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55-OR
S50
Abstracts
Thursday, October 19, 2006 4:00 PM – 5:30 PM Session VII: Groovy ASHI Scholar 55-OR
DIRECT DISCOVERY OF CLASS I HLA WEST NILE VIRUS EPITOPES Curtis P. McMurtrey,1 Wilfred Bardet,1 Annette Fleshman,1 Christina Mason,2 Angela Wahl,1 Kenneth Jackson,2 William Hildebrand.1 1Microbiology/Immunology, University of Oklahoma Health Sciences Center, Oklahoma City, OK, USA; 2Molecular Biology Proteomics Labratory, University of Oklahoma Health Sciences Center, Oklahoma City, OK, USA Aim: While there are several indirect approaches for indicating the class I epitopes unique to West Nile Virus infected cells, direct empiric detection of class I presented epitopes unique to infected cells has not been pursued. Here we describe our method for the WNV infection of HLA-A*0201 secreting cells in a hollow fiber bioreactor unit. We describe our method for infecting cells in the bioreactor and for monitoring the viral infection to ensure that we are obtaining peptide from infected cells. Methods: Our process includes the infection of cells that secrete HLA-A*0201 and B*0702, purification of the soluble HLA class I, elution of the peptides from the class I, and LC-MS analysis of peptides unique to, or increased upon, infected cells. During the infection of the cells in the bioreactor, WNV infection of these cells are monitored using immunohistochemistry and florescence in situ hybridization combined with flow cytometry. Additionally viral titer in harvests are monitored using TaqMan based RT-PCR. Scope: West Nile Virus is an intracellular pathogen that infects numerous mammals, including humans. Because WNV is an intracellular viral pathogen, cytotoxic T lymphocytes (CTL) are positioned to eliminate infected cells by recognizing class I HLA presented peptide epitopes. CTL are known to respond to WNV, and the identification of HLA class I peptide epitopes unique to WNV infected cells is positioned to aid in the development of vaccines, diagnostics, and therapeutics. Conclusions: We demonstrate the bioreactor production of class I HLA-A*0201 and B*0702 from WNV infected cells, the monitoring of infection, the purification of peptides from the class I HLA of infected cells, and the comparative LC-MS mapping of these peptides. We demonstrate the class I HLA reveal both hostencoded and virus-encoded epitopes following WNV infection.
56-OR
CHARACTERIZATION OF ENDOGENOUSLY LOADED MHC CLASS I PEPTIDES DURING INFLUENZA INFECTION Angela R. Wahl, Fredda Schafer, Wilfried Bardet, Rico Buchli, Annette Fleshman, Gillian Air, William H. Hildebrand. Microbiology and Immunology, University of Oklahoma Health Sciences Center, Oklahoma City, OK, USA Aim: The influenza virus infects 5% to 20% of the United States population every year and elicits a mild to severe respiratory illness. Natural Influenza A infection generates both strong humoral and Cytotoxic T lymphocyte (CTL) responses while vaccination mounts a strong humoral response but a weak CTL response. CTLs target and destroy influenza-infected cells by recognizing viral-derived peptides in context of the class I molecule on the surface of infected cells. Characterization of the class I ligands that mark infected cells during influenza infection is important to understand the nature of the CTL response during infection and for the development of anti-viral vaccines. Methods: Our laboratory has developed a novel hollow fiber bioreactor method to collect milligram quantities of endogenously loaded class I molecules. Infected and uninfected peptide pools are subject to mass spectrometry to identify and sequence peptides that are unique to infected cells or exhibit increased presentation. Scope: Direct epitope discovery of Influenza-infected cells has important implications for viral diagnosis, vaccine development and mediation of immune responses. Conclusions: We have identified multiple Influenza-derived HLA class I peptides including those derived from hemagglutinin and nucleoprotein molecules. In addition, we have characterized several virus induced changes in host-derived peptide presentation. These epitopes are currently being validated by peptide binding assays, ELIspot, and the staining of infected cells with TCR mimics.
Abstracts
Thursday, October 19, 2006 4:00 PM – 5:30 PM Session VII: Groovy ASHI Scholar 55-OR
DIRECT DISCOVERY OF CLASS I HLA WEST NILE VIRUS EPITOPES Curtis P. McMurtrey,1 Wilfred Bardet,1 Annette Fleshman,1 Christina Mason,2 Angela Wahl,1 Kenneth Jackson,2 William Hildebrand.1 1Microbiology/Immunology, University of Oklahoma Health Sciences Center, Oklahoma City, OK, USA; 2Molecular Biology Proteomics Labratory, University of Oklahoma Health Sciences Center, Oklahoma City, OK, USA Aim: While there are several indirect approaches for indicating the class I epitopes unique to West Nile Virus infected cells, direct empiric detection of class I presented epitopes unique to infected cells has not been pursued. Here we describe our method for the WNV infection of HLA-A*0201 secreting cells in a hollow fiber bioreactor unit. We describe our method for infecting cells in the bioreactor and for monitoring the viral infection to ensure that we are obtaining peptide from infected cells. Methods: Our process includes the infection of cells that secrete HLA-A*0201 and B*0702, purification of the soluble HLA class I, elution of the peptides from the class I, and LC-MS analysis of peptides unique to, or increased upon, infected cells. During the infection of the cells in the bioreactor, WNV infection of these cells are monitored using immunohistochemistry and florescence in situ hybridization combined with flow cytometry. Additionally viral titer in harvests are monitored using TaqMan based RT-PCR. Scope: West Nile Virus is an intracellular pathogen that infects numerous mammals, including humans. Because WNV is an intracellular viral pathogen, cytotoxic T lymphocytes (CTL) are positioned to eliminate infected cells by recognizing class I HLA presented peptide epitopes. CTL are known to respond to WNV, and the identification of HLA class I peptide epitopes unique to WNV infected cells is positioned to aid in the development of vaccines, diagnostics, and therapeutics. Conclusions: We demonstrate the bioreactor production of class I HLA-A*0201 and B*0702 from WNV infected cells, the monitoring of infection, the purification of peptides from the class I HLA of infected cells, and the comparative LC-MS mapping of these peptides. We demonstrate the class I HLA reveal both hostencoded and virus-encoded epitopes following WNV infection.
56-OR
CHARACTERIZATION OF ENDOGENOUSLY LOADED MHC CLASS I PEPTIDES DURING INFLUENZA INFECTION Angela R. Wahl, Fredda Schafer, Wilfried Bardet, Rico Buchli, Annette Fleshman, Gillian Air, William H. Hildebrand. Microbiology and Immunology, University of Oklahoma Health Sciences Center, Oklahoma City, OK, USA Aim: The influenza virus infects 5% to 20% of the United States population every year and elicits a mild to severe respiratory illness. Natural Influenza A infection generates both strong humoral and Cytotoxic T lymphocyte (CTL) responses while vaccination mounts a strong humoral response but a weak CTL response. CTLs target and destroy influenza-infected cells by recognizing viral-derived peptides in context of the class I molecule on the surface of infected cells. Characterization of the class I ligands that mark infected cells during influenza infection is important to understand the nature of the CTL response during infection and for the development of anti-viral vaccines. Methods: Our laboratory has developed a novel hollow fiber bioreactor method to collect milligram quantities of endogenously loaded class I molecules. Infected and uninfected peptide pools are subject to mass spectrometry to identify and sequence peptides that are unique to infected cells or exhibit increased presentation. Scope: Direct epitope discovery of Influenza-infected cells has important implications for viral diagnosis, vaccine development and mediation of immune responses. Conclusions: We have identified multiple Influenza-derived HLA class I peptides including those derived from hemagglutinin and nucleoprotein molecules. In addition, we have characterized several virus induced changes in host-derived peptide presentation. These epitopes are currently being validated by peptide binding assays, ELIspot, and the staining of infected cells with TCR mimics.