CIISB Research Highlights Archive

6 Oct 2022

A broad and potent neutralization epitope in SARS-related coronaviruses (PNAS)

Since the beginning of the COVID-19 pandemic, many severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants have emerged that are resistant to varying extents to neutralizing antibody responses induced by current vaccines and natural infection, especially the recent Omicron variants. Neutralizing potency and breadth for an antibody are often somewhat mutually exclusive. Here, we delineate the molecular interaction between a therapeutic antibody (ADG20) and SARS-CoV-2 receptor-binding domain (RBD) by X-ray crystallography and characterize its binding epitope. We show that this site is targeted by a few rare antibodies that have both potency and breadth. These findings provide insights into the design of more universal vaccines and broad therapeutic antibodies, which are pressingly needed.

6 Oct 2022

SARS-CoV-2 Delta and Omicron variants evade population antibody response by mutations in a single spike epitope (Nature Microbiology)

Population antibody response is thought to be important in selection of virus variants. We report that SARS-CoV-2 infection elicits a population immune response that is mediated by a lineage of VH1-69 germline antibodies. A representative antibody R1-32 from this lineage was isolated. By cryo-EM, we show that it targets a semi-cryptic epitope in the spike receptor-binding domain. Binding to this non-ACE2 competing epitope results in spike destruction, thereby inhibiting virus entry. On the basis of epitope location, neutralization mechanism and analysis of antibody binding to spike variants, we propose that recurrent substitutions at 452 and 490 are associated with immune evasion of the identified population antibody response. These substitutions, including L452R (present in the Delta variant), disrupt interactions mediated by the VH1-69-specific hydrophobic HCDR2 to impair antibody-antigen association, enabling variants to escape. The first Omicron variants were sensitive to antibody R1-32 but subvariants that harbour L452R quickly emerged and spread. Our results provide insights into how SARS-CoV-2 variants emerge and evade host immune responses.

6 Oct 2022

Posttranslational modifications optimize the ability of SARS-CoV-2 spike for effective interaction with host cell receptors (PNAS)

SARS-CoV-2 spike protein, which forms the basis for high pathogenicity and transmissibility of the virus, is a prime target for the development of both diagnostics and vaccines for the debilitating disease caused by the virus. We present a full model of spike methodically crafted and used to study its atomic-level dynamics by multiple microsecond simulations. The results shed light on the impact of posttranslational modifications on the pathogenicity of the virus. We show how glycan–glycan and glycan–lipid interactions broaden the protein’s dynamical range and thereby, its effective interaction with the surface receptors on the host cell. Palmitoylation of the spike membrane domain, however, results in a unique deformation pattern that might prime the membrane for fusion.

6 Oct 2022

Spatial- and Valence-Matched Neutralizing DNA Nanostructure Blocks Wild-Type SARS-CoV-2 and Omicron Variant Infection (ACS Nano)

Natural ligand–receptor interactions that play pivotal roles in biological events are ideal models for design and assembly of artificial recognition molecules. Herein, aiming at the structural characteristics of the spike trimer and infection mechanism of SARS-CoV-2, we have designed a DNA framework-guided spatial-patterned neutralizing aptamer trimer for SARS-CoV-2 neutralization. The ∼5.8 nm tetrahedral DNA framework affords precise spatial organization and matched valence as four neutralizing aptamers (MATCH-4), which matches with nanometer precision the topmost surface of SARS-CoV-2 spike trimer, enhancing the interaction between MATCH-4 and spike trimer. Moreover, the DNA framework provides a dimensionally complementary nanoscale barrier to prevent the spike trimer–ACE2 interaction and the conformational transition, thereby inhibiting SARS-CoV-2–host cell fusion and infection. As a result, the spatial- and valence-matched MATCH-4 ensures improved binding affinity and neutralizing activity against SARS-CoV-2 and its varied mutant strains, particularly the current Omicron variant, that are evasive of the majority of existing neutralizing antibodies. In addition, because neutralizing aptamers specific to other targets can be evolved and assembled, the present design has the potential to inhibit other wide-range and emerging pathogens.

6 Oct 2022

The mechanism of RNA capping by SARS-CoV-2 (Nature)

The RNA genome of SARS-CoV-2 contains a 5′ cap that facilitates the translation of viral proteins, protection from exonucleases and evasion of the host immune response. How this cap is made in SARS-CoV-2 is not completely understood. Here we reconstitute the N7- and 2′-O-methylated SARS-CoV-2 RNA cap (^7MeGpppA_2′-O-Me) using virally encoded non-structural proteins (nsps). We show that the kinase-like nidovirus RdRp-associated nucleotidyltransferase (NiRAN) domain of nsp12 transfers the RNA to the amino terminus of nsp9, forming a covalent RNA–protein intermediate (a process termed RNAylation). Subsequently, the NiRAN domain transfers the RNA to GDP, forming the core cap structure GpppA-RNA. The nsp14 and nsp16 methyltransferases then add methyl groups to form functional cap structures. Structural analyses of the replication–transcription complex bound to nsp9 identified key interactions that mediate the capping reaction. Furthermore, we demonstrate in a reverse genetics system that the N terminus of nsp9 and the kinase-like active-site residues in the NiRAN domain are required for successful SARS-CoV-2 replication. Collectively, our results reveal an unconventional mechanism by which SARS-CoV-2 caps its RNA genome, thus exposing a new target in the development of antivirals to treat COVID-19.

6 Oct 2022

Structure of SARS-CoV-2 membrane protein essential for virus assembly (Nature Communications)

The coronavirus membrane protein (M) is the most abundant viral structural protein and plays a central role in virus assembly and morphogenesis. However, the process of M protein-driven virus assembly are largely unknown. Here, we report the cryo-electron microscopy structure of the SARS-CoV-2 M protein in two different conformations. M protein forms a mushroom-shaped dimer, composed of two transmembrane domain-swapped three-helix bundles and two intravirion domains. M protein further assembles into higher-order oligomers. A highly conserved hinge region is key for conformational changes. The M protein dimer is unexpectedly similar to SARS-CoV-2 ORF3a, a viral ion channel. Moreover, the interaction analyses of M protein with nucleocapsid protein (N) and RNA suggest that the M protein mediates the concerted recruitment of these components through the positively charged intravirion domain. Our data shed light on the M protein-driven virus assembly mechanism and provide a structural basis for therapeutic intervention targeting M protein.

9 Sep 2022

Intermediates in SARS-CoV-2 spike-mediated cell entry (Science Advances)

SARS-CoV-2 cell entry is completed after viral spike (S) protein-mediated membrane fusion between viral and host cell membranes. Stable prefusion and postfusion S structures have been resolved by cryo-electron microscopy and cryo-electron tomography, but the refolding intermediates on the fusion pathway are transient and have not been examined. We used an antiviral lipopeptide entry inhibitor to arrest S protein refolding and thereby capture intermediates as S proteins interact with hACE2 and fusion-activating proteases on cell-derived target membranes. Cryo-electron tomography imaged both extended and partially folded intermediate states of S2, as well as a novel late-stage conformation on the pathway to membrane fusion. The intermediates now identified in this dynamic S protein-directed fusion provide mechanistic insights that may guide the design of CoV entry inhibitors.

9 Sep 2022

A public antibody class recognizes an S2 epitope exposed on open conformations of SARS-CoV-2 spike (Nature Communications)

Delineating the origins and properties of antibodies elicited by SARS-CoV-2 infection and vaccination is critical for understanding their benefits and potential shortcomings. Therefore, we investigate the SARS-CoV-2 spike (S)-reactive B cell repertoire in unexposed individuals by flow cytometry and single-cell sequencing. We show that similar to 82% of SARS-CoV-2 S-reactive B cells harbor a naive phenotype, which represents an unusually high fraction of total human naive B cells (similar to 0.1%). Approximately 10% of these naive S-reactive B cells share an IGHV1-69/IGKV3-11 B cell receptor pairing, an enrichment of 18-fold compared to the complete naive repertoire. Following SARS-CoV-2 infection, we report an average 37-fold enrichment of IGHV1-69/IGKV3-11 B cell receptor pairing in the S-reactive memory B cells compared to the unselected memory repertoire. This class of B cells targets a previously undefined non-neutralizing epitope on the S2 subunit that becomes exposed on S proteins used in approved vaccines when they transition away from the native pre-fusion state because of instability. These findings can help guide the improvement of SARS-CoV-2 vaccines.

To fully understand the potential shortcomings of SARS-CoV-2 vaccination, it is necessary to delineate the properties of the antibodies elicited, during immunization, and also infection. Through investigation of the SARS-CoV-2 spike-reactive B cell repertoire, authors identify following infection, a subset of B cells enriched and almost exclusively target a non-neutralizing S2 epitope present in aberrant forms.

9 Sep 2022

Structural insights of a highly potent pan-neutralizing SARS-CoV-2 human monoclonal antibody (PNAS)

As the coronavirus disease 2019 (COVID-19) pandemic continues, there is a strong need for highly potent monoclonal antibodies (mAbs) that are resistant against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants of concern (VoCs). Here, we evaluate the potency of the previously described mAb J08 against these variants using cell-based assays and delve into the molecular details of the binding interaction using cry-oelectron microscopy (cryo-EM) and X-ray crystallography. We show that mAb J08 has low nanomolar affinity against most VoCs and binds high on the receptor binding domain (RBD) ridge, away from many VoC mutations. These findings further validate the phase II/III human clinical trial underway using mAb J08 as a monoclonal therapy.