Immunoprotective monoclonals to B anthracis spores

炭疽芽孢杆菌孢子的免疫保护性单克隆抗体

• 批准号: 6562567
• 负责人: Alison Davis O'Brien
• 金额: $ 22.31万
• 依托单位: HENRY M. JACKSON FDN FOR THE ADV MIL/MED
• 依托单位国家: 美国
• 财政年份: 2002
• 资助国家: 美国
• 起止时间: 2002-09-30 至 2004-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6562567
• 关键词: Bacillus anthracis; anthrax; antibacterial antibody; bacterial antigens; bacterial proteins; bioterrorism /chemical warfare; enzyme linked immunosorbent assay; immunologic substance development /preparation; laboratory mouse; laboratory rabbit; macrophage; monoclonal antibody; protein engineering; protein sequence; spores

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.

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媒介。 这些蓄意的微生物破坏事件造成了众多负面后果，其中有一个是积极的结果：11 名患有典型致命性吸入性炭疽病的受害者并非全部死亡。 事实上，喹诺酮类药物和其他抗生素的积极使用加上疾病的早期识别导致 11 名患者中有 6 名存活下来。 不幸的是，数百名可能接触炭疽孢子的人需要延长抗生素治疗疗程。 剩下的一个健康问题是，接受抗生素预防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萌发。 增加疾病患者生存的可能性以及暴露者保持健康的可能性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。 最近的证据表明，针对 PA（炭疽杆菌的两个 A 亚基毒素、水肿因子和致死因子的共享 B 亚基）的抗体实际上与孢子表面结合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，使我们得出以下假设：针对 PA 和/或其他物质的单克隆抗体 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并最终杀死。 基于这一理论，我们的目标是开发针对炭疽芽孢杆菌孢子的免疫保护性单克隆抗体，从而在动物模型中提供针对炭疽的保护。 最终，我们打算将这些单克隆抗体人源化，用作短期预防剂或治疗方式。 具体目标如下。 具体目标 1 是引发针对经辐射的炭疽芽孢杆菌休眠孢子、经辐射的活化孢子、活化孢子表面蛋白提取物和重组 PA (rPA) 的小鼠抗血清。 我们将在酶联免疫吸附测定 (ELISA)、体外萌发测定和巨噬细胞测定中测试这些抗血清，以评估吞噬作用、萌发和杀孢子活性。 然后，我们将从那些血清表现出一种或多种抗孢子相关活性的动物中制备单克隆抗体。 具体目标 2 是首先在小鼠肠外孢子攻击模型中评估这些炭疽芽孢杆菌单克隆抗体预防炭疽病的能力，如果有保护作用，则在兔吸入性炭疽模型中评估这些能力。 具体目标 3 是通过 N 端测序鉴定每种 mAb 识别的孢子表面蛋白。 具体目标 4 是与 SUNOL Molecular Corporation 一起启动这些可提供保护的单克隆抗体的人源化版本的工程。