High-Affinity Interactions between Peptides and Heat Shock Protein 70 Augment CD8+ T Lymphocyte Immune Responses

Abstract

Exogenously delivered antigenic peptides complexed to heat shock proteins (HSPs) are able to enter the endogenous Ag-processing pathway and prime CD8+ CTL. It was determined previously that a hybrid peptide containing a MHC class I-binding epitope and HSP70-binding sequence Javelin (J0) in complex with HSP70 could induce cytotoxic T cell responses in vivo that were more robust than those induced by the minimal epitope complexed with HSP70. The present study introduces a novel, higher-affinity HSP70-binding sequence (J1) that significantly enhances binding of various antigenic peptides to HSP70. A competition binding assay revealed a dissociation constant that was 15-fold lower for the H2-Kb OVA epitope SIINFEKL-J1 compared with SIINFEKL-J0, indicating a substantially higher affinity for HSP70. Further, modifying the orientation of the hybrid epitope and introducing a cleavable linker sequence between the Javelin and the epitope results in even greater immunogenicity, presumably by greater efficiency of epitope processing. The enhanced immunogenicity associated with Javelin J1 and the cleavable linker is consistently observed with multiple mouse and human epitopes. Thus, by creating a series of epitopes with uniform, high-affinity binding to HSP70, successful multiple epitope immunizations are possible, with equal delivery of each antigenic epitope to the immune system via HSP70. These modified epitopes have the potential for creating successful multivalent vaccines for immunotherapy of both infectious disease and cancer.

Immunostimulatory Activity of Major Membrane Protein II fromMycobacterium tuberculosis

“…The exact mechanism of the high immunostimulating activity of the fusion protein is not fully known, but it may be associated with previous reports indicating that HSPs play a varied role in enhancing the ability of APCs to stimulate T cells (6,10,44,45). In fact, the fusion protein induced the expression of higher levels of APC-associated molecules on DC than MMP did.…”

“…Further, HSPs of both mammalian host cell and bacterial origins are reported to have chaperon activity (6,44) and can effectively prime a cytolytic response (10,45). In fact, we previously reported that HSP70 effectively induced the cross-priming of CD8 ϩ T cells through the cytosolic pathway when secreted from recombinant BCG in the phagosome of DC as part of a fusion protein (31).…”

“…In addition, HSP70, one of the heat shock proteins, plays various roles in the upregulation of the ability of APCs to stimulate T cells (6,10,44,45). Further, HSPs of both mammalian host cell and bacterial origins are reported to have chaperon activity (6,44) and can effectively prime a cytolytic response (10,45).…”

“…We sought another strategy to further enhance the T cell-stimulating activity of BCG, especially of the ability to activate IFN-␥-producing CD8 ϩ T cells quickly and strongly. To this end, we used heat shock protein 70 (HSP70) as a fusion partner (6,10,44,45). The gene encoding HSP70 of BCG was directly linked with that of MMP and extrachromosomally transformed into BCG (BCG-70 M) (31).…”

Enhanced Activation of T Lymphocytes by Urease-Deficient Recombinant Bacillus Calmette-Guérin Producing Heat Shock Protein 70-Major Membrane Protein-II Fusion Protein

“…To this end, we used heat shock protein 70 (HSP70) (21)(22)(23)(24). The gene encoding HSP70 was directly connected with that of MMP-II and was extrachromosomally transformed into BCG (production of BCG-70M).…”