Efficient discovery of SARS-CoV-2-neutralizing antibodies via B cell receptor sequencing and ligand blocking.

Although several monoclonal antibodies (mAbs) targeting severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) have been approved for coronavirus disease 2019 (COVID-19) therapy, development was generally inefficient, with lead generation often requiring the production and testing of numerous antibody candidates. Here, we report that the integration of target-ligand blocking with a previously described B cell receptor-sequencing approach (linking B cell receptor to antigen specificity through sequencing (LIBRA-seq)) enables the rapid and efficient identification of multiple neutralizing mAbs that prevent the binding of SARS-CoV-2 spike (S) protein to angiotensin-converting enzyme 2 (ACE2). The combination of target-ligand blocking and high-throughput antibody sequencing promises to increase the throughput of programs aimed at discovering new neutralizing antibodies.

Continuous flexibility analysis of SARS-CoV-2 Spike prefusion structures

With the help of novel processing workflows and algorithms, we have obtained a better understanding of the flexibility and conformational dynamics of the SARS-CoV-2 spike in the prefusion state. We have re-analyzed previous cryo-EM data combining 3D clustering approaches with ways to explore a continuous flexibility space based on 3D Principal Component Analysis. These advanced analyses revealed a concerted motion involving the receptor-binding domain (RBD), N-terminal domain (NTD), and subdomain 1 and 2 (SD1 & SD2) around the previously characterized 1-RBD-up state, which have been modeled as elastic deformations. We show that in this dataset there are not well-defined, stable, spike conformations, but virtually a continuum of states moving in a concerted fashion. We obtained an improved resolution ensemble map with minimum bias, from which we model by flexible fitting the extremes of the change along the direction of maximal variance. Moreover, a high-resolution structure of a recently described biochemically stabilized form of the spike is shown to greatly reduce the dynamics observed for the wild-type spike. Our results provide new detailed avenues to potentially restrain the spike dynamics for structure-based drug and vaccine design and at the same time give a warning of the potential image processing classification instability of these complicated datasets, having a direct impact on the interpretability of the results.

Expression and characterization of SARS-CoV-2 spike proteins.

The severe acute respiratory syndrome coronavirus 2 spike protein is a critical component of coronavirus disease 2019 vaccines and diagnostics and is also a therapeutic target. However, the spike protein is difficult to produce recombinantly because it is a large trimeric class I fusion membrane protein that is metastable and heavily glycosylated. We recently developed a prefusion-stabilized spike variant, termed HexaPro for six stabilizing proline substitutions, that can be expressed with a yield of >30 mg/L in ExpiCHO cells. This protocol describes an optimized workflow for expressing and biophysically characterizing rationally engineered spike proteins in Freestyle 293 and ExpiCHO cell lines. Although we focus on HexaPro, this protocol has been used to purify over a hundred different spike variants in our laboratories. We also provide guidance on expression quality control, long-term storage, and uses in enzyme-linked immunosorbent assays. The entire protocol, from transfection to biophysical characterization, can be completed in 7 d by researchers with basic tissue cell culture and protein purification expertise.

A Combination of Receptor-Binding Domain and N-Terminal Domain Neutralizing Antibodies Limits the Generation of SARS-CoV-2 Spike Neutralization-Escape Mutants.

Most known SARS-CoV-2 neutralizing antibodies (nAbs), including those approved by the FDA for emergency use, inhibit viral infection by targeting the receptor-binding domain (RBD) of the spike (S) protein. Variants of concern (VOC) carrying mutations in the RBD or other regions of S reduce the effectiveness of many nAbs and vaccines by evading neutralization. Therefore, therapies that are less susceptible to resistance are urgently needed. Here, we characterized the memory B-cell repertoire of COVID-19 convalescent donors and analyzed their RBD and non-RBD nAbs. We found that many of the non-RBD-targeting nAbs were specific to the N-terminal domain (NTD). Using neutralization assays with authentic SARS-CoV-2 and a recombinant vesicular stomatitis virus carrying SARS-CoV-2 S protein (rVSV-SARS2), we defined a panel of potent RBD and NTD nAbs. Next, we used a combination of neutralization-escape rVSV-SARS2 mutants and a yeast display library of RBD mutants to map their epitopes. The most potent RBD nAb competed with hACE2 binding and targeted an epitope that includes residue F490. The most potent NTD nAb epitope included Y145, K150, and W152. As seen with some of the natural VOC, the neutralization potencies of COVID-19 convalescent-phase sera were reduced by 4- to 16-fold against rVSV-SARS2 bearing Y145D, K150E, or W152R spike mutations. Moreover, we found that combining RBD and NTD nAbs did not enhance their neutralization potential. Notably, the same combination of RBD and NTD nAbs limited the development of neutralization-escape mutants in vitro, suggesting such a strategy may have higher efficacy and utility for mitigating the emergence of VOC. IMPORTANCE The U.S. FDA has issued emergency use authorizations (EUAs) for multiple investigational monoclonal antibody (MAb) therapies for the treatment of mild to moderate COVID-19. These MAb therapeutics are solely targeting the receptor-binding domain of the SARS-CoV-2 spike protein. However, the N-terminal domain of the spike protein also carries crucial neutralizing epitopes. Here, we show that key mutations in the N-terminal domain can reduce the neutralizing capacity of convalescent-phase COVID-19 sera. We report that a combination of two neutralizing antibodies targeting the receptor-binding and N-terminal domains may be beneficial to combat the emergence of virus variants.

Potent neutralization of SARS-CoV-2 variants of concern by an antibody with an uncommon genetic signature and structural mode of spike recognition.

The emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) lineages that are more transmissible and resistant to currently approved antibody therapies poses a considerable challenge to the clinical treatment of coronavirus disease (COVID-19). Therefore, the need for ongoing discovery efforts to identify broadly reactive monoclonal antibodies to SARS-CoV-2 is of utmost importance. Here, we report a panel of SARS-CoV-2 antibodies isolated using the linking B cell receptor to antigen specificity through sequencing (LIBRA-seq) technology from an individual who recovered from COVID-19. Of these antibodies, 54042-4 shows potent neutralization against authentic SARS-CoV-2 viruses, including variants of concern (VOCs). A cryoelectron microscopy (cryo-EM) structure of 54042-4 in complex with the SARS-CoV-2 spike reveals an epitope composed of residues that are highly conserved in currently circulating SARS-CoV-2 lineages. Further, 54042-4 possesses uncommon genetic and structural characteristics that distinguish it from other potently neutralizing SARS-CoV-2 antibodies. Together, these findings provide motivation for the development of 54042-4 as a lead candidate to counteract current and future SARS-CoV-2 VOCs.

Local computational methods to improve the interpretability and analysis of cryo-EM maps.

Cryo-electron microscopy (cryo-EM) maps usually show heterogeneous distributions of B-factors and electron density occupancies and are typically B-factor sharpened to improve their contrast and interpretability at high-resolutions. However, 'over-sharpening' due to the application of a single global B-factor can distort processed maps causing connected densities to appear broken and disconnected. This issue limits the interpretability of cryo-EM maps, i.e. ab initio modelling. In this work, we propose 1) approaches to enhance high-resolution features of cryo-EM maps, while preventing map distortions and 2) methods to obtain local B-factors and electron density occupancy maps. These algorithms have as common link the use of the spiral phase transformation and are called LocSpiral, LocBSharpen, LocBFactor and LocOccupancy. Our results, which include improved maps of recent SARS-CoV-2 structures, show that our methods can improve the interpretability and analysis of obtained reconstructions.

Cross-reactive coronavirus antibodies with diverse epitope specificities and Fc effector functions.

The continual emergence of novel coronaviruses (CoV), such as severe acute respiratory syndrome-(SARS)-CoV-2, highlights the critical need for broadly reactive therapeutics and vaccines against this family of viruses. From a recovered SARS-CoV donor sample, we identify and characterize a panel of six monoclonal antibodies that cross-react with CoV spike (S) proteins from the highly pathogenic SARS-CoV and SARS-CoV-2, and demonstrate a spectrum of reactivity against other CoVs. Epitope mapping reveals that these antibodies recognize multiple epitopes on SARS-CoV-2 S, including the receptor-binding domain, the N-terminal domain, and the S2 subunit. Functional characterization demonstrates that the antibodies mediate phagocytosis-and in some cases trogocytosis-but not neutralization in vitro. When tested in vivo in murine models, two of the antibodies demonstrate a reduction in hemorrhagic pathology in the lungs. The identification of cross-reactive epitopes recognized by functional antibodies expands the repertoire of targets for pan-coronavirus vaccine design strategies.

Prolonged evolution of the human B cell response to SARS-CoV-2 infection.

A comprehensive understanding of the kinetics and evolution of the human B cell response to SARS-CoV-2 infection will facilitate the development of next-generation vaccines and therapies. Here, we longitudinally profiled this response in mild and severe COVID-19 patients over a period of five months. Serum neutralizing antibody (nAb) responses waned rapidly but spike (S)-specific IgG+ memory B cells (MBCs) remained stable or increased over time. Analysis of 1,213 monoclonal antibodies (mAbs) isolated from S-specific MBCs revealed a primarily de novo response that displayed increased somatic hypermutation, binding affinity, and neutralization potency over time, providing evidence for prolonged antibody affinity maturation. B cell immunodominance hierarchies were similar across donor repertoires and remained relatively stable as the immune response progressed. Cross-reactive B cell populations, likely re-called from prior endemic beta-coronavirus exposures, comprised a small but stable fraction of the repertoires and did not contribute to the neutralizing response. The neutralizing antibody response was dominated by public clonotypes that displayed significantly reduced activity against SARS-CoV-2 variants emerging in Brazil and South Africa that harbor mutations at positions 501, 484 and 417 in the S protein. Overall, the results provide insight into the dynamics, durability, and functional properties of the human B cell response to SARS-CoV-2 infection and have implications for the design of immunogens that preferentially stimulate protective B cell responses.

Broad and potent activity against SARS-like viruses by an engineered human monoclonal antibody.

The recurrent zoonotic spillover of coronaviruses (CoVs) into the human population underscores the need for broadly active countermeasures. We employed a directed evolution approach to engineer three severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) antibodies for enhanced neutralization breadth and potency. One of the affinity-matured variants, ADG-2, displays strong binding activity to a large panel of sarbecovirus receptor binding domains and neutralizes representative epidemic sarbecoviruses with high potency. Structural and biochemical studies demonstrate that ADG-2 employs a distinct angle of approach to recognize a highly conserved epitope that overlaps the receptor binding site. In immunocompetent mouse models of SARS and COVID-19, prophylactic administration of ADG-2 provided complete protection against respiratory burden, viral replication in the lungs, and lung pathology. Altogether, ADG-2 represents a promising broad-spectrum therapeutic candidate against clade 1 sarbecoviruses.

Molecular determinants and mechanism for antibody cocktail preventing SARS-CoV-2 escape.

Antibody cocktails represent a promising approach to prevent SARS-CoV-2 escape. The determinants for selecting antibody combinations and the mechanism that antibody cocktails prevent viral escape remain unclear. We compared the critical residues in the receptor-binding domain (RBD) used by multiple neutralizing antibodies and cocktails and identified a combination of two antibodies CoV2-06 and CoV2-14 for preventing viral escape. The two antibodies simultaneously bind to non-overlapping epitopes and independently compete for receptor binding. SARS-CoV-2 rapidly escapes from individual antibodies by generating resistant mutations in vitro, but it doesn't escape from the cocktail due to stronger mutational constraints on RBD-ACE2 interaction and RBD protein folding requirements. We also identified a conserved neutralizing epitope shared between SARS-CoV-2 and SARS-CoV for antibody CoV2-12. Treatments with CoV2-06 and CoV2-14 individually and in combination confer protection in mice. These findings provide insights for rational selection and mechanistic understanding of antibody cocktails as candidates for treating COVID-19.

Continuous flexibility analysis of SARS-CoV-2 spike prefusion structures.

Using a new consensus-based image-processing approach together with principal component analysis, the flexibility and conformational dynamics of the SARS-CoV-2 spike in the prefusion state have been analysed. These studies revealed concerted motions involving the receptor-binding domain (RBD), N-terminal domain, and subdomains 1 and 2 around the previously characterized 1-RBD-up state, which have been modeled as elastic deformations. It is shown that in this data set there are not well defined, stable spike conformations, but virtually a continuum of states. An ensemble map was obtained with minimum bias, from which the extremes of the change along the direction of maximal variance were modeled by flexible fitting. The results provide a warning of the potential image-processing classification instability of these complicated data sets, which has a direct impact on the interpretability of the results.

LY-CoV555, a rapidly isolated potent neutralizing antibody, provides protection in a non-human primate model of SARS-CoV-2 infection.

SARS-CoV-2 poses a public health threat for which therapeutic agents are urgently needed. Herein, we report that high-throughput microfluidic screening of antigen-specific B-cells led to the identification of LY-CoV555, a potent anti-spike neutralizing antibody from a convalescent COVID-19 patient. Biochemical, structural, and functional characterization revealed high-affinity binding to the receptor-binding domain, ACE2 binding inhibition, and potent neutralizing activity. In a rhesus macaque challenge model, prophylaxis doses as low as 2.5 mg/kg reduced viral replication in the upper and lower respiratory tract. These data demonstrate that high-throughput screening can lead to the identification of a potent antiviral antibody that protects against SARS-CoV-2 infection. ONE SENTENCE SUMMARY: LY-CoV555, an anti-spike antibody derived from a convalescent COVID-19 patient, potently neutralizes SARS-CoV-2 and protects the upper and lower airways of non-human primates against SARS-CoV-2 infection.

SARS-CoV-2 mRNA vaccine design enabled by prototype pathogen preparedness.

A vaccine for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is needed to control the coronavirus disease 2019 (COVID-19) global pandemic. Structural studies have led to the development of mutations that stabilize Betacoronavirus spike proteins in the prefusion state, improving their expression and increasing immunogenicity1. This principle has been applied to design mRNA-1273, an mRNA vaccine that encodes a SARS-CoV-2 spike protein that is stabilized in the prefusion conformation. Here we show that mRNA-1273 induces potent neutralizing antibody responses to both wild-type (D614) and D614G mutant2 SARS-CoV-2 as well as CD8+ T cell responses, and protects against SARS-CoV-2 infection in the lungs and noses of mice without evidence of immunopathology. mRNA-1273 is currently in a phase III trial to evaluate its efficacy.

Continuous flexibility analysis of SARS-CoV-2 Spike prefusion structures.

With the help of novel processing workflows and algorithms, we have obtained a better understanding of the flexibility and conformational dynamics of the SARS-CoV-2 spike in the prefusion state. We have re-analyzed previous cryo-EM data combining 3D clustering approaches with ways to explore a continuous flexibility space based on 3D Principal Component Analysis. These advanced analyses revealed a concerted motion involving the receptor-binding domain (RBD), N-terminal domain (NTD), and subdomain 1 and 2 (SD1 & SD2) around the previously characterized 1-RBD-up state, which have been modeled as elastic deformations. We show that in this dataset there are not well-defined, stable, spike conformations, but virtually a continuum of states moving in a concerted fashion. We obtained an improved resolution ensemble map with minimum bias, from which we model by flexible fitting the extremes of the change along the direction of maximal variance. Moreover, a high-resolution structure of a recently described biochemically stabilized form of the spike is shown to greatly reduce the dynamics observed for the wild-type spike. Our results provide new detailed avenues to potentially restrain the spike dynamics for structure-based drug and vaccine design and at the same time give a warning of the potential image processing classification instability of these complicated datasets, having a direct impact on the interpretability of the results.

Broad neutralization of SARS-related viruses by human monoclonal antibodies.

Broadly protective vaccines against known and preemergent human coronaviruses (HCoVs) are urgently needed. To gain a deeper understanding of cross-neutralizing antibody responses, we mined the memory B cell repertoire of a convalescent severe acute respiratory syndrome (SARS) donor and identified 200 SARS coronavirus 2 (SARS-CoV-2) binding antibodies that target multiple conserved sites on the spike (S) protein. A large proportion of the non-neutralizing antibodies display high levels of somatic hypermutation and cross-react with circulating HCoVs, suggesting recall of preexisting memory B cells elicited by prior HCoV infections. Several antibodies potently cross-neutralize SARS-CoV, SARS-CoV-2, and the bat SARS-like virus WIV1 by blocking receptor attachment and inducing S1 shedding. These antibodies represent promising candidates for therapeutic intervention and reveal a target for the rational design of pan-sarbecovirus vaccines.

Site-specific glycan analysis of the SARS-CoV-2 spike.

The emergence of the betacoronavirus, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of coronavirus disease 2019 (COVID-19), represents a considerable threat to global human health. Vaccine development is focused on the principal target of the humoral immune response, the spike (S) glycoprotein, which mediates cell entry and membrane fusion. The SARS-CoV-2 S gene encodes 22 N-linked glycan sequons per protomer, which likely play a role in protein folding and immune evasion. Here, using a site-specific mass spectrometric approach, we reveal the glycan structures on a recombinant SARS-CoV-2 S immunogen. This analysis enables mapping of the glycan-processing states across the trimeric viral spike. We show how SARS-CoV-2 S glycans differ from typical host glycan processing, which may have implications in viral pathobiology and vaccine design.

Structural Basis for Potent Neutralization of Betacoronaviruses by Single-Domain Camelid Antibodies.

Coronaviruses make use of a large envelope protein called spike (S) to engage host cell receptors and catalyze membrane fusion. Because of the vital role that these S proteins play, they represent a vulnerable target for the development of therapeutics. Here, we describe the isolation of single-domain antibodies (VHHs) from a llama immunized with prefusion-stabilized coronavirus spikes. These VHHs neutralize MERS-CoV or SARS-CoV-1 S pseudotyped viruses, respectively. Crystal structures of these VHHs bound to their respective viral targets reveal two distinct epitopes, but both VHHs interfere with receptor binding. We also show cross-reactivity between the SARS-CoV-1 S-directed VHH and SARS-CoV-2 S and demonstrate that this cross-reactive VHH neutralizes SARS-CoV-2 S pseudotyped viruses as a bivalent human IgG Fc-fusion. These data provide a molecular basis for the neutralization of pathogenic betacoronaviruses by VHHs and suggest that these molecules may serve as useful therapeutics during coronavirus outbreaks.

Cryo-EM structure of the 2019-nCoV spike in the prefusion conformation.

The outbreak of a novel coronavirus (2019-nCoV) represents a pandemic threat that has been declared a public health emergency of international concern. The CoV spike (S) glycoprotein is a key target for vaccines, therapeutic antibodies, and diagnostics. To facilitate medical countermeasure development, we determined a 3.5-angstrom-resolution cryo-electron microscopy structure of the 2019-nCoV S trimer in the prefusion conformation. The predominant state of the trimer has one of the three receptor-binding domains (RBDs) rotated up in a receptor-accessible conformation. We also provide biophysical and structural evidence that the 2019-nCoV S protein binds angiotensin-converting enzyme 2 (ACE2) with higher affinity than does severe acute respiratory syndrome (SARS)-CoV S. Additionally, we tested several published SARS-CoV RBD-specific monoclonal antibodies and found that they do not have appreciable binding to 2019-nCoV S, suggesting that antibody cross-reactivity may be limited between the two RBDs. The structure of 2019-nCoV S should enable the rapid development and evaluation of medical countermeasures to address the ongoing public health crisis.