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1.
EuropePMC; 2021.
Preprint in English | EuropePMC | ID: ppcovidwho-318387

ABSTRACT

Potently neutralizing SARS-CoV-2 antibodies often target the receptor binding site (RBS) of spike protein but the variability of RBS epitopes hampers broad neutralization of different clades of coronaviruses and emerging drifted viruses. Here, we identified a human RBS antibody that potently neutralizes SARS-CoV and SARS-CoV-2 variants that belong to clade 1 SARS-related coronavirus. X-ray crystallography revealed coordinated recognition by the heavy chain to conserved sites and the light chain to RBS, allowing for the mimicry of ACE2 binding mode. The minimum footprints in the hypervariable region of RBS contributed to the breadth of neutralization, and the activity was further enhanced by IgG3 switching. Eventually, the coordinated binding resulted in broad neutralization of SARS-CoV and emerging SARS-CoV-2 variants of concern. Furthermore, therapeutic treatment in a hamster model provided protection at low dosage. The structural basis for broadly neutralizing activity informs the design of broad spectrum of therapeutics and vaccines.Funding: This work was supported by Japan Agency for Medical Research and Development grant JP19fk0108111 (TH, YT), JP20fk0108298 (TK, TH, KM, YT), JP20am0101093 (KM), JP20ae0101047 (KM), JP20fk0108251 (HS), and JP20am0101124 (YK), by Ministry of Education, Culture, Sports, Science and Technology grant JPMXS0420100119 (KM) and 20H05773 (TH), by The Naito Foundation (TH), and by Joint Usage/Research Center program of Institute for Frontier Life and Medical Sciences, Kyoto University (KM).Conflict of Interest: AS is an employee of Shionogi & Co., Ltd. MO is a CEO, employee, and shareholder of Trans Chromosomics, Inc. These authors acknowledge a potential conflict of interest and attest that the work contained in this report is free of any bias that might be associated with the commercial goals of the company. TO, YA, MO, TH, KM, and YT declare that an intellectual property application has been filed using the data presented in this paper. The other authors declare that they have no competing interests.Ethical Approval: Animal procedures were approved by the Animal Ethics Committee of the National Institute of Infectious Diseases, Japan, and performed in accordance with the guidelines of the Institutional Animal Care and Use Committee. In vitro escape mutation screening experiments for SARSCoV-2 were performed at the Biosafety Level-3 facility of the Research Center for ZoonosisControl, Hokkaido University, and the National Institute of Infectious Diseases following the institutional guidelines.

2.
Immunity ; 54(10): 2385-2398.e10, 2021 10 12.
Article in English | MEDLINE | ID: covidwho-1370548

ABSTRACT

Potent neutralizing SARS-CoV-2 antibodies often target the spike protein receptor-binding site (RBS), but the variability of RBS epitopes hampers broad neutralization of multiple sarbecoviruses and drifted viruses. Here, using humanized mice, we identified an RBS antibody with a germline VH gene that potently neutralized SARS-related coronaviruses, including SARS-CoV and SARS-CoV-2 variants. X-ray crystallography revealed coordinated recognition by the heavy chain of non-RBS conserved sites and the light chain of RBS with a binding angle mimicking the angiotensin-converting enzyme 2 (ACE2) receptor. The minimum footprints in the hypervariable region of RBS contributed to the breadth of neutralization, which was enhanced by immunoglobulin G3 (IgG3) class switching. The coordinated binding resulted in broad neutralization of SARS-CoV and emerging SARS-CoV-2 variants of concern. Low-dose therapeutic antibody treatment in hamsters reduced the virus titers and morbidity during SARS-CoV-2 challenge. The structural basis for broad neutralizing activity may inform the design of a broad spectrum of therapeutics and vaccines.


Subject(s)
Broadly Neutralizing Antibodies/immunology , Cross Reactions/immunology , SARS-CoV-2/immunology , Animals , Betacoronavirus/immunology , Binding Sites, Antibody , Broadly Neutralizing Antibodies/chemistry , Broadly Neutralizing Antibodies/therapeutic use , COVID-19/prevention & control , COVID-19/therapy , COVID-19/virology , Cricetinae , Humans , Immunoglobulin Class Switching , Immunoglobulin Fab Fragments/chemistry , Immunoglobulin Fab Fragments/metabolism , Immunoglobulin G/chemistry , Immunoglobulin G/immunology , Mice , Protein Domains , Spike Glycoprotein, Coronavirus/chemistry , Spike Glycoprotein, Coronavirus/immunology , Spike Glycoprotein, Coronavirus/metabolism
3.
Nucleic Acids Res ; 49(7): e40, 2021 04 19.
Article in English | MEDLINE | ID: covidwho-1050155

ABSTRACT

Generation of conditional knockout (cKO) and various gene-modified cells is laborious and time-consuming. Here, we established an all-in-one cKO system, which enables highly efficient generation of cKO cells and simultaneous gene modifications, including epitope tagging and reporter gene knock-in. We applied this system to mouse embryonic stem cells (ESCs) and generated RNA helicase Ddx1 cKO ESCs. The targeted cells displayed endogenous promoter-driven EGFP and FLAG-tagged DDX1 expression, and they were converted to Ddx1 KO via FLP recombinase. We further established TetFE ESCs, which carried a reverse tetracycline transactivator (rtTA) expression cassette and a tetracycline response element (TRE)-regulated FLPERT2 cassette in the Gt(ROSA26)Sor locus for instant and tightly regulated induction of gene KO. By utilizing TetFE Ddx1F/F ESCs, we isolated highly pure Ddx1F/F and Ddx1-/- ESCs and found that loss of Ddx1 caused rRNA processing defects, thereby activating the ribosome stress-p53 pathway. We also demonstrated cKO of various genes in ESCs and homologous recombination-non-proficient human HT1080 cells. The frequency of cKO clones was remarkably high for both cell types and reached up to 96% when EGFP-positive clones were analyzed. This all-in-one cKO system will be a powerful tool for rapid and precise analyses of gene functions.


Subject(s)
DEAD-box RNA Helicases/metabolism , Gene Knockout Techniques/methods , RNA, Ribosomal/metabolism , Animals , Cell Line , Embryonic Stem Cells , Fibroblasts , Gene Expression , Gene Knock-In Techniques , Humans , Mice , Mice, Inbred C57BL , RNA Processing, Post-Transcriptional , Ribosomes/metabolism
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