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1.
PLoS Comput Biol ; 17(12): e1009675, 2021 12.
Article in English | MEDLINE | ID: covidwho-1619980

ABSTRACT

Identifying the epitope of an antibody is a key step in understanding its function and its potential as a therapeutic. Sequence-based clonal clustering can identify antibodies with similar epitope complementarity, however, antibodies from markedly different lineages but with similar structures can engage the same epitope. We describe a novel computational method for epitope profiling based on structural modelling and clustering. Using the method, we demonstrate that sequence dissimilar but functionally similar antibodies can be found across the Coronavirus Antibody Database, with high accuracy (92% of antibodies in multiple-occupancy structural clusters bind to consistent domains). Our approach functionally links antibodies with distinct genetic lineages, species origins, and coronavirus specificities. This indicates greater convergence exists in the immune responses to coronaviruses than is suggested by sequence-based approaches. Our results show that applying structural analytics to large class-specific antibody databases will enable high confidence structure-function relationships to be drawn, yielding new opportunities to identify functional convergence hitherto missed by sequence-only analysis.


Subject(s)
Antigens, Viral/chemistry , COVID-19/immunology , COVID-19/virology , Epitopes, B-Lymphocyte/chemistry , SARS-CoV-2/chemistry , SARS-CoV-2/immunology , Amino Acid Sequence , Animals , Antibodies, Neutralizing/chemistry , Antibodies, Neutralizing/genetics , Antibodies, Viral/chemistry , Antibodies, Viral/genetics , Antibodies, Viral/metabolism , Antibody Specificity , Antigen-Antibody Complex/chemistry , Antigen-Antibody Complex/genetics , Antigen-Antibody Reactions/genetics , Antigen-Antibody Reactions/immunology , Computational Biology , Coronavirus/chemistry , Coronavirus/genetics , Coronavirus/immunology , Databases, Chemical , Epitope Mapping , Epitopes, B-Lymphocyte/genetics , Humans , Mice , Models, Molecular , Pandemics , SARS-CoV-2/genetics , Single-Domain Antibodies/immunology
2.
MAbs ; 14(1): 2002236, 2022.
Article in English | MEDLINE | ID: covidwho-1585298

ABSTRACT

Coronavirus disease 2019 (COVID-19) is an evolving global public health crisis in need of therapeutic options. Passive immunization of monoclonal antibodies (mAbs) represents a promising therapeutic strategy capable of conferring immediate protection from SARS-CoV-2 infection. Herein, we describe the discovery and characterization of neutralizing SARS-CoV-2 IgG and VHH antibodies from four large-scale phage libraries. Each library was constructed synthetically with shuffled complementarity-determining region loops from natural llama and human antibody repertoires. While most candidates targeted the receptor-binding domain of the S1 subunit of SARS-CoV-2 spike protein, we also identified a neutralizing IgG candidate that binds a unique epitope on the N-terminal domain. A select number of antibodies retained binding to SARS-CoV-2 variants Alpha, Beta, Gamma, Kappa and Delta. Overall, our data show that synthetic phage libraries can rapidly yield SARS-CoV-2 S1 antibodies with therapeutically desirable features, including high affinity, unique binding sites, and potent neutralizing activity in vitro, and a capacity to limit disease in vivo.


Subject(s)
Antibodies, Neutralizing/immunology , Antibodies, Viral/immunology , COVID-19/immunology , Cell Surface Display Techniques , Immunoglobulin G/immunology , Peptide Library , SARS-CoV-2/immunology , Single-Domain Antibodies/immunology , Spike Glycoprotein, Coronavirus/immunology , Animals , Antibodies, Neutralizing/genetics , Antibodies, Neutralizing/metabolism , Antibodies, Neutralizing/pharmacology , Antibodies, Viral/genetics , Antibodies, Viral/metabolism , Antibody Specificity , Binding Sites, Antibody , COVID-19/metabolism , COVID-19/prevention & control , COVID-19/virology , Chlorocebus aethiops , Disease Models, Animal , Epitopes , Female , Host-Pathogen Interactions , Immunoglobulin G/genetics , Immunoglobulin G/metabolism , Immunoglobulin G/pharmacology , Mesocricetus , SARS-CoV-2/pathogenicity , Single-Domain Antibodies/genetics , Single-Domain Antibodies/metabolism , Single-Domain Antibodies/pharmacology , Vero Cells
3.
Rev Med Virol ; 31(6): e2231, 2021 11.
Article in English | MEDLINE | ID: covidwho-1574317

ABSTRACT

The Spike protein is the target of both antibody-based therapeutics (convalescent plasma, polyclonal serum, monoclonal antibodies) and vaccines. Mutations in Spike could affect efficacy of those treatments. Hence, monitoring of mutations is necessary to forecast and readapt the inventory of therapeutics. Different phylogenetic nomenclatures have been used for the currently circulating SARS-CoV-2 clades. The Spike protein has different hotspots of mutation and deletion, the most dangerous for immune escape being the ones within the receptor binding domain (RBD), such as K417N/T, N439K, L452R, Y453F, S477N, E484K, and N501Y. Convergent evolution has led to different combinations of mutations among different clades. In this review we focus on the main variants of concern, that is, the so-called UK (B.1.1.7), South African (B.1.351) and Brazilian (P.1) strains.


Subject(s)
Antibodies, Monoclonal/therapeutic use , Antibodies, Neutralizing/therapeutic use , COVID-19/therapy , SARS-CoV-2/genetics , Spike Glycoprotein, Coronavirus/genetics , Antibodies, Monoclonal/chemistry , Antibodies, Monoclonal/metabolism , Antibodies, Neutralizing/chemistry , Antibodies, Neutralizing/metabolism , Antibodies, Viral/chemistry , Antibodies, Viral/metabolism , Antibodies, Viral/therapeutic use , Brazil/epidemiology , COVID-19/epidemiology , COVID-19/immunology , COVID-19/virology , COVID-19 Vaccines/administration & dosage , Gene Expression , Humans , Immune Evasion , Immunization, Passive/methods , Mutation , Phylogeny , Protein Binding , Risk Assessment , SARS-CoV-2/classification , SARS-CoV-2/drug effects , SARS-CoV-2/immunology , South Africa/epidemiology , Spike Glycoprotein, Coronavirus/immunology , United Kingdom/epidemiology
4.
Front Immunol ; 12: 793953, 2021.
Article in English | MEDLINE | ID: covidwho-1572289

ABSTRACT

Durability of SARS-CoV-2 Spike antibody responses after infection provides information relevant to understanding protection against COVID-19 in humans. We report the results of a sequential evaluation of anti-SARS-CoV-2 antibodies in convalescent patients with a median follow-up of 14 months (range 12.4-15.4) post first symptom onset. We report persistence of antibodies for all four specificities tested [Spike, Spike Receptor Binding Domain (Spike-RBD), Nucleocapsid, Nucleocapsid RNA Binding Domain (N-RBD)]. Anti-Spike antibodies persist better than anti-Nucleocapsid antibodies. The durability analysis supports a bi-phasic antibody decay with longer half-lives of antibodies after 6 months and antibody persistence for up to 14 months. Patients infected with the Wuhan (WA1) strain maintained strong cross-reactive recognition of Alpha and Delta Spike-RBD but significantly reduced binding to Beta and Mu Spike-RBD. Sixty percent of convalescent patients with detectable WA1-specific NAb also showed strong neutralization of the Delta variant, the prevalent strain of the present pandemic. These data show that convalescent patients maintain functional antibody responses for more than one year after infection, suggesting a strong long-lasting response after symptomatic disease that may offer a prolonged protection against re-infection. One patient from this cohort showed strong increase of both Spike and Nucleocapsid antibodies at 14 months post-infection indicating SARS-CoV-2 re-exposure. These antibodies showed stronger cross-reactivity to a panel of Spike-RBD including Beta, Delta and Mu and neutralization of a panel of Spike variants including Beta and Gamma. This patient provides an example of strong anti-Spike recall immunity able to control infection at an asymptomatic level. Together, the antibodies from SARS-CoV-2 convalescent patients persist over 14 months and continue to maintain cross-reactivity to the current variants of concern and show strong functional properties.


Subject(s)
Antibodies, Neutralizing/immunology , Antibodies, Viral/immunology , COVID-19/immunology , SARS-CoV-2/immunology , Spike Glycoprotein, Coronavirus/immunology , Adult , Aged , Antibodies, Neutralizing/metabolism , Antibodies, Viral/metabolism , Binding Sites, Antibody/immunology , COVID-19/virology , Cohort Studies , Cross Reactions/immunology , Female , Humans , Male , Middle Aged , Neutralization Tests/methods , Nucleocapsid/immunology , Nucleocapsid/metabolism , Protein Binding/immunology , SARS-CoV-2/physiology , Spike Glycoprotein, Coronavirus/metabolism , Time Factors
5.
Science ; 373(6556)2021 Aug 13.
Article in English | MEDLINE | ID: covidwho-1559379

ABSTRACT

The emergence of highly transmissible SARS-CoV-2 variants of concern (VOCs) that are resistant to therapeutic antibodies highlights the need for continuing discovery of broadly reactive antibodies. We identified four receptor binding domain-targeting antibodies from three early-outbreak convalescent donors with potent neutralizing activity against 23 variants, including the B.1.1.7, B.1.351, P.1, B.1.429, B.1.526, and B.1.617 VOCs. Two antibodies are ultrapotent, with subnanomolar neutralization titers [half-maximal inhibitory concentration (IC50) 0.3 to 11.1 nanograms per milliliter; IC80 1.5 to 34.5 nanograms per milliliter). We define the structural and functional determinants of binding for all four VOC-targeting antibodies and show that combinations of two antibodies decrease the in vitro generation of escape mutants, suggesting their potential in mitigating resistance development.


Subject(s)
Antibodies, Neutralizing/immunology , Antibodies, Viral/immunology , COVID-19/immunology , SARS-CoV-2/immunology , SARS-CoV-2/pathogenicity , Spike Glycoprotein, Coronavirus/immunology , Angiotensin-Converting Enzyme 2/antagonists & inhibitors , Angiotensin-Converting Enzyme 2/metabolism , Antibodies, Neutralizing/chemistry , Antibodies, Neutralizing/metabolism , Antibodies, Viral/chemistry , Antibodies, Viral/metabolism , Antibody Affinity , Antigen-Antibody Reactions , COVID-19/virology , Humans , Immune Evasion , Immunoglobulin Fab Fragments/immunology , Immunoglobulin Fab Fragments/metabolism , Mutation , Neutralization Tests , Protein Domains , Receptors, Coronavirus/antagonists & inhibitors , Receptors, Coronavirus/metabolism , SARS-CoV-2/genetics , Spike Glycoprotein, Coronavirus/chemistry , Spike Glycoprotein, Coronavirus/genetics , Spike Glycoprotein, Coronavirus/metabolism
6.
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
8.
J Immunol ; 207(10): 2405-2410, 2021 11 15.
Article in English | MEDLINE | ID: covidwho-1471046

ABSTRACT

Severe acute respiratory syndrome corona virus 2 (SARS-CoV-2) causes severe acute respiratory syndrome. mRNA vaccines directed at the SARS-CoV-2 spike protein resulted in development of Abs and protective immunity. To determine the mechanism, we analyzed the kinetics of induction of circulating exosomes with SARS-CoV-2 spike protein and Ab following vaccination of healthy individuals. Results demonstrated induction of circulating exosomes expressing spike protein on day 14 after vaccination followed by Abs 14 d after the second dose. Exosomes with spike protein, Abs to SARS-CoV-2 spike, and T cells secreting IFN-γ and TNF-α increased following the booster dose. Transmission electron microscopy of exosomes also demonstrated spike protein Ags on their surface. Exosomes with spike protein and Abs decreased in parallel after four months. These results demonstrate an important role of circulating exosomes with spike protein for effective immunization following mRNA-based vaccination. This is further documented by induction of humoral and cellular immune responses in mice immunized with exosomes carrying spike protein.


Subject(s)
Antibodies, Viral/metabolism , COVID-19 Vaccines/immunology , COVID-19/immunology , Exosomes/metabolism , SARS-CoV-2/physiology , Spike Glycoprotein, Coronavirus/metabolism , T-Lymphocytes/metabolism , Animals , Blood Circulation , Cells, Cultured , Exosomes/immunology , Healthy Volunteers , Humans , Immunization , Interferon-gamma/metabolism , Mice , Mice, Inbred C57BL , Spike Glycoprotein, Coronavirus/immunology , T-Lymphocytes/immunology , Tumor Necrosis Factor-alpha/metabolism , Vaccination
9.
Front Immunol ; 12: 757691, 2021.
Article in English | MEDLINE | ID: covidwho-1463478

ABSTRACT

The increase in confirmed COVID-19 cases and SARS-CoV-2 variants calls for the development of safe and broad cross-protective vaccines. The RBD of the spike protein was considered to be a safe and effective candidate antigen. However, the low immunogenicity limited its application in vaccine development. Herein, we designed and obtained an RBD heptamer (mHla-RBD) based on a carrier protein-aided assembly strategy. The molecular weight of mHla-RBD is up to 450 kDa, approximately 10 times higher than that of the RBD monomer. When formulated with alum adjuvant, mHla-RBD immunization significantly increased the immunogenicity of RBD, as indicated by increased titers of RBD-specific antibodies, neutralizing antibodies, Th2 cellular immune response, and pseudovirus neutralization activity, when compared to RBD monomer. Furthermore, we confirmed that RBD-specific antibodies predominantly target conformational epitopes, which was approximately 200 times that targeting linear epitopes. Finally, a pseudovirus neutralization assay revealed that neutralizing antibodies induced by mHla-RBD against different SARS-CoV-2 variants were comparable to those against the wild-type virus and showed broad-spectrum neutralizing activity toward different SARS-CoV-2 variants. Our results demonstrated that mHla-RBD is a promising candidate antigen for development of SARS-CoV-2 vaccines and the mHla could serve as a universal carrier protein for antigen design.


Subject(s)
Bacterial Proteins/metabolism , COVID-19 Vaccines/immunology , COVID-19/immunology , Carrier Proteins/metabolism , Hemolysin Proteins/metabolism , SARS-CoV-2/physiology , Spike Glycoprotein, Coronavirus/metabolism , Th2 Cells/immunology , Animals , Antibodies, Neutralizing/metabolism , Antibodies, Viral/metabolism , Broadly Neutralizing Antibodies/metabolism , Cell Line , Escherichia coli Proteins , Humans , Lymphocyte Activation , Mice , Mice, Inbred BALB C , Protein Domains/genetics , Spike Glycoprotein, Coronavirus/genetics , Spike Glycoprotein, Coronavirus/immunology
10.
Front Immunol ; 12: 751584, 2021.
Article in English | MEDLINE | ID: covidwho-1463475

ABSTRACT

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused a global pandemic of novel coronavirus disease (COVID-19). Though vaccines and neutralizing monoclonal antibodies (mAbs) have been developed to fight COVID-19 in the past year, one major concern is the emergence of SARS-CoV-2 variants of concern (VOCs). Indeed, SARS-CoV-2 VOCs such as B.1.1.7 (UK), B.1.351 (South Africa), P.1 (Brazil), and B.1.617.1 (India) now dominate the pandemic. Herein, we found that binding activity and neutralizing capacity of sera collected from convalescent patients in early 2020 for SARS-CoV-2 VOCs, but not non-VOC variants, were severely blunted. Furthermore, we observed evasion of SARS-CoV-2 VOCs from a VH3-30 mAb 32D4, which was proved to exhibit highly potential neutralization against wild-type (WT) SARS-CoV-2. Thus, these results indicated that SARS-CoV-2 VOCs might be able to spread in convalescent patients and even harbor resistance to medical countermeasures. New interventions against these SARS-CoV-2 VOCs are urgently needed.


Subject(s)
COVID-19/immunology , Mutation/genetics , SARS-CoV-2/physiology , Adult , Antibodies, Monoclonal/metabolism , Antibodies, Neutralizing/metabolism , Antibodies, Viral/metabolism , COVID-19/genetics , COVID-19/therapy , Female , Humans , Immune Evasion , Immunization, Passive , Male , Middle Aged , Spike Glycoprotein, Coronavirus/immunology , Spike Glycoprotein, Coronavirus/metabolism
11.
Front Immunol ; 12: 730766, 2021.
Article in English | MEDLINE | ID: covidwho-1463473

ABSTRACT

The SARS-CoV-2 pandemic has spread to all parts of the world and can cause life-threatening pneumonia and other severe disease manifestations known as COVID-19. This health crisis has resulted in a significant effort to stop the spread of this new coronavirus. However, while propagating itself in the human population, the virus accumulates mutations and generates new variants with increased fitness and the ability to escape the human immune response. Here we describe a color-based barcoded spike flow cytometric assay (BSFA) that is particularly useful to evaluate and directly compare the humoral immune response directed against either wild type (WT) or mutant spike (S) proteins or the receptor-binding domains (RBD) of SARS-CoV-2. This assay employs the human B lymphoma cell line Ramos, transfected for stable expression of WT or mutant S proteins or a chimeric RBD-CD8 fusion protein. We find that the alpha and beta mutants are more stably expressed than the WT S protein on the Ramos B cell surface and/or bind with higher affinity to the viral entry receptor ACE2. However, we find a reduce expression of the chimeric RBD-CD8 carrying the point mutation N501Y and E484K characteristic for the alpha and beta variant, respectively. The comparison of the humoral immune response of 12 vaccinated probands with 12 COVID-19 patients shows that after the boost, the S-specific IgG class immune response in the vaccinated group is similar to that of the patient group. However, in comparison to WT the specific IgG serum antibodies bind less well to the alpha variant and only poorly to the beta variant S protein. This is in line with the notion that the beta variant is an immune escape variant of SARS-CoV-2. The IgA class immune response was more variable than the IgG response and higher in the COVID-19 patients than in the vaccinated group. In summary, we think that our BSFA represents a useful tool to evaluate the humoral immunity against emerging variants of SARS-CoV-2 and to analyze new vaccination protocols against these variants.


Subject(s)
COVID-19/immunology , Cell Separation/methods , Flow Cytometry/methods , SARS-CoV-2/physiology , Spike Glycoprotein, Coronavirus/metabolism , Angiotensin-Converting Enzyme 2/metabolism , Antibodies, Viral/metabolism , Antibody Formation , Female , Humans , Immunization, Secondary , Immunoglobulin A/metabolism , Immunoglobulin G/metabolism , Male , Middle Aged , Mutation/genetics , Spike Glycoprotein, Coronavirus/genetics , Vaccination
12.
Front Immunol ; 12: 730404, 2021.
Article in English | MEDLINE | ID: covidwho-1459289

ABSTRACT

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virus causes a spectrum of clinical manifestations, ranging from asymptomatic to mild, moderate, or severe illness with multi-organ failure and death. Using a new machine learning algorithm developed by us, we have reported a significantly higher number of predicted COVID-19 cases than the documented counts across the world. The sole reliance on confirmed symptomatic cases overlooking the symptomless COVID-19 infections and the dynamics of waning immunity may not provide 'true' spectrum of infection proportion, a key element for an effective planning and implementation of protection and prevention strategies. We and others have previously shown that strategic orthogonal testing and leveraging systematic data-driven modeling approach to account for asymptomatics and waning cases may situationally have a compelling role in informing efficient vaccination strategies beyond prevalence reporting. However, currently Centers for Disease Control and Prevention (CDC) does not recommend serological testing either before or after vaccination to assess immune status. Given the 27% occurrence of breakthrough infections in fully vaccinated (FV) group with many being asymptomatics and still a larger fraction of the general mass remaining unvaccinated, the relaxed mask mandate and distancing by CDC can drive resurgence. Thus, we believe it is a key time to focus on asymptomatics (no symptoms) and oligosymptomatics (so mild that the symptoms remain unrecognized) as they can be silent reservoirs to propagate the infection. This perspective thus highlights the need for proactive efforts to reevaluate the current variables/strategies in accounting for symptomless and waning fractions.


Subject(s)
COVID-19 Vaccines/immunology , COVID-19/immunology , SARS-CoV-2/physiology , Antibodies, Neutralizing/metabolism , Antibodies, Viral/metabolism , Asymptomatic Diseases , COVID-19/transmission , COVID-19 Serological Testing , Centers for Disease Control and Prevention, U.S. , Humans , Immunity , United States , Vaccination
13.
Front Immunol ; 12: 707977, 2021.
Article in English | MEDLINE | ID: covidwho-1457901

ABSTRACT

The ongoing COVID-19 pandemic caused by SARS-CoV-2 is a huge public health crisis for the globe. The receptor-binding domain (RBD) of SARS-CoV-2 spike (S) protein plays a vital role in viral infection and serves as a major target for developing neutralizing antibodies. In this study, the antibody response to the RBD of SARS-CoV-2 S protein was analyzed by a panel of sera from animals immunized with RBD-based antigens and four linear B-cell epitope peptides (R345, R405, R450 and R465) were revealed. The immunogenicity of three immunodominant peptides (R345, R405, R465) was further accessed by peptide immunization in mice, and all of them could induced potent antibody response to SARS-CoV-2 S protein, indicating that the three determinants in the RBD were immunogenic. We further generated and characterized monoclonal antibodies (15G9, 12C10 and 10D2) binding to these epitope peptides, and finely mapped the three immunodominant epitopes using the corresponding antibodies. Neutralization assays showed that all three monoclonal antibodies had neutralization activity. Results from IFA and western blotting showed that 12C10 was a cross-reactive antibody against both of SARS-CoV-2 and SARS-CoV. Results from conservative and structural analysis showed that 350VYAWN354 was a highly conserved epitope and exposed on the surface of SARS-CoV-2 S trimer, whereas 473YQAGSTP479 located in the receptor binding motif (RBM) was variable among different SARS-CoV-2 strains. 407VRQIAP412 was a highly conserved, but cryptic epitope shared between SARS-CoV-2 and SARS-CoV. These findings provide important information for understanding the humoral antibody response to the RBD of SARS-CoV-2 S protein and may facilitate further efforts to design SARS-CoV-2 vaccines and the target of COVID-19 diagnostic.


Subject(s)
B-Lymphocytes/immunology , Epitopes, B-Lymphocyte/metabolism , Peptides/metabolism , Spike Glycoprotein, Coronavirus/metabolism , Amino Acid Motifs/genetics , Antibodies, Monoclonal/metabolism , Antibodies, Neutralizing/metabolism , Antibodies, Viral/metabolism , COVID-19 Vaccines , Conserved Sequence/genetics , Epitope Mapping , Epitopes, B-Lymphocyte/genetics , HEK293 Cells , Humans , Immunity, Humoral , Peptides/genetics , Protein Binding , Spike Glycoprotein, Coronavirus/genetics
14.
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
15.
Immunity ; 54(9): 2159-2166.e6, 2021 09 14.
Article in English | MEDLINE | ID: covidwho-1454205

ABSTRACT

The emergence of SARS-CoV-2 antigenic variants with increased transmissibility is a public health threat. Some variants show substantial resistance to neutralization by SARS-CoV-2 infection- or vaccination-induced antibodies. Here, we analyzed receptor binding domain-binding monoclonal antibodies derived from SARS-CoV-2 mRNA vaccine-elicited germinal center B cells for neutralizing activity against the WA1/2020 D614G SARS-CoV-2 strain and variants of concern. Of five monoclonal antibodies that potently neutralized the WA1/2020 D614G strain, all retained neutralizing capacity against the B.1.617.2 variant, four also neutralized the B.1.1.7 variant, and only one, 2C08, also neutralized the B.1.351 and B.1.1.28 variants. 2C08 reduced lung viral load and morbidity in hamsters challenged with the WA1/2020 D614G, B.1.351, or B.1.617.2 strains. Clonal analysis identified 2C08-like public clonotypes among B cells responding to SARS-CoV-2 infection or vaccination in 41 out of 181 individuals. Thus, 2C08-like antibodies can be induced by SARS-CoV-2 vaccines and mitigate resistance by circulating variants of concern.


Subject(s)
Antibodies, Monoclonal/metabolism , Antibodies, Neutralizing/metabolism , Antibodies, Viral/metabolism , B-Lymphocytes/immunology , COVID-19 Vaccines/immunology , COVID-19/immunology , Germinal Center/immunology , Lung/virology , SARS-CoV-2/physiology , Animals , Cells, Cultured , Clone Cells , Cricetinae , Disease Models, Animal , Humans , Neutralization Tests , Spike Glycoprotein, Coronavirus/immunology , Vaccination , Viral Load
16.
mBio ; 12(5): e0251021, 2021 10 26.
Article in English | MEDLINE | ID: covidwho-1450587

ABSTRACT

The sensitivity of SARS-CoV-2 variants of concern (VOCs) to neutralizing antibodies has largely been studied in the context of key receptor binding domain (RBD) mutations, including E484K and N501Y. Little is known about the epistatic effects of combined SARS-CoV-2 spike mutations. We now investigate the neutralization sensitivity of variants containing the non-RBD mutation Q677H, including B.1.525 (Nigerian isolate) and Bluebird (U.S. isolate) variants. The effect on neutralization of Q677H was determined in the context of the RBD mutations and in the background of major VOCs, including B.1.1.7 (United Kingdom, Alpha), B.1.351 (South Africa, Beta), and P1-501Y-V3 (Brazil, Gamma). We demonstrate that the Q677H mutation increases viral infectivity and syncytium formation, as well as enhancing resistance to neutralization for VOCs, including B.1.1.7 and P1-501Y-V3. Our work highlights the importance of epistatic interactions between SARS-CoV-2 spike mutations and the continued need to monitor Q677H-bearing VOCs. IMPORTANCE SARS-CoV-2, the causative agent of COVID-19, is rapidly evolving to be more transmissible and to evade acquired immunity. To date, most investigations of SARS-CoV-2 variants have focused on RBD mutations. However, the impact of non-RBD mutations and their synergy with studied RBD mutations are poorly understood. Here, we examine the role of the non-RBD Q677H mutation arising in many SARS-CoV-2 lineages, including VOCs. We demonstrate that the Q677H mutation enhances viral infectivity and confers neutralizing antibody resistance, particularly in the background of other SARS-CoV-2 VOCs.


Subject(s)
Antibodies, Neutralizing/metabolism , COVID-19/metabolism , SARS-CoV-2/pathogenicity , Spike Glycoprotein, Coronavirus/immunology , Antibodies, Viral/metabolism , HEK293 Cells , Humans , Mutation , Protein Binding , SARS-CoV-2/metabolism , Spike Glycoprotein, Coronavirus/metabolism
17.
J Immunol ; 207(10): 2581-2588, 2021 11 15.
Article in English | MEDLINE | ID: covidwho-1450886

ABSTRACT

SARS-CoV-2 is a respiratory pathogen that can cause severe disease in at-risk populations but results in asymptomatic infections or a mild course of disease in the majority of cases. We report the identification of SARS-CoV-2-reactive B cells in human tonsillar tissue obtained from children who were negative for coronavirus disease 2019 prior to the pandemic and the generation of mAbs recognizing the SARS-CoV-2 Spike protein from these B cells. These Abs showed reduced binding to Spike proteins of SARS-CoV-2 variants and did not recognize Spike proteins of endemic coronaviruses, but subsets reacted with commensal microbiota and exhibited SARS-CoV-2-neutralizing potential. Our study demonstrates pre-existing SARS-CoV-2-reactive Abs in various B cell populations in the upper respiratory tract lymphoid tissue that may lead to the rapid engagement of the pathogen and contribute to prevent manifestations of symptomatic or severe disease.


Subject(s)
Adenoids/immunology , B-Lymphocyte Subsets/immunology , B-Lymphocytes/immunology , COVID-19/immunology , Mucous Membrane/immunology , Receptors, Antigen, B-Cell/genetics , Respiratory System/immunology , SARS-CoV-2/physiology , Antibodies, Viral/metabolism , Child , HEK293 Cells , Humans , Immunologic Memory , Lymphocyte Activation , Single-Cell Analysis , Spike Glycoprotein, Coronavirus/immunology , Transcriptome
18.
Clin Immunol ; 232: 108871, 2021 11.
Article in English | MEDLINE | ID: covidwho-1446516

ABSTRACT

Despite the burgeoning field of coronavirus disease-19 (COVID-19) research, the persistence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) neutralising antibodies remains unclear. This study validated two high-throughput immunological methods for use as surrogate live virus neutralisation assays and employed them to examine the half-life of SARS-CoV-2 neutralising antibodies in convalescent plasma donations made by 42 repeat donors between April and September 2020. SARS-CoV-2 neutralising antibody titres decreased over time but typically remained above the methods' diagnostic cut-offs. Using this longitudinal data, the average half-life of SARS-CoV-2 neutralising antibodies was determined to be 20.4 days. SARS-CoV-2 neutralising antibody titres appear to persist in the majority of donors for several months. Whether these titres confer protection against re-infection requires further study and is of particular relevance as COVID-19 vaccines become widely available.


Subject(s)
Antibodies, Neutralizing/metabolism , Antibodies, Viral/metabolism , COVID-19/metabolism , Adult , Antibodies, Neutralizing/immunology , Antibodies, Neutralizing/therapeutic use , Antibodies, Viral/immunology , Antibodies, Viral/therapeutic use , Blood Donors , COVID-19/immunology , COVID-19/therapy , Female , Half-Life , Humans , Immunization, Passive , Longitudinal Studies , Male , Middle Aged , Plasma/immunology , Plasma/metabolism , SARS-CoV-2/immunology , Young Adult
19.
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
20.
Nat Commun ; 12(1): 5621, 2021 09 23.
Article in English | MEDLINE | ID: covidwho-1437680

ABSTRACT

Although serological studies have shown that antibodies against SARS-CoV-2 play an important role in protection against (re)infection, the dynamics of mucosal antibodies during primary infection and their potential impact on viral load and the resolution of disease symptoms remain unclear. During the first pandemic wave, we assessed the longitudinal nasal antibody response in index cases with mild COVID-19 and their household contacts. Nasal and serum antibody responses were analysed for up to nine months. Higher nasal receptor binding domain and spike protein-specific antibody levels at study inclusion were associated with lower viral load. Older age was correlated with more frequent COVID-19 related symptoms. Receptor binding domain and spike protein-specific mucosal antibodies were associated with the resolution of systemic, but not respiratory symptoms. Finally, receptor binding domain and spike protein-specific mucosal antibodies remained elevated up to nine months after symptom onset.


Subject(s)
Antibodies, Neutralizing/analysis , Antibodies, Viral/analysis , COVID-19/diagnosis , Nasal Mucosa/metabolism , SARS-CoV-2/immunology , Adolescent , Adult , Antibodies, Neutralizing/immunology , Antibodies, Neutralizing/metabolism , Antibodies, Viral/immunology , Antibodies, Viral/metabolism , COVID-19/blood , COVID-19/immunology , COVID-19/virology , COVID-19 Serological Testing/statistics & numerical data , Child , Humans , Immunity, Mucosal , Longitudinal Studies , Male , Middle Aged , Nasal Mucosa/immunology , Nasal Mucosa/virology , Severity of Illness Index , Viral Load , Young Adult
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