Nanobodies for the Detection and Neutralization of Listeria monocytogenes

描述(由申请人提供)：李斯特菌病是一种严重的食源性疾病，由单核细胞增生性李斯特菌引起，是一个重大的公共卫生问题。据估计，美国每年有1600例李斯特菌病病例，导致250人死亡。除了这一高死亡率外，孕期感染还可能导致流产或死产。及早检测食品供应中的病原体是保护公众健康的关键。近年来李斯特氏菌病的反复暴发凸显了开发一种快速、灵敏、廉价的生物传感器的必要性。此外，治疗受感染的孕妇需要一个疗程的高侵入性静脉注射抗生素。一种廉价而有效的预防措施可以防止李斯特菌的定居，这将是对产前护理的有力补充。我们建议使用纳米抗体来解决李斯特氏菌病防御中的这些关键漏洞。纳米抗体是从骆驼科物种(骆驼、羊驼、骆驼)独特的免疫系统中发现的重链单域抗体。纳米体生产成本低，高度稳定，具有独特的结合模式，使其独特地适合于开发新的李斯特菌生物传感器和治疗药物。本项目的目的是了解从茶树科噬菌体展示文库中提取的纳米抗体是如何与李斯特菌表面抗原结合并相互作用的，为开发新型李斯特菌生物传感器和治疗药物奠定基础。我们的初步研究已经确定了四个纳米小体，它们与表面暴露的目标结合，并且是李斯特菌毒力因子所必需的。在结构生物学和结合研究中，我们将评估从天然茶树科噬菌体展示文库到李斯特菌毒力因子In1b(AIM1)的纳米小体识别的表位范围。我们还将利用微生物学和结构研究来检查茶树科纳米抗体检测和中和李斯特氏菌的潜力(AIM 2)。

DESCRIPTION (provided by applicant): Listeriosis is a severe food-borne disease caused by the bacterium Listeria monocytogenes and is a significant public health concern. There are an estimated 1600 cases of Listeriosis per year in the US resulting in 250 deaths. In addition to this high mortality rate infection during pregnancy can lead to miscarriage or stillbirth. Early detection of the pathogen in the food supply is key to protecting public health. Repeated outbreaks of Listeriosis in recent years highlight the need for development of a rapid, sensitive and inexpensive biosensor. In addition, treatment of infected pregnant women requires a course of highly invasive intravenously administered antibiotics. An inexpensive and effective prophylactic that could prevent Listeria colonization would be a powerful addition to prenatal care. We propose using nanobodies to address these critical holes in the defense against Listeriosis. Nanobodies are heavy chain, single domain antibodies derived from the unique immune system repertoires found in Camelidae species (camels, alpacas, llamas). Nanobodies are inexpensive to produce, highly stable, and have distinctive binding modes making them uniquely suited to developing new Listeria biosensors and therapeutics. The goal of this project is to understand how nanobodies derived from a naïve Camelidae phage display library bind and interact with Listeria surface antigens, to lay the foundation for development of novel Listeria biosensors and therapeutics. Our preliminary studies have identified four nanobodies that bind to a target that is both surface exposed and required for Listeria virulence factor. Usin structural biology and binding studies we will assess the range of epitopes recognized by nanobodies derived from a combined naïve Camelidae phage display library to Listeria virulence factor InlB (AIM 1). We will also employ microbiological and structural studies to examine the potential of Camelidae nanobodies to detect and neutralize Listeria (AIM 2).