Immunoprotective monoclonals to B. anthracis spores

描述(申请人提供)：炭疽芽胞最近被用作生物恐怖主义试剂。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：并不是所有患有典型致命的吸入性炭疽热的11名受害者全部死亡。事实上，积极使用喹诺酮类药物和其他抗生素，再加上对疾病的早期识别，导致了11名患者中的6名存活。不幸的是，数百名可能接触炭疽孢子的其他人需要延长抗生素疗程。一个仍然存在的健康问题是，接受抗生素预防的人在结束治疗后可能仍然会出现吸入性炭疽病，因为休眠的活孢子会萌发。增加疾病患者存活的可能性以及接触这种疾病的患者保持健康的可能性更高的一种方法是防止具有感染性的休眠孢子萌发，然后转化为营养细胞。最近有证据表明，抗PA(炭疽杆菌两种A亚单位毒素的共同B亚基，浮肿因子和致死因子)的抗体实际上结合在孢子表面并降低孢子萌发水平，再加上甲醛灭活的孢子可以作为豚鼠炭疽的保护性疫苗，我们得出了以下假设：抗PA的单抗和/或其他孢子表面表达的抗原可以阻止孢子萌发或使孢子更容易被巨噬细胞吞噬，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胞的免疫保护性单抗，在动物模型中提供对炭疽的保护。最终，我们打算使这些单抗人性化，以用作短期预防性药物或治疗方法。具体目标如下。特异性目的1是诱导小鼠抗辐照炭疽杆菌休眠孢子、辐照激活孢子、活化孢子表面蛋白提取物和重组PA(RPA)的抗体。我们将在酶联免疫吸附试验(ELISA)、体外发芽试验和巨噬细胞试验中测试这些抗血清，以评估吞噬、发芽和杀孢子活性。然后，我们将从其血清显示一种或多种抗孢子相关活性的动物中制备单抗。具体目的2是评估这些炭疽单抗预防炭疽病的能力，首先在小鼠非肠道孢子攻击模型中，如果具有保护作用，则在吸入性炭疽模型中预防炭疽病。特异性目标3是通过N末端测序鉴定各单抗识别的孢子表面蛋白(S)。具体目标4是与Sunol分子公司一起，对那些提供保护的单抗进行人源化版本的工程设计。

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.