Ferrets have the potential to become an important animal model of influenza infection, as they most closely demonstrate the disease symptoms and pathology that occurs in humans. Ferrets are susceptible to infection with human as well as avian influenza A with the potential to cause human pandemics. However, the availability of reagents with which to analyze cellular immune responses in ferrets is limited to cross-reacting anti- mouse and anti-human antibodies as the genome of the domestic ferret has not yet been sequenced. To generate the reagents necessary to perform these studies we are generating retroviral cDNA-GFP fusion protein libraries from mRNA isolated from activated ferret PBMCs. Those clones that express membrane proteins will be sequenced and identified by homology to the feline and canine genomes. The vectors encoding those proteins of interest for use in immunological assays for analysis of ferret cellular immune responses can then be used to generate murine hybridomas by DNA immunization with the cDNA constructs subcloned into plasmid vaccine vectors, and the resulting murine monoclonal antibodies screened for antigen specificity and affinity on retrovirus transduced human cell lines and ferret cells. These reagents will then be validated for flow cytometryic analyses of the cellular immune responses of ferrets to influenza virus challenge.
Completion of the proposed research will provide the ability to perform analyses of cellular immune responses against influenza A infection and determine the correlates of immune protection against this virus in ferrets. In addition, the cDNA libraries will be useful for those interested in further studies of ferret gene expression in lymphoid tissues and PBMCs. Similar methodology would be useful for generating antibody reagents for other model organisms for which genomic sequence data is not available, such as the cotton rat.