title: Targeting HIV proteins to the major histocompatibility complex class I processing pathway with a novel gp120-anthrax toxin fusion protein.
abstract: A challenge for subunit vaccines whose goal is to elicit CD8(+) cytotoxic T lymphocytes (CTLs) is to deliver the antigen to the cytosol of the living cell, where it can be processed for presentation by major histocompatibility complex (MHC) class I molecules. Several bacterial toxins have evolved to efficiently deliver catalytic protein moieties to the cytosol of eukaryotic cells. Anthrax lethal toxin consists of two distinct proteins that combine to form the active toxin. Protective antigen (PA) binds to cells and is instrumental in delivering lethal factor (LF) to the cell cytosol. To test whether the lethal factor protein could be exploited for delivery of exogenous proteins to the MHC class I processing pathway, we constructed a genetic fusion between the amino-terminal 254 aa of LF and the gp120 portion of the HIV-1 envelope protein. Cells treated with this fusion protein (LF254-gp120) in the presence of PA effectively processed gp120 and presented an epitope recognized by HIV-1 gp120 V3-specific CTL. In contrast, when cells were treated with the LF254-gp120 fusion protein and a mutant PA protein defective for translocation, the cells were not able to present the epitope and were not lysed by the specific CTL. The entry into the cytosol and dependence on the classical cytosolic MHC class I pathway were confirmed by showing that antigen presentation by PA + LF254-gp120 was blocked by the proteasome inhibitor lactacystin. These data demonstrate the ability of the LF amino-terminal fragment to deliver antigens to the MHC class I pathway and provide the basis for the development of novel T cell vaccines.
authors.affiliation: Molecular Immunogenetics and Vaccine Research Section, Metabolism Branch, Division of Clinical Sciences, National Cancer Institute, Bethesda, MD 20892, USA.
authors.forename: T J
authors.initials: TJ
authors.lastname: Goletz
authors.forename: K R
authors.initials: KR
authors.lastname: Klimpel
authors.forename: N
authors.initials: N
authors.lastname: Arora
authors.forename: S H
authors.initials: SH
authors.lastname: Leppla
authors.forename: J M
authors.initials: JM
authors.lastname: Keith
authors.forename: J A
authors.initials: JA
authors.lastname: Berzofsky
mesh_terms: D016915:AIDS Vaccines; D000111:Acetylcysteine / Q000031:analogs & derivatives / Q000494:pharmacology; D017951:Antigen Presentation; D000942:Antigens, Bacterial; D001408:Bacillus anthracis / Q000276:immunology*; D001427:Bacterial Toxins / Q000235:genetics / Q000276:immunology*; D003546:Cysteine Endopeptidases / Q000187:drug effects; D015853:Cysteine Proteinase Inhibitors / Q000494:pharmacology; D003602:Cytotoxicity, Immunologic; D006678:HIV / Q000276:immunology*; D015699:HIV Envelope Protein gp120 / Q000235:genetics / Q000276:immunology*; D015395:Histocompatibility Antigens Class I; D009097:Multienzyme Complexes / Q000187:drug effects; D010446:Peptide Fragments / Q000235:genetics / Q000276:immunology; D046988:Proteasome Endopeptidase Complex; D011993:Recombinant Fusion Proteins / Q000276:immunology; D013602:T-Lymphocytes, Cytotoxic / Q000276:immunology
chemical_list: D016915:AIDS Vaccines; D000942:Antigens, Bacterial; D001427:Bacterial Toxins; D015853:Cysteine Proteinase Inhibitors; D015699:HIV Envelope Protein gp120; D015395:Histocompatibility Antigens Class I; D009097:Multienzyme Complexes; D010446:Peptide Fragments; D011993:Recombinant Fusion Proteins; C030325:anthrax toxin; C067713:lactacystin; D003546:Cysteine Endopeptidases; D046988:Proteasome Endopeptidase Complex; D000111:Acetylcysteine