Research Project
3138685
The HIV envelope protein (ENV-P); which is expressed on the surface of actively infected cells, appears to play an important antigenic role in the immune recognition of the infected cells. The purpose of this proposal is to define the epitopes on the ENV- P that are recognized by MHC class I and class II restricted mouse and human T cells and to examine what role, if any, the presence of a carbohydrate moiety near to a potential T cell epitope may have on its expression. Two distinct sources of ENV-P antigen will be compared. Affinity purified ENV-P obtained from mammalian cells transfected with the ENV-P gene (containing amino acids 61-531 of the mature gp120 vs. a recombinant vaccinia vector that contains the proviral gp160 gene (V-ENV-P) and expresses the mature gp120 protein on the surface of infected cells will be used as the sources of intact antigen in order to generate class I and class II-restricted ENV-P- specific mouse and human T cells. HPLC fractionated ENV-P peptides generated in vitro by trypsin and CNBr cleavage along with 20 amino acid overlapping synthetic peptides covering the entire ENV-P molecule will be prepared and used as the source of antigen in order to locate and characterize the epitopes recognized by the ENV-P-specific T cells. We will determine, by virtue of the ability of synthetic peptides to bind to isolated MHC molecules, which peptides contain potential T cell epitopes. These results will be compared with those for the actual T cell epitopes defined for T cells primed with intact ENV-P or infected with V-ENV-P. Peptides found to contain nonexpressed T cell epitopes will be utilized in studies to determine why, perhaps due to the presence of carbohydrate side chains, these epitopes are not expressed. In summary, the results of this study will provide comprehensive information about the epitopes and thereby the regions of the HIV ENV-P that are recognized by class I and class II restricted mouse and human T cells. We will be able to evaluate whether these epitopes fall in conserved or variable regions of the ENV-P molecule, whether potential epitopes are not expressed because of APC processing of the native ENV-P molecule, whether carbohydrate molecules somehow influence the recognition of particular regions of the ENV-P by T cells and whether similar ENV-P epitopes are generated by APC given isolated ENV-P vs. synthesis of ENV-P by infected APC.
