Structures of the Omicron spike trimer with ACE2 and an anti-Omicron antibody.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) Omicron variant has become the dominant infective strain. We report the structures of the Omicron spike trimer on its own and in complex with angiotensin-converting enzyme 2 (ACE2) or an anti-Omicron antibody. Most Omicron mutations are located on the surface of the spike protein and change binding epitopes to many current antibodies. In the ACE2-binding site, compensating mutations strengthen receptor binding domain (RBD) binding to ACE2. Both the RBD and the apo form of the Omicron spike trimer are thermodynamically unstable. An unusual RBD-RBD interaction in the ACE2-spike complex supports the open conformation and further reinforces ACE2 binding to the spike trimer. A broad-spectrum therapeutic antibody, JMB2002, which has completed a phase 1 clinical trial, maintains neutralizing activity against Omicron. JMB2002 binds to RBD differently from other characterized antibodies and inhibits ACE2 binding.

A Fc-enhanced NTD-binding non-neutralizing antibody delays virus spread and synergizes with a nAb to protect mice from lethal SARS-CoV-2 infection.

Emerging evidence indicates that both neutralizing and Fc-mediated effector functions of antibodies contribute to protection against SARS-CoV-2. It is unclear whether Fc-effector functions alone can protect against SARS-CoV-2. Here, we isolated CV3-13, a non-neutralizing antibody, from a convalescent individual with potent Fc-mediated effector functions. The cryoelectron microscopy structure of CV3-13 in complex with the SARS-CoV-2 spike reveals that the antibody binds from a distinct angle of approach to an N-terminal domain (NTD) epitope that only partially overlaps with the NTD supersite recognized by neutralizing antibodies. CV3-13 does not alter the replication dynamics of SARS-CoV-2 in K18-hACE2 mice, but its Fc-enhanced version significantly delays virus spread, neuroinvasion, and death in prophylactic settings. Interestingly, the combination of Fc-enhanced non-neutralizing CV3-13 with Fc-compromised neutralizing CV3-25 completely protects mice from lethal SARS-CoV-2 infection. Altogether, our data demonstrate that efficient Fc-mediated effector functions can potently contribute to the in vivo efficacy of anti-SARS-CoV-2 antibodies.

Computational redesign of Fab CC12.3 with substantially better predicted binding affinity to SARS-CoV-2 than human ACE2 receptor.

SARS-CoV-2 is responsible for COVID-19 pandemic, causing large numbers of cases and deaths. It initiates entry into human cells by binding to the peptidase domain of angiotensin-converting enzyme 2 (ACE2) receptor via its receptor binding domain of S1 subunit of spike protein (SARS-CoV-2-RBD). Employing neutralizing antibodies to prevent binding between SARS-CoV-2-RBD and ACE2 is an effective COVID-19 therapeutic solution. Previous studies found that CC12.3 is a highly potent neutralizing antibody that was isolated from a SARS-CoV-2 infected patient, and its Fab fragment (Fab CC12.3) bound to SARS-CoV-2-RBD with comparable binding affinity to ACE2. To enhance its binding affinity, we employed computational protein design to redesign all CDRs of Fab CC12.3 and molecular dynamics (MD) to validate their predicted binding affinities by the MM-GBSA method. MD results show that the predicted binding affinities of the three best designed Fabs CC12.3 (CC12.3-D02, CC12.3-D05, and CC12.3-D08) are better than those of Fab CC12.3 and ACE2. Additionally, our results suggest that enhanced binding affinities of CC12.3-D02, CC12.3-D05, and CC12.3-D08 are caused by increased SARS-CoV-2-RBD binding interactions of CDRs L1 and L3. This study redesigned neutralizing antibodies with better predicted binding affinities to SARS-CoV-2-RBD than Fab CC12.3 and ACE2. They are promising candidates as neutralizing antibodies against SARS-CoV-2.

A SARS-CoV-2 antibody broadly neutralizes SARS-related coronaviruses and variants by coordinated recognition of a virus-vulnerable site.

Potent neutralizing SARS-CoV-2 antibodies often target the spike protein receptor-binding site (RBS), but the variability of RBS epitopes hampers broad neutralization of multiple sarbecoviruses and drifted viruses. Here, using humanized mice, we identified an RBS antibody with a germline VH gene that potently neutralized SARS-related coronaviruses, including SARS-CoV and SARS-CoV-2 variants. X-ray crystallography revealed coordinated recognition by the heavy chain of non-RBS conserved sites and the light chain of RBS with a binding angle mimicking the angiotensin-converting enzyme 2 (ACE2) receptor. The minimum footprints in the hypervariable region of RBS contributed to the breadth of neutralization, which was enhanced by immunoglobulin G3 (IgG3) class switching. The coordinated binding resulted in broad neutralization of SARS-CoV and emerging SARS-CoV-2 variants of concern. Low-dose therapeutic antibody treatment in hamsters reduced the virus titers and morbidity during SARS-CoV-2 challenge. The structural basis for broad neutralizing activity may inform the design of a broad spectrum of therapeutics and vaccines.

Fab-dimerized glycan-reactive antibodies are a structural category of natural antibodies.

Natural antibodies (Abs) can target host glycans on the surface of pathogens. We studied the evolution of glycan-reactive B cells of rhesus macaques and humans using glycosylated HIV-1 envelope (Env) as a model antigen. 2G12 is a broadly neutralizing Ab (bnAb) that targets a conserved glycan patch on Env of geographically diverse HIV-1 strains using a unique heavy-chain (VH) domain-swapped architecture that results in fragment antigen-binding (Fab) dimerization. Here, we describe HIV-1 Env Fab-dimerized glycan (FDG)-reactive bnAbs without VH-swapped domains from simian-human immunodeficiency virus (SHIV)-infected macaques. FDG Abs also recognized cell-surface glycans on diverse pathogens, including yeast and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike. FDG precursors were expanded by glycan-bearing immunogens in macaques and were abundant in HIV-1-naive humans. Moreover, FDG precursors were predominately mutated IgM+IgD+CD27+, thus suggesting that they originated from a pool of antigen-experienced IgM+ or marginal zone B cells.

Engineered Ultra-High Affinity Synthetic Antibodies for SARS-CoV-2 Neutralization and Detection.

The Covid-19 pandemic is a centenarial global catastrophe. Similar events are likely to be recurring with more frequency in the future. The inability to control the virus' impact is caused by many factors, but the lack of a technology infrastructure to detect and impede the virus at an early stage are principal shortcomings. Using phage display mutagenesis, we have generated a cohort of high performance antibody fragments (Fabs) that can be used in a sensitive point of care (POC) assay and are potent inhibitors (IC50-0.5 nM) to viral entry into cells. The POC assay is based on a split-enzyme (ß-lactamase) complementation strategy that detects virus particles at low nM levels. We have shown that this assay is equally effective for detecting other viruses like Ebola and Zika. Importantly, its components can be freeze dried and stored, but becomes fully active when rehydrated.

Structures of Human Antibodies Bound to SARS-CoV-2 Spike Reveal Common Epitopes and Recurrent Features of Antibodies.

Neutralizing antibody responses to coronaviruses mainly target the receptor-binding domain (RBD) of the trimeric spike. Here, we characterized polyclonal immunoglobulin Gs (IgGs) and Fabs from COVID-19 convalescent individuals for recognition of coronavirus spikes. Plasma IgGs differed in their focus on RBD epitopes, recognition of alpha- and beta-coronaviruses, and contributions of avidity to increased binding/neutralization of IgGs over Fabs. Using electron microscopy, we examined specificities of polyclonal plasma Fabs, revealing recognition of both S1A and RBD epitopes on SARS-CoV-2 spike. Moreover, a 3.4 Å cryo-electron microscopy (cryo-EM) structure of a neutralizing monoclonal Fab-spike complex revealed an epitope that blocks ACE2 receptor binding. Modeling based on these structures suggested different potentials for inter-spike crosslinking by IgGs on viruses, and characterized IgGs would not be affected by identified SARS-CoV-2 spike mutations. Overall, our studies structurally define a recurrent anti-SARS-CoV-2 antibody class derived from VH3-53/VH3-66 and similarity to a SARS-CoV VH3-30 antibody, providing criteria for evaluating vaccine-elicited antibodies.

Structural basis for the neutralization of SARS-CoV-2 by an antibody from a convalescent patient.

The COVID-19 pandemic has had an unprecedented health and economic impact and there are currently no approved therapies. We have isolated an antibody, EY6A, from an individual convalescing from COVID-19 and have shown that it neutralizes SARS-CoV-2 and cross-reacts with SARS-CoV-1. EY6A Fab binds the receptor binding domain (RBD) of the viral spike glycoprotein tightly (KD of 2 nM), and a 2.6-Å-resolution crystal structure of an RBD-EY6A Fab complex identifies the highly conserved epitope, away from the ACE2 receptor binding site. Residues within this footprint are key to stabilizing the pre-fusion spike. Cryo-EM analyses of the pre-fusion spike incubated with EY6A Fab reveal a complex of the intact spike trimer with three Fabs bound and two further multimeric forms comprising the destabilized spike attached to Fab. EY6A binds what is probably a major neutralizing epitope, making it a candidate therapeutic for COVID-19.

Potent neutralizing antibodies against multiple epitopes on SARS-CoV-2 spike.

The severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) pandemic continues, with devasting consequences for human lives and the global economy1,2. 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.