title: CD4(+) T cells induced by a DNA vaccine: immunological consequences of epitope-specific lysosomal targeting.
abstract: Our previous studies have shown that targeting DNA vaccine-encoded major histocompatibility complex class I epitopes to the proteasome enhanced CD8(+) T-cell induction and protection against lymphocytic choriomeningitis virus (LCMV) challenge. Here, we expand these studies to evaluate CD4(+) T-cell responses induced by DNA immunization and describe a system for targeting proteins and minigenes to lysosomes. Full-length proteins can be targeted to the lysosomal compartment by covalent attachment to the 20-amino-acid C-terminal tail of lysosomal integral membrane protein-II (LIMP-II). Using minigenes encoding defined T-helper epitopes from lymphocytic choriomeningitis virus, we show that the CD4(+) T-cell response induced by the NP(309-328) epitope of LCMV was greatly enhanced by addition of the LIMP-II tail. However, the immunological consequence of lysosomal targeting is not invariably positive; the CD4(+) T-cell response induced by the GP(61-80) epitope was almost abolished when attached to the LIMP-II tail. We identify the mechanism which underlies this marked difference in outcome. The GP(61-80) epitope is highly susceptible to cleavage by cathepsin D, an aspartic endopeptidase found almost exclusively in lysosomes. We show, using mass spectrometry, that the GP(61-80) peptide is cleaved between residues F(74) and K(75) and that this destroys its ability to stimulate virus-specific CD4(+) T cells. Thus, the immunological result of lysosomal targeting varies, depending upon the primary sequence of the encoded antigen. We analyze the effects of CD4(+) T-cell priming on the virus-specific antibody and CD8(+) T-cell responses which are mounted after virus infection and show that neither response appears to be accelerated or enhanced. Finally, we evaluate the protective benefits of CD4(+) T-cell vaccination in the LCMV model system; in contrast to DNA vaccine-induced CD8(+) T cells, which can confer solid protection against LCMV challenge, DNA vaccine-mediated priming of CD4(+) T cells does not appear to enhance the vaccinee's ability to combat viral challenge.
authors.affiliation: Department of Neuropharmacology, The Scripps Research Institute, La Jolla, California 92037, USA.
authors.forename: F
authors.initials: F
authors.lastname: Rodriguez
authors.forename: S
authors.initials: S
authors.lastname: Harkins
authors.forename: J M
authors.initials: JM
authors.lastname: Redwine
authors.forename: J M
authors.initials: JM
authors.lastname: de Pereda
authors.forename: J L
authors.initials: JL
authors.lastname: Whitton
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D000914:Antibodies, Viral / Q000097:blood; D018955:CD36 Antigens / Q000502:physiology; D015496:CD4-Positive T-Lymphocytes / Q000276:immunology*; D002402:Cathepsin D / Q000378:metabolism; D002460:Cell Line; D000939:Epitopes; D008217:Lymphocytic choriomeningitis virus / Q000276:immunology*; D051907:Lysosomal Membrane Proteins; D008247:Lysosomes / Q000378:metabolism*; D008562:Membrane Glycoproteins; D051379:Mice; D008810:Mice, Inbred C57BL; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000276:immunology; D019444:Vaccines, DNA / Q000276:immunology*; D014765:Viral Vaccines / Q000276:immunology*
chemical_list: D000914:Antibodies, Viral; D018955:CD36 Antigens; D000939:Epitopes; D051907:Lysosomal Membrane Proteins; D008562:Membrane Glycoproteins; D010446:Peptide Fragments; C489452:Scarb2 protein, mouse; D019444:Vaccines, DNA; D014765:Viral Vaccines; D002402:Cathepsin D