Hsp 70 and hybrid peptides in cross-presentation and vaccinology

This study was designed to explore heat shock protein 70 (hsp70) ability to generate effective CD8+ T cell responses against intracellular pathogens, particularly HSV. Hsp70 has been shown to cross-present or cross-prime exogenous foreign antigen into the MHC class I pathway of antigen presentation, which is dependent upon the proteasome as well as the transported associated with antigen processing. In addition to cross presentation, hsp70 has also been shown to have a variety of affects upon antigen presenting (APC) cells via a specific interaction between receptors found on the APC and hsp70. Interaction between this protein and APC causes induction of various proinflammatory cytokines such as IL-1 and TNF-a in addition hsp70/ APC interaction initiates the APC to undergo a pattern of changes leading to its maturation. In terms of the immune response, hsp70 has been shown to play two roles, firstly the ability to crosspresent or cross-prime antigen and secondly hsp70 seems to function as a potent adjuvant. To begin this evaluation we used confocal microscopy and determined that in fact hsp70 does interact with APC. Then due to the very high level of conservation between hsp70 from broad phylogenetic origins we choose to determine if hsp70 from many sources had similar adjuvant properties. Using an ELISA based approach it was found that in fact hsp70 from plant, bacteria, and man all had similar abilities to induce Il-6, Il-12, and TNF-a from spleenocytes. In addition evidence generated by FACS found that the various hsp70 also caused the maturation of APC as measured by CD40 and B7.1. Hsp70 are know to be the most conserved at the N-terminal ATP binding domain. Therefore using trypsinization hsp70 was cleaved generating the ATP binding domain that was tested for adjuvant properties. Using the ATP domain in an ELISA based adjuvant tests revealed it did not have adjuvant properties. Finally, since many cognate MHC class I peptides have low affinity for hsp70 a hybrid peptide that contained a region with 20 fold increased affinity for hsp70 was produced and tested in many assays. Using hsp70 bound with the hybrid peptide to immunize mice and comparing the immune response to mice immunized with hsp70 bound to a class I epitope alone demonstrated that using hybrid peptides was in fact a better approach. In vitro follow up studies on the hybrid peptides surprisingly showed that hybrid peptides alone can gain access to the class I pathway of antigen presentation via an interaction with hsp70 on the surface of APC. This interaction was proteasome sensitive and inhibited by antibodies against hsp70.