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1.
Microbiol Spectr ; 9(2): e0135221, 2021 10 31.
Article in English | MEDLINE | ID: covidwho-1526454

ABSTRACT

The emerging new lineages of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) have marked a new phase of coronavirus disease 2019 (COVID-19). Understanding the recognition mechanisms of potent neutralizing monoclonal antibodies (NAbs) against the spike protein is pivotal for developing new vaccines and antibody drugs. Here, we isolated several monoclonal antibodies (MAbs) against the SARS-CoV-2 spike protein receptor-binding domain (S-RBD) from the B cell receptor repertoires of a SARS-CoV-2 convalescent. Among these MAbs, the antibody nCoV617 demonstrates the most potent neutralizing activity against authentic SARS-CoV-2 infection, as well as prophylactic and therapeutic efficacies against the human angiotensin-converting enzyme 2 (ACE2) transgenic mouse model in vivo. The crystal structure of S-RBD in complex with nCoV617 reveals that nCoV617 mainly binds to the back of the "ridge" of RBD and shares limited binding residues with ACE2. Under the background of the S-trimer model, it potentially binds to both "up" and "down" conformations of S-RBD. In vitro mutagenesis assays show that mutant residues found in the emerging new lineage B.1.1.7 of SARS-CoV-2 do not affect nCoV617 binding to the S-RBD. These results provide a new human-sourced neutralizing antibody against the S-RBD and assist vaccine development. IMPORTANCE COVID-19 is a respiratory disease caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The COVID-19 pandemic has posed a serious threat to global health and the economy, so it is necessary to find safe and effective antibody drugs and treatments. The receptor-binding domain (RBD) in the SARS-CoV-2 spike protein is responsible for binding to the angiotensin-converting enzyme 2 (ACE2) receptor. It contains a variety of dominant neutralizing epitopes and is an important antigen for the development of new coronavirus antibodies. The significance of our research lies in the determination of new epitopes, the discovery of antibodies against RBD, and the evaluation of the antibodies' neutralizing effect. The identified antibodies here may be drug candidates for the development of clinical interventions for SARS-CoV-2.


Subject(s)
Antibodies, Neutralizing/therapeutic use , Antibodies, Viral/therapeutic use , COVID-19/therapy , SARS-CoV-2/immunology , Spike Glycoprotein, Coronavirus/immunology , Angiotensin-Converting Enzyme 2/antagonists & inhibitors , Angiotensin-Converting Enzyme 2/genetics , Angiotensin-Converting Enzyme 2/immunology , Animals , Antibodies, Monoclonal/immunology , Antibodies, Monoclonal/therapeutic use , Antibodies, Neutralizing/immunology , Antibodies, Neutralizing/metabolism , Antibodies, Viral/immunology , Antibodies, Viral/metabolism , Binding Sites/immunology , COVID-19 Vaccines/immunology , Crystallography, X-Ray , Disease Models, Animal , Female , Humans , Immunization, Passive/methods , Immunoglobulin G/blood , Mice , Mice, Inbred C57BL , Mice, Transgenic , Protein Interaction Domains and Motifs/immunology , Viral Load/drug effects
2.
Sci Rep ; 11(1): 20738, 2021 10 20.
Article in English | MEDLINE | ID: covidwho-1475484

ABSTRACT

Monoclonal antibodies that target SARS-CoV-2 with high affinity are valuable for a wide range of biomedical applications involving novel coronavirus disease (COVID-19) diagnosis, treatment, and prophylactic intervention. Strategies for the rapid and reliable isolation of these antibodies, especially potent neutralizing antibodies, are critical toward improved COVID-19 response and informed future response to emergent infectious diseases. In this study, single B cell screening was used to interrogate antibody repertoires of immunized mice and isolate antigen-specific IgG1+ memory B cells. Using these methods, high-affinity, potent neutralizing antibodies were identified that target the receptor-binding domain of SARS-CoV-2. Further engineering of the identified molecules to increase valency resulted in enhanced neutralizing activity. Mechanistic investigation revealed that these antibodies compete with ACE2 for binding to the receptor-binding domain of SARS-CoV-2. These antibodies may warrant further development for urgent COVID-19 applications. Overall, these results highlight the potential of single B cell screening for the rapid and reliable identification of high-affinity, potent neutralizing antibodies for infectious disease applications.


Subject(s)
Antibodies, Neutralizing/chemistry , B-Lymphocytes/virology , COVID-19/blood , COVID-19/immunology , SARS-CoV-2 , Angiotensin-Converting Enzyme 2/metabolism , Animals , Antibodies, Monoclonal/immunology , Antibodies, Viral/immunology , B-Lymphocytes/immunology , Binding Sites/immunology , Biological Products , Female , HEK293 Cells , Humans , Immunoglobulin G/immunology , Immunoglobulin G/metabolism , Immunologic Memory , Mice , Mice, Inbred BALB C , Protein Binding , Spike Glycoprotein, Coronavirus , Vaccines
3.
Proc Natl Acad Sci U S A ; 118(41)2021 10 12.
Article in English | MEDLINE | ID: covidwho-1462069

ABSTRACT

We describe a general method that allows structure determination of small proteins by single-particle cryo-electron microscopy (cryo-EM). The method is based on the availability of a target-binding nanobody, which is then rigidly attached to two scaffolds: 1) a Fab fragment of an antibody directed against the nanobody and 2) a nanobody-binding protein A fragment fused to maltose binding protein and Fab-binding domains. The overall ensemble of ∼120 kDa, called Legobody, does not perturb the nanobody-target interaction, is easily recognizable in EM images due to its unique shape, and facilitates particle alignment in cryo-EM image processing. The utility of the method is demonstrated for the KDEL receptor, a 23-kDa membrane protein, resulting in a map at 3.2-Šoverall resolution with density sufficient for de novo model building, and for the 22-kDa receptor-binding domain (RBD) of SARS-CoV-2 spike protein, resulting in a map at 3.6-Šresolution that allows analysis of the binding interface to the nanobody. The Legobody approach thus overcomes the current size limitations of cryo-EM analysis.


Subject(s)
Cryoelectron Microscopy/methods , SARS-CoV-2/metabolism , Single-Domain Antibodies/metabolism , Spike Glycoprotein, Coronavirus/metabolism , Binding Sites/immunology , COVID-19/virology , HEK293 Cells , Humans , Models, Molecular , Protein Binding , Protein Domains , SARS-CoV-2/immunology , SARS-CoV-2/physiology , Single-Domain Antibodies/immunology , Spike Glycoprotein, Coronavirus/chemistry , Spike Glycoprotein, Coronavirus/ultrastructure
4.
Biochem Biophys Res Commun ; 574: 14-19, 2021 10 15.
Article in English | MEDLINE | ID: covidwho-1446453

ABSTRACT

Following the initial surges of the Alpha (B.1.1.7) and the Beta (B.1.351) variants, a more infectious Delta variant (B.1.617.2) is now surging, further deepening the health crises caused by the pandemic. The sharp rise in cases attributed to the Delta variant has made it especially disturbing and is a variant of concern. Fortunately, current vaccines offer protection against known variants of concern, including the Delta variant. However, the Delta variant has exhibited some ability to dodge the immune system as it is found that neutralizing antibodies from prior infections or vaccines are less receptive to binding with the Delta spike protein. Here, we investigated the structural changes caused by the mutations in the Delta variant's receptor-binding interface and explored the effects on binding with the ACE2 receptor as well as with neutralizing antibodies. We find that the receptor-binding ß-loop-ß motif adopts an altered but stable conformation causing separation in some of the antibody binding epitopes. Our study shows reduced binding of neutralizing antibodies and provides a possible mechanism for the immune evasion exhibited by the Delta variant.


Subject(s)
Angiotensin-Converting Enzyme 2/immunology , COVID-19/immunology , Immune Evasion/immunology , Mutation/immunology , SARS-CoV-2/immunology , Spike Glycoprotein, Coronavirus/immunology , Amino Acids/genetics , Amino Acids/immunology , Amino Acids/metabolism , Angiotensin-Converting Enzyme 2/genetics , Angiotensin-Converting Enzyme 2/metabolism , Antibodies, Viral/immunology , Binding Sites/genetics , Binding Sites/immunology , COVID-19/metabolism , COVID-19/virology , Humans , Immune Evasion/genetics , Molecular Dynamics Simulation , Mutation/genetics , Neutralization Tests , Protein Binding , Protein Domains , SARS-CoV-2/genetics , SARS-CoV-2/metabolism , Spike Glycoprotein, Coronavirus/chemistry , Spike Glycoprotein, Coronavirus/genetics
5.
Nat Commun ; 12(1): 5652, 2021 09 27.
Article in English | MEDLINE | ID: covidwho-1440473

ABSTRACT

The emergence of numerous variants of SARS-CoV-2, the causative agent of COVID-19, has presented new challenges to the global efforts to control the COVID-19 pandemic. Here, we obtain two cross-neutralizing antibodies (7D6 and 6D6) that target Sarbecoviruses' receptor-binding domain (RBD) with sub-picomolar affinities and potently neutralize authentic SARS-CoV-2. Crystal structures show that both antibodies bind a cryptic site different from that recognized by existing antibodies and highly conserved across Sarbecovirus isolates. Binding of these two antibodies to the RBD clashes with the adjacent N-terminal domain and disrupts the viral spike. Both antibodies confer good resistance to mutations in the currently circulating SARS-CoV-2 variants. Thus, our results have direct relevance to public health as options for passive antibody therapeutics and even active prophylactics. They can also inform the design of pan-sarbecovirus vaccines.


Subject(s)
Antibodies, Viral/immunology , Broadly Neutralizing Antibodies/immunology , COVID-19/therapy , Immunization, Passive/methods , SARS-CoV-2/immunology , Animals , Antibodies, Monoclonal/administration & dosage , Antibodies, Monoclonal/immunology , Antibodies, Monoclonal/isolation & purification , Antibodies, Monoclonal/metabolism , Antibodies, Viral/administration & dosage , Antibodies, Viral/isolation & purification , Antibodies, Viral/metabolism , Binding Sites/genetics , Binding Sites/immunology , Broadly Neutralizing Antibodies/administration & dosage , Broadly Neutralizing Antibodies/isolation & purification , Broadly Neutralizing Antibodies/metabolism , CHO Cells , COVID-19/epidemiology , COVID-19/immunology , COVID-19/virology , Chlorocebus aethiops , Cricetulus , Epitopes/immunology , HEK293 Cells , Humans , Mice , Middle East Respiratory Syndrome Coronavirus/genetics , Middle East Respiratory Syndrome Coronavirus/immunology , Neutralization Tests , Pandemics/prevention & control , Protein Multimerization , Receptors, Virus/metabolism , SARS-CoV-2/genetics , Sf9 Cells , Spike Glycoprotein, Coronavirus/genetics , Spike Glycoprotein, Coronavirus/immunology , Spike Glycoprotein, Coronavirus/metabolism , Vero Cells
6.
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
7.
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
8.
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
9.
Biochem Biophys Res Commun ; 571: 152-158, 2021 09 24.
Article in English | MEDLINE | ID: covidwho-1330656

ABSTRACT

Potent neutralizing antibodies (Abs) have been proven with therapeutic efficacy for the intervention against SARS-CoV-2. Majority of these Abs function by directly interfering with the virus entry to host cells. Here, we identified a receptor binding domain (RBD) specific monoclonal Ab (mAb) 82A6 with efficient neutralizing potency against authentic SARS-CoV-2 virus. As most Abs targeting the non-receptor binding motif (RBM) region, 82A6 was incapable to block the RBD-ACE2 interaction. In particular, it actively promoted the S1 subunit shedding from the S protein, which may lead to effective reduction of intact SARS-CoV-2 viruses. Importantly, it could block potential syncytia formation associated with post-infectious cell surface expression of S proteins. Our study evidenced a RBD specific Ab with unique beneficial efficacy against SARS-CoV-2 infection, which might bring informative significance to understand the collective effects of neutralizing Abs elicited in COVID-19 patients.


Subject(s)
Antibodies, Neutralizing/therapeutic use , Antibodies, Viral/therapeutic use , COVID-19/therapy , SARS-CoV-2/immunology , Spike Glycoprotein, Coronavirus/immunology , Antibodies, Monoclonal/therapeutic use , Antibody Specificity , Binding Sites/immunology , COVID-19/immunology , COVID-19/virology , Giant Cells/immunology , Giant Cells/virology , HEK293 Cells , Humans , Immunization, Passive , In Vitro Techniques , Protein Domains , Protein Subunits , SARS-CoV-2/physiology , Spike Glycoprotein, Coronavirus/antagonists & inhibitors , Spike Glycoprotein, Coronavirus/chemistry , Virus Shedding
10.
J Genet Genomics ; 48(2): 107-114, 2021 02 20.
Article in English | MEDLINE | ID: covidwho-1316536

ABSTRACT

The ongoing COVID-19 pandemic and its unprecedented global societal and economic disruptive impact highlight the urgent need for safe and effective vaccines. Taking substantial advantages of versatility and rapid development, two mRNA vaccines against COVID-19 have completed late-stage clinical assessment at an unprecedented speed and reported positive results. In this review, we outline keynotes in mRNA vaccine development, discuss recently published data on COVID-19 mRNA vaccine candidates, focusing on those in clinical trials and analyze future potential challenges.


Subject(s)
COVID-19 Vaccines/immunology , COVID-19/immunology , RNA, Messenger/immunology , SARS-CoV-2/immunology , Vaccines, Synthetic/immunology , Binding Sites/genetics , Binding Sites/immunology , COVID-19/epidemiology , COVID-19/virology , COVID-19 Vaccines/genetics , COVID-19 Vaccines/therapeutic use , Drug Development , Humans , Pandemics/prevention & control , RNA, Messenger/genetics , RNA, Messenger/metabolism , SARS-CoV-2/metabolism , SARS-CoV-2/physiology , Vaccines, Synthetic/genetics , Viral Proteins/genetics , Viral Proteins/immunology , Viral Proteins/metabolism
11.
Nat Commun ; 12(1): 4210, 2021 07 09.
Article in English | MEDLINE | ID: covidwho-1303772

ABSTRACT

Neutralizing antibodies (nAbs) to SARS-CoV-2 hold powerful potentials for clinical interventions against COVID-19 disease. However, their common genetic and biologic features remain elusive. Here we interrogate a total of 165 antibodies from eight COVID-19 patients, and find that potent nAbs from different patients have disproportionally high representation of IGHV3-53/3-66 usage, and therefore termed as public antibodies. Crystal structural comparison of these antibodies reveals they share similar angle of approach to RBD, overlap in buried surface and binding residues on RBD, and have substantial spatial clash with receptor angiotensin-converting enzyme-2 (ACE2) in binding to RBD. Site-directed mutagenesis confirms these common binding features although some minor differences are found. One representative antibody, P5A-3C8, demonstrates extraordinarily protective efficacy in a golden Syrian hamster model against SARS-CoV-2 infection. However, virus escape analysis identifies a single natural mutation in RBD, namely K417N found in B.1.351 variant from South Africa, abolished the neutralizing activity of these public antibodies. The discovery of public antibodies and shared escape mutation highlight the intricate relationship between antibody response and SARS-CoV-2, and provide critical reference for the development of antibody and vaccine strategies to overcome the antigenic variation of SARS-CoV-2.


Subject(s)
Angiotensin-Converting Enzyme 2/immunology , Antibodies, Neutralizing/immunology , Antibodies, Viral/immunology , Receptors, Virus/immunology , SARS-CoV-2/immunology , Angiotensin-Converting Enzyme 2/metabolism , Animals , Binding Sites/immunology , COVID-19/immunology , Cricetinae , Disease Models, Animal , Epitopes/immunology , Female , Humans , Male , Neutralization Tests , Receptors, Antigen, B-Cell/immunology , Spike Glycoprotein, Coronavirus/immunology
12.
Front Immunol ; 12: 660198, 2021.
Article in English | MEDLINE | ID: covidwho-1221948

ABSTRACT

The worldwide pandemic caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is unprecedented and the impact on public health and the global economy continues to be devastating. Although early therapies such as prophylactic antibodies and vaccines show great promise, there are concerns about the long-term efficacy and universal applicability of these therapies as the virus continues to mutate. Thus, protein-based immunogens that can quickly respond to viral changes remain of continued interest. The Spike protein, the main immunogen of this virus, displays a highly dynamic trimeric structure that presents a challenge for therapeutic development. Here, guided by the structure of the Spike trimer, we rationally design new Spike constructs that show a uniquely high stability profile while simultaneously remaining locked into the immunogen-desirable prefusion state. Furthermore, our approach emphasizes the relationship between the highly conserved S2 region and structurally dynamic Receptor Binding Domains (RBD) to enable vaccine development as well as the generation of antibodies able to resist viral mutation.


Subject(s)
Protein Interaction Domains and Motifs/genetics , Protein Interaction Domains and Motifs/immunology , SARS-CoV-2/immunology , Spike Glycoprotein, Coronavirus/genetics , Spike Glycoprotein, Coronavirus/immunology , Angiotensin-Converting Enzyme 2/metabolism , Antibodies, Neutralizing/immunology , Antibodies, Viral/immunology , Binding Sites/genetics , Binding Sites/immunology , COVID-19/immunology , COVID-19/pathology , Cell Line , HEK293 Cells , Humans , Protein Domains/genetics , Protein Domains/immunology , Protein Stability , SARS-CoV-2/genetics
13.
Cell Rep ; 35(6): 109109, 2021 05 11.
Article in English | MEDLINE | ID: covidwho-1201425

ABSTRACT

It is unclear whether individuals with enormous diversity in B cell receptor repertoires are consistently able to mount effective antibody responses against SARS-CoV-2. We analyzed antibody responses in a cohort of 55 convalescent patients and isolated 54 potent neutralizing monoclonal antibodies (mAbs). While most of the mAbs target the angiotensin-converting enzyme 2 (ACE2) binding surface on the receptor binding domain (RBD) of SARS-CoV-2 spike protein, mAb 47D1 binds only to one side of the receptor binding surface on the RBD. Neutralization by 47D1 is achieved independent of interfering RBD-ACE2 binding. A crystal structure of the mAb-RBD complex shows that the IF motif at the tip of 47D1 CDR H2 interacts with a hydrophobic pocket in the RBD. Diverse immunoglobulin gene usage and convergent epitope targeting characterize neutralizing antibody responses to SARS-CoV-2, suggesting that vaccines that effectively present the receptor binding site on the RBD will likely elicit neutralizing antibody responses in a large fraction of the population.


Subject(s)
Antibodies, Neutralizing/genetics , COVID-19/genetics , Immunoglobulins/genetics , Adult , Antibodies, Monoclonal/immunology , Antibodies, Neutralizing/immunology , Antibodies, Viral/immunology , Binding Sites/immunology , COVID-19/immunology , COVID-19/therapy , Epitopes/genetics , Epitopes/immunology , Female , Genes, Immunoglobulin/genetics , Genetic Variation/genetics , Humans , Immunization, Passive/methods , Immunoglobulins/immunology , Male , Middle Aged , Peptidyl-Dipeptidase A/metabolism , Protein Binding/immunology , Protein Domains/genetics , Receptors, Virus/immunology , Receptors, Virus/metabolism , SARS-CoV-2/genetics , SARS-CoV-2/immunology , SARS-CoV-2/pathogenicity
15.
J Clin Invest ; 131(7)2021 04 01.
Article in English | MEDLINE | ID: covidwho-1166659

ABSTRACT

Multiple studies have shown loss of severe acute respiratory syndrome coronavirus 2-specific (SARS-CoV-2-specific) antibodies over time after infection, raising concern that humoral immunity against the virus is not durable. If immunity wanes quickly, millions of people may be at risk for reinfection after recovery from coronavirus disease 2019 (COVID-19). However, memory B cells (MBCs) could provide durable humoral immunity even if serum neutralizing antibody titers decline. We performed multidimensional flow cytometric analysis of S protein receptor binding domain-specific (S-RBD-specific) MBCs in cohorts of ambulatory patients with COVID-19 with mild disease (n = 7), and hospitalized patients with moderate to severe disease (n = 7), at a median of 54 days (range, 39-104 days) after symptom onset. We detected S-RBD-specific class-switched MBCs in 13 of 14 participants, failing only in the individual with the lowest plasma levels of anti-S-RBD IgG and neutralizing antibodies. Resting MBCs (rMBCs) made up the largest proportion of S-RBD-specific MBCs in both cohorts. FCRL5, a marker of functional memory on rMBCs, was more dramatically upregulated on S-RBD-specific rMBCs after mild infection than after severe infection. These data indicate that most SARS-CoV-2-infected individuals develop S-RBD-specific, class-switched rMBCs that resemble germinal center-derived B cells induced by effective vaccination against other pathogens, providing evidence for durable B cell-mediated immunity against SARS-CoV-2 after mild or severe disease.


Subject(s)
B-Lymphocytes/immunology , COVID-19/immunology , SARS-CoV-2 , Adult , Aged , Antibodies, Neutralizing/blood , Antibodies, Viral/blood , Binding Sites/immunology , COVID-19/virology , Case-Control Studies , Cohort Studies , Female , Humans , Immunity, Cellular , Immunoglobulin Class Switching , Immunologic Memory , Male , Middle Aged , Pandemics , SARS-CoV-2/immunology , Severity of Illness Index , Spike Glycoprotein, Coronavirus/chemistry , Spike Glycoprotein, Coronavirus/immunology , Time Factors
16.
Biochem Biophys Res Commun ; 538: 192-203, 2021 01 29.
Article in English | MEDLINE | ID: covidwho-1125111

ABSTRACT

Immediately from the outset of the COVID-19 pandemic, researchers from diverse biomedical and biological disciplines have united to study the novel pandemic virus, SARS-CoV-2. The antibody response to SARS-CoV-2 has been a major focus of COVID-19 research due to its clinical relevance and importance in vaccine and therapeutic development. Isolation and characterization of antibodies to SARS-CoV-2 have been accumulating at an unprecedented pace. Most of the SARS-CoV-2 neutralizing antibodies to date target the spike (S) protein receptor binding domain (RBD), which engages the host receptor ACE2 for viral entry. Here we review the binding sites and molecular features of monoclonal antibodies that target the SARS-CoV-2 RBD, including a few that also cross-neutralize SARS-CoV.


Subject(s)
Angiotensin-Converting Enzyme 2/immunology , Antibodies, Neutralizing/immunology , Antibodies, Viral/immunology , Receptors, Virus/immunology , SARS-CoV-2/immunology , Spike Glycoprotein, Coronavirus/immunology , Angiotensin-Converting Enzyme 2/chemistry , Binding Sites/immunology , Humans , Protein Binding/immunology , Protein Domains/immunology , Receptors, Virus/chemistry
17.
mSphere ; 6(1)2021 02 24.
Article in English | MEDLINE | ID: covidwho-1102156

ABSTRACT

The majority of infections with SARS-CoV-2 are asymptomatic or mild without the necessity of hospitalization. It is of importance to reveal if these patients develop an antibody response against SARS-CoV-2 and to define which antibodies confer virus neutralization. We conducted a comprehensive serological survey of 49 patients with a mild course of disease and quantified neutralizing antibody responses against a clinical SARS-CoV-2 isolate employing human cells as targets. Four patients (8%), even though symptomatic, did not develop antibodies against SARS-CoV-2, and two other patients (4%) were positive in only one of the six serological assays employed. For the remaining 88%, antibody response against the S protein correlated with serum neutralization whereas antibodies against the nucleocapsid were poor predictors of virus neutralization. None of the sera enhanced infection of human cells with SARS-CoV-2 at any dilution, arguing against antibody-dependent enhancement of infection in our system. Regarding neutralization, only six patients (12%) could be classified as high neutralizers. Furthermore, sera from several individuals with fairly high antibody levels had only poor neutralizing activity. In addition, employing a novel serological Western blot system to characterize antibody responses against seasonal coronaviruses, we found that antibodies against the seasonal coronavirus 229E might contribute to SARS-CoV-2 neutralization. Altogether, we show that there is a wide breadth of antibody responses against SARS-CoV-2 in patients that differentially correlate with virus neutralization. This highlights the difficulty to define reliable surrogate markers for immunity against SARS-CoV-2.IMPORTANCE There is strong interest in the nature of the neutralizing antibody response against SARS-CoV-2 in infected individuals. For vaccine development, it is especially important which antibodies confer protection against SARS-CoV-2, if there is a phenomenon called antibody-dependent enhancement (ADE) of infection, and if there is cross-protection by antibodies directed against seasonal coronaviruses. We addressed these questions and found in accordance with other studies that neutralization is mediated mainly by antibodies directed against the spike protein of SARS-CoV-2 in general and the receptor binding site in particular. In our test system, utilizing human cells for infection experiments, we did not detect ADE. However, using a novel diagnostic test we found that antibodies against the coronavirus 229E might be involved in cross-protection to SARS-CoV-2.


Subject(s)
Antibodies, Viral/immunology , Antibody Formation/immunology , COVID-19/immunology , Coronavirus Infections/immunology , SARS-CoV-2/immunology , Adult , Antibodies, Neutralizing/immunology , Antibody-Dependent Enhancement/immunology , Binding Sites/immunology , Female , Hospitalization , Humans , Male , Neutralization Tests/methods , Nucleocapsid/immunology , Seasons , Serologic Tests/methods , Spike Glycoprotein, Coronavirus/immunology , Surveys and Questionnaires , Vaccines/immunology
18.
Nat Commun ; 12(1): 844, 2021 02 08.
Article in English | MEDLINE | ID: covidwho-1069105

ABSTRACT

There are only few data concerning persistence of neutralizing antibodies (NAbs) among SARS-CoV-2-infected healthcare workers (HCW). These individuals are particularly exposed to SARS-CoV-2 infection and at potential risk of reinfection. We followed 26 HCW with mild COVID-19 three weeks (D21), two months (M2) and three months (M3) after the onset of symptoms. All the HCW had anti-receptor binding domain (RBD) IgA at D21, decreasing to 38.5% at M3 (p < 0.0001). Concomitantly a significant decrease in NAb titers was observed between D21 and M2 (p = 0.03) and between D21 and M3 (p < 0.0001). Here, we report that SARS-CoV-2 can elicit a NAb response correlated with anti-RBD antibody levels. However, this neutralizing activity declines, and may even be lost, in association with a decrease in systemic IgA antibody levels, from two months after disease onset. This short-lasting humoral protection supports strong recommendations to maintain infection prevention and control measures in HCW, and suggests that periodic boosts of SARS-CoV-2 vaccination may be required.


Subject(s)
Antibodies, Neutralizing/immunology , Antibodies, Viral/immunology , COVID-19/immunology , Health Personnel/statistics & numerical data , SARS-CoV-2/immunology , Adult , Binding Sites/immunology , COVID-19/virology , Cell Line, Tumor , Female , Humans , Immunoglobulin A/immunology , Male , Middle Aged , Protein Binding , Receptors, Virus/metabolism , SARS-CoV-2/physiology , Spike Glycoprotein, Coronavirus/immunology , Spike Glycoprotein, Coronavirus/metabolism , Time Factors
19.
J Virol Methods ; 290: 114084, 2021 04.
Article in English | MEDLINE | ID: covidwho-1065422

ABSTRACT

The use of monoclonal neutralizing antibodies (mNAbs) is being actively pursued as a viable intervention for the treatment of Severe Acute Respiratory Syndrome CoV-2 (SARS-CoV-2) infection and associated coronavirus disease 2019 (COVID-19). While highly potent mNAbs have great therapeutic potential, the ability of the virus to mutate and escape recognition and neutralization of mNAbs represents a potential problem in their use for the therapeutic management of SARS-CoV-2. Studies investigating natural or mNAb-induced antigenic variability in the receptor binding domain (RBD) of SARS-CoV-2 Spike (S) glycoprotein, and their effects on viral fitness are still rudimentary. In this manuscript we described experimental approaches for the selection, identification, and characterization of SARS-CoV-2 monoclonal antibody resistant mutants (MARMs) in cultured cells. The ability to study SARS-CoV-2 antigenic drift under selective immune pressure by mNAbs is important for the optimal implementation of mNAbs for the therapeutic management of COVID-19. This will help to identify essential amino acid residues in the viral S glycoprotein required for mNAb-mediated inhibition of viral infection, to predict potential natural drift variants that could emerge upon implementation of therapeutic mNAbs, as well as vaccine prophylactic treatments for SARS-CoV-2 infection. Additionally, it will also enable the assessment of MARM viral fitness and its potential to induce severe infection and associated COVID-19 disease.


Subject(s)
Antibodies, Monoclonal/pharmacology , Antigenic Variation/genetics , Drug Resistance, Viral/genetics , SARS-CoV-2/genetics , Selection, Genetic , Animals , Antibodies, Monoclonal/immunology , Antibodies, Monoclonal/therapeutic use , Antibodies, Neutralizing/immunology , Antibodies, Neutralizing/pharmacology , Antibodies, Neutralizing/therapeutic use , Binding Sites/genetics , Binding Sites/immunology , COVID-19/drug therapy , COVID-19/virology , Chlorocebus aethiops , Humans , Phenotype , SARS-CoV-2/drug effects , SARS-CoV-2/immunology , Spike Glycoprotein, Coronavirus/genetics , Spike Glycoprotein, Coronavirus/immunology , Vero Cells
20.
Nature ; 588(7839): 682-687, 2020 12.
Article in English | MEDLINE | ID: covidwho-851300

ABSTRACT

The coronavirus disease 2019 (COVID-19) pandemic presents an urgent health crisis. Human neutralizing antibodies that target the host ACE2 receptor-binding domain (RBD) of the severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) spike protein1-5 show promise therapeutically and are being evaluated clinically6-8. Here, to identify the structural correlates of SARS-CoV-2 neutralization, we solved eight new structures of distinct COVID-19 human neutralizing antibodies5 in complex with the SARS-CoV-2 spike trimer or RBD. Structural comparisons allowed us to classify the antibodies into categories: (1) neutralizing antibodies encoded by the VH3-53 gene segment with short CDRH3 loops that block ACE2 and bind only to 'up' RBDs; (2) ACE2-blocking neutralizing antibodies that bind both up and 'down' RBDs and can contact adjacent RBDs; (3) neutralizing antibodies that bind outside the ACE2 site and recognize both up and down RBDs; and (4) previously described antibodies that do not block ACE2 and bind only to up RBDs9. Class 2 contained four neutralizing antibodies with epitopes that bridged RBDs, including a VH3-53 antibody that used a long CDRH3 with a hydrophobic tip to bridge between adjacent down RBDs, thereby locking the spike into a closed conformation. Epitope and paratope mapping revealed few interactions with host-derived N-glycans and minor contributions of antibody somatic hypermutations to epitope contacts. Affinity measurements and mapping of naturally occurring and in vitro-selected spike mutants in 3D provided insight into the potential for SARS-CoV-2 to escape from antibodies elicited during infection or delivered therapeutically. These classifications and structural analyses provide rules for assigning current and future human RBD-targeting antibodies into classes, evaluating avidity effects and suggesting combinations for clinical use, and provide insight into immune responses against SARS-CoV-2.


Subject(s)
Antibodies, Neutralizing/therapeutic use , Antibodies, Neutralizing/ultrastructure , COVID-19/drug therapy , COVID-19/immunology , SARS-CoV-2/immunology , Angiotensin-Converting Enzyme 2/chemistry , Angiotensin-Converting Enzyme 2/metabolism , Angiotensin-Converting Enzyme 2/ultrastructure , Antibodies, Neutralizing/chemistry , Antibodies, Neutralizing/immunology , Binding Sites/genetics , Binding Sites/immunology , Cell Line , Cryoelectron Microscopy , Humans , Models, Molecular , Mutation , Receptors, Coronavirus/chemistry , Receptors, Coronavirus/metabolism , Receptors, Coronavirus/ultrastructure , SARS-CoV-2/chemistry , SARS-CoV-2/metabolism , SARS-CoV-2/ultrastructure , Spike Glycoprotein, Coronavirus/chemistry , Spike Glycoprotein, Coronavirus/genetics , Spike Glycoprotein, Coronavirus/immunology , Spike Glycoprotein, Coronavirus/ultrastructure
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