The human immune response to vaccine antigens such as HBsAg depends on processing to antigenic fragments, binding of the fragments to the groove of the major histocompatibility antigens, and recognition of the combination of antigen + MHC by the T cell receptor. We have previously characterized a series of 12 cloned T cell lines from two HBsAg vaccine recipients. These were restricted to different MHC antigens of the donors and were presumably specific for different epitopes of HBsAg. This project is to map the epitopes recognized by these T cell clones and to determine: a) whether one epitope predominates, as has been reported for a site in the first 30 amino acids of HBsAg; b) whether different MHC types present different epitopes; c) since different T cell clones recognize the products of only one of the two antigen processing pathways, whether the two processing pathways produce different sets of antigenic fragments; and d) whether protein antigens can gain access to the nonendosomal processing pathway via membrane fusion using a fusion peptide sequence found in HBsAg. We have used PCR to cut up the gene for HBsAg into four overlapping fragments. These were cloned into the p-alpha cloning vector which grows in E. coli and yeast. So far, we have cloned all four fragments in E. coli and expressed the amino terminal fragment in yeast. The fragment was secreted, was the right MW on SDS gel, and stained in a Western blot with antibodies specific for HBsAg. This material did not stimulate a T cell clone specific for HLA DR7 and the endosomal processing pathway, and further study is underway using another T cell clone that uses DR6 and the nonendosomal processing pathway. As additional fragments become available, we will test our full panel of HBsAg specific T cell clones and hybridomas specific for HBsAg. In this way, we can analyze the effect of antigenic specificity on: vaccine responsiveness, viral protection, and disease outcome.
