Anthrax vaccination by targeting spore germination

描述(申请人提供)：萌发是炭疽芽孢杆菌生命周期的上游事件。萌发的孢子可能被认为是其生命周期中最薄弱的一环，因为停止它们的功能将阻止疾病的进一步发展。因此，针对炭疽芽孢杆菌萌发阶段表达的特定蛋白的疫苗的研制可能是遏制炭疽生长的有效策略。阻止萌发阶段将防止下游事件，包括炭疽三种天然毒素PA、LF和EF的产生，以及生物恐怖分子可能向炭疽中注入的任何其他外来毒素。 最近，我们利用蛋白质组学技术对炭疽芽孢杆菌的22个萌发相关蛋白进行了鉴定。传统的蛋白质组学主要使用2D电泳法和质谱仪来研究蛋白质。我们已经确定了这些萌发蛋白在孢子休眠到萌发的过程中存在差异表达。已经鉴定了11种已鉴定的蛋白质。它们包括1个分泌型蛋白(免疫抑制因子A)、4个潜在的膜相关蛋白(山茶素、丙氨酸外消旋酶、拉宾糖转运体和L型钙通道)、2个分子伴侣(HSP60和CPN60)、2个能量相关蛋白(三磷酸腺苷合成酶和3-磷酸甘油醛脱氢酶)和1个新的炭疽特异性蛋白。免疫抑制剂A和骆驼溶素是一种毒性很强的金属蛋白酶，在炭疽杆菌中尚未见报道。 唐博士是这项研究的合作伙伴，他建立了一套基于载体疫苗应用于皮肤的非侵入性疫苗接种系统。该系统将在这项研究中用于开发炭疽疫苗，该疫苗将专门针对两种与萌发相关的蛋白：免疫抑制剂A和骆驼溶素。我们还将比较目前在我们实验室构建的PA和LF靶向疫苗。同时，来自免疫抑制剂A和骆驼溶素的疫苗将在体外和体内进行评估，以确定它们阻止孢子萌发和对巨噬细胞的细胞毒性的效果。 这项史无前例的研究将揭示在炭疽芽孢杆菌中表达的免疫抑制剂A和骆驼溶素的病理机制和疫苗接种能力。

DESCRIPTION (provided by applicant): Germination occurs as an upstream event during the life cycle of Bacillus anthracis. Germinating spores may be considered the weakest link during its life cycle because stopping their function would prevent further development of the disease. Therefore, the development of vaccines against specific proteins expressed during the germination stage of Bacillus anthracis may be an effective strategy to halt the growth of anthrax. Blocking the germination stage would prevent downstream events including the production of anthrax's three natural toxins, PA, LF, and EF, as well as any other foreign toxins that bioterrorists may insert into anthrax. We have recently characterized twenty-two germination-associated proteins from Bacillus anthracis by using proteomic techniques. Conventional proteomics studies proteins primarily using 2D electrophoresis and mass spectrometry. We have determined these germination proteins are differentially expressed from spore dormancy to germination. Eleven identified proteins have been identified. They include one secreted protein (Immune inhibitor A), four potential membrane-associated proteins (Camelysin, Alanine racemase, Larabinose transporter, and L-type calcium channel), two molecular chaperones (HSP60 and cpn60), two energy-related proteins (ATP synthase and Glyceraldehyde 3-phosphate dehydrogenase) and one novel anthrax specific protein. Immune inhibitor A and camelysin are virulent metalloproteinases and have been unreported in Bacillus anthracis. Dr. Tang, a Co-PI in this study, has well-established a noninvasive vaccination system based on vector based vaccines applied to the skin. That system will be used in this study to develop anthrax vaccines that will specifically target two of the germination-associated proteins: immune inhibitor A and camelysin. We will also compare current PA and LF targeted vaccines that have been constructed in our laboratory. In parallel, vaccines derived from immune inhibitor A and camelysin will be evaluated in vitro and in vivo to determine their efficacies in blocking spore germination and cytotoxicity to macrophage cells. This unprecedented study will elicit the pathological mechanisms and vaccination capabilities of immune inhibitor A and camelysin expressed in Bacillus anthracis.