ABSTRACT The protective antigen (PA) component of anthrax toxin mediates entry of the toxin's lethal factor (LF) and edema factor into the cytosolic compartment of mammalian cells. The amino-terminal domain of LF (LFn; 255 amino acids) binds LF to PA, and when fused to heterologous proteins, the LFn domain delivers such proteins to the cytoplasm in the presence of PA. In the current study, we fused a 9-amino acid cytotoxic T-lymphocyte (CTL) epitope (LLO 91-99 ) from an intracellular pathogen, Listeria monocytogenes, to LFn and measured the ability of the resulting LFn-LLO 91-99 fusion protein to stimulate a CTL response against the epitope in BALB͞c mice. As little as 300 fmol of fusion could stimulate a response. The stimulation was PA-dependent and occurred with the peptide fused to either the amino terminus or the carboxyl terminus of LFn. Upon challenge with L. monocytogenes, mice previously injected with LFn-LLO 91-99 and PA showed a reduction of colony-forming units in spleen and liver, relative to nonimmunized control mice. These results indicate that anthrax toxin may be useful as a CTL-peptide delivery system for research and medical applications. All viruses and some bacterial and protozoan pathogens have evolved the ability to survive and replicate within mammalian cells. Immune recognition of these cytoplasmic pathogens depends upon the cell surface display of peptide antigens derived from pathogen-associated proteins. These peptides are presented in complexes with host class I molecules encoded by the major histocompatibility complex (MHC-I), and cytotoxic T lymphocytes (CTL) are activated following recognition of the foreign peptide in complex with MHC-I (1). Activated CTL lyse the infected cell, secrete cytokines, and then proliferate and differentiate (2, 3). Each of these steps plays an important role in clearing the host of the pathogen. Lysis of the target cell deprives the organism of its replicative niche and exposes the pathogen to elements of the humoral immune system. Secretion of cytokines has many effects, including enhancement of local immune responses. Proliferation of the CTL clone results in expansion of a set of reactive CTL to effect clearance of the pathogen from other infected cells, whereas differentiation provides a set of long-lived memory cells available to respond more quickly and effectively to subsequent challenge. Vaccines that prime these memory cells provide protection of the host upon reexposure to the pathogen. For a vaccine to mimic infection by cytoplasmic pathogens, it must introduce the target antigen(s) into the cytosol of host cells in vivo. This has been accomplished by expressing heterologous antigens by live viral or bacterial vectors, by using adjuvants, or by delivering DNA expression vectors (DNA vaccines) (4-7). Herein we describe a strategy for introducing CTL epitopes into the cytosol of host cells, in vivo, using delivery components of an intracellularly acting toxin, anthrax toxin. Anthrax toxin is composed of three proteins that act in binary combinations to elicit two toxic effects, lethality and edema (8). Lethal factor (LF) and edema factor (EF) are intracellularly acting proteins, both of which require protective antigen (PA) for their translocation to the cytosol of eukaryotic cells. Initially, LF and EF bind competitively to proteolytically activated PA (PA 63 ) at the cell surface. The protein complex is endocytosed, and LF and EF are translocated to the cytosol following endosomal acidification. There, EF expresses its adenylate cyclase activity, elevating cAMP levels, and LF expresses its as yet undefined activity, inducing lethal overproduction of certain cytokines in its target cells, macrophages (9, 10). The amino-terminal domain of LF (LFn; 255 residues) has been found to contain the information necessary for binding to PA and mediating translocation. The domain alone lacks lethal potential that depends on the putatively enzymatic carboxyl-terminal moiety (11). Recent results have demonstrated that genetically fusing LFn to heterologous polypeptides enables them to be delivered to the cytosol of cultured mammalian cells in the presence of PA To test the potential of the anthrax toxin system to mediate presentation of peptides in complex with MHC-I, we chose to fuse LFn to a CTL epitope from Listeria monocytogenes. L. monocytogenes is a facultative intracellular bacterial pathogen that survives within the cytosol of macrophages. After its entry into the cell by phagocytosis, the bacterium produces listeriolysin O (LLO), which lyses the phagosomal membrane and allows the bacterium to escape to the cytosol. LLO within the cytosol is proteolytically processed, generating short peptides, some of which are presented at the cell surface in complex with MHC-I. Processing of LLO results in the presentation of a nonameric peptide, LLO [91][92][93][94][95][96][97][98][99] MATERIALS AND METHODS Peptide Synthesis. The LLO 91-99 peptide GYKDGNEYI was synthesized on an Applied Biosystems model 432A peptide synthesizer
