Nanobodies for the Detection and Neutralization of Listeria monocytogenes

DESCRIPTION（由申请人提供）：李斯特菌病是由单核细胞增生李斯特菌引起的严重食源性疾病，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，导致250人死亡。此外，妊娠期感染的高死亡率可导致流产或死产。在食品供应中及早发现病原体是保护公众健康的关键。近年来李斯特菌病的反复暴发突出了开发一种快速、灵敏和廉价的生物传感器的必要性。此外，受感染孕妇的治疗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定植，这将是产前护理的有力补充。我们建议使用纳米体来解决这些防御李斯特菌病的关键漏洞。纳米抗体是一种重链单域抗体，来源于骆驼科物种（骆驼、羊驼、大羊驼）独特的免疫系统库。纳米体生产成本低，高度稳定，并且具有独特的结合模式，使它们特别适合开发新的李斯特菌生物传感器和治疗方法。该项目的目的是了解从naïve Camelidae噬菌体展示文库中提取的纳米体如何与李斯特菌表面抗原结合并相互作用，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种纳米体，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。利用结构生物学和结合研究，我们将评估来自naïve Camelidae噬菌体展示库的纳米体识别的表位范围到李斯特菌毒力因子InlB （AIM 1）。我们还将利用微生物学和结构研究来检验Camelidae纳米体检测和中和李斯特菌（AIM 2）的潜力。

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).