Structure-guided multivalent nanobodies block SARS-CoV-2 infection and suppress mutational escape.
Structure-guided multivalent nanobodies block SARS-CoV-2 infection and suppress mutational escape.
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DOI：
10.1126/science.abe6230
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发表时间：
2021-02-12
期刊：
影响因子：
--
通讯作者：
Schmidt FI
中科院分区：
文献类型：
--
作者：
Koenig PA;Das H;Liu H;Kümmerer BM;Gohr FN;Jenster LM;Schiffelers LDJ;Tesfamariam YM;Uchima M;Wuerth JD;Gatterdam K;Ruetalo N;Christensen MH;Fandrey CI;Normann S;Tödtmann JMP;Pritzl S;Hanke L;Boos J;Yuan M;Zhu X;Schmid-Burgk JL;Kato H;Schindler M;Wilson IA;Geyer M;Ludwig KU;Hällberg BM;Wu NC;Schmidt FI
单克隆抗体是抗击COVID-19的重要武器。然而，这些大蛋白质难以以所需的量和低成本生产。人们的注意力已经转向纳米抗体，纳米抗体被恰当地命名为单结构域抗体，更容易生产，并有可能通过吸入给药。Koenig等人描述了四种与严重急性呼吸综合征冠状病毒2（SARS-CoV-2）刺突蛋白结合并防止细胞感染的纳米抗体（参见Saelens和Schepens的观点）。结构显示，纳米抗体靶向SARS-CoV-2刺突蛋白上的两个不同表位。多价纳米抗体比单个纳米抗体更有效地中和病毒，并且结合两个表位的多价纳米抗体防止病毒逃逸突变体的出现。Science，this issue p.eabe6230;另见p. SARS-CoV-2-neutralizing nanobodies were combined to design potent multivalent nanobodies.严重急性呼吸综合征冠状病毒2（SARS-CoV-2）的全球规模和快速传播对社会、医疗保健系统和科学提出了前所未有的挑战。除了有效和安全的疫苗外，抗体相关分子的被动免疫提供了利用脊椎动物免疫系统对抗高危患者病毒感染的机会。纯重链抗体（VHH）的可变结构域，也称为纳米抗体，是此类努力中合适的先导分子，因为它们小、极其稳定、易于工程化并且在简单表达系统中生产经济。我们基于两个原则设计了中和SARS-CoV-2的改进的多价纳米抗体：（i）其表位的详细结构信息和与病毒刺突蛋白的结合模式，以及（ii）对刺突催化的病毒与细胞膜融合的机制见解。通过噬菌体展示，使用来自用RBD和灭活病毒免疫的羊驼和美洲驼的纳米抗体文库，鉴定了对SARS-CoV-2刺突的受体结合结构域（RBD）特异性的纳米抗体。四种所得的纳米抗体-VHH E、U、V和W-有效地中和SARS-CoV-2和SARS-CoV-2假型水泡性口炎病毒。X射线晶体学显示，纳米抗体结合到RBD上的两个不同表位，界面“E”和“UVW”，其可以通过纳米抗体的组合协同靶向以抑制感染。与VHH E和VHH V复合的三聚体刺突的冷冻电子显微镜（cryo-EM）显示，VHH E稳定刺突的构象，其中所有三种RBD处于“向上”构象（3-向上），这是一种通常与通过受体结合的活化相关的状态。与该观察结果一致，我们发现VHH E在不存在同源受体ACE 2的情况下触发刺突的融合活性。相比之下，VHH V使尖峰稳定在2-up构象中并且不诱导融合。在结构信息的基础上，我们设计了具有改进的中和性质的二价和三价纳米抗体。VHH最有效地抑制感染，不激活融合，并且可能通过胜过病毒与其受体的相互作用来灭活病毒粒子。然而，进化实验揭示了逃逸突变体的出现，在穗与单一氨基酸的变化，是完全不敏感的抑制VHH β。VHH VE也比单独的VHH E或VHH V更有效地中和;如通过冷冻-EM所确定的，稳定了刺突的3-up构象;并且更强烈地诱导刺突融合活性。我们的结论是，过早激活的病毒粒子上的融合机制是一个意想不到的中和机制，因为增强中和不能简单地归因于更好地阻断病毒-受体相互作用。在不存在靶膜的情况下激活尖峰可能诱导不可逆的构象变化，以呈现能量上有利的融合后构象，而不催化融合本身。VHH VE同时靶向两个独立的表位在很大程度上防止了进化实验中抗性逃逸突变体的出现。我们的结果证明了用于中和的纳米抗体的模块化组合的强度。纳米抗体对刺突的过早激活揭示了一种不寻常的中和模式，并产生了对融合机制的见解。在病毒体上，SARS-CoV-2刺突三聚体大多处于无活性构型，所有RBD处于向下构象（左）。二价纳米抗体VE的结合使刺突稳定在活性构象中，所有RBD向上（中间），引发融合后构象的过早诱导，这不可逆地使刺突蛋白失活（右）。由严重急性呼吸系统综合征冠状病毒2型（SARS-CoV-2）引起的大流行病继续蔓延，造成破坏性后果。对于被动免疫的努力，纳米抗体具有比常规抗体更大的尺寸和成本优势。在这项研究中，我们产生了四个靶向SARS-CoV-2刺突蛋白受体结合结构域的中和纳米抗体。我们使用X射线晶体学和冷冻电子显微镜来定义两个不同的结合表位。基于这些结构，我们设计了多价纳米抗体，其中和活性是单价纳米抗体的100倍以上。双互补位纳米抗体融合抑制逃逸突变体的出现。几种纳米抗体构建体通过受体结合竞争进行中和，而其他单价和双互补位纳米抗体触发刺突融合机制的异常激活。刺突蛋白中这些过早的构象变化阻止了生产性融合，并使病毒粒子无感染性。
Monoclonal antibodies are an important weapon in the battle against COVID-19. However, these large proteins are difficult to produce in the needed quantities and at low cost. Attention has turned to nanobodies, which are aptly named, single-domain antibodies that are easier to produce and have the potential to be administered by inhalation. Koenig et al. describe four nanobodies that bind to the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike protein and prevent infection of cells (see the Perspective by Saelens and Schepens). Structures show that the nanobodies target two distinct epitopes on the SARS-CoV-2 spike protein. Multivalent nanobodies neutralize virus much more potently than single nanobodies, and multivalent nanobodies that bind two epitopes prevent the emergence of viral escape mutants. Science, this issue p. eabe6230; see also p. SARS-CoV-2–neutralizing nanobodies were combined to design potent multivalent nanobodies. The global scale and rapid spread of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pose unprecedented challenges to society, health care systems, and science. In addition to effective and safe vaccines, passive immunization by antibody-related molecules offers an opportunity to harness the vertebrate immune system to fight viral infections in high-risk patients. Variable domains of heavy-chain–only antibodies (VHHs), also known as nanobodies, are suitable lead molecules in such efforts, as they are small, extremely stable, easy to engineer, and economic to produce in simple expression systems. We engineered improved multivalent nanobodies neutralizing SARS-CoV-2 on the basis of two principles: (i) detailed structural information of their epitopes and binding modes to the viral spike protein and (ii) mechanistic insights into viral fusion with cellular membranes catalyzed by the spike. Nanobodies specific for the receptor binding domain (RBD) of SARS-CoV-2 spike were identified by phage display using nanobody libraries from an alpaca and a llama immunized with the RBD and inactivated virus. Four of the resulting nanobodies—VHHs E, U, V, and W—potently neutralize SARS-CoV-2 and SARS-CoV-2–pseudotyped vesicular stomatitis virus. X-ray crystallography revealed that the nanobodies bind to two distinct epitopes on the RBD, interfaces “E” and “UVW,” which can be synergistically targeted by combinations of nanobodies to inhibit infection. Cryo–electron microscopy (cryo-EM) of trimeric spike in complex with VHH E and VHH V revealed that VHH E stabilizes a conformation of the spike with all three RBDs in the “up” conformation (3-up), a state that is typically associated with activation by receptor binding. In line with this observation, we found that VHH E triggers the fusion activity of spike in the absence of the cognate receptor ACE2. VHH V, by contrast, stabilizes spike in a 2-up conformation and does not induce fusion. On the basis of the structural information, we designed bi- and trivalent nanobodies with improved neutralizing properties. VHH EEE most potently inhibited infection, did not activate fusion, and likely inactivated virions by outcompeting interaction of the virus with its receptor. Yet evolution experiments revealed emergence of escape mutants in the spike with single–amino acid changes that were completely insensitive to inhibition by VHH EEE. VHH VE also neutralized more efficiently than VHH E or VHH V alone; stabilized the 3-up conformation of spike, as determined by cryo-EM; and more strongly induced the spike fusogenic activity. We conclude that the premature activation of the fusion machinery on virions was an unexpected mechanism of neutralization, as enhanced neutralization could not be attributed simply to better blocking of virus-receptor interactions. Activation of spike in the absence of target membranes likely induces irreversible conformational changes to assume the energetically favorable postfusion conformation without catalyzing fusion per se. Simultaneous targeting of two independent epitopes by VHH VE largely prevented the emergence of resistant escape mutants in evolution experiments. Our results demonstrate the strength of the modular combination of nanobodies for neutralization. Premature activation of spike by nanobodies reveals an unusual mode of neutralization and yields insights into the mechanism of fusion. On virions, SARS-CoV-2 spike trimers are mostly in an inactive configuration with all RBDs in the down conformation (left). Binding of bivalent nanobody VE stabilizes the spike in an active conformation with all RBDs up (middle), triggering premature induction of the postfusion conformation, which irreversibly inactivates the spike protein (right). The pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) continues to spread, with devastating consequences. For passive immunization efforts, nanobodies have size and cost advantages over conventional antibodies. In this study, we generated four neutralizing nanobodies that target the receptor binding domain of the SARS-CoV-2 spike protein. We used x-ray crystallography and cryo–electron microscopy to define two distinct binding epitopes. On the basis of these structures, we engineered multivalent nanobodies with more than 100 times the neutralizing activity of monovalent nanobodies. Biparatopic nanobody fusions suppressed the emergence of escape mutants. Several nanobody constructs neutralized through receptor binding competition, whereas other monovalent and biparatopic nanobodies triggered aberrant activation of the spike fusion machinery. These premature conformational changes in the spike protein forestalled productive fusion and rendered the virions noninfectious.
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