|Ferman Ii, Geoffrey|
Submitted to: Keystone Symposia
Publication Type: Abstract Only
Publication Acceptance Date: February 20, 2006
Publication Date: March 28, 2006
Citation: Ferman Ii, G.S., Golde, W.T. 2006. Novel approach for identification of class i restricted t cell epitopes of foot-and-mouth disease virus. Keystone Symposia. 2006 P. 63 Technical Abstract: Foot-and-Mouth disease Virus(FMDV) is the causative agent for a highly contagious and economically important disease affecting cloven hoofed animals including cattle and swine. Neutralizing antibody is the hallmark of protection against this disease, and important target epitopes of neutralizing antibody have been reported. However, the identification of T cell epitopes and in particular, epitopes for cytotoxic T-lymphocytes would be an important advance toward the design of next generation FMDV vaccines with faster kinetics of protection and broader strain coverage for this rapidly mutating virus. Methods to identify such sequences using peptide libraries to stimulate secondary, in vitro cytotoxic T cells or CD4 T memory cells have yielded minimal results. We developed replication defective human adenovirus type-5(huAd5) constructs expressing portions of the viral genome or specific viral proteins to analyze the specificity of T cell responses but crossreactivity between the huAd5 vector and endogenous, porcine adenoviruses made this approach difficult. In an alternate approach, we have isolated swine MHC class I (SLA-I)molecules expressed on mouse fibroblast cells transfected with the alpha genes of SLA-I. Cell lines transfected with the SLA class I gene product SLA-1-0401 and SLA-2-0401 were infected with an attenuated virus (leaderless FMDV). This virus utilizes heparan sulfate as a receptor for viral attachment and entry into these mouse fibroblasts. Infection of the mouse fibroblasts with the leaderless FMDV allows for the processing and presentation of viral peptides via the endogenous pathway, loading peptides into the SLA-I molecules expressed by these cells. We have shown that SLA-I molecules are expressed by flow cytometry and confocal microscopy and are not affected by the attachment and internalization of the leaderless virus. Growth curve studies indicate that the virus enters these cells but does not replicate to the same titer as the parent, virulent virus. Subsequent analysis of the peptides that are bound by the SLA-I molecules by mass spectrometry should provide critical information for the formulation of more effective vaccines that utilize a CTL response to infection.