide sufficient control against emerging infections and most of them are not stable under field conditions. Polyanhydride nanoparticles (PANs) have been shown to be safe for use in many food animals and humans. In this project, our team of experts in nanotechnology, molecular biology, vaccines and poultry diseases will develop polyanhydride nanoparticle-based vaccines (i.e., nanovaccines) as a platform technology for more efficient poultry vaccination.
Animal Health Component
Research Effort Categories

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
Knowledge Area
311 - Animal Diseases;

Subject Of Investigation
3299 - Poultry, general/other;

Field Of Science
1090 - Immunology;
Goals / Objectives
. Polyanhydride nanoparticles (PANs) have been shown to be safe for use in many food animals and humans. In this project, our team of experts in nanotechnology, molecular biology, vaccines and poultry diseases will develop polyanhydride nanoparticle-based vaccines (i.e., nanovaccines) as a platform technology for more efficient poultry vaccination. Specifically, we will test the new technology in a model system with infectious bronchitis virus (IBV) causing infectious bronchitis (IB) disease, which results in severe economic losses to the poultry industry. Specifically, we will:Aim I: Examine the safety and fate of polyanhydrides (PANs) in chickens. We hypothesize that PANs will function as a safe and well-tolerated adjuvant for subunit vaccines in poultry. To test this hypothesis, we will utilize fluorescently labeled PANs to examine cell toxicity and tissue distribution in vivo. This aim will be successfully attained when the safety and persistence of PANs are well characterized in the target host for immunization, chickens.Aim II: Decipher the immunogenicity of IBV proteins encapsulated within PANs. We hypothesize that PANs will efficiently release IBV antigens throughout the lifespan of broilers (average 8 weeks). To test this hypothesis, IBV-encapsulated PANs will be used to immunize chicken and examine their immunogenicity over 8 weeks. This aim will be successfully attained when stable and sufficient release of IBV antigens is demonstrated from the PANs.Aim III: Analyze the protective immunity of polyanhydride nanovaccine against challenge with IBV. We hypothesize that the polyanhydride nanovaccine will provide a better immunization strategy than commercial live attenuated vaccines (LAV). To test this hypothesis, we will examine the protective efficacy of the nanovaccine in a standard model system of IBV in comparison to LAV using readouts that include clinical signs, histology, virus load, and immunological parameters. This aim will be successfully attained when the developed nanovaccine is shown to be more protective than LAV in the stringent IBV challenge model. Overall, developing a novel platform technology based on polyanhydride nanovaccines will significantly improve poultry immunization programs and help in providing a sustained supply of food to consumers in the USA and worldwide, an important mission of the Nanotechnology program supported by NIFA.
Project Methods
The main objective of this project is to develop a platform technology for rapid production of effective nanovaccines against key poultry diseases. We will focus our efforts on utilizing biodegradable PANs to improve efficacy and immunogenicity of IBV vaccine constructs. Specifically, we will synthesize <100 nm PANs to examine the full potential of nanotechnology to help the poultry industry. We hypothesize that using PANs will improve the immunogenicity and efficacy of IBV vaccine constructs. To test this hypothesis, we will first evaluate the tissue distribution and safety of PANs in chicken following administration of increasing levels of PANs to ensure their practical use in the poultry industry. Next, in Aim II, we will characterize the sustained release of IBV antigens from PANs. We will evaluate the stability and biological activity of the released antigens and utilize an optimized immunization protocol to examine immunogenicity of the nanovaccine in chickens. The encapsulation of both IBV subunit proteins and whole inactivated virus into the PANs will enable the rapid production of an efficacious vaccine. Finally in Aim III, we will evaluate the protective efficacy of PAN-based vaccines in a challenge model of IB in comparison to available IB live-attenuated vaccines, the standard in commercial poultry production. Such studies will provide valuable information on the viability of using nanoparticles as a vaccine platform in poultry. The independent and logical flow of experimental outcomes from these aims will help us systematically evaluate the potential

Progress 03/15/16 to 03/14/19

Target Audience:Researchers, veterinarians, herd owners and disease control responsible agents. <!-- /* Font Definitions */ @font-face {font-family:"Cambria Math"; panose-1:2 4 5 3 5 4 6 3 2 4; mso-font-charset:0; mso-generic-font-family:roman; mso-font-pitch:variable; mso-font-signature:3 0 0 0 1 0;} @font-face {font-family:Calibri; panose-1:2 15 5 2 2 2 4 3 2 4; mso-font-charset:0; mso-generic-font-family:swiss; mso-font-pitch:variable; mso-font-signature:-536859905 -1073732485 9 0 511 0;} /* Style Definitions */ p.MsoNormal, li.MsoNormal, div.MsoNormal {mso-style-unhide:no; mso-style-qformat:yes; mso-style-parent:""; margin:0in; margin-bottom:.0001pt; mso-pagination:widow-orphan; font-size:12.0pt; font-family:"Calibri",sans-serif; mso-ascii-font-family:Calibri; mso-ascii-theme-font:minor-latin; mso-fareast-font-family:Calibri; mso-fareast-theme-font:minor-latin; mso-hansi-font-family:Calibri; mso-hansi-theme-font:minor-latin; mso-bidi-font-family:Arial; mso-bidi-theme-font:minor-bidi;} p {mso-style-priority:99; mso-margin-top-alt:auto; margin-right:0in; mso-margin-bottom-alt:auto; margin-left:0in; mso-pagination:widow-orphan; font-size:12.0pt; font-family:"Times New Roman",serif; mso-fareast-font-family:"Times New Roman";} .MsoChpDefault {mso-style-type:export-only; mso-default-props:yes; font-family:"Calibri",sans-serif; mso-ascii-font-family:Calibri; mso-ascii-theme-font:minor-latin; mso-fareast-font-family:Calibri; mso-fareast-theme-font:minor-latin; mso-hansi-font-family:Calibri; mso-hansi-theme-font:minor-latin; mso-bidi-font-family:Arial; mso-bidi-theme-font:minor-bidi;} @page WordSection1 {size:8.5in 11.0in; margin:1.0in 1.0in 1.0in 1.0in; mso-header-margin:.5in; mso-footer-margin:.5in; mso-paper-source:0;} div.WordSection1 {page:WordSection1;} --> Changes/Problems:-We attempted several times to overexpress IBV-S protein (Whole S protein) in E. coli with little success. Basically, the protein yield was low to use for polyanhydride encapsulation. To overcome the yield problem, we used the whole-inactivated IBV virus instead of E. coli purified protein. The obtained results confirmed that the project goals were accomplished despite this change in choice of IBV antigens. What opportunities for training and professional development has the project provided?In this project, a graduate student, a scientist and research assistant are working on various aspects of paratuberculosis vaccine. I work with the team members to accomplish the project goals and at the same time develop their technical, professional and presentation skills. I supported participation of team members in both local and national meetings where they presented their research findings. When experiment conclude, I will work with the students to present the project findings through scientific reports and presentations in national and international conferences. In general, training is provided in different aspects of virology and genetic manipulations (Basic bacteriology, genetics and genomics). In addition, more chances are given for training on different approaches to analyze host immune responses (Immunology). How have the results been disseminated to communities of interest?Two reports submitted. Presentation in the following meetings. Next Generation Polyanhydride Nanovaccine Platform Technology For Poultry. GRC on Nanoscale Science and Engineering for Agriculture and Food Systems. June 3-8, 2018. South Hadley, MA. Nanovaccines in Sickness and in Health. Illinois Institute of Technology Seminar. October 16, 2018. Nanovaccines for animal diseases, the Polyanhydride platform technology. European Congress on Vaccine and Vaccination 2018. October 26-27, 2018. Budapest, Hungary. Nanovaccines and Nano-adjuvants for Efficient Vaccination Programs. World Vaccine Congress., April 14-17, 2019. Washington D.C. A Novel Nanovaccine With Broad Spectrum Immunity Against Different Avian Influenza Subtypes. Annual Egyptian Veterinary Poultry Association. April 20-23. Porto Marina, Alexandria, Egypt. What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

What was accomplished under these goals? -To test for the safety of PAN (Aim I), specific pathogen-free (SPF) eggs were inoculated with PAN particles loaded with Fluorescein isothiocyanate (FITC) and hatchability rate and embryo development compared with mock (PBS-inoculated) group. Normal embryo development was observed in PAN-FITC inoculated eggs and hatchability rates of PAN-FITC inoculated eggs was identical to mock inoculated eggs (100%). Furthermore, the dosage of PAN particles (10 mg) administered in this pilot experiment was 10 times more than the normal PAN-adjuvanted vaccine dose (1 mg). This pilot study demonstrates the safety associated with administration of PANs in chicken embryos. In subsequent experiments, we prepared PAN-based vaccines and inoculated 1 day old chicks with 2 mg of PAN-Flu vaccine with no untoward effect on any of the immunized chicks. To develop PAN as novel vaccine platform technology, we estimated the immune responses (Aim II) generated from 1 day old chicks immunized with PAN-S encapsulating S1 protein. In addition, we used whole inactivated IBV virus as a rapid protocol to develop an effective vaccine against IBV. The Narasimhan lab synthesized PAN-S and PAN-IBV inactivated vaccine candidates following diacid synthesis, 20:80 CPTEG:CPH copolymer using a water-oil-oil double emulsion process. Quality control (QC) for all particles were performed on each batch consisted scanning electron microscopy (SEM) indicated the size PAN-IBV size range of 80-120 nm. In addition, the release kinetics of PAN-S indicated a 20% burst of payload after 1 day of PAN-S suspension in water while the continuous release of antigen continued up to the end of observation period at 5 days post suspension in water. For estimating protective immunity of PAN vaccine candidates (Aim III), 1 day old chicks were immunized with 2 mg PAN loaded with 20 ug IBV whole virus or S1 protein via intramuscular (IM) injection. At 3 weeks post immunization, all chicks were challenged with a virulent virus strain (Ark-DPI, 6.5E9 genome copy no/bird). At 8 days post challenge, PAN-S vaccine and MLV chicks have increased serum IgY titers relative to unvaccinated chicks while PAN-IBV (whole-inactivated virus) did not (data not shown). In addition, significant high levels of viral shedding in mock (Negative control) vaccinated chicks were detected in tracheal swabs compared to those birds vaccinated with modified live vaccine MLV (Positive control) or PAN-S (only S1 protein encapsulated PAN). Clinically, animals vaccinated with PAN-S1 didn't show the full spectrum of disease progress (coughing and sneezing) compared to un-vaccinated chicks (data not shown). These results demonstrate that PAN adjuvanted S1 vaccines can elicit protective immunity in chickens when challenged with virulent strains of IBV.


  • Type: Journal Articles Status: Submitted Year Published: 2019 Citation: 1. Thukral, A., Ross, K, Hansen, C., Phanse, Y., Narasimhan, B., Steinberg, H., Talaat, A.M.* (2018). A Single Dose Polyanhydride-based Nanovaccine Against Johnes Disease. (submitted npj.Vaccines). 2. Kingstad-Bakke, B.A.; Chandrasekar, S.S.; Phanse, Y.; Ross, K.A.; Marulasiddappa, S.; Kawaoka, Y.; Osorio, J.E.; Narasimhan, B.; Talaat, A.M. Effective Mosaic-based Nanovaccines against Avian Influenza in Poultry. (Submitted, Vaccines).