Immune-based therapy against STEC intoxication and HUS

标题：针对STEC中毒和HUS的免疫疗法 产滋贺毒素（Stx）的E.大肠杆菌（STEC）是引起暴发和散发急性病例的细菌 人类腹泻，主要是儿童，可导致溶血性尿毒综合征（HUS）， 肾衰竭和中枢神经系统异常。目前没有治疗方法，抗生素是禁忌的。我们也这么想 其他人的研究表明，抗滋贺毒素（Stx 1和Stx 2）的人单克隆抗体（HuMab）， 在动物模型中具有保护作用，并且安全，证明这些毒素确实是STEC的主要原因 相关的HUS，并且特异性HuMab是一种有效的基于免疫的疗法。然而，这样的待遇， 昂贵且麻烦，保质期有限，并且需要静脉内给药给有风险的患者， 开发HUS。在这里，我们提出了一个非常简化的策略，使用骆驼衍生（VHH）抗体交付 通过mRNA纳米颗粒来预防或阻止HUS的发展。预计这将是一个低成本和安全的 治疗，对暴露于或出现STEC感染的患者进行单次肌内（IM）注射 或者腹泻出血抗毒素产品是基于一个高度创新的VHH为基础的中和剂（VNA） 我们实验室开发的平台。将VHH连接在一起形成VNA导致增强的毒素中和 效力并允许用单一药剂靶向多种毒素。这种VNA现已成功地 产生并在动物模型中测试针对肉毒杆菌神经毒素、艰难梭菌TcdA和 TcdB、蓖麻毒素、炭疽、Stx 1和Stx 2毒素。靶向Stx 1和Stx 2的单一VNA蛋白（VNA-Stx）， 注射IP或IM，有效中和Stx 1和Stx 2。我们以前报告的数据显示：a）多个 IP或IM施用VNA-Stx蛋白保护患有腹泻的STEC感染的仔猪免于发展 全身中毒，和; B）单次IM注射具有分泌型VNA-Stx 1/Stx 2的腺病毒（Ad）载体 当给予感染的仔猪时，转基因具有同样的保护性[2]。最近，我们开发了一个 异源四聚体VNA更广泛地中和引起人类疾病的多种Stx 2天然变体。 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，并探索和评估更实用，简化VNA输送系统使用各种mRNA （目标1）。我们的假设是，mRNA技术的应用，表达一个单一的， 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 并允许更实用的肌内给药。这一假设将首先在老鼠身上进行测试 目的2：抗STEC中毒和抗STEC经口感染。该模型 导致急性腹泻症状和严重的结肠粘膜损伤，约48小时后全身性 血管介导的中毒（致死性神经系统症状），与某些事件序列非常相似 在感染STEC菌株的人中观察到，这些菌株继续发展为HUS（目标3）。

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).