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1.
PubMed; 2020.
Preprint in English | PubMed | ID: ppcovidwho-292815

ABSTRACT

Biotin-labeled molecular probes, comprising specific regions of the SARS-CoV-2 spike, would be helpful in the isolation and characterization of antibodies targeting this recently emerged pathogen. To develop such probes, we designed constructs incorporating an N-terminal purification tag, a site-specific protease-cleavage site, the probe region of interest, and a C-terminal sequence targeted by biotin ligase. Probe regions included full-length spike ectodomain as well as various subregions, and we also designed mutants to eliminate recognition of the ACE2 receptor. Yields of biotin-labeled probes from transient transfection ranged from ~0.5 mg/L for the complete ectodomain to >5 mg/L for several subregions. Probes were characterized for antigenicity and ACE2 recognition, and the structure of the spike ectodomain probe was determined by cryo-electron microscopy. We also characterized antibody-binding specificities and cell-sorting capabilities of the biotinylated probes. Altogether, structure-based design coupled to efficient purification and biotinylation processes can thus enable streamlined development of SARS-CoV-2 spike-ectodomain probes. Funding: Support for this work was provided by the Intramural Research Program of the Vaccine Research Center, National Institute of Allergy and Infectious Diseases (NIAID). Support for this work was also provided by COVID-19 Fast Grants, the Jack Ma Foundation, the Self Graduate Fellowship Program, and NIH grants DP5OD023118, R21AI143407, and R21AI144408. Some of this work was performed at the Columbia University Cryo-EM Center at the Zuckerman Institute, and some at the Simons Electron Microscopy Center (SEMC) and National Center for Cryo-EM Access and Training (NCCAT) located at the New York Structural Biology Center, supported by grants from the Simons Foundation (SF349247), NYSTAR, and the NIH National Institute of General Medical Sciences (GM103310). Conflict of Interest: The authors declare that they have no conflict of interest. Ethical Approval: Peripheral blood mononuclear cells (PBMCs) for B cell sorting were obtained from a convalescent SARS-CoV-2 patient (collected 75 days post symptom onset under an IRB approved clinical trial protocol, VRC 200 - ClinicalTrials.gov Identifier: NCT00067054) and a healthy control donor from the NIH blood bank pre-SARS-CoV-2 pandemic.

2.
Cell Rep ; 37(1): 109771, 2021 10 05.
Article in English | MEDLINE | ID: covidwho-1439919

ABSTRACT

Understanding mechanisms of protective antibody recognition can inform vaccine and therapeutic strategies against SARS-CoV-2. We report a monoclonal antibody, 910-30, targeting the SARS-CoV-2 receptor-binding site for ACE2 as a member of a public antibody response encoded by IGHV3-53/IGHV3-66 genes. Sequence and structural analyses of 910-30 and related antibodies explore how class recognition features correlate with SARS-CoV-2 neutralization. Cryo-EM structures of 910-30 bound to the SARS-CoV-2 spike trimer reveal binding interactions and its ability to disassemble spike. Despite heavy-chain sequence similarity, biophysical analyses of IGHV3-53/3-66-encoded antibodies highlight the importance of native heavy:light pairings for ACE2-binding competition and SARS-CoV-2 neutralization. We develop paired heavy:light class sequence signatures and determine antibody precursor prevalence to be ∼1 in 44,000 human B cells, consistent with public antibody identification in several convalescent COVID-19 patients. These class signatures reveal genetic, structural, and functional immune features that are helpful in accelerating antibody-based medical interventions for SARS-CoV-2.


Subject(s)
Angiotensin-Converting Enzyme 2/immunology , Antibodies, Monoclonal/chemistry , Antibodies, Monoclonal/immunology , COVID-19/immunology , COVID-19/virology , SARS-CoV-2/immunology , Spike Glycoprotein, Coronavirus/immunology , Aged , Angiotensin-Converting Enzyme 2/chemistry , Animals , Antibodies, Monoclonal/genetics , Antibodies, Monoclonal/ultrastructure , Antibodies, Neutralizing/immunology , Antibodies, Viral/immunology , Antibody Formation , B-Lymphocytes/immunology , Binding Sites , Chlorocebus aethiops , Cryoelectron Microscopy , HEK293 Cells , Humans , Immunoglobulin Heavy Chains/chemistry , Immunoglobulin Heavy Chains/genetics , Immunoglobulin Heavy Chains/immunology , Immunoglobulin Heavy Chains/ultrastructure , Immunoglobulin Light Chains/chemistry , Immunoglobulin Light Chains/genetics , Immunoglobulin Light Chains/immunology , Immunoglobulin Light Chains/ultrastructure , Male , Protein Binding , Protein Interaction Domains and Motifs , SARS-CoV-2/chemistry , Spike Glycoprotein, Coronavirus/chemistry , Vero Cells
3.
AIChE J ; 67(12): e17440, 2021 Dec.
Article in English | MEDLINE | ID: covidwho-1427045

ABSTRACT

Antiviral monoclonal antibody (mAb) discovery enables the development of antibody-based antiviral therapeutics. Traditional antiviral mAb discovery relies on affinity between antibody and a viral antigen to discover potent neutralizing antibodies, but these approaches are inefficient because many high affinity mAbs have no neutralizing activity. We sought to determine whether screening for anti-SARS-CoV-2 mAbs at reduced pH could provide more efficient neutralizing antibody discovery. We mined the antibody response of a convalescent COVID-19 patient at both physiological pH (7.4) and reduced pH (4.5), revealing that SARS-CoV-2 neutralizing antibodies were preferentially enriched in pH 4.5 yeast display sorts. Structural analysis revealed that a potent new antibody called LP5 targets the SARS-CoV-2 N-terminal domain supersite via a unique binding recognition mode. Our data combine with evidence from prior studies to support antibody screening at pH 4.5 to accelerate antiviral neutralizing antibody discovery.

4.
Semin Immunol ; 50: 101428, 2020 08.
Article in English | MEDLINE | ID: covidwho-947460

ABSTRACT

The vaccine field is pursuing diverse approaches to translate the molecular insights from analyses of effective antibodies and their targeted epitopes into immunogens capable of eliciting protective immune responses. Here we review current antibody-guided strategies including conformation-based, epitope-based, and lineage-based vaccine approaches, which are yielding promising vaccine candidates now being evaluated in clinical trials. We summarize directions being employed by the field, including the use of sequencing technologies to monitor and track developing immune responses for understanding and improving antibody-based immunity. We review opportunities and challenges to transform powerful new discoveries into safe and effective vaccines, which are encapsulated by vaccine efforts against a variety of pathogens including HIV-1, influenza A virus, malaria parasites, respiratory syncytial virus, and SARS-CoV-2. Overall, this review summarizes the extensive progress that has been made to realize antibody-guided structure-based vaccines, the considerable challenges faced, and the opportunities afforded by recently developed molecular approaches to vaccine development.


Subject(s)
COVID-19 Vaccines/immunology , COVID-19/immunology , COVID-19/prevention & control , Vaccinology/methods , COVID-19 Vaccines/therapeutic use , Humans , Primary Prevention/methods , SARS-CoV-2/immunology
5.
Cell Rep ; 33(4): 108322, 2020 10 27.
Article in English | MEDLINE | ID: covidwho-888426

ABSTRACT

Biotin-labeled molecular probes, comprising specific regions of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike, would be helpful in the isolation and characterization of antibodies targeting this recently emerged pathogen. Here, we design constructs incorporating an N-terminal purification tag, a site-specific protease-cleavage site, the probe region of interest, and a C-terminal sequence targeted by biotin ligase. Probe regions include full-length spike ectodomain as well as various subregions, and we also design mutants that eliminate recognition of the angiotensin-converting enzyme 2 (ACE2) receptor. Yields of biotin-labeled probes from transient transfection range from ∼0.5 mg/L for the complete ectodomain to >5 mg/L for several subregions. Probes are characterized for antigenicity and ACE2 recognition, and the structure of the spike ectodomain probe is determined by cryoelectron microscopy. We also characterize antibody-binding specificities and cell-sorting capabilities of the biotinylated probes. Altogether, structure-based design coupled to efficient purification and biotinylation processes can thus enable streamlined development of SARS-CoV-2 spike ectodomain probes.


Subject(s)
Antibodies, Neutralizing/immunology , Antibodies, Viral/immunology , Coronavirus Infections/immunology , Molecular Probes/immunology , Pneumonia, Viral/immunology , Spike Glycoprotein, Coronavirus/immunology , Angiotensin-Converting Enzyme 2 , Antibody Specificity/immunology , Binding Sites, Antibody/immunology , Biotinylation , COVID-19 , Cryoelectron Microscopy , Humans , Pandemics , Peptidyl-Dipeptidase A/metabolism , Receptors, Virus/metabolism
6.
Nat Microbiol ; 5(10): 1185-1191, 2020 10.
Article in English | MEDLINE | ID: covidwho-752497

ABSTRACT

Antibody-based drugs and vaccines against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) are being expedited through preclinical and clinical development. Data from the study of SARS-CoV and other respiratory viruses suggest that anti-SARS-CoV-2 antibodies could exacerbate COVID-19 through antibody-dependent enhancement (ADE). Previous respiratory syncytial virus and dengue virus vaccine studies revealed human clinical safety risks related to ADE, resulting in failed vaccine trials. Here, we describe key ADE mechanisms and discuss mitigation strategies for SARS-CoV-2 vaccines and therapies in development. We also outline recently published data to evaluate the risks and opportunities for antibody-based protection against SARS-CoV-2.


Subject(s)
Antibody-Dependent Enhancement , Betacoronavirus , Coronavirus Infections/immunology , Coronavirus Infections/therapy , Pneumonia, Viral/immunology , Pneumonia, Viral/therapy , Viral Vaccines/adverse effects , Animals , Antibodies, Monoclonal/administration & dosage , Antibodies, Neutralizing/administration & dosage , Antibodies, Viral/administration & dosage , Antibodies, Viral/immunology , Antibody-Dependent Enhancement/immunology , Betacoronavirus/immunology , COVID-19 , COVID-19 Vaccines , Coronavirus Infections/drug therapy , Coronavirus Infections/prevention & control , Humans , Immunization, Passive/adverse effects , In Vitro Techniques , Models, Immunological , Pandemics/prevention & control , Pneumonia, Viral/prevention & control , Respiratory Tract Diseases/etiology , Respiratory Tract Diseases/immunology , Risk Factors , SARS-CoV-2 , Safety , Viral Vaccines/immunology
7.
Science ; 369(6508): 1167-1168, 2020 09 04.
Article in English | MEDLINE | ID: covidwho-744814
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