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1.
Eur J Immunol ; 51(11): 2665-2676, 2021 11.
Article in English | MEDLINE | ID: covidwho-1482126

ABSTRACT

To monitor infection by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and successful vaccination against coronavirus disease 2019 (COVID-19), the kinetics of neutralizing or blocking anti-SARS-CoV-2 antibody titers need to be assessed. Here, we report the development of a quick and inexpensive surrogate SARS-CoV-2 blocking assay (SUBA) using immobilized recombinant human angiotensin-converting enzyme 2 (hACE2) and human cells expressing the native form of surface SARS-CoV-2 spike protein. Spike protein-expressing cells bound to hACE2 in the absence or presence of blocking antibodies were quantified by measuring the optical density of cell-associated crystal violet in a spectrophotometer. The advantages are that SUBA is a fast and inexpensive assay, which does not require biosafety level 2- or 3-approved laboratories. Most importantly, SUBA detects blocking antibodies against the native trimeric cell-bound SARS-CoV-2 spike protein and can be rapidly adjusted to quickly pre-screen already approved therapeutic antibodies or sera from vaccinated individuals for their ACE2 blocking activities against any emerging SARS-CoV-2 variants.


Subject(s)
Antibodies, Blocking/blood , Antibodies, Neutralizing/blood , Antibodies, Viral/analysis , COVID-19 Serological Testing/methods , COVID-19/diagnosis , Flow Cytometry/methods , Antibodies, Blocking/immunology , Antibodies, Neutralizing/immunology , COVID-19/immunology , Humans , SARS-CoV-2 , Spike Glycoprotein, Coronavirus/immunology
2.
Front Immunol ; 12: 730766, 2021.
Article in English | MEDLINE | ID: covidwho-1463473

ABSTRACT

The SARS-CoV-2 pandemic has spread to all parts of the world and can cause life-threatening pneumonia and other severe disease manifestations known as COVID-19. This health crisis has resulted in a significant effort to stop the spread of this new coronavirus. However, while propagating itself in the human population, the virus accumulates mutations and generates new variants with increased fitness and the ability to escape the human immune response. Here we describe a color-based barcoded spike flow cytometric assay (BSFA) that is particularly useful to evaluate and directly compare the humoral immune response directed against either wild type (WT) or mutant spike (S) proteins or the receptor-binding domains (RBD) of SARS-CoV-2. This assay employs the human B lymphoma cell line Ramos, transfected for stable expression of WT or mutant S proteins or a chimeric RBD-CD8 fusion protein. We find that the alpha and beta mutants are more stably expressed than the WT S protein on the Ramos B cell surface and/or bind with higher affinity to the viral entry receptor ACE2. However, we find a reduce expression of the chimeric RBD-CD8 carrying the point mutation N501Y and E484K characteristic for the alpha and beta variant, respectively. The comparison of the humoral immune response of 12 vaccinated probands with 12 COVID-19 patients shows that after the boost, the S-specific IgG class immune response in the vaccinated group is similar to that of the patient group. However, in comparison to WT the specific IgG serum antibodies bind less well to the alpha variant and only poorly to the beta variant S protein. This is in line with the notion that the beta variant is an immune escape variant of SARS-CoV-2. The IgA class immune response was more variable than the IgG response and higher in the COVID-19 patients than in the vaccinated group. In summary, we think that our BSFA represents a useful tool to evaluate the humoral immunity against emerging variants of SARS-CoV-2 and to analyze new vaccination protocols against these variants.


Subject(s)
COVID-19/immunology , Cell Separation/methods , Flow Cytometry/methods , SARS-CoV-2/physiology , Spike Glycoprotein, Coronavirus/metabolism , Angiotensin-Converting Enzyme 2/metabolism , Antibodies, Viral/metabolism , Antibody Formation , Female , Humans , Immunization, Secondary , Immunoglobulin A/metabolism , Immunoglobulin G/metabolism , Male , Middle Aged , Mutation/genetics , Spike Glycoprotein, Coronavirus/genetics , Vaccination
3.
Eur J Immunol ; 51(11): 2633-2640, 2021 11.
Article in English | MEDLINE | ID: covidwho-1344986

ABSTRACT

Here, we describe a new, simple, highly multiplexed serological test that generates a more complete picture of seroconversion than single antigen-based assays. Flow cytometry is used to detect multiple Ig isotypes binding to four SARS-CoV-2 antigens: the Spike glycoprotein, its RBD fragment (the main target for neutralizing antibodies), the nucleocapsid protein, and the main cysteine-like protease in a single reaction. Until now, most diagnostic serological tests measured antibodies to only one antigen and in some laboratory-confirmed patients no SARS-CoV-2-specific antibodies could be detected. Our data reveal that while most patients respond against all the viral antigens tested, others show a marked bias to make antibodies against either proteins exposed on the viral particle or those released after cellular infection. With this assay, it was possible to discriminate between patients and healthy controls with 100% confidence. Analysing the response of multiple Ig isotypes to the four antigens in combination may also help to establish a correlation with the severity degree of disease. A more detailed description of the immune responses of different patients to SARS-CoV-2 virus might provide insight into the wide array of clinical presentations of COVID-19.


Subject(s)
Antibodies, Viral/blood , COVID-19 Serological Testing/methods , COVID-19/diagnosis , Flow Cytometry/methods , Antigens, Viral/immunology , COVID-19/immunology , High-Throughput Screening Assays , Humans , SARS-CoV-2 , Sensitivity and Specificity , Serologic Tests
4.
Adv Sci (Weinh) ; 8(18): e2100323, 2021 09.
Article in English | MEDLINE | ID: covidwho-1316190

ABSTRACT

Blood cell analysis is a major pillar of biomedical research and healthcare. These analyses are performed in central laboratories. Rapid shipment from collection site to the central laboratories is currently needed because cells and biomarkers degrade rapidly. The dried blood spot from a fingerstick allows the preservation of cellular molecules for months but entire cells are never recovered. Here leucocyte elution is optimized from dried blood spots. Flow cytometry and mRNA expression profiling are used to analyze the recovered cells. 50-70% of the leucocytes that are dried on a polyester solid support via elution after shaking the support with buffer are recovered. While red blood cells lyse upon drying, it is found that the majority of leucocytes are preserved. Leucocytes have an altered structure that is improved by adding fixative in the elution buffer. Leucocytes are permeabilized, allowing an easy staining of all cellular compartments. Common immunophenotyping and mRNAs are preserved. The ability of a new biomarker (CD169) to discriminate between patients with and without Severe Acute Respiratory Syndrome induced by Coronavirus 2 (SARS-CoV-2) infections is also preserved. Leucocytes from blood can be dried, shipped, and/or stored for at least 1 month, then recovered for a wide variety of analyses, potentially facilitating biomedical applications worldwide.


Subject(s)
Communicable Diseases/diagnosis , Diagnostic Tests, Routine/methods , Dried Blood Spot Testing/methods , Hematology/methods , Immunophenotyping/methods , Antibodies, Viral/blood , Biomarkers/blood , Blood Specimen Collection/methods , COVID-19/diagnosis , Cell Separation/methods , Communicable Diseases/virology , Erythrocytes/virology , Flow Cytometry/methods , Humans , Leukocytes/virology , RNA, Messenger/blood , SARS-CoV-2/genetics
5.
J Immunol ; 207(2): 720-734, 2021 07 15.
Article in English | MEDLINE | ID: covidwho-1311404

ABSTRACT

Most shared resource flow cytometry facilities do not permit analysis of radioactive samples. We are investigating low-dose molecular targeted radionuclide therapy (MTRT) as an immunomodulator in combination with in situ tumor vaccines and need to analyze radioactive samples from MTRT-treated mice using flow cytometry. Further, the sudden shutdown of core facilities in response to the COVID-19 pandemic has created an unprecedented work stoppage. In these and other research settings, a robust and reliable means of cryopreservation of immune samples is required. We evaluated different fixation and cryopreservation protocols of disaggregated tumor cells with the aim of identifying a protocol for subsequent flow cytometry of the thawed sample, which most accurately reflects the flow cytometric analysis of the tumor immune microenvironment of a freshly disaggregated and analyzed sample. Cohorts of C57BL/6 mice bearing B78 melanoma tumors were evaluated using dual lymphoid and myeloid immunophenotyping panels involving fixation and cryopreservation at three distinct points during the workflow. Results demonstrate that freezing samples after all staining and fixation are completed most accurately matches the results from noncryopreserved equivalent samples. We observed that cryopreservation of living, unfixed cells introduces a nonuniform alteration to PD1 expression. We confirm the utility of our cryopreservation protocol by comparing tumors treated with in situ tumor vaccines, analyzing both fresh and cryopreserved tumor samples with similar results. Last, we use this cryopreservation protocol with radioactive specimens to demonstrate potentially beneficial effector cell changes to the tumor immune microenvironment following administration of a novel MTRT in a dose- and time-dependent manner.


Subject(s)
Cryopreservation/methods , Flow Cytometry/methods , Leukocytes, Mononuclear/immunology , Melanoma, Experimental/pathology , Myeloid Cells/immunology , Animals , CD4-Positive T-Lymphocytes/immunology , CD8-Positive T-Lymphocytes/immunology , Cell Line, Tumor , Immunophenotyping/methods , Mice , Mice, Inbred C57BL , Natural Killer T-Cells/immunology , Pandemics , Signal Transduction/immunology , Tumor Microenvironment/immunology
6.
Eur J Immunol ; 51(8): 1992-2005, 2021 08.
Article in English | MEDLINE | ID: covidwho-1251932

ABSTRACT

The phenotype of infused cells is a major determinant of Adoptive T-cell therapy (ACT) efficacy. Yet, the difficulty in deciphering multiparametric cytometry data limited the fine characterization of cellular products. To allow the analysis of dynamic and complex flow cytometry samples, we developed cytoChain, a novel dataset mining tool and a new analytical workflow. CytoChain was challenged to compare state-of-the-art and innovative culture conditions to generate stem-like memory cells (TSCM ) suitable for ACT. Noticeably, the combination of IL-7/15 and superoxides scavenging sustained the emergence of a previously unidentified nonexhausted Fit-TSCM signature, overlooked by manual gating and endowed with superior expansion potential. CytoChain proficiently traced back this population in independent datasets, and in T-cell receptor engineered lymphocytes. CytoChain flexibility and function were then further validated on a published dataset from circulating T cells in COVID-19 patients. Collectively, our results support the use of cytoChain to identify novel, functionally critical immunophenotypes for ACT and patients immunomonitoring.


Subject(s)
Data Mining/methods , Flow Cytometry/methods , Receptors, Antigen, T-Cell/immunology , Receptors, Chimeric Antigen/immunology , T-Lymphocytes/immunology , T-Lymphocytes/metabolism , COVID-19/blood , COVID-19/immunology , Cytokines/metabolism , Genetic Engineering , Humans , Immunologic Memory , Immunophenotyping , Immunotherapy, Adoptive , Receptors, Antigen, T-Cell/genetics , Receptors, Antigen, T-Cell/metabolism , Receptors, Chimeric Antigen/genetics , SARS-CoV-2/immunology
7.
Blood ; 137(26): 3656-3659, 2021 07 01.
Article in English | MEDLINE | ID: covidwho-1215090

ABSTRACT

Vaccination is crucial in combatting the severe acute respiratory syndrome coronavirus 2 pandemic. The rare complication of thrombocytopenia and thrombotic complications at unusual sites after ChAdOx1 nCov-19 vaccination is caused by platelet-activating antibodies directed against platelet factor 4 (PF4). We present a widely applicable whole-blood standard flow cytometric assay to identify the pathogenic antibodies associated with vaccine-induced immune-mediated thrombotic thrombocytopenia (VITT) after ChAdOx1 nCov-19 vaccination. This assay will enable rapid diagnosis by many laboratories. This trial was registered at www.clinicaltrials.gov as #NCT04370119.


Subject(s)
Autoantibodies/blood , COVID-19 Vaccines/adverse effects , COVID-19/prevention & control , Flow Cytometry/methods , Immunoglobulin G/blood , Platelet Activation/immunology , Platelet Factor 4/immunology , Purpura, Thrombocytopenic, Idiopathic/diagnosis , Receptors, IgG/immunology , SARS-CoV-2 , Vaccination/adverse effects , Antibody Specificity , Autoantibodies/biosynthesis , Autoantibodies/immunology , COVID-19 Vaccines/immunology , Heparin/adverse effects , Heparin/immunology , Humans , Immunoenzyme Techniques , Immunogenicity, Vaccine , Immunoglobulin G/biosynthesis , Immunoglobulin G/immunology , P-Selectin/analysis , Purpura, Thrombocytopenic, Idiopathic/etiology , Purpura, Thrombocytopenic, Idiopathic/immunology
8.
J Vis Exp ; (170)2021 04 06.
Article in English | MEDLINE | ID: covidwho-1202107

ABSTRACT

The COVID-19 pandemic has underscored the need for rapid high-throughput methods for sensitive and specific serological detection of infection with novel pathogens, such as SARS-CoV-2. Multiplex serological testing can be particularly useful because it can simultaneously analyze antibodies to multiple antigens that optimizes pathogen coverage, and controls for variability in the organism and the individual host response. Here we describe a SARS-CoV-2 IgG 3-plex fluorescent microsphere-based assay that can detect both IgM and IgG antibodies to three major SARS-CoV-2 antigens-the spike (S) protein, spike angiotensin-converting enzyme-2 (ACE2) receptor-binding domain (RBD), and nucleocapsid (Nc). The assay was shown to have comparable performance to a SARS-CoV-2 reference assay for IgG in serum obtained at ≥21 days from symptom onset but had higher sensitivity with samples collected at ≤5 days from symptom onset. Further, using soluble ACE2 in a neutralization assay format, inhibition of antibody binding was demonstrated for S and RBD.


Subject(s)
Antibodies, Neutralizing/blood , COVID-19 Serological Testing/methods , COVID-19/diagnosis , Flow Cytometry/methods , Immunoglobulin G/blood , Immunoglobulin M/blood , SARS-CoV-2/immunology , Antibodies, Viral/blood , Antibodies, Viral/immunology , COVID-19/blood , COVID-19/immunology , Humans , Spike Glycoprotein, Coronavirus/immunology
9.
Commun Biol ; 4(1): 486, 2021 04 20.
Article in English | MEDLINE | ID: covidwho-1195630

ABSTRACT

There is an ongoing need of developing sensitive and specific methods for the determination of SARS-CoV-2 seroconversion. For this purpose, we have developed a multiplexed flow cytometric bead array (C19BA) that allows the identification of IgG and IgM antibodies against three immunogenic proteins simultaneously: the spike receptor-binding domain (RBD), the spike protein subunit 1 (S1) and the nucleoprotein (N). Using different cohorts of samples collected before and after the pandemic, we show that this assay is more sensitive than ELISAs performed in our laboratory. The combination of three viral antigens allows for the interrogation of full seroconversion. Importantly, we have detected N-reactive antibodies in COVID-19-negative individuals. Here we present an immunoassay that can be easily implemented and has superior potential to detect low antibody titers compared to current gold standard serology methods.


Subject(s)
Antibodies, Viral/immunology , COVID-19/diagnosis , Flow Cytometry/methods , Nucleoproteins/immunology , SARS-CoV-2/immunology , Seroconversion , Antigens, Viral/immunology , COVID-19/epidemiology , COVID-19/virology , Humans , Immunoassay/methods , Immunoglobulin G/immunology , Immunoglobulin M/immunology , Pandemics , Reproducibility of Results , SARS-CoV-2/physiology , Sensitivity and Specificity
10.
PLoS Pathog ; 17(4): e1009448, 2021 04.
Article in English | MEDLINE | ID: covidwho-1190180

ABSTRACT

The SARS-CoV-2 infection causes severe respiratory involvement (COVID-19) in 5-20% of patients through initial immune derangement, followed by intense cytokine production and vascular leakage. Evidence of immune involvement point to the participation of T, B, and NK cells in the lack of control of virus replication leading to COVID-19. NK cells contribute to early phases of virus control and to the regulation of adaptive responses. The precise mechanism of NK cell dysregulation is poorly understood, with little information on tissue margination or turnover. We investigated these aspects by multiparameter flow cytometry in a cohort of 28 patients hospitalized with early COVID-19. Relevant decreases in CD56brightCD16+/- NK subsets were detected, with a shift of circulating NK cells toward more mature CD56dimCD16+KIR+NKG2A+ and "memory" KIR+CD57+CD85j+ cells with increased inhibitory NKG2A and KIR molecules. Impaired cytotoxicity and IFN-γ production were associated with conserved expression of natural cytotoxicity receptors and perforin. Moreover, intense NK cell activation with increased HLA-DR and CD69 expression was associated with the circulation of CD69+CD103+ CXCR6+ tissue-resident NK cells and of CD34+DNAM-1brightCXCR4+ inflammatory precursors to mature functional NK cells. Severe disease trajectories were directly associated with the proportion of CD34+DNAM-1brightCXCR4+ precursors and inversely associated with the proportion of NKG2D+ and of CD103+ NK cells. Intense NK cell activation and trafficking to and from tissues occurs early in COVID-19, and is associated with subsequent disease progression, providing an insight into the mechanism of clinical deterioration. Strategies to positively manipulate tissue-resident NK cell responses may provide advantages to future therapeutic and vaccine approaches.


Subject(s)
COVID-19/immunology , Killer Cells, Natural/immunology , Aged , Aged, 80 and over , COVID-19/mortality , Cohort Studies , Female , Flow Cytometry/methods , Humans , Interferon-gamma/metabolism , Italy/epidemiology , Lymphocyte Activation/immunology , Male , Middle Aged , Severity of Illness Index
11.
Crit Care ; 25(1): 23, 2021 01 09.
Article in English | MEDLINE | ID: covidwho-1151679

ABSTRACT

BACKGROUND: Biomarkers of disease severity might help individualizing the management of patients with the acute respiratory distress syndrome (ARDS). Whether the alveolar compartmentalization of biomarkers has a clinical significance in patients with pneumonia-related ARDS is unknown. This study aimed at assessing the interrelation of ARDS/sepsis biomarkers in the alveolar and blood compartments and explored their association with clinical outcomes. METHODS: Immunocompetent patients with pneumonia-related ARDS admitted between 2014 and 2018 were included in a prospective monocentric study. Bronchoalveolar lavage (BAL) fluid and blood samples were obtained within 48 h of admission. Twenty-two biomarkers were quantified in BAL fluid and serum. HLA-DR+ monocytes and CD8+ PD-1+ lymphocytes were quantified using flow cytometry. The primary clinical endpoint of the study was hospital mortality. Patients undergoing a bronchoscopy as part of routine care were included as controls. RESULTS: Seventy ARDS patients were included. Hospital mortality was 21.4%. The BAL fluid-to-serum ratio of IL-8 was 20 times higher in ARDS patients than in controls (p < 0.0001). ARDS patients with shock had lower BAL fluid-to-serum ratio of IL-1Ra (p = 0.026), IL-6 (p = 0.002), IP-10/CXCL10 (p = 0.024) and IL-10 (p = 0.023) than others. The BAL fluid-to-serum ratio of IL-1Ra was more elevated in hospital survivors than decedents (p = 0.006), even after adjusting for SOFA and driving pressure (p = 0.036). There was no significant association between alveolar or alveolar/blood monocytic HLA-DR or CD8+ lymphocytes PD-1 expression and hospital mortality. CONCLUSIONS: IL-8 was the most compartmentalized cytokine and lower BAL fluid-to-serum concentration ratios of IL-1Ra were associated with hospital mortality in patients with pneumonia-associated ARDS.


Subject(s)
Biomarkers/analysis , Bronchoalveolar Lavage Fluid/immunology , Pulmonary Alveoli/drug effects , Aged , Analysis of Variance , Biomarkers/blood , Bronchoalveolar Lavage/methods , Cohort Studies , Female , Flow Cytometry/methods , France , Humans , Male , Middle Aged , Pneumonia/complications , Prospective Studies , Respiratory Distress Syndrome , Statistics, Nonparametric
12.
Int J Mol Sci ; 22(5)2021 Mar 08.
Article in English | MEDLINE | ID: covidwho-1150749

ABSTRACT

Quantitative and robust serology assays are critical measurements underpinning global COVID-19 response to diagnostic, surveillance, and vaccine development. Here, we report a proof-of-concept approach for the development of quantitative, multiplexed flow cytometry-based serological and neutralization assays. The serology assays test the IgG and IgM against both the full-length spike antigens and the receptor binding domain (RBD) of the spike antigen. Benchmarking against an RBD-specific SARS-CoV IgG reference standard, the anti-SARS-CoV-2 RBD antibody titer was quantified in the range of 37.6 µg/mL to 31.0 ng/mL. The quantitative assays are highly specific with no correlative cross-reactivity with the spike proteins of MERS, SARS1, OC43 and HKU1 viruses. We further demonstrated good correlation between anti-RBD antibody titers and neutralizing antibody titers. The suite of serology and neutralization assays help to improve measurement confidence and are complementary and foundational for clinical and epidemiologic studies.


Subject(s)
COVID-19 Serological Testing/methods , COVID-19 Serological Testing/standards , COVID-19/blood , COVID-19/immunology , Neutralization Tests/methods , Neutralization Tests/standards , SARS-CoV-2/immunology , Antibodies, Neutralizing/blood , Antibodies, Neutralizing/immunology , Antibodies, Viral/blood , Antibodies, Viral/immunology , Cross Reactions , Flow Cytometry/methods , Fluorescence , Humans , Immunoglobulin G/blood , Immunoglobulin G/immunology , Immunoglobulin M/blood , Immunoglobulin M/immunology , Microspheres , Receptors, Virus/chemistry , Receptors, Virus/immunology , Spike Glycoprotein, Coronavirus/chemistry , Spike Glycoprotein, Coronavirus/immunology
13.
J Biosci Bioeng ; 131(6): 696-702, 2021 Jun.
Article in English | MEDLINE | ID: covidwho-1141952

ABSTRACT

Monoclonal antibodies are extremely valuable functional biomaterials that are widely used not only in life science research but also in antibody drugs and test drugs. There is also a strong need to develop high-quality neutralizing antibodies as soon as possible in order to stop the rapid spread of new infectious diseases such as the SARS-CoV-2 virus. This study has developed a membrane-type immunoglobulin-directed hybridoma screening (MIHS) method for obtaining high-quality monoclonal antibodies with high efficiency and high speed. In addition to these advantages, this paper demonstrates that the MIHS method can selectively obtain monoclonal antibodies that specifically recognize the functional structure of proteins. The MIHS method is a useful technology that greatly contributes to the research community because it can be easily introduced in any laboratory that uses a flow cytometer.


Subject(s)
Antibodies, Monoclonal/analysis , Antibodies, Monoclonal/immunology , Antibody Specificity/immunology , Hybridomas/metabolism , Animals , Antibodies, Monoclonal/biosynthesis , Antibodies, Monoclonal/isolation & purification , Antibodies, Neutralizing/analysis , Antibodies, Neutralizing/biosynthesis , Antibodies, Neutralizing/immunology , Antibodies, Neutralizing/isolation & purification , Cell Line, Tumor , Enzyme-Linked Immunosorbent Assay/methods , Flow Cytometry/methods , Green Fluorescent Proteins/genetics , Green Fluorescent Proteins/immunology , Green Fluorescent Proteins/metabolism , Humans , Hybridomas/cytology , Immunoglobulin Isotypes , Immunoprecipitation , Mice , Time Factors
14.
Int J Mol Sci ; 22(5)2021 Mar 08.
Article in English | MEDLINE | ID: covidwho-1134167

ABSTRACT

Quantitative and robust serology assays are critical measurements underpinning global COVID-19 response to diagnostic, surveillance, and vaccine development. Here, we report a proof-of-concept approach for the development of quantitative, multiplexed flow cytometry-based serological and neutralization assays. The serology assays test the IgG and IgM against both the full-length spike antigens and the receptor binding domain (RBD) of the spike antigen. Benchmarking against an RBD-specific SARS-CoV IgG reference standard, the anti-SARS-CoV-2 RBD antibody titer was quantified in the range of 37.6 µg/mL to 31.0 ng/mL. The quantitative assays are highly specific with no correlative cross-reactivity with the spike proteins of MERS, SARS1, OC43 and HKU1 viruses. We further demonstrated good correlation between anti-RBD antibody titers and neutralizing antibody titers. The suite of serology and neutralization assays help to improve measurement confidence and are complementary and foundational for clinical and epidemiologic studies.


Subject(s)
COVID-19 Serological Testing/methods , COVID-19 Serological Testing/standards , COVID-19/blood , COVID-19/immunology , Neutralization Tests/methods , Neutralization Tests/standards , SARS-CoV-2/immunology , Antibodies, Neutralizing/blood , Antibodies, Neutralizing/immunology , Antibodies, Viral/blood , Antibodies, Viral/immunology , Cross Reactions , Flow Cytometry/methods , Fluorescence , Humans , Immunoglobulin G/blood , Immunoglobulin G/immunology , Immunoglobulin M/blood , Immunoglobulin M/immunology , Microspheres , Receptors, Virus/chemistry , Receptors, Virus/immunology , Spike Glycoprotein, Coronavirus/chemistry , Spike Glycoprotein, Coronavirus/immunology
15.
Cytometry A ; 99(1): 11-18, 2021 01.
Article in English | MEDLINE | ID: covidwho-1086332

ABSTRACT

Cytometry is playing a crucial role in addressing the COVID-19 pandemic. In this commentary-written by a variety of stakeholders in the cytometry, immunology, and infectious disease communities-we review cytometry's role in the COVID-19 response and discuss workflow issues critical to planning and executing effective research in this emerging field. We discuss sample procurement and processing, biosafety, technology options, data sharing, and the translation of research findings into clinical environments. © 2020 International Society for Advancement of Cytometry.


Subject(s)
COVID-19/prevention & control , Containment of Biohazards/trends , Flow Cytometry/trends , SARS-CoV-2/isolation & purification , /trends , Biomedical Research/methods , Biomedical Research/trends , COVID-19/epidemiology , Containment of Biohazards/methods , Flow Cytometry/methods , Humans , Information Dissemination/methods , /methods
16.
EMBO Mol Med ; 13(3): e13549, 2021 03 05.
Article in English | MEDLINE | ID: covidwho-1038772

ABSTRACT

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


Subject(s)
Antibodies, Neutralizing/blood , Antibodies, Viral/blood , COVID-19 Serological Testing/methods , COVID-19/diagnosis , COVID-19/immunology , Flow Cytometry/methods , SARS-CoV-2/immunology , Spike Glycoprotein, Coronavirus/immunology , Adult , COVID-19/virology , COVID-19 Serological Testing/statistics & numerical data , Enzyme-Linked Immunosorbent Assay , ErbB Receptors/genetics , Female , Flow Cytometry/statistics & numerical data , Hep G2 Cells , Humans , Jurkat Cells , Male , Middle Aged , Neutralization Tests , Pandemics , Polymerase Chain Reaction , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/immunology , SARS-CoV-2/genetics , Spike Glycoprotein, Coronavirus/genetics
17.
Cytometry B Clin Cytom ; 100(1): 33-41, 2021 Jan.
Article in English | MEDLINE | ID: covidwho-1006421

ABSTRACT

Over a remarkably short period of time, a great deal of knowledge about severe acute respiratory syndrome coronavirus 2 (SARS-CoV2) infection has been acquired, through the focused and cooperative effort of the international scientific community. Much has become known about how the immune response is coordinated to fight infection, and how it becomes dysregulated in severe disease. In this review, we take an in-depth look at the many immune features associated with the host response to SARS-CoV2, as well as those that appear to mark severe disease.


Subject(s)
COVID-19/diagnostic imaging , COVID-19/immunology , Flow Cytometry/methods , Fluorescent Antibody Technique/methods , SARS-CoV-2/immunology , Biomarkers/analysis , COVID-19/pathology , COVID-19/therapy , Chemokines/analysis , Chemokines/metabolism , Cytokines/analysis , Cytokines/metabolism , Fluorescent Antibody Technique/trends , Host-Pathogen Interactions/immunology , Humans , Immunity/physiology , Metabolomics/methods , Metabolomics/trends , Risk Assessment , Severity of Illness Index
18.
Int J Mol Sci ; 21(22)2020 Nov 22.
Article in English | MEDLINE | ID: covidwho-945834

ABSTRACT

Recent advances in the field of flow cytometry (FCM) have highlighted the importance of incorporating it as a basic analysis tool in laboratories. FCM not only allows the identification of cell subpopulations by detecting the expression of molecules in the cell membrane or cytoplasm, but it can also quantify and identify soluble molecules. The proper functioning of the FCM requires six fundamental systems, from those related to the transport of events to the systems dedicated to the analysis of information. In this review, we have identified the main considerations that every FCM user must know for an optimal antibody panel design, the quality systems that must govern the FCM protocols to guarantee reproducible results in research or clinical laboratories. Finally, we have introduced the current evidence that highlights the relevance of FCM in the investigation and clinical diagnosis of respiratory diseases, establishing important advances in the basic and clinical study of diseases as old as Tuberculosis along with the recent proposals for the monitoring and classification of patients infected with the new SARS-CoV2 virus.


Subject(s)
Flow Cytometry/methods , Respiratory Tract Diseases/diagnosis , Humans
19.
Eur J Clin Microbiol Infect Dis ; 40(4): 751-759, 2021 Apr.
Article in English | MEDLINE | ID: covidwho-880323

ABSTRACT

SARS-CoV-2 has emerged as a previously unknown zoonotic coronavirus that spread worldwide causing a serious pandemic. While reliable nucleic acid-based diagnostic assays were rapidly available, only a limited number of validated serological assays were available in the early phase of the pandemic. Here, we evaluated a novel flow cytometric approach to assess spike-specific antibody responses.HEK 293T cells expressing SARS-CoV-2 spike protein in its natural confirmation on the surface were used to detect specific IgG and IgM antibody responses in patient sera by flow cytometry. A soluble angiotensin-converting-enzyme 2 (ACE-2) variant was developed as external standard to quantify spike-specific antibody responses on different assay platforms. Analyses of 201 pre-COVID-19 sera proved a high assay specificity in comparison to commercially available CLIA and ELISA systems, while also revealing the highest sensitivity in specimens from PCR-confirmed SARS-CoV-2-infected patients. The external standard allowed robust quantification of antibody responses among different assay platforms. In conclusion, our newly established flow cytometric assay allows sensitive and quantitative detection of SARS-CoV-2-specific antibodies, which can be easily adopted in different laboratories and does not rely on external supply of assay kits. The flow cytometric assay also provides a blueprint for rapid development of serological tests to other emerging viral infections.


Subject(s)
Antibodies, Viral/immunology , COVID-19 Serological Testing/methods , COVID-19/diagnosis , Immunoglobulin G/immunology , Immunoglobulin M/immunology , SARS-CoV-2/immunology , Spike Glycoprotein, Coronavirus/immunology , Angiotensin-Converting Enzyme 2 , Enzyme-Linked Immunosorbent Assay , Flow Cytometry/methods , HEK293 Cells , Humans , Reference Standards , Reproducibility of Results , Sensitivity and Specificity
20.
Front Immunol ; 11: 400, 2020.
Article in English | MEDLINE | ID: covidwho-830046

ABSTRACT

The cytotoxicity of epitope-specific CD8+ T cells is usually measured indirectly through IFNγ production. Existing assays that directly measure this activity are limited mainly to measurements of up to two specificities in a single reaction. Here, we develop a multiplex cytotoxicity assay that allows direct, simultaneous measurement of up to 23 different specificities of CD8+ T cells in a single reaction. This can greatly reduce the amount of starting clinical materials for a systematic screening of CD8+ T cell epitopes. In addition, this greatly enhanced capacity enables the incorporation of irrelevant epitopes for determining the non-specific killing activity of CD8+ T cells, thereby allowing to measure the actual epitope-specific cytotoxicity activities. This technique is shown to be useful to study both human and mouse CD8+ T cells. Besides, our results from human PBMCs and three independent infectious animal models (MERS, influenza and malaria) further reveal that IFNγ expression by epitope-specific CD8+ T cells does not always correlate with their cell-killing potential, highlighting the need for using cytotoxicity assays in specific contexts (e.g., evaluating vaccine candidates). Overall, our approach opens up new possibilities for comprehensive analyses of CD8+ T cell cytotoxicity in a practical manner.


Subject(s)
Epitopes, T-Lymphocyte/immunology , Epitopes, T-Lymphocyte/isolation & purification , Flow Cytometry/methods , T-Lymphocytes, Cytotoxic/immunology , Animals , Humans , Mice , Staining and Labeling/methods
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