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1.
EuropePMC; 2022.
Preprint in English | EuropePMC | ID: ppcovidwho-331567

ABSTRACT

While humoral immune responses to infection or vaccination with ancestral SARS-CoV-2 have been well-characterized, responses elicited by infection with variants are less understood. Here we characterized the repertoire, epitope specificity, and cross-reactivity of antibodies elicited by Beta and Gamma variant infection compared to ancestral virus. We developed a high-throughput approach to obtain single-cell immunoglobulin sequences and isolate monoclonal antibodies for functional assessment. Spike-, RBD- and NTD-specific antibodies elicited by Beta- or Gamma-infection exhibited a remarkably similar hierarchy of epitope immunodominance for RBD and convergent V gene usage when compared to ancestral virus infection. Additionally, similar public B cell clones were elicited regardless of infecting variant. These convergent responses may account for the broad cross-reactivity and continued efficacy of vaccines based on a single ancestral variant. One Sentence Summary WA1, Beta and Gamma variants of SARS-CoV-2 all elicit antibody responses targeting similar RBD epitopes;public and cross-reactive clones are common.

2.
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
3.
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.

4.
Sci Rep ; 10(1): 18149, 2020 10 23.
Article in English | MEDLINE | ID: covidwho-1387454

ABSTRACT

Antigens displayed on self-assembling nanoparticles can stimulate strong immune responses and have been playing an increasingly prominent role in structure-based vaccines. However, the development of such immunogens is often complicated by inefficiencies in their production. To alleviate this issue, we developed a plug-and-play platform using the spontaneous isopeptide-bond formation of the SpyTag:SpyCatcher system to display trimeric antigens on self-assembling nanoparticles, including the 60-subunit Aquifex aeolicus lumazine synthase (LuS) and the 24-subunit Helicobacter pylori ferritin. LuS and ferritin coupled to SpyTag expressed well in a mammalian expression system when an N-linked glycan was added to the nanoparticle surface. The respiratory syncytial virus fusion (F) glycoprotein trimer-stabilized in the prefusion conformation and fused with SpyCatcher-could be efficiently conjugated to LuS-SpyTag or ferritin-SpyTag, enabling multivalent display of F trimers with prefusion antigenicity. Similarly, F-glycoprotein trimers from human parainfluenza virus-type 3 and spike-glycoprotein trimers from SARS-CoV-2 could be displayed on LuS nanoparticles with decent yield and antigenicity. Notably, murine vaccination with 0.08 µg of SARS-CoV-2 spike-LuS nanoparticle elicited similar neutralizing responses as 2.0 µg of spike, which was ~ 25-fold higher on a weight-per-weight basis. The versatile platform described here thus allows for multivalent plug-and-play presentation on self-assembling nanoparticles of trimeric viral antigens, with SARS-CoV-2 spike-LuS nanoparticles inducing particularly potent neutralizing responses.


Subject(s)
Antigens/immunology , Betacoronavirus/metabolism , Nanoparticles/chemistry , Spike Glycoprotein, Coronavirus/immunology , Animals , Antibodies, Neutralizing/immunology , Antigens/genetics , Antigens/metabolism , Aquifex , Bacteria/enzymology , Bacterial Proteins/genetics , Betacoronavirus/isolation & purification , COVID-19 , Coronavirus Infections , Ferritins/genetics , Helicobacter pylori/metabolism , Humans , Mice , Multienzyme Complexes/genetics , Neutralization Tests , Pandemics , Pneumonia, Viral , Protein Multimerization , Recombinant Proteins/biosynthesis , Recombinant Proteins/chemistry , Recombinant Proteins/immunology , SARS-CoV-2 , Spike Glycoprotein, Coronavirus/genetics , Spike Glycoprotein, Coronavirus/metabolism , Surface Properties
5.
Science ; 373(6556)2021 Aug 13.
Article in English | MEDLINE | ID: covidwho-1295159

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.
Immunity ; 54(8): 1869-1882.e6, 2021 08 10.
Article in English | MEDLINE | ID: covidwho-1293864

ABSTRACT

Vaccine-associated enhanced respiratory disease (VAERD) was previously observed in some preclinical models of severe acute respiratory syndrome (SARS) and MERS coronavirus vaccines. We used the SARS coronavirus 2 (SARS-CoV-2) mouse-adapted, passage 10, lethal challenge virus (MA10) mouse model of acute lung injury to evaluate the immune response and potential for immunopathology in animals vaccinated with research-grade mRNA-1273. Whole-inactivated virus or heat-denatured spike protein subunit vaccines with alum designed to elicit low-potency antibodies and Th2-skewed CD4+ T cells resulted in reduced viral titers and weight loss post challenge but more severe pathological changes in the lung compared to saline-immunized animals. In contrast, a protective dose of mRNA-1273 induced favorable humoral and cellular immune responses that protected from viral replication in the upper and lower respiratory tract upon challenge. A subprotective dose of mRNA-1273 reduced viral replication and limited histopathological manifestations compared to animals given saline. Overall, our findings demonstrate an immunological signature associated with antiviral protection without disease enhancement following vaccination with mRNA-1273.


Subject(s)
COVID-19 Vaccines/immunology , COVID-19/immunology , COVID-19/prevention & control , Host-Pathogen Interactions/immunology , SARS-CoV-2/immunology , Vaccines, Synthetic/immunology , Animals , Antibodies, Neutralizing/immunology , Antibodies, Viral/immunology , Biopsy , COVID-19 Vaccines/administration & dosage , Disease Models, Animal , Humans , Immunoglobulin G , Immunohistochemistry , Mice , Outcome Assessment, Health Care , RNA, Messenger , Spike Glycoprotein, Coronavirus/immunology , T-Lymphocyte Subsets/immunology , T-Lymphocyte Subsets/metabolism , Vaccines, Synthetic/administration & dosage
7.
Vaccines (Basel) ; 9(2)2021 Jan 21.
Article in English | MEDLINE | ID: covidwho-1045354

ABSTRACT

The COVID-19 pandemic highlights an urgent need for vaccines that confer protection from SARS-CoV-2 infection. One approach to an effective COVID-19 vaccine may be through the display of SARS-CoV-2 spikes on the surface of virus-like particles, in a manner structurally mimicking spikes on a native virus. Here we report the development of Newcastle disease virus-like particles (NDVLPs) displaying the prefusion-stabilized SARS-CoV-2 spike ectodomain (S2P). Immunoassays with SARS-CoV-2-neutralizing antibodies revealed the antigenicity of S2P-NDVLP to be generally similar to that of soluble S2P, and negative-stain electron microscopy showed S2P on the NDVLP surface to be displayed with a morphology corresponding to its prefusion conformation. Mice immunized with S2P-NDVLP showed substantial neutralization titers (geometric mean ID50 = 386) two weeks after prime immunization, significantly higher than those elicited by a molar equivalent amount of soluble S2P (geometric mean ID50 = 17). Neutralizing titers at Week 5, two weeks after a boost immunization with S2P-NDVLP doses ranging from 2.0 to 250 µg, extended from 2125 to 4552, and these generally showed a higher ratio of neutralization versus ELISA than observed with soluble S2P. Overall, S2P-NDVLP appears to be a promising COVID-19 vaccine candidate capable of eliciting substantial neutralizing activity.

8.
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
9.
bioRxiv ; 2020 Aug 22.
Article in English | MEDLINE | ID: covidwho-666088

ABSTRACT

Antigens displayed on self-assembling nanoparticles can stimulate strong immune responses and have been playing an increasingly prominent role in structure-based vaccines. However, the development of such immunogens is often complicated by inefficiencies in their production. To alleviate this issue, we developed a plug-and-play platform using the spontaneous isopeptide-bond formation of the SpyTag:SpyCatcher system to display trimeric antigens on self-assembling nanoparticles, including the 60-subunit Aquifex aeolicus lumazine synthase (LuS) and the 24-subunit Helicobacter pylori ferritin. LuS and ferritin coupled to SpyTag expressed well in a mammalian expression system when an N-linked glycan was added to the nanoparticle surface. The respiratory syncytial virus fusion (F) glycoprotein trimer - stabilized in the prefusion conformation and fused with SpyCatcher - could be efficiently conjugated to LuS-SpyTag or ferritin-SpyTag, enabling multivalent display of F trimers with prefusion antigenicity. Similarly, F-glycoprotein trimers from human parainfluenza virus-type 3 and spike-glycoprotein trimers from SARS-CoV-2 could be displayed on LuS nanoparticles with decent yield and antigenicity. Notably, murine vaccination with the SARS-CoV-2 spike-LuS nanoparticles elicited ~25-fold higher neutralizing responses, weight-per-weight relative to spike alone. The versatile platform described here thus allows for multivalent plug-and-play presentation on self-assembling nanoparticles of trimeric viral antigens, with SARS-CoV-2 spike-LuS nanoparticles inducing particularly potent neutralizing responses.

10.
Nature ; 586(7830): 567-571, 2020 10.
Article in English | MEDLINE | ID: covidwho-703377

ABSTRACT

A vaccine for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is needed to control the coronavirus disease 2019 (COVID-19) global pandemic. Structural studies have led to the development of mutations that stabilize Betacoronavirus spike proteins in the prefusion state, improving their expression and increasing immunogenicity1. This principle has been applied to design mRNA-1273, an mRNA vaccine that encodes a SARS-CoV-2 spike protein that is stabilized in the prefusion conformation. Here we show that mRNA-1273 induces potent neutralizing antibody responses to both wild-type (D614) and D614G mutant2 SARS-CoV-2 as well as CD8+ T cell responses, and protects against SARS-CoV-2 infection in the lungs and noses of mice without evidence of immunopathology. mRNA-1273 is currently in a phase III trial to evaluate its efficacy.


Subject(s)
Betacoronavirus/immunology , Coronavirus Infections/immunology , Coronavirus Infections/prevention & control , Pandemics/prevention & control , Pneumonia, Viral/immunology , Pneumonia, Viral/prevention & control , Viral Vaccines/immunology , Animals , Antibodies, Neutralizing/immunology , Betacoronavirus/genetics , CD8-Positive T-Lymphocytes/immunology , COVID-19 , COVID-19 Vaccines , Clinical Trials, Phase III as Topic , Coronavirus Infections/genetics , Coronavirus Infections/virology , Female , Lung/immunology , Lung/virology , Mice , Mutation , Nose/immunology , Nose/virology , Pneumonia, Viral/virology , RNA, Messenger/genetics , RNA, Viral/genetics , SARS-CoV-2 , Th1 Cells/immunology , Toll-Like Receptor 4/agonists , Toll-Like Receptor 4/immunology , Viral Vaccines/chemistry , Viral Vaccines/genetics
11.
N Engl J Med ; 383(16): 1544-1555, 2020 10 15.
Article in English | MEDLINE | ID: covidwho-680559

ABSTRACT

BACKGROUND: Vaccines to prevent coronavirus disease 2019 (Covid-19) are urgently needed. The effect of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) vaccines on viral replication in both upper and lower airways is important to evaluate in nonhuman primates. METHODS: Nonhuman primates received 10 or 100 µg of mRNA-1273, a vaccine encoding the prefusion-stabilized spike protein of SARS-CoV-2, or no vaccine. Antibody and T-cell responses were assessed before upper- and lower-airway challenge with SARS-CoV-2. Active viral replication and viral genomes in bronchoalveolar-lavage (BAL) fluid and nasal swab specimens were assessed by polymerase chain reaction, and histopathological analysis and viral quantification were performed on lung-tissue specimens. RESULTS: The mRNA-1273 vaccine candidate induced antibody levels exceeding those in human convalescent-phase serum, with live-virus reciprocal 50% inhibitory dilution (ID50) geometric mean titers of 501 in the 10-µg dose group and 3481 in the 100-µg dose group. Vaccination induced type 1 helper T-cell (Th1)-biased CD4 T-cell responses and low or undetectable Th2 or CD8 T-cell responses. Viral replication was not detectable in BAL fluid by day 2 after challenge in seven of eight animals in both vaccinated groups. No viral replication was detectable in the nose of any of the eight animals in the 100-µg dose group by day 2 after challenge, and limited inflammation or detectable viral genome or antigen was noted in lungs of animals in either vaccine group. CONCLUSIONS: Vaccination of nonhuman primates with mRNA-1273 induced robust SARS-CoV-2 neutralizing activity, rapid protection in the upper and lower airways, and no pathologic changes in the lung. (Funded by the National Institutes of Health and others.).


Subject(s)
Betacoronavirus/immunology , Coronavirus Infections/immunology , Coronavirus Infections/prevention & control , Pandemics/prevention & control , Pneumonia, Viral/immunology , Pneumonia, Viral/prevention & control , Viral Vaccines/immunology , Animals , Antibodies, Neutralizing/blood , Antibodies, Viral/blood , Betacoronavirus/physiology , CD4 Antigens , COVID-19 , COVID-19 Vaccines , Coronavirus Infections/pathology , Coronavirus Infections/therapy , Disease Models, Animal , Dose-Response Relationship, Immunologic , Immunization, Passive , Lung/pathology , Lung/virology , Macaca mulatta , Pneumonia, Viral/pathology , Pneumonia, Viral/therapy , SARS-CoV-2 , Spike Glycoprotein, Coronavirus , T-Lymphocytes/immunology , Viral Load , Viral Vaccines/administration & dosage , Virus Replication
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