Lactobacilli surface antibody expression for in vivo protection against cholera

描述（由申请人提供）：霍乱，有时称为亚洲霍乱或流行性霍乱，是一种由产肠毒素的霍乱弧菌菌株引起的传染性胃肠炎。NIAID将霍乱弧菌定性为“B”类病原体，构成潜在的生物恐怖主义威胁。霍乱是一种水传播疾病，与食物消费有关。例如，在美洲，食用生贝类和生蔬菜与霍乱爆发有关。霍乱最严重的形式是已知最迅速致命的疾病之一，健康人的血压可能在症状出现后一小时内下降到令人窒息的水平;如果不提供医疗或补液，受感染的病人可能在三小时内死亡。在一个常见的情况下，疾病从第一次液体粪便发展到休克在4至12小时，死亡在18小时至几天，除非提供口服补液。霍乱毒素（CT）首先通过其B亚单位（CT B）附着到肠上皮细胞后发生大量液体损失，然后在其A亚单位进入细胞后通过腺苷酸环化酶的活化影响电解质平衡。目前在美国还没有批准的霍乱疫苗治疗方法，因为迄今为止的策略通常只提供可变的短期免疫力。因此，预防霍乱的公共卫生措施，如适当的污水处理和可靠的安全饮用水来源，被认为是防治这一疾病的最有效方法。 在这项R21提案中，我们将在乳鼠模型中开发和测试一种生物技术支架，用于预防霍乱感染。我们将制备表达抗CTB人单链抗体片段（scFv）的乳酸杆菌作为生物吸附剂以从肠道去除CT。我们的策略是针对这种乳酸杆菌生物吸附剂的临床应用，例如在酸奶中施用，以替代疫苗接种或用作辅助被动免疫治疗。我们预期它的预防效用和它的使用后，首次暴露于细菌的一般公众或，鉴于生物恐怖主义的威胁，武装部队人员和第一反应。 我们将在以下三个具体目标的范围内产生和测试这种新的生物技术支架：具体目标#1：从人噬菌体展示文库中选择抗CTB单链抗体片段。具体目的#2：构建表达CTB特异性scFv抗体片段的乳杆菌菌株。具体目标3：研究用表达CTB特异性scFv抗体的乳杆菌菌株在小鼠中针对霍乱弧菌感染的肠道定殖的保护作用。 公共卫生相关性：在美国，霍乱在19世纪流行，但现代污水和水处理系统几乎已经消除。然而，由于交通的改善，更多的人从美国前往非洲，亚洲或拉丁美洲的部分地区，那里正在发生流行性霍乱。到霍乱流行地区的美国旅行者可能会接触到霍乱细菌。此外，旅行者可能会将受污染的海鲜带回美国;食源性疫情是由旅行者带入美国的受污染海鲜引起的。霍乱在世界上的一些地方是一个严重的问题，来自美国的旅行者可以在旅行前接种疫苗。在这项提案中，我们将开发一种全新的方法来防止霍乱毒性。我们将开发一种针对霍乱毒素的人类抗体，并将其表达在乳酸杆菌上，并利用这种生物技术工具在肠道中定居。这将是一个宝贵的工具，为旅客和第一反应和武装部队人员。

DESCRIPTION (provided by applicant): Cholera, sometimes known as Asian or epidemic cholera, is an infectious gastroenteritis caused by enterotoxin-producing strains of the bacterium Vibrio cholerae. The NIAID has characterized V. cholerae as a category 'B'-pathogen that poses a potential bioterrorism threat. Cholera is a waterborne disease and is associated with food consumption. For example, in the Americas, consumption of raw shellfish and raw vegetables has been associated with cholera outbreaks. In its most severe forms, cholera is one of the most rapidly fatal illnesses known, and a healthy person's blood pressure may drop to hypotensive levels within an hour of the onset of symptoms; infected patients may die within three hours if medical treatment or rehydration is not provided. In a common scenario, the disease progresses from the first liquid stool to shock in 4 to 12 hours, with death following in 18 hours to several days unless oral rehydration is provided. Massive liquid loss occurs after the cholera toxin (CT) first attaches to the gut epithelia via its B subunit (CTB), and then upon cell entry of its A subunit affects the electrolyte balance by activation of adenyl cyclase. There is no currently approved vaccine treatment for cholera available in the U.S. since strategies thus far have generally provided only variable, short-term immunity. Therefore, public health measures that prevent cholera, such as adequate sewage treatment and a reliable source of safe drinking water, are considered the most effective way of combating the disease. In this R21 proposal, we will develop and test in a suckling mouse model a biotechnology scaffold for protection against cholera infection. We will prepare Lactobacilli expressing an anti-CTB human single chain antibody fragment (scFv) as a bioadsorbent to remove CT from the gut. Our strategy is aimed at the clinical application of this Lactobacilli bioadsorbent, administered for example in yogurt, to either replace vaccination or for use as an adjunct passive immune therapy. We anticipate both its prophylactic utility and its use after initial exposure to the bacteria for the general public or, given the bioterrorism threat, Armed Forces Personnel and First responders. We will generate and test this new biotechnology scaffold under the remit of the following three specific aims: Specific aim #1: Selection of anti-CTB single chain antibody fragments from a human phage display library. Specific aim #2: Construction of Lactobacilli strains expressing a CTB-specific scFv antibody fragment. Specific aim #3: Studying the protective effect of gut colonization with a Lactobacilli strain expressing a CTB- specific scFv antibody against V. cholerae infection in mice. PUBLIC HEALTH RELEVANCE: In the United States, cholera was prevalent in the 1800s but has been virtually eliminated by modern sewage and water treatment systems. However, as a result of improved transportation, more persons from the United States travel to parts of Africa, Asia, or Latin America where epidemic cholera is occurring. U.S. travelers to areas with epidemic cholera may be exposed to the cholera bacterium. In addition, travelers may bring contaminated seafood back to the United States; foodborne outbreaks have been caused by contaminated seafood brought into this country by travelers. Cholera is such a problem in parts of the world that travelers from the US can receive a vaccine prior to travel. In this proposal we will develop an entirely new approach protect against cholera toxicity. We will develop a human antibody to cholera toxin and express it on Lactobacilli and colonize the gut with this biotechnology tool. It will be an invaluable tool for the traveler and first responders and armed forces personnel.