Nanobody toolkit for human coronavirus classification

摘要 地方性人类冠状病毒（HCoV）NL 63、OC 43、229 E和HKU 1通常是 与相对温和和自限性呼吸道疾病，类似于普通感冒，在健康 个体然而，新出现的人畜共患冠状病毒（SARS-CoV、MERS-CoV和SARS-CoV-2） 可能会导致更严重的呼吸窘迫，特别是在那些有潜在健康状况的人中， 致命的冠状病毒是多种多样的，从人畜共患病宿主直接或通过 中间宿主由于人口不断增加， 生态系统的侵蚀，从而确保自然和非自然宿主之间的更多碰撞。我们的探索 一项研究计划寻求开发一个小型的免疫试剂工具包，它将构成逻辑门的基础， 对冠状病毒进行识别和分类。单一免疫试剂，对所有共有蛋白具有广泛识别 架构，将允许识别冠状病毒属。分离免疫试剂与绝对 人和人畜共患冠状病毒之间独特氨基酸变异的特异性将允许进一步的 分类为已知病原体。这样的工具包将迅速确定新出现的病毒是否是 冠状病毒是我们熟悉的，或者是一种新的属或谱系，以前从未蔓延到 并可能需要紧急跟进流行病或大流行的可能性。我们的免疫试剂 基于美洲驼单结构域抗体或纳米抗体，其在大肠杆菌中以高产率廉价生产。 大肠杆菌和高度模块化，允许融合酶报告酶的一步探测病毒感染 细胞和夹心测定的简易建立。纳米抗体序列的计算机永生化 这些在Genbank中沉积后立即可供更广泛的研究社区使用， 多克隆血清或分泌IgG的杂交瘤。单罐库技术可实现快速响应， 应对未来任何新出现的冠状病毒，确保我们更加主动而不是被动 来保护人类健康。

ABSTRACT The endemic human coronaviruses (HCoV) NL63, OC43, 229E and HKU1 have typically been associated with relatively mild and self-limiting respiratory disease resembling the common cold in healthy individuals. However, the newly emerging zoonotic coronaviruses (SARS-CoV, MERS-CoV and SARS-CoV-2) can cause more severe respiratory distress, especially in those with underlying health conditions and can be fatal. Coronaviruses are diverse and spillover from zoonotic reservoirs either directly to humans or via an intermediate host. Spillover is likely to be more frequent due to the ever-expanding human population and ecosystem erosion, thereby ensuring more collisions between natural and unnatural host. Our exploratory research proposal seeks to develop a small toolkit of immunoreagents that will form the basis of a logic gate to identify and classify coronaviruses. A single immunoreagent, with broad recognition of overall shared protein architecture, will allow the identification of the coronavirus genera. Separate immunoreagents with absolute specificity for unique amino-acid variation between the human and zoonotic coronaviruses will allow the further classification to a known pathogen. Such a toolkit will quickly establish if a newly emerging virus is a coronavirus we are familiar with or is a novel genus or lineage that has never before spilled over into the human population and may demand urgent follow up for epidemic or pandemic potential. Our immunoreagents are based on llama single domain antibodies or nanobodies that are inexpensive to produce at high yields in E. coli and highly modular, allowing fusion to enzymatic reporter enzymes for one-step probing of virus infected cells and facile establishment of sandwich assays. Immortalization of nanobody sequences in silico means these are immediately available to the wider research community following deposition in Genbank unlike polyclonal sera or hybridomas secreting IgG. Single-pot library technology enables a rapid response to be mounted against any newly emerged coronavirus in the future, ensuring we are more pro-active than re-active to help safeguard human health.