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With the ease of aersol transmission, respiratory infections are by nature highly contagious. The continuous emergence of new avian influenza viruses (AIV) is of particular concern because of the potential to infect multiple species. Genetic shifts and drifts in the genome provide ideal conditions for changing host specificity and pathogenesis. Furthermore, serotype definition and antibody protection depend on responses to two glycoproteins. Control measures in domestic animals have depended on eradication and vaccine strategies. Adaptive immunity, especially cross-reactive CD8+ T cell immunity, is critical to the resolution of infection. Memory responses are really the essence of an effective vaccine and immune protection. The availability of inbred chicken lines with well-defined MHC haplotypes has allowed for characterization of avian MHC restricted T cell immunity. Our ultimate aim is to exploit natural regulatory mechanisms to enhance cross-reactive, protective CD8+ T cell memory. Objective 1 will evaluate the in vitro and in vivo functions of the initial effector and the subsequent memory CD8+ T cell responses with respect to the kinetics and response to individual AIV proteins. Objective 2 will identify cytokines that enhance CD8+ T cell immunity, first by in vitro stimulation of memory T cells and second by administering cytokines in vivo with cDNA vaccine immunization. The functions of CD8+ T cells will be determined using well-established procedures; cytotoxic T cell assays, detection of IFN? secretion and adoptive transfer to chickens prior to AIV infection. This study will focus on identifying cytokines that induce vigorous memory CD8+ T cells.
NON-TECHNICAL SUMMARY: <p>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 being infected. 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 which given as a vaccine. Furthermore, in order to maximize the immunity, we will add proteins to the vaccine that are know to regulate the immune response in the chicken.
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APPROACH: <p>The long-term success of adaptive immunity depends on the integrity and vigor of memory cell responses. We will evaluate the CD8+ T lymphocyte responses to AIV using our established procedures. In addition to the CTL assay, our in vivo assay in which the potential for adoptively transferred immune cells to protect chicks against challenge infection will be included. CD8+ T cells have been demonstrated that respond to AIV could be detected by CTL and adoptive transfer assays. APC used to quantify CTL responses and to stimulate CD8+ T cells in these studies will be AIV infected and UV treated chicken CK cells derived from MHC matched or mismatched chicks. Our Texas isolate, Ck/TX/02, which causes mild respiratory illness will be used. Birds will be infected with Ck/TX/02 . The long-term success of adaptive immunity depends on the integrity and vigor of memory cell responses. We will evaluate the CD8+ T lymphocyte responses to AIV using our established procedures. In addition to the CTL assay, our in vivo assay in which the potential for adoptively transferred immune cells to protect chicks against challenge infection will be included. CD8+ T cells have been demonstrated that respond to AIV could be detected by CTL and adoptive transfer assays. APC used to quantify CTL responses and to stimulate CD8+ T cells in these studies will be AIV infected and UV treated chicken CK cells derived from MHC matched or mismatched chicks. <p>Our Texas isolate, Ck/TX/02, which causes mild respiratory illness will be used. Birds will be infected with Ck/TX/02. Following AIV infection, the peak initial effector response, responsible for control of acute infection, and the rebound of AIV specific T cells, the memory response, will be determined. CD8+ T cell specificity of protection will be determined following depletion of CD4+ or CD8+ T cells prior to evaluating function. We will examine all 10 AIV proteins for their in vivo capacity to stimulate CD8+ T cells using APC expressing the individual proteins. The T cells will be prepared from blood collected after AIV infection of B2/B2 at the peak effector response and after memory cells have been well established. <p>The AIV specificity will be determined with the in vitro CTL using APC expressing the individual viral proteins. The responses to varying dilutions of effector to target cells will be determined and the overall responses to each protein statistically compared. Having identified the kinetics of the CD8+ T cell responses to AIV, individual proteins that induce the response and markers that are characteristic of effector or memory (memory effector) cells, the impact of cytokines on the CD8+ T cell function and phenotype during the effector and memory cell expansion will be examined. The in vitro influence of cytokines on AIV specific CD8+ T cells will be determined before evaluating the regulation associated with selected cytokines in in vivo protection. We will concentrate on responses to APCs with SFV vectors expressing individual viral proteins shown to elicit strong CD8 responses and one viral proteins that elicits detectable but weaker responses. The practical objective is to identify enhancing cytokines.