The goal of this proposal is to determine the influence of subcellular localization and the flanking amino acids on the presentation of an HIV gp 160 epitope to cytotoxic T lymphocytes (CTL). CTL provide a critical component of the protective immune response. Their major role is the elimination of virus infected cells. A prerequisite for the recognition of infected cells is the intracellular degradation and selective presentation of viral antigens in the form of peptides associated with class I molecules at the cell surface. The limited natural selection of viral CTL epitopes is determined by intra- and extracellular mechanisms that are poorly understood. Recombinant vaccinia and sindbis virus expression systems will be used to express peptides containing a defined CTL epitope from HIV. This epitope was chosen because it is recognized by both human and murine CTL. Using molecular techniques these peptides will be localized to the cytosol and the endoplasmic reticulum (ER). Two membrane bound forms of the peptide, one on the cytoplasmic and the other on the luminal side of the ER will be expressed from minigenes. A cytoplasmic """"""""preprocessed"""""""" form of the epitope will be used as control for transport into the ER and assembly with the murine Dd class I molecule. The resulting peptides will be tested for their ability to be processed and presented to gp 160 specific CTL. These studies will provide information on the mechanism(s) and subcellular localization of antigen processing. The influence of flanking amino acid sequences on processing will be analyzed by substitution of the wild-type flanking residues to provide insight into the use of recombinant minigenes as potential vaccines. Finally, the subcellular localized antigens will be used to examine the role of the peptide transporter which is believed to be responsible for entry of peptides into the ER. Their role in antigen processing and transport of epitopes will be investigated by testing the localized peptide antigens in cells exhibiting transporter mutations and by downregulating transporter gene expression in normal cells using antisense technology.
| Stohlman, S A; Bergmann, C; Cua, D et al. (1994) Location of antibody epitopes within the mouse hepatitis virus nucleocapsid protein. Virology 202:146-53 |
| Bergmann, C; Stohlmann, S A; McMillan, M (1993) An endogenously synthesized decamer peptide efficiently primes cytotoxic T cells specific for the HIV-1 envelope glycoprotein. Eur J Immunol 23:2777-81 |
