A critical component in the defense against viruses and tumors relies on the presentation of viral or tumor derived peptides to CD8+ T-cells in the context of major histocompatibility class I molecules (MHC I). Successful presentation of such peptide antigens elicits cell mediated immune responses specific for host cells displaying these peptide antigens and thus serves as the central mechanism for irradication of abnormal (infected or transformed) cells. Clinically, the use of vaccines in order to augment immune responses has largely relied on stimulation via class II MHC molecules which primarily elicit humoral antibody responses as a result of the interaction of soluble peptide antigens, in context of these MHC class II molecules with CD4+ T-cells. The preference for class II mediated presentation of soluble antigens is based on the distinct loading pathways of class I and class II MHC molecules, which largely excludes soluble antigens from the class I loading pathway. Recently, approaches for the direct cell-surface loading of MHC class I molecules have emerged that are dependent on the presence of b2 microglobulin in the peptide innoculum, and, as we show, are based on the ability of b2 to stabilize newly emerging empty class I MHC molecules and make them receptive to peptide loading. This """"""""adjuvant"""""""" activity of b2 is dependent on its affinity for the MHC heavy chain, and has led to our studies in which we have engineered higher affinity variants of hb2 that appear to have improved abilities to stabilize cell-surface class I molecules, facilitate peptide loading, and stimulate MHC restricted, peptide specific T-cells in vitro. The high affinity variants currently being evaluated have mutations that promote either the formation of a salt bridge between residues of the MHC heavy chain and b2, or promote hydrophobic interactions and the exclusion of water molecules from the interface between the two chains, both of which should increase the strength of association between the chains. To further understand the mechanism of b2 induced peptide loading of cell-surface MHC molecules we have also generated variants of the high affinity forms of hb2 that contain a tag at their amino terminus that allows us to monitor specifically, the exchange of the exogenously added b2 with the endogenously acquired b2 on the cell surface. Our hope is that these high-affinity b2 adjuvants, and a better understanding of the mechanism of b2 induced cell-surface MHC I loading will lead to the development of novel peptide-based vaccines for the treatment of viral infections and cancers.
