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1.
Structure ; 30(5): 707-720.e5, 2022 05 05.
Article in English | MEDLINE | ID: covidwho-1829569

ABSTRACT

Because of the evolutionary variants of SARS-CoV-2, development of broad-spectrum neutralizing antibodies resilient to virus escape is urgently needed. We identified a group of high-affinity nanobodies from camels immunized with receptor-binding domain (RBD) of SARS-CoV-2 spike protein and resolved the structures of two non-competing nanobodies (NB1A7 and NB1B11) in complex with RBD using X-ray crystallography. The structures show that NB1A7 targets the highly conserved cryptic epitope shared by SARS-CoV-2 variants and some other coronaviruses and blocks ACE2 receptor attachment of the spike protein, and NB1B11 epitope overlaps with the contacting surface of ACE2 and is different from the binding site of NB1A7. These two nanobodies were covalently linked into multivalent and bi-paratopic formats, which significantly improved the avidity and neutralization potency and may further inhibit viral escape. The results contribute to the structure-guided design of antibodies against future variants of SARS-CoV-2 virus to combat coronavirus epidemics and pandemics.


Subject(s)
COVID-19 , Single-Domain Antibodies , Angiotensin-Converting Enzyme 2 , Antibodies, Neutralizing , Broadly Neutralizing Antibodies , Epitopes/metabolism , Humans , Protein Binding , SARS-CoV-2/genetics , Single-Domain Antibodies/chemistry , Single-Domain Antibodies/genetics , Spike Glycoprotein, Coronavirus/chemistry
2.
Nat Immunol ; 23(5): 768-780, 2022 May.
Article in English | MEDLINE | ID: covidwho-1751739

ABSTRACT

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection and vaccination elicit CD4+ T cell responses to the spike protein, including circulating follicular helper T (cTFH) cells that correlate with neutralizing antibodies. Using a novel HLA-DRB1*15:01/S751 tetramer to track spike-specific CD4+ T cells, we show that primary infection or vaccination induces robust S751-specific CXCR5- and cTFH cell memory responses. Secondary exposure induced recall of CD4+ T cells with a transitory CXCR3+ phenotype, and drove expansion of cTFH cells transiently expressing ICOS, CD38 and PD-1. In both contexts, cells exhibited a restricted T cell antigen receptor repertoire, including a highly public clonotype and considerable clonotypic overlap between CXCR5- and cTFH populations. Following a third vaccine dose, the rapid re-expansion of spike-specific CD4+ T cells contrasted with the comparatively delayed increase in antibody titers. Overall, we demonstrate that stable pools of cTFH and memory CD4+ T cells established by infection and/or vaccination are efficiently recalled upon antigen reexposure and may contribute to long-term protection against SARS-CoV-2.


Subject(s)
COVID-19 , SARS-CoV-2 , Antibodies, Neutralizing , Antibodies, Viral , Epitopes/metabolism , Humans , Receptors, CXCR5/metabolism , T-Lymphocytes, Helper-Inducer
3.
Int J Mol Sci ; 23(6)2022 Mar 08.
Article in English | MEDLINE | ID: covidwho-1732071

ABSTRACT

Nanobodies provide important advantages over traditional antibodies, including their smaller size and robust biochemical properties such as high thermal stability, high solubility, and the ability to be bioengineered into novel multivalent, multi-specific, and high-affinity molecules, making them a class of emerging powerful therapies against SARS-CoV-2. Recent research efforts on the design, protein engineering, and structure-functional characterization of nanobodies and their binding with SARS-CoV-2 S proteins reflected a growing realization that nanobody combinations can exploit distinct binding epitopes and leverage the intrinsic plasticity of the conformational landscape for the SARS-CoV-2 S protein to produce efficient neutralizing and mutation resistant characteristics. Structural and computational studies have also been instrumental in quantifying the structure, dynamics, and energetics of the SARS-CoV-2 spike protein binding with nanobodies. In this review, a comprehensive analysis of the current structural, biophysical, and computational biology investigations of SARS-CoV-2 S proteins and their complexes with distinct classes of nanobodies targeting different binding sites is presented. The analysis of computational studies is supplemented by an in-depth examination of mutational scanning simulations and identification of binding energy hotspots for distinct nanobody classes. The review is focused on the analysis of mechanisms underlying synergistic binding of multivalent nanobodies that can be superior to single nanobodies and conventional nanobody cocktails in combating escape mutations by effectively leveraging binding avidity and allosteric cooperativity. We discuss how structural insights and protein engineering approaches together with computational biology tools can aid in the rational design of synergistic combinations that exhibit superior binding and neutralization characteristics owing to avidity-mediated mechanisms.


Subject(s)
Binding Sites , Molecular Docking Simulation , Molecular Dynamics Simulation , Single-Domain Antibodies/chemistry , Spike Glycoprotein, Coronavirus/chemistry , Amino Acids , Antibody Affinity , Epitopes/chemistry , Epitopes/metabolism , Humans , Multiprotein Complexes/chemistry , Mutagenesis , Protein Binding , Protein Engineering , Protein Interaction Domains and Motifs , Single-Domain Antibodies/genetics , Single-Domain Antibodies/metabolism , Spike Glycoprotein, Coronavirus/genetics , Spike Glycoprotein, Coronavirus/metabolism
4.
Viruses ; 14(2)2022 01 30.
Article in English | MEDLINE | ID: covidwho-1667347

ABSTRACT

Currently, SARS-CoV-2 causing coronavirus disease 2019 (COVID-19) is responsible for one of the most deleterious pandemics of our time. The interaction between the ACE2 receptors at the surface of human cells and the viral Spike (S) protein triggers the infection, making the receptor-binding domain (RBD) of the SARS-CoV-2 S-protein a focal target for the neutralizing antibodies (Abs). Despite the recent progress in the development and deployment of vaccines, the emergence of novel variants of SARS-CoV-2 insensitive to Abs produced in response to the vaccine administration and/or monoclonal ones represent a potential danger. Here, we analyzed the diversity of neutralizing Ab epitopes and assessed the possible effects of single and multiple mutations in the RBD of SARS-CoV-2 S-protein on its binding affinity to various antibodies and the human ACE2 receptor using bioinformatics approaches. The RBD-Ab complexes with experimentally resolved structures were grouped into four clusters with distinct features at sequence and structure level. The performed computational analysis indicates that while single amino acid replacements in RBD may only cause partial impairment of the Abs binding, moreover, limited to specific epitopes, the variants of SARS-CoV-2 with multiple mutations, including some which were already detected in the population, may potentially result in a much broader antigenic escape. Further analysis of the existing RBD variants pointed to the trade-off between ACE2 binding and antigenic escape as a key limiting factor for the emergence of novel SAR-CoV-2 strains, as the naturally occurring mutations in RBD tend to reduce its binding affinity to Abs but not to ACE2. The results provide guidelines for further experimental studies aiming to identify high-risk RBD mutations that allow for an antigenic escape.


Subject(s)
Antibodies, Neutralizing/metabolism , Antibodies, Viral/metabolism , Binding Sites, Antibody/genetics , Computational Biology/methods , Mutation , SARS-CoV-2/genetics , Spike Glycoprotein, Coronavirus/genetics , Antibodies, Neutralizing/immunology , Antibodies, Viral/immunology , Epitopes/metabolism , Host Microbial Interactions/genetics , Humans , Protein Binding , SARS-CoV-2/immunology , Spike Glycoprotein, Coronavirus/immunology
5.
Viruses ; 14(2)2022 01 27.
Article in English | MEDLINE | ID: covidwho-1667340

ABSTRACT

Coronavirus disease 2019 (COVID-19), the pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), is characterized by symptoms such as fever, fatigue, a sore throat, diarrhea, and coughing. Although various new vaccines against COVID-19 have been developed, early diagnostics, isolation, and prevention remain important due to virus mutations resulting in rapid and high disease transmission. Amino acid substitutions in the major diagnostic target antigens of SARS-CoV-2 may lower the sensitivity for the detection of SARS-CoV-2. For this reason, we developed specific monoclonal antibodies that bind to epitope peptides as antigens for the rapid detection of SARS-CoV-2 NP. The binding affinity between antigenic peptides and monoclonal antibodies was investigated, and a sandwich pair for capture and detection was employed to develop a rapid biosensor for SARS-CoV-2 NP. The rapid biosensor, based on a monoclonal antibody pair binding to conserved epitopes of SARS-CoV-2 NP, detected cultured virus samples of SARS-CoV-2 (1.4 × 103 TCID50/reaction) and recombinant NP (1 ng/mL). Laboratory confirmation of the rapid biosensor was performed with clinical specimens (n = 16) from COVID-19 patients and other pathogens. The rapid biosensor consisting of a monoclonal antibody pair (75E12 for capture and the 54G6/54G10 combination for detection) binding to conserved epitopes of SARS-CoV-2 NP could assist in the detection of SARS-CoV-2 NP under the circumstance of spreading SARS-CoV-2 variants.


Subject(s)
Antibodies, Monoclonal/metabolism , Antibodies, Viral/metabolism , Biosensing Techniques/methods , Epitopes/metabolism , Nucleocapsid Proteins/metabolism , SARS-CoV-2/immunology , Viral Proteins/metabolism , Animals , Antibodies, Monoclonal/immunology , Antibodies, Viral/immunology , Epitopes/genetics , Epitopes/immunology , Humans , Immunoassay , Mice , Mice, Inbred BALB C , Nucleocapsid Proteins/genetics , Nucleocapsid Proteins/immunology , Peptides/immunology , Peptides/metabolism , Protein Binding , SARS-CoV-2/genetics , SARS-CoV-2/metabolism , Viral Proteins/immunology
6.
Signal Transduct Target Ther ; 7(1): 23, 2022 01 25.
Article in English | MEDLINE | ID: covidwho-1655541
7.
J Cell Mol Med ; 26(1): 25-34, 2022 01.
Article in English | MEDLINE | ID: covidwho-1570773

ABSTRACT

Transmission electron microscopy has historically been indispensable for virology research, as it offers unique insight into virus function. In the past decade, as cryo-electron microscopy (cryo-EM) has matured and become more accessible, we have been able to peer into the structure of viruses at the atomic level and understand how they interact with the host cell, with drugs or with antibodies. Perhaps, there was no time in recent history where cryo-EM was more needed, as SARS-CoV-2 has spread around the globe, causing millions of deaths and almost unquantifiable economic devastation. In this concise review, we aim to mark the most important contributions of cryo-EM to understanding the structure and function of SARS-CoV-2 proteins, from surface spikes to the virus core and from virus-receptor interactions to antibody binding.


Subject(s)
Angiotensin-Converting Enzyme 2/chemistry , Antibodies, Viral/chemistry , COVID-19 Vaccines/chemistry , COVID-19/prevention & control , Receptors, Virus/chemistry , Spike Glycoprotein, Coronavirus/chemistry , Angiotensin-Converting Enzyme 2/immunology , Angiotensin-Converting Enzyme 2/metabolism , Antibodies, Viral/biosynthesis , COVID-19/immunology , COVID-19/virology , COVID-19 Vaccines/administration & dosage , COVID-19 Vaccines/biosynthesis , Cryoelectron Microscopy , Epitopes/chemistry , Epitopes/immunology , Epitopes/metabolism , Humans , Models, Molecular , Protein Binding , Protein Interaction Domains and Motifs , Protein Structure, Secondary , Receptors, Virus/immunology , Receptors, Virus/metabolism , SARS-CoV-2/drug effects , SARS-CoV-2/pathogenicity , SARS-CoV-2/ultrastructure , Serine Endopeptidases/chemistry , Serine Endopeptidases/immunology , Serine Endopeptidases/metabolism , Spike Glycoprotein, Coronavirus/immunology , Spike Glycoprotein, Coronavirus/metabolism , Virion/drug effects , Virion/pathogenicity , Virion/ultrastructure
8.
Nat Immunol ; 22(12): 1503-1514, 2021 12.
Article in English | MEDLINE | ID: covidwho-1493136

ABSTRACT

Prevention of viral escape and increased coverage against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants of concern require therapeutic monoclonal antibodies (mAbs) targeting multiple sites of vulnerability on the coronavirus spike glycoprotein. Here we identify several potent neutralizing antibodies directed against either the N-terminal domain (NTD) or the receptor-binding domain (RBD) of the spike protein. Administered in combinations, these mAbs provided low-dose protection against SARS-CoV-2 infection in the K18-human angiotensin-converting enzyme 2 mouse model, using both neutralization and Fc effector antibody functions. The RBD mAb WRAIR-2125, which targets residue F486 through a unique heavy-chain and light-chain pairing, demonstrated potent neutralizing activity against all major SARS-CoV-2 variants of concern. In combination with NTD and other RBD mAbs, WRAIR-2125 also prevented viral escape. These data demonstrate that NTD/RBD mAb combinations confer potent protection, likely leveraging complementary mechanisms of viral inactivation and clearance.


Subject(s)
Antibodies, Monoclonal/immunology , Antibodies, Neutralizing/immunology , COVID-19/immunology , SARS-CoV-2/immunology , Spike Glycoprotein, Coronavirus/immunology , Amino Acid Sequence , Animals , Antibodies, Monoclonal/administration & dosage , Antibodies, Monoclonal/metabolism , Antibodies, Neutralizing/chemistry , Antibodies, Neutralizing/metabolism , Antibodies, Viral/immunology , Antibodies, Viral/metabolism , Binding Sites/genetics , COVID-19/metabolism , COVID-19/prevention & control , Disease Models, Animal , Dose-Response Relationship, Drug , Epitope Mapping , Epitopes/chemistry , Epitopes/immunology , Epitopes/metabolism , Humans , Mice, Transgenic , Neutralization Tests , Protein Binding , Protein Conformation , SARS-CoV-2/genetics , SARS-CoV-2/metabolism , Sequence Homology, Amino Acid , Spike Glycoprotein, Coronavirus/genetics , Spike Glycoprotein, Coronavirus/metabolism , Survival Analysis
9.
Nat Immunol ; 22(11): 1452-1464, 2021 11.
Article in English | MEDLINE | ID: covidwho-1454797

ABSTRACT

There is limited understanding of the viral antibody fingerprint following severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection in children. Herein, SARS-CoV-2 proteome-wide immunoprofiling of children with mild/moderate or severe coronavirus disease 2019 (COVID-19) versus multisystem inflammatory syndrome in children versus hospitalized control patients revealed differential cytokine responses, IgM/IgG/IgA epitope diversity, antibody binding and avidity. Apart from spike and nucleocapsid, IgG/IgA recognized epitopes in nonstructural protein (NSP) 2, NSP3, NSP12-NSP14 and open reading frame (ORF) 3a-ORF9. Peptides representing epitopes in NSP12, ORF3a and ORF8 demonstrated SARS-CoV-2 serodiagnosis. Antibody-binding kinetics with 24 SARS-CoV-2 proteins revealed antibody parameters that distinguish children with mild/moderate versus severe COVID-19 or multisystem inflammatory syndrome in children. Antibody avidity to prefusion spike correlated with decreased illness severity and served as a clinical disease indicator. The fusion peptide and heptad repeat 2 region induced SARS-CoV-2-neutralizing antibodies in rabbits. Thus, we identified SARS-CoV-2 antibody signatures in children associated with disease severity and delineate promising serodiagnostic and virus neutralization targets. These findings might guide the design of serodiagnostic assays, prognostic algorithms, therapeutics and vaccines in this important but understudied population.


Subject(s)
COVID-19 Serological Testing/methods , COVID-19/complications , COVID-19/immunology , SARS-CoV-2/immunology , Systemic Inflammatory Response Syndrome/immunology , Adolescent , Antibodies, Neutralizing/metabolism , Antibodies, Viral/metabolism , COVID-19/diagnosis , Child , Child, Preschool , Disease Progression , Epitopes/metabolism , Female , Hospitalization , Humans , Immunity, Humoral , Immunoglobulin A/metabolism , Immunoglobulin G/metabolism , Immunoglobulin M/metabolism , Male , Prognosis , Proteome , Severity of Illness Index , Systemic Inflammatory Response Syndrome/diagnosis
10.
Viruses ; 13(10)2021 09 22.
Article in English | MEDLINE | ID: covidwho-1438738

ABSTRACT

Antibodies targeting the spike (S) and nucleocapsid (N) proteins of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) are essential tools. In addition to important roles in the treatment and diagnosis of infection, the availability of high-quality specific antibodies for the S and N proteins is essential to facilitate basic research of virus replication and in the characterization of mutations responsible for variants of concern. We have developed panels of mouse and rabbit monoclonal antibodies (mAbs) to the SARS-CoV-2 spike receptor-binding domain (S-RBD) and N protein for functional and antigenic analyses. The mAbs to the S-RBD were tested for neutralization of native SARS-CoV-2, with several exhibiting neutralizing activity. The panels of mAbs to the N protein were assessed for cross-reactivity with the SARS-CoV and Middle East respiratory syndrome (MERS)-CoV N proteins and could be subdivided into sets that showed unique specificity for SARS-CoV-2 N protein, cross-reactivity between SARS-CoV-2 and SARS-CoV N proteins only, or cross-reactivity to all three coronavirus N proteins tested. Partial mapping of N-reactive mAbs were conducted using truncated fragments of the SARS-CoV-2 N protein and revealed near complete coverage of the N protein. Collectively, these sets of mouse and rabbit monoclonal antibodies can be used to examine structure/function studies for N proteins and to define the surface location of virus neutralizing epitopes on the RBD of the S protein.


Subject(s)
Betacoronavirus/immunology , Coronavirus Nucleocapsid Proteins/immunology , Spike Glycoprotein, Coronavirus/immunology , Animals , Antibodies, Monoclonal/immunology , Antibodies, Neutralizing/immunology , Antibodies, Viral/immunology , Binding Sites/immunology , COVID-19/immunology , Coronavirus Nucleocapsid Proteins/metabolism , Cross Reactions , Epitopes/metabolism , Humans , Mice , Neutralization Tests , Phosphoproteins/immunology , Phosphoproteins/metabolism , Protein Binding/immunology , Rabbits , SARS-CoV-2/immunology , SARS-CoV-2/pathogenicity , Spike Glycoprotein, Coronavirus/genetics , Spike Glycoprotein, Coronavirus/metabolism , Viral Envelope Proteins/metabolism
11.
Nat Commun ; 12(1): 5469, 2021 09 22.
Article in English | MEDLINE | ID: covidwho-1434103

ABSTRACT

SARS-CoV-2 remains a global threat to human health particularly as escape mutants emerge. There is an unmet need for effective treatments against COVID-19 for which neutralizing single domain antibodies (nanobodies) have significant potential. Their small size and stability mean that nanobodies are compatible with respiratory administration. We report four nanobodies (C5, H3, C1, F2) engineered as homotrimers with pmolar affinity for the receptor binding domain (RBD) of the SARS-CoV-2 spike protein. Crystal structures show C5 and H3 overlap the ACE2 epitope, whilst C1 and F2 bind to a different epitope. Cryo Electron Microscopy shows C5 binding results in an all down arrangement of the Spike protein. C1, H3 and C5 all neutralize the Victoria strain, and the highly transmissible Alpha (B.1.1.7 first identified in Kent, UK) strain and C1 also neutralizes the Beta (B.1.35, first identified in South Africa). Administration of C5-trimer via the respiratory route showed potent therapeutic efficacy in the Syrian hamster model of COVID-19 and separately, effective prophylaxis. The molecule was similarly potent by intraperitoneal injection.


Subject(s)
Antibodies, Neutralizing/pharmacology , COVID-19/drug therapy , Single-Domain Antibodies/pharmacology , Spike Glycoprotein, Coronavirus/metabolism , Administration, Intranasal , Animals , Antibodies, Neutralizing/administration & dosage , Antibodies, Neutralizing/genetics , Antibodies, Neutralizing/immunology , Cryoelectron Microscopy , Crystallography, X-Ray , Disease Models, Animal , Dose-Response Relationship, Immunologic , Epitopes/chemistry , Epitopes/metabolism , Female , Male , Mesocricetus , Neutralization Tests , SARS-CoV-2/drug effects , Single-Domain Antibodies/administration & dosage , Single-Domain Antibodies/immunology , Single-Domain Antibodies/metabolism , Spike Glycoprotein, Coronavirus/chemistry
12.
Cell Rep ; 36(13): 109760, 2021 09 28.
Article in English | MEDLINE | ID: covidwho-1401299

ABSTRACT

Many anti-severe acute respiratory syndrome coronavirus 2 (anti-SARS-CoV-2) neutralizing antibodies target the angiotensin-converting enzyme 2 (ACE2) binding site on viral spike receptor-binding domains (RBDs). Potent antibodies recognize exposed variable epitopes, often rendering them ineffective against other sarbecoviruses and SARS-CoV-2 variants. Class 4 anti-RBD antibodies against a less-exposed, but more-conserved, cryptic epitope could recognize newly emergent zoonotic sarbecoviruses and variants, but they usually show only weak neutralization potencies. Here, we characterize two class 4 anti-RBD antibodies derived from coronavirus disease 2019 (COVID-19) donors that exhibit breadth and potent neutralization of zoonotic coronaviruses and SARS-CoV-2 variants. C118-RBD and C022-RBD structures reveal orientations that extend from the cryptic epitope to occlude ACE2 binding and CDRH3-RBD main-chain H-bond interactions that extend an RBD ß sheet, thus reducing sensitivity to RBD side-chain changes. A C118-spike trimer structure reveals rotated RBDs that allow access to the cryptic epitope and the potential for intra-spike crosslinking to increase avidity. These studies facilitate vaccine design and illustrate potential advantages of class 4 RBD-binding antibody therapeutics.


Subject(s)
Broadly Neutralizing Antibodies/immunology , COVID-19/immunology , SARS-CoV-2/immunology , Antibodies, Neutralizing/immunology , Antibodies, Viral/immunology , Binding Sites/immunology , Broadly Neutralizing Antibodies/pharmacology , Cross Reactions , Epitopes/metabolism , Humans , Protein Binding , Protein Interaction Domains and Motifs , Receptors, Virus/metabolism , SARS-CoV-2/pathogenicity , Spike Glycoprotein, Coronavirus/chemistry , Spike Glycoprotein, Coronavirus/immunology
13.
J Phys Chem Lett ; 12(16): 4059-4066, 2021 Apr 29.
Article in English | MEDLINE | ID: covidwho-1387120

ABSTRACT

The spike glycoprotein (S-protein) mediates SARS-CoV-2 entry via intermolecular interaction with human angiotensin-converting enzyme 2. The receptor binding domain (RBD) of the S-protein has been considered critical for this interaction and acts as the target of numerous neutralizing antibodies and antiviral peptides. This study used the fragment molecular orbital method to analyze the interactions between the RBD and antibodies/peptides and extracted crucial residues that can be used as epitopes. The interactions evaluated as interfragment interaction energy values between the RBD and 12 antibodies/peptides showed a fairly good correlation with the experimental activity pIC50 (R2 = 0.540). Nine residues (T415, K417, Y421, F456, A475, F486, N487, N501, and Y505) were confirmed as being crucial. Pair interaction energy decomposition analyses showed that hydrogen bonds, electrostatic interactions, and π-orbital interactions are important. Our results provide essential information for understanding SARS-CoV-2-antibody/peptide binding and may play roles in future antibody/antiviral drug design.


Subject(s)
Angiotensin-Converting Enzyme 2/immunology , Antibodies, Neutralizing/metabolism , Antibodies, Viral/metabolism , Peptides/metabolism , Spike Glycoprotein, Coronavirus/metabolism , Antibodies, Neutralizing/immunology , Antibodies, Viral/immunology , Binding Sites/immunology , Epitopes/immunology , Epitopes/metabolism , Humans , Hydrogen Bonding , Models, Chemical , Protein Binding , Protein Domains , Quantum Theory , SARS-CoV-2/chemistry , Static Electricity
14.
J Proteome Res ; 19(11): 4398-4406, 2020 11 06.
Article in English | MEDLINE | ID: covidwho-1387124

ABSTRACT

Presentation of antigenic peptides by MHCI is central to cellular immune responses against viral pathogens. While adaptive immune responses versus SARS-CoV-2 can be of critical importance to both recovery and vaccine efficacy, how protein antigens from this pathogen are processed to generate antigenic peptides is largely unknown. Here, we analyzed the proteolytic processing of overlapping precursor peptides spanning the entire sequence of the S1 spike glycoprotein of SARS-CoV-2, by three key enzymes that generate antigenic peptides, aminopeptidases ERAP1, ERAP2, and IRAP. All enzymes generated shorter peptides with sequences suitable for binding onto HLA alleles, but with distinct specificity fingerprints. ERAP1 was the most efficient in generating peptides 8-11 residues long, the optimal length for HLA binding, while IRAP was the least efficient. The combination of ERAP1 with ERAP2 greatly limited the variability of peptide sequences produced. Less than 7% of computationally predicted epitopes were found to be produced experimentally, suggesting that aminopeptidase processing may constitute a significant filter to epitope presentation. These experimentally generated putative epitopes could be prioritized for SARS-CoV-2 immunogenicity studies and vaccine design. We furthermore propose that this in vitro trimming approach could constitute a general filtering method to enhance the prediction robustness for viral antigenic epitopes.


Subject(s)
Aminopeptidases/metabolism , Antigens, Viral , Epitopes , Spike Glycoprotein, Coronavirus , Antigens, Viral/chemistry , Antigens, Viral/metabolism , Chromatography, Liquid , Epitopes/chemistry , Epitopes/metabolism , HEK293 Cells , HLA Antigens/chemistry , HLA Antigens/metabolism , Humans , Peptides/analysis , Peptides/chemistry , Peptides/metabolism , Proteomics/methods , Spike Glycoprotein, Coronavirus/chemistry , Spike Glycoprotein, Coronavirus/metabolism , Tandem Mass Spectrometry
15.
Immunity ; 54(10): 2399-2416.e6, 2021 10 12.
Article in English | MEDLINE | ID: covidwho-1364126

ABSTRACT

With the emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants with increased transmissibility and potential resistance, antibodies and vaccines with broadly inhibitory activity are needed. Here, we developed a panel of neutralizing anti-SARS-CoV-2 monoclonal antibodies (mAbs) that bound the receptor binding domain of the spike protein at distinct epitopes and blocked virus attachment to its host receptor, human angiotensin converting enzyme-2 (hACE2). Although several potently neutralizing mAbs protected K18-hACE2 transgenic mice against infection caused by ancestral SARS-CoV-2 strains, others induced escape variants in vivo or lost neutralizing activity against emerging strains. One mAb, SARS2-38, potently neutralized all tested SARS-CoV-2 variants of concern and protected mice against challenge by multiple SARS-CoV-2 strains. Structural analysis showed that SARS2-38 engaged a conserved epitope proximal to the receptor binding motif. Thus, treatment with or induction of neutralizing antibodies that bind conserved spike epitopes may limit the loss of potency of therapies or vaccines against emerging SARS-CoV-2 variants.


Subject(s)
Antibodies, Neutralizing/immunology , Epitopes/immunology , SARS-CoV-2/immunology , Amino Acid Motifs , Animals , Antibodies, Monoclonal/chemistry , Antibodies, Monoclonal/immunology , Antibodies, Monoclonal/therapeutic use , Antibodies, Neutralizing/chemistry , Antibodies, Neutralizing/therapeutic use , COVID-19/prevention & control , COVID-19/virology , Epitopes/chemistry , Epitopes/metabolism , Humans , Immunoglobulin Light Chains/chemistry , Immunoglobulin Light Chains/metabolism , Mice , Neutralization Tests , Protein Domains , SARS-CoV-2/genetics , Spike Glycoprotein, Coronavirus/chemistry , Spike Glycoprotein, Coronavirus/genetics , Spike Glycoprotein, Coronavirus/immunology
16.
Nat Commun ; 12(1): 4676, 2021 08 03.
Article in English | MEDLINE | ID: covidwho-1340999

ABSTRACT

Interventions against variants of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) are urgently needed. Stable and potent nanobodies (Nbs) that target the receptor binding domain (RBD) of SARS-CoV-2 spike are promising therapeutics. However, it is unknown if Nbs broadly neutralize circulating variants. We found that RBD Nbs are highly resistant to variants of concern (VOCs). High-resolution cryoelectron microscopy determination of eight Nb-bound structures reveals multiple potent neutralizing epitopes clustered into three classes: Class I targets ACE2-binding sites and disrupts host receptor binding. Class II binds highly conserved epitopes and retains activity against VOCs and RBDSARS-CoV. Cass III recognizes unique epitopes that are likely inaccessible to antibodies. Systematic comparisons of neutralizing antibodies and Nbs provided insights into how Nbs target the spike to achieve high-affinity and broadly neutralizing activity. Structure-function analysis of Nbs indicates a variety of antiviral mechanisms. Our study may guide the rational design of pan-coronavirus vaccines and therapeutics.


Subject(s)
Broadly Neutralizing Antibodies/immunology , Epitopes/immunology , SARS-CoV-2/immunology , Single-Domain Antibodies/immunology , Angiotensin-Converting Enzyme 2/chemistry , Angiotensin-Converting Enzyme 2/metabolism , Antibodies, Monoclonal/chemistry , Antibodies, Monoclonal/immunology , Antibodies, Monoclonal/metabolism , Binding Sites , Broadly Neutralizing Antibodies/chemistry , Broadly Neutralizing Antibodies/classification , Broadly Neutralizing Antibodies/metabolism , COVID-19/drug therapy , COVID-19/prevention & control , Epitopes/chemistry , Epitopes/metabolism , Humans , Models, Molecular , Mutation , Protein Binding , SARS-CoV-2/genetics , Single-Domain Antibodies/chemistry , Single-Domain Antibodies/classification , Single-Domain Antibodies/metabolism , Spike Glycoprotein, Coronavirus/chemistry , Spike Glycoprotein, Coronavirus/genetics , Spike Glycoprotein, Coronavirus/immunology , Spike Glycoprotein, Coronavirus/metabolism , Structure-Activity Relationship
17.
Structure ; 29(7): 655-663.e4, 2021 07 01.
Article in English | MEDLINE | ID: covidwho-1263379

ABSTRACT

Emerging SARS-CoV-2 strains, B.1.1.7 and B.1.351, from the UK and South Africa, respectively, show decreased neutralization by monoclonal antibodies and convalescent or vaccinee sera raised against the original wild-type virus, and are thus of clinical concern. However, the neutralization potency of two antibodies, 1-57 and 2-7, which target the receptor-binding domain (RBD) of the spike, was unaffected by these emerging strains. Here, we report cryo-EM structures of 1-57 and 2-7 in complex with spike, revealing each of these antibodies to utilize a distinct mechanism to bypass or accommodate RBD mutations. Notably, each antibody represented an immune response with recognition distinct from those of frequent antibody classes. Moreover, many epitope residues recognized by 1-57 and 2-7 were outside hotspots of evolutionary pressure for ACE2 binding and neutralizing antibody escape. We suggest the therapeutic use of antibodies, such as 1-57 and 2-7, which target less prevalent epitopes, could ameliorate issues of monoclonal antibody escape.


Subject(s)
Angiotensin-Converting Enzyme 2/chemistry , Antibodies, Monoclonal/chemistry , Antibodies, Neutralizing/chemistry , Antibodies, Viral/chemistry , Receptors, Virus/chemistry , SARS-CoV-2/chemistry , Spike Glycoprotein, Coronavirus/chemistry , Angiotensin-Converting Enzyme 2/genetics , Angiotensin-Converting Enzyme 2/immunology , Angiotensin-Converting Enzyme 2/metabolism , Antibodies, Monoclonal/genetics , Antibodies, Monoclonal/immunology , Antibodies, Monoclonal/metabolism , Antibodies, Neutralizing/genetics , Antibodies, Neutralizing/immunology , Antibodies, Neutralizing/metabolism , Antibodies, Viral/genetics , Antibodies, Viral/immunology , Antibodies, Viral/metabolism , Binding Sites , Cloning, Molecular , Cryoelectron Microscopy , Epitopes/chemistry , Epitopes/genetics , Epitopes/immunology , Epitopes/metabolism , Gene Expression , HEK293 Cells , Humans , Models, Molecular , Mutation , Protein Binding , Protein Conformation, alpha-Helical , Protein Conformation, beta-Strand , Protein Interaction Domains and Motifs , Receptors, Virus/genetics , Receptors, Virus/immunology , Receptors, Virus/metabolism , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/immunology , Recombinant Proteins/metabolism , SARS-CoV-2/genetics , SARS-CoV-2/immunology , SARS-CoV-2/metabolism , Spike Glycoprotein, Coronavirus/genetics , Spike Glycoprotein, Coronavirus/immunology , Spike Glycoprotein, Coronavirus/metabolism
18.
Mol Cell Proteomics ; 20: 100059, 2021.
Article in English | MEDLINE | ID: covidwho-1087559

ABSTRACT

Antibodies play essential roles in both diagnostics and therapeutics. Epitope mapping is essential to understand how an antibody works and to protect intellectual property. Given the millions of antibodies for which epitope information is lacking, there is a need for high-throughput epitope mapping. To address this, we developed a strategy, Antibody binding epitope Mapping (AbMap), by combining a phage displayed peptide library with next-generation sequencing. Using AbMap, profiles of the peptides bound by 202 antibodies were determined in a single test, and linear epitopes were identified for >50% of the antibodies. Using spike protein (S1 and S2)-enriched antibodies from the convalescent serum of one COVID-19 patient as the input, both linear and potentially conformational epitopes of spike protein specific antibodies were identified. We defined peptide-binding profile of an antibody as the binding capacity (BiC). Conceptually, the BiC could serve as a systematic and functional descriptor of any antibody. Requiring at least one order of magnitude less time and money to map linear epitopes than traditional technologies, AbMap allows for high-throughput epitope mapping and creates many possibilities.


Subject(s)
COVID-19/immunology , Epitope Mapping/methods , Spike Glycoprotein, Coronavirus/immunology , Antibodies, Monoclonal/immunology , Antibodies, Monoclonal/metabolism , Antibodies, Viral/metabolism , Enzyme-Linked Immunosorbent Assay , Epitopes/metabolism , Escherichia coli Proteins/immunology , High-Throughput Nucleotide Sequencing , Humans , Immune Sera/blood , Immune Sera/immunology , Peptide Library
19.
Protein Sci ; 30(4): 716-727, 2021 04.
Article in English | MEDLINE | ID: covidwho-1080719

ABSTRACT

Infection with SARS-CoV-2 elicits robust antibody responses in some patients, with a majority of the response directed at the receptor binding domain (RBD) of the spike surface glycoprotein. Remarkably, many patient-derived antibodies that potently inhibit viral infection harbor few to no mutations from the germline, suggesting that naïve antibody libraries are a viable means for discovery of novel SARS-CoV-2 neutralizing antibodies. Here, we used a yeast surface-display library of human naïve antibodies to isolate and characterize three novel neutralizing antibodies that target the RBD: one that blocks interaction with angiotensin-converting enzyme 2 (ACE2), the human receptor for SARS-CoV-2, and two that target other epitopes on the RBD. These three antibodies neutralized SARS-CoV-2 spike-pseudotyped lentivirus with IC50 values as low as 60 ng/ml in vitro. Using a biolayer interferometry-based binding competition assay, we determined that these antibodies have distinct but overlapping epitopes with antibodies elicited during natural COVID-19 infection. Taken together, these analyses highlight how in vitro selection of naïve antibodies can mimic the humoral response in vivo, yielding neutralizing antibodies and various epitopes that can be effectively targeted on the SARS-CoV-2 RBD.


Subject(s)
Angiotensin-Converting Enzyme 2/metabolism , Antibodies, Neutralizing/metabolism , COVID-19/metabolism , SARS-CoV-2/physiology , Spike Glycoprotein, Coronavirus/metabolism , Antibodies, Neutralizing/chemistry , Binding Sites , Epitopes/chemistry , Epitopes/metabolism , Humans , Molecular Docking Simulation , Protein Binding , Protein Interaction Domains and Motifs , Spike Glycoprotein, Coronavirus/chemistry
20.
Biochem Biophys Res Commun ; 534: 457-460, 2021 01 01.
Article in English | MEDLINE | ID: covidwho-1064870

ABSTRACT

Spike and nucleocapsid proteins of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2-SP, SARS-CoV-2-NP) are the main immunogenic targets for antibodies. We herein demonstrate that the glycosylation of SARS-CoV-2-NP masks some of its antibody epitopes. In many cases, this can lead to false-negative serological tests. Deglycosylation of SARS-CoV-2-NP significantly increased the number of positive tests. The glycosylation pattern analysis of this protein revealed that the putative N-linked glycosylation sites, at the amino acid positions 48 and 270, co-located with two of the main immunodominant B cell epitopes.


Subject(s)
Enzyme-Linked Immunosorbent Assay/methods , Epitopes/immunology , Recombinant Proteins/immunology , Spike Glycoprotein, Coronavirus/immunology , Animals , CHO Cells , Cricetinae , Cricetulus , Epitopes/genetics , Epitopes/metabolism , Escherichia coli/genetics , Glycosylation , Humans , Recombinant Proteins/metabolism , Spike Glycoprotein, Coronavirus/genetics , Spike Glycoprotein, Coronavirus/metabolism
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