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，并且与更大的 发病率和死亡率，我们正面临着一场全球健康危机。不选择毒品的对策 迫切需要抵抗。这项研究计划的总体目标是开发新的基于免疫的 发展中国家和发达国家针对主要MDR病原体的预防策略。在这个项目中，我们 旨在开发新的基于益生菌酵母的免疫干预措施，以对抗艰难梭菌， 空肠弯曲菌是世界范围内细菌性肠炎和肠道炎症性疾病的主要致病菌。 我们已经开发了一种新的平台技术，通过工程益生菌对抗这些肠道病原体 酵母菌布拉酵母（Saccharomyces boulardii）分泌直接靶向细菌的多特异性单结构域抗体 在肠道感染部位的毒力因子。通过我们以前资助的卓越中心， 翻译研究（CETR）项目，我们已经将这项技术应用于目标C。艰难，而且已经 产生了组成型分泌四特异性单结构域抗体（VHH）的原型酵母菌株， 核聚变反应中和了两个主要的碳原子。艰难梭菌肠毒素TcdA和TcdB。一个最近完成的证明- 主要研究表明口服给药保护小鼠免受原发性和复发性C.艰难 感染基于这些非常令人鼓舞的结果，我们建议：1）产生铅S。布拉氏菌株 分泌针对C. difficile和C.空肠; 2） 临床前评价先导菌株减少细菌定植和预防疾病的有效性 在动物模型中;和3）开发用于干燥和包封这些先导酵母菌株的制剂， 未来可用于人类。我们设想，我们未来用于人类的最终产品将是肠溶的 包衣胶囊含有干燥的酵母菌株，类似于那些益生菌S。布拉地产品销售超过 柜台随着拟议翻译活动的完成，我们预计将产生一个最终的改进， 工程酵母菌株的产品，以防止C.艰难梭菌病和预防C.空肠 疾病对主要候选药物的成功临床前验证也将允许进一步开发和大规模应用。 规模化的努力，这些产品的最终商业化对C。difficile和C.空肠疾病，用于 目前我们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.