A Novel Probiotic-Based Nanobody Delivery Platform

项目摘要 我们的目标是产生基于乳酸乳球菌的纳米抗体表达菌株，并证明纳米抗体表达菌株的生物学活性。 产生的纳米抗体可以在体外和体内有效地中和艰难梭菌毒素。 C.艰难梭菌是一种普遍存在的厌氧革兰氏阳性细菌， 环境压力和一线抗生素，如克林霉素1。病原菌株C.艰难分泌 毒性糖基转移酶、大梭菌毒素A（TcdA）和B（TcdB）的不同组合，以及 二元毒素C.艰难梭菌转移酶（CDT）2. TcdA和TcdB破坏紧密连接并进入固有层 促进炎症。CDT与TcdA、TcdB协同作用加重C.艰难梭菌感染（CDI），诱导 细胞突起增加C.艰难梭菌，并抑制保护性宿主嗜酸性细胞反应， 结肠3.毒素酶活性损害肠上皮细胞，导致炎症性结肠炎， 严重腹泻、腹痛、流感样症状，甚至死亡1。 CDI通常发生在抗生素治疗后，当个体的内源性微生物区系改变或 严重减少。虽然现有的抗生素对许多患者有效，但持续使用抗生素会进一步增加患者的死亡率。 破坏了导致CDI复发的微生物群。因此，经常性的CDI目前是最有效的 用粪便微生物群移植（FMT）疗法治疗4 -6。然而，FMT是一种昂贵的、异构的、 定义不明确的治疗药物，由于安全性问题需要FDA特别批准7。因此，新 迫切需要预防和治疗CDI的方法。 我们最近从骆驼VHH噬菌体展示中产生了独特的纳米抗体。我们的研究表明， 鉴定的纳米抗体结合梭菌毒素A（TcdA）、B（TcdB）和C的关键构象表位。 艰难梭菌转移酶/二元毒素（CDT），具有中和肠道中CDI相关毒素的潜力 道。为了将这些纳米抗体口服递送到肠道中并改善粘膜 口服给药后的药代动力学/药效学，我们的策略是表达纳米抗体 在L.乳酸。我们相信L。lactis是一种理想的运载工具，因为它已经收到了“一般 FDA认证为“安全”状态，并且不会在人胃肠道中定植。 该方案旨在验证一种新的L.用于快速开发的基于乳酸杆菌的纳米抗体平台， 将纳米抗体递送到肠道中。与此同时，我们将使用 这个平台用于预防和治疗CDI。具体目标是：1）选择治疗 基于域映射和C.艰难梭菌毒素中和，2）产生L.乳酸菌菌株， 表达针对TcdA、TcdB或CDT的中和纳米抗体，以及3）证明组合的 菌株抑制C.小鼠模型中的艰难发病机制。

Project Summary Our objective is to produce Lactococcus lactis-based nanobody-expressing strains and demonstrate that the nanobodies produced can effectively neutralize Clostridium difficile toxins in vitro and in vivo. C. difficile is a ubiquitous anaerobic gram-positive bacterium that can sporulate and become highly resistant to environment stresses and frontline antibiotics such as clindamycin1. Pathogenic strains of C. difficile secrete varying combinations of virulent glycosyltransferases, the large clostridial toxins A (TcdA) and B (TcdB), and binary toxin, C. difficile transferase (CDT)2. TcdA and TcdB disrupt tight junctions and enter the lamina propria to promote inflammation. CDT synergizes with TcdA and TcdB to worsen C. difficile infection (CDI), inducing cell protrusions that increase adherence of C. difficile, and suppressing protective host eosinophilic response in the colon3. The toxin enzymatic activities damage intestinal epithelial cells, resulting in inflammatory colitis, severe diarrhea, abdominal pain, flu-like symptoms, and even death1. CDI often occurs following antibiotic-treatment when the endogenous microflora of individuals is altered or severely reduced. While existing antibiotics are effective in many patients, continuous antibiotic usage further damages the microbiota contributing to recurring CDI. As a result, recurrent CDI is currently most effectively treated with fecal microbiota transplant (FMT) therapy4-6. FMT, however, is an expensive, heterogeneous and poorly defined therapeutic that requires special FDA approval due to safety concerns7. As a result, new approaches are desperately needed to prevent and treat CDI. We recently generated unique nanobodies from a camelid VHH phage display. Our studies demonstrate that the identified nanobodies bind to key conformational epitopes of clostridial toxins A (TcdA), B (TcdB), and C. difficile transferase/binary toxin (CDT), with the potential to neutralize CDI-associated toxins in the intestinal tract. To orally deliver these nanobodies into the intestinal tract and improve mucosal pharmacokinetics/pharmacodynamics following oral administration, our strategy is to express the nanobodies in the context of L. lactis. We believe L. lactis represents an ideal delivery vehicle as it has received “Generally Regarded as Safe” status from the FDA and does not colonize in the human gastrointestinal tract. This proposal is designed to validate a novel L. lactis-based nanobody platform for rapid development and delivery of nanobodies into the intestinal tract. At the same time, we will validate the first drug candidate using this platform for the prevention and treatment of CDI. The specific aims are to: 1) Select therapeutic nanobodies based on domain mapping and C. difficile toxin neutralization, 2) Generate L. lactis strains that express a neutralizing nanobody against either TcdA, TcdB, or CDT, and 3) Demonstrate that combined strains inhibit C. difficile pathogenesis in a murine model.