Probiotic yeast secreting single-domain antibodies to prevent Clostridium difficile and Campylobacter jejuni disease

摘要 由于多药耐药(MDR)细菌越来越难以治疗，并与更多的 在发病率和死亡率方面，我们正面临一场全球健康危机。不选药的对策 迫切需要抵抗。这一研究计划的总体目标是开发新型的基于免疫的 发展中国家和发达国家针对主要耐多药病原体的预防战略。在这个项目中，我们 目的研制新型益生菌酵母菌免疫干预制剂，以对抗艰难梭菌和 空肠弯曲杆菌，全世界细菌性腹泻和肠道炎症性疾病的主要原因。 我们开发了一种新的平台技术，通过设计一种益生菌来对抗这些肠道病原体 酵母菌，布拉酵母，分泌多特异性单域抗体直接针对细菌 肠道感染部位的毒力因子。通过我们之前资助的英才中心 翻译研究(CETR)项目，我们已经将这项技术应用于艰难梭菌，并已经 产生了一个原型酵母菌株，它结构性地分泌一种四特异单结构域抗体(VHH) 融合，中和艰难梭菌的两种主要肠毒素TcdA和TcdB。一份最近完成的证明- 原理研究表明，口服给药可以保护小鼠免受艰难梭菌的原发和复发。 感染。在这些非常令人鼓舞的结果的基础上，我们建议：1)产生领先的布氏葡萄球菌菌株 分泌针对艰难梭菌和空肠弧菌细菌定植因子的多特异性抗体；2) 临床前评估铅细菌减少细菌定植和预防疾病的效果 在动物模型中；以及3)开发一种配方，用于干燥和包裹这些铅酵母菌株，即 兼容未来在人类身上的使用。我们设想我们未来供人类使用的最终产品将是肠道的。 含有干燥酵母菌株的包衣胶囊，类似于在非处方销售的布氏益生菌产品 柜台。随着拟议的翻译活动的完成，我们预计将产生最终的改进 用于预防艰难梭菌疾病的工程酵母菌株的产品和用于预防空肠弯曲菌的原型产品 疾病。对主要候选者的成功临床前验证还将使进一步的开发和大规模- 加大努力，使这些产品最终商业化，以对抗艰难梭菌和空肠弯曲菌疾病，用于 我们目前还没有有效的预防措施。

Abstract As multidrug-resistant (MDR) bacteria are increasingly difficult to treat and are associated with greater morbidity and mortality, we are facing a global health crisis. Countermeasures that do not select for drug resistance are urgently needed. The overall goal of this research program is to develop novel immune-based preventive strategies against major MDR pathogens in developing and developed countries. In this project we aim to develop novel probiotic yeast-based immune-interventions against Clostridium difficile and Campylobacter jejuni, the major causes of bacterial diarrheal and intestinal inflammatory diseases worldwide. We have developed a novel platform technology against these enteric pathogens by engineering a probiotic yeast, Saccharomyces boulardii, to secrete multi-specific, single-domain antibodies directly targeting bacterial virulence factors at the intestinal site of infection. Through our previously funded Centers of Excellence for Translational Research (CETR) project, we have applied this technology to target C. difficile, and have already generated a prototype yeast strain that constitutively secretes a tetra-specific, single-domain-antibody (VHH) fusion which neutralizes the two major C. difficile enterotoxins TcdA and TcdB. A recently completed proof-of- principle study has shown that oral administration protected mice from both primary and recurrent C. difficile infection. Building upon these highly encouraging results, we propose to: 1) generate lead S. boulardii strains secreting multi-specific antibodies against bacterial colonization factors of C. difficile and C. jejuni; 2) preclinically evaluate the efficacy of the lead strains on reducing bacterial colonization and preventing disease in animal models; and 3) develop a formulation for drying and encapsulating these lead yeast strains that is compatible for future use in humans. We envision that our future final products for human use will be enteric coated capsules containing dried yeast strains, similar to those probiotic S. boulardii products sold over-the- counter. With the completion of proposed translational activities, we expect to generate a final improved product of engineered yeast strains to prevent C. difficile disease and a prototype product to prevent C. jejuni disease. Successful preclinical validation of the lead candidates will also allow further development and large- scale efforts for eventual commercialization of these products against C. difficile and C. jejuni diseases, for which we currently have no effective preventative measures.