Sortase-mediated modification of aDEC205 affords optimization of antigen presentation and immunization against a set of viral epitopes

Abstract

A monoclonal antibody against the C-type lectin DEC205 (aDEC205) is an effective vehicle for delivery of antigens to dendritic cells through creation of covalent aDEC205-antigen adducts. These adducts can induce antigen-specific T-cell immune responses or tolerance. We exploit the transpeptidase activity of sortase to install modified peptides and protein-sized antigens onto the heavy chain of aDEC205, including linkers that contain nonnatural amino acids. We demonstrate stoichiometric site-specific labeling on a scale not easily achievable by genetic fusions (49 distinct fusions in this report). We conjugated a biotinylated version of a class I MHCrestricted epitope to unlabeled aDEC205 and monitored epitope generation upon binding of the adduct to dendritic cells. Our results showtransfer of aDEC205 heavy chain to the cytoplasm, followed by proteasomal degradation. Introduction of a labile dipeptide linker at the N terminus of a T-cell epitope improves proteasome-dependent class I MHC-restricted peptide cross-presentation when delivered by aDEC205 in vitro and in vivo. We also conjugated aDEC205 with a linker-optimized peptide library of known CD8 T-cell epitopes from the mouse ?-herpes virus 68. Animals immunized with such conjugates displayed a 10-fold reduction in viral load.