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1.
International Journal of Biological Macromolecules ; 2022.
Article in English | ScienceDirect | ID: covidwho-1796724

ABSTRACT

SARS-CoV-2 engages with human cells through the binding of its Spike receptor-binding domain (S-RBD) to the receptor ACE2. Molecular blocking of this engagement represents a proven strategy to treat COVID-19. Here, we report a single-chain antibody (nanobody, DL4) isolated from immunized alpaca with picomolar affinity to RBD. DL4 neutralizes SARS-CoV-2 pseudoviruses with an IC50 of 0.101 μg mL−1 (6.2 nM). A crystal structure of the DL4-RBD complex at 1.75-Å resolution unveils the interaction detail and reveals a direct competition mechanism for DL4's ACE2-blocking and hence neutralizing activity. The structural information allows us to rationally design a mutant with higher potency. Our work adds diversity of neutralizing nanobodies against SARS-CoV-2 and should encourage protein engineering to improve antibody affinities in general.

2.
Proc Natl Acad Sci U S A ; 119(1)2022 01 04.
Article in English | MEDLINE | ID: covidwho-1626013

ABSTRACT

The COVID-19 pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection has resulted in tremendous loss worldwide. Although viral spike (S) protein binding of angiotensin-converting enzyme 2 (ACE2) has been established, the functional consequences of the initial receptor binding and the stepwise fusion process are not clear. By utilizing a cell-cell fusion system, in complement with a pseudoviral infection model, we found that the spike engagement of ACE2 primed the generation of S2' fragments in target cells, a key proteolytic event coupled with spike-mediated membrane fusion. Mutagenesis of an S2' cleavage site at the arginine (R) 815, but not an S2 cleavage site at arginine 685, was sufficient to prevent subsequent syncytia formation and infection in a variety of cell lines and primary cells isolated from human ACE2 knock-in mice. The requirement for S2' cleavage at the R815 site was also broadly shared by other SARS-CoV-2 spike variants, such as the Alpha, Beta, and Delta variants of concern. Thus, our study highlights an essential role for host receptor engagement and the key residue of spike for proteolytic activation, and uncovers a targetable mechanism for host cell infection by SARS-CoV-2.


Subject(s)
Angiotensin-Converting Enzyme 2/chemistry , Angiotensin-Converting Enzyme 2/metabolism , Membrane Fusion , SARS-CoV-2 , Spike Glycoprotein, Coronavirus/chemistry , Spike Glycoprotein, Coronavirus/metabolism , Animals , COVID-19/virology , HEK293 Cells , Host-Pathogen Interactions , Humans , Mice , Protein Binding , Proteolysis , Virus Internalization
3.
Emerg Microbes Infect ; 11(1): 351-367, 2022 Dec.
Article in English | MEDLINE | ID: covidwho-1585238

ABSTRACT

The emergence of multiple severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants of concern threatens the efficacy of currently approved vaccines and authorized therapeutic monoclonal antibodies (MAbs). It is hence important to continue searching for SARS-CoV-2 broadly neutralizing MAbs and defining their epitopes. Here, we isolate 9 neutralizing mouse MAbs raised against the spike protein of a SARS-CoV-2 prototype strain and evaluate their neutralizing potency towards a panel of variants, including B.1.1.7, B.1.351, B.1.617.1, and B.1.617.2. By using a combination of biochemical, virological, and cryo-EM structural analyses, we identify three types of cross-variant neutralizing MAbs, represented by S5D2, S5G2, and S3H3, respectively, and further define their epitopes. S5D2 binds the top lateral edge of the receptor-binding motif within the receptor-binding domain (RBD) with a binding footprint centred around the loop477-489, and efficiently neutralizes all variant pseudoviruses, but the potency against B.1.617.2 was observed to decrease significantly. S5G2 targets the highly conserved RBD core region and exhibits comparable neutralization towards the variant panel. S3H3 binds a previously unreported epitope located within the evolutionarily stable SD1 region and is able to near equally neutralize all of the variants tested. Our work thus defines three distinct cross-variant neutralizing sites on the SARS-CoV-2 spike protein, providing guidance for design and development of broadly effective vaccines and MAb-based therapies.


Subject(s)
COVID-19/virology , SARS-CoV-2/immunology , Spike Glycoprotein, Coronavirus/chemistry , Spike Glycoprotein, Coronavirus/immunology , Animals , Antibodies, Neutralizing/immunology , Antibodies, Viral/immunology , COVID-19/immunology , Epitope Mapping , Female , Humans , Mice , Mice, Inbred BALB C , Neutralization Tests , SARS-CoV-2/chemistry , SARS-CoV-2/genetics , Spike Glycoprotein, Coronavirus/genetics
4.
EMBO Mol Med ; 13(12): e14544, 2021 12 07.
Article in English | MEDLINE | ID: covidwho-1478717

ABSTRACT

An essential step for SARS-CoV-2 infection is the attachment to the host cell receptor by its Spike receptor-binding domain (RBD). Most of the existing RBD-targeting neutralizing antibodies block the receptor-binding motif (RBM), a mutable region with the potential to generate neutralization escape mutants. Here, we isolated and structurally characterized a non-RBM-targeting monoclonal antibody (FD20) from convalescent patients. FD20 engages the RBD at an epitope distal to the RBM with a KD of 5.6 nM, neutralizes SARS-CoV-2 including the current Variants of Concern such as B.1.1.7, B.1.351, P.1, and B.1.617.2 (Delta), displays modest cross-reactivity against SARS-CoV, and reduces viral replication in hamsters. The epitope coincides with a predicted "ideal" vulnerability site with high functional and structural constraints. Mutation of the residues of the conserved epitope variably affects FD20-binding but confers little or no resistance to neutralization. Finally, in vitro mode-of-action characterization and negative-stain electron microscopy suggest a neutralization mechanism by which FD20 destructs the Spike. Our results reveal a conserved vulnerability site in the SARS-CoV-2 Spike for the development of potential antiviral drugs.


Subject(s)
COVID-19 , SARS-CoV-2 , Antibodies, Viral , Humans , Spike Glycoprotein, Coronavirus
5.
Nat Commun ; 12(1): 4635, 2021 07 30.
Article in English | MEDLINE | ID: covidwho-1333940

ABSTRACT

SARS-CoV-2, the causative agent of COVID-191, features a receptor-binding domain (RBD) for binding to the host cell ACE2 protein1-6. Neutralizing antibodies that block RBD-ACE2 interaction are candidates for the development of targeted therapeutics7-17. Llama-derived single-domain antibodies (nanobodies, ~15 kDa) offer advantages in bioavailability, amenability, and production and storage owing to their small sizes and high stability. Here, we report the rapid selection of 99 synthetic nanobodies (sybodies) against RBD by in vitro selection using three libraries. The best sybody, MR3 binds to RBD with high affinity (KD = 1.0 nM) and displays high neutralization activity against SARS-CoV-2 pseudoviruses (IC50 = 0.42 µg mL-1). Structural, biochemical, and biological characterization suggests a common neutralizing mechanism, in which the RBD-ACE2 interaction is competitively inhibited by sybodies. Various forms of sybodies with improved potency have been generated by structure-based design, biparatopic construction, and divalent engineering. Two divalent forms of MR3 protect hamsters from clinical signs after live virus challenge and a single dose of the Fc-fusion construct of MR3 reduces viral RNA load by 6 Log10. Our results pave the way for the development of therapeutic nanobodies against COVID-19 and present a strategy for rapid development of targeted medical interventions during an outbreak.


Subject(s)
Antibodies, Neutralizing/immunology , Antibodies, Viral/immunology , COVID-19/immunology , SARS-CoV-2/immunology , Single-Domain Antibodies/immunology , Angiotensin-Converting Enzyme 2/metabolism , Animals , Antibodies, Neutralizing/pharmacology , Antibodies, Neutralizing/ultrastructure , Antibodies, Viral/pharmacology , Antibodies, Viral/ultrastructure , Binding Sites/immunology , COVID-19/prevention & control , COVID-19/virology , Cryoelectron Microscopy , Crystallography, X-Ray , Female , Humans , Mass Spectrometry/methods , Mesocricetus , Mice, Inbred C57BL , Neutralization Tests , Protein Binding/drug effects , Receptors, Virus/metabolism , SARS-CoV-2/metabolism , SARS-CoV-2/physiology , Single-Domain Antibodies/chemistry , Single-Domain Antibodies/metabolism
6.
PLoS Pathog ; 17(3): e1009328, 2021 03.
Article in English | MEDLINE | ID: covidwho-1115314

ABSTRACT

A key step to the SARS-CoV-2 infection is the attachment of its Spike receptor-binding domain (S RBD) to the host receptor ACE2. Considerable research has been devoted to the development of neutralizing antibodies, including llama-derived single-chain nanobodies, to target the receptor-binding motif (RBM) and to block ACE2-RBD binding. Simple and effective strategies to increase potency are desirable for such studies when antibodies are only modestly effective. Here, we identify and characterize a high-affinity synthetic nanobody (sybody, SR31) as a fusion partner to improve the potency of RBM-antibodies. Crystallographic studies reveal that SR31 binds to RBD at a conserved and 'greasy' site distal to RBM. Although SR31 distorts RBD at the interface, it does not perturb the RBM conformation, hence displaying no neutralizing activities itself. However, fusing SR31 to two modestly neutralizing sybodies dramatically increases their affinity for RBD and neutralization activity against SARS-CoV-2 pseudovirus. Our work presents a tool protein and an efficient strategy to improve nanobody potency.


Subject(s)
Angiotensin-Converting Enzyme 2/immunology , Antibodies, Neutralizing/immunology , Antibodies, Viral/immunology , SARS-CoV-2/immunology , Single-Domain Antibodies/immunology , Antibodies, Neutralizing/chemistry , Antibodies, Neutralizing/genetics , Antibodies, Viral/chemistry , Antibodies, Viral/genetics , Antibody Affinity , Binding Sites , Crystallography, X-Ray , HEK293 Cells , Humans , Models, Molecular , Recombinant Fusion Proteins/chemistry , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/immunology , Single-Domain Antibodies/chemistry , Single-Domain Antibodies/genetics
7.
J Biol Chem ; 296: 100111, 2021.
Article in English | MEDLINE | ID: covidwho-1066049

ABSTRACT

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), a ß-coronavirus, is the causative agent of the COVID-19 pandemic. Like for other coronaviruses, its particles are composed of four structural proteins: spike (S), envelope (E), membrane (M), and nucleoprotein (N) proteins. The involvement of each of these proteins and their interactions are critical for assembly and production of ß-coronavirus particles. Here, we sought to characterize the interplay of SARS-CoV-2 structural proteins during the viral assembly process. By combining biochemical and imaging assays in infected versus transfected cells, we show that E and M regulate intracellular trafficking of S as well as its intracellular processing. Indeed, the imaging data reveal that S is relocalized at endoplasmic reticulum (ER)-Golgi intermediate compartment (ERGIC) or Golgi compartments upon coexpression of E or M, as observed in SARS-CoV-2-infected cells, which prevents syncytia formation. We show that a C-terminal retrieval motif in the cytoplasmic tail of S is required for its M-mediated retention in the ERGIC, whereas E induces S retention by modulating the cell secretory pathway. We also highlight that E and M induce a specific maturation of N-glycosylation of S, independently of the regulation of its localization, with a profile that is observed both in infected cells and in purified viral particles. Finally, we show that E, M, and N are required for optimal production of virus-like-particles. Altogether, these results highlight how E and M proteins may influence the properties of S proteins and promote the assembly of SARS-CoV-2 viral particles.


Subject(s)
Coronavirus Envelope Proteins/genetics , Nucleocapsid Proteins/genetics , SARS-CoV-2/growth & development , Spike Glycoprotein, Coronavirus/genetics , Viral Matrix Proteins/genetics , Virion/growth & development , Virus Assembly/physiology , Animals , Biomimetic Materials/chemistry , Biomimetic Materials/metabolism , Cell Line, Tumor , Chlorocebus aethiops , Coronavirus Envelope Proteins/metabolism , Endoplasmic Reticulum/metabolism , Endoplasmic Reticulum/ultrastructure , Endoplasmic Reticulum/virology , Gene Expression , Golgi Apparatus/metabolism , Golgi Apparatus/ultrastructure , Golgi Apparatus/virology , HEK293 Cells , Hepatocytes/metabolism , Hepatocytes/ultrastructure , Hepatocytes/virology , Host-Pathogen Interactions/genetics , Humans , Nucleocapsid Proteins/metabolism , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , SARS-CoV-2/genetics , SARS-CoV-2/metabolism , Spike Glycoprotein, Coronavirus/metabolism , Vero Cells , Viral Matrix Proteins/metabolism , Virion/genetics , Virion/metabolism , Virus Internalization , Virus Release/physiology
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