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1.
J Med Virol ; 94(4): 1606-1616, 2022 04.
Article in English | MEDLINE | ID: covidwho-1718406

ABSTRACT

The coronavirus disease 2019 (COVID-19) pandemic has sparked the rapid development of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) diagnostics. However, emerging variants pose the risk for target dropout and false-negative results secondary to primer/probe binding site (PBS) mismatches. The Agena MassARRAY® SARS-CoV-2 Panel combines reverse-transcription polymerase chain reaction and matrix-assisted laser desorption/ionization time-of-flight mass-spectrometry to probe for five targets across N and ORF1ab genes, which provides a robust platform to accommodate PBS mismatches in divergent viruses. Herein, we utilize a deidentified data set of 1262 SARS-CoV-2-positive specimens from Mount Sinai Health System (New York City) from December 2020 to April 2021 to evaluate target results and corresponding sequencing data. Overall, the level of PBS mismatches was greater in specimens with target dropout. Of specimens with N3 target dropout, 57% harbored an A28095T substitution that is highly specific for the Alpha (B.1.1.7) variant of concern. These data highlight the benefit of redundancy in target design and the potential for target performance to illuminate the dynamics of circulating SARS-CoV-2 variants.


Subject(s)
COVID-19 Nucleic Acid Testing/methods , Reverse Transcriptase Polymerase Chain Reaction , SARS-CoV-2/isolation & purification , Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization , COVID-19/epidemiology , COVID-19/virology , Coronavirus Nucleocapsid Proteins/genetics , Genetic Variation , Genome, Viral/genetics , Humans , New York City/epidemiology , Phosphoproteins/genetics , Polyproteins/genetics , RNA, Viral/genetics , SARS-CoV-2/genetics , Viral Proteins/genetics
2.
Protein J ; 39(3): 198-216, 2020 06.
Article in English | MEDLINE | ID: covidwho-1718840

ABSTRACT

The devastating effects of the recent global pandemic (termed COVID-19 for "coronavirus disease 2019") caused by the severe acute respiratory syndrome coronavirus-2 (SARS CoV-2) are paramount with new cases and deaths growing at an exponential rate. In order to provide a better understanding of SARS CoV-2, this article will review the proteins found in the SARS CoV-2 that caused this global pandemic.


Subject(s)
Betacoronavirus/chemistry , Betacoronavirus/physiology , Coronavirus Infections/virology , Pneumonia, Viral/virology , Viral Proteins/chemistry , Viral Proteins/metabolism , Amino Acid Sequence , Betacoronavirus/genetics , COVID-19 , Coronavirus Envelope Proteins , Coronavirus Infections/drug therapy , Coronavirus Infections/metabolism , Coronavirus Nucleocapsid Proteins , Drug Discovery/methods , Genome, Viral , Host-Pathogen Interactions/drug effects , Humans , Nucleocapsid Proteins/chemistry , Nucleocapsid Proteins/genetics , Nucleocapsid Proteins/metabolism , Pandemics , Phosphoproteins , Pneumonia, Viral/drug therapy , Pneumonia, Viral/metabolism , Polyproteins , Protein Interaction Maps/drug effects , SARS-CoV-2 , Sequence Alignment , Spike Glycoprotein, Coronavirus/chemistry , Spike Glycoprotein, Coronavirus/genetics , Spike Glycoprotein, Coronavirus/metabolism , Viral Envelope Proteins/chemistry , Viral Envelope Proteins/genetics , Viral Envelope Proteins/metabolism , Viral Matrix Proteins/chemistry , Viral Matrix Proteins/genetics , Viral Matrix Proteins/metabolism , Viral Nonstructural Proteins/chemistry , Viral Nonstructural Proteins/genetics , Viral Nonstructural Proteins/metabolism , Viral Proteins/genetics , Viral Regulatory and Accessory Proteins/chemistry , Viral Regulatory and Accessory Proteins/genetics , Viral Regulatory and Accessory Proteins/metabolism , Viroporin Proteins
4.
Front Immunol ; 12: 820126, 2021.
Article in English | MEDLINE | ID: covidwho-1715000

ABSTRACT

This study aims to assess the immunological response and impact on virological control of the mRNA vaccines for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) among people living with HIV (PLWH). In this single-center observational study, all PLWH were offered vaccination with mRNA1273 or BNT162b2. Both anti-N and anti-S1-receptor binding domain (RBD) antibodies were measured together with HIV-1 RNA levels after the first dose (M0) and then at 1 (M1), 2 (M2) and 6 (M6) months later. A total of 131 individuals (median age: 54 years [IQR: 47.0-60.5]; male: 70.2%; median baseline CD4 T-cell: 602/µl [IQR 445.0-825.5]; median nadir CD4 T-cells 223/µl [IQR 111.0-330.0]) were included. All participants were positive for anti-RBD antibodies at 30 days, 60 days and 6 months after the first dose, with no statistical difference between those with HIV-1 RNA below or >20 copies/ml. HIV-1 RNA data were collected for 128 patients at baseline and 30 days after the first dose; for 124 individuals, 30 days after the second dose; and for 83 patients, 6 months after the first dose. Nineteen (14.8%) of 128 had detectable HIV-1 RNA (>20 copies/ml) at M0, 13/128 (10.2%) at M1 (among which 5 were newly detectable), 15/124 (12.1%) at M2 (among which 5 were newly detectable), and 8/83 (9.6%) at M6. No serious adverse effects were reported. All participants elicited antibodies after two doses of mRNA vaccines, with only a minor impact on HIV-1 RNA levels over a 6-month period.


Subject(s)
/immunology , CD4-Positive T-Lymphocytes/immunology , COVID-19/immunology , HIV Infections/immunology , HIV-1/physiology , RNA, Viral/analysis , SARS-CoV-2/physiology , Adult , Aged , Antibodies, Viral/blood , Antibody Formation , Coronavirus Nucleocapsid Proteins/immunology , Female , Humans , Immunity, Heterologous , Male , Middle Aged , Phosphoproteins/immunology , Spike Glycoprotein, Coronavirus/immunology , Vaccination
5.
Nat Commun ; 13(1): 915, 2022 02 17.
Article in English | MEDLINE | ID: covidwho-1703249

ABSTRACT

Quantitative or qualitative differences in immunity may drive clinical severity in COVID-19. Although longitudinal studies to record the course of immunological changes are ample, they do not necessarily predict clinical progression at the time of hospital admission. Here we show, by a machine learning approach using serum pro-inflammatory, anti-inflammatory and anti-viral cytokine and anti-SARS-CoV-2 antibody measurements as input data, that COVID-19 patients cluster into three distinct immune phenotype groups. These immune-types, determined by unsupervised hierarchical clustering that is agnostic to severity, predict clinical course. The identified immune-types do not associate with disease duration at hospital admittance, but rather reflect variations in the nature and kinetics of individual patient's immune response. Thus, our work provides an immune-type based scheme to stratify COVID-19 patients at hospital admittance into high and low risk clinical categories with distinct cytokine and antibody profiles that may guide personalized therapy.


Subject(s)
Antibodies, Viral/blood , COVID-19/pathology , Cytokines/blood , SARS-CoV-2/immunology , Severity of Illness Index , Aged , Coronavirus Nucleocapsid Proteins/immunology , Disease Progression , Female , Hospitalization , Humans , Immunoglobulin A/blood , Immunoglobulin G/blood , Immunoglobulin M/blood , Immunophenotyping/methods , Machine Learning , Male , Middle Aged , Phosphoproteins/immunology
6.
Front Immunol ; 13: 776861, 2022.
Article in English | MEDLINE | ID: covidwho-1701723

ABSTRACT

Cardiovascular dysfunction and disease are common and frequently fatal complications of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. Indeed, from early on during the SARS-CoV-2 virus pandemic it was recognized that cardiac complications may occur, even in patients with no underlying cardiac disorders, as part of the acute infection, and that these were associated with more severe disease and increased morbidity and mortality. The most common cardiac complication is acute cardiac injury, defined by significant elevation of cardiac troponins. The potential mechanisms of cardiovascular complications include direct viral myocardial injury, systemic inflammation induced by the virus, sepsis, arrhythmia, myocardial oxygen supply-demand mismatch, electrolyte abnormalities, and hypercoagulability. This review is focused on the prevalence, risk factors and clinical course of COVID-19-related myocardial injury, as well as on current data with regard to disease pathogenesis, specifically the interaction of platelets with the vascular endothelium. The latter section includes consideration of the role of SARS-CoV-2 proteins in triggering development of a generalized endotheliitis that, in turn, drives intense activation of platelets. Most prominently, SARS-CoV-2-induced endotheliitis involves interaction of the viral spike protein with endothelial angiotensin-converting enzyme 2 (ACE2) together with alternative mechanisms that involve the nucleocapsid and viroporin. In addition, the mechanisms by which activated platelets intensify endothelial activation and dysfunction, seemingly driven by release of the platelet-derived calcium-binding proteins, SA100A8 and SA100A9, are described. These events create a SARS-CoV-2-driven cycle of intravascular inflammation and coagulation, which contributes significantly to a poor clinical outcome in patients with severe disease.


Subject(s)
Blood Platelets/metabolism , COVID-19/pathology , Cardiovascular Diseases/pathology , Endothelium, Vascular/metabolism , Platelet Activation/immunology , Angiotensin-Converting Enzyme 2/metabolism , COVID-19/mortality , Cardiovascular Diseases/virology , Coronavirus Nucleocapsid Proteins/immunology , Endothelial Cells/metabolism , Humans , Myocardium/pathology , Phosphoproteins/immunology , SARS-CoV-2/immunology , Spike Glycoprotein, Coronavirus/immunology , Spike Glycoprotein, Coronavirus/metabolism
7.
Front Immunol ; 13: 817905, 2022.
Article in English | MEDLINE | ID: covidwho-1699973

ABSTRACT

The duration of humoral and cellular immune memory following SARS-CoV-2 infection in populations in least developed countries remains understudied but is key to overcome the current SARS-CoV-2 pandemic. Sixty-four Cambodian individuals with laboratory-confirmed infection with asymptomatic or mild/moderate clinical presentation were evaluated for Spike (S)-binding and neutralizing antibodies and antibody effector functions during acute phase of infection and at 6-9 months follow-up. Antigen-specific B cells, CD4+ and CD8+ T cells were characterized, and T cells were interrogated for functionality at late convalescence. Anti-S antibody titers decreased over time, but effector functions mediated by S-specific antibodies remained stable. S- and nucleocapsid (N)-specific B cells could be detected in late convalescence in the activated memory B cell compartment and are mostly IgG+. CD4+ and CD8+ T cell immune memory was maintained to S and membrane (M) protein. Asymptomatic infection resulted in decreased antibody-dependent cellular cytotoxicity (ADCC) and frequency of SARS-CoV-2-specific CD4+ T cells at late convalescence. Whereas anti-S antibodies correlated with S-specific B cells, there was no correlation between T cell response and humoral immune memory. Hence, all aspects of a protective immune response are maintained up to nine months after SARS-CoV-2 infection and in the absence of re-infection.


Subject(s)
Antibodies, Neutralizing/blood , Antibodies, Viral/blood , CD4-Positive T-Lymphocytes/immunology , CD8-Positive T-Lymphocytes/immunology , Immunologic Memory/immunology , SARS-CoV-2/immunology , B-Lymphocytes/immunology , COVID-19/pathology , Cambodia , Coronavirus Nucleocapsid Proteins/immunology , Humans , Immunity, Cellular/immunology , Immunity, Humoral/immunology , Phosphoproteins/immunology , Spike Glycoprotein, Coronavirus/immunology
8.
Stem Cell Reports ; 17(3): 522-537, 2022 03 08.
Article in English | MEDLINE | ID: covidwho-1692862

ABSTRACT

Patients with coronavirus disease 2019 (COVID-19) commonly have manifestations of heart disease. Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) genome encodes 27 proteins. Currently, SARS-CoV-2 gene-induced abnormalities of human heart muscle cells remain elusive. Here, we comprehensively characterized the detrimental effects of a SARS-CoV-2 gene, Orf9c, on human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs) by preforming multi-omic analyses. Transcriptomic analyses of hPSC-CMs infected by SARS-CoV-2 with Orf9c overexpression (Orf9cOE) identified concordantly up-regulated genes enriched into stress-related apoptosis and inflammation signaling pathways, and down-regulated CM functional genes. Proteomic analysis revealed enhanced expressions of apoptotic factors, whereas reduced protein factors for ATP synthesis by Orf9cOE. Orf9cOE significantly reduced cellular ATP level, induced apoptosis, and caused electrical dysfunctions of hPSC-CMs. Finally, drugs approved by the U.S. Food and Drug Administration, namely, ivermectin and meclizine, restored ATP levels and ameliorated CM death and functional abnormalities of Orf9cOE hPSC-CMs. Overall, we defined the molecular mechanisms underlying the detrimental impacts of Orf9c on hPSC-CMs and explored potentially therapeutic approaches to ameliorate Orf9c-induced cardiac injury and abnormalities.


Subject(s)
COVID-19/pathology , Coronavirus Nucleocapsid Proteins/genetics , Genome-Wide Association Study/methods , SARS-CoV-2/genetics , Action Potentials/drug effects , Adenosine Triphosphate/metabolism , Apoptosis/drug effects , Apoptosis/genetics , COVID-19/virology , Down-Regulation , Humans , Ivermectin/pharmacology , Meclizine/pharmacology , Myocytes, Cardiac/cytology , Myocytes, Cardiac/metabolism , Phosphoproteins/genetics , Pluripotent Stem Cells/cytology , Pluripotent Stem Cells/metabolism , Protein Interaction Maps/genetics , RNA, Messenger/chemistry , RNA, Messenger/metabolism , SARS-CoV-2/isolation & purification , Signal Transduction/genetics , Transcriptome/drug effects , Up-Regulation
9.
ACS Appl Mater Interfaces ; 14(8): 10844-10855, 2022 Mar 02.
Article in English | MEDLINE | ID: covidwho-1692677

ABSTRACT

The widespread and long-lasting effect of the COVID-19 pandemic has called attention to the significance of technological advances in the rapid diagnosis of SARS-CoV-2 virus. This study reports the use of a highly stable buffer-based zinc oxide/reduced graphene oxide (bbZnO/rGO) nanocomposite coated on carbon screen-printed electrodes for electrochemical immuno-biosensing of SARS-CoV-2 nuelocapsid (N-) protein antigens in spiked and clinical samples. The incorporation of a salt-based (ionic) matrix for uniform dispersion of the nanomixture eliminates multistep nanomaterial synthesis on the surface of the electrode and enables a stable single-step sensor nanocoating. The immuno-biosensor provides a limit of detection of 21 fg/mL over a linear range of 1-10 000 pg/mL and exhibits a sensitivity of 32.07 ohms·mL/pg·mm2 for detection of N-protein in spiked samples. The N-protein biosensor is successful in discriminating positive and negative clinical samples within 15 min, demonstrating its proof of concept used as a COVID-19 rapid antigen test.


Subject(s)
Antigens, Viral/analysis , COVID-19/diagnosis , Coronavirus Nucleocapsid Proteins/analysis , Graphite/chemistry , Nanocomposites/chemistry , Zinc Oxide/chemistry , Antibodies, Immobilized/immunology , Antigens, Viral/immunology , Biosensing Techniques/instrumentation , Biosensing Techniques/methods , Coronavirus Nucleocapsid Proteins/immunology , Electrochemical Techniques/instrumentation , Electrochemical Techniques/methods , Electrodes , Humans , Immunoassay/instrumentation , Immunoassay/methods , Limit of Detection , Phosphoproteins/analysis , Phosphoproteins/immunology , Proof of Concept Study , SARS-CoV-2/chemistry
10.
Int J Mol Sci ; 23(4)2022 Feb 09.
Article in English | MEDLINE | ID: covidwho-1690219

ABSTRACT

The development of prophylactic agents against the SARS-CoV-2 virus is a public health priority in the search for new surrogate markers of active virus replication. Early detection markers are needed to follow disease progression and foresee patient negativization. Subgenomic RNA transcripts (with a focus on sgN) were evaluated in oro/nasopharyngeal swabs from COVID-19-affected patients with an analysis of 315 positive samples using qPCR technology. Cut-off Cq values for sgN (Cq < 33.15) and sgE (Cq < 34.06) showed correlations to high viral loads. The specific loss of sgN in home-isolated and hospitalized COVID-19-positive patients indicated negativization of patient condition, 3-7 days from the first swab, respectively. A new detection kit for sgN, gene E, gene ORF1ab, and gene RNAse P was developed recently. In addition, in vitro studies have shown that 2'-O-methyl antisense RNA (related to the sgN sequence) can impair SARS-CoV-2 N protein synthesis, viral replication, and syncytia formation in human cells (i.e., HEK-293T cells overexpressing ACE2) upon infection with VOC Alpha (B.1.1.7)-SARS-CoV-2 variant, defining the use that this procedure might have for future therapeutic actions against SARS-CoV-2.


Subject(s)
COVID-19/virology , Coronavirus Nucleocapsid Proteins/genetics , SARS-CoV-2/physiology , Virus Replication/physiology , Coronavirus Nucleocapsid Proteins/analysis , Giant Cells/drug effects , Giant Cells/virology , HEK293 Cells , Humans , Limit of Detection , Nasopharynx/virology , Phosphoproteins/analysis , Phosphoproteins/genetics , RNA, Antisense/pharmacology , RNA, Viral , Ribonuclease P/genetics , SARS-CoV-2/drug effects , SARS-CoV-2/genetics , Sensitivity and Specificity , Social Isolation , Viral Load , Viroporin Proteins/genetics , Virus Replication/drug effects
11.
Front Immunol ; 13: 816220, 2022.
Article in English | MEDLINE | ID: covidwho-1686484

ABSTRACT

SARS-CoV-2 variants of concern (VOCs) can trigger severe endemic waves and vaccine breakthrough infections (VBI). We analyzed the cellular and humoral immune response in 8 patients infected with the alpha variant, resulting in moderate to fatal COVID-19 disease manifestation, after double mRNA-based anti-SARS-CoV-2 vaccination. In contrast to the uninfected vaccinated control cohort, the diseased individuals had no detectable high-avidity spike (S)-reactive CD4+ and CD8+ T cells against the alpha variant and wild type (WT) at disease onset, whereas a robust CD4+ T-cell response against the N- and M-proteins was generated. Furthermore, a delayed alpha S-reactive high-avidity CD4+ T-cell response was mounted during disease progression. Compared to the vaccinated control donors, these patients also had lower neutralizing antibody titers against the alpha variant at disease onset. The delayed development of alpha S-specific cellular and humoral immunity upon VBI indicates reduced immunogenicity against the S-protein of the alpha VOC, while there was a higher and earlier N- and M-reactive T-cell response. Our findings do not undermine the current vaccination strategies but underline a potential need for the inclusion of VBI patients in alternative vaccination strategies and additional antigenic targets in next-generation SARS-CoV-2 vaccines.


Subject(s)
/immunology , Antibodies, Neutralizing/blood , CD4-Positive T-Lymphocytes/immunology , CD8-Positive T-Lymphocytes/immunology , COVID-19 Vaccines/immunology , COVID-19/immunology , Adult , Aged , Aged, 80 and over , Antibodies, Viral/blood , Antibody Affinity/immunology , COVID-19/mortality , Coronavirus M Proteins/immunology , Coronavirus Nucleocapsid Proteins/immunology , Female , Humans , Male , Middle Aged , Phosphoproteins/immunology , SARS-CoV-2/immunology , Spike Glycoprotein, Coronavirus/immunology , Vaccination
12.
Front Immunol ; 12: 793197, 2021.
Article in English | MEDLINE | ID: covidwho-1674334

ABSTRACT

Background: Despite similar rates of infection, adults and children have markedly different morbidity and mortality related to severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2). Compared to adults, children have infrequent severe manifestations of acute infection but are uniquely at risk for the rare and often severe Multisystem Inflammatory Syndrome in Children (MIS-C) following infection. We hypothesized that these differences in presentation are related to differences in the magnitude and/or antigen specificity of SARS-CoV-2-specific T cell (CST) responses between adults and children. We therefore set out to measure the CST response in convalescent adults versus children with and without MIS-C following SARS-CoV-2 infection. Methods: CSTs were expanded from blood collected from convalescent children and adults post SARS-CoV-2 infection and evaluated by intracellular flow cytometry, surface markers, and cytokine production following stimulation with SARS-CoV-2-specific peptides. Presence of serum/plasma antibody to spike and nucleocapsid was measured using the luciferase immunoprecipitation systems (LIPS) assay. Findings: Twenty-six of 27 MIS-C patients, 7 of 8 non-MIS-C convalescent children, and 13 of 14 adults were seropositive for spike and nucleocapsid antibody. CST responses in MIS-C patients were significantly higher than children with uncomplicated SARS-CoV-2 infection, but weaker than CST responses in convalescent adults. Interpretation: Age-related differences in the magnitude of CST responses suggest differing post-infectious immunity to SARS-CoV-2 in children compared to adults post uncomplicated infection. Children with MIS-C have CST responses that are stronger than children with uncomplicated SARS-CoV-2 infection and weaker than convalescent adults, despite near uniform seropositivity.


Subject(s)
COVID-19/complications , SARS-CoV-2/immunology , Systemic Inflammatory Response Syndrome/immunology , T-Lymphocytes/immunology , Adolescent , Adult , Antibodies, Viral/immunology , COVID-19/immunology , Child , Child, Preschool , Convalescence , Coronavirus Nucleocapsid Proteins/immunology , Female , Humans , Infant , Male , Middle Aged , Phosphoproteins/immunology , Spike Glycoprotein, Coronavirus/immunology
13.
PLoS One ; 17(2): e0262591, 2022.
Article in English | MEDLINE | ID: covidwho-1666759

ABSTRACT

SARS-CoV-2 Nucleocapsid (N) is the most abundant viral protein expressed in host samples and is an important antigen for diagnosis. N is a 45 kDa protein that does not present disulfide bonds. Intending to avoid non-specific binding of SARS-CoV-2 N to antibodies from patients who previously had different coronaviruses, a 35 kDa fragment of N was expressed without a conserved motif in E. coli as inclusion bodies (N122-419-IB). Culture media and IB washing conditions were chosen to obtain N122-419-IB with high yield (370 mg/L bacterial culture) and protein purity (90%). High pressure solubilizes protein aggregates by weakening hydrophobic and ionic interactions and alkaline pH promotes solubilization by electrostatic repulsion. The association of pH 9.0 and 2.4 kbar promoted efficient solubilization of N122-419-IB without loss of native-like tertiary structure that N presents in IB. N122-419 was refolded with a yield of 85% (326 mg/L culture) and 95% purity. The refolding process takes only 2 hours and the protein is ready for use after pH adjustment, avoiding the necessity of dialysis or purification. Antibody binding of COVID-19-positive patients sera to N122-419 was confirmed by Western blotting. ELISA using N122-419 is effective in distinguishing between sera presenting antibodies against SARS-CoV-2 from those who do not. To the best of our knowledge, the proposed condition for IB solubilization is one of the mildest described. It is possible that the refolding process can be extended to a wide range of proteins with high yields and purity, even those that are sensible to very alkaline pH.


Subject(s)
Antibodies, Viral/blood , Antigens, Viral/chemistry , COVID-19/blood , COVID-19/diagnosis , Coronavirus Nucleocapsid Proteins/chemistry , Immunoglobulin G/blood , Inclusion Bodies/chemistry , Protein Refolding , SARS-CoV-2/immunology , Antibodies, Viral/immunology , Antigens, Viral/immunology , COVID-19/virology , Coronavirus Nucleocapsid Proteins/immunology , Enzyme-Linked Immunosorbent Assay/methods , Escherichia coli/genetics , Escherichia coli/metabolism , Humans , Hydrogen-Ion Concentration , Hydrostatic Pressure , Immunoglobulin G/immunology , Phosphoproteins/chemistry , Phosphoproteins/immunology , Protein Structure, Tertiary , Recombinant Proteins/chemistry , Recombinant Proteins/immunology , Solubility
14.
Microb Cell Fact ; 21(1): 21, 2022 Feb 05.
Article in English | MEDLINE | ID: covidwho-1666655

ABSTRACT

We have developed a method for the inexpensive, high-level expression of antigenic protein fragments of SARS-CoV-2 proteins in Escherichia coli. Our approach uses the thermophilic family 9 carbohydrate-binding module (CBM9) as an N-terminal carrier protein and affinity tag. The CBM9 module was joined to SARS-CoV-2 protein fragments via a flexible proline-threonine linker, which proved to be resistant to E. coli proteases. Two CBM9-spike protein fragment fusion proteins and one CBM9-nucleocapsid fragment fusion protein largely resisted protease degradation, while most of the CBM9 fusion proteins were degraded at some site in the SARS-CoV-2 protein fragment. All of the fusion proteins were highly expressed in E. coli and the CBM9-ID-H1 fusion protein was shown to yield 122 mg/L of purified product. Three purified CBM9-SARS-CoV-2 fusion proteins were tested and found to bind antibodies directed to the appropriate SARS-CoV-2 antigenic regions. The largest intact CBM9 fusion protein, CBM9-ID-H1, incorporates spike protein amino acids 540-588, which is a conserved region overlapping and C-terminal to the receptor binding domain that is widely recognized by human convalescent sera and contains a putative protective epitope.


Subject(s)
Coronavirus Nucleocapsid Proteins/genetics , Escherichia coli/metabolism , Recombinant Fusion Proteins/biosynthesis , SARS-CoV-2/metabolism , Spike Glycoprotein, Coronavirus/genetics , Antibodies, Viral/immunology , Antigen-Antibody Reactions , COVID-19/pathology , COVID-19/virology , Chromatography, High Pressure Liquid , Coronavirus Nucleocapsid Proteins/metabolism , Humans , Mass Spectrometry , Phosphoproteins/genetics , Phosphoproteins/metabolism , Receptors, Cell Surface/genetics , Recombinant Fusion Proteins/analysis , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/immunology , SARS-CoV-2/isolation & purification , Spike Glycoprotein, Coronavirus/metabolism
15.
Nat Immunol ; 23(2): 275-286, 2022 02.
Article in English | MEDLINE | ID: covidwho-1661973

ABSTRACT

The humoral arm of innate immunity includes diverse molecules with antibody-like functions, some of which serve as disease severity biomarkers in coronavirus disease 2019 (COVID-19). The present study was designed to conduct a systematic investigation of the interaction of human humoral fluid-phase pattern recognition molecules (PRMs) with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Of 12 PRMs tested, the long pentraxin 3 (PTX3) and mannose-binding lectin (MBL) bound the viral nucleocapsid and spike proteins, respectively. MBL bound trimeric spike protein, including that of variants of concern (VoC), in a glycan-dependent manner and inhibited SARS-CoV-2 in three in vitro models. Moreover, after binding to spike protein, MBL activated the lectin pathway of complement activation. Based on retention of glycosylation sites and modeling, MBL was predicted to recognize the Omicron VoC. Genetic polymorphisms at the MBL2 locus were associated with disease severity. These results suggest that selected humoral fluid-phase PRMs can play an important role in resistance to, and pathogenesis of, COVID-19, a finding with translational implications.


Subject(s)
COVID-19/immunology , Immunity, Humoral , Receptors, Pattern Recognition/immunology , SARS-CoV-2/immunology , Animals , C-Reactive Protein/immunology , C-Reactive Protein/metabolism , COVID-19/metabolism , COVID-19/virology , Case-Control Studies , Chlorocebus aethiops , Complement Activation , Coronavirus Nucleocapsid Proteins/genetics , Coronavirus Nucleocapsid Proteins/immunology , Coronavirus Nucleocapsid Proteins/metabolism , Female , Glycosylation , HEK293 Cells , Host-Pathogen Interactions , Humans , Male , Mannose-Binding Lectin/genetics , Mannose-Binding Lectin/immunology , Mannose-Binding Lectin/metabolism , Phosphoproteins/genetics , Phosphoproteins/immunology , Phosphoproteins/metabolism , Polymorphism, Genetic , Protein Binding , Receptors, Pattern Recognition/genetics , Receptors, Pattern Recognition/metabolism , SARS-CoV-2/genetics , SARS-CoV-2/metabolism , SARS-CoV-2/pathogenicity , Serum Amyloid P-Component/immunology , Serum Amyloid P-Component/metabolism , Signal Transduction , Spike Glycoprotein, Coronavirus/genetics , Spike Glycoprotein, Coronavirus/immunology , Spike Glycoprotein, Coronavirus/metabolism , Vero Cells
17.
J Virol Methods ; 302: 114486, 2022 04.
Article in English | MEDLINE | ID: covidwho-1654882

ABSTRACT

BACKGROUND: Recently, the Coronavirus Disease 2019 (COVID-19) caused by SARS-CoV-2 infection has spread rapidly around the world, becoming a new global pandemic disease. Nucleic acid detection is the primary method for clinical diagnosis of SARS-CoV-2 infection, with the addition of antibody and antigen detection. Nucleocapsid protein (NP) is a kind of conservative structural protein with abundant expression during SARS-CoV-2 infection, which makes it an ideal target for immunoassay. METHODS: The coding sequence for SARS-CoV-2-NP was obtained by chemical synthesis, and then inserted into pET28a(+). The soluble recombinant NP (rNP) with an estimated molecular weight of 49.4 kDa was expressed in E. coli cells after IPTG induction. Six-week-old BALB/c mice were immunized with rNP, and then their spleen cells were fused with SP2/0 cells, to develop hybridoma cell lines that stably secreted monoclonal antibodies (mAbs) against NP. The mAbs were preliminarily evaluated by enzyme-linked immunosorbent assay (ELISA), and then used to develop a magnetic particle-based chemiluminescence enzyme immunoassay (CLEIA) for measurement of SARS-CoV-2-NP. RESULTS: mAb 15B1 and mAb 18G10 were selected as capture and detection antibody respectively to develop CLEIA, due to the highest sensitivity for rNP detection. The proposed CLEIA presented a good linearity for rNP detection at a working range from 0.1 to 160 µg/L, with a precision coefficient of variance below 10 %. CONCLUSION: The newly developed mAbs and CLEIA can serve as potential diagnostic tools for clinical measurement of SARS-CoV-2-NP.


Subject(s)
COVID-19 , Coronavirus Nucleocapsid Proteins , SARS-CoV-2 , Animals , Antibodies, Monoclonal/metabolism , Antibodies, Viral/metabolism , COVID-19/diagnosis , Coronavirus Nucleocapsid Proteins/analysis , Coronavirus Nucleocapsid Proteins/genetics , Escherichia coli/genetics , Humans , Immunoassay/methods , Luminescence , Mice , Phosphoproteins/analysis , Phosphoproteins/genetics , Sensitivity and Specificity
18.
J Proteomics ; 255: 104501, 2022 03 20.
Article in English | MEDLINE | ID: covidwho-1654819

ABSTRACT

Phosphorylation of viral proteins serves as a regulatory mechanism during the intracellular life cycle of infected viruses. There is therefore a pressing need to develop a method to efficiently purify and enrich phosphopeptides derived from viral particles in biological samples. In this study, we utilized Phos-tag technology to analyze the functional phosphorylation of the nucleocapsid protein (N protein; NP) of severe respiratory syndrome coronavirus 2 (SARS-CoV-2). Viral particles were collected from culture supernatants of SARS-CoV-2-infected VeroE6/TMPRSS2 cells by ultracentrifugation, and phosphopeptides were purified by Phos-tag magnetic beads for LC-MS/MS analysis. Analysis revealed that NP was reproducibly phosphorylated at serine 79 (Ser79). Multiple sequence alignment and phylogenetic analysis showed that the Ser79 was a distinct phospho-acceptor site in SARS-CoV-2 but not in other beta-coronaviruses. We also found that the prolyl-isomerase Pin1 bound to the phosphorylated Ser79 in NP and positively regulated the production of viral particles. These results suggest that SARS-CoV-2 may have acquired the potent virus-host interaction during its evolution mediated by viral protein phosphorylation. Moreover, Phos-tag technology can provide a useful means for analyzing the functional phosphorylation of viral proteins. SIGNIFICANCE: In this study, we aimed to investigate the functional phosphorylation of SARS-CoV-2 NP. For this purpose, we used Phos-tag technology to purify and enrich virus-derived phosphopeptides with high selectivity and reproducibility. This method can be particularly useful in analyzing viral phosphopeptides from cell culture supernatants that often contain high concentrations of fetal bovine serum and supplements. We newly identified an NP phosphorylation site at Ser79, which is important for Pin1 binding. Furthermore, we showed that the interaction between Pin1 and phosphorylated NP could enhance viral replication in a cell culture model.


Subject(s)
Nucleocapsid Proteins , Phosphopeptides , Chromatography, Liquid , Coronavirus Nucleocapsid Proteins , Humans , NIMA-Interacting Peptidylprolyl Isomerase/metabolism , Nucleocapsid Proteins/chemistry , Phosphopeptides/chemistry , Phosphoproteins , Phosphorylation , Phylogeny , Pyridines , Reproducibility of Results , SARS-CoV-2 , Tandem Mass Spectrometry
19.
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
20.
Front Immunol ; 12: 785599, 2021.
Article in English | MEDLINE | ID: covidwho-1643498

ABSTRACT

Zinc ion as an enzyme cofactor exhibits antiviral and anti-inflammatory activity during infection, but circulating zinc ion level during Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) infection is unclear. This study aimed to evaluate serum zinc ion level in Coronavirus Disease 2019 (COVID-19) patients and healthy subjects, as well as its correlation with antibodies against SARS-CoV-2. 114 COVID-19 patients and 48 healthy subjects (38 healthy volunteers and 10 close contacts of patients with COVID-19) were included. Zinc ion concentration and levels of antibodies against SARS-CoV-2 Spike 1 + Spike 2 proteins, nucleocapsid protein, and receptor-binding domain in serum were measured. Results showed that the concentration of zinc ion in serum from COVID-19 patients [median: 6.4 nmol/mL (IQR 1.5 - 12.0 nmol/mL)] were significantly lower than that from the healthy subjects [median: 15.0 nmol/mL (IQR 11.9 - 18.8 nmol/mL)] (p < 0.001) and the difference remained significant after age stratification (p < 0.001) or when the patients were at the recovery stage (p < 0.001). Furthermore, COVID-19 patients with more severe hypozincemia showed higher levels of IgG against the receptor-binding domain of SARS-CoV-2 spike protein. Further studies to confirm the effect of zinc supplementation on improving the outcomes of COVID-19, including antibody response against SARS-CoV-2, are warranted.


Subject(s)
Antibodies, Viral/blood , COVID-19/blood , COVID-19/immunology , Immunity , SARS-CoV-2/immunology , Zinc/blood , Adult , Antibodies, Viral/immunology , COVID-19/virology , Case-Control Studies , Cations, Divalent/blood , Coronavirus Nucleocapsid Proteins/immunology , Female , Humans , Immunoglobulin G/blood , Immunoglobulin G/immunology , Male , Middle Aged , Phosphoproteins/immunology , Protein Domains/immunology , Real-Time Polymerase Chain Reaction/methods , Retrospective Studies , Reverse Transcriptase Polymerase Chain Reaction/methods , SARS-CoV-2/genetics , Spike Glycoprotein, Coronavirus/immunology
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