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1.
Anal Bioanal Chem ; 414(5): 1773-1785, 2022 Feb.
Article in English | MEDLINE | ID: covidwho-1653430

ABSTRACT

Nucleic acid tests to detect the SARS-CoV-2 virus have been performed worldwide since the beginning of the COVID-19 pandemic. For the quality assessment of testing laboratories and the performance evaluation of molecular diagnosis products, reference materials (RMs) are required. In this work, we report the production of a lentiviral SARS-CoV-2 RM containing approximately 12 kilobases of its genome including common diagnostics targets such as RdRp, N, E, and S genes. The RM was measured with multiple assays using two different digital PCR platforms. To measure the homogeneity and stability of the lentiviral SARS-CoV-2 RM, reverse transcription droplet digital PCR (RT-ddPCR) was used with in-house duplex assays. The copy number concentration of each target gene in the extracted RNA solution was then converted to that of the RM solution. Their copy number values are measured to be from 1.5 × 105 to 2.0 × 105 copies/mL. The RM has a between-bottle homogeneity of 4.80-8.23% and is stable at 4 °C for 1 week and at -70 °C for 6 months. The lentiviral SARS-CoV-2 RM closely mimics real samples that undergo identical pre-analytical processes for SARS-CoV-2 molecular testing. By offering accurate reference values for the absolute copy number of viral target genes, the developed RM can be used to improve the reliability of SARS-CoV-2 molecular testing.


Subject(s)
COVID-19 Nucleic Acid Testing/methods , COVID-19/diagnosis , Genome, Viral , RNA, Viral/genetics , Reagent Kits, Diagnostic/standards , SARS-CoV-2/genetics , COVID-19/virology , COVID-19 Nucleic Acid Testing/standards , Coronavirus Envelope Proteins/genetics , Coronavirus Envelope Proteins/metabolism , Coronavirus Nucleocapsid Proteins/genetics , Coronavirus Nucleocapsid Proteins/metabolism , Coronavirus RNA-Dependent RNA Polymerase/genetics , Coronavirus RNA-Dependent RNA Polymerase/metabolism , Gene Dosage , Gene Expression , Humans , Jurkat Cells , Lentivirus/genetics , Lentivirus/metabolism , Phosphoproteins/genetics , Phosphoproteins/metabolism , RNA, Viral/metabolism , RNA, Viral/standards , Reagent Kits, Diagnostic/supply & distribution , Reference Standards , Reproducibility of Results , Spike Glycoprotein, Coronavirus/genetics , Spike Glycoprotein, Coronavirus/metabolism , Viral Genome Packaging
2.
Cell Rep Med ; 3(2): 100510, 2022 02 15.
Article in English | MEDLINE | ID: covidwho-1636907

ABSTRACT

Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) variants of concern (VOCs) exhibit escape from neutralizing antibodies, causing concern about vaccine effectiveness. However, while non-neutralizing cytotoxic functions of antibodies are associated with improved disease outcome and vaccine protection, Fc effector function escape from VOCs is poorly defined. Furthermore, whether VOCs trigger Fc functions with altered specificity, as has been reported for neutralization, is unknown. Here, we demonstrate that the Beta VOC partially evades Fc effector activity in individuals infected with the original (D614G) variant. However, not all functions are equivalently affected, suggesting differential targeting by antibodies mediating distinct Fc functions. Furthermore, Beta and Delta infection trigger responses with significantly improved Fc cross-reactivity against global VOCs compared with D614G-infected or Ad26.COV2.S-vaccinated individuals. This suggests that, as for neutralization, the infecting spike sequence affects Fc effector function. These data have important implications for vaccine strategies that incorporate VOCs, suggesting these may induce broader Fc effector responses.


Subject(s)
Antibodies, Neutralizing/immunology , Antibodies, Viral/immunology , COVID-19/immunology , Immunoglobulin Fc Fragments/immunology , SARS-CoV-2/immunology , /immunology , Adult , Aged , COVID-19/blood , COVID-19/prevention & control , COVID-19/virology , Cohort Studies , Cross Reactions , Female , HEK293 Cells , Humans , Jurkat Cells , Male , Middle Aged , Neutralization Tests , Protein Binding , Spike Glycoprotein, Coronavirus/immunology , THP-1 Cells , Treatment Outcome , Vaccination/methods
3.
Sci Rep ; 12(1): 586, 2022 01 12.
Article in English | MEDLINE | ID: covidwho-1621273

ABSTRACT

The performance of a laboratory-developed IgG/IgA flow cytometry-based immunoassay (FCI) using Jurkat T cells stably expressing full-length native S protein was compared against Elecsys electrochemiluminiscent (ECLIA) Anti-SARS-CoV-2 S (Roche Diagnostics, Pleasanton, CA, USA), and Liaison SARS-CoV-2 TrimericS IgG chemiluminiscent assay (CLIA) (Diasorin S.p.a, Saluggia, IT) for detection of SARS-CoV-2-specific antibodies. A total of 225 serum/plasma specimens from 120 acute or convalescent COVID-19 individuals were included. Overall, IgG/IgA-FCI yielded the highest number of positives (n = 179), followed by IgA-FCI (n = 177), Roche ECLIA (n = 175), IgG-FCI (n = 172) and Diasorin CLIA (n = 154). For sera collected early after the onset of symptoms (within 15 days) IgG/IgA-FCI also returned the highest number of positive results (52/72; 72.2%). Positive percent agreement between FCI and compared immunoassays was highest for Roche ECLIA, ranging from 96.1 (IgG/IgA-FCI) to 97.7% (IgG-FCI), whereas negative percent agreement was higher between FCI and Diasosin CLIA, regardless of antibody isotype. The data suggest that FCI may outperform Roche ECLIA and Diasorin CLIA in terms of clinical sensitivity for serological diagnosis of SARS-CoV-2 infection.


Subject(s)
Antibodies, Viral/blood , Flow Cytometry/methods , Immunoassay/methods , SARS-CoV-2/immunology , Spike Glycoprotein, Coronavirus/immunology , Antibodies, Viral/immunology , COVID-19 Serological Testing , Humans , Jurkat Cells , Retrospective Studies , Sensitivity and Specificity
4.
J Med Chem ; 64(19): 14332-14343, 2021 10 14.
Article in English | MEDLINE | ID: covidwho-1621195

ABSTRACT

In addition to a variety of viral-glycoprotein receptors (e.g., heparan sulfate, Niemann-Pick C1, etc.), dendritic cell-specific intercellular adhesion molecule-3-grabbing nonintegrin (DC-SIGN), from the C-type lectin receptor family, plays one of the most important pathogenic functions for a wide range of viruses (e.g., Ebola, human cytomegalovirus (HCMV), HIV-1, severe acute respiratory syndrome coronavirus 2, etc.) that invade host cells before replication; thus, its inhibition represents a relevant extracellular antiviral therapy. We report two novel p-tBu-calixarene glycoclusters 1 and 2, bearing tetrahydroxamic acid groups, which exhibit micromolar inhibition of soluble DC-SIGN binding and provide nanomolar IC50 inhibition of both DC-SIGN-dependent Jurkat cis-cell infection by viral particle pseudotyped with Ebola virus glycoprotein and the HCMV-gB-recombinant glycoprotein interaction with monocyte-derived dendritic cells expressing DC-SIGN. A unique cooperative involvement of sugar, linker, and calixarene core is likely behind the strong avidity of DC-SIGN for these low-valent systems. We claim herein new promising candidates for the rational development of a large spectrum of antiviral therapeutics.


Subject(s)
Calixarenes/chemistry , Cell Adhesion Molecules/antagonists & inhibitors , Glycoconjugates/metabolism , Glycoproteins/antagonists & inhibitors , Hydroxamic Acids/chemistry , Lectins, C-Type/antagonists & inhibitors , Phenols/chemistry , Receptors, Cell Surface/antagonists & inhibitors , Viral Proteins/antagonists & inhibitors , Antiviral Agents/chemistry , Antiviral Agents/metabolism , Antiviral Agents/pharmacology , Cell Adhesion Molecules/metabolism , Cell Line , Cytomegalovirus/metabolism , Dendritic Cells/cytology , Dendritic Cells/metabolism , Ebolavirus/physiology , Glycoconjugates/chemistry , Glycoconjugates/pharmacology , Glycoproteins/genetics , Glycoproteins/metabolism , Humans , Jurkat Cells , Lectins, C-Type/metabolism , Models, Biological , Protein Binding , Receptors, Cell Surface/metabolism , Recombinant Proteins/biosynthesis , Recombinant Proteins/chemistry , Recombinant Proteins/isolation & purification , Viral Proteins/genetics , Viral Proteins/metabolism
5.
Nat Commun ; 13(1): 19, 2022 01 10.
Article in English | MEDLINE | ID: covidwho-1616981

ABSTRACT

T cells play a vital role in combatting SARS-CoV-2 and forming long-term memory responses. Whereas extensive structural information is available on neutralizing antibodies against SARS-CoV-2, such information on SARS-CoV-2-specific T-cell receptors (TCRs) bound to their peptide-MHC targets is lacking. Here we determine the structures of a public and a private TCR from COVID-19 convalescent patients in complex with HLA-A2 and two SARS-CoV-2 spike protein epitopes (YLQ and RLQ). The structures reveal the basis for selection of particular TRAV and TRBV germline genes by the public but not the private TCR, and for the ability of the TCRs to recognize natural variants of RLQ but not YLQ. Neither TCR recognizes homologous epitopes from human seasonal coronaviruses. By elucidating the mechanism for TCR recognition of an immunodominant yet variable epitope (YLQ) and a conserved but less commonly targeted epitope (RLQ), this study can inform prospective efforts to design vaccines to elicit pan-coronavirus immunity.


Subject(s)
COVID-19/immunology , Epitopes, T-Lymphocyte/immunology , HLA-A2 Antigen/immunology , Receptors, Antigen, T-Cell/immunology , SARS-CoV-2/immunology , Spike Glycoprotein, Coronavirus/immunology , CD4-Positive T-Lymphocytes/immunology , CD4-Positive T-Lymphocytes/metabolism , CD4-Positive T-Lymphocytes/virology , CD8-Positive T-Lymphocytes/immunology , CD8-Positive T-Lymphocytes/metabolism , CD8-Positive T-Lymphocytes/virology , COVID-19/virology , Epitopes, T-Lymphocyte/metabolism , HLA-A2 Antigen/chemistry , HLA-A2 Antigen/metabolism , Humans , Immunodominant Epitopes/immunology , Immunodominant Epitopes/metabolism , Jurkat Cells , K562 Cells , Peptides/chemistry , Peptides/immunology , Peptides/metabolism , Protein Binding , Protein Conformation , Receptors, Antigen, T-Cell/chemistry , Receptors, Antigen, T-Cell/metabolism , SARS-CoV-2/metabolism , SARS-CoV-2/physiology , Spike Glycoprotein, Coronavirus/metabolism , Surface Plasmon Resonance/methods
6.
Cell ; 185(4): 603-613.e15, 2022 02 17.
Article in English | MEDLINE | ID: covidwho-1588149

ABSTRACT

SARS-CoV-2 mRNA vaccines induce robust anti-spike (S) antibody and CD4+ T cell responses. It is not yet clear whether vaccine-induced follicular helper CD4+ T (TFH) cell responses contribute to this outstanding immunogenicity. Using fine-needle aspiration of draining axillary lymph nodes from individuals who received the BNT162b2 mRNA vaccine, we evaluated the T cell receptor sequences and phenotype of lymph node TFH. Mining of the responding TFH T cell receptor repertoire revealed a strikingly immunodominant HLA-DPB1∗04-restricted response to S167-180 in individuals with this allele, which is among the most common HLA alleles in humans. Paired blood and lymph node specimens show that while circulating S-specific TFH cells peak one week after the second immunization, S-specific TFH persist at nearly constant frequencies for at least six months. Collectively, our results underscore the key role that robust TFH cell responses play in establishing long-term immunity by this efficacious human vaccine.


Subject(s)
COVID-19/immunology , COVID-19/virology , Immunity/immunology , SARS-CoV-2/immunology , T Follicular Helper Cells/immunology , Vaccination , Vaccines, Synthetic/immunology , /immunology , Adult , B-Lymphocytes/immunology , COVID-19/blood , Clone Cells , Cohort Studies , Cytokines/metabolism , Female , Germinal Center/immunology , HLA-DP beta-Chains/immunology , Humans , Immunodominant Epitopes/immunology , Jurkat Cells , Lymph Nodes/metabolism , Male , Middle Aged , Peptides/chemistry , Peptides/metabolism , Protein Multimerization , Receptors, Antigen, T-Cell/metabolism
7.
Cell Rep ; 31(11): 107772, 2020 06 16.
Article in English | MEDLINE | ID: covidwho-1385222

ABSTRACT

ISG15 is a ubiquitin-like modifier that also functions extracellularly, signaling through the LFA-1 integrin to promote interferon (IFN)-γ release from natural killer (NK) and T cells. The signals that lead to the production of extracellular ISG15 and the relationship between its two core functions remain unclear. We show that both epithelial cells and lymphocytes can secrete ISG15, which then signals in either an autocrine or paracrine manner to LFA-1-expressing cells. Microbial pathogens and Toll-like receptor (TLR) agonists result in both IFN-ß-dependent and -independent secretion of ISG15, and residues required for ISG15 secretion are mapped. Intracellular ISGylation inhibits secretion, and viral effector proteins, influenza B NS1, and viral de-ISGylases, including SARS-CoV-2 PLpro, have opposing effects on secretion of ISG15. These results establish extracellular ISG15 as a cytokine-like protein that bridges early innate and IFN-γ-dependent immune responses, and indicate that pathogens have evolved to differentially inhibit the intracellular and extracellular functions of ISG15.


Subject(s)
Cytokines/metabolism , Signal Transduction , Ubiquitins/metabolism , Animals , HEK293 Cells , Humans , Influenza, Human/immunology , Influenza, Human/metabolism , Interferon-gamma/immunology , Interferon-gamma/metabolism , Jurkat Cells , Mice , Mice, Inbred C57BL , Mycobacterium Infections/immunology , Mycobacterium Infections/metabolism , Pathogen-Associated Molecular Pattern Molecules , Typhoid Fever/immunology , Typhoid Fever/metabolism , Viral Nonstructural Proteins/metabolism
8.
Science ; 373(6559): 1109-1116, 2021 Sep 03.
Article in English | MEDLINE | ID: covidwho-1341301

ABSTRACT

The spillovers of betacoronaviruses in humans and the emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants highlight the need for broad coronavirus countermeasures. We describe five monoclonal antibodies (mAbs) cross-reacting with the stem helix of multiple betacoronavirus spike glycoproteins isolated from COVID-19 convalescent individuals. Using structural and functional studies, we show that the mAb with the greatest breadth (S2P6) neutralizes pseudotyped viruses from three different subgenera through the inhibition of membrane fusion, and we delineate the molecular basis for its cross-reactivity. S2P6 reduces viral burden in hamsters challenged with SARS-CoV-2 through viral neutralization and Fc-mediated effector functions. Stem helix antibodies are rare, oftentimes of narrow specificity, and can acquire neutralization breadth through somatic mutations. These data provide a framework for structure-guided design of pan-betacoronavirus vaccines eliciting broad protection.


Subject(s)
Antibodies, Monoclonal/immunology , Antibodies, Neutralizing/immunology , Betacoronavirus/immunology , Spike Glycoprotein, Coronavirus/chemistry , Spike Glycoprotein, Coronavirus/immunology , Viral Vaccines/immunology , Virus Internalization , Animals , Antibodies, Monoclonal/isolation & purification , Antibodies, Neutralizing/isolation & purification , Convalescence , Cricetinae , Cross Reactions , Humans , Immunoglobulin Fab Fragments/immunology , Immunoglobulin Fc Fragments/immunology , Jurkat Cells , Lung/immunology , Membrane Fusion/immunology , Neutralization Tests , Peptide Mapping , Protein Conformation, alpha-Helical , SARS-CoV-2/immunology , Spike Glycoprotein, Coronavirus/genetics , Viral Load/immunology
9.
J Clin Invest ; 131(10)2021 05 17.
Article in English | MEDLINE | ID: covidwho-1255762

ABSTRACT

BACKGROUNDRecent studies have reported T cell immunity to the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in unexposed donors, possibly due to crossrecognition by T cells specific for common cold coronaviruses (CCCs). True T cell crossreactivity, defined as the recognition by a single TCR of more than one distinct peptide-MHC ligand, has never been shown in the context of SARS-CoV-2.METHODSWe used the viral functional expansion of specific T cells (ViraFEST) platform to identify T cell responses crossreactive for the spike (S) glycoproteins of SARS-CoV-2 and CCCs at the T cell receptor (TCR) clonotype level in convalescent COVID-19 patients (CCPs) and SARS-CoV-2-unexposed donors. Confirmation of SARS-CoV-2/CCC crossreactivity and assessments of functional avidity were performed using a TCR cloning and transfection system.RESULTSMemory CD4+ T cell clonotypes that crossrecognized the S proteins of SARS-CoV-2 and at least one other CCC were detected in 65% of CCPs and unexposed donors. Several of these TCRs were shared among multiple donors. Crossreactive T cells demonstrated significantly impaired SARS-CoV-2-specific proliferation in vitro relative to monospecific CD4+ T cells, which was consistent with lower functional avidity of their TCRs for SARS-CoV-2 relative to CCC.CONCLUSIONSOur data confirm, for what we believe is the first time, the existence of unique memory CD4+ T cell clonotypes crossrecognizing SARS-CoV-2 and CCCs. The lower avidity of crossreactive TCRs for SARS-CoV-2 may be the result of antigenic imprinting, such that preexisting CCC-specific memory T cells have reduced expansive capacity upon SARS-CoV-2 infection. Further studies are needed to determine how these crossreactive T cell responses affect clinical outcomes in COVID-19 patients.FUNDINGNIH funding (U54CA260492, P30CA006973, P41EB028239, R01AI153349, R01AI145435-A1, R21AI149760, and U19A1088791) was provided by the National Institute of Allergy and Infectious Diseases, the National Cancer Institute, and the National Institute of Biomedical Imaging and Bioengineering. The Bloomberg~Kimmel Institute for Cancer Immunotherapy, The Johns Hopkins University Provost, and The Bill and Melinda Gates Foundation provided funding for this study.


Subject(s)
CD4-Positive T-Lymphocytes/immunology , COVID-19/immunology , Epitopes, T-Lymphocyte/immunology , Immunologic Memory , Receptors, Antigen, T-Cell/immunology , SARS-CoV-2/immunology , Adult , Aged , Cross Reactions , Female , Humans , Jurkat Cells , Male , Middle Aged
10.
J Clin Invest ; 131(10)2021 05 17.
Article in English | MEDLINE | ID: covidwho-1238630

ABSTRACT

BACKGROUNDRecent studies have reported T cell immunity to the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in unexposed donors, possibly due to crossrecognition by T cells specific for common cold coronaviruses (CCCs). True T cell crossreactivity, defined as the recognition by a single TCR of more than one distinct peptide-MHC ligand, has never been shown in the context of SARS-CoV-2.METHODSWe used the viral functional expansion of specific T cells (ViraFEST) platform to identify T cell responses crossreactive for the spike (S) glycoproteins of SARS-CoV-2 and CCCs at the T cell receptor (TCR) clonotype level in convalescent COVID-19 patients (CCPs) and SARS-CoV-2-unexposed donors. Confirmation of SARS-CoV-2/CCC crossreactivity and assessments of functional avidity were performed using a TCR cloning and transfection system.RESULTSMemory CD4+ T cell clonotypes that crossrecognized the S proteins of SARS-CoV-2 and at least one other CCC were detected in 65% of CCPs and unexposed donors. Several of these TCRs were shared among multiple donors. Crossreactive T cells demonstrated significantly impaired SARS-CoV-2-specific proliferation in vitro relative to monospecific CD4+ T cells, which was consistent with lower functional avidity of their TCRs for SARS-CoV-2 relative to CCC.CONCLUSIONSOur data confirm, for what we believe is the first time, the existence of unique memory CD4+ T cell clonotypes crossrecognizing SARS-CoV-2 and CCCs. The lower avidity of crossreactive TCRs for SARS-CoV-2 may be the result of antigenic imprinting, such that preexisting CCC-specific memory T cells have reduced expansive capacity upon SARS-CoV-2 infection. Further studies are needed to determine how these crossreactive T cell responses affect clinical outcomes in COVID-19 patients.FUNDINGNIH funding (U54CA260492, P30CA006973, P41EB028239, R01AI153349, R01AI145435-A1, R21AI149760, and U19A1088791) was provided by the National Institute of Allergy and Infectious Diseases, the National Cancer Institute, and the National Institute of Biomedical Imaging and Bioengineering. The Bloomberg~Kimmel Institute for Cancer Immunotherapy, The Johns Hopkins University Provost, and The Bill and Melinda Gates Foundation provided funding for this study.


Subject(s)
CD4-Positive T-Lymphocytes/immunology , COVID-19/immunology , Epitopes, T-Lymphocyte/immunology , Immunologic Memory , Receptors, Antigen, T-Cell/immunology , SARS-CoV-2/immunology , Adult , Aged , Cross Reactions , Female , Humans , Jurkat Cells , Male , Middle Aged
11.
Sci Rep ; 11(1): 10716, 2021 05 21.
Article in English | MEDLINE | ID: covidwho-1238014

ABSTRACT

SARS-CoV-2 is the virus that causes the disease called COVID-19, which has caused the worst pandemic of the century. Both, to know the immunological status of general population and to evaluate the efficacy of the vaccination process that is taking place around the world, serological tests represent a key tool. Classic serological tests, based on colorimetric techniques, such as ELISA or CLIA, continue to be the most widely used option. However, a real improvement in results is still needed. We developed a highly sensitive and specific FCM assay that allows the detection of IgG and IgA antibodies, directed against the native and functional S-protein of SARS-CoV-2 exposed on the membrane of a transfected cell line, up to 8 months after infection.


Subject(s)
Antibodies, Viral/immunology , COVID-19/immunology , Flow Cytometry , Immunoglobulin A/immunology , Immunoglobulin G/immunology , SARS-CoV-2/immunology , Spike Glycoprotein, Coronavirus/immunology , Adult , Aged , Female , Humans , Jurkat Cells , Male , Middle Aged
12.
PLoS Pathog ; 17(5): e1009576, 2021 05.
Article in English | MEDLINE | ID: covidwho-1236599

ABSTRACT

The efficient spread of SARS-CoV-2 resulted in a unique pandemic in modern history. Despite early identification of ACE2 as the receptor for viral spike protein, much remains to be understood about the molecular events behind viral dissemination. We evaluated the contribution of C-type lectin receptors (CLRS) of antigen-presenting cells, widely present in respiratory mucosa and lung tissue. DC-SIGN, L-SIGN, Langerin and MGL bind to diverse glycans of the spike using multiple interaction areas. Using pseudovirus and cells derived from monocytes or T-lymphocytes, we demonstrate that while virus capture by the CLRs examined does not allow direct cell infection, DC/L-SIGN, among these receptors, promote virus transfer to permissive ACE2+ Vero E6 cells. A glycomimetic compound designed against DC-SIGN, enable inhibition of this process. These data have been then confirmed using authentic SARS-CoV-2 virus and human respiratory cell lines. Thus, we described a mechanism potentiating viral spreading of infection.


Subject(s)
COVID-19/transmission , Lectins, C-Type/metabolism , SARS-CoV-2/metabolism , Spike Glycoprotein, Coronavirus/metabolism , Animals , Antigens, CD/metabolism , COVID-19/prevention & control , Cell Adhesion Molecules/metabolism , Cell Line , Chlorocebus aethiops , Humans , Jurkat Cells , Lung/metabolism , Mannose-Binding Lectins/metabolism , Mannosides/pharmacology , Protein Binding/drug effects , Receptors, Cell Surface/metabolism , Respiratory Mucosa/metabolism , Vero Cells
13.
Cell Death Differ ; 28(9): 2765-2777, 2021 09.
Article in English | MEDLINE | ID: covidwho-1195611

ABSTRACT

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virus is highly contagious and causes lymphocytopenia, but the underlying mechanisms are poorly understood. We demonstrate here that heterotypic cell-in-cell structures with lymphocytes inside multinucleate syncytia are prevalent in the lung tissues of coronavirus disease 2019 (COVID-19) patients. These unique cellular structures are a direct result of SARS-CoV-2 infection, as the expression of the SARS-CoV-2 spike glycoprotein is sufficient to induce a rapid (~45.1 nm/s) membrane fusion to produce syncytium, which could readily internalize multiple lines of lymphocytes to form typical cell-in-cell structures, remarkably leading to the death of internalized cells. This membrane fusion is dictated by a bi-arginine motif within the polybasic S1/S2 cleavage site, which is frequently present in the surface glycoprotein of most highly contagious viruses. Moreover, candidate anti-viral drugs could efficiently inhibit spike glycoprotein processing, membrane fusion, and cell-in-cell formation. Together, we delineate a molecular and cellular rationale for SARS-CoV-2 pathogenesis and identify novel targets for COVID-19 therapy.


Subject(s)
COVID-19/virology , Giant Cells/virology , Lymphocytes/virology , SARS-CoV-2/metabolism , SARS-CoV-2/pathogenicity , Spike Glycoprotein, Coronavirus/metabolism , COVID-19/pathology , Cell Line , Cell Line, Tumor , Giant Cells/pathology , HEK293 Cells , HeLa Cells , Humans , Jurkat Cells , K562 Cells , Lymphocytes/pathology , Virus Internalization , Virus Replication/genetics
14.
Immunohorizons ; 5(2): 70-80, 2021 02 04.
Article in English | MEDLINE | ID: covidwho-1067831

ABSTRACT

Tyrosine kinase 2 (TYK2) is a member of the JAK family of nonreceptor tyrosine kinase, together with JAK1, JAK2, and JAK3. JAKs are important signaling mediators of many proinflammatory cytokines and represent compelling pharmacological targets for autoimmune and inflammatory diseases. Pan-acting small-molecule JAK inhibitors were approved for the treatment of rheumatoid arthritis and ulcerative colitis. However, their limited selectivity among JAK members have led to undesirable side effects, driving a search toward specific JAK inhibitors. Recently, TYK2 has emerged as a target of choice for the treatment of autoimmune diseases and severe COVID-19 with an optimum balance between efficacy and safety, based on observations from human genetics studies and clinical outcomes of several agents targeting cytokine pathways for which TYK2 plays an essential role. In this article, we address selective targeting of TYK2 from the genetic sequence space through development of antisense oligonucleotides (ASOs) against TYK2 mRNA. Potent ASO candidates were identified from the screening of over 200 ASOs using locked nucleic acid gapmer design. The lead ASOs exhibited potent and selective knockdown of TYK2 mRNA and protein across a panel of model human cell lines in a dose-dependent manner, showing no reduction in the mRNA and protein expression levels of other JAK paralogs. In agreement with the depletion of TYK2 proteins, several TYK2-mediated cytokine signaling pathways, including IFN-α and IL-12, were inhibited upon ASO treatment. Our results established the TYK2 ASOs as investigational tool compound and potential therapeutic agent for the treatment of autoimmune diseases and severe COVID-19.


Subject(s)
Autoimmune Diseases/drug therapy , COVID-19/drug therapy , Janus Kinase Inhibitors/therapeutic use , Oligonucleotides, Antisense/genetics , RNA, Messenger/genetics , SARS-CoV-2/physiology , TYK2 Kinase/genetics , Disease Progression , Gene Knockdown Techniques , Humans , Interferon-alpha/metabolism , Interleukin-12/metabolism , Jurkat Cells , Molecular Targeted Therapy , Oligonucleotides, Antisense/therapeutic use , Signal Transduction
15.
EMBO Mol Med ; 13(3): e13549, 2021 03 05.
Article in English | MEDLINE | ID: covidwho-1038772

ABSTRACT

A correct identification of seropositive individuals for the severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) infection is of paramount relevance to assess the degree of protection of a human population to present and future outbreaks of the COVID-19 pandemic. We describe here a sensitive and quantitative flow cytometry method using the cytometer-friendly non-adherent Jurkat T-cell line that stably expresses the full-length native spike "S" protein of SARS-CoV-2 and a truncated form of the human EGFR that serves a normalizing role. S protein and huEGFRt coding sequences are separated by a T2A self-cleaving sequence, allowing to accurately quantify the presence of anti-S immunoglobulins by calculating a score based on the ratio of fluorescence intensities obtained by double-staining with the test sera and anti-EGFR. The method allows to detect immune individuals regardless of the result of other serological tests or even repeated PCR monitoring. As examples of its use, we show that as much as 28% of the personnel working at the CBMSO in Madrid is already immune. Additionally, we show that anti-S antibodies with protective neutralizing activity are long-lasting and can be detected in sera 8 months after infection.


Subject(s)
Antibodies, Neutralizing/blood , Antibodies, Viral/blood , COVID-19 Serological Testing/methods , COVID-19/diagnosis , COVID-19/immunology , Flow Cytometry/methods , SARS-CoV-2/immunology , Spike Glycoprotein, Coronavirus/immunology , Adult , COVID-19/virology , COVID-19 Serological Testing/statistics & numerical data , Enzyme-Linked Immunosorbent Assay , ErbB Receptors/genetics , Female , Flow Cytometry/statistics & numerical data , Hep G2 Cells , Humans , Jurkat Cells , Male , Middle Aged , Neutralization Tests , Pandemics , Polymerase Chain Reaction , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/immunology , SARS-CoV-2/genetics , Spike Glycoprotein, Coronavirus/genetics
16.
Elife ; 92020 12 15.
Article in English | MEDLINE | ID: covidwho-977809

ABSTRACT

Vaccines are powerful tools to develop immune memory to infectious diseases and prevent excess mortality. In older adults, however vaccines are generally less efficacious and the molecular mechanisms that underpin this remain largely unknown. Autophagy, a process known to prevent aging, is critical for the maintenance of immune memory in mice. Here, we show that autophagy is specifically induced in vaccine-induced antigen-specific CD8+ T cells in healthy human volunteers. In addition, reduced IFNγ secretion by RSV-induced T cells in older vaccinees correlates with low autophagy levels. We demonstrate that levels of the endogenous autophagy-inducing metabolite spermidine fall in human T cells with age. Spermidine supplementation in T cells from old donors recovers their autophagy level and function, similar to young donors' cells, in which spermidine biosynthesis has been inhibited. Finally, our data show that endogenous spermidine maintains autophagy via the translation factor eIF5A and transcription factor TFEB. In summary, we have provided evidence for the importance of autophagy in vaccine immunogenicity in older humans and uncovered two novel drug targets that may increase vaccination efficiency in the aging context.


Subject(s)
Aging/immunology , Autophagy/immunology , CD8-Positive T-Lymphocytes/immunology , Respiratory Syncytial Virus Vaccines/immunology , Spermidine/pharmacology , Adjuvants, Immunologic/pharmacology , Adult , Aged , Animals , Basic Helix-Loop-Helix Leucine Zipper Transcription Factors/metabolism , Cell Line, Tumor , Humans , Immunologic Memory/immunology , Interferon-gamma/blood , Jurkat Cells , Mice , Mice, Inbred C57BL , Mice, Knockout , Middle Aged , Peptide Initiation Factors/metabolism , RNA-Binding Proteins/metabolism , Respiratory Syncytial Viruses/immunology , Spermidine/blood , Vaccination , Young Adult
17.
Cell Death Differ ; 28(5): 1610-1626, 2021 05.
Article in English | MEDLINE | ID: covidwho-957566

ABSTRACT

The receptor-interacting serine/threonine protein kinase 1 (RIPK1) is a key mediator of regulated cell death and inflammation. Recent studies suggest that RIPK1 inhibition would fundamentally improve the therapy of RIPK1-dependent organ damage in stroke, myocardial infarction, kidney failure, and systemic inflammatory response syndrome. Additionally, it could ameliorate or prevent multi-organ failure induced by cytokine release in the context of hyperinflammation, as seen in COVID-19 patients. Therefore, we searched for a RIPK1 inhibitor and present the aromatic antiepileptic and FDA-approved drug primidone (Liskantin®) as a potent inhibitor of RIPK1 activation in vitro and in a murine model of TNFα-induced shock, which mimics the hyperinflammatory state of cytokine release syndrome. Furthermore, we detected for the first time RIPK1 activation in the respiratory tract epithelium of hospitalized patients who tested positive for SARS-CoV-2 infection. Our data provide a strong rationale for evaluating the drug primidone in conditions of hyperinflammation in humans.


Subject(s)
COVID-19/enzymology , Primidone/pharmacology , Receptor-Interacting Protein Serine-Threonine Kinases/metabolism , SARS-CoV-2/metabolism , Animals , COVID-19/drug therapy , COVID-19/pathology , Cell Death/drug effects , HEK293 Cells , HT29 Cells , Humans , Inflammation/drug therapy , Inflammation/enzymology , Inflammation/pathology , Jurkat Cells , Mice , NIH 3T3 Cells , U937 Cells
18.
Sci Rep ; 10(1): 16615, 2020 10 06.
Article in English | MEDLINE | ID: covidwho-834915

ABSTRACT

Middle East Respiratory Syndrome coronavirus (MERS-CoV) is a highly virulent pathogen that causes Middle East Respiratory Syndrome (MERS). Anti-MERS-CoV antibodies play an integral role in the prevention and treatment against MERS-CoV infections. Bioactivity is a key quality attribute of therapeutic antibodies, and high accuracy and precision are required. The major methods for evaluating the antiviral effect of antiviral antibodies include neutralization assays using live viruses or pseudoviruses are highly variable. Recent studies have demonstrated that the antibody-dependent cellular cytotoxicity (ADCC) activity of antiviral antibodies is more consistent with the virus clearance effect in vivo than neutralization activity. However, no reports evaluating the ADCC activity of anti-MERS antibodies have been published to date. Here, we describe the development of a robust and reliable cell-based reporter gene assay for the determination of ADCC activity of anti-MERS antibodies using 293T/MERS cells stably expressing the spike protein of MERS-CoV (MERS-S) as target cells and the engineered Jurkat/NFAT-luc/FcγRIIIa stably expressing FcγRIIIA and NFAT reporter gene as effector cells. According to the ICH-Q2 analytical method guidelines, we carefully optimized the experimental conditions and assessed the performance of our assay. In addition, we found that the ADCC activity of afucosylated anti-MERS antibodies is higher than their fucosylated counterparts. The establishment of this ADCC determination system provides a novel method for evaluating the bioactivity of anti-MERS antibodies and improving ADCC activity through modification of N-glycosylation of the Fc segment.


Subject(s)
Antibodies, Viral/analysis , Antibody-Dependent Cell Cytotoxicity/immunology , Coronavirus Infections/immunology , Cytotoxicity Tests, Immunologic/methods , Middle East Respiratory Syndrome Coronavirus/immunology , Middle East Respiratory Syndrome Coronavirus/metabolism , Antibodies, Neutralizing/immunology , Antibodies, Viral/immunology , Coronavirus Infections/virology , Genes, Reporter , HEK293 Cells , Humans , Jurkat Cells , Luciferases/genetics , Middle East Respiratory Syndrome Coronavirus/genetics , NFATC Transcription Factors/genetics , Receptors, IgG/genetics , Receptors, IgG/immunology , Response Elements , Spike Glycoprotein, Coronavirus/metabolism , Transfection
19.
Comput Biol Chem ; 89: 107376, 2020 Dec.
Article in English | MEDLINE | ID: covidwho-753733

ABSTRACT

Human ubiquitin carboxyl-terminal hydrolase-2 (USP2) inhibitors, such as thiopurine analogs, have been reported to inhibit SARS-CoV papain-like proteases (PLpro). The PLpro have significant functional implications in the innate immune response during SARS-CoV-2 infection and considered an important antiviral target. Both proteases share strikingly similar USP fold with right-handed thumb-palm-fingers structural scaffold and conserved catalytic triad Cys-His-Asp/Asn. In this urgency situation of COVID-19 outbreak, there is a lack of in-vitro facilities readily available to test SARS-CoV-2 inhibitors in whole-cell assays. Therefore, we adopted an alternate route to identify potential USP2 inhibitor through integrated in-silico efforts. After an extensive virtual screening protocol, the best compounds were selected and tested. The compound Z93 showed significant IC50 value against Jurkat (9.67 µM) and MOTL-4 cells (11.8 µM). The binding mode of Z93 was extensively analyzed through molecular docking, followed by MD simulations, and molecular interactions were compared with SARS-CoV-2. The relative binding poses of Z93 fitted well in the binding site of both proteases and showed consensus π-π stacking and H-bond interactions with histidine and aspartate/asparagine residues of the catalytic triad. These results led us to speculate that compound Z93 might be the first potential chemical lead against SARS-CoV-2 PLpro, which warrants in-vitro evaluations.


Subject(s)
Antiviral Agents/pharmacology , COVID-19/drug therapy , Coronavirus 3C Proteases/antagonists & inhibitors , Coronavirus Protease Inhibitors/pharmacology , SARS-CoV-2/drug effects , SARS-CoV-2/enzymology , Ubiquitin Thiolesterase/antagonists & inhibitors , Antiviral Agents/chemistry , COVID-19/virology , Cell Line, Tumor , Coronavirus 3C Proteases/metabolism , Coronavirus Protease Inhibitors/chemistry , Drug Evaluation, Preclinical , Humans , Jurkat Cells , Models, Molecular , Molecular Structure , Ubiquitin Thiolesterase/metabolism
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