A flow cytometry-based proliferation assay for clinical evaluation of T-cell memory against SARS-CoV-2.
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ABSTRACT:
SUBMITTER: Lind Enoksson S
PROVIDER: S-EPMC8484816 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Vaccines against SARS-CoV-2 have alleviated infection rates, hospitalization and deaths associated with COVID-19. In order to monitor humoral immunity, several serology tests have been developed, but the recent emergence of variants of concern has revealed the need for assays that predict the neutralizing capacity of antibodies in a fast and adaptable manner. Sensitive and fast neutralization assays would allow a timely evaluation of immunity against emerging variants and support drug and vaccine discovery efforts. Here we describe a simple, fast, and cell-free multiplexed flow cytometry assay to interrogate the ability of antibodies to prevent the interaction of Angiotensin-converting enzyme 2 (ACE2) and the receptor binding domain (RBD) of the original Wuhan-1 SARS-CoV-2 strain and emerging variants simultaneously, as a surrogate neutralization assay. Using this method, we demonstrate that serum antibodies collected from representative individuals at different time-points during the pandemic present variable neutralizing activity against emerging variants, such as Omicron BA.1 and South African B.1.351. Importantly, antibodies present in samples collected during 2021, before the third dose of the vaccine was administered, do not confer complete neutralization against Omicron BA.1, as opposed to samples collected in 2022 which show significant neutralizing activity. The proposed approach has a comparable performance to other established surrogate methods such as cell-based assays using pseudotyped lentiviral particles expressing the spike of SARS-CoV-2, as demonstrated by the assessment of the blocking activity of therapeutic antibodies (i.e. Imdevimab) and serum samples. This method offers a scalable, cost effective and adaptable platform for the dynamic evaluation of antibody protection in affected populations against variants of SARS-CoV-2.
| S-EPMC9730237 | biostudies-literature
Project description:The humoral immune response to SARS-CoV-2 results in antibodies against spike (S) and nucleoprotein (N). However, whilst there are widely-available neutralization assays for S-antibodies, there is no assay for N-antibody activity. Here we present a simple in vitro method called EDNA (electroporated-antibody dependent neutralization assay) that provides a quantitative measure of N-antibody activity in unpurified serum from SARS-CoV-2 convalescents. We show that N-antibodies neutralize SARS-CoV-2 intracellularly and cell-autonomously but require the cytosolic Fc-receptor TRIM21. Using EDNA, we show that low N-antibody titres can be neutralizing, whilst some convalescents possess serum with high titres but weak activity. N-antibody and N-specific T cell activity correlates within individuals, suggesting N-antibodies may protect against SARS-CoV-2 by promoting antigen presentation. This work highlights the potential benefits of N-based vaccines and provides an in vitro assay to allow the antibodies they induce to be tested.
| S-SCDT-EMBOJ-2021-108588 | biostudies-other
Project description:The SARS-CoV-2 virus is the causing agent of the coronavirus disease 2019 (COVID-19) pandemic responsible for millions of deaths worldwide. The development of the humoral response to the virus has been the subject of intensive research. A flow cytometry-based assay using native full-length SARS-CoV-2 Spike protein expressed in 293 T cells was recently proposed as a complementary seropositivity assay. The aim of our study was to further develop the flow cytometry assay and to standardize its parameters for reliable inter-laboratory use. We have optimized the protocol, established the Receiving Operating Characteristic (ROC) curve and tested reproducibility using pre-COVID and convalescent, SARS-CoV-2 individual plasma samples. The flow-based assay was simplified and standardized by cultivating the 293 T cells in suspension and expressing results in Mean Equivalent Soluble Fluorochrome (MESF) using an internal antibody positive control. The ROC curve was determined with an area under the curve (AUC) of 0.996 and the assay specificity and sensitivity were established at 100% and 97.7% respectively. Reproducibility was good as determined on multiple cytometers, on different days, and with data acquisition as far as 72 h post-staining. The standardized assay could be used as a high throughput confirmatory assay in flow cytometry laboratories involved in serological testing.Supplementary informationThe online version contains supplementary material available at 10.1007/s10616-021-00511-1.
| S-EPMC8720558 | biostudies-literature
Project description:BackgroundNeutralizing antibodies (NAbs) against SARS-CoV-2 have been shown to correlate with protection against infection. Simple tools such as lateral flow assays (LFA) that can accurately measure NAbs may be useful for monitoring anti-SARS-CoV-2 immunity in the future.ObjectivesWe assessed the performance of the ichroma™ COVID-19 nAb test, a rapid semiquantitative LFA, for the prediction of serum neutralizing activity against SARS-CoV-2 variants.Study designSerum samples were collected from COVID-19 recovered patients and vaccinated individuals. The result of the ichroma assay was provided as inhibition rate, and was compared to anti-SARS-CoV-2 IgG levels, and NAbs against Alpha, Delta and Omicron variants.ResultsA total of 90 sera from recovered unvaccinated patients and 209 sera from the vaccine cohort were included in this study. In post-infection samples, the ichroma inhbition rate was found to be correlated with IgG levels (ρ = 0.83), and with anti-Alpha NAbs levels (ρ = 0.78). In the vaccine cohort, a good correlation was also observed between the ichroma inhibition rate and IgG levels (ρ = 0.84), as well as NAbs against Alpha (ρ = 0.62), Delta (ρ = 0.88) and Omicron (ρ = 0.74). An ichroma inhbition rate of 77.2%, 90.8% and 99.6% accurately predicted neutralization against Alpha, Delta and Omicron variants respectively.ConclusionsThe ichroma™ COVID-19 nAb assay, with appropriate variant cut-offs, can be useful for the monitoring of anti-SARS-CoV-2 immunization and may provide a rapid prediction of protection, especially in individuals with significant levels of NAbs.
| S-EPMC9383946 | biostudies-literature
Project description:BackgroundThe novel coronavirus disease 2019 (COVID-19) is an infectious disease caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which has quickly spread worldwide since its outbreak in December 2019. One of the primary measures for controlling the spread of SARS-CoV-2 infection is an accurate assay for its diagnosis. SARS-CoV-2 real-time PCR kits suffer from some limitations, including false-negative results in the clinic. Therefore, there is an urgent need for the development of a rapid antibody test kit for COVID-19 diagnosis.MethodsThe nuclear capsid protein (N) and spike protein 1 (S1) fragments of SARS-CoV-2 were expressed in Escherichia coli, and rapid antibody-based tests for the diagnosis of SARS-CoV-2 infection were developed. To evaluate their clinical applications, the serum from COVID-19 patients, suspected COVID-19 patients, recovering COVID-19 patients, patients with general fever or pulmonary infection, doctors and nurses who worked at the fever clinic, and health professionals was analyzed by the rapid antibody test kits. The serum from patients infected with Mycoplasma pneumoniae and patients with respiratory tract infection was further analyzed to test its cross-reactivity with other respiratory pathogens.ResultsA 47 kDa N protein and 67 kDa S1 fragment of SARS-CoV-2 were successfully expressed, purified, and renatured. The rapid antibody test with recombinant N protein showed higher positive rate than the rapid IgM antibody test with recombinant S1 protein. Clinical evaluation showed that the rapid antibody test kit with recombinant N protein had 88.56 % analytical sensitivity and 97.42 % specificity for COVID-19 patients, 53.48 % positive rate for suspected COVID-19 patients, 57.14 % positive rate for recovering COVID-19 patients, and 0.5-0.8 % cross-reactivity with other respiratory pathogens. The analytical sensitivity of the kit did not significantly differ in COVID-19 patients with different disease courses (p < 0.01).ConclusionsThe rapid antibody test kit with recombinant N protein has high specificity and analytical sensitivity, and can be used for the diagnosis of SARS-CoV-2 infection combined with RT-PCR.
| S-EPMC8381135 | biostudies-literature
Project description:BackgroundAs vaccines against SARS-CoV-2 are now being rolled out, a better understanding of immunity to the virus, whether from infection, or passive or active immunisation, and the durability of this protection is required. This will benefit from the ability to measure antibody-based protection to SARS-CoV-2, ideally with rapid turnaround and without the need for laboratory-based testing.MethodsWe have developed a lateral flow POC test that can measure levels of RBD-ACE2 neutralising antibody (NAb) from whole blood, with a result that can be determined by eye or quantitatively on a small instrument. We compared our lateral flow test with the gold-standard microneutralisation assay, using samples from convalescent and vaccinated donors, as well as immunised macaques.FindingsWe show a high correlation between our lateral flow test with conventional neutralisation and that this test is applicable with animal samples. We also show that this assay is readily adaptable to test for protection to newly emerging SARS-CoV-2 variants, including the beta variant which revealed a marked reduction in NAb activity. Lastly, using a cohort of vaccinated humans, we demonstrate that our whole-blood test correlates closely with microneutralisation assay data (specificity 100% and sensitivity 96% at a microneutralisation cutoff of 1:40) and that fingerprick whole blood samples are sufficient for this test.InterpretationTaken together, the COVID-19 NAb-testTM device described here provides a rapid readout of NAb based protection to SARS-CoV-2 at the point of care.FundingSupport was received from the Victorian Operational Infrastructure Support Program and the Australian Government Department of Health. This work was supported by grants from the Department of Health and Human Services of the Victorian State Government; the ARC (CE140100011, CE140100036), the NHMRC (1113293, 2002317 and 1116530), and Medical Research Future Fund Awards (2005544, 2002073, 2002132). Individual researchers were supported by an NHMRC Emerging Leadership Level 1 Investigator Grants (1194036), NHMRC APPRISE Research Fellowship (1116530), NHMRC Leadership Investigator Grant (1173871), NHMRC Principal Research Fellowship (1137285), NHMRC Investigator Grants (1177174 and 1174555) and NHMRC Senior Principal Research Fellowships (1117766 and 1136322). Grateful support was also received from the A2 Milk Company and the Jack Ma Foundation.
| S-EPMC8641961 | biostudies-literature
Project description:BackgroundIn the context of the pandemic, the rapid emergency use authorisation of diagnostic assays for SARS-CoV-2 has meant there are few peer-reviewed published studies of clinical performance of commercial assays.AimsTo evaluate the clinical performance of AusDiagnostics respiratory multiplex tandem PCR assay including SARS-CoV-2.MethodsWe reviewed the results following implementation of AusDiagnostics respiratory multiplex tandem PCR assay including SARS-CoV-2, and compared with an in-house RT-PCR assay at our State Reference Laboratory.ResultsInitial validation using AusDiagnostics coronavirus multiplex tandem PCR assay including SARS-CoV-2 demonstrated good concordance with the State Reference Laboratory. After implementing the AusDiagnostics respiratory multiplex tandem PCR assay including SARS-CoV-2, we tested 7839 samples. 127 samples in which SARS-CoV-2 was detected using the AusDiagnostics assay were referred for testing at the State Reference Laboratory, with concordant results in 118/127 (92.9%) of samples. After resolution of discrepancies, 125/127 (98.4%) of AusDiagnostics results were determined to be true positive results. Out of 7839 samples tested for SARS-CoV-2 during this period, only 2 tests (0.02%) were indeterminate results.ConclusionThe AusDiagnostics respiratory MT-PCR assay is a reliable assay for detection of SARS-CoV-2.
| S-EPMC7236671 | biostudies-literature
Project description: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.
| S-EPMC8420214 | biostudies-literature
Project description:IntroductionEvaluation of different cell-based assays for the study of adaptive immune responses against SARS-CoV-2 is crucial for studying long-term and vaccine-induced immunity.MethodsEnzyme-linked immunospot assay (ELISpot) and intracellular cytokine staining (ICS) using peptide pools spanning the spike protein and nucleoprotein of SARS-CoV-2 were performed in 25 patients who recovered from paucisymptomatic (n = 19) or severe COVID-19 (n = 6).ResultsThe proportion of paucisymptomatic patients with detectable SARS-CoV-2 T cells was low, as only 44% exhibit a positive T cell response with the ICS and 67% with the ELISpot. The magnitude of SARS-CoV-2 T cell responses was low, both with ICS (median at 0.12% among total T cells) and ELISpot (median at 61 SFCs/million peripheral blood mononuclear cells [PBMC]) assays. Moreover, T cell responses in paucisymptomatic patients seemed lower than among patients with severe disease. In the paucisymptomatic patients, the two assays were well correlated with 76% of concordant responses and a Cohen's kappa of 55. Furthermore, in four patients SARS-CoV-2 T cells were detected by ELISpot but not with ICS. Short-term culture could improve the detection of specific T cells.ConclusionsIn patients who recovered from paucisymptomatic COVID-19, the proportion of detectable anti-SARS-CoV-2 responses and their magnitude seemed lower than in patients with more severe symptoms. The ELISpot appeared to be more sensitive than the ICS assay. Short-term culture revealed that paucisymptomatic patients had nonetheless few SARS-CoV-2 T cells at a very low rate in peripheral blood. These data indicate that various ex-vivo assays may lead to different conclusions about the presence or absence of SARS-CoV-2 T cell immunity.
| S-EPMC9449588 | biostudies-literature
Project description:Monitoring antigen-specific T cell frequency, function, and phenotype is essential to assess the host immune response to pathogens or novel vaccines. Here, we describe a rapid and simple ex vivo whole blood assay to detect and phenotype the SARS-CoV-2-specific T cell response. We detail steps for whole blood stimulation with SARS-CoV-2 spike peptide and subsequent cell fixation and cryopreservation. We further describe thawing and cell staining steps for flow cytometry analysis. This approach minimizes sample manipulation and has a quick turnaround time. For complete details on the use and execution of this protocol, please refer to Riou et al. (2021).
| S-EPMC9510067 | biostudies-literature