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Principal Investigator: JOHN H KEHRL
Organization: NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS DISEASES
Fiscal Year: 2020
Award: $45,608
Funding agency: National Institute of Allergy and Infectious Diseases
Recombinant SARS-CoV-2 spike protein was made in CHO and HEK293F cells and purified by affinity chromatography. To modify the glycosylation of the spike protein CHO cells were treated with Kifunesin, an inhibitor of class I -mannosidases. This prevents the trimming of mannose residues on glycoproteins and results in oligomannose-type glycans (referred to as, high mannose spike protein). Alternatively, the CHO purified spike protein was treated with PNGase, which removes N-linked glycans (referred to as, de-glycosylated spike protein). The purified spike proteins were coupled to beads, which were used for a flow cytometry-based assay to assess ACE2 and specific antibody binding. Both the CHO and HEK293F purified proteins efficiently bound ACE2 and were recognized by specific antibodies. For subsequent flow cytometry and imaging assays the purified spike proteins were fluorescently labeled.
To assess the cellular uptake of the SARS-CoV-2 spike protein in vivo, we injected fluorescently labeled recombinant protein intranasally, intravenously, and intradermally into C57Bl/6 mice. Most of the studies were preformed with CHO expressed protein, but similar results were found with HEK293F expressed protein. At various time points we determined the localization of the injected protein by intravital 2-photon imaging and confocal imaging of fixed and live tissues. Intranasal injection led to a rapid and persistent binding of the spike protein by alveolar and interstitial macrophages. 18 hours post injection numerous neutrophils had infiltrated the lungs. Explanted lung live cell imaging showed numerous damaged neutrophils within the lung parenchyma. We confirmed the neutrophil infiltration by flow cytometry, which also revealed an influx of monocytes and dendritic cells. A comparison between the CHO expressed protein and the high mannose version revealed that the high mannose protein recruited more neutrophils and lymphocytes into the lung. Lung imaging suggested that the high mannose protein caused more neutrophil fragmentation and damage. Analysis of the cells binding to the spike protein in vivo following intranasal injection by ex vivo flow cytometry revealed the strong uptake by lung macrophages, but also low-level binding to neutrophils, lymphocytes, monocytes, and eosinophils. Intravenous injection of the spike protein led to widespread endothelial cell uptake. This was evident in the lung, liver, spleen, heart, and intestines. In the spleen red pulp macrophages and in the liver Kupffer cells also rapidly accumulated the spike protein. Local injection of the spike protein near the inguinal lymph node led to rapid uptake by the interfollicular SignR1 positive macrophages and little uptake by the adjacent subcapsular sinus macrophages. Next, we analyzed spike protein binding to human cell lines and human peripheral blood leukocytes. In addition to the SARS-CoV-2 spike protein we also used a related Coronavirus virus spike protein that from HCoV-NL63. In contrast, to SARS-CoV-2 spike protein the HCoV spike protein bound poorly to the cell lines and to blood leukocytes. Among the cell lines tested, U937 cells bound the highest level of spike protein. Leukocytes from various donors variably bound the spike protein. Some exhibited high levels of binding while others had only low levels. B cells, neutrophils, eosinophils, monocytes, dendritic cells bound higher amounts than did CD4, CD8, and NK cells. Most of the binding depended upon the presence of cations. Based on the data that showed human neutrophils bind the spike protein and that intranasal injection causes neutrophil fragmentation we determined whether the SARS-CoV2 spike protein causes neutrophil netosis. The addition of the CHO spike protein caused a small, but reproducible increase in human neutrophil netosis, while the high mannose version caused more, and the de-glycosylated protein caused less. These results suggest that exposure of neutrophils to the SARS-CoV-2 spike protein causes neutrophil damage and low level netosis.
Based on the strong macrophage/monocyte binding and the know importance of cytokines in COVID-19 we measured select cytokines in cell supernatants of M0/M1/M2 macrophages exposed to either the CHO expressed spike protein or a HCoV-NL63 spike protein. The HCoV spike protein had little impact on cytokine production while the CHO expressed spike protein triggered in TNF-alpha, IL-10, and IL-6 production from MO and M2 macrophages; increased IL-12p70 and CXCL10 from M1; and increased CCL17 and IL1-RA from M2 macrophages. No increases in IL12p40, interferon-gamma, IL-23, or IL-1beta were found. The SARS-CoV-2 spike protein induced increases in the chemokines CXCL10 and CCL17 may help recruit leukocytes to sites of ongoing SARS-CoV2 replication. The triad of elevated CXCL10, IL-6, and IL-10 have been reported to anticipate the subsequent clinical progression of patients suffering with COVID-19.
In mice alveolar macrophages and eosinophils both express Siglec-F. Siglecs are sialic-acid-binding immunoglobulin-like lectins that are expressed predominately by immune cells. Many pathogenic viruses express sialic acids, which are recognized by one or more Siglecs. Siglec-dependent recognition of these pathogen glycans can alter immune responses, either to the advantage or to the detriment of the pathogen. To determine whether Siglec-F bound the SARS-CoV-2 spike protein we used the previously describe bead assay and measured the binding of a fluorescently labeled recombinant Siglec-F fusion protein. This assay showed strong binding of Siglec-F to both the CHO and HEK293F expressed SARS-CoV-2 spike protein, but poor binding to the high mannose and de-glycosylated CHO expressed spike proteins. Mouse Siglec-F and human Siglec-8 are functionally convergent paralogs that recognize 6'-sulfo-sialyl Lewis X as a preferred glycan ligand. Therefore, we tested human Siglec-8 as well as human Siglec-1, 2, and 5. However, none of the human Siglecs that we tested bound the spike protein. Using the same assay, we also assessed several possible endothelial cell receptors that mediate binding to cell surface glycans. However, we found little binding of PSGL-1, L-selectin, or P-selectin to the spike protein preparations. Further studies will needed to determine the binding site of the spike protein on endothelial cells and to human leukocytes.
Previous studies of SARS-CoV identified open reading frame 3a (ORF3a) as an essential factor for disease pathogenesis; an activator of NLRP3 inflammasomes, and a potential trigger for both apoptotic and necrotic cell death. ORF3a is a transmembrane protein that contains several conserved motifs including a cysteine-rich motif (amino acids 127 thru 133). SARS-CoV-2 ORF3a shares 73% amino acid homology with its counterpart in SARS-CoV, and the cysteine-rich motif is conserved. We investigated the effect of various cysteine-to-alanine substitution mutations in SARS-CoV-2 ORF3a and determined that disulfide bond formation at cysteine-133 is integral for ORF3a to oligomerize. We have previously shown that SARS-CoV-1 ORF3a oligomers insert into plasma and lysosomal membranes. Imaging studies have shown a similar localization of SARS-CoV-2 ORF3a. Compared to the wild type protein the SARS-CoV-2 C133 mutant protein less efficiently targeted these cellular membranes. Our studies indicate that by inserting into lysosomal membranes ORF3a triggers lysosomal damage, dysfunction, and cellular stress eventually triggering NLRP3 inflammasome assembly. Finally, the recent finding that lysosomal deacidification promotes SARS-CoV-2 egress from infected cells suggests the possible involvement of ORF3a in viral egress. Thus, interfering with ORF3a oligomerization and its membrane insertion represents a viable therapeutic approach to treating COVID-19 patients.
Terms: <(TNF)-α><2-photon><2019 novel coronavirus><2019-nCoV><A-152E5.3><ABCD-2><ABCD-2 protein><Affinity Chromatography><Alanine><Alveolar><Alveolar Macrophages><Amino Acids><Antibodies><Apoptotic><Assay><Asses><B blood cells><B cell><B cell differentiation factor><B cell stimulating factor 2><B cells><B-Cell Differentiation Factor><B-Cell Differentiation Factor-2><B-Cell Stimulatory Factor-2><B-Cells><B-Lymphocytes><B-cell><BCDF><BSF-2><BSF2><Binding><Binding Proteins><Binding Sites><Bioassay><Biologic Assays><Biological Assay><Blood><Blood Cells><Blood Eosinophil><Blood Neutrophil><Blood Plasma><Blood Polymorphonuclear Neutrophil><Blood Reticuloendothelial System><Blood leukocyte><Blood monocyte><Body Tissues><C57BL/6 Mouse><CC Chemokine TARC><CCL17><CCL17 gene><CD162 antigen><CD4 Cells><CD4 Positive T Lymphocytes><CD4 T cells><CD4 helper T cell><CD4 lymphocyte><CD4+ T-Lymphocyte><CD4-Positive Lymphocytes><CD40 Receptor-Associated Factor 1><CD40bp Protein><CD62L Antigens><CD62P Antigens><CD8 Cell><CD8 T cells><CD8 lymphocyte><CD8+ T cell><CD8+ T-Lymphocyte><CD8-Positive Lymphocytes><CD8-Positive T-Lymphocytes><CHO Cells><COVID-19><COVID19><CRAF1 Protein><CRG-2><CSIF><CSIF-10><CXCL10><CXCL10 gene><Cachectin><Carbohydrates><Cations><Cell Body><Cell Communication and Signaling><Cell Death><Cell Function><Cell Line><Cell Process><Cell Signaling><Cell Surface Receptors><Cell Survival><Cell Viability><Cell physiology><Cell surface><Cell-Mediated Cytolysis><Cell-Mediated Lympholysis><CellLine><Cells><Cellular Cytotoxicity><Cellular Function><Cellular Membrane><Cellular Physiology><Cellular Process><Cellular Stress><Chemokine (C-C Motif) Ligand 17><Chemotactic Cytokines><Chimera Protein><Chimeric Proteins><Chinese Hamster Ovary Cell><Clinical><Combining Site><Confocal Microscopy><Coronaviridae><Coronavirus><Coupled><Cysteine><Cytokine Synthesis Inhibitory Factor><Cytotoxic cell><D-Mannose><Data><Dendritic Cells><Disease><Disorder><Donkey><Dysfunction><Endothelial Cells><Eosinophilic Granulocyte><Eosinophilic Leukocyte><Equus asinus><Exhibits><Exposure to><Flow Cytofluorometries><Flow Cytofluorometry><Flow Cytometry><Flow Microfluorimetry><Flow Microfluorometry><Functional disorder><Fusion Protein><GMP-140><Gamma interferon><Genetic Alteration><Genetic Change><Genetic defect><Glycans><Glycoproteins><HCoV><HPGF><Half-Cystine><Heart><Hepatocyte-Stimulating Factor><Homologous Chemotactic Cytokines><Hour><Human><Human Cell Line><Hybridoma Growth Factor><IFI10><IFN-Gamma><IFN-beta 2><IFN-g><IFN-γ><IFNB2><IFNG><IFNγ><IL-1><IL-10><IL-23><IL-6><IL1><IL10><IL10A><IL6 Protein><INP10><IP-10><Image><Immune><Immune Globulins><Immune Interferon><Immune response><Immunes><Immunoglobulins><Immunological response><Inflammasome><Inflammation><Inflammatory><Inguinal Lymph Node><Inguinal lymph node group><Injections><Innate Immunity><Integral Membrane Protein><Intercrines><Interferon Gamma><Interferon Type II><Interferon-gamma><Interleukin 10 Precursor><Interleukin I><Interleukin-1><Interleukin-10><Interleukin-6><Intestinal><Intestines><Intracellular Communication and Signaling><Intravenous><Intrinsic Membrane Protein><K lymphocyte><Kupffer Cells><L-Cysteine><L-Selectin><LAM-1><LAM-1 Leukocyte Adhesion Molecule><LAP-1 Protein><LECAM-1><LECAM-3><Label><Lectin><Leu-8 Antigen><Leukocytes><Leukocytes Reticuloendothelial System><Ligand Binding Protein><Ligand Binding Protein Gene><Ligands><Link><Liver><Lung><Lung Parenchyma><Lung Respiratory System><Lung Tissue><Lymph Node Reticuloendothelial System><Lymph Node Subcapsular Sinus><Lymph node proper><Lymphatic nodes><Lymphocyte><Lymphocyte Adhesion Molecule 1><Lymphocyte Cytotoxicity><Lymphocyte-Stimulating Hormone><Lymphocytic><Lymphocytotoxicity><MGI-2><MOB-1><Macrophage Cell Factor><Macrophage-Derived TNF><Mannopyranose><Mannopyranoside><Mannose><Mannosidase><Marrow Eosinophil><Marrow Neutrophil><Marrow leukocyte><Marrow monocyte><Measures><Mediating><Mel-14 Antigen><Membrane><Metabolic Glycosylation><Mice><Mice Mammals><Mitochondria><Modern Man><Molecular><Molecular Interaction><Monocyte-Derived TNF><Murine><Mus><Mutation><Myeloid Differentiation-Inducing Protein><N-Acetylneuraminic Acids><NK Cells><Native Immunity><Natural Immunity><Natural Killer Cells><Necrosis><Necrotic><Neutrophil Infiltration><Neutrophil Recruitment><Neutrophilic Granulocyte><Neutrophilic Leukocyte><Non-Specific Immunity><Non-structural Protein><Nonspecific Immunity><Nonstructural Protein><ORFs><Open Reading Frames><P-Selectin><P-selectin glycoprotein ligand-1><P-selectin ligand protein><PSGL-1><Pathogenesis><Pathogenicity><Pathway interactions><Patients><Pattern><Peripheral Blood Cell><Physiopathology><Plasma><Plasma Serum><Plasmacytoma Growth Factor><Platelet alpha-Granule Membrane Protein><Polymorphonuclear Cell><Polymorphonuclear Leukocytes><Polymorphonuclear Neutrophils><Polysaccharides><Population><Preparation><Production><Property><Protein Binding><Protein Coding Region><Proteins><Proteomics><Pulmonary Macrophages><Pulmonary imaging><RNA Seq><RNA sequencing><RNAseq><Reactive Site><Receptor Cell><Receptor Protein><Recombinant Proteins><Recombinants><Red Pulp><Reporting><Reproducibility><Reticuloendothelial System, Serum, Plasma><Role><Route><SARS><SARS Virus><SARS corona virus><SARS coronavirus><SARS coronavirus disease><SARS-Associated Coronavirus><SARS-CoV><SARS-CoV disease><SARS-CoV-2><SARS-CoV2><SARS-Related Coronavirus><SARS-associated coronavirus 2><SARS-coronavirus-2><SARS-related coronavirus 2><SCYA17><SCYB10><SIS cytokines><Selectins><Severe Acute Respiratory Syndrome><Severe Acute Respiratory Syndrome CoV disease><Severe Acute Respiratory Syndrome Virus><Severe Acute Respiratory Syndrome corona virus><Severe Acute Respiratory Syndrome coronavirus><Severe Acute Respiratory Syndrome coronavirus disease><Severe acute respiratory syndrome coronavirus 2><Sialic Acids><Siglec-1><Signal Transduction><Signal Transduction Systems><Signaling><Site><Small Inducible Cytokine A17><Small Inducible Cytokine Subfamily A (Cys-Cys), Member 17><Spleen><Spleen Reticuloendothelial System><Splenic Red Pulp><Stellate Sinusoidal Macrophage><Strains Cell Lines><Structure of parenchyma of lung><Subcapsular Sinus><Subcellular Process><T Helper Factor><T4 Cells><T4 Lymphocytes><T8 Cells><T8 Lymphocytes><TARC><TNF><TNF A><TNF Alpha><TNF Receptor-Associated Factor 6 Gene><TNF gene><TNF receptor-associated factor 3><TNF-α><TNFA><TNFα><TQ1 Antigen><TRAF-3><TRAF3><TRAF6><TRAF6 gene><Techniques><Testing><Therapeutic><Time><Tissues><Transgenic Mice><Transmembrane Protein><Transmembrane Protein Gene><Triad><Triad Acrylic Resin><Triad resin><Tumor Necrosis Factor><Tumor Necrosis Factor-alpha><U937 Cells><Vaccine Design><Veiled Cells><Viral><Virus><Virus Replication><White Blood Cells><White Cell><Wuhan coronavirus><affinity purification><aminoacid><base><biological signal transduction><bound protein><bowel><cell mediated cytotoxicity><cell stress><cell type><cellular targeting><chemoattractant cytokine><chemokine><confocal imaging><corona virus><corona virus disease 2019><coronavirus disease 2019><cultured cell line><cytokine><disulfide bond><eosinophil><exposed human population><flow cytophotometry><gIP-10><genome mutation><glycosylation><hepatic body system><hepatic organ system><host response><human CoV><human corona virus><human coronavirus><human exposure><imaging><imaging study><immunoresponse><in vitro Model><in vivo><inhibitor><inhibitor/antagonist><insight><interferon beta 2><interleukin-23><interstitial><intra-vital microscopy><intravenous injection><intravital microscopy><lFN-Gamma><live cell image><live cell imaging><live cellular image><live cellular imaging><liver macrophage><lung imaging><lung scanning><lymph cell><lymph gland><lymph nodes><lymphnodes><lymphocyte activating factor><macrophage><membrane structure><mitochondrial><monocyte><mutant><necrocytosis><neutrophil><novel><paralog><paralogous gene><particle><pathogen><pathogenic virus><pathophysiology><pathway><peripheral blood><prevent><preventing><pulmonary><receptor><recruit><severe acute respiratory syndrome-CoV><sialic acid binding Ig-like lectin><sialoadhesin><sialyl Lewis x><siglec><social role><targeted drug therapy><targeted drug treatments><targeted therapeutic><targeted therapeutic agents><targeted therapy><targeted treatment><transcriptome sequencing><two-photon><uptake><viral multiplication><viral pathogen><viral replication><virus multiplication><white blood cell><white blood corpuscle>