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1.
J Clin Invest ; 131(20)2021 10 15.
Article in English | MEDLINE | ID: covidwho-1470547

ABSTRACT

BACKGROUNDMultisystem inflammatory syndrome in children (MIS-C) is a rare but potentially severe illness that follows exposure to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Kawasaki disease (KD) shares several clinical features with MIS-C, which prompted the use of intravenous immunoglobulin (IVIG), a mainstay therapy for KD. Both diseases share a robust activation of the innate immune system, including the IL-1 signaling pathway, and IL-1 blockade has been used for the treatment of both MIS-C and KD. The mechanism of action of IVIG in these 2 diseases and the cellular source of IL-1ß have not been defined.METHODSThe effects of IVIG on peripheral blood leukocyte populations from patients with MIS-C and KD were examined using flow cytometry and mass cytometry (CyTOF) and live-cell imaging.RESULTSCirculating neutrophils were highly activated in patients with KD and MIS-C and were a major source of IL-1ß. Following IVIG treatment, activated IL-1ß+ neutrophils were reduced in the circulation. In vitro, IVIG was a potent activator of neutrophil cell death via PI3K and NADPH oxidase, but independently of caspase activation.CONCLUSIONSActivated neutrophils expressing IL-1ß can be targeted by IVIG, supporting its use in both KD and MIS-C to ameliorate inflammation.FUNDINGPatient Centered Outcomes Research Institute; NIH; American Asthma Foundation; American Heart Association; Novo Nordisk Foundation; NIGMS; American Academy of Allergy, Asthma and Immunology Foundation.


Subject(s)
COVID-19/complications , Immunoglobulins, Intravenous/therapeutic use , Mucocutaneous Lymph Node Syndrome/immunology , Mucocutaneous Lymph Node Syndrome/therapy , Systemic Inflammatory Response Syndrome/immunology , Systemic Inflammatory Response Syndrome/therapy , COVID-19/blood , COVID-19/immunology , COVID-19/therapy , Case-Control Studies , Cell Death/immunology , Cell Lineage/immunology , Child , Child, Preschool , Fas Ligand Protein/immunology , Female , Humans , Infant , Interleukin-1beta/antagonists & inhibitors , Interleukin-1beta/blood , Leukocyte Count , Male , Mucocutaneous Lymph Node Syndrome/blood , Neutrophil Activation , Neutrophils/classification , Neutrophils/immunology , Neutrophils/pathology , Systemic Inflammatory Response Syndrome/blood
3.
Sci Adv ; 7(34)2021 08.
Article in English | MEDLINE | ID: covidwho-1365115

ABSTRACT

Novel coronavirus disease 2019 (COVID-19) severity is highly variable, with pediatric patients typically experiencing less severe infection than adults and especially the elderly. The basis for this difference is unclear. We find that mRNA and protein expression of angiotensin-converting enzyme 2 (ACE2), the cell entry receptor for the novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) that causes COVID-19, increases with advancing age in distal lung epithelial cells. However, in humans, ACE2 expression exhibits high levels of intra- and interindividual heterogeneity. Further, cells infected with SARS-CoV-2 experience endoplasmic reticulum stress, triggering an unfolded protein response and caspase-mediated apoptosis, a natural host defense system that halts virion production. Apoptosis of infected cells can be selectively induced by treatment with apoptosis-modulating BH3 mimetic drugs. Notably, epithelial cells within young lungs and airways are more primed to undergo apoptosis than those in adults, which may naturally hinder virion production and support milder COVID-19 severity.


Subject(s)
Angiotensin-Converting Enzyme 2/genetics , Apoptosis/genetics , COVID-19/genetics , Gene Expression Profiling/methods , Age Factors , Aged , Angiotensin-Converting Enzyme 2/metabolism , Animals , COVID-19/metabolism , COVID-19/virology , Cells, Cultured , Chlorocebus aethiops , Female , Humans , Infant , Lung/cytology , Lung/metabolism , Lung/virology , Male , Mice, Inbred C57BL , Middle Aged , SARS-CoV-2/physiology , Severity of Illness Index , Vero Cells , Virus Internalization
4.
Immunol Cell Biol ; 99(8): 796-799, 2021 09.
Article in English | MEDLINE | ID: covidwho-1352474

ABSTRACT

The B-cell response to COVID-19 vaccines in convalescent individuals.


Subject(s)
COVID-19 , SARS-CoV-2 , Antibodies, Viral , B-Lymphocytes/immunology , COVID-19 Vaccines , Humans , Immunologic Memory , Walking
5.
PLoS Pathog ; 17(5): e1009519, 2021 05.
Article in English | MEDLINE | ID: covidwho-1232468

ABSTRACT

SARS-CoV-2 is the novel coronavirus that is the causative agent of COVID-19, a sometimes-lethal respiratory infection responsible for a world-wide pandemic. The envelope (E) protein, one of four structural proteins encoded in the viral genome, is a 75-residue integral membrane protein whose transmembrane domain exhibits ion channel activity and whose cytoplasmic domain participates in protein-protein interactions. These activities contribute to several aspects of the viral replication-cycle, including virion assembly, budding, release, and pathogenesis. Here, we describe the structure and dynamics of full-length SARS-CoV-2 E protein in hexadecylphosphocholine micelles by NMR spectroscopy. We also characterized its interactions with four putative ion channel inhibitors. The chemical shift index and dipolar wave plots establish that E protein consists of a long transmembrane helix (residues 8-43) and a short cytoplasmic helix (residues 53-60) connected by a complex linker that exhibits some internal mobility. The conformations of the N-terminal transmembrane domain and the C-terminal cytoplasmic domain are unaffected by truncation from the intact protein. The chemical shift perturbations of E protein spectra induced by the addition of the inhibitors demonstrate that the N-terminal region (residues 6-18) is the principal binding site. The binding affinity of the inhibitors to E protein in micelles correlates with their antiviral potency in Vero E6 cells: HMA ≈ EIPA > DMA >> Amiloride, suggesting that bulky hydrophobic groups in the 5' position of the amiloride pyrazine ring play essential roles in binding to E protein and in antiviral activity. An N15A mutation increased the production of virus-like particles, induced significant chemical shift changes from residues in the inhibitor binding site, and abolished HMA binding, suggesting that Asn15 plays a key role in maintaining the protein conformation near the binding site. These studies provide the foundation for complete structure determination of E protein and for structure-based drug discovery targeting this protein.


Subject(s)
Amiloride/pharmacology , COVID-19/drug therapy , Coronavirus Envelope Proteins/metabolism , SARS-CoV-2/drug effects , SARS-CoV-2/metabolism , Amiloride/pharmacokinetics , Animals , Antiviral Agents/pharmacology , Binding Sites/drug effects , COVID-19/virology , Chlorocebus aethiops , Coronavirus Envelope Proteins/chemistry , Humans , Ion Channels/metabolism , Nuclear Magnetic Resonance, Biomolecular , Protein Binding/drug effects , Protein Conformation/drug effects , Protein Domains , Vero Cells , Virus Assembly/drug effects
6.
PLoS Pathog ; 17(2): e1009165, 2021 02.
Article in English | MEDLINE | ID: covidwho-1079380

ABSTRACT

The interactions between antibodies, SARS-CoV-2 and immune cells contribute to the pathogenesis of COVID-19 and protective immunity. To understand the differences between antibody responses in mild versus severe cases of COVID-19, we analyzed the B cell responses in patients 1.5 months post SARS-CoV-2 infection. Severe, and not mild, infection correlated with high titers of IgG against Spike receptor binding domain (RBD) that were capable of ACE2:RBD inhibition. B cell receptor (BCR) sequencing revealed that VH3-53 was enriched during severe infection. Of the 22 antibodies cloned from two severe donors, six exhibited potent neutralization against authentic SARS-CoV-2, and inhibited syncytia formation. Using peptide libraries, competition ELISA and mutagenesis of RBD, we mapped the epitopes of the neutralizing antibodies (nAbs) to three different sites on the Spike. Finally, we used combinations of nAbs targeting different immune-sites to efficiently block SARS-CoV-2 infection. Analysis of 49 healthy BCR repertoires revealed that the nAbs germline VHJH precursors comprise up to 2.7% of all VHJHs. We demonstrate that severe COVID-19 is associated with unique BCR signatures and multi-clonal neutralizing responses that are relatively frequent in the population. Moreover, our data support the use of combination antibody therapy to prevent and treat COVID-19.


Subject(s)
Antibodies, Monoclonal , Antibodies, Neutralizing , Antibodies, Viral , COVID-19 , Convalescence , SARS-CoV-2 , Spike Glycoprotein, Coronavirus , Adult , Aged , Animals , Antibodies, Monoclonal/genetics , Antibodies, Monoclonal/immunology , Antibodies, Neutralizing/genetics , Antibodies, Neutralizing/immunology , Antibodies, Viral/genetics , Antibodies, Viral/immunology , COVID-19/genetics , COVID-19/immunology , Chlorocebus aethiops , Cloning, Molecular , Epitope Mapping , Epitopes/genetics , Epitopes/immunology , Female , Humans , Immunoglobulin G/genetics , Immunoglobulin G/immunology , Male , Middle Aged , SARS-CoV-2/immunology , Spike Glycoprotein, Coronavirus/genetics , Spike Glycoprotein, Coronavirus/immunology , Vero Cells
7.
EMBO J ; 39(21): e106057, 2020 11 02.
Article in English | MEDLINE | ID: covidwho-846583

ABSTRACT

Coronavirus disease 2019 (COVID-19) is caused by SARS-CoV-2 and has spread across the globe. SARS-CoV-2 is a highly infectious virus with no vaccine or antiviral therapy available to control the pandemic; therefore, it is crucial to understand the mechanisms of viral pathogenesis and the host immune responses to SARS-CoV-2. SARS-CoV-2 is a new member of the betacoronavirus genus like other closely related viruses including SARS-CoV and Middle East respiratory syndrome coronavirus (MERS-CoV). Both SARS-CoV and MERS-CoV have caused serious outbreaks and epidemics in the past eighteen years. Here, we report that one of the interferon-stimulated genes (ISGs), cholesterol 25-hydroxylase (CH25H), is induced by SARS-CoV-2 infection in vitro and in COVID-19-infected patients. CH25H converts cholesterol to 25-hydrocholesterol (25HC) and 25HC shows broad anti-coronavirus activity by blocking membrane fusion. Furthermore, 25HC inhibits USA-WA1/2020 SARS-CoV-2 infection in lung epithelial cells and viral entry in human lung organoids. Mechanistically, 25HC inhibits viral membrane fusion by activating the ER-localized acyl-CoA:cholesterol acyltransferase (ACAT) which leads to the depletion of accessible cholesterol from the plasma membrane. Altogether, our results shed light on a potentially broad antiviral mechanism by 25HC through depleting accessible cholesterol on the plasma membrane to suppress virus-cell fusion. Since 25HC is a natural product with no known toxicity at effective concentrations, it provides a potential therapeutic candidate for COVID-19 and emerging viral diseases in the future.


Subject(s)
Antiviral Agents/pharmacology , Betacoronavirus/drug effects , Cholesterol/metabolism , Coronavirus Infections/drug therapy , Pneumonia, Viral/drug therapy , Respiratory Mucosa/virology , Steroid Hydroxylases/pharmacology , Virus Internalization/drug effects , Acetyl-CoA C-Acetyltransferase/metabolism , Animals , COVID-19 , Cell Line , Cell Membrane/drug effects , Cell Membrane/metabolism , Chlorocebus aethiops , Enzyme Activation/drug effects , Humans , Middle East Respiratory Syndrome Coronavirus/drug effects , Organoids/virology , Pandemics , Respiratory Mucosa/drug effects , SARS Virus/drug effects , SARS-CoV-2 , Vero Cells
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