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1.
J Virol ; 96(4): e0160021, 2022 02 23.
Article in English | MEDLINE | ID: covidwho-1759291

ABSTRACT

A comprehensive study of the B cell response against SARS-CoV-2 could be significant for understanding the immune response and developing therapeutical antibodies and vaccines. To define the dynamics and characteristics of the antibody repertoire following SARS-CoV-2 infection, we analyzed the mRNA transcripts of immunoglobulin heavy chain (IgH) repertoires of 24 peripheral blood samples collected between 3 and 111 days after symptom onset from 10 COVID-19 patients. Massive clonal expansion of naive B cells with limited somatic hypermutation (SHM) was observed in the second week after symptom onset. The proportion of low-SHM IgG clones strongly correlated with spike-specific IgG antibody titers, highlighting the significant activation of naive B cells in response to a novel virus infection. The antibody isotype switching landscape showed a transient IgA surge in the first week after symptom onset, followed by a sustained IgG elevation that lasted for at least 3 months. SARS-CoV-2 infection elicited poly-germ line reactive antibody responses. Interestingly, 17 different IGHV germ line genes recombined with IGHJ6 showed significant clonal expansion. By comparing the IgH repertoires that we sequenced with the 774 reported SARS-CoV-2-reactive monoclonal antibodies (MAbs), 13 shared spike-specific IgH clusters were found. These shared spike-specific IgH clusters are derived from the same lineage of several recently published neutralizing MAbs, including CC12.1, CC12.3, C102, REGN10977, and 4A8. Furthermore, identical spike-specific IgH sequences were found in different COVID-19 patients, suggesting a highly convergent antibody response to SARS-CoV-2. Our analysis based on sequencing antibody repertoires from different individuals revealed key signatures of the systemic B cell response induced by SARS-CoV-2 infection. IMPORTANCE Although the canonical delineation of serum antibody responses following SARS-CoV-2 infection has been well established, the dynamics of antibody repertoire at the mRNA transcriptional level has not been well understood, especially the correlation between serum antibody titers and the antibody mRNA transcripts. In this study, we analyzed the IgH transcripts and characterized the B cell clonal expansion and differentiation, isotype switching, and somatic hypermutation in COVID-19 patients. This study provided insights at the repertoire level for the B cell response after SARS-CoV-2 infection.


Subject(s)
Antibodies, Neutralizing/genetics , Antibodies, Viral/genetics , B-Lymphocytes/immunology , COVID-19/genetics , Immunoglobulin G/genetics , Receptors, Antigen, B-Cell/genetics , SARS-CoV-2/immunology , Antibodies, Neutralizing/immunology , Antibodies, Viral/immunology , COVID-19/immunology , Humans , Immunoglobulin G/immunology , Receptors, Antigen, B-Cell/immunology
2.
Nat Immunol ; 23(3): 423-430, 2022 03.
Article in English | MEDLINE | ID: covidwho-1713201

ABSTRACT

The global severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic requires effective therapies against coronavirus disease 2019 (COVID-19), and neutralizing antibodies are a promising therapy. A noncompeting pair of human neutralizing antibodies (B38 and H4) blocking SARS-CoV-2 binding to its receptor, ACE2, have been described previously. Here, we develop bsAb15, a bispecific monoclonal antibody (bsAb) based on B38 and H4. bsAb15 has greater neutralizing efficiency than these parental antibodies, results in less selective pressure and retains neutralizing ability to most SARS-CoV-2 variants of concern (with more potent neutralizing activity against the Delta variant). We also selected for escape mutants of the two parental mAbs, a mAb cocktail and bsAb15, demonstrating that bsAb15 can efficiently neutralize all single-mAb escape mutants. Furthermore, prophylactic and therapeutic application of bsAb15 reduced the viral titer in infected nonhuman primates and human ACE2 transgenic mice. Therefore, this bsAb is a feasible and effective strategy to treat and prevent severe COVID-19.


Subject(s)
Antibodies, Bispecific/immunology , Antibodies, Monoclonal/immunology , Antibodies, Viral/immunology , SARS-CoV-2/immunology , Animals , Antibodies, Bispecific/chemistry , Antibodies, Bispecific/genetics , Antibodies, Monoclonal/chemistry , Antibodies, Monoclonal/genetics , Antibodies, Neutralizing/genetics , Antibodies, Neutralizing/immunology , Antibodies, Viral/chemistry , Antibodies, Viral/genetics , COVID-19/immunology , COVID-19/pathology , COVID-19/prevention & control , COVID-19/virology , Cloning, Molecular , Disease Models, Animal , Dose-Response Relationship, Immunologic , Epitopes , Humans , Macaca mulatta , Mice , Neutralization Tests , Protein Engineering/methods , Structure-Activity Relationship
3.
J Clin Invest ; 132(4)2022 Feb 15.
Article in English | MEDLINE | ID: covidwho-1705312

ABSTRACT

Many SARS-CoV-2 neutralizing antibodies (nAbs) lose potency against variants of concern. In this study, we developed 2 strategies to produce mutation-resistant antibodies. First, a yeast library expressing mutant receptor binding domains (RBDs) of the spike protein was utilized to screen for potent nAbs that are least susceptible to viral escape. Among the candidate antibodies, P5-22 displayed ultrahigh potency for virus neutralization as well as an outstanding mutation resistance profile. Additionally, P14-44 and P15-16 were recognized as mutation-resistant antibodies with broad betacoronavirus neutralization properties. P15-16 has only 1 binding hotspot, which is K378 in the RBD of SARS-CoV-2. The crystal structure of the P5-22, P14-44, and RBD ternary complex clarified the unique mechanisms that underlie the excellent mutation resistance profiles of these antibodies. Secondly, polymeric IgG enhanced antibody avidity by eliminating P5-22's only hotspot, residue F486 in the RBD, thereby potently blocking cell entry by mutant viruses. Structural and functional analyses of antibodies screened using both potency assays and the yeast RBD library revealed rare, ultrapotent, mutation-resistant nAbs against SARS-CoV-2.


Subject(s)
Antibodies, Viral/immunology , Broadly Neutralizing Antibodies/immunology , COVID-19/immunology , COVID-19/virology , SARS-CoV-2/genetics , SARS-CoV-2/immunology , Animals , Antibodies, Neutralizing/blood , Antibodies, Neutralizing/genetics , Antibodies, Neutralizing/immunology , Antibodies, Viral/blood , Antibodies, Viral/genetics , Antibody Affinity , B-Lymphocytes/immunology , Binding Sites/genetics , Binding Sites/immunology , Broadly Neutralizing Antibodies/blood , Broadly Neutralizing Antibodies/genetics , COVID-19/therapy , Cloning, Molecular , Disease Models, Animal , Humans , Immunization, Passive , Immunoglobulin G/immunology , In Vitro Techniques , Lung/virology , Mice , Mice, Inbred BALB C , Mutation , Neutralization Tests , Receptors, Virus/immunology , Spike Glycoprotein, Coronavirus/genetics , Spike Glycoprotein, Coronavirus/immunology
5.
Lancet Microbe ; 2(12): e666-e675, 2021 12.
Article in English | MEDLINE | ID: covidwho-1683810

ABSTRACT

BACKGROUND: Among the most consequential unknowns of the devastating COVID-19 pandemic are the durability of immunity and time to likely reinfection. There are limited direct data on SARS-CoV-2 long-term immune responses and reinfection. The aim of this study is to use data on the durability of immunity among evolutionarily close coronavirus relatives of SARS-CoV-2 to estimate times to reinfection by a comparative evolutionary analysis of related viruses SARS-CoV, MERS-CoV, human coronavirus (HCoV)-229E, HCoV-OC43, and HCoV-NL63. METHODS: We conducted phylogenetic analyses of the S, M, and ORF1b genes to reconstruct a maximum-likelihood molecular phylogeny of human-infecting coronaviruses. This phylogeny enabled comparative analyses of peak-normalised nucleocapsid protein, spike protein, and whole-virus lysate IgG antibody optical density levels, in conjunction with reinfection data on endemic human-infecting coronaviruses. We performed ancestral and descendent states analyses to estimate the expected declines in antibody levels over time, the probabilities of reinfection based on antibody level, and the anticipated times to reinfection after recovery under conditions of endemic transmission for SARS-CoV-2, as well as the other human-infecting coronaviruses. FINDINGS: We obtained antibody optical density data for six human-infecting coronaviruses, extending from 128 days to 28 years after infection between 1984 and 2020. These data provided a means to estimate profiles of the typical antibody decline and probabilities of reinfection over time under endemic conditions. Reinfection by SARS-CoV-2 under endemic conditions would likely occur between 3 months and 5·1 years after peak antibody response, with a median of 16 months. This protection is less than half the duration revealed for the endemic coronaviruses circulating among humans (5-95% quantiles 15 months to 10 years for HCoV-OC43, 31 months to 12 years for HCoV-NL63, and 16 months to 12 years for HCoV-229E). For SARS-CoV, the 5-95% quantiles were 4 months to 6 years, whereas the 95% quantiles for MERS-CoV were inconsistent by dataset. INTERPRETATION: The timeframe for reinfection is fundamental to numerous aspects of public health decision making. As the COVID-19 pandemic continues, reinfection is likely to become increasingly common. Maintaining public health measures that curb transmission-including among individuals who were previously infected with SARS-CoV-2-coupled with persistent efforts to accelerate vaccination worldwide is critical to the prevention of COVID-19 morbidity and mortality. FUNDING: US National Science Foundation.


Subject(s)
COVID-19 , Coronavirus 229E, Human , Coronavirus NL63, Human , Coronavirus OC43, Human , Middle East Respiratory Syndrome Coronavirus , Antibodies, Viral/genetics , COVID-19/epidemiology , Cross Reactions , Humans , Pandemics , Phylogeny , Reinfection/epidemiology , SARS-CoV-2/genetics , Spike Glycoprotein, Coronavirus/genetics
6.
Signal Transduct Target Ther ; 7(1): 44, 2022 02 09.
Article in English | MEDLINE | ID: covidwho-1683982

ABSTRACT

The wide transmission and host adaptation of SARS-CoV-2 have led to the rapid accumulation of mutations, posing significant challenges to the effectiveness of vaccines and therapeutic antibodies. Although several neutralizing antibodies were authorized for emergency clinical use, convalescent patients derived natural antibodies are vulnerable to SARS-CoV-2 Spike mutation. Here, we describe the screen of a panel of SARS-CoV-2 receptor-binding domain (RBD) targeted nanobodies (Nbs) from a synthetic library and the design of a biparatopic Nb, named Nb1-Nb2, with tight affinity and super-wide neutralization breadth against multiple SARS-CoV-2 variants of concern. Deep-mutational scanning experiments identify the potential binding epitopes of the Nbs on the RBD and demonstrate that biparatopic Nb1-Nb2 has a strong escape-resistant feature against more than 60 tested RBD amino acid substitutions. Using pseudovirion-based and trans-complementation SARS-CoV-2 tools, we determine that the Nb1-Nb2 broadly neutralizes multiple SARS-CoV-2 variants at sub-nanomolar levels, including Alpha (B.1.1.7), Beta (B.1.351), Gamma (P.1), Delta (B.1.617.2), Lambda (C.37), Kappa (B.1.617.1), and Mu (B.1.621). Furthermore, a heavy-chain antibody is constructed by fusing the human IgG1 Fc to Nb1-Nb2 (designated as Nb1-Nb2-Fc) to improve its neutralization potency, yield, stability, and potential half-life extension. For the new Omicron variant (B.1.1.529) that harbors unprecedented multiple RBD mutations, Nb1-Nb2-Fc keeps a firm affinity (KD < 1.0 × 10-12 M) and strong neutralizing activity (IC50 = 1.46 nM for authentic Omicron virus). Together, we developed a tetravalent biparatopic human heavy-chain antibody with ultrapotent and broad-spectrum SARS-CoV-2 neutralization activity which highlights the potential clinical applications.


Subject(s)
Antibodies, Neutralizing/pharmacology , Antibodies, Viral/pharmacology , Immunoglobulin Fc Fragments/pharmacology , Recombinant Fusion Proteins/pharmacology , SARS-CoV-2/drug effects , Single-Domain Antibodies/pharmacology , Antibodies, Neutralizing/biosynthesis , Antibodies, Neutralizing/genetics , Antibodies, Viral/biosynthesis , Antibodies, Viral/genetics , Antibody Affinity , Enzyme-Linked Immunosorbent Assay , Epitopes/chemistry , Epitopes/immunology , Escherichia coli/genetics , Escherichia coli/metabolism , Gene Expression , Humans , Immunoglobulin Fc Fragments/biosynthesis , Immunoglobulin Fc Fragments/genetics , Models, Molecular , Neutralization Tests , Protein Binding/drug effects , Protein Conformation , Protein Interaction Domains and Motifs , Recombinant Fusion Proteins/biosynthesis , Recombinant Fusion Proteins/genetics , SARS-CoV-2/growth & development , SARS-CoV-2/immunology , Single-Domain Antibodies/biosynthesis , Single-Domain Antibodies/genetics , Spike Glycoprotein, Coronavirus/antagonists & inhibitors , Spike Glycoprotein, Coronavirus/chemistry , Spike Glycoprotein, Coronavirus/genetics , Spike Glycoprotein, Coronavirus/immunology
7.
Science ; 375(6582): 782-787, 2022 02 18.
Article in English | MEDLINE | ID: covidwho-1650668

ABSTRACT

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) Beta variant of concern (VOC) resists neutralization by major classes of antibodies from COVID-19 patients and vaccinated individuals. In this study, serum of Beta-infected patients revealed reduced cross-neutralization of wild-type virus. From these patients, we isolated Beta-specific and cross-reactive receptor-binding domain (RBD) antibodies. The Beta-specificity results from recruitment of VOC-specific clonotypes and accommodation of mutations present in Beta and Omicron into a major antibody class that is normally sensitive to these mutations. The Beta-elicited cross-reactive antibodies share genetic and structural features with wild type-elicited antibodies, including a public VH1-58 clonotype that targets the RBD ridge. These findings advance our understanding of the antibody response to SARS-CoV-2 shaped by antigenic drift, with implications for design of next-generation vaccines and therapeutics.


Subject(s)
Antibodies, Neutralizing/immunology , Antibodies, Viral/immunology , COVID-19/immunology , Cross Reactions , SARS-CoV-2/immunology , Spike Glycoprotein, Coronavirus/immunology , Adolescent , Adult , Aged , Aged, 80 and over , Antibodies, Neutralizing/chemistry , Antibodies, Neutralizing/genetics , Antibodies, Viral/genetics , Antibodies, Viral/metabolism , COVID-19/virology , Female , Humans , Male , Middle Aged , Neutralization Tests , Protein Binding , Protein Domains , Protein Interaction Domains and Motifs , SARS-CoV-2/chemistry , SARS-CoV-2/genetics , Spike Glycoprotein, Coronavirus/chemistry , Spike Glycoprotein, Coronavirus/genetics , Spike Glycoprotein, Coronavirus/metabolism
8.
Signal Transduct Target Ther ; 7(1): 18, 2022 01 19.
Article in English | MEDLINE | ID: covidwho-1639142

ABSTRACT

Emerging SARS-CoV-2 variants are the most serious problem for COVID-19 prophylaxis and treatment. To determine whether the SARS-CoV-2 vaccine strain should be updated following variant emergence like seasonal flu vaccine, the changed degree on antigenicity of SARS-CoV-2 variants and H3N2 flu vaccine strains was compared. The neutralization activities of Alpha, Beta and Gamma variants' spike protein-immunized sera were analysed against the eight current epidemic variants and 20 possible variants combining the top 10 prevalent RBD mutations based on the Delta variant, which were constructed using pseudotyped viruses. Meanwhile, the neutralization activities of convalescent sera and current inactivated and recombinant protein vaccine-elicited sera were also examined against all possible Delta variants. Eight HA protein-expressing DNAs elicited-animal sera were also tested against eight pseudotyped viruses of H3N2 flu vaccine strains from 2011-2019. Our results indicate that the antigenicity changes of possible Delta variants were mostly within four folds, whereas the antigenicity changes among different H3N2 vaccine strains were approximately 10-100-fold. Structural analysis of the antigenic characterization of the SARS-CoV-2 and H3N2 mutations supports the neutralization results. This study indicates that the antigenicity changes of the current SARS-CoV-2 may not be sufficient to require replacement of the current vaccine strain.


Subject(s)
Antibodies, Neutralizing/metabolism , Antibodies, Viral/metabolism , COVID-19 Vaccines/metabolism , COVID-19/prevention & control , Immunogenicity, Vaccine , SARS-CoV-2/immunology , Spike Glycoprotein, Coronavirus/immunology , Amino Acid Substitution , Antibodies, Neutralizing/chemistry , Antibodies, Neutralizing/genetics , Antibodies, Viral/chemistry , Antibodies, Viral/genetics , Binding Sites , COVID-19/immunology , COVID-19/virology , COVID-19 Vaccines/administration & dosage , COVID-19 Vaccines/chemistry , Epitopes/chemistry , Epitopes/genetics , Epitopes/immunology , Gene Expression , Humans , Immune Sera/chemistry , Influenza A Virus, H3N2 Subtype/chemistry , Influenza A Virus, H3N2 Subtype/genetics , Influenza A Virus, H3N2 Subtype/immunology , Influenza Vaccines/administration & dosage , Influenza Vaccines/chemistry , Influenza Vaccines/metabolism , Influenza, Human/immunology , Influenza, Human/prevention & control , Influenza, Human/virology , Models, Molecular , Mutation , Neutralization Tests , Protein Binding , Protein Conformation , Protein Interaction Domains and Motifs , SARS-CoV-2/chemistry , SARS-CoV-2/pathogenicity , Spike Glycoprotein, Coronavirus/chemistry , Spike Glycoprotein, Coronavirus/genetics
9.
MAbs ; 14(1): 2021601, 2022.
Article in English | MEDLINE | ID: covidwho-1625321

ABSTRACT

Coronavirus disease 2019, caused by SARS-CoV-2, remains an on-going pandemic, partly due to the emergence of variant viruses that can "break-through" the protection of the current vaccines and neutralizing antibodies (nAbs), highlighting the needs for broadly nAbs and next-generation vaccines. We report an antibody that exhibits breadth and potency in binding the receptor-binding domain (RBD) of the virus spike glycoprotein across SARS coronaviruses. Initially, a lead antibody was computationally discovered and crystallographically validated that binds to a highly conserved surface of the RBD of wild-type SARS-CoV-2. Subsequently, through experimental affinity enhancement and computational affinity maturation, it was further developed to bind the RBD of all concerning SARS-CoV-2 variants, SARS-CoV-1 and pangolin coronavirus with pico-molar binding affinities, consistently exhibited strong neutralization activity against wild-type SARS-CoV-2 and the Alpha and Delta variants. These results identify a vulnerable target site on coronaviruses for development of pan-sarbecovirus nAbs and vaccines.


Subject(s)
Antibodies, Viral/immunology , Antigens, Viral/immunology , Broadly Neutralizing Antibodies/immunology , COVID-19/immunology , SARS-CoV-2/immunology , Spike Glycoprotein, Coronavirus/immunology , Angiotensin-Converting Enzyme 2/chemistry , Angiotensin-Converting Enzyme 2/metabolism , Antibodies, Viral/genetics , Antibodies, Viral/metabolism , Antibody Affinity , Antibody Specificity , Antigen-Antibody Reactions , Antigens, Viral/chemistry , Antigens, Viral/genetics , Broadly Neutralizing Antibodies/genetics , Broadly Neutralizing Antibodies/metabolism , Crystallography, X-Ray , Epitopes/chemistry , Epitopes/immunology , Humans , Immunoglobulin Fragments/immunology , Molecular Docking Simulation , Monte Carlo Method , Neutralization Tests , Peptide Fragments/chemistry , Peptide Fragments/metabolism , Protein Domains , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/immunology , Recombinant Fusion Proteins/metabolism , SARS-CoV-2/genetics , Spike Glycoprotein, Coronavirus/chemistry , Spike Glycoprotein, Coronavirus/genetics
10.
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
11.
Gene ; 813: 146113, 2022 Mar 01.
Article in English | MEDLINE | ID: covidwho-1616498

ABSTRACT

Since late 2019, when SARS-CoV-2 was reported at Wuhan, several sequence analyses have been performed and SARS-CoV-2 genome sequences have been submitted in various databases. Moreover, the impact of these variants on infectivity and response to neutralizing antibodies has been assessed. In the present study, we retrieved a total number of 176 complete and high-quality S glycoprotein sequences of Iranian SARS-COV-2 in public database of the GISAID and GenBank from April 2020 up to May 2021. Then, we identified the number of variables, singleton and parsimony informative sites at both gene and protein levels and discussed the possible functional consequences of important mutations on the infectivity and response to neutralizing antibodies. Phylogenetic tree was constructed to represent the relationship between Iranian SARS-COV2 and variants of concern (VOC), variants of interest (VOI) and reference sequence. We found that the four current VOCs - Alpha, Beta, Gamma and Delta - are circulated in different regions in Iran. The Delta variant is notably more transmissible than other variants, and is expected to become a dominant variant. However, some of the Delta variants in Iran carry an additional mutation, namely E1202Q in the HR2 subdomain that might confer an advantage to viral/cell membrane fusion process. We also observed some more common mutations such as an N-terminal domain (NTD) deletion at position I210 and P863H in fusion peptide-heptad repeat 1 span region in Iranian SARS-COV-2. The reported mutations in the current project have practical significance in prediction of disease spread as well as design of vaccines and drugs.


Subject(s)
COVID-19/genetics , SARS-CoV-2/genetics , Spike Glycoprotein, Coronavirus/genetics , Antibodies, Neutralizing/immunology , Antibodies, Viral/genetics , COVID-19/epidemiology , COVID-19/metabolism , Databases, Genetic , Humans , Iran/epidemiology , Mutation/genetics , Phylogeny , Protein Binding , RNA, Viral , SARS-CoV-2/metabolism , SARS-CoV-2/pathogenicity , Sequence Analysis, DNA/methods , Spike Glycoprotein, Coronavirus/metabolism
12.
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
13.
Nat Commun ; 12(1): 6737, 2021 11 18.
Article in English | MEDLINE | ID: covidwho-1526077

ABSTRACT

Antibodies bind antigens via flexible loops called complementarity-determining regions (CDRs). These are usually 6-20 residues long. However, some bovine antibodies have ultra-long CDRs comprising more than 50 residues organized in a stalk and a disulfide-rich knob. The design features of this structural unit and its influence on antibody stability remained enigmatic. Here, we show that the stalk length is critical for the folding and stability of antibodies with an ultra-long CDR and that the disulfide bonds in the knob do not contribute to stability; they are important for organizing the antigen-binding knob structure. The bovine ultra-long CDR can be integrated into human antibody scaffolds. Furthermore, mini-domains from de novo design can be reformatted as ultra-long CDRs to create unique antibody-based proteins neutralizing SARS-CoV-2 and the Alpha variant of concern with high efficiency. Our findings reveal basic design principles of antibody structure and open new avenues for protein engineering.


Subject(s)
Complementarity Determining Regions/genetics , SARS-CoV-2/genetics , Animals , Antibodies, Viral/genetics , Antibodies, Viral/immunology , COVID-19/genetics , Cattle
14.
MAbs ; 13(1): 1987180, 2021.
Article in English | MEDLINE | ID: covidwho-1483313

ABSTRACT

The global health crisis and economic tolls of COVID-19 necessitate a panoply of strategies to treat SARS-CoV-2 infection. To date, few treatment options exist, although neutralizing antibodies against the spike glycoprotein have proven to be effective. Because infection is initiated at the mucosa and propagates mainly at this site throughout the course of the disease, blocking the virus at the mucosal milieu should be effective. However, administration of biologics to the mucosa presents a substantial challenge. Here, we describe bifunctional molecules combining single-domain variable regions that bind to the polymeric Ig receptor (pIgR) and to the SARS-CoV-2 spike protein via addition of the ACE2 extracellular domain (ECD). The hypothesis behind this design is that pIgR will transport the molecule from the circulation to the mucosal surface where the ACE ECD would act as a decoy receptor for the nCoV2. The bifunctional molecules bind SARS-Cov-2 spike glycoprotein in vitro and efficiently transcytose across the lung epithelium in human tissue-based analyses. Designs featuring ACE2 tethered to the C-terminus of the Fc do not induce antibody-dependent cytotoxicity against pIgR-expressing cells. These molecules thus represent a potential therapeutic modality for systemic administration of neutralizing anti-SARS-CoV-2 molecules to the mucosa.


Subject(s)
Antibodies, Viral , COVID-19/drug therapy , Receptors, Polymeric Immunoglobulin , SARS-CoV-2/immunology , Single-Chain Antibodies , Spike Glycoprotein, Coronavirus/immunology , Angiotensin-Converting Enzyme 2/genetics , Angiotensin-Converting Enzyme 2/immunology , Animals , Antibodies, Viral/genetics , Antibodies, Viral/immunology , Antibodies, Viral/pharmacology , CHO Cells , COVID-19/genetics , COVID-19/immunology , Cricetulus , Dogs , Female , Humans , Madin Darby Canine Kidney Cells , Mice , Mouth Mucosa/immunology , Protein Domains , Receptors, Polymeric Immunoglobulin/genetics , Receptors, Polymeric Immunoglobulin/immunology , Receptors, Polymeric Immunoglobulin/therapeutic use , SARS-CoV-2/genetics , Single-Chain Antibodies/genetics , Single-Chain Antibodies/immunology , Single-Chain Antibodies/pharmacokinetics , Single-Chain Antibodies/pharmacology , Spike Glycoprotein, Coronavirus/antagonists & inhibitors , Spike Glycoprotein, Coronavirus/genetics , Swine
15.
Signal Transduct Target Ther ; 6(1): 369, 2021 10 25.
Article in English | MEDLINE | ID: covidwho-1483125

ABSTRACT

The lung is the prophylaxis target against SARS-CoV-2 infection, and neutralizing antibodies are a leading class of biological products against various infectious viral pathogen. In this study, we develop a safe and cost-effective platform to express neutralizing antibody in the lung with replicating mRNA basing on alphavirus replicon particle (VRP) delivery system, to prevent SARS-CoV-2 infections. First, a modified VEEV replicon with two subgenomic (sg) promoters was engineered to translate the light and heavy chains of antibody simultaneously, for expression and assembly of neutralizing anti-SARS-CoV-2 antibody CB6. Second, the feasibility and protective efficacy of replicating mRNA against SARS-CoV-2 infection were demonstrated through both in vitro and in vivo assays. The lung target delivery with the help of VRP system resulted in efficiently block SARS-CoV-2 infection with reducing viral titer and less tissue damage in the lung of mice. Overall, our data suggests that expressing neutralizing antibodies in the lungs with the help of self-replicating mRNA could potentially be a promising prophylaxis approach against SARS-CoV-2 infection.


Subject(s)
Alphavirus , Antibodies, Neutralizing , Antibodies, Viral , COVID-19/therapy , Replicon , SARS-CoV-2/metabolism , Animals , Antibodies, Neutralizing/biosynthesis , Antibodies, Neutralizing/genetics , Antibodies, Viral/biosynthesis , Antibodies, Viral/genetics , COVID-19/genetics , COVID-19/metabolism , Chlorocebus aethiops , Cricetinae , Female , Mice , Mice, Inbred BALB C , RNA, Messenger/genetics , RNA, Messenger/metabolism , SARS-CoV-2/genetics , Vero Cells
16.
Nature ; 600(7889): 530-535, 2021 12.
Article in English | MEDLINE | ID: covidwho-1475311

ABSTRACT

The emergence of SARS-CoV-2 variants is jeopardizing the effectiveness of current vaccines and limiting the application of monoclonal antibody-based therapy for COVID-19 (refs. 1,2). Here we analysed the memory B cells of five naive and five convalescent people vaccinated with the BNT162b2 mRNA vaccine to investigate the nature of the B cell and antibody response at the single-cell level. Almost 6,000 cells were sorted, over 3,000 cells produced monoclonal antibodies against the spike protein and more than 400 cells neutralized the original SARS-CoV-2 virus first identified in Wuhan, China. The B.1.351 (Beta) and B.1.1.248 (Gamma) variants escaped almost 70% of these antibodies, while a much smaller portion was impacted by the B.1.1.7 (Alpha) and B.1.617.2 (Delta) variants. The overall loss of neutralization was always significantly higher in the antibodies from naive people. In part, this was due to the IGHV2-5;IGHJ4-1 germline, which was found only in people who were convalescent and generated potent and broadly neutralizing antibodies. Our data suggest that people who are seropositive following infection or primary vaccination will produce antibodies with increased potency and breadth and will be able to better control emerging SARS-CoV-2 variants.


Subject(s)
Antibodies, Neutralizing/immunology , Antibodies, Viral/immunology , Broadly Neutralizing Antibodies/immunology , SARS-CoV-2/immunology , Antibodies, Neutralizing/genetics , Antibodies, Neutralizing/isolation & purification , Antibodies, Viral/genetics , Antibodies, Viral/isolation & purification , /immunology , Broadly Neutralizing Antibodies/genetics , Broadly Neutralizing Antibodies/isolation & purification , Convalescence , Epitopes, B-Lymphocyte/chemistry , Epitopes, B-Lymphocyte/immunology , Female , Humans , Male , Neutralization Tests , Seroconversion , Single-Cell Analysis , Spike Glycoprotein, Coronavirus/chemistry , Spike Glycoprotein, Coronavirus/immunology
17.
Curr Med Sci ; 41(6): 1065-1074, 2021 Dec.
Article in English | MEDLINE | ID: covidwho-1449989

ABSTRACT

Coronavirus disease 2019 is threatening thousands of millions of people around the world. In the absence of specific and highly effective medicines, the treatment of infected persons is still very challenging. As therapeutics, neutralizing antibodies (NAbs) have great potential. Many NAbs have been reported, and most target various regions on the receptor-binding domain of the spike (S) protein, or the N-terminal domain. Several NAbs and NAb cocktails have been authorized for emergency use, and more are in clinical trials or are under development. In this review, considering the angle of binding epitopes on the S protein, we summarize the functions and the underlying mechanisms of a set of well-recognized NAbs and provide guidance for vaccine design and the combinatorial use of these antibodies. In addition, we review the NAbs and NAb cocktails that have been approved for emergency use and discuss the effectiveness of these NAbs for combating severe acute respiratory syndrome coronavirus 2 mutants.


Subject(s)
Antibodies, Neutralizing/immunology , Antibodies, Viral/immunology , Epitopes, B-Lymphocyte/chemistry , SARS-CoV-2/chemistry , SARS-CoV-2/immunology , Angiotensin-Converting Enzyme 2/chemistry , Antibodies, Neutralizing/chemistry , Antibodies, Neutralizing/genetics , Antibodies, Viral/chemistry , Antibodies, Viral/genetics , COVID-19/immunology , COVID-19/virology , Epitope Mapping , Humans , Models, Molecular , Mutation , Pandemics , Receptors, Virus/chemistry , SARS-CoV-2/genetics , Spike Glycoprotein, Coronavirus/chemistry
18.
Lancet Microbe ; 2(12): e666-e675, 2021 12.
Article in English | MEDLINE | ID: covidwho-1447264

ABSTRACT

BACKGROUND: Among the most consequential unknowns of the devastating COVID-19 pandemic are the durability of immunity and time to likely reinfection. There are limited direct data on SARS-CoV-2 long-term immune responses and reinfection. The aim of this study is to use data on the durability of immunity among evolutionarily close coronavirus relatives of SARS-CoV-2 to estimate times to reinfection by a comparative evolutionary analysis of related viruses SARS-CoV, MERS-CoV, human coronavirus (HCoV)-229E, HCoV-OC43, and HCoV-NL63. METHODS: We conducted phylogenetic analyses of the S, M, and ORF1b genes to reconstruct a maximum-likelihood molecular phylogeny of human-infecting coronaviruses. This phylogeny enabled comparative analyses of peak-normalised nucleocapsid protein, spike protein, and whole-virus lysate IgG antibody optical density levels, in conjunction with reinfection data on endemic human-infecting coronaviruses. We performed ancestral and descendent states analyses to estimate the expected declines in antibody levels over time, the probabilities of reinfection based on antibody level, and the anticipated times to reinfection after recovery under conditions of endemic transmission for SARS-CoV-2, as well as the other human-infecting coronaviruses. FINDINGS: We obtained antibody optical density data for six human-infecting coronaviruses, extending from 128 days to 28 years after infection between 1984 and 2020. These data provided a means to estimate profiles of the typical antibody decline and probabilities of reinfection over time under endemic conditions. Reinfection by SARS-CoV-2 under endemic conditions would likely occur between 3 months and 5·1 years after peak antibody response, with a median of 16 months. This protection is less than half the duration revealed for the endemic coronaviruses circulating among humans (5-95% quantiles 15 months to 10 years for HCoV-OC43, 31 months to 12 years for HCoV-NL63, and 16 months to 12 years for HCoV-229E). For SARS-CoV, the 5-95% quantiles were 4 months to 6 years, whereas the 95% quantiles for MERS-CoV were inconsistent by dataset. INTERPRETATION: The timeframe for reinfection is fundamental to numerous aspects of public health decision making. As the COVID-19 pandemic continues, reinfection is likely to become increasingly common. Maintaining public health measures that curb transmission-including among individuals who were previously infected with SARS-CoV-2-coupled with persistent efforts to accelerate vaccination worldwide is critical to the prevention of COVID-19 morbidity and mortality. FUNDING: US National Science Foundation.


Subject(s)
COVID-19 , Coronavirus 229E, Human , Coronavirus NL63, Human , Coronavirus OC43, Human , Middle East Respiratory Syndrome Coronavirus , Antibodies, Viral/genetics , COVID-19/epidemiology , Cross Reactions , Humans , Pandemics , Phylogeny , Reinfection/epidemiology , SARS-CoV-2/genetics , Spike Glycoprotein, Coronavirus/genetics
19.
Nat Microbiol ; 6(10): 1233-1244, 2021 10.
Article in English | MEDLINE | ID: covidwho-1434113

ABSTRACT

Understanding the molecular basis for immune recognition of SARS-CoV-2 spike glycoprotein antigenic sites will inform the development of improved therapeutics. We determined the structures of two human monoclonal antibodies-AZD8895 and AZD1061-which form the basis of the investigational antibody cocktail AZD7442, in complex with the receptor-binding domain (RBD) of SARS-CoV-2 to define the genetic and structural basis of neutralization. AZD8895 forms an 'aromatic cage' at the heavy/light chain interface using germ line-encoded residues in complementarity-determining regions (CDRs) 2 and 3 of the heavy chain and CDRs 1 and 3 of the light chain. These structural features explain why highly similar antibodies (public clonotypes) have been isolated from multiple individuals. AZD1061 has an unusually long LCDR1; the HCDR3 makes interactions with the opposite face of the RBD from that of AZD8895. Using deep mutational scanning and neutralization escape selection experiments, we comprehensively mapped the crucial binding residues of both antibodies and identified positions of concern with regards to virus escape from antibody-mediated neutralization. Both AZD8895 and AZD1061 have strong neutralizing activity against SARS-CoV-2 and variants of concern with antigenic substitutions in the RBD. We conclude that germ line-encoded antibody features enable recognition of the SARS-CoV-2 spike RBD and demonstrate the utility of the cocktail AZD7442 in neutralizing emerging variant viruses.


Subject(s)
Antibodies, Neutralizing/chemistry , Antibodies, Neutralizing/genetics , SARS-CoV-2/immunology , Antibodies, Monoclonal/chemistry , Antibodies, Monoclonal/genetics , Antibodies, Monoclonal/immunology , Antibodies, Neutralizing/immunology , Antibodies, Viral/chemistry , Antibodies, Viral/genetics , Antibodies, Viral/immunology , Antigenic Variation , Binding Sites , COVID-19/immunology , COVID-19/virology , Complementarity Determining Regions/chemistry , Complementarity Determining Regions/genetics , Humans , Mutation , Protein Domains , Spike Glycoprotein, Coronavirus/chemistry , Spike Glycoprotein, Coronavirus/genetics , Spike Glycoprotein, Coronavirus/immunology
20.
Front Immunol ; 12: 715464, 2021.
Article in English | MEDLINE | ID: covidwho-1430698

ABSTRACT

The mutants resulted from the ongoing SARS-CoV-2 epidemic have showed resistance to antibody neutralization and vaccine-induced immune response. The present study isolated and identified two novel SARS-CoV-2 neutralizing antibodies (nAbs) from convalescent COVID-19 patients. These two nAbs (XG81 and XG83) were then systemically compared with nine nAbs that were reconstructed by using published data, and revealed that, even though these two nAbs shared targeting epitopes on spike protein, they were different from any of the nine nAbs. Compared with XG81, XG83 exhibited a higher RBD binding affinity and neutralization potency against wild-typed pseudovirus, variant pseudoviruses with mutated spike proteins, such as D614G, E484Q, and A475V, as well as the authentic SARS-CoV-2 virus. To explore potential broadly neutralizing antibodies, heavy and light chains from all 18 nAbs (16 published nAbs, XG81 and XG83) were cross-recombined, and some of the functional antibodies were screened and studied for RBD binding affinity, and neutralizing activity against pseudovirus and the authentic SARS-CoV-2 virus. The results demonstrated that several recombined antibodies had a more potent neutralization activity against variant pseudoviruses compared with the originally paired Abs. Taken together, the novel neutralizing antibodies identified in this study are a likely valuable addition to candidate antibody drugs for the development of clinical therapeutic agents against SARS-CoV-2 to minimize mutational escape.


Subject(s)
Antibodies, Neutralizing/immunology , Antibodies, Viral/immunology , Broadly Neutralizing Antibodies/immunology , Broadly Neutralizing Antibodies/therapeutic use , SARS-CoV-2/immunology , Spike Glycoprotein, Coronavirus/immunology , Antibodies, Neutralizing/genetics , Antibodies, Neutralizing/therapeutic use , Antibodies, Viral/genetics , Antibodies, Viral/therapeutic use , Antibody Affinity/immunology , B-Lymphocytes/immunology , Broadly Neutralizing Antibodies/genetics , COVID-19/immunology , COVID-19/therapy , Cell Line , Epitopes/immunology , Humans , Immunotherapy/methods , Neutralization Tests , SARS-CoV-2/drug effects
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