title: Antigenicity and immunogenicity of an intracellular delivery system of major histocompatibility complex class I epitopes that bypasses proteasome processing.
abstract: The development of a cell-free synthetic vaccine to induce an effective cytotoxic T lymphocyte response is an important challenge in T-cell--mediated immunity. Because standard vaccinations with nominal epitopes were found to be only partially effective in vivo, the authors suggest an alternative strategy: the delivery of epitopes directly to the cell cytosol in a proteasome bypass mechanism of processing. Two model peptides, the presentation level on the cell surface of which can be directly assessed, were conjugated via a cross-linker to an internalization peptide derived from an antennapedia homeobox protein. The linker was designed to undergo spontaneous hydrolysis, after which the epitope is subsequently released. The conjugates were shown to enter RMA and P815 cells, where the epitopes were released mainly in cytosol and endogenously loaded on the major histocompatibility complex class I molecules to be presented on the cell surface. Concomitant inhibition of proteasome activity by MG132 significantly increased the presentation level of both model peptides, indicating proteasome-independent processing. This phenomenon was exploited to enhance the immunogenicity of the conjugates. Conjugates were emulsified with MG132 in incomplete Freund's adjuvant and injected into mouse footpads. Analysis of the draining lymph nodes indicated an increase in the percentage of both CD4+ and CD8+ lymphocytes. In vitro cytolytic assays implied significant, albeit moderate, priming only when the proteasome inhibitor was administered with the conjugate. This approach may be useful for the development of efficient synthetic cell-free vaccines.
authors.affiliation: Department of Immunology, The Weizmann Institute of Science, Rehovot, Israel.
authors.forename: B
authors.initials: B
authors.lastname: Tirosh
authors.forename: M
authors.initials: M
authors.lastname: Fridkin
authors.forename: E
authors.initials: E
authors.lastname: Tzehoval
authors.forename: E
authors.initials: E
authors.lastname: Vadai
authors.forename: F A
authors.initials: FA
authors.lastname: Lemonnier
authors.forename: L
authors.initials: L
authors.lastname: Eisenbach
mesh_terms: D000818:Animals; D051943:Antennapedia Homeodomain Protein; D017951:Antigen Presentation / Q000187:drug effects; D001692:Biological Transport; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D003546:Cysteine Endopeptidases / Q000378:metabolism*; D015853:Cysteine Proteinase Inhibitors / Q000494:pharmacology; D003600:Cytosol / Q000187:drug effects / Q000276:immunology; D003601:Cytotoxicity Tests, Immunologic; D000939:Epitopes / Q000276:immunology* / Q000378:metabolism; D005260:Female; D005620:Freund's Adjuvant / Q000494:pharmacology; D015395:Histocompatibility Antigens Class I / Q000276:immunology* / Q000378:metabolism; D018398:Homeodomain Proteins / Q000378:metabolism; D007976:Leupeptins / Q000494:pharmacology; D008055:Lipids; D008198:Lymph Nodes / Q000187:drug effects / Q000276:immunology; D018655:Lymphocyte Count; D051379:Mice; D008811:Mice, Inbred DBA; D009097:Multienzyme Complexes / Q000037:antagonists & inhibitors / Q000378:metabolism*; D009687:Nuclear Proteins; D009842:Oligopeptides / Q000276:immunology / Q000378:metabolism; D046988:Proteasome Endopeptidase Complex; D013602:T-Lymphocytes, Cytotoxic / Q000187:drug effects / Q000276:immunology; D014157:Transcription Factors; D022223:Vaccines, Subunit / Q000378:metabolism / Q000494:pharmacology*
chemical_list: D051943:Antennapedia Homeodomain Protein; D015853:Cysteine Proteinase Inhibitors; D000939:Epitopes; D015395:Histocompatibility Antigens Class I; D018398:Homeodomain Proteins; D007976:Leupeptins; D008055:Lipids; D009097:Multienzyme Complexes; D009687:Nuclear Proteins; D009842:Oligopeptides; D014157:Transcription Factors; D022223:Vaccines, Subunit; C114843:incomplete Freund's adjuvant; D005620:Freund's Adjuvant; D003546:Cysteine Endopeptidases; D046988:Proteasome Endopeptidase Complex; C072553:benzyloxycarbonylleucyl-leucyl-leucine aldehyde