TARGETING HIV PROTEINS TO THE MAJOR HISTOCOMPATIBILITY COMPLEX CLASS-I PROCESSING PATHWAY WITH A NOVEL GP120-ANTHRAX TOXIN FUSION PROTEIN

A challenge for subunit vaccines whose goal is to elicit CD8(+) cytoto xic T lymphocytes (CTLs) is to deliver the antigen to the cytosol of t he living cell, where it can be processed for presentation by major hi stocompatibility complex (MHC) class I molecules. Several bacterial to xins 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 a ntigen (PA) binds to cells and is instrumental in delivering lethal fa ctor (LF) to the cell cytosol, To test whether the lethal factor prote in could be exploited for delivery of exogenous proteins to the MHC cl ass I processing pathway, we constructed a genetic fusion between the amino-terminal 254 aa of LF and the gp120 portion of the HIV-1 envelop e protein, Cells treated with this fusion protein (LF254-gp120) in the presence of PA effectively processed gp120 and presented an epitope r ecognized by HIV-1 gp120 V3-specific CTL, In contrast, when cells were treated with the LF254-gp120 fusion protein and a mutant PA protein d efective for translocation, the cells were not able to present the epi tope and were not lysed by the specific CTL, The entry into the cytoso l and dependence on the classical cytosolic MHC class I pathway were c onfirmed by showing that antigen presentation by PA + LF254-gp120 was blocked by the proteasome inhibitor lactacystin. These data demonstrat e 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.