Designer probiotics for prevention of cholera

项目摘要 霍乱是一种急性肠道疾病，每年在全世界造成300万至500万例病例和10万多人死亡， 特别是在资源贫乏的环境中。人道主义危机地区往往与霍乱爆发有关 因为无法获得干净的水。霍乱治疗的主要手段是口服补液， 拯救生命的干预措施，而最好的长期预防策略是改善清洁水的获取， 改善卫生设施，基础设施改造，这些都需要数年时间才能实施。为了减缓 霍乱，特别是在新出现的疫情背景下，2013年，世卫组织开始储存口服灭活疫苗， 用于反应性疫苗接种运动的疫苗。这一战略正在证明其有效性;然而，其中一个 局限性在于，接种疫苗后，需要2周时间才能形成保护性免疫力。因此， 在这段时间里，我们需要保护。在此，作为解决这一问题的权宜之计，我们 我建议开发益生菌大肠杆菌Nissle 1917（EcN）的设计变体， 配备有蛋白质递送系统（T3 EcN）以将相关治疗剂分泌到胃肠道中 处于危险中的个体的管腔。有趣的是，先前感染霍乱可提供约5年的保护， 已经发现感染霍乱的人主要产生靶向和阻断 两种霍乱毒素亚基（CTA和CTB）、唾液酸酶和O-特异性多糖（OSP）的活性 其LPS的部分。这些观察结果有力地表明，益生菌介导的物质沉积， 这些毒力因子的活性，即，单域抗体（又名VHH），也应该采取行动，以防止 霍乱的发展。这里，在目的1中，我们提出鉴定和开发VHH的异二聚体， 压制OSP CTB和Sia这些异源二聚体将被工程化以被识别为分泌蛋白， T3 EcN是EcN的变体，其编码经修饰的III型分泌系统，其将蛋白质分泌到其细胞中。 而不是进入宿主细胞。在目标2中，使用完善的小鼠新生儿霍乱模型，我们 将研究分泌这些VHH的T3 EcN阻断霍乱发展的能力， 霍乱在肠上皮细胞中的沉积。这些探索性研究有可能导致 制定新的低成本干预措施，以遏制新出现的霍乱流行病的蔓延。

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.