The protective antigen (PA) component of anthrax toxin mediates entry of the toxin's lethal factor (LF) and edema factor to the cytosolic compartment of mammalian cells. The amino-terminal domain of LF (Lfn; 255 amino acids) is devoid of toxic activity and binds LF to PA. Heterologous proteins fused to Lfn are delivered into the cytoplasm of host cells in the presence of PA. The investigators have fused a nine-residue cytotoxic T-lymphocyte (CTL) epitope (LLO91-99) from an intracellular pathogen, Listeria monocytogenes, to Lfn and have demonstrated the ability of the resulting LFn-LLO91-99 fusion protein to stimulate a protective CTL response against the epitope in BALB/c mice. They propose to expand these studies to determine if anthrax toxin can be used as a system for priming a variety of CTL responses. They propose the following experiments: 1) They will immunize with an anthrax toxin fusion containing three separate CTL epitopes in an attempt to induce immunity similar to that seen following sublethal infection. 2) They will investigate the use of the anthrax toxin to deliver CTL epitopes from L. Monocytogenes presented by H-2 M3. Immunization with these epitopes, which contain n-formyl methionine, may be protective in mice of most MHC haplotypes. It has proven difficult to immunize with these epitopes using existing technologies. 3) They will determine if a single anthrax toxin fusion with LCMV NP is able to stimulate CTL and protect mice of different murine haplotypes against LCMV infection. Success of these experiments would suggest that the anthrax toxin system may be useful in immunizing genetically diverse populations. 4) They will incorporate epitopes from both LCMV and L.monocytogenes in the same anthrax toxin fusion to determine if a protective CTL response can be primed against multiple pathogens following immunization with a single fusion protein and 5) They will determine if addition of polycationic sequences can replace Lfn in these fusions, allowing the delivery of CTL epitopes by PA in vivo and stimulating protective CTL responses. This would greatly expand the ease with which this system could be used. The research in this proposal will allow the investigators to further establish whether anthrax toxin may be useful as a general CTL-peptide delivery system for research and medical applications.
| D'Orazio, S E; Halme, D G; Ploegh, H L et al. (2003) Class Ia MHC-deficient BALB/c mice generate CD8+ T cell-mediated protective immunity against Listeria monocytogenes infection. J Immunol 171:291-8 |
