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United States Department of Agriculture

Agricultural Research Service

Research Project: Characterization of Protective Host Responses to Avian Influenza Virus Infections in Avian Species

Location: Exotic & Emerging Avian Viral Diseases Research

2013 Annual Report

1a. Objectives (from AD-416):
1. Conduct comparative immunology studies of avian species to determine variations in protective innate defense mechanisms to avian influenza infections. 2. Characterize humoral and cellular immune responses to avian influenza viruses and identify epitopes associated with immunity. 3. Develop immunological reagents and methodologies to evaluate vaccine efficacy and protection.

1b. Approach (from AD-416):
The project plan has three interrelated objectives that are designed to increase our basic understanding of the immunological response to avian influenza virus (AIV) infection in different avian species and will result in improved vaccine development. The first objectives initiate the characterization of host cytokine expression profiles from specific innate immune cells in vitro. These profiles will then be compared with cytokine profiles obtained from avian influenza (AI) infected tissues, in vivo, for a better understanding of the overall innate immune response to avian influenza viruses (AIV). Innate immune cells will be isolated or produced for in vitro analysis from multiple sources, including: specific pathogen-free (SPF) White Leghorn (egg laying-type), SPF White Plymouth Rock (meat-type) chickens, and SPF Small Beltsville White SPF turkeys from the Southeast Poultry Research Laboratory. In addition, some experiments will utilize commercial chickens, turkeys, geese, ducks or others as needed, as sources for primary cell culture. We have in-house supplies of SPF birds and eggs, which can be used for these studies. In addition, we also have access to major histocompatibility complex (MHC)-defined birds (Avian Disease Oncology Laboratory, East Lansing, MI) which can be used for immunogenetic comparison of cytokine responses within members of Gallus gallus species. Expected outcomes for this objective will be the determination of which cytokines and transcription factors contribute to a resistant phenotype of avian species to AIV. It may be likely that different profiles are determined for different bird species. Once innate immunity profiles have been established, the cellular and humoral immune responses that contribute to natural and vaccine-induced protection will be characterized. We will utilize chickens, turkeys, ducks and other bird species in these studies. Experiments in Objective 2 will determine antibody levels following infection and vaccination, identify cellular immune responses to homologous and heterologous AI isolates, and determine putative epitopes involved with immunity. We will specifically address: (1) the induction of anti-viral antibodies that correlates with protective efficacy, decreases in virus shedding, and provide cross-reactivity to homo- and heterosubtypic isolates, (2) the induction of cellular immunity in poultry and wild birds following infection or vaccination and challenge, (3) identify T-cell epitopes to the hemagglutinin and nucleoprotein proteins of AI. Finally, we will develop immunological reagents and methodologies to evaluate vaccine efficacy and protection. Besides the usual indicators of vaccine induced protection, including survival, or decreases in shedding, assays to determine why and how a particular vaccine induces immunological protection against challenge are lacking. By incorporating cytokines and toll-like receptor agonists into vaccine formulations, their contributions to humoral and cellular immunity can be evaluated. Finally, the extent of cross protective immunity developed in vaccinated birds will be examined by utilizing antigenic cartography.

3. Progress Report:
The Immunology project has been active both nationally and internationally to meet the objectives and milestones of the project. Accomplishments included; 1) the production and characterization of individual immune cell types to examine how they immunologically react to avian influenza, 2) the protection of poultry from highly pathogenic avian influenza through application of vaccines, and 3) the study of pathology and immune responses to avian influenza viruses and vaccines in different bird species. Collaborative research continues with national and international partners to continue to study the immune response of poultry to avian influenza. University partners include, but not limited to, the University of Georgia, the University of Delaware, the University of Arkansas, and the Ohio State University. Collaborative work with industry has included projects with CEVA Biomune, and Goldsboro Milling Company. Internationally, collaboration with the US Department of Agriculture, Office of International Research Programs and the All Russian Research Institute for Animal Health has continued to support the development of vaccines and immunology against avian influenza.

4. Accomplishments
1. Vaccine protection of poultry against the 2012 H7N3 highly pathogenic avian influenza virus currently circulating in Mexico. Highly Pathogenic (HP) AI is an economically important disease of poultry with high morbidity and mortality that has significant impact on poultry production and global trade. New outbreaks of HPAI in commercial poultry represent one of the most critical diseases to contain and require reporting to the World Organization for Animal Health. Following an outbreak of HPAI in Mexico in 2012, we determined the virulence mechanisms unique to this virus and tested a number of different vaccine formulations for protection of poultry. It was demonstrated that the Mexican 2012 HPAI virus kills birds within days, but that several available vaccine viruses can provide complete protection of poultry from clinical disease but not infection. These studies provide information that can aid in the control of the virus in Mexico and help prepare the United States if the virus was introduced in our poultry population.

2. Understanding differences in pathogenicity and response to vaccination against H5N1 HPAI in domestic ducks. Domestic ducks are key hosts in the transmission of H5N1 highly pathogenic avian influenza (HPAI) viruses, and therefore are included in vaccination programs to control H5N1 HPAI. Clear differences in response to infection and vaccination were demonstrated between the two most common domestic duck species, Pekin (Anas platyrhinchos domesticus) and Muscovy (Cairina moschata), with the Muscovy ducks presenting a more severe disease than the Pekin ducks and responding poorly to vaccination. These differences were in part explained by differences observed in the in the immune response in the infected ducks. This information is fundamental for developing effective vaccination programs for controlling H5N1 HPAI in different species of ducks.

3. Contribution of dendritic cells to pathogenesis and immune response of poultry to avian influenza. Dendritic cells (DC) are professional antigen-presenting cells of the immune system that function to initiate primary immune responses, and stimulate anti-viral immunity. They are specialized at capturing and processing virus for immune presentation. The pathologic and immune characteristics of chicken DCs following infection with high and low pathogenic avian influenza viruses were determined. Chicken DCs were determined to take up avian influenza virus, and supported replication of both high and low pathogenic influenza viruses. The DCs mounted a robust antiviral immune response, including interferon alpha. These studies are critical in understanding the immunological response of birds to avian influenza for development of control strategies.

4. Maternal antibody to avian influenza virus suppresses the immune response to viral-vectored vaccines to avian influenza virus. Avian influenza is an important poultry pathogen that is often controlled through the use of vaccines, but maternal antibody that comes from the egg yolk can potentially suppress the immune response during the early life of the chick. Using a passive antibody model system to mimic maternal antibodies, two differnet viral-vectored vaccines were tested with or without maternal antibody and it was shown even for this type of vaccine that maternal antibody will at least partially suppress the immune response to this type of vaccine. It is important to get an early response to vaccines for poultry disease because the production life of chickens is so short, and this study shows that the new generation of vaccines does not resolve the vaccine suppresion issue.

Review Publications
Kapczynski, D.R., Pantin Jackwood, M.J., Guzman, S.G., Ricardez, Y., Spackman, E., Bertran, K., Suarez, D.L., Swayne, D.E. 2013. Characterization of the 2012 highly pathogenic avian influenza H7N3 virus isolated from poultry in an outbreak in Mexico: pathobiology and vaccine protection. Journal of Virology. 87(16):9086-9096. Available:

Kapczynski, D.R., Afonso, C.L., Miller, P.J. 2013. Immune responses of poultry to newcastle disease virus. Developmental and Comparative Immunology. 41(3):447-453. Available:

Faulkner, O.B., Estevez, C., Yu, Q., Suarez, D.L. 2013. Passive antibody transfer in chickens to model maternal antibody after avian influenza vaccination. Veterinary Immunology and Immunopathology. 152(3-4):341-347.

Last Modified: 05/25/2017
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