Immunological Secretomes of the Early Anthrax Infection

描述（由申请人提供）：我们计划开发靶向炭疽杆菌早期感染期间释放的蛋白质/肽（分泌组）的新型疫苗。在过去的几年中，我们建立了蛋白质组学技术来鉴定新的炭疽抗原。本实验室已经构建了几种编码这些新型炭疽抗原的载体疫苗。此外，我们还通过鉴定与萌发和早期生长相关的孢子蛋白，发表了炭疽芽孢杆菌的蛋白质组。然而，炭疽芽孢杆菌释放的蛋白质/肽（分泌组）不包括在我们和其他出版的炭疽蛋白质组。在本研究中，我们将主要利用质谱技术来鉴定炭疽的分泌体，并通过双向电泳进行蛋白质分离。我们将使用毛细管超滤（CDF）探针收集炭疽感染小鼠的分泌体，以捕获体内低丰度和分泌的蛋白/肽。CDF探针是本实验室近年来发展起来的一种从小鼠体内收集低丰度分泌蛋白的新技术。我们计划着重于炭疽感染早期阶段释放的蛋白质/肽。我们的假设是，针对炭疽感染早期阶段（上游事件）的疫苗将阻断下游事件，包括保护性抗原（PA），致死因子（LF）和水肿因子（EF）的产生。为了构建更有效的疫苗，我们将检测炭疽分泌体的细胞毒性和免疫原性。与此同时，我们将评估与目前基于PA/LF的疫苗相比，编码早期炭疽感染分泌物的疫苗的效力。最后，我们已经确定了一种新的毒素称为骆驼溶素样蛋白（CLP），发挥免疫原性，并在炭疽生命周期的早期阶段从休眠孢子中释放出来。因此，该蛋白质将作为阳性对照，以评价在拟定研究中鉴定的新抗原。

DESCRIPTION (provided by applicant): We plan to develop the novel vaccines targeting the released proteins/peptides (secretome) during the early infection of Bacillus anthracis. In the past years, we had established the proteomic techniques to identify novel anthrax antigens. Several vector-based vaccines encoding these novel anthrax antigens have been constructed in our laboratory. In addition, we have published a proteome of Bacillus anthracis by identifying spore proteins associated with germination and early outgrowth. However, the released proteins/peptides (secretome) of Bacillus anthracis are not included in our and other published anthrax proteomes. In this proposal, we will mainly employ the mass spectrometric techniques to identify the anthrax secretomes with/without protein separation by 2-D electrophoresis. We will collect the secretomes from anthrax-infected mice using capillary ultrafiltration (CDF) probes in order to capture the in vivo low abundant and secretory proteins/peptides. The CDF probes are a novel technique recently developed in our laboratory to collect low abundant and secretory proteins from mice. We plan to emphasize on the proteins/peptides released during the early stages of anthrax infection. Our hypothesis is that vaccines targeting the early stages of anthrax infection (upstream events) will block the downstream events including the production of protective antigen (PA), lethal factor (LF), and edema factor (EF). In order to construct more effective vaccines, we will determine the cytotoxicities and immunogenicities of anthrax secretomes. In parallel, we will evaluate the efficacies of vaccines which encoded secretomes of early anthrax infection as compared to current PA/LF-based vaccines. Lastly, we have identified a novel toxin called camelysin-like protein (CLP) which exerts immunogenic and is released from dormant spores during the early stage of the anthrax life cycle. This protein will thus serve as a positive control to evaluate novel antigens identified in the proposed studies.