The severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) pandemic continues, with devasting consequences for human lives and the global economy 1,2 . The discovery and development of virus-neutralizing monoclonal antibodies could be one approach to treat or prevent infection by this coronavirus. Here we report the isolation of sixty-one SARS-CoV-2-neutralizing monoclonal antibodies from five patients infected with SARS-CoV-2 and admitted to hospital with severe coronavirus disease 2019 (COVID-19). Among these are nineteen antibodies that potently neutralized authentic SARS-CoV-2 in vitro, nine of which exhibited very high potency, with 50% virus-inhibitory concentrations of 0.7 to 9 ng ml −1 . Epitope mapping showed that this collection of nineteen antibodies was about equally divided between those directed against the receptor-binding domain (RBD) and those directed against the N-terminal domain (NTD), indicating that both of these regions at the top of the viral spike are immunogenic. In addition, two other powerful neutralizing antibodies recognized quaternary epitopes that overlap with the domains at the top of the spike. Cryo-electron microscopy reconstructions of one antibody that targets the RBD, a second that targets the NTD, and a third that bridges two separate RBDs showed that the antibodies recognize the closed, 'all RBD-down' conformation of the spike. Several of these monoclonal antibodies are promising candidates for clinical development as potential therapeutic and/or prophylactic agents against SARS-CoV-2.The novel coronavirus SARS-CoV-2 1,2 has caused more than 14 million confirmed infections globally, and has caused more than 600,000 deaths. This pandemic has also put much of the world on pause, with unprecedented disruption of lives and unparalleled damage to the economy. A return to some semblance of normality will depend on the ability of science to deliver an effective solution, and the scientific community has responded admirably. Drug development is well underway, and vaccine candidates have entered clinical trials. Another promising approach is the isolation of SARS-CoV-2-neutralizing monoclonal antibodies (mAbs) that could be used as therapeutic or prophylactic agents. The primary target for such antibodies is the viral spike, a trimeric protein 3,4 that is responsible for binding of the virus to the ACE2 receptor on the host cell 1,3,5,6 . The spike protein is comprised of two subunits. The S1 subunit has two major structural elements, RBD and NTD; the S2 subunit mediates virus-cell membrane fusion after the RBD has engaged ACE2. Reports of the discovery of neutralizing mAbs that target the RBD have been published recently [7][8][9][10][11] . We now describe our efforts in isolating and characterizing a collection of mAbs that not only target multiple epitopes on the viral spike but also show very high potency in neutralizing SARS-CoV-2. Patient selectionForty patients with PCR-confirmed SARS-CoV-2 infection were enrolled in a cohort study on virus-neutralizing antibodies. Plas...

Highlights d Structures of seven NTD-directed neutralizing antibody complexes with spike or NTD d Structures define distinct recognition classes, one observed in multiple donors d Supersite is glycan free, electropositive, with mobile b-hairpin and flexible loops d Most potent NTD-directed neutralizing antibodies may target this supersite

The SARS-CoV-2 spike employs mobile receptor-binding domains (RBDs) to engage the human ACE2 receptor and to facilitate virus entry, which can occur through low pH-endosomal pathways. To understand how ACE2 binding and low pH impact spike conformation, we determined cryo-EM structures –at serological and endosomal pH– delineating spike recognition of up to three ACE2 molecules. RBDs freely adopted ‘up’ conformations required for ACE2 interaction, primarily through RBD movement combined with smaller alterations in neighboring domains. In the absence of ACE2, cryo-EM structures revealed single-RBD-up conformations to dominate at pH 5.5, resolving into a solitary all-down conformation at lower pH. Notably, a pH-dependent refolding region (residues 824-858) at the spike-interdomain interface displayed dramatic structural rearrangements and mediated RBD positioning through coordinated movements of the entire trimer apex. These findings provide insight into how receptor interactions and endosomal pH alter RBD positioning and potentially facilitate immune evasion from RBD-up binding antibody.

23The SARS-CoV-2 pandemic rages on with devasting consequences on human lives and the 24 global economy. The discovery and development of virus-neutralizing monoclonal 25 antibodies could be one approach to treat or prevent infection by this novel coronavirus. 26Here we report the isolation of 61 SARS-CoV-2-neutralizing monoclonal antibodies from 5 27 infected patients hospitalized with severe disease. Among these are 19 antibodies that 28 potently neutralized the authentic SARS-CoV-2 in vitro, 9 of which exhibited exquisite 29 potency, with 50% virus-inhibitory concentrations of 1 to 9 ng/mL. Epitope mapping showed 30 this collection of 19 antibodies to be about equally divided between those directed to the 31 receptor-binding domain (RBD) and those to the N-terminal domain (NTD), indicating that 32 both of these regions at the top of the viral spike are quite immunogenic. In addition, two 33 other powerful neutralizing antibodies recognized quaternary epitopes that are overlapping 34 with the domains at the top of the spike. Cyro-electron microscopy structures of one 35 antibody targeting RBD, a second targeting NTD, and a third bridging RBD and NTD 36 revealed recognition of the closed, "all RBD-down" conformation of the spike. Several of 37 these monoclonal antibodies are promising candidates for clinical development as potential 38 therapeutic and/or prophylactic agents against SARS-CoV-2. 39 40 Background 41A novel coronavirus, now termed SARS-CoV-2 1,2 , has caused nearly 8 million confirmed 42 infections globally, leading to about 450,000 deaths. This pandemic has also put much of the 43 world on pause, with unprecedented disruption of lives and unparalleled damage to the economy. 44A return to some semblance of normalcy will depend on science to deliver an effective solution, 45 and the scientific community has responded admirably. Drug development is well underway, and 46 vaccine candidates are entering clinical trials. Another promising approach is the isolation of 47 SARS-CoV-2-neutralizing monoclonal antibodies (mAbs) that could be used as therapeutic or 48 prophylactic agents. The primary target for such antibodies is the viral spike, a trimeric protein 3,4 49 that is responsible for binding to the ACE2 receptor on the host cell 1,3,5,6 . The spike protein is 50 comprised of two subunits. The S1 subunit has two major structural elements: RBD and NTD; the 51 S2 subunit mediates virus-cell membrane fusion after the RBD engages ACE2. Reports of 52 discovery of neutralizing mAbs that target the RBD have been published recently 7-11 . We now 53 describe our efforts in isolating and characterizing a collection of mAbs that not only target 54 multiple epitopes on the viral spike but also show exquisite potency in neutralizing SARS-CoV-2. 55 56 Patient Selection 57Forty patients with PCR-confirmed SARS-CoV-2 infection were enrolled in an observational 58 cohort study on virus-neutralizing antibodies. Plasma samples from all subjects were first tested 59 for neutralizing activity against SARS-CoV-2 pseudovir...

SummaryNumerous antibodies that neutralize SARS-CoV-2 have been identified, and these generally target either the receptor-binding domain (RBD) or the N-terminal domain (NTD) of the viral spike. While RBD-directed antibodies have been extensively studied, far less is known about NTD-directed antibodies. Here we report cryo-EM and crystal structures for seven potent NTD-directed neutralizing antibodies in complex with spike or isolated NTD. These structures defined several antibody classes, with at least one observed in multiple convalescent donors. The structures revealed all seven antibodies to target a common surface, bordered by glycans N17, N74, N122, and N149. This site – formed primarily by a mobile β-hairpin and several flexible loops – was highly electropositive, located at the periphery of the spike, and the largest glycan-free surface of NTD facing away from the viral membrane. Thus, in contrast to neutralizing RBD-directed antibodies that recognize multiple non-overlapping epitopes, potent NTD-directed neutralizing antibodies target a single supersite.