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1.
Cell ; 185(13): 2279-2291.e17, 2022 Jun 23.
Article in English | MEDLINE | ID: covidwho-1866951

ABSTRACT

The isolation of CCoV-HuPn-2018 from a child respiratory swab indicates that more coronaviruses are spilling over to humans than previously appreciated. We determined the structures of the CCoV-HuPn-2018 spike glycoprotein trimer in two distinct conformational states and showed that its domain 0 recognizes sialosides. We identified that the CCoV-HuPn-2018 spike binds canine, feline, and porcine aminopeptidase N (APN) orthologs, which serve as entry receptors, and determined the structure of the receptor-binding B domain in complex with canine APN. The introduction of an oligosaccharide at position N739 of human APN renders cells susceptible to CCoV-HuPn-2018 spike-mediated entry, suggesting that single-nucleotide polymorphisms might account for viral detection in some individuals. Human polyclonal plasma antibodies elicited by HCoV-229E infection and a porcine coronavirus monoclonal antibody inhibit CCoV-HuPn-2018 spike-mediated entry, underscoring the cross-neutralizing activity among ɑ-coronaviruses. These data pave the way for vaccine and therapeutic development targeting this zoonotic pathogen representing the eighth human-infecting coronavirus.


Subject(s)
Coronavirus 229E, Human , Coronavirus Infections , Coronavirus , Animals , CD13 Antigens/chemistry , CD13 Antigens/metabolism , Cats , Cell Line , Coronavirus/metabolism , Coronavirus 229E, Human/metabolism , Dogs , Humans , Receptors, Virus/metabolism , Spike Glycoprotein, Coronavirus/metabolism , Swine
2.
EuropePMC; 2022.
Preprint in English | EuropePMC | ID: ppcovidwho-336851

ABSTRACT

SARS-CoV-2 Omicron sublineages carry distinct spike mutations and represent an antigenic shift resulting in escape from antibodies induced by previous infection or vaccination. We show that hybrid immunity or vaccine boosters result in potent plasma neutralizing activity against Omicron BA.1 and BA.2 and that breakthrough infections, but not vaccination-only, induce neutralizing activity in the nasal mucosa. Consistent with immunological imprinting, most antibodies derived from memory B cells or plasma cells of Omicron breakthrough cases cross-react with the Wuhan-Hu-1, BA.1 and BA.2 receptor-binding domains whereas Omicron primary infections elicit B cells of narrow specificity. While most clinical antibodies have reduced neutralization of Omicron, we identified an ultrapotent pan-variant antibody, that is unaffected by any Omicron lineage spike mutations and is a strong candidate for clinical development.

3.
Sci Transl Med ; 14(646): eabn1252, 2022 May 25.
Article in English | MEDLINE | ID: covidwho-1784766

ABSTRACT

New variants of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) continue to arise and prolong the coronavirus disease 2019 (COVID-19) pandemic. Here, we used a cell-free expression workflow to rapidly screen and optimize constructs containing multiple computationally designed miniprotein inhibitors of SARS-CoV-2. We found the broadest efficacy was achieved with a homotrimeric version of the 75-residue angiotensin-converting enzyme 2 (ACE2) mimic AHB2 (TRI2-2) designed to geometrically match the trimeric spike architecture. Consistent with the design model, in the cryo-electron microscopy structure TRI2-2 forms a tripod at the apex of the spike protein that engaged all three receptor binding domains simultaneously. TRI2-2 neutralized Omicron (B.1.1.529), Delta (B.1.617.2), and all other variants tested with greater potency than the monoclonal antibodies used clinically for the treatment of COVID-19. TRI2-2 also conferred prophylactic and therapeutic protection against SARS-CoV-2 challenge when administered intranasally in mice. Designed miniprotein receptor mimics geometrically arrayed to match pathogen receptor binding sites could be a widely applicable antiviral therapeutic strategy with advantages over antibodies in greater resistance to viral escape and antigenic drift, and advantages over native receptor traps in lower chances of autoimmune responses.


Subject(s)
COVID-19 , SARS-CoV-2 , Animals , Antibodies, Neutralizing , Antibodies, Viral , Cryoelectron Microscopy , Humans , Mice , Spike Glycoprotein, Coronavirus
4.
Science ; 375(6579): 449-454, 2022 Jan 28.
Article in English | MEDLINE | ID: covidwho-1723472

ABSTRACT

Understanding broadly neutralizing sarbecovirus antibody responses is key to developing countermeasures against SARS-CoV-2 variants and future zoonotic sarbecoviruses. We describe the isolation and characterization of a human monoclonal antibody, designated S2K146, that broadly neutralizes viruses belonging to SARS-CoV- and SARS-CoV-2-related sarbecovirus clades which use ACE2 as an entry receptor. Structural and functional studies show that most of the virus residues that directly bind S2K146 are also involved in binding to ACE2. This allows the antibody to potently inhibit receptor attachment. S2K146 protects against SARS-CoV-2 Beta challenge in hamsters and viral passaging experiments reveal a high barrier for emergence of escape mutants, making it a good candidate for clinical development. The conserved ACE2-binding residues present a site of vulnerability that might be leveraged for developing vaccines eliciting broad sarbecovirus immunity.


Subject(s)
Angiotensin-Converting Enzyme 2/metabolism , Antibodies, Viral/immunology , Betacoronavirus/immunology , Broadly Neutralizing Antibodies/immunology , COVID-19/therapy , SARS-CoV-2/immunology , Spike Glycoprotein, Coronavirus/immunology , Angiotensin-Converting Enzyme 2/chemistry , Animals , Antibodies, Monoclonal/chemistry , Antibodies, Monoclonal/immunology , Antibodies, Monoclonal/metabolism , Antibodies, Monoclonal/therapeutic use , Antibodies, Viral/chemistry , Antibodies, Viral/metabolism , Antibody Affinity , Broadly Neutralizing Antibodies/chemistry , Broadly Neutralizing Antibodies/metabolism , Broadly Neutralizing Antibodies/therapeutic use , COVID-19/immunology , Cross Reactions , Cryoelectron Microscopy , Epitopes , Humans , Immune Evasion , Mesocricetus , Models, Molecular , Molecular Mimicry , Mutation , Protein Conformation , Protein Domains , Receptors, Coronavirus/chemistry , Receptors, Coronavirus/metabolism , Spike Glycoprotein, Coronavirus/chemistry , Spike Glycoprotein, Coronavirus/genetics , Spike Glycoprotein, Coronavirus/metabolism
5.
[Unspecified Source]; 2020.
Preprint in English | [Unspecified Source] | ID: ppcovidwho-292754

ABSTRACT

We used two approaches to design proteins with shape and chemical complementarity to the receptor binding domain (RBD) of SARS-CoV-2 Spike protein near the binding site for the human ACE2 receptor. Scaffolds were built around an ACE2 helix that interacts with the RBD, or de novo designed scaffolds were docked against the RBD to identify new binding modes. In both cases, designed sequences were optimized first in silico and then experimentally for target binding, folding and stability. Nine designs bound the RBD with affinities ranging from 100pM to 10nM, and blocked bona fide SARS-CoV-2 infection of Vero E6 cells with IC (50) values ranging from 35 pM to 35 nM;the most potent of these - 56 and 64 residue hyperstable proteins made using the second approach - are roughly six times more potent on a per mass basis (IC (50) ~ 0.23 ng/ml) than the best monoclonal antibodies reported thus far. Cryo-electron microscopy structures of the SARS-CoV-2 spike ectodomain trimer in complex with the two most potent minibinders show that the structures of the designs and their binding interactions with the RBD are nearly identical to the computational models, and that all three RBDs in a single Spike protein can be engaged simultaneously. These hyperstable minibinders provide promising starting points for new SARS-CoV-2 therapeutics, and illustrate the power of computational protein design for rapidly generating potential therapeutic candidates against pandemic threats.

7.
Nature ; 598(7880): 342-347, 2021 10.
Article in English | MEDLINE | ID: covidwho-1379317

ABSTRACT

SARS-CoV-2 infection-which involves both cell attachment and membrane fusion-relies on the angiotensin-converting enzyme 2 (ACE2) receptor, which is paradoxically found at low levels in the respiratory tract1-3, suggesting that there may be additional mechanisms facilitating infection. Here we show that C-type lectin receptors, DC-SIGN, L-SIGN and the sialic acid-binding immunoglobulin-like lectin 1 (SIGLEC1) function as attachment receptors by enhancing ACE2-mediated infection and modulating the neutralizing activity of different classes of spike-specific antibodies. Antibodies to the amino-terminal domain or to the conserved site at the base of the receptor-binding domain, while poorly neutralizing infection of ACE2-overexpressing cells, effectively block lectin-facilitated infection. Conversely, antibodies to the receptor binding motif, while potently neutralizing infection of ACE2-overexpressing cells, poorly neutralize infection of cells expressing DC-SIGN or L-SIGN and trigger fusogenic rearrangement of the spike, promoting cell-to-cell fusion. Collectively, these findings identify a lectin-dependent pathway that enhances ACE2-dependent infection by SARS-CoV-2 and reveal distinct mechanisms of neutralization by different classes of spike-specific antibodies.


Subject(s)
Antibodies, Neutralizing/immunology , Lectins/metabolism , SARS-CoV-2/metabolism , SARS-CoV-2/pathogenicity , Angiotensin-Converting Enzyme 2/metabolism , Animals , Cell Adhesion Molecules/metabolism , Cell Fusion , Cell Line , Cricetinae , Female , Humans , Lectins/immunology , Lectins, C-Type/metabolism , Membrane Fusion , Receptors, Cell Surface/metabolism , SARS-CoV-2/immunology , Sialic Acid Binding Ig-like Lectin 1/metabolism , Spike Glycoprotein, Coronavirus/immunology , Spike Glycoprotein, Coronavirus/metabolism
8.
Nature ; 597(7874): 97-102, 2021 09.
Article in English | MEDLINE | ID: covidwho-1309448

ABSTRACT

An ideal therapeutic anti-SARS-CoV-2 antibody would resist viral escape1-3, have activity against diverse sarbecoviruses4-7, and be highly protective through viral neutralization8-11 and effector functions12,13. Understanding how these properties relate to each other and vary across epitopes would aid the development of therapeutic antibodies and guide vaccine design. Here we comprehensively characterize escape, breadth and potency across a panel of SARS-CoV-2 antibodies targeting the receptor-binding domain (RBD). Despite a trade-off between in vitro neutralization potency and breadth of sarbecovirus binding, we identify neutralizing antibodies with exceptional sarbecovirus breadth and a corresponding resistance to SARS-CoV-2 escape. One of these antibodies, S2H97, binds with high affinity across all sarbecovirus clades to a cryptic epitope and prophylactically protects hamsters from viral challenge. Antibodies that target the angiotensin-converting enzyme 2 (ACE2) receptor-binding motif (RBM) typically have poor breadth and are readily escaped by mutations despite high neutralization potency. Nevertheless, we also characterize a potent RBM antibody (S2E128) with breadth across sarbecoviruses related to SARS-CoV-2 and a high barrier to viral escape. These data highlight principles underlying variation in escape, breadth and potency among antibodies that target the RBD, and identify epitopes and features to prioritize for therapeutic development against the current and potential future pandemics.


Subject(s)
Broadly Neutralizing Antibodies/immunology , COVID-19/virology , Cross Reactions/immunology , Immune Evasion , SARS-CoV-2/classification , SARS-CoV-2/immunology , Spike Glycoprotein, Coronavirus/chemistry , Spike Glycoprotein, Coronavirus/immunology , Adult , Aged , Animals , Antibodies, Monoclonal/chemistry , Antibodies, Monoclonal/immunology , Antibodies, Viral/chemistry , Antibodies, Viral/immunology , Antibody Affinity , Broadly Neutralizing Antibodies/chemistry , COVID-19/drug therapy , COVID-19/immunology , COVID-19 Vaccines/chemistry , COVID-19 Vaccines/immunology , Cell Line , Cricetinae , Epitopes, B-Lymphocyte/chemistry , Epitopes, B-Lymphocyte/genetics , Epitopes, B-Lymphocyte/immunology , Female , Humans , Immune Evasion/genetics , Immune Evasion/immunology , Male , Mesocricetus , Middle Aged , Models, Molecular , SARS-CoV-2/chemistry , SARS-CoV-2/genetics , Spike Glycoprotein, Coronavirus/genetics , Vaccinology
9.
Nat Struct Mol Biol ; 28(6): 478-486, 2021 06.
Article in English | MEDLINE | ID: covidwho-1226434

ABSTRACT

Three highly pathogenic ß-coronaviruses have crossed the animal-to-human species barrier in the past two decades: SARS-CoV, MERS-CoV and SARS-CoV-2. To evaluate the possibility of identifying antibodies with broad neutralizing activity, we isolated a monoclonal antibody, termed B6, that cross-reacts with eight ß-coronavirus spike glycoproteins, including all five human-infecting ß-coronaviruses. B6 broadly neutralizes entry of pseudotyped viruses from lineages A and C, but not from lineage B, and the latter includes SARS-CoV and SARS-CoV-2. Cryo-EM, X-ray crystallography and membrane fusion assays reveal that B6 binds to a conserved cryptic epitope located in the fusion machinery. The data indicate that antibody binding sterically interferes with the spike conformational changes leading to membrane fusion. Our data provide a structural framework explaining B6 cross-reactivity with ß-coronaviruses from three lineages, along with a proof of concept for antibody-mediated broad coronavirus neutralization elicited through vaccination. This study unveils an unexpected target for next-generation structure-guided design of a pan-ß-coronavirus vaccine.


Subject(s)
Antibodies, Monoclonal , Antibodies, Neutralizing , Antibodies, Viral , Betacoronavirus/immunology , Coronavirus Infections/immunology , Spike Glycoprotein, Coronavirus , Animals , Antibodies, Monoclonal/immunology , Antibodies, Monoclonal/metabolism , Antibodies, Neutralizing/immunology , Antibodies, Neutralizing/metabolism , Antibodies, Viral/immunology , Antibodies, Viral/metabolism , Coronavirus Infections/prevention & control , Cross Reactions , Epitopes , Female , HEK293 Cells , Humans , Mice , Protein Binding , Spike Glycoprotein, Coronavirus/immunology , Spike Glycoprotein, Coronavirus/metabolism
11.
Science ; 370(6515): 426-431, 2020 10 23.
Article in English | MEDLINE | ID: covidwho-889834

ABSTRACT

Targeting the interaction between the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike protein and the human angiotensin-converting enzyme 2 (ACE2) receptor is a promising therapeutic strategy. We designed inhibitors using two de novo design approaches. Computer-generated scaffolds were either built around an ACE2 helix that interacts with the spike receptor binding domain (RBD) or docked against the RBD to identify new binding modes, and their amino acid sequences were designed to optimize target binding, folding, and stability. Ten designs bound the RBD, with affinities ranging from 100 picomolar to 10 nanomolar, and blocked SARS-CoV-2 infection of Vero E6 cells with median inhibitory concentration (IC50) values between 24 picomolar and 35 nanomolar. The most potent, with new binding modes, are 56- and 64-residue proteins (IC50 ~ 0.16 nanograms per milliliter). Cryo-electron microscopy structures of these minibinders in complex with the SARS-CoV-2 spike ectodomain trimer with all three RBDs bound are nearly identical to the computational models. These hyperstable minibinders provide starting points for SARS-CoV-2 therapeutics.


Subject(s)
Antiviral Agents/chemistry , Betacoronavirus/drug effects , Drug Design , Peptidyl-Dipeptidase A/chemistry , Spike Glycoprotein, Coronavirus/antagonists & inhibitors , Amino Acid Sequence , Angiotensin-Converting Enzyme 2 , Animals , Binding Sites , COVID-19 , Chlorocebus aethiops , Coronavirus Infections , Cryoelectron Microscopy , Molecular Docking Simulation , Pandemics , Pneumonia, Viral , Protein Binding/drug effects , SARS-CoV-2 , Spike Glycoprotein, Coronavirus/chemistry , Vero Cells
12.
Cell ; 183(4): 1024-1042.e21, 2020 11 12.
Article in English | MEDLINE | ID: covidwho-773817

ABSTRACT

Analysis of the specificity and kinetics of neutralizing antibodies (nAbs) elicited by SARS-CoV-2 infection is crucial for understanding immune protection and identifying targets for vaccine design. In a cohort of 647 SARS-CoV-2-infected subjects, we found that both the magnitude of Ab responses to SARS-CoV-2 spike (S) and nucleoprotein and nAb titers correlate with clinical scores. The receptor-binding domain (RBD) is immunodominant and the target of 90% of the neutralizing activity present in SARS-CoV-2 immune sera. Whereas overall RBD-specific serum IgG titers waned with a half-life of 49 days, nAb titers and avidity increased over time for some individuals, consistent with affinity maturation. We structurally defined an RBD antigenic map and serologically quantified serum Abs specific for distinct RBD epitopes leading to the identification of two major receptor-binding motif antigenic sites. Our results explain the immunodominance of the receptor-binding motif and will guide the design of COVID-19 vaccines and therapeutics.


Subject(s)
Antibodies, Neutralizing/immunology , Epitope Mapping/methods , Spike Glycoprotein, Coronavirus/immunology , Angiotensin-Converting Enzyme 2 , Antibodies, Monoclonal/chemistry , Antibodies, Monoclonal/genetics , Antibodies, Monoclonal/immunology , Antibodies, Neutralizing/blood , Antibodies, Neutralizing/chemistry , Antibodies, Viral/blood , Antibodies, Viral/chemistry , Antibodies, Viral/immunology , Antigen-Antibody Reactions , Betacoronavirus/immunology , Betacoronavirus/isolation & purification , Betacoronavirus/metabolism , Binding Sites , COVID-19 , Coronavirus Infections/pathology , Coronavirus Infections/virology , Epitopes/chemistry , Epitopes/immunology , Humans , Immunoglobulin A/blood , Immunoglobulin A/immunology , Immunoglobulin G/blood , Immunoglobulin G/immunology , Immunoglobulin M/blood , Immunoglobulin M/immunology , Kinetics , Molecular Dynamics Simulation , Pandemics , Peptidyl-Dipeptidase A/chemistry , Peptidyl-Dipeptidase A/metabolism , Pneumonia, Viral/pathology , Pneumonia, Viral/virology , Protein Binding , Protein Domains/immunology , Protein Structure, Quaternary , SARS-CoV-2 , Spike Glycoprotein, Coronavirus/chemistry , Spike Glycoprotein, Coronavirus/genetics , Spike Glycoprotein, Coronavirus/metabolism
13.
Nature ; 583(7815): 290-295, 2020 07.
Article in English | MEDLINE | ID: covidwho-291856

ABSTRACT

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a newly emerged coronavirus that is responsible for the current pandemic of coronavirus disease 2019 (COVID-19), which has resulted in more than 3.7 million infections and 260,000 deaths as of 6 May 20201,2. Vaccine and therapeutic discovery efforts are paramount to curb the pandemic spread of this zoonotic virus. The SARS-CoV-2 spike (S) glycoprotein promotes entry into host cells and is the main target of neutralizing antibodies. Here we describe several monoclonal antibodies that target the S glycoprotein of SARS-CoV-2, which we identified from memory B cells of an individual who was infected with severe acute respiratory syndrome coronavirus (SARS-CoV) in 2003. One antibody (named S309) potently neutralizes SARS-CoV-2 and SARS-CoV pseudoviruses as well as authentic SARS-CoV-2, by engaging the receptor-binding domain of the S glycoprotein. Using cryo-electron microscopy and binding assays, we show that S309 recognizes an epitope containing a glycan that is conserved within the Sarbecovirus subgenus, without competing with receptor attachment. Antibody cocktails that include S309 in combination with other antibodies that we identified further enhanced SARS-CoV-2 neutralization, and may limit the emergence of neutralization-escape mutants. These results pave the way for using S309 and antibody cocktails containing S309 for prophylaxis in individuals at a high risk of exposure or as a post-exposure therapy to limit or treat severe disease.


Subject(s)
Antibodies, Monoclonal/immunology , Antibodies, Neutralizing/immunology , Betacoronavirus/immunology , Cross Reactions/immunology , SARS Virus/immunology , Severe Acute Respiratory Syndrome/immunology , Spike Glycoprotein, Coronavirus/immunology , Angiotensin-Converting Enzyme 2 , Animals , Antibodies, Monoclonal/chemistry , Antibodies, Monoclonal/pharmacology , Antibodies, Neutralizing/chemistry , Antibodies, Neutralizing/pharmacology , Antibodies, Viral/chemistry , Antibodies, Viral/immunology , Antibodies, Viral/pharmacology , Antibody-Dependent Cell Cytotoxicity/drug effects , Antibody-Dependent Cell Cytotoxicity/immunology , B-Lymphocytes/immunology , Betacoronavirus/chemistry , Betacoronavirus/drug effects , COVID-19 , Chlorocebus aethiops , Coronavirus Infections/immunology , Coronavirus Infections/prevention & control , Coronavirus Infections/therapy , Coronavirus Infections/virology , Cross Reactions/drug effects , Cryoelectron Microscopy , Epitopes, B-Lymphocyte/chemistry , Epitopes, B-Lymphocyte/immunology , HEK293 Cells , Humans , Immune Evasion/immunology , Immunoglobulin Fab Fragments/chemistry , Immunoglobulin Fab Fragments/immunology , Immunoglobulin Fab Fragments/pharmacology , Immunologic Memory/immunology , Killer Cells, Natural/drug effects , Killer Cells, Natural/immunology , Models, Molecular , Neutralization Tests , Pandemics/prevention & control , Peptidyl-Dipeptidase A/chemistry , Peptidyl-Dipeptidase A/metabolism , Pneumonia, Viral/immunology , Pneumonia, Viral/prevention & control , Pneumonia, Viral/therapy , Pneumonia, Viral/virology , SARS Virus/chemistry , SARS Virus/drug effects , SARS-CoV-2 , Severe Acute Respiratory Syndrome/virology , Spike Glycoprotein, Coronavirus/chemistry , Vero Cells
14.
Cell ; 181(2): 281-292.e6, 2020 04 16.
Article in English | MEDLINE | ID: covidwho-5754

ABSTRACT

The emergence of SARS-CoV-2 has resulted in >90,000 infections and >3,000 deaths. Coronavirus spike (S) glycoproteins promote entry into cells and are the main target of antibodies. We show that SARS-CoV-2 S uses ACE2 to enter cells and that the receptor-binding domains of SARS-CoV-2 S and SARS-CoV S bind with similar affinities to human ACE2, correlating with the efficient spread of SARS-CoV-2 among humans. We found that the SARS-CoV-2 S glycoprotein harbors a furin cleavage site at the boundary between the S1/S2 subunits, which is processed during biogenesis and sets this virus apart from SARS-CoV and SARS-related CoVs. We determined cryo-EM structures of the SARS-CoV-2 S ectodomain trimer, providing a blueprint for the design of vaccines and inhibitors of viral entry. Finally, we demonstrate that SARS-CoV S murine polyclonal antibodies potently inhibited SARS-CoV-2 S mediated entry into cells, indicating that cross-neutralizing antibodies targeting conserved S epitopes can be elicited upon vaccination.


Subject(s)
Betacoronavirus/metabolism , Spike Glycoprotein, Coronavirus/chemistry , Spike Glycoprotein, Coronavirus/ultrastructure , Amino Acid Sequence , Angiotensin-Converting Enzyme 2 , Antibodies, Neutralizing/metabolism , Antibodies, Neutralizing/pharmacology , Antigens, Viral/chemistry , Antigens, Viral/immunology , Antigens, Viral/metabolism , Betacoronavirus/chemistry , Cell Line , Cryoelectron Microscopy , Humans , Models, Molecular , Peptidyl-Dipeptidase A/metabolism , Receptors, Virus/chemistry , Receptors, Virus/metabolism , SARS Virus/metabolism , SARS-CoV-2 , Spike Glycoprotein, Coronavirus/immunology , Spike Glycoprotein, Coronavirus/metabolism , Virus Internalization/drug effects
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