Immune-based therapy against STEC intoxication and HUS

针对 STEC 中毒和 HUS 的免疫疗法

• 批准号: 10517289
• 负责人: Charles Bix Shoemaker
• 金额: $ 46.69万
• 依托单位: TUFTS UNIVERSITY BOSTON
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-11-19 至 2024-10-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10517289
• 关键词: Acute; Acute Diarrhea; Acute Kidney Failure; Adenovirus Vector; Adult; Age; Alpaca; Animal Model; Anthrax disease; Antibiotics; Antibodies; Appearance; Bacteria; Blood Vessels; Botulinum Toxins; Child; Clostridium difficile; Colon; Data; Data Reporting; Development; Diarrhea; Disease; Disease Outbreaks; Disease Progression; Dose; Engineering; Escherichia coli EHEC; Escherichia coli Infections; Event; Exposure to; Food; Gnotobiotic; Half-Life; Hemolytic-Uremic Syndrome; Hemorrhagic colitis; Hour; Human; Immunotherapy; Individual; Infection; Intoxication; Intramuscular; Intramuscular Injections; Investments; Kidney Failure; Laboratories; Life; Light; Link; Mediating; Messenger RNA; Modeling; Mucous Membrane; Mus; N-terminal; National Institute of Allergy and Infectious Disease; National Institute of Diabetes and Digestive and Kidney Diseases; Neurologic Symptoms; Oral; Patients; Prevention; Private Sector; Production; Proteins; Rare Diseases; Research; Research Support; Ricin; Risk; Route; Safety; Serum; Shiga Toxin; Shiga-Like Toxin I; Shiga-Like Toxin II; Source; System; Technology; Testing; Therapeutic; Time; Toxin; Transgenes; Variant; Work; acute symptom; antigen binding; antitoxin; clinical practice; comparative efficacy; cost; efficacy testing; human disease; human monoclonal antibodies; human pathogen; improved; innovation; intravenous administration; mRNA delivery; manufacture; nanoparticle; oral infection; porcine model; pre-clinical; prevent; research clinical testing; waterborne

Title: Immune-based therapy against STEC intoxication and HUS Shiga toxin (Stx) producing E. coli (STEC) are the bacteria responsible for outbreaks and sporadic acute cases of diarrhea in humans, primarily children, that can lead to hemolytic uremic syndrome (HUS) resulting in acute kidney failure and CNS abnormalities. No therapy currently exists, and antibiotics are contraindicated. We, and others, have shown that human monoclonal antibodies (HuMab) against Shiga toxins (Stx1 and Stx2) were protective in animal models and safe demonstrating that these toxins indeed are the primary cause of STEC associated HUS, and that specific HuMab is an effective immune-based therapy. However, such treatment is costly and cumbersome with limited shelf-life and requires intravenous administration to patients at risk of developing HUS. Here we propose a much-simplified strategy using camelid-derived (VHH) antibodies delivered by mRNA nanoparticles to prevent or arrest development of HUS. It is expected to be a low-cost and safe therapy, given in a single intramuscular (IM) injection to patients exposed to or presenting with STEC infection or bloody diarrhea. The antitoxin product is based on a highly innovative VHH-based neutralizing agents (VNA) platform developed at our laboratory. Linking VHHs together into VNAs leads to enhanced toxin neutralization potency and permits the targeting of multiple toxins with a single agent. Such VNAs have now been successfully generated and tested for efficacy in animal models against botulinum neurotoxin, Clostridium difficile TcdA and TcdB, ricin, anthrax, and Stx1 and Stx2 toxins. A single VNA protein targeting both Stx1 and Stx2 (VNA-Stx), injected IP or IM, potently neutralized both Stx1 and Stx2. We previously reported data showing that: a) multiple IP or IM administrations of VNA-Stx protein protected STEC-infected piglets with diarrhea from developing systemic intoxication, and; b) a single IM injection of an adenovirus (Ad) vector with a secretory VNA-Stx1/Stx2 transgene was equally protective when given to infected piglets [2]. More recently, we developed a heterotetrameric VNA that more broadly neutralizes diverse Stx2 natural variants that cause human disease. Here we propose to complete development of a VNA capable of potent neutralization of all known STEC human pathogens, and explore and evaluate more practical, simplified VNA delivery systems using various mRNA delivery technologies (Aim 1). Our hypothesis is that the application of mRNA technology to express a single, multi-specific VNA will be more effective than HuMab, simpler to manufacture at a fraction of the cost of HuMab and permit the much more practical intramuscular administration. This hypothesis will be first tested in mice against toxicosis (Aim 2) and against oral infection of STEC in the gnotobiotic (GB) piglet model. This model leads to symptoms of acute diarrhea and serious colonic mucosal damage followed ~48 hours later with systemic vascular-mediated intoxication (fatal neurologic symptoms) that closely mimic some of the sequence of events observed in humans infected with STEC strains who go on to develop HUS (Aim 3).

标题：针对 STEC 中毒和 HUS 的免疫疗法 产生志贺毒素 (Stx) 的大肠杆菌 (STEC) 是导致疫情和散发性急性病例的细菌 人类（主要是儿童）腹泻，可导致溶血性尿毒症综合征 (HUS)，从而导致急性 肾衰竭和中枢神经系统异常。目前尚无治疗方法，并且禁忌使用抗生素。我们，和 其他人已表明抗志贺毒素（Stx1 和 Stx2）的人单克隆抗体 (HuMab) 在动物模型中具有保护作用，并安全地证明这些毒素确实是 STEC 的主要原因 相关的 HUS，并且特定的 HuMab 是一种有效的基于免疫的疗法。然而这样的治疗是 昂贵且繁琐，保质期有限，需要对有风险的患者进行静脉注射 发展 HUS。在这里，我们提出了一种使用骆驼源 (VHH) 抗体的简化策略 通过 mRNA 纳米粒子来预防或阻止 HUS 的发展。预计将成为一种低成本且安全的 治疗，对暴露于或患有 STEC 感染的患者进行单次肌内 (IM) 注射 或血性腹泻。该抗毒素产品基于高度创新的 VHH 中和剂 (VNA) 我们实验室开发的平台。将 VHH 连接成 VNA 可增强毒素中和作用 效力并允许用单一药剂靶向多种毒素。此类 VNA 现已成功 生成并在动物模型中测试其对抗肉毒杆菌神经毒素、艰难梭菌 TcdA 和 TcdB、蓖麻毒素、炭疽、Stx1 和 Stx2 毒素。同时靶向 Stx1 和 Stx2 的单一 VNA 蛋白 (VNA-Stx)， 腹腔注射或肌内注射，可有效中和 Stx1 和 Stx2。我们之前报告的数据显示：a) 多个 腹膜内或肌内注射 VNA-Stx 蛋白可保护受 STEC 感染的仔猪免于出现腹泻 全身中毒； b) 单次肌内注射腺病毒 (Ad) 载体和分泌型 VNA-Stx1/Stx2 当给予受感染的仔猪时，转基因具有同样的保护作用[2]。最近，我们开发了一个 异四聚体 VNA 更广泛地中和导致人类疾病的多种 Stx2 自然变体。 在这里，我们建议完成 VNA 的开发，该 VNA 能够有效中和所有已知的人类 STEC 病原体，并探索和评估使用各种 mRNA 的更实用、更简化的 VNA 传递系统 交付技术（目标 1）。我们的假设是应用 mRNA 技术来表达单个、 多特异性 VNA 将比 HuMab 更有效，制造更简单，成本仅为 HuMab 的一小部分 并允许更实用的肌肉注射。该假设将首先在小鼠身上进行测试 在限菌 (GB) 仔猪模型中对抗中毒（目标 2）和 STEC 口腔感染。这个型号 导致急性腹泻症状和严重结肠粘膜损伤，约 48 小时后出现全身症状 血管介导的中毒（致命的神经系统症状），与某些事件序列非常相似 在感染 STEC 菌株且随后发展为 HUS 的人类中观察到这一现象（目标 3）。