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1.
Life Sci Alliance ; 5(2)2022 02.
Article in English | MEDLINE | ID: covidwho-1547941

ABSTRACT

The clinical outcome of SARS-CoV-2 infections, which can range from asymptomatic to lethal, is crucially shaped by the concentration of antiviral antibodies and by their affinity to their targets. However, the affinity of polyclonal antibody responses in plasma is difficult to measure. Here we used microfluidic antibody affinity profiling (MAAP) to determine the aggregate affinities and concentrations of anti-SARS-CoV-2 antibodies in plasma samples of 42 seropositive individuals, 19 of which were healthy donors, 20 displayed mild symptoms, and 3 were critically ill. We found that dissociation constants, K d, of anti-receptor-binding domain antibodies spanned 2.5 orders of magnitude from sub-nanomolar to 43 nM. Using MAAP we found that antibodies of seropositive individuals induced the dissociation of pre-formed spike-ACE2 receptor complexes, which indicates that MAAP can be adapted as a complementary receptor competition assay. By comparison with cytopathic effect-based neutralisation assays, we show that MAAP can reliably predict the cellular neutralisation ability of sera, which may be an important consideration when selecting the most effective samples for therapeutic plasmapheresis and tracking the success of vaccinations.


Subject(s)
Antibodies, Viral/blood , COVID-19/immunology , Microfluidics/methods , SARS-CoV-2/immunology , Adult , Aged , Angiotensin-Converting Enzyme 2/blood , Angiotensin-Converting Enzyme 2/immunology , Antibodies, Viral/immunology , Antibody Affinity , B-Lymphocytes/immunology , B-Lymphocytes/virology , COVID-19/blood , COVID-19/etiology , Cross Reactions , Female , Humans , Male , Middle Aged , Severity of Illness Index , Spike Glycoprotein, Coronavirus/blood , Spike Glycoprotein, Coronavirus/immunology , Surface Plasmon Resonance
2.
J Mater Chem B ; 9(42): 8851-8861, 2021 11 03.
Article in English | MEDLINE | ID: covidwho-1526111

ABSTRACT

Nanomaterial-based optical techniques for biomarker detection have garnered tremendous attention from the nanofabrication community due to their high precision and enhanced limit of detection (LoD) features. These nanomaterials are highly responsive to local refractive index (RI) fluctuations, and their RI unit sensitivity can be tuned by varying the chemical composition, geometry, and dimensions of the utilized nanostructures. To improve the sensitivity and LoD values of these nanomaterials, it is common to increase both dimensions and aspect ratios of the fabricated nanostructures. However, limited by the complexity, prolonged duration, and elevated costs of the available nanofabrication techniques, mass production of these nanostructures remains challenging. To address not only high accuracy, but also speed and production effectiveness in these nanostructures' fabrication, our work reports, for the first time, a fast, high-throughput, and cost-effective nanofabrication protocol for routine manufacturing of polymer-based nanostructures with high sensitivity and calculated LoD in the pM range by utilizing anodized aluminum oxide (AAO) membranes as templates. Specifically, our developed platform consists of arrays of nearly uniform polystyrene nanopillars with an average diameter of ∼185 nm and aspect ratio of ∼11. We demonstrate that these nanostructures can be produced at a high speed and a notably low price, and that they can be efficiently applied for biosensing purposes after being coated with aluminum-doped silver (Ag/Al) thin films. Our platform successfully detected very low concentrations of human C-reactive protein (hCRP) and SARS-CoV-2 spike protein biomarkers in human plasma samples with LoDs of 11 and 5 pM, respectively. These results open new opportunities for day-to-day fabrication of high aspect ratio arrays of nanopillars that can be used as a base for nanoplasmonic sensors with competitive LoD values. This, in turn, contributes to the development of point-of-care devices and further improvement of the existing nanofabrication techniques, thereby enriching the fields of pharmacology, clinical analysis, and diagnostics.


Subject(s)
Aluminum Oxide/chemistry , Biomarkers/blood , High-Throughput Screening Assays/methods , Nanostructures/chemistry , Silver/chemistry , Biosensing Techniques , C-Reactive Protein/analysis , COVID-19/diagnosis , COVID-19/virology , Dimethylpolysiloxanes/chemistry , Humans , Limit of Detection , Point-of-Care Systems , Polystyrenes/chemistry , SARS-CoV-2/isolation & purification , SARS-CoV-2/metabolism , Spike Glycoprotein, Coronavirus/blood
3.
PLoS One ; 16(9): e0257016, 2021.
Article in English | MEDLINE | ID: covidwho-1484849

ABSTRACT

BACKGROUND: Activation of the immune system is implicated in the Post-Acute Sequelae after SARS-CoV-2 infection (PASC) but the mechanisms remain unknown. Angiotensin-converting enzyme 2 (ACE2) cleaves angiotensin II (Ang II) resulting in decreased activation of the AT1 receptor and decreased immune system activation. We hypothesized that autoantibodies against ACE2 may develop after SARS-CoV-2 infection, as anti-idiotypic antibodies to anti-spike protein antibodies. METHODS AND FINDINGS: We tested plasma or serum for ACE2 antibodies in 67 patients with known SARS-CoV-2 infection and 13 with no history of infection. None of the 13 patients without history of SARS-CoV-2 infection and 1 of the 20 outpatients that had a positive PCR test for SARS-CoV-2 had levels of ACE2 antibodies above the cutoff threshold. In contrast, 26/32 (81%) in the convalescent group and 14/15 (93%) of patients acutely hospitalized had detectable ACE2 antibodies. Plasma from patients with antibodies against ACE2 had less soluble ACE2 activity in plasma but similar amounts of ACE2 protein compared to patients without ACE2 antibodies. We measured the capacity of the samples to inhibit ACE2 enzyme activity. Addition of plasma from patients with ACE2 antibodies led to decreased activity of an exogenous preparation of ACE2 compared to patients that did not have antibodies. CONCLUSIONS: Many patients with a history of SARS-CoV-2 infection have antibodies specific for ACE2. Patients with ACE2 antibodies have lower activity of soluble ACE2 in plasma. Plasma from these patients also inhibits exogenous ACE2 activity. These findings are consistent with the hypothesis that ACE2 antibodies develop after SARS-CoV-2 infection and decrease ACE2 activity. This could lead to an increase in the abundance of Ang II, which causes a proinflammatory state that triggers symptoms of PASC.


Subject(s)
Autoantibodies/blood , COVID-19/immunology , SARS-CoV-2/isolation & purification , Spike Glycoprotein, Coronavirus/blood , Angiotensin II/blood , Angiotensin II/immunology , Angiotensin-Converting Enzyme 2/genetics , Autoantibodies/immunology , Autoantibodies/isolation & purification , COVID-19/blood , COVID-19/virology , Female , Humans , Male , Peptidyl-Dipeptidase A/blood , Receptor, Angiotensin, Type 1/blood , Receptor, Angiotensin, Type 1/genetics , Receptor, Angiotensin, Type 1/immunology , Renin-Angiotensin System/genetics , Renin-Angiotensin System/immunology , SARS-CoV-2/genetics , SARS-CoV-2/immunology , Spike Glycoprotein, Coronavirus/immunology , Spike Glycoprotein, Coronavirus/isolation & purification
4.
J Clin Invest ; 131(17)2021 09 01.
Article in English | MEDLINE | ID: covidwho-1463086

ABSTRACT

Defining the correlates of protection necessary to manage the COVID-19 pandemic requires the analysis of both antibody and T cell parameters, but the complexity of traditional tests limits virus-specific T cell measurements. We tested the sensitivity and performance of a simple and rapid SARS-CoV-2 spike protein-specific T cell test based on the stimulation of whole blood with peptides covering the SARS-CoV-2 spike protein, followed by cytokine (IFN-γ, IL-2) measurement in different cohorts including BNT162b2-vaccinated individuals (n = 112), convalescent asymptomatic and symptomatic COVID-19 patients (n = 130), and SARS-CoV-1-convalescent individuals (n = 12). The sensitivity of this rapid test is comparable to that of traditional methods of T cell analysis (ELISPOT, activation-induced marker). Using this test, we observed a similar mean magnitude of T cell responses between the vaccinees and SARS-CoV-2 convalescents 3 months after vaccination or virus priming. However, a wide heterogeneity of the magnitude of spike-specific T cell responses characterized the individual responses, irrespective of the time of analysis. The magnitude of these spike-specific T cell responses cannot be predicted from the neutralizing antibody levels. Hence, both humoral and cellular spike-specific immunity should be tested after vaccination to define the correlates of protection necessary to evaluate current vaccine strategies.


Subject(s)
COVID-19 Vaccines/administration & dosage , COVID-19 , Immunity, Cellular/drug effects , SARS-CoV-2 , Spike Glycoprotein, Coronavirus , T-Lymphocytes , Adult , COVID-19/blood , COVID-19/immunology , COVID-19/prevention & control , Female , Humans , Male , Middle Aged , SARS-CoV-2/immunology , SARS-CoV-2/metabolism , Spike Glycoprotein, Coronavirus/blood , Spike Glycoprotein, Coronavirus/immunology , T-Lymphocytes/immunology , T-Lymphocytes/metabolism
5.
Sci Rep ; 11(1): 19675, 2021 10 04.
Article in English | MEDLINE | ID: covidwho-1450292

ABSTRACT

Kidney function is affected in COVID-19, while kidney itself modulates the immune response. Here, hypothesize if COVID-19 urine biomarkers level can assess immune activation vs. clinical trajectory. Considering the kidney's critical role in modulating the immune response, we sought to analyze activation markers in patients with pre-existing dysfunction. This was a cross-sectional study of 68 patients. Blood and urine were collected within 48 h of hospital admission (H1), followed by 96 h (H2), seven days (H3), and up to 25 days (H4) from admission. Serum level ferritin, procalcitonin, IL-6 assessed immune activation overall, while the response to viral burden was gauged with serum level of spike protein and αspike IgM and IgG. 39 markers correlated highly between urine and blood. Age and race, and to a lesser extend gender, differentiated several urine markers. The burden of pre-existing conditions correlated with urine DCN, CAIX and PTN, but inversely with IL-5 or MCP-4. Higher urinary IL-12 and lower CAIX, CCL23, IL-15, IL-18, MCP-1, MCP-3, MUC-16, PD-L1, TNFRS12A, and TNFRS21 signified non-survivors. APACHE correlated with urine TNFRS12, PGF, CAIX, DCN, CXCL6, and EGF. Admission urine LAG-3 and IL-2 predicted death. Pre-existing kidney disease had a unique pattern of urinary inflammatory markers. Acute kidney injury was associated, and to a certain degree, predicted by IFNg, TWEAK, MMP7, and MUC-16. Remdesavir had a more profound effect on the urine biomarkers than steroids. Urinary biomarkers correlated with clinical status, kidney function, markers of the immune system activation, and probability of demise in COVID-19.


Subject(s)
Acute Kidney Injury/pathology , Biomarkers/urine , COVID-19/immunology , Renal Insufficiency, Chronic/pathology , Acute Kidney Injury/complications , Adult , Aged , Antigens, CD/urine , Biomarkers/blood , CA-125 Antigen/urine , COVID-19/mortality , COVID-19/pathology , COVID-19/virology , Chemokines, CC/blood , Cross-Sectional Studies , Female , Humans , Interleukin-12/urine , Interleukin-6/blood , Male , Membrane Proteins/urine , Middle Aged , Procalcitonin/blood , Renal Insufficiency, Chronic/complications , SARS-CoV-2/isolation & purification , SARS-CoV-2/metabolism , Severity of Illness Index , Spike Glycoprotein, Coronavirus/blood
7.
Emerg Infect Dis ; 27(2): 663-666, 2021 02.
Article in English | MEDLINE | ID: covidwho-1389113

ABSTRACT

Antibody response against nucleocapsid and spike proteins of SARS-CoV-2 in 11 persons with mild or asymptomatic infection rapidly increased after infection. At weeks 18-30 after diagnosis, all remained seropositive but spike protein-targeting antibody titers declined. These data may be useful for vaccine development.


Subject(s)
COVID-19/immunology , Immunity, Humoral , SARS-CoV-2/immunology , Adolescent , Adult , Antibodies, Viral/blood , Antibodies, Viral/immunology , Asymptomatic Infections , COVID-19/blood , COVID-19/virology , Child , Female , Humans , Longitudinal Studies , Male , Middle Aged , Nucleocapsid Proteins/blood , Nucleocapsid Proteins/immunology , Spike Glycoprotein, Coronavirus/blood , Spike Glycoprotein, Coronavirus/immunology , Time Factors , Vietnam , Young Adult
8.
Protein Expr Purif ; 179: 105802, 2021 03.
Article in English | MEDLINE | ID: covidwho-1386444

ABSTRACT

The receptor-binding domain (RBD) of the SARS-CoV-2 spike protein is a commonly used antigen for serology assays critical to determining the extent of SARS-CoV-2 exposure in the population. Different versions of the RBD protein have been developed and utilized in assays, with higher sensitivity attributed to particular forms of the protein. To improve the yield of these high-sensitivity forms of RBD and support the increased demand for this antigen in serology assays, we investigated several protein expression variables including DNA elements such as promoters and signal peptides, cell culture expression parameters, and purification processes. Through this investigation, we developed a simplified and robust purification strategy that consistently resulted in high levels of the high-sensitivity form of RBD and demonstrated that a carboxyterminal tag is responsible for the increased sensitivity in the ELISA. These improved reagents and processes produce high-quality proteins which are functional in serology assays and can be used to investigate seropositivity to SARS-CoV-2 infection.


Subject(s)
COVID-19/blood , Protein Domains/genetics , SARS-CoV-2/isolation & purification , Spike Glycoprotein, Coronavirus/isolation & purification , Antibodies, Viral/immunology , COVID-19/genetics , COVID-19/virology , Enzyme-Linked Immunosorbent Assay , Humans , Protein Binding/genetics , SARS-CoV-2/genetics , Spike Glycoprotein, Coronavirus/blood , Spike Glycoprotein, Coronavirus/genetics
9.
PLoS One ; 16(9): e0257016, 2021.
Article in English | MEDLINE | ID: covidwho-1388955

ABSTRACT

BACKGROUND: Activation of the immune system is implicated in the Post-Acute Sequelae after SARS-CoV-2 infection (PASC) but the mechanisms remain unknown. Angiotensin-converting enzyme 2 (ACE2) cleaves angiotensin II (Ang II) resulting in decreased activation of the AT1 receptor and decreased immune system activation. We hypothesized that autoantibodies against ACE2 may develop after SARS-CoV-2 infection, as anti-idiotypic antibodies to anti-spike protein antibodies. METHODS AND FINDINGS: We tested plasma or serum for ACE2 antibodies in 67 patients with known SARS-CoV-2 infection and 13 with no history of infection. None of the 13 patients without history of SARS-CoV-2 infection and 1 of the 20 outpatients that had a positive PCR test for SARS-CoV-2 had levels of ACE2 antibodies above the cutoff threshold. In contrast, 26/32 (81%) in the convalescent group and 14/15 (93%) of patients acutely hospitalized had detectable ACE2 antibodies. Plasma from patients with antibodies against ACE2 had less soluble ACE2 activity in plasma but similar amounts of ACE2 protein compared to patients without ACE2 antibodies. We measured the capacity of the samples to inhibit ACE2 enzyme activity. Addition of plasma from patients with ACE2 antibodies led to decreased activity of an exogenous preparation of ACE2 compared to patients that did not have antibodies. CONCLUSIONS: Many patients with a history of SARS-CoV-2 infection have antibodies specific for ACE2. Patients with ACE2 antibodies have lower activity of soluble ACE2 in plasma. Plasma from these patients also inhibits exogenous ACE2 activity. These findings are consistent with the hypothesis that ACE2 antibodies develop after SARS-CoV-2 infection and decrease ACE2 activity. This could lead to an increase in the abundance of Ang II, which causes a proinflammatory state that triggers symptoms of PASC.


Subject(s)
Autoantibodies/blood , COVID-19/immunology , SARS-CoV-2/isolation & purification , Spike Glycoprotein, Coronavirus/blood , Angiotensin II/blood , Angiotensin II/immunology , Angiotensin-Converting Enzyme 2/genetics , Autoantibodies/immunology , Autoantibodies/isolation & purification , COVID-19/blood , COVID-19/virology , Female , Humans , Male , Peptidyl-Dipeptidase A/blood , Receptor, Angiotensin, Type 1/blood , Receptor, Angiotensin, Type 1/genetics , Receptor, Angiotensin, Type 1/immunology , Renin-Angiotensin System/genetics , Renin-Angiotensin System/immunology , SARS-CoV-2/genetics , SARS-CoV-2/immunology , Spike Glycoprotein, Coronavirus/immunology , Spike Glycoprotein, Coronavirus/isolation & purification
10.
Nat Commun ; 12(1): 4586, 2021 07 28.
Article in English | MEDLINE | ID: covidwho-1387355

ABSTRACT

Heterogeneous immunoassays such as ELISA have become indispensable in modern bioanalysis, yet translation into point-of-care assays is hindered by their dependence on external calibration and multiple washing and incubation steps. Here, we introduce RAPPID (Ratiometric Plug-and-Play Immunodiagnostics), a mix-and-measure homogeneous immunoassay platform that combines highly specific antibody-based detection with a ratiometric bioluminescent readout. The concept entails analyte-induced complementation of split NanoLuc luciferase fragments, photoconjugated to an antibody sandwich pair via protein G adapters. Introduction of a calibrator luciferase provides a robust ratiometric signal that allows direct in-sample calibration and quantitative measurements in complex media such as blood plasma. We developed RAPPID sensors that allow low-picomolar detection of several protein biomarkers, anti-drug antibodies, therapeutic antibodies, and both SARS-CoV-2 spike protein and anti-SARS-CoV-2 antibodies. With its easy-to-implement standardized workflow, RAPPID provides an attractive, fast, and low-cost alternative to traditional immunoassays, in an academic setting, in clinical laboratories, and for point-of-care applications.


Subject(s)
Antibodies, Viral/blood , COVID-19 Serological Testing/methods , COVID-19/diagnosis , Immunoassay/standards , Luminescent Measurements/standards , SARS-CoV-2/immunology , Spike Glycoprotein, Coronavirus/blood , COVID-19/immunology , COVID-19/virology , COVID-19 Serological Testing/instrumentation , Calibration , GTP-Binding Proteins/chemistry , Genes, Reporter , Humans , Immunoconjugates/chemistry , Limit of Detection , Luciferases/genetics , Luciferases/metabolism , Point-of-Care Testing , SARS-CoV-2/genetics
11.
Signal Transduct Target Ther ; 6(1): 300, 2021 08 11.
Article in English | MEDLINE | ID: covidwho-1351933

ABSTRACT

Elderly people and patients with comorbidities are at higher risk of COVID-19 infection, resulting in severe complications and high mortality. However, the underlying mechanisms are unclear. In this study, we investigate whether miRNAs in serum exosomes can exert antiviral functions and affect the response to COVID-19 in the elderly and people with diabetes. First, we identified four miRNAs (miR-7-5p, miR-24-3p, miR-145-5p and miR-223-3p) through high-throughput sequencing and quantitative real-time PCR analysis, that are remarkably decreased in the elderly and diabetic groups. We further demonstrated that these miRNAs, either in the exosome or in the free form, can directly inhibit S protein expression and SARS-CoV-2 replication. Serum exosomes from young people can inhibit SARS-CoV-2 replication and S protein expression, while the inhibitory effect is markedly decreased in the elderly and diabetic patients. Moreover, three out of the four circulating miRNAs are significantly increased in the serum of healthy volunteers after 8-weeks' continuous physical exercise. Serum exosomes isolated from these volunteers also showed stronger inhibitory effects on S protein expression and SARS-CoV-2 replication. Our study demonstrates for the first time that circulating exosomal miRNAs can directly inhibit SARS-CoV-2 replication and may provide a possible explanation for the difference in response to COVID-19 between young people and the elderly or people with comorbidities.


Subject(s)
COVID-19/genetics , Diabetes Mellitus/genetics , MicroRNAs/genetics , Spike Glycoprotein, Coronavirus/genetics , Adult , Age Factors , Aged , COVID-19/blood , COVID-19/pathology , COVID-19/virology , China , Circulating MicroRNA/blood , Circulating MicroRNA/genetics , Cohort Studies , Diabetes Mellitus/blood , Diabetes Mellitus/pathology , Diabetes Mellitus/virology , Exercise , Exosomes/genetics , Exosomes/metabolism , Exosomes/virology , Female , Gene Expression Regulation , HEK293 Cells , Host-Pathogen Interactions/genetics , Humans , Male , MicroRNAs/blood , Middle Aged , SARS-CoV-2/genetics , SARS-CoV-2/growth & development , SARS-CoV-2/metabolism , Spike Glycoprotein, Coronavirus/blood , Virus Replication
12.
JCI Insight ; 6(15)2021 08 09.
Article in English | MEDLINE | ID: covidwho-1350084

ABSTRACT

A subset of COVID-19 patients exhibit post-acute sequelae of COVID-19 (PASC), but little is known about the immune signatures associated with these syndromes. We investigated longitudinal peripheral blood samples in 50 individuals with previously confirmed SARS-CoV-2 infection, including 20 who experienced prolonged duration of COVID-19 symptoms (lasting more than 30 days; median = 74 days) compared with 30 who had symptom resolution within 20 days. Individuals with prolonged symptom duration maintained antigen-specific T cell response magnitudes to SARS-CoV-2 spike protein in CD4+ and circulating T follicular helper cell populations during late convalescence, while those without persistent symptoms demonstrated an expected decline. The prolonged group also displayed increased IgG avidity to SARS-CoV-2 spike protein. Significant correlations between symptom duration and both SARS-CoV-2-specific T cells and antibodies were observed. Activation and exhaustion markers were evaluated in multiple immune cell types, revealing few phenotypic differences between prolonged and recovered groups, suggesting that prolonged symptom duration is not due to persistent systemic inflammation. These findings demonstrate that SARS-CoV-2-specific immune responses are maintained in patients suffering from prolonged post-COVID-19 symptom duration in contrast to those with resolved symptoms and may suggest the persistence of viral antigens as an underlying etiology.


Subject(s)
COVID-19/immunology , SARS-CoV-2/immunology , Adult , Aged , Aged, 80 and over , Antibodies, Viral/blood , Antibodies, Viral/immunology , Antigens, Viral/blood , Antigens, Viral/immunology , COVID-19/blood , Female , Humans , Immunity , Immunity, Cellular , Male , Middle Aged , Spike Glycoprotein, Coronavirus/blood , Spike Glycoprotein, Coronavirus/immunology , T-Lymphocytes/immunology , Young Adult
13.
Cancer Cell ; 39(8): 1081-1090.e2, 2021 08 09.
Article in English | MEDLINE | ID: covidwho-1343145

ABSTRACT

As COVID-19 adversely affects patients with cancer, prophylactic strategies are critically needed. Using a validated antibody assay against SARS-CoV-2 spike protein, we determined a high seroconversion rate (94%) in 200 patients with cancer in New York City that had received full dosing with one of the FDA-approved COVID-19 vaccines. On comparison with solid tumors (98%), a significantly lower rate of seroconversion was observed in patients with hematologic malignancies (85%), particularly recipients following highly immunosuppressive therapies such as anti-CD20 therapies (70%) and stem cell transplantation (73%). Patients receiving immune checkpoint inhibitor therapy (97%) or hormonal therapies (100%) demonstrated high seroconversion post vaccination. Patients with prior COVID-19 infection demonstrated higher anti-spike IgG titers post vaccination. Relatively lower IgG titers were observed following vaccination with the adenoviral than with mRNA-based vaccines. These data demonstrate generally high immunogenicity of COVID-19 vaccination in oncology patients and identify immunosuppressed cohorts that need novel vaccination or passive immunization strategies.


Subject(s)
COVID-19 Vaccines/immunology , COVID-19/complications , COVID-19/immunology , Neoplasms/complications , Neoplasms/immunology , SARS-CoV-2/immunology , Seroconversion , Adult , Aged , Aged, 80 and over , Antibodies, Viral/blood , Antibodies, Viral/immunology , COVID-19/epidemiology , COVID-19/virology , COVID-19 Vaccines/administration & dosage , COVID-19 Vaccines/adverse effects , Female , Host-Pathogen Interactions/immunology , Humans , Immunogenicity, Vaccine , Immunoglobulin G/blood , Immunoglobulin G/immunology , Male , Middle Aged , Neoplasms/epidemiology , Neoplasms/therapy , Public Health Surveillance , Risk Factors , Spike Glycoprotein, Coronavirus/blood , Spike Glycoprotein, Coronavirus/immunology , Vaccination
15.
J Clin Invest ; 131(14)2021 07 15.
Article in English | MEDLINE | ID: covidwho-1311202

ABSTRACT

BACKGROUNDWeeks after SARS-CoV-2 infection or exposure, some children develop a severe, life-threatening illness called multisystem inflammatory syndrome in children (MIS-C). Gastrointestinal (GI) symptoms are common in patients with MIS-C, and a severe hyperinflammatory response ensues with potential for cardiac complications. The cause of MIS-C has not been identified to date.METHODSHere, we analyzed biospecimens from 100 children: 19 with MIS-C, 26 with acute COVID-19, and 55 controls. Stools were assessed for SARS-CoV-2 by reverse transcription PCR (RT-PCR), and plasma was examined for markers of breakdown of mucosal barrier integrity, including zonulin. Ultrasensitive antigen detection was used to probe for SARS-CoV-2 antigenemia in plasma, and immune responses were characterized. As a proof of concept, we treated a patient with MIS-C with larazotide, a zonulin antagonist, and monitored the effect on antigenemia and the patient's clinical response.RESULTSWe showed that in children with MIS-C, a prolonged presence of SARS-CoV-2 in the GI tract led to the release of zonulin, a biomarker of intestinal permeability, with subsequent trafficking of SARS-CoV-2 antigens into the bloodstream, leading to hyperinflammation. The patient with MIS-C treated with larazotide had a coinciding decrease in plasma SARS-CoV-2 spike antigen levels and inflammatory markers and a resultant clinical improvement above that achieved with currently available treatments.CONCLUSIONThese mechanistic data on MIS-C pathogenesis provide insight into targets for diagnosing, treating, and preventing MIS-C, which are urgently needed for this increasingly common severe COVID-19-related disease in children.


Subject(s)
COVID-19/etiology , COVID-19/physiopathology , Haptoglobins/physiology , Intestinal Mucosa/physiopathology , Protein Precursors/physiology , SARS-CoV-2 , Systemic Inflammatory Response Syndrome/etiology , Systemic Inflammatory Response Syndrome/physiopathology , Adolescent , Antigens, Viral/blood , Biomarkers/blood , COVID-19/virology , Case-Control Studies , Child , Child, Preschool , Female , Haptoglobins/antagonists & inhibitors , Humans , Infant , Infant, Newborn , Intestinal Mucosa/drug effects , Intestinal Mucosa/virology , Male , Oligopeptides/pharmacology , Permeability/drug effects , Proof of Concept Study , Protein Precursors/antagonists & inhibitors , Protein Precursors/blood , SARS-CoV-2/immunology , SARS-CoV-2/pathogenicity , Spike Glycoprotein, Coronavirus/blood , Spike Glycoprotein, Coronavirus/immunology , Systemic Inflammatory Response Syndrome/virology , Young Adult
16.
PLoS One ; 16(7): e0254367, 2021.
Article in English | MEDLINE | ID: covidwho-1304472

ABSTRACT

COVID-19 serological test must have high sensitivity as well as specificity to rule out cross-reactivity with common coronaviruses (HCoVs). We have developed a quantitative multiplex test, measuring antibodies against spike (S) proteins of SARS-CoV-2, SARS-CoV, MERS-CoV, and common human coronavirus strains (229E, NL63, OC43, HKU1), and nucleocapsid (N) protein of SARS-CoV viruses. Receptor binding domain of S protein of SARS-CoV-2 (S-RBD), and N protein, demonstrated sensitivity (94% and 92.5%, respectively) in COVID-19 patients (n = 53), with 98% specificity in non-COVID-19 respiratory-disease (n = 98), and healthy-controls (n = 129). Anti S-RBD and N antibodies appeared five to ten days post-onset of symptoms, peaking at approximately four weeks. The appearance of IgG and IgM coincided while IgG subtypes, IgG1 and IgG3 appeared soon after the total IgG; IgG2 and IgG4 remained undetectable. Several inflammatory cytokines/chemokines were found to be elevated in many COVID-19 patients (e.g., Eotaxin, Gro-α, CXCL-10 (IP-10), RANTES (CCL5), IL-2Rα, MCP-1, and SCGF-b); CXCL-10 was elevated in all. In contrast to antibody titers, levels of CXCL-10 decreased with the improvement in patient health suggesting it as a candidate for disease resolution. Importantly, anti-N antibodies appear before S-RBD and differentiate between vaccinated and infected people-current vaccines (and several in the pipeline) are S protein-based.


Subject(s)
Antibodies, Viral , COVID-19 , Chemokines , Coronavirus Nucleocapsid Proteins , Immunoglobulin G , Immunoglobulin M , SARS-CoV-2 , Spike Glycoprotein, Coronavirus , Adult , Animals , Antibodies, Viral/blood , Antibodies, Viral/immunology , COVID-19/blood , COVID-19/immunology , Chemokines/blood , Chemokines/immunology , Coronavirus Nucleocapsid Proteins/blood , Coronavirus Nucleocapsid Proteins/immunology , Female , Humans , Immunoglobulin G/blood , Immunoglobulin G/immunology , Immunoglobulin M/blood , Immunoglobulin M/immunology , Macaca mulatta , Male , Middle Aged , Phosphoproteins/blood , Phosphoproteins/immunology , Rabbits , SARS-CoV-2/immunology , SARS-CoV-2/metabolism , Spike Glycoprotein, Coronavirus/blood , Spike Glycoprotein, Coronavirus/immunology
17.
J Med Virol ; 93(4): 2262-2269, 2021 04.
Article in English | MEDLINE | ID: covidwho-1217377

ABSTRACT

This study assesses the clinical performance of three anti-SARS-CoV-2 assays, namely EUROIMMUN anti-SARS-CoV-2 nucleocapsid (IgG) ELISA, Elecsys anti-SARS-CoV-2 nucleocapsid (total antibodies) assay, and LIAISON anti-SARS-CoV-2 spike proteins S1 and S2 (IgG) assay. One hundred and thirty-seven coronavirus disease 2019 (COVID-19) samples from 96 reverse-transcription polymerase chain reaction confirmed patients were chosen to perform the sensitivity analysis. Non-SARS-CoV-2 sera (n = 141) with a potential cross-reaction to SARS-CoV-2 immunoassays were included in the specificity analysis. None of these tests demonstrated a sufficiently high clinical sensitivity to diagnose acute infection. Fourteen days since symptom onset, we did not find any significant difference between the three techniques in terms of sensitivities. However, Elecsys performed better in terms of specificity. All three anti-SARS-CoV-2 assays had equivalent sensitivities 14 days from symptom onset to diagnose past-COVID-19 infection. We also confirmed that anti-SARS-CoV-2 determination before Day 14 is of less clinical interest.


Subject(s)
COVID-19 Testing/methods , COVID-19/blood , COVID-19/virology , Coronavirus Nucleocapsid Proteins/blood , Immunoassay/methods , SARS-CoV-2/isolation & purification , Spike Glycoprotein, Coronavirus/blood , Adult , Aged , Aged, 80 and over , Antibodies, Viral/blood , COVID-19/diagnosis , COVID-19/immunology , Coronavirus Nucleocapsid Proteins/immunology , Cross Reactions , Enzyme-Linked Immunosorbent Assay , Female , Humans , Immunoglobulin A/blood , Immunoglobulin G/blood , Male , Middle Aged , Phosphoproteins/blood , Phosphoproteins/immunology , Retrospective Studies , Sensitivity and Specificity , Spike Glycoprotein, Coronavirus/analysis , Spike Glycoprotein, Coronavirus/immunology
18.
Viruses ; 13(5)2021 04 24.
Article in English | MEDLINE | ID: covidwho-1201926

ABSTRACT

As of April 2021, the COVID-19 pandemic has swept through 213 countries and infected more than 132 million individuals globally, posing an unprecedented threat to human health. There are currently no specific antiviral treatments for COVID-19 and vaccination programmes, whilst promising, remain in their infancy. A key to restricting the pandemic is the ability to minimize human-human transmission and to predict the infection status of the population in the face of emerging SARS-CoV-2 variants. Success in this area is dependent on the rapid detection of COVID-19 positive individuals with current/previous SARS-CoV-2 infection status. In this regard, the ability to detect antibodies directed against the SARS-CoV-Spike protein in patient sera represents a powerful biomarker for confirmation of infection. Here, we report the design of a proof-of-concept cell-based fluorescent serology assay (termed C19-S-I-IFA) to detect SARS-CoV-2 infection. The assay is based on the capture of IgG antibodies in the serum of COVID-19-positive patients using cells exogenously expressing SARS-CoV-2-Spike and their subsequent fluorescent detection. We validate the assay in 30 blood samples collected in Oxford, UK, in 2020 during the height of the pandemic. Importantly, the assay can be modified to express emerging Spike-variants to permit assessments of the cross-reactivity of patient sera to emerging SARS-CoV-2 strains.


Subject(s)
COVID-19 Testing/methods , COVID-19/virology , Fluorescent Antibody Technique/methods , SARS-CoV-2/isolation & purification , A549 Cells , Antibodies, Viral/blood , COVID-19/blood , COVID-19/diagnosis , COVID-19/immunology , Cross Reactions , Humans , Immunoglobulin G/blood , Pandemics , SARS-CoV-2/genetics , SARS-CoV-2/immunology , Sensitivity and Specificity , Spike Glycoprotein, Coronavirus/blood , Spike Glycoprotein, Coronavirus/genetics
19.
Transfusion ; 61(5): 1377-1382, 2021 05.
Article in English | MEDLINE | ID: covidwho-1088172

ABSTRACT

BACKGROUND: The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virus is the cause of the ongoing coronavirus disease 2019 (COVID-19) pandemic, infecting millions of people and causing more than two million deaths. The SARS-CoV-2 Spike glycoproteins mediate viral entry and represent the main target for antibody responses. Humoral responses were shown to be important for preventing and controlling infection by coronaviruses. A promising approach to reduce the severity of COVID-19 is the transfusion of convalescent plasma. However, longitudinal studies revealed that the level of antibodies targeting the receptor-binding domain (RBD) of the SARS-CoV-2 Spike declines rapidly after the resolution of the infection. STUDY DESIGN AND METHODS: To extend this observation beyond the RBD domain, we performed a longitudinal analysis of the persistence of antibodies targeting the full-length SARS-CoV-2 Spike in the plasma from 15 convalescent donors. We generated a 293T cell line constitutively expressing the SARS-CoV-2 Spike and used it to develop a high-throughput flow cytometry-based assay to detect SARS-CoV-2 Spike-specific antibodies in the plasma of convalescent donors. RESULTS AND CONCLUSION: We found that the level of antibodies targeting the full-length SARS-CoV-2 Spike declines gradually after the resolution of the infection. This decline was not related to the number of donations but strongly correlated with the decline of RBD-specific antibodies and the number of days post-symptom onset. These findings help to better understand the decline of humoral responses against the SARS-CoV-2 Spike and provide important information on when to collect plasma after recovery from active infection for convalescent plasma transfusion.


Subject(s)
Antibodies, Viral/blood , COVID-19/blood , SARS-CoV-2/metabolism , Spike Glycoprotein, Coronavirus/blood , COVID-19/therapy , Female , HEK293 Cells , Humans , Immunization, Passive , Longitudinal Studies , Male
20.
Sci Adv ; 7(7)2021 02.
Article in English | MEDLINE | ID: covidwho-1080728

ABSTRACT

Chile has one of the worst numbers worldwide in terms of SARS-CoV-2 positive cases and COVID-19-related deaths per million inhabitants; thus, characterization of neutralizing antibody (NAb) responses in the general population is critical to understanding of immunity at the local level. Given our inability to perform massive classical neutralization assays due to the scarce availability of BSL-3 facilities in the country, we developed and fully characterized an HIV-based SARS-CoV-2 pseudotype, which was used in a 96-well plate format to investigate NAb responses in samples from individuals exposed to SARS-CoV-2 or treated with convalescent plasma. We also identified samples with decreased or enhanced neutralization activity against the D614G spike variant compared with the wild type, indicating the relevance of this variant in host immunity. The data presented here represent the first insights into NAb responses in individuals from Chile, serving as a guide for future studies in the country.


Subject(s)
Antibodies, Neutralizing/blood , Antibodies, Viral/blood , COVID-19 Serological Testing , COVID-19 , Mutation, Missense , SARS-CoV-2 , Spike Glycoprotein, Coronavirus , Amino Acid Substitution , Animals , COVID-19/blood , COVID-19/genetics , Chile , Chlorocebus aethiops , Female , HEK293 Cells , Humans , Male , SARS-CoV-2/genetics , SARS-CoV-2/metabolism , Spike Glycoprotein, Coronavirus/blood , Spike Glycoprotein, Coronavirus/genetics , Vero Cells
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