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The Role of Chicken T Lymphocytes in Protective Immunity to Avian Influenza Virus

Lupiani, Blanca; Collisson, Ellen
Texas A&M University
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AIV, highly contagious and genetically variable, is a targeted pathogen for USDA funding. Outbreaks of AIV are followed by the application of stringent and costly biosecurity measures to prevent the contamination and movement of animals and humans. Because the promiscuous, zoonotic nature of AIV makes the use of live vaccines suspect, recombinant vaccines are the most realistic alternatives. However, the most effective vaccines should incorporate epitopes that induce specific CD8+ T cells. The ultimate goal of our research is to exploit mechanisms that enhance viral specific CD8+ T lymphocyte immunity to improve the efficacy of recombinant vaccines.

We will characterize the CD8+ T cell response to AIV infection and identify AIV proteins that are responsible for induction of protective CD8+ T cells. Future studies will identify cytokines that can be incorporated into vaccines to induce maximum immune responses. Specific objectives are to:

  1. Evaluate the role of chicken effector and memory CD8+ T cells in protection against AIV infection.
  2. Identify AIV proteins that induce vigorous CD8+ T cell responses.
More information

Avian influenza is a constant threat to poultry because it is constantly changing so that vaccines do not work and the virus may infect new hosts. Wild birds serve as the reservoir for AIV which can sometimes infect poultry which serve as an incubator for AIV infecting swine and humans. Because of its importance in animal and human medicine, control depends on providing a means to protect chickens from getting infected. Unlike current vaccines, those that provide protection must be effective against a broad range of genetically distinguishable strains of AIV.

In order to develop a vaccine that will protect against a broad spectrum of AIV, we will identify viral components that are both common to AIV strains and will induce protection when given as a vaccine. These proteins will be tested for the ability to induce protection when inoculated into the chicken as a vaccine.

PROGRESS: 2005/06 TO 2007/09
We have developed the tools and assays to evaluate T lymphocyte responses to avian influenza virus (AIV). This was the first year of a USDA grant to study memory responses to AIV. The B2/B2 and B19/B19 lines from Northern Illinois University are being used at this time as a source of immune cells. Kidney cells from 10-day old naive birds of each line have been expanded and frozen as a source of MHC compatible and mismatched antigen presenting cells (APC). Immune cells are collected from the peripheral blood mononuclear cells (PBMC) of AIV infected chickens 2 to 25 weeks of age and 10 days to 5 months p.i. Control PBMC are prepared from age and MHC matched birds that are not infected. We are using H5N9 or H5N3 stock viruses that have been titered in chicken 10-day old embryos. The assay identifies levels of IFNƒ× in the supernatants of induced T lymphocytes using NO secretion from monocytes. As the lymphocytes are incubated with APC prior to adding the sup to monocytes, the time of incubation with MHC matched AIV infected APC was optimized to 24 and 48 hours. PBMC are collected at 9 days p.i. in order to observe the effector response and after 4 weeks infection to evaluate the memory responses. The initial characterizations have been expanded in order to determine the CD8+ and CD4+ phenotype of responding lymphocytes for infected chickens. Because the studies will proceed to determine the responses to individual viral proteins, HA and NP genes have been cloned into the Semliki Forest virus vector system which will be used for expression in the matched and mismatched APC. In order to phenotype the responding lymphocytes, we have examined changes in cell populations with CD44 antigens using flow cytometry and ex vivo and in vitro stimulated T lymphocytes. In earlier studies, we similarly characterized the T lymphocyte responses of chickens to IBV. Our results indicate that the numbers of cells with median levels of CD44 on T lymphocytes from infected birds are increasing following at least 6 weeks of infection (detecting memory cells) and in vitro stimulation of the same T memory cell preparation by MHC matched APC expressing homologous AIV. The assays are now in place to characterize memory lymphocytes in terms of antigen specificity and phenotype. A huge problem working with AIV is that we can use only the low path viral strains which are non-invasive. In order to mimic an invasive response to proteins of high path AIV, we will use IBV as a vector for AIV proteins. IBV only infects avian species and typically progresses to tissue beyond the respiratory enteric tract. To develop this vector, we have replaced the 5 ORF of IBV with HA. The IBV genomic cDNA region with the HA insertion has been ligated into a cDNA of IBV that represents the 3' end encoding all of the structural proteins of IBV. Plasmids with fragments that cumulatively represent the entire genome have been amplified for reconstruction of a chimeric IBV with HA.

IMPACT: 2005/06 TO 2007/09
We have developed a new procedure for detecting responses of memory T lymphocytes following avian influenza virus infection. A method has been discovered that characterizes the phenotype of memory T lymphocytes specific for influenza virus. A coronavirus vector has been modified to carry the HA protein of avian influenza as first steps to develop a safe method to characterize responses to individual influenza proteins in the chicken and perhaps use as a practical vaccine for AIV.

Funding Source
Nat'l. Inst. of Food and Agriculture
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Bacterial Pathogens
Meat, Poultry, Game