Identification of HHV-1 Peptide Epitopes Using Acid Elution and LC Triple Quadrapole Mass Spectroscopy

Recent advances in T cell immunobiology have allowed identification of numerous viral, bacterial, and tumor-derived epitopes recognized by CD8+ cytotoxic T lymphocytes (CTL). Most of these short 8-12 amino acid long sequences are generated by proteolytic fragmentation in the cell cytoplasm, prior to transport into the lumen of the endoplasmic reticulum (ER) by transporters of antigenic peptides (TAP), where they associate with nascent class I heavy chains and B(second power)-microglobulin. Herpesviruses are double stranded DNA viruses which establish latent infections and are widely prevalent in the host population. We have generated a protocol to characterize T-cell epitopes presented by MHC class I molecules and recognized by CD8+ cytotoxic T lymphocytes (CTL). We propose the three following technical AIMS: (1) HLA class I transfectants of C1R (EBV-B cell) will be infected with HHV-1 resulting in cell surface expression of viral epitopes presented by a uniform population of class I alleles. (2) Identify relevant viral peptide epitopes presented in the context of HLA-A1, -A2, -A3, -A11, and -A24. (3) Validate that synthetic peptides confer sensitivity to anti-viral CTL recognition (cytolysis and/or cytokine release) and demonstrate successful in vitro sensitization of normal CTL using autologous peptide-pulsed DC.