Designer probiotics for prevention of cholera

项目总结 霍乱是一种急性腹泻疾病，每年在全球范围内造成300-500万例病例和超过10万人死亡， 尤其是在资源贫乏的情况下。人道主义危机地区往往与霍乱暴发有关。 由于获得清洁水的渠道出现故障。霍乱治疗的主要手段是口服补液，这是一种潜在的 挽救生命的干预措施，而最好的长期预防战略是改善获得清洁水和 改善卫生条件，基础设施改造可能需要数年时间才能实施。减缓……的传播速度 霍乱，特别是在新爆发的背景下，世卫组织于2013年开始储备口服死亡病例 用于反应性疫苗接种活动的疫苗。这一战略正在显示出有效性；然而，它的一个 限制是，接种疫苗后，需要2周时间才能形成保护性免疫。因此，干预措施 在此期间提供保护是必要的。在这里，作为解决这个问题的权宜之计，我们 建议开发我们已有的益生菌大肠杆菌Nissle 1917(ECN)的设计变体 配备蛋白质递送系统(T3EcN)，将相关治疗药物分泌到胃肠道 高危人群的管腔。有趣的是，先前感染霍乱可提供约5年的保护期 已发现感染霍乱的人主要产生针对和阻断霍乱病毒的抗体 霍乱毒素两个亚基(CTA和CTB)唾液酸酶和O-特异性多糖(OSP)的活性 其内毒素的部分含量。这些观察结果强烈表明，益生菌介导的阻滞剂的沉积 这些毒力因子的活性，即单域抗体(又名VHH)也应该起作用，以防止 霍乱的发展。这里，在目标1中，我们建议鉴定和开发VHH的杂二聚体，该杂二聚体 中和OSP、CTB和SIA。这些异源二聚体将被改造成分泌型蛋白质 T3EcN是ECN的一个变体，它编码一种经过修改的III型分泌系统，它将蛋白质分泌到其 包围而不是进入宿主细胞。在目标2中，使用已建立的小鼠新生儿霍乱模型，我们 将调查分泌这些VHH的T3EcN阻止霍乱发展的能力以及 霍乱在肠上皮细胞中的沉积。这些探索性研究有可能导致 开发一种新的低成本干预措施，以遏制新出现的霍乱疫情的传播。

PROJECT SUMMARY Cholera, an acute diarrheal illness, causes 3-5 million cases and over 100,000 deaths worldwide each year, particularly in resource poor settings. Areas of humanitarian crises are often associated with cholera outbreaks due a breakdown in access to clean water. The mainstay of cholera treatment is oral rehydration, a potentially life-saving intervention, while the best long-term prevention strategy is to improve access to clean water and improved sanitation, infrastructure modifications that can take years to implement. To slow the spread of cholera, particularly in the setting of emerging outbreaks, in 2013 the WHO began stockpiling oral killed vaccines for use in reactive vaccination campaigns. This strategy is demonstrating efficacy; however, one of its limitations is that post-vaccination, it takes 2 weeks to develop protective immunity. Thus, interventions that provide protection during this window are needed. Here, as a stopgap measure to address this issue, we propose to develop designer variants of the probiotic Escherichia coli Nissle 1917 (EcN) that we have equipped with a protein delivery system (T3EcN) to secrete relevant therapeutics into the gastrointestinal lumen of at risk individuals. Interestingly, prior infection with cholera provides protection for ~5 years and humans infected with cholera have been found to primarily generate antibodies that target and block the activity of the two cholera toxin subunits (CTA and CTB), sialidase and the O-specific polysaccharide (OSP) moiety of its LPS. These observations strongly suggest that probiotic-mediated deposition of agents that block the activity of these virulence factors, i.e., single domain antibodies (aka VHH), should also act to prevent the development of cholera. Here, in Aim 1, we propose to identify and develop heterodimers of VHH that neutralize OSP, CTB and Sia. These heterodimers will be engineered to be recognized as secreted proteins by T3EcN, a variant of EcN that encodes a modified type III secretion system, which secretes proteins into its surrounding rather than into host cells. In Aim 2, using the well-established mouse neonatal cholera model, we will investigate the ability of T3EcN that secrete these VHH to block the development of cholera as well as the deposition of cholera into intestinal epithelial cells. These exploratory studies have the potential to lead to the development of a new low-cost intervention to stem the spread of emerging cholera epidemics.