Maximizing CTL responses: The effects of covalent peptide linkage to beta 2 -microglobulin

Major histocompatability molecules (MHC) are involved in the display of self and non-self peptide antigens for recognition by the immune system. The density of such displayed peptides is crucial to the immune system's responsiveness; this especially becomes of value in the generation of strong T cell responses against foreign immunogenic peptides and disease. There have been multiple approaches used to increase cell-surface MHC-peptide complex density and stability, including peptide sequence modification and covalent attachment of immunogenic peptides to MHC I heavy chains. The idea behind the covalent linkage of peptides to MHC molecules is that it allows for an increase in a peptide's avidity for its relevant MHC molecule. As a result, the half-life of the complex on the cell surface and functional peptide density is increased. The ability to increase the half-life and functional peptide density of an MHC I-peptide complex increases the likelihood of eliciting stronger CTL responses to clinically important peptides and potentially to some peptides that are hampered by low or suboptimal MHC I binding affinity in their free form. We have hypothesized that the covalent linkage of the proven murine immunogenic peptide, iL3, to human Beta 2- microglobulin (hbeta2m) would increase peptide immunogenicity and therefore in vivo effectiveness as a vaccine. We have developed the iL3-L 12-hbeta2m fusion protein based on the proven mouse immunogenic peptide, iL3, which is derived from the immunogenic ribosomal protein, H2.16, expressed in a mutagenized murine fibroblastic tumor cell line, BCA-34. The iL3 fusion protein and the corresponding experimental negative control, the H-2Kd-binding NP(147-155) influenza-peptide fusion protein (NP-L12-hbeta2m), were produced in E. coli for exogenous antigen application onto dendritic cells, as well as in recombinant vaccinia virus for endogenous antigen expression. The iL3-L12-hbeta 2m fusion protein was found to productively assemble on the surface of H-2Kd-expressing mouse target cells and was capable of sensitizing target cells to lysis by iL3-specific CTL. Furthermore, mice immunized with dendritic cells pulsed with this iL3 fusion protein were able to reject a challenge with iL3+ BCA-34 cells. Collectively, this data demonstrates the potential of vaccine development that incorporates peptides covalently-linked to beta 2-microglobulin.