Immunological Secretomes of the Early Anthrax Infection

描述(申请人提供)：我们计划开发针对炭疽杆菌早期感染过程中释放的蛋白质/多肽(分泌组)的新型疫苗。在过去的几年里，我们已经建立了鉴定新的炭疽抗原的蛋白质组学技术。我们实验室已经构建了几种编码这些新的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/多肽(分泌组)不包括在我们和其他已发表的炭疽蛋白质组中。在这个方案中，我们将主要使用质谱学技术来鉴定有/没有蛋白质分离的二维电泳法的炭疽分泌体。我们将使用毛细管超滤(CDF)探针从炭疽感染的小鼠体内收集分泌体，以捕获体内低丰度和分泌性的蛋白质/肽。CDF探针是我们实验室最近发展起来的一种新技术，用于收集小鼠的低丰度和分泌性蛋白质。我们计划将重点放在炭疽感染早期阶段释放的蛋白质/肽上。我们的假设是，针对炭疽感染早期阶段(上游事件)的疫苗将阻止下游事件的产生，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。同时，我们将评估编码早期炭疽感染分泌体的疫苗与目前以PA/LF为基础的疫苗的有效性。最后，我们鉴定了一种新的毒素，称为类骆驼毒素蛋白(CLP)，它具有免疫原性，在炭疽菌生活史的早期阶段从休眠的孢子中释放出来。因此，该蛋白将作为评估拟议研究中确定的新抗原的阳性对照。

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.