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2.
Biomed Pharmacother ; 151: 113104, 2022 Jul.
Article in English | MEDLINE | ID: covidwho-1850705

ABSTRACT

The Severe Acute Respiratory Syndrome Coronavirus type 2 (SARS-CoV-2) has continuously evolved, resulting in the emergence of several variants of concern (VOCs). To study mechanisms of viral entry and potentially identify specific inhibitors, we pseudotyped lentiviral vectors with different SARS-CoV-2 VOC spike variants (D614G, Alpha, Beta, Delta, Omicron/BA.1), responsible for receptor binding and membrane fusion. These SARS-CoV-2 lentiviral pseudoviruses were applied to screen 774 FDA-approved drugs. For the assay we decided to use CaCo2 cells, since they equally allow cell entry through both the direct membrane fusion pathway mediated by TMPRSS2 and the endocytosis pathway mediated by cathepsin-L. The active molecules which showed stronger differences in their potency to inhibit certain SARS-CoV-2 VOCs included antagonists of G-protein coupled receptors, like phenothiazine-derived antipsychotic compounds such as Chlorpromazine, with highest activity against the Omicron pseudovirus. In general, our data showed that the various VOCs differ in their preferences for cell entry, and we were able to identify synergistic combinations of inhibitors. Notably, Omicron singled out by relying primarily on the endocytosis pathway while Delta preferred cell entry via membrane fusion. In conclusion, our data provide new insights into different entry preferences of SARS-CoV-2 VOCs, which might help to identify new drug targets.


Subject(s)
COVID-19 , SARS-CoV-2 , COVID-19/drug therapy , Caco-2 Cells , Drug Evaluation, Preclinical , Humans , Spike Glycoprotein, Coronavirus/metabolism
3.
Eur J Immunol ; 52(8): 1297-1307, 2022 Aug.
Article in English | MEDLINE | ID: covidwho-1782587

ABSTRACT

COVID-19, caused by SARS-CoV-2, has emerged as a global pandemic. While immune responses of the adaptive immune system have been in the focus of research, the role of NK cells in COVID-19 remains less well understood. Here, we characterized NK cell-mediated SARS-CoV-2 antibody-dependent cellular cytotoxicity (ADCC) against SARS-CoV-2 spike-1 (S1) and nucleocapsid (NC) protein. Serum samples from SARS-CoV-2 resolvers induced significant CD107a-expression by NK cells in response to S1 and NC, while serum samples from SARS-CoV-2-negative individuals did not. Furthermore, serum samples from individuals that received the BNT162b2 vaccine induced strong CD107a expression by NK cells that increased with the second vaccination and was significantly higher than observed in infected individuals. As expected, vaccine-induced responses were only directed against S1 and not against NC protein. S1-specific CD107a responses by NK cells were significantly correlated to NK cell-mediated killing of S1-expressing cells. Interestingly, screening of serum samples collected prior to the COVID-19 pandemic identified two individuals with cross-reactive antibodies against SARS-CoV-2 S1, which also induced degranulation of NK cells. Taken together, these data demonstrate that antibodies induced by SARS-CoV-2 infection and anti-SARS-CoV-2 vaccines can trigger significant NK cell-mediated ADCC activity, and identify some cross-reactive ADCC-activity against SARS-CoV-2 by endemic coronavirus-specific antibodies.


Subject(s)
COVID-19 , SARS-CoV-2 , Antibodies, Viral/metabolism , Antibody-Dependent Cell Cytotoxicity , BNT162 Vaccine , Humans , Killer Cells, Natural , Pandemics
5.
Mol Ther Methods Clin Dev ; 23: 418-423, 2021 Dec 10.
Article in English | MEDLINE | ID: covidwho-1466817

ABSTRACT

Vaccination with the adenoviral-vector-based AstraZeneca ChAdOx1 nCov-19 (Vaxzevria) vaccine is efficient and safe. However, in rare cases vaccinated individuals developed life-threatening thrombotic complications, including thrombosis in cerebral sinus and splanchnic veins. Monitoring of the applied vector in vivo represents an important precondition to study the molecular mechanisms underlying vaccine-driven adverse effects now referred to as vaccine-induced immune thrombotic thrombocytopenia (VITT). We previously have shown that digital PCR (dPCR) is an excellent tool to quantify transgene copies in vivo. Here, we present a highly sensitive dPCR for in situ quantification of ChAdOx1 nCoV-19 copies. Using this method, we quantified vector copies in human plasma 24, 72, and 168 h post vaccination and in a variety of murine tissues in an experimental vaccination model 30 min post injection. We describe a method for high-sensitivity quantitative detection of ChAdOx1 nCoV-19 with possible implications to elucidate the mechanisms of severe ChAdOx1 nCov-19 vaccine complications.

6.
Blood ; 138(22): 2256-2268, 2021 12 02.
Article in English | MEDLINE | ID: covidwho-1443788

ABSTRACT

SARS-CoV-2 vaccine ChAdOx1 nCoV-19 (AstraZeneca) causes a thromboembolic complication termed vaccine-induced immune thrombotic thrombocytopenia (VITT). Using biophysical techniques, mouse models, and analysis of VITT patient samples, we identified determinants of this vaccine-induced adverse reaction. Super-resolution microscopy visualized vaccine components forming antigenic complexes with platelet factor 4 (PF4) on platelet surfaces to which anti-PF4 antibodies obtained from VITT patients bound. PF4/vaccine complex formation was charge-driven and increased by addition of DNA. Proteomics identified substantial amounts of virus production-derived T-REx HEK293 proteins in the ethylenediaminetetraacetic acid (EDTA)-containing vaccine. Injected vaccine increased vascular leakage in mice, leading to systemic dissemination of vaccine components known to stimulate immune responses. Together, PF4/vaccine complex formation and the vaccine-stimulated proinflammatory milieu trigger a pronounced B-cell response that results in the formation of high-avidity anti-PF4 antibodies in VITT patients. The resulting high-titer anti-PF4 antibodies potently activated platelets in the presence of PF4 or DNA and polyphosphate polyanions. Anti-PF4 VITT patient antibodies also stimulated neutrophils to release neutrophil extracellular traps (NETs) in a platelet PF4-dependent manner. Biomarkers of procoagulant NETs were elevated in VITT patient serum, and NETs were visualized in abundance by immunohistochemistry in cerebral vein thrombi obtained from VITT patients. Together, vaccine-induced PF4/adenovirus aggregates and proinflammatory reactions stimulate pathologic anti-PF4 antibody production that drives thrombosis in VITT. The data support a 2-step mechanism underlying VITT that resembles the pathogenesis of (autoimmune) heparin-induced thrombocytopenia.


Subject(s)
Antigen-Antibody Complex/immunology , Autoantibodies/immunology , COVID-19/prevention & control , Capsid Proteins/adverse effects , Drug Contamination , Genetic Vectors/adverse effects , HEK293 Cells/immunology , Immunoglobulin G/immunology , Platelet Factor 4/immunology , Purpura, Thrombocytopenic, Idiopathic/etiology , SARS-CoV-2 , Spike Glycoprotein, Coronavirus/adverse effects , Adenoviridae/immunology , Animals , Antigen-Antibody Complex/ultrastructure , Autoantibodies/biosynthesis , Capillary Leak Syndrome/etiology , Capsid Proteins/immunology , Cell Line, Transformed , /immunology , Dynamic Light Scattering , Epitopes/chemistry , Epitopes/immunology , Extracellular Traps/immunology , Extravasation of Diagnostic and Therapeutic Materials/etiology , Genetic Vectors/immunology , HEK293 Cells/chemistry , Humans , Imaging, Three-Dimensional , Immunoglobulin G/biosynthesis , Inflammation , Mice , Microscopy/methods , Platelet Activation , Proteomics , Purpura, Thrombocytopenic, Idiopathic/blood , Purpura, Thrombocytopenic, Idiopathic/immunology , Sinus Thrombosis, Intracranial/diagnostic imaging , Sinus Thrombosis, Intracranial/immunology , Spike Glycoprotein, Coronavirus/immunology , Virus Cultivation
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