Designer probiotics for prevention of cholera

用于预防霍乱的设计师益生菌

• 批准号: 10092926
• 负责人: CAMMIE LESSER
• 金额: $ 24.86万
• 依托单位: MASSACHUSETTS GENERAL HOSPITAL
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-02-01 至 2023-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10092926
• 关键词: Acute Diarrhea; Address; Adenylate Cyclase; Alpaca; Antibodies; Area; Binding; Cells; Cessation of life; Cholera; Cholera Toxin; Cholera Toxin A Subunit; Collaborations; Complex; Cytosol; Deposition; Development; Diarrhea; Disease Outbreaks; Engineering; Enzyme-Linked Immunosorbent Assay; Epidemic; Epithelial Cells; Epitopes; Escherichia coli; Fibrinogen; Future; Goals; Human; Immune response; Immunity; Immunize; Immunoglobulin-Secreting Cells; Improve Access; Inactivated Vaccines; Individual; Infection; Infrastructure; Intervention; Intestines; Laboratories; Lead; Libraries; Life; Measures; Mediating; Memory B-Lymphocyte; Modeling; Modification; Mus; Neuraminidase; O Antigens; Oral; Patients; Peripheral Blood Lymphocyte; Phage Display; Prevention; Prevention strategy; Probiotics; Proteins; Rehydrations; Resources; Risk; Role; Sanitation; Savings; System; Testing; Therapeutic; Toxin; Type III Secretion System Pathway; Vaccination; Variant; Vibrio cholerae; Vibrio cholerae infection; Virulence; Virulence Factors; Water; adaptive immune response; cell killing; diarrheal disease; dimer; expectation; extracellular; follow-up; ganglioside receptor; gastrointestinal; improved; intervention cost; intestinal epithelium; mouse model; nanobodies; neonatal mice; oral vaccine; prevent; receptor; stem; sugar; uptake; vaccination strategy

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.

项目摘要 霍乱是一种急性腹泻性疾病，每年在全球造成3-5万例，超过100,000例死亡， 特别是在资源差的环境中。人道主义危机地区通常与霍乱爆发有关 由于获得清洁水的分解。霍乱治疗的主要手段是口服补液，这可能是一种潜在的 挽救生命的干预措施，而最佳的长期预防策略是改善获得清洁水和 改进的卫生，基础设施修改可能需要数年才能实施。减慢传播 霍乱，尤其是在新兴爆发的情况下，2013年，世卫组织开始库存口腔 用于反应性疫苗接种运动的疫苗。该策略正在证明功效；但是，其中之一 局限性是在疫苗接种后，需要2周才能开发保护性免疫。因此，干预措施 需要在此窗口期间提供保护。在这里，作为解决此问题的定格措施，我们 建议开发益生菌大肠杆菌Nissle 1917（ECN）的设计师变体 配备蛋白质输送系统（T3ECN），将相关的治疗剂分泌为胃肠道 处于风险个人的管腔。有趣的是，事先感染霍乱可提供约5年的保护 已发现感染霍乱的人类主要产生靶向和阻塞的抗体 两个霍乱毒素亚基（CTA和CTB），唾液酸酶和O特异性多糖（OSP）的活性 其唱片的部分。这些观察结果强烈表明，益生菌介导的药物的沉积 这些毒力因子的活性，即单域抗体（又称VHH），也应起作用以防止 霍乱的发展。在AIM 1中，我们建议识别和开发VHH的异二聚体 中和OSP，CTB和SIA。这些异二聚体将被设计为被认为被认为是分泌的蛋白 T3ECN，ECN的变体，编码修改的III型分泌系统，该系统将蛋白质分泌到其 周围而不是进入宿主细胞。在AIM 2中，使用良好的小鼠新生儿霍乱模型，我们 将研究分泌这些VHH阻止霍乱以及的T3ECN的能力 霍乱沉积到肠上皮细胞中。这些探索性研究有可能导致 开发新的低成本干预措施，以阻止新兴霍乱流行病的传播。