Augmentation of cytotoxic T cells (CTL) response using stress proteins is emerging as a new treatment for a number of tumor models. The applicants propose that a similar approach holds much promise as a vaccination mode for HIV infection. The ability of stress proteins to elicit T cell responses is known to arise from peptides bound to them and not from their own sequences. Stress proteins are thought to escort antigenic peptides bound to them to the ER, where the peptides are transferred to immature MHC class I molecules for indirect antigen presentation to CTL. While this unorthodox pathway was previously invoked for one stress protein, the applicants have now shown that the peptide binding capacity of another stress protein, BiP, can be harnessed for this purpose. Furthermore, these investigators have shown BiP to bind viral peptides in vitro and then to augment the activation of CD8+ clones directed against the virus. Now they intend to apply these findings to HIV and explore the feasibility of developing the BiP-mediated indirect presentation pathway into an anti-viral immunization method. In order to utilize """"""""re-presentation"""""""" of HIV peptides by BiP as a vaccine, better understanding of how the pathway works is necessary. Therefore, the applicants propose to first determine whether BiP can bind a spectrum of known MHC class I-restricted HIV peptides using their recently developed assays. Second, the applicants will use microscopy and biochemistry to determine if the presentation will require uptake of HSP-peptide complexes by professional antigen-presenting cells like macrophages and dendritic cells, or if it can occur at the cell surface. Third, using a non-releasing mutant BiP, the applicants will try to determine whether ATP-driven release of peptide is needed for the transfer to MHC class I proteins, and by using TAP-deficient antigen presenting cells, whether peptide needs be transported into the ER to be presented in this pathway. Fourth, the applicants proposed to assess the importance of proteolytic trimming of the peptides inducted by the BiP pathway for MHC class I protein presentation. Finally, fusion peptides will be made between good BiP binders and good HIV CTL antigens, and tested as a general means of enhancing specific CTL responses. It is hoped that the proposed in vitro experiments will define effective techniques for augmenting antigen presentation by BiP, which in turn may lead to tests in animal models and eventually clinical trials.
