Immunoprotective monoclonals to B. anthracis spores

描述（由申请人提供）：炭疽芽孢杆菌孢子最近被用作生物恐怖主义的代理人。在这些蓄意的微生物破坏事件的许多负面后果中，有一个积极的结果：11名典型的致命吸入性炭疽患者并非全部死亡。事实上，积极使用喹诺酮类药物和其他抗生素，加上早期发现疾病，导致11名患者中有6人存活。不幸的是，其他数百名可能接触到炭疽孢子的人需要延长抗生素治疗的疗程。仍然存在的健康问题是，接受抗生素预防治疗的人在治疗结束后仍可能出现吸入性炭疽，因为休眠的活孢子会发芽。增加疾病患者存活的可能性和使接触者更有可能保持健康的一种方法是防止传染性休眠孢子发芽并随后转化为营养细胞。最近有证据表明，针对PA（炭疽芽胞杆菌两种A亚基毒素的共同B亚基，水肿因子和致死因子）的抗体实际上与孢子表面结合并降低孢子萌发水平，考虑到甲醛灭活的孢子可以作为豚鼠对炭疽的保护性疫苗，这使我们提出以下假设：针对PA和/或其他孢子表面表达抗原的单克隆抗体可以阻止孢子萌发或使孢子更容易被吞噬并最终被巨噬细胞杀死。基于这一理论，我们的目标是开发针对炭疽芽孢杆菌的免疫保护性单抗，在动物模型中赋予对炭疽的保护。最终，我们打算将这些单抗人源化，作为短期预防药物或治疗方式。具体目的如下：特异性目的1是诱导小鼠抗辐照炭疽芽胞杆菌休眠孢子、辐照活化孢子、活化孢子表面蛋白提取物和重组PA （rPA）的血清。我们将在酶联免疫吸附试验（elisa）、体外萌发试验和巨噬细胞试验中测试这些抗血清，以评估吞噬、萌发和杀孢活性。然后，我们将从那些血清显示出一种或多种这些抗孢子相关活性的动物中制备单克隆抗体。具体目标2是评估这些炭疽芽胞杆菌单克隆抗体的能力，首先在小鼠肠道外孢子攻击模型中预防炭疽疾病，如果有保护作用，则在兔吸入性炭疽模型中进行。特异性目的3是通过n端测序鉴定每个单抗识别的孢子表面蛋白。具体目标4是与SUNOL分子公司一起启动这些具有保护作用的单克隆抗体的人源化版本的工程。

DESCRIPTION (provided by applicant): Bacillus anthracis spores were recently used as agents of bioterrorism. Among the many negative consequences of these deliberate instances of microbiological sabotage was one positive outcome: not all of the 11 victims with the typically lethal inhalational form of anthrax died. Indeed, the aggressive use of quinolones and other antibiotics coupled with the early recognition of disease resulted in the survival of 6 of the 11 patients. Unfortunately, hundreds of other individuals potentially exposed to the anthrax spores required an extended course of antibiotic therapy. A remaining health concern is that the people who received antibiotic prophylaxis may still present with inhalation anthrax after conclusion of their therapy as dormant viable spores germinate. One way to increase the likelihood that patients with disease will survive and that those exposed will have a higher probability of remaining healthy is to prevent the infectious dormant spores from germinating and subsequently transforming to vegetative cells. Recent evidence that antibodies against the PA (the shared B subunit for the two A subunit toxins of B. anthracis, edema factor and lethal factor) actually bind to the surface of spores and decrease the level of spore germination, taken with the fact that formaldehyde-inactivated spores can serve as a protective vaccine against anthrax in guinea pigs, led us to the following hypothesis: mAbs against PA and/or other spore-surface-expressed antigens can block spore germination or render spores more susceptible to phagocytosis and ultimately killing by macrophages. Based on this theory, our goals are to develop immunoprotective mAbs against B. anthracis spores that confer protection against anthrax in animal models. Ultimately, we intend to humanize those mAbs for use as short-term preventative agents or therapeutic modalities. The Specific Aims are as follows. Specific Aim 1 is to elicit mouse antisera against irradiated B. anthracis dormant spores, irradiated activated spores, activated spore-surface protein extracts, and recombinant PA (rPA). We will test those antisera in enzyme-linked immunosorbent assays (ELISAs), in vitro germination assays, and macrophage assays to evaluate phagocytosis, germination, and sporicidal activity. We will then prepare mAbs from those animals whose sera demonstrate one or more of these anti-spore related activities. Specific Aim 2 is to assess the capacity of these B. anthracis mAbs to prevent anthrax disease first in a mouse parenteral spore challenge model and, if protective, in a rabbit model of inhalational anthrax. Specific Aim 3 is to identify the spore-surface protein(s) recognized by each mAb through N-terminal sequencing. Specific Aim 4 is to initiate, with SUNOL Molecular Corporation, the engineering of humanized versions of those mAbs that confer protection.