ANTHRAX TOXIN FUSIONS TO STIMULATE CTL IMMUNITY

描述（改编自申请人摘要）：保护性抗原 (PA)炭疽毒素的一种成分介导毒素的致命因子进入 (LF)和水肿因子到哺乳动物细胞的胞质区室。 的 LF的氨基末端结构域（Lfn; 255个氨基酸）缺乏毒性的氨基酸残基。 活性并将LF结合至PA。 与Lfn融合的杂合蛋白是 在PA存在下递送到宿主细胞的细胞质中。 的 研究人员融合了一个9个残基的细胞毒性T淋巴细胞（CTL）表位， （LLO 91 -99）从细胞内病原体单核细胞增生李斯特菌（Listeria monocytogenes）到Lfn 并且已经证明了所得LFn-LLO 91 -99融合物 在BALB/c中刺激针对表位的保护性CTL应答的蛋白 小鼠 他们建议扩大这些研究，以确定炭疽毒素是否可以 用作引发多种CTL应答的系统。 他们建议， 以下实验：1）它们将用炭疽毒素融合物免疫 含有三个独立的CTL表位， 类似于亚致死性感染。 2)他们将 研究使用炭疽毒素递送来自L. 单核细胞增多症由H-2 M3呈递。 用这些表位免疫， 含有N-甲酰甲硫氨酸，可保护小鼠的大多数MHC 单倍型。 已经证明难以使用这些表位进行免疫， 现有技术。 3)他们将确定是否一个单一的炭疽毒素 与LCMV NP的融合能够刺激CTL并保护不同免疫缺陷的小鼠。 小鼠单倍型抗LCMV感染。 这些实验的成功 这表明炭疽毒素系统可能有助于免疫 基因多样的人群。 4)它们将结合来自 LCMV和单核细胞增生李斯特菌在同一炭疽毒素融合，以确定 如果保护性CTL应答可以针对多种病原体启动， 在用单一融合蛋白免疫后，和5）它们将 确定添加聚阳离子序列是否可以取代这些中的Lfn 融合，允许通过PA在体内递送CTL表位，并刺激 保护性CTL应答。 这将极大地扩展 这个系统可以使用。 这项提案中的研究将使研究人员能够进一步 确定炭疽毒素是否可用作通用CTL-肽 用于研究和医疗应用的输送系统。

DESCRIPTION (Adapted from the applicant's abstract): 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.