A Novel Probiotic-Based Nanobody Delivery Platform

项目摘要 我们的目标是培养基于乳酸乳球菌的纳米体表达菌株，并证明 制备的纳米体在体外和体内都能有效地中和艰难梭菌毒素。 艰难梭菌是一种普遍存在的厌氧革兰氏阳性细菌，可产生孢子并对 环境压力和一线抗生素，如克林霉素1。艰难梭菌分泌型致病菌株 毒性糖基转移酶的不同组合，大的梭状芽胞杆菌毒素A(TcdA)和B(Tcdb)，以及 二元毒素艰难梭菌转移酶(CDT)2.TcdA和TcdB破坏紧密连接进入固有层 以促进炎症。CDT与TcdA和TcdB协同作用加重艰难梭菌感染(CDI)，诱导 细胞突起增加艰难梭菌的粘附性并抑制保护性宿主嗜酸性粒细胞反应 科罗拉多3。毒素的酶活性破坏肠上皮细胞，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，但持续使用抗生素进一步 破坏微生物区系，导致CDI复发。因此，经常性的CDI目前是最有效的 采用粪便微生物区系移植(FMT)疗法4-6。然而，FMT是一种昂贵的、异质的和 由于安全问题，需要FDA特别批准的定义不明确的治疗方法7。因此，新的 迫切需要采取措施来预防和治疗CDI。 我们最近从骆驼VHH噬菌体展示中产生了独特的纳米体。我们的研究表明 已识别的纳米体与梭状芽胞杆菌毒素A(TcdA)、B(TcdB)和C的关键构象表位结合。 艰难转移酶/二元毒素(CDT)，具有中和肠道中CDI相关毒素的潜力 一条小路。将这些纳米体口服到肠道并改善粘膜 药代动力学/药效学口服后，我们的策略是表达纳米体 在乳乳杆菌的背景下。我们相信L.Lactis代表了一种理想的递送工具，因为它已经收到了 被美国食品及药物管理局认定为“安全”状态，不在人体胃肠道内定居。 该方案旨在验证一种基于乳酸乳杆菌的新型纳米体平台的快速开发和 将纳米小体送入肠道。同时，我们将验证第一种候选药物的有效性 这个防治CDI的平台。具体目标是：1)选择治疗方法 基于结构域映射和艰难梭菌毒素中和的纳米抗体，2)产生乳酸乳杆菌菌株， 表达针对TcdA、TcdB或CDT的中和纳米体，以及3)展示其组合 菌株在小鼠模型中抑制艰难梭菌的致病作用。

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.