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2024-03-07 20:49:14

指南共识：RBD快速眼动睡眠行为障碍管理指南2022年版|褪黑激素|氯硝西泮|RBD|睡眠|-健康界

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指南共识：RBD快速眼动睡眠行为障碍管理指南2022年版

2023

09/16

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神经内科高岱佺医生

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在较年轻成人中(＜40岁)，RBD最常发生于使用抗抑郁药或有发作性睡病时。RBD在儿童中罕见，大多与1型发作性睡病和使用抗抑郁药有关，偶尔与脑干肿瘤和多种神经精神疾病有关。

2022年12月，美国睡眠医学会（AASM）发布了《快速动眼期睡眠行为障碍（RBD）管理指南》，该指南为RBD患者的管理提供了临床实践建议。主要包括：（1）帮助患者保持安全的睡眠环境，特别是移走床边锐器或可能造成伤害的物体，或者在其边缘和床头板垫上垫子；（2）患有严重RBD的患者建议与他们的伴侣分开睡觉，或者在其之间放一个枕头；（3）在有条件的情况下，建议临床医生使用氯硝西泮、褪黑激素等药物治疗成人孤立性RBD或继发性RBD快速眼动睡眠行为障碍(rapid eye movement sleep behavior disorder, RBD)是一种异态睡眠，特征为在快速眼动(rapid eye movement, REM)睡眠期间肌张力降低的状态消失时，出现梦境表演行为。RBD梦境表演的严重程度不一，轻则出现良性的手部动作，重则出现暴力的挥动手臂、拳打脚踢。患者通常担心此类行为会造成或可能造成自己和/或床伴受伤而就医。RBD是α-突触核蛋白神经变性疾病的前驱综合征。因此，通常经过较长一段时间后，绝大多数RBD患者最终会出现帕金森病(parkinson disease, PD)或相关疾病[如多系统萎缩(multiple system atrophy, MSA)或路易体痴呆(dementia with Lewy bodies, DLB)]的症状和体征。出现帕金森综合征前，患者可能有轻微的感觉、运动和认知功能障碍(包括嗅觉丧失和便秘)，这与即将发生的神经系统疾病一致。2022版RBD管理指南提供了现有证据的全面更新和RBD治疗临床实践建议的综合。它旨在通过告知护理RBD患者的临床医生来优化以患者为中心的护理。本临床实践指南通过确定在特定情况下(孤立性RBD、继发性RBD、药物诱发/加重的RBD)最有效的治疗方法，为RBD的管理提供实践建议。建议1：临床医生使用氯硝西泮(相对于不治疗)治疗成人孤立性RBD。(有条件)备注：作为老年患者，在使用和给药氯硝西泮时应考虑患者的年龄可能对氯硝西泮的镇静副作用更敏感，需要更长时间代谢和消除苯二氮卓。建议2：临床医生使用速释褪黑激素(相对于不治疗)治疗成人孤立性RBD。(有条件)备注：由于褪黑激素在美国和其他几个司法管辖区不受FDA监管，不同的配方可能会导致不同品牌褪黑激素的功效不同。具有美国药典(USP)验证标志的褪黑激素标签已被证实包含标签上所述的褪黑激素量，并可提供目前可用的褪黑激素治疗方案中最一致的剂量。建议3：临床医生使用普拉克索(相对于不治疗)治疗成人孤立性RBD。(有条件)备注：普拉克索似乎对多导睡眠图(PSG)记录的周期性肢体运动升高的RBD患者最有效，这表明其疗效可能次于辅助运动活动。建议4：临床医生使用经皮给药的卡巴拉汀(相对于不治疗)治疗轻度认知障碍(MCI)成人的孤立性RBD。(有条件)建议5：临床医生使用氯硝西泮(相对于不治疗)治疗成人因医疗条件导致的继发性RBD。(有条件)备注：在使用氯硝西泮和给药时，应考虑患者的医疗状况、年龄以及氯硝西泮引起的镇静和失衡的风险。老年患者可能对氯硝西泮的镇静副作用更敏感，需要更长时间代谢和消除苯二氮卓类药物。建议6：临床医生使用速释褪黑激素(相对于不治疗)治疗成人因医疗条件导致的继发性RBD。(有条件)备注：由于褪黑激素在美国和其他几个司法管辖区不受FDA监管，不同的配方可能会导致不同品牌褪黑激素的功效不同。具有美国药典(USP)验证标志的褪黑激素标签已被证实包含标签上所述的褪黑激素量，并可提供目前可用的褪黑激素治疗方案中最一致的剂量。建议7：临床医生使用经皮给药的利伐斯的明(相对于不治疗)治疗成人因医疗条件(帕金森病)引起的继发性RBD。(有条件)建议8：由于成人的医疗条件，临床医生不要使用深部脑刺激(相对于不治疗)来治疗继发性RBD。(有条件)备注:本建议仅基于深部脑刺激对继发性快速眼动睡眠行为障碍的影响。它不适用于在帕金森氏病的运动症状的治疗中使用深部脑刺激。建议9：临床医生使用停药(相对于继续用药)来治疗成人药物性RBD。(有条件)流行病学RBD的患病率在一般人群中为0.5%-1.25%，在老年人群中约为2%。这意味着全球预计有4000万至1亿患者，但绝大多数病例未被发现在较年轻成人中(＜40岁)，RBD最常发生于使用抗抑郁药或有发作性睡病时。RBD在儿童中罕见，大多与1型发作性睡病和使用抗抑郁药有关，偶尔与脑干肿瘤和多种神经精神疾病有关。单纯性RBD是α-突触核蛋白神经病变的前驱综合征，其普遍存在于PD患者(33%-50%)、MSA患者(80%-95%)和DLB患者(80%）临床表现梦境表演 — RBD的典型症状是REM睡眠期间有反复发作的睡眠相关发声和/或复杂运动行为，与梦境相对应。神经系统表现 — 单纯性RBD患者常有符合早期神经变性疾病的特征，即轻微的进行性运动和认知功能障碍。典型表现包括轻度姿势不稳和步态异常(包括步态冻结)，与轻微帕金森综合征一致。视频多导睡眠监测 — 为确诊RBD和排除其他类似于RBD的睡眠障碍，需行视频PSG检查。治疗建立安全的睡眠环境是首要治疗目标，方法包括改变睡眠环境以及必要时给予药物治疗安全的睡眠环境 — 梦境表演的发生频率不能预测是否出现损伤，故应建议所有RBD患者及其床伴改变睡眠环境以预防损伤。轻症患者使用这种方法可能就已足够。可逆因素 — RBD患者应尽可能停用或不用已知会加重RBD的药物，包括5-羟色胺能抗抑郁药。许多药物相关梦境表演病例在停用致病药物后呈自限性。褪黑素— 对于频繁出现破坏性或损伤性行为的患者，可以选择褪黑素。其耐受性往往优于另一种一线药物氯硝西泮，尤其是对于有神经变性疾病的老年患者氯硝西泮 — 长期以来，小剂量氯硝西泮(起始剂量0.25-0.5mg，睡前口服)用于治疗RBD

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关键词:

褪黑激素,氯硝西泮,RBD,睡眠

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中科院孙兵团队等发现新冠不同突变株逃逸中和抗体的规律图谱

2021

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生物世界

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新型冠状病毒刺突糖蛋白的受体结合域（RBD）是介导病毒与宿主细胞ACE2受体的结合关键部位，也是中和抗体和疫苗作用的关键靶点。

新型冠状病毒刺突糖蛋白的受体结合域（RBD）是介导病毒与宿主细胞ACE2受体的结合关键部位，也是中和抗体和疫苗作用的关键靶点。针对RBD关键抗原改变的有效监测和对其所引起免疫逃逸的早期应对，具有重要科学意义。虽然全世界的流行病学研究已经产生了大量的病毒基因组序列，并通过GISAID计划快速共享，这些数据对于监控病毒的传播和理解其进化的机制是必不可少的。但是如何有效地从海量的流行病学数据中追踪可能会引起新冠病毒突变株的免疫逃逸和抗原变化规律依然有许多挑战。对于引入突变可能造成RBD抗原漂移的分子机制与导致抗体作用失效的关键氨基酸，依然不是很清楚。从而不能在新冠病毒的新变种迅速传播之前进行有效的预警。近日，中国科学院分子细胞科学卓越创新中心（生物化学与细胞生物学研究所）孙兵研究组与时任上海市（复旦大学附属）公共卫生临床中心卢洪洲团队及复旦大学基础医学院谢幼华团队合作，在 Genome Medicine 期刊发表了题为：Comprehensive mapping of binding hot spots of SARS-CoV-2 RBD-specific neutralizing antibodies for tracking immune escape variants 的研究论文。该研究系统性地揭示了新冠病毒（SARS-CoV-2）受体结合区域（RBD）所诱导中和抗体表位的主要分类。并深入分析了新冠病毒突变株逃逸中和抗体的变化规律及潜在的关键氨基酸位点，为监测新冠病毒突变株及预判疫苗、单抗药物以及血清学诊断的有效性提供了关键科学依据和可借鉴的检测方法。研究团队基于前期工作，从新冠病毒感染康复人群分离的93个RBD特异性抗体和结合已报道的抗体，利用丙氨酸扫描和表位竞争实验，把RBD上保护性的抗原表位系统性地分为四类。研究发现：大多数中和抗体主要识别第一类和第二类抗原表位；而能交叉结合SARS-CoV-2及SARS-CoV的中和抗体主要识别第三和第四类抗原表位，该类表位主要位于RBD core区域并显著影响RBD稳定性，其在进化上由有一定保守性，靶向该位置的交叉抗体可以限制病毒免疫逃逸。a.来自4个新冠康复患者的93个RBD特异性抗体的中和活性,b.利用单抗盘将抗原表位分为四大类, c.抗原表位与中和活性的构效关系, d.抗原表位与交叉识别SARS-CoV及SARS-CoV-2能力的构效关系, e.单克隆中和抗体对天然变异毒株的中和能力, f.新冠康复患者外周血清对天然变异毒株的中和能力。借助新冠数据共享平台，研究人员对每个RBD抗原位点上的抗体结合热点（hot spots）区域定向监测单点变异株和多点突变株对抗体逃逸的影响，鉴定出多个未报道的导致中和抗体活性减弱或消失的RBD关键自然突变位点。多点突变（如Beta株）可以显著降低大多数单克隆抗体及康复患者外周血浆的中和活性。未来的工作重点，需要警惕不同抗原表位的多点突变带来的严重危害。分子细胞卓越中心孙兵研究组博士后伊春艳和孙晓玉、上海市（复旦大学附属）公共卫生临床中心卢洪洲团队林逸骁医生以及复旦大学基础医学院谢幼华团队博士后谷陈建为该研究的共同第一作者。孙兵研究员、卢洪洲教授，谢幼华教授和凌志洋副研究员为本文共同通讯作者。论文链接：https://doi.org/10.1186/s13073-021-00985-w

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关键词:

抗体,RBD,病毒,抗原,中和

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三分钟看懂城市版图|聚焦城市RBD，带你读懂一座城市的未来|延庆|商务区|世园会|rbd_网易房产

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三分钟看懂城市版图|聚焦城市RBD，带你读懂一座城市的未来

2019-05-24 10:34:01　来源: 网易房产

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“人类为了生存来到了城市，人们为了生活的美好，留在了城市。”——亚里士多德城市，为人而生，也为人而进化。而城市与人类的关系，因为经济因素的加入，使得这二者之间呈现出了由表及里的改变。其中，休闲与旅游在人类生活中地位的提升，体现的正是人们对于从物质需求到人文内核的转变，亦是城市功能逐渐发展完善的体现，更直接催生了城市休闲商务区RBD的诞生。每座城都有自己的RBD（休闲商务区），上海RBD在黄浦区，广州RBD在天河区，深圳RBD在南山区……那么，延庆未来RBD在哪里？形成大型RBD必须有三个条件：其一，靠近但不比邻综合性枢纽；其二，位于政府主导的城市发展方向上；其三，有实力派企业推动。世园会催化交通加持，延庆新城RBD蓝图初显延庆作为北京生态皇冠上的一颗明珠，拥有优美、磅礴的天赋资源、现在，也必将成为北京城市新名片。“世园会”催化价值聚变 ,下个“RBD”就在延庆新城。自世园盛会启幕以来，板块热度高居不下，鉴证着延庆前所未有的城区蜕变，赢来城市发展又一极--正在崛起的延庆新城RBD。旅游休闲性质使城市空间从传统意义的消费场所本身转变为消费对象，功能的变迁使其成为超大能量的带动体。世园会和冬奥会两大国际盛会共同促使众多道路交通的建设，极大缩短了城市空间距离。从延庆对内交通枢纽，到对外双城切换的多维交通支点：京新高速、京藏高速、京礼高速、S2轻轨、以及2019年即将开通的京张高铁，每一条都是能量流、资金流、信息流、产业流的带动。 RBD城市风向标，指引城市发展RBD作为城市中心体系中的一个新枢纽，使原并不活跃的地区转换为新的城市休闲性商业游憩活动中心，其核心是吸引人流的商业游憩，这种功能的形成将对城市产生永久的影响。世园会作为延庆RBD轴“芯”，依托优越的自然景观资源，不仅持续吸引海量观光休闲游客，更将成为延庆新城延伸板块的驱动引擎。汇聚面积约1.9万平米世园会商业服务设施、3万多平米的碧桂园·世奥中央广场，形成延庆新城未来新的区域商业中心——世园商圈，提供商业、酒店、办公多种综合性服务功能。延庆新城也因此能畅享世园商圈辐射的繁华与商业配套。受延庆新城政策支持，根据规划，延庆新城通过发展创意产业、都市旅游、区域经济、生态居住，积极打造生态文明示范区和京西北科技创新特色发展区，而带有世园基因的延庆RBD，集文化、生态、休闲、商业、商务于一体的城市生态商务休闲区，以无可置疑的地位成为城市发展的风向标和发展样板。城市生态休闲商务区，引领投资风向标在多重利好的带动下，延庆新城板块吸引力与日俱增，不仅成为下一个城市关注焦点，更是成为商家纷纷投资的热土。各大品牌纷纷抢滩进驻，餐饮类：老舍茶馆、鼎泰楼、东来顺、麦当劳、肯德基、必胜客、吉野家在内的众多知名品牌进驻世园会。生活类：北京世园凯悦酒店、北京世园海泉湾商务酒店、北京世园璞燊（shēn）酒店已进驻世园新城，中交富力万豪酒店落户延庆城北会展板块。科技类：神州数码将凭借平台优势，为延庆区构建IT服务全价值链生态和环保产业生态圈；结合延庆区绿色发展区域战略定位优化区域产业结构；加强本地高端人才培养，促进延庆区产业升级和人才发展的良性循环。一定程度上提高了所在城区的人流活跃度，拉动区域发展。而无论从配套、交通、或自然景观来看，世园会周边板块地段优势十分明显，投资价值不言而喻，碧桂园·世奥中央广场以前瞻性战略眼光，以世园会为圆心，助力打造延庆新城——城市生态休闲商务区。依附世园会为区域打造城市生态休闲商务区，以和谐生态居住、艺术旅游休闲、高端新兴产业、现代商务服务等功能为主的现代都市生态新区；为延庆新城提供旅游购物休闲娱乐场所，为都市上班族提供休闲商务空间，为周边居民提供工作之余放松、娱乐的休闲生活。

单价4万出逃，千灯湖大平层也开始卷了？

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新冠疫苗第四针接种放开，丽珠重组新冠病毒融合蛋白疫苗有效保护奥密克戎变异株感染者，该疫苗有哪些特点？ - 知乎

新冠疫苗第四针接种放开，丽珠重组新冠病毒融合蛋白疫苗有效保护奥密克戎变异株感染者，该疫苗有哪些特点？ - 知乎首页知乎知学堂发现等你来答切换模式登录/注册医生医学疫苗新冠疫苗丽珠疫苗新冠疫苗第四针接种放开，丽珠重组新冠病毒融合蛋白疫苗有效保护奥密克戎变异株感染者，该疫苗有哪些特点？12月13日，国务院联防联控机制印发《新冠病毒疫苗第二剂次加强免疫接种实施方案（以下简称“《方案》”），强调可在第一剂次加强免疫接种基础上，在感染高风…显示全部关注者800被浏览1,503,708关注问题写回答邀请回答好问题 2020 条评论分享201 个回答默认排序爱泡温水的青蛙中山大学外科学硕士关注这个疫苗名字看起来很长，本质就是新冠病毒上的刺突糖蛋白的受体结合区（RBD），不过是人工魔改过的。根据《 Cell Research》上的研究显示，丽康V-01疫苗的原理是：在新冠病毒捕获的刺突糖蛋白的受体结合区（RBD）的 N 末端，配备干扰素-α (IFNα)，并在 RBD的C 末端进行免疫球蛋白（IgG1 Fc ）二聚化。这种改进会靶向和激活淋巴结中的树突状细胞 (DC)，增强抗原加工和呈递。在较低剂量接种，即可显示出比单纯的 RBD 更强的免疫原性，并诱导更高的 IgG抗体滴度、 CD8 + T 细胞反应[1]。这种代号为IRF的疫苗具有以下优点：强效：主要针对引流淋巴结的抗原加工和呈递，使得这种魔改的RBD（IRF）诱导的抗体的水平远高于对单体 RBD 的反应水平。安全：在低剂量 (0.01 µg)的情况下就引发了强大而持久的免疫反应。可以缓慢释放到淋巴结中，从而导致长期、有效和安全的免疫反应。耐久：在恒河猴模型中，IRF 疫苗诱导的 NAb 水平维持了 250 多天。制造和储存方便：与Fc的融合易于大规模生产和纯化，并可以在 4–25 °C 下稳定数月。当然，现在是应用到人体阶段，仅靠以上这些动物实验是无法实现的。据相关研究显示，丽康V-01疫苗的I、II、III期临床试验数据同样可观。一、I期临床试验I期临床试验于 2021 年 2 月在广东省疾病预防控制中心（中国高州）启动，共招募了180 名符合条件的受试者[2]。安全性：30 天内约有 25% 的疫苗相关总体不良事件，主要是注射部位的疼痛和瘙痒，另外是发烧、厌食、肌肉痛、头痛。有效性：第 21 天有适度的免疫反应，在第 28 天进一步增加，在第 35 天或第 49 天达到峰值。中和抗体的血清转化率在第 21 天时大多低于 70%，在第 49 天时所有接种组的血清转化率均高于 95%。V-01 在≥60 岁老年参与者中同样引起满意的体液反应，而且不良反应发生频率较低，这显示可以安全应用于老年人。二、II期临床试验II期临床试验于2021 年 3 月在高州市疾病预防控制中心启动，招募440名年轻人和440名≥60 岁的老年人，主要评估免疫原性和安全性[3]。老年人不良反应比年轻人少，最常见的不良反应为疲劳、发烧、咳嗽、头痛、肌肉疼痛和老年人会出现恶心。中和抗体几何平均滴度方面，年轻人和老年人类似，并无太大差异，都在第 35 天达到峰值，并在第 49 天保持较高水平；打两针会比打一针的中和抗体血清转化率和高，而且是大大的高；三、III期临床试验III期临床试验主要在巴基斯坦和马来西亚进行[4] [5]，招募4928名受试者：已完成两剂灭活疫苗的 18 岁或以上成年人。目的是：评估 3-6 个月内接种一剂 V-01 疫苗的有效性、免疫原性和安全性。安全性：不良事件发生率跟安慰剂没差别（19.7% VS 21.5%），主要是注射部位疼痛是，其次是瘙痒和硬结；免疫源性：接种后 14 天，V-01 疫苗组的中和效价增加11.3倍（128.3–1452.8）；3. 功效：针对Omicron 变异感染，保护效力为 47.0%，针对 Delta 变体的为79.9%。四、总结可以看出，III期临床试验的接种方案是有区别于I、II期的，主要前提就是接种2针灭活疫苗的前提，非常适合我们的国情。目前最主要的民众担心，就是病毒变异太快，最初接种的疫苗已经跟不上Omicron的步伐。III期研究显示，在接种过两剂灭活疫苗的人群中使用重组 SARS-CoV-2 融合蛋白疫苗进行加强接种，会具有 47.8% 的增量疗效。总的来说，从目前的情况来看，这款疫苗是具有进行推广的潜力，如果情况合适，还是可以接种的。五、答疑区1、这个疫苗必须要接种完灭活疫苗后，再接种才有保护效力吗？并不是。从美国临床试验数据库[6]、发表论文的I、II期临床试验数据来看，这个重组融合疫苗在不接种灭活疫苗的基础上，接种第二针开始会具有明显的抗体上升，同样具有保护效力。但第一针的保护效力过低。2、没接种过灭活疫苗，可以打吗？暂时没有相关方面的信息，也就是说暂时不可以。目前这个V01重组疫苗主要的研究方向还是作为接种灭活疫苗后的加强针。根据广东省药品监督管理局的公示，目前主要作为防疫序贯加强免疫紧急使用[7]。3、针对Omicron的效果怎么样？在接种第一剂加强针之后，抗体会在第7天开始明显增加，14天达到高峰，可以持续高水平约3个月[8]。对于Omicron BA.1，加强针后产生的抗体同样具备强大的中和能力（如下图），第14天抗体滴度可达灭活疫苗加强针的14.3 倍。总体来说，目前公布的数据还是可观的，而且对于年龄大于60岁的老年人在安全的前提下同样可以激发满意的免疫反应，这可能是紧急序贯使用的原因之一。参考^https://www.nature.com/articles/s41422-021-00531-8^https://www.tandfonline.com/doi/full/10.1080/22221751.2021.1951126^https://journals.lww.com/cmj/Fulltext/2021/08200/Immunogenicity_and_safety_of_a_recombinant_fusion.11.aspx^https://www.tandfonline.com/doi/full/10.1080/22221751.2022.2088406^https://clinicaltrials.gov/ct2/show/NCT05096832^https://covid19.trackvaccines.org/vaccines/108/^http://mpa.gd.gov.cn/gkmlpt/content/4/4021/post_4021223.html#1889^https://www.mdpi.com/2077-0383/11/14/4164编辑于 2022-12-17 11:25赞同 369116 条评论分享收藏喜欢收起Justin LiuNUS School of Medicine 关注现在疫情已经是四面开花了，第四针相当于应急接种，此时最重要的指标是起效速度，丽珠作为一款注射的重组蛋白疫苗，按官方说法受试者在使用公司疫苗序贯加强后，接种后7天，中和抗体水平即可比免前提高约20倍；14天即接近峰值，可应急接种使用；加强免疫后6个月，抗体水平仍维持在峰值的1/3以上，处在较高水平，优势明显看起来不错，但必须和两种吸入式疫苗进行比较。鼻喷苗目前只有北京能打，其他地区还仅供货给医院应急接种。对大部分人而言，需要做的是在丽珠重组蛋白 vs 康希诺腺病毒吸入之间做选择。以下信息截图自钟老昨天的讲座[1]：腺病毒疫苗起效很快，可以立刻诱发黏膜免疫，5-7天就可以达峰，作为异种加强针28天后的抗体滴度比同种加强针高11倍（116.8 vs. 10.5对于BA1.1 omicron）,而丽珠的重组蛋白疫苗仅高3倍(33 vs. 10.5)。从效力和起效速度来看，腺病毒吸入疫苗都完胜。我明早打腺病毒吸入疫苗作为第三针，到时候可以亲身获得一个数据点给大家分享一下。但从原理上而言，重组蛋白疫苗的安全性较高，参见以下数据，不良反应明显少于mRNA和腺病毒疫苗（注意图中臭名昭著的AZ疫苗采用的是大猩猩载体ChAdOx1，而非国内使用的Ad5，免疫原性差别较大）此外，黏膜免疫一部分来自recruit lung resident T cells （Trm）, 对于年轻人而言，这个可以提升对病毒的免疫，但对老年人可能会加剧免疫反应应答，有一定可能诱发ILD。[2]因此对老年人而言，如果时间允许，打丽珠疫苗较为安全。对于已在疫情第一线城市的，腺病毒吸入苗更加及时。参考^钟南山新冠（Omicron）疫情动态及应对^衰老与免疫-衰老与TRM https://mp.weixin.qq.com/s/i7Qew4PLT0sn86A3z8Fy1g编辑于 2022-12-16 19:59赞同 21192 条评论分享收藏喜欢

A novel receptor-binding domain (RBD)-based mRNA vaccine against SARS-CoV-2 | Cell Research

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A novel receptor-binding domain (RBD)-based mRNA vaccine against SARS-CoV-2

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Letter to the Editor

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Published: 05 August 2020

A novel receptor-binding domain (RBD)-based mRNA vaccine against SARS-CoV-2

Wanbo Tai1 na1, Xiujuan Zhang1 na1, Aleksandra Drelich2, Juan Shi1, Jason C. Hsu2, Larry Luchsinger1, Christopher D. Hillyer1, Chien-Te K. Tseng2, Shibo Jiang1 & …Lanying Du1 Show authors

Cell Research

volume 30, pages 932–935 (2020)Cite this article

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ImmunologyMolecular biology

Dear Editor,The pandemic of coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) highlights the need to develop effective and safe vaccines. Similar to SARS-CoV, SARS-CoV-2 recognizes angiotensin-converting enzyme 2 (ACE2) as receptor for host cell entry.1,2 SARS-CoV-2 spike (S) protein consists of S1, including receptor-binding domain (RBD), and S2 subunits.3,4 We previously demonstrated that RBDs of SARS-CoV and MERS-CoV serve as important targets for the development of effective vaccines.5,6To identify an mRNA candidate vaccine, we initially designed two mRNA constructs expressing S1 and RBD, respectively, of SARS-CoV-2 S protein (Fig. 1a). Both culture supernatants and lysates of cells transfected with S1 or RBD mRNA reacted strongly with a SARS-CoV-2 RBD-specific antibody (Supplementary information, Fig. S1a), demonstrating expression of the target proteins.Fig. 1: Design and evaluation of SARS-CoV-2 S1 and RBD mRNA vaccines.a Schematic diagram of SARS-CoV-2 S1 and RBD mRNA construction. The synthesized nucleoside-modified S1 and RBD mRNAs were encapsulated with LNPs to form mRNA-LNPs. b–j IgG and neutralizing antibodies induced in immunized BALB/c mice at different immunogen doses via intradermal (I.D.) prime and boost at 4 weeks. Sera at 10 days post-2nd immunization with SARS-CoV-2 S1 or RBD mRNA-LNP (e.g., S1-LNP or RBD-LNP) (30 μg/mouse), or empty LNP (control), were detected for SARS-CoV-2 RBD-specific IgG antibodies by ELISA (b) or neutralizing antibodies against pseudotyped (c) and live (d) SARS-CoV-2 infection. Sera at 10, 40, and 70 days post-2nd immunization with above mRNA-LNPs (10 μg/mouse) or control were detected for neutralizing antibodies against pseudotyped (e–g) and live (h–j) SARS-CoV-2 infection. The ELISA plates were coated with SARS-CoV-2 RBD-Fc protein (1 µg/ml), and IgG antibody (Ab) titer was calculated. Overall, 50% neutralizing antibody titer (nAb NT50) was calculated against SARS-CoV-2 pseudovirus infection in hACE2/293T cells, or against live SARS-CoV-2 infection by a cytopathic effect (CPE)-based microneutralization assay in Vero E6 cells. The dotted lines indicate detection limit. k Dose-dependent inhibition of sera of mice receiving a vaccine (30 μg/mouse) on SARS-CoV-2 RBD-hACE2 receptor binding in hACE2/293T cells by flow cytometry analysis. Percent (%) inhibition was calculated based on relative fluorescence intensity with or without respective serum at indicated dilutions. l–n Representative images of such inhibition by sera (1:5) of mice immunized with SARS-CoV-2 S1 mRNA-LNP (S1-LNP) (l), RBD mRNA-LNP (RBD-LNP) (m), or empty LNP control (n) are shown in blue lines with respective median fluorescence intensity (MFI) values. The binding between SARS-CoV-2 RBD-Fc protein (5 µg/mL) and hACE2 is shown in red lines. Gray shades indicate Fc-hACE2 binding. o Cross-reactivity of immunized mouse sera against SARS-CoV RBD by ELISA. SARS-CoV RBD-Fc protein-coated plates (1 µg/mL) were used to detect IgG Ab titer. p–r Cross nAb NT50 of above sera (twofold serial dilutions from 1:5) against infection of SARS-CoV pseudovirus expressing S protein of human SARS-CoV strains Tor2 (p) and GD03 (q), or palm civet SARS-CoV strain SZ3 (r) in hACE2/293T cells. Data (b, c, e–g, k–r) are presented as means ± SEM of mice (n = 5); data (d, h–j) are presented as means ± SEM of duplicate wells of pooled sera from five mice per group. Significant differences are shown as *P < 0.05; **P < 0.01; ***P < 0.001. Experiments were repeated twice with similar results.Full size imageTo detect whether S1 and RBD mRNAs durably express antigens in multiple cell types, we constructed N-terminal mCherry-tagged SARS-CoV-2 S1 and RBD mRNAs, encapsulated them with lipid nanoparticles (LNPs) (Supplementary information, Fig. S1b), and tested mCherry expression. Relative to the control, both RBD- and S1-mCherry mRNAs showed robust protein expression in cells for at least 160 h, with higher expression of the RBD construct (Supplementary information, Fig. S2a). In addition, these mRNAs expressed proteins efficiently in a variety of human (A549, Hep-2, HEP-G2, Caco-2, HeLa, 293 T), monkey (Vero E6), and bat (Tb1-Lu) cell lines (Supplementary information, Fig. S2b). Particularly, the expression of RBD-mCherry protein was higher than that of S1-mCherry protein in all cell lines tested (Supplementary information, Fig. S2b). These data indicate long-term and broad expression of mRNA-encoding proteins, particularly RBD, in target cells.We then characterized LNP-encapsulated S1 and RBD mRNAs for stability and subcellular localization. The mCherry-tagged S1 and RBD showed strong and stronger fluorescence intensity, respectively, irrespective of incubation temperature (4 or 25 °C) and culture time (0, 24, or 72 h) (Supplementary information, Fig. S3a). S1- and RBD-mCherry proteins were not colocalized with nuclei but associated with lysosomes (Supplementary information, Fig. S3b). These results suggest that LNP-encapsulated SARS-CoV-2 S1 and RBD mRNAs are stable at various temperatures and may be resistant to lysosomal degradation.We next evaluated T follicular helper (Tfh), germinal center (GC) B, and plasma cell responses induced by SARS-CoV-2 S1 and RBD mRNA-LNPs in BALB/c mice. Mice were intradermally (I.D.) prime and boost immunized with each mRNA-LNP (30 μg/mouse) or empty LNP control, and draining lymph nodes or spleens were tested for Tfh, GC B, or plasma cells 10 days post-2nd immunization (Supplementary information, Fig. S4a). The percentages of Tfh cells (Supplementary information, Fig. S5a) and GC B cells (Supplementary information, Fig. S5b) were higher or significantly higher in the lymph nodes of RBD mRNA-LNP-immunized mice than in those of S1 mRNA-LNP-immunized mice, whereas only a background level of Tfh and GC B cells was shown in the LNP control-injected mice. Plasma cells were also significantly increased in splenocytes of the vaccinated mice, as compared to the control group (Supplementary information, Fig. S5c). These data demonstrate the recruitment of Tfh, GC B, and/or plasma cells in vivo, particularly after immunization with SARS-CoV-2 RBD mRNA-LNP vaccine.We further evaluated humoral immune responses and neutralizing antibodies induced by S1 and RBD mRNA-LNPs. Mice were immunized with each mRNA-LNP at three different schedules (Supplementary information, Fig. S4a–c), and sera were collected for detection of IgG, subtype (IgG1 and IgG2a), and neutralizing antibodies. First, ELISA results revealed that S1 and RBD mRNA-LNPs (30 μg/mouse, I.D. prime and boost) induced RBD-specific IgG (Fig. 1b), IgG1 (Th2) (Supplementary information, Fig. S5d), and IgG2a (Th1) (Supplementary information, Fig. S5e) antibodies 10 days after boost immunization and that IgG antibody titer induced by RBD was significantly higher than that by S1 (Fig. 1b). Pseudovirus neutralization assay showed that S1 and RBD mRNA-LNPs elicited neutralizing antibodies against SARS-CoV-2 pseudovirus entry into human ACE2-expressing 293T (hACE2/293T) cells; particularly, RBD elicited significantly higher-titer neutralizing antibodies than S1 (Fig. 1c). Neutralizing antibodies, particularly those induced by RBD mRNA-LNP, also potently neutralized live SARS-CoV-2 infection (Fig. 1d). Next, both S1 and RBD mRNA-LNPs (10 μg, I.D. prime and boost) induced SARS-CoV-2 RBD-specific IgG (Supplementary information, Fig. S6a) and neutralizing antibodies against SARS-CoV-2 pseudovirus infection (Fig. 1e) 10 days after boost dose, and maintained at similarly high levels for 40 and 70 days post-boost immunization, while the titer of neutralizing antibodies elicited by RBD mRNA-LNP was always significantly higher than that by S1 mRNA-LNP (Fig. 1f, g; Supplementary information, S6b, c). Importantly, RBD mRNA-LNP induced antibody levels that potently neutralized live SARS-CoV-2 infection, reaching peak titer at 70 days post-2nd immunization and being significantly more potent than SARS-CoV-2 S1 mRNA-LNP-induced antibodies (Fig. 1h–j). Finally, RBD mRNA-LNP (10 μg, I.D. prime and intramuscular (I.M.) boost) also elicited significantly higher-titer RBD-specific IgG (Supplementary information, Fig. S6d) or neutralizing antibodies than S1 mRNA-LNP against SARS-CoV-2 pseudovirus (Supplementary information, Fig. S6g) and live SARS-CoV-2 (Supplementary information, Fig. S6j) infection 10 days after boost immunization, and such antibodies maintained at similar or even higher levels for at least 70 days post-boost dose (Supplementary information, Fig. S6e, f, h, i, k, l). In contrast, empty LNP control only elicited a background, or undetectable, level of antibodies incapable of neutralizing SARS-CoV-2 infection (Fig. 1b–j; Supplementary information, Fig. S6). These data suggest that RBD mRNA-LNP vaccine immunized at different immunogen doses and variant routes induced strong RBD-specific antibody responses and potent neutralizing antibodies against pseudotyped and live SARS-CoV-2 infection.To substantiate antiviral activity, we found the binding of SARS-CoV-2 RBD to ACE2 receptor in hACE2/293T cells was inhibited by serum antibodies produced from RBD or S1 mRNA-LNP-vaccinated mice. Specifically, anti-RBD antibodies potently inhibited, in a dose-dependent manner, RBD-ACE2 receptor binding, which was much stronger than anti-S1 antibodies (Fig. 1k–m), while the control LNP-induced mouse sera did not inhibit RBD-ACE2 binding (Fig. 1k, n). These data suggest that RBD mRNA-LNP-induced antibodies can potently block binding between SARS-CoV-2 RBD and its ACE2 receptor.Since SARS-CoV-2 RBD shares about 70% sequence identity with SARS-CoV RBD,7 we evaluated whether serum antibodies from SARS-CoV-2 mRNA-LNPs may cross-react with SARS-CoV RBD and neutralize SARS-CoV infection. ELISA results showed that the titer of IgG (Fig. 1o), IgG1 (Supplementary information, Fig. S5f), and IgG2a (Supplementary information, Fig. S5g) antibodies induced by SARS-CoV-2 RBD mRNA-LNP was higher, or significantly higher, than those by SARS-CoV-2 S1 mRNA-LNP in cross-reacting with SARS-CoV RBD and cross-neutralizing infection by three SARS-CoV pseudoviruses expressing S proteins of human strains Tor2 (Fig. 1p), GD03 (Fig. 1q), and palm civet strain SZ3 (Fig. 1r), respectively. These results suggest that SARS-CoV-2 RBD mRNA vaccine can elicit antibodies cross-reacting with SARS-CoV RBD and cross-neutralizing SARS-CoV infection.We also investigated SARS-CoV-2 RBD-specific T cell responses induced by S1 and RBD mRNA-LNPs in immunized mice. Splenocytes collected 10 days post-2nd immunization were stimulated with SARS-CoV-2 RBD overlapping peptides (Supplementary information, Table S1), and detected for secretion of IFN-γ (Th1), TNF-α (Th1), and IL-4 (Th2) in CD45+CD4+ T cells, as well as IFN-γ, TNF-α, and IL-4 in CD45+CD8+ T cells by flow cytometry analysis. Compared with the LNP control, immunization with RBD mRNA-LNP could significantly increase the frequency of IFN-γ-, TNF-α- or IL-4-producing CD45+CD4+ (Supplementary information, Fig. S7a–c) or CD45+CD8+ (Supplementary information, Fig. S7d–f) T cells, respectively. However, S1 mRNA-LNP could only significantly increase the frequency of TNF-α-producing CD45+CD4+ (Supplementary information, Fig. S7b) and IFN-γ- or IL-4-producing CD45+-CD8+ (Supplementary information, Fig. S7d, f) T cells, respectively. Therefore, RBD mRNA vaccine can effectively elicit RBD-specific CD45+CD4+ (Th1) and CD45+CD8+ T cell responses.As opposed to DNA, mRNA does not enter the nucleus and is not lysed by lysosomal enzymes (Supplementary information, Fig. S8),8 contributing to its high stability and translation efficiency. GC, where GC B cells interact with Tfh and B cells, is the major site for production of high-affinity antibodies.9 Here, we showed that RBD mRNA-LNP elicited strong Tfh and GC B cell responses and potent neutralizing antibodies able to inhibit the binding between SARS-CoV-2 RBD and ACE2 receptor (Supplementary information, Fig. S8), demonstrating its high potency against SARS-CoV-2 infection.The repertoire of COVID-19 vaccines currently in clinical trials include mRNA, adenovirus, and DNA-based vaccines, most of which encode SARS-CoV-2 full-length S protein.10,11,12 It has been shown that adenovirus-based ChAdOx1 nCoV-19 vaccine elicits specific IgG antibody titer of 1:400–6400 and neutralizing antibody titer of 1:5–40, whereas the DNA vaccine induces a neutralizing antibody titer of 1:74–170, against live virus infection in immunized monkeys.10,11 In addition, neutralizing antibody titer against pseudotyped SARS-CoV-2 infection ranged from 1:89–1115 in mice immunized with a full-length S-based mRNA vaccine.12 Here we found that a SARS-CoV-2 RBD-based mRNA vaccine at 30 μg/mouse elicited SARS-CoV-2 RBD-specific IgG antibody titer (~1:230,000) and neutralizing antibody titer in mice against pseudotyped and live SARS-CoV-2 infection at ~1:10,000 and 1:540, respectively. Moreover, immunization with this vaccine at a lower immunogen dose (10 μg) via variant immunization routes (I.D. prime and I.D. or I.M. boost) also induced high-titer IgG antibodies with neutralizing activity against pseudotyped and live SARS-CoV-2 infection that persisted for at least 70 days during the detection period. Thus the IgG and neutralizing antibody titers induced by the RBD-based mRNA vaccine were higher than those reported, suggesting beneficial protection against SARS-CoV-2 challenge in vivo. Future studies warrant evaluation of protective efficacy using mRNA vaccine against other reported vaccines under development. Previous studies showed that SARS-CoV full-length S protein induced harmful immune responses with enhanced infection or liver damage after virus challenge, raising safety concerns.6 In contrast, RBD-based SARS-CoV and MERS-CoV vaccines had no evidence to cause harmful immune responses, including eosinophilic immune enhancement.5,6 Although no obvious adverse effects have been reported in currently developed COVID-19 vaccines, cautions need to be paid regarding their safety. More studies will be needed to investigate vaccine-associated immunopathology in addition to evaluate their protective efficacy.Overall, this study identifies RBD as a key antigen to design effective vaccines against SARS-CoV-2, indicating great potential of RBD-based mRNA vaccine for mitigation of the COVID-19 pandemic and possible SARS-related epidemics in the future. The strategy of developing RBD-based mRNA COVID-19 vaccine, as described herein, can also be applied to develop vaccines against other emerging and reemerging coronavirus diseases in the future.

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PubMed Google ScholarContributionsW.T. and X.Z. conducted the study and analyzed the data. A.D., J.S., J.C.H. and C.K.T. performed the experiments. L.L., C.D.H. and S.J. reviewed and revised the paper. L.D. designed and supervised the study, wrote and revised the manuscript.Corresponding authorsCorrespondence to

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科学网—2022年(1)：新冠病毒变异株Omicron刺突蛋白RBD结构域与宿主ACE2蛋白的亲和力弱于目前主流的Delta毒株 - 徐志建的博文

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2022年(1)：新冠病毒变异株Omicron刺突蛋白RBD结构域与宿主ACE2蛋白的亲和力弱于目前主流的Delta毒株

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2022-1-24 16:49

|个人分类:COVID-19|系统分类:论文交流

2020年来，由新型冠状病毒（SARS-CoV-2）引起的新冠肺炎（COVID-19）疫情在全球持续蔓延。截至2021年12月末，全球累计病例已超2.7亿，死亡人数超530万。国内疫情虽已得到有效的控制，但不断出现的新冠病毒变异株，给疫情防控、疫苗和药物研发带来了严峻的挑战。 2021年11月24日，南非政府首次向世界卫生组织（WHO）报告B.1.1.529变异株，11月26日，WHO将其定性为最高级别的“值得关切的变异株（VOC）”，命名为Omicron。Omicron变异株在刺突蛋白（Spike）上拥有30余个突变，远超其他VOC变异株1。根据WHO的统计，近两个月的确诊病例中，Delta变异株的占比为99.8%2， Omicron刺突蛋白上的众多突变是否会使其取代Delta变异株而成为下一个占优势地位的变异株备受关注。因此，十分有必要研究Omicron刺突蛋白上的突变对其感染宿主和免疫逃逸能力的实际影响。基于此，2022年1月5日，中科院上海药物所徐志建/朱维良课题组、宫丽崑课题组，联合国防科技大学计算机学院吴诚堃研究团队，在Signal Transduction and Targeted Therapy上发表文章SARS-CoV-2 Omicron RBD shows weaker binding affinity than the currently dominant Delta variant to human ACE2，通过计算模拟和ELISA实验，研究了Omicron变异株刺突蛋白Spike上的受体结合域（RBD）与宿主细胞上的ACE2蛋白的亲和能力变化，并通过计算模拟预测了5个处于上市/临床的单克隆抗体与RBDOmicron结合自由能。新冠病毒刺突蛋白上的受体结合域（RBD）是病毒感染宿主细胞的关键位点，也是众多抗体作用的靶点，而Omicron变异株的RBD上有15个突变位点（图1a）。为此，研究人员首先基于天河超级计算机，通过分子动力学模拟方法分别计算了宿主细胞上的ACE2蛋白与野生型RBD（RBDWT）, Delta变异株RBD（RBDDelta）及Omicron变异株RBD（RBDOmicron）的结合自由能（图1b），发现Delta变异株的RBDDelta与ACE2结合能力显著强于RBDOmicron，而RBDOmicron与ACE2的结合能力与野生型RBDWT相当。接着他们又通过ELISA方法分别测定了ACE2与RBDWT、RBDDelta和RBDOmicron的亲和力常数（图1c），所得实验结果与计算模拟结果一致，即Omicron RBD结合ACE2的能力与野生型相当，但弱于Delta变异株的RBD。为了研究Omicron变异株的免疫逃逸能力，研究人员计算预测了3个上市抗体（Etesevimab、Bamlanivimab、Regdanvimab）和2个临床阶段的抗体（BD-368-2、Bebtelovimab）与三种RBD（RBDWT、RBDDelta、RBDOmicron）的结合自由能（图1d）。计算结果表明，2个上市抗体（Etesevimab、Bamlanivimab）和1个临床阶段的抗体（BD-368-2）与RBDOmicron的结合能力明显弱于RBDWT，第3个上市抗体Regdanvimab与RBDOmicron的结合能力也有减弱的趋势，而处于临床阶段的抗体Bebtelovimab对RBDOmicron和RBDDelta均显示出较强的结合能力。所研究的3个上市抗体的计算结果与近期文献报道的生物实验基本一致3-5。据此，研究人员指出Omicron变异株对现有多个单克隆抗体存在很高的免疫逃逸风险。作者在文中也特别指出，新冠病毒感染和复制过程受众多因素影响，且Omicron刺突蛋白三聚体与ACE2的亲和力变化趋势可能与RBD结构域的不同，加之Omicron的显著免疫逃逸风险，故应该十分重视该变异病毒株而不能掉以轻心。近日已有预印本实验结果提示Omicron全长刺突蛋白三聚体与ACE2的亲和力与Delta相当6，强于野生型6, 7。中科院上海药物所硕士研究生吴乐云、博士研究生周丽萍和工程师刘婷婷，国防科技大学计算机学院工程师莫孟霞和吴诚堃副研究员，为本文共同第一作者。中科院上海药物所徐志建、朱维良、宫丽崑研究员为本文共同通讯作者。国防科技大学龚春叶副研究员和卢凯研究员为论文的共同作者。该研究工作得到上海市自然基金委、国家重点研发计划及天河新一代超级计算机等项目的资助。图1. (a) Delta和Omicron变异株在RBD上的突变；(b)宿主ACE2蛋白与RBDWT、RBDDelta和RBDOmicron的结合自由能；(c) 宿主ACE2蛋白与RBDWT、RBDDelta和RBDOmicron的亲和力常数；(d) 单克隆抗体mAbs与RBDWT、RBDDelta和RBDOmicron的结合自由能；(e) RBD上Delta和Omicron变异株突变位点的残基能量贡献。NS：无显著性差异；*P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001。据悉，该团队还系统地调研了新冠肺炎药物及潜在靶标研究进展（药学学报, 2020）8，分别建立了基于分子对接（Acta Pharm Sin B, 2020）9和基于配体相似性的抗新冠肺炎靶标预测与药物虚拟筛选平台（Brief Bioinform, 2021）10，计算研究了新冠病毒刺突蛋白与宿主ACE2蛋白的结合机制（J. Phys. Chem. Lett., 2020）11，并研究了新冠病毒刺突蛋白上的E484K突变引起免疫逃逸的分子机制（Brief Bioinform, 2021）12。Omicron相关的部分模拟结果于2021年12月7日以预印版公开（DOI：https://doi.org/10.26434/chemrxiv-2021-n23f5）。原文链接： https://www.nature.com/articles/s41392-021-00863-2 参考文献： 1. European Centre for Disease Prevention and Control. Implications of the emergence and spread of the SARSCoV-2 B.1.1. 529 variant of concern (Omicron), for the EU/EEA. 26 November 2021. ECDC: Stockholm. 2021.2. Weekly epidemiological update on COVID-19-30 November 2021. https://www.who.int/publications/m/item/weekly-epidemiological-update-on-covid-19---30-november-2021 (accessed 30 November).3. Cameroni, E., et al., Broadly neutralizing antibodies overcome SARS-CoV-2 Omicron antigenic shift. bioRxiv 2021.4. Cao, Y., et al., B.1.1.529 escapes the majority of SARS-CoV-2 neutralizing antibodies of diverse epitopes. bioRxiv 2021.5. Liu, L., et al., Striking Antibody Evasion Manifested by the Omicron Variant of SARS-CoV-2. bioRxiv 2021.6. Mannar, D., et al., SARS-CoV-2 Omicron Variant: ACE2 Binding, Cryo-EM Structure of Spike Protein-ACE2 Complex and Antibody Evasion. bioRxiv 2021, 2021.12.19.473380.7. Yin, W., et al., Structures of the Omicron Spike trimer with ACE2 and an anti-Omicron antibody. bioRxiv 2021, 2021.12.27.474273.8. 王晓宇, et al., 抗致病性冠状病毒活性化合物及其潜在靶标蛋白研究进展[J]. 药学学报 2020, 55 (10), 2340-2357.9. Shi, Y., et al., D3Targets-2019-nCoV: a webserver for predicting drug targets and for multi-target and multi-site based virtual screening against COVID-19. Acta Pharm Sin B 2020, 10 (7), 1239-1248.10. Yang, Y., et al., Ligand-based approach for predicting drug targets and for virtual screening against COVID-19. Brief Bioinform 2021, 22 (2), 1053-1064.11. Peng, C., et al., Computational Insights into the Conformational Accessibility and Binding Strength of SARS-CoV-2 Spike Protein to Human Angiotensin-Converting Enzyme 2. J. Phys. Chem. Lett. 2020, 11, 10482-10488.12. Wu, L., et al., Exploring the Immune Evasion of SARS-CoV-2 Variant Harboring E484K by Molecular Dynamics Simulations. Brief Bioinform 2021.

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Characterization of the receptor-binding domain (RBD) of 2019 novel coronavirus: implication for development of RBD protein as a viral attachment inhibitor and vaccine | Cellular & Molecular Immunology

Characterization of the receptor-binding domain (RBD) of 2019 novel coronavirus: implication for development of RBD protein as a viral attachment inhibitor and vaccine | Cellular & Molecular Immunology

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Characterization of the receptor-binding domain (RBD) of 2019 novel coronavirus: implication for development of RBD protein as a viral attachment inhibitor and vaccine

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Published: 19 March 2020

Characterization of the receptor-binding domain (RBD) of 2019 novel coronavirus: implication for development of RBD protein as a viral attachment inhibitor and vaccine

Wanbo Tai1, Lei He2, Xiujuan Zhang1, Jing Pu1,3, Denis Voronin

ORCID: orcid.org/0000-0003-2652-07871, Shibo Jiang1,3, Yusen Zhou2 & …Lanying Du1 Show authors

Cellular & Molecular Immunology

volume 17, pages 613–620 (2020)Cite this article

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ImmunotherapyViral infection

AbstractThe outbreak of Coronavirus Disease 2019 (COVID-19) has posed a serious threat to global public health, calling for the development of safe and effective prophylactics and therapeutics against infection of its causative agent, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), also known as 2019 novel coronavirus (2019-nCoV). The CoV spike (S) protein plays the most important roles in viral attachment, fusion and entry, and serves as a target for development of antibodies, entry inhibitors and vaccines. Here, we identified the receptor-binding domain (RBD) in SARS-CoV-2 S protein and found that the RBD protein bound strongly to human and bat angiotensin-converting enzyme 2 (ACE2) receptors. SARS-CoV-2 RBD exhibited significantly higher binding affinity to ACE2 receptor than SARS-CoV RBD and could block the binding and, hence, attachment of SARS-CoV-2 RBD and SARS-CoV RBD to ACE2-expressing cells, thus inhibiting their infection to host cells. SARS-CoV RBD-specific antibodies could cross-react with SARS-CoV-2 RBD protein, and SARS-CoV RBD-induced antisera could cross-neutralize SARS-CoV-2, suggesting the potential to develop SARS-CoV RBD-based vaccines for prevention of SARS-CoV-2 and SARS-CoV infection.

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Philip V’kovski, Annika Kratzel, … Volker Thiel

IntroductionThree highly pathogenic human coronaviruses (CoVs) have been identified so far, including Middle East respiratory syndrome coronavirus (MERS-CoV), severe acute respiratory syndrome (SARS) coronavirus (SARS-CoV), and a 2019 novel coronavirus (2019-nCoV), as previously termed by the World Health Organization (WHO).1,2,3 Among them, SARS-CoV was first reported in Guangdong, China in 2002.4 SARS-CoV caused human-to-human transmission and resulted in the 2003 outbreak with about 10% case fatality rate (CFR),1 while MERS-CoV was reported in Saudi Arabia in June 2012.5 Even though with its limited human-to-human transmission, MERS-CoV showed a CFR of about 34.4%.2 The 2019-nCoV was first reported in Wuhan, China in December 2019 from patients with pneumonia,6 and it has exceeded both SARS-CoV and MERS-CoV in its rate of transmission among humans.7 2019-nCoV was renamed SARS-CoV-2 by Coronaviridae Study Group (CSG) of the International Committee on Taxonomy of Viruses (ICTV),8 while it was renamed HCoV-19, as a common virus name, by a group of virologists in China.9 The disease and the virus causing it were named Coronavirus Disease 2019 (COVID-19) and the virus responsible for COVID-19 or the COVID-19 virus, respectively, by WHO.3 As of March 5, 2020, a total of 95,333 confirmed cases of COVID-19 were reported, including 3,282 deaths, in China and at least 85 other countries and/or territories.7 Currently, the intermediate host of SARS-CoV-2 is still unknown, and no effective prophylactics or therapeutics are available. This calls for the immediate development of vaccines and antiviral drugs for prevention and treatment of COVID-19.A coronavirus contains four structural proteins, including spike (S), envelope (E), membrane (M), and nucleocapsid (N) proteins.2,10,11 Among them, S protein plays the most important roles in viral attachment, fusion and entry, and it serves as a target for development of antibodies, entry inhibitors and vaccines.1,12,13,14,15,16,17 The S protein mediates viral entry into host cells by first binding to a host receptor through the receptor-binding domain (RBD) in the S1 subunit and then fusing the viral and host membranes through the S2 subunit.16,18,19 SARS-CoV and MERS-CoV RBDs recognize different receptors. SARS-CoV recognizes angiotensin-converting enzyme 2 (ACE2) as its receptor, whereas MERS-CoV recognizes dipeptidyl peptidase 4 (DPP4) as its receptor.20,21 Similar to SARS-CoV, SARS-CoV-2 also recognizes ACE2 as its host receptor binding to viral S protein.22 Therefore, it is critical to define the RBD in SARS-CoV-2 S protein as the most likely target for the development of virus attachment inhibitors, neutralizing antibodies, and vaccines.In this study, we identified the RBD fragment in SARS-CoV-2 S protein and found that the recombinant RBD protein bound strongly to human ACE2 (hACE2) and bat ACE2 (bACE2) receptors. In addition, it blocked the entry of SARS-CoV-2 and SARS-CoV into their respective hACE2-expressing cells, suggesting that it may serve as a viral attachment inhibitor against SARS-CoV-2 and SARS-CoV infection. Moreover, we demonstrated that SARS-CoV RBD-specific polyclonal antibodies cross-reacted with SARS-CoV-2 RBD protein and inhibited SARS-CoV-2 entry into hACE2-expressing cells. We have also shown that SARS-CoV RBD-specific polyclonal antibodies could cross-neutralize SARS-CoV-2 pseudovirus infection, suggesting the potential to develop SARS-CoV RBD-based vaccine for prevention of infection by SARS-CoV-2 and SARS-CoV.Results and discussionBy alignment of the RBD sequences of SARS-CoV and SARS-CoV-2, we identified the region of SARS-CoV-2 RBD at residues 331 to 524 of S protein (Fig. 1a). We then constructed a recombinant RBD protein containing codon-optimized RBD sequences with a C-terminal Fc of human IgG1 (hFc) using pFUSE-hIgG1-Fc2 expression vector, expressed the protein in mammalian cell 293T, and purified it from cell culture supernatant using protein A affinity chromatography. Similar to SARS-CoV and MERS-CoV RBD protein controls, SARS-CoV-2 RBD protein had high expression with strong purity (Fig. 1b). Notably, only SARS-CoV-2 and SARS-CoV RBDs were recognized by SARS-CoV RBD-specific, but not MERS-CoV RBD-specific, polyclonal antibodies (Fig. 1c), whereas only MERS-CoV RBD was recognized by MERS-CoV RBD-immunized polyclonal antibodies (Fig. 1d), suggesting the cross-reactivity of SARS-CoV RBD-specific antibodies with SARS-CoV-2 RBD protein.Fig. 1Characterization of SARS-CoV-2 RBD. a Multiple sequence alignment of RBDs of SARS-CoV-2, SARS-CoV, and MERS-CoV spike (S) proteins. GenBank accession numbers are QHR63250.1 (SARS-CoV-2 S), AY278488.2 (SARS-CoV S), and AFS88936.1 (MERS-CoV S). Variable amino acid residues between SARS-CoV-2 and SARS-CoV are highlighted in cyan, and conserved residues among SARS-CoV-2, SARS-CoV, and MERS-CoV are highlighted in yellow. Asterisks represent fully conserved residues, colons represent highly conserved residues, and periods represent lowly conserved residues. The alignment was performed using Clustal Omega. SDS-PAGE (b) and Western blot (c, d) analysis of SARS-CoV-2 RBD. The protein molecular weight marker (kDa) is indicated on the left. SARS-CoV and MERS-CoV RBDs were included as controls. Antisera (1:3,000 dilution) from mice immunized with SARS-CoV RBD (c) and MERS-CoV RBD (d) were used for Western blot analysisFull size imageFour experiments were performed to detect the binding between SARS-CoV-2 RBD and hACE2 receptor. First, we tested if stably transfected hACE2/293T cells expressed hACE2 by flow cytometry analysis. Since 293T cells alone did not express either hACE2 or hDPP4, they could not be recognized by anti-hACE2 or anti-hDPP4 antibodies (Fig. 2a (left panel)). Only hACE2/293T cells, but not hDPP4/293T cells, expressed hACE2, which was recognized by an anti-hACE2 antibody (Fig. 2a (middle panel)), whereas only hDPP4/293T cells, but not hACE2/293T cells, expressed hDPP4 and was, correspondingly, recognized by an anti-hDPP4 antibody (Fig. 2a (right panel)). These data confirmed the expression of hACE2 in hACE2/293T cells and the expression of hDPP4 in hDPP4/293T cells. Second, we used these hACE2/293T cells to detect the binding of SARS-CoV-2 RBD protein to cell-associated hACE2 by flow cytometry analysis and immunofluorescence staining. Similar to SARS-CoV RBD, SARS-CoV-2 RBD bound to hACE2/293T cells expressing hACE2 (Fig. 2b (left and middle panels)), but not to hDPP4/293T cells expressing hDPP4 (Fig. 2c (left and middle panels)). Furthermore, the binding between SARS-CoV-2 RBD and hACE2-expressing 293T cells was much stronger than the binding between SARS-CoV RBD and hACE2-expressing 293T cells (Fig. 2b (left and middle panels)). MERS-CoV RBD did not bind to hACE2-expressing 293T cells (Fig. 2b (right panel)), but rather bound to hDPP4-expressing 293T cells (Fig. 2c (right panel)). The results from immunofluorescence staining revealed positive signals for both hACE2 and hFc on hACE2/293T cells treated with SARS-CoV-2 RBD and SARS-CoV RBD, both of which contained a C-terminal hFc tag, whereas hACE2/293T cells treated with MERS-CoV RBD (containing a C-terminal hFc tag) showed positive signals for hACE2, but not for hFc, indicating that there is no binding of MERS-CoV RBD to the hACE2-expressing cells (Fig. 2d). These data suggest that SARS-CoV-2 RBD and SARS-CoV RBD can bind to cell-associated hACE2, but not to hDPP4. Third, we detected the binding of SARS-CoV-2 RBD to soluble hACE2 protein (sACE2) by ELISA. The results indicated that SARS-CoV-2 RBD bound to sACE2 in a dose-dependent manner and that the binding between SARS-CoV-2 RBD and sACE2 with 50% effective dose (EC50) of 1.07 μg/ml was stronger than that between SARS-CoV RBD and sACE2 (EC50: 1.66 μg/ml). In contrast, MERS-CoV RBD did not bind to sACE2 (Fig. 2e). While neither SARS-CoV-2 RBD nor SARS-CoV RBD bound to sDPP4, MERS-CoV RBD strongly bound to sDPP4 (EC50: 0.92 μg/ml) (Fig. 2f). These data suggest that both SARS-CoV-2 RBD and SARS-CoV RBD could bind to hACE2 in solution, but not to hDPP4 in solution. Fourth, flow cytometry analysis further indicated that the binding between SARS-CoV-2 RBD and cell-associated hACE2 receptor could be significantly blocked by sACE2 protein (Fig. 2g, i), but not by sDPP4 protein (Fig. 2h, i). Taken together, the above results confirm that the identified SARS-CoV-2 RBD could bind to both cell-associated and soluble hACE2 proteins.Fig. 2Detection of SARS-CoV-2 RBD binding to human ACE2 receptor. a Flow cytometry analysis of receptor expression in stable cell lines. (left panel) 293T cells alone expressed neither human ACE2 (hACE2) receptor (orange line) nor hDPP4 receptor (cyan line); (middle panel) hACE2-expressing 293T (hACE2/293T) cells expressed only hACE2 (orange line), but not hDPP4 (cyan line); (right panel) hDPP4-expressing 293T (hDPP4/293T) cells expressed only hDPP4 (cyan line), but not hACE2 (orange line). Mock-incubated cells (gray shading) were used as control. Representative images and median fluorescence intensity (MFI) ± standard error (s.e.m.) were shown (n = 4). b, c Flow cytometry analysis of SARS-CoV-2 RBD binding to cell-associated hACE2 receptor in hACE2/293T stable cell lines. SARS-CoV-2 RBD protein bound strongly to hACE2/293T cells (b (left panel, red line)), but not to hDPP4/293T cells (c (left panel, violet line)). SARS-CoV RBD protein bound to hACE2/293T cells (b (middle panel, red line)), but not to hDPP4/293T cells (c (middle panel, violet line)). MERS-CoV RBD protein did not bind to hACE2/293T cells (b (right panel, red line)), but rather bound to hDPP4/293T cells (c (right panel, violet line)). Human IgG Fc (hIgG-Fc, hereinafter hFc) protein-incubated cells (blue line) and mock-incubated cells (gray shading) were included as controls (b, c). Representative images and MFI ± s.e.m. were shown (n = 4). d Immunofluorescence detection of SARS-CoV-2 RBD binding to cell-associated hACE2 receptor in hACE2/293T cells. SARS-CoV-2 RBD protein (green) and SARS-CoV RBD protein (green), each of which was fused with a C-terminal hFc, were stained with FITC-labeled goat anti-human IgG antibody (1:500). hACE2 was stained with a goat-anti-hACE2 antibody (5 μg/ml) and Alexa-Fluor 647-labeled anti-goat antibody (red) (1:200). Fc-fused MERS-CoV RBD protein did not bind to hACE2, so only hACE2 (red), but not RBD (green), was detected in hACE2/293T cells. Nuclei were stained with 4’,6-diamidino-2-phenylindole (DAPI, blue). Scale bar: 10 μm. Representative images are shown. e Detection of dose-dependent binding of SARS-CoV-2 RBD protein to soluble hACE2 (sACE2) receptor by ELISA. The SARS-CoV-2 RBD binding to soluble hDPP4 (sDPP4) receptor (f), and the binding of both SARS-CoV RBD and MERS-CoV RBD proteins to sACE2 (e), or sDPP4 (f), were tested. Control: hFc protein. Data are presented as mean A450 ± s.e.m. (n = 4). 50% effective dose (EC50) was calculated for the binding between SARS-CoV-2 RBD (black) or SARS-CoV RBD (red) and hACE2 protein (e, sACE2), or the binding between MERS-CoV RBD and hDPP4 protein (sDPP4, green) (f). g–i Flow cytometry analysis of inhibition of SARS-CoV-2 RBD protein binding to hACE2/293T cells by sACE2. Binding of SARS-CoV-2 RBD to hACE2/293T cells (g, h, green line) was blocked by sACE2 (g, black line), but not by sDPP4 (h, red line). hFc protein-incubated cells (blue line) and mock-incubated cells (gray shading) were included as controls (g, h). Representative images are shown. i The blocking ability of sACE2 or sDPP4, as described above, was expressed as MFI ± s.e.m. (n = 4). Low MFI correlates with high blockage. Experiments were repeated twice and yielded similar resultsFull size imageLike SARS-CoV and MERS-CoV, SARS-CoV-2 also originates from bats.22,23,24 Next, we detected the binding affinity of the identified SARS-CoV-2 RBD to bat ACE2 (bACE2) and compared this binding with that of SARS-CoV RBD. We transiently transfected a bACE2-expressing plasmid into 293T cells and included a hACE2-expressing plasmid as a control, followed by detection of fluorescence intensity 48 h later. Results indicated that SARS-CoV-2 RBD bound strongly to 293T-expressed bACE2 with intensity similar to that of its binding to 293T-expressed hACE2 (Fig. 3a, c), and that this binding occurred in a dose-dependent manner (Fig. 3e, f). In addition, the binding affinity between SARS-CoV-2 RBD and 293T-expressed bACE2 (EC50: 0.08 μg/ml) or hACE2 (EC50: 0.14 μg/ml) was significantly higher than that between SARS-CoV RBD and 293T-expressed bACE2 (EC50: 0.96 μg/ml) or hACE2 (EC50: 1.32 μg/ml) (Fig. 3b, d–f). Nevertheless, MERS-CoV RBD bound neither bACE2- nor hACE2-expressing 293T cells (Fig. 3). These data suggest that SARS-CoV-2 RBD can bind to both bACE2 and hACE2 with significantly stronger binding than that of SARS-CoV RBD to either bACE2 or hACE2, supporting the bat origin of SARS-CoV-2. These results may partially explain why SARS-CoV-2 is more transmissible than SARS-CoV.Fig. 3Comparison of SARS-CoV-2 RBD protein binding to human and bat ACE2 receptors. Flow cytometry analysis of SARS-CoV-2 RBD binding to hACE2 and bat ACE2 (bACE2) receptors in 293T cells transiently expressing hACE2 or bACE2. 293T cells were transiently transfected with hACE2 or bACE2 plasmid and incubated with SARS-CoV-2 RBD protein at various concentrations for analysis. SARS-CoV RBD and MERS-CoV RBD proteins were used as controls. Representative images of SARS-CoV-2 RBD protein (2.5 μg/ml) binding to bACE2/293T (a, black line), or hACE2/293T (c, black line), cells were shown. Binding of SARS-CoV RBD protein (2.5 μg/ml) to bACE2/293T (b, red line), or hACE2/293T (d, red line), cells were used as a comparison. MERS-CoV RBD protein (green line) and mock-incubated (gray shading) cells (a–d) were included as controls. e, f Dose-dependent binding of SARS-CoV-2 RBD protein to bACE2/293T (e), or hACE2/293T (f), cells by flow cytometry analysis. Significant differences between binding of SARS-CoV-2 RBD (black) and SARS-CoV RBD (red) to cell-associated bACE2 receptor (e), or hACE2 receptor (f) were identified based on the EC50 values. The data are presented as mean ± s.e.m. (n = 4). Experiments were repeated twice and yielded similar resultsFull size imageWe then evaluated the potential of the identified SARS-CoV-2 RBD protein as an inhibitor of viral entry. To accomplish this, we first generated a pseudotyped SARS-CoV-2 by cotransfection of a plasmid encoding Env-defective, luciferase-expressing HIV-1 (pNL4-3.luc.RE) and a plasmid expressing S protein of SARS-CoV-2 into 293T cells, followed by collection of pseudovirus-containing supernatants. We then incubated serially diluted SARS-CoV-2 RBD protein with hACE2/293T target cells, followed by the addition of pseudovirus and detection of inhibitory activity of infection. With the capacity for only one-cycle infection, S protein-expressing pseudovirus cannot replicate in the target cells.25,26 Therefore, the inhibition of pseudovirus infection represents inhibition of viral entry, as mediated by viral S protein. As expected, SARS-CoV-2 RBD protein inhibited SARS-CoV-2 pseudovirus entry into hACE2-expressing 293T cells in a dose-dependent manner with 50% inhibition concentration (IC50) as low as 1.35 µg/ml. Interestingly, it also blocked the entry of SARS-CoV pseudovirus into hACE2-expressing 293T cells with IC50 of 5.47 µg/ml (Fig. 4a). Similarly, SARS-CoV RBD protein blocked the entry of both SARS-CoV pseudovirus and SARS-CoV-2 pseudovirus into hACE2-expressing 293T cells with IC50 of 4.1 and 11.63 µg/ml, respectively (Fig. 4b). In addition, neither SARS-CoV-2 RBD nor SARS-CoV RBD blocked the entry of MERS-CoV pseudovirus into hDPP4-expressing 293T cells (Fig. 4c). MERS-CoV RBD did not block the entry of SARS-CoV-2 pseudovirus or SARS-CoV pseudovirus into hACE2-expressing 293T cells, but it did block the entry of MERS-CoV pseudovirus into hDPP4-expressing 293T cells (IC50: 22.25 µg/ml) (Fig. 4a–c). These results suggest that SARS-CoV-2 RBD protein could be developed as an effective therapeutic agent against SARS-CoV-2 and SARS-CoV infection.Fig. 4Ability of SARS-CoV-2 RBD to inhibit viral entry, as well as its cross-reactivity and cross-neutralizing activity with SARS-CoV. a Dose-dependent inhibition of SARS-CoV-2 RBD protein against pseudotyped SARS-CoV-2 entry into hACE2/293T cells. SARS-CoV and MERS-CoV RBDs, as well as hDPP4/293T cells, were included as controls. SARS-CoV-2 RBD protein inhibited entry of SARS-CoV-2 and SARS-CoV pseudoviruses into their respective target (hACE2/293T) cells (a), but not the entry of MERS-CoV pseudovirus into its target (hDPP4/293T) cells (a). SARS-CoV RBD protein inhibited both SARS-CoV-2 and SARS-CoV pseudovirus entry, but not MERS-CoV pseudovirus entry (b). MERS-CoV RBD inhibited neither SARS-CoV-2 nor SARS-CoV pseudovirus entry, but it did inhibit MERS-CoV pseudovirus entry (c). The data are presented as mean inhibition (%) ± s.e.m. (n = 4), and 50% inhibition concentration (IC50) was calculated for SARS-CoV-2 RBD (a, b, black), or SARS-CoV RBD (a, b, red), protein against SARS-CoV-2 pseudovirus and SARS-CoV pseudovirus and for MERS-CoV RBD protein (green) against MERS-CoV pseudovirus (c). d Cross-reactivity of SARS-CoV-2 RBD protein with SARS-CoV RBD-specific mouse sera by ELISA. Sera of mice immunized with mammalian cell-expressed SARS-CoV RBD protein30 were tested. Sera of mice immunized with mammalian cell-expressed MERS-CoV RBD protein31 were used as control. The data are presented as mean A450 ± s.e.m. (n = 4). The IgG antibody (Ab) titers were calculated as the endpoint dilution that remains positively detectable for SARS-CoV-2 RBD (black), or SARS-CoV RBD (red), binding to anti-SARS-CoV RBD sera (d) and for MERS-CoV RBD (green) binding to anti-MERS-CoV RBD sera (e). f Cross-neutralization of SARS-CoV RBD-immunized mouse sera against SARS-CoV-2 infection by pseudovirus neutralization assay. MERS-CoV RBD-immunized mouse sera were used as control. The data are presented as mean neutralization (%) ± s.e.m. (n = 4). 50% neutralizing antibody titers (NT50) were calculated against SARS-CoV-2 pseudovirus (black), or SARS-CoV pseudovirus (red), (f) infection in hACE2/293T target cells, as well as against MERS-CoV pseudovirus (green) (g) infection in hDPP4/293T cells. Experiments were repeated twice and yielded similar resultsFull size imageSince SARS-CoV-2 is more phylogenetically related to SARS-CoV than MERS-CoV,22 we further detected the cross-reactivity of SARS-CoV RBD-specific antibodies with SARS-CoV-2 RBD and cross-neutralizing activity of SARS-CoV RBD-specific antibodies against pseudotyped SARS-CoV-2. First, we performed an ELISA to detect the cross-reactivity of SARS-CoV RBD-immunized mouse sera with SARS-CoV-2 RBD. The results showed that SARS-CoV-2 RBD reacted strongly with anti-SARS-CoV RBD IgG with antibody titer of 1:2.4 × 104 (Fig. 4d), but it did not react with anti-MERS-CoV RBD IgG (Fig. 4e). As expected, SARS-CoV RBD reacted strongly with anti-SARS-CoV RBD IgG (antibody titer: 1:1.4 × 105) (Fig. 4d), but not with anti-MERS-CoV RBD IgG (Fig. 4e). MERS-CoV RBD did not react with anti-SARS-CoV RBD IgG (Fig. 4d), but instead reacted with anti-MERS-CoV RBD IgG (antibody titer: 1:1.3 × 105) (Fig. 4e). Second, we performed a pseudovirus neutralization assay to detect the cross-neutralizing activity of SARS-CoV RBD-immunized mouse sera against SARS-CoV-2 pseudovirus infection. Results revealed that SARS-CoV RBD-specific antisera could neutralize SARS-CoV-2 pseudovirus infection with a neutralizing antibody titer of 1:323, while these antisera could neutralize SARS-CoV pseudovirus infection with higher neutralizing antibody titer (1:1.2 × 104) (Fig. 4f). MERS-CoV RBD-inducing mouse sera only neutralized MERS-CoV pseudovirus infection in hDPP4-expressing cells with a neutralizing antibody titer of 1:4 × 104 (Fig. 4g), but failed to neutralize infection by either SARS-CoV-2 pseudovirus or SARS-CoV pseudovirus (Fig. 4f). These data suggest that SARS-CoV RBD-specific antibodies can cross-react with SARS-CoV-2 RBD and cross-neutralize SARS-CoV-2 pseudovirus infection.In summary, we have characterized the SARS-CoV-2 RBD protein which exhibits strong binding to its cell-associated and soluble ACE2 receptors with human and bat origin. This RBD protein also demonstrated significantly higher binding affinity to ACE2 than SARS-CoV RBD. SARS-CoV-2 RBD protein could block S protein-mediated SARS-CoV-2 pseudovirus and SARS-CoV pseudovirus entry into their respective ACE2 receptor-expressing target cells, suggesting the potential of SARS-CoV-2 RBD protein as a viral attachment or entry inhibitor against SARS-CoV-2 and SARS-CoV. SARS-CoV RBD-induced antibodies could cross-react with SARS-CoV-2 RBD and cross-neutralize SARS-CoV-2 pseudovirus infection, indicating that SARS-CoV RBD-specific antibodies may be used for treatment of SARS-CoV-2 infection and that either SARS-CoV RBD protein or SARS-CoV-2 RBD protein may be used as a candidate vaccine to induce cross-reactive or cross-neutralizing antibodies for prevention of SARS-CoV-2 or SARS-CoV infection. Taken together, this study provides an essential foundation for the design and development of SARS-CoV-2 RBD-based vaccines and therapeutics.Materials and methodsConstruction, expression, and purification of recombinant proteinThe construction, expression, and purification of recombinant RBD proteins of SARS-CoV-2, SARS-CoV, and MERS-CoV were performed as previously described with some modifications.27,28 Briefly, genes encoding residues 331-524 of SARS-CoV-2 S protein, residues 318-510 of SARS-CoV S protein, or residues 377-588 of MERS-CoV S proteins, were amplified by PCR using codon-optimized SARS-CoV-2 S protein (GenBank accession number: QHR63250.1), SARS-CoV S protein (GenBank accession number: AY278488.2), or MERS-CoV S protein (GenBank accession number: AFS88936.1), as respective template, and fused into pFUSE-hIgG1-Fc2 expression vector (hereinafter named hFc; InvivoGen, San Diego, CA). The RBD proteins were expressed in human embryonic kidney (HEK)293T cells, secreted into cell culture supernatants, and purified by protein A affinity chromatography (GE Healthcare, Marlborough, MA).SDS-PAGE and Western blotThe purified RBD proteins were analyzed by SDS-PAGE and Western blot as previously described.27,29 Briefly, proteins were separated by 10% Tris-glycine SDS-PAGE and stained with Coomassie brilliant blue or transferred to nitrocellulose membranes. The blots were blocked with 5% fat-free milk in PBS containing 0.5% Tween-20 (PBST) for 2 h at 37 °C and further incubated with SARS-CoV RBD-specific polyclonal antibody (mouse sera, 1:3,000),30 or MERS-CoV RBD-specific antibody (mouse sera, 1:3,000),31 overnight at 4 °C. The blots were then incubated with horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG (1:5,000, Thermo Fisher Scientific, Waltham, MA) for 1 h at room temperature and then visualized with ECL Western blot substrate reagents and Amersham Hyperfilm (GE Healthcare).Flow cytometry analysisFlow cytometry analysis was performed to detect the binding of SARS-CoV-2 RBD protein to hACE2 receptor in 293T cells stably expressing hACE2 (hACE2/293T).26,32 SARS-CoV and MERS-CoV RBDs, as well as 293T cells stably expressing hDPP4 receptor (hDPP4/293T), were used as controls. Briefly, cells were incubated with respective RBD of SARS-CoV-2, SARS-CoV, or MERS-CoV containing a C-terminal hFc at 20 μg/ml for 30 min at room temperature, which was followed by incubation with FITC-labeled goat anti-human IgG antibody (1:500; Thermo Fisher Scientific) for 30 min and analyzed by flow cytometry. The blockage of RBD-receptor binding was performed by incubation of soluble human ACE2 (sACE2; 5 μg/ml; R&D Systems, Minneapolis, MN) receptor with respective RBD of SARS-CoV-2, SARS-CoV, or MERS-CoV (20 μg/ml), followed by the same procedure as that described above. hIgG-Fc protein (hFc: 20 μg/ml), or soluble human DPP4 (sDPP4; 5 μg/ml; R&D Systems) receptor, was included as control.Detection of hACE2 protein expression in hACE2/293T, or hDPP4 protein expression in hDPP4/293T, stable cell lines was performed by flow cytometry analysis, as described above, except that the cells were sequentially incubated with hACE2- or hDPP4-specific goat antibody (0.5 μg/ml; R&D Systems) at room temperature for 20 min and FITC-labeled anti-goat IgG antibody (1:200; Abcam, Cambridge, MA) for 1 h at 4 °C.Flow cytometry analysis was also performed to detect the binding between SARS-CoV-2 RBD and hACE2, or bat-ACE2 (bACE2), receptor in transiently transfected 293T cells. Briefly, 293T cells were transfected with hACE2 or bACE2 plasmid using the calcium phosphate method, and 48 h later, they were incubated with SARS-CoV-2 RBD protein at various concentrations for 30 min at room temperature. SARS-CoV and MERS-CoV RBDs were included as controls. After staining with FITC-conjugated goat anti-human IgG antibody (1:500; Thermo Fisher Scientific), the mixture was analyzed by flow cytometry as described above.Immunofluorescence stainingThis was performed to detect the binding between SARS-CoV-2 RBD and hACE2 receptor in hACE2/293T stable cell lines.33 SARS-CoV and MERS-CoV RBDs were used as controls. Briefly, cells were sequentially incubated with Fc-fused SARS-CoV-2, SARS-CoV, or MERS-CoV RBD (20 μg/ml) and hACE2-specific goat antibody (5 μg/ml) for 30 min at room temperature. After three washes, the cells were incubated with FITC-labeled goat anti-human IgG (Fc) antibody (1:500; Thermo Fisher Scientific), or Alexa-Fluor 647-labeled anti-goat antibody (1:200 dilution; Abcam) for 30 min at room temperature. The nuclei were stained with 4’,6-diamidino-2-phenylindole (DAPI) for 5 min and mounted in VectaMount Permanent Mounting Medium. The samples were imaged on a confocal microscope (Zeiss LSM 880), and the images were prepared using the ZEN software.ELISAELISA was performed to detect the binding of SARS-CoV-2 RBD protein to sACE2 receptor, as previously described.27,32,34 SARS-CoV and MERS-CoV RBDs, as well as sDPP4 protein, were used as controls. Briefly, ELISA plates were precoated with SARS-CoV-2, SARS-CoV, or MERS-CoV RBD (1 μg/ml) overnight at 4 °C and blocked with 2% fat-free milk in PBST for 2 h at 37 °C. Serially diluted sACE2, or sDPP4, protein was added to the plates and incubated for 2 h at 37 °C. After four washes, the bound protein was detected using hACE2- or hDPP4-specific goat antibody (0.5 μg/ml, R&D system) for 2 h at 37 °C, followed by incubation with HRP-conjugated anti-goat IgG antibody (1:5,000, Thermo Fisher Scientific) for 1 h at 37 °C. The reaction was visualized by addition of substrate 3,3’,5,5’-Tetramethylbenzidine (TMB) (Sigma, St. Louis, MO) and stopped by H2SO4 (1 N). The absorbance at 450 nm (A450) was measured by an ELISA plate reader (Tecan, San Jose, CA).The cross-reactivity of SARS-CoV-2 RBD protein to SARS-CoV RBD-specific antibody was performed by coating ELISA plates with SARS-CoV-2 RBD (1 μg/ml), as well as SARS-CoV RBD or MERS-CoV RBD (as controls, 1 μg/ml), followed by sequential incubation with serially diluted SARS-CoV RBD- or MERS-CoV RBD-immunized mouse sera and HRP-conjugated anti-mouse IgG (1:5,000; Thermo Fisher Scientific) antibodies.Pseudovirus neutralization and inhibition assaysSARS-CoV-2 pseudovirus was generated, as previously described, with some modifications.25,27,29 Briefly, 293T cells were cotransfected with a plasmid encoding Env-defective, luciferase-expressing HIV-1 genome (pNL4-3.luc.RE) and a plasmid encoding SARS-CoV-2 S protein using the calcium phosphate method. SARS-CoV and MERS-CoV pseudoviruses were packaged as controls. The transfected medium was changed into fresh Dulbecco’s Modified Eagle’s Medium (DMEM) 8 h later, and pseudovirus-containing supernatants were collected 72 h later for single-cycle infection in target cells. Pseudovirus neutralization assay was then performed by incubation of SARS-CoV-2, SARS-CoV, or MERS-CoV pseudovirus with serially diluted SARS-CoV RBD- or MERS-CoV RBD-immunized mouse sera for 1 h at 37 °C, followed by addition of the mixture into hACE2/293T (for SARS-CoV-2 pseudovirus and SARS-CoV pseudovirus) or hDPP4/293T (for MERS-CoV pseudovirus) target cells. Fresh medium was added 24 h later, and the cells were lysed 72 h later in cell lysis buffer (Promega, Madison, WI). The lysed cell supernatants were incubated with luciferase substrate (Promega) and detected for relative luciferase activity using the Infinite 200 PRO Luminator (Tecan). The 50% MERS pseudovirus neutralizing antibody titer (NT50) was calculated using the CalcuSyn computer program, as previously described.29,35Inhibition of pseudovirus entry by SARS-CoV-2 RBD protein was carried out, as previously described, with some modifications.31 Briefly, SARS-CoV-2 RBD protein at serial dilutions was incubated with hACE2/293T target cells for 1 h at 37 °C. After removing medium containing the protein, the cells were infected with SARS-CoV-2 pseudovirus. SARS-CoV RBD and MERS-CoV RBD, as well as SARS-CoV pseudovirus and MERS-CoV pseudovirus, were used as controls. Fresh medium was added 24 h later, and the cells were lysed and analyzed, as described above. The 50% inhibitory concentration (IC50) of the RBD protein was calculated using the CalcuSyn computer program, as described above.Statistical analysisValues were expressed as mean and standard error (s.e.m). Statistical significance between different groups was calculated by GraphPad Prism Statistical Software. Two-tailed Student’s t test was used. ∗∗∗ represents P < 0.001.

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Download referencesAcknowledgementsThe authors thank Dr. Fang Li at the University of Minnesota for providing 293T cells stably expressing human dipeptidyl peptidase 4. This study was supported by the NIH grants (R01AI137472 and R01AI139092) and intramural funds of the New York Blood Center (VIM-NYB616 and CFM-NYB595).Author informationAuthors and AffiliationsLindsley F. Kimball Research Institute, New York Blood Center, New York, NY, USAWanbo Tai, Xiujuan Zhang, Jing Pu, Denis Voronin, Shibo Jiang & Lanying DuBeijing Institute of Microbiology and Epidemiology, Beijing, ChinaLei He & Yusen ZhouKey Laboratory of Medical Molecular Virology (MOE/NHC/CAMS), School of Basic Medical Sciences, Fudan University, Shanghai, ChinaJing Pu & Shibo JiangAuthorsWanbo TaiView author publicationsYou can also search for this author in

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PubMed Google ScholarContributionsW.T. performed the experiments, analyzed the data, and contributed to manuscript writing. W.T., L.H., Z.X., and J.P. performed the experiments. D.V. prepared the fluorescence image. L.D. conceptualized the study. S.J., Y.Z., and L.D. designed the experiments, wrote and revised the manuscript. All authors approved the final version of this manuscript.Corresponding authorsCorrespondence to

Shibo Jiang, Yusen Zhou or Lanying Du.Ethics declarations

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Reprints and permissionsAbout this articleCite this articleTai, W., He, L., Zhang, X. et al. Characterization of the receptor-binding domain (RBD) of 2019 novel coronavirus: implication for development of RBD protein as a viral attachment inhibitor and vaccine.

Cell Mol Immunol 17, 613–620 (2020). https://doi.org/10.1038/s41423-020-0400-4Download citationReceived: 03 March 2020Accepted: 06 March 2020Published: 19 March 2020Issue Date: June 2020DOI: https://doi.org/10.1038/s41423-020-0400-4Share this articleAnyone you share the following link with will be able to read this content:Get shareable linkSorry, a shareable link is not currently available for this article.Copy to clipboard

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Keywords2019 novel coronavirusSARS-CoV-2spike proteinreceptor-binding domainviral inhibitorcross-neutralization

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