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1.
Intern Med ; 61(20): 3053-3062, 2022 Oct 15.
Article in English | MEDLINE | ID: covidwho-2079926

ABSTRACT

Objective To examine the continuation of antibody prevalence status after 12 months and background factors in antibody-positive subjects following asymptomatic infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Methods We initially determined the SARS-CoV-2 anti-nucleocapsid protein immunoglobulin G (anti-N IgG) antibody prevalence in 1,603 patients, doctors, and nurses at 65 medical institutions in Kanagawa Prefecture, Japan. We then obtained consent from 33 of the 39 subjects who tested positive and performed follow-up for 12 months. Results Follow-up for up to 12 months showed that a long-term response of the anti-N IgG antibody could be detected in 6 of the 33 participants (18.2%). The proportions with hypertension, using an angiotensin-receptor blocker, and without a drinking habit were higher among the participants with a long-term anti-N IgG antibody response for up to 12 months than among those without a long-term antibody response. Conclusions The proportion of individuals with subclinical COVID-19 who continuously had a positive result for the anti-N IgG antibody at 12 months was low.


Subject(s)
COVID-19 , Immunoglobulin G , Angiotensin Receptor Antagonists , Antibodies, Viral/blood , Asymptomatic Infections/epidemiology , COVID-19/epidemiology , COVID-19/immunology , Coronavirus Nucleocapsid Proteins/immunology , Humans , Immunoglobulin G/blood , Phosphoproteins/immunology , SARS-CoV-2
2.
PLoS One ; 17(2): e0262591, 2022.
Article in English | MEDLINE | ID: covidwho-1968842

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
3.
Emerg Infect Dis ; 28(9): 1859-1862, 2022 09.
Article in English | MEDLINE | ID: covidwho-1963356

ABSTRACT

Given widespread use of spike antibody in generating coronavirus disease vaccines, SARS-CoV-2 nucleocapsid antibodies are increasingly used to indicate previous infection in serologic surveys. However, longitudinal kinetics and seroreversion are poorly defined. We found substantial seroreversion of nucleocapsid total immunoglobulin, underscoring the need to account for seroreversion in seroepidemiologic studies.


Subject(s)
COVID-19 , SARS-CoV-2 , Antibodies, Viral , COVID-19/epidemiology , Coronavirus Nucleocapsid Proteins/immunology , Humans , Kinetics , Nucleocapsid , Phosphoproteins/immunology , Seroepidemiologic Studies
4.
Sci Rep ; 12(1): 10366, 2022 06 20.
Article in English | MEDLINE | ID: covidwho-1900657

ABSTRACT

The Covid-19 pandemic, caused by SARS-CoV-2, has resulted in over 6 million reported deaths worldwide being one of the biggest challenges the world faces today. Here we present optimizations of all steps of an enzyme-linked immunosorbent assay (ELISA)-based test to detect IgG, IgA and IgM against the trimeric spike (S) protein, receptor binding domain (RBD), and N terminal domain of the nucleocapsid (N-NTD) protein of SARS-CoV-2. We discuss how to determine specific thresholds for antibody positivity and its limitations according to the antigen used. We applied the assay to a cohort of 126 individuals from Rio de Janeiro, Brazil, consisting of 23 PCR-positive individuals and 103 individuals without a confirmed diagnosis for SARS-CoV-2 infection. To illustrate the differences in serological responses to vaccinal immunization, we applied the test in 18 individuals from our cohort before and after receiving ChAdOx-1 nCoV-19 or CoronaVac vaccines. Taken together, our results show that the test can be customized at different stages depending on its application, enabling the user to analyze different cohorts, saving time, reagents, or samples. It is also a valuable tool for elucidating the immunological consequences of new viral strains and monitoring vaccination coverage and duration of response to different immunization regimens.


Subject(s)
COVID-19 , Seroconversion , Antibodies, Viral/analysis , Brazil , COVID-19/diagnosis , COVID-19/prevention & control , COVID-19 Vaccines/administration & dosage , ChAdOx1 nCoV-19/administration & dosage , Coronavirus Nucleocapsid Proteins/immunology , Humans , Immunoglobulin A , Immunoglobulin G , Immunoglobulin M , Pandemics , Phosphoproteins/immunology , SARS-CoV-2 , Spike Glycoprotein, Coronavirus/immunology , Vaccination , Vaccines, Inactivated/administration & dosage
5.
EBioMedicine ; 75: 103805, 2022 Jan.
Article in English | MEDLINE | ID: covidwho-1850947

ABSTRACT

BACKGROUND: Two doses of mRNA vaccination have shown >94% efficacy at preventing COVID-19 mostly in naïve adults, but it is not clear if the second dose is needed to maximize effectiveness in those previously exposed to SARS-CoV-2 and what other factors affect responsiveness. METHODS: We measured IgA, IgG and IgM levels against SARS-CoV-2 spike (S) and nucleocapsid (N) antigens from the wild-type and S from the Alpha, Beta and Gamma variants of concern, after BNT162b2 (Pfizer/BioNTech) or mRNA-1273 (Moderna) vaccination in a cohort of health care workers (N=578). Neutralizing capacity and antibody avidity were evaluated. Data were analyzed in relation to COVID-19 history, comorbidities, vaccine doses, brand and adverse events. FINDINGS: Vaccination induced robust IgA and IgG levels against all S antigens. Neutralization capacity and S IgA and IgG levels were higher in mRNA-1273 vaccinees, previously SARS-CoV-2 exposed, particularly if symptomatic, and in those experiencing systemic adverse effects (p<0·05). A second dose in pre-exposed did not increase antibody levels. Smoking and comorbidities were associated with 43% (95% CI, 19-59) and 45% (95% CI, 63-18) lower neutralization, respectively, and 35% (95% CI, 3-57%) and 55% (95% CI, 33-70%) lower antibody levels, respectively. Among fully vaccinated, 6·3% breakthroughs were detected up to 189 days post-vaccination. Among pre-exposed non-vaccinated, 90% were IgG seropositive more than 300 days post-infection. INTERPRETATION: Our data support administering a single-dose in pre-exposed healthy individuals as primary vaccination. However, heterogeneity of responses suggests that personalized recommendations may be necessary depending on COVID-19 history and life-style. Higher mRNA-1273 immunogenicity would be beneficial for those expected to respond worse to vaccination and in face of variants that escape immunity such as Omicron. Persistence of antibody levels in pre-exposed unvaccinated indicates maintenance of immunity up to one year. FUNDING: This work was supported by Institut de Salut Global de Barcelona (ISGlobal) internal funds, in-kind contributions from Hospital Clínic de Barcelona, the Fundació Privada Daniel Bravo Andreu, and European Institute of Innovation and Technology (EIT) Health (grant number 20877), supported by the European Institute of Innovation and Technology, a body of the European Union receiving support from the H2020 Research and Innovation Programme. We acknowledge support from the Spanish Ministry of Science and Innovation and State Research Agency through the "Centro de Excelencia Severo Ochoa 2019-2023" Program (CEX2018-000806-S), and support from the Generalitat de Catalunya through the CERCA Program. L. I. work was supported by PID2019-110810RB-I00 grant from the Spanish Ministry of Science & Innovation. Development of SARS-CoV-2 reagents was partially supported by the National Institute of Allergy and Infectious Diseases Centers of Excellence for Influenza Research and Surveillance (contract number HHSN272201400008C). The funders had no role in study design, data collection and analysis, the decision to publish, or the preparation of the manuscript.


Subject(s)
/administration & dosage , Antibody Formation/drug effects , COVID-19/prevention & control , Health Personnel , SARS-CoV-2/immunology , /immunology , Adult , Antibodies, Viral/immunology , COVID-19/epidemiology , COVID-19/immunology , Coronavirus Nucleocapsid Proteins/immunology , Female , Humans , Immunogenicity, Vaccine , Immunoglobulin A/immunology , Immunoglobulin G/immunology , Male , Middle Aged , Phosphoproteins/immunology , Spike Glycoprotein, Coronavirus/immunology
6.
BMC Microbiol ; 21(1): 351, 2021 12 18.
Article in English | MEDLINE | ID: covidwho-1840945

ABSTRACT

BACKGROUND: The 2019 novel coronavirus disease (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 virus (SARS-CoV-2) is a current worldwide threat for which the immunological features after infection need to be investigated. The aim of this study was to establish a highly sensitive and quantitative detection method for SARS-CoV-2 IgG antibody and to compare the antibody reaction difference in patients with different disease severity. RESULTS: Recombinant SARS-CoV-2 nucleocapsid protein was expressed in Escherichia coli and purified to establish an indirect IgG ELISA detection system. The sensitivity of the ELISA was 100% with a specificity of 96.8% and a 98.3% concordance when compared to a colloidal gold kit, in addition, the sensitivity of the ELISA was 100% with a specificity of 98.9% and a 99.4% concordance when compared to a SARS-CoV-2 spike S1 protein IgG antibody ELISA kit. The increased sensitivity resulted in a higher rate of IgG antibody detection for COVID-19 patients. Moreover, the quantitative detection can be conducted with a much higher serum dilution (1:400 vs 1:10, 1:400 vs 1:100). The antibody titers of 88 patients with differing COVID-19 severity at their early convalescence ranged from 800 to 102,400, and the geometric mean titer for severe and critical cases, moderate cases, asymptomatic and mild cases was 51,203, 20,912, and 9590 respectively. CONCLUSION: The development of a highly sensitive ELISA system for the detection of SARS-CoV-2 IgG antibodies is described herein. This system enabled a quantitative study of rSARS-CoV-2-N IgG antibody titers in COVID-19 patients, the occurrence of higher IgG antibody titers were found to be correlated with more severe cases.


Subject(s)
Antibodies, Viral/blood , COVID-19/diagnosis , Coronavirus Nucleocapsid Proteins/immunology , Immunoglobulin G/blood , Adolescent , Adult , Aged , Aged, 80 and over , COVID-19/immunology , Child , Child, Preschool , China , Enzyme-Linked Immunosorbent Assay , Female , Humans , Infant , Male , Middle Aged , Phosphoproteins/immunology , SARS-CoV-2 , Sensitivity and Specificity , Young Adult
7.
Viruses ; 14(5)2022 05 05.
Article in English | MEDLINE | ID: covidwho-1820421

ABSTRACT

Serological detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) nucleocapsid (N), spike (S), and neutralizing antibodies (Abs) is commonly undertaken to evaluate the efficacy of vaccination. However, the relative efficiency of different SARS-CoV-2 Ab detection systems has not been extensively investigated. Here, we evaluated serological test systems in vaccinated Japanese. SARS-CoV-2 N, S, and neutralizing Abs in sera of 375 healthy subjects a mean 253 days after vaccination were assessed. The sensitivity of Elecsys Anti-SARS-CoV-2 S (Roche S) and Anti-SARS-CoV-2 S IgG (Fujirebio S) was 100% and 98.9%, respectively, with a specificity of 100% for both. The sensitivity of Anti-SARS-CoV-2 neutralizing Ab (MBL Neu) was 2.7%, and the specificity was 100%. Fujirebio S correlated with Roche S (rho = 0.9182, p = 3.97 × 10-152). Fujirebio S (rho = 0.1295, p = 0.0121) and Roche S (rho = 0.1232, p = 0.0170) correlated weakly with MBL Neu. However, Roche S did correlate with MBL Neu in patients with COVID-19 (rho = 0.8299, p = 1.01 × 10-12) and in healthy subjects more recently after vaccination (mean of 90 days, rho = 0.5306, p = 0.0003). Thus, the Fujirebio S and Roche S results were very similar, but neither correlated with neutralizing antibody titers by MBL Neu at a later time after vaccination.


Subject(s)
Antibodies, Neutralizing , COVID-19 Vaccines , COVID-19 , Antibodies, Neutralizing/blood , Antibodies, Viral/blood , COVID-19/immunology , COVID-19/prevention & control , Coronavirus Nucleocapsid Proteins/immunology , Humans , Immunoglobulin G/blood , Japan , Phosphoproteins/immunology , SARS-CoV-2 , Spike Glycoprotein, Coronavirus
8.
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
10.
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
11.
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
12.
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
13.
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
14.
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
15.
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
16.
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
18.
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
19.
PLoS One ; 17(1): e0262868, 2022.
Article in English | MEDLINE | ID: covidwho-1643287

ABSTRACT

A serological COVID-19 Multiplex Assay was developed and validated using serum samples from convalescent patients and those collected prior to the 2020 pandemic. After initial testing of multiple potential antigens, the SARS-CoV-2 nucleocapsid protein (NP) and receptor-binding domain (RBD) of the spike protein were selected for the human COVID-19 Multiplex Assay. A comparison of synthesized and mammalian expressed RBD proteins revealed clear advantages of mammalian expression. Antibodies directed against NP strongly correlated with SARS-CoV-2 virus neutralization assay titers (rsp = 0.726), while anti-RBD correlation was moderate (rsp = 0.436). Pan-Ig, IgG, IgA, and IgM against NP and RBD antigens were evaluated on the validation sample sets. Detection of NP and RBD specific IgG and IgA had outstanding performance (AUC > 0.90) for distinguishing patients from controls, but the dynamic range of the IgG assay was substantially greater. The COVID-19 Multiplex Assay was utilized to identify seroprevalence to SARS-CoV-2 in people living in a low-incidence community in Ithaca, NY. Samples were taken from a cohort of healthy volunteers (n = 332) in early June 2020. Only two volunteers had a positive result on a COVID-19 PCR test performed prior to serum sampling. Serological testing revealed an exposure rate of at least 1.2% (NP) or as high as 5.7% (RBD), higher than the measured incidence rate of 0.16% in the county at that time. This highly sensitive and quantitative assay can be used for monitoring community exposure rates and duration of immune response following both infection and vaccination.


Subject(s)
Antibodies, Viral/chemistry , COVID-19 Serological Testing/methods , COVID-19/diagnosis , Coronavirus Nucleocapsid Proteins/immunology , SARS-CoV-2/immunology , Spike Glycoprotein, Coronavirus/immunology , Adolescent , Adult , Aged , Aged, 80 and over , Antibodies, Neutralizing/chemistry , Antibodies, Neutralizing/immunology , Antibodies, Viral/immunology , COVID-19/blood , COVID-19/epidemiology , COVID-19 Serological Testing/standards , Coronavirus Nucleocapsid Proteins/chemistry , Epidemiological Monitoring , Female , Humans , Immunoglobulin A/chemistry , Immunoglobulin A/immunology , Immunoglobulin G/chemistry , Immunoglobulin G/immunology , Immunoglobulin M/chemistry , Immunoglobulin M/immunology , Male , Middle Aged , New York/epidemiology , Phosphoproteins/chemistry , Phosphoproteins/immunology , Protein Interaction Domains and Motifs , Recombinant Proteins/chemistry , Recombinant Proteins/immunology , SARS-CoV-2/classification , Sensitivity and Specificity , Spike Glycoprotein, Coronavirus/chemistry
20.
Front Immunol ; 12: 751869, 2021.
Article in English | MEDLINE | ID: covidwho-1634057

ABSTRACT

BACKGROUND: Immunological characteristics of COVID-19 show pathological hyperinflammation associated with lymphopenia and dysfunctional T cell responses. These features provide a rationale for restoring functional T cell immunity in COVID-19 patients by adoptive transfer of SARS-CoV-2 specific T cells. METHODS: To generate SARS-CoV-2 specific T cells, we isolated peripheral blood mononuclear cells from 7 COVID-19 recovered and 13 unexposed donors. Consequently, we stimulated cells with SARS-CoV-2 peptide mixtures covering spike, membrane and nucleocapsid proteins. Then, we culture expanded cells with IL-2 for 21 days. We assessed immunophenotypes, cytokine profiles, antigen specificity of the final cell products. RESULTS: Our results show that SARS-CoV-2 specific T cells could be expanded in both COVID-19 recovered and unexposed groups. Immunophenotypes were similar in both groups showing CD4+ T cell dominance, but CD8+ and CD3+CD56+ T cells were also present. Antigen specificity was determined by ELISPOT, intracellular cytokine assay, and cytotoxicity assays. One out of 14 individuals who were previously unexposed to SARS-CoV-2 failed to show antigen specificity. Moreover, ex-vivo expanded SARS-CoV-2 specific T cells mainly consisted of central and effector memory subsets with reduced alloreactivity against HLA-unmatched cells suggesting the possibility for the development of third-party partial HLA-matching products. DISCUSSION: In conclusion, our findings show that SARS-CoV-2 specific T cell can be readily expanded from both COVID-19 and unexposed individuals and can therefore be manufactured as a biopharmaceutical product to treat severe COVID-19 patients. ONE SENTENCE SUMMARY: Ex-vivo expanded SARS-CoV-2 antigen specific T cells developed as third-party partial HLA-matching products may be a promising approach for treating severe COVID-19 patients that do not respond to previous treatment options.


Subject(s)
Adoptive Transfer , CD4-Positive T-Lymphocytes/transplantation , CD8-Positive T-Lymphocytes/transplantation , COVID-19/therapy , SARS-CoV-2/immunology , Adult , Antibodies, Viral/immunology , CD4-Positive T-Lymphocytes/immunology , CD8-Positive T-Lymphocytes/immunology , COVID-19/immunology , Cell- and Tissue-Based Therapy , Coronavirus Nucleocapsid Proteins/immunology , Epitopes, T-Lymphocyte/immunology , Female , Humans , Immunophenotyping , Leukocytes, Mononuclear/immunology , Male , Middle Aged , Phosphoproteins/immunology , Spike Glycoprotein, Coronavirus/immunology , Viral Matrix Proteins/immunology , Young Adult
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