Immune-based therapy against STEC intoxication and HUS

针对 STEC 中毒和 HUS 的免疫疗法

• 批准号: 10095464
• 负责人: Charles Bix Shoemaker
• 金额: $ 47.27万
• 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 2023-10-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10095464
• 关键词: Acute; Acute Diarrhea; Acute Kidney Failure; Adenovirus Vector; Adult; Age; Alpaca; Animal Model; Anthrax disease; Antibiotics; Antibodies; Appearance; Bacteria; Blood Vessels; Bontoxilysin; Child; Clinical Data; Clostridium difficile; Data; Data Reporting; Development; Diarrhea; Disease; Disease Outbreaks; Disease Progression; Dose; Engineering; Escherichia coli; 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; Lead; 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 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; base; clinical practice; comparative efficacy; cost; efficacy testing; human disease; human monoclonal antibodies; human pathogen; improved; innovation; intravenous administration; mRNA delivery; 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的免疫治疗 志贺毒素(STX)是引起暴发和散发性急性病例的致病菌。 人类，主要是儿童的腹泻，可导致溶血性尿毒症综合征(HUS)，导致急性 肾功能衰竭和中枢神经系统异常。目前还没有治疗方法，抗生素是禁忌。我们，还有 其他研究表明，针对志贺毒素(STX1和STX2)的人源性单抗(HuMab)是 动物模型的保护性和SAFE证明这些毒素确实是STEC的主要原因 相关的HUS，而该特异性HuMab是一种有效的基于免疫的治疗方法。然而，这样的待遇是 价格昂贵且繁琐，保质期有限，需要对有风险的患者进行静脉注射 发展中的HUS。在这里，我们提出了一种非常简化的策略，使用骆驼源性(VHH)抗体传递 通过信使核糖核酸纳米粒预防或阻止HUS的发展。预计这将是一种低成本和安全的 单次肌肉注射(IM)治疗暴露于STEC感染或出现STEC感染的患者 或者是血性腹泻。该抗毒素产品是基于一种高度创新的VHH中和剂(VNA) 我们实验室开发的平台。将VHH连接成VNAs可增强毒素中和作用 效力，并允许用一种试剂靶向多种毒素。这样的VNA现在已经成功地 产生并在动物模型中测试对肉毒杆菌神经毒素、艰难梭菌TcdA和 TcdB、蓖麻毒素、炭疽、STX1和Stx2毒素。针对STX1和STX2的单个VNA蛋白(VNA-STX)， 注射IP或IM，有效中和STX1和STX2。我们之前报告的数据显示：a)多个 腹腔或肌肉注射VNA-STX蛋白对STEC感染腹泻仔猪的保护作用 全身中毒；以及；b)单次肌注腺病毒(Ad)载体，携带分泌型VNA-STX1/Stx2 转基因对受感染的小猪同样具有保护作用[2]。最近，我们开发了一种 异四聚体VNA，更广泛地中和导致人类疾病的各种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STEC人类 病原体，并使用各种mRNA探索和评估更实用、简化的VNA递送系统 交付技术(目标1)。我们的假设是应用信使核糖核酸技术表达单个， 多特异性VNA将比HuMab更有效，制造更简单，成本仅为HuMab的几分之一 并允许更实用的肌肉内给药。这一假说将首先在小鼠身上进行检验。 在GnotoBiotic(GB)仔猪模型中抗中毒(目标2)和抗STEC口腔感染。这款车 导致急性腹泻和严重的结肠粘膜损害的症状，大约48小时后出现系统性 血管介导的中毒(致命的神经系统症状)，与某些事件的顺序非常相似 在感染STEC菌株的人类中观察，这些菌株继续发展为HUS(目标3)。