Equine infectious anemia virus (EIAV) is a hematogenously spread lentiviral infection that affects all members of the family Equidae and that has a worldwide distribution. The broad and long-term objective is to identify mechanisms of immune control of lentiviral infections. The control of clinical disease in horses and the maintenance of the inapparent carrier state during persistent infection with EIAV makes this an attractive model in which to study immune control of lentiviral infection. MHC-restricted, T-cell-mediated, viral-specific cytotoxicity has been demonstrated in EIAV. Identification of viral epitopes recognized by CD8+, class I-restricted cytotoxic T lymphocytes in carrier horses and the ability of identified epitopes to induce protective immunity in horses will be the focus of the proposed research. To identify viral epitopes recognized by CTL from carrier horses, three specific aims are proposed.
In specific aim 1, equine kidney (EK) cells with the ELA-A5 haplotype will be infected with a tissue culture adapted stain of EIAV, strain WSU5. EK cells will be lysed after 14 days in culture in order to free equine MHC class I (ELA class I) molecules and their bound peptides from the cell membrane. EK cells have been previously demonstrated not to express ELA class II antigens on their surface. ELA class I molecules and their bound peptides will be immunoaffinity purified from cell lysates; peptides will be acid-eluted from ELA class I molecules, separated by membrane centrifugation, and resolved by reverse-phase HPLC.
In specific aim 2, obtained RP-HPLC fractions will be screened for their ability to sensitize target EK cells (ELA-A5) for lysis by in-vitro stimulated CTL from carrier horses. Peptides will be further purified from fractions, sequenced and those encoded by EIAV identified. Synthetic peptides corresponding to identified EIAV peptides will be screened by limiting dilution chromium release cytotoxicity assays in order to determine which peptides are recognized by predominant CTL memory cells in carrier horses.
In specific aim 3, naive horses with an ELA-A5 haplotype will be immunized with EIAV synthetic peptides obtained in specific aim 2, and then challenged with live, homologous strain virus, in order to determine if viral peptides identified from infected EK cells and recognized by predominant CTL from carrier horses can induce protective immunity in naive horses. The candidate is a veterinarian with two years of clinical medical experience and three years of residency training in comparative anatomic pathology. The candidate's long-term career goal is to contribute to the development of vaccines that prime T cell mediated immunity and to elucidate mechanisms of immune control of lentiviral infections. The Department of Veterinary Microbiology and Pathology at Washington State University provides an environment with a history of productive collaborative research utilizing interdisciplinary expertise in lentivirology, microbiology, pathology, immunology, and molecular biology.
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